Biological Effects of Phenolic Compounds on Saccharomyces Cerevisiae

In this current grant year, it was discovered that some of the variability in response to phenolic compounds displayed by different commercial strains was possibly due to subtle differences in preparation of the synthetic juice medium, Triple M. This was confirmed using slightly different protocols and the Premier Cuvee yeast strain. Therefore, the preparation and composition of this Medium has been redesigned this year in order to avoid this problem. Analysis of the impact of phenolic compounds is continuing. To date, gallic acid appears to be uniformly stimulatory at concentrations found in wine while ferulic acid may be neutral, stimulatory or inhibitory to sugar consumption under laboratory conditions mimicking natural fermentations. The hexose transporters encoded by the FDTT9/HXT11 genes that are under multidrug resistance control were shown to be expressed under enological conditions. The presence of these genes has been shown to be stimulatory for the uptake of inhibitory drugs. They may therefore play a role in uptake or release of phenolic compounds inside of the cell by coupling such movements to the uptake of glucose. In this current grant year, we launched the comparative analysis of the impact of phenolic compounds on different yeast strains (conducted by Laura Lange). She initially found that the effects were quite variable and there appeared to be no consensus as to whether the effects of a given compound were stimulatory or inhibitory with the exception of gallic acid. In the course of her work, we discovered that the inherent variability in the composition of the Triple M medium led to variation in the response of the yeast. The pH of this medium is adjusted using ammonium hydroxide. We found that since the pH of our water supply varied, so did the ammonium content of the medium. Further analysis revealed that this difference in nitrogen content was not impacting the results. However, the medium has been redesigned to contain a constant concentration of nitrogen. Another problem with the medium was the low concentration of potassium. Previous work in my laboratory indicated that potassium deficiency could lead to a sluggish fermentation and that this effect was somewhat strain dependent. We therefore decided to use potassium hydroxide to adjust the pH of the medium to assure a reasonably high concentration of potassium. Finally, the micronutrients were prepared as a stock solution and diluted when added to the medium. We noticed that a precipitate formed in the stock upon storage and that the age of the stock solution was correlated with variability in the effect of the phenolic compounds. This makes sound physiological sense since the cofactors derived from the vitamins and minerals in this mix are critical for respiratory activity of the yeast. To eliminate this problem, we now make the micronutrient cocktail fresh each time and use the Triple M medium within 48 hours of preparation. This has eliminated the variability in the response. This important optimization of the medium slightly delayed progress on the grant, but we are now conducting the comparative studies across strains. This will not be finished as originally hoped by the end of this grant year, so will continue into the first three months of the next grant year.

Development of a New Assay to Measure Tannins in Grapes and Wines

We studied pectin’s effect on the analysis of tannins in our precipitation assay, and found that the order in which the assay components were added determined whether or not precipitation was inhibited. If pectin was mixed with tannin before tannin was exposed to protein, then there was a marked inhibition of the amount of tannin precipitated. However, if the tannin was mixed with the protein prior to the addition of pectin, then there was no effect on the amount of tannin precipitated. We used citrus pectin and carboxymethylcellulose (CMC) and determined that the amount of citrus pectin required to inhibit the tannin assay by 50%was approximately 500 ng/ml whereas CMC showed 50%inhibition at about 70 ug/ml. We extracted water-soluble and EDTA soluble pectin from grape berries and found that water-soluble pectin does not inhibit tannin precipitation, but EDTA soluble fraction can. However, EDTA soluble grape pectin has a much higher 150 (1400 ug/ml) in the assay than either citrus pectin or the CMC. Thus, since the water-soluble grape pectin was ineffective at all levels tested, and the EDTA soluble pectin was only marginally effective, this result indicates that it is highly unlikely that grape pectin will interfere with the tannin solution assay. We studied tannin development in Cabernet Sauvignon, Pinot noir, Syrah and Zinfandel. Zinfandel looked very similar to Cabernet Sauvignon but compared to Cabernet Sauvignon, Syrah had more tannin in the seeds than the skins at harvest. Even though the Syrah seeds contained more than twice as much tannin as Cabernet seeds, the wine made from the Cabernet fruit had 2.4 times as much tannin (1414 mg/1) as the wine from the Syrah fruit (585 mg/1). This points to tannin extraction as being more important for the amount in the resulting wine than the amount in the fruit at harvest. In the course of experiments to determine if the “background” absorbance in our tannin assay represented polymeric pigments, we found that protein precipitation separated the polymeric pigments into two classes that we have designated LPP for large polymeric pigments, and SPP for small polymeric pigments. We devised a combined procedure based on protein precipitation and traditional SO2 bleaching methods to allow measurements of SPP and LPP in wines along with the determination of total tannin. Thus, we have extended the scope of the solution assay and we are able to use the assay to provide a direct measure of the polymeric pigments in wines.

