Screening Yeast Strains For The Winemaking Of Aroma Enhancement From The Brewing Ecosystem Of Luzhou Flavor Liquor In South Sichuan

Wine typical aroma derives from varietal volatile compounds which were released from flavor precursors with large amounts that stored in grape skins. These precursors, mainly in glycosidic forms, can be hydrolyzed by β-glucosidase to release volatile compounds, giving wine typical aroma characteristics. Some non-Saccharomyces yeasts exhibit high level of producing β-glucosidases, and can improve the complexity and regional concept of wine aroma via mix-fermentation with Saccharomyces sp. To improve wine aroma sensory quality, some non-Saccharomyces strains with high-level of β-glucosidase activity were isolated from Luzhou-flavor liquor cellars in South Sichuan Province, and used in winemaking application to enhance aroma. The results obtained are:1.The yeast diversity of Luzhou-flavor liquor cellars in South Sichuan Province were preliminary revealed. A total of 133 strains of non-Saccharomyces were classified into 8 genuses, 13 species according to morphology by WLN agar, including Wickerhamonyces(one species), Debaryomuces(one species), Pichias(four species), Lodderomyces(one species), Zygosaccharomyces(one species), Candidas(three species), Issatchosporon(one species) and Torulaspora(one species) Wickerhamonyces and Debaryomuces were dominant strains, accounting for 36.1% and 34.6%, respectively.2. Three strains of non-Saccharomyces with high-level of β-glucosidase activity were isolated. Nineteen strains of non-saccharomyces with high-level β-glucosidase activity were rapidly screened from 133 strains by a developed “semiquantitative colorimetry” method using aesculin 96-well plates, and then were subjected to rescreen by p-NPG spectrophotometry. Finally, 3 strains L. elongisporus I, P. fermentans I, P. membranifaciens I, were remained, among which, L.elongisporus I presented highest β-glucosidase activity(106.47 U/mL·10-3).3.Enzymatic characters and tolerance under winemaking-like conditions of β-glucosidase, as well as pectinase and protease activity secreted by L. elongisporus I, P. fermentans I and P. membranifaciens I were assayed. The β-glucosidases produced by L. elongisporus I with excellent tolerance to the typical winemaking conditions was suitable for winemaking application. As for L. elongisporus I β-glucosidases, the optimum reaction temperature was 55 ℃ and the optimum reaction pH value was 5.0.4.Two strains, H5Y-28 and Z9Y-3 were identified as Lodderomyces elongisporus and Pichia fermentans, respectively. Combined with identification by WLN agar and physiology tests, H5Y-28 and Z9Y-3 were precisely identified by 26 S rRNA technology, whose result showed H5Y-28 belonged to Lodderomyce elongisporus and Z9Y-3 belonged to Pichia fermentans, with 100% and 99% similarity, respectively.5.The mix-fermentation of Lodderomyces elongisporus or Pichia fermentans with Saccharomyces yeast was evaluated. Their β-glucosidases were also applied to enhance aroma in winemaking.The results showed that mix-fermentation gave wine aroma characteristics like temperate cranberry, sweet fruit and spice, besides, the level of aroma enhancement was positive related to the inoculation proportion of non-Saccharomyces. The optimum inoculation proportion for L. elongisporus and Saccharomyces yeast was 10:1. However, wines with β-glucosidases treatment exhibited fungus, animal and vegetable characteristics.