ES2378601A1 - Employment of an avocado seed extract in the production of foodstuffs. (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Use of an avocado seed extract in the preparation of food products. The invention relates to a process for obtaining an extract with antioxidant properties from the seed of the avocado (fruit of persea americana mill.) comprising the steps: (a) extraction from the fruit crushed with a solvent selected from water, ethanol or mixtures thereof and obtaining a supernatant (b) concentration of the supernatant resulting from step (a) by vacuum distillation. The invention also relates to said extract and to food compositions such as for example meat products containing it. (Machine-translation by Google Translate, not legally binding)

Description

EMPLOYMENT OF AN AVOCADO SEED EXTRACT IN THE PREPARATION OF FOOD PRODUCTS

FIELD OF THE INVENTION

The present invention falls within the food sector and more particularly within the sector of antioxidant additives for food in general. Specifically, the invention relates to a process for obtaining an extract rich in natural antioxidants from the seed of the avocado fruit, to the extract with antioxidant properties obtained from the seed of the avocado fruit (Persea americana Mill.) And its Use in the preparation of food compositions such as meat products that have improved properties in terms of their shelf life and sensory characteristics.

BACKGROUND OF THE INVENTION

Synthetic antioxidants have been used for years as food additives to prolong the shelf life, especially of fats and products with a high lipid content, delaying their oxidation process. However, its use is increasingly restricted due to the potential risks and toxicity of these substances and the rejection of consumers (Nawar, 1996). Thus, today the trend of the food industry is focused on replacing these synthetic antioxidants with naturally occurring antioxidants, especially those from plants. A few years ago, the term antioxidant of natural origin was mainly associated with compounds such as ascorbic acid - vitamin C -, vitamin E and eventually with vitamin A. However, in recent years numerous investigations have expanded the number of species Chemicals with antioxidant activity, among which the so-called "polyphenols" stand out especially due to the presence in their chemical structure of one or more phenolic rings (Heinonen, 2007).

These phenolic compounds and, particularly those present in fruits and vegetables, have gained interest among consumers and in the scientific community because in addition to their use as antioxidants in food, numerous trials have found evidence that these compounds present, under certain conditions , antibacterial properties, as well as beneficial effects against certain cardiovascular pathologies or against certain types of cancer.

However, there are also a good number of publications and scientific trials in which the use of polyphenols as antioxidant agents did not report beneficial results or even showed themselves as pro-oxidizing agents, that is, they promote oxidation, in oxidative stability tests ( Estevez et al. 2008; Salminen et al. 2008). At present it is known that this apparent contradiction of the results obtained can be attributed to several factors that must always be taken into account when considering the use of these compounds as an antioxidant in food:

Under the term "polyphenols" a wide variety of phenolic compounds are included that are classified into: 1.-) flavonoids, formed by two aromatic rings joined by an oxygenated heterocycle, and among which are flavonols, flavones, isoflavones, anthocyanins , proanthocyanidins, flavanones, etc. and 2.-) non-flavonoids, commonly called phenolic acids, which contain an aromatic ring with different functional groups. The distribution of these compounds in plants, their polarity and solubility, as well as their antioxidant activity varies significantly among the different chemical families, so it is required a broad knowledge of what phenolic compounds and in what concentration are present in a given extract, in order to predict its potential use as an antioxidant.

In general, phenolic compounds are attributed reducing characteristics as they act as electron donor agents demonstrating their potential as free radical neutralizers, and thus acting as antioxidants. However, in more complex systems such as in a meat product, phenolic compounds are integrated into much more complex redox reactions and their reducing potential is dependent on many factors such as pH, concentration or the presence of other compounds such as metals or other antioxidants, the same compound being able to act as an antioxidant or pro-oxidant depending on the factors described above. In addition, plant phenolic compounds can also regenerate other antioxidants or act synergistically

or antagonistically with other phenolic compounds or other endogenous antioxidants such as vitamin E.

