Protein Byproducts Transformation from Environmental Burden into Value-added Products

PROTEIN BYPRODUCTS

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PROTEIN BYPRODUCTSTRANSFORMATION FROM ENVIRONMENTAL BURDEN

INTO VALUE-ADDED PRODUCTS

Edited by

Gurpreet SinGh DhillonDepartment of Agricultural

Food and Nutritional Sciences (AFNS) University of Alberta, Edmonton

AB, Canada

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v

TOC

Contents

List of Contributors ix

IGENERAL INTRODUCTION

1. Protein-Rich By-Products: Production Statistics, Legislative Restrictions,

and Management OptionsT.M. HICKS, C.J.R. VERBEEK

1. Introduction 32. Food Production Cycle and By-Products 43. Protein-Rich By-Products 64. Biosecurity and Risk Governance 105. Policy Regarding Plant and Animal By-Products 136. Current Management Options 147. Value Addition 17List of Abbreviations 17References 17

2. Agricultural-Based Protein By-Products: Characterization and Applications

G.S. DHILLON, S. KAUR, H.S. OBEROI, M.R. SPIER, S.K. BRAR

1. Introduction 212. Plant-Derived Protein By-Products 223. Animal-Derived Protein By-Products 314. Conclusions and Future Perspectives 33References 33

3. Meat Industry Protein By-Products: Sources and Characteristics

T.M. HICKS, C.J.R. VERBEEK

1. Introduction 372. The Meat Industry 383. Animal Products and By-Products 424. Characteristics of Common Protein By-Products 47

5. Innovations in By-Product Treatment and Uses 56List of Abbreviations 58References 58

4. Marine Processing Proteinaceous By-Products: A Source of Biofunctional

Food IngredientsA.C. NEVES, P.A. HARNEDY, R.J. FITZGERALD

1. Introduction 632. Fish and Shellfish Proteins 643. Biofunctional Activities 734. Bioavailability 765. Regulations for Functional Foods 776. Commercial Products Containing Marine-Derived

Bioactive Protein Hydrolysates or Peptides 787. Conclusions 78List of Abbreviations 78Acknowledgments 79References 79

IIEXTRACTION, RECOVERY,

CHARACTERIZATION, AND MODIFICATION TECHNIQUES

5. Technical Issues Related to Characterization, Extraction, Recovery,

and Purification of Proteins from Different Waste Sources

M. GONG, A.-M. AGUIRRE, A. BASSI

1. Introduction 892. Value Recovery of Protein By-Products from

Waste Materials 903. Techniques for Waste Protein Separation 954. Conclusions and Future Directions 102

vi CONTENTS

List of Abbreviations 103References 103

6. Modification of Protein Rich Algal-Biomass to Form Bioplastics and Odor Removal

K. WANG, A. MANDAL, E. AYTON, R. HUNT, M.A. ZELLER, S. SHARMA

1. Introduction 1072. Experimental 1093. Results and Discussion 1104. Conclusions 116Acknowledgments 117References 117

IIITRANSFORMATION

OF PROTEINS BY-PRODUCTS TO HIGH VALUE PRODUCTS

7. Food Industry Protein By-Products and Their Applications

L.J. YU, M.S.-L. BROOKS

1. Introduction to Food Industry By-Products 1212. Significant Sources of Food Protein By-Products 1243. Applications of Food Protein By-Products 1274. Future Directions 130List of Abbreviations 130References 131

8. Biobased Flocculants Derived from Animal Processing Protein By-Products

G.J. PIAZZA, R.A. GARCIA

1. Flocculation in Industrial Processes and Wastewater Treatment 135

2. Source of Animal By-Product Proteins 1373. Protein and Peptide Flocculants from Animal-

Processing By-Products and Other Agricultural Sources 139

4. Conclusions 144List of Abbreviations 144References 144

9. Pharmaceutical and Cosmetic Applications of Protein By-Products

Y. LUO, T. WANG

1. Introduction 1472. Sericin 1483. Whey Protein 1504. Soy Protein 1525. Zein 1556. Summary 156References 157

10. Application of Waste-Derived Proteins in the Animal Feed Industry

M. WADHWA, M.P.S. BAKSHI

1. Food-Processing Industry Wastes, Coproducts, and Residues 161

2. Animal Organic Wastes 1653. Single-Cell Protein (SCP) Production and

Utilization 1664. Poor-Quality Crop Residues (PQCRs): Processing

and Use 1715. Biofuel Coproduct–Waste Use 1726. Coproducts from Nonconventional Oilseeds

and Their Use 1767. Comparative Evaluation of Conventional and

Nonconventional Protein Supplements 1808. Industrial Wastes 1819. Conclusions and Future Prospects 182References 183

11. Novel Applications of Protein By-products in Biomedicine

M.C. GARCÍA, J.M. ORELLANA, M.L. MARINA

1. Introduction 1932. Application of Protein By-Products in

Biomedicine 1943. Animal-Origin Protein By-Products 1994. Vegetal-Origin Protein By-Products 2035. Conclusions and Future Prospects 208List of Abbreviations 208Acknowledgments 208References 208

CONTENTS vii

12. Microalgal-Based Protein By-Products: Extraction, Purification, and Applications

T. CHIONG, C. ACQUAH, S.Y. LAU, E.H. KHOR, M.K. DANQUAH

1. Background 2132. Microalgal Proteins 2153. Bioprocess Development 2194. Application of Microalgal Proteins 2255. Conclusions 230Acknowledgment 230References 230

13. Recovery and Applications of Proteins from Distillery By-Products

J.S. WHITE, J.E. TRAUB, D.L. MASKELL, P.S. HUGHES, A.J. HARPER, N.A. WILLOUGHBY

1. Introduction 2352. Distilleries and Production Processes 2373. By-Product Generation and Yield from a

Distillery 2394. By-Products as Protein Feed Ingredients 2435. Protein-Enriched By-Products 2466. Conclusions and Future Perspectives 250List of Abbreviations 251References 251

14. Recovery and Applications of Feather ProteinsN. REDDY, M.S. SANTOSH

1. Introduction 2552. Structure and Properties of Feathers and Keratin 2563. Extraction of Keratin from Feathers 2614. Applications of Feathers and Keratin 2655. Conclusions 272List of Abbreviations 272Acknowledgments 272References 273

15. Algae Derived Single-Cell Proteins: Economic Cost Analysis and Future Prospects

D.M. MAHAPATRA, H.N. CHANAKYA, T.V. RAMACHANDRA

1. Introduction 2752. Materials and Methods 279

3. Results and Discussions 2834. The Future of Algal Proteins as By-Products

from Integrated Algal Processes 296Acknowledgments 298References 299

16. Whey Proteins and their Value-Added Applications

R. SINGH, GEETANJALI

1. Introduction 3032. General Aspects of Whey Protein 3043. Value Addition to Whey Protein 3054. Concluding Remarks 310Acknowledgments 310References 310

17. Seafood Waste-Derived Peptides: Their Antioxidant Activity and Potential

as Alternative Preservatives in Fish Products

M. NIKOO, X. XU, H. AHMADI GAVLIGHI

1. Introduction 3152. Production of Antioxidant Peptides from Seafood

Waste Proteins 3163. Antioxidant Mechanisms of Seafood Waste

Peptides 3174. Examples of Antioxidant Peptides Isolated

from Waste Proteins of Fish, Molluscs, and Crustaceans 318

5. Oxidation and Protection of Fish Products 3216. Seafood Waste-Derived Peptides as Alternative

Preservatives in Fish Products 3227. Conclusions 329References 329

Index 333

ix

List of Contributors

C. Acquah Department of Chemical Engineering, Curtin University; Curtin Sarawak Research Institute, Curtin University, Miri, Sarawak, Malaysia

A.-M. Aguirre Department of Chemical and Biochemical Engineering, Faculty of Engineering, University of Western Ontario, London, Ontario, Canada

E. Ayton Department of Textiles, Merchandising & Interiors, University of Georgia, Athens, GA, United States

M.P.S. Bakshi Department of Animal Nutrition Guru Angad Dev Veterinary and Animal Science University, Ludhiana, India

A. Bassi Department of Chemical and Biochemical Engineering, Faculty of Engineering, University of Western Ontario, London, Ontario, Canada

S.K. Brar INRS, ETE, University of Quebec, QC, Canada

M.S.-L. Brooks Department of Process Engineering and Applied Science, Dalhousie University, Halifax, Nova Scotia, Canada

H.N. Chanakya Centre for Sustainable Technologies (ASTRA), Indian Institute of Science; Centre for infrastructure, Sustainable Transportation and Urban Planning [CiSTUP], Indian Institute of Science, Bangalore, Karnataka, India

T. Chiong Department of Chemical Engineering, Curtin University; Curtin Sarawak Research Institute, Curtin University, Miri, Sarawak, Malaysia

M.K. Danquah Department of Chemical Engineering, Curtin University, Miri, Sarawak, Malaysia

G.S. Dhillon Department of Agricultural, Food and Nutritional Sciences (AFNS), University of Alberta, Edmonton, AB, Canada

R.J. FitzGerald Department of Life Sciences, University of Limerick, Limerick, Ireland

M.C. García Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, Faculty of Biology, Environmental Sciences and Chemistry, University of Alcalá, Barcelona, Spain

R.A. Garcia US Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Biobased and Other Animal Coproducts Research Unit, Wyndmoor, PA, United States

H. Ahmadi Gavlighi Department of Food Science and Technology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran

Geetanjali Department of Chemistry, Kirori Mal College, University of Delhi, Delhi, India

M. Gong Department of Chemical and Biochemical Engineering, Faculty of Engineering, University of Western Ontario, London, Ontario, Canada

P.A. Harnedy Department of Life Sciences, University of Limerick, Limerick, Ireland

A.J. Harper Institute of Mechanical and Process Engineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, United Kingdom

T.M. Hicks School of Engineering, Faculty of Science and Engineering, University of Waikato, Hamilton, New Zealand

P.S. Hughes College of Agricultural Sciences, Oregon State University, Corvallis, OR, United States

x LIST OF CONTRIBUTORS

R. Hunt Algix, LLC, Meridian, MS, United States

S. Kaur Department of Biological Sciences, University of Lethbridge, AB, Canada

E.H. Khor Department of Chemical Engineering, Curtin University, Miri, Sarawak, Malaysia

S.Y. Lau Department of Chemical Engineering, Curtin University, Miri, Sarawak, Malaysia

Y. Luo Department of Nutritional Sciences, University of Connecticut, Storrs, CT, United States

D.M. Mahapatra Energy and Wetlands Research Group, Centre for Ecological Sciences, Indian Institute of Science; Centre for Sustainable Technologies (ASTRA), Indian Institute of Science, Bangalore, Karnataka, India

A. Mandal Department of Statistics, University of Georgia, Athens, GA, United States

M.L. Marina Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, Faculty of Biology, Environmental Sciences and Chemistry, University of Alcalá, Barcelona Spain

D.L. Maskell International Centre for Brewing and Distilling, Heriot-Watt University, Edinburgh, United Kingdom

A.C. Neves Department of Life Sciences, University of Limerick, Limerick, Ireland

M. Nikoo Department of Fisheries, Faculty of Natural Resources, Urmia University, Urmia, West Azerbaijan, Iran

H.S. Oberoi Department of Post-Harvest Technology Division, Indian Institute of Horticultural Research (IIHR), Bangalore, India

J.M. Orellana Animal Research Center, University of Alcalá, Barcelona, Spain

G.J. Piazza US Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Biobased and Other Animal Coproducts Research Unit, Wyndmoor, PA, United States

T.V. Ramachandra Energy and Wetlands Research Group, Centre for Ecological Sciences, Indian Institute of Science; Centre for Sustainable Technologies (ASTRA), Indian Institute of Science; Centre for infrastructure, Sustainable Transportation and Urban Planning [CiSTUP], Indian Institute of Science, Bangalore, Karnataka, India

N. Reddy Center for Emerging Technologies, Jain University, Jain Global Campus, Ramanagara District, Bengaluru, India

M.S. Santosh Center for Emerging Technologies, Jain University, Jain Global Campus, Ramanagara District, Bengaluru, India

S. Sharma Department of Textiles, Merchandising & Interiors, University of Georgia, Athens, GA, United States

R. Singh Department of Applied Chemistry, Delhi Technological University, Delhi, India

M.R. Spier Federal University of Paraná, UFPR, Post-Graduation in Food Engineering, Curitiba, Brazil

J.E. Traub Institute for Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, United Kingdom

C.J.R. Verbeek School of Engineering, Faculty of Science and Engineering, University of Waikato, Hamilton, New Zealand

M. Wadhwa Department of Animal Nutrition Guru Angad Dev Veterinary and Animal Science University, Ludhiana, India

K. Wang Department of Textiles, Merchandising & Interiors, University of Georgia, Athens, GA, United States

T. Wang Department of Nutritional Sciences, University of Connecticut, Storrs, CT, United States

J.S. White Institute for Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, United Kingdom

N.A. Willoughby Institute for Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, United Kingdom

X. Xu State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China

L.J. Yu Department of Process Engineering and Applied Science, Dalhousie University, Halifax, Nova Scotia, Canada

M.A. Zeller Algix, LLC, Meridian, MS, United States

S E C T I O N I

GENERAL INTRODUCTION1 Protein-rich by-products: production statistics, legislative restrictions, and management options 3

2 Agricultural-based protein by-products: characterization and applications 21

3 Meat industry protein by-products: sources and characteristics 37

4 Marine processing proteinaceous by-products: a source of biofunctional food ingredients 63

C H A P T E R

3Protein Byproductshttp://dx.doi.org/10.1016/B978-0-12-802391-4.00001-X Copyright © 2016 Elsevier Inc. All rights reserved.

1Protein-Rich By-Products:

Production Statistics, Legislative Restrictions, and Management

OptionsT.M. Hicks, C.J.R. Verbeek

School of Engineering, Faculty of Science and Engineering, University of Waikato, Hamilton, New Zealand

1 INTRODUCTION

Most of the world’s food is derived from ag-ricultural, horticultural, and fishery processes. With a growing population, urbanization, and increased income, the food industry has be-come increasingly market driven. As a result of globalization and reduced trade barriers, it has grown to account for approximately 10% of the world’s gross domestic product (Murray 2007). Fortunately, environmental protection and sus-tainability are currently better aligned with the worlds’ consumption of natural resources. Over the past few decades, it has tried to adopt tech-nologies to improve waste minimization and environmental performance. Although the most valuable elements are extracted from foods dur-ing harvest and processing, what remains in both the product-specific and product-nonspecific

wastes may also contain other potentially valu-able components.

Predicting future food production and associ-ated by-products is complicated and has to take into account not only changes in population size, dietary composition, land requirements, and primary resources, but also climate and environ-mental aspects (Godfray et al., 2010a). Overall, increased global demand for animal-based prod-ucts requires a substantially greater increase in plant and other feed resources, which will sub-sequently generate a much larger volume of pro-tein-rich materials than currently produced.

The quantity of food materials wasted each year is exorbitant, and urbanization and the in-creasing per capita income will see this quantity rise further through increased consumption of staple foods and through diversification into an-imal products, such as meat, fish, and dairy. This

4 1. PRODUCTION STATISTICS, LEGISLATIVE RESTRICTIONS, AND MANAGEMENT OPTIONS

I. GENERAL INTRODUCTION

will be most challenging for transitional coun-tries, which are expected to undergo a much more rapid increase in per capita meat con-sumption compared to high-income countries (ie, China will increase by ∼50%, from 49 kg in 2000 to 74 kg per capita per year in 2030 com-pared to an increase of ∼9%, from 86 kg to 95 kg per capita per year, in higher income countries) (Msangi and Rosegrant, 2011). Such nutritional transitions result in a rapid increase in animal products, putting a significant amount of pres-sure on food supply chains within transitional countries than those in the developed world.

A major facet of the problem we face, is being able to source adequate quantities of high-quali-ty protein from which to feed both humans and animals, without intensifying the overall envi-ronmental impact (van Huis, 2013). Obviously, increasing production of animal-based products will result in a much higher consumption of grain and protein feeds to feed livestock, which are es-timated to require ∼6 kg of plant protein for ev-ery kilogram of protein they produce (Pimentel and Pimentel, 2003). However, this could be bet-ter perceived by the ∼30 kg of grain required to produce 1 kg of edible boneless meat from grain-fed cattle (Foley, 2011). Conversely, while chicken and pork are more efficient converters of plant proteins, pasture-fed cattle are able to convert nonfood material into usable protein.

The technology for recovering nutrients and usable materials from industry is often feasible, but the regulations regarding what can be done with by-products of industry may not always al-low for the technology to be adopted. Despite a concerted effort to better use by-products of the agricultural and food industry to improve the management of resources, sensible legisla-tive incentives also need to be implemented. This chapter identifies areas of food production and related industries generating waste and by-products with high levels of recoverable protein, in particular, those derived from agricultural production itself. Current and future manage-ment options for the transformation and/or

disposal of these wastes and by-products are then considered in light of current legislation and technological restrictions.

2 FOOD PRODUCTION CYCLE AND BY-PRODUCTS

The modern food cycle is comprised of sev-eral stages, including agricultural production, postharvest handling and storage, food process-ing and packaging, distribution and retail, and finally, end-of-life and consumption (Fig. 1.1) (Kummu et al., 2012). Agricultural production, postharvest handling, and storage of food give rise to unintended food losses and ancillary by-products, while processing and packaging and distribution and retail result in “food waste.” Food loss, by-products, and food waste are formed at every stage of the food production process. While the generation of by-products, such as crop residues and animal by-products (ABPs) during agricultural production is consid-ered unavoidable, food losses, owing to a lack of market or degradation during handling or transportation could be avoided with care, but when considering statistics, it is often difficult to distinguish between the two.

For various reasons, approximately one-third of the food produced worldwide is wasted (Godfray

FIGURE 1.1 General food production stages, starting from agricultural production and postharvest handling and storage to processing and packaging, distribution, retail, and consumption.

2 FOOD PRODUCTION CYCLE AND BY-PRODUCTS 5


et al., 2010a; Food and Agriculture Organization of the United Nations, 2011). These wastes (and possible by-products) are created during the manufacturing processes and are often removed in order to give the product the desired sensory and nutritional qualities. Although the magnitude of food losses, by-products, and food waste var-ies depending on the product type (Table 1.1) and the stage of production considered (Table 1.2), it is strongly influenced by the technology and infra-structure available to the region.

It has been estimated that around 60 mil-lion metric tons (MMT) of ABPs are produced worldwide every year (Leoci, 2014), along with significantly higher quantities of crop residues (Santana-Méridas et al., 2012). Obviously, indus-trial processing of any food, whether it is intended for human or animal consumption (or other in-dustrial processes, such as biofuels) leads to a vast quantity of waste and by-products, typically rang-ing between 30 and 60% by weight (Table 1.1). In the case of crops, only 60% of global production is used for human consumption, mostly in the form of grains, pulses, oil plants, fruits, and vegetables, leaving 35% as by-products (used for animal fod-der) and the remaining 5% for conversion to bio-fuel and other industrial products (Foley, 2011).

In high-income regions, most food waste oc-curs during distribution and consumption, with high losses also occurring during agricultural production of plant products and fish (Table 1.2). Harvesting of crops also results in an inedible portion of the biomass (including edible prod-uct lost during harvest) contributing to what is known as crop residues. For most common edible crops, the residue-to-crop-production ratio is be-tween 0.9 and 3 to 1 (Scarlat et al., 2010). This mass is not accounted for in Table 1.2, however, typical quantities of some common food crops are given in Table 1.3. In lower-income regions, losses occur at every stage, particularly post-harvest, to a much higher degree, but occur sig-nificantly less at the consumption stage. Higher losses throughout production in low-income re-gions are an artefact of inadequate knowledge,

skills, technologies, and infrastructure to sup-port the food supply chain compared to the in-dustrialized world (Godfray et al., 2010b).

Globally, billions of tons of agro-industrial residues and by-products are generated annu-ally (Table 1.3). These include solid, liquid, and gaseous residues and can be seen as one of the most abundant, cheap, and renewable resources available (Santana-Méridas et al., 2012). Given that food waste has a typical composition of ∼30–60 wt.% starch, 10–40 wt.% lipids, and 5–10 wt.% protein (Pleissner and Lin, 2013), mil -lions of tons of protein, from plant and animal sources, could be better used. Agricultural production also has other unavoidable wastes

TABLE 1.1 Percentage of By-Products and Waste Generated During Different Production Processes

Production process

Converted to waste and by-products (%)

PLANT PRODUCTS

Cornstarch production 41–43

Fruit and vegetable processing 5–30

Potato starch production 80

Red wine production 20–30

Sugar production from sugar beet 86

Vegetable oil production 40–70

Wheat starch production 50

ANIMAL PRODUCTS

Beef slaughter 40–52

Crustacean processing 50–60

Fish canning 30–65

Fish filleting, curing, salting, smoking 50–75

Cheese production 85–90

Mollusk processing 20–50

Pig slaughter 35

Poultry slaughter 31–38

Yogurt production 2–6

Adapted from de las Fuentes et al. (2004).



associated with it, including manure and efflu-ent, which also contain high levels of recover-able protein. These by-products and wastes find new life, often as animal feed ingredients.

3 PROTEIN-RICH BY-PRODUCTS

Waste materials generated during agricul-tural production, including inedible plant and animal parts, are removed during harvesting and postharvest processing. Other unavoidable nutrient-rich wastes, such as manure and dead-stock, are also produced. Due to their high levels of recoverable protein, carbohydrate and fiber, many of the by-products and wastes of the agri-cultural industry currently find reuse as animal feeds or animal feed ingredients.

Animal feed ingredients are blended in such a way as to create a more nutritious food for live-stock. Plant-derived ingredients include grains, such as maize, barley, sorghum, oats, and wheat (which can also be used for bioethanol produc-tion), from which the by-products are often di-verted back to feed. These grain by-products include corn gluten meal, brewers and distiller’s grains, malt sprouts, brewer’s yeast, and wheat mill feed (Lefferts et al., 2006; Naik et al., 2010). More importantly, it has been assumed that by 2020, up to 10% of transportation fuels will be derived from biofuels, generating up to 100 MMT of additional protein (Scott et al., 2007). Higher value applications for inedible and nonessential amino acids derived from these by-products may eventually be commercialized, providing a feedstock for protein-based plastics,

TABLE 1.2 Combined Food Losses and Food Waste for Each Stage of the Food Production Chain, Expressed as a Weight Percentage of the (Edible Only) Incoming Resource

Agricultural production (wt.%)

Postharvest handling and storage (wt.%)

Processing and packaging (wt.%)

Distribution (wt.%)

Consumption (wt.%)

HIGH INCOME

Cereals 2 2–10 0.5–10 2 20–27

Roots and tubers 20 7–10 15 7–9 10–30

Oilseeds and pulses 6–12 0–3 5 5 4

Fruits and vegetables 10–20 4–8 2 8–12 15–28

Meat 2.9–3.5 0.6–1 5 4–6 8–11

Fish and seafood 9.4–15 0.5–2 6 9–11 8–33

Milk and dairy 3.5 0.5–1 1.2 0.5 5–15

LOW INCOME

Cereals 6 4–8 2–7 2–4 1–12

Roots and tubers 6–14 10–19 10–15 3–11 2–6

Oilseeds and pulses 6–15 3–12 8 2 1–2

Fruits and vegetables 10–20 9–10 20–25 10–17 5–12

Meat 5.1–15 0.2–1.1 5 5–7 2–8

Fish and seafood 5.1–8.2 5–6 9 10–15 2–4

Milk and dairy 3.5–6 6–11 0.1–2 8–10 0.1–4

Regions were grouped (Gustavsson et al. 2011) into medium- to high-income regions (Europe, United States, Canada, Oceania, and industrialized Asia) and low-income regions (sub-Saharan Africa, North Africa, West and Central Asia, South and Southeast Asia, and Latin America).

3 PROTEIN-RICH BY-PRODUCTS 7


TABLE 1.3 Estimates of Production By-Products and Crop Residues from Commodity Crops in Million Metric Tons (MMTs) Per Annum (Santana-Méridas et al. 2012)

Production processResidue production (MMT/year) Production process

Residue production (MMT/year)

Roots and tubers Cereals

Potato foliage, tops peels and pulps 116.7 Rice straw 457.0

Cassava peels, stalks, bagasse 82.6 Wheat straw 475.1

Fruits Barley straw 105.0

Apple pomace 20.9 Maize straw and stalks 1266.6

Orange peels, pulps and mem-branes

34.7 Maize cobsMillet

337.888.9

Legumes Banana leaves, stems/peels 183.8

Beans straw and pods 57.2 Grape pomace 20.5

Soybeans straw and pods 392.7 Slaughterhouse By-products

Oil crops Cattle

Sunflower foliage/stems 15.3 Protein meal 6.9

Olive leaves and stems 10.3 Tallow 4.2

Coconut shells, husks/fronts 18.7 Bloodmeal 0.38

Palm oil shells, husks/fronts 13.5 Sheep

Groundnuts stalks/shells 71.1 Protein meal 0.58

Rapeseed straw 73.8 Tallow 0.59

Cottonseed stalks 80.1 Bloodmeal 0.05

Tree nuts Pigs

Almond hulls and shells 0.9 Protein meal 3.7

Walnut shells 1.70 Tallow 7.6

Industrial crops Bloodmeal 0.34

Sugarcane leaves and tops 168.5 Chicken

Cotton stalks 197.6 Protein meal 5.5

Fiber crops leaves/stalks 56.9 Tallow 2.6

Vegetables Bloodmeal 0.18

Onion leaves and stems 35.0 Fish

Tomatoes leaves and stems 72.9 Protein meal 6.2

Cucumber leaves and stems 25.9

Slaughterhouse by-products calculated from the proportion of live weight in each rendering product for each species considered (Wiedemann and Yan, 2014), using the 2013 estimate of livestock slaughtered globally (Food and Agriculture Organization of the United Nations, 2013). Fishmeal estimate from 2002 (Hardy and Tacon, 2002).



biopesticides or commodity organic compounds (Naik et al., 2010; Scott et al., 2007).

Oil production by-products (oil meals and press cakes) from processing oilseeds, such as soybean, canola, sunflower seed, linseed, palm kernel and others, are also important feed in-gredients. Oil meals are obtained by solvent extraction of the oil cakes, which are obtained by pressing the seed. In 2013, 269 MMT of vari-ous oil meals were produced globally, of which 181 MMT was soymeal (United States Depart-ment of Agriculture, 2015a). In the United States alone, 36 MMT of soymeal is produced annually (United States Department of Agricul-ture, 2015a), representing more than two-thirds of the proteinaceous animal feed in the country (Lefferts et al., 2006). Other oilseed meals are lower in protein and higher in fiber and are often used for feeding ruminants. Cottonseed meal is also high in protein and is mainly used as cattle feed in the United States or as aquaculture feed. Unlike other seeds, the press cake obtained from castor seeds during castor oil production is in-edible because of its high level of phytotoxins (ricin, a toxic protein), hydrocyanides, and other allergens, however, this too has a high level of protein, ∼20–30% (Table 1.4).

Other plant ingredients may include alfalfa by-products, such as alfalfa meal, pellets, and concentrated alfalfa solubles, which are typical-ly fed to ruminants. Further, various nuts, seeds, and their by-products, such as hulls and seed screenings; legume by-products, such as bean straw meal and hulls; and even dried roots and tubers, such as sweet potatoes and chipped or pelletized cassava, find use in animal feed.

Agricultural production—specifically the production of animal-derived goods—also re-sults in by-products. In fact, around 30 wt.% of an animal produced for food is not used directly for human consumption, and downed or dead animals are another waste artefact of produc-tion. These waste materials are processed by the rendering industry, producing protein-rich products (Table 1.4). Global production of ABP

meals from rendering is in excess of 13 MMT per year (Fig. 1.2). These products include meat meal, meat and bone meal, poultry by-product meal, poultry meal, blood meal, feather meal, hydrolyzed leather and leather meal, eggshell meal, hydrolyzed hair, unborn calf carcasses, ensiled paunch, bone marrow, and dried plasma (Lefferts et al., 2006).

Other than the preceding, about 30 wt.% of the fish caught globally each year is not used directly for human consumption; instead it is used to produce protein-rich marine by-prod-ucts, in excess of 6 MMT per annum (Table 1.4). Typical animal feed ingredients derived from marine origin include fishmeal, dried fish sol-ubles, crab meal, shrimp meal, fish protein con-centrate, and other fish by-products (Lefferts et al., 2006).

Finally, animal waste has also been used as a feed ingredient, including dried ruminant waste (manure), dried poultry waste, dried poultry lit-ter, dried swine waste, undried processed animal waste products, and processed animal waste de-rivatives (Lefferts et al., 2006). According to the Association of American Feed Control Officials, in the United States, these processed animal waste products must be treated appropriately to ensure that the product is free of harmful patho-gens, pesticide residues, parasites, heavy met-als, or drug residues (Association of American Feed Control Officials, 2007). Although recycled animal wastes have been knowingly incorpo-rated into animal feed for almost 50 years, the Food and Drug Administration does not en-dorse the use of recycled animal waste (Lefferts et al., 2006). Regardless, protein content in dried manure ranges from 12 to 18 wt.% for cattle, 28 to 48 wt.% for poultry, and 22 to 25 wt.% for pigs (Chen et al., 2003), making it another source of valuable protein and nutrients.

Just as the sources of waste are diverse, so too are the wastes generated, each with a different chemical and physical makeup, directly affect-ing how they are best used (Table 1.5). Many studies focused on the valorization of these and

3 PROTEIN-RICH BY-PRODUCTS 9


TABLE 1.4 Typical Protein Content and US and Global Production Quantities in Million Metric Tons (MMTs) of Some Protein Meals Produced from the Agricultural Industry

Protein mealCrude protein (%) References

US production(MMTs)

Global production(MMTs)

PLANT PRODUCTS

Alfalfa meal 19.2 National Research Council (2001) 0.513–1.91a

Canola seed meal 37.8 National Research Council (2001) 1.07b

Castor seed cake 31–36 Annongu and Joseph (2008); Fuller et al. (1971)

Castor seed meal 20.8 Annongu and Joseph (2008)

Corn gluten meal 53.9–65.0 National Research Council (2001); Adeola (2003); Agunbiade et al. (2004)

5.9a

Cottonseed cake 21.1–57.3 Kassahun et al. (2012); Pousga et al. (2007); Khanum et al. (2007)

Cottonseed meal 34.3–44.9 National Research Council (2001); Khanum et al. (2007); El-Saidy and Gaber (2003)

0.82–1.09a,b 10.3–15.5a,c

Cow pea seed meal 32.7 El-Saidy and Saad (2008)

Linseed cake 34.7 Kassahun et al. (2012)

Linseed meal 32.6–35.4 National Research Council (2001); El-Saidy and Gaber (2003)

0.142–0.147a,b 1.02a

Peanut meal 51.8 National Research Council (2001) 0.12–0.159a,c 4.32–6.83a,b,c

Rapeseed cake 35.6 Kassahun et al. (2012)

Rapeseed meal 34.1–37.9 Khanum et al. (2007); Chu et al. (2014) 39.2b

Sesame seed cake 32.8 Kassahun et al. (2012)

Soybean cake 40.1–49.1 Agunbiade et al. (2004); Kassahun et al. (2012)

Soybean meal 44.4– 53.8 National Research Council (2001); El-Saidy and Gaber (2003); Chu et al. (2014)

39.1b 200.8b

Sunflower meal 28.4– 42.0 National Research Council (2001); El-Saidy and Gaber (2003)

0.23–0.29a,b 16.0b

ANIMAL PRODUCTS

Bloodmeal 80.2–100.5 Martínez-Llorens et al. (2008); Haughey (1976); National Research Council and Canadian Department of Agriculture (1971); Preston (2014)

Feather meal 0.63d

Hydrolyzed feather meal

81.2–92 National Research Council (2001); Preston (2014); Nengas et al. (1995)

Meat and bone meal

49.5–59.4 National Research Council (2001); National Research Council and Canadian Department of Agriculture (1971); Preston (2014); Nengas et al. (1995); Garcia and Phillips (2009); Howie et al. (1996); Kamalak et al. (2005)

1.8–2.1d,e

Meat meal 51.7–58.4 National Research Council (2001); Kamalak et al. (2005); Qiao and Thacker (2004)

2.4a

(Continued )



other waste streams in a profitable way. Obvi-ously, for protein meals that can be fed to live-stock or fish, the price for which they are sold will generally cover the cost of producing them, and in the case of ABPs, the revenue generates a

reasonable profit. However, for inedible protein meals (including meals which either have no market or limited market access), adding value through conversion into novel products is of greater necessity. The problems with imparting additional value to these products is not neces-sarily related to the scientific or technological feasibility or even cost, but are most commonly associated with the perceived risks and often re-strictive supporting legislation.

4 BIOSECURITY AND RISK GOVERNANCE

Every nation strives to maintain its biosecuri-ty to protect its ecological and economic resourc-es from disease and invasive pests. The most ef-fective means of governing the risks posed by the importation of dangerous or questionable materials, and the harm they may cause to ani-mals or humans, is to impose legal restrictions. The importance of maintaining biosecurity is most apparent when considering the risks of in-ternational trading. The introduction of invasive pests and disease through international trade could lead to adverse effects, not only on plant

Protein mealCrude protein (%) References

US production(MMTs)

Global production(MMTs)

Fish meal 59.0–68.5 National Research Council (2001); Qiao and Thacker (2004); Bimbo (2000); Trushenski and Gause (2013)

0.33a 4.1–6.2a,b,f

Poultry by-product meal

51.7–63 Preston (2014); Nengas et al. (1995); Kamalak et al. (2005); Trushenski and Gause (2013)

1.2d

Shrimp meal 22.8–50 Preston (2014); Okoye et al. (2005); Fanimo et al. (2000); Everts et al. (2003); Fanimo et al. (2006)

a Based on production statistics for 1989–1990 in Animal Feeds Compendium (1992) (Ash, 1992).b Based on a forecast for production quantities for 2014 in USDA Agricultural Statistics (2015) (United States Department of Agriculture, 2015b).c Based on production statistics for 2003–2012 in USDA Agricultural Statistics (2013) (United States Department of Agriculture, 2013).d Based on 2012 US Rendering Market Report (2013) (Swisher, 2013).e Based on US manufacturing statistics from 1992 (United States Department of Commerce, 1994).f Fishmeal production statistics 2002 (Hardy and Tacon, 2002).

TABLE 1.4 Typical Protein Content and US and Global Production Quantities in Million Metric Tons (MMTs) of Some Protein Meals Produced from the Agricultural Industry (cont.)

FIGURE 1.2 Global production estimates for animal by-product protein meals expressed in metric tons (Kaluzny 2013). Total global production ∼13 million metric tons.

4 BIOSECURITY AND RISK GOVERNANCE 11


TABLE 1.5 Residues of Food Processing and By-Products

Industry Food processed Residues and by-productsProducts from by-products

PLANTS

Grain crops

Grain, flour, bread, biscuits, crackers, cakes starch, bakery goods

Straw, stems, leaves, husks, cobs, hulls, fiber, bran, germ, gluten, steep liquor

Biomass for ethanol production

Fruits and vegetables

Tinned fruits and veg-etables, juices, vegetable oils, starches, sugars

Rotten fruits and vegetables, stem waste, pits, seeds, peels, pulp

Pectin, pigments, sweeteners, antioxi-dants, essential oils, proteins, vitamins, sterols, ethanol, yeast, enzymes

Edible oils

Oils, hydrogenated fats Press solids and oil cakes, oil water emulsions, rancid fats, shells of oilseeds

Biosurfactants

ANIMALS

Fish and seafood

Canned fish, filleted fish, smoked fish, salted fish, processed crustaceans and mollusks

Scales, fins, bones, guts, fish oil and shells

Fishmeal, fish oil, polyunsaturated fatty acids, fish protein concentrate, hydro-lysate, collagen, gela-tine, chitin, chitosan, calcium carbonate

Meat

Processed meat and poul-try products

Blood, hides, hair, heads, horns, hooves, offal, fat, meat trimmings, feathers, feet, giblets

Bloodmeal, meat meal, fat, feather meal, hydrolysate, bone meal, plasma, red blood cells, collagen, gelatine

(Continued)



and animal health, but biodiversity and food production as a whole, and should be appropri-ately managed (Maye et al., 2012).

These measures must consider not only the scientific evidence supporting such a restriction, but must also consider any reasonable precau-tions that can act to offset any deficiencies in a solely scientific approach. Hence, during the development of a new policy, a risk analysis is first performed, followed by evaluation of that risk through the lens of current legal, institu-tional, social, and economic circumstances, all of which is undertaken by the stakeholders who represent them (Mills et al., 2011). As such, risk governance deals with the management of both perceived and scientifically founded risks.

Although risk management implemented through public policy is focused at the national level, many food and natural resource policies operate at levels both below and beyond the national level (Mills et al., 2011). However, as a result of the discrepancies between each state’s local policy making and a lack of cohesive global regulations, the intersection between risk and commerce continues to be a major challenge fac-ing the international trading system.

A significant amount of trade conflict expe-rienced at the World Trade Organization has involved the United States, Canada, and/or the European Union (Hornsby, 2013). Some topics

that became the focus of either formal or infor-mal disputes have included hormone-fed beef, bovine spongiform encephalopathy (BSE), raw milk cheese, genetically modified organisms, chlorine-washed chicken, and wood packing materials. Such disputes imply the presence of a transatlantic divide over what constitutes a legitimate risk regulation, however, this is an oversimplification. Although the risk regula-tions set forth by the European Union take a precautionary approach, acting in light of scien-tific uncertainty and taking into account public concerns, the US system is based on a “sound science” approach, free from political influence, however, this has not always been the case. It has been argued that the United States used to be more precautionary than the European Union (Hornsby, 2013), but was pressured to limit the calculation of risk in public policy. The EU’s regulatory failures during food safety crises served to undermine public trust in the EU in-stitutions, resulting in the use of a precaution-ary approach (Hornsby, 2013). Overall, it has also been proposed that both regions partake in “occasional and selective application of pre-caution to different risks in different places and time” (Wiener, 2011). Nevertheless, there are some consistencies around the world regarding the safe handling, distribution, and disposal of food, animal wastes, and by-products.

Industry Food processed Residues and by-productsProducts from by-products

Dairy

Milk, butter, cream, yogurt, cheese, ice cream

Whey, wastewater Whey protein

Adapted from de las Fuentes et al. (2004) and Ramachandran et al. (2007).

TABLE 1.5 Residues of Food Processing and By-Products (cont.)

5 POLICY REGARDING PLANT AND ANIMAL BY-PRODUCTS 13


5 POLICY REGARDING PLANT AND ANIMAL BY-PRODUCTS

The degree to which protein by-products, particularly ABPs, can be used is limited by the customs, religions, and regulatory require-ments of the region. All feedstuffs imported into a country must comply with rules regarding hygiene, traceability, contaminants, labeling re-quirements, and health issues given its expect-ed use. The use of the product is then subject to more specific rules, largely limiting the use of those feedstuffs containing animal-derived products. The first diagnosis of BSE in the United Kingdom in 1986 and the subsequent publica-tion in 1996 that new variant Creutzfeldt–Jakob disease in humans had most probably arisen from exposure to BSE-infected meat, sparked a global crisis with respect to food safety and risk management.

Up until the outbreak of BSE during the 1980s, almost all protein by-products were used as feed supplements for livestock. In 1989, the practice of feeding ruminant animal protein meals to other ruminants was banned, along with the use of specified bovine offal (brain, spinal cord, oth-er organs potentially infected with BSE) (Ocker-man and Hansen, 2000). More recent infectious disease outbreaks, such as avian influenza and severe acute respiratory syndrome, have fur-ther jeopardized diplomatic relations, fright-ened the public, and caused massive economic losses by disrupting global commerce (Karesh and Cook, 2005). Since then, concern over the risks posed by ABPs, including infectious dis-eases (such as swine fever, foot and mouth) and other contaminants (such as dioxins), to hu-man and animal health, has resulted in strict regulations regarding their safe handling and disposal (Cunningham, 2003; Department for Environment Food and Rural Affairs, 2011). As such, most countries now have local regulations put in place that are typically broad in scope and directly affect any person or business that

generates, uses, disposes, stores, handles, or transports food waste containing animal prod-ucts and ABPs derived from the food processing industry.

Currently, most countries no longer allow ani-mal by-product meals containing any amount of ruminant tissue to be fed to other ruminant ani-mals, although meat and bone meals containing ruminant tissue are still able to be fed to nonru-minant animals, such as poultry, swine, pets, and aquaculture species in most countries, including New Zealand (Garcia and Phillips, 2009). To the contrary, throughout the European Union, meat and bone meals are banned from the feed of any animal that may become human food, and as a result, in the European Union, meat meal and meat and bone meal are primarily incinerated or used as an ingredient in pet food (Kirchmayr et al., 2007).

In most countries, legislation for waste dis-posal and disposal of dead animals and of slaughterhouse materials (animal rendering) is already in place. In Germany, the Animal Dis-ease Act, the Meat Hygiene Act, the Poultry Meat Act, and the Meat Hygiene Ordinance also regulate the disposal of slaughterhouse offal. To protect animal and human health, the Cana-dian Food Inspection Agency (CFIA) enforces federal regulations governing the production and use of rendered materials that may be used in animal feed. However, a policy established by the National Renderers Association, which prevented ovine material (sheep) from being used in meat and bone meals in the United States and Canada, and has been withdrawn (Malone, 2005).

Compared to Canadian and US policy, the framework of the EU regulations regarding ABPs and derived products is complex, resulting from ongoing reviews by the EU Commission. Each updated regulation is a result of the succes-sive amendment to the initial Regulation (EC) 1774/2002, most recently amended with (EU) No. 749/2011. The regulation covers the safe disposal



options available for all animal products, includ-ing meat, fish, milk, and eggs not intended for human consumption, and other products of animal origin, including hides, feathers, wool, bones, horns, and hooves. It also prohibits cater-ing waste being used as livestock feed and cov-ers disposal of fallen stock, companion animals, and wild animals if they are suspected of being diseased. The regulations also control the use of ABPs as feed, fertilizer, and technical products with rules for their transformation via compost-ing and biogas operations and their disposal via rendering and incineration (Department for En-vironment Food and Rural Affairs, 2011).

6 CURRENT MANAGEMENT OPTIONS

When looking at the existing options avail-able for management of these by-products (and/or wastes), both legal regulations and the best ecological and economical solutions need to be considered. Whether a material is deemed to be a valuable by-product (or a waste that needs to be disposed of) depends on the social, legal, and technological framework surrounding its origin. From there, the most sensible form of manage-ment becomes a compromise between what is viewed as acceptable, based on legal require-ments and local perceptions, and what is tech-nologically and financially feasible (Fig. 1.3).

Although it is most desirable to prevent waste and by-product formation, followed by reuse or recycling into other product lines, the forma-tion of by-products and waste is inevitable, and management options must be innovative and also meet local regulatory requirements. Waste management is then possible through several media: to use it in its current form, dispose of it through incineration or landfill, or add val-ue to it through bioprocessing or valorization technologies (Fig. 1.4). The choice of media used will largely depend on the cost, customs, and regulatory environment. For example,

converting the by-product to animal fodder (bio-reduction) may not be feasible in all countries.

Excess and waste food has been used as ani-mal fodder for centuries, and in many parts of the world, farmers still use waste food to feed their animals—primarily pigs and poultry. The practice of feeding waste material containing meat products to pigs was banned in the United Kingdom in 2001 (Statutory Instrument 2001, No. 1704 The Animal By-products Amendment) to prevent further spread of BSE, and soon after, a new regulation was implemented throughout the European Union (The Animal By-Products Regulation, EC No: 1774/2002) prohibiting ca-tering waste from being fed to farmed animals. This includes all waste food and used cooking oils, as well as waste from vegetarian restaurants and kitchens. Based on these laws, only certain types of waste food can be given to livestock and must first be treated appropriately.

If the by-product cannot be immediately used as it is or treated appropriately for use as an animal feed, it must be safely disposed of.

FIGURE 1.3 Forming a sensible waste management system relies on compromise between public perception, legislation, cost, and technologies.

6 CURRENT MANAGEMENT OPTIONS 15


Because of the time and expense of treating these food wastes, most end up in landfill. Currently, landfilling and incineration account for the treat-ment of greater than 95% of food waste in most European countries (Melikoglu et al., 2013). In general, using the biomass waste in the form it is in, either as an animal feed or fertilizer or as a fuel to generate electricity, is the most simplistic approach and generates a value of ∼US$70–200 per metric ton of biomass (Tuck et al., 2012).

6.1 Incineration

Incineration is the simplest means of waste disposal, with its major advantage being the significant reduction in volume of the waste stream, which is up to 90% for waste streams with high amounts of paper, cardboard, plastics, and horticultural waste (Hoornweg and Bhada-Tata, 2012). However, most food wastes are not appropriate for incineration, owing to their high moisture content. When properly equipped, an

incinerator can be used as a means of energy recovery to generate electricity. Heat released from the combustion of waste can be used to produce steam, which can turn a steam turbine, generating electricity. However, because of the increased concentration of toxins in the ash, in-cinerators must be operated alongside landfill systems in order to dispose of them. Combus-tion destroys chemical compounds and disease-causing bacteria, leaving it pathogen free, but causes serious environmental problems through the production of carbon dioxide, nitrogen ox-ides, sulfur dioxide, and trace quantities of toxic pollutants, such as heavy metals and dioxins. The remaining residues are often landfilled, ow-ing to their high heavy metal content.

6.2 Pyrolysis and Gasification

Thermochemical conversion of food and in-dustry wastes are an effective means of convert-ing energy-rich biomass into a more easily used

FIGURE 1.4 Flow diagram for conversion of agro-industrial by-products and crop residues.



liquid or gaseous intermediate. High tempera-tures can be used with minimal (gasification) or no oxygen present (pyrolysis) to break down hydrocarbon containing wastes, resulting in com-bustible syngas mixtures, containing carbon mon-oxide and hydrogen (85%), with small amounts of carbon dioxide and methane. This syngas in-termediate can be further processed to produce bio-based gasoline, diesel, or jet fuel, or be used in a fuel cell to generate electricity or steam.

6.3 Landfilling

Landfills— burying the material—are a com-mon final disposal site for waste and the resi-dues remaining from other treatment options. At atmospheric pressure, 1 metric ton of organic material generates approximately 200–500 m3 of landfill gas over a 10–20 year timeframe (Jardine et al., 2004), comprised of 60–65 % methane and 35–40 % carbon dioxide, which represents around 8% of the anthropogenic methane (CH4) emitted worldwide (Melikoglu et al., 2013). Methane has 21 times the global warming potential of carbon dioxide and can be recovered and burned (with or without energy recovery) to reduce green-house gas emissions (Hoornweg and Bhada-Tata, 2012). Other serious environmental impli-cations of landfilling include the risk of leachate (potential toxic liquid that drains from landfills) entering surrounding soils and groundwater.

Although the use of landfills is common, their use has been discouraged through the implemen-tation of landfill taxes and directives, such as the UK “Landfill Tax” in 1996 and EU Landfill Direc-tive established in 1999 (Jardine et al., 2004). Ob-viously, other disposal options are preferred to landfilling, which costs ∼US$400 per metric ton.

6.4 Bioprocessing

Around 60% of the municipal waste sent to landfill is biodegradable and mostly comprised of food waste (Hoornweg and Bhada-Tata, 2012). This makes bioprocessing, such as composting

and anaerobic digestion, sensible options for disposing of these organic waste streams.

A common means of obtaining a safe end product is achieved through composting. This involves a combination of chemical and micro-biological processes occurring throughout three stages that convert organic materials to a stable, soil-like product called compost (Som et al., 2009; Verbeek et al., 2012). Provided composting is car-ried out well, the volume and mass of the waste can be reduced by up to 40%. For composting to occur efficiently, the conditions of the compost-ing process must be maintained at an optimal level to encourage microbial growth. Because of changes in the composition of waste material with location and over time, the compost mixture needs optimization through regular adjustments. For example, if the system becomes anaerobic, of-fensive odors can be produced, and if it becomes too wet or too dry, the process will halt altogeth-er. Some of these organic waste materials require specific pretreatment before composting can oc-cur. In the United Kingdom, EU standards must be implemented over and above UK standards if the site treats category 2 ABPs, which have first been pressure rendered, or category 3 ABPs if they exclude catering waste. Exceptions apply for some types of ABPs in the United Kingdom, which can be composted in closed reactors at 70°C for more than 1 h or in housed rows of piled green-waste (windrows) at 60°C for more than 8 days under strict operating parameters with a maximum particle size of 400 mm.

Although compost is of limited value, it is still a more economic option compared to landfilling. Other bioprocesses can be employed that produce more valuable products. Biofuels can be pro-duced using fermentation, valued at US$ 200–400 per metric ton more than the initial biomass waste (Tuck et al., 2012). Anaerobic digestion is another means of disposing of organic waste materials and is carried out in an enclosed vessel. The meth-ane generated can either be flared or collected for combustion to generate heat and/or electricity, which also adds value to the waste biomass.


REFERENCES 17

The maximum value can be recovered from these waste materials by converting them into more purified streams and using them in the manufacture of lubricants, surfactants, plastics, fibers, and industrial solvents. Theoretically, all ABPs in the European Union could be combust-ed as fuel for energy, provided the EU Commis-sion formulates the appropriate rules and regu-lations, which as of yet has not been done.

Although there are many technologies cur-rently available (or in developmental stages) that aim to valorize by-products of industry, leg-islation has yet to be passed that explicitly deals with higher technology outcomes. Most current law deals with the safe handling and disposal of animals, their products, and by-products and animal feeding. Although it is necessary to con-tain health and environmental risks through appropriate legislation, it is becoming apparent that the use of ABPs and food wastes (exclud-ing crop residues and some agro-industrial by-products) for animal fodder and composting is not only obsolete, but in many nations, illegal.

7 VALUE ADDITION

Many technologies exist that aim to valorize by-products of the agricultural industry. Al-though the edible portion of these protein-rich by-products could be used for recovery of essen-tial amino acids for human consumption, or as is for use in animal feeds, higher value applica-tions for inedible and nonessential amino acids may include providing a feedstock for protein-based materials, such as plastics, and for the pro-duction of biopesticides and commodity organic compounds (Naik et al., 2010; Scott et al., 2007).

Along with more obvious uses of protein hydrolysates—animal feeds and biomass for energy recovery—protein-based meals from crop residues and agro-industrial by-products also find value addition through use in bio-logical processes. An example is the use of vari-ous oilseed cakes, which have been shown to

be ideal mediums for many types of bacteria and fungi responsible for producing a variety of enzymes, antibiotic and antimicrobial com-pounds, and bioactive metabolites (Ramachan-dran et al., 2007). Protein-based raw materials can be used for the production of 1,2-ethanedi-amine and 1,4-butanediamine from the amino acids serine and arginine, respectively (Sanders et al., 2007). Furthermore, protein-based sur-factants are valuable mild surfactants, because the structure and properties of the amino acids in the surfactants are similar to the amino acids that make up the tissue of skin.

If valorization technologies are to be imple-mented on a commercial scale, they must work within current legal constructs. However, this does not deal directly with the science involved and may inhibit progress if new legislation is not developed that more closely examines the evidence and whether risk regarding human and animal health is still an issue. In light of current legislation and potential markets for value-added commodities, it is becoming apparent that the use of protein-rich agricultural by-products for lower value applications, such as animal fodder, is no longer a sensible use of such a valuable resource.

LIST OF ABBREVIATIONS

ABP Aanimal by-productBSE Bovine spongiform encephalopathyCFIA Canadian Food Inspection AgencyEC European CommissionEU European UnionUK United KingdomUS United States

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C H A P T E R

21Protein Byproductshttp://dx.doi.org/10.1016/B978-0-12-802391-4.00002-1 Copyright © 2016 Elsevier Inc. All rights reserved.

2Agricultural-Based Protein

By-Products: Characterization and Applications

G.S. Dhillon*, S. Kaur**, H.S. Oberoi†, M.R. Spier‡, S.K. Brar§

*Department of Agricultural, Food and Nutritional Sciences (AFNS), University of Alberta, Edmonton, AB, Canada; **Department of Biological Sciences, University of Lethbridge, AB, Canada; †Department of Post-Harvest Technology division, Indian Institute of Horticultural Research (IIHR), Bangalore, India; ‡Federal University of Paraná, UFPR, Post-Graduation in Food Engineering, Curitiba, Brazil; §INRS, ETE,

University of Quebec, QC, Canada

1 INTRODUCTION

The abundant quantity of agro-industrial wastes and by-products are produced both in the organized and unorganized sectors through-out the world. Most of the time, these valu-able resources are not efficiently used or find low-value applications, such as animal feed or soil composting. These waste feedstocks or by-products can be efficiently used for the extrac-tion and production of high-value-added prod-ucts, such as amino acids, bioactive peptides, antimicrobials, enzymes, edible oils, essential oils, polyphenolic compounds, pigments, food

additives, anticarcinogenic compounds, dietary fiber, biofuels, single-cell proteins, organic acid-fermented beverages, compost, and biodegrad-able plastics.

Recently, the growing awareness of nutrition, health, and environmental consciousness of con-sumers is compelling researchers to search for sources of good quality sustainable plant proteins for food applications. With the increased industri-al demand of protein sources in the food industry, there has been a recent upsurge of research efforts to extract dietary proteins from plant- and animal-based waste feedstocks or by-products. Besides food applications, proteinaceous feedstocks find

22 2. AGRICULTURAL-BASED PROTEIN BY-PRODUCTS: CHARACTERIZATION AND APPLICATIONS

1. GENERAL INTRODUCTION

various other applications, such as biopolymers, biocomposites, bioplastics, wastewater treatment, and agriculture.

The potential agricultural-based sources for the extraction of proteins are fruit- and veg-etable-processing industries, distilleries, and oilseed by-products. Agricultural produce pro-cessing results in an abundant quantity of by-products, including protein-rich by-products. By-products resulting from the production of bioenergy are also generally rich in proteins, because of the utilization of carbohydrate and lipid fractions. These sustainable low-cost and abundant protein by-products can be consid-ered as feedstocks for various applications, such as food formulations, biomedicine, bioplastics, biopolymers, among others. This chapter mainly focuses on the plant- and animal-based protein by-products, their physicochemical and biologi-cal characterization, and finally their high-value applications.

2 PLANT-DERIVED PROTEIN BY-PRODUCTS

2.1 By-Products from Bioenergy Production, Breweries, and Wineries

Because of the quick depletion of nonrenew-able fossil fuel reserves, biofuel production is in-creasing at a fast pace. Biofuels are generally pro-duced from renewable agricultural feedstocks. Hence, by-products resulting from bioenergy (eg, bioethanol and biodiesel production) and breweries and wineries are produced in abun-dant quantities worldwide. These by-products are considered a rich source of proteins.

Corn (Zea mays L.) is one of the most impor-tant food and industrial crops in the United States. It is widely used for the production of bioethanol. In the 2014-15 marketing year, world annual production of corn was around 988.077 million metric tons (MMT), of which the US alone contributed 36.5% (http://www.barchart.com/commodityfutures/Tocom_Corn_Futures/

profile/CVF15). The production of corn is ex-pected to increase because of its utilization for bioethanol production. Distiller’s grain, which is a by-product of bioethanol manufacturing, mainly from corn and wheat, comes in different forms. Lipids and carbohydrates are used for biofuels production, and the leftover fraction, distiller’s grain, is rich in protein content. Protein is the second most abundant component of corn after starch. The protein content of different corn varieties ranges from 6% to 12% on a dry basis.

Wine is an alcoholic beverage made from fermented grapes or other fruits, such as pome-granates, berries, and apples. In 2014, the total worldwide production was 28,230,400 (liters per thousand) (Teixeira et al., 2014). The top four wine producers are France (16.54%), Italy (15.85%), Spain (13.53%), and the United States (10.77%) (www.wineinstitute.org/resources/statistics). The wine-making process generates a large amount of solid waste (up to 30% w/w of the material used), mainly consisting of organic wastes, namely fruit stems, skins, and seeds. Bioactive compounds from winery by-products have disclosed interesting health-promoting activities both in vitro and in vivo (Teixeira et al., 2014). The by-products resulting from vi-nification contain appreciable amounts of pro-tein, which currently do not find any high-value applications. Moreover, the type of by-products produced during wine making is closely de-pendent on the specific vinification procedures, which also affect the physicochemical properties of the by-products, the characteristics of which determine its further use and specific valoriza-tion path in which it could be integrated.

Similarly, biodiesel is produced from Jatro-pha oil, melon, palm oil, soybean, rapeseed oil, sunflower oil, and used oil can be used to make biodiesel. Jatropha is very important in the production of biodiesel because it is a noned-ible plant and hence does not compete with the food oils. The solid by-products obtained after oil extraction from seeds are known as oil cakes or oil meal. Their composition varies widely

http://www.barchart.com/commodityfutures/Tocom_Corn_Futures/profile/CVF15



http://www.wineinstitute.org/resources/statistics

http://www.wineinstitute.org/resources/statistics

2 PLANT-DERIVED PROTEIN BY-PRODUCTS 23


depending on the quality of seeds or nuts, grow-ing conditions, and extraction methods. Oil cakes can be either edible or nonedible. Edible cakes have a high protein content ranging from 15% to 50%. The compositions of oil cakes origi-nating from different types of plants are listed in Table 2.1. The protein content in different oil cakes ranges between 6.3% and 49.5%. Oil cakes are currently used mainly for feed applications to poultry, ruminant, and in the fish and swine industries (Franke et al., 2009; Molina-Alcaide and Yanez-Ruiz, 2008; Mushtaq et al., 2009; Soren and Sastry, 2009). Some of them are con-sidered to be suitable organic nitrogenous fertil-izers. Several cakes have been used for produc-tion of proteins, enzymes, antibiotics, vitamins, antioxidants, mushrooms, and ethanol (Bernes-son, 2007; Cervero et al., 2010; Soren and Sas-try, 2009; Vastag et al., 2011).

The by-products resulting from bioetha-nol, biodiesel, and breweries and wineries are widely used as an animal and aquaculture feed (Traub, 2014). Further development of the high-value applications of the by-products resulting from biofuels production will enhance the eco-nomic viability of the overall biofuel production process.

2.2 Oil Crops

The amino acid content of different agricul-tural by-products is provided in Table 2.2.

Canola protein (CP): Canola or rapeseed (Bras-sica napus) is an important oilseed crop in many countries and is the second most abundant source of edible oil in the world. Canola seeds contain approximately 40% oil and 17–26% pro-tein (Uppstrom, 1995). Canola meal (CM), the by-product of canola oil extraction, is highly proteinaceous and contains up to 50% protein on a dry basis. The main protein constituents of CM are napin and cruciferin, the storage pro-teins, and oleosin, a structural protein associ-ated with the oil fraction (Uppstrom, 1995). CP is currently used for human food and animal feed. However, because of its abundance, CM can be exploited for other nonfood applications, including bioflocculation.

Soy protein (SP): This protein is produced from soybeans (Glycine max) by a multistep process that removes the oil and indigestible compo-nents. Depending on the processing steps used, SP ingredients may take the form of isolated soy protein (ISP), SP concentrate, or soy flour. Cur-rently, SP find applications in human food and an-imal feed (Hertrampf and Piedad-Pascual, 2000; Montgomery, 2010). It is a potential raw material for SP-based bioflocculants. Recent studies dem-onstrated the ability of SP-based bioflocculants for the treatment of diatomite, kaolin, and kanto loam (Liu et al., 2012; Piazza and Garcia, 2010a; Seki et al., 2010). Considering the abundant pro-duction of SP, various other high-value applica-tions can also be developed.

2.3 Cereal Proteins

Barley proteins: Barley grain (Hordeum vulgare L.) is the fourth most widely grown cereal in the world next to wheat, rice, and corn (Yalçin et al., 2008). In 2009 and 2010, global production of barley was 152 and 124 MMT, respectively (FAO-STAT, 2010). In Canada, nearly 80% of the barley

TABLE 2.1 The Composition of Oil Cakes Originating from Different Types of Plant Sources

Oil cake Dry matter (%)Protein content (%)

Canola oil cake 90.0 33.9

Coconut oil cake 88.8 25.2

Cottonseed cake 94.3 40.3

Groundnut oil cake 92.6 49.5

Mustard oil cake 89.8 38.5

Olive oil cake 85.2 6.3

Palm kernel cake 90.8 18.6

Sesame oil cake 83.2 35.6

Soy bean cake 84.8 47.5

Sunflower oil cake 91.0 34.1

Adapted from Kolesarova et al. (2011).

24

2. AG

RIC

ULT

UR

AL-B

ASED

PRO

TEIN

BY-PR

OD

UC

TS: C

HA

RA

CT

ERIZA

TIO

N A

ND

APPLIC

AT

ION

S

1. GE

NE

RA

L INT

RO

DU

CT

ION

TABLE 2.2 Amino Acid Composition of Different Proteins (g Amino Acid: 100 g Unless Otherwise Mentioned)

Class Amino acid

Zein (Mossé, 1961; Pomes, 1971)

Canola (Brassica napus, cv. Altex) (Tzeng et al., 1988a, b)

Soy protein isolate (Wang et al., 2008)

Rice bran protein (RBP) (Wang et al., 1999)

Casein (Wang et al., 2010)

Barley protein fractions PGF/PF proteins (%) (Wang et al., 2010)

Tomato Seeds protein (mg/g protein) (Sarkar and Kaul, 2014)

Basic Lysine 0 6.60 5.23 ± 0.01 5.5 7.10 1.81/5.03 59.63

Arginine 1.8–4.71 7.28 7.35 ± 0.35 9.0 3.30 3.91/4.14 —

Histidine 1.1–1.32 3.18 2.81 ± 0.24 3.0 2.70 2.32/1.81 25.01

Acidic Aspartic acid (as asparagine)

4.61 (4.5) 7.79a 11.47 ± 0.71a 10.5a 6.30a (4.34/9.05) —

Glutamic acid (as glutamine)

1.5–26.9 (21.4)

20.81b 20.67 ± 0.83b 15.3b 19.0b (26.74/15.15) —

–OH Serine 5.7–7.05 4.41 5.32 ± 0.09 5.3 4.60 5.23/5.02 —

Threonine 2.7–3.45 4.81 3.98 ± 0.13 4.4 3.70 3.50/4.52 36.49

Tyrosine 5.1–5.25 3.19 3.61 ± 0.18 3.7 5.50 3.46/2.56 —

–S Methionine 2.0–2.41 2.24 0.92 ± 0.07 2.0 2.60 1.56/1.70 —

Cysteine 0.8–0.83 2.08 0.05 ± 0 2.6 0.04 1.33/0.31 —

Nonpolar Glycine 0–0.7 4.60 3.74 ± 0.27 6.1 1.60 3.54/8.31 —

Alanine 8.3–10.52 4.53 3.72 ± 0.06 6.8 2.70 4.52/9.29 —

Valine 3.1–3.98 5.65 4.28 ± 0.32 5.7 6.00 5.61/8.03 55.19

Leucine 19.3–21.1 7.47 6.79 ± 0.83 8.0 8.40 7.56/7.22 77.90

isoleucine 5–6.2 4.47 4.35 ± 0.11 3.0 4.90 3.46/3.05 49.30

Phenylalanine 6.8–7.3 4.67 5.14 ± 0.58 5.1 4.50 4.54/3.16

Tryptophan 0.16 ND ND 7.0 ND ND/3.56 12.36

Proline 9.0–10.53 6.22 5.13 ± 0.33 ND ND 16.57/8.09 —

SSA — — — — — — 30.58

AAA — — — — — — 87.32

AAA, aromatic-containing amino acids; ND, not detectable; PGF, pearled grain flour; PF, pearling flour; SSA, sulfur-containing amino acids.a Asparate + asparagine.b Glutamate + glutamine.



crop is used as livestock feed, 15% for malting, and <5% for direct human intake. Recently, there has been an increasing interest in barley as a food ingredient, which is attributed to the health ben-efits of barley β-glucan, lignans, calcium, and selenium. β-glucan, a soluble dietary fiber com-ponent, reduces blood cholesterol and glycemic index (Behall et al., 2006; Keenan et al., 2007). The approval of a health claim for soluble barley β-glucan by the US Food and Drug Administra-tion (FDA) further increased research efforts for barley food product development (FDA, 2005). After β-glucan isolation from barley grains, the remaining portions are a good source of proteins, starch, and lipids (Baik and Ullrich, 2008). Cur-rently, there is no commercial application of these fractions. Therefore, research in this direction is sought to develop their full value.

The total barley grain protein composition varies from 8% to 13% w/w, with various barley grain tissues enriched with specific protein types at different levels (Pomeranz and Shands, 1974). The major proteins in barley endosperm are hor-dein (35–45%) and glutelin (35–45%), whereas barley bran and germ are enriched in cytoplas-mic proteins (mainly albumin and globulin) (Lâsztity, 1984). These protein fractions may display different functional qualities for vari-ous applications owing to different molecular structures. Generally, barley bran is removed from barley grains to yield a bright white kernel for various food applications through a barley milling process known as pearling. The removed grain layers are called the pearling flour (PF), and the rest is called the pearled grain floor (PGF). The PF is a rich source of albumin and globulin pro-teins (Yeung and Vasanthan, 2001). However, it is an underused and inexpensive by-product.

In brewing industries, barley endosperm proteins (hordein and glutelin) are considered undesirable compounds or even contaminants and are normally precipitated out in the barley spent grain (BSG). Hordein and glutelin proteins (20–30% w/w) in BSG are almost three times that of the whole grain (Jadhav et al., 1998). BSG is

generally used as animal feed. Limited research has been carried out to evaluate barley protein fractions, focused mainly on hordein or whole-grain proteins (Bamforth and Milani, 2004; Bilgi and Çelik, 2004; Kapp and Bamforth, 2002).The thorough research to identify value-added ap-plications for barley processing by-products is desirable. This will facilitate the expanded pros-pects for barley protein fractions in food and nonfood applications.

The solubility profile of hordein indicates that it has low solubility at pH 3–8 with the least value attained at pH 5. High solubility was observed at pH 10. A similar solubility profile was observed for the glutelin and PGF protein fractions, with the minimum values obtained at pH 5. An increase in protein solubility was also observed at pH 10 (Wang et al., 2010). The pH profile indicates that these proteins are suitable for their application as flocculants for the treat-ment of wastewater streams having pH above neutral; for example, oil sand tailings with pH levels <8.5 (Table 2.3).

Zein: It is the major storage protein of corn and comprises 45–50% of the total protein in corn. Currently, zein production is nearly 500 tons/year from corn gluten meal. Zein belongs to the characteristic class of proteins known as prolamines, which occur specifically in cereals (corresponding to hordein in barley and gliadin in wheat). Zein is not suitable for human intake owing to its negative nitrogen balance and poor solubility in water. The other main disadvan-tage to utilization of zein in human foodstuffs is their poor “functionality.” Functional prop-erties refer to those attributes that provide the desired physical or sensory properties to the food. For example, proteins do not only con-tribute nutritional quality to a food, but also contribute textural properties, such as gelation (essential in meats, cheeses, gels, and so forth), adhesion (meats, bakery, pasta), emulsification (deli meats, soups, cakes), foaming or whipping (cakes, frozen desserts), and moisture sorption (intermediate moisture, shelf-stable foods).



Zein is soluble in concentrated ethanol, high concentrations of urea, high concentrations of alkali (pH 11 or higher) or anionic detergents. Currently, zein is used for making fiber, adhe-sive, coating, ceramic, ink, cosmetic, textile, chewing gum, and biodegradable plastics. These new applications of zein appear prom-ising, but require the development of low-cost manufacturing methods. Currently, zein prices vary from US$10 to $40 per kg, depending on purity, representing a very high-value addition to corn. However, it also confines more wide-spread applications of zein. This mandates find-ing practical ways to significantly lower the cost of manufacturing and to increase the zein use.

Zein is mainly rich in glutamic acid (21–26%), leucine (20%), proline (10%), and alanine (10%), but deficient in basic and acidic amino acids (Table 2.2). High content of nonpolar amino acid

residues and paucity in basic and acid amino acids accounts for the solubility behavior of zein. In whole corn, zein occurs as a heteroge-neous mixture of disulfide-linked aggregates with a average molecular weight of 44,000 Da (Pomes, 1971). The high isoelectric pH of zein (pH 5–9), indicates its potential as a flocculating agent to be used in wastewater treatments hav-ing pH levels higher than neutral. However, the poor solubility of zein restricts its use in various industrial applications. Hence, different modifi-cation techniques are sought to increase its solu-bility and extend its applications in various sec-tors, such as food, bioplastics, and biopolymers, among others.

Gluten: It is the main structural protein com-posite of wheat and other cereals, including rye and barley. The protein fraction of gluten comprises gliadins and glutenins, with gliadins

TABLE 2.3 Characteristics of Protein By-Products Derived from Different Agricultural Sources

Protein Isoelectric pH Remarks Reference

Albumin 4.7 Used for commercial applications Ge et al. (1998)

Barley proteins 5 Barley bran and germ are rich in cytoplasmic proteins (albumin and globulin)

Barley floor contains endosperm proteins (hordein and glutelin.

No commercial applicationHigh solubility at pH 10 makes it ideal for treat-

ment of wastewaters with pH above neutral

Wang et al. (2010)

Canola meal protein

4.5–5.5 (best 5) High production of canola

Casein 4.6 Chen et al. (1987)

Collagen 6 Maslennikova et al. (2013)

Corn zein 6.2 (varies between 5 and 9) Insoluble in water Shukla and Cheryan (2001)

Gelatin 4.8 Used for commercial applications Maslennikova et al. (2013)

Gluten Nearly 6 Wang et al. (2006)

Hordeins 6.5–8 Faulks et al. (1981)

Keratin 5.6–6 Cooper and Sun (1986)

Rice proteins 4.4 No current applications. Rice polishing pro-duces large quantities of wastewater rich in proteins and starch

Watanabe et al. (2011)

Soya protein 4.5–5.5 Seki et al. (2010)



containing monomeric proteins and gluten-ins containing aggregated proteins. Recently, Sapone et al. (2012) thoroughly reviewed the gluten-related disorders. Besides the well-known disorder of celiac disease (CD), there are spectrum of other acknowledged gluten-related disorders. Hence, the number of individuals preferring a gluten-free diet is much higher than the projected number of CD patients, fueling a global market of gluten-free products reaching US$2.5 billion in worldwide sales in 2010. This trend is supported by the concept that along with CD, other conditions related to the inges-tion of gluten have emerged as health concerns among many individuals in the world, which has caused emerging health care concerns and reduced its market potential as food ingredient. Because of consumer preference for gluten-free diets, gluten has become a significant by-prod-uct of grain production. Hence, new applica-tions of gluten should be explored.

Rice proteins: Rice, a cereal grain, is the most widely consumed staple food for a large part of the world’s human population, especially in Asia (Vaughan, 1994). The demand for polished rice, so-called “rinse-free rice,” has increased. As a result, rice manufacturing plants produce a large amount of rice washing drainage, which is a water pollution source. This drainage con-tains a relatively high amount of solid particles composed of starch and proteins (a by-product consisting of the inner bran layers of the kernel with part of the germ and a small portion of the starchy interior) produced during rice pol-ishing (Watanabe et al., 2006, 2009). There is no practical application of this protein-rich waste. Economical processes can be developed for the extraction of proteins and starch for various ap-plications.

2.4 Fruit Wastes and By-Products

Agro-based industries, especially apple-pro-cessing industries, are experiencing a surge in their growth around the globe. This enormous

increase in fruit processing has been generat-ing millions of tons of agro-industrial wastes (both solid and liquid by-product) worldwide. These by-products are generally rich sources of proteins and other bioactive compounds. Crude protein composition (percent DM basis) of veg-etable and fruit wastes are provided in Table 2.4.

Grapes (Vitis vinifera L.): Currently, up to 210 million tons of grapes are produced annually, with 15% of the produced grapes produced for the wine-making industry. Grapes are consid-ered as a rich source of bioactive compounds. The major by-products from wine-making ac-tivity are organic wastes (grape pomace, con-taining seeds, pulp and skins, grape stems, and grape leaves), wastewater, and inorganic wastes (diatomaceous earth, bentonite clay, and perlite) (Oliveira et al., 2013).

Grape pomace is produced during the pro-duction of must (grape juice) by pressing whole grapes. Currently, 9 million tons of grape pom-ace are produced per year in the world, which constitutes 20–30% w/w on average of the to-tal grapes used for wine production (Dwyer et al., 2014; Kataliníc et al., 2010). The moisture percentage of grape pomace varies from 50% to 72% depending on the grape variety considered and its ripening state. The insoluble residues from this material have a lignin content ranging from 16.8% to 24.2% and a protein content lower than 4%. The main polymer-type constituent of the cell walls present in grape pomace are peptic substances, ranging from 37% to 54% of cell wall polysaccharides. Cellulose is the second type of cell wall polysaccharides in abundance in grape pomace, varying from 27% to 37% (González-Centeno et al., 2010). However, this high content of nondigestible polysaccharides results in gas-trointestinal disturbances in animals. Therefore additional fermentation processes may be re-quired for its valorization to animal feed.

Considering the separate fractions of grape pomace (seeds and peels), the relative propor-tion of seeds ranges from 15% to 52% of the dry material (Ghafoor et al., 2009; Nawaz et al., 2006;



Stamatina et al., 1995). The composition of grape seeds (w/w) consists of up to 40% fiber, 16% es-sential oil, 11% protein, 7% complex phenolic compounds, like tannins, and other substances like sugars and minerals (Campos et al., 2008). Likewise, grape skins constitute 65% of the to-tal material of grape pomace on average. Grape skin has been reported as a rich source of pheno-lic compounds, even though the final yield is de-pendent on the specific vinification process and

the extraction method used. The grape pomace and stems are currently not valued as highly profitable waste and are mainly directed to com-posting or discarded in open areas, potentially causing environmental problems.

Apple (Malus domestica): According to the Food and Agriculture Organization of the United Nations (FAO), worldwide apple production exceeded 69,603,640 tons in 2008–09 (http://faostat.fao.org). The processing of apples to

TABLE 2.4 Crude Protein Composition (Percent DM Basis) of Vegetable and Fruit Wastes

Scientific name Crude protein (CP)

FRUIT WASTES

Banana peels (Wadhwa and Bakshi, 2013) Musa acuminata 8.1

Muskmelon peels (Wadhwa and Bakshi, 2013) Cucumis melo 9.5

Watermelon peels (Wadhwa and Bakshi, 2013) Citrullus lanatus 7.9

Citrus pulp (without peels) (Wadhwa and Bakshi, 2013) Citrus limetta 10.5

Apple pomace (NRC, 2001) Malus domestica 7.7

Grape pomace (Zalikaranab et al., 2007) (Vitis vinifera L.) <4%

VEGETABLE WASTES

Sugar beet leaves (Wadhwa and Bakshi, 2005) Beta vulgaris 21.9

Cauliflower leaves (Wadhwa and Bakshi, 2005) Brassica oleracea B. 17.0

Cabbage leaves (Wadhwa and Bakshi, 2005) Brassica oleracea C. 19.9

Black chick pea plant (Wadhwa and Bakshi, 2005) Cicer arietinum 13.6

Pea vines (Wadhwa and Bakshi, 2005) Pisum sativum 11.8

Radish leaves (Wadhwa and Bakshi, 2005) Raphanus sativus 19.4

Summer squash vines (Wadhwa and Bakshi, 2005) Cucurbita pepo L. 13.9

Baby corn husk (Bakshi and Wadhwa, 2012a) Zea mays Linn. 11.6

Carrot (NRC, 1989) Daucus carota 9.9

Potato (NRC, 1989) Solanum tuberosum L. 9.5

Snow peas (Bakshi and Wadhwa, 2012b) Pisum sativum var. saccharatum 23.2

Pea pods (Wadhwa and Bakshi, 2013) Pisum sativum 9.8

Carrot pulp (Wadhwa and Bakshi, 2013) Daucus carota 7.2

Bottle gourd pulp (Wadhwa and Bakshi, 2013) Lagenaria siceraria 24.3

Sugar beet pulp (NRC, 2001) Beta vulgaris 10.0

Tomato pomace (Wadhwa and Bakshi, 2013) Solanum lycopersicum 22.1

Adapted from Wadhwa and Bakshi (2013)

http://faostat.fao.org/




various products yields millions of tons of waste, for example, 25–30% solid pomace waste and 5–10% liquid sludge (Dhillon et al., 2013). Apple pomace and apple pomace sludge resulting from apple processing contains 2.9–5.7% protein (dry basis), which amounts to 28.8–33.8 g/L protein (Dhillon et al., 2011, 2013). And the apple waste also contains cull fruits (fresh fruits unsuitable for human consumption).

Citrus pulp: Pulp from citrus processing (50–70% of the fruit by weight) is the residue left after extraction of the juice. It contains 60–65% peel, 30–35% internal tissues, and up to 10% seeds (Crawshaw, 2004). Citrus pulp is usually made from oranges (60%), grapefruits, and lemons. It contains 5–10% CP and various other nutritional elements. Citrus molasses is a by-product obtained after citrus juice extraction. The fresh pulp mixed with lime is pressed to re-move moisture. The resulting liquid (press juice) is screened to remove the larger particles, ster-ilized, and concentrated. It is a thick, viscous, dark-brown to almost black liquid and is known as citrus molasses. The composition of citrus mo-lasses (60–65% sugars and 4–5% CP) is equiva-lent to sugarcane molasses.

Mango: This fruit, belonging to the genus Man-gifera, is one of the most important fruits marketed worldwide with a global production exceeding 26 million tons in 2004 (FAOSTAT, 2004). It is cul-tivated or grown naturally in more than 90 coun-tries worldwide (mainly tropical and subtropical regions) and is known to be the second largest tropical fruit crop produced in the world (Joseph and Abolaji, 1997). Mango processing generates approximately 50% of the fruit handled as waste. The edible pulp makes up 33–85% of the fresh fruit, while the peel and the kernel amount to 7–24% and 9–40%, respectively, on a fresh weight basis (Wu et al., 1993). After processing mango, the available by-products and waste includes cull fruits (fresh fruits unsuitable for human con-sumption), mango kernel meal, de-oiled mango kernel meal, and mango peels. The mango waste contains 10.3% CP (Sunita and Rao, 2003).

Pineapple (Ananas comosus): The posthar-vest processing of pineapple fruit yields skins, crowns, and waste from fresh trimmings and the pomace after extracting the juice. Raw pineapple waste (on a DM basis) contains 4–8% CP. Banana is another common fruit consumed in abundant quantities throughout the world. Banana leaves, pseudo-stems, and ripe banana peels contain about 10–17%, 3–5%, and 8% CP, respectively.

Finally, there are various other fruits that con-tribute abundant quantities of by-products dur-ing postharvest and processing and hence can be viewed as a potential source for proteins and other value-added products.

2.5 Vegetable-Processing Wastes

Baby corn (Zea mays Linn.): Thailand is the ma-jor baby corn producing and exporting country in the world. It is eaten both raw and cooked and is used as a delicacy in Asian cuisine. Only 15% is the edible baby corncob. The other 85% con-stitutes the outer peels with a silky threadlike structure called baby corn husk, which is consid-ered a waste material and a source of environ-mental pollution (Bakshi and Wadhwa, 2012a). Other by-products are tassels and green plant material.

Carrot (Daucus carota): Carrots are widely used as vegetables around the world. The post-harvesting and processing of carrot results in the production of various by-products: cull (grade-out), carrot tops, and carrot pomace after juice extraction. Carrot by-products find limited ap-plications as an animal feed.

Peas (Pisum sativum L.): Next to soybeans, groundnuts, and beans, peas are one of the most important legume crops. After harvesting sweet peas, which are eaten as a vegetable, the leftover plant is called pea vine, which contains around 11.8% CP. Pea vine protein fractionation shows that it contains the highest concentration of albumins, followed by glutelins, globulins, and prolamins. After harvesting sweet peas, the mature plant is left in the field for drying and is called pea straw.



It contains 5–10% CP. With higher protein content and less fiber, pea straw has a higher nutritive animal feed value than cereal straws. After shell-ing peas, the leftover material is empty pea pods, which contain high CP concentrations—19.8%.

Snow peas (Pisum sativum var. saccharatum): A variety of pea, but unlike sweet peas, these are valued for their pods rather than just the beans inside and are eaten whole with the pod. Snow peas are delicate and sweet in flavor. Frost-af-fected snow peas are considered unfit for human consumption, fail the quality control test, and are not exported. Cull snow peas contain high CP concentration of 23–25% with 35.8% total sugars on DM basis. These are an excellent source of vitamins A, B complex, C, and K. Also, they are rich in pigments, such as lutein and zeaxanthin, which help promote vision. An integrated pro-cess for extraction of protein and other bioactive compounds will create extra income for growers.

Potato (Solanum tuberosum L.): Potatoes are the most popular vegetable throughout the world. Along with wheat and rice, potato ranks as one of the most important staple crops in the human diet around the globe. Potatoes contain various compounds with high bioactivity, especially the potato skin. On the first cell layers under the skin are rich sources of various bioactive com-pounds. Besides starch as main component, potato tubers also contain an ample quantity of bioactive compounds, such as proteins, pep-tides, carotenoids, polyamines, polyphenols, suberins, glycoalkaloids, and dietary fiber. The fresh potatoes contain 65–75% starch (depend-ing on the variety) and 9.5% CP and other com-pounds. Most of them enhance human health and hence find potential applications in food industry. Potato processing, generally caused by potato-based fast-food products (chips and fries), results in the production of by-products, mainly peel waste which is generally used for animal feed or soil improvement. Approximate-ly 40% of potatoes are wasted, representing ap-proximately 10 tons/day of residue (Barampou-ti and Vlyssides, 2005).

Some potato species are known to have high protein content in their tubers. Among the health-promoting compounds are carotenoids, flavonoids, caffeic acid, and the tuber stor-age protein patatin, all of which exhibit activ-ity against free radicals and have antimicrobial, antiinflammatory, and antiallergic properties (Brown et al., 2007; Ritter et al., 2008). During the peak production season, it is a problem for farm-ers to dispose of the surplus and cull potatoes because of strict environmental laws. The only option for the farmers is to feed them to the live-stock. However, raw potatoes are not very pal-atable and have a laxative effect and, therefore, not considered good for animal feed.

Sweet potato: Sweet potatoes are used as vegeta-bles throughout the world. Shochu is a traditional Japanese liquor made from rice, sweet potatoes, and barley. Sweet potatoes are widely used for making this beverage and it is very popular in southern Japan. Recent increases in shochu pro-duction have resulted in an abundant quantity of distillery by-products. A small fraction of these by-products have been used as an animal feed (Mahfudz et al., 1996) and the remaining portion have been discarded into the ocean. This practise of by-product management becomes problematic from the perspective of environmental protection, and it mandates optimal treatment of distillery by-products, which is also important for the success of commercial shochu production. Hence, devel-oping new applications for the shochu distillery by-products (SDBs) originating from the sweet potato is necessary from the standpoint both of the economy and of environmental health.

