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.     

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.     

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.     

Turbidity and Haze Formation in Beer — Insights and Overview

Beer is a complex mixture of over 450 constituents. In addition, it contains macromolecules such as proteins, nucleic acids, polysaccharides and lipids. Proteins influence the entire brewing process with regard to enzymes, which degrade starch, β‐glucans and proteins; with protein‐protein linkages that stabilize foam and are responsible for mouthfeel and flavour stability; and in combination with polyphenols, thought to form haze. With this complexity, problems in processability are as various as the constituents. Several substances in beer are responsible for haze formation. Organic components such as proteins, polyphenols and carbohydrates (α‐glucans, β‐glucans) are known to form haze. In addition, inorganic particles such as filter aids and label remains can cause increased turbidity. In this article only non‐microbiological induced hazes are described. Many studies have been conducted on the identification of haze and foam active components in beer. Hence the aim of this work was to survey the different possibilities of haze formation and for haze identification. A summary is provided on methods for haze identification including dyeing methods, microscopic analyses and size exclusion chromatography.     

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.     

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.     

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.     

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.     

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.     

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.     

In-line dosing for bentonite fining of wine or juice: Contact time, clarification, product recovery and sensory effects

Field tests to evaluate in-line dosing with bentonite followed by centrifugation as an alternative to batch fining for protein haze control in white wine or juice were undertaken. The tests were performed at a commercial winery with a Sultana wine and Gordo (Muscat of Alexandria) juice and using two types of bentonite: Vitiben and SIHA-Active-Bentonite G. Fining performance was monitored by heat testing and quantification of heat unstable protein by HPLC. Heat test turbidity and heat unstable protein concentration decreased to stable values between 30 s and 2 min after bentonite injection. Sensory evaluation of Sultana wine fined with Vitiben by balanced reference duo-trio difference tests detected no difference between untreated, in-line dosed, and batch fined wine. Furthermore the volume of wine or juice occluded in lees can be substantially reduced by centrifugal compaction. However, incomplete separation of bentonite from wine or juice during centrifugation produced a carryover of 30% of the added bentonite into the clarified wine. This carryover problem may be mitigated, inter alia, by reducing operating flowrate through the centrifuge or using multiple centrifugation steps (in parallel or series). Therefore, inline dosing followed by centrifugation provides a rapid processing method for protein haze reduction in wine or juice with a decreased volume of lees. It can reduce significant value losses presently arising in the wine industry from batch fining and the resulting quality downgrades that occur in wine recovered from bentonite lees by rotary drum vacuum filtration.     

苹果汁中活性蛋白的性质及其稳定化处理研究进展

苹果汁中活性蛋白质 (HAP)是澄清苹果汁产生混浊沉淀的重要原因之一 ,疏水性氨基酸易于和多酚聚集 ,形成混浊。苹果汁中不同浓度的活性蛋白质与低分子的多酚以及简单酚所引起混浊的反应机理不同 ;活性蛋白质的种类、分子结构、反应的时间、温度、反应体系的 pH、无机离子及其离子强度对浑浊的产生有重要影响。单宁酸、膨润土、硅溶胶和聚乙烯聚吡咯烷酮 (PVPP)是有效的稳定化处理措施。     

The chemical components in malt associated with haze formation in beer

In bright beer, haze formation is a serious quality problem, which reduces beer storage and shelf life. In this study, haze‐active proteins, alcohol chill haze formation ability, α‐amylase activity, the contents of total polyphenol, protein and its fractions and amino acids were analysed using 23 barley accessions to investigate the relationship between the quality components in the malt and the haze character in beer. The results showed that there were largely genotypic variations for all examined traits among the 23 barley accessions. However, there was no significant correlation between the haze character and α‐amylase activity. All haze characteristics were significantly and positively correlated with total protein content, albumin, globulin and the hordein content, as well as the glutamic acid (glutamine), proline and phenylalanine content, and were not correlated with total polyphenols. A model describing the relationship between the chill haze in the beer and the protein content in the malt was developed. Copyright © 2016 The Institute of Brewing & Distilling     

不同黄酒部位的沉淀蛋白质氨基酸组成分析

为分析黄酒混浊与沉淀的原因,采用硅溶胶吸附分离法获得黄酒酒液和酒脚中易引起沉淀的蛋白,并一起分析测定这2个样品和酒脚的氨基酸组成。结果表明:酒液沉淀蛋白的主要氨基酸为谷氨酸、脯氨酸和苯丙氨酸,相对含量分别为41.33%、18.66%和9.56%;酒脚沉淀蛋白中主要氨基酸为谷氨酸、天门冬氨酸、亮氨酸,分别为12.39%、11.32%和8.02%;酒脚中主要氨基酸为谷氨酸、天门冬氨酸、亮氨酸,分别为16.57%、10.91%、7.14%。该结果显示黄酒所含醇溶蛋白质中的谷氨酸导致黄酒混浊、沉淀现象的主要氨基酸。     

