Vorgabe neu Journal of Applied Botany and Food Quality 81, 41 - 44 (2007) 1Humboldt University Berlin, Institute for Horticultural Sciences, Section Fruit Science / 3Section Quality Dynamics and Postharvest Physiology 2 Technical University Berlin, Institute of Food Technology and Food Chemistry, Department of Food Analysis Influence of location and fertilization on antioxidant acitivity in highbush blueberries (Vaccinium corymbosum L.) I. Eichholz1, S. Rohn2, L.W. Kroh2, S. Huyskens-Keil3 (Received February 23, 2007) Summary Highbush blueberry cultivars ‘Bluecrop’ and ‘Reka’ were growing in two variants of mulching and fertilizing systems on formerly used farmland. Fruits were harvested at two picking dates and analyzed for their content of phenolic compounds and antioxidant activity. These data were compared with samples of two forest soil locations from the Brandenburg region (Beelitz and Klaistow). The results showed significant differences between cultivars, both harvest times and different locations. The variations in fertilization and ground cover (with or without mulch) showed significant dif- ferences. Moreover, it is demonstrated that without ground cover and commercial fertilization higher contents of total phenolic compounds and an increase in antioxidant activity tendentiously occurred. This result paralleled the decline in vegetative growth and was associated with drought stress. Introduction Secondary plant metabolites, especially phenolic compounds, which are responsible for flavour and colour, are being produced by all plant material for the protection of biotic and abiotic influences. Moreover it is known that phenolic compounds block free radicals, which are activated due to oxidative stress or environmental pollution (RICE- EVANS et al., 1995). Therefore, phenols have been suggested to reduce cellular damage and play a role in preventing harmful diseases such as cancer and coronary heart disease (KALT and DUFOUR, 1997). Berry fruits are one of the richest sources of antioxidants in our diets (KÄHKÖNEN et al., 1999). Especially blueberries have received much attention due to high levels of plant phenolic compounds and thus high antioxidant activity (PRIOR et al., 1998). Blueberry bushes originally grow on heathland or forest soil. However, in Germany there are attempts to cultivate blueberries on formerly used farmland. On such location soil condition are not favourable due to high soil pH, low organic matter content and low water storability. Moreover, there is a lack of symbiosis of mycorrhiza organism, which is essential for nutrition especially for nitrogen supply (YANG et al., 2002). Therefore, soil preparation (mulching) and fertilization are important features for cultivation on farmland soil. Furthermore, the foliar applications of boron support growing and fruiting of plants, as boron is known for enhancing fruit set and yield of many crops, e.g. for apple (WÓJCIK, 2006) and black currant (WÓJCIK, 2005) as well as fruit berry number of blueberries (BLEVINS et al., 1996). Boron also plays an important role as a drought stress inhibitor. WERMINGHAUSEN (1957) reported that plants with higher boron level showed a descreased transpiration rate under drought conditions. Therefore, the aim of the present study was to investigate the effect of different nitrogen and boron supply as well as ground cover systems of formerly used farmland on changes in bioactive secondary plant metabolites of highbush blueberries. Materials and methods Plant material Plant material was obtained from the highbush blueberry cultivars ‘Bluecrop’ and ‘Reka’. Bushes were planted in 1997 in peat filled holes on formerly used farmland in Berlin-Dahlem. In 2004 soil minerals data comprised of 31 mg P 2 O 5 , 62 mg K 2 O, 33 mg MgO per 100 g soil, soil acidity was 5.58 (CaCl 2 ) with an organic matter content of 2.97%. The plants were cultivated in two ground cover (with and without pine bark mulch) and fertilization variants. Fer- tilization was conducted from May to mid July with a nutrient supply of 10 g N- 29 g P 2 O 5 - 83 g K 2 O- 13 g MgO- 66 mg B per plant in first fertigation variant (F1). Plants of second nutrient supply system (F2) received an increased nitrogen fertilization (14 g/ plant) and ad- ditionally boron foliar applications (400 mg/ plant). Ripe berries were picked at two harvest dates providing 70-80% of total crop (‘Reka’: 19/07/04 and 29/07/04; ‘Bluecrop’: 28/07/04 and 04/08/04). Samples of each variant and cultivar were frozen immediately and stored at -20°C until further analysis. These samples were compared with blueberries of two forest soil locations from the Brandenburg region, Klaistow and Beelitz. Chemical analysis Fifteen berries of each variant were carefully crushed and 1 ml of juice was filtered through filtrations tubes (20 µm pore size, Supelco, Deisenhofen, Berlin) and frozen immediately at -30°C until analysis. Antioxidant activity was determined by electron spin resonance (ESR) spectroscopy and trolox equivalent antioxidant capacity (TEAC) assay. ESR was performed as described by RÖSCH et al. (2003), using a table spectrometer Miniscope MS 100 (Magnettech, Berlin, Germany). The signal intensity of a stabilized synthetic radical (Potassium nitro- sodisulfonate (Fremy´s salt), Sigma- Aldrich, Steinheim, Germany) was obtained after 20 min, by which the time of reacting with blueberry antioxidants was completed and expressed as moles of Fremy´s salt reduced by one mole antioxidant/l. TEAC assay was carried out on a Specord 40 (Analytik Jena, Jena, Germany) spectrophotometer as described by RE et al. (1999) with ABTS (2,2´-azinobis (3-ethylbenzothiazonline-6-sulfonic acid, Sigma- Aldrich, Steinheim, Germany) as free radical and TROLOX (6- hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, Sigma- Aldrich, Steinheim, Germany) as a standard at a wavelength of 732 nm. Results were expressed as mmol TROLOX/100 ml. Total soluble phenols were analyzed photometrically with Folin- Ciocalteu reagent (Sigma-Aldrich, Steinheim, Germany) by the method of Jennings (1981), using gallic acid (Serva, Heidelberg, Germany) as a standard at a wavelength of 765 nm. Results were expressed as mg gallic acid /100 ml. Statistic calculations The statistic calculations were performed with SPSS 11.0 by SPSS Inc., Chicago, USA (2001). Significances of differences were con- ducted with a Tukey test (P< 0.05). Results and discussion Influence of location on total phenol content and antioxidant activity There were significant differences between the forest soil locations Beelitz (22.0 mmol Fremy´s salt/ l) and Klaistow (27.7 mmol Fremy´s salt/ l) when using ESR method (Fig. 1). TEAC assay determined differences between the locations Beelitz (14.4 mmol TROLOX/ 100 ml) and Klaistow (24.8 mmol TROLOX/ 100 ml), as well as Beelitz and Berlin-Dahlem (27.0 mmol TROLOX/ 100 ml). Significant differences in total phenol analysis were observed between the locations Beelitz (69.8 mg gallic acid/ 100 ml) and Berlin-Dahlem (125.9 mg gallic acid/ 100 ml). Plants of Berlin-Dahlem are more stressed by wind and unfavourable soil conditions. Furthermore open farmland berries were influenced by a high solar radiation in com- parision to forest locations. Stress as well as increased light condi- tions increase phenolic metabolism (FEUCHT and TREUTTER, 1989; HÄKKINEN, 2000) and might explain the higher levels of bioactive compounds in Berlin-Dahlem. The forest soil location Beelitz showed the lowest content of total phenols as well as of antioxidants in all analysis, probably due to a higher berry weight and higher soil nitrogen content. High