COBISS Code 1.01 DOI: 10.14720/aas.2014.103.2.12 Agrovoc descriptors: organic agriculture,farming systems,antioxidants,fruits,vegetables,drug plants,spice crops,proximate composition Agris category code: f60,q03 Antioxidant activity in selected Slovenian organic and conventional crops Manca KNAP1, Nives OGRINC2, Klemen POTOČNIK3, Rajko VIDRIH4 Received July 09, 2014; accepted September 05, 2014. Delo je prispelo 09. julija 2014, sprejeto 05. septembra 2014. ABSTRACT IZVLEČEK The demand for organically produced food is increasing. There is widespread belief that organic food is substantially healthier and safer than conventional food. According to literature organic food is free of phytopharmaceutical residues, contain less nitrates and more antioxidants. The aim of the present study was to verify if there are any differences in the antioxidant activity between selected Slovenian organic and conventional crops. Method of DPPH (2,2-diphenyl-1-picryhydrazyl) was used to determine the antioxidant activity of 16 samples from organic and conventional farms. The same varieties of crops were analysed. DPPH method was employed to measure the antioxidant activity of polar antioxidants (AAp) and antioxidant activity of fraction in ethyl acetate soluble antioxidants (EA AA). Descriptive statistics and variance analysis were used to describe differences between farming systems. Estimated differences between interactions for the same crop and different farming practice were mostly not statistically significant except for the AAp for basil and beetroot. Higher statistically significant values were estimated for conventional crops. For the EA AA in broccoli, cucumber, rocket and cherry statistically significant higher values were estimated for organic production. Key words: antioxidant activity, organic farming, conventional farming, fruits, herbs, vegetables ANTIOKSIDATIVNA UČINKOVITOST V PRIDELKIH IZ SLOVENSKE EKOLOŠKE IN KONVENCIONALNE PRIDELAVE Povpraševanje po ekološko pridelanih živilih se povečuje. Ekološki proizvodi veljajo za bolj zdrave v primerjavi s konvencionalnimi. Po navajanju drugih virov ne vsebujejo fitofarmacevtskih sredstev imajo manjšo vsebnost nitratov in vsebujejo več antioksidantov. Namen študije je bil preveriti ali obstajajo razlike v antioksidativni učinkovitosti med slovenskimi pridelki pridelanimi na ekološki in konvencionalni način. Za določevanje antioksidativne učinkovitosti smo uporabili metodo DPPH (2,2-difenil-1-pikrilhidrazil). Analiza je bila opravljena na 16 vrstah iste sorte pridelkov iz ekološke in konvencionalne pridelave. Izmerili smo polarno antioksidativno učinkovitost (AAp) in antioksidativno učinkovitost v etil-acetatu topnih antioksidantov (EA AA). Izračunali smo osnovne statistične parameter po vrstah pridelkov in načinu kmetovanja in z analizo variance ocenili razlike med načinoma kmetovanja za posamezne pridelke. Ocenjene razlike med interakcijami pridelka in načina kmetovanja večinoma niso statistično značilne. Izjema za AAp sta bazilika in rdeča pesa, kjer so bile večje vrednosti za konvencionalne pridelke. Za vrednost EA AA so bile statistično značilno večje vrednosti ocenjene za ekološki brokoli, kumaro, rukolo in češnjo. Ključne besede: antioksidativna učinkovitost, ekološko kmetijstvo, konvencionalno kmetijstvo, sadje, zelišča, zelenjava 1 University of Ljubljana, Biotechnical Faculty, Department of Food Science and Technology, Jamnikarjeva 101, Ljubljana, SI-1000, Slovenia, corresponding author, e-mail: manca.knap@gmail.com 2 Assoc. Prof., Ph. D. J. Stefan Institute, Jamova cesta 38, Ljubljana, SI-1000, Slovenia 3 Assist. Prof., Ph. D. University of Ljubljana, Biotechnical Faculty., Department of Animal Science, Groblje 