MENDELNET 2016

ANALYSIS OF RED CURRANT ( RUBRUM) AND RED GOOSEBERRY (RIBES UVA-CRISPA) VARIETIES BY INDUCTIVELY COUPLED PLASMA ATOMIC EMISSION SPECTROSCOPY

VACLAV STURSA1, PAVEL DIVIS1,2, ZUZANA JURECKOVA1, ALES MATEJICEK3 1Department of Food Chemistry and Biotechnology 2Materials Research Centre Brno University of Technology Purkynova 118, 612 00 Brno CZECH REPUBLIC 3Research and Breeding Institute of Pomology Holovousy Ltd. Holovousy 129, 508 01 Horice CZECH REPUBLIC [email protected]

Abstract: are highly valued containing many organic compounds with significant health benefits. This work is focused on determination of elemental composition of different red currant and gooseberry varieties, which is less known. An digestion method was used to decompose samples and a method for determination of 9 nutritionaly important elements by inductively coupled plasma optical emission spectrometry was optimized. From the analysed varieties ´Jesan´ and ´Rubigo´red currants and ´Hinnonmaki Rot´ and ´Krasnoslawjanskij´red gooseberries were evaluated as the best for the use in food industry in terms of elemental composition. Key Words: currant, gooseberry, fruit, elemental composition

INTRODUCTION Currants and gooseberries are known in Europe since the 14th century. They were consumed either fresh or as juices and jams, or as a fermented fruit beverages. Currant is a small shrub belonging to the family of Grossulariaceae, of the genus Ribes. The currant bush grows up to a height of 1–1.5 meters. Its leaves are yellowish-green in color and are arranged spirally on the stems in bunches of five. During each season, the shrub bears pendulous chain of small berries. The fruit has size of about 1 cm in diameter with a glossy skin and a persistent calyx at the apex, and containing 3–10 tiny . Depending on the berries colour, red, white and black currants varieties can be distinguished (Strik 2003, Djordjevic et al. 2014). Gooseberries are deciduous shrubs, fast growing under optimum conditions to 1 m tall and 1.5 m wide. The leaves are alternate, single, deeply lobed, and glossy dark green. The fruit borne singly or in pairs at the axils, is a with many minute seeds at the center. A gooseberry may be green, white, yellow, or shades of red from pink to purple to almost black. Fruits of the European gooseberry may be very large but usually they are up to 3 cm long, less in width (Strik 2003). Growing of currants and gooseberries has been recently extensively on the decline mainly due to a massive expansion of Podosphaera mors-uvae fungus, the most important disease in gooseberry and currant. Within the modern trend of consuming foods with a high content of biologically active substances the cultivation of new varieties of currants and gooseberries resistant to pests and diseases becomes again very significant. In 2005 there were nearly 40 000 hectares of orchards growing gooseberry and 163 000 hectares growing currants worldwide. Berries are good source of many nutritionally significant compounds. Whereas the organic composition of the currant and gooseberry is subject of numerous publications (Benvenuti et al. 2004, Pantelidis et al. 2007, Da Silva Pinto et al. 2010, Vagiri et al. 2013) elemental composition of different currant and gooseberry varieties is less known. Elemental analysis of food matrices can be performed

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by various spectroscopic techniques. One of the often used techniques is atomic emission spectroscopy, mainly with inductively coupled plasma. The main analytical advantages of the ICP–OES over other spectroscopic techniques are the capability for efficient and reproducible vaporization, atomization and excitation of wide range of elements in various sample matrices, minimum spectral interferences and simultaneous determination of all metallic elements and some metalloids and non-metals (Ebdon et al. 1998). This work is focused on the elemental analysis of six different varieties of red currant and gooseberries. These varieties were grown at Research and Breeding Institute of Pomology Holovousy and they are resistant for most diseases typical for currants and gooseberries. As the elemental composition of fruit is one of the important nutrition parameter used in food industry, the results of this study may help to choose the best fruit in restored breeding programme of currant and gooseberry in the Czech Republic or in other countries.

