Revisiting sugar-fat mixtures: sweetness and creaminess vary with phenotypic markers of oral sensation

Genetic variation in oral sensation presumably influences ingestive behaviors through sensations arising from foods and beverages. Here, we investigated the influence of taste phenotype [6-n-propylthiouracil (PROP) bitterness, fungiform papillae (FP) density] on sweet and creamy sensations from sugar/fat mixtures. Seventy-nine subjects (43 males) reported the sweetness and creaminess of water or milk (skim, whole, heavy cream) varying in sucrose (0-20% w/v) on the general Labeled Magnitude Scale. Sweetness grew with sucrose concentration and when shifting from water to milk mixtures--the growth was greatest for those tasting PROP as most bitter. At higher sucrose levels, increasing fat blunted the PROP-sweet relationship, whereas at lower levels, the relationship was effectively eliminated. Perceived sweetness of the mixture exceeded that predicted from the sum of components at low sucrose concentrations (especially for those tasting PROP most bitter) but fell below predicted at high concentrations, irrespective of fat level. Creaminess increased greatly with fat level and somewhat with sucrose. Those tasting PROP most bitter perceived greater creaminess in the heavy cream across all sucrose levels. Perceived creaminess was somewhat lower than predicted, irrespective of PROP bitterness. The FP density generally showed similar effects as PROP on sweetness and creaminess, (but to a lesser degree) and revealed potential taste-somatosensory interactions in weakly sweet stimuli. These data support that taste phenotype affects the nature of enhancement or suppression of sweetness and creaminess in liquid fat/sugar mixtures. Taste phenotype effects on sweetness and creaminess likely involve differential taste, retronasal olfactory, and somatosensory contributions to these perceptual experiences.     

Accumulation of phyllodulcin in sweet-leaf plants ofHydrangea serrata and its neutrality in the defence against a specialist leafmining herbivore

Among wild plants ofHydrangea serrata (Hydrangeaceae) in Japan, there are sweet plants whose leave contain a kind of isocoumarin, phyllodulcin, which happens to be 350 times as sweet as sucrose to the human tongue. In a primary beech forest in Ashu, Kyoto, the spatial distribution of sweet plants and temporal and the spatial distribution of phyllodulcin within and among plants were investigated using a high performance liquid chromatograph. The distribution of sweet plants was confined within a valley and was parapatric with non-sweet plants. A plant's characteristic phyllodulcin accumulation did not change, even when transplanted into the different habitats. The phyllodulcin content of the sweet plants varied greatly among plants, and the population mean peaked in July when the plants flowered. Within a plant, phyllodulcin content was elevated by partial defoliation. We examined the possible effect of phyllodulcin on herbivory by a specialist leafmining herbivore,Antispila hydrangifoliella (Lepidoptera: Heliozelidae). We transplanted sweet and nonsweet plants reciprocally between their original habitats, excluded attacks by parasitoids, and compared performance of the leafminer. Leafminer colonization and larval survivorship on transplanted andin situ plants was not significantly different between sites. The fact that accumulation of phyllodulcin did not augment a defensive function, at least against herbivory by the leafminer, and the sporadic distribution of phyllodulcin-accumulating plants, suggest that the genotypes synthesizing phyllodulcin emerged independently at separate localities by mutation, and that the genotypes are almost adaptively neutral in defence against the specialist herbivore.     

