ES2581585B1 - Procedure for obtaining an extract of acorn pulp, extract obtained and its use - Google Patents

Abstract

Procedure for obtaining an extract of acorn pulp. # The present invention relates to a procedure for obtaining an extract of acorn pulp, to the extract of acorn pulp obtainable by said method, to its use, in particular as an antioxidant, and to a food, cosmetic or pharmaceutical product comprising said extract.

Description

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DESCRIPTION

Procedure for obtaining an extract of acorn pulp, extract obtained and its use Field of the invention

The present invention belongs to the field of obtaining natural antioxidants. Specifically, it refers to a procedure for obtaining an extract of acorn pulp, the extract obtainable by said procedure and its use, in particular as an antioxidant.

Background of the invention

Protection against oxidative deterioration of various types of food is a clear concern in the food industry.

Antioxidants can act in various ways during oxidation, such as by sequestering metal ions, capturing free radicals, acting as reducing agents, decomposing peroxides, etc. In food systems oxidation involves the oxidation of lipids, while in systems Live free radicals can also damage proteins, DNA and other molecules.

As antioxidants, synthetic compounds such as butylhydroxytoluene (BHT) or butylhydroxyanixol (BHA) have been used in the food industry. However, there is a growing interest in replacing these synthetic antioxidants with natural antioxidants that have identical mechanisms of action. The worldwide demand for natural antioxidants has increased due to doubts about the safety of synthetic foods and food additives and the public's sensation that natural foods and food additives provide certain health benefits. Most of the natural compounds with antioxidant activity are found in plants, and many of them are polyphenolic in nature. The term polyphenols refers to aromatic compounds that contain at least two hydroxyphenolic groups in the molecule. This group of compounds includes flavonoids, a heterogeneous group of plant polyphenols (there are more than 4000) that share a benzopyran structure. Flavonoids constitute pigments (orange, red and blue colors) of various vegetables (flowers, fruits, leaves) and are important factors for growth, development and protection

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of the plants. The main flavonoids with antioxidant activity present in vegetables are: (+) - catechin, (-) - epicatechin, (-) - epigallocatechin and the corresponding esters of gallic acid: gallate of (-) - epicatechin and gallate of (-) - epigallocatechin

The raw materials of residual origin of agro-industrial processes that can serve as sources of antioxidants deserve particular attention. In this field, some materials have been considered as raw materials such as residues of potato skin, olive pomace, alpechines, grape seeds and wine bagasse and grape skins. Studies have been carried out that include the identification of active polyphenolic compounds in apple bagasse, seeds and citrus skins, coconut by-products.

The acorn is the characteristic fruit of the species of the genus Quercus. Within this genus there are numerous species that give acorns, such as holm oak, oak or cork oak. This type of tree is common in the west of the Iberian Peninsula where the largest area of pasture is concentrated. Acorn maturation occurs in autumn-winter providing an important food resource for wildlife and domestic animals. Special attention requires the use of acorns as feed for pigs, a species that has been able to obtain greater use of the nutritional values of the acorn when peeled before consumption. This peculiarity has caused that the acorn is preferably reserved for the Iberian pig and that its crusta in the pasture is centered in its fattening with acorn (montanera), fundamentally of oak (Quercus ilex). The composition of the acorn varies from one genus to another and throughout its maturation. The use of acorn in the formulation of feed as well as its importance in animal fattening, has placed this fruit among the most appreciated in the animal feed sector. However, its use is less in the human food sector.

With all this, the acquisition of an extract that encompasses all these characteristics, that is natural, that has antioxidant activity, that can be applied in foods and that preserves foods in which oxidative deterioration is included, acquires a special interest.

Object of the invention

In a first aspect, the present invention relates to a method of obtaining an extract of acorn pulp (method of the invention), which comprises the following steps:

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a) Crush the acorn pulp,

b) Add to the crushing of the acorn pulp obtained in step a) a solvent selected from the group consisting of water, acidified water, C1-C6 alcohol and mixtures thereof,

c) Stir the mixture of the crushed and the solvent obtaining two phases,

d) Separate the two phases obtained in step c) and collect the supernatant comprising the extract.

Also, in a second aspect, the present invention, refers to an extract of acorn pulp obtainable by the method according to the first aspect of the invention.

A third aspect of the invention relates to the use of acorn pulp extract according to the second aspect of the invention as an antioxidant.

Finally, a fourth aspect of the invention relates to a food, cosmetic or pharmaceutical product comprising acorn pulp extract according to the second aspect of the invention.

