Evaluating the Effects of Sterile Membrane and Other Filtration on the Sensory and Chemical Properties of Wine

The goal of our project is to evaluate the effects of sterile membrane and other filtration on the sensory and chemical characteristics of wine. To do this, we have filtered two red wines, a Cabernet Sauvignon and a Merlot, and one white wine blend through 0.45 µm PVDF and PES membrane filters and compared the sensory and chemical characteristics of these wines to unfiltered control wines. Treatments were expanded with the Merlot and white blend to also examine the effects of a pad filter and cartridge depth filter used as prefilters. We have examined changes in dissolved oxygen content, tannin content, and color during the course of filtration and found only minor changes. Sensory panels were trained for each of these wines and each of the treatments evaluated immediately after filtering and then on a regular basis for 9 weeks (for the Cabernet), 24 weeks (for the Merlot), and 20 weeks (for the white blend). While all three wines changed significantly over time in the bottle, very few significant differences were observed in aroma or mouthfeel between filtration treatments. In other words, our results thus far indicate limited impact of sterile filtration on the sensory or chemical properties of the wine, regardless of the type of filter material used. We did observe a small decrease in tannin and astringency with the pad filter for the Merlot wine, but we are continuing to investigate the significance of this change. In addition, we are preparing to evaluate the effects of filtration of both a red and white wine using a cross-flow filter, as well as evaluating the effects of the associated pumps, during the next three months.

Production and Management of Aroma Compounds by

The yeast Dekkera/Brettanomyces is commonly found in wines and is responsible for a wide array of characteristic odors. The primary aim of this project is to illuminate the factors that are most important in the survival and growth of Brettanomyces bruxellensis in wine and how these factors influence the production of off odors. The role of growth in wine and defined medium on the metabolic state of Brettanomyces cells was investigated. Three strains were analyzed during growth over 54 days in wine and a defined medium. Differences in the metabolic profile were determined and correlated with each variable, strain and growth medium. Ten Brettanomyces strains for the production of odor active compounds in defined medium with specific substrates added were also analyzed. These ten strains were chosen from approximately 100 Brettanomyces strains that were screened for differences in aroma production. The ten strains were selected for lower levels of odor production both in defined medium and wine. The analysis of the metabolic profile of Brettanomyces in wine and defined medium revealed some expected differences in lipid and carbon metabolism. Unexpected differences in amino acid and nitrogen compounds were also found. Fatty acids and lipids are involved in ethanol tolerance and general stress tolerance. The changes seen in fatty acid metabolism suggest the cells are altering membrane lipids in ways designed to stabilize the membranes under stress conditions. Fatty acids such as isovaleric and isobutyric acid are also involved in off-aroma production so this adaption to stress may be correlated with off-character formation. Proline and urea were uniquely increased in wine-grown cells. The increase in these nitrogen compounds could be due their use as nitrogen sources but proline may play another role as well. Plants and bacteria use proline as an osmoprotectant. It may play a similar stabilizing role in Brettanomyces in wine. An increase in metabolic compounds involved in the use of pentose sugars was also found in wine grown cells. Differences between strains were also observed. Surprisingly there was a consistent and significant trend showing that strain UCD 2091 was different from strains UCD 2082 and UCD 2807 in multiple metabolic pathways.

Metabolic Profiling of Grape and Wine Aromas

Most aroma compounds exist in grapes as glycosidically bound precursors and the aglycones (‘free’ aroma compounds) are released by enzymatic or acidic hydrolysis during crushing, fermentation and wine aging. We evaluated procedures for measuring the glycosidically bound volatiles using both acid and enzyme hydrolysis conditions followed by analysis of the free volatiles by Headspace Solid-Phase-Microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS). Acid hydrolysis of a mixture of standard compounds at pH 1 and 100º C for 1 hour released 20-60% of the bound volatiles, however, these conditions also resulted in significant degradation (>50%) of the free volatiles. Enzyme hydrolysis was generally more effective at releasing glycosidically bound compounds with minimal artifactual changes in the concentrations of the free volatiles. However, esterase and oxidase activity was still observed resulting in artifactual degradation of the free volatiles. Most previous procedures for evaluating volatile glycosides employ an initial sample preparation step using column chromatography to isolate the glycoside fraction from the grapes prior to hydrolysis and analysis of the free volatiles. We observed that different types of sample preparation columns were not equally effective at retaining the glycosides and no column-type was effective for all the glycosides in our mixture. Using optimized conditions obtained from the previous studies with model systems, we compared direct enzyme hydrolysis of Cabernet Sauvignon and Chardonnay grape homogenates with and without prior isolation of the glycoside fraction. Both procedures resulted in new ‘free’ volatiles. However, results varied depending on the type of treatment. This research is providing important insight into approaches for rapid estimation of ‘aroma potential’ of grapes.

