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.     

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.     

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 role of corks in oxidative spoilage of white wines

The permeability of corks to oxygen was examined by comparing the level of browning and the concentration of the antioxidants, free and total sulfur dioxide and ascorbic and erythorbic acids in wines bottled and sealed with two cork types and stored in an atmosphere of either nitrogen, oxygen or air. Three different batches of corks that had been treated with peroxide during processing, representing one cork type, and a batch of corks that had been treated with chlorine during processing representing the second cork type, were taken for the study. In bottled wines stoppered with one of the peroxide-treated corks (Cork A), there was a higher level of browning and a greater loss of the antioxidants in those wines in bottles stored under oxygen and air than that in the wines in bottles stored under nitrogen, indicating that corks of this batch were permeable to oxygen. There was no significant difference in the loss of ascorbic and erythorbic acid or the level of browning from bottled wines stored under the three gases and sealed with the chlorine-treated corks (Cork C). For the other two peroxide-treated corks (Corks B and D), there was no significant difference in the level of browning or loss of the antioxidants from bottled wines stored under the three gases and sealed with Cork B, nor was there a significant difference in the level of browning or loss of free or total sulfur dioxide from bottled wines stored under the three gases and sealed with Cork D. From comparative data, it was evident that oxidation of the wines sealed with Corks B and D was due to compounds in the corks rather than oxygen permeation through the corks, or oxygen ingress at bottling. Because the bottles of wine selected for this study were from batches that had already exhibited sporadic oxidation, the results found are indicative, but not necessarily representative, of the properties of all peroxide- and chlorine-treated cork types used in the wine industry.     

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.     

Development and evaluation of alternative processes for sterilization and deodorization of cork barks and natural cork stoppers

Cork taint, one of the most known off-flavours in wine, is usually attributed to the presence of the aromatic compound 2,4,6-trichloroanisole (2,4,6-TCA) in cork stoppers made from the bark of the cork oak, Quercus suber. There are many major chemical/biochemical pathways through which 2,4,6-TCA can be formed during cork production. The estimated incidence of cork-tainted wine bottles ranges from 2–to 7%, costing global wine industry approximately US$ 10 billion annually. During this study, a laboratory-scale system was designed and constructed, to efficiently treat cork bark and stopper samples using ozone and/or other sterilizing gases, such as steam, via sequential application of pulsed vacuum–pressure cycles. The developed physicochemical processes were studied and evaluated in order to achieve both sterilization conditions and 2,4,6-TCA removal without affecting the mechanical and bottling properties of cork. According to the results, the application of ozone alone seems to be a promising treatment method for cork barks. However, the pulsed treatment ensures both sufficient removal (99%) of both bacteria and moulds from cork stoppers when combined with ozone or steam and satisfactory deodorization of cork stoppers achieving high percentages (90%) of 2,4,6-TCA removal when combined with steam. The operating cost of each alternative process (plain or pulsed, with or without ozone and/or steam) was estimated and compared, in laboratory scale, for the selection of the most efficient process, taking into account technicoeconomical aspects.     

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.     

Effects of electron beam irradiation on microbial inactivation and quality of kimchi paste during storage

The effects of electron beam irradiation on microbial inactivation and quality of noninoculated and inoculated (Listeria monocytogenes) kimchi pastes were examined. Kimchi paste samples were irradiated at doses of 2, 4, 6, 8 and 10 kGy and stored for 21 days at 4 °C. Irradiation (10 kGy) reduced the populations of total aerobic bacteria, lactic acid bacteria, and yeast and moulds in the samples by 1.72, 2.24 and 0.86 log CFU g^?1, respectively, compared to the control. In particular, coliforms were not detected at 8 and 10 kGy, and the population of L. monocytogenes in inoculated samples was significantly decreased by 2.67 log CFU g^?1. Electron beam irradiation delayed the changes in O₂ and CO₂ concentrations, pH, acidity and reducing sugar content observed in kimchi paste during storage. These results suggest that electron beam irradiation can be used to improve the microbiological safety and shelf life of kimchi paste.     

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.     

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.     

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.     

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.     

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.     

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.     

Effect of y‐radiation on chives safety and quality

The effect of γ‐irradiation on the quality of chives was evaluated. The samples were irradiated at 1.0 and 2.0 kGy, stored at 4 °C for 10 days and used for microbiological (aerobic mesophilic, moulds and yeasts, E. coli and Salmonella sp), biochemical (vitamin C and lipoperoxide (MDA) contents and superoxide dismutase (SOD) and guaiacol peroxidase (POX) activity) and sensorial evaluation. For irradiated samples, the total counts of aerobic mesophilic and moulds and yeasts showed a reduction of 6 log cycles during storage, and colour analysis showed no significant difference (P > 0.05) for the b*‐value. The contents of vitamin C were not significantly affected by irradiation and storage time. The MDA contents and SOD activity changed insignificantly at both γ‐irradiation levels after storage, while POX was significantly increased (P ≤ 0.05) at 1 kGy. Samples irradiated at 2.0 kGy presented sensorial acceptance after the storage. These results show that γ‐irradiation increases the shelf life of chives without significant changes in their quality.     

