Pepper antioxidant composition as affected by organic, low-input and soilless cultivation

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Research ArticleReceived: 10 February 2009 Revised: 16 June 2009 Accepted: 24 June 2009 Published online in Wiley Interscience: 5 August 2009

(www.interscience.wiley.com) DOI 10.1002/jsfa.3719

Pepper antioxidant composition as affected byorganic, low-input and soilless cultivationPilar Flores,∗ Pilar Hellın, Alfredo Lacasa, Alicia Lopez and Jose Fenoll

Abstract

BACKGROUND: The aim of this work was to study the effects of organic (O), low-input (LI) and soilless (SL) cultivation on peppernutritional quality and antioxidant activity. For that, 24 commercial greenhouses were selected following strict criteria inorder to reduce the influence of environmental conditions and realistically reflect commercial production systems. Fruits wereharvested at two maturity stages (green and red) and three harvesting times during two consecutive years.

RESULTS: Pepper antioxidant activity mainly stemmed from water-soluble compounds, including organic acids and phenoliccompounds. Only some differences in sugars and malic and citric acid concentrations were detected between the O and LIsystems. Sugars, phenolic compounds, ascorbic acid and hydrophilic antioxidant activity were higher in the SL system. In spiteof these differences, overall differences between harvesting times or between years were far greater than those due to thecropping system.

CONCLUSION: The main differences in the nutritional quality of pepper fruits were observed between the soil (O and LI) and SL(the most favourable) systems. The results highlight the importance of comparing different harvesting times and years in orderto study the effect of cropping system on a specific crop.c© 2009 Society of Chemical Industry

Keywords: organic; low-input; soilless; antioxidants; nutritional quality

INTRODUCTIONThe last decade has seen increasing consumer demand for organicproducts largely due to growing awareness about health andnutrition. In general, a perception exists that organic foodsare safer, more nutritious and more environmentally friendlythan conventional foods. However, recent reviews of previousstudies on the yield and quality of organic versus conventionalvegetables have pointed to the difficulty of compiling resultsand drawing meaningful conclusions.1 For example, severalstudies have reported the beneficial effect of organic productionon the nutritional composition of fruits,2 whereas others haveshown that organic cultivation has no consistent effect on fruitnutritional composition compared with conventional practices.3

These differences observed in the literature may be partly dueto the different sampling methods used and agricultural andenvironmental factors, which can influence food quality to differingextents.4 It is clear, then, that more studies involving suitableexperimental designs and manipulation are needed to make validcomparisons. In addition, comparative studies usually do notinclude soilless cultivation practices, although such practices aregrowing in importance. Indeed, little is known about the quality offruits grown under soilless conditions compared with those grownin soil.

Pepper is cultivated in Mediterranean countries in conventional(usually following low-input guidelines), organic and soillessconditions. The fruit is considered an excellent source of bioactivenutrients such as carotenoids, vitamin C and phenolic compounds,which define its nutritional quality and antioxidant capacity.5,6

Carotenoids are recognised as powerful natural antioxidants

that help prevent a broad range of cancers7 and cardiovasculardiseases.8,9 Vitamin C is required in many biological functionssuch as the synthesis of collagen and the biosynthesis ofcertain hormones. In addition, it has the potential to counteractboth inflammation processes and oxidative damage.10 Phenolicsare the largest group of phytochemicals in plant foods thatcontribute to the antioxidant and antiproliferative activities offruits and vegetables.11 As a consequence, dietary phenolics areconsidered to play an important role in human health and diseaseprevention.12

According to Magkos et al.,13 there are three basic categoriesof comparative studies: retail market studies, farm studies andcultivation studies. Farm studies are considered to realisticallyreflect the production system as long as a thorough selectionof farms is carried out. The present work constitutes a farmstudy to compare the nutritional quality of peppers grownunder organic, low-input and soilless systems. Following Magkoset al.’s recommendations, greenhouses were selected accordingto specific rules to control, to the greatest extent possible, factorsother than the production system that could influence fruit quality.The fruit quality parameters studied included soluble sugars andnutritive compounds with proven antioxidant activity.

∗ Correspondenceto:Pilar Flores, InstitutoMurcianodeInvestigacion yDesarrolloAgrario y Alimentario (IMIDA), c/Mayor s/n, E-30150 La Alberca (Murcia), Spain.E-mail: [email protected]

Instituto Murciano de Investigacion y Desarrollo Agrario y Alimentario (IMIDA),c/Mayor s/n, E-30150 La Alberca (Murcia), Spain

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Figure 1. Daily net radiation (%) and average temperature (◦C) during 2005 (full lines) and 2006 (dotted lines) seasons.