MATERIAL AND METHODS The ICP–OES instrument was calibrated by certified solutions of metals of interest (1 g/l, Astasol, Analytika, Czech Republic). For the decomposition of fruit samples nitric acid (Analpure, Analytika) was used. In all analyses only ultrapure water prepared by ELGA station (Veolia Watter systems Ltd., UK) was used. To verify the proper function of ICP–OES instrument the quality control material METRANAL no.3 containing different metals in strawberry leaves matrix (Analytika) was used. All of the analyses were performed on an ICP–OES (Ultima 2, Horiba Jobin Yvon, ) equipped with Mainhard type nebuliser and cyclonic spray chamber. An analytical balance AND HA-202M (A&D Company, Japan) was used to weight samples. Samples were shaken on GFL 3006 shaker (Gesselschaft für Laboratortechnik GmbH, ) and heated on Gerhardt heating plate (Gerhardt Bonn, Germany). All of the currant and gooseberry varieties were grown in the Czech Republic at the Research and Breeding Institute of Pomology Holovousy Ltd. (50 22'29'' N, 15 34'38'' E, 321m alt.) in the experimental orchard. The type in the experimental orchard is heavy loamy clay soil with a minimum thickness of 60–80 cm. The bedrock consists of clay stone. Average annual temperature of the locality is 8.14 °C. Average annual rainfall is 655 mm and average rainfall during vegetation period is 379 mm. All were 3 years old and no pesticides were used during the cultivation. The samples came from the harvests in 2013 and 2014. All fruits were harvested at full maturity, stored under -18 °C prior analysis and analyzed as soon as possible. Analyzed red currant varieties were: ´Detvan´, ´Jesan´, Junnifer´, ´Losan´, ´Rovada´, ´Rubigo´ and analyzed gooseberry varieties were: ´Alan´, ´Hinnonmaki Rot´, ´Karát´, ´Karmen´, ´Krasnoslawjanskij´,´Remarka´. Approximately 10 grams of fruit was decomposed by 20 ml of concentrated nitric acid. After 24 h of nitric acid addition and continuous sample shaking the sample was heated for 60 min until the complete decomposition. After cooling down, the samples were transferred into the 100 ml volumetric flasks and filled up with water. Each of the fruit variety was decomposed and analysed three times. The quality control material samples were prepared by the same way as the fruit samples. All concentrations were expressed as the average. The standard deviation was less than 10 % for all analysed samples. The concentrations in mg/kg of fresh weight were calculated as cm = cs .V / m, where cm is the concentration of element of interest in mg/kg, cs is the concentration of element of the interest in the analysed solution (mg/l), V is the volume of analysed solution (l) and m is the weight of the sample used for the analysis (kg). Obtained data were further analyzed with the XLStat and Microsoft Excel software. Testing for significance of mean effects and interactions on all variables was calculated using ANOVA analysis of variance and Tukey´s test. Statistical significance was set at P = 0.05.

RESULTS AND DISCUSSION Before the analysis ICP–OES operating conditions were optimized by analysis of a standard solution containing 10 mg/l of each element of the interest. The standard matrix was modified to be the same as in the analysed samples. The optimal wavelengths were chosen in order to achieve the sufficient sensitivity and the least interference and they are presented in Table 1. The optimal power to plasma was 1200 W and peristaltic pump rotation 20 rpm. The optimal nebuliser pressure was estimated by measuring the ratio of the intensities of the magnesium 280 and 285 nm spectral lines and by measuring

