جداسازی، شناسایی مولکولی و ارزیابی ایمنی باکتری‌های اسید لاکتیک پروتئولیتیک به دست آمده از نمونه‌های مختلف شیر خام

نویسندگان
علی مویدی 1
ماندانا محمودی 2
مرتضی خمیری 2
شهرام لقمان 2
1 عضو هیات علمی دانشگاه علوم کشاورزی و منابع طبیعی گرگان
2 دانشگاه علوم کشاورزی و منابع طبیعی گرگان
چکیده
برخی از باکتری­های اسید لاکتیک موجود در شیر خام به­ویژه انواع پروتئولیتیک، خصوصیات تکنولوژیک مفیدی دارند و نقش مهمی در توسعه ویژگی­های محصول نهایی بازی می­کنند، اما غالبا در طی فرآوری از بین می­روند. از این رو، جداسازی و نگهداری آنها به منظور کاربردهای بعدی ارزشمند خواهد بود. در این پژوهش، پس از جداسازی باکتری­های اسید لاکتیک موجود در شیر خام گاو، گوسفند و بز بر روی محیط­های کشت MRS agar و M17 agar، جدایه­های گرم مثبت و کاتالاز منفی انتخاب شده و از نظر فعالیت پروتئولیتیکی روی محیط Skim milk agar غربال شدند. جدایه­های دارای فعالیت پروتئولیتیکی مناسب با استفاده از توالی­یابی قطعه ژنی 16S rDNA شناسایی شدند و فعالیت همولیزی و مقاومت آنتی­بیوتیکی آنها در برابر 10 آنتی بیوتیک رایج مورد بررسی قرار گرفت. آنالیز توالی­یابی با برنامه BLAST منجر به شناسایی Lactobacillus delbruekii (5 جدایه)، L. delbruekii زیرگونه bulgaricus، L. fermentum، L. reuteri، L. curvatus، Lactococcus lactis زیر­گونه lactis، و Streptococcus lutetiensis شد. از میان جدایه­های شناسایی شده، L. lactis زیر­گونه lactis پس از 48 ساعت گرمخانه­گذاری در 37 درجه سانتی­گراد با ایجاد قطر هاله 23 میلی­متر بیشترین فعالیت پروتئولیزی را نشان داد. در مورد فعالیت همولیزی، فقط Lactobacillus fermentum فعالیت آلفا-همولیزی نشان داد و سایر باکتری­ها غیر­همولیتیک بودند. تمام جدایه­های شناسایی­شده به تتراسایکلین، آمپی­سیلین، اریترومایسن، ونکومایسین و جنتامایسین حساس بودند به­جز L. reuteri و Lactococcus lactis زیرگونه lactis که به ترتیب به ونکومایسین و جنتامایسن مقاوم بودند. در مورد مقاومت به پنی­سیلین، کلرآمفنیکول، کلیندامایسین، کانامایسین و استرپتومایسین نتایج بسته به نژاد مورد بررسی متفاوت بود. نتایج این پروژه، نشان­دهنده تنوع نژادهای باکتری­های اسید لاکتیک پروتئولیتیک در شیر خام است. به علاوه، اگرچه باکتری­های اسید لاکتیک ایمن در نظر گرفته می­شوند و در لیست GRAS قرار دارند، اما نژادهای وحشی جداسازی­شده از شیر خام باید از نظر جنبه­های مرتبط با ایمنی مورد بررسی قرار گیرند.
کلیدواژه‌ها
شیر خام
ایمنی
باکتری های اسید لاکتیک
فعالیت پروتئولیتیکی

موضوعات

عنوان مقاله English

Isolation, molecular identification and safety assessment of proteolytic lactic acid bacteria obtained from different raw milks

نویسندگان English

Ali Moayedi 1
Mandana Mahmoudi 2
Morteza Khomeiri 2
Shahram Loghman 2
1 Gorgan University of Agricultural Sciences and Natural Resources
2 Ph.D. Graduate, Gorgan University of Agricultural Sciences and Natural Resources
چکیده English

Some lactic acid bacteria (LAB), in particular proteolytic strains, in raw milk have useful technological properties and play important role in developing desired characteristics in the final products. However, they are mostly lost during heating and technological processes. Therefore, it would be important to be isolated and maintained for further applications as pure adjunct cultures. In this study, after selective isolation of LAB from raw cow’s, ewe’s and goat’s milks on MRS agar and M17 agar, Gram-positive catalase-negative were selected and maintained as pure cultures. Then, the isolates were screened according to their proteolytic activities on skim milk agar (well-diffusion method). The potent Gram-positive, catalase-negative proteolytic strains were identified via 16S rDNA gene sequencing and antibiotic resistance (10 antibiotic agents, disc-diffusion method) and hemolytic activities (on blood agar containing 5% sheep blood) were evaluated. BLAST analysis resulted in identification of Lactobacillus delbruekii (five isolates), L. delbruekii subsp. bulgaricus, L. fermentum, L. reuteri, L. curvatus, Lactococcus lactis subsp. Lactis, and Streptococcus lutetiensis. L. lactis subsp. Lactis obtained from cow milk showed the highest proteolytic activity as a 23-mm halo zone was observed on skim milk agar plate after 48 h incubation at 37 °C. In case of hemolysin production, only Lactobacillus fermentum showed α-hemolytic activity and the others showed no activities. All the identified isolates were sensitive to tetracycline, ampicillin, erythromycin, vancomycin and gentamycin with the exception of L. reuteri and lactococcus lactis subsp. lactis that found to be resistant to vancomycin and gentamycin, respectively. About penicillin, chloramphenicol, clindamycin, kanamycin and gent streptomycin, the results were varied. Our results verified diversity of wild proteolytic LAB strains in raw milk, as reported before. Although LAB mainly belong to the GRAS list, strains isolated from raw milk should be re-checked for their safety-related properties.

