Yeast plays a key role in taste and aroma formation of fermented beverages with Saccharomyces cerevisiae being an undisputed leader in the field. However, a special role belongs to the Brettanomyces yeast. On the one hand, it is considered as a contaminant in beverages fermentation, on the other hand, it is generally used in traditional Belgian sorts of beer (lambic, gez, Flanders red ale and others), as well as in some red and white wines (Chardonnay, Sauvignon Blanc).
In addition, due to the development of resource-saving technologies, residual brewer's yeast of the genus Brettanomyces have interested researchers as a source of a biologically active substance beta-glucan, which is a polysaccharide contained in the yeast cell walls. Noteworthy, beta-glucan content in Brettanomyces yeast cells is higher than in Saccharomyces . For the successful application of Brettanomyces in brewing, winemaking and producing biologically active substances, it is necessary to better understand the relationship between the genotype and metabolism characteristics of the yeast genus.
Today molecular biology and microbiology methods are widely used for the yeast identification. To identify Brettanomyces yeast, we applied the PCR method using primers DB90F and DB384R , which are specific for Brettanomyces yeast. PCR products were subjected to electrophoresis in 1% agarose gel using a horizontal electrophoresis system MGU-602T (C.B.S. Scientific, USA). Ethidium bromide was used as a dye. The results were visualized in ultraviolet light and documented using a BioDoc-It Imaging System camera (UPV, USA).
To reduce the analysis time and exclude electrophoresis, a more advanced, fast and convenient real-time PCR (RT-PCR) method was used. Amplification was performed on a CFX96 Real-Time System (BIO-RAD, USA) to identify the strains under study using the mentioned primers DB90F and DB384R. However, in the experiments, a nonspecific rise in the amplification curves was observed both in the case of the studied Brettanomyces strains and in the case of the S. cerevisiae yeast. Therefore, the High Resolution Melting (HRM) method was used to refine the results. Thus, the use of HRM analysis allows to identify peaks of amplified specific DNA fragments of the yeast genus Brettanomyces without taking into account nonspecific peaks.
The work has shown that current methods of molecular biology allow to perform identification of microorganisms fast and accurately. This undoubtedly distinguishes them from the traditionally used microbiological and biochemical methods of detection and identification. In addition, the approaches of molecular biology and molecular genetic methods, in particular, open up big prospects in terms of genetic and genomic research, which is likely to help researchers not only with identification, but also with the effective management of directed biosynthetic processes.
1. Ivanova, V. A. Residual brewing yeasts as a source of beta-glucans / V. A. Ivanova, E. V. Antontceva, R. Harbah, T.V. Meledina, M. M. Shamtsyan // E3S Web of Conferences – 2020. - Vol. 164. - P. 06027.
2. Zagoruiko, V.A. Identification of the yeast species Brettanomyces bruxellensis using specific primers / V.A. Zagoruiko, I.V. Chernousova, T.K. Skorikova, S.A. Kishkovskaya, T.N. Tanashchuk, E.V. Ivanova, T.V. Chernookova, V.G. Gerzhikova, T.K. Zhilyakova, M.G. Tkachenko, E.P. Ishchuk// Viticulture and winemaking. - 2009. - V.39. - Pp. 57 – 60 (in Russian).
|Publication||IOP Conference Series: Earth and Environmental Science|
|Affiliation of speaker||ITMO University|
|Position of speaker||assistant|