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

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.     

The wine proteins

Proteins are typically present in wines in low concentrations, contributing little to their nutritive value. However, they assume a considerable technological and economical importance because they greatly affect the clarity and stability of wines. Although exhibiting a large diversity, the majority of the wine proteins are structurally related and have been identified as pathogenesis related (PR) proteins. Thus, different wines are essentially composed by identical sets of polypeptides. They derive from the grape pulp, and survive the vinification process simply because they are highly resistant to proteolysis and to the low pH characteristic of wines. There is increasing evidence suggesting that although protein-dependent, the development of turbidity in wines is controlled by a number of factors of non-protein origin, such as polyphenols, the wine pH and the presence of polysaccharides. A variety of procedures has been developed and tested for the specific removal of proteins from wines. Even though bentonite fining is nonspecific and can impair the quality of wine, it remains the only effective method to stabilize wines.     

Protein haze formation in wines revisited. The stabilising effect of organic acids

The effect on the wine protein haze potential of five organic acids commonly encountered in wines (l(+)-tartaric, l(−)-malic, citric, succinic and gluconic acids) was assessed. All five acids, tested at 20 mM, reduced dramatically the haze potential of proteins, either in wine or dissolved in water, throughout the range of pH values typical of wines (i.e., from 2.8 through 3.8). Subtle differences among the acid effects did not correlate with the number of their carboxyl groups, but were attributed to electrostatic interactions that depend upon the acid pK_a values, the protein pI values and the medium pH. These results invalidate or question the validity of all experiments on wine proteins involving wine model solutions containing organic acids. Overall, the results obtained in the present work clearly indicate that organic acids with a common occurrence in wines exhibit a stabilising effect upon the haze potential of the wine proteins.     

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.     

Fast analysis of proteins in wines by capillary gel electrophoresis

Wine proteins play an important role in different characteristics of wine (e.g., aroma and body, foaming in sparkling wines). They can also cause a number of technological problems during vinification and may be responsible for the appearance of turbidity in bottled wine. These important features of proteins in wine have made necessary the development of new and fast analytical methods that can provide deeper knowledge about these biopolymers. However, separation and characterization of wine proteins is difficult and time-consuming mainly due to their low concentration and large number of interfering compounds. Besides, long sample preparation protocols can bring about protein decomposition. This paper proposes a new and fast method for carrying out the analysis of the protein fraction of wines. The procedure consists of direct treatment of wine using a centrifugal filter device (CFD), denaturation of the proteinaceous fraction with sodium dodecyl sulfate (SDS) and 2-mercaptoethanol, and subsequent CGE analysis of SDS-proteins. Results on the molecular weight (Mw) and relative quantity of proteins of wines are attained in about 1 h with this procedure. The method is applied to analyze different wines from Canary Islands. To our knowledge, this is the first report of separation of wine proteins according to their Mw by CGE.     

Preventing protein haze in bottled white wine

Slow denaturation of wine proteins is thought to lead to protein aggregation, flocculation into a hazy suspension and formation of precipitates. The majority of wine proteins responsible for haze are grape‐derived, have low isoelectric points and molecular weight. They are grape pathogenesis‐related (PR) proteins that are expressed throughout the ripening period post véraison, and are highly resistant to low pH and enzymatic or non‐enzymatic proteolysis. Protein levels in un‐fined white wine differ by variety and range up to 300 mg/L. Infection with some common grapevine pathogens or skin contact, such as occurs during transport of mechanically harvested fruit, results in enhanced concentrations of some PR proteins in juice and wine. Oenological control of protein instability is achieved through adsorption of wine proteins onto bentonite. The adsorption of proteins onto bentonite occurs within several minutes, suggesting that a continuous contacting process could be developed. The addition of proteolytic enzyme during short term heat exposure, to induce PR protein denaturation, showed promise as an alternative to bentonite fining. The addition of haze‐protective factors, yeast mannoproteins, to wines results in decreased particle size of haze, probably by competition with wine proteins for other non‐proteinaceous wine components required for the formation of large insoluble aggregations of protein. Other wine components likely to influence haze formation are ethanol concentration, pH, metal ions and phenolic compounds.     

