YOĞURT KAYMAĞINDAN İZOLE EDİLEN YARROWIA LIPOLYTICA’NIN MOLEKÜLER İDENTİFİKASYONU VE LİPOLİTİK AKTİVİTESİ

Yıl 2022, Cilt: 46 Sayı: 2, 450 - 457, 29.05.2022

Merve Eylül Kıymacı , Duygu Şimşek , Nurten Altanlar

https://doi.org/10.33483/jfpau.1090889

Öz

Amaç: Yarrowia lipolytica enzimler, organik asitler, proteinler, yüzey aktif maddeler gibi çeşitli metabolitler üreten ve bazı biyotransformasyon ve biyoremediasyon reaksiyonlarında endüstriyel potansiyele sahip olan aerobik dimorfik bir mayadır. Endüstriyel üretimlere bir ön çalışma olması amacıyla, bu çalışmada Y. lipolytica suşunun yoğurt kremasından izole edilmesi, moleküler karakterizasyonunun yapılması ve lipolitik aktivitesinin araştırılması amaçlanmıştır.

Gereç ve Yöntem: Ankara'daki farklı yerel süt üreticilerinin sütü fermente ederek elde ettikleri ev yapımı yoğurt kaymaklarından toplam 10 örnek alınmıştır. Yarrowia türlerinin izolasyonunda Yeast-Pepton-Gliserol (YPG) sıvı besiyeri ve Yarrowia lipolytica Distinctive (YLD) agar besiyeri kullanılmıştır. Y. lipolytica'nın tür tanımlaması ITS-Polimerize zincir reaksiyonu analizi ile yapılmıştır. Lipolitik aktivite; Rodamin-B/Zeytin yağı agar besiyeri prosedürü ile belirlenmiştir.

Sonuç ve Tartışma: Ev yapımı yoğurt kaymağı örneklerinden YPG besiyerinde gelişen maya kolonileri izole edilmiştir. Maya izolatları Gram boyama yöntemi ile boyanmış ve Gram pozitif boyananlar YLD agar üzerine inoküle edilmiştir. İnkübasyon süresi sonunda YLD agar üzerinde gelişen kahverengi pigmentli koloniler Yarrowia türü olarak seçilmiştir. Moleküler karakterizasyon sonuçlarına göre kahverengi pigmentli bir koloni toplam 358 baz, %100 dizi uyum oranı ve %100 benzerlik oranı ile Yarrowia lipolytica olarak tanımlanmış ve suşun lipaz pozitif olduğu belirlenmiştir. Çalışmamızda izole edilen Y. lipolytica suşunun çeşitli özelliklerinden faydalanmak üzere, endüstriyel üretimlerde kullanılabilecek bir üretici kültür olarak geliştirilmesi, yapılacak ileriki çalışmalarla desteklenmelidir.

Anahtar Kelimeler

Endüstriyel maya, lipolitik aktivite, Yarrowia lipolytica

MOLECULAR IDENTIFICATION AND LIPOLYTIC ACTIVITY OF YARROWIA LIPOLYTICA ISOLATED FROM YOGHURT CREAM

Öz

Objective: Yarrowia lipolytica is an aerobic dimorphic yeast that produces various metabolites such as enzymes, organic acids, proteins, surfactants and has industrial potential in some biotransformation and bioremediation reactions. To be a preliminary study to industrial productions, in present study it was aimed to isolate Y. lipolytica strain from yoghurt cream, perform its molecular characterization and investigate the lipolytic activity.

Material and Method: A total of 10 samples were taken from the homemade yoghurt cream obtained by fermented milk from local dairy producers in Ankara. Yeast-Peptone-Glycerol (YPG) broth and Yarrowia lipolytica Distinctive (YLD) agar media were used for isolation of the species. Species-level identification was carried out by ITS-Polymerized chain reaction analysis. Lipolytic activity was determined with Rhodamine-B/Olive Oil Agar nutrient medium procedure.

Result and Discussion: Yeast colonies were isolated from homemade yoghurt creams by growing in YPG broth. Yeasts were stained with Gram staining method and Gram-positive stained ones were inoculated on YLD agar. At the end of the incubation period, the brown pigmented colony growing on YLD agar was selected as Yarrowia species. According to the molecular characterization results the brown pigmented colony was identified as Yarrowia lipolytica with a total of 358 bases, 100% sequence matching ratio and 100% similarity ratio and the strain was determined as lipase positive. The development of the Y. lipolytica strain isolated in our study as a producer culture that can be used in industrial production should be supported by further studies to benefit from its various properties.

