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    • 专利摘要:
    Procedure to obtain an enzymatic hydrolyzate from over-ripe grape seeds and use thereof. The present invention relates to a process based on the enzymatic hydrolysis in basic medium of the protein fraction of overripe grapes to obtain a natural enzymatic hydrolyzate of variable molecular weight. The present invention also relates to the enzymatic hydrolyzate obtained by said process and to the use thereof as a color stabilizer in red wines. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2709524A1
    申请号:ES201731214
    申请日:2017-10-16
    公开日:2019-04-16
    发明作者:Arrobas Belén Gordillo；Pulido Francisco José Rodriguez；Morgado Bruno Rodriguez；Rubio Juan Parrado；Martin Maria Lourdes Gonzalez-Miret；Gilete María Luisa Escudero；Vila Dolores Hernanz；Hierro Jose Miguel Hernandez；Bastante María Jesús Cejudo；Palacios Maria José Jara；Bueno Julio Nogales；Bocanegra Berta Baca；Granados Francisco José Rivero；Mira Francisco José Heredia
    申请人:Universidad de Sevilla；
    IPC主号:
    专利说明:

    [0001]
    [0002] Procedure to obtain an enzymatic hydrolyzate from overripe seeds of grapes and use thereof
    [0003]
    [0004] The present invention relates to a process for obtaining an enzymatic hydrolyzate from overgrowth of grapes 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 the elaboration and treatment of wines.
    [0005]
    [0006] BACKGROUND OF THE INVENTION
    [0007] 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 broths" are well known by winemakers in regions with a warm climate, so different strategies have been developed to appease their effects as far as possible. One of the possible techniques to avoid these color drops is to modulate the interactions between pigments and copigments of wine (Gomez-Mguez, M. et al, J. Food Eng. 79 (2007) 271-278). Among these, the addition of natural sources of copigments is considered an alternative to traditional vinification, generally from natural sources of wine 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
    [0008] (Gordillo, B. et al., Food Chem. 206 (2013) 249-259; Gordillo, B. et al., Food Chem.
    [0009] 141 (2013) 2184-2190).
    [0010]
    [0011] Apart from these phenolic compounds, grape seeds contain approximately 14% of proteins, which are natural biopollimers with copigmentation capacity (Fantozzi, P.J. Am. Oil Chem. Soc. 58 (1981) 1027-1031). The total content of proteins throughout the berry depends on the variety and degree of maturity (Bertazzo, A. et al., J. Mass Spectrom, 45 (201) 966-970, Lopez-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 that are 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 claim that they are likely to be potentially useful in practical applications (Gazzola, D. et al., Food Chem. 155 (2014) 132-139).
    [0012]
    [0013] In the vitivinicola 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 added to the wine for stabilize its color is not alien to this, but also improves the use of waste from the wine industry.
    [0014]
    [0015] DESCRIPTION OF THE INVENTION
    [0016]
    [0017] The present invention relates to a process based on the enzymatic hydrolysis in a basic medium of the protein fraction of overripe grapes for obtaining a natural enzymatic hydrolyzate of variable molecular weight. The utilization of seeds in advanced stage of maturity reduces the interference of the polyphenolic compounds in the hydrolytic process, because they undergo polymerization reactions with maturation, which leads to a certain insolubilization.
    [0018]
    [0019] 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.
    [0020]
    [0021] Therefore, in a first aspect, the present invention relates to a process for obtaining an enzymatic hydrolyzate from grape overgrowth characterized by comprising the following steps:
    [0022]
    [0023] (a) separation of the grape seed overrun of the grape marc, obtaining a seed meal and degreasing the seed meal by means of extraction of its lipid fraction,
    [0024] (b) treatment of the degreased seed meal obtained in step a) with an alkaline solution for the extraction of proteins, acidification of the obtained extract until obtaining a precipitate, separating the precipitate and redissolving it in water to obtain a concentrate aqueous protein,
    [0025] (c) Enzymatic hydrolysis of the aqueous protein concentrate obtained in step (b) to obtain an enzymatic hydrolyzate.
    [0026]
    [0027] In a preferred embodiment of the invention, the method comprises a step d) of centrifugation of the enzymatic hydrolyzate of step (c). After the centrifugation of step (d), on the one hand, a precipitate is obtained formed by the remains of the starting material that remains insoluble (mainly insoluble fiber and part of undigested proteins) and, on the other hand, a supernatant composed mainly for proteins, peptides and amino acids, so! as other minor components solubilized during the process (mainly phenols and sugars).
    [0028]
    [0029] In another preferred embodiment of the invention, the method comprises a step (e) of separating the supernatant (enzymatic hydrolyzate) from the precipitate obtained in step (d).
