WO2008006923A1

WO2008006923A1 - Method for preparing a condiment based on grape fermentation, condiment thus obtained and use thereof to inhibit the development of mutagens and/or carcinogens

Info

Publication number: WO2008006923A1
Application number: PCT/ES2007/000420
Authority: WO
Inventors: Lluís PUIGNOU GARCÍA; Rosa Busquets Santacana
Original assignee: Universidad De Barcelona
Priority date: 2006-07-14
Filing date: 2007-07-12
Publication date: 2008-01-17
Also published as: ES2299364B1; ES2299364A1

Abstract

The invention relates to a condiment containing 1-50% v/v ethanol and 5-100 g/l of a dry extract, which can be obtained using a method comprising the following steps: (a) freeze-drying a grape ferment or an over-extracted grape ferment, such as to eliminate the solvents and most of the volatile compounds; and (b) transforming the freeze-dried product obtained in step (a) into the condiment with the required ethanol content and the required percentage of dried extract by adding one or more ethanolic or aqueous-ethanolic solutions for food consumption and, optionally, separating the insoluble particles by means of filtration. The invention also relates to a method for inhibiting the formation of mutagens and/or carcinogens during the cooking of a protein food by macerating the protein food in the condiment prior to cooking or by rubbing the protein food with the condiment during cooking.

Description

Condiment and its use to inhibit the development of mutagens and / or carcinogens

The present invention relates to the field of food safety. More specifically, the invention relates to the prevention of the formation of toxic compounds during the process of cooking protein foods through the use of a new seasoning, and also with the preparation of this seasoning.

STATE OF THE PREVIOUS TECHNIQUE

Humans are regularly exposed to exogenous contaminants from food, for example pesticide residues used in agriculture and industry. In addition, the risk of exposure to other toxic compounds that form in food during cooking processes is also considerable. The heat treatment that is needed to transform a raw food into an edible food, improves the sensory properties and prevents the contamination of microorganisms. However, it has been widely demonstrated that said heat treatment produces the formation of endogenous toxins such as polycyclic aromatic hydrocarbons ("polycyclic aromatic hydrocarbons", PAHs), nitrosamines ("nitrosamine compounds", NOCs), heterocyclic amines and acrylamide.

Heterocyclic amines are potent mutagens and potential human carcinogens that can induce tumors in animals after long-term exposure (cf. eg E. G: Snyderwine et al., Mutation Research 1997, vol. 376, pp. 203 -210). These compounds are metabolized by humans. Both heterocyclic amines and their metabolites can be found in human hair, in urine, and also forming adducts with hemoglobin and DNA, which is considered the first step for the initiation of cancer (cf. KH Dingley et al., Cancer Epidemiologv, Biomarkers & Prevention 1999, vol. 8, pp. 507-512).

Heterocyclic amines are products of the Maillard reaction (or browning or browning reaction, "browning" in English, which is the reaction of amino acid groups of amino acids, peptides or proteins with some carbonyl groups of sugars, and which gives training result of brown-yellowish pigments), which takes place when meat or fish is heated. The levels of heterocyclic amines depend on the type of meat or fish, cooking time, temperature and the ingredients used in the cooking process (cf. R. Busquéis et al., J. Chromatoqr. B 2004, vol. 802, pp. 79-86).

Several culinary practices and ingredients have been studied to find which favor the formation of smaller amounts of heterocyclic amines. It has been found that the use of olive oil, garlic, onion, and tomato, among other ingredients, reduces the production of heterocyclic amines.

The effect of using some marinades to reduce heterocyclic amines has been described in the scientific literature (cf. e.g. L.M. Tikkanen et al., Food and Chemical Toxicoloqy 1996, vol. 34, pp. 725-730). This document describes that the mutagenic activity of cooked chicken can be reduced by maceration with a mixture of several ingredients. According to this document, because a complex mixture of several compounds was used, it was not possible to evaluate which compounds inhibited the formation of mutagenicity in the treated chicken samples. It was assumed that this effect may be caused by the presence of hydroxyanisole butylate, which is an antioxidant, and by flavonoids found in the species used.

In another study, the levels of heterocyclic amines in macerated and non-macerated fried chicken were determined (cf. C: P: Salmon et al., Food and Chemical Toxico loq and 2006, vol. 34, pp. 484-492). Huatiao, which is a wine for cooking, is present in seven of the forty-two marinade mixtures used. The article says that the macerated chicken had considerably less amount of 2-amino-1-methyl-6-phenylimdazo [4,5- £>] pyridine (PhIP) than the unmacerated chicken. However, this effect is attributed to natural sugars, and some properties of marinades such as the prevention of water loss during cooking or water retention near the surface of the meat. No correlation is established between the presence of a wine component with the prevention of the formation of mutagenic and / or carcinogenic compounds. Thus, from what is known in the state of the art, the interest in finding new methods to prevent the formation of mutagenic and / or carcinogenic compounds during the cooking process is deduced.

EXPLANATION OF THE INVENTION

The inventors have found that the formation, during cooking, of harmful mutagenic and / or carcinogenic compounds can be inhibited by macerating the food for a specific period of time before cooking, or smearing the food during cooking with the new seasoning. The seasoning is prepared from fermentation products derived from grape extracts and shows several surprising properties, since it acts as a radical inhibitor and as a shield to prevent the reaction between mutagen precursors such as amino acids and creatinine. Thus, it has been found that the seasoning is a potent inhibitor of the formation of mutagenic heterocyclic amines, in cooked foods. The seasoning is prepared by a modified procedure with respect to the traditional winemaking procedure, which decisively influences its functionality.

