Molecular Identification and Phylogenetic Analysis of Lactic Acid Bacteria Isolated from Cow Raw Milk in Al-Kut -Iraq
Keywords:
lactic acid bacteria, Molecular characterization, cow milk, Al-Kut, Iraq
Abstract
The objective of “this study was to determine the genetic variability of lactic acid bacteria (LAB) present in locally sourced cow's milk. A total of 50 raw milk samples were collected from several local markets in Al-Kut. The specimens were cultured in the De man, Rogosa, and Sharpe (MRS) medium, which is particularly formulated to promote the growth of lactic acid bacteria. The presumed isolates were then identified by the use of polymerase chain reaction (PCR), which selectively targets the 16S rRNA gene, and afterwards followed by DNA sequencing”. The results revealed that lactic acid bacteria were present in 28% (14 isolates) of the samples, including two genera and five species. Specifically, there were 2 strains of Lactococcus lactis and 12 strains of Lactobacillus spp, including 5 Lactobacillus Plantarum, 4 Lactobacillus gasseri, 2 Lactobacillus acidophilus, and 1 Lactobacillus delbrueckii. The predominant Lactic acid Bactria in the present study (Lactobacillus Plantarum) were furtherly tested for their antibacterial activity and the results showed that , Lactobacillus Plantarum exhibited potent antibacterial effects against indicator bacteria like “Staphylococcus aureus and Staphylococcus epidermidis, as well as pathogenic bacteria such as E.coli and Pseudomonas” aeruginosa.
The 16S rRNA gene sequencing and phylogenetic tree analysis indicated that all the obtained strains exhibited 99% homology with certain LAB strains published in the NCBI. Subsequently, the isolates were recorded in the NCBI.
References
1. Lavanya B, Sowmiya, S, Balaji S, Muthuvelan, B. Screening and characterization of lactic acid bacteria from fermented milk. Br J Dairy Sci. 2011;2:5-10.
2. Klaenhammer T, Altermann E, Arigoni F, Bolotin, A, Breidt F, Broadbent J, Cano R, Chaillou S, Deutscher J, Gasson M, van de Guchte M, Guzzo. Discovering lactic acid bacteria by genomics. Antonie van Leeuwenhoek. 2011;82:29-58. 10.1007/978-94-017- 2029-8_3
3. Killer J, Kopečný J, Mrázek J, Rada V, Dubná S, Marounek M. Bifidobacteria in the digestive tract of bumblebees. Anaerobe. 2010;16(2):165- 170. 10.1016/j.anaerobe.2009.07.007
4. Olofsson TC, Vásquez A. Detection and identification of a novel lactic acid bacterial flora within the honey stomach of the honeybee Apismellifera. Curr Microbiol. 2008;57(4):356-363. 10.1007/s00284- 008-9202-0
5. Hammes WP, Vogel RF. The genus Lactobacillus. In: Wood BJB, Holzapfel WH, editors. The lactic acid bacteria. 2nd ed. London: Blackie Academic and Professional; 1995. 398 p. 10.1007/978-1- 4615-5817-0_3
6. Satokari RM, Vaughan EE, Smidt H, Saarela M, Matto J, de Vos WM. Molecular approaches for the detection and identification of bifidobacteria and lactobacilli in the human gastrointestinal tract. Sys Appl Microbiol. 2003; 26:572-584. 10.1078/072320203770865882
7. Verna EC and Lucak S. Use of probiotics in gastrointestinal disorders: what to recommend. Ther Adv Gastroenterol.2010; 3(5): 307-319. 10.1177/ 1756283X10373814
8. Ouwehand A, Salminen S, Isolauri E. Probiotics: An overview of beneficial effects. Antonie Van Leeuwenhoek. 2002;82(1-4):279-289. 10.1023/a:1020620607611
9. De Preter V, Vanhoutte T, Huys G, Swings J, De Vuyst L, Rutgeerts P, Verbeke K. Effects of Lactobacillus casei Shirota, Bifidobacterium breve, and oligofructose-enriched inulin on colonic nitrogen-protein metabolism in healthy humans. Am J Physiol Gastrointest Liver Physiol. 2007;292(1):358-368. 10.1152/ajpgi.00052.2006
10. Gerald WT. Probiotics: Biology, genetics and health aspects: Minireviews. Appl Environ Microbiol. 2004;70:3189-3194. 10.1128/AEM
11. Chaucheyras DF, Durand H. Probiotics in animal nutrition and health. Benef Microbes. 2010;1(1):3-9. https://doi.org/10.3920/bm2008.1002
12. Park YW. Impact of goat milk and milk products on human nutrition. 2007CAB Reviews Perspectives in Agriculture Veterinary Science Nutrition and Natural Resources.2007;2(081).1-19. 10.10.79/PAV SNNR20072081
13. Escaren L, Salinas H, Wurzinger M, Iniguez L, Solkner J, Meza C. Dairy goat production system: Status quo, perspectives and challenges. Trop Anim Health Prod. 2013;45:17-34. 10.1007/s11250- 012-0246-6
14. Quigley L, O’Sullivan O, Stanton C, Beresford TP, Ross RP, Fitzgerald GF, Cotter PD. The complex microbiota of raw milk. FEMS Microbiol Rev. 2013; 37:664-698. 10.1111/1574-6976.12030
15. Nikolic M, Terzic-Vidojevic A, Jovcic B, Begovic J, Golic N, Topisirovic L. Characterization of lactic acid bacteria isolated from Bukuljac: A homemade goat’s milk cheese. Int J Food Microbiol. 2008;122:162-170. 10.1016/j.ijfoodmicro.2007.11.075
16. Perin LM, Nero LA. Antagonistic lactic acid bacteria isolated from goat milk and identification of a novel nisin variant Lactococcus lactis. BMC Microbiol. 2014;14:36. 10.1186/1471-2180-14-36
