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" ’ " VAJ' ‘ 5" 'p'r‘fij‘fr ' ro- -.'"'I5'.'.'n'. 9,18% 0‘6 I7 MICHIGAN STATE II LIBRARIES III/III IIIIIIIII IIIII IIIIIIIII 0563 5366 3,, III LIBRARY Mi‘higan State University This is to certify that the dissertation entitled VITAMIN B-12 FORMATION IN TEMPEH FERMENTATION BY MIXEDZCULTURE presented by SUPARMO has been accepted towards fulfillment of the requirements for PhoDo degree in FOOd Science TQM/y [putt/11¢; Dr. P. Markakis Major professor Date 8 - 10 — 1988 MS U is an Affirmative Action/Equal Opportunity Institution 042771 3ft}. MSU LIBRARIES ~— 1’ ,- RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. IVE» ‘VITAHIN 3-12 FORHKTION IN TEMPEH FERMENTED BY HIXED-CULEURE Suparno A DISSERIKTION Submitted to Michigan State university in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science 1988 'Ihreevitamin B-lZ-producirg bacteria, mm. mmmmm, mtriedinmimd- culture fermentationwith mammrtheprmtimof vitaminB-lZ-oontainingteipeh. Cultured individually on boiled-sterilized soybeans, 5W ardB.mtg:1mgrewhestat35C, while $.911mstuaedqvtiml Mammogmardfimmabletogrwm tryptose—glucose—yeastextractagaratthep-Irameofs.0to7.o, Mes.fl1mgmmflierameof5.5to7.o.30ybeans,mmid1 thebacteriagrew, wereassayed forvitaminB-lz activityusing WWastestorganisn. B-lZwasdetectedafterthe firstdayoffernenbatimardcartinxedbeirgproducedvmenthesmdy vastemimtedafterttmeédays.§.mpmmnedmehigmst1eve1 of 8-12, follwedby§.marflthenbyg.m. amalg- :ingthefermentatimtim, frunonedaytotwodays, doubled or tripledthevitaminB—lz contents, dependingmthebacteriaused. Additimofacidtothesoybeansmubitedmegmwthofthebacteria, mileacfiitionofcobaltdidmtaffectthegrowfluorthevitaminB-lz Supamo predictim. Soaking the soybeans before boiling and sterilizatim exharcedthegrowtharflB-lz production WIS-W: hxtslightly diminishedthegrwthandvitaminpmdictimby 3mm and Selim- Inmixedmlhmewithmld,msoybeam,again$.glimproduced thelargestwantityofB-lz, tonneabys.mmmam then by rm. memmam. however} reduced theirptoductimotB-uinthepresenceofmold. Soakingthesaybeans before the mind-calcite fermentatim mhanced the vitamin productim WEN. hrtdrastimllyrednedit by B-Warfi 5.91m. Wham did not maintaintheir 3-12 producing ability, even at refrigeratim tamer-aura, W 1-2 mofstorage. that mind-calm tenpeh was assayed for 8-12 activity usirq mm. which is aprotozoamspafiimmlytotme B-lZactivity, itmsfwrflthatonlyasnallfractimofthetotal B—lZneasmedbyLJgiMwastrue 3-12: S.2rg/gofterpeh(dry basis) for fim, 1.0 ng/g for gm and 0.0 for rm Wisprodmedbymimed-wlumeofg.glmwimariyaeof thethreetestedbacteriahadappeamrveardtastesimilartothatof regulartaipeh. 'IheProrteinEfficiency Ratio (PER) of maggmige ardfi.g11m-tarpehsmssimflartothatofregulartarpeh.‘fiaem of fimim-tapeh was slightly lower. ACKNOWLEDGMENTS The author expresses sincere gratitude to Dr. P. Markakis, Professor of Food Science, for the excellent guidance and patience throughout the course of the study and preparation of this manuscript. Appreciation is also extended to Drs. W.O. Song, J.J. Pestka, C.M. Stine, Professors of Food Science and Human Nutrition, and Dr. E.S. Beneke, Professor of Botany Plant Pathology, for their participation in the guidance committee. Thanks are also due to Dr. W. Bergen for the amino acid analysis of my tempeh samples. The author wishes to express his gratitude to Gadjah Mada University for providing leave of absence, and to the government of Indonesia and The Rockefeller Foundation for the financial support for this program. Finally, the author expresses his most sincere appreciation to his wife Niniek and family who have helped in far too many ways to enumerate. ii TABLE OF CONTENTS LIST OF TABLES . . . . . . LIST OF FIGURES . . . . . . INTRODUCTION . . . . . . . REVIEW OF LITERATURE . . . Legumes as human foods Food fermentations . . T O O O O O O O 0 Origin of tempeh Raw materials of tempeh . Productions and consumptions of tempeh Tempeh Fermentation . Organisms in tempeh fermentation . . . . Parameters in tempeh fermentation . . . Changes occurring during tempeh fermentation General changes . Carbohydrate changes Lipid changes . . Protein changes . ‘Vitamin changes . Toxic substance and antinutritional factors changes . Safety . . . . . Tempeh preparation . . Traditional tempeh Pilot plant method Protein quality evaluation . . . . . . Assays utilizing laboratory animals Assays utilizing amino acid profile Assays utilizing microorganism . . Assays utilizing proteolytic enzymes Cobalamins . . Chemical properties . . . . . . . . . Physical properties . . . . . . . . . preparation iii Page vi viii iv Stability . . . . Synthesis . . Biochemical functions Analytical methods. Sources . . Dietary intake Deficiency . Allowance . . Requirement . Toxicity. . . Added cobalt . . MATERIALS AND METHODS. . . . Materials . . . . . . . Microorganisms . Laboratory animals Methods . . . . . . . Total counts . . Determination of cobalamin Extraction of cobalamin ‘Vitamin B-12 assay utilizing L31§ighm§nii Standard solution . Inoculum. . . . Assay tubes . . Determination . Assay of ”true vitamin B-1 Proximate analysis: Inhibitory test . . Experiments . . . . . Effect of temperature Effect of time . . Effect of pH. . . . Effect of soaking and Starter preparation . . Determination of "true vitamin B-12' oooooooNoooo I :3 acid addition Effect of cobalt and acid addition. Effect of soaking and mixed culture Determination of protein quality. Protein Efficiency Ratio. Amino acid analysis Tryptophan analysis Sensory evaluation. . . Statistical evaluation. 44 44 44 46 46 46 47 47 48 48 49 49 50 50 51 52 52 52 53 53 53 55 55 56 57 58 58 59 60 61 62 “SULTS O C I C O O O O O O O O Inhibitory test . . . Effect of temperature Effect of pH . . . . Effect of time. . . Effect of tempeh preparation. Effect of soaking and acid addition Effect of cobalt and acid addition Effect of soaking and tempeh mold . . . . . "True vitamin 8-12“ . . Storage study of starters Sensory evaluation. . . . Tempeh composition. . . . Protein Efficiency Ratio. Amino acid analysis . . . mucms IONS . C C O O C C O O . BIBLIOGRAPHY APPENDICES Appendix A Composition of media used Appendix B Page concurrent growth with in the experiments 2 X 2 Factorial design for acid and cobalt addition . . . . . . . Appendix c 2 X 2 Factorial design for soaking and mold treatment . . . . Appendix D Questionnaire for triangle test. . . . . . . 65 65 65 69 70 74 74 77 77 79 81 84 85 86 87 91 102 105 106 107 LIST OF TABLES Table 1. 2. 10. Basal bioassay diet . . . . . . . . . . . . . . Recovery of vitamin B-12 activity in fermented- soybean extract assayed by LLleighmanii and Malena: Effect of incubation temperature on the total count of B-lz-producing bacteria grown on heat-treated soybeans . . . . . . . . . . . . . Effect of incubation temperature on vitamin B-12 production by bacteria grown on heat-treated soym C O O O O O O O O O O O O I O O O O O 0 Growth of B-12-producing bacteria on tryptose- glucose-yeast extract agar at different pH 1 avg]. s O O O O O O O O O O O O O O O O O O C Total count of vitamin B-12-producing bacteria and vitamin B-12 production during a 3-day fermentation of soybeans. . . . . . . . . . . . Growth of vitamin B-12 producing-organisms and vitamin 8-12 production on soaked, acidified and control soybeans, at 30C for 2 days. . . . Bacterial count and vitamin B-12 production on soybeans treated with acid and cobalt . . . . . Effect of soaking and mixed-culture on total count of vitamin B-12 bacteria and vitamin B-12 content of soybeans fermented for 3 days. . . . Vitamin B-12 activities of tempeh measured by Wandhleiehmaeii-------- vi Page 28 66 67 67 69 72 75 78 80 80 11. 12. 13. 14. 15. 16. 17. vii ‘Vitamin production, bacterial and mold counts of mixed-culture tempeh starters stored at room temperature (23C) . . . . . . . . . . . . . . Vitamin production, bacterial and mold counts of mixed-culture tempeh starter stored under refrigeration . . . . . . . . . . . . . . . . Sensory evaluation by the triangle test of regular tempeh and tempehs prepared by mixed- culture . . . . . . . . . . . . . . . Composition of regular tempeh and tempeh made with mixed-culture inocula . . . . . . . Protein Efficiency Ratio of regular tempeh and tempeh made with mixed-culture inocula . . . Amino acid composition of regular tempeh and tempeh prepared with mixed-culture inocula . Amino acid pattern of FAO reference and amino acid contents and scores of regular tempeh and tempeh prepared with mixed-culture inocula. . 82 83 85 86 87 89 9O LIST OF FIGURES Figure Page 1. Flow sheet of tempeh preparation methods . . 24 2. Structure of cobalamins . . . . . . . . . . . 32 3. Separation of amino acid standard . . . . . . 63 4. Standard curve for tryptophan determination . 64 5. Trends of bacterial growth and 8-12 production during a 3-day fermentation . . . . . . . . . 73 viii INTRODUCTION Tempeh is an Indonesian food made by fermenting tender-cooked soybeans with the mold Bhizgpus. The soy- beans are bound together by cottony mycelia of the mold into a firm white cake during fermentation. Sliced-tempeh fried to a crisp golden brown has an universally accept- able flavor (Steinkraus, 1960). It serves as additional source of protein to Indonesian diets, in which rice is the staple food eaten three times daily. Nutritionally, tempeh contains 45% protein (d.b.), with Protein Efficiency Ratio (PER) of 2.2 (Suparmo and Markakis, 1987), and appreciable vitamin B contents (Murata gt. a1. 1967, 1970, Roelofsen and Talens, 1964, Djurtoft and Nielsen, 1983). Autret and van Veen (1955) suggested the use of tempeh as supplementary feeding for protein-malnourished children in the region where sufficient milk can not be produced. Tempeh is also gaining its popularity among vegetarians in the United States (Shurtleff and Aoyagi, 1980). The growing number of tempeh manufacturers in the United States reached 53 in December 1983 and 900 metric tons of tempeh/year were produced (Beuchat, 1984). The tempeh fermentation was studied extensively by Steinkraus gt. 31. (1960), Hesseltine gt. g1. (1963), and Hesseltine and Wang (1967). They described the mold species to be used and reformulated the process in order to obtain optimal yield on a laboratory scale using pure mold culture. Progress in the development of larger scale tempeh processing has also been made (Martinelli gt. Q1. 1964). Steinkraus gt. 31. (1965) developed a pilot plant process for the production of dehydrated tempeh. The production of Bhizgptg gligggpgttg starter and their application in tempeh fermentation have been studied by Rusmin and Djien (1974), and Wang gt. 31. (1975). The production of tempeh in Indonesia, however, is still a household art. Instead of using mold spore preparations, small pieces of tempeh from previous fermentation are used as starter by many tempeh manufac- turers. A commercial tempeh starter in Indonesia, called nggz, is made by growing the unpurified mold culture on a layer of soaked and boiled soybeans, and sandwiched between hairy leaves of flitigggg similig or Iggtgna grandig. The leaves, which then contain mold mycelia and spores, are sun-dried and sold as starters. Liem gt. g1. (1977) reported that commercial tempeh purchased in Toronto, Canada, contained significant amount of vitamin 8-12, which is not normally present in foods from plant origin. An appreciable amount of vitamin 8-12 was also found in soybean and cowpea-tempeh in Nigeria which were prepared using QEQI. and another inoculum from West-Java, Indonesia (Djurtoft and Nielsen, 1983) and in commercial tempeh and tempeh burger purcha- sed in Tallahassee, Florida (Truesdell gt. 31., 1987). Steinkraus (1985) reported that a non-toxic strain of fi1gtg1g113 pnggmgni3g, a bacterium which grows along with the tempeh-mold during fermentation, is responsible for the vitamin 8-12 production. Abdulla gt. 31. (1981) reported that vitamin 8-12 is the nutrient least available in the diet of people who eat foods solely derived from plants. The production of vitamin 8-12 in tempeh fermentation makes tempeh not only a good source of protein but also a source of vitamin 8-12 as well. Nutritionally, tempeh becomes a more important diet for strict-vegetarians. All vitamin 8-12 found in nature is synthesized by microorganisms. Tempeh produced using pure mold culture did not contain vitamin 8-12. Some bacteria are used commercially in the production of vitamin 8-12. So far, these bacteria have not been reported present in tempeh. Besillue wetsuits and W elixaeese are among the bacteria used commercially to produce vitamin 8-12 which grow in the range of the growth conditions of tempeh mold. Chung and Fields (1986) used n.mgg3tg:13m to increase the vitamin 8-12 content in corn meal. The availability of the vitamin 8-12 in tempeh, however, has not been studied yet. Some bacteria produced various vitamin 8-12 analogs (Ellenbogen, 1984), which do not show vitamin 8-12 activity. L3gtgt3gi113§,1g1ghm3311, which is not specific for vitamin B-12, has been used officially for vitamin 8-12 determination. Another assay using protozoa anzgmgn3§ 331n33gngig, is very specific for vitamin 8-12, not responding to the presence of vitamin 8-12 analogs (Ellenbogen, 1984). The purposes of this study were: 1) to study the growth parameters of B-lZ-producing bacteria (x.pngnmgn13g, 3.3g33tgzign, and 3.911y3gg35) in the micro-environment of tempeh fermen- tation, 2) to study the production of vitamin 8-12 by the three organisms at tempeh-fermentation's conditions, 3) to produce mixed-culture starter from the three bacteria and the tempeh mold for the production of vitamin 8-12 in tempeh and to study their stability, 4) to study the protein quality of tempehs produced by the mixed-culture fermentation, 5) to evaluate the sensory quality of the tempehs and 6) to determine the amount of true vitamin 8-12. REVIEW OF LITERATURE Legumes as Human Foods Legumes are inexpensive protein sources which are especially important for people with marginal nutri- tion. In relation to the amino acids, legume proteins are valuable in their complementary effect on cereal proteins (Steinke and Hopkins, 1983). For people in developing countries, legumes also serve as sources of vitamins and minerals, especially thiamin, riboflavin, calcium and iron, while in the more developed countries they may serve as sources of complex carbohydrate and fiber, which prevent the development of some diseases caused by consumption of purified foods. Substituting soybean protein for animal protein in the diet lowers plasma cholesterol level (Carroll gt; 31. 