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     

顶空-固相微萃取/气相色谱串联质谱法测定葡萄酒中多种氯酚及氯代茴香醚

样品中氯酚经乙酰化生成乙酰氯酚后,通过优化萃取头、离子强度、温度、时间等固相微萃取条件后,建立顶空-固相微萃取-气相色谱串联质谱法同时测定测定葡萄酒中的3种氯酚2,4,6-三氯酚(TCP)、2,3,4,6-四氯酚(TeCP)、五氯酚(PCP)以及两种氯代茴香醚2,4,6-三氯苯甲醚(TCA)、五氯苯甲醚(PCA)含量的方法。结果表明：该方法检出限为0.1ng/L,回收率为86.0%～105.9%,相对标准偏差小于16.5%。该方法前处理简单、高效、检测灵敏度高,可应用于进出口葡萄酒中木塞污染物的监控。     

The sensory evaluation of 2,4,6‐trichloroanisole in wines

This work proposes a sensory method to verify the ‘cork taint’ defect in food and beverages. This off‐flavour has considerable economic impact in wine but occasionally can occur in other food and beverages. In wine, 2,4,6‐trichloroanisole (TCA) is generally considered to be the main compound responsible for this taint. It is an easily recognized compound because of its low sensorial threshold, and it is described as a mouldy and damp cardboard odour. This sensory method, developed in wine, consists of specific panel training to recognize TCA in a series of olfactory tests. The effectiveness of the panel was tested with contaminated wines in which the TCA content had been previously determined by Solid Phase Microextraction‐Gas Chromatography Mass Spectrometry (SPME‐GC/MS) analysis. This sensory method is useful to train a panel able to recognize the ‘cork taint’ defect in different situations (legal appraisals or quality assurance systems). The use of a reliable sensory assay can reduce the number of chemical analyses and the proposed method can be applied to other beverages such as beer. Copyright © 2015 The Institute of Brewing & Distilling     

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。     

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.     

Absorption of chloroanisoles from wine by corks and by other materials

Sixty 750 mL bottles of a white wine were each spiked with deuterium-labelled 2,4,6-trichloroanisole (d₅ TCA) and then sealed with a variety of wine corks. Thirty months later, approximately half of the d_5-TCA had been absorbed by each of the corks, regardless of supplier, bleaching treatment or whether the corks were natural or agglomerate. In addition to the added labelled TCA, every one of these corks also contained endogenous (i.e. unlabelled) TCA. Fifteen of the corks, mostly agglomerates, imparted some of their endogenous TCA into the wines. There was no direct relationship between the amount of endogenous TCA in the corks and that found in wines. The high variability in the distribution of endogenous TCA between wine and cork contrasts with the relatively uniform distribution of the d₅-_-TCA. This contrasting behaviour distinguishes between wines tainted prior to closure and wine tainted by corks. Natural bark corks in wine bottles can also absorb 2,3,4,6-tetrachloroanisole (TeCA) and pentachloro-anisole (PCA) from bottled wine. Bottle storage of commercial wines that had become tainted with TeCA and PCA during production resulted in the corks absorbing most of these two compounds, so that the wines were no longer tainted to a significant extent. Soaking whole corks in wine spiked with chloroanisoles, under conditions typically employed in the wine industry to test batches of corks for possible taint, resulted in most of the TCA in the wine being absorbed by the corks. Thus, if five corks are being soaked in wine in order to test for taint, four sound corks could reabsorb TCA that had leached into the surrounding wine from a single contaminated cork, reducing the concentration of TCA to a point where it escapes detection. Soaking corks singly rather than in groups of five will therefore be a more sensitive method of screening batches of corks for taint than soaking them in groups of five. Plastic materials such as the lids of glass containers used to store wine samples were also able to absorb chloroanisoles via both direct liquid contact and the vapour phase. Wine cask bladders and polyethylene film were particularly effective in removing TCA from wine.     

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.     

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.     

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.     

The analysis of 2,4,6-trichloroanisole and other chloroanisoles in tainted wines and corks

A new method has been developed for the analysis, by gas chromatography/mass spectrometry, of 2,4,6-trichloroanisole (TCA) and other chloroanisoles in cork-tainted wines, using a polydeuterated form of TCA as an internal standard. In a survey of wines presented at a wine assessment course, 4.8% (i.e. 18 bottles out of 374) were assessed by at least 20% of the participants as being affected by cork taint. TCA was present in each of these 18 wines at a concentration close to, or above the sensory detection threshold. All cases of taint seen by the participants could therefore be attributed, at least in part, to the presence of TCA, and this in turn could be attributed to the cork, since variation in apparent taint between bottles of the same wine was observed in every case. TCA was also found in the corks from the wines. Randomly selected bottles of wines considered to be affected by a high proportion of cork taint, and the corks from those bottles, were also analysed. There was considerable variation in the distribution of TCA and other chloroanisoles between wine and cork. In many cases, chloroanisoles were found only in the cork. TCA in corks was accompanied by varying amounts of 2,4– or 2,6-dichloroanisoles, 2,3,4,6-tetrachloroanisole and pentachloroanisole, indicating more than one origin for chloroanisoles in corks. No chloroanisoles, other than those derived by methylation of products formed by non-enzymatic chlorination of phenol, were detected in any of the samples.     

