Food and Chemical Toxicology 48 (2010) 34713476

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Food and Chemical Toxicology

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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
b
c

Max Rubner-Institute, Department of Safety and Quality of Fruit and Vegetables, Haid-und-Neustr. 9, 76131 Karlsruhe, Germany
Instituto de Agroqumica y Tecnologa de Alimentos, CSIC, Avda. Agustn Escardino, 7, 46980 Paterna, Spain
Chemisches und Veterinruntersuchungsamt Stuttgart, Schaandstrae 3/2, 70736 Fellbach, Stuttgart, Germany

a r t i c l e

i n f o

Article history:
Received 19 May 2010
Accepted 20 September 2010

Keywords:
Ames test
Antibacterial effect
Flavonoids
Organic cultivars
White grape skin extracts
Pesticides

a b s t r a c t
Grape skin extracts of Riesling Vitis vinifera L. grapes from conventionally or organically managed cultivars were compared on the basis of their phenolic content, antioxidant capacity, antimicrobial and antimutagenic properties and pesticide loads. Promising results on their biological properties suggest that
those extracts would be valuable as food preservatives. The antioxidant capacity of conventional extracts
was signicantly higher, according to the higher content in catechin, epicatechin and procyanidin B. Pesticide loads did not affect the antimutagenic or antimicrobial properties of the extracts. Both extracts
inhibited 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 quercetin
and its derivatives, higher antimicrobial effects against Listeria monocytogenes and Salmonella typhimurium were observed for the organic white grape skin extracts. Conventional or organic extracts did not
show 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 chrysogenum 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 gaining
more and more relevance in the food industry, following increasing
consumer awareness of diet-related health problems and consumer mistrust regarding synthetic additives, (Pokorny, 1991;
Smid and Gorris, 1999; Gram et al., 2002). Fruit skins and seeds
are rich in bioactive substances such as phenolic acids, avonoids
and 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 grape
pomace is approximately 9 million tons per year (Meyer et al.,
1997; Schieber et al., 2002), and has an increasing economical signicance. Red grape pomace and seeds have especially been often
investigated as source of anthocyanins and avonoids with impor-

Corresponding author. Present address: Food Safety and Quality Unit, Joint
Research Centre, Institute for Reference Materials and Measurements, European
Commission, Retieseweg 111, B-2440 Geel, Belgium. Tel.: +32 014 571 853; fax: +32
014 571 787.
E-mail address: margarita.corrales@ec.europa.eu (M. Corrales).
0278-6915/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2010.09.025

tant antimicrobial, antioxidant and health-promoting properties.

Those by-products have been suggested as an attractive target
for production of food additives or nutraceuticals (Lu and Foo,
1999; Jayaprakasha et al., 2003; Yilmaz and Toledo, 2004; Baydar
et al., 2004, 2006; Ozkan et al., 2004; Kammerer et al., 2004). The
characterization of white grape pomace components and a more
exhaustive knowledge of their biological properties will also help
in the valorisation of white grape by-products, thus providing
new strategies to diminish the ecological impact of polyphenol rich
wastes (Sanchez et al., 2009).
As consumers choice towards organic foods increases, a better
understanding of the inuence of agricultural practices on the biological properties of food will also assist in the marketing strategies
and decision making. White grapes of the Riesling variety which
originated in the German Rhine region are also extensively produced in the bordering Palatinate region of Germany. They produce
high quality sweet or semi-sweet white wine and the pomace is
typically 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, and
in other countries around the world. Nowadays Riesling grapes are
also cultivated under organic farming conditions. Available

