Collaborative Interlaboratory Studies for the Validation of ELISA Methods for the Detection of Allergenic Fining Agents Used in Wine According to the Criteria of OIV Resolution 427–2010 Modified by OIV–Comex 502–2012

The clarification or fining of wine removes undesired substances (mainly proteins, phenols, and tannins), which would roil the wine and cause bitterness and astringency. A common fining agent, egg white, can be directly added to wine through the inlet of a circulating pump, but more typically egg white comes as commercial preparation in powdered form (commercially named egg albumin). Skimmed milk or more frequently purified caseinates are used to remove bitterness and hardness of white wine and sherry. Both egg white and caseinates are fining agents with optimal enological properties, but their residues could represent a risk for subjects suffering from food allergy. The rules for allergen labeling were detailed in Directives 2003/89/EC, and Directive 2005/26/EC established a list of food ingredients provisionally excluded from labeling, that included wine fining agents. Extended till June 2012, wine labeling exemption can be now maintained only if (1) egg and milk derivatives are not used and cross-contamination is under control; and (2) wine clarified with such products is negative for the presence of residues using techniques with detection and quantification limits of 0.25 and 0.5 ppm, respectively. Analytical requirements were defined in the OIV resolution 427–2010 (OIV 2010) modified by OIV/COMEX 502–2012 (OIV 2012). On the basis of a previous experience, an interlaboratory collaborative trial was organized to validate a commercial ELISA kit designed to measure allergenic residues in red wine fined with egg white proteins. In the meantime, the performance of the commercial caseinate ELISA kit for white wine was rechecked according to the new limit of detection and limit of quantification values, recommended by OIV in 2012. The collaborative interlaboratory studies showed that both ELISA kits had good reproducibility, repeatability, and robustness in detecting residues of allergenic fining agents in wine, in good agreement with the requirements of the OIV resolution 427–2010 modified by OIV/COMEX 502–2012.     

Ochratoxin A removal from red wine by several oenological fining agents: bentonite,egg albumin,allergen-free adsorbents,chitin and chitosan

The ability of several oenological fining agents to remove ochratoxin A (OTA) from red wine was studied. The adsorbents tested were activated sodium bentonite, egg albumin, allergen-free adsorbents (complex PVPP, plant protein and amorphous silica (complex) and high molecular weight gelatine), and the non-toxic biodegradable polymers (chitin and chitosan). Several dosages within the oenological use range were tested and the wine pH, colour parameters and polyphenol concentration impact associated with each fining agent were studied. Generally, OTA removal achieved in all treatments was higher when the adsorbent dosage increased, but the impact on wine quality also was higher. Chitin at 50?g?hl^?1 removed 18% the OTA without affecting significantly the wine-quality parameters. At the highest dosage tested the gelatine and complex treatments achieved greater OTA removal (up to 39–40%) compared with bentonite, egg albumin and chitin. Moreover, the gelatine and the complex had a lower impact on colour parameters and polyphenol concentration compared with chitosan, whilst OTA was reduced to around 40%. Chitosan achieved the greatest OTA removal (67%), but it strongly affected the wine-quality parameters. Otherwise, bentonite showed a relative efficiency to remove OTA, but the CI value decreased considerably. The egg albumin treatment only removed OTA up to 16% and moreover affected strongly the CI value and CIELab parameters. The results of this survey showed that the non-toxic chitin adsorbent and the allergen-free adsorbents tested could be considered as alternative fining agents to reduce OTA in red wine.     

Ochratoxin A removal from red wine by several oenological fining agents: bentonite, egg albumin, allergen-free adsorbents, chitin and chitosan

