Qualitative composition of anthocyanin dye obtained from black currant berries in various methods

. Identification of anthocyanidins was carried out in five samples of anthocyanins obtained under different extraction conditions. It has been shown that anthocyanins are most fully extracted from black currant by an aqueous solution at temperatures of 70° С for 30 minutes and 100°C for 5 minutes, treatment in an ultrasonic bath for 30 minutes and in an ultrasonic homogenizer for 15 minutes. Eight anthocyanidins were identified in the samples of anthocyanins isolated by various methods. The most common were five anthocyanidins - these are cyanidin-3-glucoside, cyanidin-3-rutinoside, delphinidin-3-glucoside, delphinidin-3-rutinoside, petunidine-3-rutinoside. They were identified in all samples of anthocyanins. The proportion of the presence of one or another anthocyanidin in the studied samples depended on the method of isolation of anthocyanins. A more complete release of anthocyanidins is facilitated by the extraction of anthocyanins from blackcurrant berries at temperatures of 70° С for 30 minutes and 100°C for 5 minutes and extraction in an ultrasonic homogenizer at 25°C for 15 minutes. Under these conditions, all eight anthocyanidins are released. Based on this, a technology of the production of anthocyanin dye from berry raw materials was proposed. Developed colorant is a red liquid with a pronounced smell of black currant and a sour taste. Anthocyanin dye has an active acidity (pH) of 3.0, contains 2.99 mg/cm 3 of anthocyanins in its composition and has antiradical activity .


Introduction
Food dyes are substances of synthetic or natural origin used to impart, restore, or standardize the color and appearance of foods, thereby making foods more appealing to consumers.
Synthetic or artificial are food colorings obtained by synthesis methods and are not found in nature.Synthetic dyes have significant processing advantages over most natural dyes, they produce bright, easily reproducible colors, and they are also less sensitive to the various types of stress that the material is subjected to during the manufacturing process.Synthetic dyes also have limiting factors for their use.Studies have shown that synthetic dyes are not toxic by themselves, but when used in mixtures, a synergistic effect can be observed [1].
Consumers are showing increasing interest in food products that contain natural ingredients, including dyes, which provide final products an attractive appearance, natural smell, taste, additional nutritional value, and are generally perceived as healthy and safe [2,3].
One of the ways to solve the problem of safe food production is the creation and use of safe natural food dyes in food production technology.In addition to color, they are sources of biologically active compounds and can impart functional properties to products [4][5][6][7].
There is a tendency in the world to increase demand for red anthocyanin pigments with biological activity, since in their composition, in addition to coloring components, they contain useful biologically active substances and have many useful properties such as lower cholesterol, prevent blood clots, increase vascular elasticity, accelerate wound healing, favorably affect vision, contribute to the prevention of cancer [8][9][10][11][12].In addition, anthocyanin solutions have antiradical activity [13,14].
Anthocyanins and their anthocyanidins are a large group of flavonoids widely distributed in plants.Anthocyanins belong to the group of bioflavonoid natural dyes and contain three to six hydroxyl groups that can be methylated [15].However, the creation of anthocyaninbased food colors has limiting factors, since the extraction of pigments from natural sources, due to poor stability, is not always easy and feasible.In this regard, the purpose of this work is to determine the optimal conditions and modes for the extraction of anthocyanins from berry raw materials to ensure maximum extraction and preservation of biologically active compounds in their composition and the creation of safe natural dyes for food products based on anthocyanin extracts.

