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Keywords:

  • Aspergillus carbonarius;
  • conidia;
  • germination;
  • grapes;
  • ochratoxin A;
  • partitioning

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Aims: Aspergillus carbonarius is an important ochratoxin A (OTA)-producing fungus which is responsible for toxin contamination of grapes and wine. The objectives of this study were to examine the partitioning of OTA in mycelium and conidia of a range of A. carbonarius strains on artificial grape juice and defined media, to determine the excretion patterns of OTA from these spores, and the effect of organic acids used in wine production on OTA excretion from conidia.

Methods and Results:  The results showed that 60–70% of the OTA was accumulated in the conidia of a number of different isolates of A. carbonarius. Calculations showed that on different defined media, an amount of 0·011- to 0·1-pg OTA was present per conidium. The OTA in spores was found to be rapidly excreted into the medium during the initial few hours after conidial germination leading to an increase of OTA in must during maceration for wine production. The presence of tartaric acid inhibited OTA production, but malic acid enhanced this production during mycelial growth. These acids were also shown to affect the time course of germination and the rate of OTA excretion from conidia during germination.

Conclusions:  This study is the first to examine and show the partitioning of OTA into spores of strains of A. carbonarius and that rapid excretion of OTA from spores could be a reason for OTA accumulation in musts during wine production.

Significance and Impact of the Study:  Conidia of A. carbonarius could be a major source of OTA contamination of grapes used in wine production. This information could help in the development of effective prevention strategies to minimize wine contamination with this important mycotoxin.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Ochratoxin A (OTA) is a secondary metabolite produced by fungi belonging to Aspergillus and Penicillium genera (Magan and Olsen 2004). It has been shown to be nephrotoxic, teratogenic, immunosuppressive and carcinogenic (Cabanes et al. 2002). The presence of OTA has been detected in a range of foods and beverages (Zimmerli and Dick 1996; Serra et al. 2003) and has also been reported in the body fluids and kidneys of animals and humans (Magan and Olsen 2004).

Grapes and derived products have been reported to be contaminated with OTA (Zimmerli and Dick 1996; Burdaspal and Legarda 1999; MacDonald et al. 1999; Otteneder and Majerus 2000). Several studies have highlighted that Aspergillus section Nigri (black aspergilli) is the main group responsible for OTA contamination of grapes, must and wine (Cabanes et al. 2002; Battilani et al. 2003, 2006; Belli et al. 2004b). Within this section, the OTA-producing species are those included in the so-called Aspergillus niger aggregate and Aspergillus carbonarius (Belli et al. 2004a). Aspergillus carbonarius is considered to be the main species responsible for OTA contamination and accumulation in grapes and wine (Battilani et al. 2003). It is a very invasive species and colonizes and penetrates berries, regardless of the skin conditions (Battilani and Pietri 2002). It has been isolated from grapes in France, Spain, Italy, South America, Greece, Israel, Portugal and Australia (Varga and Kozakiewicz 2006).

The surface of grape berries represents a favourable habitat for these fungal species that have various impacts on the quality of grapes and derived products (Bae et al. 2004). Contamination with asexual spores (conidia) occurs from colonized crop debris and from the airspora. Under conducive environmental conditions, the spores germinate and attach to the surface (Barhoom and Sharon 2004). Spore germination is the first stage in the fungal colonization of such food matrices (Pardo et al. 2004) and later on for the biosynthesis of primary and secondary metabolites, including mycotoxins (Calvo et al. 2002).

There is surprisingly little information on the mycotoxin content of conidia of such species and indeed mycotoxigenic species generally. Previous studies have suggested that the spores of mycotoxigenic species can contain mycotoxins. It has been demonstrated that the conidia of Alternaria alternata contain alternariol and alternariol monomethylether (Haggblom 1987), and the conidia of Aspergillus flavus can contain aflatoxin (Wicklow and Shotwell 1983). A range of mycotoxins, including fumagillin, fumigaclavine A, fumitremorgin C, trypacidin and verruculogen, have been demonstrated to be present in the spores of Aspergillus fumigatus (Land et al. 1994; Fischer et al. 2000). In addition, the conidia of some Fusarium species contain deoxynivalenol (Miller 1992) and the conidia of Stachybotrys chartarum contain satratoxins (Sorenson et al. 1987). This is important as they can also have allergenic implications and may play an important role in the indoor air quality (Jarvis and Miller 2005).