Detailed description of the invention

In a first aspect, the present invention relates to a method of obtaining an extract of acorn pulp, comprising the following steps:

a) Crush the acorn pulp,

b) Add to the crushing of the acorn pulp obtained in step a) a solvent selected from the group consisting of water, acidified water, C1-C6 alcohol and mixtures thereof,

c) Stir the mixture of the crushed and the solvent obtaining two phases,

d) Separate the two phases obtained in step c) and collect the supernatant comprising the extract.

Thus, the first stage of the process of the invention consists in crushing the acorn pulp. Acorn pulp can be separated from other parts of the acorn, in particular from the shell, by manual or mechanical means. In stage a), the crushing of the acorn pulp is carried out by manual or mechanical means. In a particular embodiment, in step a), pulp grinding is carried out until obtaining particles of a size ranging from 0.1 and 10 mm.

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In step b), the proportions used of pulp: solvent can be modified in order to obtain extracts with greater or less concentration and therefore with different antioxidant activity. Preferably, the ratio between crushed acorn pulp and solvent (pulp: solvent) is between 1:50 (weight: volume) and 1: 2 (weight: volume). In a preferred embodiment, in step b) the proportion pulp: solvent is 1:10 (weight: volume).

The use of one or the other solvent influences the amount of compounds extracted as well as the type of compounds with antioxidant activity that is obtained due to their characteristics and their affinity for the solvent. In a preferred embodiment, in step b) the selected solvent is water and more preferably a mixture of water and a C1-C6 alcohol.

In the context of the present invention, the term "C1-C6 alcohol" refers to an alcohol that can have from one carbon to six carbons in a straight or branched chain, with at least one hydroxide function.

In a preferred embodiment of the process of the invention, the solvent is a mixture of water and a C1-C6 alcohol and the water: C1-C6 alcohol ratio is between 20:80 and 80:20 (v: v), so more preferred is between 30:70 and 70:30 (v: v) and even more preferably the water: alcohol C1-C6 ratio is 50:50 (v: v).

In a particular embodiment of the process of the invention according to any one of the particular embodiments described in the preceding paragraphs, in step b), the solvent selected is a mixture of water and ethanol, where the water: ethanol ratio is comprised between 20: 80 and 80:20 (v: v), more preferably between 30:70 and 70:30 (v: v) and more preferably still 50:50 (v: v). The extraction carried out with a mixture of water and ethanol in a 50:50 ratio is what results in an extract with a higher content of phenolic and flavonoid compounds.

The conditions of temperature, time and exposure to light in stage c) can be adjusted in a range in which the obtaining of compounds with antioxidant activity is optimal avoiding degradation. Preferably step c) is carried out at a temperature between 20 ° C and 28 ° C, preferably between 22 ° C and 26 ° C, for a time between 30 and 210 minutes, preferably between 100 and 150 minutes, and under conditions from

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reduced brightness, preferably darkness. In a particular embodiment according to any one of the particular embodiments described in the preceding paragraphs, step c) is carried out in the dark, at a temperature of 25 ° C and for a time of two hours.

By agitating stage c) two phases are obtained from which the supernatant phase that separates from the solid phase is used. In step d), the separation of the two phases formed in step c) to collect the supernatant comprising the extract is carried out by decantation or centrifugation or any other method that allows the differentiation of the phases.

The supernatant collected in step d), which comprises the acorn pulp extract, may comprise some rest of the crushing or impurities, so that in a particular embodiment, the process of the invention according to any one of the particular embodiments described in the previous paragraphs, it comprises a step e) of filtration of the supernatant collected in step d). Likewise, the supernatant collected in step d) or the filtrate obtained in step e) can be concentrated by distillation and / or can be transformed by lyophilization or any other modification that allows obtaining an extract that suits the application needs. Thus, in another particular embodiment according to any one of the particular embodiments described in the preceding paragraphs, the process of the invention comprises a stage f) of distillation of the supernatant collected in step d) or of the filtrate obtained in step e). And in another particular embodiment, the process of the invention according to any one of the particular embodiments described above, comprises a step g) of lyophilization of the supernatant collected in step d) or of the filtrate obtained in step e) or of the distillate obtained in stage f). Depending on the final extract, its conservation may be carried out under environmental conditions or it will require controlled temperature storage in an appropriate container. In a particular embodiment, the extract is preserved in dark and cooling conditions.

A second aspect of the invention relates to an extract of acorn pulp obtainable by the process of the invention according to any one of the embodiments according to the first aspect of the invention. In a preferred embodiment, the extract has a phenolic compound content between 16 and 28 mg equivalent of gallic acid per gram of sample. Preferably between 23 and 28 mg equivalents of gallic acid per gram of sample. In a preferred embodiment the extract of the invention has a content in flavonoid compounds comprised between 1 and 3.1 mg equivalent of

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Catechin per gram of sample. Preferably between 2 and 3.1 mg equivalent of catechin per gram of sample.

