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IV. PLANT DT~ ~ TO ~ A STY T~ ~

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12 Cassava Processing in Africa Olusola B. Oyewole Cassava is an important food crop in the tropics and many countries in Africa. The crop contributes significantly to the diets of over 800 million people, with per capita consumption averaging 102 kilograms per year. In some areas of Africa it constitutes over 50 percent of the daily diets of the people. Traditionally, cassava is processed before consumption. Processing is necessary for several reasons. First, it serves as a means of removing or reducing the potentially toxic cyanogenic glucosides present in fresh cassava. Second, it serves as a means of preservation. Third, processing yields products that have different characteristics, which creates variety in cassava diets. The objective of this paper is to detail the strategy and program being followed in our laboratory to utilize the knowledge of biotechnology to improve the processing of cassava in Africa. TRADITIONAL PROCESSING Processing of cassava for food involves combinations of fermenta- tion, drying, and cooking. Fermentation is an important method common in most processings. While there are many fermentation techniques for cassava, they can be broadly categorized into solid-state fermentation and submerged fermentation. Solid-state fermentation, typified by gari production, uses grated or sliced cassava pieces that are allowed to ferment while exposed to the natural atmosphere or pressed in a bag. Submerged fermentation involves the soaking of whole peeled, cut and peeled, or unpeeled cassava roots in water for various periods, as typified by the production of fufu and lalun in Nigeria. Traditionally, cassava is fermented for 4 to 6 days in order to - The support of the International Foundation for Science, Stockholm, Sweden, is gratefully acknowledged. 89

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JO FERMENTED FOODS effect sufficient detoxification of the roots. Some processors, out of economic pressure, ferment cassava for less than 2 days. Some cases of food poisioning have been attributed to this practice. Application of biotechnology to traditional cassava processing has prospects for producing safe and well-detoxified products. RESEARCH APPROACH Our approach on cassava processing research is divided into three areas: 1. Investigating the science of the traditional submerged fermenta- tion of cassava to fafu and lafun production; 2. Optimization of the processing through process controls; and 3. Improvement of traditional processing through application or biotechnological techniques. The microorganisms involved in the submerged fermentation process have been isolated and found to include Bacillus subtilis, Klebsiella sp., Candida tropicalis, C. krusei, and a wide spectrum of lactic acid bacteria, major among which are Lactobacillus plantarum and Leuconostoc mesenteroides. A microbial succession trend was found with the starch degrading Bacillus subtilis, giving way to the lactic acid bacteria and yeasts that dominate the latter part of the fermentation. The pH and titratable acidity of the fermenting cassava roots increased from 6.3+0.2, 0.08+0.03 percent to 4.0+0.3, 0.36+0.05 percent, respectively, after the 96-hour fermentation period. Organic acids produced during fermentation include lactic, acetic, propanoic, and butanoic acids, among others, and these are believed to contribute to the characteristic havor of fermented cassava products. Fermentation causes release of some bound minerals, including calcium and magne- sium. The most important contribution of fermentation is the release from the plant tissues of the enzyme linamarase, which is involved in the breakdown of the linamarin and lotaustralin (cyanogenic glucosides) of cassava, which releases hydrogen cyanide and thus detoxifies the product. PROCESS OPTIMIZATION Processing conditions for optimizing the fermentation process have been investigated in our laboratory. We found that the temperature range of 30 to 35C, with a soaking period of 48 to 60 hours, is best for submerged processing. The size to which the roots are cut, peeling or nonpeeling of roots before processing, changing or nonchanging of

