Reactivity of beer bitter acids toward the 1-hydroxyethyl radical as probed by spin-trapping electron paramagnetic resonance (EPR) and electrospray ionization-tandem mass spectrometry (ESI-MS/MS)

The iso-α-acids or isohumulones are the major contributors to the bitter taste of beer, and it is well-recognized that they are degraded during beer aging. In particular, the trans-isohumulones seem to be less stable than the cis-isohumulones. The major radical identified in beer is the 1-hydroxyethyl radical; however, the reactivity between this radical and the isohumulones has not been reported until now. Therefore, we studied the reactivity of isohumulones toward the 1-hydroxyethyl radical through a competitive kinetic approach. It was observed that both cis- and trans-isohumulones and dihydroisohumulones are decomposed in the presence of 1-hydroxyethyl radicals, while the reactivities are comparable. On the other hand, the tetrahydroisohumulones did not react with 1-hydroxyethyl radicals. The apparent second-order rate constants for the reactions between the 1-hydroxyethyl radical and these compounds were determined by electron paramagnetic resonance (EPR) spectroscopy and electrospray ionization-tandem mass spectrometry [ESI(+)-MS/MS]. It follows that degradation of beer bitter acids is highly influenced by the presence of 1-hydroxyethyl radicals. The reaction products were detected by liquid chromatography-electrospray ionization-ion trap-tandem mass spectrometry (LC-ESI-IT-MS/MS), and the formation of oxidized derivatives of the isohumulones was confirmed. These data help to understand the mechanism of beer degradation upon aging.     

Isomerization and degradation kinetics of hop (Humulus lupulus) acids in a model wort-boiling system

The rate of isomerization of alpha acids to iso-alpha acids (the compounds contributing bitter taste to beer) was determined across a range of temperatures (90-130 degrees C) to characterize the rate at which iso-alpha acids are formed during kettle boiling. Multiple 12 mL stainless steel vessels were utilized to heat samples (alpha acids in a pH 5.2 buffered aqueous solution) at given temperatures, for varying lengths of time. Concentrations of alpha acids and iso-alpha acids were quantified by high-pressure liquid chromatography (HPLC). The isomerization reaction was found to be first order, with reaction rate varying as a function of temperature. Rate constants were experimentally determined to be k1 = (7.9 x 10(11)) e(-11858/T) for the isomerization reaction of alpha acids to iso-alpha acids, and k2 = (4.1 x 10(12)) e(-12994/T) for the subsequent loss of iso-alpha acids to uncharacterized degradation products. Activation energy was experimentally determined to be 98.6 kJ per mole for isomerization, and 108.0 kJ per mole for degradation. Losses of iso-alpha acids to degradation products were pronounced for cases in which boiling was continued beyond two half-lives of alpha-acid concentration.     

Structures, sensory activity, and dose/response functions of 2,5-diketopiperazines in roasted cocoa nibs (Theobroma cacao)

The taste compounds inducing the blood-like, metallic bitter taste sensation reported recently for a dichloromethane extract prepared from roasted cocoa nibs were identified as a series of 25 diketopiperazines by means of HPLC degustation, LC-MS/MS, and independent synthesis. Among these 25 compounds, 13 cis-configured diketopiperazines, namely, cyclo(L-IIe-L-Phe), cyclo(L-Val-L-Leu), cyclo(L-Pro-L-Pro), cyclo(L-IIe-L-Pro), cyclo(L-Val-L-Tyr), cyclo(L-Ala-L-Tyr), cyclo(L-Phe-L-Ser), cyclo(L-Ala-L-IIe), cyclo(L-Leu-L-Phe), cyclo(L-Pro-L-Val), cyclo(L-Pro-L-Thr), cyclo(L-Pro-L-Tyr), and cyclo(L-Val-L-Val) were identified for the first time in cocoa. In addition, the taste recognition thresholds for the metallic as well as the bitter taste of the diketopiperazines were determined, and after quantitative analysis by using two diastereomeric diketopiperazines as the internal standards, the sensory impact of the diketopiperazines was evaluated on the basis of their dose-over-threshold (DoT) factors calculated as the ratio of the concentration and the threshold concentration of a compound. These data revealed DoT factors above 1.0 exclusively for cis-cyclo(L-Pro-L-Val), cis-cyclo(L-Val-L-Leu), cis-cyclo(L-Ala-L-Ile), cis-cyclo(L-Ala-L-Leu), and cis-cyclo(L-Ile-L-Pro), whereas all of the other diketopiperazines were present below their individual bitter taste threshold concentrations and should therefore not contribute to the cocoa taste. Because the DoT factors do not consider the nonlinear relationship between the concentration and gustatory response of an individual compound, we, for the first time, report on the recording of dose/response functions describing the human bitter taste perception of diketopiperazines more precisely.     

