Thesis_value Addition to Aflatoxin Contaminated Peanut Meal

8/10/2019 Thesis_value Addition to Aflatoxin Contaminated Peanut Meal

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ABSTRACT

OAKES, AARON JEFFREY. Development of Process to Add Value to Aflatoxin ContaminatedPeanut Meal. (Under the direction of Jack P. Davis.)

Plants are inexpensive sources of protein and make good candidates for protein extrac-

tion. Processes have been developed to extract protein from corn, wheat, oats, barley, and

peanut meal. Enzymatic hydrolysis increases protein extraction and creates peptides shown

to have bio-active properties. Peanut meal is the material that remains after commercial oil

extraction from peanuts. Aflatoxin content limits the value of peanut meal and this material

is not currently considered to be food grade by the Food and Drug Administration. Recently

a novel process was developed that simultaneously extracted protein and removed aflatoxin

from peanut meal [Seifert, 2009]. A terminal product of this process was an aqueous pro-

tein/peptide extract with negligible levels of aflatoxin. The purpose of this study was to

generate similar protein/peptide extracts and convert them to powders using industrially

relevant larger scale processing techniques.

Naturally contaminated peanut meal containing 52 g/kg aflatoxin B1 was dispersed in

water to create a 4 kg batch at 10% peanut meal to water. The pH was adjusted to either

pH 2.1 or 9.1 using 2N HCl or NaOH. The enzymes pepsin and Alcalase were added at

enzyme to protein levels of 19000 units/g and 0.6 Anson units (AU)/g of peanut meal protein

respectively, similar to the used by Seifert and others [Seifert et al., 2010].

A sodium bentonite clay was added at 0.2% (w/w) as a processing aid to bind aflatoxin.

Each treatment was also prepared without the addition of clay. Pepsin control treatments

were prepared by adjusting the pH to 2.0 but omitting the addition of enzyme, both with andwithout clay. Similarly Alcalase controls were prepared by adjusting pH 9.1, but omitting

Alcalase, both with and without the addition of clay. All eight treatments were performed in

triplicate for a total of 24 samples. Dispersions were stirred for one hour and then allowed

to settle for 40 minutes before the soluble supernatant was collected. The clay with bound

aflatoxin and peanut meal particulates were then physically removed from the water soluble

fractions by gravitational separation.

Maltodextrin with a dextrose equivalent of 12 was added to the collected supernatant

at (8% w/w). After stirring for 45 minutes the supernatant with maltodextrin was spray

dried using a Bchi B-290 lab scale spray dryer. The spray dryer inlet temperature was185C and the outlet temperature was maintained at 90C by adjusting the feed flow rate.

Powder yields from the spray dryer were affected by the treatments and ranged from 56 to

84%. Yields were calculated as the ratio of solids collected to solids sprayed. The treatment

that yielded the most protein was at pH 9.1 with the addition of Alcalase. This treatment


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had good spray drying yields at 60%, the powders contained 30% protein and no detectable

aflatoxin on a dry weight basis. The addition of clay reduced the aflatoxin content of all

powders, regardless of pH or presence of enzyme, to below 20 g/kg. Powders made from

treatments with enzyme had higher oxygen radical absorbing capacity than those without

enzyme addition. All powders contained less than 20 g/kg aflatoxin. Moderately aflatoxin

contaminated peanut meal can be used to produce protein powder containing undetectably

low levels of aflatoxin and 30% protein.


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Development of Process to Add Value toAflatoxin Contaminated Peanut Meal

byAaron Jeffrey Oakes

A thesis submitted to the Graduate Faculty ofNorth Carolina State University

in partial fulfillment of therequirements for the Degree of

Master of Science

Food Science

Raleigh, North Carolina

2011

APPROVED BY:

Timothy H. Sanders G. Keith Harris

Jack P. DavisChair of Advisory Committee


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BIOGRAPHY

Aaron Jeffrey Oakes was born during a snow storm on a cold night in December of 1982 in the

small coastal town of Brunswick, Maine. Considering his start, it is fitting that Aaron would

spend ten years of his life playing ice hockey. Growing up, Aaron was also active in BoyScouts of America eventually earning the Eagle Scout award. His scouting experience evinced

in his high school hockey career when the team elected Aaron, a junior, to be team captain.

By the conclusion of the season, Aaron earned the conferences Sportsmanship Award and

won, by popular vote, the teams Most Valuable Player Award.

After high school, Aarons athletic focus shifted to cycling while his academic focus be-

came the science behind athletics. At the University of Massachusetts - Amherst, Aaron ma-

jored in exercise science while racing for, and eventually presiding over, the UMass Bicycle

Racing Club. With graduation only months away, he discovered food science and promptly

added it as a major. In 2006, Aaron graduated from UMass, earning a Bachelors of Sciencewith a dual concentration in Food Science and Exercise Science.

Shortly after graduation, Aaron worked for Decas Cranberries Inc. grading and assuring

quality of harvested cranberries. Aaron left Decas to work as a product developer in the

R&D department at Unilever North America in Englewood Cliffs, NJ. While at Unilever,

Aaron developed a product that showcased on the supermarket shelves within 18 months,

which proved a very gratifying experience. That same year, Aaron finished 4th place in

the Mountain Bike National Championship race in the Semi-Pro category, another equally

gratifying experience given his years of hard work and dedication. The result qualified Aaron

for an upgrade to the Pro category.

In early 2008 Aaron left Unilever to race mountain bikes full time and prepare for graduate

school at North Carolina State University. By Fall 2008, he had a home in the USDA ARS

peanut lab at NCSU working under the direction of Dr. Jack Davis. Aaron is currently

juggling roles as a professional-level mountain bike and cyclocross racer, a full-time graduate

student, a part-time researcher, and a part-time coach with Wenzel Coaching.

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TABLE OF CONTENTS

List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi

List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Chapter 1 Review of the Literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1 Introduction to the peanut . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1.2.1 Health benefits of peanut consumption . . . . . . . . . . . . . . . . . . . 21.2.2 Bioactive compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.2.3 Peanut protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

1.3 Peanut Meal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61.3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61.3.2 Value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

1.3.3 Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61.4 Aflatoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71.4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71.4.2 Prevalence in crops. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71.4.3 Health concerns. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81.4.4 Prevalence in food and feed. . . . . . . . . . . . . . . . . . . . . . . . . . 81.4.5 Destruction and management of aflatoxin . . . . . . . . . . . . . . . . . 101.4.6 Prevention and management . . . . . . . . . . . . . . . . . . . . . . . . . 13

1.5 Peanut allergens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141.5.1 Mitigation strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151.5.2 Therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

1.6 Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

1.6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151.6.2 Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161.6.3 Nutritional considerations. . . . . . . . . . . . . . . . . . . . . . . . . . . 16

1.7 Spray Drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171.7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171.7.2 Drying bioactive compounds . . . . . . . . . . . . . . . . . . . . . . . . . 181.7.3 Drying method comparisons . . . . . . . . . . . . . . . . . . . . . . . . . 181.7.4 Stickiness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191.7.5 Strategies to reduce stickiness . . . . . . . . . . . . . . . . . . . . . . . . 191.7.6 Maltodextrin as a carrier . . . . . . . . . . . . . . . . . . . . . . . . . . . 201.7.7 Protein denaturation during drying . . . . . . . . . . . . . . . . . . . . . 21

1.7.8 Outlet temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Chapter 2 Amino Acid Analysis of Hydrolyzed Peanut Meal Proteins . . . . . . . . 242.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

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2.3 Materials & Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252.4 Results & Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Chapter 3 Process Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273.3 Materials & Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

3.3.1 Protein extraction from peanut meal. . . . . . . . . . . . . . . . . . . . . 283.3.2 Insoluble fraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293.3.3 Water soluble fraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

3.4 Results & Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313.4.1 Protein extraction from peanut meal. . . . . . . . . . . . . . . . . . . . . 313.4.2 Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333.4.3 Insoluble fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363.4.4 Soluble fraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Chapter 4 Process Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

4.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444.3 Materials & Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

4.3.1 Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454.3.2 Spray drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

4.4 Results & Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464.4.1 Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464.4.2 Spray drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Chapter 5 Finished Material Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 575.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575.3 Materials & Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

5.3.1 Aflatoxin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585.3.2 Turbidity of powder solutions . . . . . . . . . . . . . . . . . . . . . . . . 585.3.3 Fiber, and ash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585.3.4 Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585.3.5 Interfacial tension. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585.3.6 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 595.3.7 Protein solubility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595.3.8 Oxygen radical absorbance capacity (ORAC). . . . . . . . . . . . . . . . 605.3.9 Hygroscopicity of powders . . . . . . . . . . . . . . . . . . . . . . . . . . 60

