Synergi hydro rp 80 column

The HPLC detection of the 70% methanol extract of M. volcanica, its fractions, and its compounds was carried out using Dionex equipment (Sunnyvale, CA, USA) equipped with a P850 pump, an ASI-100 automated sample injector, an STH585 column oven (maintained at 30 °C), and a UVD170S detector. The gradient elution of samples (10 µL) was carried out using acidified water (0.1% trifluoroacetic acid, A) and methanol (B) at 0.7 mL/min, as follows: 5–25% B from 0–15 min; 25–60% B from 15–40 min; 60–100% B from 40–43 min; 100–5% B from 43–45 min; and 5% B from 45–55 min. The eluate was monitored at 225, 250, and 300 nm. The samples were mainly analyzed using a Phenomenex Synergi Hydro-RP 80 Å column (150 × 4.60 mm, 4 µm), except the two samples shown in Section 2.4, a low-purity compound 7 (300 nm) and a high-purity compound 7 (300 nm), which were analyzed using an Agilent ZORBAX Eclipse XDB-C18 80 Å column (4.6 × 150 mm, 5 µm).

A liquid chromatograpy mass spectroscopy (LC-MS) analysis was achieved using an Agilent 6410B triple quadrupole (Agilent Technologies, Wilmington, DE, USA) equipped with electrospray ionization (ESI) (Agilent Technologies, Wilmington, DE, USA), according to a manufacturer’s protocol. Briefly, 100 mg sample dissolved in 1 mL of MeOH and centrifuged. Volume of sample injection into HPLC system (1200 Series LC, Agilent Technologies, Wilmington, DE, USA) was 5 μL. 150 cm × 2 mm², 4 μm Synergi Hydro-RP 80 Å column (Phenomenex, Torrance, CA, USA) was used for LC separation at 30 °C. ESI activated at 3 kV and 380 °C as a source temperature. LC-ESI-MS was measured under the following conditions: capillary voltage = 3 kV, cone voltage = 30 kV, source offset = 30 V, nebulizer pressure = 15 bar, desolvation gas flow-rate = 650 L/h, cone gas flow-rate = 150 L/h, fragmentor voltage = 90 V, collision voltage = 20 V. 0.1% formic acid in distilled water as mobile phase A and 0.1% formic acid in acetonitrile as mobile phase B separated the sample and went into the ESI chamber at a flow rate of 0.5 mL/min for 20 min. Sample was detected by multiple-reaction monitoring mode (MRM) of monitoring the transition pairs at m/z 252.1/136.1.

The “growth in beer test” had been performed to study the beer-spoilage capability of L. brevis BM-LB13908 (Sakamoto and Konings, 2003 (link)). Approximately 10⁵ cells/mL of logarithmic growing, VBNC, and recovered cells were inoculated into finished degassed beer under sterile conditions at 26°C. Non-inoculated beer was served as control. The inoculated and non-inoculated beers were examined visually every day for turbidity for 30 days. At day 30, lactic acid and acetic acid concentrations were detected by reversed-phase high performance liquid chromatography (HPLC) and quantified by external standard method (Deng et al., 2015 (link)). Fifty μL of cell samples collected by centrifugation and filter was injected into the 1100 series HPLC system (Agilent, United States). Compounds were separated on a 250 × 4.60 mm, 4 μm Synergi Hydro-RP 80 Å column (Phenomenex, Lane Cove, Australia) at 30°C. The mobile phase was 0.43% orthophosphoric acid with a flow rate of 1.0 mL/min, and elution was monitored by 210 nm UV detection.