Stability of white wine proteins: combined effect of pH, ionic strength, and temperature on their aggregation

Protein haze development in white wines is an unacceptable visual defect attributed to slow protein unfolding and aggregation. It is favored by wine exposure to excessive temperatures but can also develop in properly stored wines. In this study, the combined impact of pH (2.5-4.0), ionic strength (0.02-0.15 M), and temperature (25, 40, and 70 °C) on wine protein stability was investigated. The results showed three classes of proteins with low conformational stability involved in aggregation at room temperature: β-glucanases, chitinases, and some thaumatin-like protein isoforms (22-24 kDa). Unexpectedly, at 25 °C, maximum instability was observed at the lower pH, far from the protein isoelectric point. Increasing temperatures led to a shift of the maximum haze at higher pH. These different behaviors could be explained by the opposite impact of pH on intramolecular (conformational stability) and intermolecular (colloidal stability) electrostatic interactions. The present results highlight that wine pH and ionic strength play a determinant part in aggregation mechanisms, aggregate characteristics, and final haze.     

Sulfate--a candidate for the missing essential factor that is required for the formation of protein haze in white wine

Protein haze formation in white wine is dependent on the presence of both wine protein and other unknown wine components, termed factor(s) X. The ability to reconstitute protein haze upon heating artificial model wine solutions (500 mg/L thaumatin, 12% ethanol, 4 g/L tartaric acid) to which candidate components were added was employed to identify factor(s) X. No protein haze was formed in the absence of additives. The individual or combined addition of caffeic acid, caftaric acid, epicatechin, epigallocatechin-O-gallate, gallic acid, or ferulic acid at typical white wine concentrations did not generate protein haze. However, PVPP fining of commercial wines resulted in a reduction in protein haze, suggesting that phenolic compounds may play a modulating role in haze formation. To elucidate the nature of the unknown factor(s) wine was fractionated and fractions were back-added to model wine and tested for their essentiality. Wine fractions were generated by ultrafiltration, reverse-phase chromatography, and mixed-mode anion-exchange and reverse-phase chromatography. The only purified fraction containing the essential component(s) was free of phenolic compounds, and analysis by mass spectrometry identified sulfate anion as the dominant component. Reconstitution with KHSO4 using either commercially available thaumatin or wine proteins confirmed the role of sulfate in wine protein haze formation. The two main wine proteins, thaumatin-like protein and chitinase, differed in their haze response in model wines containing sulfate. Other common wine anions, acetate, chloride, citrate, phosphate, and tartrate, and wine cations, Fe(2+/3+) and Cu(+/2+), when added at typical white wine concentrations were not found to be essential for protein haze formation.     

Roles of grape thaumatin-like protein and chitinase in white wine haze formation

Grape chitinase was found to be the primary cause of heat-induced haze formation in white wines. Chitinase was the dominant protein in a haze induced by treating Sauvignon blanc wine at 30 °C for 22 h. In artificial wines and real wines, chitinase concentration was directly correlated to the turbidity of heat-induced haze formation (50 °C for 3 h). Sulfate was confirmed to have a role in haze formation, likely by converting soluble aggregates into larger visible haze particles. Thaumatin-like protein was detected in the insoluble fraction by SDS-PAGE analysis but had no measurable impact on turbidity. Differential scanning calorimetry demonstrated that the complex mixture of molecules in wine plays a role in thermal instability of wine proteins and contributes additional complexity to the wine haze phenomenon.     

Saccharomyces cerevisiae mannoproteins that protect wine from protein haze: their release during fermentation and lees contact and a proposal for their mechanism of action

A fraction containing the mannoproteins released during fermentation from the winemaking strain of Saccharomyces cerevisiae, Maurivin PDM, was able to reduce the visible protein haze in white wine. This fraction of haze protective mannoprotein material (HPM) could be recovered by either ultrafiltration or ethanol precipitation. The kinetics of the release of both mannose- and glucose-containing polymers during the growth cycle of PDM were determined as a guide to the release of HPM. Active HPM was first detected in the culture supernatant when the cells were exponentially growing. HPM was also released into the medium under an environment simulating winemaking conditions by PDM cells during fermentation as well as during storage on yeast lees. Since the amounts of HPM released during fermentation are greater than those subsequently extracted from the cell wall, fermentation would be a more viable procedure than extraction from yeast cells for the commercial production of HPM. Yeast invertase, a mannoprotein with haze protective activity, was used as a model substrate to investigate the mechanism of haze protection. Invertase was found to reduce visible turbidity but not prevent protein precipitation. Invertase itself did not precipitate but remained soluble in the wine. On the basis of these observations, we propose that the mechanism of haze protection may be one of competition between HPM and wine proteins for unknown wine component(s), the latter being required for the formation of large insoluble aggregates of denatured protein. As the available concentration of these components decreases, due to the presence of HPM, the particle size of the haze decreases and thus visible turbidity declines.     