Development of an Enology Outreach Program

There has been a chronic underinvestment in extension and outreach for the wine industry. The specific goal of this proposal is the development of a comprehensive program for extension and outreach in enology. This comprehensive program includes an interactive web site that will serve both researchers in enology and winery personnel. With the plethora of information sources currently available on the internet, searching effectively and efficiently for pertinent and accurate information has become challenging. This site will function as a means to link researchers across the United States and internationally that have an active program or interest in enology. The aim of the site is to share information within the research and production communities, facilitate collaboration, eliminate unnecessary redundancy, and foster communication on issues of regional, national or global importance. The site was renamed ‘EnologyAccess’ ( http://enologyaccess.org/ ) and launched under this name with an inaugural webinar in August of 2008. In this past year we developed the interactive tools and social networking for the site (ea2) which will be launched in the next couple of months. We are actively migrating content to this site and providing links to extension specialist and researcher websites across the nation and the world. For this site to be more useful we will need to develop a stream of income that will support the site curation. This year we launched one of the signature programs of our new extension center, Wine Flavor 101. The first courses in this series were sold out and we received a response rate on the course questionnaire of over 70%. The surveys provided a wealth of ideas and constructive criticism for the programs which will assure their continued success. As we develop solid income streams we will be able to hire content curators and add more and more content to the site and offer an expanded line of programs. Progress on meeting our goals is slow but steady.

Metabolic Basis of Differing Ethanol Yields in Saccharomyces

The goal of this study is the metabolomic analysis of ten commercial wine yeast strains that show differences in fermentation behavior and in final ethanol yield. Five of these strains tend to give lower and five higher ethanol yields during grape juice fermentation. Since non-sterile cold-settled juices were to be used to mimic wine production conditions, genetically marked strains of each commercial strain were generated. These strains were marked with a drug resistance gene that encodes resistance to kanamycin or G418. Initial experiments compared the fermentation performance of these strains to their parental strains and there were no differences noted. The kanamycin resistance gene was inserted into the HO gene which is required for yeast mating and not expressed in diploid cells thus it presents an attractive location at which to insert foreign marker DNA. The strains were grown in six replicate fermentations for the metabolomic analysis. Ethanol yields and dominance of the fermentation by the strains were monitored. One strain was not able to complete the fermentation of this juice but it was still taken for metabolomic analysis as this may reveal why it eventually arrested fermentation. Comparisons of the metabolomics data for high and low ethanol-producing strains suggests that low ethanol producers maintain active growth longer than high ethanol producers thus directing more sugar carbon towards biosynthetic intermediates and cellular components than high producers. High ethanol producing strains also showed evidence of the accumulation of degradation products of plant cell wall polysaccharides. This has not been observed before and offers the possibility that some of these components may be catabolized as well at this time which would boost yields of ethanol even further. Analysis of the metabolite pool levels of strains with intermediate ethanol production is currently underway.

Metabolic Profiling of Grape and Wine Aromas

The complex aroma of wine is derived from many sources, with grape-derived components being responsible for the varietal character. The ability to monitor grape aroma compounds would allow for better understanding of how vineyard practices and winemaking processes influence the final volatile composition of the wine. Previously we developed a procedure using GC-MS combined with solid-phase microextraction (SPME) for profiling the free volatile compounds in Cabernet Sauvignon grapes. During the current funding year we have modified this procedure in order to determine the volatile glycoside content of grapes using acid and enzyme hydrolysis conditions. The results indicate acid hydrolysis at low pH and high temperature can release from 50-80%of the total aglycone present in the sample. However, extensive degradation of many free volatile aglycones also occurs, with the effect being dependent on the compound studied and the hydrolysis conditions. This makes it difficult to use acid hydrolysis conditions to predict the ?potential? volatile composition of the grapes. This information is needed to understand the effects of viticultural practices on grape aroma composition and to be able to evaluate the effects of winemaking processes on changes in grape composition throughout the winemaking process. Further study is needed in order to minimize artifactual changes occurring from acid hydrolysis of volatile glycosides in grapes while maximizing their hydrolysis prior to analysis. As recently noted by Hayasaka et al. (2010), availability of rapid, sensitive profiling methods is important in assessing the nonvolatile fraction of grapes for a wide variety of industry applications.