Thus, one aspect of the present invention is to provide a process for the preparation of a condiment with an ethanol content of 1-50% v / v and a percentage of dry extract of 5-100 g / l, which comprises the steps of: (a) lyophilize a grape fermentation or an over-extracted grape fermentation, so that the solvents and most of the volatile compounds are separated; (b) transform the lyophilized product obtained in step (a) into the seasoning with the required content of ethanol and the required percentage of dry extract, by adding one or more ethanolic or aqueous-ethanol solutions for food consumption. Preferably, in step (b) the lyophilized product is dissolved by stirring and ultrasonic treatment. Optionally, the particles and some microorganisms of the seasoning can be removed by successive filtration, eg through inert filters of 1 μm. Filtration can be carried out under pressure in the presence of nitrogen to minimize the oxidation of the condiment components.

In a preferred embodiment, grape fermentation or over-extracted grape fermentation is obtained by a winemaking process comprising a cryogenic treatment. The cryogenic treatment includes the freeze the crushed grapes (skin, pips and must) below the freezing point of the mixture, in order to extract the maximum amount of potentially active components from the grapes. It is carried out at a temperature between -15 ⁰ C and -196 ⁰ C, and is carried out before mashing the must, nuggets and skins of the crushed grapes.

The term "winemaking procedure" means here the procedure for obtaining grape fermentation, regardless of the fact that the final product is wine, champagne or champagne. With the exclusion of cryogenic treatment and additional treatment with ethanol, the vinification procedure is based on traditional methods. Thus, the temperature and duration of the contact between skins, pips and must of the grapes, type of yeast cells, number of fermentations and the feeding time depend on the type of fermented grape desired and can be easily optimized by a person skilled in The matter

By "over-extracted grape fermentation" is meant here, a grape fermented to which an enriched ethanol solution is added. This enriched solution is obtained by treating the residues of the winemaking process (pips, skins and stems) with food grade ethanol to extract those substances that have not been extracted during the winemaking process.

For example, when the over-extracted grape fermentation comes from a process for obtaining wine, it can be obtained by applying a winemaking procedure based on traditional methods, including a more efficient extraction of the components of the crushed grapes, resulting in to obtain a clarified wine. Preferably, the grape varieties used are obtained in phytosanitary controlled crops. Generally, the winemaking process includes several stages and begins with the crushing of the grapes and ends when the clarified wine is obtained. Thus, after the harvest of the grapes, the stems and foliage of the fruits are separated. Stems are scrupulously removed except in cases where varieties have a low polyphenol content. Then, the grapes are crushed to produce must mixed with seeds and berry skins. This mixture is subjected to a cryogenic treatment that is carried out at a temperature generally between -15 ⁰ C and -196 ⁰ C. Then, the mixture is further macerated to extract the components of the seeds, skin and stems when they are present. The obtained mixture is pressed before or after the alcoholic fermentation depending on the type of grape used. If pressed before fermentation, the seeds, skins and stems when present are separated from the must. If the mixture is pressed after fermentation, the seeds, skins and stems when they are present are separated from the grape fermentation. Preferably, Saccaromvces strains are used in the fermentation process since they favor enrichment of the final product with the desired components such as amino acids.

The residues (pips, skins, stems) obtained before or after pressing the mixture can be treated with food grade ethanol to extract those substances that have not been extracted during the winemaking process. The enriched ethanol solution can be added to the grape fermented to give the over-extracted grape fermentation. The enriched ethanol solution can also be used to reconstitute the lyophilized semi-solid grape fermentation obtained.

As stated above for the elimination of solvents, grape fermentation or over-extracted grape fermentation are subjected to a lyophilization process. This lyophilization step allows to concentrate the matrix of desired components and eliminate volatile compounds such as aldehydes (for example acetaldehyde and formaldehyde), some alcohols, some esters and some organic acids. Some of these compounds can promote the formation of heterocyclic amines. The process is carried out below the triple point of the water (273.16 K and 6.105 kPa), thus, the water is converted directly into steam. Also, the low temperatures and the vacuum stop the biochemical processes that can alter the stability of the raw material.

Preferably, step (b) is carried out by the addition of food grade ethanol, staple, pomace brandy, wine distillate, brandy, ethanol enriched from the winemaking process, or mixtures thereof between them or with water. In a particular embodiment, the enriched ethanolic solution obtained in the extraction of the mixture of skins, nuggets and stems is used of the winemaking procedure. A different concentration of the seasoning can be obtained depending on the volume of ethanol / water added.

Another aspect of the present invention is to provide a condiment obtainable by the procedure described above, with an ethanol content of 1-50% v / v and a dry extract of 5-100 g / l. Preferably, the ethanol content is between 1 and 40% v / v. More preferably, the ethanol content is between 1 and 5% v / v. Also preferably, the proportion of dry extract is between 5 and 30 g / l.

Another aspect of the present invention is the use of the seasoning in the process of cooking a protein food. The use of the seasoning as an inhibitor of the development of mutagens and / or carcinogens, especially heterocyclic amines, in the cooking process of a protein food is also part of the invention.

The seasoning can be applied directly to the protein food portions before and / or during the cooking process. The heat treatment used can be any of the usual ones such as frying, roasting, browning, grilling, grilling, barbecue, braising, boiling or poaching, and any heat source based on convection, induction can also be used. or radiation

A further aspect of the present invention is to provide a method for inhibiting the formation of mutagens and / or carcinogens generated during the cooking of a protein food comprising mashing the food with the seasoning of the present invention between 10 minutes and 24 hours before cooking. . It is also an aspect of the present invention to provide a method for inhibiting the formation of mutagens and / or carcinogens generated during the cooking of a protein food comprising spreading the protein food with the seasoning of the present invention during cooking. Both methods of inhibiting the formation of mutagens are especially useful for inhibiting the formation of heterocyclic amines. Examples of these heterocyclic amines are 2-amino-1, 6-dimethylimidazo [4,5-¿)] pyridine (DMIP) and 2-amino-1-methyl-6-phenolimidazo [4,5 -ib] pyridine (PhIP). The seasoning of the present invention can be used in any cooking process and for cooking any type of protein food. Preferably, the protein food is meat or fish, including crustaceans and molluscs. More preferably, the meat is veal, lamb, pork, poultry and game animals. Preferably, the amount of seasoning is between 0.1 and 30 ml / g of food.

