Assay of distinct modes of polysaccharidases dosage in vinification with cv. Malbec. Effects on microbiological evolution, color and skin depletion

In the maceration stage of winemaking, enzymes can be used to degrade the polysaccharides present in the cell walls and middle sheets, and thus facilitate the extraction of juice and the release of polyphenols and aroma precursors retained in the grape skins. This work aims to analyze the influence of two enzyme complexes produced by autochthonous yeasts on the red winemaking process, in order to evaluate their effect on the chemical composition of the wines obtained, as well as on the extraction of color and polyphenols, and the depletion of the skin. Two strains previously selected for the effect of their enzymatic complex on the color extraction and improvement in the technological properties of the grape must were used (Longhi et al., 2019). A multi-enzymatic extract from Aureobasidium pullulans m11-2 was obtained by inoculating the microorganism in a broth according to Moyo et al. (2003) with modifications (pH 3.8) and incubated with stirring at 28°C for 72 h. Pectinase, xylanase, cellulase and amylase activities were quantified by determining reducing sugars by DNS, modified by Qian Li et al. (2015). Likewise, Torulaspora delbrueckii m7-2 was used for the production of the enzyme complex during vinification. Malbec red grapes (Vitis vinifera L.) from San Rafael (Mendoza) wine region, vintage 2021, were used to conduct the vinifications. The must obtained by crushing 60 Kg of grapes was corrected in acidity, sulfited (50 ppm) and distributed in 5 L containers. Four winemaking assays were performed, in duplicate: (1) inoculation with a native strain of Sacchromyces cerevisiae (SR1), at 108 cell/mL as inoculum, conducted at 20°C (control, C); (2) sequential inoculation of T. delbrueckii m7-2, with an initial cellular concentration of 107 cells/mL, followed by SR1 inoculation at 4th day (Td); (3) cold pre-fermentation maceration (CPM, 8°C-4 days) with m11-2 enzyme extract and SR1 inoculation (Ap); and (4) CPM without enzymatic treatment and SR1 inoculation (E). Growth kinetics of total yeasts were determined on YPD and DRBC agar, and of non-Saccharomyces yeasts in lysine medium. All enzymatic activities were monitored at pH 3.80 and 20°C. The pectinolytic activity was the main one, showing a level of 1.80 U/mL in the m11-2 extract and an initial level of 1.47 U/mL for the in situ producer strain (m7-2). Microscopic observations of the extracted skins in Ap and E vinifications were carried out to evaluate the effect of the enzymatic complex m11-2 on the cell wall, and were also compared with the fresh grape skins. Differences were observed between the skins enzymatically treated (Ap) and the control (E); the former showed cell emptying, greater rupture of the epidermis layers and less firmness, unlike the control that exhibited almost intact epidermal layers. These images allowed us to know the cell morphology of the varietal cv. Malbec and the enzymatic hydrolysis of its cell walls.

Authors: Sara Jaquelina Longhi – Biotechnology Laboratory, Department of Biology and Food, Faculty of Sciences Applied to Industry, National University of Cuyo. Bernardo de Irigoyen 375, San Rafael, Mendoza, Argentina. National Council for Scientific and Technical Research (CONICET), Godoy Cruz 2290, Autonomous City of Buenos Aires, Argentina. ,María Carolina MARTÍN (1,2)  María Belén AVENDAÑO(1) María Gabriela MERÍN (1,2)   Luciana Paola PRENDES (1,2) Juliana GARAU (1,2) Vilma Inés MORATA DE AMBROSINI (1,2) (1) Biotechnology Laboratory, Department of Biology and Food, Faculty of Sciences Applied to Industry, National University of Cuyo. Bernardo de Irigoyen 375, San Rafael, Mendoza, Argentina. (2) National Council for Scientific and Technical Research (CONICET), Godoy Cruz 2290, Autonomous City of Buenos Aires, Argentina.

Email: slonghi@fcai.uncu.edu.ar

Keywords: polysaccharidases, winemaking, polyphenols, malbec, grape cell wall

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