PROCESS FOR OBTAINING A TANNIN EXTRACT ISOLATED FROM GRAPES, TANNIN EXTRACT OBTAINED AND USES THEREOF

Abstract

A process is provided for obtaining a tannin extract isolated from grapes using grape seeds. The process comprises: preparing the grape seeds as the source of tannins; preparing an aqueous solution of a polyphenol-solubilising agent selected from urea, a compound containing the SO.sub.3 group and sodium hydroxide at concentration ranging between 1% and 15% by weight; performing a single extraction in an autoclave reactor; separation and direct concentration in order to produce the isolated extract. The invention also relates to the tannin extract obtained. The new tannin extract isolated from grape seeds comprises a mixture of tannins and non-tannins, the composition whereof provides the extract with improved antioxidant and light fastness properties. The tannin extract isolated from grape seeds further comprises a colouring agent.

Claims

1. An aqueous extraction process for obtaining a tannin extract isolated from grapes which process comprises using a source of tannins, preparing an aqueous solution of a polyphenol-solubilising agent and carrying out a tannin extraction step in a reactor that comprises the source of tannins and the prepared aqueous solution at a given pressure and temperature, wherein: the source of tannins are whole grape seeds, without grinding or milling, the polyphenol-solubilising agent is selected from urea, sodium hydroxide and a compound containing the SO.sub.3 group, at a concentration in the aqueous solution between 1% and 15% by weight with respect to the weight of the whole grape seeds, and the extraction step is performed in an autoclave at a pressure ranging between 1 and 6 atmospheres, and a temperature ranging between 80 C. and 160 C.

2. (canceled)

3. The process according to claim 1, wherein the polyphenol-solubilising agent is urea whereby further extracting in the tannin extract an azo colouring agent free of banned aromatic amines showing a maximum at 352.8 nm, 2.70 A, measured by UV-Vis spectroscopy between 350-800 nm.

4. The process according to claim 1, wherein the concentration of the polyphenol-solubilising agent in the aqueous solution ranges between 2% and 8% by weight with respect to the weight of the whole grape seeds.

5. The process according to claim 1, wherein the weight ratio between the grape seeds and the water used to prepare the aqueous solution ranges between 0.5:1 and 1:4.

6. The process according to claim 1, wherein the extraction step is performed at a temperature ranging between 100 C. and 130 C., and a pressure ranging between 2.5 and 3.5 atmospheres.

7. The process according to claim 1, wherein, once the extraction process has been completed, the reactor is cleaned with new water that is susceptible to being reused to prepare a new aqueous solution of the polyphenol-solubilising agent.

8. The tannin extract according to claim 14, wherein the tannin extract comprises a mixture of pure hydrolysable tannins, pure condensed tannins that include procyanidins and non-tannins that include gallic acid.

9. The tannin extract according to claim 14, wherein, by using urea as the polyphenol-solubilising agent in the process for obtaining the extract, the tannin extract further comprises an azo colouring agent free of banned aromatic amines and shows a maximum at 352.8 nm, 2.70 A, measured by UV-Vis spectroscopy between 350-800 nm.

10. (canceled)

11. (canceled)

12. The process of claim 1, further comprising concentrating the extract by evaporation, to obtain the isolated tannin extract in a concentrated form.

13. The process of claim 1, further comprising spray drying, to obtain the isolated tannin extract in the solid state.

14. A tannin extract isolated from whole grape seeds obtainable by the process defined in claim 1.

15. A device comprising a tannin extract obtained by using a source of tannins, preparing an aqueous solution of a polyphenol-solubilising agent and carrying out a tannin extraction step in a reactor that comprises the source of tannins and the prepared aqueous solution at a given pressure and temperature, wherein: the source of tannins are whole grape seeds, without grinding or milling, the polyphenol-solubilising agent is selected from urea, sodium hydroxide and a compound containing the SO.sub.3 group, at a concentration in the aqueous solution between 1% and 15% by weight with respect to the weight of the whole grape seeds, and the extraction step is performed in an autoclave at a pressure ranging between 1 and 6 atmospheres, and a temperature ranging between 80 C. and 160 C.