17. Bergey’s manual of systematic bacteriology; Volume 3: The Firmicutes: (464-513); Springer-Verlag, New York. 2009.
18. Weisburg, W.G., S.M. Barns, D.A. Pelletier and D.J. Lane, 1991. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol., 173: 697-703.
19. CHEN Z L, CHENG C, MA K, et al. Isolation and identification of lactic acid bacteria from fermented yak milk products in tibet area[J]. Food Science, 2008, 29(12): 408-412.
20. YANG J X, CHEN Z L, YANG H Y, et al. Identification and characterization of lactic acid bacteria from chinese yak milk cheeses[J].Food Science, 2013, 34(9): 198-204.
21. LV J M, LI J B, ZHU C, et al. Isolation and identification of Lactobacillus from yak milk dregs[J]. China Dairy Cattle, 2011(20): 57-60
22. BAO X H. Diversity analysis of lactic acid bacteria in yak milk products in Gansu and Sichuan province[D]. Inner Mongolia Agricultural University, 2012.
23. BAO Q, YU J ,LIU W, et al. Isolation and identification of predominant lactic acid bacteria in traditional fermented yak milk products in Sichuan province of China[J]. Dairy Sci Technol, 2012, 92(3): 309-319.
24. Ming, L., Zhang, Q., & Le Yang, J. A. H. . Comparison of antibacterial effects between antimicrobial peptides and bacteriocins isolated from Lactobacillus plantarum on three common pathogenic bacteria. International journal of clinical and experimental medicine, 2015.8(4), 5806
25. Le, N. T. T., Bach, L. G., Nguyen, D. C., Le, T. H. X., Pham, K. H., Nguyen, D. H., & Hoang Thi, T. T. . Evaluation of factors affecting antimicrobial activity of bacteriocin from Lactobacillus plantarum microencapsulated in alginate-gelatin capsules and its application on pork meat as a bio-preservative. International journal of environmental research and public health,2019. 16(6), 1017.
26. Lei, S., Zhao, R., Sun, J., Ran, J., Ruan, X., & Zhu, Y. . Partial purification and characterization of a broad‐spectrum bacteriocin produced by a Lactobacillus plantarum zrx03 isolated from infant's faeces. Food science & nutrition, 2020. 8(5), 2214-2222.
27. Hassan, Mahreen Ul, et al. "Characterisation of bacteriocins produced by Lactobacillus spp. isolated from the traditional Pakistani yoghurt and their antimicrobial activity against common foodborne pathogens." BioMed research international2020. 7(5)3345-3355.
28. Refay, R. M., Abushady, H. M., Amer, S. A., & Mailam, M. A. Determination of bacteriocin-encoding genes of lactic acid bacteria isolated from traditional dairy products of Luxor province, Egypt. Future Journal of Pharmaceutical Sciences, 2020. 6(1), 1-14
29. Chen X, Song Y, Xu H, Menghe BL, Zhang HP, Sun ZH. Genetic relationship of Enterococcus faecalis from different sources revealed by multilocus sequence typing. J Dairy Sci. 2015;98(8):5183-5193. 10.3168/jds.2015-9571
30. Ren L, Suo H. Molecular Identification of lactic acid bacteria isolated from the traditional fermented yak yogurt in western Sichuan Region. Proceeding of 7th International Conference on Education, Management, Information and Mechanical Engineering (EMIM 2017). Adv Com Sci Res. 2017; 76: 1248-1256. 10.2991/emim17.2017.252
31. ZHANG W, LIU W, SONG Y, et al. Multilocous sequence typing of dairy-associated Leuconostoc mesenteroides population reveals clonal structure with intragenic homologous recombination[J]. Journal of Dairy Science, 2015, 98(6): 2284–2293
32. ZHANG H P. Biodiversity of lactic acid bacteria in naturally fermented dairy products[J]. Chinese Bulletin of Life Sciences, 2015, 27(7): 837-846.
