The effect of the sweetness inhibitor 2(-4-methoxyphenoxy)propanoic acid (sodium salt) (Na-PMP) on the taste of bitter-sweet stimuli

The effect of the sweetness inhibitor 2(-4-niethoxyphenoxy)propanoicacid (sodium gait) (Na-PMP) on the taste and temporal propertiesof a range of bitter-sweet stimuli was determined using a trainedsensory panel. Na-PMP was found to be an effective inhibitorof the sweetness response of all stimuli tested, reducing bothsweetness intensity and persistence. The inhibitor was foundto be specific to sweet taste, no reduction in bitterness intensityor persistence was observed at the concentrations of Na-PMPemployed in this study. The results therefore do not supportthe claim of Fuller and Kurtz (1991), that Na-PMP is a potentbitterness inhibitor, but rather support the existence of twodistinct receptor sites/loci in sweet and bitter chemoreception.     

Riboflavin-binding protein exhibits selective sweet suppression toward protein sweeteners

Riboflavin-binding protein (RBP) is well known as a riboflavin carrier protein in chicken egg and serum. A novel function of RBP was found as a sweet-suppressing protein. RBP, purified from hen egg white, suppressed the sweetness of protein sweeteners such as thaumatin, monellin, and lysozyme, whereas it did not suppress the sweetness of low molecular weight sweeteners such as sucrose, glycine, D-phenylalanine, saccharin, cyclamate, aspartame, and stevioside. Therefore, the sweet-suppressing activity of RBP was apparently selective to protein sweeteners. The sweet suppression by RBP was independent of binding of riboflavin with its molecule. Yolk RBP, with minor structural differences compared with egg white RBP, also elicited a weaker sweet suppression. However, other commercially available proteins including ovalbumin, ovomucoid, beta-lactogloblin, myoglobin, and albumin did not substantially alter the sweetness of protein sweeteners. Because a prerinse with RBP reduced the subsequent sweetness of protein sweeteners, whereas the enzymatic activity of lysozyme and the elution profile of lysozyme on gel permeation chromatography were not affected by RBP, it is suggested that the sweet suppression is caused by an interaction of RBP with a sweet taste receptor rather than with the protein sweeteners themselves. The selectivity in the sweet suppression by RBP is consistent with the existence of multiple interaction sites within a single sweet taste receptor.     

Electrochromatographic enantioseparation of amino acids using polybutylmethacrylate-based chiral monolithic column by capillary electrochromatography

This article describes the development of a polybutylmethacrylate‐based monolithic capillary column as a chiral stationary phase. The chiral monolithic column was prepared by polymerization of butyl methacrylate (BMA), ethylene dimethacrylate (EDMA), and N‐methacryloyl‐l ‐glutamic acid (MAGA) in the presence of porogens. The porogen mixture included N,N‐dimethyl formamide and phosphate buffer. MAGA was used as a chiral selector. The effect of MAGA content was investigated on electrochromatographic enantioseparation of d,l ‐histidine, d,l ‐tyrosine, d,l ‐phenyl alanine, and d,l ‐glutamic acid. The effect of acetonitrile (ACN) content in mobile phase on electro‐osmotic flow was also investigated. It was demonstrated that the poly(BMA‐EDMA‐MAGA) monolithic chiral column can be used for the electrochromatographic enantioseparation of amino acids by capillary electrochromatography (CEC). The mobile phase was ACN/10 mM phosphate buffer (45:55%) adjusted to pH 2.7. It was observed that l ‐enantiomers of the amino acids migrated before d ‐enantiomers. The separation mechanism of electrochromatographic enantioseparation of amino acids in CEC is discussed. Chirality 24:606–609, 2012. © 2012 Wiley Periodicals, Inc.     

Critical regions for the sweetness of brazzein

Brazzein is a small, heat-stable, intensely sweet protein consisting of 54 amino acid residues. Based on the wild-type brazzein, 25 brazzein mutants have been produced to identify critical regions important for sweetness. To assess their sweetness, psychophysical experiments were carried out with 14 human subjects. First, the results suggest that residues 29-33 and 39-43, plus residue 36 between these stretches, as well as the C-terminus are involved in the sweetness of brazzein. Second, charge plays an important role in the interaction between brazzein and the sweet taste receptor.     

