Red wines are usually fermented in contact with skins and seeds to extract polyphenols, including anthocyanins, that help to build and stabilize color. Concentration and composition of anthocyanins vary significantly among genera, species, and cultivars [1
]. The chemical structure of anthocyanins is also very variable and directly influences extractability, solubility, and color characteristics in juice and wine [3
]. While most cultivars of Vitis vinifera
have only simple anthocyanin glucosides and acylated derivatives [5
], species like Vitis labrusca
or other genera like Muscadinia rotundifolia
, or hybrid grapes can display a wider range of anthocyanins including diglycosides of variable composition and structure [2
]. Extraction kinetics during fermentation are strongly dependent on cultivar, wine style, fermentation conditions, pH and degree of ripeness [4
], which makes the prediction of color characteristics in the finished wine based on original grape composition very challenging. In addition to that, anthocyanins and other polyphenols in wine start to polymerize, oxidize, and react with other wine components immediately after their extraction [8
], which adds an additional challenge to any prediction model. Several polyphenol extraction approaches have been described in the literature that range from solvent based methods [9
] to combinations between physical treatments with heat, microwaves, or ultrasound with mild solvents [12
]. Some comparative studies suggest that selected extraction methods can be used to predict the amount of extractable polyphenols [4
], however, most of these projects only looked at a limited number of Vitis vinifera
cultivars and only compared finished wines with the predicted polyphenol concentrations. Anthocyanin extraction dynamics during fermentation have been described for Vitis vinifera
only by Glorie in 1993 [3
] and are usually not part of extraction prediction studies. The most frequently used extraction methods in the wine industry are the ITV Standard method [9
], the extraction according to Glorie [10
], and the method suggested by the AWRI [11
]. Previous studies found that, despite the considerable time commitment, Glorie’s method is best suited to predict the color characteristics of red wine [4
]. However, all methods lead to a significant over-extraction of phenolic substances compared to wine after fermentation, most likely because all extractions are performed with grape paste from a blender, which also leads to a complete destruction and extraction of seeds [4
]. Microwave-assisted or ultrasound-assisted extraction techniques have been described as an alternative for grapes and other polyphenol-rich material [13
]. Microwave assisted extraction can be viewed as more advanced than traditional solvent extraction methods because the matrix is heated internally and externally without a thermal gradient. Moisture inside and outside the plant cells evaporates, which produces tremendous pressure on the cell structure. When the cell walls rupture, cell material including anthocyanins leaches into the solvent until equilibrium is reached [13
]. A similar principle is found with ultrasound-assisted extraction where the treatment increases mass transfer rates by cavitation forces, where bubbles explosively collapse and generate enough force to cause cell rupture [12
]. With these physically assisted methods, solvent use and time commitment can be significantly reduced.
The goal of this study was to show color extraction dynamics of 11 grapes during fermentation and compare color characteristics and anthocyanin profiles of the wines to five of the most common extraction methods.
3. Results and Discussion
Fermentations finished within 10 days varying with sugar level, which was greatly dependent on growing region and grape cultivar. However, extended macerations continued for a total duration of 14 days to standardize the extraction prior to pressing. Table 1
shows sugar, nitrogen, and acid levels prior to fermentation and prior to diammonium phosphate additions. Some cultivars did not require nitrogen addition, so diammonium phosphate (DAP) was added to deficient trials to reach the recommended 10 mg/L nitrogen per percent sugar.
All wines finished fermentation and fermented to dry. Table 2
shows analytical data collected from wines after pressing and clarification. Since berries were destemmed manually and mashed after separating them into duplicates, the solid-to-liquid ratio was not affected by processing. As a result, the variability among experimental duplicates is minimized and reflects only minor differences and inconsistencies within clusters of one cultivar. Since these fermentations were performed on a four liter scale, inconsistencies within clusters are exaggerated compared to a large scale production where differences are neutralized in a larger volume. The rate of ethanol production and therefore polyphenol extraction depends on the initial sugar concentration but also the solid-to-liquid ratio [23
]. Total tannins and total phenols were analyzed as catechin and gallic acid equivalents, respectively. The numbers fall into the range that was previously reported for wines that were made from these cultivars.
