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Chapter 2 Food and Animal Feec! At least 25 percent of the world's population, approximately one billion people, suffer from hunger and malnutrition. It is vital, therefore, that they produce more food and improve their standard of nutrition. Food losses should be decreased through low-cost methods of food preservation, since standard modern methods of food processing such as canning, freezing, or dehydration with artificial heat make the preserved food too expensive for families on an income of US$200-$300 per year.* Most cultures have traditionally used some form of microbial process to preserve foods that would otherwise spoil. Some of these processes have also contributed to increasing the nutritive value of the final product through the increased production of essential nutrients or the synthesis of nutrients not present in the original food. Cheeses are perhaps the most widespread and best known of these foods. But there are many others, some of which are largely confined to certain parts of the world and almost totally unknown elsewhere. This section describes a number of these processes. While it is recognized that food habits and customs are among the most difficult to change, there have already been dramatic changes in some food habits of developing countries. The spread of wheat flour bread throughout much of the lowland tropics is an example of one such change, and it involves a microbial process, yeast fermentation. The purpose of this section is to bring to the attention of the reader a number of lesser-known processes that preserve or enhance the nutritive value of foods and beverages and that may merit introduction to areas where they are unknown or invite more widespread use and improvement where they are already used. None of the methods (except for large-scale single~cell protein production of animal feeds) requires large investment in capital equipment or plants. These processes also illustrate the potential contribution of microbiology toward diversifying the use of limited resources as well as the need to develop *National Academy of Sciences. 1978. Postharvest Food Losses in Developing Coun- tries. Washington, D.C. 18
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FOOD AND ANIMAL FEED 19 trained microbiologists for a variety of research and development opportu- nities. It must be stressed that food processing is always potentially hazard- ous, and new methods or products should only be marketed after careful investigation by qualified personnel. Food Preservation Three relatively low-cost methods of food preservation frequently carried out in combination are: 1) salting; 2) sun or smoke drying; and 3) acid fer- mentation. Salt is one of the best low-cost chemicals for preserving fresh foods rang- ing from vegetables to meats and fish. Added in concentrations of approxi- mately 2-2.5 percent by weight to fresh vegetables, salt promotes an an- aerobic lactic acid fermentation. The fermentation favors development of a microbial flora that converts, for example, cucumbers to pickles and cabbage to sauerkraut (see flow sheets). Pickling is the traditional method of providing a winter supply of vegetables in much of Asia and Europe. There is less need for extended storage in tropical areas where vegetables can be grown on a year-round basis, but these methods may be valuable for taking advantage of periodic surplus production and providing variety to the diet. Korean kimchi, a staple in that country, consists of mixed vegetables fermented by lactic acid bacteria. An inoculum is generally not required, as the bacteria are ubiquitous. The salt concentration, coupled with anaerobic conditions at ambient temperature, controls the development and sequence of the lactic-acid-producing organisms. The combination of salt and acid leads to products with excellent keeping quality. Fish Sauce and Paste Fish and shrimp are excellent protein foods, but they are highly perish- able. Also, many fish caught in nets are too small to be sold commercially or are species not generally consumed directly as fresh fish. Thus, low-cost methods of preserving small and surplus fresh fish and shrimp are of great importance in food distribution and consumption. Larger surplus fresh fish are often salted, sun dried, and consumed as dried fish or as fish powder. Large quantities of small fish are fermented to produce fish or shrimp sauces and pastes in Southeast Asia. The basic procedure is to mix the freshly netted small and trash fish with sea salt, in proportions that ensure that the extracted fish juices contain about 20 percent salt in the final product. Such high salt concentrations inhibit putrefaction. No inoculum is required: the microorganisms in the gut of the fish and enzymes in the fish tissues control hydrolysis (solubilization) of the fish proteins.
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20 MICROBIAL PROCESSES FLOW SHEET: Cucumber (Pickle) Fermentation Fresh Cucumbers Wash Cover with 5~ salt brine (add dill or other flavoring) Cover fermentation container to avoid evaporation and to exclude air Ferment for 1 or 2 weeks (total acid 0.6-1.09to as lactic acid, pH 3.6-3.4) FLOW SHEET: Cabbage (Sauerkraut) Fermentation Fresh Cabbage* Trim and clean Remove core Shred (to 2-5 mm width) Add salt (about 2.25~o by weight) and distribute evenly Fill into fermentation containers Salt and shredded cabbage can be mixed as fermentation container is filled. Cover top of fermentation container and use a water seal to prevent entrance of air. The seal must allow escape of CO2 gas produced during fermentation. Ferment to acidity desired For optimum keeping quality the acidity should be high (below pH 4), but the sauerkraut can be consumed earlier if desired. The fish sauces (nuloc mam in Vietnam, patis in Indonesia and the Philip- pines, and nampla in Thailand) are salty condiments adding some essential amino acids and vitamins (mainly B-complex) to the diet. Similar processes in which less protein hydrolysis occurs lead to fish or shrimp pastes. The pastes may be mixed with cereals; ragi and millet are reported to be used for this purpose. These products are of limited nutritional value (particularly for infants)—despite their protein content—because of their high salt content. *Or other green leafy vegetables.
