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II. OVERVIEW

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1 Upgrading Traditional Biotechnological Processes M. 'I. R. Nout TRADITIONAL FOOD FERMENTATION The general aims of food technology are to exploit natural food resources as efficiently and profitably as possible. Adequate and economically sound processing, prolongation of shelf life by preserva- tion and optimization of storage and handling, improvement of safety and nutritive value, adequate and appropriate packaging, and maximum consumer appeal are key prerequisites to achieving these aims. Fermentation is one of the oldest methods of food processing. The history of fermented foods has early records in Southeast Asia, where China is regarded as the cradle of mold-fermented foods, and in Africa where the Egyptians developed the concept of the combined brewery- bakery. The early Egyptian beers were probably quite similar to some of the traditional opaque sorghum, maize, or millet beers found in various African countries today (11. In technologically developed regions, the crafts of baking, brewing, wine making, and dairying have evolved into the large-scale industrial production of fermented consumer goods, including cheeses, cultured milks, pickles, wines, beers, spirits, fermented meat products, and soy sauces. The introduction of such foreign "high-tech" fermented products to tropical countries by early travelers, clergymen, and colonists was followed by an accelerated demand during the early postindependence period. Their high price ensured status, and their refined quality guaranteed continued and increasing consumption. In contrast, many of the traditional indigenous foods lack this image; some may even be regarded as backward or poor people's food. Factors contributing to such lack of appeal include inadequate grading and cleaning of raw materials, crude handling and processing tech- 11

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12 FERMENTED FOODS piques, and insufficient product protection due to lack of packaging. Such unhygienic practices are easily translated into a fear of food- borne diseases. From a nutritionist's point of view, many traditional starchy staples are deficient in energy, protein, and vitamins. Variable sensory characteristics (quality) and lack of durability (shelf life) reduce convenience to the consumer: time needs to be spent selecting products of adequate quality, whereas perishable products require frequent purchasing and result in increased wastage. In addition, ungraded heterogenous products, inconvenient unpacked bulk foods, or unattrac- tive presentation inhibit consumers to develop regular purchasing attitudes. The contrast outlined here serves as a general guideline to the major targets for upgrading the present status of traditional indigenous fermented foods. The latter are part of the regional cultural heritage; they are well known and accepted by consumers and consequently provide an appropriate basis for development of a local food industry, which not only preserves the agricultural produce but also stimulates and supports agroindustrial development. DECENTRALIZED SMALL-SCALE PROCESSES In most African countries, 70 percent or more of the population lives in rural areas. However, if the present trend in urbanization continues (urban growth rates of 5 to 10 percent annually), 50 percent of the African population will be living in cities by the year 2000. Governments become increasingly aware that rural industrialization is a worthwhile investment because it creates job opportunities, improves agricultural productivity, and helps to check urbanization. But even at the present urbanization rate, a rapidly increasing low-income population will be located in urban areas. The resultant uncoupling in place and time of primary production and food consumption necessitates the manufacture of wholesome, low-cost, nutritious products that can withstand low- hygiene handling. Agro-allied industries are closely linked to regions of primary production, and it is particularly in the field of food processing, with low-cost perishable raw materials, that establishment of a rural network of small-scale processing facilities is most appropriate. Home- or village-scale enterprises require only modest capital investment, which should be made available on a "soft loan" basis. Against this back- ground, some basic process improvements that increase the appeal of traditional fermented foods and that can be carried out by simple means will be outlined (21.

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BIOTECHNOLOGICAL PROCESSES BASIC PROCESSING OPERATIONS 13 In food manufacturing several operations are required to prepare raw materials, handle and process them into products, and finally prepare the finished product for distribution and sale by preservation and/or packaging. One might think of sorting, grading, cleaning, disinfection, grinding, or packaging. The establishment and success of some indigenous enterprises in Nigeria and Kenya show that the appeal and marketability of such products as beans, peas, gari, and spices, formerly sold in bulk, increase significantly when they have "only" been sorted, cleaned, graded, sometimes ground, labeled, and packaged in simple polythene bags. NUTRITIVE VALUE The nutritive value of traditional fermented foods needs improve- ment. The energy density of starch-based porridges is inadequate, particularly when used for weaning purposes. Root crop- or cereal- derived products have rather low protein contents, and the quality of their protein is limited by the amount of lysine present. Various antinutritional factors, including polyphenols, physic acid, trypsin inhibitors, and lectins, are present in legumes and cereals. Composite products (legume additions to starchy staples) offer an opportunity to improve protein quantity and quality. Combinations of simple unit operations, including roasting, germination, and fermenta- tion, afford increased energy density in porridges and reduce antinutri- tional factors considerably (31. STABILIZATION OF NATURAL FERMENTATIONS BY INOCULUM ENRICHMENT Most traditional fermented products result from natural fermenta- tions carried out under nonsterile conditions. The environment resulting from the chemical composition of the raw materials fermentation temperature, absence or presence of oxygen, and additives such as salt and spices causes a gradual selection of microorganisms responsible for the desired product characteristics. The main advantage of natural fermentation processes is that they are fitting to the rural situation, since they were in fact created by it. Also, the consumer safety of several African fermented foods is improved by lactic acid fermentation, which creates an environment that is unfavorable to pathogenic Enterobacteriaceae and Bacillaceae.

