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

Document Type : Research Paper

Authors

1 Ph.D. Student, Department of Animal Physiology, Faculty of Animal Science, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran

2 Associate Professor, Department of Animal Physiology, Faculty of Animal Science, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran

3 Associate Professor, Department of Food Science and Technology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran

4 Professor, Department of Food Materials and Process Design Engineering, Faculty of Food Science and Technology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran

5 Associate Professor, Department of Biology, Gorgan Branch, Islamic Azad University, Gorgan, Iran

6 Professor, Department of Animal Science, University of Mohaghegh Ardabili, Ardebil, Iran

Abstract

Introduction: 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.
Materials and methods: 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.
Results and discussion: 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.
Conclusions: 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.

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Abnous, K., Brooks, S. P., Kwan, J., Matias, F., Green-Johnson, J., Selinger, L. B., & Kalmokoff, M. (2009). Diets enriched in oat bran or wheat bran temporally and differentially alter the composition of the fecal community of rats. The Journal of nutrition, 139(11), 2024-2031. doi: 10.3945/jn.109.109470
Abushelaibi, A., Al-Mahadin, S., El-Tarabily, K., Shah, N. P., & Ayyash, M. (2017). Characterization of potential probiotic lactic acid bacteria isolated from camel milk. LWT-Food Science and Technology, 79, 316-325. doi: 10.1016/j.lwt.2017.01.041
Adetoye, A., Pinloche, E., Adeniyi, B. A., & Ayeni, F. A. (2018). Characterization and anti-salmonella activities of lactic acid bacteria isolated from cattle faeces. BMC Microbiology, 18(1), 1-11. doi: 10.1186/s12866-018-1248-y
Angmo, K., Kumari, A., Savitri, D., & Bhalla, A. K. (2016). Probiotic characterization of lactic acid bacteria isolated from fermented foods and beverage of Ladakh. LWT-Food Science and Technology, 66, 428-435. doi: 10.1016/j.lwt.2015.10.057           
Bao, Y., Zhang, Y., Zhang, Y., Liu, Y., Wang, S., Dong, X., & Zhang, H. (2010). Screening of potential probiotic properties of Lactobacillus fermentum isolated from traditional dairy products. Food Control, 21.5, 695-701. doi: 10.1016/j.foodcont.2009.10.010
Barakat, O. S., Ibrahim, G. A., Tawfik, N. F., El-Kholy, W. I., & El-Rab, G. D. (2011). Identification and probiotic characteristics of Lactobacillus strains isolated from traditional Domiati cheese. International Journal of Microbiology Research, 3(1), 59. doi: 10.9735/0975-5276.3.1.59-66
Begley, M., Hill, C., & Gahan, C. G. (2006). Bile salt hydrolase activity in probiotics. Applied and Environmental Microbiology72(3), 1729-1738. doi: 10.1128/AEM.72.3.1729-1739.2006
Boirivant, M., & Strober, W. (2007). The mechanism of action of probiotics. Current Opinion in Gastroenterology, 23(6), 679- 692. doi:10.1097/MOG.0b013e3282f0cffc
Bozkurt, M., Kucukyilmaz, K., Ayhan, V., Çabuk, M., & Çatli, A. U. (2011). Performance of layer or broiler breeder hens varies in response to different probiotic preparations. Italian Journal of Animal Science, 10, 162-169. doi: 10.4081/ijas.2011.e31
Cappuccino, J. G., & Sherman, N. (2014). Microbiology. A Laboratory Manual (10th Edition). Pearson Publishing.
Collado, M. C., Jussi, M., & Seppo, S. (2008). Adhesion and aggregation properties of probiotic and pathogen strains. European Food Research and Technology, 226, 1065-1073. doi: 10.1007/s00217-007-0632-x
El Jeni, R., Dittoe, D. K., Olson, E. G., Lourenco, J., Corcionivoschi, N., Ricke, S. C., & Callaway, T. R. (2021). Probiotics and potential applications for alternative poultry production systems. Poultry Science, 100(7), 101156. doi: 10.1016/j.psj.2021.101156
