White Wine Protein Research Articles

Influence of the structural features of commercial mannoproteins in white wine protein stabilization and chemical and sensory properties

The cloudy aspect formed in white wines due to protein instability is a visual defect. Sodium bentonite is the most commonly used fining agent to treat this instability, but has usually a negative impact on the wine’s physicochemical and sensory characteristics. Aiming to find suitable alternatives, eleven commercial mannoproteins were chemically characterized concerning their sugar composition and protein content, and their effectiveness on wine protein stabilization. Also, their effect on the amount and nature of phenolic compounds, browning potential, chromatic and sensory characteristic was evaluated. Protein stabilization effectiveness was related to their chemical composition, namely their high mannose to glucose ratio. Additionally, some mannoproteins decreased the browning potential. Thus, mannoproteins could be an effective alternative for protein stabilization, preserving or even improving wine quality.

White Wine Proteins: How Does the pH Affect Their Conformation at Room Temperature?

Our studies focused on the determination of aggregation mechanisms of proteins occurring in wine at room temperature. Even if the wine pH range is narrow (2.8 to 3.7), some proteins are affected by this parameter. At low pH, the formation of aggregates and the development of a haze due to proteins sometimes occur. The objective of this work was to determine if the pH impacted the conformational stability of wine proteins. Different techniques were used: circular dichroism and fluorescence spectroscopy to investigate the modification of their secondary and tertiary structure and also SAXS to determine their global shape. Four pure proteins were used, two considered to be stable (invertase and thaumatin-like proteins) and two considered to be unstable (two chitinase isoforms). Two pH values were tested to emphasize their behavior (pH 2.5 and 4.0). The present work highlighted the fact that the conformational stability of some wine proteins (chitinases) was impacted by partial modifications, related to the exposure of some hydrophobic sites. These modifications were enough to destabilize the native state of the protein. These modifications were not observed on wine proteins determined to be stable (invertase and thaumatin-like proteins).

Multi‐allergen quantification of fining‐related egg and milk proteins in white wines by high‐resolution mass spectrometry

A method based on High-Resolution Mass Spectrometry was developed for the simultaneous determination of fining agents containing potentially allergenic milk (casein) and egg-white (lysozyme and ovalbumin) proteins, added to commercial white wines at sub-ppm levels. Selected tryptic peptides were used as quantitative markers. An evaluation of protein digestion yields was also performed by implementing the (15)N-valine-labelled analogues of the best peptide markers identified for αS1 -casein and ovalbumin. The method was based on the combination of ultrafiltration (UF) of protein-containing wines, tryptic digestion of the dialyzed wine extracts and liquid chromatography/high resolution mass spectrometry (LC/HRMS) analysis of tryptic digests. Peptides providing the most intense electrospray ionization (ESI)-MS response were chosen as quantitative markers of the proteins under investigation. Six-point calibrations were performed by adding caseinate and egg-white powder in the concentration range between 0.25 and 10 µg/mL, to an allergen-free white wine. The following three peptide markers, LTEWTSSNVMEER, GGLEPINFQTAADQAR and ELINSWVESQTNGIIR, were highlighted as best markers for ovalbumin, while GTDVQAWIR and NTDGSTDYGILQINSR for lysozyme and YLGYLEQLLR, GPFPIIV and FFVAPFPEVFGK for caseinate. Limits of detection (LODs) ranged from 0.4 to 1.1 µg/mL. The developed method is suited for assessing the contemporary presence of allergenic milk and egg proteins characterizing egg white and caseinate, fining agents typically employed for wine clarification. The LODs of the method enable the detection of sub-ppm concentrations of residual fining agents, that could represent a potential risk for allergic consumers.

Addition of Carrageenan at Different Stages of Winemaking for White Wine Protein Stabilization

Carrageenan added at different stages of winemaking was assessed for its protein removal and impact on wine heat stability and on the chemical and sensorial profile of the wines. Carrageenan was added to a Semillon during fermentation and after fermentation and to finished wines, and the effect of each addition was compared to that of bentonite fining at the same time point. Data on protein concentration, heat stability, and bentonite requirement indicate that when added at the correct dosage carrageenan was very effective in stabilizing wines at dosages at least three times lower than those of bentonite. In addition, carrageenan treatment did not cause an increase in lees volume relative to bentonite and resulted in very similar chemical parameters to the unfined and bentonite-treated wine. Sensorially, although carrageenan-treated wine was significantly different from the unfined wine, the magnitude of difference did not vary significantly when compared to bentonite treatment. The feasibility of carrageenan use in a winery production setting will need to be determined by individual wineries, as technical issues including frothing, slower filterability, and risk of overfining will need to be considered relative to the benefits, particularly when carrageenan is used before or during fermentation.

