Pharmacology of the Umami Taste Receptor.

Umami, the fifth taste, has been recognized as a legitimate taste modality only recently relative to the other tastes. Dozens of compounds from vastly different chemical classes elicit a savory (also called umami) taste. The prototypical umami substance glutamic acid or its salt monosodium glutamate (MSG) is present in numerous savory food sources or ingredients such as kombu (edible kelp), beans, soy sauce, tomatoes, cheeses, mushrooms, and certain meats and fish. Derivatives of glutamate (Glu), other amino acids, nucleotides, and small peptides can also elicit or modulate umami taste. In addition, many potent umami tasting compounds structurally unrelated to amino acids, nucleotides, and MSG have been either synthesized or discovered as naturally occurring in plants and other substances. Over the last 20 years several receptors have been suggested to mediate umami taste, including members of the metabotropic and ionotropic Glu receptor families, and more recently, the heterodimeric G protein-coupled receptor, T1R1/T1R3. Careful assessment of representative umami tasting molecules from several different chemical classes shows activation of T1R1/T1R3 with the expected rank order of potency in cell-based assays. Moreover, 5'-ribonucleotides, molecules known to enhance the savory note of Glu, considerably enhance the effect of MSG on T1R1/T1R3 in vitro. Binding sites are found on at least 4 distinct locations on T1R1/T1R3, explaining the propensity of the receptor to being activated or modulated by many structurally distinct compounds and these binding sites allosterically interact to modulate receptor activity. Activation of T1R1/T1R3 by all known umami substances evaluated and the receptor's pharmacological properties are sufficient to explain the basic human sensory experience of savory taste and it is therefore unlikely that other receptors are involved.

New Umami Amides: Structure-Taste Relationship Studies of Cinnamic Acid Derived Amides and the Natural Occurrence of an Intense Umami Amide in Zanthoxylum piperitum.

A series of aromatic amides were synthesized from various acids and amines selected from naturally occurring structural frameworks. These synthetic amides were evaluated for umami taste in comparison with monosodium glutamate. The effect of the substitution pattern of both the acid and the amine parts on umami taste was investigated. The only intensely umami-tasting amides were those made from 3,4-dimethoxycinnamic acid. The amine part was more tolerant to structural changes. Amides bearing an alkyl- or alkoxy-substituted phenylethylamine residue displayed a clean umami taste as 20 ppm solutions in water. Ultraperformance liquid chromatography coupled with a high quadrupole-Orbitrap mass spectrometer (UPLC/MS) was subsequently used to show the natural occurrence of these amides. (E)-3-(3,4-Dimethoxyphenyl)-N-(4-methoxyphenethyl)acrylamide was shown to occur in the roots and stems of Zanthoxylum piperitum, a plant of the family Rutaceae growing in Korea, Japan, and China.

Comparative analysis of three Australian finger lime (Citrus australasica) cultivars: identification of unique citrus chemotypes and new volatile molecules.

The volatile constituents of the peel of three cultivars of Australian finger lime (Citrus australasica) were investigated: Alstonville, Judy's Everbearing and Durham's Emerald. Both qualitative and quantitative GC-MS analyses were performed on their peel solvent extract. The results showed that the unique phenotypes of finger lime are also correlated to unique molecular compositions. Each cultivar revealed a different chemotype: limonene/sabinene for cv. Alstonville, limonene/citronellal/isomenthone for cv. Judy's Everbearing, and limonene/citronellal/ citronellol for cv. Durham's Emerald. To the best of our knowledge, these chemotypes have never been reported in any other citrus species. Furthermore, the amounts of some volatile constituents (γ-terpinene, α-pinene, ß-pinene, citral), which are generally the major constituents besides limonene in lime species, were surprisingly low in the three cultivars. Comparative GC-MS analysis also showed that some volatile molecules tended to be specific to one cultivar and could therefore be considered as markers. Moreover six molecules were reported for the first time in a citrus extract and confirmed by synthesis. Heart-cutting enantioselective two-dimensional GC-MS was performed to determine the enantiomeric distribution of the major chiral constituents. The combined data on three finger lime cultivars gives evidence of their divergence from other citrus species.

50 years of mass spectrometry at Firmenich: a continuing love story.