Goktepe, I., Juneja, V. K., & Ahmedna, M. (2005). Probiotics in Food Safety and Human Health. CRC Press. doi: 10.1201/9781420027570
Gómez, N. C., Ramiro, J. M., Quecan, B. X., & de Melo Franco, B. D. (2016). Use of potential probiotic lactic acid bacteria (LAB) biofilms for the control of Listeria monocytogenes, Salmonella Typhimurium, and Escherichia coli O157: H7 biofilms formation. Frontiers in Microbiology, 7, 863. doi: 10.3389/fmicb.2016.00863
Heller, K. J. (2000). Probiotic bacteria in fermented foods: Product characteristics and starter organisms. American Journal of Clinical Nutrition, 73(2), 374s-379s. doi: 10.1093/ajcn/73.2.374s
Herreros, M. A., Sandoval, M. A., Gonzalez, H., Castro, J. M. Fresono, J. M., & Tornadijo, M. E. (2005). Antimicrobial activity and antibiotic resistance of lactic acid bacteria isolated from Armada cheese (a Spanish goats milk cheese). Food Microbiology, 22(5), 455-459. doi: 10.1016/j.fm.2004.11.007        
Jankovic, T., Frece, J., Abram, M. & Gobin, I. (2012). Aggregation ability of potential probiotic Lactobacillus plantarum strains. International Journal of Sanitary Engineering Research, 6(1), 19-24.
Jin, L. Z., Ho, Y. W., Ali, M. A., Abdullah, N., Ong, K. B., & Jalaludin, S. (1996a). Adhesion of Lactobacillus isolates to intestinal epithelial cells of chicken. Letters in Applied Microbiology, 23(3), 229-232. doi: 10.1111/j.1472-765x.1996.tb01149.x
Jin, L. Z., Ho, Y. W., Abdullah, N., Ali, M. A., & Jalaludin, S. (1996b). Antagonistic effects of intestinal Lactobacillus isolates on pathogens of chicken. Letters in Applied Microbiolog, 23(2), 67-71. doi: 10.1111/j.1472-765x.1996.tb00032.x
Jin, L. Z., Abdullah, H. Y., & Jalaludin, S. (1998). Growth performance, intestinal microbial populations, and serum cholesterol of broilers fed diets containing Lactobacillus cultures. Poultry Science, 77(9), 1259-1265. doi: 10.1093/ps/77.9.1259             
Kumar, S., Chaunhan, N., Gopal, M., Kumar, R., & Dilbaghi, N. (2015). Development and evaluation of alginate-chitosan nanocapsules for controlled release of acetamiprid. International Journal of Biological Macromolecules, 81, 631-637. doi: 10.1016/j.ijbiomac.2015.08.062     
Ludfiani, D. D., Asmara, W., Wahyuni, A. E. T. H., & Astuti, P. (2021). Identification of Lactobacillus spp. on basis morphological, physiological, and biochemical characteristic from Jawa Super Chicken Excreta. In BIO Web of Conferences, (Vol. 33, p. 06012). EDP Sciences. doi: 10.1051/bioconf/20213306012
Lutful Kabir, S. M. (2009). The role of probiotics in the poultry industry. International Journal of Molecular Sciences, 10(8), 3531-3546. doi: 10.3390/ijms10083531
Ma, K., Chen, W., Lin, X. Q., Liu, Z. Z., Wang, T., Zhang, J. B., & Yang, Y. J. (2023). Culturing the Chicken Intestinal Microbiota and Potential Application as Probiotics Development. International Journal of Molecular Sciences, 24(3), 3045. doi: 10.3390/ijms24033045
Maragkoudakis, P. A., Mountzouris, K. C., Psyrras, D., Cremonese, S., Fischer, J., Cantor, M. D., & Tsakalidou, E. (2009). Functional properties of novel protective lactic acid bacteria and application in raw chicken meat against Listeria monocytogenes and Salmonella enteritidis. International Journal of Food Microbiology, 130(3), 219-226. doi: 10.1016/j.ijffoodmicro.2009.01.027
Nikoskelainen, S., Ouwehand, A. C., Bylund, G., Salminen, S., & Lilius, E. M. (2003). Immune enhancement in rainbow trout (Oncorhynchus mykiss) by potential probiotic bacteria (Lactobacillus rhamnosus). Fish and Shellfish Immunology, 15(5), 443-452. doi: 10.1016/s1050-4648(03)00023-8
Noohi, N., Papizadeh, M., Rohani, M., Talebi, M., & Pourshafie, M. R. (2021). Screening for probiotic characters in lactobacilli isolated from chickens revealed the intra-species diversity of Lactobacillus brevis. Animal Nutrition, 7(1), 119-126. doi: 10.1016/j.aninu.2020.07.005
Ouwehand, A. C., Kirjavainen, P. V., Shortt, C., & Salminen, S. (1999). Probiotics: mechanisms and established effects. Iranian Journal of Pharmaceutical Research, 9, 34-52. doi: 10.1016/s0958-6946(99)00043-6
