CHAPTER 1

INTRODUCTION

1.1General introduction

SCP refers to the dried microbial cells or total protein extracted from pure microbial cell culture (monoculture – Algae, bacteria, filamentous fungi, yeasts, etc…), which can be used as food supplement to humans (Food Grade) or animals (Feed grade).

The term ‘microbial protein ‘ was replaced by a new term ‘single cell protein ‘ (SCP) due to single celled habit of micro-organism used as food or feed through biotechnological process. The term SCP was coined by Prof.C.L.Wilson in 1966. This term is more appropriate as most of the microorganisms grow as single or filamentous individuals. SCP contains high protein content (60 – 80% of dry cell weight), and small amounts of fats, carbohydrates, nucleic acids, vitamins, and minerals. It is also rich in essential amino acids such as Lys and Met.

Microorganisms have been employed for many years in the production of high protein food content such as cheese and fermented soybean products. Protein being the main nutritional component in both types (Haider et al., 1989). Since a large proportion of cell dry weight is accounted for protein, the nutritional value of a microbial derived food source is determined by the levels of protein produced (Patel, 1995). The development of microbial systems for use in food industry has Firstly, growth of microorganism is very much faster than of animals . Secondly, a broader range of materials may be considered as suitable substrates depending on the microorganism chosen. The two chief strategies with regard to substrate to consider low grade waste material, or to use relatively simple carbohydrate source to produce microbial material containing very high quality of protein (Reed & Nagodawithana, 1995). Microorganisms have the ability to upgrade low protein organic material to high protein food, and this has exploited on by industry. This phenomenon was employed in Germany during the first world war when the growth of Saccharomyces cerevisiae was exploited for human consumption. Moreover, Candida arborea and C. utilis were used during the second world war and about 60% of the country prewar food input was replaced (Litchfield, 1983).

As a commodity, SCP must be competitive with commercial animal and plant proteins, in terms of nutritional value and must conform with human and animal food safety requirements. The rapid growth rate and high protein content of microbes and their ability to utilize inexpensive feedstock as source of carbon and energy for growth have made microorganism’s prime candidates for use as human food and animal feed protein supplement (Rasool Shafiee et al., 2005).

1.2 HISTORICAL PERSPECTIVE

The pioneering research conducted almost a century ago by Max Delbrück and his colleaguesat the Institut für Gärungsgewerbe in Berlin, first highlighted the value of surplus brewer’syeast as a feeding supplement for animals (Delbrück M., 1910). This experience proved more than useful in the ensuing First World War, when Germany managed to replace as much as half of its imported protein sources by yeast. Since brewers yeast from beer production was not produced insufficient quantity to meet the demands as a protein feed, a very large proportion of yeast biomass was expressly produced by aerobic fermentations in a semidefined medium containing ammonium salts as the nitrogen source (Hayduck F., 1913).

After the end of World War I, German interest in fodder yeast declined, but was revived around 1936 by the ‘Heeresverwaltung’, when both brewer’s yeast, and a variety of yeast specially mass cultured, were used to supplement human and animal diets. By then the advantages of aerobic production of baker´s yeast in a rich wort had been fully recognised asa rapid means of producing food in large scale industrial installations. A radically different concept to that of agricultural production (Barnell H. R., 1974). By the begining of World War II, yeasts had been incorporated first into army diets, and later into civilian diets. Ambitious plans were laid for production of well over 100,000 tons per year. This figure never surpassed 15,000, probably because of the extensive disruption which typically accompanies wartime economies.

The Food and Agriculture Organisation of the United Nations (FAO) which brought forward the hunger and malnutrition problem of the world population in 1960, introducing the concept of the protein gap (25% of the world population had a deficiency of protein intake in their diet). Thepopulation growth predictions, moreover showed that the number of inhabitants would doublebetween 1960 and 2000, from 2.5 billion to 5 billion (the actual figure reaches 6 billion), andthe greater part of this increase would take place in those countries suffering from malnutrition. The Malthusian prospect of a limiting food supply was reinforced by fears that agricultural production would fail to meet the increasing food requirements of humanity. The resumption of peace had also procured a new atmosphere geared towards the academic study in civilian matters, and fermentation processes saw a very important period of progress. A greater involvement of private companies in the marketing of fermentation products had already begun. By the early 60´s a number of multi-national companies decided to investigate the production of microbial biomass as a source of feed protein. The basic kinetic mechanisms ruling the growth pattern of microbes had been elucidated (Monod J., 1942) and were being established for yeasts and filamentous fungi (Pirt S. J. and Kurowski W. M., 1970).

The novelty of unwanted waste product consumption added a new economic incentive to SCP production, as the idea of zero cost substrates, or even the obtainment of additional revenues through the concept of waste treatment were argued and incorporated favourably to reduce the production cost estimates. The benefits of SCP production were thus extended from the production of food to the preservation of the environment.