Changes during storage in conventional and ecological wine: phenolic content and antioxidant activity

Polyphenol content, free radical scavenging capacity, and changes during storage over 7 months in the dark were studied in ecological and conventional red and white wines. In red wines, the most changeable components during storage were the anthocyanins since during storage anthocyanins content decreased 88% in conventional wine and 91% in ecological wine. Initially, the total flavonol contents of the conventional and ecological red wines were 163.88 +/- 2.69 and 153.58 +/- 1.71 mg/L, respectively, and no significant variations occurred during storage. No differences in hydroxycinnamic acid derivatives content between conventional and ecological red and white wines were observed. The flavonol level in white wines was very low, as expected since these compounds are found in grape skin. The initial antioxidant activity was 5.37 +/- 0.14 and 5.82 +/- 0.31 mM equivalents Trolox for conventional and ecological red wines, respectively; no significant differences were observed (p = 0.2831), and these values were 7-8 times higher than the antioxidant activity observed in conventional and ecological white wine. In contrast with other studies, the total concentrations of phenolic compounds in conventional and ecological red and white wines were not related to antioxidant activity (p > 0.05). In red wines, no significant differences were observed in the antioxidant activity of ecological and conventional red wine (p = 0.28), while in white wine significant differences were observed in the antioxidant activity between conventional and ecological white wine (p = 0.006).     

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.     

A cyclic voltammetry method suitable for characterizing antioxidant properties of wine and wine phenolics

Phenolic antioxidants are ranked by reducing strength and characterized for reversibility using cyclic voltammetry at a glassy carbon electrode. Phenolics with an ortho-diphenol group show a first oxidation peak close to 400 mV (vs. Ag/AgCl) in a model wine solution (12% ethanol, 0.033 M tartaric acid, adjusted to pH 3.6), with a linear concentration dependence below 0.01 mM. Dilution of white wines 10x, and red wines 400x, gave first oxidation peak currents in the 1.5 to 2.2 microA range and 1.9 to 3.4 microC of charge passed by 500 mV, producing values for the concentrations of phenolic antioxidants with low oxidation potentials in the wines. Further peaks in the cyclic voltammograms of the diluted wines correspond to classes of phenolics with higher oxidation potentials, providing a qualitative assessment of wine phenolics based on reducing strength.     

Antioxidant capacities and phenolics levels of French wines from different varieties and vintages.

Phenolics from grapes and wines can play a role against oxidation and development of atherosclerosis. Levels of phenolics, major catechins [(+)-catechin, (-)-epicatechin, procyanidin dimers B1, B2, B3, and B4], phenolic acids (gallic acid and caffeic acid), caftaric acid, malvidin-3-glucoside, peonidin-3-glucoside, and cyanidin-3-glucoside were quantified by HPLC with UV detection for 54 French varietal commercial wines taken from southern France to study the antioxidant capacity and the daily dietary intake of these compounds for the French population. The highest antioxidant capacity was obtained with red wines and ranged from 12.8 mmol/L (Grenache) to 25.2 mmol/L (Pinot Noir). For white wines, Chardonnay enriched in phenolics by special wine-making was found to have an antioxidant capacity of 13.8 mmol/L, comparable to red wine values. For red wines classified by vintages (1996-1999) antioxidant capacities were approximately 20 mmol/L and then decreased to 13.4 mmol/L for vintages 1995-1991. Sweet white wines have 1.7 times more antioxidant capacity (3.2 mmol/L) than dry white wines (1.91 mmol/L). On the basis of a still significant French wine consumption of 180 mL/day/person, the current daily intake of catechins (monomers and dimers B1, B2, B3, and B4) averaged 5 (dry white wine), 4.36 (sweet white wines), 7.70 (rosé wines), 31.98 (red wines), and 66.94 (dry white wine enriched in phenolic) mg/day/resident for the French population. Red wine, and particularly Pinot Noir, Egiodola, Syrah, Cabernet Sauvignon, and Merlot varieties, or Chardonnay enriched in phenolics during wine-making for white varieties contribute to a very significant catechin dietary intake.     

