Phylogenetic Analysis and Extracellular Enzyme Profiles of Yeast Strains Isolated from Raspberry Fruits

Abstract

Raspberry fruit contains phenolic compounds, flavones, flavonoids, vitamins, and antioxidant substances that are important properties for health and pharmacological sciences. Edible berries provide also a suitable habitat for the growth of various microorganisms. In this study, yeast biota associated with raspberry fruits was determined by molecular identification techniques. Raspberry fruits were collected from Çanakkale, Gelibolu (Gallipoli). Yeast strains were isolated and then identified by using the analysis of ITS1-5.8S-ITS2 rDNA gene sequences. The phylo-genetic analysis of all yeast strains was carried out by using the MEGA–X phylogenetic analysis tool. The extracel-lular enzyme profiles of identified yeast species were determined by the API-ZYM kit system. The distribution of yeast species on the raspberry fruits was determined as Hanseniaspora uvarum, Metschnikowia viticola, Aureo-basidium pullulans, and Metschnikowia pulcherrima. It was observed that yeast strains belong to Metschnikowia genus were dominant on raspberry fruits. All yeast strains in Metschnikowia genus showed different enzyme pro-files against seven extracellular enzymes. These enzymes may be the discriminatory enzymes for the yeast strains in the Metschnikowia genus. When the phylogenetic relationships among all yeast strains were investigated, all strains were divided into two main clades. While the first clade consists of only Metschnikowia genus, second clade includes H. uvarum and A. pullulans yeast species. Our results indicated that restriction patterns and also extracel-lular enzyme profiles could be utilized for differentiation of yeast strains within the genus. M. pulcherrima, H. uvarum, and A. pullulans can be used for industrial applications for future researches.

Keywords

• Extracellular enzymes
• PCR-RFLP
• Raspberry
• Yeast biota

Project Number

FYL-2014-110

References

1. Abbas, A. C. (2006). Production of antioxidants, aromas, colors, flavors, and vitamins by yeasts. In A. Quarel & G. Fleet (Eds.), Yeasts in Food and Beverages, vol 2, (pp. 285-334). Springer, Verlag Berlin Heidelberg.
2. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215(3), 403 – 410. DOI: https://doi.org/10.1016/S0022-2836(05)80360-2.
3. Carvalho, C. M., Meirinho, S., Estevinho, M. L. F., & Choupina, A. (2010). Yeast species associated with honey: Different identification methods. Archivos de zootecnia, 59(225), 103-113. Retrieved from https://www.researchgate.net/publication/288352904.
4. Cheng, C. K., Demirci, A., & Catchmark, M. J. (2011). Pullulan: biosynthesis, production, and applica-tions. Applied Microbiology and Biotechnology, 92, 29–44. DOI: https://doi.org/10.1007/s00253-011-3477-y.
5. Delfini, C., & Formica, J. V. (2001). Wine microbiology science and technology. Marcel Dekker Inc., Italy.
6. Deshpande, M. S., Rale, V. B., & Lynch, J. M. (1992). Aureobasidium pullulans in applied microbiology: A status report. Enzyme and Microbial Technology, 14(7): 514–527. DOI: https://doi.org/10.1016/0141-0229(92)90122-5.
7. Dodor, D. E., & Tabatabai, M. A. (2007). Arylamidase activity as an index of nitrogen mineralization in soils. Journal Communications in Soil Science and Plant Analysis, 38(15-16), 2197-2207. DOI: https://doi.org/10.1080/00103620701549132.
8. Esen, A. (2003). Hydrolases; β-Glucosidase. In J. R. Whitaker, A. G. J. Voragen, D. W. S. Wong (Eds.), Handbook of Food Enzymology (pp. 791-803), Dekker: New York.

