Development of Conjugal Systems for Gene Transfer and Allelic Exchange in

AVF Proj. ID: 9817 / /Cat.: ,
Primary Investigator(s): Dr. David A. Millsby

Oenococcus oeni (formerly Leuconostoc oenos) is a common starter culture used to achieve the malolactic fermentation (MLF) in commercial wine production. To date, basic research on O. oeni, especially molecular analysis of genes involved in growth on grape juice, has been hampered by the lack of efficient gene transfer and allelic exchange systems. One potential mechanism for genetic manipulation of starter cultures is the use of conjugal systems indigenous to lactic acid bacteria (LAB). Previous work on the conjugal element pRSOl from the dairy starter Lactococcus lactis identified several regions involved in conjugative transfer and localized the conjugative origin of transfer (oriT). In an effort to develop a generally applicable system for gene transfer, we examined the host range of pRSOl, and several oriT-containing plasmid derivatives, with a range of LAB recipients. Conjugal transfer was demonstrated into Leuconostoc mesenteriodes subsp. cremoris, Enterococcus faecalis, Streptococcus thermophilus, Pediococcus cerevisiae and 0. oeni. Broad host range transfer of pRSOl and mobilization of oriT-containing plasmid derivatives provides a novel means for molecular analysis of a variety of LAB, such as O. oeni, that are resistant to current methods of genetic manipulation. Current work is focused on the development of the appropriate mobilization and allelic exchange vectors for genetic analysis of O. oeni.

Factors Affecting Sugar Utilization and Rate of Fermentation During Vinification

AVF Proj. ID: 9722 / /Cat.: ,
Primary Investigator(s): Linda Bissonby

The HXT Display technique has been successfully applied to cells isolated from white grape juice, and a preliminary analysis of the HXT genes expressed during grape juice fermentation has been completed. Of the 14 HXT genes that we were able to evaluate, 11 were found to be expressed in grape juice. This technique did not allow quantitation of the relative abundance of the messages for the expressed HXT genes, so we are developing an alternative method that will allow quantitation, based upon the unique sequences identified in the untranslated regions of the messages identified in the original HXT display analysis. The four genes which were not amplified in this analysis are being cloned in order to characterize the 3′ untranslated regions. The pathway for the internalization and turnover of the HXT2 protein has been evaluated. It was originally thought that if this pathway could be blocked, fermentations would not arrest prematurely. This does not appear to be a viable option for the elimination of fermentation problems. Cells not turning over Hxt2p appear to be sluggish in conducting the fermentation from the beginning. Further, blocking the pathway for internalization blocks internalization of other factors as well and seems to make the cells more sensitive to ethanol. Finally, the role of fatty acids in ethanol tolerance and stuck and sluggish fermentations has been evaluated. The requirement for unsaturated fatty acids has been demonstrated under enological conditions. Saturated fatty acids offer little benefit to the organisms. Fatty acid limitation appears to affect consumption of fructose more severely than that of glucose for reasons that are as yet obscure. This may be related to the type of HXT genes that are expressed upon fatty acid limitation. Surprisingly, fatty acid limited cultures maintained viability while stuck as long as sufficient glucose was available.

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.

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.

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.

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.

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.