3, Domžale, SI-1230, Slovenia 4 Assoc. Prof., Ph. D. University of Ljubljana, Biotechnical Faculty, Department of Food Science and Technology, Jamnikarjeva 101, Ljubljana, SI-1000, Slovenia Članek je nastal na osnovi podatkov doktorske disertacije Mance Knap. Mentor: prof. dr. Rajko Vidrih, somentorica: prof. dr. Nives Ogrinc This article is based on Doctoral dissertation Thesis of Manca Knap. Supervisor: Prof. Ph. D. Rajko Vidrih, co-supervisor: Prof. Ph. D. Nives Ogrinc 1 INTRODUCTION For a variety of reasons all crop species vary in their composition of antioxidants, and other nutritional relevant substances. Fruits, herbs and vegetables are very important for human nutrition. Besides providing energy, food of plant origin, is a rich source of dietary fibres and minerals. Food of plant origin is thus the most important source of antioxidants (Hertog and Hollman, 1996; Pietta, 2000; Chandrasekara and Shahidi, 2011). Antioxidant activity depends on different chemical attributes and may be specific to variety. It depends mainly on phenolics, which are considered more potent antioxidants as compared to vitamins (Koleva et al., 2002; Usenik et al., 2008). In foods of plant origin, there are numerous compounds that contribute to antioxidative activity and function in different ways. The most important antioxidants include asorbic acid, carotenoids and phenolic compounds. Group of phenolic compounds include monophenols with a single benzene ring, hydroxycinnamic acids, flavonoids and their glycosides which include catechins, proanthocyanidins, anthocyanins and flavonols (Gulcin, 2012). The most complex and poorly defined are high molecular weight tannins regularly present in fruit. In the above mentioned groups there are altogether few thousand of active substances which in different ways contribute to the total antioxidant activity (Hribar and Simcic, 2000). Phenols are considered polar compounds and are soluble in polar solvents like water, methanol, etc. However some of them predominantly phenols with more aromatic rings, less OH groups or deglycosylated phenols are partially soluble in nonpolar solvents. Phenolic compounds also show partial solubility in less polar solvents like n-butanol and ethyl acetate (Dhingra et al. 2014). Higher EA AA means more above mentioned phenol compounds are present in ethyl acetate fraction (Rice-Evans et al., 1996) . Soil, climate, variety, degree of ripeness and also the freshness, storage conditions can all affect the content of biologically active compounds. Type of farming system may also affect the chemical composition of foodstuffs, especially on those that originate from the use of chemical fertilizer and pesticides (Dangour et al., 2009). Organic farming represents a production system, looking for harmony between the environment and the agriculture production (Casado and de Molina, 2009; Bavec et al., 2010). It excludes the use of synthetic fertilizers and pesticides, plant growth regulators and genetically modified organisms (Singh et al., 2009). The use of pesticides was perceived to be associated with effects on health, but it was also associated with benefits like cheaper foods and higher yield (Huang, 1996; Miles and Frewer, 2001; Torjusen et al., 2001). It is widely believed that mineral fertilizers reduce antioxidant levels in plants, while organic fertilizers enhance the antioxidant levels (Dumas et al., 2003; Aldrich et al., 2010; Oliveira et al., 2013). However literature show mixed data regarding the phytochemical status of organic and conventional vegetables (Faller and Fialho, 2010; Sinkovic et al., 2015). In this study the antioxidant activity in selected organic and conventionally produced fruits, herbs and vegetables in Slovenia was measured. The aim of the study was to check if there are differences in antioxidant activity between analyzed organic and conventional crops. 