Sufficient intake of folate is critical worldwide due to low folate concentrations in daily diets. Folate supplementation in food is carried out with success in some countries, but recent data also indicate the importance of maintaining a suitable balance between folate and vitamin B12 intake due to their interdependent metabolisms. In this study, a high folate producer Lactobacillus plantarum SM39 was cultured in combination with a vitamin B12 producer, Propionibacterium freudenreichii DF13. The co-culture process was optimized to produce high levels of both vitamins in a whey permeate medium supplemented with yeast extract (SWP). Addition of 5 mg/L cobalt chloride, 15 mg/L DMBI, and 10 mg/L pABA to SWP medium followed by a two-step fermentation under optimal conditions, i.e. three days anaerobic/four days aerobic, led to high vitamin B12 and folate yields, 751 +/- 353 ng/mL and 8399 +/- 784 ng/mL, respectively. Folate yields were more than 10-fold higher than maximum values reported for natural fermentations and comparable to levels recently achieved using a genetically optimized strain. The controlled addition of pABA and cobalt offers the possibility to produce a vitamin supplement with a suitable balance of folate and vitamin B12 of 170-1. The fermented medium containing high levels of natural folate and vitamin B12 content could be used to fortify foods with natural vitamins or as a nutritional supplement. (C) 2011 Elsevier Ltd. All rights reserved.