Factors Affecting Sugar Utilization and Rate of Fermentation During Vinification

AVF Proj. ID: 9621 / /Cat.: ,
Primary Investigator(s): Linda Bissonby

In this grant year we have: 1) successfully developed the technology for the analysis of HXT gene expression; 2) developed methodology yielding high quantities of high quality mRNA from strains of Saccharomyces during grape juice fermentation; 3) begun profiling of H’XT gene expression during grape juice fermentation conducted by commercial strains; 4) confirmed the critical role of HXT2 in non-proliferative phase fermentation of glucose and fructose in grape juice; 5) defined the pathway by which the HXT2 transporter is degraded in response to nitrogen limitations. Further, we have shown that HXTl and HXT3 are expressed early during the fermentation of grape juice, with a shift to HXT2 and HXT3 upon entry into stationary phase of growth during the fermentation. We are now poised to undertake a more thorough investigation of expression of the remainder of the HXT genes during grape juice fermentation.

New Fining Techniques Utilizing Adsorbent Resins

AVF Proj. ID: 9630 / /Cat.: ,
Primary Investigator(s): Barry Gumpby

To date we have evaluated the qualitative abilities of three commercially available, organophilic adsorbent resins for the purpose of removing protein-complex precursors and colored compounds from grape juice. Data have been generated towards chemically characterizing the specific amounts of protein and other constituents removed, as well as characterizing what compounds (sugars, acids, etc.) have not been removed. Resin technology appears to be promising, as the treated juice did not retain the haze forming proteins as determined in the control sample by traditional heat stability analysis and “Coomassie blue” protein analysis. The protein results are similar to those from previous efforts in which we evaluated adsorbent resins for preliminary unstable protein removal from apple juice. Resin treated juices had measured NTU values of less than 1.0 compared to a value of over 20 NTU in the non-resin treated control juice subjected to the same heat treatment. Using the “Coomassie blue” chemical analysis for proteins, over 90% of the proteins detected by this method were removed. Phenolics levels have also been reduced in the processed juices by values of 33% or 85% depending upon which resin was used. At the same time that these constituents were being removed, other data indicate essentially no changes in original Brix or pH of the juice upon being subjected to resin treatment. In separate experiments, dark colored white grape juice samples were decolorized using the three resins. These experiments were run to 100 bed volume and the degree of decolorization was quantitatively measured as %Transmittance at 430 nm. After 100 bed volumes two resins significantly improved the transmittance of the dark colored white grape juice by 20 units of %Transmittance over the starting juice. The untreated juice in this experiment had significant color and measured only 42%T. Our pilot scale studies have been conducted using 2.54 cm (1″) x 183 cm (6′) columns so that the results better reflect what would occur during production scale operations. The columns are operated using pumped flow, again to mimic production conditions. A peristaltic pump connected to the inlet line is used for delivering all juice, conditioning, and regenerating solutions.

Rapid Analytical Methods For Measurement of Cork-Taint in Wine

AVF Proj. ID: 9634 / /Cat.: ,
Primary Investigator(s): Susan E. Ebelert, Christian E. Butzkeby

Work has been initiated to synthesize novel compounds similar in structure to TCA that can be used for antibody production. Because of the low molecular weight, TCA cannot be used directly for antibody production; a protein conjugate must be synthesized and its structure confirmed prior to antibody production and screening. The sensitivity and specificity of the antibodies and the resulting immunoassay will be confirmed independently using an automated gas chromatographic headspace analysis procedure for TCA which we have developed.

