Evaluation of probiotic properties of predominant lactic acid bacteria isolated from the gastrointestinal tract and reproductive system of Ross 308 broiler breeders

Probiotic bacteria are Gram-positive and negative-catalase with various characteristics including resistance to acid and bile salt conditions, antimicrobial characteristics, bacitracin production, and lack of capability for transferring genes resistant to antibiotics. These bacteria are a part of selected and useful bacteria in the digestive system, which leads to reinforcement of the body's immune system if they are adequately consumed (106-107 CFU/g). This research aimed to identify molecular identification and assessment of probiotic characteristics of lactic acid bacteria (LAB) isolates separated from the digestive and reproduction system of  Ross 308 broiler breeders.
Twenty Ross 308 broiler breeders were selected and samples of the vagina and ileum of them, and the cecum and feces of roosters were taken to separate LAB. The Gram and catalase test was used to approve the biochemical characteristics of LAB. The suspension containing each LAB isolate was harvested at 4  for five min (10,000×g) to assess the survival of the selected LAB isolated under conditions simulating the GI tract. Then, remained sediment in the buffer solution containing HCL (1N) reached to pH equal to two by eliminating the supernatant. After adding 0.1 % (w/v) pepsin to bacterial suspension, it was stored at 37  for three hours. After incubation, the pH of suspension with NaCl (1N) was reached to six. Then, tolerance to small intestine condition was evaluated in PBS solution (pH=7), containing Oxgall (0.3% w/v) and pancreatin (0.1% w/v) of washed suspensions of the LAB was mixed and incubated at 37 . Finally, the LAB isolate population was determined by consecutive dilution in sterile PBS and plate media on MRS agar compared to a blank sample (untreated). For molecular identification of dominant lactic acid bacteria, the DNA of the dominant lactic acid isolate was extracted by polymerase chain reaction (PCR) kit. Then, it was amplified using primers (44F; 1542 R) in temperature conditions. Afterward, for initial approval, PCR products were transferred to 1.5 % agarose gel and electrophoresis was performed in TBE buffer in the presence of positive control and negative control samples. The good diffusion method was used to determine the anti-bacterial effect of the selected LAB isolated against pathogenic factors. To assess auto-aggregation of the selected isolate, the cells obtained from its 24-hour culture were separated by refrigerated centrifugation (10 min, 4 , 6000×g) and was dissolved in phosphate buffer during two phases (pH=7.2) so that the obtained suspension had absorption equal to . Then, the suspension was put at the temperature of 37  for four hours. Then, the absorption of LAB isolate suspension in 600 nm was read. A combination of an equal volume of LAB isolates suspension and pathogenic bacteria were vortexed and incubated at 37℃ for four hours to assess auto-aggregation of isolate. The surface part of the suspensions was read at 600 nm and calculated. About 200 µl of 24 h culture of selected LAB was added to four mL of 1 % MRS agar medium to evaluate the selected LAB isolated antibiotic susceptibility. This combination was overlaid on plates containing 1.5 mL of 1.5% MRS agar, and then, discs of antibiotics including Ampicillin, Gentamicin, Streptomycin, Cefazolin, Ciprofloxacin, Penicillin, Cephalothin, Imipenem, Novobiocin, Clindamycin, Vancomycin, Ceftriaxone, and Nalidixic acid was placed on each plate. After 24 h of incubation at 37℃, the diameter of the inhibition zone (mm) was measured and reported as resistant, relatively sensitive, and sensitive. To consider the capability of hemolysis of blood, LAB isolate was streaked on the surface of a blood agar plate supplemented with 5% sheep blood. After 48 h of incubation at 37℃, the plates were considered in terms of diameter inhibition zone creation and color change in the medium.
The results of the sequencing test led to the identification of Levilactobacillus brevis. The predominant LAB isolated from the gastrointestinal tract (GIT) and reproductive tracts of Ross 308 broiler breeders and L. brevis isolated from the ileum (82.00%) (P<0.05) in the simulated conditions of the GIT compared to the L. brevis isolated from other parts GIT and the reproductive system had more survival than acid and bile. Examining the anti-bacterial effect of the aforementioned isolate showed that the L. brevis bacterium had an inhibitor influence on Shigella dysentery bacteria (87.00%) and Staphylococcus aureus (81.25%). In addition, the highest and the lowest characteristics of co-aggregation of this isolate were observed against Listeria monocytogenes (46.00%) and Salmonella typhimurium (38.00%). This isolate had 43% auto-aggregation characteristics and no hemolytic activity. Assessing antibiotic resistance of LAB isolate showed that the biggest diameter of inhibition zone of bacterium (23.5 mm) was related to Imipenem that had no significant difference with Ampicillin (22.5 mm) and Vancomycin (22.5 mm) and its lowest value (12 mm) was related to Ceftriaxone that had no significant difference with Gentamicin and Cephalotin. The results of this study showed that L. brevis bacterium can be used in the nutrition of broiler breeders as a probiotic bacterium.
The use of probiotics in the feeding of Ross 308 broiler breeders may eliminate the public health concerns of antimicrobial resistance development to some extent, as this could replace the use of antibiotics. According to antimicrobial characteristics, antibiotic resistance, hemolytic activity, auto-aggregation, and co-aggregation of predominant LAB isolate, it can be concluded that L. brevis can be useful and applicable as a probiotic supplement in producing food and pharmaceutical products for broiler breeders.