A determination of the role of fruit maturity in the sensory properties of

This project attempts to clarify the consequences for wine flavor that result from harvesting fruit at different maturities. It aims to do a complete study (from a sensory and chemical analyses point of view) from the vineyard to the resultant wines. During the current funding period we did the descriptive sensory analysis of the 2006 wines, did chemical analyses of the 2006 wines and had a winemaker panel evaluate the quality of a subset of the 2006 wines. We harvested the 2007 grapes from a single vineyard at six time-points over a fourteen week period (August 23 to November 28, 2007). The samples spanned maturity levels from what would be considered early/normal harvest (average Brix=21) to late harvest (average Brix=30.8). The fresh grapes were evaluated by a trained descriptive sensory panel and by Napa Valley winemakers. The trained panel found subtle but consistent differences among grapes harvested at different times and these results were very similar to those found in 2006. Specifically, lower Brix grapes were more sour in skin and pulp, had more vegetative skins and had greener, more bitter seeds, whereas higher Brix grapes were sweeter, more fruity, more squishy with browner and nuttier seeds and more raisin-flavored skins. The Napa winemakers each used their own method to determine whether the sample grapes were ready to be harvested and in 2006 as well as 2007 they all agreed that grapes from harvest 1 through 3 were not ready to be picked. However, for harvests 4 through 6 there were some differing clusters of winemakers. Additionally, grape berry samples were prepared and frozen for chemical analyses. There were differences between the 2006 and 2007 grapes in the concentration of tannins, anthocyanins and 2-methoxy-3isobutyl pyrazine concentrations. The 2007 wines were made and will be evaluated by a trained sensory descriptive panel (in April) and Napa Valley winemakers (in June). The descriptive analysis of the 2006 wines showed that wines made from grapes with higher initial sugar content were higher in dark fruit, dry fruit and spicy flavors and lower in sourness as well as lower in fresh and cooked vegetative flavors. Additionally, for higher initial sugar concentration musts adding water prior to fermentation had a larger effect on the wine sensory attributes than adding water after fermentation. The winemakers thought that wines made with grapes harvested between 24.7 and 26 Brix were of better quality, and would receive the highest retail price. Wine chemical analyses of 2006 and 2007 wines are on-going.

Characterization of ethyl phenols and other volatile components in Brettanomyces bruxellensis off-flavor occurrence

Brettanomyces bruxellensis contributes flavors to wine which are described as Band-aid,
burnt plastic, smoky, spicy, leathery, horse sweat, and barnyard. While winemakers
debate whether the ‘Brett’ aroma should always be considered an off-flavor, there is a
consensus reflected in the AVF funding priorities that an improved understanding of
Brettanomyces will assist the winemaking community. Building on previous work
performed at Cornell, we had proposed several microbiological and chemical studies for
the 2007-08 research cycles. Specifically, our objectives were to investigate B.
bruxellensis volatile phenol production in relation to the precursor concentration and to
the yeast growth phase. Five strains of B. bruxellensis strain were studied in model wine
with variable levels of hydroxycinnamic acid (HCA) precursors. For all strains, we
observed similar growth kinetics for concentrations up to 5 mg/L of the HCA precursors.
At 10 mg/L of HCA precursors, yeast growth was slowed during the exponential phase,
but the final yeast concentration was unchanged. Volatile phenol production was highly
correlated to HCA level for all initial precursor concentrations. The conversion
efficiency of HCA precursors (1-10 mg/L) to volatile phenols was greater than 50% in
most cases, with the exception of 4-EP production by B. bruxellensis strain 2091 (<30%).
4-EP production was more differentiated among strains and started slightly later than 4-
EG. In summary, this work has demonstrated that early detection of most strains of B.
bruxellensis before the midpoint of the exponential growth phase can allow for
intervention prior to production of volatile phenols, that at least one strain is capable of
producing detectable levels of phenols much earlier in growth, and that potential volatile
phenol production is well predicted by precursor concentration.