SDBs contain abundant amounts of health-related food ingredients, such as essential amino acids, vitamin E, dietary fiber, citric acid, yeast, and minerals. The filter cake of the sweet pota-to–fermentation residue has potential as a food material, owing to its high CP content and an amino acid balance that is superior to that of ce-real grains (Wu and Bagby, 1987).

Sugar beet (Beta vulgaris): More than 200,000 tons of red beet are produced in Western Europe

3 ANIMAL-DERIVED PROTEIN BY-PRODUCTS 31


annually, most of which (90%) is consumed as vegetable. The rest are processed into juice and food colorant, the latter commonly known as beetroot red (Henry, 1996). The pomace from the juice industry, accounting for 15–30% of the raw material is used as an animal feed or com-post (Otto and Sulc, 2001). The different wastes and by-products of sugar beet include the leaves (22% CP) and pulp, which is mainly composed of pectin (28.7% DM basis); cellulose (20% DM basis); hemicellulose (17.5% DM basis); CP (9% DM basis); and lignin (4.4% DM basis) (Ja-cob, 2009). The sugar beet pulp is dried and sold as dried sugar beet pulp or mixed with molasses to form dried molasses beet pulp.

Tomato (Lycopersicon esculentum L.): Tomatoes are grown worldwide with an annual produc-tion of nearly 100 million tons (Kalogeropoulos et al., 2012). As per statistics from the World Pro-cessing Tomato Council, more than 30 million tons of tomatoes are processed annually world-wide to produce tomato juice, ketchup, canned tomatoes, sauces, paste, puree, powder, and many other products (Zuorro et al., 2014). To-mato processing simultaneously generates large quantities of solid by-products, mainly peel and seeds, usually raising environmental concerns. Tomato waste is made up of culled tomatoes (14–20% CP) and tomato pomace (19–22% CP). The culled fruits may be damaged, diseased, too small, or misshapen and do not meet the grading standards for sale in the fresh market or for pro-cessing. Tomato pomace is a mixture of tomato peels, seeds, and small amounts of pulp that re-main after processing. Although, tomato peel is acknowledged to be high-value raw material for lycopene, tomato seeds have attracted limited attention. A recent study conducted by Sarkar and Kaul (2014) showed that tomato seeds are a potential source of protein, having all amino ac-ids significantly higher than (WHO/FAO/UNU 2007) recommendations and a high calculated PER value of 2.66 (Table 2.2). Tomato seeds have been reported to contain approximately 24.5% CP and are highest in glutamic acid and aspartic

acid (Persia et al., 2003). Unlike many other plant proteins, tomato seed has also been re-ported to have a high lysine content (Brodowski and Geisman, 1980). The net protein retention of whole tomato seed meal, defatted tomato seed meal, and tomato seed protein concentrate was studied as 2.65, 2.52, and 2.51, respectively, as compared with 2.91 for casein (Sogi et al., 2005). Nevertheless, underused tomato seeds—with high protein content and good nutritional attri-butes—can be a considered as a potential source of sustainable protein supplement for future food formulations.

2.6 Value-Added Options for Fruit and Vegetable Postharvest and Postprocessing Wastes

Fruit and vegetable wastes are highly fer-mentable and perishable, mainly because of high moisture, total soluble sugars, and CP con-tents. During the peak production or processing season, massive quantities of these valuable re-sources are available and cannot be consumed at the same pace as they become available. Thus, they become surplus and can cause environ-mental pollution. Therefore, suitable methods should be adopted to conserve such resources, which can be used for extraction of valuable components, such as proteins and other bio-products. This will also help mitigate environ-ment pollution and create extra revenues.

3 ANIMAL-DERIVED PROTEIN BY-PRODUCTS

Whey or Lactoserum: Whey (also known as lac-toserum), the major dairy by-product through-out the world, is readily available in most milk-producing areas. It has around 5% dry matter, 12–13% CP (DM basis), and 60–70% lactose (DM basis). The protein content of whey varies from 0.55–65% on dry weight basis. Recently, whey has been used for the production of various



health products, such as whey protein concen-trate, lactalbumin, and whey protein hydro-lysates. Whey protein contains around 35% β-lactoglobulin, 12% α-lactoalbumin, glycomac-ropeptide, ∼8% immunoglobulins, ∼5% serum albumin, and ∼15% minor proteins.

Casein: This is the main protein component of milk. Casein is amphiphilic in nature. The iso-electric point of casein is 4.6 (Table 2.3), above which it is negatively charged and is soluble in water (Chen et al., 1987). The conformation of casein is almost similar to the denatured globular proteins. The high number of proline residues in casein cause particular bending of the protein chain and inhibit the formation of close-packed, ordered secondary structures. Ca-sein does not have any tertiary structure, which describes its stability against heat denaturation, as there is very little structure to unfold. Also, in the absence of tertiary structure, there is substantial exposure of hydrophobic residues, which results in strong association reactions of casein and renders them partially soluble in wa-ter. The major use of casein has been as a food ingredient to augment physical properties, such as foaming and whipping, thickening and water binding, emulsification, and texture. Casein also enhances food nutrition value. Further, casein is used as wood adhesive, for leather finishing and paper coating, and in synthetic fibers, as well as plastics for buckles and buttons. However, the flocculation potential of casein has not been much explored. Few studies demonstrated the ability of casein as flocculent (Seki et al., 2004).

Gelatin: A heterogeneous mixture of water-sol-uble proteins of high average molecular weight, gelatin is derived from the collagen present in animals, such as cattle, pigs, sheep, and fish. Collagen is the major insoluble fibrous protein in the extracellular matrix and in the connective tissue of animals (Lodish et al., 2000). Gelatin has been used in the wine-fining process for the removal of the natural haze-forming constitu-ents (Cole, 1986). Similarly, Piazza and Garcia (2010b) showed that porcine gelatin promoted

clay flocculation. Flocculation was completed within 24 h irrespective of the addition of cal-cium chloride. The same research group dem-onstrated that beef-skin gelatin and hydrolyzed fish collagen were found to improve flocculation of clay suspension when pH 5.5 buffer was add-ed (Piazza and Garcia, 2010a). However, gela-tin is costly as it finds commercial applications in food and other industries. The extraction of gelatin from waste sources using economical methods will shift toward feasible flocculation processes.

Albumin: Belonging to a family of globular proteins, it is found in the blood plasma of hu-mans, animals (eg, cattle, sheep, goats, horses, chickens, and pigs, among others), and eggs. Se-rum albumin is a protein found in small quanti-ties in the plasma phase of animal blood. Serum albumin has a wide range of commercial ap-plications, depending on the grade, purity, and quality of the product. Albumin from different sources, especially bovine serum albumin, has found potential applications in medical and bio-logical laboratories. It has been widely used in various other applications, such as diagnostics, biotechnology, aquaculture, food and beverages, chemicals and enzymes, and others.

Recent studies demonstrated the flocculation potential of albumin (Seki and Suzuki, 2003; Seki et al., 2009). Seki and Suzuki (2003) carried out the flocculation of diatomite by methylated egg albumin. Diatomite is composed of approx-imately 90% SiO2 and it has a negative charge above pH 3. Egg albumin has an isoelectric point between pH 4 and 5 and is also negatively charged at near-neutral pH. Therefore, the egg albumin is not supposed to act as flocculant be-cause of similar charge with diatomite surface. Hence, methylated egg albumin with positive charge will attach strongly to the negatively charged diatomite surface even at near-neutral pH and led to improved flocculation. Modern extraction techniques allow for the extraction of albumin from hemolyzed blood as well as from high-quality plasma.


With advances in recombinant technology, it is expected that the cloning of the albumin gene may, in the near future allow albumin produc-tion that will take a significant share of the market away from the animal-sourced albumin and result in the development of various new applications.

4 CONCLUSIONS AND FUTURE PERSPECTIVES

The literature reviewed revealed that there has been increasing demand for sustainable protein sources in various food and nonfood applica-tions. The development of novel processes for the effective and efficient utilization of protein-rich agro-industrial wastes will generate an array of value-added products, thus generating extra revenues and help in waste management and environmental pollution reduction. Nowadays, protein by-products, resulting from many agri-cultural sources, has been considered as potential sources for various applications. Proteins derived from agricultural commodities, such as oilseeds, rice, barley, whey, and abattoir wastes, among others have been used successfully in various food, nonfood, and livestock feed applications.

However, for different applications, the pro-teins must be available in a reasonably concen-trated, homogeneous, and soluble form. The pu-rity, solubility, charge distribution, and molecular weight are the main attributes which affect their end-use applications. Hence, extraction and puri-fication techniques are sought for the production of high-quality proteins to use in various applica-tions. Further development of different processes for protein by-product use will enhance the eco-nomic viability of the overall production process.

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C H A P T E R


3Meat Industry Protein By-Products: Sources and CharacteristicsT.M. Hicks, C.J.R. Verbeek

School of Engineering, Faculty of Science and Engineering, University of Waikato, Hamilton, New Zealand

1 INTRODUCTION

Domesticated livestock play a significant role in maintaining the modern human lifestyle. These animals are reared and slaughtered for meat, as well as dairy and other animal prod-ucts. Global demand will continue to rise along with population growth, and as more under-developed countries adopt Westernized eating habits (Horrigan et al., 2002; Thornton, 2010).

The continual growth of this market has trig-gered some dramatic changes to what would now be considered traditional farming prac-tice. Intensive breeding of livestock is aimed at maximizing product yield and has become com-monplace in developed countries (Hazell and Wood, 2008; Pretty, 2008). To keep up with the calorific demand of high production, pasture is often supplemented with grains, such as maize or wheat. Feed may also be fortified with protein meals, such as corn gluten meal, and in countries

where there is no restriction, this can include animal by-products (ABPs), such as blood meal (Bocquier and González-García, 2010). Pre- and probiotics, growth-promoting factors, supple-ments, and additives are often used in intensive farming practices to accelerate growth, boost production, and improve animal condition.

No matter how rapid or big an animal grows, only 30–40% of the animal is used for human consumption (Ockerman and Hansen, 2000; Swan, 1999) and what remains equates to a sig-nificant volume of solid and liquid waste. These are considered an aesthetic, environmental, and economic burden, and their safe disposal can in-cur considerable cost to the meat processor.

With the expected rise in worldwide consump-tion of animal protein products, so too will there be an increase in associated waste. In a world with finite resources, the minimization, recovery, and utilization of by-products becomes increasingly important (Fallows and Wheelock, 1982).

38 3. MEAT INDUSTRY PROTEIN BY-PRODUCTS: SOURCES AND CHARACTERISTICS


This chapter evaluates various aspects of ABPs obtained from commercial slaughterhouses which are then rendered to produce higher value products. Sources of raw materials are inves-tigated, including emerging industry specific wastes, such as those from fisheries. The effect of some processing techniques and nuances of typical rendering processes on product quality, economic value, and end use are considered.

One limitation of this discussion is the intrin-sic variability of meat processing. Although not covered extensively, it is important to realize that the following aspects of this industry directly in-fluence the type of ABPs and their final uses:

• Animal production and therefore by-product quantity and quality are subject to daily and seasonal variation.

• Animal market conditions vary between different locations and countries.

• Animal composition varies both between species and within a species depending on the animal’s breed, age, gender, health, and condition.

• Raw material obtained for rendering will depend on what is considered “edible,” which is subject to the culture, customs, religion, regulations, and market demand of the considered location.

• The collection process of the rendering plant and product handling also determines the quality and end use.

Also, this chapter specifically deals with by-products from commercial slaughter of cattle, and sheep and lambs, and pork, poultry, and seafood processing. Processing of animal wastes from farms, game meats, zoo animals, and companion animals is outside the scope of this discussion.

2 THE MEAT INDUSTRY

Production and processing of animals for food result in ancillary ABPs. Continuous research behind the technology and regulatory aspects of

meat processing is aimed at improving hygienic meat collection and minimizing product loss. Slaughterhouse operations vary depending on the size and space limitations of the plant. Com-mon operations, regardless of species, include holding, stunning, killing, bleeding, hide or hair removal, evisceration, carcass washing, trim-ming, and dressing. Secondary operations may also occur on-site including cutting, deboning, and processing into retail products.

A typical process flow for slaughter of live-stock is described in Fig. 3.1, where the principle commodity is meat, however, other by-products obtained during the slaughter process are:

• edible by-products, such as offal and casings• inedible by-products, such as hides, horn,

and wool• low value by-products, such as protein meals

for animal feed• waste items, with no useful purpose to be

disposed of

Although the most valuable materials have already been removed during processing, po-tentially useful components may remain in the low-value products and waste (Jayathilakan et al., 2012). These components need to be effec-tively used to cover the overheads of slaughter-ing, pollution control, and disposal (Ockerman and Hansen, 2000; Swan, 1999). Despite their associated environmental and health risks, these waste materials are now recognized for their potential to be recycled or converted into value-added products and now represent valuable economic commodities.

2.1 Economics of By-Product Processing

Providing meat, milk, and eggs for human consumption also results in the ancillary pro-duction of edible and inedible by-products. Approximately 49% of the live weight of cattle, 44% of pigs, 37% of broilers, and 57% of most fish species cannot be sold as meat or used in meat products for human consumption and are

2 THE MEAT INDUSTRY 39


FIGURE 3.1 Process flow diagram for general meat processing.



classified as inedible by-products (Meeker and Hamilton, 2006). In the United States alone, this is estimated to correspond to the production of ∼25 million metric tons of inedible animal tissue annually (Bisplinghoff, 2006).

ABPs may represent up to 75% of an animal’s live weight, but in their unprocessed state may be worth as little as 10–20% of the animal’s total value (Scaria, 1989). However, with appropriate processing, the revenue from the end products may nearly equate to those of the meat. In some cases, the commercial value of these by-products is higher than the sum of the operating expenses and the margin required to operate profitably (Swan, 1999). Rendering is the most economi-cal method for safely handling these materials, salvaging billions of dollars of saleable product (Bisplinghoff, 2006).

The edible products are considered to be the most valuable part of the slaughtered animal. Technologies have been developed to capital-ize on the production of edible materials from normally inedible products, such as protein con-centrates from horns and hooves for use as fla-voring agents (Scaria, 1989). Classification of by-products depends on local culture, customs, and regulations, and as a result, some products can be downgraded from edible to inedible (Swan, 1999). Inedibles are then rendered into products, such as meat and bone meal (MBM), meat meal (MM), poultry meal, hydrolyzed feather meal, blood meal, fish meal, and animal fats.

2.2 Rendering

Rendering is a combination of mechanical, thermal, and sometimes chemical processes to separate biological materials (the waste from the carcass of a dead animal) into its constitu-ent components: fat, protein, and water. More importantly, it can be seen as an integrated sys-tem to safely process raw material into a ma-terial that complies with environmental and disease control legislation. The purpose of ren-dering is to:

• Sterilize by-products for safety.• Remove fat to prevent oxidation during

storage.• Dry to inhibit bacterial growth and facilitate

transportation and storage.• Create saleable products.

Rendering depends largely on the type and condition of raw material and directly influ-ences production costs, product quality, and fi-nancial success of the product (Anderson, 2006). Therefore, appropriate selection and operation is paramount for high quality.

2.2.1 Rendering SourcesBiological raw material not only includes

inedible offal, fat, and animals classified as “condemned” but also animals that died be-fore slaughtering. Rearing animals leads to a large number of on-farm livestock mortalities. These deaths include stillbirths, the unwanted offspring of livestock, culled animals, and ani-mal deaths caused by disease or age. Render-ing plants deal with the disposal of these ani-mals, along with waste from slaughterhouses (Fig. 3.2). Therefore rendering not only gener-ates value-added products, it also provides a hygienic means of disposing of fallen and con-demned animals.

2.2.2 Basic Principles of RenderingThe two basic processes occurring during ren-

dering are the separation of fat and drying the remaining protein-rich residue. Rendering in-volves crushing the raw material and cooking at 110–120°C for sterilization. Most of the fat is de-canted and the residual solids (∼20% fat) are then centrifuged. The solids are sometimes solvent extracted leaving degreased protein-meal (3–5% fat) as residue (Fallows and Wheelock, 1982).

The main factors determining product qual-ity are:

• precrushing and quantity of the raw material used

• steam pressure

2 THE MEAT INDUSTRY 41


• agitator speed and temperature• cooking or drying temperature• end-point moisture content

To obtain the highest quality products, ma-terial should be rendered as soon as possible after slaughtering to minimize bacterial deg-radation of proteins and fats. Where possible, processing should avoid introduction of ad-ditional air or moisture to minimize oxidation and hydrolysis.

Generally, rendering begins with the removal of undesirable parts from the raw materials. In

particular, paunch content must be removed and viscera washed. Failure to do so results in a green-tinted product, degrades fat quality (higher free fatty acid [FFA] content; Fernando, 1992) and increases moisture, impurity, and unsaponifiable content (Bennett, 1927). Qual-ity is also ensured by rapid cooking, allowed for by adequate heat transfer during cooking, achieved through size reduction of the raw material. The cooked mixture is separated into fat, water, and protein by screening, pressing, centrifugation, solvent extraction, and drying (Auvermann et al., 2004).

FIGURE 3.2 Sources of raw material for rendering from animal production.



Rendering can be categorized into either “edible” or “inedible.” Edible rendering only uses by-products obtained under sterile condi-tions. For example, fat trimming is ground and melted to release moisture and “edible” tallow. The three end products (protein, fat, and water) are separated by screening and sequential cen-trifugation. The protein-rich solids are dried and sold for use as animal feed; water is treated for recycling or discharge; and the edible fat is stored for refinement. Other edible products are also produced, such as gelatin from bone col-lected hygienically.

By contrast, inedible rendering processes use the remaining protein, fat, and keratin (hoof and horn). The rendering process also involves cooking, dewatering, and separating fat, pro-tein, and water but often requires some precook-ing processes. This includes the removal of skin and paunch and thorough washing of the entire carcass. The hide is not normally removed from hogs and small animals, but removal of their hair is carried out before washing and cleaning (Auvermann et al., 2004). The cleaned carcasses are then crushed, weighed, and passed through metal and nonmetal detectors to remove nearly all of the magnetic and nonmagnetic metal ma-terials (tags, hardware, and boluses).

Although edible and inedible rendering pro-cesses are similar, they differ in their raw ma-terials, end products, and sometimes equip-ment. Furthermore, different rendering systems work well for small- (poultry), medium- (swine, sheep, calves), and large-sized (cattle and horse) mortalities (Auvermann et al., 2004). Typically, all rendering systems can be classified as either wet or dry depending on whether the fat is re-moved before or after the drying operation; both can be carried out in a batch, semicontinuous or continuous mode. More recently, low-tempera-ture rendering systems have become popular, as they prevent destruction of amino acids and maintain the biological activities of lysine, me-thionine, and cystine (Swan, 1999; Auvermann et al., 2004).

3 ANIMAL PRODUCTS AND BY-PRODUCTS

3.1 Meat and Poultry By-Products

Rendered, inedible by-products are often di-vided into three categories:

1. tallows, which are the fats extracted during the rendering process

2. degreased bones for use in the production of gelatin

3. protein meals, such as MM, MBM, hoof and horn meal, and blood meal

Tallows are used in the manufacture of candles, soaps and cosmetics, paints, printing inks, water repellants, and biodiesel production (Fallows and Wheelock, 1982; Bhatti et al., 2008). In addition to gelatin production, degreased bone has been used in the manufacture of glues used in plywood and abrasive paper manufac-ture and bone flour in the production of fine china (Fallows and Wheelock, 1982).

The by-product mass produced from four commercial meat species reared in Australia, rel-ative to the supply of 1000 kg of retail portions is given and expressed as a percentage of the live weight (Table 3.1) (Wiedemann and Yan, 2014).

The most important and valuable use for the protein rich ABPs is as a feed ingredient for livestock, poultry, aquaculture, and companion animals. Although use of ABPs in feeds and fertilizers are technologically and economically viable, a growing market also exists for protein hydrolysates (Bhaskar et al., 2007), which may be used as flavor enhancers, functional ingredi-ents, or to boost the nutritional value of foods considered to have a low protein quality. Ad-ditionally, some inedible ABPs are transformed for use in pharmaceuticals and to recover amino acids for a feedstock for higher applications.

In most countries, everything produced from the animal, other than the dressed carcass, is considered a by-product. These by-products in-clude hides, skins, hair, feathers, heads, horns,

3 ANIMAL PRODUCTS AND BY-PRODUCTS 43


hoofs, feet, toe nails, bones, tendons, glands, muscle and fat tissues, shells, and the contents of the gastrointestinal tract, blood, and internal or-gans (Table 3.2). This represents ∼30–50% of the live weight of the animal and equates to the pro-duction of ∼60 million metric tons of ABPs glob-ally every year (Leoci, 2014). These ABPs can be subdivided into two categories—edible and inedible—the allocation of which depends on the country of origin.

The distribution of by-product yields varies for both species and different breeds of livestock and is probably influenced by the age, gender, and condition of the animal and slaughterhouse operation (Ockerman and Hansen, 2000; Scaria, 1989; Ockerman and Basu, 2014). This variation is likely to be greater when the product yields of

livestock from different countries are compared, as animals will be reared according to market demand and disseminated and used according to the local cultural and regulatory environ-ment. It is for this reason that factors such as the dressing percentage and retail yield should be determined based on data representative of the supply chain being investigated (Wiedemann and Yan, 2014).

3.2 Seafood By-Products

Over the past 20 years, global demand has driven a significant increase in aquaculture. The quota system for harvesting wild popu-lations, along with changes in environmental and economic perception has been driving an

TABLE 3.1 Product Mass for Four Commercial Meat Species Relative to 1000 kg of Retail Edible Product

Description Product Beef Sheep Chicken Pork

Farm-gate product Live weight 2375 2255 1712 1618

Intermediate product Hot standard carcass weight 1307 1060 1216 1229

Wholesale product Cold carcass weight 1267 1017 1179 1177

Wholesale/retail product Retail cuts 887 895 967 906

Edible offal 113 105 33 94

Retail portions (retail cuts + edible offal)

1000 1000 1000 1000

Hides 214 169 0 0

Pet food 30 34 17 84

Rendering material Unprocessed meat bone offal 989 800 593 372

Rendering products Protein meal products 257 152 154 53

Tallow 154 156 73 109

Blood meal 14 14 5 5

Sum (%LW) material to rendering 41.6 35.5 34.6 23.0

Protein meal (%LW) 10.8 6.7 9.0 3.3

Tallow (%LW) 6.5 6.9 4.3 6.7

Blood meal (%LW) 0.6 0.6 0.3 0.3

Total product from rendering (%LW) 17.9 14.3 13.6 10.3

%LW, percentage live weight.Data retrieved from Wiedemann and Yan (2014).



increase in harvest utilization, and like other meat-processing industries, is also being pushed to maximize by-product recovery and utilization. Historically, only the most desir-able portion of the fish was used, accounting for as little as ∼20–30 wt.% of the animal, and what remained was disposed of or processed into cheap animal feed (Pigott, 2000). Although the quantity used for human consumption may have improved in recent years, fish and shell-fish processing results in more than 60 wt.% by-products, including head, skin, trimmings,

fins, scale, viscera, and bones (Kristbergsson and Arason, 2007).

In 2001, total global fishery production (ex-cluding aquatic plants) was reported to exceed 130 million metric tons, with ∼38 million tons obtained from aquaculture practices (Vannuccini, 2004), and by 2012, total production had reached 158 million tons, with ∼67 million tons from aquaculture (FAO, 2014). Processing this seafood for consumption leads to the gen-eration of a large volume of fish waste, most of which is used to create fish silage, fish meal, and

TABLE 3.2 Edible and Inedible Animal By-Products (Scaria, 1989) and Estimated Proportions as Percentage Live-Weight (%LW) from Commercially Produced Livestock (Leoci, 2014) and (Meeker and Hamilton, 2006)

Edible Edible/Inedible Inedible

Liver Lungs Horn

Heart Spleen Hooves

Tongue Small intestine Teeth

Kidneys Large intestine Bile liquid

Brain Stomach Hair

Oxtail Urinary bladder Wool

Caecum Bristles

Oesophagus Foetus

Testicles

Uterus

Skin/Hide

Bone

Blood

Pancreas

Tallow

Lips

Snouts

Ears

Beef Sheep Chicken Pork Fish

Average %LW for human consumption (Leoci, 2014) 55 50 70 60

Average %LW edible and inedible ABPs (Leoci, 2014) 45 50 30 40

Average %LW not for human consumption (Meeker and Hamilton, 2006)

49 37 44 57

%LW, percentage live weight.

3 ANIMAL PRODUCTS AND BY-PRODUCTS 45


fish sauce, along with approximately 8 wt.% being discarded (Kelleher, 2005). In addition to fish processing, a large quantity of wastes from other sources are processed, such as mol-luscs, crustaceans, and cephalopods. A typical fish-processing operation involves stunning, grading, removing slime, deheading, washing, scaling, gutting, cutting of fins, meat bone sepa-ration, and preparing steaks and fillets (Fig. 3.3) (Ghaly et al., 2013).

Many other valuable materials can be extracted from fish muscle, skin, oil, bone, viscera, and the shells of shellfish and crustaceans. These are all rich in bioactive components, such as water-soluble minerals, peptides, collagen, gelatin, en-zymes, oligosaccharides, and fatty acids (Ghaly et al., 2013; Kim and Mendis, 2006). The value of these compounds is likely to increase because of growing evidence of their potential health bene-fits including antihypertensive, antioxidant, anti-microbial, anticoagulant, antidiabetic, anticancer,

immunostimulatory, calcium-binding, and hypo-cholesteremic properties and appetite suppres-sion (Harnedy and FitzGerald, 2012). As such, marine by-products should be considered valu-able resources that show promise as functional food ingredients and potential as materials for use in biomedical and nutraceutical applications (Senevirathne and Kim, 2012).

Fish meal and oil constitute the bulk of by-prod-ucts generated from fisheries. In Iceland, these by-products comprise 63 wt.% of the fish processed but only account for ∼14% of the total revenue of exported seafood products (Kristbergsson and Arason, 2007). Like other ABPs, when appropri-ately processed, inedible portions are often equal to or exceed the value of the primary edible prod-uct (Pigott, 2000). However values tend to fluctu-ate depending on the availability of other animal feeds, particularly during pandemic events or disease outbreaks, such as bovine spongiform encephalopathy (BSE) or avian flu (Kristbergsson

FIGURE 3.3 Generalized flow chart of fish-processing operations and by-products.



and Arason, 2007). In the future, an increased un-derstanding of proteins and fish oil chemistry may yield resources of greater value (Kristbergsson and Arason, 2007).

3.3 Animal By-Product Meal Quality

The quality of the final by-product meal is largely dependent on the extent of microbial degradation prior to rendering, the temperature, pressure, and rendering time and any product refinement. The value of meals is reduced by mi-crobial or thermal degradation reflected in sen-sory and aesthetic properties, often contributing a dark color and off flavors and odors.

The extent of microbial degradation is deter-mined by the type of material collected, its con-dition, and the storage and handling techniques involved prior to rendering. To minimize the extent of degradation, the entire collection and rendering process is generally carried out as quickly as is possible.

Sensory attributes, such as color, texture, flavor, odor, and physical qualities, including solubility and particle size distribution, are indicators of the overall quality of the protein meal (Auvermann et al., 2004). These physical attributes are typically the controlling factors, which dictate the overall taste and palatability of the product for use in animal feed supplements and may also influence final digestibility. As such, poor sensory quali-ties lower the value of the meal as it is more dif-ficult to implement as a feed ingredient (Baumont et al., 2000). For example, a product that is dark in color is often the result of browning reactions or oxidation between carbohydrates or lipids with proteins during heating (Papadopoulos, 1989) and is often accompanied by a burnt caramel-ized flavor and odor. Malodor is often an indi-cation of poor storage and handling conditions that have resulted in putrefaction of proteins and auto-oxidation of lipids generating offensive vola-tile compounds (den Brinker et al., 2003; Verbeek et al., 2012). Other physical properties, such as the material’s density and the overall uniformity of

particle size, may also become important depend-ing on their intended final use.

The quantity and quality of the protein is what will decide the final value of the product. Animal protein meals are an important feed in-gredient for poultry, fish, and pigs in most parts of the world, although some limitations exist to prevent outbreaks of BSE (the United States and Canada have banned specified risk material (SRM) from use in animal fodder or fertilizer ap-plications, and no animal proteins can be used as feed ingredients for other farmed animals throughout Europe). Solubility can be a useful indicator of how denatured a protein source has become during rendering and drying. Proteins are generally denatured and undergo aggrega-tion a result of the high temperatures, and as a result they tend to become fairly insoluble.

In addition to physical properties, the chemi-cal characteristics of the protein meal will also influence its value and final application. The quantity and bioavailability of both macro- and micronutrients is important for tailoring animal feed and are also indicators of quality; for exam-ple, high calcium and phosphorus content in a MM may indicate the presence of bone.

Amino acid composition is also a good indi-cator of the quality, as heat-labile amino acids, such as tyrosine, tryptophan, phenylalanine, histidine, cysteine, lysine, and methionine tend to get damaged, oxidized, or cross linked dur-ing high-temperature processing. Such changes in the primary structure of the proteins directly affects the solubility and nutritional value and will also change the behavior of the material in higher end applications.

Product quality is generally improved by em-ploying low-temperature rendering operations and is most notable for amino acid digestibility (Ockerman and Hansen, 2000). A study of New Zealand MBMs showed that on average, batch drying between 100 and 130°C was able to pro-duce products of a similar nutritional quality to those produced at 60–90°C (Hendriks et al., 2004). Although low-temperature rendering could be

4 CHARACTERISTICS OF COMMON PROTEIN BY-PRODUCTS 47


used in countries that are free of BSE, such as New Zealand and Australia, it would not be rec-ommended in countries with a known risk of BSE.

Current legislation in Canada, the United States, and the European Union acts to mini-mize the spread of BSE by prohibiting the use of SRM from cattle (such as the brain and spinal cord) in feeds or fertilizers. To ensure that any BSE prions are inactivated in the final products, SRM is first segregated from the edible and in-edible raw materials, which are then rendered under the EU guidelines of 133°C at 300 kPa, for 20 min (Taylor and Woodgate, 2003). The SRM is subsequently treated using a method deemed sufficient to deactivate BSE prions within the material, such as hyperbaric thermal or alkaline hydrolysis (Taylor and Woodgate, 2003), or is in-cinerated for disposal.

Slaughterhouses are the largest producer of ABPs and will often have processing opera-tions on site for some or all of the by-products produced. Blood may be processed on site, pre-served, aged, or refrigerated and transported by truck to a processing facility, whereas other ma-terials from slaughterhouses, processors, whole-salers, and retailers that cannot be processed on site are sent to rendering plants. The raw mate-rials are usually first collected in large storage bins or silos and may have built-in dewatering devices to drain excess water (Fernando, 1992) prior to transport to a rendering plant by truck (Goldstrand, 1992). Most waste material con-tains a large number of microbes or is easily al-tered by microbial activity. Given a typical raw material composition (∼60% water, 20% pro-tein and minerals, and 20% fat; Meeker and Hamilton, 2006), an ideal medium for microbial growth and enzymatic activity exists.

During storage and transportation, oxygen present in the blood and meat tissues is rapid-ly consumed by aerobic and facultative bacte-ria, and once consumed, the proteins begin to undergo putrefaction as anaerobic bacteria de-compose them. The formation of odorous amines during putrefaction, such as indole, skatole, pu-

trescine, and cadaverine, lowers the quality and value of the final product. Putrefaction can be prevented through the addition of a suitable pre-servative, such as formic acid, sodium chloride, and unslaked lime, or 3% sulfuric acid or potassi-um metabisulfite in the case of blood (Fernando, 1992; Hertrampf and Piedad-Pascual, 2000).

Rendering of carcasses in advanced stages of decomposition is uncommon as both hide re-moval and carcass cleaning becomes difficult. Furthermore, the high water content increases transportation cost and separation can lead to other disposal problems caused by the high lev-els of organic compounds in the water.

It is generally better to store the raw material as long as possible before size reduction as grinding of the material leads to an increase in FFA content. Preservatives, such as sodium chlorite or dilute acids (reducing the pH to 3.5–4.0), can be added to delay the increase in FFAs, but in general, it is best practice to perform rendering as soon after size reduction as is practical (Fernando, 1992).

4 CHARACTERISTICS OF COMMON PROTEIN BY-PRODUCTS

4.1 Blood Meal

Collectible blood makes up approximate 4–6% of an animal’s live weight. It is protein rich (∼17 wt.%) with a good balance of amino acids, rich in lysine, and also high in iron (Jayathilakan et al., 2012). During collection, whole blood be-gins to coagulate as fibrinogen proteins form insoluble complexes with free calcium ions. If necessary, coagulation can be inhibited through the addition of ethylenediaminetetraacetic acid disodium salt (EDTA), oxalic acid, or anhydrous sodium citrate (Duarte et al., 1999; Ockerman and Hansen, 1988). However, it is not common practice as blood is typically coagulated to aid dewatering. Blood is often aged prior to dry-ing to induce coagulation at lower tempera-ture thereby minimizing thermal degradation



(van Oostrom, 2001), although low temperature coagulation can also be achieved by adding free calcium ions in the form of 1% calcium chloride (MIRINZ, 1985).

When stored in a dry condition, blood meal is a microbiologically stable material, relying on its low moisture content and water activity for preservation (Ockerman and Hansen, 1988). Blood can be dried in a number of ways to ob-tain a stable, highly nutritive product, free of contaminants, such as wool or hair. Whole blood is strained prior to dewatering to remove debris, followed by coagulation using direct steam in-jection (∼90°C) or being passed through a heat-ed pipe (Bimbo, 2005; Macy and Butler, 1969). The coagulant is separated using a centrifuge decanter to remove up to 50%, followed by ther-mal drying. Common drying operations include direct batch drying, batch coagulation followed by batch drying, continuous coagulation prior to drying, continuous dryers, spray dryers, or other novel systems (Fernando, 1992).

Drying conditions have a significant effect on crude protein content, protein quality, and bio-availability of amino acids. Ring drying typically produces a better quality product than a flash dryer as it uses warm air rather than a heated metal surface. Because direct contact with a hot surface is avoided, heat labile amino acids (eg, lysine) are not destroyed (Bimbo, 2005). Blood meal is usually dark red-brown in color, which can be represented numerically using the 1976 Commission internationale de l'éclairage color space values (CIE L*a*b*), where L* is a measure of lightness, and a* and b* are measures of red-ness and yellowness respectively. Typically, com-mercial blood meals have a moisture content of 5–8 wt.% and a water activity of 0.3–0.45 (Roberts et al., 2005), typical characteristics of thermally dried blood meals are given (Table 3.3).

The amino acid composition of blood proteins varies between species. Notably, poultry blood has a significantly higher quantity of isoleucine compared to bovine or porcine blood; similarly, porcine blood has a significantly lower quantity

of lysine (Márquez et al., 2005). The differences in amino acid composition between species has been observed in the meals produced from spray drying, where avian blood meals have higher ar-ginine, cysteine, isoleucine, and tyrosine content compared with porcine and bovine blood meals (Kats et al., 1994). Variation in amino acid con-tent not only influences the feed application, but also higher end applications, such as the manu-facture of thermoplastics.

4.2 Meat By-Product Meals

MM and MBM are both produced by render-ing the inedible or unsold by-products from slaughterhouse operations. Most the raw mate-rials present in MM and MBM come from cattle, swine, and poultry, with most of the literature available on MBM containing material sourced from more than one species. MMs often include the addition of deadstock (animals that died before slaughter), in some cases representing greater than 10% of the total raw material ren-dered (Garcia and Phillips, 2009). It may also in-clude expired meats from retailers and materials recovered from dissolved air floatation used for treating rendering effluent.

Until recently, a policy established by the Na-tional Renderers Association prevented ovine material (sheep) from being used in MBMs in the United States and Canada, which has been withdrawn (Malone, 2005). It is unlikely the ex-clusion or inclusion of sheep in MBM has any more effect on the variation of the product com-position than the rendering process itself.

The average MBM is dark red-brown in color, however, it is known to range from tan to dark brown in color, with an average CIE L*a*b* value indicated in Table 3.5. It is generally sold based on the quantity of crude protein, which although it is typically high, can be rather variable. MBM has a high mineral content (higher than MM) and is relatively low in fat (Garcia and Phil-lips, 2009). Like most other thermally denatured proteins, those present in MBM have very low



TABLE 3.3 Typically Reported Range of Product Characteristics and Proximate Analysis of Blood Meal (National Research Council and Canadian Department of Agriculture, 1971; Martínez-Llorens et al., 2008; Haughey, 1976; Preston, 2014; Hicks et al., 2013; Howie et al., 1996; Kamalak et al., 2005; Laining et al., 2003; Marichal et al., 2000; National Research Council, 2001; Nengas et al., 1995), Amino Acid Composition (National Research Council, 2001; Knaus et al., 1998; Li et al., 2011), and Mineral Content (National Research Council and Canadian Department of Agriculture, 1971; Preston, 2014; National Research Council, 2001; Pearl, 2004)

Characteristic Value

Dry material (%) 85.2–98.5

Crude protein (%) 80.2–100.5

Ether extract (%) 0.2–1.5

Crude fiber (%) 0–6.2

Gross energy kcal/kg 4844–5660

Ash 1–8.6%

Moisture content 5–8%

Water activity 0.30–0.45

Particle size 85–200 µm

Color Dark, red-brown

CIE L*a*b* value L* = 25.2

a* = 14.4

b* = −2.9

Amino acids %CP Mineral content

Alanine 7.82 Ca 0.30–0.40%

Arginine 4.38–4.91 P 0.23–0.30%

Asparagine 4.67 Mg 0.03–0.24%

Aspartic acid 6.20 K 0.09–0.33%

Cystine 0.11–1.92 Na 0.31–0.40%

Glutamine 4.32 Cl 0.26–0.33%

Glutamic acid 6.38 S 0.32–0.77%

Glycine 3.86–4.95 Cu 9.7–10.8 mg/kg

Histidine 5.57–6.61 Fe 2453–4110 mg/kg

Isoleucine 1.18–2.54 Mn 5.2–9 mg/kg

Leucine 11.4–14.8 Se 0.77 mg/kg

Lysine 8.25–10.7 Zn 22–33 mg/kg

Methionine 0.01–1.17 Mo 0.6 mg/kg

Phenylalanine 5.83–8.20

Proline 6.29

Serine 4.49

Threonine 3.95–7.06

Tryptophan 1.30–3.89

Tyrosine 2.86

Valine 8.21–10.45

%CP, percentage of crude protein.



solubility in water (∼5.4% of the true protein, which is ∼55% of the dry mass). MBM is a het-erogeneous mixture of particles; those derived from bone are high in ash, while those from soft tissues are high in protein. Lower temperature processing typically leads to greater amino acid bioavailability, but the phenomenon apparently is not caused by improved protein solubility, as any correlation between processing parameters, such as “peak cooking temperature” and protein solubility has not yet been found (Garcia and Phillips, 2009).

As a feedstuff, MM and MBM have been thoroughly characterized including the proxi-mate composition, gross energy, and amino acid profiles, which are more useful for designing nonfeed applications. A compilation of typical values for general blends of MM and MBM meal are shown in Tables 3.4 and 3.5.

MBM can be distinguished from MM by its higher mineral content, containing much higher levels of phosphorus and calcium. More spe-cific variations exist, indicative of the raw ma-terials used, for example “poultry MM,” “por-cine MBM,” or the raw materials composition, for example “MM” or “MM with blood after solvent extraction.” In addition, mixed species MBMs can be sold. The comprehensive catalog of data from the United States and Canada has been published by the National Academy of Sci-ences and contains average values obtained for proximate analysis of these more specific ABPs, along with nutritional information. This in-cludes digestibility, mineral content, and amino acid composition (National Research Council and Canadian Department of Agriculture, 1971). More regular, but less comprehensive updates are given as part of the NRC nutritional recom-mendations for livestock (National Research Council, 1982, 2001).

As indicated in the tables, large variation can be expected for some properties of MM and MBM as they tend to vary in composition be-tween different rendering facilities. In addition, variation of product characteristics also varies

significantly within a rendering plant, indicating that obtaining MBM from the same plant does not guarantee a consistent quality (Hendriks et al., 2004). To ensure better consistency, some renderers blend MBM from different sources, resulting in a product with less variation in ash and protein content (Garcia and Phillips, 2009).

Although, MBM has been recommended for use in animal feeds as a source of protein caused by its high availability of essential amino acids, minerals, vitamin B12, MBM and other rendered protein meals have potential for use in other applications, including as fuel, phosphorus fertilizer, a source of amino acids for chemical synthesis, and for the production of biobased plastics.

4.3 Poultry By-Product Meals

By-products of poultry processing include feathers, heads and feet, viscera, and meat trim-mings. Unlike the meat components, feathers are processed separately to produce feather meal. Keratin is the main protein in feathers and is vir-tually insoluble. To improve digestibility feath-ers are rendered using a batch or continuous hydrolyzer. The material first passes through a spreader to cut up any large components such as heads or feet. Hydrolysis is carried out un-der pressure (30–50 psi) for ∼45–60 min to break peptide and disulfide bonds, as well as hydro-gen-bonding interactions between nonadjacent amino acids. After hydrolysis, the material is dried to yield feather meal, which can be ground to finer particle sizes and is an excellent source of cysteine (Meeker and Hamilton, 2006). Hoof, horn, and hair meal are produced via a similar process. The typical composition of hydrolyzed feather meal is given in Table 3.6.

Another means of improving the digestibil-ity of feather meal includes inoculation with a feather-degrading bacterium (Bacillus lichenifor-mis), which excretes a strong keratinase enzyme capable of hydrolyzing collagen, elastin, and feather keratin (Jayathilakan et al., 2012).



TABLE 3.4 Typically Reported Range of Product Characteristics and Proximate Analysis of Meat and Bone Meals (National Research Council and Canadian Department of Agriculture, 1971; Preston, 2014; Howie et al., 1996; Kamalak et al., 2005; National Research Council, 2001; Nengas et al., 1995; Garcia and Phillips, 2009), Amino Acid Composition (National Research Council, 2001; Li et al., 2011), and Mineral Content Reported by (National Research Council and Canadian Department of Agriculture, 1971; Preston, 2014; National Research Council, 2001)

Characteristic Typical values

Dry material (%) 92.9–100




Ash 20.7–52.9%

Moisture content 1.9–5.7%

pH 5.89–7.19

Protein solubility 2.2–7.2%

Particle size 25.6–800 µm

Color Light tan brown to dark brown

CIE L*a*b* value L* = 51.2

a* = 22.1

b* = 38.9


Alanine 9.19 Ca 10.60–13.50%

Arginine 6.98–7.06 P 4.73–6.50%

Aspartic acid 4.25 Mg 0.24–1.20%

Cystine 0.94–1.01 K 1.02–1.56%

Glutamine 5.40 Na 0.71–0.78%

Glutamic acid 7.79 Cl 0.44–0.80%

Glycine 16.67 S 0.39%

Histidine 1.89–2.29 Cu 1.5–10 mg/kg

Isoleucine 2.76–3.69 Fe 500–602 mg/kg

Leucine 6.13–6.85 Mn 12.3–22 mg/kg

Lysine 5.18–6.08 Zn 94 mg/kg



Proline 11.3

Serine 4.00



Tyrosine 2.79

Valine 4.20–4.29




Poultry by-product meal is produced by ren-dering necks, feet, undeveloped eggs, and vis-cera of poultry, excluding feathers. In contrast, poultry meal contains only skin, bone, and trim-mings and excludes feathers, heads, feet, and

entrails. As a result, poultry meal tends to have a lower fat and ash content than poultry by-product meal, but because of the large amount of variability within either type of meal, it is unlikely that there is any real difference in the

TABLE 3.5 Typically Reported Range of Product Characteristics and Proximate Analysis of Meat Meal (National Research Council and Canadian Department of Agriculture, 1971; Preston, 2014; Kamalak et al., 2005; National Research Council, 2001; Qiao and Thacker, 2004; Kong et al., 2014), Amino Acid Composition (Preston, 2014; National Research Council, 2001) and Mineral Content Reported by (National Research Council and Canadian Department of Agriculture, 1971; Preston, 2014; National Research Council, 2001; Kong et al., 2014)


Dry material (%) 90.2–100




Gross energy kcal/kg 4294

Ash 18.4–26.4%

Moisture content 5.4%

Color Dark, red-brown


Alanine 8.11 Ca 8.49–9.01%

Arginine 7.06–7.96 P 4.18–4.44%

Aspartic acid 7.96 Mg 0.27–0.29%

Cystine 0.93–1.12 K 0.49–0.58%

Glutamic acid 12.46 Na 0.78–1.78%

Glycine 16.38 Cl 0.44–1.39%

Histidine 2.06–2.96 S 0.48–0.53%

Isoleucine 2.81–2.96 Cu 9.7–21 mg/kg

Leucine 6.24–6.31 Fe 400–701 mg/kg

Lysine 5.38–5.46 Mn 9.5–26 mg/kg

Methionine 1.43–1.72 Se 0.45 mg/kg

Phenylalanine 3.43–3.57 Zn 114–190 mg/kg

Proline 10.45 Mo 2.4 mg/kg

Serine 4.05



Tyrosine 2.34

Valine 4.06–4.44




proximate composition despite the differences in the raw materials rendered. Further to this, there appears to be no difference in the digestibility of the protein or free amino acids present in the two meals (Watson, 2006). The composition of poultry by-product meal is given in Table 3.7.

4.4 Seafood By-Product Meals

Fish, bones, and offal from processed fish are wet or dry rendered to produce a brown protein powder known as fish meal. Fish meal can be made from various mixed species, usually from

TABLE 3.6 Typically Reported Range of Product Characteristics and Proximate Analysis of Hydrolyzed Feather Meal (Preston, 2014; National Research Council, 2001; Nengas et al., 1995), Amino Acid Composition (National Research Council, 2001; Li et al., 2011), and Mineral Content Reported by (Preston, 2014; National Research Council, 2001)



Crude protein (%) 81.2–92

Ether extract (%) 6.2–7


Ash 3–3.5%

Color Off-white to brown


Alanine 5.09 Ca 0.33–0.48%

Arginine 6.93–6.99 P 0.45–0.50%

Asparagine 2.03 Mg 0.22%


Cystine 5.07–5.09 Na 0.34%

Glutamine 3.48 Cl 0.20–0.26%


Glycine 10.90 Cu 10 mg/kg

Histidine 1.07–1.15 Fe 76 mg/kg

Isoleucine 4.62–4.8 Mn 10 mg/kg


Lysine 2.57–2.63 Zn 90–111 mg/kg



Proline 14.37

Serine 10.72



Tyrosine 2.48

Valine 7.02–7.52




the bycatch obtained during harvesting, but is also made from the by-products of dressing spe-cific species, such as menhaden, anchovy, and to a lesser extent, herring (Meeker and Hamil-ton, 2006). During a dry rendering process, fish

oil generated during cooking is not removed. The oil is removed during wet rendering opera-tions, and is the most common method used. Wet rendering is applied to fish that have a high oil content, as removal of the oil is necessary for

TABLE 3.7 Typically Reported Range of Product Characteristics and Proximate Analysis of Poultry By-Product Meal (Preston, 2014; Kamalak et al., 2005; Nengas et al., 1995; Trushenski and Gause, 2013), Amino Acid Composition (Li et al., 2011; Dust et al., 2005), and Mineral Content Reported by (Preston, 2014; National Research Council, 2001)





Crude fiber (%) 1.2–2.5

Ash 10.5–17%

Color Tan to light brown


Alanine 7.64 Ca 0.33–0.48%

Arginine 6.77–7.20 P 0.45–0.5%

Asparagine 4.25 Mg 0.22%


Cystine 1.63 Na 0.34%

Glutamine 5.51 Cl 0.2–0.26%


Glycine 14.65 Cu 10 mg/kg

Histidine 2.02–2.12 Fe 76 mg/kg

Isoleucine 3.61–3.64 Mn 10 mg/kg


Lysine 5.35–6.27 Zn 90–111 mg/kg



Proline 10.45

Serine 4.15



Tyrosine 2.86

Valine 4.49–4.81




producing a shelf-stable product (Bimbo, 2005). After cooking and oil removal, the remaining material is hydraulically pressed to remove any liquor, and the solids (press cake) are dried and ground to produce fish meal. The stick-water can be concentrated and added back to the press cake to form wholemeal, or can be sold as a separate products called fish solubles (Bimbo, 2005). The typical composition of

menhaden fish meal and shrimp meal are given in Tables 3.8 and 3.9.

The meals made from oily fish, such as her-ring, tend to have much higher fat content (∼8.5%) (Martínez-Llorens et al., 2008) com-pared to other fish species, such as anchovy or sardine (Laining et al., 2003; NRC, 2001). Fish meals can be used in all types of animal feeds, usually where the fishy odor and flavors

TABLE 3.8 Typically Reported Range of Product Characteristics and Proximate Analysis of Menhaden Fish Meal (National Research Council, 2001; Qiao and Thacker, 2004; Trushenski and Gause, 2013; Bimbo, 2000), Amino Acid Composition (National Research Council, 2001; Bimbo, 2000; Kerr et al., 1998), and Mineral Content Reported by (National Research Council, 2001; Bimbo, 2000)





Crude fiber (%) 0.9

Gross energy kcal/kg 3025–4440

Ash 19.7–21.4%

Moisture content 4.8%

Color Brown


Arginine 3.43–6.04 Ca 4.87–5.34%

Cystine 0.58–0.97 P 2.93–3.05%

Glycine 7.35 Mg 0.20%

Histidine 2.45–2.83 K 0.74%

Isoleucine 3.67–4.09 Na 0.68%

Leucine 7.02–7.22 Cl 0.80%

Lysine 7.51–7.65 S 1.16%

Methionine 2.61–2.81 Cu 7 mg/kg

Phenylalanine 3.59–3.99 Fe 562 mg/kg

Serine 4.08 Mn 32 mg/kg

Threonine 3.92–4.20 Se 2.26 mg/kg

Tryptophan 0.82–1.05 Zn 112 mg/kg

Tyrosine 1.87–2.94 Mo 1.8 mg/kg

Valine 4.57–4.82




are beneficial, such as pet food (Meeker and Hamilton, 2006). Unlike other by-product meals, fish meals appear to have significantly higher calcium, phosphorus, and selenium content, making them an ideal nitrogen and phosphorus rich fertilizer for application to se-lenium deficient soils, such as those found in New Zealand.

5 INNOVATIONS IN BY-PRODUCT TREATMENT AND USES

Utilization of ABP meals as feed ingredients has become increasingly restricted, therefore growing attention has been focused on developing novel, nonfeed applications that have a substantially higher economic value. Higher value applications

TABLE 3.9 Typically Reported Range of Product Characteristics and Proximate Analysis of Shrimp Meal (Preston, 2014; Okoye et al., 2005; Fanimo et al., 2000; Everts et al., 2003; Fanimo et al., 2006), Amino Acid Composition and Mineral Content Reported by Fanimo et al. (2006)





Crude fiber (%) 3.6–20

Ash 14–56%

Color Light tan brown


Alanine 4.47 Ca 15.60%

Arginine 3.82 P 2.00%

Aspartic acid 8.11 Mg 0.83%

Cystine 0.96 K 0.23%

Glutamic acid 10.18 Na 0.31%

Glycine 4.56

Histidine 1.58

Isoleucine 3.58

Leucine 5.00

Lysine 3.86

Methionine 1.75

Phenylalanine 7.15

Proline 3.25

Serine 3.42

Threonine 3.42

Tryptophan 1.05

Tyrosine 4.43

Valine 4.34


5 INNOVATIONS IN BY-PRODUCT TREATMENT AND USES 57


of ABPs generally require the product to already be fit for, or made fit for, human consumption or medical use. For example, blood proteins col-lected under sterile conditions are typically em-ployed as binders, natural color enhancers, emul-sifiers, fat replacers, and meat curing agents and have also found use as egg replacers, protein and iron supplements, and as a source of bioactive compounds (Ofori and Hsieh, 2012).

The degree to which ABPs can be used is of-ten limited not only by the customs, religions, and regulatory requirements of the region they are processed in, but also by the biological and physicochemical properties of the materials. This is particularly true for red blood cells and whole blood, which despite their nutritional value, are generally not used as food ingredients because of their dark red color and the metallic flavor imparted by the high iron content (Du-arte et al., 1999). As a result, the use of blood is generally restricted to products where the black color is acceptable, although the heme pigment and its chelated iron can be removed through extraction with acidified acetone or via bleach-ing with hydrogen peroxide to remove the color (Wismer-Pedersen, 1988). An alternative is to use plasma, which makes up 65–70% of blood by volume and contains 7.9% protein (3.3% al-bumins, 4.2% immunoglobulins and globulins, and 0.4% fibrinogen) and is more commonly used in food applications because of its neutral flavor and color (Duarte et al., 1999).

Blood components, which are currently iso-lated for medical uses, include fibrinogen, fi-brinolysin, serotonin, immunoglobulins, and plasminogen. Purified bovine serum albumin is used during testing for the Rh factor in human beings, as a stabilizer for vaccines, and in anti-biotic sensitivity tests. Bovine thrombin is used to promote coagulation of blood and hold skin grafts in place, and porcine plasmin enzyme is used to digest fibrin in the blood clots of heart attack patients (Bah et al., 2013).

Innovations have been made over the years to alter the apparent quality of rendered products

and include various bleaching techniques and the addition of antioxidants and adulterants. Although some of these treatments enhance the quality of the product, adulterants interfere with chemical detection methods used to classify the products, placing them higher on the quality and price scale than they should be.

Potential nonfeed uses of ABP meals exploit their high protein concentration. All ABP meals have potential as a source of biomass for biofu-el production. Poultry meal, feather meal, and MBM have been used as alternative nitrogen sources to replace yeast extract during the fer-mentation of potato starch to produce bioethanol (Izmirlioglu and Demirci, 2012). Feather meal has recently been shown to be a good source of fat for the production of biodiesel while subse-quently improving the protein concentration of the feather meal (Kondamudi et al., 2009).

The components of MBM could be separated to use the protein from particles derived from soft tissues or exploited for the high mineral concentration of the bone particles. Like other nitrogen-rich organic products, MBM has dem-onstrated an ability to control plant pathogens (Lazarovits, 2001), its protein concentrated to create an adhesive (Park et al., 2000) and as the feedstock for the production of a plastic material designed to be a pet chew toy (Garcia et al., 2004). These and other applications have potential commercial value, but with the excep-tion of fuel uses, most have only been imple-mented on a laboratory or demonstration scale (Garcia et al., 2005).

Current high-value applications of blood products include preparation of blood agar, pro-viding a source of hydrolysates for microbial use, producing commercial porphyrin deriva-tives and enzymes used in medical applications. Recent research has also shown promise for the recovery and extraction of bioactive compounds from hydrolyzed blood proteins. Some of the peptides studied have shown medical benefits including inhibition of the angiotensin I-con-verting enzyme (subsequently minimizing the



formation of compounds which cause constric-tion of blood vessels raising blood pressure and creating an antihypertensive effect), antioxidant activity, antimicrobial properties, antigenotoxic effects, mineral-binding ability, and opioid ac-tivity (Bah et al., 2013; Toldrá et al., 2012).

There are still challenges that need to be over-come in order to adopt ABP meals as a feedstock for biobased products. Many potential applica-tions for blood meal and MBM are ruled out by the potential for prion contamination of the material (Garcia and Phillips, 2009). Most of the higher end applications require sterile condi-tions for the collection of the raw material and often, subsequent thermal or chemical treat-ment to ensure its integrity. In cases where it is impractical to collect and generate the protein meal under sterile conditions, or where it is con-sidered an SRM, other novel applications can be created. Recent examples include extrudable thermoplastic materials produced from blood meal and its decolored derivative, compres-sion molding of defatted feather meal and whey blends (Sharma et al., 2008), and the incorpora-tion of hydrolysate from SRM as a curing agent to create a thermosetting epoxy resin (Mekonnen et al., 2013). Protein hydrolysates have also been shown to have flocculant activity, such as those from enzyme hydrolyzed MBM (Piazza and Garcia, 2010, 2014). In the future, amino acids derived from animal protein meals may even be used to synthesize commodity chemicals, such as acetonitrile (Lammens et al., 2012).

Although there is a significant volume of liter-ature available regarding the physical, chemical, and nutritive characteristics of ABP meals, in-cluding licensed software, such as AminoDat4.0, containing data compiled over decades by the US National Research Council, very little data exists that is relevant to the material properties and handling of such products. Furthermore, there is limited industry-wide information on the sources of raw material used to produce ABP meals, the rendering treatment used, or geo-graphic availability (Garcia and Phillips, 2009),

making economic analysis for the pilot scale and start-up phases of novel technologies difficult.

This chapter has shown that differences in the sources of raw material, how they are handled, and the rendering processes used result in sig-nificant variation in the physical, chemical, and nutritive properties of the ABP meals produced. As such, innovative technologies designed to use these protein meals will have to account for this, particularly if the technology is taken to the global stage.


ABP Animal by-productBSE Bovine spongiform encephalopathyEDTA Ethylenediaminetetraacetic acid disodium saltFFA Free fatty acidLW Live weightMBM Meat and bone mealMM Meat mealSRM Specified risk material

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C H A P T E R


4Marine Processing Proteinaceous

By-Products: A Source of Biofunctional Food Ingredients

A.C. Neves, P.A. Harnedy, R.J. FitzGeraldDepartment of Life Sciences, University of Limerick, Limerick, Ireland

1 INTRODUCTION

The total global production of fish and shell-fish has increased in recent years, reaching 158 million tons (mt) in 2012. This value was di-vided between wild capture and aquaculture, and although 91.3 mt arose from capture, the greatest increase was observed in aquaculture production. Aquaculture production has in-creased steadily at an average annual growth rate of 6.1% from 36.8 mt in 2002 to 66.6 mt in 2012 with an estimated value of US$137.7 bil-lion (FAO, 2014a). Finfish species account for 66% of total aquaculture production followed by molluscs (33.0%) and crustaceans (11%). It is estimated that 86% was destined for human consumption with the remaining 14.0% des-tined for nonfood products, such as fish meal and fish oil (FAO, 2014b). China leads in the production of fish and shellfish, reaching 43.5 mt in 2013 followed by India (4.2 mt), Vietnam (3.1 mt), Indonesia (3.1 mt), Bangladesh,

Norway, Thailand, Chile, Egypt, and Myanmar (FAO, 2014b).

During the processing of fish and shellfish for human consumption, that is, filleting, freezing, canning or curing, by-products, such as heads, bones, viscera, gills, dark muscle, belly flaps, and skin of different fish species and shells and bys-sus from shellfish or specimens below the stan-dards for trade (the most abundant by-products in this category being mussel meat, arising from undersized, cracked, and fouled mussels) are generated (FAO, 2014b; Kim and Mendis, 2006). The overall trend in the fish and shellfish indus-try suggests that in 2022, aquaculture produc-tion will reach 99.33 mt, and this will lead to a similar rate of increase in by-products generated (FAO, 2014a). Although some African countries, as well as Norway and Iceland, can use some of these by-products for human consumption by drying fish heads and processing roe with heat treatments (FAO, 2014b), the majority of the by-products are currently discarded or used as

64 4. MARINE PROCESSING PROTEINACEOUS BY-PRODUCTS: A SOURCE OF BIOFUNCTIONAL FOOD INGREDIENTS


fertilizer, animal feed, or for fish oil or glue pro-duction (Arvanitoyannis and Kassaveti, 2008; Waite, 1986).

An alternative potential use for these by-products is in the production of nutraceuticals and bioactive ingredients. At present, compo-nents derived from fish oils, such as polyun-saturated fatty acids (PUFAs), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), are used as bioactive agents for food supple-ments (Narayan et al., 2006; Zuta et al., 2003). However, marine by-products are also rich reservoirs of high-quality proteins that can act as substrates for the generation of bioactive peptides (BAPs) and amino acids (Harnedy and FitzGerald, 2012). Marine processing by-products can contain between 7% and 23% w/w protein, depending on the species of fish or shellfish (Harnedy and FitzGerald, 2012; Murray and Burt, 2001).

Biological activities, such as antihyperten-sive, antioxidant, antimicrobial, anticoagulant, antidiabetic, anticancer, immunostimulatory, calcium-binding, hypocholesteremic, and ap-petite-suppression activity have been linked to proteins and protein-derived peptides from fish and shellfish (Kim et al., 2008; Kristinsson, 2008; Okada et al., 2008; Venugopal, 2009b; Vercruysse et al., 2005). Some studies have also reported on BAPs arising specifically from by-products of the fish and shellfish processing industry (Gildberg, 2004; Harnedy and FitzGerald, 2012), such as skins, frames, heads, bones, and shells.

Gelatin is one of the marine processing pro-teinaceous components already used in the pharmaceutical, cosmetic, and food industries. Currently, gelatin is derived mainly from por-cine and bovine sources with only 1.5% coming from fish and other sources (FAO, 2014b). The use of marine-derived gelatin and its peptides has advantages, such as their preference among the Islamic and Jewish communities as these ingredients are acceptable in halal and kosher products (Karim and Bhat, 2008). Generation of food products using marine instead of bovine

gelatin could help prevent the risk of infec-tion from diseases, such as bovine spongiform encephalopathy (BSE; Karim and Bhat, 2008). Therefore, it is clear that by-products from ma-rine processing show significant promise as functional food ingredients.

However, with legislation-related challenges in handling and processing by-products in some countries, as well as challenges with the commercialization of products coming from by-product materials and their health claims, a high percentage of the by-products from the fish and shellfish industries are still currently discarded. These components cannot be re-turned to the sea because this action can disturb the equilibrium of the ecosystem. Therefore, significant funds are spent annually on incin-eration of these by-products by both the fish and shellfish industry (Kim and Mendis, 2006). This review will focus on the proteins that can be extracted from fish and shellfish sources, the peptides that may be generated and their biological activities, as well as their commercial applications in the food industry.

2 FISH AND SHELLFISH PROTEINS

2.1 Composition

Fish and shellfish proteins are classified into three main groups, that is, sarcoplasmic, myo-fibrillar and stromal proteins. The sarcoplas-mic (water or low-salt buffer soluble) proteins are present in the sarcoplasm and account for approximately 15.0–35.0% of the total muscle tissue protein in fish. These consist mainly of enzymes associated with metabolic processes, such as the conversion of glycogen to adenosine triphosphate (ATP) for the production of energy along with creatine kinase, aldolase, and glycer-aldehyde-3-phosphate dehydrogenase (Ladrat et al., 2003; Vareltzis, 2000). Pigmented proteins, such as myoglobin or hemoglobin, can also be found in some species (Belitz et al., 2004).

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These proteins differ depending on the species of fish and shellfish because their metabolic func-tions change depending on the ecosystem within which they live (FAO, 2014b). Myofibrillar pro-teins are structural proteins that are soluble in high-salt solutions and account for 65.0–75.0% of the total protein in fish muscles (Venugopal, 2009a). They are responsible for the contractile apparatus that allows movement. They are com-posed essentially of two main proteins, actin and myosin, and are similar to the composition of mammalian myofibrillar proteins. Myosin, the most abundant protein in muscle, ranges from 50.0–58.0%, while actin only accounts for 15.0–20.0% of the total muscle protein (Varelt-zis, 2000). Other structural and regulatory pro-teins associated with movement include tropo-myosin, troponin, actinin, desmin, nebulin, and C and M proteins. Stromal proteins form the connective tissues present in the skin, bone, and swim bladder, and the myocommata in muscle (FAO, 2014b). They constitute around 3.0% w/w of the total muscle protein for most fish and shellfish species; however, some species, such as shark and ray, can contain levels of stromal proteins as high as 10.0% w/w, compared with 17.0% w/w in mammalian species (FAO, 2014b). Stromal proteins predominately consist of colla-gen, with the remaining 10.0% w/w made up of elastin and gelatin (Belitz et al., 2004; Kim and Mendis, 2006; Venugopal, 2009a).

Fish skin, bones, tendons, and cartilage con-tain high quantities of collagenous proteins. There are several variants of collagen, which can be found in different amounts, in different parts of the fish. The most abundant is type I collagen, which is present in the skin and bones, whereas type II collagen is present in the cartilage. Type III collagen is also found in the skin of fish, but its amount decreases with age. Approximately 50% w/w is found in young fish and 5.0–10.0% in older fish (Van der Rest and Garrone, 1991). Other variants of collagen are found in lower amounts, and occurrence varies depending on the organ. The denaturation of collagen by

enzymatic, chemical, and heat treatment pro-duces gelatin. The type of collagen most used for the production of gelatin is type I, which contains in its structure a high percentage of the amino acids glycine (Gly), proline (Pro), and hy-droxyproline (Hyp) (Grant, 2007; Schrieber and Gareis, 2007; Van der Rest and Garrone, 1991). The basic structure of collagen is composed of three chains of polypeptides that are linked by peptide bonds forming a triple helix. Each poly-peptide has the sequence Gly-X-Y (with X cor-responding to Pro, and Y corresponding to Hyp in the majority of cases) repeated a significant number of times. These proteins are also rich in other amino acids, such as valine (Val) and ala-nine (Ala) (Kim and Mendis, 2006). When the three chains adopt a coil structure, it forms fi-brils that maintain the stability of the molecule (Djabourov, 1988; Tosh et al., 2003). Fish colla-gen can be divided in two major chains corre-sponding to an α-chain, with α1 and α2 variants and a cross-linked component (β-chain) with molecular weights of approximately 200 kDa for the β-chain and of approximately 116 and 97 kDa for the subunits of the α-chain (Gómez-Guillén et al., 2005; Nikoo et al., 2011). Type II and III collagens are homotypic molecules having identical α-chains, whereas type I col-lagen is a heterotypic molecule having distinct α-chains. Although the amino acid composition of gelatin derived from fish is quite similar to mammalian collagen, there are lower amounts of Pro and Hyp and higher amounts of serine (Ser) and threonine (Thr) in fish-derived gelatin, in particular cold-water species (Eastoe, 1957; Pati et al., 2010). These differences in amino acid composition lead to some variations in gelatin properties, such as differences in rigidity and temperature stability, and altered gelling prop-erties. However, the major variation in the prop-erties and composition of collagen depend on the species of fish (Pati et al., 2010).

Collagenous proteins can also be found in shellfish, such as in the byssus of mussels. Mussel byssus collagen is composed of two



pepsin-resistant components, each contain-ing three identical α-like chains (Benedict and Waite, 1986; Qin and Waite, 1995). One is found predominantly at the proximal end of the byssus thread (Col-P) with a molecular mass of 50 kDa, and another is found predominantly at the distal end of the thread (Col-D), with a molecular mass of 60 kDa (Qin and Waite, 1995). Both Col-P and Col-D contain high levels of Gly, glutamic acid (Glu) or glutamine (Gln) and 4-hydroxyproline (4-Hyp), followed by Pro and Ala; other amino acids are present in lower amounts (Benedict and Waite, 1986; Qin and Waite, 1995). The ami-no acid content of these types of collagen has the characteristics of types I and III, being rich in Gly, Pro, and Hyp. Although, the main amino acids present in collagen from mussel byssus are similar to those present in collagen from fish skin, there are some variations in the relative concentration of these amino acids.

Collagen in its partially hydrolyzed coiled form, gelatin, is used extensively in the cosmetic, pharmaceutical, and food industry because of its potential to improve elasticity, consistency, and stability of food (Gudipati, 2013). In 2007, gelatin production reached 326,000 t world-wide with 121,800 t being produced in Europe alone. The demand for gelatin by the food in-dustry has greatly increased, and this has led to the search for alternative sources of gelatin. Nowadays, 46% of gelatin production comes from pigskin followed by 29% from bovine hide. Pork and cattle bones account for 23.1% of gelatin production (Gómez-Guillén et al., 2009). The production of fish skin–derived gelatin, mainly from cod, salmon, and pollock, only ac-counted for around 1.5% of the total production of this product in 2007, which corresponds to approximately 4800 t worldwide. However, this percentage has been increasing in recent years, and research in other species of fish, such as ti-lapia, perch, and catfish, has been performed to access new sources of gelatin (Jamilah and Harvinder, 2002; Muyonga et al., 2004; Rawdkuen et al., 2010). Moreover, fish skins and

bones used for extraction of gelatin constitute a large part of the by-products coming from the fish industry, making the production of gelatin from these by-products very profitable. Other sources of marine-derived gelatin that have been investigated are squid and mussel byssus (Mendis et al., 2005a). However, more research needs to be carried out for these sources to be used for gelatin production. The interest in gela-tin is linked with (1) its capacity to form stable three-dimensional gels, (2) its acceptability for use in the food industry as a nontoxic ingredi-ent, (3) its solubility in biological fluids at body temperature, and (4) its ability to form film ma-terials that are strong and flexible as well as be-ing edible and biodegradable (Gudipati, 2013).

2.2 Protein Extraction Methods

To date, various methods have been used for the extraction of proteins from different species of fish and shellfish. These methods vary in pa-rameters, such as weight-to-volume ratio during extraction, number of sequential extractions em-ployed, homogenization, pH of extraction, and agitation time. Among the methods mainly de-scribed in the literature for recovery of proteins from fish are the acid and alkali-aided process-ing and the surimi (a fish-paste product) process. The pH shift processes employing both alkaline and acid are used for extraction of protein from different fish species, as well as shellfish, and consist of homogenization of the sample in an acid or alkaline solution and recovery of the protein by centrifugation where solids, such as bones, scales, and skin are removed. In some methods, mechanical extraction by mincing and/or grinding is employed prior to homog-enization (Halldórsdóttir et al., 2011; Raghavan and Kristinsson, 2009; Shaviklo and Johanns-son, 2007; Thorkelsson et al., 2008; Vareltzis and Undeland, 2008). The soluble protein is then precipitated by adjusting the pH to the isoelec-tric point of the myofibrillar proteins (around pH 5.5) to recover a protein isolate. Conversely,

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the surimi process for protein recovery con-sists of three washes with cold water with 0.2% NaCl included in the last wash, followed by re-fining and dewatering the slurry (Kristinsson et al., 2005). However, this method is less suc-cessful than the pH shift method as sarcoplas-mic proteins are lost, leading to a reduced pro-tein recovery (Kristinsson et al., 2005).

Among the food-friendly methods described in the literature for extraction of gelatin from fish skin are acid and pepsin-solubilization approaches (Jongjareonrak et al., 2005; Nagai et al., 2002). These methods begin with an alka-line treatment of the skin to remove any remain-ing muscle proteins, followed by extraction of the gelatin with either an acid solution or incu-bation with pepsin (Nalinanon et al., 2007). Ac-cording to Jongjareonrak et al. (2005), the acid method of collagen extraction from fish skin leads to a higher yield of collagen compared to the pepsin method with yields of 90% and 4.7% w/w extracted, respectively. The gelatin extrac-tion methods reported in the literature that in-volves a pH shift vary not only in extraction pH but also in the extraction temperature, which can be between 45 and 70°C (Jaswir et al., 2009; Montero and Gómez-Guillén, 2000; Zhou and Regenstein, 2005).

The formation of gelatin from collagen occurs because of the disruption of hydrogen bonds and hydrolysis of peptide bonds. The conver-sion of collagen to gelatin may be achieved by decreasing the solution pH level; its heating and cooling allows the solubilization of the col-lagen. Because of the lower content of Pro and Hyp in fish collagen compared to mammalian collagen, the gelling properties of fish, pork, and bovine gelatin differ in melting tempera-ture, for example, < 17°C for coldwater fish, 24–29°C for warmwater fish, and > 30°C for pork and bovine gelatin. Their gel strengths also differ, for example, ∼ 100 g for coldwater fish gelatin; ∼ 200 g for warmwater; and 200–300 g for pork and bovine gelatin. However, studies on extracting and characterizing collagen from

different species of fish are ongoing (Gómez-Guillén et al., 2011).

2.3 Enzymatic Hydrolysis of Food Proteins and Techno-Functional Properties

Enzymatic hydrolysis is a method which has been successfully used for modification of food proteins to improve their physicochemi-cal, techno- and biofunctional, and sensory properties without affecting their nutritive value (Kristinsson and Rasco, 2000a,b; Shahidi et al., 1995; Thorkelsson et al., 2008). The choice of enzyme, temperature, pH, and enzyme-to- substrate ratio is crucial for achievement of an appropriate degree of hydrolysis (Guérard et al., 2001; Kristinsson and Rasco, 2000a,b). Proteinases can be categorized into four major classes accord-ing to the specificity of the peptide bonds they cleave, that is, serine, thiol, carboxyl, and metallo proteinases. They can also be classified according to their hydrolysis mechanism, for example, en-doproteinases or exopeptidases. Endoproteinases cleave peptide bonds within protein molecules, whereas exopeptidases systematically remove amino acids or dipeptides from either the N- (aminopeptidases) or the C-terminus (carboxy-peptidases) by hydrolyzing the terminal peptide bonds (Kristinsson and Rasco, 2000a).

It has been reported in several studies that food proteins modified by hydrolysis have improved techno-functional properties when compared with intact proteins (Flanagan and FitzGerald, 2003; Ryan et al., 2008). Among the techno-functional properties improved on hy-drolysis by the generation of lower molecular mass peptides with less secondary structures, are solubility, viscosity, turbidity, emulsifying, foaming, gelation, heat, and pH stability. These improvements in techno-functional properties have been shown in hydrolysates from fish and shellfish when compared with their intact pro-teins. This can be of significant interest in the use of by-products from the fish and shellfish indus-try (Adler-Nissen, 1976; Gbogouri et al., 2004;



Kristinsson and Rasco, 2000a,b; Decourcelle et al., 2014; Shahidi et al., 1995). The extent of improvement in the techno-functional proper-ties of the proteins upon hydrolysis depends on the enzyme used for hydrolysis as well as the pH, time, and temperature of hydrolysis be-cause these are responsible for the variation in the degree of hydrolysis and the peptide profile.

The study and control of the process of enzy-matic hydrolysis is especially important in the fish and shellfish industry because certain fish and shellfish may contain endogenous enzymes. These enzymes are mainly associated with the fish viscera. However, there are some enzymes, such as calpains and cathepsins, which contrib-ute to the proteolytic breakdown of proteins in muscle cells (Aspmo et al., 2005), although this activity is lower in fish and shellfish muscle than in terrestrial-derived muscle (bovine and por-cine). Endogenous marine enzymes are currently used to improve the bio- and techno-functional properties of proteins in the shellfish industry, for example, in the generation of fermented shrimp paste and fermented mussel sauce (Binsan et al., 2008; Je et al., 2005; Kleekayai et al., 2015; Peralta et al., 2008; Rajapakse et al., 2005b).

2.4 Characterization of Fish and Shellfish Hydrolysates

Proteins from fish and shellfish contain se-quences of peptides encrypted within their pri-mary structure that may play an important role in metabolic regulation and may have potential benefits in controlling disease and promoting health. These BAPs are released during gastro-intestinal digestion, fermentation, or food pro-cessing and are short in length, generally having between 2 to 20 amino acids (Pihlanto-Leppälä, 2000a). The biological activities linked with these peptides vary depending on their length, on the amino-acids that are present, as well as their se-quence. The BAPs released and the properties associated with them, depend on the type of hy-drolysis, pretreatment, pH, time, temperature,

enzymes involved, and the ratio of enzyme-to-substrate used (Kim & Wijesekara 2010; Udenig-we & Aluko 2012). Controlled generation and the targeted utilization of BAPs may beneficially modulate physiological systems within the hu-man body, ultimately enhancing health and con-trolling disease.

Cardiovascular-related diseases and diabe-tes mellitus are two of the leading public health problems causing more than 4.35 million deaths in Europe annually (Petersen et al. 2005). Using marine coproduct-derived proteins to generate peptides that modulate markers associated with these conditions could potentially lead to the development of natural food-derived agents for the management of these diseases.

Several studies have been carried out using food sources, such as eggs, milk, cereals, and fish proteins (Li et al. 2004; Pihlanto-Leppälä, 2000a,b; Power et al., 2012) to generate BAPs with in vi-tro activities related to the previously mentioned health conditions. These include peptides with angiotensin-converting enzyme (ACE) and renin inhibitory activity associated with the renin-angiotensin system (Brown 2006; Deng et al., 2012; Li et al., 2004; Ni et al., 2012), dipeptidyl-peptidase IV (DPP-IV), α-glucosidase, and α-amylase inhibition, enzymes linked with the stimulation of insulin biosynthesis and secretion, reduction of glucagon release, reduction of appe-tite and glucose metabolism in general, as well as glucose absorption rate (Guasch et al., 2012; Havale and Pal, 2009; Jesson et al., 2005). Other potential bioactivities from food protein-derived peptides relate to the antioxidant and antiinflam-matory effects associated with tissue damage and regeneration (one of the main complications in patients with diabetes and cardiovascular dis-ease [CVD]) (Cui et al., 2005; Power et al., 2012) or antiobesity effects, which correlate with many other diseases (Boggiano et al., 2005).

Tables 4.1 and 4.2 list the peptides presently identified with different biological activities coming from fish and shellfish by-products, respectively. The main bioactivities currently

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TABLE 4.1 Biological Activities Associated with Protein Hydrolysates and Peptides from Fish By-Product Sources

Species name Marine source Biological activity Sequence References

Atlantic salmon (Salmo salar) Head ACE inhibitory WM, WA, VW, MW, IW, LW, LF, AW, WV, WL, FL, DW, GIG

Ohta et al. (1997); Ono et al. (2003)

Atlantic salmon (Salmo salar) Fin ACE inhibitory VWDPPKFD, FEDYV-PLSCF, FNVPLYE

Ahn et al. (2012)

Atlantic salmon (Salmo salar) Frames Antihypertensive — Ewart et al. (2009)

Atlantic salmon (Salmo salar) Skin ACE inhibitory AP, VR Gu et al. (2011)

Atlantic salmon (Salmo salar) Skin DPP IV inhibitory GPGE, GPGA Li-Chan et al. (2012)

Atlantic salmon (Salmo salar) Filleting by-products Anticancer — Picot et al. (2006)

Atlantic salmon (Salmo salar) Fish Antimicrobial AEVAPAPAAAAPAKAPK-KKAAAKPKKAGPS

Lüders et al. (2005)

Cod (Gadus macrocephalus) Frame Antioxidant, ACE inhibitory — Jeon et al. (1999)

Cod (Gadus macrocephalus) Skin Antioxidant, ACE inhibitory TGGGNV, TCSP Ngo et al. (2011)

Herring (Clupea harengus) Whole, body, head, gonads

Antioxidant — Sathivel et al. (2003)

Hoki (Johnius belengerii) Skin Antioxidant HGPLGPL Mendis et al. (2005a,b)

Hoki (Johnius belengerii) Bone Ca-binding — Jung et al. (2007)

Hoki (Johnius belengerii) Frame Antioxidant, Ca-binding GSTVPERTHPACPDFN, VLSGGTTMYASLYAE

Kim et al. (2007); Jung and Kim (2007)

Pacific whiting (Merluccius productus)

Whole fish Immunoregulatory — Duarte et al. (2006)

Pollock (Theragra chalcogramma)

Skin, frame ACE inhibitory GPL, GPM, FGASTRGA Byun and Kim (2001); Je et al. (2004)


Frame Antioxidant, Ca-binding LPHSGY, VLSGGTTMA-MYTLV

Je et al. (2005); Jung et al. (2006)


Skin Iron-chelating SCH Guo et al. (2013)


Skin Mineral chelating GPAGPHGPPG,WR, SGSTG, KIGER, MLGER, GPAG-PR, AGPAGPR, QGLIGPR, GPVGHGPPGKDG

Guo et al. (2015)

(Continued)

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Pollock (Theragra chalcogramma) Whole fish Immunoregulatory NGMTY, NGLAP, WT Hou et al. (2012)

Sardine (Harengula zunasi) Head, bone, gut Antianaemia — Shang-gui et al. (2004)

Sea Bream (Sparus autarus) Scale ACE inhibitory GY, VY, GF, VIY Fahmi et al. (2004)

Snapper (Priacanthus macra-canthus)

Skin Antioxidant — Phanturat et al. (2010)

Snapper (Lutjanus vitta) Skin Antioxidant — Khantaphant and Benjakul (2008)

Sole (Solea solea) Skin Antioxidant — Gimenez et al. (2009)

Sole (Limanda aspera) Frame Antioxidant, Antihyper-tensive

N-terminal RPDFDLEPPY, MIFPGAGGPEL

Jun et al. (2004); Jung et al. (2005); Rajapakse et al. (2002)

Tuna (Thunnus thynnus) Frame Antioxidant, Antihyper-tensive

VKAGFAWTANQQLS, GDLGKTTTSNWSPP

Je et al. (2007); Lee et al. (2010)

Yellowtail (Seriola lalandi) Bone, scale Antioxidant, ACE inhibi-tory

— Morimura et al. (2002); Ohba et al. (2003)

Adapted from Harnedy and FitzGerald, 2013a.

TABLE 4.1 Biological Activities Associated with Protein Hydrolysates and Peptides from Fish By-Product Sources (Cont.)