Pineapple stem bromelain immobilized on different supports: Catalytic properties in model wine

Bromelain from pineapple stem has been covalently immobilized on different supports to select the more efficient biocatalyst that should be applied toward unstable proteins in real white wine. In this preliminary study, catalytic properties of different immobilized bromelain forms were compared under wine-like conditions, against a synthetic substrate (Bz-Phe-Val-Arg-pNA).Covalent immobilization affected protease kinetic properties, even if all immobilized forms presented both a better substrate affinity and higher half-life (with the exception of a few procedures) with respect to the free enzyme. Stem bromelain was successfully immobilized on chitosan beads without glutaraldehyde thus yielding a food-safe and promising biocatalyst for unstable real wine future application.

Assessment of Potential Effects of Common Fining Agents Used for White Wine Protein Stabilization

A variety of fining agents are commercially available to the wine industry, including proteins and inorganic ion exchangers. These fining agents are essentially used to control the levels of phenolics in wine, but they also have the potential to interact with other wine components, most often as a side effect. They are therefore expected to influence, at least in part, the potential for wine protein haze formation. Six common fining agents—casein, egg albumin, isinglass, chitosan, chitin, and polyvinylpolypyrrolidone (PVPP)—were analyzed to assess their effects on wine protein haze-forming potential and on the levels of proteins and phenolic compounds in a Muscat of Alexandria wine. Bentonite was selected as the positive control, whereas nonfined wine was used as the negative control. Differential results were detected among the selected fining agents when compared to the controls. Egg albumin and chitosan, although incapable of stabilizing the wine, originated a small but significant decrease in the protein haze formed, whereas chitosan and PVPP were second to bentonite in removing the most polyphenols from the wine. Thus, while chitosan fining removes a fraction of polyphenols from the wine and seems to induce a small decrease in its haze-forming potential, PVPP eliminates more polyphenols while leaving its haze-forming potential unaltered. The fining agents analyzed did not significantly affect wine protein content but did remove considerable levels of polyphenols and presented no apparent effect on protein stabilization of the fined wines. Results show that these fining agents do not contribute significantly to protein stabilization in white wines, confirming that bentonite was the most effective agent in wine protein stabilization.

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.

Bromelain from pineapple stem in alcoholic–acidic buffers for wine application

Bromelain from pineapple stem has been studied in unexplored model wine buffer (pH 3.2 tartaric acid and ethanol) in order to evaluate its applicability for white wine protein stabilisation. Bromelain proteolytic activity was studied against several synthetic peptide substrates in McIlvaine buffer covering a wide pH range. Among different synthetic substrates, Bz-Phe-Val-Arg-pNA turned out to be the most suitable one to test bromelain activity at the average minimum pH value of wine (3.2). The protease activity was significantly increased by the presence of 5 mM cysteine while no influence was exerted by EDTA. The inhibiting effect of ethanol was investigated in the range 0–25% (v/v), resulting in being rather limited at the average minimum concentration found in wine (10% v/v). The kinetic parameters were estimated in both McIlvaine (as reference) and model wine buffers. This study showed that stem bromelain worked well at the average minimum pH value of wine, and had a significantly higher activity in model wine buffer ( K a increased of 42%). These results are the basis for suggesting a future biotechnological application of this protease in winemaking.

Proteins in white wines: Thermo-sensitivity and differential adsorbtion by bentonite

Abstract Protein fractions in a Chardonnay wine (invertases, glucanases, chitinases and thaumatin-like proteins) were identified using 2D-electrophoresis and mass spectrometry. The sensitivity of these fractions to heat-induced denaturation and precipitation following heat-treatments at different temperatures was studied and compared to their affinity for bentonite, a clay used to adsorb proteins and stabilise wines with regards to protein hazes. The different proteins exhibited different sensitivity with regards to heat-induced precipitation, glucanases being the most sensitive and invertases the least. Thaumatines and chitinases were characterised by a wide range of behaviours attributed to structural micro-heterogeneities. Protein depletion upon the addition of increasing bentonite doses was also dependent on the considered fraction. A good correlation was shown between protein affinity for bentonite and their sensitivity to heat precipitation. Results were discussed considering the current winemaking practices used to assess white wine stability and define bentonite doses needed to achieve their stabilisation.