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FOOD AND ANIMAL FEED Acid Milks, Yogurt, and Cereal 21 Fresh unpasteurized milk, if allowed to stand at ordinary temperature, sours naturally because the streptococci and lactobacilli present convert milk lactose to lactic acid. This results in a natural acid preservation of the nutri- ents. The acid produced results In stable products resistant to putrefaction and the development of food spoilage or some disease-producing organisms. It is, however, susceptible to some fungi and especially to Geotrichum. Modern yogurt processing involves inoculating mink with Streptococcus thermophilus and Lactobac~llus bulgaricus and incubating it at 45°C. For the production of Russian kefir, mild inoculated with kefir grains (consisting of a lactobacillus and a yeast growing symbiotically) yield an acidified, carbonated sour milk with a low alcohol content. Incubation is at room temperature. Sour milks or yogurts boiled with ground whole wheat or bulgar wheat and then sun dried yield extremely nutritious stable foods, which can be stored for years without deterioration. This is the basic process for Egyptian kishk and Greek trahana. Other ingredients such as spices, pepper, tomato, onion, or garlic, and other vegetables may also be incorporated in the prod- ucts, which are either consumed directly or used as a major protein ingredient in soups. Indian Idli (Dosai} Indian idli is a nutritious, protein-rich acidic steamed bread popular in South India. Its acidity makes it quite resistant to food spoilage and certain disease-producing organisms. In preparing idli, polished rice and dehulled black gram (Phaseolus mango, mung bean, a legume similar to split pea) are soaked separately during the day. The proportions can be any combination from 1 to 3 parts rice to 1 part black gram. Since black gram is more expensive than rice, most poorer people use higher proportions of polished rice. In the evening, the soaked rice and black gram are ground separately with a mortar and pestle. For idli, the rice is coarsely ground and the black gram finely ground; for dosai, both are finely ground. Water, in a proportion twice the weight of rice and black gram, and 1 percent salt (weight to volume) are also added. The batter is incubated overnight. During this time the batter becomes acidic (about pH 4.5) and it is leavened by carbon dioxide produced by the principal fermenting microorganism Leuconostoc mesenteroides. Streptococcus faecalis is also present and contributes to the acid content. The leavened, acidified batter is steamed in small cups to produce the idli cakes or fried like a pancake to yield dosai (Figure 2.1~. Both are tasty, nutritious foods. This process of producing wholesome protein-rich food can be adapted to other ingredients. Dehulled soybean can be substituted for black gram. Other starchy cereals could be substituted for the rice.
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22 African A idle Porridges MICROBIAL PROCESSES Naturally fermented acidic porridges are staple foods in many parts of Africa; for example, West African gari is prepared from an acid-fermented cassava porridge. During the fermentation, at an optimum temperature of 35°C, any cyanide-containing sugars present are hydrolyzed, removing the cyanide. The cassava becomes acidic, and the characteristic pleasing flavor of gari develops. The fermentation is usually complete in 3-4 days. The principal microorganisms include Corynebacterium manihot, which hydrolyzes the starch, producing lactic and formic acids—a process evolving heat. As the product becomes more acidic (about pH 4.25), a yeast-like fungus Geotrichum candidum (also found in camembert cheese) develops, oxidizing the acid and producing the gari flavor. Typically, the liquid Is pressed from the ferment and the starchy residue is either used directly as fufu or dried in a basket over a fire with continual turning until it is converted to dry gelatinized granules, which can be stored for later consumption. Related acidic porridges are made by fermentation of millet and maize (ogi), and mahewu (maize and wheat) and sorghum. The acidity protects the products from food spoilage organisms, thus providing a wholesome food that keeps well in a relatively contaminated environment. LImitations The basic limitation to the introduction of these low-cost preservation technologies to new locations or countries is cultural preference and taste. This is particularly so in the case of fish, where, apart from the sauce and paste processes in Southeast Asia, most people will only eat "conventional" varieties. Fish, particularly in tropical conditions, spoils rapidly, and eating spoiled fish can have dangerous consequences. Canning and mechanical refrig- eration are the only widely accepted methods of fish preservation added since ancient times, and both methods are too expensive for use by the poorest sections of developing countries. Where the products of fermentation technologies are acceptable, however, there may be opportunities for increasing conservation of food resources through more widespread use of such methods, particularly with better qual- ity control. There may be an important role here for the microbiologist in improving the techniques in ways appropriate to the local situation. High salt content has excellent preservative action and is valuable as a condiment, but it restricts the amount of food that can be consumed. Lower salt contents, along with sufficient acid (pH 4.5), offer a satisfactory preserva- tive action while permitting more consumption, thus contributing to better nutrition.
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FOOD AND ANIMAL FEED 23 FIGURE2.1 Steamed Indian idli cakes or same dough fried as a pancake (dosai). (Photograph courtesy of K. H. Steinkraus) Generally, production of acidic cereal porridges like ogi and mahewu pro- ceeds better at temperatures of about 50°C, which are favorable for rapid development of Lactobacillus delbrueckii. Fermentation time is shorter and other undesirable microorganisms have less chance of developing. Research Needs Research on the improvement and popularizing of fermented foods should be centered on: · Studying acidic fermentations for their realiability in areas where the are not traditionally used; and · Undertaking socioeconomic research to determine if consumers will accept the new products and, if not, how to encourage them to do so.
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24 MICROBIAL PROCESSES Improving Nutritional Value Beers and wines make a valuable contribution to the proper nutrition of people subsisting on low incomes in the developing world. The fermentation processes involved raise the vitamin, protein, and, in some cases, the essential amino acid content of starchy substrates such as cassava, rice, maize, millet, sorghum, and other cereal grains. Native maize or sorghum (kaffir) beers, indigenous rice wines, and alco- holic rice pastes bear little physical resemblance to Western beers and wines. The indigenous wines and beers are generally cloudy, opalescent, effervescent beverages, because of their content of microorganisms and substrate residues. All alcoholic beverages and foods provide a similar euphoria, depending on their alcoholic content, but Western beers and wines often provide the con- sumer with an excess of calories. Most of the indigenous products provide essential nutrition to consumers in the form of vitamins, protein, amino acids, and calories. In addition to the food value in the basic ingredients, the microorganisms synthesize from these ingredients essential amino acids, protein, and vitamins that are consumed with the product. These organisms may also utilize a portion of the starch, reducing total solids and increasing the percentage of protein in the product, converting a low-protein food such as cassava to an acceptable staple in the diet. A few of these fermentation processes will be described, and similar or related commodities can be produced wherever they might add valuable nutri- tion to the diet. Two basic processes are used. The first involves germination (malting) of the grain, which produces enzymes (amylases) that transform a portion of the starch to sugars (glucose and maltose). The sugars are then fermented by yeasts such as Saccharomyces cerevisiae to ethyl alcohol. The yeast, however, also grows and synthesizes amino acids, proteins, and vitamins from the grain constituents. To retain all the nutrients in the beer or wine, it is essential that the products not be clarified or faltered. The second process involves a starch-digesting mold (Amylomyces rouxii) and a yeast (Endomycopsis burtonii). The combination results in hydrolysis of the starch to sugars, which are then fermented to alcohol. An example of the first process is kaffir (sorghum) beer. Kaffir beer is an alcoholic beverage with a pleasantly sour taste and the consistency of a thin gruel. It is the traditional beverage of the Bantu people of South Africa, and the alcohol content may vary from 1 to 8 percent. Kaffir beer is generally made from kaffircorn (Sorghum caffroru m), mat, and unmelted kaffircom meal. Maize or millet (EIeusine coracana) may be substituted for part or all of the kaffircorn depending on the relative cost. Even cassava and plantains may be used, though with these the procedure would not be the same as with grain.