Oxidation of wine, Control for Quality

There was extensive progress towards unraveling the key reactions in the oxidation of ethanol with hydrogen peroxide. Iron and its oxidation state are proving to be critical to the reaction. Paradoxically, the addition of oxygen to a simple Fenton reaction nearly stops the oxidation of ethanol. When sulfites are present, phenolics seem to enhance the oxidation, also unexpected because phenolics act as antioxidants on their own. The new understanding of oxidation revealed here is beginning to suggest means to better control oxidation and the next few months of this project should give some more direct guidance.

Evaluation of a Quality Control Tool for Predicting the Distribution of 2,4,6 -Trichloroanisole (TCA) in Cork Populations and Bottled Wines

2,4,6-trichloroanisole (TCA) transferred from natural cork closures to bottled wine was studied. The corks used in the study were all imported into California from Portugal. Ten different bales, provided by seven different cork suppliers, previously studied for their Releasable TCA (RTCA) in part one of same study, were used on a commercial bottling line with a California Chardonnay. After nine months of storage under normal commercial conditions, 100 bottles from each of the ten cork bales were opened and wine from each bottle analyzed for TCA. Results were presented in part two of same study. After twenty months, 60 bottles from each of the ten cork bales were opened and wine from each bottle analyzed for TCA. For each cork bale, the occurrence of bottles

A Determination of the Role of Fruit Maturity in the Sensory Properties of Paso Robles Cabernet Suavignon Wines

This project attempts to clarify the consequences for wine flavor that result from harvesting fruit at different maturities. It aims to do a complete study (from a sensory and chemical analyses point of view) from the vineyard to the resultant wines. During the 2006 harvest we harvested from a single vineyard at six time-points over a ten week period (September-November 2006). The samples spanned maturity levels from what would be considered early/normal harvest (average Brix=22) to late harvest (average Brix=30). The grapes were evaluated by a trained descriptive sensory panel and by Napa Valley winemakers. The trained panel found subtle but consistent differences among grapes harvested at different times. The Napa winemakers each used their own method to determine whether the sample grapes were ready to be harvested and that over the period of observation the grapes became more likely to be ready to pick. Additionally, grape berry samples were prepared and frozen for chemical analyses. The wines were made and will be evaluated by a trained sensory descriptive panel (in April) and Napa Valley winemakers (in June). Wine chemical analyses are on-going.

Construction of Low H2S Producing Strains of Wine Yeast

The aim of this one year project was to evaluate the impact of altered alleles derived from the low-sulfide producing strain UCD932 for their potential to reduce sulfide formation in high-sulfide producers. UCD932 is a native wine isolate previously shown to not produce sulfide under any enological circumstances evaluated. However, it is not as strong of a fermentor as other strains and has limited commercial applicability. The sequences of the genes of the sulfate reduction pathway were analyzed in UCD932 and genetic crosses were used to define those genes associated with the low sulfide production trait. In most crosses only one gene, subsequently identified as MET10UCD932 seemed to be required to reduce sulfide production while in other crosses it seemed that more than one gene was required. UCD932 contains mutations in CYS4HOM6MET5and MET6 in addition to the mutation in MET10. Allele swap technology, the replacement of one form of a gene by a natural variant from another strain, was used to evaluate the role of these genes in sulfide production in UCD522. UCD522 is a well known high sulfide producer. Replacement of MET10UCD522 with MET10UCD932eliminated H2S production by UCD522. Replacement of other alleles appeared to have no effect. Similarly, replacement of the MET10 genes of two native high sulfide producers, UCD940 and UCD950, with MET10UCD932also eliminated sulfide production in those two genetic backgrounds in both synthetic and actual grape juice. Thus, allele swap of the existing MET10 gene of a commercial or native strain with the MET10 gene from UCD932 is an effective strategy to reduce sulfide production. Further tests demonstrated that this allele did not affect other enological properties of the strains such as sulfite tolerance, fermentation rate or progression, or aroma profile with the exception of the loss of sulfides. A patent application has been submitted for the use of the MET10UCD932 allele to reduce sulfide formation. In one other high sulfide producing strain, UCD939, replacement of the existing MET10 gene with MET10UCD932 appeared to be a lethal event. Thus there are likely combinations of genes of this pathway that do not permit cell growth or that are toxic. An important goal for this grant was the analysis of the potential impact on strain competitiveness due to the change in allele at MET10. To test this, mixtures of equal concentrations of modified and unmodified UCD522 were inoculated into synthetic and actual juices. The two strains can be easily distinguished on BiGGY agar as one, MET10UCD932 gives white colonies on this medium and the other, MET10UCD522, gives tan colonies. At the end of fermentation, the relative percentage of the strain carrying MET10UCD932 had decreased from 50%to 20-25%indicating that there is an advantage in the wild to hydrogen sulfide formation. This is not surprising and explains why so many strains have evolved to produce sulfide. It also suggests that while the percentage of the allele swap strain decreased, it is still competitive and would likely dominate a fermentation when used following normal inoculation practices.