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14 FERMENTED FOODS In addition, the variety of microorganisms present in a fermented food can create rich and full flavors that are hard to imitate when using pure starter cultures under aseptic conditions. However, natural fermentation processes tend to be difficult to control if carried out at a larger scale; moreover, the presence of a significant accompanying microflora can accelerate spoilage once the fermentation is completed. Particularly with increased holding periods between product fermentation and consumption when catering for urban markets, uncontrolled fermentations under variable conditions will cause unacceptable wastage by premature spoilage.- Techniques to stabilize fermentations operating under nonsterile conditions would therefore be appropriate in the control of natural fermentations. For this purpose the use of pure culture starters, obtained either by laboratory selection procedures or genetic engi- neering, offers no realistic solutions because they are expensive and require sterile processing conditions. A more feasible approach is to exploit the ecological principle of inoculum enrichment by natural selection. This can be achieved by the sourdough process, in which some portion of one batch of fermented dough is used to inoculate another batch. This practice is also referred to as `'back-slopping" or inoculum enrichment. The resulting starters are active and should not be stored but used in a continuous manner. Sourdoughs from commercial sources, having been maintained by daily or weekly transfers during 2 or more years, contain only two or three microbial species, although they are exposed to a wide variety of potential competitors and spoilage-causing microorganisms each time the sourdough is mixed with fresh flour for a transfer. It can take as long as 10 weeks of regular transfers before a sourdough population becomes stabilized. Such populations could contain a yeast, Saccharo- myces exiguous, and one or two Lactobacillus species, namely Lb. brevis var. linderi II and Lb. sanfrancisco. Although the mechanism of the stable coexistence of sourdough populations is not yet fully understood, lack of competition for the same substrate might play an important role. Other factors besides substrate competition, such as antimicrobial substances produced by lactic acid bacteria, might play an important role in the stability of such stable populations, obtained by "back-slopping" (4~. Similar experiments in the field of tempe manufacture showed that the first stage of the tempe process soaking of soybeans can be rendered more predictable in terms of acidification of the beans, by simple inoculum enrichment. Depending on soaking temperatures, stable soaking water populations were obtained after 30 to 60 daily transfers, containing Leuconostoc spp. at 14 and 19C, yeasts and Lactobacillus spp. at 25C, Lactobacillus spp. at 30C, or Pediococcus

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BIOTECHNOLOGICAL PROCESSES 15 and Streptococcus spp. at 37 and 45C. Tempe made with well-acidified beans contained fewer undesirable microorganisms and was more attractive (51. Based on the same principle of inoculum enrichment, the intrinsic microbiological safety of composite meals of cereals and legumes can be improved significantly by lactic fermentation (6~. This offers interesting possibilities in the manufacture of food for vulnerable consumer groups, such as infants, malnourished patients, and the elderly (7~. Although development of such gradually evolved and stable fermen- tation starters will be an attractive proposition for use in small-scale fermentations under nonsterile conditions, they will not be the most appropriate in all cases. This is exemplified by the sauerkraut (lactic acid fermented cabbage) fermentation, during which flavor development is determined by a succession of Leuconostoc and Lactobacillus species occurring during the course of the fermentation. Practical experience in the sauerkraut industry in the Netherlands has shown that carryover of previous sauerkraut into a fresh batch of cabbage will cause a rapid domination of homofermentative Lactobacillus spp., which should normally only dominate during the final stage of fermentation. The result is an excessively sour-tasting product that lacks the flavor otherwise produced by the heterofermentative Leuconostoc and Lacto- bacillus spp. In the exercise of upgrading traditional food fermentation techniques, it would therefore be worthwhile to investigate the effect of inoculum enrichment on product characteristics and consumer acceptance. MULTISTRAIN DEHYDRATED STARTER A different tool to stabilize fermentations under nonsterile conditions is the use of multistrain dehydrated starters, which can be stored at ambient temperatures, enabling more flexibility. Such homemade starters are widely used in several Asian food fermentations. Examples are the manufacture of tempe (mainly from soybeans) and tape (from glutinous rice or cassava). Indonesian traditional tempe starters (usar) are essentially molded hibiscus leaves that carry a multitude of molds, dominated by Rhizopus spp., including the Rh. oryzae and Rh. microsporus varieties. Instead of using usar, Indonesian tempe produc- tion is increasingly carried out with factory-prepared "pure" starters consisting of granulated cassava or soybean fiber carrying a mixed population of Rhizopus species (51. These starters are more homoge- nous and their dosage is convenient, but because they are manufactured under nonsterile conditions, some are heavily contaminated with