Therefore, in view of the above, it is clear that there is still a need to provide new effective natural extracts with antioxidant capacity, while not pro-oxidant, to be used in the food sector and extend the half-life of the same. In this sense the inventors have discovered a new extract obtained from the seed of the fruit of various varieties of avocado (Persea americana Mill.) Useful for its use in the elaboration in general of food products, and meat products in particular. This extract has proven its effectiveness as an antioxidant agent in raw and heat treated meat products, improving their shelf life and oxidative stability, being also a product that does not produce significant changes in the color or taste of food.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Evolution of the TBA-RS content of fresh hamburgers subjected to refrigeration and made with a base formula (Control) and with the addition of an extract obtained from the seed of the fruit of the Persea americana Mill. Figure 2 Evolution of the content in TBA-RS of fresh, cooked and cooked and refrigerated hamburgers, made with a base formula (Control) and with the addition of an extract obtained from the seed of the fruit of the Persea americana Mill. Figure 3. Evolution of the carbonyl content of cooked hamburgers subjected to refrigeration and made with a base formula (Control) and with the addition of an extract obtained from the seed of the fruit of the Persea americana Mill. Figure 4. Evolution of the a * value of the CIELa system * b * of cooked hamburgers subjected to refrigeration and made with a base formula (Control) and with the addition of an extract obtained from the seed of the fruit of Persea amer icana Mill.

DESCRIPTION OF THE INVENTION

The present invention is therefore related to an extract with antioxidant activity obtained from the seed of the fruit of Persea americana Mill. Useful for its use in the general production of food products, and meat products in particular.

The extract is obtainable by a process, hereinafter the method of the invention, which comprises the following steps:

(a) extraction from the crushed seed with a solvent selected from water, ethanol for food use or mixtures thereof and obtaining a supernatant

(b) concentration of the supernatant resulting from step (a) by vacuum distillation.

The avocado (Persea americana Mill.) Is a plant of the family of Lauraceae native to Central America and Mexico. The international trade in avocado fruit has grown in recent years, ceasing to be an exotic fruit to be incorporated into the diet of many countries, thanks to its wide possibilities for fresh consumption and for processing. The edible part of the fruit is the pulp, which according to our investigations represents 68% -75% of the total weight of the fruit depending on the variety of avocado; At present, the rest of the inedible parts, the seed and the shell, do not undergo any type of industrial use.

The process of the invention comprises firstly the collection of the fruits, obtaining the seed and cutting; In the present invention, different varieties of avocados present in the national and international market have been evaluated, among which the Hass varieties (the most frequent in the international market), Fuerte and Criolla, without major differences in content were obtained and antioxidant effect. The crushed avocado seed is then contacted with the selected solvent, which in a particular embodiment is a mixture of ethanol for food use and water in the ratio of 7: 3. The relative amount of seed crushing and solvent can vary between wide margins depending in part on the need to obtain a more or less concentrated extract depending on the application after which it is intended. In a particular embodiment, the ratio of seed crushing: solvent, the solvent being a mixture of ethanol water in the ratio 7: 3, is 1: 5. The solvent is for food use since the extract obtained is useful in the preparation of food products.

The type of solvent used determines the type of phenolic compounds extracted in each case and their concentration due to an obvious issue of polarity or chemical affinity. However, the inventors have shown that the extraction of phenolic compounds with water, or ethanol or with mixtures of both, leads to extracts with similar antioxidant capacity and proven efficacy, although the mixture of ethanol: water and in particular in a 7: 3 ratio is It has proven as the solvent that provides the highest degree of extraction. The choice of the type of solvent in particular of the amount of ethanol is largely determined by the use to which the extract of the invention is intended as set forth below.

Step a) of the process is preferably carried out in a conventional homogenization system provided with blades and provided with means that allow maintaining the temperature at or below 35 ° C, such as a cold water bath. A homogenate is obtained, which is generally separated in a solid and a supernatant. Said separation can be done by any conventional procedure, for example, by centrifugation followed by filtration or decantation.

The supernatant resulting from stage a) is generally filtered and can then be subjected to the next stage b) or alternatively it can be stored refrigerated after filtration until such time as stage b) is carried out.

Step b) comprises subjecting the supernatant from step a) to vacuum distillation which can be carried out by any conventional method preferably by vacuum column or in a rotary evaporator. The supernatant is concentrated by removal of the solvent at low temperature as a result of the vacuum which allows to preserve the antioxidant activity of the extracted compounds. The type of solvent used in the process of the invention influences the time required for stage b) (the higher the water content or if it is only water).

The process of the invention comprises an additional step of characterization and titration of the antioxidant activity according to the determination of the total phenolic compounds carried out according to the Folin-Ciocalteau method (see Examples 1 and 2)

The process of the invention has the advantage of being effective, fast, and low cost in addition to being environmentally friendly due to the type of solvents used, and allows obtaining an extract of the seed of the fruit of the Persea americana Mill. antioxidant properties, which constitutes another aspect of the present invention, hereinafter extract of the invention.

