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

Abstract

The present invention relates to a colourant composition based on anthocyanins, comprising an extract of seedless Berberis vulgaris L. fruit and an extract of the skin of Myrtus communis L. fruit; to a method for preparing anthocyanin-based colourant compositions; and to the colourant composition obtained using the method. The present invention also relates to the uses of the colourant composition as a food colourant and, optionally, as a preservative and an antioxidant, and to food products comprising the colourant composition.

Description

[0001] DESCRIPTION

[0002] Coloring composition based on anthocyanins from wild fruits, preparation method and uses

[0003] TECHNICAL FIELD

[0004] The present invention falls within the technical field of colorants, particularly food colorants, and also, due to the possible applications of the coloring compositions of the present invention, within the technical field of food preservatives and antioxidants.

[0005] BACKGROUND OF THE INVENTION

[0006] In the food industry, it is common to use different food additives - mostly of synthetic origin - in food formulation in order to guarantee adequate organoleptic properties in accordance with consumer standards (e.g., colorings), guarantee hygienic and sanitary quality (e.g., preservatives with antimicrobial properties), prevent alterations due to oxidation processes (e.g., antioxidants), etc. throughout the entire shelf life of these foods, as well as trying to extend that shelf life as much as possible to reduce food waste.

[0007] All authorized food additives listed in the positive list of additives in Regulation (EU) No 1129 / 2011 are safe, provided they are used in accordance with current regulations, and are subject to periodic re-evaluations by the European Food Safety Authority (EFSA). However, many synthetic additives are subject to some controversy, such as azo dyes and certain synthetic preservatives and antioxidants.

[0008] Azo dyes have been the subject of many studies, one of the most controversial being the so-called "Southampton Study," in which a combination of food dyes and sodium benzoate was mixed with food and administered to a group of children aged 3 to 9. When the behavioral 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 the dyes.Such is the impact on the health of children consuming these colorants that in 2008 the European Parliament approved Regulation (EC) No 1333 / 2008, which states that the possible adverse effects on activity and attention in children must be clearly indicated on the label, along with the name or E number of each colorant listed in the Regulation, which includes the following: tartrazine (E-102); quinoline yellow (E-104); sunset yellow (E-110); carmoisine (E-122); Ponceau 4R, cochineal red A (E-124); Allura red AC (E-129).

[0009] There are many known adverse effects and / or contraindications associated with the use of these food colorings. For example, tartrazine (e.g., E-102; ADI of 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. Sunset Yellow FCF (e.g., E-110; ADI of 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, learning disabilities in offspring, as well as immunomodulatory and xenoestrogenic effects. Allura Red AC (E-129; ADI of 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 (E-129; ADI of 7 mg / kg body weight), has been re-evaluated in the European Union twice, concluding that there is a possibility that it may be genotoxic at high doses., E-122; ADI of 4 mg / kg body weight) is a colorant whose use is prohibited in the United States.

[0010] On the other hand, erythrosine (e.g., E-127; ADI of 0.1 mg / kg body weight), which is a synthetic polyiodized 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 children's behavior and thyroid function due to its high iodine content.

[0011] There are also other colorants widely used in infant foods belonging to the group of blue colorants derived from triarylmethane, such as Patent Blue V (e.g., E-131; ADI of 1 mg / kg body weight), Brilliant Blue FCF (e.g., E-133; ADI of 6 mg / kg body weight), known in the United States as “FD&C Blue No. 1”, Green S (e.g., E-142; ADI of 5 mg / kg body weight), Rapid Green (e.g., E-143; ADI of 12.5 mg / kg body weight), or Brilliant Black (e.g., E-151; ADI of 1 mg / kg body weight), which are widely used in the production of sweets, jams, marmalades, jellies and ice creams and require special control due to the possible adverse effects they can cause in the population, particularly in the infant population.

[0012] Other examples of additives subject to control are the antioxidant additives BHA (e.g., E-320) and BHT (e.g., E-321), both synthetic antioxidant additives. BHA is an antioxidant composed of two isomers (2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole) that can be used in many foods, with an ADI of 1 mg / kg body weight. BHT is a fat-soluble antioxidant that can be used alone or in combination with BHA, and has an ADI of 0.5 mg / kg body weight. EFSA established these ADI values ​​based on studies in rodents that showed that consuming high concentrations of these compounds could lead to forestomach hyperplasia, growth retardation, increased mortality, and certain behavioral effects in rat pups.

[0013] 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 food (e.g., the reaction between sorbic acid and nitrites and / or sulfites, yielding compounds with mutagenic activity). On the other hand, the quantities of benzoic acid and benzoates (e.g., E-210) are also limited to an ADI of 5 mg / kg, given that they can promote the presence of benzene compounds. Finally, sulfites (e.g., E-220 to E-228) are widely used additives in the food industry, but they are on the list of allergens that must be declared on food labels because they can cause problems in sensitive individuals, asthmatics, etc.

[0014] In this context, the need to provide new additives or coloring compositions of natural origin that do not pose a health risk, without compromising the organoleptic and hygienic-sanitary quality of food, is evident. Furthermore, there is a growing societal demand for increasingly natural foods that do not contain food additives such as colorings, preservatives, and antioxidants, or, if they do contain them, that these additives be of natural origin.

[0015] Furthermore, the food industry must use two or more food additives in the formulation of many foods to guarantee their hygienic and sanitary quality (use of antimicrobials), organoleptic quality (use of food colorings and antioxidant additives), and to be able to market foods that are durable, safe, and appealing to consumers. Currently, there are various food additives of natural origin on the market that could be incorporated into food formulations, such as antioxidants (e.g., ascorbic acid, tocopherols, rosemary extract), antimicrobials (e.g., niacin), or colorings with reddish-purple hues (e.g., E-120 - Carmine, E-163 - anthocyanins), although their use is not authorized in all foods or at any concentration.

[0016] However, no single ingredient can replace the function of coloring agents, antioxidants, and preservatives. Furthermore, it's important to note that the food industry has not yet succeeded in formulating magenta-purple foods without artificial colorings, especially in foods with a pH range where anthocyanins (E 163) exhibit a reddish hue (e.g., pH 3-4). This represents a significant challenge for the food industry, as consumers today demand more natural foods and, therefore, foods free of artificial colorings.The present invention comprises obtaining and applying a previously undescribed natural composition, where its chemical composition and physicochemical properties give it, without resorting to the use of azo-type colorants, authorized according to current regulations, to achieve in foods with acidic pH (pH 3 - 4) chromatic characteristics currently available on the market, in relation to the range of purples-magentas.

[0017] To date, different fruits have been investigated in search of possible coloring sources, such as fruits like Crataegus monogyna Jacq., Sorbus aria L., Prunus avium L., Fragaria vesca L. and Vaccinium myrtillus L., their anthocyanin profile, color characteristics and some of their bioactive properties such as antioxidant, antibacterial and antifungal have been reported, showing the potential of these fruits to be used as sources of natural colorants in the development of healthier food products (Dias et al., Food & Function 2016, 7, 4523; Vega et al., Food Chemistry 2023, 414, 135669; Tamayo-Vives et al., Foods 2023, 12, 2427.).

[0018] Regarding Berberis vulgaris L, it is now known for the use of berberine, a natural alkaloid present in high quantities in this plant, especially in its stems and roots, which has shown to have a wide use in the treatment of hepatic oxidative stress, Alzheimer's, idiopathic infertility due to male factor, weight loss, prevention of diabetes and cardiovascular problems (Fatehi-Hassanabad et al., Journal of ethnopharmacology 2005, 102(1), 46-52; Fatehi et al., An International Journal Devoted to Pharmacological and Toxicological Evaluation of Natural Product Derivatives 2005, 19(3), 222-225; Abd El-Wahab et al., BMC complementary and alternative medicine 2013, 13, 1-12). EP2007429 also describes oral compositions with beneficial cardiovascular effects, comprising berberine or extracts containing it, among other ingredients.The same applies to EP3406144, based on compositions containing berberine chloride, among other ingredients, for oral administration in the control of hyperlipidemia and cardiovascular risk factors. However, this alkaloid lacks coloring activity, making it unsuitable as a replacement for the aforementioned synthetic colorants. On the other hand, patent W02010109286A1 describes a non-hygroscopic crystalline preparation rich in colored phenolic compounds obtained from plants for use in beverages, mentioning several botanical species, including Berberis spp. However, the combination of these species to obtain the preparation is not mentioned, nor is the botanical species Myrtus spp. included in the invention description.

