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Characterization of phenolic content, in vitro biological activity, and pesticide

loads of extracts from white grape skins from organic and conventional cultivars

Margarita Corrales a,⇑, Avelina Fernandez b, Maria G. Vizoso Pinto a, Peter Butz a, Charles M.A.P. Franz a,Eberhard Schuele c, Bernhard Tauscher a

a Max Rubner-Institute, Department of Safety and Quality of Fruit and Vegetables, Haid-und-Neustr. 9, 76131 Karlsruhe, Germanyb Instituto de Agroquímica y Tecnología de Alimentos, CSIC, Avda. Agustín Escardino, 7, 46980 Paterna, Spainc Chemisches und Veterinäruntersuchungsamt Stuttgart, Schaflandstraße 3/2, 70736 Fellbach, Stuttgart, Germany

a r t i c l e i n f o

 Article history:

Received 19 May 2010Accepted 20 September 2010

Keywords:

Ames testAntibacterial effectFlavonoidsOrganic cultivarsWhite grape skin extractsPesticides

a b s t r a c t

Grape skin extracts of Riesling  Vitis vinifera L. grapes from conventionally or organically managed culti-vars were compared on the basis of their phenolic content, antioxidant capacity, antimicrobial and anti-mutagenic properties and pesticide loads. Promising results on their biological properties suggest thatthose extracts would be valuable as food preservatives. The antioxidant capacity of conventional extractswas significantly higher, according to the higher content in catechin, epicatechin and procyanidin B. Pes-ticide loads did not affect the antimutagenic or antimicrobial properties of the extracts. Both extractsinhibited the growth of Gram-positive foodborne pathogens such as  Staphylococcus aureus,  Enterococcus

 faecalis and  Enterococcus faecium  to similar extents. Possibly as a result of higher amounts of quercetinand its derivatives, higher antimicrobial effects against Listeria monocytogenes and Salmonella typhimuri-

um  were observed for the organic white grape skin extracts. Conventional or organic extracts did notshow remarkable antimutagenic effects when tested against the mutagen IQ by means of the Ames test.Due to the presence of fungicides, the conidial germination of  Penicillium expansum,  Penicillium chrysog-

enum  and   Aspergillus niger,  were inhibited by 95% by conventional GSE, while negligible effects were

observed with organic grape extracts. The latter, however, showed inhibitory effects against  Trichoderma

viridie and Aspergillus versicolor . 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Natural products obtained from fruit and vegetables are gainingmore and more relevance in the food industry, following increasingconsumer awareness of diet-related health problems and con-sumer mistrust regarding synthetic additives, (Pokorny, 1991;Smid and Gorris, 1999; Gram et al., 2002). Fruit skins and seedsare rich in bioactive substances such as phenolic acids, flavonoidsand vitamins (Harbone, 1994; Bravo, 1998; Alberto et al., 2002).

Grape by-products represent approx. 20% of the total weight of the processed grapes. After grape harvesting, the estimated grapepomace is approximately 9 million tons per year (Meyer et al.,1997; Schieber et al., 2002), and has an increasing economical sig-nificance. Red grape pomace and seeds have especially been ofteninvestigated as source of anthocyanins and flavonoids with impor-

tant antimicrobial, antioxidant and health-promoting properties.Those by-products have been suggested as an attractive targetfor production of food additives or nutraceuticals (Lu and Foo,1999; Jayaprakasha et al., 2003; Yilmaz and Toledo, 2004; Baydaret al., 2004, 2006; Ozkan et al., 2004; Kammerer et al., 2004). Thecharacterization of white grape pomace components and a moreexhaustive knowledge of their biological properties will also helpin the valorisation of white grape by-products, thus providingnew strategies to diminish the ecological impact of polyphenol rich

wastes (Sanchez et al., 2009).As consumer’s choice towards organic foods increases, a better

understanding of the influence of agricultural practices on the bio-logical properties of food will also assist in the marketing strategiesand decision making. White grapes of the Riesling variety whichoriginated in the German Rhine region are also extensively pro-duced in the bordering Palatinate regionof Germany. They producehigh quality sweet or semi-sweet white wine and the pomace istypically employed by local producers to obtain mainly spirits.