Compared with the methods known in the state of the art to inhibit the formation of mutagens and / or carcinogens during the cooking of a food, the present invention represents an important improvement in food safety. Through regular use of the seasoning, the health risk related to exposure to these genotoxic compounds can be reduced extraordinarily without changing the usual diet too much, introducing new and pleasant sensory properties very close to the natural components of the raw material. In addition, due to the intrinsic characteristics of condiments (eg high polyphenol content), additional benefits such as prevention of heart disease are associated with their consumption. Thus, the use of the seasoning of the present invention implies an increase in food safety and an improvement in dietary habits, providing a lower probability of suffering genetic alterations due to exposure to heterocyclic amines.

For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the 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. The summary of this application is incorporated here by reference. The following examples 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 the temperature profiles obtained during cooking chicken fillets. FIG. 2 shows the formation of DMIP in chicken cooked on the grill, before cooking, with the seasoning of the present invention at different proportions of ethanol / water, compared to chicken treated only with ethanol / water mixtures and chicken without any treatment before of cooking.

FIG. 3 shows a comparison of the formation of PhIP in grilled chicken, untreated versus treated with condiments based on wine extract dissolved in ethanol / water at different percentages and treated only with ethanol / water mixtures.

ADDITIONAL DESCRIPTION OF THE FIGURES

FIG. 1 describes the temperature profile obtained during the grilling process of the chicken fillet. The vertical axis "T ( ⁰ C)" indicates temperature at ⁰ C. The horizontal axis "t (min)" indicates the time in minutes. The cooking was carried out at 181, 1-212.4 ⁰ C (temperature range in the central part of the pan) for 4.5 minutes on each side of the fillet. The temperature was measured at different points in the pan and the fillet during cooking with K-type probes (see instruments and accessories). (1) Temperature measured at 1 mm below the surface of the meat that is not in contact with the pan at the beginning of the cooking process. (2) Temperature in the central and inner part of the fillet. (3) Temperature 1 mm below the surface of the meat that is in contact with the pan at the beginning of the cooking process. (4) Temperature of the surface of the pan in the most distant part of the heat source. (5) Temperature in the central part of the surface of the pan above the heat source.

FIG.2 compares the formation of DMIP in grilled chicken without having been treated with seasoning (represented as "N") and treated with condiments based on wine extract dissolved in ethanol / water at different percentages (represented as "C" to the liquid to treat food containing lyophilized grape fermented in ethanol / water medium). "R" indicates that the liquid for treating the food was a reference medium consisting of ethanol / water without lyophilized grape fermentation. The amounts of DMIP found (vertical axis) have been expressed in nanograms of DMIP per gram of cooked chicken "ng / g". FIG. 3 compares the formation of PhIP in untreated grilled chicken (represented as "N") and treated with condiments based on lyophilized grape fermentation dissolved in ethanol / water at different percentages (represented as "C" and corresponding to the liquid to treat food containing the lyophilized grape fermented in ethanol / water medium). "R" indicates that the liquid for treating the food was a reference medium consisting of ethanol / water without lyophilized grape fermentation. The concentrations of PhIP found (vertical axis) have been expressed in nanograms of PhIP per gram of cooked chicken "ng / g".

DETAILED DESCRIPTION OF THE SPECIFIC EMBODIMENTS

materials

The solvents and reagents used were of analytical quality or HPLC quality. Buffer solutions and standards were filtered through 0.45 µm nylon filters and samples, through 0.22 µm nylon filters before injection into the HPLC system. Ethyl acetate, acetonitrile, methanol, acetic acid, aqueous ammonia solution (32%), ammonium acetate and sodium hydroxide were purchased from Merck (Merck, Germany). Sodium carbonate and food grade ethanol were obtained from Panreac (Panreac, Spain). The following heterocyclic amines were purchased from Toronto Research Chemicals (Toronto, Canada): 2-amino-1, 6- dimethylimidazo [4,5-¿)] pyridine (DMIP), 2-amino-1-methyl- 6-phenylimidazo [4,5-

¿>] Pyridine (PhIP) and 2-amino-1-trideuterometyl-6-phenylimidazo [4,5-ιb] pyridine (D ₃ - PhIP) -Concentrated solutions of about 130 μg / g in methanol were prepared. The standards were prepared from the concentrated solutions. For the characterization of the seasoning, gallic acid (Merck, Germany), Folin-Ciocalteu reagent (Panreac, Spain), 2,2'-azino-bis (3- ethylbenzothiazolin-6-suifonic acid) (diamonic salt) (Sigma salt) (Sigma salt) was used , Germany), potassium persulfate (Aldrich, Germany) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Aldrich, Germany). Ninhydrin solution, amino acid standards and lithium citrate buffer solutions were obtained from Biochrom (Biochrom, England). In the purification of heterocyclic amines formed in cooked foods, Isolute ^® diatomaceous earth was used, which was obtained from International Sorbent Technology (IST, England) as well as solid phase extraction cartridges; PRS (500mg) and C-iβ (100mg) that were obtained from Vary (Vary, United States). The Visiprep ™ and Vísídry ™ vacuum systems from Supelco (Supelco, United States) were used for solid phase extraction procedures and for drying samples containing heterocyclic amines obtained from meat fillets.

Instruments and accessories

The condiments were obtained by means of a drying procedure carried out in a Telstar Liolabor lyophilizer model (Telstar, Spain), which had a maximum condenser capacity of 12 Kg / ice, 0.65 m ² of useful evaporation surface and 4 useful plates (45 cm x 36 cm).

