Determination of Some Probiotic Properties of Lactic Acid Bacteria Isolated From Different Food Products

Abstract

Lactic acid bacteria (LAB) are a group of commercially important organisms that play an important role in the fermentation process of hexose sugars into lactic acid and widely used in industrial applications and as probiotics since they were designated as “generally recognized as safe” organisms. In order to isolate lactic acid bacteria (LAB), different food samples were collected and 14 bacterial strains were purified from those samples. Gram staining, catalase test, methylene blue staining was used for morphologic identification, RAPD-PCR, and 16S rRNA gene sequencing methods were used for molecular identification. The isolates were characterized for antibiotic susceptibility, survival under pepsin, pancreatin, bile salts, low pH, bacteriocin production, and hemolytic activity. According to the 16s rRNA gene sequence analysis isolates were designated as Lactobacillus sakei MH1, Lactococcus garvieae MH3, Enterococcus faeceium MH5, Pediococcus acidilactici MH10, Pediococcus acidilactici MH11, Pediococcus acidilactici MH12, Pediococcus acidilactici MH13. Disc diffusion test results show that all of the isolates were resistant to kanamycin, and most of resistant to gentamicin. MIC test results show that all of the isolates were resistant to streptomycin. No strains showed antimicrobial effect against indicator microorganisms according to the bacteriocin production test. All strains were able to survive in pancreatin and different bile salt (%0.3, %0.5, %1) concentrations. According to these results, our isolates are probiotic potential candidates for the application in the food and pharmaceutical industries, however, isolates should be screened for furtherprobiotic selection criteria.

Keywords

• Lactic Acid Bacteria
• Probiotic
• Antibiotic Susceptibility

Farklı Gıda Ürünlerinden İzole Edilen Laktik Asit Bakterilerinin Bazı Probiyotik Özelliklerinin Belirlenmesi

Öz

Laktik asit bakterileri (LAB), heksoz şekerlerinin laktik aside fermantasyonu sürecinde önemli bir rol oynayan ticari olarak önemli bir organizma grubudur ve “genel olarak güvenli kabul edilen” olarak tanımlandıkları için endüstriyel uygulamalarda ve probiyotik olarak yaygın olarak kullanılan organizmalardır. Laktik asit bakterilerini (LAB) izole etmek için farklı gıda örnekleri toplanmış ve bu örneklerden 14 bakteri suşu saflaştırılmıştır. Morfolojik tanımlama için gram boyama, katalaz testi, metilen mavisi boyama, moleküler tanımlama için RAPD-PZR ve 16S rRNA gen dizileme yöntemleri kullanılmıştır. Ayrıca izolatlar, antibiyotik duyarlılığı, pepsin, pankreatin, safra tuzları, düşük pH, bakteriyosin üretimi ve hemolitik aktivite altında hayatta kalma açısından karakterize edilmiştir. 16S rRNA gen dizileme sonuçlarına göre izolatlar Lactobacillus sakei MH1, Lactococcus garvieae MH3, Enterococcus faeceium MH5, Pediococcus acidilactici MH10, Pediococcus acidilactici MH11, Pediococcus acidilactici MH12, Pediococcus acidilactici MH13 olarak belirlendi. Disk difüzyon testi sonuçları, izolatların tamamının kanamisine, çoğunun ise gentamisine dirençli olduğunu göstermektedir. MİK test sonuçları izolatların tamamının streptomisine dirençli olduğunu göstermektedir. Tüm suşlar bakteriyosin üretim testine göre indikatör mikroorganizmalara karşı antimikrobiyal etki göstermemiştir. Tüm suşların, pankreatin ve farklı safra tuzu (%0.3, %0.5, %1) konsantrasyonunda hayatta kalabildiği tespit edilmiştir. Bu sonuçlara göre izolatlarımız gıda ve ilaç endüstrilerinde uygulama için probiyotik potansiyel adaylardır, ancak izolatların diğer probiyotik seçim kriterleri açısından taranması gerekmektedir.