5.4 Results & Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

5.4.1 Aflatoxin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 605.4.2 Turbidity of powder solutions . . . . . . . . . . . . . . . . . . . . . . . . 625.4.3 Fiber and ash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 625.4.4 Sugar content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 625.4.5 Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

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5.4.6 Surface tension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 665.4.7 SDS-PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 695.4.8 Protein solubility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 695.4.9 Oxygen radical absorbance capacity . . . . . . . . . . . . . . . . . . . . . 695.4.10 Hygroscopicity of powders . . . . . . . . . . . . . . . . . . . . . . . . . . 70

Chapter 6 Concluding remarks & future work . . . . . . . . . . . . . . . . . . . . . . 746.1 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 746.2 Future work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

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LIST OF TABLES

Table 1.1 Nutritional Properties of Raw Peanuts [America, 2008,Ahmed and Young,1982] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Table 1.2 Mineral Content of Raw Peanuts[Ahmed and Young, 1982] . . . . . . . 3Table 1.3 Amino acids composition of peanut protein adapted from [Lusas, 1979] 5Table 1.4 Peanut meal pricing adapted from [Seifert, 2009] . . . . . . . . . . . . . . 7Table 1.5 LD50 of AFB1 for several species adapted from [Newberne and Butler,

1969, Wogan, 1966]. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Table 1.6 Levels of total aflatoxin contamination permissible by the FDA adapted

from [FDA, 2008] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Table 1.7 Legal limits of aflatoxin in peanuts: adapted from [Reddy et al., 2010] . 10Table 1.8 AFB1 contamination levels and frequency in market products adapted

from [Li et al., 2009, Reddy et al., 2010, Turner et al., 2002,Williams et al.,2004] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Table 1.9 Spray dried heat-sensitive or bioactive compounds . . . . . . . . . . . . . 18Table 1.10 Temperatures at which microbial death or enzyme inactivation rate changes 22Table 1.11 Spray dried protein products. . . . . . . . . . . . . . . . . . . . . . . . . . 23

Table 3.1 Yields as ratio of solubles collected to total batch size, solids, and proteincontents of water soluble fractions collected by centrifugation and settling 35

Table 3.2 Dry peanut meal particle size distribution . . . . . . . . . . . . . . . . . . 35

Table 4.1 Treatment names and summary of conditions . . . . . . . . . . . . . . . . 45

Table 5.1 Concentration of sugars in soluble fractions and feed stocks: mean of 3replicates 1 standard error . . . . . . . . . . . . . . . . . . . . . . . . . . 66

Table 5.2 Hygroscopicity of powders expressed as g water uptake per 100 g pow-der solids after 1 week at 20C 82% relative humidity: means of 3 repli-cates 1 standard error . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

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LIST OF FIGURES

Figure 1.1 Geography of populations at risk of chronic exposure to unregulatedaflatoxin exposure LAC, Latin America and the Caribbean: Adapted

from[Williams et al., 2004] . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Figure 2.1 Amino acid composition of water soluble controls (A), hydrolysatesafter 240 minutes (B), and unhydrolyzed peanut meal (C) Points aremeans of 3 1 standard deviation Adapted from [Seifert, 2009] . . . . 26

Figure 3.1 Soluble solids content of water soluble fraction collected as affected bymeal to solvent ratio of dispersion . . . . . . . . . . . . . . . . . . . . . . 32

Figure 3.2 Soluble protein content of supernatant as affected by meal to solvent ratio 33Figure 3.3 Soluble protein content of water supernatant per gram peanut meal as

affected by meal to solvent ratio . . . . . . . . . . . . . . . . . . . . . . . 34Figure 3.4 Volume of soluble fraction with increasing settling time for 5, 10, 15,

and 20% dispersions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36Figure 3.5 Vacuum oven dial setting and corresponding measured air or water

temperature inside the oven. . . . . . . . . . . . . . . . . . . . . . . . . . 38Figure 3.6 Bchi B-290 spray dryer components weighed for yield and hold-up

calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40Figure 3.7 Effects of spray dryer inlet temperature and maltodextrin content on

powder yield during spray drying of water soluble fractions collectedfrom 10% dispersions at pH 8.2. . . . . . . . . . . . . . . . . . . . . . . . 41

Figure 3.8 Effects of spray dryer inlet temperature on powder yield for pepsin andAlcalase treated peanut meal solubles . . . . . . . . . . . . . . . . . . . . 42

Figure 4.1 pH of dispersions before (pHi), and after (pHf) the application of treat-ments acid/base, enzyme, and clay . . . . . . . . . . . . . . . . . . . . . 48

Figure 4.2 Mass of solubles collected after settling . . . . . . . . . . . . . . . . . . . 49Figure 4.3 Solids content of solubles collected after settling (%) . . . . . . . . . . . 50Figure 4.4 Solids collected in the soluble fraction as % solids * mass of soluble

fraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51Figure 4.5 Solids content of insoluble fraction. . . . . . . . . . . . . . . . . . . . . . 52Figure 4.6 Solids collected in each section of spray dryer as percentage of total

solids sprayed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54Figure 4.7 Quantity of solids fed to spray dryer as feed stock quantity * percent

solids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Figure 5.1 Aflatoxin content of insoluble materials, powders, and feed stocks onsolids basis by treatment. . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Figure 5.2 Absorbance of hydrated powder solutions (15, 1, 0.5, and .1% w/v) at500 nm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

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Figure 5.3 Visual appearance of pH2 & clay, Pep & clay, pH8 & clay, Alcalase &clay soluble fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Figure 5.4 Fiber and ash content of insoluble fractions on dry weight basis . . . . 65Figure 5.5 Protein content of powders and feed stocks by treatment on solids basis

(g/ 100g) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

Figure 5.6 Interfacial tension of feed stocks over time, the blue line represents valuefor water at 25 C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68Figure 5.7 SDS-PAGE hydrated powders and feed stocks at the same solids con-

tents for pH2, Pep, pH8, and Alc treatments . . . . . . . . . . . . . . . . 70Figure 5.8 BCA protein solubility of powder protein in McIlvaine buffers at pH 3.0,

5.0, 7.0 in g/mL: means of 3 replicates 1 standard error . . . . . . . 71Figure 5.9 Oxygen radical absorbance capacity of powders and soluble fractions

(mMol TE): means of 3 replicates 1 standard error . . . . . . . . . . . 72

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CHAPTER

ONE

REVIEW OF THE LITERATURE

1.1 Introduction to the peanut

The earliest recorded history ofArachis hypogaea L., the plant we know as the peanut, dates

back to the early 1500s [Hammons, 1982]. South America is credited as the origin of wild

peanuts, and peanut cultivation originates in the area that is now southern Bolivia - north-

western Argentina [Hammons, 1982]. Cultivated peanuts are generally divided into four

market types: Spanish, runner, Virginia, and Valencia[Henning et al., 1982]. These types are

characterized by the number and arrangement of branches on the plant [Wynne and Coffelt,

1982]. In the United States peanuts are grown in Virginia, North Carolina, South Carolina,

Texas, Oklahoma, New Mexico, Mississippi, and Arkansas, but the most productive region

includes Georgia, Florida, and Alabama [Henning et al., 1982]. Most of these peanuts are

grown in Georgia, and 25% of the US crop is grown in Texas [APC, 2010]. Finished peanut

production exceeds 3 109 pounds annually [NAS, 2009]. The majority (80%) of these peanuts

are runner type, which are primarily used for peanut butter production [APC, 2010]. Peanuts

as a snack nut are the most commonly consumed of all snack nuts [ Mintel, 2009]. The virginia

type peanuts are the largest seed size and are used as a snack nut [APC, 2010]. A far less

prevalent snack nut is the valencia type, which only accounts for about 1% of the US peanut

crop [APC, 2010]. Four percent of the US crop are spanish type peanuts which are used for

candy and confections [APC, 2010]. The popularity of peanuts is due to their pleasing flavor,

texture, and favorable nutritional properties [Mintel, 2009].

Roasting and salting are done to improve the flavor of peanuts and this is the most widely

used method of preparation [Singh and Singh, 1991]. Whole peanuts are also prepared by

boiling, fermented and fried, coated with flour and fried, and as peanut butter[Singh and

Singh, 1991].

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1.2 Nutrition

Raw peanuts contain a healthy balance of micro and macro-nutrients [Ahmed and Young,

1982]. Peanuts are composed of 15-21% carbohydrates by weight [America, 2008]. The prin-

ciple carbohydrates in peanuts are fructose, glucose, inositol, and sucrose, which typically

comprise 2.7, 1.9, 1.3, and 149mg per 100 g of peanuts respectively [Ahmed and Young, 1982].

