The role of processing on ochratoxin A content in Italian must and wine: a study on naturally contaminated grapes

Available data show that ochratoxin A (OTA) is a possible contaminant of wine and its concentration is higher in red wines than in white and rosé. The aim of this work was to study the fate of OTA during the main stages of the winemaking process (crushing, maceration, alcoholic fermentation, malo-lactic fermentation, bottle-aging) and the influence of technological treatments on OTA concentration in order to identify the critical process steps. Attention was focused on red winemaking, in different wineries in the south of Italy, with two naturally OTA contaminated grape varieties ('Negroamaro' and 'Primitivo') and on different vintages (2001-2002-2003). The results show that no OTA is produced during winemaking, but each operation during winemaking can modify OTA content. The OTA present in grapes to a certain degree is released to the juice during crushing. Maceration increases the OTA content, while alcoholic and malo-lactic fermentation cause a reduction in OTA in the wine.     

Vinification Effects on Changes in Volatile Compounds of Wine

Alcohols, esters and fatty acids in wines from red, rose and carbonic maceration of Monastrell grapes were measured during yeast fermentation. Isolated volatile compounds were analyzed by gas chromatography alone and with-mass spectrometry. Increases in these compounds was most important during the first days of fermentation and concentrations varied between vinifications. The lower fermentation temperature and shorter skin contact time of rose vinification produced higher concentrations of esters and acids than in red wine. The intracellular fermentation in carbonic maceration vinification resulted in formation of almost all esters and fatty acids before alcoholic fermentation started. The final wine was closer to rose than red wine in aromatic composition.     

European research on ochratoxin A in grapes and wine

European wine production represents about 70% of world production and thus is an important export commodity. Ochratoxin A (OTA) was first detected as a wine contaminant in 1996 and the role of Aspergillus section Nigri and A. carbonarius in OTA production discovered in Europe in 1999. Subsequently Europe-wide surveys have shown that A. carbonarius is predominantly responsible for OTA contamination of grapes, wine and vine fruits. Analyses of wine samples throughout Europe have shown that there is a gradient in OTA concentration with a decrease from red, to rose and to white wines. The latitude of production is an important factor in determining risk from OTA contamination. Some geographic regions in Southern Europe are more prone to contamination with the toxigenic species and OTA. Ochratoxin A has also been found in much higher concentrations (max. 53 mug/kg) in dried vine fruit than in wine suggesting that A. carbonarius can dominate the drying vine fruit ecosystem. There is a significant lack of knowledge in Europe on conducive climatic conditions preharvest and their relationship with levels of risk from OTA contamination in grapes and their fate in wine production. This needs to be integrated with cultivation system to maximise the prevention of OTA entering this food chain.     

Managing ochratoxin A risk in the grape-wine food chain

The main source of ochratoxin A (OTA) in the wine food chain is the infection of grapes by "black aspergilli" in the field. OTA-producing black aspergilli include principally Aspergillus carbonarius, followed by A. niger and possibly A. tubingensis. They are opportunistic fungi that develop particularly on damaged berries at ripening, although they may occur and form OTA on grapes from veraison to harvest. Climatic conditions (high humidity and temperature) and geographical location are important factors favouring OTA accumulation in grape berries. The severity of aspergillus rot is influenced by excessive irrigation and rainfall prior to harvest, which causes berry splitting. In addition, berry wounds caused by insect attack provide preferential entries for black aspergilli. High OTA levels occur in grapes severely damaged by the grape moth, Lobesia botrana, particularly in Mediterranean areas. Some grape varieties display greater susceptibility to aspergillus rot due to intrinsic genetic characteristics and bunch conformation (i.e. compact>sparse). Control measures for toxigenic mycoflora in the vineyards must consider these critical control points. Proper fungicidal and insecticidal treatments can reduce OTA contamination. Nevertheless, knowledge about the fate of OTA and its distribution in wine and winery by-products is important to manage OTA risk in contaminated stock. In our wine-making experiments, only 4% of the OTA present in grapes remained in the wine--the majority is retained in pressed grape pomaces. OTA concentration remained unchanged in wine after a 1-year aging as well as in all liquid fractions collected during vinification (i.e. must, free run wine, and wine after first and second decantation). Activated carbon can reduce OTA levels in wine but negatively affects wine quality.     

