WO2011030007A1

WO2011030007A1 - Polymerization method, in particular for polymerizing oenologic additives, and polymers produced by said method

Info

Publication number: WO2011030007A1
Application number: PCT/FR2009/051708
Authority: WO
Inventors: Marc Bonneau
Original assignee: Calone-Bonneau, Marguerite
Priority date: 2009-09-10
Filing date: 2009-09-10
Publication date: 2011-03-17
Also published as: EP2475707A1

Abstract

The invention relates to a method for developing novel non-allergenic oenologic polymers, which consists of the polymerization, conducted in a plurality of steps, of natural materials that are used in oenology, particularly caseins, ovalbumins, gelatins, lysozymes, isinglasses, glutens, enzymes, and tannins. The polymers and copolymers according to the invention can be used, alone or variously combined, in the treatment, clarification, and preservation of plant-based beverages and in particular of grape musts, still or sparkling wines, fruit juices, beers, ciders, and vinegars. Said novel oenologic polymers, insoluble and functional in an aqueous medium, do not release allergenic residues in the beverages in which the same are used, and are sufficiently safe for the operators and for consumers.

Description

POLYMERIZATION METHOD, IN PARTICULAR AUXILIARY

ONEOLOGICS AND POLYMERS OBTAINED BY THIS PROCESS

The present invention relates to a method of polymerization, including oenological auxiliaries and polymers obtained by this process, as well as the characteristics and industrial applications of new non-allergenic processing aids.

These auxiliaries, obtained by the stable chemical bond between a central particulate core, organic or mineral, and peripheral biological molecules of animal, plant or microbial origin, constitute new stable and functional polymers and copolymers in an aqueous medium. These polymers and copolymers are characterized by the fact that they are functional in aqueous media and that they do not leave any contaminants residues, toxic or allergenic, in drinks where they are used as processing aids.

The present invention more particularly describes a process for obtaining polymerized organic macromolecules which have specific bonding, clarification and preservation activities for beverages of plant origin and their fermented derivatives, with fruit, with grape must. , still and sparkling wines, beers, vinegars, ...) and for natural beverages, flavored and dietary waters. The various technical operations of sticking, clarifying, filtering and stabilizing wine are essential steps in obtaining drinks with satisfactory physical and organoleptic characteristics for the consumer.

Bonding is defined as "the incorporation into the wine of substances capable of flocculating by causing the particles in suspension". The bonding, clarification and preservation products currently used are:

- of mineral origin: bentonite, silica gel, kaolin

- of animal origin: albumin, casein, gelatines, fish glues ...,

- Of vegetable origin: tannins, glutens, prolamines, gums ...,

- of microbial origin: enzymes, mannoproteins, cell extracts and metabolites,

- Of synthetic origin: PolyVinylPolyPyrrolidone (PVPP).

The interest of the professionals for the products of collage of biological origin remains strong because most of the alternative products proposed do not make it possible to obtain results as varied and complete as those observed with the glues of animal origin. The plant necks are a new generation of clarification and maturing products for musts and wines. It should be noted that the glues of animal and vegetable origins currently used have the major characteristic of being soluble and diffusible in the treated wine where they persist in the residual state at the end of technological treatment.

The presence of these technological residues is an adulteration of the treated drink and a pathogenic risk for the allergic consumer.

European Directive 2007/68 / EC on food-borne allergic risks from animal glues requires that wines clarified by certain allergenic glues such as ovalbumin and casein are subject to specific labeling intended to warn the consumer . The absence of native or altered allergenic residues in a finished food product is now required for Public Health purposes.

The polymerization process described hereinafter, illustrated by numerous examples, is new as regards its application in the oenological sector. Previous publications, reported hereinafter, which describe apparently similar processes and products, have not given rise to any oenological use because of their final residual toxicity.

U.S. Patent No. 4,479,970 A describes a method of treating wine using a technological compound obtained by cross-linking gelatin with glutaraldehyde. Alternatively, the gelatin may be bound to a carrier matrix.

GB Patent No. 2,028,340 A discloses a process for treating beer with a technological compound obtained by crosslinking gelatin or fish glue on a carrier matrix consisting of cellulose, diatomite or albumin using a ligand agent such as than glutaraldehyde. The process of assembling these different substances describes a few steps that are not sufficient to ensure the complete absence of toxic technological residues in the final compound or in the treated drink.

