ES2990137A1 - Coloring composition based on anthocyanins from wild fruits, preparation method and uses - Google Patents

Abstract

Colouring composition based on anthocyanins from wild fruits, method of preparation and uses. The present invention relates to a colouring composition based on anthocyanins comprising an extract of the seedless fruit of Berberis vulgaris L. and an extract of the skin of the fruit of Myrtus communis L., as well as to the method of preparing colouring compositions based on anthocyanins, and to the colouring composition obtained by said method. Additionally, the present invention relates to the uses of said colouring composition as a food colouring, and optionally also as a preservative agent and as an antioxidant agent, as well as to food products comprising said colouring composition. (Machine-translation by Google Translate, not legally binding)

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, in particular 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 the formulation of foods in order to guarantee adequate organoleptic properties 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 shelf life of said foods, in addition to trying to extend said shelf life as much as possible to reduce food waste.

All authorised food additives on the positive list of additives in Regulation (EU) No 1129/2011 are safe when used in accordance with current regulations and are subject to regular 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 Southampton Study, in which a group of children aged 3 to 9 years were given a combination of food colouring and sodium benzoate mixed with their food. When the behaviour 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 impact on health that the consumption of these dyes can have on children that in 2008 the European Parliament approved Regulation (EC) No 1333/2008, indicating that the possible negative effects on children's activity and attention must be clearly indicated on the label, together with the name or E number of each dye listed in said Regulation, including the following: tartrazine (E-102); quinoline yellow (E-103); 104); orange yellow (E-110); chamoisine (E-122); Ponceau 4R, cochineal red A (E-124); Allura red AC (E-129).

There are many known adverse effects and/or contraindications resulting from the use of these food colorings. For example, tartrazine (i.e., E-102; accepted daily intake (ADI) 7.5 mg/kg body weight), known in the United States as “FD & C Yellow No. 5”, has been linked to cases of urticaria, purpuric lesions, anaphylaxis, etc. The dye sunset yellow (i.e., E-110; ADI 2.5 mg/kg body weight), known in the United States as “FD & C Yellow No. 6”, has also been linked to genotoxicity in murine 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 body weight), known in the United States as “FD & C Red No. 40”, has been re-evaluated in the European Union twice, concluding that there is a possibility that it may be genotoxic at high doses. Carmoisine (i.e., E-122; ADI 4 mg/kg body weight) is a colorant whose use is banned 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 which is obtained by iodination of fluorescein - a dye banned in the European Union -, has been linked to alterations in childhood behavior and thyroid function due to its high iodine content.

There are also other colorants widely used in foods for infants 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, jams, jellies and ice creams and require special control due to the possible adverse effects they may cause in the population, in particular, in the child population.

Other examples of additives that are 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 on the basis of studies in rodents which showed that the consumption of high concentrations of these compounds could lead to forestomach hyperplasia, growth retardation, increased mortality and certain effects on the behaviour of rat pups.

There are also certain limitations on the quantity of preservative additives in food, 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 it has been incorporated into the food (e.g., reaction between sorbic acid and nitrites and/or sulphites, giving 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, sulphites (i.e., E-220 to E-228) are widely used additives 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 people, asthmatics, etc.