Investigation of Mechanisms for Perception of Astringency

Astringency has been shown to have carry-over effects which influences the perception of astringency of red wine and astringent compounds. To quantitatively measure this effect, trained judges rated intensity of astringency continuously while repeatedly sipping wine. When red wines were sipped 4 times at 25 second intervals between sips, the astringency intensity increased significantly with each sip. At each sip, astringency increased, reached a maximum intensity about 15-16 seconds after the sip was taken, then decreased slowly until the next sip was taken and again astringency increased. For two of the wines the increase with each sip was significant, whereas the other two the increase in astringency between the 3rd and 4th sip was not significant. To see what happened when more sips were taken, astringency was continuously rated over 8 sips for alum and tannic acid. The 4th sip of tannic acid was not more significantly intense than the 3rd , although the astringency continued to slowly rise. The practical consequences of these results are that most evaluations of red wines are rendered invalid due to the carry-over phenomenon demonstrated in this study. Astringency of red wines evaluated either by a winemaker during winemaking or for blending or by wine show judges is influenced by the wines tasted before it. To quantify the effects of physiological differences on perception of astringency the saliva flow rates of the judges in the red wine and alum/tannic acid studies above were determined. Individuals with low flow rates of saliva perceived astringency of red wine and of the alum and tannic acid more intensely and longer than high-flow subjects. This physiological difference in perception of astringency is of great significance in understanding how astringency is perceived. Since saliva flow rate varies with the size of the person generally speaking, the practical value of this information may lie in marketing wine, where it may account for preferences of “smaller” consumers. In evaluations of the viscosity of saliva-tannin mixtures, it was shown that viscosity of saliva decreases when tannin is added, analogous to sipping red wine. This is consistent with the observations of the effect of salivary flow status. If astringency is felt as the friction resulting from precipitation of saliva proteins by tannins, than high-flow subjects can restore lubrication better and hence perceive lower levels of astringency than low-flow individuals.

Studies on the Interactions of Flavor Compounds with Nonvolatile

Flavor and aroma are important factors in influencing food and beverage (i.e., wine) choices. However, knowledge of flavor concentration alone is not sufficient to determine the perceived sensory aroma intensity. This is due to the presence of interactions between flavors and nonvolatile food components which can alter flavor volatility and release. We used NMR techniques to study the mechanisms of binding between flavor compounds and polyphenols, the main nonvolatile constituents of wine. Our results showed that the interactions are dependent on the structure of both the flavor compounds and the polyphenols. The interactions are principally due to hydrophobic interactions between the aromatic rings of the flavors and the polyphenols. However hydrogen-bonding effects help to stabilize the complex and enhance the specificity. By understanding the mechanisms of these interactions and their effects on flavor perception we will be better able to optimize grape and wine composition and winemaking procedures to improve wine flavor. This proposal is a continuation of a previously funded project. During previous years we focused on developing analytical methods (Gas Chromatographic and NMR) and sensory procedures for measuring odorant interactions in model solutions (Objective b and c). Results have been published (Mialon and Ebeler, 1997) and presented at national meetings of the ASEV (June 1996 & June 1998). An oral presentation outlining recent progress was presented at the National Meeting of the American Chemical Society in New Orleans, LA in August, 1999. A manuscript has been accepted for publication as a result of this research.