Effect of fungi contamination in the chemical composition of Hungarian wines

The present study shows the effect of different types of mold, especially microorganisms isolated from the corks, on Tokaj white wines. The transformation in wine chemical composition induced by contamination with molds such as Penicillium chrysogenum, Penicillium expansum and Phanerochaete spp. was different in each type of mold. Molds can contaminate wine either by transferring from grapes contaminated or by non-hygienic conditions during wine technology. As a result of contamination, molds modify wine chemical composition in terms of total and volatile acidity, residual sugars, alcohol volume, total solids and ash.     

E‐beam irradiation affects the maximum specific growth rate of Bacillus cereus

When conventional preservative treatments are applied, such as heat or acid, the maximum specific growth rate (μ_max) of survivors is the same as that of untreated cells. However, when new nonthermal technology is applied, the effects of it on the kinetics of the microorganism can be unpredictable. In this sense, Cabeza et al. (2010) reported longer doubling times after irradiating with accelerated electron beam. The aim of this work was to study the effect of electron beam irradiation on the μ_max of Bacillus cereus and compare it with a conventional inactivation treatment (heat). To prove this, μ_max was estimated in ham at 12 °C and in TSB at 22 °C after 0, 2, 3 or 4 log reduction by irradiation; likewise, μ_max was estimated in whole milk at 12 °C and in TSB at 22 °C after the same log reduction using heat treatments. Our findings show that irradiation affected the μ_max of survivor cells. Irradiation intensity was inversely proportional to μ_max, such that greater intensity was associated with lower μ_max. At the same time, growth temperature had an effect on the decrease in μ_max: the radiation‐induced reductions in μ_max were greater at 12 °C than at 22 °C. In summary, E‐beam irradiation decreases the μ_max of B. cereus, while heat treatment does not. This suggests that the shelf life of irradiated foods must be longer than that of heat‐preserved foods after the application of a similar inactivation treatment.     

CONTAMINATION OF GRAINS BY MYCOTOXIN-PRODUCING MOLDS AND MYCOTOXINS AND CONTROL BY GAMMA IRRADIATION

Ninety random grain samples were collected and analyzed for mycotoxins, and the effect of gamma irradiation on the production of mycotoxins in grains was studied. Aspergillus, Penicillium, Mucor, Rhizopus, Fusarium, Alternaria, Scopulariopsis and Cladosporium were the most common fungal genera isolated from grains. Aspergillus flavus, Aspergillus niger, Aspergillus candidus, Aspergillus ochraceus, Penicillium citrinum, Penicillium expansum, Penicillium citreonigrum, Penicillium purpurogenum, Penicillium griseofulvum and Penicillium verrucosumwere the most common Aspergillus and Penicillium species in grains. Out of 120 Aspergillus and Penicillium isolates, 80 were mycotoxin producers. Analysis of grains revealed the occurrence of aflatoxin B₁ ochratoxin A, cycolopiazonic acid and citrinin. Of the 90 samples, 67 were positive for one or more mycotoxin. Irradiation of grains at dose of 2.0 and 4.0 kGy decreased significantly the total fungal counts compared with unirradiated controls. After 100 days of storage at room temperature, the unirradiated grains were contaminated with high concentrations of mycotoxins as compared with irradiated 4.0‐kGy samples. Mycotoxin production in grains decreased with increasing irradiation doses and was not detected at 6.0 kGy over 100 days of storage.     

Moulds and ochratoxin A on surfaces of artisanal and industrial dry sausages

The use of moulds as a seasoning for sausage can have both desirable and undesirable consequences. The desirable consequences are the creation of a successful product that appeals to consumers. The undesirable consequences are due to the growth of undesirable moulds that produce highly toxic secondary metabolites referred to as mycotoxins. The aim of the paper was to investigate the presence of moulds producing ochratoxin A (OTA) on the surface of sausages from northern Italy. A total of 757 mould strains were isolated from sausage casings. The most frequently identified species were Penicillium nalgiovense, Penicillium oxalicum, Eurotium amstelodami, Penicillium olsonii, Penicillium chrysogenum, Penicillium verrucosum, Penicillium viridicatum, and Eupenicillium crustaceum. Aspergillus ochraceus was detected in only one production lot. Approximately 45% of these samples were positive for the presence of OTA. On the casings of the investigated sausages, the lowest and highest OTA values were 3 and 18 μg/kg, respectively. The OTA concentration was reduced to below the limit of detection (LOD) by brushing and washing the sausages prior to sale. From these data it appears that the presence of OTA on the surface of sausage (on the casings) is not indicative of any health risk for human consumption of sausage, since OTA was not identified inside the dry meat.