§§ WWINIWIHIIWIWWIMIIHHWIIIHIWI THS .~ . ‘ ‘l’u' . [Munuugmfiatugpm ’ ‘ 3' " F A 3'13" t .. - u E. 4‘“; ~ “ :16 ; inn-«"3 7." '3. This is to certify that the EVALUATION thesis entitled OF TEMPEH PREPARED FROM GERMINATED SOYBEANS presented by Suparmo has been accepted towards fulfillment of the requirements for M. S. degree in Food Science Mejia? HIM?” 0-7 639 72.5% flaw/WA; Major professor MS U is an Affirmative Action/Equal Opportunity Institution MSU LlBRARlES .—:_—_ RETURNING MATERIALS: Place in book drooflto remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. n A Alb. “k .2 ‘ g", \ Rm. . h f}!!- 9"” 450 0023 r: :C’°"a“ ’3’ * i. '- 5 5. :~ a l '7‘ 4*; (I?! J g I‘. is} :‘ .EVALUATION OF TEMPEH PREPARED FROM GERMINATED SOYBEANS By Suparmo A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition l984 ABSTRACT EVALUATION OF TEMPEH PREPARED FROM GERMINATED SOYBEANS By Suparmo Tempeh is a traditional Indonesian food made from soybeans fermented by a mixed culture of Rhizopus species. The tempeh fermentation improves the acceptance and the digestibility of the soybeans. The objective of this research was to combine the tempeh fermentation with soybean germination in order to further improve the nutritive value of tempeh. Germination for 24 hours reduced the oligosaccharide, phytic acid and crude fat content, and slightly increased the total and soluble protein content. The combination of soybean germination and tempeh fermentation reduced more oligosaccharides and phytic acid than tempeh fermentation alone, while further decrease of fat content and further increase of total and soluble protein were also observed. Lectin activity was not observed in any of the tempeh samples. The PER of tempeh prepared from germinated soybeans was 2.25, while that of regular tempeh was 2.18. Tempeh from germinated soybeans could not be differentiated in taste and appearance. ACKNOWLEDGMENTS The author would like to thank his major professor, Dr. P. Markakis, for his encouragement and excellent guidance throughout the course of this study and assistance in the preparation of this manuscript. Appreciation is also expressed to Drs. R. Herner and D.R. Dilley of the Department of Horticulture, and Drs. C.M. Stine and M.A. Uebersax of the Department of Food Science and Human Nutrition, for their participation in my graduate committee. The author wishes to express his gratitude to the Government of Indonesia and Gadjah Mada University for providing a leave of absence, and to The Rockefeller Foundation for the financial support for this program. Finally, the author is deeply grateful to his parents, Padmomartono, his wife, Niniek, and his daughters Dian and Dani for their support, encouragement and understanding. TABLE OF CONTENTS Page LIST OF TABLES. . . . . . . . . . . . . . . . . . . VI LIST OF FIGURES . . . . . . . . . . . . . . . . . . VII INTRODUCTION. . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . 6 Tempeh. . . . . . . . . . . . . . . . . . 10 Raw materials . . . . . . . 13 Organisms in tempeh fermentation. . . . . . 15 Growth requirements . . . . 16 Changes occurring during tempeh fermentation. . 20 General . . . . . . . . . . . . . . . . . 20 Carbohydrate. . . . . . . . . . . . . . . . 23 Lipid . . . . . . . . . . . . . . . . . . . 25 Protein . . . . . . . . . . . . . . . . . . 29 Vitamin . . . . . . . . . . . . . . . . . . 31 Phytic acid . . . . . . . . . . . . . . . . 32 Aflatoxin . . . . . . . . . . . . . . . . . 33 Soybean lectin. . . . . . . . . . . . 34 Protein quality evaluation. . . . . . . . . . . 36 MATERIALS AND METHODS . . . . . . . . . . . . . . . 39 Germination of soybeans . . . . . . . . . . . . 39 Tempeh preparation. . . . . . . . . . . . 39 Protein and soluble protein . . . . . . . . . . 41 Extraction of crude fat . . . . . . . . . . . . 41 Oligosaccharide determination . . . . . . . . . 41 Phytic acid determination . . . . . . . . . . . 43 Hemagglutinin assay . . . . . . . . . . . . . . 45 Protein efficiency ratio. . . . . . . . . . . . 48 RESULTS AND DISCUSSION. . . . . . . . . . . . . . . 51 Germination . . . . . . . . . . . . . . . . . 51 Tempeh fermentation . . . . . . . . . 51 Total nitrogen and soluble. nitrogen . . . . . . 52 IV Page Crude fat . . . . . . . . . . . . . . . . . . . 55 Oligosaccharides. . . . . . . . . . . . . . . . 57 Phytic acid . . . . . . . . . . . . . . . . . . 59 Protein efficiency ratio. . . . . . . . . . . . 62 Lectin activity . . . . . . . . . . . . . . . . 64 CONCLUSION. . . . . . . . . . . . . . . . . . . . . 66 BIBLIOGRAPHY. . . . . . . . . . . . . . . . . . . . 67 Table TO ll 12 VI LIST OF TABLES Proximate composition of soybeans and seed parts. Nutritional composition of tempeh before and after fermentation . . . . . . . . Raw materials and the correspondent tempeh names. . . . . . . . . . . .' Chemical characteristics of oils from tempeh and unfermented soybeans . . . . . . Amino acid composition of tempeh and soybeans. Amino acid pattern required for growth and maintenance of rat and man . . . . . Total and soluble nitrogen in germinated soybeans and tempeh prepared from germinated soybeans . . . . . . . . . Crude fat in germinated soybeans and tempeh prepared from germinated soybeans. Oligosaccharides in germinated soybeans and tempeh . . . . . . . . . . . . . Phytic acid content in germinated soybeans and tempeh prepared from germinated soybeans Protein efficiency ratio of regular tempeh and tempeh prepared from germinated soybeans Lectin titer and the amount of germinated soybeans needed to agglutinate 50% of RBC suspension . . . . . Page 14 14 27 30 38 53 56 6O 61 63 6S Figure l Changes in soluble carbohydrate and reducing sugar contents during germination of soybeans. 2 Changes in raffinose and stachyose contents during germination of soybeans . 3 Drawing of Rhizopus sp.. 4 Changes occurring during tempeh fermentation 5 Structure of stachyose 6 Free fatty acid (FFA) content, temperature and total bacterial plate count of soybeans inoculated with R. oligosporus at 32°C 7 Proposed structure for phytic acid 8 Standard curve for Fe determination. 9 The HPLC separation of oligosaccharides. LIST OF FIGURES VII Page 11 13 17 21 24 28 33 46 58 INTRODUCTION Soybeans are an inexpensive protein-rich food which has been widely consumed in the Orient for thousands of years. They are of special value for supplementary feeding in many countries where milk and meat cannot be produced sufficiently. According to Autret and van Veen (I955), soybeans can satisfy the nutritional needs of infants, children, pregnant and nursing women. Soy milk is popular in China. It was adopted by the FAO in its program to meet the protein needs for protein malnutrition in infants and young children in many developing countries. It is also useful as a milk substitute, especially for infants and children who are allergic to milk. As in other countries in south-east Asia, rice is a staple food in Indonesia. It is eaten daily, two or three times a day. Since foods of animal origin are not sufficiently available, they are not consumed daily, and vegetables are the main supplement commonly eaten along with rice. Soybean products like tempeh (fermented soy- beans) and tofu (soybean curd) are supplements frequently used. Nutritionally, these supplements add protein to the rice diet. Lysinemand‘threonine are the first and second limiting amino acids in rice, while sulfur containing 1 amino acids are the limiting factors in soybeans. The supplementation of a small amount of soybeans to rice slightly improves the chemical score of the amino acids in rice by increasing the threonine content. Because of the sulfur amino acid deficiency in soy, sulfur amino acids become the first limiting as the proportion of soy protein increases (Jansen, T972). Germination and fermentation of beans are two popular processes which can improve both the sensory values such as texture, flavor, aroma and color, and the nutritional quality of soybeans. Everson _t _l, (l943) reported that germinated soybeans were superior to the ungerminated ones when they were fed to laboratory animals. Germination reduced growth inhibiting factors and increased the protein efficiency ratio of the soybeans. Fermentation of food, especially high-protein food, is commonly practiced in Asia. Whitaker (1978) mentioned the following advantages and reasons for fermenting food: improving flavor, aroma, texture, color, facilitate solubilization, improving digestibility, nutrition, less cooking and removing toxic substances. Another reason why fermentation is so popular in Asia is that this process is relatively simple and inexpensive, and it can be performed with simple household utensils available locally. It can be performed by people in rural areas who have no scientific background about the process of fermentation. The tempeh fermentation is traditionally performed in Indonesia. It consists of two steps: the lactic acid fermentation and the mold fermentation. The lactic fermentation appears to facilitate the subsequent growth of the fungus, while the mold fermentation changes the soybeans to a cake-like solid, in which the soybeans are bound together by cottony mycelia. Tempeh has a pleasant odor and a relatively mild taste (Steinkraus et 11., l960). The tempeh fermentation was studied extensively by Steinkraus 33 31. (l960), Hesseltine ;t__l. (l963), and Hesseltine and Wang (T967). They described the mold species to be used and reformulated the process in order to have optimal yield in a laboratory scale. Progress in the development of larger scale tempeh processing has also been made (Martinelli gt 31., 1964). Steinkraus _t _l. (1965) developed a pilot plant process for the production of dehydrated tempeh. The production of Rhizopus oligosporus spores and their application in