EXPERIMENTALStudy siteThe study was carried out during two consecutive growing seasons(2005 and 2006) on a 460 ha site located in southeast Spain (37◦

51′ N, 0◦ 48′ E, 50 m above sea level) with a Mediterranean climate.In this area the soil is classified as calcic xerosol. Daily atmosphericaverage temperature and net radiation are presented in Fig. 1.These data were acquired from the Agricultural InformationSystem of Murcia of the Institute of Agricultural and Food Researchand Development of Murcia (SIAM-IMIDA). Pepper (Capsicumannuum L. cv. Quito) fruits were supplied by 24 commercialgreenhouses (eight per treatment) following organic (O), low-input (LI) or soilless (SL) farming systems. Greenhouses wereselected according to specific rules to control factors other thanthe production system that could influence fruit quality. Thusneighbouring greenhouses were selected, pepper seedlings weresupplied by the same nursery 45–60 days after sowing, andplanting was carried out at the beginning of December. Theorganic greenhouses were registered in the Organic AgricultureCouncil of Murcia Region and the low-input greenhouses inthe Integrated Production Register and/or subjected to privateprotocols (EUREP-GAP, UNE 155.000).

Crop managementThe low-input and soilless greenhouses were fertilised withsynthetic fertilisers including KNO3, NH4NO3, Ca(NO3)2, Mg(NO3)2,NH4H2PO4 and microelements (Fe, Zn and Mn). Additionally, thelow-input systems received HNO3 and H3PO4 and the specificapplication of organic manure (sheep manure) to the soil. Inboth production systems, pests were managed according toan integrated production protocol for pepper crop14 by theapplication of synthetic pesticides, sulfur and natural enemies.Soil preparation in organic greenhouses involved biofumigation

and solarisation during the summer months before transplantingwith pepper plants from prior seasons used as cover crop. Inthese organic greenhouses, radish (Raphanus sativus), zucchini(Cucurbita pepo) and/or cucumber (Cucumis sativus) were used forcrop rotation. Additional fertilisation was performed with solublefertilisers of organic origin, including organic manure extracts,amino acids from seaweed and microelements. Pest managementincluded the use of insect traps, sulfur and natural enemies.Finally, all greenhouses were similarly irrigated by drip irrigation.Total fruit yield from each plot was determined by farmers usingcommercial-scale harvesting equipment. The values for both yearsranged from 6.2 to 9.1 kg m−2, from 7.3 to 10.0 kg m−2 and from9.5 to 17.3 kg m−2 for organic, low-input and soilless cultivationrespectively.

Harvesting and sample preparationThe experimental design included a total of 24 greenhouses(eight greenhouses per treatment), each of them divided intotwo subplots. Fruits were harvested at two stages of ripening:totally developed green fruits (without brown or red parts) andfully mature red fruits (without green or brown parts). Thesewere collected from ten randomly selected plants per subplot(resulting in 16 replicates per treatment, with ten fruits perreplicate) at three harvesting times (HT1, HT2 and HT3) throughoutthe 2005 and 2006 growing seasons (in February, May and July).The peppers were washed with deionised water and the seedswere removed. Fruits belonging to the same subplot were cutinto small pieces and mixed to constitute a sample. The peppersamples were homogenised with liquid N2 in a vibrating Micro-Mill‘Pulverisette 0’ (Fritsch, Idar-Oberstein, Germany). Aliquots of 5 gwere homogenised with 5 mL of water in a Polytron PT-MR 3100(Kinematica AG, Littau, Switzerland). Ethyl acetate (10 mL) wasadded and the mixture was homogenised again. The homogenate

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Effect of cultivation system on pepper antioxidant composition www.soci.org

was centrifuged at 8000 × g for 10 min at 4 ◦C. The top phase wastransferred to a test tube and the pellet was resuspended in 5 mLof ethyl acetate, centrifuged and the two resulting phases wereseparated. This step was repeated twice. The organic fractionswere combined and used for the determination of chlorophylls,total carotenoids and antioxidant activity in the lipophilic fraction.The aqueous phase was used for the determination of solublesugars, organic acids, total phenolic compounds and antioxidantactivity in the hydrophilic fraction.

Soluble sugarsSugars in the aqueous phase were analysed using a Hewlett-Packard Model 1100 high-performance liquid chromatography(HPLC) system (Waldbronn, Germany) equipped with a quaternarypump and a refractive index detector. Separations were performedon a 300 mm × 7.8 mm i.d. CARBOSep CHO-682 LEAD column(Transgenomic, San Jose, CA, USA) with ultrapure water as mobilephase at a flow rate of 0.4 mL min−1. Standard solutions of glucose,fructose and sucrose (Sigma, Steinheim, Germany) were injectedat concentrations of 1–10 g L−1 to obtain the linearity of thedetector response and the detection limits of the studied sugars.

