Role of Microorganisms in Tempeh Manufacture
―Isolation of vitamin B12 producing bacteria―
By NORIYUKI OKADA
Department of Applied Microbiology, National Food Research Institute (Tsukuba, Ibaraki, 305 Japan)
Tempeh originated hundreds of years ago (Plate 1-b ). The first group of microoganisms in Central and East Java, and is now Indo or the lactic acid producing group works in nesia's most popular fermented food. It is a modest way, playing an important part made of cooked soybeans bound together by at this stage. As shown in Plate 1-c, the a white mycelium of Rhizovus mold into com liquid becomes viscous during soaking, in pact cakes and is usually served as thin sliced dicating the occurrence of an extensive gro,vth and fried. It can be an ideal main course at of microorganisms. From this liquid, lactic meals in place of meat, chicken, or fish, and acid bacteria were detected about 1os;ml in can be the protein backbone of a vegetarian number and its pH value was lower than 4 diet, because tempeh contains 20 % protein, a as a result of propagation of lactic acid high amount of unsaturated lipids, no choles bacteria:;J. The decrease of pH value is very terol and vitamin B12• The last one is often important for the later fungal fermentation lacking in vegetarian diets. Although the stage, because the low pH prevents the growth essential microorganism in the tempeh fermen of undesirable bacteria. After soaking· the tation is a fungus or a mold, other two hulls are removed by rubbing the soaked beans microbial groups are also concerned in the between hands or by stamping them with feet tempeh manufacture; a lactic acid producing in water (Plate 1-d). The loosened hulls are group and a vitamin B,2 producing group. floated away with water, then the cotyledons This paper will present the role of these are boiled shortly, cooled, surface dried, and microorganisms in tempeh manufacture as inoculated with a starter (Plate 1-e, f) made well as isolation results of vi tamin B 12 pro of tapioca starch residues (Plate 1-g) con ducing bacteria, and prospects of the utiliza taining Rhizopus spores. The inoculated tion of isolates are also discussed. cotyledons are packed into perforated poly ethylene bags (Plate 1-h), or traditionally Manufacture of tempeh wrapped in banana leaves (Plate 1-i). Incuba tion is done on shelves at room temperature There are many variations in Indonesian (an annual average temperature at Bogor is manufacturing process01 . The process essen about 27°C) for 1 to 2 days (Plate 1-j) until tially requires those steps ; hydration of soy the cotyledons are completely overgrown with beans, partial cooking, acidification, dehulling, the mold myceli um (Plate 1-k, 1) . surface drying, inoculation with a starter, The occurrence at soybeans during fermen packing in a container, and incubation. The tation is as follows. For the first few hours, process seen at Bogor was as follows. there is a lag time during which germination Soybeans without dirt and stones are of spores starts and rapid mold growth fol cooked partially with drum cans (Plate 1-a), lows though no clear changes can be seen. then they are soaked in water through night When the growth of mold becomes evident, 311
Plate 1. Manufacture of tempeh a : Cooking of soybeans, b: Soaking, c: Soybeans after soaking, d: Dehulling, e: Mixing of starter, f : Soybeans after inoculation, g: Ragi tempeh ( tempeh starter) , h: Packing, i: Packing ( traditional method), j: Incubation, k, I: Tempeh (final product) . 312 JARQ Vol. 22, No. 4, 1989