The ability of several oenological fining agents to remove ochratoxin A (OTA) from red wine was studied. The adsorbents tested were activated sodium bentonite, egg albumin, allergen-free adsorbents (complex PVPP, plant protein and amorphous silica (complex) and high molecular weight gelatine), and the non-toxic biodegradable polymers (chitin and chitosan). Several dosages within the oenological use range were tested and the wine pH, colour parameters and polyphenol concentration impact associated with each fining agent were studied. Generally, OTA removal achieved in all treatments was higher when the adsorbent dosage increased, but the impact on wine quality also was higher. Chitin at 50 g hl(-1) removed 18% the OTA without affecting significantly the wine-quality parameters. At the highest dosage tested the gelatine and complex treatments achieved greater OTA removal (up to 39-40%) compared with bentonite, egg albumin and chitin. Moreover, the gelatine and the complex had a lower impact on colour parameters and polyphenol concentration compared with chitosan, whilst OTA was reduced to around 40%. Chitosan achieved the greatest OTA removal (67%), but it strongly affected the wine-quality parameters. Otherwise, bentonite showed a relative efficiency to remove OTA, but the CI value decreased considerably. The egg albumin treatment only removed OTA up to 16% and moreover affected strongly the CI value and CIELab parameters. The results of this survey showed that the non-toxic chitin adsorbent and the allergen-free adsorbents tested could be considered as alternative fining agents to reduce OTA in red wine.     

Precipitation of salivary proteins after the interaction with wine: the effect of ethanol, pH, fructose, and mannoproteins

Astringency is a complex sensation mainly caused by the precipitation of salivary proteins with polyphenols. In wine it can be enhanced or reduced depending on the composition of the medium. In order to investigate the effect of ethanol, tartaric acid, fructose, and commercial mannoproteins (MPs) addition on the precipitation of salivary proteins, the saliva precipitation index (SPI) was determined by means of the sodium dodecyl sulphate polyacrylamide gel electrophoresis of human saliva after the reaction with Merlot wines and model solutions. Gelatin index, ethanol index, and Folin-Ciocalteu index were also determined. As resulted by Pearson's correlation, data on SPI were well correlated with the sensory analysis performed on the same samples. In a second experiment, increasing the ethanol (11%-13%-17%), MPs (0-2-8 g/L), fructose (0-2-6 g/L) level, and pH values (2.9-3.0-3.6), a decrease in the precipitation of salivary proteins was observed. A difference in the SPI between model solution and red wine stated that an influence of wine matrix on the precipitation of salivary proteins occurred. PRACTICAL APPLICATION: Results provide interesting suggestions for enologists, which could modulate the astringency of red wine by: (i) leaving some residual reducing sugars (such as fructose) in red wine during winemaking of grapes rich in tannins; (ii) avoiding the lowering of pH; (iii) adding commercial mannoproteins or promoting a "sur lie" aging; and (iv) harvesting grapes at high technological maturity in order to obtain wines with a satisfactory alcoholic content when possible.     

Clarifying effect of different fining agents on mulberry wine

Fining experiments have been conducted on mulberry wine, in which five fining agents (bentonite, gelatin, casein, egg albumin and PVPP) were investigated. The effect of these agents on polyphenol content, colour intensity, antioxidant activity and aromatic profile has been determined. In general, all treatments improved the limpidity of the mulberry wines to some extent. The wine was mostly negatively affected by gelatin and led to the greatest reduction of phenol content (16.61%), colour intensity (32.26%) and antioxidant activity. Concerning aroma, the addition of PVPP, casein and bentonite both significantly altered the aroma profile. It is noteworthy that egg albumin led to the lowest diminution total phenol value (5.74%) and improvement of antioxidant capacity and colour among the tested fining agents. Furthermore, the aroma profile remained mostly unaffected by egg albumin. As consequence, the optimal fining agent for mulberry wine was egg albumin.     

Fining of Red Wines with Pomace Cell Wall Material: Effect on Wine Phenolic Composition