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TABLE 4.2 Biological Activities Associated with Protein Hydrolysates and Peptides from Shellfish Sources


Clam (Meretrix lusoria) Muscle ACE inhibitory VRK, YN Tsai et al. (2008)

Krill (Mesopodopsis orientalis) Fermented product Antioxidant — Faithong et al. (2010)

Krill (Euphausiacea) Muscle ACE inhibitory KLKFV Kawamura et al. (1992)

Lobster (Pleuroncodes planipes) Shell waste Anticancer — Kannan et al. (2011)

Mussel (Mytilus edulis) Fermented sauce Antihypertensive EVMAGNLYPG Je et al. (2005)

Mussel (Mytilus edulis) Fermented sauce Antioxidant HFGBPFH Rajapakse et al. (2005a,b)

Prawn (Penaeus japonicus) Muscle Antioxidant IKK, FKK, FIKK Suetsuna et al. (2000)

Oyster (Crassostrea gigas) Fermented sauce Antihypertensive Not given (MW 592.9 Da) Je et al. (2005)

Oyster (Crassostrea gigas) Muscle Antimicrobial Liu et al. (2008)

Oyster (Crassostrea gigas) Muscle ACE inhibitory FY, AW, VW, GW Katano et al. (2003)

Oyster (Pinctada fucata martencii) Muscle ACE inhibitory VVYPWTQRF Wang et al. (2008)

Oyster (Crassostrea talienwhanensis Crosse) Muscle HIV-1 protease inhibitory LLEYSI, LLEYSL Lee and Maruyama (1998)

Oyster (Crassostrea gigas) Muscle Anticancer HFNIGNRCLC Cheong et al. (2013)

Shrimp (Penaeus setiferus) Shell waste Anticancer — Kannan et al. (2011)

Shrimp (Penaeus setiferus) Fermented product ACE inhibitory SV, IF Kleekayai et al. (2015)

Shrimp (Penaeus setiferus) Fermented product Antioxidant WP Kleekayai et al. (2015)

Shrimp (Penaeus aztecus) Head Appetite suppressant — Cudennec et al. (2008)

Shrimp (Acetes chinensis) Whole shrimp ACE inhibitory FCVLRP, IFVPAF, KPPETV, YLLF, AFL

Hai-Lun et al. (2006)

Shrimp (Plesionika izumiae Omori) Whole shrimp Antihypertensive VWYHT, VW Nii et al. (2008)

Shrimp (Acetes vulgaris / Acetes sp.) Fermented product Antioxidant — Faithong et al. (2010)

Shrimp (Metapenaeus monoceros) Shell waste Antioxidant — Manni et al. (2010)

Shrimp (Litopenaues vannamei) Cephalothorax ACE inhibitory — Benjakul et al. (2009a,b)

Shrimp (Litopenaues vannamei) Cephalothorax Antioxidant — Benjakul et al. (2009a,b)

Squid (Dosidicus gigas) Skin Antioxidant FDSGPAGVL, NGPLQAGQPGER Mendis et al. (2005a,b)

Squid (Dosidicus eschrichitii Steenstrup) Skin Antioxidant — Lin and Li (2006)

Squid (Dosidicus gigas) Skin Antioxidant — Gimenez et al. (2009)

Squid (Dosidicus gigas) Muscle Antioxidant NADFGLNGLEGLA, NGLEGLK Rajapakse et al. (2005a,b)

Adapted from Harnedy and FitzGerald, 2013a.



associated with peptides from fish and shellfish by-products are related to CVD. These include ACE inhibitory, anticoagulant, antihypertensive, and antioxidant activities, which make the pep-tides generated from marine by-products good prospective ingredients for the management of conditions such as diabetes and cardiovascular-related diseases.

There are a number of well-characterized proteolytic enzymes arising from plant, animal, and microbial sources used for the generation of BAPs. Among the most commonly used en-zymes for the generation of BAPs from fish and shellfish are enzymes, such as pepsin (from por-cine stomach), trypsin, and chymotrypsin (from bovine pancreas), pronase E (from Streptomyces griseus), Alcalase (from Bacillus licheniformis), Neutrase (from Bacillus amyloliquefaciens), col-lagenase (from Clostridium histolyticum), and pa-pain (Carica papaya). However, autolysis and fer-mentation processes can also generate peptides with biological activities caused by the action of endogenous proteolytic enzymes, such as calci-um-activated calpains and lysosomal cathepsins (Bauchart et al., 2007; Bragadóttir, 2001; Roh et al., 2009). The fermentation of fish and shellfish for subsequent use in the food industry is largely carried out in Asian countries for flavoring and food supplementation applications (Thorkels-son and Kristinsson, 2009; Venugopal, 2008). However, the possible presence of halophilic pathogenic bacteria, the high salt content, and the formation of biogenic amines such as hista-mine, raises some consumer safety and legal is-sues on the potential hazards of consuming such fermented products. Legal issues are also raised for the exploitation of fish protein hydrolysates generated through the use of some alkali and acid treatment methods, because these chemi-cal processes may represent a hazard for human health. Therefore, the use of enzymatic hydroly-sis with the proteolytic enzymes described ear-lier, under a controlled environment, is the most promising for the generation of BAPs from fish and shellfish by-products for use in products for

human consumption, because it leads to a more consistent molecular-weight profile and peptide composition. Furthermore, in vitro enzymatic hydrolysis with carefully controlled conditions takes, in general, significantly less time to reach the same degree of hydrolysis than fermenta-tion processes, which can take up to 18 months, a process which is often used in the generation of natural condiments (Samaranayaka and Li-Chan, 2011). Hydrolysates generated through controlled enzymatic hydrolysis and fermen-tation processes from fish and shellfish by-products are reported to have bioactive prop-erties, such as antioxidant, ACE inhibitory, calcium-binding, antihypertensive, anticoagu-lant, anticancer, antimicrobial, HIV-1 protease inhibitor, antianemia, and appetite suppressant activity (Harnedy and FitzGerald, 2013a).

The hydrolysates used for food and dietary purposes need to be characterized by their pep-tide size (Gauthier et al., 1986) because low-molecular-mass peptides, mainly di- and tripep-tides, appear to have higher biofunctional value (Fahmi et al., 2004; Vijayalakshmi et al., 1986). Peptides with greater than 20 amino acid residues are reported to be associated with improvements in techno-functionality (Gauthier et al., 1986). Therefore, the separation and identification of the peptides and amino acids composing the protein hydrolysates must be studied in order to obtain a better knowledge about their composi-tion. Among the methods used for this purpose include reversed-phase high-performance liquid chromatography (RP-HPLC) which has been used to separate peptides from protein hydro-lysates based on differences in hydrophilicity and hydrophobicity (Lemieux et al., 1991). In the analysis of peptides on the basis of their molecu-lar mass, the most commonly used techniques include size-exclusion chromatography (SEC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fekete et al., 2012). Moreover, specific quantification and character-ization of the amino acids within peptides is gen-erally carried out by HPLC mass spectrometry

3 BIOFUNCTIONAL ACTIVITIES 73


(MS), HPLC tandem mass spectrometry (MS/MS), or a combination of ultra-performance liquid chromatography (UPLC) with MS/MS (Dillen and Cuyckens, 2012; Le Maux et al., 2014; Zhu and FitzGerald, 2010).

3 BIOFUNCTIONAL ACTIVITIES

The increasing rates of obesity in the mod-ern world and conditions linked with it, such as type 2 diabetes and CVD, has raised obesity to a global epidemic level (WHO, 2013b). As mentioned previously, peptides derived from marine by-products have shown several bioac-tivities mainly related to CVD as presented in Tables 4.1 and 4.2. The health benefits of fish and shellfish by-products for the control of type 2 diabetes and hypertension and their potential as antioxidant agents will be outlined herein.

3.1 Type 2 Diabetes

Type 2 diabetes is a form of diabetes melli-tus, a condition characterized by an insulin defi-ciency or resistance (Yang et al., 2011). This form of diabetes, also called non–insulin-dependent diabetes, correlates with an insulin resistance, which means that the pancreas can produce and release insulin but this process occurs in an inefficient manner or in insufficient quanti-ties. In contrast, in type 1 diabetes, also called insulin-dependent diabetes, the pancreas fails to produce and release insulin (Yang et al., 2011). Type 2 diabetes is the more common of the two types accounting for more than 90.0% of cases (Wild et al., 2004). The incidence of type 2 diabe-tes has been increasing in developed countries. This is associated with increased obesity and unhealthy lifestyles. The hyperglycemia caused by insulin deficiency can lead to complications, such as heart, kidney, and mouth diseases; loss of vision; and nerve damage (Wild et al., 2004). Worldwide, 170 million individuals are affected by type 2 diabetes, and it is expected that this

number will increase by 50.0% in 2030 (Ginter and Simko, 2013). The countries with the highest prevalence of type 2 diabetes are Saudi Arabia, the United States, Switzerland, and Austria with more than 10.0% of the adult population affect-ed by this disease (Ginter and Simko, 2013).

To date, the management and control of type 2 diabetes has targeted the inhibition of enzymes, such as DPP-IV, α-glucosidase, and α-amylase. These enzymes are linked with the stimulation of insulin biosynthesis and secretion, the reduc-tion of glucagon release, the reduction of appe-tite and glucose metabolism in general, as well as the glucose absorption rate (Guasch et al., 2012; Havale and Pal, 2009; Jesson et al., 2005). DPP-IV degrades the two incretin hormones, glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1). These incretins enhance insulin secretion from pancreatic β-cells in the presence of glucose. By inhibiting the action of DPP-IV, the incretin hormones are maintained in the circulation and continue to facilitate insu-lin production (Nongonierma and FitzGerald, 2013). In vivo studies have shown that by inhibiting DPP-IV, it is possible to increase cir-culating GLP-1 in the body by 4–6 times (Nauck and El-Ouaghlidi, 2005). Consequently, DPP-IV inhibitors are used to control type 2 diabetes (Nauck and El-Ouaghlidi, 2005). Some synthetic drugs with DPP-IV inhibitory activity, such as diprotin A and B, sitagliptin, vildagliptin, and saxagliptin, are now being used for the treat-ment of type 2 diabetes. However, adverse ef-fects, including headache, nausea, skin and mucosal infection, and even pancreatic cancer have been associated with the use of these drugs ( Matveyenko et al., 2009). Therefore, the use of natural sources of DPP-IV inhibitors may be ben-eficial. Some protein hydrolysates derived from marine sources have been reported to have in vitro DPP-IV inhibitory activity with IC50 values ranging from 0.138 to 1.200 mg mL−1 ( Harnedy and FitzGerald, 2013b; Li-Chan et al., 2012; Pascual et al., 2007). Some peptide sequenc-es have been identified from marine sources,



such as salmon-skin gelatin, for example, Gly-Pro-Ala-Glu (372.4 Da) and Gly-Pro-Gly-Ala (300.4 Da), with DPP-IV IC50 values of 49.6 and 41.9 µM, respectively (Li-Chan et al., 2012). The currently identified DPP-IV inhibitory peptides from fish and shellfish by-products are summa-rized in Tables 4.1 and 4.2, respectively.

Although, the in vitro studies mentioned earlier show the potential of fish and shellfish peptides in the control of type 2 diabetes, in vivo studies need to be carried out before these peptides are used in humans. However, some in vivo studies with other components from fish and shellfish sources have shown other activities related with the control of diabetes. For exam-ple, investigations with fish oil have shown the ability to regulate adiponectin in mice, which correlates with insulin synthesis (Neschen et al., 2006). Furthermore, the antidiabetic prop-erties of insulin derived from spotted dogfish (Scyliorhinus canicula) and the hammerhead shark (Sphyrna lewini) were tested in humans showing metabolic actions similar to mamma-lian insulin (Anderson et al., 2002).

3.2 Hypertension

CVD, such as hypertension, atherosclerosis, heart failure, and ischemia or reperfusion inju-ries, as well as complications arising from these CVDs, such as chronic renal failure, are increas-ing rapidly worldwide and contribute to an esti-mated 17 million deaths annually (WHO, 2013a). Hypertension alone accounts for 9.4 million deaths worldwide every year (WHO, 2013a). By 2025, an estimated 1.56 billion adults will suffer from hypertension and are at risk of developing other CVDs (WHO, 2013a). These diseases are highly linked with smoking, high cholesterol, diabetes, obesity, and physical inactivity with Europe, East and Central Asia, and North Amer-ica reporting the highest number of deaths re-lated to CVDs (Lawes et al., 2008; WHO, 2013a).

Several pathways are involved in the regula-tion of blood pressure, including the kinin-nitric

oxide, the neutral endopeptidase, the endothelin- converting enzyme, and the renin-angiotensin systems. Manipulation of the renin-angiotensin pathway is most commonly targeted for the treatment and prevention of hypertension. This is mainly by inhibiting ACE. This enzyme is a carboxydipeptidase, which converts angioten-sin I into a potent vasoconstrictor, angiotensin II, in the renin-angiotensin system (RAS) and inac-tivates bradykinin (a potent vasodilator), in the kinin nitric oxide system (KNOS) (Ni et al., 2012). Inhibition of ACE may reduce vasoconstriction, leading to lowering of blood pressure. Synthetic ACE inhibitors including captopril, enalapril, al-acepril, and lisinopril are currently used because of their high effectiveness in the treatment and prevention of hypertension. However, they can cause adverse side effects. Several natural com-ponents have been studied to potentially over-come this problem. Among these components is ACE inhibitory peptides derived from fish and shellfish proteins. Hydrolysates from protein-rich by-products of the fish and shellfish pro-cessing industry, such as cod frames and skins, pollock frames and skins, sea bream scales, and yellowtail bones and scales, along with clam, krill, oyster, and shrimp have been shown to have ACE inhibitory activity in vitro, with IC50 values ranging from 0.036 to 1.090 mg mL−1 (Benjakul et al., 2009a,b; Hai-Lun et al., 2006; Katano et al., 2003; Kawamura et al., 1992; Tsai et al., 2008; Wang et al., 2008). Some studies have identified the peptides with high activity. These peptides are generally short, mostly di- and tri-peptides, containing no more than nine amino acids. Among the most potent are the di- and tri-peptides, Gly-Tyr, Val-Tyr, Gly-Phe, and Val-Ile-Tyr from sea bream protein hydrolysates having IC50 values ranging from 7.5 to 708 µM (Fahmi et al., 2004) along with Val-Arg-Lys and Tyr-Asn from clam protein hydrolysates having IC50 val-ues ranging from 51 to 700 µM (Tsai et al., 2008). Results from in vitro studies in combination with simulated gastrointestinal digestion suggest the potential of these peptides as antihypertensive

3 BIOFUNCTIONAL ACTIVITIES 75


agents in vivo. The ACE inhibitory peptides currently identified from fish and shellfish by-products are summarized in Table 4.1 and 4.2, respectively.

The in vitro ACE inhibitory activity of the peptides from fish and shellfish makes them good potential sources of ingredients for the control of CVDs. However, in vivo studies are required to prove the effectiveness of these pep-tides in humans. Some in vivo studies have been performed to show the antihypertensive activ-ity of fermented mussel sauce, tuna frames, and shrimp in spontaneously hypertensive rats (Cao et al., 2010; Je et al., 2005; Lee et al., 2010). Fur-thermore, the anticoagulant potential of clam and yellowfin sole frame hydrolysates has been shown in human plasma (Jung et al., 2007; Raj-apakse et al., 2005a).

3.3 Oxidative Stress

Oxidation is a vital metabolic process that re-leases reactive oxygen species, which are reac-tive molecules containing oxygen, such as O−2 and OH−. When this process is uncontrolled or the free radicals generated are not eradicated, an imbalance of the normal redox reactions occurs, leading to oxidative stress, which is ultimately toxic to the cell (Di Bernardini et al., 2011). Oxi-dative stress has implications in the develop-ment of several diseases, such as CVDs, diabe-tes, neurodegenerative diseases, and cancer. The oxidation process also plays a role in food dete-rioration through lipid and protein peroxidation (Di Bernardini et al., 2011; Schurink et al., 2007; Viljanen et al., 2004). The use of antioxidants in food has been shown to have a beneficial effect in promoting health, however, the synthetic an-tioxidants used to date to retard lipid oxidation, including butylated hydroxyanisole (BHA), bu-tylated hydroxytoluene (BHT), tert-butylhydro-quinone (TBHQ), and propyl gallate, can have some adverse effects (Wanasundara and Sha-hidi 2005). Therefore, the use of natural sources of antioxidants would be beneficial for the food

industry as they may represent no hazard for human health. Therefore, fish- and shellfish-derived protein hydrolysates show potential as sources of antioxidant BAPs. Fish by-products, such as skin from cod, hoki, herring, pollock, snapper, sole, bones and frames from yellow-tail, as well as shellfish by-products from krill, mussel, prawn, shrimp, and squid have been re-ported to show antioxidant potential (Tables 4.1 and 4.2). Among the peptides identified to have antioxidant activity are peptides rich in amino acids such as histidine (His), leucine (Leu), tyro-sine (Tyr), methionine (Met), and cysteine (Cys) (Mendis et al., 2005b; Sarmadi and Ismail, 2010). Moreover, peptides with the repeating sequence Gly-Pro, such as His-Gly-Pro-Leu-Gly-Pro-Leu and Gly-Glu-(Hyp)-Gly-Pro-(Hyp)-Gly-Pro-(Hyp)-Gly-Pro-(Hyp)-Gly-Pro-(Hyp)-Gly (Kim et al., 2001; Mendis et al., 2005b) are also known to have peroxidation inhibiting activity. Other peptides rich in hydrophobic amino acids, such as Gly, Leu, Val, Ala, Pro, and Hyp have also been shown to inhibit oxidation processes. Among these are peptides, such as Thr-Gly-Gly-Gly-Asn-Val and Phe-Asp-Ser-Gly-Pro-Ala-Gly-Val-Leu from fish skin gelatin (Mendis et al., 2005a). The antioxidant peptides identified from fish and shellfish by-products are summarized in Tables 4.1 and 4.2 respectively. Again, animal and human studies need to be carried out to test the effectiveness of these antioxidant peptides in vivo in order to validate their utilization for the enhancement of human health.

3.4 Other Bioactivities

Other biological activities linked with the con-ditions mentioned earlier include an appetite-suppression activity found in hydrolysates from sardine, cod, blue whiting, and shrimp by-products (Tables 4.1 and 4.2). The appe-tite-suppression activity was attributed to the presence of gastrin and cholecystokinin-like molecules in protein hydrolysates from these sources. These peptides, which regulate the



secretion of gastric acid and pancreatic enzymes, are linked with appetite suppression (Cudennec et al., 2008; Ravallec-Plé and Van Wormhoudt, 2003). Other activities were found in fresh wa-ter clam and salmon frame hydrolysates (Lin et al., 2010; Wergedahl et al., 2004), such as those with the capacity of reducing acyl coenzyme A (acyl CoA; cholesterol acyltransferase) activity and with high bile acid-binding capacity. Some calcium and iron-binding peptides have been found in shrimp, hoki frames, and Alaskan pollock skin and backbone. These metal chelat-ing activities are important in many metabolic processes, including oxygen transport, energy production, cellular proliferation, and nutrient absorption. (Guo et al., 2013; Huang et al., 2011; Jung and Kim, 2007; Jung et al., 2006). Antico-agulant activity had been reported in peptides extracted from starfish, Echiuroidea worm, and blue mussel (Jo et al., 2008; Jung and Kim, 2009; Koyama et al., 1998), as well as from proteins from blood clam shell, yellowfin sole, and gran-ulated clam (Jung et al., 2002, 2007; Rajapakse et al., 2005a). Other biological activities, such as antimicrobial (Liu et al., 2008), antiviral (Zeng et al., 2008), (Song et al., 2012), antican-cer (Hsu et al., 2011), and HIV-1 protease inhibi-tors (Lee and Maruyama 1998) were reported in several fish and shellfish protein hydrolysates (Tables 4.1 and 4.2).

4 BIOAVAILABILITY

BAPs can be released from food proteins dur-ing gastrointestinal digestion and through fer-mentation, enzymatic hydrolysis, and during food processing. Research effort in the field of BAPs has increased in the last few years. Several studies report that fish and shellfish have poten-tial as a source of BAPs for the generation of bio-functional foods (Harnedy & FitzGerald 2013a). However, more studies need to be carried out in order to determine if BAPs from fish and shell-fish sources (Tables 4.1 and 4.2) maintain their

activities when subjected to different food pro-cessing techniques.

To present their biological effects in the hu-man body, the peptides must maintain their biological activity after passing through the gastrointestinal tract, resisting the action of the gastrointestinal enzymes. A simulated gastroin-testinal digestion (SGID) approach is often car-ried out in vitro to test the biological activity of peptides after being submitted to gastrointesti-nal enzymes. These enzymes are specifically the gastric enzyme, pepsin, and the pancreatic en-zyme preparation, Corolase PP (composed of the three major pancreatic enzymes trypsin, chymo-trypsin, and elastase). Moreover, the hydrolysis of proteins by these enzymes alone can gener-ate BAPs. This has been shown in studies using an SGID approach for the generation of BAPs from different food sources such as soy, whey, pea, flaxseed, and fish gelatin (Guo et al., 2015; Hernández-Ledesma et al., 2004; Lo et al., 2006; Marambe et al., 2011; Vermeirssen et al., 2004). Furthermore, in vitro SGID has shown that the activity of the gastrointestinal enzymes has the potential of increasing or decreasing the potency of the BAPs generated by controlled enzymatic hydrolysis (Nongonierma and FitzGerald, 2013). For this reason, testing the stability of BAPs with in vitro SGID has good potential to assess the bioavailability of these peptides after consump-tion. However, some fish- and shellfish-derived BAPs have been shown to be resistant to SGID, such as the ACE-inhibitory peptide Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe from oyster, which maintained its activity after incubation with pepsin, trypsin, and α-chymotrypsin (Wang et al., 2008).

The physical and chemical instability, the possibility of aggregation, and large molecular size of the BAPs may lower their permeability through biological membranes to eventually reach the bloodstream. For example, the per-meability of BAPs though the gastrointestinal tract is linked with three main transport routes: (1) PepT1 carrier-mediated transport, which

5 REGULATIONS FOR FUNCTIONAL FOODS 77


transports di- and tripeptides through H+ cou-pling; (2) tight-junction paracellular diffusion, which transports oligopeptides in their intact form through tight junction pores; (3) endocy-tosis–exocytosis, which transports basic and hydrophobic peptides (Norris and FitzGerald, 2013). However, endocytosis–exocytosis–me-diated transport of peptides may hydrolyze them into amino acids when inside the cell be-fore reaching the bloodstream. This makes the PepT1 carrier-mediated transport and tight-junction paracellular diffusion potentially bet-ter routes of transport of BAP into the blood-stream. Moreover, whole peptides and single amino acids are less easily absorbed across the gastrointestinal membrane than short pep-tides with two to six amino acids (Grimble et al., 1986). Fish- and shellfish-derived BAPs have been reported not only to resist gastroin-testinal digestion but are also capable of being assimilated into the bloodstream. For example, in vivo animal studies with spontaneously hypertensive rats have shown that the antihy-pertensive effect of peptides derived from fish, such as bonito, mackerel, salmon, sardine, sea cucumber, sole and tuna, and shellfish, such as mussel, oyster, and shrimp, is maintained after the digestion and assimilation processes. Moreover, Ohara et al. (2007) showed that Ala-Hyp-Gly and Ser-Hyp-Gly remained intact 4 h after being administrated, which indicates that these peptides also resisted digestion and were able to be assimilated into the bloodstream.

However, there are several strategies that can be used to improve the bioavailability of marine-derived BAPs. Among these, modifica-tion of the N- and C-terminals; structural modi-fication, including alkylation and glycosylation; and encapsulation have also been shown to improve the bioavailability of peptides. More-over, peptides containing a high percentage of Pro and Hyp amino acids seem to be more resis-tant to hydrolysis by gastrointestinal enzymes (FitzGerald and Meisel, 2000). Some of these approaches have already been used for the im-

provement of the bioavailability of BAPs from fish and shellfish. For example, the encapsula-tion of tuna peptides in liposomes was shown to be effective in the preservation of their antihy-pertensive capacity in spontaneously hyperten-sive rats (Hwang et al., 2010).

5 REGULATIONS FOR FUNCTIONAL FOODS

Functional foods are foods that claim to have some or several health benefits by preventing disease or enhancing a specific body function. The functional food industry is growing with world sales expected to reach US$67 billion in 2016 (Kissinger, 2015) with Asian countries and the Central and Northern European countries being the biggest consumers when compared with Mediterranean countries (Van Trijp, 2007). Although the acceptance of functional foods has been increasing by consumers, it is highly dependent on their age, gender, and occupa-tion (Van Trijp, 2007). The health benefits most often claimed are related to the gastrointesti-nal and cardiovascular systems; regeneration and protection of tissues, such as skin, mus-cle, bone, and cartilage; along with relaxing and immune-regulatory properties. However, health claims must follow strict legislation and regulations that depend on the type of claim and in which country that it is issued. For ex-ample, in the European Union, the European Food Safety Authority (EFSA) is the regulatory body for the evaluation of health claims. In Ja-pan, where the concept of functional foods was born, it is regulated by the Japanese Ministry for Health under the Foods for Specific Health Use (FOSHU) initiative; in China by the State Food and Drugs Administration (SFDA); in Australia and New Zealand, by the Food Standard Australia New Zealand (FSANZ); and in the United States, by the Food and Drug Adminis-tration (FDA) (Shimizu, 2014). Moreover, each country may have specific directives in relation



to functional food claims. These regulatory bod-ies aim to assure consumers that only standard-ized nutritional claims or specifically authorized health claims may be presented on food labels. Recently, for example, in Japan new categories such as qualified, standardized, and reduction of disease risk FOSHU claims have been added to facilitate the health claims process.

6 COMMERCIAL PRODUCTS CONTAINING MARINE-DERIVED BIOACTIVE

PROTEIN HYDROLYSATES OR PEPTIDES

Food products with health claims have been commercialized all over the world following different approvals from the countries in which they are produced and sold. Some products containing fish-protein hydrolysates or fish-derived peptides associated with health claims are already commercially available. Examples of these products include PeptACE, Vasotensin, Levenorm, Peptide ACE 3000, Tensiotin, Lapis Support, Valtyron, Wakame jelly peptide, and Peptide Nori S, all claiming to be antihyperten-sive; Stabilium 200, AntiStress 24, and Protizen, claiming to have relaxing properties; Collagen HM for improving joint health; Glycollagen, marine cartilage powder, and Protein M+ claim-ing to be joint pain soothers; PeptiBal that claims to have immunomodulatory properties; Seacure claiming to improve gastrointestinal health; and Peptide N and Nutripeptin, which claim to lower glycemic index; and Fortidium Liquamen, which also claims to lower glycemic index, as well having antioxidant and antistress proper-ties (Harnedy and FitzGerald, 2013a).

Moreover, in the pharmaceutical industry, gelatin from fish is already used for the produc-tion of hard and soft gel capsules (Bae et al., 2007; Gudipati, 2013; Haug and Kurt, 2009) and as a cross-linking medical glue (Gaissmaier and Ahlers, 2009).

7 CONCLUSIONS

The exploitation of marine resources for extraction of BAPs has intensified in recent years, and fish and shellfish processing pro-tein coproducts have been identified as valu-able sources of BAPs. Several studies have been carried out in the extraction and hydrolysis of proteins from fish and shellfish coproducts as well as on their purification and characteriza-tion. There are numerous possibilities yet to be studied about coproducts from different spe-cies of fish and shellfish, different extraction and purification methods, and different poten-tial biological activities. Although valuable re-search has been recently carried out, the use of fish and shellfish coproducts as biofunctional food ingredients still needs more work in ad-dressing the large-scale production of these products and their gastrointestinal stability, bioavailability, and compatibility with different food matrices and efficiency in in vivo. Finally, marketing and economic studies are required to inform consumers of the benefits of fish- and shellfish-derived BAP components.


ACE Angiotensin-converting enzymeATP Adenosine triphosphateBAP Bioactive peptideBHA Butylated hydroxyanisoleBHT Butylated hydroxytolueneBSE Bovine spongiform encephalopathyCol-D Distal collagenCol-P Proximal collagenCVD Cardiovascular diseasesDHA Docosahexaenoic acidDPP-IV Dipeptidyl-peptidase IVEFSA EUROPEAN Food Safety AuthorityEPA Eicosapentaenoic acidFAO Food and Agriculture Organization of the

United NationsFDA Food and Drug AdministrationFOSHU Foods for specific health useFSAI Food Safety Authority of IrelandFSANZ Food Standard Australia New Zealand


REFERENCES 79

GIP Glucose dependent insulinotropic peptideGLP-1 Glucagon-like peptide-1IC50 50% enzymatic inhibitionKNOS Kinin nitric oxide systemMS HPLC-mass spectrometryMS/MS HPLC-tandem mass spectrometryPUFA Polyunsaturated fatty acidsRAS Renin-angiotensin systemRP-HPLC Reversed-phase high-performance liquid

chromatographySDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel

electrophoresisSEC Size-exclusion chromatographySGID Simulated gastrointestinal digestionUPLC Ultra-performance liquid chromatographyWHO World Health Organization

AcknowledgmentsAdriana CunhaNeves was funded under the Sea Change Strategy with the support of the Marine Institute and the Irish Department of Agriculture, Food and the Marine, funded under the National Development Plan 2007-2013 (Grant-Aid Agreement No. MFFRI/07/01). Pádraigín A Harnedy was funded under the National Development Plan 2007-2013, through the Food Institutional Research Measure, administered by the Department of Agriculture, Food and the Marine, Ireland under grant issue 13/F/467.

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S E C T I O N II

EXTRACTION, RECOVERY, CHARACTERIZATION, AND MODIFICATION

TECHNIQUES5 Technical issues related to characterization, extraction,

recovery, and purification of proteins from different waste sources 89

6 Modification of protein rich algal-biomass to form bioplastics and odor removal 107

C H A P T E R


5Technical Issues Related

to Characterization, Extraction, Recovery, and Purification of Proteins from Different

Waste SourcesM. Gong, A.-M. Aguirre, A. Bassi

Department of Chemical and Biochemical Engineering, Faculty of Engineering, University of Western Ontario, London, Ontario, Canada

1 INTRODUCTION

Proteins, as one of the building blocks for ani-mals and human bodies, have diverse functions, such as catalyzing metabolic reactions, record-ing genetic information, transporting materials through membranes, and even storing energy. However, proteins from varied sources are often discarded into waste treatment plants or directed to the environment without any treatment, pos-ing challenges for future waste processing and representing a source for potential value-added products, such as animal feed, human food, or bioactive peptides with several applications in health. Hence, much attention has been focused on protein recovery from waste feedstock.

According to the Food and Agriculture Orga-nization of the United Nations (FAO), there is an expected increase in the production and demand of protein meals in the next decade. A forecast for these parameters is presented in Fig. 5.1. From a worldwide perspective, it is expected that the supply of protein meals satisfies the demand for the next 10 years (lines representing total world production and consumption are overlapped in Fig. 5.1); but for local economies, the scenario may be quite different. Developing countries will provide most of the production of protein meals, which may translate into the coverage of their internal consumption, but given that the developed countries will have a demand higher than their supply, then challenges may appear in

90 5. TECHNICAL ISSUES

II. EXTRACTION, RECOVERY, CHARACTERIZATION, AND MODIFICATION TECHNIQUES

protein meal distribution around the globe. For this reason, the recovery of protein from waste potentially represents an attractive alternative, especially in countries where the demand is higher than the supply.

Table 5.1 presents the main sources of pro-tein in an average human diet. Most of the pro-tein consumed by humans comes from cereals, followed by animal protein sources, such as meat, milk, and fish. Thousands of tons of food are produced annually to guarantee global con-sumption. The processing of large amounts of food requires large amounts of water, and a con-siderable portion of the protein originally in the food ends as part of the wastewater. The protein concentration reported in wastewater for some food industries is provided in Table 5.1. The re-covery of the protein from waste streams may represent an economic relief to the industries as it can be sold as a coproduct and it will, at the same time, reduce the expenses of wastewater disposal as the organic load is considerably reduced.

2 VALUE RECOVERY OF PROTEIN BY-PRODUCTS FROM WASTE

MATERIALS

Protein waste can fall into two major catego-ries: aqueous solutions and mainly solids. In a very broad sense, proteins are grouped into globular or fibrous proteins. Most studies have been devoted to the globular or readily water-soluble proteins of separation technologies. The globular proteins are a group of diverse protein molecules that form “spheres” and are known for being water soluble when contact-ing with water. Few processes for globular pro-tein recovery have been adequately adjusted to large-scale production (Thomson, 1984). Substantial amounts of globular protein exist in a wide range of processing wastewater (eg, meat and fish processing, dairy and soy process-ing, and starch-containing wastewater), where-as the solid protein (or fibrous protein) can come from algae, cellulose waste, and seafood- or

FIGURE 5.1 Protein meal production and consumption forecast. Adapted from OECD and FAO (2014).

2 VALUE RECOVERY OF PROTEIN BY-PRODUCTS FROM WASTE MATERIALS 91


dairy-processing waste. The fibrous proteins are not water-soluble as their function is to normally play supportive structural roles in the cells. These proteins are also important and hence, their recovery from solid wastes, in addition to the globular proteins, is vital.

2.1 Proteins from Wastewater

Various kinds of compounds are found in wastewater, and different indicators of quality are used as a way of standardizing the extent of pollution of the water. Chemical oxygen de-mand (COD) indirectly measures the amount of organic compounds in water, and volatile sus-pended solids (VSS) are obtained from the loss on ignition of total suspended solids. High levels of organic compounds, including proteins, are present in wastewater from food industries and are generally decomposed by microorganisms, depleting the oxygen dissolved in the water and imposing a risk for aquatic life. Therefore, any attempt to reduce the content of protein in wastewater will be beneficial for the environ-ment and it may represent a source of profit for the industries.

2.1.1 Fishery ProcessingIndustrial fishery-processing wastewater has

become an increasing problem for the environ-ment, especially in countries where the fishery industry is an important part of the economy. Negative effects have been reported in some areas in Galicia (northwest of Spain) and re-gions in Chile. In fish meal production, high COD (30–120 g COD/L), and high VSS (5–40 g VSS/L) wastewater was generated at an aver-age flow rate of 1100 m3/h for a plant capacity of 100-ton fish/h (Afonso and Bórquez, 2002). It was reported that 45% of processed shrimp waste was composed of the exoskeleton and cephalothorax (Venugopal and Shahidi, 1995), which represent 50–70% of the raw material mass and contain valuable components, such as protein, chitin, and pigments (De Holanda and Netto, 2006). To avoid environment pollution, effective treatment must be done before efflu-ent is discharged. Simultaneously, valuable pro-teins can be concentrated and recycled into the by-product production process by proper treat-ment; both economically benefit the company for higher productivity and reduce the difficulty of treating wastewater (Guerrero et al., 1998).

TABLE 5.1 Protein Sources and Concentration in Wastewater Effluents

Protein sourceHuman diet protein supply (%)

World production (1000 tons, 2010) Protein concentration in wastewater

Cereals 40.4 2,476,416 Soy: 4.0 g/L (Cassini et al., 2010)

Meat 17.8 2,96,107 4.2–10.0 g/L (Ruiz et al., 1997)

Milk, excluding butter 10.1 719 0.6–0.9 g/L (Seesuriyachan et al., 2009)

Fish, seafood 6.4 59,873 (aquaculture) Surimi: 2–5 g/LFish fillets: 3–6 g/LShellfish and crustaceans: 0.1–6 g/L

(Afonso and Bórquez, 2002)

Vegetables 5.7 1,044,380 —

Pulses 4.9 68,829 —

Eggs 3.4 69,092 —

Oil crops 3.4 170,274 —

Starchy roots 2.8 747,740 Potato: 1.8% (Zwijnenberg et al., 2002)

Other sources 5.1 —



2.1.2 Meat ProcessingHigh-COD wastewater is common in meat

production, where blood and body wash water can be the main source of the organic load. The meat-processing wastewater, especially blood, has a high protein content, between 15% and 20% w/w (Liu, 2002). In Mexico only, about 6 million cattle are consumed annually, generating 91 mil-lion liters of blood (Gómez-Juárez et al., 1999). However, merely 15% blood is used for animal feed and the rest is discarded into municipal sewers and landfills, which is a significant waste of high-quality proteins and represents a high demand for COD removal. Therefore, meth-ods for collection and processing of blood into dried blood plasma and red cells was proposed and has achieved commercial success (Gómez-Juárez et al., 1999; Selmane et al., 2008). It was re-ported that ultrafiltration or spray drying can be employed to concentrate blood effectively (Dai-lloux et al., 2002). The North American Spray Dried Blood and Plasma Producers Association (NASDBPP) was established to enhance safe, high-quality blood-derived protein production for commercial livestock and companion animal feeds. APC Inc., DuCoa, and Harimex Inc. are some members of NASDBPP, which is devoted improving and standardizing the animal health supplement industry.

For meat processing, at least 20 L of water is required to process 1 broiler chicken, and more than 25 million chicken are processed daily in the United States. This effluent contains 35% protein (Lo et al., 2005). Similarly, mechanically deboned chicken meat or beef bones also gen-erate tremendous volume of wastewater with high protein content (Selmane et al., 2008). En-zymatic hydrolysis was suggested to recover proteins without destroying the functional-ity of the proteins (Fonkwe and Singh, 1996), while precipitation is the only well-proven large-scale food proteins-processing method (Thomson, 1984).

As the market for meat is expanding, high-quality animal feed generates increased

attention. The proteins recovered from various sources are becoming an important portion of animal feeds. Protein hydrolysates can be used for flavoring and incorporated into aquaculture feeds (De Holanda and Netto, 2006). In fact, up to 50% of traditional fish meal can be replaced by single-cell protein (SCP) without much re-duction in the growth performance of fish (Al-Hafedh and Alam, 2013).

2.1.3 Dairy ProcessingExtensive research has been performed to re-

cover valuable proteins from dairy wastewater. Dairy proteins are valuable products with excel-lent nutritional and functional properties. From an industrial and environmental perspective, the recovery of these proteins is favorable and re-sults in substantial financial advantages (Cassini et al., 2010). Therefore, their applications for high value-added food additives, nutraceuticals, and therapeutics have been explored (Chollangi and Hossain, 2007). The major protein in milk is casein and it contributes to almost 80% of total protein in dairy effluents. Lactic acid fermenta-tion has been used to recover casein from dairy wastewater. Acid fermentation reduces the pH of the media, causing biocoagulation. This pro-cess was employed to separate colloidal proteins from the wastewater stream, followed by solid–liquid separation (Seesuriyachan et al., 2009). Adsorption, membrane technology (ultrafiltra-tion), and chromatography are other common methodologies used for recovering protein.

Apart from dairy industry, whey wastewater is also generated from industrial soy protein iso-late (SPI) production. For each ton of SPI, about 20 tons of whey wastewater is produced, which contains approximately 4 g/L protein (Jiang et al., 2011). Similar to the dairy wastewater, biochemical, membrane separation, and electro-chemical technologies can be applied to recover protein. However, Jiang et al. (2011) also stated that more commercially, operationally, and en-vironmentally friendly techniques are needed to recover target proteins.

2 VALUE RECOVERY OF PROTEIN BY-PRODUCTS FROM WASTE MATERIALS 93


2.1.4 Silk ProcessingLarge amounts of sericin, which is a protein

produced by silkworms, is present in silk waste-water. About 5 × 104 tons are produced world-wide annually in the degumming process of natural silk yarn and tissues. With a COD higher than 6000 mg/L, the demand for biological treat-ment plants is significant. In the silk industry, the degumming process washes out sericin: This globular and water-soluble protein with a mo-lecular weight ranging from 10 to more than 300 kDa accounts for 20–30% of the total raw silk co-coon weight. An effective enzymatic hydrolysis process was developed by Wu et al. (2008) us-ing a common protein hydrolysate protease P [“Amano” 6 (EC 3.4.21.63, Aspergillus melleus)] to produce bioactive peptides from sericin waste-water. The bioactive peptides may have applica-tions in the food, cosmetics, and pharmaceutical industries (Wu et al., 2008). However, further de-velopment in simplifying separation is needed.

2.1.5 Starch Wastewater and Potato Processing

Starch wastewater is another most com-mon wastewater source in the food industry. It is produced in the starch separation process from corn, sweet potato, or tapioca, with con-siderable amounts of fine suspended solids (Mu et al., 2014). Most soluble suspended solids, in-cluding valuable proteins, in these wastewaters are normally discarded directly into the envi-ronment; however, after appropriate recovery processing, these solids could be used in human food, animal feeds, and coating adhesives (Liu et al., 2013).

As an essential crop, approximately 360 mil-lion tons of potatoes were produced in 2013 across the world (OECD and FAO, 2014). Peel-ing and slicing produces a tremendous amount of wastewater, containing potato protein, soluble starch, and other organic substances. The subsequential COD can reach as high as 20,000 mg/L, which indicates that the discharge of the wastewater would cause environmental

problems and result in abundant loss of the po-tato proteins (Liu et al., 2013). Potato proteins are superior in quality than most major plant proteins. They are rich in amino acids and have a protein quality close to those of a whole egg (Knorr, 2007). Considering the quantity and quality of potato protein and even though peels and other solid potato fragments can be readily removed by screening or settling, the study of recovering dissolved and suspended solids in waste effluent is still inadequate. Simple heat or biological treatments for recovery of proteins can consume large amounts of energy and do not provide satisfactory separation efficiency. Acid precipitation is an alternative method of recovery; however, these methods exhibit poor functional properties of protein as they are of-ten cooked and damaged leading Gonzalez and coworkers to study the use of hydrocolloids as complexion agents (Gonzalez et al., 1991). More recently, foaming (Liu et al., 2013) and mem-brane technologies (Šárka et al., 2009) have been developed as more advanced recovery meth-ods for proteins. Further, Knorr researched the quality and acceptability of the potato proteins, which are satisfactory alternatives for the poten-tial products (Knorr, 2007).

2.1.6 Antibiotic ProcessingOutside food processing, high organic load

waste streams are often generated from biosyn-thesis of antibiotics which has 10–40 g/L COD. Because of toxic residues, treating these waste-water can be extremely difficult and expensive, therefore, recovering proteins became of interest (Li et al., 2004).

2.2 Proteins from Solid Waste

Besides wastewater, other waste products also exist in the form of solid, or sludge. It is ex-pensive to treat intact excess sludge, and solu-bilization of intracellular materials is the first step for protein recovery. Chemical, physical, or biological methods can be applied to disrupt



sludge floc, such as alkali treatment and ultra-sound. In previous studies, the total suspended solids decrease both in size and amount, as the dissolved organic were released into aqueous phase (Zhang et al., 2007). Therefore, the organ-ics were transferred to water for protein recov-ery while fewer BOD exist in the sludge; the treated solids occupy less volume and would be easier and less expensive for further treatment (Hwang et al., 2008).

Studies have reported that about one third of the food produced for human consumption is lost or wasted, which corresponds to 1.3 billion tons/year. This food has considerable amounts of protein and other compounds that can be re-covered. Fig. 5.2 presents the food loss or waste per person and per year in the world and also by different country groups (FAO, 2011). It can be seen that most of the waste happens at the pro-duction and retail stages. If proper waste collec-tion programs were developed, then this waste could be an attractive source for protein recovery.

Waste solids from a variety sources, such as rice straw, kiwifruit peels, pineapple waste, cap-sicum powder, and Chinese cabbage have been proposed for protein recovery (Zhao et al., 2010).

2.2.1 Algae WasteAlgae are photosynthetic organisms that

can grow on wastewater and consume nitrates, phosphates, and carbohydrates. As the water is cleaned, the algae can be settled as excess sludge, removed for biofuel production, and discarded as animal feed. Microalgae, considered a source of several valuable compounds used in nutri-tional supplements, natural dyes, and skin care products, are especially rich in proteins and have been extensively studied for resolving the world food shortage. The common fresh water micro-algae Chlorella vulgaris is famous for its high oil content and fast growth. It has a well-established safety criterion (Suetsuna and Chen, 2001) as an edible food in Japan; moreover, its proteins are reported to have excellent emulsifying proper-ties, which would find valorization into food supplements, pharmaceutics, and/or cosmetics (Ursu et al., 2014).

The high protein content in the algae cells has raised interest on recovering SCPs or functional peptides from waste algae biomass or even de-fatted algae biomass. SCP generally accounts for sources of mixed protein, which is extract-ed from pure or mixed cultures. Antioxidant

FIGURE 5.2 Food loss and waste every year. Adapted from FAO (2011).

3 TECHNIQUES FOR WASTE PROTEIN SEPARATION 95


or anticancer peptides may be of interest for medicine production, and SCP is a high-quality protein additive for animal feed or human food (Moraine et al., 1979).

The residual from the microalgae Botryococ-cus braunii gives a protein concentrate that can develop ultrafine biopolymer fibers with many end-use applications (Verdugo et al., 2014), and the C. vulgaris waste might become a new pro-tein source for antioxidative peptides (Sheih et al., 2009). In fact, more than 50% (dry mass) of algae consist of high-quality protein (Sheih et al., 2009). Approximately 80% of the protein nitrogen can be washed into water from hex-ane-defatted Spirulina platensis biomass (Gerde et al., 2013). Compared with other sources of protein production, the unit operation for algal protein is greatly reduced, remaining merely concentration and final processing as the major cost (Walker et al., 2005). Solubilization of pro-teins in alkaline conditions and isoelectric point precipitation are popular methods for recover-ing protein from microalgae, and the addition of enzyme under alkaline conditions can increase protein extraction yield to 80% from 30% from microalgae meals (Sari et al., 2013).

2.2.2 Cellulose WasteThermomonospora curvata is a cellulose-

degrading bacteria used for treating more than one-half of the municipal solid waste in the United States. It has been implicated as one of the major cellulose (especially paper) degraders for municipal solid cellulose waste (Crawford et al., 1973). More applications were found, and it can now be used for converting cellulosic pa-per industry waste to valuable microbial protein such as additives in human food and drinks, waterproof adhesives, or industrial enzymes (Mekonnen et al., 2014).

2.2.3 Meat and Fish WasteLow-value animal-processing waste (eg, fish

filleting or poultry bone residue) is usually un-desirable for human consumption because of the

undesirable textures, colors, and strong flavors (Hultin and Kelleher, 2000). As a typical meat-processing technique, mechanically deboned turkey residue retains a valuable amount of pro-tein. This part of the protein serves as a valu-able source of human food, but sometimes is disposed as waste. Enzymatic hydrolysis is a more effective method over alkaline extraction for this kind of protein. Both the collagenous and noncollagenous proteins (such as blood and bone marrow) can be treated and can potential-ly reduce the volume of waste. Other poultry-processing wastes are quite similar, for example, enzymatic hydrolysates from broiler chicken heads are reported to have superior organo-leptic and physicochemical properties (Fonkwe and Singh, 1996).

Significant amounts of marine-processing by-products from onboard- and onshore-processing plants, including substandard muscles, viscera, heads, skins, fins, frames, trimmings, and shell-fish and crustacean shell waste could be gener-ated for bioactive peptide production (Harnedy and FitzGerald, 2012).

Fish filleting leads to 50% of the total weight of the raw fish material processed into waste. In rare cases, the fish meal would be collected for animal feed production, but usually it is simply discarded, leading to severe odor and pollution. The solid waste contains almost the same pro-tein content as fish flesh. Different approaches, such as acid extraction, precipitation, and en-zymatic hydrolysis, have been carried out to recover these high nutritive value proteins for human consumption (Batista, 1999).

3 TECHNIQUES FOR WASTE PROTEIN SEPARATION

To efficiently recover protein fractions from waste streams, proper concentration and sepa-ration techniques are necessary. Moreover, as proteins naturally occur as mixtures with varied characteristic properties, further fractionation



would be desirable for more specific application and allow larger margin for profit. Meanwhile, undesirable components, such as flavors and nucleic acids, must be removed for use in the food industry (Thömmes et al., 2001). Therefore, improving and simplifying the protein recovery processes on a large scale have become of in-terest. Procedures have been developed—from alkaline–acid extraction, precipitation, and floc-culation to enzymatic hydrolysis and foaming.

3.1 Centrifugation

Centrifugation is one of the conventional methods for the primary recovery of proteins. It is suitable for large-scale production of larger particles or microorganisms (eg, baker’s yeast) (Thömmes et al., 2001), and it is usually com-bined with other treatments. Although it is a simple process, the significant energy consump-tion makes it expensive and less interesting for current large-scale production. Moreover, it might be very efficient in the large scale for the clarification of comparatively large particles, for example, yeast cells, but the performance would show rapid reduction with the decreasing par-ticle size (Thömmes, 1997).

3.2 Alkaline/acid Precipitation

As a simple physicochemical method for re-covering protein, the alkaline or acid extraction method typically consists of solubilizing crude protein-containing materials by increasing or decreasing pH followed by centrifugation or other methods to obtain the protein-containing aqueous phase, and precipitation of the protein from the aqueous phase and harvesting (Hultin and Kelleher, 2000).

Rapid and reliable protein extraction was proposed to treat yeast cells under mild alkaline conditions for electrophoretic analysis (Kush-nirov, 2000). Under defined alkaline conditions, highly purified DNA can be recovered after a series of steps (Marko et al., 1982). To achieve a

high yield of starch and protein while keeping the damage acceptable, Lee et al. (2007) devel-oped optimum alkaline extraction conditions for both protein and starch, namely pH 9 at 30°C, and achieved a maximum starch yield of 95% and protein yield of 68% (Lee et al., 2007). It was also suggested that low ionic strength may be useful for the separation of different myofibril-lar proteins (Stefansson and Hultin, 1994). On the acid side, pH 4 to 4.5 was most effective for protein recovery under heated (80–90°C) conditions, and the yields can be improved, providing better fluid dynamics (Meister and Thompson, 1976). Boles et al. (2000) applied acid precipitation followed by dialysis for the con-centration and purification of proteins from beef bones. To separate colloidal proteins from the wastewater, acid fermentation was also studied as a biocoagulation process in dairy wastewater treatment under different solid retention times (Seesuriyachan et al., 2009).

Precipitation is often combined with acid or alkaline treatments. On a large scale, it is the only economical technology for the aqueous food protein processing (Thomson, 1984). Hydro-gen chloride (HCl) aqueous solution is widely used for adjusting pH for higher protein recov-ery yield, as pH is a significant factor on yield. This is because pH is a significant factor on yield whereas temperature shows little effect within the range of 4 to 25°C (Gonzalez et al., 1991). The protein recovery efficiency was the highest at pH 3.3 when treating the supernatant of disrupted excess sludge (Hwang et al., 2008). With proper pH, about 85% of the proteins can be recovered by isoelectric precipitation when combined with ultrafiltration in treating traditional fish and meat products (Afonso and Bórquez, 2002). Methods to treat casein from milk and for soya protein isolates by isoelectric precipitation were successfully developed (Thomson, 1984). Elec-trical coagulation can be applied for egg-pro-cessing-wastewater treatment, yielding a high-quality water (more than 90% removal of COD, turbidity, and total suspended solids), while the



protein recovered showed better digestibility than liquid whole egg (Xu et al., 2002).

Considering the complex composition of po-tato juice waste, a combination of thermal co-agulation and acidic precipitation was reported to efficiently recover protein (Cheng et al., 2010). However, its applications to animal feed were severely limited because of the complete loss of the protein functionality (Ursu et al., 2014), which is a major drawback for precipitation. Moreover, it is hard to recover protein from whey wastewaters without special treatment (Zall et al., 1983). Organic solvents can also be combined with this technology for industrial en-zyme production, but it can lead to inactivation of the material (Thomson, 1984). Despite these drawbacks, precipitation is a desirable process because of the simplicity and low cost, with po-tential for further development combining salt, acid, ethanol, ammonium sulfate, and carboxy-methyl cellulose complexion using continuous precipitation (Waglay et al., 2014).

3.3 Flocculation and Coagulation

Flocculation is a process where compounds in a liquid form flocs, which precipitates easier. This technique is widely employed because it is less expensive in both fixed and operating cost, and it gives higher quality to the clarified effluent (Moraine et al., 1979). Different types of coagulants can be categorized as inorganic, polymeric, and organic bioflocculants (Sarkar et al., 2006). In bioflocculants, the dominant fac-tors for flocculating activity are the molecular weight and functional groups; low molecular weights are usually effective with the types that have amino and carboxyl groups, whereas high molecular weights are more common for poly-mers, such as proteins and polysaccharide bio-flocculants.

The optimal coagulant concentration for protein recovery and water treatment depends on various factors, including total solids, inlet protein concentration of wastewater, and fat

content. More than 97% turbidity was removed, and more than 95% protein and fat were recov-ered for all coagulants studied (Xu et al., 2001). Similarly, 83% protein recovery and 97% tur-bidity reduction has been achieved, using chi-tosan as the protein recovery agent (Wibowo et al., 2005). The production yield was increased by complexion with alginate and by adjusting complex concentration and treatment time. Wa-glay reported that (NH4)2SO4 gave the highest yield (98.6%) for the recovery of protein among FeCl3, MnCl2, ethanol, and (NH4)2SO4, whereas FeCl3 offered the best purification of water (Wa-glay et al., 2014). In addition, activated charcoal treatment can be applied after flocculation to remove color and the odor, which is of great in-terest in the food and nutraceutical industries (Sarkar et al., 2006).

3.4 Solvent Extraction

Solvent extraction processes have been used on the production scale for solid–liquid separa-tions. Increased correlations have been deter-mined for organic chemicals. Solvent extraction can provide a product that has good nutritional properties and that is free of nucleic acids with almost no odor and taste. However, the func-tional properties of the proteins were denatured during the process, which significantly limited its applications. By adding proper polymers, two-phase aqueous systems can be formed for the purification of enzymes and cell proteins. Therefore, to effectively solubilize cells and tissues, as well as to preserve the bioactivity of proteins, a mixture of a zwitter ionic and a nonionic surfactant—DPS/B30—was success-fully developed. This reagent proved its ability to harvest proteins while preserving their struc-tures, which is highly attractive in the food in-dustry, diagnostics, theragnostic, and regenera-tive medicine (Hwang et al., 2012). Additional steps, such as column chromatography, centrifu-gation, and other separation steps can be elimi-nated by employing solvent extraction, making



the process more economical. However, rela-tively low purification factors may be a problem, and added polymers may affect the final prod-uct quality (Thomson, 1984).

3.5 Enzymatic Hydrolysis

Enzymatic hydrolysis for protein recovery is a complex process because of enzyme-specific reactions to break different peptide bonds. It was suggested by Fonkwe and Singh (1996) that better functionality maintenance of the recovered proteins usually depends on tem-perature, pH, agitation, protease specificity, concentration of enzyme and substrate, and the ratio between the last two parameters. Enzy-matic hydrolysis is a simple, efficient method with milder conditions that would not destroy the proteins recovered. It has some advantages over the traditional acid–alkaline extraction method of recovering proteins from poultry waste: the collagenous, noncollagenous, and other proteins (such as blood and bone mar-row) would be hydrolyzed, which would lead to a higher protein yield and reduce the vol-ume of waste (wastewater and collagen/bone slurry) generated (Fonkwe and Singh, 1996). In addition, in the food industry, the main obsta-cle for hydrolysate has been bitterness. Because there are no commercially available methods for bitter peptide removal, the strict control of proteolytic reactions can prevent or minimize peptide formation (Linder et al., 1995).

Recovery of the protein from the waste by enzymatic hydrolysis has been widely studied. A suitable enzyme for the production reaction can be chosen by the pH-stat enzymatic hydro-lysis method (Wu et al., 2008). The relative im-portance of pH, temperature, time, and enzyme and protein concentrations was determined using degree of hydrolysis and yield (Linder et al., 1995). A special antioxidant activity–rich protein can be isolated. Its optimal operation conditions with shortened processing time were at temperatures higher than 50°C, with a higher

enzyme-to-substrate ratio of 0.5 % w/w (Sow-mya et al., 2012). De Holanda also measured the optimal hydrolysis conditions, estimated as pH 8.4, temperature 84.0°C and enzyme-to-substrate ratio of 3.0% w/w, which is quite similar to the conditions reported by Linder et al. (1995). For human nutrition and health, biologically active hydrolysates and bioactive peptides with free radical scavenging activity can be produced from algae protein waste by pepsin protease. And the antioxidant activity was proven by Sheih et al. (2009). Moreover, the enzymatic hydrolysis of sericin could further enhance the antioxidant ac-tivity of native sericin (Wu et al., 2008). Some of the proteins recovered from enzymatic hydroly-sis showed higher nitrogen and ash content than that obtained by alkaline extraction, but still showed similar characteristics (De Holanda and Netto, 2006).

3.6 Foaming

Proteins are known to enhance and to stabi-lize foams. Beginning in the 1970s, foam sepa-ration has been used for protein recovery from liquid waste streams. Also known as adsorptive bubble separation, it uses foam bubbles to concen-trate and raise the liquid to the top of the col-umn as surface active materials are adsorbed to the gas–liquid interface of the bubbles (proteins, polysaccharides, and fatty acids). The foamabili-ty of the liquid can be measured using the meth-od described by Coffmann and Garciaj (1977). Foaming can be applied in the fields of metallur-gical, fish, food, and biochemical processes. The optimal foaming operation is correlated with air flow rate, liquid height, the influent protein con-centration, pH, influent volume, foaming time, and other process variables (Chan et al., 2007).

Maximum protein recovery was obtained at 200 mg/L initial protein concentration, although the process time decreased with increasing air flow rate (Aksay and Mazza, 2007). The protein enrichment ratio was inversely proportional to the volume of foam generated and the protein



recovery percentage (Chan et al., 2007). Further developments of the process can be achieved by applying an inclined foam separation column, which had higher enrichment ratios than the vertical separation columns (Mu et al., 2014). In addition, two-stage foam separation technology can be applied with which a protein recovery of 82.95% was achieved by Jiang et al. (2011) for soybean protein wastewater. The efficiency is still relatively low; however, being relatively in-expensive cost, foaming would be suitable as the first stage of a multistage concentration process (Chan et al., 2007).

3.7 Ultrafiltration Membranes

Ultrafiltration (UF) has been established as a major operation process in the concentration of macromolecules, in particular in the food in-dustry and in the dairy industry for the recovery and fractionation of milk components (Baldasso et al., 2011). UF is a very attractive alternative membrane separation process, as no tempera-ture control or extreme pH are involved, which makes the concentration process more eco-nomical and would not denature the proteins, which would lead to the loss of functionality. Meanwhile, organic load, turbidity, and protein content reduction in wastewater can be simul-taneously accomplished. For example, highly functional properties and composition have been shown in the proteins recovered by UF compared to those proteins in regular surimi in terms of gel hardness, elasticity, color, and water retention (Lin et al., 1995). UF membranes with a nominal molecular weight cutoff of 10 or 20 kDa were commonly used to retain proteins and re-move the lactose and small molecules from whey wastewater (Arunkumar and Etzel, 2015). A sta-ble performance of 100% lactose reduction and 95% protein recovery can be expected for a 10 kDa membrane (Chollangi and Hossain, 2007). However, according to Cassini and cowork-ers, the 5 kDa membrane presented better re-sults, with the lowest permeate flux reduction

and the best final wastewater quality (Cassini et al., 2010).

The major problems encountered in mem-brane technology include flux decline, fouling, and sterilization. These problems are outstand-ing when handling food proteins because of their colloidal characteristics, high suspended solids and fats, and the favorable condition for microbial growth. To resolve this problem, pre-treatments such as reverse osmosis (RO) is often combined with UF for separating water from salts and other solutes. The importance of pre-treatment was confirmed by Zhang and Kutowy (1997) in their investigation on the feasibility of recycling chiller water from membranes made of different polymeric materials. As a result, the permeate water that passed through a cross flow reverse osmosis membrane system was found to have very good quality and can be recycled or reused (Sarkar et al., 2006). A membrane sys-tem, including RO and UF was evaluated for the treatment of oxytetracycline (OTC) waste liquor. The COD level was significantly decreased from 10,000 to less than 200 mg/L, whereas OTC was more than three times concentrated in the retentate (Li et al., 2004).

A new generation of membranes with high flux/rejection characteristics have been devel-oped, which have to a great extent increased the probability of water reuse and recycling. The method of generating an electric field of appro-priate polarity across the membrane has been proposed to inhibit the formation of deposits (Thomson, 1984).

3.8 Electrical Processes

Electrodialysis (ED) is an electrochemical process that arranges ion-exchange membranes alternately in a direct current field under the in-fluence of electrical potential difference. It has been used to reduce salt levels in the food in-dustry. Current supply, electrodes, ion exchange membranes, solvents, and electrolytes are the key elements complementary for ED (Xu and



Huang, 2008a). A typical ED cell consists of a se-ries of anion- and cation-exchange membranes arranged in an alternating pattern between an-odes and cathodes in each individual cell. The voltage gradient is maintained across this an-nular film of liquid. The ion concentrations in-crease in alternating compartments during the ED with a simultaneous decrease of ions in other compartments. Hence, it is capable of fractionat-ing protein mixtures, depending on the motili-ties of different components (Walha et al., 2007). Notably, the outlet fractions are at low ionic strength and are ideal for further processing by UF and other techniques (Thomson, 1984).

Güven and coworkers demonstrated that whey wastewater could be treated by electro-chemical oxidation with reduced pollution load in the final effluent (Güven et al., 2008). The production of pharmaceuticals on a sub-stantial scale was evaluated in industry scale (Thomson, 1984). A recent invention explored a bipolar membrane for ED development (Xu and Huang, 2008a). Moreover, a continuous pro-duction-scale electrophoretic separator has been developed and work has been done on the hy-drodynamics of the system for further scale-up (Thomson, 1984).

The performance and the economic cost of UF, RO, and ED were compared; ED appeared to be an economic desalination process for Gabes brackish groundwater because of low energy consumption (Walha et al., 2007). Because of the significant short total residence time—around 1 min—and the high flow rates, high protein yields and minimal biological activity loss can be obtained. The system is thus useful for purifying high-value proteins, such as enzymes or func-tional peptides (Thomson, 1984). However, it is not as economically competitive as other mem-brane separations, such as RO and UF because of the high cost of electrodes and ion exchange membranes (especially bipolar membranes) and relatively short lifetime of membranes when placed in a high-density electrical field (Xu and Huang, 2008b).

3.9 Adsorption Chromatography

Since the 1950s, highly purified functional proteins can be produced on the laboratory scale by ion-exchange chromatography (Peterson and Sober, 1956). Protein from different sources, es-pecially from waste streams, often contain a large variety of dissolved compounds in com-paratively low concentrations; therefore con-centration was indicated as necessary prior to fractionation. The complexity of the purifica-tion made chromatographic techniques the only methods that could guarantee the desired frac-tionation resolution (Thömmes, 1997).

The basic operating principle for chromatog-raphy is to distribute the solutes through the solid and liquid phases. As the extent of adsorp-tion depends on the nature of the adsorbent, the characteristics of the dissolved solutes, and the precise environmental conditions, various reten-tion times can be obtained and hence the sepa-ration. Ion-exchange or affinity techniques have been widely applied for their higher resolutions (Thomson, 1984). The molecular sieve proper-ties for liquid chromatography have been dis-cussed, rigid silica skeleton have the advantage of dimensional stability in any eluent whereas organic gels do not have the problem of irrevers-ible protein adsorption (Eltekov et al., 1973).

Immobilized metal ion affinity (IMA) adsorp-tion is one of the most powerful methods for protein fractionation. The metal atoms or ions are immobilized on a polymer, dominating the interactions at the active adsorption sites. The proteins are therefore separated from heavy metals by affinity. Different types of metal ions, such as zinc, copper, and zeolites can easily be immobilized on the gel derivatives to form IMA adsorbents that can easily being regenerated. No damage to the protein functions was ob-served during the adsorption–desorption pro-cess (Porath and Olin, 1983). Interestingly, the maximum adsorption was obtained at the iso-electric point (pI), so various proteins adsorbed to adsorbents could be desorbed at pI by specific



eluents in order to achieve satisfactory separa-tion (Chiku et al., 2003).

There are several different options for contin-uous operation of liquid chromatography, from serious columns of chromatography to packed bed or fluidized bed operations. The feasibility of protein-binding ligands introduced to cross-flow filtration membranes has also been dis-cussed for the purification of enzymes. Among them, packed bed or fluidized bed operations have garnered the most attention. Packed bed adsorption consists of frontal chromatogra-phy, equilibration, sample application, wash, elution, and cleaning. The main operation dif-ference between this and fluidized bed ad-sorption is the direction of liquid flow and the mobility of the adsorbents. Many parameters, such as mass transfer, equilibrium of protein–ligand interaction, adsorbent particle size, bed height, flow rate, feedstock viscosity, and den-sity would influence the process performance (Thömmes, 1997).

Because they eliminate using centrifugation or UF for cell removal, fluidized or expanded beds are the option for integrating unit op-erations. However, conventional fluidized bed chromatography has a low capacity because of fluid dynamics. Also its extensive back mix-ing results in a longer process time and hence, higher cost. These problems can be solved by stabilizing the adsorbents at fixed positions or by creating a high-density adsorbent with well-defined density and size (Hansson et al., 1994). Expanded bed chromatography was used for the recovery and purification of protein, achieving three times faster results and three times more concentrated solution with higher yield (Johans-son et al., 1996). The purification of C-phycocy-anin and allophycocyanin from S. platensis was also carried out by expanded beds. Combined with conventional gel filtration and ion ex-change chromatography methods, high yield was achieved for the extraction and adsorption by expanded bed adsorption, reducing both op-eration time and costs (Bermejo et al., 2006).

Adsorption chromatography is a very promis-ing process; however, partly because of the inher-ent cost of adsorptive processes and the partial unsuitability of the support materials for large-scale use (Thomson, 1984), further development is necessary and other separation technologies are under study. The combination of different techniques may further enhance the protein re-covery and waste treatment ability and efficiency, which was also suggested by Jhaveri in their eval-uation of precipitation, ion-exchange chromatog-raphy, UF and foaming for the treatment of and protein recovery from clam-processing effluents (Jhaveri, 1984). It also should not be forgotten that each addition to the sequence of operation steps would increase the total production cost, therefore the number of purification steps and the total operation time should be minimized.

3.10 Continuous Liquid–Solid Fluidized Beds for Protein Recovery

In addition to the expanded fluidized bed technologies for protein separation mentioned earlier, the first biotechnological process using a continuous liquid–solid circulating fluidized bed (LSCFB) system was developed by Lan et al. (2002a,b) for continuous recovery of proteins from unclarified cheese whey, at the Univer-sity of Western Ontario, Canada. This technol-ogy is currently being commercialized by Renix Inc. (London, Ontario) as uninterrupted ion ex-change (UIX). Fig. 5.3 provides a schematic of the ion exchanger system.

The system consists of a riser, a downcomer, a liquid–solid separator, top and bottom connec-tion lines between the riser and the downcomer, and optional top and bottom washing sections. The downcomer is filled with the ion exchange resin, preselected to have the correct ion exchange functionality for the proteins of interest and as well as a terminal liquid falling velocity, in liquid higher than any particulates to be encountered in the feed stream. The system is fed at the down-comer inlet (Fig. 5.3), initially with clear water,



at a liquid velocity sufficient to fluidize the bed and carry any contaminant (nonresin) particles upward of the bed. Fluidization achieves a high mass transfer rate and prevents nonresin solids from being trapped in the bed. Resin is allowed to tumble at a predetermined rate from the bottom of the downcomer, past the feed wash zone to the bottom of the riser. In the riser, dilute regenerant (eg, NaOH or NaCl) is fed in upflow from the bottom, driving the resin upward and fluidizing it slightly, while recharging it in cocurrent flow with the correct counter-ion. The system estab-lishes a slowly rotating packed bed regime for the resin. The combination of the resin and wash water at the bottom of the downcomer creates a “dynamic seal” (Lan et al., 2002a,b), because its main function is to keep the liquid streams of the riser and downcomer separate from each

other. The advantages of this technology are that all of the steps of a classical ion exchange cycle are performed simultaneously and continuously without having to change feed streams or physi-cally move the columns and “dirty” feeds can be used with no prefiltration. This technology is especially amenable to recovery of proteins from waste streams.

4 CONCLUSIONS AND FUTURE DIRECTIONS

Because of environmental pollution, strategies to reduce the amount of waste production are needed. These strategies should represent an eco-nomical profit to companies in order to motivate the research and implement the technologies.

FIGURE 5.3 Schematic of the liquid–solid circulating fluidized bed (LSCFB) ion exchanger system. (1) Riser, (2) top wash water section, (3) liquid–solid separator, (4) top connecting pipe, (5) particle circulation rate measuring device, (6) downcomer, (7) distributor, (8) bottom wash water section, (9) bottom connecting pipe. Adapted from Lan et al. (2002b).


REFERENCES 103

The proteins present in waste are expensive com-pounds that once recovered have many inter-esting applications and increasing potential for industry development. Currently, it is of great in-terest to investigate a simple, inexpensive separa-tion technology for recovery of valuable proteins from various waste sources. This can be either in the form of aqueous solutions or solids. Although further improvements are needed, different ap-proaches can be applied, which can decrease treatment cost, increase value-added by-product production, and eliminate environmental pollu-tion from food waste processing.


COD Chemical oxygen demandDPS/B30 A mixture of a zwitter-ionic surfactant, N-dec-

yl-N,N-dimethyl-3-ammonio-1-pro-panesulfo-nate (DPS) and a nonionic surfactant, Brij, 30 (B30)

ED ElectrodialysisFAO Food and Agriculture Organization of the

United NationsIMA Immobilized metal ion affinityLSCFB Liquid–solid circulating fluidized bedNASDBPP North American Spray Dried Blood and Plas-

ma Producers AssociationOTC OxytetracyclinepI Isoelectric pointRO Reverse osmosisSCP Single-cell proteinSPI Soy protein isolateUF UltrafiltrationUIX Uninterrupted ion exchangeVSS Volatile suspended solids

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C H A P T E R


6Modification of Protein Rich

Algal-Biomass to Form Bioplastics and Odor Removal

K. Wang*, A. Mandal**, E. Ayton*, R. Hunt†, M.A. Zeller†, S. Sharma*

*Department of Textiles, Merchandising & Interiors, University of Georgia, Athens, GA, United States; **Department of Statistics, University of Georgia, Athens, GA,

United States; †Algix, LLC, Meridian, MS, United States

1 INTRODUCTION

Proteins are by-products from the agricultur-al and horticultural industries (Verbeek and van den Berg, 2010). Because proteins are naturally occurring polymers and do not require polymer synthesis, they can become value-added prod-ucts when converted into plastics. However, unlike synthetic polymers that contain identi-cal monomers, proteins have 20 different amino acids that form their polypeptide chain. The protein is folded into secondary, tertiary, and quaternary structures and is stabilized by intra-molecular interactions, such as hydrogen bond-ing, electrostatic interactions, and hydrophobic interactions. The amino acid sequence of the protein will define its final properties as a poly-mer (Rouilly and Rigal, 2002; Swain et al., 2004;

Verbeek and van den Berg, 2010). Plant proteins used for plastic production include corn zein, wheat gluten, peanut protein, and soy protein (Swain et al., 2004). Animal proteins, such as keratin, gelatin, collagen, and whey protein, have also been used to produce plastics (Mekon-nen et al., 2013).

To improve their functional properties, it is necessary to modify the proteins. Protein modification includes the denaturation of pro-teins. Proteins are denatured either by thermal or chemical treatment. Denaturation allows the protein to unfold and realign in a new three-dimensional configuration that is stabilized by new intramolecular interactions (Verbeek and van den Berg, 2010). Depending on their prima-ry structure, denaturation can cause proteins to become available to form cross-links.

108 6. MODIFICATION OF PROTEIN RICH ALGAL-BIOMASS TO FORM BIOPLASTICS AND ODOR REMOVAL


Soy protein was used to make the first bio-polymers, and this set the precedent for making polymers based on agricultural materials. For example, in the 1930s, Henry Ford manufactured automobile body parts from a mixture of soy protein and phenol-formaldehyde resin (Swain et al., 2004). Soy protein is isolated from the soy-bean through processing. Soy protein products include soy flour, soy concentration, and soy iso-late. Soy isolates have the highest concentration of protein (more than 90% dry weight) (Swain et al., 2004).

Soy protein plastics have moderate strength and good biodegradability, but they are also typically brittle (Zhang et al., 2003; Mo and Xiuzhi, 2001). Denaturation through chemical modification to the soy protein could improve its ability to form plastics with better mechani-cal properties. Urea is a common chemical used for the denaturation of protein. In one study, authors Mo and Sun showed that the tensile strength and Young’s modulus increased with elevated urea concentration for urea-modified soy protein isolate plastics. Furthermore, com-patibilizers or cross-linking agents can be added to protein bioplastics to improve the mechani-cal properties of protein bioplastics (Mo and Xiuzhi, 2001). Park et al. (2000) showed that soy isolate protein cross-linked with glutaraldehyde significantly improved the tensile strength and elongation at break with increasing concentra-tions of glutaraldehyde.

The primary disadvantage of using soy pro-tein and other terrestrial proteins for the produc-tion of bioplastics is the feedstock. Also plastic production competes with the food industry for its food applications. It becomes advantageous to invest in other agricultural coproducts and waste products as sources of bioplastic proteins. For example, feather meal protein, a by-product of rendering or recycling of animals and waste, becomes a value-added product when consid-ered for bioplastic conversion. Sharma et al. (2008) reported a 9.2 MPa stress at break, 1.40% strain at break, and 2.20 GPa Young’s modulus

for compression-molded feather meal–protein plastic. Feather meal–protein plastic has high stiffness and low extensibility and is compara-ble to other bioplastics made from unplasticized proteins. Modification to feather meal protein using thermal denaturation requires pressure and temperature. Differential scanning calorim-etry (DSC) analysis indicated a peak denatur-ation temperature of feather meal protein occur-ring at 134°C (Sharma et al., 2008).

Algae offers an alternative for bioplastic pro-duction because of their protein content, high biomass yield, ability to be cultivated in their natural environment, potential cost effectiveness, and minimized effect on the food chain (Rajen-dran et al., 2012; Becker 2007). Typically 10–47% of green algae’s dry weight is protein (Fleur-ence, 1999). Euglena gracilis has a protein content of 39–61% of dry matter (Becker, 2007). Unlike soy protein isolate or feather meal protein, it is not economical or technically feasible to isolate the protein from the alga biomass. Therefore, harvested and dried algae (refined to a particle size approximately <150 µm) are added whole in bioplastic and thermoplastic blend formulations.

Zeller et al. (2013) investigated bioplastics and thermoplastic blends from spirulina and chlorella microalgae. Spirulina and chlorella mi-croalgae went through modification by denatur-ation and thermoplastic blending. DSC indicated that spirulina had a peak denaturation at 100°C and chlorella had a peak denaturation at 110°C. Dynamic mechanical analysis (DMA) helped in optimizing and/or identifying that glycerol-plasticized spirulina and chlorella bioplastics (20% glycerol by weight) exhibited satisfactory viscoelastic properties. Tensile testing indicated that spirulina bioplastics and chlorella bioplas-tics had low extension and high modulus. The mechanical properties of algae bioplastics were comparable to soy protein isolate, feather meal, and duckweed. The authors also studied the influence of blending with polyethylene on me-chanical performance spirulina- and chlorella-based thermoplastic blends (Zeller et al., 2013).

2 EXPERIMENTAL 109


Zeller et al. (2013) proved that algae are a feasible alternative for bioplastic and thermo-plastic production. Although they investigated chlorella and spirulina, the study in this chapter investigated the protein modification of catfish algae (planktonic algae)—considered waste and a nuisance for catfish farms—and Nannochlo-ropsis (microalgae). The latter is a protein-rich coproduct and has been scaled commercially because of its ability to accumulate high levels of polyunsaturated fatty acids. Algae bioplastics and their thermoplastic blends were developed through thermomechanical processing and were evaluated for their thermal and dynamic mechanical properties. Additionally, one of the drawbacks to the protein modification of algae is odor, which either occurs naturally within the algae or is generated through thermoplas-tic processing because of heat and pressure. To commercialize these algae bioplastics, the odor-causing volatiles must be removed. A systematic design of experiment approach was undertaken to determine the influence of the factors, such as types of algae, scavenger materials (adsorbents), synthetic resin, and compatibilizer on the odor of plastics.

2 EXPERIMENTAL

2.1 Materials

The Solix microalgae and the catfish algae dry powder received from Algix, Inc. (Merid-ian, Mississippi), were used in this research. Solix microalgae is Nannochloropsis, which is mostly found in marine environments, but also occurs in fresh and brackish water (Fawley and Fawley, 2007). The algae of the Nannochloropsis genus have a diameter of about 2–3 µm and a very simple ultrastructure with reduced struc-tural elements compared to neighboring taxa (Kandilian et al., 2013). The catfish algae are planktonic algae that grow in fishponds and are usually eaten by catfish. They are microscopic

free-floating plants that are usually suspend-ed in the top few feet of water where there is enough sunlight for their photosynthesis. These algae are mainly composed of green al-gae, blue–green algae, diatoms, and euglenas. The ultrahigh molecular weight polyethylene (UHMW-PE) powder and syntactic polypropyl-ene granules were received from Sigma-Aldrich (St. Louis, Missouri). The polyethylene powder had particle sizes of 53–75 µm, and the polypro-pylene was processed via cryogenic grinding to similar particle sizes.

2.2 Bioplastics Processing

For preparing the algae bioplastic, thermo-mechanical compression molding of the sam-ples was performed using a 24-ton benchtop press (Carver Model 3850, Wabash, Indiana) with electrically heated and water-cooled plat-ens. The stainless steel molds can form two rectangular flex bars for DMA at one time. Each different weight ratio formulation was thor-oughly hand mixed. Compression molding of samples used a 20-min cook time at ±150°C followed by a 10-min cooling period, and both were performed under pressure greater than 24,000 Pa.

2.3 Thermal Properties Analysis

Thermal analysis can provide information about the changes of the samples through the heating process. Thermal gravimetric analysis (TGA) was performed using a Mettler Toledo TGA/SDTA851e. DSC was performed using a Mettler Toledo DSC821e. TGA was performed from 25 to 500°C under N2 environment with a heating rate of 10°C/min. DSC was performed from –50 to 250°C under N2 environment with a heating rate of 20°C/min. All samples were prepared with sample weights between 3 and 8 mg. For the plastic samples, fine pieces were cut from DMA flex bars before running on TGA and DSC.



2.4 Mechanical Properties Analysis

DMA was used to characterize the viscoelas-tic behavior of the algae bioplastics. DMA was performed using a dual-cantilever setup at a frequency of 1 Hz on a DMA 8000 instrument from PerkinElmer, Inc. (Waltham, Massachu-setts) for specimens with dimensions in millime-ters: 9 (width) × 2.5 (thickness) × 12.5 (length). All samples were run with a displacement of 0.05 mm from room temperature to 160°C at a temperature ramp of 2°C/min. All samples were run in duplicate.

2.5 Odor Panel Test

The algae bioplastics odor testing factors are listed in Table 6.1. The percentage of algae was kept constant at 50% by weight. If either scav-enger or compatibilizer or both of the two were present, 5% of each was added to the formula-tions, and consequently, the percentage of resin was decreased by 5 or 10%, respectively. All the samples for odor testing were conditioned at 25°C and 55% relative humidity for 24 h to be assessed by the panelists.

3 RESULTS AND DISCUSSION

3.1 Thermal Analysis of Algae Powder

Through TGA, the changes in physical and chemical properties of materials were measured as a function of increasing temperature (with constant heating rate). The TGA results of Solix microalgae and catfish algae powder showed two-step degradation (Fig. 6.1). The first one around 50–100°C represents the bound water

TABLE 6.1 Algae Bioplastics Odor Testing Factors and Level

Factor

Levels

− +

A = Algae Catfish algae Solix microalgae

B = Scavenger Activated carbon

Zeolite

C = Resin Polyethylene Polypropylene

D = Compatibilizer Absent Present

FIGURE 6.1 TGA of Solix and catfish algae powder.

3 RESULTS AND DISCUSSION 111


and low volatiles loss. The second degrada-tion occurring from 175 to 375°C and peaking at 300°C represents carbohydrate and protein burning because it occurs in the range where carbohydrates (eg, hemicelluloses, cellulose, and starch) and protein are typically degraded (Sharma et al., 2008). Solix microalgae has a greater and broader degradation peak, which may be caused by its higher content of lipids than the catfish algae.

DSC reveals the difference in the amount of heat required in increasing the temperature of a sample and reference. For the DSC results, a strong denaturing peak begins at around 20°C and ends at about 150°C (Fig. 6.2) for both So-lix and catfish algae powder. The peak centers around 90°C. At 150°C, both the algae proteins are maximally denatured. The TGA result indi-cates that degradation can occur from around 175°C, whereas the DSC result indicates that proteins are maximally denatured at 150°C. Therefore, both kinds of algae powder are best processed at 150°C, which do not risk their

degradation at higher temperatures, yet yield-ing their maximum denaturation.

3.2 Thermal and Mechanical Analysis of Algae Bioplastics

Figs. 6.3 and 6.4 show the TGA of algae bioplas-tics made of Solix and catfish algae and their ther-moplastic blends (50/50 w/w) with UHMW-PE and isotactic polypropylene (PP). Both the Solix-based bioplastic and the catfish-based bioplastic showed three-step degradation in the TGA ther-mograph. The first degradation, which happened at 25–100°C, was caused by bound water loss and volatiles. The second degradation, which took place around 300°C, again represents the degra-dation of the two kinds of algae, with Solix-based bioplastic showing a broader peak. The third deg-radation peaks occur above 400°C because of the PP or polyethylene (PE) degradation.

Fig. 6.5 shows the DSC of Solix- and catfish-based bioplastics. The endothermic peak at around 75°C represents the algae denaturing.

FIGURE 6.2 DSC of Solix and catfish algae powder.



The melting point of PE can be observed at around 130°C whereas the melting point of PP is near 165°C.

Figs. 6.6 and 6.7 show DMA results of vis-coelastic properties of pure algae bioplastics

compared to PE and PP, and PE/PP thermo-plastic algae blends. From Fig. 6.6, we can see that the 100% Solix microalgae bioplastic has the lowest modulus and highest tan delta, meaning it is soft and flexible, whereas the

FIGURE 6.3 TGA of Solix algae based bioplastics and their thermoplastic blends.

FIGURE 6.4 TGA of catfish algae based bioplastics and their thermoplastic blends.



100% catfish algae bioplastic has the highest modulus and lowest tan delta, meaning it is hard and stiff. PP behaves stiffer than polyeth-ylene. The DMA of PE/PP blended algae bio-plastics again show that the combination of the two relatively stiff materials, catfish algae and

PP, leads to the highest modulus and lowest tan delta. However, the blend of the two flex-ible materials, Solix and PE, shows the low-est modulus and highest tan delta. The 50–50 catfish-PE and 50–50 Solix-PP formulations have medium modulus and tan delta. Between

FIGURE 6.5 DSC of Solix and catfish algae based bioplastics and their thermoplastic blends.

FIGURE 6.6 DMA of pure algae bioplastics compared to PE and PP.



these two, catfish-PE is better because its mod-ulus and tan delta values are both higher than those of Solix-PP blend.

3.3 Odor of Algae Bioplastics

The experimental design and panelist re-sponses are provided in Table 6.2. The design used is a resolution IV half fraction of 24 full fac-torial design, given by D = ABC. The response was categorical in nature—1: almost no odor; 2: less odor; 3: medium odor; 4: more odor; and 5: serious odor.