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CASSAVA PROCESSING 91 fermentation water at intervals during processing, and the age of roots all affect the characteristics of the final product. In addition, the protein contents of products can be improved by cofermentation with legumes such as soybeans and cowpea. BIOTECHNOLOGICAL INVESTIGATIONS The overall goal of our biotechnological investigations is to develop an appropriate starter culture for cassava processing that will effectively produce linamarase enzymes for detoxifying cassava, break down starch to the simple sugars needed for acid production, improve the protein content of the products, reduce processing time, and yield products with stable desired qualities. The following summarizes our current findings: The microorganisms involved in fermentation have been charac- terized. Characterized isolates were used as single and multiple starter cultures for cassava fermentation. This has made it possible to understand the roles of each of the microorganisms implicated in the natural fermentation process. Bacillus subtilis and Klebsiella spp. contribute significantly to the rotting of cassava roots. In addition, B. subtilis produces amylase enzymes that are necessary for the break- down of starch to sugars, which are needed for the growth of other fermenting microorganisms, including the tactics. Yeasts play a major role in odor development and, where high yeast biomass is encouraged, protein-enriched products are not. Lactic acid bacteria convert cassava sugars to lactic and other acids that contribute to the flavor in addition to having preservative effects. Appropriate starters have been developed that can produce amy- lase and linamarase enzymes necessary for starch breakdown and cyanogenic glucoside hydrolysis; two major biochemical processes needed in cassava processing. For this, the lactic acid bacteria were investigated since they were the predominant microbial group present at the beginning of fermentation and which persist and survive the acidic conditions that prevail in cassava fermentation. To date, we have found strains of Lactobacillus plantarum that are capable of producing amylase and linamarin. The linamarase produced has been purified, and it exhibits optimal activity at pH 5 to 8 and temperature of 30 to 40C. Prospects for cassava processing using a selected single culture with properties for starch hydrolysis, cyanide detoxification, and acid production have thus evolved. .. To initiate genetic manipulation of cassava lactic acid bacteria,

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92 FERMENTED FOODS the plasmid profiles of the lactobacilli isolated from cassava were studied. The presence of plasmids among cassava lactobacilli has been confirmed. Further research is needed to investigate the correlation between possession of plasmids and linamarase production in order to establish prospects for genetic manipulation. FUTURE RESEARCH Beyond the selection of appropriate starter cultures for cassava fermentation, it will be necessary to improve the starter culture. Genetic manipulation of the starter culture offers the best hope for improved cassava processing, with higher economic returns and improved stable qualities. Cassava processing could also be enhanced by using biotechnological principles to modify structural and processing characteristics of cassava cultivars to meet specific product requirements. The linamarase elaborated by cassava plant tissues and fermenting microorganisms has been found to be unstable under high acidic conditions characteristic of the latter part of natural fermentation. Techniques for increasing the stability of linamarase enzyme to acidic conditions could be investigated. The usefulness of cassava fermenting microorganisms could be further investigated for the production of other economically viable products such as acidulants and antimicrobial agents. A biotechnological approach could be investigated for the treatment of odorous fermented cassava water and cassava root peels.

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13 Improving the Nutritional Quality of Ogi and Gari T. G. Sokari Ogi is a blancmange-like product processed by fermenting the slurry from wet-milled maize (or sorghum or millet). Used as both a weaning food for infants and as a breakfast food by adults, ogi is one of the most important food items in Nigeria. Yet it is nutritionally inferior to maize, which is deficient in certain essential amino acids, because of the maize-milling process that is an integral part of ogi production. Cassava, another very important food crop, has the problem of possible nutritional complications because it contains the cyanogenic glucosides linamarin and lotanstralin. Although the cyanogens in cassava are hydrolyzed to hydrogen cyanide during processing by the endogenous enzyme linamarase (1,2), not all processes are equally effective. It has even been suggested that traditional processing techniques are unlikely to remove all the cyanide from cassava (3,41. In view of this, studies were undertaken to increase the protein content of ogi relatively inexpensively and to develop a technique for processing cassava into gari that would eliminate cyanogens from the product or reduce them to innocuous levels. PROCESSING OF OGI The traditional technique for processing maize into ogi is summarized in Figure 1. Also shown in Figure 1 is an alternative to this procedure; a 20-minute boiling step is substituted for the normal 24- to 28-hour steeping of maize prior to wet milling (51. Cowpea can be combined with maize to increase the protein content of ogi. Substituting 20 minutes of boiling for the traditional 24 to 28 hours of steeping prior to wet milling maize reduced processing time from 72-76 hours to about 24 hours. There was, however, no significant 93