Mass spectrometry (MS) has been intensively used in the field of flavor and fragrance since its beginning in the 1950s, and it remains an essential technique for current and future research in this field. After a short historical section on the introduction and development of MS at Firmenich, this work reviews the main applications of MS-based techniques published by Firmenich researchers over the past 5 years. It exemplifies the use of gas chromatography (GC)-MS for the discovery of new odorant - hence volatile - molecules in a broad range of natural products, such as fruits, meats, and vegetables. Non-volatile compounds play a major role in taste attributes and are also possible precursors of odorant molecules. Their identification by liquid chromatography (LC)-MS in the context of malodor generation from sweat is a typical example of such a relationship. With their high selectivity and sensitivity, GC-MS and LC-MS instruments are used in the fields of flavor and fragrance not only for identification, but also as unique tools for the accurate quantitation of compounds in complex matrices. This is particularly important for regulatory analyses such as dosage of potential allergens in perfumes and for the development of delivery systems. Finally, because of the rapid response time of MS, the kinetics of processes such as the release of flavors in the mouth during food consumption can be monitored by direct sampling into the mass spectrometer.

Identification of a new lactone contributing to overripe orange aroma in Bordeaux dessert wines via perceptual interaction phenomena.

Recent studies have demonstrated the existence of a typical sensory concept for Bordeaux dessert wines, including the world famous wines of Sauternes. Volatile compounds from several chemical families (thiols, aldehydes, and lactones) were identified and correlated with aromatic typicality in these wines. However, these studies were unable to indicate "key" aromas of overripe fruits, especially overripe orange. The alternative strategy developed in this research combined both analytical and sensory studies of fractions of dessert wine extracts obtained by semipreparative high-performance liquid chromatography (HPLC). Multidimensional gas chromatography coupled to olfactometry and mass spectrometry (MDGC-O/MS) was applied to some of the HPLC fractions recalling "overripe fruit", and a new lactone, 2-nonen-4-olide, was identified. Reconstitution and omission tests using the HPLC fractions highlighted the importance of specific compounds, particularly 2-nonen-4-olide, in the expression of overripe orange notes. Although this lactone presents minty and fruity odors, its key contribution to the typical aroma of orange in Bordeaux dessert wines was revealed through perceptual blending.

Quantification of Hydrogen Sulfide and Methanethiol and the Study of Their Scavenging by Biocides of the Isothiazolone Family.

A sensitive analytical method for the quantification of malodourous hydrogen sulfide (H2 S) and methanethiol (CH3 SH) was developed and validated. The method consisted of solid-phase microextraction with concomitant in-fibre derivatisation of the mercaptan using N-ethylmaleimide. The adducts were analysed by GC-MS using a triple quadrupole in the selected reaction monitoring mode. The limits of detection of solutions of H2 S and CH3 SH in water were 100 and 10 ng L-1 , respectively, below their odour-perception thresholds. The analytical method was used to show the scavenging effect of 1,2-benzisothiazol-3(2H)-one (Proxel) and 2-methyl-4-isothiazolinone. The mechanism by which H2 S and CH3 SH are trapped with Proxel was studied by ultraperformance liquid chromatography (UPLC)-MS.

Identification and quantitation of new glutamic acid derivatives in soy sauce by UPLC/MS/MS.

Glutamic acid is an abundant amino acid that lends a characteristic umami taste to foods. In fermented foods, glutamic acid can be found as a free amino acid formed by proteolysis or as a non-proteolytic derivative formed by microorganisms. The aim of the present study was to identify different structures of glutamic acid derivatives in a typical fermented protein-based food product, soy sauce. An acidic fraction was prepared with anion-exchange solid-phase extraction (SPE) and analyzed by UPLC/MS/MS and UPLC/TOF-MS. α-Glutamyl, γ-glutamyl, and pyroglutamyl dipeptides, as well as lactoyl amino acids, were identified in the acidic fraction of soy sauce. They were chemically synthesized for confirmation of their occurrence and quantified in the selected reaction monitoring (SRM) mode. Pyroglutamyl dipeptides accounted for 770 mg/kg of soy sauce, followed by lactoyl amino acids (135 mg/kg) and γ-glutamyl dipeptides (70 mg/kg). In addition, N-succinoylglutamic acid was identified for the first time in food as a minor compound in soy sauce (5 mg/kg).

Quantification of selected furocoumarins by high-performance liquid chromatography and UV-detection: capabilities and limits.