1978). Despite all of the beneficial attributes, legumes contain a wide range of antinutritional factors such as protease and amylase inhibitors, haemaglutinins, phytate, tannins, flatus factors and may also contain aflatoxin. For the consumption of legumes, suitable means must be used in order to remove or inactivate the antinutritional factors and maximize the good traits. Food Fermentations Various types of fermentation have been used by man around the world since prehistoric times. Foods were fermented for many reasons including improvement in sensory properties (flavor, taste and texture) and for food preservation. As a result of fermentation, the foods are enhanced in their nutritive values by the synthesis of vitamins, by breakdown of complex chemical components into simpler materials, which improve their digestibility, and the degradation of anti-nutritive factors and toxic materials. In natural fermentations, mixed cultures of dif- ferent species or genera of microorganisms perform biochemical changes that produce the desirable changes. Whitaker (1978) reported that there are two known major types of food fermentations: occidental type and oriental type. In the occidental cultures, the raw material for fermentation of high protein foods derived from animal sources such as dairy and meat products, while in the oriental cultures legumes and cereals are the major sources. Another major difference is in the microor- ganisms used. Occidental cultures used mainly bacteria and yeast while in oriental cultures many fermentations involve the use of filamentous fungi. Fermented foods derived from legumes and cereals are important diet in South East Asia. Soybeans (g1yging n3x.L.) are good source of protein that are widely used in the Orient. Soy sauce, miso, tempeh, sufu and natto are examples of fermented soybeans which originated in the Orient. (Hesseltine and Wang, 1967 and Whitaker, 1978). These fermented foods produce very fine flavors which complement and enhance the taste of the predomin- ant staple dishes consisting of rice and vegetables, which would otherwise be less palatable. Starting with the discovery by Robert Koch who, about one hundred years ago, published a reliable isolation method to chose a single species of organism, fermentation practices have changed drastically (Slater, 1981). The ease of isolating appropriate organism led to the development of monoculture fermentation which is ' consistent in quality and production rate. Brewing is among the processes that gained an advantage from pure culture fermentation. For specific purposes, however, when changes are expected beyond those which can be achieved by single culture, mixed culture fermentation is more desirable. Japanese soy sauce is produced in a two step fermentation processes using Agpgtg1113§_ gtyz3g_ followed by Egdigggggtg ggy3g fermentation in brine. Spontaneous alcohol fermentations are often started by successions of yeast species, in which those sensitive to alcohol predominate, such as fi3nggn13gpgz3 gni11gmgg§11 and 9333133 pg1ghgttim3, followed by species such as Saseharomxsee roeei. S-serexieiae1_ When the alcohol content rises above 10%, the predominant species are fireerexieiae var ellipsoideue or S-oxiformie- A high concentration of malic acid in wine is undesirable and a malolactic fermentation is carried out by lactic acid bacteria. An example of mixed-culture in dairy fermenta- tion is yogurt fermentation by .Laetobasillue pulsarisue and Strentoeoesue thermophilue (Harrison. 1978)- There is a spectrum of microbial communities, ranging from '1oose' to 'tight' association, a few of which appear to be obligatory. In loose association, the environment supports the growth of more than one popula- tion constituting a community. At different times and under different conditions, the components of the popula- tion inevitably interact. At the time when a particular nutrient is present in limiting concentration or a metabolite is concentrated in the closed environment, it affects the rate of growth of certain members of the community. Meers (1973) classified microbial interaction into six distinct catagories: (l) ggtpgtitign which occurs when the growth of two or more species is limited by a common factor such as the substrate, (2) ptg33t1gn marked by the collapse of the population of one species caused by another species. An example of this predator-prey relationship is the use of ciliate protozoa in removing bacteria in a suspension of waste water. (3) pgtggitism as in phage-infected bacteria culture. This relationship has not been exploited. (4) 33gng31igm is an interaction in which the growth of one organism interferes with the growth of another. The form of interference could be a change in temperature, pH, or toxic metabolite produc- tion. (5) ngntt31igm, which rarely occurs, is the coexistence of two or more species without any interac- tion, and (6) ggmmgng31i§m and mut3311gm involve one organism giving benefit to another. In commensalism, the benefit is one sided, whereas in mutualism, all component species derive benefit from the association. TEMPEH Tempeh, fermented soybeans by 33133333 g1igggpgtgg, has a very attractive flavor and appearance, requires only soybeans, which are an inexpensive substrate, and takes only a short time to ferment. The most common and popular material used for making tempe is kg3g131 (soy- beans) and the word "tempe" then refers to soybean tempe 10 and was introduced to the Western world and written in English as "tempeh". In Indonesia, tempeh is still considered to be an inferior food as it is very inexpensive and sold by street vendors. It is very rarely served in big res- taurants or hotels. However, there has been an improve- ment in Indonesian people's attitude toward tempeh as local experts keep campaigning for the wholesomeness of tempeh as a good source of protein, vitamin and calories. Origin of tempeh Shurtleff and Aoyagi (1985), found that the earliest known reference mentioning tempeh is "serat centini", written around 1815 in central Java by Rangga Sutrasna. The document contains a line mentioning "onions and uncooked tempe", and for that reason the origin of tempeh fermentation is speculated to have originated in Java. There is another speculation that tempeh fermenta- tion was introduced along with the introduction of soybeans to Java by Chinese merchants in about 2,000 years ago. The method then underwent some modification, including the introduction of new microorganisms used in the fermentation to suit Javanese tastes and better adapt to the Indonesian climate. 11 Raw materials of tempeh The word tempe (pronounced tem-pay) collectively refers to fermented foods, typically legumes, bound together into compact cakes by mold mycelia from 33133333 genera. An additional descriptive word is added to indicate the material from which the tempeh was prepared. Tempeh from soybeans, (3333131), is called tgmpg_kg3g131, from velvet beans, (hgng3k), it is tgmpg_tgng3k, from peanut press-cake (3333311), it is tgmpg_t3ngk11, from tofu by-product, it is tg33g_gg333g and from coconut press-cake it is tgmpg_tgnggtgk. The most popular material used for tempeh in Indonesia is soybeans. For that reason the word tempeh usually refers to soybean tempeh. Commercial fresh soybean tempeh in the U.S. should contain no less than 39 % solids and 18 % protein, no more than 3 % hulls, and no foreign or added substan- ces (Shurtleff and Aoyagi, 1980). Many cereals and beans have been tried in the making of tempeh in laboratories. Wheat has been tried by Wang and Hesseltine (1966), wheat, oats, rye, barley, rice and a combination of rice or wheat with soybeans by Hesseltine gt. 31. (1970), red kidney beans by (Gomez and Kothary, 1979), cowpeas by (Djurtoft, 1983 and 1984), and chickpea, horsebeans by (Rae and Robinson, 1977). 12 Fermented insoluble fraction of soybeans by Emilia eitenhila instead of Rhianna 911mm. called oncom, is very popular in West Java. It has an orange or pinkish appearance rather than white. Productions and consumptions of tempeh In Indonesia, tempeh producers are usually small family-run cottage industries, which mostly produce around 10 kg (21 lbs) a day with a retail price about US$ 0.25 per kg (Shurtleff and Aoyagi, 1979). In Indonesia, tempeh is the nation's most popular soyfood, the annual production in 1983 was 154,000 metric tons and had a retail value of USS 85.5 million and made up 64% of the soybean production and imports. Tempeh was produced by 41,200 small companies involving 128,000 workers. Most of those establishments were family-run and still use traditional procedures and equipment (Winarno and Reddy, 1986). Shurtleff and Aoyagi (1984) reported that there were 53 tempeh factories in the United States with a total production of 9000 metric tons 1983. The largest factory produced 7,000 lbs of tempeh a day. 13 TEMPEH FERMENTATION Organisms in Tempeh Fermentation An impure culture of mold is used for prepari- ng traditional tempeh fermentation. The inoculum is taken from pieces of a previous fermentation or its wrapper, which usually consists of leaves. A 'tradi- tional inoculum is available commercially as a dry spore preparation on dry hairy 31313333 t1113gg333 leaves. Tempeh starters are now also available in powder form prepared from a pure culture of 33133333 31133333333. Tempeh starters sold in the U.S. are in this form. Principal microorganisms in Indonesian tempeh were studied by Hesseltine gt.31. (1963). They isolated some strains of Rhizopus and identified them as: 33133333 mas. B-etolonifer. R-arrhizse. B-Iomoeaeneie. . 3.333133133333333, and 3.313313333. It was concluded that 3.31133333333 is the principal species used in Indonesia for making tempeh. Liem gt. 31. (1977) found that some commercial tempeh contains vitamin 8-12. They isolated a bacterium 3133313113 33g3m3313g, which is responsible for the presence of vitamin 8-12 in the tempeh. Wang gt. 31. (1975) and Rusmin and Djien (1976) successfully developed a method for mass production of 33133333 31133333333 spores and their application. 14 Certain bacteria acidify soybeans during soaking (Steinkraus gt. 31., 1960 and Steinkraus, 1985), while other organisms, may grow along with the tempeh mold in traditional tempeh fermentation. Aware of the role of the bacteria during soaking, traditional tempeh makers usually add water from previous soaking into the new batch of soaking water to ensure good acidification. Some bacteria grow along with the tempeh mold during the fermentation. Typically they are spore former bacilli (Steinkraus gt. 31., 1960, Sudarmadji and Markakis, 1977). Liem gt. 31. (1977) reported that 3133313113 3333333133 was found in commercial tempehs and vwas responsible for the production of vitamin 8-12 in tempeh. Parameters in tempeh fermentation For a good tempeh fermentation, two conditions should be fulfilled (Steinkraus gt._31. 1960). First, the soybeans need to be bound together into compact cakes by the growth of the mold mycelia. Second, the soybeans must undergo a partial digestion by the mold enzymes, thus the tempeh fermentation is centered in the mold growth. Since tempeh mold does not grow well on unskinned beans, dehulling is an essential step in tempeh fermentation. Oxygen is essential to the mold. When the layer of the 15 fermenting beans is thicker than 2 inches or about 5 cm, mold grows less heavily in the center than it does in the thinner layer. To make a good tempeh, the thickness should not be more than 3 cm ( Steinkraus gt. 31., 1960: Martinelly gt. 31., 1964). The growth of the mold on various soybean sizes was studied by Robinson and Kao (1977). 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 as containers for tempeh fermentation. However, if the amount of aeration is in excess, the soybeans at the surface will be dehydrated and sporulation will start producing undesirable black spores and poor appearance. It is obvious that high relative humidity is absolutely needed (Steinkraus gt. 31., 1964). Temperature is a critical factor for microbial growth. Temperature slightly above room temperature is best for tempeh fermentation. An optimum fermentation temperature of 37C was reported by Steinkraus gt. 31. (1960). 