The hypothesis of this work is to select qualified Lactobacillus spp. bacteria to propose as probiotic, and spotlight on particular properties such as S-layer and other properties that make Lactobacillus suitable candidates for probiotics.

Lactobacillus isolation from food sources was performed following (Perelmuter et al., 2008); one g of food sample was mixed in 10mL of sterile normal saline then, stirred vigorously and let the suspension settle for 10 min at room temperature. One hundred μL of the suspension was spread on previously prepared and sterile MRS medium (Oxoid LTD, limited, Hamshire, UK), all inoculated plates were incubated at 37 °C under aerobic conditions for 24 – 48 h. The growth colonies were purified and picked up for further analysis.

Lactobacillus isolates were tested to be based on utilizing various carbohydrates according to (Kandler and Weiss, 1986). The biochemical identification of the obtained isolates at the genus/species level was performed according to established phenotypic criteria, and for acid and gas production from glucose, arginine hydrolysis, and fermentation of L-arabinose, lactose, mannitol, melibiose, raffinose, ribose, sucrose, sorbitol, xylose, and salicin (Merk) as described by (Samells et al., 1995).

The minimal inhibitory concentration (MIC) of bile salt was evaluated for each of the Lactobacillus isolates, Then 20 μL of bacterial growth, 1 x 10⁸ (cfu.mL^-1), of each collected isolates were transferred to 980 μL of newly prepared sterile MRS broth containing 3% bile salts and incubated at 37 °C for 2 h.

For bacteria survival rate examination under the acidic condition at 37 °C, all isolates were tested following (Perdigon et al., 2002); about 20 μL of 1 x 10⁸ (cfu.mL^-1) of each fresh isolates were moved to 980 μL of an acidic buffer consisting of 3.5 g of D-glucose, 2.05 g from NaCl and 0.6 g of KH₂PO₄, 0.11 g of CaCl₂ and 0.37 g of KCl per liter, with adjusted pH to 2.0 and 4.5. Then, isolates were examined for tolerance to bile salt and acid when samples were taken at 0 min and 2 h and plated on freshly prepared sterile MRS agar plates after making of serial dilution, and plates were incubated at 37 °C for 48 h, the plates were enumerated by choosing a couple of plates (30 – 300 colony) for each dilution.

Lactobacillus antimicrobial activity was determined through the agar spot method, following (Casey et al., 2004; Baccigalupi et al., 2005), with minor modifications; briefly, about 5 μL of (1 x 10⁸ cfu.mL^-1) of freshly prepared growth to each obtained isolate was plated into agar spots made previously on the MRS agar plate. All inoculated plates were incubated at 37 °C for the development of spots. Each of the indicator bacteria (bacteria spp. used is mentioned below) was inoculated on Luria broth and incubated overnight at 37 °C with shaking. Then, the bacteria were mixed with 15 mL of Luria soft agar (0.7% agar, w/v) and dispensed onto MRS agar plates that were full of grown colonies of Lactobacillus. All plates were incubated overnight at 37 °C and the inhibition zones were subjected to measure and record (Bauer et al., 1996). The indicators were Shigella dysentery, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, and Escherichia coli.

Anti-Bacterial sensitivity of lactobacilli in vitro was studied via the disc diffusion method according to the guidelines of the National Committee for Clinical Laboratory Standards (Wayne, 2015). It was studied against eight antibiotics: Ampicillin 25 μg, Chloramphenicol 50 μg, Gentamycin 10 μg Carbenicillin 100 μg, Nalidixic acid 30 μg, Methenamine Mandelate 3 μg, and Cortimoxazole 25 μg.

