White Wine Protein Research Articles

White Sparkling Wine Proteins and Glycoproteins and Their Behavior in Foam Expansion and Stability

The volume and stability of wine foams are influenced by many components of the matrix, especially proteins. However, the synergistic or inhibiting effects among these protein fractions, as well as their interactions with other wine components, are still under study. The present research aims to understand the individual and cooperative effects of different wine proteins and glycoproteins on the volume and stability of foams. To address this objective, different protein fractions were purified from a Chardonnay white wine and tested in different model wine conditions (with/without ethanol), along with a commercial yeast-based oenological additive. Different fractions were considered, including total protein fraction (FT), Mannoproteins (MP), and non-mannosylated proteins (NMP), as well as a protein fraction soluble in ammonium sulfate (FSA). These protein fractions were characterized, and their foaming properties were evaluated using a modified Rudin apparatus. The results showed that FT exhibited higher foam expansion (FE%) compared to its subfractions (NMP and MP) that, when tested individually, did not guarantee optimal foam formation. This suggests that foaming properties are enhanced when both glycosylated and non-glycosylated proteins are present in the system. Additionally, the foaming behavior was influenced by the presence of ethanol in the model wine. The FSA fraction demonstrated high foam expansion and stability, with ethanol enhancing foam expansion but reducing stability. A commercial yeast-based oenological additive, mainly containing glycoproteins, was also tested and behaved similarly to MP. This study provides valuable insights for sparkling wine producers to optimize practices for enhancing product quality and confirm previous research regarding the role of the synergy between MP and NMP in wine foam formation and stability.

Quantification of thaumatin-like proteins in white wine using MALDI-TOF mass spectrometry

The main soluble proteins in bottled wine are pathogenesis-related proteins. Their concentration is influenced by the harvesting technique used, the time of the juice exposition to grape skins and possible microbial infection of grapes. The most typical are namely chitinases and thaumatin-like proteins (TLPs), which accumulate in grapes on ripening. They show a low molecular weight of 20–35 kDa and are resistant to proteolysis as well as the acidic pH of wine. Chitinases are considered the primary cause of heat-induced haze formation because of their irreversible denaturation and aggregation. This process can additionally be affected by the non-protein wine components. We focused on the development of a fast quantification method for wine TLPs using matrix-assisted laser desorption/ionization mass spectrometry. White wine samples were analyzed directly, after dialysis or they were subjected to protein concentrating procedures before the measurements. MS-based quantification was achieved by comparing the peak areas of an internal standard (cytochrome c, thaumatin or myoglobin) and TLPs in the acquired mass spectra. The content of TLPs in commercial wines was determined at milligrams per liter. Chitinases could not be quantified in this way because of a low concentration in the analyzed bentonite-fined wines resulting in missing spectral signals. Anyway, this procedure becomes an alternative to common quantification methods based on liquid chromatography.

A Minimally Invasive Approach for Preventing White Wine Protein Haze by Early Enzymatic Treatment.

Protein stability in bottled white wine is an essential organoleptic property considered by consumers. In this paper, the effectiveness of an early enzymatic treatment was investigated by adding a food-grade microbial protease at two different stages of winemaking: (i) at cold settling, for a short-term and low temperature (10 °C) action prior to alcoholic fermentation (AF); (ii) at yeast inoculum, for a long-lasting and medium temperature (18 °C) action during AF. The results reveal that protease sufficiently preserved its catalytic activity at both operational conditions: 10 °C (during cold settling) and 18 °C (during AF). Furthermore, protease addition (dosage 50–150 μL/L) raised the alcoholic fermentation rate. The treatment at yeast inoculum (dosage 50 μL/L) had a remarkable effect in preventing haze formation, as revealed by its impact on protein instability and haze-active proteins. This minimally invasive, time and resource-saving enzymatic treatment, integrated into the winemaking process, could produce stable white wine without affecting color quality and phenol content.

Advances in White Wine Protein Stabilization Technologies.

