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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
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.
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.
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 19°C, yeasts and Lactobacillus spp. at 25°C, Lactobacillus spp. at 30°C, or Pediococcus
BIOTECHNOLOGICAL PROCESSES 15 and Streptococcus spp. at 37° and 45°C. 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
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
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.
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. .
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.
2 Genetic improvement of Microbial Starter Cultures Susan K. HarIander Fermentation has been used for preserving food for hundreds of years and virtually every culture has, as part of its diet, a variety of fermented milk, meat, vegetable, fruit, or cereal products. Microorgan- isms, including bacteria, yeasts, and mold, produce a wide range of metabolic end products that function as preservatives, texturizers, stabilizers, and flavoring and coloring agents. Several traditional and nontraditional methods have been used to improve metabolic properties of food fermentation microorganisms. These include mutation and selection techniques; the use of natural gene transfer methods such as transduction, conjugation and transformation; and, more recently, genetic engineering. These techniques will be briefly reviewed with emphasis on the advantages and disadvantages of each method for genetic improvement of microorganisms used in food fermentations. TRADITIONAL GENETIC IMPROVEMENT STRATEGIES Mutation and Selection In nature, mutations (changes in the chromosome of an organism) occur spontaneously at very low rates (one mutational event in every 106 to 107 cells per generation. These mutations occur at random throughout the chromosome, and a spontaneous mutation in a metabolic pathway of interest for food fermentations would be an extremely rare event. The mutation rate can be dramatically increased by exposure of microorganisms to mutagenic agents, such as ultraviolet light or various chemicals, which induce changes in the deoxyribonucleic acid (DNA) of host cells. Mutation rates can be increased to one mutational event in every 10' or 102 cells per generation for auxotrophic mutants, and one in 103 to 1os for the isolation of improved secondary metabolite 20
MICROBIAL STARTER CULTURES 21 producers. A method of selection is critical for effective screening of mutants as several thousand individual isolates may need to be evaluated to find one strain with improved activity in the property of interest. Mutation and selection techniques have been used to improve the metabolic properties of microbial starter cultures used for food fermentations; however, there are severe limitations with this method. Mutagenic agents cause random mutations, thus specificity and preci- sion are not possible. Potentially deleterious undetected mutations can occur, since selection systems may be geared for only the mutation of interest. Additionally, traditional mutation procedures are extremely costly and time-consuming and there is no opportunity to expand the gene pool. In spite of these limitations, mutation and selection techniques have been used extensively to improve industrially im- portant microorganisms and, in some cases, yields of greater than 100- times the normal production level of bacterial secondary metabolites have been achieved. Natural Gene Transfer Methods The discovery of natural gene transfer systems in bacteria has greatly facilitated the understanding of the genetics of microbial starter cultures and in some cases has been used for strain improvement. Genetic exchange in bacteria can occur naturally by three different mechanisms: transduction, conjugation, and transformation. Transduction Transduction involves genetic exchange mediated by a bacterial virus (bacteriophage). The bacteriophage acquires a portion of the chromosome or plasmid from the host strains and transfers it to a recipient during subsequent viral infection. Although transduction has been exploited for the development of a highly efficient gene transfer system in the gram-negative organism Escherichia coli, it has not been used extensively for improving microorganisms used in food fermentations. In general, transduction efficiencies are low and gene transfer is not always possible between unrelated strains, limiting the usefulness of the technique for strain improvement. In addition, . . . . ~ . . · . . . . . . ~ . . ~ bacteriophage nave not been isolated ano are not well cnaraclerlzea for most strains. Conjugation Conjugation, or bacterial mating, is a natural gene transfer system that requires close physical contact between donors and recipients and is responsible for the dissemination of plasmids in nature. Numerous
22 FERMENTED FOODS genera of bacteria harbor plasmid DNA. In most cases, these plasmids are cryptic (the functions encoded are not known), but in some cases important metabolic traits are encoded by plasmid DNA. If these plasmids are also self-transmissible or mobilizable, they can be trans- ferred to recipient strains. Once introduced into a new strain, the properties encoded by the plasmid can be expressed in the recipient. The lactic acid bacteria naturally contain from one to more than ten distinct plasmids, and metabolically important traits, including lactose- fermenting ability, bacteriophage resistance, and bacteriocin produc- tion, have been linked to plasmid DNA. Conjugation has been used to transfer these plasmids into recipient strains for the construction of genetically improved commercial dairy starter cultures. There are some limitations in the application of conjugation for strain improvement. To exploit the use of conjugative improvement requires an understanding of plasmid biology and, in many cases, few conjuga- tive plasmids encoding genes of interest have been identified or sufficiently characterized. Conjugation efficiencies vary widely and not all strains are able to serve as recipients for conjugation. Moreover, there is no opportunity to expand the gene pool beyond those plasmids already present in the species. Transformation Certain microorganisms are able to take up naked DNA present in the surrounding medium. This process is called transformation and this gene transfer process is limited to strains that are naturally competent. Competence-dependent transformation is limited to a few, primarily pathogenic, genera, and has not been used extensively for genetic improvement of microbial starter cultures. For many species of bacteria, the thick peptidoglycan layer present in gram-positive cell walls is considered a potential barrier to DNA uptake. Methods have been developed for enzymatic removal of the cell wall to create protoplasts. In the presence of polyethylene glycol, DNA uptake by protoplasts is facilitated. If maintained under osmotically stabilized conditions, transformed protoplasts regenerate cell walls and express the transformed DNA. Protoplast transformation procedures have been developed for some of the lactic acid bacteria; however, the procedures are tedious and time-consuming, and frequently parameters must be optimized for each strain. Transformation efficiencies are often low and highly variable, limiting the application of the technique for strain improvement. Electroporation The above mentioned gene transfer systems have become less popular since the advent of electroporation, a technique involving the
