Matrix effect during the application of a rapid method using HS-SPME followed by GC–ECD for the analysis of 2,4,6-TCA in wine and cork soaks

An off-flavor in wine known as ‘cork taint’ is of concern in the wine and cork industry. Cork taint imparts a musty flavor to the wine and is primarily due to the presence of 2,4,6-trichloroanisole [2,4,6-TCA] in cork stoppers. During this study, an instrumental method for 2,4,6-TCA analysis was developed and evaluated using headspace solid-phase microextraction (HS-SPME) and gas chromatography coupled with an electron capture detector (GC–ECD). 2,3,6-Trichlorotoluene [2,3,6-TCT] was assayed as the internal standard. The method was developed in synthetic wine and was applied in commercial wine samples, as well as in cork soaks obtained by the extraction of TCA from cork stoppers and cork barks using synthetic wine. The method performance was evaluated through the estimation of its linearity (R² > 0.99), repeatability (RSD value = 5.72%) and sensitivity (recovery > 86%, LOD = 0.177–0.368 ng/L) in different types of samples. Due to the complexity of the samples used, the study has been especially focused on the matrix effects that were identified causing significant bias to the quantitative analysis of 2,4,6-TCA in cork soaks, where there is a lack of previous studies.     

Presence of cork-taint responsible compounds in wines and their cork stoppers

 Chloroanisoles [2,4-dichloroanisole, 2,4,6-trichloroanisole (TCA), 2,3,4,6-tetrachloroanisole and pentachloroanisole], chlorophenols [2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol and pentachlorophenol (PCP)] and guaiacol were detected in red and white cork-tainted bottled wines. These compounds were also found in the cork stoppers from those bottles. A significant correlation was found between TCA in wines and TCA in cork stoppers, and between TCA in wine and intensity of cork taint. At low levels of TCA, the presence of guaiacol or PCP were also found to influence in cork taint. Received: 29 November 1999     

Migration of 2,4,6-trichloroanisole from cork stoppers to wine

The migration of 2,4,6-trichloroanisole (TCA) from cork stoppers to wine is studied under different experimental conditions. Corks that were either naturally contaminated or spiked with a TCA solution were immersed in an ethanol–water mixture (12% v/v) for 6 days or were used as a closure for bottled wines over periods of 1, 4 and 8 months. The TCA content was determined after the stated periods using headspace solid-phase microextraction and gas chromatography electron capture detection. Three different spiking procedures were tested: the injection of a TCA solution into the cork, the immersion of the cork in a TCA in hexane solution, and cutting off a thin slab to inject different quantities of a TCA solution into it before rejoining the two parts. Only the first of these procedures was subsequently used as the immersion technique failed to retain sufficient quantities of TCA and the slab technique retained too much. Corks spiked with 120 ng TCA gave 8% migration after 6 days for corks used in the immersion experiments and less than 1% migration for corked bottles kept for 1 month. When the contact time was 4 and 8 months, migration was 4 and 8% respectively for corks spiked with 1 g TCA. It was concluded that the contact surface, temperature and time all favour TCA migration. Under the same conditions, wine bottled with naturally contaminated cork stoppers showed TCA concentrations which varied depending on the characteristics of every individual cork stopper.     

Screening of musty-earthy compounds from tainted cork using water-based soaks followed by headspace solid-phase microextraction and gas chromatography–mass spectrometry

Compounds responsible for musty-earthy off-flavours in cork, namely 2-methylisoborneol (MIB), geosmin (GSM), 2,4,6-trichloroanisole (TCA) and 2-methoxy-3-isopropylpyrazine (IPMP), were determined from tainted cork using water-based soaks followed by headspace solid-phase microextraction and gas chromatography–mass spectrometry (HS-SPME–GC–MS). The influence of the fibre coating used and of the extraction time and temperature were investigated for the joint analysis of MIB, GSM, TCA and IPMP. Considering the obtained results, an extraction time of 30 min at a temperature of 50 °C were fixed as experimental conditions, using a divinylbenzene/carboxen-polydimethylsiloxane/polydimethylsiloxane (DVB/CAR/PDMS) fibre. Quality parameters of the chromatographic method were obtained and good recoveries (117–128%) were found in spiked aqueous cork macerates. Using the same experimental conditions, the presence of guaiacol, 2,3,4,6-tetrachloroanisole (TeCA), pentachloroanisole (PCA), 2,4,6-tribromoanisole (TBA) and 2-methoxy-3,5-dimethylpyrazine (MDMP) could also be evaluated in a single chromatogram. From all the compounds analyzed in tainted samples, TCA and guaiacol were the only contaminants present and only TCA concentrations were found above its perception threshold in water.     