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FOOD AND ANIMAL FEED 25 The kaffircorn grain is steeped for 6-36 hours. It is then drained and placed in layers and germinated by periodic moistening for 4-6 days. Germi- nation continues until the bud is about 2.5 cm long and the material is then sun dried. The essential steps are: mashing, souring, boiling, conversion, straining, and alcoholic fermentation. Mashing is carried out in hot (50°C) water. Proportions of malted to unmelted grains vary, but 1: 4 is satisfactory. Approximately 4 liters of water are added for about every 2 kg of grain. Souring begins immediately due to the presence of lactobacilli (~Lactobacillus delbrueckiiJ, at a tem- perature of 50°C. Souring is complete in 6-15 hours. Water is added and the mixture is boiled. It is then cooled (to 40°-60°C) and more malt is added. Conversion (starch hydrolysis) proceeds for 2 hours and then the mash is cooled (25°-30°C). Yeasts present in the malt are responsible for the natural fermentation, although Saccharomyces cerevisiae isolated from kaffir beer can be inoculated. Kaffir beer is ready for consumption in 4-8 hours, while it is still actively fermenting. The ethanol content is generally from 2 to 4 percent. The beer also contains from 0.3 to 0.6 percent lactic acid and from 4 to 10 percent solids. Production of acetic acid by Acetobacter species is the principal cause of spoilage. Daily consumption of 3 liters of kaffir beer, made from approximately 0.5 kg of grain, is not unusual for a workingman. The improvement in the vitamin content of a diet that includes beer compared with a diet in which the knifer corn is consumed directly is shown in Table 2.1. TABLE 2.1 Comparison of Diet with and without Maize Beer Amount of Food Eaten (g) Diet without Diet with Kaffir Beer Beer Food Item Maize, wholemeal 350 137.5 Maize, 60~o extraction 35 0 137.5 Maize beer 5 pints (2840 ml) Vegetables 130 130 Sweet potatoes 470 470 Kidney beans 30 30 Vitamin B. 0.002 0.00195 Riboflavin 0.00113 0.00232 Nicotinic acid 0.0117 0.0203 Calories 3016 2979 Source: B. S. Platt. 1964. Biological ennoblement: improvement of the nutritive value of foods and dietary regimes by biological agencies. Food Technology (Chicago) 18:665.
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26 MICROBIAL PROCESSES The caloric content of the two diets is quite similar, only 37 calories being lost in the diet containing beer. The most notable improvement is the doubl- ing of the riboflavin and the near doubling of nicotinic acid in the diet containing beer, because of synthesis of these vitamins during malting arid fermentation. Pellagra, which is relatively common in people subsisting on maize, is never noted in those consuming usual amounts of kaiser beer. An example of the second process is Indonesian tape ketan, which is closely related to indigenous rice wine. It is a sweet-sour, alcoholic paste in which a starch-digesting mold (Amylomyces rouxii) and at least one yeast (Endomycopsis burtonii) hydrolyze steamed rice starch to maltose and glu- cose and then produce ethanol and organic acids, which provide an attractive flavor and aroma. Fermentation is complete in 2-3 days at 30°C. If yeasts of the genus Hc~nsenula are present, the acids and ethanol are esterified, produc- ing highly aromatic esters. The inocula are obtainable in the markets of Indonesia as a product called ragi (in Thailand, luk-paeng). Ragi is a white dried-rice flour cake about 2.5 cm in diameter (Figure 2.2) containing a variety of molds and yeasts, FIGURE 2.2 Indonesian ragi cakes used for inoculum for tape ketan and tape ketclla. (Photograph courtesy of C. W. Hesseltinc)
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FOOD AND ANIMAL FEED 27 including those described above. Housewives prepare steam-soaked glutinous rice, inoculate it with the powdered ragi, place the inoculated rice into earth- enware jugs with added water and allow the mixture to ferment for 3-5 days. The liquid portion is then consumed and additional water is added. Fermenta- tion continues for 3-5 days' after which the liquid portion is again drunk. This is repeated until all the rice is fermented. Any residual dregs are sun dried and used as a type of ragi. Hence, there is no loss of nutrients. Detailed studies have been made of the biochemical and nutritional changes that occur during tape fermentations. Most of the starch is hydro- lyzed to sugars, which, in turn, are fermented to ethanol and organic acids. Lysine, the main limiting amino acid in rice, is selectively synthesized by the microorganisms so that it increases by 15 percent. Thiamine, which is very low in polished rice(O.04 mg/100 g), is increased threefold (to 0.12 mg/100 g) by the action of the microorganisms. Up to 8 percent ethanol is produced; this selves as calories for consumers. Also, it probably contributes to destruc- tion of disease-producing and food-spoiling organisms that might be present in the fermentation water. Through the loss of total solids resulting from utilization of the starch, the protein content of tape ketan is increased to as much as 16 percent (dry basis) compared with 7-8 percent in rice. The tape ketan process is a simple way of raising the protein quality in starch substrates and also of producing thiamine, which may be deficient in predominantly polished-rice diets. Protein enhancement is all the more important in the case of tape ketella, which is also produced in Indonesia. Tape ketella is a sweet-sour alcoholic food made from cassava tubers. The tubers are peeled, steamed, and cut into pieces about 5 x 5 cm. They are then carefully inoculated on all surfaces with powdered ragi. A mold (Amylomyces rouxii) and yeasts of the genera Endo- mycopsis or Hansenula, along with related types, overgrow the cassava, utiliz- ing a portion of the starch for energy. Cassava contains as little as 1 or 2 percent protein and by itself is clearly unable to contribute to proper human protein nutrition, even though it can provide sufficient calories. Consumption of a portion of the cassava as tape ketella, which may contain 8 percent protein, can have a beneficial effect on nutrition. Limitations In establishing these fermentations in areas of the world where they are unknown, the proper cultures should be obtained from either culture collec- tions or scientists who have done research on the products. Acceptance of new foods will be more difficult. Technical studies must be accompanied by socioeconomic studies of the potential role of these products in the particular society.
