NATTO BACILLUS CONTAINS A LARGE AMOUNT OF WATER-SOLUBLE VITAMIN K (MENAQUINONE-7)

Abstract

Bacillus natto (Bacillus subtilis natto) was cultivated, and an analysis was conducted after performing lysozyme treatment and water extraction of the culture supernatant and the B. subtilis natto cells. The intracellular existence of a large amount of water-soluble vitamin K (Menaquinone-7: MK-7) was established. The existence of small amounts of other types of vitamin K2 including MK-4 and PK was also confirmed in the culture solution (water-soluble fractions). The amount of water-soluble menaquinone-7 in Bacillus was 85 µg/g wet weight of the bacteria, and the amount was equivalent to more than 100 times as much as that contained in the culture solution (0.02 µg). Gel filtration using Sephacryl S-200HR revealed that the molecular weight of the water-soluble menaquinone-7 is approximately MW 200,000, and isoelectric focusing revealed a behavior similar to that of protein, with a pI of about 4.2. A rabbit antibody was prepared with this water-soluble vitamin K as the antigen. By using the Ouchterlony method, the antibody showed a reaction (precipitation line) with the water-soluble menaquinone-7 prepared from both the intracellular fraction and the extracellular culture solution, and it was found through the antigen–antibody reaction that the menaquinone-7 disappears from the supernatant of the reactant (the water-soluble menaquinone-7 is thus neutralized). Based on these results, it was inferred that the vitamin K produced by B. subtilis natto becomes water-soluble by forming an intracellular complex with protein and is released in the extracellular fraction during the culture process.