In contrast, the process which is the subject of the present invention comprises several successive steps, including a final purification step intended to eliminate the toxic residues of the ligands and to ensure the perfect innocuity of the polymers obtained.

DE Patent No. 25 38 251 A1 describes a process for the treatment of beverages with the aid of a technological compound obtained by crosslinking gelatin or casein.

US Patent 4,490,399 A discloses a method of treating wine with a technological compound obtained by crosslinking gelatin on a silica matrix using a ligand agent such as glutaraldehyde, to obtain an insoluble final product.

In contrast, the method, object of the present invention uses neither gelatin nor silica and develops formulations and composite and original techniques for the preparation of support cores and functional peripheral layers.

WO 01/3781 2 A discloses an insoluble pharmaceutical composition comprising polymers such as proteins, PVPP and alginate, crosslinked with a crosslinking ligand such as glutaraldehyde. Similarly, US Pat. No. 5,935,442 A describes the preparation of particles intended for analytical chromatography which comprise a macromolecular support nucleus of various natures (agarose, alginate, albumin, starch, cellulose, dextrans, gelatins, gum arabic, silica). , activated by a ligand agent (cyanogen bromide, divinylsulfon, epichlorohydrin, glutaraldehyde, hydrazine, periodate, tosylate).

EP 0 648 480 A describes a macromolecular compound based on collagen, made insoluble by crosslinking the protein with carbodiimide or glutaraldehyde.

WO 01/68798 discloses a composition for the stabilization and clarification of wine comprising cross-linked PVPP.

Unlike the 4 patents cited above, the composite polymers, objects of the present invention, are specifically developed for exclusive oenological applications. To this end, they comprise, on the surface of various carrier nuclei activated or crosslinked by various ligand agents, one or more fixed layers of functional biological molecules, developed according to various and original formulations. The process according to the invention makes it possible to prepare polymers with guaranteed safety.

The non-allergenic oenological compositions according to the invention constitute a timely technical innovation for winemaking professionals and a satisfactory solution for the current regulations relating to food safety.

In general, the invention relates to a process for the preparation, in particular of oenological auxiliaries, consisting of a polymerization of natural molecules and which comprises the following steps:

- preparation of a macromolecular support nucleus,

chemical activation of said nucleus by ligand agents,

anchoring by covalent bonds oriented on said activated nucleus of one or more peripheral layers of natural molecules, and - neutralization and removal of residues of said ligand reagents.

The polymerization process is characterized in that:

the step of preparing said macromolecular support nucleus is carried out using flocculable particles of a biological or mineral nature,

the step of chemical activation of said support nucleus is carried out by means of ligand reagents, homo or hetero-bifunctional, comprising at least two opposite reactive groups connected by several constituent atoms,

the step of anchoring by covalent moieties oriented on said support core activated by said ligand reagents, one or more layer (s) peripheral (s) of natural molecules is carried out so as to obtain functional molecules,

the step of neutralizing and eliminating the residues of said residual ligand reagents is carried out by specific washing and inerting.

The natural molecules that make up the peripheral layers are:

- Of animal origin, including caseins, gelatins, enzymes, lysozymes, bioactive peptides;

- Of plant origin, including glutens, gliadins, enzymes, lectins, tannins, gums arabic;

or of microbial origin, especially enzymes such as pectinases, glucanases, oxidases, catalases.

The nature, the density and the oriented presentation of the natural peripheral molecules condition the functional properties and the specific performances of the various polymerized technological aids according to the invention. The process makes it possible to prepare and obtain, from inorganic and organic compounds authorized by the Oenological Codex, new purified, insoluble and non-allergenic technological polymers that can be used in the manufacture of beverages and their food derivatives: still wines, sparkling wines , ciders, beers, juices, sodas ....

