Çiğ Süt, Peynir Örneklerinden İzole Edilen Lactococcus. lactis ssp. lactis Suşlarının ve Kefir Süpernatanlarının Bazı Biyokimyasal Özellikleri, Antimikrobiyel Etkinliği ve Antibiyotiklere Karşı Dirençlığının Araştırılması

Bu çalışmada çiğ koyun sütünden yapılan peynir örneklerinden ve inek sütünden izole edilen Lactococcus lactis ssp. lactis (L. lactis ssp. lactis) izolatlarının ve kefir süpernatanlarının (kefir mikroflorasi) bazı biyokimyasal özellikleri, antibiyotiklere karşı dirençliliği ve antibakteriyal etkileri incelenmiştir. Araştırmada 30 kefir süpernatantı ve 60 L. lactis ssp. lactis izolatı analiz edilmiştir. 30 izolat çiğ inek sütlerinden ve 30 izolat koyun peyniri örneklerinden elde edilmiştir. Kefir süpernatanlarının E. faecalis’e karşı antimikrobiyal etki göstermezken, B. cereus’a karşı en etkili olduğu gözlenmiştir. Ayrıca şiddete sırasına göre E. coli ve S. aureus’a etkili olduğu saptanmıştır. Kefir süpernatantlarinda antibiyotiklere karşı en yüksek direnç kloramfenikol ve en düşük direnç ise eritromisin’de gözlenmiştir. Çiğ koyun peyniri örneklerinden ve çiğ inek sütlerinden izole edilen L. lactis ssp. lactis izolatlarının en yüksek antimikrobiyal etkisi E. faecalis’e karşı gözlenmiştir. Ayrıca S. auerus ve L. sakei’ye karşı antimikrobiyal aktiviteleri E. coli göre daha yüksek olduğu görülmüştür. Çiğ koyun peyniri örneklerinden izole edilen L. lactis ssp. lactis izolatların antibiyotiklere karşı %43.3 streptomysine, %13.3 tetrasikline ve %22.2 eritromisine dirençli bulunmuştur. Çiğ inek süt örneklerinden elde edilen L. lactis ssp. lactis izolatların’da ise %40 streptomysine, %40 tetrasikline, %16.7 ampisiline ve %23.3 eritromisine karşı dirençli bulunmuştur. Sonuç olarak, koyun peyniri örneklerinden izole edilen L. lactis ssp. lactis izolatlarının daha yüksek antimikrobiyal aktivite ve laktik asit üretimine sahip olduğu saptanmıştır. Çiğ inek sütü ve çiğ koyun peynirlerden elde edilen tüm izolatlar arasında, 7 adetinde Tet(S) (%11.7) , 6 adetinde Str(A) (%10) ve 1 adetinde Str(B) (%1.7) direnç geni PCR metoduyla ile saptanmıştır.

Investigation of Some Biochemical Properties, Antimicrobial Activity and Antibiotic Resistances of Kefir Supernatants and Lactococcus lactis ssp. lactis Strains Isolated from Raw Cow Milk and Cheese Samples

In this study some biochemical characteristics, antibiotic resistance profiles and antibacterial activity of kefir microfloras (kefir supernatants)and Lactococcus lactis ssp. lactis (L. lactis ssp. lactis) bacteria isolated from cheese made from sheep milk and raw cow milk were investigated.In this study, 30 kefir supernatants and 60 isolates of L. lactis ssp. lactis were analyzed. 30 isolates obtained from raw cow milk and 30isolates obtained from raw sheep cheeses. In kefir supernatants the highest antimicrobial activity was observed against B. cereus, followedby E. coli, S. aureus whereas no antimicrobial activity was observed against the E. faecalis. Kefir supernatants exhibited highest resistance tochloramphenicol and lowest resistance to erythromycin. The highest antimicrobial activity in L. lactis ssp. lactis isolates obtained from rawsheep cheese and raw cow milk samples were found against E. faecalis followed by S. aureus, L. sakei and E. coli respectively. The isolatesobtained from raw sheep cheese samples 43.3% were resistant to streptomycin, 13.3% tetracycline and 22.2% erythromycin. The isolatesobtained from raw cow milk samples 40% were resistant to streptomycin, 40% tetracycline, 16.7% ampicillin and 23.3% erythromycin. Theresults showed that L. lactis ssp. lactis bacteria isolated from cheese samples derived from raw sheep milk demonstrated better antimicrobialactivity and lactic acid production. Among all isolates obtained from raw cow milks and raw sheep cheeses Tet(S) resistance gene wasconfirmed in 7 (11.7%), Str(A) in 6 (10%) and Str(B) in 1 (1.7%) isolates by PCR method.