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M. Corrales et al. / Food and Chemical Toxicology 48 (2010) 34713476

experimental data suggest that cultivar practices may inuence the

content of avonoids and other secondary metabolites in fruits.
However, data on this are still contradictory and further research
is needed. For example, Malusa et al. (2004) reported higher polyphenol content in organic grapes compared to conventional ones,
whereas Vian et al. (2006) found a higher content of anthocyanins
in conventionally grown grapes when compared to the organically
grown ones.
White grape pomace has been insufciently studied when
compared to the red varieties and grape seeds, probably due to
their lower contents of anthocyanins and proanthocyanidins.
However, white grape pomace is also a rich source of avonoids
and stilbenes with important health-promoting attributes (Birt
et al., 2001; Mennen et al., 2004; Bertelli and Dipak, 2009). Additionally, white grape pomace does not possess the red/purple colour characteristic of red grape varieties extracts and the high
astringency of seed extracts. All these characteristics allow the
use of white grape skin extracts as potential additives or nutraceuticals for a wider range of products than their red grape skin
extract equivalents.
Therefore, this work aimed at assessing relevant chemical differences between extracts from organically and conventionally
grown grape skin Riesling Vitis vinifera L. variety. Flavonoid content was analysed by means of HPLC, total phenolics by the FolinCiocalteu method and the antioxidant capacity as Trolox
equivalents. Relevant biological properties such as the antimutagenic capacity and the antimicrobial properties against food
borne pathogen bacteria and moulds were compared on the basis of the pesticide content of the extracts. Furthermore a thorough characterization of the organic samples provides data on
the marketability of these extracts as food additives. Differences
between organic and conventionally grown samples can also
provide insight into the effects of the use of pesticides, which
may mask natural biological properties of the white grape
pomace.
2. Materials and methods
Analytical grade reagents, solvents and chemicals were obtained from Sigma
Aldrich (Taufkirchen, Germany) and Merck (Darmstadt, Germany). Standards used
for identication and quantication purposes with HPLC were as follows: catechin,
epicatechin, epicatechingallate, procyanidin B (extrasynthese, Lyon, France), quercetin, quercetin-3-O-rhamnoside, hyperoside, kaempferol (Sigma, Taufkirchen, Germany). More than 500 standards for pesticide analysis were provided by the Dr.
Ehrenstorfer GmbH (Augsburg, Austria) company and by Sigma Aldrich (Taufkirchen, Germany).
Samples of Riesling Vitis vinifera L. grapes were collected in 2005 from
two adjacent elds cultivated according to the German legislation for conventional or organic farming (meaning no plant protection with synthetically
produced chemicals; no utilisation of easily soluble mineral fertilisers; intensive humus management; crop rotation; no application of synthetic chemical
growth 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. Grapes
were separated into three different pomace fractions: skins, stems and seeds.
For this work, skins were lyophilised and then milled with a coffee grinder
prior to extraction.
2.1. Determination of nitrogen content
The total nitrogen content in the samples was estimated 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 a
desiccator to room temperature. The dish was heated at 103 C until water was
evaporated (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 and re-ashed at 550 C to constant
weight. The ashes were cooled in a desiccator to room temperature and weighted to
the nearest 0.1 mg.