Padmavathi, T., Bhargavi, R., Priyanka, P. R., Niranjan, N. R., & Pavitra, P. V. (2018). Screening of potential probiotic lactic acid bacteria and production of amylase and its partial purification. Journal of Genetic Engineering and Biotechnology, 16(2), 357-362. doi: 10.1016/j.jgeb.2018.03.005
Palaniyandi, S. A., Damodharan, K., Suh, J. W., & Yang, S. H. (2017). In vitro characterization of Lactobacillus plantarum strains with inhibitory activity on enteropathogens for use as potential animal probiotics. Indian Journal of Microbiology, 57, 201-210. doi: 10.1007/s12088-017-0646-4
Pelicano, E. R. L., Souza, P. A., Souza, H. B. A., Figueiredo, D. F., & Boiago, M. M. (2005). Intestinal mucosa development in broiler chickens fed natural growth promoters. Brazilian Journal of Poultry Science, 221-229. doi: 10.1590/S1516-635x200500040005
Pourjafar, H., Noori, N., Gandomi, H., Basti, A. A., & Ansari, F. (2020). Viability of microencapsulated and non-microencapsulated. Biotechnology Reports, 25, e00432. doi: 10.1016/j.btre.2020.e00432
Pumriw, S., Luang-In, V., & Samappito, W. (2021). Screening of probiotic lactic acid bacteria isolated from fermented Pak-Sian for use as a starter culture. Current Microbiology, 78(7), 2695-2707. doi: 10.1007/s00284-021-02521-w
Rao, K. P., Chennappa, G., Suraj, U., Nagaraja, H., Raj, A. P., & Sreenivasa, M. Y. (2015). Probiotic potential of Lactobacillus strains isolated from sorhumbased traditional fermented food. Probiotics and Antimicrobial Protein, 7, 146-156. doi: 10.1007/s12602-015-9186-6
Rojo-Bezares, B., Saenz, Y., Poeta, P., Zarazang, M., Ruiz-Larrea, F., & Torres, C. (2006). Assessment if antibiotic susceptibility within lactic acid bacteria strains isolated from wine. International Journal of Food Microbiology, 111, 234-240. doi: 10.1016/j.ijfoodmicro.2006.06.007
Rushdy, A. A., & Eman, Z. G. (2013). Antimicrobial compounds produced by probiotic Lactobacillus brevis isolated from dairy products. Annals of Microbiology, 63, 90-81. doi: 10.1007/s13213-012-0447-2
Saarela, M., Mogensen, G., Fonden, R., Matto, J., & Mattila-Sandholm, T. (2000). Probiotic bacteria: safety, functional and technological properties. Journal of Biotechnology, 84(3), 197-215. doi: 10.1016/S0168-1656(00)00375-8
Van Coillie, E., Goris, J., Cleenwerck, I., Grijspeerdt, K., Botteldoorn, N., Van Immerseel, F., & Heyndrickx, M. (2007). Identification of lactobacilli isolated from the cloaca and vagina of laying hens and characterization for potential use as probiotics to control Salmonella Enteritidis. Journal of Applied Microbiology, 102(4), 1095-1106. doi: 10.1111/j.1365-2672.2006.03164.x
Vinderola, C. G., & Reinheimer, J. A. (2003). Lactic Acid Starter and Probiotic Bacteria: A Comparative “in Vitro” Study of Probiotic Characteristics and Biological Barrier Resistance. Food Research International, 36(9-10), 895-904. doi: 10.1016/S0963-9969(03)00098-x
Wang, W., Chen, L., Zhou, R., Wang, X., Song, L., Huang, S., & Xia, B. (2014a). Increased proportions of Bifidobacterium and the Lactobacillus group and loss of butyrate-producing bacteria in inflammatory bowel disease. Journal of Clinical Microbiology, 52(2), 398-406. doi: 10.1128/JMC.01500-13
Wang, L., Fang, M., Hu, Y., Yang, Y., Yang, M., & Chen, Y. (2014b). Characterization of the most abundant Lactobacillus species in chicken gastrointestinal tract and potential use as probiotics for genetic engineering. Acta Biochimica et Biophysica Sinica, 46(7), 612-619. doi: 10.1093/abbs/gmu037
Yousefi-Kelarikolaei, K., Mohiti-Asli, M., Hosseini, S. A., & Yousefi-Kelarikolaei, H. (2013). Effect of antibiotic, probiotic, prebiotic and multi-enzyme in pelleted diet on the performance of broilers. Animal Production Research1(4), 63-72.
Zhang, Y., Zhang, l., Du, M., Yi, H., Guo, C., Tuo, Y., Han, X., Li, J., Zh, L., & Yang, L. (2011). Antimicrobial activity against Shigella sonnei and probiotic properties of wild lactobacilli from fermented food. Microbiological Research, 167(1), 27-31. doi: 10.1016/j.micres.2011.02.006