By the mid 60´s, some quarter of a million tons of food yeast were being produced in different parts of the world and the Soviet Union alone planed an annual production of 900,000 tons by 1970 of

food and fodder yeast, to compensate agricultural protein production deficits (Bunker H. J., 1966). By 1980, SCP production processes were operating on a large scale in developed countries, and plans to extend SCP production to underdeveloped countries were being made.

But a number of technical and political developments that occurred in the 80´s conditioned the expansion of the promising SCP industry. Marked improvements in plant breeding and crop production on a global basis allowed for a continued increase in agricultural output, beyond the expected ceilings (Fig. 1). Local effects such as agricultural reform implemented in China, also resulted in marked agricultural output. Finally, the prospect of the end of the cold war could first be foreseen at this time, with important liberation of agricultural reserve stocks for market trading [13]. This trend was later materialised by the General Agreement on Tariffs and Trade (GATT) signed in Marrakesh, commiting 118 countries to a new open trade world market in 1994 [14]. This treaty effectively de-regulated the world distribution of goods, opening new market areas and connecting countries with surpluses, and countries with deficits. This treaty had an immense effect on agricultural product trade worldwide.

Fig. 1. World production of the main agricultural crops in metric tons. Data obtained from Food and Agriculture Organisation of the United Nations (FAO) [13].

In view of these developments, the price of the majour agricultural crops did not experience the increases expected in previous decades, and the market price of protein of plant origin continuously decreased in constant currency terms. This effectively outmarketed SCP (Fig. 2).

Fig. 2. Real Prices of agricultural exports from industrial and developing countries, (1955-1996). Source: Food and Agriculture Organisation of the United Nations (FAO), based on World Bank data [13].

In view of these developments, many industrial SCP processes were discontinued, leaving behind them a wealth of skill and knowledge, which have been successfully benchmarked in other fermentation processes. Specific research in the field also declined in consonance with the market trend (Fig. 3).

Fig. 3. Number of scientific papers cited including ‘SCP’ or ‘Single Cell Protein’ in their titleor key words. Data from Cambridge Scientific Abstracts database (CSA) [15].

The most notable example of the evolution of SCP processes into new products is perhaps that led by Rank Hovis McDougall (RHM) in cooperation with ICI, founding Marlow Foods (now part of the AstraZeneca group), a company which started producing myco-protein and fungal protein based products under the commercial trademark Quorn(http://www.quorn.com).The products were initially derived for human consumption in 1964 from the batch cultivation of Fusarium venenatum (formerly F. graminearum) strain A3/5 on starch and other waste products. The myco-protein production process experienced an evolution of 20 years and an estimated R+D expenditure of $ 40 million, before unrestricted clearence by the UK Ministry of Agriculture, Fisheries and Foods was granted in 1985. The myco-protein is now produced in continuous culture and the biomass is manipulated to achieve a texture and taste which resemble meat products, covering a market as a meat alternative for vegetarian formulations. Quornproducts are currently the only SCP-based products exclusively directed at human consumption in the market.

1.3 Sources of SCP

There are two sources of SCP. One is photosynthetic organisms and the second one is non-photosynthetic organisms.

1.3.1 Photosynthetic organisms :

Both algae and bacteria are among the photosynthetic microorganisms that are used for single cell protein production.

1.3.1.1. Algal protein

The protein content of algae is approximately 50 gm per 100 gm algal mass (dry weight). In addition to protein, they contain abundant amount of β-carotene , vitamin K and little quantity of vitamin C. But they are poor sources of vitamin of the B group. Moreover, protein content lower level of sulpher containing amino acid. Also the digestibility is low and the test can be unpleasant. Sources of algal protein are as follows.

Chlorella sp. Scenedesmus acutus Spirulina maxima

1.3.1.2 Bacterial protein

The photosynthetic bacteria that are used for single cell protein production include bacteria of the genus Rhodopseudomonas which have been grown on sewage or industrial waste in Japan for use as an animal feed. These bacteria grow in mixed cultures with nitrogen fixing and other aerobic bacteria. They must be supplied with organic substances as carbon and energy sources, and will not grow using only carbon dioxide and light as the algae do. The culture densities of the bacteria are in the range of 1 to 2 grams of dry material per litre, and the problems of separation and concentration that occur with the algae are also present in this system.

1.3.2 Non-photosynthetic organisms :

The non photosynthetic organism that are grown to produce SCP include bacteria and moulds, yeast and other fungi. These organisms are aerobic and must have oxygen to grow. They also require an organic carbon and energy source, together with sources of nitrogen, phosphorus, sulpher and mineral element.

1.3.2.1 Bacterial protein and actinomycetous protein :

Bacteria are widely used as a source of SCP because of their short life cycle (20 – 30 min.) and capacity to utilize a wide range of organic substrates as a source of energy. Actinomycetes also utilize these renewable sources as bacteria. The bacterial groups of major interest in this regard are hydrogen, methanol or methane, and paraffins utilizing bacteria. Sources of bacterial protein are as follows.

Methylococcus caplulates Pseudomonas sp. Hyphomicrobium sp. Actinobacter sp. Flavobacterium.