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

In addition, the food industry has to resort to the use of two or more food additives in the formulation of many foods to ensure their hygienic-sanitary quality (use of antimicrobials), organoleptic quality (use of food colorings and antioxidant additives) and to be able to market foods that are long-lasting, safe and appetizing for the consumer. Nowadays, there are different food additives of natural origin on the market that could be incorporated into food formulation, such as antioxidants (e.g., ascorbic acid, tocopherols, rosemary extract), antimicrobials (e.g., niacin) or colorants with red-violet tones (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 single ingredient that can replace the function of colouring additives, antioxidants and preservatives. On the other hand, it is also important to highlight that today the food industry has not managed to formulate foods with magenta - purple colouring without the use of artificial colourings, especially in foods with a pH range where anthocyanins (E 163) have a reddish colour (e.g. pH 3 - 4). This is a major handicap for the food industry, since today consumers demand more natural foods and therefore lacking artificial colourings in their formulation. The present invention comprises the obtaining and application of a natural composition, not previously described, where its chemical composition and physical-chemical properties confer on it, without resorting to the use of azo-type dyes, authorized according to current regulations, to achieve in foods with acidic pH (pH 3 - 4) some chromatic characteristics currently available on the market, in relation 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 such 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 (Diaset al., Food & Function2016, 7, 4523; Vegaet al., Food Chemistry2023, 414, 135669; Tamayo-Viveset al., Foods2023, 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 been 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 ethnic pharmacology 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 at the cardiovascular level, which comprise berberine or extracts containing it, among other ingredients. The same is true of EP3406144, based on compositions containing berberine chloride, among other ingredients, in oral form, for use in the control of hyperlipidemia and cardiovascular risk factors. However, this alkaloid lacks coloring activity, so it is not suitable for replacing the synthetic colorants mentioned above. On the other hand, patent WO2010109286A1 describes a non-hygroscopic crystalline preparation rich in colored phenolic compounds obtained from plants for use in beverages, in which a series of botanical species are mentioned, including Berberis spp. However, the combination of these species with each other to obtain the preparation is not mentioned, nor is the botanical species of 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 damage, cardiovascular diseases, obesity, diabetes and osteoporosis (lmranet al. Food science & nutrition 2021, 9 (10), 5854-5868). However, myrcetine is also an alkaloid that lacks coloring activity, so it is not of interest to replace existing synthetic dyes. Finally, it is worth mentioning 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 be used 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, an anthocyanin-based coloring composition is provided comprising an extract of the seedless fruit of Berberis vulgaris L. and an extract of the fruit skin of Myrtus communis L.

The term "anthocyanin-based" as it relates to the coloring compositions of the invention means that said coloring compositions comprise anthocyanins, preferably naturally occurring anthocyanins.

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 be that fruit of Berberis vulgaris L. in an optimal state of ripeness, that is, that fruit that has a Brix degree (°Brix) value between 9 and 20° Brix, preferably between 11 and 18° Brix, where 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. 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 "fruit skin 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 be that fruit of Myrtus communis L. in an optimal state of ripening, that is, that fruit that has a Brix degree value (°Brix) between 5 and 11 ° Brix, preferably between 6 and 9 ° Brix, where 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 replace the function of colouring, antioxidant and preservative additives on its own. This is an existing problem in the state of the art that the inventors have managed to solve by means of the colouring compositions based on anthocyanins of the present invention, which arise from the unexpected effects derived from the combination of specific extracts from these plants. These compositions, which comprise extracts from 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 colouring capacity and, in addition, also preservative (e.g., antimicrobial and antifungal) and antioxidant capacity. Regarding 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. Regarding 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, polyphenol oxidase or lipoxygenase).

On the other hand, regarding the coloring capacity, the compositions of the present invention advantageously provide coloration 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 colorant, even for food coloring in food products intended for populations especially sensitive to the known problems derived from the use of artificial colorants, such as the child population. Furthermore, in a particular way, the coloring compositions of the present invention manage 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 these ranges, which solves the gap existing in the market regarding 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 woody, thorny shrub with yellow flowers and 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 are typically up to 1 cm in size and bluish-black in colour. The seedless fruit extracts of Myrtus communis L. used in the context of the present invention typically include a plurality of anthocyanins which may be selected, but are not limited to, 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. in the Natural Park of l'Albufera (Valencia) and the Municipal Natural Site of the Sierra de la Murta (Alcira). In order to access these plant genetic resources, a permit to access plant genetic resources for non-commercial research purposes was initially obtained (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, a permit to access 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 colouring composition may comprise an amount of the extract of the seedless fruit 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 the 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 colouring 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 colouring 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 a quantity of said extract of the peel 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 quantities by weight of the extracts of the seedless fruit of Berberis vulgaris L. and of the peel of the fruit of Myrtus communis L. comprised by any one of these colouring compositions of the invention will in no case exceed 100% by weight relative to the total weight of the colouring 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 may also comprise an encapsulating agent. Examples of an encapsulating agent include, but are not limited to, a maltodextrin or a cyclodextrin.

