TARHANADAN İZOLE EDİLEN LAKTOBASİLLER TARAFINDAN ÜRETİLEN BAKTERİYOSİNLERİN KARAKTERİZASYONU

Yıl 2020, Cilt: 45 Sayı: 4, 786 - 799, 21.06.2020

Halil İbrahim Kaya , Ömer Şimşek

https://doi.org/10.15237/gida.GD20027

Öz

Tarhana Anadolu’da kış için hazırlanan ve sık tüketilen fermente bir gıdadır. Fermente tarhana hamuru laktik asit bakterileri (LAB) ve maya türlerinden oluşan mikrofloraya sahiptir. Bu florada bazı LAB’de bakteriyosin üretimiyle antimikrobiyal aktivite gösterirler. Çalışmamızın amacı tarhanadan izole edilmiş L. namurensis PFC70, L. plantarum PFC74 ve L. paralimentarius PFC97 suşlarının bakteriyosinlerinin belirlenmesi ve karakterizasyonudur. PFC70, PFC74 ve PFC97 suşlarının Micrococcus luteus DSM1790 suşuna karşı 400, 1600, 1600 AU/mL antimikrobiyal aktiviteye sahip olduğu belirlenmiştir. Suşların kültür üst sıvılarındaki metabolitlerin yüksek sıcaklığa ve proteaz enzimlerine karşı hassas, düşük pH koşullarında stabil, bakteriyosin tabiatında olduğu anlaşılmıştır. Üretici hücrelerin genomunda yapılan PZR taramasında, PFC74 bakteriyosininin plantarisin benzeri olduğu belirlenmiştir. Bakteriyosinler, amonyum sülfat çöktürmesi, katı faz ekstraksiyonu ve ters faz sıvı kromotografisi ile saflaştırılmış ve trisin-SDS PAGE ile moleküler büyüklükleri 5 kDa altında olduğu tespit edilmiştir. PFC70 ve PFC74 bakteriyosini bakteriyosidal, PFC97 ise bakteriyostatik etkili bulunmuştur. Bu sonuçlar PFC70 suşunun yeni bir bakteriyosin üreticisi olduğunu göstermiştir.

Anahtar Kelimeler

Tarhana, Laktik Asit Bakterisi, Bakteriyosin, Antimikrobiyal

Destekleyen Kurum

Pamukkale Üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimi

Proje Numarası

2010 BSP 021

Teşekkür

Bu çalışmayı 2010 BSP 021 proje numarası ile destekleyen Pamukkale Üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimi’ne teşekkür ederiz.

CHARACTERIZATION OF BACTERIOCINS PRODUCED BY LACTOBACILLI ISOLATED FROM TARHANA

Öz

Tarhana is a fermented food that has been consumed often, produced for winter in Anatolia. Fermented tarhana dough microflora includes lactic acid bacteria (LAB), yeast species and some LAB exhibit antimicrobial activity due to bacteriocin production. The purpose of study was determination and characterization of bacteriocins of L. namurensis PFC70, L. plantarum PFC74, L. paralimentarius PFC97 strains which were isolated from tarhana. Strains had 400, 1600, 1600 AU/mL antimicrobial activity against Micrococcus luteus DSM1790 respectively. The relevant methabolites of strains at the culture supernatant was to be bacteriocin nature with low pH stability, high temparature and protease enzymes sensitivity. Producer cell genome showed that PFC74 bacteriocin was similiar with plantaricin. Bacteriocins were purified by ASP, SPE, RPLC and thereby molecular weights were determined under 5 kDa with Tricine-SDS-PAGE. PFC70 and PFC74 bacteriocin were found to be bacteriocidal, while PFC97 was bacteriostatic. Results indicated that PFC70 was novel bacteriocin producer. 