The instruments used in the characterization of the seasoning and in the quantification of heterocyclic amines in chicken were: Unicam UV-Vis spectrophotometer model 4-100 (Thermo Elemental, Spain). Amino acid analyzer Pharmacia LKB Biotechnology model Alpha Plus (LKB, England). Alliance 2690 liquid chromatograph (Waters, United States) with quaternary pump and automatic injector. Mass spectrometer

LCQ ™ (ThermoFinnigan, United States) with ion trap analyzer. For the determination of heterocyclic amines a source of electrospray ionization was used.

The cooking temperature control was carried out with K-type thermocouple probes, Kapton model, for food measurements (CoIe Parmer, United States), whose working range was from -250 to 404 ⁰ C. The temperature monitoring was carried out by a PCMCIA card with six channels and a TC6 Thermocouple PC cad 2.2 Normadics program. Inc. (Colé Parmer, United States). The samples were crushed by a crusher

Microtron®MB 550 (Kinematica, Switzerland) and Uϊtra-turrax ^® T ~ 25 basic homogenizer provided with a disperser arm model 18-G (IKA instruments, Germany). The heat source used for cooking the meat was a Teka IZ622 Model 412-10282 ceramic hob (Teka Industrial, Spain). Seasoning Preparation

To obtain the condiments, the grape fermentation obtained by the winemaking procedure described above from grapes of the Merlot variety harvested in the DO Penedés, vintage 2004, was distributed in polypropylene or amber glass containers (30-180 ml) . This initial product was lyophilized under the following conditions: chamber and pump vacuum 0.22 mm Hg (29.3 Pa) and 0.13 mm Hg (17.3 Pa), respectively. The condenser and plate temperatures at the beginning and end of the lyophilization cycle were -55 ⁰ C and 58 ⁰ C (condenser), and - 50 ⁰ C and 40 ⁰ C (plates). The duration of the lyophilization cycle was 48 hours. Applying the described conditions a lyophilizate of grape fermentation was obtained with a yield of 28 g / l. The lyophilized samples were reconstituted with a hydroorganic mixture consisting of analytical grade water and food grade ethanol. The proportion of ethanol in the applications tested was 0, 5, 13, 30 and 50%. The product obtained was stable and colored depending on the amount of natural components. However, in order to avoid undissolved particles, the product was treated with ultrasound for a few minutes and filtered through membranes with a pore size of 1 μm. The composition of the condiments tested, expressed in terms of dry extract (lyophilized) per liter of seasoning, comprised the range of 5-100 g / l. A reddish and transparent liquid was obtained, with pleasant flavor and aroma and closely related to its primary components.

Seasoning characterization:

1. Determination of the total polyphenol content

The total polyphenol content was carried out following the spectrophotometric method called Folin-Ciocalteu (cf. Official Journal of the European Communities. 1990 No 2676/90 of September 17, 1990). Said method consists of an oxidation of the phenols of the wine matrix by means of the phosphomolibed and phosphotungstic acids present in the reagent called Folin-Ciocalteu. The quantification was performed by external calibration using 6 concentration levels. Gallic acid was chosen as the calibration standard. The concentration range used was 0 to 8 ppm gallic acid (^ = O, 999). For the present test, 0.25 ml of appropriately diluted wine sample or gallic acid standard, 12.5 ml of ultrapure water, 1.25 ml of Folin-Ciocalteu reagent and 5 ml of a 20% carbonate solution were mixed. sodium, all this is diluted to a total volume of 25 ml with ultrapure water. These solutions were homogenized and the reaction took place for 30 minutes. After the reaction time, absorbance readings were made at 750 nm on a Unicam UV-Vis model 4-100 spectrophotometer. The determinations were made in triplicate. As an example, the following table shows the content of total polyphenols in the different samples: raw material (Merlot wine); seasoning C, with 13% ethanol in water and 2.5% dry extract; seasoning C (x2), with 13% ethanol in water and 5% dry extract; seasoning C (x2), subjected to three freezing cycles (-18 ⁰ C) - defrosting (25 ⁰ C). Table 1 shows the antioxidant activity values for the different samples. The wine studied has been made with grapes of the Merlot variety from the 2004 harvest in D. O Penedés. Polyphenols were determined by mass of dry extract. The results have been expressed in mg equivalent of gallic acid per gram of dry extract obtained after the lyophilization process.

Table 1 :

Seasoning

Commodity Condiment Condiment C (x2) +

Total polyphenols premium C C (x2) heat treatment

Half

(mg equivalent

87.8 82.0 80.8 82.1 gallic acid / g dry extract) Standard deviation

2.6 1.5 1, 3 0.3 relative (%) n = 3 determinations

The results obtained show that the lyophilization process and the new reconstitution medium did not significantly affect the polyphenolic content. It was also shown that polyphenols were conserved in the freeze-thaw cycles, which is a valuable stability data for the conservation of the products obtained.

2. Determination of antioxidant activity

The test for the determination of the equivalent antioxidant activity in Trolox based on the use of persulfate (TEAC) was performed to measure the antioxidant activity of the condiments and test the stability of said parameter in the wine matrices subjected to temperature cycles (freezing and defrosting) (cf. R. Re et al., Free Radie. Biol. Med. 1999, vol. 26, pp. 1231-1237). The present method comprises the generation of the colored radical cation of 2,2'-azino-bis (3-ethylbenzothiazolin-6-sulfonic acid) (ABTS) using potassium persulfate as the oxidizing agent. The ABTS ⁺ radical cation could be reduced by hydrophobic or hydrophilic antioxidant compounds, present in the medium, which would cause an inhibition of absorbance. The quantification of the antioxidant activity was performed using an external calibration line of 6 concentration levels. For quantification, recently prepared calibration standards of Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid were used. The working range was 0-1500 μM Trolox (1 ^ = 0.997). To generate the ABTS radical cation, ABTS and sodium persulfate were dissolved in concentrations of 7 mM and 2.45 mM, respectively. The reaction took place at room temperature, in the dark and under nitrogen atmosphere for 12-16 h. The solution of the ABTS radical cation was diluted with ethanol until obtaining an absorbance of 0.7 to 734 nm at 3O ⁰ C. The samples were diluted with ethanol until obtaining an inhibition of the absorbance comprised between 20-80% of the diluted solution of the ABTS radical cation.

