Red Wine Tannin Interaction with Polysaccharides

The objectives of this proposal are to do the following:

  1. Investigate polysaccharide interaction with tannins in wine.
  2. Determine how intermolecular interactions between macromolecules in wine influence wine mouthfeel.

Following the first two years of activities, excellent progress has been made. Ten wineries were recruited and ten wines were provided with all different winemaking processes. Laboratory activities have made significant progress, with polysaccharides and tannins extraction from wine and chemical characterization. Interactions between tannins from red wines and polysaccharides have been characterized by dynamic light scattering. The effect of pH and ethanol content on the interactions between tannin and polysaccharide has been investigated.

Impact of Pre-Fermentation Cold Soak Conditions on Microbial Populations and Consequences for Wine Aroma

Wine aroma is one of the most important components of wine quality and can be impacted by grape variety, viticultural practices, and winemaking procedures. One particular practice that is employed during Pinot noir production to impact wine aroma is cold soaking. In this process grapes are held at cold temperatures to prevent growth of Saccharomyces cerevisiae and delay the beginning of alcoholic fermentation. Recent research has demonstrated that yeast naturally present during the cold soak can impact wine aroma and flavor (Hall et al. 2017). This research builds off these results and investigates how cold soak conditions could be manipulated to encourage or discourage growth of certain yeast and the consequences for wine aroma. Specifically, ways a winemaker may manage a cold soak (temperature, SO2, yeast diversity) were investigated for their impact on yeast populations and production of volatile aromas. Pinot noir wines were produced where the grapes were cold soaked for six days at two different temperatures (6 or 10?C) with the addition of 0, 50, or 100 mg/L SO2. Six non-Saccharomyces yeast species commonly isolated from grapes were inoculated and their populations monitored throughout the cold soak. Wine was also produced from grapes that did not undergo cold soak. Temperature and SO2 concentration impacted the growth of non-Saccharomyces yeast during the six day cold soak in a species specific manner. The highest populations observed were in the cold soak at 10?C when no SO2 addition had been made. Here H. uvarum increasing in population from approx. 103 cfu/mL to almost 108 cfu/mL by the end of the cold soak. As increasing concentrations of SO2 were added to the grapes prior to cold soak the growth of the non-Saccharomyces yeast including H. uvarum decreased. When 50 mg/L SO2 was added only low populations of H. uvarum, T. delbrueckii, and L. thermotolerans were detected at the end of the 6?C cold soak while at 10?C only H. uvarum was detected. When 100 mg/L SO2 was added there were few culturable yeast present in the cold soaks at either 6 or 10?C. Overall, increasing SO2 was more effective at minimizing H. uvarum growth than decreasing the temperature as there was still significant growth of H. uvarum at 6?C when no SO2 addition was made.

All wines made from grapes that underwent cold soak had significantly higher color and polymeric pigment content than wine made from grapes that did not undergo cold soak with only small differences in color and polymeric pigment content being noted between wines made from grapes cold soaked under different SO2 and temperature conditions. Initial volatile aroma analysis demonstrated significant differences between the concentration of a number of esters in wines made from grapes that were or were not cold soaked. In particular, wines made from grapes cold soaked with no SO2 additions had lower concentrations of certain ethyl esters.

Identifying Compound(s) Responsible for Off-flavors associated with “Stressed Vine Syndrome” in Pinot Noir

One of the increased concerns of wine industry is related to vine stress. Although the off-flavor descriptors vary from winery to winery, the frequent descriptors used in the wineries include “tequila”, “shellfish”, “peanut”, “ashtray”, “dry weed”, “herbaceous’, “flint” and other descriptors. In young wine, the taint smells like “bay leave”, and the wines do not age well. There were observations from wineries that taint could be related to compromised or nutritionally imbalanced fruits from stressed vines, induced by drought, nitrogen deficit, or a combination of many factors, but the exact cause(s) has never been studied or documented. This research is aimed at identifying the chemical nature of these off-flavors using gas chromatography/olfactometry, GC-MS and sensory analysis. Once the chemical nature of the off-flavor is identified, viticulture and enology remedies could be further investigated. The objective for the first year is to identify wine sensory characteristics that define “stress vine syndrome” through sensory evaluation and to identify wines for further analysis.

Several wines have been identified from industrial collaborators. Rollin Soles from ROCO winery identified a matured vineyard with full cover crop and dry farming practice to give a wine with bay leaf aromatic (2012 vintage) and the taint intensified with aging. Another vineyard with young vine also repeated to give dried herb tequila aromatics. Gary Horner from Erath winery also identified several wines with stressed vine aromatics including Pinot noir, Pinot gris and Chardonnay. More off-flavor wines are being recruited.

