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Production of microbial protein foods on edible substrates, food by-products, and ligno-cellulosic wastes


Preface
Introduction
Contributions to the solution of nutritional problems
Development of protein-rich vegetarian meat substitutes in the western world
References
Discussion summary


K.H. Steinkraus

Department of Microbiology, Cornell University, New York State Agricultural Experiment Station, Geneva, New York, USA


Preface


Dr. Noel Vietmeyer of the Board on Science and Technology for International Development, National Academy of Sciences (US), reported discussing the miracle winged bean plant with an influential Filipino family. When he showed them that the beans were already growing on a fence in the servants' quarters, they were disappointed and said, "It's only a poor man's crop." Dr. Vietmeyer commented, "Some of the Third World's best crops may be waiting in the poor man's garden, ignored by science. Merely to have survived as useful crops suggests that the plants are inherently superior. They are already suited to the poor man's small plot and to his mixed farming, his poor soil, his diet, and the way of life of his family and village" (1).

His statement recalled my experiences working with the indigenous fermented foods in Indonesia. It has only been since the Western world began to research and use Indonesian tempeh that the topic has gained enough prestige to encourage Indonesian scientists to research their own foods. An Indonesian scientist told me that, in the past, he would not have had the nerve to approach his administration with the suggestion that he work on this familiar fermented food because he would have been refused permission Only recently have the West and the developing world begun to realize the gold mine of information that is available regarding indigenous methods the developing countries themselves have evolved at the village level to feed people on minimal incomes. We have much to learn about how to utilize our bioresources to greatest advantage in feeding the world of the future. It would be a great mistake not to make maximal use of available village-level technology. However, it would also be a mistake not to recognize the part prestige plays in acceptance of foods, not only for the rich but also for the poor As soon as the Western world adopts a poor man's or a village food, that food takes on enhanced prestige and it tends to be accepted much more widely than before.


Introduction


Hunger and Poverty in the World Today

Hunger and poverty go hand-in-hand in this world where vast millions must support their families on less than US$1 per day. The poor are generally vegetarians, consuming on the average a pound or pound and a half of cereal grains per person per day. They need nutritious, low-cost meat substitutes. As the population reaches 8,000 million in the 21st century, much of the world may be compelled to become vegetarians. There are already an estimated 10 million vegetarians, mainly young adults, in the United States. If vegetarian foods can be formulated with the flavour, texture, and nutritive value of meat, they will likely become acceptable as staples in the diet.

Protein-Calorie Malnutrition

Protein-calorie malnutrition is a serious problem in the developing world today. Insufficient protein and calories not only stunt physical growth but also retard brain growth and mental development. In addition, malnutrition also results in low resistance to infectious disease. This leads to frequent, disabling sickness and partly accounts for the high death rates and low productivity in the developing world (2,3).

Vitamin Deficiencies

Other forms of malnutrition are also widespread in the developing world, among them vitamin A deficiency leading to xerophthalmia and tragic blindness in children, thiamine deficiency resulting in beri-beri, including the rapidly fatal infantile beri-beri; niacin deficiency causing pellagra in some areas; vitamin D deficiency resulting in rickets; iron deficiency anemia; and iodine deficiency resulting in goiter. Vitamin C and riboflavin deficiencies are also found in the developing world.

No country can hope to sustain rapid economic development unless it is accompanied by, and preferably preceded by, adequate nutrition for its people. Malnourished people, perhaps with their potential mental development impaired, and highly susceptible to infectious disease, generally lack the vigour and high level of intelligence a nation needs to develop quickly. Unfortunately, the problem is further compounded by the lack of universal education in the developing world.


Contributions to the solution of nutritional problems


The Green Revolution

What can be done to alleviate the nutritional problems that face the developing world? The Green Revolution has resulted in a vast increase in worldwide productivity of rice and wheat. It has enabled mankind to continue to feed a burgeoning population up to now, but it has not relieved the plight of the millions of hungry and malnourished in the developing world. The basic problem remains essentially one of economics. The food is generally available if the people have the money to buy it, and farmers the world over will produce more food if they can sell it for a profit. However, we have no way at present to improve the economic status of millions of malnourished people, unless the world decides to use the US$350,000 million spent each year on armaments (US$100,000 million of which is spent by Third World countries) to improve the economic and nutritional status of the poor (4).

