Myriam Troccaz

Olfactory Perception of Cysteine−S-Conjugates from Fruits and Vegetables

Volatile sulfur compounds have a low odor threshold, and their presence at microgram per kilogram levels in fruits and vegetables influences odor quality. Sensory analysis demonstrates that naturally occurring, odorless cysteine- S-conjugates such as S-( R/ S)-3-(1-hexanol)- l-cysteine in wine, S-(1-propyl)- l-cysteine in onion, and S-(( R/ S)-2-heptyl)- l-cysteine in bell pepper are transformed into volatile thiols in the mouth by microflora. The time delay in smelling these volatile thiols was 20-30 s, and persistent perception of their odor occurred for 3 min. The cysteine- S-conjugates are transformed in free thiol by anaerobes. The mouth acts as a reactor, adding another dimension to odor perception, and saliva modulates flavors by trapping free thiols.

Nonvolatile S-alk(en)ylthio-L-cysteine derivatives in fresh onion (Allium cepa L. cultivar).

The L-cysteine derivatives (R)-2-amino-3-(methyldisulfanyl)propanoic acid (S-methylthio-L-cysteine), (R)-2-amino-3-(propyldisulfanyl)propanoic acid (S-propylthio-L-cysteine), (R)-2-amino-3-(1-propenyldisulfanyl)propanoic acid (S-(1-propenylthio)-L-cysteine), and (R)-2-amino-3-(2-propenyldisulfanyl)propanoic acid (S-allylthio-L-cysteine) were prepared from 3-[(methoxycarbonyl)dithio]-L-alanine, obtained from the reaction of L-cysteine with methoxycarbonylsulfenyl chloride. The occurrence of these S-(+)-alk(en)ylthio-L-cysteine derivatives in onion (Allium cepa L.) was proven by using UPLC-MS-ESI(+) in SRM mode. Their concentrations in fresh onion were estimated to be 0.19 mg/kg S-methylthio-L-cysteine, 0.01 mg/kg S-propylthio-L-cysteine, and 0.56 mg/kg (S-(1-propenyllthio)-L-cysteine, concentrations that are about 3000 times lower than that of isoalliin (S-(1-propenyl-S-oxo-L-cysteine). These compounds were treated with Fusobacterium nucleatum, a microorganism responsible for the formation of mouth malodor. These L-cysteine disulfides were demonstrated to predominantly produce tri- and tetrasulfides. Isoalliin is almost entirely consumed by the plant enzyme alliin lyase (EC 4.4.1.4 S-alk(en)yl-S-oxo-L-cysteine lyase) in a few seconds, but it is not transformed by F. nucleatum. This example of flavor modulation shows that the plant produces different precursors, leading to the formation of the same types of volatile sulfur compounds. Whereas the plant enzyme efficiently transforms S-alk(en)yl-S-oxo-L-cysteine, mouth bacteria are responsible for the transformation of S-alk(en)ylthio-L-cysteine.

Properties of Recombinant Staphylococcus haemolyticus Cystathionine β‐Lyase (metC) and Its Potential Role in the Generation of Volatile Thiols in Axillary Malodor

Enzymes implicated in cysteine and methionine metabolism such as cystathionine β‐lyase (CBL; EC 4.4.1.8), a pyridoxal‐5′‐phosphate (PLP)‐dependent carbon–sulfur lyase, have been shown to play a central role in the generation of sulfur compounds. This work describes the unprecedented cloning and characterization of the metC‐cystathionine β‐lyase from the axillary‐isolated strain Staphylococcus haemolyticus AX3, in order to determine its activity and its involvement in amino acid biosynthesis, and in the generation of sulfur compounds in human sweat. The gene contains a cysteine/methionine metabolism enzyme pattern, and also a sequence capable to effect β‐elimination. The recombinant enzyme was shown to cleave cystathionine into homocysteine and to convert methionine into methanethiol at low levels. No odor was generated after incubation of the recombinant enzyme with sterile human axillary secretions; sweat components were found to have an inhibitory effect. These results suggest that the generation of sulfur compounds by Staphylococci and the β‐lyase activity in human sweat are mediated by enzymes other than the metC gene or by the concerted activities of more than one enzyme.