A first embodiment of the polymerization process according to the invention is characterized in that the flocculable particles used in the step of preparing the macromolecular macromolecule are aggregated proteins (caseins, albumins, gelatins, gluten, ...), and in that the ligands used in the chemical activation step are selected from the group consisting of aldehydes, pyrrophenyl chloroformate, N-hydroxysuccinimide chloroformate, and the like. chloro-2-3-epoxypropane, aminobenzyloxymethyl, carboxymethyl hydrazide, aminoalkyl, or carbonyl diimidazole. This embodiment of the polymerization process describes the preparation of fixed and activated macromolecular protein nuclei, on the surface of which are anchored, by oriented covalent chemical moieties, various functional protein glues, hitherto used in enology in soil form. ble, such as albumin, casein, or gelatin. This first mode of the process leads to the realization of three types of homopolymers and six types of copolymers:

a-1). Nucleus (aggregated and activated albumins) - layer (functional oriented albumin),

a-2). Core (ines album aggregated and activated) -layer (functional oriented casein),

b-1). Nucleus (Ag Regulated and Activated Casein) -layer (functional oriented casein),

b-2). Core (aggregated and activated caseins) - layer (functional oriented albumin).

These four variants of polymers according to the invention constitute a range of specific glues adapted to each type of wine to be treated.

According to a first example provided by way of indication and not limitation, the manufacture of the casein-type polymer casein functional (variant b-1 above) comprises:

a) -preparation of the aggregated macromolecular nucleus: poured into a beaker

100g of oenological-grade casein powder and 2 liters of buffered water-pH 7.2 at 25 ° C. The powder is totally dissolved by stirring with a mixer. 200 ml of a solution of acetaldehyde (C2H O) at 5% in pH8 buffer are then introduced dropwise with the aid of a dosing pump and with constant stirring. As soon as the granules are visible in the medium, the addition of acetaldehyde is stopped and the mixture is allowed to settle for 2 hours at room temperature.

b) - chemical activation of the nucleus by ligands: after sedimentation of the granules at the bottom of the beaker, the supernatant is removed and the 200 ml of the sediment of the granules thus obtained are taken up in 200 ml of a 5% solution of glutaraldehyde (C ₅ H ₈ O 2) in buffer at pH 7.4 with gentle stirring for 1 hour. The casein granules thus activated are then allowed to sediment for 1 hour, the supernatant is siphoned off and the activated granules are washed twice in succession with 800 ml of buffered water at pH 7.4.

c) covalent anchoring on the activated nucleus of a peripheral layer of oriented and functional casein molecules: the 200 ml of activated granule sediment and washed are resuspended in 400ml of a new solution of casein at 5% buffered water - pH7.2 at 25 ° C, with gentle stirring for 2 hours. It is allowed to settle again, and the supernatant is removed.

d) - purification and inerting: the casein polymers thus obtained are washed twice with 1 liter of a solution of glycine (C2H ₅ NO 2) -NaCl buffer, pH 6.5. After sedimentation and further washing with 400 ml of buffered water - pH7.2, the casein polymers are dried under vacuum at 5 ° C. and stored as a yellow powder in airtight bags under inert atmosphere at 5 ° C.

The insoluble nature of the cashein-casein granular homopolymer obtained according to the invention is characterized in the following way: a sample of 2 mg of polymers is suspended in a volume of 20 ml of alcoholic water (ethanol 10% VA /) and buffered pH6, and incubated, with gentle shaking, for 24 hours at 15 ° C. After decantation, the absence of residual casein is verified on a sample of 0.1 ml of supernatant by an enzyme immunoassay based on bovine anti-casein antibodies, the specific detection threshold of which is 2 ppm. The results obtained during five successive titrations prove to be negative, thus indicating the absence of 2ppb of allergenic caseins in the solution treated with the polymer.

During its oenological application, the casein-casein polymer is resuspended in cold water with stirring for 10 minutes. The gluing treatment is carried out on the basis of incorporation in the wine at a rate of 50 to 100 g of polymer hydrate per 100 liters of must or white or rosé wines to be treated. The wine is fined in 2 hours with intermittent and moderate stirring. The adsorption of the polyphenols by the polymer thus makes it possible to lighten the colored wines, to reduce the maderised taste and to stabilize the young wines against oxidase breakage. The bottling and the final filtration of the wine eliminates all the precipitates of the collage, and leads to a drink devoid of unstable tannins.

