The complexity of protein haze formation in wines

The mechanism responsible for protein haze formation in wines remains essentially to be elucidated. Current knowledge suggests the absolute requirement of one or more as yet unknown non-proteinaceous wine components (termed the X factor) for protein precipitation in wines. Using the single grape variety Arinto wine, naturally containing 280 mg protein/l, a series of heat stability tests were performed over a range of wine-relevant pH values (from 2.8 to 3.8). The results obtained indicate the existence of at least two different mechanisms responsible for the heat-induced precipitation of the Arinto wine proteins: one occurring only at the higher pH values, that appears to result from isoelectric precipitation of the proteins; another prevailing at the lower pH values, but possibly operating also at other pH values, that depends on the presence of the X factor. Therefore, conclusive evidence is provided for the existence of the X factor, here defined as one or more low molecular mass wine components that sensitise proteins for heat-induced denaturation at low wine pH values and whose presence is a pre-requisite for the precipitation of proteins in wines under these circumstances. The chemical nature of protein aggregation was further analysed as a function of pH. Neither of the two proposed mechanisms responsible for the heat-induced precipitation of the wine proteins is electrostatic in nature, lectin-mediated or divalent cation-dependent. Both mechanisms show minimum turbidity at pH 7, but increased turbidity towards lower and higher pH values.     

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.     

Protein stabilisation of Chardonnay wine using trisacryl and bentonite: a comparative study

The stabilisation of a Chilean Chardonnay wine by SP-Trisacryl-M and bentonite was investigated, evaluating protein, polyphenol and polysaccharide adsorption, turbidity and wine quality. The wine could be stabilised by adding at least 0.3 kg m⁻³ of bentonite or 12 kg m⁻³ of trisacryl, removing 95% and 76% of the wine proteins, respectively. The protein adsorption data for bentonite and trisacryl were fitted using the Freundlich isotherm. The wine protein adsorption isotherm on trisacryl was unfavourable. Protein removal from Chardonnay by trisacryl in a packed column at continuous operation was about 50% during the first 70 bed volumes (BV) of treated wine and decreased progressively until the end of the treatment (100 BV). The adsorbents showed a higher selectivity for proteins than for polyphenols and polysaccharides. A sensorial panel could not detect statistically significant differences between the bentonite and trisacryl treatments of wine at P  ≤ 0.05.     

Evaluation of pathogenesis-related protein content and protein instability of seven white grape (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.) clones from Casablanca Valley,Chile

Thaumatin-like proteins and chitinases are the main pathogenesis-related (PR) proteins found in grapes, grape juice and wine and are responsible for protein haze formation in bottled white wine during storage and transport. We have studied the effect of the content of both thaumatin-like proteins and chitinases on protein instability of Sauvignon blanc (clones 1, 107 and 242) and Chardonnay (clones 4, 5, 75 and Mendoza) grape juices from both a warm and a cold production zone in the Casablanca Valley, Chile. The PR proteins were identified and quantified by reversed-phase liquid chromatography (RP-HPLC). Protein instability was determined using a heat test and was expressed in nephelometric turbidity units (NTUs). Thaumatin-like (TL) proteins were identified as the major PR proteins present in all grape juices studied. Three TL proteins were identified and named Vitis vinifera thaumatin-like proteins 1, 2 and 3 (VVTL1, VVTL2 and VVTL3). Chitinase A (ChitA) was identified in the Sauvignon blanc and Chardonnay grape juices, and chitinase B (ChitB) was found only in Chardonnay grape juices. Significant differences in the protein content and in the type of protein were observed between grapes from different production zones and between grapes of different varieties, respectively.     