The extract of the present invention, obtainable by the method according to any one of the embodiments of the first aspect of the invention, is effective as an antioxidant agent and in the preservation of food, cosmetic and pharmaceutical products. Thus, a third aspect of the invention relates to the use of acorn pulp extract according to the second aspect of the invention as an antioxidant. In addition, a fourth aspect of the invention refers to a food, cosmetic or pharmaceutical product comprising the extract according to any one of the embodiments of the second aspect of the invention.

The incorporation of the extract into a product is carried out in multiple ways, such as by addition within a formulation, by spraying on the product or any other method that allows the correct distribution of the extract and its antioxidant action. The concentration of extract to be used is determined by factors specific to the extract, such as antioxidant capacity, as well as those attributable to the food product such as type of product, content and type of fat, concentration in anti-and pro-oxidants, etc. Likewise, the technological processes that can be carried out after the addition of the extract as well as the preservation characteristics of the food product must be taken into account.

Examples

Below are some concrete examples of realization of the invention that serve to illustrate the invention.

Example 1. Obtaining the acorn pulp extract by using solvent mixture.

To obtain the extract, the acorn pulp (Gen. Quercus) was obtained in two stages of ripening, green and ripe. The pulp was obtained with the help of a knife separating them from the shell of the acorn. Subsequently, they were crushed by means of crushing in a conventional chopper. The extraction of compounds with antioxidant properties was carried out by using two solvents and mixing both. Thus, distilled water, ethanol and two mixtures of water were used: ethanol in proportion 50:50 and 70:30 (vol: vol), which gave rise to four different extracts for the different stages of

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maturation. A proportion 1:10 (g: mL) of pulp and solvent used, presented the best results in previous experiences, which is why it was used. The solvent and pulp mixtures were kept under stirring for two hours at a controlled temperature of 25 ° C under conditions of reduced luminosity. Once the agitation time elapsed, the phases were separated by centrifugation, obtaining two perfectly differentiated phases. The supernatant phase was filtered through Whatman No. 54 paper filter and kept in an opaque container at refrigeration temperature.

Example 2. Characterization of four acorn pulp extracts by quantifying their total phenolic content.

The quantification of total phenols of acorn pulp extracts was carried out following the procedure described by Singleton et al. (1999). 50 pL of each of the extracts were deposited in a microtiter plate well to which 20 pL of the Folin-Cicalteau reagent and 50 pL of 20% sodium carbonate (w / v) were added. The mixture was incubated for one hour at room temperature, after which the absorbance at 765 nm was measured against a blank where the sample was replaced by distilled water. The quantification of the measurements was performed against a standard curve of gallic acid. The results were expressed as equivalent mg of gallic acid per gram of sample and are reflected in Table 1.

Example 3. Characterization of four extracts of acorn pulp by quantifying their flavonoid content.

The quantification of the flavonoid content of the extracts obtained from the acorn pulp was carried out following the method proposed by Zhishen et al. (1999). This procedure is based on the formation of chelates between AlCh and flavonoids in the sample, which have a pink coloration. Thus, 400 pL of sample was deposited in a test tube to which 60 pL of 10% NaNO2, 60 pL of 20% AlCh and 400 pL of 1M NaOH were added. After stirring, its absorbance at 510 nm was measured. The quantification of the measurements carried out was done against a catechin standard curve. The results were expressed in mg of equivalent catechin per gram of sample and are reflected in Table 1.

Table 1. Content in total phenolic compounds (mg Eq gallic acid / g sample) and flavonoid content (mg Eq. Catechin / g sample) of acorn pulp extracts

obtained using as solvent: water, ethanol, water mixture: 50:50 ethanol and water mixture: 70:30 ethanol.

 Water Method of extraction Ethanol 50:50 70:30 SEM Sig.

 Total Phenolic Compounds

 17.00 c 16.15 d 27.51 to 23.69 c 0.26 ***

 Flavonoids

 2.15 b 1.19 c 3.04 a 1.41 b 0.21 ***

n = 5 determinations

Sig .: Level of significance ANOVA test; ***: p <0.001. a, b, c, d: In the same row averages

5 with different letters imply statistically significant differences (Tukey test, p <0.05)

The antioxidant capacity evaluation of acorn pulp extracts was carried out following two synthetic radical reduction methodologies, FRAP method (iron reducing antioxidant power, Ferric ion Reducing Antioxidant Power) and ABTS method (acid 2.2 '-azino-bis- (3-ethylbenzothiazoline) -6-sulfonic acid), as shown in examples 4 and 5. In these tests the antioxidant power of the sample is evidenced by changes in the coloration of a solution containing a radical liable to be reduced In both cases, the response obtained is extrapolated to a calibration curve with Trolox, so the results are expressed in TEAC (antioxidant activity equivalent to Trolox).

