Interactions between yeast lees and wine polyphenols during simulation of wine aging. II. Analysis of desorbed polyphenol compounds from yeast lees

In the first part of this work, the analysis of the polyphenolic compounds remaining in the wine after different contact times with yeast lees during simulation of red wine aging was undertaken. To achieve a more precise view of the wine polyphenols adsorbed on lees during red wine aging and to establish a clear balance between adsorbed and remnant polyphenol compounds, the specific analysis of the chemical composition of the adsorbed polyphenolic compounds (condensed tannins and anthocyanins) after their partial desorbtion from yeast lees by denaturation treatments was realized in the second part of the study. The total recovery of polyphenol compounds from yeast lees was not complete, since a rather important part of the initial wine colored polyphenols, especially those with a dominant blue color component, remained strongly adsorbed on yeast lees, as monitored by color tristimulus and reflectance spectra measurements. All anthocyanins were recovered at a rather high percentage (about 62%), and it was demonstrated that they were not adsorbed in relation with their sole polarity. Very few monomeric phenolic compounds were extracted from yeast lees. With the use of drastic denaturing treatments, the total recovery of condensed tannins reached 83%. Such tannins extracted from yeast lees exhibited very high polymeric size and a rather high percentage of galloylated residues by comparison with initial wine tannins, indicating that nonpolar tannins were preferentially desorbed from yeast lees by the extraction treatments.     

Impact of oxygen consumption by yeast lees on the autolysis phenomenon during simulation of wine aging on lees

Potential oxygen consumption by lees, more precisely by nonviable yeasts, during wine aging was recently described. Additionally, yeast autolysis is described as the main mechanism of degradation of lees during wine aging. Thus, to understand the effect of oxygen consumption by yeast lees during wine aging, an accelerated wine aging methodology was tested. Wine aging in the presence of yeast lees was studied both in the presence and in the absence of oxygen. Different markers of yeast autolysis were followed to find a relationship between oxygen consumption by yeast lees and changes in the final wine composition after aging. No differences for compounds tested were found in the wine and in the lees except among sterol compounds in lees: in the presence of oxygen, the concentration of ergosterol in lees was significantly lower than that in the absence of oxygen. It was hypothesized that ergosterol could be oxidized under the influence of oxygen, but none of the known products of ergosterol oxidation were recovered in the corresponding yeast lees. In addition, the decrease of ergosterol content in yeast lees cannot account for the total amount of oxygen consumed by yeast lees during such wine aging.     

Effect of aging on lees and of three different dry yeast derivative products on Verdejo white wine composition and sensorial characteristics

A study was made of the effect of aging on lees and of three different commercial yeast derivative products of different composition and degree of purification on the phenolic compounds, color, proteins, polysaccharides, and sensorial characteristics of white wines. The results obtained showed that the lees and yeast derivative products can interact or adsorb some of the phenolic compounds present in wines, reducing their concentration. This reduction depends on the treatment applied, the phenolic compound analyzed, and the stage of vinification or aging process. The use of lees and yeast derivative products can reduce the color intensity and the browning of the wines immediately following treatment. The monosaccharide and polysaccharide content of yeast derivative products depends on the manufacturing process and degree of purification of the product, both of which have an influence on wine treatments. After 6 months in the bottle, both the aging on lees and the treatment with commercial yeast derivative products gave rise to wines with better sensorial characteristics than in the case of the control wines.     

Inhibition of trypsin by condensed tannins and wine

Phenolic compounds are abundant vegetable secondary metabolites in the human diet. The ability of procyanidin oligomers and wine polyphenols to inhibit trypsin activity was studied using a versatile and reliable in vitro method. The hydrolysis of the chromogenic substrate N-benzoyl-d,l-arginine-p-nitroanilide (BApNA) by trypsin was followed by spectrophotometry in the presence and absence of condensed tannins and wine. A clear relationship between the degree of polymerization of procyanidins and enzymatic inhibition was observed. Trypsin activity inhibition was also detected in several types of wine. In general, the inhibition increased with the concentration of phenolic compounds in wines. These results may be relevant when considering these compounds as antinutritional factors, thereby contributing to a reduced absorption of nutrients.     