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Ondes N, Ozpinar H: Occurrence of ESBL-producing enterobacteriaceae in red meat samples. Kafkas Univ Vet Fak Derg, 22 (1): 79-83, 2016. DOI: 10.9775/kvfd.2015.13944
Tekiner IH, Ozpinar H: Occurrence and characteristics of extended spectrum beta-lactamases-producing Enterobacteriaceae from foods of animal origin. Braz J Microbiol, 47, 444-451, 2016. DOI: 10.1016/j. bjm.2015.11.034
Devirgilis C, Barile S, Caravelli A, Coppola D, Perozzi G: Identification of tetracycline and erythromycin-resistant Gram-positive cocci within the fermenting microflora of an Italian dairy food product. J Appl Microbiol, 109, 313-323, 2010. DOI: 10.1111/j.1365-2672.2010.04661.x
Danielsen M, Wind A: Susceptibility of Lactobacillus spp. to antimicrobial agents. Int J Food Microbiol, 82, 1-11, 2003. DOI: 10.1016/ S0168-1605(02)00254-4
Temmerman R, Pot B, Huys G, Swings J: Identification and antibiotic susceptibility of bacterial isolates from probiotic products. Int J Food Microbiol, 81, 1-10, 2003. DOI: 10.1016/S0168-1605(02)00162-9
Boziaris IS, Adams MR: Transient sensitivity to nisin in cold shocked Gram negatives. Lett Appl Microbiol, 31, 233-237, 2000. DOI: 10.1046/j.1365-2672.2000.00804.x
Arias CA, Murray BE: The rise of the Enterococcus: beyond vancomycin resistance. Nat Rev Microbiol, 10, 266-278, 2012. DOI: 10.1038/nrmicro2761
Dogan M, Ozpınar H: Investigation of probiotic features of bacteria isolated from some food products. Kafkas Univ Vet Fak Derg, 23 (4): 555- 562, 2017. DOI: 10.9775/kvfd.2016.17273
Joshi VK, Sharma S, Rana NS: Production, purification, stability and efficacy of bacteriocin from isolates of natural lactic acid fermentation of vegetables. Food Technol Biotechnol, 44, 435-439, 2006.
Kim DH, Chon JW, Kang IB, Kim H, Kim HS, Song KY, Seo KH: Growth inhibition of Cronobacter sakazakii in experimentally contaminated powdered infant formula by kefir supernatant. J Food Prot, 78, 1651-1655, 2015. DOI: 10.4315/0362-028X.JFP-15-119
Kim DH, Jeong D, Kim H, Kang IB, Chon JW, Song KY, Seo KH: Antimicrobial activity of kefir against various food pathogens and spoilage bacteria. Korean J Food Sci Anim Resour, 36 (6): 787-790, 2016. DOI: 10.5851/kosfa.2016.36.6.787
Medrano M, Fernando Perez P, Abraham AG: Kefiran antagonizes cytopathic effects of Bacillus cereus extracellular factors. Int J Food Microbiol, 122, 1-7, 2008. DOI: 10.1016/j.ijfoodmicro.2007.11.046
Sarkar S: Biotechnological innovations in kefir production: a review. Brit Food J, 110 (3): 283-295, 2008. DOI: 10.1108/00070700810858691
Chifiriuc MC, Cioaca AB, Lazar V: In vitro assay of the antimicrobial activity of kephir against bacterial and fungal strains. Anaerobe, 17, 433- 435, 2011. DOI: 10.1016/j.anaerobe.2011.04.020
Magalhães KT, de Melo Pereira GV, Campos CR, Dragone G, Schwan RF: Brazilian kefir: Structure,microbial communities and chemical composition. Braz J Microbiol, 42, 693-702, 2011. DOI: 10.1590/S1517- 83822011000200034
Klijn N, Weerkamp AH, de Vos WM: Detection and characterization of lactose-utilizing Lactococcus spp. in natural ecosystems. Appl Environ Microbiol, 61 (2): 788-792, 1995.
