Fungal flora and ochratoxin a production in grapes and musts from france

Eleven samples of grapes and musts used in red table wines were investigated for the occurrence of potential ochratoxin A (OTA)-producing molds. From these samples, 59 filamentous fungi and 2 yeasts were isolated. Among the 30 genera isolated, Deuteromycetes were the most frequent (70%) followed by Ascomycetes (10%). Six of the eleven grapes samples were contaminated by potentially ochratoxinogenic strains (Penicillium chrysogenum and Aspergillus carbonarius). When cultivated in vitro on solid complex media, the 14 strains of A. carbonarius produced OTA. No other species produced OTA under the same conditions. Among must samples, eight of eleven were found to be contaminated by OTA (concentrations from <10 to 461 ng/L). There is a strong correlation between the presence of ochratoxin-producing strains on grapes and OTA in musts. These findings should be connected with the OTA contamination of human blood in these areas and in France.     

trans-Resveratrol, quercetin, (+)-catechin, and (-)-epicatechin content in south Italian monovarietal wines: relationship with maceration time and marc pressing during winemaking

The concentrations of trans-resveratrol, (+)-catechin, (-)-epicatechin, and quercetin were evaluated by means of high-performance liquid chromatography-diode array detection in red wines obtained from Aglianico, Piedirosso, and Nerello Mascalese grapes. The trans-resveratrol and epicatechin concentrations did not differ significantly between experimental wines. The concentration of quercetin in Nerello Mascalese wines was more than twice that observed in Aglianico and Piedirosso wines. Nerello Mascalese wines also significantly differed from other wines in the (+)-catechin content, which was significantly higher than those found in the other two wines. During maceration, the maximum extraction of trans-resveratrol was reached after 12 days for Aglianico and Piedirosso, after which a decline was observed. On the contrary, in the case of Nerello Mascalese, the concentration of trans-resveratrol increased steadily throughout the whole maceration process. After 2 days of maceration, the maximum concentration of quercetin was observed in Aglianico must, whereas the maximum quercetin extraction was reached after 12 days for Piedirosso and 17 days for Nerello Mascalese. The maximum levels of (+)-catechin and (-)-epicatechin were generally observed after 12 days of maceration for all wines, although a decline of (-)-epicatechin occurred after maximum extraction in Aglianico and Piedirosso wines. Following marc pressing, a significant increase in the concentration of trans-resveratrol for Aglianico, (+)-catechin and (-)-epicatechin for Piedirosso, and (-)-epicatechin for Nerello Mascalese was observed.     

Effect of the maceration technique on the relationships between anthocyanin composition and objective color of Syrah wines

The effects of two different vinification techniques, traditional fermentation and carbonic maceration, on the anthocyanin composition and color of young red wines, made with Syrah grapes grown in a warm climate, were compared. Tristimulus colorimetry was applied to study the color of wines during the vinification, and a high-performance liquid chromatography (HPLC) procedure was used for the analysis of anthocyanins. Carbonic maceration led to wines with lower anthocyanin content, mainly monoglucosides, and total phenols. This was related to lighter wines, less saturated, but more colorful (higher chroma C*ab values), and hues hab similar to those of the Syrah wines made by traditional vinification. Thus, the lightness L* had much more influence on the saturation s*uv of the wines obtained by carbonic maceration than the chroma (s*uv = C*uv/L*). From a study of the color-composition relationships using linear and multiple regression, better relationships were found for the wines from traditional vinification, where the chromatic parameters L*, hab, and s*uv could be predicted from the 3-monoglucosides of delphinidin, petunidin, peonidin, and malvidin concentrations (R > 0.9). However, a good prediction of the chroma C*ab from the anthocyanin composition was not possible. On the contrary, C*ab was the best predicted parameter from the anthocyanins monoglucosides (R > 0.9) in the carbonic maceration wines.     

