Fruits of new selection forms and varieties of snowball tree for manufacture of products of therapeutic and prophylactic purpose

Mykhailo Kotsiubynskyi Vinnytsia State Pedagogical University, Ostrozkoho st., 32, Vinnytsia, 21100, Ukraine. Tel.: +38-043-226-52-20. Е-mail: shevchukoksana8@gmail.com Moskalets, T. Z., Moskalets, V. V., Vovkohon, А. H., & Knyazyuk, O. V. (2019). Fruits of new selection forms and varieties of snowball tree for manufacture of products of therapeutic and prophylactic purpose. Regulatory Mechanisms in Biosystems, 10(4), 432–437. doi:10.15421/021964

Recently, both abroad and in Ukraine the number of works on the use of natural plant resources as food has been increasing (Moskalets & Rybalchenko, 2016;Sіzaуа et al., 2017). In this regard, special attention is paid to a comprehensive study and further use in economic activity of nutrionally valuable fruit and berry plants. The snowball tree (V. opulus) is not an exception (Česonienė et al., 2010;Kucharska et al., 2015;Popova, 2017). In addition, it performs the most important soil protection functions, and is broadly used as an agro-forest ameliorant (Shchetilina et al., 2016).
Fruits of the snowball tree can be maintained well in long term storage, in which their biologically active substances are preserved, which is important in practical terms for their processing. However, unfortunately, snowball tree fruits are underused in the production of products at catering establishments, despite their high biological value (Tsuchiya, 2015;Saltan et al., 2016;Sarıözkan et al., 2017). Broader and more rational use of snowball tree fruits is possible only after complex assessment of their chemical composition. Today, fruits of the snowball tree are of great interest as raw materials in the manufacture of products with target function action. The use of snowball tree fruits as a highly efficient raw material for processing and a wide range of uses in nutrition, including as food additives is described in works by the domestic and foreign scientists: Kucharska et al. (2015), Golubieva et al. (2016), Shchetilina (2016), Jurikova et al. (2017), Popova (2017), Sіzaуа et al. (2017), Polka et al. (2019).
Specialists of the Ivano-Frankivsk Association Cаrраtfoodindustry have developed receipes and technologies for various snowball tree fruit-based products and semi-finished products of therapeutic and prophylactic purpose. On the basis on the snowball tree, the scientists of the Ivano-Frankivsk National Medical University are developing new high-performance drugs. Scientists of the Academician Z. О. Mаnіv Department of Economic Theory of the King Danylo Halytsky Ivano-Frankivsk University together with specialists from the food industry and forestry are investigating the prospects for the use of wild fruits, berries and mushrooms in the Prykarpattia region in the production of medical-prophylactic products (Sіzaуа et al., 2017;Moskalets et al., 2019;Polka et al., 2019). The possibility and perspective of using squeezes, powders from the snowball tree in the production of desserts, bakery products, kissels, spices and colourings have been shown repeatedly (Shchetilina et al., 2016;Goszcz et al., 2017;Konarska & Domaciuk, 2018).
The "State Register of Plant Varieties Suitable for Dissemination in Ukraine" as of August 2019 included 7 varieties of V. opulus, which opens up opportunities of its industrial cultivation and provision as a raw material to the processing and pharmaceutical industries. In this regard, search, selection and creation of new high-performance varieties of snowball tree with high consumer and biochemical properties is relevant.
Therefore the objective of our research was the survey and development of large-fruited and highly productive varieties of snowball tree with high quality indicators of fruits, suitable for industrial plantations for obtaining valuable raw materials for production of products of functional purpose, including powdered food additives, syrups, marmaledes and drugs. Study goals were: -to develop large-fruited and high-performance varieties of snowball tree; -to determine the organoleptic, biochemical, technological indicators and nutritional properties of its fruits; -to ascertain the suitability of raw fruit for the production of functional products, including powdered food additives, syrups, marmalades and drugs. The objects of our study were plants of selected forms and varieties of the snowball tree, fruits and products of their processing.

Materials and methods
The studies were conducted in 2017-2019 in the Institute of Horticulture of the National Academy of Agrarian Sciences of Ukraine (the soil was dark grey podzolic, the content of easily hydrolysed nitrogen equaled 89, mobile phosphorus -153, exchangeable potassium -111 mg/kg). We studied the collections and selected elite forms of snowball tree. As a standard (st) we took the regional variety of Ukrainian breeding Velykoplidna. Care of the experimental plants was carried out using common agricultural measures. Protection measures against pests and pathogens were not taken.
