Cork taint of wines: role of the filamentous fungi isolated from cork in the formation of 2,4,6-trichloroanisole by o methylation of 2,4,6-trichlorophenol

Abstract

Cork taint is a musty or moldy off-odor in wine mainly caused by 2,4,6-trichloroanisole (2,4,6-TCA). We examined the role of 14 fungal strains isolated from cork samples in the production of 2,4,6-TCA by O methylation of 2,4,6-trichlorophenol (2,4,6-TCP). The fungal strains isolated belong to the genera Penicillium (four isolates); Trichoderma (two isolates); and Acremonium, Chrysonilia, Cladosporium, Fusarium, Mortierella, Mucor, Paecilomyces, and Verticillium (one isolate each). Eleven of these strains could produce 2,4,6-TCA when they were grown directly on cork in the presence of 2,4,6-TCP. The highest levels of bioconversion were carried out by the Trichoderma and Fusarium strains. One strain of Trichoderma longibrachiatum could also efficiently produce 2,4,6-TCA in liquid medium. However, no detectable levels of 2,4,6-TCA production by this strain could be detected on cork when putative precursors other than 2,4,6-TCP, including several anisoles, dichlorophenols, trichlorophenols, or other highly chlorinated compounds, were tested. Time course expression studies with liquid cultures showed that the formation of 2,4,6-TCA was not affected by a high concentration of glucose (2% or 111 mM) or by ammonium salts at concentrations up to 60 mM. In T. longibrachiatum the O methylation of 2,4,6-TCP was catalyzed by a mycelium-associated S-adenosyl-L-methionine (SAM)-dependent methyltransferase that was strongly induced by 2,4,6-TCP. The reaction was inhibited by S-adenosyl-L-homocysteine, an inhibitor of SAM-dependent methylation, suggesting that SAM is the natural methyl donor. These findings increase our understanding of the mechanism underlying the origin of 2,4,6-TCA on cork, which is poorly understood despite its great economic importance for the wine industry, and they could also help us improve our knowledge about the biodegradation and detoxification processes associated with chlorinated phenols.

FIG. 1.

Evolution of bacterial populations (gray bars) and fungal populations (solid bars) during the manufacture of agglomerated cork stoppers. The samples correspond to several stages of the manufacturing process described in Materials and Methods, as follows: raw cork (bars A), boiled cork (bars B), mold growth room stage (bars C), stacked planks exposed to the ambient environmental conditions (bars D), granulated cork (bars E), glued agglomerated cork stoppers (bars F), and final cork stoppers (SO₂ treated) (bars G). The results are the means of four independent experiments.

FIG. 2.

HPLC analysis of 2,4,6-TCA and 2,4,6-TCP levels in culture supernatants of T. longibrachiatum. (A) Culture at zero time; (B) 24-h culture; (C) 72-h culture; and (D) 72-h culture containing exogenous pure 2,4,6-TCA. Peaks corresponding to 2,4,6-TCA and 2,4,6-TCP are indicated by arrows. mAU, milliunits of absorbance.

FIG. 3.

Time course of bioconversion of 2,4,6-TCP, production of 2,4,6-TCA, and growth curves for T. longibrachiatum cultures grown in MEB (A), in MEB supplemented with 111 mM glucose (B), in MEB supplemented with 50 mM ammonium acetate (C), and in MEB containing 111 mM glucose and 50 mM ammonium acetate (D). Growth (•) was estimated by determining mycelial dry weight. Concentrations of 2,4,6-TCP (▴) and 2,4,6-TCA (▵) in supernatants were determined by HPLC. Each data set is the average data for duplicate samples obtained in three independent experiments.

FIG. 4.

Growth curves, levels of 2,4,6-TCP and 2,4,6-TCA, and cell-associated methyltransferase activity in liquid cultures of T. longibrachiatum grown in the presence (A) or in the absence (B) of 2,4,6-TCP (10 μg ml⁻¹). Growth (•) was measured by determining mycelial dry weight. Concentrations of 2,4,6-TCP (▴) and 2,4,6-TCA (▵) in supernatants were determined by HPLC after extraction from culture medium. SAM-dependent methyltransferase activity (○) was estimated by using mycelia. Each data set is the average data for duplicate samples obtained in three independent experiments.