The “stressed vine” off-flavor was characterized by a sensory panel consisted of six winemakers from the Willamette Valley who have been involved with “stressed vine” off flavors previously and five OSU researchers involved in the project. The panel was able to identify the “stressed vine” off-flavor from the wines collected from industry. The off-flavor was also compared with wine standards comprised of tequila, peanut shell, bay leaf, agave and herbal characteristics. The off-flavored wine and the standards will be used for further sensory training and analysis.

Some preliminary chemical analyses were also performed in these wines including higher alcohols, esters, volatile phenols and TDN (kerosene aroma). The analyses were also performed on 20 normal Pinot noir wines. Data are being analysis to compare the off-flavored wine with the normal wines. GC/olfactometry will be performed next to identify the off-flavor compound(s) in “vine stressed” wines.

Investigating Fruitiness Perception in Red and White wines

This report details activities that occurred from August 2017 to January 2017. Several accomplishments were achieved during these first 6 months. We successful made wine, Pinot noir and Pinot gris, that contained no aroma compounds. This was achieved by altering some winemaking productions and an addition of resin that specifically absorbs aroma compounds. There is enough wine to serve as our base wine for the entirety of the study.  Fruit standards for red wines were developed and used for three sensory panels that investigated fruitiness in red wine. After 3 training sessions panelists were found to be consistent with their standard evaluations. The first sensory panel validated previous research focused on the impact of β-­damascenone, lactones, furaneols and red and black-berry associated esters to fruitiness in red wine. Our results for furaneols and β-dsamscenone do agree with previous work. The 2nd and 3rd sensory panels investigated the same compounds as the 1st sensory panel but at different concentrations and combinations. We have found that when norisoprenoids, β-damascenone and β-ionone, are at low concentrations and when furaneol compounds are at high concentrations, there is an impact to perception of red fruit aromas. However this only occurs if all other compounds are at lower concentrations. Once these compounds are in combination with higher concentrations of other compounds, the tested esters and lactones, there is a shift from red fruit aromas to dark fruit aromas. As we anticipated dark fruit aromas appear to be due to a combination of many compounds, with no one compound class dominating over another. We still have 1 more sensory panel to conduct to complete the investigation into the impact of norisoprenoids, furaneols, lactones and red and black berry associated esters on fruity aroma perception. It is our intent to begin focusing on the effect of acetate esters and volatile fatty acids to fruity perception of red wine. We also are prepared to begin investigating fruitiness in white wine, focusing on terpenes and esters.

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 this may not be due to the filtration treatments. In addition, we have evaluated the effects of filtration of both a red and white wine using a cross-flow filter. No effects on sensory characteristics were found for the white wine. Cross-flow filtration of the red wine seemed to stabilize the flavor profile of the wine, compared to the control that took on earthy characteristics, due possibly to growth of another organism. We also studied the effects of the three types of pumps typically used in wineries and found no significant effects of pumping either for short or extended periods of time. Finally, we evaluated the use of various gases for rinsing bottles prior to filling and their effect on the bottle shock phenomena observed. No differences were seen between bottles rinsed with argon, nitrogen, or carbon dioxide. Differences were observed when oxygen was introduced into the bottles.

Assessments of Difficult to Ferment Juices

The major goal of this project is to uncover the causes of chronically difficult to ferment juices. These juices are defined as those not due to fermentation management failures and inattention to nutritional needs of the yeast and maintenance of permissive growth and fermentation conditions. These juices are often derived from the same vineyard or block of a vineyard, and other similarly managed vineyards and blocks display normal fermentation kinetics. One class of these difficult to ferment juices is characterized by a high proline to arginine ratio. We have confirmed in several yeast strains that mannitol accumulates within the yeast in these juices and that this is associated with the presence of oxidative stress. To date all of the commercial strains tested are sensitive to these juices and reduce fermentation capacity. We have also confirmed the inhibitory role of previously identified lactic acid bacteria in yeast fermentation but have also discovered that these bacteria are efficient at inducing the establishment of the [GAR+] prion in wine strains. This prion is a protein conformational change that is inherited by progeny cells during cell division, thus once cells in the population have changed to establish the prion, subsequent generations will also be in the prion state without the need for continued induction. We have identified several other genera of lactic acid bacteria as well as acetic acid bacteria from arrested wines that are also capable of inducing the [GAR+] prion in wine yeast. We have received samples from over 53 wineries that have suspected bacterial inhibition of fermentation and have been able to isolate [GAR+] pion-inducing bacteria from many of these wines. Further we have shown that bacteria isolated from stuck wines, when grown in growth-permissive media then removed from the medium via filtration the inducer is still present in the filtrate and capable of  inducing the prion in wine strains of S. cerevisiae. Thus the inducer may be present in the absence of viable bacteria if the fermentation had bacteria present at some point. Controlling the presence of these bacteria is therefore important under production conditions.