16. The device of claim 15, wherein the device comprises leather.

17. The device of claim 15, wherein the device is a food.

18. The device of claim 15, wherein the device is a cosmetic.

19. The device of claim 15, wherein the device is a drug.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0055] In order to contribute to a better understanding of what has been explained above, a set of drawings is attached wherein, schematically and only as a non-limiting example, an embodiment is represented.

[0056] FIG. 1 shows the chromatogram, obtained by means of reverse-phase high-performance liquid chromatography (HPLC), of the extract obtained according to the first and second aspects of the invention, wherein the source of tannins are whole grape seeds and the polyphenol-solubilising agent used in the extraction step is sodium metabisulfite according to Example 1 of the invention.

[0057] FIG. 2 shows the chromatogram, obtained by means of reverse-phase high-performance liquid chromatography (HPLC), of an extract obtained according to the first and second aspects of the invention, wherein the source of tannins are whole grape seeds and the polyphenol-solubilising agent used in the extraction step is urea according to Example 2 of the invention.

[0058] FIG. 3 shows the chromatogram, obtained by means of reverse-phase high-performance liquid chromatography (HPLC), of the extract obtained according to reference Example 3, wherein the source of tannins is ground grape pomace and the polyphenol-solubilising agent used in the extraction step is sodium metabisulfite.

[0059] FIG. 4 shows the scanning graph of the extract, obtained from a sample extract according to Example 2 of the invention (), as compared to the scanning graph of a conventional brown colouring agent () obtained between 350-800 nm by means of UV-Vis spectrophotometry.

[0060] FIG. 5 shows the chromatogram, obtained by means of HPLC-DAD at 500 nm, of a sample extract obtained according to Example 2 of the invention within the 200-800 nm range.

[0061] FIG. 6 shows a comparative assay of the artificial light fastness according to the IUF 402 standard, wherein we may observe the stability of the extract obtained according to Example 1 and Example 2 of the invention.

[0062] In order to contribute to a better understanding of what has been explained above, some examples are attached wherein, schematically and only as non-limiting examples, specific embodiments of the invention are represented.

EXAMPLES

Example 1: Tannin Extract Isolated from Grape Seeds

Chemical Extraction

[0063] For 100 kg of whole dried grape seeds, 5 kg of sodium metabisulfite and 100 litres of distilled water were used. The aqueous solution contained 5% of sodium metabisulfite.

[0064] All the components were introduced into an autoclave reactor and the aqueous extraction was performed therein, keeping the temperature at 120 C. and the pressure at 3 atmospheres for 1 hour.

[0065] Once the specified time had elapsed, a tannin extract isolated from grape seeds in the liquid state suitable for the tanning of skins was obtained.

Example 2: Tannin Extract Isolated from Grape Seeds

Chemical Extraction

[0066] For 100 kg of whole dried grape seeds, 5 kg of urea and 100 litres of distilled water were used. The aqueous solution contained 5% of urea.

[0067] All the components were introduced into an autoclave reactor and the aqueous extraction was performed therein, keeping the temperature at 120 C. and the pressure at 3 atmospheres for 1 hour.

[0068] Once the specified time had elapsed, a tannin extract isolated from grape seeds in the liquid state suitable for the tanning of skins was obtained.

Example 3 (Reference Example): Tannin Extract Isolated from Grape Pomace

Chemical Extraction

[0069] For 200 kg of pomace, 2 kg of sodium metabisulfite and 400 litres of distilled water were used.

[0070] All the components were introduced into an autoclave reactor and the aqueous extraction was performed therein, keeping the temperature at 136 C. and the pressure at 5 atmospheres for 3 hours.

[0071] Once the specified time had elapsed, the tannin extract isolated from pomace was obtained.