Lipids make up 44-56% of peanuts by weight and this oil is primarily composed of mono- and

poly-unsaturated fatty acids [USDA, 2010]. Only about 6.8% of the weight of raw peanuts

is saturated fatty acids [USDA, 2010]. The relative amounts of each fatty acid in peanuts

varies depending on cultivar, growing location, and maturity level of the peanut [Ahmed

and Young, 1982]. Peanuts also contain a variety of vitamins and minerals [USDA, 2010].

Nutritional content varies by peanut cultivar and generally these nutritive differences are

small [Ahmed and Young, 1982]. Protein content of raw peanuts ranges from 22-30% [USDA,

2010].

Table 1.1. Nutritional Properties of Raw Peanuts [America, 2008, Ahmed and Young, 1982]

Nutrient Contentmg per 100g

Vitamin A 26Vitamin E 46Niacin 12.8-16.7Pyridoxine 0.30Riboflavin 0.13Thiamin 0.99

Vitamin C 5.8

1.2.1 Health benefits of peanut consumption

The American Heart Association (AHA) estimates that one in three Americans has some

type of cardiovascular disease (CVD), which was involved in 56% of all deaths in 2005 [ AHA,

2009]. Links between diet and risk for developing CVD are well established [Hu and Willett,

2002].Increasing intake of fruits and vegetables decreases the risk of developing coronary heart

disease (CHD) [Liu, 2001]. Similarly, the relative risk of mortality from stroke, ischemic heart

disease, and CVD is significantly reduced by consuming greater than 3 serving of fruits and

vegetables daily [Bazzano et al., 2002,Joshipura, 2001].

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Table 1.2. Mineral Content of Raw Peanuts [Ahmed and Young, 1982]

Nutrient Content mg per 100g

Calcium 82.6Copper 1.2

Iron 1.6Magnesium 174.1Manganese 1.8Phosphorus 438.1Potassium 626.3Sodium 6.3Zinc 6.1

Foods other than fruits and vegetables have similar health protective benefits. Diets low

in saturated fatty acids and high in unsaturated fatty acids reduce total blood cholesterol con-centrations while improving the ratios of high density lipoprotein to low density lipoproteins,

thus reducing risks for CVD and CHD [Hu and Willett, 2002,Wu et al., 2009]. The lowest rates

of mortality were strongly associated with high consumption of cereals, legumes, fish, oils,

and wine [Menotti, 1999]. Peanuts are about 50% lipid, the ratio of saturated to unsaturated

fatty acids are consistent with blood lipid profile improving diets. Moreover, Kris-Etherton

and others [Kris-Etherton et al., 1999] reviewed equations used to predict the blood choles-

terol and lipoprotein levels in subjects consuming nuts and found that the actual reductions

in cholesterol were 25% greater than predicted. This indicates that non-lipid components in

nuts are likely responsible for part of the reductions. The same authors later confirmed thatindeed bioactive compounds in nuts positively affect blood lipids and can prevent CHD [Kris-

Etherton et al., 2008]. Hamsters that consumed diets containing peanut oil, peanut flour, or

whole peanuts had lower instances of risk factors for CVD than those consuming the control

diet [Stephens et al., 2010]. This study was the first to indicate non-lipid peanut components

have a favorable impact on risk factors for CVD.

1.2.2 Bioactive compounds

Bioactive compounds, also called phytochemicals, are found in plant foods and provide

health benefits greater than those provided by the basic nutrition of the food in which they arefound [Leonora et al., 2008]. Examples of bioactive compounds found in foods are phenolic

compounds, which include flavonoids, resveratrol, and phytoestrogens [Kris-Etherton et al.,

2002]. Bioactive compounds have many benefits to human health, and can serve many func-

tions within the body including antigens, enzyme inhibitors, and hormones which can reduce

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both hypertension and inflammation [Havsteen, 2002]. A survey of the diet of Finnish people

indicated that those with the highest intakes of flavonoids had lower mortality rates [Knekt

et al., 2002]. Specifically, high levels of quercetin and kaempferol were associated with de-

creased mortality from ischemic heart disease[Knekt et al., 2002]. Rates of death from cere-

brovascular disease and cancer were lowest among those with highest kaempferol, herperetin,

and naringenin intakes, while reduction of rheumatoid arthritis, type 2 diabetes, cataract, and

asthma all relate to high levels of flavonoid intakes [Knekt et al., 2002]. Whole peanuts con-

tain several classes of bioactive compounds like flavonoids, phenolic acids, plant sterols and

stilebens [Leonora et al., 2008]. Peanut kernels contain, trans-resveratrol, dihydroquercetin,

p-hydroxybenzoic acid, p-coumaric acid; the skins, stems, and other parts not typically con-

sumed, are also concentrated sources of bioactive compounds [Leonora et al., 2008]. Recent

research had demonstrated that fat-free peanut flour can reduce the incidence of atheroscle-

rosis in hamsters[Stephens et al., 2010]. This suggests that non-lipid components found in

peanuts may have heart health benefits.

1.2.3 Peanut protein

Raw peanuts contain 22-30% protein depending on peanut variety [USDA, 2010]. The vast

majority (90%) of this protein is globular and classified as either arachin or conarachin

[Cherry, 1990,Lusas, 1979]. Globular means that in water the tertiary structure of the proteins

is roughly spherical [Damodaran, 1996]. Arachin proteins are storage proteins whereas the

conarachin proteins are present in the cellular cytoplasm of the peanut [Cherry, 1990]. The

other 10% of peanut proteins are albumins, meaning water soluble [Lusas, 1979, Damodaran,

1996].

Proteins are polymers of amino acids, and peanut proteins are composed of mostly glu-

tamic acid, arginine, and aspartic acid (Table 1.3). A nutritionally complete protein is one

containing all the essential amino acids: isoluecine, methionine, tryptophan, leucine, pheny-

lalanine, valine, lysine, threonine, and histidine [Manore and Thompson, 2000]. Peanuts

contain all the essential amino acids, but they are not present in large enough quantities for

peanut protein to be considered a complete protein (Table1.3). In peanuts lysine, methionine,

and threonine are considered the limiting amino acids because they are found in insufficient

quantities [McOsker, 1962]. Supplementing diets with these amino acids improves protein ex-

change ratio and provides sufficient amino acids for optimal growth [McOsker, 1962,Manore

and Thompson, 2000].

One third of all children under 5 years of age in the world are malnourished [Latham,

1997]. Protein deficiency is the major contributing factor to this malnourishment affecting

approximately 200 million people [Latham, 1997]. Inexpensive sources of protein are needed

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Table 1.3. Amino acids composition of peanut protein adapted from [Lusas, 1979]

Amino Acid gram per 16 g Nitrogen

Glutamic Acid 19.9

Aspartic Acid 14.1Arginine 11.3Leucine 6.7Glycine 5.6Phenylalanine 5.2Serine 4.9Valine 4.5Proline 4.4Alanine 4.2Isoleucine 4.1Tyrosine 4.1Lysine 3.0Threonine 2.5Histidine 2.3Cystine 1.3Tryptophan 1.0Methionine 0.9

to ensure adequate nutrition for populations in these parts of the world. The proteins avail-

able from various plant sources can provide balanced and adequate quantities of amino acids

for human health [Young and Pellett, 1994]Peanuts can be processed to a variety of protein-rich food ingredients [ Diarra et al.,

2005, Cherry, 1990]. Many commercially available food ingredients are derived from peanuts

and peanut protein like flours, flakes, concentrates, and isolates[Lusas, 1979]. Peanut flour

for example, contains 60% protein and can be used to fortify breads, cereals, corn chips, and

donuts[Ayres and Davenport, 1977]. Several processes can be used to produce peanut milk

which can be consumed as a beverage or further processed into cheese, yogurt and spread

type products [Diarra et al., 2005]. Peanuts are abundant with roughly four billion pounds

produced annually in the US, and relatively inexpensive [Ash and Dohlman, 2008]. Unfortu-

nately many peanuts, especially those grown in developing countries, are often contaminatedwith aflatoxin.

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1.3 Peanut Meal

1.3.1 Introduction

World-wide, peanuts are produced primarily for oil; however, in the US peanuts are primar-

ily consumed directly and generally only substandard peanuts are used for oil production.Aflatoxin contaminated peanuts are discolored, and less dense than uncontaminated peanut

seeds and subsequently discarded during peanut sorting [Whitaker, 1997]. These discarded

substandard nuts are generally pressed then solvent extracted for oil. The virtually fat-free

material remaining after the oil is extracted from peanuts is called peanut meal [Mcwatters

and Cherry, 1982]. Peanut meal is effectively twice as concentrated in water soluble com-

pounds compared to whole seeds because by weight roughly half of a peanut is oil. This

means peanut meal contains 45 to 60 percent protein and the whole seed only contains 22 to

30 percent protein [Mcwatters and Cherry, 1982].

