Doğal Fermente Gıdalardan İzole Edilen Muhtemel Laktik Asit Bakterilerinin Antimikrobiyal Aktiviteleri ve Laktik Asit Üretim Düzeylerinin İncelenmesi

Yıl 2022, Cilt: 36 Sayı: 1, 25 - 40, 01.06.2022

Özüm Özoğlu , Mehmet Gumustas , Sibel A. Özkan , Evrim Güneş Altuntaş

https://doi.org/10.20479/bursauludagziraat.943244

Öz

Laktik asit bakterileri pek çok fermente ve probiyotik gıdanın bünyesinde yer alan faydalı bakterilerdir. Bu bakterileri önemli kılan özellikleri arasında GRAS (Generally Recognized As Safe) statüde olmaları ve ürettikleri antimikrobiyal metabolitler bulunmaktadır. Laktik asit bakterilerinin ürettiği antagonistik etkiye sahip en önemli metabolit bu gruba adını veren laktik asit olup; bunun yanısıra hidrojen peroksit, asetik asit, diasetil, bakteriyosin vb. metabolitler ile de antagonistik etkiye neden olmaktadırlar. Bu çalışmada çeşitli peynir, sucuk ve kefir örneklerinden izole edilen 23 adet muhtemel laktik asit bakteri izolatının Salmonella Enteritidis ATCC 13076, Streptococcus mutans ATCC 25175, Escherichia coli ATCC 25922, Bacillus subtilis ATCC 6633 ve Staphylococcus aureus ATCC 43300 (metisilin ve oksasilin dirençli) patojenleri üzerindeki inhibisyon etkisi Agar Spot Yöntemi ile araştırılmıştır. Test sonucunda pozitif etki gösteren izolatların süpernatantları ile Kuyu Difüzyon denemesi gerçekleştirilmiştir. Son olarak izolatların ürettiği laktik asit miktarları HPLC yöntemi ile de belirlenmiştir. Agar Spot Testi’ne göre izolatların neredeyse tamamı tüm patojenler üzerinde etkinlik gösterirken, patojenler üzerindeki inhibisyon etkiyi gösteren zon çaplarının 1-24.5 mm aralığında değiştiği tespit edilmiştir. 24.5 mm zon çapı ile maksimum etki 31 nolu izolat tarafından E.coli ATCC 25922 üzerinde gözlemlenirken; 1 mm zon çapı ile minimum etki 431 nolu izolat tarafından S. Enteritidis ATCC 13076 test mikroorganizmasına karşı gözlemlenmiştir. Kuyu Difüzyon Testi’nde ise hiçbir pozitif sonuç elde edilememiş ve bu nedenle bakterilerin antagonistik etkilerinin daha çok laktik asit üretiminden kaynaklandığı düşünülmüştür. Yapılan kromatografik çalışma ile laktik asit bakterilerinin ürettiği laktik asit miktarının 0.13-5.52 mmol aralığında olduğu gözlenmiştir. Çalışmadan elde edilen sonuçlar laktik asit bakterilerinin bakteriyosin gibi önemli protein bazlı inhibitörleri üretmese bile, ürettiği temel metabolit olan laktik asit ile etkin bir antimikrobiyal aktivite kapasitesine sahip olduğunu göstermektedir.

Anahtar Kelimeler

Antimikrobiyal aktivite, laktik asit, laktik asit bakterisi, HPLC

Destekleyen Kurum

Ankara Üniversitesi Bilimsel Araştırmalar Proje Koordinatörlüğü

Proje Numarası

17H0415001

Teşekkür

Çalışmada kullanılan patojen suşların temininde sağladıkları destek için Ankara Üniversitesi Eczacılık Fakültesi Farmasötik Mikrobiyoloji Anabilim Dalı Öğretim Üyeleri Doç. Dr. Banu KAŞKATEPE ve Doç. Dr. Müjde ERYILMAZ’a teşekkür ederiz.

Investigation of Antimicrobial Activities and Lactic Acid Production Levels of Presumptive Lactic Acid Bacteria Isolated From Naturally Fermented Foods

