METHOD FOR PRODUCING A PRODUCT FOR THE TARTARIC STABILIZATION OF WINE, STABILIZATION PRODUCT AND STABILIZATION PROCESS USING SAID PRODUCT

DESCRIPTION Field of application

The present invention concerns a method for producing a product for the tartaric stabilization of wine, stabilization product and stabilization process using said product.

The technology in question is suitable for use advantageously in the wine sector in order to prevent tartaric precipitations which form in white, red and rose wines .

State of the art

As is known, for the commercial distribution of wines in bottles, the wine must not only be clear at the time of bottling, but must also remain in this condition over the course of time, in particular in the case of wines which are intended to be stored for long periods, for example in a wine cellar.

In order for the wine to retain its clear characteristics over time, specific stabilization treatment methods are used.

According to the state of the art numerous treatment methods for the stabilization of wines are known.

In the wine sector the precipitation of tartaric or protein salts present in the wine is frequently referred to by the expression "tartaric or protein casse".

In fact, for example during the conservation of white wines, the protein casse produces cloudiness or specific deposits, usually of whitish colour, which appear in the bottle when the wine conservation temperature is high and/or following enrichment of the wine with tannin originating from the stopper.

One of the known solutions for the stabilization of protein casse consists in the treatment of musts and wines with betonite, but the doses necessary for obtaining the desired stabilizing effect may be sufficiently high to alter the organoleptic properties of the wines and musts thus treated.

In order to eliminate the proteins responsible for the formation of the casse, other experiments have been carried out, based on yeasts or enzymatic methods for example using proteases or particular enzymes able to catalyze rupture of the peptide bond between amino acids in the proteins, but the results are not yet satisfactory.

It is known that potassium, tartaric acid and calcium are present in a high concentration in wines, musts, grape juice in general or other beverages. In

addition, during the fermentation phase, potassium bitartrate, also called tartar, is formed. Furthermore, during fermentation, calcium tartrate may also be formed.

Wine, thus being a solution supersaturated with potassium bitartrate, is subject to precipitation of this salt. Likewise the presence of calcium may constitute a further risk for the precipitation of the tartaric acid salt.

Total precipitation of the abovementioned salts in crystalline form is a phenomenon which generally requires a lot of time and is influenced by various factors. These factors include, for example, the quantity of ethanol present in the wine, the temperature at which the wine is stored and the acidity of the wine itself.

It must be pointed out in particular that the abovementioned salts are less soluble in ethanol than in water. Therefore, at the same time as formation of ethanol due to fermentation of the wine, and also thereafter, slow and gradual precipitation of the salts occurs. The presence of crystals or deposited solids due to this phenomenon represents a serious problem, since they are generally not particularly liked by the consumer who is therefore discouraged from drinking the wine. The precipitation of the potassium bitartrate and calcium tartrate salts, in fact, may generally continue even

after bottling of the wine, with particularly negative consequences for the product's image. It is therefore necessary to subject the wine to tartaric stabilization processes so as to prevent precipitation of the abovementioned troublesome salts.

A first known method for the tartaric stabilization of wines employs a reduction in the temperature, which facilitates precipitation of potassium bitartrate, but not the calcium tartrate. This property is today still the property most commonly exploited using both conventional systems and continuous systems.

Once it has precipitated, the bitartrate must be removed by means of filtration in order to eliminate the crystals which have thus formed.

This known method has a number of drawbacks in that it does not demonstrate a very high degree of efficiency and in some cases is entirely ineffective and also causes the elimination during filtration of some substances which have a beneficial effect on the quality of the wine .

An improvement to this method envisages adding to the wine large quantities of crystals so that the low- temperature treatment becomes more effective and lasts a shorter amount of time. The filtration operation is, however, necessary in order to be able to eliminate the

crystals added and those which are newly formed.

A second known method envisages treating the wine using membrane-filter electrodialysis, as described, for example, in the patents FR2709308, FR2192170 and IT971999.

Finally, methods for the tartaric stabilization of wine, which employ ion-exchange- resins, are known.