Effect of flash release treatment on phenolic extraction and wine composition

The flash release (FR) process, consisting of rapidly heating the grapes and then applying strong vacuum, has been proposed to increase the polyphenol content of red wines. Its impact on polyphenol extraction kinetics and on the polyphenol composition of red juice and wines was studied over two seasons on different grape varieties (Grenache, Mourvedre, Carignan). The FR process allows fast extraction of all phenolic compounds (hydroxycinnamic acids, flavonols, anthocyanins, catechins, proanthocyanidins) and can be used to produce polyphenol-enriched grape juices. However, the concentration of all polyphenols dramatically decreased throughout fermentation when pressing was achieved immediately after FR. The FR wines made with pomace maceration were also enriched in polyphenols compared to the corresponding control wines. Increasing the duration of high-temperature exposure in the FR treatment further increased extraction of phenolic compounds but also accelerated their conversion to derived species. The tannin-to-anthocyanin ratio was particularly low in the wine fermented in the liquid phase, higher after FR than in the control, and even higher after longer heating. FR resulted in an increased tannin-to-anthocyanin ratio and an increased conversion of anthocyanins to tannin-anthocyanin adducts showing the same color properties as anthocyanins. The tannin-to-anthocyanin ratio was particularly low in the wine fermented in the liquid phase that also contained larger amounts of orange sulfite bleaching-resistant pigments.     

Genetic determinants of the release of mannoproteins of enological interest by Saccharomyces cerevisiae

Cell wall mannoproteins released by Saccharomyces cerevisiae during wine fermentation and aging have recently attracted the attention of enologists and researchers in enology due to their positive effect over a number of technological and quality properties of the wines, including protein and tartaric stability, aroma and color stability, astringency, mouthfeel, malolactic fermentation, or foam properties of sparkling wines. This work has investigated the effect of deletions involving genes related to cell wall biogenesis on the release of mannoproteins, as well as the effect of the released mannoproteins on wine protein stability. When available, the phenotypes have been studied in different genetic backgrounds, in haploid or diploid strains, and in homo- or heterozygosis. Strains deleted for GAS1, GPI7, or KNR4 release higher amounts of mannoproteins and polysaccharides to the medium. These increased amounts of mannoproteins and polysaccharides lead to a stronger stability of Sauvignon Blanc wines against protein haze.     

Evolution of 3-mercaptohexanol, hydrogen sulfide, and methyl mercaptan during bottle storage of Sauvignon blanc wines. Effect of glutathione, copper, oxygen exposure, and closure-derived oxygen

The effects of wine composition and postbottling oxygen exposure on 3-mercaptohexanol (3-MH), hydrogen sulfide (H2S), and methyl mercaptan (MeSH) were investigated. A Sauvignon blanc wine with initial copper concentration of 0.1 mg/L was treated with copper sulfate and/or glutathione (GSH) prior to bottling to give final concentrations of 0.3 and 20 mg/L, respectively. The wines were bottled with a synthetic closure previously stored in either ambient air or nitrogen to study the effect of the oxygen normally present in the closure. Bottled wines were stored for 6 months in either air or nitrogen to study the effect of oxygen ingress through the closure. Copper addition resulted in a rapid initial decrease in 3-MH. During storage, a further decrease of 3-MH was observed, which was lower with GSH addition and lowered oxygen exposure. H2S accumulated largely during the second 3 months of bottle storage, with the highest concentrations attained in the wines treated with GSH and copper. Lower oxygen from and through the closure promoted H2S accumulation. The concentration of MeSH was virtually not affected by the experimental variables at 6 months, although differences were observed after 3 months of storage. The implications for wine quality are discussed.     

Relationship between red wine grade and phenolics. 2. Tannin composition and size

Commercial red wines ( Vitis vinifera L. cv. Shiraz) produced during the 2009 vintage underwent winemaker assessment for allocation grade soon after production. The wines were then subjected to phenolic analysis to measure wine color (total anthocyanin, SO(2) nonbleachable pigment, and wine color density) and tannins (concentration, composition, and average degree of polymerization). A positive relationship was found between wine phenolic concentration and projected bottle price. Tannin compositional analysis suggested that there was specifically a relationship between wine grade and skin-derived tannins. These results suggest that maximization of skin tannin concentration and/or proportion is related to an increase in projected wine bottle price.     