2.3. Determination of potassium content

Twenty-ve grams of dry grape skins were weighted and 150 ml of de-ionized
hot 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. Samples
were ltered and the potassium content was measured using a selective ionic electrode which was previously calibrated (Dr. W. Ingold AG, Urdorf, Schweiz). Results
were expressed in g potassium kg1.
2.4. Sugar content in Brix
The sugar content in the methanolic extracts was measured in Brix using a Kruess 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% methanol
in aliquots of 30 mL. All extractions were assisted by an ultrasound generator (Bandelin, Sonorex RK 100H, Walldorf, Germany) for 9 min. After each extraction, samples were centrifuged at 9000 rpm for 10 min, the supernatants were collected,
ltered (0.45 lm) (PTFE lter, Whatman, US) and lyophilised. The solid extracts
were weighed and dissolved in distilled water to a concentration of 5%, 10% and
20% (w/v) for the antimicrobial assays.
2.6. Solid-phase extraction
For avonoid extraction and identication, ltered methanolic supernatants
(described above) were evaporated and dissolved in 10 mL acidied water
(pH 1.5). The solution was re-extracted with 100 mL ethyl acetate. Supernatants
were collected, evaporated and dissolved in 5 mL water (pH 7). Extracts were then
applied to Chromabond C18 cartridges (Varian, Frankfurt am Main, Germany). Cartridges were rst activated with 2 mL methanol and 1 mL water. Flavonoids were
eluted with 10 mL of ethyl acetate. Extracts were concentrated and diluted in methanol for LC-DAD/ESI-MS analysis.
2.7. Analysis of avonoids by LC-DAD/ESI-MS
Supernatants resulting from the solid-phase extraction were dissolved in 2 mL
methanol. Solutions were membrane-ltered (0.45 lm) (PTFE lter, Whatman, US)
and analysed in an Agilent Technologies LC/MSD Series 1100 (binary solvent delivery, autosampler, UVVis Diode Array Detector (DAD), electrospray ionization (ESI);
Agilent Technologies, Palo Alto, CA)0 mass spectrometer. The separation was performed with an Aqua Column (80 A
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% acetic
acid in water (v/v) and solution B: 0.5% acetic acid, 50% water and 49.5% acetonitrile
(v/v/v). For avonoid analysis, the gradient was as follows: 1024%B (20 min), 24
30%B (20 min), 3055%B (20 min), 6075%B (15 min). The injection volume was
20 lL and the ow rate 1.0 mL min1. The detection was monitored at 320 nm
(Kammerer et al., 2004).
The mass spectrometer was tted with an ESI source in negative mode. The column eluate was recorded in the range m/z 501000. The mass spectrometer was
programmed to do an MS2 scan of the most abundant ion in the full mass. Nitrogen
was used both as drying gas at ow rate of 11.0 L min1, and as nebulising gas at a
pressure of 60psi. The nebuliser temperature was set to 350 C. Peak identication
and quantication took place by MS and by comparison of the retention times with
commercially available standards.
2.8. Antioxidant capacity
The ABTS radical cation assay described by Miller et al. (1993) and later improved by Re et al. (1999) was used for the determination of the polar antioxidant
capacity. A stock solution of 5 mM ABTS (2,20 -azino-di 3-ethylbenzothiazoline-6sulphonic acid) was diluted in water and preincubated for at least 12 h with
140 mM (nal concentration) of K2S2O8 to produce the radical cation ABTS+. The
ABTS+ solution was then diluted in 5 mM saline phosphate buffer pH 7.4 until
absorbance readings reached a value of 1.5 at 735 nm. An aliquot extract of
100 lL, was 100-fold diluted in buffer mixed with 2.9 mL ABTS and set 15 min at
30 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 used
to calculate the antioxidant activity of the samples, expressed as mmol TROLOX
equivalents (TE) g1
extract .
2.9. Total phenolic content
Total phenolic content was determined using the FolinCiocalteau reagent (Singleton and Rossi, 1965). An extract aliquot of 125 ll was mixed with 625 ll of FolinCiocalteau reagent (previously diluted 10-fold with distilled water at 45 C) and

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M. Corrales et al. / Food and Chemical Toxicology 48 (2010) 34713476