    [0030]
    [0031] In another preferred embodiment of the invention, the method comprises a step (f) of clarification of the enzyme hydrolyzate supernatant obtained in step (e) by filtering it. Preferably the filtration is carried out with a filter of 0.2 p, m.
    [0032]
    [0033] 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 elimination of polyphenols must be done at this point and not before , since the adsorption phenomena do not happen selectively and it is necessary that the medium be filtered and clarified.
    [0034]
    [0035] In a preferred embodiment of the invention, after the removal of the polyphenols, the enzymatic hydrolyzate is centrifuged (step (h)) to separate the PVPP if present and other insoluble particles.
    [0036] In another preferred embodiment, after said centrifugation, the enzymatic hydrolyzate is concentrated until obtaining a solid paste (step (i)).
    [0037]
    [0038] In another preferred embodiment, the solid paste obtained in step (i) is subjected to a drying step (j) by lyophilization thereof.
    [0039]
    [0040] In step (a), the overripe grape seed is separated from the pomace preferably by physical methods such as sieving, since they have a smaller size than the residues of skins and scrapes present in the pomace. Once the overripe grape seed has been separated, the flour of the same is preferably prepared by trituration and sieving of the overripe grape seed separated from the pomace. Degreasing is preferably carried out by Sohxlet, with apolar organic solvents, such as hexane. More preferably, Sohxlet extraction 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 per gravimetry can be determined.
    [0041]
    [0042] In a preferred embodiment, the extraction in an alkaline medium of step (b) is carried out with a NaOH solution, more preferably 10 M NaOH.
    [0043]
    [0044] In a preferred embodiment, the acidification of step (b) is carried out with HCl, more preferably 6M HCl.
    [0045]
    [0046] In another preferred embodiment, the acidification of step (b) is carried out until pH between 2.5 and 3.5, preferably 3.0, obtaining an acidic protein precipitate (insoluble protein fraction) which is preferably recovered by centrifugation.
    [0047]
    [0048] In another preferred embodiment, once the extraction has been carried out in an alkaline medium and prior to the acidification, the obtained extract is left to decant until the supernatant shows no turbidity (approximately 3 days). The supernatant is the one that is subsequently subjected to acidification.
    [0049]
    [0050] In another preferred embodiment, the pH at which the enzymatic hydrolysis of the stage is carried out (c) is alkaline, more preferably the pH ranges between 8 and 9. The temperature at which the hydrolysis step 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 (agroindustrial residue of the overripe grape seed) and in turn to maintain the proteolytic activity of the chosen enzymes during the duration of the process. This temperature achieves a situation of compromise between the activity of the enzyme and the solubility of the product, since if the temperature of 60 ° C is reached, the enzyme decreases its activity after 30 minutes.
    [0051]
    [0052] The enzymatic hydrolysis is preferably carried out in a period between three hours and three and a half hours (depending on the scale of the process) in continuous agitation.
    [0053]
    [0054] Enzymes that are effective in the hydrolysis process described in the method of the present invention (step c)) are proteolytic enzymes, preferably of endoprotease action, commonly used in the hydrolysis of proteins. 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, non-specific and well known and characterized from a scientific point of view for optimal parameters of pH and temperature.
    [0055]
    [0056] In the present invention, grape pomace is understood as the solid residue obtained as a by-product after the vinification process.
    [0057]
    [0058] In the present invention, seed of overripe grape is understood to be that seed from grapes that have greatly exceeded the technological maturity necessary to produce table wines, reaching a degree of sugars higher than 25 ° Brix ", and which are used for the elaboration of generous wines.
    [0059]
    [0060] In the present invention, "generous wine" is understood to mean those with a high alcohol content (from 15 °) because they are obtained from grapes with a high concentration of sugars and / or that involve the addition of extra vinyl alcohol prior to aging. in wood.
    [0061]
    [0062] 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.
    [0063]
    [0064] A final aspect of the invention relates to the use of overripe grape seeds obtained as a by-product of the production of generous wines for obtaining an enzymatic hydrolyzate as defined in the second aspect of the present invention.
    [0065]
    [0066] Throughout the description and the claims the word "comprises" and its variants do not intend to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will be apparent in part from the description and in part 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.
    [0067]
    [0068] BRIEF DESCRIPTION OF THE FIGURES
    [0069]
    [0070] FIG. 1: shows a graph representing the increase in antioxidant activity (p, mol TE / L) of an anthocyanin solution (Mv-3glc) after the addition of increasing concentrations of the enzymatic hydrolyzate of the present invention.
    [0071]
    [0072] FIG. 2: shows a graph representing the increase in the coloring intensity of an anthocyanin solution (Mv-3glc) after the addition of increasing concentrations of the enzymatic hydrolyzate of the present invention.