Polyphenol content and total antioxidant activity of vini novelli (young red wines)

Eight commercial Italian vini novelli (red wines prepared by carbonic maceration and supposed to be consumed within three months from their wine-making) were evaluated for their total antioxidant activity. The wines had an average total phenol content (1605.4 +/- 337.4 mg/L gallic acid equivalents) lower than that of wines prepared by traditional maceration and consumable after aging (2057. 3 +/- 524.0 mg/L gallic acid equivalents). The average flavanol content (424.7 +/- 121.3 mg/L catechin equivalents) and the total antioxidant activity (16.8 +/- 3.8 mmol/L Trolox equivalents) of vini novelli were higher than the corresponding values (382.7 +/- 174.5 mg/L catechin equivalents and 12.3 +/- 3.3 mmol/L Trolox equivalents) found for aged wines. Three couples of experimental wines were prepared from the same grapes by traditional or carbonic maceration. These wines showed a different phenolic pattern, anthocyanins being more abundant in vini novelli. However, the average total antioxidant activities of the wines were similar, suggesting that aging (and not the wine-making technique) is the main factor influencing the antioxidant activity of red wines.     

Occurrence of ochratoxin A- and aflatoxin B1-producing fungi in Lebanese grapes and ochratoxin a content in musts and finished wines during 2004

This paper reports the results of an extensive survey on the occurrence of filamentous fungi isolated from wine-grapes in Lebanon and to test their ability to produce ochratoxin A (OTA) and aflatoxin B1 (AFB1) on CYA culture medium, in order to assess their potential for producing these mycotoxins on grapes. From the 470 grapes samples taken during season 2004, 550 fungi strains were isolated with 490 belonging to Aspergillus spp. and 60 belonging to Penicillium spp. All these isolated fungi starins were tested for their ability to produce OTA and AFB1. Aspergillus carbonarius shows that it is the only species able to produce OTA with a production percentage reaching 100% and a maximum concentration of 52.8 microg/g of Czapek yeast extract agar (CYA). In its turn, Aspergillus flavus was considered as the only AFB1-producing species with production percentage of 45.3% and a maximum concentration reaching 40 microg/g CYA. A total of 47 handmade musts produced from the collected grapes were also analyzed in order to correlate the presence of OTA in must and the occurrence of filamentous fungi on grapes; 57.4% were contaminated with OTA at low level with concentrations ranging between 0.011 and 0.221 microg OTA L(-1). The analysis of these must samples was not performed with regard to AFB1. Seventy samples of finish red wine were also assayed for OTA content. The results showed that 42 of the tested samples (60%) were found to be positive for OTA with low levels (0.012-0.126 microg OTA L(-1)).     

Characterization of the mean degree of polymerization of proanthocyanidins in red wines using liquid chromatography-mass spectrometry (LC-MS)

An HPLC-MS method for the characterization of proanthocyanidins (PA) has been refined. Further application to red wines provided interesting conclusions about the composition of the flavanol fraction and PA extractability during winemaking. The yield in PA extraction increases with the length of the postfermentative maceration (PFM), as well as the mean degree of polymerization (mDP) of wine flavanols. In early winemaking events mostly monomers to trimers are extracted from grape solids, whereas PFM is required for the significant extraction of higher oligomers. Nevertheless, at the end of a regular process of elaboration the mDP is not very high and does not usually exceed a value of 2.3, dimers and trimers being the predominant flavanols in red wines. With regard to groups of compounds, gallocatechins and prodelphinidins (located in the skins) are extracted rapidly in the first stages of the winemaking. On the contrary, long postfermentative macerations are required for the extraction of galloyled derivatives from the seeds. PA extractability is also dependent on the grape variety used for winemaking. Thus, wines made with Graciano grapes were found to require a longer process of PFM than those made from Tempranillo grapes to obtain similar yields in the extraction of flavanols.     

Removal of ochratoxin A in red wines by means of adsorption treatments with commercial fining agents

The presence in wine of the fungal metabolite, ochratoxin A (OTA), represents a serious risk for consumer health. A variety of fining agents, including activated carbon, silica gel, potassium caseinate, egg albumin, and gelatin, was evaluated in relation to their abilities to remove OTA in fortified wines. Freundlich adsorption isotherms were used to model the adsorption behavior between ochratoxin A and the fining agent. Potassium caseinate and activated carbon were found to be the best fining agents that could be used to remove OTA in wine. Potassium caseinate removed up to 82% of OTA when used at 150 g/hL, whereas activated carbon showed the highest specific adsorption capacity due to a high surface area per mass and low adsorption of total polyphenols.     