Riesling is the predominantly grown grape variety in Germany,being also common in the neighbouring French region Alsace, andin other countries around the world. Nowadays Riesling grapes arealso cultivated under organic farming conditions. Available

0278-6915/$ - see front matter    2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.fct.2010.09.025

⇑ Corresponding author. Present address: Food Safety and Quality Unit, JointResearch Centre, Institute for Reference Materials and Measurements, EuropeanCommission, Retieseweg 111, B-2440 Geel, Belgium. Tel.:+32 014 571 853; fax: +32014 571 787.

E-mail address:  margarita.corrales@ec.europa.eu (M. Corrales).

Food and Chemical Toxicology 48 (2010) 3471–3476

Contents lists available at   ScienceDirect

Food and Chemical Toxicology

j o u r n a l h o m e p a g e :  w w w . e l s e v i e r . c o m / l o c a t e / f o o d c h e m t o x

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experimental data suggest that cultivar practices may influence thecontent of flavonoids and other secondary metabolites in fruits.However, data on this are still contradictory and further researchis needed. For example, Malusa et al. (2004) reported higher poly-phenol content in organic grapes compared to conventional ones,whereas Vian et al. (2006) found a higher content of anthocyaninsin conventionally grown grapes when compared to the organicallygrown ones.

White grape pomace has been insufficiently studied whencompared to the red varieties and grape seeds, probably due totheir lower contents of anthocyanins and proanthocyanidins.However, white grape pomace is also a rich source of flavonoidsand stilbenes with important health-promoting attributes (Birtet al., 2001; Mennen et al., 2004; Bertelli and Dipak, 2009). Addi-tionally, white grape pomace does not possess the red/purple col-our characteristic of red grape varieties’ extracts and the highastringency of seed extracts. All these characteristics allow theuse of white grape skin extracts as potential additives or nutra-ceuticals for a wider range of products than their red grape skinextract equivalents.

Therefore, this work aimed at assessing relevant chemical dif-ferences between extracts from organically and conventionallygrown grape skin Riesling   Vitis vinifera L. variety. Flavonoid con-tent was analysed by means of HPLC, total phenolics by the Fo-lin–Ciocalteu method and the antioxidant capacity as Troloxequivalents. Relevant biological properties such as the antimuta-genic capacity and the antimicrobial properties against foodborne pathogen bacteria and moulds were compared on the ba-sis of the pesticide content of the extracts. Furthermore a thor-ough characterization of the organic samples provides data onthe marketability of these extracts as food additives. Differencesbetween organic and conventionally grown samples can alsoprovide insight into the effects of the use of pesticides, whichmay mask natural biological properties of the white grapepomace.

2. Materials and methods

Analytical grade reagents, solvents and chemicals were obtained from Sigma–Aldrich (Taufkirchen, Germany) and Merck (Darmstadt, Germany). Standards usedfor identification and quantification purposes with HPLC were as follows: catechin,epicatechin, epicatechingallate, procyanidin B (extrasynthese, Lyon, France), quer-cetin, quercetin-3-O-rhamnoside, hyperoside, kaempferol (Sigma, Taufkirchen, Ger-many). More than 500 standards for pesticide analysis were provided by the Dr.Ehrenstorfer GmbH (Augsburg, Austria) company and by Sigma Aldrich (Taufkir-chen, Germany).

Samples of Riesling   Vitis vinifera L. grapes were collected in 2005 fromtwo adjacent fields cultivated according to the German legislation for con-ventional or organic farming (meaning no plant protection with syntheticallyproduced chemicals; no utilisation of easily soluble mineral fertilisers; inten-sive humus management; crop rotation; no application of synthetic chemicalgrowth regulators). They were situated in the Palatine county in Germany.The selection of neighboring parcels allowed the comparison between organic

and conventional cultivars under similar soil and climate conditions. Grapeswere separated into three different pomace fractions: skins, stems and seeds.For this work, skins were lyophilised and then milled with a coffee grinderprior to extraction.

 2.1. Determination of nitrogen content 

The total nitrogen content in the samples wasestimated by the Kjedahl method(Amtliche Sammlung, 2002). Results were expressed in g nitrogen kg1.