The objectives of the present work were (i) to quantify the partitioning of OTA into spores, mycelium and medium for different isolates of A. carbonarius; (ii) to investigate the effect of the medium type on OTA accumulation in the conidia of an isolate of this species; (iii) to examine the OTA excretion ability of conidia of A. carbonarius during germination; and (iv) to determine the effect of organic acids present in grapes and wine on the rate of excretion of the OTA from spores.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Fungal isolates

In this study, five isolates of A. carbonarius from wine grapes were used. These were ITA1102 (Italy), IS10.8 (Israel), PORT219 (Portugal), GR458 (Greece) and 2Mu134 (France). For comparison of the partitioning of OTA, an isolate of Aspergillus ochraceus (ITA703) was also included. These isolates have all been previously demonstrated to produce OTA on an artificial grape juice medium and Czapek yeast extract agar (CYA) (Mitchell et al. 2004; Belli et al. 2004b). The identity of all isolates have been checked and confirmed by Cabi Biosciences, Egham, Surrey, UK (Dr Z. Lawrence).

Inoculum preparation

The isolates were grown on CYA agar for 7 days at 25°C to obtain heavily sporulating culture. The spores were collected in sterile NaCl solution (0·9%). Spore concentration was counted using a Thoma chamber, and the final concentration was adjusted to 106 spores ml−1 except for the germination study, where a higher inoculum concentration was required (about 109 spores ml−1).

Culture conditions for OTA content of spores

Two experimental studies were conducted. The first involved the use of four isolates of A. carbonarius to examine the partitioning of OTA between spores, mycelium and medium. This was carried out on an artificial grape juice agar medium representative of mid-veraison (Mitchell et al. 2004). The basal medium had a water activity (aw) of 0·987. This was also modified to 0·95 aw with glycerol. This water availability level has been shown to be optimum condition for OTA production (Mitchell et al. 2004). Twenty millilitres of the agar media was poured in each Petri plate (polystyrene 15 × 94 mm; Fisher Bioblock Scientific, Illkirch, France). The solidified media surfaces were carefully covered with sterile cellophane discs (8·5 cm, P400; Cannings, Bristol, UK). The plates were centrally inoculated with 3-mm agar discs taken from the media of the same composition containing a spore lawn of each isolate previously grown for 24 h. The colonies were allowed to grow for up to 20 days at 20 and 25°C. This was done to enable enough biomass/spores to be produced under the test conditions for OTA extraction. In all the cases, 15–20 Petri plates per treatment were used to enable whole colonies to be used for replicated quantification of biomass, spores and OTA content.

The second experiment involved a comparison of different defined nutrient media with one isolate only (2Mu134). For this study, CYA, Czapek dox agar (CZ), malt extract agar (MEA), synthetic medium (SM), grape synthetic medium (SGM) and grape juice agar (GJA) medium were used. CYA, CZ, MEA and SM were prepared according to Cahagnier et al. (1998). SGM was prepared according to Mitchell et al. (2004). GJA medium was prepared by adding agar (1·5%) to sterilized red grape juice. d(+) glucose, agar (Difco), yeast extract (Difco), malt extract (Difco), peptone (Difco) and sucrose were purchased from Fisher Bioblock Scientific, Illkirch, France. d(-) fructose, l(-) tartaric acid, l(-) malic acid, (NH4)2HPO4, KH2PO4, K2HPO4, MgSO4.7H2O, NaCl, CaCl2, CuCl2, FeSO4.7H2O, ZnSO4.7H2O, MnSO4.H2O, CuSO4.5H2O, Na2B4O7. H2O, (NH4)6 Mo7O24.4H2O, NH4NO3 and (+) catechin hydrate were purchased from Sigma Aldrich, Saint Quentin Fallavier, France. Twenty millilitre of the agar media was poured in each Petri plate (polystyrene 15 × 94 mm; Fisher Bioblock Scientific, Illkirch, France). Each medium was centrally inoculated with 2 μl of the adjusted spore suspension (106 spores ml−1) and incubated for 7 days at 25°C.