In a second aspect of the invention, there is provided a method of preparing an anthocyanin-based colouring composition, more preferably, a method of preparing an anthocyanin-based colouring 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 an extract of the seedless fruit 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) mix 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 ultrasound probe. Said extraction with an ultrasound probe can be carried out, for example, by means of an ultrasound processing equipment, such as the Fisherbrand™ Model 705 Sonic Dismembrator equipment, preferably at an ultrasound power of 325 W - 375 W. In another embodiment, said hydroalcoholic extraction of step a) can be carried out with a proportion (solid/liquid, S/L) 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 acid pH, more preferably in a hydroalcoholic medium that provides a pH between 2 and 4, still 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 one portion of the seedless fruit of Berberís vulgaris L. (i.e., skin of the ripe fruit of Berberís vulgaris L.) is subjected to hydroalcoholic extraction to obtain an extract of the seedless fruit of Berberís 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 seedless fruit of Berberis vulgaris L. (solid) to hydroalcoholic solvent (liquid) of between 20 g/L and 28 g/L and/or, optionally, in a hydroalcoholic medium (e.g., a medium consisting of ethanol and water) providing an acidic pH, more preferably in a hydroalcoholic medium providing a pH between 2 and 4, still more preferably in a hydroalcoholic medium providing a pH between 3.0 and 3.5. These pH values can preferably be achieved by acidification of the medium with citric acid, e.g. with 5 M citric acid.

In step b) of the process, at least a portion of Myrtus communisL. peel (i.e., peel of the ripe fruit of Myrtus communisL.) is subjected to hydroalcoholic extraction to obtain an extract of the skin of the fruit of Myrtus communisL. In a preferred embodiment, said hydroalcoholic extraction is preferably carried out with an ultrasound probe. Said extraction with an ultrasound probe can be carried out, for example, with an ultrasound processing equipment, such as the Fisherbrand™ Model 705 Sonic Dismembrator equipment, preferably at an ultrasound 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 proportion (solid/liquid, S/L) of said Myrtus communisL. fruit peel. (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 term “about” when preceding and referring to a numerical value is understood in the context of the present invention to disclose that particular numerical value and further designates any value within a range formed by that numerical value ± 5%, more preferably a range defined by the numerical value ± 2%. By way of illustration, the term “about 1” should be interpreted as “within the range of 0.95 to 1.05”, preferably “within the range of 0.98 to 1.02”.

The term “range between” in the context of the present invention is understood to include both the numerical values between the two numerical values present at the two extremes of the range and the two numerical values forming each of the two extremes themselves, unless otherwise indicated. That is to say, for example, “within the range from 0.95 to 1.05” should be interpreted to include 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 seedless portion of the fruit of Berberis vulgaris L. and/or said skin portion 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 skin 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 stage, prior to step a), wherein the seed is separated from the skin and pulp in the fruit of Berberis vulgaris L. and wherein the skin is separated from the pulp and seed of the fruit of Myrtus communis L., preferably the mature 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 the seedless fruit of Berberis vulgaris L. and the amount of the extract of the fruit skin of Myrtus communis L. that are mixed in step c) of the process can each be independently comprised between 40 - 60% by weight, with respect to the total weight of the coloring composition. Preferably, the amount of the extract of the seedless fruit of Berberis vulgaris L. and the amount of the extract of the fruit skin of Myrtus communis L. that are mixed in step c) of the process can each be independently comprised between 45 - 55% by weight, with respect to the total weight of the coloring 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 fruit skin of Myrtus communis L. mixed in step c) shall in no case exceed 100% by weight with respect to the total weight of the colouring composition.