Assays

Chromatography of the Extracts Obtained According to Examples 1 to 3

[0072] The extracts obtained Examples 1 to 3 were analysed by means of high-performance column chromatography with reverse-phase separation HPLC-DAD. A column of the Xbridge Phenyl type was used. For the chromatographic analysis, a 1:25 aqueous solution of the extracts obtained in the examples was prepared. Subsequently, the extracts were filtered to 0.45 m in order to be analysed. 25 l of the extract were injected. The chromatogram capture wavelength was 271.1 nm. See FIGS. 1 to 3, which correspond, respectively, to Examples 1 to 3.

[0073] For the analysis, a new spectral library was generated wherein the spectra of the different compounds present in commercial Mimosa, Quebracho Tree, Tara and Chestnut Tree tannin samples were incorporated. The spectra of commercial polyphenol standards with different chemical natures, including the condensed, or catechinic, types and the hydrolysable type, or gallotannins, were also incorporated. Below we show a list of the standards studied by means of the chromatographic separation method defined in the invention:

TABLE-US-00001 Retention Compound CAS N.sup.o Category MW time (min) (R.sup.2) (+)-Catechin 154-23-4 Flavan-3-ol 290.3 11.8 0.9983 ()-Epicatechin 490-46-0 Flavan-3-ol 290.3 16.1 0.9985 ()-Epigallocatechin 970-74-1 Flavan-3-ol 306.3 10.3 0.9998 ()-Epicatechin gallate 2157-08-5 Flavan-3-ol 442.4 22.7 0.9979 ()-Epigallocatechin 959-51-5 Flavan-3-ol 458.4 17.3 n.c. gallate.sup.(1) Procyanidin B1 20315-25-7 Procyanidin Dimer 578.5 10.9 0.9999 Procyanidin B2 29106-49-8 Procyanidin Dimer 578.5 15.4 0.9503 Procyanidin A2 20315-25-7 Dimeric Catechin 576.5 23.7 0.9535 Penta-O-galloil-B-D- 14937-32-7 Gallotannin 940.7 25.1 n.c. glucose.sup.(1) Summary of the standards analysed by means of reverse-phase HPLC-DAD. .sup.(1)n.c. (no correlation).

[0074] The compounds identified in the extracts obtained according to Example 1, Example 2 and reference Example 3 were then compared.

[0075] Table 1 below lists the compounds identified in the tannin extract isolated from grape seeds according to Example 1:

TABLE-US-00002 % Surface area/ Total RT Surface surface Peak (min) Identification area area 1 2.97 Gallic Acid standard 590,723 16.1 2 3.46 Catechinic tannin from 43,598 1.2 Quebracho Tree 1 3 3.87 Catechinic tannin from Mimosa 1 51,787 1.4 4 4.17 Catechinic tannin. (Same 753,881 20.5 chromophore as Procyanidin B2) 5 5.93 Catechinic tannin. (Same 62,630 1.7 chromophore as (+)-Catechin) 6 6.54 Catechinic tannin from Mimosa 2 55,427 1.5 7 8.07 Catechinic tannin from Mimosa 3 193,423 5.3 8 8.91 Catechinic tannin. (Same 124,261 3.4 chromophore as Procyanidin B2) 9 11.71 Catechinic tannin. (Same 92,394 2.5 chromophore as ()-Epicatechin) 10 11.96 Catechinic-hydrolysable tannin. 32,164 0.9 (Same chromophore as ()- Epigallocatechin gallate) 11 12.88 Catechinic-hydrolysable tannin. 138,372 3.8 (Same chromophore as ()- Epicatechin gallate) 12 13.63 Catechinic tannin from Mimosa 4 26,330 0.7 13 13.83 Gallic tannin from Tara 1 38,722 1.1 14 14.67 Catechinic-hydrolysable tannin. 60,524 1.6 (Same chromophore as ()- Epigallocatechin gallate) 15 15.99 ()-Epicatechin standard 38,685 1.1 16 16.25 Catechinic tannin. (Same 6,683 0.2 chromophore as ()-Epicatechin) 17 17.01 Tannin derived from gallic acid 46,040 1.3 18 18.57 Catechinic-hydrolysable tannin. 22,905 0.6 (Same chromophore as ()- Epicatechin gallate) 19 19.07 Catechinic-hydrolysable tannin. 63,283 1.7 (Same chromophore as ()- Epigallocatechin gallate) 20 21.78 ()-Epicatechin gallate standard 23,363 0.6 21 33.17 Catechinic tannin from 502,689 13.7 Quebracho Tree 2

