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VII. COMMERCIALIZATION

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24 Commercialization of Fermented Foods in Sub-Saharan Africa Nduka Okafor Fermented foods form an important part of the diets of people throughout the world, and the people of sub-Saharan Africa are no exception. In many parts of the world, as urbanization increases, the preparation of fermented foods moves from the small-scale household level to large-scale operations. Under these new conditions the foods are prepared with better scientific knowledge. For this reason large- scale factory procedures may differ from traditional approaches. For example, cheese that used to be produced with protease present in rennet may now be produced with protease produced by fungi. With this in mind, a review was carried out in 1981 (1) to learn the extent to which some important fermented foods of sub-Saharan Africa had progressed toward commercialization. The stage that each food had attained was measured on a scale of 8, as shown in Table 1. The purpose of this paper is to indicate to what extent various sub- Saharan fermented foods have progressed in the past decade toward being industrialized and to examine the role, if any, that modern techniques of biotechnology, particularly genetic engineering, have played in commercialization. INDUSTRIALIZATION OF FERMENTED FOODS Table 1 lists the fermented foods about which information is available, including those reviewed earlier (11. A review of the extent of progress toward industrialization of alcoholic beverages of sub-Saharan Africa was recently published (2) and is incorporated here into Table 1. The following conclusions can be drawn: · In 1981 the following foods had been produced on an industrial or 165

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166 FERMENTED FOODS TABLE 1 Fermented Foods of Africa South of the Sahara Food Region Processing Level of Advance Microorganisms CASSAVA-BASED 1981 and 1991 Garri West Africa; Pulp fermented 1,4,6,7 8 Zaire Foo-foO (4) Nigeria Whole roots 0 1 Cornebacterium fermented Bacillus Lactic acid bacteria Chikwangue Zaire Whole roots 0 fermented Lafun Nigeria Flour from chips 0 Kokonte Ghana Flour from chips 0 Cingwada East, Central & Flourfrom chips 0 South Africa CEREAL-BASED NON-ALCOHOLIC Ogi Nigeria, Benin Republic Koko (aflate) (5) Ghana Fermented ground 1,2,4,6 8 cereal Fermented ground 1 cereal 7,(8?) 1 Lactic acid bacteria Mahewu (Mogow) South Africa Fermented ground 1,2,4,5, cereal 6,7,8 Injera (10) Ethiopia Fermented ground 1,2 1.2 Entero bacteria ceae, cereal Lactic acid bacteria MILK-BASED Ayib (16) Ethiopia Cheese-like — 1,2 Lactic acid bacteria and yeasts Nono Nigeria Fermented milk — 1 Lactobacillus bulgaricus L. plantarum, L. helvebcus Streptococcus cremoris Fermented milk (3) Zimbabwe ``Lacto" (3) Zimbabwe ALCOHOLIC BurukutulPito West Africa Fermented milk Fermented milk — 8 Fermentation of 1,2 malted sorghum Sorghum (Kaffir) South Africa Fermentation of 1,2,4,5, beer malted sorghum 6, 7, 8 Merissa (2) Sudan Fermentation of 0 malted sorghum Bussa (2) Kenya Fermentation of 1,2 malted sorghum PALM-BASED Palm wines East, West, Central and South Africa 1,2 Lactococcus spp. Lactococcus spp. Spontaneous fermentation of palm sap