Organic acidsSamples were analysed by HPLC with a UV–visible photodiodearray detector. Separations were performed on a 250 mm × 3 mmi.d., 3 µm Prontosil 120-3-C18 column (Bischoff Chromatography,Leonberg, Germany) with 50 mmol L−1 phosphoric acid as mobilephase at a flow rate of 0.5 mL min−1. Readings were taken at195 nm. Standard solutions of ascorbic acid, malic acid and citricacid (Sigma) were injected at concentrations of 0.05–1 g L−1.

Total phenolic compoundsThe total phenolic content was determined using the Sin-gleton and Rossi15 colorimetric procedure with phospho-tungstic–phosphomolybdic acid reagent. The phenolic concen-tration was calculated by recording the optical density of eachsample at 660 nm in a Shimadzu UV-2401PC spectrophotometer(Kyoto, Japan) and comparing it with a standard curve (50–200 mgL−1) of gallic acid (Fluka, Steinheim, Germany).

Chlorophylls and total carotenoidsChlorophylls were determined in the organic phase at two differentwavelengths, 663 and 645 nm. The chlorophyll concentration wasmeasured by the method of Arnon,16 with equations correctedaccording to Nagata and Yamashita.17 Total carotenoids weredetermined in the organic phase by measuring the optical densityat 450 nm in a spectrophotometer. Quantification was performedby comparison with a standard curve (1–5 mg L−1) of β-carotene(Fluka).

Hydrophilic and lipophilic antioxidant activitiesHydrophilic (HAA) and lipophilic (LAA) antioxidant activitieswere determined using the 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) decoloration method based on the abilityof antioxidant molecules to reduce ABTS•+ to ABTS. Bothactivities were extracted and measured according to Cano et al.18

The ABTS•+ radical was generated daily from ABTS (Fluka) byhorseradish peroxidase (Sigma) in 5 mmol L−1 sodium phosphatebuffer (pH 7.4) and ethanol/phosphoric acid (7 g L−1) for HAAand LAA respectively. A dose–response curve was derived for

ascorbic acid (Sigma) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (Sigma) by plotting the absorbance at770 nm as a percentage of the absorbance of the uninhibitedradical cation solution (blank). The absorbance was recordedwith a spectrophotometer. All samples were analysed in triplicateand antioxidant activity was expressed as ascorbic equivalentantioxidant capacity (AEAC index) for HAA and Trolox equivalentantioxidant capacity (TEAC index) for LAA.

Statistical analysisData were subjected to analysis of variance (ANOVA) with maineffects (cropping system, harvesting time and year) and theirtwo- and three-way interactions using the SPSS 15.0 softwarepackage (Chicago, IL, USA). Tukey’s multiple range test was usedto determine differences between means, while Pearson’s test wasapplied to detect significant bivariate correlations.

RESULTSClimatic dataData collected during the two growing seasons by SIAM-IMIDApointed to differences between 2005 and 2006 as regards dailynet radiation and average temperature (Fig. 1). In general, netradiation was higher in 2005 than in 2006. As far as temperaturewas concerned, lower values were registered in 2005 than in 2006during the period preceding the first harvesting (60–120 daysafter transplanting), but thereafter the average temperature wasgenerally higher in 2005 than in 2006.

Water contentThe cropping system (CS) had no effect on the water content ofgreen or red peppers (Tables 1 and 2). For both ripening stages,peppers from the first and second harvesting times (HT) showedlower water content than fruits of the third harvesting (Table 2).The year (Y) significantly affected this parameter in green fruitsbut not in red fruits (Table 1). In addition, significant CS × HT andHT × Y interactions were observed in both years as well as CS × Yand CS × HT × Y interactions in 2006.

Soluble sugar contentIn green peppers, glucose and fructose concentrations dependedon the cropping system (Table 1). The LI system produced thelowest concentrations in fruits, whereas no differences werefound between the O and SL systems (Table 2). The harvestingtime had a significant (P < 0.001) effect on the soluble sugarcontents (Table 1). Glucose and fructose concentrations had fallenby the end of the growing period, while sucrose increased inconcentration as the season progressed (Table 2). In addition,sugar concentrations were significantly (P < 0.001) higher in 2006than in 2005. Finally, the effect of cropping system was significantlydependent on harvesting time (significant CS × HT interaction)for all soluble sugars and also depended on the year (CS × Y) inthe case of sucrose.