In general, the steps of the preparation method indicated above are identical and applicable, with the appropriate specific proteins, to the manufacture and characterization of the homopolymers a1 and b1, and of the copolymers a2 and b2, all objects of the invention. 'invention.

Furthermore, the process followed and the techniques used can be developed and industrially produced from polymers and copolymers insoluble in aqueous medium and free of residual contaminants.

According to a second exemplary embodiment according to the invention, the copolymer is formed of a macromolecular nucleus of aggregated ovalbumin and of an anchored peripheral layer of lysozyme. Lysozyme (E1 105) is an enzyme protein It has a mucamidal activity, extracted from egg yolk and used for its antibacterial activity in food production.

Residual traces in treated beverages are known to be lysozyme for allergic consumers of egg proteins.

a) Preparation of the nucleus: the granular nucleus of ovalbumin is obtained by driping from the micro-pipette 100 ml of ovalbumin in 10% solution (Weight / Volume) in PBS buffer - pH 6.5 at 30 ° C. in a beaker containing 500 ml of water at 90 ° C. with stirring. In contact with water at 90 ° C, the ovalbumin micro-droplets flocculate rapidly as fine granules. After lowering the temperature to 30 ° C., the ovalbumin granules are collected by sieving.

b) activation: the granules are resuspended in 100 ml of a 5% glutaraldehyde fixative solution (C 5 H 8 O 2) in bicarbonate buffer pH 8 at 25 ° C. with gentle stirring for 2 hours. After sedimentation, the supernatant liquid is allowed to settle and is removed.

c) anchoring: the activated ovalbumin granules thus obtained are washed twice with water in 500 ml of water or they are resuspended in 200 ml of a buffered solution-pH 7 of 5% lysozyme. After a further incubation for 2 hours at 25 ° C. under mild agitation, the granules of copolymers obtained are collected.

d) purification and inerting: the granules are washed in 200 ml of glycine buffer

(C2H5NO2) - NaCl pH 6.5. After sedimentation and two successive washes with 200 ml of buffered water - pH 7.2, the albumin-lysozyme copolymers are dried under vacuum at 5 ° C., and are stored in the form of a fine pale yellow granulate, in sachets under an inert atmosphere. 5 ° C.

The activity and the insoluble nature of the ovalbumin-lysozyme copolymer obtained is controlled as follows: a sample of 10 mg of copolymer is suspended in a volume of 100 ml of white wine containing 5 × 10 ³ malolactic bacteria and is incubated with moderate shaking for 4 hours at 15 ° C. After decantation of the copolymer, the number of viable malolactic bacteria remaining on a 0.5 ml aliquot of supernatant is evaluated by culture on nutrient agar and the presence of residual lysozyme is investigated by immunoenzymatic technique with anti-lysozyme antibody. (detection threshold of 2ppb). The results obtained in successive titrations prove to be negative, and confirm the antibacterial activity of the copolymer and the absence of residual lysozyme in the treated wine. This copolymer is used, in fractional additions, at doses of 25 to 50 g / hl. Musts or wines in which it limits native bacterial flora and lactic acid bacteria fermentations.

It is then removed from the treated drink by decantation-filtration.

- A second indicative but non-exclusive embodiment of the polymerization process is characterized in that the flocculable particles used in the preparation step of the macromolecular support core are chosen from the group comprising bentonite, kaolin, bentonite, zeolite , active charcoal, latex, or polyvinylpolypyrrolidone, and characterized in that the ligands used in the chemical activation step are chosen from the group comprising aldehydes (acetaldehyde, formaldehyde, lutaraldehyde, etc. ), amino-alkyl, aminobenzyloxymethyl, bis-sulfosuccinimidyl suberate, dimethyladipimate, carboxymethyl hydrazide, carbonyl di-imidazole, N-hydroxysuccinimide chloroformate, p-nitrophenyl chloroformate.

A first indicative but non-exclusive embodiment of the method according to this method describes the preparation of a multilayer copolymer of the bentonite-albumin-enzyme type.

The enzymes useful in wine fermentation are pectinases, beta-glucanases, glucose oxidases and fungal catalases, sold in the form of powders or soluble granules. Accidental inhalation or skin contact with enzymes may cause sensitization that generates allergic reactions in operators.