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28 Research bleeds SIlCROBlAL PROCESSES Small-scale laboratory studies are needed in applying these processes in a new environment. Production of Meat-Like Flavors Shoyu (soy sauce) and miso (soybean paste) are made by similar processes from soybeans by using koji prepared with the molds Aspergillus some and A. oryzue. The koji process for culturing microorganisms for various purposes is described below. Both products are salty and are used to add flavor to vegetables, fish, and meat. Miso exists in many colors and flavors and is a paste, whereas shoyu is a liquid from which the insoluble solids have been removed. Koji is generally made from rice, although some forms involve the use of barley or soybeans. The finished koji is added to soaked, pressure-cooked, whole soybeans. When these products are mashed together, a salt-tolerant yeast, Saccharomyces rouxii, and considerable amounts of salt (4-13 percent weight to weight) are added. The salt is added for flavor and to retard growth of toxin-forming bacteria. The mash is then placed in tanks made of concrete or wood and containing several tons of substrate. The mash in the tanks is allowed to ferment from a few days to a number of months, depending on the type of miso desired. On completion of the fermentation, miso is either ground into a uniform paste with the consistency of peanut butter or pack- aged directly. Traditionally, miso is used as a flavoring base for soup eaten at breakfast. To this base, vegetables and seafood are added. Miso imparts a meat-like flavor to the soup and is added to fish and meats before baking or broiling. Currently, it is also being incorporated into sauces for pizza and spaghetti and is used as an ingredient in some commercially prepared salad dressings. Shoyu manufacture differs from miso in that wheat that has been cleaned, roasted, and crushed is used in place of rice to make the koji. Whereas miso requires the use of whole soybeans, modern shoyu manufacture utilizes de- fatted soybean flakes, which are moistened and blended in a ratio of 55 percent soybean flakes to 45 percent crushed wheat. This mixture of wheat and soybeans is inoculated with selected strains of a mold, Aspergillus oryzue or A. some, and transformed to koji as it becomes overgrown with mold (Figure 2.3~. During molding the temperature is held below 40°C, and about 3-4 days are required for completion of the process. At this time, about an equal amount of brine is added to the koji and the mixture is placed in large tanks and inoculated with a yeast, Saccharomyces rouxii~ and a bacterium of the Lactobacillus species. Depending on the temperature, the mash is allowed
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36 MICROBIAL PROCESSES FLOW SHEET: Indonesian Tempeh Fermentation Whole, clean soybeans Soak overnight (or 24 hours) to hydrate soybeans and allow bacterial fermentation and acidification* Dehull by hand or by passing through machine to loosen hulls Remove hulls by flotation on water Boil cotyledons for 60 minutes Drain and cool and allow surface moisture to evaporate Inoculate with tempeh mold + Ferment small packets of inoculated soybeans wrapped in banana leaves or in clean shallow covered pans t Incubate at a temperature of 30 - 35 C until soybeans are completely covered with mold mycelium (generally 24 to 36 hours) Tempeh~cakes can be sold on the market or used in home by slicing thin strips and deep frying or cutting into chunks and cooking in soups Research Needs Research requirements include: · Establishing a small company or laboratory with microbiological know- how to produce a dry, pure tempeh culture inoculum in small packages for distribution at low cost. This is important for the production of tempeh in small cottage industries that do not have technically trained people. · Standardizing the inoculum, as has been done with baker's yeast, to yield a product that will produce a uniform tempeh under standard condi- tions of time and temperature. The inoculum should have good keeping prop- erties and should not require refrigeration. · Conducting research on the control of mold contamination in rural environments. · Adapting simple fermentation equipment for tempeh production at the local level in countries unfamiliar with its production. · Studying sociocultural aspects of introducing tempeh and tempeh-like products to people unfamiliar with the product or with soybeans. *In a temperate climate it may be necessary to add 0.5 percent vinegar during the cook- ing to increase the acidity.