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COMMERCIALIZATION OF FERMENTED FOODS MISCELLANEOUS Iru (dawadawa) (10) Nigeria Fermented seeds 0 8 Lactic acid bacteria of Parkia Ogili (17) Nigeria Fermented seeds 0 1,2 Lactic acid bacteria of castor oil Ugha (Ukraka) (6) Nigeria Fermented seeds 0 1,2 Bacillus of oil-bean Fura (Ghussab) Mali Millet and cheese O Asami East, Central Fermented milk O South Africa 167 Key: 1 = Organisms isolated 2 = Role(s) of organism(s) determined 3 = Selection and genetic improvement of organisms 5 = Improvement in raw material used 6 = Laboratory simulation of fermented food production 7 = Pilot plant production 8 = Industrial plant production semiindustrial scale: ogi, garri, palm wine, mahewu, and sorghum (kaffir) beer. Two new products are now being produced on an industrial or semiindustrial scale. The first is a Nigerian condiment known as dawa- dawa. It is being produced under the trade name of Dadwa by the firm of Cadburys in Nigeria from Parkia seeds as in the traditional fermentation. The second is a Zimbabwean fermented milk product known as Lacto. It is similar to the traditional fermented milk of Zimbabwe (31. · The organisms involved in the fermentation of several foods that were unknown in 1981 have now been identified. They are foo-foo (4), kokonte (5), ugba (ukpaka) (6), and ogili (7,81. The case of dawa-dawa is interesting. In 1981 the organisms involved were unknown; in 1991 not only are they known (9), but the food itself has been commercialized. · Some foods not previously recorded have been added: tej from Ethiopia (10~; nono, a milk-based product from Nigeria; and Zimbabwean fermented milk (31. DISCUSSION AND CONCLUSIONS As can be seen, very little has changed in the progress of the fermented foods of Africa toward industrial production. The 1990s are the era of biotechnology, especially genetic engineering. Fermented foods are brought about by microorganisms, and one would expect that these organisms would be subjected to the technology of gene cloning to improve their activity in the fermentation of foods.

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168 FERMENTED FOODS For example, the fermentation of most carbohydrate foods such as cassava or maize is brought about by lactic acid bacteria. One would therefore have expected that these organisms would be targeted for improvement by gene cloning. Only one example of the advantage of the use of this technique will be given. In garri fermentation lactic acid bacteria play an important part in producing the flavor of the food (111. Yet these organisms cannot split starch. If the amylase gene can be cloned into a lactic acid bacterium involved in garri fermentation, it is conceivable that fermentation will occur faster. If the gene for linamarase production can also be simultaneously cloned, then not only will the fermentation be faster but detoxification also will occur (12~. The only work having any relationship to gene cloning in organisms involved in fermentation was the isolation of plasmids from cassava fermenting organisms by Nwankwo et al. (131. They found that they could not transfer the plasmids to E. cold and there the work ended. The lack of ability to exploit this new technique in an area of vital importance to Africa south of the Sahara is a clear example of (an almost?) missed opportunity in an age when seemingly everyone is cloning a gene from one source or another. Nevertheless, there have been some developments in other directions. For example, Ofuya and Nnajiofor (14) have developed a starter culture for garri that should prove useful in the commercialization of the food. Also, Ofuya and Fiito (15) have developed a rapid method for assessing the quality of garri based on an iodine reaction. REFERENCES 1. Okafor, N. 1981. A scheme for the improvement of fermented foods of Africa, south of the Sahara. Pp. 61-69. In: Global Impacts of Applied Microbiology. S. O. Emejuaiwe, O. Ogunbi, and S. O. Sanni (Edgy. London: Academic Press. 2. Okafor, N. 1990. Traditional alcoholic beverages of tropical Africa: Strategies for scale-up. Process Biochemistry International 25:213-220. 3. Feresu, S. B., and M. I. Muzondo. 1990. Identification of some lactic acid bacteria from two Zimbabwean fermented milk products. World Journal of Microbial Biotechnology 6:178-186. 4. Okafor, N., C. O. Oyolu, and B. C. Ijioma. 1984. Microbiology and biochemistry offoo-foo production. Journal of Applied Microbiol- ogy 55:1-13. 5. Mensah, P., A. M. Tomkins, B.S. Drasar, and T. J. Harrison.