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the ratio of signal and background (SBR). The results are presented in Figure 1 and it can be seen that the optimal pressure was 0.29 MPa. The plasma gas flow was adjusted according to the manufacturer's recommendations to 13 l/min. The optimal flow of additional (auxiliary) gas was different for minor (0.2 l/min) and major elements (0.8 l/min) as it is illustrated in Figure 2. The alkali and alkaline-earth metals have low ionization energy and they emit radiation already in the initial radiation zone which is located under the edge of the ICP torch. Addition of auxiliary gas lifts the plasma above the injector tube and it allows better monitoring of the radiation emission. After the ICP–OES conditions have been optimized, the correct settings of the ICP–OES instrument were verified by analysis of quality control material. The recoveries ranged from 92 to 105% (Table 1) which is acceptable. Results from the elemental analysis of red currant are summarized in Table 2. Large differences were observed in metal contents in currant. While , copper and were present in quantities less than 1 mg/kg, sodium, iron and magnesium up to 50 mg/kg and calcium with phosphorous up to 700 mg/kg. All currants were an excellent source of potassium (up to 1800 mg/kg). The difference between content of different currant varieties was statistically significant (p < 0.05) which can be related to different pomological characteristic of analysed cultivars. From the nutritional point of view the ´Jesan´ and ´Rubigo´ varieties were evaluated as the best as they contained significantly higher concentrations of all major elements. In an average it can be concluded that consumption of 100 g of red currants examinated in this study cover about 1–5% of the recommended dietary allowance of potassium, calcium, phosphorous, sodium, iron, manganese, magnesium, copper and zinc for woman and men (Driskell 2009). Table 1 ICP–OES chosen wavelengths and results from the analysis of QCM material QCM certified ICP-OES wavelenght value measured value recovery Zn 206.191 27.1 ± 1.8 24.9 ± 1.4 92 P 213.618 n.a. * * Mn 257.610 187 ± 18 179 ± 15 96 Fe 259.940 912 ± 90 872 ± 43 96 Mg 285.213 4210 ± 420 4191 ± 249 99 Cu 324.750 8.68 ± 0.76 8.14 ± 0.55 94 Ca 422.673 n.a. * * Na 588.900 n.a. * * K 766.490 21200 ± 2100 22319 ± 1712 105 Legend: n.a. – not available Results from the elemental analysis of red gooseberry cultivars are summarized in Table 3. The fruits of red gooseberry contained highest concentration of phosphorous, potassium, calcium and magnesium while other elements concentration was comparable to concentrationfound in red currants varieties. The difference between mineral content of differentgooseberry varieties was statistically significant (p < 0.05). In terms of the total mineral content it can be concluded that ´Hinnonmaki Rot´ and ´Krasnoslawjanskij´ varieties are the most promising for use in the food industry. Consumption of 100 g of these gooseberry varieties can cover up to 8 % of the recommended dietary allowance of minerals for woman and men (Driskell 2009) The comparison of red currant and gooseberry mineral composition with mineral composition of other small fruit species like strawberry, raspberry, elderberry, coenelian cherry and sea buckthorn is presented in Table 4 (Strik 2007, Hegedus et al. 2008, Kalyoncu et al. 2009, Charlrbois et al. 2010, Bal et al. 2011,Vagiri et al. 2013, Cetkovska et al. 2015, Divis et al. 2015). It can be seen that only cornelian cherry contains significantly higher concentration of major elements, however in general it can be said that the nutritional value of red currant and gooseberry is comparable with other small fruits.

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Figure 1 Effect of the additional gas flow to the intensity of measured signal (I). Additional gas flow increases from 0.1 to 0.8 l/min

Figure 2 Effect of the pressure applied on the nebuliser

Legend: SBR – signal to background ratio The comparison of red currant and gooseberry mineral composition with mineral composition of other small fruit species like strawberry, raspberry, elderberry, coenelian cherry and sea buckthorn is presented in Table 4 (Strik 2007, Hegedus et al. 2008, Kalyoncu et al. 2009, Charlrbois et al. 2010, Bal et al. 2011,Vagiri et al. 2013, Cetkovska et al. 2015, Divis et al. 2015). It can be seen that only cornelian cherry contains significantly higher concentration of major elements, however in general it can be said that the nutritional value of red currant and gooseberry is comparable with other small fruits. As it was mentioned above only few studies was published before dealing with elemental analysis of currant and gooseberry. Hegedus et al. 2008 analysed ´Detvan´,´Jonkheer van Tets´ and ´Rondom´ red currant varieties grown in Hungary, while Nour et. al analysed ´Abundent´, ´Houghton Castle´ and ´Rosu Timpuriu´ red currants varieties grown in Romania. In comparison with this study, red currant varieties grown in Hungaria and Romania contained higher concentration of major elements, mainly calcium, potassium and magnesium. Other elements concentration was comparable with results published in this study. The differences in elemental composition can be caused by different soil type used for the cultivation and by different climatic conditions in each region. The measured elemental