set 3 min at room temperature. After this, 500 ll of sodium carbonate (0.6 M; 45 C)
were added to the mixture and incubated for 15 min at 45 C. The absorbance was
measured spectrophotometrically at 750 nm.
A gallic acid hydrate solution (Roth, Karlsruhe, Germany) was used as standard
for the calibration curve and the results were expressed as lmol of gallic acid equivalents (GAE) g1
extract .
2.10. Antimutagenic activity
The antimutagenic activity was tested according to Edenharder et al. (1994),
following a modication of the method proposed by Maron and Ames (1983). A
mixture containing 500 ll of isotonic KCl, 12 lL of a solution of the mutagen IQ
(Toronto research Chemicals Inc., Downsview, Ontario, Canada) in DMSO (resulting
in a average of 700 His + revertants per plate), different amounts (from 150 to
500 ll) of the extract resuspended in sterile water and adjusted to pH 7.4 (the volume up to 500 ll was lled with isotonic KCl), 500 ll of the mammalian metabolic
activator 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. Revertants were counted after cultivation for 24 h at 37 C. Two replicates were analyzed
at each dose. A decrease in the number of revertant cells means antimutagenic effects against IQ.
As control, toxicity against S. typhimurium TA98 and S. typhimurium TA102 was
assayed. In this case, colonies were grown in the presence of histidine, without the
mutagen IQ. Two pomace concentrations were tested on top agar plates with a total
number of colonies of approx. 300. Colonies were counted after cultivation for 24 h
at 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 enterica 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 DSM
20409 were growth in the Man, Rogosa and Sharpe broth (MRS broth, Merck,
Darmstadt, Germany) at 37 C. Stock cultures were maintained at 20 C in
15% (v/v) glycerol.
Penicillium chrysogenum DSM 844, Penicillium expansum DSM 62841, Aspergillus
niger, DSM 1988, Aspergillus versicolor DSM 63292 and Trichoderma viridie DSM
63065 were cultured and grown in malt extract agar plates (12 g L1 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 for
screening the antagonistic activity of the extracts. Standard I or MRS agar
(12 g L1) plates were overlayed with Standard I or MRS soft agar (7.5 g L1) previously inoculated with ca. 1  106 CFU mL1 of overnight cultures of the corresponding indicator bacterial strain. Wells were cut from the soft agar layer
surface with the back of a sterile Pasteur pipette, and 20lL of each extract concentration (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 bacterial
growth were measured in mm.

2.13. Effect of grape skin extracts on conidia germination and germ tube length
The effect of grape skin extracts (GSE) on the germination of conidia was carried
out according to the method described by Droby et al., 1997). The spore concentration was determined with a Neubauer counting chamber (Optik Labor, Hecht, Germany) and adjusted to 5  105 spores mL1. Aliquots of 90 lL of the spore
suspension were mixed with 10 lL of GSE (10% w/v) and the conidia germination
determined after 24 h incubation at 30 C.

2.14. Analysis of pesticide loads

Pesticide residues were measured using the QuEChERS pesticide multiresidue
method, as described by Anastassiades et al. (2003, 2007) and EU standarisation
methods (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 dissolved
in 10 mL distilled water, extracted with 10 mL acetonitrile by vigorous shaking.
Thereafter, a salt-mixture containing 4 g MgSO4, 1 g NaCl and 1 g Na3citrat 2H20
and 0.5 g Na2H citrate sesquidydrate) was added and mixed well. Samples were
centrifuged at 3000 g/min for 5 min. Clean-up was carried out by dispersive solid-phase extraction (D-SPE), which involved the mixing of an extract aliquot with
25 mg PSA (sorbent) and 15 mg MgSO4 (drying agent). Samples were centrifuged at
3500 rpm for 3 min. Supernatants were directly amenable to both liquid chromatographic (LC)- and gas chromatographic (GC)-analysis. LC-MS and GC-MS analysis
followed different methods according to the CEN norm EN 15662.