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COMMERCIALIZATION OF FERMENTED FOODS 169 1991. Antimicrobial effects of fermented Ghanaian maize dough. Journal of Applied Bacteriology 70:203-210. 6. Obeta, J. A. N. 1983. A note on the microorganisms associated with the fermentation of the seeds of the African oil bean tree. Journal of Applied Bacteriology 54:433~35. 7. Ogundana, S. K. 1980. The production of ogiri: Nigerian soup condiment. Lebensmittel Wissenschaff und Technologia 13:33~336. 8. Onunkwo, A. U. 1982. Some edible fermentation products of Nigeria. M.Sc. thesis, University of Strathclyde, Glasgow. 9. Odunfa, S. A. 1981. Microorganisms associated with the fermen- tation of the African locust bean, Parkia:filicoidea, during iru prepara- tion. Journal of Plant Foods 3:245-250. 10. Girma, M., B. A. Gashe, and B. Lakew. 1989. The effect of fermentation on the growth and survival of Salmonella typhimurium, Staphylococcus aureus, Bacillus cereus, and Pseudomonas aeroginosa in fermenting tef (Eragrostis tefJ. Mircen Journal of Applied Microbiol- ogy 5:61-66. 11. Okafor, N., and J. Uzuegbu. 1987. Studies on the contributions of microorganisms on the organoleptic properties of garri, a fermented food derived from cassava (Manihot esculenta Crantz). Journal of Food Agriculture 2:99-105. 12. Okafor, N., and A. O. Ejiofor. 1990. Rapid detoxification of cassava mash fermenting for garri production following inoculation by a yeast simultaneously producing linamarase and amylase. Process Biochemistry International 25:82-86. 13. Nwankwo, D., E. Anadu, and R. Usoro. 1989. Cassava fer- menting organisms. Mircen Journal of Applied Microbiology 5:169- 179. 14. Ofuya, C. O., and C. Nnajiofor. 1989. Development and evalua- tion of a starter culture for the industrial production of garri. Journal of Applied Microbiology 66:37~2. 15. Ofuya, C. O., and J. Fiito. 1989. A rapid method for determining the quality of garri based on iodine reduction test. Letters in Applied Microbiology 9:153-155. 16. Ashenafi, M. 1990. Effect of curd cooking temperatures on the microbiological qualities of ayib, a traditional cottage cheese. World Journal of Microbial Biotechnology 6: 159-162. 17. Odibo, F. J. C., and A. I. Umeh. 1989. Microbiology of the fermentation of Telfaria seeds for ogiri production. Mircen Journal of Applied Microbiology and Biotechnology 5:217-222.

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25 Biotechnology for Production of Fruits, Wines, and Alcohol J. Maud Kordylas Fermentation is biotechnology in which desirable microorganisms are used in the production of value-added products of commercial importance. Fermentation occurs in nature in any sugar-containing mash from fruit, berries, honey, or sap tapped from palms. If left exposed in a warm atmosphere, airborne yeasts act on the sugar to convert it into alcohol and carbon dioxide. The making of wines and beers uses this biotechnology under controlled conditions. Alcoholic beverages have been produced for centuries in various societies. They are often central to the most valued personal and social ceremonies of both modern and less literate societies. In such traditional ceremonies as childnaming, marriage feasts, and funerals, alcoholic beverages are often present. In Africa, maize, millet, bananas, honey, palm and bamboo saps, and many fruits are used to ferment nutrient beers and wines. The best known being kaffir beer and palm wines. Industrial fermentation processes are conducted with selected micro- organisms under specified conditions with carefully adjusted nutrient concentrations. The products of fermentation are many: alcohol, glycerol, and carbon dioxide are obtained from yeast fermentation of various sugars. Butyl alcohol, acetone, lactic acid, monosodium glutamate, and acetic acid are products of bacteria action; citric acid, gluconic acid, antibiotics, vitamin Be, and riboflavin are some of the products obtained from mold fermentation. YEASTS Yeasts, the main microorganisms involved in alcoholic fermentation, are found throughout the world. More than 8,000 strains of this vegetative microorganism have been classified. About 9 to 10 pure 170