 2.2. Determination of ashes content 

Twenty grams of dry skins were weight into a platinum dish and cooled in adesiccator to room temperature. The dish was heated at 103 C until water wasevaporated (ca. 2 h). Samples were reduced to ashes in a furnace at 550 ± 5 C(5 h).The ashes were moistened with 5 ml water andre-ashedat 550 C to constant

weight. The ashes were cooledin a desiccator to room temperature andweighted tothe nearest 0.1 mg.

 2.3. Determination of potassium content 

Twenty-five grams of dry grape skins were weighted and 150 ml of de-ionizedhot water were added. Samples were vigorously shaken for 15 min at approx. 95 C.Then, 250 mL of water were added and the samples were cooled at 20 C. Sampleswere filtered and the potassium content was measured using a selective ionic elec-trode which was previously calibrated (Dr. W. Ingold AG, Urdorf, Schweiz). Resultswere expressed in g potassium kg1.

 2.4. Sugar content in  Brix

The sugar content in the methanolic extracts was measured in Brix usinga Kru-ess AR4 ABB refractometer (Analysesystemen GmbH, Burlandigen, Germany).

 2.5. Extraction of the methanolic fraction

Five grams of milled grape skins were extracted with 100 mL of 60% methanolin aliquots of 30 mL. All extractions were assisted by an ultrasound generator (Ban-delin, Sonorex RK 100H, Walldorf, Germany) for 9 min. After each extraction, sam-ples were centrifuged at 9000 rpm for 10 min, the supernatants were collected,filtered (0.45 lm) (PTFE filter, Whatman, US) and lyophilised. The solid extractswere weighed and dissolved in distilled water to a concentration of 5%, 10% and20% (w/v) for the antimicrobial assays.

 2.6. Solid-phase extraction

For flavonoid extraction and identification, filtered methanolic supernatants(described above) were evaporated and dissolved in 10 mL acidified water(pH 1.5). The solution was re-extracted with 100 mL ethyl acetate. Supernatantswere collected, evaporated and dissolved in 5 mL water (pH 7). Extracts were thenapplied to Chromabond C18 cartridges (Varian, Frankfurt am Main, Germany). Car-tridges were first activated with 2 mL methanol and 1 mL water. Flavonoids wereelutedwith10 mL of ethyl acetate. Extracts were concentratedand diluted in meth-anol for LC-DAD/ESI-MS analysis.

 2.7. Analysis of flavonoids by LC-DAD/ESI-MS 

Supernatants resulting from the solid-phase extraction were dissolved in 2 mL methanol. Solutions were membrane-filtered (0.45 lm) (PTFE filter, Whatman, US)and analysed in an Agilent Technologies LC/MSD Series 1100 (binary solvent deliv-ery, autosampler, UV–Vis Diode Array Detector (DAD), electrospray ionization (ESI);Agilent Technologies, Palo Alto, CA) mass spectrometer. The separation was per-formed with an Aqua Column (80 ÅA

0

250 4.6 mm i.d.; 5 lm), operated at 20 C,using a binary gradient method with two mobile phases: Solution A: 0.5% aceticacid in water (v/v) andsolution B: 0.5% acetic acid, 50% waterand 49.5%acetonitrile(v/v/v). For flavonoid analysis, the gradient was as follows: 10–24%B (20 min), 24–30%B (20 min), 30–55%B (20 min), 60–75%B (15 min). The injection volume was20lL and the flow rate 1.0 mL min1. The detection was monitored at 320 nm(Kammerer et al., 2004).

The mass spectrometer wasfitted with an ESI source in negative mode. The col-umn eluate was recorded in the range  m/z  50–1000. The mass spectrometer wasprogrammed to do an MS2 scan of the most abundant ion in the full mass. Nitrogenwas used both as drying gas at flow rate of 11.0 L min1, and as nebulising gas at apressure of 60psi. The nebuliser temperature was set to 350 C. Peak identificationand quantification took place by MS and by comparison of the retention times withcommercially available standards.