Thus, we must look for alternate ways of increasing the food supply or modifying the distribution of cereals and legumes between animals and man.

Increased Utilization of Cereals for Feeding Humans

For example, on a worldwide basis, about 400 pounds of cereal grains are available per person per year (5). In the developing world, cereal grains are generally consumed by humans. In the United States, about 2,000 pounds of cereal grains are available per person per year. Of this, about 200 pounds are consumed directly in foods such as bread, cereals, etc. The rest is used for animal feeds and alcoholic beverage production. If Americans alone became vegetarians, releasing the grain now fed to animals, we could feed approximately another 800 million people a basic cereal diet.


Development of protein-rich vegetarian meat substitutes in the western world


Spun Soy Protein Analogues

Americans and other Westerners are not likely to become vegetarian at this point, but there have been some interesting trends in the West that will eventually favour our becoming more vegetarian. This has included the development of meat analogues, principally from soybean (6).

Meat analogue is an industrial term for meat substitutes or synthetic meats made principally from plant proteins. The basic technique is to extract soybean protein and concentrate it to above 90 per cent purity. The protein is then extruded through platinum dies and chemical baths to form very fine filaments similar to hair, which are then combined to form a fibrous meat-like texture. Meat flavours and fats are added. Synthetic bacon bits ("BaCOS," General Mills, Inc., Minneapolis, Minnesota) have been on the market for some time. Synthetic hamburger bits used widely in chili and other soup-like dishes are also on the market, and synthetic roast beef, ham, chicken, etc., have been developed.

Extruded Soy Nuggets

Even the large meat companies have been developing meat analogues. Swift and Co. (Chicago, Illinois) has evolved a process whereby soybean, usually in the form of grits or soy protein concentrate, is tempered with water, mixed with desirable flavours, and processed through a machine (Wenger Extruder, for example) in which the thick dough is exposed to high pressure and temperature. As the material emerges from the extruder, it develops a puffed structure, or emerges as a chewy, meat-like nugget. There is little question that these meat analogues will be an important addition to future diets.

All of this has taken place in the West, where meat consumption is a large and very important part of the diet. Wider use of lower-cost meat analogues may reduce the need for real meats. It is also a way of directly consuming legumes in a form acceptable to meat-eating consumers.

Miller, Rank, Hovis, MacDougall Mould-Mycelium-Based Meat Analogues

The Miller, Rank, Hovis, MacDougall Research Group in England developed an alternative method of producing meat analogues. In their process, they grow an edible mould (Fusarium sp.) on low-cost starchy substrates, adding inorganic nitrogen (for synthesis of protein) and minerals to produce a type of single-cell protein (SCP). The mould mycelium, which provides the fibrous meat-like texture, is grown in tanks, recovered by filtration, and meat flavours and fats are added (7,8). The process is particularly adapted to production of synthetic chicken breast meat. It has been licensed by a large US company, and it is likely that the mould-mycelium meat analogues will eventually appear on the American market. They are already being market-tested in Europe.

These mould-mycelium-based meat analogues are produced by highly sophisticated technology. They are entirely beyond the economic means of the poor in the developing world at present. This is true also of all canned, frozen, and most dehydrated foods that are so important in the developed world. Thus, we must look elsewhere if we hope to contribute to improved nutrition among the poor.

Significantly related to future food and feed production is microbial farming, or SCP production.

SCP Production on Hydrocarbons

SCP production on inedible substrates such as hydrocarbons is one of the great developments in modern applied microbiology (9). Single-cell protein consists of cells of bacteria, yeasts, mould, or algae containing, respectively, up to 80 per cent, 50 per cent, 40 per cent, 40 per cent protein on a dry weight basis. SCP production requires no arable land; it can be produced in the desert. While grasses such as elephant grass and alfalfa double their cell mass in two to three weeks, bacteria and yeasts double their cell mass within two to four hours. Thus, 1,000 kg of yeast can produce 12,000 kg of new cells containing 6,000 kg of protein in a 24-hour period. The selected micro-organisms use hydrocarbons as a source of energy for growth, and inorganic nitrogen for synthesis of protein. Their remaining nutrient requirements are minerals and a sufficient supply of oxygen (10).