Öz

Lactic acid bacteria are useful bacteria which are included to composition of many fermented and probiotic food. Being GRAS (Generally Recognized As Safe) statute and the antimicrobial metabolites produced by them are among the properties which make these bacteria significant. The most important metabolite with antagonistic effect produced by lactic acid bacteria is lactic acid that gives the name of these bacteria, besides LAB can have antagonistic effect by producing hydrogen peroxide, acetic acid, diacetyl, bacteriocin etc. In this study, inhibition effects of 23 presumptive lactic acid bacteria isolates that isolated from various cheese, sucuk and kefir samples on Salmonella Enteritidis ATCC 13076, Streptococcus mutans ATCC 25175, Escherichia coli ATCC 25922, Bacillus subtilis ATCC 6633 and Staphylococcus aureus ATCC 43300 (methicillin and oxacillin resistant) were investigated by Agar Spot method. According to the assay, Well Diffusion Test was performed with supernatant of the isolates showed positive results. Finally, the quantification of lactic acid was detected by HPLC method. As a result of Agar Spot Test, while all of the isolates were effective on all pathogens, the inhibition zone diameters were ranged between 1-24.5 mm. While the maximum effect was observed by isolate 31 on E.coli ATCC 25922 with a zone diameter of 24.5 mm, the minimum effect was observed by isolate 431 against S. Enteritidis ATCC 13076 test microorganism with a zone diameter of 1 mm.Then, due to not to have any positive results from Well Diffusion Assay, it has been considered the antagonistic effect of the bacteria were originated from lactic acid production. It was observed that lactic acid produced by LAB was ranged between 0.13-5.52 mmol by chromatographic study. Even if the LAB do not produce protein-based inhibitors such as bacteriocin, the results shows LAB has an effective antimicrobial activity capacity with lactic acid, the main metabolite of LAB.

