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.     

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.     

两种澄清剂对4种鲜葡萄酒的下胶效果分析

选用保香皂土和蛋清作为下胶澄清剂对4种鲜葡萄酒下胶,分析2种下胶澄清剂的下胶量对4种鲜葡萄酒热稳定性及感官质量（包括外观、香气和口感）的影响。结果表明,鲜酒轻柔桃红葡萄酒、鲜酒轻柔红葡萄酒和鲜酒浓甜红葡萄酒选用保香皂土下胶,下胶量为1.0 g/L时,3款葡萄酒的热稳定性均合格且风味物质损失最少,感官评分分别为92、91、93;鲜酒轻柔干红葡萄酒选用蛋清下胶,下胶用量为0.07 g/L时,轻柔干红葡萄酒热稳定性合格且风味物质损失最少,感官评分为93分。     

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.     

Effects of Fining Agents on Antioxidant Capacity of Red Wines

The antioxidant capacity of wine depends not only on the grape variety but also on the wine processing steps. The aim of this study was to determine the effects of different fining agents, used at different concentrations, on the antioxidant status of fined wines. The results demonstrated that the use of a combination of gelatin and Kieselsol led to the highest total phenol value (3,491 mg/L GAE) and antioxidant activities (29%) among the tested fining agents. Wines were mostly negatively affected by the use of egg white as an agent and led to the lowest value of total phenol (3,038 mg/L GAE) and the lowest rate of antioxidant activity (26%). Significant differences (p<0.05) were determined between gelatin, egg white, and the control groups. The results of the grouping of analyzed parameters in n‐dimensional space, with different fining agents at different concentrations, demonstrated the importance of a low concentration of fining agents for high antioxidant activity and total phenols.     

Lysozyme in wine: A risk evaluation for consumers allergic to hen's egg

Lysozyme used in wine production could present a risk for consumers allergic to hen's egg. Thus, precautionary labeling of lysozyme on wines has been adopted within the European Community by updating Annex IIIa by Directive 2007/68/EC on November 27, 2007. Since no scientific data is known about the actual amounts and risks of lysozyme in wines, various in vitro efforts and skin prick tests were applied in this study to evaluate the presence of lysozyme in wines and the reactivity of those residues in allergic individuals and to fulfill the claim of updating Annex IIIa announced in Directive 2003/89/EC. Depending on the wine's color (red or white wine) and fining with bentonite, which is known as an important step to remove unstable proteins mainly from white wines, diverse results were obtained concerning the amounts of lysozyme in finished wines and their in vitro and in vivo reactivity in humans allergic to hen's egg.     

Residual Amounts of Proteinaceous Additives in Table Wine

Radioactive acetic anhydride was used to label casein, isinglass, egg white, and a pectic enzyme complex. The pectic enzyme was added to three different grape juices which were fermented, bentonite fined and filtered in an approximation of industrial practice. Three wines were fined using isinglass or egg white (red wines) or casein (white wines) applied at three rates each, and finished as above. Residual amounts of each additive were determined by scintillation counting of samples taken at each step. All of the pectic enzyme remained in the finished wines. Residual fining agents decreased with the application rate to levels showing much variation in the red wines and less in the whites.     

Collaborative Tests of ELISA Methods for the Determination of Egg White Protein and Caseins Used as Fining Agents in Red and White Wines

Methods are needed for detecting allergenic proteins in wine after fining processes, both for legal requirements and to estimate any risk for allergic individuals. Two of the critical allergens in this case are the protein fraction from egg white and caseins. Requirements for methods were set up both by the International Organisation of Vine and Wine (OIV) and the European Commission and included collaborative tests to determine repeatability, reproducibility, limit of detection (LOD), and recovery. Two collaborative tests with 18 participants from all over the world were conducted using commercial enzyme-linked immunosorbent assay kits to quantify egg white protein and casein. For each analyte, nine red and white wines (RW and WW, respectively) with different analyte concentrations were prepared and sent to the participants. Resulting sets of data were analyzed according to the AOAC by eliminating outliers and calculating the performance statistics for each assay. For wines with no or very low analyte concentrations, the values were extrapolated from the calibration curve and the resulting LODs are in accordance with the performance criteria set by the OIV method OIV-MA-AS315-23: R2012. For casein in WW and egg white protein in RW, all Horwitz ratio values for samples with concentrations above the required limit of quantification (0.5 mg/L) were below 2. While the recovery of egg white protein from RW is 90 % or more, the recovery of casein from WW is between 73 and 76 %, which is attributable to the instability/solubility of casein in wine.     