Heat-unstable protein removal by different bentonite labels in white wines

The relationship between heat-unstable wine proteins and bentonite fining is still a matter of debate. Some authors have suggested that bentonite removes protein according to matrix parameters, but others have described it as selective for some protein classes. The aim of our work was to characterize the fining effects of five different types of activated sodium bentonite on two international white wines (Chardonnay and Sauvignon blanc). The physical properties of the bentonite samples and the general wine characteristics were determined, and protein removal was evaluated with gradient SDS-PAGEs, protein quantification and subsequent band identification by MALDI-TOF mass spectrometry. For the Chardonnay and Sauvignon blanc tested in the study, fining with specific bentonite labels to remove select residual proteins, some of which are potential allergens, was very promising. The removal of a single protein by bentonite affected the wine’s thermal stability.     

An invertase fragment responsible for improving the protein stability of dry white wines

The thermoprotective effect of yeast parietal mannoproteins improves the protein stability of white wines aged on their lees (sur lie). The substance responsible for this is an N‐glycosylated, 31.8 kDa mannoprotein that corresponds to a parietal invertase fragment of Saccharomyces cerevisiae. This mannoprotein is released into the wine during autolysis of the lees by the combined action of β‐glucanases from the cell wall and the yeast's vacuolar protease. This mannoprotein may be obtained industrially by extracting yeast mannoproteins using enzymatic digestion of the cell wall with commercially prepared β‐glucanases (Glucanex^TM ‐Novo‐Nordisk). A wine's susceptibility to protein breakdown may be considerably reduced by adding this mannoprotein extract. As a result, less bentonite is needed to stabilise the wine. © 1999 Society of Chemical Industry     

改型膨润土对红葡萄酒澄清过程中蛋白质和色素的影响

利用改型膨润土对红葡萄酒原酒进行澄清,主要考察了膨润土添加量、不同澄清时间以及不同pH条件下葡萄酒的澄清效果,结果表明:膨润土的最佳添加量为0.4g/L时即可达到良好的澄清效果;膨润土对葡萄酒的澄清在最初的8h内作用明显并且应尽可能在较低的pH条件下进行,这样就会有更多的蛋白质带正电荷,与带负电荷的膨润土表面产生电荷吸引,从而使蛋白质沉淀,有利于保持酒体的长期稳定。     

Optimal Conditions for Controlling Haze-Forming Wine Protein with Bentonite Treatment: Investigation of Matrix Effects and Interactions Using a Factorial Design

Protein instability in white wine can result in unsightly haze formation, and therefore, its prevention by adsorption of haze proteins onto bentonite is an important unit operation in commercial wine production. Optimisation of this process is challenging due to the performance impact of environmental factors and matrix effects which are difficult to control and study in wine systems. These issues are addressed in the present study; the effect of different factors on adsorption behaviour of a purified thaumatin-like grape protein (VVTL1) by sodium bentonite in a chemically defined model wine solution was investigated using a factorial design with surface response analysis. Bentonite adsorption of VVTL1 was well characterised by a multi-factor Langmuir adsorption model. The main effects of pH, temperature, potassium concentration as well as the pH*potassium matrix interaction all had a significant effect (p?<?0.05) on the adsorption capacity, as did the aging of bentonite slurry before use. Observations support the hypothesis that VVTL1 adsorption onto sodium bentonite is affected by steric mass action and local interactions of exposed protein charge, with pH and temperature effects related to changes in protein conformation under those conditions. Variation in potassium concentration can cause similar effects and influence adsorption capacity by affecting bentonite swelling and charge potential, providing a greater surface area for adsorption. From a processing perspective, results suggest bentonite treatment efficiency will be optimised by treating wines at higher temperatures rather than during cold storage, at the lower pH and before cold (tartrate) stabilisation.     

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.     