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FOOD AND ANIMAL FEED Single-Cell Protein Production 37 Single-cell protein (SCP) refers to the cells of yeasts, bacteria, fungi, and algae grown for their protein content. Cells of these microorganisms also contain carbohydrates, fats, vitamins, and minerals. SCP products are used either for animal feed or human food. They are potentially very important sources of amino acids, proteins, vitamins, and minerals that can be prepared from otherwise inedible or low-quality waste material. Yeasts Yeasts in baked and fermented food products have a long history of hu- man consumption. Dried brewer's yeast, a by-product of the brewing indus- try, has an established use in animal feed formulations. It is also used in hu- man nutrition as a "health food" dietary supplement. In recent years SCP processes have been practiced on a commercial scale based on the growth of yeasts in deep-tank, agitated, and aerated cultures. Examples of raw materials used in these processes and yeasts that utilize them are molasses, Saccharomyces cerevisiae; e-paraffin hydrocarbons, Saccharo- mycopsis lipolytica; and cheese whey, Klayreromyces fragilis. The Symba process, developed in Sweden, utilizes starchy wastes by combining two yeasts, Saccharomycopsisfbuligera and Candida utilis. Bacteria Large-scale propagation of bacteria as a source of animal feed protein has been considered only in the last decade. A large-scale (75,000 t) facility for producing the methanol-utilizing bacterium Methylophilus methylotrophus is being constructed in the United Kingdom, and a large pilot plant for growing a stable mixed culture of methane-utilizing bacteria is being operated in The Netherlands. The conversion of cellulosic materials such as bagasse from sugar cane processing to SCP by bacteria of the genera Cellulomonas and Alcaligenes has been investigated on a laboratory and small pilot-plant scale at Louisiana State University. Plans call for commercial-scale production. (The microbio- logical utilization of cellulose is discussed in Chapter 8 of this report.) Advantages claimed for bacteria over yeasts for production of SCP include more rapid generation and a higher content of crude protein and certain essential amino acids, particularly methionine. However, bacterial cells are smaller than yeasts and may be more costly to harvest unless the cells can be flocculated to give a higher solids slurry prior to centrifugation. Further (apart from fermented milks and cheeses, which often contain as many bac- teria as 5 x 109 per g of foodstuff), bacteria as such have had only a brief
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38 MICROBIAL PROCESSES history of use as either animal feed or hum auk food. Food and drug regulatory agencies in most countries will have to be convinced of the safety of bacterial products before permitting their use. Fungi People have eaten higher fungi, particularly mushrooms, since ancient times. Recently, a different method of growing fungal mycelium, including mushroom mycelium, has been developed on a large pilot-plant or com- mercial scale in deep-tank, aerated, and agitated cultures. Typical raw mate- rials and organisms are shown in Table 2.2. Problems encountered with production of fungal mycelium as a source of SCP include slow growth rates and the consequent need to maintain sterile conditions over an extended time to prevent overgrowth by bacterial and wild yeast contaminants. This increases costs for fungal mycelium production. In recent laboratory-scale studies, Chaetomium cellulolyticum, ~ thermo-tol- erant cellulolytic fungus, has shown promise in the conversion of cellulose to SCP. Care must be taken to use strains of fungi that do not produce mycotoxins that affect domestic livestock or human beings. Algae Algae are of interest as a source of SCP because they grow well in open ponds and utilize carbon dioxide as a carbon source and sunlight as an energy source for photosynthesis. Algae of the genera Chlorella and Scenedesmus have been grown for food use in Japan. Spirulzna species have been eaten for many years by inhabitants of the northern shores of Lake Chad in Africa and by the Aztec Indians in Mexico, where they are now being grown on a pilot- plant scale in the alkaline waters of Lake Texcoco. Spirulina is a particularly TAB Ll 2.2 Raw Materials Used in Growing ~ ungi Commercially Raw Materials Fungal Species Cane and beet molasses Maize syrups, denture (Glucose), cheese whey, and canning wastes Coffee-processing wastes Maize wet-milling waste Maize and pea-canning wastes Agaricus campestris Morchella esculenta, M. hortensis, M. crassipes (Morel mushrooms) Trichoderma spp. Gliocladium spp. Trichoderma reesei
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FOOD AND ANIMAL FEED 39 attractive algal source of SCP because of its high nutritive value and because the large cell filaments make it relatively easy to harvest by fine mesh nets or filtration. Further discussions of algae are in the section on wastes in Chapter 7 and in the NAS publication Underexploited Tropical Plants with PromisingEconomic Value. Limitations SCP production is capital-intensive and, with the exception of algal pro- duction by photosynthesis, energy-intensive. Processes that must be con- ducted under sterile conditions require stainless-steel equipment that can be cleaned and sterilized. They also require provisions for sterilizing the growth medium and recovering the SCP product without introducing other microbial contaminants, particularly human pathogens. Trained personnel are needed to supervise and maintain quality control of production. At present, SCP processes for the production of animal feed are the most attractive, since conventional animal feedstuffs such as soybean meal and fish meal must be imported to many tropical and subtropical countries at prevail- ing international prices. In using SCP for animal feed, however, there is a large loss of conversion efficiency as opposed to direct human use. For human food applications, the use of microorganisms is limited to those such as Saccharomyces cerevisiae and Candida utilis that are accepted by regulatory and public health authorities as safe for human food use. Even those organ- isms, if they are to be consumed as a significant portion of the protein in the diet, must be processed further to reduce nucleic acid contents to below the levels that could lead to kidney stone formation or gout. To achieve economies of scale, an SCP production facility should have a capacity of at least 50,000 t per year unless operated as a waste-treatment facility in a food-processing plant. This implies that sufficient raw materials will be available in close proximity to the SCP plant to meet these production requirements. For raw materials, carbohydrates such as sugars or sugar-containing by- products, wastes, and starches are likely to be available in semitropical and tropical countries in the quantities required for an economically viable scale of production. In general, concentrations of utilizable carbohydrates in wastes should be sufficiently high that handling of large volumes of dilute materials is avoided. A portion of the crude molasses produced from sugar cane operations could be diverted to SCP production (yeasts), if a source of nitrogen were added to provide a source of protein and vitamins for animal feeds, particu- larly poultry rations.