Comparative Study of Protein Stabilization in White Wine Using Zirconia and Bentonite: Physicochemical and Wine Sensory Analysis

A semi-industrial application of the continuous stabilization of white wine protein using a column packed with zirconia was studied and compared to the traditional bentonite treatment using a Macabeu white wine. Physicochemical and wine sensory properties were evaluated using a rating system and triangle tests. Continuous protein stabilization was analyzed in three residence times, and the equivalent of 300 BV of wine was used for both treatments. Wine protein content was reduced by 21%, 40%, and 42% using the continuous process with residence times of 7.5, 15, and 30 min, respectively, and by 61.4% using the bentonite treatment. The wines obtained from the packed column were protein stable up to 25, 75, and 175 BV for residence times of 7.5, 15, and 30 min, respectively. The amount of polyphenol removed was less than 10%, and similar amounts were removed from the wine regardless of residence time, while 20.6% of polyphenol was removed using bentonite. The physicochemical and sensory properties of wine treated with bentonite were similar to those of wine treated with zirconia.

Proteolytic activity of Leuconostoc oenos

The proteolytic activity of Leuconostoc oenos protease on white wine proteins and polypeptides was studied. Comparison of the peptide profiles before and after protease action demonstrated the presence of two new and an increase in two other peptide peaks. In total 56.7 mg l−1 of peptides was liberated by action of the Lc.oenos proteases. Essential amino acids for Lc. oenos growth were liberated as free amino acids, and arginine, which has a stimulatory effect on Lc. oenos growth, was quantitatively the more important amino acid obtained by protease activity. Thus proteolytic activity serves to provide the cells with essential growth factors.

White wine continuous protein stabilization by packed column.

Protein stabilization is an important stage in the production of white wine. This paper studies white wine protein stabilization using a continuous process with zirconium oxide (powder and pellets) packed in a column. The results show that the total proteins decrease by 50 and 70% for the pellet and powdered zirconium oxides, respectively. Treatment with all zirconium oxides improves wine stability. The effect of the heat regeneration process on both zirconium oxide forms is to increase the adsorption capacity. The wine treated with powdered zirconium oxide after the regeneration is the most effective for preventing protein haze. The protein profile of wine after treatment shows that the 20-50 kDa and 50-70 kDa fractions are the ones removed preferentially, while the 15 kDa fraction and the ones higher than 70 kDa are removed the least. The results show that the protein fraction with a molecular weight of 15 kDa does not affect the protein instability of the wines studied. The protein fraction with a molecular weight higher than 70 kDa seems to influence protein instability. The physicochemical properties of wine after treatment were not affected, and the values obtained were like those of the standardized range.

White Wine Protein Stabilization by a Continuous Process Using a Packed Column

An alternative method of protein stabilization for a Muscat wine was studied at laboratory scale using zirconium oxide as adsorbent material in a packed column in continuous regime. Protein removal for ZrO₂ was about 70% during the first 50 bed volumes (BV) and decreased progressively until the end of the treatment (100 BV). The first fraction of treated wine (first 50 BV) was protein stable, after which it was protein unstable. The protein fraction of 20 to 30 kDa appeared to be responsible for the protein instability of the wine. After ZrO₂ regeneration, the wines obtained were protein stable during all treatments, since the adsorption capacity of the packed column increased. No relation was found between the protein stability of the wine and polysaccharide removal by the ZrO₂ in the original form or after regeneration.

A proteolytic effect of Oenococcus oeni on the nitrogenous macromolecular fraction of red wine

The proteolytic activity of Oenococcus oeni protease on the release of individual amino acids and peptides from a red wine macromolecular nitrogenous fraction was evaluated. 148.7 mg l −1 of individual amino acids were released and a difference of 109.5 mg l −1 with respect to the amino acids liberation from white wine was observed. Stimulatory amino acids for O. oeni growth, glutamic acid and proline, were the most important amino acids obtained by the protease activity. In the HPLC and spectral analysis of peptides before and after the protease activity of O. oeni, several changes have been observed. Three new peaks, six higher and one lower have been found in the chromatogram obtained after the enzyme action. The specific activity of O. oeni protease is higher on the red than on the white wine proteins and this is important considering the lower protein concentration in red wine.

A proteolytic effect ofOenococcus oenion the nitrogenous macromolecular fraction of red wine

The proteolytic activity of Oenococcus oeni protease on the release of individual amino acids and peptides from a red wine macromolecular nitrogenous fraction was evaluated. 148.7 mg l−1 of individual amino acids were released and a difference of 109.5 mg l−1 with respect to the amino acids liberation from white wine was observed. Stimulatory amino acids for O. oeni growth, glutamic acid and proline, were the most important amino acids obtained by the protease activity. In the HPLC and spectral analysis of peptides before and after the protease activity of O. oeni, several changes have been observed. Three new peaks, six higher and one lower have been found in the chromatogram obtained after the enzyme action. The specific activity of O. oeni protease is higher on the red than on the white wine proteins and this is important considering the lower protein concentration in red wine.