Effect of addition of commercial grape seed tannins on phenolic composition, chromatic characteristics, and antioxidant activity of red wine

The effect of addition of grape seed tannins on the phenolic composition, chromatic characteristics, and antioxidant activity of red wine was studied. Two highly pure commercial grape seed tannins (GSE100 and GSE300) were selected, and their phenolic compositions were determined. Two types of red wines were made with Castela?o/Tinta Miu?da (3/2, w/w) grapevine varieties by fermentation on skin using two different maceration times, which correspond to the wines rich and poor in polyphenols, respectively. Each of these wines was used for experimentation with the addition of GSE100 and GSE300 before and immediately after alcoholic fermentation. Phenolic composition, chromatic characteristics, and antioxidant activity of the finished red wines were analyzed by HPLC-DAD, CIElab 76 convention, and DPPH radical test, respectively. The results showed that the addition of grape seed tannins had obvious effects of increasing color intensity and antioxidant activity only in the wines poor in polyphenols. Although GSE300 contained much higher amounts of di- and trimer procyanidins and a lower amount of polymeric proanthocyanidins, it provided effects of increasing the color intensity and antioxidant activity of the wines poor in polyphenols similar to those of GSE100. Furthermore, GSE100 released more gallic acid to wines than GSE300, although no gallic acid was detected in GSE100. Tannins added after alcoholic fermentation had a better effect on phenolic composition of red wine than tannins added before alcoholic fermentation.     

Changes in the sotolon content of dry white wines during barrel and bottle aging

GC-MS in electron ionization mode (EI) was used as a simple, sensitive method for assaying sotolon [4,5-dimethyl-3-hydroxy-2(5) H-furanone] in various dry white wines. The impact of barrel-aging conditions, that is, whether yeast lees were present or not, on the formation of sotolon in dry white wines was then studied. The sotolon content was highest in dry white wines aged in new barrels without lees, often exceeding the perception threshold (8 microg/L). These results demonstrated that yeast lees were capable of minimizing the formation of sotolon in dry white wines during aging. The sotolon and oxygen contents of several bottle of the same white wine were also compared 7 years after bottling. At the range of dissolved oxygen concentrations generally measured, between 5 and 100 microg/L, the sotolon content remained below its perception threshold in wine. The perception threshold was exceeded only in wines with oxygen concentrations above 500 microg/L. The presence of dissolved oxygen in the wine samples analyzed also resulted in a decrease in their free sulfur dioxide content.     

Influence of wine turbidity on the accumulation of volatile compounds from the oak barrel

In this work the influence of the turbidity of a red wine aged in oak barrels on the accumulation of volatile compounds has been studied. From the results obtained, it may be concluded that the wine turbidity had an effect on the concentration of many of the oak volatile compounds, especially furanic and phenolic compounds. In all cases the concentrations of furanic aldehydes and vanillin were significantly higher (p < 0.01) in the filtered wine than in the unfiltered wine. However, in the unfiltered wine the concentrations of gamma-butyrolactone and eugenol were higher. This difference in the concentrations would be due to the different compositions of the wines and the binding of compounds to some components of the lees. Likewise, it has been observed that the composition of the lees is fundamental in the binding of volatile compounds to the lees as the fermentative lees behave differently from the lees from the wine aging in barrels.     

Synthesis and stable isotope dilution assay of ethanethiol and diethyl disulfide in wine using solid phase microextraction. Effect of aging on their levels in wine

Ethanethiol and diethyl disulfide (DEDS) most often occurred at levels above their olfactive threshold in wines with nauseous sulfur-linked smells. As ethanethiol is very oxidizable and chemically reactive, a stable isotopic dilution analysis of both ethanethiol and its disulfide in wines using solid phase microextraction and GC-MS was developed. The latter involved the determination of the proportion of DEDS formed by oxidation of the thiol during the analysis conditions, which was obtained by the use of two differently labeled disulfide standards. An original synthesis of labeled ethanethiol standards in conditions minimizing oxidation was developed, and the corresponding labeled diethyl disulfides were obtained from these thiols. This analytical method was used to follow the levels of these sulfur compounds during aging in a young red wine spiked with ethanethiol and added with enological tannins, with or without oxygen addition. The total levels of these two sulfur compounds were shown to decrease steadily after 60 days of aging, up to 83%. The effect of oxygen sped this decrease, but the effect of enological tannins was very slight. Residual ethanethiol was detected in its disulfide form from approximately 36% in the nonoxygenated wines to 69% in the oxygenated samples.     