    [0073]
    [0074] FIG. 3: shows a graph representing the color degradation (%) of an anthocyanin solution (Mv-3glc) after the addition of increasing concentrations of the enzymatic hydrolyzate of the present invention, during a storage period of 30 days.
    [0075]
    [0076] EXAMPLES
    [0077]
    [0078] The invention will now be illustrated by means of tests carried out by the inventors, which highlights the effectiveness of the product of the invention.
    [0079] Example 1: Preparation of an enzymatic hydrolyzate from seed overgrowth of grapes according to the method of the present invention
    [0080]
    [0081] The agroindustrial protein residue that is used in the invention process is seed overgrowth of grape (Vitis vinifera L.) from pomace obtained from the elaboration of traditional warm 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 composition of amino acids are presented, by way of example, in Tables 1 and 2, respectively.
    [0082]
    [0083] Table 1. Average nutritional composition of grape seeds (Zhou et al., 2011).
    [0084]
    [0085]
    [0086]
    [0087]
    [0088] Table 2. Average amino acid composition of grape seed (Wu and Lu, 2004).
    [0089]
    [0090]
    [0091]
    [0092]
    [0093] First, the overripe grape seed is separated from the pomace by sieving, since they are smaller in size than the residues of skins and scrapes present in the pomace. After separation, it undergoes a conditioning process that includes the crushing and subsequent sieving until obtaining dry seed flour.
    [0094]
    [0095] Subsequently, the lipid fraction of the seed meal is extracted by 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. In Table 3 the chemical characterization of the degreased flour obtained is shown.
    [0096]
    [0097] Table 3. Chemical characterization of the degreased flour of over-ripe grape seed used in the invention for obtaining the low molecular weight enzymatic hydrolyzate.
    [0098]
    [0099]
    [0100]
    [0101]
    [0102] The obtaining of a protein concentrate from the degreased flour of overripe grape seed is carried out by means of an extraction in an alkaline medium at constant pH 10.5 with 10 M NaOH, for 3 hours, in a ratio of 1:10 (w / v ). After the 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).
    [0103]
    [0104] The protein extract obtained in the previous step is subjected to enzymatic hydrolysis in a conditioned bioreactor to maintain the pH constant throughout the hydrolysis process. This constant pH ensures an optimal activity of the enzyme. The hydrolytic process for obtaining the protein hydrolyzate from the grape seed protein extract is carried out under the following conditions:
    [0105] Substrate: protein extract obtained in the previous stage: 10% (w / v; 100 g seeds / L in deionized water)
    [0106] Enzyme: endoproteases (trypsin and chymotrypsin); 0.3% (v / v; 3 ml / L in deionized water)
    [0107] Temperature: 55 ° C
    [0108] pH: 8.5
    [0109] Constant agitation: 300 rpm
    [0110] Reaction time: 3 hours
    [0111]
    [0112] Once the enzymatic hydrolysis has proceeded for a preferable period of time equal to 3 hours, the hydrolyzate obtained in said step is centrifuged.
    [0113]
    [0114] After centrifugation, on the one hand, there is obtained a precipitate formed by the remains of the starting material that 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 so! as other minor components solubilized during the process (mainly phenols and sugars). Table 4 shows the distribution of the molecular weights of the protein enzymatic hydrolyzate obtained.
    [0115]
    [0116] Table 4. Distribution of the molecular weights of the enzymatic protein hydrolyzate obtained from grape seed, according to the method of invention.
    [0117]
    [0118]
    [0119]
    [0120]
    [0121] Said supernatant is clarified by passing it through a filter of 0.2 p, m and is subjected to the elimination of polyphenols by adsorption with PVPP (20% w / v; 20 g PVPP / L).
    [0122]
    [0123] After the elimination of the polyphenols, the enzymatic hydrolyzate is centrifuged to separate the PVPP and other insoluble particles, concentrated until obtaining a solid paste and lyophilized.
    [0124]
    [0125] Example 2: Effect of the enzymatic hydrolyzate obtained in example 1 as color stabilizer of red wine
    [0126]
    [0127] This example demonstrates the effectiveness of the invention method for obtaining a water-soluble enzymatic hydrolyzate from overripe grape seed whose antioxidant and copigmenting properties allow its use in the vinyl industry, specifically in the vinification of red wines as agents color stabilizers of natural origin and agents that improve biofunctional properties.
    [0128]
    [0129] The distribution of the molecular weights of the enzymatic hydrolyzate obtained from degreased meal of overgrowed grape seed and its protein fraction of low molecular weight (<1 kDa) are shown in Table 4 above.