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16 FERMENTED FOODS spoilage-causing bacteria and yeasts. This requires quality monitoring of the inoculum and of the fermentation process in which it is used. Other examples of durable home-prepared starter materials used in Asian food fermentations are Indonesian ragi and Vietnamese men tablets (81. Depending on their specific purpose, these dehydrated tablets, prepared from fermented rice flour, contain mixed populations of yeasts, molds, and bacteria. Ragi tablets can be stored up to 6 months and constitute a convenient starter material for application in home and small-scale industrial fermentations of rice or cassava, for example. Especially in the fermentation of neutral pH, protein-rich substrates, such as legumes, one should be extremely careful with the use of substandard inoculum. If the process lacks factors that control micro- bial development, pathogens may survive or produce toxins in such products. Tempe manufacture is a good example of a process with intrinsic safety. The preliminary soaking of the beans results in an acidification that inhibits the multiplication of bacterial contaminants during the mold fermentation stage. Also, antimicrobial substances of Rhizopus oligosporus would play a protective role against outgrowth of several genera of microorganisms. Moreover, near-anaerobic condi- tions and microbial competition during the fermentation stage, and the usual cooking or frying of tempe prior to consumption, strongly reduce the chances of food-borne illness (51. Nevertheless, the introduction of fermentation processes in regions where they are not traditionally mastered requires adequate guidance, supervised processing, and monitoring of product safety. ENZYME PRODUCTION BY KOJ/ TECHNIQUE Not only microorganisms but also enzymes play an important role in the manufacture of traditional fermentation processes. In cassava processing the naturally occurring enzyme linamarase is able to degrade potentially toxic cyanogenic glycosides (e.g., linamarin). This enzymatic detoxification has always been an integral part of traditional cassava fermentations, such as in Sari and lalun. Under certain conditions the detoxification of linamarin is accelerated by linamarase addition (91. It is conceivable that there will be commercial applications for the enzymatic process of linamarin decomposition, which could be used to detoxify cassava without having to ferment it; the result would be a neutral and bland-havored product. Enzyme sources for African traditional beer brewing are mostly germinated sorghum and millet varieties, whereas sorghum and millet malts possess adequate diastatic power with cx-amylase, resulting in

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BIOTECHNOLOGICAL PROCESSES 17 poor conversion of dextrins into maltose (101. The availability of cheap technical-grade B-amylase preparations could lead to the development of novel brewing processes utilizing home-grown starch sources instead of imported barley malt. In East Asia, koji is used as a source of enzymes in the manufacture of soy sauce and rice wine. Koji is obtained by solid-substrate fermentation of cereals or soybeans with fungi (e.g., Aspergillus o~yzoe and Asp. soyne). Depending on the particular substrate to be degraded, selected strains of molds are used, often as mixed cultures. Their enzymes include amylases, proteases, and cellulolytic enzymes. During fermentation the enzymes are accumulated into the koji. The enzymes produced are subsequently extracted from the koji using brine solutions. Koji fermentations are carried out in East Asia at a small home scale, as well as in the large-scale industrial manufacture of soy sauce and rice wine ( 1 1 ). Although mycotoxin-producing molds such as Aspergillus Jqavus and Asp. parasitious occur in koji as natural contaminations, they have not been observed to produce aflatoxins under the given conditions. The principle of fungal solid-substrate fermentation may be used to prepare enzyme concentrations for conversion of starch, detoxification of cyanogenic glycosides, and other applications. DRY MATTER BALANCE Food fermentation is advantageously used for food preservation and to obtain desirable flavor and digestibility. However, some processes are rather wasteful. For instance, prolonged soaking and microbial respiration of organic matter may lead to considerable losses of valuable raw material dry matter. Examples can be found in the traditional process of ogi manufacture (fermented maize cake) and the tempe process, during which up to 30 percent of the raw material may be lost by leaching during soaking steps. Encouraging research has been carried out by Banigo et al. ( 12) in the field of Nigerian ogi manufacture, aimed at reducing these raw material losses by omitting soaking stages. It would certainly be worthwhile to investigate dry matter balances of traditional fermentations with a view to reducing losses of raw material by implementing "dry" instead of"wet" processing. IMPLEMENTATION No matter how much research is carried out on improved traditional processes or novel products, the ultimate aim is implementation.

