Page 276 - Demo

280 PROCEEDINGS OF INTERNATIONAL SCIENTIFIC CONFERENCE ON APPLIED BIOTECHNOLOGYbasal fermentation medium and incubated for 48 hours in an incubator shaker at 30%u00b0C and 150 rpm. Cells were harvested by centrifugation at 9000 rpm for 10 minutes, washed twice with phosphate-buffered saline, lyophilized, and stored at 4%u00b0C until further use.2.3. Extraction and purification of vitamin K2Extraction and purification of vitamin K2 were performed as described by Collins [6], with modifications. Approximately 100 mg of lyophilized cells was shaken with 20 mL of a chloroform/methanol solvent mixture (2:1, v/v) for 2 hours. The solvent phase, obtained by centrifugation at 9000 rpm for 5 minutes to remove cell debris, was evaporated to dryness under reduced pressure at 40%u00b0C using a rotary evaporator and then resuspended in chloroform. The extract was applied as a uniform streak to a silica gel 60 F254 TLC plate, with a mobile phase of hexane/diethyl ether mixture (85:15, v/v). The vitamin K2 band in the extract, aligned with the menaquinone-4 standard (Sigma-Aldrich) under ultraviolet light, was removed, dissolved in chloroform, and stored at -20%u00b0C until analysis.2.4. Analysis of vitamin K2 by UV spectrophotometerThe purified extract was diluted in methanol, and its absorbance was measured at a wavelength of 248 nm using a UV spectrophotometer. The concentration of vitamin K2 was calculated by applying the optical density (OD) value to a standard curve equation for menaquinone-4, as described by Song et al. (2014) [7]. Each experiment was conducted in triplicate.3. RESULTS3.1. Selection of Bacillus subtilis strain producing vitamin K2