Anahtar Kelimeler

Antimicrobial activity, lactic acid, lactic acid bacteria, HPLC

Proje Numarası

17H0415001

Kaynakça

• Aka-Gbezo, S., Konan, A. G., N’Cho, M., Achi, P., Koffi-Nevry, R., Koussemon-Camara, M., & Bonfoh, B. 2017. Screening of antimicrobial activity of lactic acid bacteria isolated from anango baca slurry, a spontaneously fermented maize product used in Côte d’Ivoire. International Journal of Biological and Chemical Sciences, 11(6): 2616–2629.
• Ali, A. A. 2010. Beneficial role of lactic acid bacteria in food preservation and human health: A review. Research Journal of Microbiology, 5(12): 1213–1221.
• Altuntaş, E. G., Ayhan, K. ve Okcu, G. 2010. Çiğ Süt ve Peynir Örneklerinden İzole Edilen Latik Asit Bakterilerinin Antimikrobiyel Aktiviteleri. GIDA, 35(3): 197–203.
• Axelsson, L. and Ahrné, S. 2000. Lactic Acid Bacteria: Applied Microbial Systematics, Ed: Priest, F.G.and Goodfellow, M., Springer, Netherlands, pp. 367–388.
• Bungenstock, L., Abdulmawjood, A. and Reich, F. 2020. Evaluation of antibacterial properties of lactic acid bacteria from traditionally and industrially produced fermented sausages from Germany. PLoS ONE, 15(3): 1–15.
• Campana, R., Van Hemert, S. and Baffone, W. 2017. Strain-specific probiotic properties of lactic acid bacteria and their interference with human intestinal pathogens invasion. Gut Pathogens, 9(1): 1–12.
• Çelik, Ş. ve Uysal, Ş. (2009). Beyaz Peynirin Bileşim, Kalite, Mikroflora ve Olgunlaşması. Atatürk Üniv.Ziraat Fak. Derg., 40(1): 141–151.
• Cizeikiene, D., Juodeikiene, G., Paskevicius, A. and Bartkiene, E. 2013. Antimicrobial activity of lactic acid bacteria against pathogenic and spoilage microorganism isolated from food and their control in wheat bread. Food Control, 31(2): 539–545.
• Cosansu, S., Kuleasan, H., Ayhan, K. and Materon, L. 2007. Antimicrobial activity and protein profiles of Pediococcus spp. isolated from Turkish “sucuk.” Journal of Food Processing and Preservation, 31(2): 190–200.
• Daouadji, S. D., Abbouni, B., Bouricha, M., and Lamine, M. 2020. Antibacterial activity of lactic acid bacteria isolated from milk and traditional fermented dairy products of south Algeria against multidrug resistance pathogenic bacteria. South Asian Journal of Experimental Biology, 10(5): 322-331.
• De Vuyst, L. and Leroy, F. 2007. Bacteriocins from lactic acid bacteria: Production, purification, and food applications. Journal of Molecular Microbiology and Biotechnology, 13(4): 194–199.
• Dinev, T., Beev, G., Tzanova, M., Denev, S., Dermendzhieva, D. and Stoyanova, A. 2018. Antimicrobial activity of lactobacillus plantarum against pathogenic and food spoilage microorganisms: A review. Bulgarian Journal of Veterinary Medicine, 21(3): 253–268.
• Doetsch, R. N. 1981. Determinative Methods of Light Microscopy.: Manual of Methods for General Bacteriology, Eds: Gerdhardt, P., Murray, R. G. E., Costilow, R. N., Nester, E. W., Wood, W. A., Krieg, N. R. and Phillips, G. B., American Society for Microbiology, (pp. 21–31).
• Dowarah, R., Verma, A. K., Agarwal, N., Singh, P. and Singh, B. R. 2018. Selection and characterization of probiotic lactic acid bacteria and its impact on growth, nutrient digestibility, health and antioxidant status in weaned piglets. PLoS ONE, 13(3).
• Durlu-Ozkaya, F., Xanthopoulos, V., Tunail, N. and Litopoulou-Tzanetaki, E. 2001. Technologically important properties of lactic acid bacteria isolates from Beyaz cheese made from raw ewes’ milk. Journal of Applied Microbiology, 91(5): 861–870.
• Erdoğmuş, S. F. ve Bostancı, B. 2020. Kefir Örnekleri̇nden Lakti̇k Asit Bakteri̇leri̇ni̇n İzolasyonu, İdenti̇fi̇kasyonu ve Anti̇mi̇krobi̇yal Etki̇leri̇nin Değerlendi̇ri̇lmesi̇. GIDA, 45(1): 72–80.
• Erginkaya, Z., Güven, M., Kavas, C., Kabak, B. ve Karaca, B. O. 2003. Farklı Laktik Asit Kültürleriyle Üretilen Yoğurtlarda Laktik Asit Bakterilerinin Aspergillus flavus Üzerine Antifungal Etkisi. Gıda Mühendisliği Dergisi, 7(15): 31–36.
• Ertekin, Ö. ve Çon, A. H. 2014. Farklı Gıdalardan İzole Edilen Laktik Asit Bakterilerinin Endüstriyel ve Probiyotik Özellikleri. Akademik Gıda, 12(4): 6–16.
• Gomez, J. S., Parada, R. B., Vallejo, M., Marguet, E. R., Bellomio, A., Perotti, N. and de Carvalho, K. G. 2021. Assessment of the bioprotective potential of lactic acid bacteria against Listeria monocytogenes in ground beef. Archives of Microbiology, 0123456789.
• González, L., Sandoval, H., Sacristán, N., Castro, J. M., Fresno, J. M. and Tornadijo, M. E. 2007. Identification of lactic acid bacteria isolated from Genestoso cheese throughout ripening and study of their antimicrobial activity. Food Control, 18(6): 716–722.
• Gumustas, M., Kurbanoglu, S., Uslu, B. and Ozkan, S. A. 2013. UPLC versus HPLC on drug analysis: Advantageous, applications and their validation parameters. Chromatographia, 76(21–22): 1365–1427.
• Gumustas, M., Uslu, B. and Ozkan, S. A. 2017. The Role and the Place of High-Performance Liquid Chromatography for the Determination of Fermented Dairy Products: Soft Chemistry and Food Fermentation, Eds: Grumezescu, A.M. and Holban, A.M., Elsevier, (pp. 421–464).