16 Temperatures as low as 25C were used to produce an acceptable tempeh. Such fermentation requires as long as 5 days for completion while fermentation at 37C requires only 1 day. Later investigation by Hesseltine gt._g1. (1963) showed that Rhizopus strains that produce satisfactory tempeh can grow from 14C to 44c except 3.3t313n1figz, which has a maximum temperature of no more than 35C. For good tempeh fermentation, it is essential to ensure that the beans are acidified to a sufficient degree at the start of fermentation ( Steinkraus gt. 31., 1960). Low pH inhibits the growth of most contaminating bacteria which would spoil the tempeh but does not interfere with the growth of the mold. The mold growth is inhibited when the pH drops below 3.5. CHANGES OCCURRING DURING TEMPEH FERHENTATION General changes As the tempeh mold grows, the temperature of the mass increases. The increasing temperature of the fer- menting soybean mass is indicative of the growth rate of the mold. Steinkraus gt. _1. (1960) reported that during the first 20 hours, spores germinate, the temperature rises gradually. During the following five hours, when growth is accelerated, the temperature may reach 43-44C, 17 and then gradually fall as the growth subsides. By this time, the beans are already knitted into a compact mass by mold mycelia. Beyond that stage, the mold sporulates and N33 is produced, due to protein breakdown. During the period of most rapid mold growth, the soluble solids rises from 13 to 22%. The soluble nitro- gen increases from 0.5% to nearly 2%, while the total nitrogen remains relatively constant, about 7.5%. The pH, which initially is 5.0 rises to 7.6. The change from 6.0 to 6.7 occurs during the period of most rapid mold growth. Optimum quality tempeh has pH 6.3 to 6.5. Hurata gt. 31. (1967) reported the following changes: - There is no large difference in protein and ash content between tempeh and unfermented soybeans. During fermentation the fiber is slightly increased. - Fat content decreases, but the acid value increases. - Free amino acid content increases. Carbohydrate changes The carbohydrate content of soybeans is about one third of their dry weight and is distributed as follows: 7% in the hull, 26% in the cotyledon, and 1 % in the hypocotyl. The carbohydrate content consists of soluble 18 sugars (stachyose 3.8%, raffinose 1.1% and sucrose 5%) and polysaccharides. Reducing sugars in dormant seeds are very low (Abrahamsen and Sudia, 1966 and East gt. 31., 1973). Reducing substances decrease very slowly during the entire fermentation. They do not regain their initial levels even though the breakdown of higher carbohydrates continues, presumably because the sugars are used by the mold. Suparmo and Markakis (1987) reported that fermen- tation for 36 hours reduced sucrose, raffinose and stachyose to about half of their initial contents. Lipid changes The ether-extractable component of soybeans was found to decrease from 22.3% to 19.6% during tempeh fermentation (Suparmo and Markakis, 1987). An increase in free fatty acids was noticed by (Sudarmadji and Markakis, 1978) and was attributed to the action of mold lipase. Hurata 3t. 31. (1967) reported that tempeh fermentation slightly increased oleic acid and decreased linoleic acid. Sudarmadji and Markakis (1978) studied the changes of free fatty acids during the entire fermentation along with changes of bacterial plate count and temperature. They distinguished three phases in tempeh fermentation: the main phase which lasted about 30 hours at 32C, 19 during which microbial growth, lipolysis and temperature increased and the result was a product of high sensory quality: the transition phase, lasting 24 hours after the first phase, when mold growth and lipolysis subsided, the temperature decreased, and the product was still in acceptable condition: and the deterioration phase when bacterial growth and lipolysis reappeared and the tempeh quality deteriorated rapidly. Protein changes According to Steinkraus gt. 31. (1960), during tempeh fermentation, soluble nitrogen including NH3, increases due to protein breakdown. Stilling and Hackler (1965) stated that most amino acids either remain un- changed or decline. A notable exception was tryptophan, which increased significantly during the first 24 hours at 37C, but declined thereafter. Murata 3t. 31. (1967) made a comparison of amino acid composition between soybeans and tempeh from Indonesia and soybeans and tempeh prepared in the laboratory. They reported that, in general, most amino acids were not changed by fermenta- tion. It was observed that the tryptophan content of the samples from Indonesia increased by about 20% and alanine in tempeh prepared in the laboratory also increased by about 20 %. The protein efficiency ratio (PER) was not 20 significantly changed by tempeh fermentation (Hurata gt. 31., 1971, and Suparmo and Markakis, 1987). Vitamin changes According to Murata (1967) tempeh fermentation resulted increased riboflavin, vitamin 8-6, nicotinic acid and pantothenic acid contents, but thiamin was little altered. Later, Murata 3t. 31. (1970) reported that biotin and folacin increased several folds during tempeh fermentation. Sanke gt. 31. (1971) reported that 3.311gggpgggg synthesized folate compounds in a synthetic folate free medium. Toxic substance and antinutritional factors changes Despite their richness in protein, legumes also contain anti-nutritional factors such as trypsin inhibitors, flatus factors, phytic acid, and lectins. Legumes are also susceptible to damage during storage due. to microbial growth that could lead to the production of toxic substances, such as mycotoxin. Van Veen gt. 31. (1968) reported that 3.31133333333 reduced the aflatoxin B-l content of peanut presscake by about 70%, while M331113 gitgphi13, which is used to prepare "oncom", another Indonesian fermented food, reduced the toxin by about 50%. The fermentation also reduced the phytic acid 21 content by about 40% (Sudarmadji and Markakis, 1977). The raffinose and stachyose content of soybeans decreased by more than 50% as a result of tempeh fermentation, according to Shallenberger 3t. 31. (1967), and Suparmo and Markakis (1987). Safety There have been no reports of food poisoning due to consuming soybean tempeh. However, there were deaths as a result of eating contaminated tempeh bongkrek. Tempeh bongkrek, made from coconut presscake, and contaminated by Egggggngngg ggggygngngng, may contain toxic substanc- es, such as heat-stable bongkrekic acid and heat-labile toxoflavin (van Veen, A.G. and Martens, W.K. 1935). The production of tempeh "bongkrek", made from coconut presscake, has therefore been discouraged. Tempeh Preparation There are two major tempeh preparation methods available today. The traditional methods which is used for making commercial tempeh in Indonesia and the pilot plant method introduced by Steinkraus (1961), which is used for making commercial tempeh in the U.S. The two methods are shown in Figure 1. The traditional method, may differ from place to place in the length of soaking 22 time, boiling time, and the fermentation time, but the principle steps are the same. Traditional Tempeh preparation Soybeans are cleaned with water and then soaked in water at least overnight. Foam appears on the surface of the soaking water, a result of lactic fermentation. Soaking water from a previous batch is added to the next batch in order to enhance the lactic fermentation. In certain places, the soybeans are boiled and dehulled before soaking. The seed coats of the soaked soybeans are separa- ted from the cotyledons by hand and eventually washed off by water. The dehulled soybeans are then cooked by boiling until soft. The cooked beans are drained using woven-bamboo baskets and the beans are spread on a flat surface to surface-dry and cool. Excess water would favor bacterial growth which may interfere with the tempeh fermentation. After the beans cool, they are inoculated by a traditional starter or tempeh from a previous batch and wrapped in banana leaves or perforated plastic bags, into approximately a quarter pound per package. The packages are then packed in baskets, and stored or incubated until the temperature rises to about 40C. It needs about 10 hrs of incubation to reach that 23 temperature. The packages are then spread out on shelves and the fermentation continues at room for another 10 hrs or longer. If the packages are left in the basket too long, the mold mycelia will not cover the beans and a poor quality tempeh will be produced. Pilot plant method Steinkraus 3t. 31. (1965) developed a pilot plant method of tempeh preparation, which involves size grading of the beans, dry dehulling, hydration, cooking, drain- ing, cooling, inoculation with a pure mold starter and fermentation at optimum conditions (35-38C, RH 75-85% for 18 hr). The graded soybeans are heated at 93C for 10 min to loosen the seed coats, cooled, and then passed through a properly spaced Burr mill to crack the beans. The hulls are separated from the cotyledons by passing the cracked beans over a gravity separator or by using an aspirator to blow the hulls off the cotyledons. The dehulled beans are hydrated by soaking them in acidified water for 2 hr at 25C or 30 min at 100C. The acid addition is intended to Control bacterial growth that might otherwise contaminate tempeh. The soaked beans are boiled for 90 to 120 min in the soaking water. The beans are drained and surface dried as they cool. 24 Iraditional Method SOYBEANS Soaking Overnight Wet Dehulling Boiling 30 minutes Cooling/Draining Inoculating (traditional inoculum) Wrapping Incubation RT-36 hrs Harvesting EEHEEH Pi1ot plant method §OY§EAN§ Dry Dehulling Acid addition (optional) Boiling 30 minutes Cooling/Draining Inoculation (pure mold inoculum) Package/Tray Incubation 37'C-18 hrs Harvesting TEMPEH Figure 1. Flow sheet of tempeh preparation methods 25 Pulverized tempeh mold (3.311g3333133) culture contain- ing both mycelia and spores are used to inoculate the beans. The inoculated beans are spread out on perforated trays covered with waxed paper to prevent dehydration and excessive aeration. The trays are put in an incubation room which is maintained at 35-37C and 75-78% relative humidity. The incubation lasts about18 hr. As soon as the mold grows covering the beans, tempeh should be harvested immediately. Delay of harvest may result in the development of an off-flavor in the tempeh, making it unfit for consumption. Protein Quality Evaluation The primary function of dietary proteins is to _ supply a mixture of amino acids of a proper pattern for the synthesis and maintenance of tissue proteins. The nutritional quality of protein is determined by the quantity, availability and proportion of the essential amino acids (EAA) comprising it. Protein quality can be determined using laboratory animals (bioassays), amino acid profiles, microbiological assays, and in yittg proteolytic enzyme assays (Hsu gt. 31. 12131. Assays using human subjects is impractical because of the biological variability among subjects, high expense and various difficulties involved in such studies. 26 Assays utilizing laboratory animals The relative efficiency of protein in providing the required amino acid pattern available to animals will determine the magnitude of measurable biological respon- ses which is dependent upon the EAA pattern, especially the most limiting amino acid in the protein. Protein nutritional values are obtained by feeding experimental animals, usually growing rats, with the test protein under controlled experimental conditions and measuring the responses of the whole-animal in terms of body weight gain or body nitrogen. The most common bioassay is called protein efficiency ratio (PER) which is indicative of the growth promoting capacity of a protein expressed as the ratio of weight gain in grams of the experimental animal per gram of protein consumed. The greater the PER value, the better the protein quality. The PER method (AOAC, 1980) specifies that the diet should have 1.6% nitrogen, or 10 % protein, that 10 weanling rats be used, that the duration of the experi- ment is a 28 day period immediately following 2days of acclimatization, that there be a concurrent casein control group included in the study. The diet is formu- lated as shown in Table 1 and the PER is calculated as: 27 g of weight gain PER = --------------------- g of protein consumed. adjusted PER = ------3;E---- X PER of test protein PER of casein Derse (1960 and 1962), who evaluated the PER method on various proteins, recommended the use of the bioassay as the official method. Hackler gt. 31. (1984) reevalua- ted the AOAC official method for PER and recommended that the PER method should be changed from a 28 to 21 day feeding test, immediately following the 2-days acclima- tion period. For protein sources that contain less than 12% crude protein, the highest percent crude protein possible should be used. Under these conditions, refere- nce casein standard diet should be made at the same level as the test protein diets. The PER has many limitations. It relates protein quality only to growth, makes no allowance for maintenance, the value of PER is not proportional with the quality of the protein (PER of 2.00 does not indicate protein quality twice as good a PER of 1.00), and as the assay takes a long time, it is imprac- tical for industrial needs. Despite the limitations, the PER method is the simplest among the bioassays and it is being used as an official method for regulatory purposes in the U.S. 28 Table 1. Basal Bioassay Diet 1.60 X 100 s = ..................... % N of sample S x % ether extract Corn Oil = s - ................... 