[0076] Table 2 below shows the compounds identified in the tannin extract isolated from grape seeds according to Example 2:

TABLE-US-00003 % Surface area/ Total RT Surface surface Peak (min.) Identification area area 1 2.97 Gallic Acid standard 842,937 12.2 2 4.39 Catechinic tannin. 11,218 0.2 (Same chromophore as ()-Epicatechin) 3 4.57 Catechinic tannin. 6.173 0.1 (Same chromophore as Procyanidin A2) 4 13.79 Gallic tannin from Tara 1 42,495 0.6 5 16.25 ()-Epicatechin standard 14,799 0.2 6 17.00 Gallic tannin from Tara 2 35,618 0.5 7 17.68 Catechinic tannin. 11.152 .02 (Same chromophore as Procyanidin A2) 8 20.87 Ellagic Acid 26,598 0.4 9 33.20 Catechinic tannin from 5,452,782 79.0 Quebracho Tree 2

[0077] The present invention refers to the chromatographic analysis of an aqueous polyphenolic extract that includes tannins, non-tannins and unidentified compounds differentiated as such on the basis of the HPLC-DAD assay of the diluted polyphenolic extract using the specified assay method. Tannins or polymers with a tanning capacity are considered to be those polymer molecules with a molecular weight greater than 500. A semi-quantitative estimate of the content of each component is included, calculated from the surface area percentage of each peak with respect to the total surface area. In Example 1 and Example 2, we may observe the peaks identified due to their spectral similarity with the substances present in the spectral library. The peaks identified in the chromatogram belong to the Grape Tannin library entries corresponding to the polyphenol standards cited in Table 1, samples of commercial vegetable tannin from Tara and commercial vegetable extracts from Mimosa, Quebracho Tree and Chestnut Tree.

[0078] Condensed tannins are polymers formed by units of anthocyanidin (flavonoid). Since they may be hydrolysed into their constituent anthocyanidins when treated with strong acids, they are sometimes known as proanthocyanidins.

[0079] The invention presents tanning polyphenolic extracts whose tannin and non-tannin composition is mostly determined by means of the specified chromatographic separation method (80.8% of the total compounds for Example 1; 93.1% of the total compounds in Example 2). In fact, the presence of non-tannins expressed as a percentage is justified on the basis of the monomeric units identified through the retention times and the UV-Vis spectra of the corresponding standards (gallic acid, ()-epicatechin, ()-epicatechin gallate). Said substances are not considered to be tannins since their molecular weight is less than 500.

Example 1

[0080] In the invention, the group of tannins or polyphenols with tanning capacity that have been referenced as condensed or catechinic (52.1%) include the following group of compounds: [0081] The set of tannins separated in the chromatogram with respect to the total surface area of the compounds identified includes chromophoric polymers of the compound Procyanidin B2, which represent a total of 23.9%. 2 tannins are detected that contain the same chromophore group as procyanidin B2, since they present the same UV-Vis spectrum. [0082] 4 catechinic tannins present in the commercial Mimosa tannin at 8.9% are detected, corroborated because they have the same retention times and UV-Vis spectra as the peaks corresponding to the chromatogram of the commercial sample used as a standard. [0083] 4 catechinic tannins present in the commercial Quebracho Tree tannin at 14.9% are detected, corroborated because they have the same retention times and UV-Vis spectra as the peaks corresponding to the chromatogram of the commercial sample used as a standard. [0084] 1 catechinic tannin present at 1.7% is detected which contain the same chromophore group as the compound (+)-catechin, since it presents the same UV-Vis spectrum. [0085] 1 catechinic tannin present at 2.7% is detected which contain the same chromophore group as the compound ()-epicatechin, since it presents the same UV-Vis spectrum.