Harvesting procedures for spores and mycelial biomass

In experiment 1, the entire mycelial colony was removed together with the cellophane layer. This was suspended in 10 ml of sterile water containing a wetting agent (Tween 80; 0·1%) to wet the spores (Ramos et al. 1999). To obtain spores, the contents were filtered through sterile glass wool, and the filtrate was centrifuged to obtain a spore pellet. Other colonies were destructively sampled for the total fungal biomass measurement. A 9-mm cork borer was used to remove agar discs from the inner and outer regions of the medium. The spore pellets, colonies and medium were weighed and then extracted for OTA. In all cases, at least five replicates per treatment were used for OTA quantification of each isolate.

For a comparison of the effect of growth media, the spores were isolated according to the method of Stormer et al. (1998). NaCl solution (0·9%, 10 ml) was added to the treatment Petri plate and the spores were collected from the mycelial mat using a Pasteur pipette. The suspension contained approximately 95% spores. Hyphal fragments were removed by filtration through a gauze filter. The spores were counted using Thoma chamber and filtered through a 0·45 μm Millipore filter (Millipore Corporation, Bellerica, MA, USA) to obtain a final spore sample in 3 ml of distilled water. Other Petri plates were sampled for the determination of OTA production capacity by A. carbonarius (2Mu134) on the different growth media used. For this, three agar plugs were removed from the inner and outer area of each colony using a cork borer (9 mm).

Conidial germination studies

One millilitre of spore suspension (109 spores ml−1) was inoculated into 250-ml Erlenmeyer flasks containing 50 ml of CYA medium and incubated on a rotary shaker (240 rev min−1) at 25°C. The germination study was monitored using a Nikon Microscope. The spores were considered to have germinated when the germ tube was equal to or longer than the diameter of the spores. One millilitre of the medium was removed periodically every 2 h after starting the experiment, examined under the microscope to determine the germination state and filtered through a 0·45 μm Millipore filter (Millipore Corporation, Bellerica, MA, USA). Filtrate and spores (recuperated by 3 ml of distilled water from the filter) were then extracted for OTA.

OTA extraction

Extraction of OTA from spores and filtrate

OTA was extracted according to the method described by Stormer et al. (1998). In this method, the spores and the filtrate were added 1 ml of 1-mol l−1 HCl and extracted three times with 3-ml chloroform, evaporated under vacuum to dryness. The extract was immediately dissolved in 1-ml methanol, filtered (Millex® HV 13 mm; Millipore Corporation, Bellerica, MA, USA) directly into amber HPLC vials and stored at 4°C until HPLC analysis was performed.

Extraction of OTA from agar plugs

OTA was extracted from agar plugs using the method developed by Bragulat et al. (2001). The plugs were weighed and placed into 2-ml Eppendorf tubes. Methanol (1 ml) was added, and the tubes were allowed to stand for 60 min, centrifuged at 16 000 g for 15 min. The supernatant was filtered (Millex® HV 13 mm; Millipore Corporation, Bellerica, MA, USA) directly into amber HPLC vials and stored at 4°C until HPLC analysis was performed.

Detection and quantification of OTA by HPLC

The production of OTA was detected and quantified by HPLC with fluorescence detection (λex = 332 nm; λem = 466 nm), using the analytical column 150 × 4·6-mm Uptisphere 5-μm C18 ODB (octadecylsilyl silica gel column) fitted with a guard column of 10 × 4 mm. The mobile phase was (water–acetic acid 0·2%, acetonitrile 59 : 41) at a flow rate of 1·0 ml min−1 and the column temperature was at 30°C. Kroma 3000 (BIO-TEK, Milan, Italy) was the data acquisition system. Injections were made with an autoinjector (BIO-TEK) and the injection volume was either 50 or 80 μl. OTA was identified by its retention time (15 min) according to a standard obtained from Sigma (Steinheim, Germany) and quantified by measuring the peak area. The detection limit was 0·025 μg l−1.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Partitioning of OTA into spores, biomass and medium in different isolates of Aspergillus carbonarius