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40 MICROBIAL PROCESSES Many cities in tropical and semitropical countries have breweries that pro- duce residues of reasonably uniform composition throughout the year. In addition to recovery of brewer's yeast, already widely used as animal feed, the remaining liquid waste—after hydrolysis to simple sugars—can be used as a potential carbon and energy source for SCP production. However, carbon-to- nitrogen ratios of the material may have to be adjusted to favorable ranges for yeast growth. In the case of starchy crops such as cassava, large quantities must be available at one site to provide a sufficient source of raw material for economic SCP production. Coffee-processing wastes contain soluble carbohydrates and have a him chemical oxygen demand (COD) and soluble solids content. Pilot-scale opera- tions in Guatemala have shown that growing Tr~choderma species in these wastes reduces the COD considerably and yields an SCP product of interest for use in animal feed. Microorganisms require sources of nitrogen, phosphorus, and mineral salts for growth, in addition to a carbon and energy source. The availability of ammonium salts such as ammonium sulfate or diammonium phosphate may be a problem in some countries. The same can be said for sources of phosphorus. A feed-grade source of phosphoric acid or soluble phosphates should be used because of the presence of arsenic and fluoride in crude phosphates. Other minerals are usually present in the water supply. To reduce contamination to a low level, the microorganism used should multiply (grow) rapidly at an acid level of pH 4.5 or below. Operations under these conditions will allow the use of clean, aseptic conditions without the need for sterile facilities. SCP production (except from algae) requires aera- tion to achieve suitable yields. Air should be filtered to remove contaminants; Power costs for aeration, fluid handling, and steam for cleaning, recovery, and drying the product can be significant factors in the total energy costs. Water requirements for SCP production are considerable for both process- ing and cooling. The growth of microorganisms produces heat, which must be removed to maintain the growth temperature within the preferred range of 30°-35°C. Cooling water temperatures should be at least 10°C lower than the growth temperature. Location of an SCP plant near the ocean would permit the use of seawater for cooling. Also, wastewater is produced from SCP operations and must either be disposed of or, preferably, treated and recycled in large operations. Three SCP processes appear to have potential for further development. These processes, although known, are not used in tropical and semitropical regions to the extent that they milt be. They are given in Table 2.3. All of these processes can be carried out in either a batch or continuous mode of operation. They should be operated under clean, aseptic conditions, but they do not require tight control over sterility throughout the process.
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41 TABLE 2.3 Production of SCP from Various Substrates Substrate Organisms Conditions Cane molasses Candida utilis C. tropicalis Rhodotorula gracilis R. pilimanae R. rubra Temperature: 30°-34° C pH 4.0-4.5 Coffee wastes Candida spp. Trichoderma spp. Temperature: 30°-35° C pH 2.0-4.0 Starchy materials, especially cassava Symba processes: mixed culture of Saccharo- mycopsis fibuligera and Candida utilis Temperature 3 0° - 34° C pH 4.0-5.0 Research Neects In many countries there is need to: · Conduct feasibility studies to determine where there is an appropriate mix of underutilized residue, technical competence, and need or economic opportunity to use the SCP produced; · Conduct studies on the use of yeasts, e.g., genus Rhodotorulc~, which have relatively rapid growth rates (2- to 2.5-hour generation times), tem- perature over the range 28° to 34°C and pH tolerance between 3.5 and 5.0; · Institute animal feeding studies using dried SCP as a component of poultry and swine rations; · Investigate a wider range of thermotolerant organisms, particularly yeasts, for their utility In producing SCP from coffee-processing wastes; · Evaluate nutritional and safety factors for animal feed applications of certain thermotolerant fungi including Sporotrichum thermophile and Paecilo- myces species that grow on cassava; arid · Develop thermotolerant strains of microorganisms to reduce the requirement for cooling water in semitropical and tropical regions. References and Suggested Reading Food Preservation Akinrele, I. A. 1963. Further studies on the fermen tation of cassava. Research Report No. 20. Lagos, Nigeria: Federal Institute of Industrial Research. Morcos, S. R.; Hegasi S. M.; and el-Damhougy, S. T. 1973. Fermented foods in common use in Egypt. I. The nutritive value of kishk. Journal of the Science of Food and Agriculture 24:1153-1156.
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42 MICROBIAL PROCESSES Mukerjee, S. K.; Albury, M. N.; Pederson, C. S.; van Veen, A. G.; and Steinkraus, K. H. 1965. Role of Leuconostoc mesenteroides in leavening the batter of idli, a fermented food of India. Applied Microbiology 13: 227-231. Pederson, C. S., and Albury, M. N. 1969. The Sauerkraut fermentation. New York Agricultural Experiment Station Technical Bulletin 824. Geneva, New York: New York State Agricultural Experiment Station. Schweigert, F.; Van Berge, W. E. L.; Wiechers, S. G.; and de Wit, J. P. 1960. The produc- tion of mahewu. Report No. 167. Pretoria, South Africa: Council for Science and Industrial Research. Stamer, J. R. 1975. Recent developments in the fermentation of sauerkraut. In Lactic acid bacteria in beverages and food, J. G. Carr; C. V. Cutting; and C. S. Whiting, eds., pp. 267-280. New York: Academic Press. Steinkraus, K. H.; van Veen, A. G.; and Thiebeau, D. P. 1967. Studies on idli-an Indian fermented black gram-rice food. Food Technology (Chicago) 21:916-919. van Veen, A. G. 1965. Fermented and dried seafood products in Southeast Asia. In Fish as Food, G. Borgstrom, ea., Volume 3, pp. 227-250. New York: Academic Press. ; Hackler, L. R.; Steinkraus, K. H.; and Mukerjee, S. K. 1967. Nutritive value of idli, a fermented food of India. Journal of Food Science 32:339-341. Improving Nutritional Value Cronk, T. C.; Steinkraus, K. H.; Hackler, L. R.; and Mattick, L. R. 1977. Indonesian tape ketan fermentation. Applied Environmental Microbiology 33:1067-1073. Ellis, J. J.; Rhodes, L. J.; and Hesseltine, C. W. 1976. The genus Amylomyces. Mycologia 68:13 1-143. Ko, S. D. 1972. Tape fermentation. Journal of Applied Microbiology 23:976-9 78. Novellie, L. 1968. Kaffir beer brewing: ancient art and modern industry. Wallerstein Laboratories Communications 31:17-32. Platt, B. S. 1946. Fermentation and human nutrition. Proceedings of the Nutrition Society 4: 132-140. . 1955. Some traditional alcoholic beverages and their importance in indigenous African communities. Proceedings of the Nutrition Society 14:115-124. . 1964. Biological ennoblement: improvement of the nutntive value of foods and dietary regimes by biological agencies. Food Technology (Chicago) 18:662-670. ; and Webb, R. A. FebruaTy 1948. Microbiological protein and human nutrition. Chemistry and Industry 7:88-90. Schwartz, H. M. 1956. Kaff~rcorn malting and brewing studies. I. The kaffir beer brewing industry in South America. Journal of the Science of Food and Agriculture 7:101-105. Production of Meat- Like F favors Ebine, H. 1972. Miso. In Proceedings of the Inte?national Symposium on Conversion and Manufacture of Foodstuffs by Microorganisms, pp. 127-139. Tokyo: Saikon Publish- ing Company. Hesseltine, C. W., and Shibasaki, K. 1961. Miso III. Pure culture fermentation with Saccharomyces rouxii. Applied Microbiology 9:515-518. , and Wang, H. L. 1967. Traditional fermented foods. Biotechnology and Bio- engineering 9:275-288. National Academy of Sciences. 1975. The winged bean: a high-protein crop for the tropics. Report of an Ad Hoc Panel of the Advisory Committee on Technology Innovation, of the Board on Science and Technology for InternationaI Development. Washington, D.C.: National Academy of Sciences. 1979. Tropical legumes: resources for the future. Report of an Ad Hoc Panel of the Advisory Committee on Technology Innovation, of the Board on Science and Technology for International Development. Washington, D.C.: National Academy of Sciences.
