Thermal treatment of aqueous solutions of xylose and primary amino acids led to rapid development of a bitter taste of the reaction mixture. To characterize the key compound causing this bitter taste, a novel bioassay, which is based on the determination of the taste threshold of reaction products in serial dilutions of HPLC fractions, was developed to select the most intense taste compounds in the complex mixture of Maillard reaction products. By application of this so-called taste dilution analysis (TDA) 21 fractions were obtained, among which 1 fraction was evaluated with by far the highest taste impact. Carefully planned LC-MS as well as 1D and 2D NMR experiments were, therefore, focused on the compound contributing the most to the intense bitter taste of the Maillard mixture and led to its unequivocal identification as the previously unknown 3-(2-furyl)-8-[(2-furyl)methyl]-4-hydroxymethyl-1-oxo-1H,4H-quinolizinium-7-olate. This novel compound, which we name quinizolate, exhibited an intense bitter taste at an extraordinarily low detection threshold of 0.00025 mmol/kg of water. As this novel taste compound was found to have 2000- and 28-fold lower threshold concentrations than the standard bitter compounds caffeine and quinine hydrochloride, respectively, quinizolate might be one of the most intense bitter compounds reported so far.

A fast and precise proton nuclear magnetic resonance (qHNMR) method for the quantitative determination of low molecular weight target molecules in reference materials and natural isolates has been validated using ERETIC 2 (Electronic REference To access In vivo Concentrations) based on the PULCON (PULse length based CONcentration determination) methodology and compared to the gravimetric results. Using an Avance III NMR spectrometer (400 MHz) equipped with a broad band observe (BBO) probe, the qHNMR method was validated by determining its linearity, range, precision, and accuracy as well as robustness and limit of quantitation. The linearity of the method was assessed by measuring samples of l-tyrosine, caffeine, or benzoic acid in a concentration range between 0.3 and 16.5 mmol/L (r(2) ≥ 0.99), whereas the interday and intraday precisions were found to be ≤2%. The recovery of a range of reference compounds was ≥98.5%, thus demonstrating the qHNMR method as a precise tool for the rapid quantitation (~15 min) of food-related target compounds in reference materials and natural isolates such as nucleotides, polyphenols, or cyclic peptides.

Weight-conscious subjects and diabetics use the sulfonyl amide sweeteners saccharin and acesulfame K to reduce their calorie and sugar intake. However, the intrinsic bitter aftertaste, which is caused by unknown mechanisms, limits the use of these sweeteners. Here, we show by functional expression experiments in human embryonic kidney cells that saccharin and acesulfame K activate two members of the human TAS2R family (hTAS2R43 and hTAS2R44) at concentrations known to stimulate bitter taste. These receptors are expressed in tongue taste papillae. Moreover, the sweet inhibitor lactisole did not block the responses of cells transfected with TAS2R43 and TAS2R44, whereas it did block the response of cells expressing the sweet taste receptor heteromer hTAS1R2-hTAS1R3. The two receptors were also activated by nanomolar concentrations of aristolochic acid, a purely bitter-tasting compound. Thus, hTAS2R43 and hTAS2R44 function as cognate bitter taste receptors and do not contribute to the sweet taste of saccharin and acesulfame K. Consistent with the in vitro data, cross-adaptation studies in human subjects also support the existence of common receptors for both sulfonyl amide sweeteners.

No abstract

Aimed at elucidating intense bitter-tasting molecules in coffee, various bean ingredients were thermally treated in model experiments and evaluated for their potential to produce bitter compounds. As caffeic acid was found to generate intense bitterness reminiscent of the bitter taste of a strongly roasted espresso-type coffee, the reaction products formed were screened for bitter compounds by means of taste dilution analysis, and the most bitter tastants were isolated and purified. LC-MS/MS as well as 1-D/2-D NMR experiments enabled the identification of 10 bitter compounds with rather low recognition threshold concentrations ranging between 23 and 178 micromol/L. These bitter compounds are the previously unreported 1,3-bis(3',4'-dihydroxyphenyl) butane, trans-1,3-bis(3',4'-dihydroxyphenyl)-1-butene, and eight multiply hydroxylated phenylindanes, among which five derivatives are reported for the first time. In addition, the occurrence of each of these bitter compounds in a coffee brew was verified by means of LC-MS/MS (ESI-) operating in the multiple reaction monitoring (MRM) mode. The structures of these bitter compounds show strong evidence that they are generated by oligomerization of 4-vinylcatechol released from caffeic acid moieties upon roasting.