Figure 1 compares the partitioning of OTA into spores, mycelium and substrate for four isolates of A. carbonarius at 20°C and 0·95 aw. This shows that for three of the four isolates, the OTA content per gram was highest in the spores, then the mycelium and finally the substrate. The only exception was the isolate from Greece (GR458) where there was more OTA in the substrate than the mycelium. Table 1 compares an isolate of A. carbonarius with one of A. ochraceus and shows that there is a significant difference in OTA partitioning. In the A. ochraceus isolate, most of the OTA was present in the substrate and some in the mycelial biomass. Very little was found in the spores.

image

Figure 1.  Comparison of the ochratoxin A content of four isolates of Aspergillus carbonarius grown on an artificial grape juice medium at 20°C and 0·95 aw for 20 days. All data are means of five replicates. Bars indicate standard errors. bsl00003, ITA1102; bsl00005, IS10·8; bsl00068, PORT219; bsl00021, GR458.

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Table 1.   Comparison of ochratoxin (μg g−1) partitioning into spores, mycelium and substrate by the isolates of Aspergillus carbonarius (PORT219) and Aspergillus ochraceus (ITA703) at 25°C and 0·95 water activity
 Aspergillus carbonariusAspergillus ochraceus
  1. *Mean ± SD.

Spores5·08 ± 0·72*0·06 ± 0·06
Mycelium2·00 ± 0·64 0·15 ± 0·05
Medium0·24 ± 0·031·05 ± 0·1

Figure 2 shows that OTA production was affected when A. carbonarius (2Mu134) was grown on the different media (CYA, CZ, MEA, SM, SGM and GJA). Aspergillus carbonarius (2Mu134) produced a higher quantity of OTA in CYA (13·3 μg g−1) followed by SM medium (5·1 μg g−1). Lower production was observed in CZ media, 0·4 μg g−1. Nutritional changes in the medium also had an effect on the OTA partitioning into conidia. Figure 2 shows that the partitioning of OTA into spores was higher in SM medium (0·1-pg OTA per conidium) followed by MEA and CYA (0·079 and 0·027 pg per conidium, respectively). Lower OTA partitioning was observed in the CZ medium.

image

Figure 2.  Comparaison of the ochratoxin A production and accumulation in the conidia of Aspergillus carbonarius (2Mu134) grown on different media at 25°C for 7 days. CYA, Czapek yeast extract agar; CZ, Czapek dox agar; MEA, malt extract agar; SM, synthetic medium; SGM, grape synthetic medium; GJA, grape juice agar medium. Bars indicate standard errors. bsl00024, OTA production capacity; □, pg OTA/conidium.

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OTA excretion ability of conidia of Aspergillus carbonarius 2Mu134 during germination

A germination study of A. carbonarius conidia was conducted in order to follow the changes in the OTA content during this early growth stage. Table 2 lists the OTA detected in the spores and the medium during germination. At 0-h incubation, the OTA concentration present in the spores was 0·029 μg per 107spores (the initial OTA concentration in the spore suspension was 0·031 μg per 107spores) and none detected in the medium. Between 0- and 2-h incubation, OTA in the conidia began to be excreted into the medium where it reached about 0·078 μg ml−1 (Table 2). After this, the OTA levels began to increase in both the spores and the medium reaching after 4 h of incubation a level in the spores (0·034 μg per 107spores) and the medium (0·105 μg ml−1) higher than the initial value present in the suspension (Table 2). Between 4 and 6 h of incubation, the OTA in the spores again started to be excreted into the medium reaching a maximum concentration of 0·140 μg ml−1. After this, the OTA level in the medium started to decrease owing to adsorption on the spores and remained relatively constant in both the spores and medium until the germination time (28 h) (Table 2).

Table 2.   Ochratoxin A (OTA) evolution (mean ± SD) during conidial germination of Aspergillus carbonarius (2Mu134) conidia on Czapek yeast extract agar (CYA) medium at 25°C
Time (h)μg OTA per 107sporesμg OTA per ml medium
  1. *Data were analysed statistically by the analysis of variance and Duncan’s multiple range test. Values in each column with different superscripts differ at P < 0·05.