The absorbance inhibition assays were performed in 3 ml (1cm wide) quartz cuvettes, as specified below. The initial absorbance of 2.970 .mu.l of diluted solution of radical cation ABTS was measured at 30 ⁰ C and was again made the measurement 5 minutes after adding 30 .mu.l of diluted sample or standard Trolox to the solution of radical cation ABTS appropriately diluted The mixture was stirred and kept for 5 minutes in a bath at 30 ⁰ C. The reaction time selected was 5 minutes because it was previously proven that the suppression of absorbance remained stable at this time. The determinations were made in quadruplicate. The antioxidant activity is shown below, expressed in Trolox equivalents per gram of dry extract obtained after a lyophilization process, found in different matrices obtained from wine and reconstituted with an ethanol / water mixture: raw material (Merlot wine); C seasoning, with 13% ethanol and 2.5% dry extract; C (x2) seasoning, with 13% ethanol and 5% dry extract; C (x2) seasoning subjected to 3 freezing cycles (-18 ° C) - defrosting (25 ⁰ C). The solution used to reconstitute the different amounts of dry extract was 13% ethanol in water. The antioxidant activity of the different matrices, detailed in Tables 2 and 3. have been expressed in mmol Trolox / I and in mmol of Trolox / g of dry extract (obtained after lyophilization process).

Table 2

Activity Subject Condiment Condiment Condiment C (x2) + raw antioxidant C C (x2) heat treatment

Half

23.7 22.0 40.5 37.7 mmol Trolox / L

Relative standard deviation 13.9 10.1 8.2 11, 1%) n = 4 determinations

Table 3

Half millimoles Trolox / g 0.84 0.78 0.75 0.70 dry extract

Relative standard deviation 13.9 10.1 8.2 11, 1%) n = 4 determinations It was found that the antioxidant activity was not significantly altered in all the samples analyzed (raw material and condiments). In addition, it should be noted that the antioxidant activity did not vary due to thermal freeze-thaw cycles, which is an important stability data related to the preservation of the products.

3. Determination of free amino acids and total amino acids

The amino acid determination was performed following the method developed by Moore et al. (cf. S. Moore et al., Anal. Chem. 1958 vol. 30, pp. 1185-1190). The identification and quantification of amino acids has been based on chromatographic separation in a stationary phase ion exchange column polystyrene-divinylbenzene with sulfonate groups that act as strong cation exchangers. The dimensions of the column were 4.6x 200 mm and 5μm in particle size (Biochrom, England). The elution of the amino acids from the column was performed with lithium citrate buffer solutions of increasing pH throughout the elution program. The temperature was conveniently controlled. The eluted amino acids reacted in continuous flow with ninhydrin at 135 ⁰ C. When the eluted compound was an amino acid, the derived compound absorbed at 570 nm. When the eluted compound was imino acid, the derived compound absorbed at 440 nm. In general, the detection limits (signal to noise ratio = 3) were found around 1 nmol in the conditions presented. The contents of free amino acids found in the condiments have been determined in triplicate, using the method of analysis described and cited above, and quantified by external calibration using norleucine as an internal standard. All the seasonings presented had the same amino acid profile since they originated in the same raw material and were produced following the same winemaking process, therefore, amino acid concentrations have been expressed for a generic seasoning. Table 4 shows the content of free amino acids expressed per gram of dry extract. Low resolution was obtained between the chromatographic peaks of proline and glycine, therefore in Table 4 the concentration corresponding to the sum of the two signals is presented. Additionally, the determination of total amino acids in the same seasoning has been performed. For this, 4 ml of seasoning was hydrolyzed with 4 ml of 12 M hydrochloric acid for 16 h at 112 ⁰ C. evaporated the solvent in vacuo and reconstituted with 25 ml of water. The determination was made in triplicate. Norleucine was used as an internal injection standard. The quantification was performed by external calibration. Table 5 shows the concentrations of the total amino acids found in the seasoning expressed by mass of dry extract.

Table 4

Half

Deviation

Amino acids (Aa) v 'μmol Aa / g standard ammonium relative extract (%) dry

Taurine 2.6 30.5

Aspartic acid 2.5 6.9

4-Hydroxypropylene 1, 8 1, 5

Threonine 1, 1 24.9

Serine 1, 4 11, 4

Glutamic Acid 6.0 1, 6

Glutamine 0.6 29.9

Prol¡na + glycine 209.6 41, 3

Alanine 6.7 1, 1

Valine 1, 1 5.0

Cysteine 1, 1 9.7

Methionine 0.6 22.0

Isoleucine 0.7 6.0

Leucine 1, 4,6,6

Tyrosine 0.5 5.7

Phenylalanine 1.0 6.6

Y-aminobutyric acid 2.8 0.2

Ammonium 12.4 15.7

Ornithine 0.9 0.5

Lysine 2.4 8.4

Histidine 1, 2.6

Tryptophan 0.2 5.9

Arginine 1, 9 3.5 n = 3 determinations Table 5

Half

Deviation μmol Aa / g

Amino acids (Aa) standard relative extract dry (%)