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FOOD AND ANIMAL FEED 43 Shibasaki, K., and Hesseltine, C. W. 1962. Miso-fermentation. Economic Botany 16:180-195. Shurtleff, W., and Aoyagi, A. 1977. The book of miso. Brookline, Massachusetts: Autumn Press. Yokotsuka, T. 1960. Aroma and flavor of Japanese soy sauce. Advances in Food Re- search 10: 75-134. 1972. Shoyu. In Proceedings of the International Symposium on Conversion and Manufacture of Foodstuffs by Microorganisms, pp. 117-125. Tokyo: Saikon Publish- ing Company. Koji Method of Producing Enzymes Hesseltime, C. W. 1972. Solid-state fermentations. Biotechnology and Bioengzneering 14:517-532. ; Swain, E. W.; and Wang, H. L. 1976. Production of fungal spores as inoculum for oriental fermented foods. Developments in Industrial Microbiology 17:101-115. Nakano, M. 1972. Synopsis on the Japanese traditional fermented foodstuffs. In Waste recovery by microorganisms, pp. 27-28. Kuala Lumpur: United Nations Educational, Scientific, and Cultural Organization, distributed in the United States by UNIPUB, New York. Sakaguchi, K. 1972. Development of industrial microbiology in Japan. In Proceedings of the International Symposium on Conversion and Manufacture of Foodstuffs by Microorganisms, pp. 7-10. Tokyo: Saikon Publishing Company. Indonesian Tempeh Hesseltine, C. W. 1965. A millennium of fungi, food and fermentation. Mycologia 57:149-197. Liem, I. T. H.; Steinkraus, K. H.; and Cronk, T. C. 1978. Production of vitamin B-12 in tempeh-a fermented soybean food. A pplied and Environmental Microbiology 34:773-776. Roelofsen, P. A., and Talens, A. 1964. Changes in some B vitamins during molding of soybeans by Rhizopus oryzoe in the production of tempeh kedelee. Journal of Food Science 29:224-226. Steinkraus, K. H.; Bwee Hwa, Y.; Van Buren, J. P.; Provvidenti, M. I.; and Hand, D. B. 1960. Studies on tempeh-an Indonesian fermented soybean food. Food Research 26:777-788. ; Van Buren, J. P.; Hackler, L. R.; and Hand, D. B. 1965. A pilot-plant process for the production of dehydrated tempeh. Food Technology (Chicago) 19:63-68. van Veen, A. G.; Graham, D. C. W.; and Steinkraus, K. H. 1968. Fermented peanut press cake. Cereal Science Today 13: 96-99. Wang, H. L.; Swain, E. W.; and Hesseltine, C. W. 1975. Mass production of Rhizopus oligosporus spores and their application in tempeh fermentation. Journal of Food Science 40: 168-1 70. Single-Cell Protein Production Aguirre, F.; Moldonado, O.; Rolz, C.; Menche, J. F.; Espinosa, R.; and Cabrera, S. 1976. Protein from waste-growing fungi on coffee waste. Chemical Technology 6:636-642. Baens-Arcega, L. 1969. Philippine contribution to the utilization of microorganisms for the production of foods. In Biotechnology and Engineering Symposium, Second International Conference on Global Impacts of Applied Microbiology, Addis Ababa, Ethiopia, Elmer L Gaden, Jr., ea., pp. 53-62. New York: John Wiley and Sons. Brook, E. J.; Stanton, W. K.; and Wallbridge, A. 1969. Fermentation methods for pro- tein enrichment of cassava. Biotechnology and Bioengineering 1 1 :1271-1284. Davis, P., ed. 1974 Single-cell protein. New York: Academic Press. Khor, G. L.; Alexander, J. C.; Santos-Nunez, J.; Reade, A. E.; and Gregory, K. F. 1976. Nutritive value of thermotolerant fungi grown on cassava. Canadian Institute of Food Science and Technology Journal 9:139-216.