Structure determination and sensory analysis of bitter-tasting 4-vinylcatechol oligomers and their identification in roasted coffee by means of LC-MS/MS

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.     

Molecular definition of black tea taste by means of quantitative studies, taste reconstitution, and omission experiments

Recently, bioresponse-guided fractionation of black tea infusions indicated that neither the high molecular weight thearubigens nor the theaflavins, but a series of 14 flavon-3-ol glycopyranosides besides some catechins, might be important contributors to black tea taste. To further bridge the gap between pure structural chemistry and human taste perception, in the present investigation 51 putative taste compounds have been quantified in a black tea infusion, and their dose-over-threshold (Dot) factors have been calculated on the basis of a dose/threshold relationship. To confirm these quantitative results, an aqueous taste model was prepared by blending aqueous solutions of 15 amino acids, 14 flavonol-glycosides, 8 flavan-3-ols, 5 theaflavins, 5 organic acids, 3 sugars, and caffeine in their "natural" concentrations. Sensory analyses revealed that the taste profile of this artificial cocktail did not differ significantly from the taste profile of the authentic tea infusion. To further narrow the number of key taste compounds, finally, taste omission experiments have been performed, on the basis of which a reduced recombinate was prepared containing the bitter-tasting caffeine, nine velvety astringent flavonol-3-glycosides, and the puckering astringent catechin as well as the astringent and bitter epigallocatechin-3-gallate. The taste profile of this reduced recombinate differed not significantly from that of the complete taste recombinate, thus confirming these 12 compounds as the key taste compounds of the tea infusion. Additional sensory studies demonstrated for the first time that the flavanol-3-glycosides not only impart a velvety astringent taste sensation to the oral cavity but also contribute to the bitter taste of tea infusions by amplifying the bitterness of caffeine.     

Sensory activity,chemical structure,and synthesis of Maillard generated bitter-tasting 1-oxo-2,3-dihydro-1H-indolizinium-6-olates

Thermal treatment of aqueous solutions of xylose, rhamnose, and l-alanine led to a rapid development of a bitter taste of the reaction mixture. To characterize the key compounds causing this bitter taste, the recently developed taste dilution analysis (TDA), which is based on the determination of the taste threshold of reaction products in serial dilutions of HPLC fractions, was performed to locate the most intense taste compounds in the complex mixture of Maillard reaction products. By application of this TDA, 26 fractions were obtained, among which seven fractions were evaluated with a high taste impact. LC/MS and NMR spectroscopy as well as synthetic experiments revealed the 1-oxo-2,3-dihydro-1H-indolizinium-6-olates 1-5 as the key compounds contributing the most to the intense bitter taste of the Maillard mixture. Calculation of the taste impact of these compounds based on a dose/activity relationship indicated that these five compounds already accounted for 56.8% of the overall bitterness of the Maillard mixture, thus demonstrating this class of 1-oxo-2,3-dihydro-1H-indolizinium-6-olates as the key bitter compounds. First synthetic studies on the relationship between the chemical structure and the human psychobiological activity of 1-oxo-2,3-dihydro-1H-indolizinium-6-olates revealed that substitution of the furan rings of 1 by 5-methylfuryl moieties (compounds 3-5) or by 5-(hydroxymethyl)furyl groups (compound 6) led to a significant increase of the bitter threshold. In contrast, the substitution of the oxygen atoms in the furan rings of 1 by sulfur atoms induced a significant decrease of the detection threshold of the 1-oxo-2,3-dihydro-1H-indolizinium-6-olate; for example, the thiophene derivative 7 showed the extraordinarily low bitter detection threshold of 6.3 x 10(-5) mmol/kg (water).     

Characterization of an intense bitter-tasting 1H,4H-quinolizinium-7-olate by application of the taste dilution analysis, a novel bioassay for the screening and identification of taste-active compounds in foods

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.     