This "microbial farming" was so promising that it was estimated that by the 1980s, 3 per cent of the total protein produced in the world would be in the form of SCP (11). Initially, it was assumed that hydrocarbon-grown SCP would be used primarily as animal feed, thus releasing vast quantities of cereal grains and legumes, for example soybean, for use in feeding humans. Unfortunately, the cost of petroleum unexpectedly rose so high that production of SCP on hydrocarbons can no longer compete with the cost of producing soybeans or fishmeal.

SCP Production on Ligno-Cellulose

Because of the limited supply of petroleum for energy and its consequent cost, it is unlikely that it can serve as a substrate for economical SCP production in the future. However, production of SCP on ligno-cellulose, the world's largest reserve supply of renewable carbohydrate, could become an efficient alternative.

Ruminant Production of Protein

Cellulose cannot be digested by man, but, as a major component of fibre, it does play a role in the motility of the gastro-intestinal tract. At present, the major practical converters of cellulose to useful products such as milk and meat are the ruminants - sheep, goats, and cattle. They have micro-organisms in their rumens that can hydrolyze cellulose to glucose which, in turn, is used by the microorganisms for energy to synthesize proteins from inorganic nitrogen that can be supplied in forms such as urea. Minerals supply the other growth requirements for these micro-organisms. The animal subsequently digests the microorganisms and synthesizes milk and meat proteins, which serve as major foods in the Western world. Thus, the ruminants themselves are SCP fermenters.

Hydrolysis of cellulose outside the ruminant is, at present, too slow to be a practical method of producing SCP.

However, many laboratories are working on the problem and it is likely that cellulose hydrolysis may become rapid enough to permit cellulose to be utilized as a major energy source for SCP production in the future.

Processes have already been developed to raise the protein content of straw to as high as 30 per cent by growing a cellulolytic mould on it. This improves the straw as an animal feed (12)

Mushroom Production on Ligno-Cellulose

It is possible, also, to use cellulose or ligno-cellulosic wastes such as waste paper, cotton waste, straw, wheat, or rice bran and go directly to a food. This idea has already been developed to a high degree in Asia in the production of mushrooms such as Volvariella volvacea, the padi mushroom, and Pleurotus ostreatus, the oyster mushroom, on cellulosic and ligno-cellulosic wastes (13-15). Mushroom contain 2 to 5 per cent protein on a fresh weight basis, but from 30 to 47 per cent on a dry weight basis (16)

As much as 1.25 kg of fresh mushrooms can be produced on 1 kg of straw. In Hong Kong there is an estimated 30,000 tons of cotton waste per year. This could serve as a substrate for producing approximately an equal weight of fresh mushrooms.

The padi mushroom is grown by many farmers in Asia, using rice straw as a substrate. Thus, the Asians have demonstrated to the world a practical way to transform ligno-cellulosic wastes directly into highly acceptable food for man. They are literally growing a type of microbial protein (SCP) directly on cellulosic waste as a nutritious, delicious food.

The padi and oyster mushrooms can be grown under rather simple conditions. Paper or cotton substrates are shredded. Straw can be trimmed, coarse ground, or used directly. Five per cent wheat or rice bran and 5 per cent CaCO3 are added, along with sufficient water to raise the moisture content to about 60 per cent. This requires that approximately 1,500 ml of water be added per kg of ligno-cellulosic waste. The substrate should then be steamed for 30 minutes.

Alternatively, the substrate can be composted in heaps where microbial activity results when the temperature rises to about 55C. The substrate is then cooled and inoculated with the mushroom spawn. The spawn is the desired mushroom species grown on soaked, sterilized wheat, corn, or rice straw, Approximately 160 9. of spawn are added to each kg of starting (dry weight) substrate. Within a few weeks, under tropical temperatures and humidities, several flushes of fresh mushrooms are produced (15).