Confusing tastes and smells: how odours can influence the perception of sweet and sour tastes

This study investigated the relationship between perception of an odour when smelled and the taste of a solution to which the odour is added as a flavorant. In Experiment 1 (E1) sweetness, sourness, liking and intensity ratings were obtained for 20 odours. Taste ratings were then obtained for sucrose solutions to which the odours had been added as flavorants. Certain odours were found to enhance tasted sweetness while others suppressed it. The degree to which an odour smelled sweet was the best predictor of the taste ratings. These findings were extended in Experiment 2 (E2), which included a second tastant, citric acid, and employed four odours from E1. The most sweet smelling odour, caramel, was found to suppress the sourness of citric acid and, as in E1, to enhance the sweetness of sucrose. Again, odours with low sweetness suppressed the sweetness of tasted sucrose. The study demonstrated that the effects of odours on taste perception are not limited to sweetness enhancement and apply to sour as well as sweet tastes. The overall pattern of results is consistent with an explanation of the taste properties of odours in terms of prior flavour-taste associations.     

Enantioanalysis of Pipecolic Acid with Stochastic and Potentiometric Microsensors

Stochastic and potentiometric microsensors based on porphyrins and polymeric surfactants such as polysodium N‐undecanoyl‐ l ‐leucylvanilate and polysodium 相似文献   

Sweet taste receptor gene variation and aspartame taste in primates and other species

Aspartame is a sweetener added to foods and beverages as a low-calorie sugar replacement. Unlike sugars, which are apparently perceived as sweet and desirable by a range of mammals, the ability to taste aspartame varies, with humans, apes, and Old World monkeys perceiving aspartame as sweet but not other primate species. To investigate whether the ability to perceive the sweetness of aspartame correlates with variations in the DNA sequence of the genes encoding sweet taste receptor proteins, T1R2 and T1R3, we sequenced these genes in 9 aspartame taster and nontaster primate species. We then compared these sequences with sequences of their orthologs in 4 other nontasters species. We identified 9 variant sites in the gene encoding T1R2 and 32 variant sites in the gene encoding T1R3 that distinguish aspartame tasters and nontasters. Molecular docking of aspartame to computer-generated models of the T1R2 + T1R3 receptor dimer suggests that species variation at a secondary, allosteric binding site in the T1R2 protein is the most likely origin of differences in perception of the sweetness of aspartame. These results identified a previously unknown site of aspartame interaction with the sweet receptor and suggest that the ability to taste aspartame might have developed during evolution to exploit a specialized food niche.     

Reduced sweetness of a monellin (MNEI) mutant results from increased protein flexibility and disruption of a distant poly-(L-proline) II helix

Monellin is a highly potent sweet-tasting protein but relatively little is known about how it interacts with the sweet taste receptor. We determined X-ray crystal structures of 3 single-chain monellin (MNEI) proteins with alterations at 2 core residues (G16A, V37A, and G16A/V37A) that induce 2- to 10-fold reductions in sweetness relative to the wild-type protein. Surprisingly, no changes were observed in the global protein fold or the positions of surface amino acids important for MNEI sweetness that could explain these differences in protein activity. Differential scanning calorimetry showed that while the thermal stability of each mutant MNEI was reduced, the least sweet mutant, G16A-MNEI, was not the least stable protein. In contrast, solution spectroscopic measurements revealed that changes in protein flexibility and the C-terminal structure correlate directly with protein activity. G16A mutation-induced disorder in the protein core is propagated via changes to hydrophobic interactions that disrupt the formation and/or position of a critical C-terminal poly-(L-proline) II helix. These findings suggest that MNEI interaction with the sweet taste receptor is highly sensitive to the relative positions of key residues across its protein surface and that loss of sweetness in G16A-MNEI may result from an increased entropic cost of binding.     