Influence of yeast macromolecules on sweetness in dry wines: role of the saccharomyces cerevisiae protein Hsp12

Yeast autolysis during lees contact influences the organoleptic properties of wines especially by increasing their sweet taste. Although observed by winemakers, this phenomenon is poorly explained in enology. Moreover, the compounds responsible for sweetness in wine remain unidentified. This work provides new insights in this way by combining sensorial, biochemical and genetic approaches. First, we verified by sensory analysis that yeast autolysis in red wine has a significant effect on sweetness. Moderate additions of ethanol or glycerol did not have the same effect. Second, a sapid fraction was isolated from lees extracts by successive ultrafiltrations and HPLC purifications. Using nano-LC-MS/MS, peptides released by the yeast heat shock protein Hsp12p were distinctly identified in this sample. Third, we confirmed the sweet contribution of this protein by sensorial comparison of red wines incubated with two kinds of yeast strains: a wild-type strain containing the native Hsp12p and a deletion mutant strain that lacks the Hsp12p protein (Δ°HSP12 strain). Red wines incubated with wild-type strain showed a significantly higher sweetness than control wines incubated with Δ°HSP12 strains. These results demonstrated the contribution of protein Hsp12p in the sweet perception consecutive to yeast autolysis in wine.     

Formation pathways of ethyl esters of branched short-chain fatty acids during wine aging

The particular behavior during wine aging of fermentative branched fatty acid ethyl esters, related to yeast nitrogen metabolism, compared that of their straight-chain analogues, related to yeast lipid metabolism, was first checked in 1-5 year aged Muscadet wines. Quantitative SIDA measurements showed that the levels of the former increased, whereas those of the latter decreased. Then, three hypothetical pathways suggested in the literature to explain these variations of branched esters were investigated. Two Muscadet and Sylvaner wines were spiked with levels of deuterated isobutanoic acid and its ethyl ester, similar to those of their natural analogues, then they were submitted to model aging. Quantitative SIDA measurements on the formation of these natural and labeled ethyl esters from the corresponding acids revealed that the behavior of the natural and labeled compounds were similar. The acid levels were much higher than the ester levels in the initial young wine, and a significant upward trend of their esterification ratios to those of the acid-ester equilibrium was observed with aging. Thus, this equilibrium proved to be the most effective in generating the branched fatty acid ethyl esters during wine aging. In contrast, the formation of these acids by Strecker-type degradation of wine amino acids in the conditions of the model aging or by hydrolysis of their glycoconjugates proved to be ineffective.     

Characterization of the nitrogen compounds released during yeast autolysis in a model wine system

The nitrogen composition of wines aged with yeast for a long period of time, as in the case of sparkling wines, depends on the composition of the base wine and on the compounds released by the yeast. In this paper, the release of the different classes of nitrogen compounds during autolysis of one of the strains of yeast used in the manufacture of sparkling wines has been studied. The yeast, Saccharomyces bayanus, was suspended in a model wine buffer, pH 3.0 and 10% ethanol, and incubated at 30 degrees C. Samples of the autolysate were taken after 4, 24, 48, 72, 168, and 360 h of autolysis. An electrophoretic and chromatographic study was conducted of the proteins, peptides with molecular weights higher and lower than 700 Da, and amino acids released during the autolysis. Using SDS-PAGE, it was observed that it was predominantly polypeptides with molecular weights lower than 10 000 that were released. Through HPLC of the fraction lower than 10 000 Da, it was observed that it is polypeptides with molecular weights of between 10 000 and 700 Da that are released first and that these later break up to give rise to peptides with molecular weights lower than 700 Da, which in turn break down into amino acids. This indicates that the nature of the nitrogen compounds present in wines aged with yeast depends on the aging time, being less polymerized as the aging time increases.     

Study of wine tannin oligomers by on-line liquid chromatography electrospray ionization mass spectrometry.

Thiolysis of a wine tannin fraction yielded trihydroxylated flavanol units (as previously observed in grape skins) in addition to the well-known procyanidins (dihydroxylated units), usually described in the literature for grape condensed tannins. To determine how they occur in condensed tannins, the wine fraction was analyzed by liquid chromatography coupled to electrospray ionization mass spectrometry. Thus, various series of ion peaks containing a variable number of trihydroxylated units were detected as monocharged ions from dimers up to pentamers. From pentamers, oligomers were found as doubly charged ions. Heptamer species corresponded to the highest mass detected. These results showed that wine condensed tannins consist of, besides procyanidins, mixed tri- and dihydroxylated flavanol units and also of pure trihydroxylated flavanol units. These new data should be taken into account to interpret organoleptic properties of wines.     

