Isolation, Identification and Determination of Saccharomyces cerevisiae Yeast Species from The Wines Made by Spontaneous Fermentation Using Papazkarası Grapes from Thrace Region

Öz

Especially in large-scale commercial wine production, fermentation is usually carried out by using the species of Saccharomyces cerevisiae yeast cultures. Standard wine yeast starter cultures are brought from abroad and used in industrial winemaking in our country. Thus, in course of time, the wines of all producers began to show similar properties. Today, studies focus on terroir wines production with local characteristics by using the countries' own local wine yeasts as a starter. Within the scope of this study Papazkarası grapes were collected from 4 vineyards with different characteristics, such as viticulture practices, microclimatic conditions and locations in the borders of Edirne and Kırklareli. In addition, the grapes were also collected from the vineyard in Tekirdağ Viticulture Research Institute. Spontaneous fermentation was done by crushing the collected grapes in the laboratory. At the end of the fermentation, yeast isolations were made from the musts to YPD medium and a total of 66 isolates were obtained as a result of purification. Fermentation rate, H2S production amount, growth at high temperature, growth at high sugar concentration, resistance to high ethanol, ability to grow at low pH values, resistance to SO2, and volatile acid analysis were done to determine whether they meet the technological requirements for basic winemaking, together with two commercial wine yeasts. DNA sequencing analyzes were made by selecting 15 of the isolates that were found to have the necessary characteristics and it was determined that they belonged to the Saccharomyces cerevisiae species. DNA fingerprinting analysis was performed using delta 12-21 primers to determine the strain differences of the isolates determined to belong to the same species. By DNA fingerprint analysis, it was determined that 9 out of 15 isolates were different strains.

Anahtar Kelimeler

• Papazkarası
• S. cerevisiae
• Wine
• DNA
• Yeast

Isolation, Identification and Determination of Saccharomyces cerevisiae Yeast Species from The Wines Made by Spontaneous Fermentation Using Papazkarası Grapes from Thrace Region

Öz

Günümüzde, özellikle büyük çaplı ticari şarap üretiminde, fermantasyon genellikle Saccharomyces cerevisiae türü maya kültürleri kullanılarak gerçekleştirilmektedir. Standart şarap mayası starter kültürleri Uluslararası büyük maya üreticisi firmalardan alınarak ülkemizde, şarap yapımında kullanılmaktadır. Bu da, üreticilerin şaraplarının zamanla birbirine benzemesine yol açmaktadır. Günümüzde yapılan çalışmalar, ülkelerin kendi yerel şarap mayalarını starter olarak kullanarak yerel özellikler taşıyan teruar şaraplarının üretimine yoğunlaşmaktadır. Bu çalışma kapsamında, Edirne ve Kırklareli sınırlarında bulunan, yapılan bağcılık uygulamaları, mikroklimatik şartları, konumları gibi özellikleri farklılık taşıyan 4 bağdan ve Tekirdağ Bağcılık Araştırma Enstitüsündeki bir bağ parselinden olmak üzere toplam 5 farklı lokasyondan Papazkarası çeşidine ait üzümler önolojik olgunluk aşamasında toplanmıştır. Toplanan üzümler laboratuvara getirilerek ezilmiş ve sonrasında spontan fermantasyona bırakılmıştır. Fermantasyon sonunda fermante olmuş şıralardan, YPD besiyerlerine, ekim yapılmıştır. Besiyerlerinde gelişen kolonilerin saflaştırması işlemi sonucunda toplam 66 adet izolat elde edilmiştir. İzolatların, 2 adet ticari şarap mayası ile birlikte, temel şarap yapımı için gereken teknolojik özellikleri karşılayıp karşılamadıklarını belirlemek için fermantasyon hızı, H2S üretim miktarı, yüksek sıcaklıkta gelişebilme, yüksek şeker konsantrasyonunda gelişebilme, yüksek etanole dayanıklılık, düşük pH değerlerinde gelişebilme, SO2’ye dayanıklılık, uçar asit miktarı analizleri uygulanmıştır. Gerekli özelliklere sahip olanlardan 15 tanesi seçilerek DNA dizileme analizleri yapılmış ve Saccharomyces cerevisiae türüne ait oldukları tespit edilmiştir. Aynı türe ait oldukları belirlenen izolatların alt tür bazında birbirinden farklı olup olmadıklarını belirlemek üzere delta 12-21 primerleri kullanılarak DNA parmak izi analizi yapılmıştır. Bu izolatlara yapılan DNA parmak izi analizlerinde 9 adet farklı suş tespit edilmiştir.