100 S X % ash Salt Mixture USP = 5 - --------------- 100 Vitamin Mix = 1 % S x % crude fiber Cellulose = 1 - .................... 100 S x % moisture Water = 5 — .................... Corn starch or sucrose to make 100 (AOAC, 1980) Assays utilizing amino acid profile The test protein is chemically digested and subjected to ion exchange chromatography. An Amino acid profile is obtained from the chromatogram of the test protein digest. By comparing the profile to the amino acid profile of a reference protein, the limiting essential amino acid of the test protein can be determined. The "Chemical Score" (Mitchell and Block, 1946) compares the sample's EAA pattern to amino acid pattern of whole-egg protein while the "Amino Acid Score" (FAO/WHO, 1973) use the pattern of human-amino-acid requirements as the reference. McLaughlan 3t. 31. (1959) recommended a 29 simplified score using only three amino acids: lysine, methionine and cystine, since these were found to be the most commonly deficient in proteins. In finding the relationship between the chemical score and their nutri- tive values for the rat, Mitchell and Block (1946) found that the % deficit in the limiting essential amino acid is inversely correlated to the biological value of protein. These chemical methods are valuable tools for screening the quality of proteins. However, the chemical methods do not indicate the availability of the amino acids since they assume that all amino acids in the sample are 100% available, any toxic materials which affect the bioassay fail to appear in the chemical methods. Assay utilizing microorganisms The growth of the microorganism Igtzghymgng pytifgzmig, which has approximately the same amino acid requirements for growth as man (Evancho gt. 31., 1977) may be used in assaying protein quality. The sample protein is hydrolyzed and serves as nitrogen source for the medium in which the microorganism is cultured. The rate of bacterial growth is indicative of the protein quality and is compared to the growth rate when a high quality protein such as egg protein or casein is the 30 source of the amino acids. In this manner, an index is developed whereby proteins are ranked relative to the reference or standard protein. The assay provides rapid estimation of protein quality which is comparable to the PER. Assays utilizing proteolytic enzymes Multi-enzyme mixtures, which digest protein similar to the 1n-yiyg rat digestibility, have been used to estimate one aspect of the protein quality, protein digestibility. Hsu gt. 31. (1977) determined the 1n-yitt3 protein digestibility using a multi-enzyme system con- sisting of trypsin, chymotrypsin, and peptidase. Hsu gt. 31. (1978) developed a rapid estimate of protein quality, C-PER. The calculation was based on the data of in-yittg protein digestibility, the EAA profile and the FAO/WHO (1973) standard for the EAA. The assay was designed to estimate protein quality from samples having rat PERs ranging from 0.67 to 3.22 with a standard error of about i 0.36 PER units. 31 COBALAHINS The anti-pernicious anemia factor from the liver was identified and purified by Smith (1948), and crys- tallized by Rickes gt. g_. (1948). The compound was called vitamin 8-12. Chemical properties Vitamin B-12 is a member of a group of compounds collectively called cobalamins. The structure of cobala- mins is shown in Figure 2. The molecule is composed of a central cobalt atom coordinated by a nearly planar base structure, corrin. Corrin consists of four pyrrole nuclei joined in a large ring containing six conjugated double bonds. The structure is very similar to that of porphyrin except that the methene bridge joining rings A and D is missing. In cyanocobalamin, the axial coordina- tion sites are occupied by a base, dimethylbenzimidazole, and a cyano group. Coenzyme cobalamin, the predominant form in foods, has adenine nucleoside, instead of the cyano group. The cyanocobalamin is an artifact, it is the product of the extraction process. Other compounds containing bases other than dimethylbenzimidazole are known and are termed B-12 analogs. Cobomide 32 V'T' __________ O E E g ‘ \ CH, '3 HN-co-crgnp CH c - - . o I H CH CO NH 0 9H, a CH,\\ :- a : HC'Z-CH, /—_—i—L— --------- / CH °v° : <" ’ / °°// 0 °”/ N cm i i...._.._/ K 5 : 5,6-Dimeihylbenz- V ”0.0.1: 0 H _| imidozoi R Designation -CN VflomfiiBu (Cyanocobolomini -OH Vitamin 8,2. (Hydroxocobolomin i - CH1 Methylcobolomin OH OH -CH Coenzyme 8,, 3 (S'—Deoxyodenosyl - 0 Nfi Cobolomino. Cobomomido) m /N NH; Figure 2. Structure of cobalamins 33 Physical Properties Cyanocobalamin occurs as a tasteless, odorless red crystalline powder or as red needle-like crystals. In its anhydrous state, it is very hygroscopic, but the moisture can be removed by heating at 105C under reduced pressure. Cyanocobalamin is soluble in water (1.25 g/ 100 ml at 25C), alcohols, phenols and other polar compounds with hydroxyl groups, but is insoluble in most organic solvents. The ultraviolet and visible absorption spectra of cobalamin have maxima at 278, 361, and 550 nm. Stability Coenzyme B-12 is unstable toward light and acid/ base hydrolysis. The first reaction to take place is the cleavage of the cobalt-carbon bond. Cyanocobalamin is the most stable form of all cobalamins. An aqueous solution of cyanocobalamin at pH 4-7 can be autoclaved at 120C and in dry form it stands heating at 100C for a few hours (Ellenbogen, 1984). Vitamin B-12 in foods is stable during heating and thermal processing (Hozova and Sorman, 1979). During boiling of the foods, the vitamin is lost through leach- ing into the cooking liquid or drippings. 34 Synthesis Vitamin B—12 (cobalamins) are synthesized in nature exclusively by microorganisms, such as (33311133 W. mm 21123235. 3121;151:113 mummies. Bronisznibagterium Wail. B-shermii. and Wm denitrifisans The produc- tion of commercial vitamin B-12 through fermentation with £.fit39§3n331§h11 ATCC 6207 or 2.3hgtm3n11 ATCC 13673 fermentation is accomplished in a two stage process: 2-4 days anaerobic fermentation followed by 3-4 days of aerobic fermentation. The 5'deoxyadenosylcobinamide is produced during the anaerobic process with added cobalt, while the aerobic process mainly produces 5,6-dimethyl- benzimidazole (Crueger and Crueger, 1984). Biosynthesis of the vitamin is believed to run parallel with the biosynthesis of porphyrin and chloro- phyll up to the formation of urophorphyrinogen III. The conversion from urophorphyrinogen II into the corrin ring system is not yet understood. Biochemical functions There are two specific biochemical reactions involv- ing cobamide coenzyme: (1) reactions requiring adenosyl- cobalamin and (2) reactions requiring methyl cobalamin (Hogenkamp, 1968, Stadtman, 1971, and Ellenbogen, 1984). 3S Reactions requiring adenosyl cobalamin are reduc- tion reactions from ribonucleotide triphosphate to deoxyribo-nucleotide triphosphate and rearrangement reactions. The rearrangement reactions exchange a hydrogen atom from one carbon atom to an adjacent one. The migrating hydrogen attaches to the 5'carbon Of the adenosyl group before it is transferred to another group. All of the rearrangement reactions occur in the energy pathways of bacteria. Vitamin B-12 affects the metabol- ism of folacin and may be essential in certain reactions in which folacin serves as a coenzyme (Nixon and Bertino, 1970). So far there is no known meaningful reaction requiring analogs. These substances have questionable health benefits. Analytical methods Microbiological methods for assaying vitamin B-12 have high sensitivities, they detect vitamin B-12 as low as 0.01 nanogram/ml. L33t333311133 1313333311, Escherichia 921i mutant. Egglsna grasilis and gshrgmenas m31ngmgng13 are the microorganisms used for the assay. L.1g13hmgnii and 3.3311 mutant respond to analogs and several substances that are structurally related to cobalamins. The Association of Official Analytical 36 Chemists and the United States Pharmacopeia (AOAC, 1980) recommend L.1313333n11 ATCC 7830 as the assay organism, whereas the British Analytical Methods Committee recom- mends the use of Q.m31ngmgngi3 (Analytical Methods Committee, 1956). The radioisotope dilution assay, which is available in the form of commercial kits, provides an alternative test which is very convenient, rapid and is not affected by inhibitors such as antibiotics or other drugs. This method is particularly important for clinical assessment of vitamin B-12 in serum. Raven 3t. 31. (1972) compared the assays utilizing L.1313h33311, E.gtg31113 and the radioisotope to determine the vitamin content of sera, and found that the 3.31331113 bioassay was the best assay to distinguish between normal and pernicious anemia. The results with the radioisotope dilution assays tend to be higher than those with microbial assays (Mollin gt. 31., 1976). Mollin 3t. 31. (1980) conducted interlaboratory comparisons of microbiological and radioisotope dilution (RID) methods for vitamin B-12 analysis in normal and abnormal sera. In general, RID produced higher results than did the microbiological assay, while excellent correlation, reproducability and recovery were obtained between the microbiological assays using 3.33331113 and 1.1313333311. Murphy gt. _1. (1986) used the difference between the value obtained using 37 L.1313nm3n11 and 3.31331113 to express the amount of analogs. Chen and McIntyre (1974) introduced a radiomet- ric microbiological assay (RMA) for measuring the levels of vitamin B-12, folate, and niacin in biological fluids and food. In the presence of 14C-labelled substrate, the test organisms readily produced 14cc; which was directly proportional to the amount of the respective vitamin present. Sources All vitamin B-12 found in nature has been produced by microorganisms. Since plants cannot synthesize it, B-12 is found primarily in animals products. Animals absorb the vitamin after it has been synthesized by bacteria in their gut from plant food they eat. Excellent sources of B-12 are animal organ meats, especially liver, kidney, and heart which contain 50- 500 mcg per 100 g. Egg-yolk, clams, oysters, and crabs are moderate sources, 5-50 mcg per 100 9, while meats, such as beef, lamb, pork and chicken, and dairy products, are rather poor sources of the vitamin and supply only 0.2-5 mcg per 100 g. Many fermented foods have been reported to contain significant amounts of vitamin B-12. Vitamin B—12 producing bacteria, as a contaminant, grow along with 38 other organisms active during fermentation. 3133313113 pnggmgn13 is the organism in tempeh (Liem gt. 31., 1977), and Bacillus magnum in kimchi (R0 st- 9.1.. 1979) - Dietary intake. The major sources of vitamin B-12 are foods from animal origin. Diets strictly derived from vegetables, although rich in many vitamins, are deficient in B-12. Chung 3t. 31. (1961) reported that low intake of vitamin B-12 related to low-cost diets. As foods derived from animals are more expensive, poor people usually have low intake of the vitamin. People in developing countries consume the vitamin in much lower quantities than do people in developed countries (FAO,1970). Intake level for the majority range from 0.64 to 3 mcg per person per day. The average American diet supplies approximately 5 to 15 mcg of B-12 / day. (Reizentein gt. 31., 1966). Abdulla 3t. 31. (1981) studied nutrient intake and health status of vegans in Sweden and reported that the diets were composed of cereals, fruits, vegetables and fermented vegetable products. Fish, meats and other animal products, such as eggs and dairy products, were absent in the diets. The daily intake of vitamin B-12 was about 0.3 to 0.4 mcg, which is approximately one-tenth of that in normal mixed diets containing animal products. 39 Analysis of the food items, including fermented vegetab- les, yielded vitamin B-12 values between 10 and 70 ng / 100 g wet weight. The small amount of the vitamin may have been due to contamination with B-12-producing microorganisms present in food. Deficiency Deficiency of vitamin B-12 can result from vitamin malabsorption, which is caused either from lack of gastric intrinsic factor or from small intestinal bac- terial overgrowth and nutritional unavailability. Only the deficiency due to nutritional unavailability will be discussed in this part. Severe megaloblastic anaemia due to nutritional unavailability is infrequent in temperate zones, but it is a common cause of maternal death in the tropics (FAO, 1970). No accurate figures are available on the prev- alence of megaloblastic anaemia in different countries. The vitamin B-12 stores in the human body exceed the daily requirements a thousand-fold, which explains the rarity of clinical vitamin B-12 deficiency due to dietary insufficiency. Sanders gt. 31. (1978) reported that vegan subjects who had been on a diet for an average of 7 years had plasma B-12 levels in the normal range. 