Removal of Ochratoxin A (OTA) from Naturally Contaminated Wines During the Vinification Process

In this study, 28 red and 18 white musts, from seven different local wineries in Turkey, were investigated to determine their ochratoxin A (OTA) content following the 2006 harvest period. The removal conditions of OTA during the main stages of the wine making processes (alcoholic fermentation, malolactic fermentation, racking, fining and storage) were investigated using HPLC techniques. In addition, the removal of OTA from two samples of red musts containing more than 1.0 μg/L of OTA and one sample of white must containing 0.83 μg/L of OTA were studied. The percentage of OTA removal in the final wine was found to be 41.8% (Sample A) and 53.7% (Sample B) in red wine and 44.7% in the white wine. The activated carbon had no greater effect on removing OTA than the other fining agents. The wine bottle ageing processes decreased the OTA content of the red wine between 11.8% and 17.6% during 12 and 18 months of storage, respectively. The 24 month ageing period did not affect the OTA level of the red wine further compared to the 18 month period.     

Antioxidant activity and phenolic compounds in organic red wine using different winemaking techniques

Abstract: Wine phenolic composition depends on the grapes used to make wine and on vinification conditions. The occurrence of these biological compounds has stimulated numerous studies focused on understanding the mechanisms that influence their concentrations in wine. This article studied the effect of different vinification techniques on the antioxidant activity and on the phenolic compounds of red wine made from the variety of Monastrell grapes obtained by organic culture. To this purpose, 3 different vinification procedures were carried out: vinification after prolonged maceration, vinification with the addition of enological enzymes, and traditional vinification procedures (used as control).The results showed similar values of antioxidant activity in all 3 types of wine elaborated and found no differences in the concentrations of the different types of phenolic compounds in wine made with the 3 different methods. The evolution of antioxidant activity and phenolic compounds tested in wines during 3 mo of storage showed a similar pattern. Organic wine has acquired an important role in the economic world and its important, working in oenology to research in this field.     

Detection of ochratoxin A and cis- and trans-resveratrol in red wines and their musts from Calabria (Italy)

The natural occurrence of ochratoxin A (OTA) and cis- and trans-resveratrols in red wines has been widely reported. The aim of this work was to evaluate the ochratoxin A (OTA) and both cis- and trans-resveratrol content of red wine (from must to wine) in a pilot-scale vinification process in Calabria (Italy). Eleven samples were collected at different stages of vinification and analysis was carried out by HPLC. Wine from manufacturer 3 contained the highest amount of trans-resveratrol (3.41?mg?l^?1). This wine was characterized by an Aglianico–Magliocco grape variety. Interestingly, data regarding OTA showed that the value of this contaminant was low in all analyzed samples and, in each case, below the legal limit (2.0?mg?l^?1 (ppb)). Overall, the results demonstrated the high quality of wines produced in Calabria.     

OCHRATOXIN A IN TURKISH WINES

In this research, 47 wine samples produced from 2001 to 2002 harvest period in four Turkish regions were analyzed for the presence of ochratoxin A (OTA). Based on the results, the OTA levels of red wines are generally higher than those of white and rose wines. However, the wines from two wine regions (Aegean and Thrace) are much more contaminated with OTA compared with the others.     

Influence of yeast strain on ochratoxin A content during fermentation of white and red must

The aim of this work was to examine whether the yeast strains, responsible for alcoholic fermentation, have an influence on the concentration of ochratoxin A (OTA) in wine. Before the fermentation, OTA was added to musts up to a concentration of about 2 microg/l. OTA content was determined in white and red wines resulting from respective musts and in methanolic extract of the yeast lees (MEL). Data showed a significant reduction of OTA at the end of alcoholic fermentation. However, depending on the yeast strain involved in the fermentation, there was a difference in the content of OTA in the wines. The percentage of OTA removal during the fermentation was between 46.83% and 52.16% in white wine and between 53.21% and 70.13% in red wine. The absence of degradation products suggested an adsorption mechanism. OTA concentration in MEL resulting from red must fermentation was higher than in white. A significant amount of OTA was not recovered either from wine or from MEL.     