 00·029b* ± 0·0020·000f
 20·016ef ± 0·0010·078d ± 0·002
 40·034a ± 0·0040·105b ± 0·012
 60·014f ± 0·0020·140a ± 0·016
 80·019ef ± 0·0020·093cb ± 0·006
100·021cd ± 0·0030·080cd ± 0·006
120·022cd ± 0·0030·076ed ± 0·003
170·023c ± 0·0020·071ed ± 0·007
280·023cd ± 0·0010·063e ± 0·004

The effect of tartaric and malic acids on the OTA excretion ability of conidia

The addition of 8 g l−1 in the culture media of both malic and tartaric acid decreased the germination time of conidia: 12 and 17 h in the presence of malic and tartaric acid, respectively, compared with 28 h for the control. The ability of OTA excretion from conidia was affected too; OTA was not excreted by the conidia in the presence of the two acids (Fig. 3a) during germination. Indeed, it was always present in the conidia (Fig. 3b).

image

Figure 3.  Effect of organic acids (8 g l−1) on the ochratoxin A excretion ability of conidia of Aspergillus carbonarius (2Mu134) during germination on Czapek yeast extract agar (CYA) medium at 25°C. (a) Ochratoxin A (OTA) evolution in the medium (□, Tartaric acid; x, malic acid; ○, control) and (b) OTA evolution in conidia (◊, Tartaric acid; x, malic acid; ○, control). Bars indicate standard errors.

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The effect of inoculum on the germination time of Aspergillus carbonarius conidia

The germination time was markedly affected by the initial inoculum concentration. The germination process occurs after 11 h of incubation at 25°C in CYA medium at a concentration of 107 spores ml−1. At a higher conidial concentration (109 spores ml−1), the germination time was around 28 h.

Dependence of OTA production on weak organic acid present in grapes

Malic and tartaric acid have an influence on the biosynthesis of OTA by A. carbonarius when grown on CYA medium. Malic acid stimulated the biosynthesis of OTA, which increased most rapidly in the medium supplied with 2·5 g l−1 and continued to increase with increasing acid concentration up to 5 g l−1. After that, it remained unchanged at concentration greater than 5 g l−1 (Fig. 4a). In contrast, OTA production was decreased in the presence of tartaric acid (Fig. 4b). The decrease was a function of the concentration of tartaric acid. Thus, no significant decrease was found at concentrations >8 g l−1 (Fig. 4b).

image

Figure 4.  Influence of malic (a) and tartaric (b) acids on the ochratoxin A production by Aspergillus carbonarius (2Mu134) grown on Czapek yeast extract agar (CYA) medium at 25°C for 7 days. Bars indicate standard errors.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

This study has shown for the first time that the partitioning of OTA into spores is significantly higher than that secreted into the medium by the isolates of A. carbonarius. Indeed, this is very different from other OTA-producing species, such as A. ochraceus. It is well known that the production of mycotoxins is dependent on the growth substrate and the environmental conditions (Ferreira 1967; Lai et al. 1970;Muhlencoert et al. 2004; Kokkonen et al. 2004). In our study, A. carbonarius produced a higher quantity of OTA in CYA followed by SM. Lower production was observed on CZ medium, which has the same composition of CYA but without yeast extract.

Results also showed that the nutritional status affected the partitioning of OTA into the spores of A. carbonarius. This OTA may be present in the outer layer of the spores as demonstrated by Stormer et al. (1998) for citrinin. In the case of A. carbonarius (2Mu134), when the spores were subjected to washing by 7 ml of distilled water (three times) just after the filtration through 0·45-μm Millipore filter, OTA levels in the conidia were decreased from 0·0425 to 0·00863 pg per conidium. This decrease may be attributed to the release of the accumulated OTA from the outer layer of the spores. Other studies have demonstrated the presence of mycotoxins in airborne dust (Skaug 2003; Jarvis and Miller 2005) and fungal conidia (Stormer et al. 1998; Fischer et al. 2000; Skaug et al. 2001). Conidia collected from the cultures of Penicillium verrucosum and A. ochraceus contained 0·4–0·7 and 0·02–0·06-pg OTA per conidium, respectively (Skaug et al. 2001). Moreover, a recent study has shown that approximately 60% of the mycotoxins found in the culture extracts of 205 Penicillium and Aspergillus species were also found in the conidial extracts indicating that a series of metabolites and mycotoxins are not only excreted by the fungi into the substrate, but can also be expected to be attached to or present in the conidia (Fischer et al. 2000).