Anahtar Kelimeler

Tarhana, Lactic acid bacteria, bacteriocin, antimicrobial

Proje Numarası

2010 BSP 021

Kaynakça

• Ahmad, V., Khan, M.S., Jamal, Q.M.S., Alzohairy, M.A., Karaawi, M.A.A., Siddiqui, M.U. (2017). Antimicrobial potential of bacteriocins: in therapy, agriculture and food preservation. Int. J. Antimicrob. Agents, 49,1–11.
• Arief, I., Budiman, C., Jenie, B., Andreas, E., Yuneni, A. (2015). Plantaricin IIA-1A5 from Lactobacillus plantarum IIA-1A5 displays bactericidal activity against Staphylococcus aureus. Benef. Microbes. 6:603-613.
• Barbosa, M.S., Todorov, S.D., Ivanova, I.V., Belguesmia, Y., Choiset, Y., Rabesona, H., Chobert, J.M., Haertlé, T., Franco, B.D.G.M. (2016). Characterization of a two-peptide plantaricin produced by Lactobacillus plantarum MBSa4 isolated from Brazilian salami. Food Control. 60:103-112.
• Beasley, S. S. ve Saris, P. E. J. (2004). Nisin-producing Lactococcus lactis strains from human milk. Appl. Environ. Microbiol. 70:5051-5053.
• Delves-Broughton, J., Blackburn, P., Evans, R.J., Hugenholtz, J. (1996). Applications of the bacteriocin, nisin”. Antonie Van Leeuwenhoek. 69,(2),193-202.
• de Vuyst, L. ve Leroy, F. (2007). Bacteriocins from lactic acid bacteria: production, purification and food applications. J. Mol. Microbiol. Biotechnol. 13:194–199.
• Franz, C.M. A. P., Du Toit, M., von Holy, A., Schillinger, U., Holzapfel, W. H. (1997). Production of nisin-like bacteriocins by Lactococcus lactis strains isolated from vegetables. J. Basic. Microbiol. 37:197-196.
• Hill, C., Nes, I.N., Ross, R.P. (2011). Bacteriocins. The 10th LAB Symposium: Thirty years of Research on Lactic acid bacteria, August 28-September 1, 2011, Netherlands, 37-56p.
• Hu, Y., Liu, X., Shan, C., Xia, X., Wang, Y., Dong, M., Zhou. J. (2017). Novel bacteriocin produced by Lactobacillus alimentarius FM-MM4 from a traditional Chinese fermented meat Nanx Wudl: Purification, identification and antimicrobial characteristics. Food Control. 77:290-297.
• Jozala, A. F., Novaes, L. C. L., Cholewa, O., Moraes, D., Penna, T. C. V. (2005). Increase of nisin production by Lactococcus lactis in different media. African J Biotechnol. 4:262-265.
• Juturu, W. ve Wu, J.C. (2018). Microbial production of bacteriocins: Latest research development and applications. Biotechnol. Adv. 36,2187–2200.
• Klaenhammer, T.R. (1993). Genetics of bacteriocins produced by lactic acid bacteria. FEMS Microbiol. Rev. 12,39-85.
• Laemmli, U.K. (1970). Cleavage of the structural proteins during the assembly of the head of bacteriophage T4. Nature. 227:680-685.
• Lü, X., Hu, P., Dang, Y., Liu, B. (2014a). Purification and partial characterization of a novel bacteriocin produced by Lactobacillus casei TN-2 isolated from fermented camel milk (Shubat) of Xinjiang Uygur Autonomous region, China. Food Control. 43:276-283.
• Lü, X., Yi, L., Dang, J., Dang, Y., Liu, B. (2014b). Purification of novel bacteriocin produced by Lactobacillus coryniformis MXJ 32 for inhibiting bacterial foodborne pathogens including antibiotic-resistant microorganisms. Food Control. 46:264-271.
• Macwana, S.J. ve Muriana, P.M. (2012). A ‘bacteriocin PCR array’ for identification of bacteriocin-related structural genes in lactic acid bacteria. J Microbiol Methods. 88:197–204.
• Milioni, C., Martínez, B., Degl’innocenti, S., Turchi, B., Fratini, F., Cerri, D., Fischetti, R. (2015). A novel bacteriocin produced by Lactobacillus plantarum LpU4 as a valuable candidate for biopreservation in artisanal raw milk cheese. Dairy Sci. Technol. 95 (4):479-494.
• Mills, S., Serrano, L.M., Griffin, C., O’Connor, M.P., Schaad, G., Bruining, C., Hill, C., Ross, R.P., Meijer, W.C. (2011). Inhibitory activity of Lactobacillus plantarum LMG p-26358 against Listeria innocua when used as an adjunt starter in the manufacture of cheese. Microb Cell Fac.10(1):7.