1.3.2.2 Yeast protein:

Modern technology for producing yeast single cell protein has largely developed since World War ǁ. Today, yeast products for human or animal consumption are produced on a commercial scale in many countries. In addition , baker’s yeast which is grown on molasses , is sold as a food flavoring and nutritive ingradient in addition to being used as a leavening agent. Sources of yeast protein are as follows:

Saccharomyces cerevisiae Candida utilis Candida guillienmondis Kluyveromyces fragilis

1.3.2.3 Moulds and other fungi

The moulds Fuserium and Rhizopus were grown by fermentation during World War ǁ.

The inoculums of aspergillus oryzae or Rhizopus arrhizus is chosen because of their nontoxic nature ( Riviere, 1977). Saprophytic fungi grow on complex organic compound and render them into simple forms. As a result of growth, high amount of fungal biomass is produced. Mycelial yield very widely depending upon organisms and substrates. Strains of some specie of moulds, Aspergillus niger, A. fumigates, Fuserium graminearum are very hazardous to human ,therefore, use of such fungi should be avoided or toxicological evolution should be done, before recommending to use as SCP. Sources of Moulds and other fungi are as follows :

Penicillum cylopium Trichoderma harzianum Paecilomyces varioti Chetomium cellulolyticum

1.4 Properties of SCP

One of the main advantages of SCP compared to other types of protein is the small doubling time of cells (td) as shown in Table 1.1 (Cleanthis J, (1987).

Table 1.1: Mass doubling time

Organism Mass doubling Bacteria and yeast 10-12 minMold and algae 2-6 hGrass and some plants 1-2 weekChickens 2-4 weekPigs 4-6 weekCattle 1-2 monthPeople 0.2-0.5 year

It is assumed that the growth occurs without any restriction. Other advantages of SCP over conventional protein sources are:

a. it is independent of land and climate; b. it works on a continuous basis; c. it causes less pollution. d. Protein synthesis is much more rapid than higher living systems.e. Microbes have short generation time.f. Easily modifiable genetically for determining the amino acid composition.g. Microbes have high protein content.h. Microbes can be grown on media containing cheap sources of C and N.

There are five factors that impair the usefulness of SCP:

a. non digestible cell wall (mainly algae); b. high nucleic acid content; c. unacceptable coloration (mainly with algae); d. disagreeable flavour (part in algae and yeasts); e. cells should be killed before consumption.

Thus SCP is treated with various methods in order to:

1. kill the cells; 2. improve the digestibility; 3. reduce the nucleic acid content.

1.5 Choice of organism

Selection of an appropriate microorganism is essential to the success of any single-cell protein production undertaking. The microorganism must be one that is edible and can serve as a feedstock for humans and/or livestock. Of course, its culture must be technologically and economically feasible. The key criteria used in selecting suitable strains for SCP production should consider the following:

the organisms must grow rapidly and vigorously; culture of the organism should involve the use of relatively simple

growth units and inexpensive nutrient sources (e.g., commercial crop fertilisers);

The substrates to be used as carbon energy and nitrogen source and the need for nutrient supplementation;

High specific growth rates, productivity and yields on a given substrate. Ideally, the organism could be grown in open culture, or at least as an enrichment culture;

pH and temperature tolerance; Aeration requirements and foaming characteristics. Safety and acceptability – non pathogenic, absence of

toxins. Ease of recovery. Protein, RNA and nutritional composition of the product. Structural properties of the final product Because single-cell protein production is only marginally economically

feasible, the least “permissible” condition is the need to culture the organism under sterile conditions, i.e., as a completely pure culture;

In general, fungi have the capacity to degrade a wider range of complex plant materials, particularly plant polysaccharides. They can tolerate low pH which contributes to reducing fermenter infections. Growth of fungi as short, highly branched filaments rather than in pellets is essential in order to optimize growth rate. But strains of some specie , Aspergillus niger, A. fumigates, Fuserium graminearum are very hazardous to human ,therefore, use of such fungi should be avoided or toxicological evolution should be done, before recommending to use as SCP.

Bacteria, in general, have faster growth rates than fungi and grow at higher temperatures, thereby reducing fermenter cooling requirements. Bacterial fermentations are easier to aerate. Protein content in bacterial can range from 60-65%.However the high nucleic acid content of bacterial proteins renders them less desirable as feedstuff for man and animal. Additionally, some groups of bacteria are characterised by the possession of endotoxins. The endotoxins could be incorporated in the feedstuff product. There is also a possibility that certain bacterial feedstuffs can promote allergenic reactions in humans who handle or ingest them. Finally, the much smaller size of bacteria makes them more difficult to harvest than yeasts.

Most of the work on single-cell protein production has been focused on the yeast. The yeast meets most of the requirements named in the preceding paragraph. Yeast fermentations are easier to aerate. Not only is the yeast easily grown, it also is a good food and fodder yeast. Although sterility is necessary, purity of culture is not essential.