The extract of the present invention has demonstrated its efficacy as an antioxidant agent in particular in raw and heat treated meat products tested (see example 4 and Figures 1 to 4). The results obtained have shown that the extract also improves the visual appearance of the meat products during their preservation, as a result of better color stability compared to a control lot as well as an effective inhibition against lipid oxidative processes and / or of the proteins of the meat products, improving the useful life and oxidative stability of the same, all this, without the extract producing significant changes in the color or flavor of the food due to its incorporation in the products. Therefore, an additional advantage of the extract of the invention is the ability not to alter the organoleptic, color, and taste properties of the food compositions to which it is incorporated which evidently enhances its acceptance by the consumer.

Therefore, in another aspect the invention relates to a process for preparing a food product comprising the addition to the base food product of the extract of the invention.

The addition can be carried out by any conventional technique. The form of incorporation depends for example on the type of base food product, and in particular, in the case of meat products of the type of meat product on which it is added. Thus, in a preferred embodiment, the addition is carried out by direct incorporation of the extract into the composition of a meat product. In another particular embodiment, the addition is carried out by direct incorporation to the surface of the meat product by spray micro-spraying. Typically before micro-spraying the extract is filtered with a suitable membrane filter (0.65 microns). Direct incorporation can be done, for example, in the case of non-minced meat products such as fresh or marinated meat packaged in modified atmospheres.

As mentioned above, the choice of the type of solvent, and in particular the amount of ethanol in the case of an ethanol: water mixture, is largely determined by the type of food product to which the extract is subsequently added. of the invention as well as by the technique with which the addition of the extract is carried out. In general, to obtain meat products in which water is added in the preparation of its composition, such as: cooked sausages, or ham, preferably an aqueous extract diluted in the product's own formulation water is used, whereby Water is preferably used in the process of obtaining the extract. However, in order to obtain meat products whose production does not add water to the composition, it is preferable to use ethanol or a mixture of ethanol: water with a higher proportion of ethanol in the process of the invention thus facilitating the concentration step a empty. It should be noted, however, that any solvent can be used in the process of the invention regardless of the specific application and the manner thereof. The possible variations in the degree of extraction of bioactive compounds depending on the use of a greater or lesser proportion of water: ethanol are minimized by the additional step of the process of the invention of characterization and titration of the antioxidant activity in which the extracts obtained they are adjusted until the concentration of antioxidant polyphenols required in each case is presented. Similarly, other parameters, such as intrinsic variability in the composition of wild fruits (caused by harvest year, location, soil type, ripening state ...) also make this stage of characterization and titration of antioxidant activity is must be carried out prior to its use in food products.

In a further aspect the invention relates to a food product comprising the extract of the invention. Said food product may be any type of food, food ingredient, or beverage, without any limitation, such as, for example, sauces, pre-cooked foods, soups, milk, yogurt, cheeses, bread, biscuits, cookies, etc. . In a particular embodiment said composition is a meat product, such as for example a raw meat product, a heat treated meat product, a cured raw meat product, a marinated meat product, among others.

The appropriate amount of extract for each food product and in particular for each meat product, can vary within wide ranges, and can be determined in each case easily by the person skilled in the art. Said quantity depends above all on the nature of the food composition, and in particular on the meat product to which it is intended, in particular on the composition, technological treatment, content and type of fat present therein, etc., in a preferred embodiment. The extract is present in the food product, and in particular in a meat product, in an amount characterized by being able to inhibit the oxidative processes of lipids and / or proteins. In a particular embodiment, the amount of extract in a meat product is such that the total polyphenol content it presents is 200400 mg of gallic acid equivalents per kg of meat product, said content of the phenolic compounds having been determined according to the Folin method. Ciocalteau (see Example 1).

The inventors have carried out comparative tests that show the antioxidant efficacy of the extract of the invention incorporated into meat products (see Example 4). Thus, figures 1 and 2 show the results of the evolution of the TBA-RS content of fresh and cooked hamburgers subjected to refrigeration. The determination of the content in TBA-RS (thiobarbituric acid reactive substances) is one of the most widespread methods for the study of lipid oxidation in food (Raharjo and Sofos, 1993; Estévez et al., 2009). This determination was made following the method described by Tarladgis et al. (1960). The rationale for this method is that malondialdehyde (a product resulting from lipid oxidation) reacts with thiobarbituric acid in an acidic medium giving rise to a colored pigment that is spectrophotometrically measurable at 532 nm; therefore an increase in the content in TBA-RS is related to an increase in the development of oxidative processes of food lipids.

