Bacterial Strains

The study provides a safety assessment of 65 LAB strains potentially useful as probiotics and other feed additives. Fifty-seven Lactobacillus strains [Lactobacillus plantarum (n = 26), Lactobacillus fermentum (n = 7), Lactobacillus casei (n = 3), L. rhamnosus (n = 3), Lactobacillus reuteri (n = 3), Lactobacillus brevis (n = 3), Lactobacillus buchneri (n = 2), Lactobacillus salivarius (n = 2), Lactobacillus agilis (n = 2), Lactobacillus acidophilus (n = 1), Lactobacillus johnsonii (n = 1), Lactobacillus diolivorans (n = 1), Lactobacillus delbrueckii (n = 1), Lactobacillus paracasei (n = 1), Lactobacillus farraginis (n = 1)], six Pediococcus strains [Pediococcus pentosaceus (n = 5), Pediococcus acidilactici (n = 1)], and two Enterococcus strains [one Enterococcus durans strain and one Enterococcus faecium strain] were selected for this study (Supplementary Table 1). A total of 47 strains are available at the culture collections: 42 strains at the Collection of Industrial Microbial Cultures (KKP), located at the prof. Waclaw Dabrowski Institute of Agricultural and Food Biotechnology (IAFB) in Warsaw (Poland), four strains at the Polish Collection of Microorganisms (PCM), located at the Institute of Immunology and Experimantal Therapy in Wroclaw (Poland) and one strain from American Type Culture Collection (ATCC). The rest 18 strains were isolated from fermented or fresh vegetables and fruits (n = 14) or probiotic drinks (n = 4). The isolates were identified by nucleotide sequence analysis of the gene encoding 16S rRNA. LAB strains belonging to the L. plantarum phylogenetic group (L. plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum) were differentiated by multiplex PCR using species-specific primers amplified the fragment of the recA gene encoding the recombinase A (22). The strains isolated from the same sources were typed by RAPD-PCR (Random Amplified Polymorphic DNA) with primers RP and PRIMO2 (23) in order to confirm their intraspecies diversity (data not shown). All strains were stored in a liquid nitrogen atmosphere in MRS (deMan- Rogosa-Sharpe) broth (Oxoid) supplemented with glycerol (15% v/v). Before the antibiotic susceptibility assay, LAB strains were cultivated in MRS agar (Oxoid) at 37°C for 24–48 h in 5% CO₂. After incubation, the colonies were suspended in 0.85% NaCl solution to prepare the inoculum for the broth microdilution test.