nitrogen levels decrease the content of phenolic com- pounds (FEUCHT and TREUTTER, 1989; HÄKKINEN, 2000). Berry weight again is negative correlated with the content of phenolic compounds (CONNOR et al., 2002b; MOYER et al., 2002). However, variation between locations is depending on local micro climate (temperature, irradition, moisture) and soil conditions in blueberry cultivation (JONES, 1999 in HOWARD, 2003; HÄKKINEN, 2000; MOYER et al., 2002). In contrast, PRIOR et al. (1998) reported no differences between the blueberry growing locations Orgeon, New Jersey and Michigan. Influence of cultivation on total phenol content and antioxidant activity ESR data ranged from 23.6 to 28.9 mmol Fremy´s salt/l, for TEAC method 24.5 to 27.5 mmol TROLOX/ 100 ml and for total phenol content from 120.1 to 134.4 mg gallic acid/ 100 ml (Fig. 2). The present results demonstrate that blueberries without ground cover, lower nitrogen and boron fertilization (F1oM) had higher contents of total phenolic compounds and an increase in antioxidative activity. This result is assumed to be related to a stress mediated process, i.e. the non covered soil variant resulted in a low organic matter content, high soil ph and thus insufficient waterstorage. This might also explain the decline in vegetative growth. In contrast the F2oM variant revealed a lower content of bioactive compounds due to additional boron application. Boron could have restricted the influx of substrate into the pentose-phosphate pathway and the synthesis of phenols (LEE and ARONOFF, 1967 in MONDY and MUNSHI, 1993). MONDY and MUNSHI (1993) reported a decreased phenol concentration in potato tubers after boron treatment. Also AL-YOUSIF et al. (1994) determined a reduced phenol content in date palm and sorghum leaves due to an increased boron level. Fig. 2: Influence of cultivation on total phenol content and antioxidant activity; F1M= commercial fertilization with mulch, F1oM= commercial fertili- zation without mulch, F2M= increased nitrogen and boron supply with mulch; F2oM= increased nitrogen and boron supply without mulch: Tukey test (P < 0.05) F1M F1oM F2M F2oM cultivation systems 5 10 15 20 25 30 35 m m ol F re m ys ' sa lt /l Antioxidant Activity (ESR) b a b b 24.5 23.928.9 23.6 F1M F1oM F2M F2oM cultivation system 5 10 15 20 25 30 35 m m ol T R O L O X / 10 0 m l Antioxidant Activity (TEAC) a a a a 25.6 25.627.5 24.5 F1M F1oM F2M F2oM cultivation system 20 40 60 80 100 120 140 160 180 m g ga ll ic a ci d / 10 0 m l Phenol content a a a a 125.7 134.4 123.3120.1 Fig. 1: Influence of location on total phenol content and antioxidant activity, Tukey Test (P < 0.05) Berlin-Dahlem Beelitz Klaistow location 5 10 15 20 25 30 35 m m ol F re m ys ' sa lt /l Antioxidant Activity (ESR) ab b a 25.4 27.722.0 Berlin-Dahlem Beelitz Klaistow location 5 10 15 20 25 30 35 m m ol T R O L O X / 10 0 m l Antioxidant Activity (TEAC) b a a 27.0 24.814.4 Berlin-Dahlem Beelitz Klaistow location 20 40 60 80 100 120 140 160 180 m g ga ll ic a ci d / 10 0 m l Phenol content b a ab 125.9 94.869.8 42 I. Eichholz, S. Rohn, L.W. Kroh, S. Huyskens-Keil Influence of cultivar and harvest time on total phenol content and antioxidant activity Comparision of cultivars showed statistic differences in antioxidant activity at the first harvest date between ‘Bluecrop’ (22.3 mmol Fremy´s salt/l) and ‘Reka’ (28.5 mmol Fremy´s salt/l) by ESR method (Fig. 3). In TEAC assay differences were found between cultivars of the second harvest date (‘Bluecrop’: 30.9 mmol TROLOX/ 100 ml; ‘Reka’: 22.3 mmol TROLOX/ 100 ml). In respect to total phenols significant differences were observed for the first harvest date between ‘Bluecrop’ (105.2 mg gallic acid/ 100ml) and ‘Reka’ (132.7 mg gallic acid/ 100ml) as well as for the second date (‘Bluecrop’: 164.9 mg gallic acid/ 