Let πij denote the probability that bioplastic samples in the ith experimental setting (ith row of Table 6.2) will have odor in category j. Here j can be 1 (no odor) to 5 (strong odor), that is, j = 1, 2, …, J = 5. Clearly, we have πi1+ πi2+ πi3+ πi4 + πi5 = 1 as the odor of the bioplastic sample must take a value between 1 and 5. Let yij be the number of the observations falling into category j for experimental setting i, and let ∑=

=n yi ijj

J

1 be

the total number of bioplastic samples taken in the ith experimental setting. Clearly, in our study, ni = 5 for all i. Then we have Eq. 6.1:

∼ π π πn(Y ,Y ,...,Y ) Multinomial( ; , , ..., )J i i i iji1 i2 i 1 2

We consider ordinal multinomial data where there is a natural order to the categories. An or-dered response can be conveniently modeled with the cumulative probabilities:

γ π π π= + + ⋅⋅⋅+ij i i ij1 2 (6.1)

From now on, we will denote our covariates A, B, C, and D by the vector x. The cumulative probabilities of Eq. 6.1 are linked to the covari-ates by the link function g(•) as follows:

γ θ β= −g x( )ij j iT

(6.2)

Note that the vector xi does not include an intercept term, and the coefficient vector β is same for all groups j = 1, …, 5, so that the pre-dictors have same effects on the response cat-egories. This model is called a cumulative link model (Agresti, 2012). There are several stan-dard choices of the link function g(•) and logit and probit are most popular among them. The breakpoints (θj) have a nice interpretation in terms of an unobserved continuous variable that might be thought of as the real underlying latent response. Simply put, it can be thought that the observed bioplastic sample will fall in category 1 if the underlying latent variable

ni=∑j=1Jyij

(Yi1, Yi2,..., YiJ)∼Multinomi-al (ni; πi1, πi2, ..., πij)

γij=πi1+πi2+⋅⋅⋅+πij

g(γij)=θj−xiTβ

FIGURE 6.7 DMA of PE/PP blended algae bioplastics.



is less than θ1, in category 2 if the underlying latent variable is more than θ1 but less than θ2, and so forth. As x changes, these cutpoints will move together to change the relative probabili-ties of the five response categories. This latent variable explanation for the model is displayed in Fig. 6.8 and different cutpoints for the latent variable Zi are also plotted. Here, assume that the distribution of β−Z xi i

T is normal, or in other words, consider the probit link for Eq. 6.2. Five categories of response is possible, depend-ing on the position of Z relative to the cutpoints θj. As xi changes, the cutpoints will move to-gether to change the relative probabilities of the five responses.

Here we consider logit links such that,

µ µµ

=−

g( ) log1

(6.4)

γθ β

θ β{ }

{ }=−

+ −

x

xwhich implies

exp

1 expij

j iT

j iT

(6.5)

for j = 1, …, J−1 and γij = 1. As there are five cat-egories of responses, from no odor to serious odor, we have J = 5. Here the log-likelihood is proportional to

∑∑= π==

l y log( )ij ijji 1

3

1

4

(6.6)

We used the statistical software R to fit the model and the output is given in Tables 6.3 and 6.4:

Zi−xiTβ

g(µ)=logµ1−µ

which implies γij=expθj−xiTβ-1+expθj−xiTβ

l=∑i=14∑j=13yijlog(πij)

TABLE 6.2 Algae Bioplastics Odor Panel Testing Results

Exp. A B C D Rating Rating scales

Response Total

yi1 yi2 yi3 yi4 yi5 ni

1 + + + + 3,4,3,3,3 1: Almost no odor 0 0 4 1 0 5

2 + + − − 5,4,3,4,5 0 0 1 2 2 5

3 + − + − 3,5,2,2,3 2: Mild odor 0 2 2 0 1 5

4 + − − + 4,4,2,3,4 3: Medium odor 0 1 1 3 0 5

5 − + + − 2,2,1,2,1 2 3 0 0 0 5

6 − + − + 2,2,3,3,2 4: Strong odor 0 3 2 0 0 5

7 − − + + 1,1,1,2,1 5: Serious odor 4 1 0 0 0 5

8 − − − − 2,2,2,2,2 0 5 0 0 0 5

FIGURE 6.8 Latent variable (Zi) view of an ordered mul-tinomial response (grid moves as x changes).

TABLE 6.3 Coefficients of Statistical Model

Factors Estimate Std. error z value Pr(>|z|)

A 2.89085 0.64019 4.516 6.31E-06

B 0.84123 0.35653 2.360 0.018299

C –1.47642 0.42566 –3.469 0.000523

D –0.02378 0.34984 –0.068 0.945812



We can see that the p values corresponding to the factors A and C are very small. We can safely conclude that these two factors are sta-tistically significant. Factor B is marginally sig-nificant and factor D does not have any effect at all.

Table 6.5 gives the estimated probabilities for all possible combinations. From there, we can make recommendations for future experiments.

The results indicate that factor A (algae) and C (resin) are significant in affecting algae plastic

odors. It is obvious that Solix microalgae, which has high levels of polyunsaturated fatty acids, has more serious odor than the catfish algae. It seems that blending with PP creates less odor than blending with PE. Factor B, types of ab-sorbent, has some effect on removing the odor. Activated carbon is more effective than zeolite in absorbing odorous volatile compounds and alleviating the smell. Factor D, the absence of compatibilizer, does not have much influence on odor removal.

4 CONCLUSIONS

The thermal analysis of algae powder and bioplastics and its thermoplastic blends helped in determining processing conditions. TGA re-sults showed that the Solix and catfish algae degradation happened from 175 to 375°C and

TABLE 6.4 Threshold Coefficients of Statistical Model

Parameters Estimate Std. error z value

θ1 –4.2758 0.9773 –4.375

θ2 0.3620 0.5410 0.669

θ3 3.3091 0.8462 3.911

θ4 5.4514 1.0816 5.040

TABLE 6.5 Estimated Probabilities

Setup Estimated probabilities

A B C D No odor Mild odor Medium odor Strong odor Serious odor

−1 −1 −1 −1 0.1147 0.8158 0.0656 0.0034 0.0005

−1 −1 −1 +1 0.1196 0.8139 0.0628 0.0033 0.0004

−1 −1 +1 −1 0.7128 0.2833 0.0037 0.0002 0.0000

−1 −1 +1 +1 0.7224 0.2739 0.0035 0.0002 0.0000

−1 +1 −1 −1 0.0235 0.6898 0.2660 0.0182 0.0025

−1 +1 −1 +1 0.0246 0.6983 0.2574 0.0174 0.0024

−1 +1 +1 −1 0.3157 0.6637 0.0194 0.0010 0.0001

−1 +1 +1 +1 0.3261 0.6543 0.0186 0.0009 0.0001

+1 −1 −1 −1 0.0004 0.0392 0.4005 0.4299 0.1299

+1 −1 −1 +1 0.0004 0.0411 0.4104 0.4235 0.1247

+1 −1 +1 −1 0.0076 0.4339 0.4962 0.0545 0.0077

+1 −1 +1 +1 0.0080 0.4453 0.4872 0.0522 0.0074

+1 +1 −1 −1 0.0001 0.0075 0.1199 0.4270 0.4455

+1 +1 −1 +1 0.0001 0.0079 0.1249 0.4334 0.4337

+1 +1 +1 −1 0.0014 0.1267 0.6087 0.2229 0.0402

+1 +1 +1 +1 0.0015 0.1321 0.6124 0.2156 0.0384


REFERENCES 117

peaked at 300°C, representing carbohydrate and protein burning. Additionally, DSC results indi-cated that proteins were maximally denatured at 150°C, suggesting the best processing tempera-ture at 150°C to yield maximum denaturation. DMA concluded that the 100% Solix microalgae bioplastic was soft and flexible whereas the 100% catfish algae bioplastic was hard and stiff. More-over, by blending with PE or PP, the modified thermoplastic blends using algae could lead to a range of mechanical properties for targeted appli-cations. The odor panel sensory study is a great tool to gauge human perception to determine suitability of algae-based plastics in applications such as packaging and other consumer prod-ucts. The headspace solid-phase microextraction (SPME) and GC/MS procedures could be used to determine which products produce objectionable odors and to address their remediation.

AcknowledgmentsOur research was partially supported through the National Security Agency grant H98230-12-10251.

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S E C T I O N III

TRANSFORMATION OF PROTEINS BY-PRODUCTS TO HIGH VALUE PRODUCTS

7 Food industry protein by-products and their applications 121

8 Biobased flocculants derived from animal processing protein by-products 135

9 Pharmaceutical and cosmetic applications of protein by-products 147

10 Application of waste-derived proteins in the animal feed industry 161

11 Novel applications of protein by-products in biomedicine 193

12 Microalgal-based protein by-products: extraction, purification, and applications 213

13 Recovery and applications of proteins from distillery by-products 235

14 Recovery and applications of feather proteins 255

15 Algae derived single-cell proteins: economic cost analysis and future prospects 275

16 Whey proteins and their value-added applications 303

17 Seafood waste-derived peptides: their antioxidant activity and potential as alternative preservatives in fish products 315

C H A P T E R


7Food Industry Protein By-Products

and Their ApplicationsL.J. Yu, M.S.-L. Brooks

Department of Process Engineering and Applied Science, Dalhousie University, Halifax, Nova Scotia, Canada

1 INTRODUCTION TO FOOD INDUSTRY BY-PRODUCTS

1.1 Significance of Food Industry

Globally, the food industry is one of the most important sectors. It generates significant rev-enue and employment throughout countries in Europe, the United States, and Canada. Food processing and the manufacturing are essential activities that result in the creation of many food products that can be subsequently exported to other countries. There are a huge variety of food products that make up the various subsectors of the food industry, including the meat, bakery, and dairy sectors.

In Europe, the food industry is the second largest manufacturing sector, equal to €1017 bil-lion for the EU-27 countries (EU, 2013a). Here, the employment in the food industry represents about 15% of the total manufacturing sector. Of the subsectors in the European food industry, the meat sector is the largest, representing 20% of total turnover. Together, the top five subsectors

(meat sector, bakery products, dairy products, drinks, and other food products) represent 76% of the total turnover and more than four-fifths of the total number of employees (EU, 2013a).

In the United States, the food industry ac-counted for 14.7% of the value of shipments from all US manufacturing plants in 2011. The meat industry was the largest single component of the food-manufacturing sector and associated with 24% of food manufacturing shipments in 2011. Other important subsectors include dairy (13%), beverages (12%), grains and oilseeds (12%), fruits and vegetables (8%), and other food products (11%) (USDA, 2013).

In Canada, the food-processing industry is the largest of all manufacturing sectors, ac-counting for the largest share (15.9%) of the manufacturing sector’s GDP in 2012. It also ac-counted for the largest share (16.7%) of the jobs in the manufacturing sector. The industry con-tinues to grow and the value of shipments more than doubled since 1992, reaching $93.7 billion in 2012 (AAFC, 2014). Inside the food industry, processed meat, dairy, and beverage products

122 7. FOOD INDUSTRY PROTEIN BY-PRODUCTS AND THEIR APPLICATIONS

III. TRANSFORMATION OF PROTEINS BY-PRODUCTS TO HIGH VALUE PRODUCTS

accounted for more than half of the total ship-ment values (C$93.7 billion) in 2012. The grain and oilseed milling industry, as well as the bak-ery and tortilla manufacturing industry, were also significant, with shares of 9.7 and 9.0%, re-spectively (AAFC, 2014).

1.2 Food Industry Wastes and Environmental Aspects

The food industry generates significant volumes of food production wastes every year. For example, in Canada, during 2009, approximately 40% of the food produced was disposed of as waste, which is equivalent to CAD $27 billion (approximately 2% of Canada’s GDP), or in other terms, representa-tive of 70% of Canada’s agri-food exports, or 1.1 times the value of Canada’s agri-food imports in 2009 (Uzea et al., 2014). The food industry waste comes from various points along the food value chain, from farm, packaging/processing, trans-portation/distribution, retailing, food service, to households. Among this, 18% of the waste comes from food processing and packaging, which could be caused by many reasons, result-ing in waste from necessary processing opera-tions, such as washing, peeling, seed removal, whey separation from casein, and waste from unnecessary losses, such as equipment defi-ciencies, employee behavior, contamination, customers’ rejections, quality of ingredients, and food safety issues (Uzea et al., 2014). Food industry wastes include both solid and liquid forms. Solid waste is mainly organic leftovers, such as seeds, bran, peels from plant origin, and bones and skins from animal origin. Liquid waste, also called wastewater, is organic water rich in nutrients, such as protein, sugar, and minerals (Aggelopoulos et al., 2014).

For food production solid waste, regulations about limiting organic matter being dumped into landfills can be strict. For example, in the United Kingdom, the granular solid waste can only be accepted at a landfill if the waste meets a total organic carbon (TOC) content of 6% or

a loss on ignition (LOI) of 10%, since waste so-lidification may be hampered by high organic content (Environment Agency, 2010). The organ-ic matter issues of solid waste can be managed by anaerobic digestion, which can be used for industrial or domestic purposes to manage or-ganic waste and/or to produce fuels (DeBruyn and Hilborn, 2007).

The vast quantities of industrial food waste produced every year are a great burden to so-ciety because of the costs associated with waste handling and disposal and treatment, as well as potential environmental contamination caused by discharged waste streams (Stantec, 2012). In many countries, such as Canada, this has led to stricter discharge limits and enforcement of discharge regulations and has also resulted in companies being subject to escalating sur-charges from publicly owned treatment works (Stantec, 2012). The purpose of these regulations is to push the food industry to reduce, renovate, and/or treat their wastes before discharging. The biochemical oxygen demand (BOD) mea-surement is one of the most commonly used pa-rameters for assessing the environmental impact of wastewater and is used as a gauge of the ef-fectiveness of wastewater treatment. BOD is de-fined as the amount of dissolved oxygen needed by aerobic biological organisms in a body of water to break down the organic material pres-ent in a water sample, at a specific temperature and specified period. The BOD5, is expressed in milligrams of oxygen consumed per liter of sample during 5 days of incubation at 20°C. It is often used as a robust surrogate of the degree of organic pollution in water and is listed as a conventional pollutant in the US Clean Water Act (Sawyer, 2003). Publicly owned treatment works in Canada that receive food-processing wastewater with BOD5 values greater than 300 mg/L will add an additional surcharge for treatment. In addition, companies are fined by an environmental enforcement agency, when wastewater that exceeds their permitted BOD5 discharge level, is discharged into a receiving

1 INTRODUCTION TO FOOD INDUSTRY BY-PRODUCTS 123


water treatment facility (Stantec, 2012). Other measures of pollutant-loading, such as total suspended solids (TSS), phenols, total phospho-rous (TP), and total Kjeldahl nitrogen (TKN), are also used for evaluating discharge fees based strength (Stantec, 2012).

A typical example of food production liq-uid waste is the cheese whey generated from dairy processing. Cheese whey has a BOD5 value of 30–50 g/L, which is much higher than the 300 mg/L additional charge limit set for the wastewater system. This higher value of BOD5 is mainly owing to its high lactose con-tent (4.5–5% w/v), soluble proteins (0.6–0.8% w/v), lipids (0.4–0.5% w/v), and mineral salts (8–10% of dried extract). Whey also contains appreciable quantities of other components, such as lactic (0.05% w/v) and citric acids, nonprotein nitrogen compounds (urea and uric acid), and B group vitamins (Kosseva and Webb, 2013).

1.3 Food Industry By-Products

Food wastes are residues of high organic load, which are removed from the production process as undesirable materials, and can be ei-ther solid or liquid in form. However, there are many potential benefits that can be achieved from the reutilization of food wastes, such as the reduction in waste disposal costs and ex-tra revenue from the creation of value-added products. To reflect this, food wastes are in-creasingly referred to as food by-products. Food by-products can be defined as substrates de-rived from food processing, from which func-tional compounds can be recovered, facilitating the development of new products with a mar-ket value (Galanakis, 2012). Examples of new products include functional foods and bioac-tive ingredients, animal feed, components of microbial media, energy from biogas and bio-diesel, and other value-added products (EU, 2013b; Ghaly et al., 2013; Ramakrishnan et al., 2013a; Lowrey et al., 2014).

Food production by-products can be divided into two main groups, depending on whether the food originated from plants or animals, and a myriad of value-added products can be created from the by-products from either ori-gin. For example, cereal sources, such as rice bran, wheat bran, and oat mill by-products, provide a good matrix for the extraction of functional ingredients, including nutritional proteins (Prakash, 1996) and dietary fibers (Hu et al., 2009). Root sources, such as potato peels and sugar beet molasses, have been investigat-ed for the extraction of phenols (Oreopoulou and Tzia, 2007) and organic acids (Fischer and Bipp, 2005), respectively. Process by-products from oil crops, such as sunflower seed resi-dues and soybean waste have been targeted for the recovery of polyphenols (Copeland and Belcher, 2001) and albumin (Jishan et al., 2009), among others. In addition, by-products from fruit and vegetable processing, such as fruit peel and pomace, have been used to extract com-pounds, such as pectin (Wang et al., 2007), poly-phenols (Rupasinghe et al., 2012), and β-carotene (Chantaro et al., 2008). Bioactive molecules that are extracted from food by-products have great potential for use in functional foods and nutri-tional supplements (Celli et al., 2014). Animal-sourced by-products can also be obtained from meat, seafood, and dairy processing, from which, proteins (Tahergorabi et al., 2011; Ramak-rishnan et al., 2013a), amino acids (Ramakrish-nan et al., 2013b), oils (Ghaly et al., 2013), and lactose (Bund and Pandit, 2007) are examples of potential high-value products.

Food industry protein by-products, either from animal or plant originated food waste, are promising sources of value-added prod-ucts. Particularly as they can be developed into products that have applications in many different areas including the food, nutraceu-tical and pharmaceutical, animal feed, and biofuel industries. In addition to the extra rev-enue obtained from the food industry protein by-products, the reduction in costs associated



with disposal and treatment of the food waste make it very attractive to develop appropri-ate technologies for the recovery of valuable proteins and creation of new products. The following sections will examine protein by-products from animal sources (milk, meat, and fish) as these are significant sectors in the food industry, and then discuss their applications and future needs.

2 SIGNIFICANT SOURCES OF FOOD PROTEIN BY-PRODUCTS

2.1 Milk Proteins

Whey is a major by-product of cheese mak-ing. Normally, 10 L of milk can produce 1 kg of cheese and generate about 9 L of whey. There are two major types of whey: sweet rennet whey and sour or acid whey. The former is a by-product in making cheddar and other types of sweet cured cheese, while the latter is a by-product from the production of cottage cheese and similar products. Both sweet and acid whey contain 60–70 g/L of total solids, 44–52 g/L of lactose and 6–10 g/L of proteins (Jelen, 2011). The main differences are the higher levels of calcium, phosphate, lactic acid, and lactate con-tents in the acid whey, with the calcium content being 10 times higher than in the sweet whey. In addition, sweet whey also contains glycomac-ropeptides (Božanic et al., 2014). Globally, most the whey produced is in the form of sweet whey, and in the United States, for example, about 94% of the whey produced is sweet whey, with the remaining 6% as acid whey (American Dairy Products Institute, 2002).

The disposal of large volumes of whey pro-duced from cheese-making has traditionally been a problem for the dairy industry because of the high pollutant-loading of the whey. However technologies have been developed to obtain value-added products from this by-product stream. The main industrial processing

of whey is by drying to produce whey powder (WP, with 13–15% protein, 70–80% lactose, and 1–8% minerals), which accounts for 70% of the annual whey production. Comparatively less whey protein concentrate (WPC, with 65–80% protein, 4–21% lactose, and 3–5% minerals) and whey protein isolate (WPI, with 88–92% protein, <1% lactose, and 2–4% minerals) are produced every year. To manufacture WP, any fat from the whey must be removed, followed by heat treat-ment, evaporation, then lactose crystallization and spray drying (Jelen, 2011).

Whey proteins in the form of WP, WPC, and WPI, are used as additives in many types of foods including meats, dairy, and baked goods because of their ability to hold water, form gels, bind ingredients, and act as emulsi-fiers (Morr, 1982; Ha and Zemel, 2003). Whey proteins have great nutritional benefits because of their unique fractions. Whey protein is typi-cally a mixture of β-lactoglobulin (β-lg, ∼65%), α-lactalbumin (α-la, ∼25%), bovine serum albu-min (∼8%), and immunoglobulins (Onwulata et al., 2011). Whey protein and its fractions can be separated using membrane filtration tech-niques based on molecular weight differences. During this process, lactose, salts, and other low molecular weight materials pass through a membrane as the permeate, while higher mo-lecular weight components, such as protein are concentrated (Ramos et al., 2012). The remaining permeate can be used to produce lactic acid, bio-ethanol, or lactose (Jelen, 2011). Ion exchange is another separation technique for whey protein and its fractions. Here, the whey protein frac-tions can be separated from the whey according to the surface charge characteristics of the mol-ecule (ie, the ζ-potential). This method uses mild pH adjustments to activate and subsequently deactivate the attraction between the ion exchange resin and the protein molecules. Ion exchange is generally more selective in what is retained during the process (Doultani et al., 2003). More recently, ion exchange membrane chromatography has been investigated as a promising method for

2 SIGNIFICANT SOURCES OF FOOD PROTEIN BY-PRODUCTS 125


whey protein isolation. It combines the advan-tages of membrane technique and ion exchange, such as short processing times, mild treatment conditions and highly specific separations (Santos et al., 2012).

Whey protein fraction, β-lg, is a good source of cysteine, and has been implicated in hy-drophobic ligand transport and uptake, en-zyme regulation, and acquisition of passive immunity in infants (Kontopidis et al., 2004; Onwulata et al., 2011). The α-la protein has branched-chain amino acids, which are used by the muscles for energy and protein synthesis, and contain bioactive peptides with antihyper-tensive, antimicrobial, antioxidative, and an-titumor activities (Kamau et al., 2010). Bovine serum albumins contribute to osmotic pressure of blood and play an important role in trans-port, distribution, and metabolism of ligands (Onwulata et al., 2011). The health benefits of whey protein and its appeal to consumers con-tinue to increase; this gives great opportunities to apply whey protein products into nutraceu-tical and pharmaceutical industry, as discussed later in this chapter.

2.2 Meat Proteins

The annual global meat production reached 296 million tons in 2010 (FAO, 2013), with pork production as the highest (109 million tons); followed by poultry (99 million tons), beef and buffalo (68 million tons), and sheep and goat (13 million tons). The meat industry generates significant amounts of by-products, and most of the by-products are produced during slaugh-tering. The slaughtering by-products include bones, skin, blood, entrails, fatty tissues, feet, skulls, and so forth, and vary with type of ani-mals. For example, by-products of pigs, cattle, and lambs represent 52.0, 66.0, and 68.0% of the live weight respectively, of which nearly 40% are edible and 20% are inedible (Jayathilakan et al., 2012; Mirabella et al., 2014). Therefore, meat industry professionals try to find new

recovery and utilization methods for these by-products, which otherwise could be significant losses for the economy and serious risks to the environment.

The poultry sector, as an example, is the most dynamic sector in meat industry. Globally, poul-try meat production increased from 69 million tons in 2000 to 94 million tons in 2008, which corresponds to an increase of 35%. This growth is continuing despite consumer scares and re-gional trade restrictions linked to the spread of various diseases, such as the outbreaks of the avian influenza and Newcastle disease (Lasekan et al., 2013). Poultry processing typi-cally includes the following steps. The birds are first inspected by a veterinarian. After inspec-tion, the birds are put on a killing line and are hung upside down by their feet by shackles on a conveyor. Once the birds are shackled, stun-ning is carried out using an electrically charged water bath, gas inhalation, or a blow to the head using a blunt object. Then slaughtering can be performed manually or automatically by using a circular knife system. The birds should bleed thoroughly and the blood is collected as a by-product for further processing. After bleeding, the birds are scalded using steam or hot water. Scalding loosens the feathers and facilitates plucking. Feathers are removed by a plucking machine and are collected as an animal by-product. Following scalding and plucking, the head, feet, and inedible organs are removed as animal by-products. The carcasses are then rinsed, cooled, weighed, inspected, categorized, packaged, and stored (FAO, 2010a).

Thus, a number of by-products can be ob-tained from poultry processing. Blood, as a by-product, represents about 2–6% of the live bird weight (Piazza et al., 2011), and if further pro-cessed, should be collected in a separate tank, then cooled and preserved. It can then be fil-tered and spray-dried to produce blood meal (with a crude protein content of 60–80%), which can be used for feeding fish, pets, and other animals. Animal blood can also be used for



making human foods, such as blood sausage in some countries (Mandal et al., 1999; Ofori and Hsieh, 2011). Feathers, with a crude protein con-tent of more than 90%, constitute 5–7% of the live bird weight (Grazziotin et al., 2008; Taskin and Kurbanoglu, 2011). The low-value proteins from feathers can be hydrolyzed and used in pet food or animal feed. Poultry feet, with a crude protein content of 16% (Okanovic et al., 2009), can either be used for human food or separated for animal consumption. Those used for human food should be approved during the inspection process and are heat treated to remove skin and nails before packing (FAO, 2010a).

In general, meat by-products obtained from animals are rich in proteins and are also good sources of minerals and B-complex vitamins. For example, animal liver is especially rich in vitamin A and has a higher proportion of unsaturated fat-ty acids than saturated fatty acids (FAO, 2010a). Animal blood is rich in protein, zinc, iron, vita-min B2, and has a higher value of trivalent iron and other nutrients. It is easily digested and ab-sorbed by the body and therefore a good source for patients, children, and elderly people (Mandal et al., 1999; Toldra et al., 2012). In addition to the consumption of meat-processing by-products for nutritive purposes, modern technologies have led the application of the meat by-products be-yond general nutrition. The applications of meat protein by-products to other areas will be pre-sented in Section 3, which follows.

2.3 Seafood Proteins

Seafood by-products have recently attracted much attention because of their natural abun-dance and underutilized bioactive materials. Various bioactive materials, such as peptides, gelatin, collagen, chitin, chitosan, calcium, and fish oil have been isolated from the seafood by-products and have been reported to have antihypertensive, antitumor, anticancer, anti-bacterial, antioxidant and anti-HIV activities (Senevirathne and Kim, 2012).

The fish-processing industry is a major ex-porter of seafood products in many countries including China, Norway, and the United States (FAO, 2010b). Globally, about 154 mil-lion tons of fish were produced in 2011, with a value of US$217.5 billion (FAO, 2012). Typically, two-thirds of the fish is processed before sale. Processing the fish involves stunning the fish, grading, slime removal, scaling, wash-ing, deheading, gutting, cutting of fins, slicing into steaks and fillets, meat bone separation, packaging, labeling, and distribution (Ghaly et al., 2013). During these processes, 20–80% of by-products are generated depending on the lev-el of processing and type of fish (AMEC, 2003).

The solid by-products from fish processing include the heads, tails, frames, and skin. Fish frames contain significant amounts of muscle proteins that can be recovered from this process-ing by-product. These muscle proteins are high-ly nutritious, easily digestible, and nutritionally superior to those of plant sources. Furthermore, they have a better balance of essential amino acids compared to other animal protein sources (Friedman, 1996). The fish proteins can be ex-tracted by chemical or enzymatic processes. In the chemical method, salts including NaCl and solvents, including isopropanol, are used (Sikor-ski and Naczk, 1981), whereas in the enzymatic extraction, enzymes such as Alcalase and Neu-trase are applied (Liaset et al., 2000). These fish proteins can be used as a functional ingredient in many food products because of their unique properties, such as water-holding capacity, oil absorption, gelling activity, foaming capacity, and emulsifying properties. They can also be used in milk replacers, infant formulas, and as bakery substitutes.

Amino acids can also be produced from fish protein by-products by enzymatic or chemical processes. Enzymatic hydrolysis of protein sub-strates uses protease enzymes, such as alcalase, chymotrypsin, trypsin, and pepsin (Kim and Wijesekara, 2010), whereas chemical hydrolysis of protein substrates uses acid or alkali to extract

3 APPLICATIONS OF FOOD PROTEIN BY-PRODUCTS 127


amino acids (Kristinsson and Rasco, 2000). The extracted amino acids can be applied in animal feed or used in the production of pharmaceuti-cals (Ikeda, 2003; Ghaly et al., 2013), as discussed in Section 3.

3 APPLICATIONS OF FOOD PROTEIN BY-PRODUCTS

3.1 Whey Proteins

Whey proteins are extremely important com-pounds in whey, because of their high nutri-tional value. They are composed of mainly β-lg, α-la, albumin, and immunoglobulin. These ther-mally sensitive fractions are high in concentra-tions of essential amino acids, including lysine, cysteine, and methionine, as well as cystine. The ratio of cysteine and methionine determines the ability of the body to use the proteins and has been found to be about 10 times higher in whey proteins than in casein. Thus, the whey proteins have higher biological value when compared to casein, or other animal-derived proteins (Božanic et al., 2014). Whey proteins are also rich in bioactive peptides (Madureira et al., 2010). These bioactive peptides may affect the major body systems including the cardiovascular, di-gestive, immune, and nervous systems and give beneficial health effects, such as antimicrobial, antioxidative, antihypertensive, or antitumor ac-tivities (Jensen and Larsen, 1993; Bounous, 2000; Davis et al., 2002; Micke et al., 2002; Crowely and Brown, 2007). Examples of nutraceutical and pharmaceutical applications using whey protein by-products are given in Table 7.1.

Whey proteins also possess many functional properties that are desirable in food systems (Morr and Ha, 1993; Zayas, 1997). Important functional properties of whey proteins include water holding capacity, emulsification, gel for-mation, and texture improvement (Mangino, 1984; Van den Hoven, 1987; Sherwin, 1995). The gel formation property of these proteins increas-

es the water-holding capacity and is also consid-ered very important to the consumer acceptabil-ity of many foods, such as processed meat, dairy, and bakery products (Van den Hoven, 1987; Sherwin, 1995; Ha and Zemel, 2003). Whey pro-tein by-products have been successfully used in processed meats, where sweet whey, WPCs, and WPIs are among the most common whey prod-ucts used. The addition of these by-products to processed meats results in improved flavor, func-tionality, and increased yields (Keaton, 1999). In general, the following recommendations (as per-centage of finished product basis) can be consid-ered guidelines for the addition of whey protein by-products: Sweet whey for 2.5–3.5%, WPC for 2.5–3.5%, and WPI for 1.0–2.0%. However, the proportion of these whey protein by-products that would be used in practice would be depen-dent on the processed meat composition and the individual functionality of the specific whey ingredient to be used (USDEC, 1997). As an-other food application, whey proteins have also been proposed as dough-enhancing additives, to provide a protective effect on the gluten net-work in frozen dough and improve the quality (Jacobson, 1997).

The conversion of whey proteins into edible packaging products (edible film and coatings) is an interesting application of a food protein by-product, especially with increasing public awareness of environmental issues around the disposal of nonbiodegradable packaging. In a review by Ramos et al. (2012), recent advances in the field are discussed. However, the basic principle used to generate a film or coating from whey proteins involves gelation, which can be facilitated by various methods, including the addition of chemicals, change in net charge, or increase in hydrostatic pressure. Each of these processes results in protein aggregation and eventual gel formation as the initial proteins un-dergo partial or complete unfolding. Producing whey protein-based packaging would be very desirable as a renewable, sustainable packaging alternative, however, further research efforts are



TABLE 7.1 Some Application Examples of Protein By-Products in Pharmaceutical, Nutraceutical, and Food Systems

Origin of waste Type of protein by-product Characteristics Applications References

Whey WPC Antitumor Cancer patients Bounous (2000)

Whey Whey protein (Fresenius Kabi, Germany) or Immunocal (Immunotec, Europe)

Increase plasma glutathione levels

HIV patients Micke et al. (2002)

Whey β-Lactoglobulin (β-lg), α-lactalbumin (α-la)/Alfa-Laval Food Engineering AB, Sweden)

Nutrition and beyond Nutraceutical Jensen and Larsen (1993)

Whey WPI/Bipro/Davisco International Foods Inc., USA

Nutrition and beyond Nutraceutical Davis et al. (2002)

Whey Deflavored whey protein/Kraft Foods Holding Inc., USA

Nutrition and beyond Nutraceutical Crowely and Brown (2007)

Whey Sweet whey, WPC, WPI Flavor, aroma, water binding, gelation, emulsification

Processed meats Keaton (1999)

Whey Whey powder Aroma, texture, stability, water binding

Bakery (pastry) Božanic et al. (2014)

Whey Whey powder Air incorporation Bakery (Glazes) Božanic et al. (2014)

Whey Whey powder Aroma, acid, fruit stability

Dairy (ice cream) Božanic et al. (2014)

Whey WPI Gel formation Edible film and coating

Ramos et al. (2012)

Meat Liver extract Nutritional supplement Nutraceutical Devatkal et al. (2004)

Meat Blood Nutritional ingredient Deli meat (sausages) Ghost (2001)

Meat Blood plasma Gelation, foaming Deli meat (cooked ham)

Autio et al. (1985)

Meat Gelatin Stabilizer Frozen desert Jamilah and Harvinder (2002)

Fish Protein hydrolytes/Seacure, USA and Canada

Regulate bowel functions

Dietary supplement Chalamaiah et al. (2012)

Fish Protein hydrolytes/Amizate, North America

Metabolic recovery Sports nutrition Chalamaiah et al. (2012)

Fish Protein hydrolytes/Molval, UK Promote cardiovascular health

Dietary supplement Chalamaiah et al. (2012)

Fish l-glutamate Flavor enhancer Food flavoring industry

Ikeda (2003)

Fish d,l-Methionine Animal nutritional supplement

Animal feed Ikeda (2003)

Fish l-Glycine/Nutropin Human growth hormone

Pharmaceutical Arakawa et al. (2007)

3 APPLICATIONS OF FOOD PROTEIN BY-PRODUCTS 129


needed to ensure its commercial success. Other examples of whey protein by-products in food applications are shown in Table 7.1.

3.2 Meat Proteins

Traditionally, meat by-products that are rich in proteins (eg, blood, skin, bones, meat trim-mings, feet, and internal organs of harvested an-imals), are consumed either by humans or used as pet food, animal feed, or fertilizer (Toldra et al., 2012). However, blood plasma, gelatin, and liver, as well as other meat by-products, have also been used to extract bioactive peptides and other value-added components (Jayathilakan et al., 2012).

Animal blood, which has a high level of pro-tein and heme iron, is an important nutritional by-product for humans. Animal blood has long been consumed in the forms of blood sausages, blood pudding, blood curd, and blood cake (Ghost, 2001; Wan et al., 2002). Blood is also used in food as an emulsifier, stabilizer, and nutrition-al ingredient (Silva and Silvestre, 2003). For feed applications, blood is mostly used in the form of blood meal, which serves as a protein, lysine, and mineral supplement, and a vitamin stabiliz-er. Blood plasma, as a component from animal blood, has good gelation and foaming proper-ties because of its high content—60%—of albu-min (Silva and Silvestre, 2003). Therefore, it has been used in cooked ham, hot dogs, and bakery products (Autio et al., 1985; Ghost, 2001).

Hides and bones are meat by-products that contain large quantities of collagen. Gelatin is produced by the controlled hydrolysis of a water-insoluble collagen derived from hides and bones. The processing of gelatin from hides and bones consists of three major steps. The first step is the elimination of noncollagenous ma-terial from the raw material. This is followed by controlled hydrolysis of collagen to gelatin. The final step is recovery and drying of the fi-nal product (Jayathilakan et al., 2012). Gelatin has been used in jellied desserts because of its

melt-in-the-mouth feeling. It is also added to a range of meat products, including meat pies, and widely used as a stabilizer for ice cream and other frozen desserts. High-bloom gelatin works as a protective colloid to ice cream, yo-gurt, and cream pies, to prevent the formation of ice crystals and the recrystallization of lactose during storage (Jamilah and Harvinder, 2002). In addition, gelatin is applied in the pharma-ceutical industry as a binding and compound-ing agent in the manufacture of tablets (Hidaka and Liu, 2003). Being a protein in nature, it is used as a plasma expander for blood in cases of very severe shock and injury. Gelatin is an excellent emulsifier and stabilizing agent for many emulsions and foams and therefore has applications in cosmetic products and printing (Arvanitoyannis, 2002).

The liver is the largest gland found in ani-mals and a by-product of meat processing. Liver extract has been used by the pharmaceu-tical industry as a source of vitamin B12 and as a nutritional supplement to treat various types of anaemia (Colmenero and Cassens, 1987; Devatkal et al., 2004). In addition, the liver can be a source of heparin, although heparin can also be extracted from the lungs and the lining of the small intestines. Heparin is an important pharmaceutical compound that is used as an anticoagulant to prevent blood clotting during surgery (Jayathilakan et al., 2012). Further ex-amples of meat protein by-products applied in different areas are given in Table 7.1.

3.3 Fish Proteins

Fish proteins are highly nutritious and easily digestible. Fish proteins contain many bioactive peptides that can be used as functional ingre-dients in food products, owning to properties such as water-holding capacity, oil absorption, gelling activity, foaming capacity, and emulsifi-cation (Ghaly et al. 2013). Fish proteins can be used as protein supplements in bakery prod-ucts, soups and infant formulas, because of the



rich amino acid composition. Also, various com-mercial nutraceuticals are produced from fish protein hydrolysates, which serve as dietary supplements to support healthy body functions and prevent aging diseases, such as cardiovas-cular disease and hypertension (Chalamaiah et al., 2012).

Commercial amino acids from fish by-prod-ucts are used as food additives, animal feed, and for pharmaceutical applications. The larg-est usage of amino acids is the food-flavoring industry, which uses monosodium glutamate, alanine, aspartate, and arginine to improve the flavor of food products. The animal feed indus-try also uses large amounts of amino acids to improve the nutritional quality of animal feed (Ghaly et al., 2013), and commonly used amino acids include lysine, methionine, threonine, and tryptophan among others. Some amino ac-ids, such as arginine, glycine, glutamate, and histidine, are widely used in protein-based pharmaceuticals as an excipient for drug de-velopment. In addition, amino acids can be used in pharmaceutical formulations and as a substrate for the microbial production of an-tibiotics and biopharmaceuticals (Arakawa et al., 2007; Ramakrishnan et al., 2013b). Since 1996, amino acids have become increasingly more valuable (Ikeda, 2003) and the demand is high. For example, amino acids are being pro-duced at the multimillion-ton scale, with the fermentative production of l-glutamate and l-lysine in 2013 estimated at more than 5 mil-lion tons (Wendisch, 2014). Further examples of the applications of fish protein by-products are shown in Table 7.1.

4 FUTURE DIRECTIONS

Food-processing protein by-products need not be an environmental burden; indeed, they are a promising source of value-added prod-ucts. With the increasing public awareness of health and nutrition issues, food-processing

protein by-products, such as whey proteins, are becoming more valuable as food ingredi-ents, biodegradable packaging, dietary sup-plements, and pharmaceuticals, because of their unique bioactive and functional proper-ties, resulting in positive benefits to human health and the environment. However, some protein by-products, including those derived from meat processing, have not been fully de-veloped in terms of their potential for value addition. This is evident as the two major com-mercial application areas for meat by-products are still as animal feed and direct human con-sumption as food. It is possible that this may be partly caused by religious reasons and health concerns discouraging the use of animal-derived compounds, such as gelatin, as food additives. Thus, it is necessary to explore meat by-products for other potential applications, such as in the cosmetics and energy industries, and more research is needed to develop novel, efficient, cost-effective technologies to ensure that food by-products can be fully used so as to maximize economic value and minimize en-vironmental impact.


α-la α-Lactalbuminβ-lg β-LactoglobulinAAFC Agriculture and Agri-Food CanadaBOD Biochemical oxygen demandEU European UnionFAO Food and Agriculture OrganizationGDP Gross domestic productLOI Loss on ignitionNaCl Sodium chlorideTKN Total Kjeldahl nitrogenTOC Total organic carbonTP Total phosphorousTSS Total suspended solidsUSDA United States Department of AgricultureUSDEC US Dairy Export CouncilWP Whey powderWPC Whey protein concentrateWPI Whey protein isolate


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http://www.ers.usda.gov/topics/food-markets-prices/processing-marketing/manufacturing.aspx



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http://sites.ivey.ca/agri-food/files/2014/07/Provision-Addressing-Food-Waste-In-Canada-EN.pdf















C H A P T E R

135Protein Byproductshttp://dx.doi.org/10.1016/B978-0-12-802391-4.00008-2 2016 Published by Elsevier Inc.

8Biobased Flocculants Derived

from Animal Processing Protein By-Products

G.J. Piazza, R.A. GarciaUS Department of Agriculture, Agricultural Research Service, Eastern Regional

Research Center, Biobased and Other Animal Coproducts Research Unit, Wyndmoor, PA, United States

1 FLOCCULATION IN INDUSTRIAL PROCESSES AND WASTEWATER

TREATMENT

1.1 Description of a Flocculant

There are a wide variety of situations in which small particles are suspended in a liquid; the muddy water produced by a storm is a familiar example. In some of these situations, even if the liquid is not disturbed, the particles will remain in suspension for a long time. One approach to removing particles from a suspension involves the addition of a substance called a flocculant.

Flocculants promote coalescence of the par-ticles into discrete flocs (Krishnan and Attia, 1988). Depending on floc density relative to that of the suspending liquid, the flocs may settle, producing a sediment, or float, producing a cream. When used in conjunction with filtration

or centrifugation, the flocculants are referred to as filtration- or centrifugation-aids (Lewellyn and Avotins, 1988). The delineation between a floc-culant and a coagulant is not absolute; generally, a coagulant is a substance that destabilizes a sus-pension (Bratby, 2006), and a flocculant causes the destabilized particles to coalesce. However, many substances used for particle–liquid sepa-ration can both destabilize colloidal forces and aid in particle coalescence. Therefore throughout the remainder of this discussion, the word floc-culant will often be used to describe a substance that has been shown to promote particle–liquid separation without regard to the mechanism of separation.

Flocculants contribute extensively to a variety of important processes, such a water and waste-water clarification (Maximova and Dahl, 2006), dewatering in mineral operations, paper man-ufacture, and concentration during chemical

136 8. BIOBASED FLOCCULANTS DERIVED FROM ANIMAL PROCESSING PROTEIN BY-PRODUCTS


production (Svarovsky, 2000). Flocculants are applied directly to soil to prevent erosion during agricultural and construction operations and are also applied to the walls of unlined canals to de-crease water loss (Sojka et al., 2007). A newer use for flocculants is an aid in the harvest of cultured microorganisms from their liquid medium as a step in the conversion of the microorganisms to specific substances or as use as biomass for con-version to biofuel (Schlesinger et al., 2012; Lee et al., 2013; Milledge and Heaven, 2013). Floc-culants have been also used to help purify sub-stances from plant biomass (Kholkin et al., 1999).

1.2 Environmental Impacts of Synthetic Organic Polymeric Flocculants

A major class of flocculants is polymeric flocculants. These are synthesized from petro-chemical precursors and may contain neutral, positively, or negatively charged functional groups. Polymeric flocculants can be extremely effective at low concentrations. However, it is becoming increasingly evident that the syn-thetic polymeric flocculants must eventually be replaced by renewable alternatives (Salehizadeh and Shojaosadati, 2001). In addition to lack of renewability, synthetic polymeric flocculants have a variety of other environment drawbacks. They may contain traces of toxic monomer or may degrade to toxic by-products (Bolto and Gregory, 2007). Positively charged synthetic polymeric flocculants can harm fish by bind-ing to their gills and suffocating them (Bratby, 2006). The formation of the potent carcinogen N-nitrosodimethylamine (NDMA) has been detect-ed in water treated with the positively charged flocculant poly(diallyldimethylammonium chlo-ride) (pDADMAC) and chlorine (Letterman and Pero, 1990; Gumbi et al., 2013). Concerns about toxicity have caused the US Bureau of Reclama-tion to ban the use of anionic polyacrylamide (PAM) in unlined canals, and in Germany, sludge treated with PAM has been banned from land applications (Kutti et al., 2011).

1.3 Biobased Flocculants

Numerous renewable materials have been tested for flocculant activity. Flocculation has been measured with derivatives of amylopec-tin, carboxymethylcellulose, guar gum, starch, and glycogen (Pal et al., 2006). Pectin, chitosan, starch, or other polysaccharides, which are ob-tained from food production, have been used as sources of bioflocculants (Salehizadeh and Shojaosadati, 2001; Renault et al., 2009; Ghimici and Nichifor, 2010; Ho et al., 2010). Certain mi-crobial fermentation products were also found to have flocculant activity, and these products were often found to be biomacromolecules (Zhang et al., 2010; Patil et al., 2011). Suspen-sions of chitosan, starch xanthate, cellulose xan-thate, and acid-hydrolyzed cellulose microfibrils have been tested for control of soil erosion (Orts et al., 2000). In general, renewable materials make poor or mediocre flocculants either be-cause they have insufficient water solubility or because they lack enough charge density to neu-tralize charged colloidal material when used at levels that are sufficient for synthetic flocculants (Bo et al., 2012; Li et al., 2014). Flocculant activ-ity can be enhanced by synthesizing derivatives of biobased flocculants with high charge density (Shogren, 2009; Kutti et al., 2011).

1.4 Protein Flocculants

Proteins have the potential to be excellent flocculants of charged suspended or colloidal material. Some proteins have relatively high molecular weight (MW) that promotes bridging, which enhances flocculation when a poly-electrolyte brings together colloidal particles (Bratby, 2006). The net charge of the protein de-pends on the medium pH. The transition pH be-tween negative to positive is given by the protein isoelectric point. At pH values above their iso-electric point, the proteins have a net negative charge. At pH values below their isoelectric point, the net charge on the protein is positive,

2 SOURCE OF ANIMAL BY-PRODUCT PROTEINS 137


largely the result of protonation of histidine and glutamate.

It was recognized more than 50 years ago that gelatin (a derivative of the protein collagen) floc-culated colloidal mineral suspensions (Kragh and Langson, 1962). The literature now contains numerous examples of flocculation promoted by other proteins. For example, a flocculant pro-tein was identified and purified from the seeds of Moringa oleifera (Gassemschmidt et al., 1995). Methylated egg albumin, casein, and soy pro-tein have been shown to flocculate diatomite (Seki et al., 2010). A gelatin protein was an ef-fective flocculant in waste drilling water (Wang et al., 2011).

The relatively high cost of proteinaceous ma-terial compared to other biobased substances has limited research on the industrial use of pro-teins. Recently, however, the demand and sup-ply balance in agriculture has been changed by the dramatic increase in the use of fats and car-bohydrates for the production of biodiesel and bioethanol, respectively, and this has caused a concomitant relative increase in protein pro-duction without an increase in market absorp-tion. To increase the use of protein, industrial or nonfood applications have been sought. One of the most successful programs has been the application of protein as a component of adhe-sives in manufactured wood products (Liu and Li, 2007).

The circumstances that contribute to the ex-cess supply of animal by-product proteins are unique to these agricultural products. First, some animal protein sources, such as chicken blood, simply have no current commercial use. Second, in recent years new limitations on the use of animal proteins in animal feed have been put in place because of the out-break of bovine spongiform encephalopathy (BSE), commonly known as mad cow disease (Franco, 2006). A possible new use of animal blood and rendered protein meals is as a com-ponent of flocculants. Research in this area is described subsequently.

2 SOURCE OF ANIMAL BY-PRODUCT PROTEINS

2.1 Rendered Protein Meals

Renderers collect and process unmarketable animal tissue, primarily the bones and offal from slaughtered livestock, the whole carcasses of deadstock, and meat products that have ex-ceeded their sell-by dates (Garcia et al., 2006). The rendering of these tissues yields purified fats, as well as a class of products called ren-dered protein meals (RPMs), which includes spe-cific products, such as meat and bone meal (MBM), blood meal, and feather meal. In the United States, nearly all RPM are used as ingredients in farm and companion animal feed; no other applica-tion of these materials is commercially signifi-cant. It has become apparent that reliance on such a narrow customer base exposes renderers to substantial risk. The occurrence of BSE in Eu-rope massively disrupted the rendering indus-try there, as regulations proscribed almost all use of RPM in animal feed (US Department of Agriculture, 2008). This event, as well as other, similar threats, demonstrates the need to diver-sify the uses of RPM.

Rendered protein meals are attractive pro-tein sources. They are dry and stable at room temperature. MBM, the most abundant RPM, is relatively inexpensive; in 2013 MBM prices were in the $400–500/metric ton range in the United States (Swisher, 2014). The meals are produced year-round and are available in all parts of the United States. They are highly concentrated pro-tein feedstocks; MBM, feather meal, and blood meal typically contain 50–55, 80, and 85% crude protein, respectively (National Renderers Asso-ciation, 2003; Garcia et al., 2006).

The critical disadvantage of RPM is the very low solubility of the proteins (Garcia et al., 2006; Garcia and Phillips, 2009; Piazza and Garcia, 2010a). An insoluble substance will not func-tion as a flocculant. When protein is extracted from MBM using mild conditions (using water



or dilute salt solution), typically only 5–10% of the protein is solubilized. Using an aggressive protein solubilizing solution designed specifical-ly for recalcitrant proteins causes approximately 25% of MBM protein to be solubilized.

Although it would be preferable to solubilize intact (the reason for preferring intact proteins will be discussed in section 3.2) proteins from RPM in high yield, a method for achieving this is yet to be developed. Hydrolyzing protein into smaller peptides typically increases the pro-tein’s solubility. Our laboratory has investigated both alkaline and enzymatic methods for hydro-lyzing RPM (Garcia et al., 2011). Alkaline hydro-lysis has the advantages of simplicity and low cost and the disadvantages of destroying certain amino acids and producing a hydrolysate with a high salt content. Enzymatic hydrolysis, con-versely, does not result in the destruction of any amino acids and does not produce a high salt hydrolysate. Enzymes are, however, relatively expensive, and they require careful control over reaction conditions in order to perform well.

Under the conditions used in our investi-gations, the hydrolysis reactions were slow, requiring approximately 8 h to achieve a high conversion of insoluble protein to soluble hy-drolysate. It was hypothesized that hydrolysis would initially produce large soluble peptides, which would be progressively reduced in size as the hydrolysis reaction proceeded; this would allow the MW distribution in the hydrolysate to be controlled by stopping the reaction when the desired average MW was achieved. Analysis of samples taken from the reactions at various time points revealed, however, that the solubilized material was reduced to small peptides long before a high level of conversion was achieved. Hydrolysis with trypsin was somewhat of an exception to this pattern. Although the other enzymes used in the research (subtilisin, Versa-zyme, Flavourzyme) hydrolyze peptide bonds more or less randomly, trypsin only hydro-lyzes peptide bonds between specific pairs of amino acids. The result is that at the end of a

long hydrolysis reaction, hydrolysate produced using trypsin has a greater proportion of large peptides relative to hydrolysates produced using nonspecific protease enzymes or alkali (Garcia et al., 2011; Piazza and Garcia, 2015).

2.2 Animal Blood

Blood from swine and cattle is collected from slaughter operations by renderers and trans-formed into a substance called blood meal. Blood meal is relatively valuable; in 2013 it sold for approximately $1100/metric ton in the United States (Swisher, 2014) and is used primarily as a lysine supplement in feed. Although chicken blood is similar in composition to the blood of swine and cattle, it is not processed into blood meal. The reasons for this are not entirely clear, but dilution with other plant wastewater streams and the smaller volume of blood pro-duced by a chicken-processing plant relative to beef- and pork-processing plants appear to be factors. Most chicken processors have no customers for their blood, and the blood is es-sentially an expensive waste disposal problem. Some chicken processors pay a renderer to take the blood. Renderers will often mix the blood into other material that is being processed into MBM or feather meal (US Department of Agri-culture, 2008). Recent research has cast doubt about the nutritional value of adding blood to feather meal, finding that in some cases it depresses the availability of certain nutrients (Sulabo et al., 2013). Chicken processors, without a rendering service to take their blood, handle it along with other liquid and solid waste. The liquid portion greatly increases the oxygen demand of the plant’s wastewater stream, re-sulting in significant expense (Mountney and Parkhurst, 1995; Piazza et al., 2011).

Blood is a rich source of protein. The blood of vertebrates is approximately 17% protein; on a dry basis, it is > 90% protein (Duarte et al., 1999). All of the protein in fresh blood is soluble. As will discussed later, hemoglobin (HEM), which


3 PROTEIN AND PEPTIDE FLOCCULANTS 139

is contained inside the red blood cells (RBCs), is a good flocculant.

Blood, however, is a particularly unstable raw material. The normal blood coagulation process greatly increases the difficulty of recovering the HEM and must be avoided. Even when coagu-lation is prevented, microbial proliferation in the blood results in the release of toxic hydro-gen sulfide (Lapin and Koburger, 1974; Persson et al., 1990). Further, the RBCs lyse progressively as they are stored, releasing the HEM into the se-rum (Viscor et al., 1987). Preserving intact RBCs in uncoagulated blood would provide an oppor-tunity to harvest relatively concentrated packets of HEM, eliminating the need to evaporate the water in the serum and removing the undesir-able dissolved serum components.

If commercialized, poultry blood process-ing to produce HEM flocculant would likely be conducted at a centralized facility that receives blood from several slaughterhouses. Such an en-terprise would be more likely to succeed if the energy and equipment required to collect and store the blood prior to processing were kept to a minimum. Ideally, the blood would be collected without regard to sterility and held at ambient temperature during collection at poultry plant, transportation, and intake at the destination. Our investigations found that many anticoagu-lant treatments used for mammalian blood, in-cluding salts of heparin, citrate, or oxalate were inadequate for preventing chicken blood coagu-lation, particularly when the blood is not refrig-erated. Ethylenediaminetetraacetic acid (EDTA), however, prevented coagulation. Further, over a period of 4 days of unrefrigerated storage, blood treated with adequate concentrations of EDTA did not produce significant H2S, and lysis of the RBCs was minimal (Garcia et al., 2014).

Recovery of HEM from preserved blood can be accomplished by washing the RBCs to remove the serum, lysing the cells, and then removing the solid debris of the lysed cells. There are many possible ways to accomplish the cell lysis. The classical method involves incubating the cells in

a hypotonic solution so that they swell and burst. From a process engineering standpoint, this method is not advantageous because it involves the addition of a great deal of water, which will have to later be removed. Ultrasonic processing has long been used in laboratories to break open plant and microbial cells: ultrasound is applied in batch mode with treatment times ranging from a few minutes to more than an hour. Ani-mal cells, including RBCs, lack a cell wall and are consequently less resistant to physical stress. Our investigations demonstrated that ultrasonic pro-cessing could lyse the much more fragile blood cells very quickly; just 300 ms of ultrasound ex-posure was adequate to release 90% of the HEM from the cells (Garcia et al., 2015). The short treat-ment time allows the application of ultrasound in continuous mode, with blood flowing rapidly through an ultrasonic-processing chamber.

3 PROTEIN AND PEPTIDE FLOCCULANTS FROM ANIMAL-

PROCESSING BY-PRODUCTS AND OTHER AGRICULTURAL SOURCES

3.1 Influence of Calcium Ion and pH on Flocculation

Negatively charged synthetic polymeric floc-culants have less deleterious environmental effects than positively charged synthetic floc-culants. Many negatively charged substances are flocculated by the addition of a negatively charged flocculant when a bridging ion is si-multaneously added. The most commonly used bridging ion is calcium ion. Natural water sourc-es often contain levels of calcium ion that are sufficient for bridging, and no external source of calcium ion is needed.

The net charge of a protein is tunable by the pH value of the medium. As discussed in section 1.4, the transition pH from positive to negative charge is termed the protein isoelectric point. This point is different for each protein type because



the isoelectric point depends on each protein’s unique amino acid composition. For most pro-teins, the isoelectric point lies between pH 5.5 and 8. It was initially assumed by us that it would be wise to use a protein as a flocculant at a medium pH near 7, which would cause most proteins to have a large population of nega-tively charged side chains. Thus a laboratory test for flocculation was developed at neutral pH using finely divided and negatively charged clay particles, a commercial sample of anionic polyacrylamide (APAM) and calcium chloride (Piazza and Garcia, 2010a). Protein flocculants were tested at pH 7 with and without the addi-tion of calcium chloride. An extract of MBM and gelatin showed only weak flocculation activity whereas two soybean protein extracts and whey protein showed no flocculant activity. Other ex-periments showed that all of these proteins were able to bind to the clay particles, although bind-ing was tighter with the proteins from MBM and gelatin. Surprisingly, the addition of calcium chloride did not affect flocculation by MBM; its addition to gelatin provided modest stimulation at low gelatin concentrations. The addition of calcium chloride to higher concentration of gel-atin provided modest inhibition of flocculation compared to no calcium chloride controls. These observations suggested that bridging through the calcium ion is at most a minor contributor to the flocculation of clay by proteinaceous materi-als. Thus, it was hypothesized that clay floccu-lation was occurring mostly by charge neutral-ization of the negatively charged clay particles by the cationic groups in MBM and gelatin. To gain experimental evidence for this hypothesis, flocculation experiments were repeated with the addition of buffered solutions at pH 5.5, 7.0, and 10.0. The best flocculation was observed in the pH 5.5 buffered solutions. At this pH value, pro-teins will have higher levels of positive charge. Two major contributors to protein positive charge are lysine (pKa 10.5) and arginine (pKa 12.5). However, the pKa of these materials are so high that lowering the pH from, for example, 7.0

to 5.5 will have no effect on their contribution to protein charge. In contrast, the solution pKa of histidine is 6. Although in proteins it is not un-common to find histidines with pKa values ap-proaching 7 (Miyagi and Nakazawa, 2008), the pKa of most protein histidines lie in the range of 6–7. Thus, reducing medium pH from 7.0 to 5.5 will result in the protonation of most histi-dine groups, adding greatly to protein positive charge. At pH 5.5, protonation of glutamic acid may also contribute to positive charge in a pro-tein. The solution pKa of glutamic acid is 4.5, but in proteins about 5% of glutamic acids have pKa values greater than 5.5 (Forsyth et al., 2002). At pH 5.5, a majority of these high pKa glutamic acids are in their protonated, noncharged state, which causes a decrease in negative charge, and thus an increase in the net positive charge of a protein.

3.2 Agricultural Proteins and Protein Hydrolysates

Investigations of protein/peptide flocculation were expanded by examining clay flocculation by hydrolysates of blood meal, feather meal, and MBM (Piazza and Garcia, 2010b). As noted pre-viously, protein meals have very low solubility, but solubility can be improved through hydroly-sis. Hydrolysates were prepared using alkaline hydrolysis and hydrolysis using the proteases, Versazyme, Flavourzyme, and Alcalase. Com-mercial peptone and hydrolysates of fish colla-gen and bovine collagen (termed gelatin) were also tested. Different concentrations of the hy-drolysates were tested with and without the addition of calcium chloride. The hydrolysates were also tested at neutral pH and in the pres-ence of pH 5.5 buffer. Flocculation of clay was usually enhanced by high concentration of hy-drolysate and also enhanced by the addition of pH 5.5 buffer. Fig. 8.1 shows the 24 h activity of the hydrolysates on suspensions of clay. The no-flocculant control (CON) (see Fig. 8.1 legend for description of all abbreviations) is shown as the


first entry on the left side of the graphs was set at 100%. Low percent readings correspond to low levels of suspended clay and thus are indicative of flocculation. Fig. 8.1A shows the results with a low flocculant level (0.5 g/L). Ten of eighteen tested materials showed higher flocculation ac-

tivity at pH 5.5 than at pH 7.0. For the remainder of the samples, there was statistically no differ-ence in flocculation at the two pH values. Some of the tested samples, such as FV, PED, HFC, and KRT, showed no sensitivity to pH because the samples lacked significant flocculation ac-tivity. When the flocculant tests were repeated with a high flocculant level (2.5 g/L), many of the hydrolysates showed improved flocculation activity (Fig. 8.1B). The behavior of the gelatins (G125 and G175) was unusual as their flocculant activity at pH 5.5 was lower than the pH 7.0 floc-culant activity. This result seems to contradict the thesis that positively charged materials are better flocculants. However, there is an explana-tion for the behavior of the gelatins that does not invalidate the thesis that positive charge is an important factor in flocculant activity. Concen-tration curves for the gelatins showed that the gelatins exhibited high flocculation activity only over a very narrow concentration range. The optimal concentration for the gelatins at pH 5.5 was lower than the optimal concentration at pH 7.0. At either pH, when the gelatin concentration is increased above the optimal value, the clay particles become coated by the gelatin, which causes the surface of the clay particles to be pre-dominantly positively charged. The positively coated clay particles repulse each other, and their suspension is stabilized. Flocculation was better at pH 7 than at pH 5.5 under the condi-tions shown in Fig. 8.1B only because the gelatin concentration had significantly surpassed the pH 5.5 optimum value, but was closer to the pH 7 optimum value. Thus, the data in Fig. 8.1 sup-ports the thesis that the best flocculant activity is shown at lower pH values where the flocculants tends to have more positive charge.

Over the course of testing many proteinaceous samples, it was apparent that samples having lower MW tended to be poorer flocculants than those with higher MW. To provide additional support for the hypothesis that protein/peptide size is an important factor in flocculation effec-tiveness, MBM was subjected to proteolysis by


FIGURE 8.1 Influence of protein/peptide additives upon flocculation of clay (kaolin) at (A) pH 5.5 () and (B) pH 7.0 (○). The charts show the percent of clay remaining in suspen-sion after 24 h compared to controls which are set at 100% for pH 5.5 and pH 7.0. Graph A, 5 mg (0.5 g/L) additive; Graph B, 25 mg (2.5 g/L) additive. Abbreviations: CON, control (no ad-ditive); BA4, blood meal, 4 h alkaline hydrolysis; BA8, blood meal, 8 h alkaline hydrolysis; BA16, blood meal, 16 h alkaline hydrolysis; FA4, feather meal, 4 h alkaline hydrolysis; FA8, feather meal, 8 h alkaline hydrolysis; FA16, feather meal, 16 h alkaline hydrolysis; MA4, meat and bone meal, 4 h alkaline hydrolysis; MA8, meat and bone meal, 8 h alkaline hydroly-sis; MA16, meat and bone meal, 16 h alkaline hydrolysis; BV, blood meal, 8 h Versazyme hydrolysis; FALC, feather meal, 4 h alcalase hydrolysis; FV, feather meal, 8 h Versazyme hydroly-sis; MV, meat and bone meal, 8 h Versazyme-hydrolysis; PED, peptone enzymatic digest; HFC, hydrolyzed fish collagen; G125, bovine skin gelatin, bloom 125; G175, bovine skin gela-tin, bloom 175; KRT, keratin and hydrolyzed keratin.



trypsin, a selective protease, and by subtilisin, a nonselective protease (Piazza and Garcia, 2015). Samples were withdrawn at progressively longer times, and the hydrolysis reaction was quenched. The MW distributions of the hydro-lysates were analyzed by gel filtration chroma-tography. The hydrolysates were tested for their ability to flocculate clay. As the hydrolysis time increased, the hydrolysates from both enzymatic treatments lost flocculation activity. The loss in activity corresponded to lower average MW of the hydrolysates. Trypsin hydrolysates had rela-tively high clay flocculation activity up to 7 h. In contrast, subtilisin hydrolysates had high kaolin flocculation activity from 1 h or less. Thus, non-selective subtilisin hydrolysis destroyed floccu-lation activity faster than selective trypsin hydro-lysis, and because selective hydrolysis produced higher MW fragments, the results support the notion that high MW materials are better floccu-lants than low MW material. A surprising finding was that at very long hydrolysis times (30–48 h), the subtilisin hydrolysates showed low floccula-tion activity, whereas those from trypsin showed almost no flocculation activity. The subtilisin hydrolysates from the 30–48 h time range were extensively degraded and contained high levels of free amino acids and small peptides. Thus, a reasonable explanation for the weak flocculant activity is that those amino acids and small pep-tides with positive charge were able to neutral-ize the negative clay surface charge by densely binding to the clay particle surface. The larger fragments produced from longer time trypsin hydrolysis could also neutralize some negative clay surface charge, but their larger size preclud-ed sufficiently dense binding on the surface of the clay particles.

3.3 Animal Blood and Blood Fractions as Flocculants

Hydrolyzed fractions of blood meal showed relatively high levels of clay flocculant activity (BA4, BA8, and BA16, Fig. 8.1). This observa-tion inspired further research on the flocculation

activity of blood. Chicken blood, bovine blood (BB), and porcine blood were studied. The more significant observations were combined in a pat-ent application which was subsequently granted (Piazza and Garcia, 2012). Chicken blood was studied first. Flocculation by chicken blood was slightly stimulated by the addition of calcium chloride and was markedly enhanced at lower pH values. Flocculation activity was retained through deliberate heating, spray drying, and freeze drying; although prolonged heating did reduce flocculation activity. The extent of floc-culation with chicken blood was about the same when sulfuric acid, citric acid, or phosphoric acid was used to reduce pH.

An estimate of production costs of a dried chicken blood flocculant was made (Piazza et al., 2011). Analysis showed that the highest cost production step was dehydration of the blood. If the blood was obtained without charge and re-moval of the blood saved the poultry slaughter-house the expense of treating bloody wastewater, the chicken blood flocculant was economically competitive to a polyacrylamide flocculant.

Flocculation using BB and porcine blood were not influenced by the use of different an-ticoagulants (Piazza and Garcia, 2012). Floccu-lation by BB and porcine blood was greatly en-hanced by the addition of pH 5.5 buffer to lower blood pH. The influence of calcium ion and pH on the flocculant activity of blood was entirely consistent with the influence of these factors on gelatin, and protein hydrolysates, that is, little influence by calcium ion and stimulation by low pH. Overall, the experimental observations sug-gested that proteins in blood are major contribu-tors to the flocculant action of blood.

The influences of sodium chloride and pH on clay flocculation by BB and the protein HEM were determined (Piazza et al., 2015). HEM is the most abundant protein in blood, and in the work described in the following section, HEM was found to be a good clay flocculant. Sodium chloride increased clay flocculation by BB and HEM. Sodium chloride shields electrostatic in-teractions between the individual clay particles


allowing easier flocculation. The effects of pH were as expected from prior work, with lower pH values favoring flocculation by BB and HEM. Clay flocculation by HEM and BB were compared to flocculation by pDADMAC, cat-ionic PAM, and APAM. The lowest effective con-centration of BB and HEM was similar to that of cationic and APAM. Approximately one-tenth the amount of the densely charged cationic floc-culant pDADMAC was needed for effective clay flocculation, which demonstrates the overriding importance of flocculant charge in the floccula-tion process.

3.4 Discovery of Blood Flocculant Proteins

Research was conducted that identified spe-cific proteins in BB that act as flocculants (Piazza et al., 2012). Whole blood and serum were frac-tionated using size-exclusion chromatography (SEC). Samples were collected at sequential timed intervals, and these samples were tested in clay flocculation assays. Those SEC fractions showing flocculation activity were fractionated by sodium dodecyl sulfate (SDS)-polyacryl-amide gel electrophoresis (PAGE). Stained PAGE bands were excised, digested with enzyme, and the MW of the peptide fragments were de-termined by matric-assisted laser desorption ionization coupled to time-of-flight analyzers (MALDI TOF) mass spectrometry (MS). The sequence confirmation of some peptides was accomplished by TOF/TOF-tandem MS (MS/MS). The combined spectra were analyzed by the Mascot search engine (Matrix Science, Bos-ton, Massachusetts) using two databases. Com-mercial samples of the identified proteins were tested in clay flocculation assays.

Flocculant assays were done using clay-tested BB, BB plasma, and commercial samples of BB proteins, as identified earlier over a wide concen-tration range. From this data, the required con-centration of flocculant to give suspended clay at or below 1 g/L in 1 and 5 h was determined (Table 8.1). For BB, the required concentration

was 88 mg/L at 1 and 5 h. BB plasma was un-able to satisfy the criteria in 1 h and for 5 h, the required concentration was 150 mg/L. Thus, whole blood is a more effective flocculant than plasma when compared on a dried mass basis.

Commercial samples of putative flocculant pro-teins were obtained. These were assayed for floc-culant activity. The third entry in Table 8.1 is HEM, and its required concentration for clay flocculation was 30 mg/L in 1 and 5 h. Several commercial proteins that were identified in blood plasma also exhibited flocculant activity: α-2-macroglobulin, fibrinogen, and γ-globulin. Flocculant assays were also performed on Cohen fraction III, which con-tains γ + β globulins, and Cohen fraction IV-4, which contains α + β globulins. Cohen fraction III exhibited flocculant activity, whereas Cohen frac-tion IV-4 did not, which strengthens the assign-ment of flocculant activity to γ-globulin.

Blood plasma and serum also exhibited clay flocculant activity. A major protein component of blood plasma is bovine serum albumin (BSA). SEC chromatography of BB plasma showed a low MW fraction, which contained flocculant activity. When analyzed by PAGE, this fraction showed a highly stained band and several ad-jacent weaker bands. The mobility of the highly stained band corresponded to that of authentic BSA, and the presence of BSA was confirmed by MS analysis. However, when a commercial sample BSA was tested, no flocculant activity was observed. Thus there must be an unidenti-fied substance in BB plasma that is the source of plasma flocculant activity.

The last two entries in Table 8.1 are porcine skin gelatin and APAM. They are presented for comparison. Porcine skin gelatin was identi-fied from prior research as having significant flocculation activity (Piazza and Garcia, 2010b). Indeed, gelatin was an effective clay flocculant at 75 mg/L for both 1 and 5 h. APAM showed flocculant activity at 5 h at 29 mg/L, which is only slightly lower than the lowest effective con-centration of hemoglobin. At 1 h, APAM did not meet the sedimentation criteria because PAM’s flocculant action is relatively slow.




4 CONCLUSIONS

Flocculants prepared from animal-processing by-products are renewable and biodegradable. The flocculants vary significantly in activity depending on the starting by-product. To date, animal by-product flocculants with the highest activity are from animal blood, protein compo-nents of animal blood, and gelatin. The mag-nitude of the flocculant activity of animal by-products is equivalent to that of some synthetic organic polymeric electrolytes.


APAM Anionic polyacrylamideBB Bovine bloodBSE Bovine spongiform encephalopathyEDTA Ethylenediaminetetraacetic acidHEM Hemoglobin

MALDI TOF Matrix-assisted laser desorption ionization coupled to time-of-flight analyzer

MBM Meat and bone mealMS Mass spectrometryMS/MS Tandem MSPAGE Polyacrylamide gel electrophoresisPAM PolyacrylamidepDADMAC Poly(diallyldimethylammonium chloride)RBC Red blood cellRPM Rendered protein mealSEC Size-exclusion chromatography

ReferencesBo, X., Gao, B., Peng, N., Wang, Y., Yue, Q., Zhao, Y., 2012.

Effect of dosing sequence and solution pH of floc prop-erties of the compound bioflocculant-aluminum sulfate dual-coagulant in kaolin-humic acid solution treatment. Bioresour. Technol. 113, 89–96.

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TABLE 8.1 The Minimal Concentration Required for Flocculation using Blood Proteins, Gelatin, and APAM (anionic polyacrylamide)

Blood fraction or samplea

1 h 5 h

Required concentration of flocculant (mg/L)b

Bovine blood (BB) 88c 88

BB plasma NSd 150

BB HEM 30 30

BB α-2-macroglobulin 60 30

BB fibrinogen 150 120

Cohen fraction III (BB γ + β globulins) 60 60

Cohen fraction IV-4 (BB α + β globulins) NS NS

BB γ-globulin 60 30

BSAe NS NS

Type A gelatin–porcine skin 75 75

APAMf NS 29

a Tests were performed in 17.8 mM, pH 5.5 MES [2-(morpholino)ethanesulfonic acid] buffer.b Required concentration of flocculants to give suspended clay at or below 1 g/L in 1 and 5 h. All mass values refer to the mass of dry samples.c Entries are generated from data obtained over a wide range of flocculant concentration.d NS, no satisfaction of sedimentation criteria at any tested concentration.e BSA, bovine serum albumin.f APAM was tested in 0.2 mM calcium chloride.












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C H A P T E R


9Pharmaceutical and Cosmetic

Applications of Protein By-ProductsY. Luo, T. Wang

Department of Nutritional Sciences, University of Connecticut, Storrs, CT, United States

1 INTRODUCTION

Proteins are the building blocks of every cell in the human body, and they constitute a major part of the food system to supply energy and nutrition. However, some of the proteins are not considered as nutritious or valuable as others, and some protein fractions must be removed from food products to preserve the quality of the final products. These proteins are generally treated as by-products or wastes in the food in-dustry. It is estimated that by 2050, a world with 2.3 billion more people than today will have a 70% increase in food demand, and the protein supply will be pivotal in feeding the world (Aiking, 2011). As the demands for proteins are recently and dramatically increased in the food industry, plenty of protein by-products are being produced from different sources during food processing, which are primarily used only as animal feed or simply wasted. Although a lot

of efforts have been made to develop new pro-cessing technologies to reduce the generation of protein wastes, how to use these protein by-products and wastes is more challenging and equally, if not more, important to ensure the sus-tainability of the protein supply.

Besides their nutritional value as food prod-ucts, proteins have secondary and tertiary structures that convey special physicochemical properties and therefore have received increas-ing attention for novel applications in many other fields other than the food industry. For example, the gelation property makes certain proteins (eg, soy protein) excellent candidates for the development of moisturizing and wound dressing systems. The self-assembly capability makes some protein molecules (eg, zein) prom-ising biomaterials for nanoscale drug delivery systems. It becomes increasingly important to explore new applications of protein by-products to fully use their properties in different fields.

148 9. PHARMACEUTICAL AND COSMETIC APPLICATIONS OF PROTEIN BY-PRODUCTS


The following chapter details the cosmetic and pharmaceutical applications of four main pro-tein by-products: sericin, whey protein, soy protein, and zein.

2 SERICIN

2.1 Introduction

Silk derived from the silkworm Bombyx mori is a natural protein that is mainly composed of two fractions, that is, fibroin and sericin (Aramwit et al., 2012). Fibroin, accounting for 70–75% of silk protein, is a fibrous protein that presents as a delicate twin thread linked by disulfide bonds. Although sericin only accounts for 25–30% of silk protein, it plays an important role in help-ing the formation of a cocoon by enveloping the fibroin fiber with successive sticky layers. Because of the unique properties, such as water absorbency, luster, dyeing affinity, thermotoler-ance, and insulation (Mondal et al., 2007), fibro-in has been extensively studied for fabrication of silk fibers and widely used in textile appli-cations. To manufacture lustrous silk from the dried cocoons of the silkworm, sericin is often selectively removed from silk protein by a de-gumming process. The removed sericin is con-sidered a waste material in the textile industry and mostly discarded in the wastewater. It is es-timated that of the 1 million tons of fresh cocoon production worldwide, approximately 50,000 tons of sericin is present in the wastewater every year, leading to environmental contamination because of the high oxygen demand for its deg-radation by microbes (Aramwit et al., 2012). In recent years, because many beneficial physical and biological properties are being discovered in silk sericin, its recovery from wastewater has received increasing attention for reuse of sericin as a value-added product. A great number of re-covery methods have been reported to effective-ly recover sericin in the silk degumming waste (Vaithanomsat and Kitpreechavanich, 2008; Chen et al., 2011; Wu et al., 2014), including both

physical (ultrafiltration, filtration, electrolysis, freezing-thawing, and biosorption) and chemi-cal (pI-centrifugation, coagulation, salting out, and denaturation by solvents) methods.

2.2 Structure and Composition of Sericin

Sericin is a highly hydrophilic protein consist-ing of 18 amino acids, among which serine, aspar-tic acid, and glycine are the three most abundant amino acids (Tokutake, 1980). The strong hydro-philicity of sericin is attributed to about 45.8% polar hydroxyl amino acids, especially the serine accounting for 32% in its total amino acids pro-file (Padamwar and Pawar, 2004). The secondary structure of sericin varies as the environmental condition changes. Mostly, sericin is more pres-ent in an amorphous random coil and less in a β-sheet organized structure. But as the repetitive absorption of moisture and mechanical stretch-ing, the random coil structures easily changes to β-sheet structure (Voegeli, 1993). Usually, sericin remains in a partially unfolded state with 63% random coil and 35% β-sheet, without α-helix content (Tsukada and Bertholon, 1981).

2.3 Bioactivity of Sericin

The bioactivities of sericin have been draw-ing attention since the 1990s, when researchers began looking into developing value-added products from sericin waste protein. The an-tioxidant activity of sericin was reported by Kato et al. (1998), who demonstrated, for the first time, the evidence of antioxidant action of sericin against lipid peroxidation and the inhibi-tory effect against tyrosinase activity. Later on, the antioxidant potentials of sericin protein in various assays were reported, including hydro-gen peroxide-induced oxidative stress in skin fibroblasts by lowering the activities of oxida-tive enzymes, such as catalase and lactate de-hydrogenase (Dash et al., 2008) and various free radicals (Fan et al., 2009). The antioxidant activ-ity of sericin also contributed to its anticancer

2 SERICIN 149


effects owing to its capability of reducing oxida-tive stress and suppressing endogenous tumor cytokines in several cancer models, such as skin tumorigenesis (Zhaorigetu et al., 2003b) and colon cancer (Zhaorigetu et al., 2007). Because of the protective effects against lipid peroxida-tion, sericin has been recently tested in alco-hol-induced hepatic injury mouse models (Li et al., 2008). By oral gavage of 0.75 g/kg body weight of sericin solution, sericin was able to significantly increase the ethanol oxidation rate in the liver and simultaneously elevate the ac-tivity of antioxidant enzymes, and thus prevent peroxidative deterioration of structural lipids in membranous organelles. In addition, sericin also demonstrated strong antioxidant activity on ul-traviolet B (UVB)-induced acute damage and tumor promotion in mouse skin (Zhaorigetu et al., 2003a), suggesting its promising potential for cosmetic applications.

2.4 Pharmaceutical and Cosmetic Applications of Sericin

Nowadays, continuous discovery of func-tional and nutritional properties of sericin opens many new applications to use and fully trans-form this waste protein into a number of diver-sified value-added products, especially in the fields of cosmetics and pharmaceuticals where silk-based biomaterials are popular (Altman et al., 2003; Leal-Egana and Scheibel, 2010).

Because the strong hydrophilicity of sericin can help maintain a moist environment and ab-sorb excess exudates from wounds, it becomes a promising wound-dressing agent for both cos-metic and pharmaceutical applications. Sericin-based wound-dressing formulations, such as films, membranes, hydrogel, porous scaffolds, and cream have been reported by various studies (Zhang, 2002). Furthermore, sericin cream–treat-ed wounds of skin excision on the dorsal area of rats were shown to heal faster with lower levels of inflammatory mediators [ie, interleukin (IL)-1β and tumor necrosis factor (TNF)-α released from

monocytes and macrophages] compared with the control (Aramwit et al., 2009). Owing to the abun-dance of hydroxyl, carboxyl, and amino functional groups in sericin, it can be blended and interacted with different biomaterials for more profound wound-dressing applications (Akturk et al., 2011; Kanokpanont et al., 2012). Furthermore, the abil-ity to absorb moisture also makes sericin a good candidate for cosmetic applications, such as a moisturizer (Padamwar et al., 2005), and in anti-aging (Gorouhi and Maibach, 2009) and antiwrin-kle (Sasaki et al., 2000) formulations.

Although silk-based biomaterials have been recently explored in the field of drug delivery for pharmaceutical applications (Numata and Kaplan, 2010; Yucel et al., 2014), such drug delivery systems are mainly fabricated from fibroin be-cause of its capability to form nanoparticles (NPs) by self-assembly. Unlike fibroin, which contains almost identical hydrophobic and hydrophilic domains and is considered as amphiphilic co-polymer, sericin mainly consists of hydrophilic amino acids and thus cannot self-assemble into NPs alone. As a result, sericin does not receive as much as attention in drug delivery as fibroin. Nevertheless, sericin is rich in polar side chains made of hydroxyl, carboxyl, and amino groups and thus it can be easily formulated with other biomaterials by cross-linking, copolymerization, and blending, which offer a new range of fea-tures for pharmaceutical applications, such as drug delivery (Cho et al., 2003) and tissue engi-neering (Mandal and Kundu, 2009a; Kundu and Kundu, 2012). It was reported that sericin was able to self-assemble with poloxamers to form NPs in the range of 100–110 nm (Mandal and Kundu, 2009b). The prepared sericin–poloxamer NPs showed high encapsulation efficiency for both hydrophobic and hydrophilic drugs and enhanced cytotoxicity against macrophage che-motactic factor (MCF)-7 cells compared with free drug. Recently, desolvation technique has been studied to successfully prepare sericin NPs with a diameter of 100–150 nm for gene deliv-ery application (Das et al., 2014). During the



preparation, the cross-linking agent, glutaralde-hyde, was required to maintain the morphology and shape of spherical sericin NPs. The pre-pared sericin NP was further modified by poly l-lysine to become positively charged to encap-sulate DNA for gene delivery. However, because of the use of toxic cross-linkers, this process is not a desired technique for fabrication of sericin NPs for pharmaceutical applications. Develop-ment of safe and nontoxic sericin NPs is yet to be invented.

3 WHEY PROTEIN

3.1 Introduction

Whey is the coproduct during cheese-making and the casein manufacturing process in the dairy industry. Whey protein is the collection of globular proteins isolated from whey. Since the 17th century through the late 20th century, whey was mainly considered a waste material by the dairy industry and disposed without con-sideration of reuse. Since the early 21st century, as the technology of recovery, separation, and purification of whey protein has significantly advanced and the demands for more nutritional ingredients have increased, whey protein has gone from being a waste product to a highly valued product with nutritional and functional properties, especially during the last 25 years (Smithers, 2008). Nowadays, the production

and consumption of dairy products, including milk, cheese, casein, and other products, have been rapidly increasing at the annual rate of up to 4.1% in developed countries (Gerosa and Skoet, 2012), which has resulted in the concomi-tant increase of whey production. Therefore, it is important to explore new applications of whey protein to fully use its nutritional and functional properties.

3.2 Structure and Composition of Whey Protein

Whey protein is generally considered a group of soluble milk proteins that are separated from casein precipitation by adjusting the pH of milk to 4.6. Whey protein is a globular pro-tein and typically consists of β-lactoglobulin (∼65%), α-lactalbumin (∼25%), bovine serum albumin (∼8%), and immunoglobulins, as well as some minor functional constituents, includ-ing lactoferrin, lactoperoxidase enzymes, and glycomacropeptide (de Wit, 1998). The soluble fraction after precipitation of casein from milk also contains lactose and fat. Based on the pro-tein purity, whey protein solid can be catego-rized into several groups: whey powder, whey protein concentrate and isolate, and hydrolyzed whey protein concentrate and isolate, as shown in Table 9.1. Whey protein is well known for its high protein quality score because it contains the complete profile of essential amino acids,

TABLE 9.1 The Composition of Different Whey Protein Products (Gangurde et al., 2011)

Product Protein concentration (%) Lactose (%) Fat (%)

Whey powder 11–14.5 63–75 1–1.5

Whey protein concentrate (WPC) 25–89 (most commonly available at 80%) 4–52 1–9 (as protein concentration increases, fat, lactose and mineral content decreases)

Whey protein isolate (WPI) 90–95 0.5–1 0.5–1

Hydrolyzed whey protein concentrate

>80 (hydrolysis used to cleave peptide bonds and reduce allergic potential)

<8 <10 (varies with protein concentration)

Hydrolyzed whey protein isolate >90 0.5–1 0.5–1

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and is an especially rich source of leucine and cysteine, both of which are of particular inter-est for health benefits (Ha and Zemel, 2003). Because of its high portion of β-lactoglobulin, whey protein can form gels upon certain trigger-ing conditions, including heating and salt addi-tion (Foegeding et al., 2002). By modifying and understanding the unique microstructures and networks of whey protein gels, numerous ap-plications have been recently developed (Ryan et al., 2013).