Materials and methods
Frozen black currant (Ríbes nígrum) was used as objects to isolate anthocyanins.The anthocyanins were prepared by extracting crushed berry raw materials with distilled water, in the temperature range from 25 to 70°С for 30 min, in the temperature range 100°С for 5 min and using indirect and direct ultrasonic-assisted extraction.
Sonorex RK100H ultrasonic bath (Bandelin, Germany) was used for indirect ultrasoundassisted extraction.Direct ultrasound-assisted extraction was performed in a Sonopuls Vitrasoris homogenizer (Bandelin, Germany).Samples were processed using an ultrasonic bath at the exposure frequency of 35 kHz and the power of 80 W in the temperature range 25°С for 30 min, using an ultrasonic homogenizer of 20 kHz and 75 W in the temperature range 25°С for 15 min.
The content of anthocyanins in solutions was determined in accordance with the method described in the paper of Ivanova et al. [16].
Analysis of the anthocyanins were carried out by HPLC on a Shimadzu LC-20 Prominence HPLC liquid chromatograph (Shimadzu, Japan) equipped with an ultraviolet sensor and a Shodex ODP-40 4E column.Analytical separation of anthocyanins was carried out using a column Shodex, Japan (250 mm×4.6 mm).The column temperature was maintained at 30°C.The mobile phase A was acetonitrile, and the mobile phase B was water.The gradient program was as follows: 0.01-4 min, 100 % A, 4-60 min, 100-25 % A; 60-75 min, 25-0 % A, 75-120 мин 0 % А.The injection volume was 20 μL, the mobile phase flow was 1.0 mL min and the detection wavelength on PDA was 510 nm.
MS analysis was performed on an ion trap amaZon SL (BRUKER DALTONIKS, Germany) equipped with an ESI source in negative ion mode.The optimized parameters were obtained as follows: ionization source temperature: 120°C, gas flow: 4 L/min, nebulizer gas (atomizer): 7.3 psi, capillary voltage: 4500 V, end plate bend voltage: 1500V, fragmentary: 280 V, collision energy: 60 eV.An ion trap was used in the scan range m/z 100−1.700for MS and MS/MS.The mass spectrometer was used in the scan range m/z 50-2000 for MS and MS/MS.A capture rate was one spectrum for MS and two spectra for MS/MS.Data collection was controlled by Windows software for BRUKER DALTONIKS.All experiments were repeated three times.A two-stage ion separation mode (MS/MS mode) was implemented [17,18].