9. Esteve-Zarzoso, B., Belloch, C., Uruburu, F., & Querol, A. (1999). Identification of yeasts by RFLP analy-sis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers. International Journal of Systematic Bacteriology, 49(1), 329–337. DOI: https://doi.org/10.1099/00207713-49-1-329.
10. Fleet, G. H., Charoenchai, C., Henschke, P. A., & Todd, B. E. N. (1997). Screening of non-Saccharomyces wine yeasts for the presence of extracellular hydrolytic enzymes. Australian Journal of Grape and Wine Research, 3(1), 2–8. DOI: https://doi.org/10.1111/j.1755-0238.1997.tb00109.x.
11. Garcia-Martos, P., Marin, P., Hernandez-Molina, J. M., Garcia-Agudo, L., Aoufi, S., & Mira, J. (2001). Extracellular enzymatic activity in 11 Cryptococcus species. Mycopathologia, 150(1), 1-4. DOI: https://doi.org/10.1023/A:1010868223582.
12. Gaur, R., Singh, R., Gupta, M. &Gaur, M. K. (2015). Aureobasidium pullulans, an economically important polymorphic yeast with special reference to pullulan. African Journal of Biotechnology, 9(47), 7989-7997. DOI: https://doi.org/10.5897/AJB10.948
13. Gibson, B. R., Pham, T., Wimalasena, T., Box, W. G., Koivuranta, K., Storgards, E., & Smart, K. A. (2011). Evaluation of ITS PCR and RFLP for differentiation and identification of brewing yeast and brewery ‘wild’ yeast contaminants. Journal of The Institute of Brewing, 117(4), 556–568. DOI: https://doi.org/10.1002/j.2050-0416.2011.tb00504.x.
14. Göktaş, A. (2011). Ahududu ve böğürtlen yetiştiriciliği. Isparta Meyvecilik Araştırma İstasyonu Müdürlüğü, Yayın No: 38. Retrieved from https://arastirma.tarimorman.gov.tr.
15. Guillamon, J. M., Sabate, J., Barrio, E., Cano, J., & Querol, A. (1998). Rapid identification of wine yeast species based on RFLP analysis of the ribosomal internal transcribed spacer (ITS) region. Archives of Microbiology, 169(5), 387–392. DOI: https://doi.org/10.1007/s002030050587.
16. Halvorsen, B. L. K., Holte, M. C. W., Myhrstad, I., Barikmo, E., Hvatttum, S. F., Remberg, A. B., Wold Haffner, H., Baugerod, L. F., Andersen, J., Moskaug, D. R., & Blomhoff, J. R. (2001). A Systematic screening of total antioxidants in dietary plants. The Journal of Nutrition, 132(3), 461-471. DOI: https://doi.org/10.1093/jn/132.3.461.
17. Hierro, N., Gonzalez, A., Mas, A., & Guillamon, J. M. (2004). New PCR-based methods for yeast identification. Journal of Applied Microbiology, 97, 792–801. DOI: https://doi.org/10.1111/j.1365-2672.2004.02369.x.
18. Johnson, E. A. (2013a). Biotechnology of non-Saccharomyces yeasts—the Ascomycetes. Applied Microbi-ology and Biotechnology, 97(2), 503–517. DOI: https://doi.org/10.1007/s00253-012-4497-y
19. Johnson, E. A. (2013b). Biotechnology of non-Saccharomyces yeasts—the Basidiomycetes. Applied Micro-biology and Biotechnology, 97(17), 7563–7577. DOI: https://doi.org/10.1007/s00253-013-5046-z.
20. Johnson, E. A., & Echavarri-Erasun, C. (2011). Yeast Biotechnology. In P.C. Kurtzman, J.W. Fell & T. Boekhout (Eds.), The Yeasts, a Taxonomic Study, (pp. 22-44). Elsevier B.V.
21. Kallscheuer, N., Menezes, R., Foito, A., Da Silva, M. H., Braga, A., Dekker, W., Sevillano, D. M., Rosado-Ramos, R., Jardim, C., Oliveira, J., Ferreira, P., Rocha, I., Silva, A. R., Sousa, M., Allwood, J. W., Bott, M., Faria, N., Stewart, D., Ottens, M., Naesby, M., Dos Santos, C. N., & Marienhagen, J. (2019).
22. Identification and microbial production of the raspberry phenol salidroside that is active against Huntington’s disease. Plant Physiology, 179(3), 969-985. DOI: https://doi.org/10.1104/pp.18.01074.