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BIOTECHNOLOGY 171 strains, with their subclassifications, are used for the fermentation of grain mashes. These belong to the type Saccharomyces cerevisiae. Each strain has its own characteristics and imparts its special properties to a distillate when used in fermentation. A limited number of yeasts in the classification Saccharomyces ellipsoides are used in the fermentation of wines from which brandy is distilled. The strains used in the fermentation of grain mashes are also used in the fermentation of rum from sugarcane extracts and in beer production. Since yeasts function best in slightly acid medium, the mash, juice, sap, or extract prepared for fermentation must be checked for adequate acidity. If acidity is insufficient, acid or acid-bearing material are added. For distilled liquors, fermentation is carried out at 24° to 29°C for 48 to 96 hours, when the mash or must is ready for distillation. The alcohol content of the fermented must is about 7 to 9 percent. RAW MATERIALS Cereals and Starchy Roots For most distilled liquors, the raw material used is a natural sugar as found in honey, ripe fruit, sugarcane juice, palm sap, beet root, milk, or a substance of amylaceous (starchy) nature that can be easily converted into simple sugars using enzymes present in cereals or through the addition of suitable malted cereal. Maize or corn is the most important grain used as fermentable starchy cereal. Starchy roots and tubers are also used. Industrial production of alcohol from cassava in Brazil has been described by De Menezee (11. The alcohol produced is concentrated in a second distillation column to 97.2 percent and is further dried to 99.9 percent and blended with gasoline for energy purposes. Malt is important in distilled liquor. In addition to converting starches from other carbohydrates to sugars, malt contains soluble proteins that contribute flavor to the distillate obtained from the fermentation of grain malt mixtures. Sugarcane Grown throughout the tropics and semitropics, sugarcane and its products, including cane juices, molasses, and sugar are used to make rum and an alcohol derived from rum. Pressed juice from sugarcane can be used as the base raw material for fermentation, or the juice can be concentrated for sugar production with the molasses residue from sugar crystallization used as a base for alcohol fermentation. Molasses

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172 FERMENTED FOODS contains about 35 percent sucrose and 15 percent reducing sugars. This gives molasses its principal value as an industrial raw material for fermentation to produce rum. Two or 3 liters of molasses produces 1 liter of rum. Acetone and butanol also are produced from molasses by fermentation with Clostridium bacteria. Food yeast Torulopsis utilis, is prepared from molasses, as are baker's and brewer's yeasts (21. Coconut Palm The coconut palm finds many uses on the tropical islands of the Pacific. Toddy is produced by tapping the unopened flower spathe of the coconut palm. The spathe is bruised slightly by gentle tapping with a small mallet and is tied tightly with fiber to prevent it from opening. It is bent over gradually to allow the toddy to flow into a receptacle. About 5 centimeters is cut from the tip of the spathe after about 3 weeks. Thereafter, a thin slice is shaved off once or twice a day and the exuding sap is collected. Palms are tapped for 8 months of the year and rested for 4 months. The average daily yield per palm is about 2 liters. The yield per spathe varies from 15 to 80 liters, and an average palm can yield 270 liters during 8 months of tapping. The fresh sweet toddy contains 15 to 20 percent total solids, of which 12 to 17.5 percent Is sucrose. Toddy ferments rapidly due to naturally occurring yeasts. Fermented toddy contains about 6 percent alcohol. After 24 hours the toddy contains 4 to 5 percent acetic acid and is unpalatable as a beverage. It can be used for the production of vinegar. Fermented toddy can be distilled to produce arrack. Freshly fermented toddy is used instead of yeast in bread making. Constant tapping of coconut palms for toddy eliminates the nut crop. In 1952 in wine distilleries in Sri Lanka, over 49 million liters of toddy was fermented to give 4.5 million proof liters of arrack (21. Oil Palm By tapping the male inflorescence of the oil palm, a sweet sap is obtained. The leaf subtending the immature male inflorescence is removed to provide access, the inflorescence is excised, and thin slices are cut once or twice daily. The exuding sap is funneled into a calabash or a bottle. The fresh sap contains 15 percent sugar. Tapping is done daily for 2 to 3 months, yielding about 3.5 liters of sap per day. The sap ferments by the action of bacteria and natural yeast to produce a beverage with a milky flocculent appearance and a slight sulfurous odor known as palm wine. Palm wine is produced and marketed in considerable quantities in Nigeria.