The evaluation of remedial treatments for wine affected by Harmonia axyridis

This study evaluated the efficacy of a range of commercially available fining agents and other interventions on reducing 2‐isopropyl‐3‐methoxypyrazine (IPMP) concentration and taint characteristics of white and red wine affected by Harmonia axyridis (HA). Wines fermented in the presence of HA beetles were treated with bentonite, activated charcoal, oak chips, deodorized oak chips and either ultraviolet (red wine) or visible (white wine) light. IPMP concentrations were lowered by activated charcoal in white wine and deodorized oak in red wine. Treatment with oak chips reduced the intensity of HA‐associated sensory attributes (‘ladybug taint’) in both white and red wines, while other applications generally had no effect on white wine and limited effect on red wines.     

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.     

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     

Comparison of fining red wines with purified grape pomace versus commercial fining agents: effect on wine chromatic characteristics and phenolic content

The large quantity of grape pomace (seeds, skin, and peduncle) produced during the winemaking process can be a problem and the search for procedures which could permit their valorisation is considered an important issue. This study investigates the use of purified grape pomace as a fining agent for reducing the level of some wine phenolic compounds, especially tannins. For this, purified grape pomace was applied to three young red wines of different phenolic composition and the results were compared with the effect of some common commercial fining agents. The results demonstrated that grape pomace can be used to decrease the wine tannin content with similar results to those obtained with casein, while also reducing anthocyanin content although to a similar extent to when bentonite is used. The effect of purified Monastrell grapes pomace is similar in the three different studied wines although it affected the most to wines with high monomeric anthocyanin content.     

Use of patatin, a protein extracted from potato, as alternative to animal proteins in fining of red wine

The use of plant-derived proteins as wine fining agent has gained increased interest owing to the potential allergenicity of animal proteins in susceptible subjects. Patatin P is the name of a family of glycoproteins that can be recovered from potato aqueous by-product. In this study, a comparative fining trial simulating industrial procedures with 10, 20 and 30?g/hL of commercial preparations of patatin, potassium caseinate, gelatin and egg albumin on an Aglianico (Vitis vinifera L.) red wine was performed. Color indexes and phenolics were analyzed by spectrophotometric methods and HPLC. The potential astringency has been evaluated by an index based on the ability of wine to precipitate salivary proteins (SPI, Saliva Precipitation Index). Patatin is a suitable alternative to animal proteins used as fining agent because: (i) a decrease in total phenolics and tannins after the treatments with 10, 20 and 30 g/hL of commercial preparation containing P was detected; (ii) Patatin, as well as all the fining agents used in this experiment, is able to diminish astringency and the content of red wine phenolics able to react with salivary proteins. Considering all concentrations tested, the effectiveness in reducing proteins reactive towards wine polyphenols was patatin?=?gelatine > egg albumin > casein (p?<?0.05); (iii) at each concentration considered, the treatment with patatin causes no depletion of chromatic characteristics of red wine although a significant slight loss of individual anthocyanins was observed.     

Detection of Traces of Ovalbumin and Casein in White and Red Wines by Quantitative Western Blotting

Fining of wine with agents containing cow's milk or hen's egg white is a common and traditional procedure. In light of increasing food allergies all over the world, the presence of fining residues has been subject of intense debate. Switzerland does not make exception, and since 2009 the Federal Department of Home Affairs has modified its food regulations stating that the labels must show if traces of fining agents are present. Nevertheless, the application of this regulation is not based on an official analytical method. In this study we show that immunoblotting is an efficient technique to detect and quantify ovalbumin and casein residues in bottled wine. We showed that final filtration is an essential step to remove finings in red wine, and that overfining of white wine may result in fining residues in finished products. Finally, for the first time in Switzerland, 22 samples were taken by food safety inspectors and officially analyzed for the regional food control authority of the Canton of Vaud. These samples were allergen free, but a larger study is currently planned in collaboration with other regional authorities of Switzerland to complete these results and make a complete picture of the Swiss wine production.     

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.     

Spectral evaluation of total phenolic components in Vitis vinifera: Grapes and wines

Whereas high levels of flavonoid extractives are responsible for the characteristic λ_Dmax at about 280 nm in the ultra-violet spectra of red wines, there is wide variability in the spectral features of natural white wines, in which the phenolic content is often minimal. For the latter, λ_Dmax may occur anywhere within the range 265–285nm, with relatively strong absorbance at 320 nm arising from the presence of hydroxycinnamate esters. Massive fining of many white wines and juices with polyvinylpyrrolidone (10% w/v) has shown residual non-phenolic u.v. absorbing constituents to be sufficiently uniform to enable direct spectral measurement of total hydroxycinnamates in white juices, white wines and rosés as (E₃₂₀-1.4) absorbance units. For white grape juices and white wines, total hydroxycinnamates are directly quantitated as mg litre^?1 ‘caffeic acid equivalents’ and total flavonoid extractives may also be estimated. Total phenolic components of red wines are accounted as (E₂₈₀-4) absorbance units. The spectral interpretations enable comparative estimation of flavonoid extractives in all natural wine types.     