Figure 3 shows the results of the evolution of the carbonyl content of fresh hamburgers under refrigeration. The increase of carbonyls is a measure that indicates the development of oxidative processes of food proteins. The measurement of carbonyl content is one of the most widely used and widespread methods for determining the oxidation of meat and meat products (Estévez et al., 2009). This determination of the carbonyl content shown in this example of the test of the antioxidant activity of the seed extract of the fruit of the Persea americana Mill. Was carried out according to the method described by Oliver et al. (1997), whose basis is that the carbonyls (products resulting from the oxidation of the proteins) react with the DNPH reagent (2,4-dinitrophenylhydrazine) producing a spectrophotometrically measurable pigment proportional to its concentration. Figure 4 shows the evolution of the a * value of the CIELa * b * system of cooked hamburgers under refrigeration. Instrumental color measurement is widely disseminated in the study of the visual characteristics of meat and meat products (Faustman and Cassen, 1990). In this test, the instrumental color of the hamburgers was determined by a Minolta colorimeter model Chroma Meter CR-300 equipped with diffuse xenon lamp illumination and with a measuring area of 8mm. A D65 illuminant with observation geometry with an angle of 0 ° was used. The device was calibrated using a white calibration plate model CR-A43. The measurements were made in the CIEL * a * b * (CIE, 1976) color space from which the chromaticity coordinates L * (luminosity), a * (red-green axis) and b * (yellow-blue axis) are obtained . The results in Figure 4 show the evolution of the a * value (range -60, pure green; +60, pure red) of the CIELa * b * system, which is a measure that mainly indicates the evolution of the red color of food, this fact being of special interest in meat products. The reduction of the red coloration of meat and meat products during refrigeration is related to the oxidation of pigments present in meat (myoglobin).

Finally, an additional advantage of the present invention is that the extract is obtained from a commercial fruit of wide distribution and accessibility, and of which Spain is the 9th largest producer in the world and the first in Europe.

The following are illustrative examples of the invention that are set forth for a better understanding of the invention and in no case should they be construed as limiting the scope thereof.

EXAMPLES Example 1: In vitro characterization of the antioxidant potential of the seed of the Persea americana Mill fruit as well as its functional components by means of techniques for determining total phenolic compounds.

The antioxidant potential of the fruits was characterized by techniques that quantify the total phenolic compound content and antioxidant activity in vitro:

1.1. Determination of total polyphenols: The determination of phenolic compounds in the seed extracts of the fruit of the Persea americana Mill. Was carried out using the Folin-Ciocalteu method described by Singleton and Rossi (1965) with modifications Pantelidis et al. (2007). The results are shown in Table 1. The rationale for this determination is based on the phenolic compounds of the fruits oxidized by the Folin-Ciocalteu reagent, giving a blue coloration directly proportional to the polyphenol content. Briefly, a 0.5 ml aliquot of the ethanol extract was taken in a test tube; subsequently 2.5 mL of 10% Folin-Ciocalteu reagent (in water) and 2.0 mL of 7.5% sodium carbonate (w / v) were added. The mixture was incubated at room temperature in the dark for 60 minutes. After this period of time the absorbance was measured immediately at 740 nm, against a blank in which the sample was replaced by the same amount of water. Quantitative measurements were performed by contrasting the results with a calibration curve using gallic acid as standard. The results were expressed as mg of gallic acid equivalents (GAE) / 100 g of fruit.

1.2 Tests of antioxidant activity in vitro using radical scavenging techniques The determination of the antioxidant activity in vitro of fruits was carried out by testing with the radical, 2,2-diphenyl-1-picrylhydrazyl -DPPH-and with the Determination of the equivalent antioxidant capacity of TROLOX (TEAC) using the ABTS radical. These techniques are based on the ability of substances with antioxidant activity to reduce a synthetic radical that has a spectrophotometrically measurable coloration, its in vitro antioxidant activity being proportional to the discoloration produced to the radical.