tempeh fermentation has been studied by Wang et 31. (I975). Wang and Hesseltine (l966) tried to make wheat tempeh and Hesseltine £3.11- (l970) introduced a new fermented cereal product using mold isolated from tempeh. All of the tempeh-like products were reported to have a pleasant, mild taste and a desirable color. Biochemical changes occurring during mold fermentation have been described by many researchers. There are changes in protein, soluble protein and amino acids composition (Steinkraus t 1., 1960; Hesseltine t 1., 1963; Stilling and Hackler, 1965; and Murata t al., 1967), carbohydrates (Hesseltine gt gt., 1963), lipids Steinkraus t 1., 1960; Nagenknecht gt gl., 1961; Murata _t gl., 1967; and Sudarmadji and Markakis, 1978), vitamins (Roelofsen and Talens, 1964; Murata gt gl., 1967; Robinson and Kao, 1977; and Liem gt gt., 1977), phytate and phytase (Sudarmadji and Markakis, 1977). There are some contradictory results regarding the nutritive value of tempeh fed to rats. Rats on tempeh diets ate more, gained more weight and had higher PERs than did the rats eating autoclaved-unfermented soybeans (Gyorgy gt gt., 1964; Kao and Robinson, 1978). However, Smith gt gt. (1964) and Hackler _t gt. (1964) reported that rats fed tempeh showed a small reduction in growth and protein efficiency compared with autoclaved and dehulled full-fat soybean meal. When methionine was supplemented to the diets, the rate of rat growth and protein efficiency values increased significantly (Smith t 1., 1964). Hackler _t _t. (1964) reported that acceptance of tempeh containing diets by rats was decreased with each 12-hour increment in fermentation time. Either mold or something elaborated by the mold depressed the acceptance of the diets containing the fermented soybeans. Germination has been reported to improve protein quality, digestibility and reduce certain anti-nutritional factors. It was the purpose of this research to combine germination and tempeh fermentation in an effort to improve the overall quality of tempeh. LITERATURE REVIEW Soybeans (Glycine max. t.) are a rich source of protein, yet the direct utilization of the beans is limited because of the presence of anti-nutritional factors and undigestible constituents. The composition of soybeans is presented in Table 1. Germination generally increases the nutritive value of seeds. Nhyte (1973) pointed out that during germination, stored materials are converted into more usable forms for the plant and eventually for man (Wang and Fields, 1978). Chen _t gt. (1975) reported that the first stage of seed germination involves the breakdown of seed reserves for their utilization by the growing root and shoot. During germination, some constituents were degraded, whereas others were synthesized. Protein and oil are major sources of energy for the developing embryo (Hsu gt _t., 1973). Abrahamsen and Sudia (1966) reported that protein decreases and amino acids increase as seeds germinate. Oil is depleted rapidly and is broken down into glycerol and fatty acids which finally give rise to carbo- hydrates via the glyoxylate cycle followed by reversed glycolysis. Soluble carbohydrates are also an important Table l. Proximate composition of soybeans and seed parts (% dry basis).* . Protein Fract1on (% N x 5.25) Fat Carbohydrate Ash Nhole bean 4O 21 34 4.9 Cotyledon 43 23 29 5.0 Hull 8.0 1 86 4.3 Hypocotyl 41 11 43 4.4 *From Wolf and Cowan (1971). energy source during the early stages of germination. Everson _t _t. (1943) reported that germinated soybean protein is distinctively superior to that of raw beans. The nutritive value of both improved by heat treatment (autoclaved). The PER ranged from 0.5 for the mature raw seed to 1.4 for the germinated seeds, to 1.7 for the auto- claved seeds and to 1.9 for the autoclaved germinated seeds. Freed and Ryan (1978) reported that some proteinase inhibi- tors disappear completely during germination, while certain new inhibitors appear. Increases in lysine and tryptophan and decrease of prolamine occurred during germination of cereal grains (Tsai _t _t., 1975; Dalby and Tsai, 1976). The interest in the changes of carbohydrates, espe- cially the oligosaccharides, raffinose and stachyose, is due to the belief that they are primarily responsible for the flatulence often experienced by persons consuming soybean-based foods (East t 1., 1972; Abrahamsen and Sudia, 1966; Hsu _t _t., 1973). Reddy _t _t. (1980) studied the flatulence in rats following ingestion of cooked and germinated black gram and a fermented product of black gram and rice blend. The maximum hydrogen production was obtained with 60% cooked black gram cotyledons in the diet. Germinated black gram seeds and fermented steamed product significantly produced lower flatus than the cooked black gram products. A positive significant correlation was found between oligosaccharides of the raffinose family present in black gram and hydrogen production by rats. Rackis _t _t. (1970) reported that an 11 tittg assay using intestinal bacteria showed that toasted, dehulled, defatted soybean meal contains a gas-producing factor and a gas- inhibiting factor. The oligosaccharides - sucrose, raffi- nose and stachyose - are associated with the gas-producing factor when incubated in thioglycollate media with anaerobic bacteria of the intestinal tract of dogs. The phenolic acids of soybeans, syringic and ferulic acids, are effective gas inhibitors jg_tjttg_and in the intestinal tract of dogs. The lipids, proteins and water-insoluble polysaccharides of soybean meal have no gas activity. Abrahamsen and Sudia (1966) studied the soluble carbo- hydrates in germinating soybean seeds. They found that the most rapid decline in total soluble carbohydrate in soybean cotyledons and embryo axis occurred during the first three days of germination. Sucrose, stachyose and raffinose were the predominant soluble carbohydrates in the cotyledon of ungerminated soybean seeds in a ratio of approximately 7:3:1, respectively. By day 1 there was approximately a 50% reduction in raffinose with only slight decrease in sucrose and stachyose. Between day 1 and day 2 there was a sharp decrease in stachyose; raffinose also continued to decrease during this interval. By day 2 stachyose and raffinose were almost completely depleted. Sucrose showed a moderate decrease between day 1 and day 2 with a marked decrease '10 between day 2 and day 3. The predominant sugar in the embryo axis of ungerminated soybean seed was stachyose followed by sucrose and raffinose. Between day 0 and day 1, both stachyose and raffinose decreased rapidly while the sucrose content increased during this interval. It showed that there was a much earlier utilization of stachyose in the embryo axis of the germinating soybean seedling than in the cotyledon. While stachyose was depleted rapidly, a synthesis of sucrose occurred. Since sucrose may be a hydrolysis product of raffinose and stachyose, the accumu- lation of sucrose may be due to partial hydrolysis of stachyose during this interval. Several unidentified oligosaccharides were present during the first two days in both embryo and cotyledon. Changes in total soluble carbohydrate is presented in Figure 1, while Figure 2 shows raffinose and stachyose during germination of soybeans. East _£._l- (1972) confirmed previous observations and pointed out that, during normal germination, the content of monosaccharides is increased, sucrose shows an initial stationary period followed by a decrease, and raffinose and stachyose experience a general decrease. Tempeh Tempeh is a traditional Indonesian food, made mostly from soybeans, fermented by a mixed culture of several species of Rhizopus. Raw tempeh has the appearance of a Sugars (mg/100mg soybean seedling). 4 .. ’— 2 reducing S—ug/ O L I O 1 2 3 Germination time (days). Figure 1. Changes in soluble carbohydrate and reducing sugar during sovbean germination (Adapted fromzAbrahamsen and Sudia, 1966). 12 4 J. .40 (I) f: m m .D T A o (I) 3 _,. Of —30 9—1 Q (3 0 Q% 2%\ oo «5&0 o 23 ®® a 20 m 2- _ (I) o C: 'r-i LH “-11 m L. w 1— ~10 o E3 1 I I ‘1 O 24 48 72 96 120 Germination time ( hours). Figure 2. Changes of raffinose and stachyose content during germination of soybeans. (East gt gt., Mg of stachyose per g of soybeans 13 cake-like solid mass of soybeans, held together by cottony mold mycelia. It is eaten, after cooking as a soup or frying, by millions of people in Indonesia and Surinam. Tempeh is also manufactured in Holland, Canada and the United States (Shurtlef and Aoyagi, 1978). The nutritional composition of tempeh is presented in Table 2. Raw Material The world tempeh (tempe) was originally applied to all cake-like products fermented by mold. There are many materials commonly used to make tempeh. Table 3 shows the raw materials used to prepare tempeh and the corresponding types produced from them. They are fermented using the same starter inoculum. The most popular type is tempe kedele, which is called tempe for simplicity and written tempeh in English. Tempe bongkrek is not allowed to be produced since many deaths occurred as a result of eating tempe bongkrek contaminated with Pseudomonas cocovenenans. In some places, soybeans are mixed with cheaper materials, such as cooked rice, rice bran, tofu by-products, or peanut press—cake in order to produce tempeh more economically. A similar tempeh-type product prepared from insoluble fraction of ground soybeans fermented with Neurospora sitophila, oncom, is very p0pular in West Java. It has a 14 Table 2. Nutritional composition of tempeh before and after fermentation*. Component Before fermentation After fermentation Crude protein (%) Crude fiber (%) Crude fat (%) Ash (%) PER Niacin (us/9) Riboflavin (ug/g) Thiamin (ug/ Vitamin B-6 ng %) Vitamin B-12 (mg %) Pantothenic