Dimitrellou D, Kandylis P, Kourkoutas Y, Koutinas AA, Kanellaki M: Cheese production using kefir culture entrapped in milk proteins . Appl Biochem Biotechnol, 176 (1): 213-230, 2015. DOI: 10.1007/s12010- 015-1568-4
Cheirsilp B, Shimizu H, Shioya S: Enhanced kefiran production by mixed culture of Lactobacillus kefiranofaciens and Saccharomyces cerevisiae. J Biotechnol, 100, 43-53, 2003. DOI: 10.1016/S0168-1656 (02)00228-6
Zhou J, Liu X, Jiang H, Dong M: Analysis of the microflora in Tibetan kefir grains using denaturing gradient gel electrophoresis. Food Microbiol, 26, 770-775, 2009. DOI: 10.1016/j.fm.2009.04.009
Zycka-Krzesinska J, Boguslawska J, Aleksandrzak-Piekarczyk T, Jopek J, Bardowski JK: Identification and characterization of tetracycline resistance in Lactococcus lactis isolated from Polish raw milk and fermented artisanal products. Int J Food Microbiol, 211, 134-141, 2015. DOI: 10.1016/j.ijfoodmicro.2015.07.009
Klare I, Konstabel C, Werner G, Huys G, Vankerckhoven V, Kahlmeter G, Hildebrandt B, Muller-Bertling S, Witte W, Goossens H: Antimicrobial susceptibilities of Lactobacillus, Pediococcus and Lactococcus human isolates and cultures intended for probiotic or nutritional use. J Antimicrob Chemother, 59, 900-912, 2007. DOI: 10.1093/ jac/dkm035
Clinical and Laboratory Standards Institute: Performance standards for antimicrobial disk susceptibility test; approved standard. CLSI document M02-A11, 32 (1): 2012.
Atanasova J, Moncheva P, Ivanova I: Proteolytic and antimicrobial activity of lactic acid bacteria grown in goat milk. Biotechnol Biotechnol Equip, 28 (6): 1073-1078, 2014. DOI: 10.1080/13102818.2014.971487
Facklam R, Elliott JA: Identification, classification, and clinical relevance of catalase-negative,gram-positive cocci, excluding the streptococci and enterococci. Clin Microbiol Rev, 8 (4): 479-495, 1995.
Buyukyoruk S, Cibik R, Cetinkaya F, Soyutemiz GE, Goksoy EO, Kirkan S: Isolation,phenotypic and molecular identification of Lactococcus lactis isolation from traditionally produced village cheeses. Asian J Anim Vet Adv, 9 (16): 2154-2158, 2010. DOI: 10.3923/javaa.2010.2154.2158
Khemariya P, Singh S, Nath G, Gulati AK: Isolation,identification,and antibiotic susceptibility of nis+ Lactococcus lactis from dairy and nondairy sources. Czech J Food Sci, 31, 323-331, 2013.
Guessas B, Kihal M: Characterization of lactic acid bacteria isolated from Algerian arid zone raw goats’ milk. Afr J Biotechnol, 3 (6): 339-342, 2004. DOI: 10.5897/AJB2004.000-2062
Rattanachaikunsopon P, Phumkhachorn P: Lactic acid bacteria: their antimicrobial compounds and their uses in food production. Ann Biol Res, 1, 218-228, 2010.