1.2.1 Determination of the antioxidant activity by the radical method - 2,2-diphenyl-1-pyrilhydrazyl (DPPH)

The antioxidant activity of the ethanolic extract of the seed of the fruit of Persea americana Mill. Was determined using the DPPH radical technique described by Kähkönen and Heinonen (2003); The results are shown in Table 1. Prior to the performance of this test, prior determinations were carried out in order to adjust the concentration of the extracts to the test conditions. Seed extracts from Persea americana Mill. Were diluted in a proportion of

1:20 Once the extracts were prepared at the different concentrations, 33 �L of each sample were added to the cuvette VIS and 2000 �L of the DPPH free radical (ABS 1,200) was added, mixed with Pasteur pipette, allowed to stand for 6 minutes in the dark and were read at a wavelength of 517 nm. The antioxidant activity of the samples was calculated by means of a Trolox standard curve, the results were expressed in mM Trolox per gram of sample (mM Trolox / g of sample).

1.2.2. Determination of the equivalent antioxidant capacity of Trolox (TEAC) by ABTS radical The analysis of the equivalent antioxidant capacity of Trolox (TEAC) of the seed extract of the fruit of Persea americana Mill. Was performed using the 2.2 'acid radical method -azinobis- (3-ethylbenzothiazoline-6-sulfonic) - ABTS- according to the method described by Re et al. (1999). First, the ABTS + cation radical was generated in vitro by reacting equal amounts (5 mL) of 7 mM ABTS (in water) and 2.45 mM of potassium persulfate (in water). The mixture was then incubated at room temperature and in the dark for 15 hours (a dark navy solution is obtained). This solution was subsequently diluted with ethanol until the absorbance reached (initial ABTS + Abs). One milliliter of the diluted ABTS + solution (initial absorbance: 0.700 at 734 nm) was mixed with 10 μL of a diluted extract (1:10) of the seed of the Persea americana Mill fruit. The reaction mixture was incubated in the dark at temperature ambient for 6 minutes. The absorbances of the ABTS + radical solution and of the test samples (fruit extracts + ABTS +) were measured immediately at 734 nm, against an ethanol blank. It was calculated as a percentage of inhibition according to the following formula:

% inhibition = [(Abs ABTS + initial - Abs ABTS + remaining) / Abs ABTS + initial] * 100

The inhibition percentages obtained for the problem samples are extrapolated in the calibration curve which allows us to present the results as TEAC (Trolox equivalent antioxidant activity) that is, the antioxidant activity equivalent to that of the Trolox (mM Trolox / milligram extract fresh fruit).

1.3 Determination of antioxidant capacity by reducing Copper (II) (CUPRAC): This method consists in measuring the antioxidant capacity to reduce the copper ion and It is applicable to assess the antioxidant capacity of both food polyphenols as of vitamin C and E (Apak et al., 2004).

The results are shown in Table 1. In a threaded test tube 1 mL of each reagent (0.2M copper chloride, 7.5 mM Neocuproine in ethanol and ammonium acetate), 0.1 mL of each previously diluted seed extract was added (1: 150). It was mixed and allowed to stand for 30 minutes in the dark. The samples are

5 read on a spectrophotometer at an absorbance of 450 nm. To express the results, a standard curve with 5 Trolox points (0.25, 0.5, 1, 1.5, and 2 mM) was prepared. The antioxidant capacity was established in Trolox equivalents (TEAC) expressed in mM Trolox per gram of sample (mM Trolox / g sample).

10 Table 1. Total phenolic compounds (CFT) content, antioxidant activity against DPPH and ABTS radicals and antioxidant activity according to the CUPRAC method of the ethanol extract from the seed of the Persea americana fruit.

CFT1 DPPH2 ABTS3 CUPRAC4

 Half of. Half of. Half of. Half of.

Seed extract

6081 1446 130 39 158 26 275 59

from Persea americana

15 1 Compound Total phenolics (see Example 1.1.) Results expressed as mg GAE / 100 g fruit 2 Test inhibition of DPPH radical (see Example 1.2.1) Results expressed as mM TEAC / g fruit

20 3 ABTS radical inhibition test (see Example 1.2.2) Results expressed as mM TEAC / g fruit 4 Antioxidant capacity according to the CUPRAC method (see Example 1.3) Results expressed as mM TEAC / g fruit D.E .: standard deviation

25

Example 2: Procedure for obtaining an extract with antioxidant activity from Persea americana Mill

The procedure began with washing and obtaining by peeling the seed

30 of the fruit of Persea americana Mill. Obtained from a national distributor (Eurobanan S.L.) at its optimum ripeness and from the Hass varieties (the most frequent in the international market), Fuerte and Creole.