acid (mg %) Phytic acid (%) Antitryptic act. (TIU/mg) Flatus factor (raffinose family sugars) 4:. N m—‘d-POONNVN-bw—‘m _.a \l U'INNOS—‘OCDONU‘I-‘(TIOCDN ow 0100 —l 5 d N¢owmomoou1m4>xowo *From: Reddy t al., 1981. Dry basis. Table 3. Raw materials and the correspondent tempeh name. Raw material Name Velvet beans Tempe benguk Peanut press-cake Tempe bungkil Soybeans Tempe kedele Water insoluble fraction of Tempe gembus ground soybeans Coconut press-cake Tempe bongkrek 15 pinkish appearance rather than white. Hesseltine and Wang (1967) developed new fermented tempeh-type product prepared from wheat, rye, oats, barley, rice and a combination of rice or wheat with soybeans. The products were reported to possess a very acceptable mild taste. Orggnisms in Tempeh Fermentation A traditional tempeh fermentationis prepared with impure cultures of mold. The inoculum is taken from pieces of a previous fermentation or its wrapper, which is usually leaves. A traditional inoculum is also available commer- cially as a dry spore preparation on dry hairy Hibiscus tiliaceous leaves. Tempeh starters are now available in powder form prepared from a pure culture of a mold, usually Rhizopus oligosporus. Hesseltine gt_gt. (1963) has isolated some strains of Rhizopus from Indonesian tempeh. They were identified as: Rhizopus stolonifer, R. oryzae, R. arrhizus, R. achlamy- dosgorus, R. nigricans, R. formosaensis. It was concluded that 3. oligosporus is the principal species used in Indo- nesia for making tempeh. Wang t 1. (1975) successfully developed a method for mass production of Rhizgpus oligosporus spores and its application. 16 Classification of Rhizopus (Frazier, 1957) Division : Thallophyta Subdivision : Eumycetes (true fungi) Class : Phycomycetes (non septate) Subclass : Zygomycetes Order : Mucorales Genus : Rhizopus Genus Rhizopus has the following characteristics: non septate; has stolon and rhizoids; sporangiophores arises from the nodes where rhizoids are formed; large sporangia, usually black; hemispherical columella; thick cottony mycelia (Figure 3). Growth Requirements Hesseltine (1963) studied the growth requirements of 4 species of RhiZOpus which were suitable for making tempeh. Carbon sources supporting the growth of the 4 species were: soybean oil, xylose, glucose, galactose, trehalose, cello- biose, but not raffinose, lactose and inuline, while the best nitrogen sources were asparagine and ammonium sulfate. Steinkrause gt gt. (1960) reported that, in order to be complete, the tempeh fermentation required that two conditions be fulfilled. First, the soybeans had to be bound into a compact cake by the growth of mold mycelia. Second, the soybeans had to undergo a partial digestion by the mold enzymes, thus the tempeh fermentation was centered in the mold growth. Since tempeh mold would not grow well on the unskinned beans, skin removal is an essential step in tempeh fermentation. 17 I Sporangium Sporangiospore apophysis ----- columella -------- . .° --- spores sporangiophore---- 0 growth habit node -—- stolon ----- rhizoid Figure 3. Rhizopus sp. 18 Oxygen is essential to mold. When the layer of the fermenting beans was thicker than 2 inches or about 5 cm, mold grew less heavy in the center than it was in the thinner layer. To make a good tempeh, the thickness should not be more than 3 cm (Steinkraus t 1., 1960; Martinelly _t gt., 1964). Robinson and Kao (1977) studied the mold fermentation on various sizes of soybean products. The mold did not grow on soybean flour and was unable to grow well on small grits, because there was too little oxygen below the surface of the mass to support mold growth. The best fermentation result was obtained when the diameter of the grits was between 0.2 to 0.4 cm. In order to get the right amount of aeration, perforated metal trays or plastic bags can be used. However, if the amount of aeration is in excess, the soybeans at the surface will dry out and sporulation will start producing undesirable black spores and poor appearance. It is obvious that high relative humidity is absolutely needed (Steinkraus _t _t., 1960; Martinelli gt gt., 1964). Temperature is a critical factor for microbial growth. Temperature slightly above room temperature is best for tempeh fermentation. Traditional tempeh makers in Indonesia start to mix the inoculum when the cooked soybeans cool down 0 to about 50 C. During mixing and wrapping the inoculated beans cool further to room temperature. For incubation, the 19 wrapped, inoculated soybeans are packed in baskets with the temperature about 35°C. The temperature will rise to about 40°C when the mold is growing overnight. The following morning, the tempeh should be taken out from the baskets and put in racks for further incubation at room temperature until the tempeh is ready for marketing. If the tempeh is not cooled when the temperature is high, the mold will stop growing and bacteria will grow very quickly producing a poor appearance and a bad odor. Steinkraus gt _t. (1960) reported that the optimum temperature for tempeh mold is 37°C. Temperatures as low as 25°C were used to produce an acceptable tempeh. Such fermentation required as long as 5 days for completion while fermentation at 37°C required only 1 day. Later investigations by Hesseltine gt_gt. (1963) showed that Rhizopus strains that produce satisfactory tempeh can grow from 14°C to 44°C except 3. stolonifer, which has a maximum temperature of no more than 35°C. Some of the R. oligosporus isolates grow poorly at 50°C. Temperature of 31°C was the best for making tempeh, except for R. stolonifer which required a temperature below 31°C. At optimum tem- perature, 22 to 24 hours was needed to complete the mold growth and produce good tempeh. For good tempeh fermentation, it is essential to ensure that the beans are acidified to a sufficient degree at the start of fermentation. The pH of soaked and cooked soybeans 20 in acidified water (30 ml lactic acid/3000 ml water/1000 g beans) was about 5. This pH was sufficiently low to inhibit growth of most contaminating bacteria which would spoil the tempeh but did not interfere with the growth of the mold. The mold growth is inhibited when the pH drops below 3.5. Changes Occurring During Temgeh Fermentation General Changes Steinkraus _t _t. (1960) studied general changes occurring during tempeh fermentation. By referring to Figure 4, the changes can be followed. The increasing temperature of fermenting bean mass was indicative of the relative growth rate of the mold. The first 20 hr, during which time the germination of spores took place, the temperature rose gradually. After 4 to 5 hr of accelerated growth, the temperature reached 43-44OC and then gradually decreased as the mold growth subsided. At this stage, the beans were already bound into compact mass by mold mycelia. Following the mold growth, sporulation and NH3 production due to protein breakdown appeared. During the period of most rapid mold growth, the soluble solids rose from 13 to 22% and continued to rise up to 27.5%. At this stage, the sporulation and NH3 production would be considered too far advanced. The soluble nitrogen rose from 0.5% to nearly 2% while the total nitrogen remained relatively constant, about 7.5%. Hd .Aoomp ..~m um mzmcxcwmpmv cowumucmscmw cmqemu wzu mcwcav mcwcczuuo mmacmsu .v wgzmwu muse: mu Om mo 00 mm Om me oq mm Om mm ON ma A: m o 1 O mmuqdumndm tchztmm lllHHHUl i “ manzaom . O.m ON .n m.m O - 0.0 2% m m cg: m.© . 1 e O.N . 0mm” 3 ll 8 0 ml m.\. D n o.m mm a S 0 ml. v 04V \/ X Hm 22 The pH, which initially was 5.0, rose to 7.6. The change from 6.0 to 6.7 occurred during the period of most rapid mold growth. Optimum quality tempeh has pH 6.3 to 6.5. Reducing substances decreased very slowly during the entire fermentation. They did not regain their initial level even though breakdown of higher carbohydrates continued. There was an increase of fiber from 3.7% dry basis in hydrated, peeled, cooked soybeans to 5.8% in the tempeh. Fermented soybeans freed from their superficial mold layer, showed a reduction in total fiber to 2.8%. The mold mycelium, removed from the tempeh, contained 7.1% fiber, which was responsible for the increase in fiber content. Later investigations by Murata _t_gt, (1967) showed the following general changes: - There were no large differences in protein and ash content between tempeh and unfermented soybeans. - During fermentation the fiber was slightly increased. - Fat content decreased but the acid value increased noticeably. - Free amino acids increased. - Riboflavin, vitamin B6, nicotinic acid and panto- thenic acid increased during fermentation, although thiamin was altered slightly. 23 Carbohydrate Changes The carbohydrate content of soybeans is about one third of their dry weight. It consists of polysaccharides, stachyose (3.8%), raffinose (1.1%), and sucrose (5%). About 7% of the total carbohydrate is located in the hull, 26% in the cotyledon, and 1% in hypocotyl. Reducing sugars in dormant seeds are very low (Gould and Greenshields, 1964; Abrahamsan and Sudia, 1966; East gt_gt., 1972 and Hsu _t _t., 1973). Soluble carbohydrate increases, while reducing sugars decline during fermentation._ The level of reducing sugars remains low even though complex carbohydrates continue to breakdown, presumably because the sugars are used by the mold. The oligosaccharides, stachyose and raffinose are of particular interest because they are considered responsible for the flatulence often experienced by people who eat soybeans (East gt gt,, 1972; Abrahamsen and Sudia, 1966). Stachyose as shown in Figure 5 is a tetrasaccharide, made up of three simple sugars: galactose, glucose, and fructose, which are joined in such a manner that upon hydrolysis, raffinose, manninotriose, galactobiose, melibiose, sucrose, glucose, galactose and fructose may be formed. The structure of stachyose and raffinose can be cleaved by acid or enzymes. a-Galactosidase liberates .wmox;umum do weapozcum .m means; 1 m m o w m o < a m 14 . mmoszm5 s8- 50‘ 00 o ‘2 R6— // (040- .; e r-i :3 g 4 $1M)— :>. - H ' u m U a m E ““ E(£20 232- S... m 0 15 3O 45 6O 75 9O —- - -—> Incubation time (hours). Figure 6. Free fatty acid (FFA) content, temperature and total bacterial plate count of soybeans inoculated with 3.011gogporus at 32°C. (Sudarmadji and Markakis, 1978). 