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The influence of some fermentation parameters on vitamin B12 formation by strains of Citrobacter freundii and Klebsiella pneumoniae isolated from Indonesian tempeh samples during tempeh fermentation was investigated. A decrease in fermentation temperature from 32 to 24 degrees C led to a decrease in vitamin B12 formation. Inoculation of soybeans with different numbers of cells of C. freundii at the beginning of solid-substrate fermentation showed that only the velocity of vitamin formation and not the final amount of vitamin formed depended on the number of cells. The addition of cobalt and 5,6-dimethylbenzimidazole increased the vitamin B12 content of tempeh. Nevertheless, levels of incorporation of the two precursors into the vitamin B12 molecule were very low. Neither C. freundii nor K. pneumoniae possessed the genes encoding the enterotoxins Shiga-like toxin SLT IIA, heat-labile enterotoxin LT Ih, and heat-stable enterotoxin ST Ih, as indicated by PCR. This result supports the suggested use of these two strains to form vitamin B12 during tempeh fermentation in Indonesia.

Vitamin B12 production by Citrobacter freundii or Klebsiella pneumoniae during tempeh fermentation and proof of enterotoxin absence by PCR