Purpose: The goal of this study was to determine the mode of protective effect from bioactive molecules produced by L. helveticus (LH-2) and L. acidophilus (La-5) against Salmonella Typhimurium infection.
Methods: Bioactives were produced after 48 hours fermentation in milk or whey protein-based media for LH-2 and La-5, respectively. Colonic carcinoma HT-29 cells were grown in Transwell inserts for 40 days until polarized (~120 ohm×cm2). Cell membrane integrity was measured through transephithelial electrical resistance (TEER). The nontoxic doses of bioactives were determined using trypan blue exclusion/SRB assay. Bioactives were pre-incubated 24 h prior to Salmonella infection and co-incubated during Salmonella infection. Lactate dehydrogenase (LDH) activity was tested using Abcam® LDH-cytotoxicity assay kit. Apoptosis was measured by flow cytometry with BDTM APO-Direct kit. Finally, an invasion assay was carried out using chicken hepatoma LMH cells as an in vitro model for Salmonella presence in poultry.
Results: The protein concentration of 370μg/ml LH-2 and 1.5mg/ml La-5 were nontoxic for the epithelial cells. LH-2 and La-5 exhibited protective effects of TEER (P < 0.05), LDH and apoptosis (P < 0.01) against S. Typhimurium on polarized HT-29 cells. In infected polarized HT-29 cells, LDH was reduced up to 49.1% in presence of LH-2 and up to 46.8% when La-5 was present. In terms of apoptosis, compared with positive control 62.5% and 75.8% less cells underwent apoptosis with LH-2 and La-5 treatment, respectively. Salmonella invasion of LMH cells was reduced less than one log cycle.
Significance: These data suggest that LH-2 and La-5 bioactives reduce the amount of cells undergo apoptosis following Salmonella infection.