VITAMIN B12 PRODUCTION BY Propionibacterium shermanil In Tempeh Warawut Krusong, Busaba Yongsmith* and Priscilla C. Sanchez**

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1 VITAMIN B12 PRODUCTION BY Propionibacterium shermanil In Tempeh Warawut Krusong, Busaba Yongsmith* and Priscilla C. Sanchez** Department of Agro-Industry, Faculty of Agricultural Technology, King Mongkut's Institute of Technology Ladkrabang ~*Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand ***Institute of Food Science and Technology, University of the Philippines at Los Banos, College, Laguna, Philippines ABSTRACT Propionibacterium shennanii 1250 was able to be used as vitamin B 12-producer in tempeh, a fermented soybean. Factors affecting the increase of vitamin B 12 content in tempeh by incorporation of P. Shermanii 1250 were investigated. The cobalt supplementation (3 ppm), moisture content (55%), incubation temperature (35" C) and concentration of P. shermanii 1250 inoculum ( %) were the optimum Tempeh, a fermented soybean food, is pro- conditions for vitamin B 12 production by P. shemwnii duced through fermentation of soybean inoculated 1250 in the product. It was found that vitamin B 12 with the mold Rhizopus oligosporus. It contains yield was correlated with cellular yields of P.. significant quantity of vitamin B 12 due to bacterial shermanii 1250 during fermentation. The highest cofermentation. However, a mixed culture containing yield of vitamin B 12 (308.4 ng/100g) was obtained Klebsiella pheumoniae, Bacillus megaterium or in tempeh at 48 hr. Moreover, propionic acid was Streptomyces olivaceus and the tempeh mold can detected by gas chromatography during fermentation. produce vitamin B 12(Supermo and Markakis, 1987). Significant increase of propionic acid was Moreover, increase in vitamin B 12during fermenta- obtained after 24 h of fermentation which also correlated with the growth of P. shermanii Netion of tempeh like products made from cowpea <Djurtoft and Nielsen, 1983), from Lathyrus sativus vertheless, the fungalstatic property of propionic acid did not strongly affect the growth of Rhhopus oligosporus, the tempeh mold. Therefore, it can be concluded that high vitamin B 12-tempeh could be obtained by this mixed fermentation process. INTI<U()IJCTIUN Vitamin B 12 is essential in the human diet. Manifestation of deficiency in vitamin B 12 include pernicious anemia. neurological symtoms. pancytopemia, hypothermia and servere weight loss as reported by Davis et ai. (1981). It is unique among the vitamin B12 in being produced exclusively by microorganisms. Bacteria of the genera Propionibacterium, Pseudomonas, Clostridium and some Streptomyces produce vitamin B 12 (Florent and Ninet, 1979). Production of vitamin B 12 by molds is controversial. No reports have been found of Rhizopus species producing vitamin B 12 or B 12-like activity (Liem et ai., 1977). seeds (Moslehuddin and Hang, 1987) and in other products such as natto (Okada et ai., 1983) and Kimchi (Ro et ai., 1979) have been reported Propionibacteria are gram-positive, non motile, catalase positive, nonspore forming, facultative anaerobic bacteria which was first isolated from Swiss (Emmenthaler) cheese (Reinbold. 1985). Propionibaclerium species are widely used in the industrial production of vitamin B 12. In addition, P. shermanii is reported as the main culture which is successively conducted in the production of vitamin B 12 in submerged fermentation with two modes of

2 aeration (Florent and Ninet, 1979). Moreover, vitamin B 12production by Propionibacterium have been carried out by several investigators (Leviton and Hargrove, 1952; Ro et al ; Yongsmith and Apiraktivongse, 1983). The purpose of this study was to investigate the vitamin B 12 production in tempeh by mixed fermentation with R. oligosporus and P. shermanii 1250 and to optimize its bio-process of vitamin B 12 production. ~MATERIALS Microorganisms AND METHODS Rhizopus oligosporus : the Rhizopus was prepared as powdered et al. (1991). inoculum as reported by Krusong Propionibacterium shermanii 1250: the broth inoculum of P. shermanii 1250 was prepared in 50 ml complete medium (Yongsmith and Chutima, 1983). After incubation at 35 C for 24 h, P. shermanii 1250 was measured and diluted to optical density (0.0.) 0.8 at 660 nm, and used as inoculum. High vitamin Bl2-tempeh preparation As reported by Krusong et al. (1991), high vitamin B 12-tempeh was prepared by soaking soybean in distilled water acidified with I% of 80% lactic acid for 2 h at 70. C, dehulling by hand, and then drying at 80. C until the surface appeared dry. Fifty grams of beans were placed in petri dishes and autoclaved for 15 min at 121. C. After inoculation with 0.5% R. oligosporus powdered inoculum and 2.0% P. sherman ii, the inoculated beans were incubated at 35. C for 24 h. of fermentation. supplementation, cobalt sulfate (CoS04.7HzO)at the concentration of 0,0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 ppm were supplemented into the sterilized pretreated soybean before inoculation of cultures; moisture content, the sterilized pretreated soybeans with optimum concentration ofcos04.7hzo was adjusted to 45, 50, 55, 60% before inoculation of cultures; incubation temperature, the sterilized pretreated soybeans with optimum concentration of CoS04.7HzO and moisture content was inoculated and incubated at 30, 32, 35 and 40 C; concentration of P. shermanii 1250 inculum, the sterilized pretreated soybeans with optimum concentration of CoS04.7HzO and moisture content was inoculated with 0.5% R. oligosporus powdered inoculum and 1.0,2.0,3.0,4.0 and 5.0% P. shermanii inoculum and, then, incubated at optimum temperature. After 24 h of fermentation, all samples with three replications were balanced by steaming for 30 min, dried at 80. C and pulverized at 80. Cand pulverized. Vitamin BIz in samples were analysed. Time course fermentation of high vitamin B12- tempeh The 50 g of sterilized pretreated soybeans, placed in petri dish, with optimum concentration of CoS04.7HzO and moisture content were inoculated with appropriated concentration of P. shermanii 1250 inoculum and 0.5% R. oligospours powdered inoculum and incubated at optimum temperature for.24,48, 72and96h. The dried pulverized offerrnented product with three replications were analysed for vitamin BI2, ph, total acidity and soluble carbohydrate. Microbial count Optimization of vitamin BI2 production in tempeh Factors affecting the vitamin B 12 production in tempeh by the incorporation with P. shermanii 1250 were investigated as followed: cobalt During the fermentation of high vitamin B 12- tempeh, number of the Propionibacterium was determined. It was done on sample plate in sodium lactate agar (Reinbold, 1985) incubated anaerobically (GasPak Anaerobic Jar, BBL) at 35 C for 6 days. Selected colonies from the sodium lactate agar plates were observed by Gram staining.

3 Vitamin B 12 determination ph and total acidity measurements RESULTS Soluble carbohydrate Determination of propionic acid

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