Grape Proteins Research Articles

Holy Grail possibly made from clay.

The main goal of the work was to find biochemical protein markers specific for grapes and wine in ancient amphorae shards and fermentation pools. Grape-specific proteins are more reliable markers than tartaric acid and other small organic acids (tartaric acid natural source are not only grape but also apple, mango, and other plants). The Yavne winery (located in the Central District of Israel) is stated to be the largest known wine production complex from the Byzantine period (ca. 1500 years ago). The site has been excavated recently, and a number of wine jar have been recovered. We have applied our ethylene vinyl acetate (EVA) (EVA studded with strong cation and anion exchangers) diskettes to the inner surface of a number of jars, thus capturing residual grape proteins therein. Via mass spectrometry analyses, we have been able to identify four grape and three yeast proteins. This has been possible because the EVA films, applied to such surfaces, are able to harvest and concentrate any trace species, rendering them amenable to instrumental analysis. Our analysis makes it possible to propose an explanation for the Holy Grail phenomenon as a dish in which wine or water begins to smell pleasant. We attribute this to the slow release of terpenes, aldehydes, and ketones from the clay walls of pottery. After digital modeling, we identified that "scallop-shaped" niches in winery were used for the condensation of high percentage alcohol by passive evaporation from fermentation tanks.

Interactions between Starmerella bacillaris and Saccharomyces cerevisiae during sequential fermentations influence the release of yeast mannoproteins and impact the protein stability of an unstable wine

Wine protein haze formation is a problem due to grape proteins aggregation during wine storage. The cell wall components of wine yeasts, particularly high molecular weight mannoproteins, have a protective effect against haze formation, although their involvement remains poorly understood. This study aimed at characterizing glycosylated proteins released by Starmerella bacillaris and Saccharomyces cerevisiae during single and sequential fermentations in a synthetic must, and testing their impact on wine protein stability. Mannoproteins-rich extracts from sequential fermentations showed an increase in the low MW polysaccharide fraction and, when added to an unstable wine, had a greater effect on protein stability than S. cerevisiae extracts. Shotgun proteomics approaches revealed that the identified cell wall proteins exclusively found in sequential fermentations were produced by both S. bacillaris (MKC7, ENG1) and S. cerevisiae (Bgl2p). Moreover, sequential fermentations significantly increased the expression of Scw4p and 1,3-beta-glucanosyltransferase (GAS5), produced by S. cerevisiae. Finally, some of the key proteins identified might play a positive role in increasing wine protein stability.

Proteomics and glycoproteomics of beer and wine.

Beer and wine are fermented beverages that contain abundant proteins released from barley or grapes, and secreted from yeast. These proteins are associated with many quality attributes including turbidity, foamability, effervescence, flavour and colour. Many grape proteins and secreted yeast proteins are glycosylated, and barley proteins can be glycated under the high temperatures in the beer making process. The emergence of high-resolution mass spectrometry has allowed proteomic and glycoproteomic analyses of these complex mixtures of proteins towards understanding their role in determining beer and wine attributes. In this review, we summarise recent studies of proteomic and glycoproteomic analyses of beer and wine including their strategies for mass spectrometry (MS)-based identification, quantification and characterisation of the glyco/proteomes of fermented beverages to control product quality.

Optimization of sauce formulation from sea grape (Caulerpa racemosa) protein hydrolysate using response surface methodology

Sea grape (Caulerpa racemosa) is an edible green alga which contains a high amount of protein with a seafood flavor, which can be released via protein hydrolysis. Our present work was designed to determine the optimal concentration of palm sugar and carboxymethylcellulose (CMC) in sea grape protein hydrolysate sauce. The process optimization was performed to evaluate multiple parameters such as viscosity, color properties, and hedonic score. Five different levels of palm sugar and CMC were designed using a central composite design to yield sauce with a maximum hedonic score. We developed different mathematical models in this work, where viscosity, color properties (L*, a*, b*), and hedonic score were modeled using 2FI (two-factor interaction), quadratic, and linear models, respectively. The numerical optimization revealed that the predicted optimal solution was palm sugar and CMC concentration with generated viscosity, L*, a*, b*, and hedonic score of 0.893 poise, 23.296, 7.300, 19.565, and 3.522, respectively, and a desirability value of 0.839. GC-MS data associated with referred information represented that sea grape sauce had four compounds with a typical seafood flavor, namely, nonanal, hexanal, (E)-2-octenal, and octanoic acid; some native palm sugar flavors; and Maillard reaction products (MRP). Our study suggests that sauce is a potential value-added product from sea grape protein hydrolysate.