 2.8. Antioxidant capacity

The ABTS radical cation assay described by Miller et al. (1993) and later im-proved by Re et al. (1999) was used for the determination of the polar antioxidantcapacity. A stock solution of 5 mM ABTS (2,20-azino-di 3-ethylbenzothiazoline-6-sulphonic acid) was diluted in water and preincubated for at least 12 h with140 mM (final concentration) of K2S2O8  to produce the radical cation ABTS+. TheABTS+ solution was then diluted in 5 mM saline phosphate buffer pH 7.4 untilabsorbance readings reached a value of 1.5 at 735 nm. An aliquot extract of 100lL, was 100-fold diluted in buffer mixed with 2.9 mL ABTS and set 15 min at30 C, then absorbance was measured at 735 nm. A calibrated curve of TROLOX(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) as standard was usedto calculate the antioxidant activity of the samples, expressed as mmol TROLOXequivalents (TE) g1

extract.

 2.9. Total phenolic content 

Total phenolic content was determined using the Folin–Ciocalteau reagent (Sin-

gleton and Rossi, 1965). An extract aliquot of 125 ll was mixed with 625 ll of Fo-lin–Ciocalteau reagent (previously diluted 10-fold with distilled water at 45 C) and

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set3 min at room temperature. Afterthis,500ll of sodium carbonate (0.6 M;45 C)were added to the mixture and incubated for 15 min at 45 C. The absorbance wasmeasured spectrophotometrically at 750 nm.

A gallic acid hydrate solution (Roth, Karlsruhe, Germany) was used as standardfor the calibrationcurveand theresultswereexpressed aslmol of gallicacid equiv-alents (GAE) g1

extract.

 2.10. Antimutagenic activity

The antimutagenic activity was tested according to  Edenharder et al. (1994),following a modification of the method proposed by  Maron and Ames (1983). Amixture containing 500 ll of isotonic KCl, 12 lL of a solution of the mutagen IQ (Toronto research Chemicals Inc., Downsview, Ontario, Canada) in DMSO (resultingin a average of 700 His + revertants per plate), different amounts (from 150 to500ll) of the extract resuspended in sterile water and adjusted to pH 7.4 (the vol-ume up to 500 ll was filled with isotonic KCl), 500 ll of the mammalian metabolicactivator S-9 (containing two cofactors), and 100 ll of a suspension of  Salmonella

typhimurium TA98 in the stationary phase, were plated on top agar plates. Rever-tants were counted after cultivation for 24 h at 37 C. Two replicates were analyzedat each dose. A decrease in the number of revertant cells means antimutagenic ef-fects against IQ.

As control, toxicity against S. typhimurium TA98 and S . typhimurium TA102 wasassayed. In this case, colonies were grown in the presence of histidine, without themutagen IQ. Twopomace concentrations were tested on top agar plates witha totalnumber of colonies of approx. 300. Colonies were counted after cultivation for 24 hat 37 C. Two replicates were analyzed at each dose.

 2.11. Bacterial and fungal strains and growth conditions

Listeria monocytogenes  Scott A,   L. monocytogenes   ATCC 19115,   Salmonella ent-

erica   serovar   typhimurium   (S. typhimurium) ATCC 14028,   Staphylococcus aureus

ATCC 25923,  Escherichia coli   ATCC 25922 were cultured in Standard I (Merck)broth at 37 C.   Enterococcus faecium  DSM 13590 and   Enterococcus faecalis   DSM20409 were growth in the Man, Rogosa and Sharpe broth (MRS broth, Merck,Darmstadt, Germany) at 37 C. Stock cultures were maintained at   20 C in15% (v/v) glycerol.

Penicillium chrysogenum DSM 844, Penicillium expansum DSM 62841, Aspergillus

niger,   DSM 1988, Aspergillus versicolor   DSM 63292 and   Trichoderma viridie   DSM63065 were cultured and grown in malt extract agar plates (12 g L 1 malt extract).Two-week-old cultures were used to prepare spore suspensions.

 2.12. Screening for antagonistic activity

The agar spot test as described by   Uhlmann et al. (1992)  was used forscreening the antagonistic activity of the extracts. Standard I or MRS agar(12gL 1) plates were overlayed with Standard I or MRS soft agar (7.5g L 1) pre-viously inoculated with ca. 1 106 CFU mL 1 of overnight cultures of the corre-sponding indicator bacterial strain. Wells were cut from the soft agar layersurface with the back of a sterile Pasteur pipette, and 20lL of each extract con-centration (5%, 10% and 20% (w/v) was inoculated in each well. After diffusion,plates were incubated at 37 C for 24 h. Inhibition zones with no bacterialgrowth were measured in mm.