The pre-cut seeds were mixed with a 7: 3 water: ethanol mixture in a 1: 5 ratio in a canister for nalgene centrifuge.

The extraction was carried out in a Sorvall Omni Mixer blade homogenization system for 5 minutes at 3000 rpm. During the chopping procedure, the centrifuge canister was placed in ice water to prevent the temperature of the content from rising from 35 ° C. After the chopping process, the canister with the homogenate was subjected to centrifugation (2000 rpm 5 minutes) to separate the solids resulting from the homogenization process.

The supernatant obtained was passed through a filter paper and cooled to its concentration by vacuum distillation

Once the avocado hydro-alcoholic supernatant of the previous phase was obtained, it was subjected to a concentration by vacuum distillation. The concentration under vacuum and low temperatures allows to preserve the antioxidant activity of the extracted compounds. The concentration process was carried out on a rotary vacuum evaporator (Heidolph model W2000) coupled to a Büchi V700 vacuum pump with Büchi V-850 vacuum controller.

The characterization and titration of the antioxidant activity was then carried out to the level determined for its application in meat products by determining the total phenolic compounds (Folin-Ciocalteau method)

As described in the "state of the art" section of this report, the efficacy of antioxidant extracts in meat systems depends on multiple factors such as the product matrix, the concentration of phenolic compounds or the presence of other antioxidants. For this reason, a series of initial tests were carried out to determine the optimum level of incorporation of the extract with antioxidant activity obtained from the seed of the Persea americana Mill fruit. On the other hand, due to the nature of the source of extraction of the active compounds (fruits) it is necessary to characterize the efficacy of the extracts obtained, as there may be variations depending on the place or time of collection. After a series of initial experimental tests on various meat products, it was determined that the optimum utilization of the seed extract of the Persea americana Mill fruit is between 200-400 ppm (parts per million; mg / kg of meat product), expressed in equivalents of gallic acid as described in Example 1.1.

Example 3: Application in meat products by incorporation into the formula or by direct spray micro spraying

The form of application of the extract of the seed of the fruit of Persea americana Mill. It varied depending on the type of meat product to which it was intended. The efficacy of the extract has been tested by direct surface application by spray micro-spraying prior filtration with 0.65micron membrane filter; This method of application has been tested on non-minced products such as fresh or marinated meat packaged in modified atmospheres.

The results obtained in a fresh and heat treated meat product incorporated directly into the mass of the product at a concentration of 300 ppm are shown below.

Example 4: Test conditions of the data presented in Figures 1 to 4

The data shown in Figures 1 to 4 correspond to an example of the use of the seed extract of the fruit of Persea americana Mill. Made in hamburgers (control batches and with addiction of the extract of the present invention) fresh and cooked that were submitted to a cooling period. The manufacturing and storage procedure is briefly described below.

Preparation of hamburgers: The initial composition of hamburgers per kilo of fresh dough was as follows:

600 grams of pork loin

125 grams of pork bacon

250 grams of water

25 grams of salt The processing of the product began with a rough chopping of the meat. In the bowl of a cutter, the chopped meat, bacon, salt and concentrated extract (except in the control lot) of the seed of the Persea americana Mill fruit (300 ppm relative to the final product) diluted in the water were added of the product formula. The dough was subjected to a finely chopped process for 6 minutes in a vacuum cutter until a fine and homogeneous paste was obtained; later hamburgers were made

5 (weight 100 grams) by means of an apparatus for this purpose.

Burger cooking: Part of the hamburger obtained (control batches and extract of the invention) underwent a cooking process in a convection oven (180ºC / 15 minutes).

10 Burger cooling period: Subsequent to making the burgers (fresh and cooked) they underwent a 15-day commercial refrigeration process, at a temperature of 2 ° C under fluorescent lighting with a 12-hour light / dark cycle . The objective of this procedure was to simulate the

15 storage conditions of a commercial linear. On days 1, 5, 10 and 15 of the cooling period burgers were extracted and stored at -80 ° C until analysis to track oxidative phenomena during the cooling period.