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94 FERMENTED FOODS Maize Cowpea 1 1 , Clean Clean and Dehull ,--"' 1 1 Boil ~ Steep (20 min) (24-48h) """ 1 Boil (30 mint 1 1 ~ Wet Mill Wet Mill Boil Water ~l it, (discard) Wet Sieve Wet Sieve Slurry Fermejtation Ogi - ~ Maize: Cowpea Slurry (up to 30% cowpea w/w) Fermentation (24-48h) Enriched Ogi - - Slurry FIGURE 1 Processing of maize into ogi including cowpea protein enrichment; dotted lines show a bypass by which processing time can be reduced. difference ~ p> 0.05) in the aroma, color, taste, and overall acceptability between the products obtained by the short-time processing and traditional processing (51. The same was also true for unenriched and protein-enriched ogi except for color (61. CYANIDE REDUCTION DURING CASSAVA PROCESSING Two foods processed from cassava (gari and ijapu) were studied. Adding water to grated cassava at the 75 percent (v/w) level and heating at 50C for 6 hours resulted in linamarin reduction of >99 percent (Figure 21. The pH of the mash fell from 6.4 to 6.3 during the period (71. After dewatering, the mash was adjusted to a pH below 4 by equilibrating with a 3-day fermented cassava liquor (40 percent, v/w) at 50C for 12 to 18 hours. The equilibrated mash was then dewatered and toasted (Figure 31. A panel of tasters who were familiar with gari but otherwise untrained could not differentiate between the product and traditionally processed gari. Both sets of products were equally acceptable to the panel.

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96 Peels (discard) ~ Cassava ~ Peel 1 Boil (1 00C/25 min) 1 Slice Soak (24-28h) Rinse Ijapu FIGURE 3 Processing of ijapu and garifrom cassava. FERMENTED FOODS - ~ Grate ~ Add Water (75% v/w, 50C/6h) Dewater/ferment (3 days/30C) it\ Toast Dewater Dewatered Mash Fermented ,1~ Liquor ~ Mash + Liquor (40% v/w) (50C/12-1 8h) 1 Spent Liquor ~ Dewater (discard) ,[ Toast Gary Gary ( t r a d i t i o n a I p r o c e d u r e ) ( s e m i c o n t i n u o u s process) The modified procedure for processing cassava into gari reduced the processing time from >96 hours to about 24 hours. Cyanogens were not detectable in the product by the method of Cooke (8,91. The study of the traditional production of ijapu (Figure 3) was intended to aid in understanding the loss of cyanogens during cassava processing. About 54 percent of the cyanogens in raw cassava were lost after boiling peeled cassava, but a substantial proportion remained in the water (Table 11. After slicing the boiled cassava and steeping the slices, a substantial proportion of the cyanogens was again lost. TABLE 1 Cyanide Content of Cassava During Processing into Ijapu Cyanide Content (ppm) Material analyzed pH Total Free HCN Bound Cyano Unprocessed peeled cassava 6.3 76.1+15.3 5.5+2.2 2.9+0.4 70.6 2.6 Boiled cassava Boil water "Ijapu" (after 24 hours steeping) ND Steep water 6.0 35.1 +8.7 (53.9) 1 2.8+2.1 1 1.8+1.4 (84.5) 4.0 1 6.2+3.9 2.4+ 1.2 2.0+0.7 1.1 +0.2 0.5+0.2 4.2+1.4 3.1+1.4 7.6 (89.2) 7.8 5.7+3.4 5.6+3.4 32.7 0.4 (53.7) 11.6 0.6 Note: Cyano, Cyanohydrin; numbers in parenthesis, percent loss; ND, not determined