Evaluation of bitter masking flavanones from Herba Santa (Eriodictyon californicum (H. and A.) Torr., Hydrophyllaceae)

Products made from Herba Santa (Eriodictyon californicum (H. & A.) Torr.) have been used as bitter remedies for some pharmaceutical applications for many years, but they are actually too aromatic to be useful for many food or pharmaceutical applications. In sensory studies flavanones homoeriodictyol (1), its sodium salt (1-Na), sterubin (2), and eriodictyol (4) could significantly decrease the bitter taste of caffeine without exhibiting intrinsic strong flavors or taste characteristics. Further investigations on 1-Na elicited a broad masking activity between 10 and 40% toward different chemical classes of bitter molecules (e.g. salicin, amarogentin, paracetamol, quinine) but not toward bitter linoleic acid emulsions. For caffeine and amarogentin, dose-response studies were performed; the masking activity toward bitter taste for both compounds reached a plateau at higher concentrations of 1-Na. Due to these facts, homoeriodictyol sodium salt (1-Na) seems to be a very interesting new taste modifier for food applications and pharmaceuticals.     

Evolution of the taste of a bitter Camembert cheese during ripening: characterization of a matrix effect.

The objective of this study was to characterize the effect of ripening on the taste of a typically bitter Camembert cheese. The first step was to select a typically bitter cheese among several products obtained by different processes supposed to enhance this taste defect. Second, the evolution of cheese taste during ripening was characterized from a sensory point of view. Finally, the relative impact of fat, proteins, and water-soluble molecules on cheese taste was determined by using omission tests performed on a reconstituted cheese. These omission tests showed that cheese taste resulted mainly from the gustatory properties of water-soluble molecules but was modulated by a matrix effect due to fat, proteins, and cheese structure. The evolution of this matrix effect during ripening was discussed for each taste characteristic.     

茶叶添加方式对酿造啤酒理化特性及感官品质的影响

在啤酒酿造工艺的煮沸和主发酵阶段分别添加乌龙茶,探讨茶叶不同添加方式对啤酒理化特性、抗氧化能力、啤酒贮存稳定性以及感官特性的影响。结果显示,与未添加茶叶的啤酒产品(对照组CG)相比,在煮沸阶段(煮沸添加组BG)和主发酵阶段(主发酵添加组MG)分别添加0.3g/L的茶叶均提高了酵母发酵速率;添加茶叶的两个茶啤酒产品(煮沸添加组和主发酵添加组)的DPPH自由基清除能力分别提升至82.74%和89.21%、ABTS+自由基清除能力分别提升至41.53%和51.49%、铁离子还原能力分别提升至36.49和43.83 mg/L,且成品茶啤酒贮藏期间的抗老化能力提高;茶啤酒中总酚以及儿茶素(EGC、C、EGCG、EC、GCG、ECG)和咖啡碱(CAF)含量升高,其中,主发酵添加组茶啤酒样品总酚和EGCG含量显著高于对照组和煮沸添加组啤酒(P0.05),分别达到734.40和8.43 mg/L。进一步的感官品评结果显示添加茶叶提高了啤酒产品的茶香气和茶滋味,其中主发酵添加组啤酒的茶香气、茶滋味及酒体协调性最好。由此可知,在啤酒酿造工艺中添加茶叶提高了酵母发酵速率,增强了啤酒中酚类物质含量及其抗氧化和抗老化性能,同时也为啤酒增添了新的茶风味产品。     

Broad tuning of the human bitter taste receptor hTAS2R46 to various sesquiterpene lactones, clerodane and labdane diterpenoids, strychnine, and denatonium

Sesquiterpene lactones are a major class of natural bitter compounds occurring in vegetables and culinary herbs as well as in aromatic and medicinal plants, where they often represent the main gustatory and pharmacologically active component. Investigations on sesquiterpene lactones have mainly focused on their bioactive potential rather than on their sensory properties. In the present study, we report about the stimulation of heterologously expressed human bitter taste receptors, hTAS2Rs, by the bitter sesquiterpene lactone herbolide D. A specific response to herbolide D was observed i.a. for hTAS2R46, a so far orphan bitter taste receptor without any known ligand. By further investigation of its agonist pattern, we characterized hTAS2R46 as a bitter receptor broadly tuned to sesquiterpene lactones and to clerodane and labdane diterpenoids as well as to the unrelated bitter substances strychnine and denatonium.     