The polymerization process is carried out as follows:

a) preparation of a hydrated and activated bentonite as a flocculable support: bentonite is a powder of alumina, with an average particle size of 100, which swells

25% in water. The hydrated bentonite is prepared by pouring with stirring 100 g of powder in 1 liter of buffered water at pH 6.5 and at a temperature of 50 ° C. and incubated for 2 hours. The gel obtained is filtered through a 45 micron porosity clarifying plate filter. The wet gel retentate is collected and gradually incorporated in suspension, with gentle stirring, in 2 liters of a 5% (W / V) solution of ovalbumin in pH 4.5 buffer. The adsorption phase of the proteins on the granular support is carried out for two hours at 5 ° C., by intermittent stirring every 10 minutes.

b) activation: the preparation is filtered on plates of porosity 45 μ and the retentate of bentonite-ovalbumin adsorbed is resuspended, by incorporations successive aliquots of 10 g in 200 ml of a solution of glutaraldehyde (C5H8O2) at 2% in buffered water pH 6.5.

The oval binder adsorbed on the bentonite is crosslinked by the aldehyde, with gentle stirring, for 60 minutes. The crosslinked bentonite-ovalbumin compound is then filtered on a 45μ porosity plate filter. If it is washed twice to remove excess residues of aldehyde then filtered again on 45μ porosity disk. c) anchoring: the activated and washed retentate is taken up in 500 ml of a buffered solution co m po rta ntun enzymatic mixture, equal parts, pectinases (EC3.2.1 .1 5) and beta- (1 - 3; 1-6) -giucanases) at 5g / l. The oriented covalent bonding of the enzymatic molecules to the activated ovalbumin molecules continues for 60 minutes with gentle agitation.

d) purification: the bentonite-albumin-enzyme copolymer thus obtained is centrifuged (7500 rpm for 20 minutes) then resuspended and washed twice in 1 liter of glycine buffer (C2H5NO2) -NaCl-pH 6.5 to neutralize residual reactive groups of the aldehyde. The copolymer is centrifuged again (7500 rpm for 20 minutes) and then dried under vacuum at -25 ° C. and it is preserved in the form of a pale yellow granular powder in hermetic bags under an inert atmosphere at 5 ° C.

The insoluble nature of the bentonite-ovalbumin-pectinase copolymer thus obtained is characterized in the following manner: a sample of 10 mg of copolymer is suspended in a volume of 100 ml of wine and is then incubated, with moderate stirring, for 2 hours at 1 5 ° C. After decantation, the presence of ovalbumin residues and allergenic pectinases is characterized on a sample of 10 ml of wine supernatant treated with an enzyme immunoassay technique using anti-ovalbumin and anti-pectinase antibodies, the specific detection thresholds of which are 2 ppb. The results of five successive titrations are negative and confirm the absence of 2ppb. of allergenic residues of ovalbumin and pectinases in the wine clarified by the copolymer according to the invention. The SDS-Page-immunoblot assays, which have a detection limit of 0.02ppb, demonstrate that musts and red and white wines clarified by the bentonite-ovalbumin-pectinase copolymer are devoid of ovalbumin residues and pectinases and are not allergenic to the consumer.

A second non-limiting example of embodiment of the method according to this second mode concerns the preparation of a multilayer bentonite-albumin-plant lectin copolymer.

Lectins are naturally occurring proteins or glycoproteins with one or more specific binding sites with a carbohydrate. They bind so Specific to oligosaccharides, by hydrophobic interactions and by hydrogen bridges between carbohydrate hydroxyls and amino acid NH 2, OH and carbonyl functions of the protein. Concanavalin A ConA), a lectin extracted from the bean genus Canavalia ensiformis, binds in particular D-mannose and D-glucose. The preparation of a bentonite-albumin-Con.A copolymer, supplied as a non-limiting indication, is carried out following the steps of the method already described:

1). Creation of the initial bentonite-albumin nucleus,

2). Activation of albumin by glutaraldehyde, (C5H8O2) in 1% solution in pH 8.5 buffer

3). Peripheral anchoring of ConA lectin in 2% buffered solution.

4). Washing and inerting.