[0086] In the invention, the group of tannins or polyphenols with tanning capacity that have been referenced as catechinic-hydrolysable (8.6%) includes the following group of compounds: [0087] 3 catechinic hydrolysable tannins present at 4.2% are detected which contain the same chromophore group as the compound ()-Epigallocatechin gallate, since they present the same UV-Vis spectrum. [0088] 2 catechinic hydrolysable tannins present at 4.4% are detected which contain the same chromophore group as the compound ()-Epicatechin gallate, since they present the same UV-Vis spectrum.

[0089] In the invention, the group of tannins or polyphenols with tanning capacity that have been referenced as hydrolysable (2.3%) includes the following group of compounds: [0090] 1 hydrolysable tannin present in the commercial Tara tannin at 1.1%, corroborated because it has the same retention time and UV-Vis spectrum as one of the peaks of the chromatogram of the commercial sample used as a standard. [0091] 1 hydrolysable tannin derived from gallic acid present at 1.3% that contains the same chromophore group as gallic acid, since they present the same UV-Vis spectrum.

[0092] In the invention, the group of non-tannins without tanning capacity that have been referenced as non-tannins (17.8%) includes the group of compounds identified as having the same retention times and UV-Vis spectra as the gallic acid, ()-epicatechin and ()-epicatechin gallate standards.

Example 2

[0093] In the invention, the group of tannins or polyphenols with tanning capacity that have been referenced as condensed or catechinic (79.2%) includes the following group of compounds: [0094] 1 catechinic tannin present in the commercial Quebracho Tree tannin at 79.0% is detected, corroborated because it has the same retention time and UV-Vis spectrum as one of the peaks of the chromatogram of the commercial sample used as a standard. [0095] 1 catechinic tannin present at 0.2% is detected that contains the same chromophore group as the compound ()-Epicatechin, since they present the same UV-Vis spectrum. [0096] 2 tannins present at 0.3% is detected that contain the same chromophore group as the compound Procyanidine A2, since they present the same UV-Vis spectrum.

[0097] In the invention, the group of tannins or polyphenols with tanning capacity that have been referenced as hydrolysable (1.1%) includes the following group of compounds: [0098] 2 hydrolysable tannins present in the commercial Tara tannin at 1.1% are detected, corroborated because they have the same retention times and UV-Vis spectra as two of the peaks of the chromatogram of the commercial sample used as a standard.

[0099] In the invention, the group of non-tannins without tanning capacity that have been referenced as non-tannins (12.8%) includes the group of compounds identified as having the same retention times and UV-Vis spectra as the gallic acid, ()-epicatechin and ellagic acid standards.

[0100] It is worth noting that the compounds identified in the tannin extract isolated from grape seeds in both Example 1 and Example 2 include a mixture of different types of tannins which comprises pure hydrolysable tannins, pure condensed tannins that includes procyanidins and non-tannins that include gallic acid and epicatechin.

[0101] Moreover, in one embodiment, the tannin extract isolated from grape seeds comprises a mixture of pure hydrolysable tannins, pure condensed tannins, tannins of the catechinic-hydrolysable type and non-tannins that include gallic acid.

[0102] The combination of the different types of tannins and the presence of gallic acid provide the extract with improved properties as compared to the properties of extracts in the prior art. Moreover, the extract may also contain a colouring agent in the UV region, which provides it with a multifunctionality that has not been previously described in the prior art.