Claims (14)

1. Procedure for obtaining an extract with antioxidant properties from the seed of the fruit of Persea americana Mill. Which comprises the following stages:

(to)

extraction from the crushed seed with a solvent selected from water, ethanol for food use or mixtures thereof and obtaining a supernatant

(b)

 concentration of the supernatant resulting from step (a) by vacuum distillation.

2.

The process according to claim 1, wherein the solvent used is a mixture of ethanol and water in the ratio of 7: 3.

3.

The method according to claim 1 or 2, wherein step (a) is carried out

in a homogenization system and at a temperature equal to or less than 35 ° C.

Four.

The process according to any one of the preceding claims, wherein the concentration of the extract is carried out in a vacuum column or in a rotary evaporator.

5.

The process according to any of the preceding claims, comprising an additional step of characterization and titration of the antioxidant activity according to the determination of the total phenolic compounds carried out according to the Folin-Ciocalteau method.

6.

 Seed extract of the fruit of Persea americana Mill. With antioxidant properties obtainable by the method according to any one of the preceding claims.

7.

Process for preparing a food product comprising the addition to a base food product of an extract according to claim 6.

8.

Method according to claim 7, wherein the food product is a meat product.

9.

Process according to claim 8, wherein the addition is carried out by direct incorporation of the extract into the composition of the meat product.

10.

Method according to claim 8, wherein the addition is carried out by

5 Direct incorporation to the surface of the meat product by spray micro-spraying. 11. Food product comprising the extract according to claim 6. 12. Food product according to claim 11, characterized in that it is a meat product. 13. Food product according to claim 12, wherein the meat product is selected from the group consisting of a raw meat product, a meat product 15 heat treated, a cured raw meat product, a marinated meat product and mixtures thereof. 14. Food product according to claims 12 or 13, wherein the amount of The extract is such that the total polyphenol content it presents is 200-400 mg of gallic acid equivalents per kg of meat product, said content of the phenolic compounds having been determined according to the Folin-Ciocalteau method. SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 201031391 SPAIN Date of submission of the application: 09.17.2010 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: See Additional Sheet RELEVANT DOCUMENTS

Category

Documents cited Claims Affected

X

MX JL05000056 A (RESEARCH AND ASSISTANCE CENTER IN TECHNOLOGY AND DESIGN) 01.06.2007, pages 4,6,7. 1,3,4,6,7,11

Y

KR 20090055387 A (IND ACADEMIC COOP) 02.06.2009, (summary) [online] [retrieved on 18.11.2011] Recovered from EPO EPODOC Database. 1-7.11

Y

GB 2371545 A (SUPERVITAMINS SDN BHD) 31.07.2002, page 3, lines 8-17; page 5, line 14; page 6, lines 15-20; page 7, lines 1-2.25-28; page 12, Example IV; claims 1.7. 1-7.11

TO

JP 11246425 A (SHISEIDO CO LTD) 14.09.1999, (summary) [online] [retrieved on 18.11.2011] Recovered from EPO EPODOC Database. 1-7.11

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 23.11.2011

Examiner A. Sukhwani Page 1/4

REPORT OF THE STATE OF THE TECHNIQUE Application number: 201031391 CLASSIFICATION OBJECT OF THE APPLICATION A23L1 / 30 (2006.01) A23L1 / 314 (2006.01) A23L3 / 3472 (2006.01) A61K36 / 54 (2006.01) Minimum documentation sought (classification system followed by classification symbols) A23L, A61K Electronic databases consulted during the search (name of the database and, if possible, search terms used) INVENES, EPODOC, WPI, X-FULL, NPL, CAPLUS, FSTA, AGRICOLA, CABA, CROPU, SCISEARCH, INTERNET State of the Art Report Page 2/4  WRITTEN OPINION Application number: 201031391 Date of Written Opinion: 23.11.2011  Statement Novelty (Art. 6.1 LP 11/1986) Claims 1 -14 YES Claims NO Inventive activity (Art. 8.1 LP11 / 1986) Claims 8 -10, 12 -14 YES Claims 1 -7, 11 NO The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published.  Considerations: The present invention aims at a process for obtaining an extract with antioxidant properties from the seed of the Persea americana Mill fruit comprising the steps (claim 1): a) extraction from the crushed seed with a solvent selected from water, ethanol for food use, or mixtures thereof, and obtaining a supernatant. b) concentration of the supernatant resulting from step a) by vacuum distillation. The solvent used is a mixture of ethanol and water in the ratio of 7: 3 (reiv. 2). Step a) is carried out in a homogenization system and at a temperature equal to or less than 35 ° C (reiv. 3).  The concentration of the extract is carried out in a vacuum column or in a rotary evaporator (reiv. 4). The antioxidant activity is determined according to the total phenolic compounds and carried out according to the Folin-Ciocalteau method (reiv. 5). The seed extract of the fruit of Persea americana with antioxidant properties obtainable by the above procedure (reiv. 6) as well as the procedure for preparing a food product comprising the addition of the extract (reiv. 7), is also protected. that the food product is a meat product (8), in which the incorporation of the extract is directly in the composition of the meat product (reiv. 9) or is indirect on its surface by spray micro-spraying (reiv. 10) . Finally, the food product comprising the extract (reiv. 11) and which is a meat product (reiv. 12), selected from raw, heat treated, cured, marinated or mixtures thereof (reiv. 13), is protected. the amount of the extract being such that the polyphenol content is 200 to 400 mg of gallic acid equivalents per kg of meat product (reiv. 14). State of the Art Report Page 3/4  WRITTEN OPINION Application number: 201031391 1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