Precipitation of salivary proteins after the interaction with wine: the effect of ethanol, pH, fructose, and mannoproteins

Astringency is a complex sensation mainly caused by the precipitation of salivary proteins with polyphenols. In wine it can be enhanced or reduced depending on the composition of the medium. In order to investigate the effect of ethanol, tartaric acid, fructose, and commercial mannoproteins (MPs) addition on the precipitation of salivary proteins, the saliva precipitation index (SPI) was determined by means of the sodium dodecyl sulphate polyacrylamide gel electrophoresis of human saliva after the reaction with Merlot wines and model solutions. Gelatin index, ethanol index, and Folin-Ciocalteu index were also determined. As resulted by Pearson's correlation, data on SPI were well correlated with the sensory analysis performed on the same samples. In a second experiment, increasing the ethanol (11%-13%-17%), MPs (0-2-8 g/L), fructose (0-2-6 g/L) level, and pH values (2.9-3.0-3.6), a decrease in the precipitation of salivary proteins was observed. A difference in the SPI between model solution and red wine stated that an influence of wine matrix on the precipitation of salivary proteins occurred. PRACTICAL APPLICATION: Results provide interesting suggestions for enologists, which could modulate the astringency of red wine by: (i) leaving some residual reducing sugars (such as fructose) in red wine during winemaking of grapes rich in tannins; (ii) avoiding the lowering of pH; (iii) adding commercial mannoproteins or promoting a "sur lie" aging; and (iv) harvesting grapes at high technological maturity in order to obtain wines with a satisfactory alcoholic content when possible.     

Interactions between whey proteins and salivary proteins as related to astringency of whey protein beverages at low pH

Whey protein beverages have been shown to be astringent at low pH. In the present study, the interactions between model whey proteins (β-lactoglobulin and lactoferrin) and human saliva in the pH range from 7 to 2 were investigated using particle size, turbidity, and ζ-potential measurements and sodium dodecyl sulfate-PAGE. The correlation between the sensory results of astringency and the physicochemical data was discussed. Strong interactions between β-lactoglobulin and salivary proteins led to an increase in the particle size and turbidity of mixtures of both unheated and heated β-lactoglobulin and human saliva at pH ∼3.4. However, the large particle size and high turbidity that occurred at pH 2.0 were the result of aggregation of human salivary proteins. The intense astringency in whey protein beverages may result from these increases in particle size and turbidity at these pH values and from the aggregation and precipitation of human salivary proteins alone at pH <3.0. The involvement of salivary proteins in the interaction is a key factor in the perception of astringency in whey protein beverages. At any pH, the increases in particle size and turbidity were much smaller in mixtures of lactoferrin and saliva, which suggests that aggregation and precipitation may not be the only mechanism linked to the perception of astringency in whey protein.     

发酵酒储藏期间浑浊沉淀类型及澄清措施

发酵酒（黄酒、葡萄酒、果酒）组成成分复杂,酒体稳定性较差,经过澄清处理后在储藏期仍然容易产生浑浊沉淀。浑浊沉淀现 象对酒体的感官鉴赏、商品价值及企业名誉等方面有着不利的影响。 因此,为提高酒体稳定性,延长货架期,探究发酵酒浑浊沉淀产 生的原因并提供解决方案是目前急需攻克的技术难题,同时也是研究热点。 该文对发酵酒储藏期间浑浊沉淀产生的类型、形成机理 及不同澄清措施的研究进展进行了综述。通过对3种发酵酒浑浊沉淀类型及澄清措施的探讨,为发酵酒进一步提高稳定性、改善感官 品质、延长货架期提供参考意义。     

Influence of Botrytis cinerea infection on Champagne wine proteins (characterized by two-dimensional electrophoresis/immunodetection) and wine foaming properties

Proteins are implicated in the foam stabilization of Champagne wines. They may have a grape, yeast, bacteria or fungal origin. Botrytis cinerea is a widespread fungal pathogen, which is the causal agent for gray mold. The first part of this work showed the deleterious effect of the presence of this fungus on the foaming properties of a champenois base wine. Foamability and foam stability were reduced, respectively, by 47.7% and 33.3% in the botrytized wine, as compared to the healthy wine. In a second part, SDS–PAGE and two-dimensional electrophoresis (2-DE), coupled with immunodetection, were used to study (thoroughly) the protein patterns of both wines. With 2-DE and silver-staining detection, the disappearance of numerous spots, located in an acidic pH range, was observed. Indeed, the number of spots detected was about two times more abundant in the healthy wine than in the botrytized one, suggesting that a proteolysis occurred. On the other hand, the presence of new proteins, likely fungal proteins, proteins secreted by the plant as a response to B. cinerea infection, or even protein fragments resulting from partial proteolysis, was detected in the botrytized wine. All these modifications of the wine protein content were undoubtedly due to the presence of B. cinerea and this might be a reason for the loss of foaming properties of Champagne base wines, though no relationship between these two phenomena can be established from the results obtained.     