40 Few cases of vitamin B-12 deficiency have been reported in vegans due to apparent dietary deficiencies. Megaloblastic anaemia due to vitamin B-12 deficiency results either from malabsorption alone or in conjunction with dietary inadequacies in communities where the intake of animal protein is low for economic, religious or other reasons. Dietary inadequacy of vitamin B-12 alone may cause mild deficiency symptoms, but rarely leads to megaloblastic anaemia. Deficiency of folate in combinaé tion with B-12 is more common in pregnancy, when the folate requirement is increased, especially when the turnover of tissue cells increases. The classical example of megaloblastic anemia from vitamin B-12 is pernicious anaemia. In developed countries, B-12 deficiency is very rare. Reported cases came from strict vegetarian groups, especially among infants breast-fed by vegan mothers (Anon, 1979). Strict vegetarian lactating mothers, who had mild anemia, had low values of B-12 in both plasma and milk. The breast-fed infants presented characteristic syndromes which included, in addition to anemia and megaloblastic anemia, pigmentation of skin, apathy, retardation and involuntary movements. All of these signs were corrected rapidly by small doses of B-12. 41 Vitamin B-12 deficiency, usually in association with folate deficiency, is a frequent complication of pregnancy in India and South-East Asia and may lead to vitamin B-12 deficiency in breast-fed infants (FAO, 1970). Deficiency of either B-12 or folate results in defective synthesis of DNA (Waxman gt. 31., 1969). In folate deficiency, the defect in DNA synthesis is the result of inadequate amounts of the vitamin, whereas in B-12 deficiency the defect is due to failure to utilize 5'methyltetra-hydrofolate, which requires B-12 for its conversion back into the folate pool. Allowance Parenteral administration of 0.5 to 1 mcg of the vitamin per day maintains patients with pernicious anemia in complete hematologic remission (Herbert, 1968). More recent turn-over studies in B-12 deficient patients show daily losses of vitamin B-12 in the range of 0.25 to 1.05 mcg/ day (FAG/WHO, 1970). On the basis of these findings, and assuming absorption of fifty percent of quantities of dietary B-12 up to 3 mcg, the recommended dietary allowances for adolescents have been set at 3 mcg/day. 42 Requirement Man and other animals require vitamin B-12. The vitamin is present in animal tissues in the coenzyme form in very low concentration. Methyl cobalamin and 5'- adenosyl cobalamin are the coenzyme form for methionine synthetase and methylmalonyl-CoA mutase, respectively. The average American diet supplies approximately 5 to 15 mcg of B-12 / day. (Reizentein et al., 1966). Parenteral administration of 0.5 to 1 mcg of the vitamin per day maintains patients with pernicious anemia in complete hematologic remission (Herbert, 1968). More recent turn- over studies in B-12 deficient patients show daily losses of vitamin B-12 in the range of 0.25-to-l.05 mcg / day (FAD/WHO, 1970). On the basis of these findings, and assuming absorption at fifty percent of quantities of dietary B-12 up to 3 mcg, the recommended dietary a1- lowances for adolescents have been set at 3 mcg/day. Toxicity Toxicity caused by ingestion of vitamin B-12 is very rare. Ingestion of far in excess of need appears to be without hazard (Goodhart and Shile, 1973). 43 55131991522311; There were poisonings among beer drinkers in Quebec, Canada and Omaha, Nebraska. It was thought that cobalt added for foam stabilization of the beerswas the cause of the poisoning. Cobalt had been used as a foam stabilizer for about two months before the accidents occurred. The consumption of the beer that caused poisoning averaged 24 pints per person per day and contained about 8 mg of cobalt. Cobalt was suspected to have aggravated thiamin deficiency as a result of high alcohol consumption (Morin and Daniel, 1967 and Taskar and Senecal, 1967). Since 1966, addition of cobaltous salts to food in the U.S. has been banned. Foods con- taining any added cobaltous salts are deemed to be adulterated ( 31 F.R. 8788, 1966). MATERIALS AND METHODS MATERIALS The soybean cultivar Corsoy, grown in Michigan, was used for tempeh preparations and as a substrate for vitamin B-12 fermentations, in all of this work. The culture media used are listed in Appendix A. iMicroorganisms Vitamin B-12-producing organisms: ‘3133313113 3333333133, a non-pathogenic strain, was kindly supplied by Dr. K.H. Steinkraus of Cornell University. 33311133 Wm NRRL B-938 and W eliyaseus NRRL B-1125 were obtained from The Northern Regional Research Laboratory, Peoria, Ill. The three organisms were grown and maintained on slants of tryptone-glucose-yeast extract agar (TGY). All cultures were maintained at 4C. Before each experiment, each organism was transferred to a fresh TGY agar slant and incubated at 30C for 48 hr. Bacterial suspensions for inoculation were prepared by harvesting the cells with sterile distilled water. The 44 45 count number of the bacterial suspension was estimated by direct count, using a hemacytometer. The suspension 3 - 104 per ml. Tempeh mold. .Bhizgnus eliggsngrns NRRL 2710. was obtained from the Northern Regional Research Laboratory, was diluted to adjust the count to 10 Peoria, Ill. The culture was maintained on potato dextrose agar (PDA) slant at 4C. The organism was transferred to a Sabouraud-dextrose agar slant (Difco) and grown at 30C for 5 days before it was used for tempeh making. The mold spore suspension was prepared by adding sterile distilled water into the slant. The suspension was subjected to hemacytometer spore count and the dilution was made to adjust the count to 102 - 103 spores per ml. The cobalamin assay organisms, Lastgbasillns leishmanii ATCC 7830 and 99hr9mgna§ 33133333313 ATCC 11532 were purchased from American Type Culture Collection, Rockville, Maryland. L.1313333311 was maintained in a stab culture on vitamin B-12 culture agar (Difco) at 4C The culture was transferred twice in L33t333311133 medium broth (Difco) and incubated at 30C for 24 hr before it was used for vitamin B-12 analyses. 3.33133333313 ATCC 11532 was maintained in single strength 3333333333 liquid medium, to which 0.2 ng vitamin B-12 per ml was added. The organism was transferred in this medium in 5-7 day intervals and 46 incubated at 27C, 1 foot below a 60 W lamp. Laboratory animals Fifty male albino rats, age 21 days, which were provided by the Harlan Sprague Dawley Company, were used in estimating the Protein Efficiency Ratio ( PER ) of the tempeh samples. METHODS Total counts The number of bacteria and mold in tempeh samples were estimated by means of total viable counts. One g of sample was weighed and placed in a sterile 2 x 25 cm test tube and homogenized (Brickman homogenizer) in 9 ml sterile water at medium speed for one minute. The slurry was then decimally diluted. Measured amounts of the diluent were added into empty, sterile plates and melted PCA medium (45C) was added into the plates. After the medium solidified, the plates were incubated at 30C for 24-48 hr, the colonies were counted on the plate containing the highest dilution in which 30 to 300 colonies appeared. The estimated count was the count of colonies in the plate multiplied by the dilution factor. In order to avoid overlapping mold and bacteria colonies, mold counts were conducted on acidified PCA 47 medium and the bacteria count on PCA containing 0.1% NaCl. The number of bacteria and mold in inoculum suspension was estimated utilizing a hemacytometer. DETERMINATION OF COBALAHIN The apparent vitamin B-12 in this study was determined by a microbiological method employing ‘L333333311133 1313333311 ATCC 7830 (AOAC, 1980). The "true vitamin B-12" activity was determined utilizing 3333333333 33133333313 according to the Analytical Methods Committee of the Society for Analytical Chemistry (1965). Extraction of cobalamin Two extraction solvents, bisulfite extracting solution (containing 1.3 g of NaZHPO4, acid and 1 g of anhydrous sodium metabisulfite, per 100 1.2 g of citric m1) and sodium cyanide aqueous solution (1%), were compared. The method using sodium cyanide, which produced higher results, was chosen for extracting vitamin B-12 as preparation for both microbial assays. A l g of sample was weighed and put into a 2 x 25 cm test tube. Into the tube, 30 ml of distilled water and 0.1 ml of freshly prepared 1 % aqueous NaCN solution were 48 added and homogenized (Brickman) at medium speed for 1 min. The pH was adjusted to 4.6 - 5.0 with 1 N HCl addition. The mixture was let to stand at room tempera- ture for 30 min with occasional shaking and its pH was readjusted, if necessary. The flask was then placed in a boiling water-bath and let to stay there for 30 min after the mixture temperature reached 90C. The flask was cooled, the mixture was transferred into a 100 ml volumetric flask and the volume was brought up to mark with water. The extract was clarified by centrifugation and the clear supernatant was designated as assay solu- tion ready for the assay. Vitamin B-12 assay utilizing Lngignggnii The medium utilized in the test :35 B-12 Assay Medium USP from Bacto. This medium is free from vitamin B-12 but contains all the other factors necessary for the growth of L.1g1gnmgnii ATCC 7830. Addition of vitamin B-12 to the medium in specified increasing concentrations gives an increasing growth response by the test organism which was measured by titration with 0.1N NaOH. Standard solutions A 100 ng/ml stock cyanocobalamin standard solution was prepared in 25% alcohol. An intermediate solution 49 (1 ng/ml) was prepared by diluting the stock solution. Both solutions were then stored in darkness at 4C. For each assay, a working solution of 0.02 ng/ml was freshly prepared. Inoculun Cells from a stock culture of L.1gi§nmanii were transferred into a sterile tube containing 10 ml liquid culture medium. The tube was incubated at 30C for 24 hr. The culture was then aseptically transferred into a sterile centrifuge tube, centrifuged for 15 min and the supernatant was decanted. The cells were washed 3 times with 10 ml of sterile 0.9% NaCl solution and finally were suspensed in the NaCl solution and designated as inoculum. The inoculum suspension was barely cloudy. Assay tubes L.1gighmanii is very sensitive to minute amounts of growth factors and to many detergents. The tubes for the assay were meticulously cleaned with sodium lauryl sulfate and then filled with distilled water and steamed for 2 hr. Duplicate tubes containing appropriate amounts of a standard cyanocobalamin solution were prepared as fol- lows: 0.0 ml (for uninoculated blanks), 0.0 (for inocu 50 lated blanks), 1.0, 2.0, 3.0, 4.0, and 5.0 ml of standard solution was added. An appropriate triplicate amount (1.0-4.0 m1) of assay solution was put in similar test tubes. To each tube containing standard solution or assay solution, distilled water was added to make 5.0 ml. Five ml of basal medium stock solution was then added to each tube. The tubes were plugged and sterilized at 121C for 10 min and then cooled. Aseptically the tubes were inoculated with 1 drop of the inoculum. The tubes were incubated up to 72 hr, and the content were titrated with 0.1N Na OH to pH 6.8. Determination A concentration-respond standard curve was prepared by plotting titration values, expressed in ml of 0.1N NaOH, against amounts of cobalamin in the reference tubes. The amount of vitamin B-12 in each test sample tube was determined by interpolation from the standard curve . Assay of "true vitamin B-12' using O.ma1hamensis Sample extracts containing about 0.5 ng of vitamin B-12 per ml were added into test tubes. To each of these test tubes was added 1 ml of 5 X strength 9.3g1hgmgngig 51 medium and 3 ml of water. The tubes were sterilized at 121C for 10 min. One drop of a 5-day culture was used to inoculate each assay tube. Great care was given to maintain organisms uniformly suspended during inOcula- tion. Following inoculation, the tubes were placed in a shaking machine at 30C and shaken in darkness for 72 hours. The tubes of assay cultures were then steamed at 100C for 5 min and 5 ml of water was added to each tube. Growth was determined spectrophotometrically. A plot of concentration-respond standard curve was constructed each time an assay was run using the same medium with levels of 0.0, 0.05, 0.1, 0.2, 0.4, and 0.8 ng of vitamin B-12 per tube. Proximate analysis Protein determination was performed by micro- Kjeldahl according to AOAC (1970). A factor of 6.25 was used to convert nitrogen content to protein. Crude fat was determined by extracting 2 g of finely ground dried sample with diethyl ether in a Goldfisch extraction apparatus (Labconco) for 4 hr and the extract was dried at 100C for 30 min, and weighed. Ash and Moisture were determined according to the AOAC methods (1970). Car- bohydrate was calculated as the difference of 100 and the total of moisture, protein, fat, and ash. 52 Inhibitory test This test was designed to examine whether the metabolites produced by mold or vitamin B-12 producing bacteria inhibit the growth of assay organisms. Each type of the vitamin B-12-producing bacteria and the tempeh mold were grown on sterilized boiled-soybeans and incubated at 30C for 36 hr. After the incubation, each sample was extracted by the vitamin B-12 extraction procedure and the extract was diluted with water 100- fold. Vitamin B-12 assays were conducted for the follow- ing samples: (1) 1 ml of the diluted extract alone, (2) 1 ml of the diluted extract + 4 ml of 0.02 ng/ml of vitamin B-12 solution, and (3) 4 ml of 0.02 ng/ml of vitamin B-12 solution (as control). Comparison of the results allowed a judgement whether there was inhibition. EXPERIMENTS Effect of temperature One hundred g portions of sterilized soybeans- (pH 6.5) were separately inoculated with each of the three vitamin B-lZ-producing bacteria and incubated at 30C, 35C, and 40C for 36 hr. Samples were taken at the end of the incubation for bacterial plate counts and vitamin B-12 analysis. 