Anahtar Kelimeler

• Papazkarası
• S. cerevisae
• Şarap
• DNA
• Maya
• Karakterizasyon

Kaynakça

1. Ali, M.N., Khan, M.M. (2014). Screening, identification and characterization of alcohol tolerant potential bioethanol producing yeasts. Current Research in Microbiology and Biotechnology, 2(1):316-324.
2. Antia, E.U., Akan, D.O., Stephen, U.N., Eno-Ibanga, K.C., Akpan, G.N. (2018). Isolation andscreening of yeast isolates indigenouspalm wine for ethanol production. Philippine Journal of Science, 147(3): 411-417.
3. Bagder, S. (2008). Türkiyede değişik şarap bölgelerinden izole edilmiş şarap mayalarının teknolojik özellikleri. (Master Thesis) Ankara University, Graduate School of Natural and Applied Science, Ankara.
4. Bilgin, A.N. (2015). Türkiye Bağlarından Şarap/Alkol Fermentasyonuna Uygun Mayaların Seçilmesi ve Moleküler Karakterizasyonu (TAGEM-13/AR-GE/10), Boğaziçi University, İstanbul.
5. Blaiotta, G., Pepe, O., Mauriello, G., Villani, F., Andolfi, R., Moschetti, G. (2002). 16S-23S rDNA intergenic spacer region polymorphism of Lactococcus gavriae, Lactococcus raffinolactis and Lactococcus lactis as revealed by PCR and nucleotide sequence analysis. Systematic and Applied Microbiology., 25(4):520-527.
6. Bokulich, N.A., Thorngate, J.H., Richardsone, P.M., Mills, D.A. (2013). Microbial biogeography of wine grapes is conditioned by cultivar, vintage, and climate. Proceedings of the National Academy of Sciences, 111(1): 139 - 148.
7. Budroni, M., Ladu, G., Zara, G., Zara, S., Farris, A.G. (2006). Molecular and Enological Characterization of Autochthonous Saccharomyces Cerevisiae Strains İsolated from Grape-Musts and Wines Cannonau. ISEIMA’06 First International Symposioum Environment Identities and Mediterranean Area, 9-12, July, Corte-Ajaccio, France.
8. Chambers, P.J., Pretorius, I.S. (2010). Fermenting knowledge: the history of winemaking, science and yeast research. EMBO Reports, 12(11):914-920.