Degradation of white wine haze proteins by Aspergillopepsin I and II during juice flash pasteurization

Bentonite is commonly used to remove grape proteins responsible for haze formation in white wines. Proteases potentially represent an alternative to bentonite, but so far none has shown satisfactory activity under winemaking conditions. A promising candidate is AGP, a mixture of Aspergillopepsins I and II.; a food grade, well characterized and inexpensive protease, active at wine pH and at high temperatures (60-80°C). AGP was added to two clarified grape juices with and without heat treatments (75°C, 1min) prior to fermentation. AGP showed some activity at fermentation temperatures (≈20% total protein reduction compared to control wine) and excellent activity when combined with juice heating (≈90% total protein reduction). The more heat stable grape proteins, i.e. those not contributing to wine hazing, were not affected by the treatments and therefore accounted for the remaining 10% of protein still in solution after the treatments. The main physicochemical parameters and sensorial characteristics of wines produced with AGP were not different from controls.     

Evaluation of the astringency of commercial tannins by means of the SDS–PAGE-based method

The astringency of wines enriched with commercial tannins (CTs) was evaluated by a method based on the SDS–PAGE electrophoresis of salivary proteins after the reaction of saliva with wine. Nineteen CTs tested in synthetic wine at the same pH (3.6) and concentration (1 g/l) gave different values of saliva precipitation index (SPI). The effect of CTs addition was investigated in four wines. Results showed that the wine matrix influenced the astringent capacity of CTs and that became less pronounced as wine polyphenolic complexity increased. For some types of wine, astringency was not affected, indicating that the effect of CTs utilisation is not easily predictable by classical methods. The ability to objectively evaluate the astringency provided by CTs with the SDS–PAGE-based method would supply producers and winemakers with a useful tool to manage the processing conditions and thus to improve the quality of wine.     

The development of esters in filtered and unfiltered wines that have been aged in oak barrels

Esters are quite important for the aroma of aged red wine as they contribute to the fruity complex nature of this wine. This research paper measured the development of esters during maturation of Merlot red wines. These wines had different amounts of turbidity. The wine was aged for 18 months either after filtration with diatomaceous earth or without filtration. Both American and French oak barrel were used for storage. The results obtained showed that the unfiltered wine had a greater concentration of esters at the end of the storage period than the filtered wine. Fatty acid and ethanol esters decreased more than isoamyl acetate, independently of filtration. Ethyl octanoate and ethyl decanoate decreased both in filtered and unfiltered wine. Isoamyl acetate was formed in unfiltered wine and ethyl butyrate and ethyl hexanoate decreased in filtered wine while they remained constant in unfiltered wine.     

Changes in aliphatic ester composition of white wines during exposition to light. An HS-SPME-GC-MS study

The irradiation of four Italian white wines with a visible light comparable to that encountered in the market place showed a modification in the amount of aliphatic esters in the wines. Aliphatic esters are responsible for fruity aromas in wines. The first wine studied was the Gewürztraminer, a white wine from Alto Adige (Northern Italy). The amounts of the esters presents in the wine decreased. All the curves follow a pseudo-first-order kinetics. This behaviour can be explained assuming a photooxidation process catalysed by riboflavin. When Arneis Blangè was irradiated, ethyl acetate showed an increase after one day of irradiation; its content then decreased and after five days' irradiation its concentration reached a maximum. Conversely, the concentration of other aliphatic esters decreased after one day's irradiation and then increased progressively to reach a maximum after five days' irradiation. The same behaviour was observed with Pecorino wine. In this case, the concentration of the aliphatic esters decreased until the second day of irradiation but increased after five days' irradiation. In the case of the irradiation of Lighea Zibibbo all the esters showed an increase in concentration after one day of irradiation. The concentration decreased after two days' irradiation, and then increased to reach a maximum after seven days of irradiation. The increase in the amount of esters in some wines and, by contrast, the decrease in the same compounds in Gewürztraminer can be explained by considering that, during the irradiation, the pH of Gewürztraminer did not change, while the pH of Arneis Blangè changed following a curve similar to that showed by the concentration of esters. The pH change can be explained assuming the presence of a photocatalytic process induced by the presence of metals in the wines and influencing the amount of tartaric acid.     