In use, the copolymer obtained is resuspended in water (1 M / 10 V) and used at a dose of 100 g per 100 L of white or red grape juice. The copolymer, incubated for 2 hours at 10 ° C. with slow stirring, fixed sugars present in the r isin and d im in ue the final concentration after fiitration. The insoluble copolymer, characterized by its capacity to bind sugars, constitutes a new oenological compound according to the invention which makes it possible, according to the doses used, to reduce the sugar content of musts and fruit juices and consequently to reduce ui re the final re q ual d alcohol beverages from the alcoholic fermentation of musts previously treated with this copolymer.

In general, the steps of the process according to the invention are similar and applicable to the manufacture of various copolymers with one or more peripheral layers, formed either of bentonite-proteins or of bentonite-proteins. plants of either bentonite-microbial proteins, and all objects of the invention.

A third nonlimiting embodiment of the method according to this second embodiment relates to the preparation and characteristics of a copolymer formed between alginates and glues of plant origin. Some vegetable proteins extracted from legumes or cereals, the different tannins (gallic tannins, ellagic tannins, and condensed tannins,) prepared from various plants and mannoproteins from yeasts are authorized as glues for musts and wines ( Regulations EC 2165/2005 and EC 1493/1999).

The application of the preparation method according to the invention to vegetable proteins intended for bonding makes it possible to obtain non-allergenic oenological auxiliaries for the consumer. A first indicative and nonlimiting variant of the preparation of an alginate-gluten copolymer is described below: an aliquot of 5 g of sodium alginate (E 401) is removed and dispersed with strong stirring in a mixer in 1 1 liter of an aqueous 5% (W / V) gluten solution at pH 7.2 and 20 ° C. A solution of 1 liter of calcium chloride at 10 g / liter is prepared in a beaker. Then, by dropwise distribution, the 5% gluten solution is added to the calcium chloride bath. Under the coagulating and sequestering action of calcium chloride, each drop of the alginate -gluten mixture immediately forms a gelled and coagulated minibill. The beads thus formed are washed twice in succession in one liter of distilled water. The beads are then immersed for 60 minutes in a 5% solution of glutaraldehyde (C5H8O2) at pH 8.5. The beads are then drained on sieves and then immersed in 1 liter of a new aqueous solution of gluten at 5% (W / V) pH 7.2 for 60 minutes at 25 ° C. The beads are again drained on sieves and then washed in 1 liter of glycine buffer (C2H5NO2) -NaCl pH7. The beads thus treated constitute an alginate-gluten copolymer with a functional surface which is then dried under vacuum at 5 ° C. and then kept in sealed bags under an inert atmosphere at 5 ° C.

The safety test of the alginate-gluten copolymer obtained according to the invention is carried out using anti-gluten antibodies and according to the immunoenzymatic technique already described. This copolymer is used alone or in combination with filtration aids and is an effective and non-allergenic alternative for the refining of wines.

A variant of the above process also applies to the preparation of an alginate-tannin copolymer: a sample of 5 g is taken. of sodium alginate (E 401) which is dispersed with strong stirring in a mixer in 1 liter of an aqueous solution of 5% (W / V) gallic tannin at pH 7.2 and 20 ° C. A solution of 1 liter of calcium chloride at 10 g / liter is prepared in a beaker.

Then, by dropwise distribution, the 5% tannin solution is added to the calcium chloride bath. Under the sequestering action of calcium chloride, each drop of the alginate - tannin mixture immediately forms a gelled and coagulated minibilla. The beads thus formed are washed twice in succession in one liter of distilled water. The beads are then immersed for 60 minutes in a 5% solution of formaldehyde (CH 2 O) at pH 8.5. The beads are then drained on sieves and then washed in 1 liter of glycine buffer (C2H5NO2) - NaCl-pH7. The beads thus treated constitute a functional surface alginate-tannin copolymer which is then dried under vacuum at 5 ° C. then kept in airtight bags under inert atmosphere at 5 ° C. This copolymer is used at a rate of 300 to 500 gr of beads per hectolitre of wine to be glued.

More generally, the alginate-protein and alginate-tannin copolymers obtained according to the process which is the subject of the invention can be prepared with various biological molecules of animal, plant, fungal (mannoprotein) or bacterial origin used as oenological auxiliaries and they are new non-allergenic oenological compounds.