[0019] 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 various cardiovascular, gastrointestinal, dermatological, and neurological diseases, through several studies focused primarily on essential oils extracted from the plant, mostly from its leaves. In particular, there are several publications regarding 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 (Imran et al. Food science & nutrition 2021, 9(10), 5854-5868).However, myrcetin is also an alkaloid that lacks coloring activity, so it is not of interest as a replacement for existing synthetic dyes. Finally, it should be mentioned that patents US2013281548A1 and US2008255226A1 describe a composition of anthocyanin extracts of plant origin, in which starch is used as an edible carrier, or cysteine ​​is used to improve its bioavailability, respectively. Although they present a list of possible fruits to use for obtaining anthocyanins, such as Myrtus communis L., these fruits are not included in the examples or the claims, nor is the combined use of Myrtus communis with Berberis vulgaris.

[0020] DETAILED DESCRIPTION OF THE INVENTION In a first aspect, a coloring composition based on anthocyanins is provided comprising a seedless fruit extract of Berberis vulgaris L. and an extract of the fruit peel of Myrtus communis L.

[0021] The expression “based on anthocyanins”, referring to the coloring compositions of the invention, means that said coloring compositions comprise anthocyanins, preferably anthocyanins of natural origin.

[0022] The expression 'seedless fruit of Berberis vulgaris L.”, in the context of the present invention, refers to the mature, seedless fruit of Berberis vulgaris L. The term “ripe fruit of Berberis vulgaris L.” is understood to mean The fruit of Berberis vulgaris L. at its optimal stage of ripeness, that is, the fruit with a Brix value (°Bhx) between 9 and 20 °Bhx, preferably between 11 and 18 °Bhx, where these Brix values ​​can be determined by any known method, although preferably they are determined using an Atago refractometer at 20 °C according to Official Method 932.14C, AOAC, 2005. In the context of the present invention, the terms “seedless fruit” and “seedless fruit” are used interchangeably. Furthermore, in the context of the present invention, the term “fruit” is understood to include one or more fruits.

[0023] The expression “skin of the fruit of Myrtus communis L.”, in the context of the present invention, refers to the skin of the ripe fruit of Myrtus communis L. “Ripe fruit of Myrtus communis L.” is understood to be that fruit of Myrtus communis L. in an optimal state of ripeness, that is, that fruit which has a Brix (°Bhx) value between 5 and 11 °Bhx, preferably between 6 and 9 °Bhx, where these Brix scale values ​​can be determined by any known method, although preferably they are determined using an Atago refractometer at 20 °C according to official method 932.14C, AOAC, 2005.

[0024] As already mentioned, there is currently no single ingredient that can replace the function of coloring, antioxidant, and preservative additives. This is a problem in the prior art that the inventors have solved with the anthocyanin-based coloring compositions of the present invention, which arise from the unexpected effects of combining specific extracts from these plants. These compositions, comprising seedless fruit extracts of Berberis vulgaris L. and fruit peel of Myrtus communis L., are particularly suitable for food use and successfully eliminate the need for multiple synthetic additives in food products, as they possess coloring, preservative (e.g., antimicrobial and antifungal), and antioxidant properties.Regarding antimicrobial capacity, these compositions significantly inhibit the growth of foodborne pathogenic bacteria (e.g., Salmonella sp., Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, E. coli, among others) and Aspergillus-type fungi. Regarding 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 polyphenol oxidase or lipoxygenase).

[0025] On the other hand, with regard to coloring capacity, the compositions of the present invention advantageously provide coloring with shades from red to magenta, with a characteristic and recognizable chromatic profile, due to the synergistic effect of both extracts, while also presenting a safety profile suitable for use as a food coloring, even for food coloring in food products intended for populations especially sensitive to the known problems derived from the use of artificial colorings, such as the infant population.Furthermore, in particular, the coloring compositions of the present invention achieve 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, thus solving the existing gap in the market regarding natural coloring additives that can provide this specific coloration, characteristic of forest fruits, without requiring the use of synthetic colorants.That is to say, in a particularly advantageous way, 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 currently available commercial colorants, and represent a natural alternative for use in food formulations, allowing the desired color range to be obtained without the need to resort to the use of artificial colorants such as azo dyes.

[0026] Berberis vulgaris L. is a thorny shrub with yellow wood and flowers, whose fruits are small, oblong berries that turn reddish when ripe. The seedless fruit extracts of Berberis vulgaris L. used in the context of the present invention typically include a plurality of anthocyanins that may be selected, without limitation, from 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.

[0027] 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 color. 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, 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.

[0028] 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 Albufera Natural Park (Valencia) and the Sierra de la Murta Municipal Natural Area (Alcira).To access these plant genetic resources, permission was initially obtained for access to plant genetic resources for non-commercial research purposes (references PN-NC_032021 and PN-NC_022022, which correspond to ABSCH-IRCC-ES-257749-1 and ABSCH-IRCC-ES-262067-1, respectively) and subsequently, permission for access for commercial purposes after signing two agreements to obtain prior informed consent and establish the mutually agreed conditions with the Valencian Government and the Government of Castilla-La Mancha (reference PN-CM_102024, which corresponds to ABSCH-IRCC-ES-276420-1 and the due diligence declaration with registration number: R EGAG E25e00001596748).

[0029] In one embodiment, the coloring composition may comprise an amount of seedless fruit extract of Berberis vulgaris L. comprising between 40% and 60% by weight of the total weight of the composition, more preferably between 45% and 55% by weight of the seedless fruit extract of Berberis vulgaris L. In another embodiment, the coloring composition may comprise an amount of fruit peel extract of Myrtus communis L. comprising between 40% and 60% by weight of the total weight of the composition, more preferably between 45% and 55% by weight of the fruit peel extract of Myrtus communis L. In particular, the coloring composition may comprise an amount of seedless fruit extract of Berberis vulgaris L.comprising between 40% and 60% by weight of the total weight of the composition and / or an amount of said extract of the peel of the fruit of Myrtus communis L. comprising between 40% and 60% by weight of the total weight of the composition. It is also evident that the sum of the amounts by weight of the extracts of the seedless fruit of Berberis vulgaris L. and of the peel of the fruit of Myrtus communis L. comprising any one of these coloring compositions of the invention shall in no case exceed 100% by weight of the total weight of the coloring composition.

[0030] The coloring composition may be in liquid form (e.g., as 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.

[0031] In one particular embodiment, the coloring composition may be in the form of a lyophilized powder and further comprise an encapsulating agent. Examples of encapsulating agents include, but are not limited to, maltodextrin or cyclodextrin.

[0032] In a second aspect of the invention, a method for preparing an anthocyanin-based coloring composition is provided, more preferably a method for preparing an anthocyanin-based coloring composition according to the first aspect of the invention. This process comprises the following steps: a) subjecting at least a portion of the seedless fruit of Berberis vulgaris L. to hydroalcoholic extraction to obtain a seedless fruit extract of Berberis vulgaris L., b) subjecting at least a portion of the peel of the fruit of Myrtus communis L. to hydroalcoholic extraction treatment to obtain a peel extract of the fruit of Myrtus communis L., and c) mixing a quantity of the seedless fruit extract of Berberis vulgaris L. resulting from step a) and a quantity of the peel extract of Myrtus communis L. resulting from step b).

[0033] In step a) of the process, at least a portion of the seedless fruit of Berberis vulgaris L. (e.g., the peel 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 using an ultrasound probe. This extraction with an ultrasonic probe can be carried out, for example, using an ultrasonic processing unit, such as the Fisherbrand™ Model 705 Sonic Dismembrator, preferably at an ultrasonic power of 325 W - 375 W. In another embodiment, this hydroalcoholic extraction of step a) can be carried out with a ratio (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.Particularly preferred, said hydroalcoholic extraction of step a) of the process of the invention is carried out in a hydroalcoholic medium (e.g., a medium formed by ethanol and water) that provides an acidic pH, more preferably in a hydroalcoholic medium that provides a pH between 2 and 4, and even more preferably in a hydroalcoholic medium that provides a pH between 3.0 and 3.5. These pH values ​​can preferably be achieved by acidifying the medium with citric acid, e.g., with 5 M citric acid.

[0034] In a particular embodiment, in step a) of the process, at least a portion of seedless fruit of Berberis vulgaris L. (e.g., the peel of the ripe fruit of Berberis vulgaris L.) is subjected to hydroalcoholic extraction to obtain a seedless fruit extract of Berberis vulgaris L., wherein said hydroalcoholic extraction is preferably carried out using an ultrasonic probe, with a solid / liquid ratio (S / L) of said seedless fruit portion 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) that provides an acidic pH, more preferably in a hydroalcoholic medium that provides a pH between 2 and 4, even more preferably in a medium hydroalcoholic 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.