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BIOTECHNOLOGY 173 The sap may be boiled to produce dark-colored sticky sugar or jaggery, which does not keep well. About 9 liters of juice produces 1 kilogram of jaggery. The fermented sap also yields yeasts and vinegar. A mean annual yield of 4,000 liters of sap per hectare of 150 palms has been recorded in eastern Nigeria. This was estimated to have a value more than double that of oil and kernels from similar palms. Tapping, however, reduces the fruit yield. Sap can also be obtained by tapping the crown of the tree laterally or by felling the palm and drilling a hole through the growing point. Both these methods are very wasteful since they kill the plant. The Palmyra palm yields about 2 liters of palm sap per day. Large palms with several tapped inDorescences give as much as 20 liters per day. A single palm of this type is estimated to produce 12,000 liters of sap during its tapping life. Fruits Grapes are the most common fruit used as raw material for alcoholic fermentation. They are used in distilled liquor to make brandy. Historically, wine is the product of fermentation of grape species Vitis vinifera. The high sugar content of most V. vinifera varieties at maturity is the major factor in their selection for use in much of the world's wine production. Their natural sugar content provides the necessary material for fermentation. It is sufficient to produce a wine with an alcohol content of 10 percent or higher. Wines containing less alcohol are unstable because of their sensitivity to bacterial spoilage. The grape's moderate acidity when ripe is also favorable to wine making. The fruit has an acidity of less than 1 percent, calculated as tartaric acid, the main acid in grapes, with a pH of 3.1 to 3.7. The flavor of grapes varies from neutral to strongly aromatic, and the pigment pattern of the skin varies from light greenish-yellow to russet, pink, red, reddish violet, or blue-black. Grapes also contain tannins needed to give bite and taste in the flavor of wines and to protect them from bacteria and possible ill effects if overexposed to the air. Other fruits can be used to produce wine. When fruits other than grapes are used, the name of the fruit is included, as in papaya or pineapple wine. Apples and citrus fruits with sufficient fermentable sugars- are crushed, and the fermentable juices are either pressed out for fermentation or the entire mass is fermented. Tropical fruits such as guava, mangos, pineapple, pawpaw, ripe banana, ripe plantain, tangerine, and cashew fruit also contain fermentable sugars with levels varying from 10 to 20 percent. Overripe plantain pulp was reported to contain 16 to 17 percent fermentable sugar, with the skin containing as much as 30 percent (3~. The tropical climate prevailing in Africa is ideal for the growth and

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BIOTECHNOLOGY 179 carambola somehow continued to flower and set fruit during the rainy season as long as there was periodic sunlight. Quality was high in guavas and soursop harvested at the beginning of the rains. The fruits were large and well formed and had good flavor. Most of the fruits harvested at the ends of the dry and rainy seasons were smaller, malformed, or diseased. This may be due to the effects of too little or too much water on the health of the plants. Too little water may have affected the activities of microorganisms in the soil, and too much water may have reduced air supply to microorganisms in the soil and leaching of nutrients from the soil. Diminished microbial activity may have affected the well-being of the plants. These assump- tions might, however, need to be confirmed through controlled experi- ments. The 180-square meter backyard plot yielded sufficient quantities of fruits guava, soursop, and carambola to provide raw materials for processing to make jams available on the local market throughout 1989 and thereafter. Carambola yields were also sufficient for wine making. The amount of pawpaw harvested from the backyard was not sufficient, however, for both jam production and wine making. More pawpaw was therefore purchased from the local market to supplement the amount harvested. The quantity of mango obtained from the one mango tree was also not sufficient to keep up with the demand for mango jam on the market. More was obtained from the local market. Table 2 shows total yields for guava, soursop, carambola, and pawpaw harvested from 1986 to 1990. Although two of the four pawpaw trees died, total yields of fruits from the backyard continued to increase over the years. Yields from crops interplanted among the fruit trees, including pepper, cocoyam, plantain, and winged beans, and from the one avocado tree that started bearing fruit in 1990, when added to those obtained from trees in Table 2, provided an overall yield of over 1 ton from the backyard plot in 1989 and again in 1990. Wine Production Wine of acceptable quality were produced from pawpaw, pineapple, and carambola. The wines made were either dry, semidry, or sweet. TABLE 2 Fruit Yields (kilograms), 1986-1990 Fruit 1986 1987 1988 1989 1 990 Guava 49.0 131.2 217.5 208.3 295.0 Soursop — 173.0 151.5 397.0 286.3 Carambola — 0.4 63.6 221.5 279.0 Pawpaw — 28.3 100.9 72.6 40.0 Total 49.0 332.9 533.5 899.4 900.3