9. Charoenchai, C., Fleet, G.H., Henschke, P. (1998). Effects of temperature, pH, and sugar concentration on the growth rates and cell biomass of wine yeasts. American Journal of Enology and Viticulture, 49(3): 283-288.
10. Cocolin, L., Pepe, V., Comitini, F., Comi, G., Ciani, M. (2004). Enological and genetic traits of Saccharomyces cerevisiae isolated from former and modern wineries. Fems Yeast Research, 5(3): 237-245.
11. Cordente, A.G., Heinrich, A., Pretorius, I.S., Swiegers, J.H. (2009). Isolation of sulfite reductase variants of a commercial wine yeast with significantly reduced hydrogen sulfide production. Fems Yeast Research, 9(3): 446-459.
12. Çavdaroğlu, Ç. (2017). Physilogical Traits of Saccharomyces cerevisiae Strains Isolated From Traditional Wines in Turkey. (Master Thesis), Middle East Technical University, Graduate School of Natural and Applied Science, Ankara.
13. Çelik, Z.D., Erten, H., Darıcı, M., Cabaroğlu T. (2017). Molecular characterization and technological properties of wine yeasts isolated during spontaneous fermentation of Vitis vinifera L.cv. Narince grape must grown in ancient wine making area Tokat, Anatolia. 40th World Congress of Vine and Wine, Sofija, Bulgaria, 29 May - 02 June 2017. Doi: 10.1051/bioconf/20170902017.
14. Furdíková, K., Makyšová, K., Špánik, I. (2017). Effect of indigenous S. cerevisiae strains on higher alcohols, volatile acids and ssters in wine. Czech Journal of Food Sciences, 35(2):131-142.
15. Garofalo, C., Tristezza, M., Grieco, F., Spano, G., Capozzi, V. (2016). From grape berries to wine: population dynamics of cultivable yeasts associated to ‘‘Nero di Troia’’ autochthonous grape cultivar. World Journal of Microbiology and Biotechnology, 32(4):59-69. DOI 10.1007/s11274-016-2017-4.
16. Gerőcs, G., Barná, K.N., Pál, S., Szőke, B., Májer, J., Farkas, T., Olasz, F. (2020). Isolation and characterization of yeast strains from Badacsony, Hungary. Indian Journal of Experimental Biology, 58:461-473.
17. Guimarães, T.M., Moriel, D.G., Machado, I.P., Picheth, C.M.T.F., Bonfim, T.M.B. (2006). Isolation and characterization of Saccharomyces cerevisiae strains of winery interest. Brazilian Journal of Pharmaceitical Sciences, 42(1): 119-126.
18. Iranzo, J.F.U., Perez, A.I.B., Canas, P.M.I. (1998). Study of oenological characteristics and enzymatic activities of wine yeasts. Food Microbiology, 15, 399-406.
19. Jolly, N.P., Pretorius, I.S., Augustyn, O.P.H. (2006). The role and use of Non-Saccharomyces yeasts in wine production. South African Journal for Enology and Viticulture, 27(1): 15-39.
20. Kuchen, B., Maturano, Y.P., Mestre, M.V., Combina, M., Toro, M.E., Vazquez, F. (2019). Selection of native non-Saccharomyces yeasts with biocontrol activity against spoilage yeasts in order to produce healthy regional wines. Fermentation, 5(3):60-75. Doi:10.3390/fermentation5030060.
21. Legras, J-L., Karst, F. (2003). Optimisation of interdelta analysis for Saccharomyces cerevisiae strain characterisation. FEMS Microbiology Letters, 221:249-255.
22. Lopes, C.A., Rodríguez, M.E., Sangorrín, M., Querol, A., Caballero, A.C. (2007). Patagonian wines: the selection of an indigenous yeast starter. Journal of Industrial Microbiology and Biotechnology, 34:539-546.
23. Lopes, C.A., Rodríguez, M.E., Querol, A., Bramardi, S., Caballero, A.C. (2006). Relationship between molecular and enological features of Patagonian wine yeasts: relevance in selection protocols. World Journal of Microbiology and Biotechnology, 22:827-833.
24. Lu, Y., Sun, F., Wang, W., Liu, Y., Wang, J., Sun, J., Mu, J., Gao, Z. (2020). Effects of spontaneous fermentation on the microorganisms diversity and volatile compounds during ‘Marselan’ from grape to wine. Lwt-Food Science and Technology, 134:110193.
25. Lu, Y., Voon, M.K.W., Huang, D., Lee, P.R., Liu, S.K. (2016). Combined effects of fermentation temperature and pH on kinetic changes of chemical constituents of durian wine fermented with Saccharomyces cerevisiae. Applied Microbiology and. Biotechnology.101(7):3005-3014. DOI 10.1007/s00253-016-8043-1.
26. Nikolaou, E., Soufleros, E.H., Bouloumpasi, E., Tzanetakis, N. (2006). Selection of indigenous Saccharomyces cerevisiae strains according to their oenological characteristic and vinification results. Food Microbiology, 23:205-211.
27. Nurgel, C. (2000). Emir ve Kalecik karası üzümlerinin şaraba işlenmesinde maya florasındaki gelişmeler ve fermantasyonda kullanılan mayaların kalite üzerine etkileri. (Phd Thesis) Çukurova University, Adana.
28. Orlić, S., Oćić, N., Jeromel, A., Huić, K., Redžepović, S. (2005). Selection of indigenous Saccharomyces cerevisiae strains from Kutjevo wine growing area at the laboratory scale. Agriculturae Conspectus Scientificus, 70(3):93-97.
29. OIV (2021). SO2 and Wine: A Review (1st ed.). Paris: OIV publications.
30. Ough, C.S., Amerine, A.M. (1988). Methods for Analysis of Musts and Wines (2nd Edt.) içinde (365). New York: Wiley.
31. Özdinç, N., Velioğlu, H.M. (2022). Ekmek mayası (Saccharomyces cerevisiae) fabrika artıklarından ham invertaz enzimi üretimi. Journal of Tekirdag Agricultural Faculty, 19(2):456-464.
32. Parish, M.E., Carrroll, D.E. (1987). Fermentation characteristics of Saccharomyces cerevisiae isolates from Vitis rotundifolia grapes and musts. American Journal of Enology and Viticulture, 38(1):45-48.
33. Pérez-Coello, M.S., Briones-Pérez, A.I., Ubeda-Iranzo, J.F., Martin-Alvarez, P.J. (1999). Characteristics of wines fermented with different Saccharomyces cerevisiae strains isolated from the La Mancha region. Food Microbiology, 16:563-573.
34. Tahmaz, H., Yüksel Küskü, D., Söylemezoğlu, G., Çelik, H. (2022). Vitis labrusca L. Genotiplerinin fenolik bileşik ve antioksidan kapasite içerikleri. Journal of Tekirdag Agricultural Faculty, 19(2):318-331.
35. Tristezza, M., Fantastico, L., Vetrano, C., Bleve, G., Corallo, D., Grieco, F., Mita, G., Grieco, F. (2014). Molecular and technological characteri-zation of Saccharomyces cerevisiae strains isolated from natural fermentation of Susumaniello grape must in Apulia, Southern Italy. International Journal of Microbiology, 2014(10):1-11 Doi: 10.1155/2014/897428.
36. Valero, E., Cambon, B., Schuller, D., Casal, M., Dequin, S. (2007). Biodiversity of Saccharomyces yeast strains from grape berries of wine-producing areas using starter commercial yeasts. FEMS Yeast Research, 7(2):317-29.
37. Varela, C., Borneman, A.R. (2016). Yeasts found in vineyards and wineries. Yeast, 34(3):111-128. Doi:10.1002/yea.3219.