5α-Androst-16-en-3α-ol β-D-glucuronide, precursor of 5α-androst-16-en-3α-ol in human sweat.

5α‐Androst‐16‐en‐3α‐ol (α‐androstenol) is an important contributor to human axilla sweat odor. It is assumed that α‐andostenol is excreted from the apocrine glands via a H2O‐soluble conjugate, and this precursor was formally characterized in this study for the first time in human sweat. The possible H2O‐soluble precursors, sulfate and glucuronide derivatives, were synthesized as analytical standards, i.e., α‐androstenol, β‐androstenol sulfates, 5α‐androsta‐5,16‐dien‐3β‐ol (β‐androstadienol) sulfate, α‐androstenol β‐glucuronide, α‐androstenol α‐glucuronide, β‐androstadienol β‐glucuronide, and α‐androstenol β‐glucuronide furanose. The occurrence of α‐androstenol β‐glucuronide was established by ultra performance liquid chromatography (UPLC)/MS (heated electrospray ionization (HESI)) in negative‐ion mode in pooled human sweat, containing eccrine and apocrine secretions and collected from 25 female and 24 male underarms. Its concentration was of 79 ng/ml in female secretions and 241 ng/ml in male secretions. The release of α‐androstenol was observed after incubation of the sterile human sweat or α‐androstenol β‐glucuronide with a commercial glucuronidase enzyme, the urine‐isolated bacteria Streptococcus agalactiae, and the skin bacteria Staphylococcus warneri DSM 20316, Staphylococcus haemolyticus DSM 20263, and Propionibacterium acnes ATCC 6919, reported to have β‐glucuronidase activities. We demonstrated that if α‐ and β‐androstenols and androstadienol sulfates were present in human sweat, their concentrations would be too low to be considered as potential precursors of malodors; therefore, the H2O‐soluble precursor of α‐androstenol in apocrine secretion should be a β‐glucuronide.

Behavioral response of the malaria mosquito, Anopheles gambiae, to human sweat inoculated with axilla bacteria and to volatiles composing human axillary odor

The responses of Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) to odors from male and female axillary sweat incubated with human axilla bacteria were recorded in a dual-choice olfactometer. Staphylococcus epidermidis was selected for its low odor-producing pattern, Corynebacterium jeikeium for its strong N&agr;-acylglutamine aminoacylase activity liberating carboxylic acids including (R)/(S)-3-hydroxy-3-methylhexanoic acid (HMHA) and Staphylococcus haemolyticus for its capacity to liberate sulfur-containing compounds including (R/S)-3-methyl-3-sulfanylhexan-1-ol (MSH). Anopheles gambiae behavioral responses were evaluated under (i) its responsiveness to take off and undertake sustained upwind flight and (ii) its discriminating capacity between the two olfactometer arms bearing a test odor in either one or both arms. Experiments were conducted in the presence of carbon dioxide pulses as a behavioral sensitizer. Anopheles gambiae clearly discriminated for the olfactometer arm conveying odor generated by incubating any of the three bacteria species with either male or female sweat. Whereas An. gambiae did not discriminate between male and female sterile sweat samples in the olfactometer, the mosquito consistently showed a preference for male sweat over female sweat incubated with the same bacterium, independent of the species used as inoculum. Sweat incubated with C. jeikeium rendered mosquitoes particularly responsive and this substrate elicited the strongest preference for male over female sweat. Tested on their own, neither HMHA nor MSH elicited a clear discriminating response but did affect mosquito responsiveness. These findings serve as a basis for further research on the odor-mediated anthropophilic host-seeking behavior of An. gambiae.