Evaluation of Vitamin K (menaquinone-7) stability and secretion in glucose and glycerol-based media by Bacillus subtilis Natto

Menaquinone-7 (MK-7) is a type of vitamin K. MK-7 is produced via bacterial fermentation, and in order to reach the desired product purity, several downstream processing steps, including extraction, drying, ultrasonication, must be carried out. These processes, however, need to be carried out in such fashion to ensure the least amount of vitamin K lost and maximum recovery into the end-product. Therefore, in this study, drying, storage at 4 and -20 °C, and ultrasonication steps were evaluated under different conditions. Static liquid fermentations were conducted in McCartney bottles to explore the maximum MK-7 secretion potentials in different glycerol and glucose-based media compositions that were optimized in our previous studies. Maximum 32.5±0.4 mg l^-1 and 14.6±0.4 mg l^-1 concentrations were achieved in glycerol and glucose-based media, respectively. Concentrations of MK-7 produced under same fermentation conditions in 30 ml McCartney amber bottles with 3 ml of media in them and in 75 ml culture tubes with 20 ml media were compared. Also, ultrasonication, drying, and storage conditions were investigated to ensure the least amount of vitamin K is lost. Results showed that drying under forced air flow was the fastest, thus, demonstrated a better preservation of the vitamin, and should replace vacuum drying. Ultrasonication for 15 min seems to be safe and sufficient for phase transition for analysis. Also, storage at refrigerated temperatures seems to preserve MK-7 at least for one week. Furthermore, fermentations in McCartney bottles indicated how MK-7 concentrations are distributed in different zones of the static liquid broth. Culture tube results provided the conclusion as of how the metabolism of MK-7 changes in static fermentation when the scale-up process begins from McCartney bottle to culture tubes. Results in general showed a clearer road map to ensuring better quality and preservation of the valuable end-product, and illuminated more the path to further scaling up of the fermentation process for commercial production of MK-7.