Fining is a winemaking technique used to remove unwanted wine components that affect clarification, astringency, color, bitterness, and aroma. One of the objectives of fining is often to reduce the wine tannin content due to its effect on wine astringency. Proteinaceous agents are commonly used with this objective, but problems related with their possible allergenic properties or the excessive enrichment of wine with proteins, which may cause stability and turbidity problems, have led to the search for new fining agents. In this way, the cell wall material from processed grape pomace could be a good alternative since it has a high affinity for tannins. In this work, the effect of cell wall material from processed pomace from Monastrell and Cabernet Sauvignon grapes on the reduction of the phenolic content of red wines is studied and the results are compared with those obtained with commercial fining products. Also, the varietal effect due to the different composition of the cell wall material from these two varieties has been evaluated. The concentration and composition of wine anthocyanins and tannins, before and after the fining process, were analyzed by HPLC and their molecular mass distribution by SEC. The results showed that, at laboratory scale, pomace cell walls have a fining effect that exceeds that of most the protein-based fining agents, even when used at their highest recommended doses. The cell wall material significantly reduced the wine phenolic content, the reduction ranging from 48 to 68% for anthocyanins and from 44 to 64% for tannins, although varietal differences exist regarding the cell wall-binding capacity. Monastrell cell walls exhibited the highest capacity and could be used at much lower doses than those used in this study, reducing the formation of lees and the wine adsorbed on them. The results indicate that this material could be an appropriate alternative for protein-based fining agents in red wine and their use would avoid allergen-related effects.     

Superficial Charge Density of Fining Agents: Influence of pH,Dose, and Temperature

The effects of pH, dose, and temperature on the superficial charge density of fining agents were determined in a model solution. This study included three gelatines and one egg's albumin, all of which are common fining agents, as well as three wheat glutens (one native and two hydrolyzed). Wheat glutens have been allowed to be used as fining agents only since 2005, and there are no preliminary studies examining them. The results of this study showed that the superficial charge density was mainly pH-dependent (p < 0.01), and that pH showed a greater effect at a higher degree of protein hydrolysis. For Gel 3, albumin and the wheat glutens, a larger protein concentration, decreased the superficial charge density. Gel 2, which was soluble in hot water, had the highest superficial charge density (>0.831 meq g^?1), which indicated that it would be more reactive than the other fining agents. Between pH 3.4 and 4.0, Gluten 1 and Gel 3 were more highly charged (superficial charge density >0.3 meq g^?1) than Gel 1 or the hydrolysed glutens. These data are useful for wine producers.     

Influence of fining agents on the sensorial characteristics and volatile composition of mead

Mead, one of the oldest fermented drinks, is derived from the fermentation of diluted honey by yeasts. In the context of wine production, several procedures are applied to stabilize the beverage and to improve its organoleptic properties. This study aims to evaluate the impact of adding fining agents on the production of mead. In general, the best results were obtained for the samples containing just one fining agent instead of two combined. However, the best performance was obtained for the combined fining agents (bentonite + gelatine + egg albumin). Tannins decreased significantly the content of volatile compounds. On the other hand, silica appears to be the best fining agent, resulting in the lowest loss of volatile compounds. Thirty‐six volatile compounds were determined by gas chromatograph–flame ionization detector and gas chromatography–mass spectrometry, including alcohols (42.5%), carbonyl compounds (40.4%), acetates (14.4%) and esters (1.8%). Eleven volatile compounds had odour activity values >1, representing those with a major impact on the aroma of mead. Significant differences (p  < 0.05) were found in 10 volatile compounds independently to the type of treatment used and no differences (p  > 0.05) were observed for remaining compounds. Copyright © 2017 The Institute of Brewing & Distilling     

Evaluation of the astringency of commercial tannins by means of the SDS–PAGE-based method

The astringency of wines enriched with commercial tannins (CTs) was evaluated by a method based on the SDS–PAGE electrophoresis of salivary proteins after the reaction of saliva with wine. Nineteen CTs tested in synthetic wine at the same pH (3.6) and concentration (1 g/l) gave different values of saliva precipitation index (SPI). The effect of CTs addition was investigated in four wines. Results showed that the wine matrix influenced the astringent capacity of CTs and that became less pronounced as wine polyphenolic complexity increased. For some types of wine, astringency was not affected, indicating that the effect of CTs utilisation is not easily predictable by classical methods. The ability to objectively evaluate the astringency provided by CTs with the SDS–PAGE-based method would supply producers and winemakers with a useful tool to manage the processing conditions and thus to improve the quality of wine.     

