Sample collection: Thirty broiler chickens (Gallus gallus domesticus) were collected from Rayerbazar, Farmgate, New Market, and Azimpur in Dhaka City, Bangladesh. Lactobacillus species isolation: The Lactobacillus strains were isolated from jejunum, ileum, and caecum of chicken. Two cm portion of digestive tract was mixed with sterile saline buffer (0.85%, pH 7.0) and homogenized using a stomacher. Decimal dilution of these samples was inoculated on selective de Man, Rogosa and Sharpe (MRS) agar medium (HiMedia, India) at 45°C for 48 h under anaerobic conditions at anaerobic jar (Oxoid, UK). Then colonies with different morphology were randomly selected from the highest dilutions of each MRS agar plate and then sub cultured to acquire pure isolates. Strain identification and preservation: Citrate utilization, indole, nitrate reduction and motility test; arginine and gelatin hydrolysis were performed. Sugar fermentation was determined in carbohydrate consumption broth (HiMedia, India) and supplemented with 1% sugar. Seventeen sugars (arabinose, fructose, galactose, glucose, lactose, maltose, mannitol, mannose, melibiose, raffinose, rhamnose, ribose, salicin, sorbitol, sucrose, trehalose and xylose) were subjected to a fermentation test under anaerobic condition; each tube was topped up with two drops of sterile liquid paraffin after inoculation. The isolates were identified using standard morphological, cultural and biochemical reactions12. Gram positive and catalase, oxidase negative isolates were stored at “20°C in MRS broth supplemented with 20% (v/ v) glycerol. Reference microorganisms: Lactobacillus acidophilus ATCC 314, Lactobacillus rhamnosus ATCC 7469 and were used as reference microorganisms and as positive control. Escherichia coli ATCC 8739, Bacillus subtilis ATCC 11774 was used as negative control. Inhibitory substances tolerance: The probiotic characteristic was tested by using the sensitivity or resistance to low pH (2.5), sodium chloride salt tolerance (6.5%) and bile salt (2%) (Oxgall, Oxoid, UK). Acid and bile salt resistance can be considered important properties of probiotic lactic acid bacteria11,13. The physiological concentration of bile salts in the small intestine is between 0.2 and 2.0. The isolated lactobacilli were subjected to primary screening for acid, sodium chloride salt and bile salt tolerance in MRS broth adjusted to pH 2.5, 3, 4, 5 with 1N HCl 1 to 8% and 0.5, 1.0, 1.5, 2.0% with bile salt respectively. The determination of growth was performed by 1.0% bacterial suspension inoculated in MRS broth and observed after 18 h after anaerobic incubation at 45°C. The experiment was performed in duplicate and the mean values were calculated. Gastric juice tolerance: Twenty five isolated lactobacilli were subjected to primary screening to gastric juice tolerance (0.5% Bile Salt, 0.2% NaCl, 0.32% pepsin and pH 2.5) in MRS broth; control with MRS broth medium pH 6.2. The determination of tolerance was performed by 1.0% bacterial culture inoculated in MRS broth and the growth was observed after 18 h after anaerobic incubation at 45°C. Then absorbance was taken at 600 nm of 18 h culture. Heat tolerance: Four Lactobacillus isolates was inoculated at 1% in MRS broth medium and incubated at different temperatures such as 15, 20, 25, 30, 37, 40, 45 and 50°C for 18 h and the growth was monitored by measuring the absorbance value of broth at 600 nm. Temperature tolerance was observed at 55, 60 and 65°C for 10, 20 and 30 min in heating water bath (Clifton, England) with 2% Lactobacillus cultures in MRS broth medium. Proteolytic activity: The proteolytic activity was determined on skim milk agar. The strains were inoculated by 3 mm diameter and incubated for 24 h at 37°C. The proteolysis activity is characterized by the observation of a clear zone surrounding the colonies15. Antimicrobial activity: Agar spot test method16-17 and agar well diffusion method18 were used to detect of inhibitory activity of Lactobacillus spp. These assays were performed in duplicate. For the agar spot test, overnight culture of Lactobacillus spp. were spotted (3 mm) into the surface of MRS agar (HiMedia, India) plates and incubated in anaerobic jar for 48 h at 45°C to allow colonies to develop. Approximately 5×107 colony forming unit (cfu) of test microorganisms was swabbed in the plate in which Lactobacillus spp. was grown. After incubation for 24 h at 45°C, the radius of the clear inhibition zone around Lactobacillus spp. was recorded. For agar well diffusion method 4 wells in each plate of 4 mm in diameter were cut into tryptone soya agar (TSA, Oxoid,UK) plate by using a sterile borer and 100 µl of the cell free supernatant (centrifugation at 10,000×g for 5 min, 4ºC ) of the isolates were placed into different well. The cell-free supernatant was adjusted to pH 6.2 using 1N NaOH and it was used as crude bacteriocin The plates were preinoculated at room temperature for the diffusion and incubated aerobically overnight at 45°C. The plates were examined for zones of inhibition. Test organisms: For the detection of antimicrobial activity of Lactobacillus isolates against pathogenic microorganisms Listeria monocytogenes (2) ATCC 19112, Staphylococcus aureus ATCC 9144, Shigella flexneri (2b) ATCC 12022, Klebsiella pneumoniae ATCC 13883, Escherichia coli (environmental isolate), Vibrio parahaemolyticus ATCC 17802, Bacillus cereus ATCC 10876, Aspergillus niger, Acetobacter spp. Saccharomyces cerevisiae, Candida spp. Antibiotic susceptibility: Antibiotic susceptibility of strains of lactobacilli was determined in vitro using the Kirby-Bauer agar disc diffusion method19. The commercial antibiotic discs used in this study were AM = ampicillin (10 µg), AK = amikacin (30 µg), CIP = ciprofloxacin (5 µg), DO = doxycycline (30 µg), E = erythromycin (15 µg), CN = gentamycin (10 µg), IMP = imipenem (10 µg), NA = nalidixic acid, N = neomycin (10 µg), F = nitrofurantoin (300 µg), TE = tetracycline (30 µg), VA = vancomycen (antibiotic disks were obtained from Emapol, Poland). MRS cultures were suspended at approximately 108 cfu/ ml (McFarland standard 0.5) on Mueller-Hinton agar plates incubated for 24 h at 37°C. For susceptibility tests, clear zone recommended for consideration of susceptible or resistance of an organism.