Intensification of extraction of phytocomponents from berry raw materials

The paper describes the methods of extraction of biologically active agents of berry raw materials. The purpose of the study was to select the parameters of phytocomponents extraction during ultrasonic treatment of berry raw materials. The batch method of maceration using water-ethanol extractants was studied. The raw material was irradiated with Volna-M ultrasonic device. The kinetics of extracting phenolic compounds from fresh black currant berries (Ribes nigrum L.) was studied. Physical-chemical and microbiological indicators were determined using standard methods. Composition and quantitative content of flavonoids and tanning agents in extracts were determined by colorimetric and high performance liquid chromatography techniques. Mathematical statistics and analysis were used in data processing. The diffusion properties of extracts were calculated in a computer program. The experiments were performed in five-fold repetition, and the data were analyzed to determine their validity in application packages. It was found that the duration of extraction during ultrasonic treatment is reduced by an average of 60 times, while increasing the yield of extractive substances. The obtained kinetic dependencies of diffusion characteristics of the raw material reveal that more complete extraction of flavonoid compounds takes place with water-ethanol extractants (40% wt.) at irrigation module 1 to 15 and 1 to 20. The analysis of physical and chemical composition of solutions showed high content of biologically active substances. The extract is recommended as components of functional products.


Introduction
The extraction of natural components of vegetable raw materials for their use in technology of enriched and functional products becomes a relevant task for the food industry. When developing formulations of new functional soft drinks, dairy and other products, biologically active compounds are introduced into their composition as functional components. Market promotion and marketing of such products are successful in positioning additives as natural derived from natural raw materials. Phytocomponents become popular ingredients of products for sports and preventive nutrition, energy drinks [1]. Bioflavonoids such as anthocyanins, polyphenols and other compounds are widely used as functional components. Currently, the antioxidant, anti-inflammatory and adaptogenic function of phenolic compounds affecting the body has been established, so they are used in food and pharmaceutical industry, agriculture [2][3][4].
Berry raw material contains various biologically active compounds with high antimicrobial, antioxidant and nutritional value [4]. The study of phytoncidal activity of plant raw materials  [5][6][7][8][9] shows the dependence of antiviral and antimicrobial effects of phytopreparations on the content of flavonoid compounds. The use of preparations with high phytoncidal activity obtained from plant raw materials allows slowing down the growth of microorganisms to a greater extent than antibiotics. The studies to obtain active compounds of plant origin for prophylactic and therapeutic preparations and ingredients are quite promising and relevant.
In the current foreign economic situation, the Russian market is limited by the supply of phytoextracts from global manufacturers of pharmaceutical substances and phytonutrients. In Russia, researchers study the types of domestic raw materials with high content of active substances. Extensive research is aimed at studying the properties and composition of natural biologically active ingredients in fruit and berry raw materials [10,11]. Berry raw materials are a source of natural and available active phytocomponents that can be used to enrich mass consumption products. The desired components are traditionally extracted by different solvent extraction. The studies of selective extraction processes present a particular interest, since plant raw materials contain active substances of different classes and functional orientation. The determination of parameters of extraction processes is aimed at ensuring selectivity of extraction and preservation of biologically active food ingredients during processing and storage. The design of extraction methods should provide the largest number of environmentally friendly extracts, with minimal processing time for raw materials.
New high-efficiency and intensive methods [12] for obtaining extracts are based on changing thermodynamic parameters of extraction system and massoutput conditions. The intensification is provided by using active hydrodynamic modes of raw material treatment, for example electrophysical treatment. In a number of works, the prospects of using ultrasonic radiation of heterogeneous systems have been proved [12][13][14][15]. Pressure alteration in extractor volume makes it possible to increase the degree of convective mass transfer at diffusion of substances from particles. There are scientific studies and tested recommendations concerning the methods of intensive treatment of plant raw materials and determining parameters of extraction affecting its diffusion properties. The effect of the nature of the extractant, irrigation module, temperature, raw material properties, processing time and other parameters were studied experimentally. The methods of intensive action on the extraction system can lead to increased output of ballast substances into extract from fruit and berry raw materials. It is also possible to reduce the antioxidant activity of extracts due to oxidative processes in active treatment zones. In the conditions of electrophysical treatment of berry raw material when threshold values of power and intensity are reached, the destructive effects of cell structures destruction are demonstrated. This increases the yield of fiber, pectin, protopectin and other substances, as well as the destruction of active components. It is necessary to find rational intensive technological parameters of the extraction process in order to ensure quality and preserve the phytocomponents of berry raw materials. This leads to empirical studies and a synthesis of their results.

Problem statement
An important task for the production of enriched and functional products is the production of available phytoextracts from berry raw materials with increased content of vitamins, bioflavonoids, mineral substances and other nutrients. The methods of extraction of active components of berry raw materials are long-term, their disadvantage is the destruction of useful substances and transition to ballast compound solution. The use of active approaches with electrophysical treatment reduces the activity of phytocomponents due to oxidation and heating of the extracted system. The purpose of the studies was to determine the parameters of the extraction process of phytocomponents during ultrasonic treatment of berry raw materials in order to reduce the processing time compared to conventional methods of extraction and increase the yield of active components.

Object of the study
The object of the study was the technological extraction of black currant berries (Ribes nigrum L.), Dachnica variety, harvested in 2019 in Kemerovo region, under ultrasonic treatment.