Degustation evaluation of fruits was performed by organoleptic properties (visual appearance, consistency, taste and aroma of berries) (Syedova, 1995;Syedova & Ogoltceva, 1999) according to a 9-point scale: 9fruits above excellent quality, 8 -fruits of excellent quality, 7 -fruit above good quality, 6 -good quality fruits, 5 -fruits above satisfactory quality, 4 -fruits of satisfactory quality, 3 -fruits of poor quality, 2 -fruits of very poor quality, 1 -the fruits are completely unsuitable for consumption. Phenological observations on plant growth and development, as well as biometric parameters (weight of the fruit, bunches, quantity of fruits in a bunch, etc.) were performed and determined by methods of the State Commission of Ukraine for Testing and Protection of Plant Varieties. The fruits were harvested by hand, productivity was determined by weighing.
The solids' content was determined by weighing according to SSU 7804:2015 (2015); content of total sugars -photocolorimetric method on the photocalorimeter KFK-3-01 -to SSU ISO 4954:2008(2009; organic acids -by titration with 0.1 n NaOH with recalculation for malic acid -to SSU 4957:2008 (2009); vitamin С -titrimetric method by restoration of Tillman's reagent, by extraction from weighed amount fruits using acid solution followed by filtration of the resulting substrate according to SSU 7803:2015 (2015); content of β-carotene -by paper chromatography followed by photoelectrocolorimetry to SSU 4305:2004 (2005); content of polyphenolic compounds -using the Folin-Denis reagent spectrophotometric method on the spectrophotometer Spekol 1500 at a wavelength of 270 nm with a photometric accuracy of 0.004 to SSU ISO 4373:2005(2006; content of Р-active compoundsspectrophotometrically, antioxidant activity -were determined using Blisar A analyzer of antioxidant activity recalculated into gallic acid (Kucherenko, 2001); pectic substances -spectrophotometrically to SSU ISO 8069:2015(2017. Biochemical analyses were performed in 4 replication in each variant of the experiment. The differences between the values in the control and experimental groups were determined using ANOVA, where the differences were considered significant at P < 0.05 (with Bonferroni correction). The results were defined as means ± standard error (x ± SE). The standard error of the mean (SD) was calculated using analysis of variance taking into account the Bessel's correction, because a small sample was taken (n < 50).

Results
The results of our studies showed that the greatest weight of the fruits belongs to the elite selected forms: Uliana, Ania (weight of the fruit equaled 1.71-1.80 g) with a sweet taste without bitterness and organoleptic evaluation at 8.3-8.7 points, with the weight of bunches of 22.5, 23.5 g аnd average quantity of fruits in a bunch -59-67, respectively (P < 0.05). At the same time the average weight of the fruit of the control variety Velykoplidna was 1.24 g, weight of a bunch -115 g and average quantity of fruits in a bunch of 79. It should be noted that over the years of the study the average weight of the fruit in the varieties Bagriana, Koralova, Ukrainochka, Rubinova was within 1.02-1.20 g with average weight of the bunch of 76-103 g аnd quantity of fruits in a bunch 55-73. The smallest mass of fruits belonged to the following forms: ЕF-7-31-12, ЕF-2-56-13, ЕF-5-12-2012 (0.65-0.70) and variety Kоrаlоvа (0.92), however, with significant productive potential with weight of bunches equaling 63-103 g аnd quantity of fruits in a bunch of 29-61 (P < 0.05).
Degustation evaluation (8.8-9.0 points) of selection forms ЕF-5-12-2012 аnd ЕF-7-31-12 with strongly sweet fruits with insignificant bitterness and bright colour, as well as of sweet forms without bitterness (Аnia, Uliana, ЕF-3-10-2010) with degustation value of 8.3-8.9 points, was higher than for the varieties Bagriana, Koralova and higher than the control variety Velykoplidna sweet without bitterness according to the tasting evaluation, which is at the level of (8.1 points). Differences in degustation value of fruit of snowball tree are important in developing and forming features of their processing products. Fruits of the snowball tree are material for diverse use by the content of the separate components: juice, puree, seeds, peel. Technological properties of snowball tree fruits play an important role during their processing: determine the output of the product, waste content, and quality of processing products. The consistency of the fruits from the studied varieties of snowball tree is dense, juicy, with easily separated seeds.
