3.7. Extraction of the Intracellular Metabolites

B. subtilis was precultured in LB liquid medium with a rotary speed of 150 rpm at 37 °C for 12 h. Then, the precultured bacteria cells were transferred to a fresh LB liquid medium, and the initial cell density was adjusted to 0.3–0.4 at 600 nm. The cells were cultivated at 37 °C on a rotary shaker at 150 rpm in 250 mL cotton-plugged flasks containing 100 mL of LB liquid medium either with or without CGA. Cell samples were collected at 2, 4, and 8 h either with or without CGA. First, bacteria cells were harvested by centrifugation at 12,000 rpm for 5 min and washed with PBS three times to remove the residual culture medium, followed by washing with ultrapure water to remove the salts from PBS. Then, we put the cells on dry ice and add 2 mL of 80% (v/v) methanol (pre-chilled to −80 °C). Then, cells were broken the cells using sonication under 10 °C and incubated the lysate at −80 °C for 2 h. Then, the metabolite-containing samples were harvested by centrifugation at 12,000 rpm for 10 min at 4–8 °C, and we collected the metabolite-containing supernatant to a new 1.5-mL tube and prepared for analyses. Three replicates were performed for each sample.

This experiment was analyzed using TSQ Quantiva (Thermo, Waltham, CA, USA). Samples were separated using a Synergi Hydro-RP column (2.0 × 100 mm, 2.5 μm, Phenomenex, Torrance, CA, USA). A binary solvent system (mobile phase A, 10 mM tributylamine adjusted with 15 mM acetic acid in water; mobile phase B, methanol) was used. This analysis focused on the TCA cycle, the glycolysis pathway, the pentose phosphate pathway, amino acids, and purine metabolism. A 25 min gradient with a flow rate of 250 μL/min was applied as follows: 1–5 min at 5% B; 5.1–20 min, 5–90% B; 20.1–25 min, 90% B. Positive-negative ion switching mode was performed for data acquisition. The resolution for Q1 and Q3 were both 0.7 FWHM. The source voltage was 3500 v for the positive and 2500 v for the negative ion mode. The source parameters were as follows: spray voltage: 3000 v; capillary temperature: 320 °C; heater temperature: 300 °C; sheath gas flow rate: 35; auxiliary gas flow rate: 10. Data analysis and quantitation were performed using the TraceFinder 3.2 software (Thermo Fisher, Waltham, CA, USA).

An in-house database for endogenous metabolites identification was created using Library Manager 2.0 (Thermo Fisher Scientific, CA, USA). Most reference spectra in the internal metabolite library were acquired from chemical standards. In some cases, standard metabolites were not accessible but were observed in biological samples. MS/MS spectra of these compounds were confirmed manually according to Metlin (www.metlin.scripps.edu) or HMDB (www.HMDB.ca) and also saved in Library Manager for reference. All of the areas of acquired peaks were normalized against the internal standard for further data processing.