Anahtar Kelimeler

• Laktik Asit Bakterileri
• Probiyotik
• Antibiyotik Duyarlılığı

Kaynakça

1. Agheyisi, R. 2008. The probiotics market: Ingredients, supplements, foods, Report code: FOD035B, BCC Research, Wellesley, MA, USA.
2. Ammor, M.S., Belén Flórez, A. and Mayo, B. 2007. Antibiotic resistance in non-enterococcal lactic acid bacteria and bifidobacteria, Food Microbiology, 24 (6), 559-570.
3. Bintsis, T. 2018. Lactic acid bacteria as starter cultures: An update in their metabolism and genetics, AIMS Microbiology, 4 (4), 665-684.
4. Bove, P., Gallone, A., Russo, P., Capozzi, V., Albenzio, M., Spano, G. and Fiocco, D. 2012. Probiotic features of Lactobacillus plantarum mutant strains, Applied Microbiology and Biotechnology, 96 (2), 431-41.
5. Charteris, W.P., Kelly, P.M., Morelli, L. and Collins, J.K. 1998. Antibiotic susceptibility of potentially probiotic Lactobacillus species, Journal of Food Protection, 61 (12), 1636-43.
6. Coppola, R., Succi, M., Tremonte, P., Reale, A., Salzano, G. and Sorrentino, E. 2005. Antibiotic susceptibility of Lactobacillus rhamnosus strains isolated from Parmigiano Reggiano cheese, Le Lait, 85 (3), 193-204. de Melo Pereira, G.V., de Oliveira Coelho, B., Magalhaes Junior, A.I., Thomaz-Soccol, V. and Soccol, C.R. 2018. How to select a probiotic? A review and update of methods and criteria, Biotechnology Advances, 36 (8), 2060-2076.
7. Diosma, G., Romanin, D.E., Rey-Burusco, M.F., Londero, A. and Garrote, G.L. 2014. Yeasts from kefir grains: isolation, identification, and probiotic characterization, World Journal of Microbiology and Biotechnology, 30 (1), 43-53.
8. Dunne, C., O'Mahony, L., Murphy, L., Thornton, G., Morrissey, D., O'Halloran, S., Feeney, M., Flynn, S., Fitzgerald, G.,Daly, C., Kiely, B., O'Sullivan, G.C., Shanahan, F. and Collins, J.K. 2001. In vitro selection criteria for probiotic bacteria of human origin: correlation with in vivo findings, The American Journal of Clinical Nutrition, 73 (2), 386-392.