The Extraction of Condensed Tannins in Red Wine Production

Over 25 wines were fermented on -20 scale using Cabernet Sauvignon grapes from four different vineyards in California. For each vineyard, a control fermentation was compared with treatments employing maceration treatments including oak extract addition, extended maceration, heat at the end, color enzyme addition, and rotary fermentation. All wines were analyzed for phenolic composition by several different assays. Many of the treatments had small or variable effects, but heat at the end appears to consistently increase phenolic levels. Wines from this and the previous years’ experiment will be offered for tasting at an event in the winter of 2001.

Biological Effects of Phenolic Compounds on Saccharomyces cerevisiae

Plant phenolic compounds are biologically active, affecting many functions of eukaryotic cells. Grapes are rich in a variety of phenolic compounds, many of which have been shown to possess activity in mammalian systems. The effects of phenolic compounds on the metabolic activities of the yeast Saccharomyces have not been thoroughly studied. The goal of this work is to investigate the effect of phenolic compounds on growth and fermentation rates in Saccharomyces cerevisiae under enological conditions and to evaluate the role of these compounds as antioxidants in yeast.

Hard and Soft Tannins: Sensory Definition and Chemical Interpretation

Twelve 1998 Cabernet Sauvignon wines have been obtained from wineries (with minimal oak contact) to select those perceived to be “hard” and “soft”. Winemakers have been interviewed in an attempt to define how each makes the judgement. Although ‘hard” or “soft” are customarily defined by mouthfeel, the contribution of other wine sensory properties is not known. These factors could include color, the aroma and flavor of the wine. More subtle, yet important, we do not know how each individual integrates the perception of taste and mouthfeel. Ten winemakers participated in a “sorting” test in which they were asked to taste the 12 wines and group them on the basis of their perceived similarity. The wines were served in dark beakers, so no information from smell or color was available for this task. Each winemaker used his/her own criteria for the grouping. (For these tests, dark glasses were used to remove color cues). Students at UCD did the same similarity sorting tasks and then were trained for time-intensity (TI) studies in which the intensity of each attribute was rated continuously over time. For each wine, the trained judges took one sip of wine and initiated rating intensity; at a prompt they swallowed the wine, then 25 sec after the first sip, they took a second sip, while continuing to rate the intensity. The procedure of rating for two successive sips was used to include the carryover effects that occur under standard tasting conditions in which several sips are taken, without rinsing. Bitterness, astringency and sourness were evaluated in duplicate, using blue glasses and testing under red light.

Investigation of Mechanisms for Perception of Astringency

Astringency intensity increases progressively with sips, a protocol for measuring astringency in the sensory laboratory was developed which would optimally mimic normal wine drinking behavior. Sip volumes and times between sips of typical consumers were quantified. On the average, 10 ml of wine were sipped at 25 sec intervals, with the wine held in the mouth 3 sec before being swallowed. Using this information, the protocol for rating astringency continuously through four sips was implemented. Judges sipped 10-ml of red wine, swallowed 6 sec later, and sipped three more sips at 25 sec intervals to mimic a realistic pattern of wine consumption by normal consumers. Upon successive sips, a consistent increase in astringency was found. After each sip, the intensity of astringency increased continuously to a maximum (FIG. 2.2), and then decreased slightly, but the initial intensity values before ingestion increased with each sip. The first two sips produced larger increases in astringency for the more astringent wines. However for sips 3 and 4, the absolute increase in astringency per sip becomes more similar. These results have important implications for wineries testing the effects of variables such as maturity on astringency. Sipping several samples in a row can not provide valid information about the effect of the treatment. Thorough rinsing between wines is essential or a time intensity method could be used. It has been hypothesized that sensory perception of astringency is the result of tannins binding to salivary proteins. As a consequence, the effectiveness of salivary lubrication is decreased, and astringency is perceived as the friction between two “unlubricated” surfaces. In previous sensory studies, we demonstrated that increasing viscosity lowers astringency. This was hypothesized to occur due to restoration of lubrication. In the research conducted this year, the change in viscosity of saliva upon addition of tannin was measured. The viscosity of saliva decreased after reacting with tannin. Future research is needed to determine if the observed small viscosity change corresponds to a significant change in perception of astringency.

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.