For extraction of S-layer, Lactobacillus spp. was cultivated anaerobically in MRS broth at 37 °C. In general, 100 mL of pre-warmed MRS medium was inoculated 1: 100 (v/v) with an overnight culture and cultivated until the optical density at 600 nm reached 0.4 and 0.7. Cells were harvested by centrifugation at 12000 ×g for 15 min at 4 °C. The cells were washed twice with 100 mL of ice-cold water. The cell pellet was extracted with 0.1 volume of 4 M guanidine hydrochloride (pH 7) for one hour at 37 °C and centrifuged at 12000 ×g for 15 min. The supernatant, containing S-layer protein monomers, was dialyzed against water at 4 °C for 16 – 24 h (Boot et al., 1993). The dialyzed extracts were analyzed by SDS-PAGE (Smit et al., 2001). SDS-PAGE of protein samples was carried out using Precision Plus Protein Standard (low molecular weight marker (10 – 250 kDa) – Hi-media). The samples were run on 12% polyacrylamide gel at 100 V. Protein bands were visualized by Coomassie blue staining. Protein concentration was determined according to Bradford's method (Bradford, 1976). For normalization of the measured absorption values, BSA (Hi-media) was used. Finally, all samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) following Laemmli method on a vertical Slab gel (Laemmli, 1970); Acrylamide gel in form of resolving 12% and stacking gel 4% was prepared and the samples were run at 100 V. Protein bands were seen after gel stained with Coomassie Brilliant Blue stain (Merck) R-250 (Dunn, 1993).

Based on the isolation of Lactobacillus spp., the bacteria were positive to grow on MRS selective media, and when examined under the light microscope, all isolates acquired blue-purple stain hence they react positively when stained with Gram. Also, based on biochemical tests displayed in Table 1, all isolates were negatively reacting with Catalase. Results of gas production from glucose to all inoculated test tubes after observation for 2 – 3 days showed that all isolates were positive for gas production from glucose fermentation. Findings of Carbohydrates fermentation analysis were listed in positive and negative reactions. Outcomes of testing the ability of isolates to grow at different temperatures showed the ability of all isolates to grow at 45 ºC whereas no growth was seen at 10 °C and 15 °C.

From the different concentrations of bile salts taken, 3% bile salts in MRS broth was the minimal inhibitory concentration of bile from most Lactobacillus isolates as shown in Table 2 and Figure 1, hence the survival of lactobacillus post 2 h. Incubation in MRS containing 3% bile salts was analyzed and the results are shown in Table 2, isolate of L. delbrueckii showed an excellent survival of 89%, LGG showed the ratio of survival 91%. Also, lactobacillus survival rates in acidic buffer with pH 4.5 and 2.0 were examined by the difference in viable cell counts following 0 min 2 h of incubation as shown in Table 3 and Figure 2. The isolated L. rhamnosus 6.90% at pH 4.5, 5.12% at pH 2.0, showed the highest survival in acidic pH, immediately followed by L. plantarum, L. delbrueckii, and L. fermentum5.70, 5.40 and 4.03% at pH 4.5, 4.90, 4.31 and 2.33% at pH 2.0 respectively.

Based on bile salt and acid tolerance: different concentrations of bile salt in MRS broth were prepared and tested on lactobacillus isolates to determine the minimal inhibitory concentration of bile salt tolerance as shown in Table 2 and Figure 1. Results showed that MIC was 3%. The vitality of lactobacillus post 2 h, incubation in MRS containing 3% bile salts that L. delbrueckii showed an excellent survival ratio of 89%, LGG showed a ratio of survival 91%. Also, lactobacilli survival rates in acidic buffer with pH 4.5 and 2.0 were examined by the difference in viable cell counts following 0 min 2 h, incubation as shown in Table 3 and Figure 2.

The isolated L. rhamnosus showed the highest survival in acidic pH; 6.90% at pH 4.5, 5.12% at pH 2.0, immediately followed by L. plantarum, L. delbrueckii, and L. fermentum showed; 5.70, 5.40 and 4.03 % at pH 4.5, 4.90, 4.31 and 2.33% at pH 2.0, respectively. Lactobacillus bacteria colonized the hindgut region in the human host and should be able to survive with the acid condition and bile salt before reaching the target tissue.