2.15. Statistical analysis

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

3. Results and discussion

Grape composition depends on a series of interactions between
genetic characteristics, environmental conditions and cultural
practices (Bourn and Prescott, 2002). The composition determines
the biological properties of the grapes, and has been extensively
investigated 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 white
grape pomace. Riesling cultivars used in this study were collected
at neighboring parcels and were grown under similar soil and climate conditions. The choice of agricultural methods (organic vs.
conventional) resulted in some differences in the chemical composition of grape skin extracts, conrming available results on red
and white grapes (Dani et al., 2007).
During conventional farming, fertilizers containing potassium
and inorganic nitrogen are normally used. Fertilizers dissolve readily in soil water, yielding plants with high quantities of nitrogen
and potassium content (Worthington, 1991). This might explain
the higher nitrogen content in conventional extracts than in organic ones (Table 1). These results are consistent to the study of GardeCerdn et al., 2009, where higher ammonium nitrogen, amino
nitrogen and assimilable nitrogen content in conventional Monastrell grapes, a red grape variety, was reported in comparison to organic ones. Additionally, potassium is involved in many plant
physiological reactions including osmoregulation, protein synthesis, and enzyme activation, which might lead to the high content
in ashes in conventional extracts (Table 1).
Phenolic compounds play a crucial role in natural plant defence
mechanisms against herbivores, pathogen stress and UV radiation
(Harborne and Williams, 2000). A screening of the individual composition of secondary metabolites in organic and conventional
Riesling grape skin extracts was carried out. Levels of quercetin
and kaempferol were signicantly higher in organic samples
(P < 0.05). In contrast, the content of the avonoids, catechin, epicatechin and procyanidin B1 was higher in the conventional grape
skin extracts (Fig. 1). According to this, the antioxidant capacity in
conventional samples (26.8 0.7 mmol TE g 1
extract ) was higher than
in organic ones (15.93 3.46 mmol TE g 1
extract ). These results support the ones of Dani et al. (2007), who reported a higher antioxidant capacity in conventionally grown Niagara grapes. However,
when comparing the avonoid content of organic and conventional
grapes, studies are inconsistent. Some authors measured a higher
content of avonoids in conventional grapes (Vian et al., 2006)
whereas others in organic ones (Malusa et al., 2004).
Our results showed that the antioxidant capacity and polyphenol content of conventional or organic extracts were not directly
correlated. The antioxidant capacity of conventionally grown grape
skin extracts was signicantly higher than in the organic samples,
but in contrast the total phenolic content in conventional extracts
(31.27 4.71 GAE g1) 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)
Potassium (g kg1)
Brix
Ashes (%)

1.11 0.11
2.26 0.48
16.60 0.82
5.78 0.11

1.06 0.02
1.80 0.03
16.83 0.63
4.80 0.26

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M. Corrales et al. / Food and Chemical Toxicology 48 (2010) 34713476

Epicatechin gallate

Procyanidin B1

Dimer catechin

Hyperoside

Quercetin

Quercetin-3-O-rhamnoside

Epicatechin

(+)-Catechin

Conventional
Kaempferol

Organic
0

10

15

20

25

30

Concentration [mg 100g-1]

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

(48.14 0.04 GAE g1). Other compounds, namely specic avonoids or even pesticide residues, might be responsible for the higher antioxidant capacity of conventional extracts. A similar trend
was described in the studies of Dani et al. (2007) on conventionally
and organically grown Niagara grape juices. Furthermore, studies
of Oliva et al. (2009) suggested that some pesticide residues present in food products may yield antioxidant capacity. For example,
they demonstrated that the presence of famoxadone, kresoximmethyl and quinoxyfen increased the antioxidant capacity of
grapes measured as Trolox equivalents. The presence of these pesticides and other pesticide residues in our conventional extracts
(Table 2) might explain the higher antioxidant capacity estimated
in this study. Nonetheless, the sensitivity of antioxidant capacity
assays towards pesticides is still not fully understood and needs
further research.
Flavonoids determine the level of susceptibility or tolerance to
fungal infections and pests in plants (Usenik et al., 2004). Their
presence in grape skin extracts may determine their antibacterial
properties. Nevertheless, despite the high content of avonoids in
grapes, 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 carbonarius, Aspergillus niger and Ulocladium (Tournas and Katsoudas,
2005). In order to combat these pests, pesticides are generally used
during conventional farming practices which may remain in the
grape by-products, as parent compounds or as their degradation
products. Antibacterial activity of the pesticides and their degradation products has been demonstrated to differ between pesticide
families (Virag et al., 2007). As a result, the antimicrobial properties of conventional and organic grape skin extracts could be affected and were thus compared in this study.
Despite the presence of pesticide residues in conventional Riesling white grape skins, both extracts (organic and conventional)
inhibited the growth of L. monocytogenes, S. aureus, E. faecalis and
E. faecium to a similar extent and results were not signicantly