Rodríguez-Figueroa JC, González-Córdova AF, Astiazaran-García H, Vallejo-Cordoba B: Hypotensive and heart rate-lowering effects in rats receiving milk fermented by specific Lactococcus lactis strains. Br J Nutr, 109, 827-833, 2013. DOI: 10.1017/S0007114512002115
Kim SY, Kim JE, Lee KW, Lee HJ: Lactococcus lactis ssp. lactis inhibits the proliferation of SNU-1 human stomach cancer cells through induction of G0/G1 cell cycle arrest and apoptosis via p53 and p21 expression. Ann N Y Acad Sci, 1171, 270-275, 2009. DOI: 10.1111/j.1749-6632.2009.04721.x
12. Hladíková Z, Smetanková J, Greif G, Greifová M: Antimicrobial activity of selected lactic acid cocci and production of organic acids. Acta Chim Slov, 5 (1): 80-85, 2012. DOI: 10.2478/v10188-012-0013-3
Aziz T, Khan H, Bakhtair SM, Naurin M: Incidence and relative abundance of lactic acid bacteria in raw milk of buffalo, cow and sheep. J Anim Plant Sci, 19 (4): 168-173, 2009.
Wassie M, Wassie T: Isolation and identification of lactic acid bacteria from raw cow milk. Int J Adv Res Biol Sci, 3 (8): 44-49, 2016. 11.
Dimitrellou D, Kandylis P, Mallouchos A, Komaitis M, Koutinas AA, Kourkoutas Y: Effect of freeze-dried kefir culture on proteolysis in fetatype and whey-cheeses. Food Chem, 119, 795-800, 2010. DOI: 10.1016/j. foodchem.2009.06.052
Dallas DC, Citerne F, Tian T, Silva VLM, Kalanetra KM, Frese SA, Robinson RC, Mills DA, Barile D: Peptidomic analysis reveals proteolytic activity of kefir microorganisms on bovine milk proteins. Food Chem, 197, 273-284, 2016. DOI: 10.1016/j.foodchem.2015.10.116
Rodrigues KL, Caputo LRG, Carvalho JCT, Evangelista J, Schneedorf JM: Antimicrobial and healing activity of kefir and kefiran extract. Int J Antimicrob Agents, 25 (5): 404-408, 2005. DOI: 10.1016/j.ijantimicag. 2004.09.020
Ghoneum M, Gimzewski J: Apoptotic effect of a novel kefir product, PFT, on multidrug-resistant myeloid leukemia cells via a hole-piercing mechanism. Int J Oncol, 44, 830-837, 2014. DOI: 10.3892/ijo.2014.2258
Guzel-Seydim ZB, Kok-Tas T, Greene AK, Seydim AC: Review: Functional properties of kefir. Crit Rev Food Sci Nutr, 51, 261-268, 2011. DOI: 10.1080/10408390903579029
Nielsen B, Gürakan GC, Ünlü G: Kefir: A multifaceted fermented dairy product. Probiotics Antimicrob Proteins, 6, 123-135, 2014. DOI: 10.1007/ s12602-014-9168-0
Otles S, Cagindi O: Kefir: A probitic dairy composition,nutritional and therapeutic aspect. PJN, 2, 54-59, 2003. DOI: 10.3923/pjn.2003.54.59
Marsh AJ, O’Sullivan O, Hill C, Ross RP, Cotter PD: Sequencingbased analysis of the bacterial and fungal composition of kefir grains and milks from multiple sources. PLoS One, 8 (7): e69371, 2013. DOI: 10.1371/ journal.pone.0069371
Lopitz-Otsoa F, Rementeria A, Elguzabal N, Garaizar J: Kefir: A symbiotic yeast bacteria community with alleged healthy capabilities. Rev Iberoam Micol, 23 (2): 67-74, 2000.