Structures of the Neisseria meningitides methionine‐binding protein MetQ in substrate‐free form and bound to l‐ and d‐methionine isomers

The bacterial periplasmic methionine‐binding protein MetQ is involved in the import of methionine by the cognate MetNI methionine ATP binding cassette (ABC) transporter. The MetNIQ system is one of the few members of the ABC importer family that has been structurally characterized in multiple conformational states. Critical missing elements in the structural analysis of MetNIQ are the structure of the substrate‐free form of MetQ, and detailing how MetQ binds multiple methionine derivatives, including both l ‐ and d ‐methionine isomers. In this study, we report the structures of the Neisseria meningitides MetQ in substrate‐free form and in complexes with l ‐methionine and with d ‐methionine, along with the associated binding constants determined by isothermal titration calorimetry. Structures of the substrate‐free (N238A) and substrate‐bound N. meningitides MetQ are related by a “Venus‐fly trap” hinge‐type movement of the two domains accompanying methionine binding and dissociation. l ‐ and d ‐methionine bind to the same site on MetQ, and this study emphasizes the important role of asparagine 238 in ligand binding and affinity. A thermodynamic analysis demonstrates that ligand‐free MetQ associates with the ATP‐bound form of MetNI ～40 times more tightly than does liganded MetQ, consistent with the necessity of dissociating methionine from MetQ for transport to occur.     

Activity and Stability of a New Sweet Protein with Taste-modifying Action, Curculin

Curculin elicited a sweet taste. After the sweetness of curculindiminished, application of deionized water or an acid to thetongue induced a sweet taste. The maximum sweetness of curculinitself was equivalent to thesweetness of 0.35 M sucrose. Themaximum sweetness induced by 0.02 M citric acid or deionizedwater after curculin dissolved in a buffer of pH 6.0 was heldin mouth for 3 min was also equivalent to that of 0.35 M sucrose.The sweetness induced by deionized water was completely suppressedby the presence of 1 mM CaCl₂ or MgCl₂, while that induced byan acid was not suppressed by the presence of divalent cations.Based on these results, the mechanism of the taste-modifyingactivity was discussed. Stability of curculin was examined undervarious conditions. The taste-modifying activity of curculinwas unchanged when curculin was incubated at 50°C for 1h between pH 3 and 11.     

Simvastatin abolishes nitric oxide‐ and reactive oxygen species‐induced cyclooxygenase‐2 expression by blocking the nuclear factor κB pathway in rabbit articular chondrocytes

Nitric oxide (NO) and reactive oxygen species (ROS) have been shown to be linked with numerous diseases, including osteoarthritis (OA). Our study aimed to examine the effect of simvastatin on NO‐ or ROS‐induced cyclooxygenase‐2 (COX‐2) expression in OA. Simvastatin has attracted considerable attention since the discovery of its pharmacological effects on different pathogenic processes, including inflammation. Here, we report that simvastatin treatment blocked sodium nitroprusside (SNP)‐ and interleukin 1 beta (IL‐1β)‐induced COX‐2 production. In addition, simvastatin attenuated SNP‐induced NO production and IL‐1β‐induced ROS generation. Treatment with simvastatin prevented SNP‐ and IL‐1β‐induced nuclear factor kappa B (NF‐κB) activity. Inhibiting NO production and ROS generation using N‐acetylcysteine (NAC) and NG‐monomethyl‐ l ‐arginine ( l ‐NMMA), respectively, accelerated the influence of simvastatin on NF‐κB activity. In addition, NAC blocked SNP and simvastatin‐mediated COX‐2 production and NF‐κB activity but did not alter IL‐1β and simvastatin‐mediated COX‐2 expression. l ‐NMMA treatment also abolished IL‐1β‐mediated COX‐2 expression and NF‐κB activation, whereas SNP and simvastatin‐mediated COX‐2 expression were not altered compared with the levels in the SNP and simvastatin‐treated cells. Our findings suggested that simvastatin blocks COX‐2 expression by inhibiting SNP‐induced NO production and IL‐1β‐induced ROS generation by blocking the NF‐κB pathway.     

Effect of chemical and physical parameters on the production of L-asparaginase from a newly isolated Serratia marcescens SK-07