100ml; ‘Reka’: 95.8 mg gallic acid/ 100ml). Variation in bioactive compounds of different cultivars are affected genetically, also described by PRIOR et al. (1998), CONNOR et al. (2002a; 2002b), MOYER et al. (2002) and HOWARD et al. (2003). Comparision of harvest times showed differences in the cultivar ‘Reka’ (first date: 28.5 mmol Fremy´s salt/l; second date: 23.2 mmol Fremy´s salt/l) using ESR method. Significances in TEAC assay of harvest times occurred at both harvest dates for ‘Bluecrop’ (first date: 23.1 mmol TROLOX/ 100ml; second date: 30.9 mmol TROLOX/ 100 ml) and ‘Reka’ (first date: 25.9 mmol TROLOX/ 100 ml; second date: 22.3 mmol TROLOX/ 100ml). Total phenol content differed significantly between harvest time and cultivar, i.e. ‘Bluecrop’ (first date: 105.2 mg gallic acid/ 100 ml; second date: 164.9 mg gallic acid/ 100 ml) as well as of ‘Reka’ (first date: 132.7 mg gallic acid/ 100 ml; second date: 95.8 mg gallic acid/ 100 ml). However, antioxidant activity (TEAC) and total phenol analysis showed that bioactive components of the medium-ripened cultivar ‘Bluecrop’ were significantly higher in the second harvest in comparision to the first harvest, whereas in contrast the first harvest of the early-ripened cultivar ‘Reka’ had higher levels of total phenols and antioxidants. The results of the ESR method showed a similar trend. Total phenolic content and antioxidant activity of blueberries decrease during fruit ripening (KALT, 2003). Although all berries were harvested at a fully ripe stage, fruits of ‘Bluecrop’ second crop showed a lower sugar/acid ratio, i.e. an earlier ripening stage (10.3) in comparison to the first harvest date (16.0). Moreover, in the cultivar ‘Bluecrop’ increased level of phenolic compounds and antioxidants of the second harvest date may also be caused by the decline in fruit weight from 1.9 g to 1.4 g, respectively. This is also consistent with findings by HOWARD et al. (2003), CONNOR et al. (2002b) and MOYER et al. (2002). References BLEVINS, D.G., SCRIVNER, C.L., REINBOTT, T.M., 1996: Foliar boron increases berry number and yield of two highbush blueberry cultivars in Missouri. J. Plant Nutr. 19, 99-113. CONNOR, A., LUBY, J., TONG, C.B.S., 2002a: Variations and heritability estimates for antioxidant activity, total phenol content, and anthocyanin content in blueberry progenies. J. Amer. Soc. Hort. Sci. 127, 82-88. CONNOR, A., LUBY, J., TONG, C.B.S., FINN, C.E., HANCOCK, J.F., 2002b: Genotypic and environmental variation in antioxidant activity, total phenol content, and anthocyanin content among blueberry cultivars. J. Amer. Soc. Hort. Sci. 127, 89-97. FEUCHT, W., TREUTTER, D., 1989: Phenolische Naturstoffe. Obst- und Garten- bauverlag, München. HÄKKINEN, S., 2000: Flavonols and phenolic acids in berries and berry products. PhD Thesis. Kuopio University Publications, D. Medical Sciences 221. HOWARD, L.R., CLARK, J.R., BROWNMILLER, C., 2003: Antioxidant capacity and phenolic content in blueberries as affected by genotpye and growing season. J. Sci. Food Agric. 83, 1238-1247. JENNINGS, A.C., 1981: The determination of dihydroxy phenolic compounds in extracts of plant tissues. Anal. Biochem. 118, 396-398. KÄHKÖNEN, M.P., HOPIA, A.I., VUORELA, H.J., RAUHA, J.-P., PIHLAJA, K., KUJALA, T.S., HEINONEN, M., 1999: Antioxidant activity of plant extracts containing phenolic compounds. J. Agric. Food Chem. 47, 3954-3962. KALT, W., DUFOUR, D., 1997: Health functionality of blueberries. HortTech- nol. 7, 216-221. KALT, W., 2003: Oxygen radical absorbing capacity, anthocyanin and phenolic content of highbush blueberries (Vaccinium corymbosum L.) during ripening and storage. J. Amer. Soc. Hort. Sci. 128, 917-923. MONDY, N.I., MUNSHI, C.B., 1993: Effect of boron on enzymatic discoloration and phenolic and ascorbic acid contents of potatoes. J. Agric. Food