发酵枸杞酒沉淀物成分鉴定及稳定性提升研究

发酵枸杞酒营养丰富,风味独特,市场前景广阔,但在货架期内易出现沉淀,影响消费者的偏爱。本研究以发酵枸杞酒及其沉淀物作为研究对象,采用物理化学方法对枸杞酒沉淀物进行定性、定量研究,探究了添加不同澄清剂（皂土、明胶、蛋清粉和蛋白酶等）对枸杞酒化学组成及稳定性的影响。结果表明,发酵枸杞酒沉淀物主要包括蛋白质、有机酸、多糖、多酚和金属元素等,其中,蛋白质占24%～34%,有机酸占22%～28%,多糖占15%～23%。添加不同种类的澄清剂对枸杞酒沉淀组分的影响不同,皂土类能够降低蛋白含量,其中皂土NC处理后的枸杞酒与对照组相比蛋白含量降低了28.13%。而明胶类能降低有机酸和多糖含量,其中明胶W处理后的枸杞酒多糖含量下降为初始对照的53.84%。添加明胶类澄清剂效果较好,可以减少枸杞酒的沉淀量,提高枸杞酒的稳定性。     

Protein evolution during the early stages of white winemaking and its relations with wine stability

Background and Aims: Grape proteins are responsible for the appearance of haziness in white wines during storage after bottling. However, only a few studies have approached the analysis of the fate of must proteins throughout the alcoholic fermentation. This study aimed to systematically investigate the daily variations in protein type and content during the fermentation in order to understand its influence on hazing potential and to attain some basic information to improve the practical management of grape proteins involved in the hazing of white wines. Methods and Results: The evolution of total soluble protein and individual protein fractions was studied in samples taken before, during and after alcoholic fermentation of a white grape must. The results were then related to variations in protein instability as measured by the heat test. Both the quantity of soluble protein and the protein instability increased during fermentation and then decreased after 1‐month storage of the wine. Protein composition did not vary during fermentation as assessed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and anion exchange chromatography (AEC). However, variations in the relative proportions of the six protein fractions obtainable by AEC were noted in the different samples. The contribution of each AEC protein fraction to wine instability was determined by considering both the intrinsic instability and the relative quantity of each of the individual protein fractions in the wine. It was demonstrated that the grape thaumatin‐like protein VVTL1, as identified by mass spectrometry, showed the largest increase during fermentation and accounted for almost 40% of the heat‐induced haze of the final wine. Moreover, the decreased protein instability noted after one month storage of the wine could be attributed to the stabilizing effect of polysaccharides released by the yeast cells. Conclusions: The quantity and relative proportion of soluble proteins vary during and after the alcoholic fermentation, as does their heat instability in wine. Grape VVTL1, constituting a large proportion of the total proteins in wine, seems to play a major role in protein haze formation. The release of yeast polysaccharides is related to an increased heat stability of total wine protein, despite the increase in the relative proportion of their most unstable component VVTL1. Therefore, the hazing potential of a white wine seems to be affected by variations in the relative proportions of its macromolecular components occurring in the early stages of winemaking. Significance of the Study: This study addressed for the first time the issue of the protein changing during the fermentation of white wine. The results obtained here offer useful information to aid understanding of the contribution of individual proteins to white wine instability, which can be applied for the improvement of the winemaking process.     

Fractionation and partial characterization of protein fractions present at different stages of the production of sparkling wines

Soluble proteins from sparkling wines (var. Chardonnay) manufactured industrially following the Champenoise method were analysed by analytical anion-exchange FPLC (fast protein liquid chromatography). Samples of the must, the unfined wine, the fined wine and the sparkling wines after 3, 6, 9, 12, 15 and 18 months of ageing with yeast lees were taken. Eight main must protein fractions were collected and characterized by isoelectric focusing and capillary electrophoresis. Six fractions consisted of proteins with isoelectric points (pIs) from 4.27 to 3.04. Two fractions, according to their UV spectra, seemed to contain phenolic compounds. A comparison of FPLC profiles of the must, the unfined wine and the fined wine indicated that both fermentation and stabilization processes decrease the concentration of those proteins with less affinity for the anion-exchange column and with a higher pI. No changes in the protein profiles of sparkling wines were observed during the first 18 months of ageing with yeast..     

