Food Safety and Functional Characterization of Enterococci Strains Isolated from Sourdough Collected from Bursa Region

Year 2024, Volume: 38 Issue: 2, 341 - 355, 15.12.2024

Ozen Sokmen , Gamze Düven , Mustafa Ay , Sine Özmen Toğay

https://doi.org/10.20479/bursauludagziraat.1486640

Abstract

In this study, the antibiotic resistance and antimicrobial activity of isolates obtained from 30 sourdough samples collected from the Bursa region were examined to determine the food safety and functional characteristics of the enterococci isolated from these samples. Additionally, the presence of virulence genes and antibiotic resistance genes, as well as resistance to low pH and bile, were investigated. A total of 178 lactic acid bacterial strains were isolated from the sourdough samples, and seven of these were identified as belonging to the Enterococcus genus. These strains exhibited antimicrobial activity against test bacteria (Salmonella typhimurium ATCC 14028, Escherichia coli ATCC 25922, Streptococcus mutans, Streptococcus aureus 6538, Enterococcus faecalis ATCC 29212, Enterococcus faecium M74, Streptococcus pyogenes ATCC 6538, Listeria innocua ATCC 33090, Listeria monocytogenes ATCC 19111 ) and were found to be safe in terms of carrying virulence (agg, gelE, cylM, cylB, cylA) and antibiotic resistance genes (vanA, vanB, tetM, ermB, aac(6’)aph(2’’)-la). When the isolates' resistance to low pH and bile was examined, it was found that they remained viable at pH 4 after 3 hours, but did not survive at pH 2 and pH 3 after 3 hours. When examining their resistance to 0.5% (w/v) bile concentration, it was determined that they remained viable at levels of 6.2-7.4 log CFU/g after 24 hours.

Keywords

Sourdough, enterococcus, antibiotic resistance, virulence genes, antimicrobial activity, survival at pH and bile salt

Project Number

FHIZ-2022-1160

Bursa Bölgesinden Toplanan Ekşi Mayalardan Elde Edilen Enterokok İzolatlarının Gıda Güvenliği ve Fonksiyonel Karakterizasyonu

Abstract

Bu çalışmada Bursa bölgesinden toplanan 30 adet ekşi mayadan izole edilen enterokokların gıda güvenliği ve fonksiyonel karakteristiğinin belirlemesi amacıyla örneklerden elde edilen izolatların antibiyotik direnç ve antimikrobiyal aktivitesinin tespiti, virülens gen ve antibiyotik direnç genlerinin aranması, düşük pH ve safraya karşı direnç özelliklerinin belirlenmesi çalışmaları yapılmıştır. Çalışmada, ekşi maya örneklerinden toplam 178 adet laktik asit bakteri suşu izole edilmiş ve bunların 7 tanesinin enterekok cinsine ait olduğu tespit edimiştir. Bu suşların test bakterilerine (Salmonella typhimurium ATCC 14028, Escherichia coli ATCC 25922, Streptococcus mutans, Streptococcus aureus 6538, Enterococcus faecalis ATCC 29212, Enterococcus faecium M74, Streptococcus pyogenes ATCC 6538, Listeria innocua ATCC 33090, Listeria monocytogenes ATCC 19111 ) karşı antimikrobiyal aktiviteye sahip olduğu tespit edilmiş ve virülans (agg, gelE, cylM, cylB, cylA) ve antibiyotik direnç genlerini (vanA, vanB, tetM, ermB, aac(6’)-aph(2’’)-la) taşıma açısından güvenli bulunmuştur. İzolatların düşük pH ve safra direnç özelliklerinin incelendiğinde, pH 4’de 3. saatin sonunda canlılıklarını koruduklarını fakat pH 2 ve pH 3’ de 3. saatin sonunda enterokokların canlı kalamadığı tespit edilmiştir. %0.5 (w/v) safra derişimine direnç özelliklerini gösteren sayım sonuçları incelendiğinde 24. saatin sonunda 6.2-7.4 log-kob/g düzeylerinde canlı kaldıkları belirlenmiştir.

Keywords

Ekşi maya, enterokok, antibiyotik direnç, virülens gen, antimikrobiyal aktivite, pH ve safra direnci

Ethical Statement

Bu çalışma etik kurul izni gerektirmemektedir. Araştırma ve yayın etik ilkelerine uygun olarak yürütülmüş ve bu makaleyi hazırlayan yazarlar araştırmaya eşit katkıda bulunmuştur. Yazarlar arasında herhangi bir çıkar çatışması bulunmamaktadır.

Supporting Institution

Uludağ Üniversitesi

Project Number

FHIZ-2022-1160

Thanks

FHIZ-2022-1160 No`lu Proje ile Bursa Uludağ Üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimi teşekkürlerimizi sunarız.

