Origin and incidence of 2-methoxy-3,5-dimethylpyrazine, a compound with a "fungal" and "corky" aroma found in cork stoppers and oak chips in contact with wines.

This study identifies a previously isolated bacterium as Rhizobium excellensis, a new species of proteobacteria able to form a large quantity of 2-methoxy-3,5-dimethylpyrazine (MDMP). R. excellensis actively synthesizes MDMP from L-alanine and L-leucine and, to a lesser extent, from L-phenylalanine and L-valine. MDMP is a volatile, strong-smelling substance detected in wines with cork stoppers that have an unpleasant "corky", "herbaceous" (potato, green hazelnut), or "dusty" odor that is very different from the typical "fungal" nose of a "corked" wine that is generally due to 2,4,6-trichloroanisole (TCA). The contamination of cork by MDMP is not correlated with the presence of TCA. It appears possible that R. excellensis is the microorganism mainly responsible for the presence of this molecule in cork bark. However, other observations suggest that MDMP might taint wine through other ways. Oak wood can also be contaminated and affect wines with which it comes into contact. Nevertheless, because 93% of the MDMP content in wood is destroyed after 10 min at 220 °C, sufficiently toasted oak barrels or alternatives probably do not represent a major source of MDMP in most of the cases. Due to MDMP's relatively low detection threshold estimated at 2.1 ng/L, its presence in about 40% of the untreated natural cork stoppers sampled at concentrations above 10 ng/cork suggests that this compound, if extracted from the stoppers, may pose a risk for wine producers.

Identification of a new source of contamination of Quercus sp. oak wood by 2,4,6-trichloroanisole and its impact on the contamination of barrel-aged wines.

Thanks to practical experience in various wineries in recent years, it is now clear that, similarly to the well-known phenomenon in corks, there are several sources of unpredictable contamination of oak wood by 2,4,6-trichloroanisole (TCA). TCA affects staves in the same barrel very sporadically, with extremely limited contaminated areas on the surface that may reach several millimeters in depth. The precise origin of the TCP and TCA in oak wood is not known at this stage. Available data indicate that the phase where stavewood is naturally dried and seasoned is the source of these undesirable organochlorine contaminants. The strictly chemical formation of 2,4,6-trichlorophenol (TCP), derived from organochlorine biocides, was demonstrated to be impossible under traditional cooperage conditions, and its accumulation remained highly improbable. Similarly to previous discoveries in corks, all the analyses of oak wood suggested that the TCP was of biochemical origin. The capacity to biomethylate chlorophenols is well-known and relatively widespread among the usual microflora in stavewood, but the precise origin of the intermediary leading to TCP formation is still unknown. One probable hypothesis is that this reaction involves chloroperoxidase (CPO). Several ideas have been proposed, but the microorganisms responsible for the formation of the TCA precursor in oak wood have not yet been identified. The extent of this problem is still severely underestimated by coopers and barrel-users, due to the extremely unpredictable, localized contamination of the staves.

Impact of toasting oak barrels on the presence of polycyclic aromatic hydrocarbons in wine.

Toasting Quercus sp. oak wood is one of the key stages in manufacturing barrels intended for aging wines and spirits. During this operation, the increase in temperature causes variable modifications in the physical structure and, more importantly, the chemical composition of the wood. Polycyclic aromatic hydrocarbons (PAH) are high-risk molecules likely to be formed during toasting of the wood and later extracted by wine or spirits in direct contact with the barrel. In the context of an analysis of all potential sources of risk associated with the manufacture of barrels for winery use [prevention policy defined using a Hazard Analysis Critical Control Point (HACCP) approach], we carried out a preliminary study to provide a more accurate assessment of potential risks related to the presence of PAH in cooperage and winemaking. Wood toasted to different levels under different conditions, as well as wines aged in barrels made using different methods, was analyzed by gas chromatography and mass spectrometry (GC-MS) for the identification of the main PAH present, quantification of each of the molecules extracted, and estimation of any possible toxicological risks, via a comparison of values with those measured in other types of food. The results clearly showed that the heating processes associated with barrel production actually resulted in the formation of various molecules in the PAH family. However, only a minority of the target PAH presented high toxicity, particularly carcinogenic potential. Because of the specific toasting process used, benzo[a]pyrene, the best-known, and one of the most dangerous, contaminants, was not significantly present in toasted barrel wood. In view of the PAH concentrations in wood and the low solubility of these compounds, their extraction in wine is apparently relatively slow and limited. Finally, comparing the overall PAH concentrations, and particularly those of the most toxic compounds, with estimated absorption from food or the environment, we found it was obvious that the contribution of toasted barrels to the total amount was extremely low and should not, therefore, be considered a major health concern.

Rapid headspace solid-phase microextraction/gas chromatographic/mass spectrometric assay for the quantitative determination of some of the main odorants causing off-flavours in wine.

In this study we present a rapid and simultaneous assay method using headspace (HS) solid-phase microextraction (SPME)/gas chromatography (GC)/electron impact (EI) mass spectrometry (MS) (selected ion monitoring) for contaminants causing the principal organoleptic defects of wine (2,4,6-trichloroanisole, 2,3,4,6-tetrachloroanisole, pentachloroanisole, 2,4,6-tribromoanisole, 1-octen-3-ol, geosmin, 2-methylisoborneol, 3-isopropyl-2-methoxypyrazine, fenchol, fenchone, 2-methoxy-3,5-dimethylpyrazine, 4-ethylphenol, 4-ethylguaiacol, 4-vinylphenol, 4-vinylguaiacol, 3-isobutyl-2-methoxypyrazine, guaiacol and ethyl acetate). The method was validated according to protocols NF ISO 5725-1, 2 and NF V03-110. Its characteristics (limit of detection (LOD), limit of quantification (LOQ), uncertainties) were determined after having optimised the SPME parameters. The target contaminants were quantified in the wines below their threshold of perception with a satisfactory relative standard deviation for all the analytes except ethyl acetate (RSD=36%); for that, the assay method permits clear differentiation of the wines that are at risk of presenting an acescent character, i.e. containing more than 120mgL(-1) ethyl acetate. The target volatile and odorous substances were determined at concentrations significantly below their threshold of perception in a hydroalcoholic context and their threshold of recovery in wines.

Identification and responsibility of 2,4,6-tribromoanisole in musty, corked odors in wine.

In this work, gas phase chromatography analysis coupled with selective selected ion monitoring (SIM) identified 2,4,6-tribromoanisole (TBA) in wines found on tasting to have significant "musty or corked" character, although they did not contain noteworthy quantities of chloroanisoles or chlorophenols, the contaminants generally reported to cause this type of defect. The perception thresholds were studied, together with contamination conditions during winemaking, storage, and bottle-aging. A "musty" off-odor was perceptible on smelling wine containing as little as 4 ng L(-)(1) TBA, and spoilage may be detected by retro-olfaction at even lower concentrations. TBA, produced by O-methylation of its direct precursor, 2,4,6-tribromophenol, generally comes from sources in the winery environment. This paper is the first to identify the sources of a large number of cases of wines polluted during storage in premises where the atmosphere was contaminated with TBA used recently to treat wood, or originating from much older structural elements of the winery, or from used wooden containers. In certain cases, although the initial source had been eliminated, residual pollution adsorbed on walls could be sufficient to make a building unsuitable for storing wooden barrels and plastics, as well as corks, which have been found to be particularly susceptible to contamination by the TBA in the winery atmosphere.