Changes in the polyphenolic and volatile contents of "fino" sherry wine exposed to ultraviolet and visible radiation during storage

Experiments of accelerated oxidation of "fino" sherry wines have been conducted at 25 degrees C and under the influence of UV-visible radiation (a xenon lamp of 1500 W). With the aim of determining the contribution of UV-vis radiation to the browning phenomenon, two types of glass bottles were employed: topaz bottles (with low values of transmittance in the UV-visible range) and transparent bottles. To identify significant differences between the wine before and after being subjected to the influence of the UV-vis radiation, the values of absorbance at 420 nm and the concentrations of various polyphenolic and volatile compounds were submitted to a multivariate variance analysis. Both factors considered (time and type of bottle) had a statistically significant effect on the values of absorbance at 420 nm and on the concentration of most of the polyphenolic compounds, whereas only the "time" factor was significant for volatile compounds. All wines showed losses in several polyphenolic compounds, which were more severe for the wines bottled in transparent glasses. However, these wines exhibited a lower degree of visual browning (abs 420 nm). In the case of volatile compounds, most of these presented increases during storage exposed to the influence of the UV-vis radiation.     

White wine with red wine-like properties: increased extraction of grape skin polyphenols improves the antioxidant capacity of the derived white wine.

Lower antioxidant activity in white wines in comparison to red wines lies in the low grape-skin-derived polyphenol content. This paper reports the analysis of the antioxidant capacities of white wine samples obtained along two different processing procedures directed to enrich the wine with polyphenols. White wine samples derived from whole squeezed grapes stored for increasing periods of time (up to 18 h) contained increasing concentrations of polyphenols (from 0.35 to 0.55 mmol/L) and, in parallel, exhibited increased capacity to scavenge free radicals and to inhibit copper ion-induced low-density lipoprotein (LDL) oxidation. However, addition of increasing concentrations of alcohol (up to 18%) to the whole squeezed grapes remarkably augmented the extraction of grape skin polyphenols into the wine up to 1.25 mmol/L, resulting in an increased capacity of the wine to scavenge free radicals and to inhibit LDL oxidation, to an extent similar to that of red wine. The extent of LDL oxidation inhibition was directly related to the wine polyphenolic content (r = 0.986). It is concluded that processing white wine by imposing a short period of grape skin contact in the presence of alcohol leads to extraction of grape skin polyphenols and produces polyphenol-rich white wine with antioxidant characteristics similar to those of red wine.     

Occurrence of pyranoanthocyanins in sparkling wines manufactured with red grape varieties

The anthocyanin pigments in rosé (Vitis vinifera cv. Garnacha) and blanc de noir (V. vinifera cv. Monastrell) base and sparkling wines were studied by LC-DAD/ESI-MS. Anthocyanins of grape origin and pyranoanthocyanins resulting from C-4/C-5 cycloaddition of the former ones with pyruvic acid, acetaldehyde, 4-vinylphenol, 4-vinylguaiacol, and 4-vinylcatechol were identified in the different wines. Rosé wines presented a higher total pigment content than blanc de noir wines. Pyranoanthocyanins represented 68.9-76.0% of total pigment content in rosé wines and 49.4-60.7% in blanc de noir wines. Malvidin 3-glucoside-pyruvate was the most abundant pigment in both rosé and blanc de noir base wines. Important qualitative and quantitative changes were observed in terms of the anthocyanin and pyranoanthocyanin pigments after the second (bottle) fermentation and 9 months of aging on yeast lees, but not after a further time (3-9 additional months) of aging on lees. Evaluation of the wine color characteristics was consistent with a greater color stability for the rosé sparkling wines that could be associated with the high content, structural diversity, and spectroscopic features of the pyranoanthocyanins present in these wines.     

Model aging and oxidation effects on varietal, fermentative, and sulfur compounds in a dry botrytized red wine