The unstable proteins in white wine cause haze in bottles of white wine, degrading its quality. Thaumatins and chitinases are grape pathogenesis-related (PR) proteins that remain stable during vinification but can precipitate at high temperatures after bottling. The white wine protein stabilization process can prevent haze by removing these unstable proteins. Traditionally, bentonite is used to remove these proteins; however, it is labor-intensive, generates wine losses, affects wine quality, and harms the environment. More efficient protein stabilization technologies should be based on a better understanding of the main factors and mechanisms underlying protein precipitation. This review focuses on recent developments regarding the instability and removal of white wine proteins, which could be helpful to design more economical and environmentally friendly protein stabilization methods that better preserve the products´ quality.

Fluorescence sensing technology for the rapid detection of haze-forming proteins in white wine

The methods currently available for determining haze proteins in wine are time-consuming, expensive, and often not sufficiently accurate. The latter may lead to bentonite over-fining of a wine, which might strip wine phenolics and aroma compounds, or wine under-fining, which increases the risk of protein instability. In this work, an efficient and rapid fluorescence-based technology to detect haze-forming proteins in white wines was developed. A fluorescent compound was synthesised to selectively bind haze-forming proteins. Studies involving HPLC demonstrated a linear dependence over a range of relevant haze protein concentrations and a low detection limit of 2 mg/L. Forty-eight control and bentonite fined wines were analysed to validate the analytical performance of the fluorescent dye in the detection of haze-forming proteins. The method can be deployed rapidly, without sample preparation, presenting an opportunity to use in routine testing and overcome limitations of the “heat test” currently used in the wine industry.

White wine protein instability: a review

The presence of protein compounds in wine leads to decreased organoleptic quality of finished products such as clarity and hue. These compounds have important nutritional values. A large part of the proteins present in wine are closely related to the pathogenesis process. Disturbance in wine samples occurs as a consequence of the presence of several non-protein factors such as the presence of phenolic compounds, metals and pH. To date, a wide range of procedures are known for the protein of wine precipitation in order to determine the total protein content of wine samples, using solutions such as: trichloroacetic acid (TCA), acetone, ethanol, etc. Numerous techniques specific to the process of protein stability are also known and developed. The best known techniques for determining protein stability are: thermal with the addition of chemicals, bentonite, tannin, ProtoCheck, acetone respectively combined (hot test + tannin, etc.).

Effect of Pre-Fermentative Maceration and Fining Agents on Protein Stability, Macromolecular, and Phenolic Composition of Albariño White Wines: Comparative Efficiency of Chitosan, k-Carrageenan and Bentonite as Heat Stabilisers.

In this work, the effect of pre-fermentative skin maceration (PFSM) on the chemical composition of the macromolecular fraction, polysaccharides and proteins, phenolic compounds, chromatic characteristics, and protein stability of Albariño monovarietal white wines was studied. PFSM increased the extraction of phenolic compounds and polysaccharides and reduced the extraction of pathogenesis-related proteins (PRPs). PFSM wine showed significantly higher protein instability. Sodium and calcium bentonites were used for protein stabilisation of wines obtained with PFSM (+PFSM) and without PFSM (−PFSM), and their efficiencies compared to fungal chitosan (FCH) and k-carrageenan. k-Carrageenan reduced the content of PRPs and the protein instability in both wines, and it was more efficient than sodium and calcium bentonites. FCH was unable to heat stabilise both wines, and PRPs levels remained unaltered. On the other hand, FCH decreased the levels of wine polysaccharides by 60%. Sodium and calcium bentonite also decreased the levels of wine polysaccharides although to a lower extent (16% to 59%). k-Carrageenan did not affect the wine polysaccharide levels. Overall, k-carrageenan is suitable for white wine protein stabilisation, having a more desirable impact on the wine macromolecular fraction than the other fining agents, reducing the levels of the wine PRPs without impacting polysaccharide composition.

Use of grape seeds to reduce haze formation in white wines

Grape pathogenesis-related (PR) proteins in white wine can induce haze and hinder the sale of the product. Bentonite is used to remove proteins and “heat-stabilise” wine however it is non-selective and can reduce wine quality. Grape seed powder (GSP) has previously been shown to remove PR proteins and reduce haze formation on a lab scale, however the effect on wine sensory properties was unknown and crucial to the evaluation of GSP as a bentonite alternative. Semillon (SEM) and Sauvignon Blanc (SAB) juices (20L in triplicate) were treated with GSP at two doses, Low (7.5 g/L) and High (15 g/L), prior to fermentation. GSP treatment reduced the concentration of wine PR proteins by up to 57% and 37% for SEM and SAB, respectively, and reduced the amount of haze formed in a heat test by up to 75% and 80%, respectively. Sensory analysis conducted by a trained panel showed that for both wine types the high GSP treatments were rated deeper in colour and higher in bitterness than the bentonite controls, with the low GSP treatment having a similar but less pronounced effect on these attributes. The GSP-treated SAB wine showed greater tropical fruit aroma, and pungency, compared to the bentonite control. Use of GSP can reduce the amount of bentonite needed to stabilize wines and may provide a sustainable and effective alternative to bentonite, notably for textural white wine styles.