Detection, Enumeration, Viability and Vitality of Srains of Lactobacillus and Pediococcus in Wine

The bacterial genera Lactobacillus and Pediococcus are regarded as spoilage organisms in winemaking. Changes in winemaking practices have led to increased incidence of wine spoilage post fermentation by these bacteria. The recent trend to longer hang time for flavor development in grapes, coupled with higher juice pH values and reduced concentrations of sulfur dioxide, have exposed wine to increased spoilage by these bacteria. Our program has focused on raising specific antibodies to these bacteria, attaching a fluorescent dye to the antibody and identifying these bacteria from other material in the cell. We have made significant progress in purifying the antibodies to reduce cross reactions with other bacteria.

We have, however, encountered some unexpected problems with regard to both progress of achieving the research objectives and project personnel. The adsorption process reported last year to reduce cross reactions has proved fairly satisfactory, but we have observed batch to batch variations, i.e. different antisera bleeds from different chickens respond differently to the adsorption process. This has slowed progress as the process has to be refined each time to meet the changes in the antisera. The eggs that were a potentially rich source of antibodies were lost when the cold box deep freeze could not withstand the 113oF temperatures Fresno endured last summer with the result that the eggs spoiled. A graduate student, Tiffany Otto, had been using re-constituted juice concentrate and observed some discrepancies in antibody attachment. It has been hypothesized that this interference could be caused by polymerized phenolics adhering to the cell surface and interfering with the antibody-antigen binding process. This was unexpected since the problem had not been encountered when using an Oenococcus oeni specific antibody in Chardonnay fermentations. This problem is being addressed using fresh juice.

Identification of Factors that Influence the Level of Large and Small Polymeric Pigments in Grapes and Wines

Our earlier estimates of how much of the fruit tannin could be bound by the insoluble matrix of the berry were made using an analysis conducted in wine with alcohol present. Since we subsequently found that alcohol has the effect of reducing the tannin binding by berry cell walls, our previous work underestimated the magnitude of this phenomenon. The most important research accomplishment from work during the 2005 season was the successful development of a method to measure the capacity of grape berry cell walls to bind tannin in the absence of ethanol. A search for a suitable commercial tannin preparation that could be used for such an assay was ultimately unsuccessful, and we were required to purify tannin from grape seeds and skins for use in the analysis. The total binding ability of grape berry cell walls is the product of the binding capacity per mg of cell wall material and the total amount of cell wall present (mg/berry). We measured both of these parameters in Cabernet Sauvignon fruit grown at Davis during normal ripening and during extended hang time. The results indicate that the amount of cell wall material in the skins and pulp of the berry do not change dramatically during normal ripening and extended hang time, but that the tannin binding capacity of mesocarp cell walls increase during ripening and show a decline during extended hang time. The reduced tannin binding by cell walls late in the ripening period may favor better tannin extraction from more mature fruit. Further targeted work would be needed to characterize this phenomenon.

We also studied the effect of drying on the mass of cell wall material and its tannin binding capacity in Cabernet Sauvignon fruit severed from the vine and hung in the canopy. Brix increased dramatically after cutting the clusters from the vine concomitant with a drastic loss in fresh weight of the berries. Despite these radical changes in solute concentration and water content, the mass of cell wall material and its tannin binding capacity appeared to change very little and remained similar to control fruit left on the plant for extended ripening.