Residues of spiroxamine in grapes following field application and their fate from vine to wine

Dissipation of the fungicide spiroxamine in grapes of two vine varieties, Roditis and Cabernet Sauvignon, exposed to field treatments was evaluated. Vines of a grape vineyard located in central Greece were sprayed once or twice with a commercial formulation of the fungicide at 30 g a.i./hL. Residues in grapes, must, and wine were determined by gas chromatography/IT-MS after extraction with cyclohexane-dichloromethane (9:1), with a limit of quantitation 0.02 mg/kg in grapes and 0.012 mg/kg in wine. Under field conditions, spiroxamine dissipation on grapes was faster during the first 2 weeks and then slower to the sixth week. About 7 days after application, half of the initial spiroxamine concentration remained on the grapes; the respective proportion at 42 days was about 10%. At 14 and 35 days, residues were lower than 0.44 and 0.22 mg/kg, respectively, values below the maximum residue levels set by the European Union (1 mg/kg). Spiroxamine residues transferred from grapes into the must and through the vinification process into the wine were also studied. Mean transfer factors of 0.26 and 0.55 were found from grapes into wine for the wines obtained without maceration and with maceration, respectively. Residues in wine, prepared from grapes with a spiroxamine content of 0.11-0.20 mg/kg, varied from <0.026 to 0.09 mg/kg. Spiroxamine diastereomer B was found to dissipate slower than diastereomer A in the field as well as during the vinification process.     

Ochratoxin a contamination in italian wine samples and evaluation of the exposure in the italian population

The scope of this study was to evaluate the exposure of the Italian population to ochratoxin A (OTA) attributable to wine consumption. With this aim 1166 wine samples (773 red wines, 290 white, 75 rose, and 28 dessert wines), collected in 19 different Italian regions and mostly produced between 1988 and 2004, were analyzed for OTA content. The obtained results are reported by year of harvest, geographical area of production, and type of wine. Red wine showed the highest maximum level of contamination (7.50 ng/mL), even though rose wines were characterized by a higher mean value (0.01 ng/mL). A gradually increasing mean concentration was also observed from the north (0.05 ng/mL) to south of Italy (0.54 ng/mL). Exposure calculations, performed using two different consumption databases, indicate a daily intake for consumer only of 0.59 up to 1.24 ng/(kg of b.w.)/day and of 0.33 up to 0.90 ng/(kg of b.w.)/day for the total population. Even in the worst case, corresponding to the calculation of the intake for consumers only in southern Italy and Islands and considering the mean consumption data increased by 1 standard deviation, a quite low exposure (1.68 ng/(kg of b.w.)/day, accounting for 9.8% of TDI) was obtained. Considering the overall OTA dietary exposure, obtained exposure rates indicate that wine did not pose a risk to the Italian population health.     

Changes of ochratoxin A in grapes inoculated with Aspergillus carbonarius and subjected to chamber-drying under controlled conditions

The production pattern for ochratoxin A in grapes inoculated with Aspergillus carbonarius and changes in its concentration during raisining of Merlot, Syrah, Tempranillo, and Cabernet Sauvignon red grapes and Pedro Ximenez white grape were studied. Grapes were chamber-dried under controlled temperature and humidity conditions, with and without dipping pretreatments in alkaline emulsions of olive oil or ethyl oleate. Based on the results for the grapes that developed the fungus (Merlot and Pedro Ximenez), a temperature of 50 °C in the absence of dipping stopped ochratoxin A production and even degraded part of the toxin already formed. Both dipping pretreatments facilitated removal of the toxin and led to its virtually complete disappearance. However, dipping in the ethyl oleate emulsion caused substantial changes in the sensory characters of the musts obtained from the raisins, so it should be avoided to ensure the expected quality in the sweet wines elaborated from them.     

