Mrs agar plate

The antagonistic activity of L. pentosus ŁOCK 0979 against the indicator fungi was tested using the double-layer method described by Lipińska et al. [13 ]. First, 10 μL of overnight bacterial culture was dropped on MRS agar plates (Merck or BTL) supplemented with 1% (m/v) polyols, galactosyl-polyols, or galactose, separately. The control group consisted of MRS agar plates (Merck) with lactobacilli colonies cultured with neither polyols nor gal-polyols. After 18–24 h, the plates were overlaid with Sabouraud 4% dextrose agar (Merck) inoculated with an indicator fungal strain (10⁵–10⁶ spores × mL⁻¹). Indicator strain inhibition zones around Lactobacillus sp. colonies were measured after 24–72 h of cultivation at 30 °C. The results were given as fungal inhibition diameters minus the diameter of Lactobacillus sp. colonies.

The modified well diffusion agar method previously described by Nami et al.^{21 (link)} was used to determine the antagonistic effect of E. faecalis KUMS-T48 cell-free supernatant on five indicator pathogens including Listeria monocytogenes (PTCC 1163), Escherichia coli (PTCC 1276), Klebsiella pneumoniae (ATCC 43,816), Yersinia enterocolitica (ATCC 2715), and Bacillus subtilis (ATCC 19,652). For this, 50 μL of the filtrate (through 0.2 µm filter) of an overnight culture of E. faecalis KUMS-T48 in MRS broth at 37 °C was added to 7 mm diameter wells on MRS agar plates (Sigma-Aldrich, USA), which were previously surface cultured with indicator pathogens and incubated overnight at 37 °C. After overnight incubation of plates at 37 °C, the clear zones around of each well were measured and considered as positive antibacterial activity. According to the diameter of the inhibition zone (average of two perpendicular diameters), the anti-pathogenic activity was divided into strong (diameter ≥ 20 mm), moderate (10 mm < diameter < 20 mm), and weak (diameter ≤ 10 mm). In order to determine the primary ingredient involved in the antagonistic properties of the cell-free supernatant, the neutralized form (adjusted to pH 7.2 by adding 1 M NaOH) and treated forms of the cell-free supernatant with catalase and proteinase K enzymes were also subjected to antibacterial tests.

The acid resistance of the selected LAB was determined according to the method described by Liong and Shah [30 (link)] with some modifications. After cultivation in MRS broth at 37 °C, the recovered cell was resuspended in an equal volume of MRS broth (final pH 1.5 and 2.0) and incubated at 37 °C for 2 h. For bile tolerance, 100 μL culture was dropped onto MRS agar plates containing 0%, 0.5%, 1%, and 3% (w/v) of bile (Sigma Chemical Co., St. Louis, MO, USA), and incubated at 37 °C for 24–48 h [31 (link)]. Acid and bile tolerance were determined based on the survival rate. The survival rate was calculated according to the following: $$Survivalrate(\%):(logA1/A0)\times 100\%.$$
A1: Total viable count of probiotic strains after treatment, A0: Total viable count of probiotic strains before treatment.