An plates

Biolog AN plates (Biolog Inc., Hayward, CA, USA) were used to evaluate the catabolic profiles of L. crispatus BC3, BC4, BC6, BC7, BC8, L. gasseri BC9, BC11, BC13, BC14, and L. vaginalis BC16 according to the manufacturers’ instructions. Cells were initially grown in MRS supplemented with 0.05% L-cysteine for 24 h, then Biolog AN plates were inoculated with 150 mL of bacterial suspensions adjusted to 65% transmittance as recommended by the manufacturer. Positive reactions were automatically recorded using a microplate reader with a 590 nm wavelength filter. Negative control measurement was obtained from a well containing water [19 (link)].

The carbon-source utilization profiles of the LAB strains were determined by Biolog System (Biolog, Inc., Hayward, CA, United States) using 95 different carbon sources. Before being used for inoculating Biolog AN plates (Biolog Inc., Hayward, CA, United States), strains were grown twice on MRS broth for 24 h, and the cells harvested by centrifugation (8,000 × g for 10 min) and washed twice in sterile phosphate buffer 50 mmol/l pH 7.0. Then, cells were re-suspended into sterile physiological solution. Each well of the Biolog AN plates was inoculated with 100 μl bacterial suspensions adjusted to 65% transmittance as recommended by the manufacturer. Positive reactions were automatically recorded using a microplate reader with a 590-nm wavelength filter after 24 h of incubation at 30°C. Three separate experiments were carried out. Similarities between the fermentation profiles were investigated through permutation analysis, using PermutMatrixEN software (Caraux and Pinloche, 2005 (link)).

BIOLOG^® AN plates (BIOLOG^® Inc., Hayward, CA, USA) were used to identify the substrate utilization pattern of the isolates [37 ]. The technology can also be used to determine substrate utilization patterns of microbial communities [63 (link)]. In the present study, the BIOLOG^® AN type plate was used to determine the carbohydrate preference of the Lactobacillus strains. The procedure followed the manufacturers’ guide with a minor modification. Both strains were inoculated in de Man, Rogosa and Sharpe medium (MRS, Carl Roth GmbH + Co. KG, Germany) and incubated in anaerobic jars (Merck KGaA, Germany) with Anaerocult C (Merck KGaA, Germany) overnight. The cultures were then washed with Phosphate Buffered Saline (PBS), pH 7.4, for three times and diluted to 10⁷ cells/mL. A total of 100µL bacterial suspension was pipetted into each well of BIOLOG^® AN plate in triplicate. The plates were incubated in anaerobic jars with Anaerocult C at 37 °C for 24 h and optical density was read with a microtiter plate reader (Tecan Infinite200Pro, Germany) at OD_590nm.