Studies on the Interaction of Flavor Compounds with Non-Volatile Components of Wine

The overall goal of this research is to understand how polyphenols, important nonvolatile constituents of grapes and wines, influence the volatility, intensity, and release of flavor aroma compounds in wines. Preliminary studies suggest that polyphenolics interact with flavor compounds in model solutions. In initial studies, we have shown that the structure of the polyphenol as well as the structure of the odorant significantly affect the interaction. The information obtained from this work will lead to new insights into the way in which polyphenol interactions influence wine aroma and taste as well as wine color, haze formation, and astringency perception.

The Extraction of Condensed Tannins in Red Wine Production

The winemaking portion of the experiment was completed on time on the commercial scale and on the small scale at UCD. Samples were collected and prepared for analysis. The various chemical analyses are still underway due to the large number of samples and the demand for analysis equipment by other projects. In summary, approximately 300 samples have been analyzed to date, all for phenolics by normal and reverse phase HPLC, as well as spectral measures of co-pigmentation. These have included all samples from the commercial wines, but not those from the small scale analyses. Since each of those procedures yields many results, the challenge now is to try an interpret the data to the point where it can be summarized in simple and understandable terms. Data collected so far shows some expected trends, but also some unexpected ones, in particular the increase in tannin levels during aging. With the large amount of data collected, additional analysis is needed to look for interactions and consistency. Additional data still must be collected and analyzed on the small scale wines. This data will be used to plan for the third year’s experiments during summer 1999.

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

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.

Adaptation of a Simple Assay to Measure Tannins in Grapes and Wines

Premium red wine production is dependent upon proper extraction of pigments and tannins from grape berry skins and seeds during winemaking. While many methods are available to estimate the level of polymeric phenols in grapes and wines, none of the simple ones give an independent estimate of the amount of tannin present. Our aim was to provide a simple tannin assay that would be suitable for analysis of grape berries during maturation as well as for monitoring tannin extraction during fermentation and extended maceration. We have taken a tannin assay that was originally developed for measuring tannins in persimmon and adapted it so that it can be used for measuring tannins in grapes and wines. We studied many of the compounds in wine that could potentially interfere with this analysis and have found that while some can indeed inhibit the assay at very high concentrations, they do not interfere at the levels found in wines. Thus, the plate binding assay has been successfully adapted. In the course of working with the plate binding assay we realized that with some simple modifications we might be able to develop a much simpler solution assay that would not require a specialized piece of equipment like a microtiter plate reader. Although our initial research objectives were only related to adapting the plate binding assay, we were able to quickly demonstrate the feasibility of a solution assay, and we have now successfully developed a new assay method for tannins. The solution assay is simpler than the plate binding method we originally set out to adapt for grapes and wine. It is also much less variable than the plate-binding assay and is useful over a much wider range. Because it requires only a spectrophotometer and a small table-top centrifuge rather than a specialized piece of equipment like a microtiter-plate reader, this analysis will be suitable for use in many winery laboratories. One of our primary objectives in this project was to provide a simple tannin assay. The new analysis that we have developed meets that objective better than the plate binding assay ever could have.

Alternatives to Bentonite

MALDI-TOF mass spectrometry has been shown to be an excellent method for the direct analysis of the composition of wine haze. Previously developed methods allow a subtractive evaluation of wine components, assay of the wine pre and post haze treatment, but are not useful for the analysis of the haze itself. MALDI-TOF in contrast to SDS-PAGE is not specific for protein and can be used to simultaneously assess the presence of non-proteinaceous material in haze. It also allows accurate investigation of low molecular weight components, poorly resolved by other techniques. The MALDI-TOF analysis has demonstrated that the two commonly used methods to induce haze in wine as a means of assessing protein stability, the heat/chill and ethanol assays, destabilize specific components within the wine, but do not yield hazes of an identical composition to each other or to hazes forming naturally in wine under commercial conditions. Phenolic compounds were found to play a role in haze formation in wines made from Muscat of Alexandria but not in wines made from Sauvignon blanc. More wines from each varietal need to be evaluated before more conclusive statements can be made. It appears that the difference is due either to the greater level of phenolic compounds in Muscat or to differences in the peptide fractions between the two varietals.