黄酒中蛋白质分布及含量与酒质稳定性关系的研究

对多种黄酒中的蛋白质进行分离和检测，研究了黄酒中蛋白质分布及含量与酒质稳定性关系。结果表明：高分子蛋白质在酒中的大量存在必然影响黄酒非生物稳定性，导致浑浊沉淀；黄酒的稳定性与酒中所含的蛋白质总量没有直接因果关系，而与黄酒中存在的某类特定蛋白质相关，此类蛋白质是小分子蛋白质，在一定条件下发生凝聚反应形成各种高分子的蛋白质分布于酒中导致浑浊；寻找一种高选择性吸附剂，并结合冷处理工艺是提高黄酒稳定性、解决黄酒沉淀的策略。     

白葡萄酒蛋白质稳定的新展望--酶解法

白葡萄酒易出现蛋白质沉淀，而传统除去蛋白质的方法无非是在葡萄酒中加入明胶或膨润土将葡萄酒中的蛋白质除掉。当然这是保持了葡萄酒的稳定性，但另一方面将葡萄酒中稳定性蛋白质也除掉了，这就降低了葡萄酒的营养价值。使用蛋白质酶解法可将葡萄酒中蛋白质水解成小肽，这样既可使得葡萄酒具有很好的稳定性，又可以提高葡萄酒的营养价值。     

The use of thaumatin and bovine serum albumin as proteins in model wine solutions in bentonite fining

This study examined the viability of using thaumatin and bovine serum albumin (BSA) as proteins in model wine solutions for bentonite fining studies and compared them with unfined New Zealand sauvignon blanc (SB) wine. Bentonite fining trials were performed on model wine solutions and unfined SB wines (pH range 3.5–4.3). Thaumatin was more readily adsorbed onto bentonites of all types than BSA and its adsorption onto bentonite was less affected by the pH of the solution. Specifically, the amount of BSA adsorbed onto bentonite decreased significantly as the pH of the solution approached the isoelectric point (pI) of BSA while thaumatin was adsorbed at that pH due to its higher pI. Changing pH affected protein adsorption of real wine less noticeably than of BSA and thaumatin, and decreasing pH increased protein adsorption in contrast to the model solutions. Neither of the model solutions can fully represent the response of real wine to bentonite fining but they are simple and cost effective to prepare and reacted to changes in bentonite concentration similar to real wine. Thaumatin is potentially a better protein to use in simple model solutions for wine stabilisation studies like filtration where molecular weights are important consideration.     

复合澄清剂对李子酒澄清效果的研究

为提高发酵李子酒的澄清效果,以新鲜李子为原料酿制成李子酒,以明胶、皂土为澄清剂,分别进行单一澄清处理和复合澄清处理,对原酒及处理后酒样的透光率、可溶性固形物、总糖、总酸、总酚、蛋白质及稳定性进行检测。结果表明,18 ℃避光密封静置4 d条件下,当明胶0.4 g/L和皂土0.02 g/L复合澄清处理时,李子酒的澄清效果最好,测得透光率为90.7%;可溶性固形物、总糖、总酸、总酚、蛋白质含量分别为4.8%、24.3 g/L、9.38 g/L、492.9 mg/L、411.4 μg/mL,营养成分损失较少,而且其酒体稳定清亮,无沉淀产生。     

Charakterisierung der elektrischen Eigenschaften und der Molmassen der Eiweißstoffe von Weißweinen

Characterization of the electrical properties and of the molecular weights of the proteins of white wines The electrical properties and the molecular weights of the proteins of the dry white wine Dimjat were investigated. The protein substances of the wine are aggregates consisting of 4 monomer proteins with molecular weights between about 28000 and 160000. The protein with the lowest molecular weight represents the major part. There are different reasons for the aggregation. This causes different properties of the aggregates and determines their reactions during the technological treatment. The protein fractions of the untreated wine have isoelectric points between 3.8 and 5.8. The protein-clouding is mainly caused by the protein-fractions with lower isoelectric points and lower molecular weights. The protein stability of the wine can therefore only be attained by such a technological treatment that leads to the removal or quantitative reduction of those protein fractions which are responsible for clouding. The protein stability is reached by the bentonitetreatment while the other treatments (heat, cold, and potassiumhexacyanoferrate (II)) we have examined were not successful.     