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IMPROVING OGI AND GARI 97 Much of the loss could, however, be accounted for in the steep water, and the proportion lost depended on the duration of steeping and the cassava:water ratio (Tables 1 and 21. ROLE OF FERMENTATION The reduction of >99 percent in the linamarin content of grated cassava within 6 hours of adding water, with little or no change in the pH of the mash, would imply that fermentation had nothing to do with the detoxication. Linamarin breakdown is essentially a hydrolytic process catalyzed by the endogenous enzyme linamarase (1,21. The results of the present study indicate that the addition of water aids in the hydrolytic process. Apparently not all of the water normally in raw cassava tuber is available for hydrolysis. During the boiling of cassava for processing into ijapu, linamarase would be inactivated. Yet a substantial proportion of the linamarin in cassava was still lost, appearing to a large extent in the water used for boiling and for steeping (Tables 1 and 2~. This would suggest that leaching could be an important factor in cyanide loss during cassava processing. This would be true not only during the boiling and steeping of cassava for ijapu production but also during the dewatering of grated cassava for gari production. CONCLUSION Cassava detoxication during processing is essentially an enzymic hydrolysis of cyanogens in cassava (1,2,81. Fermentation has little role TABLE 2 Effect of Sliced Boiled Cassava: Steep Water Ratio on Cassava Detoxification . Ratio (w/v) Material analyzed pH Total Free HCN Bound Cyano 1:1 Unboiled cassava 6.4 90.2 4.5 3.0 85.7 1.5 Boiled cassavaa ND 51.7 1.5 0.5 50.3 1.0 (42.7) (41.3) Boil water 6.1 20.2 0.5 0.3 19.7 0.2 -Sliced, boiled ND 5.6 1.1 0.6 4.5 0.6 cassavab (93.8) (94 7) Steep water 4.3 25.5 5.7 4.5 19.7 1.3 1:2 Sliced boiled ND 5.7 1.7 0.6 4.0 1.2 cassavab (93.7) (95.3) Steep water 4.3 21.3 1.9 1.4 19.4 0.5 1:3 Sliced, boiled ND 4.2 1.6 0.5 2.6 1.0 cassavab (95.3) (97.0) Steep water 4.2 11.5 2.1 1.1 9.4 1.0 a Prior to slicing and soaking in water. b after 24 hours soaking; other notes as in Table 1.

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110 FERMENTED FOODS TABLE 3 Lactic Acid and Volatile Fatty Acid Content of Field Collected Kawal, Sigda, and Furundu (Mean and Range in 9 100 9-' Dry Matter) Acid Kawal Sigda Lactic Acetic Propionic Isobutyric n-Butyric Isovaleric n-Valeric Total VFA Furundu 0.21 (0 03 0.51 ) 5.08 (2.1 2~.75) 0.90 (0 51 - 1.59) 0.24 (0.04 0.38) 2.94 (1.18 4.73) 0.22 (0.06 0.60) 0.18 (0.01~.61 ) 9.56 (4.7~1 2.1 ) 3.07 (2 8~3.35) 1.10 (1 .0~1 . 19) 0.04 (0.03~.05) <0.01 0.08 (0.02~. 1 5) 0.02 (0.01~.03) <0.01 1.24 (1 .12 - 1 .38) 0.50 (0.0~1 .67) 1.59 (1 .22-2.05) 0.09 (0.02~.25) <0.01 0.24 (0.05 0~73) 0.17 (0.02~.35) 0.01 (0.01~.02) 2.17 (1 .602.63) the eleventh day of the latter fermentation, VFA mainly n-butyric (8 percent), acetic (5 percent) and n-propionic (2 percent) comprised 15 percent of the fermentation mixture. However, the pH had not changed by more than 0.5 unit from the initial value. On the other hand, by the fifth day of the sigda fermentation, when a total acid concentration of 6 percent had been attained, the pH of the fermentation mixture had fallen to 4.0 from an initial value of about 6.0. This difference in the course of fermentation is almost certainly attributable to the stronger buffering capacity of the substrate of the kawal fermentation, C. obtusifolia leaves, which possess approximately double the calcium content of sesame seedcake. Conditions in kawal do not, therefore, favor the selection of acidoduric lactic acid bacteria. In addition to these bacteria, the two yeast species present in unfermented sesame seedcake proliferated during the initial period of fermentation. Concomitantly, starch levels were observed to fall rapidly from 2 percent in the unfermented substrate to zero after the first two days of fermentation. As the only amylolytic organisms present, the yeasts presumably were responsible for degradation of starch, rendering it available to the lactic acid bacteria. The poorly fermentative yeasts Torulopsis sp. and Debaryomyces sp. isolated in the final stages of the fermentation can utilize lactic acid aerobically and may cause the decline in concentration of the compound during this period. The addition of kambo did not appear to have any significant effect on the course of the sigda fermentation, and it was concluded that this supplementation was probably practiced mainly on organoleptic grounds. The VFA, primarily n-butyric and acetic acids, which are the principal products of the kawal fermentation, are characteristic of clostridial fermentation of plant material as is the accumulation of