کلیدواژه‌ها English

Raw milk
Proteolysis
lactic acid bacteria
Safety
[1] Tulini, F.L., Hymery, N., Haertlé, T., Le Blay, G., and De Martinis, E.C.P. 2016. Screening for antimicrobial and proteolytic activities of lactic acid bacteria isolated from cow, buffalo and goat milk and cheeses marketed in the southeast region of Brazil. Journal of Dairy Research, 83, 115-124.
[2] Shahrampour, D., Khomeiri, M., Kashiri, M., Razavi, S. A. 2019. Evaluation of Antibacterial and Antifungal activity of Indigenous Lactobacillus plantarum isolated from various foods. Journal of Food Science and Technology, 85 (15): 327-336. (In Persian)
[3] Zoumpopoulou, G., Tzouvanou, A., Mavrogonatou, E., Alexandraki, V., Georgalaki, M., Anastasiou, R., Papadelli, M., Manolopoulou, E., Kazou, M., & Kletsas, D., Papadimitriou, K., and Tsakalidou, E. 2017. Probiotic Features of Lactic Acid Bacteria Isolated froma Diverse Pool of Traditional Greek Dairy Products Regarding Specific Strain-Host Interactions. probiotics and antimicrobial proteins. 10 (2): 313–322.
[4] Abushelaibi, A., Al-Mahadin, S., El-Tarabily, K., Shah, N.P. and Ayyash, M. 2017. Characterization of potential probiotic lactic acid bacteria isolated from camel milk. LWT - Food Science and Technology, 79: 316-325
[5] Aspri, M., Bozoudi, D., Tsaltas, D., Hill., C. 2017. Raw donkey milk as a source of Enterococcus diversity: Assessment of their technological properties and safety characteristics. Food Control, 73: 81-90.
[6] Badis, A., Guetarni, D., Boudjema, M., Henni, D. E., Kihal, M. 2004. Identification and technological properties of lactic acid bacteria isolated from raw goat milk of four Algerian races. Food Microbiology. 21 (5): 579-588.
[7] Savijoki, K., Ingmer, H., and Varmanen, P. 2006. Proteolytic systems of lactic acid bacteria. Applied Microbiology and Biotechnology. 71: 394-406.
[8] Georgala, A.K., Tsakalidou, E., Kandarak, I., Kalantzopoulos, G. 1997. An Index of Proteolysis Degree in Ewes' Milk and Ewes' Milk Yoghurt, by single Strains and Combinations of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, Isolated from Traditional Greek Yoghurt. Food Science and technology international. 3 (3): 259-263.
[9] Soleymanzadeh, N., Mirdamadi, S., and Kianirad, M. 2016. Antioxidant activity of camel and bovine milk fermented by lactic acid bacteria isolated from traditional fermented camel milk (Chal). Dairy Science & Technology. 96: 443-457.
[10] Franciosi, E., Settanni, L., Cavazza, A., and Poznanski, E. 2009. Biodiversity and technological potential of wild lactic acid bacteria from raw cows’ milk. International Dairy Journal. 19: 3–11.
[11] Delavenne, E., Mounier, J., Déniel, F., Barbier, G., and Le Blay, G. 2012. Biodiversity of antifungal lactic acid bacteria isolated from raw milk samples from cow, ewe and goat over one-year period. International Journal of Food Microbiology. 155: 185–190.
[12] Medina, R., Katz, M., Gonzalez, S, Oliver, G. 2001. Characterization of the Lactic Acid Bacteria in Ewe’s Milk and Cheese from Northwest Argentina. Journal of Food Protection, 64 (4): 559–563
[13] Wouters, J., Ayad E., Hugenholtz, J., Smit, G. 2002. Microbes from raw milk for fermented dairy products. International Dairy Journal. 12 (2-3): 91-109.
[14] Forghani, F., Nazemi, A., Sharifi, S., Eskandari., M. 2010. Isolation and Molecular identification of Lactic Acid Bacteria of Raw Milks from Central Alborz. Journal of Microbial Biotechnology, 5 (2): 21-28. (In Persian)
[15] Leite, A. M. O., Miguel, M. A. L., Peixoto, R. S., Ruas-Madiedo, P., Paschoalin, V. M. F., Mayo, B., and Delgado, S. 2015. Probiotic potential of selected lactic acid bacteria strains isolated from Brazilian kefir grains. Journal of Dairy Science. 98:3622–3632.
[16] CLSI. 2013. “Disc diffusion supplemental tablesʼʼ Performance standards for antimicrobial susceptibility testing. Adapted in part from CLSI document M100-S23 (M02-A11); Wayne, PA 19807: Clinical and Laboratory Standards Institute.
