ES2990137B2 - Coloring composition based on anthocyanins from wild fruits, preparation method and uses - Google Patents

Description

DESCRIPTION

Coloring composition based on anthocyanins from wild fruits, preparation method and uses

Technical field

The present invention falls within the technical field of colorants, particularly food colorants, as well as, due to the possible applications of the coloring compositions of the present invention, within the technical field of food preservatives and antioxidants.

Background of the invention

In the food industry, it is common to use different food additives - mostly of synthetic origin - in food formulations in order to guarantee adequate organoleptic properties and in accordance with consumer standards (e.g., colorants), guarantee hygienic-sanitary quality (e.g., preservatives with antimicrobial properties), avoid alterations due to oxidation processes (e.g., antioxidants), etc. throughout the entire shelf life of said foods, in addition to trying to prolong said shelf life as much as possible to reduce food waste.

All authorized food additives listed on the positive list of additives in Regulation (EU) No. 1129/2011 are safe, provided they are used in accordance with current regulations, and are subject to periodic re-evaluation by the European Food Safety Authority (EFSA). However, many synthetic additives are controversial, such as azo dyes and some synthetic preservatives and antioxidants.

Azo dyes have been the subject of many studies, one of the most controversial being the so-called "Southampton Study", in which a group of children aged 3 to 9 years old were given a combination of food colouring and sodium benzoate mixed with food. When the behavioural patterns of these children were compared to a placebo group, clear evidence of symptoms associated with attention deficit hyperactivity disorder (ADHD) was found in the group of children who had consumed these dyes. Such is the health impact that the consumption of these dyes can have on children that in 2008 the European Parliament approved Regulation (EC) No 1333/2008, stating that the possible negative effects on children's activity and attention must be clearly indicated on the label, next to the name or E number of each colouring listed in said Regulation, which include the following: tartrazine (E-102); Quinoline yellow (E-104); Sunset yellow (E-110); Chamoisin (E-122); Ponceau 4R, Cochineal red A (E-124); Allura red AC (E-129).

There are many known adverse effects and/or contraindications derived from the use of these food colorings. For example, tartrazine (i.e., E-102; ADI of 7.5 mg/kg body weight), known in the United States as "FD & C Yellow No. 5", has been associated with cases of hives, purpuric lesions, anaphylaxis, etc. Sunset yellow (i.e., E-110; ADI of 2.5 mg/kg body weight), known in the United States as "FD & C Yellow No. 6", has also been associated with genotoxicity in mouse models, with learning deficits in offspring, as well as with immunomodulatory and xenoestrogenic effects. Allura Red AC (i.e., E-129; ADI 7 mg/kg bw), known in the United States as "FD & C Red No. 40", has been re-evaluated in the European Union twice, concluding that it may be genotoxic at high doses. Carmoisine (i.e., E-122; ADI 4 mg/kg bw) is a colorant banned for use in the United States.

On the other hand, erythrosine (i.e., E-127; ADI of 0.1 mg/kg body weight), which is a synthetic polyiodinated xanthene dye known in the United States as "FD & C Red No. 3" and obtained by iodination of fluorescein - a dye banned in the European Union -, has been linked to alterations in children's behavior and thyroid function due to its high iodine content.

There are also other colorants widely used in foods for infant use belonging to the group of blue colorants derived from triarylmethane, such as patent blue V (i.e., E-131; ADI of 1 mg/kg body weight), brilliant blue FCF (i.e., E-133; ADI of 6 mg/kg body weight), known in the United States as "FD & C Blue No. 1", green S (i.e., E-142; ADI 5 mg/kg body weight), fast green (i.e., E-143; ADI of 12.5 mg/kg body weight), or brilliant black (i.e., E-151; ADI of 1 mg/kg body weight), which are widely used in the production of sweets, preserves, marmalades, jellies and ice creams and require special monitoring due to the potential adverse effects they may cause in the population, particularly in children.

Other examples of additives subject to control are the antioxidant additives BHA (i.e., E-320) or BHT (i.e., E-321), both synthetic antioxidant additives. BHA is an antioxidant composed of two isomers (2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole) that can be used in many foods, with an ADI of 1 mg/kg body weight. BHT is a fat-soluble antioxidant that can be used alone or in combination with BHA, and has an ADI of 0.5 mg/kg body weight. EFSA established these ADI values based on studies in rodents showing that consumption of high concentrations of these compounds could lead to forestomach hyperplasia, growth retardation, increased mortality and certain behavioral effects in rat pups.

There are also certain limitations on the quantity of preservative additives in foods, such as sorbic acid and its sorbates (e.g., E-200), whose toxic potential lies not so much in the compound itself, but in its transformation products once incorporated into the food (e.g., reaction between sorbic acid and nitrites and/or sulfites, producing compounds with mutagenic activity). On the other hand, the quantities of benzoic acid and benzoates (i.e., E-210) are also limited to an ADI of 5 mg/kg, taking into account that they can promote the presence of benzene compounds. Finally, sulfites (i.e., E-220 to E-228) are additives widely used in the food industry, but are on the list of allergens that must be declared on food labels, because they can cause problems in sensitive individuals, asthmatics, etc.

In this context, the need to provide new additives or coloring compositions of natural origin that do not pose a risk to health, without compromising the organoleptic and hygienic-sanitary quality of food, is evident. Furthermore, there is a growing demand in society for increasingly natural foods, foods that do not contain food additives such as colorings, preservatives, and antioxidants, or, if they do contain them, that are of natural origin.

Furthermore, the food industry must resort to the use of two or more food additives in the formulation of many foods to guarantee their hygienic and sanitary quality (use of antimicrobials), organoleptic quality (use of food colorings and antioxidant additives), and to be able to market long-lasting, safe, and palatable foods for the consumer. Today, there are various natural food additives on the market that could be incorporated into food formulations, such as antioxidants (e.g., ascorbic acid, tocopherols, rosemary extract), antimicrobials (e.g., niacin), or colorings with red-violet hues (e.g., E-120 - carmine, E-163 - anthocyanins), although their use is not authorized for incorporation into all foods or in any concentration.

However, there is no ingredient that can alone replace the functions of coloring additives, antioxidants, and preservatives. Furthermore, it is also important to highlight that the food industry has not yet been able to formulate foods with magenta-purple coloring without the use of artificial colorings, especially in foods with a pH range where anthocyanins (E 163) have a reddish color (e.g., pH 3-4). This represents a significant handicap for the food industry, since consumers today demand more natural foods and therefore foods free of artificial colorings in their formulation. The present invention comprises obtaining and applying a previously undescribed natural composition whose chemical composition and physical-chemical properties allow it to achieve, without resorting to the use of azo-type colorings authorized under current regulations, the color characteristics currently available on the market in foods with acidic pH (pH 3-4), with respect to the purple-magenta range.

To date, different fruits have been investigated in search of obtaining possible coloring sources, as has been the case of fruits such as Crataegus monogyna Jacq., Sorbus aria L., Prunus avium L., Fragaria vesca L. and Vaccinium myrtillus L., having reported their anthocyanin profile, their color characteristics and some of their bioactive properties as antioxidant, antibacterial and antifungal, evidencing the potential of these fruits to be used as sources of natural colorants in the development of healthier food products (Dia et al., Food & Function 2016, 7, 4523; Vega et al., Food Chemistry 2023, 414, 135669; Tamayo-Vive et al., Foods 2023, 12, 2427.).

Regarding Barberis vulgaris L., it is known today for the use of berberine, a natural alkaloid present in high quantities in this plant, especially in its stems and roots, which has shown to have a wide use in the treatment of hepatic oxidative stress, Alzheimer's, idiopathic male factor infertility, weight loss, prevention of diabetes and cardiovascular problems (Fatehi-Hassanabad et al., Journal of ethnopharmacology 2005, 102 (1), 46-52; Fatehi et al., An International Journal Devoted to Pharmacological and Toxicological Evaluation of Natural Product Derivatives 2005, 19 (3), 222-225; Abd El-Wahabet al., BMC complementary and alternative medicine 2013, 13, 1-12). EP2007429 also describes compositions for oral administration with beneficial effects on cardiovascular health, comprising berberine or extracts containing it, among other ingredients. The same is true of EP3406144, based on compositions containing berberine chloride, among other ingredients, administered orally, for use in controlling hyperlipidemia and cardiovascular risk factors. However, this alkaloid lacks coloring activity, making it unsuitable as a replacement for the aforementioned synthetic colorants. Furthermore, patent WO2010109286A1 describes a non-hygroscopic crystalline preparation rich in colored phenolic compounds obtained from plants for use in beverages. Several botanical species are mentioned, including Berberis spp. However, the combination of these species to obtain the preparation is not mentioned, nor is the botanical species Myrtus spp. included in the description of the invention.

On the other hand, the composition of the Myrtus communis L. plant is known in terms of bioactive compounds (Messaoud and Boussaid, Chemistry & biodiversity 2011, 8 (2), 300-310) and anthocyanins (Maldini, Phytochemical Analysis 2011, 27 (5), 249-256), as well as its use in the treatment of different cardiovascular, gastrointestinal, dermatological or neurological diseases, through several studies focused mainly on essential oils extracted from the plant, mostly from its leaves. In particular, there are different publications referring to myrcetin, present mainly in the leaves and roots of plants belonging to the Myrtus family, with great therapeutic potential against cancer, liver injuries, cardiovascular diseases, obesity, diabetes and osteoporosis (lmranet al. Food science & nutrition 2021, 9 (10), 5854-5868). However, myrcetin is also an alkaloid that lacks coloring activity, so it is not of interest to replace existing synthetic colorants. Finally, it should be mentioned that patents US2013281548A1 and US2008255226A1 describe a composition of anthocyanin extracts of plant origin, in which starch is used as an edible vehicle, or cysteine is used to improve its bioavailability, respectively. Although they present a list of possible fruits to use to obtain anthocyanins, such as Myrtus communis L., they are not included in the examples or in the claims, nor the combined use of Myrtus communis together with Berberis vulgaris.

Detailed description of the invention

In a first aspect, there is provided an anthocyanin-based coloring composition comprising an extract of seedless fruit of Berberis vulgaris L. and an extract of the skin of the fruit of Myrtus communis L.

The expression "anthocyanin-based" in relation to the coloring compositions of the invention means that said coloring compositions comprise anthocyanins, preferably anthocyanins of natural origin.

The expression "seedless fruit of Berberis vulgaris L." in the context of the present invention refers to the ripe seedless fruit of Berberis vulgaris L. "ripe fruit of Berberis vulgaris L." is understood to mean that fruit of Berberis vulgaris L. in an optimal state of ripeness, that is, that fruit that has a Brix degree value (°Brix) between 9 and 20° Brix, preferably between 11 and 18° Brix, wherein these Brix scale values can be determined by any known method, although, preferably, they are determined using an Atago refractometer at 20 °C according to official method 932.14C, AOAC, 2005. In the context of the present invention, the expressions "seedless fruit" and "seedless fruit" are used completely interchangeably. Furthermore, in the context of the present invention, the term “fruit” is considered to include one or more fruits.