The juiciness of the fruit of almost all the samples did not exceed 70-75%. The lowest juice output (60-64%) was obtained from samples of snowball trees ЕF-2-56-13, ЕF-3-10-2010, ЕF-7-31-12, Ukrainochka, Rubinova. Waste (seeds and peel) is from 8-15%. According to these characteristics, the fruits of the varieties Ania, Uliana were superior to the control: juice output was higher by 3-15%, the pulp -5-17%. According to the high juiciness indicator (76-81%) and due to the pleasant sweet taste with a slight bitterness, the use of the fruits of the abovementioned varieties in the production of fruit juices is quite promising. All the investigated fruits of snowball tree were characterized by low content of organic acids -1.34-2.29% (P < 0.05). The presence of organic acids in the fruits of snowball tree not only increases their resistance to pathogens and pests, but also enlarges the technological capabilities of their processing.
The most acid-containing fruits are those of snowball trees ЕF-3-10-2010, ЕF-2-56-13 (1.94-2.29%), varieties Uliana, Ania (1.89-2.26%), compared with the control -variety Velykoplidna, 1.79%. Low-acid fruits were characteristic of the varieties: Rubinova, Kоrаlоvа, Ukrainochka, ЕF-5-12-2012 (1.12-1.52%). The highest content of sugars was observed in the fruits of the varieties Ukrainochka, Rubinova, Bаhrianа, Kоrаlоvа, ЕF-7-31-12 (9.1-12.6%), which is significantly higher than the content of total sugars in the fruits of the control variety Velykoplidna (8.7%). The sugar-acid index of the studied fruits ranged from 2.97-8.29 (P < 0.05), and in varieties Velykoplidna, Аnia, Uliana, ЕF-3-10-2010, ЕF 2-56-13 was less than 5. Statistically reliable differences were determined by biochemical indicators of snowball tree fruits (Table 2). Therefore, the maximum amount of solids was found in the fruits of ЕF-3-10-2010, ЕF-5-12-2012, ЕF-7-31-12, ЕF-2-56-13 Kоrаlоvа (21.4-24.7%), and minimal in fruits of Uliana and Velykoplidna varieties equaling 14.3-16.8%. A quite high content of polyphenols аnd Р-active substances (618-950 mg%) was observed in the fruits (6,020-11,053 mg/kg), which indicates the possibility of use of fruits as raw materials for the production of therapeutic and prophylactic products. Our results showed that the fruits of all the samples contained no less than 40 mg/100 g of ascorbic acid (P < 0.05). As the results of our research show, the fruits of V. opulus are rich in pectin substances (from 4.6-13.0 mg/kg). This amount of pectin substances in its fruits is of interest due to their preventive properties. Carotene content in the studied snowball tree fruits was insignificant, equaling 2.1-5.3 mg/kg (P < 0.05). Among the tested varieties particular attention is drawn to the form ЕF-2-56-13 with a low sugar-acid index (2.97), high content of titrated acids (2.29%) but with a very bitter taste of the fruit, and therefore high in glycoside viburnin.  Note: * -P ˂ 0.05 compared to the control (standard); SE -standard error of the mean; taste evaluation of fruits performed according to organoleptic parameters on a set of indicators taste and aroma of berries. The results of our research on determining the antioxidant activity of the investigated snowball tree fruits showed a sufficiently high level. On average, this indicator, depending on the variety, varies from 332-526 mg%. The highest value of antioxidant activity was exhibited by the fruits of the ЕF 3-10-2010, Kоrаlоvа, Bаhrianа (519-540 mg%) varieties, somewhat inferior to the fruits of the varieties Ukrainochka, Velykoplidna (387-392 mg%). Research results of chemical composition of snowball tree fruits showed that due to their content of nutrients and bioactive substances they are a promising raw material for the production of functional foods.
