ES2709524B2 - Procedure to obtain an enzymatic hydrolyzate from overgrown grape seeds and use thereof - Google Patents

Description

DESCRIPTION

Procedure to obtain an enzymatic hydrolyzate from overgrown grape seeds and use thereof

The present invention relates to a process for obtaining an enzymatic hydrolyzate from overripe grape seeds and use of said hydrolyzate as a color stabilizer in red wines. Therefore, the present invention could be framed within the wine industry in the technical sector of wine making and treatment.

BACKGROUND OF THE INVENTION

The cultivation of the vine and the elaboration of wines face problems related to high environmental temperatures. In dry springs and hot summers, the period between the color of grape change and its optimum industrial maturity decreases, while the gap between the maturity of the pulp and the solid parts increases. This makes it difficult for the grapes to reach the correct phenolic maturity, producing poor and irregular colors. These problems of "color drops" are well known to winemakers in regions of warm weather, so different strategies have been developed to appease their effects as much as possible. One of the possible techniques to avoid these color drops it is modular the interactions between pigments and co-pigments of wine (Gómez-Míguez, M. et al, J. Food Eng. 79 (2007) 271-278). Among these, the addition of natural sources of co-pigments is considered an alternative to Traditional winemaking, usually from natural sources of oenological origin such as pomace, skins or wood chips.These sources are rich in polyphenolic compounds, which have traditionally been considered the best copigments for anthocyanins.

(Gordillo, B. et al. Food Chem. 206 (2013) 249-259; Gordillo, B. et al., Food Chem.

141 (2013) 2184-2190).

Apart from these phenolic compounds, grape seeds contain approximately 14% protein, which are natural biopolymers with co-pigmenting capacity (Fantozzi, P. J. Am. Oil Chem. Soc. 58 (1981) 1027-1031). The total protein content in the whole berry depends on the variety and the degree of maturation (Bertazzo, A. et al. J. Mass Spectrom. 45 (201) 966-970; López-Miranda, S. et al. Food Chem. 127 (2011) 481-486), although they are mostly concentrated in the endosperm of the seeds. The types of proteins found in grape seeds are similar to those present in other plant species, having multimers with wide ranges of molecular weights. They are usually stabilized by non-covalent interactions between different subunits, and recent studies state that they are likely to be potentially useful in practical applications (Gazzola, D. et al. Food Chem. 155 (2014) 132-139).

In the wine industry different aggregates have been used to improve the stability and color in the wine, but the method proposed by the present invention uses a source from the industry itself, which not only implies that the hydrolyzate that is added to the wine to stabilize its color is not alien to it, but also improves the use of waste from the wine industry.

DESCRIPTION OF THE INVENTION

A subject of the present invention is a process based on the enzymatic hydrolysis in basic medium of the protein fraction of overripe grape seeds to obtain a natural enzymatic hydrolyzate of variable molecular weight. The use of seeds in an advanced state of maturity reduces the interference of polyphenolic compounds in the hydrolytic process, because they undergo polymerization reactions with maturation, which leads to a certain insolubilization.

Through the process of copigmentation, the peptides protect the color of the compounds of anthocyanin origin present in red wines through the phenomenon of copigmentation, so that the enzymatic hydrolyzate obtained can be used as a color stabilizer in red wines.

Therefore, in a first aspect, the present invention relates to a process for obtaining an enzymatic hydrolyzate from overripe grape seeds characterized by comprising the following steps:

(a) separation of the overripe grape seed from the grape pomace, obtaining a seed meal and degreasing the seed meal by extracting its lipid fraction,

(b) treatment of the degreased seed meal obtained in step a) with an alkaline solution for the extraction of proteins, acidification of the extract obtained until obtaining a precipitate, separation of the precipitate and redisolution thereof in water to obtain a concentrate aqueous protein,

(c) Enzymatic hydrolysis of the aqueous protein concentrate obtained in step (b) to obtain an enzymatic hydrolyzate.

In a preferred embodiment of the invention, the process comprises a step d) of centrifugation of the enzymatic hydrolyzate of step (c). After centrifugation of step (d), a precipitate formed by the remains of the starting material remains insoluble (mainly insoluble fiber and part of undigested proteins) and, on the other hand, a supernatant composed mainly of by proteins, peptides and amino acids, as well as other minor components solubilized during the process (mainly phenols and sugars).

In another preferred embodiment of the invention, the process comprises a step (e) of separating the supernatant (enzymatic hydrolyzate) from the precipitate obtained in step (d).

In another preferred embodiment of the invention, the process comprises a step (f) of clarification of the enzyme supernatant hydrolyzate obtained in step (e) by filtration thereof. Preferably the filtration is carried out with a 0.2 p, m filter.

