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.

Chiral Terpenes – Quantitation, Threshold Determination and Sensory Impact on Aromatic White Wines

Objective 1 – Development of methodology using multi-dimensional gas chromatography-mass spectrometery to measure chiral terpenes (limonene, linalool, citronellol, α-terpineol and α-pinene) in aromatic white wines.  

Since September 2013 method development has been ongoing. Specifically chemical synthesis of dueterated-limonene and α-pinene has been conducted. Synthesis of d2-α-pinene was successful. D2-limonene is proving more problematic and this synthesis is ongoing. These deuterated compounds are used for internal standards, and more accurate measurements are achieved when the internal standards have similar structure to the target compounds. Preliminary work has shown that the internal standards that can be purchased do not work well for limonene and pinene, therefore we are attempting chemical synthesis of these compounds. For all other compounds and standards have been composed into a working standard for the method and method development is ongoing. At the present time, method development is proceeding on schedule and is anticipated to finish the end of February.

Additional wines have been collected over the last few months to build up a substantial collection of aromatic white wines from regions around the globe. Areas in which aromatic white wines have been obtained include Washington, Oregon, New York, California, New Zealand, Australia, Germany, Austria and Italy.

Objective 2 –  Once quantitation of enantiomers is achieved the impact of the enantiomers will be investigated through a series of sensory testsThis objective is slated to begin during Spring/summer 2014.

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 Head. -Phase-Microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS). Acid hydrolysis of a mixture of standard compounds at pH 1 and 100°C for 1 hour released 20-60{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} of the bound volatiles, however, these conditions also resulted in significant degradation (>50{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23}) 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.

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 Head. -Phase-Microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS). Acid hydrolysis of a mixture of standard compounds at pH 1 and 100°C for 1 hour released 20-60{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} of the bound volatiles, however, these conditions also resulted in significant degradation (>50{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23}) 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.

Adaptive Evolution of Commercial Wine Strains for Reduced Ethanol Production

The goal of this project is to use the process of adaptive evolution to generate strains with reduced levels of production of ethanol. A corollary of this grant is to use this analysis to understand the basis of the appearance of increased ethanol yields evident in some commercial wine strains. The presence of external aldehydes was used as the driving force to evolve wine yeast towards less efficient conversion of sugar carbon to ethanol. 5-hydroxymethlyfurfural and furfural, two aldehydes normally present in the winery environment, were selected for this study. Strains were screened for initial resistance to HMF and furfural and were categorized as resistant, partially resistant to one or both compounds and sensitive to one or both compounds. Seven strains were selected for the adaptive evolution study and grown for 250 generations in the presence of aldehydes. Strains evolved in one of three directions: no change in ethanol yield per input carbon (one strain), slight to modest increases in ethanol level (five strains) and a reduction in ethanol yield (one strain). Aldehydic stress is common in the winery environment and these results offer credence to the belief that more tolerant robust strains develop the capacity for higher ethanol yields. The only strain that showed reduced ethanol yields was a vineyard, not winery, isolate that had no inherent tolerance to HMF. Thus it is possible to use adaptive evolution in the presence of aldehydic stress to generate strains with a reduced tendency to produce ethanol. However many wine strains have naturally evolved tolerances to aldehydes and this seems to correlate with increased not decreased ethanol production. Under natural conditions the genetic tendency appears to be in the direction of reduction of biosynthetic activities that consume reducing power in favor of the reduction of catabolic and environmental aldehydes.

Vegetative Aroma: Sensory Definition, Chemical Interpretation (and ultimately) Causal Explanation

During the past year we completed sensory studies (descriptive analysis) and chemical analyses on wines made from grapes obtained from four different Cabernet Sauvignon vineyard blocks that were previously identified as producing wines with high levels of vegetal sensory characters.

Within each of the four vineyard sites, cluster microclimates were varied to yield different canopy densities and cluster exposures (with the main effect being differences in light intensity). Vegetal sensory properties (i.e., cooked veggie, bell pepper, and olive aromas) were primarily influenced by vineyard location, however canopy manipulations and row orientation were also important. Measurable levels of 2-methoxy-3-isobutylpyrazine (MIBP) were only found in the wines from one site (Gonzalez; Viticultural Region I) which had the lowest average temperatures of the sites studied.

These wines also were characterized as most vegetal in the sensory descriptive analysis studies. Finally, wines from a separate cluster thinning trial were made and we propose to do sensory analysis of these wines during 2006/07.

Ester Formation by Saccharomyces cerevisiae During Fermentation

During 2005/06 we monitored kinetics of ester production for seven different commercial yeast strains. Using headspace SPME combined with gas chromatographic analysis we observed significant differences among the strains with respect to rate of ester accumulation and maximum and final levels of esters formed. Interestingly, patterns of ester production also depended on the individual esters monitored. For example, production of ethyl acetate varied significantly with yeast strain, while production of hexyl acetate was similar among the strains studied. These results will now be combined with biotechnological tools that will allow us to monitor gene expression and enzyme activity in these strains. These studies will allow us to better understand the factors influencing flavor formation in Saccharomyces cerevisiae fermentations.

