Ten strains of Geotrichum candidum were studied on a liquid cheese model medium for the production of sulfur compounds which contribute to the aroma of cheeses. The volatile components produced by each cultured strain were extracted by dynamic headspace extractions, separated and quantified by gas chromatography (GC), and identified by GC-mass spectrometry. It was shown that four strains of this microorganism produced significant quantities of S-methyl thioacetate, S-methyl thiopropionate, S-methyl thiobutanoate, S-methyl thioisobutanoate,S-methyl thioisovalerate, andS-methyl thiohexanoate. This is the first example of the production of these compounds by a fungus. In addition, dimethyldisulfide, dimethyltrisulfide, dimethylsulfide, and methanethiol, which are more commonly associated with the development of cheese flavor in bacterial cultures, were also produced by G. candidum in various yields, depending on the strain selected. The potential application of these strains in cultured microbial associations to produce modified cheeses with more desirable organoleptic properties is discussed.

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The odor characteristics of individual components present in a library comprised of S-methyl thioesters were determined independently by two laboratories using similar but not identical techniques. The odor potency was assessed by values of best estimate-GC-lower amount detected by sniffing (BE-GC-LOADS). For small and medium chain S-methyl thioesters, these values were found to increase from 6 ng for S-methyl thiobutanoate to 90 ng for S-methyl thiostearate. All assessors detected a "green", "floral", or "pineapple" odor for S-methyl thiohexanoate and described thioesters containing a 2-6 carbon chain length as "cheesy". The results of this preliminary analysis were confirmed by a more extensive study of selected compounds, namely S-methyl thioacetate, S-methyl thiopropionate, S-methyl thiobutanoate, and S-methyl thiohexanoate, using a trained panel of 18 subjects. The subjects confirmed the presence of the "green" and "fruity" notes in the odor of S-methyl thiohexanoate. The analysis also revealed a significant difference in the odor of S-methyl thiopropionate relative to that of S-methyl thioacetate and S-methyl thiobutanoate. When "cheesy" characteristics were mentioned, the majority of panelists clearly associated the flavor of S-methyl thiopropionate with Camembert with almost 20% of all the descriptors given referring specifically to this cheese variety as compared to about 2 and 5% in the case of S-methyl thioacetate and thiobutanoate, respectively. Prompted by this observation, two samples of Camembert prepared from unpasteurized and pasteurized milk were analyzed and relatively large amounts of S-methyl thiopropionate were found in the former but not in the latter cheese. The results obtained in the course of this work suggest that the sensory analysis of combinatorial libraries is a useful new approach in the search for new commercial flavors and/or identification of characteristic flavors in foods.

In this study, an analysis of the transition between the inactive ("closed") and active ("open") conformations of Candida rugosa lipase in solution is performed using irreversible enzyme inhibitors, cyclic saligenin phosphates. It is shown that >90% inhibition of the enzyme activity toward water-soluble substrates (esterolytic activity) can be achieved with as little as 0.3 mol of the inhibitor per mole of enzyme, whereas activity toward emulsified substrates decreases by approximately 20% under the same conditions. It is also shown that short-term exposure of this inhibited enzyme preparation to an interface leads to a significant increase in esterolytic activity, which even exceeds that of the untreated control. These experimental observations suggest that the inhibitors interact predominantly, if not exclusively, with the open form of the enzyme and that any transitions occurring between the two conformers of the enzyme in solution, in the absence of an interface, are extremely slow. This conclusion is verified by separating the open and closed forms of the enzyme by hydrophobic interaction column chromatography on phenyl-sepharose. Fractions enriched with the respective conformations of the enzyme are further purified using gel-permeation chromatography. On the basis of the elution pattern from this step, and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the open (active in the absence of interface) form of the lipase is found to be present in solution as a dimer, whereas the closed form appears to be a monomer. The latter form of the enzyme may be activated by up to 60-fold when exposed to triolein.

A novel approach to enzymatic biotransformations in aqueous‐organic two‐phase systems was developed where the aqueous phase was contained within permeable polymeric capsules suspended in organic solvent. Microencapsulated β‐glucosidase, used as a model enzyme, was shown to retain its catalytic activity for a considerable time and was repeatedly used in batch experiments after recharging the microcapsules with solid glucose. The reaction conditions for the synthesis of hexyl β‐[D]‐glucopyranoside were optimized with regard to the polymer composition of the microcapsules, pH, and the volume ratio of aqueous to organic phases. The potential for further improvement in the efficiency of the system was demonstrated by designing a bioreactor which incorporated units for product recovery and recycling of the organic solvent. Other advantages of the proposed methodology include facile control over the size and composition of the microcapsules, and mild reaction conditions during their preparation. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 385–390, 1998.