MICROBIAL STARTER CULTURES 23 application of high-voltage electric pulses of short duration to induce the formation of transient pores in cell walls and membranes. Under appropriate conditions, DNA present in the surrounding medium may enter through the pores. Electroporation is the method of choice for strains that are recalcitrant to other gene transfer techniques; although optimization of several parameters (e.g., cell preparation conditions, voltage and duration of the pulse, regeneration conditions, etc.) is still required. GENETIC ENGINEERING Genetic engineering provides an alternative method for improving microbial starter cultures. This rapidly expanding area of technology provides methods for the isolation and transfer of single genes in a precise, controllable, and expedient manner. Genes that code for specific desirable traits can be derived from virtually any living organism (plant, animal, microbe, or virus). Genetic engineering is revolutionizing the science of strain improvement and is destined to have a major impact on the food fermentation industry. Although much of the microbial genetic engineering research since the advent of recombinant DNA technology in the early 1970s has focused on the gram-negative bacterium Escherichia coli, significant progress has been made with the lactic acid bacteria and yeast. Appropriate hosts have been identified, multifunctional cloning vectors have been constructed, and reliable, high-efficiency gene transfer procedures have been developed. Further, the structural and functional properties, as well as the expression in host strains, of several important genes have been reported. Engineered bacteria, yeast, and molds could also be used for the production of other products, including food additives and ingredients, processing aids such as enzymes, and pharmaceuticals. Prerequisites Metabolism and Biochemistry of the Host A necessary prerequisite for the application of genetic engineering to any microorganism is a fundamental understanding of the metabolism and biochemistry of the strain of interest. Although for hundreds of years the metabolic potential of microbial starter cultures has been exploited, in many cases little is known about specific metabolic pathways, the regulation of metabolism, or structural and functional relatic~nshins of critical genes involved in metabolism. This information
24 FERMENTED FOODS is essential for the design of genetic improvement strategies, as it provides the rationale for selection of desirable genets) and assures that once inserted into a new host, the genets) will be appropriately expressed and regulated as predicted. Transformable Hosts Plasmid-free, genetically characterized and highly transformable hosts, coupled with multifunctional expression vectors, provide the necessary tools for transfer, maintenance, and optimal expression of cloned DNA in microbial starter cultures. Many microbial starter cultures harbor plasmid DNA, and although most plasmids remain cryptic, resident plasmids interfere with identification of plasmid- containing transformants. Use of plasmid-free hosts also eliminates plasmid incompatibility problems and the possibility of cointegrate formation between transforming and endogenous plasmids. It is im- portant to note that plasmid-free strains are used for the development of model systems; however, ultimately it will be necessary to engineer commercial strains. Vector Systems A vector can be defined as a vehicle for transferring DNA from one strain to another. Plasmids are frequently used for this purpose because they are small autonomously replicating circular DNA forms that are stable and relatively easy to isolate, characterize, and manipulate in the laboratory. Native plasmids do not naturally possess all of the desirable features of a vector (e.g., multiple cloning sites, selectable markerts), ability to replicate in several hosts, and so forth). Therefore, genetic engineering is frequently used to construct multifunctional cloning vectors. Although antibiotic resistance markers greatly facili- tate genetic engineering in microbial systems, vectors derived solely from food-grade organisms may be critical in obtaining regulatory approval for use of the organisms, as antibiotic resistance determinants may not be acceptable in food systems. An alternative vector strategy involves the development of linear fragments of DNA that are capable of integrating into the host chromosome via homologous recombination. Although transformation frequencies are very low, the advantage of the integrative vector is that transformed genetic information is targeted to the chromosome where it will be more stably maintained. Insertion sequences (IS elements) naturally present in the chromosome that can transpose chromosomal DNA to plasmids could be used as an alternative strategy for developing integrative vectors for some strains of lactic acid bacteria.
MICROBIAL STARTER CULTURES Efficient Gene Transfer Systems 25 Once genefs) have been identified and cloned into the appropriate vector in the test tube, they must be introduced into a viable host. Since the recombinant DNA is a naked DNA molecule, gene transfer systems based on protoplast transformation and electroporation are most applicable in genetic engineering experiments. High transforma- tion efficiencies (greater than 104 to 105 transformants per kilogram of DNA) greatly facilitate screening and identification of appropriate transformants. Electroporation is the transformation procedure of choice for most microbial strains. Expression Systems Transfer of structural genes to a new host using genetic engineering does not guarantee that the genes will be expressed. To optimize expression of cloned genes, efficient promoters, ribosome-binding sites, and terminators must be isolated, characterized, and cloned along with the genets) of interest. Identification of signal sequences essential for secretion of proteins outside the cell may be useful for situations where microbial starter cultures are used to produce high- value food ingredients and processing aids. Secretion into the medium greatly facilitates purification of such substances. Properties of Interest Several properties could be enhanced using genetic engineering. For example, bacteriocins are natural proteins produced by certain bacteria that inhibit the growth of other often closely related bacteria. In some cases, these antimicrobial agents are antagonistic to pathogens and spoilage organisms commonly found as contaminants in fermented foods. Transfer of bacteriocin production to microbial starter cultures could improve the safety of fermented products. Acid production is one of the primary functions of lactobacilli during fermentation. Increasing the number of copies of the genes that code for the enzymes involved in acid production might increase the rate of acid production, ensuring that the starter will dominate the fermentation and rapidly destroy less-aciduric competitors. Certain enzymes are critical for proper development of flavor and texture of fermented foods. For example, lactococcal proteases slowly released within the curd are responsible for the tart flavor and crumbly texture of aged cheddar cheese. Cloning of additional copies of specific proteases involved in ripening could greatly accelerate the process. An engineered Saccharomyces cerevisiae (baker's yeast), which is more efficient in leavening of bread, has been approved for use in the
26 FERMENTED FOODS United Kingdom and is the first strain to attain regulatory approval. This strain produces elevated levels of two enzymes, maltose permease and maltase, involved in starch degradation. Limitations There are a number of issues that must be resolved before genetically engineered starter cultures could be used in food. Engineered strains will need to be approved for use by appropriate regulatory agencies. To date, no engineered organisms have been approved in the United States, and specific criteria for approval have not been established by the Food and Drug Administration. The public must be assured that the products of biotechnology are safe for consumption. If consumers have the perception that the products are not safe, the technology will not be utilized. Although genetic engineering is probably safer and more precise than strain- improvement methods used in the past, most U.S. consumers are not aware of the role of bacteria in fermented foods and do not have a fundamental understanding of recombinant DNA technology, and they may be unwilling to accept the technology. This may be less of a problem in developing countries where improved microbial starter cultures could provide significantly safer and more nutritious foods with longer shelf life and higher quality. Another limitation is that genetic improvement of microbial starter cultures requires sophisticated equipment and expensive biological materials that may not be available in developing countries. Where equipment and materials are available in industrialized countries, there may be little incentive for researchers to improve strains that would probably not be used in their own countries. Genetic improvement of microbial starter cultures is most appropriate for those fermentations that rely solely or primarily on one microorgan- ism. In many cases, our knowledge about the fermentation is limited, making selection of the target strain very difficult. Since many food fermentation processes are complex and involve several microorgan- isms, genetic improvement of just one of the organisms may not improve the overall product.
3 Sudan's Fermented Food Heritage Hamid A. Dirar If we accept the idea that Africa is the birthplace of Man, it would seem logical that the first human or humanoid to consume a fermented food would have lived there. That fermented product could have been a piece of meat or some kind of berry picked up or stored by a hunter- gatherer. Later, and after those early men, or rather women, developed the taste for such goods they began to intentionally store fresh food items to undergo spontaneous fermentation. Should this be the case, one would expect to find in Africa today a diverse array of fermented food products. Unfortunately, we know very little about African fermented foods because no genuine attempt has been made by any African scientist to document all the fermented foods of his or her country. For at least one African country, the Sudan, I set out 6 years ago to collect, confirm, reconfirm, sift, and classify information on all fermented foods in the country. The major source of information was the elderly rural women of Sudan. The list of fermented foods and beverages, which now includes 60 different items, will make the basis for a book that should be ready for publication within a year. In the following sections I discuss some of the important aspects that came out of this personal initiative, which was not in any way sponsored by any agency, except perhaps some help from Band Aid of Britain. FERMENTED FOODS The Sudanese seem to bring just about anything edible or barely edible to the forge of the microbe, to the extent that one could confidently say: food in Sudan is fermented. The raw materials to be fermented include the better-known products such as sorghum, millet, milk, fish, and meat. Also, a number of unorthodox raw materials are 27
28 FERMENTED FOODS fermented: bones, hides, skins, hooves, gall bladder, fat, intestines, caterpillars, locusts, frogs, and cow urine. The bulk of these foods is poured into the bowl of sorghum porridge, being either a sorghum (or millet) staple or its sauce and relish. The few remaining ones are alcoholic or nonalcoholic beverages, the most important of which are prepared from sorghum. In other words, every fermented food item orbits around the sorghum grain. Sorghum-Based Foods Sorghum fermented foods and drinks are the most sophisticated and are prepared by the most complicated procedures. Compared with similar sorghum products of Africa and indeed of the whole world, the Sudan's sorghum products stand out as unique in many respects: · The Sudan seems to have the greatest number of fermented sorghum products. There are about 30 such products that are basically different from one another. · There is a wide use of sorghum malt in the preparation of food and drink. Throughout Africa sorghum malt is more commonly used in the preparation of beers. In Sudan, however, while malt is used in three major beer types, it is also used to make some seven solid food products. This situation does not seem to hold true for other African countries, judging by the literature. · The making of bread-type foods from sorghum is not common in Africa. The Sudan, however, has about 12 sorghum breads (discs, sheets, flakes). Close scrutiny of these breads reveals an influence from the Middle East; some of these breads carry names and are prepared by methods used for similar products in the Arab World. · A comparison of the procedures followed in the preparation of some sorghum food products in Sudan with procedures for making similar products in other African countries suggests that the art of making these products traveled from Sudan to West Africa and perhaps to East Africa, too. In some cases the product travelled carrying the same Arabic-Sudanese name. This suggests that sorghum food culture is more ancient than in other areas of Africa, and this food evidence may be taken to strengthen previous hypotheses that the origin of sorghum domestication is somewhere in northeast Africa. Dairy Products The most common fermented milk product of Sudan is rob. Milk is fermented overnight, and the resulting sour milk is churned to give
SUDAN'S FERMENTED FOODS 29 butter; the remaining buttermilk is rob. The principal aim behind rob production is the need to facilitate the extraction of butter from the milk. The butter (furssah) is later boiled to give butter oil or ghee, which can be stored for use in the lean season. Rob production is in the hands of animal-owning nomadic tribes, and the bulk of it is produced during the rainy season (July-October). Huge amounts of rob are thrown away during this season as useless after the butter has been removed. Some women, however, allow the souring process to proceed further after butter extraction until the curd is separated from the whey. They then collect the curd and sun dry it to give a kind of granular cheese called kush-kush that is turned into sauce for sorghum porridge in later months. Another kind of sour milk is fermented camel milk, called gariss. This is probably the only fermented food product invented by men. Gariss is prepared by camel boys who depend on it as their major nourishment when they roam with their herds into remote areas. The milk is fermented in a skin bag hitched to the saddle of a camel that is allowed to go about its business as usual grazing, sleeping, walking, trotting, etc. This product, unlike rob, is fermented for consumption and no butter is removed from it. A third indigenous dairy product is biruni, also called leben-gedim, which is a fermented unchurned milk ripened for up to 10 years! A related product, but not ripened, is mish, which is made by prolonged fermentation to the extent that maggots thrive in it. The product is consumed whole, with the maggots included. These two products are closely related to Egyptian mish (11. Dairy products that have entered the Sudan from Egypt within the last century are jibnabeida (white cheese), zabadi (yogurt), and black cumin-flavored mish. These products are strictly confined to urban communities, where the Egyptian influence is more strongly felt. Fish Products Southeast Asia takes all the fame in the literature concerning the production of fermented fish products. But if one sorts out all the various products of that corner of the world carrying a confusing array of names, one finds that the products boil down to four major categories: sauces, pastes, dried fish, and whole salted fish. These four types of fermented fish products are also found in the Sudan, only they are all prepared from freshwater Nile fish. This situation has not been reported for other African or Arab countries. The Sudanese fish products include kejeik (large sun-dried split fish); fessiekh (salted fermented whole tiger fish); mindeshi (pounded small fish paste, fermented, and may be dried later); and terkin or melaha (fermented fish sauce or paste- not dried).
30 FERMENTED FOODS Meat Products While some urban people in Sudan make very thin strips of red beef and dry them in the sun to give shermout, the traditional rural product is a truly fermented one. Thick strips of fat-bearing meat are hung on a rope indoors and left to undergo fermentation and slow drying to give a proteolytic product, shermout. The Sudanese also ferment the sheath of fat surrounding the stomach to give the strongest-smelling product of all, miriss. Others ferment the small intestines to give musran. The clean small intestines may also first be sun dried together with strips of the lungs, heart, kidneys, liver, etc., and then all pounded together and mixed with some potash and molded into a fist-sized ball and allowed to slowly ferment and dry, to give twini-digla. The large intestine is cleaned and stuffed with fat and hung to ferment and dry for a month, to give the sausage called skin. Beirta is prepared from he-goat meat. Small pieces of muscle meat, lungs, kidneys, liver, heart, etc., are mixed with milk and salt, packed into a clay pot, and allowed to undergo some sort of pickling, presumably. Um-tibey is best prepared from gazelle's meat. The rumen is carefully emptied and then stuffed with the vertebrae of the neck, cut-up heart, kidneys, liver, etc. The rumen is next tied and hung high to undergo fermentation. The whole thing may then be cooked by burying it in hot ashes and embers. Fresh bones may be fermented in a number of ways. The large bones, with pieces of attached meat and tendons, may simply be thrown on a thatched roof to ferment slowly for weeks or even months to give the product called adum (bone). The meshy ball bone endings of the ball and socket joints may be pounded fresh and fermented into a paste called dodery. The vertebrae of the backbone may be chopped into smaller pieces that are sun dried, pounded with stones, mixed with a little water and salt, molded into a ball, and allowed to ferment and dry to give kaidu-digla (bone ball). The fresh hide, skin, or hoof may be buried in mud or moist ash to undergo fermentation. The fermented product can then be cut into strips or pieces and sun dried and stored. The gall bladder is removed full with its gall juice. Some sorghum flour or grains are added to the juice to absorb it and then hung to undergo slow drying. The product, itaga, is later pounded into a sort of spice usually consumed with fatty meat dishes. Vegetable Products A number of fermented vegetable products are produced in rural Sudan. Interestingly, these products can be grouped into either meat
SUDAN'S FERMENTED FOODS 31 substitutes or -sour milk (rob) substitutes, the two major flavors of sauces in the country. Kawal (2,3) is the major meat substitute. It is a strong-smelling product derived by fermentation of the pounded green leaves of the wild legume Cassia obtusifolia, which grows during the rainy season. The product is used in the preparation of sauces to completely replace meat or for use as a meat extender. Its protein is of high quality, rich in the sulfur amino acids. Furundu, a similar meat substitute, is prepared from the seeds of red sorrel Hibiscus sabdariffa. Sigda is another meat substitute and is prepared by fermentation of sesame oilseed presscake. All these products are dried after fermentation in the form of hard, irregular, small balls and may keep for a year or so. Other ill-defined but related products are kerjigil (from a mixture of pumpkins, sesame, and cowpea) and teshnuti (from okra seeds). Sour milk (rob) substitutes are made from oil-bearing seeds in a manner analogous to the use of soybeans to give dairy product analogs. Rob-heb is made from the seeds of the watermelon. Rob-ful is made from peanuts. In either case the seeds are pounded into a paste that is allowed to undergo a souring fermentation. When mixed with water and turned into sauce the product has the color (off white) and taste (sour) of the sour milk sauce called mulah-rob. A related product is um-zummatah, obtained by the souring fermentation of watermelon juice. The same name is sometimes given to the sour steep water, also called mayat-aish, of fermented whole sorghum or millet grain. Alcoholic Products Opaque beers are commonly brewed in Africa but procedures vary. The brewing of merissa in Sudan is probably the most complicated and advanced of all (4,51. The unique features of this brewing method include the use of only a small amount (5 percent) of sorghum malt as an enzyme preparation, rather than a substrate. Malt constitutes 25 to 100 percent of the substrate in the brewing of most African and European beers. Another unique feature is the use of a caramelized sorghum product, called surij, in the process. Third, there is a special starter activation step during the process that is lacking from other African brewing procedures. Also, the brewer women seem to be aware of the properties of enzymes and microbes as well as those of the acids produced during fermentation. This explains the unique treatment of the substrate, where parts of it are half cooked, others fully cooked, and yet others overcooked to meet enzyme requirements for a mixture of raw and gelatinized starch and to effect sterilization of products when needed. The merissa process has been well recognized as a complex process that deserves further investigation. Clear beers are not common in Africa, and the literature gives reports
32 FERMENTED FOODS only on otika of Nigeria and amgba of Cameroon (6,71. The Sudan has a clear sorghum (or millet) beer called assaliya (or um-bilbil). A look at the production of these three beers reveals that the assaliya process, involving some 40 steps, is far more complicated than the otika or amgha procedures, which involve fewer than 20 steps. It is suggested that the art of brewing clear beers traveled to West Africa from Sudan. Amgba of Cameroon is even called bilbil. In Sudan there are perhaps 30 to 50 opaque beer types with different but related brewing methods. The area seems to be a center of diversity of sorghum beers, and perhaps the art of brewing of opaque beers traveled to East Africa from this region. The traditional wines of Sudan are the date wines. The palm wine of West Africa is not known in Sudan nor is lagmi, the wine obtained by fermentation of the sap of the date palm as practiced in northwest Africa. Only the fruit of the date palm is fermented in the Sudan, and the bulk of wines thus made are produced and consumed in the Northern Province where most of the date palms exist. At least 10 different date wines are produced, the most important of which are sherbet, nebit, and dakkai (81. In the Southern Sudan a kind of mead is produced by fermentation of diluted wild bee's honey. The product, called duma, is primed by a specially prepared starter culture called duma-grains (iyal-duma). FERMENTED FOODS AND SURVIVAL STRATEGIES A careful examination of fermented food products of Sudan would immediately suggest a close link between food fermentation and food shortage in this part of the world. First, about 80 percent of these foods, particularly the marginal ones using bones, intestines, fat, etc., are found in western Sudan in the Kordofan and Darfur regions, the traditional famine areas. Second, most of the foods are preserved by both fermentation and drying, which means that they are intended for long storage and that food shortages or even famine are anticipated. In other words, the inventors of such foods have the experience of repeated famines. Further, practically all fermented sauce ingredients are produced during the late months of the rainy season, which shows that, unless a person secures all of his or her food requirements from this short season, he or she will probably suffer greatly in the remaining 9 months of the year. The harsh environment has actually dictated the need to ferment and dry anything that might prevent starvation. To live on the edge of the desert must have been a great force in sharpening the sense for survival and creativity.
SUDAN'S FERMENTED FOODS 33 The strong link between many fermented foods and food shortages is also revealed by the fact that if a family became rich it would drop a number of fermented foods from its menu, not because of social pressure but because there was no longer any need for them now that ample supplies of meat, milk, poultry, etc., were available. Poor people who ferment bones, hides, locusts, etc., do so not because they relish these foods but because it is part of the coping strategy they follow to deal with the vagaries of a capricious environment. The first victims of any famine are the children, among whom death exacts a great toll. Babies and children die in the laps of women more than they do in the laps of men. Maternal compassion must be the greatest impetus behind the rural woman's desperate attempts to save her child that propel her to look for an insect, a piece of hide, a frog, or a bone as savior. Many fermented foods are thus famine foods, and rural women must be credited with their invention. These women must have saved thousands of children from certain death during famines. Their vital role must be recognized and hailed. BIOTECHNOLOGY AND FERMENTED FOODS This relationship has not been discussed widely in the literature. One can imagine, however, that biotechnology can be of help in the improvement of fermented foods at three levels: · Raw materials. Fermented foods are produced from either animal or plant starting materials, and the availability of these substrates will of course aid in the production of fermented foods. Biotechnological methods to improve animal and plant production have been dealt with by experts in those fields on many occasions. Only a special reminder should be made not to neglect certain wild plants and marginalized crops the so-called lost crops of Africa (e.g., sorrel and okra). Attempts to restore the forest cover should give some attention to trees that bear fruits used during famines or even trees that host caterpillars. · Fermentation engineering. Recent developments in biotechnology have -given rise to great innovations in bioreactor designs. Most of these designs deal with liquid reaction media, but it should not be forgotten that a great number of fermented foods are produced through a solid-substrate fermentation in which the fermenting paste is frequently hand mixed. Bioreactors to simulate such a process are needed for the modernization of such traditional fermented foods. ~ Microbiology and enzymology. There are many opportunities for biotechnological innovations in the microbiology of fermented foods.
34 FERMENTED FOODS First, all the microorganisms involved should be isolated, character- ized, and preserved as a germplasm collection. Second, the metabolic role of each of the strains involved should be clearly identified, and their full potential, even in other fields of biotechnology, should be studied. The powerful technique of monoclonal antibodies for the characterization of different strains of the same species can be of great help in this area. Many of these organisms have the enzyme complement to produce vitamins and amino acids in fermented foods. This potential can be improved through the technique of recombinant DNA technology to produce strains that are capable of producing and releasing the required amino acid or vitamin into the food. To avoid food losses due to spoilage-causing organisms and to avoid possible development of food-poisoning microbes, it is possible to genetically engineer a strain required for a process as a pure culture. Such a strain may bring about all the changes required in the food and grow at a convenient temperature. REFERENCES 1. Abdel-Malek, Y. 1978. Traditional Egyptian dairy fermentations. Global Impacts of Applied Microbiology 5: 198-208. 2. Dirar, H. A. 1984. Kawal, a meat substitute from fermented Cassia obtusifolia leaves. Economic Botany 38:342-349. 3. Dirar, H. A., D. B. Harper, and M. A. Collins. 1985. Biochemical and microbiological studies on kawal, a meat substitute derived by fermentation of Cassia obtusifolia leaves. Journal of the Science of Food and Agriculture 36:881-892. 4. Dirar, H. A. 1976. The art and science of merissa fermentation. Sudan Notes and Records 57:115-129. 5. Dirar, H. A. 1978. A microbiological study of Sudanese merissa brewing. Journal of Food Science 43:1683-1686. 6. Ogundiwin, J. O. 1977. Brewing otika ale from guinea corn in Nigeria. Brewing and Distilling International 7~61:40-41. 7. Chevassus-Agnes, S., J. C. Favier, and A. Joseph. 1976. Techno- logie traditionelle et valeur nutritive des "bieres" do sorgho du Cameroon. Cahier de Nutrition et de Dietetique 11~2~:89-104. 8. All, M. Z., and H. A. Dirar. 1984. A microbiological study of Sudanese date wines. Journal of Food Science 49:459-460, 467.
4 ·esser-Known Fermented Plant Foods Kofi E. Aidoo In many parts of the world, fermented foods form an important part of the diet. These foods are made from plant and animal materials in which bacteria, yeasts, and molds play an important role by modifying the material physically, nutritionally, and organoleptically. Fermented plant foods may be classified into groups as (a) those made from cereal grains (maize, sorghum, millet, rice, wheat), such as pozol (Mexico), kenkey, ogi, and injera (Africa); (b) those made from pulses, nuts, and other seeds, such as ontjom (Indonesia) and dawadawa (Savannah Africa); (c) those from tubers (cassava, aroids, potatoes), such as gari (Africa) and farinha puba (Brazil, Peru, and Ecuador); (d) those from fruits and vegetables, such as gundruk (Nepal) and kimchi (Korea, East Asia); and (e) beverages derived from tree saps, such as nipa wine (Far East? and pulque (Mexico). Most traditional fermented plant foods are prepared by processes of solid-substrate fermentation in which the substrate is allowed to ferment either spontaneously (usually African or Latin American processes) or by adding a microbial inoculum (Far East, South Asia, and Southeast Asia). Cereal grains account for more than 60 percent of food materials used in the preparation of indigenous fermented foods in Africa. Although maize is a comparatively well-researched crop, no significant research has been done on some of the important traditional crops, such as sorghum and millet (1~. Tef (Eragrostis tef), a staple food grain of Ethiopian subsistence farmers, is still relatively less well known. Many indigenous fermented foods, some of which long predate recognition of the existence of microorganisms, are eaten in various parts of the world. Increasing interest in this field is reflected in the range of publications (2-101. This paper presents information on some of the lesser-known fermented plant foods that are still produced and 35
36 FERMENTED FOODS marketed on a small scale and that serve as a staple diet for millions of people in developing countries. REGIONAL PERSPECTIVES Cereals are major staples in many developing countries, and the fermentation of cereal grains to prepare a variety of foods has a long history. Fermented products from maize are usually found in Africa and Central and South America and those from sorghum (guinea corn) and millet in Africa and South Asia. Food fermentations based on rice are practiced in India, China, Southeast Asia, and the Far East, while those from wheat are particularly important in the Middle East, Turkey, and the Far East (111. Fermented foods from tubers are usually found in Africa, among the Andean Indians and in the South Pacific, and the process of detoxification of the tuber before fermentation is still carried out by soaking in water. Chica, an alcoholic beverage made from maize in Peru since pre- Hispanic times, also is produced from potato, oca (Oxalis tuberosa), arracacha (Arracacia xanthorrhiza), maca (Lepidium evenii), and other Incan crops that science has almost totally neglected. Although cassava and sweet potatoes provide nourishment for more than 500 million people, only a small proportion of this highly perishable staple crop is used in food fermentations in Africa and Latin America. Legumes account for a substantial amount of food protein intake in developing countries. Of the total world production of over 58 million metric tons in 1990, developing countries produced 62 percent, together with 54 percent of world nut production (121. Fermented products from legumes are not as popular in Africa or Latin America as in the Far East and South and Southeast Asia, where soybean, for instance, is used extensively in the production of fermented products such as soy sauce, miso, and tempe, and black gram dhal for the production of idli and dosa. Fermented seed products, however, are often used as condiments in Savannah Africa. In the tropics, highly perishable foods such as fruits and vegetables may be preserved as fermented products. Some fermented vegetables provide vitamins, particularly during long cold months in the northern parts of East Asia, and others are consumed as part of traditional family life in Southeast Asia. In Mexico refreshing beverages are prepared from a variety of fruits, including pineapples, apples, and oranges.
LESSER-KNOWN FOODS PRODUCTS FROM CEREAL GRAINS Ahai 37 Ahai is a sweet, malty-tasting beverage brewed from maize in Southern Ghana and is usually served as a welcome drink and at outdoor ceremonies, wakes, and funerals. Whitby (13) has reported that the traditional method of preparing ahai is much the same as for pito, an acid-alcohol beer brewed from sorghum or millet in West Africa, except that ahai is not boiled again after fermentation. So far, no studies have been made on the microbiological, biochemical, and nutritional changes that take place during ahai production. Ting Ting is a staple food for a large proportion of the population of Botswana. It is prepared from maize by natural fermentation. In other regions it is prepared from sorghum or millet. Moss et al. (14) made an extensive study of tiny fermentation and noted that the success of the fermentation depends on a number of factors, among which temperature is very important. The microbiology of tiny fermentation is well documented, but further studies need to be carried out, particularly on the nutritional value. Ting may be similar, nutritionally, to other acid-fermented cereal gruels like kenkey (West Africa), kisra (Sudan), and pozol (Mexico). Maasa Mausa is a snack food made from millet or sorghum and is very popular in Savannah Africa, particularly during Ramadan. The method of preparation of mnasa has been reported (9), but there is no information on the microbiology and biochemistry of this fermented product. There are hundreds of fermented products from cereal grains in the tropical regions of the world that require extensive studies on methods of preparation and biochemical, microbial, and nutritional changes. These include the West African fura or fula, jamin-bang of the Kaingang Indians of Brazil, and the Maori's kaanja-kopawai, a process of fermenting maize in water prior to eating. The Maoris claim koanja- kopawai is health giving, and many of the older people attribute their age to this part of their diet.
38 PRODUCTS FROM ROOT TUBERS Farinha puba FERMENTED FOODS Farinha puba is a coarse flour made from cassava and is found in the Amazonian regions of Brazil, Peru, and Ecuador. Woolfe and Woolfe (15) presented an outline on the preparation of Farinha puba, which is also known as farinha de mandioca in Brazil. They noted that the technology was exported to West Africa in the nineteenth century and presumably adapted locally to give the gari process. Gari, a popular West African staple food that is also eaten in other tropical African countries, is prepared by fermenting cassava; details of improved methods of production are given by Steinkraus et al. (61. The processes involved in the production of farinha puba and gari are similar, but unlike gari very little information has been published on the methods of production and on the microbiology, nutritional values, and toxicological problems offarinha puba. It has been reported that cassava fermentation as practiced in Africa, Asia, and Latin America (16) is an unreliable detoxification method, and the process further reduces the already low protein content. Other studies have shown that cassava fermentation for gari production does not totally eliminate the cyanide content but reduces it by at least 65 percent (17,18~. Fatalities from cassava poisoning appear to be rare, but long- term toxic effects, (e.g., goiter and cretinism) in cassava-consuming populations may be more serious, especially in the Amazon, where the pressed-out juices are used for making soups and stews (151. In Africa the pressed-out juice is often used for the production of cassava starch for laundry purposes. The use of pure microbial cultures under controlled fermentation conditions might bring about not only complete hydrolysis of the poisonous glycoside but also an enhanced fermenta- tion process. Kokonte Kokonte, another important cassava-based staple, is eaten by millions of people in Savannah Africa. Like many other fermented foods, kokonte (Ghana) is known by various names such as ilalun (Nigeria) and icingwadal (East Africa). The method of preparation of kokonte has been reported, but further studies need to be done, particularly on microflora and production of mycotoxins during fermentation (19,201.
LESSER-KNOWN FOODS 39 Masato (masata) Masato, or cassava beer, is an alcoholic beverage produced from cassava in the Amazon. It has an alcohol content of 6 to 12 percent by volume and is offered to guests as a sign of hospitality. It is considered an offense to refuse a drink (15~. In Brazil it is called kaschiri and in Mozambique masata. Preparation of masato is similar to that of chica by the Andean Indians. As a first step of fermentation, cassava is chewed and spat out by women. In Mozambique women chew the yucca plant to produce a similar product. So far, no scientific account of the masato fermentation process has been published. Studies on improving the traditional methods of production are necessary to save this ancient art of the Andean Indians from extinction. Chuno Chuno is a food product from potato prepared by the inhabitants of the high Andes of Peru, Chile, Ecuador, Colombia, and Bolivia. An outline of the method of production has been reported, but the microorganisms involved in the fermentation are still not known (91. The Incan anu (Tropaecolum tuberssum) is a tuber that must be fermented before being eaten baked, fried, or added to stew (211. The crop is cultivated in Colombia, Peru, and Bolivia and is also grown as a flowering ornament in Britain and the United States. The fermentation involved during 'icuring" has not been reported. PRODUCTS FROM LEGUMES, PULSES, AND OTHER SEEDS In Savannah Africa, fermented products from legumes and other seeds are important food condiments and are generally strong smelling. Quite often seeds that are used for fermentation are inedible in their raw unfermented state. Fermentation of the West and Central African iru or dawadawa is similar to the Japanese natto, and there is adequate literature on the preparation, biochemistry, microbiology, and industrialization of iru. Other indigenous products that are receiving some attention include ugba (African oil bean seed), ogiri (seeds of watermelon), ogiri-igbo (castor oil seed), and ogiri-nwan (fluted pump- kin beans). Lupins (Lupinus mutabilis), which are native to the Andes, contain bitter alkaloids and can cause toxicity problems. Lupin seeds are
40 FERMENTED FOODS debittered by soaking them in running water, a process similar to the Maoris' process for corn fermentation and the Ichunol methods of Peru and Bolivia. So far, no report has been published on the debittering of lupine by fermentation, but the soaking may involve some fermentation. Kenima is a Nepalese fermented product from legumes. There is no published information on the method of preparation, microbiology, and nutritional value. PRODUCTS FROM FRUITS AND VEGETABLES Colonche is a sweet fizzy beverage produced in Mexico by fermenting the juice of tunas (fruits of the prickly pear cacti, mainly Opuntia species). Tepache is also a refreshing beverage prepared originally from maize but from various fruits and is consumed throughout Mexico. Although some studies have been made on these products (22), it appears that more work is needed, particularly on the biochemical and nutritional changes that take place during the preparations. The Nepalese pickle or gundrak is a fermented dried vegetable served as a side dish with the main meal and is also used as an appetizer in the bland starchy diet. Several hundred tons of gundrak is produced annually, and production is still at the household level. Dietz (23) reported on the method of preparation and the role of gundrak in the diet of Nepalese people. It has been found that a disadvantage of the traditional process is loss of 90 percent of the carotenoids. Improved methods and further studies might help reduce vitamin loss. COMMERCIALIZATION To industrialize some of these fermented plant foods from traditional processes, extensive studies must be made to determine the essential microorganisms, optimum fermentation conditions, biochemical changes, nutritional profile, and possible toxicological problems associ- ated with certain plant materials or the fermented product itself. Commercial or large-scale processes for indigenous fermented foods need to be adapted to specific local circumstances. Advantages of industrialization include a product with an extended shelf life, maximum utilization of raw materials, production of important by-products, and bioenrichment or fortification of a product for specific consumers such as special diets, weaning foods and exclusion of or reduction in the
LESSER-KNOWN FOODS 41 levels of mycotoxins. Mycotoxins appear to be a major problem in some fermented products, particularly those of cereal and root tuber Orlgln. Studies in Japan on okara, a by-product of the tofu industry, have shown that fermenting it with tempe fungus could result in a product that is useful as a high-fiber, low-energy food material (24~. REFERENCES 1. Cross, M. 1985. Waiting for a green revolution. New Scientist 1486 30. 2. Hesseltine, C. W. 1965. A millennium of fungi, food and fermentation. Mycologia 57: 149-197. 3. Hesseltine, C. W. 1983. The future of fermented foods. Nutrition Review 41:293-301. 4. Rose, A. H. 1982. Economic Microbiology. Fermented Foods, Vol. 7, London: Academic Press. 5. Steinkraus, K. H. 1983. Fermented foods, feeds and beverages. Biotechnology Advances 1 :31~6. 6. Steinkraus, K. H. 1983. Handbook of Indigenous Fermented Foods. New York: Marcel Dekker. 7. Beuchat, L .R. 1983. Indigenous fermented foods. Pp. 477-528 in: Biotechnology, Vol. 5. H. J. Rehm and G. Reed (Eds.) Weinheim: Verlag Chemie. 8. Wood, B. J. B. 1985. Microbiology of fermented foods. London: Elsevier Applied Science Publishers. Vols. 1 and 2. 9. Campbell-Platt, G. 1987. Fermented Foods of the World: A Dictionary and Guide. London: Butterworths. 10. Berghofer, E. 1987. Use of non-European fermented foods in Austrian market. Ernahrung 11~1~:1~22. 11. Hesseltine, C. W. 1979. Some important fermented foods of mid-Asia, the Middle East and Africa. Journal of the American Oil Chemists Society 56:367-374. 12. FAO. 1991. Food and Agriculture Organization Quarterly Bulle- tin of Statistics 4~11. 13. Whitby, P. 1968. Foods of Ghana. Food Research Institute Report 1: 1-31. 14. Moss, M. O., S. F. Mpuchane, and O. M. Murphy, 1984. Ting- a fermented maize meal product of southern Africa. Proceedings of the Institute of Food Science and Technology 17: 139-148. 15. Woolfe, M., and J. Woolfe. 1984. Some traditional processed
42 FERMENTED FOODS foods of South America. Proceedings of the Institute of Food Science and Technology (U.K.) 17:131-138. 16. Seneviratne, G. 1985. Making cassava a safer food. Development Forum, UNDESI/DPI and UNU, 13: 13. 17. el Tinay, A. H., P. L. Bureng, and E. A. E. Yas. 1984. Hydrocyanic acid levels in fermented cassava. Journal of Food Tech- nology 19:197-202. 18. Ayernor, G. S. 1985. Effect of the resting of cassava on product yield and cyanide detoxification. Journal of Food Technology 20:89- 96. 19. Aidoo, K. E. 1986. Lesser-known fermented plant foods. Tropi- cal Science 26:249-258. 20. Aidoo, K. E. 1991. Postharvest storage and preservation of tropical crops. Pp. 747-764 in: Mycotoxin and Animal Foods. J. E Smith and R.S. Henderson, Eds. Boca Raton, Fla.: CRC Press. 21. Vietmeyer, N. 1984. Lost crops of the Incas. Ceres 17~31:37~0. 22. Ulloa, M. 1980. Indigenous fermented beverages of Mexico. Pp. 45~9 in: Global Impact of Applied Microbiology. S. O. Emejuaiwe, O. Ogunbi, and S. O. Sanni Eds., London: Academic Press. 23. Dietz, H. M. 1984. Fermented dried vegetables and their role in nutrition in Nepal. Proceedings of the Institute of Food Service and Technology (U.K.) 17:208-213. 24. Matsuo, M. 1989. Morphological and physicochemical properties and composition of Okara fermented with Rhizopus oligosporus. Journal of the Japanese Society of Nutrition and Food Science 42(2): 173-178. 25. Uchimura, T., V. V. Garcia, and D. M. Flores. Microbiological studies on fermented rice cake, 'puto' and the application of puto making using cassava flour. Tropical Root Crops: Postharvest Physiol- ogy and Processing. I. Uritani and E. D. Reyes (Eds.~. Tokyo: Japanese Science Society Press. 26. Mabbett, T. 1991. Local strains make good. African Farming and Food Processing Jan/Feb: 25-26.
5 Lactic Acid Fermentations Keith H. Steinkraus Lactic acid bacteria perform an essential role in the preservation and production of wholesome foods. The lactic acid fermentations are generally inexpensive, and often little or no heat is required in their preparation, making them fuel efficient as well. Foods fermented with lactic acid play an important role in feeding the world's population on every continent. Lactic acid bacteria perform this essential function in preserving and producing a wide range of foods: fermented fresh vegetables such as cabbage (sauerkraut, Korean kimchi); cucumbers (pickles); fermented cereal yogurt (Nigerian ogi, Kenyan uji); sourdough bread and bread- like products made without wheat or rye flours (Indian idli, Philippine puto); fermented milks (yogurts and cheeses); fermented milk-wheat mixtures (Egyptian kishk, Greek trahanas); protein-rich vegetable protein meat substitutes (Indonesian tempe); amino acid/peptide meat- flavored sauces and pastes produced by fermentation of cereals and legumes (Japanese miso, Chinese soy sauce); fermented cereal-fish- shrimp mixtures (Philippine balao balao and burong dalag); and fermented meats (e.g., salami). Lactic acid bacteria are generally fastidious on artificial media, but they grow readily in most food substrates and lower the pH rapidly to a point where competing organisms are no longer able to grow. Leuconostocs and lactic streptococci generally lower the pH to about 4.0 to 4.5, and some of the lactobacilli and pedicocci to about pH 3.5, before inhibiting their own growth. In addition to producing lactic acid, lactobacilli also have the ability to produce hydrogen peroxide through oxidation of reduced nicotinamide adenine dinucleotide (NADH) by flavin nucleotide, which reacts rapidly with gaseous oxygen. Flavoproteins, such as glucose oxidase, also generate hydrogen peroxide and produce an antibiotic effect on other organisms that might cause food spoilage; the lactobacilli themselves are relatively resistant to hydrogen peroxide. 43
44 FERMENTED FOODS Streptococcus lactis produces the polypeptide antibiotic nisin, active against gram-positive organisms, including S. cremoris, which in turn produces the antibiotic diplococcin, active against gram-positive organisms such as S. Iactis. Thus, these two organisms compete in the fermentation of milk products while inhibiting growth of other gram- positive bacteria. Carbon dioxide produced by heterofermentative lactobacilli also has a preservative effect in foods, resulting, among others, from its flushing action and leading to anaerobiosis if the substrate is properly protected. Brining and lactic acid fermentation continue to be highly desirable methods of processing and preserving vegetables because they are of low cost, have low energy requirements for both processing and preparing foods for consumption, and yield highly acceptable and diversified flavors. Depending on the salt concentration, salting directs the subsequent course of the fermentation, limiting the amount of pectinolytic and proteolytic hydrolysis that occurs, thereby controlling softening and preventing putrefaction. Lactic acid fermentations have other distinct advantages in that the foods become resistant to microbial spoilage and toxin development. Acid fermentations also modify the flavor of the original ingredients and often improve nutritive value. Because canned or frozen foods are mostly unavailable or too expensive for hundreds of millions of the world's economically deprived and hungry people, acid fermentation combined with salting remains one of the most practical methods of preservation, often enhancing the organoleptic and nutritional qualities of fresh vegetables, cereal gruels, and milk-cereal mixtures. SAUERKRAUT Lactic acid fermentation of cabbage and other vegetables is a common way of preserving fresh vegetables in the western world, China, and Korea (where kimchi is a staple in the diet). It is a simple way of preserving food: the raw vegetable is sliced or shredded, and approximately 2 percent salt is added. The salt extracts liquid from the vegetable, serving as a substrate for the growth of lactic acid bacteria. Anaerobic conditions should be maintained, insofar as possi- ble, to prevent the growth of microorganisms that might cause spoilage. The sequence of organisms that develop in a typical sauerkraut fermentation is as follows: Leuconostoc mesenteroides initiates the growth in the shredded cabbage over a wide range of temperatures and salt concentrations. It produces carbon dioxide and lactic and acetic acids, which quickly lower the pH, thereby inhibiting development of undesirable microorganisms that might destroy crispness. The carbon dioxide produced replaces the air and facilitates the anaerobiosis
LACTIC ACID FERMENTATIONS 45 required for the fermentation. The fermentation is completed in sequence by Lactobacillus brevis and Lb. plantarum. Lb. plantarum is responsible for the high acidity. If the fermentation temperature or salt concentration is high, Pecicoccus cerevisiae develops and contributes to acid production. As would be expected, the rate of completion of the fermentation depends on the temperature and salt concentration. At 7.5°C fermenta- tion is very slow: under these circumstances, L. mesenteroides grows slowly, attaining an acidity of 0.4 percent in about 10 days and an acidity of 0.8 to 0.9 percent in a month. Lactobacilli and pediococci cannot grow well at this temperature, and the fermentation may not be completed for 6 months. At 18°C a total acidity (as lactic acid) of 1.7 to 2.3 percent will be reached, with an acetic to lactic acid ratio of 1:4, in about 20 days. At 32°C a similar activity will be reached in 8 to 10 days, with most of the acid being lactic acid produced by the homofermentative bacteria Lb. plantarum and P. cerevesiae. Increasing the salt concentration to 3.5 percent results in 90 percent inhibition of growth and acid production for both L. mesenteroides and Lb. brevis. The ratio of nonvolatile to volatile acid produced has a marked effect on flavor, Lb. brevis producing a harsh, vinegar-like flavor and L. mesenteroides a mild, pleasantly aromatic flavor. The homofermenters Lb. plantarum and P. cerevesiae yield unacceptable products. KOREAN KIMCHI Korean kimchi differs from sauerkraut in two respects: it has, optimally, much less acid and it is carbonated. Chinese cabbage and radish are the major substrates; garlic, green onion, ginger, leaf mustard, hot pepper, parsley, and carrot are minor ingredients. Kimchi is available year-round, is served three times daily, and is a diet staple along with cooked rice and certain side dishes. It accounts for about an eighth of the total daily food intake of an adult. Its popularity is largely due to its carbonation derived from fermentation with natural microflora. Salting of the cabbage can be done at 5 to 7 percent salinity for 12 hours or 15 percent salinity for 3 to 7 hours, followed by rinsing and draining. Optimum salt concentration during kimchi fermentation is approximately 3 percent. Lower temperatures (about 10°C) are pre- ferred to temperatures above 20°C. Optimum acidity of kimchi is 0.4 to 0.8 percent lactic acid with a pH between 4.2 and 4.5; higher acidity makes it unacceptable. Organisms isolated from kimchi include L. mesenteroides, S. faecilis, Lb. brevis, Lb. plantarum, and P. cer- evesiae.
46 PICKLED VEGETABLES FERMENTED FOODS Pickling of cucumbers and other vegetables is widely practiced today. Although a variety of techniques are used, placing cucumbers in a 5 percent salt brine is a satisfactory method. The cucumbers absorb salt until there is an equilibrium between the salt in the cucumbers and the brine. Acidity reaches 0.6 to 1.0 (as lactic acid) with a pH of 3.4 to 3.6 in about 2 weeks, depending on the temperature. In Malaysia the most common vegetables pickled are cucumbers, ginger, onion, leek, chili, bamboo shoots, and leafy tropical vegetables like mustard leaves. Young unripe fruits commonly pickled include mangoes, papaya, pineapple, and lime. In Egypt carrots, cucumbers, turnips, cauliflower, green and black olives, onions, and hot and sweet peppers are among the vegetables pickled. They are used as appetizers and served with practically every meal. INDIAN IDLI AND DOSA Indian idli is a small, white, acidic, leavened, steam-cooked cake made by lactic fermentation of a thick batter made from polished rice and dehulled black gram dhal, a pulse (Phaseolus mango). The cakes are soft, moist, and spongy and have a pleasant sour flavor. Dosa, a closely related product, is made from the same ingredients, both finely ground. The batter is generally thinner, and dosa is fried like a pancake. Idli fermentation is a process by which leavened bread-like products can be made from cereals other than wheat or rye and without yeast. The initial step in the fermentation is to wash both rice and black gram dhal. They are then soaked for 5 to 10 hours and drained. The coarsely ground rice and black gram are then combined with water and 1 percent salt to make a thick batter. The batter is fermented in a warm place (30 to 32°C) overnight, during which time acidification and leavening occur. The batter is then placed in small cups and steamed or fried as a pancake. The proportions of rice to black gram vary from 4:1 to 1:4, depending on the relative cost on the market. Idli and dosa are both products of natural lactic acid fermentation. L. mensenteroides and S. faecalis develop during soaking, then continue to multiply following grinding. Each eventually reaches more than 1 x 109 cells per gram, 11 to 13 hours after formation of the batter. These two species predominate until 23 hours following batter formation. Practically all batters would be steamed by then. If a batter is further incubated, the lactobacilli and streptococci decrease in numbers and P. cerevisiae develops. L. mesenteroides is the microor- ganism essential for leavening of the batter and, along with S. faecalis,
LACTIC ACID FERMENTATIONS 47 is also responsible for acid production. Both functions are essential for producing a satisfactory idli. In idli made with a 1:1 ratio of black gram to rice, batter volume increased about 47 percent 12 to 15 hours after incubation at 30°C. The pH fell to 4.5 and total acidity rose to 2.8 percent (as lactic acid). Using a 1:2 ratio of black gram to rice, batter volume increased 113 percent and acidity rose to 2.2 percent in 20 hours at 29°C. Reducing sugars (as glucose) showed a steady decrease from 3.3 milligrams per gram of dry ingredients to 0.8 milligrams per gram in 20 hours, reflecting their utilization for acid and gas production. Soluble solids increased, whereas soluble nitrogen decreased. Flatulence-causing oligosaccha- rides, such as stachyose and raffinose, are completely hydrolyzed. A 60 percent increase in methionine has been reported during fermentation. The increase would be of considerable nutritional impor- tance if true, but the results conflict with earlier findings. Thiamine and riboflavin increases during fermentation and phytate phosphorous decreases have also been reported. PHILIPPINE PUTO Philippine puto is a leavened steamed rice cake made from year-old rice grains that are soaked, ground with water, and allowed to undergo a natural acid and gas fermentation. Part of the acid is neutralized with sodium hydroxide during the last stage of fermentation. Puto is closely related to Indian idli, except that it contains no legume. SOURDOUGH BREADS AND RELATED FERMENTATIONS There is a close relationship between yeasts and lactic acid bacteria in sourdough breads, soy sauce, miso, and kefir. Sourdough leaven contains both yeasts and lactobacilli. The method of preparing such leavens is ancient. Wheat, rye, or other cereal grain flour is mixed with water and incubated for a few days in a warm place. Initially, a wide range of microorganisms develop, but eventually the lactic acid bacteria predominate because of their acid production. Yeasts also can survive, because they tolerate acid well. More flour is added to make a dough. This dough is then subdivided and used to make a batch of bread, while the rest of the dough is kept for future bread making. Wherever sourdough leavens have been studied, the organisms found have been similar. The essential microorganisms in sourdough are a Lactobacillus sp. and a yeast, Torulopsis holmii. Saccharomyces inusitatus also has
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
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 35°C. 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.
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.
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.
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
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.
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
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 -80°C. 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
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 30°C. 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
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.