Transformation ability of fungi isolated from cork and grape to produce 2,4,6-trichloroanisole from 2,4,6-trichlorophenol

The ability of eight fungal strains to transform 2,4,6-trichlorophenol (TCP) to 2,4,6-trichloroanisole (TCA) was studied. These fungi were isolated from cork, belonging to the genera Penicillium, Aspergillus, Trichoderma and Chrysonilia, and from grapes Botrytis cinerea. All, except Chrysonilia, produced TCA when grown directly on cork in the presence of TCP, Aspergillus and Botrytis cinerea being the ones with the highest level of production. It is the first time that Botrytis cinerea, a microorganism often present on grapes and in winery environments, has been shown to transform TCP into TCA. This result can partially explain the wine cork taint before being bottled.     

Migration of phenolic compounds from different cork stoppers to wine model solutions: antioxidant and biological relevance

Considering the enological interest of cork, this study aimed to identify and quantify the phenolic compounds able to migrate from different classes (natural cork stoppers “Flor” and “Third” quality and microagglomerate cork stopper) of cork stoppers into bottled wine model solutions. Another aim was to evaluate some antioxidant and biological features of cork phenolics that migrated into the wine model solutions. The main phenolic acids and aldehydes detected were as follows: gallic and protocatechuic acid detected both around 3.5 mg/L and vanillin and protocatechuic aldehyde detected around 2.5 and 1.5 mg/L after 27 months of bottling, respectively. Trace amounts of more complex polyphenols, namely hydrolysable tannins (castalagin/vescalagin and mongolicain A/B), were also detected. Two antioxidant features of the wine model solutions bottled with different wine cork stoppers were studied, namely the antiradical capacity and the reducing capacity, being the natural cork stoppers the ones with the higher activities. The intestinal absorption of the compounds in each wine model solution after 27 months in bottled was evaluated. The simpler phenolic compounds were able to cross Caco-2 cell model. The antiproliferative activity of the same wine model solutions was also evaluated against gastric and breast cancer cells. All samples were active against the two cell lines, which highlight the possible health outcomes of wine sealed with cork stoppers.     

Origin and fate of 2,4,6-trichloroanisole in cork bark and wine corks

2,4,6-Trichloroanisole (TCA), which is a major cause of cork taint in bottled wine, is already present in the bark of living cork trees to the extent that it can account for the majority of incidences of cork taint in bottled wine. Other post-harvest sources of TCA are known and may add to the forest-derived TCA in cork. Both the origin of TCA in the bark in the forest, and the means by which additional TCA can accumulate in the corks during manufacture, have been examined. TCA can originate from 2,4,6-trichlorophenol (TCP) produced from naturally-occurring phenol and chlorine from sanitisers and cleaning products, and town water. Also, chlorophenol biocides have accumulated in the environment due to the large quantities used in previous times – TCP has been a minor impurity in pentachlorophenol biocides and a major ingredient in other preparations. There is some evidence that chlorophenols were used in pest management in the forest prior to restrictions on the use of these materials. The factors affecting the uptake and loss of TCA by the bark on the tree and by corks during production, and through to their use in the bottling of wine have been considered in this review.     

Fungal strains isolated from cork stoppers and the formation of 2,4,6-trichloroanisole involved in the cork taint of wine

Cork taint is mainly due to 2,4,6-trichloroanisole (TCA) produced through the activity of undesirable fungal strains. We observed that CFU mould number in TCA-containing stoppers was not quantitatively different to that of the stoppers not containing TCA (ca. 10(5)CFU/g). In contrast more fungi diversity was observed in TCA-containing stoppers. Penicillium spp (Penicillium chrysogenum, Penicillium glabrum), Aspergillus spp (Aspergillus niger and Aspergillus oryzae), Chrysonilia sitophila, Mucor racemosus, Paecilomyces sp. and Trichoderma viride were found in TCA-containing stoppers, while C. sitophila and Penicillium sp. were the main fungi in the stoppers devoid of TCA. Conidia were numerous close to the lenticels and present from the lateral surface through to the centre of the stoppers. Strains of Aspergillus, Mucor, Paecilomyces, Penicillium and Trichoderma isolated from TCA-containing stoppers were able to convert 2,4,6-trichlorophenol (TCP) in TCA in resting cell or growing conditions. The best yields of conversion were obtained by green fungi Paecilomyces sp. and P. chrysogenum, 17% and 20%, respectively. Chysonilia sitophila and Penicillium sp. did not produce TCA from TCP in our conditions.     