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.

High-proline proteins in experimental hazy white wine produced from partially botrytized grapes.

Undesirable effects of the pathogen Botrytis cinerea include reduced quality and quantity of wine grapes. Winemaking is also complicated by the formation of a protein haze in white wines and oxidative browning of red wines. We analyzed proteins in experimental Moravian white wines characterized by their instability and haze formation in bottles during storage despite prior bentonite treatment. To study the relationship of wine proteins and haze, we carried out proteomics on hazy and clear white wines produced with partly or largely botrytized grapes and standard reference wines. Wine proteins were identified after their extraction, electrophoresis, and tryptic digestion by reversed-phase liquid chromatography of peptides, coupled with tandem mass spectrometry. Plant defense proteins, yeast glycoproteins, and various enzymes from Botrytis, particularly hydrolases, were found. As the content of the known haze-active thaumatin-like proteins and chitinases was visually low on stained gels (missing bands) compared to previous studies with unfined wines, other proteins are discussed in terms of the haze formation. As the main novelty, this work reveals the role of high proline-containing proteins in the propensity of white wines to turbidity following prior Botrytis damage of grapes.

The use of chitosan as alternative to bentonite for wine fining: Effects on heat-stability, proteins, organic acids, colour, and volatile compounds in an aromatic white wine

This investigation focuses on the use of chitosan treatment simulating a bentonite fining in order to detect any modification of the wine haze potential by simultaneously evaluating the secondary effects on untargeted fixed and volatile compounds. A significant removal of chitinases was observed after fining an aromatic white wine with 1 g/L of a fungoid chitosan. Even if the more stable thaumatin like protein fractions were not significantly affected, the heat stability of the fined chitosan wine samples were highly improved in the 55–62 °C range. Among the secondary effects of a fining treatment, a reduction of tartaric acid, malic acid, potassium and iron was showed. All the free terpenols with the exception of α-terpineol were reduced in significant amounts, whilst the glycosylated forms and the fermentative aroma compounds were not affected by a 1 g/L chitosan addition.

The impact of wine components on fractionation of Cu and Fe in model wine systems: Macromolecules, phenolic and sulfur compounds

A variety of techniques have been developed with the ability to measure different forms of metals in wine with the ultimate aim of providing a more accurate indicator of metal induced spoilage of wine. This study was conducted in order to identify which wine components influence the measurement of Cu and Fe in their fractionated and/or electrochemically active forms. The measurement techniques involved detection of labile Cu by stripping potentiometry and fractionation of Cu and Fe by sequential solid phase extraction, with detection by inductively coupled plasma-optical emission spectroscopy. The wine components assessed included those extracted from wine (red wine tannin, white wine protein, white wine polysaccharide, red wine polyphenol, white wine polyphenol), and commercially available monomeric compounds, including phenolic compounds and sulfur-containing compounds. For Cu, only hydrogen sulfide, which is known to induce the formation of Cu(I) sulfide, showed any appreciable influence on the fractionation and electrochemical detection of Cu. This form of Cu was also identified as the major component of red and white wines. For Fe, the fractionation was different for red versus white wine, and influenced significantly by extracted red wine polyphenol, (−)-epicatechin, gallic acid and tartaric acid. The wine components showed more influence on Fe at pH4.00 compared to pH3.25. These results enable a targeted use of these techniques in the assessment of metal-induced spoilage of wine.