 2.13. Effect of g rape skin extracts on conidia germination and germ tube length

The effectof grape skin extracts (GSE)on the germinationof conidia was carriedout according to the method described by Droby et al., 1997). The spore concentra-tion was determined with a Neubauer counting chamber (Optik Labor, Hecht, Ger-

many) and adjusted to 5

10

5

spores mL 

1

. Aliquots of 90lL of the sporesuspension were mixed with 10 lL of GSE (10% w/v) and the conidia germinationdetermined after 24h incubation at 30 C.

 2.14. Analysis of pesticide loads

Pesticide residues were measured using the QuEChERS pesticide multiresiduemethod, as described by Anastassiades et al. (2003, 2007) and EU standarisationmethods (2007) in combination with LC-MS/MS and GC-MS/MS. For this purpose,5 g of ground grape skins (raw material) and grape skin extracts were dissolvedin 10 mL distilled water, extracted with 10 mL acetonitrile by vigorous shaking.Thereafter, a salt-mixture containing 4 g MgSO4, 1 g NaCl and 1g Na3citrat 2H20and 0.5 g Na2H citrate sesquidydrate) was added and mixed well. Samples werecentrifuged at 3000 g/min for 5 min. Clean-up was carried out by dispersive so-lid-phase extraction (D-SPE), which involved the mixing of an extract aliquot with25 mg PSA (sorbent) and 15 mg MgSO4 (drying agent). Samples were centrifuged at3500 rpm for 3 min. Supernatants were directly amenable to both liquid chromato-

graphic (LC)- and gas chromatographic (GC)-analysis. LC-MS and GC-MS analysisfollowed different methods according to the CEN norm EN 15662.

 2.15. Statistical analysis

Experiments were carried out in triplicate and results were tested for statisticalsignificance by t -test using the SSPS Statistical program (Version 11.5). Differenceswere considered statistically significant at the  P  < 0.05 level.

3. Results and discussion

Grape composition depends on a series of interactions betweengenetic characteristics, environmental conditions and culturalpractices (Bourn and Prescott, 2002). The composition determinesthe biological properties of the grapes, and has been extensivelyinvestigated in the red varieties and seeds ( Jayaprakasha et al.,2003; Yilmaz and Toledo, 2004; Baydar et al., 2004; Ozkan et al.,2004; Baydar et al., 2006), but very scarcely investigated in whitegrape pomace. Riesling cultivars used in this study were collectedat neighboring parcels and were grown under similar soil and cli-mate conditions. The choice of agricultural methods (organic  vs.conventional) resulted in some differences in the chemical compo-sition of grape skin extracts, confirming available results on redand white grapes (Dani et al., 2007).

During conventional farming, fertilizers containing potassium

and inorganic nitrogen are normally used. Fertilizers dissolve read-ily in soil water, yielding plants with high quantities of nitrogenand potassium content (Worthington, 1991). This might explainthe higher nitrogen content in conventional extracts than in organ-icones (Table 1). These results are consistent to the study of Garde-Cerdán et al., 2009, where higher ammonium nitrogen, aminonitrogen and assimilable nitrogen content in conventional Monast-rell grapes, a red grape variety, was reported in comparison to or-ganic ones. Additionally, potassium is involved in many plantphysiological reactions including osmoregulation, protein synthe-sis, and enzyme activation, which might lead to the high contentin ashes in conventional extracts (Table 1).