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18 FERMENTED FOODS Unfortunately, a wide gap exists between research data published in scientific journals and the practice of food processing. Much attention should be given to the extent of usefulness of new products to the end user. To this effect, not only should the sensory, nutritional, and other quality characteristics of newly developed products or processes be taken into account, but they should also be integrated with sound price calculations, market surveys, and extension efforts. Only a competitive process has good chances of being implemented. In conclusion, the importance of a business-oriented approach and close contact between researchers and food processors, working together toward mutual benefit, must be stressed. REFERENCES 1. Hesseltine, C. W. 1981. Future of fermented foods. Process Biochemistry 16:2-13. 2. Bruinsma, D. H., and M. J. R. Nout. 1990. Choice of technology in food processing for rural development. Paper presented at the symposium `'Technology and Rural Change in Sub-Saharan Africa," Sussex University, Brighton, U.K., Sept. 27-30, 1989. In: Rural Households in Emerging Societies: Technology and Change in Sub- Saharan Africa. M. Haswell, and D. Hunt (Eds.~. New York: Berg Publishers. 3. Nout, M. J. R. 1990. Fermentation of infant food. Food Labora- tory News 6~2120:10-12. 4. Spicher, G. 1986. Sour dough fermentation. Chemie Mikrobiolo- gie Technologie der Lebensmittel 10~3/4~:65-77. 5. Nout, M. J. R., and F. M. Rombouts. 1990. Recent developments in tempe research. Journal of Applied Bacteriology 69~51:609-633. 6. Nout, M. J. R. 1991. Ecology of accelerated natural lactic fermentation of sorghum-based infant food formulas. International Journal of Food Microbiology 12~2/31:217-224. 7. Mensah, P., A. M. Tomkins, B. S. Drasar, and T. J. Harrison. 1991. Antimicrobial effect of fermented Ghanaian maize dough. Journal of Applied Bacteriology 70~3~:203-210. 8. Hesseltine, C. W., R. Rogers, and F. G. Winarno. 1988. Microbiological studies on amylolytic Oriental fermentation starters. Mycopathologia 101~31:141-155. 9. Ikediobi, C. O., and E. Onyike. 1982. The use of linamarase in gari production. Process Biochemistry 17:2-5. 10. Nout, M. J. R., and B. J. Davies. 1982. Malting characteristics of finger millet, sorghum and barley. Journal of the Institute of Brewing 88: 157-163. .

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BIOTECHNOLOGICAL PROCESSES 19 11. Fukushima, D. 1989. Industrialization of fermented soy sauce production centering around Japanese shoyu. Pp. 1-88 in: Industrializa- tion of Indigenous Fermented Foods. K. H. Steinkraus (Ed.~. New York: Marcel Dekker, Inc. 12. Banigo, E. O. I., J. M. de Man, and C. L. Duitschaever. 1974. Utilization of high-lysine corn for the manufacture of ogi using a new, improved processing system. Cereal Chemistry 51:559-572.

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48 FERMENTED FOODS been isolated and identified in sourdough leaven. The lactobacillus species has a preference for maltose and uses the maltose phosphorylase pathway to metabolize the sugar, whereas T. holmii grows on glucose but not on maltose, so that both develop in a dough where the amylases hydrolyze starch to maltose. The basic biochemical changes that occur in sourdough bread fermentation are (1) acidification of the dough with lactic and acetic acids produced by the lactobacilli and (2) leavening of the dough with carbon dioxide produced by the yeast and the lactobacilli. Typical flavor and aroma development can be traced to biochemical activities of both lactobacilli and yeasts. The chewy characteristic of sourdough bread may be due to the production of bacterial polysaccharides by the lactobacilli. NIGERIAN OGI (KENYAN Udl) Nigerian ogi is a smooth-textured, sour porridge with a flavor resembling that of yogurt. It is made by lactic acid fermentation of corn, sorghum, or millet. Soybeans may be added to improve nutritive value. Ogi has a solids content of about 8 percent. The cooked gel- like porridge is known as "pap." The first step in the fermentation is steeping of the cleaned grain for 1 to 3 days. During this time the desirable microorganisms develop and are selected. The grain is then ground with water and filtered to remove coarse particles. After steeping, the pH should be 4.3. Optimum pH for ogi is 3.6 to 3.7. The concentration of lactic acids may reach 0.65 percent and that of acetic acid 0.11 percent during fermentation. If the pH falls to 3.5, it is less acceptable. Ogi is a naturally fermented product. A wide variety of molds, yeasts, and bacteria are present initially. Lb. plantarum appears to be the essential microorganism in the fermentation. Following depletion of the fermentable sugars, it is able to utilize dextrins from the corn. Saccharomyces cerevisiae and Candida mycoderma contribute to the pleasant flavor. NIGERIAN GARI Nigerian gari is a granular starchy food made from cassava (Manihot utilissima or M. esculenta) by lactic acid fermentation of the grated pulp, followed by dry-heat treatment to gelatinize and semidextrinize the starch, which is followed by drying. Cassava tubers are washed, peeled, and grated. An inoculum of 3-day-old cassavajuice or fermented