The importance of the phenomenon we are addressing in this project can be easily seen when we compare the magnitude of the total cell wall binding capacity (skin + mesocarp) with the amount of tannin we find in grape berries. At the point of normal harvest in this experiment the total tannin binding capacity of the fruit was calculated to be 0.98 mg/berry. Data for Syrah fruit typically show about 2 mg of tannin in the fruit whereas Pinot noir is usually slightly lower at 1.6 mg/berry and Cabernet Sauvignon is usually higher around 2.6 mg/berry. Of course this number varies with the average seed number per berry, so these values are taken from our previous work simply to illustrate the fact that the total binding capacity of the cell wall material in a berry can be well over half (61%) of the amount of tannin found in the fruit for a variety like Pinot noir. Taken together this work suggests that grape berry cell walls may play an important role in tannin extraction from fruit during fermentationgrape berry cell walls may play an important role in tannin extraction from fruit during fermentation

Determination of the Basis of Low H2S Production in Saccharomyces cerevisiae

The completion of the sequence of the genome of the yeast Saccharomyces allowed identification of every gene in this organism. Mutations of each gene have been constructed and the entire set of mutations is available for analysis. The aim of this project is to identify every mutation that results in sulfide formation in wine yeast and those that suppress it. If the mutations suppressing sulfide formation could be identified, the altered alleles of the genes involved could be transferred to other genetic backgrounds resulting in commercial strains with reduced ability to produce hydrogen sulfide during wine production. The goal of this study was to identify genes leading to reduced sulfide formation and to confirm the role of those genes in sulfide formation in genetic crosses.

This study has focused on strain UCD932. This strain is a low sulfide producer under all conditions evaluated. We discovered that this strain carries two independent mutations affecting sulfide production. One leads to reduced production of sulfide and the other to increased consumption of reduced sulfide. The additive effect of these genes is to keep sulfide levels low in the cell. Over-expression of these modified alleles did not result in sulfide reduction in high sulfide-producing strains indicating that these altered alleles are not dominant in the presence of a wild type copy of the gene. Genetic crossing against high sulfide producers indicated that both genes could reduce sulfide formation to some extent with the greatest reduction occurring when both were present simultaneously. Thus this project has been successful and the goals of the research have been met. The next phase of this work will be the use of these altered alleles in the genetic modification of commercial strains. This will allow us to develop strains with reduced sulfide formation potential to meet the overall aim of my laboratory ?which is the elimination of sulfide production by yeast as a cause of hydrogen sulfide during wine production.

Metabolism of Dekkera/Brettanomyces Isolated from Wine

We tentatively have identified potential aroma producing compounds that are made by the five different Brettanomyces isolates without the specific substrate added. These results can be interpreted as the production of compounds that are not affected by substrate availability. Most of the isolates produced the fruity compound, ethyl acetate, without additional substrate but only strain 2058 produced 2-methyl-butanoic acid, described as sweaty. Strain 2082 was the only one to produce the isoamyl alcohol (cleaning fluid) and isoamyl acetate (banana) compounds with no additional substrate added. Strain 2091 was the only one to produce ethyl laurate, a soapy compound, in the defined medium alone.

Adding the different substrates allows us to define some compounds that are affected by substrate as well as a group that are specific to a combination of substrate and Brettanomyces strain. For example, 4-ethylphenol and 4-vinylphenol production are characteristic of the coumaric acid substrate, while isovaleric acid was characteristic of tryptophan addition. Some compounds were produced by specific isolates only in the presence of a certain substrate. For instance strain 2091 only produced 2-methylbutanoicacid in the presence of phenylalanine.

Addition of amino acids to wines inoculated with Brettanomyces appears to have little or no affect on the growth of the yeast. We were able to determine numbers of cells by QPCR even though the cells of strain 2091 did not grow on plates. This may indicate that the cells were in a viable but not culturable state or that the QPCR detected cells that were not living. If we are able to detect products of metabolism in the wines where we could not plate the Brettanomyces it could mean that the cells are still capable of metabolism even if they cannot grow on the plating medium.