23. Kähkönen, M. P., Hopia, A. I., Vuorela, H. J., Rauha, J. P., Pihlaja, K., Kujala, T. S., & Heinonen, M. (1999). Antioxidant activity of plant extracts containing phenolic compounds. Journal of Agricultu-ral and Food Chemistry, 47(10) 3954-3962. DOI: https://doi.org/10.1021/jf990146l.
24. Krisch, J., Galgóczy, L., Papp, T., & Vagvolgi, C. (2009). Antimicrobial and antioxidant potential of waste products remaining after juice pressing. Annals of the Faculty of Engineering Hunedoara-Journal of Engineering, 8(4), 131–134. Retrieved from https://www.academia.edu/22636802.
25. Kumar, S., Stecher, G., Li, M., Knyaz, C., & Tamura, K. (2018). MEGA X: Molecular Evolutionary Ge-netics Analysis across computing platforms. Molecular Biology and Evolution, 35(6),1547-1549. DOI: https://doi.org/10.1093/molbev/msy096.
26. Lee, B. H. (1996). Fundamentals of Food Biotechnology, VCH Publishers, USA.
27. Leff, J. W., & Fierer, N. (2013). Bacterial communities associated with the surfaces of fresh fruits and vegetables. PLoS ONE, 8(3), e59310. DOI: https://doi.org/10.1371/journal.pone.0059310.
28. Liu, H. M., Guo, J., Cheng, Y. J., Liu, P., Long, C. A., & Deng, B. X. (2010). Inhibitory activity of tea polyphenol and Hanseniaspora uvarum against Botrytis cinerea infections. Letters in Applied Micro-biology, 51, 258–263. DOI: https://doi.org/10.1111/j.1472-765X.2010.02888.x.
29. Lopez, V., Querol, A., Ramon, D., & Fernandez-Espinar, M. T. (2001). A simplified procedure to analyze mitochondrial DNA from industrial yeasts. International Journal of Food Microbiology, 68(1-2), 75–81. DOI: https://doi.org/10.1016/S0168-1605(01)00483-4.
30. Masneuf-Pomarede, I., Bely, M., Marullo, P. & Albertin, W. (2016). The genetics of non-conventional wine yeasts: current knowledge and future challenges. Frontiers in Microbiology, 6, 1563. DOI: https://doi.org/10.3389/fmicb.2015.01563
31. Nikolaou, E., Andrighetto, C., Lombardi, A., & Nikolaos, T. (2007). Heterogeneity in genetic and pheno-typic characteristics of Saccharomyces cerevisiae strains isolated from red and white wine fermenta-tion. Food Control, 18, 1458–1465. DOI: https://doi.org/10.1016/j.foodcont.2006.11.004.
32. Nikolaou, E., Soufleros, E. H., Boulompasi, E., & Tzanetakis, N. (2006). Selection of indigenous Saccha-romyces cerevisiae strains according to their oenological characteristics and vinification results. Food Microbiology, 23(3), 205-211. DOI: https://doi.org/10.1016/j.fm.2005.03.004.
33. Peter, G., Tornai-Lehoczki, J., Suzuki, M., & Dlauchy, D. (2005). Metschnikowia viticola sp. nov., a new yeast species from grape. Antony van Leeuwenhoek, 87(2), 155-160. DOI: https://doi.org/10.1007/s10482-004-2842-6.
34. Puupponen-Pimia R., Nohynek, L., Hartmann-Schimidlin, S., Kahkonen, M., Heinonen, M., & Maatta-Riihinen, K. (2005). Berry phenolics selectively inhibit the growth of intestinal pathogens. Journal of Applied Microbiology, 98, 991–1000. DOI: https://doi.org/10.1111/j.1365-2672.2005.02547.x.
35. Riaz, M., Ahmad, M., & Rahman, N. (2011). Anti-microbial screening of fruit, leaves, root, and stem of Rubus fruticosus. Journal of Medicinal Plant Research, 24 (5), 5920–5924. Retrieved from https://academicjournals.org/journal/JMPR/article-abstract/615921A41070.
36. Rodriguez-Vico, F, Clemente-Jimenez, J. M., Mingorance-Cazorla, L., Martinez-Rodriguez, S., & Las He-ras-Vazquez, F. J. (2003). Molecular characterization and oenological properties of wine yeasts iso-lated during spontaneous fermentation of six varieties of grape must. Food Microbiology, 21, 149–155. DOI: https://doi.org/10.1016/S0740-0020(03)00063-7.