Structure-sweetness relationship in thaumatin: importance of lysine residues

To clarify the structural basis for the sweetness of thaumatin I, lysine-modified derivatives and carboxyl-group-modified derivatives were prepared by chemical modification followed by chromatographic purification. The sweetness of derivatives was evaluated by sensory analysis. Phosphopyridoxylation of lysine residues Lys78, Lys97, Lys106, Lys137 and Lys187 markedly reduced sweetness. The intensity of sweetness was returned to that of native thaumatin by dephosphorylation of these phosphopyridoxylated lysine residues except Lys106. Pyridoxamine modification of the carboxyl group of Asp21, Glu42, Asp60, Asp129 or Ala207 (C-terminal) did not markedly change sweetness. Analysis by far-UV circular dichroism spectroscopy indicated that the secondary structure of all derivatives remained unchanged, suggesting that the loss of sweetness was not a result of major disruption in protein structure. The five lysine residues, modification of which affected sweetness, are separate and spread over a broad surface region on one side of the thaumatin I molecule. These lysine residues exist in thaumatin, but not in non-sweet thaumatin-like proteins, suggesting that these lysine residues are required for sweetness. These lysine residues may play an important role in sweetness through a multipoint interaction with a putative thaumatin receptor.     

Synthesis and Taste of Some Dihydrochalcone Glycosides

Hesperetin-7-β-maltoside (V), -7-β-cellobioside (VI) and -7-β-lactoside (VII) were prepared by the coupling of hesperetin with the α-acetobromo derivatives of the appropriate disaccharides, followed by saponification. V was showed to be as sweet as glucose.

Naringenindihydrochalcone-4′-β-sophoroside (VIII), -4′-[β-d-glucosyl (1→2) β-d-galactoside] (IX) and also hesperetindihydrochalcone-4′-β-kojibioside (X), -4′-β-maltoside (XI), -4′-β-cellobioside (XII) and -4′-β-lactoside (XIII) were prepared by the catalytic reduction of the appropriate flavanone-7-β-glycosides in alkaline medium.

Their relative sweetness values were discussed in comparison with dihydrochalcones of naringin and neohesperidin.     

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.     

Affinity Chromatography of Acid Proteases on SPI Coupled to PAB-Cellulose

Tastes of Leu-Lys-Tyr (LKY) analogues, a series of potent angiotensin I converting enzyme (ACE) inhibitory peptides were evaluated. Some of these analogues were found to be sweet, such as Val–Lys-Tyr and Ala-Orn-Tyr. Furthermore, the structural requirements for sweetness or decreasing the bitterness were investigated by considerations of the structure–taste relationship with LKY analogues.     

Solution structure, backbone dynamics, and stability of a double mutant single-chain monellin. structural origin of sweetness

Single-chain monellin (SCM), which is an engineered 94-residue polypeptide, has been characterized as being as sweet as native two-chain monellin. Data from gel-filtration high performance liquid chromatography and NMR has proven that SCM exists as a monomer in aqueous solution. In order to determine the structural origin of the taste of sweetness, we engineered several mutant SCM proteins by mutating Glu(2), Asp(7), and Arg(39) residues, which are responsible for sweetness. In this study, we present the solution structure, backbone dynamics, and stability of mutant SCM proteins using circular dichroism, fluorescence, and NMR spectroscopy. Based on the NMR data, a stable alpha-helix and five-stranded antiparallel beta-sheet were identified for double mutant SCM. Strands beta1 and beta2 are connected by a small bulge, and the disruption of the first beta-strand were observed with SCM(DR) comprising residues of Ile(38)-Cys(41). The dynamical and folding characteristics from circular dichroism, fluorescence, and backbone dynamics studies revealed that both wild type and mutant proteins showed distinct dynamical as well as stability differences, suggesting the important role of mutated residues in the sweet taste of SCM. Our results will provide an insight into the structural origin of sweet taste as well as the mutational effect in the stability of the engineered sweet protein SCM.     

Dihydroisocoumarins and phthalide from wood samples infested by fungi

Wood samples, infested by fungi during storage, were shown to contain, besides the known 5-methyl-mellein, additional (3R)-8-hydroxy-3-methyl-3,4-dihydroisocoumarins substituted by 7-methyl, 5-formyl, 5-carboxy, 5-hydroxy, 5-methoxy, 6-methoxy-5-methyl and 6,7-dimethoxy-5-methyl groups, as well as 6-formyl-7-hydroxy-5-methoxy-4-methylphthalide. Several 2-methylchromanones were synthesized in order to show that this class of compounds can be distinguished from 3-methyl-3,4-dihydroisocoumarins by MS.     