A fourth non-limiting example of realizing the process according to this second mode concerns the preparation and characteristics of a stable copolymer formed between an organic polyvinylpolypyrrolidone (PVPP) core and an anchored tanned peripheral layer.

PolyvinylPolyPyrrolidone (PVPP) constitutes the macromolecular support nucleus on which the various polyvinyl phenols, such as anthocyanins, catechins, flavonols and phenolic acids, are selectively adsorbed by formation of hydrogen bonds. A 20% suspension of PVPP in water (200 g of powder in 1 liter of buffered water at pH 6.5 and 10 ° C.) is prepared and slowly mixed with gentle stirring at 1 ° C. liter of mixture, in equal parts, of a 10% (W / V) aqueous solution of gall-nut tannin and a 10% aqueous solution (W / V) of oak tannin. The adsorption phase of the tannins on the PVPP is then carried out for 2 hours at 10 ° C., with intermittent stirring. The preparation is then centrifuged (5000 rpm / 20 min) and the PVPP-tannin pellet is resuspended, by successive incorporation of 10 g aliquots, into one liter of a solution of tresyl chlorate 2-trifluoroethane sulfonyl - (C2H2CIF3O2S ) at 5% in phosphate buffer pH 8.5.

The anchoring of the tannins on the surface of the PVPP is continued for 60 minutes under gentle agitation, followed by centrifugation (5000 rpm / 20 min) and 2 washes of the PVPP-tannin pellet in 1 liter of water. The compound is then dried under vacuum at 5 ° C. It is stored in powder form in a sealed bag under an inert atmosphere at 5 ° C.

The PVPP-tannin complex thus formed applies directly to white and rosé wines at a dose of 40 g per hectolitre. After incubation for 3 hours at 10 ° C., the PVPP-tan-ins complex binds the wine proteins in excess, and in particular natural oxidation enzymes such as laccase (EC 1 .10.3.2) and tyrosinase. (EC 1 .14.18.1) present in weathered grape juice. The PVPP-tannin-enzyme complex thus obtained is completely removed by clarifying filtration of the treated wines. This complex has the characteristics of insolubility and non-allergenicity of oenological products according to the invention. An alternative embodiment of a PVPP-based compound according to the invention relates to the preparation and properties of a PVPP-tannin-protein multilayer copolymer.

100 g of a PVPP-tannin compound prepared as above are removed and suspended in 1 liter of a 5% (w / v) solution of ovalbumin pH 6.5 buffer.

The adsorption phase of the ovalbumin on the tannins is carried out for 2 hours at 10 ° C., by intermittent stirring every 10 minutes. The PVPP-tannin-ovalbumin complex is filtered on fried glass or is resuspended. by successive additions of 5 g aliquots in 1 liter of a 1% solution of glutaraldehyde (C5H8O2) in pH 8.5 phosphate buffer with slow stirring for 60 minutes. After anchoring the surface molecules of ovalbumin, the PVPP-tannin-ovalbumin copolymer thus obtained is filtered and washed twice in 1 liter of glycine-NaCl buffer - pH 6.5 and then drained and dried under vacuum. It is stored in a bag under an inert atmosphere at 5 ° C. It is used for the bonding and clarification of wines and it has the immunological safety properties of the compounds according to the invention.

Generally speaking, according to the nature of the support nucleus used, depending on the nature of the ligand reactant and the nature of the molecules fixed on the surface, the manufacturing process according to the invention makes it possible to obtain a range of various oenological polymers which are within the scope of the present invention as functional and non-allergenic polymers. These are indicative and non-limiting examples:

agarose-albumin copolymers, agarose-casein copolymers, and agarose-gelatin copolymers, whose manufacturing uses, for the preparation of the support nucleus and for the anchoring of the functional molecules, to cyanogen bromide reagent ligands, or aldehyde type, or epichlorohydrin type, or dimethyladipimate type, or bis-sulfosuccinimidyl suberate type.

the agarose-lectin and agarose-enzyme copolymers whose production uses, for the preparation of the support nucleus and for the anchoring of the functional molecules, to ligand reagents of p-nitrophenyl chloroformate type or N-hydroxysuccinimide chloroformate type or type epichlorohydrin, or periodate type.

the dextran-protein copolymers and the cellulose-protein copolymers whose manufacture uses, for the preparation of the support nucleus and for the anchoring of the functional proteins, ligand type aminobenzyloxymethyl or carboxymethyl hydrazide or amino-alkyl type reagents, or carbonyl di-imidazole type. The acrylate-protein copolymers and the latex-protein copolymers whose manufacture uses, for the preparation of the support nucleus and the anchoring of the functional proteins, to reactants ligands type aldehydes.