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

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

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

In a third aspect of the invention, there is provided a colouring composition obtained or obtainable by the method of the second aspect of the invention, defined above. This colouring composition comprises an extract of the seedless fruit of Barberis vulgaris L. and an extract of the skin of the fruit of Myrtus communis L. In one embodiment, the colouring 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 colouring composition may comprise an amount of the fruit skin extract 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 peel 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 colouring composition may comprise an amount of the extract of the seedless fruit 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 peel 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 Barberis vulgaris L. and of the peel of the fruit of Myrtus communis L. comprised by any of these coloring compositions of the invention shall in no 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 further comprise an encapsulating agent. Examples of an encapsulating agent include, but are not limited to, a maltodextrin or a cyclodextrin.

In a fourth aspect, there is provided the use of the colouring composition, defined according to the first or third aspect of the invention, as a food colouring, preferably, as a food colouring under pH conditions comprised between 2.5 and 6.5, and still more preferably as a food colouring under pH conditions comprised 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 colouring composition of the first or third aspect of the invention as a food colouring, preferably, as a food colouring under pH conditions comprised between 2.5 and 6.5, or more preferably under pH conditions comprised 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 colorant, preferably, as a food colorant 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, still more preferably, as a food colorant, preservative and antioxidant under pH conditions comprised 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 colouring composition according to the first or third aspect of the invention. The term “food product” is understood to mean 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, flavoured fermented milks, dairy desserts, ice creams, popsicles, flavoured 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 of about 3.0 to 3.5 or, more preferably, a food matrix with a pH value of about 3.0 to 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 candies (e.g., jelly beans or lollipops). "Food matrix" in the context of the present invention is understood to mean the food product as such, before 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 colour of natural colourings 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 colouring compositions of the present invention make it possible to obtain magenta colours, which currently require the combination of different synthetic colourings with blue tones, since the food industry does not currently have natural or synthetic colouring additives that have 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 of between about 5.5 and 6.0 or, more preferably, a food matrix with a pH value of between 5.5 and 6.0. It is known that some dairy desserts, such as ice creams (cream-based), have a pH of around 5.5 - 6.0, such 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 derive to a purple hue and then to a dark blue one. However, the coloring compositions of the present invention allow to obtain 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 experimentally demonstrated 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 BV2 extract in freeze-dried powder, 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 powdered MC2 extract, 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 provided by it, but should not be considered as limiting the scope thereof. Thus, it is possible that the invention may be put into practice in a manner other than that specifically described in the following examples.

Example 1 - Colouring composition, in fluid form, of seedless fruit extracts of Berberís vulgaris L. and fruit peel of Mvrtus communis L.

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

First, the fruits of Berberís vulgaris L. were selected. with an optimum state of maturity (11.0 - 19.0° Brix, determined by an Atago refractometer at 20 °C according to the official method 932.14C (AOAC, 2005), and the seeds were separated, thus obtaining the fruit (skin and pulp) of Berberís vulgaris L. without seeds. Next, 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 (corresponds to 0.149 mm or 0.0059 inches). The extract of the seedless fruit of Berberis vulgaris L. 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., ratio of freeze-dried seedless fruit extract of Berberis vulgaris L. by weight to the volume of hydroalcoholic medium), for a time between 1.5 and 4 minutes, using a mixture of ethanol and water (80%: 20%, v/v) acidified with citric acid to pH 3 as extraction solvent. This allowed to obtain a hydroalcoholic extract of seedless fruit of Berberis vulgaris L. rich in anthocyanins (extract BV1).

The BV1 extract had a total content of 9.15 mg cya-3-gluE/g BV1 extract (where “cya-3-gluE” means “cyanidin-3-glucoside equivalents”) of monomeric anthocyanins, analysed using 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 change to a hemiacetal form where they are colourless. 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 major anthocyanins were delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunidin-3-O-glucoside, pelargonidin-3-O-glucoside and malvidin-3-O-glucoside. The colour of the BV1 extract was determined using a Hunter Color Flez CiE Illuminate C, 2° and 45/0° geometry colourimeter, using cylindrical glass cuvettes with a diameter of 5 cm and a height of 1.3 cm, 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 of the fruit skin of Myrtus communis L. (hereinafter, “MC1 extract”)