Development of a quantitative real-time PCR assay for the detection of Aspergillus carbonarius in grapes

Aspergillus carbonarius is the main species responsible for the production of ochratoxin A (OTA) in wine grapes. To monitor and quantify A. carbonarious in grapes, a quantitative real-time PCR assay was developed as a possible tool for predicting the potential ochratoxigenic risk. DNA extraction from grape berries was performed by using conventional extraction and clean up through EZNA Hi-bond spin columns. A TaqMan probe was used to quantify A. carbonarius genomic DNA in grape berries samples. An exogenous internal positive control was used to overcome DNA recovery losses due to matrix inhibition. The quantification of fungal genomic DNA in naturally contaminated grape was performed using the TaqMan signal versus spectrophotometrically measured DNA quantities (Log10) calibration curve with a linearity range from 50 to 5 x 10(-4) ng of DNA. A positive correlation (R2=0.92) was found between A. carbonarious DNA content and OTA concentration in naturally contaminated grape samples. This is the first application of TaqMan real-time PCR for identifying and quantifying A. carbonarius genomic DNA occurring in grapes. The rapid DNA extraction method for grapes, together with the commercial availability of reagents and instrumentation, allows to perform a remarkable number of reproducible assays (96-well format) in less than 4 h.     

Effects of pulsed electric fields on the extraction of phenolic compounds during the fermentation of must of Tempranillo grapes

The effects of a pre-treatment of the grape skin with electric field treatments (PEF) at 5 and 10 kV/cm on the evolution of colour, anthocyanin and index of total polyphenols during the Tempranillo grape vinification and on the wine characteristics after fermentation have been investigated.Results showed that the permeabilization of the grape skin by application of a PEF treatment at room temperature caused an increment of the colour intensity, anthocyanin content and of total polyphenolic index with respect to the control during all the vinification process. Generally along the maceration process the values of these three parameters increased when the electric field strength raised from 5 to 10 kV/cm.Colour intensity and anthocyanin content of the wine significantly increased when the PEF treatment was applied at 10 kV/cm. However, the total phenolic index increased considerably with the application of a PEF treatment at 5 kV/cm. A further increment on the electric field strength did not appreciably augment this attribute. PEF did not affect the ratio between the components of the red wine colour (tint and yellow, red and blue components) and other wine characteristics such as alcohol content, total acidity, pH, reducing sugar concentration and volatile acidity.These results indicate that the PEF treatment previous to the maceration step in the winemaking process of the red wine can contribute to reduce the duration of the maceration during vinification or to increase the quantity of these attributes in the final wine.

Industrial relevance

This study investigates the effect of the application of a PEF pre-treatment of the grape skin on the evolution of colour, anthocyanin and index of total polyphenols during the Tempranillo grape vinification. Results obtained indicate that the application of a PEF treatment preceding the maceration step in the winemaking process of red wine could contribute to reduce the duration of the maceration during vinification or to increase the quantity of these parameters in the final wine. Latter effect could be of interest in grapes poor in polyphenols.     

Fenhexamid residues in grapes and wine

The behaviour of the fungicide, fenhexamid, on grapes, and during wine-making, as well as its effect on the microflora of alcoholic and malolactic fermentation has been studied. After treatment, the residue on grapes decreased rapidly to one-third of the initial level after the first week, while it remained constant during the following two weeks. At harvest, in the wine obtained by vinification without skins, the fungicide residue decreased on average by 49% , while in the wine obtained by vinification with skins, the decrease was on average 62% . The presence of this fungicide on grapes and in the wine did not affect alcoholic and malolactic fermentation, nor did fermentation cause any degradation of the fungicide. A simple and rapid gas chromatograhic method (GC-NPD) for the determination of fenhexamid residues in grapes, must and wine is described.     