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.     

Wine bottle closures: physical characteristics and effect on composition and sensory properties of a Semillon wine 1. Performance up to 20 months post-bottling

A Semillon wine was bottled using 14 different closures: a screw‐cap type, two grades of conventional natural cork, two ‘technical cork’ closures (natural cork with a synthetic component), and 9 closures manufactured from synthetic polymer material. Closure performance was evaluated for physical aspects (e.g. extraction force and energy, change in closure diameter, and ease of closure reinsertion), and for wine composition and sensory properties. Wine under the screw cap closure retained the greatest concentration of sulfur dioxide (SO₂) and ascorbic acid and had the slowest rate of browning. For other closures the trend of SO₂ loss relative to the screw cap closure was apparent from an early stage of testing, and was most evident in the group of synthetic closures, intermediate in the conventional corks, and least evident in the technical cork closures. The loss of SO₂ was in general highly correlated with an increase in wine browning (OD₄₂₀) and the concentration of SO₂ in the wine at six months was a strong predictor of future browning in the wine, particularly after eighteen months. Neither the concentration of dissolved oxygen at bottling (0.6–3.1 mg/L), nor the physical closure measures were predictors of future browning. For several closures upright storage tended to accelerate loss of SO₂ from the wine, but in many cases this effect was marginal. The closures differed widely in regard to physical characteristics, and in general synthetic corks appeared least ‘consumer‐friendly’ in terms of extraction forces, energies, and ease of closure re‐insertion, but there was a trend for natural cork closures to exhibit larger variability in physical characteristics than technical cork and synthetic closures. Sensory analysis indicated large differences in wine flavour properties, with closures which tended to result in the best retention of free SO₂ having wine sensory scores for ‘citrus’ that were generally high whilst scores for the attributes ‘developed’/‘oxidised’ were low. The situation was reversed for wine under closures that performed poorly in the retention of free SO₂. It was found that below a critical level of free SO₂ remaining in the wine, closures exhibited substantially higher ‘oxidised’ aroma. Whilst trichloroanisole‐type (TCA) taint was a noticeable problem for some cork and technical cork closures, any plastic‐type taint appeared not to be a problem with most synthetic closures.     

固相微萃取-气相色谱法测定葡萄酒及软木塞中2，4，6-三氯苯甲醚（TCA）

采用固相微萃取-气相色谱法测定葡萄酒及软木塞中的TCA含量,样品试液经固相微萃取装置萃取、富集后,直接无分流进样,经毛细管柱DB-5分离后,用ECD检测器检测。结果表明,该方法检出限为0.1 ng/L,回收率在90%～106%之间,相对标准偏差(n=5)小于5%。     

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.     

Permeation of 2,4,6-trichloroanisole through cork closures in wine bottles

Deuterium-labelled trichloroanisole (d₅-TCA) added to the surface of wine corks in bottles did not contaminate wine after more than three years of bottle storage. Most of the added d₅-TCA was lost, presumably by evaporation. That remaining was largely confined to the outer portion of the closures. Under the conditions of this study, the cork closures were highly effective barriers to the transmission of exogenous TCA.     

顶空固相微萃取与气相色谱-电子捕获技术联用检测软木塞中2,4,6-三氯苯甲醚

建立利用顶空固相微萃取与气相色谱电子捕获技术联用分析检测葡萄酒软木塞中2,4,6-三氯苯甲醚含量的方法。该方法测定软木塞中2,4,6-三氯苯甲醚的相关系数（R²）为0.999 9,精密度好（相对标准偏差不大于5.6%）,回收率为101%～108%,检出限为0.20 pg/L、定量限为0.66 pg/L。利用该方法测定了11 种不同类型的软木塞中2,4,6-三氯苯甲醚的含量,证实了所建方法适用于葡萄酒软木塞中痕量2,4,6-三氯苯甲醚含量的测定。     

葡萄酒及软木塞中TCA的检测分析方法研究现状及应用前景

软木塞污染已经成为葡萄酒行业最为严重的问题之一,而导致软木塞和葡萄酒污染的最主要物质之一就是TCA。本文主要介绍葡萄酒和软木塞中TCA 的检测方法和萃取浓缩方法,同时也对TCA 检测的应用前景和发展趋势进行了展望。     

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.     

Identification of 2,4,6-trichloroanisole (TCA) causing a musty/muddy off-flavor in sake and its production in rice koji and moromi mash

2,4,6-Trichloroanisole (TCA), which has been identified as the main component responsible for the cork taint in wine, was detected in sake samples having a musty/muddy off-flavor by stir bar sorptive extraction (SBSE). We confirmed that TCA is one of the components causing this off-flavor in sake, as in other alcoholic beverages, from a sensory analysis showing the correlation between TCA concentration and the intensity of the musty/muddy off-flavor. We investigated the route of TCA production in the rice koji preparation process and in the moromi mash process for sake brewing. We found that TCA is produced mainly by the biomethylation of 2,4,6-trichlorophenol (TCP) by rice koji in brewing and that TCP originates from the wooden tools used in preparing rice koji.