Example 4. Evaluation of the antioxidant capacity of four acorn pulp extracts by utilizing the ABTS radical.

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The method proposed by Re et al. (1999). Thus, the ABTS radical was obtained from the equal mixture of a solution of 7nM ABTS and 2.45 nM potassium persulfate. This solution was kept at room temperature for 16 hours in dark conditions for the generation of the radial. The solution was diluted with ethanol to obtain an absorbance of 0.7 (± 0.1) at 734 nm. 245 pL of the solution with the ABTS radical was deposited in a well of a microtiter plate and its absorbency was measured at 734 nm. Then 5pL of the extract to be tested was added and, after one minute, the measurement was repeated at 734 nm. The results obtained faced a Trolox calibration curve. The results were expressed as mg TEAC / g sample and are reflected in Table 2.

Example 5. Evaluation of the antioxidant capacity of four acorn pulp extracts using the FRAP method.

This method proposed by Benzie and Strain (1996), determines the ferric reduction capacity of a sample at low pH, transforming the tripyridyltriazine complex (TPTZ) with iron (III) to its ferrous form. Thus, the FRAP reagent was prepared from 2.5 mL of a 10 mM TPTZ solution together with 2.5 mL of a 20 mM FeCl3-6H2O solution and 25 mL of 0.3 mM acetate buffer and pH 3.6. An absorbance reading was carried out at 593 nm of 200 mL of the FRAP reagent to which 7 qL of extract to be tested were subsequently added 10 with absorbency measurements taken after 30 minutes. The results obtained faced a Trolox calibration curve. The results were expressed as mg TEAC / g sample and are reflected in Table 2.

Table 2. Antioxidant capacity measured by the ABTS method and by the FRAP method (mg TEAC / g sample) of acorn pulp extracts obtained using as solvent: water, ethanol, water mixture: 50:50 ethanol and water mixture: 70:30 ethanol.

 Water Method of extraction Ethanol 50:50 70:30 SEM Sig.

 ABTS

 25.33 b 24.67 b 44.07 to 29.45 b 2.31 ***

 FRAP

 32.83 b 35.02 b 62.67 a 49.61 b 2.83 ***

n = 5 determinations

Sig .: Level of significance ANOVA test; ***: p <0.001. a, b: In the same row, means with different letters imply statistically significant differences (Tukey test, p <0.05).

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Claims (14)

5 10 fifteen twenty 25 30 35 1. - Procedure for obtaining an extract of acorn pulp, which comprises the following stages: a) Crush the acorn pulp, b) Add to the crushing of the acorn pulp obtained in step a) a solvent selected from the group consisting of water, acidified water, C1-C6 alcohol and mixtures thereof, c) Stir the mixture of the crushed and the solvent obtaining two phases, d) Separate the two phases obtained in step c) and collect the supernatant comprising the extract. 2. - Method according to claim 1, comprising a step e) of filtering the supernatant collected in step d). 3. - Method according to claim 1 or 2, comprising a step f) of distillation of the supernatant collected in step d) or of the filtrate obtained in step e). 4. - Method according to any one of claims 1 to 3, comprising a step g) of lyophilization of the supernatant collected in step d) or of the filtrate obtained in step e) or of the distillate obtained in step f). 5. - Method according to any one of claims 1 to 4, wherein in step a) the acorn pulp is crushed to obtain a particle size between 0.1 mm and 10 mm. 6. - Method according to any one of claims 1 to 5, wherein the crushed ratio of acorn pulp: solvent is comprised between 1:50 (weight: volume) and 1: 2 (weight: volume). 7. - Method according to any one of claims 1 to 6, wherein the solvent selected is a mixture of water and a C1-C6 alcohol. 8. - Procedure according to claim 7, wherein the water: C1-C6 alcohol ratio is between 20:80 and 80:20 (v: v). 9. - Method according to claim 7 or 8, wherein the solvent selected in step b) is a mixture of water and ethanol. 10. - Method according to any one of claims 1-9, wherein step c) is carried out at between 20 and 28 ° C, for between 30 and 210 minutes. 11. - Acorn pulp extract obtainable by the method according to any one of claims 1 to 10. 12. - Extract according to the previous claim, which has a content in phenolic compounds comprised between 16 and 28 mg equivalent of gallic acid per gram of sample. 13. - Extract according to claim 11 or 12, which has a content in flavonoid compounds comprised between 1 and 3.1 mg equivalent of catechin per gram of sample. 14. - Use of the acorn pulp extract according to any one of claims 11 to 13 as an antioxidant. 5 15.- Food, cosmetic or pharmaceutical product comprising pulp extract acorn according to any one of claims 11 to 13.