The developing countries in Asia are already expanding their own use of mushrooms in the diet. Taiwan is producing canned mushrooms for export. Last year Americans consumed 163,000 metric tons of mushrooms, 22 per cent of which were imported (17)

I have discussed the production of SCP on inedible substrates such as hydrocarbons and cellulose and the production of mushrooms on ligno-cellulosic wastes. What remains to be discussed is the growth of microbial protein on edible substrates and the conversion of byproducts, such as oilseed press-cakes, to food by means of fermentation.

Again, it was Asia that taught the world how to convert vegetable protein to meat-like flavours in the form of soy sauce (shoyu) and Japanese soybean paste (miso) (18, 19).

Production of Indonesian Tempeh

Asians, particularly the Indonesians, have introduced meat-like textures into vegetable substrates. A prime example is Indonesian tempeh in which soybeans are soaked, dehulled, briefly cooked, cooled, inoculated with the mould Rhizopus oligosporus, wrapped in wilted banana or other large leaves, and fermented from 36 to 48 hours. During this time the white mould-mycelium knits the soybean cotyledons into a tight cake that can be sliced thin and deep-fat fried or cut into chunks and used in soups (20 - 22). Tempeh is a major meat substitute in Indonesia, and it is produced daily by small factories in the villages.

Containing nearly 47 per cent protein, it is very nutritious and, in fact, kept thousands of Westerners alive in Japanese prisoner-of-war camps during World War I I. The mould not only introduces texture, but it also solubilizes the proteins and lipids, making them more digestible. It releases a peppery flavour that adds to the nutty flavour of the soybean substrate. The mould doubles the riboflavin content, increases the niacin level by almost seven times, decreases pantothenate slightly, and, unfortunately, decreases thiamine content, but surprisingly vitamin B12 is found in nutritionally significant amounts (23).

One of the problems of vegetarian diets is that vegetable foods generally do not contain significant vitamin B12. It was found that a bacterium sometimes present in the mould is responsible for the vitamin B12 in tempeh (24). If the fermentation is carried out with pure mould, the tempeh does not contain B12. If the bacterium is present, the tempeh will contain as much as 150 mg B12 per g. Thus, this single food provides both protein and vitamin B12 for vegetarians.

There are at least five vegetarian communes in the United States today (for example, The Farm, Summertown, Tennessee) where tempeh has been adopted as the major protein source, replacing meat in the diet. In California, Nebraska, and Canada (Toronto), there are at least six small factories producing tempeh commercially. The acceptance of this Indonesian food technology in the United States and Canada suggests that the technology could also be extended to developing countries, thus improving the diversity and nutritive value of the diets of the poor.

It has already been demonstrated that the tempeh process can be used to introduce texture into other substrates made, not only from soybeans, but from wheat and other cereals as well (25). A bacterium has also been used to raise the content of vitamin B12 in Indian idli, which is made by fermenting a batter of ground soaked rice and black gram dahl with Leuconostoc mesenteroides (26).

There is a similarity between the Miller, Rank, Hovis, MacDougall meat analogue process discussed above and tempeh production In both cases, the texture is derived from mould mycelium, but the former process is sophisticated and relatively costly, while the latter is low-cost technology.

Fast-Cooking Foods

Fast-cooking foods are appreciated world-wide because fuel is costly. Tempeh fermentation reduces the cooking time for soybeans from six hours of boiling to ten minutes, or four to five minutes of deep-fat frying at 190 C.

Increasing the Protein Content of High-Starch Substrates SCP can easily be produced by growing suitable organisms on a starch substrate to which minerals and inorganic nitrogen are added. This is the basic process used by Miller, Rank, Hovis, and MacDougall to produce mould-mycelium for their meat analogues.

