Preparations derived from Lentinula edodes (Berk.) Pegl. are widely used as dietary supplements, they contain compounds with immune system enhancing and chemopreventive properties. These preparations contain a blend of minerals and vitamins, including vitamins A, D, B-1, B-2, C, and niacin, but only scant vitamin B-12. Our goal was to optimize the growth conditions of submerged mycelial cultures of L edodes in order to obtain a new dietary supplement enriched in vitamin B12. We designed a biotechnological process in which cobalamin precursors, cobalt chloride and the methyl donors betaine, methionine, and choline, were added to the culture medium. The vitamin B-12 content in mycelial extracts was determined by RP-HPLC, while total Co2+ content was determined by HPIC. At the optimal Co2+ concentration (40 mu g/mL), the vitamin B-12 level in L. edodes mycelium reached 95 mu g/g of dry weight. All methyl donors had a positive effect on cobalamin biosynthesis, at least doubling its concentration in mycelia as compared mycelia grown in non-enriched medium. The amount of cobalamin in submerged cultivated mycelia of L edodes was 10,000-fold higher than that recorded for fruiting bodies. Our results suggest that these optimized culture conditions could be applied to obtain a new cobalamin-enriched dietary supplement derived from L edodes. (c) 2008 Elsevier Inc. All rights reserved.