Cysteic acid 6.7 7.3

Bullfighting 4.0 9.7

Aspartic acid 7.9 1, 8

4-Hydroxyproline 4.3 17.2

Threonine 5.6 4.6

Serine 8.1 4.2

Glutamic Acid 5.5 10.0

Proline 203.2 28.0

Glycine 14.7 2.7

Alanine 12.2 6.8

Valine 4.1 7.0

Cysteine 1, 0 34.3

Methionine 0.5 40.7

Isoleucine 3.0 5.3

Leucine 3.1 6.6

Tyrosine 1, 7 4.9

Phenylalanine 3.3 17.1

Ammonium 30.4 7.0

Usina 4.1 5.6

Histidine 2.0 12.6

Tryptophan 0.25 57.7

Arginine 2.7 13.3 n = 3 determinations

4. Determination of acetaldehyde

The quantification of acetaldehyde was carried out following the method developed by Mamede et al. (cf. ME O, Mamede et al., Food Chem. 2006 vol. 96, pp. 586-590) based on trapping volatile compounds in a Purge and trap system / dynamic head space system, equipped with a Tenax trap, and gas chromatography coupled to mass spectrometry for separation and determination. An HP-INNOWax capillary column (Hewlett-Packard, USA) 30 mx 0.25 mm I. D coated with crosslinked polyethylene glycol was used.

Table 6

Acetaldehyde Wine Lyophilized wine mg / l 100 not detectable Standard deviation 12.7 relative (%) n = 3 determinations

Seasoning application in a frying process (without the use of oil or fat) of chicken breast.

The effect of the seasoning was tested on chicken breast because this is the type of meat where the highest levels of PhIP and DMIP heterocyclic amines have been found. First, the chicken breasts were filleted, using a knife and scalpel. The fillets were cut following previously established standard dimensions to be able to control and ensure the reproducibility of heat and matter transport. In the cooking tests, the dimensions of the fillets adopted have been: 6 cm x 5 cm and 5 mm thick. The application of the seasoning to the meat, prior to cooking, was done by pouring 5 ml of seasoning on each chicken fillet. For each experimental condition tested, two chicken fillets were used, using a total of 10 ml of seasoning. In order to keep the meat in contact with the seasoning, the fillets were humidified with the seasoning in a plastic bag (food grade polypropylene) provided with a tight seal. The air in the bag was displaced by pressure to ensure a homogeneous impregnation of the food. In this way, a thin reddish film was observed covering the surface of the meat. The reddish color mentioned was due to the presence of tannins, which are natural pigments that mainly originate from the seeds and skin of the grapes, and which were present in the seasoning used in the example specific. Although the contact time between the meat and the seasoning can be varied, in the specific embodiment 1 hour was chosen. It should be specified that, in the case of seasonings rich in tannins, the contact time may influence the coloration acquired by the meat after the cooking process. Additionally, the contact time and the composition of the seasoning used have an influence on the organoleptic properties of the cooked meat, in all cases acquiring aromas and flavor typical of the seasoning used.

Grilled chicken breast was obtained under controlled conditions of time and temperature. The formation of the genotoxic compounds studied is highly dependent on the temperature reached in the fillet and the cooking time, so it was very important to control these variables. In this way, it could be demonstrated that the decrease in the generation of heterocyclic amines studied in fillets cooked under the same conditions, was due only to the use of the seasoning.

The exhaustive temperature control was carried out using Kapton model K type thermocouple probes for food measurements (CoIe Parmer, USA). The thermocouples were calibrated just before each test by performing boiling analytical quality water temperature measurements (100 ° C). Once calibrated, the thermocouples were inserted into the raw chicken breast fillets; Two of the probes were located 1mm below each of the sides of the fillet that come into direct contact with the heat source during (FIG. 1, thermocouples 1 and 3). This is the area where heterocyclic amines are formed in a greater amount. The position of these probes is decisive to know the maximum temperature to which the meat is subjected. The third thermocouple was inserted in the central-inner part of the fillet (FIG. 1, thermocouple 2). Finally, the fourth and fifth thermocouples monitored the temperatures in the part furthest from the center and in the central part of the surface of the pan (FIG. 1, thermocouples 4 and 5) to record the maximum temperature range where the Ia took place meat cooking. The heat source was a ceramic hob that heated by convection. The dimensions of the pan where the griddle cooking tests were carried out were 27 x 27 cm. This pan was made of aluminum covered by a non-stick teflon tricapa. This way you managed to perform a cooking process commonly used and with precise control of the temperature.

The cooking tests were started when the central and exterior temperatures of the pan were constant. In the specific example, we worked at power 9 of the ceramic hob convection plate (maximum power). Under these conditions, the central and exterior temperatures of the pan (starting at 25 ° C) reached a constant temperature after 20 minutes of maintaining the pan over the heat source. The tests were carried out in triplicate. The cooking time control was performed using a conventional stopwatch. In the specific embodiment presented, the cooking time was 9 minutes (4.5 min on each side of the steak). The central temperature of the pan was maintained 181, 1-212.4 ⁰ C and the outside temperature of the pan was maintained within the range 157.0-170.4 ⁰ C throughout the cooking. No type of oil or fat was used for cooking the food or any additive. In FIG. 1 the temperature profiles obtained in the specific embodiment are shown.

In parallel, a control trial was performed without using the seasoning of the present invention and another control trial replacing the seasoning with a mixture of ethanol / water in the same proportion as in the seasoning.