In red peppers, glucose and fructose concentrations wereabout 1.8-fold higher than in green fruits, whereas the sucroseconcentration was lower (Table 2). The O system producedsignificantly lower concentrations of the major sugars (glucose andfructose) compared with the LI and SL systems. Similar to the resultsfound in green peppers, glucose and fructose concentrations inred peppers also decreased as the growing period progressed. Nosignificant differences between the results for 2005 and 2006 weredetected (Table 1).

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Table 1. Significance values (P) according to ANOVA test for effects of cropping system (CS), harvesting time (HT) and year (Y) on fruit qualityparameters: water content (Water) glucose (Gluc), fructose (Fruct), sucrose (Sucr), malic acid (Malic), ascorbic acid (Ascorb), citric acid (Citric), totalphenolic compounds (Phenol), chlorophylls (Chll), total carotenoids (β-Carot), antioxidant activity in hydrophilic fraction (AAH) and antioxidantactivity in lipophilic fraction (LAA)

Water Gluc Fruct Sucr Malic Ascorb Citric Phenol β-Carot Chll AAH LAA

Green peppers

CS 0.183 0.002 0.003 0.745 0.001 0.384 0.000 0.390 0.879 0.822 0.018 0.052

HT 0.006 0.000 0.000 0.000 0.000 0.000 0.001 0.000 0.000 0.000 0.000 0.661

Y 0.000 0.000 0.000 0.000 0.000 0.421 0.022 0.108 0.000 0.000 0.418 0.000

CS × HT 0.002 0.007 0.020 0.020 0.000 0.329 0.191 0.047 0.015 0.023 0.010 0.030

CS × Y 0.089 0.325 0.266 0.021 0.010 0.046 0.891 0.000 0.166 0.037 0.239 0.000

HT × Y 0.000 0.000 0.000 0.014 0.000 0.036 0.133 0.000 0.001 0.000 0.000 0.000

CS × HT × Y 0.618 0.012 0.027 0.025 0.000 0.609 0.024 0.119 0.040 0.002 0.056 0.194

Red peppers

CS 0.111 0.000 0.000 0.001 – 0.000 0.000 0.000 0.469 0.629 0.000 0.583

HT 0.000 0.012 0.001 0.673 – 0.000 0.000 0.000 0.000 0.000 0.837 0.000

Y 0.244 0.081 0.456 0.077 – 0.373 0.000 0.000 0.049 0.000 0.000 0.162

CS × HT 0.000 0.072 0.318 0.365 – 0.002 0.001 0.018 0.001 0.042 0.001 0.000

CS × Y 0.004 0.055 0.022 0.175 – 0.001 0.016 0.001 0.584 0.641 0.000 0.000

HT × Y 0.006 0.063 0.764 0.201 – 0.395 0.162 0.000 0.067 0.000 0.000 0.000

CS × HT × Y 0.000 0.113 0.026 0.072 – 0.012 0.073 0.049 0.129 0.042 0.005 0.000

Organic acidsThe organic acids detected in green peppers were ascorbic(vitamin C), malic and citric acids (Table 3). The cropping systemhad a significant effect on malic and citric acid concentrations,whereas no differences in ascorbic acid concentration weredetected among agricultural systems (Table 1). For malic acid theeffect of cropping system depended on the harvesting time andyear, and HT × Y and CS × HT × Y interactions were also detected.In general, organic acid concentrations decreased significantlyduring the growing period, while the values for 2005 were lowerthan those for 2006, except in the case of ascorbic acid, for whichno differences were detected (Table 3).

In red peppers, malic acid was not detected, while the citric acidconcentration was higher (about 4.0-fold) than in green fruits. Thecropping system had a significant effect on both ascorbic and citricacid concentrations (Tables 1 and 3), with SL peppers showing thehighest ascorbic acid concentration and, similar to the results forgreen fruits, red peppers from the LI system showing the highestvalue of citric acid. The effect of cropping system on both organicacids depended on the harvesting time and year (Table 1). Similarto green peppers, red fruits showed decreasing ascorbic and citricacid concentrations from HT1 to HT3 (Table 3), while citric acidconcentrations were lower in 2005 than in 2006.

Total phenolic compoundsThe effect of cropping system on total phenolic compounds wassignificant only in red peppers (Table 1), with SL fruits showingthe highest value (Table 3). At both green and red maturity stagesthe phenolic concentration decreased from HT1 and HT2 to HT3and, unlike the other compounds, values for 2005 were higher(about 1.4-fold) than those for 2006. In addition, ANOVA detectedsignificant CS × HT, CS × Y and HT × Y interactions (Table 1).