Kinetics of sugars, organic acids and acetaldehyde during simultaneous yeast-bacterial fermentations of white wine at different pH values

Successfully inducing malolactic fermentation in the production of grape wines can be challenging, especially in wines with multiple inhibitors such as low pH values and high ethanol concentrations. In the present study, the kinetics of several chemicals of enological relevance was studied in Chardonnay vinified by traditional, consecutive alcoholic (AF) and malolactic fermentations (MLF), and simultaneous AF/MLF, where bacteria were co-inoculated with yeast. The Chardonnay must was adjusted to four pH values (3.2, 3.35, 3.5 or 3.65), and the concentrations of sugars, organic acids as well as acetaldehyde were followed throughout the fermentations. The degradation of glucose and fructose was slower at the lowest must pH value (3.2) but independent from the time point of bacterial inoculation. In all cases, malolactic conversion was faster after yeast-bacterial co-inoculation and was completed in simultaneous treatments at pH values of 3.35-3.65, and consecutive treatments at pH 3.5 and 3.65. No statistically significant difference was observed among the final acetic acid concentrations among all inoculation and pH treatments, but there was a trend towards higher acetic acid residues in wines produced by co-inoculation, especially at high pH values. Overall, simultaneous AF/MLF allowed for greatly reduced fermentation times, but the must pH remained a strong factor for fermentation success and determined the final concentration of various wine components. The time point of inoculation influenced formation and degradation kinetics of organic acids and acetaldehyde considerably, and these are of relevance for vinification decisions.     

Fining white wine with plant proteins: effects of fining on proanthocyanidins and aroma components

Plant-derived insoluble proteins (wheat gluten, and isolates from pea, lentil, and soybean) were used as fining agents in model white wine (made from Catalanesca grapes) after cold stabilization. Plant proteins were effective in giving a fast and remarkable decrease in turbidity. GC/MS and HPLC/MS approaches indicated that individual proteins had a different impact on the levels of compounds relevant to wine stability. Protein stability of wine was not affected by fining with plant proteins. Lentil proteins and gluten gave the best removal of monomeric and dimeric flavonol. Both caused a decrease in the total content of fermentative aroma compounds, such as ethyl esters, acetate esters, and alcohols. Lentil proteins had the highest impact on the aroma components, giving a marked decrease in aroma components. Gluten may thus be regarded as giving the best balance between fining efficacy and retention of aroma compounds. Also, gluten in the treated wines remained well below the suggested threshold for gluten-free foods. This study provides a methodological frame for thorough characterization of the impact of specific interventions on key wine components.     

葡萄与葡萄酒中总酚测定方法的研究进展

酚类物质是葡萄酒的骨架成分,也是葡萄酒保健功能的主要作用物质,所以准确测定酚类物质总量对评判葡萄酒的保健功能和判断葡萄酒品质有重要的意义。文章综述高锰酸钾法、福林酚法、普鲁士蓝法、香草醛—盐酸(硫酸)法、高效液相色谱法、蛋白质沉淀法在酚类物质测定中的应用情况,并分析其优缺点,为筛选和优化葡萄与葡萄酒总酚测定方法的研究工作提供参考。     

Protein removal from a Chardonnay juice by addition of carrageenan and pectin

Backgrounds and Aims: Bentonite is commonly added to white wines to remove the grape proteins responsible for haze formation. Despite being effective, this technique has drawbacks; thus, new solutions are desirable. The ability of carrageenan and pectin to remove heat‐unstable grape proteins, and the impact that such addition has on the physicochemical and sensorial profile of a wine were assessed. Methods and Results: Carrageenan and pectin were added separately or in combination to a Chardonnay juice prior to fermentation. Both adsorbents removed proteins (up to 75%), thus increasing wine protein stability. Carrageenan was more effective than pectin at increasing wine protein stability. Conclusions: Pectin and carrageenan removed protein and partially stabilized the samples of the wine. Significance of the Study: Pre‐fermentation addition of pectin or carrageenan may provide the wine industry with an alternative protein stabilization procedure.