In another preferred embodiment of the invention, the process comprises a step (g) of removing polyphenols from the clarified enzymatic hydrolyzate obtained in step (f). Preferably, the removal of polyphenols is carried out by adsorption with polyvinylpolypyrrolidone (PVPP), more preferably with PVPP 20% w / v (20 g PVPP / L of the hydrolysis medium.) This removal of polyphenols has to be done at this point and not before , since the adsorption phenomena do not occur selectively and it is necessary that the medium is filtered and clarified.

In a preferred embodiment of the invention, after removal of the polyphenols, the enzymatic hydrolyzate is centrifuged (step (h)) to separate the PVPP if present and other insoluble particles.

In another preferred embodiment, after said centrifugation, the enzymatic hydrolyzate is concentrated until a solid paste is obtained (step (i)).

In another preferred embodiment, the solid paste obtained in step (i) is subjected to a step (j) of drying by lyophilization thereof.

In step (a), the overripe grape seed is separated from the pomace preferably by physical methods such as sieving, since they are smaller than the remains of skins and scrapes present in the pomace. Once the overripe grape seed is separated, the flour thereof is preferably prepared by crushing and sieving the overripe grape seed separated from the pomace. Degreasing is preferably carried out by Sohxlet, with apolar organic solvents, such as hexane. More preferably, the extraction by Sohxlet is carried out for approximately 24 hours, depending on the scale of the process and the amount of sample to be degreased. The organic solvent is subsequently evaporated and the amount of lipids can be determined by gravimetry.

In a preferred embodiment, the extraction in alkaline medium of step (b) is performed with a solution of NaOH, more preferably 10 M NaOH.

In a preferred embodiment, the acidification of step (b) is performed with HCl, more preferably 6M HCl.

In another preferred embodiment, the acidification of step (b) is carried out up to pH between 2.5 and 3.5, preferably 3.0, obtaining an acidic protein precipitate (insoluble protein fraction) which is preferably recovered by centrifugation.

In another preferred embodiment, once the extraction has been carried out in an alkaline medium and prior to acidification, the extract obtained is left to decant until the supernatant does not show turbidity (approximately 3 days). The supernatant is the one that is subsequently subjected to acidification.

In another preferred embodiment, the pH at which the enzymatic hydrolysis of the stage is performed (c) it is alkaline, more preferably the pH varies between 8 and 9. The temperature at which the hydrolysis stage is carried out is between 50 and 55 ° C, more preferably at 55 ° C, all with the objective to solubilize the protein of the starting material (protein agroindustrial residue of overripe grape seed) and in turn maintain the proteolytic activity of the enzymes chosen for the duration of the process. This temperature reaches a compromise between the activity of the enzyme and the solubility of the product, since if the temperature is reached at 60 ° C, the enzyme decreases its activity after 30 minutes.

Enzymatic hydrolysis is preferably performed in a period between three hours and three and a half hours (depending on the scale of the process) under continuous agitation.

Enzymes that are effective in the hydrolysis process described in the method of the present invention (step c)) are proteolytic enzymes, preferably endoprotease-acting, commonly used in protein hydrolysis. These include trypsin, chymotrypsin and combinations of both, which are stable to the conditions of the hydrolysis method described in the invention and are not inactivated at the pH used. Such enzymes (endoproteases) are commercial, nonspecific and well known and scientifically characterized for optimal pH and temperature parameters.

In the present invention, grape pomace is understood as the solid residue obtained as a by-product after the vinification process.

In the present invention, supermature grape seed is understood as that seed from grapes that have widely exceeded the technological maturity necessary to produce table wines, reaching a degree of sugars greater than 25 ° Brix ”, and which are used for processing of generous wines.

In the present invention, generous wine is understood as those with a high alcohol content (from 15 °) for being obtained from grapes with a high concentration of sugars and / or that entail the addition of extra wine alcohol prior to aging. in wood.

Another aspect of the invention relates to the use of the enzymatic hydrolyzate obtained by the method described in the first aspect of the invention as a color stabilizer in red wines.

A final aspect of the invention relates to the use of overripe grape seeds obtained as a byproduct of the production of generous wines for obtaining an enzymatic hydrolyzate as defined in the second aspect of the present invention.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: shows a graph representing the increase in antioxidant activity (p, mol TE / L) of an anthocyanic solution (Mv-3glc) after the addition of increasing concentrations of the enzymatic hydrolyzate of the present invention.

FIG. 2: shows a graph representing the increase in the dye intensity of an anthocyanic solution (Mv-3glc) after the addition of increasing concentrations of the enzymatic hydrolyzate of the present invention.

FIG. 3: shows a graph representing the color degradation (%) of an anthocyanic solution (Mv-3glc) after the addition of increasing concentrations of the enzymatic hydrolyzate of the present invention, during a storage period of 30 days.