Effects of fining with different bentonite labels and doses on colloidal stability and colour of a Valpolicella red wine

This study addressed four different labels of activated sodium bentonite during the fining of a young Valpolicella red wine. Preliminary tests determined the bentonite dose (0.15, 0.30 or 0.50 g L^?1) that would achieve colloidal stability, and this dose was further applied during laboratory‐scale trials. The bentonites were characterised by physico‐chemical parameters, and the effects of the label and dose on the wine colloidal stability, proteins, colour indices and phenolic compounds were measured. The results demonstrated that 0.50 g L^?1 for every bentonite label provided colloidal stabilisation without harshly affecting the colour. Unlike to what happens in white wines the least charged bentonite labels were effective at stabilising the red wine colloidal state by partially reducing its protein content. Simultaneously, the most negatively charged clay samples determined the largest depletions on the total polyphenols, anthocyanins, tannins and polymeric pigments. The differentiated action of clays with different surface charge density among white and red wines may be explained by the interaction mediated by positively charged anthocyanins towards either protein or tannin depletion. Although pioneering, these results may move towards the definition of a safe, allergen‐free, and effective adjuvant for colloidal stabilisation targeted to wine type.     

Effects of protein separation conditions on the functional and thermal properties of canola protein isolates

Canola meal protein isolates were prepared from defatted canola meal flour using alkaline solubilization and acid precipitation. A central composite design was used to model 2nd-order response surfaces for the protein yield and the functional properties of protein isolates. The solubilization pH and precipitation pH were used as design factors. The models showed that the protein yield and functional properties of isolates, such as water absorption and fat absorption, were sensitive to both solubilization pH and precipitation pH, whereas the emulsification was sensitive to only solubilization pH. Gel electrophoresis analysis of protein fractions gave evidence to the compositional changes between proteins isolated under different conditions. Differences in glass transition temperatures suggest that proteins tend to be more denatured when solubilized at highly alkaline conditions. These conformational and compositional changes due to different protein separation conditions have contributed to the changes in functional properties of protein isolates. PRACTICAL APPLICATION: Protein isolation conditions may be determined primarily through optimization of total protein yield. Improvements in protein functional properties may be achieved with a relatively small sacrifice in yield by altering isolation conditions.     

Protein stabilisation of white wines using zirconium dioxide enclosed in a metallic cage

Backgrounds and Aims: White wines are stabilised by removing the heat unstable proteins through adsorption by bentonite. Bentonite fining is not an efficient wine processing step and can also remove other wine components. Alternative absorbents are thus sought; zirconium dioxide (zirconia) is recognised as a promising candidate. The aim of this work was to assess the viability of zirconia treatments to stabilise white wines, with particular attention on process development. Methods and Results: Effective treatment was achieved by enclosing zirconia pellets into a metallic cage submerged in the wine. With this method, the wine could be treated with the adsorbent for the time required for protein stabilisation, and then removed without further manipulation. Zirconia treatments of three unstable wines partially or fully stabilised them without detectable modifications of their physicochemical parameters and colours, apart from the removal of metals and some acids, particularly when wines were treated for long times and with high dosages of the adsorbent. A simple and inexpensive zirconia regeneration method was also developed. Conclusions: The zirconia application to wine was very effective in removing proteins, and the proposed regeneration procedure could facilitate the uptake and development of zirconia‐based solutions for the wine industry. Significance of the Study: This study confirmed the effectiveness of zirconia in removing wine proteins and demonstrated that the proposed method of application has the potential to become a viable alternative to bentonite.