3.3 Bioactivity of Whey Protein

The bioactivities of whey protein have been well documented for decades, and many novel functionalities are still being discovered, especially for whey protein hydrolysates. Whey protein has been proven to possess a diverse ar-ray of functional properties (Krissansen, 2007), such as promoting bone growth and muscle strength, enhancing immunoregulation, low-ering blood and liver cholesterol, and having antioxidant, antiinflammatory, and anticancer activities. Each component in whey protein offers unique contributions. For example, β-lactoglobulin and α-lactalbumin, the two major components in whey protein, are well-known for their strong antimicrobial and immune-modulating activity in tissues (Krissansen, 2007), while bovine serum albumin, a good source of essential amino acids, appears to inhibit the growth of estrogen-responsive breast cancer (Laursen et al., 1990). Consumption of whey powder or whey protein concentrate is also as-sociated with intake of lactose, which is enzy-matically hydrolyzed to galactooligosaccharide and used by bifidobacteria, thus contributing to gastrointestinal health (Shah, 2000). Further-more, the abundance of branched-chain amino acids, such as leucine and valine, is another ma-jor contribution to the health benefits of whey protein. These branched-chain amino acids from whey protein can stimulate insulin secretion by triggering muscle protein synthesis, which

is sensed by the insulin-signaling pathway and thus reduces postprandial blood glucose (Nilsson et al., 2007). Recently, the bioactivity of whey protein hydrolysates has become an active research area. Different enzyme-treated hydro-lysates have been characterized and evaluated mainly for their antioxidant activities (Athira et al., 2014; O’Keeffe and FitzGerald, 2014).

3.4 Pharmaceutical and Cosmetic Applications of Whey Protein

Research endeavors on the pharmaceutical and clinical efficacy of milk consumption have been ongoing. Many studies showed positive association of milk consumption and the main-tenance of healthy life, which further triggered intensive research into the effects of milk com-ponents on health, where whey protein plays an important role (Luhovyy et al., 2007). By realizing the health benefits of whey protein as a nutritional food, scientists are beginning to explore its new applications in pharmaceu-ticals, such as dietary interventions to pre-vent chronic diseases and the development of drug delivery systems from whey protein formulations.

Dietary intervention of whey protein has been recently found to regulate weight gain and body composition in both animal experi-ments and human clinical trials. By studying the Wistar rats, which were fed a high-fat diet, Belobrajdic et al. (2004) demonstrated that di-etary intervention of whey protein concen-trate significantly reduced energy intake and visceral, subcutaneous, and carcass fat, and more importantly, dramatically reduced plas-ma insulin concentration and increased insulin sensitivity, which all together contributed to less weight gain. A more recent study on the similar Wistar rat model compared the weight-gain regulatory effects between casein and whey protein isolate and concluded that whey protein isolate had the predominant influence, accounting for 70% of the overall effect on body



weight gain (Royle et al., 2008). Another study further proved the effects of whey protein on body weight gain and composition in a double-blind and randomized clinical trial in obese adults (Baer et al., 2011). The study pointed out that the effects of whey protein were more sig-nificant than soy protein supplementation. A recent review on epidemiological studies also indicated that whey protein supplements have desirable effects on metabolic disorders and the musculoskeletal system (Graf et al., 2011). The underlying mechanisms may be the improve-ment of antioxidant defenses and the reduc-tion of lipid and protein oxidation (Haraguchi et al., 2011), however, the specific mechanisms on how whey protein alters body weight and metabolic disease are yet to be investigated.

Another emerging application of whey pro-tein in pharmaceuticals and cosmetics is the de-velopment of drug delivery systems for bioactive compounds. Because of the special rheological properties of whey protein, various environ-mental conditions, such as ionic strength, heat-ing, and pH, can induce the formation of whey protein gels with different microstructures. Al-though the gelation of whey protein has been extensively studied as a tool to modify food tex-ture and as a delivery system for food nutrients, its applications in the pharmaceutical and bio-medical fields is just starting to emerge in recent five years. For example, novel water-insoluble whey protein aerogels with nanostructures were recently prepared by CO2 supercritical drying technique and showed great potential as drug carriers (Betz et al., 2012). Hydrogel particles pre-pared with whey protein and other biopolymers, such as alginate, are also receiving increasing at-tention for their applications in pharmaceuticals. Whey protein–alginate composite hydrogel mic-roparticles prepared by a cold gelation technique have been tested in animal models for oral deliv-ery of insulin (Deat-Laine et al., 2013), showing great future clinical applications for high loading capacity, excellent mucoadhesive properties, and enhanced bioavailability.

4 SOY PROTEIN

4.1 Introduction

Soybean is a species of legume native to East Asia and has a long history as a major protein source in Asia. However, long ago, soybean was not considered suitable for direct human consumption and only used as a cheap protein source for animal feeds. In the United States until the 1920s, soybean was grown only for its industrial by-product. During the past four decades, soybeans became a major source of edible oil, while soy protein was disregard-ed as a by-product from soy oil manufacture (Horan, 1974). As more and more nutritional values of soy protein are being discovered, it has been processed into various food prod-ucts for human consumption, such as bakery products, breakfast cereal, meat food products, and so on (Singh et al., 2008). Nevertheless, the application of soy protein in the cosmetic and pharmaceutical areas is still to be explored so as to fully realize its health benefits.

4.2 Structure and Composition of Soy Protein

Soybean contains approximately 40% protein and 20% oil on an average dry matter base. Soy protein is the major aqueous extract from soy-bean and collected by acidification at pH 4.5–4.8 (Nishinari et al., 2014). After oil removal from soybean, the remaining material is referred to as defatted flakes. There are three forms of soy pro-tein products, depending on the protein content, which varies from 50% to more than 90% (Singh et al., 2008) (Table 9.2): soy flours or grits, soy protein concentrates, and soy protein isolates. Soy flours or grits are the least refined forms of soy protein, prepared by grinding soybean flakes, and can be classified into different groups based on fat content (Table 9.2). Soybean protein concentrates are more refined products with 70% or more protein content and are prepared

4 SOY PROTEIN 153


from defatted flakes or flour by removing the oligosaccharides and some minor components. Soy protein isolates, being the most refined with higher than 90% protein content, are prepared by removing the water-insoluble polysaccha-rides and low-molecular-weight components that are present in concentrates. In general, soy protein represents a mixture of four protein frac-tions categorized by sedimentation coefficients: 2, 7, 11, and 15S, among which 7S (glycinin, MW = 360,000 Da, approximate 60%) and 11S (β-conglycinin, MW = 180,000 Da, approximate 40%) are the two major fractions (Kinsella, 1979; Teng et al., 2009). All the essential amino acids are present in 7 and 11S, and they are an espe-cially good resource of lysine, although relative-ly low in methionine (Song et al., 2011). In addi-tion to being a good source of highly digestible protein, soy protein is also rich in isoflavones (up to 1 g/kg) and many minerals, such as po-tassium, phosphorus, calcium, and magnesium (Singh et al., 2008).

4.3 Bioactivity of Soy Protein

As soy protein contains all nine essential ami-no acids as well as isoflavones, its nutritional properties and health benefits have been widely studied and well documented in recent decades (Slavin, 1991; Friedman and Brandon, 2001; Xiao, 2008). The reported bioactivities and

health benefits of soy protein include lower-ing blood cholesterol, preventing and treating cancers, and alleviating heart disease, osteo-porosis, and menopausal symptoms. Particu-larly, by assessing the recently published work of 22 randomized trials, the American Heart Association has reported that daily consump-tion of soy protein together with isoflavones is associated with beneficial effects on cardio-vascular health (Sacks et al., 2006a, 2006b). In contrast, soy protein supplementation was reported not to confer lipid-lowering effects or cardiovascular benefits in postmenopausal women in a 1-year cohort study (Campbell et al., 2010). Nevertheless, the efforts of ex-ploring the health benefits of soy protein diets have continued to grow (Messina, 2010). For example, diets supplemented with soy pro-tein have been recently studied for their pro-tective effects on neurological damage and focal cerebral ischemia in experimental stroke animal models (Lovekamp-Swan et al., 2007; Cheatwood et al., 2011). Despite the numerous health-promoting attributes of soy products, a recent report revealed that although consum-ers showed some preferences for soy-based food products, they do not seem to recognize soy protein as the link to the health benefits of soy foods (Chang et al., 2012).

Separation of soluble protein hydrolysates from food-processing waste (eg, industrial

TABLE 9.2 Proximate Composition of Commercial Forms of Soybean Protein (Singh et al., 2008)

Product Protein (%) Fat (%) Moisture (%)

Soy flours and grits

Full fat 41.0 20.52 —

High fat 46.0 14.5 6.0

Low fat 52.5 4.0 6.0

Defatted 53.0 0.6 6.0

Lecithinated 51.0 6.5 7.0

Soy protein concentrate 66.2 0.3 6.7

Soy protein isolate 92.8 <0.1 4.7



effluents from a soymeal concentrate plant) is also considered as a novel approach to recover and use valuable components from food wastes. Meanwhile many new technologies are being de-veloped and commercialized (Galanakis, 2012). Membrane technology, especially ultrafiltration, has been extensively studied as a key technol-ogy with a high recovery rate in obtaining soy protein hydrolysates (Moure et al., 2005; Moure et al., 2006). The recovered soy protein fractions, mainly hydrolysates and peptides, have been extensively studied for their enhanced bioactivi-ties, especially antioxidant (Peñta-Ramos and Xiong, 2002) and angiotensin-converting en-zyme inhibitory activities (Chiang et al., 2006).

4.4 Pharmaceutical and Cosmetic Applications of Soy Protein

Because of the notable bioactivities of soy protein, it has been widely studied as a func-tional ingredient or dietary supplement in daily diets to promote health and improve subhealth conditions, as previously discussed. However, the pharmaceutical and cosmetic applications have not attracted much interest until recent years. Because of its major fractions—7S and 11S—soy protein is well-known for its film-forming property during a two-step process (Subirade et al., 1998), that is, a preheating step to unfold protein molecules and a cooling and dehydration step to form disulfide cross-linking and hydrophobic bonds. Although the major applications of soy protein–based films were proposed as edible coating and food packag-ing (Cho et al., 2007), Chen et al. (2008) inves-tigated the potential of soy protein isolate film as a drug delivery system and elucidated the release kinetics of drugs from films in a simu-lated gastrointestinal condition. And in a recent study, soy protein isolate film was investigated as a matrix for wound-dressing applications (Peles and Zilberman, 2012). The study demon-strated that by the appropriate selection of plas-ticizer and cross-linking agent, the film with an

optimal mechanical property was successfully fabricated and exhibited great features for use in burn and ulcer dressings, as well as in cosmetic formulations.

In addition to film-based drug delivery sys-tems, soy protein–based nano- or microparticles have received increasing attention. Because soy protein components exist as globular molecules, with both a hydrophobic core (nonpolar amino acids) and hydrophilic shell (polar amino acids) in an aqueous environment, the fabrication of soy protein spherical particles can be achieved by the formation of aggregates via a two-step method, that is, desolvation followed by a cross-linking process (Lazko et al., 2004; Weber et al., 2000). The application of soy protein particulate sys-tems as carriers for pharmaceutical and nutra-ceutical compounds have been proposed (Teng et al., 2012; Chien et al., 2014). Recently, a novel drug delivery system has been synthesized by conjugating folic acid, a target-specific ligand for cancer cells, onto soy protein NPs to enhance encapsulation capability and delivery specific-ity (Teng et al., 2013b). By using curcumin as a model drug, the study suggested that both the encapsulation efficiency and release profile were significantly improved for folic acid conjugated soy protein NPs, and that the anticancer efficacy was dramatically increased by 93% against Caco-2 cells, compared with unconjugated soy protein NPs. Furthermore, the formation of complex NPs or microspheres using soy protein and other mac-romolecules, including zein (Chen and Subirade, 2009), alginate (Zheng et al., 2007), and chitosan (Teng et al., 2013a) has also been studied. Soy protein matrix incorporating anthocyanins, natural polyphenols modulating skin hyperpig-mentation, for topical applications as skin for-mulations (Plundrich et al., 2013) has also been researched. The incorporation was created by a freeze-drying process, and the prepared matrix was able to effectively inhibit tyrosinase activity, showing potential to inhibit melanin biosyn-thesis and thus slow down the polymerization of brown pigments for skin benefits against

5 ZEIN 155


hyperpigmentation. In addition, isoflavones present in soy protein were also reported to exert beneficial effects in preventing skin aging in post-menopausal women (Daniel et al., 2001).

5 ZEIN

5.1 Introduction

Zein is the major storage protein (accounting for 35–65%) in corn and has been found exclu-sively in the endosperm (Luo and Wang, 2012). Zein was first discovered in 1897 because of its unique solubility in aqueous alcohol solutions (Shukla and Cheryan, 2001). Zein has a negative nitrogen balance; it is deficient in basic and acid-ic amino acids, especially tryptophan and lysine. The poor water solubility and imbalanced amino acid profile make zein a less than ideal protein for human consumption. Originally, the primary production of zein came from the by-products of starch and oil during the wet milling process and zein protein was usually incorporated into animal feed. Zein was considered a waste pro-tein and did not receive much attention until recent years when novel applications are taking advantage of its unique physicochemical and biological properties, especially in the field of pharmaceuticals.

5.2 Structure and Composition of Zein

Zein is comprised of a mixture of various polypeptides and proteins, which are defined by their solubility, charges, and molecular weights. So far, three fractions have been identi-fied as α-, β-, and γ-zein, with α-zein being the major fraction in corn (about 75–85%). The av-erage molecular weight of zein is in the range of 21–25 kDa with a small subfraction of 10 kDa. Regarding the tertiary structures of zein, a distorted cylinder model with 9–10 adjacent and antiparallel repeating helices was first pro-posed by Argos et al. (1982) and then revised by Matsushima et al. (1997), who corrected the

shape of repeating helices to rectangular prism and further elucidated the dimensions of this model (Matsushima et al., 1997). The bottom and top surfaces of this model have a hydro-philic structure while the other four surfaces are highly hydrophobic, explaining the two-dimen-sional aggregation of zein molecules rather than monomers. The molecular structures have been reviewed in detail by Luo and Wang (2014).

5.3 Bioactivity of Zein

Because of the imbalanced profile of amino acids and poor water solubility, the biological activity of zein as a whole protein has been rare-ly reported and only limited to its antioxidant activity. In 1991, Wang et al. first reported that although zein had strong antioxidant activity against methyl linoleate in a powder model sys-tem (Wang et al., 1991), this activity was attrib-uted to the phenol compounds contaminating zein protein, such as tocopherols. This has been further confirmed in a recent study investigat-ing the antioxidant activities of pure zein (>97% protein content) under different conditions (Zhang et al., 2011). The native pure zein had very limited antioxidant activity, however, the antioxidant activities of zein were significant-ly improved after being treated under strong acidic (pH 2.7) or basic (pH 12.5) conditions for 24 h, which may be attributed to the deamida-tion of glutamine to glutamic acid–glutamate. To increase the antioxidant activity of zein and expand its nutritional applications, enzymatic hydrolysis has been performed to produce an-tioxidant hydrolysate from zein. Alcalase and papain were reported to be an effective enzyme to treat zein, and the resultant hydrolysates showed the potent ability to stabilize both wa-ter- and ethanol-soluble free radicals and su-peroxide ions, although these effects varied with molecular weight and hydrophobicity of the hydrolysate (Kong and Xiong, 2006; Tang et al., 2010). The in vitro digestion by pepsin and pancreatin of Alcalase-treated zein hydrolysate



further modified the antioxidant activity that has been linked to promote the health of human digestive tract (Zhu et al., 2008).

The biological activity of papain-treated zein hydrolysate has recently been discovered in vivo for pharmaceutical applications. Zein hy-drolysate strongly simulated glucagon-like pep-tide-1 (GLP-1) secretion in enteroendocrine cells when administrated in the duodenal or jejunal part of small intestine in rats (Hira et al., 2009). Later on, it was reported that through the same mechanism, zein hydrolysate was also shown to decrease plasma dipeptidyl peptidase-IV activ-ity and thus affect glucose tolerance (Mochida et al., 2010). A recent follow-up study by the same research group demonstrated that oral ad-ministration of zein hydrolysate had potent ef-fects on simulating the secretion of both GLP-1 and glucose-dependent insulinotropic polypep-tide (GIP), hence improving glucose tolerance in male normal rats and diabetic Goto-Kakizaki rats (Higuchi et al., 2013). These results together suggested that oral consumption of zein-based hydrolysate has great potential for prevention and treatment of diabetes.

5.4 Pharmaceutical and Cosmetic Applications of Zein

Besides the preceding biological activities, the major pharmaceutical application of zein has been focused on the development of zein-based drug delivery systems. The unique solubility and molecular structure make zein an ideal biomaterial to fabricate particulate delivery systems for both drugs and nutrients. The fabri-cation of zein colloidal particles is considered a liquid–liquid dispersion or antisolvent method (Zhong and Jin, 2009). This method is based on the sudden change of its solubility by the rapid decrease of alcohol concentration, resulting in the self-assembly and solidification of zein par-ticles. The dimension of the particles (usually in the range of 200–1000 nm) is dependent on various parameters, including the concentration

of residual solvent, the initial concentration of zein, and the shear force used to homogenize the dispersion. So far, many research efforts have been conducted to develop zein-based particu-late systems for various applications, especially for the encapsulation and delivery of hydropho-bic bioactive compounds, such as essential oils (Xiao et al., 2011; Wu et al., 2012), fat-soluble vitamins (Luo et al., 2012a), polyphenols (Zou et al., 2012), antimicrobials (Zhang et al., 2010), and anticancer drugs (Lai and Guo, 2011). Re-cently, increasing attention has been drawn to developing oral drug delivery systems from zein for pharmaceutical applications. Howev-er, the delivery systems made from zein alone are not ideal for oral consumption, because the particulate structure will decompose when zein is digested in the gastrointestinal tract. Thus, zein-polysaccharide complex systems are cur-rently being extensively studied. The most stud-ied complex systems are zein-chitosan (Luo et al., 2011; Luo et al., 2012a; Luo et al., 2013b) and zein-alginate (Laelorspoen et al., 2014; Hu and McClements, 2015) complex or core–shell particles. Several recent studies have demon-strated that zein NPs have good biocompat-ibility and low toxicity when tested in various cell lines, and that zein NPs can be internalized into the cytoplasm, indicating their potential for intracellular drug delivery (Xu et al., 2011; Luo et al., 2013a). Nevertheless, to fully explore its pharmaceutical applications, more cellular and animal-based evaluations are needed.

6 SUMMARY

Proteins are a major category of industrial waste. The proteins discussed in this chapter are just some examples from diverse resources of un-derused proteins. For example, how to recover and use the visceral waste proteins from the seafood and meat industries is just beginning to receive more attention. But much more intensive studies need to be conducted in the future. Fortunately,


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the pace of research in this field is continuously accelerating year by year, and the manufacturers in the food, textile, and agricultural industries have begun to show their increasing interest in recovering waste proteins from by-products. The exploration of novel applications of waste-derived proteins in the pharmaceutical and cosmetic areas will have a huge effect on transforming them from an environmental burden into value-added prod-ucts. But the rapid development of this research area will be contingent on advancing technologies of recovering this protein with more efficiency and less hazardous residues.

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C H A P T E R


10Application of Waste-Derived Proteins in the Animal Feed

IndustryM. Wadhwa, M.P.S. Bakshi

Department of Animal Nutrition Guru Angad Dev Veterinary and Animal Science University, Ludhiana, India

A major gap exists between the demand and supply of feedstuffs for feeding to livestock in the developing countries. To bridge the gap be-tween demand and supply, there are only two options: increasing the efficiency of nutrient utilization from existing feed resources or tap new nonconventional feed resources (NCFRs) for feeding to livestock, poultry, and fish. The first option has little scope, as efficiency of nu-trient utilization cannot be increased beyond certain limits. The second option of using NCFR has great potential, especially in developing countries. The wastes, coproducts, and resi-dues—animal organic wastes, poor quality crop residues, single-cell proteins, biofuel coproducts and waste, coproducts from nonconventional oilseeds, and industrial waste—can become NC-FRs with proper processing.

Major constraints in using these residues, wastes, and by-products are short shelf lives

and the fact that they can contain antinutritional factors and may require dehydration (because of high moisture content) and detoxification. There is an urgent need for processing techniques that are economical and practicable.

1 FOOD-PROCESSING INDUSTRY WASTES, COPRODUCTS, AND

RESIDUES

Apricot wastes: After processing apricots (Prunus armeniaca L.), the stones, pits, and seeds containing kernels are discarded as waste, lead-ing to environmental pollution. The apricot kernels are rich in crude protein (CP; 23–31.4%) and ether extract (EE; 45–53.4%) (Hallabo et al., 1977). After oil extraction from apricot ker-nels, the remaining residue (press cake) contains about 50% CP, which, after removal of the bitter

162 10. APPLICATION OF WASTE-DERIVED PROTEINS IN THE ANIMAL FEED INDUSTRY


component (amygdalin, a poisonous glycoside) by distillation can be used as protein source in livestock feed.

Banana leaf waste: Banana leaves contain about 10–17% CP, 4.3% digestible crude protein (DCP) and 43.1% total digestible nutrients (TDNs) (Reddy and Reddy, 1992). Fresh banana foliage supplementation up to 15% in the ration of lac-tating Friesian cows did not alter milk produc-tion (El-Ghani, 1999). Because the moisture con-tent is very high (82–85%), these can be ensiled either after wilting or mixing with straw, stover, or even with dried broiler litter and can be fed to lactating animals without any adverse effect on milk production.

Cashew apple meal (CAM): Sundried CAM, containing 6.5–11.4% CP incorporated in the concentrate mixture at 10% level on an N-basis, was fed to lactating Gir cows for 90 days. The average daily milk production (5.19 vs 5.17 kg) was comparable with the control. The study sug-gested that CAM can be incorporated in concen-trate mixture at 10% level without any adverse influence on milk production (Sundaram, 1986). CAM can be incorporated in broiler or layer ra-tion without affecting their performance.

Passion fruit wastes: The rind contains 11.1–15.5% CP on dry matter (DM) basis (Pruthi, 1960). Chopped and dried rinds of passion fruit mixed with molasses could be used as feed for cattle (Otagaki and Matsumoto, 1958).

Baby corn husk: Fresh baby corn husk con-tained 11.7% CP and 1.8% EE. The fresh or en-siled baby corn husk was highly acceptable and palatable, comparable with the conventional maize fodder (Bakshi and Wadhwa, 2012a).

Bottle gourd pulp: Bottle gourd (Lagenaria sicer-aria) is extensively used as a vegetable. The resi-due after extraction of juice (which has medici-nal properties) is called bottle gourd pulp. It can be conserved by sun drying and then ground for feeding to animals. It is a rich source of protein (24.3%) and can be incorporated up to 50% in the concentrate mixture of adult ruminants (Wadh-wa et al., 2015).

Empty pea pods: After shelling peas, the left-over material is empty pea pods, which contain 19.8% CP and 1.0% EE. These are rich in total soluble sugars (TSS; 35.8%), total phenolics (9.4%), and macro- and microminerals. Pea pods are relished by ruminants, are highly palatable with high nutritive value, and can be fed as a complete feed (Wadhwa et al., 2006).

Sarson saag waste: Sarson saag (an Indian dish) is prepared by steam cooking leaves of Brassica campestris (mustard), Spinacia oleracea (spinach), and Trigonella foenum-graecum (fenugreek) in a 95:4:1 ratio. The chopped leaves, after thorough washing, are steam cooked; the pulp is used for human consumption. The leftover fibrous frac-tion is a waste, called sarson saag waste. It is dis-carded in landfills, posing a threat to the envi-ronment. Sarson saag waste contains 14.5% CP. Adult buffaloes can consume 50–55 kg of fresh sarson saag waste in a day. Sarson saag waste supplemented with a mineral mixture is highly palatable, can serve as an excellent source of nu-trients for ruminants, and can be fed as a com-plete feed (Bakshi et al., 2005).

Snow peas: These are eaten whole with pod as a salad. Frost-affected snow peas are considered unfit for human consumption and therefore dis-carded. Cull snow peas contain 23–25% CP, 1.0% EE, and 35.8% total sugars on DM basis. Adult male buffaloes consume 50–55 kg/day, indicat-ing very high palatability. Fresh or sun-dried cull snow peas can be effectively used in the diet of livestock, without affecting the health of the animals (Bakshi and Wadhwa, 2012b).

Tomato pomace: The mixture of skin, core, and seeds left after the extraction of pulp from the tomato-processing industry is called tomato pom-ace, which contains 19–22% CP and 11–13% EE (Ajila et al., 2012). Tomato pomace can be fed fresh or after sun drying. In multiparous dairy cows (26 kg milk/day), dried tomato pomace could be included up to 32.5% in the concentrate mixture without any adverse effect on health, milk yield, and DMI (Belibasakis, 1990). The sun-dried, ground tomato pomace could replace

1 FOOD-PROCESSING INDUSTRY WASTES, COPRODUCTS, AND RESIDUES 163


the concentrate mixture completely in the diet of male buffaloes without affecting DMI, digestibil-ity of nutrients, urinary purine derivatives, mi-crobial protein synthesis, and volatile fatty acid production in the rumen (Bakshi et al., 2015).

Cabbage, cauliflower, radish, and sugar beet waste: The cauliflower (Brassica oleracea Botrytis) leaves with stem (obtained after removing curd for hu-man consumption), cabbage (Brassica oleracea Cap-itata) leaves, radish leaves, sugar beet (Beta vulgaris L.) leaves contain 17.0, 19.9, 19.4, and 21.9% CP re-spectively. A feeding trial on goat bucks revealed that these vegetable wastes were highly palatable and their nutritive value was comparable to green oat fodder (Wadhwa and Bakshi, 2005).

Pineapple waste: A study was conducted in Philippines on 5000 feedlot beef cattle, raised on the fresh as well as ensiled pine apple wastes containing peel, pomace, and crown. About 90% of waste material and 10% concentrate mixture when fed to growing finishing steers achieved a growth rate of 600–700 g/day.

Egusi seed meal: It is a wild member of the gourd family, looks almost identical to watermelon, but is filled with very dry, bitter flesh. Its seeds are the true delicacy. Seeds contain 50% edible oil and 30% protein. The seeds make an excellent di-etary supplement in many parts of Africa, where farmers lack access to meat or dairy products. Af-ter soaking, fermenting, or boiling, the seeds take on different flavors and are frequently added to thicken soups and stews. And the seeds can also be roasted and ground into a spread like peanut butter. Seeds are also often shelled and eaten as a snack. With further preparation, egusi-seed meal can be pressed into patties to be used like a meat substitute, and its oil can be used for cooking. The egusi can also be an important supplemen-tary baby food, helping prevent malnutrition. Blending the seeds with water and honey pro-duces a milky liquid that can be used for feeding babies if breast milk is unavailable (Akobundu et al., 1982; Davert, 2010). The egusi seed meal can also be safely incorporated in the ration of livestock and poultry.

Spent coffee grounds (SCGs): The residual sludge (75–80% moisture), after extraction of instant coffee is called spent coffee grounds. It is a mixture of coffee pulp and husk constitutes about 40% of whole beans. The chemical com-position of finely ground samples revealed that it contains 31.33% cell solubles, 12.55% CP, and 15.57% EE. But the N-solubility was very low, which can be assigned to denaturation of pro-teins as the coffee beans were roasted at 200oC for 20 min. Likewise the low in vitro pepsin and DM digestibility were possibly caused by the formation of intramolecular linkages during roasting, which prevented or hampered enzy-matic hydrolysis, thereby indicating that SCGs may have limited value as an animal feed (Sikka et al., 1985). A feeding trial on white Yorkshire fattening pigs revealed that SCGs beyond 10% in the isonitrogenous diet depressed both average daily gain (ADG) and feed conversion efficiency (Sikka and Chawla, 1986). Langar et al. (1989) re-vealed that SCGs at 12% level in the urea straw mixture naturally fermented for 12 days, fed ad libitum to the adult male buffaloes, depressed the daily DM intake, digestibility of nutrients and N-retention. It was concluded that it was not suitable as a feed ingredient for animal feeding.

1.1 Soy-Processing Industry Wastes and By-Products

Soy sauce cake: Soy sauce is made from a fermented paste of boiled soybeans, roasted, crushed wheat grains, brine, and Aspergillus ory-zae or Aspergillus sojae, Saccharomyces cerevisiae, and Lactobacillus sp. The fully fermented grain slurry is placed into cloth-lined containers and pressed to separate the solids from the liquid soy sauce. The isolated solids are used as an animal feed, while the liquid soy sauce is pasteurized to eliminate any active yeast and molds remaining in the soy sauce and can be filtered to remove any fine particulates. It usually has a water con-tent of 30%, so it dries well in a drier. Dried soy sauce cake contains 89% DM. Soy sauce cake is



rich in protein (25.1%); fat (21.1%); lipophilic vitamins, such as vitamin E (21.5 mg/100 g) and vitamin K1 (42.1 g/100 g); and isoflavones (Table 10.1). It can be fed in amounts up to 4 kg to lactating dairy cows producing 25 kg milk/day without any adverse effect on milk yield and its quality; while it can be fed at 20–30% in the diet of hogs and 3–7% in the diet of poultry (Anonymous, 2008; Ha et al., 2015).

Tofu waste: By-products from tofu process-ing, sometimes known as soybean-curd lees or tofu-cake, are leftover when tofu (soybean curd) is made from soybeans. The filtrate made from milled and boiled soybean mash, which con-tains protein and fat, is called soymilk, while tofu wastes are the residue. It is a good source of protein, fat, and has very high TDN value for cattle (Table 10.1). Fresh raw tofu wastes (soy-bean curd) deteriorate very quickly, because of the high moisture content (around 80%). There-fore, drying or ensiling is required to avoid de-terioration. Ensiling is the most convenient way of preserving tofu wastes at present. Tofu waste is mixed with either beet pulp and/or rice straw in 50:50 ratio and packed into drum silos. Good silage made from tofu wastes is better than the raw wastes in both digestibility and palatability (Amaha et al., 1996).

Soybean nugget waste: This waste has a very high CP content with very low EE and neutral detergent fiber (NDF) content (Table 10.1). The

in vitro and in sacco DM digestibility was very high (90.39% and 98.04%) (Multani et al., 1990).

Whey (also known as Lactoserum): It is a highly nutritious by-product resulting after the pro-duction of cheese, curd cheese, or casein from milk, which can be used well when fed to ani-mals in a variety of forms, such as liquid whey, condensed whey, dried whey, or as dried whey products. Ruminants can consume up to 30% of their DM intake as liquid whey without im-paired performance (Anderson, 1975; Anderson et al., 1974), while swine may experience diar-rhea when more than 20% of their DM is liquid whey (Modler, 1987). Horvowska and Nierodzik (1975) reported that with a 5% addition of con-densed whey to the broiler feed mixture, car-cass quality was improved and feed costs were reduced by 10%. Kurnick et al. (1955) reported that with 4% of the carbohydrate replaced with dried whey, the whey-containing feed showed significantly better growth, earlier sexual matu-rity, and more eggs from the poultry. Fermented, ammoniated condensed whey is an acceptable liquid protein supplement for ruminants. Small amounts of dried whey or partially delactosed whey in nonruminant rations often increase weight gains, feed efficiency, protein digest-ibility, and fat digestibility, as well as mineral absorption and retention. Including 10% or more dried whey or partially delactosed whey in high-concentrate rations of lactating cows

TABLE 10.1 Nutritional Characteristics of the By-Products and Wastes of the Food-Processing Industry

CP EE CF NDF Ca NFE TDN References

Noodle waste 8.99–10.40 11.31–17.31 2.32 10.58 3.79 73.59 — Multani et al., 1990

Tofu residues 24.88–32.0 10.75–16.69 15.05–26.0 — 4.28 21.03–40.72 86.67–88.39 Inoue et al., 1989; Amaha et al., 1996

Soybean sauce cake

25.1–30.30 8.59–21.1 15.66 — — 28.79 66.06 Anonymous, 1988, 2008; Ha et al., 2015

Soybean nugget waste

50.68 0.69 — 5.33 — — — Multani et al., 1990

SB by-products 41.28 17.44 8.38 4.22 28.66 92.42 Amaha et al., 1996

2 ANIMAL ORGANIC WASTES 165


usually prevents most of the milk fat depression experienced with such rations without reducing concentrate consumption. Rumen butyrate usu-ally is increased when rations contain whey or whey products. Adding whey to grass and le-gume silages improved the silage quality and digestibility, and ammonia nitrogen concentra-tions were reduced in silage when whey was added to urea-treated corn silage. Growth rates have been favorable when calves were fed milk replacers containing up to 89% dried whey.

2 ANIMAL ORGANIC WASTES

The philosophy behind feeding processed animal wastes is based on coprophagy within the same or other animal species always exist-ing in nature. Rabbits, rats, poultry, and pigs are the most typical examples, because in specific nutritional situations, they consume their own excreta in substantial quantities to meet their re-quirements of nutrients missing from their diets. This phenomenon is attributable to the animals’ instinct to search for nutrients created by the en-dogenous synthesis of the enteric microflora.

Poultry excreta (litter and droppings): It is a rich source of Ca and N, used extensively as soil conditioner, and has been exploited for its nutritional worth. The value of poultry lit-ter as a feedstuff has been reported to be more than three times its value as fertilizer (Fontenot and Jurubescu, 1980). The nutrients in poultry excreta can be used to feed a variety of animal species. The CP content in the litter varies from 13% to 31% (Cross, 1995). In poultry droppings (PD), true protein fractions are made up of un-digested dietary protein, endogenous metabolic fecal protein, spilled mash, and products of cae-cal fermentation. About 40–60% of total N is made up of NPN, of which uric acid is the major component (Flachowsky, 1997). The amino acid (AA) profile of PD is equivalent to that of barley and rich source of macro- and microelements. The chemical composition of poultry litter is

governed by the type and thickness of the origi-nal bedding spread in a poultry house, density of birds per unit space, type and age of birds, composition and spillage of feed, level of feed-ing, and the life of the litter (Bakshi and Wad-hwa, 2004). Apparently, the organic matter, CP and crude fiber contents will be highly variable. The nutritive value of rice husk- and sawdust-based litter was assessed on adult male buffa-loes by replacing 0–100% in an isonitrogenous concentrate mixture. The nutrient digestibilities and N balance was comparable with the control up to 50% replacement, but beyond this level, the nutrient utilization was depressed signifi-cantly (Bakshi et al., 1996a).

Poultry litter contain number of pathogens (Clostridium, Corynebacterium, Mycobacterium, Bacillus, Staphylococcus, Streptococcus, Salmonel-la or E.coli), which may have adverse effect on the health of animals (Alexander et al., 1968). Processing (sun drying, solar drying, dry heat-ing, ensiling, and deep stacking) of poultry lit-ter has been shown to eliminate not only most of these pathogens, but has also been shown to improve the nutritive value of excreta. Deep stacking was observed to be the most effec-tive and economical method for the elimina-tion of pathogens (Sandhu et al., 1993; Kaur et al., 1997a, 1997b; Bakshi and Fontenot, 1998; Teli et al., 2002). Pathogens, such as Escherichia coli and Salmonella were completely eliminated within 3 days, without any adverse effect on the in sacco degradability of nutrients but to be on the safer side 28 days period of deep stacking has been recommended (Bakshi et al., 1995). Deep-stacked poultry litter (DSPL) containing concentrate mixtures supplemented with natu-rally fermented wheat straw (WS) gave better results as compared to uromol-impregnated WS (Bakshi et al., 1996b, 1997). Subsequent studies with DSPL at a 40% level in the isonitrogenous concentrate mixture supplemented with FWS did not have any adverse effect on the nutri-ent utilization or growth rate of young buffalo calves (Teli et al., 1999). Similarly, soybean meal



in ewes’ rations could be replaced completely by DSPL on a nitrogen basis without affecting the nutritive value of the rations (Bakshi and Fontenot, 1998). Studies also revealed that sun-dried poultry droppings (DPD) could be used efficiently (30% level, N basis, in the basal mix-ture) by the buffalo calves (Chahil et al., 1998). Incorporation of DPD at a 20% level in the basal mixture did not have any adverse effect on the milk yield of buffaloes (Teotia et al., 1988). These studies showed the potential utility of poultry excreta as source of nitrogen and minerals with quality roughage, such as FWS, in the different categories of ruminants’ rations.

Cattle dung: The low CP, high CF, and low ME contents of ruminant waste limit its utility as a feed ingredient.

Pig manure: Eggum and Christensen (1974) found that about 60% CP in pig manure was available with its biological value of 70%. Ami-no acid analyses indicated that pig manure pro-tein is rich in lysine (5.24% lysine, compared to barley, 3.65% lysine) and other essential AAs (methionine, threonine), which usually limit the feeding ration for pigs. Processed pig waste was found to contain on an average 1600 IU/kg of vi-tamin A (Hennig and Poppe, 1977). Hennig et al. (1972) fed pelleted diets containing 40% dried swine waste to bulls with ADG of 1.1 kg. Fla-chowsky (1975, 1997) fed pelleted diets contain-ing 30% and 50% solid material from semiliquid swine waste to cattle for 252 days. The animals gained 1.18 and 1.0 kg daily on the respective experimental diets as compared to 1.23 kg/day in control group. The sundried swine excreta was observed to be a good source of nutrients, namely CP (17.88%), Ca (1.97%), and phospho-rus (1.08%). It was concluded that sun-dried swine waste can be incorporated in the concen-trate mixture up to 30% on N basis without any adverse effect on the health or nutrient utiliza-tion in adult male buffaloes (Bakshi and Wad-hwa, 2014).

Animal waste may include harmful residues of pesticides; medicinal drugs; trace minerals

and heavy metals; and pathogenic bacteria, parasites, and molds. No chlortetracycline was detected in the kidney, muscle, and liver of any of the 12 steers fed the litter. No residues of ni-carbazin or amprolium were found in any of the tissues of 24 cattle fed litter. The levels of arsenic residues observed by Webb and Fontenot (1975) after a 5-day withdrawal were 0.2 ppm or lower, much below the tolerance level of 1 ppm (FDA).

3 SINGLE-CELL PROTEIN (SCP) PRODUCTION AND UTILIZATION

SCP is also called biomass, bioprotein, or mi-crobial protein and refers to proteins extracted from pure microbial cell cultures. Microbial pro-tein or SCP has various benefits over animal and plant proteins in that its requirement for growth is neither seasonal or climate dependent; it can be produced all year round. It does not require a large expanse of land as in plant or animal protein production. It has high protein content with wide AA spectrum, low fat content, high-er protein-to-carbohydrate ratio than forages, can be grown on waste, and is environmentally friendly as it helps in recycling waste (Adedayo et al., 2011). It is produced using bacteria, fungi, algae, or yeasts. Filamentous fungi (Aspergillus, Fusarium, Rhizopus, and so forth), algae (Spiru-lina, Chlorella, and so forth), many bacterial spe-cies (Bacillus, Lactobacillus, Pseudomonas, and so forth), and yeasts (S. cerevisiae) are extensively used for SCP production (Bhalla et al., 1999). The protein content varies between 50% and 55% (yeast SCP) and 80–82% (bacterial SCP). Algal SCP has limitations, such as the need for warm temperatures and plenty of sunlight in addition to carbon dioxide, and also that the algal cell wall is indigestible. Bacteria are ca-pable of growth on a wide variety of substrates, have a short generation time, and have high protein content. Their use is somewhat limited by poor public acceptance of bacteria as food, small size, and difficulty of harvesting and high

3 SINGLE-CELL PROTEIN (SCP) PRODUCTION AND UTILIZATION 167


content of nucleic acid on dried weight basis. Yeasts are probably the most widely accepted and used microorganism for SCP. The CP con-tent of yeast SCP is comparable with SBM and fish meal; SCP also contains fats; carbohydrates; nucleic acids (Table 10.2); vitamin B-complex, namely thiamine, riboflavin, glutathione, folic acid; phosphorus and potassium; and is rich in lysine, but poor in methionine and cysteine. Bacterial SCP has higher protein, but is poor in sulfur-containing AAs; it has high nucleic acid content (Kurbanoglu, 2001). It has a good poten-tial for a supplemental protein source for feed-ing livestock (Feedipedia, 2015). This could de-velop because microbes can be used to ferment some of the waste materials, such as agricultural by-products; vegetable and fruit wastes; wood and wood-processing wastes; food, cannery, and food-processing wastes; and residues from alcohol production or from human and animal excreta. In nearly all instances where a high rate of production would be achieved, the SCP will be found in rather dilute solutions, usually less than 5% solids. Besides SCP biomass, some or-ganisms also produce other useful by-products, such as vitamins and growth hormones (such as indole acetic acid as a growth promoter) (Dhev-endaran et al., 2004; Jitendra and Ashish, 2012).

Fruit- and vegetable-processing wastes as sub-strate for SCP production: These are useful sub-strates for production of SCP. In addition to solv-ing waste disposal and the associated pollution, the global shortage of protein-rich food and feed is expected to be controlled to some extent. SCP can be produced from dried and pectin-extracted apple pomace by using Trichoderma viride and

Aspergillus niger (Devarajan et al., 2004; Ven-druscolo et al., 2008; Villas-Bôas et al., 2003). Bhalla and Joshi (1994) reported a 200% increase in CP enrichment in apple pomace by using a combination of Candida utilis (Henneberg), syn. Pichia jadinii and A. niger. A fivefold increase in CP content with Rhizopus oligosporus (Albu-querque et al., 2006), 100% increase in CP, and 60% increase in mineral contents with C. utilis (Villas-Boas et al., 2002) have been observed. Pomegranate waste, orange waste, banana waste, and watermelon waste were used as sole carbon sources for preparation of fermentation media on which strains of yeasts—S. cerevisiae—have been used to produce SCP (Uchakalwar and Chandak, 2014). Citrus peel juice has also been used to generate SCP using Fusarium. Po-tato peels supplemented with ammonium chlo-ride have also been used for the production of SCP by using a nontoxic fungi Pleurotus ostrea-tus. Similarly, waste from orange-, sugarcane-, and grape-processing industries have also been used for the production of SCP (Gautam and Guleria, 2007). Papaya-processing waste (PPW) served as substrate for S. cerevisiae growth. The product contained 45% CP. The commercial feed of shrimp could be replaced up to 50% by SCP obtained from PPW. The 50% inclusion of PPW diets were comparable to commercial feed in weight, growth, feed conversion ratio (FCR), and survival rate of shrimp (Kang et al., 2010). Mango-peel extract has been used for SCP pro-duction using Pichia pinus yeast. In this study, a maximum yield of 6.2 g/L was observed at the third day of growth, and cells contained 62.2% CP, 39% true protein, and 12.9% nucleic

TABLE 10.2 Composition of the SCP (% Dry Weight)

Parameter Yeast Bacteria Fungi Algae Reference

Ash 5–9.5 3–7 9–14 8–10 Miller and Litsky, 1976

Protein 45–55 50–65 30–45 40–60

Nucleic acid 6–12 8–12 7–10 3–8

Fat 2–6 1.5–3 2–8 7–20



acids (Rashad et al., 1990). Oboh et al. (2012) re-vealed that S. cerevisiae fermentation of the peels caused a significant increase (P < 0.05) in the protein (4.3–6.7% to 12.8–19.7%), fat (8.4–12.6% to 13.6–14.8%), total phenol content (1.0–2.7 to 3.7–5.4 mg/g), and antioxidant activities. Con-versely, there was a significant (P < 0.05) de-crease in the carbohydrate (65.5–73.8% to 57.2–64.2%), crude fiber (9.6–12.2% to 1.1–3.1%), and phytate (0.31–39 to 0.28–0.37 g/100 g) content. Therefore, cheap, nonpathogenic and aerobic S. cerevisiae could be used to enhance the nutritive value and antioxidant properties of citrus peels; however, fermented shaddock and orange peels show the highest nutritive and antioxidant po-tentials, while grape fruit peel showed the least potential. The fermented peels could be a source of nutrient and nutraceuticals for livestock. Mondal et al. (2012) evaluated cucumber and orange peels for the production of SCP using S. cerevisiae by submerged fermentation. Results revealed that cucumber peel generates higher CP (53.4%) than that of orange (30.5% CP)/100 g of substrate used. The percentage of protein in SCP was much lower (17.47%) when S. cerevi-siae was grown on supplemented fruit hydroly-sate medium that contained inorganic nitrogen sources but was devoid of glucose. Addition of glucose to the supplemented fruit hydrolysate medium enhanced the protein content (60.31%) within the yeast cell. Thus the SCP production by yeast depends on the substrates and media composition. Maragatham and Panneerselvam (2011) used papaya fruit extract (9.6% saccha-ride, 0.2% CP, and 7.0% TSS) as substrate for SCP production using S. cerevisiae. The dry biomass had 34.0% CP, 40.0% saccharide, 0.003% lipids, 9.54% moisture, and 0.14% total ash. Khan et al. (2010) produced SCP from S. cerevisiae by us-ing fruit wastes and observed that banana skin generates the highest amount of CP, followed by that of pomegranate rind, apple waste, mango waste, and sweet orange peel, respectively with 58.62%, 54.28%, 50.86%, 39.98%, and 26.26% CP/100 g of substrate used. Dhanasekaran et al.

(2011) produced SCP from pineapple waste us-ing yeast.

Dried poultry manure (DPM) as substrate for SCP production: SCP was produced from DPM using seven strains of yeast (C. utilis, Candida tropicalis, and three strains of S. cerevisiae, Saccharomyces uvarum, and Rhodotorula). Fermentation of DPM increased the CP content from 19.1% to 24.9%, NPN content decreased from 9.6% to 8.7%, uric acid content decreased from 7.2% to 0.3%, and EE increased from 1.7% to 2.4%. Amino acids of fermented DPM were greatly increased than those of the DPM except for glycine, histidine, and tyrosine. The nutritive value of the DPM was improved when it was used as a substrate for SCP production, and it could be included in amounts up to 9% in broiler diets without adverse effect on the growth performance of broiler chickens up to 4 weeks of age (El-Deek et al., 2009).

Potential constraints: The factors that impair the usefulness of SCP are nondigestible cell wall (mainly algae) and high nucleic acid content (6–10%), which elevates serum uric acid levels and results in kidney stone formation (Nasseri et al., 2011). The levels need to be limited in the diets of monogastric animals. About 70–80% of the total N is represented by AAs whereas the rest occur in nucleic acids. Unacceptable color-ation (mainly with algae), disagreeable flavor (part in algae and yeasts), and cells should be destroyed before consumption.

Nutritional value of SCP: For the assessment of the nutritional value of SCP, factors such as nutri-ent composition, AA profile (Table 10.3), vitamin and nucleic acid content, palatability, allergies, and gastrointestinal effects should be taken into consideration (Lichtfield, 1968). Also long-term feeding trials should be undertaken for toxico-logical effects and carcinogenesis (Israelidis). Nutritive values of SCP vary with the microor-ganisms used and the method of harvesting, dry-ing, and processing (Mahajan and Dua, 1995).

Ruminants: The AA profile of many SCPs is favorable, is similar to that of fish meal

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TABLE 10.3 Comparative Amino Acid Composition of SCP (g AA/ 100 g Protein) By Using Different Substrates and Microorganisms

Fungi Bacteria yeast Algae

SPI–HEssential AAPleurotus florida

Paecilomyces variotii

Lactobacillus acidophilus 3205

Cellulomonas sp.

Kluyveromyces fragilis Candida utilis Spirulina

CP% 62.8 57–63 — — 45–56.2 49.8 — —

Threonine 0.6 4.6 4.8 4.37 5.8 3.4 6.2 3.97

Valine 6.6 5.1 6.4 6.79 5.4 4.05 7.1 4.31

Cysteine — 1.1 — — — 2.4 1.23

Methionine 2.1 1.5 2.1 1.69 1.9 1.55 2.5 1.34

Isoleucine 7.3 4.3 6.4 4.2 4.0 3.2 6.7 4.39

Leucine 6.8 6.9 8.2 8.66 6.1 4.4 9.8 7.91

Phenylalanine 4.3 3.7 4.2 3.69 2.8 3.0 5.9 4.99

Histidine 19.8 — 2.5 2.96 — 1.6 2.2 2.57

Lysine 9.5 6.4 10.3 8.0 6.9 7.6 4.8 6.16

Arginine 8.3 — 4.9 6.18 — 3.8 7.3 7.41

Tryptophan — 1.2 — — 1.4 — 0.3 1.26

Substrate Wheat straw pretreated with 2% NaOH at 100°C

Spent sulfite liquor

— Rice straw Cheese whey Bagasse pith pretreated with NaOH at room temp.

— —

Reference Ahmadi et al., 2010

Erdman et al., 1977

Han, 1974 Kurbanoglu, 2001

Becker and Venketaraman, 1982

Ishihara et al., 2003

SPI-H, Soy protein isolate hydrolysate.



(Nitayavardhana et al., 2013; Alriksson et al., 2014), and seems to be better used by ru-minant animals. Feeding SCP up to 20% of the diet to calves or up to 15% in the diet of lambs or 75% of dietary protein in lambs did not affect daily gain and dressing percentage. Milk pro-duction and milk production efficiency was in-creased when SCP replaced groundnut meal in lactating goat diets. SCP was able to replace up to 55% of the fish meal and SBM in swine diets, while still maintaining satisfactory performance (Feedipedia, 2015).

Broilers: Machalek et al. (1988) reported that the replacement of 5% and 10% SBM in the fin-isher broiler diets with a similar amount of yeast protein concentrate Vitex (48% CP) increased growth while FCR, dressing percentage, and carcass quality were similar. Samanta and Mon-da (1990) indicated that growth, feed intake (FI), FCR, and carcass dressing percentage were not different among broiler chickens (0–6 weeks) fed diets containing fish meal replaced by 0–75% yeast. Onifade et al. (1998) reported that feeding yeast to chicks improved body weight gain (BWG) and FCR. Also, Kanat and Calialar (1996) reported that active dry yeast effectively increases BWG without affecting FCR of broiler chicks. Supplementation of yeast improved FCR but not growth rate, and heart weights were not influenced. Moreover, Spring (2002) and Santin et al. (2003) revealed that yeast can im-prove the immune status of birds and reduce the toxic effects of aflatoxin. Gao et al. (2008) indi-cate that dietary supplemental yeast at 2.5 g/kg improved growth performance. Dietary yeast affected immune functions, digestibility of Ca and phosphorus, and intestinal mucosal morphology of broilers. Shahzad and Rajoka (2011) replaced SBM with biomass produced by Aspergillus terreus at 30% and 60% on protein basis and the bird’s response of weight gain, feed consumption, feed efficiency, FCR, pro-tein efficiency ratio, and net protein utilization were not affected adversely up to 30% level in growing broiler chicks. Bananas contain 0.95%

CP, but when subjected to microbial (Aspergillus niger or Aspergillus foetidus) fermentation, the CP level increased to 21.5%, and up to 20% could be used in broiler diets. The best growth in poultry occurred at the 5% and 10% levels. As the di-etary level of SCP increased in broiler diets the gain, feed conversion, and FI decreased (Jassim et al., 1986; Pirmohammadi et al., 1999).

Layers: Tollba and El-Nagar (2008) indicated that supplementing the laying diet with live yeast ingredients improved BWG, mortality rate, FI, and FCR. Farhoomand and Dadvend (2007) demonstrated that S. cerevisiae supple-mented at 100 g/kg diet significantly improved FCR, decreased abdominal fat, intestine length, and increased BWG, dressing yield, and liver and spleen weights compared to control diet or the other levels.

Najib et al. (2014) supplemented the diets of laying hens with 0–15% of SCP yeast. The SCP yeast contained 48% CP (1.2% lysine), 3380 kcal true ME/kg, 6.41% EE (43.2% oleic acid). Perfor-mance of the hens in terms of hen-day egg pro-duction, egg mass, egg weight, and feed conver-sion was improved significantly (P < 0.05) when 5% yeast was included in the ration. However, there was a clear indication that addition of 15% SCP may be harmful to the birds. It was con-cluded that adding 5% SCP, produced from date waste to the poultry diet, produced no adverse effect to the performance of the birds and may be included in their diet. Layer performance was optimized when they were fed at 2.5% of DM or 10% of dietary protein.

Aquaculture production: Yeast SCPs with ex-cellent nutrient profiles and capacity to be mass produced economically have been added to aquaculture diets as partial replacement for fish meal (Olvera-Novoa et al., 2002; Li and Gatlin, 2003). Some yeast strains with probi-otic properties, such as S. cerevisiae (Oliva-Teles and Goncalves, 2001) and Debaryomyces hansenii (Tovar et al., 2002), boost larval survival either by colonizing the gut of fish larvae, thus trigger-ing the early maturation of the pancreas, or via

4 POOR-QUALITY CROP RESIDUES (PQCRS): PROCESSING AND USE 171


the immune-stimulating glucans derived from the yeast cell wall (Campa-Córdova et al., 2002; Burgents et al., 2004). However, many of SCP supplements are deficient in sulfur-containing AAs, particularly methionine (Oliva-Teles and Goncalves, 2001), which restricts their extensive use as the sole protein source.

There are various feed formulations that re-place fish meal with yeast SCP and give up to a 75% positive growth response: P. variotii fed to Lepidocephalus thermalis (Patil and Jadhav, 2014; Davies and Wareham, 1988); Oregon Moist Pel-let fed to Oncorhynchus kisutch and Salmo gaird-neri (Mahnken et al., 1980); juvenile Nile tilapia (Al-Hafedh and Alam, 2013); Puntius vitatus (Anithakumari, 2000); Xiphophorus maculates (Mithun, 2005); Barbus schwancfeldi (Jagadam-biga, 2005); Carassius auratus, Cyprinus carpia, Helostoma temminckii, Catla, and Pecilia sphenops fed on SCP diets (Dhevendaran et al., 2013). Per-era (1995, cited in Feedipedia, 2015) reported reduced gains in trout fed SCP. When SCP was included in prawn diets, there was an increase in gains and feed conversion (Manju and Dhev-endaran, 1997).

4 POOR-QUALITY CROP RESIDUES (PQCRS): PROCESSING AND USE

The conventional cereals, millet straws, and stovers are deficient in most nutrients [3.5–4.5% CP; 7.5–8.5% acid detergent lignin (ADL)], but still constitute the bulk of dry matter consumed by ruminants under field conditions in most of the developing countries. These simply serve as rumen fillers in low-milk-producing animals. The nutritional worth of these PQCRs can be improved considerably by natural fermentation with urea or by inoculation with pure lignino-lytic fungi.

Naturally fermented straw: In the Northern states of India, especially Haryana, Punjab, and Eastern UP, more than 80% of the rice straw is burnt, causing environmental pollution and

various health problems in humans. Natural fermentation of such crop residues with urea will help in using rice and other straws effec-tively in ruminants. Wheat or rice straw and urea (96.5:3.5) moistened to 40% stacked for 9 days (FWS) could meet the basal energy and protein requirements for maintenance of adult ruminants and partly the production require-ments of 6–9 month-old buffalo calves (Bakshi et al., 1986, 1987; Bakshi and Langar, 1994a; Kaur et al., 2007, 2008; Wadhwa et al., 2010a). The natural fermentation technology involves a combination of treatments, namely, using chaffed straw (physical), release of ammonia by the hydrolysis of urea, and its utilization by the microbes for their proliferation, thereby enrich-ing the straw with microbial protein (biological). An increase in stack temperature to 55°C (physi-cal) facilitates the penetration of NH3 into cell wall and breaks the alkali labile lignocellulose bond (chemical) (Bakshi and Wadhwa, 1999). The CP content increased from 3.5% to 6.5% to 7.5%. A 396-day lactation trial revealed that ad lib feeding of FWS with low protein concentrate resulted in higher milk production without any adverse effect on the quality of milk and con-ception rate in buffaloes. It was concluded that production can be economized (60–65 paise/kg milk) by feeding FWS and about 60–70% of the oilseed cakes can be spared for feeding to nonruminants (Lamba et al., 2002). The NPN-rich nonconventional feedstuffs, such as DSPL or uromol bran, could be incorporated in the concentrate mixture and fed along with FWS without any adverse effect on the nutrient uti-lization, health, or productive performance of buffalo calves (Bakshi et al., 1996a). The process has universal application on all cereal straws, stovers, and millet straws and stalks (Bakshi and Langar, 1994a). Natural fermentation eliminates pathogenic microorganisms. The technology is highly applicable under field conditions.

Microbial treatment: Fourteen strains of lig-ninolytic fungi were tested for upgrading the nutritive value of straws with minimum



pretreatment without exogenous nutrients, ex-cept Coprinus cinereus, which required N and phosphorus exogenously (Wadhwa and Bak-shi, 1997). Heterobasidion annosum (Bakshi et al., 2001), Phellinus linteus (Gangwar et al., 2003), Phialophora hoffmannii (Lamba et al., 2003), and Cyathus stercoreus (Bakshi et al., 2011) or a blend of Pycnoporus sanguineus–Oideodendron echinula (Wadhwa et al., 2008) were observed to be the most effective. The increase in in sacco DM and CP degradability up to 5–10 days was associ-ated with linear increase in DM loss in all the cases. To achieve the optimum degradability with minimum nutrient losses, the fermentation should be stopped within 6–8 days. Shrivas-tava et al. (2014) fermented WS with Crinipellis sp. RCK-1 for 5 days. The fermented straw was enriched with essential AAs and fungal protein and had 36.74% OM digestibility and 5.38 MJ ME/kg DM. The calves fed on fermented straw-based diets exhibited significantly higher DM intake and digestibility and TDNs and had higher ADG.

Uromol-impregnated wheat straw (UmIWS): This contains a urea and molasses mixture (urea-to-molasses ratio, 3.5:10.5) cooked for 30 min at 100° ± 10°C, diluted with 40 L of water, sprinkled on 96.5 kg WS, mixed and sun dried. The CP content in WS and UmIWS was 3.2% and 10.5% respectively. The UmIWS supple-mented with minerals and vitamin A fed ad lib could meet the basal maintenance requirement of adult ruminants (Bakshi and Langar, 1994b; Wadhawan et al., 1995). The technology is also applicable under field conditions. Use of molas-ses and cooking for 30 min adds to the cost.

Nonconventional straws: Straws of fodder crops after thrashing seeds are either burnt or ploughed in the field, but keeping in view the current shortage of feedstuffs, these were evalu-ated as potential livestock feed. Straws of four different fodder crops, namely, Trifolium resupi-natum (shaftal), Trifolium alexandrium (berseem), Medicago sativa (alfalfa), and Lolium perenne (rye grass) were evaluated on 16 male buffaloes for

their nutritional worth. The CP and NDF con-tent varied between 6.4% and 8.7% and 65% to 81%, respectively. The digestibility of nutrients was almost same in T. resupinatum and L. pe-renne, but significantly higher as compared to other straws. The efficiency of microbial protein synthesis indicated that organic matter of straws of M. sativa and T. alexandrium was used more efficiently for synthesis of microbial protein. The values for N retention and apparent biological value were highest for L. perenne, although com-parable with that of M. sativa and T. alexandrium. It was concluded that all the straws could be fed exclusively to adult ruminants without any deleterious effect on the health of the animals (Kaushal et al., 2006). These straws had much better nutritive value than conventional straws, stovers, or stalks such as that of wheat, rice, maize, pearl millet, and so forth.

5 BIOFUEL COPRODUCT–WASTE USE

Dried distillers’ grain with solubles (DDGS): In the biofuel industry, starch is extracted from ce-reals by fermentation to produce bioethanol, and DDGS is a coproduct. Distillers’ grain (DG) raw materials used for distillation include maize, wheat, rice, tapioca, and sweet potato. The nu-tritional characteristics depend on the raw ma-terials used (Table 10.4). Distillers’ grain has a relatively high CP content. DDGS contains 27–35% protein, but low energy content and is used in livestock, poultry, and fish feeds. It is cheaper than maize and SBM, which are primarily sub-stituted by DDGS in animal diets (Hoffman and Baker, 2011). The amount of DDGS that can be introduced into animal rations depends on the nutrient composition of the individual ingredi-ent and unique limiting factors for the various species being fed. Arora et al. (2008) found that 1 kg of DDGS can displace 1.2 kg of maize in a typical beef ration. Hoffman and Baker (2011) found that in aggregate (including major types

5 BIOFUEL COPRODUCT–WASTE USE 173


of livestock or poultry), a metric ton of DDGS can replace, on average, 1.22 metric tons of feed consisting of maize and SBM. In general, studies show that DDGS can account for approximately 30–40% in growing beef cattle rations and up to 40% in finishing beef cattle, up to 30% for lactat-ing dairy cows; up to 20% of growing-finishing lamb diets and 25% of lactating ewe diets will provide good performance results. Some stud-ies have shown that DDGS can be added at lev-els up to 50% of the ration of growing finishing lambs without affecting growth performance or carcass quality and can be supplemented up to 20% for farrow-to-finish hogs (Vander Pol et al., 2006; Anonymous, 2012). Conserva-tively, DDGS can be added at 5–8% of starter diets for broilers and turkeys and 12–15% of diets for layers and growing-finishing diets for broilers, ducks, and turkeys when diets are not formulated on a digestible AA basis. The DDGS diet achieves excellent performance and egg and meat quality. Recent research studies have shown that DDGS can be added to poultry di-ets at 25% for layers and broilers to achieve ex-cellent performance, and egg and meat quality

provided that accurate nutrient profiles specif-ic to the DDGS source are used, and diets are formulated on a digestible AA basis. Adding 10% DDGS to all aquaculture feeds can result in excellent performance (Anonymous, 2012). For normal inclusion levels of DDGS in animal diets, the limiting essential AAs are lysine and tryptophan for maize DDGS and lysine and threonine for wheat DDGS.

If DDGS is fed to cattle, the sulfur content should be determined and considered along with the feeding level and sulfur contributions from other dietary ingredients to ensure that total dietary sulfur content does not exceed 0.40%. The sulfur content of DDGS may also contribute to an increased animal risk of mul-berry heart disease, which is a vitamin E or selenium deficiency or a combination of both. High dietary sulfur and/or sulfate (Halvorson et al., 1962) or cysteine (Lowry and Baker, 1989) concentrations can be antagonistic to the uti-lization and bioavailability of high levels of selenium, in the form of selenate or selenite (Halvorson and Monty, 1960; Ardüser et al., 1985) and vitamin E (dl-α-tocopheryl acetate)

TABLE 10.4 Nutritional Characteristics of the By-Products and Wastes from the Biofuel Industry

CP EE CF Ca NFE TDN References

DDGS

Maize 25.0–32.0 8.8–12.4 5.4–10.4

0.17–0.26 — 85.0–90.0 Stein et al. (2005, 2006)

Wheat 38.48–40.67 4.63 6.0 0.10 — — Lan et al. (2008)

Rice 32.76 2.73 11.11 1.21 52.25 — Ha et al. (2015)

Barley hulled 17.7 2.5 5.7 — — — Anonymous (2012)

Oats 16.0 6.3 2.0 — — — Anonymous (2012)

Sorghum 31.5–32.7 8.0 34.8* — — — Urriola et al. (2007)

Sweet potato 20.46 6.97 27.35 5.09 40.13 60.73 Ha et al. (2015)

Tapioca 12.16 4.63 39.36 12.80 31.05 44.48 Ha et al. (2015)

Spent brewer’s grains

18.75–29.03 6.95 22.0–22.94 0.26–6.45 34.77–49.52 54.6–71.14 Ha et al. (2015); Virk and Chopra (1979)

* NDF.



(Boyazoglu et al., 1967). In addition to sulfur, it will increase N and P excretion, which may have a negative effect on animal health. There-fore, while formulating diets with DDGS, sul-fur level must be considered.

Like other nutrients, the AA composi-tion of DDGS varies with the substrate used (Table 10.5). The standardized ileal digestibility of lysine is the most variable compared with all other essential AAs (Fastinger and Mahan, 2006;

Stein et al., 2006; Pahm et al., 2008). The greater variation in lysine digestibility compared with the digestibility of other AAs is because lysine is the most sensitive AA to heat damage and the extent of heat damage varies among DDGS sources (Cromwell et al., 1993; Stein et al., 2006). Most AAs in DDGS have a digestibility that is approximately 10% units less compared to corn, which may be a result of the greater concen-tration of dietary fiber in DDGS compared to

TABLE 10.5 Concentration and Standardized Ileal Digestibility (SID) of Crude Protein (CP) and Amino Acids (AA) in Distillers’ Dried Grains with Solubles (DDGS) Fed to Growing Pigs (Stein and Shurson, 2009)

Concentration of CP and AA (%) SID of CP and AA (%)

ItemCorn DDGS

Sorghum DDGS

Wheat DDGS

Corn DDGS

Sorghum DDGS

Wheat DDGS

CP 27.27 31.50 40.67 72.8 71.4 72.2

INDISPENSABLE AA

Arginine 1.16 1.06 1.53 81.2 78.2 85.5

Histidine 0.72 0.68 0.92 77.5 70.6 77.4

Isoleucine 1.00 1.31 1.35 75.2 72.7 79.6

Leucine 3.12 4.02 2.66 83.7 76.3 82.9

Lysine 0.78 0.66 0.65 62.0 62.0 56.6

Methionine 0.55 0.51 0.53 82.0 75.4 81.1

Phenylalanine 1.32 1.62 1.92 81.0 75.8 86.3

Threonine 1.06 1.03 1.21 70.8 68.6 74.9

Tryptophan 0.21 0.34 0.40 70.2 70.4 85.7

Valine 1.34 1.59 1.70 74.5 72.3 81.9

DISPENSABLE AA

Alanine 1.90 2.79 1.48 78.0 73.4 68.0

Aspartic acid 1.82 2.09 1.92 68.6 68.0 56.7

Cystine 0.53 0.47 0.73 73.3 65.6 75.2

Glutamic acid 4.28 6.08 9.81 80.4 75.5 86.3

Glycine 1.02 0.99 1.62 63.4 66.9 67.8

Prolamine 2.06 2.41 4.11 74.3 83.1 81.0

Serine 1.16 1.35 1.88 75.6 72.5 77.0

Tyrosine 1.01 — — 80.9 — —

Corn DDGS data (34 sources) from Stein et al. (2005, 2006), Urriola et al. (2007), Pahm et al. (2008); Sorghum DDGS data (1 source) from Urriola et al. (2007); Wheat DDGS data (2 sources) from Widyaratne and Zijlstra (2007); hulled Lan et al. (2008).

5 BIOFUEL COPRODUCT–WASTE USE 175


corn. However, except for lysine, variability in AA digestibility among DDGS sources is within the normal range of variation observed in other feed ingredients. Sources of DDGS that have a low lysine digestibility often have low lysine content. As a result, the ratio of lysine to CP pro-vides an estimate of the relative lysine digest-ibility among DDGS sources (Stein, 2007). Ami-no acid digestibility in sorghum DDGS and in wheat DDGS is similar to the values measured in corn DDGS (Urriola et al., 2007; Widyaratne and Zijlstra, 2007; Lan et al., 2008).

The shelf life of wet DG is very small and challenging to store on the farm because it spoils rapidly. Fresh DG should be used within a few days of arrival or may be stored anaerobically or ensiled, either alone or in combination with other feedstuffs. Wet DG is an effective source of protein and energy and performance of feed-lot cattle was improved. But the effects depend on level of DG, rumen degradable protein, NDF, and fat content in the DG. Despite added fi-ber from DG, dietary roughage should not be eliminated, and for optimal cattle performance the concentration and source of roughage need to be considered in wet DG diets. Feeding wet DG at concentrations greater than 10–15% of the dietary DM might increase urinary N excretion and ammonia and nitrous oxide emissions.

Spent brewer’s grains (SBGs): This high-moisture by-product of breweries is quite rich in protein (27–33% on a DM basis). Fresh SBGs are highly palatable and relished by animals. SBGs are relatively rich in rumen undegradable protein because of the heat applied during the brewing process, but it can be detrimental for monogastric animals. Therefore, it is often used in high-yielding dairy cows. The effective nitro-gen degradability of SBGs reported in feed tables and in the scientific literature is about 41–49% (Batajoo and Shaver, 1998; Sauvant et al., 2004; Nishiguchi et al., 2005; Promkot et al., 2007; Volden, 2011), much lower than those of SBM and cereal by-products (Sauvant et al., 2004; Nishiguchi et al., 2005; Volden, 2011). Nitrogen

degradability depends on the amount of heat used during the drying process. In one study, the amount of bypass protein doubled when the temperature increased from 50 to 135°C (Pereira et al., 1998). Heating also decreased the protein solubility while increasing the insoluble ADF-bound nitrogen fraction (Enishi et al., 2005). Values for the intestinal digestibility of nitrogen range from 74% (Yue et al., 2007) to 84% (Sauvant et al., 2004), which is much lower than the values reported by these authors for SBM, corn gluten meal (CGM), and maize DG (more than 90%). As usual for cereal grains and their by-products, lysine is the first limiting AA in SBGs used for high-yielding ruminants, so it needs to be blend-ed with sources of bypass protein rich in lysine. Virk and Chopra (1979) reported that the fresh SBGs contain 14.6% DCP and 54.6% TDN for buffaloes. It is usually recommended to include SBGs up to 20–25% of the concentrate DM, and up to 15–20% of the total dietary DM. Howev-er, up to 30% has also been recommended, and it has been shown that this rate does not affect milk production in dairy cattle (Ewing, 1997). No difference in performance was observed when dried, rewetted, or wet SBGs were fed to lactating dairy cattle (Porter et al., 1977). Dried SBGs were found to successfully replace part of the forage in the diet (Younker et al., 1998). How-ever, replacing SBM with wet or dried brewer’s grains in lactating dairy cattle was detrimental to performance (Hoffman and Armentano, 1988). SBGs silage could be included at up to 15% in dairy diets with no effect on nutrient intake, pro-duction and quality of milk (Geron et al., 2010). SBGs silage was found to be a satisfactory re-placement for maize silage in rations for lactat-ing dairy cattle (Münger and Jans, 1997). Inclu-sion rates as high as 40% have been considered acceptable (Ewing, 1997). Up to 24% DM SBGs in male growing cattle did not alter rumen fer-mentation (pH, efficiency of microbial protein synthesis, and NH3) and OM digestibility (Geron et al., 2008). In a trial with finishing beef cattle fed with 0.5–1 kg or 1–2 kg of ensiled brewer’s



grains, intake and growth performance were low-er in the second group, while the performance of animals in the first group was similar to those fed a control diet based on maize silage. Using a pH stabilizer in cattle fed 1 to 2 kg of ensiled brewer’s grains resulted in similar performance and carcass quality as in the control group (Mo-rel and Lehmann, 1997). SBGs were found to be a satisfactory replacement for CGM in rations for growing heifers (Lopez-Guisa and Satter, 1991). Virk et al. (1981) conducted a 120-day growth trial on growing buffalo calves and reported that replacing 50% of the concentrate mixture with sun-dried SBGs nitrogen basis does not show any adverse effect on ADG of buffalo calves (512 vs 532 g/day with leguminous fodder and 623 vs 632 g/day with nonleguminous fodder). Fur-ther studies on lactating buffaloes on the same feeding regimen for 150 days did not show any adverse effect on milk yield (7.5 vs 7.6 kg/day) and its composition (Virk et al., 1981).

6 COPRODUCTS FROM NONCONVENTIONAL OILSEEDS

AND THEIR USE

To meet the ever-increasing protein demand for feeding different categories of animals, nonconventional oilseeds, such as neem, cas-tor, karanj, and Jatropha, are being explored for their nutritional worth for animals. Their chemi-cal composition (Table 10.6), methods of detoxi-fication, and level of incorporation are discussed briefly.

Maize germ meal or maize oil cake: It is a by-product obtained after extraction of maize oil from full-fat maize germ, a coproduct of the starch industry. It is a good source of protein Like other maize by-products, maize germ meal tends to be poor in lysine (about 4% of the pro-tein), although richer than maize grain (3%). Maize germ meal is particularly rich in phos-phorus, as the germ contains much of the phos-phorus in the grain (Widmer et al., 2007).

Ruminants: Maize germ meal included at up to 45% in the diet does not show any adverse effect on nutrient digestibility (Watanabe et al., 2014), and it increased body and carcass weights, with-out affecting carcass yields of Nelore cross–Lim-ousin bulls 320 kg (Miotto et al., 2009). Complete dietary replacement of maize by maize germ meal did not have any effect on the carcass com-position, except that Longissimus dorsi muscle (meat) was enriched with conjugated linoleic acid and transvaccenic acid (7.69%) with a low n-6:n-3 ratio in heifers (Kazama et al., 2008) and lambs (Urbano et al., 2014).

Poultry: The apparent metabolizable energy (AME) value for maize germ meal (protein 11%, fat 9% as fed) for broilers was about 15.9 MJ/kg DM (Brito et al., 2005c). The optimum dietary inclusion rate of maize germ meal was 21–22% until the birds were 38 days of age, and it then replaced all of the maize in the final phase (Ew-ing, 1997; Brito et al., 2005a). Similar optimal in-clusion rates were observed when maize germ meal replaced sorghum (Stringhini et al., 2009). The inclusion of 50% maize germ meal (protein 12%, fat 10% DM) in layer diets (30–60 weeks) did not affect laying performance and quality of both eggs and shells (Brito et al., 2005b). For hens that have moulted, in the second egg production cycle the recommended inclusion rate was 16%, replacing 25% maize (Brito et al., 2009).

Palm kernel cake (PKC): It is produced after the extraction of oil from the kernels of the palm fruits. PKC is also known as palm kernel meal (PKM) or palm kernel expeller (PKE). Two types of oil extraction process are employed: screw press (expeller) or solvent extraction. The CP content is considered to be more than sufficient to meet the requirement of most ruminants. PKC has a good AA profile, with availability between 62% and 87% (Yeong et al., 1981). Limiting AAs are lysine, methionine, and tryptophan. PKC also contains high residual fat (about 10%), car-otene, and vitamin E (about 0.3 IU/kg), which can act as a natural antioxidant. Recommended levels of PKC feeding are 30–80% for growing

6 COPRODUCTS FROM NONCONVENTIONAL OILSEEDS AND THEIR USE 177


beef cattle and 20–50% for goats, while for lac-tating dairy cattle it is 20–50%. Recommended levels of PKC in feed for poultry and freshwater fish are no more than 10%. The optimum level of PKC in feed for ruminant animals is 30% (Wan Zahari et al., 2012).

Camelina sativa meal: It is also called false flax (gold of pleasure) and is an oilseed crop of the Brassica (Cruciferae) family. Camelina is not a food crop; however, coproducts (ie, meal) obtained after oil extraction from the seed are valuable as an animal feed (Pilgeram et al., 2007). Studies on using camelina meal

in the diet of beef heifers (Moriel et al., 2011), dairy cows (Hurtaud and Peyraud, 2007; Halmemies-Beauchet-Filleau et al., 2011), and ewes (Szumacher-Strabel et al., 2011) have been reported. Studies conducted on feeding camelina meal to broilers and egg-laying hens show that the meal can be included in broiler and layer diets up to 10% without compromis-ing bird performance, while potentially increas-ing the omega-3 fatty acid content threefold in the meat (Aziza et al., 2010a) and eightfold in eggs (Cherian et al., 2009). In addition, dietary camelina meal at a 10% level led to significant

TABLE 10.6 Chemical Composition of Nonconventional Oilseed By-Products and Protein Supplements

Coproducts CP CF NDF ADF EE Ash References

Palm kernel cake 14.5–19.5 13.0–20.0 66.8–78.9 52.9 1.5–8.0 2.0–10.0 Wong and Wan Zahari, 1997; Siew, 1989

Camelina meal 36.2 8.4 41.8 — 6.0–12.0 6.0–7.0 Aziza et al., 2010a,b; Cherian et al., 2009

Castor oilseed cake 44.52 — 28.35 12.60 7.24 8.82 Wadhwa et al., 2010a,b

Neem seed cake

Expeller pressed 12.4–19.6 17.9 — — 1.8–3.30 13.9–14.3 Bedi et al., 1975a; Nath et al., 1978

Solvent extracted 17.9–18.98 25.9–30.1 62.20 47.15 0.4–1.04 5.5–23.1 Christopher et al., 1976; Garg, 1989; Wadhwa et al., 2010b

Corn gluten meal 58.90 — 32.90 13.10 3.02 1.48 Wadhwa et al., 2010b

Maize oil cake/Corn germ meal

22.0–31.0 8.5–11.7 29.4–61.1 — 3.0–10.0 1.6–2.8 Anderson et al., 2012; Li et al., 2013

Deoiled J. curcas kernel meal

62.4 — 18.0 1.21 9.10 Makkar and Becker, 2009

Deoiled J. platyphylla kernel meal

66.4 1.14 9.0 Makkar et al., 2011

Karanj cake

Expeller pressed 24.1–28.7 3.9–10.7 18.2 10.6 6.1–14.2 3.2–7.1 Gupta et al., 1981; Ravi et al., 2000; Prabhu, 2002; Panda, 2004

Solvent extracted 30.0–34.0 5.0–5.6 18.0–28.0 1.7–20.0 0.1–2.2 4.4–6.9 Konwar et al., 1984; Gupta et al., 1981; Soren et al., 2007



reduction in lipid oxidation products and an improvement in γ-tocopherol content and an-tioxidant activity in the dark meat (Aziza et al., 2010b). Using coproducts from biofuel production, such as camelina meal, can reduce feed cost while promoting environmental equi-librium and sustainability (Cherian, 2012).

Castor (Ricinus communis L.) seed meal: On av-erage, castor seeds contain 46–55% oil by weight (Ogunniyi, 2006). Castor cake is poisonous and allergenic to animals because of the presence of three antinutritional compounds: ricin (a heat labile toxic protein), ricinine (a toxic alkaloid), and a stable allergen known as CB-1A (Gardner et al., 1960; Ogunniyi, 2006; Gowda et al., 2009). Castor meal contains 27% CP and its fiber con-tent is higher than the other nonedible cakes and meals. Castor meal is deficient in methio-nine, lysine, and tryptophan (Abbeddou and Makkar, 2012). Ricinus communis meal cooked at 100°C for 50 min could be considered for addi-tion at up to 15% in chick diets. The addition of lime at 4% was also promising when fed at up to 10% and 15% in the diet of sheep and beef cattle, respectively.

Crambe meal: It contains epiprogoitrin, a thio-glucoside, which undergoes a hydrolysis reac-tion sequence, initiated by the thioglucosidase enzyme system which suppresses thyroidal io-dine uptake and causes thyroid hyperplasia and hypertrophy (Gould and Gumbmann, 1980). Thus, feeding raw crambe meal with intact glucosinolates and active thioglucosidase can reduce palatability and cause growth inhibi-tion and pathological changes in body organs (Carlson and Tookey, 1983). In addition to a significant amount of CP in crambe meal, pro-tein efficiency tests showed that its protein is of good nutritional quality, with a well-balanced AA profile (Ghandi et al., 1994). Sodium carbon-ate treatment left 1.7% epiprogoitrin in crambe meal, thus reducing its content by 82% (Mus-takas et al., 1976), while only 0.6% remained in ferrous sulfate-treated meal (Kirk et al., 1971). Heat-carbonate-treated dehulled-meal from

Crambe abyssinica has been shown to have ac-ceptable palatability and can replace up to two-thirds of SBM in the supplement for beef cattle. Water washing of crambe meal after inactivation of thioglucosidase resulted in 20–25% DM loss, but the resulting meal contained 50% CP and 0.6% residual epiprogoitrin (Baker et al., 1977).

Azadirachta indica A. Juss seed meal: It is com-monly known as neem. The cake obtained after the extraction of oil is bitter in taste. The toxic-ity of azadirachta cake is caused by the presence of azadirachtin, limonoids (tetra nortri terpe-noid), isoprenoids, and nimbidin, a sulfurous compound (Yakkundi, 1997; Usman et al., 2005; Saxena et al., 2010). Bedi et al. (1975a) observed poor palatability, depressed growth rate, and re-duced nutrient digestibility in cross-bred calves fed concentrate mixtures containing 25 and 57% neem seed cake (NSC). When NSC was substi-tuted at rates of 25 and 50% digestible crude protein (DCP) for GNC in concentrate mixtures, loss of body weight with poor palatability was noted in buffalo calves, and there was signifi-cantly depressed nutrient digestibility, espe-cially at the higher level of incorporation (Bedi et al., 1975b), indicating that untreated NSC was not suitable even for maintenance of animals. Uko et al. (2006) incorporated up to 30% raw full-fat azadirachta kernels into cockerel chick diets. Feed intake and BWG were depressed in-dependently of the inclusion level and starting from 15% in the diet, anemia and leucocytosis occurred. Deoiled azadirachta meal included up to 10% in the diet of in-lay Japanese quails reduced feed efficiency (but intake, egg produc-tion, and quality were not affected) and caused adverse effects in liver and kidney tissues with long-term feeding (Elangovan et al., 2000). Wa-ter washing is one of the successful methods to detoxify azadirachta meal (Agrawal et al., 1987). Ammoniation of azadirachta cake was found to result in a detoxified product suitable for ani-mal feeding (Nagalakshmi et al., 1999). Feeding of water-washed neem seed cake up to 45% of concentrate did not show any adverse effects on

6 COPRODUCTS FROM NONCONVENTIONAL OILSEEDS AND THEIR USE 179


intake or digestibility. There was higher weight gain and nitrogen retention in growing buffalo calves (Nath et al., 1983; Agrawal et al., 1987). Up to 25% of concentrate can be fed to grow-ing goats (Verma et al., 1995). Nath et al. (1989) conducted a 300-day lactation trial on 32 cross-bred milch cows. The control group was given a concentrate mixture containing 40% GNC, 30% maize, 27% wheat bran, 2% mineral mixture, and 1% common salt. In the experimental group, the GNC was replaced with WWNSKC. The re-sults showed that there was no adverse effect on DM intake, digestibility of nutrients, milk yield, butter fat content, and organoleptic evaluation of milk and reproductive ability of the cows in the two groups. The nitrogen balance was high-er (P < 0.05) in the WWNSKC group due to less excretion of urinary nitrogen and a concomitant decrease in blood urea nitrogen. Water washing, methanol extraction, urea and alkali treatments of Azadirachta indica meal gave promising results when fed to farm animals.

Karanj Pongamia pinnata (L.) seed cake: The karanj seeds yield nonedible oil, which has me-dicinal properties (Wani and Sreedevi, 2011). The leftover is karanj cake, which contains an-tinutritional factors, such as phytates, tannins, protease inhibitors, glabrin, and a fat-soluble constituent, karanjin (a furano-flavonoid) (Vinay and Sindhu Kanya, 2008). The antinutritional factors of karanj cake are soluble in oil; there-fore, complete removal of oil from cake appears to be more effective than other treatment meth-ods. When untreated karanj expeller cake was fed at 10% of the diet, it depressed weight gain in chicks (Natanam et al., 1989a) and decreased egg production of layers (Natanam et al., 1989b). Long-term feeding at 20% and 24% cake or meal in lamb concentrate had deleterious effects on lamb performance, especially spermatogenesis (Singh et al., 2006). Water-washed Pongamia pin-nata meal can be incorporated at up to 13.5% of the concentrate in lamb diet (Vinay and Sindhu Kanya, 2008). Water-washed or deoiled karanj cake could replace SBM or groundnut cake up

to 50% on an N-basis without any adverse ef-fect on nutrient metabolism, growth, or health of animals (Dutta et al., 2012).