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112 FERMENTED FOODS ammonia nitrogen. However, all attempts to isolate clostridial species from the fermentation mixture were unsuccessful, indicating that the microbial origin of VFA must be sought elsewhere. The formation of acetic acid can probably be ascribed to the heterofermentative B. subtilis, the co-dominant microorganism, whereas propionic acid is probably an end product of anaerobic fermentation by Propionibacte- rium sp. for which lactate is a preferred substrate. Utilization of lactate in this way could explain the low level of lactate in kawal, despite the substantial population of Lactobacillus plantarum. The microbial pathway leading to formation of n-butyric acid is difficult to define, although its production may be characteristic of fermentation by this type of mixed culture as a whole rather than that by any single microorganism. n-Propanol (2.3 percent), n-butanol (0.1 percent), and ethanol (0.1 percent) were also detected in kawal toward the end of fermentation, though all were lost from the product during the drying phase. Formation of such alcohols is probably due to anaerobic fermentation of carbohydrate by the yeast species present. The identification of Bacillus sp. as the principal microorganism in furundu when considered in the context of a final pH of 6.2 and the presence of both VFA and lactic acid in the fermentation mixture suggest that the furundu fermentation may be intermediate in character between those of sigda and kawal. Further investigation of the furundu fermentation would be most instructive in this respect. CONCLUSIONS The sigda and furundu fermentations appear quite unlike the tradi- tional oilseed fermentations practiced in Nigeria and elsewhere in West Africa where foods such as ogili and ogiri are fermented from castor oil seed (Ricinus communist and melon seed (Citrullus vulgarisJ. There are even variations of the fermentation which use sesame seed and karkade seed known as ogiri-sara and red sorrel, respectively. During these West African fermentations, the pH increases to over 9.0 and ammonia production is frequently observed in the later stages. The fermentations are dominated by Bacillus sp., frequently Bacillus subtilis, an organism associated with spoilt sigda in Sudan. The principal reason for the difference would appear to be in the preparation of the seeds prior to fermentation, which in West Africa involves boiling for several hours in water until soft. Such pretreatment may alter the course of fermentation by two mechanismsfirst, by rendering protein and polysaccharide more available for degradative attack by microorganisms, and second, by effectively eliminating much of the heat-sensitive indigenous microflora. The removal of amylolytic yeasts

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FERMENTATIONS OF SUDAN 113 may well favor the selection of amylase-producing bacteria such as Bacillus sp. rather than lactic acid bacteria incapable of utilizing starch. The three fermentations studied appear to afford a route by which unpalatable plant material or oilseed cake of little economic value can be converted into acceptable meaty-tasting food that is particularly rich in sulphur amino acids, which tend to be deficient in diets where access to meat or fish is limited. Phytic acid present in seeds can frequently hinder absorption of minerals in the gastrointestinal tract. As fermentation of plant products has been shown to reduce physic acid levels substantially, it is likely that the bioavailability of minerals in both sesame and karkade seed is increased in sigda and furundu. REFERENCES 1. Dirar, H. A. 1984. Kawal meat substitute from fermented Cassia obtusifolia leaves. Economic Botany 38:342-349. 2. 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. 3. Elfaki, A. E., H. A. Dirar, M. A. Collins, and D. B. Harper. 1991. Biochemical and microbiological investigations of sigda- a Sudanese fermented food derived from sesame oilseed cake. Journal of the Science of Food and Agriculture 57.