White Wine Protein Analysis by Capillary Zone Electrophoresis

Capillary zone electrophoresis was evaluated as a method for the analysis of wine proteins. Seven wines from three different varieties, Chardonnay, Sauvignon blanc, and Muscat of Alexandria, were used in this systematic analysis of the effect of variables of capillary electrophoresis conditions on wine protein profiles. While there was some differences in the magnitude of protein peaks as a function of vintage and winemaking conditions, each of the varieties displayed a distinct and distinguishable protein profile. If wine is analyzed directly, both proteinaceous and nonproteinaceous UV-absorbing materials are detected. The protein fraction can be obtained from wines by ultrafiltration and analyzed separately. Resuspension of the retentate proteins following ultrafiltration under conditions allowing sample stacking yielded the greatest number and sharpest separation of peaks. The electrophoresis running buffer impacted protein profiles and number of peaks, as expected. Protein peaks were found to be protease-sensitive, suggesting that wine proteins are not intrinsically resistant to protease degradation. Hazes induced by heat/chill treatment effectively stripped the wines of protein content. Capillary zone electrophoresis represents an excellent tool in the analysis of wine proteins and has the potential to differentiate wine varietals.

Protein Fraction Analysis of White Wine by FPLC

This paper proposes a method, using FPLC, for separating white wine proteins into several fractions. Gel filtration followed by cationic exchange were used for quantifying and separating the different protein fractions of wine. Four peaks were detected by gel filtration, but SDS-PAGE indicate that only peak 2 and peak 3 were proteins. Subsequent analysis of peaks 2 and 3 by cationic exchange chromatography and SDS-PAGE indicate that peak 2 is a single protein of 60 KDa and peak 3 is an ensemble of several proteins of similar molecular weights and different electric charges. We have also studied the fate of proteins throughout the winemaking process, and this study shows that total protein content decreases. This decrease was much greater in the proteins with high positive electric charge.

Proteolytic activity of Leuconostoc oenos: Effect on proteins and polypeptides from white wine

The proteolytic activity of Leuconostoc oenos protease on white wine proteins and polypeptides was studied. Comparison of the peptide profiles before and after protease action demonstrated the presence of two new and an increase in two other peptide peaks. In total 56.7 mg l −1 of peptides was liberated by action of the Lc.oenos proteases. Essential amino acids for Lc. oenos growth were liberated as free amino acids, and arginine, which has a stimulatory effect on Lc. oenos growth, was quantitatively the more important amino acid obtained by protease activity. Thus proteolytic activity serves to provide the cells with essential growth factors.

Proteins in white wine, I: Procyanidin occurrence in soluble proteins and insoluble protein hazes and its relationship to protein instability

The presence of procyanidins in wine protein fractions was established by a chemical assay and by pyrolysis DI-EI MS/MS of proteins. The chemical assay, involving acid-catalysed oxidative degradation of the procyanidins, gave cyanidin which was specifically quantified by HPLC. The assay was calibrated with purified grape seed tannin (GST); procyanidin was expressed in μg GST. Cyanidin yields were little altered by the presence of a large excess of protein. The limit of detection of the assay was 1 μg GST and the limit of quantification 3.5 μg GST. Both heat-induced and natural hazes isolated from various white wines were all shown to contain procyanidins, with a content ranging from < 0.02 to 4.9% (w/w). Although a crude soluble protein isolate from white wine contained a detectable amount of procyanidins, none were found in fractions separated chromatographically from this isolate. This observation, and the absence of procyanidins in resolubilised heat-induced haze, demonstrated that procyanidins are only weakly associated with both heat-induced hazes and soluble wine proteins. Nevertheless, procyanidins appear to be implicated in the formation of protein hazes in wine, because many soluble wine proteins that had been rendered free of procyanidins would precipitate to give haze in wine but not in model wine which was devoid of phenolic compounds.

Proteins in white wine, II: Their resistance to proteolysis is not due to either phenolic association or glycosylation

Soluble wine proteins were separated by anion exchange chromatography. When the fractionated proteins were treated in model wine solution with an active peptidase preparation over six weeks at 15°C, all but one of the separated proteins were totally resistant to proteolysis. Because all the separated proteins contained no associated phenolic compounds and because, from neutral sugar analysis, most contained less than one mole of carbohydrate per mole of protein, neither phenolic association nor glycosylation are responsible for their proteolytic resistance.