Phenolic compounds play a crucial role in natural plant defencemechanisms against herbivores, pathogen stress and UV radiation

(Harborne and Williams, 2000). A screening of the individual com-position of secondary metabolites in organic and conventionalRiesling grape skin extracts was carried out. Levels of quercetinand kaempferol were significantly higher in organic samples(P  < 0.05). In contrast, the content of the flavonoids, catechin, epi-catechin and procyanidin B1 was higher in the conventional grapeskin extracts (Fig. 1). According to this, the antioxidant capacity inconventional samples (26.8 ± 0.7 mmol TE g 1

extract) was higher thanin organic ones (15.93 ± 3.46 mmol TE g 1

extract). These results sup-port the ones of  Dani et al. (2007), who reported a higher antioxi-dant capacity in conventionally grown Niagara grapes. However,when comparing the flavonoid content of organic and conventionalgrapes, studies are inconsistent. Some authors measured a highercontent of flavonoids in conventional grapes (Vian et al., 2006)

whereas others in organic ones (Malusa et al., 2004).Our results showed that the antioxidant capacity and polyphe-

nol content of conventional or organic extracts were not directlycorrelated. The antioxidant capacity of conventionally grown grapeskin extracts was significantly higher than in the organic samples,but in contrast the total phenolic content in conventional extracts(31.27± 4.71 GAEg1) was lower than in organic ones

 Table 1

Chemical characteristics conventional and organic grape skin extracts (Riesling  Vitis

vinifera L.).

Parameter Conventional GSE Organic GSE

Nitrogen (g kg1) 1.11 ± 0.11 1.06 ± 0.02Potassium (g kg1) 2.26 ± 0.48 1.80 ± 0.03Brix 16.60 ± 0.82 16.83 ± 0.63

Ashes (%) 5.78 ± 0.11 4.80 ± 0.26

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(48.14± 0.04 GAE g1). Other compounds, namely specific flavo-noids or even pesticide residues, might be responsible for the high-er antioxidant capacity of conventional extracts. A similar trendwas described in the studies of Dani et al. (2007) on conventionallyand organically grown Niagara grape juices. Furthermore, studiesof  Oliva et al. (2009) suggested that some pesticide residues pres-

ent in food products may yield antioxidant capacity. For example,they demonstrated that the presence of famoxadone, kresoxim-methyl and quinoxyfen increased the antioxidant capacity of grapes measured as Trolox equivalents. The presence of these pes-ticides and other pesticide residues in our conventional extracts(Table 2) might explain the higher antioxidant capacity estimatedin this study. Nonetheless, the sensitivity of antioxidant capacityassays towards pesticides is still not fully understood and needsfurther research.

Flavonoids determine the level of susceptibility or tolerance tofungal infections and pests in plants (Usenik et al., 2004). Theirpresence in grape skin extracts may determine their antibacterialproperties. Nevertheless, despite the high content of flavonoids ingrapes, they possess high levels of sugars, nutrients and water

which make them very susceptible for mould contamination,mostly caused by Botrytis cinerea, Alternaria spp. and Cladosporium

spp.   and less commonly by  Fusarium, Penicillium, Aspergillus car-

bonarius, Aspergillus niger  and Ulocladium (Tournas and Katsoudas,2005). In order to combat these pests, pesticides are generally usedduring conventional farming practices which may remain in thegrape by-products, as parent compounds or as their degradationproducts. Antibacterial activity of the pesticides and their degrada-tion products has been demonstrated to differ between pesticidefamilies (Virag et al., 2007). As a result, the antimicrobial proper-ties of conventional and organic grape skin extracts could be af-fected and were thus compared in this study.

Despite the presence of pesticide residues in conventional Ries-ling white grape skins, both extracts (organic and conventional)

inhibited the growth of  L. monocytogenes,  S. aureus, E. faecalis andE. faecium  to a similar extent and results were not significantly

different (P  > 0.05). The antimicrobial activity of the extractsdemonstrated here, supported previous results of various studies( Jayaprakasha et al., 2003; Yilmaz and Toledo 2004; Baydar et al.,2004; Ozkan et al., 2004; Baydar et al., 2006). Only L. monocytoge-

nes was stronger inhibited by organic GSE. This could be explained

by the higher content of quercetine and its derivatives, which werereported to possess anti-listerial activity when used in their pure

0 5 10 15 20 25 30

Kaempferol

(+)-Catechin

Epicatechin

Quercetin-3-O-rhamnoside

Quercetin

Hyperoside

Dimer catechin

Procyanidin B1

Epicatechin gallate

Concentration [mg 100g-1]

Conventional

Organic

Fig. 1.   Comparison of flavonoid content in conventional and organic grape skin extracts (Riesling Vitis vinifera L.). (a) Paired samples are significant differences (P  < 0.05); (b)non significantly different (P  > 0.05).