Jatropha curcas L. meal (physic nut): Among dif-ferent species, J. curcas is the most studied feed for poultry, followed by pigs and cattle. Its seeds contain 25–35% oil and de-shelled seeds contain 55–60% oil (Makkar and Becker, 2009; King et al., 2009), which is mainly used for biodiesel production. The kernel meal obtained after oil extraction is an excellent source of nutrients and contains 60–66% CP; while Jatropha pro-tein isolate has about 81-85% CP (Makkar and Becker, 2009). The contents of essential AAs, ex-cept lysine, are higher in Jatropha kernel meal than in SBM. However, antinutrients, for exam-ple, a trypsin inhibitor, lectin, and phytate, and phorbol esters are present in the meal (Makkar et al., 2011). Rumen microbes cannot degrade phorbol esters (Makkar and Becker, 2010), and they cause severe toxic symptoms in ruminants and nonruminants. A new, nontoxic species of Jatropha, Jatropha platyphylla has been identi-fied, which is free of phorbol esters (Makkar et al., 2011). Heat-labile antinutrients, protease inhibitors, and lectins are easy to inactivate by moist heating, and phytase could be incorpo-rated into the diet for degradation of phytate. Detoxified J. curcas kernel meal (DJKM) and heat-treated J. platyphylla kernel meal (H-JPKM) contain approximately 9–10% phytate, which can be eliminated by adding 1500 FTU phy-tase per kilogram of diet. DJKM, H-JPKM, and detoxified J. curcas protein isolate (DJPI) can replace 50, 62.5, and 75% of fish meal protein, respectively, in fish diets without compromis-ing their growth performance and nutrient uti-lization (Kumar et al., 2011). In addition, DJKM could also replace 50% of fish meal protein with-out adversely affecting growth and nutrient uti-lization in shrimp. No mortalities, unaffected hematological values, and no adverse histopath-ological alterations in stomach, intestine, and liver of fish suggested that they were in normal health (Kumar et al., 2010; Kumar, 2011). DJKM



has also been fed to turkeys with no significant difference in FI and weight gain compared with the SBM-containing diet (Boguhn et al., 2010). In pigs, the average weight gain and feed-to-gain ratio were similar for DJKM-fed groups and the SBM-based control group. In addition, the se-rum and hematological parameters did not dif-fer among the groups, and values were within the normal range (Wang et al., 2011). Overall, the DJKM can replace SBM protein in fish, shrimp, turkey, and pig diets by as much as 50% (Makkar et al., 2012).

7 COMPARATIVE EVALUATION OF CONVENTIONAL AND

NONCONVENTIONAL PROTEIN SUPPLEMENTS

The relative proportion of rumen degradable protein (RDP) and rumen undegradable protein (RUDP) plays an important role in productive and reproductive performance of high-yielding dairy cattle. Comparative evaluation of con-ventional protein supplements, such as solvent-extracted groundnut cake and mechanically extracted mustard cake (MC), with nonconven-tional protein supplements, such as mechani-cally extracted castor oilseed cake (COSC), un-decorticated solvent-extracted neem seed cake (NSC), and CGM, revealed that RUDP content of groundnut cake, MC, and sunflower cake var-ies between 23% and 26% of CP. Cottonseed cake (CSC) and CGM varies between 46% and 78% of CP. The higher concentration of RUDP in the CSC and CGM is mainly because of higher pro-lamins and glutelins (Wadhwa et al., 1993). The groundnut and MC have significantly higher intestinal digestion as compared to the RUDP fraction of these supplements. However, no such differences were observed between ruminally exposed or unexposed samples of neem seed cake and CGM, indicating higher potential of these nonconventional protein supplements as

rumen bypass proteins (Wadhwa et al., 2007). Irrespective of protein supplements, the degrad-ability of globulin was highest, followed by al-bumin, glutelin, and prolamin (Wadhwa et al., 2010b). The polypeptides in the globulin, pro-lamin, and glutelin fraction of groundnut cake with a molecular weight (MW) 35, 15, 16, and 19 kDa, respectively, were highly resistant to micro-bial degradation even after 24 h or rumen incu-bation. The degradation pattern of MC revealed that subunits of globulin, prolamin, and glutelin with MW of 14–33, 14, and 31 kDa, respectively, were highly resistant to microbial degradation. NSC subunits with MW 124 (in globulin), 30 (in albumin), 66 (in prolamin), and 54 kDa (in glute-lin) were resistant to microbial degradation. The subunits in the prolamin fraction of CGM with MW 18, 24, and 43 kDa and that in the glutelin fraction with MW of 22 and 60 kDa were found to be highly resistant to microbial degradation. The results conclusively revealed that in major-ity of the cases, the low-MW polypeptides in the tested protein supplements showed resistance to rumen microbial fermentation, even after 48 h of rumen incubation (Wadhwa et al., 2012).

In another study, the conventional protein supplements, such as MC, deoiled mustard cake (DMC), deoiled groundnut cake (DGNC), SBM, and CSC, were compared with noncon-ventional protein supplements CGM, maize oil cake (MOC), tomato pomace (TP), and SBGs. The CP content varied from 18.2% (TP) to 66% (CGM). TP had the highest EE (11.0%) and lignin (13.5%) content. The in vitro meth-ane production was the lowest (P < 0.01) from TP followed by that from CSC, SBG, MC, and CGM. The digestion kinetic parameters for CP revealed that the RUDP as percent of total protein was the highest (P < 0.01) in CGM. It was followed by TP and was lowest in DGNC. It was concluded that the protein supplements with high RUDP value, such as TP, CSC, SBG, and CGM had low methane production poten-tial (Lamba et al., 2014).

8 INDUSTRIAL WASTES 181


8 INDUSTRIAL WASTES

Panax ginseng meal: It is a traditional herbal medicine that has been used therapeutically for more than 2000 years. It is the most valuable of all medicinal plants, especially in Korea, China, and Japan. The name panax means “all healing,” and has possibly stemmed from traditional be-lief that the various properties of ginseng can heal all aspects of the illness encountered by the human body (ie, it acts as a panacea for the human body). Among medicinal herbs, Korean ginseng is well known for its positive effects on growth, high blood pressure, stress, and cancer (Joo, 1982). After the solubles are extracted with solvents, such as water or alcohol (70%–75%), a residue is produced, known as ginseng meal. It contains 16.06% CP, 2.0% EE, 45% NDF, and 60% NFE on a DM basis. It also contains saponins. Studies revealed that it increased the milk yield and milk quality of dairy cattle (Joo et al., 1975) and also the growth rate of chicks (Yildirim and Erener, 2010). Maeng et al. replaced alfalfa hay (0–15%) with ginseng meal and observed that in vitro DM digestibility was not affected, but CP digestibility was depressed without affecting VFA production, suggesting that ginseng meal could be used as a feed resource for ruminants. Some livestock farmers raising Korean native cattle feed them medicinal herbs, including gin-seng meal, to improve the carcass quality.

The dietary composition and feeding strate-gies may offer practical and efficient solutions for reducing body fat deposition in modern poultry strains (Fouad and El-Senousey, 2014). Ginseng, the root of Panax ginseng C.A. Meyer, is a well-known traditional herbal drug in East Asian countries, which has been used to treat several heart and metabolic diseases (Yan et al., 2011a). Ginseng has many bioactive components, such as saponins, that inhibit lipogenesis in chickens (Qureshi et al., 1983). Dietary supplementation with wild ginseng adventitious root meal at 1% and 2% increased egg production and reduced

serum cholesterol concentrations in laying hens. The inclusion of ginseng adventitious root meal reduced saturated fatty acid (SFA) and in-creased polyunsaturated fatty acid (PUFA) and PUFA:SFA ratio in egg yolk (Yan et al., 2011b). This reduced the availability of blood triglycer-ide for transport and deposition in the abdomi-nal areas of poultry. Yan et al. (2011a) showed that the inclusion of wild ginseng adventitious root meal use of WGM at the 0.1% level could enhance growth performance in broilers; and led to a significant reduction in abdominal fat depo-sition at 0.3% in the diets of broiler chickens.

Condensed molasses fermentation solubles (CMS): It is the organic residue of microbial fer-mentation, produced in making monosodium glutamate from raw sugar and molasses. CMS contains 42.0% moisture, 45.0% CP, 1% CF, and no crude fat. Chloride is the most common min-eral (3.04%) followed by potassium (0.74%) and sulfur (0.67%). Of trace minerals, lead, cadmi-um, and chromium are present at levels of 0.356, 0.037, and 0.330 ppm, respectively, which are be-low the minimum permitted levels for livestock feed. The level of total AAs is 9.88%. Of these, 5.52% is glutamic acid, 1.47% alanine acid, and 1.20% aspartic acid. Particularly, high levels of nonprotein nitrogen (32.0%) in CMS have at-tracted ruminant nutritionists to study whether it can replace the NPN in conventional feeds. The viscosity of CMS is 28.0, while that of mo-lasses is 10.0. Earlier, most of the CMS produced in Korea was dumped in the sea as waste, at a cost of approximately US$3.5 million. Now it is used in Korea as a feed under the brand name Molatein. It has a pleasant flavor from the fer-mentation of molasses. Supplementing the diet (roughage concentration 22:78) of Korean native cattle (Hanwoo) with CMS at 0–4% level indi-cated that CMS supplementation up to 4% level did not affect FI, feed conversion efficiency, and average daily weight gain. Consequently, CMS supplementation was more economical at 2% than at 4% (Ha et al., 2015). It is also being used



in the diet of beef cattle (Dehaan, 1993). A study was performed on Holstein cows (same parity, calving intervals, lactation periods, and milk yields) that suggested that up to 4% CMS may be used as a supplement in the diet of lactating dairy cattle, as it did not show any adverse effect on milk yield or its quality (Ha et al., 2015).

Guar meal: While manufacturing guar gum, the germ and the husks are removed by grind-ing and dry heating to obtain the guar “splits” (endosperm), which are subjected to various treatments for getting guar gum. Besides gum, the other by-product is called guar meal, which consist of germs and husks in approximately a 25:75 ratio (Lee et al., 2004). The regular guar meal (Churi) contains 40–45% CP and 12.7% CF, whereas guar meal (korma) contains 50–55% CP and 7.2% CF on a DM basis. As per Indian Standards (BIS, 1987), the guar meal for feeding livestock should contain minimum 45% CP, 3% EE, and maximum 8% CF. Its lysine (1.72% on DM basis) and methionine plus cysteine content (0.96% on DM basis) are comparable to that of groundnut meal but much lower than that of SBM. Guar meal contains about 12% gum resi-due (Lee et al., 2005), which increases viscosity in the intestine and reduces digestibility and growth (Lee et al., 2009). It also contains tryp-sin inhibitors, saponin, hemagglutinins, hy-drocyanic acid, and polyphenols (Verma and McNab, 1982; Gutierrez et al., 2007). However, antitrypsic activity was found to be lower than in heat-treated SBM and is not the main cause of the antinutritional effects of guar meal in poul-try (Lee et al., 2004).

Earlier studies revealed that raw guar meal can constitute up to 25% of cattle rations. Pro-cessed meal can be used as the sole protein com-ponent of cattle diets (Göhl, 1982). Grewal et al. (2014) reported that guar meal (guar-korma, roasted guar-korma, and guar-churi) could re-place 70–80% SBM in the concentrate mixture without any adverse effect on FI and nutrient utilization in male buffaloes. Salehpour and Qazvinian (2012) replaced cottonseed meal with

guar meal at 0–100% level in isocaloric and iso-nitrogenous rations of Holstein lactating cows indicated that with up to 50% replacement in rations, there was no significant effect on DM in-take, milk yield, and fat corrected milk yield and milk composition.

Different methods such as steam pelleting, toasting, water treatment, and methionine sup-plementation failed to improve performance in broilers, whereas the addition of cellulase, hemicellulase, or β-mannanase, using combi-nations of heat treatments (autoclaving), and enzyme treatment improved feed utilization (with or without affecting BWG) (Patel and Mc-Ginnis, 1985; Lee et al., 2003, 2004, 2005, 2009). Fermentation with A. niger or Fusarium sp. was also found to be useful (Nagra et al., 1998a,b). However, even for treated guar meal, the feed-ing threshold should remain as low as 5% to avoid problems (Lee et al., 2005). Shahbazi (2012) incorporated guar meal at 0–5% level in isocaloric and isonitrogenous diets, either with or without β-mannanase. It was concluded that adding 5% guar meal to laying hens’ diets has adverse effects on their productive perfor-mance, and it seems that hens can tolerate guar meal in the diet up to 2.5% without any det-rimental effects on egg production, egg mass, and feed efficiency.

9 CONCLUSIONS AND FUTURE PROSPECTS

A number of food-processing and agro-in-dustrial wastes are being tapped for incorpora-tion in animal feed: biofuel coproducts, brewery waste, and nonconventional oilseed cakes and meals. Some of these are rich in proteins, soluble sugars, digestible fiber, minerals, and vitamins, and they have great potential for feeding to ani-mals. They do not have any adverse effect on the productive and reproductive performance of animals. But caution is required while feed-ing such products, as some of the wastes have


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C H A P T E R


11Novel Applications of Protein By-products in Biomedicine

M.C. García*, J.M. Orellana**, M.L. Marina**Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, Faculty of Biology, Environmental Sciences and Chemistry,

University of Alcalá, Barcelona, Spain; **Animal Research Center, University of Alcalá, Barcelona, Spain

1 INTRODUCTION

The food industry produces a large amount of waste ranging from 5% to 90% depending on the processed raw material. The lack of infra-structure and processes to handle this material along with high processing costs, make these residues a major disposal problem for indus-trial processing plants. In most cases, these by-products have two main destinations: landfill and incineration. Inappropriate management of landfill results in emissions of greenhouse gases, methane and carbon dioxide. Conversely, incineration involves the formation and release of pollutants and secondary wastes, such as di-oxins, furans, acid gases, and particulates. Thus, both destinations produce environmental pollu-tion and health risks. To contribute to environ-mental sustainability and protection, it is neces-sary to recover these residues and look for new opportunities of application (Deng et al., 2012).

Some food by-products are processed into low-market-valued products, such as animal feed or fertilizers. Nevertheless, these food by-products contain valuable substances, and their processing as cheap products does not guaran-tee the efficient use of these natural resources. Thus, a more attractive approach from the eco-nomic point of view is to recover the high-add-ed-valued constituents present in food by-prod-ucts. Fiber, phenol, antioxidants, flavonoids, and phytosterols are some examples of valuable sub-stances that have been extracted from food by-products. Additionally, some food by-products contain high protein content. These products are considered cheap sources of proteins that beyond their nutritional properties, could also exert therapeutic capabilities. Proteins extracted from these by-products could be useful in bio-medicine if suitable processing is developed. Conversely, nutritional research and product development is focused on the production of

194 11. NOVEL APPLICATIONS OF PROTEIN BY-PRODUCTS IN BIOMEDICINE


products that help to reduce or control diet-related diseases, such as hypertension, cancer, atherosclerosis, and so forth, and protein by-products could be cheap sources of compounds with these therapeutic functionalities.

2 APPLICATION OF PROTEIN BY-PRODUCTS IN BIOMEDICINE

Although there are some examples of proteins in protein by-products exerting bioactive prop-erties, in most cases, proteins are not bioactive. Nevertheless, peptides with biological activities can be released from protein sequences. Pep-tides from food protein hydrolysates exerting beneficial biological functionalities are called bioactive peptides. Bioactive peptides have been defined as “specific protein fragments that have a positive impact on body functions or conditions and may ultimately influence health” (Korkonen and Pihlanto, 2006). Biological activity of bioactive peptides seems to be related to their molecular weight with peptides weighing less than 1 kDa the most usual. Bioactive peptides contain from 2 to 20 amino acids, although there are some

exceptions, such as anticancer lunasin with 43 amino acids. These peptides can be of interest not only for the food industry but also for the pharmacological industry.

Bioactive peptides can be produced from pro-teins by microbial fermentation, in vivo diges-tion or in vitro enzymatic proteolysis. In some cases, autolysis is also possible. The enzymatic hydrolysis of native proteins is the most efficient technology to recover bioactive peptides from protein by-products. In this case, protease and hydrolysis conditions selection is of significant importance. Commercially available proteases, such as alcalase, flavourzyme, thermolysin, or trypsin are the most usual options. Alterna-tively, noncommercial proteases isolated from other organisms, such as plants or animals are a cheaper option. Although less frequently oc-curring, bioactive peptides can also appear as independent entities, and hence do not require any process for their release from large protein fragments.

According to the BIOPEP database, there are close to 3000 different bioactive peptides with more than 37 different types of bioactivi-ties (Minkiewicz et al., 2008). Fig. 11.1 shows

FIGURE 11.1 Distribution of bioactivities within bioactive peptides in the BIOPEP database (2998 peptides in February 2015).

2 APPLICATION OF PROTEIN BY-PRODUCTS IN BIOMEDICINE 195


the distribution of bioactivities within bioactive peptides. The most frequently occurring bioac-tive peptides show antioxidant, antibacterial, or angiotensin-converting enzyme (ACE) inhibitor activities.

Bioactive peptides have many advantages over synthetic drugs because they have low tox-icity, different bioactivities, exhibit less side ef-fects, and are more bioavailable. Nevertheless, the complexity of the system to produce bioac-tive peptides from natural sources and the high cost of purification are the main factors limiting the large-scale production and commercializa-tion of these peptides. One promising approach to reduce costs is the use of food by-products. Moreover, this approach also promotes an ef-ficient waste disposal and use of protein re-sources. Table 11.1 shows the potential bioac-tivities and hydrolysis conditions employed for the recovery of bioactive peptides from protein by-products. Moreover, Table 11.2 groups the potential bioactivities and extraction conditions employed for the recovery of bioactive proteins from protein by-products. Among bioactive peptides and proteins obtained from food by-products, the presence of antihypertensive, an-tioxidant, antibacterial, and anticancer ones is remarkable.

Antihypertensive peptides are the best-known class of bioactive peptides. Antihyper-tensive peptides inhibit the in vitro activity of ACE, which is involved in the main system reg-ulating blood pressure—the renin-angiotensin system. This enzyme hydrolyzes the peptide angiotensin I, present in the bloodstream, to the vasoconstrictor peptide angiotensin II, which results in an increase in blood pressure. More-over, the presence of this peptide also causes the increase in the retention of salts and water in kidneys, which results in an increase in blood pressure. ACE also catalyzes the hydrolysis of bradykinin, which is a potent vasodilator. The ACE inhibition potency is expressed as the in-hibitor concentration needed for a 50% inhibi-tion of ACE activity (Puchalska et al., 2015).

Although there are different synthetic drugs to treat hypertension, including synthetic ACE inhibitors, most of them can cause side effects, such as skin rashes, cough, angioedema, taste disturbances, reduced renal function, increased potassium levels, fetal disturbances, and so forth. ACE inhibitor peptides could be an alter-native or a complementary approach to reduce hypertension without side effects.

Antioxidant peptides and proteins contribute to the prevention of oxidative stress. Oxidative stress is caused by free radicals, such as reac-tive oxygen species. These radicals can damage DNA, lipids, and proteins when the body’s an-tioxidant defense system fails, causing aging or several diseases, such as cancer, diabetes, or ath-erosclerosis. Indeed, environmental conditions, way of life, or different pathologies can result in an imbalance between natural antioxidants and free radicals, leading to oxidative stress. Syn-thetic antioxidants show strong activity against several oxidation systems but, in many cases, they are limited or forbidden because of carci-nogenicity and health risks. Natural antioxidant peptides may protect the body from free radicals and retard their deterioration. In some cases, hy-drolysates show higher antioxidant activity than isolated peptides because of a synergic relation-ship among antioxidants (García et al., 2013; Puchalska et al., 2014).

There is an increasing resistance of bacteria to drugs (eg, antibiotics). This is a significant prob-lem which requires new antimicrobial strategies against bacteria. Antimicrobial peptides can be effective against antibiotic-resistant bacteria. The main mode of action is the disintegration of the lipid bilayer of target-cell membranes. Antibac-terial peptides are usually positively charged to bind with these anionic phospholipid-rich mem-branes. Moreover, antibacterial peptides have both hydrophobic and hydrophilic sites that are soluble in aqueous solutions and enter lipid-rich membranes (Izadpanah and Gallo, 2005).

Although in less extension, anticancer ca-pacity has also been found among peptides

196

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TABLE 11.1 Potential Bioactivities and Hydrolysis Conditions Employed for the Recovery of Bioactive Peptides from Protein By-Products

By-product Enzyme Hydrolysis conditions Peptides Potential bioactivities References

ANIMAL ORIGIN PROTEIN BY-PRODUCTS

Egg yolk by-product Pepsin, trypsin, and chymotrypsin

E/S ratio, 10 U/mg sample; 37°C; pH, 8.3; 4 h (trypsin and chymotrypsin)

E/S ratio, 20 U/mg sample; 37°C; pH, 3.5; 4 h (pepsin)

— Antioxidant and antimicrobial

Zambrowicz et al., 2012

Egg yolk by-product Pepsin E/S ratio, 20 U/mg sample; 37°C; pH, 3.5; 2 h

YINQMPQKSREYINQMPQKSREAVTGRFAGHPAAQYIEAVNKVSPRAGQF

Antioxidant, antidiabetic, and antihypertensive

Zambrowicz et al., 2015

Egg white by-product Protease isolated from Cucurbita ficifolia

E/S ratio, 1000 U/mg sample; 4 h

LAPSLPGKPKPD Antihypertensive Eckert et al., 2014

Egg white by-product Protease isolated from Cucurbita ficifolia

E/S ratio, 200 U/mg sample; 37°C; pH, 8.0; 5 h

SWVEDILN

Antihypertensive Pokora et al., 2014

Salmon pectoral fin Pepsin E/S ratio, 1:100 (w/w); 8 h — Antioxidant, hepatoprotective, and antiinflammatory

Je et al., 2013

Tuna dark protein Alcalase E/S ratio, 1%; 55–60°C; pH, 9–10.5; 1 h

— Antioxidant Saidi et al., 2014

Tuna cooking juice Gamma irradiation — — Antioxidant Choi et al., 2012

Silver carp by-products Pepsin 37°C; pH, 2.0 — Antioxidant Zhong et al., 2011

Anchovy wastewater Protamex E/S ratio, 0.02 g/g sample; 55°C; pH, 6.5; 6 h

GLSRLFTALK Antimicrobial Tang et al., 2015

Shrimp by-products Alcalase E/S ratio, 100 U/ g sample; 50°C; pH, 8.0; 30 min

— Antioxidant Sila et al., 2014

Shrimp by-products Alcalase E/S ratio, 0.5%; 50°C — Antioxidant Sowmya et al., 2014

Shrimp shell Cryotin E/S ratio, 0.1 g/kg sample; 40°C, pH, 11.0; 1 h

— Anticancer Kannan et al., 2011

Crayfish by-products Autodigestion Lactic acid fermentation

37°C; pH, 6.0; 10–12 h (autodigestion)

— — Cremades et al., 2003

Shrimp shell Bacillus cereus E/S ratio, 20; 40°C, pH, 8.0; 3 h — Antioxidant Laila et al., 2010

Blue mussel by-products Protamex E/S ratio, 0.1%; 45°C; 1 h — Anticancer Beaulieu et al., 2013

Echinoderm by-products Alcalase E/S ratio, 0.75%; 55°C; pH, 8.0; 16 h

— Antioxidant Mamelona et al., 2010

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Bovine blood Pepsin in alcoholic medium

23°C; pH, 5.5 TKAVEHLDDLPGA-LSELSDLHAHKLRVD-PVNFKLLSHSLLLDDLPGALSELSDLHA-HKLRVDPVNFKLLSHSLKLLSHSLLLSHSL

Antimicrobial and antihypertensive

Adje et al., 2011

Bovine blood — — VNFKLLSHSLLVT-LASHL

Antimicrobial Hu et al., 2011

Pigskin and fish bone Protease from Bacillus

E/S ratio, 20: 1; 60°C; pH, 8.0; 1 h

— Antioxidant and antihypertensive

Morimura et al., 2002

Bovine Achilles tendon Collagenase from Alcaligenes odorans

37°C; pH, 7.5; 24 h AKGANGAPGIA-GAPGFPGARGPS-

GPQGPAGPPPAGNPGADGQP-

GAKGANGAP

Antihypertensive Banerjee and Shanthi, 2012

Buffalo horn — — QYDQGVYEDCTDCGNAADNANELFPPN

Antioxidant Liu et al., 2010

Cracklings and chicken feathers

Alcalase — — Antioxidant Flaczyk et al., 2003

Caprine whey 25 different microflora from french cheeses

E/S ratio, 1:8; 37°C; 24 h WLAHK Antihypertensive Didelot et al., 2006

Sheep cheese whey Protease from Bacillus

E/S ratio, 2%; 45°C; pH, 8.0; 4 h

LAFNPTQLEGQCHV Antioxidant and antihypertensive

Folmer Correa et al., 2014

VEGETAL ORIGIN PROTEIN BY-PRODUCTS

Rice by-product Alcalase, neutrase, and flavourzyme

Fermentation with Bacillus spp.

E/S ratio, 500 U/g sample, pH 8 and 55°C (alcalase), pH 6.5 and 40°C (neutrase), pH 6.0 and 60°C (flavourzyme); 2 h

Microbial fermentation at 37°C; 72 h

VVVAGGGGGGGGYSYQDVYNKSYQDVYNASLQEQEQGQVQKSYQDISVSAYKSYQDVYNQYKSYQDVYNYPGLSNFQQQYYPGLSNVGVALKSYQDISVSAKSYQDVYNVAESSQYKSYQDVYN

Antioxidant Dei Piu et al., 2014

Brewers’ spent grain Alcalase — — α-glucosidase inhibitor Lin et al., 2012

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Peanut meal Alcalase, flavourzyme, and pepsin

4 h — Antioxidant Kane et al., 2012

Peanut meal Alcalase E/S ratio, 8.3 mAU/g protein; 50°C; 1 h

— Antihypertensive White et al., 2013

Sesame meal Protease A Amano 2G

E/S ratio, 2%; 50°C; pH, 7.0, 30 min

— Antioxidant Das et al., 2012

Sunflower meal Alcalase and flavourzyme

E/S ratio, 0.3 AU/g sample; 50°C; pH, 8.0; 1 h (alcalase); E/S ratio, 100 LAPU/g sample; 50°C; pH, 7.0; 2 h (flavourzyme)

— Antihypertensive Megías et al., 2009

Rapeseed meal Alcalase E/S ratio, 0.3 AU/g sample; 50°C; pH, 8.0; 1 h

— Antihypertensive HIV protease inhibitor

Pedroche et al., 2004

Yust et al., 2004

Seaweed (P. columbina) by-products

Trypsin and alcalase E/S ratio, 5%; 50°C; pH, 8.0; 4 h

— Immunosuppressive, antihypertensive, and antioxidant

Cian et al., 2012a

Seaweed (P. columbina) by-products

Fungal protease + flavourzyme

E/S ratio, 50 g/Kg; 55°C; pH, 4.3; 3 h (fungal protease); E/S ratio, 20 g/Kg; 55°C; pH,7.0; 3 h (flavourzyme)

— Immunomodulatory, antihypertensive, and antioxidant

Cian et al., 2012bCian et al., 2013

Seaweed (C. vulgaris) by-products

Pepsin E/S ratio, 2%; 50°C; pH,2.0; 15 h

VECYGPNRPQF Antihypertensive, antioxidant, and anticancer

Chuan-Sheih et al., 2009

Chuan-Sheih et al., 2010

Peach, cherry, apricot, plum, and olive seeds

Thermolysin and alcalase

E/S ratio, 0.03–0.1g/g sample; 50°C; pH,8.0; 2–7 h (thermolysin)

E/S ratio, 0.1–0.3 AU/g sample; 50°C; pH, 8.0–8.5; 2–24 h (alcalase)

LYSPHLYTPHLAGNPENELLNDENLPLLLLPGANH

Antihypertensive and antioxidant

Esteve et al., 2015, González-García et al., 2014; Gar-cía et al., 2015; Vásquez-Villan-ueva et al., 2015

Apricot seeds Alcalase E/S ratio, 2:100; 55°C; pH, 8.0; 1 h — Antihypertensive Zhu et al., 2010

Wool waste Keratinolytic microorganisms

— — Antioxidant Fakhfakh et al., 2013

Okara Pepsin and pancreatin

E/S ratio, 1:20; 37°C; pH,2.0; 60 min (pepsin); E/S ratio, 1:20; 37°C; pH,7.5; 195 min (pancreatin)

TIIPLPV Antioxidant Jiménez-Escrig et al., 2010

E/S, Enzyme-to-substrate ratio.

TABLE 11.1 Potential Bioactivities and Hydrolysis Conditions Employed for the Recovery of Bioactive Peptides from Protein By-Products (cont.)

3 ANIMAL-ORIGIN PROTEIN BY-PRODUCTS 199


from protein by-products. Novel therapeutic means against cancer are needed because con-ventional anticancer treatments cause adverse effects on normal cells and because of the fre-quent development of multidrug resistance by cancer cells. Anticancer peptides have shown a great potential as they have demonstrated cancer selective toxicity and high tissue pen-etration. Anticancer peptides are similar to antibacterial peptides and they usually consist of basic and hydrophobic residues that elec-trostatically interact with negatively charged components of plasma membrane of cancer cells. Not all is known about the mechanism of action of anticancer peptides. In some cases, anticancer peptides show membrane-lytic ef-fect, but in others they could induce apoptosis via disruption of the mitochondrial membrane (Tyagi et al., 2015).

3 ANIMAL-ORIGIN PROTEIN BY-PRODUCTS

Domestic and industrial use of animal prod-ucts (meat, milk, and eggs) results in large amounts of residues that, in some cases, have been demonstrated to be rich sources of proteins with the potential to obtain bioactive peptides.

3.1 Egg-Protein By-Products

Egg is a product containing many substances that are interesting from the nutritional and bio-logical point of view. Egg is widely used to ex-tract substances, such as lecithin, ovotransferrin, lysozyme, or cystatin on an industrial scale. As a consequence, many egg by-products rich in pro-teins are generated. An example is the isolation of lecithin from egg yolk. Egg yolk by-products

TABLE 11.2 Potential Bioactivities and Extraction Conditions Employed for the Recovery of Bioactive Proteins from Protein By-Products

Protein by-product Extraction/purification procedure Protein

Potential bioactivity References

Syzygium cumini seeds

Phenol extraction, extraction with tris-HCl buffer, anion-exchange chromatography, and size-exclusion chromatography

Lactoferrin Antibacterial Binita et al., 2014

Passion fruit seeds

Extraction with phosphate buffer, size-exclusion chromatography, cation-exchange chromatography, and reversed-phase chromatography

Two proteins (12.088 and 11.930 kDa)

Antifungal Agizzio et al., 2003, 2006

Passion fruit seeds

Ion-exchange chromatography on Q-Sepharose column, hydrophobic interaction chromatography, ion-exchange chromatography on DEAE cellulose and size-exclusion chromatography

Passiflin (67 kDa) Antifungal Lam and Ng, 2009

Peach seeds — PR-A (∼300 kDa) and PR-B (∼10 kDa) proteins

Antiinflammatory Arichi et al., 1985a, b

Turmeric waste grits

Extraction with hot water and addi-tion of polyvinyl pyrrolidone to remove polyphenols and size-exclusion chromatography

β-turmerin protein (34 kDa)

Antioxidant Smitha et al., 2009



resulting from this procedure have a 60% protein fraction and are mainly constituted by phosvi-tin, a strong antioxidant protein. This protein by-product has been hydrolyzed with pepsin, trypsin, and chymotrypsin, and hydrolysates have been investigated for their antioxidant and antimicrobial activities. All hydrolysates show antioxidant capacity although trypsin and chy-motrypsin endopeptidases show extracts with higher antioxidant capacity than extracts ob-tained with pepsin. Moreover, trypsin hydroly-sate shows weak antimicrobial capacity (Zam-browicz et al., 2012). The same authors have recently obtained various peptides from egg yolk by-products with multiple bioactivities (antioxi-dant, antidiabetic, and antihypertensive) by their digestion with pepsin (Zambrowicz et al., 2015). Peptides presented molecular weights from 1 to 2 kDa and corresponded to fragments of apoli-poprotein B (YINQMPQKSRE, YINQMPQK-SREA), vitellogenin-2 (VTGRFAGHPAAQ), and apovitellin-1 (YIEAVNKVSPRAGQF).

Conversely, egg white by-products resulting during the isolation of lysozyme and cystatin with ethanol have also been explored for the presence of bioactive peptides. This by-product is mainly constituted by ovalbumin and ovotransferrin. This protein waste was hydrolyzed with an un-conventional protease isolated from Asian pump-kin (Cucurbita ficifolia). This enzyme is cheaper than the usual commercial proteases showing a full enzyme activity at a wide pH range (Eckert et al., 2014). After ultrafiltration, size-exclusion chromatography, and reversed-phase chroma-tography, it was possible to obtain a peptide with highly strong ACE inhibitor activity (IC50 value of 1.97 µmol/L). Additionally, the same group also isolated two tetrapeptides from ovalbumin show-ing ACE inhibitor activity: SWVE (IC50 = 33.88 µg) and DILN (IC50 = 73.44 µg) (Pokora et al., 2014).

3.2 Marine-Protein By-Products

Processing of fish involves stunning, grad-ing, slime removal, deheading, washing, scaling,

gutting, cutting fins, meat bone separation, and so forth. Marine by-products are constituted by substandard mussels, viscera, heads, skins, fins, frames, trimmings, shellfish, crustaceous shells, cooking juices, and so forth. In some cases, these by-products can reach 50–70% of the raw mate-rial. This residue has been proposed for the pro-duction of proteins and peptides with different therapeutic properties: antihypertensive, anti-oxidant, Ca-binding, antimicrobial, human im-munodeficiency virus (HIV) protease inhibitor, appetite suppression, and so forth. Many differ-ent fish and seafood species by-products have been investigated. Furthermore, different bioac-tive peptides extracted from marine by-products have been commercialized as nutraceuticals. Recently, production and characterization of hy-drolysates from fish and seafood by-products have been thoroughly reviewed by Harnedy and FitzGerald (2012), Chalamaiah et al. (2012), and Ghaly et al. (2013). Most recent works on this item have been mainly focused on exploring the bio-activities of new fish by-products and improving technologies to extract bioactive peptides from shrimp and other seafood by-products.

3.2.1 Fish-Protein By-ProductsMany different bioactive peptides have been

identified in by-products from carp, tuna, yel-lowtail, cod, sole, sardine, and so forth. In this chapter, we have included the most recent works devoted to the extraction of bioactive peptides from fish by-products. Salmon pectoral fin–protein by-products were employed for the production of bioactive peptides by pepsin hy-drolysis. Obtained peptides exerted antioxidant, antiinflammatory, and hepatoprotective effects (Je et al., 2013). Another example of protein by-product recovery is the dark part of tune muscle. This by-product is derived from the processing of canned tuna and contains approximately 25% protein. To revalorize this waste, Saidi et al. (2014) digested these proteins with alcalase and fractionated the resulting peptide extract by ul-trafiltration and size-exclusion chromatography.

3 ANIMAL-ORIGIN PROTEIN BY-PRODUCTS 201


Both the whole peptide extract and the frac-tions demonstrated antioxidant properties when using different antioxidant assays (radi-cal-scavenging assays, reducing-power assay, ion-chelating activity assay, and inhibition of lipid autooxidation assay). Tuna cooking juice is another by-product generated during canning of tuna. This wastewater contains 4% protein and has been hydrolyzed for its better utiliza-tion. Choi et al. (2012) proposed the hydrolysis of proteins by irradiation with ionizing energy. Ionizing energy is capable of scission water mol-ecules, creating H+ and OH– that enable protein hydrolysis. Some advantages of radiation hy-drolysis are simple processing, simultaneous sterilization, and no enzyme consumption.

Silver carp leftovers (frame, dark muscle, cut-offs, viscera, skin, scales, small bones, and fins) were also employed to produce a protein iso-late. Proteins were solubilized at basic pH, and the protein isolate was separately hydrolyzed with seven proteases (alcalase, flavourzyme, neutrase, papain, pepsin, protamex, and tryp-sin). Treatment with pepsin yielded the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging capacity, and the fraction with pep-tides below 1 kDa in weight contained the most potent antioxidant peptides (Zhong et al., 2011).

Anchovy-cooking wastewater is a by-product from the production of boiled-dried anchovies, a traditional Chinese food. The hydrolysis of this wastewater with protamex enzyme produced peptides with antimicrobial properties. More-over, an antimicrobial peptide with 1104 Da mo-lecular weight was isolated (Tang et al., 2015).

3.2.2 Seafood-Protein By-ProductsAbout 45% of processed seafood comes from

shrimp, and processing results in the produc-tion of waste residue composed of exoskeleton and cephalothorax that can represent 50–70% of shrimps. This waste has been employed for the extraction of carotenoprotein, which is a com-plex constituted by carotenoids linked to a high-density lipoprotein.

Sila et al. (2014) prepared a protein isolate from shrimp waste with an 80.8% protein level by extraction using alcalase at a 1000 U/g sam-ple ratio for 2 h. Afterward, they hydrolyzed this isolate using alcalase enzyme at a 100 U/g sam-ple ratio for 30 min. The resulting peptides dem-onstrated antioxidant capabilities when using different antioxidant assays, including the ca-pacity to protect a supercoiled DNA strand from scission by peroxyl and hydroxyl radicals into the nicked circular form. Sowmya et al. (2014) optimized the hydrolysis of shrimp waste using alcalase to obtain highly antioxidant peptides. Optimization was carried out using fraction-ally factorial design. To improve hydrolysis of proteins and increase the amount of generated peptides, other enzymes have also been tried. As an example, cryotin produced eightfold more soluble peptides than alcalase during the extrac-tion of proteins from shrimp shells (Kannan et al., 2011). In this case, peptides resisting gas-trointestinal digestion present in fractions < 10 kDa and from 10 to 30 kDa demonstrated an-ticancer activity by the clear inhibition of the growth of both colon and liver cancer cells.

Carotenoproteins have also been hydrolyzed using enzymes extracted from fish by-products. Fish viscera are a rich sources of digestive en-zymes, such as trypsin. In fact, trypsin has been isolated from cuttlefish, mandarin fish, salmon, the Amazonian tambaqui, yellowfin tuna, sil-ver mojarra, sardine, gray triggerfish, striped seabream, bogue, Sardinella, barbel, and so forth. Carotenoproteins were extracted from black ti-ger shrimp shells using trypsin from bluefish viscera. A protein isolate containing 70.20% protein and mainly constituted by a 45 kDa protein was obtained (Klomkao et al., 2009). More recently, trypsin from barbel viscera was purified and employed to recover carotenopro-teins from shrimp shells obtaining a protein isolate with similar protein content (1.09%) (Sila et al., 2012).

The crayfish is a crustacean similar to a lob-ster and its by-products (head, thorax, and



claws) have been employed to obtain bioac-tive peptides. A controlled autodigestion and fermentation with lactic acid bacterium were the two procedures employed for the release of peptides. The controlled autodigestion process produced the extract with the best nutritional properties, suitable for patients with malnutri-tion (Cremades et al., 2003).

Shrimp shell is also a major source of the polysaccharide chitin. Usually, chitin isolation consists of deproteinization using alkali solu-tions. Nevertheless, the extracted protein from this process is of limited use because some ami-no acids are lost, resulting in a lower nutritional and functional value and because there is a race-mization of L-amino acids to form D-amino ac-ids that are not absorbed by humans. Moreover, this process can cause a partial deacetylation of chitin and hydrolysis of the polymer. Enzymatic hydrolysis has rinsed as an alternative to alka-line deproteinization. Alcalase and pancreatin have been used for this purpose. Although alca-lase was the enzyme yielding the highest recov-ery (∼65%) of proteins, it was lower than that obtained by alkaline extraction (de Holanda and Netto, 2006). The protease from Bacillus cereus has also been employed for the deproteiniza-tion and extraction of chitin. In this case, around 88.8% deproteinization, which is similar to the deproteinization obtained by alkaline extrac-tion, was reached. Moreover, these hydrolyzed proteins showed remarkable antioxidant capac-ity (Laila et al., 2010).

Blue mussel by-products (shells, damaged mussels, and substandard mussels) present a protein content ranging from 10% to 23% and have also been employed to obtain bioactive peptides. For that purpose, protamex, employed as an enzyme for antiproliferative activity against four different carcinoma cells, was as-sayed. After fractionation using 50 kDa, 1 kDa, and 200 Da cutoff filters, the fraction retained in the 50 kDa filter showed the highest capacity to inhibit the proliferation of all cancer cell lines even at a concentration of 11 µg/mL. Taking

into account that the US National Cancer Insti-tute indicates that an extract, to be commercially valuable, must present a biological efficiency at concentrations below 100 to 150 µg/mL, it was possible to conclude that this fraction had a strong potential as anticancer agent (Beaulieu et al., 2013).

3.2.3 Other Marine By-ProductsEchinoderm by-products (viscera of Atlantic

sea cucumber and green sea urchin) have also been used to obtain antioxidant peptides using alcalase (Mamelona et al., 2010).

3.3 Other Animal-Protein By-Products

The use of animal by-products, such as scales, horns, hoofs, skin, bones, meat trimmings, or blood have been limited because of the negative perception of consumers since they were consid-ered a source of bovine spongiform encephalop-athy and chemical contaminants. Nevertheless, these by-products represent an environmental pollution problem, and different strategies have been developed for their reuse. These animal wastes are rich sources of proteins and they have been proposed as a starting material for the production of bioactive peptides. A wide range of enzymes derived from microorganisms (alca-lase, flavourzyme, neutrase, collagenase, pro-teinase K), animals (pepsin, trypsin), or plants (ficin, bromelain, papain, and so forth) have been employed for this purpose. Moreover, different functionalities were observed among bioactive peptides: antihypertensive, antithrombotic, opi-oid, antioxidant, antimicrobial, and so forth. Re-cently, detailed review articles have been pub-lished on the bioactive peptides derived from animal by-products (di Bernardini et al., 2011; Lafarga and Hayes, 2014; Mora et al., 2014).

Animal blood is generally discarded although it contains large amounts of proteins. In a recent work, Adje and coworkers hydrolyzed bovine hemoglobin with pepsin and 30% (v/v) etha-nol and isolated 12 fractions by reversed-phase

4 VEGETAL-ORIGIN PROTEIN BY-PRODUCTS 203


high-performance liquid chromatography (RP-HPLC). One peptide with 40 amino acids from the central part of the hemoglobin α-subunit and three derived peptides were identified in some of these fractions. These peptides dem-onstrated both antimicrobial and ACE inhibi-tor properties (Adje et al., 2011). The same year, other authors isolated peptide VNFKLLSHSLL-VTLASHL with antimicrobial activity from the same central part of the hemoglobin α-subunit. In this case, authors did not hydrolyze blood proteins, and this peptide was isolated directly from lysed erythrocytes by ion exchange and re-versed-phase chromatography (Hu et al., 2011).

Collagen is the most abundant protein in ver-tebrates and it is considered a very useful source of bioactive peptides with diverse capacities (antihypertensive, antithrombotic, treatment of type 2 diabetes, and so forth). Hydrolysis of ker-atin and collagen is difficult because of their sta-ble structure. Hence, they have not been widely employed for the production of bioactive pep-tides. Morimura et al. (2002) developed a meth-od for the extraction of collagen. This method involves extraction of fat and inorganic compo-nents in the initial step, followed by the extrac-tion of proteins under acidic conditions. More-over, they optimized a hydrolysis process trying 16 different commercial enzymes observing that Bacillus sp. enzyme yielded the highest degrada-tion efficiency (83%). Pigskin and fish bone hy-drolysates obtained using this enzyme showed significant antiradical activity and potential for reducing blood pressure (IC50 = 0.16 mg/mL for fish bone and 0.41 mg/mL for pigskin). More recently, collagen from the Achilles tendon was hydrolyzed with a bacterial collagenase, and peptides with antihypertensive capacity were isolated (Banerjee and Shanthi, 2012).

Other animal by-products used for the pro-duction of bioactive peptides are horns. Horns have been extensively employed in traditional Chinese medicine. Some authors have studied the presence of antioxidant peptides in water buffalo horns. Three peptides with antioxidant

properties and less than 1.5 kDa were isolat-ed and identified by mass spectrometry (Liu et al., 2010). Other examples are cracklings and chicken feathers that have been hydrolyzed with alcalase to obtain peptides with antioxidant properties (Flaczyk et al., 2003).

Conversely, whey is a waste by-product from the dairy industry and it represents 80% of milk. Whey is a by-product obtained from the pro-duction of cheese and caseins and it contains 20% milk proteins. Different bioactive peptides can be released during manufacture of cheese because of the peptidases used in milk clotting (Corrons et al., 2012). For example, peptides re-leased in the whey obtained in the production of mozzarella cheese demonstrated antioxidant and antiproliferative effects on cancer cells (de Simone et al., 2009). Gastrointestinal digestion of whey proteins has also demonstrated a release of peptides with the capacity to prevent food intake and control type 2 diabetes (Jakubowicz and Froy, 2013). In vitro enzymatic hydrolysis of whey proteins has also been employed for the production of bioactive peptides. Different commercial (eg, chymotrypsin, trypsin, pepsin, thermolysin) and noncommercial enzymes have been used, and peptides with antioxidant and antihypertensive activities have been obtained (Didelot et al., 2006; Folmer Correa et al., 2014).

4 VEGETAL-ORIGIN PROTEIN BY-PRODUCTS

4.1 Cereal-Protein By-Products

Rice by-products are produced during mill-ing of rice. This by-product is composed of bran, broken kernels, bran oil, wax, and hulls. Dei Piu et al. (2014) demonstrated the antioxi-dant properties of peptides that were obtained by hydrolysis of rice starch industry by-prod-ucts. This study employed different proteolytic enzymes (alcalase, neutrase, and flavourzyme) and different microbial whole cells of Bacillus



spp. The highest antioxidant capacity was ob-served in the extract obtained by fermentation with Bacillus licheniformis and Bacillus pumilus, followed by the neutrase hydrolysate and the alcalase hydrolysate. After fractionation by size-exclusion chromatography, most active peptides were observed in fraction A14 from the alcalase hydrolysate and fractions AG 4 and 5 from the hydrolysate obtained with B. pumilus.

Brewers’ spent grain is the main by-prod-uct obtained from the brewing industry. It has a high protein content (∼24%). Hydrolysis of these proteins with alcalase resulted in peptides with α-glucosidase inhibitory activity. The frac-tion below 5 kDa of the hydrolysate was capable of inhibiting 56.41% of α-glucosidase. These peptides have the ability to delay digestion and absorption of carbohydrates and are suitable for patients with type 2 diabetes (Lin et al., 2012).

4.2 Oil-Processing By-Products

Seed meal is a by-product derived from oil extraction that presents high protein content. Among seed components, proteins have normal-ly been the less studied fraction. In many cases, the most appreciated part of the seed is the oil, and proteins are considered a residue produced from the extraction of this lipid fraction. Some ex-amples of seed meals that have been employed for the production of bioactive peptides are pea-nut, sesame, sunflower, and rapeseed meals. Pea-nut meal is a high-protein by-product resulting from the extraction of peanut oil. The high afla-toxin level in this by-product limits its applica-tion. Kane et al. (2012) used three different en-zymes (alcalase, flavourzyme, and pepsin) for the hydrolysis of peanut meal, observing the lowest degree of hydrolysis with pepsin. Resulted pep-tides demonstrated antioxidant capacity. More re-cently, White et al. (2013) developed a pilot-scale process to extract proteins, sequester aflatoxin, and produce bioactive peptides from peanut meal. Hydrolysis of peanut meal with alcalase enzyme produced peptides with ACE inhibitor

capacity; the highest ACE inhibition was in the fraction containing peptides below 3 kDa.

Sesame meal was used for the production of antioxidant peptides by hydrolysis with Protease A Amano 2G (Das et al., 2012). Differ-ent hydrolysis parameters were optimized to maximize peptide yield and antioxidant activ-ity. Moreover, two different reactor modes were tried: a conventional batch reactor and an enzy-matic membrane reactor running in a continuous mode. Significantly higher hydrolysis degrees were obtained when working in the continuous mode probably because of the continuous with-drawal of products from the reactor.

Sunflower meal, produced during sunflower oil extraction, is usually employed for animal feeding. Nevertheless, it contains 30% protein, and within these proteins, there are peptides with ACE inhibitor activity. These peptides were released from native proteins by sequential hy-drolysis with alcalase and flavourzyme enzymes (Megías et al., 2009).

Rapeseed meal peptides obtained by hydroly-sis with alcalase have also demonstrated antihy-pertensive properties (Pedroche et al., 2004). The same group also observed that this hydrolysate contains peptides with the ability to inhibit HIV protease. Inhibition activity was demonstrated by measuring growth of Escherichia coli express-ing HIV protease (Yust et al., 2004).

4.3 Seaweed-Protein By-Products

Some seaweed is appreciated for its content in gelling substances, such as alginate, agar, or carrageenans, collectively known as phycocol-loids. They are used as food additives and extrac-tion involves successive washings with cold and hot water. The first cold-water wash is consid-ered a waste in which many proteins, including phycobiliproteins remain. This waste from Por-phyra columbina seaweed has been hydrolyzed with trypsin and alcalase. Both the protein ex-tract and the hydrolysates showed immunosup-pressive activity. The hydrolysates also showed



antihypertensive and antioxidant capacities (Cian et al., 2012a). The same group employed the proteins remaining after the final extrac-tion of phycocolloids from the same seaweed to obtain peptides with immunomodulatory, antihypertensive, and antioxidant activities (Cian et al., 2012b, 2013). For that purpose, they employed a sequential hydrolysis with a fungal protease and with flavourzyme enzyme.

Chlorella vulgaris is another seaweed used in Taiwan to produce algae essence by water ex-traction. Derived from this industrial process is a high-protein by-product (more than 50% pro-tein content) that is employed as low-cost ani-mal feed. This abundant subproduct has been hydrolyzed with different commercial enzymes (papain, pepsin, alcalase, and flavourzyme), and the bioactive properties of derived peptides have been studied (Chuan-Sheih et al., 2009). A pep-tide with 11 amino acids (VECYGPNRPQF) and high antihypertensive activity (IC50 = 29.6 µM) was isolated from the pepsin hydrolysate. The same group also reported the antioxidant activ-ity and anticancer properties of this peptide on gastric cancer cells (Chuan-Sheih et al., 2010).

4.4 Fruit Stones

Processing of fruits involves canning, drying, freezing, and preparation of juices, jams, and jel-lies. This processing generates a residue rang-ing from 5% to 50% of the raw material. Gener-ated residues are made up of effluents and solid residues. Solid residues are mainly made up of leaves, stems, stones, and skins (Datt et al., 2008). Fruit stones store proteins that together with lip-ids and carbohydrates, are the plant food reserves in the first stages of its growth. Taking into ac-count that fruit seeds have high protein content, their possible application for obtaining bioactive proteins and peptides constitutes an attractive strategy for the revalorization of this residue.

Different proteins with antimicrobial proper-ties have been isolated from plant seeds: chitin-ases, β-1,3-glucanases, defensins, thionins, lipid

transfer proteins, and 2S albumins. These pro-teins play an important role in the protection of plants from microbial infection. In some cases, the protein fraction of fruit seeds is appreciated in alternative medicine, such as proteins present in the Syzygium cumini seed. Syzygium cumini seed extracts are known for their hypoglycemic, antiinflammatory, antibacterial, anti-HIV activ-ity, antidiarrheal effects, and antiarthritic activi-ties (Binita et al., 2014). These seeds contain from 6.3% to 8.5% protein content and, among them, it is possible to highlight the presence of lacto-ferrin. Lactoferrin is a mammalian origin pro-tein with antibacterial activity that was purified from S. cumini seeds using different chromato-graphic techniques.

Antimicrobial proteins have also been ob-served in passion fruit seeds. Agizzio et al. (2003, 2006) found two proteins (12.088 and 11.930 kDa) from passion fruit seeds that showed in vitro antifungal activity by inhibiting the growth of the phytopathogenic fungi Fusarum oxysporum and Colletotrichum lindemuthianum and the yeast Saccharomyces cerevisiae. These two proteins presented sequence homologies with 2S albumins. Later on, Lam and Ng (2009) iso-lated a dimeric protein (67 kDa) called passiflin from passion fruit seeds that exerted antifungal properties. Its N-terminal amino acid sequence was remarkably similar to that of antifungal bo-vine β-lactoglobulin. Moreover, it also inhibited proliferation of breast cancer cells.

Japanese researchers also described thera-peutic properties in some proteins from peach seeds. They reported that peach seed proteins PR-A and PR-B of approximately 300 and 10 kDa, respectively, showed antiinflammatory properties. Both PR-A and PR-B proteins and also the whole protein extract demonstrated an-tiedema, antigranulation, and antiwrithing ac-tivities in in vivo experiments with rats (Arichi et al., 1985a, b).

Peach (Prunus persica L. Batsch), apricot (Prunus armeniaca L.), plum (Prunus domestica L.), cherry (Prunus cerasus L.), and olive (Olea europeae)



seeds have high protein contents. These seed proteins have been extracted (González-García et al., 2014; Esteve et al., 2015; García et al., 2015; Vásquez-Villanueva et al., 2015) with Tris-HCl buffers (pH 7.5) containing sodium dodecylsul-fate and dithiothreitol with previous extraction of fat with hexane. Extractions were accelerated by using high-intensity focused ultrasounds, and solubilized proteins were precipitated with cold acetone. Protein isolates with protein con-tents higher than 90% were obtained from these seeds. These isolates were digested with differ-ent enzymes under optimized conditions: alca-lase, thermolysin, neutrase, PTN, flavourzyme, and protease P. Hydrolysis degrees ranged from 46% to 91%, observing the highest amount of peptides when using thermolysin and alcalase.

The antioxidant capacity of digested extracts was evaluated using up to five different in vi-tro antioxidant assays covering different anti-oxidant mechanisms: radical (ABTS [2,2’-azino-bis{3-ethylbenzothiazoline-6-sulfonic acid}], hydroxyl, and DPPH) scavenging capac-ity, reduction capacity (ferric reduction power [FRAP] assay), and inhibition of lipid peroxida-tion (Fig. 11.2). Enzymes yielding the extracts with the highest antioxidant capacity were, in general, alcalase and thermolysin. Apricot, plum, and olive seed proteins hydrolyzed with alcalase showed, in general, the highest antioxi-dant capacities whereas cherry seed hydrolysate seemed to be the least antioxidant. When ther-molysin enzyme was employed, cherry seed hydrolysate kept on yielding the lowest antioxi-dant capacity whereas peach seed hydrolysate showed the highest antioxidant capacity for the ABTS radical scavenging assay and the lipid peroxidation assay.

Regarding the ACE inhibitor capacity, all seeds yielded the highest capability when they were hy-drolyzed with thermolysin enzyme. IC50 values of hydrolysates from seeds of the Prunus species were similar and ranged from 0.25 to 0.30 mg/mL. The IC50 value obtained with the apricot seed hydrolysate (0.26 ± 0.02 mg/mL) was compared

with the observed by Zhu et al. (2010). The authors employed six different proteases: flavourzyme, neutrase, protamex, alcalase, proleather, and pa-pain. The highest ACE inhibitor capability was observed with alcalase (IC50 = 0.378 ± 0.015 mg/mL). According to these results, thermolysin is a most suitable enzyme to obtain potential antihy-pertensive peptides from apricot seeds, followed by alcalase.

Peptides extracts were fractionated using ul-trafiltration filters with 3 and 5 kDa molecular weight cutoffs. In most cases, antioxidant ca-pacity in fractions was lower than that obtained in whole peptide extracts (Esteve et al., 2015; González-García et al., 2015; Vásquez- Villanueva et al., 2016; García et al., 2015). This is probably because of a synergic effect that is usual among antioxidant compounds. The highest ACE in-hibitor capacity was observed, in all cases, in the fraction below 3 kDa of the thermolysin ex-tract. Peptides in most active fractions were next identified by electrospray quadrupole time-of-flight mass spectrometry coupled to RP-HPLC and to hydrophilic interaction chromatography ( HILIC). All identified peptides contained a high amount of hydrophobic amino acids, which is a typical feature of ACE inhibitor and antioxidant peptides (Erdmann et al., 2008; Sarmadi and Is-mail, 2010). Prunus seeds showed common pep-tides but olive seeds did not show any peptide in common with the Prunus species. Furthermore, a study on the stability of peptides to a simu-lated gastrointestinal digestion was also carried out for peach, olive, and plum seed peptides. The results demonstrated that gastrointestinal enzymes did not modify or slightly reduced the capacities of bioactive peptides.

4.5 Other Vegetal-Origin Protein By-Products

Wool waste is mainly composed of keratin and is produced during wool processing. One limitation for its use is its resistance to con-ventional enzymes, such as pepsin, trypsin, or



papain. The use of keratinolytic microorganisms enabled an increase in the digestibility of this waste and produced peptides with antioxidant activity (Fakhfakh et al., 2013).

Okara is the residue obtained in the manu-facture of soybean milk and tofu from soybean. This residue can contain from 24.5 to 37.5 g pro-teins/100 g and has been employed as a source of bioactive peptides. Peptides below 1 kDa

released after in vitro gastrointestinal digestion showed antioxidant and ACE inhibitor activi-ties. Moreover, a peptide derived from lipoxy-genase with significant antioxidant activity was identified (Jiménez-Escrig et al., 2010).

Turmeric (Curcuma longa) is a plant used to extract curcumin, appreciated as a spice, color-ing agent, and for its flavor. Turmeric waste grits are a by-product produced in the isolation of

FIGURE 11.2 Comparison of antioxidant capacity evaluated using four different in vitro assays (ABTS radical-scav-enging assay, hydroxyl radical-scavenging assay, FRAP assay, and lipid peroxidation inhibition assay) of hydrolysates obtained from cherry, apricot, peach, plum, and olive seed proteins using alcalase and thermolysin enzymes. (Data from Esteve et al., 2015; García et al., 2015; González-García et al., 2014; Vásquez-Villanueva et al., 2015).



curcumin from turmeric. This by-product con-tains β-turmerin (34 kDa) protein. Smitha et al. (2009) purified and characterized this protein and observed a high antioxidant capacity. The protein extraction from turmeric waste grits in-volved treatment with hot water and polyvinyl pyrrolidone to remove polyphenols.

5 CONCLUSIONS AND FUTURE PROSPECTS

A lot of concern rests on the residues derived from the food-processing industry because they can produce environmental pollution and health risks. Additionally, the sustainable exploitation of food resources is an important aspect in view of the growing world population. Conversely, there is a demand for functional foods that have an active role in the prevention of certain dis-eases, especially those derived from aging. The reuse and revalorization of protein by-products is an arising trend that can solve these problems. Protein by-products have been employed to ob-tain bioactive peptides and proteins. Hydrolysis of protein by-products with commercial prote-ases is the most usual strategy to obtain bioac-tive peptides. The most abundant bioactive pep-tides exerted antihypertensive and antioxidant activity. The most explored protein by-products are of marine origin.

Future work should focus on protein by-prod-ucts that have not been explored yet or that need to be further explored. Taking into account the increased world consumption of plant foods in comparison with animal foods, future research should be directed to the exploitation of protein by-products from vegetal origin.


ABTS 2,2’-Azino-bis(3-ethylbenzothiazoline-6- sulfonic acid)

ACE Angiotensin-converting enzymeDPPH 2,2-Diphenyl-1-picrylhydrazyl

FRAP Ferric reduction powerHILIC Hydrophilic interaction chromatographyHIV Human immunodeficiency virusRP-HPLC Reversed-phase high-performance liquid chro-

matography

AcknowledgmentsThis work was supported by the Ministry of Economy and Competitiveness (ref. AGL2012-36362) and the Comunidad Autónoma de Madrid and European funding from FEDER program (S2013/ABI-3028, AVANSECAL).

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C H A P T E R


12Microalgal-Based Protein By-Products: Extraction,

Purification, and ApplicationsT. Chiong*,**, C. Acquah*,**, S.Y. Lau*,

E.H. Khor*, M.K. Danquah**Department of Chemical Engineering, Curtin University, Miri, Sarawak, Malaysia; **Curtin Sarawak Research Institute, Curtin University, Miri, Sarawak, Malaysia

1 BACKGROUND

Microalgae have been identified as an alterna-tive protein source in response to insufficient pro-tein supply from conventional sources, resulting from increasing world population (Becker, 2007). Microalgae biotechnology has therefore cap-tured the interests of many researchers and is emerging as a sustainable value-adding process for the development of biochemical and biomol-ecules for a wide range of applications to meet vital consumer needs. Apart from playing a role in cosmeceuticals, nutraceuticals, and func-tional foods (Borowitzka, 2013), mass culturing of microalgal species has also been explored for wastewater treatment (Pires et al., 2013; Razzak et al., 2013) and carbon dioxide fixation (Pires et al., 2012; Razzak et al., 2013). Microalgae are photosynthetic microorganisms that can grow

rapidly in harsh conditions because of their unicellular or simple multicellular structure (Li et al., 2008; Mata et al., 2010). They are the most widely distributed organisms in the world and can be found in almost every water body (Garcia-Moscoso et al., 2013). More than 50,000 species of microalgae are in existence, but only a small fraction has been well studied and classi-fied, with a much lower number being cultivated on a large scale (Rui, 2008).

The majority of the commercially available re-combinant proteins are expressed from bacterial (Andersen and Krummen, 2002; Swartz, 2001), yeast (Kushnirov, 2000; Zhang et al., 2011; Cereghino et al., 2002), and mammalian cell culture (Bandaranayake and Almo, 2014; Schutt et al., 1997). Bacterial and yeast fermentations have often been the expression systems of choice for the production of recombinant proteins.

214 12. MICROALGAL-BASED PROTEIN BY-PRODUCTS: EXTRACTION, PURIFICATION, AND APPLICATIONS


Fermentation systems are easily developed, and the production cost is low once the initial capital investment to procure fermentation and purification units is covered. However, bacterial fermentation is limited in application as bacte-ria are unable to perform posttranscriptional and posttranslational modifications essential for the production of functional eukaryotic pro-tein. High intracellular levels of heterologous proteins also tend to result in the formation of protein aggregates as insoluble inclusion bodies. In addition, the presence of bacterial endotoxins and proteases makes purification difficult and can lead to adverse effects in humans (Walker et al., 2005). Most of the problems associated with bacteria expression systems are overcome by the use of yeast. Yeast folds and assembles mammalian proteins effectively and carries gly-cosylation. However, the glycan chains synthe-sized by yeast and mammalian cells are very different (Schillberg et al., 2003). The pattern of glycosylation does not commensurate that of higher organisms and usually involves hyper-glycosylation (Walker et al., 2005). Mammalian cell cultures are expensive to maintain and scale up and are susceptible to pathogenic contamina-tions (such as viruses and prions) or oncogenic DNA sequences, thus raising safety concerns es-pecially for therapeutic application in humans (Schillberg et al., 2003).

As genetically accessible photosynthetic or-ganisms, microalgae are now recognized as a potential alternative for recombinant protein expression (Specht et al., 2010). Microalgae are capable of high-level protein expression in low-cost growth media and are easy to culture un-der various growth conditions using minimal energy (He, 2004). Microalgae expression sys-tems are inherently faster to develop, requir-ing only a few weeks from the generation of initial transformants to scaling up of produc-tion volumes (Mayfield et al., 2003; Mayfield et al., 2007). This is considerably shorter than with higher plants; 2 and 3 years for tobacco and corn, respectively (Manuell et al., 2007).

Recombinant proteins can be expressed from the nuclear, chloroplast, and mitochondrial genomes of some microalgal species (Gong et al., 2011). Both the chloroplast and nuclear genome of microalgae can be genetically trans-formed, making the production of a variety of transgenic proteins in a single organism pos-sible (Mayfield et al., 2003). Microalgae can be grown in closed bioreactors, reducing the risk of contamination by airborne contaminants, and also protecting the environment from any potential flow of transgenes into the surround-ing ecosystem (Specht et al., 2010). The majority of green microalgae are categorized as general-ly recognized as safe (GRAS). This means they are safe for consumption, and therefore po-tentially a source therapeutic proteins for oral delivery with minimal purification (Mayfield et al., 2007; Specht et al., 2010).

Proteins make up a large fraction of the bio-mass of actively growing microalgae, but they are generally undervalued compared to minor products, such as omega fatty acids (González López et al., 2010). Table 12.1 compares the pro-tein contents of various microalgae species with other human food sources.

Production of intracellular metabolites from microalgae involves extensive upstream and downstream processing steps that start with microalgal cultivation, algal biomass recovery, or harvesting, followed by further processing, such as dewatering, drying, cell disruption, extraction, and product purification (Show et al., 2015). The intrinsic rigidity of the micro-algae cell wall often inhibits the total extrac-tion of specific intracellular components. A cell disruption operation is thus required to permit complete access to the internal components and facilitate the extraction process (Safi et al., 2014). A variety of disruption methods are available to disintegrate microalgae cell walls and mem-branes to liberate intracellular contents (Show et al., 2015). Table 12.2 presents some cell disrup-tion methods for microalgae protein production along with protein yields.

2 MICROALGAL PROTEINS 215


2 MICROALGAL PROTEINS

A variety of recombinant proteins have been produced experimentally from the nuclear and chloroplast genomes of transgenic Chlamydomo-nas reinhardtii. This includes monoclonal anti-bodies, vaccines, hormones, and pharmaceutical

proteins (Gong et al., 2011). A large single-chain (lsc) antibody, namely HSV8-lsc, was expressed in the chloroplast of C. reinhardtii by Mayfield et al. (2003), and a high level of protein accumu-lation was achieved by synthesizing the lsc gene in the chloroplast codon bias, and by driving expression of the chimeric gene using either of

TABLE 12.1 Comparison of Various Protein Sources and Their Relative Percentage Amount

Category Source Protein content (%)

Conventional source Bakers’ yeast 39

Meat 43

Milk 26

Rice 8

Soybean 37

Microalgae sources Anabaena cylindrical 43–56

Aphanizomenon flos-aquae 62

Arthrospira platensis 52–55

Chaetoceros calcitrans 34–38

Chlamydomonas reinhardtii 48

Chlorella vulgaris 51–58

Chlorella pyrenoidosa 57

Dunaliella salina 57

Dunaliella oculata 49

Euglena gracilis 39–61

Haematococcus pluvialis 51–53

Isochrysis galbana 45–51

Nannochloropsis oculata 44–49

Porphyridium cruentum 28–39

Prymnesium parvum 28–45

Scenedesmus obliquus 50–56

Scenedesmus quadricausa 47

Scenedesmus dimorphus 8–18

Spirulina maxima 60–71

Spirulina platensis 46–63

Synechococcus sp. 63

Spirogyra sp. 6–20

Tetraselmis maculate 52

Adapted from Spolaore et al. (2006); Harun et al. (2010); Safi et al. (2014); Natrah et al. (2007).



two C. reinhardtii chloroplast promoters and 5_ and 3_RNA elements. This lsc antibody contains the entire immunoglobulin A (IgA) heavy chain fused to the variable region of the light chain, and accumulates as a fully soluble protein in the chloroplast. Chlamydomonas reinhardtii is also capable of synthesizing and assembling a full-length IgG1 human monoclonal antibody (mAb) in transgenic chloroplasts (Tran et al., 2009). The antibody, 83K7C, derived from human IgG1 is directed against anthrax antigen 83 (PA83). It has been shown to block the effects of anthrax toxin in animal models (Tran et al., 2009). The microalgae-expressed 83K7C binds to PA83

in vitro with similar affinity to the mammalian-expressed 83K7C antibody. A bioactive mam-malian protein called bovine mammary-associated serum amyloid (M-SAA) was expressed in Chlam-ydomonas chloroplast, and the accumulation of this soluble protein was above 5% of total protein (Manuell et al., 2007). The chloroplast-expressed M-SAA contains the correct amino terminal se-quence with minimal posttranslational modifi-cation and is able to stimulate mucin production in human gut epithelial cell lines.

Chlamydomonas has also been used for vaccine production. An expression vector, pACTBVP1, containing the fusion of food-mouth-disease

TABLE 12.2 Various Cell Disruption Methods for Microalgae Protein Production

Protein extraction % Protein yield References

Two passes of high-pressure homogeniza-tion at 2700 bar

Porphyridium cruentum 90.0%Arthrospira platensis 78.0%Chlorella vulgaris 52.8%Nannochloropsis oculata 52.3%Haematococcus pluvialis 41.0%

Safi et al. (2014)

High pressure homogenization (150 MPa, 6 passes)

91% extraction from Nannochloropsis sp. Grimi et al. (2014)

Alkaline (pH12) treatment of freeze-dried biomass at 40°C for 1 h

P. cruentum 80.3%A. platensis 69.5%C. vulgaris 43.3%N. oculata 33.3%H. pluvialis 27.5%

Safi et al. (2013)

Electroextraction using repetitive 2-ms-long pulses of alternating polarities with certain field strengths

Nannochloropsis salina 400% higher soluble protein recovery than the negative control (unpulsed suspension of microalgae)

Coustets et al. (2013)

Flash hydrolysis in subcritical water within 10s residence time

More than 60% of dry weight Garcia-Moscoso et al. (2013)

Enzyme assisted extraction at alkaline pH 50–80% w/w Sari et al. (2013)

Bead milling for 30 min followed by ion exchange chromatography

64% w/w in algae soluble isolate Schwenzfeier et al. (2011)

Milling with a pestle and mortar in the presence of an inert ceramic powder (aluminum oxide) for 5 min

30–55% of dry weight González López et al. (2010)

Extraction using ultrapure water for 12 h and grinding with a Potter homog-enizer. Protein precipitation using 25% trichloroacetic acid-to-homogenate (2.5:1 v/v)

Hillea sp. 15.3%Dunaliella tertiolecta 11.4%Skeletonema costatum 11.1%Amphidinium carterae 10.2%Isochrysis galbana 10.1% dry matter

Barbarino and Lourenço (2005)

2 MICROALGAL PROTEINS 217


virus (FMDV) VP1 gene and cholera toxin B (CTB) subunit gene was transferred into the chlo-roplast genome of C. reinhardtii by biolistic meth-od (Sun et al., 2003). The CTBVP1 fusion protein expressed in the chloroplast accounted for up to 3% of total soluble protein. It also retained both GM1-ganglioside binding affinity and antigenic-ity of the FMDV VP1 and CTB proteins. Classical swine fever virus (CSFV) structural protein E2 has also been established using C. reinhardtii (He et al., 2007). E2 protein accumulated up to 1.5–2% of total soluble protein based on enzyme-linked immunosorbent assay (ELISA) quantification. The protein immunogenicity test revealed that subcutaneous immunization of extracts of E2 in-duced significant serum antibody against CSFV. However, oral immunization of naive mice with these extracts did not result in detectable serum responses, suggesting that large doses of antigen or strong mucosal adjuvants might be needed. In another study by Surzycki et al. (2009), VP28 protein from the white spot syndrome virus was expressed in the chloroplast of C. reinhardtii. They also reported that the key factors affect-ing the level of recombinant protein expres-sion in the chloroplasts are codon optimization, protease activity, protein toxicity, and transfor-mation-associated genotypic modification. The accumulated VP28 protein yield was reported to be more than 20% of total cell protein.

The utilization of fusion proteins in microal-gal chloroplast as a method to increase accumu-lation of recombinant proteins that are difficult to express was demonstrated by Muto et al. (2009). The recombinant reporter protein, lucif-erase, accumulated to significantly higher levels than luciferase expressed alone. Near-wild type levels of functional Rubisco holoenzyme were generated following the proteolytic removal of the fused luciferase, while luciferase activity for the fusion protein was almost 33 times great-er than luciferase expressed alone. The study also showed that engineered proteolytic process-ing sites can be used to liberate the exogenous protein from the endogenous fusion partner,

allowing for the purification of the intended ma-ture protein. Franklin et al. (2002) showed the importance of codon optimization of reporter genes to achieve high levels of recombinant protein expression. They synthesized a gene en-coding green fluorescent protein (GFP) de novo and optimized its codon usage to reflect that of major C. reinhardtii chloroplast-encoded pro-teins. GFP accumulated in the chloroplast trans-formed with the codon-optimized green fluores-cent protein cassette (GFPct) was approximately 80-fold more than nonoptimized ones.

The diatom Phaeodactylum tricornutum has also been proven to successfully express monoclonal human IgG antibody against the hepatitis B sur-face protein (Hempel et al., 2011). The antibody CL4mAb was expressed and assembled in the endoplasmatic reticulum of P. tricornutum and accumulated up to 8.7% of total soluble protein in 2 days of induction. In addition, this work also demonstrated the possibility of expression of hepatitis B surface antigen (HBsAg) in a microal-gal system, with an accumulation of 0.7% of total soluble protein. HBsAg gene was also introduced into the cell of Dunaliella salina (Geng et al., 2003) by using electroporation, and the expression was stably maintained for at least 60 generations in medium devoid of chloramphenicol.

Phycobiliproteins, which are considered ideal marker pigments for understanding the distri-bution and trophic dynamics of picoplankton populations, can be found in cyanobacteria (blue–green algae). These highly fluorescent proteins were extracted from a Synechococ-cus CCMP 883 cyanobacteria culture by using a buffer composed of 3% 3-[(3-cholamidopro-pyl) dimethyammonio]propanesulfonic acid (CHAPS) and 0.3% asolectin combined with nitrogen cavitation (Viskari and Colyer, 2003). The extraction efficiency by this method was greater than 85% in less than 3 h. The complete resolution of the protein components of phy-cobilosome from cyanobacterium Synechocystis 6803 alongside their molecular mass detection and determination has also been demonstrated



through the combined use of high-performance liquid chromatography (HPLC) coupled on-line with electrospray ionization mass spectrometry (Zolla and Bianchetti, 2001). This method also allowed simultaneous separation and identifi-cation of phycobiliproteins, phycocyanin, and allophycocyanin from linker proteins. Simo et al. (2005) also showed that capillary electro-phoresis–mass spectrometry (CE–MS) with opti-mized operating parameters was able to analyze the main proteins (namely, allophycocyanin-α chain, allophycocyanin-β, C-phycocyanin-α, and C-phycocyanin-β) found in Spirulina platensis. The operating parameters include composition of the separation buffer, electrospray conditions, and washing routine between runs (Table 12.3).

2.1 Proteins as By-Products from Microalgal Bioprocesses

The inherent superior characteristics of mi-croalgae over other autotrophs enhance their

cultivation for carbon sequestration and the de-velopment of a wide range of biochemicals. Mi-croalgae species often possess carbon concentrat-ing mechanisms that allow growth under varied CO2 concentrations and growing conditions to augment intracellular concentrations of dis-solved inorganic carbon (Zeng et al., 2011, 2012; Menetrez, 2012). Microalgae account for more than 40% of global carbon fixation (Hannon et al., 2010), resulting in the conversion of inor-ganic carbons into beneficial biochemical organ-ic compounds rich in oil, lipids, proteins, and carbohydrates (Zeng et al., 2011, 2012). Extracted lipids from microalgae may exist as omega oils for food and pharmaceutical applications or true lipids for the production of biodiesel. Proteins and carbohydrates extracted can be used for the production of cosmetics, food and feed proteins, pharmaceutical products, and bioethanol (Soh et al., 2014; Menetrez, 2012).

The presence of different intracellular com-ponents of commercial value necessitates the

TABLE 12.3 Protein Production in Microalgae

Protein expressed Function Microalgae References

83K7C Full-length IgG1 human monoclonal antibody against anthrax protective antigen 83

Chlamydomonas reinhardtii

Tran et al. (2009)

M-SAA Bovine mammary-associated serum amyloid; stimulate mucin production in human gut epithelial cell lines

C. reinhardtii Manuell et al. (2007)

VP28 White spot syndrome virus protein 28 C. reinhardtii Surzycki et al. (2009)

Human glutamic acid decarboxylase 65 (hGAD65)

Autoantigen in type 1 (insulin-dependent) diabetes

C. reinhardtii Wang et al. (2008)

VEGF Treatment for pulmonary emphysema C. reinhardtii Rasala et al. (2010)

Green fluorescent protein (GFP) Reporter gene C. reinhardtii Franklin et al. (2002)

CL4mAb Monoclonal human IgG antibody against hepatitis B virus surface protein

Phaeodactylum tricornutum

Hempel et al. (2011)

HBsAg Vaccine against hepatitis B P. tricornutumDunaliella salina

Hempel et al. (2011)Geng et al. (2003)

Phycobiliproteins Ideal marker pigments; colorants in food and cosmetics

SynechococcusSynechocystisSpirulina platensis

Viskari and Colyer (2003)Zolla and Bianchetti (2001)Simo et al. (2005)

3 BIOPROCESS DEVELOPMENT 219


establishment of biorefineries. Microalgal bio-refineries have the potential to maximize profit margins through the diversification of bioprod-uct portfolios. For example, the production of microalgae biomass to generate 4 billion m3 of biodiesel contains ∼40% of proteins as by-products (Wijffels and Barbosa, 2010). This amounts to more than 3 billion tons of proteins as compared to an estimated 18 million tons of soy-bean containing ∼40% protein imported into the European market as of 2008 (Wijffels and Barbosa, 2010). In addition, the market prices of recombinant proteins and industrial en-zymes are in the region of US$5–10,000/kg and $25–50/kg, respectively (Hannon et al., 2010). Barreiro et al. (2014) demonstrated that the ex-traction of proteins from Nannochloropsis gadi-tana and Scenedesmus almeriensis led to an im-provement in the quality and yield of biocrude as the main product. Furthermore, the therapeu-tic efficacy of microalgae protein waste gener-ated from the production of algae essence from Chlorella vulgaris has been studied in the produc-tion of anticancer peptides (Sheih et al., 2009). Remarkably, its antioxidant activity toward per-oxyl radicals and low-density lipoprotein were promising. Lipid-extracted microalgae biomass generally contains the following compositions: protein, carbohydrate, and obstinate contents of residual biomass in the proportion 52, 25, and 23%, respectively (Brentner et al., 2011). These components can be collectively recovered for the production of methane gas (Brentner et al., 2011; Hernández et al., 2014) and biohydrogen gas (Nobre et al., 2013) to power energy systems.

Techniques used in extracting microalgae intracellular components are expected to be be-nign to obtain quality yields of products and by-products. Among all the known techniques for extracting microalgae proteins, as discussed later in Section 3.4, enzymatic and continuous-flow techniques, such as pulsed electric field, supersonic flow fluid processing, and ultra-sound are thought of as the most appropri-ate for quality multicomponent derivation

(Vanthoor-Koopmans et al., 2013). Ionic liquids and surfactants are also preferred for the sepa-ration of multicomponents, especially when the protein by-product is of interest. Character-istics of ionic liquids that make them suitable for multicomponent separations are low melt-ing temperature, recyclability, and the propen-sity to separate hydrophilic from hydrophobic compounds. Conversely, surfactants are largely employed for specific proteins to retain their functionality (Vanthoor-Koopmans et al., 2013). Efficient extraction of protein (30–60% dry weight nitrogen) to yield lipid-rich biofuel inter-mediates from Scenedesmus sp. by means of flash hydrolysis has also been demonstrated (Garcia-Moscoso et al., 2013).

3 BIOPROCESS DEVELOPMENT

3.1 Transgenic Modifications

A number of factors are considered in the se-lection of techniques for the production of pro-teins. These include the protein nature, yield requirement, duration, ease of manipulation, complexity of purification, and production cost (Barzegari et al., 2010). Microalgae-based pro-teins, just as other microbial cell proteins, are in-tracellular and thus require transgenic modifica-tions to enable their expression into the growth medium. Also, mechanisms that will lyse the cell membranes to enable release of proteins into the surrounding medium can be employed (Chisti and Moo-Young, 1986; Geciova et al., 2002). These alternative mechanisms are discussed in Section 3.4. The nature of the microalgal cell wall has an influence on the type of lysis method and parameters for external expression of cytoplas-mic proteins (Geciova et al., 2002).

Advances in genetic modification have led to the successful modification of the genomes of various fermentation systems. Expression of recombinant proteins in microalgae can occur in the genomes of the nucleus or plastids (Cadoret



et al., 2008, 2012; Rasala et al., 2010) with either having pros and cons. The characteristic features of nuclear transgenesis include posttranslational modification, production of glycosylated pro-teins, and regulated and tissue-specific expres-sions (Rasala et al., 2010). However, they are faced with poor yields and transgenic silencing (Cadoret et al., 2012; Rasala et al., 2010, 2012). A mechanism to debottleneck issues of transgene silencing in the nucleus of C. reinhardtii for the selection marker Ble has been developed (Rasala et al., 2012). Contrary to nuclear transgenesis, plastid genomes, such as the chloroplast, have a higher protein yield, strong endogenous pro-moters, allow the insertion of genes by homolo-gous recombination into the genome, and en-able gene silencing (Cadoret et al., 2012; Gimpel et al., 2015). However, the chloroplast requires cell lysis to enable extraction of expressed pro-teins and is unable to produce glycosylated pro-teins (Rasala and Mayfield, 2011). Transgenic modifications are largely employed during the synthesis of recombinant proteins and the rapid enhancement of protein metabolism in micro-algal systems. The use of Agrobacterium tumefa-ciens, rodlike Gram-negative bacteria, has been applied for transgenic modification of wild-type C. reinhardtii strains (Kumar et al., 2004). The developed protocol reportedly yielded a higher transformation frequency by 50-fold when com-pared to glass-bead techniques. Unlike other techniques that required the use of microalgae species deficient in cell wall (such as D. salina and Porphyridium cruentum), the use of A. tume-faciens was possible for microalgae with intact cell walls and was devoid of contamination.