[0103] Table 3 below lists the compounds identified in the tannin extract isolated from grape pomace according to reference Example 3:

TABLE-US-00004 % Surface area/ Total RT Surface surface Peak (min.) Identification area area 1 2.39 Catechinic tannin from 619,151 13.1 Quebracho Tree A 2 2.56 Catechinic tannin from 921,371 19.5 Quebracho Tree B 3 3.05 Catechinic tannin from 58,790 1.2 Quebracho Tree C 4 4.42 Catechinic tannin. 97,276 2.1 (Same chromophore as Procyanidin B2) 5 5.3 Catechinic tannin from 91,454 1.9 Mimosa 1. 6 6.44 Catechinic-hydrolysable 186,980 4.0 tannin. (Same chromophore as ()-Epicatechin gallate) 7 13.09 Catechinic tannin from 14,589 0.3 Mimosa 2 8 15.1 Gallic tannin from Tara 1 81,810 1.7 9 16.77 Catechinic tannin from 29,581 0.6 Mimosa 3 10 19.94 Ellagic Acid 45,466 1.0 11 33.03 Catechinic tannin from 244,369 5.2 Quebracho Tree 2

[0104] Table 4 is a summary table of the contents and types of tannins and non-tannins in the extracts obtained according to Examples 1 to 3:

TABLE-US-00005 Reference Example 1 Example 2 Example 3 % Surface area/ % Surface Total surface % Surface area/ area/Total Tannins area Total surface area surface area Catechinic 52.1 79.5 36.4 Hydrolysable 2.3 1.1 1.7 Catechinic- 8.6 0 4.0 Hydrolysable Total 63.0 80.6 42.1 % Surface area/ Total surface % Surface area/ % Total Non-Tannins area Total surface area surface area Gallic Acid 16.1 12.2 0 Ellagic Acid 0 0.4 1.0 Catechinic 1.7 0.2 7.7 Total 17.8 12.8 8.7 % Surface area/ Total surface % Surface area/ % Total Not identified area Total surface area Surface area Total 19.2 6.6 49.2

[0105] The percentage of catechinic tannins obtained from grape seeds is almost twice as much in one embodiment (Example 1) as in the prior art (reference Example 3) and more than twice as much (Example 2) as in the prior art (reference Example 3). The percentage of non-tannins that include gallic acid is similar in the two examples, 1 and 2, according to the invention, whereas they do not appear in reference Example 3. Although they do not have tanning power, the presence of non-tannins contributes to improving the solubility of tannins, increasing the penetration rate and separating the fibres from the skins. Therefore, an adequate balance between tannins and non-tannins in the extract isolated from grape seeds is responsible for the improved properties of the extract obtained according to the invention. Said balance is maintained in the extract obtained in Example 2, which further comprises the azo colouring agent.

UV-Vis Scan of the Extract Obtained in Example 2

[0106] The reference colouring agent used was Dark Brown CA.

[0107] The extract obtained according to Example 2 was compared to the reference colouring agent by means of a UV-Vis scan obtained between 350-800 nm using UV-Vis spectroscopy. The graph obtained is represented in FIG. 4.

[0108] In said FIG. 4, we may observe a single maximum corresponding to the reference colouring agent, Dark Brown CA, at 472.85 nm () and a maximum for the extract obtained according to Example 2 at 352.8 nm (), 2.70 A, which is in the UV region.

Total Chromatogram of the Extract Obtained in Example 2

[0109] A new HPLC analysis of the extract obtained in Example 2 was performed, and said chromatogram was obtained within the 200-800 nm range.

[0110] In the total chromatogram of the extract, only one high-magnitude peak (1) was identified, with a spectrum similar to that obtained by means of UV-Vis (which represents the overall absorption of all the polyphenols present in the sample). See FIG. 5.

[0111] Using the spectrum of the compound with RT=33.2 (1), it was determined that one of the polyphenolic compounds present in the tannin extract isolated from grape seeds presents significant absorption within the visible range. This fact enhances the degree of fixation that said compound undergoes as compared to skins tanned with the tannin extract isolated from grape seeds obtained according to Example 2.

Light Fastness

[0112] In order to determine the light fastness of the extracts obtained according to the present invention, a comparative assay of the artificial light fastness according to the IUF 402 standard was performed. See FIG. 6, where 1 is leather tanned with Mimosa extract, 2 is leather tanned with Chestnut Tree extract, 3 is leather tanned with Quebracho Tree extract, 4 is leather tanned with Tara extract, 5 is leather tanned with grape seed extract using sodium metabisulfite (Example 1), 6 is leather tanned with grape seed extract using urea (Example 2).