References

• Akabanda, F., Owusu-Kwarteng, J., Tano-Debrah, K., Parkouda, C. and Jespersen, L. 2014. The use of lactic acid bacteria starter culture in the production of Nunu, a spontaneously fermented milk product in Ghana. International Journal of Food Sciences, 2014(2014): 721067
• Altuntaş, E.G., Ayhan, K., Okcu, G., Erkanlı, K., Balci, M. and Sonakın, S.S. 2010. Antimicrobial activities of lactic acid bacteria isolated from raw milk and cheese samples. GIDA - Journal of Food, 35(3): 197-203.
• Aslam, M., Diarra, M. S., Checkley, S. and Bohaychuk, V. 2012. Characterization of antimicrobial resistance and virulence genes in Enterococcus spp. isolated from retail meats in Alberta, Canada. International Journal of Food Microbiology, 156(3): 222–230.
• Bagci, U., Ozmen Togay, S., Temiz, A. and Ay, M. 2019. Probiotic characteristics of bacteriocin-producing Enterococcus faecium strains isolated from human milk and colostrum. Folia Microbiologica, 64(6): 735 750.
• Bakırcı, F. ve Köse, E. 2017. Ekşi hamurlardan laktik asit bakterileri ve mayaların izolasyonu ve tanımlanması. Akademik Gıda, 15(2): 149-154.
• Bellici, A. E., Karasu-Yalcin, S., Eryasar-Orer, K. and Yalçın, E. 2019. MALDI-TOF/TOF mass spectrometry for determination of yeast diversity in traditional cornelian cherry tarhana produced with different cereal/pseudocereal flours. Annals of Microbiology, 69, 613-625.
• Corsetti, A., Settanni, L., Valmorri, S., Mastrangelo, M. and Suzzi, G. 2007. Identification of subdominant sourdough lactic acid bacteria and their evolution during laboratory-scale fermentations. Food Microbiology, 24(6): 592-600.
• Coşansu, S., Kuleaşan, H., Ayhan, K. and Materon, L. A. 2007. Antimicrobial activity and protein profiles of Pediococcus spp. isolated from Turkish “SUCUK”. Journal of Food Processing and Preservation, 31: 190 200.
• Eaton, T. J. and Gasson, M. J. 2001. Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates. Applied and Environmental Microbiology, 67(4): 1628-1635.
• Foulquie Moreno, M. R., Sarantinopoulos, P., Tsakalidou, E. and De Vuyst, L. 2006. The role and application of enterococci in food and health. International Journal of Food Microbiology, 106(1): 1–24.
• Franz, C. M. A. P., Holzapfel, W. H. and Stiles, M. E. 1999. Enterococci at the crossroads of food safety? International Journal of Food Microbiology, 47(1-2): 1–24.
• Franz, C. M. A. P., Stiles, M. E., Schleifer, K. H. and Holzapfel, W. H. 2003. Enterococci in foods – a conundrum for food safety. International Journal of Food Microbiology, 88 (2-3): 105–122.
• Hajikhani, R., Beyatlı, Y. ve Aslim, B. 2007. Antimicrobial activity of Enterococcus strains isolated from white cheese. International Journal of Dairy Technology, 60: 105-108.
• Hammad, A. M., Hassan, H. A. and Shimamoto, T. 2015. Prevalence, antibiotic resistance and virulence of Enterococcus spp. in Egyptian fresh raw milk cheese. Food Control, 50: 815-820.
• Harris, L. J., Daeschel, M. A., Stiles, M. E. and Klaenhammer, T. R. 1989. Antimicrobial activitiy of lactic acid bacteria against Listeria monocytogenes. Journal of Food Protection, 52(6): 384-387.
• Hugas, M., Garriga, M. and Aymerich, M. T. 2003. Functionality of enterococci in meat products. International Journal of Food Microbiology, 88(2-3): 223-233.
• Klein, G. 2003. Taxonomy, ecology and antibiotic resistance of enterococci from food and gastro-intestinal tract. International Journal of Food Microbiology, 88(2-3): 123-131.
• Leisner, J., Hansen, M., Larsen, M., Hansen, L., Ingmer, H. and Sørensen, S. 2011. The genome sequence of the lactic acid bacterium, Carnobacterium maltaromaticum ATCC 35586 encodes potential virulence factors. International Journal of Food Microbiology, 152(3): 107–115.
• Leroy, F., Verluyten, J., Messens, W. and Vuyst, L. D. 2002. Modelling contributes to the understanding of the different behaviour of bacteriocin-producing strains in a meat environment. International Dairy Journal, 12(2-3): 247-253.
• Lian, W., Hsiao, H. and Chou, C. 2003. Viability of microencapsulated Bifidobacteria in simulated gastric juice and bile solution. Short communication. International Journal of Food Microbiology, 86(3): 293–301.
• 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 Suppl 2, S51–S62.
• Nespolo, C. R. and Brandelli, A. 2010. Production of bacteriocin-like substances by lactic acid bacteria isolated from regional ovine cheese. Brazilian Journal of Microbiology, 41(4): 1009-1018.