It is known from the article by Amati, Ferrarini and Barbieri entitled "Autoarrichimento dei mosti con membrane permeo-selettive" [Self-enrichment of musts using permeable selective membranes] (Industrie delle Bevande 1997, Istituto di Ind. Agrarie, Univ. degli Studi di Bologna, vol. 26, No. 147, pages 23-25, 40, xp- 000983024) to enrich certain fractions of the must by means of nanofiltration so as to separate a permeate from a retentate, following which the permeate undergoes an electrodialysis treatment before being combined again with the retentate. The patents DE 2548066 and DE 2553416 teach the removal of the tartrate from the wine by performing ultrafiltration of the wine and then carrying out a tartaric stabilization process on the permeate alone.

This latter solution has a number of drawbacks. In fact treatment with resins applied to all of the wine also results in the removal of aromatic and colouring

substances, influencing negatively the quality of the wine itself.

On the other hand electrodialysis, owing to the limitations of the currently available membranes, is very problematic since it requires decidedly sophisticated equipment .

All of the abovementioned solutions are not satisfactory in that they are both fairly costly and time-consuming and moreover modify the organoleptic properties of the wine treated.

A further method consists in adding to the wine metatartaric acid which prevents crystallization of the tartaric salts under the effect of cold.

The protective effect, however, disappears when the metatartaric acid hydrolyzes, and this process occurs all the more rapidly the higher the conservation temperature.

This method therefore is effective only for a limited period of time and therefore may be applied only to wines intended for consumption and moreover envisages the addition to the wine of a .foreign compound which is initially absent.

In order to overcome the abovementioned problems, more recently wine stabilization methods which use mannoproteins have been used.

As is known, the wall of yeast cells has a thickness

of about 70 mm and represents 15 to 25% of the dry weight of the cell (J. S. D. Bacon, V. C. Farmer, D. Jones, I. F. Taylor, biochemistry. J. 114,557 (1969) and is an important source of polysaccharides. It in fact consists of 90% polysaccharides, the remainder consisting of proteins and lipids. Its composition varies depending on the yeast strain. The polysaccharides in the wall of the yeasts consist mainly of glucans, mannoproteins and chitin.

As is known, the practice of refining the wines on the fermentation lees has highlighted several chemical/physical phenomena which are associated with the polysaccharides .

In particular, it has been possible to observe that the polysaccharides of yeasts, such as those in the grape, participate in formation of the body and structure of wines and also bond with the tannins, reducing their aggressive action.

More recently (April 2006) the EC has authorised the use of mannoproteins in the wine sector.

Mannoproteins are compounds which are obtained industrially from the lysis of wine yeasts. The aim of using these substances is to replicate in a standardised manner, by means of the simple addition of mannoproteins inside a vat, the process which occurs with refinement on

lees .

The use of mannoproteins for the tartaric (and also protein) stabilization of wines is described in particular in patent EP 789750.

According to the preamble of this patent, some recent tests have shown that the mannoproteins extracted using the heat from the cellular walls of yeasts which belong to the species Saccharomyces cerevisiae have an effect which inhibits crystallisation of the tartaric salts .

The process for obtaining the mannoproteins consists in solubilizing them using heat, in particular at a temperature of 100 ⁰ C in an aqueous means, and retrieving them both directly by means of lyophilization and by means of precipitation of the ethanol and centrifugal drying of the precipitate.

These mannoproteins did not have a particularly high stabilization efficiency for most of the wines.

The patent EP 789750 describes a treatment for stabilizing wine against the effects of tartaric salts and proteins, which envisages adding mannoproteins extracted from the cell wall of the yeasts by means of enzymatic digestion.

More particularly, the tartaric stabilization treatment described in the abovementioned patent uses

mannoproteins extracted from the cell walls of yeasts by means of enzymatic digestion using a mixture of Beta 1,3 and Beta 1,6 glucanase. The yeast used belongs in particular to the species Saccharomyces cerevisiae and the quantity of mannoproteins used in the wine is preferably less than 30 g/hl.

Differently, the patent EP 1240306 describes a wine stabilization technique which uses an oligonucleotide or a polynucleotide.

Numerous wine treatment methods are known, in particular where enzymatic preparations with a pectolytic activity and β-glucanase are used, with the aim of freeing the mannoproteins from the yeasts, reducing the polysaccharides with a high molecular weight, such as pectins and glucans, in order to improve the fattiness of the wines, stabilize further the aromas and colour, and facilitate the clarification operations which may be less intense and therefore have a less invasive effect on the characteristics of the wine.

Hitherto the use of glucans in vinification processes and in particular for the stabilization of wine was not recommended. In fact, the glucans released by the yeasts may act as filler colloids, slowing down filtration, in particular in the case of young wines. In particular, as is known, the glucans produced by Botrytis

cinerea are an important obstacle to filtration.

