Centrifugal evaporator

P. aeruginosa KCTC 2513, P. aeruginosa CCARM 0223, and P. aeruginosa CCARM 0224 were purchased from the Korean Collection for Type Cultures (KCTC). P. aeruginosa strains were cultured in nutrient broth (NB; Difco, Detroit, MI, USA) agar under aerobic conditions at 30 °C. Then, the selected LAB was grown in MRS broth for 48 h at 30 °C to produce a mass amount of metabolites. Culture medium was then centrifuged for 10 min at 12,000 rpm, and the supernatant was filtered using a 0.22 μm membrane filter. Subsequently, the filtrates were used directly as samples for antimicrobial activity screening, and filtrates concentrated using a centrifugal evaporator (EYELA, Tokyo, Japan) at 45 °C were utilized for further antimicrobial and antibiofilm tests.

To assess antimicrobial activity, we employed the indicator strains Staphylococcus aureus KCTC 3881 and Staphylococcus aureus CCARM 3089. S. aureus KCTC 3881 was sourced from the Korean Collection for Type Cultures (KCTC), and S. aureus CCARM 3089 was obtained from the Culture Collection of Antimicrobial Resistant Microbes (CCARM). Nutrient broth (NB; Difco, Detroit, MI, USA) was used under aerobic conditions at 37 °C for S. aureus KCTC 3881 and S. aureus CCARM 3089 as indicators. The selected LAB for the study was cultured in MRS broth at 37 °C for 48 h to produce mass metabolites for experiments. Subsequently, centrifugation was performed at 3500 rpm for 15 min at 4 °C. The supernatants were filtered through a 0.22 μm membrane filter to obtain the cell-free supernatant (CFS). The CFS underwent liquid–liquid fractionation with ethyl acetate (1:1; v/v) to obtain the ethyl acetate fraction (EA). The concentrated fractions, obtained using a centrifugal evaporator (EYELA, Tokyo, Japan) at 45 °C, were utilized for further assessments.

To produce sufficient quantities of the two PSG derivatives for the purpose of elucidating their chemical structure, multiple scale-up reactions were carried out as follows; the molar concentrations of PSs, UDP-Glc and MrSGT enzyme were maintained in the proportions described above, while scaling up the total volume of the reaction mixture to 1 mL. At the end of the reaction, reaction mixtures from more than five batches were pooled and extracted by ethyl acetate partitioning. The solvent layer obtained was evaporated to dryness using a centrifugal evaporator (EYELA, Tokyo, Japan) set at 40 °C, and reconstituted in 5 mL of the mobile phase used for the HPLC–MS/MS analyses. The extracts were immediately loaded onto a reverse-phase C₈ cartridge of the CombiFlash Rf medium-pressure liquid chromatography (MPLC) system (Teledyne ISCO, Lincoln, NE, USA), and the flow rate was set at 8 mL/min. The eluents passing through a UV detector were all automatically fractionated over a 50-min running time. The fractions confirmed to contain the PSG derivative with a good purity (>97 %) by the tracing HPLC–MS/MS analyses, were pooled and freeze-dried. Their structures were further confirmed by Varian INOVA 500 nuclear magnetic resonance (NMR, Varian Inc., Palo Alto, CA, USA) spectroscopic analysis together with high resolution (HR) LCT-premier XE MS (Waters, Milford, MA, USA) analysis.