Moment of Bentonite Addition, Co-Addition of Tannins, and Bentonite Type Affect the Differential Affinity of Pathogenesis-Related Grape Proteins towards Bentonite during Fermentation.

To test the effect of the moment of bentonite addition, co-addition of tannins, and bentonite type on the differential affinity of pathogenesis-related (PR) proteins towards bentonite during grape must fermentation, three separate experiments were set up. PR proteins in the obtained wines were analyzed by reverse phase and size exclusion high-performance liquid chromatography (HPLC). The most significant reduction of bentonite dose and PR protein concentration was achieved by applying bentonite in the last third of fermentation. Particular thaumatin-like proteins (TLP) and proteins with lower molecular mass in general were more affected than others, while TLPs were more affected than chitinases. Exogenous enological tannins interacted with particular PR proteins, mostly TLPs, and lowered the total bentonite dose required. The combined application of tannins and bentonite in fermentation removed more PR proteins than bentonite alone, but did not achieve a synergistic effect in reducing the bentonite dose. Various bentonite types, including two Na-activated bentonites, an activated Na bentonite with specifically adsorbed silica, and an active Na-Ca bentonite, exhibited differential affinity towards different PR proteins. The results obtained could be used in developing wine fining protocols which combine treatments with complementary affinity for adsorption and removal of PR proteins, and in this way achieve greater efficiency of bentonite fining by reducing its total dose, which is of significant interest to the wine industry.

White Wine Protein Instability: Mechanism, Quality Control and Technological Alternatives for Wine Stabilisation—An Overview

Wine protein instability depends on several factors, but wine grape proteins are the main haze factors, being mainly caused by pathogenesis-related proteins (thaumatin-like proteins and chitinases) with a molecular weight between 10~40 kDa and an isoelectric point below six. Wine protein stability tests are needed for the routine control of this wine instability, and to select the best technological approach to remove the unstable proteins. The heat test is the most used, with good correlation with the natural proteins’ precipitations and because high temperatures are the main protein instability factor after wine bottling. Many products and technological solutions have been studied in recent years; however, sodium bentonite is still the most efficient and used treatment to remove unstable proteins from white wines. This overview resumes and discusses the different aspects involved in wine protein instability, from the wine protein instability mechanisms, the protein stability tests used, and technological alternatives available to stabilise wines with protein instability problems.

Influence of the reducing environment in the misfolding of wine proteins.

While proteins are present in wine at low concentration, and are largely associated with undesirable haze formation in white wines, certain types or fractions make direct and indirect contributions to sensory quality and physical stability. The proteins found in wine represent a small subclass of the total pool of grape proteins that remain soluble in the non-physiological conditions of the wine matrix which is characterised by the presence of alcohol, high acidity, and relatively high levels of phenolic compounds. Although initially stable in these conditions, during storage of white and rosé wines proteins undergo changes leading to haze formation which is considered one of the most relevant non-microbiological defects, and which makes the wine commercially unacceptable. This phenomenon involves the two most abundant proteins present in wines: thaumatin-like proteins and chitinases, both belonging to pathogenesis-related proteins of the grape berry. Haze formation is often triggered by thermal fluctuations occurring during storage of white wines, although the presence of other non-protein-related factors seems to be necessary. Here, we review the characteristics of these two protein families and the factors that influence their solubility with a focus on the disulfide bonds reduction as a possible trigger for the onset of their aggregation.

Chitinases and thaumatin-like proteins in Sauvignon Blanc and Chardonnay musts during alcoholic fermentation

Protein precipitation, also referred to as protein instability, may lead to haziness in bottled wines and result in significant commercial losses. To avoid problems of this nature, fining finished wines with clay (bentonite) is the most commonly applied methodology. However, bentonite fining reduces yield and may affect wine quality. Protein haze has been primarily linked to grape pathogenesis-related proteins, in particular chitinases and thaumatin-like proteins. To better understand the persistence of these proteins during fermentation, reverse phase chromatography was used to monitor the evolution of total grape proteins as well as of chitinases and thaumatin-like proteins during alcoholic fermentation. The data confirm a previously reported significant decrease in total protein content during fermentation. This reduction in total protein levels was observed throughout fermentation, and was affected by factors such as fermentation temperature, yeast strain or grape cultivar. However, significant changes in the concentration of free chitinases were observed in a yeast strain-dependent manner. The data thus confirm the correlation between the levels of yeast cell wall chitin and changes in chitinase concentration, and suggest that it is primarily the amount of lateral chitin, and not the chitin in bud scars, that is responsible for this activity.