53 Effect of time Each 100 g portion of sterilized soybeans was inoculated with each one of the vitamin B-lZ-producing bacteria and incubated at 30C for 72 hr. Samples were harvested every 12 hr and were subjected to bacterial counts and vitamin B-12 assays. Effect of pH Due to the difficulty of adjusting the pH of soybean substrate, the effect of pH was studied by growing the vitamin B-lz-producing bacteria on Tryptose- glucose-yeast extract agar slants. The media were ad- justed to pH of: 4.5: 5.0; 5.5; 6.0; 6.5 and 7.0 by adding 1 N of HCl or 1 N of NaOH. Nine tubes were inoculated with each bacterium and then incubated at 30C for 48 hr. Tubes showing visible growth were considered positive. The effect of pH was expressed as the fraction of positive test to total tubes. Effect of soaking and acid addition In the traditional method, soybeans are soaked overnight or longer before dehulling, while in the pilot plant method, instead of soaking overnight, vinegar or lactic acid is added to acidify the soybeans. 54 Three samples, designated as soaked, acid-added, and control were prepared as follows: A 100 g sample of soybeans was soaked overnight in 300 ml of distilled water and about 25 ml of soak-water from a previous batch was added. The next morning, the soaking water was discarded, the soybeans were boiled for 30 min in dis- tilled water, and then dehulled. The soybeans were designated as soaked soybeans. The acid-added sample was prepared by soaking 100 g of soybeans in 300 ml 0.9% lactic acid overnight at room temperature, the acidified water was then drained from the beans, the beans were peeled and the skins were separated. The skinned beans were returned to the acidified soak water and boiled for 30 min. At the end of boiling the water was drained and the beans were allowed to cool and surface-dry. At this stage the lactic acid content of the beans was 0.9-1.2 g/ 100 g and the pH was 5.2-5.3. Controls were prepared by soaking the soybeans in tap water for 1 hr to hydrate the soybeans followed by boiling them for 30 min in distilled water. The soybeans were dehulled and designated as control. The three samples, soaked, acid-added and control, were then sterilized and subjected to mixed-culture fermentation. The tempehs were analyzed for vitamin B-12 content and mold and bacterial counts. 55 Effect of cobalt and acid addition The effect of cobalt and acid addition on bacterial growth were studied in a factorial design as shown in Appendix B. Solution of cobalt chloride (CoClz) (10 ug Co++ per ml) and lactic acid (0.9%) were prepared and 1 ml of each solution was added to 100 g of sterilized soybeans fitting the design. The pH of the acidified soybeans was 5.0 and that without acid was 6.0. The soybeans were then inoculated with vitamin B-12- producing bacteria and incubated at 30C for 2 days. At the end of the fermentation, the soybeans were subjected to bacterial counts and vitamin B-12 analysis. Effect of soaking and mixed culture The effect of soaking and concurrent mold growth on the bacterial growth and vitamin B-12 production were studied in a factorial design as shown in Appendix C. Two samples, designated as soaked and unsoaked soybeans were prepared as follows: A 100 g of soybeans were soaked overnight in 300 ml of distilled water and about 25 ml of soak-water from a previous batch. The next morning, the soaking water was discarded, the soybeans were boiled for 30 min in distilled water. The soybeans were dehulled and designated as soaked soybeans. The unsoaked soybeans were prepared by soaking the soybeans in distilled water only 56 for 1 hr to hydrate the soybeans followed by boiling them for 30 min in distilled water. The soybeans were dehulled and designated as unsoaked soybeans. For mold treatment, each sample was divided into two portions. One portion was inoculated with 1 ml of mold-spore suspension and the other was given 1 ml of distilled water (without mold). The four samples were inoculated with vitamin B-12- producing bacteria and incubated at 30C for three days. At the end of fermentation, the samples were subjected to bacterial counts and vitamin B-12 analysis. Starter preparation In a preliminary study, rice-based starters were prepared according to the method of Wang gt. a1. (1975). It was found out that when mixed cultures of the mold and bacteria were inoculated on sterile rice, the tempeh mold was overgrown by bacteria. The result did not show a good mold-spore formation as when the mold grows alone on rice substrate. Mixture of rice and soybean (1:1) substrate did not improve the growth of the mold in the mixed culture. The mixed-cultures were then grown on sterilized soybean substrate. A 100 g of boiled soybeans were put in 400 ml beaker, covered by aluminum foil and sterilized at 15 lb 57 for 30 min. After cooling, the soybeans were inoculated with 1 ml of bacterial suspension and 1 ml of mold suspension. The cultured soybeans were lightly packed in Petri dishes and incubated at 30C for 6 days. After incubation, the soybean mass was freeze-dried and ground into powder. The powder were designated as a starter. Each starter was divided into two parts: one part was stored at room temperature and the other in a refrigera- tor (2-4C). For evaluation of the starter, once a month samples were taken for mold and bacterial counts and for examin- ing their performance in tempeh fermentation and vitamin B-12 production. Determination of "true vitamin B-12' "True vitamin B-12" was determined employing Q.mgln§mgn§i§. The determination of the true B-lz was performed along with the determination of apparent vitamin B-12 employing L.lgighmanii which serves as control. 58 DETERMINATION OF PROTEIN QUALITY Protein Efficiency Ratio The Protein Efficiency Ratio (PER) was determined according to the AOAC method (1980). Five diets were prepared using the following protein sources: 1. Casein (reference protein) 2. Tempeh fermented with mold 3. Tempeh fermented with mold 4. Tempeh fermented with mold 5. Tempeh fermented with mold All diets were standardized to meet position: Protein Fat/oil Salt mix USP 10 % 8 % 5 % only (as control) + S-oliyageus + E-mmgnia + B-megaterim the following com- Vitamin mix = 1 % Cellulose = 1 % 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 were from United States Biochemical Corporation, Cleveland, Ohio. Fifty male rats, age 21 days and average weight of 44 g, were used in the assay. The animals were kept in individual cages, and allowed to acclimate for 4 days on rat chow diet. At the beginning of the test, each ten 59 rats were randomly assigned into one group of treatment and both diets and water were given ad 11bitgm. The experiment was terminated after 28 days from the beginn- ing of the assay, during which time body weight and diet consumed by individual rats were recorded every 4 days. The PER for each group were calculated as the ratio of weight gain per protein intake. Corrected PER were calculated by multiplying the PER of treatments with 2.50/PER of casein. Amino acid analysis Amino acid composition was determined according to the HPLC method of Cohen gt. a1. (1986). Tempeh sample was hydrolyzed in 6N HCl at 110C for 24 hr. The hydroly- sate was cleared by filtration. The sample was dried under vacuum. The sample derivatization reagent consisted of ethanol-triethylamine (TEA)-water7phenylisothiocyanate (PITC) (7:1:1:1) and was made fresh daily. Phenyl- thiocarbamate (PTC)-amino acids were formed by adding 20 ul of the reagent to the dried samples. The mixtures were then sealed in vacuum vials for 20 min at room tempera- ture, in which time the reaction of free amino acids with the PITC was completed. The reagent was then removed 60 under vacuum. A model ALC 204 liquid chromatograph (Water Assoc.), which consisted of two solvent delivery systems, model M600A, a fixed-wavelength UV detector (254 nm), model M440, and a sample auto-injector, model M7103, was used. Amino acids were separated by a 15cm x 3.9 mm Pico- Tag analytical column. The solvent system consisted of (A) an aqueous buffer and (B) 60 % acetonitrile in water. The buffer (pH=6.4) was 0.14 M sodium acetate containing 0.5 ml TEA /L of Na acetate solution. A chromatogram of amino acid standards is shown in Figure 3. Tryptophan analysis The determination of tryptophan was performed spectrophotometrically according to the method of Spies (1967). A sample containing 3 mg of protein was weighed in a 5 ml screw-cap type vial. The vial was placed at 4C overnight. A 100 mg of pronase was weighed into a 15 ml centrifuge tube and then placed at 4C overnight. Ten ml of 0.1M sodium phosphate buffer (pH 7.5) was added to the pronase and was shaken gently for 15 min. The mixture was centrifuged to obtain a clear solution. A 0.1 ml of the pronase solution was added into the sample vial and the mixture of sample and enzyme was incubated at 40C for 24 hr. Blanks, without sample, were 61 prepared along with the samples and used to determine the tryptophan content of the enzyme. After incubation, each sample was cooled in ice and a 0.9 ml of sodium phosphate buffer (pH 7.5) was added to each vial. After the content of the vial was mixed, it was put into a 50 ml stoppered flask containing 30 mg p-dimethylaminobenz-aldehyde (DAB). Nine ml of 21.2 N sulfuric acid was added into the flask. The flask was gently swirled and then placed in the dark for 6 hr at 25C. A 0.1 ml of 0.045% Na-nitrite solution was added into the flask. After it was let to stay for 30 min, the % transmittance (%T) of the mixture was measured at 590 nm. The regression line of tryptophan concentration vs log of transmittance is described as follows: Y = 1.9807 - 0.0048 X and r = 0.9954. The standard curve is presented in Figure 4. Sensory evaluation Triangle tests were performed to determined the taste difference among tempehs prepared with mixed cultures and regular tempeh. A sample of the question- naire is presented in Appendix D. 62 Statistical calculation Statistical calculations were conducted using the "Statgrafic" statistical computer program. WII . 63 *1 D— Figure Eluent Eluent ml/min. UV 254 3. A: 0.14M sodium acetate, B: 60% acetonitrile in water; Column nm. Identity of peaks: 1. Asp 7. Thr 13. Met 2. Glu 3. Ser 4. Gly 8. Ala 9. Pro lO.--- l4. Cys 15. Ile l6. Leu 5.. SEPARATION OF AMINO ACID STANDARDS 0.5 ml TEA, pH 6.4 flow—rate: 1.0 Pico-Tag analysis column; detector: 5. His 6. Arg 11. Tyr 12. Val 17. Phe 18. Lys IOO 53 8 ‘Cmosilbn ol 3 64 0.6 Y - 0.015 + 0.004 X r - 0.9978 0.5 - 0.4 ~ 0.3 - 590 02 OJ ' l_ 1 _L I J o 20 40 so so 100 120 140 Tryptophan (meg) Figure 4. Standard curve for tryptophan determination 65 RESUDTS AND DISCUSSIONS Inhibitory test The recovery of the analysis of vitamin B-12 in fermented-soybeans extract was presented in Table 2. All tests, four samples and two methods, gave recovery approaching 100%. Beck (1983) suggested that recovery less than 80% indicates the presence of inhibitor. The extract of the fermented soybeans, which contain metabo- lites of the vitamin B-12-producing bacteria, did not contain inhibitor that interfere with the vitamin deter- mination. Effect of Temperature The effect of temperature on bacterial growth and vitamin B-12 production on heat treated soybeans is presented in Tables 3 and 4, respectively. The total count and the vitamin content of the cultured soybeans were significantly different at the three selected temperatures, 30C, 35C and 40C. At 40C all bacteria grew more slowly and produced less vitamin than at the other two temperatures. 65 Table 2. 