Method for the gas chromatographic assay with mass selective detection of trichloro compounds in corks and wines applied to elucidate the potential cause of cork taint

To investigate the role of trichloro compounds as a potential cause of "cork taint" in wine, an assay for trichloroanisole (TCA) and trichlorophenol (TCP) in corks and wine was developed utilizing solid phase extraction on a C(18) cartridge followed by gas chromatography with mass selective detection. Recovery and imprecision for TCA were 86-102 and 1.6-5.8%, respectively, and for TCP 82-103% and 1.7-3.9%, respectively. Limits of detection and quantitation were 0.1 and 2 ng/L, respectively, for TCA, and 0.7 and 4 ng/L, respectively for TCP. A survey of 2400 commercial wines revealed a higher incidence of cork taint in white wine than in red and in wines utilizing composite cork closures; wines from central Europe and Spain had higher overall rates of contamination and those from Canada and Italy the lowest. Significant but modest associations were found between the TCA and TCP contents of the wines and corks, but many wines exhibiting cork taint had low or undetectable concentrations of TCA. Over a 12-month period, experimentally bottled wines exhibited a slow increase in TCA and TCP content while cork closures manifested a decrease; most bottles showing cork taint contained low levels of TCA, and TCP concentrations were well below the sensory threshold. Neither compound was cytotoxic to human cell lines in culture up to final concentrations of 500 ng/mL. It was concluded that these two trichloro compounds are, at most, minor components of cork taint in commercial wines.     

Heat-induced gelation of chicken Pectoralis major myosin and beta-lactoglobulin

The denaturation, aggregation, and rheological properties of chicken breast muscle myosin, beta-lactoglobulin (beta-LG), and mixed myosin/beta-LG solutions were studied in 0.6 M NaCl, 0.05 mM sodium phosphate buffer, pH 7.0, during heating. The endotherm of a mixture of myosin and beta-LG was identical to that expected if the endotherm of each protein was overlaid on the same axis. The maximum aggregation rate (AR(max)) increased, and the temperature at the AR(max) (T(max)) and initial aggregation temperature (T(o)) decreased as the concentration of both proteins was increased. The aggregation profile of <0.5% myosin was altered by the presence of 0.25% beta-LG. Addition of 0.5-3.0% beta-LG decreased storage moduli of 1% myosin between 55 and 75 degrees C, but increased storage moduli (G') when heated to 90 degrees C and after cooling. beta-LG had no effect on the gel point of > or =1.0% myosin, but enhanced gel strength when heated to 90 degrees C and after cooling. After cooling, the G' of 1% myosin/2%beta-LG gels was about 1.7 times greater than that of gels prepared from 2% myosin/1% beta-LG.     

Polyphenol compounds and anti-inflammatory activities of Korean black raspberry ( Rubus coreanus Miquel) wines produced from juice supplemented with pulp and seed

Three types of Korean black raspberry wine were produced via alcoholic fermentation from juice, juice-pulp, and juice-pulp-seed, respectively. These wines were compared in terms of their anti-inflammatory activities and polyphenol contents. The total content of polyphenol compounds in wines was increased by 22.4% after supplementation with pulp and by 56.7% after supplementation with both pulp and seed. The reduction rate of NO evolution was highest in the order juice-pulp-seed wine, juice-pulp wine, and juice wine. Addition of the juice-pulp-seed wine at a level of 62.5-500 mg/L decreased the NO evolution rate by 40.5-94.2%. Eight fractions were obtained from juice-pulp-seed wine via ethyl acetate extraction and silica gel chromatography. Of these, the AF fraction, which exhibited the highest in vitro anti-inflammatory activity, exerted inhibitory effects on ear edema, writhing response, and vein membrane vascular permeability in mice. 3,4-Dihydroxybenzoic acid accounted for 37.6% of the total polyphenol content in the AF fraction.     

Determination of stilbenes (delta-viniferin, trans-astringin, trans-piceid, cis- and trans-resveratrol, epsilon-viniferin) in Brazilian wines

Phenolics from grapes and wines can play a role against oxidation and development of atherosclerosis. Stilbenes have been shown to protect lipoproteins from oxidative damage and to have cancer chemopreventive activity. We describe a method for the direct determination of stilbenes in several red wines using high-performance liquid chromatography with UV detection. In a survey of 12 commercial wines from the south of Brazil (Rio Grande del Sul), levels of delta-viniferin are reported for the first time in different varieties of red wines. Brazilian red wine contains trans-astringin, trans-piceid, trans-resveratrol, cis-resveratrol (in high quantity: 5 times more than the trans form), epsilon-viniferin, and a compound isolated for the first time in wine, trans-delta-viniferin. Isolation and identification of delta-viniferin was achieved by NMR after extraction and fractionation of red wine phenolics. delta-Viniferin contributes, as well as cis-resveratrol and trans-piceid, to a significant proportion of stilbenes in wine dietary intake, particularly with Merlot varieties containing an average level of 10 mg/L for delta-viniferin, 15 mg/L for cis-resveratrol, and 13 mg/L for trans-piceid. The total stilbene intake from wine origin was estimated for the Brazilian population as 5.3 mg/day per person (on the basis of a regular wine consumption of 160 mL/day). delta-Viniferin can contribute to around 20% of total stilbenes in wine (average of 6.4 mg/L in red Brazilian wines). It would be important in the future to investigate the origins of the differences in wine stilbene levels in relation to the vine varieties, and the bioavailability of the newly extracted stilbene delta-viniferin in plasma after consumption of different types of wines.     