Chem. 41, 554-556. MOYER, R.A., HUMMER, K.E., FINN, C.E., FREI, B., WROLSTAD, R.E., 2002: Anthocyanins, phenolics, and antioxidant capacity in diverse small fruits: Vaccinium, Rubus, and Ribes. J. Agric. Food Chem. 50, 519-525. PRIOR, R.L., CAO, G., MARTIN, A., SOFIC, E., MCEWEN, J., O´BRIEN, C., LISCHNER, N., EHLENFELDT, M., KALT, W., KREWER, G., MAINLAND, M., 1998: Antioxidant capacity as influenced by total phenol and anthocyanin content, maturity, and variety of Vaccinium species. J. Agric. Food Chem. 46, 2686-2693. RE, R., PELLEGRINI, N., PROTEGGENTE, A., PANNALA, A., YANG, M., RICE-EVANS, C., 1999: Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Rad. Biol. Med. 26, 1231-1237. RICE-EVANS, C.-A., MILLER, N.J., BOLWELL, P.G., BRAMLEY, P.M., PRIDHAM, J.B., 1995: The relative antioxidant acitivies of plant-derived polyphenolic flavonoids. Free Radical Res. 22, 375-383. RÖSCH, D., BERGMANN, M., KNORR, D., KROH, L.W., 2003: Structure-anti- oxidant efficiency relationships of phenolic compounds and their contribution to the antioxidant activity of Sea Buckthorn juice. J. Agric. Food Chem. 51, 4233-4239. WERMINGHAUSEN, B., 1957: Nährstoffmangelerscheinungen im Obstbau und ihre Behebung. Obst- und Gartenbauverlag, München. WÓJCIK, P., 2006: Effect of postharvest sprays of boron and urea on yield and Fig. 3: Influence of cultivar and harvest time on total phenol content and antioxidant activity, Tukey (P < 0.05) 1.harvest time 2.harvest time Bluecrop Reka cultivar 5 10 15 20 25 30 35 m m ol F re m ys ' sa lt /l Antioxidant Activity (ESR) a b b b 22.3 28.525.0 23.2 1.harvest time 2.harvest time Bluecrop Reka cultivar 5 10 15 20 25 30 35 m m ol T R O L O X / 10 0 m l Antioxidant Activity (TEAC) a b bc c 23.1 25.930.9 22.3 1.harvest time 2.harvest time Bluecrop Reka cultivar 20 40 60 80 100 120 140 160 180 m g ga ll ic a ci d / 10 0 m l Phenol content a b cc 105.2 132.7164.9 95.8 Antioxidant activity in blueberries 43 fruit quality of apple trees. J. Plant Nutr. 29, 441-450. WÓJCIK, P., 2005: Response of Black Currant to boron fertilization. J. Plant Nutr. 28, 63-72. YANG, W.Q., GOULART, B.L., DEMCHAK, K., LI, Y., 2002: Interactive effects of mycorrhizal inoculation and organic soil adjustments on nitrogen acquisition and growth of highbush blueberry. J. Amer. Soc. Hort. Sci. 127, 742-748. Addresses of the authors: 1 Humboldt Universität zu Berlin, Institut für Gartenbauwissenschaften, Fachgebiet Obstbau, Albrecht-Thaer-Weg 3, D-14195 Berlin 2 Technische Universität Berlin, Institut für Lebensmitteltechnologie und Lebensmittelchemie, Fachgebiet Lebensmittelanalytik, TIB 4/3-1, Gustav- Meyer-Allee 25, D-13355 Berlin 3 Humboldt Universität zu Berlin, Institut für Gartenbauwissenschaften, Lehr- und Forschungsgebiet Produktqualität/Qualitätssicherung, Lentzeallee 75, D- 14195 Berlin 44 I. Eichholz, S. Rohn, L.W. Kroh, S. Huyskens-Keil << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /All /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Warning /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJDFFile false /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /ColorConversionStrategy /LeaveColorUnchanged /DoThumbnails false /EmbedAllFonts true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveEPSInfo true /PreserveHalftoneInfo false /PreserveOPIComments false /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile () /PDFXOutputCondition () /PDFXRegistryName (http://www.color.org) /PDFXTrapped /Unknown /Description << /FRA /ENU (Use these settings to create PDF documents with higher image resolution for improved printing quality. The PDF documents can be opened with Acrobat and Reader 5.0 and later.) /JPN /DEU /PTB /DAN /NLD /ESP /SUO /ITA /NOR /SVE >> >> setdistillerparams << /HWResolution [2400 2400] /PageSize [612.000 792.000] >> setpagedevice