:Valutazione dell'effetto chiarificante e della potenziale allergenicità di proteine di origine vegetale in vino rosso

Wine transmits sensations that are expressive of a history which is dependent on its origin and cultivation methods. Clarification, one of the main winemaking operations, is used to make the wine clean and stable by adding proteins with absorbent power that interact with colloids, which are responsible for turbidity and wine instability. Whichever method is used, clarification improves the appearance and taste of wine. It decreases astringency by lowering the concentration of tannins concentration and stabilizes the colour of red wine. In the field of clarifying agents, animal gelatine is the most used because of its efficiency, usefulness and low cost. This capacity to interact with colloids and wine tannins is made by the physical- chemical properties of this animal substance. The use of bovine gelatine in enology has already been abandoned, since 1997, as a precaution, Reg.CE n. 2087/97, after the outbreak of BSE (Bovine spongiform encephalopathy) which can cause a human degenerative pathology of the central system of the brain called Creutzfeldt Jacob disease. The diffusion of this pathology has resulted in particular attention in the use of animal protein adjuvant and has stimulated new studies of clarifying agents derived from different sources. The research in this field is focused firstly on wheat proteins (gliadins and glutenins) which could be used in the winemaking process, because of the way they interact (also at low concentration) with phenolic compounds and tannins forming a stable and insoluble complex. Wheat gluten is composed of a prolamin fraction called gliadin which is rich in proline, and useful for the absorption of polyphenols. The amino acid composition of proteins play an important role in the polyphenols’ absorption through the formation of stable compounds; moreover it is already known that proteins rich in proline are able to bind tannin compounds more strongly. During my PhD program I have focused my studies on the characterisation of new vegetable clarifying agents, like wheat proteins, corn and legume proteins to evaluate the technological efficiency in red wine, protein residuals in treated wines and their allergic properties. At the beginning vegetable clarifying agents already permitted by Reg. CE 2165/2005 were taken into account and the study was planned as follow: 1. Retail (commercial) products are being bought (Gluvital 21000, GBS-P51, ProGel 21110, Isolated gluten, Corn Gluten, SoyPro 900, bean CV4, peas ProFlo, and Farilup); wheat and corn protein extracts are being produced in our laboratory. Moreover conventional clarifying agents, like gelatine, egg albumin, caseinate, were used as a comparison in our experimental tests. 2. All samples were analysed with chemical and physical assays; the protein content of each sample was evaluated which allowed us to find the degree of purity of our clarifying agents. 3. The qualitative evaluation of protein patterns was carried out using SDS-PAGE analysis and has showed different protein molecular weights of our clarifying agents. 4. Policlonal antibodies (able to bind protein clarifying agents) were used; these being anti-gliadin, anti-High molecular weigh glutenin subunits, anti-HMW-LMW glutenin subunits, anti-egg albumin, anti-soy, anti-legume. 5. These polyclonal antibodies (above) were checked to determine their clarifying agent’s specificity as a background in order to carry out experimental tests focused on the research of allergenic residual proteins in wine. 6. The water and hydro-alcoholic solubility of each clarifying agent has been evaluated (12% ethanol, pH 3.5) to determine any potential allergenic risk. Our tests have shown better solubility of hydrolysed clarifying proteins when using a hydro-alcoholic solution. 7. We had evaluated the solubility of each clarifying agent in a hydro-alcoholic solution (12% ethanol, pH 3.5) containing tannins in increased concentration (0.1-0.5-1-2-4 g/L). Using this method it was shown that an increase in tannins solution caused an increase of the precipitate formed by interaction between tannins and proteins with a consequent decrease in clarifying agent residual in solution. 8. For each sample the technological quality (decrease of turbidity and phenolic compounds) was evaluated. We have carried out some micro-clarification with young red wine, Merlot and Merlot/Cabernet cultivar (2005/2007) by using conventional clarifying agents (like gelatine and egg albumin) and corn, wheat proteins. Our results have shown that vegetable proteins work as well as conventional clarifying proteins. Moreover vegetable proteins cause a decrease of turbidity and tannins and they act less strongly on antocianin compounds that are responsible for the colour of red wine. These results led us to the conclusion that some vegetable proteins should be used as clarifying agents. These proteins can be very useful in winemaking operations but they also can act as potential allergens if they’re still in wine. The European Food Safety Authority (EFSA) have proposed a risk assessment of these new clarifying agents and have also enacted some regulations about food labelling (Dir. 68/2007/CE) integration the appendix 3 bis Dir. 13/2000/CE that lists food allergens. Present in this list are gluten and derived products, egg and egg based products, fish and fish products based, milk and milk products based, dioxide sulphites and sulphur at 10 mg/L highest concentration. As some paper described we had analysed these clarifying agents: egg albumin, wheat gluten and legume proteins (Weber et al., 2007; Rolland et al., 2008). 9. Four different protein assays were evaluated (TCA method, Ethanol precipitation, Acetone precipitation and KDS) in order to find the most efficient. Our results show that KDS precipitation allows us to understand wine protein patterns better. 10. Micro-clarification was made with different amounts of clarifying agents (up to more than the legal quantity permitted, 50g/hL), to evaluate the minimum protein concentration detectable by using specific antibodies. 11. Moreover we are carrying out immunological tests with commercial wine treated with egg albumin for a more sensitive detection of residual proteins in wine. As described by Weber et al., 2007 the amount of clarifying agent used in winemaking operations, less than 50 g/hL, is not detectable in the wine (with immunoenzymatic and biochemical analysis); however, when using the highest amount , all clarifying proteins seem to be detectable. As found by our research, vegetable proteins could be good clarifying agents; they have a technological efficiency as well as conventional proteins, but sometimes they could give a potential allergenic risk. Corn prolammins protein could be an interesting proposition in the winemaking operation and free from risk, incidently, they aren’t allergenic and so they are not listed in the appendix 3 bis. For this reason wine treated with this product may not be listed