Determination of free proline and monosaccharides in wine samples by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD)

A sensitive and selective analytical method for the simultaneous separation and quantitative determination of proline and free monosaccharides in wine samples by high-performance anion-exchange chromatography coupled with pulsed amperometric detection is described. Under optimized experimental conditions, a complete separation was obtained in less than 30 min, using an isocratic elution with 10 mM NaOH and 1 mM Ba(OAc)(2). No postcolumn addition of strong bases to the eluent for enhancing detection sensitivity was needed. Upon 25-fold sample dilution and purification to avoid interference of tannins, pigments, and phenolic compounds, the fingerprinting of common monosaccharides (i.e., arabinose, glucose, fructose, galactose, and xylose) and proline in wines, musts, and vinegars can be easily accomplished. The method allows high recovery and satisfies the necessary requirements for accuracy, repeatability, and sensitivity. Values obtained for proline content ranged from 470 to 1190 mg/L in "Aglianico" red wines (mean value, 870 +/- 192 mg/L, n = 21) and from 168 to 286 mg/L in white wines (mean value, 208 +/- 32 mg/L, n = 11). Lower levels were found in musts of red and white grapes, 550 and 87 mg/L, respectively. The lowest content of proline, ca. 10 mg/L, was found both in white and red vinegars.     

Immunochemical and mass spectrometry detection of residual proteins in gluten fined red wine

Recently, wheat gluten has been proposed as technological adjuvant in order to clarify wines. However, the possibility that residual gluten proteins remain in treated wines cannot be excluded, representing a hazard for wheat allergic or celiac disease patients. In this work, commercial wheat glutens, in both partially hydrolyzed (GBS-P51) and nonhydrolyzed (Gluvital 21000) forms, were used as fining agents in red wine at different concentrations. Beside immunoenzymatic analyses using anti-gliadin, anti-prolamin antibodies and pooled sera of wheat allergic patients, a method based on liquid chromatography coupled to mass spectrometry has been proposed to detect residues of gluten proteins. Residual gluten proteins were detected by anti-prolamin antibodies, anti-gliadin antibodies and sera-IgE only in the wine treated with GBS-P51 at concentration 50, 150, and 300 g/hL, respectively, whereas no residual proteins were detected by these systems in the wine treated with Gluvital 21000. In contrast liquid chromatography-mass spectrometry analyses allowed the detection of proteins in red wines fined down to 1 g/hL of Gluvital 21000 and GBS-P51. Our results indicate that MS methods are superior to immunochemical methods in detecting gluten proteins in wines and that adverse reactions against gluten treated wines cannot be excluded.     

Evolution and occurrence of 1,8-cineole (eucalyptol) in Australian wine

A new method has been developed for the quantitation of 1,8-cineole in red and white wines using headspace solid-phase microextraction (SPME) combined with stable isotope dilution analysis (SIDA) and gas chromatography-mass spectrometry (GC-MS). An extensive survey of Australian wines (44 white and 146 red) highlighted that only red wines contained significant amounts of 1,8-cineole (up to 20 μg/L). Hydrolytic studies with limonene and α-terpineol, putative precursors to 1,8-cineole, showed a very low conversion into 1,8-cineole (< 0.6%) over a 2 year period, which does not account for the difference between white and red wines. 1,8-Cineole was chemically stable in model wine solution over 2 years, and absorption from a Shiraz wine by bottle closures was most evident for a synthetic closure only (14% absorption after 1 year). Two commercial ferments at two different locations were monitored daily to investigate the evolution of 1,8-cineole throughout fermentation. Both ferments showed daily increases in 1,8-cineole concentration while in contact with grape solids, but this accumulation ceased immediately after pressing. This observation is consistent with the extraction of 1,8-cineole into the ferment from the solid portions of the grape berries.