[0035] In step b) of the process, at least a portion of the peel of Myrtus communis L. (i.e., peel of the ripe fruit of Myrtus communis L.) is subjected to hydroalcoholic extraction to obtain an extract of the peel of the fruit of Myrtus communis L. In a preferred embodiment, said hydroalcoholic extraction is preferably carried out with an ultrasound probe. This ultrasound probe extraction can be performed, for example, with an ultrasound processing unit, such as the Fisherbrand™ Model 705 Sonic Dismembrator, preferably at an ultrasound power of approximately 450 W - 500 W, more preferably approximately 500 W. In another embodiment, the hydroalcoholic extraction of step b) is carried out with a solid / liquid (S / L) ratio of the Myrtus communis L. fruit peel (solid) 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.In a particularly preferred embodiment, said hydroalcoholic extraction of step b) of the process of the invention is carried out in a hydroalcoholic medium providing a pH between 5 and 7, preferably in a hydroalcoholic medium providing 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 providing a pH between 5 and 7, more preferably in a medium formed by ethanol and water providing a pH of approximately 6.

[0036] The expression “approximately”, when preceding and referring to a numerical value in the context of the present invention, is understood to disclose that particular numerical value and, furthermore, to designate any value within a range consisting of that numerical value ± 5%, more preferably a range defined by the numerical value ± 2%. By way of illustration, the expression “approximately 1” should be interpreted as “within the range between 0.95 and 1.05”, preferably “within the range between 0.98 and 1.02”.

[0037] The expression “range between”, in the context of the present invention, is understood to include both the numerical values ​​between the two numerical values ​​at the extremes of the range and the two numerical values ​​that form each of the extremes themselves, unless otherwise indicated. That is to say, for example, “within the range between 0.95 and 1.05” should be interpreted to include both the values ​​within the range formed by the maximum value of 1.05 and the minimum value of 0.95, and specifically 1.05 and 0.95.

[0038] In a particular embodiment, in step b) of the process, at least a portion of the peel of the fruit of Myrtus communis L. (i.e., the peel of the ripe fruit of Myrtus communis L.) is subjected to hydroalcoholic extraction to obtain an extract of the peel of the fruit of Myrtus communis L., wherein said hydroalcoholic extraction is preferably carried out using an ultrasonic probe, with a solid / liquid (S / L) ratio of said peel of the fruit of Myrtus communis L. (solid) to hydroalcoholic solvent (liquid) of between 18 g / L and 20 g / L and / or, optionally, in a hydroalcoholic medium providing a pH between 5 and 7, more preferably in a hydroalcoholic medium providing a pH of approximately 6. The process according to this second aspect of the invention may further optionally comprise, prior to step a) and / or step b), subjecting said portion of the fruit devoid of seeds of Berberis vulgaris L.and / or said portion of peel of the fruit of Myrtus communis L, respectively, to a freeze-drying stage.

[0039] Steps a) and b) of the process of the invention are based on the use of the seedless fruit of Berberis vulgaris L. and the peel of the fruit of Myrtus communis L. Such plant material can be obtained from the fruit of each of these two shrubs by means known in the art. In a particularly preferred embodiment, the process of the invention comprises a preliminary step, prior to step a), in which the seed is separated from the peel and pulp in the fruit of Berberis vulgaris L. and in which the peel 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.

[0040] In a particular embodiment, the amount of seedless fruit extract of Berberis vulgaris L. and the amount of fruit peel extract of Myrtus communis L. mixed in step c) of the process may each independently be between 40 and 60% by weight of the total weight of the coloring composition. Preferably, the amount of seedless fruit extract of Berberis vulgaris L. and the amount of fruit peel extract of Myrtus communis L. mixed in step c) of the process may each independently be between 45 and 55% by weight of the total weight of the coloring composition. It is also evident that the sum of the weights of the seedless fruit extract of Berberis vulgaris L. and the fruit peel extract of Myrtus communis L.mixed in step c) shall in no case exceed 100% by weight with respect to the total weight of the coloring composition.

[0041] Preferably, the seedless fruit extract of Berberis vulgaris L. resulting from step a), and / or the fruit peel extract of Myrtus communis L. resulting from step b), can be subjected independently to a freeze-drying step prior to step c). This allows both extracts to be mixed directly in freeze-dried powder form in step c), thus facilitating a more homogeneous mixture. Optionally, the freeze-dried mixture resulting from step c), obtained from step c), can be subsequently encapsulated in the presence of at least one encapsulating agent. This encapsulating agent must be compatible with the pH of the food matrix into which the coloring composition of the present invention is to be incorporated once obtained.

[0042] In another embodiment, the seedless fruit extract of Berberis vulgaris L. resulting from step a), and / or the fruit peel extract of Myrtus communis L. resulting from step b), can be subjected independently to a freeze-drying step prior to step c), and, optionally, the freeze-dried seedless fruit extract of Berberis vulgaris L. resulting from said freeze-drying and / or the freeze-dried fruit peel extract of Myrtus communis L. resulting from said freeze-drying can be encapsulated independently in the presence of at least one encapsulating agent, before carrying out step c). Said encapsulating agent must be compatible with the pH of the food matrix into which the coloring composition of the present invention is to be incorporated once obtained.Preferably, the seedless fruit extract of Berberis vulgaris L. resulting from step a) and the fruit peel extract of Myrtus communis L. resulting from step b) can be subjected independently to a freeze-drying step prior to step c) and, optionally, the resulting freeze-dried seedless fruit extract of Berberis vulgaris L. and the resulting freeze-dried fruit peel extract of Myrtus communis L. can then be encapsulated in the presence of at least one encapsulating agent, also prior to step c). This allows both extracts to be freeze-dried and encapsulated independently, so that in step c) they would already be mixed in the form of a freeze-dried and encapsulated powder, facilitating the production of a homogeneous encapsulated mixture.

[0043] In another embodiment, the mixture resulting from step c) can be subjected to a subsequent freeze-drying step. In this way, the extracts resulting from steps a) and b) can be mixed in liquid form (e.g., in the presence of a quantity of the solvent used in the hydroalcoholic extraction), and the mixture obtained in step c) can then be freeze-dried. After this freeze-drying step, the mixture resulting from step c), in freeze-dried form, can optionally be encapsulated, preferably microencapsulated, in the presence of at least one encapsulating agent. This encapsulating agent must be compatible with the pH of the food matrix into which the coloring composition of the present invention is to be incorporated once obtained.

[0044] Examples of suitable encapsulating agents include, but are not limited to, cyclodextrins and maltodextrins. Encapsulation, particularly microencapsulation, can be carried out using methods known in this field of the art, including, for example, spray-drying (micro)encapsulation. Such spray-drying can be performed using any known equipment designed for this purpose, such as the Buchi Mini Spray Dryer, model B-290.

[0045] In a third aspect of the invention, a coloring composition obtained or obtainable by the method of the second aspect of the invention, defined above, is provided. This coloring composition comprises a seedless fruit extract of Berberis vulgaris L. and a fruit peel extract of Myrtus communis L. In one embodiment, the coloring composition may comprise an amount of the seedless fruit extract of Berberis vulgaris L. comprising between 40% and 60% by weight of the total weight of the composition, more preferably between 45% and 55% by weight of the seedless fruit extract of Berberis vulgaris L. comprising between 45% and 55% by weight of the total weight of the composition. In another embodiment, the coloring composition may comprise an amount of the fruit peel extract of Myrtus communis L.comprising between 40% and 60% by weight of the total weight of the composition, more preferably, an amount of Myrtus communis L. fruit peel extract comprising between 45% and 55% by weight of the total weight of the composition. In particular, the coloring composition may comprise between 40% and 60% by weight of Berberis vulgaris L. seedless fruit extract of the total weight of the composition and / or between 40% and 60% by weight of said Myrtus communis L. fruit peel extract of the total weight of the composition. It is also evident that the sum of the weights of the Berberis vulgaris L. seedless fruit extract and the Myrtus communis L. fruit peel extract...comprised of any one 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.

[0046] The coloring composition, according to the third aspect of the invention, may be in liquid form (e.g., as 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.

[0047] In one particular embodiment, the coloring composition may be in the form of a freeze-dried powder and further comprise an encapsulating agent. Examples of encapsulating agents include, but are not limited to, maltodextrin or cyclodextrin. In a fourth aspect, the coloring composition, as defined in the first or third aspect of the invention, is provided for use as a food coloring, preferably as a food coloring under pH conditions between 2.5 and 6.5, and even more preferably as a food coloring under pH conditions between 3.0 and 6.0. This use may further include use as a preservative (e.g., as an antibacterial and / or antifungal agent).In a particularly preferred embodiment, the coloring composition of the first or third aspect of the invention is provided for use as a food coloring, preferably as a food coloring under pH conditions between 2.5 and 6.5, or more preferably under pH conditions 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, the coloring composition of the first or third aspect of the invention is provided for use as a food coloring, preferably as a food coloring under pH conditions between 2.5 and 6.5, or more preferably under pH conditions between 3.0 and 6.0, as a preservative (e.g., as an antibacterial and / or antifungal agent), and as an antioxidant.