Factors Affecting Sugar Utilization and Rate of Fermentation

AVF Proj. ID: 9515 / /Cat.: ,
Primary Investigator(s): Linda F. Bissonby

Protracted or stuck fermentations are a recurring problem in the wine industry. Nutritional supplementation often stimulates fermentation and can reduce the incidence of problem fermentations. Fermentation rate decreases as a consequence of a drop in sugar transport capacity. Sugar transporters are the key site of control of flux through glycolysis. The goal of this research program is to determine which glucose transporters are expressed during grape juice fermentation and to define the mechanisms of regulation of transporter activity. In addition, we are also investigating nutritional and environmental factors that affect fermentation rate. Last year we discovered that a suboptimum potassium to proton ratio results in a sluggish fermentation. Potassium is know to be stimulatory to fermentation rate and it has been postulated that this is a cell surface phenomenon, that is, that potassium directly stimulates glucose transport. Given that some rootstocks now used commercially in California result in low petiole potassium, we proposed investigating the effect of the potassium to proton ratio in natural juices on fermentation rate using commercial yeast strains. We also explored the effect of other ions on fermentation rate. We discovered that the potassium effect is not mediated at the level of the cell surface, and that once the fermentation is stuck, addition of potassium is unable to re-initiate fermentation. Re-inoculation also does not re-initiate fermentation even with the addition of potassium.

Factors Affecting the Formation of Hydrogen Sulfide and Acetic Acid

AVF Proj. ID: 9516 / /Cat.: ,
Primary Investigator(s): Roger B. Boulton, Christian Butzkeby

The formation of hydrogen sulfide during fermentation was followed by direct headspace sampling and quantification by gas chromatography with flame photometric detection. The formation of acetic acid was followed in the same fermentations but by sampling and capillary electrophoresis. Juice samples were analyzed for individual amino acids and correlations between these values, calculated free amino nitrogen (FAN) content and the formation of acetic acid and hydrogen sulfide were performed. The formation of acetic acid was very low (mean-0.09 mg/L, sd = 0.10 mg/L.) and could not be correlated with any of the compositional factors. The formation of hydrogen sulfide was variable (mean = 37 ug/L of juice, sd = 64 ug/L), ranging from none to 300 ug/L. The effect of yeast strain showed a wide variation in a single juice (mean = 226 ug/L, sd = 128 ug/L) with the cultures of Fermevin, Prisse du Mousse, Premier Cuvee being below the mean and those of Montrachet, Pasteur Champagne, Enoferm ICV-D46 and Lavin 7IB being above the mean. In the temperature series, the production of hydrogen sulfide increased from 68 ug/L at 15°C to 164 ug/L at 30°C and then fell to 56 ug/L at 35°C. Statistical analysis (principal component analysis) found the formation of sulfide during fermentation to be positively correlated (CC=+0.56) with the FAN content and (CC=+0.61) with the total nitrogen content. With respect to individual amino acids, alanine (CC=+0.72), glutamine (CC=+0.71), gamma-amino butyric acid (CC+0.68), glycine (CC=+0.66) and methionine (CC=+0.63) were the most strongly correlated with sulfide formation. These results with California juices are contrary to the widely-held belief that sulfide production is caused by low levels of free amino nitrogen.

Factors Affecting Sugar Utilization and Fermentation Rates

AVF Proj. ID: 9416 / /Cat.: ,
Primary Investigator(s): Linda F. Bissonby

In this grant year, the investigation of the genetic and environmental factors that influence fermentation rate was continued. A more detailed analysis of the effect of limitation of individual amino acids was conducted. This analysis revealed that yeast cells are particularly sensitive to lysine limitation, moderately sensitive to adenine and leucine levels, but fairly insensitive to tryptophan, histidine and uracil concentrations. The levels of an amino acid that support maximal growth rate seem to be lower than the concentrations required for maximal fermentation rate, for some but not all nitrogen sources. In addition to analysis of nitrogen levels and reduced fermentation rate, we also discovered that an imbalance of potassium to hydrogen ion concentration would lead to a stuck fermentation. Maximum fermentation rate is equivalent in low pH juices and in juice-like synthetic media of differing potassium ion concentrations. However, in low potassium juices stuck fermentations develop. Sugar transporters, the HXT proteins, are degraded under conditions leading to arrest of fermentation. Analysis of degradation of one of these transporters, the HXT2 protein, revealed dramatic instability. This protein is degraded rapidly upon cessation of growth and upon nutrient limitation. There are now 14 HXT genes known in Saccharomyces. Not all of these transporters is expressed during grape juice fermentation. We are still in the process of determining which ones are expressed under these conditions.