Investigation of 2-Sulfanylethyl Acetate Cysteine-S-Conjugate as a Potential Precursor of Free Thiols in Beer

The compounds 2-sulfanylethyl acetate (2SEA, roasted- or burnt-like flavor) and 3-sulfanylpropyl acetate (also with roasted- or burnt-like flavor), mainly issued from the Ehrlich pathway (in this case, the catabolism by yeast of cysteine and homocysteine, leading to the formation of 2-sulfanylethan-1-ol and 2-sulfanylpropan-l-ol, which are subsequently acetylated), are found in all fresh beers, at levels strongly depending on the yeast strain used in fermentation. Yet, even in the absence of yeast, their production is still seen to occur in the bottle during the first months of storage. As for other thiols, S-cysteine adducts have been suspected to be hydrolyzed during beer aging. The commercially unavailable S-cysteine conjugate of 2SEA (Cys-2SEA, 93% purity), never described before in the literature, was synthesized by acetylation of the S-cysteine conjugate of 2-sulfanylethan-l-ol (Cys-2SEol). Cys-2SEA was solubilized in model aqueous media (pH 4.2) and in beer before aging. 2SEA was further extracted and analyzed by gas chromatography coupled to a pulsed flame photometric detector (GC-PFPD), and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI(+)MS/MS) allowed us to quantify the undegraded Cys-2SEA (specific m/z ions at 120, 148, and 191). Cys-2SEA was shown to be relatively stable in both media, although some beer constituents were noted as being able to slightly facilitate the chemical degradation (molar conversion into free thiol up to 0.3% at room temperature). A modified Strecker degradation mechanism is proposed here, explaining the release of thiols from the Schiff bases created between the cysteine adducts and beer diketones. Up to now, tests have failed to reveal evidence for the presence of Cys-2SEo1 and Cys-2SEA in hops by direct HPLC-ESI(+)-MS/MS analyses, although 2SEol was released from cysteine-S-conjugate extracts in the presence of apotryptophanase.