2 MATERIALS AND METHODS 2.1 Plant material Herbs (basil, parsley, celery) and vegetables (broccoli, beetroot, carrot, cherry tomato, cucumber, eggplant, tomato and rocket) were grown in experimental field, where mineral fertilizers had not been used for more than 30 years. The same varieties of crops were used in both farming systems. Crop varieties used in this study are presented in Table 1 and Table 2. Basic soil cultivation, sowing, and harvesting dates and methods were identical for organic and conventional experimental plot. Organic crops were only irrigated while conventional crops were fertilized with Plantella extra plus NPK (15:15:15), according to instructions given by the manufacturer, on the 14th, 21st, 28th, 35th and 42nd day during growth period. The climatic conditions, variety, irrigation, ripening time, and storage conditions were the same for the crops grown organically and conventionally. Samples of same variety of fruits (apple, cherry, pear and raspberry) were obtained from known organic and conventional farms in Slovenia. Organic fruits derived from certified organic productions possessed certification for organic farming, according to the Institute of Certification (KON-CERT Maribor, Slovenia). Conventional fruit samples were from conventional farms. Samples were harvested in the year 2012 when they were in commercial maturity stage. Samples were cleaned, each plot stored separately in a cooling room at + 4 °C and 95% relative humidity until the analyses in laboratory were performed. 2.2 Sample preparation Samples were prepared in three repetitions no later than 12 h after harvest. They were washed, dried out in air and cut into small pieces. Ten gram of each sample were homogenised with 20 g of 2 % methaposphoric acid, using an Ultra-turrax T 25 (IKA, Germany). Methaposphoric acid was used to get low pH which stabilises ascorbic acid in samples (Osborn-Barnes and Akoh, 2003). The obtained homogenates were immediately frozen at - 20 °C till further use. Samples were thawed before use and centrifuged at 1700 xg for 5 minutes (Rotanta 460R; Hittich, Germany). The supernatants were transferred into micro centrifuge tubes and centrifuged again at 16 x g for 5 minutes (Centrifuge 5415c; Eppendorf, Germany). Finally, supernatants were filtered through 0.45 ^m filter and used as a sample. 2.3 Determination of antioxidant activity The antioxidant activity of fruits, herbs and vegetables from organic and conventional farming was measured by means of free radical DPPH (2,2-diphenyl-1-picryhydrazyl) as previously reported by Brand-Williams et al. (1995) and Shyu and Hwang (2002) with some modifications. This method is based on the reduction of the stable DPPH radical (2,2-dipenyl-1-picrylhydazyl) in to DPPH2. A solution of 4 mg DPPH /100 ml of ethyl acetate was used to determine antioxidant activity of fraction in ethyl acetate soluble antioxidants (EA AA) and a solution of 4 mg DPPH /100 ml of methanol was used for polar antioxidant activity (AAp). For the determination of EA AA 5 ml of supernatant and 5 ml of the ethyl acetate were thoroughly mixed and upper layer (ethyl acetate fraction) was used for the essay. For AAp 60 or 100 ^l of supernatant was mixed with 1.5 ml freshly prepared DPPH. After stirring, the tubes were left in the dark for 30 minutes. The absorbance of the samples was measured at 517 nm after the reaction time. All samples were analysed as triplicate. For EA AA ethyl acetate was used as a blank and for AAp methanol was used as a blank. The antioxidant activity was expressed as millimol DPPH equivalents per 100 g of fresh weight (mmol DPPH/100 g FW). 