66 Recovery of vitamin B-12 activity in fermented-soybean extract assayed by Lleishmanii and Q malhamensis Vitamin B-12 activity (ng/g) and recovery (%) by L-leishmanii by Q-malbamensis (Avgis.d.) Rec. (Avg:s.d.) Rec. Regular tempeh B-12 Std 0.08:0.012 0.30:0.010 Extract 0.00:0.009 0.00:0.002 Extract + B-12 Std 0.09:0.010 113 0.31:0.014 103 K-nnenmgnia-tempeh B-12 Std 0.08:0.012 0.30:0.010 Extract 0.17:0.026 0.00:0.052 Extract + B-12 Std 0.27:0.035 108 0.3010.076 100 B-megaterium-tempeh B-12 Std 0.08:0.012 0.30:0.010 Extract 0.30:0.045 0.34:0.042 Extract + B-lz Std 0.3910.041 103 0.62:0.049 97 S-Qlixaeeus-tempeh B-12 Std 0.08:0.012 0.30:0.010 Extract 0.52:0.062 0.01:0 Extract + B-lZ Std 0.59:0.076 98 0.31:0.006 100 67 Table 3. Effect of incubation temperature on the total count of B-12-producing bacteria grown on heat-treated soybeans for 2 days. (avg. of logs) Organism 30C 35C 40C K-eneumgniae 6.7 7.2 6-5 B-msgaterium 6-6 6.9 6-5 §.9111a§gg§ 5.9 5.7 5.0 Table 4. Effect of incubation temperature on the B-12 production by bacteria grown for 2 days on heat-treated soybeans (ng/g (d.b.). (Avg i s.d.) Organism 30C 35C 40C K.pnggmggia§ 46.9il.35 53.0:2.51 43.212.08 B.m§g§§g;igg 79.212.37 88.013.53 79.912.49 §.gliyg§gg§ 146.316.75 86.814.46 41.415.68 68 At 35C 3.9nggmgnigg and B.mgg§§grigm grew better than at 30C. At the temperatures of 30C and 40C, the growth of the two organisms were statistically not different. The growth of 5.911yggggg at 35C and 40C were significantly different, but at 30C and 35C were not different statis- tically. The temperatures for maximum growth of the three organisms agree with the optimum temperatures of the three species cited in Bergey's Manual. The fi.gliy§ggg§ count at all tested temperatures was significantly lower than that of B.mgg§tgrigm, and K.pnggmgnige. This organism, which also has the highest potential of vitamin B-12 production, would be the preferred organism for mixed culture with tempeh mold as the bacterium might not compete excessively with mold. B.m§g§;grigm is the second best vitamin B-12 producer. Its growth and vitamin production was slightly affected in the range of tempera- ture encountered in the tempeh fermentation. There were significant differences in the B—12 production by different bacteria and at different temp- eratures. Incubation at 35C significantly enhanced the B-12 production by §.pgggmgnig§ and §.meg§terigm. Production of the vitamin by the same bacteria at 30C and 40C were not different statistically. §.gliy§§gg§, which produced the highest level of vitamin B-12 among the three bacteria, synthesizes the vitamin much better 69 at 30C than at the other two temperatures. Effect of pH The effect of pH on the growth of vitamin B-12- producing bacteria is presented in Table 5. All of the tested bacteria grew well in the range of pH 5.5-7.0. While x.pnggmgni§g and B.m§gatgzigm grew very well in the pH range of 5.0-7.0 and $.9112agggg grew well only in the range of 5.5 to 7.0.' Table 5. Growth of B-12-producing bacteria on tryptose-glucose-yeast extract agar at different pH levels. Organism 4.0 4.5 5.0 5.5 6.0 6.5 7.0 §.pnggmggia 0/9 2/9 9/9 9/9 9/9 9/9 9/9 B-megaterium 0/9 3/9 9/9 9/9 9/9 9/9 9/9 5.911yggggg 0/9 0/9 2/9 9/9 9/9 9/9 9/9 *) Total No. of positive tube / replication. The pH range for tempeh fermentation was reported to be 2.5 to 7.0 (Steinkraus, 1960). Traditional tempeh preparation with soaking, lowers the pH of soybeans to 4 - 5, while acid addition in the pilot-plant procedure 70 reduces the pH of the soybean even lower. It has been proved that tempeh mold can grow on cooked-unacidified soybeans. Steinkraus (1983) suggested that tempeh manufacturers acidify the beans before fermentation to prevent the growth of toxic-producing organisms. Regarding the pH requirement, B.mggatgrigm and K.pnenmgni§g seem to be better choices than 5.9112gggg5. Organisms growing at lower pH can start producing the vitamin earlier during the tempeh fermentation. B.mgggt§rigm is clearly more promising than K.pnggmgniag since the Bacillus produces more vitamin than the Klgbsiglla. §.gliy§ggg§, which has higher pH require- ment, would grow late in the process of tempeh fermenta- tion, perhaps waiting for the pH to rise. Furthermore when the soybean pH increases, the temperature also increases away from the optimal temperature of 5.9112ggggg. However, §.Qliygggg§ is more powerful vitamin B-12 producer that the other two organisms. Effect of Time The effect of time on the growth of vitamin B-12- producing organisms and the production of vitamin B-12 is presented in Table 6. When grown on heat-treated soybean at 30C, vitamin B-12 was detected after one day of fermentation and the B-12 content still increased when 71 the study was terminated after three days. 5.9;iyggggg produced the highest level of vitamin B-12 during the three-day test period, followed by B.mggg§grigm and then SW. The vitamin B-lZ-producing organisms grew logarith- mically, while the vitamin contents increased linearly during the 3-day fermentation. When vitamin content at four observation points was plotted against fermentation time, the vitamin production by the three organisms were best expressed in regression lines as shown in Figure 5. The slope, which represents the vitamin B612 production rate, shows that $.911239ggs is superior compared to the other two organisms when grown on soybean substrate at pH 6.6 and at 30C. The rate ratio is : K : B : S = 28 : 45 : 90. The slow growth (and vitamin production) in the first day of fermentation is probably due to the lag phase of the bacteria. The tempeh fermentation usually lasts from 20 to 48 hours. After 48 hours, off odor develops and the tempeh is not fit for consumption. Vitamin B-12 fermen- tation by 5.9111ggggg for 1 day produced vitamin up to 50 ng/g dry soybeans. Lengthening the fermentation time by 1 day increased the vitamin to 150 ng/g dry soybeans. The other two bacteria only doubled the vitamin content when the fermentation was prolonged from 24 hours to 48 hours. 72 Table 6. Total count of vitamin B-lZ-producing organisms and vitamin B-12 production during a 3-day fermentation of soybeans Vit.B12 (ng/g d.b.) Organism Time Count (Day) (Avg of Log) (Avg 3 s.d) §.pn§umgnig 0 1.9 5.1 i 0.06 1 3.8 23.6 i 1.14 2 5.4 52.6 t 1.54 3 8.8 89.0 i 5.25 B.mggatg;igm 0 1.9 9.1 i 0.03 1 4.7 36.7_i 4.13 2 7.1 83.4 i 5.47 3 9.2 144.4 i 8.87 §.91iygggg§ 0 1.6 9.9 i 0.77 1 3.6 51.8 i 2.16 2 5.6 144.2 i 9.41 3 7.0 277.5 110.59 The B-12 values under 10 represent negligible amount. 73 1° , 300 K Y-to+2.2X r-aoau) O~B Y-2.1+2.4x nation '3 :':;:’:xx "098" 8 Y-LTOLBX 7.0.8968 ' " ”0.9856 8_ 260-8 Y--18.43900X r-0.0749 » 8 //// 7. ‘ B 200‘ _ °r v / rv-DCOO § OI I I no I (D ~— -‘ .a “3 8 l \‘ tn / . ‘ .% 0 1 2 8 O I 2 3 Time (day) Time (dew Bacterial growth B-12 production Figure 5. Trends of bacterial growth and B-12 production during a 3-day fermentation. (K = 5.9neumoniae B = §.megaterium and S = §.olivaceus) 74 Effect of Tempeh Preparation Certain steps of the tempeh preparation may have an effect on the growth and vitamin B-12-producing capabili- ties of the tested organisms. Such preparation steps are: soaking (lactic fermentation), concurrent mold growth (tempeh fermentation) and acid addition. Cobalt, an element of vitamin B-12, is added to enhance the vitamin synthesis in commercial vitamin B-12 production. In the following experiments, the interaction between the two fermentations (lactic and tempeh) and between two added substances (acid and cobalt) are studied. The effect of soaking and acid addition Soaking soybeans overnight or longer facilitate the growth of tempeh mold. During soaking, lactic fermenta- tion acidifies the soybeans from neutrality down to pH 4. According to Steinkraus (1960) replacing the soaking treatment with acid addition produces the same result. In our experiments, soaking never acidified the beans to pH 4.0. Even by adding soak water from a previous soaking operation did not lower the pH to below 5.5. The effects of soaking and acid addition to soybeans on the growth of vitamin B-lZ-producing bacteria and vitamin B-12 production are presented in Table 7. Acid addition diminished the growth of §.pgggmggiae and 75 B.mgga§g;igm, and fully stopped the growth of §.gliyag§g§. It also inhibited the production of vitamin B-12 by the three organisms. Soaking of the soybeans did not significantly affect the growth of K.pnggmgniag, but it significantly reduced the growth of B.mgg§§grium and fi.gliyaggg§. Soaking slightly increased the production of vitamin B-12 by K.pn§gmgnia. However, soaking reduced the production of the vitamin by B.mggatgrium and by Selim Table 7. Growth of vitamin B-12-producing organisms and Vitamin B-12 production on soaked (S), acidified (A) and control soybeans (C), at 30C for 2 days. Organism Tmt Count Vit.B-12(ng/g d.b.) (Avg of Logs) (avg.¢s.d.) K.pgggmgni§g C 7.0 47.2 i 1.15 S 7.2 64.4 i 4.07 A 2.8 11.8 i 1.23 B.mggatgrigm C 7.3 81.2 i 2.17 S 7.0 72.8 i 4.76 A 2.9 11.2 i 1.22 S. 'va 3 C 6.2 159.5 i 6.07 s 5.7 81.4 i 1.30 A 1.6 11.2 i 1.21 76 The previous experiment (effect of pH) did not show that pH 5.5 suppresses the growth of B.mgg§§grium and $.gliyagegs. The growth of the natural flora during soaking may consume nutrients or produce metabolites which adversely affect the growth of B.mgg3§§rigm and fi.gliy§g§g§ but not that of K.pnggmgniag. This experi- ment was followed by an attempt to explore whether B.nggg§gzigm and 5.9113gggns were inhibited by products formed during soaking. The two organisms were streaked on (1) soaked-soybean extract agar tubes and on (2) soaked-soybean-extract + Tryptose-Glucose-Yeast extract (TGY) tubes. The extract concentration was the same on both media. Visible observations showed B.mggatgzigm_and §.glig§g§g§ grew well on the extract + TGY, but they grew poorly on the extract alone. It was clear that the inhibition was not because of adverse metabolites formed during soaking. Rather an element that is critical to the growth of $.9112agggg and B.mggatgrinm was depleted during soybean soaking. Supplying the element by adding tryptose-glucose-yeast extract to the medium restored the growth of 5.9112ggggs and fi.m§ggtg;igm. Longer period of fermentation reStores the growth of these two vitamin B-lZ-producing bacteria and the vitamin B-12 production. It seems that both organisms might have long 77 lag-phase (as indicated in the regression lines) that differentiate the growth of both organisms on soaked and unsoaked soybeans. Acid addition inhibits the growth of the tested organisms since it lowers the pH of the substrate beyond their tolerance limits. The effect of Co++ and acid addition The effect of cobalt and acid addition are presented in Table 8. The effect of cobalt addition to the soybean substrate on bacterial growth and vitamin B-12 production was not significant, while the lactic acid addition strongly reduced the growth of the bacteria. The cobalt addition did not reduce the strong effect of the acid addition to the bacteria. Effect of soaking and concurrent growth with tempeh mold The effect of soaking and mold presence on bacterial growth and vitamin B-12 production by vitamin B-lZ-producing bacteria is presented in Table 9. In a three-day fermentation, soaking of the soybeans clearly enhanced the growth of E.pnggmgnigg and the vitamin B-12 production by this bacterium, but did not significantly affect fi.m§gatgrium, and diminished the growth and vitamin B-12 production by §.olivaggus. In concurrent 78 growth with the mold. S-glixaeeus and Benegaterium grew more poorly and produced less vitamin B-12 compared to growing in the absence of the mold. K.pn§gmgniag did not appeared to be greatly affected by the presence of mold. S-elixaseue produced the largest quantity of B-12 both in the absence or presence Among the three bacteria, of the mold. Table 8. Bacterial counts and vitamin B-12 productions on soybeans treated with acid (A) and cobalt (C) A C Count Vitamin (ng/g d.b.) (Avg.of Log) (Avgis.d.) Control + - 1.9 9.90 i 0.04 + + 1.5 11.60 i 0.37 - - 2.1 11.87 i 0.45 - + 2.4 12.53 i 0.09 E.pgggmggig§ + - 2.7 11.40 i 0.24 + + 2.7 11.03 i 0.66 - - 8.0 47.27 i 2.71 - + 8.6 45.17 i 3.69 B.mgg§;g;igm + - 2.8 11.50 i, 0.45 + + 2.1 11.17 1 0.69 - - 7.8 63.03 i 4.87 - + 8.4 56.73 i 4.76 fi.glig§ggg§ + - 2.2 10.93 i 0.62 + + 2.4 10.97 i 0.26 - - 7.0 108.73 1 9.54 - + 7.2 116.63 1 13.72 The low values of the vitamin (up to 10) represent negligible amount. 79 On unsoaked soybeans, the growth and the vitamin production by K.pnggmgnigg was slightly better in the presence of B.Qligg§pgrgg. However, the enhancement was not as great as that from soaking. It is more likely that the symbiosis, as reported by Steinkraus (1983), may occur as a result of soaking rather than concurrent growth with the mold. Combination of soaking and concur- rent mold growth indicates that only soaking has a favorable effect on the growth and vitamin B-12 produc- tion by K.pnggmgniag, while the relationship between the bacterium and the mold appears to be rather competitive. The concurrent mold growth slightly suppressed the growth of B.mgg§§gzign and _§.Qliyaggg§. The combination of soaking and concurrent mold growth intensify the depletion of nutrients necessary for both organiSms. As a result, both growth and vitamin production decrease considerably. "True vitamin B-12" The vitamin B-12 activity of tempeh measured by Q.m§1n§mgn§i§ and L.lgignmanii are presented in Table 10. The vitamin B-12 activity measured by L.L§1§hm§nii showed that mixed-culture of mold and §.olivagegs produced the highest amount of vitamin B-12 followed by §.m§gatgrigm 80 Table 9. Effect of soaking (S) and mixed-culture (M) on total count of Vit B-12 bacteria and vitamin B-12 content (ng/g d.b.) of soybeans fermented for 3 days. Organism S M pH Bact Count Vit B12 (Avg.log) (avg.is.d.) 53.2 143.9 60.7 102.1 K-nneumeniae + I+ I I Lbbm +-+ H4+H4+ HO‘UIIh UIONUI hNO‘P homegateriun 156.7 155.5 102.6 24.6 000th W‘DUI-b + I+-I I I+F+H4+ -I~I0I chum fi-elixaeeus 392.9 256.2 256.2 43.4 hfldm #000 DWOQ p +~+ mmUlOt mounds GOtUIOI O... U'IUHN UIO‘ON \lem + I+-I +-+ II 888. I+P+H4+ Table 10. Vitamin B-12 activity of tempeh measured by Q.malhamen§i§ and L-leighaaaii (nq/q d-b-) 0 malbaaensis L-leishmaaii (Avg i s.d.) (Avg 1 s.d.) K.pneumogiae 0 63.6 i 2.86 B.mgg§tgrigm 5.15 i 0.10 88.1 i 5.01 §.Qli!