Chlorophylls and total carotenoidsNo significant effect of cropping system on chlorophylls orcarotenoids was detected in green or red peppers. However, these

parameters were significantly affected by both the harvestingtime and year (Table 1). In general, chlorophyll and β-caroteneconcentrations decreased as the growing period progressed,except in red peppers where chlorophylls increased from HT1to HT3 (Table 3). In green peppers, chlorophyll values were higherin 2005 than in 2006, while in red peppers the opposite wasthe case. These results were inversely correlated with those ofcarotenoid content.

HAA and LAAIn green and red peppers, HAA was 100–250-fold higher than LAA(Table 4). The O system showed the lowest HAA at both maturitystages. The effect of harvesting time was significant only in greenpeppers, which showed a decrease in HAA at HT2 (Tables 1 and 4).A significant effect of year was observed in red peppers, withhigher values in 2005 than in 2006.

The cropping system had no effect on LAA in green or redpeppers. Only an effect of year and harvesting time on greenand red fruits respectively was observed. Multiple significantinteractions between the main factors (CS, HT and Y) were detectedby ANOVA (Table 1).

CorrelationsSignificant correlations (P < 0.01) according to Pearson’s testwere detected between HAA and water-soluble compounds(organic acids and phenolic compounds) (Table 5). In greenpeppers, ascorbic acid showed the highest correlation, followed byphenolics. However, the correlation between HAA and phenolics inred fruits was higher than that observed for ascorbic acid. Despitethe citric acid concentration being much lower in green than inred peppers (Table 3), the only correlation between citric acid andHAA was observed at the first maturity stage. Finally, LAA valueswere significantly correlated with those of chlorophylls (in greenand red peppers) and total carotenoids (in red peppers only).

In addition, several significant correlations were observedbetween pigment content (chlorophylls and β-carotene) and an-


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Table 2. Main effects of cropping system, harvesting time and yearon water content (%), glucose, fructose and sucrose (mg g−1 FW) ofgreen and red peppers

Water content Glucose Fructose Sucrose

Green peppers

Cropping system

O 94.4 ± 0.2 15.0 ± 0.5b 16.1 ± 0.6b 4.9 ± 0.5

LI 94.7 ± 0.2 13.6 ± 0.4a 14.2 ± 0.5a 4.8 ± 0.3

SL 95.0 ± 0.2 14.8 ± 0.4b 15.6 ± 0.5b 4.7 ± 0.3

Harvesting time

HT1 94.4 ± 0.1a 15.1 ± 0.5b 16.4 ± 0.6b 3.9 ± 0.3a

HT2 94.6 ± 0.2a 15.7 ± 0.4b 16.6 ± 0.6b 4.8 ± 0.3ab

HT3 95.1 ± 0.2b 12.0 ± 0.2a 12.4 ± 0.2a 5.7 ± 0.4b

Year

2005 93.5 ± 0.1 12.2 ± 0.2 11.9 ± 0.2 3.6 ± 0.2

2006 95.7 ± 0.1 16.2 ± 0.4 18.0 ± 0.4 5.8 ± 0.3

Red peppers

Cropping system

O 92.7 ± 0.3 24.0 ± 0.6a 26.3 ± 1.0a 1.9 ± 0.1a

LI 92.2 ± 0.2 27.5 ± 0.3b 31.7 ± 0.4b 1.7 ± 0.2a

SL 92.4 ± 0.3 26.7 ± 0.3b 30.6 ± 0.4b 2.4 ± 0.1b

Harvesting time

HT1 91.8 ± 0.2a 27.4 ± 0.6b 31.6 ± 0.6b 2.1 ± 0.2

HT2 92.1 ± 0.3a 25.7 ± 0.3a 29.2 ± 0.5a 2.1 ± 0.1

HT3 93.1 ± 0.2b 26.1 ± 0.3a 29.4 ± 0.5a 1.9 ± 0.2

Year

2005 92.0 ± 0.3 26.1 ± 0.4 30.7 ± 0.6 1.8 ± 0.1

2006 92.6 ± 0.2 26.7 ± 0.3 29.5 ± 0.4 2.2 ± 0.1

Values are mean ± standard error. For each main factor (croppingsystem and harvesting time), different letters in a column indicatesignificant differences according to Tukey’s test.

tioxidant constituents (Table 6). In green peppers the chlorophyllconcentration was negatively correlated with malic acid and sol-uble sugar (glucose, fructose and sucrose) concentrations andpositively correlated with ascorbic acid. In red fruits, only the phe-nolic concentration showed a significant and negative correlationwith chlorophyll content. In contrast, β-carotene was positivelycorrelated with most of the other antioxidant constituents in bothgreen and red peppers, apart from sucrose in red fruits, whichshowed a negative and significant correlation with this pigmentcontent.