1.5.1 Selection of Saccharomyces cerevisiae

Saccharomyces cerevisiae (brewer’s and baker’s yeast) has been used in classical food fermentation applications (beer, bread, yeast extract/vitamins, wine, saké, distilled spirits). Also it is used in the production of fuel alcohol, glycerol, invertase and animal

feeding (R. F. Beudeker et al., (1990).Microbial inoculants, which are used as a process aid, generally have a higher value. In this case, the objective of the production process is to optimiseyield of viable cells of defined biological activity with good shelf life characteristics. Saccharomyces cerevisiae, is categorised primarily as a microbial inoculant.

Since this yeast is a rich source of B vitamins and chromium, it has been studied extensively for its medicinal properties. So, it has been reported the use of yeasts (Saccharomyces boulardii or Saccharomyces cerevisiae, a commercial baker's yeast) as a potential biotherapeutic agent in combination with standard antibiotics for the treatment of Clostridium difficile-associated diarrhea and colitis (D. J. Kovacs et al., 2000). As a source of B vitamins, Saccharomyces cerevisiae strains can relieve stress, depression, irritability, and fatigue and also help to reduce some effects of aging. In the same way, as a source of biotin, they can strengthen hair and nails, and treat cradle cap and diabetes neuropathy. As a source of chromium, these yeasts can reduce blood sugar levels in people with type 2 diabetes, reduce risk of high cholesterol in blood, and aid in the treatment of chronic acne and furunculosis. Chromium can be difficult for the body to absorb, but it is more easily absorbed

when taken with brewer's yeast (J. Hegoczki et al., 1997). In addition since brewer's yeast also has several other minerals including selenium, zinc, phosphorus and magnesium, it is often used for loss of appetite (Y. C. Li, 1994)

Modern technology for SCP production originated in Great Britain in 1879 with the introduction of aerated vats for producing baker’s yeast (S. cerevisiae). The centrifuge was first used about 1900 in the US for separating baker’s yeast cells from the medium.

Table 1.2: Developments in single cell protein production by S. cerevisiae: ancient times to present time.Period Technical development

2500 BC Top fermenting yeast recovered for baking

1781 Compressed yeast prepared from brewer’s yeast (UK, Netherland, Germany)

1860 Vienna process aerated, malted grain mash substrate (Austria)

1868 Introduction of compressed yeast manufacturing into US (Fleischmann)

1879 Continuous aeration (UK)

1900 Centrifuge used for yeast separation (US)

1914- 18 Incremental feeding, molasses, ammonium salts (Germany)

1936 Heiskenskjold process using sulphite liquor (Finland, Germany) and Scholler- Tornnesch process for fodder yeast from wood sugar (Germany)

1959 Continuous production of baker’s yeast on commercial scale (UK)

1983- 5 High cell density, direct dry process for SCP production from ethanol and carbohydrates (US)

1.6 SUBSTRATES FOR SCP PRODUCTION

The choice of substrates that are normally abundant and in proximity to the production plant has determined the design and strategy of SCP processes.

The most widespread and commonly used substrates for SCP production have been those where the carbon and energy source is derived from carbohydrates. This is due to the fact that their building blocks (mono and disaccharides) are natural microbial substrates, and that carbohydrates are a renewable resource which is widely distributed.

1.6.1 Molasses

Molasses is a by-product of the sugar manufacturing process. Besides its high sugar content, molasses contains minerals, organic compounds and vitamins which are valuable nutrients in fermentation processes (Olbrich H, 1973). In fact, about 9% of the dry matter in yeast grown on molasses has been estimated to originate from substances other than sucrose (Olbrich H, 1973). Nevertheless, biomass production from molasses requires supplementation with a suitable nitrogen source, as well as phosphorus. The traditional nitrogen sources used are ammonia or ammonium salts, and phosphorus can be added in the form of salts.

Baker´s yeast was the first microorganism to be produced in aerobic stirred fermentation onmolasses as it is still produced today (Chen, S. L. and Chinger M, 1985). However, this yeast has seldom been destined as food, but rather for baking purposes.

1.6.2 Starch

A cheaper, more amenable SCP substrate of carbohydrate origin is starch. This very abundant carbohydrate may be obtained from bulb plants of tropical and temperate regions, or from rice, maize and cereals. In tropical countries, cassava has been proposed as a good source of starch for SCP processes (Forage, A. J. and Righelato, R. C., 1979).

The Symba process developed in Sweden (Jarl, K., 1969) utilized starchy wastes combining two yeasts in sequential mixed culture: The amylase producing Endomycopsis fibuligira, and the fast growing Candida utilis. The process consists of three phases: The incoming starch waste from potato tubers is fed through heat exchangers and sterilised. The medium is then fed to a first bioreactor where the starch hydrolysing yeast grows and hydrolyses starch. The hydrolysed solution is then fed to a second reactor where culture conditions favour the proliferation of C. utilis.