2.4 Statistical analysis The data were analysed using SAS/STAT statistical software (SAS, 2012). Analysis of variance was performed using the GLM (General Linear Models) procedure. Statistical model included farming system, crop species and interaction between farming system and crop species as fixed effects (statistical model (1)). In statistical model yijk is dependent variable (AAp or AE AA), ^ is estimated overall mean, Fi is farming system fixed effect i=1,2 (organic farming, conventional farming), Cj is crop fixed effect j=1,2,3,...,16 (basil, apple, ...), FCij is interaction between Fi and Cj and eijk is residual. Differences between LSMs (Least Square Means) for the same crop in different farming system were estimated using t-test and significance level was set atp < 0.05. yljk = ¡a + F+Cj+FQj+e ijk •••(1) 3 RESULTS AND DISCUSSION 3.1 Descriptive statistic grown according to organic farming system are r-prt j • .. + r- -j ... •.,.• r shown in the Table 1 and according to The descriptive statistic for antioxidant activities of , - ° n • j i ... iui 4- J4 j conventional farming system in the Table 2. fraction in ethyl acetate soluble antioxidants and ° J antioxidant activities of polar antioxidants of crops Table 1: Descriptive statistics for antioxidant activities of fraction in ethyl acetate soluble antioxidants (EA AA) and polar antioxidants' activities (AAp) of crops produced in organic farming Farming Crop Variety EA AA AAp (mmol/100 g FW) (mmol/100 g FW) M SD Min Max M SD Min Max Organic Apple Malus domestica^ 33 'Idared' 0.018 0.31 0.35 0.35 0.008 0.34 0.36 crops Basil Ocimum basilicum 0.33 0.024 0.30 0.34 0.93 0.007 0.92 0.93 Beetroot Beta vulgaris 'Rote0 06 Kugel' Brassica oleracea0 14 'Corvet' 014 0.032 0.04 0.10 0.82 0.492 0.26 1.17 Broccoli 0.020 0.12 0.16 0.86 0.046 0.83 0.92 Carrot Daucus carota 'Kuroda' 0.14 0.040 0.11 0.19 0.18 0.003 0.18 0.18 Cherry Prunus avium 'Burlat' 0.29 0.024 0.26 0.31 0.44 0.015 0.43 0.46 Cherry Lycopersicon tomato esculentum var. cerasiforme 0.13 0.013 0.12 0.14 0.31 0.008 0.31 0.32 'Gardener's Delight'' Cucumber Cucumis sativus 'Darina' 0.10 0.011 0.09 0.11 0.13 0.004 0.12 0.13 Eggplant Solanum melongena 'Halflange Violette' 0.19 0.020 0.17 0.21 0.80 0.018 0.78 0.81 Parsley Petroselinum crispum 0.11 0.010 0.10 0.12 0.58 0.044 0.53 0.62 'Italian Parsley' Pear Pyrus communis 'Conference' 0.14 0.044 0.11 0.17 0.23 0.008 0.22 0.23 Pepper Capsicum anuum 'California 0.11 0.051 0.05 0.15 0.38 0.017 0.36 0.40 Wonder' Raspberry Rubus idaeus 'Willamette'' 0.30 0.023 0.27 0.31 0.80 0.009 0.79 0.81 Rocket Eruca sativa 0.13 0.009 0.12 0.13 0.73 0.055 0.67 0.76 Tomato Lycopersicon esculentum 0.08 0.027 0.05 0.10 0.34 0.015 0.32 0.35 'Volovsko srce' Subtotal 0.18 0.092 0.04 0.35 0.56 0.314 0.12 1.17 AAp - polar antioxidants' activity; EA AA - antioxidant activity of fraction in ethyl-acetate soluble antioxidants; FW - fresh weight; M - average value; SD - standard deviation; Min - minimum; Max - maximum The AAp of the organic samples varied from the EA AA ranged from 0.08 mmol DPPH/100 g FW lowest at 0.13 mmol DPPH/100 g FW (cucumber) (tomato) to 0.33 mmol DPPH/100g FW (basil and to 0.93 mmol DPPH/100 g FW (basil), while the apple). Table 2: Descriptive statistics for antioxidant activities of fraction in ethyl acetate soluble antioxidants (EA AA) and polar antioxidants' activities (AAp) of crops produced according to conventional farming Farming Crop Species/Variety EA AA AAp (mmol/100 g FW) (mmol/100 g FW) M SD Min Max M SD Min Max Conventional Apple Malus domestica^ ^ 'Idared' 0.023 0.28 0.32 0.51 0.039 0.47 0.55 crops Basil Ocimum basilicum 0.36 0.009 