DISCUSSIONWhen the effect of cropping system on sugar concentrationswas analysed, the greatest differences found between treatmentsrepresented a maximum increase of around 13% (comparing LIwith O red peppers), whereas differences among harvesting timesor between years reached around 25%. In addition, the effect ofcropping system was different in green and red fruits. Moreover,this effect was in most cases highly dependent on harvestingtime and/or year. The most important factor determining peppersugar concentrations was maturity stage (green or red). Higherglucose and fructose concentrations in red than in green pepperscan be attributed both to hexose accumulation during maturation

through the enzyme acid invertase and to sucrose breakdownduring the last ripening phase through sucrose synthase.19

Among the organic acids detected in pepper fruits, ascorbic acidis of particular relevance, since it is involved in important physio-logical processes such as collagen and hormone biosynthesis andbecause of its protective role in inflammatory processes through itsantioxidant properties.10,20 Several authors have reported highercontents of vitamin C in organically grown vegetables comparedwith conventional crops.2 However, in pepper, differences be-tween cropping systems were evident only in red fruits, where SLpeppers showed higher values than peppers cultivated in soil (Oand LI) systems, while no differences were detected between Oand LI peppers. In the same way the phenolic content differed onlyin red peppers, SL again producing the highest values. As far asβ-carotene was concerned, no differences between cropping sys-tems were detected in green or red fruits. The increases in overallnutrient concentration between conventional and organic cropsobserved by other authors have been attributed to differences inwater content.21 According to other authors, the higher nutrientcontent in organically grown vegetables is mainly attributable tolower nitrogen (N) availability.13 In pepper we found no differencesin fruit water content among cropping systems. In addition, ourfindings showed no differences in plant N content between O andLI crops (personal communication). Such differences in the resultsmay be attributed to several causes. For example, in previousstudies, conventional systems using high fertilisation rates wereusually compared with organic systems in which deficiencies in Nwere the principal problem for plant growth,22 probably because,while organic manures usually contain the necessary amount of Nfor plant growth, only part of this nutrient is immediately availableto plants.23 In our study, soil cropping systems including syntheticfertilisation (conventional) were subjected to low-input protocolsaccording to the present-day environment-friendly trend. In addi-tion, organic crops from previous comparative cultivation studiesusually only included applications of organic manures. However,in commercial organic greenhouses, such as those selected in thepresent study, plants are also fertilised with several N sources oforganic origin (water-soluble extract from organic manure, aminoacids from seaweed, etc.) and organic crops are not under limitingN conditions, as reflected by the results for plant N content (datanot shown). On the other hand, previous studies on pepper haveshown that increasing N concentration in the growth mediumdoes not lead to modifications in the ascorbic acid and pheno-lic concentrations or the water content of fruits.5 Therefore anyincrease in N availability in LI relative to O systems would notnecessarily have affected the pepper nutrient content.

When comparing soil (O and LI) with SL cultivation, we foundhigher values of phenolics and ascorbic acid in soilless-grownfruits. Little is known about the comparative effects of soiland soilless systems on fruit quality. Increases in mineral andcarbohydrate fruit concentrations in soilless over soil culture havebeen reported.24 The same authors attributed the higher fruitquality and yield observed in closed soilless systems to betternutrition control and improved water use efficiency compared withconventional culture. In tomato, only slight differences in mineral,sugar and ascorbic acid levels were observed between the twoproduction methods.25 In cherry tomato and lettuce, differencesin the evolution of ascorbic acid and mineral concentrationsduring storage were observed between soil- and soilless-grownproducts.26

According to Chassy et al.,27 pepper composition is littleinfluenced by cropping systems and shows a different response


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Table 3. Main effects of cropping system, harvesting time and year on malic acid (Malic), ascorbic acid (Ascorb), citric acid (Citric), total phenoliccompounds (Phenol) (mg·g−1 FW), total chlorophylls (Chll) and total carotenoids (β-Carot) (µg·g−1 FW) of green and red peppers

