بررسی تنوع ژنتیکی لاکتوباسیلوس‌های جدا شده از نمونه‌های شیر گاو به روش RAPD-PCR
محورهای موضوعی : فصلنامه کیفیت و ماندگاری تولیدات کشاورزی و مواد غذایی
حکیمه قلعه خانی
1
(کارشناسی ارشد، گروه میکروبیولوژی، واحد کرمان، دانشگاه آزاد اسلامی، کرمان، ایران)
معصومه حاجی رضایی
2
(استادیار، گروه میکروبیولوژی، واحد کرمان، دانشگاه آزاد اسلامی، کرمان، ایران)
آرتادخت توکلی
3
(استادیار، گروه میکروبیولوژی، واحد کرمان، دانشگاه آزاد اسلامی، کرمان، ایران)
کلید واژه: تنوع ژنتیکی, genetic diversity, لاکتوباسیلوس, Lactobacilli, RAPD-PCR, درخت فیلوژنی, Phylogeny Tree,
چکیده مقاله :
محصولات لبنی مناطق مختلف می توانند منبعی برای سویه های جدید لاکتوباسیلوس باشند، بنابراین شناسایی مولکولی و بررسی تنوع ژنتیکی آنها می تواند گام موثری در شناسایی ذخیره لاکتوباسیلوس های بومی با خصوصیات عملکردی ویژه و به کارگیری آنها در محصولات لبنی صنعتی شود. هدف از این تحقیق جداسازی لاکتوباسیلوس ها از فلور موجود در شیر گاو شهرستان نرماشیر و بررسی تنوع ژنتیکی آنها می باشد. بدین منظور تعداد 21 نمونه شیر از مناطق مختلف این شهرستان جمع آوری و با روش های مرسوم فنوتیپی و بیوشیمیایی لاکتوباسیلوس ها جداسازی شدند. به منظور غربالگری و بررسی تنوع ژنتیکی لاکتوباسیلوس های جدا شده، پنج پرایمر RAPD به طور تصادفی انتخاب گردید و پس از PCR، تجزیه و تحلیل تنوع ژنتیکی و فیلوژنتیکی با استفاده از نرم افزار Pyelph انجام و درخت فیلوژنی رسم شد. تجزیه خوشه ای داده های مولکولی توانست جدایه ها را در فاصله ژنتیکی 10 به هفت گروه مجزا تقسیم بندی کند. جدایه های K3 و K4 در گروه یک و جدایه های K21 و K13 و K11 در گروه چهار قرار گرفتند. بقیه جدایه ها در گروه های مجزا قرار گرفتند. از آنجائیکه جدایه های K3 و K4 از یک ناحیه و جدایه های K21 و K13 نیز از یک ناحیه جمع آوری شده اند، قرار گرفتن آنها در یک گروه منطقی به نظر می رسد. نتایج بدست آمده از این تحقیق بیانگر تنوع ژنتیکی بالای جدایه های بومی شهرستان نرماشیر و مناسب بودن تکنیک RAPD-PCR برای مطالعات مشابه می باشد.
The use of lactobacilli in the production of fermented foods has several thousand years old due to their ability to make the desired changes in the taste and texture of the food, as well as their importance in the human health. Different dairy products can be a source of new strains of Lactobacillus. Therefore, molecular identification and their genetic variation can be an effective step in identifying native lactobacilli reserves with specific functional characteristics and applying them to industrial dairy products. The purpose of this study is to isolate lactobacilli from flora in cow's milk of Narmeshir city and study their genetic diversity. For this purpose, 21 milk samples were collected from different regions of the city, and Lactobacilli wereisolated by the conventional phenotypic and biochemical methods. In order to screen and investigate the genetic diversity of isolated lactobacilli, five RAPD primers were randomly selected. After doing PCR, the analyses of genetic and phylogenetic diversity were performed using Pyelph software and phylogeny trees were drawn. Cluster analysis of molecular data was able to divide the isolates into seven distinct groups at a genetic distance of 10. K3 and K4 isolates were in group 1 and isolates K21, K13, and K11were in group 4. The remaining isolates were placed in separate groups. Since K3 and K4 isolates were from a region and K21 and K13 isolates were also collected from a region, their placement seems to be logical in a group.