Purification of heterocyclic amines

Sample treatment: The cooked meat fillets were crushed with a common crusher. The crushed meat was stored in closed polypropylene containers in a freezer (-18 ⁰ C) until analysis. Extraction, purification and preconcentration of heterocyclic amines was performed using the solid phase extraction method developed by F. Toribio et al. (cf. F. Toribio et al. J. Chromatoqr. A 1999, vol. 836, pp. 223-233). For each determination, 3 g of meat cooked grilled and crushed were used. To these were added 10 ml of 1 M sodium hydroxide and the mixture was homogenized with a U! Tra-turrax ^® T-25 basic arm. The homogeneous crushed sample was dispersed in Isolute ^® diatomaceous earth (13g), which is a very porous and inert support that allows increasing the surface of the sample in contact with the extracting solvent in the next liquid-liquid extraction stage. 75 ml of ethyl acetate were necessary of to pack the dispersed sample and to elute the analytes. Heterocyclic amines were retained in a PRS ion exchange cartridge (500mg) previously conditioned with 0.1M hydrochloric acid (5ml), water (1OmI) and methanol (5ml). Subsequently the cartridge was washed with methanol: water (4: 6, 15ml) and water (2ml). Elution of heterocyclic amines was performed with 0.5 M ammonium acetate at pH = 8.5 (2OmI) toward a cartridge C _E (100mg) that had been conditioned with MeOH (5ml) and water (5ml) . The cartridge was washed with water (5ml). Finally, the analytes were eluted with a MeOH: NH ₃ 9: 1 solution (0.8 ml). The purified sample was evaporated to dryness with a gentle stream of nitrogen and reconstituted with 100 µl of a methanolic solution containing D ₃ -PhIP as internal standard. The solid phase extraction and evaporation of the samples has been carried out using vacuum systems. The purified samples were filtered through 0.22 μm nylon filters before being injected into the chromatographic system.

Quantification of heterocyclic amines

The analysis of heterocyclic amines in foods is hindered by the low levels of concentration in which they are formed and by the complexity of the matrix of the sample. However, the coupling of the liquid chromatography to the mass spectrometry allows a reliable quantification of the analytes after having performed the treatment of the previously described sample. To quantify heterocyclic amines in grilled chicken of this specific embodiment, standard addition has been used. 60 µl of a methanolic solution of the analytes at concentration 1.8 µg / g were added to a purified sample in parallel with four other non-added samples. The chosen contact time was 1 h. The relationship between the amount of amines found with the amount of added amines allowed to obtain the recovery value of each analyte in each of the sample treatments. The recovery values served to convert the amount of heterocyclic amines found in the purified samples into the concentration of these in the cooked meat. For the quantification of the analytes in the purified samples, the ratio of areas between the analytes and the internal standard became a ratio of concentrations between the analytes and the concentration of the internal standard by means of the calibration line. Known the concentration of internal standard with which the purified samples and evaporated to dryness, 0.1669 μg / g D ₃ -PhIP, the concentration of the analytes in the purified samples was obtained. These concentrations were corrected by the estimated recovery value since the heterocyclic amines were not fully recovered throughout the purification procedure, and were expressed in terms of the amount of each heterocyclic amine per gram of chicken fillet (treated with the seasoning) and fried on the grill.

The separation and detection methods applied in the specific embodiment are presented below. These methods are based on previous work carried out by Barceló-Barrachina et al. (cf. J. Chromatogr. B, 2004, vol. 802 pp. 45-59; J. Chromatoαr. A .. 2004, vol. 1023, pp. 67-78).

Analytical Conditions:

Heterocyclic aromatic amines were separated by reversed phase liquid chromatography using a Symmetry ^® C ₈ column (5μm, 2.1x150mm). An Alliance ^® 2690 quaternary pump (Waters, USA) was used to carry out the separation. The separation took place at a flow rate of 0.3 ml / min. The constituents of the mobile phase were: acetonitrile (A) and a 3OmM acetic acid-ammonium acetate buffer solution adjusted to pH = 4.5 with ammonia (B). The elution gradient was: 0-0.5 min, 5% A in B; 0.5-15 min, 5-20% A in B; 15-18 min, 20-60% A in B; 18-24 min, 60% A in B; 24-30 min 60-5% A in B: 30-35 min 5% A in B. The injection volume was 5 μl.

The analyte detection and identification system has been a mass spectrometer with an LCQ ™ ion trap analyzer (ThermoFinnigan, San José, USA). The optimal working conditions were: spray voltage, 3 kV; nebulizer gas, 90 au; auxiliary gas, 60 au; heated capillary temperature, 280 ⁰ C; capillary voltage, 30.5 V; lens tube voltage, 13 V; 1.5 m / z insulation band; activation time 30 ms; activation parameter Q (AQ) ", 0.45. The acquisition mode was a product ion scan. The collision energies and the m / z scan intervals are shown in Table 7. Table 8 specifies the assignments of the ions obtained in the MS / MS spectrum operating in mode of Ion scan product. These data which have served to identify the analytes.

Table 7. MS / MS acquisition parameters used in the quantification.

0-9.90 DMIP 163 46 148 [140-170]

17.50- PhIP 225 51 210 [200-230]

21, 20

D ₃ -PhIP 228 51 210 [200-235]

Table 8. Assignment of the main ions obtained in MS / MS (product ion scavenging) from the heterocyclic amines studied.

Ion Precursor Ions Product

Compound m / z Assignment m / z Assignment (Abundance,%)

DMIP 163 [M + H] ⁺ 148 (100) [M + H-CH ₃ r 146 (40) [M + H-NH ₃ ] ⁺

PhIP 225 [M + H] ⁺ 210 (100) [M + H-CH ₃ ] ^{+ "} 208 (20) [M + H-NH ₃ ] ⁺

D ₃ -PhIP 228 [M + H] ⁺ 211 (20) [M + H-NH ₃ ] ⁺ 210 (100) [M + H-CD ₃ ] ^{+ '}

The results obtained in the control tests and in the tests using seasoning are shown in Tables 9 and 10. In the specific embodiment, the following seasonings have been used: seasoning A, with 0% ethanol in Water; seasoning B, with 5% ethanol in water; seasoning C, with 13% ethane! in water; seasoning D, with 30% ethanol in water; seasoning E, with 50% ethanol in water. All these condiments contained 2.5% of dry extract obtained by lyophilization of the fermented grape. The content of heterocyclic amines have been expressed in terms of ng of analytes per gram of grilled chicken, treated or not treated with seasoning before grilling. The standard deviation obtained from the independent analyzes is also indicated. The contents of PhIP and DMIP found in the grilled chicken that had been previously treated with seasoning were compared with the natural formation of these compounds when the chicken fillets were cooked on the plate without using seasoning. In parallel, complementary experiments were carried out as a control by treating chicken fillets with ethanol / water mixtures of a composition coinciding with that of the condiments studied in the specific embodiment. In this way, the effect of condiments on the formation of heterocyclic amines could be tested, ruling out the effect of the hydroalcoholic matrix on the inhibition of the formation of heterocyclic amines.