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16 Continuous Production of Soy Sauce in a Bioreactor Takashi Hamada, Yaichi Fukushima, and Hiroshi Motai Soy sauce is a traditional all-purpose seasoning with a salty taste and sharp flavor. In the conventional method of brewing soy sauce (Figure 1), cooked soybeans and roasted wheat are mixed with spores of Aspergillus species and fermented in solid culture for 2 days to produce koji. The koji is then mixed with brine to make moromi, the mash that ferments to produce soy sauce. Over time the soybeans and wheat are hydrolyzed by enzymes such as proteinases, peptidases, and amylases. During the first stage of moromi fermentation, Pediococcus halophilus grows and produces lactic acid, which lowers the pH. Accompanying the decrease in pH, vigorous alcohol fermentation by Zygosaccharomyces rouxii occurs. As a result, 2 to 3 percent ethanol and many kinds of aroma components are produced by this yeast. At the same time, phenolic compounds such as 4-ethylguaiacol (4EG) and 4-ethylphenol, which add characteristic aroma to soy sauce, are produced by other types of yeasts such as Candida versatilis and Candida etchellsii. It takes over 6 months for the entire fermentation and aging of the moromi mash. Therefore, shortening this period is important and new processes for soy sauce brewing are desirable. This paper describes the continuous production of soy sauce in a bioreactor system, which consists of reactors containing immobilized glutaminase and immobilized cells of P. halophilus, Z. rouxii, and C. versatilis. MANUFACTURING PROCESSES The processes for soy sauce production using the conventional and bioreactor methods are shown in Figure 1. The bioreactor method differs from the conventional one in the following ways: (a) proteases 114

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CONTINUOUS PRODUCTION OF SOY SAUCE Conventional Method | Soybeans Wheat | Steam Roast 1 ~ Starter | Koli ~ Salt Soln. | | Morom i Mash | ! . (n c o Go o 115 Bioreactor Method I Defatted I [Wheat | I Soybean I ' I Steam Roast Hydrolysis and Fermentation Lactic Acid and Alcohol Fermentation Aging Culture LContinuous culture Broth I of KoliMold Hydrolysis (40 to 50C for 3 days) | MoromiMash | Filtration | Raw Liquid | l Salt l | Immol Sized | | Glutaminase | Immobilized Lactic Acid Bacteria P. halophilus cat Filtration ~ Immobilized Yeast Z. rouxii, C. versatilis Pasteurization | Soy Sauce | ! Pasteurization | Soy Sauce | FIGURE 1 Manufacturing processes for soy sauce by conventional and bioreactor methods. from continuous submerged culture are used (1), (b) fermentation is carried out in the liquid state, and (c) the fermentation period is considerably shorter. It takes several months for the conventional fermentation but only about 2 days for the bioreactor method. In the bioreactor method, raw liquid was successively passed through, first, a glutaminase reactor to increase glutamic acid; second, a P. halophilus reactor to carry out lactic acid fermentation; and, third, a Z. rouxii reactor to carry out alcohol fermentation and a C. versatilis reactor to produce phenolic compounds such as 4-ethylguaiacol. Two reactors containing immobilized yeast cells were set in parallel, and the flow rate of the feed solution to the Z. rouxii and C. versatilis

