Protein haze is an esthetic problem in white wines that can be prevented by removing grape proteins that have 13 survived the winemaking process. The haze-forming proteins are grape pathogenesis-related proteins that are highly stable during 14 winemaking, but some of them precipitate over time and with elevated temperatures. Protein removal is currently achieved by 15 bentonite addition, an inefficient process that can lead to higher costs and quality losses in winemaking. The development of 16 more efficient processes for protein removal and haze prevention requires understanding the mechanisms that are the main 17 drivers of protein instability and the impacts of various wine matrix components on haze formation. This review covers recent 18 developments in wine protein instability and removal and proposes a revised mechanism of protein haze formation. 24 by land area in the world. 1,2 Furthermore, much value is added 25 in the form of winemaking to over half the world's grapes, with 26 the production of 252 million hectoliters of wine in 2012. 2 The 27 contribution of the wine sector to the world economy in 2013 28 reached a value of U.S.$277.5 billion, 3 with a large proportion 29 of the wine exported. Thus, a substantial volume of wine is 30 subject to potentially damaging conditions during trans-31 portation and storage, such as inappropriate temperature or 32 humidity, that can cause deleterious modifications of the 33 organoleptic features of the wine. 4 f1 34Wine clarity, especially that of white wines (Figure 1), is 35 important to most consumers and is also one of the 36 characteristics that is most easily affected by inappropriate 37 shipping and storage conditions. For this reason, securing wine 38 stability prior to bottling is an essential step of the winemaking 39 process and presents a significant challenge for winemakers. A 40 stable white wine is one that is clear and free from precipitates 41 at the time of bottling, through transport and storage, to the 42 time of consumption. Hazy wine and the presence of 43 precipitates are most commonly caused by three factors: 44 microbial instability, tartrate instability, and protein heat 45 instability. 5 Microbial stability is achieved prior to bottling by 46 sulfur dioxide addition and filtration; 6 tartrate stability is 47 achieved by either cold stabilization, ion exchange resins, or 48 emr00 | ACSJCA | JCA10.0.1465/W Unicode | research.3f (R3.6.i7:4236 | 2.0 alpha 39) 2014/12/19 13:33:00 | PROD-JCAVA | rq_4578338 | 4/10/2015 14:19:11 | 11 | JCA-DEFAULT 55 lees through processing using rotary drum vacuum filtration, 56 specialized lees filtration equipment, or centrifugation 57 processes that are considered laborious and that can potentially 58 degrade wine quality. 8−10 Quality degradation and loss of wine 59 through bentonite usage has been estimated to cost the global 60 wine industry around U.S.$1 billion per year. 11 Other issues 61 and costs related to bentonite use include tank downtime for 62 bentonite treatment, ...

A thermal unfolding study of thaumatin-like protein, chitinase, and invertase isolated from Vitis vinifera Sauvignon blanc and Semillon juice was undertaken. Differential scanning calorimetry demonstrated that chitinase was a major player in heat-induced haze in unfined wines as it had a low melt temperature, and aggregation was observed. The kinetics of chitinase F1 (Sauvignon blanc) unfolding was studied using circular dichroism spectrometry. Chitinase unfolding conforms to Arrhenius behavior having an activation energy of 320 kJ/mol. This enabled a predictive model for protein stability to be generated, predicting a half-life of 9 years at 15 degrees C, 4.7 days at 30 degrees C, and 17 min at 45 degrees C. Circular dichroism studies indicate that chitinase unfolding follows three steps: an initial irreversible step from the native to an unfolded conformation, a reversible step between a collapsed and an unfolded non-native conformation, followed by irreversible aggregation associated with visible haze formation.

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

Consumers expect white wines to be clear. During the storage of wines, grape proteins can aggregate to form haze. These proteins, particularly chitinases and thaumatin-like proteins (TL-proteins), need to be removed, and this is done through adsorption by bentonite, an effective but inefficient wine-processing step. Alternative processes are sought, but, for them to be successful, an in-depth understanding of the causes of protein hazing is required. This study investigated the role played by ionic strength (I) and sulfate toward the aggregation of TL-proteins and chitinases upon heating. Purified proteins were dissolved in model wine and analyzed by dynamic light scattering (DLS). The effect of I on protein aggregation was investigated within the range from 2 to 500 mM/L. For chitinases, aggregation occurred during heating with I values of 100 and 500 mM/L, depending on the isoform. This aggregation immediately led to the formation of large particles (3 μm, visible haze after cooling). TL-protein aggregation was observed only with I of 500 mM/L; it mainly developed during cooling and led to the formation of finite aggregates (400 nm) that remained invisible. With sulfate in the medium chitinases formed visible haze immediately when heat was applied, whereas TL-proteins aggregated during cooling but not into particles large enough to be visible to the naked eye. The data show that the aggregation mechanisms of TL-proteins and chitinases are different and are influenced by the ionic strength and ionic content of the model wine. Under the conditions used in this study, chitinases were more prone to precipitate and form haze than TL-proteins.

A method to fractionate grape and wine proteins by hydrophobic interaction chromatography (HIC) was developed. This method allowed the isolation of a thaumatin-like protein in a single step with high yield and >90% purity and has potential to purify several other proteins. In addition, by separating HIC fractions by reverse phase HPLC and by collecting the obtained peaks, the grape juice proteins were further separated, by SDS-PAGE, into 24 bands. The bands were subjected to nanoLC-MS/MS analysis, and the results were matched against a database and characterized as various Vitis vinifera proteins. Moreover, either directly or by homology searching, identity or function was attributed to all of the gel bands identified, which mainly consisted of grape chitinases and thaumatin-like proteins but also included vacuolar invertase, PR-4 type proteins, and a lipid transfer protein from grapes.

Grape thaumatin-like proteins (TLPs) play roles in plant-pathogen interactions and can cause protein haze in white wine unless removed prior to bottling. Different isoforms of TLPs have different hazing potential and aggregation behavior. Here we present the elucidation of the molecular structures of three grape TLPs that display different hazing potential. The three TLPs have very similar structures despite belonging to two different classes (F2/4JRU is a thaumatin-like protein while I/4L5H and H2/4MBT are VVTL1), and having different unfolding temperatures (56 vs. 62°C), with protein F2/4JRU being heat unstable and forming haze, while I/4L5H does not. These differences in properties are attributable to the conformation of a single loop and the amino acid composition of its flanking regions.

Residual proteins in finished wines can aggregate to form haze. To obtain insights into the mechanism of protein haze formation, a reconstitution approach was used to study the heat-induced aggregation behavior of purified wine proteins. A chitinase, four thaumatin-like protein (TLP) isoforms, phenolics, and polysaccharides were isolated from a Chardonnay wine. The same wine was stripped of these compounds and used as a base to reconstitute each of the proteins alone or in combination with the isolated phenolics and/or polysaccharides. After a heating and cooling cycle (70 °C for 1 h and 25 °C for 15 h), the size and concentration of the aggregates formed were measured by scanning ion occlusion sensing (SIOS), a technique to detect and quantify nanoparticles. The chitinase was the protein most prone to aggregate and the one that formed the largest particles; phenolics and polysaccharides did not have a significant impact on its aggregation behavior. TLP isoforms varied in susceptibility to haze formation and in interactions with polysaccharides and phenolics. The work establishes SIOS as a useful method for studying wine haze.