Ochratoxin A in wine and grape juice sold in Canada.

Ochratoxin A (OTA) was determined in 251 samples of wines and grape juice collected over 3 years in Canada. In total, 25/84 samples of red wine, 22/96 samples of white wine, 3/46 red grape juices and 1/25 white grape juices contained OTA levels above the limit of quantitation (LOQ). Canadian wines, when compared with imported products, showed both a lower OTA occurrence, noted as positive (19 versus 48% above the limit of detection (LOD) for wines), and a lower level of OTA contamination (upper bound mean of 17.5 versus 163pg ml(-1) for wines). Wines from the USA contained no quantifiable levels of ochratoxin A. OTA was found in Canadian and US grape juice samples, with 12.9% above the LOD and an upper bound mean of 13.3pg ml(-1). It was extracted from a wine or grape juice sample by passing it through an immunoaffinity column. The sample matrix was washed off the column with water. OTA was eluted from the column with methanol and quantitatively determined by liquid chromatography using a fluorescence detector. The presence of OTA was confirmed by esterification with boron trifluoride-methanol. The LOQ of OTA was estimated as 20 pg ml(-1) in white wine (S/N 10:1) and 40 pg ml(-1) in red wine, white grape juice and red grape juice (S/N 20.1). The LOD was estimated as 4pgml(-1) for white wine and 8pgml(-1) for red wine and white and red grape juices (S/N 3:1).     

Quality of base and sparkling wines as influenced by the type of fining agent added pre-fermentation

A comparison of the effect on base wines of pre-fermentative clarification between a fining mixture (potassium caseinate, bentonite and cellulose microcrystalline) and bentonite was carried out. Fining agents were added to two grape juices from different cultivars: Macabeo and Parellada. These varietal wines are used for making sparkling wine within the appellation (certified brand of origin) Cava. Vinification was done in parallel on an industrial scale (100 000 l). The use of the fining mixture in grape juice made the fermentations more complete and gave wines with less browning ability, more foam stability time and a lower content of nitrogenous fraction, polyphenols and some volatile compounds than the wines treated with single bentonite. Sensorial analysis showed that the wines produced with the use of different fining agents had different organoleptic characteristics. In addition, the effect of fining agent added pre-fermentatively was observed on some components of sparkling wines.     

Determination of residues of propamocarb in wine by liquid chromatography-electrospray mass spectrometry with direct injection.

A simple, sensitive and reliable liquid chromatography-mass spectrometry method with direct injection of diluted samples is reported for the determination of propamocarb residues in wine. Red and white wines were diluted 40- and 20-fold, respectively, using water. Liquid chromatography was performed with a mobile-phase gradient and detection was by electrospray mass spectrometry in a positive ionization mode. Propamocarb was detected as the protonated molecular species at m/z 189. Using matrix-matched calibrant solutions, a calibrated range equivalent to 0.05-2.0 mg kg(-1) in red and white wines and limits of detection of 0.025 mg kg(-1) for white wine and of 0.05 mg kg(-1) for red wine (0.00125 microg ml(-1) of sample solution injected) were readily achievable. Recovery of propamocarb hydrochloride from wine spiked before dilution was in the range 91-115%. The chromatograms were free of isobaric interferences. In a small wine survey (72 samples), no residues of propamocarb were detected above 0.1 mg kg(-1).     

Repercussion of the clarification treatment agents before the alcoholic fermentation on volatile composition of white wines

Albariño must was clarified with two fining agents, as bentonite and silica gel. Alcoholic fermentation was carried out at semi-industrial scale, and the concentration of free monoterpenes, norisoprenoids, C₆-alcohols, higher alcohols, fatty acids, ethyl esters, and acetates was then investigated. The use of bentonite allows loss of several volatile compounds compared to wine control. In a second step, the effect of clarification–stabilization treatments with silica gel on aromatic substances was compared in order to study this influence in white wines elaborated from different aromatic varieties (Albariño and Loureira). The results showed that the use of these fining agent plays a significant role in the wine volatile composition.