The extraction of proteins from natural sources can be expensive, tedious, nonexistent for some proteins (such as single-chain fv frag-ments), and prone to external contamination (Barzegari et al., 2010). Most studies on the ex-pression of recombinant proteins from eukary-otic microalgae have been centered on C. rein-hardtii, owing to the fact that all its genomes (nuclear, chloroplast, and mitochondria) are

well characterized (Pourmir et al., 2013) and are commercially viable. Other commercially viable species include P. tricornutum (diatom), D. salina, Coccomyxa sp., and Chlorella ellipsoidea (Mathieu-Rivet et al., 2014). Analogous to C. reinhardtii, D. salina also has competitive biological and techni-cal features, such as inexpensive media require-ments, rapid growth, ease of manipulation, and high capacity for posttranslational modifica-tions (Barzegari et al., 2010). To establish a com-mercially viable protein production system from microalgae, some pertinent issues ought to be addressed. Mathieu-Rivet et al. (2014) discussed challenges masking the protocols, which are es-sential to increase the output of recombinant proteins as compared to the traditional systems such as the Chinese hamster ovary (CHO) cell lines. Transgenic modifications of microalgae for commercial production of proteins are possible, owing to the ability to identify the appropriate genes that lead to the production of specified proteins (Cadoret et al., 2012). That notwith-standing, there is still no commercial produc-tion of recombinant proteins from microalgae on the market (Cadoret et al., 2012). Optimiza-tion of production yield for commercial produc-tion of recombinant protein can be achieved by engineering the codon region to replace an endogenous psbA gene with a vector driven by the psbA promoter and 59 untranslated regions (UTRs) (Manuell et al., 2007). Nevertheless, the absence of the endogenous psbA genes ren-dered the microalgae nonviable for large-scale processes because of the consequential lack of photosynthesis (Gimpel et al., 2015). Remark-ably, Gimpel et al. (2015) first reported the pro-duction of the recombinant protein, bovine milk amyloid A (MAA), in microalgae using psbA coding sequences to regenerate photosynthesis, which is a remedy to the previously discussed hurdle in the expression of recombinant pro-teins from microalgae. The developed proto-col yielded MAA proteins at 46-fold higher in amount than the natural MAA protein source, bovine colostrum, and is also expected to reduce



economics by 60 times compared to the natural sources currently commercially available (Gim-pel et al., 2015). In addition, Eichler-Stahlberg et al. (2009) successfully demonstrated that the incorporation of endogenous intron sequences into the expression cassettes of C. reinhardtii nu-cleus increased the yield of luciferase proteins. Hempel and Maier (2012) engineered the diatom P. tricornutum to secrete proteins into the culture medium, thereby circumventing extraction pro-cesses. Recombinant proteins produced within diatoms have high purities because diatoms do not naturally secrete much proteins. Hence, this discounts the need for purification processes (Hempel and Maier, 2012). Fig. 12.1 shows the bioprocess steps for the development and pro-duction of recombinant proteins.

3.2 Photobioreaction

Large quantities of microalgal biomass can be obtained from controlled photobioreaction under optimal conditions. However, biochemi-cal compositions of microalgae may vary in response to seasonal changes and environmen-tal parameters, such as medium temperature, salinity, light, nutrient supply, and production technology (Buono et al., 2014). The efficiency of such photobioreaction systems depends on pho-ton-to-biomass conversion (Liao et al., 2014). There are two types of systems for microalgae cultivation. These are open systems (such as raceway ponds, shallow ponds, and circular tanks) and closed photobioreactors (such as flat and tubular photobioreactors). While open tank systems are inexpensive, growth is easily affected by variations in environmental param-eters, and this leads to a decrease in the com-position of preferred compounds, loss of carbon dioxide and water through evaporation, and culture contamination. Closed photobioreactor systems cultivate microalgae cells at set param-eters to attain the desired composition (Harun et al., 2010; Buono et al., 2014; Liao et al., 2014). They are, however, expensive to operate, have a

high capital cost, and require more research to optimize yield-to-cost ratio (Harun et al., 2010; Buono et al., 2014). Liao et al. (2014) discussed various approaches proposed for the enhance-ment of light absorption and consequent bio-mass conversion. These include (1) shortening the light path, (2) enlarging the surface area–to-volume ratio, (3) directing light into the photobi-oreactors, and (4) applying a light-to-dark cycle.

3.3 Culture Harvesting and Dewatering

Microalgal harvesting and dewatering are re-garded as one of the challenging and expensive steps in the production of microalgal biomass. Such processes may involve centrifugation, floc-culation, floatation, filtration and screening, gravitational sedimentation, and electropho-retic techniques (Keris-Sen et al., 2014; Udu-man et al., 2010). The selection of an efficient and versatile technique is proposed to be one that is applicable to most microalgae species in order to obtain high dry biomass weights at an affordable cost (Danquah et al., 2009). Notably, the choice of optimal techniques for microalgae harvesting and dewatering has been observed to differ, owing to the vast differences in the biological and physicochemical characteristics (such as cell membrane charge, conformation, and particulate cell size) of microalgae species (Uduman et al., 2010). Research advances are now being focused on establishing technologies capable of extracting intracellular components directly by circumventing the processes of de-watering (Keris-Sen et al., 2014).

3.4 Cell Lysis Techniques

There exist variations in the cell walls of dif-ferent microalgal species with some having frag-ile structures and others having thick structures. Disruption of the microalgal cell wall can be achieved by either mechanical or nonmechanical means to extract endogenous proteins. Mechani-cal techniques include milling with either glass



FIGURE 12.1 Schematic bioprocess flow for the production of recombinant proteins. Adapted from Mayfield et al. (2007); Gong et al. (2011).



beads or silicon carbon whiskers, high-pressure homogenization, and ultrasonication. Nonme-chanical techniques include chemical treatments with solvents, such as sodium dodecyl sulfate; extraction using electrical fields; physical disrup-tion, such as flash freeze-thaw; and enzymatic treatments, such as lyticase enzyme (Coustets et al., 2015; Chisti and Moo-Young, 1986; Zhou et al., 2014; González López et al., 2010). Fig. 12.2 gives an illustration of a plethora of mechanisms for producing and extracting proteins. The choice of the appropriate technique and efficiency of protein release is dependent on the mechanical ri-gidity and chemical characteristics of the cell wall (Safi et al., 2014; Barbarino and Lourenço, 2005)

3.4.1 ElectroporationElectroporation is the formation of irrevers-

ible pores in the cytoplasmic membrane of cells when subjected to high and pulsating electric fields. This then causes the cell membrane to lose its inherent functional properties for elec-trical resistance, membrane potential, and as a medium barrier (Sheng et al., 2011). The key parameters in protein extraction using this technique are the field strength and pulse du-ration (Coustets et al., 2015). Cell membrane permeation is achieved at a determined criti-cal field effect and is inversely proportional to the size of the microalgae and its organelles (Coustets et al., 2015). The cytoplasm of a living

FIGURE 12.2 An illustration of different techniques for protein extraction. Section A shows two broad classes of proteins and Section B shows their mechanism of extraction.



cell possesses complex electrical signal circuits and bioelectric interactions essential for regener-ation, morphogenesis, and left–right patterning, thus facilitating the manipulation of organelles by external electric fields (Esser et al., 2010). In addition, the cytoplasmic membrane is made electrically transparent at externally applied frequencies > ∼100 kHz (Esser et al., 2010). Benefits derived by using an electroextraction approach for protein release from microalgae are the capacity to retrieve higher yield of pro-teins; assurance of protein stability; avoidance of chemical additives for protein extraction; environmental friendliness; absence of damage effects on microalgal vacuoles; and convenience for large-scale production (Coustets et al., 2013; Coustets et al., 2015).

3.4.2 UltrasonicationUltrasonication is one of the known tech-

niques for cell wall disruption. It is energy ef-ficient, readily scalable, and can be operated continuously. In addition, it possesses the ca-pacity to disrupt cell walls without the need for addition, utilization, and subsequent separa-tion of beads, which can prolong the processing time, as well as increase processing cost (Wang et al., 2014a; Gerde et al., 2012). Application of ultrasonication for cell wall lysis is based on ei-ther low-frequency nonfocused ultrasound (LF-NFU) treatment within the range of 20–40 kHz or high-frequency focused ultrasound (HFFU) treatment in megahertz (Wang et al., 2014a). Quantification of disrupted cell wall can be per-formed with the use of cell pigments, flow cy-tometry, and microscopic cell counting (Gerde et al., 2012). Wang et al. (2014a) demonstrated the effect of LFNFU and HFFU for cell disrup-tion of Scenedesmus dimorphus and N. oculata. It was observed that cell disruption by HFFU was more energy efficient than LFNFU (Wang et al., 2014a). However, the use of ultrasonica-tion has been reported unsuitable for the extrac-tion of bioactive proteins from microalgal spe-cies (González López et al., 2010).

3.4.3 MillingMilling is one of the widely used mechanisms

for cell lysis and is suitable for commercial-scale protein extraction. Milling can be done through the use of glass beads and silicon carbide. The ef-ficiency of cell wall disruption using bead mill-ing is affected by the suspension feed rate in a continuous process, residence time distribution in a batch process, agitator speed and design, milling chamber design, biomass concentration, bead diameter, bead density and filling (Postma et al., 2014). The location of the intracellular component of interest determines the size and loading of beads (Geciova et al., 2002). Although an increase in the bead loading reflects positive-ly on cell disruption, it generates more heat and consumes more power. Predefined optimum loading for bead loading has been reported to be 80–85% (Geciova et al., 2002). Cell wall dis-ruption efficiency of 54.4 ± 1.8% was achieved for Synechocystis PCC 6803 cyanobacteria spe-cies after a period of 10 min bead milling (Zhou et al., 2014). Zhou et al. (2014) demonstrated the superior characteristics of carbide whiskers over other mechanical techniques (sonication, glass beads, and freezing and thawing). A cell wall disruption efficiency of 93.3 ± 2.3% was achieved in 6 min for Synechocystis PCC 6803 cyanobacteria species to enable protein extrac-tion (Zhou et al., 2014).

3.4.4 Solvent ExtractionWater has been used to demonstrate success-

ful protein extractions from various microalgae species by means of osmosis (Barbarino and Lourenço, 2005). About 19–25% of dissolved proteins per dry weight were estimated to have dissolved from P. cruentum and Arthrospira pla-tensis. However, N. oculata and C. vulgaris, which have a cellulose and hemicelluloses cell wall, and Haematococcus pluvialis also having a thick trilaminar cell wall made of cellulose and spo-ropollenin, had only 6–10% of proteins being released (Safi et al., 2014). Also, an ionic liquid extraction method demonstrated the generation

4 APPLICATION OF MICROALGAL PROTEINS 225


of protein from Chlorella pyrenoidosa cells with a yield of 12.1% (Wang and Zhang, 2012). The optimum extraction was achieved when the ionic liquid was prepared via a mixture of 3-(dimethylamino)-1-propylamine and for-mic acid. Chemical reagents have also been employed in the treatment of a myriad of mi-croalgae for protein extraction. A. platensis, C. vulgaris, N. oculata, H. pluvialis, and P. cruentum were treated with NaOH solution at a pH of 12 to yield soluble proteins (Safi et al., 2014).

3.4.5 Freeze–Thaw ExtractionFreeze–thaw extraction was used for the ex-

traction of protein in C. pyrenoidosa cells but dis-played a low extraction yield of 3.2% under op-timum freezing conditions of −20°C for 80 min and thawing conditions of 60°C cycles (Wang and Zhang, 2012). However, a combination of a freeze–thaw operation with ultrasonication yielded up to 22.9% for identical microalgae spe-cies. This represents a significant improvement over ultrasonication (16%) and freeze–thaw operations as standard extraction techniques (Wang and Zhang, 2012). The cell wall of the cyanobacteria Synechocystis was disrupted for 45 min at an efficiency of 43.3 ± 2.5% through a freeze–thaw mechanism (Zhou et al., 2014).

3.5 Protein Purification and Quantification

Microalgae are GRAS, hence they largely do not require intensive, complex, and costly pu-rification processes as compared to other culti-vation systems, such as bacteria. However, ow-ing to the sensitive and specific applications of protein molecules, purification technologies are required to ensure the absence of any possible molecular contaminants. Protein purification can be performed by (1) precipitation at the iso-electric points, pI (2) concentration using tan-gential ultrafiltration, (3) HPLC, (4) foam frac-tionation, and (5) dye ligand chromatography (Ursu et al., 2014; Agyei et al., 2013). Table 12.4

presents a summary of purification technologies employed for the production of proteins from different microalgal cultivation systems.

The quantitative amount of proteins from microalgal biomass can be estimated using the following techniques: (1) a colorimetric meth-od with fluorescence based on chromophore binding redox reaction; (2) analysis of elemen-tal concentration of nitrogen and subsequent conversion to protein by Kjeldahl or Lowry method; and (3) a crude protein analysis, N × 6.25, where N is the total nitrogenous content (% of dry weight) and 6.25 the conversion fac-tor (González López et al., 2010; Barbarino and Lourenço, 2005). Colorimetric methods are de-pendent on the appearance of a chromophore and sensitivity interferences. They have accura-cies that are a function of the sample pretreat-ment method. Nitrogen elemental analyses are less prone to interferences. Nevertheless, nitro-gen-to-protein conversion factors are required for elemental nitrogen analysis (González López et al., 2010). Crude protein analysis method leads to overestimation of protein concentra-tion in microalgae, which is caused by the pres-ence of nonprotein nitrogenaceous components (Barbarino and Lourenço, 2005).

4 APPLICATION OF MICROALGAL PROTEINS

4.1 Natural Pigment

Phycobiliproteins are a basic class of natu-ral pigments (NPs) identified from microalgae, which have received great attention. Phycobili-proteins play an important role in the pigmenta-tion metabolism of microalgae and also exhibit some useful biological functions, such as anti-oxidative, anticarcinogenic, antiinflammatory, antiobesity, antiangiogenic, and neuroprotective properties (Cuellar-Bermudez et al., 2015). Phy-coerythrin (PE) is a red-colored phycobilipro-tein found in the chloroplast of red microalgae.

226

12. MIC

RO

ALG

AL-B

ASED

PRO

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BY-PR

OD

UC

TS: EX

TR

AC

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AT

ION

, AN

D A

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TIO

NS

III. TR

AN

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TABLE 12.4 Microalgal Protein Expression Systems and Purification Technologies

Microalgae bioreactor

Expression medium for protein

Plasmid type/expression vector Protein expressed

Purification Technologies Application References

Schizochytrium sp.

Endoplasmatic Reticulum

pCL0143, pCL0154, pCL0161, pCL0160, pCL0153 (Note: different plasmids for different HA source strains)

Hemagglutinin proteins of influenza virus

Centrifugation and filtration techniques.

Vaccine for influenza virus

Bayne et al. (2013)

Chlamydomonas reinhardtii

Chloroplast pTJ322-XR, pTJ322-optXR, and pTJ322-16S/optXR

Xylose reductase protein from the filamentous fungus Neurosporacrassa

Centrifugation and affinity gel purification

Production of xylitol Pourmir et al. (2013)

C. reinhardtii Nuclear pgLUC, pNSgLUC, pcCAgLUC

Recombinant Gaussialuciferase

Nickel affinity chromatography

Bioluminescence Lauersen et al. (2013)

C. reinhardtii Chloroplasts pCG2-FLAG Mutated E7 protein of Human Papillomavirus (HPV) type 16, E7GGG

Ni-NTA and anti-FLAG M2 Affinity gel

Vaccine against Human Papillomavirus (HPV)-related lesions

Demurtas et al. (2013)

C. reinhardtii Chloroplasts pJAG9 for a-pfs25 and pJAG15 for a-pfs28

Surface protein 25 (Pfs25) and 28 (Pfs28) of Plasmodium falciparum

Affinity purification with anti-FLAG M2affinity resin

Production of malaria transmission blocking vaccines

Gregory et al. (2012)

diatom Phaeodactylum tricornutum

Endoplasmatic Reticulum

pPha-DUAL[2xNR] MonoclonalIgG1 antibody (CL4mAb)

Protein A-Sepharose beads

Bind against Hepatitis B Virus surface protein

Hempel et al. (2011)

C. reinhardtii Chloroplast pD1-Kan Domain 14 of human fibronectin

FLAG affinity chromatography

Fibronectins enhances cell adhesion, migration, growth and differentiation

Rasala et al. (2010)

4 A

PPLICA

TIO

N O

F MIC

RO

ALG

AL PR

OT

EINS

227

III. TR

AN

SFOR

MA

TIO

N O

F PRO

TE

INS B

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DU

CT

S TO H

IGH

VA

LU

E PR

OD

UC

TS

Microalgae bioreactor

Expression medium for protein

Plasmid type/expression vector Protein expressed

Purification Technologies Application References

C. reinhardtii Chloroplast pD1-Kan Human vascular endothelial growth factor

FLAG affinity chromatography

Treatment for pulmonary emphysema, erectile dysfunction, and depression


C. reinhardtii Chloroplast pD1-Kan High mobility group protein B1

FLAG affinity chro-matography

Cell mediation in wound healing such as activation of endo-thelial cells and innate immune cells.


C. reinhardtii Chloroplast pKB101 vector C-terminal domains of proteins from a rodent Plasmodium species fused to the algal granule bound starch synthase (GBSS)

French press disruption technique followed by sedimentation and Percoll gradient centrifugation

Production of antimalaria starch-bound vaccines

Dauvillée et al. (2010)

C. reinhardtii Chloroplast p322 Bovine mammary-associated serum amyloid (M-SAA)

Reverse-phase chromatography with octyl-Sepharose resin

Provides a physical block in mammalian gut to avert bacterial and viral infection

Manuell et al. (2007)



B-phycoerythrin (BPE) and R-phycoerythrin (RPE) are two main classes of PE found in red microalgae. The unique physical properties of PE make it suitable for applications in clinical re-search and molecular biology. Its highly fluores-cent properties exhibit high photostability with a fluorescent quantum yield independent of pH, which minimizes interferences from Rayleigh and Raman scatter (Sekar and Chandramohan, 2008; Pumas et al., 2012).

PE can be used as label for biological mol-ecules, as a reagent in fluorescence immunoas-says, flow cytometry, fluorescence microscopy, and diagnostics (Spolaore et al., 2006). Phyco-erythrin has been extracted and purified from several microalgae species such as P. cruentum (Kathiresan et al., 2007), Phormidium sp. A27DM (Parmar et al., 2011) and the thermophilic cya-nobacterium Leptolyngbya sp. KC45 (Pumas et al., 2012). Pumas and colleagues screened cyanobacteria species from hot spring to obtain PE with a higher temperature stability than PE extracted from mesophilic species such as P. cruentum. Kathiresan et al. (2007) investigated the effect of macronutrients on the production of PE by P. cruentum. The result showed that PE production is mainly affected by phosphate, nitrate, chloride, and sulfate concentrations. Meanwhile, Hernandez-Mireles and Rito-Palo-mares (2006) developed a simplified process model that incorporated isoelectric precipita-tion and an aqueous two-phase system (ATPS) for the fractionation of the cell homogenate of P. cruentum to produce high-purity BPE. The result demonstrated an improvement in protein purity up to 5.9 times compared with the purity of the initial crude extract, contributing to overall BPE recovery of 72%.

C-phycocyanin (C-PC) is another phycobili-protein with great commercial value as a natu-ral colorant in the nutraceutical, cosmetic, and pharmaceutical industries. Phycocyanin has been found to exhibit significant antioxidative, antiinflammatory, hepatoprotective, and radi-cal-scavenging properties (Romay et al., 2003).

Basically, there are three main classes of phy-cocyanins. These are C-phycocyanin (C-PC), R-phycocyanin (R-PC), and allophycocyanin. C-PC is the main phycobiliprotein in most blue-green algae. The pigment has a single visible absorbance maximum between 615 and 620 nm, a maximum fluorescence emission at around 640 nm, and molecular weight between 70 and 110 kD. The pigment is composed of two equal subunits of α and β, but may vary among differ-ent species. Both α and β subunits contain only the phycocyanobilin chromophore. C-PC pig-ment is widely used as natural dye for various purposes because of its deep and intense blue color. They are well suited as fluorescent re-agents without any toxic effect for immunologi-cal analysis because they have a broad excitation spectrum and fluorescence with a high quantum yield (Hardy, 1986). Purification of phycocyanin from crude algae extracts is usually obtained by a combination of different techniques, such as ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration chromatog-raphy (Kuddus et al., 2013).

4.2 Nutraceutical and Pharmaceutical Applications

Purified C-PC from spirulina has nutraceuti-cal and pharmaceutical potentials in the area of immunomodulation, anticancer, antiviral, and cholesterol-reduction effects (Belay, 2002). It is known that the human body has a self-mod-ulating system to control the production and activity of individual cytokines to ensure that the immune system functions favorably. C-PC plays an important role in balancing immune cells and cytokines to prevent excess produc-tion that could lead to impaired immune system (Belay, 2002). The antioxidative and radical-scavenging activities of peptides from algae protein are well documented (Sheih et al., 2009; Samarakoon and Jeon, 2012). Samarakoon and Jeon (2012) reported that microalgae are com-posed of excellent nutritional quality compared

4 APPLICATION OF MICROALGAL PROTEINS 229


to macroalgae as well as conventional plants in daily diet. Enhanced radical-scavenging activi-ties have been reported in selenium-enriched C-PC obtained from A. platensis grown in a sele-nium (Se)-enriched medium (Huang et al., 2007). They reported that increased Se concentration in Se–PC is directly proportional to its scavenging activity, especially on superoxide and hydrogen peroxide radicals.

McCarty (2007) proposed that phycocyano-rubin, a reduced form of chromophore phy-cocyanobilin found in blue–green algae, has been investigated to be a potential inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. The result was sup-ported by some rodent studies with versatile antiinflammatory and cytoprotective activities through oral administration of phycocyanoru-bin (Romay et al., 2003; Riss et al., 2007). Hence, sufficient intake of PhyCB-enriched spirulina extracts may have a significant potential for can-cer prevention (McCarty, 2007).

4.3 Microalgal Protein As Fish Meal

Fish meal is a globally traded commod-ity that has been extensively used as feed for shrimp, farmed fish, and other animals. The rapid growth of the global aquaculture industry has resulted in greater demands for processed fish meal and its constituent ingredients. Any increase in the market price of these ingredients has a significant negative effect on the operating costs of aquaculture. Currently, the production of fish meal from plant protein concentrates is expensive, and fish are unable to absorb much of the protein from soybean meal in their di-ets. Thus, seeking alternative protein sources in fish diets is critical to the economic success of the aquaculture industry. Partial replace-ment of fish meal with microalgae is possible for providing protein-source vitamins and es-sential fatty acid contents for cultured fish, es-pecially in tropical regions where microalgae are found in abundant amounts (Ayoola, 2010;

El-Hindawy et al., 2006). The use of microalgae as fish feed inputs has been studied with en-couraging results. Ju et al. (2012) evaluated the effect of partial replacement of fish meal protein by a defatted microalgae meal on Pacific white shrimp Litopenaeus vannamei. The result showed that the defatted microalgae can be used as fish feed additive at 3% to stimulate shrimp growth and improve feed utilization. In addition, the microalgae protein improved the shrimp quality through enriched accumulation of astaxanthin, a natural dietary component. A 90-day experi-ment was conducted to investigate the effects of partial replacement of fish meal with dried microalgae (Chlorella sp. and Scenedesmus sp.) in Nile tilapia diets. The results were positive in terms of fish growth performance, feed efficien-cy, and body composition (Tartiel et al., 2008). Thus, the replacement of fish meal with alterna-tive protein sources, particularly microalgae, in aquaculture feed is biologically and economi-cally feasible.

4.4 Enzyme (Haloperoxidase) Extracted from Microalgae

Haloperoxidase, known as redox enzyme, cat-alyzes the oxidation of halides, such as chloride, bromide, or iodide, in the presence of hydrogen peroxide. Haloperoxidases are found to be in-volved in the biosynthesis of halogenated marine metabolites. These metabolites have been found to exhibit biological benefits, including antifun-gal, antibacterial, antiviral, and antiinflammatory properties (Wang et al., 2014b). Two types of hal-operoxidases have been discovered: vanadium-dependent haloperoxidase (vHPO) (Almeida et al., 2001; Leblanc et al., 2006) and heme-depen-dent haloperoxidase (heme-HPO) (Vaillancourt et al., 2006). Both types of haloperoxidase possess different reaction mechanisms and active sites. Recently, research has focused on vHPO because of the stability of its oxidation state during the synthesis of halogenated species (Suthiphong-chai et al., 2008; Wang et al., 2014b). In addition,



vHPOs possess several positive properties in contrast to other halogenating enzymes. These include resistance to high temperatures, ability to halogenate a broad range of organic compounds, and sustained bioactivity under oxidative condi-tions in the presence of organic solvents. These important properties make vHPOs good candi-dates for commercial biotransformation. Cart-er-Franklin and Butler (2004) and Butler and Carter-Franklin (2004) reported the application of marine algal vBPOs in the chemoenzymatic catalysis of bromonium-assisted cyclization of terpenes and ethers. According to them, vHPOs have the ability to halogenate a range of organic compounds in a stereospecific manner. The en-zyme is also capable of carrying out sulfoxida-tion reactions with enantioselectivity on methyl phenyl substrate in vitro (ten Brink et al., 2001).

5 CONCLUSIONS

Although the use of microalgae proteins as food supplements and in aquaculture has been in exis-tence for a long time, the advent of newer biotech-nologies and the expansion of microalgal research have increased the prospects in mass commercial microalgae protein production. Also, microalgae, having been regarded as safe phytoplanktons, have comparable and in some instances, superior characteristics over current conventional cultiva-tion systems and protein sources. This puts them in a unique position of significance to herald cheap and rapid commercial protein production as main products or by-products. In addition, endogenous and recombinant proteins extracted from microalgae have a myriad of applications in the cosmetic, food, and nutraceutical industries. Among the various microalgal lysis techniques, electroextraction has a significant potential in terms of rapidity and extraction efficiency.

Despite the various biotechnological advanc-es in the upstream and downstream processing of microalgae proteins, there still remain some drawbacks that hinder full-scale production of

commercial proteins. Notable among them are the lack of a standardized protocol for the pro-duction of recombinant proteins in microalgae, the resistance of commercial protein companies to switch to microalgal cultivation systems, heavy microalgal culture dewatering require-ments, and the lack of genomic knowledge of most microalgae species to explore protein pro-duction potential. Further and a more extensive research and development targeted at establish-ing a robust and cost-effective bioprocess with adaptable capacity to produce both endogenous and extracellular proteins from microalgae is re-quired to realize full-scale production potential.

AcknowledgmentThe authors wish to thank Curtin University for providing the financial support for this research through the Curtin Sarawak Research Institute (CSRI) Academic Grant Scheme.

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C H A P T E R


13Recovery and Applications of Proteins From Distillery

By-ProductsJ.S. White*, J.E. Traub*, D.L. Maskell**,

P.S. Hughes†, A.J. Harper‡, N.A. Willoughby**Institute for Biological Chemistry, Biophysics and Bioengineering, School of

Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, United Kingdom; **International Centre for Brewing and Distilling, Heriot-Watt University, Edinburgh, United Kingdom; †College of Agricultural Sciences, Oregon State University, Corvallis,

OR, United States; ‡Institute of Mechanical and Process Engineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, United Kingdom

1 INTRODUCTION

Distillation has existed for well over 1000 years. The earliest records of whisky dis-tillation in Scotland are attributed to the tax re-cords of the day, the Exchequer Rolls, which in-dicated that Friar John Cor bought enough malt to make 1500 bottles, suggesting that production was already well established by that date. Today, under the EU Commission Regulation (EC) No. 110/2008 (EU, 2008a), the definition of whisky or whiskey includes the following description:

A spirit drink produced exclusively by:

(i) distillation of a mash made from malted cereals with or without whole grains of other cereals, which has been:

- saccharified by the diastase of the malt contained therein, with or without other natural enzymes,- fermented by the action of yeast;

(ii) one or more distillations at less than 94.8% vol., so that the distillate has an aroma and taste derived from the raw materials used,

(iii) maturation of the final distillate for at least three years in wooden casks not exceeding 700 litres capacity.

This prevents most Indian whisky from being sold in Europe because molasses from sugar-cane is often used as the substrate to make ex-tra neutral alcohol, which is then blended with grain spirits (Maitlin, 2014). This also provides for a distinct difference from other ethanol-based fermentations and distillations, such as neutral spirit (used as the base for other potable

236 13. RECOVERY AND APPLICATIONS OF PROTEINS FROM DISTILLERY BY-PRODUCTS


alcohols, such as vodka and gin) and also more importantly, fuel ethanol.

EU regulations allow for country-specific legislation and both the Scotch Whisky Regu-lations (2009) and Irish Whiskey Act (1980) prescribe whiskey production in these respec-tive countries. The US Code of Federal Regu-lations, 27 CFR 5.22 (CFR, 2008), documents the various types of whiskey produced in the United States. Canadian whisky is defined in Canada under the Food and Drugs Act (Food and Drug Regulations, 2014). The cereal grains and products are country specific (Table 13.1). In the United Kingdom, Scotch whisky now ac-counts for a quarter of food and drink exports, with 106 malt whisky distilleries and seven grain distilleries in Scotland with the capacity to produce 750 million liters of pure alcohol (MLPA) per year (Gray, 2014). For 2013, actual production was 295 and 364 MLPA for malt and grain distilleries, respectively (Gray, 2014). Scotch malt whisky, as the name suggests, is (and must be) produced solely from malted barley, while other whiskies are produced from a range of cereal grains (Table 13.1). In the United Kingdom, grain whisky is made from wheat or maize (corn in the United States)

with barley included at approximately 10% as a source of starch-saccharifying enzymes.

India is the largest distilled spirits producer with annual production of 1560 MLPA of potable alcohol of which 56% is used to produce whisky (Maitlin, 2014). However, nearly 70% of this spirit originates from sugarcane molasses, which is more similar to rum production. And because ba-gasse, the main sugarcane residue, is low in pro-tein (less than 2%; da Penha et al., 2014), it is not included in detail in this review. Corn, rye, wheat, barley, and malted barley are used as substrates for American whiskey, and production volume is on par with Scotland, although finding exact pro-duction volumes for the United States is difficult. The Distilled Spirits Council of the United States provides statistics based on distilled spirits sales by volume for the range of spirits produced in the United States, but this reflects the actual cases of bottled whiskey sold and cannot be directly used to predict production volume. The largest num-ber of distilleries are located in Indiana, Illinois, Kentucky, Ohio, Tennessee, and Virginia (Buglass et al., 2011), although a craft distilling boom has led to 305 distilleries reported in the United States in 2013 (Kornstein and Luckett, 2014). For Kentucky, where 95% of bourbon is produced, there are

TABLE 13.1 Whiskey Production by Country, Feedstock, Category, and Estimated Annual Production Volume

Country Feedstock Categorya MLPA

Scotland Malted barley, wheat, corn Single malt, single grain, blended, blended malt, blended grain

659b

India Sugarcane, rice, millet, barley (malt), sorghum, corn

Blended whiskies of variable categories 874c

United States Malt (barley or rye), barley, wheat, corn, rye

Bourbon, corn, rye, straight, blended straight, blended, bonded (or bottled in bond)

576d

Ireland Malted barley, barley, corn, wheat Single malt; pure pot still; poitín, blended, single grain <100

Japan Malt, corn, rye Malt, grain, blended <100

Canada Malted barley, barley, wheat, corn, rye Canadian, rye <100

a Feedstocks and categories based on Buglass et al. (2011).b Data from Gray (2014).c Estimated based on Maitlin (2014). Grain whisky is at most 500 MLPA.d Based on 144 MLPA in Kentucky in 2013 (Kornstein and Luckett, 2014).MLPA, million liters of pure alcohol.

2 DISTILLERIES AND PRODUCTION PROCESSES 237


approximately 23 distilleries with production of 1.2 million barrels or 144 MLPA in 2013 (assuming 200 L/barrel and alcohol diluted to a maximum of 60% ABV before the onset of maturation; Korn-stein and Luckett, 2014). Bourbon accounts for ap-proximately half of the Tennessee whiskey sales, so if this is assumed to be half of the total volume, whiskey production in the United States can be estimated as 576 MLPA. This is higher than the previously reported figure for American whiskey, which was stated as being more or less stabilized at 400 MLPA for many years (Buglass et al., 2011). It may be explained by the recent surge in the number of craft distillers (Lyons, 2014).

The range of whiskies leads to a number of by-products and terminologies. Even the spelling requires a note of caution: whisky is used for Scot-land, Japan, Canada, and India, whereas whiskey is preferred in the United States and Ireland. In-deed the term coproducts is also used interchange-ably with by-products—by-products will be the preferred term in this review as it is the legal term used in the Scotch whisky industry. By-products are clearly defined by European legislation under Article 5 of Directive 2008/98/EC (EU, 2008b). Whisky by-products are generally used as animal feed, an end use that is now under increased com-petition from similar products from fuel ethanol plants. Indeed, the production volume from fuel ethanol distilleries is well in excess of that from whisky distilleries (Tables 13.1 and 13.2) and this highlights the need for alternative markets. The US market for dried distillers’ grains with solu-bles (DDGS) from corn bioethanol plants is also

saturated, and as the number of plants continues to increase, new markets are required to maintain the contribution of DDGS to overall plant profit-ability (Rosenstrater, 2011). Novel uses could be realized if characterization of these streams was readily available. In this review the generation, protein content, and composition of whiskey and fuel ethanol by-products produced by grain distilleries will be discussed. A detailed under-standing of the composition of by-products will enable development of strategies to separate the protein components and maximize their use as feed ingredients. Brazil is the second largest fuel ethanol producer and sugarcane is the feedstock. This process is quite distinct from grain ethanol with sugarcane bagasse, the fibrous residue after sugar extraction, as the main by-product. Bagasse is low in protein and minerals, of low nutritive value, and is mainly burnt as fuel (Lopes Silva et al., 2014), so is not included in this review. The main focus is on by-products from malt and grain whiskey and grain bioethanol plants.

2 DISTILLERIES AND PRODUCTION PROCESSES

The basic process steps involved in whiskey and grain fuel ethanol production are similar in that they require the release of fermentable sugars from grain-based feedstocks, before subsequent fermentation and distillation. In the case of whis-key, the choice of cereal substrates, distillation steps (eg, number of distillations and use of pot

TABLE 13.2 The Main Fuel Ethanol-Producing Countries, Feedstocks, and 2014 Production Volumes

Country/Region Feedstock Volume (MLPA)

United States Corn 54,131

Brazil Sugarcane 23,432

Europe Wheat, corn 5,470

Rest of world Various 9,975

MLPA, million liters of pure alcohol.Data from RFA (2015).



still versus Coffey or other continuous column stills), maturation and finishing, all affect the qual-ity and classification of the final product. Whiskey production can be viewed as a fine art with many differences between countries and categories in addition to distillery-specific variations. It is not the purpose of this review to discuss all of them; moreover, the aim is to provide a general overview of how by-products are generated and how spe-cific processes affect these. A good introduction to whiskey production and the distinct categories is provided by Buglass et al. (2011); Buxton and Hughes (2014); and Russell and Stewart (2014). For fuel ethanol production, there are also a num-ber of process options, depending on the type of grain and how it is processed before fermentation (De Nicola et al., 2011; US Grains Council, 2012; RFA, 2015). The key point is that all of these steps affect the range of by-products generated in a dis-tillery. A basic overview of the steps involved in a distillery is provided in Fig. 13.1. The different process options available at each step can be sum-marized as follows:

1. Substrate: The choice of feedstock depends on the final product. For whiskey, malted barley or rye, or whole grains, including

barley, rye, corn, and wheat are used. US bioethanol plants use corn, whereas feed-grade wheat is also used in Europe. This review only focuses on grain distilleries, so processes for other feedstocks, such as sugarcane in Brazil, are not included.

2. Sorting and milling: The grains are first mechanically sorted to remove any debris, including stones, metal, small grain particles, and dust. The grain is then milled (although in some cases left whole) to a desired grist size depending on the subsequent cooking and mashing steps. The process in a wet-grind corn bioethanol plant is quite different and yields a wide range of by-products (US Grains Council, 2012).

3. Starch extraction and sugar conversion: The grains undergo a specific heating, cooking, and mashing regime to extract and convert the starch to fermentable sugars. Detailed reviews of the various cooking, steeping, and mashing protocols in whisky production can be found in textbooks (Buglass et al., 2011; Russell and Stewart, 2014), and similar resources are available for bioethanol production (De Nicola et al., 2011; US Grains Council, 2012). For Scotch whisky, the enzymes must be

FIGURE 13.1 The basic steps involved in a distillation process with the generation of by-products highlighted. The solid lines signify steps and processes that are common to all distilleries whereas optional steps are highlighted as dotted lines. There are a number of recycle steps that may be involved, but for simplicity these are not included here.

3 BY-PRODUCT GENERATION AND YIELD FROM A DISTILLERY 239


provided by the malt, whereas for other processes, exogenous enzymes can be added. These enzymes include amylases to aid starch conversion and β−glucanases, especially in wheat-based processes where viscosity problems caused by β−glucans may be encountered further downstream (Bringhurst and Brosnan, 2014). The sugar-rich liquid (termed the wash) is either then separated for fermentation or an all-grain process (grains-in mash) is used where no solid–liquid separation takes place prior to fermentation and distillation. This step has an obvious effect on the generation and quality of by-products. For example, in Scotch malt whisky production, the draff (the malted barley residue remaining after starch extraction) is separated prior to fermentation of the wash, whereas in grain distilleries, an all-grain process is often used, with either no solids separation or the removal of only coarse grains taking place prior to fermentation and distillation (Bringhurst and Brosnan, 2014). For American and Irish whiskies, an all-grain process is also preferred (Buglass et al., 2011).

4. Fermentation: The sugars in the wash or grains-in mash are fermented by yeast (Saccharomyces cerevisiae) to produce ethanol. The exact strains are country and distillery specific (Russell and Stewart, 2014) and lactic acid bacteria are often present (Wilson, 2014). Indeed in American whiskey production, “sour mashes” are encouraged with spent mash from a previous distillation and/or lactic acid bacteria directly added (Buglass et al., 2011; Lyons, 2014). The fermented wash/mash is then transferred to the stills with no prior separation steps.

5. Distillation: Ethanol is separated from the fermented wash/mash by distillation. In the case of fuel ethanol, ethanol is distilled using continuous columns to approximately 95% and the remaining water removed with molecular sieves (RFA, 2015). For whiskey production, distillation using pot stills

(essential for Scotch malt whisky and Irish pot still whiskey) is under fine control so that esters and higher alcohols are imparted into the final whiskey product. The use of copper pot stills plays an important role in whiskey aroma, including for the removal of undesirable sulfur notes (Harrison et al., 2011). For grain whiskey, Coffey stills (or other continuous column stills) are used, and the spirit is “purer” with less congeners (Buxton and Hughes, 2014). The type of distillation and the use of copper pot stills and “sacrificial” copper in grain whisky affects the application of by-products. Most importantly, the presence of copper restricts the use of pot ale syrup, dark grains, and DDGS from whisky distilleries in feeds for certain animals that are sensitive to copper, such as sheep, and with maximum levels of trace elements in feeding stuffs set by Directive No. 1334/2003 in Europe (EU, 2003a).

6. The main by-products are generated after mashing and distillation. The range of by-products generated and terminologies are discussed in more detail in the following section.

3 BY-PRODUCT GENERATION AND YIELD FROM A DISTILLERY

In Scotland, malt and grain whisky distill-eries produce different by-products (Table 13.3). The typical layout of these distilleries and gen-eration of by-products is compared in Fig. 13.2. For malt distilleries, the draff is separated from the wort before fermentation with pot ale result-ing from the first distillation step and spent lees from the second. For every one liter of alcohol produced, approximately 2.5 kg of draff (as-suming the draff generated matches malt used and spirit yield is 407 L of alcohol produced per ton of malt according to current yield from Gray, 2014), at least 8 L of pot ale and 10 L of spent lees and washings are generated (based on

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TABLE 13.3 Description and Use of By-Products from Distilleries and Approximate Yield Based on Production of 1 L of Alcohol

Type Description Use

BY-PRODUCTS FROM MALT WHISKEY

Draff(2.5 kg)

The grain residue remaining after starch conversion and extraction for fermentation. Equivalent to spent grain from breweries.

Moist feed for cattle and sheep.

Pot ale(8 L)

The residue remaining in the pot still after first distillation step. It contains 4–5% solids and consists of yeast, barley, and yeast residues, proteins, carbohydrates, and a variable amount of copper.

Spread on land as fertilizer, disposed of in sea or water courses, added to AD plant, or concentrated to pot ale syrup.

Spent lees and washings(10 L)

The liquid residue from subsequent distillations and wash-ings. Dilute solution of organic acids and alcohols.

Treated in bioplants.

Pot ale syrup(0.8 L)

Pot ale concentrated to 40–50% solids by evaporation. Feed for cattle and pigs. Restrictions on use in sheep.

Dark grains/Distillers pellets(0.8 kg)

Animal feed that is produced from combining draff and pot ale syrup that is dried and pelleted.

Feed for cattle and horses.

BY-PRODUCTS FROM GRAIN WHISKEY AND FUEL ETHANOL

Stillage or spent wash The liquid residue remaining after distillation. Similar to pot ale but also contains cereal residues from grains-in process.

Generally undergoes solid–liquid separation into thin stillage/centrate and wet distillers’ grains.

Thin stillage or centrate Liquid fraction from stillage/spent wash centrifugation. Disposal to sea, concentrated by evaporation to syrup, treatment and methane generation by AD or portion is backset to the steep liquor.

Concentrated syrup or solubles

Thin stillage concentrated by evaporation. Differs from pot ale syrup in that it does not contain yeast residue.

Used directly as a feed or mixed with WDG and dried to form DDGS.

WDG or DDG WDG is the solids fraction separated from the stillage. Simi-lar to draff but also contains yeast. WDG can be dried to 10% moisture to form DDG.

Used directly as a livestock feed or dried to form DDG or DDGS if solubles are added back in.

DDGS(0.9 kg for whisky and 0.75 kg

for corn fuel ethanol)

DDGS is produced from WDG dried with solubles to 10% moisture.

Used as an animal feed for cattle with some applica-tions for pigs and chickens.

Corn oil(0.02 kg)

Extracted from thin stillage from US dry-grind ethanol plants.

Converted to biodiesel or used as an animal feed ingredient.

Grain whiskey and dry-grind corn fuel ethanol distilleries generate similar by-products and these are grouped together. The products from wet-grind corn bioethanol plants are not included. Yields are included where they are known with exact calculations and assumptions explained elsewhere in this review. By-products that are marketed as feeds are highlighted in bold. AD, anaerobic digestion; DDG, dry distillers’ grain; DDGS, dried distillers’ grain with solubles; WDG, wet distillers’ grain.

3 BY-PRODUCT GENERATION AND YIELD FROM A DISTILLERY 241


3.3 t pot ale and 3.9 t spent lees and washings/t malt processed; Pass and Lambert, 2003). In some cases, pot ale syrup is evaporated and sold as pot ale syrup or the syrup is dried with draff to produce dark grains. The yield of dark grains from a malt distillery is estimated as 0.8 kg/L al-cohol (based on 0.31 t dark grains/t malt accord-ing to Pass and Lambert, 2003, and spirit yield of 407 L alcohol/t malt according to Gray, 2014).

In Scotch whisky grain distilleries, solid–liquid separation after sugar extraction is not so common compared to malt whiskey distilleries (separation of larger grain particles may occur, but this is mixed back with the centrifuged sol-ids after distillation). All of the grain, yeast, and liquid residues are collected after distillation as spent wash (or stillage). White soft winter wheat is the main feedstock with grain distilling bar-ley included as an enzyme source (Bringhurst and Brosnan, 2014). One distillery is known

to use maize, although other grain distilleries have kept the capability to switch grains if re-quired. The spent wash is generally centrifuged with part of the centrate (the liquid fraction also known as thin stillage) backset as process liquor for grains steeping. The rest is concentrated by evaporation to form concentrated syrup (or sol-ubles) and sold directly as a feed or mixed and dried with the solids fraction (distillers’ grains) to form DDGS. This is typical for all-grain mash processes and similar to whiskey production in Ireland and United States. For a grain whiskey process based on wheat feedstock, the volume of DDGS generated can be estimated as 0.9 kg/L alcohol (based on 0.327 t dry by-product/t grain according to data in Pass and Lambert, 2003, and corrected for 10% moisture in DDGS and spirit yield of 386 L/t grain according, Gray, 2014).

The by-products from the grain whiskey pro-cess are very similar to those from wheat and

FIGURE 13.2 Layout of Scotch malt and grain whisky distilleries and generation of by-products (adapted from Buglass et al., 2011). The main by-products marketed as feed ingredients are highlighted in bold.



dry-grind corn fuel ethanol plants, with wet distillers’ grains (WDG) or DDGS as the main by-product. For corn fuel ethanol plants, recov-ery of corn oil from the thin stillage also occurs, with 85% of dry mills extracting oil for use as an animal feed ingredient or feedstock for biodiesel production (RFA, 2015). As a rule of thumb, dry-grind corn ethanol plants produce 11 L ethanol and 8 kg distillers’ grain (Rosenstrater, 2011) and 0.23 kg corn oil (based on RFA assumptions; RFA, 2015) per bushel (25.4 kg) of corn. In con-trast to dry-grind plants, wet-grind fuel ethanol plants generate very different by-products with the corn undergoing a number of fractionation steps prior to starch hydrolysis and fermenta-tion (US Grains Council, 2012). The main dif-ference between the two processes is the initial treatment of the grain. In dry milling, the corn is milled and processed into ethanol with the by-products generated after distillation. For wet milling, the corn is steeped under acidic condi-tions prior to separation into distinct products, such as corn oil, corn germ meal, corn gluten meal, corn gluten feed, starch, ethanol, and high fructose corn syrup (US Grains Council, 2012). According to RFA assumptions, 89% of corn fuel ethanol plants are based on the dry-grind pro-cess, and daily output figures for 2015 showed that DDGS production was more than eight times higher than combined total of corn gluten feed and corn gluten meal. As the by-products from the wet-grind process have stable markets and new corn ethanol plants are based on the dry-grind technology, the wet-grind process is not discussed further in this review.

In Scotland, malt distilleries have much lower operating capacities than grain distilleries and this is reflected in the total volume of by-products generated. In Scotland, the average capacity of a malt distillery is 3 MLPA and ranges from 40,000 to 10.8 million L, whereas the seven grain dis-tilleries range in capacity from 18 to 110 million L (Gray, 2014). This has an effect on the end use of the by-products; processes that are eco-nomically viable for producing large volumes of

by-products may not be suitable for smaller dis-tilleries. The high water content of by-products (draff is approximately 74–80% moisture and pot ale 95% water) means high energy costs involved in drying and evaporation and investment and en-ergy costs may not be feasible at small scale. Dark grains plants, which produce pelleted feeds from draff and pot ale, often service several malt distill-eries ( Stewart, 2014). For distilleries in remote lo-cations, this is not an option. In contrast, a single grain distillery will generally have a purpose-built feed plant generating DDGS through dewatering, evaporation, and drying steps (Stewart, 2014).

The end use of by-products has an effect on distillery economics, sustainability, and over-all contribution to local farming economies. For dry-grind corn bioethanol plants, the generation of DDGS is closely linked to distillery income. A typical dry mill earned 27% of its gross revenue from the sale of distillers’ grain and corn distillers’ oil in 2013 (US Grains Council, 2012). April 2015 Iowa sale prices for DDGS and ethanol were £1.06 and £2.78 for 8 kg of DDGS and 10.6 L of etha-nol, which is generated from 1 bushel of corn (ap-proximately 25.4 kg; USDA, 2015). Corn DDGS also plays an important role in replacing the feed corn that has been redirected from use as a feed for livestock to fuel ethanol plants (US Grains Council, 2012). For Scottish distilleries, by-prod-ucts also generate income, but this is less than 6% of the new-make spirit sales (estimated from Gray, 2014). New-make spirit from malt and grain distilleries sell at £230 and £115/L, which is much higher than US fuel ethanol (currently at £0.26/L based on April 2015 exchange rates and US fuel ethanol selling price of $1.47/gal; USDA, 2015).

The profitability of Scotch by-products could be viewed as less of a concern as ensuring that their end use is sustainable and fits with the Scotch Whisky Association (SWA) environmen-tal strategy (SWA, 2009). Of recent concern to farmers and feed manufacturers is that there has been a shift away from animal feed products to investment in energy from by-products from processes, such as anaerobic digestion (AD) and

4 BY-PRODUCTS AS PROTEIN FEED INGREDIENTS 243


combustion in combined heat and power (CHP) plants. This enables distilleries to improve their carbon reductions and feeds directly into the SWA sustainability targets of 20% reduction in reliance on fossil fuels by 2020 and 80% reduc-tion by 2050 (SWA, 2009). To preserve the pro-tein component of these cereal grains for the food chain, newer higher-value processes are required. These processes can be designed to fit in with energy-generating processes, such as AD and combustion if the protein fraction is first extracted. This will also enable distillers to contribute to the increasing demand for protein while also securing increased income from their by-products. Alternative feed uses for US DDGS are also required with the US market currently saturated and dependent on export markets for the surplus (US Grains Council, 2012).

4 BY-PRODUCTS AS PROTEIN FEED INGREDIENTS

The use of distillery by-products in animal feed is not a new idea. The earliest record for Scotch production also records the number of

cattle and pigs fed with the by-products (Pass and Lambert, 2003). The continued application of by-products in feed is important in satisfy-ing the increased demand for proteins in animal and fish feeds. The worldwide need for protein as food and feed ingredients is driven by an in-creasing population and demand for fish and meat products. Sustainable, local sources of feed have been advocated by the Food and Agricul-ture Organization of the United Nations (FAO). Currently, the European Union imports 68% of its protein feed requirement corresponding to 28 million tons of protein-rich feed imported into the European Union in 2011–2012 (FEFAC, 2014). The main sources of protein feeds used in the Eu-ropean Union in 2013 are shown in Fig. 13.3. The coproducts from food and bioethanol were 18 million tons (12% of total feed) of which 485,000 t was imported DDGS (FEFAC, 2014). The by-products from whisky and fuel ethanol distill-eries play an important role in the feed industry. Adding value to these products and developing low-cost sustainable production methods are necessary to make them economically attrac-tive for distilleries and to ensure the continued availability of the protein component of these

FIGURE 13.3 Range of feed ingredients used in the 154 million tons of compound feeds consumed in Europe in 2013 (data from FEFAC, 2014).



grains for feed purposes. With feed cereals ac-counting for nearly half of feed ingredients, the generation of feed by-products from bioethanol plants will also play an important role in ensur-ing that the protein component of these grains will continue to be available for the food chain if the original grain is redirected from feed to bio-ethanol production.

The nutritional composition of commonly available whiskey and fuel ethanol by-products are detailed in Tables 13.4 and 13.5. The high moisture content of some of these feeds means that they are of low value, restricted use, short shelf life, and not suitable for transporting long distances. In the case of syrups, viscosity and flow problems mean that they are less attractive for farmers. For example, draff has a high water content (between 74% and 80%), which means it is prone to microbial contamination within days and requires ensiling if it is to be stored for lon-ger periods (Stewart, 2014). Draff is of low val-ue and often provided to local farmers for free. However, it makes an important contribution to

the local farmers and economy (Bell et al., 2012). The decision on whether the draff is provided to farmers (often on a free basis) as WDG or dried and sold as DDGS or dark grains comes down to the economies of drying, which is decided on scale of production. For bourbon produc-tion in Kentucky, just over 147,000 tons of WDG is produced each year and 60% is sold (mostly as DDGS, but some WDG) while the remain-der is given away as WDG to farms within an hour’s drive (Kornstein and Luckett, 2014).

The other malt whiskey feed by-product, pot ale must be concentrated by evaporation to 40–50% solids and used as cattle or pig feed. Evaporation is energy intensive, the equipment used is prone to fouling and the process con-centrates everything in the pot ale, including undesirable components, such as phytate and copper. Where facilities exist, draff is dried with pot ale syrup to approximately 10% moisture to produce dark grains (or malt distillers’ pel-lets). Dark grains are similar to wheat and corn DDGS although they also contain significant

TABLE 13.4 Composition of Commercially Available Solid By-Products from Distilleries

Producta Draff Dark grains Vitagold Vivergo EU DDGS US DDGS

DM (%) 20–24 90 35 92 90 89

CP (%) 22–24 24 36 35 29 30

Oil 9 9 10 7 10 11

NDFb 62 41 36 32 36 34

Starch 1.7 4.0 4.0 2.0 1.0 9.3

Sugar 2.0 2.0 1.5 1.0 1.0 1.7

Lysine 3.9 4.5 3.0 2.0 2.7 3.0

Methionine 1.5 1.8 1.8 1.4 1.9 2.0

Cysteine 2.1 2.1 2.0 1.8 1.5 2.0

Histidine 2.1 2.6 3.0 2.3 2.6 2.7

Threonine 3.8 4.5 3.6 3.2 3.7 3.7

a Components are expressed as %DM with amino acids as %CP. Data for draff and dark grains (distillers’ barley pellets) from Scottish malt distilleries; Vitagold (wet grains) from Scottish wheat distillers; Vivergo (wheat DDGS) from the Vivergo Fuels bioethanol plant in Yorkshire, England; and DDGS and imported maize distillers from outside the United Kingdom from Trident Feeds (www.tridentfeeds.co.uk/products/). Average composition for US corn DDGS was obtained from Feedipedia (www.feedipedia.org).b NDF is neutral detergent fiber and roughly equivalent to hemicellulose, true cellulose, and lignin.CP, crude protein; DDGS, dried distillers’ grain with solubles; DM, dry matter.

http://www.tridentfeeds.co.uk/products/

http://www.feedipedia.org/

4 BY-PRODUCTS AS PROTEIN FEED INGREDIENTS 245


concentrations of copper from the pot ale, re-stricting their use compared to the fuel ethanol distillery equivalents.

The number of dark grains plants and pot ale evaporation facilities in Scotland is rapidly di-minishing with energy generation by CHP and AD becoming an attractive option. The SWA published a guide for distillers (SWA, 2012) high-lighting the use of AD and CHP for by-product processing and energy generation. The grain component (draff from malt distilleries or the centrifuged solids; WDG from grain distilleries) can be combusted in CHP, the energy for which can come from conversion of pot ale or thin still-age (centrate) to methane by AD. A good exam-ple of this implementation is the conversion of the CoRD (originally formed by the Combination of Rothes Distillers Limited more than 100 years ago) dark grains plant. The dark grains plant was established in 1970 to service a group of distill-eries on Speyside with the capacity to process 90,000 t draff and 300,000 t pot ale. In 2013, it was converted to a biomass-fired CHP process and the Helius CoRDe Limited plant has an annual

input of 130,000 t of wet draff and 40,000 t of wood chips (Sherrard, 2014). It also includes a pot ale process plant which has an annual capac-ity to process 430,000 t pot ale and pressed draff liquor to produce 44,000 t of syrup as feed.

When used as feed, the generation of by-products from whisky distilleries makes an im-portant contribution to the farming economy as highlighted by a report commissioned by the Scottish government (Bell et al., 2012). If eco-nomically feasible methods to extract the protein components were available, this would enable use in feeds to be maintained while allowing the other components to be used in energy genera-tion. Based on the data presented in this review, the volume of protein available in the differ-ent by-products can be estimated (Table 13.6). For Scotland, there is 175 t of protein available in by-products from malt and grain distilleries. The current use of grain-based distillery by-products is dominated by ruminants as they are able to cope better with the cellulosic and fiber content of the by-products while syrups are also fed to pigs. If the protein component could be

TABLE 13.5 Composition of Commercially Available Syrup-Based By-Products from Distilleries

Producta Spey syrup Proflo syrup Distillers’ solubles

DM (%) 42 24 44

CP (%) 32 35 20

Oil 1.0 4.5 11

NDF 1.0 10 NR

Starch 1.0 2.0 NR

Sugar 2.0 5.0 NR

Lysine 6.5 3.2 5.7

Methionine 1.1 1.5 1.5

Cysteine 2.1 1.5 NR

Histidine 3.2 3.2 2.7

Threonine 5.6 3.2 4.3

a Data for Spey syrup (pot ale syrup from Scotch malt distilleries) and Proflo syrup (syrup from Vivergo Fuels wheat bioethanol plant in Yorkshire, England) available from Trident Feeds (www.tridentfeeds.co.uk/products/). Data for distillers’ solubles (corresponding to concentrated solubles from corn fuel ethanol plant) was obtained from Belyea et al. (1998).CP, crude protein; DDGS, dried distillers’ grain with solubles; DM, dry matter; NDF, neutral detergent fiber (roughly equivalent to hemicellulose, true cellulose, and lignin); NR, not reported.

http://www.tridentfeeds.co.uk/products/



exploited, this would open up new markets, in-cluding applications in aquaculture.

5 PROTEIN-ENRICHED BY-PRODUCTS

Processes for adding value to distillery by-products have been led by the US corn fuel ethanol industry. Approximately 40% of the energy use of a bioethanol plant is in producing DDGS (Burton et al., 2014), and with increased energy costs, pro-cesses that either reduce the cost of dewatering, drying, or pelleting or add value to the product are required to ensure the continued sustainabil-ity of the by-product markets. The main progress has focused on reducing the water content of wet grains (modified WDG) and by increasing the use of DDGS feeds for nonruminant animals (US Grains Council, 2012). Research into applications of DDGS has expanded its use in poultry, swine, and aquaculture (US Grains Council, 2012). Fur-ther information on the literature surrounding the use of distillers’ grains in dairy, beef, swine, poultry, sheep, horses, companion animals, and aquaculture feeds is available from the University of Minnesota’s website (www.ddgs.umn.edu).

The main restriction on the incorporation of the solid by-products, namely DDGS, and draff in feeds, in particular for use in monogastrics and aquaculture, is the high fiber content con-tributed by the nonstarch polysaccharides. A number of physical, thermal, chemical, and bio-logical treatment methods have been developed to reduce the fiber content while increasing the corresponding crude protein levels. The sim-plest technique is reduction in the fiber content by milling and sieving. This process has been developed for both corn (Srinivasan et al., 2005) and wheat DDGS (Randall and Drew, 2010). A wet fractionation process was also developed for wheat DDGS that involved extracting the protein by mixing with water and precipitating the protein by autoclaving (Reveco et al., 2012). The dried protein-enriched product was tested in trout feed, and inclusion levels up to 30% did not impair feed performance.

The main proteins in cereal residues are insol-uble in water and salts, and chemical extraction involves treatment under acidic or alkali condi-tions with solvent addition. Zein is the main pro-tein in corn DDGS (Anderson et al., 2012), gluten in wheat DDGS (Villegas-Torres et al., 2015), and hordein and glutenin in draff (or spent grain)

TABLE 13.6 Worldwide Production of Distillery By-Products and Crude Protein Content

Country By-product Yield (1000 dry t/y)a Protein (1000 t/y)b

SOURCE: WHISKY/WHISKEY

Scotland (malt) Draff 162 37

Pot ale syrup 99 32

Scotland (grain) DDGS 295 106

United States DDGS 410 121

Ireland/Canada/Japan (per country) DDGS 72 21

SOURCE: BIOETHANOL

Europe DDGS 3,938 1,134

United States DDGS 38,974 11,497a Yield was estimated based on the MLPA in Tables 13.1 and 13.2 and by-product yields in Table 13.3. The same yield conversion factor of 0.8 kg/L alcohol was used for DDGS, apart from Scotland where the higher figure of 0.9 was used to reflect the yield from wheat feedstocks.b Protein yield was calculated based on the data in Tables 13.4 and 13.5.DDGS, dried distillers’ grain with solubles; MLPA, million liters of pure alcohol.

http://www.ddgs.umn.edu/

5 PROTEIN-ENRICHED BY-PRODUCTS 247


(Celus et al., 2006) with the albumins and globu-lins already solubilized from the grains during the preceding steeping, mashing, and fermenta-tion steps. Extraction methods include chemical treatment with alkali (Vieira et al., 2014; Celus et al., 2007) and/or solvents, such as propanol or ethanol (Anderson et al., 2012), physico-chemical treatments, such as ultrasound and calcium carbonate (Tang et al., 2009), and en-zymatic processes (Treimo et al., 2009; Niemi et al., 2013) or a combination of chemical and en-zymatic treatments (Bals et al., 2008; Cookman and Glatz, 2009). Two commercial technologies for zein extraction exist: One is used prior to fer-mentation (Prairie Gold, Inc., Bloomington, Illi-nois) and the other is used downstream (POET, LLC, Sioux Falls, South Dakota). The corn oil and protein extraction (COPE) process was de-veloped at the University of Illinois, licensed by Prairie Gold, and uses in-house ethanol to ex-tract zein from corn before the dry-grind ethanol fermentation. The product Amazein is marketed as a high-quality protein with advantages over DDGS-derived zein as it has not been through the ethanol production process. The POET pro-cess uses different starch and sugar extraction technology for fuel ethanol production and the DDGS produced (POET’s Dakota Gold) is mar-keted as a higher value product because of the less severe processing. Zein is extracted from this and marketed as INVIZ zein; a natural, non-toxic edible protein product that can be used as a biobased alternative in industrial applications.

Fractionation methods are interesting as they also allow the remaining material to be contin-ued to be used as fuel for CHP or further conver-sion of the carbohydrate components to biofuel. Although more than 70% of the protein can be extracted using these methods (Villegas-Torres et al., 2015), the use of solvents and enzymes are costly and the economic feasibility of this has not been demonstrated at large scale apart from the two zein processes highlighted. The costs involved in processing would need to be met by higher value of the concentrated protein

product with applications in sectors other than livestock and pig or chicken feed.

An ideal application of by-products is as a protein feed ingredient in aquaculture. Fish meal is traditionally used as the main protein source. However, the global fish meal output has remained static at 6–7 million t/year for the last 20 years (FAO, 2009). To meet the demands of an expanding aquaculture market, fish feed manufacturers are seeking alternative protein feed ingredients. The increased demand for fish feed, which is driven by both demand from aquaculture and also other feed sectors, such as swine and normal fluctuations in price caused by environmental events, has meant that the price of fish meal has approximately doubled ev-ery 8 years. The volatility in fish meal price and soybean meal (the main protein source in animal feed) is compared in Fig. 13.4. On a protein ba-sis, the price of fish meal is currently four times that of soybean meal, at $3170 and $810/t of pro-tein for fish meal and soybean meal, respectively (Index Mundi, 2015). DDGS from US bioethanol plants currently sells at US$177/t (USDA, 2015), corresponding to US$656/t protein (assuming 10% moisture and 30% crude protein [CP]). It is clear that if proteins from by-products could be used in fish feed as a replacement for fish meal, this would be a more lucrative application.

Distillers’ by-products are not known to con-tain antinutritional factors, such as trypsin in-hibitors and gossypol, although the presence of mycotoxins may be of concern (US Grains Council, 2012). To be used in fish feed as a fish meal replacer, the by-products would need to have protein concentrations at least similar to fish meal (ie, >65%). It is clear from Tables 13.5 and 13.6 that the currently available by-products do not meet this criterion and would require further steps to concentrate the protein to make them attractive alternatives. Another barrier to the use of distillers’ by-products in fish feed is the amino acid profile. Fish have very specific nutrient requirements, including essential ami-no acids. The amino acid requirements of the



main fish species farmed in Europe and amino acid content in fish meal and soybean meal is summarized in Table 13.7. What is interesting from this is that it is clear that lysine levels (of-ten the first limiting amino acids in feed diets) in distillery by-products are less then fish require-ments, except in the case of dark grains and pot ale syrup from malt distilleries, that is, only

malted barley by-products provide adequate ly-sine. In all cases, the methionine levels are less than required, although this is typical of plant protein sources.

Distillery by-products have been used in aquaculture (Randall and Drew, 2010; Shur-son, 2012; Øverland et al., 2013; Magalhães et al., 2015). However, the use of the grain-based

TABLE 13.7 Amino Acid Requirement of Important European Aquaculture Species (As % Minimum of Dietary Protein) and Amino Acid Composition of Fish Meal and Soybean Meal

Amino acid Atlantic salmon Rainbow trout European seabass Fish meal SBM

Arginine 3.7 4.7 4.6 5.8 7.4

Histidine 1.8 1.6 1.6 2.2 2.6

Isoleucine 1.8 1.9 2.6 4.3 4.6

Leucine 3.2 3.3 4.3 7.0 7.5

Lysine 4.1 4.2 4.8 7.5 6.1

Methionine 2.3 2.3 2.3 2.8 1.4

Phenylalanine 2.8 2.8 2.6 3.8 5.0

Threonine 0.5 1.9 2.7 4.1 3.9

Tyrosine 0.5 0.5 0.6 1.1 1.3

Valine 3.0 3.0 2.9 4.9 4.8

SBM, soybean meal.Data obtained from Feedipedia (www.feedipedia.org) and Food and Agriculture Organization of the United Nations (FAO), Aquaculture Feed and Fertilizer Resources Information System (www.fao.org/fishery/affris/species-profiles/en/).

FIGURE 13.4 Price of fish meal (Peru fish meal and pellets, 65% protein) and soybean meal (CME soybean meal futures, min. 48% protein) (data from Index Mundi, 2015) .

http://www.feedipedia.org/

http://www.fao.org/fishery/affris/species-profiles/en/

5 PROTEIN-ENRICHED BY-PRODUCTS 249


by-products is restricted by the high fiber con-tent (at the expense of protein) and also the presence of phytate. High levels of structural fiber are responsible for limited tolerance of rainbow trout for DDGS in their diet (Stone et al., 2005; Reveco et al., 2012). Supplementa-tion with essential amino acids, such as lysine and methionine, is required to achieve higher incorporation levels (Cheng et al., 2003; Cheng and Hardy, 2004a), whereas phytase application may also be recommended to release phospho-rous and other essential micronutrients bound to phytate (Cheng and Hardy, 2004b). The effect of DDGS on feed production such as pelleting has also been assessed (Ayadi et al., 2011).

Technology to preprocess barley to extract the protein for aquafeed prior to ethanol fermenta-tion has been developed and is being commercial-ized by Montana Microbial Products, LLC with technology developed along with USDA Agri-cultural Research Services (Barrows et al., 2009). An enzymatic process is used to concentrate the protein in the barley from 12% to 55% with the starch extracted to be used in bioethanol produc-tion. This barley protein concentrate has been tested in fish feed use. However, this technology is only suitable for fuel ethanol production and could not be applied to Scottish whisky as cur-rent legislation does not allow for preprocessing of the grain with exogenous enzymes.

Research into distillery by-products has mainly focused on the solids residue. Adding value to the liquid residues, either pot ale from malt whiskey production or centrate, thin still-age, or solubles from grain and fuel ethanol dis-tilleries is more of a challenge. During DDGS production, the solubles must be evaporated before codrying with the wet grains whereas pot ale has to be evaporated prior to sale as pot ale syrup for pigs. These liquid residues are low in solids; typically at less than 5%. The two by-products differ in that pot ale also contains the yeast solids whereas during thin stillage pro-duction, the yeast is transferred to the solid wet grain stream. If methods independent of heating

and evaporation could be developed to concen-trate the protein component of these streams, it would be an obvious advantage for distilleries and reduce the energy requirements and carbon footprint of feed production. Extraction of the protein component would also allow the water component of these streams, containing soluble polysaccharides, to be further processed. Anaer-obic digestion is an attractive option for whiskey distilleries (SWA, 2012), and it is envisioned that protein removal from the liquid streams would enhance the AD process while also providing a novel protein by-product. Recent work under-taken by the authors at Heriot-Watt University has led to the development of a process to ex-tract concentrated protein streams from pot ale, allowing a dried protein product to be used in salmon feed. The effect of this process on anaer-obic digestion of the pot ale stream is of particu-lar interest with expected benefits resulting from the reduction in protein loading. This technol-ogy is currently being commercialized by Hori-zon Proteins Ltd. (www.horizonproteins.com), a spin-off company from Heriot-Watt University.

Feed applications are mainly concerned with the nutrient composition, digestibility, function-ality, and palatability of the feed produced from by-products. Brewing by-products [brewers’ spent grain (BSG) and associated proteins and hydrolysates] have been demonstrated to have additional benefits, from both a technofunction-al and health perspective with the potential to be used in human foods (Celus et al., 2007; McCarthy et al., 2013; Connolly et al., 2014, 2015). It is fea-sible that the same techniques and properties can be obtained with distillery by-products. However, for DDGS, there are a number of com-pounds that are highlighted by the FAO that are of potential concern for use in human diets: the amount of yeast cells (nucleic acids), bacteria, low lysine, metal contamination, and antibiotic residues (Makkar, 2012). Antibiotic residues are of concern for US corn ethanol where they are used to reduce bacterial contamination during the fermentation (US Grains Council, 2012).

http://www.horizonproteins.com/



Applications of by-products in feeds are strict-ly regulated. Legislation on the use and market-ing of all feeds in the United States and European Union defined strict guidelines for safety, purity, and traceability. Feed regulations in the United States have been recently updated and expanded under the Food and Drug Administration (FDA) Food Safety Modernization Act (FDA, 2011), which was signed into law in January 2012. This Act requires the FDA to inspect feed produc-tion facilities on a regular basis; monitor myco-toxin levels; test DDGS for antibiotic residues and potential microbial contamination, such as Escherichia coli and Salmonella; use pest control programs; and include good manufacturing practices (US Grains Council, 2012). In the Eu-ropean Union, the marketing and use of feeds is regulated by Directive No. 767/2009 (EU, 2009) and all approved feed components are listed in the EU feed catalog under Regulation No. 68/2013 (EU, 2013). It should be noted that pro-cesses that alter by-products from their approved description and application may require addi-tional approval before they can be used as feeds within the European Union. Article 24(6) of the 2009 feed regulations states that “the person who, for the first time, places on the market a feed material that is not listed in the Catalogue shall immediately notify its use to the representatives of the European feed business sectors referred to in Article 26(1). The representatives of the European feed business sectors shall publish a Register of such notifications on the Internet and update the Register on a regular basis.”

To be marketed as a feed, only the nutritional value of the by-product is taken into account. If additional claims are to be made against a prod-uct, such as added health benefits or animal per-formance, the by-product is classified as a feed additive and must comply with Regulation (EC) No. 1831/2003 (EU, 2003b) or gain approval by submitting a food additive petition (FAP) to the FDA for marketing as a feed additive in the United States. Any additional benefits and effect on health and safety of both humans handling the feed and animal consumers must be scientifically

verified. It has been suggested that it would take 3 years to prepare the dossier at a cost of €1 mil-lion before authorization for the use of the feed additive for specific species and usage within the European Union is granted (Burton et al., 2014). This is obviously a very costly and lengthy pro-cess compared to the marketing of feeds, although feed additives will obviously have a much higher selling price than feed ingredients.

6 CONCLUSIONS AND FUTURE PERSPECTIVES

New applications of distillery by-products are required to ensure that the protein from the ce-real grains and yeast continues to be available for the food chain. Distillery by-products are mainly used as livestock feed, with the high fiber content restricting the levels that can be incorporated into other diets. Wider applications and higher value by-products could result if the protein component could be separated and marketed as a higher-val-ue feed ingredient with the by-product residues being used in other energy-generating processes, such as AD or CHP plants. Integrated solutions that are feasible for a range of distillery capacities are required; a current issue is that technology that is feasible at large-capacity bioethanol and grain distilleries is not economically feasible for smaller-sized whiskey producers. Processes need to be low in energy, for example, dewatering and drying of DDGS accounts for 40% of the energy use of a bioethanol plant (Burton et al., 2014); low cost; robust enough to handle variations in by-product composition; and applicable for the range of distillery capacities. In particular, novel processing solutions are required for the liquid-based streams, which are often disposed of at a cost to distilleries.

There is worldwide need for protein as food and feed ingredients, and this is driven by an increasing population and demand for fish and meat products. With the natural limit on fish meal production levels reached; alternative


REFERENCES 251

protein sources are necessary to meet the pre-dicted increased demand from aquaculture. Pro-tein from distillery by-products could be used as a replacement for fish meal or soybean meal in feed formulations. In Scotland alone, approxi-mately 170 thousand tons of protein is available from Scotch whisky by-products. If the appropri-ate fractionation technology was available, this could be used as a local feed ingredient for the salmon aquaculture sector, reducing the need for imported fish meal and soybean meal. More than 12 million tons of protein is available annually from grain whisky and bioethanol distilleries. With both sectors set to expand, appropriate techniques to recover the protein for food is per-tinent, especially in light of replacing grains that have been diverted from the table to the still.


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C H A P T E R


14Recovery and Applications

of Feather ProteinsN. Reddy, M.S. Santosh

Center for Emerging Technologies, Jain University, Jain Global Campus, Ramanagara District, Bengaluru, India

1 INTRODUCTION

Poultry feathers are inevitably generated as by-products and are mostly disposed as waste. Although universally available at almost no cost and composed of more than 90% protein (kera-tin), a highly valuable biopolymer, there is lim-ited use of feathers for industrial applications. About 96 million tons of poultry are produced worldwide annually with chicken accounting for about 85 million tons, turkey for about 5.6 million tons, and duck meat for about 4.4 mil-lion tons (http://faostat3.fao.org/home/E). Feathers in chicken account for about 10% of their body weight, which means that about 8.5 million tons of feathers are generated every year exclusively from chicken. The availability of 8.5 million tons of a high protein-containing by-product is phenomenal because the produc-tion of protein fibers (wool and silk) across the world is only about 2.5 million tons. In addition, proteins are considerably expensive compared to carbohydrate and synthetic polymers. Wool has a selling price between $1.50 and 8/kg and

silk sells from $8 to 32/kg. Although protein sources other than wool and silk, such as colla-gen, soy proteins, or albumin are available, they have limited quantities and/or are expensive or probably major sources of food and therefore not ideally suited for industrial applications. In addition to their low cost and large availability, feathers have low density (0.9 g/cm3) and hol-low honeycomb-like structures that assist in noise absorption. Because feathers are inevitably generated, they are renewable and sustainable sources and do not require additional resources. Despite these characteristic advantages, most of the feathers generated are disposed of as waste or in some countries used as animal feed (Zhang et al., 2012). However, using feathers as feed could lead to diseases such as bird flu and there-fore, feather feed has been banned in several countries. In addition, feed applications provide minimal value addition.

Although feathers are not used on a large scale for industrial applications, researchers have demonstrated the possibility of using feather and feather keratin to develop various

http://faostat3.fao.org/home/E

256 14. RECOVERY AND APPLICATIONS OF FEATHER PROTEINS


products. Feathers in their native form have been used as reinforcement for composites and to develop thermoplastics (Reddy et al., 2013a,b, 2014). Keratin extracted from feathers and other sources have been used to prepare hydro-gels and nano- and microparticles for medical applications, as a sizing agent for textile yarns, and as fertilizer (Reddy et al., 2015). Products developed from feathers and keratin have been demonstrated to be suitable for electrical, auto-motive, food, pharmaceutical, environmental, and other applications.

The limited use of feathers for industrial applications is mainly caused by two reasons. First, keratin in feathers is highly cross-linked through disulfide bonds that are difficult to break. This makes it difficult to dissolve feathers using normal solvents. Feathers can be hydro-lyzed to extract keratin, but hydrolysis causes considerable decrease in the molecular weight of keratin, resulting in poor properties of products developed. Second, feathers are nonthermoplas-tic and do not melt, which restricts their use to commodity products developed through extru-sion and other techniques. Despite these limita-tions, the increasing need to find renewable and sustainable sources and reduce dependence on petroleum-based products, growing environ-mental regulations on the use of synthetic poly-mer based products will lead to interest in using renewable and biodegradable sources, such as feathers. This chapter provides an overview of the structure of feathers, methods used to extract keratin from feathers, and the potential products that can be developed. Instead of the traditional characterization and analysis of the structure of keratin, we have included the recent findings on the structure and the properties of the feathers and keratin to provide a better understanding of the opportunities for application of feather keratin. Potential applications of feather keratin have been included, but the reader should refer to the original publication for in-depth informa-tion on the process of developing and properties of the products discussed in this chapter.

2 STRUCTURE AND PROPERTIES OF FEATHERS AND KERATIN

Feathers are generally classified as penna-ceous or plumulaceous depending on their location on the body of the bird. Pennaceous feathers are found distally, whereas plumula-ceous feathers are proximal to the body. Fur-ther, feathers can be divided as rachis, barbs, barbules, and the calamus (Fig. 14.1) (Maderson et al., 2009). Rachis (about 6 µm in diameter in most birds) is composed mainly of the cortex containing β-keratin and is responsible for the tensile strength of the feathers. Keratin in the rachis is arranged in the form of fine fibrils and microfibrils that are intricately bonded to the matrix (Lingham-Soliar et al., 2010). Filaments (fibrils) in the cortex are axially oriented and have an average diameter of about 6 mm, but filaments that are circumferentially oriented have also been observed. A schematic depiction (Fig. 14.2) of the arrangement of the fibrils along the rachis reveals the longitudinal and structural arrangement that was confirmed when the sur-face of the feathers were exposed to prolonged biodegradation and the degraded feathers were observed in a scanning electron microscope (SEM) (Lingham-Soliar et al., 2010). Fibrils on the surface had a diameter of about 500 nm, and those beneath had diameters of 100 nm. In each fiber, periodic nodes were seen at intervals of about 70 µm with each node terminating into a characteristic hook or ring (Lingham-Soliar et al., 2010). Fibrils are embedded in a matrix consisting of 3 nm filaments that are assembled into the form of a sheet (Fraser and Parry, 2011a,b). X-ray diffraction studies have shown that the fil-aments in feathers have a helical structure with four repeating units per turn with a pitch length of 9.5 nm. In each repeating unit, there are a pair of twisted β-sheets, with the twist in the sheets being opposite to that in the helix (Fig. 14.2). Each β-sheet consists of 32 residues of keratin (100 residues total). Further studies have shown that the inner surface of the β-sheet consists of

2 STRUCTURE AND PROPERTIES OF FEATHERS AND KERATIN 257


FIGURE 14.1 Schematic of the structure of feathers with dark gray (red in the web version) indicating β-keratin and α-keratin indicated in gray (blue in the web version). For detailed description of the diagram, please refer to Maderson et al. (2009). Reproduced with permission from John Wiley and Sons.



a high concentration of hydrophobic residues whereas charged and cysteine residues lie on the outside (Fraser and Parry, 2008).

The unique and exceptional structure of feather keratin leads to the distinct properties of feathers. Arrangement of the fibrils with the repeat units (nodes) creates a dog-bone shape similar to the “brick-bridge” architecture seen in a nacre (Fig. 14.3). These nodes connect the fiber system by bridging gaps in the matrix and provide high strength, toughness, and resis-tance to the axial fracture. The interfacial matrix present between the fibers provides very high ductility and the ability to absorb and dissipate large amounts of energy before failure. The dog-bone-shaped fibrils minimize fiber pull out and increase the transmission of forces similar to steel-reinforced concrete (Lingham-Soliar, 2014).

Based on X-ray diffraction analysis and the amino acid sequence in feathers, a new model for the repeating unit in feather keratin (twisted β-sheet) has been proposed (Fig. 14.4) (Fraser and Parry, 2009). In the new model, it has been suggested that β-sheets are assembled from dimers and the sheets are perpendicular to the diad. Further, a high degree of specificity was found to be introduced during the assembly of the sheets. Such specific arrangements and sequence regularities lead to the internalization of the amino acid residues and protection from aqueous environments.

Raman spectra obtained from feather and films made from feather keratin were stud-ied to understand the structural changes that occurred during keratin extraction and film formation. It was confirmed that the major

FIGURE 14.2 Schematic depiction of the parts and the structural arrangement of fibrils inside the barbs in chicken feathers. Reproduced from Lingham-Soliar (2014) with permission from Springer.

2 STRUCTURE AND PROPERTIES OF FEATHERS AND KERATIN 259


component of the feathers was the β-sheet structure (1668 cm−1) with a significant amount of sheets with β-turns (1682 cm−1). However, the follicle region of the feathers was found to predominantly contain α-helical struc-tures. Keratin extracted from the feather using sodium sulfide retained the β-sheet structure, but the amount of disordered regions in the keratin increased as suggested by the differ-ences in the peak at 1228 cm−1 (Fig. 14.5). Lower levels of disulfide cross-linking was observed in the keratin compared to native feathers because of the destruction of the bonds during extraction (Church et al., 2010). A character-istic 13C NMR spectrum of feathers (Fig. 14.6) had peaks between 0 and 50 ppm mainly caused by the alkyl side-chain resonance from the amino acids (Fig. 14.6). A distinct peak at 40 ppm is thought to be caused by the β car-bon in cross-linked cystine and another peak

at 172–173 ppm was suggested to be caused by the carbonyl resonance related to glycine and the β-sheet conformation of the feather (Zhang et al., 2012). Regarding physical structure, a two-dimensional net structure was detected for feather keratin as early as 1950s. The net struc-ture has been corroborated in recent studies, and it has been observed that the keratin fila-ments are arranged side to side in an orthogo-nal pattern (Fraser and Parry, 2011a,b).

Genetically, up to 149 β-keratin genes and about 33 α-keratin genes have been identified in the new chicken genome assembly. Although, the number of α-keratin genes is considerably much lower than the β-keratin genes, the α-keratin genes are thought to replace some important functions of the β-keratin genes during the for-mation of appendages (Ng et al., 2014). Most of the β-keratin genes are located on chromosome 27 (63 genes), chromosome 25 (13 genes), and

FIGURE 14.3 SEM images and schematic representation of the nodes in feather barbs that is similar to the brick-bridge mortar structure and responsible to provide the excellent mechanical properties to the feathers. (A) shows that the corticle cells in the barbules have nodes, (B) is a 3D depiction of the fiber bundling, and (C) shows the brick-bridge mortar structure between the barbules and the matrix and the crack stopping mechanism. Reproduced from Lingham-Soliar (2014) with permission from Springer.


FIGURE 14.5 Raman spectra of the rachis and calamus (A), barb and barbule (B) and follicle sheath (C) in feathers and in the extracted keratin (right image). Reproduced from Church et al. (2010) with permission from Elsevier.

FIGURE 14.4 Models developed to depict the repeating unit in feather keratin. Spheres represent location of groups on the side chains and different chemical bonds. Please refer to the original article for detailed color description of the model. Top and bottom rows represent two possible configurations. Projections down the x-axis are (A) and (D), y-axis are (B) and (E), and z-axis are (C) and (F). Reproduced from Fraser and Parry (2009) with permission from Elsevier.

3 EXTRACTION OF KERATIN FROM FEATHERS 261


some β-keratin genes are also found on chro-mosome 1 (1 gene), chromosome 2 (13 genes), chromosome 7 (1 gene), and chromosome 10 (6 genes) (Ng et al., 2014). However, some β-keratin genes have been found to be expressed only in specific parts of the feathers, such as the bar-bules (Kowata et al., 2014).

