Characteristics of Lactic Acid Bacteria Intended for Probiotic Use from Indigenous and Commercial Pig Feces in Thailand

Lactic acid bacteria (LAB) are currently applied as probiotics due to their ability to survive in gut environment and antagonistic effects toward enteric pathogens. Difference of species and strains of LAB directly relate to host type in each geographical area. However, there has still been inconsistency between genetic and phenotypic identifications. This study aimed to determine and validate identification methods for probiotic candidate selection between phenotypic; morphological and biochemical characteristics, protein profile analysis, genetic analyses; complete 16S rRNA gene sequencing and antimicrobial susceptibility using EFSA criteria.

60 fecal samples were obtained from antibiotic-free healthy fattening indigenous pigs (N=30) and commercial pigs (N=30). LAB were isolated from the feces and screened for probiotics by acid and bile tolerance. The selected LAB were identified according to phenotypic and biochemical characterizations (Gram’s staining, motility test, catalase production, ability to grow at 50ºC and 21 types of sugar assimilations), whole-cell protein profile by SDS-PAGE and complete 16S rRNA gene sequencing. The identified LAB were test suitability to use as probiotics following EFSA criteria using agar-disk diffusion test and minimum inhibitory concentrations (MICs).

A total of 204 LAB were obtained from all pigs. 34 strains were selected as putative probiotics by screening criteria including acid and bile tolerance. According to SDS-PAGE and 16S rRNA gene sequencing, they were identified as Enterococcus faecium (N = 11), Enterococcus hirae (N =9), Lactobacillus agilis (N = 3), Lactobacillus plantarum (N = 4), Pediococcus acidilactici (N = 1), and Pediococcus pentosaceus (N = 6). After screening antimicrobial susceptibility by agar-disk diffusion test and confirming by MICs, only five isolates (1 P. acidilactici, 1 P. pentosaceus and 3 L. plantarum) from 34 selected LAB were suitable to use as probiotic.

This study recognizes that mannitol assimilation is the biochemical marker for differentiation of E. faecium and E. hirae, and growth ability at 50ºC to separate P. pentosaceus from P. acidilactici. By using SDS-PAGE, whole-cell protein patterns of LAB species-specific were demonstrated into different clades which agree with the phylogenetic tree of 16S rRNA gene sequencing; therefore it can be used as a consensual phenotypic screening tool for LAB species identification. The identification results reveal that E. faecium and E. hirae were the most prevalence putative probiotics in feces of indigenous pigs, whereas P. pentosaceus were the most common in feces of commercial pigs.

Disclosure of Interest: None Declared.