1.3.2 Value

Peanut meal is used as a protein source in animal feeds and the quality as a feed ingredient

has been studied. The quality of eggs improves with protein supplementation from peanut

meal [Pesti et al., 2003]. For broiler chickens however, soy protein meal is a better protein

source than peanut meal [Costa et al., 2001]. Pigs fed nine diets had no differences in growth

when on a peanut meal based diet compared to the control soy bean meal diet [Shelton et al.,

2001].

1.3.3 PotentialCottonseed, soybean, pea, cashew, flax, pecan, and even tomato seed meals can all be used to

produce high protein food ingredients [Lawhon et al., 1972,Liadakis et al., 1995,Manak et al.,

1980, McWatters and Cherry, 1977, Oomah et al., 1994, Piva et al., 1971].

Currently, peanut meal is not considered a food grade material by the US Food and Drug

Administration (FDA), likely due to the aflatoxin contamination commonly found in peanut

meal. Because peanut meal is not food grade, literature on protein extraction from peanut

meal for food purposes is limited; however, protein extraction from whole peanuts has been

investigated [Rustom et al., 1991, Cherry, 1990]. Protein extraction yields can be improved

by manipulating the solids to solvent ratio, pH of the solvent, temperature of solvent, andduration of the extraction [Rustom et al., 1991]. Enzymatic hydrolysis can further increase the

amount of solids extracted from peanut [Rustom et al., 1993]. Protein can be extracted from

whole peanuts and subsequently spray dried to create high protein food ingredients [Lawhon

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Table 1.4. Peanut meal pricing adapted from [Seifert, 2009]

Use Aflatoxin Level (g/kg) Price ($/ton)

Dairy Cattle Feed < 20 210

Animal Feed 20 300 175Fertilizer > 300 95

et al., 1981]. However, the value and potential uses of peanut meal are limited by the aflatoxin

contamination levels (Table3.2).

1.4 Aflatoxin

1.4.1 Introduction

Toxic metabolites of fungi known as mycotoxins are prevalent in a variety of crops, feeds, and

foods [Diener et al., 1982].Aspergillus flavusis the most common mycotoxin producing fungus

found in peanuts; this is true across various climates and geographic regions [Diener et al.,

1982]. Aflatoxin is produced by the fungi Aspergillus flavus and Aspergillus parasiticus: hence

the name afla-toxin [Eaton and Gallagher, 1994]. Aflatoxin B1 (AFB1) is the most common of

the four forms of aflatoxin, B1, B2, G1, and G2 [Diener et al., 1982].

1.4.2 Prevalence in crops

Many agricultural commodities are often contaminated with aflatoxin producing fungi in-

cluding coffee, corn, wheat, rice, soybean, sunflower, cotton, almond, pistachio, and peanut

[Soliman, 2002, Bhat et al., 2010, Yazdanpanah et al., 2005]. Humidity, temperature, and time

influence growth ofA. parasiticusandA. flavus[Rustom, 1997,Diener et al., 1982]. On peanuts

between 6 and 46CA. parasiticuscan produce aflatoxin, and between 12 and 42CA. flavus

produces aflatoxin as long as a minimum moisture content of 8% is met [Rustom, 1997, Di-

ener et al., 1982]. Drought stresses also make peanuts more susceptible to contamination

likely due to decreased physiological activity within the peanut[Diener et al., 1982]. Peanut

cultivar also effects contamination levels; newer cultivars are more resistant to fungal growth

while native, or unimproved, cultivars are more susceptible to contamination [Mutegi et al.,

2009].

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Table 1.5. LD50 of AFB1 for several species adapted from[Newberne and Butler, 1969,Wogan,1966].

Animal LD50 (mg/kg)

Cat 0.6

Dog 0.5-1.0Duckling 0.3-0.4Guinea Pig 1.4Hamster 10.2Mouse 9.0Pig 0.6Rabbit 0.3Rat 1.0-17.9Trout 0.5

1.4.3 Health concerns

The carcinogenic effects of aflatoxin in animals are well established and highly species de-

pendent [Eaton and Gallagher, 1994]. The lethal orally administered dose of aflatoxin B1

required to kill 50% of a test population (LD50) for several species is summarized in table1.5.

Since aflatoxin, specifically aflatoxin B1 (AFB1), is one of the most carcinogenic chemicals

studied, the FDA, World Health Organization (WHO), and European Union (EU) limit con-

centrations in foods intended for animal feed (Table 1.6) [Eaton and Gallagher, 1994, FDA,

2008].

Numerous epidemiological studies have established the connection between aflatoxinconsumption and incidence of liver cancer in humans [Eaton and Gallagher, 1994, Rustom,

1997, Wild and Gong, 2010, Williams et al., 2004]. Acute aflatoxin poisoning is rare, 25% of

these cases are fatal, and chronic exposure rates are high, especially in developing coun-

ties [Williams et al., 2004]. As such, many countries established legal limits for aflatoxin con-

centrations allowed in foods, specifically peanuts, intended for human consumption (Table

1.7) [Reddy et al., 2010]. Developing countries have especially high incidences of aflatoxicosis

because regulation and testing is prohibitively expensive, and food is scarce so uncontami-

nated alternatives are not available [Williams et al., 2004].

1.4.4 Prevalence in food and feed

There is a high prevalence of contaminated food being sold (Table1.8)[Li et al., 2009, Reddy

et al., 2010, Turner et al., 2002]. Consequently, levels of exposure are especially high in the

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Table 1.6. Levels of total aflatoxin contamination permissible by the FDA adapted from [FDA,2008]

Peanut products intended for Action levelg/kg

Dairy animals 20Immature animals 20Breeding beef cattle, breeding swine, or mature poultry 100Finishing swine of 100 pounds or greater 200

Figure 1.1. Geography of populations at risk of chronic exposure to unregulated aflatoxinexposure LAC, Latin America and the Caribbean: Adapted from [Williams et al., 2004]

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Table 1.7. Legal limits of aflatoxin in peanuts: adapted from [Reddy et al., 2010]

Agency/Country Aflatoxin Upper limitg/kg

Australia Total 15Brazil B1 and G1 30Bulgaria Total 15Canada Total 15China B1 20Egypt Total 10Hungary Total 15India Total 30Japan B1 10Kenya Total 20Korea B1 10Russia B1 5Taiwan Total 15Turkey B1 5United States Total 20

affected countries with rates of exposure exceeding 90% of those populations [Williams et al.,

2004].

1.4.5 Destruction and management of aflatoxin

The destruction of aflatoxin in contaminated food and feeds has been thoroughly investi-

gated [Bullerman and Bianchini, 2007, Ciegler, 1978, Piva et al., 1995]. The treatments can be

classified by mode of action as either chemical, mechanical, or radiative.

Chemical

Various chemical treatments of naturally and artificially contaminated foodstuffs effectively

reduce aflatoxin contamination levels [Piva et al., 1995, Rhee et al., 1977]. During processing

of peanut protein isolate or concentrate from contaminated peanuts, most (85%) aflatoxin

remains with the protein fraction [Rhee et al., 1977]. However, the addition of methylamineat levels up to 1.25%, hydrogen peroxide, or ammonia gas reduces the aflatoxin remaining

in the resultant protein concentrates [Rhee et al., 1977]. Similarly, aflatoxin can be com-

pletely destroyed during processing of protein isolate/concentrate with the addition of ace-

tone at >65%, isopropyl alcohol at 80%, benzoyl peroxide at 0.5%, or sodium hypochloride

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Table 1.8. AFB1 contamination levels and frequency in market products adapted from [Liet al., 2009, Reddy et al., 2010, Turner et al., 2002,Williams et al., 2004]

Country Commodity Frequency of aflatoxin Contamination level% g/kg

Bangladesh Peanuts 65

Botswana Peanuts 12-329Botswana Peanut Butter 0.3-23Brazil Peanut Products 67 43-1100Brazil Peanuts 27 43-1100China Peanut butter 82 1 - 79Cyprus Peanut butter 56.7 > 10Egypt Peanut and watermelon seeds 82Gambia Peanuts 162Ghana Peanuts 13-32Guinea Peanuts 61 1-112India Groundnut 21 > 30

Japan Peanut butter 2.6Nepal Peanuts > 30Senegal Peanut Oil 85 40Sudan Peanut and peanut butter 87.4-197.3Thailand Peanut oil 102Uganda Peanut, cassava 12 > 100

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at 0.2%[Natarajan et al., 1975, Rhee et al., 1977, Stoloff et al., 1976]. In addition to reducing

contamination as processing aids, hydrogen peroxide, sodium hypochlorite, and ammonia

are effective as treatments when applied directly to foodstuffs [Piva et al., 1995,Paulsen et al.,

1976]. Calcium hydroxide, formaldehyde, and sodium bisulfite also effectively destroy afla-

toxin [Piva et al., 1995]. Treatment with ozone is effective, but dependent on temperature and

treatment time; in some foods, like corn, undesirable oxidation of fatty acids can limit the

potential use of this treatment [Dollear, 1968,Jr. and King, 2002, Proctor et al., 2004]. Indeed,

while many chemical treatments are effective, safety and health concerns, as well as cost,

prohibit commercial application of these treatments [Piva et al., 1995].