According to the results of the comprehensive evaluation of the varieties and elite forms of fruits of V. opulus for food production: for the production of syrups, mors, juices from snowball tree fruits, the following varieties are recommended: Kоrаlоvа, Bаhrianа, Ania, Uliana, ЕF-3-10-2010, ЕF-5-12-2012, ЕF-7-31-12, having high organoleptic, technological characteristics and high content of anthocyanins, since colour depends on it, product yield and nutritional value; for the production of sauces we recommend fruit of the varieties Velykoplidna, Ania, Uliana, ЕF-3-10-2010, ЕF-2-56-13, which have a high content of organic acids,which means that the number of acidifiers added to the product can be reduced or that they need not be added at all; fruits of the varieties Kоrаlоvа, Bаhrianа, Ania, ЕF-3-10-2010 are recommended for production of gelling and candy products (jellies, candy bars, chocolates) due to the high solids content and natural pectin, to obtain powders and teas, fruits and leaves of all the studied varieties of V. opulus are recommended, which are also sources of antioxidants, flavonoids and other biologically active substances; in the manufacture of pharmaceuticals of natural origin (cardiovascular and antispasmodic action) fruits with high glycoside content, particularly those of elite form ЕF-2-56-13, are promising raw materials.

Discussion
In the Institute of Horticulture of the NAAS and its network, work on creating collections аnd separate selective breeds of not commonly used forms, in particular snowball tree, has been going on for a long time. The most important qualitative indicator for any plant is weight of the fruit. Currently 7 varieties of snowball tree are included in the State Register of Plant Varieties Suitable for Dissemination in Ukraine (August 2019), though, results of analytical and synthetic breeding and the conducted biometric and biochemical studies showed that varieties Uliana, Ania and EF-3-10-2010 are promising new high-yielding elite forms which are at the level not inferior to the control (standard) by elements of productivity.
Our studies have shown clear differences in varieties and elite forms in the biochemical composition of fruits, their taste properties, which in turn encourages their further use for particular nutritional or pharmaceutical purposes. This is very important because the hallmark of modern society is healthy food, the leading role in which belongs to consumption of food rich in vitamins, macro and microelements composition. Such requirements are met by leaves, fruits and seeds of the snowball tree (Karaçelik et al., 2015;Goszcz et al., 2017;Konarska & Domaciuk, 2018;Kozlowska et al., 2018). Studying and conducting comparative assessment of differences in degustation evaluation of fruits of the snowball tree are important for developing features of its processing products. According to the literature, Maksimenko et al. (2014), Zhbanova & Maslennikov (2015, organic acids in ripe fruits of snowball tree are represented mainly by malic, citric, caprylic, formic, valeric, isovaleric, chlorogenic acids, and also traces of quinic, caffeic and acetic acids were detected. Savieliev et al. (2010), Golubieva et al. (2016), Zhidekhina et al. (2016 reported that green fruits (unlike ripe fruits) are rich in quinic and caffeic acids, the content of which significantly reduces during ripening. Ersoy et al. (2017), Goszcz et al. (2017), Cory et al. (2018), Kızılay (2019) found that the fruits of the snowball tree contain triterpene compounds: α-and β-amyrin, alanolic and hedrogenic acids and their acetyl derivatives, ursolic acid, as well as steroids represented by phytosterol. Česonienė et al. (2010), Shchetilina et al. (2016) studied a broad range of biologically active substances, including antioxidants, providing new opportunities of using raw materials from the snowball tree in production of pharmaceuticals. According to the literature data Konarska & Domaciuk (2018), sugars are the main source of energy necessary for different physiological processes in the cell. They determine nutritional characteristics of both fresh fruits of the snowball tree and products of their processing. Content and composition of sugars in fruits of V. opulus is represented by the total and invert sugars, as well as glucose, fructose and sucrose. The conducted studies revealed differences between the varieties of snowball tree in the content of total sugars. Studies by Shchetilina et al. (2016) (Skrovankova et al., 2015;Ceylan et al., 2018). Pectic substances are involved in flavouring, and also have the properties of preserving natural colour and aroma of the fruits in jelly (Golubieva et al., 2016;Cory et al., 2018;Polka et al., 2019). Studies by Golubieva et al. (2016), Cory et al. (2018), Polka et al. (2019) revealed possibilities of using pectic substances of the fruits as a gelling component in public catering, in the food industry, etc. In addition, pectins can be used as an alcohol adsorbent (Popova, 2017;Kozlowska et al., 2018;Zakłos-Szyda et al., 2019). Konarska & Domaciuk (2018), Jurikova et al. (2019) do not indicate fruits of snowball tree as fruits of high vitamin value. However, in the fruits of all the samples we found not less than 400 mg/kg of ascorbic acid, indicating a high nutritional value and technological parameters, allowing them to be used for production of functional syrups, juices, desserts, etc. Vitamin C is a strong antioxidant, but cannot accumulate in the human body, which is why it is important to consume vitamin C-containing foods regularly. Analysis of vitamin composition and daily vitamin requirements showed that the inclusion of snowball tree fruits in the diet can satisfy over 50% of the body's need for vitamin С and 1-15% of the body's need for carotene -content in fruits 2.1-5.3 mg/kg for the daily requirement for humans 4-5 mg/kg.