§§§g§ 1.04 i 0.18 176.4 i 5.60 81 and B-nnmeniae- However. Q-malhemensis. the more specific organism, indicates that B.mgggtgrigm_produces more "true vitamin B-12" activity than §.gliy§ggg§_while tempeh produced by mixed-culture of mold and E.pn§ymgnia§ did not show any vitamin B-12 activity. The discrepancies between the vitamin B-12 activi- ties measured by the two methods might be attributed to the presence of vitamin B-12 analogs or precursors. The synthesis of vitamin B-12 in the commercial production takes much longer time. Cobalt and 5,6, dimethyl benz- imidazole is added late in the fermentation to suppress the amount of vitamin B-12 analogs. This experiment also elucidated the inaccuracy of vitamin B-12 analysis employing L.1§ignmanii, especially when a great quantity of analogs is present. Storage study of starters The results of storage study of the mixed culture starters are presented in Table 11 and 12. Storage of starters markedly reduced the viability of the vitamin B-lZ-producing organisms. The reduction of both bacterial count and the capability to produce vitamin B-12 markedly decreased after one month storage, both at room and refrigeration temperatures. The bacterial count and the vitamin production capability continued to decrease 82 Table 11. Vitamin Production (ng/g d.b.), Bact. & Mold Counts of Mixed-Culture Tempeh Starters stored at Room Temperature (23C) T O Bact.Count Mold Count Vit. B-12 (Avg of log) (Avg of log) (Avgis.d.) 0 K 8.0 6.3 63.6 i 2.86 1 K 6.1 5.1 40.1 i 4.00 2 K 5.3 4.7 35.1 i 1.99 3 K 4.7 4.7 32.8 1 1.79 4 K 4.4 4.3 30.2 i 1.45 0 B 8.7 5.6 88.1 i 5.01 1 B 5.7 4.9 51.5 1 0.98 2 B 4.9 4.4 29.4 1 6.41 3 B 4.3 4.3 15.3 i 4.95 4 B 4.1 4.1 14.2 i 4.69 0 S 5.3 6.4 178.1 i 4.10 1 S 4.4 5.4 59.5 i 9.57 2 S 3.8 4.9 27.4 i 4.90 3 S 3.1 4.5 16.3 i 3.21 4 S 3.1 4.2 14.6 i 4.29 T=storage time (month): O= B-l2 organisms; blew ktw: S=§oelixaseu_- presented in AverageiS.D. 83 Table 12. Vitamin production (ng/g d.b.), bact. & mold counts of mixed-culture tempeh starters stored under refrigeration (4C) T O Bact.Count Mold Count Vit B-12 (Avg of log) (Avg of log) (Avg i s.d.) 0 K 8.0 6.3 63.6 i 2.86 1 K 5.2 5.3 35.1 i 7.04 2 K 4.5 5.2 27.0 i 1.96 3 K 4.3 4.9 19.1 i 4.26 4 K 4.2 4.9 18.1 i 3.08 0 B 8.7 5.6 88.1 i 5.01 1 B 7.2 5.2 81.6 i 4.10 2 B 6.2 5.0 67.3 i 2.03 3 B 5.7 5.0 51.9 i 2.70 4 B 5.5 4.8 49.1 i 0.73 0 S 5.3 6.4 176.4 1 5.60 1 S 4.7 5.5 96.6 i 4.20 2 S 4.4 5.2 67.5 i 4.82 3 S 4.2 4.9 41.0 i 4.41 4 S 4.2 4.7 35.1 i 6.50 T = Storage Time (Month): 0 a B-12 organisms K = 18211330201238 = 8w; 8 = S-eLizaseus presented in Average:S.D. 84 until 3 months of storage and leveled off for the next month of storage. Mold count decreased during the entire storage time in a much slower rate. At the end of 4-month storage, the starters still have the capability to produce a good quality of tempeh. Refrigeration temperatures preserved the mold viability better than room temperature. The presence of §.m§gatgrium lowered the mold count sig- nificantly in the starter. During the entire storage, again the §.Qliyaggn§ showed the superiority in the vitamin production compared to the other two organisms. Sensory evaluation A triangle test was performed by 15 panelists who .are familiar with the taste of tempeh. The test was performed twice and 26 completed questionnaires were collected. Table 13 summarizes the triangle test results. In general, no statistical difference was found between the taste of regular tempeh and any of the tempeh produced by mixed culture. The tempeh prepared with the three mixed-cultures was not identified by the panelists. Tempeh prepared with K.pngumgniag was the most difficult to identify followed by B.mggatgrigm and §.Qlivgcegs. 85 Tempeh Composition Composition of regular tempeh and tempeh made with mixed-culture inoculums are presented in Table 14. In general, the composition of tempehs made with mixed- culture inocula were very similar to that of regular tempeh. Suparmo and Markakis (1987) reported that the protein content of tempeh was slightly higher than that of soybeans. The increase of protein content was at the expense of fat and carbohydrate, which were utilized for energy during the fermentation by the mold. Table 13. Sensory evaluation by the triangle test of regular tempeh and tempeh prepared by mixed culture. ~ Correct False Total Lemmas-tempeh 4 22 26 law-tempeh 7 19 26 Lem-tempeh 9 17 26 14 correct identification out of 26 indicates significant different at p = 0.05. 86 Table 14. Compositions of regular tempeh and tempehs made with mixed-culture inocula1 Moisture Protein Fat CHO Ash Tempeh % %2 % %3 % Regular (mold only) 66.5 16.5 6.3 9.7 1.0 Mixed-E.pngumgniag 67.2 16.5 6.5 8.8 1.0 Mixed-B.mgga§grigm 67.2 16.1 6.5 9.1 1.1 Mixede§.gliygggg§ 67.7 15.8 6.2 9.3 1.0 1) average of triplicate analysis. 2) proteinsN x 6.25 3) determined by difference. Protein Efficiency Ratio (PER) The PERs of regular tempeh and tempeh made with mixed-culture inocula are presented in Table 15. The casein diet had significantly higher PER than all of the tempeh diets. The PER of §.Qlig§g§g§ and K.pngumgnigg- tempehs did not differ significantly from that of the regular tempeh. The PER of tempeh prepared with B.mgg§tgrigm mixed-culture was not significantly dif- ferent from that of the K.pn§umgniag-tempeh but was different from that of regular tempeh and tempeh prepared with §.glivag§gs mixed culture. 87 Table 15. Protein Efficiency Ratio (PER) of regular tempeh and tempehs prepared with mixed-culture inocula. Diet PER i 8.0. Adjusted PER Casein 2.13 i 0.20 2.50a* Tempeh (regular) 1.93 i 0.14 2.26b Tempeh (§.gliy§ggg§) 1.90 i 0.11 2.22b Tempeh (K.pnggmgnia) 1.79 i 0.12 2.10bc Tempeh (fi.m§ggtgrigm) 1.66 i 0.13 1.95c Different subscript indicates significant different at p=0.05. Amino acid analysis The amino acid composition of regular tempeh and tempeh made with mixed-culture inocula are presented in Table 16. The FAO amino acid pattern and the amino acid scores of the tempeh are presented in Table 17. Essential amino acids constitute less than half of the total amino acids in the tempeh. The sum of the total amino acids did not account for 100% of the analyzed protein, probably because of decomposition to NH3 or incomplete hydrolysis. 88 Methionine and cysteine (sulfur-containing amino acids) are the limiting amino acids in all of the tempeh samples. The scores were 71, 69, 63, and 74 for regular tempeh, K-tempeh, B-tempeh and S-tempeh, respectively. The score in B-tempeh was the lowest, which agrees with the result in the bioassay test. Soybeans, the raw material of tempeh, have the same limiting amino acid with a score of 74 (FAO, 1973). 89 Table 16 Amino acid composition of regular tempeh (R) and tempeh prepared with mixed-culture inocula (K,B, and S) ( g / 169 N ). R Tempeh K B S Tempeh Tempeh Tempeh NOn-essential amino acids 11.2 11.1 9.4 15.9 15.3 15.6 5.4 5.5 5.2 4.1 4.1 4.0 2.2 2.4 2.4 6.7 6.7 6.5 4.1 4.0 3.8 5.2 5.1 5.1 54.8 54.2 52.0 4.0 4.2 4.0 3.0 3.1 2.9 4.5 4.7 4.7 1.2 1.0 1.2 1.3 1.2 1.4 5.0 5.0 5.0 8.3 8.3 8.3 5.3 5.5 5.1 1.2 1.3 1.2 6.7 6.7 6.6 40.5 41.0 40.4 Aspartate 11.2 Glutamate 15.8 Serine 5.3 Glycine 4.2 Histidine 2.4 Arginine 6.6 Alanine 4.2 Proline 5.4 55.1 Essential amino acids Threonine 4.1 Tyrosine 3.1 Valine 4.7 Methionine 1.1 Cystine 1.4 Isoleucine 5.0 Leucine 8.2 Phenylalanine 5.5 Tryptophan 1.2 Lysine 6.6 40.9 Total 96.0 90 Table 17. Amino acid pattern of FAO reference and amino acid content (mg/g of protein) and score (%) of regular tempeh and tempeh prepared with mixed-culture inocula ( R.,K.,B., and S.) FAO S Pattern ------------------------ c s** c s c s c s Isoleucine 40 50 125 50 125 50 125 50 125 Leucine 70 82 117 83 119 83 119 83 119 Threonine 40 41 103 40 100 42 105 40 100 Valine 50 47 94 45 90 47 94 47 94 Methionine + Cystine 35 25 71 24 69 22 63 26 74 Phenylalanine + Tyrosine 60 85 142 83 138 86 143 80 133 Tryptophan 10 12 120 '12 120 13 130 12 120 Lysine 55 66 120 67 122 67 122 66 120 * R, K., B., and S. are regular tempeh and tempeh prepared from mixed culture of mold and K. pg_umgniag, B.megatsrium and 3 211232395 reSPectiveIY- ** C 2 content: S = score = (C/FAO pattern) CONCEUSIONS All three tested vitamin B-lZ-producing organisms were able to grow at the temperature range of tempeh fermentation. fi.gliygg§g§ grew in the pH range 5.5 to 7.0 while K.pnggmgnia§ and fi.mgga§grigm grew at pH 5.0 to 7.0. When grown on heated soybeans at pH 6.6 and at 30C, fi.glix;§§g§ produced the highest amount of vitamin B-12, followed by B.mgga§grigm and K.pnggmgnigg. Prolonging the tempeh fermentation by 1 day, doubled or tripled the vitamin content of the tempeh, depending on the organism. Acid addition, as practiced in the pilot plant method of tempeh production, inhibited the growth of vitamin B-lZ-producing bacteria. Cobalt addition did not affect the growth or the vitamin production by the vitamin B-lZ-producing organisms. Soaking enhanced the growth and vitamin production by §.pnggmgnia§, but slightly diminished the growth and vitamin B-12 produc- tion by EM and 9.2113395- Tempeh starters produced by mixed culture of 3.911gggpgrgg and vitamin B-lZ-producing organisms lost their vitamin B-lZ-producing capabilities when stored at 91 92 room temperature for over 2 months. Mixed culture starters ( mold with B.mgggtgrigm_ or with $.911yagggfi) stored in a refrigerator (2-4C) for 4 months still had the vitamin B-lZ—producing capability. 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Defective DNA synthesis in human megaloblastic bone marrow: Effects of homocysteine and methionine. J. Clin.Invest., 48, 284. Whitaker, J.R. 1978. Biochemical changes occurring during the fermentation of high-protein foods. Food Winarno, F.G., and Reddy, N.R. 1986. Tempe. in Legume- based fermented foods. Reddy, N.R., Pierson, M.D., and Salunkhe, D.K. ed. C.R.C. Press, Inc. Boca Raton, Fl. APPENDICES APPENDIX A COMPOSITION OF MEDIA USED IN THE EXPERIMENTS 1. Tryptone-glucose-yeast extract agar. 2. 3. “no Yeast extract (Difco) 3 Tryptone (Difco) 5 D-glucose (Mallinckrodt Chem.Works) 1 Agar (Difco) 15 g Distilled water to make 1000 ml. Ochromonas medium Plate Bacto-vitamin free casamino acids (Difco) 10 Bacto-dextrose (Difco) 20 Diammonium Hydrogen Citrate 1 Monopotassium phosphate 0 Magnesium sulfate 0 Calcium carbonate 0 Ethylenediamine tetra acetic acid 0 Manganese sulfate 0.1 zinc sulfate 0. Thiamine Ferrous sulfate 20 Cobalt sulfate 6 Boric acid 12 Cupric sulfate 800 Potassium Iodine 20 Sodium molybdate 0.1 DL-Tryptophane 0.2 DL-Methionine 0.4 L-Cystine 0.2 g Choline chloride 4 mg Inositol 20 mg 2 0 1 (IOIEEIE W2 Inuamnnuamnn “lmhfldfimhg “I ihfl PAB mg Biotin 2 mcg Tween Final reaction of the medium was pH 5.8 at 25'C Count Agar (PCA) Beef Extract (Difco) 3 g Tryptone (Difco) 5 g d-Glucose 1 g Agar 15 g To rehydrate the medium, 24 g of the medium was suspended in 1000 m1 of cold distilled water. 102 4. 103 Potato Dextrose Agar, PDA Potatoes, Infusion from 200 g Dextrose (Difco) 20 g Agar (Difco) 15 g To hydrate the medium, 39 g of PDA was suspended in 1000 ml of cold distilled water and heated to boiling to dissolve the medium completely. To prepared for mold count, the medium was acidified to pH 3.5 by adding sterile 10 % tartaric acid solution. Sabouraud Dextrose Agar Neopeptone (Difco) 10 g Dextrose (Difco) 40 g Agar (Difco) 15 9 To hydrate the medium, 65 g of the medium was completely suspended in 1000 ml of cold distilled water, distributed in tubes or flasks and sterilized at 121'C for 15 min. The final reaction of the medium was pH 5.6. Micro Assay Culture Agar (Difco) Yeast extract 20 g Proteus Peptone 5 g Dextrose 10 g Monopotassium Phosphate 2 g Sorbitan Monooleate Complex 0.1 g Agar 10 g To hydrate the medium, 47 g of the medium was suspended in 1000 ml of cold distilled water and heated to boiling for 3 min to dissolve the medium. The medium ‘was distributed to tubes and sterilized at 121°C for 15 min. lulaighmanli was maintained in stab of cultures in the medium at 4°C. Transfer was made bimonthly for stock culture. Micro Inoculum Broth (Difco) Yeast Extract (Difco) 20 g Proteus Peptone (Difco) 5 g Dextrose (Difco) 10 g Monopotassium Phosphate 2 g Sorbitan Monooleate Complex 0.1 g To hydrate the medium, 37 g of the medium was dissolve in 1000 ml of distilled water. The medium was distributed in tubes in 10 ml quantities and sterilized at 121C for 15 min. The medium was employed in preparing the inoculum of Mali used in vitamin B-12 determination. 104 8. B-12 Assay Medium.USP Vitamin Free Casamino Acids (Difco) 15 g Dextrose (Difco) 40 g Aspargin (Difco) 0.2 g Sodium Acetate 20 g Ascorbic Acids 4 g l-Cystine, Difco 0.4 g dl-Tryptophane 0.4 g Adenine Sulfate 0.02 g Guanine Hydrochloride 0.02 g Uracil 0.02 g Xanthine 0.02 g Riboflavin 0.001 g Thiamine Hydrochloride . 0.001 g Biotin 0.008 mg Niacin 0.002 g p-Amino Benzoic Acid (Difco) 0.002 9 Calcium Pantothenate 0.001 g Pyridoxine Hydrochloride 0.004 g Pyridoxal Hydrochloride 0.004 g Pyridoxamine Hydrochloride 0.0008 g Folic Acid 0.0002 g Monopotassium Phosphate 1 g Dipotassium Phosphate 1 9 Magnesium Sulfate 0.4 g Sodium Chloride 0.02 g Ferrous Sulfate 0.02 g Manganese Sulfate 0.02 g Sorbitan Monooleate Complex 2 g The medium is free of vitamin B-12, but contains all the other factors necessary for the growth of Luigignmgnii ATCC 7830. A single strength of assay medium was prepared by dissolving 85 g of the medium in 1000 m1 of distilled water and heated to boiling for 2-3 min. To each assay tube, a 5 ml of the medium was added and the volume was made to 10 ml by the sample and added water. The assay tubes were sterilized at 121°C for 5 min. APPENDIX C 2 x 2 FACTORIAL DESIGN FOR SOAKING AND HOLD TREATMENTS Run Design Observations Tmt.Combintn. Bact. Count Vitamin Soak Mold Rep Avg Rep Avg ‘ 1 2 3 1 2 3 106 III/7W} IfitiIiII/m 35366 6 NH Ufis Two ll!“ llTiilI/IW 31