1-Guandalini S, Pensabene S, Abu M, Gobio L. Lactobacillus GG Administered in Oral Rehydration Solution to Children with Acute Diarrhea: A Multicenter Eur-opean Trial. Journal of Pediatric Gastroe-nterology and Nutrition. 2000;30(1):54-60. 2-Bazarcheh Shabestari N, Hajirezaei M. Molecular identification of lactobacilli isolated from traditional cheeses of Ban-dar Abbas city. New Cellular and Molec-ular Biotechnology Journal. 2020;10(39): 79-90. [In persian] 3-Moreno MF, Sarantinopoulos P, Tsakali-dou E, De Vuyst L. The role and applica-tion of enterococci in food and health. International journal of food microbe-ology. 2006;106(1):1-24. 4-Klewicki R, Klewicka E. Antagonistic activity of lactic acid bacteria as prob-iotics against selected bacteria of the Enterobaceriacae family in the presence of polyols and their galactosyl deriva-tives. Biotechnology letters. 2004;26(4): 317-320. 5-Reid A, Rawls JF, Pierce NE. Animals in a bacterial world, a new imperative for the life sciences. Proceedings of the Nati-onal Academy of Sciences. 2013;110(9): 3229-3236. 6-Sanders ME, Klaenhammer TR. Invited Review: The scientific basis of Lactobac-illus acidophilus NCFM functionality as a probiotic. Journal of dairy science. 2001; 84(2):319-33. 7-Torres-Llanez MJ, Vallejo-Cordoba B, Diaz-cinco ME, Mazorra-Manzano MA, Gonzalez-Cordova AF. Characterization of the natural microflora of artisanal Mexican Fresco cheese. Food Control. 2006; 17:683-690. 8-Soleymani Fard F, Ghobadi Dana M, Piravi Vanak Z. Screening local Lactob-acilli from Iran in terms of production of lactic acid and identification of superior strains. Biological Journal of Microorg-anisms. 2015;15:155-166. [In Persian] 9-Roy D, Ward P, Vincent D, Mondou F. Molecular identification of potentially probiotic lactobacilli. Current microbio-logy. 2000;40(1):40-46. 10-Visintin S, Alessandria V, Valente A, Dolci P, Cocolin L. Molecular identifi-cation and physiological characterization of yeasts, lactic acid bacteria and acetic acid bacteria isolated from heap and box cocoa bean fermentations in West Africa. International journal of food microbio-logy. 2016;216:69-78. 11-Anderson AC, Sanunu M, Schneider C, Clad A, Karygianni L, Hellwig E, Al-Ahmad A. Rapid species-level identifi-cation of vaginal and oral lactobacilli using MALDI-TOF MS analysis and 16S rDNA sequencing. BMC microbiology. 2014;14(1):312. 12-Soto A, Martín V, Jiménez E, Mader I, Rodríguez JM, Fernández L. Lactobacilli and bifidobacteria in human breast milk: influence of antibiotherapy and other host and clinical factors. Journal of pediatric gastroenterology and nutrition. 2014; 59(1):78. 13-Tafvizi F, Tajabadi ebrahimi M, Hey-dari Nasrabadi M, Bahrami H. Detection of genetic diversity of lactobacillus spec-ies isolated from different traditional dairy products by RAPD markers. New Cellular and Molecular Biotechnology Journal. 2011;1(3):33-42. [In Persian] 14-Hedaiati Marzoni H, Samiezadeh lahiji-Genetic HA. Diversity Assessment of Lines and Varieties in Winter Rapeseed (Brassica napus L.) using RAPD and SSR Molecular Markers. Journal of Crop Bre-eding. 2016;8(17):139-131. [In Persian] 15-Safari S, mehrabi AA. Genetic Relatio-nships of Rapeseed Cultivars Revealed by RAPD Markers. Journal of Crop Bree-ding. 2017;8(19):177-170. [In Persian] 16-Asadi F, Naderi Shahab MA, Mirzaii nadoshen H. Genetic diversity of poplar clones using random amplification poly-morphism DNA (RAPD PCR). Pajouhe-sh-Va-Sazandegi. 2001;14(1):36-44. [In Persian] 17-Dashti H, Naghavi MR, Shah Nejat Boshehri AL, Shirani H. Evaluation of genetic diversity of wheat germplasm using markers RAPD. Modern genetic journal. 2009;4(3):55-62. [In Persian] 18-Behnam B, Bahman Yazdi S, Abd mM-ishani S, Talei AR, Shah Nejat Boshehri AL. Apllication of the Random Amplified Polymorphic (RAPD) technique as a DNA marker for detection of polymer-phism among Italian durum wheat cultiv-ars. Oolom zeraei Iran. 2000;2(2):29-34. [In Persian] 19-Hajirezaei M, Baghizade A, Javadi GH, Sadeghizadeh M. Genetic diversity asse-ssment of a few numbers of pistachio cultivars in Kerman province based on RAPD markers. Iranian journal of boil-ogy. 2008;22(3):462-469. [In Persian] 20-Zamani ZA, Sarkhosh A, Fatahi Mogh-adam MR, Ebadi A. Evaluation of genetic diversity among a number of pomegr-anate genotypes using RAPD markers. Iranian Journal of Agricultural Sciences. 