The taste and aroma of snowball tree fruits are quite specific due to the presence of valeric acid, essential oils and glycosides. Glycoside of vyburnin provides the fruits with a specific smell. This property, on the one hand, according to some authors (Kucharska et al., 2015;Zakłos-Szyda et al., 2019) impedes the distribution of the plant in both industry and private sector gardens. However, national breeders are working on developing new varieties of snowball tree of high quality parameters of fruits and low bitterness. One of the important glycosides of snowball tree is iridoid viburnin. Česonienė et al. (2010), Skrovankova et al. (2015), Tsuchiya (2015), Jurikova et al. (2019) demonstrated that in pharmacy on the basis of iridoids of natural origin, preparations of cardiovascular and antispasmodic group can be produced, and the results of recent studies (Stępień, et al., 2010;Kucharska et al., 2015;Skrovankova et al., 2015) have revealed its antitumour activity. Furthermore, (Kraujalytė et al., 2013;Kucharska et al., 2015;Konarska & Domaciuk, 2018;Jurikova et al., 2019) have confirmed that biologically active substances of V. opulus manifest hemostatic, antiseptic, tonic effect on the human body, and also increase the tone of the uterine muscles and prevent the appearance of varicose veins.
Among the tested varieties particular attention is drawn to the elite form ЕF-2-56-13 with a low sugar-acid index (2.97), high content of titrated acids (2.29%) but with a very bitter taste of the fruit, and also high content of glycoside viburnin. And this, in turn, is of great research interest, because information about the presence of this glycoside in biomass of other species of plants in the scientific literature is absent. At the same time there are some reports that this iridoid was found not only in the bark but also in the fruits and leaves of V. opulus. Due to the unfavourable environmental situation, the risk of developing cardiovascu-lar disease, diseases of the nervous system, lungs, and blood increase every year. This is due to the provision and accumulation of free radicals in the human body (Skrovankova et al., 2015;Jurikova et al., 2019). For prevention of this tendency, introduction of antioxidants, substances that protect it from the negative impact of the environment, into the human body is necessary. The results of our research on determining the antioxidant activity of the investigated fruits of the snowball tree revealed them to have a high antioxidant level. The results showed that by the value of total antioxidant activity,which is characterized by a number of nutrients and biologically active substances, the fruits V. opulus constitute a valuable raw material for obtaining healthy and functional foods. The functional properties of snowball tree fruits were generally confirmed by the content of antioxidants: vitamin С, β-carotene, pectin and flavonoids, providing 20-60% of the human body's daily requirements for them.
The modern food industry is paying more attention to healthy food with a balanced composition of nutrients, including functional products (Česonienė et al., 2010;Maksimenko et al., 2014;Kucharska et al., 2015;Tsuchiya, 2015;Zhbanova & Maslennikov, 2015). Analysis of the horticulture industry in general demonstrates that today the snowball tree is a nichе culture both in Ukraine and around the world. Domestic scientists and market analysts (Popova, 2017;Sіzaуа et al., 2017;Moskalets et al., 2019), have distinguished some important factors of the expediency of cultivating snowball tree.
Some scientists, in particular Ersoy et al. (2017), Ceylan et al. (2018), Cory et al. (2019) are paying special attention to а separate group of imported pectin-containing goods, dried fruits and berries, fruits distillates, fruits and berry wines. The state statistics does not distinguish processing of fruits as a separate sphere. According to the official data of State Statistics , a general trend towards reduction of the number of processing enterprises in Ukraine and people employed in this sphere is observed. In 2014, 335 enterprises were engaged in the processing and preservation of fruits and vegetables. The programme of development of horticulture spheres for the period to 2025 aims at stabilization and further increase in the production of fruits and berries; saturation of the domestic food market with competitive products and their wide export; increase in production of normatively safe products due to transition from industrial-chemical methods of farming to biological methods, which ultimately opens up even more opportunities for growing and distributing V. opulus.
