Isolation of Lactic Acid Bacteria
Various samples were taken from different fermented products in west of Algeria (olives, cow’s and camel’s milk, butter, maize silage and beer drech), and used as bacterial sources. The dilution plate method, on acidified deMan Rogosa and Sharpe (MRS, pH 5.4) agar medium, was used to select LAB and promoting the selection of the bacilli form. Plates were incubated at 37°C for 24h to 48h under anaerobic conditions (anaerobic jar) and all candidates that corresponded to LAB appearance were selected and purified by sub culturing, then used to verify their probiotic capacity.

Molecular Identification of the Isolates
Total DNA Extraction
Total DNA was extracted from 2ml overnight cultures. After centrifugation at 12000g for 5min, cells were collected and washed twice, with sterile distilled water. The pellets were resuspended in 400µl of lysis buffer (2% Triton X-100, 10mM NaCl, 10mM Tris-HCl, pH 8, 1 mM EDTA, 3% SDS) containing lysozyme 3 mg/ml, and then incubated for 1 h at 37°C. Suspensions were grinded for 2min with 0.6g of sterile beads and 400µl of phenol /chloroforme (V/V). Phases were separated by centrifugation for 5 min at 12000g, then, in a new Eppendorf microtube, the aqueous phase was carefully mixed with 1 µl of RNase (10 mg/ml) and incubated for 15min at 37°C. Equal quantity of phenol/ chloroform was added to the aqueous phase, then, the mixture was vortexed and centrifuged for 5min at 12000g. The aqueous phase was transfered in another Eppendorf in order to precipitate DNA with 40µl of sodium acetate 3M pH 7 and 800µl of absolute cold ethanol with incubation for 30min on ice, followed by a centrifugation at 4°C for 10min at 12000g. DNA was washed with ethanol 70% and centrifuged at 12000g for 5min, then, dried during 5 min and resuspended in 50µl of Tris-EDTA. Finally, Total DNA was stored at –20°C.

PCR Amplification of 16S rRNA Gene
PCR amplification of the 16S rRNA gene was carried out using primers: BSF8-20 (5’-AGA GTT TGA TCC TGG CTC AG -3’) and BSR1541-20 (5’-AAG GAG GTG ATC CAG CCG CA- 3’), with a total volume of 50 ìl containing: 2.5µl of DNA, 25µl of Mix DNA enzyme (2X), 5µl of each primer, and 12.5µl of sterile water. PCR conditions were: initial DNA denaturation at 94°C for 2 min followed by 29 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s and elongation at 72°C for 1 min, then a final cycle at 72°C for 5 min. Amplicons were purified using Wizard SV Gel and PCR Clean-Up System and Sequencing. The purified PCR products were sequenced then the obtained sequences were compared to those in the GenBank database using the BLAST algorithm.

Acidity and Artificial Gastric Conditions Tolerance
Overnight cultures were centrifuged at 12000 g for 10min then the pellets were washed three times in sterile phosphate saline buffer (PBS) pH 7.4, and cultured on MRS agar pH 3.5. The viability of cells was recorded after 24h of incubation in anaerobic atmosphere at 37°C.

Isolates were tested for their capacity to grow after both 2h and 18h of incubation, on a broth simulating gastric conditions (NaCl 125 Mm/L, KCl 7 Mm/L, NaHCO₃ 45 Mm/L, pepsin 3 g/L). Overnight cultures were centrifuged at 12000 g for 15min at 4°C, then washed twice with phosphate buffer (50 Mm/L, pH 6.5), and resuspended in 3ml of the same buffer. One milliliter of this suspension was pelleted at 12000g for 5min at 4°C, and finally resuspended in 10 ml of artificial gastric solution. The final pH was adjusted to pH 2 and pH 3, and anaerobic conditions were maintained.^10,11

Bile Tolerance
Two different methods were used to verify the strains resistance to bile
The first method consisted in inoculating bacteria on MRS broth supplemented with 0.3% natural bovine bile pH 4 for 24h at 37°C in anaerobic conditions. The same bacteria were inoculated for the second time on MRS agar pH 5.8 for 72h, to evaluate the tolerance on bile salt.¹²

In the second method, the isolates were inoculated in MRS broth (pH 4 supplemented with 0.3% of bile salt) and incubated at 37°C in anaerobic conditions, once for 3h, and once for 24h. The suspensions were re-suspended in MRS broth without bile (pH 5.8), to evaluate the survival percentage of LAB by the measure of the optical density (OD) at 600 nm.