12 the temperature of soybeans rises upper than vitamin B, 2 >. We examined V. 8 12 content the surrounding temperature. The extensive of tempeh as compared with other fermented growth of mold makes soybeans form into a foods in the tropics0 >. Table 1 shows the compact mass with secretion of enzymes such V. B, 2 content of some fermented foods of as proteases and lipases. Some of the fungal the tropics. The daily requirement of V. B, 2 hyphae penetrate into the soybean cotyledons for man is estimated to be 3 µg. The test and secrete enzymes there too. Thus the soy samples which were detected an appreciable beans become soft and easily digestible as amount of V. B12 were fish sauce (3-month the protein and lipids are decomposed to solu fermentation, Thailand) 2.4 µg/1 00 g, fer ble matter. The tempeh is now ready to eat. mented shrimp (Thailand) 2.5 µg/100 g, ka However the mold continues to grow even pi (Thailand) 5.3 µg/100 g, tempeh (Indo after this stage, and spore formation and nesia) 4.6 µg/100 g, thua-nao (Thailand) ammonia production due to protein breakdown 1.5 pg/100 g. Hov,,ever V. B 12 detected in fish appear finally. The pH value below 4 at the sauce, fermented shrimp and ka-pi may not beginning rises to 6 to 6.5 at its optimum be produced through fermentation, since they quality, and goes beyond 7.5 at the over ripe are made of animal materials. Those soy stage. fermented products such as tempeh and thua
nao are worthy of study, because V. B 12 de Vitamin B,2 content of tempeh as tected in them is considered to increase thxough fermentation, since soybeans do not compared with other fermented contain any V. B,/''· foods in the tropics Production mode of vitamin B,2 in It is already known that tempeh contains tempeh Although vitamin B,t was detected in Table 1. V. B,2 content of fermented foods tempeh, not all the tempeh samples contained
V.812 such an amount of V. B,1 • The detectable No. Sample name Country (µg/ 100 amount was greatly dependent upon the fresh g fresh) ness of samples. Only a few amount was l Soy sauce mush Tha
Table 2. V. B12 content of tempeh from Indonesia and tempeh prepared at Japanese laboratory
Sample V. 812 Note no. (µg/100 g fresh) 1 4.6 Transported slowly from Indonesia•> (Smell was not so good). 2 o. 7 Transported quickly from lndonesiab> (Smell was good). 3 0.05 Prepared with tempeh starter of Indonesia. 4 0.06 Prepared with Rhizopus otigosporns NRRL 2710 5 0.02 6 0.03 a) : It was exposed around 25'C for 3 days before analysis. b) : It was brought from Indonesia as quickly as possible. However. it was exposed around 25'C for 8 hr before analysis.
leasing ammonia that invites an increase of and enzymes, and V. B,2 producing bacteria pH value. Consequently, bacteria that usually are to make tempeh grade up to a substitutive favor neutral pH grow at the later stage of food for meat by adding V. B, 2 • fermentation, and some of them contribute to yield V. B 12 • Isolation and identification of vita Thus tempeh is produced through coopera min B ,2 producing bacteria tive works of microorganisms: lactic acid bacteria are to prepare a condition for the In order to obtain V. B12 producing micro fungal growth by lowering pH, a mold is organisms, bacteria were isolated from those to nit and to solubilize soybeans by myceria samples that were collected in Bogor and its
Table 3. V. B12 productivity of bacteria isolated from tempeh Isolate Isolate Isolate Isolate Isolate V B a> V. B12"' V. B12"' no. • 12 no. no. no. no. 3 > 66 120 0. 4 169 72 232 l. 0 309 181 4 >165 126 0.5 170 0.9 235 0.9 310 105 5 88 132 >165 171 0.3 238 l. 3 315 170 7 143 133 39 172 103 240 10 327 33 JO >110 137 4 177 0.4 251 0. 2 328 >110 12 >110 141 0.4 178 l. 0 252 30 330 > 110 13 >165 142 0.3 180 0. 8 254 1.1 333 > 66 16 58 143 0.3 186 31 256 0.8 336 > 66 22 1350 144 5 187 0. 5 263 0.9 337 380 25 81 145 o. 4 189 0. 7 264 l. 0 348 0.5 31 >110 146 0.3 190 0.9 266 44 355 > 110 32 >110 150 >110 191 54 267 0.6 362 83 36 253 151 0.4 192 l. 0 270 510 363 >110 39 299 152 0.9 197 >110 271 0.6 366 51 70 92 153 0.6 199 0.4 273 0.9 368 120 72 56 154 0.6 200 188 274 0. 7 372 178 81 99 155 247 201 0.6 275 125 373 l. 0 84 >110 156 >110 205 145 276 97 375 0. l 90 78 157 0.9 210 178 289 125 380 o. 5 91 >110 158 0.4 217 0. 7 290 68 387 0.4 95 122 159 0.3 222 0.4 291 112 389 0. 2 110 0. 1 160 0.6 224 0.8 307 168 114 0. 7 162 0. 7 228 168 308 171
