Development, Validation, and Application of an Ultra-High Performance Liquid Chromatography–Diode Array Detection (UHPLC-DAD) Method to Assess Menaquinone-7 Production by Staphylococci

Vitamin K2, consisting of various menaquinones (MK-n), provides health benefits beyond the coagulation role of vitamin K1 and is primarily produced by bacteria in fermented foods or the human gut. Although Bacillus and some lactic acid bacteria are established producers, staphylococcal synthesis remains poorly characterized. This study optimized an extraction procedure for vitamin K2 from staphylococcal biomass and developed a rapid, 10-min ultra-high-performance liquid chromatography method with diode array detection (UHPLC-DAD) to quantify MK-7, the most bioavailable form. The method was validated for linearity, accuracy, and intra- and inter-day precision using Staphylococcus carnosus IMDO-S10, Staphylococcus shinii IMDO-S216, and Staphylococcus equorum IMDO-S257. Staphylococcus carnosus IMDO-S10 produced the highest MK-7 levels (1688 nmol/g cell dry mass), while MK-7 production by S. shinii IMDO-S216 decreased under high aerobic conditions. The validated UHPLC-DAD method enables screening of staphylococcal strains and monitoring of MK-7 production during growth, supporting applications in food fermentation.