10 0‘ J.\ N of bacterial plate count Log. 29 high sensory quality. The second phase was a transition phase lasting 24 hr after the first phase, when microbial growth and lipolysis subsided the temperature decreased and the product was still in good condition. The third phase was a deterioration phase. In this phase, the bacterial growth and lipolysis reappeared and the tempeh quality deteriorated rapidly. Protein Changes Steinkraus _t _t. (1960) showed that during tempeh fermentation, soluble nitrogen including NH3 increased due to protein breakdown. In general, most amino acids either remained unchanged or declined according to Stilling and Hackler (1965). A notable exception was tryptophan, which was significantly higher in tempeh fermentation for 24 hr at 37°C, but declined thereafter. During fermentation ammonia increased significantly, possibly from amino acids deamination. A comparison of amino acids composition between soybeans and tempeh made in Indonesia, and soybeans and tempeh prepared in the laboratory was made by Murata gt gt. (1967). The amino acids composition is shown in Table 5. In general most amino acids were not changed by fermentation. It was observed that the tryptophan content of the samples from Indonesia increased about 20% and alanine in tempeh prepared in the laboratory also increased about 20%. 30 Table 5. Amino acid composition of tempeh and soybeans (mg/9N)* . . Indonesian Soybeans Harosoy Soybeans** Am1no ac1ds Soybeans Tempeh Soybeans Tempeh Aspartic acid 744 756 704 673 Threonine 278 282 241 218 Serine 270 268 323 273 Glutamic acid 1050 1000 1110 974 Proline 342 309 342 307 Glycine 292 275 263 257 Alanine 250 228 280 338 Cystine 113 121 98 80 Valine 328 345 336 319 Methionine 77 81 61 61 Isoleucine 338 356 301 310 Leucine 525 565 518 492 Tyrosine 171 161 188 178 Phenylalanine 302 302 384 307 Tryptophan 67 87 63 66 Lysine 392 410 ‘384 330 Histidine 160 167 187 171 Arginine 491 440 392 373 * Adapted from Murata _£._l- (1967). **Prepared in Food and Nutritional Laboratory, Osaka City University. Fermentation was done according to Stein- kraus method, 48 hr. 31 Changes in the amino acids of tempeh from Indonesia and of tempeh prepared in the laboratory were not always parallel. During the fermentation, the amount of free amino acids increased progressively. After 48 hr of fermentation, the amount of individual free acids increased 85 times over those of the unfermented soybeans. Robinson and Kao (1977) reported that the water soluble protein increased two to three times after tempeh fermenta- tion. The increase resulted from the proteolytic activity of the mold, which partially hydrolyzed the insoluble protein to soluble protein. From the amino acids study, it was found that the essential amino acids remained unchanged after fermentation. Vitamin Changes The nutritional value of tempeh and utilization of protein from tempeh might be affected by its content of B vitamins. A sharp increase of riboflavin, nicotinic acid, vitamin B6 and pantothenic acids during tempeh fermentation were reported (Steinkraus t al., 1960; Roelofsen and Talens, 1964; Murata gt gt., 1967; Liem t 1., 1977). Robinson and Kao (1978) reported that water soluble vitamins increased during tempeh fermentation. 32 Phytic Acid Change Phytic acid (inositol hexaphosphate, with the proposed structure shown in Figure 7), is one of many anti-nutritional factors present in plant origin foods. It has been demon- strated that the presence of phytic acid in diets may reduce the availability of some essential, di- and tri- valent metals (Welch and House, 1982; House _t _t., 1982). The interaction between phytic acid and mineral ions to form insoluble complexes appears to be the major factor responsible for the adverse nutritional effects observed in high phytate diets. During tempeh fermentation, the Rhizopus oligosgorus used in this fermentation produced phytase which reduced about one-third of the phytic acid content (Sudarmadji and Markakis, 1977). Other molds, such as Neurospora sitophila, which is used to prepare oncom, a tempeh-like product from peanut press cake (Fardiaz and Markakis, 1981), Mucor dispersus and Actinomucor elegans (Wang gt gt., 1980) were also reported to produce phytase. Changes in Aflatoxin Content No aflatoxin presence in tempeh prepared from soybeans were reported. However, cases have been reported of tempeh and oncom prepared from peanut press-cake which contained aflatoxin at a significant level. Van Veen gt gt. (1968) 33 OH OH O—P-O -O-P-O at F as OH OH O-P—O __O-P-O , _ no as Figure 7. Proposed structure for phytic acid ( Wheeler and Ferrel, 1971 ). 34 reported that Neurosgora sitophila reduced the aflatoxin B1 of peanut press cake by about 50%, while Rhizopus oligo- sporus reduced it by about 70%. Soybean Lectin Early investigators in nutrition were fully aware that certain legumes were very poor sources of protein unless subjected to heat treatment. Heat labile substances can interfere with the normal growth of animals, and can even cause death after prolonged ingestion. Crude trypsin was not effective in counteracting this inhibitor of growth (Liener, 1953; Honavar _t _t., 1962). Liener (1953) reported that a protein other than trypsin inhibitor and having hemagglutinating properties was lethal when injected into rabbits. The name "Soyin" was proposed for this protein which was characterized by marked hemagglutinating action and was devoid of either urease or antitryptic activity. Since then the names agglutinin, hemagglutinin, phyto- hemagglutinin were also proposed for this factor. Later, this substance was known to be able to distinguish among blood groups, and for this reason the term lectin (Latin, legere, to select or pick out) was suggested. Lectins from plant, as well as from other sources as snails and fishes, have become the subject of intense activity since several groups of investigators showed certain lectins not only have high specifity in agglutinating erythrocytes, but also 35 preferentially agglutinate malignant cells (Lis and Sharon, 1973). Chemically, lectin is a glycoprotein with molecular weight about 96,000. A unique feature of its composition is the high madbse and glucosamine content, which are up to 10 percent (Wada _t _t., 1966). Lis gt gt. (1966) reported four distinct soybean hemagglutinins chromatographically separable. Catsimpoolas and Meyer (1969) reported that at least four different forms of lectins were separated from soybean and their isoelectric points were 5.85, 6.00, 6.10, and 6.20. Lis and Sharon (1973) later reported that soybean lectin is comprised of four apparently identical subunits, each of molecular weight 30,000 i 500, and is therefore a tetramer. Biologically, lectin was thought to be active in sugar transport. Most of lectin in seeds was found in the cotyledons, but lectin was also detected in the embryo axis and the seed coat. Soybean lectin was detected to be present in all of the tissue of young seedlings, but decreased as the plant matured and was not detectable in plants older than two to three weeks (Pueppke t 1., 1978). Bhuvaneswari t al. (1977) linked lectin with the symbiosis between legumes and Rhizobia. Soybean lectin was found to bind to living cells 15 of the 22 Rhizobium japonicum strains tested. The lectin did not bind to Rhizobia strains which do not nodulate soybean. The binding of 36 lectins to the bacteria was shown to be specific and reversible. Soybean lectin can be inactivated by heating. Maximum stability toward thermal inactivation was obtained in the region of pH 6 to 7. It is also inactivated by some chemicals such as urea, guanidine and quadrivalent and trivalent metallic ions. Pepsin addition readily inacti- vated soybean lectin, but trypsin was found not to be effective for this purpose (Liener, 1958). The method in quantitative assay for measuring cell agglutination seems to be in the improvement process. Visual estimation of the degree of agglutination either in the test tube or with microtitrator were used. The agglutinating activity is measured by serial dilution of the lectin, with visual estimation of the end point where no agglutination is observed (Liener, 1953). This method was reported as very precise, but does not permit the detection of small differences in hemagglutinating activity. Liener (1955) developed a quantitative procedure in which the degree of agglutination is evaluated photometrically by measuring the absorbance of unsedimented cells. Protein Quality Evaluation Protein quality in human nutrition is mainly related to the efficiency with which food proteins are used for the synthesis and maintenance of tissue protein. This quality 37 is a function of the essential amino acid (EAA) and the digestibility of the proteins. The closer the EAA pattern to a reference EAA pattern (Table 6) required for human growth and maintenance, the better the quality of the protein. Usually proteins from animal origin have better digestibility than plant proteins. There are two types of tests used to determine the quality of protein, tg vitro tests and i vivo tests. The tg vitro tests are designed to provide a rapid estimate of the nutritive value of protein. The i vivo tests involve either animals or humans. Protein efficiency ratio (PER) is the most common 1 vivo test used for the nutritional evaluation of proteins or foods. 