a) : V. 812 productivity (ng/cells in 5 ml of culture). 314 JARQ Vol. 22, No. 4, 1989
10 Table 4. ldenfitication of isolate no. 22
Characteristics J(lebsiella Isolate pneumo11iae no. 22 8 ~ ,;; Gram test ... Motility Nitrate reductase + + l·hS production 0 I Urease + + Phenylalanine deamination 21 Lysine clecarboxylase + + Arginine cli hydrolase I Ornitihine decarboxylase t::..~t:.=L::.. l::,.__J 2 4 6 8 10 /J- Galactosiclase + + Days Utilization of: Citrate + + Fig. 1. Changes of V. 812 content Malonate + + Samples were put in the plastic bag Fermentation of : and placed at 30'C. D-Glucose + + 0 : Indonesian tempeh, t::,. : T empeh Aclonitol c10> + prepared with Rhizopus oligosJ,rns L-Arabinose + + NRRL 2710. Inositol + + Raffinose + + D-Sorbitol + + Lactose + + L- Rhamnose + + Sucrose + + ~84 a) : differs among strains. 60 Q surrounding towns·>. Table 3 shows V. B12 productivity of the respective isolates and 43 Fig. 2 shows the distribution of productivity of them. Sixty-nine out of 397 isolates showed 40 - 2§... ~ some degrees of response to Lactobacillits 3 -~ leichmannii ATCC 7830. This microorganism '-- requires a very careful ha.ndling·1 >, though it ....0 0 is generally used for a V. B assay. One .D ,, 12 =::, ,..;;.;.. isolate showed V. B, 2 productivity at the level z 20 of more than 1,000 ng/(cells in 5 ml of cul ture) . Thirty-eight isolates showed 100 ng to 1,000 ng (the same as t he above unit), ,....._9 22 isolates showed 10 ng to 100 ng, and 9 isolates showed 1 ng to 10 ng. The known ....L. V. B1~ producer, Bacillus •megateriit1n JAM <0.l 0.1 I - I 0- I 00- > IOOO 1166 used as a control microorganism showed -I 10 100 IOOO a value of 402 ng (the same as the above V. B 12 productivity (ng/cclls in 5 ml of culture) unit). Vitamin B, 2 productivity by various Fig. 2. Distribution of V. 812 productivity microorganisms seen in the textbook is 2.3 to 315
Table 5. Representative vitamin B,2 producing bacteria in tempeh
V. 8 2 producer Representative Sampling 1 Isolate V.812 No. numbers/ Total V. B,a producing produc. place no. numbers bacteria tivity•> Pasar, Bogor 14/42 J(lebsiella pneumoniae 22 1350 Resembles either !(. terrigena 5 88 or /(. />lanticola /(. pneumoniae sub. ozaenae 31 >110 2 Tegallega, Bogor 6/31 Resembles I(. Lerrigena 81 99 I(. /mewnoniae 91 >110 3 Semplak, Bogor 1/31! G(+), rod (Bacillus sp.) 95 122 4 Cipanas, Pacet 3/12 /(. pneumoniae 132 >165 Escherichia coli 133 39 5 Cianjur :vis Resembles either /(. terrigena 150 >110 or /(. /Jlanticota !(. pnezimoniae 155 247 6 Caringin, Cicurug 2/ 14 K. pnemnoniae 169 72 7 Kongsi, Cicurug I/ 8 X . Jmeumoniae 172 103 8 Caringin, Cicurug 5/22 G( + ) , rod ( Bacillus sp.) 186 31 Resembles 1(. pla11ticola 191 54 9 Pasar, Bogor 3/ 15 Resembles JC planlicola 200 188 Resembles K terrigena 210 178 10 Rawasigdin, Cicurug 2/10 G( +), rod ( Bacillus sp.) 228 168 11 Bababan, Ciparay, Bandung 2/ 13 Enlerobacter cloacae 252 30 12 Sukasari, Bogor 5/ 17 Resembles !(. terrigena 266 44 [(. pneumoniae 270 510 13 Sukasari, Bogor 3/ 16 K. pnewnoniae 275 125 14 Abesin, Bogor 5/13 I(. pneumomae 307 168 Resembles /(. terrige11a 308 171 15 Menteng, Bogor 5/11 /(. pnewmoniae 328 >UO Resembles E<. pltmtico7a 330 >110 16 Kedungwaringin, 2/ 25 J(. fmeumoniae 337 380 Kedunghalang 17 Ciomas, Ciomas 4/ 7 G(+). rod ( Bacillus sp.) 362 83 G(-), oxidase ( + ) , rod 366 51 !(. /me1mt01iiae 368 120 18 Ciluar, Keclunghalang 2/ 9 J(. pu.eumoniae 372 178 a) : ng/ cells in 5 ml of culture.