Challenging the Limit of Detection for Egg Allergen Detection in Red Wines by Surface Plasmon Resonance Biosensor

The improvement of a surface plasmon resonance (SPR)-based immunoassay for the detection of traces of egg-based fining agents in red wines is herein described. The latter represents an extension of a previously developed direct assay targeted to the detection of ovalbumin (OVA) as marker of the presence of egg white powder residues, a typical fining agent utilized by the winery industry. In this paper, a suitable pre-treatment procedure was optimized for the sensitive detection of OVA at sub-ppm levels in red wines fortified with egg-white powder, by using an immunoassay proved to be reliable for both white and roseé wines. A red wine from Chianti grapes selected as reference matrix was artificially contaminated with egg-white powder before undergoing different sample purification. Several purification strategies were investigated and tested in order to challenge the limit of detection (LOD) obtained with the methods currently in use for egg allergen detection. Finally, the optimized two-step pre-treatment, combining polyvinylpolypyrrolidone-based purification and size exclusion chromatography, enabled to achieve an LOD in red wine as low as 0.2 μg/mL. The optimized SPR-based method met the method performance criteria issued by the International Organization of Vine and Wine (OIV) concerning the minimum sensitivity required for the analyses of potentially allergenic fining agent proteins in wines, confirming the biosensor as promising tool to monitor the residual contamination level of fined red wines.     

Impact of wine manufacturing practice on the occurrence of fining agents with allergenic potential

Proteinogenic wine fining agents are hidden allergens and could present a risk for consumers with allergies. Therefore, the European Parliament adopted Directive 2003/89/EC amending Directive 2000/13/EC to declare ingredients, contaminations and processing aids that are known to trigger allergic reactions. The Amendment Regulation (EU) 1266/2010 excluded the labelling of wines which are processed with hen’s egg and products thereof until 30 June 2012 to get more scientific findings. After 1 July 2012 wine fining agents have to be declared if above 0.25 mg l^–1 (Regulation (EU) 579/2012 in conjunction with article 120 g of Regulation (EU) 1234/2007). The Organisation International de la Vigne et du Vin (OIV) advises this limit of detection (LOD) for potential allergenic residues of proteins. Wine fining agents are processing aids and according to the wine producer’s knowledge will be removed after coagulation by filtration or other production steps. Due to lack of scientific data, residues of fining agents in the final product could not be excluded. In this risk assessment, highly sensitive ELISA methods for ovalbumin of known origin for wine have been developed. The objective was to investigate the presence of allergen residues in wine after certain technological treatments were applied to remove the wine fining agents. For all developed ELISA methods the LODs are in the low µg l^–1 range between 5 and 10 µg l^–1 fining agent, whereas the LOQ varies between 5 and 80 µg l^–1 fining agent. The results of the investigation of well-known wines and fining agents demonstrate that white wines fined with white or ovalbumin from hen’s egg could retain allergens. The use of certain technological procedures during wine processing leads to different results. In white wine, bentonite or sheet filtration followed by sterile filtration lead to wines containing no detectable amounts of ovalbumin. In red wine, especially the final sterile filtration removes the fining agents.     

Red wine astringency: a review

The chemical and physical basis for red wine astringency is reviewed. Models describing the physiological foundation of astringency are presented and compared. The phenolic and other red wine components that evoke astringency are described, together with their sensory properties and the factors that affect their perception. The paper also presents a detailed account of the chemical properties of saliva and the possible modes of interaction between these and red wine phenolics that lead to the necessary physical changes in saliva required to elicit astringency. Reasons for differences in astringency perception across oral sites and amongst individual tasters are also discussed. It is concluded that whilst great advances have been made in the field of red wine phenolic chemistry in recent years, a better understanding of the effect of wine polyphenol-salivary protein interaction on the rheological properties of saliva is required in order to gain a comprehensive understanding of red wine astringency.     

Effect of aging in new oak, one-year-used oak, chestnut barrels and bottle on color, phenolics and gustative profile of three monovarietal red wines

This study provides specific information about the influence of storage in bottle and in 225-L barrels made from oak (new and used for 1 year) and chestnut wood on color indexes, spectrophotometric evaluable polyphenols, anthocyanins, phenolic acids and gustative attributes of three monovarietal red wines (Piedirosso, Cabernet Sauvignon and Merlot). The results of the analysis of variance show that wood type has a significant influence on chromatic characteristics, on vanilline reactive flavans, on low molecular weight phenolics and on astringency of wines. The effect depends on the type of wine. Therefore, both traditional and alternative wood containers could be used as an instrument to regulate the polymerization, oxidation and copigmentation reaction of wine phenolics and the sensory properties of red wine. The maturation in chestnut wood could be an interesting challenge to widen the supply of red wines maturated in wood, but its use needs more care than oak.     