38 Table 6. Amino acid patterns required for growth and maintenance of rat and man* Rat Man Growth Maintenance Growth Maintenance Histidine 1.9 2.2 _1.6 -- Isoleusine 5.0 4.7 4 1 2.8 Leusine 6.3 4.3 9.4 3.8 Lysine 8.2 3.4 6 1 3.4 Phenylalanine 6.6 6.3 7.4 3.8 + Tyrosine Methionine 4.6 4.4 3.4 3.7 + Cystine Threonine 4.6 4.6 5.2 2.0 Valine 5.1 4.7 5.5 2.8 Tryptophan 1.0 1.0 1.0 1.0 *From Jansen (1978). MATERIALS AND METHODS Germination of Soybeans The soybeans variety Corsoy 79C 82 were supplied by the Michigan Crop Improvement Association, East Lansing. Unbroken seeds were washed and soaked in five times the volume of freshly boiled and cooled water overnight. Petri dishes, 9 cm in diameter, lined with water saturated filter paper were used for sprouting the beans. Soaked soybeans were put in the Petri dishes making one layer on the filter paper, covered and incubated in the dark at room temperature (about 23°C). To avoid mold growth during germination, all samples were washed with water at intervals of 12 hr. Samples were harvested at 6 hr intervals for 24 hr, washed with water, steamed (100°C) for 10 minutes and then frozen until they were used for tempeh fermentation or analyzed. Tempeh Preparation The tempeh used in this experiment were prepared by a method that is practiced in many Indonesian villages. Soybeans or germinated soybeans were soaked overnight in tap water at room temperature. About 100 9 portions of 39 4O soybeans or germ, soybeans were soaked overnight in 300 ml tap water containing about 10 m1 of the liquor of a previous fresh soybean soaking. This treatment favored some lactic acid fermentation in the soybeans. The hull was removed in running water and the dehulled soybeans were boiled for 90 minutes. The purpose of the boiling is to partially cook and sterilize the beans. The boiled soybeans were drained and cooled. After the temperature reached about 60°C, the hot mass was inoculated with mold spores. Culture of Rhizopus oligosporus NRRL 2710 was used in this experiment. Mold culture was grown and stored on Sabouraud Dextrose Agar which provides good mycelia growth and grown on Potato Dextrose Agar to produce spores for inoculation. After one week on Potato Dextrose Agar, the mycelia and spores were harvested with 3 m1 sterile water for each slant. The suspension contained about 106 spores per m1, and 3 ml of it was used to inoculate 100 g of germinated soybean mass. The inoculated mass was packed in disposable Petri dishes and incubated at 30-31°C for 36 hr. The tempeh produced was then steamed for 10 min and frozen until used for analysis. For analysis, the tempeh was dried in a vacuum oven at 70°C and reduced to fine powder using an Arthur Thomas mill. 41 Protein and Soluble Protein Protein determination was performed by micro-Kjeldahl according to AOAC (1970) and soluble protein determination was done according to the method of Rhee gt gt. (1981) as follows: 0.1 g of finely-ground sample (No. 60 sieve) was extracted with 9.9 m1 of 0.1 M phosphate buffer, pH 7.2 for 1 hr, at room temperature followed by centrifugation. An aliquot of the supernatant was used for the nitrogen deter- mination. A factor of 6.25 was used to convert nitrogen content to protein. Extraction of Crude Fat About 2 g of finely ground dried sample was extracted with diethyl ether in a Goldfisch extraction apparatus (Labconco.) for 4 hr. The extract was dried at 100°C for 30 min, and weighed. Oligosaccharides Determination Oligosaccharides were determined utilizing HPLC according to the method of Conrad and Palmer (1976). The high pressure liquid chromatograph consisted of a Model M-45 solvent delivery system, 4.2 mm i.d. X 30 cm long uBondapak/Carbohydrate analytical column, and a model R401 differential Refractometer detector, all from Waters Associates, Inc., Milford, MA. The responses were recorded on a Kontes recorder lOO. 42 Four extraction solvents were tried, namely hot alcohol 80%, hot alcohol 60%, hot water and cold water. The best extraction was obtained with 60% hot alcohol. Extraction with hot water resulted in a very viscous solution. Two grams of sample were first extracted with 15 ml of hexane twice. The clear supernatant was discarded and the residue was allowed to dry under the hood overnight. To the residue 20 m1 of 60% alcohol was added, mixed for 2 hr in a 80°C waterbath, and centrifuged for 15 min. The supernatant was saved and the precipitate was extracted again, twice with 10 ml of 60% alcohol. The three supernatants were combined and heated in a boiling waterbath for 30 min, cooled and a few drops of 10% lead acetate was added to precipitate the protein which was not coagulated by the heat treatment. After the contents of the tube were mixed well they were allowed to stand for a few minutes and then centrifuged. The supernatnat was saved and a few drops of 10% oxalic acid was added to remove the excess of lead. The tube was centrifuged again and the supernatant was saved. To remove the color in the supernatant, 1 g of activated carbon was added and let stand for 30 min. The tube was centrifuged, the supernatant was saved and the precipitate was washed with 10 ml of alcohol 60%. After centrifugation, the supernatant was combined with the previous supernatant and the mixture was concentrated in a flash-evaporator at 40°C until about 2 ml of the solution was obtained. The 43 concentrated solution was transferred into a 5 ml volumetric flask, and brought to volume with water. Prior to HPLC analysis, the concentrated extract was filtered through a .22 u-pore diameter membrane filter utilizing a Swinnex syringe filter (Millipore Corp.). A 20 ul of water-clear extract from the filtration was injected into the HPLC. A degassed mixture of acetonitrile and water (80:20, v/v) was used as solvent at a flow rate of 2.3 ml per minute. The detector attenuation was 8x for sucrose and 2x for raffinose and stachyose, and the recorder chart speed was 1 cm per minute. Identification and quantification of oligosaccharides were based on a comparison of retention times and area of those obtained with a standard mixture containing known amounts of sucrose, raffinose, stachyose and inositol. Standard curves were also made from known concentration mixtures. Phytic Acid Determination The determination of phytic acid consisted of the extraction of phytic acid, iron chelation and determination of the chelated iron according to the method of Wheeler and Ferrel (1971) with a slight modification. One gram of finely ground sample (50 mesh) was extracted with 50 ml 3% Trichloroacetic acid (TCA) in 125 m1 Erlenmeyer flask for 90 minutes using a mechanical shaker. For better 44 extraction, the flask was occasionally swirled by hand. The suspension was centrifuged and 10 m1 of the supernatant was transferred into a 40 m1 conical bottom centrifuge tube. Four ml of FeCl3 solution (2 mg Fe3+ per m1 of 3% TCA) was added to the aliquot by blowing rapidly from the pipet. The tube and content were heated in a boiling water for 45 minutes. The tube was centrifuged for 15 minutes and the clear supernatant was decanted carefully. The precipitate was washed twice by dispersing well in 25 ml 3% TCA, heated in boiling water bath for 5 minutes and centrifuged. The washing was repeated once more with water. The precipitate was dispersed in 0.5 ml H20 and mixed with 3 ml 1.5 N NaOH. After mixing well, the volume was brought to approximately 30 ml with water and heated in a boiling water bath for 30 minutes. The tubes were centrifuged for 15 minutes and the supernatant was decanted carefully. The precipitate was washed twice by dispersing well in 20 ml of water and then centrifuged. The Fe(0H)3 precipitate was dissolved in 40 m1 of hot HNO3 and transferred into 100 ml volumetric flask. The flask was cooled to room temperature and the volume was brought to mark with water. The iron was then determined according to AOAC (1975). Ten m1 of aliquot from the previous step was transferred into a 25 m1 volumetric flask and then 1 m1 of 10% hydroxyla- mine solution was added. The flask was rotated and let it stand a few minutes, and 9.5 ml 2 M NaOAc solution and 45 1 ml o-phenanthroline solution (0.1 g/100 ml) was added, The volume was then brought to mark, mixed a few minutes and its absorbance was measured at 510 nm against a water blank with a Beckman DU Spectrophotometer (Beckman Instruments, Inc., Fullerman, CA). A standard curve was previously prepared using a series of solution containing 0 to 2.4 ug Fe per ml. The following relationship was obtained. A = 0.224 C - 0.001675 510 (r = 0.9995) where C is the concentration of Fe in g/ml and A510 is the absorbance at wave length of 510 nm. This equation was plotted and the curve in Figure 8 was obtained. The phytate phosphorus content was calculated based on the assumption that the molecular ratio of ironzphosphorus is 4:6. Hemagglutination Assay A quantitative determination of lectin is based on the agglutination of 50% of countable red blood cells (RBC) according to the method of Coffey (personal communication). This method consists of the extraction of lectin, the preparation of a RBC suspension, the agglutination of the cells and the counting of the remaining suspended cells, utilizing the Coulter Counter (Coulter Electronics). 46 0.8 - 0.7 - (16- 0.5 - A 510 0.4 _ g a l 1 1 O 0.8 1.6 2.4 3.2 Concentration, pg/ml Figure 8. Standard curve for Fe determination. 