[0113] The results of the assay confirm that the skin tanned with the extract isolated from grape seeds according to the invention, Examples 1 and 2, with and without a colouring agent, respectively, presents improved light stability. It is surprising that the presence of a colouring agent in the extract (see column 6) does not interfere with the light stability of the tanned skin even after 36 hours have elapsed (last row). Therefore, the light fastness of the tannin extract isolated from grape seeds according to the invention presents an improved light fastness as compared to the extracts in the prior art.

Determination of Certain Aromatic Amines Derived from Azo Colouring Agents (ISO 17234:2015) in Skin Tanned Using the Tannin Extract Isolated from Grape Seeds According to Example 2

[0114] Compliance with the ISO 17234-1:2015 standard (Second edition 2015 Apr. 1) to determine the presence of certain banned aromatic amines in azo colouring agents, which makes them suitable or not for general use, is well-known to persons skilled in the art.

[0115] This assay analyses the presence of certain analytes, i.e. certain aromatic amines, with a limit of detection of 30 mg/kg, in a tanned sample according to said ISO 17234-1:2015 standard. As a result of said analysis, the authors of the present invention confirm that the colouring agent is free of banned aromatic amines, which are listed in Table 5. Below we list the banned aromatic amines according to said ISO 17234-1:2015 standard in Table 5:

TABLE-US-00006 BANNED AROMATIC AMINES RESULTS 4-aminobiphenyl (CAS No. 92-67-1) Not detected.sup.(1) benzidine (CAS No. 92-87-5) Not detected.sup.(1) 4-chloro-o-toluidine (CAS No. 95-69-2) Not detected.sup.(1) 2-naphthylamine (CAS No. 91-59-8) Not detected.sup.(1) o-amino-azotoluene (CAS No. 97-56-3) Not detected.sup.(1) 5-nitro-o-toluidine (CAS No. 99-55-8) Not detected.sup.(1) 4-chloroaniline (CAS No. 106-47-8) Not detected.sup.(1) 4-methoxy-m-phenylenediamine (CAS No. 615- Not detected.sup.(1) 05-4) 4,4-diaminophenylmethane (CAS No. 101-77-9) Not detected.sup.(1) 3,3-dichlorobenzidine (CAS No. 91-94-1) Not detected.sup.(1) 3,3-dimethoxybenzidine (CAS No. 119-90-4) Not detected.sup.(1) 3,3-dimethylbenzidine (CAS No. 119-93-7) Not detected.sup.(1) 4,4-methylene di-o-toluidine (CAS No. 838-88-0) Not detected.sup.(1) p-cresidine (CAS No. 120-71-8) Not detected.sup.(1) 4,4-methylene-bis-(2-chloroaniline) (CAS No. Not detected.sup.(1) 101-14-4) 4,4-oxydianiline (CAS No. 101-80-4) Not detected.sup.(1) 4-4-thiodianiline (CAS No. 139-65-1) Not detected.sup.(1) o-toluidine (CAS No. 95-53-4) Not detected.sup.(1) 4-methyl-m-phenylenediamine (CAS No. 95-80- Not detected.sup.(1) 7) 2,4,5-trimethylaniline (CAS No. 137-17-7) Not detected.sup.(1) o-anisidine (CAS No. 90-04-0) Not detected.sup.(1) 4-aminoazobenzene (CAS No. 60-09-3) Not detected.sup.(1) 2,4-xylidine (CAS No. 95-68-1) Not detected.sup.(1) 2,6-xylidine (CAS No. 27-62-7) Not detected.sup.(1) .sup.(1)No banned aromatic amines were detected in the azo colouring agent. Limit of detection <30 mg/kg.