The performance of HPLC-UV as a means of quantifying selected furocoumarins in essential oils has been evaluated, based on a ring test validation approach. Accuracy profiles were generated, to determine bias and statistical confidence associated with determination at different concentrations, along with lower limits of quantification (LOQ). From these findings, it can be concluded that the method described may only be used in simple cases (essential oils), to measure individual furocoumarin compounds at concentrations greater than 10mg/l; the non compound-specific nature of detection by absorption in the UV range is unable to overcome the effect of interferences arising from chromatographic coelutions, such as those encountered in the analysis of complex commercial fragrance mixtures. The use of an algorithmically calculated 'spectral similarity' function, with reference to authentic standards, may be used to improve reliability in assignment and quantification.

Identification and synthesis of new volatile molecules found in extracts obtained from distinct parts of cooked chicken.

Several chicken parts (skin, fat, juice) were cooked in different ways (roasting, simmering) and investigated separately for their volatile composition. In-depth GC/MS analysis of the separate fractions revealed several unknown molecules. Mass spectra interpretation allowed us to identify nine molecules for the first time in chicken, including cyclic aldehydes, cyclic ketones, and new δ-lactones containing an unsaturated linear chain. Identification was confirmed by chemical synthesis followed by comparison of the mass spectra and linear retention indices. The natural occurrence of five of these molecules is reported here for the first time in a natural product.

Discovery of new lactones in sweet cream butter oil.

Sweet cream butter oil was analyzed to identify new volatile compounds that may contribute to its flavor, with an emphasis on lactones. The volatile part of butter oil was obtained by using short-path distillation. As some previously unknown lactones were detected in this first extract, it was fractionated further. The fatty acids were removed, and the extract was fractionated by flash chromatography. Three lactonic fractions possessing a creamy, buttery, and fatty character were investigated in depth by gas chromatography (GC) and mass spectrometry (MS) (EI and CI) and high-resolution GC-time-of-flight MS. Many lactones were identified by their mass fragmentation and by comparison with reference materials synthesized during this work. Six γ-lactones, five δ-lactones, and one ε-lactone were identified for the first time in butter oil, seven of them for the first time in a natural product. The possible contribution of these new lactones to the aroma of butter oil is briefly discussed.

Easy access to aroma active unsaturated γ-lactones by addition of modified titanium homoenolate to aldehydes.

The homo-Reformatsky reaction, in which a metal homoenolate of an ester is added to an aldehyde, was adapted to produce γ-lactones from unsaturated, enolizable aldehydes. By use of titanium homoenolate, 11 different γ-lactones were synthesized in one step with moderate to good yields from readily available aldehydes. In particular, this procedure allowed the rapid preparation of a series of C(12) unsaturated γ-lactones differing in the position and configuration of the double bond. These reference compounds will be used to identify previously unknown lactones in butter oil. The chromatographic, spectral, and sensory descriptions of the synthesized lactones are provided.

Controlled release of volatile secondary and tertiary alcohols by neighboring group participation: stepwise cyclization and re-opening of 2,2'-bis(carbamoyl)dibenzoates at neutral pH.

Bioactive, volatile, secondary and tertiary fragrance alcohols are efficiently released by intramolecular neighboring-group-assisted hydrolysis of 2,2'-bis(carbamoyl)dibenzoates at neutral pH. The stepwise cyclization of 2,2'-[(methylimino)bis(propane-3,1-diylcarbamoyl)]dibenzoates is followed by the re-opening of the intermediately formed diphthalimide and proceeds in an overall four-step consecutive reaction sequence. Kinetic rate constants for all four reaction steps could be determined pairwise by reversed-phase HPLC. At neutral pH, secondary alcohols were released by one order of magnitude faster than the tertiary alcohols, and the rate constants for the re-opening of the diphthalimides were found to be in the same order of magnitude as the release of the tertiary alcohols. Dynamic headspace analysis on a dry cotton surface finally confirmed the efficient release of tertiary alcohols under mild reaction conditions generally encountered for applications in functional perfumery.

Odorous impact of volatile thiols on the aroma of young botrytized sweet wines: identification and quantification of new sulfanyl alcohols.