• Halkman, A. K. ve Ayhan, K. 2005. Mikroorganizma Sayımı: Gıda Mikrobiyolojisi ve Uygulamaları, Ed: Halkman, A.K., Başak Matbaacılık Ltd. Şti., Ankara, (p. 18).
• Halkman, A. K., Yetişmeyen, A., Halkman, Z., Yıldırım, M., Yıldırım, Z. ve Çavuş, A. 1994. Kaşar Peynir Üretiminde Starter Kültür Kullanımı Üzerinde Araştırmalar. TÜBİTAK Türk Tarım ve Ormancılık Derg, 18(5): 365–377.
• Harrigan, W. F. and McCance, M. E. (1976). Laboratory methods in food and dairy microbiology, Academic Press Inc, London, 425p.
• Hori, C., Yamazaki, T., Ribordy, G., Takisawa, K., Matsumoto, K., Ooi, T., Zinn, M., and Taguchi, S. 2019. High-cell density culture of poly(lactate-co-3-hydroxybutyrate)-producing Escherichia coli by using glucose/xylose-switching fed-batch jar fermentation. Journal of Bioscience and Bioengineering, 127(6): 721–725.
• ICH Official web site : ICH. (n.d.). https://www.ich.org/(07.05.2021).
• Kıvanç, M. ve Erikçi, Ş. Y. 2018. Sofralık Fermente Zeytinlerden (Olea Europaea L.) İzole Edilen Laktik Asit Bakterilerinin Antimikrobiyal Aktivitesinin ve Bazı Metabolik Özelliklerinin Belirlenmesi. Anadolu Üniversitesi Bilim ve Teknoloji Dergisi C- Yaşam Bilimleri ve Biyoteknoloj, 7(1): 41–51.
• Kivanç, M., Yılmaz, M., and Çakır, E. 2011. Isolation and identification of lactic acid bacteria from boza, and their microbial activity against several reporter strains. Turkish Journal of Biology, 35(3): 313–324.
• König, H., Unden, G.,and Fröhlich, J. 2017. Biology of Microorganisms on Grapes, in Must and in Wine: Vol. XXII (2nd ed.), Springer International Publishing, 522p.
• Mathialagan, M., Thangaraj Edward, Y. S. J., David, P. M. M., Senthilkumar, M., Srinivasan, M. R., and Mohankumar, S. 2018. Isolation, Characterization and Identification of Probiotic Lactic Acid Bacteria (LAB) from Honey Bees. International Journal of Current Microbiology and Applied Sciences, 7(4): 894–906.
• Nebbia, S., Lamberti, C., Lo Bianco, G., Cirrincione, S., Laroute, V., Cocaign-Bousquet, M., Cavallarin, L., Giuffrida, M. G. and Pessione, E. 2021. Antimicrobial potential of food lactic acid bacteria: Bioactive peptide decrypting from caseins and bacteriocin production. Microorganisms, 9(1): 1–19.
• Orji, J. O., Amaobi, C. B., B., M. I., Uzoh, C. V. and Emioye, A. A. 2020. Antagonistic effect and bacteriocinogenic activity of Lactic Acid Bacteria isolated from Sorghum bicolor - based ‘ ogi ’ on food borne bacterial pathogens from cabbage. African Journal of Clinical and Experimental Microbiology, 21(1): 45–52.
• Ringø, E., Van Doan, H., Lee, S. H., Soltani, M., Hoseinifar, S. H., Harikrishnan, R. and Song, S. K. 2020. Probiotics, lactic acid bacteria and bacilli: interesting supplementation for aquaculture. Journal of Applied Microbiology, 129(1): 116–136.
• Rodríguez, J. M., Martínez, M. I. and Kok, J. 2002. Pediocin PA-1, a wide-spectrum bacteriocin from lactic acid bacteria. In Critical Reviews in Food Science and Nutrition, 42(2): 91–121.
• Sari, M., Suryanto, D. and Yurnaliza. 2018. Antimicrobial activity of lactic acid bacteria isolated from bekasam against staphylococcus aureus ATCC 25923, escherichia coli ATCC 25922, and salmonella sp. IOP Conference Series: Earth and Environmental Science, 130(1).
• Schillinger, U. and Lücke, F. K. 1989. Antibacterial activity of Lactobacillus sake isolated from meat. Applied and Environmental Microbiology, 55(8): 1901–1906.
• Stupar, J., Holøymoen, I. G., Hoel, S., Lerfall, J., Jakobsen, A. N. and Rustad, T. 2021. Diversity and antimicrobial activity towards listeria spp. and escherichia coli among lactic acid bacteria isolated from ready-to-eat seafood. Foods, 10(271):17p..
• Teuber, M. 2008. Lactic Acid Bacteria: Biotechnology: Second Edition, Eds: Rehm, H. J. and Reed G., Wiley-Blackwell, pp. 325–366.
• Ucar, R. A., Pérez-Díaz, I. M., and Dean, L. L. 2020. Gentiobiose and cellobiose content in fresh and fermenting cucumbers and utilization of such disaccharides by lactic acid bacteria in fermented cucumber juice medium. Food Science and Nutrition, 8(11): 5798–5810.
• Yang, Q., Wei, C., Guo, S., Liu, J., and Tao, Y. 2020. Cloning and characterization of a l-lactate dehydrogenase gene from Ruminococcaceae bacterium CPB6. World Journal of Microbiology and Biotechnology, 36(12): 1–10.
• Yildirim Kumral, A., Kumral, N. A. ve Gürbüz, O. 2020. Candida boidinii’nin Farklı Suşlarının Deltamethrini Parçalama Potansiyellerinin İn-vitro Koşullarda Belirlenmesi. B Bursa Uludag Üniv. Ziraat Fak. Derg., 34(2): 337–349.
• Zhang, J. and Li, X. 2018. Novel strategy for phenyllactic acid biosynthesis from phenylalanine by whole cell recombinant Escherichia coli coexpressing l-phenylalanine oxidase and l-lactate dehydrogenase. Biotechnology Letters, 40(1): 165–171.
• Zheng, J., Wittouck, S., Salvetti, E., Franz, C. M. A. P., Harris, H. M. B., Mattarelli, P., O’Toole, P. W., Pot, B., Vandamme, P., Walter, J., Watanable, K., Wuyts, S., Felis, G. E., Gänzle, M. G. and Lebeer, S. 2020. A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. International Journal of Systematic and Evolutionary Microbiology, 70(4): 2782–2858.