Malic Ascorb Citric Phenol Chll β-Carot

Green peppers

Cropping system

O 1.39 ± 0.08b 1.50 ± 0.08 0.84 ± 0.05a 0.46 ± 0.02 65.6 ± 2.2 29.1 ± 1.1

LI 1.23 ± 0.04a 1.58 ± 0.04 1.14 ± 0.05b 0.46 ± 0.01 67.3 ± 1.6 28.0 ± 0.7

SL 1.13 ± 0.05a 1.60 ± 0.05 0.96 ± 0.04a 0.47 ± 0.01 67.2 ± 1.7 27.8 ± 0.7

Harvesting time

HT1 1.29 ± 0.06b 1.80 ± 0.05b 1.13 ± 0.06b 0.48 ± 0.01b 80.5 ± 1.2b 31.6 ± 0.7b

HT2 1.16 ± 0.04a 1.45 ± 0.05a 0.96 ± 0.05a 0.51 ± 0.01b 57.9 ± 1.8a 26.4 ± 0.6a

HT3 1.22 ± 0.05ab 1.45 ± 0.04a 0.92 ± 0.03a 0.40 ± 0.01a 62.5 ± 1.2a 26.5 ± 0.8a

Year

2005 1.03 ± 0.03 1.54 ± 0.05 0.95 ± 0.04 0.48 ± 0.01 75.4 ± 1.0 25.6 ± 0.6

2006 1.39 ± 0.04 1.59 ± 0.03 1.05 ± 0.04 0.45 ± 0.01 59.5 ± 1.4 30.4 ± 0.6

Red peppers

Cropping system

O ND 1.89 ± 0.05a 4.24 ± 0.12a 0.62 ± 0.02a 4.3 ± 0.6 108.3 ± 5.7

LI ND 1.95 ± 0.03a 4.56 ± 0.09b 0.64 ± 0.02a 4.0 ± 1.0 113.4 ± 4.1

SL ND 2.13 ± 0.02b 4.13 ± 0.07a 0.71 ± 0.02b 4.6 ± 1.0 112.9 ± 3.3

Harvesting time

HT1 ND 2.17 ± 0.04c 4.59 ± 0.10b 0.79 ± 0.01c 1.2 ± 0.0a 126.2 ± 4.6c

HT2 ND 1.98 ± 0.03b 4.09 ± 0.08a 0.67 ± 0.01b 3.5 ± 0.4a 112.7 ± 3.8b

HT3 ND 1.86 ± 0.03a 4.31 ± 0.09a 0.51 ± 0.02a 8.6 ± 1.7b 96.0 ± 3.2a

Year

2005 ND 2.06 ± 0.03 4.06 ± 0.07 0.77 ± 0.01 1.2 ± 0.0 117.1 ± 3.6

2006 ND 1.97 ± 0.03 4.54 ± 0.07 0.57 ± 0.02 6.9 ± 1.0 107.9 ± 3.2

Values are mean ± standard error. For each main factor (cropping system and harvesting time), different letters in a column indicate significantdifferences according to Tukey’s test. ND, not detected.

from other crops to nutrient bioavailability. These authors alsodemonstrated the importance of comparing the effects ofcropping systems in different years owing to annual variations. Inthe present study a general seasonal decrease was observed infruit metabolite concentrations that can be attributed both to thenatural increase in production during the growing period (leadingto metabolite dilution in fruits) and to the natural decrease inplant energy and nutrient reserve in the last phenological stages.Also, differences between years were observed for most of themetabolites studied, presumably due to year-to-year changes inenvironmental conditions (light, temperature, etc.).

Antioxidant activity in vegetables constitutes a measurementof phytochemical contents with antioxidant properties, which arebelieved to mitigate the effects of oxidative stress in diseasedevelopment and the aging process.28 In fruits the most abundantantioxidants are vitamin C, carotenoids and phenolic compounds.In particular in peppers, HAA was significantly correlated with bothascorbic acid and phenolic contents, all of which increased from thegreen to the red stage. Vitamin C and phenolic antioxidant activityis based on electron donation and metal ion chelation in the caseof some phenolic compounds. In particular, phenolics have beenshown in several fruits to be correlated with antioxidant capacityto a greater extent than vitamin C.29 As far as the effect of croppingsystem on antioxidant activity was concerned, organically grownpeppers showed the lowest HAA values at both green and redmaturity stages. However, this parameter seemed to be moreaffected by the harvesting time. In addition, an interaction between

cropping system and other factors was observed depending onthe maturity stage. As far as LAA was concerned, it represented avery low proportion of total pepper antioxidant activity. In greenpeppers it was significantly associated with changes in chlorophyllsand also in carotenoids. Dietary chlorophylls and their derivativesare known to possess antioxidant activity through their ability toreduce free radicals.30 In red peppers the observed increase incarotenoids and decrease in chlorophylls compared with greenpeppers led to higher LAA values. Finally, decreasing chlorophylland increasing β-carotene contents, which are associated with theripening process,31 were correlated with increasing antioxidantlevels and sugar concentrations, except in the case of sucrose,whose concentration decreased, as it is broken down during thelast phases of ripening through sucrose synthase.32