Identification of bitter off-taste compounds in the stored cold pressed linseed oil

Whereas freshly pressed linseed oil provides a delicate nutty flavor, a lingering bitter off-taste is developing upon storage at room temperature. Using a sensory guided fractionation approach, the key bitter compound was identified in stored linseed oil, and its structure was determined as the methionine sulfoxide-containing, cyclic octapeptide cyclo(PLFIM OLVF) by means of FTIR, LC-MS, NMR spectroscopy, and amino acid analysis. Although this peptide is known in the literature as cyclolinopeptide E, the bitter taste activity of this compound has not previously been described. Sensory evaluation revealed a recognition threshold concentration of 12.3 micromol/L water.     

Structural and sensory characterization of compounds contributing to the bitter off-taste of carrots (Daucus carota L.) and carrot puree

Sequential application of solvent extraction, gel permeation chromatography, and HPLC in combination with taste dilution analyses revealed that not a sole compound but a multiplicity of bitter tastants contribute to the bitter off-taste of cold-stored carrots and commercial carrot puree, respectively. Among these bitter compounds, 3-methyl-6-methoxy-8-hydroxy-3,4-dihydroisocoumarin (6-methoxymellein), 5-hydroxy-7-methoxy-2-methylchromone (eugenin), 2,4,5-trimethoxybenzaldehyde (gazarin), (Z)-heptadeca-1,9-diene-4,6-diin-3,8-diol (falcarindiol), (Z)-heptadeca-1,9-diene-4,6-diin-3-ol (falcarinol), and (Z)-3-acetoxy-heptadeca-1,9-diene-4,6-diin-8-ol (falcarindiol 3-acetate) could be identified on the basis of MS as well as 1D- and 2D-NMR experiments. Due to the low concentrations of <0.1 mg/kg and the high taste thresholds found for eugenin and gazarin, these compounds could be unequivocally excluded as important contributors to the bitter taste of carrots. Calculation of bitter activity values as the ratio of their concentration to their bitter detection threshold clearly demonstrated that neither in fresh and stored carrots nor in commercial carrot puree did 6-methoxymellein contribute to the bitter off-taste. In contrast, the concentrations of falcarindiol in stored carrots and, even more pronounced, in carrot puree were found to be 9- and 13-fold above its low bitter detection concentration of 0.04 mmol/kg, thus demonstrating that this acetylenic diol significantly contributes to the bitter taste of the carrot products investigated.     

Evolution of chemical and sensory properties during aging of top-fermented beer

The aging and consequent changes in flavor molecules of a top-fermented beer were studied. Different aging conditions were imposed on freshly bottled beer. After 6 months of aging, the concentration changes were recorded for acetate esters, ethyl esters, carbonyls, Maillard compounds, dioxolanes, and furanic ethers. For some flavor compounds, the changes with time of storage were monitored at different temperatures, either with CO(2) or with air in the headspace of the bottles. For some molecules a relationship was determined between concentration changes and sensory evaluation results. A decrease in volatile esters was responsible for a reduced fruity flavor during aging. On the contrary, various carbonyl compounds, some ethyl esters, Maillard compounds, dioxolanes, and furanic ethers showed a marked increase, due to oxidative and nonoxidative reactions. A very high increase was found for furfural, 2-furanmethanol, and especially the furanic ether, 2-furfuryl ethyl ether (FEE). For FEE a flavor threshold in beer of 6 mug/L was determined. In the aged top-fermented beer, FEE concentrations multiple times the flavor threshold were observed. This was associated with the appearance of a typical solvent-like flavor. As the FEE concentration increased with time at an almost constant rate, with or without air in the headspace, FEE (and probably other furanic ethers) is proposed as a good candidate to evaluate the thermal stress imposed on beer.     

Orosensory-directed identification of astringent mouthfeel and bitter-tasting compounds in red wine

Application of sequential solvent extraction, followed by HPLC combined with the taste dilution analysis, enabled the localization of the most intense velvety astringent, drying, and puckering astringent, as well as bitter-tasting, compounds in red wine, respectively. Isolation of the taste components involving gel adsorption chromatography, ultrafiltration, and synthesis revealed the identification of 26 sensory-active nonvolatiles, among which several hydroxybenzoic acids, hydroxycinnamic acids, flavon-3-ol glycosides, and dihydroflavon-3-ol rhamnosides as well as a structurally undefined polymeric fraction (>5 kDa) were identified as the key astringent components. In contradiction to literature suggestions, flavan-3-ols were found to be not of major importance for astringency and bitter taste, respectively. Surprisingly, a series of hydroxybenzoic acid ethyl esters and hydroxycinnamic acid ethyl esters were identified as bitter compounds in wine. Taste qualities and taste threshold concentrations of the individual wine components were determined by means of a three-alternative forced-choice test and the half-mouth test, respectively.     