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44 MICROBIAL PROCESSES Lewis, C. W. 1976. Energy requirements for single-cell protein production. Journal of Applied Chemistry and Biotechnology 26:568-576. Litchfield, J. H. 1974. The facts about food from unconventional sources. (chemical Processing (London) 20: 1 1-18. 1977. Single-cell proteins. Food Technology (Chicago) 31 :175-179. Mateles, R. I., and Tannenbaum, S. R., eds. 1968. Single-cell protein. Cambridge, Massa- chusetts: Massachusetts Institute of Technology Press. Moo-Young, M. 1977. Economics of SCP production. Process Biochemistry (London) 12:6-10. ; Chahal, D. S.; Swan, J. E.; and Robinson, C. W. 1977. SCP production by Chae- tomium cellulolyticum, a new thermotolerant cellulolytic fungus. Biotechnology and Bioengineering 19:527-538. National Academy of Sciences. 1975. Underexploited tropical plants with promising economic value. Report of an Ad Hoc Panel of the Advisory Committee on Tech- nology Innovation, Board on Science and Technology for International Development, Commission on International Relations. Washington, D.C. Peppler, H. J., ed. 1978. Microbial technology. New York: Krieger Publishing Com- pany. Ratledge, C. 1975. The economics of single-cell protein production. Chemistry and Industry (London) 21:918-920. Richmond, A., and Vonshak, A. 1978. Algae-an alternative source of protein and bio- mass for arid zones. Arid Lands Newsletter 9 :1-7. Storasser, J.; Abbott, J. A.; and Battey R. F. 1970. Process enriches cassava with protein. Food Engineering, May: 112-116. Tannenbaum, S. R., and Wang, D. I. C., eds. 1975. Single-cell protein II. Cambridge, Massachusetts: Massachusetts Institute of Technology Press. Waslien, C. I. 1975. Unusual sources of protein for man. CRC (Critical Reviews in Food Science and flut7ition 5: 77-151. Wiken, T. O. 1972. Utilization of agricultural and industrial wastes by utilization of yeasts. In Proceedings of the Fourth International Fermentation Symposium, Kyoto, Japan, pp. 569-576. Osaka: Society of Fermentation Technology. Sources of Cultures Food Preservation American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. Improving Nutritional Value American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. Centraalbureau voor Schimmelcultures, P.O. Box 273, 3740 AG, Baarn, The Nether- lands. National Collection of Yeast Cultures, Lyttell Hall, Nutfield, Redhill, Surrey RH1 4HY England. Production of Meat-Like Flavors American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. Centraalbureau voor Schimmelcultures, P.O. Box 273, 3740 AG, Baarn, The Nether- lands. Koji Method of Producing Enzymes American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A.
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FOOD AND ANIMAL FEED 45 Centraalbureau voor Schimmelcultures, P.O. Box 273, 3740 AG, Baarn, The Nether- lands. Indonesian Tempeh Centraalbureau voor Schimmelcultures, P.O. Box 273, 3740 AG, Baarn, The Nether- lands. American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A. Single-cell Protein Production American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. Research Contacts Food Preservation O. Kandler, Botanisches Institut, Der Universitat Munchen, 800 Munchen 19, Menzinger Strasse 67, Federal Republic of Germany. Carl S. Pederson, Professor of Microbiology, Ementus. Cornell University, Geneva, New York 14456, U.S.A. Keith H. Steinkraus, Department of Food Science and Technology, New York State Agricultural Experiment Station, Geneva, New York 14456, U.S.A. Reese Vaughn, Professor Emeritus, Department of Food Science and Technology, Uni- versity of California, Davis, California 95616, U.S.A. Improving Nutritional Value Clifford W. Hesseltine, Northern Regional Research Laboratory, 1815 N. University, Peoria, Illinois 6 1604, U.S.A. Keith H. Steinkraus, Department of Food Science and Technology, New York State Agricultural Experiment Station, Geneva, New York 14456, U.S.A. Production of Meat-Like Flavors Hideo Ebine, National Food Research Institute, Ministry of Agriculture and Forestry, Yatabe-machi Ibaraki-ken, 300-31, Japan. Clifford W. Hesseltine, Northern Regional Research Laboratory, 1815 N. University, Peoria, Illinois 61604, U.S.A. Nakano Masahiro, Meiji Daigaku, Ikuta Kosha, Ikuta 5158, Kawasakishi, Kanagawa-ken 214, Japan. Shinshu Miso Research Institute, Minamiagata-machi 1014, Nagano-shi 380, Japan. William Shurtleff, New-Age Foods Study Center, 790 Los Palos Manor, Lafayette, Cali- fornia 94549, U.S.A. Tamotsu Yokotsuka, Kikkoman Shoyu Co., Ltd., 339 Noda, Noda-shi, Chiba-ken, Japan. Koji Method of Producing Enzymes Hideo Ebine, National Food Research Institute, Ministry of Agriculture and Forestry, Yatabemachi, Ibaraki-ken, 300-31, Tokyo, Japan. Clifford W. Hesseltine, Northern Regional Research Laboratory, 1815 N. University, Peoria, Illinois 61604, U.S.A. William Shurtleff, New-Age Foods Study Center, 790 Los Palmos Manor, Lafayette, California 94549, U.S.A. Ta~notsu Yokotsuka, Kikkoman Shoyu Co. Ltd., 339 Noda, Noda-shi, Chiba-ken, Japan.
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46 Indonesian Tempeh MICROBIAL PROCESSES Clifford W. Hesseltine, Northern Regional Research Laboratory, 1815 N. University, Peoria, Illinois 61604, U.S.A. Keith H. Steinkraus, Department of Food Science and Technology, New York State Agricultural Experiment Station, Geneva, New York 14456, U.S.A. A. G. van Veen, Division of Nutritional Sciences, Cornell University, Savage Hall, Ithaca, New York 14853, U.S.A. Single-Cell Protein Production Allen I. Laskin, EXXON Research and Engineering Company, P.O. Box 45, Linden, New Jersey 07036, U.S.A. John H. Litchfield, Battelle Columbus Laboratories, 505 King Avenue, Columbus, Ohio 43201, U.S.A. Jacques C. Senez, Centre National de la Recherche Scientifique, Laboratoire de Chemie Bacterienne, 31 Chemin Joseph-Aiguier 13274, Marseille 2, France. Steven R. Tannenbaum, Massachusetts Institute of Technology, Cambridge, Massachu- setts 02139, U.S.A.
Representative terms from entire chapter: