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VI. HUMAN HEALTH, SAFETY, AND NUTRITION
22 Nutrition and Safety Considerations O. Paredes Lopez Fermentation was one of the first methods used by Man to produce and preserve foods. Microbial fermentations have played an important role in food processing for thousands of years. Fermentations provide a way to preserve food products, to enhance nutritive value, to destroy undesirable factors, to make a safer product, to improve the appearance and taste of some foods, to salvage material otherwise not usable for human consumption, and to reduce the energy required for cooking (1~. Preservation of foods by salting is an age-old practice; while preventing the growth of pathogenic microbes, it allows the develop- ment of harmless, halotolerant ones that produce desirable censorial changes in the substrate (2~. Traditional fermented foods may be divided into two broad cate- gories: (a) submerged culture-fermentations (SCFs) and (b) solid- substrate fermentations (SSFs). In SCFs microbial activity occurs at a relatively low biomass concentration in the liquid phase, while in SCFs microbial growth and product formation occur on the surfaces of solid substrates (3,4~. Some examples of traditional fermented foods for SCFs are pulque and tesguino, soy sauce, fish sauce, kaffir beer, and palm and rice wines. Examples of SSFs are tempe, miso, pozol, oncom, and natto. One of the major characteristics that distinguishes SSFs from SCFs is that SSF processes usually occur at low-moisture contents (e.g., 10 to 20 percent), conditions under which water activity favors the development of filamentous fungi. However, for many indigenous fermentations the microbial interactions are complex and mixed fungal-bacterial, fungal-yeast, and yeast-bacterial combinations occur (51. These interactions play an important role in the nutritional, safety, and sensory characteristics of the end product (61. 153
154 FERMENTED FOODS EFFECT OF FERMENTATION ON NUTRITIONAL COMPOSITION Changes in Proximate Composition and Soluble Components During fermentation the microorganisms secrete hydrolytic enzymes into the substrate and assimilate some of the fatty acids, amino acids, and simple sugars thus liberated. These are converted into microbial structural components and secondary metabolites. Lactic acid fermen- tation is an ancient process whereby a varied group of bacteria ferment carbohydrates, producing lactic acid as the major end product. This type of fermentation is used for the production of dairy products, sauerkraut, bread, meat, and silage. In particular, traditional SSFs of legumes, cereals, and starchy substrates have been associated in many regions of the world with the activity of lactic acid bacteria (7~; during fermentation lactic acid accumulates, with a concomitant increase in acidity and a decrease of dry matter yields. The higher pH values of fermented legumes, compared to other materials under similar conditions, have been attributed to their higher protein content (8,91. It seems that the only fermented food showing significant changes In its crude composition is pozol. The fermentation mixture contains Agrobacterium azotophilum, which is capable of fixing nitrogen (10~. Due to the crude methods of analysis, the proximate composition of foods does not change much during fermentation. However, there is almost always a high increase in the soluble fraction of a food during fermentation. The proteolytic activity of bacteria in traditional fermentations degrades complex proteins into simpler proteins, pep- tides, and amino acids. The bacteria used in natto fermentation cause substantial increases in the level of free amino acids and soluble carbohydrates. On the other hand, Rhizopus spp., used in the fermenta- tion of various types of tempe, are highly hydrolytic, and outstanding increases in soluble fat, protein, and carbohydrate are observed. Free fatty acids, including the essential fatty acids, linoleic and linolenic acids, may increase in these indigenous fermented foods (11,121; this increase is thought to be of nutritional significance. The increase in soluble solids is a nutritionally desirable event, as the food is effectively digested prior to consumption. In some cases the microorganisms are capable of producing pectinases and cellulases, softening the texture of the food and liberating sugars that would otherwise be unavailable to the human digestive system. Consequently, fermented foods are expected to be more digestible than their unfer- mented counterparts. Changes in Composition of Amino Acids and Vitamins Methionine, the limiting amino acid in legumes, has been reported to increase during tempe kedele production, and lysine, the limiting
NUTRITION AND SAFETY CONSIDERATIONS 155 amino acid in cereals, increases during fermentation with Rhizopus spp. (11. During kocho production, an acidic fermentation, the essential amino acid content is considerably enhanced. On the other hand, during tape' ketan and enjera production, the levels of some essential amino acids fall, whereas others remain unchanged (111. In general, most traditional fermented foods exhibit slight changes in essential . · . amino aclos. Interestingly, isolation of improved strains of Aspergillus niger for an SSF process allowed 200 to 300 percent lysine overproduction compared to the parent strain (51. However, it should be emphasized that bioavailability and balance of amino acids are more important than their total content. Hence, biological experiments to assess their nutritional value are warranted. Traditional fermentations dramatically improve the vitamin content of a wide variety of substrates. Of all the foods investigated, only enjera showed a decline in vitamin content (1,131. Changes in Unwanted Components Unwanted components, such as physic acid, trypsin inhibitor, flatus factors, and lectins, may be present in high concentrations in several desirable foods. Phytic acid and trypsin inhibitor interfere with digestion by binding enzymes. Phytic acid may also bind minerals, reducing their bioavailability. Lectins are capable of binding to the intestinal wall and thus interfering with nutrient absorption. Presoaking and cooking of foods can reduce the levels of some, but not all, of these antinutritional factors. However, microorganisms have the capacity to hydrolyze them, reducing their levels even further (14~. Hence, bacteria, yeasts, and fungi that degrade antinutrients at a fast rate and at early stages of fermentation need to be identified or developed (11. Changes in Biological Value Since fermentation increases the quantity of soluble proteins in foods, it may improve the amino acid profile, and because it reduces the levels of certain antinutritional factors that interfere with digestion, it would not be unreasonable to suggest that fermented foods will be more efficiently utilized by the human digestive system. Single- as well as mixed-culture fermentations of pearl millet by yeasts improve starch and protein digestibility (15~. Enjera is one of the few traditional fermented foods that shows a decline in protein efficiency ratio (PER), probably due to a decline in the essential amino acid content (161. Also, increases in PER values of some indigenous fermented foods can be obtained by incorporating soybeans into cereal-based substrates.
156 FERMENTED FOODS SAFETY ASPECTS OF TRADITIONAL FERMENTED FOODS Because many fermented foods are produced using fungi, the risk of mycotoxin contamination is high. During natural fermentations, food-poisoning flora and coliforms may also grow with the tactics. These microorganisms need to be eliminated to make fermented foods safe for consumption (161. Several factors contribute to the safety of fermented foods: (a) Soaking and cooking. Washing, soaking, and cooking treatments reduce the in situ microbial contaminants. (b) Salting. Various fermented foods are made with the addition of salt, which acts as a preservative. (c) Acid formation. Many indigenous fermentations are carried out by acid-producing microorganisms, where these organic acids (e.g., lactic, acetic, fumaric acids) act as preserva- tives or as bacteriostatic agents. An inhibitory pH for bacterial growth is considered to be 3.6 to 4.1. (d) Antibiotic production. Molds used in some traditional fermentations produce antimicrobial glycopeptides. (e) Low moisture content. In the case of SSF processes, the low water activity may be an important preservative factor. and (f) Reduction of aflatoxin by some microorganisms. Rhizopus and Neurospora species, among others, are reported to decrease aflatoxin content of contami- nated substrates. Despite these factors, it has been reported that the sanitary quality of some Oriental fermented foods is poor (17,181. Safe products are usually obtained when the following recommendations are observed: (a) appropriate soaking of the beans in acid at a low pH; (by adequate cooking time; (c) using hygienic conditions during production, handling, and storage; and (d) good refrigeration of products (5°C) between production and consumption. In summary, production of foods with high nutritional and sensory values, and free of microbiological health risks, is a key component of any policy aimed at upgrading the social role of traditional fermented foods in less developed countries. REFERENCES 1. Paredes-Lopez, O., and G. I. Harry. 1988. Food biotechnology review: Traditional solid-state fermentations of plant raw materials. Application, nutritional significance, and future prospects. Critical Reviews in Food Science and Nutrition 27:159-187. 2. Beuchat, L. R. 1978. Traditional fermented food products. Pp. 22~253 in: Food and Beverage Microbiology, L. R. Beuchat (Ed.), Westport, Conn.: The AVI Publishing Co. 3. Tengerdy, R. P. 1985. Solid substrate fermentation. Trends in Biotechnology 3:9~99.
NUTRITION AND SAFETY CONSIDERATIONS 157 4. Paredes-Lopez, O., and A. Alpuche-Solis. 1991. Solid substrate fermentation. A biotechnological approach to bioconversion of wastes. Pp. 117-145 in: Bioconversion of Waste Materials to Industrial Prod- ucts, Vol. 1, A. M. Martin (Ed.), London: Elsevier, Applied Science Publication. 5. Rogers, P. L. 1989. Principles and applications of bioprocess technology in the food industry. Pp. 223-239 in: Biotechnology and the Food Industry, P. L. Rogers and G. H. Fleet (Eds.~. New York: Gordon and Breach, Science Publishers. 6. Hall, R. J. 1989. Application of biotechnology to traditional fermentations. Pp. 241-277 in: Biotechnology and the Food Industry. P. L. Rogers and G. H. Fleet (Eds.~. New York: Gordon and Breach Science Publishers. 7. Fukushima, D. 1985. Fermented vegetable protein and related foods of Japan and China. Food Reviews International 1: 149-209. 8. Zamora, A., and M. L. Fields. 1979. Nutritive quality of fermented cowpeas and chickpeas. Journal of Food Science 44:23= 237. 9. Paredes-Lopez, O., J. Gonzalez-Castaneda, and A. Carabez- Trejo. 1991. Influence of solid substrate fermentation on the chemical composition of chickpea. Journal of Fermentation and Bioengineering 71:58-62. 10. Cravioto, O. R., Y. O. Cravioto, G. Massiew, and J. Guzman. 1955. El pozol, forma indigene de consumir el maiz en el sureste de Mexico y su aporte de nutrientes a la dicta. Ciencia (Mexico) 15:27- 30. 11. Steinkraus, K. H. 1983. Indonesian tempe and related fermenta- tions. Pp. 217-251 in: Handbook of Indigenous Fermented Foods, Microbiology Series, Vol. 9, K. H. Steinkraus (Ed.~. New York: Marcel Dekker. 12. Paredes-Lopez, O., G. I. Harry, and R. Montes-Rivera. 1987. Development of a fermentation procedure to produce a tempe-related food using common beans as substrate. Biotechnology Letters 9:333- 333. 13. Soni, S. K., and D. K. Sandhu. 1989. Fermentation of idli: Effects of changes in raw materials and physical-chemical conditions. Journal of Cereal Science 10:227-238. 14. Mital, B. K., and S. K. Garga. 1990. Tempe Technology and food value. Food Reviews International 6:213-224. 15. Khetarpual, N., and B. M. Chauhan. 1990. Fermentation of pearl millet flour with yeasts and lactobacilli: In vitro digestibility and utilization of fermented flour for weaning mixtures. Plant Foods and Human Nutrition 40:167-173. 16. Wang, H. L., and C. W. Hesseltine. 1981. Use of microbial cultures: Legume and cereal products. Food Technology 33~11:79-83.
158 FERMENTED FOODS 17. Tanaka, N., S. K. Kovats, J. A. Guggisberg, L. M. Meske, and M. P. Doyle. Evaluation of the microbiological safety of tempe made from unacidified soybeans. Journal of Food Protection 48:438~41. 18. Samson, R. A., J. A. van Kooij, and E. de Boer. 1987. Microbiological quality of commercial tempe in the Netherlands. Journal of Food Protection 50:92-94.
23 Mycotoxic Flora of Some Indigenous Fermented Foods Felixtina E. ,Ionsyn Fermented foods have a wide usage in Sierra Leone as baby/weaning foods. Ogi (fermented maize/sorghum) and foofoo pap (fermented cassava) are examples. Foofoo is also one of the two staples of the Creoles that is now widely used by other tribes especially when rice is scarce. Ogiri (fermented sesame seeds) is a favorite condiment used mostly by the poor as a low-cost protein substitute. Several studies ( 1-4) have shown that toxigenic fungi do not participate in the fermentation processes but contaminate the product during or after the fermentation. It has been demonstrated ( 1~) that at times the substrate for fermentation (maize, sesame seeds) has had prior exposure to myco- toxin. In the case of maize, an aflatoxin B. level of 200 ~g/kg was reduced to 58 ~g/kg in the resulting fermented mashogi (51. The long cooking period (6 hours) of sesame seeds before fermentation accounts for the loss of mycotoxins. Studies carried out by Ogunsanwo et al. (6) have shown that losses of 64 percent aflatoxin B. and 83 percent aflatoxin Go could be observed in ogiri product prepared from Aspergil- luspavus-contaminated melon seeds. In Sierra Leone, ogiri is produced by moist solid fermentation of sesame seeds, a process similar to Nigerian ogiri, which is made from fermented melon seeds (Citrullus vulgaris) (7) and Dawa-dawa from fermented locust beans (Parkiafilicoidea) (81. Traditionally, the boiled seeds are wrapped in jute bags and allowed to ferment for 4 to 5 days before smoke treatment is applied. In such processes whitish threads are observed after day 2 and molds become obvious after 3 to 6 days This study was funded by the International Foundation for Science, Stockholm, Sweden. 159
160 FERMENTED FOODS (31. Microscopic examination of these whitish threads revealed the presence of toxigenic and nontoxigenic Aspergilli and Penicillia species. Detection of the corresponding mycotoxins of these toxigenic fungi in the fermented, marketed, and stored ogiri (4) led to the present study to design appropriate fermentation and storage techniques to reduce the risk of mycotoxin contamination. MATERIALS AND METHODS Fermentation Process Sesame seeds were soaked overnight and pounded in a mortar to dehull. The seeds were then washed and boiled for 6 hours. The boiled seeds were divided into three portions. One portion was transferred to a clean dry nylon fiber bag; the other was placed in a clean dry jute bag. Both were tightly wrapped. The third was placed in a plastic bowl with a tight-sealed lid. Three replicates of each of the nylon fiber and jute bag arrays were made. These were divided into three groups. Group one was left to ferment for 5 days without smoke treatment. Group two received early smoke treatment, from day 2 until day 5. Group three was smoked consistently from day 3 to day 8, and thereafter on alternate days for 2 weeks. Marketing and Storage The three common methods for wrapping ogiri are (a) the use of dried banana leaves Musa sapientum, (b) the use of fresh or smoked leaves of the plant Newbouldia laevis, and (c) the use of small plastic wraps. Leaf and plastic-wrapped ogiri samples bought from the local markets were examined immediately under a stereo microscope. Samples with no obvious fungal presence were selected. Three experimental designs were set up as follows: (a) a set of six samples (three from each type of leaf wrap) was smoked consistently for a week, (b) another set of six (two from each type of leaf and plastic wrap) remained unsmoked and stored at room temperature, and (c) the three types of wraps (minus ogiri) were placed in sterile plastic petri dishes and stored at room temperature. Determination of Mycotoxins Twenty gram samples from each experimental design (jute and nylon fiber bags) were analyzed for aflatoxin using the method of Kellert and
MYCOTOXIC FLORA 161 Spott (91. The modified method of Nowotny et al. (10) was used to screen 10-g samples for the other mycotoxins. RESULTS The use of clean dry nylon fiber bags proved very effective. Fermentation was observed to last 3 or 4 days. No fungal growth was noticed on the outside of the bag or on the fermented product even on day 3 before smoke treatment. Using jute bags, fermentation lasted 5 to 6 days, and evidence of fungal contamination was obvious between days 2 and 3 of the fermentation. But when the jute bags received smoke treatment from day 2 to the final day of fermentation, no fungal contamination was observed. Whitish threads observed on jute bags on day 3 disappeared when smoke treatment was applied. The use of plastic bowls for fermentation was highly unsuitable because the process took longer 2 weeks. When ogiri was smoked for 2 weeks, it had a very appealing aroma and texture. In contrast, the end product from the plastic bowl experiment lacked the characteristic ogiri aroma. When ogiri samples from both the jute and nylon fiber bags were assayed for mycotoxins, there was no evidence of contamination. Effect of the Types of Wraps Samples wrapped in dry leaves of the banana plant were less susceptible to fungal attack than ogiri wrapped in leaves of Newbouldia laevis. However, regular smoke treatment reduced the incidence of fungal contamination of ogiri in both types of leaf wraps. Plastic- wrapped samples had no observable fungi even up to 2 weeks of incubation but were devoid of the pleasant aroma characteristic of the smoked product. DISCUSSION It has been clearly demonstrated in this study that the use of clean dry nylon fiber bags instead of jute bags for the fermentation and early smoke treatment of the fermenting mash contributed significantly to the exclusion of fungi and thereby reduced the risk of mycotoxin contamination during ogiri production. Further related studies on methods of improving fermentation techniques on other products are now being considered.
162 REFERENCES FERMENTED FOODS 1. Jonsyn, F. E. 1988. Mycopathologia 104:123-127. 2. Jonsyn, F. E. 1989. Mircen Journal 5:547-562. 3. Jonsyn, F. E. 1990. Mycopathologia 110:113-117. 4. Jonsyn, F. E. 1991. In press. 5. H. G. Muller, personal communication. 6. Ogunsanwo, B. M., O. O. Faboya, O. R. Idowo, T. Ikotun, and D. A. Akano. 1989. Die Nahrung 33 :983-988. 7. Odunfa, S. A. 1981. Journal of Plant Foods 3:245-250. 8. Antai, S. P., and M. H. Ibarahim. 1986. Journal of Applied Bacteriology 61: 145-148. 9. Kellert, M., and H. J. Spott. 1980. Bundesgesundheitsblatt 23(1/2): 13-21. 10. Nowotny, P., W. Baltes, W. Kroenert, and R. Weber. 1983. Chemie Mikrobiologie Technologie Der Lebersmitteln 8:2~28.