[0048] In a particularly preferred embodiment, the colorant composition, according to the first or third aspect of the invention, is provided for use as a colorant, preservative, and antioxidant, more preferably as a food colorant, preservative, and antioxidant, and even more preferably as a food colorant, preservative, and antioxidant under pH conditions between 3 and 3.5 or between 5.0 and 6.0. Such pH conditions may correspond, for example, to the pH of a food matrix.

[0049] According to a fifth aspect of the invention, a food product comprising a coloring composition according to the first or third aspect of the invention is provided. "Food product" means 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). Such food product may include, but is not limited to, gelatins, flavored fermented milks, dairy desserts, ice cream, popsicles, flavored soft drinks, and confectionery such as gummies or lollipops, etc.

[0050] In a preferred embodiment, the food product further comprises a food matrix having a pH value between approximately 3.0 and 3.5, or more preferably, a food matrix with a pH value between 3.0 and 3.5. Examples of food products comprising such a food matrix with a pH value of approximately 3.0–3.5 include, but are not limited to, gelatins, flavored fermented milks, juices, nectars, chicken, soft drinks, and confectionery (e.g., gummies or lollipops). In the context of the present invention, "food matrix" refers to the food product as such, prior to the introduction or addition of the coloring composition of the present invention.

[0051] In fact, it is well known that the pH of the food matrix significantly influences the color of natural colorants currently used in the food industry, such as E-163, leading to reddish hues at pH 3, with L* values ​​of 20.17, a*: 44.29, and b*: 32.82 according to the CIELAB system. In this respect, the colorant compositions of the present invention are particularly advantageous, allowing for the production of magenta colors, which currently require the combination of various synthetic colorants with blue tones, since the food industry does not currently have access to natural or synthetic color additives that exhibit a magenta-purple hue.

[0052] The CIELAB system, L* is a model that allows colors to be classified according to 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).

[0053] In another preferred embodiment, the food product further comprises a food matrix with a pH value between approximately 5.5 and 6.0 or, more preferably, a food matrix with a pH value between 5.5 and 6.0. It is known that some dairy desserts, such as cream-based ice creams, have a pH of around 5.5–6.0, so that it is not possible to impart a magenta-purple hue even with the natural colorant E-163, due to the hue it exhibits in that pH range, more particularly at pH 6, with L* values: 11.14, a*: 31.72, Σ>*: 4.47, corresponding to a dark maroon color, which gradually shifts to a purple hue and then to a dark blue.However, the coloring compositions of the present invention allow obtaining magenta-purple colors within that pH range of approximately 5.5–6.0, with CIELAB values, for example, of L*: 7.06, a*: 14.05, b*: -0.74 or L*: 14.69, a*: 14.98, b*: -1.14, as demonstrated experimentally in the examples described below. Throughout the description and claims, the word “comprises” and any variation thereof are not intended to exclude other technical features, ingredients, or steps.

[0054] BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Fig. 1 - Anthocyanin profile of the lyophilized powder BV2 extract, obtained by ultra-high efficiency liquid chromatography coupled to an ultrasensitive diode detector (UHPLC-DAD, after identification carried out by HPLC-DAD-MS).

[0056] Fig. 2 - Anthocyanin profile of the MC2 extract in lyophilized powder, obtained by UHPLC-DAD, with prior identification carried out by HPLC-DAD-MS.

[0057] 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.

[0058] EXAMPLES

[0059] The following examples are provided to illustrate the invention and the technical effects it provides, but should not be considered as limiting its scope. Therefore, it is possible that the invention may be implemented in a manner different from that specifically described in the following examples.

[0060] Example 1 - Coloring composition, in liquid form, of extracts of seedless fruit of Berberis vulgaris L. and peel of the fruit of Myrtus communis L.

[0061] Preparation and characterization of the seedless fruit extract of Berberis vulgaris L. (hereinafter, “BV1 extract”)

[0062] First, Berberis vulgaris L. fruits at optimal maturity (11.0–19.0 ​​°Bhx, determined using an Atago refractometer at 20 °C according to official method 932.14C (AOAC, 2005)) were selected, and the seeds were removed, thus obtaining seedless Berberis vulgaris L. fruit (peel and pulp). This fruit was then subjected to freeze-drying, specifically a 5-day freeze-drying cycle at -80 °C (± 5 °C) at 0.029 mbar, followed by particle size homogenization to approximately 0.150 mm using an IKA Multidrive Basic (BS000) mill. Subsequent particle size verification was performed using a 100-mesh (US STD. Sieve) sieve (corresponding to 0.149 mm or 0.0059 inches). The extract of the seedless fruit of Berberis vulgaris L.The extract was then obtained by hydroalcoholic extraction under ultrasonic power conditions (Fisherbrand™, Sonic Dismembrator, Model 705) of 325–375 W, with a solid-to-liquid ratio of 20–28 g / L (i.e., the weight of freeze-dried seedless Berberis vulgaris L. fruit extract relative to the volume of hydroalcoholic medium), for a time of 1.5–4 minutes, using an ethanol-water mixture (80%:20%, v / v) acidified with citric acid to pH 3 as the extraction solvent. This yielded a hydroalcoholic extract of seedless Berberis vulgaris L. fruit rich in anthocyanins (extract BV1).

[0063] Extract BV1 contained a total monomeric anthocyanin content of 9.15 mg cya-3-gluE / g extract BV1 (where “cya-3-gluE” means “cyanidin-3-glucoside equivalents”), as analyzed by the differential pH spectrophotometric method. In this analytical method, the sample is placed in a solution with pH 1 so that the anthocyanins are in their oxonium form, their most stable form, and in another solution with pH 4.5 where the anthocyanins are converted to a colorless hemiacetal form. Quantification is performed based on the difference in absorbance between the two solutions, measured at the wavelength of highest absorption of the predominant anthocyanin (500–560 nm).

[0064] Extract BV1 was also 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 rate 0.75 mL / min, trifluoroacetic acid (0.1%) / acetonitrile. This analysis 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 color of extract BV1 was determined using a Hunter ColorFlex CiE Illuminate C, 2 colorimeter. o and 45 / 0° geometry, using cylindrical glass cuvettes 5 cm in diameter and 1.3 cm high, and the following values ​​were obtained, expressed in the CIELAB system: L* 23.44, a*: 48.00 and b* 2.94 at pH 3.

[0065] Preparation and characterization of the extract of the peel of the fruit of Myrtus communis L. (hereinafter, “MC1 extract”)

[0066] On the other hand, Myrtus communis L. fruits at optimal maturity (6.0–9.0 °Brix, determined using an Atago refractometer at 20 °C according to official method 932.14C (AOAC, 2005)) were selected, and the peels were separated from the pulp and seeds, thus obtaining Myrtus communis L. fruit peel devoid of pulp and seeds. This peel was then subjected to freeze-drying, specifically a 5-day freeze-drying cycle at -80 °C (± 5 °C) at 0.029 mbar, followed by particle size homogenization (0.150 mm) using an IKA Multidrive Basic (BS000) mill. The extract of the peel of the fruit of Myrtus communis L. was obtained by hydroalcoholic extraction under conditions of ultrasonic power (Fisherbrand™, Sonic Dismembrator, Model 705) of approximately 450 - 500 W, with a solid-liquid ratio of 18 - 22 g / L (e.g., ratio of the extract of the seedless fruit of Myrtus communis L.The extract was lyophilized (by weight relative to the volume of hydroalcoholic medium) for a period of 18 to 22 minutes, using a mixture of ethanol and water (80%:20%, v / v) with a pH value of 6 as the extraction solvent. This allowed obtaining a hydroalcoholic extract of Myrtus Communis L. rich in anthocyanins (extract MC1).

[0067] The results showed that extract MC1 had a total monomeric anthocyanin content of 26.34 mg cya-3-gluE / g extract MC1. Extract MC1 was also analyzed by IIHPLC 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. The major anthocyanins were determined to be delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunidin-3-O-glucoside, and malvidin-3-O-glucoside. The color of extract MC1 was determined using a Hunter ColorFlex CiE Illuminate C, 2 colorimeter. oand 45 / 0° geometry, using cylindrical glass cuvettes 5 cm in diameter and 1.3 cm high, 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.