Effect of wine closure oxygen permeability on volatile sulfur compounds in Pinot noir and Chardonnay wines during post-bottle aging

It is generally assumed that bottled wine is a dynamic system. A proper wine bottle closure will allow a dynamic and healthy gas exchange between the wine the air. The amount of oxygen ingression through the wine closure can directly affect wine aging and flavor development. Too much oxygen ingress can oxidize the wine during post-bottle aging, whereas too little oxygen ingress may slow flavor development. It is a concern that screw caps with too low oxygen permeability may even induce ?reduced? sulfur off-flavor due to accumulation of volatile thiols. To test this hypothesis, natural cork (up and down), synthetic, and screw caps with Saran-Tin, Saranex and low density polyethylene (LDPE) lines with various oxygen permeability were investigated on both Pinot noir and Chardonnay wines over two years storage. For Chardonnay wine, the LDPE screw cap gave the highest dissolved oxygen, lowest free SO2, and highest absorbance at 420 nm. The Saran-Tin screw cap gave the lowest dissolved oxygen. Similar trends were observed for Pinot noir wines, with Saran-Tin screw cap gave the lowest dissolved O2 and highest free SO2 and total SO2. Decreasing trend was observed for some sulfur compounds during 2 years of storage. Methanethiol had the highest decrease in wines with LDPE screw cap and synthetic closures, correlated with high oxygen permeability of these closures and low SO2 content in the wines. Similar pattern was observed for H2S. On the other hand, a decreasing trend was observed for ethylthiol acetate due to hydrolysis, and was not affected by closure types. Other sulfur compounds remained stable or decreased slightly, and there was no elevated sulfur compound for any screw cap closures after two years of storage. In addition, we did not detect any significant amount of dimethyl disulfide and dimethyl trisulfide in any of the experimental wines. Expert sensory panel did not pick up sulfur off-flavor in the experimental wines, and trained sensory panelist did not pick up any aroma difference between the Saran-Tin and LDPE screw capped wines. These results suggest the original wine status was probably more important, and the data does not support the hypothesis that dimethyl disulfide could converted to methanethiol under low oxygen conditions.

Development of an Enology Outreach Program

There has been a chronic underinvestment in extension and outreach for the wine industry. The specific goal of this proposal is the development of a comprehensive web site that will serve both researchers in enology and winery personnel. With the plethora of information sources currently available on the internet, searching effectively and efficiently for pertinent and accurate information has become challenging. This site will function as a means to link researchers across the United States and internationally that have an active program or interest in enology. The aim of the site is to share information within the research and production communities, facilitate collaboration, eliminate unnecessary redundancy, and foster communication on issues of regional, national or global importance. The site was renamed “Enology Access” and launched under this name with an inaugural webinar in August of 2008. This current year focused on analysis of site software packages with the goal of making sure that the interactive types of programming that we are planning could be supported by the software. Joomla was found to have the best capabilities including being able to interact with other types of programming. Videos, text content and an interactive comprehensive glossary have been uploaded to the site. Other materials are under development and will be uploaded soon. Researchers and institutions are being invited to register on the site and many have already done so. Webinars and live seminar broadcasting has been successfully implemented via the Adobe Connect software maintained on campus. Thus the site has been successfully launched and content is continuing to be developed. In the next year we will develop more interactive educational capabilities.

Oxidation of Wine: Control for Quality

Very good progress has been made in understanding the Fenton reaction. Data collected in Denmark in collaboration with Skibsted revealed the identity of major free radical in wine, demonstrating that the Fenton reaction is important and the role of sulfite in slowing this reaction. Studies of additives to prevent oxidation are currently underway. This research has stimulated many other efforts in wine oxidation around the globe.

Development of an Enology Outreach Program

There has been a chronic underinvestment in extension and outreach for the wine
industry. The specific goal of this proposal is the development of a comprehensive web
site that will serve both researchers in enology and winery personnel. With the plethora
of information sources currently available on the internet, searching effectively and
efficiently for pertinent and accurate information has become challenging. This site will
function as a means to link researchers across the United States and internationally that
have an active program or interest in enology. The aim of the site is to share information
within the research and production communities, facilitate collaboration, eliminate
unnecessary redundancy, and foster communication on issues of regional, national or
global importance. In the current funding year an Implementation and Advisory
Committee for the Enology Extension Online web site was formed. The committee
consists of: Dr. James Harbertson (Washington State University), Dr. James Osborne
(Oregon State University), Dr. Brent Trela (Texas Tech University), Dr. Trevor Phister
(North Carolina State University) and Dr. Bruce Zoecklein (Virginia Tech). Dr. Bisson is
chairing the group and the committee is assisted by Kay Bogart. The Implementation and
Advisory Committee has designed the scope of the site, which is described in detail in the
request for continuing funding. In addition the group considered the best mechanisms for
creation and maintenance of the site. The site has been named ?Enology Extension
Online? and will be launched in April 2008.