Improving grape phenolic content and wine chromatic characteristics through the use of two different elicitors: methyl jasmonate versus benzothiadiazole

Benzothiadiazole (BTH) and methyl jasmonate (MeJ) have been described as exogenous elicitors of some plant defense compounds, polyphenols among them. The objective of this study was to determine whether the application of BTH or MeJ to grape clusters at the beginning of the ripening process had any effect on the accumulation of the main flavonoid compounds in grapes (anthocyanins, flavonols, and flavanols) and the technological significance of these treatments in the resulting wines. The results obtained after a 2 year experiment indicated that both treatments increased the anthocyanin, flavonol, and proanthocyanidin content of grapes. The wines obtained from the treated grapes showed higher color intensity and total phenolic content than the wines made from control grapes. The exogenous application of these elicitors, as a complement to fungicide treatments, could be an interesting strategy for vine protection, increasing, at the same time, the phenolic content of the grapes and the resulting wines.     

Levels of stilbene oligomers and astilbin in French varietal wines and in grapes during noble rot development

Phenolics from grapes and wines can play a role against oxidation and development of atherosclerosis. Stilbenes have been shown to have cancer chemopreventive activity and to protect lipoproteins from oxidative damage. A method for the direct determination of stilbene oligomers (viniferin and pallidol) as well as astilbin in different types of wine using high-performance liquid chromatography with UV detection is described. In a survey of 21 commercial wines from the south of France, levels of pallidol and viniferin are reported for the first time in different types of wines. Viniferin was found to be present only in red and botrytized sweet white wines with levels between 0.1 and 1.63 mg/L; pallidol was not found in dry and sweet white wines but only in wines made by maceration with stems, with levels between 0.38 and 2.22 mg/L. Highest levels of astilbin were found in Egiodola (15.13 mg/L), Merlot (11.61 mg/L), and Cabernet Sauvignon (8.24 mg/L) for red wines and in Sauvignon (5.04 mg/L) for white varietal wines. Astilbin levels are highest for recent vintages, but pallidol is not found in older vintages. During noble rot development in Sauvignon or Sémillon grapes from the Sauternes area, levels of trans-astringin, trans-resveratrol, trans-piceid, and pallidol are quite low (<0.5 mg/kg of grapes). Viniferin and astilbin levels become optimum at 2 and 30 mg/kg, respectively, during spot grape and speckle grape stages.     

Pulsed electric field-assisted vinification of aglianico and piedirosso grapes

Pulsed electric field (PEF) treatments were applied to increase the polyphenolic content of fresh red wines made from Aglianico and Piedirosso grapes. Prior to the fermentation/maceration step, the grape skins were treated at different PEF intensities (field strengths from 0.5 to 1.5 kV/cm and energy inputs from 1 to 50 kJ/kg), with their permeabilization being characterized by electrical impedance measurements. Furthermore, the release kinetics of the total polyphenols and anthocyanins were characterized during the maceration stage by spectroscopic and Folin-Ciocalteu colorimetric methods, respectively. Finally, the fresh wine, obtained after pressing, was characterized for total acidity, pH, reducing sugar, color intensity, total polyphenols, anthocyanins content, antioxidant activity, and volatile compound composition. PEF treatment on Aglianico grapes induced a significantly higher release of polyphenols (+20%) and anthocyanins (+75%), thus improving the color intensity (+20%) and the antioxidant activity of the wine (+20%) while preserving the other organoleptic characteristics. In contrast, there was only a minor impact on the polyphenolic release kinetics of Piedirosso grapes, despite the significant degree of cell membrane permeabilization.     

Assay of ochratoxin A in wine and beer by high-pressure liquid chromatography photodiode array and gas chromatography mass selective detection.