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LACTIC ACID FERMENTATIONS 49 mash liquor is added. The pulp is placed in a cloth bag, excess water is squeezed out, and the pulp undergoes an anaerobic acid fermentation for 12 to 96 hours. Optimum temperature is 35C. When the phi of the mash reaches 4.0, with about 0.85 percent total acid (as lactic acid), the gari has the desired sour flavor and a characteristic aroma. In village processes, further moisture may be removed, and the pulp is then toasted (semidextrinized) in shallow iron pots and dried to less than 20 percent moisture. Village-processed gari has a carbohydrate content of about 82 percent with 0.9 percent protein. Lactic, acetic, propionic, succinic, and pyruvic acids have been identified in Sari, with aldehydes and esters providing the aroma. For consumption the gari is added to boiling water, in which it increases in volume by 300 percent to yield a semisolid plastic dough. The stiff porridge is rolled into a ball (10 to 30 grams wet weight) with the fingers and dipped into stew. PHILIPPINE BALAO BALAO Balao balao is a lactic acid fermented rice-shrimp mixture, generally prepared by blending cooked rice, whole raw shrimp, and solar salt and then allowing the mixture to ferment for several days or weeks, depending on the salt content. The chitinous shell becomes soft, and when the fermented product is cooked, the whole shrimp can be eaten. With a salt concentration of 3 percent added to the rice-shrimp mixture, the pH falls to an organoleptically desirable value of 4.08, with titratable acidity reaching 1.32 percent acid (as lactic acid) in 4 days. Balao balao made with 3 percent salt is best in color, odor, flavor, texture, and general acceptability and is the least salty. Balao balao offers a basic method of preservation for cereal-shrimp-fish mixtures. When properly packed to exclude air, sufficient acid is produced to preserve the products without resorting to high-temperature cooking. MEXICAN PULQUE Pulque is a white, acidic, alcoholic beverage made by fermentation of juice of Agave species, mainly A. atrovirens or A. americana, the century plants. It has been a national Mexican drink since the time of the Aztecs. Pulque plays an important role in the nutrition of low- income people in the semiarid regions of Mexico. The essential microorganisms in the pulque fermentation are Lb. plantarum, a heterofermentative Leuconostoc, Sac. cerevisiae, and Zymomonas mobilis.

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so FERMENTED FOODS The heterofermentative Leuconostoc plays the essential role of producing dextrans, which contribute a characteristic viscosity to pulque and also increase the acidity of the agave juice very rapidly, inhibiting growth of other less desirable bacteria. Lb. plantarum contributes to the final acidity of pulque. Sac. cerevisiae appears to be a major producer of ethanol, but Z. mobilis is considered to be the most important ethanol producer in pulque. Under anaerobic conditions, Zymomonas transforms 45 percent of the glucose to ethanol and carbon dioxide. It also produces some acetic acid, acetylmethylcarbinol, and some slime gums, which may contribute to the viscous nature of traditional pulque. Soluble solids in the fresh agave juice decrease from 25-30 percent to 6.0 percent in pulque. The pH falls from 7.4 to 3.5-4.0. Total acid increases from 0.03 percent to 0.4-0.7 percent (as lactic acid). Sucrose decreases from 18.6 percent to less than 1 percent. Ethanol increases from O percent to 4-6 percent (v/v). The B vitamins are present in nutritionally important quantities, with ranges reported as follows (in milligrams per 100 grams): thiamine, 5 to 29; niacin, 54 to 515; riboflavin, 18 to 33; pantothenic acid, 60 to 335; p-aminobenzoic acid, 10 to 12; pyridoxine, 14 to 23; and biotin, 9 to 32. EGYPTIAN KISHK, GREEK TRAHANAS, AND TURKISH TARHANAS Egyptian kishk, Greek trahanas, and Turkish tarhanas are mixtures of sheep's milk yogurts and parboiled wheat. Tomato, tomato paste, or onion are sometimes added. In all cases the milk or buttermilk undergoes a typical lactic acid fermentation in which the pH ranges from 3.5 to 3.8 and titratable acidity is 1.3 to 1.8 percent (as lactic acid). Proportions of wheat to yogurt range from 2:1 to 1:3. The wheat is parboiled at some stage in the process. In its simplest form the wheat is added directly to the yogurt and the mixture is boiled until the wheat has absorbed the free moisture. The mixture is cooled and formed into biscuits that are sun dried. If the wheat is ground prior to mixing with the yogurt, the fines are discarded because they harden the final product. In Egypt the principal microorganisms reported in kishk are the heterofermentative Lb. brevis and the homofermentative Lb. cased and Lb. plantarum. In Cyprus sheep's milk yogurt contains principally S. thermophilus and Lb. bulgaricus. Dried kishk and trahanas are not hygroscopic and can be stored in open jars for several years without deterioration. They also are well balanced nutritionally.