[0068] Preparation of the coloring composition in liquid form comprising the seedless fruit of Berberis vulgaris L. and the fruit peel of Myrtus communis L. (hereinafter referred to as coloring composition “BV1 :MC1”)

[0069] Finally, both individual extracts, BV1 and MC1, were mixed in quantities corresponding to 50%:50% (w / w) ± 10% of Berberis vulgaris L. and Myrtus communis L., resulting in a coloring composition according to the invention, in liquid form, more particularly in the form of a hydroalcoholic solution, comprising skin extracts of Berberis vulgaris L. and Myrtus communis L. (coloring composition BV1:MC1).

[0070] Example 2 - Coloring composition, in the form of a freeze-dried powder, of extracts of seedless fruit of Berberis vulgaris L. and peel of Myrtus communis L. The individual extracts BV1 and MC1 were obtained as described in Example 1, which were used as described below:

[0071] Preparation and characterization of the seedless fruit extract of Berberis vulgaris L. in the form of a lyophilized powder (hereinafter, “BV2 extract”)

[0072] Starting with extract BV1, the ethanol was evaporated using a sample concentrator (SpeedVac SPD130DLX; 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).

[0073] The lyophilized extract BV2 had an extraction yield of 65.8% and a moisture content of 15.14 g water / 100 g extract. Its chemical composition revealed a total polyphenol content of 159.84 mg GAE / g dry extract (where “GAE” stands for “gallic acid equivalents”), as measured by the Fast Blue BB method (Palombini et al., 2016). The Fast Blue BB method involves reacting the sample with the Fast Blue BB reagent (e.g., 4'-amino-2',5'-diethoxybenzanilide) at a concentration of 0.1% (w / v) in 5% (v / v) NaOH. In an alkaline environment, the phenolic groups of the polyphenols form a stable azo complex, producing a color that is then measured spectrophotometrically at a wavelength of 420 nm.

[0074] Of this total polyphenol value corresponding to 159.84 mg GAE / g dry extract, hydroxybenzoic acids are the major 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, and it also contains 5.50 mg QE / g (where “QE” means “quercetin equivalents”) of dry extract of flavonols, all of which were measured following the QUENCHER (QUIck, Easy, New, CHEap and Reproducible) methodology, where a small amount of sample, previously homogenized to a particle size of 0.037 mm, is subjected to direct contact with the reagents specific to each determination.This methodology allows for the precise and reliable measurement of both soluble and insoluble compounds, and enables subsequent spectrophotometric analysis at the wavelengths of greatest absorption for 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 for each phenolic group.Furthermore, by high-performance liquid chromatography coupled to an ultraviolet-visible detector (HPLC-UV / Vis), after extraction in metaphosphoric acid (4.5%) and separation and quantification by IIV-VIS (Thermo Separation Spectra Series LIV100) 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.

[0075] 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 ionization electrospray mass spectrometer (Linear Ion Trap LTQ XL, Thermo Scientific, C18 Water Spherisorb ODS2 column), quantifying a total of 5 anthocyanins by ultra-fast high-efficiency liquid chromatography coupled to a diode 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.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).

[0076] The inventors analyzed the coloring activity of the BV2 extract, observing that it exhibited 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 ​​above 7.5. Specifically, the BV2 extract at pH 4.5 had a color close to the standard RAL 02020 29 color of the RAL design color system, while at pH 6.5 it had a color close to the S8010 Y10R standard of the NCS 2050 color system (Natural Color System is an international color system for design, architecture, production, research, and education). BV2 demonstrated a chromatic profile with a characteristic color that evolved with pH, ​​in aqueous solution at a concentration of 20 mg / mL (Table 1):

[0077] Table 1

[0078] On the other hand, the preservative activity (antibacterial and antifungal) of BV2 was analyzed, which allowed us to conclude that BV2 presents inhibitory activity against bacteria involved in foodborne illnesses and which are mandatory to analyze 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 showed inhibitory activity against the growth of three Gram-negative and three Gram-positive bacteria, exhibiting minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of 10 / 20, 5 / 10, 2.5 / 10, 5 / 10, and 1.25 / 20 mg / mL of extract against the reference Gram-negative bacterial strains Yersinia enterocolitica (ATCC 8610), Enterobacter cloacae (ATCC 49741), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 9027), and Salmonella enterocolitica (ATCC 13076), respectively, and minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of 1.25 / 10, 5 / 20, and 2.5 / 20 mg / mL against the bacterial strains Gram positive reference Bacillus cereus (ATCC 11778), Listeria monocytogenes (ATCC 19111) and Staphylococcus aureus (ATCC 25923), respectively.On the other hand, the extract also exhibits antifungal activity with minimum inhibitory concentrations (MICs) 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 bacterium was added to each well, ensuring the presence of 1.5 x 10⁻⁶ CFU / mL. 5Colony-forming units (CFU) per well were incubated at 37 °C for 24 hours. After incubation with iodonitrotetrazolium chloride (INT), used as a stain, the minimum inhibitory concentration (MIC) was determined. This was the minimum concentration that inhibited bacterial growth, visible as a yellow coloration that did not change to pink. Bacteria from the wells that remained yellow were subsequently plated onto solid medium and incubated at 37 °C for 24 hours. The lowest concentration with no bacterial growth was determined as the minimum bactericidal concentration (MBC). For antifungal activity, the minimum inhibitory concentration (MIC) was determined using a binocular microscope.

[0079] The antioxidant activity of the BV2 extract was also evaluated using two in vitro chemical antioxidant activity methods (Folin-Ciocalteu and DPPH) and one in vitro biological antioxidant activity method (OxHLIA). In the Folin-Ciocalteu method, the reducing compounds present in the sample react with the phosphomolybdotungstic acid in the Folin-Ciocalteu reagent, generating a medium blue coloration at a wavelength of 750 nm. In the DPPH method, the DPPH (2,2-diphenyl-1-picrihydrazil) solution reacts with the antioxidant molecules in the sample, causing a change in color from purple to yellow. This color change can be monitored by spectrophotometric readings at 517 nm.The OxHLIA method, on the other hand, is an in vitro biological method in which sheep blood erythrocytes, diluted in PBS, are brought into contact with the corresponding fruit extract and with AAPH (2,2'-azobi(2-methylpropionamidine) dihydrochloride), which generates free radicals. The optical density is then measured at 690 nm every 10 minutes until complete hemolysis is observed.

[0080] Extract BV2 showed high antioxidant activity with values ​​of 88.03 mg GAE / g dry extract using the Folin-Ciocalteu method and 111.37 mg TE / g dry extract using the DPPH method (where “TE” stands for “Trolox equivalents”). For OxHLIA, an IC50 of 125 pg / mL was obtained for antihemolytic activity over 60 minutes. The term “IC50” means “median inhibitory concentration,” that is, the concentration of inhibitor that causes a 50% decrease in the analyzed activity. Finally, although the consumption of Berberis vulgaris L. fruit is considered safe and has been traditionally consumed for generations in the Iberian Peninsula, primarily in Spain, toxicity studies were conducted.Thus, the possible toxicity of the fruit in non-tumor cells of pig liver (PLP2) was evaluated by monitoring cell growth by phase contrast microscopy, obtaining as a result the concentration of the sample capable of inhibiting 50% of the net cell growth, which in this case was a concentration greater than 400 pg / mL of extract, showing zero hepatotoxicity.

[0081] Preparation and characterization of the extract of the peel of the fruit of Myrtus communis L. in the form of a lyophilized powder (hereinafter, “MC2 extract”)

[0082] Starting with extract MC1, the ethanol was evaporated using a sample concentrator (SpeedVac SPD130DLX; 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 peel of the fruit of Myrtus communis L. (extract MC2).

[0083] The lyophilized extract MC2 had an extraction yield of 57.6% and a moisture content of 9.22 g water / 100 g extract. Its chemical composition revealed a total polyphenol content of 271.18 mg GAE / g dry extract, with hydroxybenzoic acids being the most abundant at 44.96 mg GAE / g dry extract. Additionally, the extract contained 20.33 mg / g dry extract of flavonols and 17.78 mg / g dry extract of hydroxycinnamic acids. These flavonols were determined using the QUENCHER method, as described in the section on the preparation and characterization of BV2.

[0084] 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.

[0085] In addition, total monomeric anthocyanins were determined by the differential pH method (see description of this method in Example 1) obtaining a total of 47.51 mg cya-3-gluE / g dry extract. In addition, 9 individual anthocyanins (Figure 2) were identified by HPLC-DAD-ESI-MS (Dionex Ultimate 3000 UPLC, Thermo Scientific, coupled to an ionization electrospray 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).

