Significance of Oak Ellagitannin Chemical Structure to Wine Oxidation

The pathway of wine oxidation, as currently understood, encompasses the cascade of reactions incited by the oxidation of phenols in the presence of oxygen, eventually coming to the conversion of ethanol into acetaldehyde. While it is evident that wines consume oxygen over time and acetaldehyde becomes increasingly apparent with age, the rate of oxidation can vary unpredictably among different wines, and it was hypothesized that different phenolic structural features, particularly those of oak ellagitannins, are the reason for such variability in oxidation. It was originally proposed that ellagitannins and other phenols with distinct functionalities be studied for their effects on oxygen consumption and acetaldehyde production. However, in light of recent studies conducted by our laboratory demonstrating that the input of oxygen does not guarantee the output of acetaldehyde, it was decided that attempting to study the pathway of oxidation in its entirety, from oxygen to acetaldehyde, would not be an effective approach.

Given the complexity of wine oxidation, a more sensible strategy would be to study the effects of phenolic structure on individual reactions rather than the pathway as a whole. In the first step of wine oxidation, the oxidation of phenols is coupled to the reduction of oxygen by iron, which acts as a shuttle for electrons between phenols and oxygen. A more specific hypothesis now is phenolic structure affects their reactivity with iron, subsequently affecting oxygen consumption and the remainder of the wine oxidation pathway. The initial reactions of wine oxidation may be characterized by the redox cycling of iron between its two oxidation states: Fe(II) and Fe(III). The addition of electrons to oxygen occurs with the oxidation of Fe(II) to Fe(III), and in the opposite direction, the loss of electrons from phenols takes place with the reduction of Fe(III) to Fe(II). The ratio of Fe(II) to Fe(III) should thus depend on the relative reaction rates of Fe(II) with oxygen and that of Fe(III) with phenols.

A quick and simple spectrophotometric method for iron speciation, employing the complexing agent Br-PADAP, is currently being optimized and validated, to be used not only to assess differential rates of iron reduction by structurally diverse phenols, but also by the industry to more generally measure the “redox status” of their wines. Our laboratory’s modified version of the Br-PADAP assay is simple and inexpensive, requiring a sample volume of only 200 μL and a reaction time of 10 min, and is done directly in a cuvette. Validation of the assay is currently underway; difficulty lies in the fact that there does not exist a standard method for iron speciation to compare, thus alternative methods of validation are being considered. This research would not only improve management of oxidation, but also furnish a more complete understanding of phenolic oxidation, with the ultimate goal being the prediction of wine aging based on phenolic content and composition.

Investigating Fruitiness Perception in Red and White Wines

This report details activities that occurred from February 2017 – January 2018. We have had a very exciting accomplishment with the adaptation of a chemometric method that can calculate chemical interactions resulting in specific sensory perceptions. This method, fuzzy set qualitative comparative analysis (fsQCA), overcomes the issues with traditional correlation analysis that made determining aroma chemical interactions very difficult. To date we have investigated 43 different compound combinations and their impact to fruit aroma in Pinot noir wine. By applying fsQCA we have found 15 compound sets that result in red fruit aroma in Pinot noir wine and 2 compound sets that result in dark fruit aroma in Pinot noir wine. The necessary and sufficient conditions found in these sets are supported by other work, but our results are the first to show the multiple combinations of compounds that can result in specific fruity aromas. We have also investigated 20 compound combinations for fruitiness in white wine. We have yet to apply fsQCA to this data but in using more traditional correlation analysis (canonical variate analysis) we find that terpenes and certain esters are important for “stonefruit” and “citrus” aromas and thiols are the drivers of more “grapefruit” aromas. In our last year we aim to continue investigating different compounds in both red and white. We are also incorporating nonvolatile factors into the analysis. In red wine we are investigating the effect of phenolic composition on fruitiness perception and in white
wine we are changing the residual sugar concentration to determine its impact on fruity perception in white wines. We will also apply fsQCA analysis to the data from our white wine sensory panels, determining the necessary and sufficient conditions for specific fruitiness in white wines.

Winery Cleaning and Sanitization, Monitoring Methodology and Efficacy of Cleaning and Sanitization Chemistries.

This project analyzed the ability of cleaners and sanitizers frequently used in the wine industry to inactivate microbial populations in solution (planktonic) and stationary (biofilm) physiologies. Cleaning and sanitizing agents were used at manufacturer’s specified concentrations for planktonic cells grown in 96-well plates. The most effective treatments for the inactivation of planktonic cells were used in the biofilm trials. In these trials, biofilms were treated with the chemicals (cleansers and sanitizers) and subsequently stained to identify remaining biomass using crystal violet. Next, the same chemicals were tested for biofilms grown on stainless steel winery tank material. Stainless steel coupons were suspended vertically in pipet tip containers and incubated with various winery microbes in 50% water/50% grape juice medium for two weeks to develop biofilms. The coupons were cleaned with water, and then submerged in the cleaning or sanitizing agents for various time periods. The ability of the chemicals to eradicate the biofilms was measured using ATP swabs and cell culturing, the two techniques most frequently employed in wineries to examine microbial load in quality control applications. Remaining work involves scaling the stainless-steel trials to larger red wine fermentations, and the development of an optimized cleaning/sanitizing recommendation for the wine industry.

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.