To routinely assay the concentrations of ochratoxin A (OTA) in wines and beers, two new methods were developed and evaluated. The first utilized solid-phase extraction on a C(18) cartridge to achieve a 100-fold sample concentration followed by high-performance liquid chromatography on a C(18) column with gradient elution and quantitation at 333 nm by means of a photodiode array detector. Positive confirmation can be carried out by purity and match-factor analysis as well as peak shift following esterification with BF(3). Total run time is 28 min. The limits of detection (LOD) and quantitation (LOQ) are 0.05 and 0.10 microg/L, respectively. Recovery and imprecision ranged from 83 to 94% and from 4.0 to 8.9%, respectively. With a throughput of 35 assays per working day, this method is ideal for routine OTA analysis. It was used to survey the concentrations of OTA in 942 wines (2 of which gave values between 0.1 and 0.2 microg/L) and 107 beers (2 of which gave values between 0.05 and 0.1 microg/L). OTA was detected more frequently in red than white wines, with the highest incidence in red wines from Spain and Argentina. There was no association between OTA and country of origin or beverage type among the beers analyzed. The second method utilized gas chromatography with mass selective detection monitoring eight specific ions, preceded by extraction in dichloromethane and derivatization with bis[trimethylsilyl]trifluoroacetamide. LOD and LOQ were 0.1 and 2 microg/L, respectively; recovery and imprecision were 69-75 and 9.0-11.1%, respectively. The method is not suitable for routine quantitation but is potentially useful as a confirmatory tool for samples with OTA > or =0.1 microg/L.     

Influence of grape maturity and maceration length on color, polyphenolic composition, and polysaccharide content of cabernet sauvignon and tempranillo wines

The aim of this paper was to study how maturity and maceration length affect color, phenolic compounds, polysaccharides, and sensorial quality of Cabernet Sauvignon and Tempranillo wines at three stages of grape ripening. Ripeness increased color extractability, phenolic compounds, and polysaccharide concentrations. Moreover, the proanthocyanidin mean degree of polymerization (mDP) and the percentage of prodelphinidins also increased with maturity, whereas the percentage of galloylation decreased. In general, wines from riper grapes contain higher proportions of skin proanthocyanidins. Color and anthocyanin concentration decreased when the maceration was longer, whereas polysaccharide and proanthocyanidin concentrations did the opposite. It was also detected that the mDP and the percentage of prodelphinidins decreased when the maceration was extended, whereas the percentage of galloylation increased. These data seem to indicate that proanthocyanidin extraction from seeds is clearly increased throughout the maceration time.     

Development of a sensitive enzyme-linked immunosorbent assay for the determination of ochratoxin A

Polyclonal antibodies for ochratoxin A (OTA) were generated from rabbits after the animals had been immunized with either OTA-gamma-globulin or OTA- keyhole limpet hemocyanin (KLH). A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA (ciELISA) were used for the characterization of the antibodies and for analysis of OTA in various agricultural commodities. The antibody titers in the serum of rabbits immunized with OTA-gamma-globulin were considerably higher than those in rabbits immunized with OTA-KLH. The antibodies from the rabbits immunized with OTA-gamma-globulin were further characterized. In the cdELISA, the concentrations causing 50% inhibition (IC(50)) of binding of OTA-horseradish peroxidase to the antibodies by OTA, ochratoxin B (OTB), and ochratoxin C (OTC) were found to be 0.90, 110, and 0.54 ng/mL, respectively. When 10 to 250 ng/g of standard OTA was spiked to soybean samples and then extracted with 50% aqueous methanol, the recovery rate of OTA was found to be 85.9% in the cdELISA. Analysis of OTA in various agricultural commodities showed that 12 of the 20 examined samples were contaminated with OTA at levels from 16 to 160 ng/g. The efficacy of cdELISA was also confirmed by the high-performance liquid chromatography method.     

Quantitation of ochratoxin A in South African wines

The natural occurrence of the carcinogenic mycotoxin ochratoxin A (OTA) in wines sold in local retail outlets in South Africa and Italy was investigated by HPLC analysis with fluorescence detection following cleanup by immunoaffinity column. All 24 local South African wines tested (15 white and 9 red) were found to contain detectable levels (>0.01 microg/L) of OTA, with a mean of 0.16 microg/L in the white wines and a mean of 0.24 microg/L in the red wines. Results were subsequently confirmed by LC-MS analysis using positive ion electrospray ionization with collision-induced dissociation of the protonated molecular ion [M + H](+) at m/z 404 and selected reaction monitoring of the resultant product ions [M + H - H(2)O - CO](+) at m/z 358 and [M + H - H(2)O](+) at m/z 386. Comparison with the fluorescence method gave a significant correlation (r = 0.87; p < 0.01). Although OTA contamination was present in all of the South African samples analyzed, levels were well below the suggested European Union limit of 0.5 microg/kg. The highest level found in a locally purchased wine was 0.39 microg/L in a blend of local and imported Spanish red wine. Of the eight Italian wines analyzed, only two red wines were contaminated above the suggested maximum level.     