    [0130]
    [0131] The biological and copigmenting properties, as well as color stabilizers, of the enzymatic hydrolyzate obtained by the method of the invention are evaluated in model solutions with anthocyanins characteristic of the wine grape and red wine (commercial malvidin 3-glucoside standard). In particular, the test consists of measuring the modification of such properties before and after the addition of the hydrolyzate enzymatic to the anthocyanin solution (150 mg / L), at a protein concentration similar to those used in vinification (150, 300 and 600 mg / L).
    [0132]
    [0133] Regarding the biological properties of the low molecular weight fraction (<1kDa), it showed antioxidant activity dependent 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 equivalent Trolox antioxidant activity (TEAC Test: 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 anthocyanin solution (^ mol TE / L).
    [0134] As for the copigmenting and stabilizing properties of the color of the enzyme hydrolyzate of the invention, it has the capacity to increase the coloring intensity of the anthocyanin solutions (Figure 2) as well as to reduce its color degradation during a storage period of 30 days ( Figure 3). The colorimetric properties of the anthocyanin solutions and their variations, before and after adding the protein preparation, were evaluated by Differential Colorimetr according to the methodology described in Gordillo et al. (Gordillo, B. et al., 60 (2012) 2896-2905; Gordillo, B. et al., 63 (2015) J. Agric. Food Chem. 7645-7653).
    权利要求:
    Claims (23)
    [1]
    A process for obtaining an enzymatic hydrolyzate from grape overgrowth characterized by comprising the following stages:
    (a) separation of the grape seed overrun of the grape marc, obtaining a seed meal and degreasing the seed meal by extracting its lipid fraction;
    (b) treatment of the defatted seed meal obtained in step (a) with an alkaline solution for the extraction of proteins, acidification of the obtained extract until obtaining a precipitate, separation of the precipitate and redissolution thereof in water to obtain a aqueous concentrated protein;
    (c) Enzymatic hydrolysis of the aqueous protein concentrate obtained in step (b) to obtain an enzymatic hydrolyzate.
    [2]
    2. Process 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 enzyme hydrolyzate supernatant.
    [3]
    3. Method according to claim 2, characterized in that it comprises a step (e) of separation of the enzymatic hydrolyzate supernatant from the precipitate obtained in step (d).
    [4]
    4. Method according to claim 3, characterized in that it comprises a step (f) of clarification of the enzyme hydrolyzate supernatant obtained in step (e) by filtering it.
    [5]
    5. Method according to claim 4, characterized in that the clarifying step (f) is carried out with a filter of 0.2 p, m.
    [6]
    6. Process according to claim 4 or 5, characterized in that it comprises a step (g) of eliminating polyphenols from the clarified enzymatic hydrolyzate obtained in step (f).
    [7]
    7. Process according to claim 6, wherein the elimination of polyphenols is carried out by adsorption with polyvinylpolypyrrolidone.
    [8]
    8. Process according to any 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]
    9. Process 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).
    [10]
    10. Process according to claim 9, characterized in that it comprises a step (j) of lyophilization of the solid paste obtained in step (h).
    [11]
    11. Process according to any of the preceding claims, characterized in that the over-ripe grape flour is prepared in step (a) by crushing and sieving the seed previously separated from the pomace.
    [12]
    12. Process according to any of the preceding claims, characterized in that the degreasing of the overripe grape flour of stage (a) is carried out by means of Sohxlet and with apolar organic solvents.
    [13]
    13. Process according to any of the preceding claims, characterized in that the extraction in an alkaline medium of step (b) is carried out with a solution of NaOH.
    [14]
    14. Process according to any of the preceding claims, characterized in that the acidification of step (b) is carried out with a solution of HCl.
    [15]
    15. Process 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]
    16. Process according to any of the preceding claims, characterized in that the pH at which the enzymatic hydrolysis of stage (c) is carried out is alkaline.
    [17]
    17. Process according to claim 16, characterized in that the pH at which the enzymatic hydrolysis of stage (c) is carried out is between 8 and 9.
    [18]
    18. Process 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]
    19. Process according to any of the preceding claims, characterized in that the enzymatic hydrolysis of step (c) is carried out with endoprotesases.
    [20]
    20. Method according to claim 19, characterized in that the endoprostases are selected from trypsin, chymotrypsin and combinations of both.
    [21]
    21. Enzymatic hydrolyzate obtained by the process described in any of claims 1-20.
    [22]
    22. Use of the enzymatic hydrolyzate described in claim 21 as a color stabilizer in red wines.
    [23]
    23. Use of overripe grapes seeds to obtain an enzymatic hydrolyzate.
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    CN110423263B|2019-09-08|2020-12-22|浙江理工大学|Method for extracting protein from grape seeds|
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