Enological tannins affect acetaldehyde evolution,colour stability and tannin reactivity during forced oxidation of red wine

The addition of enological tannins is a practice admitted and widely used to improve wine phenolic compositions. Although wine ageing is an oxidative process involving phenolic compounds and acetaldehyde production and consumption, the effect of enological tannins on the acetaldehyde and chromatic and phenolic changes of a red wine during oxidation is still not known. In this study, three enological tannins preparations containing gallotannins GT, ellagitannins ET and condensed tannins CT were added to a young commercial Lambrusco red wine in order to obtain wines with three different phenolic compositions. Samples were then treated with hydrogen peroxide to trigger oxidation. All added tannins have determined a production of acetaldehyde greater than control. Acetaldehyde was then consumed in 30 days. The samples treated with CT and ET showed, after oxidation, an increase in colour intensity, determined as the sum of 420, 520 and 620 nm absorbances. A simultaneously increase in polymeric pigments was detected. No significant variation of the Saliva Precipitation Index (SPI), an index useful to give an indirect measure of wine astringency, was observed after 30 days of treatment of CT and ET samples. Results highlight that the addition of CT and ET can help to stabilise colour without affecting wine reactivity towards salivary proteins. A significant role of acetaldehyde has been even detected. These are the first data showing the effect of different enological tannins on the production and consumption of acetaldehyde formation during an oxidation process.     

Validation by a Collaborative Interlaboratory Study of an ELISA Method for the Detection of Caseinate Used as a Fining Agent in Wine

The clarification or fining of wine removes undesirable substances such as proteins, phenols, and tannin compounds that would cloud the wine and cause bitterness and astringency. Caseinates have useful fining properties, but their residues could present a risk for allergic subjects. A commercial kit that was developed to detect caseinates in food has been examined for its applicability to a wine matrix; it is sensitive to caseinates at concentrations as low as 1 ppm. The general characteristics of the caseinate assay, described below in detail, are as follows. It is a sandwich enzyme-linked immunosorbent assay (ELISA): the microplate is first coated with a specific anti-casein antibody; and after incubation with the wine sample, a secondary anti-casein antibody conjugated with horseradish peroxidase is added to form a sandwich. The antibodies have been tested for their immunoreactivity and the reproducibility of antigen recognition has been measured. An interlaboratory collaborative trial was organized to evaluate the performance of this ELISA method and its ease of use by laboratories routinely dealing with wine/food analyses. The results satisfy the criteria established by the International Organisation of Vine and Wine in the Compendium of International Methods of Analysis MA-EAS1-07-ETCOL.     

Degree of hydrolysis of some vegetable proteins used as fining agents and its influence on polyphenol removal from red wine

The increasing demand for economic and efficient wine fining leads to the search for alternatives to traditionally used proteins, preferably new products with the side effect of non-allergenicity. A comparative fining trial on laboratory scale with equal quantities of vegetable proteins, partly used for the first time as fining agents for wine in Germany, was accomplished. Spectrum of wheat, rice, maize and potato proteins was expanded by enzymatic hydrolysis, additionally to evaluate influence of protein structure on fining effects. Proteins were modified by two different enzymes, proline-specific endopeptidase (PeP) and alcalase. Molecular weight distributions and degree of hydrolysis (DH) were compared to color intensity and HPLC data of phenolic marker substances primarily responsible for bitterness and astringency. With a total of 20 different varieties of fining agents, statistical evaluation was performed for a rapid exploratory analysis of taste improvement without the need for sensorial tests. Overall, influence on phenol composition and color intensity decreased with the degree of proteolysis. The effect of maize proteins and corresponding hydrolyzates was rather low, whereas maize proteins were nearly not influenced by the use of 2 different peptidases. Wheat proteins with highest proline content (about 9–10%) and molecular weight fractions of 14 and 30–45 kDa had a pronounced effect on color intensity. Rice and potato proteins with only half of proline content and molecular weight fractions mainly ranging between 10 and 32 kDa showed less effect on wine color, but preferably on phenolic marker compounds mainly responsible for bitter and astringent taste sensations.     

