This research investigated yeast populations and diversity during pre-fermentation cold maceration and alcoholic fermentation of Vitis vinifera L. cv. Pinot noir grapes from a commercial vineyard (Dayton, OR). Fermentations were conducted at the Oregon State University research winery in 100 L tanks while grapes from the same vineyard lot were fermented at a commercial winery. Samples were taken daily during pre-fermentation maceration (9°C) and alcoholic fermentation (27°C) and plated on WL and lysine media to determine Saccharomyces and non-Saccharomyces populations and diversity. Total non-Saccharomyces populations increased from 1 x 10³ cfu/mL to 1 x 10⁵ cfu/mL during pre-fermentation cold maceration and reached a maximum of 1 x 10⁷ cfu/mL during alcoholic fermentation. Thirteen distinct yeast species were tentatively identified based on appearance on WL media and were initially screened for β-glucosidase activity using 4-methyllumbelliferyl-β-D-gluconopyranoside (4-MUG) plates. The identity of the isolates screening positive for β-glucosidase activity was determined by sequence analysis of the D1/D2 domain of the 26S rDNA gene. The five isolates identified were Metschnikowia pulcherrima, Hanseniaspora uvarum, Kluveromyces thermotolerans, and two Saccharomyces cerevisiae isolates. β-glucosidase activity was further characterized and quantified using a liquid media representing grape must conditions (pH 3.5, 20° Brix) at two temperatures (25°C and 8°C). While increasing sugar concentration suppressed the β-glucosidase activity of H. uvarum (-99%), β-glucosidase activity still remained relatively high for M. pulcherrima, S. cerevisiae isolate 1, and S. cerevisiae isolate 2. At 8°C, β-glucosidase activity was reduced for M. pulcherrima compared to activity at 25°C, but activity increased for K. thermotolerans, S. cerevisiae isolate 1, and S. cerevisiae isolate 2.
The yeast isolates possessing β-glucosidase activity were used in fermentations of Vitis vinifera L. cv. Pinot Noir grapes. The grapes were treated with high hydrostatic pressure (HHP) to inactivate naturally occurring yeast and bacteria. All yeast isolates grew during pre-fermentation cold maceration (7 days at 9°C) and populations increased 3 to 4 logs. Following pre-fermentation cold maceration, all ferments were warmed to 27°C and inoculated with S. cerevisiae RC212. Alcoholic fermentations were all complete within eight days and after pressing wines were analyzed for volatile aroma compounds by SPME-GC-MS. The presence of different yeast isolates during pre-fermentation cold maceration resulted in wines with unique aroma profiles. Ethyl ester concentrations were highest in the wine that did not undergo a pre-fermentation cold maceration, while concentrations of branch-chained esters were higher in the treatments with yeast present during pre-fermentation cold maceration. Pre-fermentation cold maceration with yeast isolates demonstrating β-glucosidase did not affect the concentration of β-damascenone or β-ionone. Wines that had undergone pre-fermentation cold maceration with S. cerevisiae isolate 1, S. cerevisiae isolate 2, and a combination of all isolates resulted in over twice the concentration of β-citronellol over wines that did not undergo a pre-fermentation cold maceration.