The expression "skin of the fruit of Myrtus communis L." in the context of the present invention refers to the skin of the ripe fruit of Myrtus communis L. "ripe fruit of Myrtus communis L." is understood to mean that fruit of Myrtus communis L. in an optimal state of ripeness, that is, that fruit that has a Brix degree value (°Brix) comprised between 5 and 11° Brix, preferably between 6 and 9° Brix, wherein these values of the Brix scale can be determined by any known method, although, preferably, they are determined using an Atago refractometer at 20 °C according to official method 932.14C, AOAC, 2005.

As already indicated, there is currently no ingredient that can alone replace the functions of coloring additives, antioxidants, and preservatives. This is a problem existing in the state of the art, which the inventors have successfully solved with the anthocyanin-based coloring compositions of the present invention, derived from the unexpected effects derived from the combination of specific extracts from these plants. These compositions, which comprise extracts of the seedless fruit of Berberis vulgaris L. and the skin of the fruit of Myrtus communis L., are also particularly suitable for food use and successfully avoid the need to use multiple synthetic additives in food products, since they have coloring capacity and, in addition, also preservative (e.g., antimicrobial and antifungal) and antioxidant capacity. With respect to the antimicrobial capacity, these compositions significantly inhibit the growth of foodborne pathogenic bacteria (e.g. Samonella sp., Listeria monocytogenes, Staphylococcus aureus, Bacilus cereus, Pseudomonas aeruginosa, E. coli, among others) and Aspergillus type fungi. With respect to the antioxidant capacity, the compositions of the present invention significantly inhibit oxidative processes in food through different mechanisms of action, e.g. by sequestering free radicals, inhibiting their generation or propagation (oxidative stress) or inhibiting free radical generating enzymes (such as, for example, polyphenoloxidase or lipoxygenase).

On the other hand, with respect to the coloring capacity, the compositions of the present invention advantageously provide coloring with shades from red to magenta, with a characteristic and recognizable chromatic profile, due to the synergistic effect of both extracts, while presenting a safety profile suitable for use as a food coloring, even for food coloring in food products intended for populations especially sensitive to the known problems derived from the use of artificial colorings, such as children. Furthermore, in a particular way, the coloring compositions of the present invention are able to provide a stable magenta or purple hue when used in environments with a pH between 3.0 and 6.0, more particularly, 3.0 - 3.5 or 5.5 - 6.0, such as a food matrix with a pH within those ranges, which solves the gap existing in the market with regard to natural coloring additives that can provide this specific coloration, typical of forest fruits, without requiring the use of synthetic colorants. That is to say, in a particularly advantageous manner, the anthocyanin-based coloring compositions of the present invention have a coloring capacity that remains stable within a pH range higher than that provided by the commercial colorants currently available, and represent a natural alternative for use in food formulations, which allows obtaining the desired color range without having to resort to the use of artificial colorants such as azo dyes.

Berberis vulgaris L. is a thorny shrub with wood and yellow flowers, the fruits of which are small oblong berries that turn reddish when ripe. Extracts of the seedless fruit of Berberis vulgaris L. used in the context of the present invention typically include a plurality of anthocyanins which may be selected, but are not limited to, two or more of delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunidin-3-O-glucoside, pelargonidin-3-O-glucoside, malvidin-3-O-glucoside and malvidin-O-deoxyhexosyl-pentoside.

Myrtus communis L. is a shrub with aromatic leaves and white flowers and its fruits typically have a size of up to 1 cm and a blue-black color. The extracts of seedless fruits of Myrtus communis L. used in the context of the present invention typically include a plurality of anthocyanins which may be selected, in a non-limiting manner, from two or more of delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunidin-3-O-glucoside, malvidin-O-dihexoside, malvidin-3-O-glucoside, petunidin-3-O-pentoside and malvidin-O-deoxyhexosyl-pentoside.

These botanical species are two wild species endemic to the Iberian Peninsula. For the present invention, samples of Berberis vulgaris L. were collected in the Serranía de Cuenca (Carrascosa, Cuenca) and Beteta (Cuenca), and of Myrtus communis L. were collected in the Albufera Natural Park (Valencia) and the Sierra de la Murta Municipal Natural Park (Alcira). In order to access these plant genetic resources, permission was initially obtained to access plant genetic resources for non-commercial research purposes (references PN-NC_032021 and PN-NC_022022, which correspond to ABSCH-IRCC-ES-257749-1 and ABSCH-IRCC-ES-262067-1, respectively) and subsequently, permission to access them for commercial purposes after signing separate agreements to obtain prior informed consent and establish the mutually agreed conditions with the Generalitat Valenciana and the Government of Castilla-La Mancha.

In one embodiment, the coloring composition may comprise an amount of the extract of seedless fruits of Berberis vulgaris L. comprised between 40% and 60% by weight, relative to the total weight of the composition, more preferably, an amount of the extract of seedless fruit of Barberis vulgaris L. comprised between 45% and 55% by weight, relative to the total weight of the composition. In another embodiment, the coloring composition may comprise an amount of the extract of the skin of the fruit of Myrtus communis L. comprised between 40% and 60% by weight, relative to the total weight of the composition, more preferably, an amount of the extract of the skin of the fruit of Myrtus communis L. comprised between 45% and 55% by weight, relative to the total weight of the composition. In particular, the coloring composition may comprise an amount of the extract of the seedless fruit of Berberis vulgaris L. between 40% and 60% by weight, relative to the total weight of the composition and/or an amount of said extract of the skin of the fruit of Myrtus communis L. comprised between 40% and 60% by weight, relative to the total weight of the composition. It is also evident that the sum of the amounts by weight of the extracts of the seedless fruit of Berberis vulgaris L. and of the skin of the fruit of Myrtus communis L. comprised by any one of these coloring compositions of the invention will in no case exceed 100% by weight relative to the total weight of the coloring composition.

The coloring composition may be in liquid form (e.g., in the form of a hydroalcoholic solution) or in the form of a lyophilized powder. In a preferred embodiment, the coloring composition may be in the form of a lyophilized powder, more preferably, in the form of an encapsulated lyophilized powder, and even more preferably, in the form of a microencapsulated lyophilized powder.

In a particular embodiment, the coloring composition may be in the form of a lyophilized powder and also comprise an encapsulating agent. Examples of encapsulating agents include, but are not limited to, maltodextrin or cyclodextrin.

In a second aspect of the invention, there is provided a method for preparing an anthocyanin-based coloring composition, more preferably, a method for preparing an anthocyanin-based coloring composition according to the first aspect of the invention. Said process comprises the following steps:

a) subjecting at least a portion of the seedless fruit of Berberis vulgaris L. to hydroalcoholic extraction to obtain a seedless fruit extract of Berberis vulgaris L.,

b) subjecting at least a portion of the skin of the fruit of Myrtus communis L. to a hydroalcoholic extraction treatment to obtain an extract of the skin of the fruit of Myrtus communis L., and

c) mixing a quantity of the extract of the seedless fruit of Berberis vulgaris L. resulting from step a) and a quantity of the extract of the skin of the fruit of Myrtus communis L. resulting from step b).

In step a) of the process, at least a portion of the seedless fruit of Berberis vulgaris L. (i.e., skin of the ripe fruit of Berberis vulgaris L.) is subjected to hydroalcoholic extraction to obtain a seedless fruit extract of Berberis vulgaris L. In a preferred embodiment, said hydroalcoholic extraction is preferably carried out with an ultrasonic probe. Said extraction with an ultrasonic probe can be carried out, for example, by means of ultrasonic processing equipment, such as the Fisherbrand™ Model 705 Sonic Dismembrator equipment, preferably at an ultrasonic power of 325 W - 375 W. In another embodiment, said hydroalcoholic extraction of step a) can be carried out with a (solid/liquid, S/L) ratio of said portion of the seedless fruit of Berberis vulgaris L. (solid) with respect to hydroalcoholic solvent (liquid) of between 20 g/L and 28 g/L. Particularly preferably, said hydroalcoholic extraction of step a) of the process of the invention is carried out in a hydroalcoholic medium (e.g., a medium formed by ethanol and water) that provides an acidic pH, more preferably in a hydroalcoholic medium that provides a pH between 2 and 4, even more preferably in a hydroalcoholic medium that provides a pH between 3.0 and 3.5. These pH values can preferably be achieved by acidifying the medium with citric acid, e.g. with 5 M citric acid.

In a particular embodiment, in step a) of the process, at least a portion of fruit devoid of seeds of Berberis vulgaris L. (i.e., skin of the ripe fruit of Berberis vulgaris L.) is subjected to hydroalcoholic extraction to obtain an extract of fruit devoid of seeds of Berberis vulgaris L., wherein said hydroalcoholic extraction is preferably carried out with an ultrasound probe, with a proportion (solid/liquid, S/L) of said portion of the fruit devoid of seeds of Berberis vulgaris L. (solid) relative to hydroalcoholic solvent (liquid) of between 20 g/L and 28 g/L and/or, optionally, in a hydroalcoholic medium (e.g., a medium formed by ethanol and water) that provides an acidic pH, more preferably in a hydroalcoholic medium that provides a pH between 2 and 4, even more preferably in a hydroalcoholic medium that provides a pH between 3.0 and 3.5. These pH values can preferably be achieved by acidifying the medium with citric acid, e.g. with 5 M citric acid.

In step b) of the process, at least a portion of Myrtus communis L. skin (i.e., skin of the ripe Myrtus communis L. fruit) is subjected to hydroalcoholic extraction to obtain an extract of the skin of the Myrtus communis L. fruit. In a preferred embodiment, said hydroalcoholic extraction is preferably carried out with an ultrasonic probe. Said extraction with an ultrasonic probe can be carried out, for example, with ultrasonic processing equipment, such as the Fisherbrand™ Model 705 Sonic Dismembrator equipment, preferably at an ultrasonic power of approximately 450 W - 500 W, more preferably, approximately 500 W. In another embodiment, said hydroalcoholic extraction of step b) is carried out with a (solid/liquid, S/L) ratio of said Myrtus communis L. fruit skin. (solid) with respect to hydroalcoholic solvent (liquid) of between 18 g/L and 22 g/L, more preferably, between 18.5 g/L and 21.0 g/L. Particularly preferably, said hydroalcoholic extraction of step b) of the process of the invention is carried out in a hydroalcoholic medium that provides a pH between 5 and 7, preferably in a hydroalcoholic medium that provides a pH of approximately 6. In a particular embodiment, said hydroalcoholic extraction of step a) of the process of the invention is carried out in a medium formed by ethanol and water that provides a pH between 5 and 7, more preferably in a medium formed by ethanol and water that provides a pH of approximately 6.

The expression “approximately”, when preceding and referring to a numerical value, is understood in the context of the present invention to disclose that particular numerical value and, in addition, to designate any value comprised within a range formed by that numerical value ± 5%, more preferably a range defined by the numerical value ± 2%. By way of illustration, the expression “approximately 1” should be interpreted as “within the range comprised between 0.95 and 1.05”, preferably, “within the range comprised between 0.98 and 1.02”.