[17] Vuillemard, J. C., Amiot, J, and Gauthier, S. 1986. Evaluation de l’activite proteolytique de bacteries lactiques par une methode de diffusion sur plaque. Microbiology-Aliments-Nutrition 3 327–332.
[18] Lawalat, H. J., Satiman, U. 2015. Identification of Lactic Acid Bacteria Proteolytic Isolated from An Indonesian Traditional Fermented Fish Sauce Bakasang by Amplified Ribosomal DNA Restriction Analysis (ARDRA). International Journal of ChemTech Research, 8 (12): 630-636.
[19] Hafeez, Z., Cakir-Kiefer, C., Roux, E., Perrin, C., Miclo, L., Dary-Mourot, A. 2014. Strategies of producing bioactive peptides from milk proteins to functionalize fermented milk products. Food Research International. 63, 71-80.
[20] Nielsen, M. S., Martinussen, T., Flambard, B., Sørensen, K. I., Otte, J. 2009. Peptide profiles and angiotensin-I-converting enzyme inhibitory activity of fermented milk products: effect of bacterial strain, fermentation pH, and storage time. International Dairy Journal, 19(3), 155-165.
[21] Barrangou, R., Lahtinen, S. J., Ibrahim, F., Ouwehand, A. C. Genus Lactobacillus. In: Lahtinen et al. (Editors). Lactic acid bacteria; Microbiological and functional aspects. 4nd edition. CRC press. 2012. P, 81.
[22] Tan, P. S. T., Poolman, B., Konings, W. N. 1993. Proteolytic enzymes of Lactococcus lactis. Journal of Dairy Research. 60: 269-286.
[23] Hidalgo-Morales, M., Robles-Olvera, V., García, H. S. 2005. Lactobacillus reuteri β-galactosidase activity and low milk acidification ability. Canadian journal of microbiology, 51(3), 261-267.
[24] FAO/WHO, 2002. Joint FAO/WHO Working Group Report on Drafting Guidelines for the Evaluation of Probiotics in Food London, Ontario, Canada, April 30 and May 1, 2002.
[25] Tejero-Sariñena, S., Barlowb, J., Costabile, A., Gibson, G.R., and Rowland, I. 2012. In vitro evaluation of the antimicrobial activity of a range of probiotics against pathogens: Evidence for the effects of organic acids. Anaerobe. 18: 530-538.
[26] Owusu-Kwarteng, J., Tano-Debrah, K., Akabanda, F., and Jespersen, L. 2015. Technological properties and probiotic potential of Lactobacillus fermentum strains isolated from West African fermented millet dough. BMC Microbiology. 15: 261-270. .
[27] Mahmoudi, I., Ben Moussaa, O., Moulouk Khaldi, T. E., Kebouchib, M., Soligot, C., 341 Le Roux, Y. and Hassouna, M. 2016. Functional in vitro screening of Lactobacillus 342 strains isolated from Tunisian camel raw milk toward their selection as probiotic. Small 343 Ruminant Research, 137: 91-98.
[28] Maragkoudakisa, P. A., Zoumpopouloua, G., Miarisa, C., Kalantzopoulosa, G., Potb, 345 B. and Tsakalidoua, E. 2006. Probiotic potential of Lactobacillus strains isolated from 346 dairy products. International Dairy Journal, 16: 189-199.
[29] Zhoua, J.S., Pillidgec, C.J., Gopalc, P.K., and Gill, H.S. 2005. Antibiotic susceptibility profiles of new probiotic Lactobacillus and Bifidobacterium strains. International Journal of Food Microbiology, 98: 211 – 217.
[30] Ammor, M.S., Flórez, A. B., Van Hoek, A.H.A.M., Reyes-Gavilan, C.G.D.L., Aarts, H.J. M., Margolles, A., & Mayo, B. (2008). Molecular characterization of intrinsic and acquired antibiotic resistance in lactic acid bacteria and bifidobacteria. Journal of Molecular Microbiology and Biotechnology. 14: 6–15.
[31] Abriouel, H., Muñoz, M.C.C., Lerma, L.L., Montoro, B.P., Bockelmann, W., Pichner, R., Kabisch, J., Cho, G.S., Franz, C.M.A.P., Gálvez, A., and Benomar, N. 2015. New insights in antibiotic resistance of Lactobacillus species from fermented foods. Food Research International, 78: 465-481.
[32] Ammor, M.S., Flórez, A. B., Mayo, B. 2007. Antibiotic resistance in non-enterococcal lactic acid bacteria and bifidobacteria. Food Microbiology, 24: 559-570.
مجله علوم و صنایع غذایی ایران
دوره 16، شماره 89
تیر 1398
صفحه 59-69