The analysis of natural cork stoppers in transversal sections as an effective tool to determine the origin of the taint by 2,4,6-trichloroanisole

The distribution of several chlorophenols (CPs) and chloroanisoles (CAs), including 2,4,6-trichloroanisole (TCA), the main compound responsible for fungal taint of wines, was analyzed in four independent batches of natural cork stoppers that were dissected into three transversal slices. The contaminants were homogeneously distributed in the slices analyzed. All the stoppers were contaminated with at least one single CP or CA, although most of the corks contained several CPs and CAs. Pentachlorophenol was the more abundant contaminant, detected in 89.2% of the corks analyzed. The analysis of releasable CPs and CAs showed that most of the contaminants present in the stoppers cannot be released into wine. The same compounds were analyzed in a different batch of natural cork stoppers that had been used to close wine bottles in a winery whose facilities were contaminated with CPs and CAs. In this case, the highest amounts of contaminants were accumulated on the heads of the stoppers in contact with the winery environment. Bottled corks were able to efficiently absorb deuterium-labeled TCA (TCA-d ₅) and/or deuterium-labeled pentachloroanisole (PCA-d ₅) from an artificially tainted wine during 40 months. In this case, most of the TCA-d ₅ and PCA-d ₅ was located in the basal slices of the closures in direct contact with wine. These data, and also data from other authors, indicate that the distribution in transversal slices of the CPs and CAs contaminating cork stoppers is in direct relationship with the origin of the taint, and therefore that the analysis of cork stoppers by transversal sections could be an effective tool to clarify the origin of the taint.     

Search for the contamination source of butyltin compounds in wine: agglomerated cork stoppers

A possible butyltin contamination source in wine was studied in this paper. Agglomerated cork stoppers, which were produced in Portugal, Spain, and Italy, used in wine bottles were examined. The domestic cork products, cork granules, and mucus used for cork products were also analyzed. The levels of mono- and dibutyltin compounds in corks were found in the range from <0.0024 to 3.3 and from <0.0029 to 6.7 microg of Sn/g, respectively. A low level of tributyltin contamination was also found in 2 of 31 tested samples. The presence of butyltin compounds in agglomerated cork stoppers was confirmed by GC-MS. Experimental results indicated that all overseas agglomerated cork stoppers studied contained mono- and/or dibutyltins. Butyltins were not detected in cork granules, mucus, most of the natural cork stoppers, and domestic agglomerated cork products. The concentrations of mono- and dibutyltins increased with the time in a 30-day experiment, showing that butyltin compounds can leach from agglomerated cork to the wine. When the butyltin concentrations in wine samples were compared with their levels in the corresponding agglomerated cork stoppers, a correlation was found. The potential harm of such food contamination was evaluated by the toxic research of butyltin compounds using Daphnia sp. as the experimental model.     

葡萄酒和软木塞中2,4,6-三氯苯甲醚检测方法的研究进展

本文介绍了葡萄酒和软木塞中2,4,6-三氯苯甲醚（2,4,6-trichloroanisole,TCA）的来源及影响,分析了2,4,6-三氯苯甲醚的检测难点,总结了目前检测2,4,6-三氯苯甲醚方法的总体趋势及现状。按照前处理方法的原理分类,介绍了前处理方法的定义、特点,并概述和讨论了前处理方法在萃取葡萄酒和软木塞中2,4,6-三氯苯甲醚中的应用;根据仪器检测方法的不同特点,总结和讨论了仪器检测方法在分析检测葡萄酒和软木塞中2,4,6-三氯苯甲醚的应用。前处理方法和仪器检测方法相结合,达到了浓缩和检测葡萄酒和软木塞中2,4,6-三氯苯甲醚的目的。     