Total Polyphenol Index (TPI)

[0116] The TPI, or total polyphenol index, is expressed as mg of gallic acid equivalents per gram of dry husk, is measured. This is a classic, robust spectrophotometric index for which the method described by Singleton et al. (Singleton, V. L.; Rossi, J. A., Jr., Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 1965, 16, (3), 144-58) was used. 5 ml of the sample diluted in milli-Q water, 100 l of the Folin-Ciocalteu reagent and 1 ml of a previously prepared 20 solution of sodium carbonate (20% Na.sub.2CO.sub.3 in Milli-Q H.sub.2O) were introduced into a test tube. The mixture was vortex-agitated and allowed to rest at room temperature in the dark for 30 min, sufficient time for the Folin-Ciocalteu reagent to be reduced by the phenolic compounds under alkaline conditions, developing a blue colour. The spectrophotometric measurement was performed at 760 nm. Milli-Q water was used as the measurement blank. The total polyphenol concentration was calculated by means of a calibration curve for gallic acid (y=0.0735x-0.0044: R.sup.2=0.9985), the concentration range whereof was (3-20 mg.Math.l-125). The results were expressed as gallic acid equivalents (mg.Math.l-1 GAE). The concentrations of the final extracts were expressed as mg of gallic acid per gram of dry husk (mg gallic acid/g dry weight).

[0117] Comparative data of the total polyphenol index provided by ES2443547 and the present invention are included below:

[0118] In ES2443547 (Example 1): [0119] Total Polyphenol Index 18.31 mg GAE/g dry husk/1.83% (g GAE/100 g dry husk) [0120] Gallic acid: 103 ppm (0.074 mg/g)/0.0074% (g GAE/100 g dry husk) [0121] Catechin: 1692 ppm (1.21 mg/g)/0.121% (g GAE/100 g dry husk) [0122] Epicatechin: 920 ppm (0.66 mg/g)/0.066% (g GAE/100 g dry husk)

[0123] The extract composition determined by means of the Folin-Ciocalteu method correspond to the 1.57%-2.43% interval of gallic acid.

[0124] Non information is provided regarding the type of polyphenolic compounds at the polymer level.

[0125] On the other hand, the polyphenolic extract is prepared using solvents different from water as the extraction media for the polyphenolic compounds.

[0126] The extraction yield according to the process disclosed by ES2443547 was 6.9% and the final extract only had obtained a total polyphenol content of 29% determined by means of High-Performance Liquid Chromatography (HPLC).

[0127] In the Present Invention (Example 1): [0128] Total Polyphenol Index 8.23% of total polyphenols/dry seed or 82.31 mg GAE/g dry seed. [0129] Gallic acid: 0.07 mg/g/0.0070% of gallic acid over dry seed; [0130] ()-Epicatechin: 0.027 mg/g dry seed/0.0027% over dry seed;

[0131] No presence of catechin is detected in the chromatogram of the sample Example 1. The total polyphenol content determined by means of High-Performance Liquid Chromatography (HPLC-DAD) is as high as, and higher than, 50% in a one-pot extraction, and in a single aqueous extraction step.

Characteristics of the Grape Seed Extract Obtained

[0132] The nature of the tannins is a mixture of the pure condensed type (catechinic) and the pure hydrolysable type (pyrogallic);

[0133] The extract comprises non-tannins that include gallic acid; their presence is believed to be responsible for a better antioxidant action and a better light fastness.

[0134] The extract is soluble in a cold aqueous medium;

[0135] It has applications as a tanning agent for hides and skins;

[0136] It has applications as an antioxidant agent in food, cosmetics and pharmaceutical products;

[0137] It has applications as a colouring agent in tanning, food, cosmetics and pharmaceutical products;

[0138] It presents improved light fastness, between the extract from Chestnut Tree 2 and the extract from Tara 4;

[0139] The colour of the tanned product is similar to that obtained with the Chestnut Tree extract.

[0140] Although we have referred to a specific embodiment of the invention, it is evident to persons skilled in the art that the process described is susceptible to numerous variations and modifications, and that all the details mentioned above may be replaced with other technically equivalent ones, without going beyond the scope of protection defined by the attached claims.