Peptide homologs, isosteres, and isomers: a general approach to structure-activity relationships

A general approach to study peptide structure is presented using three areas of ongoing research in our laboratories. The first involves the molecular basis for taste of peptide derivatives. We synthesized dipeptides based on L -aspartyl-α-aminocycloalkane carboxylic acid methyl ester. A homologous series of cycloalkane derivatives was studied. The cyclopropane, cyclobutane, and cyclopentane derivatives are sweet, the cyclohexane and cycloheptane peptides are bitter, and the cyclooctane homolog is tasteless. The related acyclic analog L -aspartyl-aminoisobutyric acid methyl ester is sweet, while the L -aspartyl diethyl glycine carboxylic acid methyl ester is tasteless. A model is presented to explain these experimental observations. The second area involves depsipeptides as isosteric replacements of α-hydroxy acids for amino acid residues in peptide chains. We have synthesized sequentially defined polydepsipeptides as model systems for polypeptides. A detailed analysis of the conformational order for these polydepsipeptides is presented. The third area involves partial retro–inverso peptide modifications of isomeric cyclic enkephalin analogs, which illustrate the relationship between the modification and biological activity. We are probing the intramolecular hydrogen-bonding features for these biologically active molecules. From such findings we are relating the structural and conformational preferences deduced from spectroscopy and molecular mechanics to biological activity.     

The anatomy of mammalian sweet taste receptors

All sweet‐tasting compounds are detected by a single G‐protein coupled receptor (GPCR), the heterodimer T1R2‐T1R3, for which no experimental structure is available. The sweet taste receptor is a class C GPCR, and the recently published crystallographic structures of metabotropic glutamate receptor (mGluR) 1 and 5 provide a significant step forward for understanding structure‐function relationships within this family. In this article, we recapitulate more than 600 single point site‐directed mutations and available structural data to obtain a critical alignment of the sweet taste receptor sequences with respect to other class C GPCRs. Using this alignment, a homology 3D‐model of the human sweet taste receptor is built and analyzed to dissect out the role of key residues involved in ligand binding and those responsible for receptor activation. Proteins 2017; 85:332–341. © 2016 Wiley Periodicals, Inc.     

The cysteine-rich region of T1R3 determines responses to intensely sweet proteins

A wide variety of chemically diverse compounds taste sweet, including natural sugars such as glucose, fructose, sucrose, and sugar alcohols, small molecule artificial sweeteners such as saccharin and acesulfame K, and proteins such as monellin and thaumatin. Brazzein, like monellin and thaumatin, is a naturally occurring plant protein that humans, apes, and Old World monkeys perceive as tasting sweet but that is not perceived as sweet by other species including New World monkeys, mouse, and rat. It has been shown that heterologous expression of T1R2 plus T1R3 together yields a receptor responsive to many of the above-mentioned sweet tasting ligands. We have determined that the molecular basis for species-specific sensitivity to brazzein sweetness depends on a site within the cysteine-rich region of human T1R3. Other mutations in this region of T1R3 affected receptor activity toward monellin, and in some cases, overall efficacy to multiple sweet compounds, implicating this region as a previously unrecognized important determinant of sweet receptor function.     

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.     

Inhibition of free radical-induced peroxidation of rat liver microsomes by resveratrol and its analogues