In order to evaluate the levels of total phenols and total tannins after fermentation, it is important to know the relationship between extractable surface area (berry skin) and average berry volume. This information is commonly provided by the 100-berry-weight which leads to the average weight per berry and the weight of the skin in fresh and dried condition. This information is provided in Table 3
for all grape cultivars studied. Because of large differences in overall berry volume, the average weight per berry does not correlate with the fresh skin weight. In fact, berry weight shows much more variability than skin weight. The total amount of extractable anthocyanins and polyphenols shows a similar pattern to the concentrations that were actually analyzed in the wines after fermentation of the skins. However, some variability is caused by a changing solid-to-liquid ratio based on berry volume.
Although the amounts of extractable polyphenols and anthocyanins vary significantly among grape species and cultivars, the overall extraction pattern follows the same general trend. Figure 1
shows the extraction of brown, red, and blue color during fermentation and maceration of all studied cultivars. The percentage of brown relative to total color is decreasing after fermentation started and reaches a minimum after three to five days. The slow increase after that until day 14 is probably due to the slow development of polymeric pigments. Red color is constantly extracted in the first two to three days and stays at a stable level until day 14. Blue color remains stable throughout fermentation, with the exception of Noble that shows a decrease in blue color in the beginning of fermentation.
While the percentage of color does not reveal major differences, the total color shows more variability among grape varieties. Figure 2
summarizes the total color of all wines observed, expressed as the sum of photometric absorbances at 420, 520, and 620 nm. Noble as the only M. rotundifolia
behaves differently due to a much higher overall extraction level, so Figure 2
a shows all the wines except Noble improving the visibility of differences.
It was described before that anthocyanins are not all equally well extractable from berry skin [3
]. The differences in extractability of anthocyanins show in the total time it takes to reach the color extraction maximum. While some cultivars like Cabernet Franc and Mourvedre need seven days or more to reach that point, color extraction of Concord peaks after only three days. Previous studies associated the extractability with the level of ripeness [4
], in this study however, the grape species and cultivar also has an effect. The other main difference among samples is the rate of color loss after reaching that maximum. Although Concord color is extracted very fast, it also shows the fastest rate of loss with more than 40% until the end of maceration on day 14. The reasons for that are speculative at this point but might be related to the anthocyanin spectrum and the presence or absence of stabilizing factors like polysaccharides and other colorless polyphenols. The biggest color losses in the first 14 days of our experiments can be observed in North American Vitis
species and hybrids. Concord (40.2%), Chambourcin (35.8%), and Marquette (26.5%) seem to have a less stable color than most Vitis vinifera
grapes which lost an average of 15.1% in the same 14 days. There is of course variability among European grapes as well, a trend however can be hypothesized based on the present data.
After 14 days of fermentation and extended maceration, all grapes were pressed, clarified and stabilized by sulfur dioxide addition. Since any physical force changes the extraction kinetics and SO2
has a bleaching effect on monomeric anthocyanins [24
], the total color change was further monitored throughout the process.
and Table 4
illustrate the main findings. Figure 3
does not include data from the M. rotundifolia
grape Noble due to differences that qualified the data points as outliers and changed the scaling. The boxplots show brown, red, blue, and total color analyzed at the appropriate wavelengths after fermentation, after pressing, and after sulfur dioxide addition. The color loss is most prominent in the blue spectrum but is very consistent across the whole range. Table 4
shows the individual color losses at each step of the winemaking process, including the days up to the color extraction maximum at each wavelength.
In order to predict the color characteristics and the amount of extractable phenolic material, the most common extraction procedures were performed and compared to the wines after fermentation. The main differences between traditional extraction techniques are pH, time, solvent strength, and temperature. For these experiments, we selected a large variation among these factors with ethanol being the main solvent and pH adjustments ranging from pH 1 to native pH around 3.9. Since extracted anthocyanins and total phenolics were quantified as malvidin-3-glucoside and gallic acid equivalents, respectively, the results can be directly compared to the wines after fermentation. Table 5
shows the extraction results for all grape cultivars.
