Mrs agar

Sodium alginate with a medium viscosity and high mannuronic acid without inulin were purchased from Hi-media (Mumbai). They were prepared in distilled water and autoclaved at 121 °C for 15 min. The low-molecular-weight chitosan (deacetylated chitin, Hi-Media, Laboratories Mumbai), was prepared in distilled water and pure hydrochloric acid (Loba Chemie, Pvt Ltd.—Mumbai, India). Xanthan gum, gum acacia, and carrageenan were purchased from Loba Chemie, Pvt Ltd.—Mumbai, India. Starch, sodium caseinate, pepsin, trypsin, calcium chloride, sodium chloride, sodium bicarbonate, trisodium citrate, sodium hydroxide, potassium chloride, phosphate buffer saline (pH 7.2), and hydrochloric acid were purchased from Hi-media Laboratories (Mumbai, India). Glacial acetic acid with molar mass of 60.05 g mol⁻¹ was bought from Merck (Darmstadt, Germany). Pancreatin, peptone, and bile salt were obtained from Loba Chemie, Pvt Ltd. (Mumbai, India). The De Man Rogosa and Sharpes (MRS) broth and MRS agar used in this work were purchased from Hi-media Laboratories (Mumbai). Indicator microorganisms used for the antimicrobial activity; i.e., Escherichia coli (MTCC No-432), Staphylococcus aureus (MTCC No-96), and Bacillus cereus (MTCC No-430), were procured from the Institute of Microbial Technology (IMTECH; Chandigarh, India).

L. reuteri KUB-AC5 isolated from chicken intestine samples was obtained from the stock culture of Department of Biotechnology, Faculty of Agro-industry, Kasetsart University, Thailand (Nitisinprasert et al., 2000 ). This strain was preserved at –80 °C in MRS medium (Difco) containing 20% (v/v) glycerol. Culture was propagated twice in MRS medium at initial pH 6.5 and 37 °C for 18–24 h. All cultivations started with the approximate concentration of 10⁹ CFU/ml at late logarithmic phase of L. reuteri as the inoculum for further investigating inhibition effects (Sobanbua et al., 2020 ). It was cultured with two independent replicates at 37 °C for 24 h in a modified MRS medium (Hamsupo et al., 2005 ) with either 2% (w/v) sucrose or 2% (w/v) glucose. Samples were taken at different time points (i.e., 0, 3, 6, 9, 12, 15, 18, 21 and 24 h) for further viable cell count and pH measurement. The viability of L. reuteri KUB-AC5 was determined by the standard plate count method. Aliquots of one mL of each sample were diluted by nine mL of sterile 0.85% NaCl solution in serial decimal dilutions. It was afterwards plated in MRS agar (Himedia) and incubated under anaerobic conditions at 37 °C for 48 h. The cell count was performed as log of colony forming units per milliliter (log CFU/ml). For pH measurement, it was performed by a digital pH meter (Mettler Toledo S220).

Bacterial isolation from various lugri samples was conducted using serial dilution and spread plate technique (Zommiti et al., 2018 (link)). 100 μl of the lugri samples were serially diluted from 10^–1 to 10^–7 in sterile normal saline (0.85% NaCl). Aliquots of 100 μl from serial dilutions were spread plate on de-Mann Rogosa Sharpe (MRS) agar (Hi-media Lab., Mumbai, India). The plates were incubated under aerobic conditions for 48 h at 37°C. Colonies with unique morphologies were further streaked on MRS agar to obtain the pure cultures. The glycerol stock (25% v/v) of each isolate was prepared and stored at −80°C for long term use.
The unique morphotypes were further estimated qualitatively for their growth at different pH (2–4) ranges and bile salt concentrations (0.3–3.0%) on the MRS agar plate at 37°C. Based on their qualitative assay, the primary identification of bacterial strains was carried out using gram staining and catalase test. For the catalase test, 3.0% of hydrogen peroxide was added to bacterial cultures. The observation of effervescence indicated the presence of a catalase enzyme. The reference type strain, Lacticaseibacillus rhamnosus (ATCC 53103) was used as a positive control for comparison in all the experiments.

The AFM₁ standard was obtained from Sigma Aldrich (St. Louis, MO, USA). High-performance liquid chromatography (HPLC) grade solvents were obtained from Merck (Darmstadt, Germany).
The isolates Lactobacillus plantarum 49, L. fermentum 111, and L. paracasei 108 were examined separately for the removal of AFM₁. These isolates were recovered from fruit processing by-products, identified with a partial 16S rRNA gene sequence analysis and characterized as potential candidates for use as probiotics [17 (link)]. Stocks were stored at −20 °C in de Man, Rogosa, and Sharpe (MRS) broth (HiMedia, Mumbai, India) with glycerol (20 mL/100 mL; Sigma-Aldrich, St. Louis, MO, USA). Working cultures were maintained aerobically on MRS agar (HiMedia, Mumbai, India) at 4 °C and transferred to a new media monthly. Prior to use in assays, each isolate was cultivated anaerobically (Anaerobic System Anaerogen, Oxoid, Hampshire, UK) in MRS broth at 37 °C for 20–24 h (to reach the stationary growth phase), harvested by centrifugation (4500× g, 15 min, 4 °C), washed twice, and resuspended in phosphate buffer solution (PBS; 50 mM K₂HPO₄/KH₂PO₄; pH 6.9) to obtain cell suspensions with an optical density reading at 660 nm (OD₆₆₀) of 0.5. This suspension had viable counts of approximately 1.1 × 10⁹ CFU/mL for each isolate when plated in MRS agar.

AgNO₃, nutrient agar, nutrient broth, MRS agar, Mueller–Hinton agar, UIM medium, methyl red (MR) medium, egg yolk agar and Simmon citrate medium from Himedia Laboratories. Carbohydrate discs, H₂O₂, Gramme stain and Ziehl-Nelseen stain. Sterile physiological solution (NS; 0.9 w/v% sodium chloride; pH 4.5–7.0). Tubes, absorbent cotton, swabs and loops. Deionized water (DW) was used to prepare the culture media and wash the silver nanoparticles.