Changes in pathogenesis-related proteins and phenolics in Vitis vinifera L. cv. ‘Sauvignon Blanc’ grape skin and pulp during ripening

Grape proteins, particularly pathogenesis-related (PR) proteins, are responsible for white wine haze formation which is of great concern to winemakers. Phenolic substances in grapes have also been reported with involvement in protein haze formation by interaction with proteins. The protein and phenolic substances in grapes are important in determining their concentrations in juice and resultant wine, and thus consequent wine protein stability. This study investigated the changes in these haze formation related components in ‘Sauvignon Blanc’ grape skin and pulp during ripening. The phenolic content on per berry basis generally increased in grape skin and pulp, but the phenolic concentration on per gram of berry basis showed a decreasing trend in grape skin, suggesting the accumulation effect of phenolics in skin is less than the dilution effect caused by berry growth. Tannin was only detected in grape skin but not in the pulp and the changes in tannin were similar to total phenolics. PR proteins were synthesized and accumulated along with the increase of total soluble solids (TSS) in both skin and pulp from véraison until harvest during ripening. The increase of PR proteins in grapes during ripening was also reflected in corresponding juice. The results suggested most of PR proteins in juice is likely extracted from grape pulp, and the riper the grapes the more PR proteins may be extracted into juice. The higher level of phenolics in riper grapes could increase the extraction of phenolics into juice and thus decrease the final concentration of PR proteins.

Monitoring the impact of an aspartic protease (MpAPr1) on grape proteins and wine properties.

The perception of haze in wine is brought about when pathogenesis-related proteins become unstable and aggregate, subsequently resulting in crosslinking until it develops into light-dispersing particles. Elimination of these proteins is usually achieved via bentonite fining, which, although effective, suffers from several drawbacks. The utilization of proteases has been proposed as an ideal alternative. In a previous study, an aspartic protease (MpAPr1) from the yeast Metschnikowia pulcherrima was purified and shown to be partially active against grape proteins in synthetic medium. In this study, the effects of pure MpAPr1 supplemented to Sauvignon Blanc juice on subsequent fermentation were investigated. The juice was incubated for 48h and thereafter inoculated with Saccharomyces cerevisiae. Results revealed that the enzyme had no observable effects on fermentation performance and retained activity throughout. Protein degradation could be detected and resulted in a significant modification of the wine composition and an increase in the presence of certain volatile compounds, especially those linked to amino acid metabolism.

Molecular and Macromolecular Changes in Bottle-Aged White Wines Reflect Oxidative Evolution-Impact of Must Clarification and Bottle Closure.

Chardonnay wines from Burgundy, obtained from musts with three levels of clarification (Low, Medium and High) during two consecutive vintages (2009 and 2010) and for two kinds of closures (screw caps and synthetic coextruded closures) were analyzed chemically and sensorially. Three bottles per turbidity level were opened in 2015 in order to assess the intensity of the reductive and/or oxidative aromas (REDOX sensory scores) by a trained sensory panel. The chemical analyses consisted in polyphenols and colloids quantification, followed by a proteomic characterization. For the two vintages, the REDOX sensory scores appeared to be driven both by the type of closure and to a lesser extent by the level of must clarification. Vintages and must racking prefermentative operations were also distinguished by chemical analyses. All white wines from the lowest must turbidity had the lowest REDOX sensory scores. Such wines exhibited lower concentrations in tyrosol and grape reaction product and higher concentrations in colloids with relatively low molecular weights. Among these macromolecules, grape proteins were also quantified, two of them exhibiting concentrations in bottled wines, which were statistically correlated to oxidative evolution in white wines.

Allergic and intolerance reactions to wine.