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116 TABLE 1 Conditions of Fermentation in Each Reactor FERMENTED FOODS Column Packed Gel Volume Volume Residence Tempera- Reactor Carrier (L) (L) Time, hours sure, C Aeration,vvm Glutaminase Chitopearl 1.8 0.6 0.7 40 P. halophilus AS 7.5 5.0 6.1 27 Z. rouxii Al - 27.0 8.0 25.5 27 0.005 C. versatilis Al 1.0 0.2 10.7 27 0.08 AS, Alginate-colloidal silica. Al, Alginate. reactors was set in a ratio of 10 to 1. Carrier, packed gel volume, and operating conditions such as residence time, temperature, and aeration in each reactor are shown in Table 1. CONTINUOUS FERMENTATION A profile of continuous fermentation by immobilized cells of P. halophilus, Z. rouxii, and C. versatilis is shown in Figure 2. The fermentation continued for over 100 days without any microbial contamination. A consistent increased level of glutamic acid (in the range of 0.3 to 0.4 percent) was found in the effluent from glutaminase reactor, with a residence time of 0.7 hours. Lactic acid was produced by immobilized cells of P. halophilus in quantities of 0.7 to 1.0 percent at a residence time of about 6 hours, and consequently the pH declined to 4.9 to 5.0, similar to that of conventionally brewed soy sauce. Ethanol was produced constantly by immobilized cells of Z. rouxii in quantities of 2.5 to 2.7 percent at a residence time of about 26 hours. This is the standard ethanol content in soy sauce. About 10 ppm (parts per million) of 4-ethylguaiacol was produced by immobilized cells of C. versatilis at a residence time of about 10 hours, and the final 4- ethylguaiacol content after mixing the two fermented liquids from the reactors of Z. rouxii and C. versatilis was about 1 ppm, which is the optimum concentration in conventional soy sauce. The total residence time for lactic acid and alcohol fermentation was about 30 hours in this system. This was considerably shorter than the conventional fermentation period of 3 to 4 months required to produce the same amounts of lactic acid and ethanol. High numbers of viable cells were present in the gel and liquid in each reactor. The number was 10- to 100-fold higher in moromi mash. The shortening of the fermentation period in the bioreactor method is possibly due to the high density of immobilized cells in the gel and free cells in the liquid. The main chemical components of the fermented liquid from the bioreactors were examined, including lactic acid, glucose, ethanol, and nitrogenous compounds.

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CONTINUOUS PRODUCTION OF SOY SAUCE 1.0 0.8 _ ~ ~ C' ct c, 0.6 Cat Ct , ' 0.4 0.2 o 3.0 ^ _ o ct5 2.0 _ ~ 1.0 o 15 Q Q - c~ 10 111 5 o PROPERTIES 117 _ ~ _ t~ 5.0 I _ ~ _ _ 1 1 1 1 1 _ err 1 1 1 1 1 _ o 20 40 60 80 100 Time (days) FIGURE 2 Profile of continuous fermentation of soy sauce by a bioreactor system. (a), lactic acid; (a), glutamic acid (values indicate the increase in the amount of glutamic acid); (v) pH; (a), ethanol; (a), glucose; (a), 4-KG after passing through the C. versatilis reactor; (a), 4-KG in the final product. 6.0 4.0 The organic acids and aroma components in the bioreactor soy sauce were examined. The proportions of organic acids except citric acid were not much different between the bioreactor soy sauce and the conventional one, although the former was a little lower in acetic acid and succinic acid. It appears that the high residual content of citric

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118 FERMENTED FOODS acid in the bioreactor soy sauce arises from the inability of P. halophilus to utilize citric acid. Aroma components present in both the bioreactor and conventional soy sauces were not qualitatively different. However, the former was higher in isoamyl alcohol and acetoin and lower in isobutyl alcohol, ethyl lactate, 4-hydroxy-2(or51-ethyl-5(or21-methyl- 3~2H)-furanone, and 4-hydroxy-5-methyl-3~2H)-furanone. To evaluate the aesthetic qualities of the bioreactor-produced soy sauce, sensory tests were carried out. For example, the intensity of the alcoholic, fresh, sweet, acid, and sharp odors as well as the special higa (baking aroma) and bushoshu (foul fermented aroma) were compared between the bioreactor and conventional soy sauces. The odors are important for the quality of soy sauce. Although the bioreactor soy sauce was a little weaker in aroma and fresh odor than the conventional soy sauce, the quality of the former was generally judged to be similar to that of the latter. The total time required for the production of soy sauce by the bioreactor system, including enzymatic hydrolysis of the raw materials, fermentation with immobilized whole cells, and the refining process, is only about 2 weeks (24. This is considerably shorter than the 6 months with the conventional method of soy sauce brewing consisting of koji making, fermentation and aging of moromi, and refining. From these results we conclude that the quality of the bioreactor soy sauce was very similar to that of the conventional soy sauce from both chemical and sensory evaluations and that the bioreactor system is practical for the production of soy sauce. REFERENCES 1. Fukushima, Y., H. Itch, T. Fukase, and H. Motai. 1989. Applied Microbiology and Biotechnology 30:60~608. 2. Hamada, T., M. Sugishita, Y. Fukushima, and H. Motai. 1991. Process Biochemistry 26:39~5.