 Table 2

Pesticide content in conventional and organic grape skin extracts (Riesling   Vitis

vinifera L.).

Pesticide content ConventionalGSE(mg kg1extract)

OrganicGSE(mg kg1extract)

Maximumresiduelevel (MRL)

(mg kg

1grape

)Boscalid (Fung.) 4.3 <DL 2.0Carbendazim (Fung.) 0.004 <DL 0.3Cyazofamid (Fung.) 0.49 <DL 0.5Cyprodinil (Fung.) 1.4 <DL 2.0Diuron (Herb.) 0.01 <DL 0.05Dimethomorph (Fung.) 1.3 0.05 2.0Famoxadone (Fung.) 1.6 <DL 2.0Fenarimol (Fung.) 0.06 <DL 0.3Fenhexamid (Fung.) 2.8 0.1 5.0Fludioxinil (Fung.) 2.4 0.01 2.0Fluquinconazol (Fung.) 1.9 <DL 0.05Folpet (Fung.) 0.009 0.01 5.0Methoxyfenozid (Fung.) 0.17 0.01 1.0Myclobutanil (Fung.) 1.2 0.01 1.0Penconazole (Fung.) 0.07 0.03 0.2Pendimethanil (Fung.) 0.01 <DL 0.05

Pyraclostrobin (Fung.) 1.6 0.005 1.0Quinoxyfen (Fung.) 0.007 0.03 1.0Tebuconazole (Fung.) 0.08 0.01 1.0Tebufenozide (Insec.) 0.19 0.01 2.0

DL: Detection limit.The results here expressed have a default expanded uncertainty lower than 50%(corresponding to a 95%) accordingto the recommendation of the Codex Committeeon pesticide residues (CCPR 2005, ALINORM 05/28/24).

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forms, as well as in wine concentrates (Rodríguez-Vaquero et al.,2007). In contrast to Baydar et al. studies, neither conventionalnor organic GSE showed antagonistic activity against the Gram-negative bacteria S . typhimurium or  E. coli (P  > 0.05) (Table 3).

Antimutagenicity and toxic effects of the white grape skin ex-tracts were tested against S. typhimurium  strains (Table 4). Whenthe mutagen IQ was present, the 50% inhibitory dose was notreached at the concentrations of white grape skins tested, showinga negligible antimutagenic effect of both extract types in the testwith   S. typhimurium   TA98. IQ rest mutagenicity was, however,slightly lower in the presence of the conventionally grown whitegrape extracts. Noticeable toxicity levels were only achieved atthe highest concentration tested (500ll plate1), with both strainsbeing more sensitive to the organic white grape skin extracts.

Those extracts were rich sources of quercetin and its derivatives,which could have exerted a certain toxic effect on Gram-negativebacteria at higher doses.

Furthermore,the natural grape skinextractswere tested fortheirinhibitory activity towards different moulds. Remarkable differ-ences between the fungistatic effect of conventional and organicwhite grape skin extracts were observed. The growth of P. chrysoge-

num, P. expansum, and A. niger  was inhibited by 95% with conven-tional GSE, whereas the fungistatic effect of organic extracts wasnegligible (P  < 0.05) (Fig. 2). In contrast, no significant differencesbetweenconventionaland organicGSEwere found fortheinhibitionof T. viridieand A.versicolor (P  > 0.05). Certain fungistatic effects havebeen attributedto isolatedflavonoidssuchas (+) –catechin, kaempf-erol and quercetin (Lattanzio et al., 1994). The presence of theseflavonoids is likelyto be responsible for the inhibitory effectof bothtypes of grape extracts against T. viridie and A. versicolor . The pesti-cide traces which were determined in the extracts are shown inTa-ble 2. The levels of pesticides found in conventional samples weresignificantly higher than in organic ones (P  > 0.05). The pesticidesidentified were mainly fungicides, which explain the higher fungi-static effect determined in conventional extracts. Contrarily, thequantified pesticide residues in grape skin extracts did not affectthe growth of foodborne pathogens (Table 3). Comparable resultswereachievedbyNgetal.,2005),whoreportedthatthecombinationof somepesticides didnot inhibit a range of bacteriaof publichealthsignificance and could even enhance their growth.