59 mg/L1 >. The highest value obtained by this either [(, terr-igena or K. vlnnticola, one was study was 1,350 ng/ (cells in 5 ml of culture) identified as IC pneimwniae sub. ozaenae. sp., which corresponds to 0.27 mg/l. two were identified as Enterobctcter clocicae Identification tests were performed on those and Escherichia coli, four were unidentified isolates that produced V. B 12 more than 10 ng/ Gram-positive rods (Bctcillus sp.) , and one ( cells in 5 ml of culture) s> . Table 4 shows the was unidentified Gram-negative rod which identification test on isolate no. 22. Table 5 differs from l(lebsiellci sp. in the oxidase test. shows the summary of results. Most of V. B ,:: producing bacteria were identified as l(lebsiella Prospects vneumoniae, already published by Shurtleff and Aoyagi 12> , namely 13 out of 33 isolates Indonesia has not only a climate of high were Klebsiella vneumoniae. However, other temperature and high humidity where molds bacteria than Klebsiellci pneumoniae were also propagate well and where tempeh is easily found. Twelve isolates were closely resembled produced without failures, but also is an 316 JARQ Vol. 22, No. 4, 1989
Islamic society where most people are vege tarians who are tend to be short of an animal References factor or vitamin B 12• It is possible to men tion that the health of people, in particular 1) F lorent, J. & Ni net, L.: Vitamin B,z, In that of poor people, has been sustained by Micrnbial technology. eds. Peppler, H.J. & tempeh which supply not only cheap protein Perlman, D., USA, 497- 519 (1979) . 2) Hasegawa, K. et al.: Induction of a high but also an animal factor or V. B 12 that is vitamin B,2 productive natto strain through usually taken from other animals through protoplast fusion. Nippon Shoknhin l(ou110 meat diet. Gakkwishi, 35, 154-159 (1988) [In Japanese with Eng·lish summary]. The V. B, 2 poducing bacteria isolated from tempeh are expected to be utilized for other 3) Manabe, M. et al. : The screening and the utilization of fermentation microorganisms. fermented foods. For instance, Japanese natto In The final report of science and technology. which is also made of soybeans may be pos The Science and Technolog·y Agency, Tokyo, sible to be improved the V. B 12 content. To 75-89 (1987) [In Japanese]. examine this possibility, investigations are 4) Okada, N. & Ohta, T.: Studies on the fac now underway from two points of view. tors affecting the standard curve of vitamin B12 assay. Rev. Nat. Food Res. Inst., 42, Whether Japanese natto bacteria have V. B 12 H7- 103 (1982) [In Japanese with English producing ability or not, and are there any summary).
useful methods of transferring V. B 12 pro 5) Okada, N. et al. : On the vitamin Bu values ducing ability to a natto bacterium? detected in natto by applying n micro On the first question, it was found that any biological assay method. Rev. Nat. Food Res. Inst., 43, 121-125 (1983) [In Japanese with Japanese natto bacteria have not V. Brn pro English summary]. ducing ability•>. In old reference, it was 6) Okada, N. et al.: Vitamin B12 content of reported that natto contained V. B 1~ and fermented foods in the tropics. Rep. Nat. 43, 126- 129 (1983) . natto bacterium had V. B 12 producing ability. Foocl Res. inst., 7) Okada, N. et al.: Isolation of bacteria However, we have found that the V. B 12 pro producing V. B12 from fermented soybean ducing ability is missing in natto bacteria. tempeh of Indonesia. Rev. Nat. Food Res. On the second question, we are now under Inst., 46, 15- 20 ( 1985). investigation by the cell fusion method which 8) Okada, N. et al. : Survey of vitamin Bu is one of the promising methods of gene trans producing bacteria isolated from Indonesian fer in improvement of microorganisms for tempeh. Rep. Nat. Food Res. hist., 47, 49- 56 (1985). practical use. Intraspecific cell fusion between 9) Okada, N.: Tempeh-An Indonesian soy mutants of Bacillus subtilis IAM 1207 was so fermented food- . Shoku1·yo, 27, 65-93 (1988) far successfu11-o,11 >. Most of V. B,~ producing [In Japanese] . bacteria found in tempeh were Klebsiella sp. 10) Okada, N. et al.: Protoplast fusion of whose genus is different from Bacillus natto Bacillus subtilfa and B . 1neucite1·ium.-On the tonicity of the semi-synthetic protoplast re and that means almost impossible to perform version medium- . Rep. Nat. Food Res. Inst., cell fusion between them. However, other 53, 10-16 (1989) [In Japanese with English bacteria than [(lebsiella sp. were also found summary]. in tempeh as V. B 12 producers. Among them 11) Okada, N. et al.: Marker expression by there were Bacillus sp. that are worthy to be mutants of Bacilli on a semi-synthetic focused on for cell fusion counterparts. Re protoplast reversion medium. Rep. Ncit. Food Res. Inst., 53, 17-22 (1989) [In Japanese cently a success of interspecific cell fusion with English summary]. between Bacillits natto and Bacilliis -mega 12) Slrnrtleff, W. & Aoyagi, A.: Tempeh pro teriu1n was reported2>, that is very encourag duction. New-Age Foods, USA, 60-67 (1980) . ing us to continue our works. (Received for publication, August 8, 1988)