The use of glucans is instead known in the area of pharmaceutical compositions on account of its absorption characteristics, as described in the international patent WO 02/12348.

The cell walls of single-cell plants and organisms consist mainly of polysaccharides with a three- dimensional structure, partially associated with proteins .

The polysaccharides form a three-dimensional network which allows one to obtain from the cell walls particles which are able to retain some initial properties such as, for example, the capacity for selective absorption.

The final properties of the particles depend on the starting product or, for example, the type of yeast, the strain used, the culture conditions and the methods used during the isolation process which must not alter the functional properties of the particles which are to be subsequently used.

For example a low number of amino acids reduces the protein content in the cell wall.

As is known, yeasts are single-cell organisms with a rigid cell wall consisting of polysaccharides, with a thickness of about 70 run, which represents about 15 to 25% of the dry weight of the yeast and the composition of

which depends on the strain used and the culture methods.

The main components of the yeast cell wall, expressed as a percentage by weight, are: mannans (a mannose polymer) 30%; glucans 30%; proteins 15%; lipids 10% and chitin 2%.

Glucan is a glucose polymer and is composed of a fraction insoluble in an alkaline environment, representing about 80 to 85% of the glucan in the cell wall, and a fraction soluble in an alkaline environment.

The results of an electron microscope analysis of the biosynthesis processes of glucans in Candida albicans showed the development of reticular connections in the cell wall, in particular in the form of triple helices of micro fibrils with a diameter of approximately 2 nm connected together so as to form fibrils with a diameter of 4-8 nm, resulting in the formation of a structure with interfibrillar spaces having a size of about 100-200 nm and giving rise to pores which are present in the cell walls and which form the structural basis of the glucan absorption capacity.

The chemical procedures principally used to isolate and purify the components in the cell wall such as, for example, the use of sodium hydroxide as a reagent for the isolation of mannoproteins, or treatment of the whole cells with pure water at a temperature above 135°C in

order to obtain a fraction highly contaminated by mannoproteins, or also the enzymatic methods for releasing the mannoproteins, have hitherto influenced the characteristics of the glucan particles, changing their reticular structure with respect to the active porous structure originally present in the cell wall.

An approach commonly used to free glucan from mannoproteins and proteins of another type consists in the extraction using 2% sodium dodecyl sulphate (SDS) at boiling point, with or without reducing agents such as mercaptoethanol .

Alternatively, enzymatic methods were used to obtain release of mannoproteins. In this connection, protease and glucanase were used, modifying the protein component of mannans and glucans which fixes the mannoprotein.

The glucan free of mannans is further purified by means of procedures which include treatment with acetic acid or hydrochloric acid.

As mentioned, the procedures illustrated for isolating and purifying cell wall components have a varying effect on the formation of the polymers, which is reflected by the occurrence of an increase in the quantity of soluble glucan and disorders in the structure of the insoluble glucan fraction.

In particular, the pores in the structure of the

glucans thus obtained are not active or are not free from physical or chemical bonding material.

The patent WO 02/12348 discloses an isolation process which is able to obtain glucan particles retaining the original porous structure, using cell walls, in particular yeast cell walls. This patent indicates an advantageous use of this glucan as an absorption agent in the pharmaceutical field.

Disclosure of the invention

The main object of the present invention is therefore that of overcoming the drawbacks of the solutions of the known type mentioned above, by providing a method for preparing a product for the tartaric stabilization of wine, a product for tartaric stabilization of wine and a process for tartaric stabilization of the wine, which are extremely effective.

A further object of the present invention is to provide a process which allows a reduction in the time needed for tartaric stabilization of wine.

A further object of the present invention is to provide a process for the tartaric stabilization of wine which can be easily used for the stabilization of different types of wines, i.e. red, white and rose wines.

A further object of the present invention is to provide a process for the tartaric stabilization of wine,

which can be easily applied.

A further object of the present invention is to provide a process for the tartaric stabilization of wine, which uses natural and biological substances.

A further object of the present invention is to provide a process for the tartaric stabilization of wine, which does not alter the organoleptic properties of the wine treated.

A further object of the present invention is to provide a process for the tartaric stabilization of wine, which complies with legislation governing tartaric stabilization in the wine sector and is entirely reliable .