Table 2
Pesticide content in conventional and organic grape skin extracts (Riesling Vitis
vinifera L.).
Pesticide content

Conventional
GSE
(mg kg1extract)

Organic
GSE
(mg kg1extract)

Maximum
residue
level (MRL)
(mg kg1grape)

Boscalid (Fung.)
Carbendazim (Fung.)
Cyazofamid (Fung.)
Cyprodinil (Fung.)
Diuron (Herb.)
Dimethomorph (Fung.)
Famoxadone (Fung.)
Fenarimol (Fung.)
Fenhexamid (Fung.)
Fludioxinil (Fung.)
Fluquinconazol (Fung.)
Folpet (Fung.)
Methoxyfenozid (Fung.)
Myclobutanil (Fung.)
Penconazole (Fung.)
Pendimethanil (Fung.)
Pyraclostrobin (Fung.)
Quinoxyfen (Fung.)
Tebuconazole (Fung.)
Tebufenozide (Insec.)

4.3
0.004
0.49
1.4
0.01
1.3
1.6
0.06
2.8
2.4
1.9
0.009
0.17
1.2
0.07
0.01
1.6
0.007
0.08
0.19

<DL
<DL
<DL
<DL
<DL
0.05
<DL
<DL
0.1
0.01
<DL
0.01
0.01
0.01
0.03
<DL
0.005
0.03
0.01
0.01

2.0
0.3
0.5
2.0
0.05
2.0
2.0
0.3
5.0
2.0
0.05
5.0
1.0
1.0
0.2
0.05
1.0
1.0
1.0
2.0

DL: Detection limit.

The results here expressed have a default expanded uncertainty lower than 50%
(corresponding to a 95%) according to the recommendation of the Codex Committee
on pesticide residues (CCPR 2005, ALINORM 05/28/24).

different (P > 0.05). The antimicrobial activity of the extracts

demonstrated 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. monocytogenes was stronger inhibited by organic GSE. This could be explained
by the higher content of quercetine and its derivatives, which were
reported to possess anti-listerial activity when used in their pure

M. Corrales et al. / Food and Chemical Toxicology 48 (2010) 34713476

forms, as well as in wine concentrates (Rodrguez-Vaquero et al.,

2007). In contrast to Baydar et al. studies, neither conventional
nor organic GSE showed antagonistic activity against the Gramnegative bacteria S. typhimurium or E. coli (P > 0.05) (Table 3).
Antimutagenicity and toxic effects of the white grape skin extracts were tested against S. typhimurium strains (Table 4). When
the mutagen IQ was present, the 50% inhibitory dose was not
reached at the concentrations of white grape skins tested, showing
a negligible antimutagenic effect of both extract types in the test
with S. typhimurium TA98. IQ rest mutagenicity was, however,
slightly lower in the presence of the conventionally grown white
grape extracts. Noticeable toxicity levels were only achieved at
the highest concentration tested (500 ll plate1), with both strains
being more sensitive to the organic white grape skin extracts.

Table 3
Antibacterial effect of conventional and organic grape skin extracts (Riesling Vitis
vinifera L.).
Strains
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

Conventional GSE
20%a

10%

Organic GSE
5% 20% 10%

+
+
++
+
+



+
+
+
+
+




+
+





+
++
++
+
+



+
+
+
+
+



5%

+
+





Inhibition zone (diameter in mm): 0 (), 13 (+), 46 (++), 710 (+++).

a
Concentration % w/v.

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.

Inhibitory dose 50%: ID50

Rest mutagenicity by 500 ll plate1 (%)
Antimutagenic potence
Toxicity TA98 (%)
Toxicity TA102 (%)

Conventional GSE

Organic GSE

n.r.
70
Inactive
17
22

n.r.
100
Inactive
49
72

ID50: inhibitory dose 50% (n.r. = ID50 not reached at 500 ll plate1).
Antimutagenic potence:
ID50 less than 25%, inactive.
ID50 between 300500 ll plate1: weak.
ID50 between 150300 ll plate1: medium.
ID50 between up to 150 ll plate1: strong.