Development of a Spectrophotometry Assay for Arginine, a Precursor to Ethyl

Ethyl carbamate is a natural fermentation byproduct but a potential carcinogen at very high doses. The major precursor to ethyl carbamate in wine is arginine, a natural amino acid in grapes. In order to minimize ethyl carbamate levels in wine, we developed a simple, rapid method for determining the arginine concentration in grape juice, that is suitable for use at a commercial winery. The new spectrophotometry assay for arginine utilizes techniques adapted from ion exchange chromatography. In our method development, we combined the application of a selective strong cation exchange resin with the NOPA assay that we developed in the previous AVF project “Rapid Analysis for Yeast Available Nitrogen Compounds in Must”. We have been able to demonstrate the isolation and quantification of arginine from various model and natural juices. The accuracy and reliability of our assay was evaluated by correlation to a HPLC reference method, and by measuring the recovery of arginine in spiked model and natural juices. The assay will be tested on commercial musts during the 1998 crush, and an analytical procedure will be made available to the California wine industry as soon as our method validation is complete. Recommendations on critical arginine levels in juices have already been made in our “Ethyl Carbamate Preventative Action Manual”.

Effects of Brettanomyces in Winemaking: Chemical, Microbiological and Sensoriale

Historically, winemakers have viewed Brettanomyces sp. as producing spoilage in wines. Despite traditionally negative connotations surrounding the yeast, some are now questioning whether or not subtle “Brett” character, in some cases, may play a positive role in flavor and bouquet development. In addition to potentially contributing to complexity, limited Brettanomyces activity may play a role in accelerated aging in young red wines. Of the nine species of Brettanomyces isolated from wine and juice, B. intermedius is the most frequently identified. This suggests the existence of several, to many, strains that may be involved in the winemaking process. Prior to this study, a comprehensive evaluation of inter- and intrastrain variability has not been undertaken. The objective of this two-year project is to study inter- and intrastrain differences between ten genetically characterized strains of the single species, Brettanomyces intermedius, in aging red wine (Pinot Noir). The project includes comparison of population dynamics as well as chemical and sensorial changes brought about during the growth phase of the respective strains. The goals of the first year were to characterize population changes often strains of B. intermedius in Pinot Noir wine incubated at 20°C and begin GC/MS identification and quantification of primary metabolites. Presently, we are in week 45 of the growth studies inoculated at, <10 CFU/ml. We have observed that three of the ten strains reached maximum cell density at 41 days post-inoculation. Five strains took 60 to 140 days to complete their growth cycle. Final population densities, in these cases, paralleled those of the early group. Two of the ten strains grow very slowly. At present (day 195), both are approaching stationary phase. Depending upon strain and replication, cell density ranges from 28,000 to 50,000 CFU/ml. CG/MS analysis of weekly samples is approximately 60%complete. In every case, 4-ethyl phenol was not detected until cell density reached relatively high numbers. These preliminary results appear to contradict the belief that 4-ethyl phenol can be used as a monitoring tool for population density estimates in the earliest stages of growth. Work also continues on monitoring ieto-glucosidase activity in inoculated wines. Initial results suggest potentially significant enzyme activity in model systems. Follow-up assays are currently being carried out in experimental lots. Upon completion of the growth cycles, each lot was cold-clarified, bottled and stored for upcoming sensory examination. Year two objectives: At four, and nine months post-bottling, each lot will be examined sensorially for differences. Initial sensory comparison of each lot will be carried out using Duo-Trio paired comparison tests. Where differences are noted, descriptive analysis will be performed using a twelve member trained panel.

Factors Affecting Sugar Utilization and Rate of Fermentation During Vinification

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.

Identification of Yeast Strain Genetic Factors in the Formation of Volatile Sulfur

To date we have confirmed the high degree of variation in H2S formation among commercial yeast strains and natural isolates. In general, all display an increase in H2S formation upon nitrogen and pantothenate limitation, but the magnitude of the effect varies widely among the strains. Some are far more sensitive to limiting nutrients than are others. The degree of response is not well-correlated with the intrinsic sulfite reduction activity as determined from color on BiGGY agar suggesting that factors other than the activity of this pathway are critical in hydrogen sulfide production. Glutathione addition to the medium did not appear to impact H2S formation. Internal glutathione levels were manipulated using two metabolic inhibitors, which seemed to have an effect on lag phase but not total amount of H2S formed once cells had adapted to the inhibitor. This adaptation would be prevented in the presence of both inhibitors, which is currently being investigated. This work has provided an important foundation for comparison of the protein and gene expression profiles of strains grown under conditions of low and high hydrogen sulfide formation.

Measurement of Volatile Aldehydes in Grapes, Wines, and Musts

Volatile, saturated aldehydes (C1-C9) in wine or aqueous ethanol solutions were derivatized with cysteamine to form the corresponding thiazolidine derivative. Following extraction into chloroform and gas chromatographic analysis, a limit of quantitation of 1 mg/mL was achieved using a nitrogen phosphorous detector. Spiked recoveries in wine were 112% with an overall coefficient of variation of 16%. The derivatization procedure was used to determine aldehyde levels in several wines: acetaldehyde was observed in the highest concentrations in these wines, however, other aldehydes were also often present. During the current year the focus has been on decreasing the variability of the analytical method.