The content of pesticides in the extracts was determined on thegrape weight basis and their content was below the maximum res-idue levels (MRLs) established for grapes by regulatory organisms(BGBI.I.S.1962, 2379, 2007; EEC Regulation 2092/91). Organic ex-tracts presented only traces of pesticide residues, likely due tothe proximity of conventional farming cultivars, and possible phys-ical or mechanical transport. Nevertheless, pesticide levels werebelow to the values established by the EEC Regulation (1991) fororganic agricultural practices, and their influence on the biologicalactivity was negligible.

4. Conclusions

Extracts from organic Riesling white grape skins contained lev-els of quercetin and quercetin derivatives which were higher thanthose of conventional ones. Their total phenolic content was alsohigher, but their total antioxidant capacity was slightly lower thanin conventional grape skins. This is probably due to their pesticideloads and higher flavonoid content in conventional grape skins.The organic extracts showed fungistatic effects against   T. viridie

and   A. versicolor , and inhibited the growth of Gram positive

0%

20%

40%

60%

80%

100%

P. chrysogenum T. v iridie P. expansum A. niger A.versicolor  

   %    I  n   h   i   b   i   t   i  o  n

Conventional

Organic

Fig. 2.  Fungistatic effect of conventional and organic grape skin extracts 10% (w/v) (Riesling  Vitis vinifera L.).

 Table 4

Inhibitory dose 50% (ID50), rest mutagenicity by 500 ll plate1 (%) and antimutagenic

potence tested on S. typhimurium TA98 with conventional or organic white grape skin

extracts in the presence of the mutagen IQ. Toxicity (%) of c with histidine, in absence

of IQ, of conventional or organic white grape skin extracts against the  S. typhimurium

TA98 and TA102 strains.

Conventional GSE Organic GSE

Inhibitory dose 50%: ID50   n.r. n.r.Rest mutagenicity by 500 ll plate1 (%) 70 100Antimutagenic potence Inactive InactiveToxicity TA98 (%) 17 49Toxicity TA102 (%) 22 72

ID50: inhibitory dose 50% (n.r. = ID50 not reached at 500 ll plate1).Antimutagenic potence:ID50 less than 25%, inactive.ID50 between 300–500 ll plate1: weak.ID50 between 150–300 ll plate1: medium.ID50 between up to 150ll plate1: strong.

 Table 3

Antibacterial effect of conventional and organic grape skin extracts (Riesling  Vitis

vinifera L.).

Strains Conventional GSE Organic GSE20%a 10% 5% 20% 10% 5%

L. monocytogenes Scott A + +     + +  

L. monocytogenes ATCC 19115 + + + ++ + +S. aureus ATCC 25923 ++ + + ++ + +E. faecium DSM 13590 + +     + +  

E. faecalis DSM 20409 + +     + +  

S. typhimurium ATCC 14028  

E. coli ATCC 25922  

Inhibition zone (diameter in mm): 0 (), 1–3 (+), 4–6 (++), 7–10 (+++).a Concentration % w/v.

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microorganisms. They did not show antimutagenic effects in theAmes test, but were more toxic against some  Salmonella  strainsthan conventional extracts. The low pesticide levels recorded inthe extracts did not mask some of their natural biological proper-ties, but yielded a remarkably higher fungistatic activity in conven-tional extracts. According to European guidelines, pesticides tracesfound in the extracts were below the MRL and would not pose arisk for consumers’ health. Thus, these results suggest that extractsfrom organically or conventionally grown white grape skins pos-sess positive biological properties, that would be helpful in pre-venting mould growth and oxidation in foods, and could then beused as food preservatives and antioxidants to increase foodshelf-life.

Conflict of Interest

The authors declare that there are no conflicts of interest.

 Acknowledgments

M. Corrales and M.G.Vizoso-Pinto thank the German AcademicExchange Service (DAAD) and Konrad Adenauer Stiftung, respec-tively for a doctoral fellowship. A. Fernandez thanks the ConsoliderProject Fun-C-Food CSD2007-00063 from the Spanish Ministry of Science and Innovation. We thank Claudia Hoffmann for providingthe organic and conventional Riesling grape pomace.

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