The activated carbon-protein copolymers, the zeolite-protein copolymers and the alumina-protein copolymers whose production uses, for the preparation of the support nucleus and for the anchoring of the functional proteins, to ligands reagents type aldehydes.

These various polymeric compounds achievable by the chemist are within the scope of the present invention as non-allergenic insoluble polymers with oenological applications.

In general, the present invention describes, according to various possible variants, a process for obtaining technological polymers with specific functional activities and without allergenic toxicity in an aqueous medium.

The present invention also relates to the industrial use of the technological polymers obtained according to the process and their implementation, alone or in various combinations, in the gluing, clarification and preservation of beverages of vegetable origin, in particular grape must, still and sparkling wines, juices, beers, ciders and vinegars.

The use of these oenological polymers is characterized in that the drinks thus treated are devoid of residual technological contaminants detectable by immunoassay, and by the fact that their use is free of allergenic toxicity for operators and consumers.

Claims

1. A method of polymerization, especially oenological auxilaries, consisting of a polymerization of biological molecules which comprises the following steps:

- preparation of a macromolecular support nucleus,

chemical activation of said support nucleus by ligand reagents,

anchoring by covalent bonds on said activated support nucleus of one or more peripheral layer (s) of natural molecules, and

neutralization and elimination of the residues of said ligand reagents;

characterized in that

the step of anchoring by covalent bonds oriented, on said support core activated by said ligand reagents, with one or more layer (s) peripheral (s) of natural molecules is carried out so as to obtain functional molecules,

the step of neutralizing and removing the residues of said residual ligand reagents is carried out by specific washes and inertings.

2. Polymerization process according to claim 1, characterized in that the flocculable particles used in the preparation step of said support core are caseins, albumins or aggregated gelatins, or aggregated vegetable proteins.

3. Polymerization process according to claim 1, characterized in that the flocculable particles used in the step of preparing said support core are chosen from the following compounds: bentonite, kaolin, zeolite, activated carbon, polystyrene, latex, or polyvinylpolypyrrolidone.

4. Polymerization process according to one of claims 1 to 3, characterized in that the ligands used during the chemical activation step are chosen from the following compounds: aldehydes, amino-alkyl, aminobenzyloxymethyl, bis-sulfosuccinimidyl suberate, dimethyladipimate, carboxymethyl hydrazide, carbonyl di-imidazole, tresyl-2-trifluoromethanesulfonyl chlorate, N-hydroxysuccinimide chloroformate, p-nitrophenyl chloroformate, epichlorohydrin, the periodate.

5. Polymerization process according to one of claims 1 to 4, characterized in that said immobilized peripheral layers consist of orientated and functional biological molecules.

6. Polymerization process according to claim 5, characterized in that said biological molecules are of animal origin, in particular caseins, gelatins, enzymes, lysozymes, bioactive peptides.

7. The method of polymerization according to claim 5, characterized in that said biological molecules are of plant origin, including glutens, gliadins, enzymes, lectins, tannins, gums arabic;

8. Polymerization process according to claim 5, characterized in that said biological molecules are of microbial origin, in particular enzymes such as pectinases, glucanases, oxidases, catalases.

9. Technological polymers obtained by the implementation of the process according to one of claims 1 to 8, provided with insolubility and specific functional activities in aqueous medium.

10. Use of in-use heating appliances, as specified in claim 9, alone or in various combinations, for the binding, clarification and preservation of beverages of vegetable origin, in particular grape musts , still and sparkling wines, juices, beers, ciders and vinegars.

1 1. Use of oenological polymers according to claim 10, characterized in that the drinks thus treated are free of residual technological contaminants detectable by immunoanalyses, and in that their use is free of allergenic toxicity for operators and consumers.