Antibacterial and Cytotoxic Effect of Bacteriocin Produced by Lactiplantibacillus plantarum

Abstract

Lactobacillus spp. was isolated from 253 children's stool samples to achieve the bacteriocin antibacterial activity and cytotoxic effect. Lactobacillus spp. isolates were identified by morphological and biochemical tests, then screened primarily and secondarily for bacteriocin production using pathogenic bacterial isolates gained from diarrheal patients as indicators (Salmonella typhi, Shigella spp., and Aeromonas hydrophilia). Molecular detection for the 16S rRNA gene responsible for this bacterium, sequencing, and BLASTN analysis were performed, the sequences were submitted to GenBank. The bacteriocin produced under the optimum conditions was extracted and purified using ammonium sulfate, ion exchange, and gel filtration chromatography (Sephadex G-150) methods. Minimum inhibitory concentration (MIC) and MTT assay were determined to investigate bacteriocin antibacterial activity against the three pathogenic indicators and anticancer activity against colon cancer cells (Caco-2), respectively. One hundred and eleven Lactobacillus spp. isolates were obtained based on morphological and biochemical tests; of them, ninety-seven (87.4%) isolates exhibited antibacterial activity in primary screening; however, only eighteen of the ninety-seven isolates (18.6%) demonstrated bacteriocin production effectiveness in secondary screening. The molecular diagnosis of these18 isolates revealed that only 15 belonged to Lactobacillus spp. Their sequencing analysis showed that two isolates displayed 100% similarity with reference Lactiplantibacillus plantarum, four isolates showed 100% similarity with reference Limosilactobacillus fermentum, and 2 isolates exhibited 100% similarity with reference Lacticaseibacillus rhamnosus. Lactiplantibacillus plantarum (SMHA16) isolate showed the most bacteriocin production activity (160AU/mL) and higher production at pH 6.0, 32°C, and 48 hrs. of incubation. The bacteriocin extracted from this isolate was purified, and the final purification fold was 21-fold with a 48.1% yield. Bacteriocin antibacterial activity (MIC) against Shigella spp. was 256 µg/mL, while against Salmonella typhi and Aeromonas hydrophila was 512 µg/mL. The cancer cells (Caco2) revealed less viability at 400 μg/mL of bacteriocin concentration (IC50 value of 135.9 μg/ml), while the effect was less on the normal cells (IC50 value of 157.9 μg/ml), which indicated the anticancer activity of bacteriocin against colon cells. This work indicates the promoting role of bacteriocin as antibacterial and anticancer agent.