Evaluation of the Influence of Common Viticultural Practices on the Chemical and Sensory Characteristics of Wines

The goal of this project is to find critical viticultural parameters that affect the chemical and sensory characteristics of the final wine. Once these parameters are identified, winemakers and vineyard managers can use computational methods developed in our lab in order to suggest the best vineyard practices to use to achieve specific target characteristics in their final wine. We are taking two complementary approaches to this problem. First, we are developing tools for searching through large existing databases of viticultural information to find the most critical factors. A Decision Tree Analysis algorithm has been developed in our lab for this purpose, and several decision metrics have been evaluated. Second, we are producing Cabernet Sauvignon wines from existing viticultural trials at the Oakville Experimental Station. For the past two harvests, we have produced 37 wines from grapes harvested from vines differing in parameters such as rootstock, irrigation, trellis, pruning, row orientation, and vine density. Chemical analysis of the wines from the first harvest has been completed, and the data has been analyzed. Especially interesting results have been found on the effects of trellis system and crop load on the phenolic profile of the completed wines. Sensory analysis of these wines has also been initiated.

PDF: Evaluation of the Influence of Common Viticultural Practices on the Chemical and Sensory Characteristics of Wines

Investigation of Mechanisms for Perception of Astingency

Astringency intensity increases upon successive sips of the same wine or astringent stimuli OR of wines tasted at a session. To determine which rinse best reduces this carryover effect, astringency of red wine was rated continuously while wine was sipped, spit (at 10s) and rinses (or crackers) were sipped (or chewed) at 20s and spit at 30s. Overall, pectin was the most effective rinse for reducing astringency between samples and minimizing this carry-over phenomena. Pectin reduced astringency intensity from 30 s after the wine was sipped (which was 10 s after the rinse was expectorated) more than CMC, Polycose or water. Although a high concentration of pectin was the MSOT effective rinse, it was not signficantly better than a low concentration of pectin or a high concentration of CMC. In another test, Pectin reduced astringency after 30s more than unsalted crackers or water. Although water lowered astringency from 30 to 40s more than crackers, after 40s crackers were more effective. For routine tests in a winery or at competitions, rinsing with a pectin solution (4 to 5 g/L) between wines should greatly increase the reliability of the tasting.

The influence of sweetness and of flavor (vegy or fruity) on perception of astringency was examined in red wine. Astringency intensity was significantly lower in the sweetened wine, than the base wine or flavored wines. Since aspartame was used, and no increase in viscosity occurred upon sweetening, the suppression of astringency is solely due to cognitive interaction. When fruitiness was rated in sweetened and flavored wines, a similar cognitive enhancement was observed in that the sweetened (unflavored) wine was rated fruitier than a fruity-flavored wine or the base wine.

Consistent with previous studies, astringency was rated less intense when artificial saliva containing salts was introduced at a medium flow rate (5 ml/min) than at a low flow rate (o.5 ml/min). When wine was sipped repeatedly, the maximum intensity (IMAX) of astringency increased with each sip at low and medium flow rates of introduction of the artificial saliva, although it decreased upon successive sips at high flow rates (8 ml/min). When 3g/L gelatin was added to the artificial saliva the same effect was observed. However, there was no difference in astringency IMAX between artificial saliva with and without protein at either flow rate for any sip, which indicates that further research is needed to understand perception of astringency.

PDF: Investigation of Mechanisms for Perception of Astingency

Investigation of Mechanisms for Perception of Astingency

The mechanism by which astringency is perceived is unknown. Two different mechanisms have been proposed.

  1. The ASTM defines astringency as the puckering or constricting of the oral epithelial tissue, suggesting that a morphological change occurs when an astringent system is evaluated.
  2. In contrast, others hypothesize that the rough feeling of astringency occurs when salivary proteins bind to tannins, oral lubrication decreases and friction (roughness) is perceived.

Preliminarily, from our study of the morphological changes in the epithelial mucosa, it appears that astringency does not result from “constriction of the oral tissue”. No obvious differences in distances between epithelial cells were observed after application of astringents. However, it is possible that the loose cells on the surface slough off after binding with tannins, perhaps resulting in perception of astringency, but this has not been substantiated.

Consistent with the second hypothesis, in previous studies, it has been shown that individuals with low flow rates of saliva perceive astringency more intensely and longer than high-flow subjects. To confirm this effect of salivary flow rate independent of variation in use of the intensity rating scale, artificial saliva was introduced to the judge’s mouth at different flow rates. Astringency was rated lower at high flow rates of application than when lower rates of application were used. In both cases, comparison of individuals with different flow rates and with the application of artificial saliva at varying flow rates, the presumption has been that astringency decreases at a higher flow rate due to restoration of lubrication. However, no difference in astringency was found between application of artificial saliva with protein vs. an artificial saliva containing no protein. These results suggest that higher salivary flow rates may dilute or more thoroughly flush the mouth, and suggest that further research into the mechanism of astringency is needed.

The persistence of astringency has been recognized but only recently has the carry-over effect been documented in which each sip of wine influences the astringency perceived in subsequent sips or wines. For meaningful evaluations of astringent red wines during winemaking, blending or in competitions a tasting protocol to remove or reduce the buildup of astringency must be developed. To do this, astringency was rated continuously while red wines were sipped, spit, and judges rinsed with one of 5 solutions. A pectin rinse reduced astringency intensity significantly more than the other rinses. We are presently investigating the most effective way to prevent the increase in intensity for wines varying in astringency level and to define the minimum time to recommend between wines.

PDF: Investigation of Mechanisms for Perception of Astingency