Materials and methods
The studies were carried out using standard methods of canning and nonalcoholic industry. The composition and quantitative content of flavonoids and tanning agents in the extracts were determined by colorimetric and high performance liquid chromatography techniques. Mathematical statistics and regression analysis were used in data processing. The quality indices of berries and extracts were determined by organoleptic, physicochemical indices of raw materials safety. Microbiological indices were determined according to GOST ISO 7218-2015 "Microbiology of foodstuff and animal feed. General requirements and recommendations for microbiological research".
The extraction of berry raw material was carried out by periodic method in maceration device. The raw material was irradiated with Volna-M ultrasonic device. The aqueous solution of ethanol of 40% wt served the extractant. Berries were ground to an average size of 2-5 mm.
The kinetics of extraction of flavonoid compounds from blackcurrant berries was studied by changing the concentration of rutin using the developed computer program [16]. Diffusion properties were calculated based on molecular diffusion coefficient D. The dimensionless indicator Z proposed by Lysyansky V.M. from differential equations of convective diffusion was thus used. For spherical raw material particles, the expression is as follows: (1) In formula (1), the parameter Z is a simplex of the average concentration by particle volume; Bi -Bio criterion; µ -tabulated value, function of Bio criterion; τ -extraction duration; q -irrigation module.
Expression (1) was solved using numerical methods for the molecular diffusion coefficient. The calculation formula for currant berries with the average particle size r, mm is as follows: The expression (2) was used to calculate discrete values of the coefficient D and construct dependence diagrams.

Results
The studies were carried out on the temperature change of the extractant in the zone of the ultrasonic radiator of the Volna-M device. The irradiated system was heated to a boiling point of 100 °С at an intensity of 100% (up to 3 W/cm 2 ) for 10 minutes. At an irradiation intensity of up to 2 W/cm 2 , intensive mixing and circulation of the solution was observed. The frequency of the radiation control system in 22 kHz is selected based on the condition of reducing the hydrodynamic resistance during mass transfer in the solid-liquid system and the earlier received recommendations [13]. At these parameters, after 3 minutes, the sediment began to form in the system.
Empirical studies of the kinetics of phenolic compounds recovery from raw materials were carried out in five-fold repetition with variation of the following parameters. The value of the irrigation module was 1:10, 1:15, 1:20. In the maceration device, the extraction was carried out without treatment and under ultrasonic treatment at an intensity of up to 2 W/cm 2 , at a frequency of 22 kHz. The samples were taken at 1 hour intervals (without treatment) and after 5 minutes (at irradiation) until the equilibrium state in the extracted system. The concentration of active components in the solutions was within standard rutin samples using colorimetric analysis methods [14]. The results were processed using statistical analysis in application packages (MS Excel). The data are presented as graphs in Figures 1 and 2.
Content and composition of active phytocomponents in obtained extraction are received on the basis of extraction-spectrophotometric methods. The content of BAS-phytoncides was determined in the extracts (Table 1).   The study of microbiology and antibacterial activity of black currant berries extracts was studied with respect to the following strains: Staphylococcus aureus, Escherichia coli, Shigella flexneri, Salmonella typhi. The analyses of growth zone inhibition results in sowing cups were studied. In the experiments it was found that the extracts do not show any sensitivity to microorganisms Bacillus and Helicobacter pylori.

Discussion
The study of the influence of ultrasonic treatment parameters of the Volna-M device showed that at a duration of more than 10 minutes and intensity of up to 3 W/cm 2 the system heats up to the boiling point. This results in the oxidation of tanning agents and formation of insoluble components with proteins. It is recommended to limit the intensity of ultrasonic irradiation to 2 W/cm 2 , at a frequency of 22 kHz. Ballast substances of their berries are prevented from entering the extract.
The diffusion curves of extracts without treatment and with ultrasonic irradiation were obtained. Their comparison shows that the values of equilibrium concentrations of extractive substances in solution (by rutin) decrease from 5 hours to 5 minutes through treatment. The data are comparable to the results of the work [15] and confirm the increase in the intensity of extraction processes. The comparison of diffusion curves at different irrigation module values shows that for water-ethanol solvents (40% wt.), large values are observed at 1 to 15 and 1 to 20.  The analysis of physical and chemical indices in Table 1 revealed that the yield of extractive substances was increased by 18% under ultrasonic irradiation. Flavonoids -by 9.7%. The yield of tanning agents and ascorbic acid was increased by 14%. The overall positive effect is associated with intense mixing and cavitation bubble formed on the surface of berry particles. Their collapse reduces the total resistance to mass transfer and increases the transition of extractive substances into the extractant.
High content of raw materials in extracts of phytocomponents -flavonoids, tanning agents and ascorbic acid, combined with water-ethanol solutions as an extractant, causes bactericidal effect on the strains of certain types of pathogenic, opportunistic and undesirable microflora.

Conclusion
The methods of extracting biologically active components from fresh black currant berries (Ribes nigrum L.) were studied. The parameters of extraction of phytocomponents during ultrasonic treatment of berry raw materials were investigated. It is found that the ultrasonic treatment reduces the process time compared to conventional extraction methods by up to 60 times and increases the yield of active phytocomponents. Process diffusion curves are constructed using the developed computer program. The periodic maceration process using water-ethanol extractants (40% wt.) is recommended at the irrigation module of 1 to 15 and 1 to 20. In order to prevent ballast substances from entering the extract, it is necessary to limit the intensity of ultrasonic irradiation to 2 W/cm 2 , at a frequency of 22 kHz. The analysis of physical and chemical composition of extracts showed high content of biologically active substances. The extract is recommended as components of functional products.