Indonesians successfully grow edible microbes on starch substrates to produce tape ketan and tape ketella, grown on rice and cassava, respectively. The major fermenting organisms are Amylomyces rouxii, a mould, and at least one yeast, such as Endomycopsis burtonii. They grow compatibly in the substrate and not only increase the protein content but triple the thiamine level and synthesize lysine, the first limiting amino acid in the starchy substrate. The acids, alcohols, and esters produced during fermentation add a flavour highly acceptable to the consumer (27). The protein content of tape ketan may be as high as 16 per cent; in tape ketella it is from 4 to 8 per cent. Unfermented cassava contains only 1 or 2 per cent protein, not an adequate amount to meet human protein requirements, yet millions of the world's very poor use cassava as a major staple food. In the form of tape ketella, the protein quantity and quality of cassava are both improved. This process could usefully be expanded and extended to other countries.

Utilization of Food Processing and Agricultural Wastes to Produce High-Quality Foods

Indonesian ontjom and bongkrek

The Indonesians have also developed methods to convert food processing by-products, such as peanut and coconut press-cakes, which the Western world has traditionally fed to animals, to high-quality foods called ontjom and bongkrek. They have done this by using the basic tempeh process. The press-cakes are hydrated, coarse ground, steamed, cooled, and inoculated with either the tempeh mould or Neurospora intermedia. The mould grows over the particles, knitting them into tight cakes that can be sliced or cut into chunks and used in soups (28). These products are low-cost, protein-rich meat analogues. The basic changes are similar to those that occur during tempeh fermentation. In addition, it has been found that the content of aflatoxin, always present in peanut press-cake, is reduced (29). The strains of Neurospora intermedia also contain cellulases that reduce the natural fibre content of the peanut or coconut press-cake.

These indigenous fermented food processes offer a unique opportunity for increasing the quantity and quality of protein in areas of the world where the staple food is largely comprised of starch. They not only contribute to Western food science, but are also suitable, at the village level, for low-cost production of foods with acceptable flavours, textures, and nutritive values.


References


1. N. Vietmeyer, 'Poor People's Crops," Agenda 1 (8): 12, U.S. Agency for International Development, Washington, D.C. (1978).

2. A.M. Raimbault, E, Berthet, M.T. Villod, and H. Dupin, "Food-Nutrition-Health and Development" in Children in the Tropics, edited by the International Children's Centre, Paris, pp. 2 - 58, 1977.

3. D.M. Hegsted, "Protein-Calorie Malnutrition," Amer. Sci.66: 61 (1978).

4. R.R. Jones, "1,000 Million Dollars Every Day," Industrial Research/Development, p.9, June 1978.

5. L.R. Brown, and E.P. Eckholm, "The Changing Face of Global Food Scarcity," Social Ed. 38: 640 (1974).

6. A.K. Smith and S.J. Circle, "Protein Products as Food Ingredients," in A.K. Smith and S.J. Circle (eds.) Soybeans: Chemistry and Technology. I. Proteins, p. 365, The AVI Publishing Co., Westport, Connecticut, 1972

7. A. Spicer "Synthetic Proteins for Human and Animal Consumption," Vet. Record 89: 482 (1971)

8. A. Spicer, "Protein Production by Micro-Fungi," Trop. Sci. 8: 239 11971)

9. C.A. Shacklady, "Single-Cell Proteins from Hydrocarbons," Outlook on Agric. 6: 102 (1970).

10. E,S, Lipinsky and J.H. Litchfield, "Algae, Bacteria, and Yeasts as Food or Feed," Critical Rev. Food Technol. 1: 581 (1970).

11. J. Wells, "Analysis of Potential Markets for Single Cell Proteins," paper presented at the Symposium on Single-Cell Protein, American Chemical Society Annual Meeting, Philadelphia, 9 April 1975.

12. I. el Rawi and K.H. Steinkraus, Unpublished Data, 1976.

13. S-T. Chang, The Chinese Mushroom (Volvariella volvacea) - Morphology, Cytology, Genetics. Nutrition and Cultivation, The Chinese University of Hong Kong Press, 1972.

14. S-T. Chang, "Cultivation of the Straw Mushroom (Volvariella volvacea) ," Unesco/UNEP/ICRO/CSCHK/CUMK Regional Training Course on Cultivation of Edible Fungi (Mushrooms) Laboratory Manual, The Chinese University of Hong Kong Press, 1977.