It is understood that the expression "range comprised between", in the context of the present invention, includes both the numerical values comprised between the two numerical values present at the two extremes of the range and the two numerical values that form each of the two extremes in themselves, unless otherwise indicated. That is, it should be interpreted, for example, that "within the range comprised between 0.95 and 1.05" includes both the values included within the range formed by the maximum value of 1.05 and the minimum value of 0.95, such as 1.05 and 0.95 specifically.

In a particular embodiment, in step b) of the process, at least a portion of the skin of the fruit of Myrtus communis L. (i.e., skin of the ripe fruit of Myrtus communis L.) is subjected to hydroalcoholic extraction to obtain an extract of the skin of the fruit of Myrtus communis L., wherein said hydroalcoholic extraction is preferably carried out with an ultrasound probe, with a solid/liquid (S/L) ratio of said skin of the fruit of Myrtus communis L. (solid) with respect to hydroalcoholic solvent (liquid) of between 18 g/L and 20 g/L and/or, optionally, in a hydroalcoholic medium that provides a pH between 5 and 7, more preferably in a hydroalcoholic medium that provides a pH of approximately 6.

The process according to this second aspect of the invention may further comprise, optionally, before step a) and/or step b), subjecting said portion of the seedless fruit of Berberis vulgaris L. and/or said portion of the skin of the fruit of Myrtus communis L., respectively, to a freeze-drying step.

Step a) and step b) of the process of the invention are based on the use of the seedless fruit of Berberis vulgaris L. and the peel of the fruit of Myrtus communis L. Said plant material can be obtained from the fruit of each of these two shrubs by means known in the art. In a particularly preferred embodiment, the process of the invention comprises a preliminary step, prior to step a), wherein the seed is separated from the peel and pulp in the fruit of Berberis vulgaris L. and wherein the peel is separated from the pulp and seed of the fruit of Myrtus communis L., preferably the ripe fruit of Berberis vulgaris L. or Myrtus communis L., manually and/or by mechanical means.

In a particular embodiment, the amount of the extract of Berberis vulgaris L. seedless fruit and the amount of the extract of the peel of the fruit of Myrtus communis L. that are mixed in step c) of the process may each independently be between 40 - 60% by weight, relative to the total weight of the coloring composition. Preferably, the amount of the extract of Berberis vulgaris L. seedless fruit and the amount of the extract of the peel of the fruit of Myrtus communis L. that are mixed in step c) of the process may each independently be between 45 - 55% by weight, relative to the total weight of the coloring composition. It is also evident that the sum of the amounts by weight of the extracts of Berberis vulgaris L. seedless fruit and of Myrtus communis L. fruit peel. mixed in step c) will not in any case exceed 100% by weight with respect to the total weight of the coloring composition.

Preferably, the seedless fruit extract of Berberis vulgaris L. resulting from step a) and/or the fruit peel extract of Myrtus communis L. resulting from step b) can be subjected, independently of each other, to a lyophilization step prior to step c). This allows both extracts to be mixed directly in the form of lyophilized powder in step c), thus facilitating the obtaining of a more homogeneous mixture. Optionally, the mixture resulting from step c), already in lyophilized form, can be subsequently encapsulated in the presence of at least one encapsulating agent. Said encapsulating agent must be compatible with the pH of the food matrix into which the coloring composition of the present invention is to be incorporated once obtained.

In another embodiment, the extract of the seedless fruit of Berberis vulgaris L. resulting from step a) and/or the extract of the skin of the fruit of Myrtus communis L. resulting from step b) can be subjected, independently of each other, to a lyophilization step prior to step c) and, optionally, the encapsulation of the lyophilized extract of the seedless fruit of Berberis vulgaris L. resulting from said lyophilization and/or of the lyophilized extract of the skin of the fruit of Myrtus communis L. resulting from said lyophilization can be carried out independently in the presence of at least one encapsulating agent, before carrying out step c). Said encapsulating agent must be compatible with the pH of the food matrix into which the coloring composition of the present invention is to be incorporated once obtained. Preferably, the seedless fruit extract of Berberis vulgaris L. resulting from step a) and the fruit peel extract of Myrtus communis L. resulting from step b) can be subjected independently of each other to a lyophilization step prior to step c) and, optionally, the resulting lyophilized extract of seedless fruit of Berberis vulgaris L. and the resulting lyophilized extract of the fruit peel of Myrtus communis L. can then be encapsulated in the presence of at least one encapsulating agent, also prior to step c). This allows both extracts to be lyophilized and encapsulated independently, such that in step c) they would already be mixed in the form of a lyophilized and encapsulated powder, facilitating the obtaining of a homogeneous encapsulated mixture.

In another embodiment, the mixture resulting from step c) can be subjected to a subsequent lyophilization step. In this way, the extracts resulting from steps a) and b) can be mixed in liquid form (i.e., in the presence of a quantity of the solvent used in the hydroalcoholic extraction, for example), and the mixture obtained in step c) itself can be lyophilized. After this subsequent lyophilization step, the mixture resulting from step c), already in lyophilized form, can optionally be encapsulated, preferably microencapsulated, in the presence of at least one encapsulating agent. Said encapsulating agent must be compatible with the pH of the food matrix into which the coloring composition of the present invention is to be incorporated once obtained.

Examples of suitable encapsulating agents include, but are not limited to, cyclodextrins and maltodextrins. Encapsulation, particularly microencapsulation, can be carried out by methods known in the art, including, for example, (micro)encapsulation by spray-drying. Such spray-drying can be carried out using any equipment known for this purpose, such as, for example, the Mini Spray Dryer, model 8-290, from Buchi.

In a third aspect of the invention, there is provided a coloring composition obtained or obtainable by the method of the second aspect of the invention, defined above. This coloring composition comprises an extract of seedless fruit of Barberis vulgaris L. and an extract of the skin of the fruit of Myrtus communis L. In one embodiment, the coloring composition may comprise an amount of the seedless fruit extract of Barberis vulgaris L. comprised between 40% and 60% by weight, relative to the total weight of the composition, more preferably, an amount of the seedless fruit extract of Barberis vulgaris L. comprised between 45% and 55% by weight, relative to the total weight of the composition. In another embodiment, the coloring composition may comprise an amount of the skin extract of the fruit of Myrtus communis L. between 40% and 60% by weight, relative to the total weight of the composition, more preferably, an amount of the extract of the skin of the fruit of Myrtus communis L. comprised between 45% and 55% by weight, relative to the total weight of the composition. In particular, the coloring composition may comprise an amount of the extract of the fruit without seeds of Barberis vulgaris L. comprised between 40% and 60% by weight, relative to the total weight of the composition and/or an amount of said extract of the skin of the fruit of Myrtus communis L. comprised between 40% and 60% by weight, relative to the total weight of the composition. It is also evident that the sum of the amounts by weight of the extracts of the fruit without seeds of Barberis vulgaris L. and of the skin of the fruit of Myrtus communis L. comprised by any of these coloring compositions of the invention will not in any case exceed 100% by weight with respect to the total weight of the coloring composition.

The coloring composition, according to the third aspect of the invention, may be in liquid form (e.g., in the form of a hydroalcoholic solution) or in the form of a lyophilized powder. In a preferred embodiment, the coloring composition may be in the form of a lyophilized powder, more preferably, in the form of an encapsulated lyophilized powder, and even more preferably, in the form of a microencapsulated lyophilized powder.

In a particular embodiment, the coloring composition may be in the form of a lyophilized powder and may also comprise an encapsulating agent. Examples of encapsulating agents include, but are not limited to, maltodextrin or cyclodextrin.

In a fourth aspect, there is provided the use of the coloring composition, defined according to the first or third aspect of the invention, as a food coloring, preferably as a food coloring under pH conditions of between 2.5 and 6.5, and even more preferably as a food coloring under pH conditions of between 3.0 and 6.0. Said use may additionally comprise the use as a preservative (e.g., as an antibacterial and/or antifungal agent). In a particularly preferred embodiment, there is provided the use of the coloring composition of the first or third aspect of the invention as a food coloring, preferably as a food coloring under pH conditions of between 2.5 and 6.5, or more preferably under pH conditions of between 3.0 and 6.0, and also as a preservative (e.g., as an antibacterial and/or antifungal agent). In a more particularly preferred embodiment, there is provided the use of the coloring composition of the first or third aspect of the invention as a food coloring, preferably as a food coloring under pH conditions comprised between 2.5 and 6.5, or more preferably under pH conditions comprised between 3.0 and 6.0, as a preservative (e.g., as an antibacterial and/or antifungal agent), and as an antioxidant.

In a particularly preferred embodiment, there is provided the use of the colorant composition according to the first or third aspect of the invention as a colorant, preservative and antioxidant, more preferably as a food colorant, preservative and antioxidant and, even more preferably, as a food colorant, preservative and antioxidant under pH conditions between 3 and 3.5 or between 5.0 and 6.0. Said pH conditions may correspond, for example, to the pH of a food matrix.

According to a fifth aspect of the invention, there is provided a food product comprising a coloring composition according to the first or third aspect of the invention. The term "food product" refers to a product intended for human and/or animal consumption. In a preferred embodiment, it refers to the product or products intended for human consumption, in particular, for the consumption of children and young adults (between 18 and 24 years of age). Said food product may comprise, but is not limited to, gelatins, flavored fermented milks, dairy desserts, ice cream, popsicles, flavored soft drinks, sweets such as jelly beans or lollipops, etc.

In a preferred embodiment, the food product further comprises a food matrix with a pH value between about 3.0 and 3.5, or more preferably, a food matrix with a pH value between 3.0 and 3.5. Examples of food products comprising such a food matrix with a pH value of about 3.0-3.5 include, but are not limited to, jellies, flavored fermented milks, juices, nectars, chickens, soft drinks, and confectionery (e.g., jelly beans or lollipops). In the context of the present invention, the term "food matrix" refers to the food product as such, prior to the introduction or addition of the coloring composition of the present invention.

In fact, it is known that the pH of the food matrix significantly conditions the color of the natural colorants currently used in the food industry, such as E-163, leading to reddish tones at pH 3, with values of L* 20.17, a*: 44.29, b*: 32.82 according to the CIELAB system. In this sense, in a particularly advantageous way, the coloring compositions of the present invention make it possible to obtain magenta colorations, which currently require the combination of different synthetic colorants with blue tones, since the food industry does not currently have natural or synthetic coloring additives that present the magenta-purple hue.

The CIELAB L* system is a model that allows colors to be classified based on their luminosity (parameter "L*") and their chromatic coordinates, where "a*” corresponds to the red/green coordinates (+a indicates red, -a indicates green), and "b*” corresponds to the yellow/blue coordinates (+b indicates yellow, -b indicates blue).

In another preferred embodiment, the food product further comprises a food matrix with a pH value between about 5.5 and 6.0 or, more preferably, a food matrix with a pH value between 5.5 and 6.0. It is known that some dairy desserts, such as ice creams (cream-based), have a pH around 5.5 - 6.0, so that it is not possible to provide a magenta-purple hue even with the natural colorant E-163, due to the hue it presents in that pH range, more particularly at pH 6, with values of L*: 11.14, a*: 31.72, b*: 4.47, corresponding to a dark garnet color, which progressively turn into a purple hue and then into a dark blue. However, the coloring compositions of the present invention allow obtaining magenta-purple colorations at that pH range around 5.5 - 6.0, with CIELAB values, for example, of L*: 7.06, a*: 14.05, b*: -0.74 or L*: 14.69, a*: 14.98, b*: -1.14, as demonstrated experimentally in the examples described below.