Characterization of Aroma Volatiles and their Glycosidic Precursors in Grapes and Wines

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 grapes and wines. We have also recently developed a method for monitoring the ‘aroma potential’ of grapes and wines without the need for initial isolation of the glycoside precursor fraction. However, this method still depends on indirect measurement of the glycosides and acid or enzymatic hydrolysis is needed to release the volatile aglycone which can result in artefact formation. In the current project we validated a novel method using UHPLC-qTOF MS/MS for direct analysis of intact aroma glycosides in grapes with minimal artifactual changes in composition. Eighteen monoterpene glycosides were identified including a monoterpene trisaccharide glycoside, which is tentatively identified here for the first time in any plant. Additionally, while previous studies have identified monoterpene malonylated glucosides in other grapevine tissue, we tentatively identify them for the first time in grape berries. Finally, we observed that depending on the glycoside monitored, there is differential accumulation of monoterpene glycosides during maturation of Muscat of Alexandria berries. This work sheds important insight into possible biochemical changes in glycosylation during grape berry maturation. In addition, this research will allow us to better understand the effects of viticultural and winemaking practices on grape and wine components that affect flavor.

Tannin Structure-Activity Relation to Red Wine Astringency

The objectives of this proposal have been to do the following:

In a companion project being submitted to the Agricultural Research Institute (California State University research initiative)

II. Conduct extended sensory studies with the University of California, Davis, on a subset of the above wines to determine the relationship between sensory and instrumental analysis of red wine mouthfeel.

These objectives are consistent with the highest priority research objective as outlined by the American Vineyard Foundation.

The overall purpose of this proposal is to determine the role of grape and wine production practices on tannin structure and perception.  In combination, tannin activity will be monitored by high performance liquid chromatography (HPLC) so that a comparison can be made between human perception and HPLC measurement.

Following the second year of activities, excellent progress has been made.  The major activity during the second year of this study has been to monitor the development of berries in approximately 75 Napa Valley blocks.  Monitoring the tannin activity in these blocks is expected to provide information on the role that grape production practices can have in overall grape and corresponding wine tannin.

Red Wine Tannin Interaction with Polysaccharides

The objectives of this proposal are to do the following:

I. Determine variation in tannin activity as a function of polysaccharides structure from yeast and/or grape in red wine.

II. Relate the polysaccharide-tannin interaction variation to the wine mouthfeel.

In a companion project funded by the Agricultural Research Institute (California State University research initiative)

III. Improve the understanding of tannin-polysaccharide interactions consequences on red wine stability.

These objectives are consistent with the highest priority research objective as outlined by the American Vineyard Foundation.
The overall purpose of this proposal is to determine the role of polysaccharides from yeast and/or grape in finished red wine on tannin structure and activity. In combination, competitive interactions between tannins and proteins or polysaccharides will be elucidated by spectrophotometry after variation of matrix parameters, so that an elaboration of a functioning interactions model can be made.

Improvement of Wine Quality: Tannin and Polymeric Pigment Chemistry

Seven vintages of UC Davis Oakville Cabernet Sauvignon spanning 35 years was selected for analysis by a complimentary suite of mass spectrometric techniques. The vintages from 1974 to 2009 selected by informal sensory evaluation and basic wine chemistry to be of a representative vertical style were: 1974, 1981, 1988, 1994, 2001, 2003 and 2009. All wines were produced at UC Davis for research purposes and stored in the UC Davis cellar together. This sample was determined to be the most controlled and uniform sample of wines varying by vintage only as could be obtained for the project. Al basic wine chemistry was obtained, pH, ethanol concentration etc. as were various assays for comparison to mass spectrometric data.

The following objective were submitted for the grant proposal under which this work was peformed.

1) Observe the evolution of pigmented tannin throughout aging

a. Employ our method of complimentary mass spectrometric techniques (ICR, QTOF, QTrap) for comprehensive identification of wine matrix compounds.

b. Observe the changes in relative abundance, depletion and accumulation in pigmented tannin composition (35 Year Vertical)

c. Correlate pigmented tannin structural analysis with well-known molecular characteristics (mDP, mass recovery) and newly developed sensory representative analyses (“grippiness”) to better convey the impact of these discoveries

2) Apply existing synthetic technologies.

a. Development of a library of standards for quantitation and calibration.

b. Postulate wine pigment precursors for examination of mechanistic pathways.

c. Employ the standards to quantitate the classes of polymeric pigment in wine.

Wine Research Assays

Phloroglucinolysis was performed on the wines used for the study (Table 1) as well as their extracts (Table 2). The mass recovery demonstrates typical value dropping off well below 80{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} after 7 years. Molecular mass after 50{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} elution also follows expected patterns of increase with age. This indicates overall larger tannin in the older wines as supported by McRae et al 2012. The molecular mass is a measure of intact tannins, as opposed to the mDP which measures only those tannins which could be cleaved by phloroglucinol. The inconsistency between these two values is reconciled by the mass recovery, indicating that more complexity of the tannin structure is a factor in the older wines, for instance additional interflavan bonds preventing phloroglucinol attack.