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LACTIC ACID FERMENTATIONS OTHER FOODS 51 Lactic acid fermentation also plays an essential role in the production of Indonesian tempe, a vegetable (soybean) protein meat substitute the texture of which is provided by mycelium of Rhizopus oligosporus, which overgrows and knits the soaked, partially cooked cotyledons into compact cakes that can be sliced thinly and deep fried or cut into chunks and used in soups in place of meat. The essential part played by lactobacilli occurs during the initial soaking when the pH falls from about 6.5 to between 4.5 and 5.0. The lower pH facilitates growth of the mold and prevents development of undesirable bacteria that might spoil the tempe. In Chinese soy sauce (Japanese shoyuJ and Japanese miso and related meat-flavored, amino acid peptide sauces and pastes, the essential microorganism for amylolytic, proteolytic hydrolysis of the soybean-wheat or soybean-rice or barley substrates is Aspergillus oryzae. Following overgrowth of the substrate by the mold, the koji is subsequently allowed to ferment in approximately 19 percent salt brine for the sauces and 6 to 13 percent salt for the pastes. Lactobacilli grow and lower the pH to about 4.5, which then allows the osmophilic yeast Sac. rouxii to grow and produce some ethanol. The ethanol combines with organic acid in the substrate, producing esters that contribute to the agreeable havor and aroma. Given the fact that these acid fermentation techniques are simple, effective, and inexpensive, their application in developing countries should be encouraged.

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6 Mixed-Culture Fermentations Clifford W. Hesseltine Mixed-culture fermentations are those in which the inoculum always consists of two or more organisms. Mixed cultures can consist of known species to the exclusion of all others, or they may be composed of mixtures of unknown species. The mixed cultures may be all of one microbial group all bacteria or they may consist of a mixture of organisms of fungi and bacteria or fungi and yeasts or other combina- tions in which the components are quite unrelated. All of these combinations are encountered in Oriental food fermentations. The earliest studies of microorganisms were those made on mixed cultures by van Leeuwenhoek in 1684. Micheli, working with fungi in 1718, reported his observations on the germination of mold spores on cut surfaces of melons and quinces. In 1875 Brefeld obtained pure- culture of fungi, and in 1878 Koch obtained pure cultures of pathogenic bacteria. The objective of both Brefeld's and Kochts studies was to identify pathogenic microorganisms. They wanted to prove what organism was responsible for a particular disease. Thus, part of Koch's fame rests on his discovery of the cause of tuberculosis. An early paper on mixed-culture food fermentation was an address by Macfadyen (1) at the Institute of Brewing, in London, in 1903 entitled, "The Symbiotic Fermentations," in which he referred to mixed-culture fermentations as "mixed infections." Probably this expression reflected his being a member of the Jenner Institute of Preventive Medicine. About half of his lecture was devoted to mixed- culture fermentations of the Orient. Among those described were Chinese yeast, koVi, Tonkin yeast, and ragi. Mixed cultures are the rule in nature; therefore, one would expect this condition to be the rule in fermented foods of relatively ancient origin. Soil, for example, is a mixed-organism environment with protozoa, bacteria, fungi, and algae growing in various numbers and kinds, depending on the nutrients available, the temperature, and the s2