Throughout the description and claims, the word "comprises" and any variations thereof are not intended to exclude other technical features, ingredients or steps.

Description of the drawings

Fig. 1- Anthocyanin profile of the lyophilized powder extract BV2, obtained by ultra-high performance liquid chromatography coupled to an ultra-sensitive diode array detector (UHPLC-DAD, after identification carried out by HPLC-DAD-MS).

Fig. 2- Anthocyanin profile of the lyophilized MC2 extract powder, obtained by UHPLC-DAD, with prior identification carried out by HPLC-DAD-MS.

Fig. 3- Anthocyanin profile of the BV2:MC2 composition in lyophilized powder, obtained by UHPLC-DAD, with prior identification carried out by HPLC-DAD-MS.

Examples

The following examples are provided for the purpose of illustrating the invention and the technical effects it provides, but they should not be construed as limiting its scope. Thus, it is possible that the invention may be practiced in a manner different from that specifically described in the following examples.

Example 1 - Coloring composition, in fluid form, of extracts of seedless fruit of Berberís vulgaris L. and skin of the fruit of Mvrtus communis L.

Preparation and characterization of the seedless fruit extract of Berberís vulgaris L. (hereinafter, “BV1 extract”)

First, the fruits ofBerberís vulgarisL. were selected. with an optimal ripeness state (11.0 - 19.0° Brix, determined by an Atago refractometer at 20 °C according to official method 932.14C (AOAC, 2005), and the seeds were separated, thus obtaining the fruit (skin and pulp) of Berberis vulgaris L. without seeds. Then, said fruit was subjected to freeze-drying, specifically, to a 5-day freeze-drying cycle at - 80 °C (± 5 °C) at 0.029 mbar, and subsequent homogenization of the particle size, until reaching approximately 0.150 mm, using an IKA Multidrive Basic mill (BS000) and subsequent verification by granulometry through a sieve with a Mesh number (U.S. STD. Sieve) of 100 (corresponding to 0.149 mm or 0.0059 inches). The extract of the seedless fruit Berberis vulgarisL. was then obtained by hydroalcoholic extraction under ultrasonic power conditions (Fisherbrand™, Sonic Dismembrator, Model 705) of 325 - 375 W, with a solid-liquid ratio of 20 - 28 g/L (i.e., proportion of lyophilized Berberis vulgarisL. seedless fruit extract by weight with respect to the volume of hydroalcoholic medium), for a time between 1.5 and 4 minutes, using an ethanol-water mixture (80%:20%, v/v) acidified with citric acid to pH 3 as extraction solvent. This allowed to obtain a hydroalcoholic extract of Berberis vulgarisL. seedless fruit rich in anthocyanins (extract BV1).

The BV1 extract had a total monomeric anthocyanin content of 9.15 mg cya-3-gluE/g BV1 extract (where “cya-3-gluE” stands for “cyanidin-3-glucoside equivalents”), analyzed by the differential pH spectrophotometric method. In this analytical method, the sample is placed in a solution with pH 1 so that the anthocyanins are in the oxonium form, this being their most stable form, and in another solution with pH 4.5 where the anthocyanins pass into a hemiacetal form and are colorless. The quantification is carried out from the difference in absorbance between both solutions, measured at the wavelength of greatest absorption of the majority anthocyanin (500-560 nm).

Likewise, the BV1 extract was analyzed by ultra high performance liquid chromatography (UHPLC-DAD) under the following conditions: Agilent LC-1290II, Poroshell 120 SB-C18 column 4.6 mm x 75 mm 2.7 pm, 35 °C, flow 0.75 mL/min, trifluoroacetic acid (0.1%)/ Acetonitrile. Through this analysis, it was determined that the majority anthocyanins were delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunidin-3-O-glucoside, pelargonidin-3-O-glucoside and malvidin-3-O-glucoside. The color of the BV1 extract was determined using a Hunter Color Flez CiE Illuminate C colorimeter, 2° and geometry 45/0°, using cylindrical glass cuvettes of 5 cm diameter and 1.3 cm height, and the following values were obtained, expressed in the CIELAB system: L*: 23.44, a*: 48.00 and b*: 2.94 at pH 3.

Preparation and characterization of the extract from the skin of the fruit of Myrtus communis L. (hereinafter, “MC1 extract”)

On the other hand, the fruits of Myrtus communis L. were selected. with an optimal ripeness state (6.0 - 9.0 ° Brix, determined by Atago refractometer at 20 °C according to official method 932.14C (AOAC, 2005), and the skins were separated from the pulp and the seed, thus obtaining Myrtus communis L. fruit skin devoid of pulp and seeds. Next, said skin was subjected to lyophilization, specifically, to a 5-day cycle of lyophilization at - 80 °C (± 5 °C) at 0.029 mbar, and subsequent homogenization of the particle size (0.150 mm) using an IKA Multidrive Basic mill (BS000). The extract of the Myrtus communis L. fruit skin was obtained by hydroalcoholic extraction under ultrasonic power conditions (Fisherbrand ™, Sonic Dismembrator, Model 705) of approximately 450 - 500 W, at a solid-liquid ratio of 18 - 22 g/L (i.e., ratio of freeze-dried seedless fruit extract of Mydus communis L. by weight to the volume of hydroalcoholic medium) for 18 to 22 minutes, using an ethanol-water mixture (80%:20%, v/v) with a pH value of 6 as extraction solvent. This made it possible to obtain a hydroalcoholic extract of Mydus communis L. rich in anthocyanins (extract MC1).

The results showed that the MC1 extract had a total monomeric anthocyanin content of 26.34 mg cya-3-gluE/g MC1 extract. The MC1 extract was also analyzed by UHPLC, under the following conditions: Agilent LC-1290II, Poroshell 120 SB-C184.6 mm x 75 mm column, 2.7 |jm, 35 °C, flow rate 0.75 mL/min, trifluoroacetic acid (0.1%)/ Acetonitrile. The major anthocyanins were determined to be delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunidin-3-O-glucoside, and malvidin-3-O-glucoside. The color of the MC1 extract was determined using a Hunter Color Flez CiE Illuminate C colorimeter, 2° and geometry 45/0°, using cylindrical glass cuvettes of 5 cm diameter and 1.3 cm height, and the following values were obtained, expressed in the CIELAB system: L*: 6.38, a*: 29.60 and b*: 8.35 at pH 3 and L*: 7.65, a*: 22.40 and b*: 3.00 at pH 6.

Preparation of the coloring composition in liquid form comprising the seedless fruit of Barberis vulgaris L. and the peel of the fruit of Myrtus communis L. (hereinafter referred to as the coloring composition "BV1:MC1")

Finally, both individual extracts, BV1 and MC1, were mixed in quantities corresponding to 50%:50% (pip) ± 10% of Berberis vulgaris L. and Mydus communis L., giving rise to a coloring composition according to the invention, in liquid form, more particularly in the form of a hydroalcoholic solution, comprising peel extracts of Berberis vulgaris L. and Myrtus communis L. (coloring composition BV1:MC1).

Example 2 - Coloring composition, in the form of a lyophilized powder, of extracts of the seedless fruit of Barberis vulgaris L. and the peel of Mvrtus communis L.

Individual extracts BV1 and MC1 were obtained as described in Example 1, which were used as follows:

Preparation and characterization of the seedless fruit extract of Berberis vulgaris L. in the form of lyophilized powder (hereinafter, “BV2 extract”).

From the BV1 extract, the ethanol was evaporated using a sample concentrator (SpeedVac SPD130DLX equipment; Thermoscientific) at 65 °C and 2.6 torr of pressure, corresponding to 346.6 Pa. The resulting ethanol-free extract was then frozen at -80 °C and subjected to lyophilization (lyophilization equipment: Freezone; 4.5 L; LABCONCO) at the same temperature and 0.029 mbar of pressure, corresponding to 2.9 Pa, and subsequent homogenization using an IKA Multidrive Basic mill (BS000), until a homogeneous powder with a particle size of 0.150 mm was obtained, thus obtaining a freeze-dried extract rich in anthocyanins from the seedless fruit of Berberis vulgaris L. (extract BV2).

The lyophilized extract BV2 presented an extraction yield of 65.8% and a moisture content of 15.14 g of water/100 g of extract. Within its chemical composition, it was determined that the extract presented a total of polyphenols, measured by the Fast Blue BB method (Palombini, et al., 2016), of 159.84 mg GAE/g dry extract (where “GAE” means “gallic acid equivalents”). The Fast Blue BB method is based on subjecting the sample to a reaction with the fast blue BB reagent (i.e., 4'-amino-2',5'-diethoxybenzanilide) at a concentration of 0.1% (w/v), in the presence of 5% (v/v) NaOH. When in an alkaline medium, the phenolic groups of the polyphenols form a stable azo complex, which generates a color that is then measured by spectrophotometry at a wavelength of 420 nm.

Of this total polyphenol value corresponding to 159.84 mg GAE/g dry extract, hydroxybenzoic acids are the majority component, with 44.96 mg GAE/g dry extract, followed by hydroxycinnamic acids with 25.58 mg FAE/g (where “FAE” means “ferulic acid equivalents”) of dry extract, it also contains 5.50 mg QE/g (where “QE” means “quercetin equivalents”) of dry extract of flavonols, all of these being measured following the QUENCHER methodology (QUIck, Easy, New, CHEap and Reproducible), which starts from a small amount of sample, previously homogenized to a particle size of 0.037 mm, which is subjected to direct contact with the reagents of each determination. This methodology allows to measure both soluble molecules and insoluble compounds in a precise and reliable way, and allows a subsequent spectrophotometric analysis. wavelengths of greatest absorption of each phenolic group, specifically at 280 nm for hydroxybenzoic acids, 320 nm for hydroxycinnamic acids and 370 nm for flavonols, respectively, these being the wavelengths of greatest absorption of each phenolic group. Likewise, by high performance liquid chromatography coupled to an ultraviolet - visible detector (HPLC-UV / Vis), after extraction in metaphosphoric acid (4.5%) and separation and quantification by UV-VIS (Thermo Separation Spectra Series UV100) at a wavelength of 215 nm using a Sphreclone ODS column (Phenomenex), three major organic acids present in the extract were identified, specifically 0.573 mg / g dry extract of ascorbic acid, 5.95 mg / g dry extract of oxalic acid and 446.34 mg / g dry extract of citric acid.