Mechanical

Commonly used physical food processing methods also reduce aflatoxin levels [Bullerman

and Bianchini, 2007]. Many processes used in food preparation are moderately effective at

destroying aflatoxin: washing wheat, washing and cooking rice, pressure cooking beans,frying tortilla chips, and baking bread will reduce the aflatoxin levels in the finished product

[Bullerman and Bianchini, 2007]. Aflatoxin contamination in coffee beans is reduced during

the traditional roasting process which subjects beans to 180 C for 18 min [Soliman, 2002].

Similarly, cooking rice, or steaming wheat prior to bread baking reduces aflatoxin levels;

wet heating methods are generally more effective than dry heating [Hwang and Lee, 2006,Je

et al., 2005]. The efficacy of thermal destruction of AFB1 in peanut and cotton seed meals

is strongly dependent on moisture content of the meals [Mann, 1967]. Longer cook times

and higher temperatures, more effectively reduce aflatoxin levels [Yazdanpanah et al., 2005].

Aflatoxin B1 begins to decompose at 150 C, but dry heating as high as 237 to 306 C are

required to completely decompose AFB1 [Rustom, 1997].

Radiative

Gamma radiation applied to infested cereals completely destroys mycotoxin producing fungi

at doses as low as 6 kGy and prevents regrowth for at least 100 days after treatment [ Aziz

et al., 2006]. This treatment is effective for contaminated or potentially contaminated foods

and feeds. Even at a lower dose of 4 kGy, fungi are nearly eliminated; such a treatment could

provide extra assurance of safety for potentially contaminated food and feeds [Aziz et al.,

2006]. Similarly, a 5 kGy dose will reduce, and 10 kGy will completely remove, the fungalload in herbs and prevent regrowth after 30 days of storage [Aquino et al., 2010]. The same 10

kGy dose administered to peanuts completely inhibits fungal growth, while higher doses (15

- 30 kGy) will destroy the aflatoxin produced by those fungi in peanuts [ Prado et al., 2003].

Ultraviolet (UV) radiation degrades AFB1, and contaminated peanut oil treated with UV

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light for 2h destroys 40 to 45% of AFB1 present[Rustom, 1997]. Ultraviolet radiation from

sunlight degrades 50% of AFB1 in naturally contaminated peanut product [Rustom, 1997].

While these chemical and physical methods to mitigate aflatoxin contamination occur-

rence and levels are effective, their implementation is difficult due to cost and safety con-

cerns [Piva et al., 1995].

1.4.6 Prevention and management

Preventing growth and aflatoxin production in food reduces total contamination levels and

provides an alternative strategy for ameliorating potential contamination. Where fungal

growth is unavoidable, due to a drought for example, aflatoxin contamination can be reduced

by applying to the soil a strain ofA. flavusthat does not produce mycotoxins[Dorner, 2008].

The atoxigenicA. flavusout-competes the naturally occurring toxin producing A. flavus, thus

reducing overall contamination[Dorner, 2009].

Segregation

Peanuts infested with fungi may have a dark and wrinkled skin; however, these nuts comprise

only a small percentage of peanuts in a typical lot [Cucullu et al., 1966]. These contaminated

nuts may have very high (6,000 g/kg) concentrations of aflatoxin [Cucullu et al., 1966].

Another smaller subset, usually less than one percent, of nuts do not show visible wrinkling

or discoloration and can have even higher levels of aflatoxin up to 1,100,000 g/kg [Cucullu

et al., 1966]. Both subsets typically represent less than ten percent of a total lot, but contain

nearly all of the aflatoxin and are less dense than intact kernels [Cucullu et al., 1966]. Indeed,

removing this subset of highly contaminated nuts is one of the most effective strategies toreduce aflatoxin contamination. After harvest, peanuts are sorted by kernel size and density

[Dorner, 2008, Whitaker and Dickens, 1979]. The sorting process allows testing of individual

lots, which can then be rejected if contamination is detected [Dorner, 2008, Whitaker and

Dickens, 1979]. Sorting is generally effective, and the chance of accepting a contaminated lot

is often less than 5% [Whitaker, 1997, Whitaker and Dickens, 1979, Dickens, 1977]. Sorting

peanuts by color can remove the majority (70 to 90%) of aflatoxin contaminated peanuts

[Whitaker, 1997, Dorner, 2008]. Since seed discoloration is strongly associated with AFB1

contamination rates, color sorting blanched peanuts is even more (up to 91%) effective than

sorting unblanched peanuts by color[Dorner, 2008].

Sequestrants

Good farming practices, sorting, and processing may not eliminate 100% of aflatoxins present.

Human exposure to aflatoxin can be limited by: improving post-harvest storage conditions

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for susceptible food, and using sequestrant clays to mitigate absorption [Groopman et al.,

2008]. Many types of sequestrants effectively absorb mycotoxins, especially AFB1, in vivo

and in vitro [Ramos et al., 1996]. Among the most effective seqrestrants are activated char-

coal, bentonite, zeolite, diatomaceous earth, and hydrated sodium calcium aluminosilicates

(HSCAS) [Ramos et al., 1996, Modirsanei et al., 2008]. The most extensively studied seques-

trants are the HSCAS [Phillips, 1999], which have a very high capacity to tightly bind afla-

toxins in vitro [Grant and Phillips, 1998].

Despite extensive study, the discrepancy between sorbent sequestering capacity for AFB1

in vitro, and the in vivo applications is limiting [Moschini et al., 2008, Diaz et al., 2004]. Nev-

ertheless, sorbent clays, in particular sodium bentonite, effectively limit deleterious effects

of aflatoxin consumption in poultry and livestock [Ramos et al., 1996]. Broiler chickens are

commonly used to evaluate in vivo efficacy of sequestrants [Ramos et al., 1996]. Even in

the absence of added aflatoxin, broiler chickens consuming sodium bentonite clay improved

weight gain, feed consumption, protein efficiency ratio, and protein digestibility [Pasha et al.,

2008]. Inclusion of only 0.3 or 0.5% sodium bentonite in feed can prevent the negative health

effects associated with high levels (1,000 g/kg) of aflatoxin consumption fed to broilers [Ker-

manshahi et al., 2009,Rosa et al., 2001]. Bentonite clays also protect piglets and sheep from the

deleterious effects of aflatoxin ingestion [Schell et al., 1993, Thieu et al., 2008, Khadem et al.,

2007]. Sodium bentonite clays are widely used because they are effective and inexpensive

to use [Khadem et al., 2007, Diaz et al., 2004]. Sodium bentonite can be used to effectively

remove aflatoxin during protein extraction from peanut meal [Seifert et al., 2010]. This novel

approach allows utilization of peanut meal as an inexpensive source of protein.

1.5 Peanut allergens

Many methods for removal or destruction of aflatoxin are available, but peanut allergies pose

a growing concern for many people. Peanut allergy is one of the most common and severe

food allergies in the United States affecting more than 3 million Americans [Sicherer and

Sampson, 2009,Sicherer et al., 2003]. Children with peanut allergic parents are ten times more

likely to be allergic than the general population [Sicherer and Sampson, 2009]. An allergy is

characterized by an "adverse immune reaction", usually to a protein [Sicherer and Sampson,

2009]. The major food allergens are water-soluble glycoproteins between 10 and 70 kDa, and

relatively stable to heat, acid, and proteases making them difficult to destroy [Sicherer andSampson, 2009]. The major peanut allergen is the 63 kDa glycoprotein known as Ara h1

[Maleki et al., 2000b]. A second major peanut allergen, Ara h2, may effect more people than

Ara h1 and is a smaller glycoprotein approximately 15 kDa [Burks et al., 1992, Koppelman

et al., 2004].

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1.5.1 Mitigation strategies

Heating typically reduces the allergenicity of proteins by denaturing them [Wal, 2003]. It

is however, exceedingly difficult to destroy the major allergenic proteins in peanuts because

they have a high resistance to heat and a specific tertiary structure which protects the binding

sites from degradation [Kopper et al., 2005, Maleki et al., 2000b]. Furthermore, the roastingprocess, which is used extensively in the US, increases the IgE-binding of peanuts [ Maleki

et al., 2000a]. Conversely, boiling peanuts apparently decreases allergenicity, which is likely

due to the migration of proteins from the nuts to the cooking water [ Mondoulet et al., 2005].

Because protein destruction by heating is ineffective, enzymatic hydrolysis may be used to

reduce allergenicity of peanut proteins by altering structure [Sen et al., 2002]. Unfortunately,

allergenic peanut proteins, in particular Ara h1, resist enzymatic hydrolysis by pepsin [Maleki

et al., 2000b].