Chitosan beads from microbial and animal sources as enzyme supports for wine application

Chitosan beads from a microbial source, i.e. Aspergillus niger (An), were produced by precipitation method for the first time and compared with supports prepared from an animal source, i.e. shellfish derived chitosan with three different molecular weights (low (LMW), medium (MMW) and high (HMW)). The produced beads were used as enzyme carriers to be applied in a continuous packed-bed reactor (PBR) for white wine protein stabilization.For this purpose, the beads were crosslinked with glutaraldehyde (GDH) and stem bromelain was immobilized on the carrier surface as a model enzyme, following two different procedures (i.e., crosslinking with glutaraldehyde (GDH) and direct linkage (DL)).Drop-like beads with an average diameter of 3.0–3.5 mm and a moisture content of 86–94% were obtained. The morphology of the produced beads in dried state, in terms of shape and surface, was studied by means of a scanning electron microscope (SEM), evidencing the obtainment of nearly spherical or oval particles. The efficacy of the crosslinking procedure and of bromelain immobilization was demonstrated by means of SEM investigation and infrared spectroscopy (FT-IR) analysis, revealing a rougher surface. Various initial protein concentrations ranging between 0.45 and 18.00 mgBSAeq mL−1 were tested in order to identify the optimal amount and to evaluate the influence of the initial concentration on the total protein loading.Stem bromelain proved to be more active when immobilized by DL on An beads and was efficient in reducing white wine hazing potential continuously, as verified with a laboratory bench-scale PBR.

Immobilised native plant cysteine proteases: packed-bed reactor for white wine protein stabilisation.

This research presents a feasibility study of using a continuous packed-bed reactor (PBR), containing immobilised native plant cysteine proteases, as a specific and mild alternative technique relative to the usual bentonite fining for white wine protein stabilisation. The operational parameters for a PBR containing immobilised bromelain (PBR-br) or immobilised papain (PBR-pa) were optimised using model wine fortified with synthetic substrate (Bz-Phe-Val-Arg-pNA). The effectiveness of PBR-br, both in terms of hazing potential and total protein decrease, was significantly higher than PBR-pa, in all the seven unfined, white wines used. Among the wines tested, Sauvignon Blanc, given its total protein content as well as its very high intrinsic instability, was selected as a control wine to evaluate the effect of the treatment on wine as to its soluble protein profile, phenolic composition, mineral component, and sensory properties. The treatment in a PBR containing immobilised bromelain appeared effective in decreasing both wine hazing potential and total protein amount, while it did not significantly affect the phenol compounds, the mineral component nor the sensory quality of wine. The enzymatic treatment in PBR was shown to be a specific and mild technique for use as an alternative to bentonite fining for white wine protein stabilisation.

Protein/Polysaccharide Interactions and Their Impact on Haze Formation in White Wines

Proteins in white wines may aggregate and form hazes at room temperature. This was previously shown to be related to pH-induced conformational changes and to occur for pH <3.5. The aim of the present work was to study the impact of wine polysaccharides on pH-induced haze formation by proteins but also the consequences of their interactions with these proteins on the colloidal stability of white wines. To this end, model systems and purified global pools of wine proteins and polysaccharides were used first. Kinetics of aggregation, proteins involved, and turbidities related to final hazes were monitored. To further identify the impact of each polysaccharide, fractions purified to homogeneity were used in a second phase. These included two neutral (mannoprotein and arabinogalactan) and two negatively charged (rhamnogalacturonan II dimer (RG-II) and arabinogalactan) polysaccharides. The impact of major wine polysaccharides on wine protein aggregation at room temperature was clearly less marked than those of the pH and the ionic strength. Polysaccharides modulated the aggregation kinetics and final haziness, indicating that they interfere with the aggregation process, but could not prevent it.

The challenging SO2-mediated chemical build-up of protein aggregates in wines

Despite the extensive research performed during the last decades, the multifactorial mechanism responsible for white wine protein haze formation is not fully characterized. A model is proposed, which is essentially based on two postulates: the experimental identification of sulfur dioxide as the non-proteinaceous factor, and the inference from reliable data available in the literature of the dynamic chemistry played by wine protein sulfhydryl groups. Unlike other reducing agents, addition of SO2 to must/wine upon heating cleaves intraprotein disulfide bonds, hinders thiol-disulfide exchange during protein interactions, and leads to formation of novel interprotein disulfide bonds. These bonds are ultimately responsible for wine protein aggregation following a nucleation-growth kinetic model, as shown by Dynamic Light Scattering experiments. The model was tested in wine model solution (using total and fractionated wine proteins) and validated under real wine conditions. The results achieved may open the way to develop techniques that will find wide application in the wine industry.