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180 FERMENTED FOODS Although no controlled organoleptic assessment was organized to evaluate the acceptability of the wines, reactions from random individu- als who tasted the wines were favorable. Marketing trials will be conducted. Alcohol Production Juice extracted from the crushed pineapple core and the inner seeded cover contained sufficient sugar to produce 6.5 to 7 percent alcohol after fermentation. With the addition of extra sugar, however, the alcohol content was increased to 10 percent. A total of 25 liters of over 90 percent concentration alcohol was distilled from 200 liters of discarded wines and 100 liters of fermented pineapple waste extract. Portions of the alcohol were used to carry out experiments to produce aperitif drinks with guava, pineapple, passion fruit, carambola, and ginger. The experiments are still in progress. BIOTECHNOLOGY PRODUCTION SYSTEM The integrated bioechnology research and development system is shown in Figure 1. The broken-line arrows indicate units not yet included but for which information has been collected to enable their future integration into the system. The chickens are needed to produce manure for the composting process, with meat and eggs as additional marketable products. Wastewater from fruit processing would be recycled to provide water for irrigation and for comporting to econo- mize on the use of potable water for those processes. From the data collected and from experience gained through the project, the integrated biotechnology production system has many advantages: · It is environmentally sound: Wastes generated from fruit pro- cessing and from the backyard plot are recycled through the composting process to produce organic fertilizer. · Labor requirements have not been excessive: Once the necessary conditions are met and controls applied for microorganisms to grow and multiply, the productive processes for wine and alcohol production, for comporting, and for nutrient release for plant nourishment are carried out with little or no supervision. · Energy requirements are low: Apart from the energy needed for production of jams and for pasteurization and to run the small-scale equipment used in processing, the integrated production system needs limited amounts of energy input to function. The microbial processes generate their own energy. The need for air conditioning to maintain

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BIOTECHNOLOGY Meat Eggs Chlickens 1 1 Manure i: _ _ Composting ~ ~ Peels and other Waste Products Fermentation ^~ / / Distillation 1 _ Wastewater - - / / / Pineapple Core Plus Inner Cur Fer~at~on Wines /1 Carambola Wine, Rejected Papaya Wine, Wines Pineapple Wine, / (sold on order) Alcohol Research and Development Vodka ~ for Aperitif Wines and Liquors Promising Products Guava Aperitif Ginger Aperitif Passion Fruit Aperitif Not yet added onto the system 181 ''~Fruit Trees \ 1/ _ Processing / Juices Jellies Guava Jelly, Soursop Jelly (on the market) \* Research / and Development \ \ \ Pulp Ja s Guava Butter Pineapple Jam Pawpaw-Ginger Jam Pineapple-Ginger Jam Mango Jam Carambola Jam (on the market) FIGURE 1 An integrated research and development system using biotechnology in the production of fruits, wines, and alcohol.

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182 FERMENTED FOODS constant environmental temperatures will likely add to the energy costs. · The system is sustainable: The interdependency of the microbial subprocesses provides sustainable support to each other with limited input required from outside. Funds generated from the sale of products (jams, wines, apertif drinks) are used to support needed research and to purchase equipment and supplementary produce required to sustain the production of marketable products. · Only practical research is undertaken: Experiments carried out are those needed to solve immediate problems arising from the production system. These are carried out either to improve the quality of a product, to formulate new products from raw materials or by- products generated within the system, or to enhance marketability of a product. · Realistic data is collected for feasibility reports. Production and trial marketing of products from the system have enabled real data to be collected. These are being used to evaluate the system economically and to produce a feasibility report based on actual figures to make decisions on establishing an industry based on the prototype research and development unit. · Valuable experience has been gained: The project has provided valuable experience in the management of a small enterprise. CONCLUSIONS AND RECOMMENDATIONS A good number of efficient microbial processes are available. Sufficient knowledge has been accumulated and information provided on their management and control. If properly selected, synchronized, and integrated, the activities of microorganisms from such processes may be harnessed and used. Their exploitation may be a more promising alternative to large-scale industrial technologies imported from devel- oped countries, which developing countries in Africa cannot afford, sustain, or manage. The priority for research is, therefore, on selecting the right types of microbial processes that can be put together to form sustainable productive systems, with research trials carried out on prototypes to determine the most economically viable combinations to be adopted for commercial exploitation. REFERENCES 1. De Menezee, I. J. B. 1978. Alcohol Production from Cassava. Pp. 41~5 in: Cassava Harvesting and Processing. International Devel- opment Research Center, Ottawa, Canada.