1.5.2 TherapiesThe most commonly employed strategy for patients with peanut allergy is to simply avoid

contact with peanuts [Sicherer and Sampson, 2009]. This strategy is limited because it is im-

possible to completely control the environment and incidental contact with peanut protein is

nearly unavoidable. Sub-lingual immunotherapy (SLIT) is the gradual deliberate exposure of

patients to minute amounts of the allergenic proteins to build a general tolerance to avoid an

adverse acute allergic response [Sicherer and Sampson, 2009]. This strategy is limited by the

varying responses to the treatment as some patients have an adverse reaction to even minute

doses and never develop a tolerance[Sicherer and Sampson, 2009]. Another approach is pep-

tide immunotherapy which involves creating a vaccine composed of small peptides[Sichererand Sampson, 2007]. The peptides generically contain all amino acid sequences so antigen

presenting cells are provided with information to create antibodies for all possible peanut al-

lergens, and without binding to the mast cells responsible for adverse reaction [Sicherer and

Sampson, 2007]. The therapeutic peanut peptides can be generated by enzymatic hydrolysis

of peanut proteins.

1.6 Hydrolysis

1.6.1 Introduction

Hydrolysis is the chemical process by which a molecule is cleaved into two parts by the

addition of a molecule of water. One fragment of the parent molecule gains a hydrogen

ion (H+) from the additional water molecule. The other group reacts with the remaining

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hydroxyl group (OH ). This reaction is catalyzed by strong acids, strong bases, or enzymes.

Hydrolysis of proteins produces peptides and free amino acids. The degree of hydrolysis

(DH) refers to the percentage of peptide bonds broken and is often measured by the amount

of acid or base required to maintain a given pH [Panyam and Kilara, 1996]. Another way to

determine DH is a spectrophotometric trinitrobenzenesulfonic (TNBS) acid method [Adler-

Nissen, 1976] More extensively hydrolyzed proteins have higher DH values and a greater

proportion of lower molecular weight peptides than the same unhydrolyzed protein.

1.6.2 Uses

Industrially, proteins are hydrolyzed to improve functionality and nutritional quality or to

improve the value of waste proteins, as in slaughterhouse waste for example [Bhaskar et al.,

2007]. Hydrolysis typically increases protein solubility across a pH range, while decreas-

ing the functionality associated with protein unfolding [Beuchat et al., 1975, Ruz-Henestrosa

et al., 2007]. This is probably due to the loss of aliphatic structure of the protein molecules.Many functional properties are highly dependent on DH; foaming, gelation, and emulsifica-

tion properties are generally hindered by smaller peptides and improved by larger ones [ Pa-

nyam and Kilara, 1996]. For example, the hydrolysis of whey proteins improves heat stability,

reduces allergenicity, and produces bioactive peptides[Foegeding et al., 2002]. Using hydrol-

ysis to create a specific DH can be used for tailoring amounts and size of peptides for special

diets, and altering the functional properties of gelation, foaming and emulsification [Foeged-

ing et al., 2002]. Despite reducing emulsifying and foaming capacity, hydrolysis improves free

radical scavenging ability of quinoa and soy protein [Monu, 2003, Peta Ramos and Xiong,

2002].

1.6.3 Nutritional considerations

Limited hydrolysis of soy protein isolates results in increasing antioxidant activity [Peta

Ramos and Xiong, 2002]. Hydrolyzed proteins, especially small (di- and tri-peptides) pep-

tides are more quickly absorbed through the small intestine than free amino acids [ Webb,

1990]. Hydrolysis can be used to improve the amino acid profile of mixtures of proteins

from several sources, providing an opportunity to inexpensively create high quality protein

foods [Radha et al., 2008]. Peanut protein hydrolysates have balanced amino acid profiles

consistent with the United Nations Food and Agriculture Organization and World HealthOrganization (FAO/WHO) guidelines for human health[Kain et al., 2009]. In addition to im-

proving the nutritional properties of proteins, hydrolysis can also be used to create bioactive

compounds and nutraceutical ingredients.

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Enzymatic hydrolysis is the most commonly used method to create angiotensin 1 con-

verting enzyme (ACE) inhibitory peptides, which play an important role in regulating blood

pressure, a major health concern [Guang and Phillips, 2009a]. ACE inhibitory peptides can

be isolated from peanut flour using the enzyme Alcalase[Guang and Phillips, 2009b]. Peanut

protein hydrolysates can be used as natural antioxidants with reported free radical scaveng-

ing properties similar to butylated hydroxy-toluene and ascorbic acid [Chen et al., 2007]. The

excellent nutritional properties of hydrolyzed peanut proteins provide incentives to further

investigate the production and properties of these hydrolysates.

1.7 Spray Drying

1.7.1 Introduction

Dry food materials are less expensive to transport and store because they are more compact,

and weigh less than their full-moisture counterparts. Spray drying is one of the most widelyused drying processes in the food and pharmaceutical industries [Masters, 1991]. Spray

drying is a commonly used drying process because it can be operated continuously with high

throughput [Masters, 1991]. The drying process consists of three main steps: atomization of

the liquid feed material, dehydration of atomized droplets, and separation of the dry particles

from the drying gas[Masters, 1991].

In spray drying, atomization refers to the process by which liquid feed material is sprayed

into a fine mist, which greatly increases the surface area of the solution to be dried. Droplets

are typically between 5 m and 200 m, and upon contact with heated drying gas the droplets

dry very quickly. The dry particles are separated from the drying medium by centrifugal

force in an apparatus, typically called a cyclone. Outlet temperature is the temperature of the

drying air measured at or just before the cyclone [Masters, 1991].

Heated air is pumped into the drying chamber and the inlet is the point at which the

atomizer enters the drying chamber [Masters, 1991]. The inlet air temperature can exceed 400C, but the outlet temperature is lower and primarily a function of feedstock moisture content

and feed flow rate[Masters, 1991, Samborska et al., 2005]. Changing spray dryer process pa-

rameters or feedstock properties will influence the finished powder characteristics [Masters,

1991,Maa, 1998,Goula and Adamopoulos, 2004]. Thermal and evaporative efficiencies as well

as product yields are influenced by spray dryer parameters [Goula and Adamopoulos, 2003].

Yield can be expressed as the ratio of solid material collected to the amount of solid mate-

rial fed into the spray dryer. Spray drying conditions must be carefully chosen to maximize

yields for each dryer layout. By manipulating process parameters according to feed liquid

characteristics and dryer layout, many heat sensitive and even bioactive compounds can be

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Table 1.9. Spray dried heat-sensitive or bioactive compounds

Compound Reference

ACE inhibitory peptides (alfalfa) [Kapel et al., 2006]

ACE inhibitory peptides (shrimp) [He et al., 2008]-galactosidase [Branchu et al., 1999]Insulin [Sthl et al., 2002]Lysozyme [Elkordy et al., 2002]Lysozyme and Tripsin [Hulse et al., 2009]Soy protein isolate [Boatright and Hettiarachchy, 1995]Tripsin [Millqvist-Fureby et al., 1999]

successfully spray dried.

1.7.2 Drying bioactive compounds

Spray dried bioactive compounds can retain the same functional properties from lab scale to

pilot scale[He et al., 2008]. This consistency across scales may explain the popularity of spray

drying. Model enzymes and proteins like tripsin and insulin can be successfully spray dried

and they are commonly used in pharmaceutical research [Sthl et al., 2002, Millqvist-Fureby

et al., 1999, Hulse et al., 2009]. A variety of other heat sensitive food and pharmaceutical

materials have been successfully spray dried (Table1.9).

1.7.3 Drying method comparisons

Spray drying has been compared to other commonly used drying methods. Freeze drying is

used to dry heat sensitive materials because of the low temperatures used. Spray drying can

be 30 to 50 times less expensive than freeze drying and an acceptable alternative [Masters,

1991]. In many cases the dry product produced by spray drying has better functional proper-

ties than when produced by freeze drying, for example faba bean powder has better solubility

and emulsion capacity when produced by spray drying than by freeze drying [Cepeda et al.,

1998]. Spray drying reduces the anti-nutritional trypsin inhibitors, tannins, and phytates in

faba bean protein powder, as well as freeze drying does [Otegui et al., 1997]. Most of the

functional properties of those protein powders were comparable, but the spray dried powder

had better gelation properties [Otegui et al., 1997]. Spray dried potato protein powder has

comparable quality to vacuum freeze dried powder, but with a lighter color that is known to

improve consumer acceptability [Claussen et al., 2007,Lkra et al., 2008]. Shrimp hydrolysate

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paste produced by vacuum drying at 80 C had a lower concentration of luecine tyrosine

serine, but more aspartic acid, glutamic acid, arginine than the spray dried powder [Bueno-

Solano et al., 2009]. Freeze dried soy protein isolates are typically less soluble than those

produced by spray drying [Boatright and Hettiarachchy, 1995]. Similarly, peanut protein

powders produced by spray drying have better water holding, oil binding, emulsion and

foaming capacity compared to peanut protein powder prepared by vacuum oven drying [Yu

et al., 2007]. Despite the fact that spray drying preserves functionality of the dried material,

this method has some limitations.