9. El-Jeni, R., El Bour, M., Calo-Mata, P., Böhme, K., Fernández-No, I.C., Barros-Velázquez, J. and Bouhaouala-Zahar, B. 2016. In vitro probiotic profiling of novel Enterococcus faecium and Leuconostoc mesenteroides from Tunisian freshwater fishes, Canadian Journal of Microbiology, 62 (1), 60-71.
10. EUCAST. 2019. The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 9.0. http://www.eucast.org (Erişim Tarihi: 21 Haziran 2021).
11. FAO and WHO. 2002. Guidelines for the evaluation of probiotics in food. https://www.who.int/foodsafety/fs_management/en/probiotic_guidelines.pdf (Erişim Tarihi: 20 Haziran 2021).
12. Felsenstein, J. 1985. Confidence Limits on Phylogenies: An Approach Using the Bootstrap, Evolution, 39 (4), 783-791.
13. Fleet, G. and Balia, R. 2006. The public health and probiotic significance of yeasts in foods and beverages. in: Yeasts in food and beverages, Springer, 381-397.
14. Giridhara Upadhyaya, P.M., Ravikumar, K.L. and Umapathy, B.L. 2009. Review of virulence factors of Enterococcus: an emerging nosocomial pathogen, Indian Journal of Medical Microbiology, 27 (4), 301-5.
15. Horáčková, Š., Žaludová, K. and Plocková, M. 2012. Stability of selected lactobacilli in the conditions simulating those in the gastrointestinal tract, Czech Journal of Food Sciences, 29, 30-35.
16. Jensen, H., Grimmer, S., Naterstad, K. and Axelsson, L. 2012. In vitro testing of commercial and potential probiotic lactic acid bacteria, International Journal of Food Microbiology, 153 (1), 216-222.
17. Kim, H., Shin, M., Ryu, S., Yun, B., Oh, S., Park, D.-J. and Kim, Y. 2021. Evaluation of probiotic characteristics of newly isolated lactic acid bacteria from dry-aged hanwoo beef, Food Science of Animal Resources, 41 (3), 468-480.
18. Koch, S., Hufnagel, M., Theilacker, C. and Huebner, J. 2004. Enterococcal infections: host response, therapeutic, and prophylactic possibilities, Vaccine, 22 (7), 822-830.
19. Kumar, S., Stecher, G. and Tamura, K. 2016. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets, Molecular Biology and Evolution, 33 (7), 1870-4.
20. Lertworapreecha, N., Poonsuk, K. and Chalermchiakit, T. 2011. Selection of potential Enterococcus faecium isolated from Thai native chicken for probiotic use according to the in vitro properties, Songklanakarin Journal of Science & Technology, 33 (1).
21. Liong, M.T., Lee, B.H., Choi, S.B., Lew, L.C., Lau, A.S.Y. and Daliri, B.M.E., 2015. Cholesterol-lowering effects of probiotics and prebiotics. In: Venema, K., Carmo, P.A., Probiotics and Prebiotics, 429–447.
22. Liu, J., Wang, Y., Li, A., Iqbal, M., Zhang, L., Pan, H., Liu, Z. and Li, J. 2020. Probiotic potential and safety assessment of Lactobacillus isolated from yaks, Microbial Pathogenesis, 145, 104213.
23. Maragkoudakis, P., Zoumpopoulou, G., Miaris, C., Kalantzopoulos, G., Pot, B. and Tsakalidou, E. 2006. Probiotic potential of Lactobacillus strains isolated from dairy products, International Dairy Journal, 16, 189-199.
24. Marchesi, J.R., Adams, D.H., Fava, F., Hermes, G.D., Hirschfield, G.M., Hold, G., Quraishi, M.N., Kinross, J., Smidt, H. and Tuohy, K.M. 2016. The gut microbiota and host health: a new clinical frontier, Gut, 65 (2), 330-339.
25. Marco, M.L., Pavan, S. and Kleerebezem, M. 2006. Towards understanding molecular modes of probiotic action, Current Opinion in Biotechnology, 17 (2), 204-210.
26. Mathur, H., Beresford, T.P. and Cotter, P.D. 2020. Health benefits of lactic acid bacteria (LAB) fermentates, Nutrients, 12 (6), 1679.
27. Morandi, S., Brasca, M., Andrighetto, C., Lombardi, A. and Lodi, R. 2006. Technological and molecular characterisation of Enterococci isolated from north–west Italian dairy products, International Dairy Journal, 16 (8), 867-875.
28. Mundy, L., Sahm, D. and Gilmore, M. 2000. Relationships between enterococcal virulence and antimicrobial resistance, Clinical Microbiology Reviews, 13 (4), 513.