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BIOTECHNOLOGY 183 2. Purseglove, J. W. 1985. Tropical Crops. In: Monocotyledons. England: Longman. 3. Kuboye, A. O., A. B. Oniwinde, and 1. A. Akinrele. 1978. Production of Alcoholic Beverages from Ripe Pineapples, Plantain, and Bananas, Vol. 2, Pp. 78-80. Nigerian Institute of Food Science and Technology. Lagos, Nigeria.

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26 Future Directions Leslie Fook-Min Yong The preparation of fermented foods predates the recorded history of Man. Early humans used observation of the apparent effects of microbial alteration of food characteristics to develop processes for food fermentation. The resultant fermented products normally have a different texture and flavor compared to the unfermented starting materials, thus making them more palatable and digestible and pro- longing their shelf life. Technical progress was initially slow, as reflected in the long fermentation periods required; it was incremental to the technical know-how and basic scientific information then available. It is probably fair to say that in the very early days brew-masters were more artisans than technologists. With the rapid advancement in understanding of the basic sciences of microbiology and biochemistry, coupled with the introduction of new equipment, the developed nations have forged ahead in improving the safety and efficiency of the bioprocesses used to manufacture traditional fermented foods, such as cheese fermentation. "OLD" AND "NEW" BIOTECHNOLOGY With the rapid progress in the biological sciences, both basic and applied aspects, it has been possible to gain a better understanding of the mystery that has surrounded fermentation processes. The types of microorganisms involved has been isolated and identified, and the physiology and metabolism of these organisms have been studied. Hence, traditional fermented foods can now be made better, faster, and more economically. The application of available knowledge to In this paper I draw on my experience working with soy sauce fermentation and then proceed to discuss the production of flavor and fragrance materials by microbial fermentation. Experience gained from this traditional fermented condiment has enabled me to develop novel bioprocesses for the production of aroma chemicals. 184

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FUTURE DIRECTIONS 185 improve traditional food fermentations in developed countries has far outpaced that in developing countries. The terms "old biotechnology" and "new biotechnology" have been used "old" to mean the undirected manipulation of microorganisms and plants, such as by mutagenesis and selection of the better strains. In this old biotechnology I would like, for convenience, to include directed control of the physical and chemical environments of the fermentation process, which could result in better performance of the useful microbes. Though mutation increases the ability to select better strains, there can, of course, be little directed alteration of genetic material. The new biotechnology, such as recombinant DNA techniques, overcomes this problem. The new biotechnology can, of course, be of tremendous help in producing superstrains of microbes that could enable accelera- tion of fermentation processes, provide more efficient utilization of raw materials, and produce better-quality products. How best can developing nations apply these biotechnologies to traditional fermented foods? Should it be application of the `'old" before the "new," "new" without the "old," or "old" and "new" simultaneously? In their enthusiasm to promote the new biotechnology for traditional fermented food applications, scientists from developed countries should not forget the different environments that exist in developed and developing countries. In developed countries the old biotechnology is already well understood and practiced efficiently in fermented food industries. Developing countries may need to acquire a better under- standing of the old biotechnology before efficiently absorbing and implementing the new biotechnology to its fullest. APPLICATION OF BIOTECHNOLOGY Preparation of traditional fermented foods is more complex and time consuming than that involved in the production of single chemical substances. For example, in soy sauce fermentation more than one type of microorganism is involved, whereas in citric acid fermentation only one species of fungus is normally used. How can developing countries apply new knowledge in the old and new biotechnologies to their own complex traditional food fermentations? Take soy sauce fermentation as an example of a traditional fermenta- tion process conducted in a developed country, such as Japan compared with that in a country like Malaysia. The technology in use in Japan is sophisticated, very advanced, and highly productive and mechanized. The microbes used have been selected over the years for their performance in producing a better-quality product. The cottage industry