Those extracts were rich sources of quercetin and its derivatives,

which could have exerted a certain toxic effect on Gram-negative
bacteria at higher doses.
Furthermore, the natural grape skin extracts were tested for their
inhibitory activity towards different moulds. Remarkable differences between the fungistatic effect of conventional and organic
white grape skin extracts were observed. The growth of P. chrysogenum, P. expansum, and A. niger was inhibited by 95% with conventional GSE, whereas the fungistatic effect of organic extracts was
negligible (P < 0.05) (Fig. 2). In contrast, no signicant differences
between conventional and organic GSE were found for the inhibition
of T. viridie and A.versicolor (P > 0.05). Certain fungistatic effects have
been attributed to isolated avonoids such as (+) catechin, kaempferol and quercetin (Lattanzio et al., 1994). The presence of these
avonoids is likely to be responsible for the inhibitory effect of both
types of grape extracts against T. viridie and A. versicolor. The pesticide traces which were determined in the extracts are shown in Table 2. The levels of pesticides found in conventional samples were
signicantly higher than in organic ones (P > 0.05). The pesticides
identied were mainly fungicides, which explain the higher fungistatic effect determined in conventional extracts. Contrarily, the
quantied pesticide residues in grape skin extracts did not affect
the growth of foodborne pathogens (Table 3). Comparable results
were achieved by Ng et al., 2005), who reported that the combination
of some pesticides did not inhibit a range of bacteria of public health
signicance and could even enhance their growth.
The content of pesticides in the extracts was determined on the
grape weight basis and their content was below the maximum residue levels (MRLs) established for grapes by regulatory organisms
(BGBI.I.S.1962, 2379, 2007; EEC Regulation 2092/91). Organic extracts presented only traces of pesticide residues, likely due to
the proximity of conventional farming cultivars, and possible physical or mechanical transport. Nevertheless, pesticide levels were
below to the values established by the EEC Regulation (1991) for
organic agricultural practices, and their inuence on the biological
activity was negligible.

4. Conclusions
Extracts from organic Riesling white grape skins contained levels of quercetin and quercetin derivatives which were higher than
those of conventional ones. Their total phenolic content was also
higher, but their total antioxidant capacity was slightly lower than
in conventional grape skins. This is probably due to their pesticide
loads and higher avonoid content in conventional grape skins.
The organic extracts showed fungistatic effects against T. viridie
and A. versicolor, and inhibited the growth of Gram positive

100%

% Inhibition

80%
60%
40%
20%

Conventional
Organic

0%
P. chrysogenum T. viridie

P. expansum

3475

A. niger

A. versicolor

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

3476

M. Corrales et al. / Food and Chemical Toxicology 48 (2010) 34713476

microorganisms. They did not show antimutagenic effects in the

Ames test, but were more toxic against some Salmonella strains
than conventional extracts. The low pesticide levels recorded in
the extracts did not mask some of their natural biological properties, but yielded a remarkably higher fungistatic activity in conventional extracts. According to European guidelines, pesticides traces
found in the extracts were below the MRL and would not pose a
risk for consumers health. Thus, these results suggest that extracts
from organically or conventionally grown white grape skins possess positive biological properties, that would be helpful in preventing mould growth and oxidation in foods, and could then be
used as food preservatives and antioxidants to increase food
shelf-life.
Conict of Interest
The authors declare that there are no conicts of interest.
Acknowledgments
M. Corrales and M.G.Vizoso-Pinto thank the German Academic
Exchange Service (DAAD) and Konrad Adenauer Stiftung, respectively for a doctoral fellowship. A. Fernandez thanks the Consolider
Project Fun-C-Food CSD2007-00063 from the Spanish Ministry of
Science and Innovation. We thank Claudia Hoffmann for providing
the organic and conventional Riesling grape pomace.
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