1.6.3 Glucose

The Quornmyco-protein production process is currently supported on glucose, nearly allof which is obtained from maize, but it has been reported earlier to use wheat-starch, a by-product of the production of wheat gluten (the protein fraction) and wheat flour (Trinci, A. P. J., 1994). This means that the process may be applied with various sources of starch as the carbon source (Steinkraus, K. H., 1986).

1.6.4 Whey

Whey is a residual liquid obtained after the removal of protein and fat from milk. Whey traditionally originates from the curding process in cheese production, but can now be obtained after ultrafiltration procedures for the production of spreading cheeses, where the protein fraction corresponding to

actalbumins and lactoglobulins is incorporated to the casein fraction, and all the proteins are in native form.

Since it is derived from milk, the processing of whey as food technology additive for direct human consumption appears an obvious outlet, but various features hinder this application. The principal sugar, lactose, is in a concentration which is too low to make transport or concentration viable in economic terms. In addition, it presents digestibility difficulties for adults, since the capacity to assimilate lactose in humans diminishes on maturity, especially in African and Asian populations.

Whey has been presented as an extremely suitable substrate for the production of SCP. In 1956 The French dairy company Fromageries Bel pioneered a project to produce yeast from whey, using lactose assimilating Kluyveromyces marxianus (formerly K. fragilis). In 1983, the company was processing 8000 tons of yeast in continuous culture (Moulin G. et al., 1983).

1.6.5 Alkanes

Alkanes were considered as an attractive substrate for SCP production, particularly in the former Soviet Union, where the structural deficit in feed protein was compensated by the availability of oil. A large number of microorganisms are able to assimilate n-alkanes in liquid culture include yeasts Candida, Pichia, Saccharomyces and fungi Aspergillus, Fusarium, Penicillium and 1-alkenes in liquid culture include yeast Candida, Rhodotorula and fungi Cephalosporium, Fusarium (Rivière J., 1977).

The toxicity of the substrate has raised suspicions on the safety of continuous feeding with SCP containing trace amounts of alkanes. SCP production plants using alkanes are not currently in operation.

1.6.6 Methanol

Methanol is a by-product of the petrochemical industry and has been used as a substrate for a number of SCP production systems. Methanol tolerance (up to 6 g l-1) and assimilation is a specialized prerequisite which may be found in yeast species such as Hansenula, Pichia, Candida and Torulopsis. The advantages of methanol over other petrochemical by-products are many, but the principal one resides in the volatile nature of the substrate, allowing it to be lost in the drying process(Faust, U. and Präve, P., 1983).

1.6.7 Cellulose

Cellulose from agriculture and forestry sources constitutes the most abundant renewable resource in the planet. The productivity of forests and woodlands, amounts to 40% of the world net productivity, while cultivated land amounts to a mere 6%. Most of this productivity is in the form of cellulose and lignin (approximately 30% of most woody plants is composed of lignin).

SCP production from these substrates, as in any other case, requires their breakdown into assimilable forms, which can be rapidly taken up in solution by the growing organism. The substrate must therefore be subjected to pretreatments in a number of steps which include milling and chemical or enzymatic hydrolysis.

1.6.8 sulfite liquor

Spent sulfite liquor has been used as a substrate for fermentations since 1909 in Sweden, and later in many other parts of the world. The first organism to be used was Saccharomyces cerevisiae, although this organism is unable to metabolise pentoses which are found in considerable amounts in this waste product. Later, other organisms better suited for the assimilation of all the sugar monomers were chosen. Namely Candida tropicalis and Candida utilis. Nevertheless, microorganisms are susceptible to sulfite, which is removed previous to the fermentation process (Webb, F. C. 1964).

1.7 SCP CONSUMPTION AND USES

1.7.1 Protein requirement

Protein requirement is very important to our body but it should be in optimum level. Excess or less amount of protein can be detrimental to our body. How much protein is required that depends on weight of body, work, age, etc.

In inactive lifestyle, the recommended dietary allowance (RDA) for a sedentary individual is

about 0.8 grams per kilogram of body weight (www.smart-strength-training.com).. So, if weight of body is 90kg then protein requirement = 90 * 0.8 = 72g per day protein is required. If lifestyle is active or hard working then protein required is 1.4g/kg to 1.8 g/kg of body weight daily.

1.7.2 NUTRITITIONAL VALUE

For the assessment of the nutritional value of SCP, factors such as nutrient composition, amino acid profile, vitamin and nucleic acid content as well as palatability, allergies and gastrointestinal effects should be taken into consideration (9). Also long term feeding trials should be undertaken for toxicological effects and carcinogenesis. Table 1.3 shows the average cell composition of the major groups of micro-organisms.

Table 1.3: The average cell composition of the major groups of micro-organisms. (% dry weight)

Component Fungi Algae Yeast Bacteria Protein 30-45 40-60 45-55 50-65Fat 2-8 7-20 2-6 1.5-3.0Nucleic acid 7-10 3-8 6-12 8-12

The vitamins of micro-organisms are primarily of the B type, B12 occurs mostly in bacteria, while vitamin A is usually found in algae. Table 1.4 shows the vitamin content of various food micro-organisms.

