Measurement of the antimicrobial ability and pressure resistance of surfactin

We selected three common gut pathogen species isolated from the giant panda to detect antimicrobial activity, i.e., E. coli, Staphylococcus aureus, and Salmonella enterica. We also used reference strains, including E. coli (CCTCC AB 212358), Staphylococcus aureus (CCTCC AB 91053), and Salmonella enterica (CCTCC M 95026), purchased from the China Center for Type Culture Collection (CCTCC). The antimicrobial abilities of surfactin were assessed using the Oxford Cup method [14]. Briefly, 50 μL of each pathogen was coated on LB solid medium and diluted to 1 × 10⁷ CFU/mL with the Oxford Cup. Ten microliters of the HPLC-purified extract was diluted and added at the midpoint of the Oxford Cup. The samples were then cultivated at 37°C for 24 h, and the inhibition zone was measured. Methanol was used as a control. Inhibition zones for each group were measured three times to reduce experimental error.

For analysis of resistance to temperature, pH, and proteases, 0.4 mg/mL surfactin extract samples were processed at 60–121°C in a water bath for 30 min, at pH 1–12 overnight (with the pH adjusted using HCl or NaOH), or with 1–3 mg/mL trypsin (pH = 8) or pepsin (pH = 2; Sangon Biotech Co., Ltd., Shanghai, China) for 4 h. After treatment, the processed surfactin extract was adjusted to the control environment (37°C, pH = 7), and antimicrobial activity was then assessed as previously described.