The percentage of the growth was calculated as follows:

Survivalrate = OD of MRS with bile / OD of MRS without bile × 100
Bile resistance corresponds to a percentage superior to 50%.^10,13

Antibiotic Susceptibility
To test isolates susceptibility to antibiotics, the disc diffusion method was employed. Overnight cultures were inoculated on Muller Hinton agar. After solidification, different antibiotic discs including: vancomycin (30µg), pristinamycin (15µg), nalidixic acid (30µg), oxacilin1 (1µg), rifampicin (30µg), streptomycin (10µg), penicillin (6µg), imipenem (10µg), cefazolin (30µg), piperacilin (75µg), gentamicin (10µg), cefsulodin (30µg) and colistin (50µg), were placed on the medium surface. The plates were then incubated at 37°C for 24h to 48h on anaerobic conditions. Results were expressed as sensitive (S) or resistant (R) according to the National Committee for Clinical Laboratory Standards¹⁴.

Antibacterial Activity
To detect the antibacterial activity, the LAB cultures were confronted to various pathogenic bacterial strains: Escherichia coli, Staphylococcus aureus (ATCC 43300), Staphylococcus aureus (ATCC 25923), Pseudomonas sp. and Listeria ivanovii. The LAB were inoculated using spot-on-the-lawn technique, on plates containing 15 ml of MRS agar, and then incubated at 37°C for 18h in anaerobic conditions. The pathogenic cultures used as indicators and inoculated in appropriate semi-liquid media at a rate of 1 % (Muller-Hinton for E. coli and Staphyloccocus, Brains Heart Infusion for Listeria and King A for Pseudomonas), were used to overlay the LAB colonies, and then incubated for the second time aerobically at 37°C. The antibacterial activity was determined by the presence of a clear zone around the colonies.^15,16

In Vitro Adherence Test to Epithelial Cells
Intestinal Epithelial Cell Suspension
Chicken intestines were used for this assay. After washing them several times in the PBS buffer until clarification, the intestinal segment was opened and held in cold PBS 0.1 M, pH 7.2, supplemented with 1% penicillin-streptomycin for 1 h under shaking. Intestinal epithelial cells were collected by scraping the intestinal wall in Dulbecco’s Modified Eagle Medium (DMEM) pH 7 supplemented with 1% penicillin-streptomycin, and held for 30 min in this medium at 4°C under agitation. Cell suspensions were centrifuged at 6000 g for 10min, the pellet was washed three times in cold PBS pH 7, then resuspended in DMEM and conserved at 4°C.^16,17

Bacterial Suspensions
Overnight cultures of LAB, were centrifuged at 12000g for 10 min and washed twice in sterilized cold PBS 0.1 M, pH 7.2, then held in the same buffer.

Adherence Assay
For adhesion assay, 1ml of epithelial cells (10⁵ 10⁶ cell/ ml) was mixed with 1ml of bacteria cells (10⁷ ­10⁸ CFU/ ml) and incubated at 37°C for 24h. This mixture was pelleted at 6000 g for 10 min and washed three times with PBS then filtered on 0.45µm sterile filter to remove nonadherent bacteria. Finally, the filter was washed in 10ml of DMEM. The In vitro adhesion of LAB to epithelial cells was observed directly with photonic microscopy using oil immersion, after staining with methylen blue. The adherence was also examined by Analytic Scanning Electron Microscopy (JEOL JSM-6610LA) after dehydratation only in a graded series of ethanol, without fixation step^{16, 17}.