As an example, the most significant results obtained for DMIP and PhIP amines are shown in Tables 9, 10 and in Figures 2 and 3.

Table 9. Effect of the different condiments, including control tests, on the formation of DMIP during the cooking of the chicken fillets.

ng DMIP / g grilled chicken ± s Composition of

. . treated with solvent mixture treated with control seasoning (no seasoning)

11.9 ± 2.1 * 11.9 ± 2.1 *

100% H ₂ O 4.3 ± 3.2 14.0 ± 2.0

5% EtOH: 95% H ₂ O 2.6 ± 0.8 16.0 ± 2.2

13% EtOH: 87% H ₂ O 5.9 ± 4.5 10.2 ± 1.4

30% EtOH: 70% H ₂ O 12.6 ± 1, 5 9.9 ± 0.6 * values obtained after cooking the food without any previous treatment. Table 10. Effect of different seasonings on the formation of PhIP during the cooking of chicken fillets.

ng PhIP / g grilled chicken ± s

Composition of the treated with solvent mixture treated with control seasoning (without seasoning)

34.1 ± 5.0 * 34.1 ± 5.0 *

100% H ₂ O 17.1 ± 7.4 55.0 ± 6.0

5% EtOH: 95% H ₂ O 13.7 ± 2.0 61, 4 ± 9.2

13% EtOH: 87% H ₂ O 31, 5 ± 7.2 64.8 ± 7.1

50% EtOH: 50% H ₂ Q 35.0 ± 13.0 70.1 ± 5.9 * values obtained after cooking the food without any previous treatment.

The use of the condiments presented in the specific embodiment has a notable effect on the formation of PhIP and DMIP. It has been observed that the reconstitution solvent affected the formation of both compounds since during the cooking process it acts as a vehicular medium of potential precursors of the heterocyclic amines. Those condiments with low ethanol content have more effectively inhibited the formation of DMIP and PhIP. DMIP was reduced by up to 78% using the seasoning with 5% ethanol in water and 2.5% dry extract (lyophilized from fermented grapes), and in the case of PhIP, up to 60% with the same type of seasoning, using contact times of one hour.

The treatment of the meat with the hydroorganic medium of the same solvent composition as in the condiments (control test using ethanol / water solutions) did not cause a significant inhibitory effect. Therefore, the use of condiments is primarily responsible for minimizing the formation of the culinary toxins studied.

Claims

1. Procedure for the preparation of a condiment with an ethanol content of 1-50% v / v and a percentage of dry extract of 5-100 g / l, comprising the steps of:

(a) lyophilize a grape fermentation or an over-extracted grape fermentation, so that the solvents and most of the volatile compounds are separated;

(b) transform the lyophilized product obtained in step (a) into the seasoning with the required content of ethanol and the required percentage of dry extract, by adding one or more ethanolic or aqueous-ethanol solutions for food consumption, and optionally separating insoluble particles by filtration.

2. Preparation process according to claim 1, wherein grape fermentation or over-extracted grape fermentation is obtained by a winemaking process comprising a cryogenic treatment being carried out at a temperature between -15 ⁰ C and -196 ⁰ C , said treatment is performed before the maceration of the must, nuggets and skin of the crushed grape.

3. Preparation process according to claim 2, wherein the over-extracted grape fermentation is obtained by adding an ethanol enriched solution to the grape fermentation.

4. Method according to claim 3, wherein the ethanol enriched solution is obtained by treating the residues of the vinification process with food grade ethanol.

5. Preparation process according to any one of claims 1-4, wherein step (b) is carried out by the addition of food grade ethanol, staple, pomace brandy, wine distillate, brandy, ethanol enriched from the winemaking process , or mixtures thereof or mixtures with water.

6. Seasoning obtainable by the method of any of claims 1-5.

7. Seasoning according to claim 6, wherein the ethanol content is between 1% and 40% v / v.

8. Seasoning according to claim 7, wherein the ethanol content is between 1% and 5% v / v.

9. Seasoning according to any of claims 6-8, wherein the proportion of dry extract is between 5 g / l and 30 g / l.

10. Use of the seasoning defined in any of claims 6-9, as an inhibitor of the development of mutagens and / or carcinogens in the cooking process of a protein food.

11. Use according to claim 10, wherein the mutagens and / or carcinogens are heterocyclic amines.

12. Use of the seasoning defined in any of claims 6-9, in the cooking of protein foods.

13. Method for inhibiting the development of mutagens and / or carcinogens that are generated during the cooking of a protein food, which comprises mashing the food with the seasoning defined in any of claims 6-9 between 10 minutes and 24 hours before Ia cooking.

14. Method for inhibiting the development of mutagens and / or carcinogens that are generated during the cooking of a protein food, which comprises spreading the protein food with the seasoning of any of claims 6-9 during cooking.

15. Method according to any of claims 13-14, wherein the mutagens and / or carcinogens are heterocyclic amines.

16. Method according to any of claims 13-15, wherein the protein food is selected from meat and fish.

17. Method according to claim 16, wherein the meat is selected from veal, lamb, pork, poultry and game birds.

18. Method according to any of claims 13-17, wherein the proportion of seasoning is between 0.1 and 30 ml of seasoning per g of food.