The extraction of anthocyanins and proanthocyanidins from grapes to wine during fermentative maceration is affected by the enological technique

The effect of three enological techniques (low temperature prefermentative maceration, must freezing with dry ice, and the use of a maceration enzyme) on the extraction of anthocyanins and proanthocyanidins from must to wine during fermentative maceration was studied to determine the extent to which these compounds are extracted and to assess the changes on their qualitative composition due to enological technique applied. The results showed that the dry ice treatment led to wines with high color intensity and high anthocyanin content, the maximum rate of extraction being observed the first 6 days of fermentative maceration. Regarding the effect of the different techniques on the quantitative and qualitative composition of proanthocyanidins, only the dry ice treatment seemed to favor the extraction of high molecular weight skin proanthocyanidins. The low temperature prefermentative maceration treatment led to the highest concentration of proanthocyanidins at the moment of pressing; however, this treatment, contrary to expectations, led to wines with the highest content of seed-derived proanthocyanidins. The use of the maceration enzyme also increased the concentration of proanthocyanidins during all of the fermentative process, as compared to a control wine, although the increase was not only due to skin proanthocyanidins but also seed proanthocyanidins. We have demonstrated in this study that maceration enzymes also facilitate seed phenolic extraction.     

Evolution of chlorpyrifos, fenarimol, metalaxyl, penconazole, and vinclozolin in red wines elaborated by carbonic maceration of Monastrell grapes

The influence of the different steps involved in the wine-making process on the disappearance of chlorpyrifos, fenarimol, metalaxyl, penconazole, and vinclozolin in red wines elaborated by carbonic maceration of Monastrell grapes was studied. The initial levels of the residues in grapes ranged from 0.28 mg/kg (penconazole) to 1 mg/kg (chlorpyrifos). Ten days after the beginning of maceration, the compound that had decreased least was chlorpyrifos (83% of initial value remaining), whereas metalaxyl, the least persistent of the residues, had decreased to 49% of its initial value. In the free-run juice, on the other hand, the highest percentage remaining corresponded to metalaxyl (10%) and the lowest to chlorpyrifos (0. 1%). After pressing, the percentages of initial values eliminated in pomace varied from 82.7% for chlorpyrifos to 17.7% for metalaxyl, whereas in the press juice the opposite was the case (37% metalaxyl and 2% chlorpyrifos remaining). In finished wine, there were residues of all the pesticides, with the exception of chlorpyrifos, metalaxyl (21%) being the most persistent. The percentages eliminated in the lees varied from 1.5 to 2.5% of the initial value.     

Effect of flash release treatment on phenolic extraction and wine composition

The flash release (FR) process, consisting of rapidly heating the grapes and then applying strong vacuum, has been proposed to increase the polyphenol content of red wines. Its impact on polyphenol extraction kinetics and on the polyphenol composition of red juice and wines was studied over two seasons on different grape varieties (Grenache, Mourvedre, Carignan). The FR process allows fast extraction of all phenolic compounds (hydroxycinnamic acids, flavonols, anthocyanins, catechins, proanthocyanidins) and can be used to produce polyphenol-enriched grape juices. However, the concentration of all polyphenols dramatically decreased throughout fermentation when pressing was achieved immediately after FR. The FR wines made with pomace maceration were also enriched in polyphenols compared to the corresponding control wines. Increasing the duration of high-temperature exposure in the FR treatment further increased extraction of phenolic compounds but also accelerated their conversion to derived species. The tannin-to-anthocyanin ratio was particularly low in the wine fermented in the liquid phase, higher after FR than in the control, and even higher after longer heating. FR resulted in an increased tannin-to-anthocyanin ratio and an increased conversion of anthocyanins to tannin-anthocyanin adducts showing the same color properties as anthocyanins. The tannin-to-anthocyanin ratio was particularly low in the wine fermented in the liquid phase that also contained larger amounts of orange sulfite bleaching-resistant pigments.