From harvest until wine arrives to the consumer, oxygen plays a crucial role in the definition of the final aroma. In the present research, the effect of the model oxidative aging on a dry red Botrytis wine, such as Italian Amarone, was considered. Amarone wine was submitted to model oxidative aging and then analyzed with two different approaches (SPE-GC-MS and HS-SPME/GC-MS). The same sampling plan was adopted to study the model aging of the same Amarone wine in anaerobic conditions. The HS-SPME/GC-MS method was applied to investigate for the first time the effect of the oxidative aging on a vast number of fermentative sulfur compounds. This research highlighted peculiar evolutions for several volatile compounds. In particular, benzaldehyde showed a sensitive increment during the oxidative aging, with a rate much higher than that reported for non-Botrytis red wines. On the other hand, several sulfides (dimethyl sulfide, 3-(methylthio)-1-propanol, etc.) disappeared after just 15 days of oxidative aging. A wine oxidation marker such as 3-(methylthio)-propanal was not found in any of the oxidized wines; conversely methionol-S-oxide was tentatively identified. This evidence has not been mentioned in the literature. A possible involvement of grape withering process and Botrytis in these mechanisms was supposed: a dry red wine, produced from the same but without any grape withering process and Botrytis infection (e.g., Bardolino wine), was submitted to oxidative aging and analysis. This red wine showed an evolution similar to those reported in the literature for dry red wines but significantly different from the Amarone wine.     

Extraction of phenolics and changes in antioxidant activity of red wines during vinification.

The moderate consumption of alcoholic beverages has been associated with protection against the development of coronary heart disease. Although alcohol itself can help prevent coronary heart disease through a number of mechanisms, red wine appears to offer protection above and beyond that attributable to alcohol alone. Red wine is a complex fluid containing grape, yeast, and wood-derived phenolic compounds, the majority of which have been recognized as potent antioxidants. The aim of this study was to investigate the major phenolic contributors to the antioxidant activity of wine. To this end, four wines were followed during the first 7-9 days of vinification. Individual phenolic compounds were quantified by HPLC, and antioxidant activity was determined by electron spin resonance spectroscopy. The extraction of the phenolics was found to be influenced by vinification procedure, grape quality, and grape variety. Although fermenting wines reached a total phenolic content comparable to that of a bottled wine after 9 days of vinification, the antioxidant activity was significantly lower than that of a finished wine. This suggests that the larger polyphenolic complexes and condensation products that appear during aging make a sizable contribution to the overall antioxidant activity of red wines.     

Evaluation of the combined effects of enzymatic treatment and aging on lees on the aroma of wine from Bombino bianco grapes

In this study, two different doses of commercial beta-glucanase enzyme preparation were tested to verify their effect on wines aged on lees. These wines were compared with two samples with no enzymatic treatment. The former was aged on lees (control), and the latter was readily filtered off from the yeast cell biomass (standard). Analysis of variance (one-way ANOVA), the Tukey test, and principal component analysis (PCA) were applied to all of the samples, which were analyzed for aroma composition, along with galacturonic acid, total acidity, pH, and color. Results showed a large number of statistically significant differences among samples. In general, wines treated with beta-glucanase were characterized by higher concentration of many volatile compounds. The presence of lees and even more the exogenous enzymatic action enhanced almost all volatile compounds. Besides the high presence of ethyl esters, it is worth mentioning the behavior of hexanol and trans-3-hexenol, which are strongly enhanced by the presence of lees and by enzymatic treatments.     

Ellagic acid and flavonoid antioxidant content of muscadine wine and juice

Antioxidant properties of flavonoids and ellagic acid were characterized in eight wines and juices produced by various processing methodologies from red and white muscadine grape cultivars (Vitis rotundifolia). Juices and wines were produced by hot- and cold-pressed techniques, and additional wine was produced following on-hull fermentation for 3, 5, and 7 days. Chromatographic conditions were developed to simultaneously separate anthocyanins, ellagic acid, and flavonols and correlated to a measurement of overall antioxidant capacity (AOX), and their changes were monitored after storage for 60 days at 20 and 37 degrees C. Regression coefficients between concentrations of individual polyphenolics and AOX ranged from 0.55 for ellagic acid to 0.90 for kaempferol. Both red and white wines had higher AOX values after storage than juices made from an identical grape press, despite lower concentrations of individual polyphenolic compounds. Red wines fermented on-hull had higher initial concentrations of antioxidant polyphenolics as compared to a corresponding hot-pressed juice, but changes in AOX during storage were more affected by time than by storage temperature despite lower concentrations of flavonoids and ellagic acid present at 37 degrees C as compared to 20 degrees C. Oxidative or polymerization reactions significantly decreased levels of monomeric anthocyanins during storage with the greatest losses observed for delphinidin and petunidin 3,5-diglucosides. Processing methods for muscadine wine and juice production were important factors influencing concentrations of antioxidant flavonoids and ellagic acid, while the role of fermentation and time had the greatest influence on retention of AOX properties during storage.