Vanillic acid

The high purity (≥97%) standards, including ascorbic acid, vanillic acid, syringic acid, epicatechin, daidzin, glycitin, ferulic acid, genistin, daidzein, glycitein, quercetin, and genistein were purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Other reagents of analytical grade. Soybeans (Liaodou, yellow seed coat, mainly planted in northern China, weigh 21.6 g of one hundred seeds) were obtained from a local market. R. oligosporus RT-3 isolated, screened and identified from Tempeh in our laboratory was used for soybean fermentation (11 (link), 12 ). The strain was cultivated on potato dextrose agar at 30°C for 3 days. Then, spores produced were scraped in water (1 × 10⁷ spores/mL).

The minimum inhibitory concentration (MIC) using the broth micro-dilution method according to Clinical and Laboratory Standard (CLSI, 2015) were tested in three assays to test antibacterial activities including (1) levofloxacin against 96 clinically isolated strain of PA; (2) 13 plant extracts against PA1803 and ATCC 27853; (3) Chlorogenic acid (CA) and its metabolites including quinic acid, caffeic acid, p-coumaric acid, dihydrocaffeic acid, shikimic acid, benzoic acid, hippuric acid, isoferulic acid, ferulic acid, gallic acid, p-hydroxybenzoic acid, syringic acid, vanillic acid (Shanghai yuanye biotechnology Co. Ltd) against PA, MSSA, MRSA, E. coli E+, E. coli E−, KPN E−, KPN E+, ISAB, EF, and AREF (5 strains, respectively). Chemicals (analytical standard, purity > 99%, Sigma-Aldrich Trading Co. Ltd.) were dissolved in sterile water or DMSO. The same amount of DMSO served as the negative control. Briefly, two-fold dilutions of chemicals were prepared with LB medium in tubes and each tube was inoculated with 500 μL overnight bacteria culture, approximately 5 × 10⁵ CFU/mL. Then, the tubes were incubated at 37 °C for 24 h. The MIC was defined as the lowest concentration (mg/mL) of chemicals that completely inhibited visible growth of bacteria.

The HCT116 cells were grown in RPMI with penicillin (100 units/mL)-streptomycin (100 units/mL) (Invitrogen, Carlsbad, CA, USA) and 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT, USA). The Hep3B, A549, and HUVEC cells were maintained in DMEM medium supplemented as above. All cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The hypoxic culture was kept in a gas-controlled chamber (Thermo Electron Corp., Marietta, OH, USA) maintained at 1% O₂, 94% N₂, and 5% CO₂ at 37 °C. The MG-132 and cycloheximide (CHX) were bought from Sigma Chemical Co (St Louis, MO, USA). The mTOR inhibitor (rapamycin) and MEK inhibitor (PD98059) were obtained from Calbiochem (San Diego, CA, USA). Vanillic acid was purchased from Yuanye Biotechnology Co. Ltd. (Shanghai, China) and its structure is shown in Figure 1A. The purity of Vanillic acid was more than 98% in HPLC analysis.