Resveratrol (3,5,4'-trans-trihydroxystilbene) is a natural phytoalexin present in grapes and red wine, which possesses a variety of biological activities including antioxidative activity. To find more efficient antioxidants by structural modification, resveratrol analogues, that is, 3,4-dihydroxy-trans-stilbene (3,4-DHS), 4,4'-dihydroxy-trans-stilbene (4,4'-DHS), 4-hydroxy-trans-stilbene (4-HS) and 3,5-dihydroxy-trans-stilbene (3,5-DHS), were synthesized and their antioxidant activity studied for the free radical-induced peroxidation of rat liver microsomes in vitro. The peroxidation was initiated by either a water-soluble azo compound 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH) or Fe(2+)/ascorbate, and monitored by oxygen uptake and formation of thiobarbituric acid reactive substances (TBARS). It was found that all of these trans-stilbene derivatives are effective antioxidants against both AAPH- and iron-induced peroxidation of rat liver microsomes with an activity sequence of 3,4-DHS>4,4'-DHS>resveratrol>4-HS>3,5-DHS. The remarkably higher antioxidant activity of 3,4-DHS is discussed.     

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.     

The water-sweet aftertaste of neohesperidin dihydrochalcone and thaumatin as a method for determining their sweet persistence

The water–sweet aftertaste produced in humans in responseto tasting intensive sweeteners such as neohesperidin dihydrochalconeand thaumatin was studied. This water–sweet aftertasteincreased with sweetener concentration and diminished with time.The decay in the sweet intensity–time relationships fitteda negative exponential function in a pattern similar to thatwhich occurs when other methods for determining persistenceare employed. Persistence time contants (T) were dependent uponthe maximal perceived sweet intensity (Ip-max) observed at theinitial time of tasting. The use of this procedure is proposedfor determining persistence of intensive sweeteners under circumstanceswhere controlled pH and temperature are desired.     

Crystal structure of Mabinlin II: a novel structural type of sweet proteins and the main structural basis for its sweetness

The crystal structure of a sweet protein Mabinlin II (Mab II) isolated from the mature seeds of Capparis masaikai Levl. grown in Southern China has been determined at 1.7A resolution by the SIRAS method. The Mab II 3D structure features in an "all alpha" fold mode consisting of A- and B-chains crosslinked by four disulfide bridges, which is distinct from all known sweet protein structures. The Mabinlin II molecule shows an amphiphilic surface, a cationic face (Face A) and a neutral face (Face B). A unique structural motif consisting of B54-B64 was found in Face B, which adopts a special sequence, NL-P-NI-C-NI-P-NI, featuring four [Asn-Leu/Ile] units connected by three conformational-constrained residues, thus is called the [NL/I] tetralet motif. The experiments for testing the possible interactions of separated A-chain and B-chain and the native Mabinlin II to the sweet-taste receptor were performed through the calcium imaging experiments with the HEK293E cells coexpressed hT1R2/T1R3. The result shows that hT1R2/T1R3 responds to both the integrated Mabinlin II and the individual B-chain in the same scale, but not to A-chain. The sweetness evaluation further identified that the separated B-chain can elicit the sweetness alone, but A-chain does not. All data in combination revealed that the sweet protein Mabinlin II can interact with the sweet-taste receptor hT1R2/T1R3 to elicit its sweet taste, and the B-chain with a unique [NL/I] tetralet motif is the essential structural element for the interaction with sweet-taste receptor to elicit the sweetness, while the A-chain may play a role in gaining a long aftertaste for the integrate Mabinlin II. The findings reported in this paper will be advantage for understanding the diversity of sweet proteins and engineering research for development of a unique sweetener for the food and agriculture based on the Mabinlin II structure as a native model.     

Effect of repeated presentation on sweetness intensity of binary and ternary mixtures of sweeteners

The purpose of the present study was to determine the effect of repeated presentation of the same sweet stimulus on sweetness intensity ratings. The sweet stimuli tested in this study were binary and ternary blends of 14 sweeteners that varied widely in chemical structure. A trained panel evaluated the sweetness intensity over four sips of a given mixture presented at 30 s intervals. The individual components in the binary sweetener combinations were intensity-anchored with 5% sucrose, while the individual sweeteners in the ternary mixtures were intensity-anchored with 3% sucrose (according to formulae developed previously). Each self-mixture was also evaluated (e.g. acesulfame-K-acesulfame-K). The main finding of this study was that mixtures consisting of two or three different sweeteners exhibited less reduction in sweetness intensity over four repeated sips than a single sweetener at an equivalent sweetness level. Furthermore, ternary combinations tended to be slightly more effective than binary combinations at lessening the effect of repeated exposure to a given sweet stimulus. These findings suggest that the decline in sweetness intensity experienced over repeated exposure to a sweet stimulus could be reduced by the blending of sweeteners.     