Evaluating potential alternatives to potassium caseinate for white wine fining: Effects on physicochemical and sensory characteristics

The use of potassium caseinate for wine fining can cause allergic reactions and must be included on wine bottle labels. The focus of this research was to determine if non-allergenic pea protein or polyvinylpolypyrrolidone (PVPP) are possible alternatives for wine fining. Trials were performed to compare the effects of these potential fining agents on phenolic compounds, browning potential, turbidity and sensory attributes. The results indicate that flavonoid and non-flavonoid phenols decreased (p < 0.05) in the wines treated with potassium caseinate, pea protein, and PVPP. All fining agents decreased (p < 0.05) wine colour. Potassium caseinate was the most effective fining agent for reducing browning potential. Pea protein and potassium caseinate resulted in increased clarity of the final wines. No sensorial differences (p < 0.05) were detected among the wines prepared with the different fining agents. This study provides information that could be used to select an appropriate alternative to potassium caseinate.     

Effects of fining agents on trans-resveratrol concentration in wine

Resveratrol has been identified as a wine component related to moderate wine consumption and a reduction in serum cholesterol levels. Processes such as wine fining that result in loss of resveratrol during winemaking are therefore of interest, and led to these present studies. A number of agents were compared and were found to lower resveratrol levels in all wines to some extent. Results from two studies (1996 and 1997) are reported. The standard addition method was used in combination with High Pressure Liquid Chromatography to calculate resveratrol levels. In Study 1 (1996), recommended maximum levels of all fining agents (Level 3), bentonite, egg white, gelatin + kieselsol and polyvinylpolypyrrolidone (PVPP), lowered resveratrol levels significantly compared to controls (Level 0). Nevertheless, addition of fining agents at Level 0 resulted in resveratrol levels that were significantly higher than those at Level 3, but resveratrol levels in wine from Level 0 were not significantly different from Level 1. In Study 2 (1997), carbon + egg white, and gelatin + kieselsol fining was studied, and their effects differed according to grape variety. Least removal of resveratrol by carbon fining occurred in wine from Cabernet Sauvignon (Vitis vinifera) whereas most removal occurred in wines from Cynthiana (Vitis aestivalis) and Noble (Vitis rotundifolia). Resveratrol levels of control wine were significantly higher than resveratrol levels of wine treated with recommended maximum addition of fining agent in all varieties. Taken overall, any addition of any fining agent lowered resveratrol levels in all wines to some extent, but complex interactions between fining agent and wine variety resulted in different regression trends. While recognising the constraints set by our particular data that could reflect unique circumstances, we are nevertheless able to infer from these trends that low levels of fining agents can be used without statistically significant loss of resveratrol.     

Optimisation of the Amido Black assay for determination of the protein content of grape juices and wines

While the heat/chill stability of white wines cannot be predicted from the total protein concentration, this information is useful in assessing the effectiveness of fining treatments. We have adapted the Amido Black assay for use in grape juice and wine. The response of bovine serum albumin (BSA) was similar in water, model wine and model juice. The linear range of the assay (<100 mg l^?1) extends beyond protein concentrations typically found in grape juices and wines. The limit of quantification was 11.1 mg l^?1 for BSA in model wine and 7.6 mg l^?1 in model juice, while the limit of detection was found to be less than 3 mg l^?1 in either solution. In contrast to other methods of protein determination, this assay is not affected by common wine phenolic and pectic compounds or by glutathione. The mean response of BSA was similar in model juice and red and white grape juices. The mean response of BSA in red wine was lower than in white wine and model wine (p < 0.001), and there was considerable variation in response among wines. Protein concentrations determined using this method were reproducible (CV < 10%). This optimised assay can be used to monitor protein levels in grape juices and wines. © 2001 Society of Chemical Industry