Table 1.4 Vitamin content of various food micro-organisms (mg/100 g dry weight) (15)

Vitamin Morchell hortensis Candida utilis Saccharomyces. cerevisiae

Methylomonas methanica

Thiamine 0.52 0.53 5.36 1.81Riboflavin 1.31 4.50 3.6-4.2 4.82Niacin 12.4 41.73 80-100 1.5-9Pyridoxine 2.62 3.34 2.5-10 14.3Pantotheric acid 12.6 3.72 10 2.42Choline 4.61 - - 968.0Folic acid 1.09 2.25 1.5-8.0 -Inositol 1.78 - - -Biotin 0.015 0.23 0.5-1.8 -Vitamin B12 0 0 0 0.96P-aminobenzoic acid - 1.7 0.9-10 -

SCP is normally considered as a source of protein. However, like any other biologicalmaterial, it also contains nucleic acids, carbohydrate cell wall material, lipids, minerals andvitamins. Nevertheless, these contributions are given little importance by nutritionists, whogenerally value SCP in terms of Kjeldhal nitrogen x 6.25 (standard factor relating aminonitrogen to protein content). However, about 10-15 % of the total nitrogen in fungi and yeastsis in the form of nucleic acids. These are not metabolized in the same way as proteins butfollow a different route. This aspect is of importance with respect to SCP formulations, andwill be dealt with separately below.

Amino N, therefore represents approximately 80% of total microbial nitrogen, and iscomposed of all essential amino acids required for human growth and nutrition (Table 1.5).

In order to be most useful for nutrition, to be most useful for nutrition, not only must all the amino acid essential to human nutrition be present, but they should be present in a certain ratio is not correct, the biological value of the protein will be lowered. The ratio generally accepted as more or less ideal is that published by the Food and Agriculture Organization (FAO) and reproduced in part in Table 1.5.

Table 1.5: Daily requirements (g) of essential aminoacids for the human adult Data retrieved from FAO (http://www.fao.org).

Essential aminoacids FAO recommendation MinimumPhenylalanine 2.2 1.1Methionine 2.2 1.1Leucine 2.2 1.1Valine 1.6 0.8Lysine 1.6 0.8Isoleucine 1.4 0.7

Threonine 1.0 0.5Tryptophan 0.5 0.25Total 12.7 6.35

With respect to egg albumin, which is considered a well balanced source of essential aminoacids for human nutrition, fungal SCP compares well, except that it is defficient in sulfurcontaining amino acids. However, they are relatively rich in lysine and threonine with respectto other traditional protein sources of agricultural origin, such as wheat (Table 1.6) [29].

Table 1.6: Essential aminoacid content of wheat, egg albumin and some fungal SCP sources(g per 16g N). Data from reference 29.

Amino acid Wheat Egg white S. cerevisiae C.lipolytica P.notatumLysine 2.8 6.5 7.7 7.8 3.9Threonine 2.9 5.1 4.8 5.4 ---Methionine 1.5 3.2 1.7 1.6 1.0Cystine 2.5 2.4 --- 0.9 ---Tryptophan 1.1 1.6 1.0 1.3 1.25Isoleucine 3.3 6.7 4.6 5.3 3.2Leucine 6.7 8.9 7.0 7.8 5.5Valine 4.4 7.3 5.3 5.8 3.9Phenylalanine 4.5 5.8 4.1 4.8 2.8

1.7.2.1 Protein digestibility and protein effici

The protein value of SCP has been determined through three basic parameters generally used in feed evaluation: the total quantity of microbial nitrogen ingested (I), the nitrogen of faeces (F) and urine (U). From these parameters, Digestibility, Biological Value and Protein efficiency can be calculated.

Digestibility (D) is the percentage of the total nitrogen consumed which is absorbed from the digestive tract.

D = 100 x ( I - F / I )

Biological Value (BV) is the percentage of the total nitrogen assimilated which is retained by the organism, taking into account the simultaneous loss of endogenous nitrogen through excretion in urine.

BV = 100 x ( I - [ F + U ] ) / ( I - F )

Protein Efficiency (PE) is the proportion of nitrogen retained when the protein under test isfed and compared with that retained when a reference protein, such as egg albumin, is fed.Yeast and fungal SCP products register high digestibility values, and these can besubstantially increased when supplemented with methionine (Table 1.7).

Table 1.7. Nutritional parameters of yeast foods in rats. Data from reference 29.