MX JL05000056 A (RESEARCH AND ASSISTANCE CENTER IN TECHNOLOGY AND DESIGN) 01.06.2007

D02

KR 20090055387 A (IND ACADEMIC COOP) 06.02.2009

D03

GB 2371545 A (SUPERVITAMINS SDN BHD) 07/31/2002

D04

JP 11246425 A (SHISEIDO CO LTD) 14.09.1999

2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement  NOVELTY  The documents cited D01, D02 and D04 refer to polyphenols extracted from Persea americana seed, and D03 discloses procedures for obtaining from plants comprising alcohol / water extraction steps followed by distillation. The process claimed with the extraction steps from the crushed seed with a solvent selected from water, ethanol, or mixtures thereof, and concentration of the supernatant by vacuum distillation is not disclosed, since apolar solvents are used in the cited documents or polar methanol type; or when referring to ethanol, antioxidant extraction from avocado seed is not done. Therefore, in view of document D01 to D04, it can be concluded that claims 1–14 are novel in accordance with Article 6 LP 11/86.  INVENTIVE ACTIVITY The object of obtaining an extract with antioxidant properties from the seed of the Persea americana fruit, which comprises extraction with water, ethanol or mixtures and concentration of the supernatant by vacuum distillation or rotary evaporator, is evident to the person skilled in the art. in view of documents D01, D02 and D03. Indeed,

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D01 refers to the process of obtaining polyphenol extract from avocado seed and its uses in food and cosmetics (pages 4, 6, 7). The process involves extraction stages with apolar and polar solvents, and evaporation stage of the solvent in a rotary evaporator, technical characteristics similar to claims 1, 3, 4 of the application under study. Also disclosed is the extract obtained and its use in a food product as well as the food product containing the extract obtained, therefore, affects the inventive activity of claims 1, 3, 4, 6, 7, 11. But in addition,

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D02 refers to a food composition comprising avocado extracts prepared from skin, seed or pulp and showing excellent antioxidant activity, and extracted with methanol (summary).

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D03 discloses a process for recovering plant components, without destroying them, which comprises, among other steps, the extraction of polar components with low alcohols or mixtures of these with water followed by a distillation that can be in a vacuum (page 3, lines 8-17; page 6, lines 15-20; claim 1); the extraction is done at room temperature (page 5, line 14); The solvent can be removed by distillation under vacuum or with an evaporator (page 7, lines 1-2, 25-28) and ethanol is also mentioned as solvent (page 12, Example IV). The components can be extracted from the avocado (claim 7).

In view of these documents, the person skilled in the art could, without any inventive effort, extract antioxidants from the crushed avocado seed, after extraction with ethanol / water and concentrate the supernatant by vacuum distillation or evaporator, put that the extraction of antioxidants from the avocado seed with polar solvent and subsequent concentration, whether by distillation or with an evaporator, is already disclosed in the documents cited D01 to D03. The proportion of ethanol / water or the determination of the total phenolic compounds according to the Folin-Ciocalteau method, object of claims 2 and 5, are also obvious in this sector of the art. Therefore, in view of documents D01 to D03, it can be concluded that claims 1-7, 11 lack inventive activity according to Article 8 LP 11/86. State of the Art Report Page 4/4