Hypersensitivity reactions to alcoholic beverages (particularly red wine) are relatively frequent, affecting 10% of the general population. Hypersensitivity reactions due to alcoholic drinks, mainly in the form of airway reactions (rhinitis and asthma), occur significantly more frequently in persons with pre-existing rhinitis and asthma. In terms of pathogenesis, it has to be differentiated between immunologic, mainly IgE-mediated, hypersensitivity reactions (wine allergies), and intolerance reactions in which no causative allergen-specific immune mechanisms can be detected. Allergens responsible for wine allergy could be: grape (Vitis vinifera) proteins (particularly the major allergen lipid transfer protein Vit v1), proteins and ingredients used for the fining of wines such as fish gelatin or isinglass (swim bladder of the fish huso, family of sturgeons), ovalbumin, dairy (casein) products, gum arabic, enzymes (lysozyme, pectinase, glucanase, cellulase, glucosidase, urease, aromatic enzymes), molds (particularly Botrytis cinerea) responsible for the noble rot in wines, yeasts and proteins from insects that contaminated the mash. Type 1 allergic reactions (positive prick tests) have been described for inorganic components like ethanol, acetaldehyde, acetic acid and sulfites, but no specific IgE could be detected in the serum. Ethanol, acetaldehyde and acetic acid, flavonoids (anthocyanins and chatechines), sulfites, histamine and other biogenic amines are the main causative agents of intolerance reactions (pseudoallergic reactions) to wine. After a short historic review of viticulture and the importance of wine in classical antiquity, we go into the chemical processes of alcoholic fermentation and the genetically inherited “flush syndrome” caused by an acetaldehyde dehydrogenase 2 polymorphism, subsequently we focus on the different etiologic factors of allergies and intolerance reactions to wine. The most frequent intolerance reactions to sulfites occur particularly after the ingestion of white wine and in asthma patients. Intolerance reactions to histamine and other biogenic amines occur mainly after ingestion of red wine and in persons with diamine oxidase (DAO) deficiency.

Reduced product loss associated with inline bentonite treatment of white wine by simultaneous centrifugation with yeast lees

Removal of residual grape protein from white wine is a routine winemaking practice for prevention of protein heat instability that can otherwise result in haze formation and consumer product rejection. Protein removal is typically achieved by batch treatment with bentonite, however considerable costs ($0.5–1.0 billion per annum worldwide) are experienced through product loss within the bentonite sediment. Continuous inline bentonite dosing followed by centrifugation is an alternative contacting approach, however previous attempts have resulted in significant carryover of bentonite into the clarified stream. This issue is addressed in the present work, whereby an 84% reduction in carryover was achieved by undertaking bentonite inline dosing in conjunction the simultaneous centrifugal removal of yeast lees following primary alcoholic fermentation. Economic modelling suggests the cost of wine losses associated with bentonite treatment may be decreased by up to 82.5% using this protocol, representing significant opportunity to reduce processing costs of industrial-scale wine production.

Characterisation of the enzymatic properties of MpAPr1, an aspartic protease secreted by the wine yeast Metschnikowia pulcherrima.

MpAPr1, encoding an acid protease from the wine yeast Metschnikowia pulcherrima IWBT Y1123, was previously isolated and shown to display potential activity against casein and grape proteins. However, its characterisation remained partial. MpAPr1 was cloned into the pGAPZαA vector and transformed into Komagataella pastoris X33 for heterologous expression. After verification of activity, the enzyme properties were characterised. Protease activity within the concentrated supernatant was retained over a pH range of 3.0 to 5.0 and between 10 °C and 50 °C. Optimal conditions for protease activity were found at 40 °C and pH 4.5. Activity was mostly unaffected by the presence of metal ions with the exception of Cu2+ and Ni2+ . Furthermore, proteolytic activity was retained in the presence of sugar and ethanol. pH and temperature conditions for MpAPr1 expression in K. pastoris were optimised. Purification was achieved by means of cation exchange chromatography and kinetic parameters (Km and Vmax ) were determined. MpAPr1 activity against grape proteins was confirmed, but the extent of the degradation was dependent on the nature of these proteins and the environmental conditions. Overall, the results suggest that MpAPr1 could be applied in food biotechnology processes such as winemaking. © 2017 Society of Chemical Industry.

