Abstract:

【Objective】In order to screen safe lactic acid bacteria (LAB) strains with probiotic properties, LAB were isolated in this study for further developing probiotics and providing resources for animal related products such as feed additives. 【Method】 A total of 13 samples of wild boar feces were collected from Greater Khingan Mountains in China, and the samples were quickly returned to the laboratory at 4°C for isolation and purification of LAB. The genomic DNA of the isolated Gram-positive bacteria was extracted by bacterial genomic DNA extraction kit. After 16S rDNA identification, the sequence was compared with information in GenBank database using BLAST, and the classification status of each strain was determined. The tolerance capacity of LAB was evaluated under acidic pH condition (pH 3.0) and bile salt (0.3%). The LAB was cultured overnight, observed and determined at different time, and then the autoaggregation ability was evaluated. The strains cultured overnight were mixed with Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium at room temperature for coaggregation test. In vitro, the adhesion ability of LAB to Caco-2 cells and IPEC-J2 cells was measured and evaluated. The anti-pathogenic activities were detected by measuring the bacterial inhibition rings on plates of LAB against E. coli, S. aureus, and S. typhimurium. Evaluation of the safety of LAB strains by in vivo and in vitro tests. LAB isolates were cultured in MRS medium for 18-24 h at 37℃. Streak plate methods were performed on sheep blood agar plates to analyze hemolytic activity. The absence/presence of virulence factor genes for the isolated strains was performed using PCR amplification with primers. After 21 days of continuous LAB supplementation, the parameters of general health status including body weight gain and organ index were calculated to assess the safety of the LAB in vivo. 【Result】Three LAB strains isolated from wild boar feces were Enterococcus mundtii, Enterococcus durans, and Lactobacillus mucosae, with the excellent tolerance to acid and bile salt. The results showed that these three strains showed strong adhesion and aggregation ability, and the antimicrobial effect of L. mucosae on the three pathogenic bacteria was better than others. The result of safety evaluation demonstrated that these strains were free of hemolytic activity, and no virulence genes could be detected. The percentage of body weight gain of the mice treated with L. mucosae was significantly higher (P<0.01) than that of the control mice on day 21. 【Conclusion】Taken together, the results indicated that three probiotic strains, especially L. mucosae, had good probiotic properties and safety, so this study provided a scientific basis for further development of probiotic preparations.

Key words: wild boar, lactic acid bacteria, aggregation, adhesion ability, safety