On the other hand, the fruits of Myrtus communisL. were selected. with an optimal state of maturity (6.0 - 9.0 ° Brix, determined by Atago refractometer at 20 °C according to the official method 932.14C (AOAC, 2005), and the skins were separated from the pulp and the seed, thus obtaining Myrtus communisL. fruit skin devoid of pulp and seeds. Next, said skin 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 (0.150 mm) using an IKA Multidrive Basic mill (BS000). The extract of the Myrtus communisL. fruit skin was obtained by hydroalcoholic extraction under ultrasound power conditions (Fisherbrand ™, Sonic Dismembrator, Model 705) of approximately 450 - 500 W, with 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 volume of hydroalcoholic medium) for a time between 18 and 22 min, using a mixture of ethanol and water (80%:20%, v/v) with a pH value of 6 as extraction solvent. This allowed to obtain an anthocyanin-rich hydroalcoholic extract of Mydus Communis L. (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-C column, 184.6 mm x 75 mm, 2.7 µm, 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 colour of the MC1 extract was determined using a Hunter Color Flez CiE Illuminate C, 2° colorimeter with 45/0° geometry, using cylindrical glass cuvettes with a diameter of 5 cm and a height of 1.3 cm, 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 colouring composition in liquid form comprising the seedless fruit of Barberis vulgaris L. and the skin of the fruit of Myrtus communis L. (hereinafter referred to as the “BV1:MC1” colouring composition)

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 colouring 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. (colouring composition BV1:MC1).

Example 2 - Colouring composition, in the form of lyophilised powder, of extracts from the seedless fruit of Barberis vulgaris L. and the skin of Mvrtus communis L.

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

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, 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 lyophilized powder extract rich in anthocyanins from the seedless fruit of Berberis vulgaris L. (extract BV2).

The freeze-dried extract BV2 presented an extraction yield of 65.8% and a humidity 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 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 value of polyphenols corresponding to 159.84 mg GAE/g dry extract, hydroxybenzoic acids turn out to be 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, in addition it 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), where a small amount of sample is started, previously homogenized to a particle size of 0.037 mm, which is subjected to direct contact with the reagents for each determination. This methodology allows to measure both soluble molecules and insoluble compounds accurately and reliably, and allows a subsequent spectrophotometric analysis at the 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 means of 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 ultra-fast 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 color variability 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 of 1 to 3.5, turning to a pale pink color (L *: 13.97, a *: 5.21, b *: 13.01) between pH values of 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 color RAL 0202029 of the RAL design color system, and at pH 6.5 it had a color close to the standard S8010 Y10R 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 of mandatory analysis in food products: For this test the reference strains were used: 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 presented inhibitory activity for the growth of three Gram-negative bacteria and three Gram-positive bacteria, presenting minimum inhibitory concentrations and minimum bactericidal concentrations (MIC/MB) 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/MB) of 1.25/10, 5/>20, 2.5/20 mg/mL against the strains reference bacteria Gram-positive Bacillus cereus (ATCC 11778), Listeria monocytogenes (ATCC 19111) and Staphylococcus 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 as Aspergillus brasiliensis and Aspergillus fumigatus, respectively. For this purpose, the BV2 extract was serially diluted in a microplate. For each dilution, the inoculum of a reference bacteria was added to each well, ensuring the presence of 1.5 x 105 colony forming units (CFU) per well. They were incubated at 37 °C for 24 hours and, after incubation with iodonitrotetrazolium chloride (INT), used as a dye, the minimum inhibitory concentration (MIC) was determined. This was 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 h. 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 an in vitro biological antioxidant activity method (OxHLIA). In the Folin-Ciocalteu method, the reducing compounds present in the sample are reacted with the phosphomolybdotungstic acid of the Folin-Ciocalteu reagent, generating a blue coloration measurable at a wavelength of 750 nm. In the DPPH method, the DPPH (2,2-diphenyl-1-picrihydrazyl) solution reacts with the antioxidant molecules in the sample, losing its color, which goes from purple to yellow, in such a way that said loss of color can be monitored by spectrophotometric readings at 517 nm. The OxHLIA method, on the other hand, is an in vitro biological method, in which erythrocytes extracted from sheep blood, diluted in PBS, are placed in contact with the corresponding fruit extract and with AAPH (2,2'-azobi(2-methylpropionamidine) dihydrochloride), which generates free radicals. Subsequently, the optical density is 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 for 60 minutes. The expression “IC50” means “medium inhibitory concentration”, that is, it refers to the inhibitor concentration 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 carried out. Thus, the possible toxicity of the fruit in non-tumorous pig liver cells (PLP2) was evaluated, by monitoring cell growth by contrast microscopy. phases, 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, showing 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, 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 lyophilized powder extract rich in anthocyanins from the skin of the fruit of Myrtus communis L. (MC2 extract).