0.35 0.37 1.68 0.072 1.60 1.73 Beetroot Beta vulgaris 'Rote0 07 Kugel' Brassica oleracea0 09 'Corvet' 009 0.009 0.06 0.08 1.25 1.326 0.10 2.70 Broccoli 0.011 0.08 0.10 0.74 0.020 0.71 0.75 Carrot Daucus carota 'Kuroda' 0.11 0.022 0.09 0.13 0.15 0.011 0.14 0.16 Cherry Prunus avium 'Burlat' 0.20 0.018 0.18 0.22 0.45 0.002 0.45 0.45 Cherry Lycopersicon tomato esculentum var. cerasiforme 0.12 0.021 0.11 0.15 0.42 0.034 0.38 0.45 'Gardener's Delight'' Cucumber Cucumis sativus 'Darina' 0.05 0.035 0.02 0.08 0.08 0.003 0.07 0.08 Eggplant Solanum melongena 'Halflange Violette' 0.24 0.006 0.23 0.24 0.55 0.005 0.55 0.56 Parsley Petroselinum crispum 0.10 0.032 0.06 0.12 0.45 0.017 0.43 0.46 'Italian Parsley' Pear Pyrus communis 'Conferense' 0.14 0.037 0.10 0.17 0.23 0.021 0.21 0.25 Pepper Capsicum anuum 'California 0.14 0.026 0.12 0.16 0.34 0.018 0.32 0.35 Wonder' Raspberry Rubus idaeus 'Willamette'' 0.26 0.029 0.24 0.30 0.68 0.014 0.66 0.69 Rocket Eruca sativa 0.06 0.022 0.04 0.08 0.50 0.009 0.49 0.51 Tomato Lycopersicon esculentum 0.08 0.016 0.07 0.10 0.17 0.014 0.16 0.18 'Volovsko srce' Subtotal 0.16 0.094 0.02 0.37 0.56 0.489 0.07 2.70 AAp - polar antioxidant activity; EA AA - antioxidant activity of fraction in ethyl-acetate soluble antioxidants; FW - fresh weight; M - average value; SD - standard deviation; Min - minimum; Max - maximum The AAp of the conventional samples varied from the lowest value of 0.07 mmol DPPH/100 g FW (cucumber) to 2.70 mmol DPPH/100 g FW (beetroot), while the EA AA ranged from 0.02 mmol DPPH/100 g FW (cucumber) to 0.37 mmol DPPH/100g FW for basil. The mean AAp for cherry was very similar between the farming systems (organic, 0.44 mmol DPPH/100 g FW; conventional, 0.45 mmol DPPH/100 g FW). The mean AAp for pear were equal (0.23 mmol DPPH/100 g FW). The mean EA AA for pear (0.14 mmol DPPH/100 g FW) and tomato (0.08 mmol DPPH/100 g FW) were unchanged between the farming system. organic farming uses less phytopharmacheuticals and plant therefore develop several defense mechanisms (Koleva et al., 2002; Borguini et al., 2013). On the other hand, Lamperi et al. (2008) and Cardoso et al. (2011) argue that there is no difference in the antioxidant activity between products from different farming systems. There are a number of reviews that compare nutritional quality of organically versus conventionally grown foods (Weibel et al., 2000; Brandt and Molgaard, 2001; Bourn and Prescott, 2002). Weibel et al. (2000) showed that fruit quality of organic apples were either similar or slightly better than that of conventional ones. According to literature data, the organic crops presented higher antioxidant activity than conventional ones (Worthington, 2001; Wang et al., 2008; Aldrich et al., 2010; Arbos et al., 2010; Crinnion, 2010; Lairon, 2010; Borguini et al., 2013). This can be attributed to the fact that 3.2 Analysis of variance In AAp 75 % and for EA AA 94 % oftotal variability were explained with statistical model (1). Effect of farming system was not statistically significant in AAp but it was in EA AA. In both antioxidant activity measurements crop was highly statistical significant. Interaction between farming system and crop was statistically significant for EA AA, but in AAp interaction has a trend that approached significance (Table 3). This shows that variability between crops was higher than variability between farming systems. Table 3: F-values and /»-values for effects included in statistical model (1) for AAp and EA AA Effect AAp EA AA F-value /»-value F-value /-value Farming system 0.00 0.949 7.65 0.008 Crop 10.74 <0.001 60.84 <0.001 Farming system and crop interaction 1.76 0.061 2.54 0.005 ° 0.20 G E ♦ f i i i * f * I i i s/yss s//s s * s/ s s