Is wine savory? Umami taste in wine

Umami is an important taste element in natural products like wine. The umami taste has distinctive properties that differentiate it from other tastes, including a taste-enhancing synergism between two umami compounds, L-glutamate and 5’-ribonulceotides, and a prolonged aftertaste. In human taste cells, taste buds transduce the chemicals that elicit the umami tastes into membrane depolarization, which triggers release of transmitter to activate gustatory afferent nerve fibers. Umami taste stimuli are primarily received by type II receptor cells which contain the T1R and T2R families of G protein-coupled taste receptors. The taste sensation of umami requires protein hydrolysis which renders free glutamic acid. The main components of the nitrogen fraction of musts and wines are amino acids, peptides, proteins and ammonium ion. Their presence in wine is from amino acids of grapes, enzymatic degradation of grape proteins, excretion by living yeasts at the end of fermentation and to proteolysis during yeast autolysis. Thus, amino acids are important contributors of the wine savory taste and flavor.

Relationship of Soluble Grape-Derived Proteins to Condensed Tannin Extractability during Red Wine Fermentation.

In red winemaking, the extractability of condensed tannins (CT) can vary considerably even under identical fermentation conditions, and several explanations for this phenomenon have been proposed. Recent work has demonstrated that grape pathogenesis-related proteins (PRPs) may limit retention of CT added to finished wines, but their relevance to CT extractability has not been evaluated. In this work, Vitis vinifera and interspecific hybrids (Vitis ssp.) from both hot and cool climates were vinified under small-scale, controlled conditions. The final CT concentration in wine was well modeled from initial grape tannin and juice protein concentrations using the Freundlich equation (r2= 0.686). In follow-up experiments, separation and pretreatment of juice by bentonite, heating, freezing, or exogenous tannin addition reduced protein concentrations in juices from two grape varieties. The bentonite treatment also led to greater wine CT for one of the varieties, indicating that prefermentation removal of grape protein may be a viable approach to increasing wine CT.

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.

Wine Protein Haze: Mechanisms of Formation and Advances in Prevention

Protein haze is an aesthetic problem in white wines that can be prevented by removing the grape proteins that have survived the winemaking process. The haze-forming proteins are grape pathogenesis-related proteins that are highly stable during winemaking, but some of them precipitate over time and with elevated temperatures. Protein removal is currently achieved by bentonite addition, an inefficient process that can lead to higher costs and quality losses in winemaking. The development of more efficient processes for protein removal and haze prevention requires understanding the mechanisms such as the main drivers of protein instability and the impacts of various wine matrix components on haze formation. This review covers recent developments in wine protein instability and removal and proposes a revised mechanism of protein haze formation.

β-Glucosidase removal due to bentonite fining during wine making

Grape proteins are discussed to cause haze formation in bottled wine leading to economic problems. A common way to remove these proteins is bentonite fining. Although the efficiency of bentonite treatment is well known, the underlying molecular mechanism of the fining process in wine is not well understood. Two white wine varieties, Riesling and Pinot gris, were treated in the must stage with different types of bentonite to investigate their efficiencies. In all cases, bentonite treatment significantly reduced the overall protein concentration based on SDS-PAGE analysis, albeit individual proteins were differently affected. However, our data indicate that one protein band was completely removed independent of the bentonite type. Mass spectrometry analysis identified the major protein as glucan endo-1,3-β-glucosidase of Vitis vinifera, which was accompanied by low amounts of several other proteins. Using molecular modeling, we propose that this enzyme with its positively charged surface is adsorbed on the negatively charged lamella of bentonite during the fining process.

Effect of pH on the protein profile and heat stability of an Italian white wine

A large amount of research is currently being conducted on wine pH because of its effect on the formation of turbidity in unfined white wines. The haze-forming tendency and the protein profile of an Italian white wine (Erbaluce di Caluso D.O.C.G.), as affected by pH ranging from 3.00 to 3.60, a common pH range in wine, were analysed. The results indicated increased heat stability of the wine at lower pH, which was revealed by a lack of haze formation even above 70°C. The pH increase was accompanied by a progressive shift in the haze formation temperature from 50–60°C to 70–80°C. The electrophoretic protein pattern of the wine was characterised, and proteins derived from both yeast and grape tissues were identified. The statistical analysis of band intensities revealed that the solubility of five yeast and grape protein bands was modified by the pH shift. Most of the proteins involved were determined to be glycosylated, a feature that has been speculated to have an influence on the differential solubility of wine proteins in response to pH.