Isolation, identification and investigation of genetic diversity of lactic acid bacteria (LABs) in human breast milk
Subject Areas : Molecular MicrobiologyElaheh Hasanvand 1 , Behrooz Doosty 2 , kamran samiei 3
1 - Student of MSC, Department of Biology, Khorramabad Branch, Islamic Azad University, Khorramabad, Iran.
2 - Associted Professor, Department of Biology, Khorramabad Branch, Islamic Azad University, Khorramabad, Iran.
3 - Department of Genetic and Plant Breeding , Faculty of Agricultural Scince, Islamic Azad University Kangavar Branch. Iran/
Keywords: Breast milk, Probiotics, Lactobacillus, PCR, 16SrRNA,
Abstract :
Background and Objective: Breast milk is rich in nutritional components and has a high biological value due to the presence of probiotic bacteria. Given the importance of lactic acid bacteria (LABs) and their positive effects on the immune system, the present study was conducted to isolate, identify, and investigate the genetic diversity of probiotic bacteria in breast milk using REP and BOX molecular markers. Materials and Methods: Breast milk samples were collected from different areas of Lorestan province and after culturing the bacteria in each sample, different techniques (gram test, catalase, and antibiogram) were used for their initial confirmation. Genetic distances between 20 selected samples were estimated using REP-PCR and BOX-PCR markers. Based on the initial results, 3 isolates were selected from heterogeneous groups and the type of bacteria was determined using 16SrRNA gene amplification, sequencing, and alignment. Results: The results showed that the studied bacteria were coccobacilli, gram-positive and catalase-negative. Based on the obtained grouping, the studied bacteria were placed in 5 different groups with a minimum of 23 and a maximum of 86% polymorphism, and the formed groups had relative correspondence with geographical distances. The results of 16SrRNA gene sequencing indicated the identification of 3 bacterial species, Limosilactobacillus fermentum, Apilactobacillus kunckeei and Ligilactobacillus apodemi in breast milk samples. Conclusion: Breast milk contains a wide range of lactic acid bacteria, and the use of DNA-based molecular markers in grouping and identifying this group of bacteria will be efficient.
References
1. Pradhan D, Mallappa RH, Grover S. Comprehensive approaches for assessing the safety of probiotic bacteria. Food Control. 2020;108:106872.
2. Selma-Royo M, Lerma JC, Cortes-Macias E, Collado MC, editors. Human milk microbiome: from actual knowledge to future perspective. Seminars in perinatology; 2021: Elsevier.
3. Eriksen KG, Christensen SH, Lind MV, Michaelsen KF. Human milk composition and infant growth. Current opinion in clinical nutrition and metabolic care. 2018;21(3):200-6.
4. Plaza-Díaz J, Fontana L, Gil A. Human milk oligosaccharides and immune system development. Nutrients. 2018;10(8):1038.
5. Mosca F, Giannì ML. Human milk: composition and health benefits. La Pediatria Medica e Chirurgica. 2017;39(2).
6. Mitchell CM, Mazzoni C, Hogstrom L, Bryant A, Bergerat A, Cher A, et al. Delivery mode affects stability of early infant gut microbiota. Cell Reports Medicine. 2020;1(9).
7. Yang B, Chen Y, Stanton C, Ross RP, Lee Y-K, Zhao J, et al. Bifidobacterium and Lactobacillus composition at species level and gut microbiota diversity in infants before 6 weeks. International journal of molecular sciences. 2019;20(13):3306.
8. Lyu L, Zhou X, Zhang M, Liu L, Niu H, Zhang J, et al. Delivery mode affects intestinal microbial composition and the development of intestinal epithelial cells. Frontiers in Microbiology. 2021;12:626144.
9. Granger CL, Embleton ND, Palmer JM, Lamb CA, Berrington JE, Stewart CJ. Maternal breast milk, infant gut microbiome and the impact on preterm infant health. Acta Paediatrica. 2021;110(2):450-7.