1.7.4 Stickiness

Sticky foods are particularly difficult to spray dry and this problem has been investigated

[Chen et al., 2007]. Despite the difficulty, many sticky foods can be successfully dried in-

cluding: sweet potatoes, honey, tomato pulp, and sugar solutions [Goula and Adamopoulos,

2008,Truong et al., 2005, Grabowski, 2006]. Stickiness during drying is due to the dried mate-rial having a critical viscosity decrease from 1010 1012 Pas to 106 108 Pas, or exceeding the

glass transition temperature (Tg) by more than 20C [Downton, 1982, Bhandari et al., 1997].

The presence of organic acids and small molecular weight carbohydrates in feedstocks leads

to stickiness during spray drying [Jayasundera et al., 2009]. Stickiness is difficult to measure

objectively; several methods have been developed for both powders and carbohydrate solu-

tions [Boonyai et al., 2004, Adhikari et al., 2007,Aguilera et al., 1995]. Powder stickiness is the

main cause of decreased yields during spray drying [Jayasundera et al., 2009, Adhikari et al.,

2007].

1.7.5 Strategies to reduce stickiness

The two main causes of low yields are dripping, indicating insufficient drying, or caking due

to over-drying [Meng et al., 2007]. The latter refers to stickiness and both causes are remedied

by either adjusting spray dryer conditions or modifying the feed material [Meng et al., 2007].

Spray dryer conditions like temperatures, air and feed flow rates, as well as the physical

design of the dryer can be adjusted to reduce stickiness[Maa, 1998, Meng et al., 2007]. For

example, using dehumidified air improves the yield of spray dried tomato pulp, an otherwise

prohibitively sticky material [Goula and Adamopoulos, 2008]. Over-drying can be prevented

by maintaining droplet temperatures less than 20

C above Tgof the feed stock [Truong et al.,2005]. Optimal drying conditions for spray drying can be predicted based on estimated T gof

the feed stock [Truong et al., 2005].

Additionally, the composition of the feed stock can be modified to improve yields by

adding carrier agents [Jayasundera et al., 2009]. The carrier agent either modifies the sur-

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face composition, or changes the bulk Tg [Jayasundera et al., 2009]. For some products the

quantity of carrier agent necessary would alter bulk composition so much that consumer

acceptability would decrease or the standard of identity would no longer be met; for exam-

ple, spray dried honey is only 50% honey [Wang and Langrish, 2009]. Consequently surface

active compounds have been investigated as carrier agents which can be used in very low

concentrations [Wang and Langrish, 2009]. These compounds work by forming a non-sticky

surface layer on the surface of the drying droplets [Wang and Langrish, 2009]. Adding sur-

face active proteins to sucrose solution in a ratio of 0.5 : 99.5 (0.5% of bulk on solids basis)

protein to sucrose causes spray drying yields to improve from zero to as much as 85% [Ad-

hikari et al., 2009a]. The protein forms a non-glassy skin around the sucrose droplet reducing

stickiness resulting in successful spray drying [Adhikari et al., 2009a]. Similarly, tripsin was

predominant the surface of droplets mostly composed of carbohydrates [Millqvist-Fureby

et al., 1999]. Other surface active compounds like small molecular weight surfactants can

also be used to selectively control the surface composition of drying droplets to improve

yields [Adler et al., 2000, Bosquillon et al., 2004]. However, in sugar rich solution containing

surface active protein, small molecular weight surfactants can drastically reduce yields by

displacing the non-sticky forming protein layer from the surface of the droplets [Adhikari

et al., 2009c].

Another strategy for increasing yields of sticky solutions is to increase the Tg of the feed

material [Bhandari et al., 1997]. High molecular weight additives effectively increase the Tgso

hotter drying temperatures can be used without stickiness problems [Bhandari and Howes,

1999]. That is, for example, rather than lowering the dryer temperature to match the feed, the

feed can be modified to by increasing Tg.

1.7.6 Maltodextrin as a carrier

Maltodextrin can be used as an additive to increase Tg of feed solutions containing sticky

materials [Adhikari et al., 2004].

Typically, maltodextrins with low dextrose equivalent (DE) values are used because low

DE maltodextrins have the highest molecular weight, which increases Tg of the feed stock

more than would the same mass of a maltodextrin with a high DE [Avaltroni et al., 2004,

Bhandari and Howes, 1999]. Powder yields and properties such as solubility, and solids

content, of many food powders are increased by the addition of maltodextrin to feed stock

prior to spray drying[Goula and Adamopoulos, 2008, Grabowski, 2006, Namaldi et al., 2006,

Abdul-Hamid et al., 2002, Kurozawa et al., 2009]. Food powders with low hygroscopicity are

desirable and the addition of maltodextrin to feed solutions prior to spray drying decreases

hygroscopicity of the resultant powders [Goula and Adamopoulos, 2008, Kurozawa et al.,

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2009]. In both spray dried chicken hydrolysates and tomato pulp adding more maltodextrin

to the feed solution prior to spray drying resulted in powders that were less hygroscopic

[Goula and Adamopoulos, 2008, Kurozawa et al., 2009]. Similarly, the DE of the maltodextins

used affected hygroscopicity of the tomato powder such that DE 6, 12, and 21 were least,

middle, and most hygroscopic respectively, and in chicken hydrolysate maltodextrins reduced

hygroscopicity more than gum arabic at the same usage levels [Goula and Adamopoulos,

2008,Kurozawa et al., 2009]. Maltodextrin is commonly used for microencapsulation because

it provides stability to sensitive materials during spray drying and storage [Gharsallaoui

et al., 2007,Gibbs et al., 1999].

1.7.7 Protein denaturation during drying

Maltodextrin inhibits protein denaturation during thus preserving enzyme activity after

spray drying [Namaldi et al., 2006,Baeza and Pilosof, 2002,Anandharamakrishnan et al.,

2007]. Carbohydrates reduce protein denaturation during drying possibly by forming hy-drogen bonds with the proteins to replace the water-protein hydrogen bonds [Carpenter and

Crowe, 1989]. A more likely mechanism is that the protein loses conformational freedom due

to the high solute concentration caused by the addition of carbohydrates [Prestrelski et al.,

1993]. Because denaturation is a loss of tertiary structure, the lack of conformational freedom

preserves the tertiary structure of the proteins [Prestrelski et al., 1993]. Another way to re-

duce conformational freedom is to increase the concentration of protein in solution. Protein

solutions with high protein concentrations retain tertiary structure after spray drying better

than solution with low protein concentrations[Millqvist-Fureby et al., 1999]. In summary the

factors total solids content, outlet temperature, and carrier concentration have the greatest

impact on protein stability during spray drying [Sloth et al., 2008].

1.7.8 Outlet temperature

The maximum temperature drying particles are assumed to reach during spray drying is

the measured outlet temperature [Masters, 1991]. Outlet temperature is a function of other

process variables and is the best predictor of residual enzyme activity and microbial inacti-

vation during spray drying [Samborska et al., 2005, Sthl et al., 2002, Elizondo and Labuza,

1974, Labuza et al., 1970]. Microbial inactivation or microbial death rate can be described by

the pseudo-z value defined as the change in temperature required to cause a 10-fold increasein microbial death [Teixeira et al., 1995]. During spray drying of lactate dehydrogenase outlet

temperatures above 89C resulted in a decrease in pseudo-z value. This means above 89C(the

pseudo-z) a small increase in outlet temperature causes a greater loss of activity than the same

temperature increase when below 89C [Matzinos and Hall, 1993]. This critical outlet temper-

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Table 1.10. Temperatures at which microbial death or enzyme inactivation rate changes

Material Outlet(C)

Effect Reference

-amylase > 80 denaturation [Samborska et al., 2005]Whey protein > 90 denaturation [Anandharamakrishnan et al., 2007]Insulin > 120 denaturation [Sthl et al., 2002]Lactate dehydrogenase > 89 change pseudo-z [Matzinos and Hall, 1993]L. bulgaricus < 70 kill [Teixeira et al., 1995]S. cerevisiae < 84 < 4 log kill [Elizondo and Labuza, 1974]S. cerevisiae < 80 4 log kill [Labuza et al., 1970]

ature can be identified for microbial death rates and protein denaturation rates [ Samborska

et al., 2005,Teixeira et al., 1995,Sthl et al., 2002,Anandharamakrishnan et al., 2007,LiCari andPotter, 1970, Costa et al., 2002]. The critical temperatures are those above which denaturation

inactivation of a desirable compound are too great, or below which there is a sharp decline

in microbial death rate (Table 1.10). Similar breaks in death rates of microbes have been

found for various microorganisms, (Table1.10). An outlet temperature of 90C is commonly

used to spray dry proteins (Table1.11). An outlet temperature of 90C is expected to cause a

minimum 4 log reduction of microorganisms without reducing solubility or denaturing the

protein. These considerations perhaps explain why this temperature is commonly used.