[0086] Extract MC2 was found to exhibit color variability depending on pH, and stability was observed from an intense purple color (L*: 77.42, a*: 34.61, b*: 15.45) at pH 3.0 to a bluish-purple color (L*: 4.22, a*: 13.71, b*: 0.05) at pH 6. Specifically, extract MC2 had a color close to the RAL 010 20 25 color standard of the RAL Design color system at pH 4.5.

[0087] MC2 demonstrated a characteristic color profile that evolved with pH, ​​in aqueous solution at a concentration of 4 mg / mL (Table 2):

[0088] Table 2

[0089] 1 RAL Design System, 2 NCS 2050 System, 3 RAL Classic System.

[0090] The MC2 extract also exhibited antimicrobial activity against five Gram-negative and three Gram-positive bacteria used as reference strains, showing minimum inhibitory concentrations and minimum bactericidal concentrations (MIC / MBC) 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 / MBC of 0.6 / 20 mg / mL extract against the Gram-positive strains Bacillus cereus (ATCC 11778), Listeria monocytogenes (ATCC 19111), and Staphylococcus. aureus (ATCC 25923), respectively. On the other hand, the extract also showed antifungal activity with an MIC of 1.25 mg / mL against the fungus Aspergillus brasiliensis.For this purpose, the same procedure was used as described above for extract BV2.

[0091] Antioxidant activity was determined using the in vitro chemical methods Folin-Ciocalteu and DPPH, and the in vitro biological method OxHLIA, as described above, in the section relating to the preparation and characterization of extract BV2 in Example 2. Antioxidant activities of 96.81 mg GAE / g and 73.90 mg TE / g were observed for extract MC2 using the Folin-Ciocalteu and DPPH methodologies, respectively, and an IC50 of 59 pg / mL was obtained for antihemolytic activity over 60 minutes using the OxHLIA method.

[0092] Finally, although the consumption of Myrtus communis L. fruit is considered safe, and its wild fruit has been traditionally consumed for generations, toxicity studies were conducted. Specifically, the potential toxicity of the fruit was evaluated in non-tumor pig liver cells (PLP2), revealing no hepatotoxicity (> 400 pg / mL extract).

[0093] Preparation and characterization of the coloring composition in the form of a lyophilized powder comprising the seedless fruit extract of Berberis vulgaris L. and the fruit peel extract of Myrtus communis L. (hereinafter referred to as the coloring composition “BV2:MC2”)

[0094] Once the individual BV2 and MC2 extracts were obtained from each fruit of the different plant species, a coloring composition according to the invention (BV2:MC2 coloring composition) was obtained in the form of a freeze-dried powder by mixing both extracts BV2 and MC2 in proportions of 50:50 w / w ± 10%. This coloring composition, suitable for use as a food ingredient in the form of a freeze-dried powder, exhibited the desired color characteristics at pH 3.5, as well as suitable functional and organoleptic properties.

[0095] Regarding the BV2:MC2 composition, it is worth highlighting the total amount of polyphenols (281.59 mg GAE / g dry extract) measured by the Fast Blue BB method, with hydroxybenzoic acids being the most abundant at 86.29 mg GAE / g dry extract. Furthermore, this extract contains a total of hydroxycinnamic acids of 32.28 mg FAE / g dry extract and flavonols of 14.77 mg QE / g dry extract, measured using the QUENCHER methodology described above.

[0096] 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.

[0097] In addition, total monomeric anthocyanins were determined by the differential pH method (see description of this method in Example 1) obtaining a total of 39.91 mg cya-3-gluE / g dry extract.Furthermore, 11 individual anthocyanins were identified by HPLC-DAD-MS-ESI analysis (Dionex Ultimate 3000 UPLC, Thermo Scientific, coupled to a Linear Ion Trap LTQ XL mass spectrometer, Thermo Scientific, C18 Water Spherisorb ODS2 column), which are shown in Figure 3 with their corresponding chromatographic peaks and their retention value Rt, and 5 anthocyanins were quantified by UHPLC-DAD (Agilent LC-1290II, Poroshell 120 SB-C18 column, 4.6 mm x 75 mm, 2.7 pm, 35 °C, flow rate 0.75 mL / min, trifluoroacetic acid (0.1%) / Acetonitrile), delphinidin-O-glucoside (6.70 mg / g dry extract), cyanidin-3-O-glucoside (1.66 mg / g dry extract), petunidin-3-O-glucoside (6.21 mg / g dry extract), pelargonidin-3-O-glucoside (0.12 mg / g dry extract) and malvidin-3-O-glucoside (7.16 mg / g dry extract).

[0098] This composition also exhibited a characteristic color profile due to the unique composition of monomeric anthocyanins obtained by combining the seedless fruit extracts of Berberis vulgaris L. and the bark extract of Myrtus communis L., according to the present invention. Specifically, thanks to the synergistic combination of these extracts from both fruits, the coloring compositions of the present invention yield a characteristic magenta color that is particularly stable at a pH of 3–3.5 and a purple color that is stable at pH values ​​of 5.5–6.0.

[0099] Specifically, this BV2:MC2 composition exhibited color variability in aqueous solution depending on the pH. An unexpected stability of a reddish-purple color (L*: 6.90, a*: 22.09, b*: 5.12) was observed at pH 3.5, a purple color (L*: 7.06, a*: 14.05, Σ>*: -0.74) at pH 5.5, and a grayish-green color was evident at pH values ​​above 7.5 (L*: 5.45, a*: 0.52, Σ>*: 2.02). Specifically, the BV2:MC2 extract had a color close to RAL 8022 of the RAL Classic color system at pH 6.5 to 7.0.

[0100] The BV2:MC2 extract showed a characteristic color profile that evolved with pH, ​​in aqueous solution at a concentration of 8 mg / mL (Table 3): Table 3

[0101] 1 RAL Classic System.

[0102] On the other hand, the BV2:MC2 composition showed preservative activity (antibacterial and antifungal) against 5 Gram-negative bacteria and 3 Gram-positive bacteria used as reference strains, showing MIC / MBC of 10 / 20 mg / mL for Yersinia enterocolitica (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 showed MIC / MBC of 0.6 / 20 against Listeria monocytogenes (ATCC 19111) and Staphylococcus aureus (ATCC 25923), and 0.6 / 20 against Bacillus cereus (ATCC 11778). Furthermore, the extract also exhibited antifungal activity with an MIC of 5 mg / mL of extract against Aspergillus fumigatus. The same procedure described above for both extract BV2 and extract MC2 was used for this purpose.

[0103] Furthermore, the antioxidant activity of this BV2:MC2 composition was confirmed using the in vitro Folin-Ciocalteu and DPPH chemical methods, as described above in the section on the preparation and characterization of the BV2 extract in Example 2, yielding values ​​of 60.31 mg GAE / g and 164.42 mg TE / g dry extract, respectively. Additionally, an IC50 of 29 pg / mL was obtained for antihemolytic activity over 60 minutes using the OxLHIA methodology.

[0104] Example 3 - Coloring composition in the form of a lyophilized powder of seedless fruit extracts of Berberis vulgaris L. and fruit peel of Myrtus communis L. in the form of a microencapsulated lyophilized powder

[0105] The 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 Berberis vulgaris L. in the form of a microencapsulated lyophilized powder (hereinafter, “BV3 extract”)

[0106] Starting from extract BV2, encapsulation was carried out by spray-drying (Buchi, Mini Spray Dryer, model B-290), with maltodextrin as the 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).

[0107] Extract BV3 showed an encapsulation extraction yield of 60.4%, and a total amount of 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 extract BV2 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 GAE / 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.

[0108] Additionally, a total monomeric anthocyanin content 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). Furthermore, a total of 5 anthocyanins were quantified by ultra-high-performance liquid chromatography coupled to a diode 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. 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).

[0109] 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

[0110] 1 RAL Classic System, 2 Sherwin-Williams System, 3 PPG / Johnstone's System, 4 Isomat System, 5 Dulux Trade System, 6 RAL Design System.

[0111] In aqueous solution, the BV3 extract presented a desired intense reddish color (L*: 19.43, a*: 41.43, o*: 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*: 17.46, o*: 9.20) that resembles the standard LPC 0416 color of the Isomat system.

