Allergens in Wine and it Detection Methods

Wine is derived from the fermentation and later processed with the appropriate preservatives and additives. The use of several products are permitted during winemaking. The use of certain additives (malic acid, calcium carbonate, potassium bicarbonate) can improve certain characteristics of the wine such as reducing or increasing the acidity, improve wine taste. Other additives such as yeast nutrients can help with the rapid fermentation and the use of fining agents can ensure and enhance the stability of the final product.  While some of the additives are present in the bottled wine and while in some it is removed after treatment without leaving any residue in the final beverage.

Allergy is caused in individuals whose immune system tends to overreact to compounds which has no immunological response in majority of the population. The allergic reaction is stimulated by the proteins in the food product which causes the immunological reaction. Allergens are the compound which brings about the immunological response, the allergen is usually a heat sensitive water soluble protein within the range of 10-100 kDa with no commonality in structure or amino acid sequence having multiple IgE binding sites. A food allergy resulting in an IgE mediated immune response is the most severe and life threatening. Allergic reaction is associated with symptoms like itchy skin, rashes, inflammation, runny nose, respiratory distress and in some cases can even cause death. Modern food products are packed with various additives and preservatives which can cause allergic reactions in any individual. This prompts for screening of food products for potential allergens, in case of allergy causing compound presence in the food it has to be mentioned on the product label so that the individual can identify and avoid consuming the allergenic food. Strict avoidance needs to be followed by individuals with food allergies and food labels is used to indicate to these individuals the presence or absence of allergens.

Due to the growing number of allergic reactions, it is crucial to have fast, reliable methods of allergen detection in processed food products. Identification and labeling the allergenic food components is mandatory in various industrialized countries around the world. The presence of undeclared allergenic food pose risk for those who suffer from food allergies. Some of the additives and processing aids used include proteins that includes important allergens such as milk proteins and egg white proteins. Milk and egg components have been used for the winemaking process as fining agents which according to the wine producer’s knowledge, is removed after coagulation by filtration. Fining the wine aims to soften or reduce the astringency or the bitterness of the wine and it is also done to remove protein that can potentially form haze or in some cases it is added to stabilize and reduce the color by absorption and precipitation of polymeric phenolic compounds and tannins. Some of the common proteins used for fining are included in the list of allergens in the EU regulation that includes milk and egg proteins, animal gelatine, fish gelatine/isinglass and those derived from wheat and white lupin. The proteinaceous substances that are used as a fining agents during wine making, removes the unwanted insoluble particles and undissolved microscopic particles (colloidal material) from the must or wine in order to improve stability. The use of an egg allergen (lysozyme) included as wine additives helps to control the fermentation processes and to avoid spoiling during winemaking. Even if the processing aids are filtered and removed in the final product, the trace elements must be excluded in order to avoid allergic reaction to sensitized individuals. Hence, reliable detection and quantification methods for food allergens are important to ensure compliance with food labeling.

Currently, the methods used to detect the potential allergens are the markers that indicates that presence of the allergens or the allergenic ingredient itself. In order to detect the allergens, the common analytical methods used include the immunochemical assays like ELISA (Enzyme Linked Immunoassay), DNA based method like polymerase chain reaction (PCR) and mass spectrometry. The choice of allergen detection method depends upon the purpose, type of sample, food matrix, turn-around time, availability of equipment and cost

Egg white proteins are one of the most common fining agents used for the red wines to remove phenolic compounds associated with the astrigiency in red wines. The main allergens of egg white are ovomucoid, ovalbumin, ovotransferrin and lysozyme. The ELISA test is the most important method used to detect the allergens because of its specificity and sensitivity and also because the equipment required is not expensive.  There are various ELISA methods used to detect the presence of proteinaceous fining agent residues in wines and most of these assays are based on specific in-house antibody production which are difficult to implement elsewhere. Hence, commercially available kits are used by the laboratories for wine analysis and control.

ELISA Working and Principle

ELISA is an antibody based detection method for the identification of protein or other protein in the food product. Three types of ELISA test kit are available as well flow, lateral flow and sandwich format with sandwich method being the most commonly used technique for allergen detection in food industry. The analysis takes one-two hours to complete and it qualitative and quantitative test method. Lateral flow ELISA devices are qualitative and semi-quantitative with a reader available for many allergenic food. The main advantages of ELISA is its sensitivity, it can detect for allergenic compounds in ppm range, it is fairly rapid compared to other methods, equipment needs are less and the kit requires low skill level to operate. Cross reactivity is the main disadvantage with ELISA and the analyst needs to understand which kits to use as some milk kits detect casein while others detect whey proteins.

Detection of allergens in food using ELISA requires the preparation of the microtitre plate well. Primary antibody is immobilized on the microtitre plate well. To this 100 µl of sample containing the antigen is added and allowed for the primary antibody and antigen from the sample to bind and form an antibody-antigen complex. This is followed by a washing procedure to remove the unbound sample material. An enzyme conjugate is added to microtitre well containing the bound antibody-antigen complex, this results in the formation of antibody-antigen-antibody complex and to this a substrate or chromogen is added. The chromogen reacts with the enzyme conjugate to produce blue color, to which absorbance is then measured at 450 nm.  The international organization of vine and wine (OIV) has also recommended the use of ELISA with a limit of detection (LOD) of <0.25mg/L and a limit of quantification (LOQ) of <0.5 mg/L (OIV 2014).

Multiplexed approach for detecting allergens using Immunochemical methods

Beads are labeled with antibodies for selected antigens like for whey or casein. Beads are then mixed and exposed to antigens for binding following which a second antigen specific labeled antibody is added for detection. The beads are then made to flow through an optical path of two lasers, where the first laser identifies bead by color and correlates it with specific antigen while the second laser determines whether recognition event has occurred and how many times it has occurred. This test method can give results both in qualitative and quantitative.

PCR Based Analysis

Useful in situation when ELISA is not applicable. Not practical for in-plant use as it involves expensive equipment and a dedicated lab. Also the test does not prove presence or absence of protein/allergen Detects only DNA sequences indicative of allergenic species and has no role in allergenic protein detection. The absence of DNA does not necessarily indicate absence of proteins as well. The equipment is costly and requires skilled personnel. In protein enriched isolates or concentrates DNA has been proven to be an alternative molecular marker for the presence of an allergenic food. This method is based on heat stable DNA polymerase amplifies DNA fragment. Methods applying detection of coding sequences of allergenic proteins allow direct analysis of allergenic food components. The detection of allergenic protein is performed through amplification of its specific DNA fragment in PCR reaction. The PCR reaction allows obtaining qualitative results pointing to the presence of sequence coding for given allergen. In the PCR-ELISA method the detection of an allergen does not require gel electrophoresis. The amplified DNA fragment is labeled with digoxygenin, with which it can be easily determined using ELISA test. PCR method can be used in raw and cooked products and it is not affected by the heating process because DNA typically remains intact after being exposed to the cooking temperatures of most food.

Mass Spectroscopy

Detects proteins and peptides and it involves various steps like extraction, cleanup, ionization, separation of ionized protein/peptide, detection. MS has a high degree of sensitivity and resolving power and it provides protein composition, structure and sequence information. The main disadvantages of this method is that it requires high expertise, cost of equipment is high, the analysis is laborious and time consuming and it is not recommended for routine analyses.

This article is based on the paper, Impact of wine manufacturing practice on the occurrence of fining agents with allergenic potential.

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