A further object of the present invention is to provide a method for producing a product for the tartaric stabilization of wine, which can be easily implemented.

A further object of the present invention is to provide a product for the tartaric stabilization of wine able to ensure stabilization which has a lasting effect over time .

Brief description of the drawings

The technical characteristics of the present invention, in accordance with the aforementioned objects, may be determined from the contents of the claims below and the advantages thereof will emerge more clearly from

the detailed description which follows provided with reference to the graphs illustrating the results of tests for tartaric stabilization using the product according to the invention compared with other commercial products of a known type.

Detailed description of a preferred embodiment of the present invention

With the process for treating wine according to the present invention it is possible to perform the tartaric stabilization of wine.

More particularly, this process for treating wine envisages initially producing glucan particles retaining active pores and a fibrous structure of the original cells.

This production is performed by means of a method which also forms the subject of the present invention, comprising a step which involves isolating, from yeast cell walls, glucan particles substantially retaining the structural porosity and fibrosity characteristics of the original reticular structure.

This isolation step envisages: a) extracting mannoproteins from suspensions with water at a temperature above boiling point; b) removing the contaminated protein fraction by means of protease or non-denaturing chemical means;

c) removing the contaminated lipid fraction by means of lypase or solvent extraction.

The steps for isolating said glucan particles in accordance with the abovementioned procedure may be also be performed in an order different from that described above without thereby departing from the scope of protection defined by the present patent.

Depending on the starting material one or more of the steps of the abovementioned procedure may be eliminated.

The glucan particles isolated by means of the abovementioned steps have a near-intact base structure and suitably activated pores .

The patent WO 02/12348 discloses an isolation process by means of which it is possible to obtain glucan particles retaining the original porous structure, using cells, cell walls, fragments of cell walls of single-cell organisms such as yeasts or fungi or residual cell-wall matter of plants containing glucan.

The isolation process, with regard to its aforementioned general characteristics and particular characteristics, is described in the patent WO 02/12348, the contents of which is to be regarded as incorporated herein as a reference source, in particular with reference to the text as published on 14.2.2002 under

number WO 02/12348 A2, page 5, line 23, to page 9, line 24, or with reference to the contents of Claims 6 to 15.

The glucan particles thus obtained have an on average high molecular weight for example greater than 100,000 Daltons and dimensions ranging between 0.1 and 25 micrometres .

The production method according to the present invention then envisages dispersing the active glucan particles thus isolated in a solvent, in particular consisting of an aqueous solution of citric acid, and then performing microfiltration of the particles using a filter having a mesh size smaller than 0.65 microns.

At this point the permeate is selected for extraction of a product consisting of active glucans with a molecular weight of less than 100,000 Daltons.

Surprisingly, the soluble particle components and the insoluble glucan components which pass through the abovementioned filter prove to be particularly effective if used in the wine sector for the tartaric stabilization of wine .

The glucan particles thus obtained are particularly active for the purposes of stabilization despite their low molecular weight.

Preferably, the active glucans with a molecular weight of less than 100,000 Daltons are obtained by

carrying out on the permeate obtained with microfiltration of the abovementioned dispersion using a filter with a mesh size smaller than 0.65 microns a first ultrafiltration step using a 50,000 Dalton membrane and then preferably a further ultrafiltration step using a 30,000 Dalton membrane.

The active glucan particles thus obtained form the subject of a specific claim in the present patent and may be marketed for oenological purposes for achieving the tartaric stabilization of wine.

The conductivity in wines is closely linked to the quantity of salts which remain dissolved without precipitating. Therefore, a high conductivity indicates a lack of precipitation and therefore tartaric stability.

Moreover, high conductivity values which remain even after long periods of time indicate a continued tartaric stability which is the desired goal in oenology.

Graphs 1 and 2 show the progression of the conductivity values in microsiemens for a time period expressed in days (standardised values) in the case of a red wine and white wine treated with different stabilizing products. In particular, the graphs allow a comparison of the behaviour of stabilizing products of the known type, such as mannoproteins, with the glucan according to the present invention indicated with two

different concentration values in a (for example aqueous) solution of 0.1 g/1 and 0.3 g/1.

The finding according to the invention therefore achieves the predefined objects.

Obviously, during its practical implementation, it may also assume forms and configurations different from that described above without thereby departing from the present scope of invention. Moreover all the details may be replaced by technically equivalent elements and the forms, dimensions and materials used may be of any nature according to requirements.