[0112] The BV3 extract also exhibited antimicrobial activity against 5 Gram-negative and 3 Gram-positive bacteria used as reference strains, showing 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. Furthermore, the microencapsulated extract exhibited antifungal activity with a minimum inhibitory concentration of 10 mg / mL against the fungus Aspergillus fumigatus. The same procedure described above for both extract BV2 and extract MC2 was used for this purpose.On the other hand, antioxidant activity was evaluated using the Folin-Ciocalteu and DPPH methods (see descriptions of these methods in the section on the preparation and characterization of extract BV2 in Example 2), yielding antioxidant activity values ​​of 46.54 mg TE / g and 18.43 mg GAE / g using the DPPH and Folin-Ciocalteu methods, respectively. An IC50 value of 293 pg / mL was also determined using the OxHLIA in vitro biological antioxidant activity method (see description of this method in the section on the preparation and characterization of extract BV2 in Example 2). Preparation and characterization of the Myrtus communis L. fruit peel extract in the form of a microencapsulated lyophilized powder (hereinafter, “MC3 extract”).

[0113] Starting from the MC2 extract, encapsulation was carried out by spray-drying (Buchi, Mini Spray Dryer, model B-290), with maltodextrin as the 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).

[0114] Extract MC3 exhibited an encapsulation yield of 69.6% and a total polyphenol content of 112.09 mg GAE / g microencapsulated extract (determined by the Fast Blue BB method). Hydroxybenzoic acids were the most abundant, at 38.65 mg GAE / g microencapsulated extract, followed by hydroxycinnamic acids at 6.83 mg GAE / g and flavonols at 7.03 mg GAE / g microencapsulated extract. All these values ​​were determined using the QUENCHER methodology, as described above in the section on the preparation and characterization of BV2.

[0115] A total of 21.06 mg cya-3-gluE / g microencapsulated extract monomeric anthocyanins was also determined using the differential pH spectrophotometric method (see description of the differential pH method in Example 1). In addition, a total of 4 anthocyanins were quantified by UHPLC-DAD under the following conditions: Agilent LC-1290II, Poroshell 120 SB-C18 column, 4.6 mm x 75 mm, 2.7 pm, 35 °C, flow rate 0.75 mL / min, trifluoroacetic acid (0.1%) / Acetonitrile. Thus, the following anthocyanins were quantified: delphinidin-3-O-glucoside (8.81 mg / g dry extract), cyanidin-3-O-glucoside (1.44 mg / g dry extract), petunidin-3-O-glucoside (8.30 mg / g dry extract) and malvidin-3-O-glucoside (8.64 mg / g dry extract).

[0116] 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):

[0117] Table 5

[0118] 1 RAL Classic System.

[0119] A range of very intense purples with a characteristic color was obtained. Thus, MC3 exhibited a color not currently found among the natural food coloring options available on the market. It was also verified that the MC3 extract was stable up to pH 7.0, above which olive green colorations were observed (L* 2.40, a* 3.48, and b* 0.22), with a color similar to RAL 9005 of the RAL Classic color system, while the unencapsulated MC2 extract began to degrade at pH 6.5 (L* 3.29, a* 7.87, and b* 0.59).

[0120] Furthermore, the preservative activity of the MC3 extract was confirmed, showing antimicrobial activity against 5 Gram-negative bacteria and 3 Gram-positive bacteria used as a 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 MIC / 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 exhibited MIC / MBC values ​​of 5 / 20, 20 / 20, and 10 / 20 mg / mL against Bacillus cereus (ATCC 11778), Listeria monocytogenes (ATCC 19111), and Staphylococcus aureus (ATCC 25923), respectively. Furthermore, the microencapsulated extract showed antifungal activity against Aspergillus fumigatus with a minimum inhibitory concentration (MIC) of 10 mg / mL.For this purpose, the same procedure described above was used for both extract BV2 and extract MC2. Furthermore, 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 methods (see descriptions of these methods in the section on the preparation and characterization of extract BV2 in Example 2), respectively. An IC50 value of 315 pg / mL was also determined using the OxHLIA in vitro biological antioxidant activity method (see descriptions of this method in the section on the preparation and characterization of extract BV2 in Example 2). Preparation of the colorant composition in the form of a microencapsulated lyophilized powder comprising the seedless fruit extract of Berberis vulgaris L. and the fruit peel extract of Myrtus communis L.(hereinafter referred to as the coloring composition “BV3:MC3”).

[0121] Finally, both individual extracts, BV3 and MC3, were mixed in quantities corresponding to 50%:50% w / w ± 10% of Berberis vulgaris L. and Myrtus communis L., resulting in a coloring composition according to the invention, in the form of a microencapsulated lyophilized powder, comprising extracts of the seedless fruit of Berberis vulgaris L. and the peel of the fruit of Myrtus communis L. (coloring composition BV3:MC3). The color profile of the BV3:MC3 extract at different pH values ​​at a concentration of 13 mg / mL of aqueous solution is shown in the following table (Table 6).

[0122] Table 6

[0123] 1 NSC2050 System, 2 Pantone® C (Coated) System, 3 RAL Effect System, 4 RALCIAssic System, 5 RAL Design System

[0124] Axis mp I or 4 - Coloring composition of extracts of the seedless fruit of Berberis vulgaris L. and the peel of the fruit of Myrtus communis L. in the form of encapsulated lyophilized powder (BV4:MC4)

[0125] Starting from the coloring 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 the encapsulating agent at a temperature of 120 °C, aspiration at 95%, with a feed flow of 5 mL / min, an air flow of 7.88 L / min and a proportion of 35% - 45% of encapsulating agent, obtaining a coloring composition in the form of a microencapsulated lyophilized powder, comprising extracts of the seedless fruit of Berberis vulgaris L. and the peel of the fruit of Myrtus communis L. (hereinafter referred to as “coloring composition BV4:MC4”), with a particle size of 0.5 - 25 pm determined by Mastersizer 3000 (Malvern, UK).

[0126] The encapsulated BV4:MC4 composition showed an encapsulation yield of 69.64%. It was determined to contain a total of 83.39 mg GAE / g dry extract polyphenols using 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. This method allowed the identification of hydroxybenzoic acids as the most abundant, totaling 37.63 mg / g dry extract, followed by hydroxycinnamic acids at 7.48 mg / g dry extract and flavonols at 5.76 mg / g dry extract, measured following the QUENCHER methodology, as described above in the section relating to the preparation and characterization of BV2.

[0127] Additionally, a total monomeric anthocyanin content 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-129011, Poroshell 120 SB-C18 column, 4.6 mm x 75 mm, 2.7 pm, 35 °C, flow 0.75 mL / min, fluoroacetic 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).

[0128] A BV4:MC4 composition microencapsulated as 35% - 45% by weight of maltodextrin (i.e., 35 - 45 g maltodextrin / 100 g microencapsulated sample) as the encapsulating agent, in addition, 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):

[0129] Table 7

[0130] 1 NCS 2050 System, 2BS 2660 System. The BV4:MC4 extract yielded a characteristic color, distinct from those obtained with the BV or MC extracts individually. Specifically, an intense magenta color was obtained at pH 4.5, a color similar to S7020-R20B of the NCS 2050 color system was observed at pH 5.5, and a color close to S8010-R10B of the same system was observed at pH 6.0. Furthermore, the extract was experimentally identified as stable up to pH 7, above which greenish colorations were obtained (L* 12.03, a* -0.89, and b* 8.69).Likewise, the BV4:MC4 extract exhibited preservative activity with antimicrobial activity against 5 Gram-negative and 3 Gram-positive bacteria used as reference strains, MIC / MBC of 10 / 20, 5 / 20, 10 / 20, 2.5 / 20 and 20 / 20 mg / mL against the Gram-negative bacteria Enterobacter cloacae (ATCC 49741), Escherichia coli (ATCC 25922), Pseudomonas aeruginosas (ATCC 9027), Salmonella enterocolitica (ATCC 13076) and Yersinia enterocolitica (ATCC 8610), respectively, and MIC / MBC of 2.5 / 20, 10 / 20 and 10 / 20 mg / mL against the Gram-positive bacteria Bacillus cereus (ATCC 11778), Listeria monocytogenes (ATCC 11778) and Listeria monocytogenes (ATCC 11778). 19111) and Staphylococcus aureus (ATCC 25923), respectively. On the other hand, the BV4:MC4 extract showed antifungal activity with a minimum inhibitory concentration of 20 mg / mL against the fungus Aspergillus fumigatus. For this, the same procedure described above was used for both the BV2 and MC2 extracts.Furthermore, this microencapsulated BV4:MC4 composition exhibited an antioxidant activity of 28.91 mg GAE / g and 45.84 mg TE / g microencapsulated extract using the Folin-Ciocalteu and DPPH methodologies, respectively, as described above in the section on the preparation and characterization of the BV2 extract in Example 2. An IC50 value of 228 pg / mL was also determined using the OxHLIA in vitro biological antioxidant activity method (see description of this method in the section on the preparation and characterization of the BV2 extract in Example 2).