On the other hand, the extract presented a total of 12.42 mg cya-3-gluE/g extract of total monomeric anthocyanins determined by the differential pH spectrophotometric method (see description of the differential pH method used in Example 1). Six individual anthocyanins were identified by HPLC-DAD-MS-ESI (Dionex Ultimate 3000 UPLC, Thermo Scientific, coupled to an electrospray ionization mass spectrometer (Linear Ion Trap LTQ XL, Thermo Scientific, C18 Water Spherisorb ODS2 Column), and a total of five anthocyanins were quantified by ultrafast high performance liquid chromatography coupled to a diode array detector (UHPLC-DAD), under the following conditions: Agilent LC-1290II, Poroshell 120 SB-C18 column, 4.6 mm x 75 mm, 2.7 pm, 35 °C, flow rate 0.75 mL/min, trifluoroacetic acid (0.1%)/Acetonitrile. These anthocyanins were delphinidin-3-O-glucoside (2.90 mg/g dry extract), cyanidin-3-O- glucoside (2.20 mg/g dry extract), petunidin-3-O-glucoside (1.21 mg/g dry extract), pelargonidin-3-O-glucoside (0.55 mg/g dry extract) and malvidin-3-O-glucoside (2.46 mg/g dry extract) (Figure 1).

The inventors analyzed the coloring activity of the BV2 extract, observing that it presented a variability of color depending on the pH, as well as the stability of an intense red color (L*: 12.87, a*: 37.05, b*: 17.83) between pH values from 1 to 3.5, going on to take a pale pink color (L*: 13.97, a*: 5.21, b*: 13.01) between pH values from 4 to 6 and a greenish yellow color (L*: 52.41, a*: -0.88, b*: 23.25) at pH values higher than 7.5. Specifically, the BV2 extract at pH 4.5 had a color close to the standard RAL 0202029 color of the RAL design color system, and at pH 6.5 it had a color close to the S8010 Y10R standard of the NCS 2050 color system (Natural Color System is an international color system for design, architecture, production, research and education).

BV2 was shown to have a chromatic profile with a characteristic color that evolved with pH, in aqueous solution at a concentration of 20 mg/mL (Table 1):

Table 1:

1 RAL Design System, 2 BS Other System, 3 NCS 2050 System.

On the other hand, the preservative activity (antibacterial and antifungal) of BV2 was analyzed, which allowed to conclude that BV2 presents inhibitory activity against bacteria involved in foodborne diseases and required analysis in food products: For this test the reference strains used were: Enterobacter cloacae (ATCC 49741), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 9027), Salmonella enterocolitica (ATCC 13076), Yersinia enterocolitica (ATCC 8610), Bacillus cereus (ATCC 11778), Listeria monocytogenes (ATCC 19111) and Staphylococcus aureus (ATCC 25923). Specifically, the BV2 extract showed inhibitory activity for the growth of three Gram-negative bacteria and three Gram-positive bacteria, presenting minimum inhibitory concentrations and minimum bactericidal concentrations (MIC/MBC) of 10/20, 5/10, 2.5/10, 5/10 and 1.25/20 mg/mL of extract against the Gram-negative reference bacteria strains Yersinia enterocolilica (ATCC 8610), Enterobacter cloacae (ATCC 49741), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 9027) and Salmonella enterocolilica (ATCC 13076), respectively, and minimum inhibitory concentrations and minimum bactericidal concentrations (MIC/MBC) of 1.25/10, 5/20, 2.5/20 mg/mL against the strains Gram-positive reference bacteriaBacillus cereus(ATCC 11778),Listeria monocytogenes(ATCC 19111) andStaphylococcus aureus(ATCC 25923), respectively. On the other hand, the extract also presents antifungal activity with minimum inhibitory concentrations (MIC) of 5 and 10 mg/mL of extract against some fungi present in food, such asAspergillus brasiliensisandAspergillus fumigatus, respectively. For this purpose, the BV2 extract was serially diluted in a microplate. For each dilution, an inoculum of a reference bacterium was added to each well, ensuring the presence of 1.5 x 105 colony forming units (CFU) per well. The wells were incubated at 37 °C for 24 hours. After incubation with iodonitrotetrazolium chloride (INT), used as a dye, the minimum inhibitory concentration (MIC) was determined. This is the minimum concentration that inhibits the growth of bacteria visible by yellow coloration and that did not change to pink. The bacteria in the wells that remained yellow were subsequently seeded on solid medium and incubated at 37 °C for 24 hours. The lowest concentration without bacterial growth was determined as the minimum bactericidal concentration (MIC). In the case of antifungal activity, the minimum inhibitory concentration (MIC) was determined by binocular microscope.

The antioxidant activity of the BV2 extract was also evaluated using two in vitro chemical antioxidant activity methods (Folin-Ciocalteu and DPPH) and one in vitro biological antioxidant activity method (OxHLIA). In the Folin-Ciocalteu method, reducing compounds present in the sample react with phosphomolybdotungstic acid in the Folin-Ciocalteu reagent, generating a measurable blue color at a wavelength of 750 nm. In the DPPH method, the DPPH (2,2-diphenyl-1-picryhydrazyl) solution reacts with the antioxidant molecules in the sample, changing its color from purple to yellow. This color loss can be monitored by spectrophotometric readings at 517 nm. The OxHLIA method, on the other hand, is an in vitro biological method in which red blood cells extracted from sheep blood, diluted in PBS, are brought into contact with the corresponding fruit extract and AAPH (2,2'-azobi(2-methylpropionamidine) dihydrochloride), which generates free radicals. Optical density is then measured at 690 nm every 10 minutes until complete hemolysis is observed.

The BV2 extract showed high antioxidant activity with values of 88.03 mg GAE/g dry extract using the Folin-Ciocalteu methodology and 111.37 mg TE/g dry extract using the DPPH methodology (where “TE” means “Trolox equivalents”). In the case of OxHLIA, an IC50 of 125 pg/mL was obtained for antihemolytic activity over 60 minutes. The expression “IC50” means “median inhibitory concentration”, that is, it refers to the concentration of inhibitor that causes a 50% decrease in the analyzed activity. Finally, although the consumption of Berberís vulgaris L. fruits is considered safe, and has been traditionally consumed for generations in the Iberian Peninsula, mainly in Spain, toxicity studies were conducted. Thus, the possible toxicity of the fruit in non-tumoral cells of pig liver (PLP2) was evaluated by monitoring cell growth by phase contrast microscopy, obtaining as a result the concentration of the sample capable of inhibiting 50% of net cell growth, which in this case was a concentration greater than 400 pg/mL of extract, evidencing zero hepatoxicity.

Preparation and characterization of the extract of the skin of the fruit of Myrtus communis L. in the form of lyophilized powder (hereinafter “MC2 extract”)

From the MC1 extract, the ethanol was evaporated using a sample concentrator (SpeedVac SPD130DLX equipment; Thermoscientific) at 65 °C and 2.6 torr of pressure, corresponding to 346.6 Pa. The resulting ethanol-free extract was then frozen at - 80 °C and subjected to lyophilization (lyophilization equipment: Freezone; 4.5 L; LABCONCO) at the same temperature and 0.029 mbar of pressure, corresponding to 2.9 Pa, and subsequent homogenization using an IKA Multidrive Basic mill (BS000), until a homogeneous powder was obtained (particle size of 0.150 mm), thus obtaining a freeze-dried extract rich in anthocyanins from the skin of the fruit of Myrtus communis L. (extract MC2).

The lyophilized extract MC2 presented an extraction yield of 57.6% and a moisture content of 9.22 g of water/100 g of extract. Within its chemical composition, it was determined that the extract presented a total of 271.18 mg GAE/g of dry extract in polyphenols, with hydroxybenzoic acids being the most present with a value of 44.96 mg GAE/g of dry extract. In addition, this extract presented a quantity of flavonols of 20.33 mg/g of dry extract and a total of hydroxybenzoic acids of 17.78 mg/g of dry extract. These flavonols were determined following the QUENCHER methodology, as described above in the section on the preparation and characterization of BV2.

On the other hand, by HPLC-UV/Vis, after extraction in metaphosphoric acid (4.5%) and separation and quantification by UV-VIS (Thermo Separation Spectra Series UV100) at a wavelength of 215 nm using a Sphreclone ODS column (Phenomenex), three organic acids present in the extract were identified, specifically 4.50 mg of ascorbic acid/g dry extract, 6.37 mg of citric acid/g dry extract and 7.16 mg of oxalic acid/g dry extract.

Likewise, the total monomeric anthocyanins were determined by the differential pH method (see description of this method in Example 1) obtaining a total of 47.51 mg cya-3-gluE/g dry extract. In addition, 9 individual anthocyanins (Figure 2) were identified by HPLC-DAD-ESI-MS (Dionex Ultimate 3000 UPLC, Thermo Scientific, coupled to an electrospray ionization mass spectrometer (Linear Ion Trap LTQ XL, Thermo Scientific, C18 Water Spherisorb ODS2 column), and a total of 4 anthocyanins were quantified by UHPLC-DAD, under the following conditions: Agilent LC-1290II, Poroshell 120 SB-C18 column, 4.6 mm x 75 mm, 2.7 pm, 35 °C, flow rate 0.75 mL/min, trifluoroacetic acid (0.1%)/acetonitrile: delphinidin-3-O-glucoside (9.41 mg/g dry extract, cyanidin-3-O-glucoside (1.63 mg/g dry extract), petunidin-3-O-glucoside (9.41 mg/g of dry extract) and malvidin-3-O-glucoside (10.30 mg/g of dry extract).

The MC2 extract was found to exhibit color variability depending on the pH, and stability was evident from a deep purple color (L*: 11.42, a*: 34.61, b*: 15.45) at pH 3.0 to a bluish-purple color (L*: 4.22, a*: 13.71, b*: 0.05) at pH 6. Specifically, the MC2 extract had a color close to the RAL 0102025 color standard of the RAL Design color system at pH 4.5).

MC2 was shown to have a characteristic chromatic profile that evolved with pH, in aqueous solution at a concentration of 4 mg/mL (Table 2):

Table 2:

1 RAL Design System, 2 NCS 2050 System, 3 RAL Classic System.

The MC2 extract also showed antimicrobial activity against five Gram-negative bacteria and three Gram-positive bacteria used as reference strains, presenting minimum inhibitory concentrations and minimum bactericidal concentrations (MIC/MBC) of 10/20 mg/mL extract against the Gram-negative bacteria Enterobactercloacae (ATCC 49741), Pseudomonas aeruginosas (ATCC 9027) and Yersinia enterocolitica (ATCC 8610), and 5/20 and 2.5/20 against the Gram-negative bacteria Escherichia coli (ATCC 25922) and Salmonella enterica (ATCC 13076), respectively, as well as MIC/MBC of 0.6/20 mg/mL extract against the Gram-positive bacteria strains Bacillus cereus (ATCC 11778), Listeria monocytogenes (ATCC 19111). and Staphylococcus aureus (ATCC 25923), respectively. Furthermore, the extract also displayed antifungal activity with an MIC of 1.25 mg/mL against the fungus Aspergillus brasiliensis. The same procedure described above for the BV2 extract was used.

Antioxidant activity was determined by the in vitro chemical methods Folin-Ciocalteu and DPPH, and the in vitro biological method OxHLIA, as described above, in the section relating to the preparation and characterization of the BV2 extract in Example 2. Antioxidant activities of 96.81 mg GAE/g and 73.90 mg TE/g were observed for the MC2 extract by the Folin-ciocalteu and DPPH methodologies, respectively, and an IC50 of 59 pg/mL was obtained for the antihemolytic activity over 60 days by the OxLHIA methodology.