• Moreno, F.M.R., Callewaert, R., Devreese, B., van Beeumen, J., de Vuyst, L. (2003). Isolation and biochemical characterization of enterocins produced by enterococci from different sources. J Appl Microbiol. 94: 214-229.
• O’Shea, E.F., Cotter, P.D., Ross, R.P., Hill, C. (2013). Strategies to improve the bacteriocin protection provided by lactic acid bacteria. Curr. Opin. Biotechnol. 24,130–134.
• O’sullivan, L., Ross, R.P., Hill, C. (2002). Potential of bacteriocin-producing lactic acid bacteria improvements in food safety and quality. Biochimie. 84:593-604.
• Özel, S. (2012). Tarhana hamuru fermantasyonunun mikrobiyal taksonomik yapısının ve populasyon dinamiğinin belirlenmesi. Pamukkale Üniversitesi, Fen Bilimleri Enstitüsü, Gıda Mühendisliği Anabilim Dalı, Yüksek Lisans Tezi, Denizli, Türkiye, 153s.
• Parente, E. ve Ricciardi, A. (1999). Production, recovery and purification of bacteriocins from lactic acid bacteria. Appl Microbiol Biotechnol. 52:628-638.
• Saavedra, L. ve Sesma, F. (2011). Purification techniques of bacteriocins from lactic acid bacteria and other gram-positive bacteria. In: Procaryotic antimicrobial peptides: from genes to applications. Djemal Drider, Sylvie Rebuffat (Editors). Springer, New York, Chapter 7:99-115.
• Schagger, H ve von Jagow, G. (1987). Tricine-sodium dodecyl slphate-poly-acrylamide gel electrophoresis for the separation of protein in the range from 1 to 100 kDa. Anal Biochem. 166:368-379.
• Simha, B.V., Sood, S.K., Kumariya, R., Garsa, A.K. (2012). Simple and rapid purification of pediocin PA-1 from Pediococcus pentosaceous NCDC 273 suitable for industrial application. Microbiol Res. 167:544-549.
• Tagg, J.R. ve McGiven, A.R. (1971). Assay system for bacteriocins. Appl Microbiol., 21:943.
• Takala, T. M. ve Saris, P. E. J. (2007). Nisin: Past, present and future. In: Research and Applications of Bacteriocins, Margaret A. Riley, and Osnat Gillor, (Editors) Horrizon Bioscience, Wymondham, 181-213.
• Todorov, S.D. (2009). Bacteriocins from Lactobacillus plantarum-production, genetic organization and mode of action. Braz J Microbiol. 40:209–221.
• Twomey, D., Ross, R.P., Ryan, M., Meaney, B., Hill, C. (2002). Lantibiotics produced by lactic acid bacteria: structure, function and applications. In: Lactic acid bacteria: genetics, metabolism and applications, R.J. Sizezen, J. Kok, T. Abee, G. Schaafsma (Editors), Kluwer Academic Publishers, Dordrecht, 165-185.
• Van Belkum, M.J., Hayema, B.J., Geis, A., Kok, J., Venema, G. (1989). Cloning of two bacteriocin genes from a lactococcal bacteriocin plasmid. Appl Environ Microbiol. 55: 1187-1191.
• Wang, J., Zhang, S., Ouyang, Y., Li, R. (2019). Current developments of bacteriocins, screening methods and their application in aquaculture and aquatic products. Biocatalysis and Agricultural Biotechnology. 22,101395.
• Wen, S.L., Philip, K., Ajam, N. (2016). Purification, characterization and mode of action of plantaricin K25 produced by Lactobacillus plantarum. Food Control. 60: 430-439.
• Woraprayote, W., Pumpuang, L., Tosukhowong, A., Roytrakul, S., Pres, R.H., Zendo, T., Kenji, S., Benjakul, S., Visessanguan, W. (2015). Two putatively novel bacteriocins active against Gram-negative food borne pathogens produced by Weissella hellenica BCC 7293. Food Control. 55: 176-184.
• Zhu, X., Zhao, Y., Sun, Y., Gu, Q. (2014). Purification and characterisation of lantaricin ZJ008, a novel bacteriocin against Staphylococcus spp. from Lactobacillus plantarum ZJ008. Food Chem. 165:216-223.
• Zou, J., Jiang, H., Cheng, H., Fang, J., Huang, G. (2018). Strategies for screening, purification and characterization of bacteriocins. Int. J. Biol. Macromol. 117,781–789.