47 Extraction A 5 gram of fresh sample (germinated soybeans or tempeh) was extracted with 25 ml of phosphate buffered saline (PBS) in a Waring blender. The PBS contained sodium 180 meq/L; potassium 5.1 meq/L; chlorine 153 meq/L; EDTA 1 mmol/L and was free from sodium azide. To avoid heat denaturation, the mixing was stopped at one minute intervals, and cooled in ice. The aliquot was centrifuged at 40,000 x g for 30 minutes, and the supernatant was saved for the assay. For quantitation purpose, the amount of nitrogen in the extract was determined using the micro Kjeldahl method. Preparation of Red Blood Cells (RBC) Fresh blood was collected from healthy pigs. The RBC was separated from the serum by centrifugation. The serum was decanted and the precipitate (RBC) was mixed with PBS, centrifuged and the supernatant was removed. The precipi- tate was washed again twice with PBS, using the same procedure. To have a better cell suspension for the assay, the RBC were diluted in 10 volumes of PBS and 9 parts of this suspension was mixed with 1 part of 10% trypsin solution. The mixture was incubated at 37°C in a water bath for 1 hr. After incubation, the mixture was centrifuged, the supernatant was discarded and the RBC was washed 3 times with PBS. For the assay, the RBC was then diluted to 7 give a Coulter count 4 x 10 cells with millipore-filtered PBS. 48 'Agglutination Assay Serial two-fold dilution of an aliquot of bean extract were made starting with 25 ul of each sample. Each of the dilutions was added to 2 m1 of RBC suspension and incubated for 1 hr. Samples were spinned at 400 x g for 45 seconds and resuspended by shaking. Samples were allowed to stand for 15 min. A 25 ul sample was drawn off from the midpoint of the tube and added to 10 ml PBS. Single erythrocytes were counted using a Coulter Counter at settings of 1/amplification and 1/aperture current = 1/2 with matching switch 20K and gain = 7. Duplicate samples were used and 2 counts were made for each duplicate. The agglutination strength was expressed as the inverse of the amount of ug protein in the extract per ml required to agglutinate 50% of the countable single cells. Protein Efficiency Ratio (PER) Test Diet The PER test was performed according to the AOAC method (1970). Three diets were prepared using the following protein sources: 1. Casein (as control) 2. Regular tempeh 3. Tempeh prepared from germinated soybeans The nitrogen content of samples was determined by the Micro-Kjeldahl method, and the fat content by the Goldfisch 49 diethyl ether extraction method. Protein = 6.25xN. All feeds were standardized to meet the following composition: Protein = 10% Vitamin mix = 1% Fat/oil = % Cellulose = 1% Salt Mix USP = 5% Corn starch + sucrose (1+1) to make 100% The vitamin mix AOAC was from Teklad Test Diets and the salt mix and vitamin free casein from United States Bio- chemical Corporation, Cleveland, Ohio. Experimental Animals Thirty one male rats, age 21 days and average weight 43 g from Harlan Sprague Dawley, Inc. were supplied by the Spartan Research Animal Inc., Haslett, Michigan. The animals were allowed to acclimate for 4 days on rat chow diet. At the beginning of the test, ten rats were, assigned into one group of treatment. The average weight of the rats was 64 g. Assay Period and Calculation of PER The rats were kept in individual cages and both diets and water were given gg libitum. Body weight and diet consumed were recorded every 4 days. The experiment was terminated after 28 days from the beginning of the assay. Weight gain and protein (N x 6.25) intake per rat for each group were calculated and the PER for each group were calculated as the ratio of weight gain per protein intake. SO Corrected PER were also calculated by multiplying the PER of treatments with 2.50/PER of casein. RESULTS AND DISCUSSION Germination Six hours of germination produced only a little elonga- tion of radicle inside the testa which was hardly noticeable but the rupture of the testa occurred after 12 hours of germination. All radicles appeared after 18 hours of germination. Germination for 24 hours resulted in a 1 cm rootlets and the cotyledon became softer. Tempeh Fermentation The first step in tempeh preparation is soaking the soybeans overnight (or longer) to let lactic acid fermen- tation occur by microorganisms which naturally occur in soybeans. Because of steaming after each sample drawing from germination, these microorganisms were destroyed and soaking the samples in tap water did not result in a good lactic acid fermentation. Good lactic acid fermentation is indicated by the formation of foam on the surface, and was obtained by adding soaking water from fresh soybeans. Dehulling soaked germinated soybeans by hand was easier than soaked ungerminated ones. 51 52 There was no difference in mold growth on germinated or ungerminated soybeans. Mold growth was noticeable after 10 to 12 hours incubation, and after 20 hours mold appeared throughout the soybeans, at which time the temperature of the beans was higher than the incubator temperature. Steinkraus gt gt. (1960, 1965) and Sudarmadji and Markakis (1978) reported that during the tempeh fermentation by 3. oligosporus the temperature rose to a peak of 40 to 45°C. A satisfactory tempeh, with all of the soybeans completely covered with thick mycelium of 3. oligosporus, was obtained after 32 to 36 hours of incubation. Tempeh from germinated soybeans can not be differentiated visually from tempeh of ungerminated soybeans. Fermentation in small (9 cm diameter) Petri dishes was better than in 14 cm diameter Petri dishes. In 14 cm diameter Petri dishes, mycelial growth was very slow in the middle. Lack of air may be the cause of the slow growth. Poor mycelial growth was reported by former researchers. Total Nitrogen and Soluble Nitrogen in Germinated Soybeans and Tempeh Prepared from Germinated Soybeans The results of total and soluble nitrogen analysis from germinated soybeans and tempeh prepared from germinated soybeans are presented in Table 7. The shorter time of germination did not seem to significantly alter the total 53 Table 7. Total and soluble nitrogen in germinated soybeans and tempeh. Hours of Total N Soluble N germination (g/100 9 sample) (g/100 9 sample) Germinated O 6.9 1.1 soybeans 6 6.9 1.1 12 7.0 1.1 18 7.0 1 l 24 7.0 1.2 Tempeh from 0 7.5 2.2 germinated 6 7.6 2.3 soybeans 12 7.6 2.5 18 7.6 2.5 24 7.6 2.6 54 nitrogen in germinated soybeans, but as the time of germina- tion increased, the germinated soybeans showed an increase in total nitrogen content. Hsu _t _t. (1973) reported that during germination, protein, oil and carbohydrates are sources of energy for the developing embryo. Soluble carbohydrates are important energy sources during early stages of germination, while protein nitrogen decreases and amino nitrogen increases as seeds germinate with the latter form being transported to the growing parts. The total nitrogen in the whole growing seed should remain the same as long as the growing parts are not removed. The soaking and boiling steps of tempeh preparation result in some loss of water soluble compounds, such as sugars and amino acids. Steinkraus _t _t. (1960) reported that the total nitrogen during tempeh fermentation remained relatively constant, about 7.5%. Murata _t gt. (1967) reported a slightly higher protein content in tempeh than in soybeans for both tempeh produced in Indonesia and tempeh prepared in Osaka University: The data from this research showed an increase in total nitrogen during tempeh fermentation of ungerminated and germinated soybeans, confirming the Japanese report. The loss of non-nitrogen compounds during tempeh preparation seems to be much higher than the loss of nitrogen compounds. Non-nitrogen compounds are utilized by the mold more than nitrogen 55 compounds during the tempeh fermentation. The soluble nitrogen content did not increase signifi- cantly during germination, but it was double after the tempeh fermentation. It seems that the increase of amino nitrogen during germination, as reported by Hsu gt gt. (1973) has not taken place during the short period of germination. The increase of soluble protein during tempeh fermentation has been previously observed by Steinkraus t l. (1960). Crude Fat in Germinated Soybeans and Tempeh Prepared from Germinated Soybeans The crude fat content of germinated soybeans and tempeh prepared from germinated soybeans is presented in Table 8. In general, the crude fat content decreased with increasing germination time and underwent further decrease during the tempeh preparation of the germinated soybeans. Both germination and mold growth seem to utilize fat for energy need. The tempeh preparation appeared to result in higher fat decrease (12-15%) than the germination of the soybeans (4%). Soaking and boiling may have contributed to the fat decrease in preparing tempeh. These data agree with those presented by Murata t l. (1967). 56 Table 8. Crude fat in germinated soybeans and tempeh prepared from germinated soybeans (% dry basis). Samples Germination Crude fat time (hrs) Germinated O 22.3 soybeans 6 21.9 12 21.9 18 21.7 24 21.5 Tempeh from O 19.6 germinated 6 18.7 soybeans 12 18.7 18 18.4 24 18.1 57 Oligosaccharides The HPLC separation of (a) a standard mixture containing sucrose, raffinose, stachyose and inositol; (b) the oligosaccharides of germinated soybeans; and (c) the oligosaccharides of tempeh, are shown in Figure 9. It may be noted that the higher the retention time of the sugar the broader the corresponding peak. A big difference in peak height between sucrose and both raffinose and stachyose was observed among samples, especially when germinated soybeans were analyzed. Sucrose and inositol which had retention times of 3.2 and 3.9 min, respectively, do not separate very well using this column. The peak of inositol appeared in the extract of tempeh, but it was very close to that of sucrose. Because of the unsatisfactory separation and the poor replication of the inositol peaks, the quantity of inositol is not reported. No inositol peak appeared in the chromato- gram from germinated soybean samples. One peak with retention time of 5.2 min appeared in the extract of tempeh. Shallenberger gt gt. (1967) reported that during the tempeh fermentation, a reducing disaccharide appeared which attained maximum concentration after about 35 hr of fermentation and was absent after 60 hr. They identified it as melibiose. Stachyose was reported to fully disappear after three days of germination (Abrahamsen and Sudia, 1966; and East .gmasmu to uumcuxm u u .mcmwnxom umumcwsgmm we uocguxm u m .mmozcumpm ecu mmocwemmc .Popwmocw .mmocuam mcwcmmucou weapst unaccapm u < .mmcwcmguummomwpo mo :owumcmamm 04a: asp .m mczmwd U m < Ammuscwev MES. onHzmme A ON 2 OH m 0 ON 2 3 m 0 ON 2 S m 0 (all? 1 11 (1111! 