Cork-borne bacteria and yeasts as potential producers of off-flavours in wine

Bacteria and yeasts were found to be present within cork lenticels, covered by mucous or fibrous substances. They survived heating, peroxide treatment and contact with the alcohol and sulfur dioxide of wine. 187 bacteria and 36 yeast strains were isolated from cork stoppers of wine bottles and, during various stages of production, from corkwood and new cork stoppers. After culturing, a number of isolates showed the ability to modify the aroma of model systems consisting of dilute or full strength wine and pulverised cork. The aromas produced by isolates of varying cork origin are tabled. A small number of isolates methylated 2,4,6-trichlorophenol, yielding 2,4,6-trichloroanisole, responsible for the typical cork taint. During the boiling of cork slabs, the internal temperature on the inside of a box made from cork slices did not exceed 87°C.     

Multitoxin evaluation in fermented beverages and cork stoppers by liquid chromatography–tandem mass spectrometry

A total of twenty‐eight mycotoxins were surveyed in wine (red, white and rose), cider (white and rose) and their cork stoppers from eight countries. Toxins of different fungi genera were detected as follows: Alternaria (ATs: alternariol – AOH; alternariol methyl – AME) and Penicillium/Aspergillus (ochratoxin A – OTA; penicillic acid – PAC). Toxins and levels varied with the sample types and country of origin. Wine presented contamination of OTA, AOH and AME. OTA was detected in forty‐one wine samples with levels ranging from 0.01 to 0.86 μg L^?1, below EU legislation. AOH and AME were detected in thirty‐three and eight of wines samples, respectively, at levels from 0.2 to 13.3 μg L^?1, while no contamination was detected in ciders up to the method LOQs. Regarding the cork stoppers toxins detected, they were AOH, AME and PAC. Corks of red wine from different countries had levels of OAH and AME ranging from 5.0 to 101.0 and 2.5 to 5 μg g^?1, respectively. It is necessary to pay more attention on the corks processing and cork type used in the bottles as, different from the ordinary ones, the ground bark and compressed type did not have toxins detected.     

Effect of wine style on the perception of 2,4,6-trichloroanisole,a compound related to cork taint in wine

2,4,6-Trichloroanisole (TCA) is a fungal metabolite that can contaminate cork stoppers and wines producing a very unpleasant mouldy odour (cork taint). Sensory control is very widely used for the detection of possible TCA presence in corks and wine, but the correct identification of TCA, especially at low concentrations, can be difficult. The aim of this study was to show how wine styles can affect the ability of the same panel to perceive TCA. For this purpose, a panel was selected, according to the panelists’ sensitivity to the TCA stimulus. The triangle test, a sensory difference test, was carried out by the panel with both white and red wines, using samples both free from TCA and spiked with known quantities of this contaminant. The results showed that the panelists identified the difference caused by the added TCA at different significance levels, depending on the wine style, so TCA detection was influenced by wine style for the selected panel.     

Compounds causing cork taint and the factors affecting their transfer from natural cork closures to wine – a review

The compounds causing cork taint and the factors affecting their transmission from cork to wine are discussed. These factors include: the solubilities of the taint compounds in wine, their affinity for the surface and the interior parts of the cork; their location on the surface of and within the closure; the rates at which they can migrate through the cork matrix; the volume of wine in contact with a closure(s); and whether taint transmission is taking place in bottled wine or with corks soaked in wine for screening purposes. 2,4,6‐Trichloroanisole (TCA) has been the primary topic of investigations reported in the general literature and is therefore the main focus of this article.     

Yield and quality in the production of cork stoppers

Bottle stoppers of natural cork are produced from cork planks as cylinders which are bored in the axial direction. The process was followed for the production of 38 mm×24 mm (length×diameter) stoppers, using cork planks of different quality grades. The mass yield is on average 24% of the initial cork with net productions of 98 stoppers/kg and 747 stoppers/m². The net yields are higher when using the best quality raw-material due to an increasing number of refused stoppers with the decrease in quality of the cork planks. The quality profiles of the stoppers are determined by the quality of the raw-material: i.e. ist grade cork planks produce 82% of high quality stoppers (superior and 1st class) and 5th grade planks only 22%. The results obtained allowed to establish correlations between the number of stoppers produced and raw-material parameters, namely the area and dry weight, which can be used to estimate production from a given lot of cork planks.     