Aims: The objective of this study is to optimize the levels of carbon and nitrogen sources of the medium in shake flask experiments and evaluate the effect of pH and dissolved oxygen (DO) on the production of l ‐asparaginase from a newly isolated Serratia marcescens SK‐07 in a batch bioreactor. Methods and Results: Central composite rotatable design (CCRD) was applied to optimize the levels of carbon and nitrogen sources of the medium in shake flask experiments. The optimal levels of l ‐asparagine, glucose, yeast extract and peptone were found to be 4·93, 3·81, 3·65 and 1·47 g l^?1, respectively, and maximal l ‐asparaginase production of 25·02 U mg^?1 was obtained under these conditions. Among the carbon sources tested, l ‐asparagine was identified to be the most favourable carbon source for enhanced production of l ‐asparaginase. The maximum l ‐asparaginase production of 29·89 U mg^?1 was achieved in a batch bioreactor at initial pH of 6·5 (uncontrolled) and DO level of 40% in the culture. Conclusions: We have isolated, screened and identified the potential micro‐organism, S. marcescens, for the production of l ‐asparaginase. An overall 5·55‐fold increase in the production was achieved under optimal levels of carbon and nitrogen sources, DO level and at initial pH of 6·5 (uncontrolled). Significance and Impact of the Study: The experiments illustrate the importance of statistical method for optimization of carbon and nitrogen sources and study the effect of physical process parameters on the production of l ‐asparaginase in shake flask and bioreactor, respectively. This study would be helpful for bioprocess development of bacterial l ‐asparaginase production.     

The Structure-taste Relationships of Aspartyl Dipeptide Esters

The molecular features common to sweet-tasting dipeptide esters are described. The molecular features of sweet amino acids were represented by the Fischer projection formulas and sweet peptides were related to the sweet amino acids through the Fischer projection formulas of the peptides. It was concluded that a peptide is sweet when it takes the formula 5a, whereas when it takes the formula 5b it is not sweet. It was also concluded that a third binding site (R₁ in 5a) besides the postulated AH–B system in a sweet molecule is necessary for an intense sweetness potency. The location of the site in the molecule relative to the AH–B system is important, as well as the shape and size of this site, because the third binding site is considered to participate in hydrophobic interaction with a similar binding site on the taste receptor. Increased sweetness is observed when these requirements are satisfied.     

Obese Women Have Lower Monosodium Glutamate Taste Sensitivity and Prefer Higher Concentrations Than Do Normal‐weight Women

The goal of this study was to determine whether obese women exhibit altered umami and sweet taste perception compared to normal‐weight women. A total of 57 subjects (23 obese and 34 normal weight) participated in a 2‐day study separated by 1 week. Half of the women in each group were evaluated using monosodium glutamate (MSG; prototypical umami stimulus) on the first test day and sucrose on the second test day; the order was reversed for the remaining women. We used two‐alternative forced‐choice staircase procedures to measure taste detection thresholds, forced‐choice tracking technique to measure preferences, the general Labeled Magnitude Scale (gLMS) to measure perceived intensity of suprathreshold concentrations, and a triangle test to measure discrimination between 29 mmol/l MSG and 29 mmol/l NaCl. Obese women required higher MSG concentrations to detect a taste and preferred significantly higher MSG concentrations in a soup‐like vehicle. However, their perception of MSG at suprathreshold concentrations, their ability to discriminate MSG from salt, and their preference for sucrose were similar to that observed in normal‐weight women. Regardless of their body weight category, 28% of the women did not discriminate 29 mmol/l MSG from 29 mmol/l NaCl (nondiscriminators). Surprisingly, we found that, relative to discriminators, nondiscriminators perceived less savoriness when tasting suprathreshold MSG concentrations and less sweetness from suprathreshold sucrose concentrations but had similar MSG and sucrose detection thresholds. Taken together, these data suggest that body weight is related to some components of umami taste and that different mechanisms are involved in the perception of threshold and suprathreshold MSG concentrations.     

Molecular mechanisms of the action of miraculin,a taste-modifying protein

Miraculin (MCL) is a homodimeric protein isolated from the fruits of Richadella dulcifica, a shrub native to West Africa. Although it is flat in taste at neutral pH, MCL has taste-modifying activity in which sour stimuli produce a sweet perception. Once MCL enters the mouth, strong sweetness can be detected for more than 1 h each time we taste a sour solution. While the human sweet taste receptor (hT1R2–hT1R3) has been identified, the molecular mechanisms underlying the taste-modifying activity of MCL remain unclear. Recently, experimental evidence has been published demonstrating the successful quantitative evaluation of the acid-induced sweetness of MCL using a cell-based assay system. The results strongly suggested that MCL binds hT1R2–hT1R3 as an antagonist at neutral pH and functionally changes into an agonist at acidic pH. Since sweet-tasting proteins may be used as low-calorie sweeteners because they contain almost no calories, it is expected that MCL will be used in the near future as a new low-calorie sweetener or to modify the taste of sour fruits.     