Anahtar Kelimeler

Industrial yeast, lipolytic activity, Yarrowia lipolytica

Kaynakça

• 1. Barth, G., Gaillardin, C. (1997). Physiology and genetics of the dimorphic fungus Yarrowia lipolytica. FEMS Microbiolgy Reviews, 19(4), 219–237. [CrossRef]
• 2. Fickers, P., Benetti, P.H., Wache, Y., Marty, A., Mauersberger, S., Smit, M.S., Nicaud, J.M. (2005). Hydrophobic substrate utilisation by the yeast Yarrowia lipolytica, and its potential applications. FEMS Yeast Research, 5(6-7), 527–543. [CrossRef]
• 3. Barth, G., Gaillardin, C. (1996). Nonconventional Yeasts in Biotechnology, Springer-Verlag, Berlin, Heidelberg, New York, p. 313-388. [CrossRef]
• 4. Fickers, P., Benetti, P.H., Wache, Y., Marty, A., Mauersberger, S., Smit, M.S. (2005). Hydrophobic substrate utilisation by the yeast Yarrowia lipolytica, and its potential applications. FEMS Yeast Research, 5, 527-543. [CrossRef]
• 5. Aloulou, A., Rodriguez, J.A., Puccinelli, D., Mouz, N., Leclaire, J., Leblond, Y., Carrière, F. (2007). Purification and biochemical characterization of the LIP2 lipase from Yarrowia lipolytica. Biochimica et Biophysica Acta, 1771(2), 228–237. [CrossRef]
• 6. Gonçalves, F.A.G., Colen, G., Takahashi, J.A. (2014). Yarrowia lipolytica and its multiple applications in the biotechnological industry. The Scientific World Journal, 476207. [CrossRef]
• [7] Ratledge, C. (2010). Single cell oils for the 21st century. AOCS Press, Champaign, p. 1-20. [CrossRef]
• 8. Sarda, L., Desnuelle, P. (1958). Actions of pancreatic lipase on esters in emulsions. Biochim Biophys Acta, 30, 513-521. [CrossRef]
• 9. Treichel, H., Oliveira, D., Mazutti, M.A., Luccio, M.D., Oliveira, J.V. (2010). A Review on microbial lipases production. Food Bioprocess Technology, 3, 182-196. [CrossRef]
• 10. Darvishi, F., Nahvi, I., Zarkesh-Esfahani, H., Momenbeik F. (2009). Effect of plant oils upon lipase and citric acid production in Yarrowia lipolytica yeast. BioMed Research International, article ID 562943. [CrossRef]
• 11. Fickers, P., Nicaud, J.M., Gaillardin, C., Destain, J., Thonart, P. (2004). Carbon and nitrogen sources modulate lipase production in the yeast Yarrowia lipolytica. Journal of Applied Microbiology, 96(4), 742-749. [CrossRef]
• 12. Fickers, P., Marty, A., Nicaud, J.M. (2011). The lipases from Yarrowia lipolytica: genetics, production, regulation, biochemical characterization, and biotechnological applications. Biotechnology Advances, 29(6), 632-644. [CrossRef]
• 13. Carreira, A., Ferreira, L. M., Loureiro, V. (2001). Brown pigments produced by Yarrowia lipolytica result from extracellular accumulation of homogentisic acid. Applied and environmental microbiology, 67(8), 3463–3468. [CrossRef]
• 14. Kumar, D., Kumar, L., Nagar, S., Raina, C., Parshad, R., Gupta, V. K. (2012). Screening, isolation, and production of lipase/esterase producing Bacillus sp. strain DVL2 and its potential evaluation in esterification and resolution reactions. Archives of Applied Science Research, 4(4), 1763-1770. [CrossRef]
• 15. Vakhlu, J., Kour, A. (2006). Yeast lipases: enzyme purifi cation, biochemical properties and gene cloning. Electronic Journal of Biotechnology, 9(1), 69-85. [CrossRef]
• 16. Babu, I.S., Rao, G.H. (2007). Lipase production by Yarrowia lipolytica NCIM 3589 in solid state fermentation using mixed substrate. Research Journal of Microbiology, 2, 469-474. [CrossRef]
• 17. Jaeger, K.E., Eggert, T. (2002). Lipases for biotechnology. Current Opinion in Biotechnology, 13, 390-397. [CrossRef]
• 18. Louhasakul, Y., Cheirsilp, B. (2022). Potential use of industrial by-products as promising feedstock for microbial lipid and lipase production and direct transesterification of wet yeast into biodiesel by lipase and acid catalysts. Bioresource Technology, 348, 126742. [CrossRef]
• 18. Fraga, J.L., Souza, C.P.L., Pereira, A.D.S., Aguieiras, E.C.G., de Silva L.O., Torres, A.G., Freire, D.G., Amaral, P.F.F. (2021). Palm oil wastes as feedstock for lipase production by Yarrowia lipolytica and biocatalyst application/reuse. 3 Biotech, 11(4), 191. [CrossRef]
• 19. Theron, C.W., Vandermies, M., Telek, S., Steels, S., Fickers, P. (2020). Comprehensive comparison of Yarrowia lipolytica and Pichia pastoris for production of Candida antarctica lipase B. Scientific Reports, 10(1), 1741. [CrossRef]
• 20. Kuncharoen, N., Techo, S., Savarajara, A., Tanasupawat, S. (2020). Identification and lipolytic activity of yeasts isolated from foods and wastes. Mycology, 11(4), 279-286. [CrossRef]
• 21. de Souza, C.E.C., Ribeiro, B.D., Coelho, M.A.Z. (2019). Characterization and application of Yarrowia lipolytica lipase obtained by solid-state fermentation in the synthesis of different esters used in the food industry. Applied Biochemistry and Biotechnology, 189(3), 933-959. [CrossRef]
• 22. Yan, J., Han, B., Gui, X., Wang, G., Xu, L., Yan, Y., Madzak, C., Pan, D., Wang, Y., Zha, G., Jiao, L. (2018). Engineering Yarrowia lipolytica to simultaneously produce lipase and single cell protein from agro-industrial wastes for feed. Scientific Reports, 8(1), 758. [CrossRef]
• 23. Darvishi, F., Nahvi, I., Zarkesh-Esfahani, H., Momenbeik, F. (2009). Effect of plant oils upon lipase and citric acid production in Yarrowia lipolytica yeast. J Biomed Biotechnol. 2009, 562943. [CrossRef]
• 25. Salihu, A., Alam, M.Z., AbdulKarim, M.I., Salleh, H.M. (2012). Lipase production: an insight in the utilization of renewable agricultural residues. Resources, Conservation & Recycling, 58, 36-44. [CrossRef]