Vitamin B12 enrichment of corn meal by mixed cultures of lactic acid producing Rhizopus and Propionibacterium shermanii ATCC 13673

Corn meal, a source of carbohydrate with high essential fatty acid, low protein and non-detectable vitamin B12 contents, has been used in the feedstuff. Vitamin B12 enrichment of corn meal was, therefore, investigated by using L(+)-lactic acid producing Rhizopus strain in combination with a lactic acid utilizing strain of vitamin B12 producing Propionibacterium shermanii ATCC 13673 in solid state cultivation. A total of 40 strains of Rhizopus was selected from various sources, 14 strains were screened as high acid producing strains. Among these 7 strains produced high amount of lactic acid in solid state culture using corn meal as substrate. R. oryzae TISTR 3211 was selected, as co-culture with P. shermanii ATCC 13673, based on the criteria in high accumulation of glucose, low sporulation in solid state culture with high capability on acid production from raw starch. Factors affecting on vitamin B12 production by the mixed cultures were investigated using basal medium in tight vinyl plastic bag. The basal medium consisted of 10 g of corn meal, 1 g CaCO3 and 0.24 mg CoSO4.7H2O at 52 percent initial moisture content. Supplementation with organic nitrogen source was essential for vitamin B12 production. Acid hydrolysate of casein of 0.125 g nitrogen resulted in the maximum vitamin B12 production. Addition of DMI, a precursor of vitamin B12, up to 10 mg did not show considerably increase on vitamin B12 production, however, DMI over 20 mg severely decreased vitamin B12 production due to its toxicity on Rhizopus growth. Increasing in inoculum size of P. shermanii ATCC 13673 extensively enhanced vitamin B12 production. Supplementation with 0.24 mg CoSO4.7H2O resulted in maximum vitamin B12 production. Moreover, CaCO3 played an important role on vitamin B12 production since lactic acid, an excellent growth supporting substrate for P. shermanii ATCC 13673, was less produced by R. oryzae TISTR 3211 in the medium without CaCO3. After 7 days of fermentation in the optimum culture medium, vitamin B12 and protein contents of corn meal (per 100 g of dry solid) produced by mixed cultures were increased from 3.69 micro g and 9.03 g to 64.48 micro g and 19.69 g, respectively. These were enough amount to be used as feed supplement.