1 1‘ 4 1 {idlilSi-llJ ll‘li 4 1 4‘ 1 4 d 1 ‘Ji < 1 Q 144 {U 1‘1qu 1A1 d 41 1‘ 41d 1 14 d (Ali‘l _ n _ . . _ _ _ _ _ _ . _ . _ . _ . . . _ I. u _ t. t. S I f. _ U U 1 8 3 _ . f. E. B I: 3 _ _ 8 _ 8 3 T: 1» _ 3 _ 3 U1 T. T... _ J .4 . 14 ,A U 0 9 I. . _ T... O O U S T: O . O S S .4 I. U _ U 3 8 _ 9 I. . l . 3 U 8 \.l \l _ U: 0 S T3 _ A S 1 T1. 9 9 O a 9 I. 3. 1 s S 3 u _ . 1. 3 n a U. 0 _ _ 3 8 3 \l S .A S _ . U U l \/ n O a . . nu n O 9 3 S 9 9 S 9 .4 I a T..S .4 1 a 1 3 O u n : I. r. 3 a S z 0 3 O 0 \l 3 U a S I U U . U n T..O B . n 8 .48 Z Z 1 - 9 1+ 0 a 3. . 1 I. \l T.. VA VA 3 I. O U S 0 U 9 S S ( l.\ n O U 14 O O 8 I z 3 T. I 14 A z a A A I. a X U 8 a O U VA n U U U .4 /1\ 9 1+ 1+ ( 1 bl 1: .L T3 T3 1 O J 1 VA 0 U 0 O U U U l.\ 14 8 1+ 14 VA /.\ mm 59 _t _t., 1972). Germination for short periods (up to 24 hr) reduced the stachyose content by less than 15%. However, this limited germination may be more valuable if it is used in the preparation of tempeh. Combination of short periods of germination and tempeh fermentation reduced stachyose by almost 90%, far more than the tempeh fermen- tation alone (Table 9). Compared to stachyose, sucrose and raffinose disappeared more gradually, especially during the tempeh fermentation. This is probably due to the formation of raffinose and sucrose from the hydrolysis of stachyose. 'Phytic Acid The effect of germination and tempeh fermentation on the phytic acid content of soybeans is presented in Table 10. Phytic acid was not altered during germination up to 12 hr. A 6.4% decrease in phytic acid occurred after the seeds were germinated for 18 hr; a further reduction of 13.2% was observed after 24 hr germination. Phytates are considered the main storage form of phosphorus in almost all seeds (Asada t 1., 1969; Mandal t 1., 1972; and Erdman, 1979). In the ripening process, phosphorus is transported to the seeds. Most of the transported phosphorus is deposited in the form of phytic acid (Asada t 1-, 1969). Mandal t 1. (1972) studied germination of mung beans and reported that phytase was 60 Table 9. Oligosaccharides in germinated soybeans and tempeh prepared from germinated soybeans (% dry basis, average of triplicates). Samples Germination Sucrose Raffinose Stachyose time (hr) Germinated O 5.61 0.21 3.82 soybeans . 6 4.73 0.20 3.61 12 4.42 0.15 3.36 18 3.55 0.13 3.48 24 3.46 0.11 3.30 Tempeh from 0 2.99 0.09 1.83 germinated 6 2.76 0.08 1.14 soybeans 12 1.90 0.06 0.72 18 1.74 0.05 0.50 24 1.39 0.05 0.49 61 Table 10. Phytic acid content in germinated soybeans and tempeh prepared from germinated soybeans (% dry basis). Samples Gigmln?::?n Phytic acid I II Average Germinated O 1.3 1.3 1.3 soybeans 6 . 1.3 1.3 1.3 12 1.3 1.3 l 3 18 1.2 l 2 1.2 24 1.1 1.2 l 2 Tempeh from 0 0.8 0.8 0.8 germinated 6 0.8 0.8 0.8 soybeans 12 0.8 0.8 0.8 18 0.6 0.7 0.7 24 0.5 0.6 0.6 62 not detected in the cotyledon of ungerminated beans but appeared in germination. Tempeh fermentation appeared to be effective in reducing the phytic acid present in soybeans. About 40% of the phytic acid disappeared as a result of the 36 hr tempeh fermentation of ungerminated soybeans and an additional 20% was lost as a result of germinating the soybeans for 24 hr. It was found that some microorganisms produce enzymes which have phytase properties. Sudarmadji and Markakis (1977) reported that the phytic acid content of soybeans was reduced by about one-third during tempeh fermentation. The reduction of the phytic acid was due to phytase elaborated by the Rhizopus oligosporus used in the tempeh fermentation. Wang gt gt. (1980) reported that two strains of 3. oligosporus, one strain of 3. Chinensis and eight strains of Aspergillus oryzae produced both extra and intra- cellular phytase. Neurospora sitoghila and 3. oligosporus were also reported to reduce phytic acid during oncom preparation (Fardiaz and Markakis, 1981). Protein Efficiengy Ratio of the Tempeh The summary of the utilization of proteins from regular tempeh and tempeh from germinated soybeans by weanling rats is shown in Table 11. The PER of tempeh prepared from germinated soybeans (24 hr germination) were slightly higher 63 Table 11. Protein efficiency ratio of regular tempeh and tempeh prepared from germinated soybeans by weanling rats . Casein Regular tempeh Tempeh prepared from germinated soybeans Average daily gain, 9 3.51iO.34 2.94:0.35 2.88:0.34 Average feed intake, g 14.0:1.06 l3.4:0.91 12.73:0.86 PER2 2.51:0.25 2.19:0.15 2.26:0.14 Corrected PER 2.50 2.18 2.25 Protein Quality 100 87.3 90.0 (Sample PER/ Casein PER x 100) 1Each datum represents an average of 10 rats. Protein (NX6.25) content of the diets was 10%. 2PER i standard deviation. 64 than the PER from regular tempeh (0 hr germination). How- ever, these differences were not significant statistically. Rats on tempeh prepared from germinated soybeans diet ate and gain less than did rats on regular tempeh diet. Either the germination process or the combination of germination and tempeh fermentation may have depressed the acceptance of the diets by rats. Hackler gt gt. (1964) reported that the depressing effect may be caused by the mold or something elaborated by the mold used in tempeh fermentation. Lectin Activity The summary of the agglutination assay is presented in Table 12. There was no agglutination activity observed in any of the tempeh samples. The tempeh preparation steps, boiling and steaming, must have denatured the protein and inactivated the lectin of soybeans. The amount of extract needed to agglutinate 50% of RBC decreased with germination. However, since the protein solubility increased with germination, the amount of protein in the extract also increased, a fact explaining the higher agglutinating power of germinated soybeans. The agglutination titer of the extracts were the same. 65 1 Table 12. Lectin agglutination titer Germination Extract Protein Protein Agglutination time (hr) needed for in the needed titer 50% RBC extract for 50% agglutination (mg/ml) RBC (ul) agglutination (U9) 0 230 0.38 87 0.011 ' 6 228 0.39 89 0.011 12 210 0.45 95 0.011 18 180 0.50 90 0.011 t 24 165 0.57 94 0.011 1Agglutination titer = the inverse of the protein concen- tration in the extract at initial RBC count 107. 50% RBC agglutination. The CONCLUSION Germination up to 24 hours markedly reduced the amount of oligosaccharides (sucrose, raffinose and stachyose) in soybeans; it also slightly reduced the crude fat content. The protein content slightly increased during germination. This increase can be explained by the decrease in the percentage concentration of carbohydrate and fat. Phytic acid content slightly decreased, but the strength of lectin remained constant. The changes occurring during germina- tion are considered to be desirable. The tempeh fermentation caused further decrease in oligosaccharides, crude fat and phytic acid with a corresponding increase in protein content. The soybean lectins were inactivated by either the boiling or the fermentation used in the preparation of tempeh. Although the combination of soybean germination and tempeh fermentation resulted in better removal of some anti-nutritive factors (phytic acid and oligosaccharides) than the tempeh fermentation alone, it did not signifi- cantly increase the utilization of the protein by growing rats. The PER of regular tempeh and tempeh prepared from germinated soybeans were almost the same. 66 67 BIBLIOGRAPHY 68 BIBLIOGRAPHY Abrahamsen, M., and Sudia, T.W. 1966. Studies on the soluble carbohydrates and carbohydrate precursors in germinating soybean seeds. Am. J. of Botany. 53(2): 108. Aman, P. 1979. Carbohydrates in raw and germinated seeds from mung beans and chick peas. J. Sci. Food Agric. 30:869. Asada, K., Tanaka, K., and Kasai, Z. 1969. Formation of phytic acid in cereal grains. Ann. N.Y. Acad. Sci. 165:801. A.O.A.C. 1975. Official Methods of Analysis, 12th ed. Association of Analytical Chemists, Washington, D.C. Autret, M., and van Veen, A.G. 1955. Possible source of proteins for child feeding in underdeveloped countries. Am. J. of Clinical Nutrition 3:234. Bhuvaneswari, T.V., Pueppke, S.G., and Bauer, W.D. 1977. Role of lectins in plant - microorganism interaction I: Binding of soybean lectin to Rhizobia. Plant Physiol. 60:486. Catsimpoolas, N., Campbel, T.G., and Meyer, E.W. 1969. Protein subunit in dormant and germinating soybean seeds. Biochem. Biophys. Acta. 168:122. Catsimpoolas, N., and Meyer, E.W. 1969. Isolation of soybean hemmaglutinin and demonstration of multiple forms isoelectric focusing. Arch. Biochem. Biophys. 132:279. Chen, L.H., Wells, C.E., and Fordham, J.R. 1975. Germinated seeds for human consumption. J. Food Sci. 40:1290. Chen, P.S. 1970. Soybeans for health, longevity and economy. 3rd ed. Provoker Press, St. Catherine, Ont., Canada. 