33. Liasi, S. A., Azmi, T. I., Hassan, M. D., Shuhaimi, M., Rosfarizan, M., & Ariff, A. B. (2009). Antimicrobial activity and antibiotic sensitivity of three isolates of lactic acid bacteria from fermented fish product, Budu. Malaysian Journal of Microbiology, 5(1), 33-37.
2. Klaenhammer T, Altermann E, Arigoni F, Bolotin, A, Breidt F, Broadbent J, Cano R, Chaillou S, Deutscher J, Gasson M, van de Guchte M, Guzzo. Discovering lactic acid bacteria by genomics. Antonie van Leeuwenhoek. 2011;82:29-58. 10.1007/978-94-017- 2029-8_3
3. Killer J, Kopečný J, Mrázek J, Rada V, Dubná S, Marounek M. Bifidobacteria in the digestive tract of bumblebees. Anaerobe. 2010;16(2):165- 170. 10.1016/j.anaerobe.2009.07.007
4. Olofsson TC, Vásquez A. Detection and identification of a novel lactic acid bacterial flora within the honey stomach of the honeybee Apismellifera. Curr Microbiol. 2008;57(4):356-363. 10.1007/s00284- 008-9202-0
5. Hammes WP, Vogel RF. The genus Lactobacillus. In: Wood BJB, Holzapfel WH, editors. The lactic acid bacteria. 2nd ed. London: Blackie Academic and Professional; 1995. 398 p. 10.1007/978-1- 4615-5817-0_3
6. Satokari RM, Vaughan EE, Smidt H, Saarela M, Matto J, de Vos WM. Molecular approaches for the detection and identification of bifidobacteria and lactobacilli in the human gastrointestinal tract. Sys Appl Microbiol. 2003; 26:572-584. 10.1078/072320203770865882
7. Verna EC and Lucak S. Use of probiotics in gastrointestinal disorders: what to recommend. Ther Adv Gastroenterol.2010; 3(5): 307-319. 10.1177/ 1756283X10373814
8. Ouwehand A, Salminen S, Isolauri E. Probiotics: An overview of beneficial effects. Antonie Van Leeuwenhoek. 2002;82(1-4):279-289. 10.1023/a:1020620607611
9. De Preter V, Vanhoutte T, Huys G, Swings J, De Vuyst L, Rutgeerts P, Verbeke K. Effects of Lactobacillus casei Shirota, Bifidobacterium breve, and oligofructose-enriched inulin on colonic nitrogen-protein metabolism in healthy humans. Am J Physiol Gastrointest Liver Physiol. 2007;292(1):358-368. 10.1152/ajpgi.00052.2006
10. Gerald WT. Probiotics: Biology, genetics and health aspects: Minireviews. Appl Environ Microbiol. 2004;70:3189-3194. 10.1128/AEM
11. Chaucheyras DF, Durand H. Probiotics in animal nutrition and health. Benef Microbes. 2010;1(1):3-9. https://doi.org/10.3920/bm2008.1002
12. Park YW. Impact of goat milk and milk products on human nutrition. 2007CAB Reviews Perspectives in Agriculture Veterinary Science Nutrition and Natural Resources.2007;2(081).1-19. 10.10.79/PAV SNNR20072081
13. Escaren L, Salinas H, Wurzinger M, Iniguez L, Solkner J, Meza C. Dairy goat production system: Status quo, perspectives and challenges. Trop Anim Health Prod. 2013;45:17-34. 10.1007/s11250- 012-0246-6
14. Quigley L, O’Sullivan O, Stanton C, Beresford TP, Ross RP, Fitzgerald GF, Cotter PD. The complex microbiota of raw milk. FEMS Microbiol Rev. 2013; 37:664-698. 10.1111/1574-6976.12030
15. Nikolic M, Terzic-Vidojevic A, Jovcic B, Begovic J, Golic N, Topisirovic L. Characterization of lactic acid bacteria isolated from Bukuljac: A homemade goat’s milk cheese. Int J Food Microbiol. 2008;122:162-170. 10.1016/j.ijfoodmicro.2007.11.075
16. Perin LM, Nero LA. Antagonistic lactic acid bacteria isolated from goat milk and identification of a novel nisin variant Lactococcus lactis. BMC Microbiol. 2014;14:36. 10.1186/1471-2180-14-36
17. Bergey’s manual of systematic bacteriology; Volume 3: The Firmicutes: (464-513); Springer-Verlag, New York. 2009.
18. Weisburg, W.G., S.M. Barns, D.A. Pelletier and D.J. Lane, 1991. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol., 173: 697-703.