Domestic and foreign scientists (Shchetilina et al., 2016;Ersoy et al., 2017;Sіzaуа et al., 2017), despite considerable experience, have underestimated the value of snowball tree fruits for processing. Using its fruits as a food was shown by Golubieva et al. (2016) . To date, the use of its raw materials in other industries is at a low level. Use of this plant in both industrial and ornamental horticulture is described in the works by Česonienė et al. (2010), Ecevit-Genç & Yildirim (2018). The main factors which limit its distribution are: strong bitterness of fruits and technological difficulties in growing the plants. Some authors (Ersoy et al., 2017;Sіzaуа et al., 2017) note the lack of high-yielding varieties of snowball tree and the bitter taste of the fruits of wild plants, which holds back the distribution of the plant both in industrial production and in private sector gardens.
However, currently 9 varieties of universal purpose count as national achievements: 5 selections of Mliiv SRS Institute of Horticulture of the National Academy of Agrarian Science (Ukrainochka, Rubinova, Koralova, Bahriana, Velykoplіdna), 2 -selection of M. M. Gryshko National Botanical Garden of the National Academy of Science (Berehyni, Nasoloda), 2 -Institute of Horticulture of the National Academy of Agrarian Science of Ukraine (Ania аnd Ulian). These are high-yielding varieties (14-20 t/hа) with high qualitative indicators of fruits and low bitterness, especially the latter two. In recent years, work on creation of plantations of V. opulus has begun, particularly, the Deliatyn Forestry (Ivano-Frankivsk Oblast) (Sіzaуа et al., 2017) with the total yield of 100 tons of fruits of snowball tree/year, Svirzh Forestry (Lviv Oblast, area 0.6 hа), Brody Forestry (Lviv region 0.5 ha) and others . Today, snowball tree fruits are intended for systematic consumption in all age groups in the healthy population. Consumption of these products reduces the risk of diseases related to food, preventing deficiency or replenishing nutrients in the human body, which preserves and improves health due to the presence of physiologically functional ingredients in their composition.

Conclusions
Thus, the study revealed that the fruits of selective breeding forms and varieties of snowball tree grown in the conditions of the Forest-Steppe of Ukraine have high organoleptic and biochemical properties. Content of organic acids, carotene, P-active compounds and antioxidants in snowball tree fruits is characterized by their high consumer properties, giving them more potential use in the food, processing, pharmaceutical industries in the production of healthy and functional foods, medical drugs. In particular, for the production of syrups and morses from snowball tree fruits of the following varieties are recommended: Kоrаlоvа, Bаhrianа, ЕF-3-10-2010, ЕF-5-12-2012, ЕF-7-31-12, having high organoleptic (tasting evaluation 8.8-9.0 points), technological characteristics (weight of the fruit equaling 0.65-1.20 g, weight of bunch 81-103 g) and high content of anthocyanins, since colour depends on it, product yield and its nutritional value. For the production of sauces, fruits of the following varieties are recommended: Velykoplidna, Ania, Uliana, ЕF-3-10-2010, ЕF-2-56-13, which have a high content of organic acids (1.89-2.29%), which permits the elimination or reduction in the number of acidifiers added to the product; fruits of the varieties of snowball tree Kоrаlоvа, Bаhrianа, Ania, ЕF-3-10-2010 are recommended for production of jelly, marmalade, candy and lactic acid fermented dairy products (jellies, sweets, cheese desserts, yoghurts, kefir) and powdered food additives due to the high content of solids (19.9-24.7%) and natural pectin (9.1-13.0 mg/kg), which reduces the quantitative content of pectin added to the product and allows candied mass of required density to be obtained; to obtain powders and teas, fruits and leaves of all the examined varieties of V. opulus are recommended, as they are sources of antioxidants, flavonoids and other biologically active substances; in the manufacture of pharmaceuticals of natural origin (cardiovascular and antispasmodic preparations) promising glycoside-containing materials are fruits of the elite form ЕF-2-56-13.