Finally, although consumption of Myrtus communis L. fruits is considered safe, and its wild fruit has been traditionally consumed for generations, toxicity studies were conducted. Thus, the potential toxicity of the fruit in non-tumorous pig liver cells (PLP2) was evaluated, revealing no hepatotoxicity (>400 pg/mL extract).

Preparation and characterization of the coloring composition in the form of lyophilized powder comprising the extract of the seedless fruit of Berberís vulgaris L. and the extract of the skin of the fruit of Myrtus communis L. (hereinafter referred to as the coloring composition “BV2:MC2”)

Once the individual BV2 and MC2 extracts of each fruit of the different plant species were obtained, a coloring composition according to the invention (BV2:MC2 coloring composition) was obtained in the form of a lyophilized powder by mixing both BV2 and MC2 extracts in proportions of 50:50 w/w ± 10%. This coloring composition, suitable for use as a food ingredient in the form of a lyophilized powder, had the desired color characteristics at pH 3.5, as well as suitable functional and organoleptic properties.

It is worth highlighting, with respect to the BV2:MC2 composition, the total amount of polyphenols (281.59 mg GAE/g dry extract) measured by the Fast Blue BB method, with hydroxybenzoic acids being the most present with a value of 86.29 mg GAE/g dry extract. In addition, this extract presents a total of hydroxycinnamic acids of 32.28 mg FAE/g dry extract and flavonols of 14.77 mg QE/g dry extract, measured according to the QUENCHER methodology described above.

On the other hand, by HPLC-UV/Vis, after extraction in metaphosphoric acid (4.5%) and separation and quantification by UV-VIS (Thermo Separation Spectra Series UV100) at a wavelength of 215 nm using a Sphreclone ODS column (Phenomenex), three organic acids present in the extract were identified, specifically 2.58 mg of ascorbic acid/g dry extract, 6.61 mg of oxalic acid/g dry extract and 230.20 mg of citric acid/g dry extract.

Likewise, the total monomeric anthocyanins were determined by the differential pH method (see description of this method in Example 1) obtaining a total of 39.91 mg cya-3-gluE/g dry extract. Furthermore, 11 individual anthocyanins were identified by HPLC-DAD-MS-ESI analysis (Dionex Ultimate 3000 UPLC, Thermo Scientific, coupled to a Linear Ion Trap LTQ XL electrospray ionization mass spectrometer, Thermo Scientific, Spherisorb ODS2 C18 Water Column), which are shown in Figure 3 with their corresponding chromatographic peaks and retention value Rt, and 5 anthocyanins were quantified by UHPLC-DAD (Agilent LC-1290II, Poroshell 120 SB-C18 column, 4.6 mm x 75 mm, 2.7 pm, 35 °C, flow rate 0.75 mL/min, trifluoroacetic acid (0.1%)/Acetonitrile), delphinidin-O-glucoside (6.70 mg/g dry extract), cyanidin-3-O-glucoside (1.66 mg/g dry extract), petunidin-3-O-glucoside (6.21 mg/g dry extract), pelargonidin-3-O-glucoside (0.12 mg/g dry extract) and malvidin-3-O-glucoside (7.16 mg/g dry extract).

Said composition also had a characteristic color profile due to the unique composition of monomeric anthocyanins achieved by combining the seedless fruit extracts of Berberis vulgaris L. and Myrtus communis L. peel according to the present invention. Specifically, thanks to the synergistic combination of these extracts of both fruits, the coloring compositions of the present invention make it possible to achieve a characteristic magenta color that is particularly stable at a pH of 3-3.5 and a purple color that is stable at pH values of 5.5-6.0.

Specifically, this BV2:MC2 composition showed color variability in aqueous solution depending on the pH, although an unexpected stability of a reddish purple color (L*: 6.90, a*: 22.09, b*: 5.12) was observed at pH 3.5, a purple color (L*: 7.06, a*: 14.05, b*: -0.74) at pH values of 5.5, and a grayish green color was evident at pH values above 7.5 (L*: 5.45, a*: 0.52, b*: 2.02). Specifically, the BV2:MC2 extract had a color close to RAL 8022 of the RAL Classic color system at pH 6.5 to 7.0.

The BV2:MC2 extract was shown to have a characteristic chromatic profile that evolved with pH, in aqueous solution at a concentration of 8 mg/mL (Table 3):

Table 3:

1 RAL Classic System.

On the other hand, the composition BV2:MC2 showed preservative activity (antibacterial and antifungal) against 5 Gram-negative bacteria and 3 Gram-positive bacteria used as reference strains, presenting MIC/MBC of 10/20 mg/mL for Yersinia enterocolilica (ATCC 8610), 5/20 mg/mL for Enterobacter Cloacae (ATCC 49741) and Pseudomonas aeruginosa (ATCC 9027) and 2.25/20 for the Gram-negative bacteria Escherichia coli (ATCC 25922) and Salmonella enterica (AtCc<13076). For Gram-positive bacteria, it presented MIC/MBC of >0.6/20 against Listeria monocytogenes (ATCC 19111) and Staphylococcus aureus (ATCC 25923), 0.6/20 for Bacillus cereus (ATCC 11778). On the other hand, the extract also presented antifungal activity with MIC of 5 mg/mL of extract against Aspergillus fumigatus, respectively. For this purpose, the same procedure as described above for both the BV2 extract and the MC2 extract was used.

Furthermore, the antioxidant activity of this BV2:MC2 composition was confirmed by the in vitro Folin-Ciocalteu and DPPH chemical methods, as described above in the section on the preparation and characterization of the BV2 extract in Example 2, obtaining values of 60.31 mg GAE/g and 164.42 mg TE/g dry extract, respectively. In addition, an IC50 of 29 pg/mL was obtained for the antihemolytic activity over 60 days using the OxLHIA methodology.

Example 3 - Coloring composition in the form of lyophilized powder of extracts of fruit without seeds of Berberís vulgaris L. and skin of the fruit of Mvrtus communis L. in the form of microencapsulated lyophilized powder

Individual extracts BV2 and MC2 were obtained as described in Example 2, which were used as follows:

Preparation and characterization of the seedless fruit extract of Berberís vulgaris L. in the form of microencapsulated lyophilized powder (hereinafter, “BV3 extract”)

From the BV2 extract, encapsulation was carried out by spray-drying (Buchi, Mini Spray Dryer, model B-290), with maltodextrin as encapsulating agent, at a concentration of 35% - 45% by weight (g/100 g), temperature of 120 °C, aspiration at 95%, feed flow of 5 mL/min and an air flow of 7.88 L/min, thus obtaining the microencapsulated extract from Berberis vulgaris l. (BV3 extract) with a particle size of 1 - 44 pm determined by Mastersizer 3000 (Malvern, UK).

The BV3 extract presented an encapsulation extraction yield of 60.4%, and an amount of total phenolic compounds of 61.06 mg GAE/g microencapsulated extract determined by the Fast Blue BB method under the conditions described above in the section corresponding to the preparation and characterization of the BV2 extract in Example 2. Within these phenolic compounds, hydroxybenzoic acids were the most representative with 36.47 mg GAE/g microencapsulated extract, followed by an amount of hydroxycinnamic acids and flavonols of 10.15 mg FAE/g and 2.41 mg QEq/g, respectively, all of these being measured following the QUENCHER methodology, as described above in the section relating to the preparation and characterization of BV2.

Likewise, a quantity of total monomeric anthocyanins of 5.57 mg cya-3-glu/g microencapsulated extract was obtained, determined by the differential pH spectrophotometric method (see description of the differential pH method in Example 1). In addition, a total of 5 anthocyanins were quantified by ultra high performance liquid chromatography coupled to a diode array detector (UHPLC-DAD), under the following conditions: Agilent LC-1290II, Poroshell 120 SB-C18 column, 4.6 mm x 75 mm, 2.7 pm, 35 °C, flow 0.75 mL/min, trifluoroacetic acid (0.1%)/ Acetonitrile. Specifically, the following anthocyanins were quantified: delphinidin-3-O-glucoside (1.91 mg/g dry extract), cyanidin-3-O-glucoside (1.53 mg/g dry extract), petunidin-3-O-glucoside (0.77 mg/g dry extract), pelargonidin-3-O-glucoside (0.28 mg/g dry extract) and malvidin-3-O-glucoside (1.66 mg/g dry extract).

Regarding the chromatic profile of BV3, a particular microencapsulated sample was studied with 40% by weight of maltodextrin (i.e., 40 g maltodextrin/100 g microencapsulated sample) as an encapsulating agent, which presented a characteristic chromatic profile that evolved with the pH, in aqueous solution with a concentration of 20 mg/mL (Table 4):

Table 4:

1 RAL Classic System, 2 Sherwin-Wiliams System, 3 PPG/Johnstone's System, 4 lsomat System, 5 Dulux Trade System, 6 RAL Design System.

In aqueous solution, the BV3 extract presented a desired intense reddish color (L*: 19.43, a*: 41.43, b*: 27.46) at a pH of 3. At pH 5.5 (approximate pH of cream-type ice cream), a slight purple color was observed (L*: 33.18, a*: 17.466b*: 9.20) that resembles the standard color LPC 0416 of the lsomat system.

The BV3 extract also showed antimicrobial activity against 5 Gram-negative bacteria and 3 Gram-positive bacteria used as reference strains, presenting MIC/MBC of 5/20 mg/mL for the Gram-negative bacteria Enterobacter cloacae (ATCC 49741) and Escherichia coli (ATCC 25922) and 10/20, 2.5/20 and 10/10 mg/mL for the Gram-negative bacteria Pseudomonas aeruginosa (ATCC 9027), Salmonella enterica (ATCC 13076) and Yersinia enterocolitica (ATCC 8610), respectively, and MIC/MBC of 2.5/20, 5/20 and 5/20 mg/mL against the Gram-positive bacteria Bacillus cereus (ATCC 11778) and Listeria monocytogenes (ATCC 19111). and Staphylococcus aureus (ATCC 25923), respectively. Furthermore, the microencapsulated extract exhibited antifungal activity with a minimum inhibitory concentration of 10 mg/mL against the fungus Aspergillus fumigatus. For this purpose, the same procedure as described above for both the BV2 extract and the MC2 extract was used. Furthermore, the antioxidant activity was evaluated using the Folin-Ciocalteu methods and the DPPH methodology (see description of these methods in the section on the preparation and characterization of the BV2 extract in Example 2), presenting antioxidant activity values of 46.54 mg TE/g and 18.43 mg GAE/g using the DPPH and Folin-Ciocalteu methodologies, respectively. An IC50 value of 293 pg/mL was also determined using the in vitro biological antioxidant activity method OxHLIA (see description of this method in the section on preparation and characterization of BV2 extract in Example 2).