In conclusion, the cropping system had little effect on pepperfruit nutritional composition when the two soil systems (O and LI)were compared, since only some differences in sugar, malic acidand citric acid concentrations were detected. Fruit antioxidantactivity was mainly associated with water-soluble compounds,including organic acids and phenolic compounds. Althoughno significant differences were detected in the metabolitesassociated with fruit antioxidant activity (ascorbic acid, phenolics,chlorophylls and carotenoids), a slight increase in most hydrophiliccompounds in LI relative to O fruits was observed, leading toan increase in HAA. When soil- and soilless-grown fruits werecompared, higher sugar, phenolic, ascorbic acid and HAA levelswere observed in SL fruits. Despite these differences due to


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Table 4. Main effects of cropping system, harvesting time and yearon antioxidant activity in hydrophilic (HAA, expressed as AEAC) andlipophilic (LAA, expressed as TEAC) fractions of green and red peppers

HAA LAA

Green peppers

Cropping system

O 2.23 ± 0.12a 0.010 ± 0.001

LI 2.55 ± 0.09b 0.009 ± 0.001

SL 2.62 ± 0.11b 0.008 ± 0.000

Harvesting time

HT1 2.97 ± 0.11b 0.009 ± 0.001

HT2 2.21 ± 0.08a 0.008 ± 0.001

HT3 2.36 ± 0.11a 0.009 ± 0.000

Year

2005 2.59 ± 0.09 0.011 ± 0.001

2006 2.44 ± 0.09 0.007 ± 0.000

Red peppers

Cropping system

O 4.69 ± 0.17a 0.058 ± 0.003

LI 5.27 ± 0.20b 0.061 ± 0.002

SL 6.19 ± 0.23c 0.061 ± 0.002

Harvesting time

HT1 5.49 ± 0.25 0.057 ± 0.002a

HT2 5.56 ± 0.24 0.058 ± 0.002a

HT3 5.44 ± 0.17 0.067 ± 0.002b

Year

2005 7.31 ± 0.15 0.061 ± 0.002

2006 4.01 ± 0.06 0.060 ± 0.001

Values are mean ± standard error. For each main factor (croppingsystem and harvesting time), different letters in a column indicatesignificant differences according to Tukey’s test.

Table 5. Significance values (P) according to Pearson’s test for correla-tions between antioxidant activity (AAH and LAA) and concentration ofantioxidant constituent, including total phenolic compounds (Phenol),malic acid (Malic), ascorbic acid (Ascorb), citric acid (Citric), chlorophylls(Chll) and total carotenoids (β-Carot)

Correlation Green peppers Red peppers

HAA–Phenol 0.467∗∗ 0.571∗∗

HAA–Malic −0.322∗∗ –

HAA–Ascorb 0.660∗∗ 0.367∗∗

HAA–Citric 0.436∗∗ −0.148

LAA–Chll 0.480∗∗ 0.235∗∗

LAA–β-Carot 0.036 0.500∗∗

Chll–β-Carot 0.414∗∗ 0.044

∗∗ Correlation significant at 0.01 level (two-tailed).

the effect of cropping system, the overall differences were fargreater among harvesting times or between years. In addition,multiple interactions (CS × HT, CS × Y and CS × HT × Y)were detected, probably owing to the fact that plant age andenvironmental conditions differently affected plants grown underdifferent cropping systems, depending on growth water and thenutrient status of the plants. These results highlight the importance

Table 6. Significance values (P) according to Pearson’s test forcorrelations between pigment content (chlorophylls (Chll) and totalcarotenoids (β-Carot)) and total phenolic compounds (Phenol), malicacid (Malic), ascorbic acid (Ascorb), citric acid (Citric), glucose (Gluc),fructose (Fruct) and sucrose (Sucr)

Green peppers Red peppers

β-Carot Chll β-Carot Chll

Phenol 0.066 0.094 0.417∗∗ −0.419∗∗

Malic 0.127∗ −0.207∗∗ – –

Ascorb 0.222∗∗ 0.269∗∗ 0.285∗∗ −0.091

Citric 0.272∗∗ 0.122 0.272∗∗ 0.126

Gluc 0.130∗ −0.200∗∗ 0.275∗∗ −0.056

Fruct 0.175∗∗ −0.261∗∗ 0.308∗∗ −0.105

Sucr 0.203∗∗ −0.236∗∗ −0.305∗∗ −0.085

∗,∗∗ Correlation significant at 0.05 or 0.01 level respectively (two-tailed).

of comparing different harvesting times and years in order to studythe effect of cropping system on a specific crop.

ACKNOWLEDGEMENTSThe authors thank Mar Davo, Marcos Ruiz and Cristobal Marınfor technical assistance and the Ministerio de Educacion y Ciencia(research project RTA2005-00224-02-00) and the European SocialFund for financial support.

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Pepper antioxidant composition as affected by organic, low-input and soilless cultivation