A possible function of OTA, which may be beneficial to spores of A. carbonarius, could be protection against UV radiation. This has previously been described for dityrosine in the outer layer of yeast spores and similarly suggested for citrinin in the spores of P. verrucosum (Stormer et al. 1998). These compounds have absorption maxima at 300–330 nm, which corresponds to the part of the total spectrum at sea level that is responsible for the mutagenic action of sunlight (Stormer et al. 1998).

In the present work, A. carbonarius conidia have been shown to excrete their accumulated OTA into the medium during the first few hours of conidial germination. Stormer et al. (1998) suggested that citrinin could prevent the germination of the spores of P. verrucosum until it has been released. Similarly, this could be suggested for OTA in the spores of A. carbonarius. Moreover, OTA in the spores reached a level higher than the initial OTA concentration present in the spore suspension after 4 h of germination (Table 2). This OTA increase could be explained by the fact that OTA was initially present inside the spores and started to be excreted. The decrease in the OTA level in the medium after 6 h of incubation (Table 2) was attributed to adsorption on the spores. In fact, Bejaoui et al. (2005) demonstrated that A. carbonarius conidia have the capacity to adsorb OTA until the appearance of well-developed mycelium. Furthermore, no OTα, a degradation product of OTA, was detected in the medium during this study.

The OTA accumulation pattern and its excretion from A. carbonarius conidia may have significant relevance when applying the Hazard Analysis and Critical Control Point (HACCP) approach during grape processing. In fact, the spores of A. carbonarius are always present in the field. Contamination with spores occurs from colonized crop debris and from the airspora. The presence of the conidia on grapes and in must could be an important source of OTA contamination as they have the capacity to excrete their accumulated OTA into the substrate. Additionally, it has been shown that OTA is produced in the field and that A. carbonarius is responsible (about 41–96%) for this production (Varga and Kozakiewicz 2006). However, OTA has also been observed to increase in must during the maceration step (Varga and Kozakiewicz 2006). The OTA releasing phenomenon observed during the first hours of conidial germination may raise the question about the contribution of A. carbonarius conidia to the OTA increase in must during maceration.

Other studies have shown that trans-Resveratrol (30 μg g−1) and piceatannol (2 μg g−1) addition to the synthetic must medium triggers OTA production by A. carbonarius (Bavaresco et al. 2003). Molina and Giannuzzi (2002) showed that aflatoxin production by Aspergillus parasiticus was inhibited in the presence of propionic acid, the inhibitory effect being attributed to the undissociated fraction of the organic acid. In the present work, it is clearly demonstrated that tartaric acid has a repressive and malic acid a supporting effect on the ochratoxigenic ability of A. carbonarius (2Mu134). These acids were also shown to affect the time course of germination and the OTA excretion ability of conidia during germination.

To our knowledge, this is the first study to examine the partitioning and accumulation of OTA in spores, mycelium and medium, and the changes which occur in OTA during the germination of fungal conidia. Ochratoxin accumulation by the conidia has been shown to be affected by the media composition. In addition to the health problems associated with inhalation exposure to the fungal conidia, conidia has been shown to excrete their accumulated OTA in to the medium during the first hours of conidial germination which could be the reason for an increase in toxin in must during maceration. The OTA excretion ability of conidia was decreased in the presence of organic acids present in grapes and wine. This could be an advantage as malic and tartaric acids are commonly used to adjust the acid balance of grape juice and wine (Coloretti et al. 2002).

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

This work was supported by grants from the European Union (QLK1-CT-2001-01761) and French “Ministère de la jeunesse de l’éducation et de la recherche” (AQS N°:02 PO571).

The authors are grateful to the Lebanese National Council for Scientific Research for according a PhD scholarship to Mr Ali Atoui.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References
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