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186 FERMENTED FOODS soy sauce fermentation in Malaysia is highly labor intensive and usually relies on "natural" inoculation of raw materials using unwashed trays for previous fermentations rather than using a separately prepared inoculum of Aspergillus oryzoe. The equipment used in Japan to conduct the fermentation is state- of-the-art machinery with microprocessor or computer control to provide the optimum conditions for microbial growth and activity. The microorganisms used have been manipulated by mutagenesis to give better performance, such as better enzymatic activity to give better hydrolysis of proteinaceous matter in defatted soybean meal as well as better flavor production. In comparison, the average process used in Malaysia could be considered primitive. This disparity is attributable to a better understanding of the theoretical and practical bases of soy sauce fermentation by scientists and technicians in Japan's soy sauce factories. The old biotechnology involved in this type of traditional fermentation is well understood in Japan, and the Japanese are now able to make better use of the new biotechnology such as the directed alteration of genetic material of the mold (Aspergillus oryzoe), yeast (Saccharomyces rouxii), and bacteria (Pediococci) used in soy sauce fermentation so as to improve their fermentative qualities. Necessary Prerequisites For developing countries to make full use of the available biotechnol- ogies in their traditional food fermentations, an understanding and acquisition of expertise in the following areas are essential. Art of fermentation A clear understanding by the master brewer of every step used in the fermentation is needed. This is the art of fermentation. Although the master brewers might not have scientific backgrounds, they could normally ensure a proper fermentation as a result of years of experience. Without a knowledge of the art of traditional food fermentation, a scientist cannot provide a scientific explanation for the process and proceed to provide assistance in improvement of the process. Microbiology It is essential to know which microorganisms involved in food fermentations are useful and how the physiology and metabolism of these microbes are affected by the physical and chemical environments of fermentations, as well as how their microbial activities in turn affect the fermentation processes. Microorganisms normally break down

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FUTURE DIRECTIONS 187 carbohydrates, proteins, and lipids present in the raw materials to be fermented by releasing enzymes into the medium. As the raw materials are hydrolyzed, the environment is changed, as sometimes reflected by a drop in pH value. Moreover, the breakdown products such as peptides and amino acids can be further converted into smaller volatile molecules that are odoriferous and hence improve the flavor characteristics of the fermented foods. Upstream and downstream processing Normally raw materials are pretreated before fermentation. It is important to comprehend how such pretreatment could affect the fermentation process. In soy sauce fermentation, whole soybeans are steamed to make the soy protein more easily hydrolyzable by the proteases of Aspergillus oryzoe. In so doing, too much moisture is introduced and-wheat flour must be added to lower the moisture content to a level that does not favor early bacterial growth and hence prevents spoilage of the fermentation. Downstream processing does not affect the bioprocess involved. However, it could alter the normal organoleptic properties of the product, especially when downstream processing involves heating, such as in the pasteurization of soy sauce. Heating causes a change in the flavor of soy sauce due to nonenzymic browning reactions, which could result in the production of pyrazine compounds. Biochemistry An understanding of the biochemical activities of the microbes actively participating in the fermentation could help to explain the change In the texture of the raw material as well as the origin of flavoring substances often present in fermented foods. Flavor and texture are important properties of fermented foods. Elucidation of flavor production in such fermentations could result in the development of processes for producing of flavoring materials by fermentation, as in the production of cheese flavors by Penicillium roquefortii. Fermentation equipment and techniques Practical experience in the use of both solid-state and submerged culture fermentation equipment is very useful. Normal training includes submerged culture bioreactors but not solid-state fermenters. It is useful to know both types of fermentations because traditional food fermentations often involve solid state fermentation. In soy sauce fermentation an initial solid-state fermentation is followed by a sub- merged fermentation step. Systems that measure and control pH,

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188 FERMENTED FOODS dissolved oxygen, temperature, and moisture help to make these bioprocesses more efficient and reduce the time required for production of a quality product. CONCLUSIONS For developing countries, future directions in applying biotechnology to traditional fermented foods should be: (1) training of a pool of technicians in the art and science of traditional food fermentations and (2) investigations by local scientists into the scientific basis of indigenous food fermentations. Theoretical basic science education, such as the microbiology and biochemistry of food fermentations, could be taught in schools; so could the use of modern bioreactor systems. However, the application of such biotechnological knowledge to actual commercial fermentations can come about only after a practical experience in a fermented food factory for a period of time. The approach to be taken in applying biotechnology to traditional food fermentations should be that of finding solutions to existing bioprocessing problems and not trying to find problems with newly acquired biotechniques. Only after the old biotechniques of fermentation have been success- fully used can industries in developing countries look forward to using the new biotechniques of recombinant DNA to improve the genetic constitution of the microorganisms involved.