Transformation ability of fungi isolated from cork and grape to produce 2,4,6-trichloroanisole from 2,4,6-trichlorophenol

The ability of eight fungal strains to transform 2,4,6-trichlorophenol (TCP) to 2,4,6-trichloroanisole (TCA) was studied. These fungi were isolated from cork, belonging to the genera Penicillium, Aspergillus, Trichoderma and Chrysonilia, and from grapes Botrytis cinerea. All, except Chrysonilia, produced TCA when grown directly on cork in the presence of TCP, Aspergillus and Botrytis cinerea being the ones with the highest level of production. It is the first time that Botrytis cinerea, a microorganism often present on grapes and in winery environments, has been shown to transform TCP into TCA. This result can partially explain the wine cork taint before being bottled.     

Influence of vision systems,black and white,colored and visual digitalization,in natural cork stopper quality estimation

Quality classification of wine natural cork stoppers is related to presence of discontinuities in the cork tissue. Automated image analysis of stoppers based on black and white cameras is used industrially for commercial classification but recently color has been introduced in image processing. This paper compares the performance of three image vision systems regarding classification accuracy of cork stoppers of good, medium and inferior quality: black and white, three‐band RGB color and manual detection by digitalization in color image. A canonical discriminant analysis approach was used to compare the discriminating power between cork stopper quality in each vision system. Good discriminant results were obtained with the area of pores expressed either in total or as ratio, mean or maximum value. The use of color slightly enlarges the range of cork inspection systems and automated systems have a similar accuracy of classification to visual inspection. Copyright © 2007 Society of Chemical Industry     

Sorption and diffusion properties of volatile phenols into cork

The sorption and diffusion properties of seven common volatile phenols in hydro-alcoholic medium placed in contact with natural cork were investigated to determine the influence of cork closures on the concentration of these compounds in wine. Weighted cork samples were immersed in model wine solutions containing selected concentrations of each compound and were sampled over time. Sorption coefficients ranged between 125 and 306 mg of aroma compound per kg of cork, while diffusion coefficients varied from 0.8 to 4.1 × 10⁻¹¹ m²/s. Sorption isotherms of guaiacol and 4-propylguaiacol, respectively, the lowest and highest sorbed compounds of an homologous series of guaiacol, accurately fit Henry’s model. Hydrophobicity of aroma compounds seems to be a determining factor in both diffusion and sorption. This could indicate a preferential pathway for diffusion through suberin, the more hydrophobic constituent of cork.     

Determination of Chloroanisoles and Chlorophenols in Cork and Wine by using HS‐SPME and GC‐ECD Detection

Cork taint is an off‐flavor problem in wine, the main reason being the presence of 2,4,6‐trichloroanisole (TCA) in the cork stopper. In addition to the TCA, the presence of other chloroanisole and chlorophenol family compounds (the perception limits of which are very low) can also result in, or contribute to, cork taint problem. In this study, the levels of 2,4‐dichloroanisole (DCA), 2,4,6‐trichloroanisole (TCA), 2,3,4,6‐tetrachloroanisole (TeCA), pentachloroanisole (PCA), 2,4,6‐trichlorophenol (TCP), 2,3,4,6‐tetrachlorophenol (TeCP) and penthaclorophenol (PCP) were assayed in cork stoppers (natural, agglomerated and colmate) and in red wine samples from different wineries in Turkey using HS‐SPME and GC‐ECD detection. The performance parameters for all chloroanisole and chlorophenol compounds were as follows: recovery 92.48–102.53%, R 0.992‐0.996. The LOQ values were DCA (8.4 ng/L), TCA (0.8 ng/L), TeCa (0.6 ng/L), PCP (0.8 ng/L), TCP (0.8 ng/L), TeCP (1.2 ng/L), and PCP (1.1 ng/L) respectively. In cork stoppers, the amounts of 2,4,6 TCA ranged between 5.4–130.6 ng/g. The 2,3,4,6 TeCA ranged between 1.12‐8.2 ng/g and the PCA ranged between nd (not detected)‐11.01 ng/g. In the wine samples, 2,4,6 TCA ranged between 1.42–70.2 ng/L. The 2,3,4,6 TeCA ranged between nd‐15.1 ng/L and the PCA ranged from nd‐5.16 ng/L. The results indicated that there was a significant correlation between the TCA in wines and the TCA in cork stoppers.