Amiloride reduces the sweet taste intensity by inhibiting the human sweet taste receptor

In mammals, sweet taste perception is mediated by the heterodimeric G-protein-coupled receptor, T1R2/T1R3. An interesting characteristic of this sweet taste receptor is that it has multiple ligand binding sites. Although there have been several studies on agonists of sweet taste receptors, little is known about antagonists of these receptors. In this study, we constructed a cell line stably expressing the human sweet taste receptor (hT1R2/hT1R3) and a functional chimeric G-protein (hGα16gust44) using the Flp-In system for measuring the antagonistic activity against the receptor. This constructed cell line responded quite intensely and frequently to the compounds applied for activation of hT1R2/hT1R3. In the presence of 3 mM amiloride, the responses to sweet tastants such as sugar, artificial sweetener, and sweet protein were significantly reduced. The inhibitory activity of amiloride toward 1 mM aspartame was observed in a dose-dependent manner with an IC₅₀ value of 0.87 mM. Our analysis of a cell line expressing hT1R3 mutants (hT1R3-A733V or hT1R3-F778A) made us to conclude that the target site of amiloride is distinct from that of lactisole, a known sweet taste inhibitor. Our results strongly indicate that amiloride reduces the sweet taste intensity by inhibiting the human sweet taste receptor and also that this receptor has multiple inhibitor binding sites.     

Negative modulation of the GABAAρ1 receptor function by l‐cysteine

l ‐Cysteine is an endogenous sulfur‐containing amino acid with multiple and varied roles in the central nervous system, including neuroprotection and the maintenance of the redox balance. However, it was also suggested as an excitotoxic agent implicated in the pathogenesis of neurological disorders such as Parkinson′s and Alzheimer′s disease. l ‐Cysteine can modulate the activity of ionic channels, including voltage‐gated calcium channels and glutamatergic NMDA receptors, whereas its effects on GABAergic neurotransmission had not been studied before. In the present work, we analyzed the effects of l ‐cysteine on responses mediated by homomeric GABA_Aρ1 receptors, which are known for mediating tonic γ‐aminobutyric acid (GABA) responses in retinal neurons. GABA_Aρ1 receptors were expressed in Xenopus laevis oocytes and GABA‐evoked chloride currents recorded by two‐electrode voltage‐clamp in the presence or absence of l ‐cysteine. l ‐Cysteine antagonized GABA_Aρ1 receptor‐mediated responses; inhibition was dose‐dependent, reversible, voltage independent, and susceptible to GABA concentration. Concentration‐response curves for GABA were shifted to the right in the presence of l ‐cysteine without a substantial change in the maximal response. l ‐Cysteine inhibition was insensitive to chemical protection of the sulfhydryl groups of the ρ1 subunits by the irreversible alkylating agent N‐ethyl maleimide. Our results suggest that redox modulation is not involved during l ‐cysteine actions and that l ‐cysteine might be acting as a competitive antagonist of the GABA_Aρ1 receptors.

     

Structure‐function relationships of brazzein variants with altered interactions with the human sweet taste receptor

Brazzein (Brz) is a small (54 amino acid residue) sweet tasting protein with physical and taste properties superior to other non‐carbohydrate sweeteners. In an investigation of sequence‐dependent functional properties of the protein, we used NMR spectroscopy to determine the three‐dimensional structures and dynamic properties of two Brz variants: one with a single‐site substitution (D40K), which is three‐fold sweeter than wild‐type Brz, and one with a two‐residue insertion between residues 18 and 19 (ins₁₈RI₁₉), which is devoid of sweetness. Although the three‐dimensional folds of the two variants were very similar to wild‐type Brz, they exhibited local conformational and dynamic differences. The D40K substitution abolished the strong inter‐stand H‐bond between the side chains of residues Gln46 and Asp40 present in wild‐type Brz and increased the flexibility of the protein especially at the mutation site. This increased flexibility presumably allows this site to interact more strongly with the G‐protein coupled human sweet receptor. On the other hand, the Arg‐Ile insertion within Loop9–19 leads to distortion of this loop and stiffening of the adjacent site whose flexibility appears to be required for productive interaction with the sweet receptor.