Results and Discussion
Identification of anthocyanidins by high performance liquid chromatography and MS spectrometry was carried out in five anthocyanin samples obtained under different extraction conditions, since the type of solvent and extraction method can affect the composition of anthocyanidins in the dye [19].The conditions for extracting anthocyanins from black currant berries and their content in the samples are presented in Table 1.From the results presented in Table 1 can be seen that anthocyanins are most fully extracted from black currant with water solution at temperatures of 70 and 100°C for 30 min (samples 2 and 3), treatment in an ultrasonic bath (sample 4) for 30 min and in an ultrasonic homogenizer for 15 min (sample 5).The content of anthocyanins in these samples is 3.73, 4.09, 3.98, 3.43 mg/ml, respectively.The lowest content of anthocyanins is observed in the sample extracted at a temperature of 25°C for 30 min, it is 2.96 mg/ml.HPLC anthocyanin profiles (510 nm) of black currant (Ríbes nígrum) extracts are shown in Figures 1 a-e  It can be seen that anthocyanidins are found in all samples of anthocyanins in the region of 510 nm.
Mass spectra of the studied samples of anthocyanins isolated by various methods are shown in Figures 2 a-e   Figure 2 shows that a qualitative analysis of the anthocyanin fraction shows that black currant extracts are dominated by cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside, delphinidin-3-O-glucoside, delphinidin-3-O-rutinoside.
Identified anthocyanidins in anthocyanin samples and molecular major fragments are presented in Table 2.In samples of anthocyanins isolated by various methods, eight anthocyanidins were identified.The most common were four anthocyanidins -these are cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside, delphinidin-3-O-glucoside, delphinidin-3-O-rutinoside.They were identified in all samples of anthocyanins.This information is in good agreement with the results of studies by several scientific groups [20][21][22].They determined that these 4 anthocyanidins are the main components of black currant berries extracts.
The proportions of the presence of the anthocyanidins in the studied samples depended on the method of isolation of anthocyanins.In all samples, high proportions of release of such anthocyanidins as cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside are observed.The amount of petunidin-3-O-rutinoside and malvidin-3-O-glucoside remains small in all methods of isolation.The amount of other anthocyanidins present determines the way anthocyanins are isolated.Isolation of anthocyanins using an ultrasonic bath caused a decrease in the content of delphinidin-3-O-rutinoside in the samples.In this sample, and the sample of anthocyanins extracted at a temperature of 25°C for 30 min, a decrease in the proportion of delphinidin-3-O-glucoside is observed.In samples 2-5, the presence of peonidin-3-rutinoside was observed.In sample 1, this anthocyanidin is present in very small amounts.Anthocyanidin pelargonidin-3-O-bettagalactoside was contained in samples 2, 3 and 5.In samples of anthocyanins 4 and 1 the content of this anthocyanidin was low.
Identification of anthocyanidins by high performance liquid chromatography and MS spectrometry showed that the composition of anthocyanidins in anthocyanin samples depends on the method of its isolation.A more complete release of anthocyanidins is facilitated by the extraction of anthocyanins from black currant at temperatures of 70°C for 30 min, 100°C for 5 min and direct-assisted ultrasonic extraction for 15 min.Under these conditions, all eight anthocyanidins are released.The results obtained are consistent with the data in Table 1, which shows that anthocyanins are most completely released under these extraction conditions.
Since the extraction of anthocyanins from black currant with water at a temperature of 70°C for 30 min and direct-assisted ultrasonic extraction at a temperature of 25°C for 15 min contribute to the complete release of anthocyanidins into the solution, a technology for obtaining anthocyanin dye from black currant, including the extraction of anthocyanins in specified conditions.
The technology of the production of anthocyanin dyes included defrosting, grinding black currant, extracting anthocyanins under specified conditions, and subsequent filtration.Organoleptic and physico-chemical parameters of anthocyanin dye isolated from black currant using water and direct-assisted ultrasonic extraction are presented in Table 3.
Anthocyanin dyes are water-soluble natural dyes that have acidic taste, dark red color and a smell of black currant.pH of the dyes is 3.0 and 3.2, respectively.Anthocyanin dyes have antiradical activity since they contain biologically active compounds such as anthocyanins.The expiration dates of the dyes is 3 months at temperatures of 0-5 °C or 12 months at -18 °C.
Anthocyanin dyes in the food industry are mainly used for coloring flour and confectionery products since their color is one of the main criteria for the organoleptic attractiveness of the product for the consumer [23][24].Pastilles were chosen for coloring in this work.Marshmallow was prepared by the traditional method according to a standard recipe.Agar-agar was used as a gelling agent.Anthocyanin dye was added as a solution during preparation in the amount of 4, 8 and 12 % by weight of the product.The best organoleptic and physico-chemical parameters have marshmallow containing 12 % anthocyanin dye in its composition.In this regard, the addition of anthocyanin dye gives the marshmallow a rich pink hue and a pronounced currant taste and smell of berry raw materials.

Conclusion
Determination of the qualitative composition of anthocyanidins contained in anthocyanin solutions showed that their content depends on the method of extracting pigment from berry raw materials.All eight anthocyanidins are contained in anthocyanin solutions extracted from blackcurrant berries at temperatures of 70°C for 30 minutes and 100°C for 5 minutes, as well as using direct ultrasonic extraction at 25°C for 15 minutes.
The organoleptic, physicochemical characteristics and safety indicators of anthocyanin dyes isolated from black currant berries were determined during aqueous extraction at a temperature of 70°C for 30 minutes and direct ultrasonic extraction at a temperature of 25°C for 15 minutes.The developed dyes are water-soluble natural dyes that have a sweet taste, dark red colour, and a distinct smell of berries.Anthocyanin dyes are safe for consumers and have antiradical activity since they contain biologically active compounds -anthocyanins.
The developed anthocyanin dyes were used to colour pastille products.The most successful in terms of organoleptic indicators was marshmallow with the addition of 12% anthocyanin dye.The addition of anthocyanin dye to the marshmallow made it possible to obtain a product that is safe for consumer health with improved organoleptic characteristics and a pronounced functional focus.
A grant from the state support for the creation and development of advanced engineering schools was provided, on 21 st of June, 2022 No. VF

Table 1 .
Conditions for extracting samples and the content of anthocyanins in them

Table 2 .
Anthocyanidin peaks appearing in black currant extracts «+» -the presence of anthocyanidin in the sample, «-» -low content of this anthocyanidin in the sample or its complete absence

Table 3 .
Organoleptic and physico-chemical parameters of anthocyanin dyes isolated from black currant by various methods