The purposeful induction of malolactic fermentation (MLF) in wines such as Pinot Noir and Chardonnay is an established commercial wine making practice in Oregon. This induction is not always successful, especially with white wines, such as Chardonnay. A study was initiated to examine the compatibility of yeasts commonly used in Oregon winemaking with various strains of malolactic bacteria. In preliminary and pilot plant scale experiments, the yeast strain found to be most conducive to malolactic fermentation by lactic acid bacteria was Montrachet (Red Star). The malolactic bacterial strains that were best able to complete malolactic fermentation in various wines, fermented by different yeast strains, were the two Oregon commercial strains, ER1A and Ey2d, and the Pinot Noir juice isolate, DAPN85A. Sensory analysis of aroma by difference from control test was done on Chardonnay wine fermented by 4 different yeast strains and 3 different malolactic bacterial strains. In all cases, there was an overall significant difference in malolactic fermented wine aroma when compared to control wines. Organic acid analyses by high pressure liquid chromatography (HPLC) and analyses of volatile compounds by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) were done on selected Chardonnay wines. Propionic acid was found to diminish in malolactic fermented wines while acetic acid content increased. Isobutanol and isobutyraldehyde increased significantly in MLF wines, compared to the controls. Chemical analyses of MLF and control wines suggested two possible chemical reactions resulting from the MLF. The first was the reduction of isobutyraldehyde to isobutanol, and the second was the hydrolysis of isobutyl acetate to isobutyraldehyde and acetate. On all GC chromatograms of wines, where MLF had occurred, there was an unidentified peak close to the retention time of isoamyl acetate. This peak was not evident in wines where MLF had not occurred. Eight compounds were tentatively identified by GC-MS in malolactic fermented wines which were not found in the control wines. These were 4-methyl-3-pentanoic acid, methyl acetate, ethyl hexanoate, hexyl acetate, 1,12-tridecadiene, hexadecanoic acid, and a compound which was tentatively identified as farnesol, or 1,2-benzenedicarboxylic acid. The latter four compounds had identity fits of less than 900 from the mass spectral analysis. Whether any of these eight compounds match the unknown "ML peak" found in the GC chromatograms is unknown. / Graduation date: 1989

In western Oregon the titratable acidity of grapes at harvest may in some seasons be higher than desirable for making quality wine, due to the retention of malic acid. The purposes of this study were 1) to investigate the effects of a vineyard cultural practice, cluster exposure at veraison by basal leaf removal, as a means of reducing the malate content and titratable acidity of grapes, and 2) to develop a rapid, simple, and inexpensive test procedure by which smaller wineries and vineyards could evaluate the effects of their own field experiments on the malate and tartrate content of their grapes. 1. At veraison, clusters of Chardonnay grapes were a) exposed to the sun by removal of all leaves opposite or below the clusters, b) treated as in a) but shaded with shadecloth, c) exposed to the sun by tying back leaves opposite or below the clusters, or d) left untreated as a control. Clusters of White Riesling were exposed to the sun by similar leaf removal a) 10 days before veraison, b) 10 days after veraison, or c) untreated. Clusters of Pinot Noir were exposed a) at veraison, b) 2 weeks after veraison, or c) untreated. Exposed clusters received 3 to 3.5 times more light than shaded clusters and up to 32% more heat, with temperature differences between exposed and shaded treatments being most pronounced during cool, sunny weather. None of the treatments had any effect on juice or berry malate, tartrate, or potassium content; however, exposed clusters of Pinot Noir had a lower pH (.03) and higher titratable acidity (.06%) than the control at harvest. Cluster exposure of Chardonnay increased sunburning of grapes, and cluster exposure of Pinot Noir at veraison caused a 1% reduction in juice soluble solids concentration at harvest. The detrimental effects of cluster exposure by basal leaf removal at veraison, as well as the lack of any major effect on the acid content of the berries, suggest that the practice has no value for acid reduction during a warm, dry maturation season in western Oregon. 2. A rapid, simple procedure for the estimation of the malate and tartrate content of grape juice is described. The procedure, which requires only a pH meter for instrumentation, does not directly measure malate and tartrate but instead measures their buffering effect. Samples are titrated between pH 2.70-3.00 and pH 4.50-4.80 and the titrant volumes required are compared to two sets of empirically derived standard curves. The malate and tartrate composition of the sample may be determined by a graphical or algebraic method. The use of the estimation method, its advantages, and its limitations are illustrated with different viticultural trials. The estimation error (estimated value - measured value) was influenced by many factors including maturity, season, vineyard location, and cultivar. Standard deviations of the estimation error for malate and tartrate in mature grapes were equal to 9% and 15%, respectively, of the mean malate and tartrate concentrations in pooled Pinot Noir and Chardonnay samples from different vineyards and years. The estimation error is probably due to interference from other buffers present in juice. Although not as accurate as existing analytical methods, the estimation method appears potentially useful for determining relative effects of treatments in vineyard trials where analytical equipment is unavailable or for monitoring malate decline during maturation of grapes. / Graduation date: 1987

Hen egg white lysozyme is a hydrolytic enzyme effective at preventing the growth of Gram positive bacteria by degrading the bacterial cell wall to a point of cell lysis. Investigating lysozyme as a processing tool in wine to control the growth of lactic acid bacteria and malolactic fermentation has significant commercial interest. In this project, the interactions of lysozyme with wine components and wine was evaluated along with the efficacy of lysozyme in preventing malolactic fermentation (MLF) in Oregon Pinot Noir and Chardonnay. The information from this work, together with results from similar projects, will allow the development of guidelines for lysozyme use in commercial wine. Interactions of lysozyme with wine components were evaluated by measurement of enzymatic activity in the presence of wine acids, ethanol, and phenolics. Enzyme inhibition was observed, to various degrees, with all wine components. Crude grape tannin altered the availability of free enzyme by complexing to lysozyme and forming a precipitate. In a model wine system, lysozyme activity was reduced by 50% when tannin was present. Lysozyme addition to red wine resulted in a reduction in pigmented compounds and detectable sensory differences. Wine trials evaluated the efficacy of lysozyme in completely preventing malolactic fermentation (MLF) and terminating MLF midway through fermentation in Oregon Pinot Noir and Chardonnay. Vintages from 1993 and 1994 were treated without SO₂, with SO₂, with SO₂ plus a starter culture of Leuconostoc oenos. Each lot was divided into 0 ppm lysozyme (control), 250 ppm lysozyme, 500 ppm lysozyme, and 1000 ppm lysozyme. Lactic acid bacteria were enumerated monthly, for ten months. Lysozyme prevented malolactic fermentation in all wines at the treatment levels of 500 and 1000 ppm. In the 1993 Pinot Noir, 250 ppm lysozyme prevented MLF but only delayed MLF in the 1994 vintage. Lysozyme effectively terminated MLF at a concentration between 200 and 300 ppm in both Pinot Noir and Chardonnay. / Graduation date: 1996

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. / Graduation date: 2013