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MIXED-CULTURE FERMENTATIONS 53 pH of the soil. Soil microorganisms relate to each other some as parasites on others, some forming substances essential to others for growth, and some having no effect on each other. ADVANTAGES Mixed-culture fermentations offer a number of advantages over conventional single-culture fermentations: Product yield may be higher. Yogurt is made by the fermentation of milk with Streptococcus thermophilus and Lactobacillus bulgaricus. Driessen (2) demonstrated that when these species were grown sepa- rately, 24 mmol and 20 mmol, respectively, of acid were produced; together, with the same amount of inoculum, a yield of 74 mmol was obtained. The number of S. thermophilus cells increased from 500 x 106 per milliliter to 880 x 106 per milliliter with L. bulgaricus. The growth rate may be higher. In a mixed culture one microorgan- ism may produce needed growth factors or essential growth compounds such as carbon or nitrogen sources beneficial to a second microorgan- ism. It may alter the pH of the medium, thereby improving the activity of one or more enzymes. Even the temperature may be elevated and promote growth of a second microbe. Mixed cultures are able to bring about multistep transformations that would be impossible for a single microorganism. Examples are the miso and shoyu fermentations in which Aspergillus oryzae strains are used to make koVi. Koji produces amylases and proteases, which break down the starch in rice and proteins in soybeans. In the miso and shoyu fermentations, these compounds are then acted on by lactic acid bacteria and yeast to produce flavor compounds and alcohol. In some mixed cultures a remarkably stable association of microor- ganisms may occur. Even when a mixture of cultures is prepared by untrained individuals working under unsanitary conditions, such as in ragi, mixtures of the same fungi, yeasts, and bacteria remain together even after years of subculture. Probably the steps in making the starter were established by trial and error, and the process conditions were such that this mixture could compete against all contaminants. Compounds made by a mixture of microorganisms often comple- ment each other and work to the exclusion of unwanted microorgan- isms. For example, in some food fermentations yeast will produce alcohol and lactic acid bacteria will produce lactic acid and other organic acids and change the environment from aerobic to anaerobic. Inhibiting compounds are thus formed, the pH is lowered, and anaerobic conditions are developed that exclude most undesirable molds and bacteria.

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54 FERMENTED FOODS Mixed cultures permit better utilization of the substrate. The substrate for fermented food is always a complex mixture of carbohy- drates, proteins, and fats. Mixed cultures possess a wider range of enzymes and are able to attack a greater variety of compounds. Likewise, with proper strain selection they are better able to change or destroy toxic or noxious compounds that may be in the fermentation substrate. Mixed cultures can be maintained indefinitely by unskilled people with a minimum of training. If the environmental conditions can be maintained (i.e., temperature, mass of fermenting substrate, length of fermentation, and kind of substrate), it is easy to maintain a mixed- culture inoculum indefinitely and to carry out repeated successful fermentations. Mixed cultures offer more protection against contamination. In mixed-culture fermentations phage infections are reduced. In pure- culture commercial fermentations involving bacteria and actinomy- cetes, invariably an epidemic of phage infections occurs, and the infection can completely shut down production. Since mixed cultures have a wider genetic base of resistance to phage, failures do not occur, often because if one strain is wiped out, a second or third phage- resistant strain in the inoculum will take over and continue the fermentation. In such processes, especially with a heavy inoculum of selected strains, contamination does not occur even when the fermentations are carried out in open pans or tanks. Mixed-culture fermentations enable the utilization of cheap and impure substrates. In any practical fermentation the cheapest substrate is always used, and this will often be a mixture of several materials. For example, in the processing of biomass, a mixed culture is desirable that attacks not only the cellulose but also starch and sugar. Cellulolytic fungi along with starch- and sugar-utilizing yeasts would give a more efficient process, producing more product in a shorter time. Mixed cultures can provide necessary nutrients for optimal performance. Many microorganisms, such as the cheese bacteria, which might be suitable for production of a fermentation product, require growth factors to achieve optimum growth rates. To add the proper vitamins to production adds complications and expense to the process. Thus, the addition of a symbiotic species that supplies the growth factors is a definite advantage. DISADVANTAGES Mixed-culture fermentations also have some disadvantages. Scientific study of mixed cultures is difficult. Obviously, it is more

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MIXED-CULTURE FERMENTATIONS 55 difficult to study the fermentation if more than one microorganism is involved. That is why most biochemical studies are conducted as single-culture fermentations because one variable is eliminated. Defining the product and the microorganisms employed becomes more involved in patent and regulatory procedures. Contamination of the fermentation is more difficult to detect and control. When two or three pure cultures are mixed together, it requires more time and space to produce several sets of inocula rather than just one. One of the worst problems in mixed-culture fermentation is the control of the optimum balance among the microorganisms involved. This can, however, be overcome if the behavior of the microorganisms is understood and this information is applied to their control. The balance of organisms brings up the problem of the storage and maintenance of the cultures. Lyophilization presents difficulties because in the freeze-drying process the killing of different strains' cells will be unequal. It is also difficult, if not impossible, to grow a mixed culture from liquid medium in contrast to typical fermentations on solid mediums, without the culture undergoing radical shifts in population numbers. According to Harrison (3), the best way to preserve mixed cultures is to store the whole liquid culture in liquid nitrogen below -80C. The culture, when removed from the frozen state, should be started in a small amount of the production medium and checked for the desired fermentation product and the normal fermentation time. Subcultures of this initial fermentation, if it is satisfactory, may then be used to start production fermentations. FUTURE Mixed-culture fermentations will continue to be used in traditional processes such as soybean and dairy fermentations. As noted above, the extensive uses of mixed-culture fermentations for dairy and meat products are well known as to the type of cultures used and the fermentation process. However, there are a large number of food fermentations based on plant substrates such as rice, wheat, corn, soybeans, and peanuts in which mixed cultures of microorganisms are used and will continue to be used One example of the complex sequential interaction of two fermenta- tions, and which employs fungi, yeast, and bacteria, is the manufacture of miso. This Oriental food fermentation product is based on the fermentation of soybeans, rice, and salt to make a paste-like fermented food. Miso is used as a flavoring agent and as a base for miso soup. There are many types of miso, ranging from a yellow sweet miso