Qualitative psychophysical studies on the gustatory effects of the sweet tasting proteins thaumatin and monellin

The gustatory effects of the sweet tasting proteins thaumatinand monellin were studied aftei application to small areas onthe anterior third of the tongue or to single fungiform papillae.The sweet sensation caused by thaumatin and monellin developedmore slowly, but reached a higher intensity and had a longerduration than that given by sucrose. Also, the response evokedby these sweet tasting proteins was more pronounced at the lateraledges, whereas that evoked by sucrose was stronger at the tipof the tongue. The taste modifier, miraculin, had no noticeableeffect on the sweet taste elicited by thaumatin, monellin andsucrose. Gymnemic acid abolished the sweet taste of all threecompounds. Experiments with time intervals of less than one minute betweenstimuli showed strong crossadaptation between thaumatin andmonellin, between the two proteins and sucrose, and betweenthe two proteins and miraculin-induced sweet taste of citricacid. While the differences in response to the sweet tasting proteinsand sucrose may be taken as evidence in favor of the existenceof more than one kind of sweet receptor, the cross-adaptationnoted between the various substances tested, would seem to indicatethat, at some point, they engage a common neural mechanism. ¹On leave from Dept. of Prosthetics, Faculty of Odontology,Karolinska Institutet. Present address: Dept. of Histology,Karolinska Institutet, S-104 01 STOCKHOLM, Sweden.     

Gustatory sensation of l- and d-amino acids in humans

Amino acids are known to elicit complex taste, but most human psychophysical studies on the taste of amino acids have focused on a single basic taste, such as umami (savory) taste, sweetness, or bitterness. In this study, we addressed the potential relationship between the structure and the taste properties of amino acids by measuring the human gustatory intensity and quality in response to aqueous solutions of proteogenic amino acids in comparison to d-enantiomers. Trained subjects tasted aqueous solution of each amino acid and evaluated the intensities of total taste and each basic taste using a category-ratio scale. Each basic taste of amino acids showed the dependency on its hydrophobicity, size, charge, functional groups on the side chain, and chirality of the alpha carbon. In addition, the overall taste of amino acid was found to be the combination of basic tastes according to the partial structure. For example, hydrophilic non-charged middle-sized amino acids elicited sweetness, and l-enantiomeric hydrophilic middle-sized structure was necessary for umami taste. For example, l-serine had mainly sweet and minor umami taste, and d-serine was sweet. We further applied Stevens’ psychophysical function to relate the total-taste intensity and the concentration, and found that the slope values depended on the major quality of taste (e.g., bitter large, sour small).     

Recognition of Superpotent Sweetener Ligands by a Library of Monoclonal Antibodies

Monoclonal antibodies (mAb) made to the superpotent guanidino sweet tasting ligand, N-(p-cyanophenyl)-N-(diphenylmethyl)-guanidineacetic acid were examined for their molecular recognition specificities using 14 different sweetener analogues in a competitive radioimmunoassay. The effects of variations in pH on ligand binding was also examined by radioimmunoassay. Photoaffinity labelling of the binding site was accomplished using a radiolabelled azido-derivative of the parent ligand, and L-chain or H-chain labelling was easily identified in several different mAb. For two of the mAb examined in this study (NC6.8 and NC10.14), the analogue binding studies are in agreement with the known Fab-ligand crystal structures. Monoclonal antibodies to this family of sweet tasting compounds may be useful probes for the study of sweet taste chemistry and identification of novel sweet taste ligands from combinatorial chemical libraries. © 1997 John Wiley & Sons, Ltd.