• Nueno-Palop, C. and Narbad, A. 2011. Probiotic assessment of Enterococcus faecalis CP58 isolated from human gut. International Journal of Food Microbiology, 145(2–3): 390–394.
• Oladipo, I. C., Sanni, A. I. and Swarnakar, S. 2014. Virulence potential of Enterococcus gallinarum strains isolated from selected Nigerian traditional fermented foods. Journal of BioScience and Biotechnology, 3(2): 97-104.
• Onur, M. ve Önlü, H. 2022. Farklı Gıda Ürünlerinden İzole Edilen Laktik Asit Bakterilerinin Bazı Probiyotik Özelliklerinin Belirlenmesi. European Journal of Science and Technology, 32, 562 – 572.
• Peters, J., Mac, K., Wishmann-Shauer, H., Klein, G. and Ellerbroek, L. 2003. Species distribution and antibiotic resistance patterns of enterococci isolated from food of animal origin in Germany. International Journal of Food Microbiology, 88, 311-314.
• Pieniz, S., de Moura, T. M., Vaz Cassenego, A. P. and reazza, R., Guedes Frazzon, A. P., Flavio Anastacio de Oliveira Camargo, F. A. and Brandelli, A. 2015. Evaluation of resistance genes and virulence factors in a food isolated Enterococcus durans with potential probiotic effect. Food Control, 51, 49-54.
• Reviriego, C., Eaton, T., Martín, R., Jiménez, E., Fernández, L., Gasson, M. J. and Rodríguez, J. M. 2005. Screening of virulence determinants in Enterococcus faecium strains isolated from breast milk. Journal of Human Lactation, 21(2): 131-138.
• Saavedra, L., Taranto, M. P., Sesma, F. and de Valdez, G. F. 2003. Homemade traditional cheeses for the isolation of probiotic Enterococcus faecium strains. International Journal of Food Microbiology, 88(2-3): 241-245.
• Sáez, G. D., Saavedra, L., Hebert, E. M. and Zárate, G. 2018. Identification and biotechnological characterization of lactic acid bacteria isolated from chickpea sourdough in northwestern Argentina. LWT, 93, 249-256
• Sakandar, H. A., Hussain, R., Kubow, S., Sadiq, F. A., Huang, W. and Imran, M. 2019. Sourdough bread: A contemporary cereal fermented product. Journal of Food Processing and Preservation, 302, 103–113.
• Salvucci, E., LeBlanc, J. G. and Pérez, G. T. 2016. Technological properties of Lactic acid bacteria isolated from raw cereal material. LWT - Food Science and Technology, 70, 185-191.
• Sarantinopoulos, P. and righetto, C., Georgalaki, M. D., Rea, M. C., Lombardi, A., Cogan, T. M., ... and Tsakalidou, E. 2001. Biochemical properties of enterococci relevant to their technological performance. International Dairy Journal, 11(8): 621-647.
• Şener, A. 2009. Serbest ve Mikroenkapsüle Probiyotik Bakterilerin Ticari Dondurma Üretiminde Kullanılabilirliği Üzerine Bir Araştırma, Doktora Tezi, Hacettepe Üniversitesi Fen Bilimleri Enstitüsü, Ankara, 139s.
• Şener, A. and Temiz, A. 2008. The effects of Certain Prebiotics on the Resistance to Acid, Bile and Cold Storage of Lactobacillus acidophilus and Bifidobacterium bifidum. Presented at the First European Food Congress, Ljubljana, Slovenia.
• Tan, Q., Xu, H., Aguilar, Z. P., Peng, S., Dong, S., Wang, B., Li, P., Chen, T., Xu, F. and Wei, H. 2013. Safety Assessment and Probiotic Evaluation of Enterococcus Faecium YF5 Isolated from Sourdough. Journal of Food Science, 78, M587-M593.
• Toğay, S. Ö. 2010. Doğal fermente gıdalar ve anne sütünden enterokokların izolasyonu, karakterizasyonu ve bunların probiyotik kültür olarak kullanılabilme potansiyelinin araştırılması. Hacettepe Üniversitesi Fen Bilimleri Enstitüsü Dergisi, 12(1): 19-25.
• Tuncer, Y., Özden, B. ve Avşaroğlu, M. D. 2008. Bozanın Bazı Mikrobiyolojik Özelliklerinin ve Laktik Asit Bakterisi İzolatlarının Antibakteriyel Aktivitelerinin Belirlenmesi. Süleyman Demirel Üniversitesi, Fen Bilimleri Enstitüsü Dergisi, 12(1): 19-25.
• Valenzuela, A. S., Benomar, N., Abriouel, H., Cañamero, M. M. and Gálvez, A. 2010. Isolation and identification of Enterococcus faecium from seafoods: Antimicrobial resistance and production of bacteriocin-like substances. Food Microbiology, 27(7): 955-961.
• Weckx, S., Allemeersch, J., Van der Meulen, R., Vrancken, G., Huys, G., Vandamme, P., Van Hummelen, P. and De Vuyst, L. 2009. Development and validation of a species-independent functional gene microarray that targets lactic acid bacteria. Applied and Environmental Microbiology, 75(20): 6488–95.
• Yangılar, F., Gülhan, B. and Kılıçgün, H. 2023. Determination of Antimicrobial Properties of Endemic Black Sakı Apple Vinegar Produced by Traditional Method Using Different Yeast Raw Materials. Bursa Uludag Üniv. Ziraat Fak. Derg., 37(1): 79-99.