Most extraction methods used here show the tendency to over-extract anthocyanins while underrepresenting total polyphenols. In order to see correlations and trends, statistical analyses, shown in Table 6
, used correlation coefficients and calculated if the observation is significantly similar. The results indicate that most extraction methods correlate significantly with the phenolic composition of the finished wines. The color extraction discussed above however makes it a relatively weak correlation. While anthocyanins and other polyphenols in wine start to polymerize, oxidize, and react with other wine components immediately after their extraction, the extracts do not have the time to mimic that before they are analyzed. Table 6
therefore includes the mentioned color extraction maximum as well, which shows an improved correlation. In fact, when the extracts are compared to the peak color intensity, some of the correlations are highly significant with correlation coefficients above 85%. This observation illustrates the difficulties of predicting color characteristics in real wine fermentations. The factor time and the reactivity of wine polyphenols cannot be factored into the prediction model because it varies with grape cultivar, production method, and the overall redox potential throughout the process.
The microwave and ultrasound assisted extraction methods show no significant correlation with color characteristics but are highly significant for total phenolics. The explanation for this observation is most likely the pretty severe change in extraction conditions compared to wine. Both methods use physical force to break cell walls and could lead to a change in extraction kinetics. Especially for acylated anthocyanins, which were shown be extracted easier than mono-glucosides due to their higher solubility in water [4
], a destruction of cell wall material favors their extraction. In addition, acylated anthocyanins are more entrapped in the matrix or form hydrogen bonds with polysaccharides, which can inhibit their extraction [12
]. Microwave and ultrasound assisted extractions have the potential to release more phenolic material than solvent based methods by disrupting these structures.
The overall differences in extraction between the methods are best visualized in an overlay of LC chromatograms, shown in Figure 4
. For this figure, Chambourcin was chosen because, being a hybrid, the grape has one of the broadest anthocyanin spectrums in this study. Although not all compounds are baseline separated with this quick method, it is obvious that anthocyanins in the second half of the chromatogram are not or only poorly extracted by some of the methods used here. The method that stands out is the AWRI extraction. It was originally not designed to predict anthocyanins and although it shows a good correlation with the red wines after fermentation, it does not nearly extract as much anthocyanins as other methods. The spectrum however and the ratio between single anthocyanins is close to the corresponding wine. Ultrasound and microwave assisted extraction on the other hand severely over-extract color compounds as shown in Figure 4
. The ratio is not close to the real wine, which explains the non-existing correlation shown in Table 6
It can still be used to have a complete spectrum of anthocyanins but is not useful for the prediction of color characteristics. The grape crushing method suggested in our study should be considered, since it was less destructive than other methods. In previous studies, the grape material is often completely destroyed including seed material, which increases the rate of polyphenol extraction and could be less representative of an actual wine fermentation. Figure 5
summarizes these observations in a Principle Component Analysis. Most extraction methods are positively correlated, which implies that the quality of their prediction is similar. The vectors of anthocyanins extracted by microwave and ultrasound are located at a 90 degree angle to the other extractions, showing that there is no correlation between them.
The overall goal from a winemaker’s perspective when evaluating red grapes before harvesting is to predict the phenolic profile and color characteristics of the finished product and determine the harvest date accordingly. This study aimed to provide insight into different laboratory-based extraction methods and compare the extracts to wines that were fermented on the skins. Most previous publications used a limited number of extraction methods and looked primarily at Vitis vinifera grapes while we included hybrids and North American grape cultivars as well. During fermentation, all cultivars reached a color extraction maximum after a few days and then started to lose color. This presents the main challenge with predicting color characteristics, because most methods show much better correlations with the wine when they are compared to the maximum color intensity and not the finished wine after processing. The level of anthocyanin extraction as well as color loss due to oxidation, polymerization, pressing, and sulfur addition depends, among previously reported characteristics like the level of ripeness and the grape cultivar. Varieties with a more complex anthocyanin spectrum display a different extraction pattern that cannot be mimicked accurately with some of the extraction methods. Because most methods tend to over-extract phenolic material, the correlation with the peak of color intensity is much better.
Despite the large time commitment, the extraction suggested by Glorie is still the best way to predict extractable anthocyanins, mostly because the method compares different pH conditions in one assay. The ITV Standard method also correlated well with the color extraction maximum but had a larger discrepancy with the wines after processing. Physically invasive methods like ultrasound or microwave assisted extractions are great for extracting more anthocyanins but over-extract to an extent where the spectrum is too different from the corresponding wine to be useful for a prediction model. The correlation with total phenols on the other hand shows promising results. Ultrasound assisted extraction, for example, does not use harsh solvent conditions and consumes very little time and energy, which potentially makes it a very convenient method to predict total extractable phenolics as a cost-effective alternative for smaller wineries.