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composition of gooseberry can be compared only with the results published by United States Department of Agriculture (USDA 2014). The data published by USDA are consistent with the results measured in this work and varies relatively little. Table 2 Elemental composition of different red currant varieties. Average value in mg/kg from two seasons. Values in the same line with different letters are significantly different at p<0.05. ´Detvan´ ´Jesan´ ´Junnifer´ ´Losan´ ´Rovada´ ´Rubigo´ Zn 1.09a 0.86b 0.61c 1.11a 0.83b 0.58c P 156cd 252a 206bc 176c 229b 262a Mn 0.29bc 0.34b 0.21c 0.41a 0.22c 0.33b Fe 2.8c 4.1ab 3.2bc 4.9a 2.9c 3.8b Mg 30a 27b 27b 25c 21d 28b Cu 0.75b 0.79b 0.78b 0.72b 0.87a 0.93a Ca 91c 147a 92c 115b 148a 151a Na 43a 46a 38b 26c 21d 28c K 1298cd 1469b 1272d 1291cd 1340c 1782a Table 3 Elemental composition of different red gooseberry varieties. Average value in mg/kg from two seasons. Values in the same line with different letters are significantly different at p<0.05. ´Alan´ ´Hinnonmaki Rot´ ´Karát´ ´Karmen´ ´Krasnoslawjanskij´ ´Remarka´ Zn 0.75c 2.65a 0.70c 1.65b 2.65a 0.80c P 305c 327c 205d 441b 568a 173d Mn 0.65cd 0.73c 0.51d 1.23b 1.55a 0.43de Fe 4.7c 9.1b 3.2d 7.5b 11.2a 3.9d Mg 79bc 84b 55c 108b 149a 42c Cu 0.40bc 0.30c 0.31c 0.48b 0.61a 0.31c Ca 323c 335bc 223d 380b 660a 134c Na 24a 11c 16b 10c 20ab 10c K 1023c 2825a 899d 1992b 2909a 944cd Table 4 Elemental composition of different small fruits in mg/kg strawberry raspberry elderberry blueberry cornelian cherry sea buckthorn Zn 0.8−1.1 2.7−4.0 5.1−11.3 1.6 0.5−4.4 n.a. P 210−241 290−351 390−1131 120 606 82−206 Mn 2.8−3.4 1.6−7.0 0.6−9.5 3.4 0.7−1.9 n.a. Fe 2.8−9.5 5.2−7.0 16−30 2.8 0.5−1.8 4−15 Mg 121−154 176−222 50−739 60 72−715 40−240 Cu 0.5 0.9−1.1 1.1−2.0 0.6 0.5−3.6 n.a. Ca 160−312 219−250 380−1528 60 517−1560 64−256 Na 10−26 10−51 60−146 10 n.a. 7−125 K 1405−1530 1510−1718 2800−5494 770 4225−14301 62−806

CONCLUSION An inductively coupled plasma optical emission spectrometry was preferably applied to get information about elemental composition of 6 different red currant and gooseberry varieties. It was demonstrated, that minor and major elements is better to analyze separately in two steps because they require different settings of spectrometer. Despite of different settings for minor and major elements an inductively coupled plasma optical emission spectrometry has still many advantages over other spectroscopic methods. This study confirmed that red currant and gooseberry are good sources of minor and major metals. From the analyzed varieties ´Jesan´ and ´Rubigo´ red currants and ´Hinnonmaki Rot´ and ´Krasnoslawjanskij´ red gooseberries showed favorable elemental composition and they can be recommended for breeding programme of currants and gooseberries in the Czech Republic.

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ACKNOWLEDGEMENTS The research was financially supported by the project NP Mze ČR QI111A141 and infrastructure of the projects REG LO1211 with financial support from National Programme for Sustainability I (Ministry of Education, Youth and Sports) and CZ.1.05/2.1.00/01.0012 was used during this work.

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