Therefore, tolerance of Lactobacillus to acid and bile salt equally is an important issue to select Lactobacillus bacteria as a probiotic.

Our result of this study agreed with the outcomes of other researches, they reported a survival rate of Lactobacillus isolates in pH 2.5 (Kim et al., 2007; Perelmuter et al., 2008). Nevertheless, the survival rate of Lactobacillus has not been more than 0.2%.

The Findings of antimicrobial activity are presented in Table 4; all collected Lactobacillus isolates showed antagonistic activity in various degrees when examined against gram-negative bacteria and gram-positive bacteria.

The standard strain LGG and the Lactobacillus isolates showed the highest inhibitory activity against Staphylococcus aureus, and Shigella dysenteriae whereas, only L. fermentum, L. fermentum, and L. rhamnosus displayed the highest inhibitory activity against E. coli. Strain LGG and the collected isolate L. rhamnosus belong to the same species, but they showed a difference in their antimicrobial activity since antimicrobial characteristics should be of a specific strain. Once they colonize the hindgut of the human intestine, they loss their antimicrobial activity in the acidic environment (Perelmuter et al., 2008; Enan et al., 2013). They are isolated, identified, and characterized seven small peptides by LGG that have a mass of about 1 kDa and that exert a bactericidal action against both Gram-positive and Gramnegative bacteria (Lu et al., 2009). While Lactobacillus spp. suppose to be getting their opportunity to display their antimicrobial activity due to production elements as; lactic acid, H₂O₂, and bacteriocins other antibacterial molecules (Eschenbach et al., 1989; Kučerová et al., 2007).

The result of this study agrees with that recorded by (Perelmuter et al., 2008; Casy et al., 2004), they had been observed a range of lactobacilli antimicrobial responses towards E. coli and bacteria besides, they approved the inhibitory activity of Lactobacillus spp. affected by the acidic environment. These results are following those reported by (Rodes et al., 2013).

Outcomes of antibacterial sensitivity of Lactobacillus isolates were revealed in Table 5: all collected isolates of food source were resistant to Chloramphenicol 50 μg whereas, all of them showed high sensitivity to; Ampicillin 5 μg, Nitrofurantoin 50 μg, Gentamicin 10 μg, Carbenicillin 100 μg, Nalidixic Acid 30 μg, Cotrimoxazole 25 μg and Methenamine Mandelate 3 mg. The safety of Lactobacillus isolates must be carefully evaluated. One safety aspect is the transfer or acquisition of antibiotic resistance. Because nonpathogenic enteric bacteria may also be a source for resistance genes that may spread to potential pathogens. Therefore, investigation activities to obtain Lactobacillus isolates for probiotic uses should include nonpathogenic as well as pathogenic bacteria (Katla et al., 2001). Lactobacillus isolates were collected in this research qualified to use as probiotics because they exhibited minor antibiotic resistance. Thus, it is most important for the selection of probiotic strains without any highly transferable antibiotic resistance or especially virulence mechanisms (Klein et al., 2000). Results obtained in this study agree with the findings reported by (Katla et al., 2001; Klein et al., 2000).

The result of the detection S-layer protein profile through SDS-PAGE has demonstrated in Figure 3: SDS-PAGE protein of Lactobacillus isolates was quite similar except the L. rhamnosus produced patterns containing band about 37 – 40 kDa., according to the previous studies, one dominant band of 43 – 46 KDa, which is known as the Sprotein (Boot et al., 1993; Smit et al., 2001). In our study, S-layer extraction was carried out according to Boot's method (Boot et al., 1993). The S-layer proteins characterized from the genus Lactobacillus range from 25 to 71 kDa in size (Sára and Sleyrtr, 2000). The Putative S-layer protein was detected by whole-cell SDS-PAGE analysis. S-layer analysis revealed the presence of a unique and single S-layer with an oblique structure. The whole-cell protein patterns of the Lactobacillus isolates are fairly homogeneous with some variability, primarily localized in the molecular mass region estimated 30 – 70 kDa.