Organism Digestibility (%) Biological Value Protein efficiencyS. cerevisiae 81 59 ---C.utilis 85-88 32-48 0.9C.utilis + 0.5% DL-methionine

90 90 2.3

In order to obtain best results in the uses of SCP as feed, it is normally necessary toundertake specific pretreatments which improve the digestion and acceptability of theproduct. Treatment is directed towards killing cells, and liberating the internal contents.Autolysis has been the most popular method in the commercial production of yeast extract.The procedure involves heating the concentrated cell suspension after harvesting, to 45-50 oCfor 24h at pH 6.5. Under these conditions, the internal enzymes hydrolyse the cell wall in part,and also attack proteins, resulting in smaller better digestible peptides. However, the processmust be carefully monitored, as many of the resulting peptides may confer undesired tastesand smells to the product, thus limiting its application.

myco-protein presents very high Protein Efficiencyvalues, reaching 75% with respect to egg albumin. In experimental tests where myco-proteinwas supplemented with 0.2% methionine, this value rose to 100%. Thus, myco-protein couldbe used as a total replacement for the human diet, in comparison with a mere 10%replacement considered as safe for yeast protein [22, 23].

1.7.2.2 Reduction of RNA content

Nucleic acids are a necessary component of all cells, but present relatively high levels inrapidly dividing cells. Thus, the nucleic acid content of yeast (around 10% of dry wheight) isapproximately five times greater than in the average mammalian organ.

Ingestion of RNA from non-conventional sources should be limited to 50g per day. Ingestion of purine compounds arising from

RNA breakdown, leads to increased plasma levels of uric acid, which can cause gout and kidney stones.

High content of nucleic acids causes no problems toanimals since uric acid is converted to allantoin which is readily excreted in urine. Nucleic acid removal is not necessary from anima feeds but is from human foods.

A temperature hold at 64C inactivates fungal proteases and allows RNA-ases to hydrolyse RNAwith release of nucleotides from cell to culture broth.A 30 min stand at 64C reduces intracellular RNAlevels in Fusarium graminearum from 80mg/g to2mg/g.Alkaline hydrolysis destroys RNA but also diminishes the nutritive value of the proteincomponent. A compromise method which is effective consists of an incubation at pH 9.5followed by a heat shock which precipitates the protein. Sodium chloride extraction follows.In some yeast products, thermal shock at 60 oC is applied followed by pancreatic ribonuclease,reducing the nucleic acid content from 9% to 2%. Similar results have been achieved by aseries of short heat bursts which activate intracellular ribonucleases in yeast [38].

1.8 Formulation of SCP

Generally, single-cell protein is initially produced as a wet paste and then is subsequently converted into dry powder form. One of the advantages of the powder formulation is a higher chemical

stability than the solution. This dry powder, similar in appearance and feel to flour, lacks the texture and food-like sensation to the mouth necessary to make an attractive food. Moreover, when placed in water, the powdered single cell protein rapidly reverts back to single-cell form. To overcome this disadvantage, we converted our obtained SCP to other edible form like chocolate. Chocolate powder serves multiple purpose of cross linking agent, taste maker, and flavoring agent. Liquid chocolate can also be used for the same. Within economic constraints some other flavoring agents or fruits can be used. Flavoring agent should be such that it adds to the nutritive value along with taste and fragrance Chocolates are consumed by people of all ages from childhood to adulthood. In an stage of life, protein intake is necessary so converting SCP to chocolate form will increase the consumption of protien.

1.9 THE ECONOMICS OF SCP PRODUCTION AND MARKETING.

In the case of SCP production, the need for accurate cost estimations is very relevant, sincein the majority of cases the product is competing against protein sources of plant origin, andthe profit margins are predictably low. In other cases, such as that of myco-proteinprocess, fungal protein is competing against meat as a meat substitute, but an added economiceffort is required to promote the product against such an established competitor, and the addedcost must be compensated for in the production economy. Thus, in all cases, product costestimation is a central element in the food and feed market industry.

1.9.1 Parameters affecting economic viability

Several parameters are used as key elements in the estimation of economic viability. Theywill be briefly outlined, but the reader is referred to specialysed references where full detailsof this complex area, together with application of these concepts to different SCP processesare presented [90-92].

1.9.1.1 Total Product Cost

Includes all the costs incurred, and it may be divided by the annualproduction in order to estimate the cost per unit of product. It is normally broken down intoManufacturing cost + General expenses. The former includes all aspects directly related toproduction, such as Direct operating costs, Labour and supervision and Utilities. GeneralExpenses include such concepts as administration and R + D, and marketing.

Sometimes detailed information on these elements is not readily available. However,empirical formulae which relate the unknown values of some parameters to other obtainableones are used in order to build an approximate picture which enbraces all these elements.

1.9.1.2 Capital Investment

All of the funds required to build start and test the production facilitybefore the product is put to the market are included. This parameter may be further subdividedinto Fixed capital, or capital invested in hardware, land and equipment, and working capital,which includes inventory of raw materials, products and supplies, receivable and payableaccounts.

1.9.1.3 Profitability

The easiest way to calculate this parameter is to calculate the return on theinvestment as a percentage.

Despite the elaborate skills with which cost estimation may be carried out, it is stillvulnerable to deviations which are sometimes strong, due to the appearance of unaccountedvariables. One such variable of technical nature mentioned already can be the appearance ofhighly branched colony mutants in the myco-protein production process.