An overview of ochratoxin A in beer and wine

Ochratoxin A (OTA) is a mycotoxin produced mainly by several fungal species of the genera Aspergillus and Penicillium. This mycotoxin has been shown to be nephrotoxic, hepatotoxic, teratogenic and carcinogenic to animals and has been classified as a possible carcinogen to humans. OTA occurs in a variety of foods, including beer and wine. Reports on OTA occurrence in beer indicate that this is a worldwide problem due to the widespread consumption of this beverage. At present, the European Union (EU) has not set a maximum allowable limit (MAL) for this mycotoxin in beer, although there is a limit in barley and malt. Studies carried out in different countries agree in the high proportion of samples contaminated with OTA although levels are, usually, below 0.2 ng/ml. OTA occurrence has been related to the contamination of malt barley with ochratoxigenic species, particularly Penicillium verrucosum. OTA produced in grains is carried to wort and, although fermentation decreases the concentration, the toxin is not eliminated. Reducing the fungal contamination of malt barley is the most promising strategy for reducing OTA in beer. With regard to wine, surveys on the presence of OTA have been conducted worldwide. The proportion of wines in which OTA is detected is very high (above 50%) in some countries (especially in the Mediterranean basin) although only a few wines contained concentrations exceeding the MAL laid down by the EU (2.0 ng/ml). A gradient of concentration is usually recognized; OTA levels decrease in the order red, rose, and white wine but also with increasing latitude of the producing countries. OTA presence in wines is due to the black aspergilli, mainly A. carbonarius, which can grow on grapes in the vineyards and produce the toxin. At grape crushing, the juice can be contaminated with the toxin which is carried over into wine, where it persists due to its stability. Pre- and post-harvest treatments are being investigated to diminish contamination of wines as much as possible.     

Detection of ochratoxin A and cis- and trans-resveratrol in red wines and their musts from Calabria (Italy)

The natural occurrence of ochratoxin A (OTA) and cis- and trans-resveratrols in red wines has been widely reported. The aim of this work was to evaluate the ochratoxin A (OTA) and both cis- and trans-resveratrol content of red wine (from must to wine) in a pilot-scale vinification process in Calabria (Italy). Eleven samples were collected at different stages of vinification and analysis was carried out by HPLC. Wine from manufacturer 3 contained the highest amount of trans-resveratrol (3.41 mg l(-1)). This wine was characterized by an Aglianico-Magliocco grape variety. Interestingly, data regarding OTA showed that the value of this contaminant was low in all analyzed samples and, in each case, below the legal limit (2.0 mg l(-1) (ppb)). Overall, the results demonstrated the high quality of wines produced in Calabria.     

Purification and structural characterisation of lipid transfer protein from red wine and grapes

Lipid transfer proteins (LTP) play a major role in plant defence and are of particular interest due to their known ability to cause allergic reactions. These proteins are expressed in grapes and also remain detectable after vinification, especially in red wine. However, it remains unknown whether the protein undergoes any changes during the vinification process. Here, we present a purification method for LTPs from Dornfelder grapes and wine. By liquid-chromatography–mass spectroscopy (LC–MS/MS) we identified LTPs from two different species (Vitis vinifera and Vitis aestivalis). Additionally, the purified LTPs were characterised using spectrometric methods, confirming their high purity and structural stability during vinification. We conclude that LTPs are resistant to the alcohol content (13.5 vol%), acidic milieu of wine and other ingredients present during the vinification process, indicating that the allergenic potential of grape LTP is not diminished by the vinification process.     

Influence of sour rotten grapes on the chemical composition and quality of grape must and wine

This study evaluated the effect of grape sour rot on wine fermentation and characterized the chemical composition and the sensory changes in wines produced from rotten musts. Microvinifications were performed during two vintages using healthy Trincadeira and Cabernet Sauvignon red grape varieties to which were added grapes affected by sour rot. Increasing sour rot percentages, up to 50%, contributed to a clear decrease in free run must and final wine yields and induced significant changes in grape must chemical composition expressed by the increase in sugar content, total acidity, volatile acidity, anthocyanins, total phenols, and color intensity. After malolactic fermentation, wines from rotten grapes showed higher values of alcohol content, dry extract, reducing sugar content, total and volatile acidity, anthocyanins, total phenols, and color intensity. Despite the higher levels of reducing sugars, the microbial stability was similar to that of healthy wines. The sensorial evaluation, after malolactic fermentation, showed that both types of wine were not statistically different regarding color, aroma, taste, and overall quality. During 6–8 month storage, wines from rotten grapes showed a significant higher percentage of color loss, suggesting that sour rot is responsible for the decrease in color stability. Nevertheless, the results of sensorial analysis demonstrated that the fermentation of grape musts containing up to 30% sour rot yields wines with similar or even higher-quality scores than wines made with healthy grapes.     