The freeze-dried 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 polyphenols of 271.18 mg GAE/g dry extract, with hydroxybenzoic acids being present in the highest quantity with a value of 44.96 mg GAE/g dry extract. In addition, this extract presented a quantity of flavonols of 20.33 mg/g dry extract and a total of hydroxycannamic acids of 17.78 mg/g 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, total monomeric anthocyanins were determined using 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. Furthermore, 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 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 colour variability depending on pH, with stability from a deep purple colour (L*: 11.42, a*: 34.61, b*: 15.45) at pH 3.0 to a bluish-purple colour (L*: 4.22, a*: 13.71, b*: 0.05) at pH 6. Specifically, the MC2 extract had a colour close to the RAL 0102025 colour standard of the RAL Design colour 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/MB) of 10/20 mg/mL extract against the Gram-negative bacteria Enterobacter cloacae (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/MB 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. On the other hand, the extract also presented antifungal activity with MIC of 1.25 mg/mL extract against the fungus Aspergillus brasiliensis. For this purpose, the same procedure was used as described above for the BV2 extract.

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 on preparation and characterization of BV2 extract in Example 2. Antioxidant activities of 96.81 mg GAE/g and 73.90 mg TE/g were observed for MC2 extract by the Folin-Ciocalteu and DPPH methodologies, respectively, and an IC50 of 59 pg/mL was obtained for antihemolytic activity over 60 days by the OxLHIA methodology.

Finally, although the consumption of Myrtus communis L. fruits is considered safe, and its wild fruit has been traditionally consumed for generations, toxicity studies were carried out. Thus, the possible toxicity of the fruit in non-tumorous pig liver cells (PLP2) was evaluated, showing zero 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 “BV2:MC2” coloring composition)

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

It is worth highlighting, with regard to the BV2:MC2 composition, the total quantity of polyphenols (281.59 mg GAE/g dry extract) measured by the Fast Blue BB method, with hydroxybenzoic acids being present in the highest quantity 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 following 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, total monomeric anthocyanins were determined using 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, C18 Water Spherisorb ODS2 column), which are shown in Figure 3 with their corresponding chromatographic peaks and their Rt retention value, 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 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).

The composition also had a characteristic colour profile due to the unique composition of monomeric anthocyanins achieved by combining the extracts of the seedless fruit of Berberis vulgaris L. and the peel of Myrtus communis L. according to the present invention. In particular, thanks to the synergistic combination of these extracts of both fruits, the colouring compositions of the present invention allow achieving a particularly stable characteristic magenta colour at a pH of 3 - 3.5 and a stable purple colour at pH values of 5.5 - 6.0.

Specifically, this BV2:MC2 composition showed colour variability in aqueous solution depending on pH, although an unexpected stability of a reddish purple colour (L*: 6.90, a*: 22.09, b*: 5.12) was observed at pH 3.5, a purple colour (L*: 7.06, a*: 14.05, b*: -0.74) at pH values of 5.5, and a greyish green colour was evident at pH values higher than 7.5 (L*: 5.45, a*: 0.52, b*: 2.02). Specifically, the BV2:MC2 extract had a colour close to RAL 8022 of the RAL Classic colour 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 BV2:MC2 composition presented 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 Ersinia 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 was used for both the BV2 extract and the MC2 extract.