Preparation and characterization of the extract of the skin of the fruit of Myrtus communis L. in the form of microencapsulated lyophilized powder (hereinafter “MC3 extract”)

From the MC2 extract, encapsulation was carried out by spray-drying (Buchi, Mini Spray Dryer, model B-290), with maltodextrin as encapsulating agent, at a concentration of 25% - 45% (g/100 g), temperature of 140 °C, aspiration at 95%, feed flow of 5 mL/min and an air flow of 7.88 L/min, thus obtaining the microencapsulated extract from M. communis (MC3 extract) with a particle size of 0.5 - 40 pm determined by Mastersizer 3000 (Malvern, UK).

The MC3 extract presented an encapsulation yield of 69.6% and a total polyphenol content of 112.09 mg GAE/g microencapsulated extract (determined by the Fast BlueBB method), with hydroxybenzoic acids being the majority with 38.65 mg GAE/g of microencapsulated extract, followed by hydroxycinnamic acids with 6.83 mg FAE/g and flavonols with 7.03 mg QE/g microencapsulated extract, respectively. All these values were determined following the QUENCHER methodology, as described above in the section on the preparation and characterization of BV2.

A total of 21.06 mg cya-3-gluE/g microencapsulated extract of monomeric anthocyanins was also determined by the differential pH spectrophotometric method (see description of the differential pH method in Example 1). In addition, a total of 4 anthocyanins were quantified by UHPLC-DAD, under the following conditions: Agilent LC-1290II, Poroshell 120 SB-C18 column, 4.6 mm x 75 mm, 2.7 pm, 35 °C, flow rate 0.75 mL/min, trifluoroacetic acid (0.1%)/Acetonitrile. In this way, the following anthocyanins were quantified: delphinidin-3-0-glucoside (8.81 mg/g dry extract, cyanidin-3-0-glucoside (1.44 mg/g dry extract), petunidin-3-0-glucoside (8.30 mg/g dry extract) and malvidin-3-0-glucoside (8.64 mg/g dry extract).

Regarding the chromatic profile of MC3, a particular microencapsulated sample was studied with 55% - 65% by weight of maltodextrin (i.e., 55 - 65 g maltodextrin/100 g microencapsulated sample) as an encapsulating agent, which presented a characteristic chromatic profile that evolved with the pH, at a concentration of 13 mg/mL of aqueous solution (Table 5):

Table 5:

1 RAL Classic System.

A range of very intense purples with a characteristic color was obtained. Thus, MC3 presented a color that is not currently found in commercially available natural food coloring options. It was also verified that the MC3 extract was stable up to pH 7.0, from which olive green colors were observed (L* 2.40, a* 3.48 and b* 0.22), with a color similar to RAL 9005 of the RAL Classic color system, while the unencapsulated MC2 extract began to degrade at pH 6.5 (L* 3.29, a* 7.87 and b* 0.59).

In addition, the preservative activity of the MC3 extract was confirmed, presenting antimicrobial activity against 5 Gram-negative bacteria and 3 Gram-positive bacteria used as reference, specifically with MICs of 10 and 20 mg/mL against the Gram-negative bacteria Enterobacter cloacae (ATCC 49741) and Pseudomonas aeruginosas (ATCC 9027), respectively, and MICs/MBCs of 5/20, 2.5/20 and 10/20 mg/mL against the Gram-negative bacteria Salmonella enterica (ATCC 13076), Escherichia coli (ATCC 25922) and Yersinia enterocolitica (ATCC 8610), respectively. Regarding Gram-positive bacteria, it presented MIC/MBC of 5/20, 20/20 and 10/20 mg/mL against the bacteria Bacillus cereus (ATCC 11778), Listeria monocytogenes (ATCC 19111) and Staphylococcus aureus (ATCC 25923), respectively. Furthermore, the microencapsulated extract presented antifungal activity against the fungus Aspergillus fumigatus with a minimum inhibitory concentration (MIC) of 10 mg/mL. For this purpose, the same procedure as described above for both the BV2 extract and the MC2 extract was used. On the other hand, it was determined that the extract had an antioxidant activity of 41.57 mg GEA/g microencapsulated extract and 83.90 mg TE/g microencapsulated extract using the Folin-ciocalteu and DPPH methodologies (see description of these methods in the section on the preparation and characterization of the BV2 extract in Example 2), respectively. An IC50 value of 315 pg/mL was also determined using the OxHLIA in vitro biological antioxidant activity method (see description of this method in the section on the preparation and characterization of the BV2 extract in Example 2).

Preparation of the coloring composition in the form of a microencapsulated lyophilized powder comprising the extract of the seedless fruit of Berberís vulgaris L. and the extract of the skin of the fruit of Myrtus communis L. (hereinafter referred to as the coloring composition “BV3:MC3”)

Finally, both individual extracts, BV3 and MC3, were mixed in amounts corresponding to 50%:50% w/w± 10% of Berberis vulgarisL. and Myrtus communisL., giving rise to a coloring composition according to the invention, in the form of a microencapsulated lyophilized powder, comprising extracts of the seedless fruit of Berberis vulgarisL. and the skin of the fruit Myrtus communisL. (coloring composition BV3:MC3), the chromatic profile of the BV3:MC3 extract at different pH at a concentration of 13 mg/mL of aqueous solution is shown in the following table (Table 6).

Table 6:

1 NSC2050 System, 2 Pantone® C(Coated) System, 3 RAL Effect System, 4 RALClassic System, 5 RAL Design System

Example 4 - Coloring composition of extracts of the seedless fruit of Berberís vulgaris L. and the skin of the fruit of Mvrtus communis L. in the form of encapsulated lyophilized powder (BV4:MC4)

Starting from the BV2:MC2 coloring composition, obtained as described in Example 2, the encapsulation process was carried out by spray-drying (Buchi, Mini Spray Dryer, model B-290) with maltodextrin as an encapsulating agent at a temperature of 120 °C, aspiration at 95%, with a feed flow of 5 mL/min, an air flow of 7.88 L/min and a proportion of 35% - 45% of encapsulating agent, obtaining a coloring composition in the form of microencapsulated lyophilized powder, comprising extracts of the seedless fruit of Berberis vulgaris L. and the skin of the fruit of Myrtus communis L. (hereinafter referred to as “BV4:MC4 coloring composition”), with a particle size of 0.5 - 25 pm determined by Mastersizer 3000 (Malvern, UK).

The encapsulated BV4:MC4 composition showed an encapsulation yield of 69.64%. It was determined to contain a total of 83.39 mg GAE/g of dry extract in polyphenols using the Fast Blue BB method under the conditions described above in the section on the preparation and characterization of the BV2 extract in Example 2, which allowed the identification of hydroxybenzoic acids as the majority with a total of 37.63 mg/g of dry extract, followed by hydroxycinnamic acids with 7.48 mg/g of dry extract and flavonols with a total of 5.76 mg/g of dry extract, measured using the QUENCHER methodology, as described above in the section on the preparation and characterization of BV2.

Likewise, a quantity of total monomeric anthocyanins of 12.78 mg cya-3-gluE/g microencapsulated extract was determined, analyzed by the differential pH spectrophotometric method (see description of the differential pH method in Example 1). In addition, 5 anthocyanins were quantified by UHPLC-DAD (Agilent LC-1290II, Poroshell 120 SB-C18 column, 4.6 mm x 75 mm, 2.7 pm, 35 °C, flow rate 0.75 mL/min, trifluoroacetic acid (0.1%)/ Acetonitrile): delphinidin-3-O-glucoside (4.17 mg/g dry extract), cyanidin-3-O-glucoside (1.17 mg/g dry extract), petunidin-3-O-glucoside (3.70 mg/g dry extract), pelargonidin-3-O-glucoside (0.03 mg/g dry extract) and malvidin-3-O-glucoside (4.15 mg/g dry extract).

A microencapsulated BV4:MC4 composition as 35%-45% by weight of maltodextrin (i.e., 35-45 g maltodextrin/100 g microencapsulated sample) as encapsulating agent, additionally, presented the following characteristic color profile, achieved between pH 3.5 and 6, in aqueous solution, at a concentration of 13 mg/mL (Table 7):

Table 7:

1 NCS 2050 System, 2 BS 2660 System.

The BV4:MC4 extract allowed to obtain a characteristic color and different from those obtained with the BV extract or MC extract individually, specifically an intense magenta color was obtained at pH 4.5 and at pH 5.5 it presented a color similar to S7020-R20B of the NCS 2050 color system and at pH 6.0 a color close to S8010-R10B of the previous color system. It was also experimentally identified that the extract was stable up to pH 7, from which greenish colors were obtained (L * 12.03, a * -0.89 and b * 8.69). Likewise, the BV4:MC4 extract presented preservative activity with antimicrobial activity against 5 Gram-negative bacteria and 3 Gram-positive bacteria used as reference strains, MIC/MBC of 10/20, 5/20, 10/20, 2.5/20 and 20/20 mg/mL against the Gram-negative bacteria Enterobacter cloacae (ATCC 49741), Escherichia coli (ATCC 25922), Pseudomonas aeruginosas (ATCC 9027), Salmonella enterocolitica (ATCC 13076) and Yersinia enterocolitica (ATCC 8610), respectively, and MIC/MBC 2.5/20, 10/20 and 10/20 mg/mL against the Gram-positive bacteria Basillus cereus (ATCC 11778), Listeria monocytogenes (ATCC 11779), Yersinia enterocolitica (ATCC 8610), and Yersinia enterocolitica (ATCC 8610). monocytogenes (ATCC 19111) and Staphylococcus aureus (ATCC 25923), respectively. On the other hand, the BV4:MC4 extract presented antifungal activity with a minimum inhibitory concentration of 20 mg/mL against the fungus, Aspergillus fumigatus. For this purpose, the same procedure described above for both the BV2 extract and the<m>C2 extract was used. Furthermore, this microencapsulated BV4:MC4 composition exhibited an antioxidant activity of 28.91 mg GAE/g and 45.84 mg TE/g microencapsulated extract using the Folin-Ciocalteu and DPPH methodologies, respectively, as described above in the section relating to the preparation and characterization of the BV2 extract in Example 2. A 1C50 value of 228 pg/mL was also determined using the OxHLIA in vitro biological antioxidant activity method (see description of this method in the section relating to the preparation and characterization of the BV2 extract in Example 2).

Example 5 - Prototype of gelatin formulated with a coloring composition of the present invention (BV2:MC2) vs. prototype of gelatin formulated with BV2 only (comparative) vs. prototypes formulated with synthetic commercial colorants (comparative)

Prototype of gelatin formulated with BV2 only

This prototype was formulated, per 100 g of gelatin, with 85 - 90% by weight of water, 8 - 10% by weight of sugar as sweetener, 1.53 - 2.5% by weight of gelatin (as base gelling ingredient), 0.1 - 0.5% by weight of E330, 0.30 - 0.35% by weight of BV2 extract, and flavoring. A gelatin with a pH of 3 - 3.5 was obtained, with CIELAB parameters of L*: 38.35, a*: 39.79, b*: 15.21, which correspond to a red hue (color close to RAL 0204040 of the RAL Design system or 21105 of the Federal Standard 595C system of US Federal Standard color guides), where the CIELAB parameters were influenced by the food matrix of the gelatin formulation.