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56 FERMENTED FOODS (prepared by a quick fermentation) to a dark, highly flavored miso. The type depends on the amount of salt, the ratio of cereals to soybeans, and the duration of the fermentation. The miso fermentation begins with the molding of sterile, moist, cooked rice that is inoculated with dry spores of Aspergillus oryzoe and A. soyae. The inoculum consists of several mold strains combined, with each strain producing a desired enzymefs). The molded rice is called koVi and is made to produce enzymes to act on the soybean proteins, fats, and carbohydrates in the subsequent fermentation. After the rice is thoroughly molded, which is accomplished by breaking the koji and mixing, the koVi is harvested before mold sporulation starts, usually in 1 or 2 days. The koji is mixed with salt and soaked and steamed soybeans. This mixture is inoculated with a new set of microorganisms, and the four ingredients are now mashed and mixed. After the production of koji with molds, the paste is placed in large concrete or wooden tanks for the second fermentation. The inoculum consists of osmophilic yeasts Saccharomyces rouxii and Candida versatilis and one or more strains of lactic acid bacteria, typically Pediococcus pentosaceus and P. halophilus (4~. Conditions in the fermentation tanks are anaerobic or nearly so, with the temperature maintained at 30C. The fermentation is allowed to proceed for varying lengths of time, depending on the type of miso desired, but it is typically 1 to 3 months. The fermenting mash is usually mixed several times, and liquid forms on the top of the fermenting mash. The initial inoculum is about 105 microorganisms per gram. Typically, 3,300 kg of miso with a moisture level of 48 percent is obtained when 1,000 kg of soybeans, 600 kg of rice, and 430 kg of salt are used. When the second fermentation is completed, aging is allowed to take place. A number of other mixed-culture fermentations are similar to the miso process, including shoyu (soy sauce) and sake (rice wine). A legitimate question can be asked as to the future prospects for the use of mixed cultures in food fermentations. What will be the effect of genetic engineering on the use of mixed cultures? Would engineered organisms be able to compete in mixed culture? Many laboratories are busy introducing new desirable genetic material into a second organism. The characteristics being transferred may come from such diverse organisms as mammals and bacteria and may be transferred from animals to bacteria. In general, the objective of this work involves introduction of one desirable character, not a number. For instance, strains of Escherichia cold have been engineered to produce insulin. However, I suspect that it may be a long time, if ever, before a single organism can produce the multitude of flavors found in foods such as cheeses, soy sauce, miso, and other fermented foods used primarily as condiments. The reason for this is the fact that a flavoring agent

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MIXED-CULTURE FERMENTATIONS 57 such as shoyu contains literally hundreds of compounds produced by the microorganisms, products from the action of enzymes on the substrate, and compounds formed by the nonenzymatic interactions of the products with the original substrate compounds. To put such a combination of genes for all these flavors into one microorganism would, at present, be almost impossible. Second, the cost of producing the food, which is relatively inexpensive as now produced, would become economically prohibitive. The use of mixed cultures in making fermented foods from milk, meat, cereals, and legumes will continue to be the direction in the future. Harrison (3), in his summary of the future prospects of mixed-culture fermentations, very succinctly concluded as follows: No claim'for novelty can be made for mixed cultures: They form the basis of the most ancient fermentation processes. With the exploitation of monocultures having been pushed to its limits it is perhaps time to reappraise the potential of mixed culture systems. They provide a means of combining the genetic properties of species without the expense and dangers inherent in genetic engineering which, in general terms, aims at the same effect. REFERENCES 1. Macfadyen, A. 1903. The symbiotic fermentations. Journal of the Federal Institutes of Brewing 9:2-15. 2. Driessen, F. M. 1981. Protocooperation of yogurt bacteria in continuous culture. Pp. 99-120 in: Mixed Culture Fermentations. M. E. Bushell and J. H. Slater, Eds. London: Academic Press. 3. Harrison, D. E. F. 1978. Mixed cultures in industrial fermentation processes. Advances in Applied Microbiology 24:129-164. 4. Hesseltine, C. W. 1983. Microbiology of oriental fermented foods. Annual Reviews of Microbiology 37:575-601.

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