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 relating to 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 by the OxLHIA methodology.

Example 3 - Coloring composition in the form of lyophilized powder of extracts of the seedless fruit of Berberis vulgaris L. and the 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 described below:

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, the 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. (extract BV3) 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 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 creams), a slight purple color was observed (L*: 33.18, a*: 17.466b*: 9.20) which resembles the standard color LPC 0416 of the Isomat 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), Listeria monocytogenes (ATCC 19111) and Staphylococcus aureus (ATCC 25923), respectively. In addition, the microencapsulated extract presented 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 was used for both the BV2 extract and the MC2 extract. On the other hand, 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 methodOxHLIA (see description of this method in the section on preparation and characterization of the BV2 extract in Example 2).

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

From the MC2 extract, the 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 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 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 0.75 mL/min, trifluoroacetic acid (0.1%)/Acetonitrile. Thus, 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 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 colour was obtained. Thus, MC3 presented a colour that is currently not found in the natural food colouring options available on the market. It was also found that the MC3 extract was stable up to pH 7.0, from which olive green colours were observed (L * 2.40, a * 3.48 and b * 0.22), with a colour similar to RAL 9005 of the RAL Classic colour system), while the non-encapsulated 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. Likewise, 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 described above was used for both the BV2 extract and the MC2 extract. On the other hand, the extract was determined to have 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 preparation and characterization of the BV2 extract in Example 2), respectively. An IC50 value of 315 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 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 “BV3:MC3” coloring composition).

Finally, both individual extracts, BV3 and MC3, were mixed in quantities corresponding to 50%:50% w/w ± 10% of Berberís vulgaris L. and Myrtus communis L., giving a colouring composition according to the invention, in the form of a microencapsulated lyophilized powder, comprising extracts from the seedless fruit of Berberís vulgaris L. and the skin of the fruit Myrtus communis L. (colouring composition BV3:MC3). The colour profile of the BV3:MC3 extract at different pH levels at a concentration of 13 mg/mL of aqueous solution is shown in the following table (Table 6) (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 from the seedless fruit of Berberis vulgaris L. and the skin of the fruit of Mvrtus communis L. in the form of encapsulated lyophilized powder (BV4:MC4)

From the colouring composition BV2:MC2, 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 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 colouring composition in the form of microencapsulated lyophilized powder, comprising extracts from the seedless fruit of Berberis vulgaris L. and the skin of the fruit of Myrtus communis L. (hereinafter referred to as “colouring composition BV4:MC4”), with a particle size of 0.5 - 25 pm determined by Mastersizer 3000 (Malvern, UK).

The encapsulated BV4:MC4 composition presented an encapsulation yield of 69.64%. It was determined to contain a total of 83.39 mg GAE/g dry extract in polyphenols using the Fast Blue BB method under the conditions described above in the section on the preparation and characterisation 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 dry extract, followed by hydroxycinnamic acids with 7.48 mg/g dry extract and flavonols with a total of 5.76 mg/g dry extract, measured following the QUENCHER methodology, as described above in the section on the preparation and characterisation 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 an encapsulating agent, additionally presented the following characteristic chromatic 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 colour different from those obtained with the BV extract or MC extract individually, specifically an intense magenta colour was obtained at pH 4.5 and at pH 5.5 it presented a colour similar to S7020-R20B of the NCS 2050 colour system and at pH 6.0 a colour close to S8010-R10B of the previous colour system. It was also experimentally identified that the extract was stable up to pH 7, from which greenish colours 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/MB 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/MB 2.5/20, 10/20 and 10/20 mg/mL against the Gram-positive bacteria Basilus cereus (ATCC 11778), Listeria 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 was used for both the BV2 extract and the<m>C2 extract. Furthermore, this microencapsulated BV4:MC4 composition exhibited an antioxidant activity of 28.91 mg GAE/g and 45.84 mg TE/g microencapsulated extract by the Folin-Ciocalteu and DPPH methodologies, respectively, as described above in the section on the preparation and characterization of the BV2 extract in Example 2. A 1C50 value of 228 pg/mL was also determined using the in vitro biological antioxidant activity methodOxHLIA (see description of this method in the section on 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 a sweetener, 1.53 - 2.5% by weight of gelatin (as a base gelling ingredient), 0.1 - 0.5% by weight of E330, 0.30 - 0.35% by weight of BV2 extract, and flavouring. A gelatin with a pH of 3 - 3.5 was obtained, 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 the 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 analysed is determined, of: Enterobacteria (absence/g) and Salmonella (absence/25 g), or maximum of 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 flavouring. 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 was 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).