69 Coffey, D. Agglutination assay. Personal communication. Conrad, E.C., and Palmer, J.K. 1976. Rapid analysis of carbohydrates by High Pressure Liquid Chromatography. Food Tech. 84:92. Dalby, A., and Tsai, C.Y. 1976. Lysine and tryptophan increases during germination of cereal grains. Cereal Chem. 53(2):222. Djien, K.S., and Hesseltine, C.W. 1961. Indonesian Fermented Foods. Soybean Digest 22(1):14. East, J.W., Nakayama, T.0.M., and Parkman, S. 1972. Changes in stachyose, raffinose, sucrose and monosaccharides during germination of soybeans. Crop Sci. 12:7. Erdman, J.R. 1979. Oilseed phytase: nutritional implica— tions. J. Am. Oil Chemists Soc. 56:736. Evancho, G.M., Hurt, H.D., Devlin, P.A., Landers, R.E., and Ashton, D.H. 1977. Comparison of Tetrahymena pyriformis W and rat bioassay for the determination of protein quality. J. Food Sci. 42:444. Everson, G.J., Steenbook, H., Cederquist, D.C., and Parsons, H.T. 1944. The effect of germination, the stage of maturity and the variety upon nutritive value of soybean protein. J. Nutrition 27:225. Fardiaz, D., and Markakis, P. 1981. Degradation of phytic acid in oncom (fermented peanut press-cake). J. Food Sci. 46:523. Fardiaz, 0., and Markakis, P. 1980. Biochemical changes during fermentation of peanut press cake (oncom). Ph.D. Thesis, Michigan State University, East Lansing. Folkes, B.F., and Yemm, E.W. 1958. Respiration and the metabolism of amino acids and proteins in germinating grains. New Phytologists. 57:106. Fordham, J.R., Weels, C.E., and Chen, L.H. 1975. Sprouting of seeds and nutrient composition of seeds and sprouts. J. Food Sci. 40:552. Frazier, W.C. 1967. Food Microbiology. McGraw-Hill Book Co. 7O Freed, R.C. and Ryan, 0.5. 1978. Changes in kunitz trypsin inhibitor during germination of soybeans: an immunoelectrophoresis assay system. J. Food Sci. 43: 1316. Gould, M.F., and Greenshield, R.N. 1964. Distribution and changes in the galactose-containing oligosaccharides in ripening and germinating bean seeds. Nature 202: 108. Gyorgy, P., Murata, K., and Ikehata, H. 1964. Antioxidants isolated from fermented soybeans (tempeh). Nature 203:870. Hackler, L.R., Steinkraus, K.H., van Buren, J.P., and Hand, 0.8. 1964. Studies on the utilization of tempeh protein by weanling rats. J. Nutrition 82:452. Hesseltine, C.W., Smith, M., Bradle, B., and Djien, K.S. 1963. Investigation of tempeh, an Indonesian food. Developments of Industrial Microbiol. 4:275. Hesseltine, C.W., and Wang, H.L. 1967. Traditional fermented foods. Biotech. and Bioeng. 9:275. Hesseltine, C.W., Smith, M., and Wang, H.L. 1970. New fermented cereal products. Development in Industrial Microbiol. 8:179. Honavar, P.M., Shih, C.V., and Liener, I,E. 1962. Inhibi- tion of the growth of rats by purified hemagglutinin fractions isolated from Phaseolus vulgaris. J. Nutrition 77:109. House, W.A., Welch, R.N., and van Campen, D.R. 1982. Effect of phytic acid on the absorption, distribution and endogenous excretion of zinc in rats. J. Nutrition 112:941. Hsu, H.W., Vavak, D.L., Satterlee, L.D., and Miller, G.A. 1977. A multienzyme technique for estimating protein digestibility. J. Food Sci. 42:1269. Hsu, S.H., Hadly, H.H., and Hymowitz, T. 1973. Changes in carbohydrate contents of germinating soybean seeds. Crop. Sci. 13:407. ' Jansen, G.R. 1978. Biological evaluation of protein quality. Food Technology. 32(12):52. 71 Kao, C., and Robinson, R.J. 1978. Nutritional aspects of fermented foods from chick pea, horse bean and soy- beans. Cereal Chem. 55:(4):512. Kohle, H., and Kauss, H. 1979. Improved analysis of hemagglutination of lectin activity. Anal. Biochem. 103:227. Ku, S., Wei, L.S., Steinberg, M.P., Nelson, A.I., and Hymowitz, T. 1976. Extraction of oligosaccharides during cooking of whole soybeans. J. Food‘Sci. 41: 361. Liener, I.E. 1953. Soyin, a toxin protein from the soybeans. J. Nutrition 49:523. Liener, I.E. 1958. Inactivation studies on the soybean hemagglutinin. J. Biol. Chem. 59:401. Lis, H., Fridman, C., Sharon, N., and Katchaski, E. 1966. Multiple hemagglutinins in soybeans. Arch. Biochem. Biophys. 117:301. Lis, H., and Sharon, N. 1972. In Methods in Enzymology (Ginzburg, V., ed.). Academic Press, New York, Vol. 28, pp. 360-368. Lis, H., and Sharon, N. 1973. The biochemistry of plant lectins (phytohemagglutinin). Annual Review of Biochem. 42:541. Makower, R.V. 1970. Extraction and determination of phytic acid in beans (Phaseolus vutgaris). Cereal Chem. 47: 288. Mandal, N.C., Burman, S., and Biswas, 8.8. 1972. Isolation, purification and characterization of phytase from germinating mung beans. Phytochem. 11:495. Martinelli, A., Filho, A., and Hesseltine, C. W. 1964. Tempeh fermentation; package and tray fermentations. Food Tech. 18:167. Mital, B.K., and Steinkraus, K.H. 1975. Utilization of oligosaccharides by lactic acid bacteria during fermentation of soymilk. J. Food Sci. 40:46. Murata, K., Ikehata, H., and Miyamoto, T. 1967. Studies on the nutritional value of tempeh. J. Food Sci. 32: 580. 72 Oppenheimer, 5.8;, and Odencrantz, J. 1972. A quanti- tative assay for measuring cell agglutination: Agglutination of sea urchin embryo and mouse teratoma cells by Concavalin A. Exp. Cell Res. 73:475. Pueppke, S.G., Bauer, W.D., Keegstra, K., and Ferguson, A.L. 1978. Role of lectins in plant-microorganism interaction II. Distribution of soybean lectin in tissue of Glycine max (L) merr. Plant Physiol. 61: 779. Rackis, J.J., Sessa, D.J., Stegerda, F.R., Shimizu, T., Anderson, J., and Pearl, S.L. 1970. Soybean factors relating to gas production by intestinal bacteria. J. Food Sci. 35:634. Reddy, N.R., and Salunkhe, D.K. 1980. Changes in oligo- saccharides during germination and cooking of black gram/rice blend. Cereal Chem. 57(5):356. Reddy, N.R., Pierson, M.D., Sathe, S.K., Salunkhe, D.K., and Beuchat, L.R. 1981. Legume-based fermented foods: Their preparation and nutritional quality. CRC Critical Reviews in Food Science and Nutrition. Vol. 17:335. Rhee, K.C., Kuo, C.K., and Lucas, E.W. 1981. In Protein functionality in foods. Cherry, J.P. ed. A.C.S. Symposium Series 147, American Chemical Society, Washington, D.C., p. 56. Robinson, R.J., and Kao, C. 1977. Tempeh and misc from chick pea, horse bean and soybeans. Cereal Chem. 54: 1192. Roelofsen, P.A., and Talens, A. 1964. Changes in some B-vitamins during molding of soybeans by Rhizopus oryzae in the production of tempeh kedelee. J. Food Sc1. 29:224. Ryan, C.A. 1973. Proteolytic enzymes and their inhibitors in plant. Ann. Rev. Plant. Physiol. 24:173. Shallenberger, R.S., Hand, 0.8., and Steinkraus, K.H. 1967. Changes in sucrose, raffinose and stachyose during tempeh fermentation. ARS. 74-41, USDA. p. 68. Shurtleff, N., and Aoyagi, A. 1979. The book of tempeh. Harper and Row, Publisher, New York. 73 Smith, A.K., Rackis, J.J., Hesseltine, C.W., Smith, M., Robbins, D.J., and Booth, A.N. 1964. Tempeh: Nutritive value in relation to processing. Cereal Chem. 41:173. Steinkraus, K.H., Hwa, Y.B., van Buren, J.P., Providenti, M.I., and Hand, 0.8. 1960. Studies on tempeh. An Indonesian fermented food. Food Res. 25:777. Steinkraus, van Buren, J.P., Hackler, L.R., and Hand, 0.8. 1965. A pilot plant process for the production of dehydrated tempeh. Food Tech. 19(1):63. Steinkraus, K.H., Lee, C.Y., and Buck, P.A. 1965. Soybean fermentation by the ontjom mold Neurospora. Food Tech. 19:1301. Steinkraus, K.H. 1978. Tempeh. An Asian example of appropriate/intermediate food technology. Food Tech. 32:79. Stilling, B.R., and Hackler, L.R. 1965. Amino acid studies on the effect of fermentation time and heat processing of tempeh. J. Food Sci. 30:1043. Sudarmadji, S., and Markakis, P. 1975. Certain chemical and nutritional aspects of soybean tempe. Ph.D. Thesis, Michigan State University, East Lansing. Sudarmadji, S., and Markakis, P. 1977. The phytate and phytase of soybean tempeh. J. Sci. of Food and Agric. 28:381. Sudarmadji, S., and Markakis, P. 1978. Lipid and other changes occuring during the fermentation and frying of tempeh. Food Chem. 3:165. Tsai, C.Y., Dalby, A., and Jones, R.A. 1975. Lysine and tryptophane increases during germination of maize seed. Cereal Chem. 52:356. van Buren, J.P., Hackler, L.R., and Steinkraus, K.H. 1972. Solubilization of soybean tempeh constituents during fermentation. Cereal Chem. 49:208. van Veen, A.G., Graham, D.C., and Steinkraus, K.H. 1968. Fermented peanut presscake. Cereal Science Today. 13: 96. 74 van Veen, A.G., and Steinkraus, K.H. 1970. Nutritive value and wholesomeness of fermented food. J. Agr. Food Chem. 18:576. Wada, S., Pallansch, M.J., and Liener, I.E. 1958. Chemical composition and end group of the soybean hemagglutinin. J. Biol. Chem. 233:395. Wagenknecht, A.C., Mattick, L.R., Lewin, L.M.. Hand, 0.8., and Steinkraus, K.H. 1961. Changes in soybean lipids during tempeh fermentation. J. Food Sci. 26: 373. Wheeler, E.L., and Ferrel, R.E. 1971. A method for phytic acid determination in wheat and wheat fractions. Cereal Chem. 48:312. Wang, Y.Y.D., and Fields, M.L. 1978. Germination of corn and sorghum in the home improved nutritive value. J. Food Sci. 43:1113. Wang, W.L., and Hesseltine, C.W. 1966. Wheat tempeh. Cereal Chem. 43:563. Wang, H.L., Ruttle, D.I., and Hesseltine, C.W. 1968. Protein quality of wheat and soybeans after Rhyzopus oligosporus fermentation. J. Nutrition 96:109. Wang, H.L., Swain, E.W., and Hesseltine, C.W. 1975. Mass production of Rhizopus oliggsporus spores and their application in tempeh fermentation. J. Food Sci. 40:168. Wang, H.L., Swain, E.W., and Hesseltine. C.W. 1980. Phytase of molds used in oriental food fermentation. J. Food Sci. 45:1262. Welch, R.M., and House, W.A. 1982. Availability to rats of zinc from soybean seeds as affected by maturity of seed, source of dietary protein, and soluble phytate. J. Nutrition 202:879. Whitaker, J.R. 1978. Biochemical changes occuring during the fermentation of high protein foods. Food Tech. 32:175. Wolf, W.J., and Cowan, J.C. 1971. Soybeans as a food source. CRC Press, Cleveland, Ohio. 11111111111111lllllll’ 11111111111111