Identification of beer bitter acids regulating mechanisms of gastric acid secretion

Beer, one of the most consumed beverages worldwide, has been shown to stimulate gastric acid secretion. Although organic acids, formed by fermentation of glucose, are known to be stimulants of gastric acid secretion, very little is known about the effects of different types of beer or the active constituents thereof. In the present study, we compared the effects of different beers on mechanisms of gastric acid secretion. To investigate compound-specific effects on mechanisms of gastric acid secretion, organic acids and bitter compounds were quantified by HPLC-DAD and UPLC-MS/MS and tested in human gastric cancer cells (HGT-1) by means of a pH-sensitive fluorescent dye which determines the intracellular pH as an indicator of proton secretion. The expression of relevant genes, coding the H(+)/K(+)-ATPase, ATP4A, the histamine receptor, HRH2, the acetylcholine receptor, CHRM3, and the somatostatin receptor, SSTR2, was determined by qPCR. Ethanol and the organic acids succinic acid, malic acid, and citric acid were demonstrated to contribute to some extent to the effect of beer. The bitter acids comprising α-, β-, and iso-α-acids were identified as potential key components promoting gastric acid secretion and up-regulation of CHRM3 gene expression by a maximum factor of 2.01 compared to that of untreated control cells with a correlation to their respective bitterness.     

Multivariate modeling of aging in bottled lager beer by principal component analysis and multiple regression methods

Data collected from the sensory test score evaluation of bottled lager beer, together with the chemical components related to aging, including carbonyl compounds, higher alcohols, unsaturated fatty acid, organic acids, alpha-amino acids, dissolved oxygen, and staling evaluation indices, including lag time of electron spin resonance (ESR) curve, 1,1'-diphenyl-2-picrylhydrazyl (DPPH) scavenged amounts, and thiobarbituric acid (TBA) values, were used to predict the extent of aging in bottled lager beer, using both multiple linear regression and principal component analysis methods. Carbonyl compounds, higher alcohols, and TBA value were significantly and positively correlated with sensory evaluation of staling flavor. While lag time and DPPH scavenging amount were negatively correlated with taste test score. Multiple regression analysis was used to fit the sensory test data using the above chemical compound aging related parameters and evaluation indices as predictors. A variable selection method based on high loadings of varimax rotated principal components was used to obtain subsets of the predominant predictor variables to be included in the regression model of beer aging, so as to eliminate the multicollinearity of the original nine variables. It was found that staling extent was influenced significantly by higher alcohols, TBA value, and DPPH scavenging amount, and the multicollinearity of the regression model was found to be weak by examining the variance inflation factors of the new predictor variables. A mathematic model of the organoleptic test score for beer aging using these three predictors was obtained by multiple linear regression, showing that the major contributors to the sensory taste of beer aging were higher alcohols, TBA index, and DPPH scavenging amount, with the adjusted R(2) of the model being 0.62.     

Oxidative degradation of lipids during mashing

Although hardly any polyunsaturated fatty acids (PUFAs) are present in the endproduct, the ingredients used for the production of beer contain a high concentration of PUFAs, such as linolic and linolenic acid. These compounds are readily oxidized, resulting in the formation of lipid-derived products that reduce the taste and quality of beer enormously. During mashing relatively high amounts of PUFAs are exposed to atmospheric oxygen at a relatively high temperature. This makes mashing a critical step in the brewing process with regard to the formation of lipid-derived off-taste products. F1 phytoprostane (PPF1) changes in antioxidant capacity and monohydroxy fatty acids (OH-FAs) were used as markers for the detection of oxidative damage to fatty acids during mashing. The pattern of OH-FA formation indicates that enzymatic oxidation of PUFAs is more important than nonenzymatic oxidation during the mashing process. Nevertheless, substantial nonenzymatic radical formation is evident from the increase of specific OH-FAs and PPF1s. It was found that a low oxygen tension reduces oxidative damage and gives a high antioxidant capacity of the mashing mixture. This indicates that mashing should be done under low oxygen pressure.