3 EXTRACTION OF KERATIN FROM FEATHERS

Keratin is extracted from feathers through the cleavage of the intra- and intermolecular disul-fide bonds and hydrogen bonds using reduc-tion, oxidation, sulfolysis, and enzymatic and chemical and/or physical treatments. Chemical extractions employ toxic and expensive chemi-cals and moreover destroy vital amino acids and may need multiple extraction steps (Eslahi et al., 2013). To avoid chemical extractions, enzy-matic approaches (using keratinase) and hot water extraction have been used. The influence of enzyme loading, type of surfactant, concen-tration of reducing agent, and other parameters were varied using protease (Savinase) as the enzyme (Eslahi et al., 2013). Before the enzy-matic treatment, feathers were washed with a

nonionic detergent and calcium carbonate. For keratin extraction, feathers were treated with a commercially available protease (Savinase) with or without a reducing agent. The addition of the surfactant and increasing concentration of the enzyme up to a certain level lead to a substan-tial increase in enzyme activity. However, the reducing agent was found to decrease the activ-ity. Similarly, increasing hydrolysis time up to 4 h increased the total protein extracted beyond which the protein content remained stable. Up to 21.6% weight loss was obtained for the feathers, which corresponded to a keratin yield of 17.7%, and the proteins obtained had molecular weight of 11–28 kDa (Eslahi et al., 2013). In another study, a feather-hydrolyzing enzyme, Serratia sp. HPC 1383, was isolated from tannery sludge and used to dissolve feathers. Up to 83.6% of the feathers could be solubilized by optimizing the treatment of feathers using the enzyme. Fig. 14.7 shows the digital picture of feathers before and after enzyme treatment, which shows the com-plete dissolution (Khardenavis et al., 2009). Although high dissolution of feather could be achieved, it required considerably longer time (up to 4 days) for complete digestion.

Feathers were solubilized using various reducing agents (thioglycolate, potassium cya-nide, or sodium sulfide) to obtain a keratin yield as high as 50% when sodium sulfide was used as the reducing agent (Gupta, 2012a, 2012b). Sodium sulfide has been demonstrated to be an highly effective reducing agent to extract kera-tin from feathers with the advantage that the reducing agent gets oxidized and is removed, resulting in chemical residue–free keratin (Poole and Church, 2015). For extraction, feathers were treated with sodium sulfide under nitrogen atmosphere at 30°C for 1 h. Extracted keratin had a molecular weight of 10 kDa (68–70%) and 15–19% of 20 kDa dimers. Films made from the sodium sulfide–extracted keratin had excellent mechanical properties and was considered to be suitable for industrial applications (Poole and Church, 2015). In another study, to prepare

FIGURE 14.6 13C NMR spectra of feather keratin. Re-produced from Zhang et al. (2012) with permission from John Wi-ley and Sons.



keratin nanoparticles through spray drying, feathers were dissolved using a aqueous solvent system consisting of 8 M urea, ethylenediami-netetraacetic acid (EDTA, 3 mM), the reducing agent mercaptoethanol (125 mM), hydroxyl-methylaminomethane (200 mM) as the buffer and sodium dodecyl sulfate (SDS) as the surfac-tant (Rad et al., 2002). Extracted keratin could be made into sponges and nanoparticles.

Instead of conventional approaches using alkali and reducing agents to extract keratin, ionic solvents (Table 14.1), which are considered to be green, nonvolatile, nonflammable, chemi-cally and thermally resistant, and also having good solubility to keratin have been explored for keratin extraction (Wang and Cao, 2012). Some of the ionic liquids used for dissolving keratin include 1-butyl-3-methylimisdazolium chloride (BMIMCl) and 1-hydroxyethyl-3-methylimidazolium bis/trisfluoromethane-sulfonyl) amide. Using the latter solvent, the highest keratin yield of 21.75% was obtained with a molecular weight of about 10 kDa (Wang and Cao, 2012). Recently, a very high yield of about 75% keratin was obtained using [Bmim]

cl as the solvent along with sodium sulfite and water as additives to assist in dissolution (Ji et al., 2014). Dissolved keratin was precipi-tated using water, and keratin was purified so that the solvent could be recycled, leading to

TABLE 14.1 Various Ionic Liquids Used to Dissolve Keratin and Their Corresponding Dissolution Rate (Wang and Cao, 2012)

Ionic liquid Dissolution rate (%)

1-Allyl-3-methylimidazolium chloride [Amim]Cl

96.06

1-Butyl-3-methylimidazolium chloride [Bmim]Cl

96.13

1-Butyl-3-methylimidazolium bromide [Bmim]Br

84.18

1-Butyl-3-methylimidazolium nitrate [Bmim]NO3

85.11

1-Butyl-3-methylimidazolium Trifluoromethanesulfonate [Bmim]CF3SO3

5.2

1-butyl-3-methyl-imidazolium hydrogen sulfate [Bsmim]HSO4

82.6

FIGURE 14.7 Digital pictures showing the dissolution of feathers after treating with the enzyme. Reproduced from Khardenavis et al. (2009) with permission from Elsevier.

3 EXTRACTION OF KERATIN FROM FEATHERS 263


a completely green process of keratin extrac-tion (Ji et al., 2014). SEM images of the surface showed progressive degradation with increas-ing time (Fig. 14.8) (Ji et al., 2014). However, the extracted keratin displayed characteristic Fou-rier transform infrared (FTIR) peaks at 1646, 1524, and 1233 cm−1 belonging to the vibrations from the peptide bonds. This suggests that the extracted keratin contained β-sheets similar to that in native feathers (Ji et al., 2014). It was suggested that sodium sulfite was necessary as a reducing agent to obtain high yields of keratin from feathers.

In another approach of extracting keratin from feathers, feathers were immersed in a solu-tion containing 0.125 mol/L of sodium bisulfite, 0.05 mol/L of the detergent sodium dodecyl sul-fate, and 2.0 mol/L urea. The mixture was heated to different temperatures (30–100°C) for various periods of time (15, 30, 45 min). Extracted ker-atin was further lyophilized to obtain the pro-teins in the form of a sponge. The molecular weight of the extracted keratin was about 14.4 kDa, similar to that of native feather. The yield of keratin was dependent on the time and tem-perature of extraction and yields of about 48% were obtained (Zhou et al., 2014).

In a three-step method, keratin films for controlled release were prepared by treating feathers with various chemicals. Feathers were immersed in 150 mL solution containing urea

(0.33 mol/L), SDS (0.05 mol/L), 2-mercaptoeth-anol (0.085 mol/L), and tris buffer. The mixture was heated at 70°C for 2 h under nitrogen at-mosphere (Yin et al., 2013). A considerably high yield of 93% keratin was obtained using this approach. Extracted keratin contained alanine (29.5%), cysteine (14.4%), proline (10.1%), and serine (7.0%) as the major components. Further analysis showed that hydrophobic amino acids accounted for about 61% and hydrophilic amino acids were 39%. Films developed from the kera-tin were useful for controlled release applica-tions (Yin et al., 2013).

A unique approach of high-density steam flash explosion (HDSFE) was used to pretreat feathers and improve their digestibility up to 93% (Zhao et al., 2012). A considerable increase in the solubility of the feathers in various sol-vents was observed after HDSFE (Fig. 14.9). In addition, change of pressure also led to con-siderable variations in the amino acid content (Table 14.2), molecular weight, and the amount of α- and β-helix content. Unlike previous approaches of chemical extraction of keratin, HDSFE preserved the molecular weights with two types of proteins (10 and 30 kDa) similar to those found in native keratin. Interestingly, a decrease in cysteine content was observed, whereas no major changes were seen in the con-tent of other amino acids. It was concluded that HDSFE was a rapid, economical and ecofriendly

FIGURE 14.8 SEM images of the surface of the feathers before and after treating with the ionic liquid for 20 and 40 min. Reproduced from Ji et al. (2014) with permission from Elsevier.



FIGURE 14.9 Solubility and digestibility of feathers at different pressures and chemicals used for pretreatment. Reproduced from Zhao et al. (2012) with permission from Royal Society of Chemistry.

TABLE 14.2 Comparison of the Amino Acid Content Between Untreated and Feathers Exposed to Various Levels of High-Pressure Treatment (Zhao et al., 2012)

Amino acids Control

Pressure (MPa)

1.4 1.6 1.8 2.0

Aspartic acid 5.42 5.37 5.64 5.32 4.88

Glutamic acid 8.41 8.59 8.72 8.41 8.25

Serine 9.33 9.53 9.41 8.99 8.69

Histidine 0.50 0.55 0.60 0.53 0.48

Glycine 7.16 8.14 7.97 7.88 7.97

Threonine 4.04 3.99 4.01 3.86 3.75

Arginine 4.96 5.15 5.11 5.00 4.94

Alanine 3.31 3.90 3.80 3.73 3.84

Tyrosine 4.15 4.11 4.10 3.99 3.93

Cysteine 5.07 2.16 2.21 1.66 1.16

Valine 6.06 6.49 6.48 6.41 6.32

Methionine 0.41 0.46 0.50 0.49 0.47

Phenylalanine 3.18 3.81 3.66 3.56 3.62

Isoleucine 3.51 3.83 3.84 3.78 3.77

Leucine 6.16 7.04 6.92 6..74 6.68

Lysine 1.12 1.11 1.18 1.10 1.05

Proline 10.03 12.13 10.13 10.68 11.52

4 APPLICATIONS OF FEATHERS AND KERATIN 265


method of increasing the solubility of feather and obtaining a high yield of keratin for various applications (Zhao et al., 2012).

4 APPLICATIONS OF FEATHERS AND KERATIN

Feathers in their raw form and keratin extracted from feathers have been used to develop various products. The simplest and most straightforward method of using feathers is to incorporate feathers as reinforcement in composites (Fig. 14.10). Whole feathers, feather fibers (barbs), powdered quill, and many other forms have been used to develop feather-based composites, mainly with polypropylene as the matrix (Reddy and Yang, 2007; Huda and Yang, 2008, 2009). As seen in Table 14.3, composites developed using feathers as rein-forcement had properties similar to that of jute

fiber–reinforced composites. However, feather-based composites have low density and high sound absorption, preferable for automobile and other applications. Although most reports on developing composites from feathers have used feathers as reinforcement, it has also been demonstrated that feathers can be used as matri-ces when glycerol was used as the plasticizer under high-temperature compression (Reddy et al., 2013a,b). Alternatively, feathers can be grafted with synthetic monomers and con-verted into thermoplastics and used as matrices (Reddy et al., 2013a,b). Several researchers have extruded composite structures from feathers through compounding and extrusion, mainly using high-density polyethylene and other syn-thetic polymers as matrices (Reddy et al., 2015).

Generally, feather films have poor mechani-cal properties and are cross-linked using physical, chemical, or enzymatic approaches. Acetylation, grafting, or other modifications have also been made to modify feathers and

FIGURE 14.10 Digital image of a composite containing raw feathers (40%) as reinforcement and polypropylene (60%) as matrix.



obtain films (Hu et al., 2011; Shi et al., 2014). Feather keratin made into films after chemical modifications has shown good tensile proper-ties and also excellent elongation and therefore high flexibility (Fig. 14.11), a feature not seen in most biopolymeric films. In addition to use for food applications, feather films have been considered to be suitable as scaffolds for tissue

engineering and have promoted the attach-ment and proliferation of mouse fibroblast cells (Fig. 14.12) (Reddy et al., 2013b). Films made from keratin extracted (sodium sulfide) from feathers was found to have tensile properties better than most biopolymer films and similar to that of some commercially available synthetic polymer-based films

TABLE 14.3 Comparison of Composites Reinforced with Feather Fiber and Quill with Jute Fiber Reinforced Composites (Huda and Yang, 2008, 2009)

CompositeThickness (mm)

Density (g cm−3)

Flexural strength (MPa)

Modulus of elasticity (MPa)

Impact resistance (J m−1)

Noise reduction coefficient

Feather fiber 4.2 0.36 4.2 ± 0.2 380 ± 31 41 ± 7.6 0.17

Feather fiber 3.2 0.47 5.6 ± 0.7 548 ± 82 30 ± 4.1 0.09

Quill 3.2 0.47 9.8 ± 1.0 805 ± 48 56 ± 5.0 0.11

Jute 3.2 0.47 9.0 ± 0.8 1315 ± 42 82 ± 5.0 0.04

FIGURE 14.11 A flexible thermoplastic film made from grafted feather keratin. Reproduced from Shi et al. (2014) with permission from American Chemical Society.



Biodegradable printed circuit boards (PCBs) using feathers as reinforcement, feather-based dielectric materials, and electrodes from car-bonized feathers have been developed for elec-tronic applications. High moisture regain of keratin was considered to be perfect to obtain composites with an ideal dielectric constant

of 1. Because PCBs form a major portion of electronic waste, attempts have been made to use biodegradable materials, such as chicken feathers to develop PCBs. Successful demon-stration of the feasibility of using feather-based PCBs in electronic devices has also been done (Fig. 14.13).

FIGURE 14.12 Attachment and proliferation of mouse fibroblasts on feather keratin films. Panel (A) shows NIH3T3 cell attachment after 4 hours, (B) shows proliferation of the cells after 4 days, (C and D) are the confocal images depicting the spreading of the cells on the butyl acrylate and butyl methacrylate modified feather films, respectively. Reproduced from Reddy et al. (2013b) with permission from Elsevier.



As biofertilizer, feathers were able to improve the amount of proteins in banana from 15.1 to 16 mg/g and the amino acids increased from 2.0 to 2.96 mg/g (Gurav and Jadhav, 2013). Similarly, an increase in the rate of seed germination and height of rye grass was seen when feather was added as fertilizer (Fig. 14.14). Higher content of nitrogen in feathers was considered to be suitable for developing flame retardants. When applied onto fabrics along with boric acid, feathers pro-vided higher flame retardancy than commercially available flame retardants (Wang et al., 2014).

Another extensively studied application for feather keratin is to develop nano- and micropar-ticles. Because proteins such as feather keratin are biocompatible, it would be ideal to develop nano- and microparticles from feather keratin. In addition, keratin is considerably more stable than cereal proteins and therefore expected to be useful in developing nanoparticles without the need for cross-linking (Reddy et al., 2015). A unique method of dissolving feather was devel-oped and the keratin extracted was made into nanoparticles that could enter various organs in mice. However, a majority of the nanoparticles

were found to be located in the kidney and liver (Fig. 14.15). Chemical modifications to increase the biocompatibility and in vivo stability to reduce clearance from the body are required to make feather keratin–based nanoparticles use-ful for in vivo applications.

Keratin protein has been used as absorbents for air filtration and wastewater treatment in various forms, such as nonwoven fabrics, loose fibers, membranes and colloids, short fibers, or particles (Pollard et al., 1992). Based on a com-bined mechanism of physisorption (trapping of pollutants in a porous network) and chemisorp-tion (occurs at chemical functionalities present on keratin proteins), pollutants from a solution or atmosphere are removed using keratin mate-rials. The abundant peptide bonds and amino acid side-chain residues in feather keratin offer active absorption sites for removal of metal ions and organic volatiles such as formaldehyde (Gupta et al., 2012a,b).

Electrospun hybrid membranes based on chicken feather keratin consisting of synthetic polyurethane as a support matrix and keratin biofiber or resin as active sites were used for the

FIGURE 14.13 Digital image of the biodegradable composite (A), circuit (B), composite etched with the circuit (C), and use of the PCB composite in a telephone (D). Reproduced from Zhan and Wool (2013) with permission from Elsevier.



removal of toxic metal ions, such as Cr6+, Cr3+, Cu2+ from wastewater. For Cr6+, the keratin/poly-amide nanofibrous membrane adsorption capac-ity increased from 2.1 to 55.9 mg/g, whereas it lowered to around 0.7 mg/g for Cr3+, and finally in the case of Cu2+, the adsorption capacities for nanofiber mats containing 50, 70, and 90% keratin were found to be 62, 90, and 103 mg/g, respectively (Aluigi et al., 2011) (Fig. 14.16). Like-wise, to remove arsenite (As3+), nonwoven filters immobilized keratin coated with acid-neutral-ized Bauxsol were tested at pH values 4–5 and found that only 53.3% As3+was removed from a

water solution when the initial concentration of arsenic was 104 ppb. Conversely, 96–100% Pb2+ and Cu2+ were removed from water with an ini-tial metal concentration of 5–510 ppb (Kar and Misra, 2004). In another study, Cu and Zn were removed from aqueous solutions with ion con-centration of 10–100 ppm using chicken feather particles treated with NaOH and anionic surfac-tant dodecyl sulfate solutions. It was observed that alkali-treated feathers adsorb more metal ions than those treated with the anionic surfac-tant, followed by the untreated feather (Sayed et al., 2005). Similarly, alkali-treated chicken

FIGURE 14.14 Seed germination (%) (A) and grass height (B) of rye grass seeds before and after treating the soil with the feather hydrolysate as fertilizer. Reproduced from Gousterova et al. (2012) with permission from University of Tehran.



feathers immobilized on a silica surface removed around 72–94% metals (Ca, Mg, Fe, and Mn) from water at a pH of 6–9 within 20–30 min. Keratin fiber was able to remove Cu, Pb, and Hg (three-metal) solution systems under acidic conditions (pH 1.9–5.9), in the order of Pb (100%) > Hg (90–98%) > Cu (56–62%). Similarly, in a five-metal solution system Cu, Pb, Zn, Cd, and Ni at pH 4 to 6 the removal was in the order Pb (70–84%) > Cu (38–49%) > Cd (10–17 %) > Ni (7–11%) > Zn (0–4%). The metal ion removal order is attributed to the atomic size of the ion; the higher the atomic size, the greater is the absorption. Organic dyes such as brilliant blue, tartrazine, malachite green, and erythrosine were removed to an extent of 70–80% from wastewater using chicken feathers (Jin et al., 2013).

Over the past century, keratin-based bioma-terials have been integrated largely with vari-ous biological activities, and one such important activity is their use in drug delivery. Keratin materials are known to regulate cellular rec-ognition and behavior through self-assembled structures. Hence, applications of keratin bio-materials are evident in tissue engineering, trauma care, drug delivery, medical devices,

and wound healing. The ability of protein-based biomaterials to function as a synthetic extracel-lular matrix facilitating cell–cell and cell–matrix interactions have made proteinaceous macro-molecules ideal candidates for use in biomedical applications (Moll et al., 1982). In spite of several proteins being investigated for biobased appli-cations, keratin-based materials have been more promising because of their natural abundance, mechanical durability, intrinsic biocompatibil-ity, and biodegradability (Yu et al., 1993).

The spontaneous self-assembly of keratin solutions have shown reproducible architecture, dimensionality, and porosity while possess-ing cell-binding motifs, such as leucine-aspartic acid-valine (LDV) and glutamic acid-aspartic acid-serine (EDS), which are capable of support-ing cellular attachment together with cellular proliferation, infiltration, and attachment. The regenerated keratin biomaterials have proven advantageous in the control of specific biologi-cal functions in a variety of tissue-engineering applications. Keratin films extracted from feather, wool, and human hair have been tested for cell compatibility by cultivation of mouse fibroblasts on the surface of films (Yu et al., 1993). The growth

FIGURE 14.15 Ability of keratin nanoparticles to enter various organs in mice. Reproduced from Xu et al. (2014) with permission from American Chemical Society.



FIGURE 14.16 SEM images and diameter distributions of keratin nanofibers obtained from 20 wt.% of keratin un-treated and formaldehyde treated after 24-h water immersion. Reproduced from Aluigi et al. (2013) with permission from Elsevier.



of cells on keratin substrate proved to be more adhesive to cells and more supportive of cellu-lar proliferation. Further, addition of chitosan to keratin films increased its mechanical strength and demonstrated antibacterial properties. By adding a cell adhesion peptide, Arg-Gly-Asp-Ser (RGDS), at the free cysteine residues of reduced keratin extracts, RGDS keratin films proved to be excellent substrates for mammalian cell growth (Yamauchi et al., 2003; Schweizer et al., 2006). In addition, structural properties of keratin were increased by blending with poly(ethylene oxide) (PEO) enabling the use of keratin materials as scaffolds for cell growth, wound dressings, and drug delivery membranes (Yamauchi et al., 1996; Ghosh et al., 2014).

Keratin-based hydrogels are shown to be neu-roinductive and capable of facilitating regenera-tion in a peripheral nerve injury model in mice (Tonin et al., 2007). The in vitro activity of Schwann cells was enhanced using keratins by inducing cellular proliferation and migration, and also by upregulating specific gene expressions required for significant neuronal functions. Keratin gel-filled conduits served as a neuroinductive provi-sional matrix when translated into a mouse tibial nerve injury model, mediating axon regeneration and improved functional recovery compared to sensory nerve autografts (Schweizer et al., 2006). In a lethal liver injury, keratin hydrogels have been exposed as a hemostatic agent in a rabbit model. Keratin-based biomaterials further have to be translated into the human clinical setting to elucidate the mechanisms by which these mate-rials regulate hemostasis and nerve regeneration. Hence, further research is very much necessary to exploit the potential of keratin-based biomaterials to be used in drug delivery application.

5 CONCLUSIONS

Recent developments in the extraction of feather keratin, such as HDSFE enable us to obtain very high yields (93%) at an economical

price using environmentally friendly tech-niques. Unlike conventional approaches of chemical extraction, it is also now possible to preserve the native structure of feather to a large extent in the extracted keratin enabling us to develop products with good properties. Products developed from feathers have been found suitable for application in the electron-ics, automotive, thermoplastic, food, medi-cal, textile, and many other industries. Even with such vast possibility for applications, it is difficult to comprehend that feathers are not being commercially used and are mostly discarded as waste. Although the properties of the products developed from feathers may be inferior to that of commercially available products, the low cost and sustainable and renewable nature make feathers an enviable choice for developing bioproducts. Chemical modifications, blending with other biopoly-mers or synthetic polymers and developing new technologies for processing of feathers could be considered to commercialize feather-based products.


FTIR Fourier transform infrared spectroscopyHDSFE High-density steam flash explosionkDa KilodaltonPCBs Printed circuit boardsppb Parts per billionPEO Polyethylene oxideSEM Scanning electron microscopeSDS Sodium dodecyl sulfate

AcknowledgmentsThe authors thank the Center for Emerging Technolo-gies, Jain University for their support to publish this work. Narendra Reddy acknowledges the support from the Ministry of Science and Technology, Department of Biotechnology, Government of India through the Ramalingaswami Fellowship. MS Santosh thanks the De-partment of Science and Technology for their support through the Young Scientist Project Grant.


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C H A P T E R

275Protein Byproductshttp://dx.doi.org/10.1016/B978-0-12-802391-4.00015-X Copyright © 2016 Elsevier Inc. All rights reserved.

15Algae Derived Single-Cell

Proteins: Economic Cost Analysis and Future Prospects

D.M. Mahapatra*,**, H.N. Chanakya**,†, T.V. Ramachandra*,**,†

*Energy and Wetlands Research Group, Centre for Ecological Sciences, Indian Institute of Science, Bangalore, Karnataka, India; **Centre for Sustainable Technologies (ASTRA), Indian Institute of Science, Bangalore, Karnataka, India;

†Centre for infrastructure, Sustainable Transportation and Urban Planning [CiSTUP], Indian Institute of Science, Bangalore, Karnataka, India

1 INTRODUCTION

Greenhouse gas (GHG) emissions as car-bon dioxide, methane from fossil-based fuel burning, and to lesser extent from unmanaged wastewaters has brought about a serious threat to the existing climatic patterns, leading to glob-al warming that is one of the burning environ-mental problems today. An estimated transition of 0.6°C with a variation from 0.4 to 0.8°C, has been noticed in the global surface temperature (IPCC, 2001). To combat this, there have been ex-tensive research operations aimed at developing a technology that biologically sequesters carbon and thus aids in GHG mitigation (Ramachandra and Mahapatra, 2015). The algal cultures have

been used as a promising substrate for C cap-ture (Chanakya et al., 2012, 2013) and for vari-ous other utilities as a feed for human beings, cattle and aquaculture, conditioning of soil, aeration in wastewater ponds, and as biofertiliz-ers (Becker, 1994) growing in diverse habitats as fresh, alkaline, and marine waters, and so forth (Carlsson et al., 2007; Schenk et al., 2008).

Globally, protein resources are very scant, as it is difficult to fix and transform atmospheric N into ammonia and then proteins (Chanakya and Sharatchandra, 2008). World demands for dietary and feed proteins have resulted in a boost in the expedition for alternatives to plant proteins as a supplemental nutrient. From 1996 onward, unconventional and alternate sources

276 15. ALGAE DERIVED SINGLE-CELL PROTEINS: ECONOMIC COST ANALYSIS AND FUTURE PROSPECTS


of proteins have been looked at. New sources from algae, bacteria, and fungi—the single-cell proteins (SCP)—were derived directly from biomass. Although biomass from the aforemen-tioned sources are being considered as alterna-tives to conventional food and feed sources, large-scale SCP production has yet to be done. Yeasts (eg, Saccharomyces and Candida) were among the first organisms to be used as SCPs, however higher nucleic acid content in SCP re-stricts its utilization as a food or feed. Higher nucleic acids in these protein diets lead to kid-ney stones and even gout because of uric acid precipitation. Enzymatic processes, mechani-cal disruption, autolysis (Curran et al., 1990), and chemical processing can be adopted for decreasing the nucleic acid content, but the pro-cess becomes uneconomical and less feasible (Parajo and Santos, 1995). Many other organ-isms have been also used for the production of SCPs, such as bacteria (eg, Alcaligens and Cellu-lomonas), molds (Fusarium, Rhizopus, and Tricho-derma), and algae (Chlorella and Spirulina). These organisms can grow and pick their nutrients from diverse sources, such as agricultural and municipal wastewaters, and also aid in the en-vironmental degradation of various pollutants (Huang and Kinsella, 1987). Table 15.1 lists the biochemical composition of various groups of organisms, which based on their protein con-tent can be used as SCP. However the presence of any toxic and carcinogenic metabolites from these substrates that are biosynthesized in vivo or accumulated from the environment need to be screened. Fig. 15.1 shows various species grown on different substrates used for SCP production.

Yeasts have been traditionally well known as a food source for their use in the preparation of beverages through conventional fermentation. Although yeast cells have a larger size (good harvestability), high lysine content, and the ca-pacity to grow at acidic conditions, two of the most crucial factors that limit the use of yeast SCP as a food or feed is their high nucleic acid content and low cell wall digestibility ( Alvarez and Enriquez, 1988), because of a complex and thick cell wall made up of mannoprotein ( Rumsey et al., 1990). Moreover a lower growth rate, relatively low protein content, and essential amino acids, such as methionine, also restrict their use as SCPs. Compared to yeast bacteria, they are usually high in protein (50–65%) with a faster growth, but small cell size and density makes harvesting practically complicated and uneconomic. Bacteria have a relatively higher nucleic acid content that needs further process-ing and has lower acceptability as a food source because bacteria are considered harmful, caus-ing several diseases. These constraints are con-trary to fungus, especially filamentous ones that can be easily harvested. They also have a lower growth rate, relatively low protein content, and restricted acceptability as a food source.

Algae have been known to be one of the prim-itive food sources in various parts of the globe. Algae possess variable amount of proteins and high cellulosic algae are especially not pre-ferred for SCPs. Globally, >10,000 tons/year of microalgal biomass is being produced through photoautotrophic cultivation ( Richmond, 2004; Chisti, 2007). The bioprocessing in microalgal biotechnology primarily involves sun drying

TABLE 15.1 Microbial Macromolecular Biochemical Compositions (Becker, 2004; Miller and Litsky, 1976)

Organisms Protein Lipids Nucleic acid Ash content

Algae 40–60 7–20 3–8 8–10

Bacteria 50–65 1–3 8–12 3–7

Yeast 45–55 2–6 6–12 5–17

Fungus 30–45 2–8 7–10 9–14

1 INTRODUCTION 277


to remove moisture (Borowitzka, 1999) and the bulk of the algal biomass (>75 %) is either marketed in the form of dried powders, cap-sules, tablets, pills, or in the form of pastilles ( Becker, 1994; Radmer, 1996). Furthermore, the seed algal biomass that is collected and main-tained can be stored by drying or freeze drying and can be kept for long periods (Anupama and Ravindra, 2000). This biomass is extensively cultivated in natural waters, artificial ponds and photobioreactors. The two most important species that are largely cultivated are Chlorella and Spirulina (Borowitzka, 1999). Compared to other protein sources, algal proteins are of a high quality enriched with other nutrient sup-plements, which is necessary as food and feed,

especially for the larval stages of fish culture. In all these screening procedures, high growth yield, total protein (40–75%), and low nucleic acid contents (<10%) are crucial (Molina Grima et al., 2003).

Protein production was practiced earlier with the help of voluminous fermenters (Anupama and Ravindra, 2000), however, these methods involved the requirement of cooling systems because of the heat generated, which is caused by enhanced metabolism linked to a high oxy-gen transfer rate and high respiration rates. These operations for protein productions are governed by many factors. The economic fac-tors are energy input, cost investment, op-eration and management (OM) costs, waste

FIGURE 15.1 SCP production in various organisms on diverse substrates. Compiled from Miller and Litsky (1976); Becker (1994), Borowitzka (1997); Anupama and Ravindra (2000); and Molina Grima et al. (2003).



management, safety protocols, and market de-mand (Borowitzka, 1997). The most significant cost is of the substrate starting from the naive biomass till its purification (Molina Grima et al., 2003). Simple unit operations for upstream and downstream processing with economic raw material concentration and purification can sub-stantially improve the cost effectiveness of the process. As processes are scaled up, the manu-facturing cost significantly comes down.

The factors that add to the raw materials costs are site procurement costs, production of raw materials, installed capacity and operation, and substrate to protein yield. In the biomass growth and separation processes, the energy required for supplying compressed air, cooling, steriliza-tion, and drying forms the next set of costs dur-ing manufacturing (Molina Grima et al., 2003). The locations and sites with readily and cheaply available energy sources, such as electricity and fossil fuels, are suitable and preferred over other locations. The capital cost involves the costs for the apparatus and its installation of the full ca-pacity process. The scale of the plant is crucial, as small-scale plants with simpler processing of raw materials are profitable. To achieve higher productivities in fermenters, there is a greater investment in energy to attain the optimum con-ditions. The cost incurred during the bioprocess is covered by the product from the firm. The cost of the product depends upon the bioprocess and the resultant quality of the final commodity. The commodity protein product’s acceptability as a food source for human beings not only depends on its nutritional value and safety but also the beliefs of the consumers for the target goods.

Agricultural and municipal water by-prod-ucts and wastewaters can be sustainably used for the production of proteins of feed quality (Borowitzka, 1995) and can also abate significant pollutant levels from these water courses. This helps improve the environmental quality and the local microclimate by reducing GHG emis-sions, nutrient capture and recycling, and qual-ity protein production. Nutrient (C, N, and P)

sources from agricultural runoff and urban wastewaters are the most abundant renewable source that can be potentially used as feed for microbes for protein-rich biomass production. Direct agricultural products, such as cellulosic substances, also contain lignin, hemicellulose, and starch in complex forms that are difficult to break down. Furthermore, naive cellulose as a substrate to be used for protein production re-quires a pretreatment either through chemical (acid catalyzed hydrolysis) or enzymatic pro-cesses (cellulase or carbohydrase) (Anupama and Ravindra, 2000).

Regarding economics, soaring fuel prices, present-day energy costs, and the costs of pure substrates put limitations on the cost-reduction strategies of SCP production. Therefore, SCP as the sole product for these bioprocesses is no lon-ger sustainable as these commodities failed to yield returns for the investment alone and were a major financial setback for investments in SCP. However, algal SCP production with waste-water treatment through algal pond processes can significantly decrease SCP production cost, which is a major economic advantage.

Because of its higher surface productiv-ity per unit area, algal bioprocesses form an efficient SCP crop, which is called algal agri-culture or algiculture. Algal pond processes in wastewater treatment (Harun et al., 2010) help transform wastewater nutrients to algal biomass ( Mahapatra et al., 2013b) and conse-quently SCP. Earlier algal species were pro-moted for their growth in oxidation ponds (Oswald et al., 1957), high-rate oxidation ponds (Oswald and Gotaas, 1957). Algal pond treat-ment processes have effluents with almost no nutrients composed of algal solids (Mahapatra et al., 2011a,b,c, 2013b). For water reuse and re-cycling as per environmental standards, pond treatment has to be devoid of any suspended solids and thus requires clarification and dis-posal of algal solids. Hence, there is a potential scope for converting the algal solid removal costs into algal SCP feed–based utilities.

2 MATERIALS AND METHODS 279


The agricultural wastewater can be very high in nutrients, such as N and P, compared to do-mestic sewage (Wilkie and Mulbry, 2002). One of the best ways to recover these nutrients is through treatment of the wastewaters through algiculture (culture through algae). Few ex-periments that have been conducted on algal growth in these agricultural wastewaters have showed promising results in large biomass production and N and P removal (Gonzalez et al., 1997; An et al., 2003). Nearly, 80% remov-al or nitrates as nitrogen source have been ob-served in the culture Botryococcus braunii (green algae) in animal wastewaters (An et al., 2003). Similarly studies carried out by using filamen-tous algae (Microspora willeana, Ulothrix sp. and Rhizoclonium hierglyphicum) have shown higher nutrient uptake rates from agricultural farm (Mulbry and Wilkie, 2001; Mulbry et al., 2008). A large number of algal-based studies involv-ing higher nutrient wastewaters from various sources are now targeted for high biomass productivities and lipid production (Soydemir et al., 2015; Lee et al., 2016), but studies on the production of protein production from such wastewaters are scanty.

In the past, a few of the algal species were tested for their nutrient uptake, biomass produc-tion, and protein content— Spirulina ( Watanabe et al., 1995; Lee et al., 1987) and Chlorella ( William et al., 1994). The bottlenecks in considering these algal species as a food or for animal feed is their lower photosynthetic capacity to fix C and low digestibility as an animal feed. Conversely, Euglena sp. have showed higher nutritional qual-ity and high-quality proteins in terms of higher amino acid content compared to the preceding species (Nakano et al., 1995; Chae et al., 2006). The enzymatic decomposability of Euglena in laboratory conditions proved to be higher than casein (the milk protein) that makes it an at-tractive fodder for mammals (Miyatake and Nakano, 1998; Ogbonna et al., 1999). Moreover euglenoids have the ability to grow in diverse habitats and are tolerant of extreme conditions,

such as low redox (Tucci et al., 2010; Mahapatra et al., 2013b) and acidic (Yamane et al., 2001) and saline conditions (Wen and Zhi-Hui, 1999).

In this context, the present study investi-gates the growth and kinetics of members of euglenophyceae for their ammonia-to-protein conversion efficiency, biomass and protein pro-ductivity in wastewaters, in a laboratory-scale open reactor. This study also demonstrates eco-nomic means of transforming the urban waste-water into value-added SCP through algal pond processes and thus estimates the cost and eco-nomics for production of a unit of SCP under present environmental conditions.

2 MATERIALS AND METHODS

2.1 Batch Studies

2.1.1 Laboratory Culturing and Growth Environment

Members of Euglenophyceae were isolated from wastewaters taken from an urban agricul-tural wastewater farm and were initially cul-tured in Bolds basal media. The algae grown in these cultures were concentrated to a cell density of 106/mL with a centrifuge. The algal inoculum was then added to the open rooftop photobiore-actors operating at a natural diurnal cycle.

A representative volume of algal broth was collected daily for the period of growth. The al-gal broth was centrifuged, after which the sus-pension volume was used for nutrient analysis and the pellet was used for gravimetry, elemen-tal composition, and subsequent protein de-termination and macromolecular composition analysis through spectroscopic analysis.

The specific growth rate was measured, tak-ing a culture time of 10 days (Eq. 15.1):

= −d C C T TSGR(/ ) (ln / )/0 f i (15.1)

where Tf –Ti (d) gives the time between the two observations

SGR (/d)=(ln C/C0)/Tf−Ti



C and C0 (g/L) gives the concentrations of biomass at Tf (the final day of exponential phase) and Ti (first day)

Biomass productivity was measured (Eq. 15.2):

= − ∆g d C C TBP( /L/ ) ( )/0 (15.2)

whereC (g/L) gives concentration of biomass at the

end of the cultivation (exponential phase)C0 (g/L) gives concentration of biomass at the

starting of the batch cultures∆T gives the duration of cultivation

2.1.2 Feed Wastewater, Nutrient, and Protein Analysis

The feed for the batch reactors was carried out with urban wastewaters collected from the nearby locality. The treatment parameters and nutrient (C, N, and P) analysis were per-formed as per standard protocols (APHA, 1998) and using HACH reagents. The total elemental analysis of the biomass was carried out by an el-emental analyzer (CHNS; LECO, True Spec). All experiments were carried out in triplicate. Re-sults were analyzed using Origin Pro software. Linear correlation tests, ANOVA analysis, and Tukey’s post hoc analysis were used to identify any significant variations wherever applicable.

The protein measurements were as per the total Kjeldahl nitrogen (TKN) and the total ni-trogen (TN) protocol following persulfate diges-tion, where the biomass sample is first digested and forcibly converted to nitrates and then ana-lyzed for total nitrogen content and total pro-teins. The protein productivity was measured (Eq. 15.3):

= − ∆g L d P P TP ( / / ) ( )/PROD 0 (15.3)

2.1.3 Nitrogen Budget AnalysisAnalysis of the nitrogen budget was recorded

for the growth period of the algal culture. Input N in the form of Org N and Amm.-N, TN, and

N in the algal biomass were determined. The N in the biomass and sludge and the culture broth was measured. The N budget (NBUDGET), nutri-tional value (NNUTRITION VALUE), protein retention (PRETENTION), and protein conversion efficiency (PCONVERSION ) are provided (Eqs. 15.4–15.7):

= −N N NBUDGET INPUT LOST (15.4)

N=[(N N )/N ] 100

RETENTION

INFLUENT EFFLUENT INFLUENT− × (15.5)

= −×

N[N N

/N ] 100

NUTRITION VALUE

UPTAKE ACCUMULATED(MIN)

UPTAKE (15.6)

= − ×P [(P P )/P ] 100CONVERSION BULK BIOMASS BULK (15.7)

2.1.4 Proximate and Ultimate AnalysisThe proximate analysis was conducted with

the help of gravimetry and by drying in a muffle furnace at >550°C temperature for determina-tion of ash content and volatile solids. Drying overnight at 110 ± 2°C was performed for deter-mination of moisture content. The higher heat-ing values of the algal sample were calculated using both Dulong’s formula and the modified Dulong’s formula to check whether there was any significant impact of N, which is a protein source for SCP (Eqs. 15.8 and 15.9):

= + −−HHV(MJ kg ) 0.3338C 1.422[H (O /8)]1 (15.8)

= + − −

−HHV(MJ kg )0.335C 1.423H 0.154O 0.145N

1

(15.9)

whereC, H, O, and N are the mass percentages

of carbon, hydrogen, oxygen, and nitrogen, respectively.

The ultimate analysis for the elemental con-tent of the algal SCP samples was carried out using Thermo Scientific Flash 2000 Organic Elemental Analyzer (SCP sample size 2–5 mg;

BP (g/L/d)=(C−C0)/∆T

PPROD(g/L/d)=(P−P0)/∆T

NBUDGET=NINPUT−NLOST

NRETENTION=[(NINFLUENT−NEFFLUENT)/NINFLUE

NT] × 100

NNUTRI-TION VALUE=[NUPTAKE

−NACCUMULAT-ED (MIN)/NUPTAKE] × 100

PCONVERSION=[(PBULK−PBIOMASS)/PBULK] × 100

HHV (MJ kg-1)=0.3338C+1.422[H−(O/8)]

HHV (MJ kg-1)=0.335C+1.423H−0.154O−-

0.145N

2 MATERIALS AND METHODS 281


finely grounded powder). The method involves combustion of the SCP samples in an oxygen environment where the elemental C, S, and N are converted into CO2, SO2, N2, and NOx. These gases are then passed through the IR cells for de-termination of C and S contents and through a thermal conductivity cell with detectors (TCD) for determination of N content. The highest de-gree of accuracy is ensured by complete conver-sion of the sample to elemental gases without dilution or splitting. Oxygen is measured in a separate furnace that was used to pyrolyze the SCP sample (2–5 mg in silver weighing cap-sules) in an inert atmosphere (He) at 1060°C to produce CO detected in a self-integrating, steady-state thermal conductivity analyzer. The oxygen content detection was made through a microprocessor based on the initial weights.

2.1.5 SEM-EDXA Analysis and Biovolume Calculation

Scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDXA) was performed for the fixed algal cells and crushed SCP samples. The pretreatment and fixing was performed:

1. Fixing the algal cells in 2.5% glutaraldehyde2. Dehydrating with ethanol (from 30% until

reaching 100% concentration)3. Drying the algal cells4. Mounting on carbon tapes (over aluminum

stub), gold-sputtered5. Observation and imaging using FEI ESEM

Quanta 200

Elemental detection was performed with an EDS Detector EDAX Genesis with working resolution of 0.5 nm at >10 kV, 2.5 nm at 1 kV, and 3.5 nm at 500 V, with accelerating voltage, 2–30 kV. Elemental analysis was done per meth-ods followed by Mahapatra et al. (2014). Bio-volume calculation was performed as per the measurements and geometry of the algal cells observed through light microscope and electron microscopy.

2.1.6 ATR-FTIR Analysis for Macromolecular Composition

Macromolecular composition for determina-tions of biochemicals was performed by attenu-ated total reflectance–Fourier transform infrared spectroscopy (ATR–FTIR) (Alpha Bruker). Algal pellets were vacuum dried at –10°C and were then analyzed with ATR–FTIR spectroscopy in the absorbance mode (1800–800/cm) wave numbers at 128 scans with a spatial resolution of 2/cm. The algal biochemical compositions, especially amide I and II bands, were monitored throughout the growth period, and the collected data were processed in Origin Pro software with an initial baseline correction, scaled up to amide Imax (Stehfest et al., 2005). P/L and P/C were de-termined from the area under the curve.

2.2 Field Investigations

2.2.1 Study AreaField observations and exploratory record-

ings were carried out at Vpuram Agricultural Farm. This farm consists of a large agricultural area and a wastewater treatment farm involv-ing pond systems with initial facultative ponds (∼5 ha; 4 m deep) and final maturation ponds (2.5 ha; 2 m deep) as shown in Fig. 15.2. Dur-ing the study period, the mean residence time of the treatment varied from 8 to 14 days, caused by variations in wastewater flow at an average loading of ∼20–30 MLD. The facultative ponds have two identical units with a surface area of >10,000 m2 and volume of >4,00,000 m3. The maturation pond has two identical units with a surface area of ∼50, 000 m2 and volume of >1,00,000 m3.

2.2.2 Economics and Cost-Based Analysis of Protein Production from Wastewater Bioprocesses

Total capital cost involved infrastructure, operation and maintenance (OM), and land cost. The infrastructure cost included the cost



FIGURE 15.2 Study area and Euglenophycean members growing in treatment ponds. (A) Vpuram Agricultural Farm and treatment ponds; (B) Algal growth in ponds; (C) Euglenophycean cells in ponds; (D) Bright field light microscopic view of euglenophycean cells; and (E) scanning electron microscopy (SEM) micrograph of euglenoids.

3 RESULTS AND DISCUSSIONS 283


of the concrete impoundment, lightings, screen, sludge removal facility, and offices. The OM costs included the wages of human resources, energy requirement, repair, and dosing chemi-cals. The lifetime of the treatment ponds was estimated to be 30 years. The total annual cost was calculated as a product of capital recovery factor and the initial capital cost plus the OM cost. The downstream processing of the algal biomass consists of drying and milling. Power cost was calculated at a rate of 4.8 INR/kWh. Repair costs for civil work, and mechanical and electrical equipment are estimated annually as a certain factor per percentage multiplied by total construction cost. The annual repair cost for civil work was calculated at 0.4 and 0.15% of the capi-tal cost for conventional wastewater treatment systems. The annual repair cost for mechanical and electrical equipment was calculated at 3% of the total construction cost. The service life of STPs was estimated at 30 years, which served as the period for lifecycle cost. The total annual cost was calculated (Eq. 15.10):

= × +AC RF IC OMTOTAL CAPITAL TOTAL (15.10)

whereACTOTAL is the total annual costRFCAPITAL is the capital recovery factorICTOTAL is the initial cost (eg, for construction,

land) OM is the operation and maintenance cost (eg, human resource, power and energy, re-pair, chemicals)

3 RESULTS AND DISCUSSIONS

3.1 Batch Studies

During the batch reactor studies, the initial feed concentrations given by TOC, TN, and TP were 250, 32, and 18 mg/L respectively. The pro-tein source, Amm.-N, was 25 mg/L, accounting for ∼78% of the total N source. It was consid-ered that these nutrients were sufficient for the

growth and development of the batch algal cul-ture (Mahapatra et al., 2014). The pH, DO, elec-trical conductivity, and TS were 6.8, 0.2 mg/L, 1200 µS/cm, and 1400 mg/L, respectively. Dur-ing the exponential growth, the pH was ∼8.5, electrical conductivity was ∼977 µS/cm, and DO levels were ∼18 mg/L.

3.1.1 Biomass Production and Nutrient Removal

The dry cell weights of the cultured biomass steadily increased from day 2 to day 8, after which the weights declined. The maximum bio-mass was ∼1.3g/L (Fig. 15.3A), indicating the ability of these algal members to grow mixotro-phically in wastewaters (Mahapatra et al., 2014; Mahapatra, 2015; Soydemir et al., 2015; Lee et al., 2016). Similar biomass densities 1.2–1.8 g/L were observed in 8 days in the two-phase photo-period studies on wastewaters (Lee et al., 2016). Much higher biomass densities (3.5–12.5 g/L in heterotrophic conditions; 1.6–6.2 g/L in photo-heterotrophic conditions) were observed in the growth and culture studies of Euglena gracilis (Schwarzhans et al., 2015). The presence and growth of these photoflagellates are proxies for high organic matter in wastewaters (Mahapatra et al., 2013a,b). Mixotrophy is advantageous compared to phototrophy as it can phototro-pically consume both inorganic C in the form of dissolved CO2, carbonates and bicarbonates during the daytime and osmotrophically con-sume dissolved organic carbons and volatile fatty acids present in the wastewater during the night time. Euglenophycean members also procure their nutrition through phagotrophy (Mahapatra et al., 2013d,e) and help in saving treatment time of wastewaters, where there is a dependence on bacteria in the initial stages for its extracellular enzymatic decomposition of complex organic macromolecules into sim-pler forms. Such beneficial algal processes save treatment time, cost, and increase treatment efficiency (Mahapatra et al., 2013a,b,c,d,e,f). Many studies have reported the efficiency

ACTOTAL=RFCAPITAL × ICTOTAL+OM



FIGURE 15.3 (A) Variation of growth rate with decrease in TOC; (B) amide I variations (ATR-FTIR); (C) transitions in macromolecular composition ratios; and (D) variations of protein production with increase in biomass during the batch cul-ture. ATR-FTIR, attenuated total reflectance–Fourier transform infrared spectroscopy.



of mixotrophic growth in wastewater treat-ment. A mean specific growth rate of 0.26/day was observed during the batch growth phase, which is comparatively lower than ear-lier studies on euglenoids grown on glucose (Schwarzhans et al., 2015).

The carbon removal was measured in terms of TOC decrease in days. The batch process showed ∼90% removal of organic C (TOC) where the bulk of the C removal (∼85%) took place in the first 4 days (Fig. 15.3A). Similar re-sults were also found in other algal batch studies grown in (1) wastewater, where the C removal in terms of COD by Scenedesmus sp. was 92.3% under the two-phase photoperiod studies (Lee et al., 2016); and (2) sago starch factory wastewa-ter, where C removal by Spirulina sp. was 98% (Phang et al., 2000). This is attributed to the mix-otrophic growth and rapid soluble C consump-tion by algal biomass. The incremental biomass growth was significantly correlated with per-centage TOC removal (R2 = 0.85; p < 0.005).

A higher TN and NH4─N removal efficiency of ∼93 and 98% was observed during the batch growth period attributed to rapid uptake of N sources from wastewater for the growth and de-velopment of algal biomass (Table 15.2). Similar results were also found on algal batch cultures in wastewaters with ∼96% N removal with Scenedes-mus sp. (Lee et al., 2016) and 99.9% of Amm.-N removal by Spirulina sp. (Phang et al., 2000). Only ∼65% of TP removal was observed during the culture period owing to incomplete uptake of P in wastewaters possibly due to high P enrich-ment in wastewaters and recalcitrant forms of P in wastewaters (Table 15.2) opposing to other studies (Lee et al., 2016) where ∼98% of TP re-moval occurred in a 2 stage photoperiod system through Scenedesmus sp. On the contrary ∼85% of inorganic P removal (Table 15.2) occurred within the growth period attributable to the large biomass stock at the end of the growth phase, which is lower than 99.4 % inorganic P removal by Spirulina sp. grown in sago starch processing wastewater (Phang et al., 2000).

3.1.2 N Fixation, Budget, and SCP Production

The wastewater N mainly in the form of Amm.-N and organic N are mostly up taken by algae. The correlation between increase in algal biomass with percentage removal in TN (r = 0.98; p < 0.0001) and Amm.-N (r = 0.95; p < 0.005) sug-gests rapid N uptake and fixation in algae. From a mass balance perspective in the batch culture, ∼33 mg/L of TN was fed into the system consist-ing of ∼25 mg/L of Amm.-N. At the end of the batch studies, the TN and Amm.-N composition was ∼2.28 and ∼0.44 mg/L respectively. The mean biomass N content was ∼3.36 g/100 g of dry biomass. From a net algal biomass of ∼0.9 g, the average N content in the biomass was found to be ∼30 mg. The total crude protein yield in the biomass at the end of the growth phase was re-corded to be ∼187 mg/L, and the crude protein productivity was ∼20 mg/L per day. The whole cell SCP productivity was ∼112 mg/L per day, which is high compared to earlier studies on Spirulina (85.7 mg/L per day; Ayala and Vargas, 1987) cultured in agro-wastewaters and low (320 mg/L per day; Andrade and Costa, 2007) when the same species is cultured in molasses under heterotrophic growth conditions.

The protein concentration in the biomass var-ied from ∼50 to ∼240 mg/L on a day-to-day basis (Fig. 15.3D). The highest accumulation of proteins was observed on the fourth day possibly because of a regime shift from mixotrophic to photoauto-trophic mode of nutrition as ∼85% of the soluble C was already consumed through mixotrophy. The NBUDGET and the NRETENTION in the reactor was found to be 30.7 mg per unit volume (1 L) and 92.98% respectively. If the entire N composition in wastewater was decomposition products of pro-teins, the protein conversion efficiency was 93.3%. The NNUTRITION VALUE was observed to be 31.96%. If these batch systems are carried out at a scalable area, the average biomass productivity, consider-ing a maximum depth of 50 cm, can be potentially ∼45 g/m2 per day with potential crude protein productivity of ∼10 g/m2 per day.

286

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TABLE 15.2 Percentage Removal of Nutrients During The Batch Algal Process

Time (days)

Total nitrogen (mg/L) Ammonium-N (mg/L) Total phosphate (mg/L) Inorganic phosphate (mg/L)

TN (mg/L)

Daily % rem. rate

TN % rem.

Amm.-N (mg/L)

Daily % rem. rate

Amm.-N % rem.

TP (mg/L)

Daily % rem. rate

TP % rem.

IP (mg/L)

Daily % rem. rate

IP % rem.

1 32.50 — — 25.00 — — 16.25 — — 16 — —

2 32.00 1.54 1.54 23.20 7.20 7.20 16.125 0.77 0.77 15.5 3.13 3.13

3 30.60 4.38 5.85 15.80 31.90 36.80 16 0.78 1.55 10.67 31.13 33.28

4 25.10 17.97 22.77 12.90 18.35 48.40 14.75 7.81 9.29 10.02 6.14 37.38

5 13.60 45.82 58.15 6.13 52.48 75.48 10.5 28.81 35.60 8.87 11.48 44.56

6 7.32 46.18 77.48 2.43 60.36 90.28 7.25 30.95 55.73 4.73 46.67 70.44

7 2.88 60.66 91.14 0.42 82.92 98.34 5.5 24.14 66.56 2.45 48.20 84.69

8 2.28 20.83 92.98 0.44 −6.02 98.24 5.49 0.18 66.63 2.44 0.41 84.75



The FTIR spectroscopic results showed dis-tinct bands for various functional moieties. The assignment of bands was based on studies re-lated to functional groups in algal biomass fol-lowing Stehfest et al. (2005). Among the distinct bands, ∼1655, 1740, and ∼1150 − 950 cm−1 are for proteins (amide I), ester or fatty acid, and carbohydrates, respectively. Fig. 15.3B provides transitions in the protein signatures through FTIR spectra with the culture time from day 1 to day 9. The amide I band in the FTIR spectra increased within the culture period indicating an increase in the protein content of the algal biomass (Fig. 15.3B). The compositional changes were traced by the protein (amide I)/carbohy-drate (P/C) and protein (amide I)/lipid (P/L) changes in the algal members (Fig. 15.3C). Cul-tures experienced a decrease in the P/L band ratio from an initial value of 47 to 6, which was attributable to a relative increase in the lipid con-tent similar to earlier studies on Chlamydomonas reinhardtii (Dean et al., 2010). This is contrary to studies on Pediastrum duplex where the P/L ratio increased to 5 (Dean et al., 2012). However, no significant difference was observed in P/C ra-tio at the beginning and the end of the culture. An increase in the P/C during day 3 is attribut-able to rapid utilization and transformation of soluble C in wastewaters, mostly as acetate and butyrate and bulk N fixation. Similar studies showed an increase in P/C ratio: 1.66 folds in P. duplex in lakes (Dean et al., 2012).

3.1.3 Biomass Composition and Elemental Analysis

The ultimate and proximate analyses of the dried algae samples are shown in Table 15.3. The composition analysis shows a high C and O con-tent, with N content varying from ∼4.2 to ∼6.4% of the biomass on a dry weight basis. The proxi-mate analysis reveals two distinct stages: (1) a dehydration stage during which when dried in an oven up to ∼105°C, the weight loss is only primarily attributed to removal of moisture (Salmon et al., 2009); and (2) a devolatilization

stage in which furnace drying from 150 to 550 °C (Silva et al., 2012) that mostly involves the re-moval of algal volatiles is where most of the weight removal took place (∼50–60%). Further-more, the valorization in terms high heat values (HHV) varied from ∼13 to 14 MJ/kg, which is lower compared to other studies (Mahapatra et al., 2013f). This reveals a great deal of poten-tial for SCP in the valorizable biomass as a nutri-ent source. No significant difference (R2 = 0.53; p >0.05 = 0.72) between the HHV were obtained by using Dulong’s formula and its modified ver-sion that includes N.

Elemental analysis of the dried and ground SCP from the culture studies in biomass and fixed algal cells of these heterokonts showed rel-atively high concentration of N (∼9.7%) in the crushed and ground SCP biomass compared to only ∼2.4% on the cell surface of the algal cells. However, an area scan on the spirally oriented strip structures (Mahapatra et al., 2013c) of these

TABLE 15.3 Ultimate and Proximate Analysis of Whole Cell Algal Proteins (SCP)

SCP

Analysis Laboratory Field studies

Ultimate analysis (%)

Sulfur 0.32 0.54

Carbon 37.2 42.4

Hydrogen 6.11 6.23

Nitrogen 4.19 6.43

Oxygen 44.3 46.3

Proximate analysis (%)

Moisture 5 8

Ash 14 8

Volatiles 72 84

Fixed carbon 9 6

HHV (MJ/kg) 13.23a 14.78a

13.67b 14.94b

a Following Eq. 15.1 (Dulong’s formula).b Following Eq. 15.2 (modified Dulong’s formula).HHV, high heat values.



algal cell surfaces showed a high N content at-tributed to N rich proteinaceous features on the cell surface. The crushed algal cells showed ∼70 and ∼16% by weight of C and O com-pared to ∼80 and 13% of C and O in the naïve fixed cells. Among the other mineral ions were Na, Mg, P, Al, K, Ca, Si, and S, which were pres-ent in minimal concentrations as also observed in earlier studies (Mahapatra et al., 2014), shown in Fig. 15.4. The electron microscopy studies re-vealed varied and interesting surface morpholo-gies of these heterokonts, owing to their varied movement patterns within a large cell surface area for the essential exchange of ions and nutri-ent intake (Mahapatra et al., 2013c,d).

3.2 Field Studies and Cost Estimations

During the field investigations, heterokonts, as members of euglenophyceae, were found to dominate the facultative ponds during all sea-sons. The dry weight of algal biomass varied from 0.4 to 0.26 g/L where the mean biomass density was 0.35 g/L. Table 15.4 shows the sea-sonal productivities and yield of algal biomass and SCP in the Vpuram treatment ponds.

Although the installed capacity of the pond unit was ∼67 MLD, only a meagre amount, ranging from 20 to 34 MLD, was fed into ponds that are made up of a mix of agricultural farm wastewater and municipality wastewater. High-er algal biomass (0.4 g/L) densities were ob-served during the pre-monsoon warm periods, showing an area productivity of ∼25 g/m2 per day and a potential yield of ∼300 tons during the growing season.

A detailed morphological characteriza-tion was performed to understand the cellular characteristics of the algal cells sprawling in the wastewater. The euglenoids were found to be in various shapes, sizes, and morphologies and ranged from 104 to 106 order of cells. The shapes varied from spherical, oval, elongated, and fusiform to C-shaped. During locomotion, the cells were elongated, and during resting or

dormant stages, they were often round. These cells were found to have characteristic pyre-noids with paramylon (β-1,3-glucan) that were either round, ovate, rods or in the form of rings that have essential roles in C capture mecha-nisms for enhanced photosynthetic produc-tivity (Mahapatra et al., 2013c,e). The cellular biovolume was calculated considering the cells as spherical. The various characteristics of the heterokonts are provided in Table 15.5. The cell protein density and biovolume were found to be 3.04 × 105 mg/m3 and 0.22 µm3/µm3 of the unit volume.

The cost and economic analysis calculations performed for the Vpuram pond systems were based on the seasonal observations of algal productivity and protein content in the algal biomass. Although the treatment plant was orig-inally meant for treatment as aerated lagoons, it was found to function as nonaerated facultative algal ponds. The treatment facility provides the land area for the algal growth; the wastewater from the agricultural fields and municipalities as the medium for growth; a residence time vary-ing from 8 to 12 days, which is roughly equal to the batch cycle of the euglenophycean members tested in the laboratory shown in the earlier sec-tion involving batch studies; and plentiful solar illumination for the growth of the algal species. Furthermore, the transparency of the algal broth varied from 0.2 to 1.2 m, indicating variations in water turbidity. However, near the inflows, there was absolutely no scope for sunlight pen-etration because of the high quantum of solids in the influx wastewater. Under high irradiance levels, the mean areal productivity observed was ∼0.22 ton/HAc per day in the present continuous system yielding ∼80 ton/year. The biomass productions, the extract of the protein crude, and the purified protein from the biomass were considered in the calculations and were the major contributors to the final cost of the pro-teins. The economic evaluation of the integrated bioprocess (water treatment and algal biomass production) considered costs related to capital



FIGURE 15.4 Scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDXA) (A) crushed algae (B) fixed algal cell.



investments and operating costs; all prices were for the financial year 2015.

The Vpuram treatment plant has the poten-tial to produce more than 100 tons of algal bio-mass annually, with the help of open facultative ponds running on its full installed capacity. The

biomass from the facultative pond effluent can be harvested using a pump with the help of a disk stack centrifuge for the continuously flow-ing system. Because the pond is built on a slope of the catchment to an artificial water body, the entire feed for the algal growth is commuted

TABLE 15.4 Characteristics of Algal Biomass Growth, Productivity and SCP Yield In Various Seasons During Field Survey

Seasons

Volumetric loading (MLD)

Biomass density (g/L)

Surface biomass productivity (g/m2/day) N content

True protein productivity (g/m2/day)

Protein yield (tons/ha)

Total SCP productivity (tons)

Pre-monsoon 20 0.4 25 6.5 1.63 1.95 300

Post-monsoon 23 0.38 23.75 5.7 1.35 1.62 285

Monsoon 34 0.26 16.25 6.2 1.01 1.21 195

Average 25.67 0.35 21.67 6.13 1.33 1.59 260

TABLE 15.5 Cellular Characterization of Algal Cells

Single cell protein characteristics Features/values

Cell nature Algae-heterokonts

Cell count (C) 4.6 × 104 – 6.9 × 106

aCell shape—spherical, oval, elongated, fusiform, c shape Spherical: diameter 20–100 µm

Cell volume—the volume of a cell 4208–525950 µm3

Unit cell volume—the real volume of a million cells, in mm3 265.08 mm3

Packed cell volume (Vc)—centrifuged volume in ml of the cells contained in 1 l. of the sample after centrifugation at 5000 rpm

5.4–7.2 mL

Cell weight—dry wt. of the cells in µg per million cells 0.8 µg/million cells

Cell density—dry wt. of the cells in mg/m3 of real volume 8 × 105 mg/m3

Cell protein density—dry wt. of total crude proteins in mg/m3 of the real volume

3.04 × 105 mg/m3

Cell Index- the packed volume per million cells (Vc/C) 1.85 mL/106 cells

Cell color Pea green to bottle green

Cell types (stages) Round, ovoid (mature)

Cell storage product Starch: paramylon bodies (round/oval/ring shaped)

Cell surface charge—zeta potential –9 to –16 mV

Bio-volume—volume of cells compared to volume of water they are occupied in 1 µm3.

0.22 µm3

a Assuming the cells to be spherical in shape.



through a natural gravity flow, incurring no expenses for pumping the wastewater media or the growth media. The cost of the infrastruc-ture and the land acquisition are the only direct costs. The present costs have been estimated us-ing various sources (Borowitzka, 1999; Molina Grima et al., 2003; Moo-Young, 1977; Venkata-raman, 1995; Grover, 2011). The fixed cost is as-sociated with the costs of important equipment. The direct production cost covers raw materials, chemical consumption, labor wages, charges on various utilities, overhead, and waste manage-ment cost. Here, marketing costs and invest-ments are not considered because the protein in the form of whole-cell SCP is planned to be di-rectly sold for further extraction of value-added chemicals and high-value metabolites.

Commercial use of algal species especially for protein production needs specific desirable characteristics and growth environments. Thus, it becomes vital to select the culture systems for the growth of algae. Certain algal species have affinities for specific substrates or growth condi-tions for higher productivity and avoiding com-petition. In the present case, the euglenoids were found to be abundant in these wastewaters ow-ing to higher availability of organic matter, high-er residence time (Mahapatra et al., 2013b), and low redox potential (Mahapatra et al., 2013a,b), which was difficult to maintain in closed labo-ratory conditions. Similarly, the cultivation of Dunaliella salina (Mohn and Cordero-Contreras, 1990) required brine and open ponds and grows abundantly near saline conditions. Contrary to this, because of high species turnover, Haemato-coccus fails to grow consistently in fully open ex-tensive cultures and needs a closed mechanism for growth.

The type and nature of the culture also governs the harvesting mechanisms; for a pond, processes with a lower abundance of algal population floc-culation or filtration separation is more economi-cal, but in cases of thick or abundant growth cul-tures, low-volume algal reactor centrifugation is more feasible. In the present case, growing the

euglenoids in phototrophic-heterotrophic mode for which an array of various harvesting strate-gies may be required. For obtaining high-value chemicals from algal by-products as the spent biomass after wastewater treatment and evalua-tion of its commercial potential, the unit process can be broken down: (1) culture, (2) harvesting and concentration, (3) separation processes, and (4) packaging and marketing.

The continuous algal systems in the Vpuram treatment ponds were provisioned with con-crete ponds because the culturing process needs a physical infrastructure such as concrete or earthworks, provisions for mixing, liners, pumps, and so forth. This involves several vital inputs to sustain algal system growth, such as nutrients, water, energy sources, such as carbon, and human resources, labor and monitoring and observation services. After sufficient growth of the algal systems, harvesting and concentrating becomes crucial. This process requires equip-ment, such as harvesters, pumps, and some-times essential chemical flocculants. In addition, harvesting requires power and labor. The down-stream processing after harvesting of algal bio-mass involves suitable driers, extractors, a large number of chemicals for separation processes of the desired product, large vessels and storage containers, as well as labor and energy. Before the product can be sent off from the production unit, there are certain basic protocols for quality control (QC) and quality assurance (QA). The fi-nal step of the bioprocess is packaging and mar-keting after QC and QA, which is not discussed here.

The cost analysis and economics of the Vpuram algal culture ponds for the production of euglenoids as SCP is provided in Table 15.6. These observations are made from the faculta-tive lagoons of the algal wastewater treatment–coupled growth ponds that have two units each of ∼ 5 ha spanning an area of >10 ha, as against the total treatment farm spanning ∼18 ha. These ponds with depths of ∼4 m usually run under low-flow conditions where



the system is mixed and churned with natural forces such as wind. This provides a favorable environment for the growth of euglenoids be-cause of the presence of higher organic matter and low turbulent conditions, with a low redox (reducing) environment. Euglenoids have a

unique ability of locomotion in groups during the day and move to deeper zones during the dark periods. These unicellular flagellates are predominantly found ∼10 cm below the sur-face of the ponds during peak sunshine hours and are therefore easier to harvest from the

TABLE 15.6 Cost Economics of Large-Scale Algal Production in Vpuram Ponds

Characteristics Vpuram algal ponds

Algal cultivation units (ponds) 2

Total cultivation area 10 ha

Pond depth 4 m

Algal cell density (harvesting) 0.35 g/L

Rate of harvesting 20 m3/year

Efficiency of harvesting 75%

Proportion of pond harvested 25% (vol. basis)

Proportion of medium recycled 95%

Rate of evaporation 4 cm/day

Algal productivity 20 g/m2/day

N concentration in ponds 20–40 mg/L

P concentration in ponds 4–16 mg/L

Cellular N content (avg.) 5.50%

Cellular P content (avg.) 1%

Annual growth period 360 days

Cost estimations

Land acquisition cost INR 12,000,000/ha (USD 180,000)

Infrastructure development cost (earthworks) INR 2,000,000/ha (USD 300,000)

Culture system costs INR 20,000,000/ha (USD 3,000,000)

Harvesting system costs INR 101,250,000/ha (USD 1,518,750)

O&M costs (15% of infra, culture and harvesting costs) INR 20,300,000/ha (USD 304,500)

Contingency (5% of total costs) INR 7,780,000/ha (USD 116,700)

Power costs INR 9,850,000/ha (USD 147,750)

SCP produced (tons)/ha: 2365.2

Total cost INR 173,180,000/ha (USD 25,977,750)

Commodity cost

Cost of SCP production/kg INR 73.20 (USD 1.098)

Cost of crude protein extract/kg INR 1,331.30 (USD 19.97)

Cost of protein extract/kg with costs for rapid drying and packaging INR 1464.40 (USD 21.97)

P, phosphorus; SCP, single-cell protein; N, nitrogen.



40–50 cm tip of the pond through an algal cap-ture mechanism (Mahapatra et al., 2013b). The harvesting frequency can be once every 8 days as per the earlier batch growth experiments. The concentration of the biomass after harvest-ing can be performed through centrifugation. The construction and infrastructure cost were estimated through local surveys. The charge for equipment, such as pumps, centrifuges, and so forth, were based on current market prices. The capital costs of the algal culturing farm coupled with a wastewater treatment system were fixed considering the life of the treatment biopro-cess as 30 years. The investments on energy were as per the charges in Indian plant set up’s ( Grover, 2011). The price of consumption of a unit of power (1 kWh) was estimated at INR 4.5 ( Ramachandra and Mahapatra, 2015). Table 15.6 provides the basic features and the character-istics of the algal production system for SCP production. The cost of the powdered SCP from

the dried algal biomass was calculated to be INR 73/kg. The price of algal heterokonts, SCP from the Vpuram treatment farm appears to be economical compared to other studies except the one conducted by Benemann et al. (1982), which is provided in Box 15.1.

The cost effectiveness in this integrated ap-proach is essentially because of negative in-vestments for culture resources as nutrients, water, flocculants, chemicals, and so forth. SCP production via solid-state fermentation (SSF), however, has a definite edge over the earlier submerged fermentation (SmF) process ( Pandey and Soccol, 1998), in terms of better growth rates, biomass production, and bioconversion stabil-ity. But specific grain size, nutrient content (C/N ratio), and water content are critical for its op-eration (Nigam and Singh, 1994; Pandey and Soccol, 1998). Perera et al. (1995) reported a two to five times higher cost involved in SmF compared to SSF and the cost of aquaculture feed available

BOX 15.1

S C P P R O D U C T I O N A S W H O L E C E L L B I O M A S S

1. Heterokonts—members of Euglenophyceae cultivated in urban wastewater treatment ponds at Vpuram, Mysore, with an area productivity of ∼16–25 g/m2 per day (INR 73/kg; present study, 2015)

2. Microalgae cultivated in various scenarios (Slade and Bauen, 2013)• Raceway ponds (400 ha) with an area

productivity of 10–20 g/m2 per day (INR 30–133/kg)

• Photobioreactor (10 ha) with an area productivity of 20–40 g/m2/day (INR 281–740/kg)

3. Mixed algal species cultivated in algal tanks (20,000 L) (INR 2700/kg; Fulks and Main, 1991)

4. Dunaliella cultivated in raceway ponds (2 ha) with a areal productivity of 4 g/m2 per day (INR 450/kg; Mohn and Cordero-Contreras, 1990)

5. Spirulina cultivated in raceway ponds (5 ha) with a areal productivity 3.2 g/m2 per day (INR 990/kg; Jassby, 1988)

6. Microalgae cultivated in ponds (800 ha) with a areal productivity 17 g/m2 per day (INR 18/kg; Benemann et al., 1982)

7. Chlorella cultivated in raceway ponds (>1 ha) with a areal productivity ∼27.5 g/m2 per day (INR 1035/kg; Kawaguchi, 1980)

Current prices in INR.



in the market. When compared to SCP produc-tion by pond-based processes, as in the present study, the cost is 13–16 times lower than the costs involved for SSF processes. A considerably high-er cost, however, of INR 1331/kg of crude protein extract from the algal bioprocess was observed in the present study because of the high extraction costs. The same crude proteins cost INR 1464/kg, if drying and packaging costs are included in the commodity cost. Apart from protein being a key composition of the whole-cell algal SCP, the algal cells were found to have 18–26% of to-tal lipids and ∼40–50% crude carbohydrates. In addition to good protein (Chae et al., 2006), car-bohydrates, and lipids (Mahapatra et al., 2013a), euglenoids have been reported to have essential vitamins, minerals, and metabolites, such as par-amylon (β-1, 3-glucan), α-tocopherol, wax esters (Krajcovic et al., 2015), w-3 poly unsaturated fatty acids (Schwarzhans et al., 2015), and are of high commercial values.

The cost estimations shown in Box 15.1 were based on the ground data on seasonal produc-tivities; the protein content in various seasons were calculated and the total biomass produc-tivity was calculated based on various assump-tions from the production perspectives. Among the studied parameters, the biomass produc-tivity and the growth period were the most crucial and sensitive. A 50% increase in the al-gal productivity from ∼20 to 30 g/m2 per day would result in a 28% decrease in the cost of algal biomass. Likewise, a decrease in the an-nual growth period to 265 days would increase the cost of the product by 38%. The cost of the unit algal biomass produced as SCP is sensitive to the transitions in productivity attributable to a higher capital and fixed cost (algal treat-ment pond units, infrastructure for harvesting, human resources and services, and so forth). Box 15.1 does not show cost requirements for nutrients, water, flocculants, and so forth, as there was a continuous influx of nutrient laden wastewater and the algal biomass unique loco-motion patterns in groups avoided the cost that

were supposed to be incurred for flocculants. In present conditions during the field studies, it was observed that there were no requirements for the addition of organic and inorganic nutri-ents to the systems that would have otherwise increased the process cost. Some of the studies have attempted to increase the productivity of euglenoid species as E. gracilis through supply-ing CO2 (Chae et al., 2006). However the cost of pumping CO2 might drive back the benefits of getting a higher productivity. For increased pro-ductivity in pure cultures of Spirulina, there is an alkaline pH requirement that can be obtained by addition of more CO2/bicarbonates, which incurs additional cost. In the same way, the ad-dition of brine (>200 g/L) to cultures of D. salina ensures halophilic conditions but might increase the production cost by pumping offshore and thus this algal population is recommended to be cultured near saltwater lakes or the sea.

The sensitivity analysis (Fig. 15.5) shows al-gal biomass productivity as one of the crucial parameters in attaining stable revenue through algal proteins as by-products. Therefore, any process factor that increases the biomass pro-ductivity at a small operating cost or incremen-tal capacity can be vital for the plant. The selec-tion of a suitable location for algal cultures is one among the many necessary factors because microclimatic conditions support good species growth for longer durations. As in the present study, euglenoids proliferated in the wastewater treatment ponds with a gravity flow, low turbu-lence, and the cultures were mixed by regular wind. The average insolation in the region was ∼5–5.5 kWh/m2 per day with a million lux in-tensity at the peak sunshine period. A high water-retention time of 8–12 days also provided the algal species with ample time to attain the exponential stage and avoided cell washouts. The selection of an appropriate species is the key for any bioprocess that possesses high growth rate, higher yield of the desired product, and adaptation to microclimatic conditions (Borow-itzka, 1995). The advantage of such species is



their ability to fix C by heterotrophy during the dark periods or in the absence of light (Chae et al., 2006; Krajcovic et al., 2015; Schwarzhans et al., 2015), which provides their high com-mercial value. During diurnal cycles and in the presence of potential C sources apart from the nutrients, euglenoids tend to fix C and grow mixotrophically (Mahapatra et al., 2013a,b). Although euglenoids have been targeted as a source of paramylon and starch (Krajcovic et al., 2015), euglenoids can fix proteins as SCP (Chae et al., 2006), lipids, (Ramachandra et al., 2013; Mahapatra et al., 2013a, 2014; Ramachandra and Mahapatra, 2015) and other essential metabo-lites of high commercial value in varying envi-ronments (Krajcovic et al., 2015).

Among the most cost and energy intensive process during the downstream processing of algal biomass is harvesting and cell concentra-tion. Thus algal species with characteristics that offer an advantage in collection and harvesting are very desirable. In the present study the eu-glenoids had a larger cell size 40–100 µm with a higher cellular granularity (specific gravity). The details of the cellular characteristics are pro-vided in Table 15.5. During the day, these photo-flagellates move in masses, where they wander about in the growth medium for light and nu-trition. Often they are found a few centimeters below the surface of the water at higher cell den-sities (>106 cells/mL). During night at low-light conditions, these algae descend down to the

FIGURE 15.5 Sensitivity analysis of the algal single-cell protein (SCP) commodities.



bottom of the ponds and are usually observed in large clumps that can be sustainably harvested through pumping from the bottom. This useful feature can be employed for harvesting algae that can save a significant capital and OM cost involved in harvesting and concentration. In some cases, where there is a lower productivity in large open ponds, such as with the production of D. salina in Australian shorelines, the lower productivity is more than compensated with an efficient harvesting strategy ( Borowitzka, 1997). The strategies for harvesting can be directed de-pending on the usage and utility of the algal bio-mass and the desired product. If the product is supposed to be used as food, feed, or nutraceu-ticals, then chemical flocculation, for example with FeCl3, Al(OH)3, is not advisable.

Extraction and purification methods of the desired algal product differ from product to product and are vital cost components of the whole production process. Product recovery from cellular biomass is an extremely challeng-ing separation process as observed in yeast, where the thick cell wall is difficult to break down for the recovery of pure proteins ( Anupama and Ravindra, 2000), similarly because of the pres-ence of a thick and resistant cell wall, the extrac-tion of astaxanthin from Haematococcus pluvialis requires a mechanical or an enzymatic treatment resulting in higher separation process cost. Con-trary to this case, the cell walls of β-carotene–producing D. salina are extremely delicate, shear sensitive, and easily damaged, resulting in fast annihilation of β-carotene because of oxidation (Mohn and Cordero-Contreras, 1990). Conse-quently, sensitive methods of recovery are es-sential in such cases, depending on the nature and type of the algal cells. In the present study, the euglenoids does not possess a cell wall, mak-ing it easier for cellular metabolite extraction. The euglenoids as SCP has the advantage of easy digestibility in food and feed because of the absence of a cell wall (Chae et al., 2006).

From a cost perspective, the integration of wastewater treatment with algal bioprocess

development resulted in a net gain of revenue. The net annual cost of the treatment plant was negative, amounting to INR 5.3 million as shown in Table 15.7. The net annual cost includes the values of the benefits drawn from the algal bio-process: the cost of treatment of a million liters per day if an activated sludge process is being used (INR 12 per treating 1000 L; Grover, 2011) plus the algal whole-cell biomass as value-add-ed by-product from the system (INR 73/kg, as per earlier calculations), plus the treated recycla-ble water, which in being sold to industries at a price of INR 4/kL. The annual resource recovery amounts to ∼INR 7 million.

The environmental aspects of the integra-tion of treatment ponds with algal productions showed a higher potential of nutrient removal and a comparatively low energy use as shown in Table 15.7. From the societal point of view, this type of pond integration system is benefi-cial as there is a low malodor generation because of aerobic conditions maintained by the algal population except at the inflows. The pond area becomes a community space and is aesthetically pleasing. Such plants are simple and easy to op-erate and also provide the local community with a scope of employment (0.1–0.3 ha of land area is required to treat one MLD of wastewater in the agricultural farm).

4 THE FUTURE OF ALGAL PROTEINS AS BY-PRODUCTS FROM INTEGRATED ALGAL PROCESSES

The main objective of present-day research and development is to tune and rapidly build up proliferating algal biomass with high feed and/or food quality and other commercial value that can be economically cultivated ap-plying principles of sustainability by integrat-ing it with other environmental processes as wastewater treatment, flue gas reduction and so forth. The crucial nexus hovers over select-ing dominant algae and introspecting their

4 THE FUTURE OF ALGAL PROTEINS AS BY-PRODUCTS FROM INTEGRATED ALGAL PROCESSES 297


credibility to maintain stable populations over seasons. This involves their abilities to with-stand grazers and out-compete other algal species and parasitic organisms with efficient environmental tolerance. Furthermore, the algal species must have very specific quali-ties for economical harvesting and further

downstream processes. On a whole, the can-didate species should have abilities to under-go open cultivation conditions, with various environmental stresses, endurance to biotic challenges, dynamic stability, and promising growth and productivity in the present agro-economic setup, especially in the tropics.

TABLE 15.7 Combined Valuation of Wastewater Treatment and Algal Production

Evaluationapproaches Unit Vpuram algal ponds

Cost economics

Investment

Construction costs (land + infrastructure costs) INR/MLD 2,800,000

Operation and maintenance (OM) INR/MLD 14,40,000

Miscellaneous costsa INR/MLD 220,000

Value of benefits

Water treatment (ASP-based process) INR/MLD 12,000

Algal SCP INR/MLD 3171

Recyclable water INR/MLD 4000

Total INR/MLD 19171

Annual resource recovery (potential) INR/year 6,997,415

Net annual cost = OM – Res. recovery INR/year –5,337,415

Environmental aspects

Chemical oxygen demand(COD) % Removal 79

Biochemical oxygen demand(BOD) % Removal 82

Total suspended solids (TSS) % Removal 93

Turbidity (NTU) % Removal 95

Nitrogen (TN) % Removal 36

Phosphorus (P) % Removal 70

Social aspects

Public participation Qualitative measure Yes

Community size served Population/MLD 13,000

Aesthetics Measured level of nuisance from odor Moderate

Human resources to operate plant Staff/MLD 0.2

Level of education/awareness Operational requirements Good

Availability of open space Hectare/MLD 0.1–0.3

aMisc cost involved in algal solids collection, removal, drying and powdering.



Although there are several reports of com-mercially important algal strains grown in large raceway ponds (Borowitzka, 1999), the economic practicability of the processes becomes the ma-jor limitation in the production of SCP to be used as food or feed. In this context, integrating vari-ous environmental bioprocesses, such as waste-water nutrient removal, C mitigation, biomass (food and/or feed) production, purification of water, recycling (reuse), and seeking a common solution for large-scale biomass production as economically and environmentally promising, paves a path for a win–win condition. Among the examples of successful algal bioprocesses are the large-scale Spirulina cultivation as a source of proteins (Jassby, 1988) and the growth of N-rich Anabaena cylindrica, which has inher-ent N2 fixing machinery that helps in recycling N for its N requirement, giving it a competitive advantage over others in low-N environments. Plus it can be used both for human food and animal feed. Interestingly, biflagellates, such as Chlamydomonas sp. And heterokonts, such as euglenoids and Micracitinium sp., naturally oc-cur and often predominate in wastewater treat-ment ponds. Based on characteristic features, morphology, and the growth environment, they are unaffected by zooplanktons and grazers and have a unique ability to form clumps, and thus can be separated with the help of bioflocculation (Mahapatra et al., 2011a, 2013a,b, 2014). Many algae have been targeted and are being cultivat-ed in raceway ponds because of their high lipid content that can be either used for production of biofuels or high-value omega-3 fatty acids, which have been reported in a few species of diatoms (Ramachandra et al., 2009; Mahapatra and Ramachandra, 2013). Conversely, Botryococ-cus brauni (An et al., 2003), albeit slow growing, has the advantage of accumulating substantial amounts of cellar biomass as hydrocarbons that are not vulnerable to be lost or affected by predators because of their storage in extracellu-lar vesicles; B. brauni is being examined for the production of biofuel.

Numerous investigations on the character-istic qualities of diverse algal species reveals stronger possibilities for strain improvement through high throughput mutagenesis and se-lection and various molecular methods of ge-netic engineering that helps in increasing the quantities of the desired proteins and commodi-ties and thus helps in the commercial viability of mass production. Moreover, the traditional phe-notypic selections and genetic recombinations can also be used for strain improvement and commercialization, although a great number of trials are required for assessing the expres-sion of the desired proteins and characters for long-term use and viability. Genetic engineer-ing approaches can be applied for the isolation of mutant genes, which are responsible for the production of essential and high-value amino acids, such as phenylalanine and tryptophan, coupled with high protein yields. A host of dif-ferent recombination techniques can possibly be used to integrate these select genes into the cell genome (Sahm, 1995; Hill, 1994). Hence, rigor-ous research and development are necessary for realizing success in the commercial viability of algal food or animal feed and other high-value byproducts and chemicals, overcoming innu-merable constraints in algal selection, engineer-ing, cultivation, and harvesting for fostering sustainability and a clean green future.

AcknowledgmentsThe authors are grateful to Department of Biotechnology (DBT); The Ministry of Science and Technology (DST); Min-istry of Environment and Forests (MoEF), Government of India and Indian Institute of Science for providing the fi-nancial and infrastructural support. The authors earnestly acknowledge Raykar (IAS) for permitting us to study the algal wastewater system dynamics. The authors sincerely acknowledge the laboratory facilities at Aquatic Ecology and Molecular Ecology laboratories in the Centre for Ecological Sciences (CES), Inorganic and Physical Chemistry (IPC), Biochemistry (BC) and Molecular Biophysics Unit (MBU) at IISc, for their help during the experiments on extraction pro-cedures. We thank the Central facilities and Institute Nano Initiative (INI) for Electron Microscopy/EDXA and FTIR analysis respectively.


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Protein Byproducts Transformation from Environmental Burden into Value-added Products

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