Prototypes of gelatin formulated with synthetic commercial colorants

For comparison with the gelatin offer found on the market, other prototypes formulated with commercial colorants were made: E-124 (synthetic Ponceau 4R 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, varying only the % of colorant incorporated in 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 colourings, totally different shades were obtained (reddish colour in the case of the prototypes formulated individually with E-124 or E-163; bluish colour 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 hue at these pH values.

Comparative study of the colour profile and CIELAB parameters of the colourant E-163, Berberis vulgaris L. (BV3) extract in the form of microencapsulated freeze-dried powder, Mvrtus communis L. extract in the form of microencapsulated freeze-dried powder (MC3), colouring composition BV3:MC3 and microencapsulated colouring composition BV4:MC4In Table 9, samples corresponding to the current marketable colourant E-163 from different natural sources such as purple carrot, red cabbage, radish and grapes have been analysed 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 colouring additives with magenta-purple hues, so it is necessary to resort to the use of a mixture of synthetic dyes with blue hues such as E-131 (patent blue), E-132 (indigotine), E-133 (brilliant blue FCF) with E-163 (anthocyanin) or other dyes with red hues such as E-122 (azorubine), E-124 (Ponceau 4R), etc. The E-163 dye, available on the market, and analysed in Table 9, provides a red-garnet colour, but fails to achieve a magenta-purple hue, unlike what happens with the compositions of the present invention.

Regarding 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 in vitro biological OxHLIA method. Likewise, it was shown that the commercial natural dye 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 Basilus cereus (ATCC 11778) and Listeria monocytogenes (ATCC 19111), respectively. This dye also had antifungal activity with a minimum inhibitory concentration of 20 mg/mL against the fungus Aspergillus fumigatus. These data demonstrate the superiority in preservative capacity of the present invention.

With the present invention, a series of colouring compositions based on extracts of natural origin with colouring power in different presentations (hydroalcoholic extract, lyophilized extract in powder form and microencapsulated extract in powder form) are achieved which 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 from this (e.g., reduction of ADHD, allergic reactions, etc.).

Study of the antimicrobial and antifungal activity of the 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. Colouring composition based on anthocyanins, characterised 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 the seedless fruit of Berberis 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. 3. Composition according to the preceding claim, comprising an amount of said extract of seedless fruit of Berberis vulgaris L. 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 the form of lyophilized powder. 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 an 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 of preparing a coloring composition based on anthocyanins, characterized in that it comprises: a) subjecting at least a portion of the seedless fruit of Berberis vulgaris L. to hydroalcoholic extraction to obtain an extract of the seedless fruit of Berberis vulgaris L., b) subjecting at least a portion of the fruit peel of Myrtus communis L. to hydroalcoholic extraction to obtain an extract of the fruit peel of Myrtus communis L., and c) mix 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 vulgaris L and the amount of the extract of the fruit skin of Myrtus communis L 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 seedless fruit of Berberis vulgaris L and the amount of the extract of the fruit skin of Myrtus communis L 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 Berberis vulgaris L. and/or said portion of skin of the fruit of Myrtus communis L., respectively, is 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 freeze-drying 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 the lyophilized extract of the fruit skin of Myrtus communis L. resulting from said lyophilization is carried out independently, in the presence of at least one encapsulation 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.