Studies on Stuck Fermentations and on Factors that Control the Rates of

AVF Proj. ID: 9636 / /Cat.: ,
Primary Investigator(s): Robert K. Mortimerby

We have found that the factor(s) that determine whether or not a fermentation will stick are vineyard-determined. If grapes from a given vineyard are crushed and the resultant must is placed in more than one fermentation tank, if one tank sticks all tanks from the same must also will stick. Stuck fermentations occur for all types of must and also occur for both inoculated and natural fermentations. We have found that viabilities of Saccharomyces cerevisiae cells in stuck fermentations are very low and this appears to be the reason the fermentations have stopped. We have also found that dilution of a stuck wine with water or even with a dry wine followed by addition of live yeast results in a reinitiation of fermentation which continues to dryness. We have considered six models that have been proposed to explain stuck fermentations. These models are: 1) nutrient depletion, 2) excess temperature during fermentation, 3) killer yeast, 4) toxin introduced with the grapes, 5) toxin produced by another microorganism and 6) toxin produced by the wine yeast. The dilution experiments eliminate model 1 and occurrence of stuck fermentations in temperature-controlled fermenters eliminates model 2. Experiments done by others eliminate model 3. The dilution experiments and the low viabilities of the wine yeast cells point to the presence of a toxin in the stuck wine. This toxin could be a fungicide brought in with grapes (model 4) or a toxin produced by another microorganism (model 5) or by the wine yeast (model 6). We believe model 5 may occur but is exceptional. Some fermentations become infected with acetobacter and the acetic acid kills or inhibits the wine yeast. We saw no other microorganisms in the 14 fermentations we studied and volatile acidity levels were near the normal range. We considered model 4 to be unlikely because one would expect the fungicide to act from the beginning of fermentation and this does not appear to be the case. Fermentation kinetics are normal until the time of sticking. This leaves model 6, a toxin produced by the wine yeast. This model was proposed several years ago by a group in Bordeaux and the toxin was proposed to be medium chain fatty acids, octanoic, decanoic and dodecanoic acid. These compounds are extremely toxic and are produced during fermentation. Several experiments done by others seem to lend strong support to this model. The goal of the study supported by AVF was to test this specific model. We studied 4 stuck fermentations and did gas chromatographic analyses of the stuck wine. We found that the concentrations of octanoic and decanoic acid were at or below normal levels in these stuck wines. This eliminates this specific model. We now believe that there may be another toxic compound produced by wine yeast and that this kills the cells and stops the fermentation. We are also now reconsidering model 4. If the fungicide brought in with the grapes needs to be modified during the fermentation process to be toxic to wine yeast this would explain the lag in onset of the stuck state. Glenn Andrade of Sutter Home Winery (personal communication) has found a correlation between spraying of the grapes with a fungicide and the occurrence of stuck fermentations.

Studies on the Interaction of Flavor Compounds With Non-Volatile

AVF Proj. ID: 9637 / /Cat.: ,
Primary Investigator(s): Susan Ebelerby

During the previous year of this proposal we focused on developing sensitive gas chromatographic headspace procedures for quantifying odorant/polyphenol interactions in model solutions. We are currently using these procedures to evaluate the effects of polyphenol structure on the strength of odorant interactions. The main focus this year has been on developing and evaluating sensory procedures for measuring these interactions. Although previous studies of flavor interactions in other foods and beverages have largely focused on measuring aroma (nasal) intensity only, we wanted to also be able to quantify oral (retronasal) aroma stimulation, similar to that experienced when wine is consumed. We have used a time-intensity procedure and model solutions to show that retronasal aroma perception is significantly affected by the chemical/physical nature of the aroma compound, by the nature of the matrix, and by individual judge factors such as salivary flow rate. We feel that this procedure now provides us with a promising tool for the sensory evaluation of matrix effects. This, in combination with the analytical headspace procedures, will provide extensive information about the importance of odorant/polyphenol interactions in wine.

