Ringer s solution

Microbial enumeration was conducted according to the method described by Li et al. (2019) (link). Samples (25 g) were added aseptically to 225 mL of sterile Buffered Peptone Water (Merck, Darmstadt, Germany), and homogenized for 2 min within a stomacher (Interscience, Saint Nom la Bretèche, France). Decimal dilutions were prepared in Ringer’s solution (LabM, Bury, United Kingdom) for microbial enumeration. Then aliquots of 0.1 mL were spread on the following growth media for the following microbial viable counts: Aerobic plate counts on Plate Count Agar (PCA, LabM) incubated at 30°C for 3 days; Xerophilic fungi on Dichloran 18% Glycerol Agar (DG18, Hopebio), incubated at 25°C for 5 days; Yeasts and molds on Rose Bengal (RB, Lang Bridge), incubated at 25°C for 5 days.

The quantitative adhesion assay was performed as described previously, with minor modifications [26 (link)]. HT-29 cells were seeded in 24-well plates at a density of 35 × 10⁴ cells per well and incubated for 14 days to form a monolayer. A day prior to the treatments, the cell monolayer was washed with PBS and fresh medium without antibiotics was added. The next day, 10⁷ CFU/mL of viable Lactobacillus AGR 4 or L. casei ATCC 393 cells were added to each well, with each strain being tested in duplicate. After 4 h of co-incubation at 37 °C, the cells were washed with PBS and lysed with 1% Triton X-100 (Sigma-Aldrich). The lysates were serially diluted in Ringer’s solution (Lab M, Lancashire, UK), plated on MRS agar, and incubated at 37 °C for 72 h. For the calculation of adhesion values the following formula was applied: % Adhesion = (V_B/V_A) × 100, where V_A is the initial viable count of bacteria tested (10⁷ CFU/mL), and V_B is the viable bacteria count, obtained from the HT-29 cells. Colony forming units per milliliter (CFU/mL) was used as viable count measure that was determined with the formula: CFU/mL = (no. of colonies × dilution factor)/volume of culture plate.

The TSB and TSA for bacterial growth were purchased from Lab M Ltd. (Lancashire, UK). Both types of growth media were prepared under aseptic conditions, using standard procedures. The BSM used consisted of distilled water, 1 g/L K₂HPO₄, 1 g/L KH₂PO₄, 1 g/L KNO₃, 1 g/L (NH₄)₂SO₄, 0.1 g/L MgSO₄·7H₂O, 0.1 g/L NaCl, and 10 mL/L of trace element solution. The trace element solution contained: 2 mg/L CaCl₂, 2 mg/L CuSO4·5H2O, 2 mg/L MnSO₄·5H₂O, 2 mg/L ZnSO₄·5H₂O, 2 mg/L FeSO₄, and 2 mg/L (NH₄)₆Mo₇O₂₄·4H₂O. BSM salts were purchased from BDH Chemicals Ltd. (Poole, UK). The Ringer’s solution was purchased from Lab M Ltd. (Lancashire, UK) and a quarter strength tablet was dissolved in 500 mL of deionized water. All media were sterilized in an autoclave (Priorclave Ltd, London, UK) at standard conditions (121 °C for 15 min).

The assay was performed as described before, with minor modifications (Plessas et al., 2020 ). Briefly, human colon adenocarcinoma HT-29 cells were seeded in 24-well plates at a density of 40 × 10⁴ cells per well and incubated for 14 days to form a monolayer. The cells were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium enriched with GlutaMAX^TM, 10% fetal bovine serum (FBS), 100 μg/mL streptomycin and 100 U/mL penicillin (Thermo Fisher Scientific, Waltham, MA, United States) and incubated at 37°C, 5% CO₂ in a humidified atmosphere. 10⁷ or 10⁸ CFU/mL of viable L. pentosus L33 or L. rhamnosus GG cells were added to each well. After 4 h of co-incubation at 37°C, the cells were washed with PBS and lysed with 1% Triton X-100 (Sigma-Aldrich, Taufkirchen, Germany). The lysates were serially diluted in Ringer’s solution (Lab M, Lancashire, United Kingdom), plated on 2% MRS agar, and incubated at 37°C, until the formation of visible colonies. For the calculation of adhesion values the following formula was applied: % Adhesion = (V_B/V_A) × 100, where V_A is the initial viable count of bacteria tested, and V_B is the viable bacteria count attached on HT-29 cells. Colony forming units per milliliter (CFU/mL) was used as viable count measure that was determined using the formula: CFU/mL = (number of colonies × dilution factor)/volume of culture plate.

Tryptone soya agar (TSA) and tryptone soya broth (TSB) were purchased from Lab M Ltd. (Lancashire, UK) and prepared according to the manufacturer’s protocol. Both TSB and TSA media contain peptone (20 g/L) and glucose (2.5 g/L). All basal salts used in the preparation of BSM (low nitrogen content) were obtained from BDH Chemicals Ltd. (Dorset, UK) and prepared accordingly: 1 L of distilled water, 1 g/L K₂HPO₄, 1 g/L KH₂PO₄, 1 g/L KNO₃, 1 g/L (NH₄)₂SO₄, 0.1 g/L MgSO₄.7H₂O, 0.1 g/L NaCl, and 10 mL/L trace elements. The trace elements solution contained 2 mg/L CaCl₂, 2 mg/L CuSO₄.5H₂O, 2 mg/L MnSO₄.5H₂O, 2 mg/L ZnSO₄.5H₂O, 2 mg/L FeSO₄, and 2 mg/L (NH₄)₆Mo₇O₂₄.4H₂O. Ringer’s solution (Lab M, Lancashire, UK) was used as a saline solution for the analysis of viable cells during the cultivation process. To prepare this solution, a ¼-strength tablet was left to completely dissolve in 500 mL of deionised water with constant stirring. All media used in this study were sterilised by autoclaving (Priorclave Ltd., London, UK) for 15 min at 121 °C.

The TSB and TSA for bacterial growth were purchased from Lab M Ltd. (Lancashire, UK). Both of these growth media were prepared using aseptic techniques under standard conditions. The BSM used consisted of distilled water, 1 g/L K₂HPO₄, 1 g/L KH₂PO₄, 1 g/L KNO₃, 1 g/L (NH₄)₂SO₄, 0.1 g/L MgSO₄7H₂O, 0.1 g/L NaCl, and 10 mL/L of a trace element solution that contained 2 mg/L CaCl₂, 2 mg/L CuSO₄5H₂O, 2 mg/L MnSO₄5H₂O, 2 mg/L ZnSO₄5H₂O, 2 mg/L FeSO₄, and 2 mg/L (NH₄)₆Mo₇O₂₄ 4H₂O. The BSM salts used were obtained from BDH Chemicals Ltd. (Poole, UK). The Ringer’s solution used throughout the study was purchased from Lab M Ltd. (Lancashire, UK). A 1/4 strength tablet was added to 500 mL of deionized water and dissolved for regular colony counting (Section 2.4). All of the media mentioned were autoclaved (Priorclave Ltd., London, UK) at 121 °C for 15 min. From 25 mL starter flasks of TSB grown at 30 °C, 1 mL of inocula were dropped into 250 mL TSB media conical flasks to establish a narrow starting range of CFUs for comparison.