29. Muñoz-Quezada, S., Chenoll, E., Vieites, J.M., Genovés, S., Maldonado, J., Bermúdez-Brito, M., Gomez-Llorente, C., Matencio, E., Bernal, M.J., Romero, F., Suárez, A., Ramón, D. and Gil, A. 2013. Isolation, identification and characterisation of three novel probiotic strains (Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036) from the faeces of exclusively breast-fed infants, British Journal of Nutrition, 109 (2), 51-62.
30. Osmanagaoglu, O., Kiran, F. and Ataoglu, H. 2010. Evaluation of in vitro probiotic potential of Pediococcus pentosaceus OZF isolated from human breast milk, Probiotics and Antimicrobial Proteins, 2 (3), 162-74. Patil, M., Pal, A., Anand, T. and Ramana, K. 2010. Isolation and characterization of lactic acid bacteria from curd and cucumber, Indian Journal of Biotechnology, 9.
31. Reis, N.A., Saraiva, M.A., Duarte, E.A., de Carvalho, E.A., Vieira, B.B. and Evangelista-Barreto, N.S. 2016. Probiotic properties of lactic acid bacteria isolated from human milk, Journal of Applied Microbiology, 121 (3), 811-20. Saitou, N. and Nei, M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees, Molecular Biology and Evolution, 4 (4), 406-425.
32. Schultz, M. 2008. Clinical use of E. coli Nissle 1917 in inflammatory bowel disease, Inflammatory Bowel Diseases, 14 (7), 1012-1018.
33. Sharma, C., Gulati, S., Thakur, N., Singh, B.P., Gupta, S., Kaur, S., Mishra, S.K., Puniya, A.K., Gill, J.P.S. and Panwar, H. 2017. Antibiotic sensitivity pattern of indigenous Lactobacilli isolated from curd and human milk samples, 3 Biotech, 7 (1), 53.
34. Soccol, C.R., Vandenberghe, L.P.d.S., Spier, M.R., Medeiros, A.B.P., Yamaguishi, C.T., Lindner, J.D.D., Pandey, A. and Thomaz-Soccol, V. 2010. The potential of probiotics: a review, Food Technology and Biotechnology, 48 (4), 413-434.
35. Tamura, K., Nei, M. and Kumar, S. 2004. Prospects for inferring very large phylogenies by using the neighbor-joining method, Proceedings of the National Academy of Sciences of the United States of America, 101 (30), 11030.
36. Turchi, B., Mancini, S., Fratini, F., Pedonese, F., Nuvoloni, R., Bertelloni, F., Ebani, V.V. and Cerri, D. 2013. Preliminary evaluation of probiotic potential of Lactobacillus plantarum strains isolated from Italian food products, World Journal of Microbiology and Biotechnology, 29 (10), 1913-1922.
37. Vinderola, C.G. and Reinheimer, J. 2003. Lactic acid starter and probiotic bacteria: Comparative “in vitro” study of probiotic characteristics and biological barrier resistance, Food Research International, 36, 895-904.
38. Vizoso Pinto, M.G., Franz, C.M., Schillinger, U. and Holzapfel, W.H. 2006. Lactobacillus spp. with in vitro probiotic properties from human faeces and traditional fermented products, International Journal of Food Microbiology, 109 (3), 205-14.
39. Xia, A.-N., Meng, X.-S., Tang, X.-J., Zhang, Y.-Z., Lei, S.-M. and Liu, Y.-G. 2021. Probiotic and related properties of a novel lactic acid bacteria strain isolated from fermented rose jam, LWT, 136, 110327.
40. Yang, J., Cao, Y., Cai, Y. and Terada, F. 2010. Natural populations of lactic acid bacteria isolated from vegetable residues and silage fermentation, Journal of Dairy Science, 93 (7), 3136-3145.
41. Yang, S. C., Lin, C. H., Sung, C. T., 2014, Antibacterial activities of bacteriocins: application in foods and pharmaceuticals, Front Microbiol., 5, 241.
42. Yılmaz, H. K. & Derya İpek, K. (2021). Probiyotikler ve Kadın Sağlığı Üzerine Etkileri. Avrupa Bilim ve Teknoloji Dergisi, (23), 518-523. DOI: 10.31590/ejosat.827669
43. Yoğurtçu, N.N. (2011). “Tulum peynirinden enterokok suçlarının izolasyonu ve antibiyotik duyarlılıklarının belirlenmesi”, Yüksek lisans tezi, SDÜ Fen Bilimleri Enstitüsü, Isparta, 70.
44. Zhou, J.S., Pillidge, C.J., Gopal, P.K. and Gill, H.S. 2005. Antibiotic susceptibility profiles of new probiotic Lactobacillus and Bifidobacterium strains, International Journal of Food Microbiology, 98 (2), 211-7.
45. Zoumpopoulou, G., Pot, B., Tsakalidou, E. and Papadimitriou, K. 2017. Dairy probiotics: Beyond the role of promoting gut and immune health, International Dairy Journal, 67, 46-60.
46. Zucko, J., Starcevic, A., Diminic, J., Oros, D., Mortazavian, A.M. and Putnik, P. 2020. Probiotic – friend or foe?, Current Opinion in Food Science, 32, 45-49.