Detection of ochratoxin A in tropical wine and grape juice from Brazil

BACKGROUND: Ochratoxin A (OTA) is the main mycotoxin found in grapes, wines and grape juices and is considered one of the most harmful contaminants to human health. In this study, samples of tropical wines and grape juices from different grape varieties grown in Brazil were analysed for their OTA content by high‐performance liquid chromatography. RESULTS: The detection and quantification limits for OTA were 0.01 and 0.03 µg L^?1 respectively. OTA was detected in 13 (38.24%) of the samples analysed, with concentrations ranging from < 0.03 to 0.62 µg L^?1. OTA was not detected in any of the grape juice samples. Most of the red wine samples proved to be contaminated with OTA (75%), while only one white wine sample was contaminated. However, the OTA levels detected in all samples were well below the maximum tolerable limit (2 µg L^?1) in wine and grape juice established by the European Community and Brazilian legislature. CONCLUSION: The results of this study indicate a low risk of exposure to OTA by consumption of tropical wines and grape juices from Brazil. © 2012 Society of Chemical Industry     

Survey: Ochratoxin A in European special wines

The occurrence of Ochratoxin A (OTA) was examined in 121 special wines made using different winemaking techniques and from many European origins. The wine groups with the highest OTA content and occurrence, above 90%, were those were the must was fortified before fermentation (mean: 4.48 μg/l) and those made from grapes dried by means of sun exposure (mean: 2.77 μg/l). Fortified wines with long aging in wooden casks were about 50% contaminated, with OTA levels below 1.00 μg/l. Wines affected by noble rot, late harvest wines and ice wines did not contain OTA. Overall, 19.8% of the wines studied contained OTA levels above the maximum permissible limit for the European Union (2 μg/kg) in wine (excluding liqueur wines).     

新疆葡萄酒中赭曲霉毒素A含量分析

赭曲霉毒素A（OTA）是葡萄及其深加工产品中主要的真菌毒素,同时也被国际癌症研究机构（IARC）定为2B类致癌物。 采用 酶联免疫法（ELISA）商业试剂盒对新疆四大产区葡萄酒中OTA含量进行调研分析,研究不同产区葡萄酒中OTA含量的差异;同时, 对不同葡萄品种酿造葡萄酒过程中发酵葡萄汁和葡萄酒的OTA含量进行测定,分析酿造过程中OTA的变化规律。 结果显示,34份葡 萄酒样品中OTA含量均未超过2 μg/L;其中,焉耆盆地和吐哈盆地葡萄酒中OTA含量较低,平均含量分别为0.19 μg/L、0.20 μg/L;在 白葡萄酒酿造过程中OTA含量呈显著的下降趋势,而红葡萄酒酿造中OTA含量呈先升高后降低的趋势。     

Application of high‐power ultrasounds during red wine vinification

Wine colour is one of the main organoleptic characteristics influencing its quality. It is of special interest in red vinifications due to the economic resources that wineries have to invest for the extraction of the phenolic compounds responsible for wine colour, compounds that are mainly located inside the skin cell vacuoles, where the volatile compounds are also found. The transfer of phenolic compounds from grapes to must during vinification is closely related to the type of grapes and the winemaking technique. During traditional winemaking, grapes are crushed and skin macerated for several days, with pumps overs to facilitate the colour extraction. To increase this extraction, some chemical (maceration enzymes) or physical technologies (thermovinification, cryomaceration, flash‐expansion) can be applied. In this work, a new methodology has been tested. This methodology consists in the application of high‐power ultrasounds to crushed grapes to increase the extraction of phenolic compounds. Crushed grapes were treated with this non‐thermal technology and vinified, with 3, 6 and 8 days of skin maceration time, and the results were compared with a control vinification, where crushed grapes were not subjected to any treatment and were skin macerated during 8 days. The wine chromatic characteristics (determined spectrophotometrically) and the individual phenolic compounds (anthocyanins and tannins, determined by HPLC) were followed during the maceration period, at the end of alcoholic fermentation and after two months in bottle. Also, the wine volatile compounds were determined by GC‐MS. The wines made with ultrasound‐treated grapes showed differences with the control wine, especially regarding total phenol content and tannin content. The wines elaborated with sonicated grapes and with only three days of skin maceration time presented similar concentration of anthocyanins and twice the concentration of tannins than control wines elaborated with 8 days of skin maceration.