Lipase-Assisted Generation of 2-Methyl-3-furanthiol and 2-Furfurylthiol from Thioacetates RACHID BEL RHLID,* WALTER MATTHEY-DORET,IMRE BLANK, LAURENT B. FAY, AND MARCEL A. JUILLERAT Nestle ´ Research Center, Nestec Ltd., Vers-chez-les-Blanc, P.O. Box 44, 1000 Lausanne 26, Switzerland Enzymatic hydrolysis of S-3-(2-methylfuryl) thioacetate and S-2-furfuryl thioacetate using lipase from Candida rugosa produced 2-methyl-3-furanthiol and 2-furfurylthiol, respectively. When reactions were carried out at room temperature and pH 5.8, 2-methyl-3-furanthiol was produced in a optimal yield of 88% after 15 min of reaction, whereas 2-furfurylthiol was obtained in a yield of 80% after 1 h of reaction time. Enzymatic hydrolysis was also performed in n-hexane, n-pentane, and water/propylene glycol mixture. The reaction rates in these media were slower as compared to those in aqueous medium; however, the reaction yields were quite similar. As expected, the stability of the generated 2-methyl-3-furanthiol and 2-furfurylthiol was better in n-hexane, n-pentane, and the water/propylene glycol mixture as compared to that in water or phosphate buffer. KEYWORDS: Thiols; thioacetates; 2-methyl-3-furanthiol; 2-furfurylthiol; lipase; hydrolysis; flavor INTRODUCTION Natural flavors are defined as biologically derived aroma chemicals generated by microbial fermentation and/or by the action of endogenous or technical enzymes (1). Among these biocatalysts, lipases and esterases have received special attention because of their effectiveness in regio- and enantioselective esterification and transesterification of organic acids and alcohols (2). Moreover, the use of lipases and esterases in different media, such as organic solvents and supercritical fluids, has been extensively studied (3-5). From an industrial point of view, the organic media exhibit many advantages as compared to aqueous systems: a decrease in microbial contamination, the possibility of dissolving apolar solutes, a reduction of water- dependent side reactions, the enhancement of the thermal stability of enzymes, and finally the easy recovery of products from low-boiling solvents. Because of their stability, effective- ness, and ease to use, lipases and esterases have been considered in many industrial applications such as dairy products (6). These enzymes are also the main biocatalysts used in the production of numerous flavoring compounds, particularly esters that are impact aroma compounds in fruit flavors (7-9). However, only a few studies deal with thioesters, which are used as aroma molecules in savory, baked, and dairy goods (10- 12). Among these thioester derivatives, thioacetates have received little attention, and very few studies deal with the generation and enzymatic hydrolysis of thioacetates to produce thiols (13). These thiols are important constituents of food aroma, and in many cases they have been proven to be impact odorants (14, 15). They especially seem to impart the charac- teristic cooked and roasted notes to meats (16) and to roasted coffee (17). However, thiols are very unstable molecules and easily oxidize to the corresponding disulfides upon storage, even at low temperature (18, 19). To overcome this drawback, enzyme-assisted formation of thiols from thioacetates may be an interesting approach if the reaction rate and yield are high. In fact, thiols could be stored in their more stable thioacetate form, and enzymatic hydrolysis could be achieved just before their use. In this study, thiols were generated by enzymatic hydrolysis of thioacetates in aqueous and organic media using lipase from Candida rugosa as biocatalyst and S-3-(2-methylfuryl) thio- acetate and S-2-furfuryl thioacetate as model substrates. MATERIALS AND METHODS Materials. All chemicals were of analytical grade purchased from Aldrich (Buchs, Switzerland; S-2-furfuryl thioacetate), from Oxford Chemicals [Hartlepool, U.K.; 2-methyl-3-furanthiol and S-3-(2-methyl- furyl) thioacetate], or from Merck (Darmstadt, Germany; sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, and anhydrous sodium sulfate). Lipase from C. rugosa (EC 3.1.1.4, type VII) was purchased from Sigma (Buchs, Switzerland). Immobilized lipase from C. rugosa (30 units/g of solid) on Sol-Gel-AK support was purchased from Fluka (Buchs, Switzerland). Organic solvents were of analytical grade purified by distillation using a Vigreux column (1 m × 1 cm). Enzymatic Hydrolysis of Thioacetates. Different amounts of enzyme were added to 10 mL of a solution of thioacetate (0.064 mmol) in distilled water or phosphate buffer (0.2 M). Enzymatic reactions were performed at different temperatures (4, 23, and 37 °C) with gentle magnetic stirring. Samples were withdrawn at various time intervals (from 1 min to 72 h). A solution of benzyl mercaptan in diethyl ether * Author to whom correspondence should be addressed [telephone (+41) 21 785 8634; fax (+41) 21 785 8549; e-mail rachid.bel-rhlid@rdls.nestle.com]. J. Agric. Food Chem. 2002, 50, 4087-4090 4087 10.1021/jf0202335 CCC: $22.00 © 2002 American Chemical Society Published on Web 06/01/2002