EXAMPLES

The invention will now be illustrated by tests carried out by the inventors, which demonstrates the effectiveness of the product of the invention.

Example 1: Preparation of an enzymatic hydrolyzate from overripe grape seeds according to the process of the present invention

The protein agroindustrial residue that is used in the process of the invention is supermature grape seed ( Vitis vinífera L.) from pomace obtained from the production of traditional generous wines of warm climate, which meet the minimum sanitary hygienic quality to be reused with industrial interest The average nutritional composition of grape seeds and their average amino acid composition are presented, by way of example, in Tables 1 and 2, respectively.

Table 1. Average nutritional composition of grape seeds (Zhou et al., 2011).

Table 2. Average grape seed amino acid composition (Wu and Lu, 2004).

First, the overripe grape seed is separated from the pomace by sieving, as they are smaller than the remains of skins and scrapes present in the pomace. After separation, it is subjected to a conditioning process that includes crushing and subsequent sieving until dry seed meal is obtained.

Subsequently, the lipid fraction of the seed meal is extracted by means of a Sohxlet, for 24 hours, with apolar organic solvents such as hexane. The organic solvent is subsequently evaporated and the amount of lipids determined by gravimetry. Table 3 shows the chemical characterization of the degreased flour obtained.

Table 3. Chemical characterization of the degreased flour of overripe grape seed used in the invention for obtaining the low molecular weight enzyme hydrolyzate.

The obtaining of a protein concentrate from the degreased flour of overripe grape seed is carried out by means of an extraction in alkaline medium at a constant pH 10.5 with 10 M NaOH, for 3 hours, in a ratio 1:10 (w / v ). After alkaline extraction, the suspension is allowed to decant for 3 days and the supernatant Subsequently, the supernatant is acidified to pH 3.0 with 6 M HCl, obtaining an acidic protein precipitate (insoluble protein fraction) which is recovered by centrifugation (3000 g., 10 min).

The protein extract obtained in the previous step is subjected to enzymatic hydrolysis in a bioreactor conditioned to keep the pH constant throughout the hydrolysis process. This constant pH ensures optimal enzyme activity. The hydrolytic process for obtaining protein hydrolyzate from grape seed protein extract is carried out under the following conditions:

Substrate: protein extract obtained in the previous stage: 10% (w / v; 100 g of seeds / L in deionized water)

Enzyme: endoproteases (trypsin and chymotrypsin); 0.3% (v / v; 3 ml / L in deionized water)

Temperature: 55 ° C

pH: 8.5

Constant agitation: 300 rpm

Reaction time: 3 hours

Once the enzymatic hydrolysis has proceeded for a preferable period of time equal to 3 hours, the hydrolyzate obtained in said step is centrifuged.

After centrifugation, a precipitate formed by the remains of the starting material remains insoluble (mainly insoluble fiber and part of undigested proteins) and, on the other hand, a supernatant composed mainly of proteins, peptides and amino acids , as well as other minor components solubilized during the process (mainly phenols and sugars). Table 4 shows the molecular weight distribution of the protein enzyme hydrolyzate obtained.

Table 4. Distribution of the molecular weights of the protein enzyme hydrolyzate obtained from grape seed, according to the method of the invention.

Said supernatant is clarified by passing it through a 0.2 p, m filter and is subjected to the elimination of polyphenols by adsorption with PVPP (20% w / v; 20 g PVPP / L).

After removal of the polyphenols, the enzymatic hydrolyzate is centrifuged to separate the PVPP and other insoluble particles, concentrated until a solid paste is obtained and lyophilized.

Example 2: Effect of the enzymatic hydrolyzate obtained in example 1 as a red wine color stabilizer

This example demonstrates the effectiveness of the method of the invention for obtaining a water-soluble enzyme hydrolyzate from overripe grape seed whose antioxidant and co-pigmenting properties allow its use in the wine industry, specifically in the vinification of red wines as agents Natural color stabilizers and agents that improve biofunctional properties.

The molecular weight distribution of the enzymatic hydrolyzate obtained from degreased flour of overripe grape seed and its low molecular weight protein fraction (<1 kDa) are shown in Table 4 above.

The biological and co-pigmenting properties, as well as color stabilizers, of the enzymatic hydrolyzate obtained by the method of the invention, are evaluated in model solutions with anthocyanins typical of the wine grape and red wine (commercial 3-glucoside malvidin pattern). Specifically, the test consists in measuring the modification of such properties before and after the addition of the hydrolyzate. enzymatic to the anthocyanin solution (150 mg / L), at a concentration of proteins similar to those that would be used in vinification (150, 300 and 600 mg / L).