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Table 1.11. Spray dried protein products

Product Inlet(C)

Outlet(C)

Reference

Alfalfa protein hydrolysate 170 90 [Kapel et al., 2006]Bovine plasma 60-110 [Matzinos and Hall, 1993]Bovine serum albumin 150 90 [Adler et al., 2000]Chicken hydrolysate 180 91-102 [Kurozawa et al., 2009]Faba bean 180 100 [Cepeda et al., 1998]Faba bean protein 190 122 [Otegui et al., 1997]Herring hydrolysates 180 100 [Hoyle and Merritt, 1994]Insulin 100-220 50-150 [Sthl et al., 2002]Milk emulsions 150 90 [Sliwinski et al., 2003]Shrimp peptides 170 90 [He et al., 2008]Sweet potato puree 150-220 100 [Grabowski, 2006]Pea protein 190 86 [Sumner et al., 1981]Potato protein 90 [Claussen et al., 2007]Soy protein isolate 210-215 80-85 [Boatright and Hettiarachchy, 1995]Tilapia hydrolysate 180 90 [Abdul-Hamid et al., 2002]Whey protein 180,200 80,100 [Anandharamakrishnan et al., 2007]

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CHAPTER

TWO

AMINO ACID ANALYSIS OF HYDROLYZED PEANUT MEAL

PROTEINS

2.1 Abstract

This work was a contribution to a larger study accepted for publication to the Journal of

Food Biochemisty. The amino acid content of proteins influences the solubility and thus

functionality of those proteins. Peanut meal protein was hydrolyzed for 240 min using three

commercially available food grade proteases. The amino acid contents of the water soluble

hydrolysates were compared to each other, control samples, and unhydrolyzed peanut pro-

tein. Enzymatic hydrolysis increased solubility of all hydrolysates and negligibly effected the

relative solubility of individual amino acids.

2.2 Introduction

Amino acids are largely responsible for the functional properties and nutritional quality

of proteins. The sequence and quantity of amino acids determines the tertiary structure.

Tertiary structure determines the solubility of the protein. Solubility is the basis of other

functional properties. Antioxidant activity is one such functional property. All 20 amino

acids can potentially quench free radicals. The most reactive amino acids (AAs) contain

nucleophillic-sulfur containing or aromatic side chains. Among the most reactive AAs are

cysteine and methionine which are sulfur containing AAs, and tryptophan, tyrosine, andphenylalanine which have aromatic side chains. Amino acids are classified as either essential

or non-essential to human health. The relative amounts of these essential amino acids in a

protein can be used to calculate the nutritional quality of that protein containing those amino

acids.

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2.3 Materials & Methods

Samples of interest were completely hydrolyzed in triplicate for 18 h at 110 C in the pres-

ence of 6 N HCl containing 0.1% phenol. Aliquots of the final hydrolysates were then deriva-

tized using AccQFluorTM reagent (Waters Corp., Milford, MA) as outlined in the manual

(WAT052874, Rev 0). Derivatives were analyzed using a Summit Model HPLC (Dionex Corp.,

Sunnyvale, CA) equipped with a Waters AccQTag (C18, 4m, 150 mm x 3.9mm) column. Elu-

ant A was an aqueous phosphate buffer solution containing triethylamine (purchased from

Waters Corp.) and diluted with water. Eluant B was Acetonitrile:water at a ratio of 60:40,

v:v. The gradient for the analysis started at 0% B then increased to 2.5% in 0.5 min, to 7%

in 15 min, to 10% in 19 min, to 33% in 32 min and held for 1 min. Eluant B was increased

in 1 min to 100%, held for 3 min, and then decreased to 0% in 3 min. The column was then

re-equilibrated at 0% B for 13 min. Total run times were 50 min at 37 C with a flow rate of 1.0

mL/min. The injection volume was 20 mL. Fluorescence detection with excitation/emission

wavelengths set to 250/395 nm was used.A standard mixture of amino acids was spiked with an internal standard of-aminobutyric

acid and hydrolyzed using the same methods as the samples. Values from this standard runs

were used to calculate response factors for each amino acid. Samples were spiked with

an equivalent amount of internal standard and response factors were used to quantify the

amount of each amino acid in the samples. As a control, a standard reference sample of

peanut butter, SRM 2387 (NIST, Washington, DC) was treated as a sample and hydrolyzed

and derivatized with every sample set.

Means of individual amino acid concentrations were compared using Tukey Tests for both

unhydrolyzed control samples and samples hydrolyzed for 240 min. Statistical analyses wereperformed using SAS (Cary, NC).

2.4 Results & Discussion

Amino acid composition of the unhydrolyzed controls were compared to those of the 4 h hy-

drolysates for each of the three proteases, and the amino acid composition of the dry, unhy-

drolyzed, defatted peanut meal (Figure2.1). Cysteine, methionine, and tryptophan were not

measured. The oxidative conditions for this analysis normalized glutamine and asparagine

with glutamic acid and aspartic acid respectively. Alcalase and flavourzyme controls had

higher concentrations of each amino acid than pepsin, but this difference was not significant

(Figure 2.1A). For the hydrolyzed samples (240 min) Alcalase produced significantly (p 1680

53.4 1680-6009.5 600-500

27.6 500-1755.1 < 175

Soluble fractions collected by centrifugation and settling had slightly different solids and

protein contents. The biggest difference between separation methods was yield, which was

defined as the ratio of soluble material collected to the total batch size (Table 3.1). The

protein and solids contents of the soluble fractions collected by gravitational separation and

centrifugation were very similar; however, the absolute amount of protein, as the product of

yield and protein or solids, was much greater when centrifugation was used. This suggests

that using settling as a method to separate insoluble from soluble fractions will generate

material analogous to the material that would be generated if high through put continuous

centrifugation equipment were used. However, in an industrial setting, centrifugation should

be used to increase the amount of soluble solids collected.

Peanut meal particle size distribution was determined to help with the selection of equip-

ment for industrial scale separation. The majority of particles in this sample of peanut meal

passed through openings 600 m but not 1680 m in size. The apparent bi-modality of the

size distribution is likely due to the large differences between opening sizes with the screens

used when passing from one screen to another (Table3.2).

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Settling Time (hours)

Volume

ofSolubleMaterial(mL)

510

1520

1 2 3 4

0

25

50

75

100

Figure 3.4. Volume of soluble fraction with increasing settling time for 5, 10, 15, and 20%dispersions

3.4.3 Insoluble fractions

Due to the scale and equipment used in processing the recovery of insoluble material was

nearly 100%. The moisture content of the insoluble fractions ranged from 10.4 to 12.7%. The

insoluble material containing hydrolyzed proteins can potentially be used as a value added

animal feed ingredient.

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3.4.4 Soluble fraction

Drying soluble fractions increased concentration of active/soluble components namely pro-

teins and peptides. Though not specifically investigated in this work, hydrolysis of peanut

proteins can be used to generate ACE inhibitory peptides. For these and other bio-active

peptides to have value as food ingredients they must be concentrated. Spray drying is a fast,continuous, high volume, and relatively inexpensive method to create powders. Commer-

cial spray drying typically utilizes liquid feeds between 30 and 50% solids [Masters, 1991].

The total soluble solids content of the soluble fractions ranged from 3 to 6%, so additional

concentration prior to spray drying was needed.

Vacuum oven drying

Vacuum oven drying was investigated as an inexpensive method to concentrate soluble solids

prior to spray drying. For better repeatability, oven dial settings and resultant air temperature

and water temperatures were first modeled (Figure 3.5). The advantage of using a vacuum

oven is that low temperatures can be used. However, the rate of evaporation, termed water

flux, was limited by air flow. Water flux is defined as mass of water loss per surface area per

second (Equation3.1). Mean water flux (N) of all trails was 3 104 kg/m2 s. High oven and

sample temperatures increased N, but this plateaued at about 6 104 kg/m2 s. The plateau

of N was attributed to the formation of a film at the surface of the drying liquid. Film forma-

tion was only observed during drying of samples which did not have protease added. This

difference between protease hydrolyzed treatments and control treatments without protease

would be enough to warrant the use

Thesis_value Addition to Aflatoxin Contaminated Peanut Meal

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