15. G. Eger, G. Eden, and E. Wissig, "Pleurotus ostreatus - Breeding Potential of a New Cultivated Mushroom," Theoret. Appl. Genet. 47: 155 (1976).

16. R.H. Kurtzman, "Mushrooms as a Source of Food Protein," in M. Friedman (ed.), Nutrition and Clinical Nutrition. I. Protein Nutritional Quality of Foods and Feeds, Part 2, pp. 305 - 318, Marcel Dekker, Inc., New York, 1975.

17. W.A. Hayes, "Edible Mushrooms," in L.R. Beuchat (ed.), Food and Beverage Mycology, pp. 301 333, The AVI Publishing Co., Westport, Connecticut, 1978.

18. T. Yokotsuka, "Aroma and Flavor of Japanese Soy Sauce," in Advances in Food Research, Vol. 20, pp. 75 - 134, Academic Press, New York, 1960.

19. K. Shibasaki and C.W. Hesseltine, "Miso Fermentation,'' Economic Botany 16: 180 (1962).

20. A.G. Van Veen and G. Schaefer, "The Influence of the Tempeh Fungus on the Soya Bean," Doc. Neer. Indones. Morbis Trop. 2: 270 (1950).

21. K.H. Steinkraus, Yap Bwee Hwa, J.P. Van Buren, M.I. Provvidenti, and D.B. Hand, "Studies on Tempeh - An Indonesian Fermented Soybean Food," Food Res. 25: 777 (1960).

22. C.W. Hesseltine, M. Smith, D. Bradle, and K.S. Djien, "Investigations of tempeh an Indonesian Soybean Food," Deve. Ind. Microbiol. 4: 275 11963).

23. K.H. Steinkraus, D.B. Hand, J.P. Van Buren, and L.R. Hackler, "Pilot Plant Studies on Tempeh," in Proceedings of a Conference on Soybean Products for Protein in Human Foods, p. 75, Northern Utilization and Research and Development Division, U.S. Dept. of Agric., Peoria, Illinois, 13-15 Sept. 1961.

24. I.T.H. Liem, K.H. Steinkraus, and T.C. Cronk, "Production of Vitamin B-12 in Tempeh - A Fermented Soybean Food," Appl. Environ. Microbiol. 34: 777 (1977).

25. H.L. Wang and C.W. Hesseltine, "Wheat Tempeh" Cereal Chem. 43: 563 (1966).

26. L.J. Parekh and K.H. Steinkraus, Unpublished Data, 1977.

27. T.C. Cronk, K.H. Steinkraus, L.R. Hackler, and L.R. Mattick, "Indonesian Tape Ketan Fermentation," Appl. Environ. Microbiol. 33: 1067 (1977).

28. A.G. van Veen and K.H. Steinkraus, "Nutritive Value and Wholesomeness of Fermented Foods," Agric. Food Chem. 18: 576 (1970) .

29. A.G. van Veen, D.C.W. Graham, and K.H. Steinkraus, "Fermented Peanut Press Cake," Cereal Sci. Today 13: 96 (1968).


Discussion summary


With regard to the fermented foods produced in Indonesia, it was pointed out that, while the fungi used may be fairly rich in lysine, they are likely to be poor in methionine. Nevertheless, as cereal diets tend to be lysine deficient, an improvement in nutritional value may be produced by fungal lysine.

Successful fungal fermentation of cassava requires the addition of inorganic nitrogen. Because Neurospora, the organism used to ferment a cassava/groundnut mixture, provides virtually no protein, the protein value of the mixture depends entirely on the amount of groundnut present.

The question of mycotoxin contamination of Indonesian fermented foods was raised, Although this problem had not been reported before, recent evidence suggests that it may have occurred. Contamination can happen if a pseudomonad, which produces highly toxic metabolites, intrudes during fermentation of tempeh. This dangerous possibility should be prevented by standardized procedures for traditional fermentation methods, using high" quality inocula from recognized, competent centres.


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