Method for protein stabilization of wines

A process for the production of a biological product having a stabilization effect on the protein precipitations of white wines, characterized in that it consists in carrying out an enzymatic digestion of invertase of Saccharomyces cerevisiae by hydrolysis to obtain the biological product of stabilization.

FIELD OF THE INVENTION
 The French present invention has for its object a product for the protein
 stabilization of wines.
 In patent application No. 2,726,284 in the name of the Faculty of Oenology
 of Bordeaux, there have been described and claimed mannoproteins extracted
 enzymatically, whose action against precipitations of tartaric and protein
 salts is particularly effective.
 More particularly, these mannoproteins were obtained by enzymatic digestion
 of the cell walls of yeast, under the action of .beta.1-3 and .beta.1-6
 glucanases, said yeasts being, in one embodiment, the species
 Saccharomyces cerevisiae.
 It has also been determined that the mannoprotein of a molecular mass of
 31,800 Daltons, called MP32, was more particularly effective, for protein
 stabilization.
 Also, tests have shown that MP32 is a fragment of invertase.
 SUMMARY OF THE INVENTION
 The invention thus relates to a procedure for obtaining mannoproteins
 enzymatically, from invertase, for the protein stabilization of white
 wines, which consists in carrying out an enzymatic digestion of invertase
 of Saccharomyces cerevisiae by hydrolysis.
 This enzymatic digestion is carried out in the presence of .beta. 1-3 and
 .beta. 1-6 glucanase and the active proteases, is acid media.
 The hydrolysate recovered is subjected to a physical treatment comprising
 particularly a dialysis against water, through a membrane with a cutoff
 threshold of 6,000 to 8,000 Daltons, followed by lyophilization with
 centrifugation and filtration prior to dialysis.
 The invention also relates to the choice of cortexes of yeast adapted to
 have an invertase activity such as to generate mannoproteins of a
 molecular mass suitable to stabilize white wines.
 The invention also relates to a process for treating a white wine for its
 protein stabilization, which consists in adding to this wine a hydrolysate
 of invertase of Saccharomyces cerevisiae, more particularly a quantity of
 the order of 25 g/hl of lyophilisate.
 The invention also relates to the stabilization product obtained.

DETAILED DESCRIPTION OF THE INVENTION
 It is known that invertase 2 or 4 of Saccharomyces cerevisiae has a
 molecular mass of 270,000 Daltons, which is much greater than the
 molecular mass of MP32.
 It is also known that invertase comprises particularly the following amino
 acid sequences:
 Lysine-Valine-Phenylalanine-Tryptophane-Tyrosine-Glutamine-Proline-Serine-G
 lutagine-Lysine.
 Surprisingly, the applicants have carried out a sequencing of the
 mannoprotein MP32 and have noted 100% homology with the portion of the
 sequence contained in the invertase.
 So as to verify that mannoprotein MP32 is indeed a fragment of invertase of
 Saccharomyces cerevisiae, it has been proposed to carry out the two
 following preparations:
 Inv1, 30 to 50 units/mg of insoluble commercial invertase SIGMA.RTM.,
 hydrolyzed in 5% of Glucanex.RTM., of the firm NOVO, and
 Inv2, 400 units/mg of soluble commercial invertase SIGMA.RTM., hydrolyzed
 in 5% of Glucanex.RTM., of the firm NOVO.
 After digestion, the hydrolysates Inv1g and Inv2g are centrifuged, filtered
 and dialyzed with a membrane having a cutoff threshold of the order of
 6,000 to 8,000 Daltons, the hydrolysate thus obtained is lyophilized.
 There is then added 25 g/hl of each of the preparations to a wine so as to
 carry out a comparative test of the effect of stabilization of each of
 these preparations.
 The results are shown in the diagram of FIG. 1, each wine having been
 subjected to a warm treatment, 80.degree. C., for 30 minutes so as to
 evaluate the protein stability.
 It has been determined that the mannoprotein extracted by an enzymatic
 method from the cell walls of yeast digested with Glucanex, has a
 stabilizing effect, because less bentonite is needed for an improved NTU
 turbidity index.
 At a low concentration of invertase, the preparation of Inv1g is even
 better than the preparation extracted enzymatically from yeast cell walls.
 There is noted an increased stabilization as soon as the concentration of
 invertase increases, even with the non-hydrolyzed preparation.
 The stability is very greatly improved when, with a high initial
 concentration of invertase, Inv2 is hydrolyzed in Glucanex (Inv2g).
 Thus, the richer the preparation is in invertase, the more active is its
 hydrolysate from Glucanex.
 It is no longer necessary to add bentonite, and the formation of cloudiness
 at the end of the test under heat, is substantially zero.
 By capillary electrophoresis, it can be determined that the content of MP32
 increases parallel to the stabilizing activity. The more MP32 there is,
 the more the stabilizing activity is substantial.
 It might be thought that the liberation of the invertase fragment of
 Saccharomyces cerevisiae in the course of autolysis of yeast, containing
 the suitable amino acid sequence, is due to the action of proteases.
 Given the physical-chemical parameters of wines, pH 3 to 3.8, the only
 Saccharomyces cerevisiae proteases adapted to be active are:
 protease A, SIGMA.RTM. and
 carboxypeptidase Y, SIGMA.RTM..
 The following tests were carried out:
 In a citrate buffer solution of 10 ml, there is hydrolyzed 100 mg of
 purified invertase Inv2, for 15 hours at 20.degree. C., with:
 5 mg of Glucanex,
 2.5 mg of carboxypeptidase Y,
 0.3 mg of protease A.
 In parallel, a similar hydrolysis is carried out, but with inactivation of
 these same enzymes with heat (1minute at 100.degree. C.).
 Then, a dialysis against water of the different preparations is conducted,
 with a membrane having as before a cutoff threshold of 6,000 to 8,000
 Daltons, then they are lyophilized.
 Identically, specimens of wine receive the preparations at the rate of 25
 g/hl, then the wines are subjected to a test under heat so as to determine
 the turbidity index.
 In the diagram of FIG. 3, the abbreviations correspond to the following
 products:
 g, Glucanex
 gc, Glucanex, but previously inactivated at 100.degree. C. for one minute,
 CY, carboxypeptidase Y,
 Cyc, carboxypeptidase Y, but previously inactivated at 100.degree. C. for
 one minute,
 PA, protease A, and
 PAc, protease A, but previously inactivated at 100.degree. C. for one
 minute.
 It is seen that the enzymatic hydrolysis of a preparation of invertase
 produces products having stabilizing action on white wine.
 Particularly noteworthy is the effectiveness of Glucanex and of protease A
 to generate mannoproteins MP32. Protease A is the most effective.
 This is moreover confirmed by tests shown in FIG. 4, which show the content
 of MP32 of the different preceding hydrolysates.
 It should also be noted that the partial deactivation by heat has a
 negative effect on the results obtained.
 It has thus been shown that the process according to the invention provides
 a stabilizing action on proteins of white wines.