Other very important variables are more conventional, but they can make or brake a business venture, in the same way as they influence private family economies. Labour costs, fuel prices or interest rates are but a few variables which can unpredictably change as a consequence of local or globaldevelopments.

1.9.2 Practical aspects of economic viability

In the case of SCP production, the raw material accounts for62% of the total product cost, followed by fixed charges attributed to the production process,with 19% [75]. Thus, the main influencing factor has been the cost of the substrate, and thisexplains the quest for the processing of different substrates

Besides the largest elements influencing Total Product Cost, there is a myriad of detailswhich can cut the cost of production. Small though their contribution may seem, the additiveeffects of all the adequate measures may represent the difference between favourable andunfavourable economic balance. Some are describe below:

Higher process temperatures leading to greater productivities may result in reduced reactorcooling costs.

Fine adjustment of the aeration levels to values still higher to the critical oxygenconcentration limit, below which the organism no longer supports an oxidative metabolicpattern, reduces aeration consumption, as well as foaming and evaporation of the medium.

Fine adjustment of the medium required to sustain growth results in savings in some growthfactors, such as vitamins, which are expensive. Cheaper sources of vitamin, where they arefound in impure mixtures (yeast extract, soya bean extract, etc) often make all the differencein the cost of the supplement. Many processes sacrifice part of the biomass to make an extract

which is fed back as a source of growth factors.

Care in the choice of the nitrogen source may be relevant. Some sources of nitrogen (i.e.urea) contain higher amounts of nitrogen per unit weight than ammonium salts. The savingscome through transport costs. Hydrated forms of salts are not recommended for the same reason.Ssavings can also be made in pH adjustment reagents.

1.9.3 Advantages and constraints of SCP as a market product

Besides the aspects cited above, the variability in the market price of other products againstwhich SCP is competing, clearly determines the market price and hence the profitability.Most of the SCP products that appeared in the food and feed market from 1960 till 1994 were destined for use as protein factors, in formulations. This means, that they were wholesaleproducts which competed against other sources of protein which could be equally substitutedin the formulations, without apparent changes in the final product.

One direct competitor for SCP in western countries was brewers yeast. Identical in almostevery feature, brewers yeast had a bitter taste which carried through to feed formulations, asthe only differing characteristic from SCP yeast. However, this competitor was a by-product,the production of which was independent from the market strategy of the producers. This leadto policies of high turnover, low stockage of the by-product and consequently low marketprices. Another competitor was excess bakers yeast. Thus, yeast and fungal SCP had to fall inthe by-product market.

One common feature of SCP processes was that they often eliminated waste products, thuscovering the function of expensive waste treatment installations. This led to the logic that thesubstrate may not only be provided at low prices or free, but received with payments by SCPproducers. In the case of public wastes, an environmental quota could be payed to SCPproducing companies. Such payments would add to those for the final product, with importantrepercussions on profitability.

Though these reasonings made some sense, market reality proved to be very different. Sincea profit was expected to materialise from SCP production, the wastes which the processconsumed passed on to become substrates, and little interest was payed on their potentialenvironmental hazard once consumed. The use of wastes, in addition brought additionalproblems in cases where the interest in waste treatment prevailed: The production volumeswere not determined by the market demand of the product, but by the need to eliminate thewaste. In those instances, waste treatment was the product and SCP was a true by-product,which accumulated until buyers could negotiate bargain sales which liberated stock capacityfor the producer. Processes using whey and sulfite liquor were examples vulnerable to theseconstraints [16, 91]

1.10 FUTURE PROSPECTS

SCP has a proven record as a source of protein which may be obtained with largeproductivities in compact installations. The daunting prospects of world famine in the 60's

lead to great expectations about the social and economic relevance of microbial proteins as afood source. These expectations were happily not fulfilled due to the opening of trade barriersand marked improvements in the breeding and production of plant protein sources.

Nevertheless, there are success stories which paved the way for a new market of specialisedfoods of microbial origin. The compactness and high degree of control achieved in processessuch as QuornTM myco-protein production process provide a high degree of safety to theconsumer, against the uncertainties which regularly surround meat products. Lowerinternational resonance have constantly arisen, and it is likely that more such cases willcontinue to surface in the future, requiring a higher degree of control on food quality foranimal and human consumption. Substitution of animal products by foods of plant origin isnot totally exempt from these dangers, as the use of pesticides or the appearance ofcarcinogens such as nitrosamines have already been reported in the past.

SCP products for animal and human nutrition are safer in this respect, since the componentsfrom which they are produced are easily controlled, and their genetic background is wellknown. They represent a line of non-conventional substitutes which will continue to have amarket for these reasons.

Our view is that there is a market for products of microbial origin, aimed at animal anddirect human consumption as substitutes for meat or even fish, given the increasing depletionof fish stocks. Aside from this view, the problem of increasingworld population and limited food production, may not demand SCP production at this time,but remains as a latent issue. The continued research on the production of microorganisms foranimal and human consumption will undoubtedly find application in the future. The potential of these techniques toimprove the characteristics of foods is considerable and holds a positive prospect [96].