The Extraction of Condensed Tannins in Red Wine Fermentations

AVF Proj. ID: 9638 / /Cat.: ,
Primary Investigator(s): Andrew Waterhouseby

The first year of this project was designed to survey the effects of a large number of different pomace maceration practices in commercial-scale fermentations on the extraction of anthocyanins and tannins. This year of the project focused on Pinot Noir and observed variables included cold soak, manual vs. mechanical punch down, and pump over vs. punch down. The wines were prepared at a cooperating winery according to an agreed-to protocol. The phenolic composition of the musts/wines were analyzed using both spectral (at the cooperating winery) and chromatographic methods (at UC Davis and ETS), including a new Silica gel based procedure which separates phenolics based on size. Five analyses were carried out on 9 wines although the focus was on 5 Pinot Noir wines from the Widoe’s Vineyard. The major difference was that the treatments of both mechanical punch down and pump over increased both colored and uncolored tannin, while the cold soak decreased these components. The pump over regime also increased monomelic color. The goals in this project included the following: 1) Develop a method for sample preparation and an HPLC method for the determination of condensed tannins. 2) Assist in the production of four lots of wine in each of two wineries. Production lots will include four fermentations each of a single vineyard Pinot Noir and Cabernet Sauvignon (fermentation conditions to be decided at a later date and with input from each winery.) 3) Through the course of each fermentation, pull samples and analyze condensed tannins by HPLC and information on color colorimetrically. Additional analyses (acetaldehyde, sugar, SO2) will be performed at the principal investigators’ discretion. 4) Conduct tastings of finished wines with production staff to associate style preferences with chemical data.

Volatile Sulfur Compounds: Incidence and Factors Affecting Their Formation in California Wines

AVF Proj. ID: 9639 / /Cat.: ,
Primary Investigator(s): Roger B. Boulton, Linda F. Bissonby

In this grant period, we have continued the analysis of hydrogen sulfide formation among 30 different yeast strains. The scope of this work makes it very long term, but it is essential to analyze a collection of wine strains rather than one or two in order to define the factors responsible for hydrogen sulfide formation. Most importantly, we are interested in defining the genes that lead to a reduction in hydrogen sulfide formation. Preliminary data suggests that these genes are dominant, that is, will convert a high producer of H2S into a low producer in single copy. This means that the gene can be introduced into any wine strain and result in reduced production of H2S. Initial studies indicate that the levels of sulfite reductase are similar across the wine strains used in this study, consistent with recent results presented by the Australian Wine Research Institute for a much smaller set of strains.

Studies on Stuck Fermentations and on Factors that Control the Rates of

AVF Proj. ID: 9636 / /Cat.: ,
Primary Investigator(s): Robert K. Mortimerby

We have found that the factor(s) that determine whether or not a fermentation will stick are vineyard-determined. If grapes from a given vineyard are crushed and the resultant must is placed in more than one fermentation tank, if one tank sticks all tanks from the same must also will stick. Stuck fermentations occur for all types of must and also occur for both inoculated and natural fermentations. We have found that viabilities of Saccharomyces cerevisiae cells in stuck fermentations are very low and this appears to be the reason the fermentations have stopped. We have also found that dilution of a stuck wine with water or even with a dry wine followed by addition of live yeast results in a reinitiation of fermentation which continues to dryness. We have considered six models that have been proposed to explain stuck fermentations. These models are: 1) nutrient depletion, 2) excess temperature during fermentation, 3) killer yeast, 4) toxin introduced with the grapes, 5) toxin produced by another microorganism and 6) toxin produced by the wine yeast. The dilution experiments eliminate model 1 and occurrence of stuck fermentations in temperature-controlled fermenters eliminates model 2. Experiments done by others eliminate model 3. The dilution experiments and the low viabilities of the wine yeast cells point to the presence of a toxin in the stuck wine. This toxin could be a fungicide brought in with grapes (model 4) or a toxin produced by another microorganism (model 5) or by the wine yeast (model 6). We believe model 5 may occur but is exceptional. Some fermentations become infected with acetobacter and the acetic acid kills or inhibits the wine yeast. We saw no other microorganisms in the 14 fermentations we studied and volatile acidity levels were near the normal range. We considered model 4 to be unlikely because one would expect the fungicide to act from the beginning of fermentation and this does not appear to be the case. Fermentation kinetics are normal until the time of sticking. This leaves model 6, a toxin produced by the wine yeast. This model was proposed several years ago by a group in Bordeaux and the toxin was proposed to be medium chain fatty acids, octanoic, decanoic and dodecanoic acid. These compounds are extremely toxic and are produced during fermentation. Several experiments done by others seem to lend strong support to this model. The goal of the study supported by AVF was to test this specific model. We studied 4 stuck fermentations and did gas chromatographic analyses of the stuck wine. We found that the concentrations of octanoic and decanoic acid were at or below normal levels in these stuck wines. This eliminates this specific model. We now believe that there may be another toxic compound produced by wine yeast and that this kills the cells and stops the fermentation. We are also now reconsidering model 4. If the fungicide brought in with the grapes needs to be modified during the fermentation process to be toxic to wine yeast this would explain the lag in onset of the stuck state. Glenn Andrade of Sutter Home Winery (personal communication) has found a correlation between spraying of the grapes with a fungicide and the occurrence of stuck fermentations.