2006;37(5):865-873. [In Persian] 21-Sheini Mehrabzadeh R, Khosravi A, Hashemi A, Jamali H. Investigation of genetic diversity of Mycobacterium fortu-itum clinical isolated by RAPD-PCR. Jo-urnal of Microbial World. 2014;6(4):281-289. [In Persian] 22-Siavoshi F, Shokouhfard M, Malekza-deh R, Dinparast Jadid N, Massarrat S, Emrani A. Genotype Variation in H. Pyl-ori Isolates from Iranian Patients by RAPD-PCR. Govaresh. 2004;9(1):7-11. [In Persian] 23-Baghbani-arani F, Tajbakhsh M, Hashe-mi SA, Rajai B, Siadat SD, Aghasadeghi MM, Sadat SM. Molecular Typing of Salmonella Paratyphi B and Salmonella paratyphi C Isolatedfrom Clinical Samp-les in Iran .Journal of Fasa University of Medical Sciences. 2012;1:19-25. [In Pers-ian] 24-Asadi H, Yari R, Zargar M. Genetic Variation of Escherichia coli Bacteria Isolatedfrom Urinary Tract Infections Using RAPD-PCR. Journal of Mazanda-ran University of Medical Sciences. 2016; 26(134):114-123. [In Persian] 25-González-Rey C, Belin AM, Jörbeck H, Norman M, Krovacek K, Henriques B, Källenius G, Svenson SB. RAPD-PCR and PFGE as tools in the investigation of an outbreak of beta-haemolytic Strepto-coccus group A in a Swedish hospital. Comparative immunology, microbiology and infectious diseases. 2003;26(1):25-35. 26-Mokhtazi Zenozi P, Hejazi MA, Lotfi H, Eslami. 2011. Using RAPD-PCR tech-nique to study the genetic diversity of lactobacilli with probiotic potential. 20th International Congress on Food Techno-logy, 25 July. Tehran. Iran. 86. [In Pers-ian] 27-Kafili T, Razavi S, Imam Juma Z, Sale-hi Jozani Gh, Naqavi M. Comparison of Molecular Methods of RAPD-PCR and DGGE-PCR in Identifying Isolated Lact-obacillus during Liquvan Cheese Ripe, Iranian Journal of Biosystem Enginee-ring. 2009;(40)1:87-91. [In Persian] 28-Sanders M. Considerations for Use of Probiotic Bacteria to Modulate Human Health. The Journal of Nutrition. 2000; 130(2):384-390. 29-Pisano MB, Patrignani F, Cosentino S, Guerzoni ME, Franz CM, Holzapfel WH. Diversity and functional properties of Lactobacillus plantarum-group strains is-olated from Italian cheese products. Dairy Science & Technology. 2011;91(1):65-76. 30-Hejazi MA, Lotfi H, Zanjani B, Maleki Barzegari A. Isolation, biochemical and molecular identification of bacteria with probiotic potential from traditional dairy products in Harris and Sarab regions. Journal of Food Research. 2010;1(3):1-19. [In Persian] 31-Hasani M, Farajniya S, Hesari J, Mosavi. 2008. Isolation and identification useful practical properties two species Lactobacillus from traditional Lighvan cheese. 18 the International congress food science and technology. 15 October. Mashhad. Iran. 108. [In Persian] 32-Ahamdi SM, Khamiri M, Khosroshahi A, Kashani Negad M. Isolation and ident-ification of Lacic acid bacteria from Ligh-van cheeses. Journal of Agricultural Scie-nces and Natural Resources. 2008;3(16): 80-91. 33-Pourahmad R, Assadi MM. Use of iso-lated autochthonous starter cultures in yogurt production. International journal of dairy technology. 2007;60:259-262. 34-Ehsani A, Mahmoodi R, Hashemi M, Raeisi M. Isolation and identification of lactobacillus in traditional cheeses of West Azarbaijan province. Iranian Jour-nal of Medical Microbiology. 2014;9:38-43. [In Persian] 35-Swearingen PA, O'sullivan DJ, Warth-esen JJ. Isolation, characterization, and influence of native, nonstarter lactic acid bacteria on Cheddar cheese quality. Journal of Dairy Science. 2001;84(1):50-9. 36-Ghobadi Dana M, Hatef Soleimanian A, Yakhchali B. Isolation and molecular id-entification of lactobacilli in some native dairy products. Journal of Research and Innovation in Food Science and Techno-logy. 2012;2:99-116. [In Persian] 37-Rivas B, Marcobal A, Munoz R. Devel-opment of a multilocus sequence typing method for analysis of Lactobacillus pla-ntarum strains. Microbiology. 2006;152: 85-93. 38-Woon PY, Tateno M. Osoegawa KBact-erial artificial chromosome libraries for mouse sequencing and functional anal-ysis. Genome Research. 2000;52(120): 126-139. 39-Sánchez I, Sesena S, Poveda JM, Cab-ezas L, Palop L. Genetic diversity, dyna-mics, and activity of Lactobacillus com-munity involved in traditional processing of artisanal Manchego cheese. Internati-onal journal of food microbiology. 2006; 107(3):265-273. 40-Abriouel H, Martin A, Maqueda M, Valdivia E. Biodiversity of the microbial community in a Spanish farmhouse chee-se as revealed by culture-dependent and culture-independent methods. Internation-al Journal of Food Microbiology. 2008; 127(3):200-208.
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