Regarding the biological properties of the low molecular weight fraction (<1kDa), it showed antioxidant activity depending on its concentration (Figure 1). The antioxidant activity of the anthocyanin solutions before and after the enzymatic hydrolyzed addition was determined according to the FRAP spectrophotometric method (Benzie and Strain, 1996), expressed as an equivalent Trolox antioxidant activity (TEAC Assay: Antioxidant Capacity as Trolox Equivalents or, in English , Trolox Equivalent Antioxidant Capacity), considered as the p, mol of Trolox with the same antioxidant capacity as 1 L of anthocyanic solution (p, mol TE / L).

Regarding the color-stabilizing and co-stabilizing properties of the enzymatic hydrolyzate of the invention, it presented the ability to increase the dye intensity of anthocyanic solutions (Figure 2) as well as decrease its color degradation during a 30-day storage period ( Figure 3). The colorimetric properties of anthocyanic solutions and their variations, before and after adding the protein preparation, were evaluated by Differential Colorimetry according to the methodology described in Gordillo et al. (Gordillo, B. et al. 60 (2012) 2896-2905; Gordillo, B. et al. 63 (2015) J. Agríe. Food Chem. 7645-7653).

Claims (23)

1. Procedure for obtaining an enzymatic hydrolyzate from overripe grape seeds characterized by comprising the following steps: (a) separation of the overripe grape seed from the grape pomace, obtaining a seed meal and degreasing the seed meal by extracting its lipid fraction; (b) treatment of the degreased seed meal obtained in step (a) with an alkaline solution for the extraction of proteins, acidification of the extract obtained until obtaining a precipitate, separation of the precipitate and redisolution thereof in water to obtain a aqueous protein concentrate; (c) Enzymatic hydrolysis of the aqueous protein concentrate obtained in step (b) to obtain an enzymatic hydrolyzate. 2. Method according to claim 1, characterized in that it comprises a step (d) of centrifugation of the enzymatic hydrolyzate obtained in step (c) to obtain a precipitate and the supernatant enzyme hydrolyzate. 3. Method according to claim 2, characterized in that it comprises a step (e) for separating the enzymatic hydrolyzate from the precipitate obtained in step (d). 4. Method according to claim 3, characterized in that it comprises a step (f) for clarifying the enzymatic hydrolyzate supernatant obtained in step (e) by filtration thereof. 5. Method according to claim 4, characterized in that the clarification step (f) is carried out with a 0.2 p, m filter. Method according to claim 4 or 5, characterized in that it comprises a step (g) of elimination of polyphenols from the clarified enzymatic hydrolyzate obtained in step (f). 7. Method according to claim 6, wherein the elimination of polyphenols is carried out by adsorption with polyvinylpolypyrrolidone. Method according to any one of claims 6 or 7, characterized in that it comprises a step (h) of centrifugation of the clarified enzymatic hydrolyzate obtained in step (g). 9. Method according to claim 8, characterized in that it comprises a step (i) of concentration until obtaining a solid paste of the enzymatic hydrolyzate obtained in step (h). Method according to claim 9, characterized in that it comprises a step (j) of lyophilization of the solid paste obtained in step (h). Method according to any one of the preceding claims, characterized in that the supermature grape flour is prepared in step (a) by crushing and sieving the seed previously separated from the pomace. Method according to any one of the preceding claims, characterized in that the degreasing of the supermature grape flour of step (a) is carried out by Sohxlet and with apolar organic solvents. 13. Method according to any of the preceding claims, characterized in that the extraction in alkaline medium of step (b) is carried out with a NaOH solution. 14. Method according to any of the preceding claims, characterized in that the acidification of step (b) is carried out with a solution of HCl. 15. Method according to any of the preceding claims, characterized in that the acidification of step (b) is carried out up to pH between 2.5 and 3.5. 16. Method according to any of the preceding claims, characterized in that the pH at which the enzymatic hydrolysis is carried out in step (c) is alkaline. 17. Method according to claim 16, characterized in that the pH at which the enzymatic hydrolysis is carried out in step (c) is between 8 and 9. 18. Method according to any of the preceding claims, characterized in that the enzymatic hydrolysis of step (c) is carried out at a temperature between 50 and 55 ° C. 19. Method according to any of the preceding claims, characterized in that the enzymatic hydrolysis of step (c) is carried out with endoprotesases. 20. Method according to claim 19, characterized in that the endoprotesases are selected from trypsin, chymotrypsin and combinations of both. 21. Enzymatic hydrolyzate obtained by the method described in any of claims 1-20. 22. Use of the enzyme hydrolyzate described in claim 21 as a color stabilizer in red wines. 23. Use of overripe grape seeds mentioned in claim 1 for obtaining an enzymatic hydrolyzate.