37. Romano, P., Capece, A, Siesto, G., & Romaniello, R. (2009). Restriction analysis of rDNA regions to dif-ferentiate non-Saccharomyces wine species in mixed cultures. Journal of Engineering and Technolo-gy Research, 1(4), 068-071. Retrieved from https://academicjournals.org/journal/JETR/article-abstract/6075D985435
38. Sena, R. F., Costelli, M. C., Gibson, L. H., & Coughlin, R. W. (2006). Enhanced production of pullulan by two strains of A. pullulans with different concentrations of soybean oil in sucrose solution in batch fermentations. Brazilian Journal of Chemical Engineering, 23(4), 507–515. DOI: http://dx.doi.org/10.1590/S0104-66322006000400008.
39. Sherman, F., Fink, G. R., & Hicks, J. B. (1986). Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
40. Spadaro, D., Vola, R., Piano, S., & Gullino, M. L. (2002). Mechanisms of action and efficacy of four iso-lates of the yeast Metschnikowia pulcherrima active against postharvest pathogens on apples. Post-harvest Biology and Technology, 24(2), 123–134. DOI: https://doi.org/10.1016/S0925-5214(01)00172-7
41. Sipiczki, M. (2020). Metschnikowia pulcherrima and Related Pulcherrimin-Producing Yeasts: Fuzzy Species Boundaries and Complex Antimicrobial Antagonism. Microorganisms, 8(1029), 1-19. DOI: https://doi.org/10.3390/microorganisms8071029
42. The Huntington's Disease Collaborative Research Group. (1993). A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. Cell, 72(6), 971-83. DOI: https://doi.org/10.1016/0092-8674(93)90585-e
43. Tournas, V. H., Katsoudas, E. (2005). Mould and yeast flora in fresh berries, grapes and citrus juices. In-ternational Journal of Food Microbiology, 105(1), 11-17. DOI: https://doi.org/10.1016/j.ijfoodmicro.2005.05.002
44. Velićanski, A., Cvetković, D., & Markov, S. (2012). Screening of antibacterial activity of raspberry (Rubus ideaus L.) fruit and pomace extracts. Acta Periodica Technologica, 43, 305–313. DOI: https://doi.org/10.2298/APT1243305V
45. Vero, S., Garmendia, G., Gonzalez, M. B., Garat, M. F., & Wisniewski, M. (2009). Aureobasidium pullu-lans as a biocontrol agent of postharvest pathogens of apples in Uruguay. Biocontrol Science and Technology, 19(10), 1033-1049. DOI: https://doi.org/10.1080/09583150903277738.
46. Wang, Q., Laamanen, J., Uosukainen, M., & Valkonen Jari, P. T. (2005) Cryopreservation of In Vitro-grown shoot tips of raspberry (Rubus idaeus L.) by encapsulation–vitrification and encapsulation–dehydration. Plant Cell Reports, 24, 280–288. DOI: https://doi.org/10.1007/s00299-005-0936-x
47. Wang, X., Li, S., Yang, X., Yang, S., & Zhu, M. (2012). Technology prospecting on enzymes: application, marketing, and engineering. Computational and Structural Biotechnology Journal, 2(3), 1-11. DOI: https://doi.org/10.5936/csbj.201209017.
48. Weber, C., & Liu, R. H. (2002). Antioxidant capacity and anticancer properties of red raspberry. Acta Hor-ticulturae, 585, 451−455. DOI: https://doi.org/10.17660/ActaHortic.2002.585.73
49. White, T. J., Bruns, T., Lee, S. & Taylor, J. (1990). Amplification and direct sequencing of fungal riboso-mal RNA genes for phylogenetics. In M. A. Innis, D. H. Gelfand, J. J. Sninsky & T.J. White (eds), PCR Protocols: A Guide to Methods and Applications (pp. 315-322.), Academic Press, San Diego, California, USA. Retrieved fro https://www.researchgate.net/publication/223397588.