The alphaproteobacterial Roseobacter clade (Rhodobacterales) is one of the most important global players in carbon and sulfur cycles of marine ecosystems. The remarkable metabolic versatility of this bacterial lineage provides access to diverse habitats and correlates with a multitude of extrachromosomal elements. Four non-homologous replication systems and additional subsets of individual compatibility groups ensure the stable maintenance of up to a dozen replicons representing up to one third of the bacterial genome. This complexity presents the challenge of successful partitioning of all low copy number replicons. Based on the phenomenon of plasmid incompatibility, we developed molecular tools for target-oriented plasmid curing and could generate customized mutants lacking hundreds of genes. This approach allows one to analyze the relevance of specific replicons including so-called chromids that are known as lifestyle determinants of bacteria. Chromids are extrachromosomal elements with a chromosome-like genetic imprint (codon usage, GC content) that are essential for competitive survival in the natural habitat, whereas classical dispensable plasmids exhibit a deviating codon usage and typically contain type IV secretion systems for conjugation. The impact of horizontal plasmid transfer is exemplified by the scattered occurrence of the characteristic aerobic anoxygenic photosynthesis among the Roseobacter clade and the recently reported transfer of the 45-kb photosynthesis gene cluster to extrachromosomal elements. Conjugative transmission may be the crucial driving force for rapid adaptations and hence the ecological prosperousness of this lineage of pink bacteria.

Besides undesirable changes in the attractive aroma, a significant decrease in the intensity of the bitterness as well as a change of the taste into a lingering, harsh bitterness has long been known as a shelf-life limiting factor of beer. Multiple studies have demonstrated that the aging of beer induces a decrease of the total amount of cis- and trans-iso-alpha-acids, the well-known bitter principles of beer. Although the trans-iso-alpha-acids exclusively, not the cis-iso-alpha-acids, were found to be degraded upon storage of beer, the key transformation products formed exclusively from the trans isomers in beer are not known. In the present study, suitable model experiments followed by LC-MS/MS and sophisticated NMR spectroscopic experiments, including the measurement of residual dipolar couplings (RDCs) in gel-based alignment media as well as a novel broadband and B(1)-field-compensated incredible natural abundance double-quantum transfer experiment (INADEQUATE) pulse sequence, enabled the identification of a series of previously unknown trans-specific iso-alpha-acid transformation products, namely, tricyclocohumol, tricyclocohumene, isotricyclocohumene, tetracyclocohumol, and epitetracyclocohumol, respectively. HPLC-MS/MS analysis of these compounds, which exhibit the aforementioned harsh lingering bitter taste and have threshold concentrations ranging from 5 to 70 micromol L(-1), confirmed their generation during aging of beer and, for the first time, explained the storage-induced changes of the beer's bitter taste on a molecular level.

The aim of the present study was to apply an activity-guided screening procedure to coffee brew to identify a key chemopreventive compound by means of in vitro antioxidant tests as well as cell culture experiments and to prove the in vivo activity of that compound by an animal feeding experiment. Solvent fractionation, followed by multiple-step ultrafiltration, revealed that the polar coffee compounds with molecular weights below 1 kDa show the major inhibitory effect on the in vitro peroxidation of linoleic acid as well as the predominant chemopreventive enzyme modulating activity on the NADPH-cytochrome c reductase (CCR) and glutathione S-transferase (GST) in human intestinal Caco-2 cells. To identify the chemical structure of the most active antioxidants and chemopreventive compounds, the polar compounds were further separated by HPLC techniques, followed by the activity-guided screening of the individual HPLC fraction. These experiments demonstrated 5-chlorogenic acid to be the most powerful antioxidant in vitro, whereas, in contrast, chemopreventive effects on the GST activity were found for the N-methylpyridinium ion, the structure of which was elucidated by LC-MS and NMR experiments and confirmed by synthesis. The in vivo activities of coffee beverage and N-methylpyridinium ions were tested in a 15-day feeding experiment on rats. In the liver, feeding of 4.5% coffee beverage resulted in increases of GST and UDP-GT activities by 24 and 40% compared to animals fed the control diet (p > 0.05), respectively. Plasma total antioxidant capacity and plasma tocopherol were elevated in animals fed the coffee beverage and the N-methylpyridinium-containing diet. In summary, the results demonstrating a strong in vitro antioxidant activity for coffee were confirmed by the feeding study. Surprisingly, feeding of N-methylpyridinium also resulted in an increased total antioxidant capacity in the plasma. The data indicate that the mode of action demonstrated for N-methylpyridinium in biological systems is different from that in foods.