Precipitation of proteins from potato juice with bentonite

The recovery of proteins from potato juice by treatment with bentonite has been investigated. All proteins can be precipitated from potato juice by acidification and addition of bentonite. The acid-coagulatable protein fraction is adsorbed less by bentonite than the acid-soluble protein fraction. Maximum adsorption of the acid-soluble fraction occurs at pH 5.0. The working conditions recommended for obtaining a protein-free potato juice are acidification to pH 4.5 and addition of bentonite to obtain a weight ratio of soluble protein: bentonite of 0.9. At the natural pH of potato juice (pH 5.8-6.0), adsorption of potato proteins on bentonite is irreversible. About 62% of the adsorbed protein can be recovered by alkali treatment at pH 13.     

Effect of using bentonite during fermentation on protein stabilisation and sensory properties of white wine

The bentonite use to remove proteins from white wine is a widespread practice that prevents protein haze formation after bottling. However, an excess of bentonite can have negative effects on both the aromatic profile of young white wines and the quality of the foam of sparkling wines. Therefore, the optimisation of bentonite amount to be used and the moment of its application during winemaking to minimise wine quality losses are of great interest for winemakers. This paper analyses how applying an equal bentonite dose at different stages (must clarification; beginning, middle and end of fermentation) on two scales (industrial and pilot) affects the protein content and stability, physical–chemical characteristics, aromatic profile and foam quality of the obtained wines. No important differences in the oenological parameters were observed between industrial and pilot scales, whereas the scale of the experimental treatments affected protein stability, aroma composition and foam quality of the wines.     

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.     

The effect of bentonite fining at different stages of white winemaking on protein stability

Background and Aims: Bentonite fining to remove protein is the most widely used treatment to prevent protein haze in white wines. Bentonite can be added at different stages during winemaking. This study aimed to determine the best time to add bentonite. Methods and Results: Unstable juices were vinified after bentonite fining of juice, or with bentonite added either early or late in fermentation. Different addition rates of bentonite were used and bentonite was either added in one or two additions. Fermentation rates were observed and protein stability and bentonite fining rates of the resultant wines were determined. Conclusions: Adding bentonite during fermentation or fining finished wines was the most efficient option in terms of amounts of bentonite required. Fermenting with bentonite present also may increase fermentation rates. Significance of the Study: Using the least bentonite necessary for heat stability is important for winemakers for quality, cost and environmental reasons. This study describes ways in which bentonite addition rates can be minimised.     

Fractionation of Sauvignon wine macromolecules by ultrafiltration and diafiltration: impact of protein composition on white wine haze stability

After ultrafiltration (UF) thermally unstable compounds of Sauvignon blanc wines were concentrated in the retentate fractions of 10–30 kDa increasing heat‐induced haze 8.9‐fold, while concentrating haze stabilising species into the retentate fractions of 100–300 kDa with a 5.3‐fold reduction of heat‐induced haze compared to unfiltered wine. Proteins having molecular weights of 18, 23, 33, 35, 40 and 60 kDa were identified as the main haze promoting agents for Sauvignon blanc, where the proteins of 56, 69 and 72 kDa were identified as thermally stable. Potassium sulphate (100 mg L^?1) and magnesium sulphate (25 mg L^?1) did not affect wine haziness at experimental diafiltration (DF) conditions using model wine solutions. Combination of UF and DF is a useful separation method of wine proteins, which is helpful to quantify haziness of individual species and to reveal the interactions between species involving protein haze.     

啤酒混浊蛋白组分的分离鉴定

采用双向电泳结合基质辅助激光解吸/ 电离飞行时间质谱(MALDI-TOFMS)技术分析确定引起啤酒混浊的蛋白组分。结果表明,仅有少量的蛋白组分参与啤酒混浊的形成。聚丙烯酰胺凝胶电泳分析发现MW 40kD 左右,25～29kD 和6.5～17kD 作为啤酒混浊蛋白组分,可能主要来自麦芽中的水溶蛋白,小部分来自麦芽醇溶蛋白。双向电泳分析证实了大部分的麦芽蛋白在酿造过程中发生降解和变性,并结合质谱技术鉴定得到BTI-CMe、germin E(Hordeum vulgare)和 protein Z 3 种组分可以抵抗酿造过程的热变性和水解作用,将成为啤酒混浊形成重要的促进因子。