Specific extraction of volatile thiols using sodium p-hydroxymercuribenzoate revealed the presence of three new sulfanylalcohols in wines made from Botrytis-infected grapes: 3-sulfanylpentan-1-ol (II), 3-sulfanylheptan-1-ol (III), and 2-methyl-3-sulfanylbutan-1-ol (IV). The first two have citrus aromas, whereas the third is reminiscent of raw onion. In addition, 2-methyl-3-sulfanylpentan-1-ol, which has a raw onion odor, was tentatively identified. Like 3-sulfanylhexan-1-ol (I), already reported in Sauternes wines, compounds II, III, and IV were absent from must. They were found in wine after alcoholic fermentation, and their concentrations were drastically higher when Botrytis cinerea had developed on the grapes. In the commercial botrytized wines analyzed, the mean levels of II, III, and IV were 209, 51, and 103 ng/L, respectively. Despite their low odor activity values, sensory tests showed additive effects among I, II, and III, thus confirming their olfactory impact on the overall aroma of botrytized wines.

From linden flower to linden honey. Part 2: Glycosidic precursors of cyclohexa-1,3-diene-1-carboxylic acids.

The presence of two unusual, recently identified terpene acids, i.e., 4-(1-hydroxy-1-methylethyl)cyclohexa-1,3-diene-1-carboxylic acid (1) and 4-(1-methylethenyl)cyclohexa-1,3-diene-1-carboxylic acid (2), was now also confirmed in (Swiss) linden honey, after solid-phase extraction and HPLC purification. NMR Spectroscopy, in combination with UPLC/MS analysis, showed the presence of several glycosides of 1, which accounted for ca. 0.6 weight-% of the honey, as quantified by UPLC-UV. The major 'glycoside' of 1, compound 5, could be isolated and identified by 2D-NMR experiments as the corresponding beta-gentiobiosyl ester (rather than the classical compound with a glycosidic bond between an aglycone OH group and the sugar). The same diglycosides found in linden honey were also detected in linden nectar; also, chestnut and fir honeys contained these glycosides in minor quantities, but not colza, acacia, or dandelion honeys (Table 2).

Stereoisomeric distribution of 3-mercaptohexan-1-ol and 3-mercaptohexyl acetate in dry and sweet white wines made from Vitis vinifera (Var. Sauvignon Blanc and Semillon).

The enantiomeric distribution of 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA) in Vitis vinifera wines was determined by combining two techniques: specific purification of volatile thiols from the wines using p-hydroxymercuribenzoate and separation of the chiral molecules by gas-phase chromatography on a cyclodextrin capillary column. The R and S enantiomer ratios of these two thiols in dry white Sauvignon blanc and Semillon wines are approximately 30:70 for A3MH and 50:50 for 3MH. However, in sweet white wines made from grapes affected by "noble rot" due to the development of Botrytis cinerea on ripe grapes, the proportion of the R and S forms of 3MH is in the vicinity of 30:70. During alcoholic fermentation, a change in the ratio of the two enantiomers of 3MH in dry white wines was observed. At the beginning of fermentation (around density 1.08), the S form represented over 60%; then, at lower density, as fermentation proceeded, the enatiomeric ratio approached 50:50. The ratio of the two 3MHA enantiomers remained constant throughout fermentation. On the contrary, the distribution of the two 3MH enantiomers changed very little during fermentation of the botrytized sweet wines. The perception thresholds for the R and S forms of 3MH in hydroalcoholic model solution are similar (50 and 60 ng/L). These two enantiomers have quite different aromas: The R form is fruitier, with a zesty aroma reminiscent of grapefruit, while the S form smells more of passion fruit. The perception thresholds of the R and S enantiomers of 3MHA are slightly different (9 and 2.5 ng/L). The less odoriferous R form is reminiscent of passion fruit, while the S form has a more herbaceous odor of boxwood.

Quantification of total furocoumarins in citrus oils by HPLC coupled with UV, fluorescence, and mass detection.

Furocoumarins or psoralens represent a class of photosensitizers whose use level is likely to be restricted to 1 ppm in cosmetic products by the EU. A reversed-phase HPLC method was developed to separate the 15 main furocoumarins present in citrus oils. Quantification by UV, fluorescence, or mass detectors was compared in terms of linearity and limit of detection. Cold-pressed oils of different citrus species were analyzed using this method. This method could be implemented in quality control laboratories equipped with an HPLC system and a UV diode array detector. Because of possible coelutions, the UV-spectral data should be carefully examined to avoid misleading interpretations of peaks.