DOI: 10.18150/mzlvwt
title: Immunomodulatory Properties of Live and Thermally-Inactivated Food-Origin Lactic Acid Bacteria – In Vitro Studies

description: In this study, the strain-specific immunomodulatory properties of live and thermally inactivated (TI) lactic acid bacteria (LAB) isolated from traditional Polish fermented foods were investigated to evaluate their potential as probiotics and postbiotics. Each strain was tested in at least three independent biological repetitions, divided into live and TI samples.Five LAB strains of different origins were isolated from traditional Polish fermented foods and obtained from the culture collection at Warsaw University of Life Sciences. The commercial probiotic strains Lacticaseibacillus rhamnosus GG and Lactiplantibacillus plantarum 299v served as reference strains. The bacteria were cultured in MRS broth and tested in both live and TI forms, with TI cultures prepared by heating at 90°C for 10 minutes.The immunomodulatory activity of these strains was assessed by measuring cytokine production in THP-1 macrophages. These cells were derived from the THP-1 monocyte line, cultured in RPMI 1640 medium with supplements at 37°C in 5% CO₂, and differentiated into macrophages using PMA (50 nM) for 48 h, then washed and maintained in antibiotic-free medium. Selected LAB strains (live and TI, 10⁶ CFU/ml) were added to macrophage cultures and incubated for 24 h. Some samples were additionally stimulated with LPS (1 µg/ml) after 4 h of incubation. Controls included macrophages without bacteria, with or without LPS. After 24 hours, supernatants were collected, and cytokine levels (IL-1β, IL-6, TNF-α, IL-10) produced by macrophages were measured using DuoSet ELISA Kits. Absorbance was recorded at 450 nm and corrected at 540 nm using a BioTek Synergy H1 microplate reader.The impact of LAB on epithelial barrier integrity was assessed using transepithelial electrical resistance (TEER) measurements in Caco-2 monolayers, which mimic mature intestinal enterocytes. Caco-2 cells were cultured in DMEM with supplements at 37°C in 5% CO₂. Cells were seeded onto polyester membrane inserts (0.4 μm pore size) and incubated for 21 days to form a differentiated monolayer, with the culture medium refreshed every other day. 24 h before bacterial exposure, the medium was replaced with an antibiotic-free medium. At time 0, baseline TEER was measured using a Millicell-ERS voltohmmeter. LAB strains (live or TI, 10⁶ CFU/well) or bacterial-free medium were added to the apical compartment, and TEER was measured at 8, 20, and 24 hours post-incubation. After 24 h, LPS (1 µg/mL) was added to the basolateral compartment, and additional TEER measurements were taken at 26, 30, and 34 hours (2, 6, and 10 hours post-LPS stimulation). Changes in TEER were expressed as the TEER Ratio, normalized to baseline values using the equation: TEER Ratio = (resistance – background resistance) × membrane area) / initial TEER.The antioxidant properties of bacterial metabolites were evaluated using ABTS and DPPH radical scavenging assays. LAB cultures (live and TI) were centrifuged, filtered, and the cell-free supernatants were used for analysis. For the ABTS assay, the radical solution (7 mM ABTS, 2.45 mM potassium peroxydisulfate) was diluted to an absorbance of 0.7 ± 0.02 at 734 nm, and mixed with sample dilutions (50 µL sample + 150 µL ABTS solution). Absorbance was measured after 6 minutes at 734 nm using a SpectraMax iD3 microplate reader. For the DPPH assay, the radical solution (DPPH in methanol, absorbance 1.1 ± 0.05 at 517 nm) was prepared and stored in the dark. Samples (10 µL bacterial supernatant + 190 µL DPPH solution) were incubated in the dark at 150 rpm for 30 minutes, after which absorbance was measured at 517 nm. Radical scavenging activity (%) was calculated as: Scavenging effect (%) = (Ac - As) / Ac × 100, where As is the sample absorbance and Ac is the control absorbance.Organic acids and sugars produced by the LAB strains were analyzed using high-performance liquid chromatography (HPLC). Samples were prepared similarly to those for antioxidant assays. The analysis was conducted using an HPLC system (Shimadzu, USA), equipped with an Aminex HPX-87H column (300 × 7.8 mm, Bio-Rad) maintained at 40°C, with a 10 mM H₂SO₄ mobile phase and a 0.5 mL/min flow rate. Separation was monitored using UV/Vis detection at 210 nm and RI detection, with quantification based on external standard curves (0.12–40 µg per injection of each analyte). Linear regression equations were generated from calibration curves relating peak area to analyte concentration.The dataset includes:Raw and calculated cytokine data from ELISA assays -&gt;The raw data from the microplate reader, including standard curves, supplemented with the plate layout and a description of the dilution rate (DR) for each sample, are provided in the files titled 'Raw [cytokine] plate layout...', while the concentrations calculated based on standard curves according to the manufacturer’s protocol and the dilution factors of individual samples are presented in the file 'Calculated cytokines...'.Raw and calculated TEER measurements from Caco-2 monolayer experiments -&gt; The raw data are provided in the file titled 'TEER raw results...', while the calculated data, processed according to the equation, are presented in the file 'TEER calculated...'.Antioxidant activity data (ABTS and DPPH assays) -&gt; The results are provided in the files titled 'ABTS results...' and 'DPPH results...'.Organic acid and sugar profiles from HPLC analysis -&gt; The results, containing the concentrations of analyzed compounds expressed in µg/mL, are provided in the file titled 'Organic acids...'.