Studies on the Interaction of Flavor Compounds With Non-Volatile

AVF Proj. ID: 9637 / /Cat.: ,
Primary Investigator(s): Susan E. Ebelerby

During the previous year of this proposal we focused on developing sensitive gas chromatographic headspace procedures for quantifying odorant/polyphenol interactions in model solutions. We are currently using these procedures to evaluate the effects of polyphenol structure on the strength of odorant interactions. The main focus this year has been on developing and evaluating sensory procedures for measuring these interactions. Although previous studies of flavor interactions in other foods and beverages have largely focused on measuring aroma (nasal) intensity only, we wanted to also be able to quantify oral (retronasal) aroma stimulation, similar to that experienced when wine is consumed. We have used a time-intensity procedure and model solutions to show that retronasal aroma perception is significantly affected by the chemical/physical nature of the aroma compound, by the nature of the matrix, and by individual judge factors such as salivary flow rate. We feel that this procedure now provides us with a promising tool for the sensory evaluation of matrix effects. This, in combination with the analytical headspace procedures, will provide extensive information about the importance of odorant/polyphenol interactions in wine.

The Extraction of Condensed Tannins in Red Wine Fermentations

AVF Proj. ID: 9638 / /Cat.: ,
Primary Investigator(s): Andrew L. Waterhouseby

The first year of this project was designed to survey the effects of a large number of different pomace maceration practices in commercial-scale fermentations on the extraction of anthocyanins and tannins. This year of the project focused on Pinot Noir and observed variables included cold soak, manual vs. mechanical punch down, and pump over vs. punch down. The wines were prepared at a cooperating winery according to an agreed-to protocol. The phenolic composition of the musts/wines were analyzed using both spectral (at the cooperating winery) and chromatographic methods (at UC Davis and ETS), including a new Silica gel based procedure which separates phenolics based on size. Five analyses were carried out on 9 wines although the focus was on 5 Pinot Noir wines from the Widoe’s Vineyard. The major difference was that the treatments of both mechanical punch down and pump over increased both colored and uncolored tannin, while the cold soak decreased these components. The pump over regime also increased monomelic color. The goals in this project included the following: 1) Develop a method for sample preparation and an HPLC method for the determination of condensed tannins. 2) Assist in the production of four lots of wine in each of two wineries. Production lots will include four fermentations each of a single vineyard Pinot Noir and Cabernet Sauvignon (fermentation conditions to be decided at a later date and with input from each winery.) 3) Through the course of each fermentation, pull samples and analyze condensed tannins by HPLC and information on color colorimetrically. Additional analyses (acetaldehyde, sugar, SO2) will be performed at the principal investigators’ discretion. 4) Conduct tastings of finished wines with production staff to associate style preferences with chemical data.