Growth Profiling and Metabolite Analysis of Lactococcus lactis Strains

Lactococcus lactis 9 k, L. lactis 9 k-1, L. lactis 9 k-2, L. lactis 9 k-3, and L. lactis 9 k-4 were cultured to OD₆₀₀ of 0.8 in the M17G medium and diluted to an optical density of OD₆₀₀ of 0.2. Afterward, 20 μl cultures were incubated into 200 μl M17G and SA media in a 96-well plate. The corresponding bacteria were also incubated into the 100 ml of media in shake flasks. The growth profiles were monitored by measuring OD₆₀₀ for 16 h at 30 °C by using a Bioscreen machine (Lab-systems, Helsinki, Finland) [23 (link)]. Ten milliliter suspension from shake flask was centrifuged when the cells were cultured for 6, 10, and 14 h, and the CDW was calculated using the previously described method [11 (link)]. The residual glucose in different strains was detected using commercial kits following the manufacturers’ instructions (Thermo Fisher Scientific, Waltham, USA). The ATP concentration was determined through bioluminescence assay with recombinant firefly luciferase and its substrate d-luciferin by using the Molecular Probes’ ATP Determination Kit (Thermo Fisher Scientific, Waltham, USA). One milliliter cultures of different strains were centrifuged at 6000×g for 5 min before the measurement of ATP concentration. Subsequently, 2 ml of 0.015 g/ml trichloroacetic acid (TCA) was added to the cells and vortexed for 3 min to extract ATP from cells. Afterward, 1 ml of supernatant from vortex was collected and diluted to the concentration of TCA lower than 0.001 g/ml with Tris–acetate buffer (PH 7.8). The extrication of ATP can be used for ATP concentration detection [42 (link), 43 (link)]. Each sample was analyzed in triplicate.