A total anthocyanin content of 0.04 mg cya-3-gluE/g gelatin was also obtained, determined by the differential pH spectrophotometric method (see description of the differential pH method in Example 1). On the other hand, a shelf life of 15 days was determined (i.e., period in which the absence of microbial growth per gram of material analyzed is determined, of: Enterobacteriaceae (absence/g) and Salmonella (absence/25 g), or maximum mesophilic aerobes (103 c.f.u./g) and Listeria monocytogenes (100 c.f.u./g), according to Regulation (EC) No 2073/2005.

Prototype of gelatin formulated with BV2:MC2

This prototype was formulated, per 100 g of gelatin, 85 - 90% by weight of water, 8 - 10% by weight of sugar as sweetener, 1.53 - 2.5% of gelatin (as base gelling ingredient), 0.1 - 0.5% by weight of citric acid (E330), with 0.20 - 0.25% by weight of the BV2:MC2 composition (the BV2:MC2 composition contains 50%:50% w/w ± 10%), and flavoring. A gelatin with a pH of 3 - 3.5 and CIELAB parameters in the final product of L*: 23.69, a*: 35.50, b*: 10.49 were obtained, which correspond to a magenta hue (color close to S 5040-R10B of the NCS 2050 system or 7421 C of the Pantone® C color system).

Gelatin prototypes formulated with synthetic commercial colorants

For comparison with the available gelatins on the market, other prototypes were made using commercial colorants: E-124 (Ponceau 4R synthetic colorant) for the red hue, and E-163 (natural colorant, anthocyanins from purple carrot, radish, or grape juice) and E-133 (brilliant blue FCF) for the purple hue. In both cases, the base formulation was the same, with the only variation being the percentage of colorant incorporated into the mixture: 0.05-0.1% by weight was added in the case of the synthetic colorant E-124; 0.15-0.25% by weight in the case of the commercial natural colorant E-163; or 0.15-0.25% by weight of the mixture of commercial synthetic colorants in a 1:4 ratio (E133:E124).

Table 8:

1 RAL Design System, 2 NCS 2050 System, 3 Isomat System, 4 RAL Effect System, 5 DIN 6164 System.

As can be seen from the CIELAB values obtained at pH 3.5 (Table 8), when using commercial food colorings, completely different shades were obtained (reddish coloration in the case of the prototypes formulated individually with E-124 or E-163; bluish coloration in the case of the prototype formulated with the combination of E-124 and E-163) to those observed with the compositions of the present invention, illustrated by the gelatin prototype formulated with BV2:MC2, which led to a magenta shade at these pH values.

Comparative study of the chromatic profile and CIELAB parameters of the colorant E-163, extract of Berberís vulgaris L. (BV3) in the form of a microencapsulated lyophilized powder, extract of Mvrtus communis L. in the form of a microencapsulated lyophilized powder (MC3), colorant composition BV3:MC3 and colorant composition BV4:MC4 microencapsulated In Table 9, samples corresponding to the current colorant E-163 available on the market, from different natural sources, such as purple carrot, red cabbage, radish and grape, have been analyzed against samples of BV3, MC3, BV3:MC3 and the BV4:MC4 composition according to the present invention:

Table 9:

NCS 2050 System, 2 DIN 6164 System, 3 Pantone® U Syntheme (Uncoated), 4 RAL Design System, 5 Dulux Trade System, 6 Pantone® C System (Coated), 7 RAL Effect System

The food industry does not currently have natural origin coloring additives that have magenta-purple hues, so it is necessary to resort to the use of a mixture of synthetic colorants with blue hues such as E-131 (patent blue), E-132 (indigotin), E-133 (brilliant blue FCF) with E-163 (anthocyanin) or other colorants with red hues such as E-122 (azorubine), E-124 (Ponceau 4R), etc. The colorant E-163, available on the market, and analyzed in Table 9, provides a red-garnet coloration, but does not achieve a magenta-purple hue, unlike what happens with the compositions of the present invention.

In relation to the antioxidant capacity of the commercial dye E163, it was found that it presented DPPH and Folin-Ciocalteu values of 48.25 mg TE/g and 49.22 mg TE/g extract, respectively, determined as described in the section relating to the preparation and characterization of the BV2 extract in Example 2. An IC50 value of 225 pg/mL of extract was also determined using the OxHLIA in vitro biological method. Likewise, it was shown that the commercial natural colorant E-163 had preservative activity, showing antimicrobial activity against 4 bacteria used as reference strains, with MIC/MBC of 20/>20, 10/>20 and 2.5/20 mg/mL against the Gram-negative bacteria Enterobacter cloacae (ATCC 49741), Escherichia coli (ATCC 25922) and Salmonella enterica (ATCC 13076), respectively, and 2.5/20 and 10/>20 mg/mL against the Gram-negative bacteria Basillus cereus (ATCC 11778) and Listeria monocytogenes (ATCC 19111), respectively. Said dye also exhibited antifungal activity with a minimum inhibitory concentration of 20 mg/mL against the fungus Aspergillus fumigatus. These data demonstrate the superior preservative capacity of the present invention.

With the present invention, a series of coloring compositions based on extracts of natural origin with coloring power in different presentations are obtained (hydroalcoholic extract, lyophilized extract in powder form and microencapsulated extract in powder form) that may serve as an alternative in the food industry for the formulation of a wide range of foods whose food matrix has a pH between 2.5 and 6.0, preferably 3.0 - 3.5 and/or 5.5 - 6, providing magenta - purple shades without the need to resort to the use of synthetic additives, with the potential health benefit derived therefrom (e.g., reduction of ADHD, allergic reactions, etc.).

Study of the antimicrobial and antifungal activity of extracts BV2, MC2, BV3 and MC3, of the compositions BV2:MC2 and BV4:MC4, and of the dye E-163

Table 10 summarizes the results obtained in the antimicrobial activity tests of the extracts BV2, MC2, BV3 and MC3, and the compositions BV2:MC2 and BV3:MC3, obtained as described in the previous Examples, as well as the results obtained with the commercial dye E-163. All these results were obtained by the method described in Example 2 of the present invention:

Table 10:

Claims (28)

1. Coloring composition based on anthocyanins, characterized in that it comprises an extract of the seedless fruit of Berberis vulgaris L. and an extract of the skin of the fruit of Myrtus communis L. 2. Composition according to claim 1, comprising an amount of said extract of Berberis vulgaris L. seedless fruit comprised between 40% and 60% by weight, relative to the total weight of the composition, and/or an amount of said extract of the skin of the fruit of Myrtus communis L. comprised between 40% and 60% by weight, relative to the total weight of the composition. 3. Composition according to the preceding claim, comprising an amount of said extract of Berberis vulgaris L. seedless fruit comprised between 45% and 55% by weight, relative to the total weight of the composition, and/or an amount of said extract of the skin of the fruit of Myrtus communis L. comprised between 45% and 55% by weight, relative to the total weight of the composition. 4. Composition according to any of claims 1-3, which is in liquid form or in lyophilized powder form. 5. Composition according to any of claims 1-3, which is in liquid form or in the form of a hydroalcoholic solution. 6. Composition according to any of claims 1-4, which is in the form of encapsulated lyophilized powder. 7. Composition according to claim 6, wherein the encapsulated lyophilized powder is microencapsulated. 8. Composition according to claim 6 or 7, further comprising at least one encapsulating agent. 9. Method for preparing a coloring composition based on anthocyanins, characterized in that it comprises: a) subjecting at least a portion of seedless fruit of Berberis vulgaris L. to hydroalcoholic extraction to obtain a seedless fruit extract of Berberis vulgaris L., b) subjecting at least a portion of Myrtus communis L. fruit peel to hydroalcoholic extraction to obtain an extract of the Myrtus communis L. fruit peel, and c) mixing a quantity of the extract of the seedless fruit of Berberis vulgaris L. resulting from step a) and a quantity of the extract of the skin of the fruit of Myrtus communis L. resulting from step b). 10. Method according to claim 9, wherein the amount of the extract of the seedless fruit of Berberis vulgarisL and the amount of the extract of the skin of the fruit of Myrtus communisL that are mixed in step c) are each independently comprised between 40-60% by weight, relative to the total weight of the coloring composition. 11. Method according to claim 9 or 10, wherein the amount of the extract of the fruit without seeds of Berberís vulgarisL and the amount of the extract of the skin of the fruit of Myrtus communisL that are mixed in step c) are each independently comprised between 45-55% by weight, relative to the total weight of the coloring composition. 12. Method according to any of claims 9-11, wherein, before step a) and/or step b), said portion of fruit devoid of seeds of Berberís vulgaris L. and/or said portion of skin of the fruit of Myrtus communis L., respectively, are subjected to a freeze-drying step. 13. Method according to any of claims 9-12, wherein, in step a) and/or in step b), said hydroalcoholic extraction is carried out with an ultrasound probe. 14. Method according to any of claims 9-13, wherein, in step a), said hydroalcoholic extraction is carried out with a proportion of said fruit devoid of Berberis vulgarisL seeds with respect to hydroalcoholic solvent of between 20 and 28 g/L. 15. Method according to any of claims 9-14, wherein, in step a), said hydroalcoholic extraction is carried out in a hydroalcoholic medium that provides an acidic medium, preferably in a hydroalcoholic medium that provides a pH between 2 and 4, more preferably in a hydroalcoholic medium that provides a pH between 3 and 3.5. 16. Method according to any of claims 9-15, wherein, in step b), the hydroalcoholic extraction treatment is carried out in a hydroalcoholic medium that provides an acidic pH, preferably in a hydroalcoholic medium that provides a pH between 5 and 7, more preferably in a hydroalcoholic medium that provides a pH between 5.5 and 6.5. 17. Method according to any of claims 9-16, wherein the extract of the seedless fruit of Berberis vulgaris L. resulting from step a) and the extract of the skin of the fruit of Myrtus communis L. resulting from step b) are each independently subjected to a lyophilization step prior to step c). 18. Method according to claim 17, wherein the encapsulation of the lyophilized extract of the seedless fruit of Berberis vulgaris L. resulting from said lyophilization and of the lyophilized extract of the skin of the fruit of Myrtus communis L. resulting from said lyophilization are carried out independently in the presence of at least one encapsulating agent, before carrying out step c). 19. Method according to claim 17, wherein the mixture resulting from step c) is encapsulated in the presence of at least one encapsulating agent. 20. Coloring composition based on anthocyanins, characterized by being obtained by the method defined according to any of claims 9-19. 21. Use of the coloring composition defined according to any of claims 1-8 or 20, characterized by being a food coloring. 22. Use of the composition according to claim 21, as a food coloring under pH conditions between 3.0 and 6.0. 23. Use of the composition according to any of claims 21-22, as a food coloring under pH conditions between 3.0 and 3.5. 24. Use of the composition according to any of claims 21-23, as a food coloring under pH conditions between 5.5 and 6.0. 25. Use of the composition according to any of claims 21-24, further comprising use as a preservative. 26. Use of the composition according to claim 25, wherein said use as a preservative comprises use as an antibacterial and/or antifungal agent. 27. Use of the composition according to any of claims 21-26, wherein said use further comprises use as an antioxidant agent. 28. Food product characterized in that it comprises the coloring composition defined according to any of claims 1-8 or 20.