[0131] 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)

[0132] Prototype of gelatin formulated with BV2 only

[0133] 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 the main gelling agent), 0.1–0.5% by weight of E330, 0.30–0.35% by weight of BV2 extract, and flavoring. A gelatin with a pH of 3–3.5 was obtained, along with CIELAB parameters of L* 38.35, a* 39.79, and b* 15.21, corresponding to a red hue (a color close to RAL 020 40 40 of the RAL Design system or 21105 of the US Federal Standard 595C 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). Furthermore, a shelf life of 15 days was determined., period in which the absence of microbial growth per gram of analyzed material is determined, of: Enterobacteriaceae (absence / g) and Salmonella (absence / 25 g), or maximum of mesophilic aerobes (10. 3 cfu / g) and Listeria monocytogenes (100 cfu / g), according to Regulation (EC) No 2073 / 2005.

[0134] Prototype of gelatin formulated with BV2:MC2

[0135] This prototype was formulated, for every 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 the base gelling ingredient), 0.1 - 0.5% by weight of citric acid (E330), with 0.20 - 0.25% by weight of the composition BV2:MC2 (the composition BV2:MC2 contains 50%:50% w / w ± 10%), and flavoring. A gelatin with a pH of 3 - 3.5 was obtained and CIELAB parameters in the final product of L*: 23.69, a*: 35.50, £>*: 10.49, which correspond to a magenta tone (color close to S 5040-R10B of the NCS 2050 system or 7421 C of the Pantone® C color system).

[0136] Gelatin prototypes formulated with synthetic commercial colorants

[0137] For comparison with the gelatin products available on the market, other prototypes were formulated with commercial colorants: 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 in the percentage of colorant incorporated into the mixture: 0.05–0.1% by weight was added for the synthetic colorant E-124; 0.15–0.25% by weight for the commercial natural colorant E-163; or 0.15–0.25% by weight for the mixture of the commercial synthetic colorants in a 1:4 ratio (E133:E124).

[0138] Table 8

[0139] 1 RAL Design System, 2 NCS 2050 System, 3 Isomat System, 4 RAL Effect system, 5 DIN 6164 system.

[0140] As can be seen from the CIELAB values ​​obtained at pH 3.5 (Table 8), the commercial food colorings produced completely different shades (reddish coloration in the case of the prototypes formulated individually with E-124 or E-163; bluish coloration in the case of the prototype formulated with the combination of E-124 and E-163) compared to those observed with the compositions of the present invention, illustrated by the gelatin prototype formulated with BV2:MC2, which resulted in a magenta hue at these pH values. Comparative study of the chromatic profile and CIELAB parameters of the colorant E-163, Berberis vulgaris L. extract (BV3) in the form of a microencapsulated lyophilized powder, and Myrtus communis L. extract.in the form of microencapsulated freeze-dried powder (MC3), coloring composition BV3:MC3 and microencapsulated coloring composition BV4:MC4. In Table 9, samples corresponding to the current commercially available colorant E-163, from different natural sources, such as purple carrot, red cabbage, radish and grape, have been analyzed against samples of BV3, MC3, BV3:MC3 and composition BV4:MC4 according to the present invention.

[0141] Table 9

[0142] 1 NCS 2050 System, 2 DIN 6164 system, 3 Syntheme Pantone® U (Uncoated), 4 RAL Design System, 5 Dulux Trade System, 6 Pantone® C (Coated) System, 7 RAL Effect System

[0143] The food industry currently lacks naturally occurring coloring additives with magenta-purple hues, making it necessary to use mixtures of synthetic colorants with blue tones, such as E-131 (patent blue), E-132 (indigotine), and E-133 (brilliant blue FCF), with E-163 (anthocyanin), or other colorants with red tones, such as E-122 (azorubine) and E-124 (Ponceau 4R). The commercially available colorant E-163, analyzed in Table 9, provides a reddish-maroon color, but does not achieve a magenta-purple hue, unlike the compositions of the present invention.

[0144] Regarding the antioxidant capacity of the commercial colorant E163, it was found to have 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 extract BV2 in Example 2. An IC50 value of 225 pg / mL of extract was also determined by the in vitro biological OxHLIA method. Furthermore, it was shown that the commercial natural colorant E-163 had preservative activity, exhibiting 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 Bacillus cereus (ATCC 11778) and Listeria monocytogenes (ATCC 19111), respectively.The dye also exhibited antifungal activity with a minimum inhibitory concentration of 20 mg / mL against the fungus Aspergillus fumigatus. These data demonstrate the superior preservative capacity of the present invention.

[0145] With the present invention, a series of coloring compositions based on extracts of natural origin with coloring power are obtained in different presentations (hydroalcoholic extract, freeze-dried extract in powder and microencapsulated extract in powder) that can 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 color tones 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.).

[0146] Study of the antimicrobial and antifungal activity of extracts BV2, MC2,

[0147] BV3 and MC3, from compositions BV2:MC2 and BV4:MC4, and from colorant E-163

[0148] 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 preceding Examples, as well as the results obtained with the commercial dye E-163. All these results were obtained using the method described in Example 2 of the present invention.

[0149] Table 10

Claims

CLAIMS 1. Coloring composition based on anthocyanins, characterized in that it comprises a seedless fruit extract of Berberis vulgaris L. and an extract of the fruit peel of Myrtus communis L.

2. Composition according to claim 1, comprising an amount of said seedless fruit extract of Berberis vulgaris L. comprising between 40% and 60% by weight, relative to the total weight of the composition, and / or an amount of said fruit peel extract of Myrtus communis L. comprising 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 seedless fruit extract of Berberis vulgaris L. comprising between 45% and 55% by weight, relative to the total weight of the composition, and / or an amount of said fruit peel extract of Myrtus communis L. comprising 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 a freeze-dried 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 encapsulated freeze-dried powder.

7. Composition according to claim 6, wherein the encapsulated freeze-dried powder is microencapsulated.

8. Composition according to claim 6 or 7, further comprising at least one encapsulating agent.

9. Method for preparing a coloring composition based on anthocyanins, characterized in that it comprises: a) subjecting at least a portion of seedless fruit of Berberis vulgaris L. to hydroalcoholic extraction to obtain a seedless fruit extract of Berberis vulgaris L., b) subjecting at least a portion of the fruit peel of Myrtus communis L. to hydroalcoholic extraction to obtain an extract of the fruit peel of Myrtus communis L., and c) mixing a quantity of the seedless fruit extract of Berberis vulgaris L. resulting from step a) and a quantity of the fruit peel extract of Myrtus communis L. resulting from step b).

10. Method according to claim 9, wherein the amount of seedless fruit extract of Berberis vulgaris L. and the amount of fruit peel extract of Myrtus communis L. that are mixed in step c) are each independently between 40-60% by weight, with respect to the total weight of the coloring composition.

11. Method according to claim 9 or 10, wherein the amount of seedless fruit extract of Berberis vulgaris L. and the amount of fruit peel extract of Myrtus communis L. that are mixed in step c) are each independently between 45-55% by weight, with respect to the total weight of the coloring composition.

12. Method according to any of claims 9-11, wherein, prior to step a) and / or step b), said portion of seedless fruit of Berberis vulgaris L. and / or said portion of fruit peel 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 ratio of said seedless fruit of Berberis vulgaris L. 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 providing an acidic medium, preferably in a hydroalcoholic medium providing a pH between 2 and 4, more preferably in a hydroalcoholic medium providing 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 providing an acidic pH, preferably in a hydroalcoholic medium providing a pH between 5 and 7, more preferably in a hydroalcoholic medium providing a pH between 5.5 and 6.

5.

17. Method according to any of claims 9-16, wherein the seedless fruit extract of Berberis vulgaris L. resulting from step a) and the fruit peel extract of Myrtus communis L. resulting from step b) are each independently subjected to a freeze-drying step prior to step c).

18. Method according to claim 17, wherein the encapsulation of the freeze-dried seedless fruit extract of Berberis vulgaris L. resulting from said freeze-drying and of the freeze-dried fruit peel extract of Myrtus communis L. resulting from said freeze-drying is carried out independently, in the presence of at least one encapsulating agent, before carrying out step c).

19. Method according to claim 17, wherein the mixture resulting from step c) is encapsulated in the presence of at least one encapsulation agent.

20. Coloring composition based on anthocyanins, characterized in 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 in that it is used as 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 such use further comprises use as an antioxidant.

28. Food product characterized in that it comprises the coloring composition defined according to any of claims 1-8 or 20.

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