10. Mohammadi F, Eshaghi M, Razavi S, Sarokhalil DD, Talebi M, Pourshafie MR. Characterization of bacteriocin production in Lactobacillus spp. isolated from mother's milk. Microbial pathogenesis. 2018;118:242-6.
11. Norouzi J, Hanafari A, Beiglari Sh. Isolation and identification of Lactic Acid Bacteria in the people's mouth and studying on their inhibitory effect on some entropathogenic bacteria. Journal of Microbial World. 2009 Sep;1(1):29-38. [In Persian]
12. Tahmourespoor S, Tahmourespour A, Kermanshahi RK. Anti adhesive effect of Lactobacillus rhamnosus as a probiotic on oral Streptococci. Journal of Microbial World. 2014 Jun;7(2):128-137. [In Persian]
13. de Almeida Júnior WLG, da Silva Ferrari Í, de Souza JV, da Silva CDA, da Costa MM, Dias FS. Characterization and evaluation of lactic acid bacteria isolated from goat milk. Food control. 2015;53:96-103.
14. Martı́n Ro, Langa S, Reviriego C, Jiménez E, Marı́n MaL Olivares M, et al. The commensal microflora of human milk: new perspectives for food bacteriotherapy and probiotics. Trends in Food Sci and Tech. 2004; 15(3-4): 121-7.
15. Gevers D, Huys G, Swings J. Applicability of rep-PCR fingerprinting for identification of Lactobacillus species. FEMS microbiology letters. 2001;205(1):31-6.
16. Lee CM, Sieo CC, Cheah YK, Abdullah N, Ho YW. Discrimination of probiotic Lactobacillus strains for poultry by repetitive sequenced‐based PCR fingerprinting. Journal of the Science of Food and Agriculture. 2012;92(3):660-6.
17. Collins M, Rodrigues U, Ash C, Aguirre M, Farrow J, Martinez-Murcia A, et al. Phylogenetic analysis of the genus Lactobacillus and related lactic acid bacteria as determined by reverse transcriptase sequencing of 16S rRNA. FEMS Microbiology letters. 1991;77(1):5-12.
18. Li S, Watanabe K, Hsu C, Chao S, Yang Z, Lin Y, et al. Bacterial composition and diversity in breast milk samples from mothers living in Taiwan and mainland China. Front Microbiol 8: 965. 2017.
19. Boix-Amorós A, Collado MC, Mira A. Relationship between milk microbiota, bacterial load, macronutrients, and human cells during lactation. Frontiers in microbiology. 2016;7:492.
20. Turnbaugh PJ, Hamady M, Yatsunenko T, Cantarel BL, Duncan A, Ley RE, et al. A core gut microbiome in obese and lean twins. nature. 2009;457(7228):480-4.
21. Kumar H, Du Toit E, Kulkarni A, Aakko J, Linderborg KM, Zhang Y, et al. Distinct patterns in human milk microbiota and fatty acid profiles across specific geographic locations. Frontiers in microbiology. 2016;7:1619.
22. Damaceno QS, Souza JP, Nicoli JR, Paula RL, Assis GB, Figueiredo HC, et al. Evaluation of potential probiotics isolated from human milk and colostrum. Probiotics and antimicrobial proteins. 2017;9:371-9.
23. Abdi M, Lohrasbi V, Asadi A, Esghaei M, Jazi FM, Rohani M, et al. Interesting probiotic traits of mother's milk Lactobacillus isolates; from bacteriocin to inflammatory bowel disease improvement. Microbial Pathogenesis. 2021;158:104998.
24. Mosca F, Giannì ML. Human milk: composition and health benefits. La Pediatria Medica e Chirurgica. 2017;39(2).
25. Jost T, Lacroix C, Braegger CP, Rochat F, Chassard C. Vertical mother–neonate transfer of maternal gut bacteria via breastfeeding. Environmental microbiology. 2014;16(9):2891-904.
26. Armand M, Gasemi MF, Fazeli MR, Mirpour, M. Optimization of biomass production by probiotic bacterium Lactobacillus rhamnonsus at pilot-plant scale. Journal of Microbial World. 2020 Sep;13(3):202-214. [In Persian]