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Milk-based fining materials that are used during winemaking may trigger allergic reactions. If they are present in the final product and depending on a country’s legislation, these substances may have to be declared on the wine label. This labeling helps consumers with allergies avoid wines that may trigger their allergic symptoms. This study investigated the utility of several procedures for removing allergenic residues remaining in wine after the use of milk protein-containing fining agents. Winemaking practices are different depending on country and areas, and because of these different standards, a fining trial was conducted to study the efficiency of various filtration methods and of other treatments for reducing the levels of milk-derived allergens, even in a worst-case scenario involving the use of high amounts of fining agents. ELISA was used to detect milk proteins in wine, and in vivo allergy tests were also conducted. All filtration methods used in this study removed casein from both red and white wines to levels undetectable by the ELISA. Other removal methods evaluated in this study were equally efficient, apart from flash pasteurization or silica sol, when a large amount of casein was applied. However, an additional sterile filtration after flash pasteurization or silica sol fining decreased casein proteins to undetectable levels as well.

Fining of wine with proteinogenic fining agents such as casein from cow's milk is a traditional and commonly used technique all over the world. Casein and other proteins from cow's milk are well-known food allergens, which pose a risk for allergic consumers. Temporary regulations exempting the labeling of milk and products thereof in wine expired. Since July 1, 2012, these fining agents have to be declared on the wine label under Regulation (EU) No. 579/2012 in conjunction to article 120g of Regulation (EU) No. 1234/2007 if exceeding the threshold of 0.25 mg/L allergenic protein. The aim of the presented study was to develop sensitive ELISA methods for the detection of casein in white and red wines and to investigate the risk of allergenic residues in fined wines. In this context it was shown that the used substance for calibration is highly relevant. Casein wine fining agents of different commercial producers were investigated by LDS-PAGE and immunoblot. In addition to casein, they contain other milk proteins, which are potentially allergic and therefore have to be incorporated in the development of antibodies for an ELISA method to be set up. An indirect ELISA for the investigation of white wine was developed. The LOD is 0.1 mg/L. For red wine the LOD is 0.2 mg/L in an indirect sandwich ELISA setup. The LOD of the indirect sandwich ELISA for white wine depends on the calibration standard. It is 0.1 mg/L for the fining agent casein and 0.01 mg/L for casein from a chemical trader. It is also shown that the use of different technological procedures during winemaking leads to no detectable amounts of casein in various wine samples.

Potential residues of the potent allergen lysozyme used as a microbial stabilizing agent in wine production might pose a serious health thread to susceptible individuals. Therefore, EU legislation requires the labeling of the allergenic agent, if it is present in the final product. To allow for product testing, an indirect ELISA method to be specifically used in wine analysis was developed and validated. Furthermore, trial wines treated with defined amounts of lysozyme were subjected to an array of different filtration and other enological processing regimes in order to evaluate their potential to deplete the allergen content of the wines. By these means, processing methods ought to be identified that can be integrated in a good manufacturing practice guideline to enable wine producers to utilize lysozyme in their cellars and still provide wines free of allergenic residues. However, among the enological procedures under scrutiny, only bentonite fining proved to be capable of significantly reducing the allergenic residues.

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.

Allergenic fining agents and processing aids from hen's egg and cow's milk used in wine production are hidden allergens and could demonstrate a health threat for allergic persons. Hence, the European parliament adopted Directive 2003/89/EC amending 2000/13/EC to declare ingredients, contaminations and processing aids which are known to trigger allergic reactions. The Amendment Directive 415/2009/EC excludes the labeling of wine which are processed with eggs or milk and products thereof until the 31st of December 2010. In 2006 Rolland et al. did a double-blind, placebo-controlled trial and basophil activation analysis about potential food allergens in wine including 5 egg-allergic and 1 milk-allergic patients. Among others they collected 24 egg white-fined or whole egg-added red wines, 34 milk-fined white wines and 25 casein-fined white wines. In this study no anaphylaxis was induced by wine consumption. But adverse reactions against treated wines could not be excluded. Due to the rarity of adult milk or egg-allergic patients reliable statistical analysis of allergic reactions against milk or egg-fined wines is inhibited. Casein, ovalbumin/hen's egg white and lysozyme products of different commercial producers were investigated by SDS-PAGE and immunoblot. These investigations show that every product contains beside casein, ovalbumin or lysozyme other milk or egg allergens respectively in different amounts. The investigations show the necessity to have analytical methods for determination of residues of fining agents or stabilizers in wine which are able to detect not only casein, ovalbumin and lysozyme but also take into account the other milk and egg allergens. This could be guaranteed by certain antibodies used in ELISA systems binding not only to casein, ovalbumin or lysozyme but also to the other allergens in processed casein, ovalbumin or lysozyme for wine production. Therefore polyclonal antibodies raised against fining agents casein and ovalbumin and stabilizer lysozyme of one fining agent producer were used in this study.

Zusammenfassung Die Schwefelbestimmungsmethode nach W. ZIMMERMANN wurde untersucht, und es wurde gefunden, dass die Genauigkeit nicht so gross ist wie oft angegeben wird. Der Fehler durfte im Aufschluss liegen. Bei der neulich von ZIMMERMANN veroffentlichten Anderung der Methode wird der Fehler durch Anwendung einer Titrationsmethode, die zu hohe Werte gibt, ungefahr kompensiert.