Impact of bentonite additions during vinification on protein stability and volatile compounds of Albariño wines.

Today, bentonite continues to be one of the most used products to remove proteins in white wines in order to avoid their precipitation in bottles. However, excessive use of bentonite has negative effects on the aroma of final wine, so the optimization of the dose and the time of its application are important for winemakers. This paper analyzes how applying an equal dose of bentonite at different stages (must clarification; beginning, middle, and end of fermentation) affects the macromolecular profile, protein stability, physical-chemical characteristics and aromatic profile of the wine obtained. The results showed the addition during fermentation (especially in the middle and at the end) reduced the total dose required for protein stabilization of Albariño wines and maintained the sensory characteristics of this variety.

Advantages of the KDS/BCA Assay over the Bradford Assay for Protein Quantification in White Wine and Grape Juice

The present study compared the performance of two colorimetric protein assays, the Bradford and the potassium dodecyl sulfate/bicinchoninic acid (KDS/BCA) assays, for use in wine and grape juice analysis. The Bradford assay was affected by protein type, whereas the KDS/BCA assay had lower protein-to-protein variation. Bovine serum albumin and lysozyme yielded an absorbance (562 nm) vs. protein concentration slope (dose–response curve) similar to that of wine proteins. In the Bradford assay, the presence of 12% ethanol and 200 mg/L of wine polyphenols decreased the protein absorbance by 28 and 16%, respectively, whereas in the KDS/BCA assay such interference was not significant. Among 64 white wines, the correlation between protein haze potential, determined by a heat test, and protein content was better for the KDS/BCA assay. This study confirmed the superiority of the KDS/BCA assay over the Bradford assay for quantifying protein in white grape juice and wine, and it yielded better predictive value with respect to the risk of white wine protein instability.

Effect of wine inhibitors on the proteolytic activity of papain from Carica papaya L. latex.

The influence of potential inhibitors naturally present in wine on the proteolytic activity of papain from Carica papaya latex was investigated to evaluate its applicability in white wine protein haze stabilization. Enzymatic activity was tested against a synthetic tripeptide chromogenic substrate in wine-like acidic medium that consisted of tartaric buffer (pH 3.2) supplemented with ethanol, free sulfur dioxide (SO2 ), grape skin and seed tannins within the average ranges of concentrations that are typical in wine. The diagnosis of inhibition type, performed with the graphical method, demonstrated that all of tested wine constituents were reversible inhibitors of papain. The strongest inhibition was exerted by free SO2 , which acted as a mixed-type inhibitor, similar to grape skin and seed tannins. Finally, when tested in table white wines, the catalytic activity of papain, even when if it was ascribable to the hyperbolic behavior of Michaelis-Menten equation, was determined to be strongly affected by free SO2 and total phenol level.

Effect of free and immobilised stem bromelain on protein haze in white wine

Background and Aims: Several studies have investigated the effectiveness of several proteolytic enzymes in free form as a treatment alternative to bentonite fining for wine stabilisation. This work tested the feasibility of stem bromelain, free or immobilised on a chitosan support, to reduce white wine protein haze potential. Methods and Results: The kinetic behaviour of stem bromelain, in free form and covalently immobilised on chitosan beads, was studied in 12 unfined white table wines fortified with a tripeptide chromogenic substrate. The effectiveness of the protease on wine hazing potential was determined by treating the wines in a laboratory-scale stirred reactor, and the heat test was applied to evaluate the decrease in nephelometric turbidity units. Immobilised protease was active towards the synthetic substrate and in reducing hazing potential of the wine. The effect of wine components on bromelain proteolytic behaviour, as investigated by principal component analysis, was much more noticeable for the free enzyme (in decreasing order of importance: pH, total phenolic substances, free and total sulfur dioxide) than for the immobilised form. Conclusions: An amount of 10 g/L biocatalyst (bromelain immobilised on chitosan beads), after a 24-h treatment in a laboratory-scale stirred reactor, revealed a high capacity to reduce wine haze potential (approximately 70%), which was unaffected by wine composition. Significance of the Study: Immobilised bromelain appears a suitable treatment alternative to bentonite fining for white wine haze stabilisation.