19. CHEN Z L, CHENG C, MA K, et al. Isolation and identification of lactic acid bacteria from fermented yak milk products in tibet area[J]. Food Science, 2008, 29(12): 408-412.
20. YANG J X, CHEN Z L, YANG H Y, et al. Identification and characterization of lactic acid bacteria from chinese yak milk cheeses[J].Food Science, 2013, 34(9): 198-204.
21. LV J M, LI J B, ZHU C, et al. Isolation and identification of Lactobacillus from yak milk dregs[J]. China Dairy Cattle, 2011(20): 57-60
22. BAO X H. Diversity analysis of lactic acid bacteria in yak milk products in Gansu and Sichuan province[D]. Inner Mongolia Agricultural University, 2012.
23. BAO Q, YU J ,LIU W, et al. Isolation and identification of predominant lactic acid bacteria in traditional fermented yak milk products in Sichuan province of China[J]. Dairy Sci Technol, 2012, 92(3): 309-319.
24. Ming, L., Zhang, Q., & Le Yang, J. A. H. . Comparison of antibacterial effects between antimicrobial peptides and bacteriocins isolated from Lactobacillus plantarum on three common pathogenic bacteria. International journal of clinical and experimental medicine, 2015.8(4), 5806
25. Le, N. T. T., Bach, L. G., Nguyen, D. C., Le, T. H. X., Pham, K. H., Nguyen, D. H., & Hoang Thi, T. T. . Evaluation of factors affecting antimicrobial activity of bacteriocin from Lactobacillus plantarum microencapsulated in alginate-gelatin capsules and its application on pork meat as a bio-preservative. International journal of environmental research and public health,2019. 16(6), 1017.
26. Lei, S., Zhao, R., Sun, J., Ran, J., Ruan, X., & Zhu, Y. . Partial purification and characterization of a broad‐spectrum bacteriocin produced by a Lactobacillus plantarum zrx03 isolated from infant's faeces. Food science & nutrition, 2020. 8(5), 2214-2222.
27. Hassan, Mahreen Ul, et al. "Characterisation of bacteriocins produced by Lactobacillus spp. isolated from the traditional Pakistani yoghurt and their antimicrobial activity against common foodborne pathogens." BioMed research international2020. 7(5)3345-3355.
28. Refay, R. M., Abushady, H. M., Amer, S. A., & Mailam, M. A. Determination of bacteriocin-encoding genes of lactic acid bacteria isolated from traditional dairy products of Luxor province, Egypt. Future Journal of Pharmaceutical Sciences, 2020. 6(1), 1-14
29. Chen X, Song Y, Xu H, Menghe BL, Zhang HP, Sun ZH. Genetic relationship of Enterococcus faecalis from different sources revealed by multilocus sequence typing. J Dairy Sci. 2015;98(8):5183-5193. 10.3168/jds.2015-9571
30. Ren L, Suo H. Molecular Identification of lactic acid bacteria isolated from the traditional fermented yak yogurt in western Sichuan Region. Proceeding of 7th International Conference on Education, Management, Information and Mechanical Engineering (EMIM 2017). Adv Com Sci Res. 2017; 76: 1248-1256. 10.2991/emim17.2017.252
31. ZHANG W, LIU W, SONG Y, et al. Multilocous sequence typing of dairy-associated Leuconostoc mesenteroides population reveals clonal structure with intragenic homologous recombination[J]. Journal of Dairy Science, 2015, 98(6): 2284–2293
32. ZHANG H P. Biodiversity of lactic acid bacteria in naturally fermented dairy products[J]. Chinese Bulletin of Life Sciences, 2015, 27(7): 837-846.
33. Liasi, S. A., Azmi, T. I., Hassan, M. D., Shuhaimi, M., Rosfarizan, M., & Ariff, A. B. (2009). Antimicrobial activity and antibiotic sensitivity of three isolates of lactic acid bacteria from fermented fish product, Budu. Malaysian Journal of Microbiology, 5(1), 33-37.
Published
2023-10-16
How to Cite
Hasan Ikareim Idbeis. (2023). Molecular Identification and Phylogenetic Analysis of Lactic Acid Bacteria Isolated from Cow Raw Milk in Al-Kut -Iraq. Central Asian Journal of Medical and Natural Science, 4(5), 623-631. https://doi.org/10.17605/cajmns.v4i5.1884
Section
Articles
