Non-acidic compounds induce the intense sweet taste of neoculin, a taste-modifying protein

Neoculin, a sweet protein found in the fruit of Curculigo latifolia, has the ability to change sourness into sweetness. Neoculin turns drinking water sweet, indicating that non-acidic compounds may induce the sweetness. We report that ammonium chloride and certain amino acids elicit the intense sweetness of neoculin. Neoculin can thus sweeten amino acid-enriched foods.     

Identification and modulation of the key amino acid residue responsible for the pH sensitivity of neoculin, a taste-modifying protein

Neoculin occurring in the tropical fruit of Curculigo latifolia is currently the only protein that possesses both a sweet taste and a taste-modifying activity of converting sourness into sweetness. Structurally, this protein is a heterodimer consisting of a neoculin acidic subunit (NAS) and a neoculin basic subunit (NBS). Recently, we found that a neoculin variant in which all five histidine residues are replaced with alanine elicits intense sweetness at both neutral and acidic pH but has no taste-modifying activity. To identify the critical histidine residue(s) responsible for this activity, we produced a series of His-to-Ala neoculin variants and evaluated their sweetness levels using cell-based calcium imaging and a human sensory test. Our results suggest that NBS His11 functions as a primary pH sensor for neoculin to elicit taste modification. Neoculin variants with substitutions other than His-to-Ala were further analyzed to clarify the role of the NBS position 11 in the taste-modifying activity. We found that the aromatic character of the amino acid side chain is necessary to elicit the pH-dependent sweetness. Interestingly, since the His-to-Tyr variant is a novel taste-modifying protein with alternative pH sensitivity, the position 11 in NBS can be critical to modulate the pH-dependent activity of neoculin. These findings are important for understanding the pH-sensitive functional changes in proteinaceous ligands in general and the interaction of taste receptor-taste substance in particular.     

Taste-modifying sweet protein, neoculin, is received at human T1R3 amino terminal domain

This study examines taste reception of neoculin, a Curculigo latifolia sweet protein with taste-modifying activity which converts sourness to sweetness. Neoculin tastes sweet to humans, but not to mice, and is received by the human sweet taste receptor hT1R2-hT1R3. In the present study with calcium imaging analysis of HEK cells expressing human and mouse T1Rs, we demonstrated that hT1R3 is required for the reception of neoculin. Further experiments using human/mouse chimeric T1R3s revealed that the extracellular amino terminal domain (ATD) of hT1R3 is essential for the reception of neoculin. Although T1R2-T1R3 is known to have multiple potential ligand-binding sites to receive a wide variety of sweeteners, the present study is apparently the first to identify the ATD of hT1R3 as a new sweetener-binding region.     

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.     

The structure of monellin and its relation to the sweetness of the protein.

The sweet protein monellin [1-3] has been shown to consist of two non-identical subunits of 50 and 42 amino acid residues, which were separated electrophoretically and chromatographically. Automatic sequential Edman degradation gave the complete sequence of the longer subunit, and a partial sequency of the shorter one. It was found that the sweetness of monellin requires the undissociated molecule. The individual subunits were not sweet, neither did they block the sweet sensation of sucrose or monellin. Blocking of the single SH of monellin abolished its sweetness as did reaction of the single methionyl residue with CNBr. Since the cysteinyl and methionyl residues appear to be adjacent, it is suggested that this part of the molecule is essential for its sweetness.     

Neoculin, a taste-modifying sweet protein, accumulates in ripening fruits of cultivated Curculigo latifolia

Neoculin is a sweet protein with a taste-modifying activity of converting sourness to sweetness. It occurs in the fruits of Curculigo latifolia, a wild plant found in tropical Asia. We successfully cultivated the plant and evaluated the production of neoculin. The neoculin content of the fruit was high for 10 weeks after flowering, following which the yield decreased gradually. The optimal period for harvesting the fruits with sensory activity coincided with this 10-week peak period during which the amount of neoculin was 1-3mg in the whole fruit and 1.3mg/g of pulp. Immunohistochemical staining showed that neoculin occurred in the whole fruit, especially at the basal portion. Although it is known that neoculin comprises an acidic subunit (NAS) with an N-glycosylated moiety and a basic subunit (NBS), protein gel blot analysis revealed the presence of a non-glycosylated NAS species. This suggests the presence of multiple NAS-NBS heterodimers in our cultivar.     

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.     

Methyl 2,3-Di-O-(L-α-aminobutyryl)-α-D-glucopyranoside,a New Sweet Substance,and Tastes of Related Compounds of Neutral Amino Acids and D-Glucose Derivatives

Methyl 2,3-di-(O-(L-phenylalanyl)-±-D-glucopyranoside and other O-aminoacyl sugars composed of neutral amino acids were synthesized to discover their tastes. Among them, phenylalanine derivatives were strongly bitter [the bitterness of methyl 2,3-di-O-(L-phenylalanyl-L-phenylalanyl-L-phenylalanyl)-±-D-glucopyranoside are equal to that of strychnine], on the other hand, O-aminoacyl sugars composed of amino acids having short side chain were very sweet; The sweetness of methyl 2,3-di-O-(L-±-aminobutyryl)-±-D-glucopyranoside is more than 50 times as strong as that of sucrose.     

Enhancing effects of NaCl and Na phosphate on human gustatory responses to amino acids

The effects of salts on sweet taste of amino acids (glycine,alanine and serine) were examined by the psychophysical method.There was no difference in quality of sweetness between theamino acids alone and the amino acids in the presence of saltssince low concentrations of NaCl and Na phosphate having nosalty taste were used. The sweetness of the amino acids wasgreatly increased with an increase of NaCl concentration. Thesweetness of 100 mM amino acids in the presence of 30 mM NaClwas equivalent to that of 500–600 mM amino acids containingno salt. On the other hand, Na phosphate little affected sweetnessof the amino acids. Significance of the present results in foodscience was discussed.     

Taste enhancements between various amino acids and IMP

It is well known that a strong synergistic interaction of umami occurs between L-alpha-amino acids with an acidic side chain, such as L-Glu or L-Asp, and 5'-mononucleotides, such as inosine 5'-monophosphate (IMP). We tested taste interactions between various L-alpha-amino acids and IMP by the psychophysical method and found that taste enhancement occurred when IMP was added to several sweet amino acids, such as L-Ala, L-Ser and Gly. The enhanced quality of taste was recognized as umami, and was not blocked by the sweetness inhibitor +/-2-(p-methoxyphenoxy)propanoic acid. The total taste intensities of various concentrations of the amino acid and IMP mixtures were measured using magnitude estimation. The results showed that the potentiation ratios were larger than 1 in the cases of L-Ala, L-Ser and Gly. However, the ratio was approximately 1 in the case of D-Ala, which had an enhanced taste of sweetness. Thus the umami taste enhancement of several sweet L-alpha-amino acids by IMP was synergistic rather than additive as that of acidic amino acids.     

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).     

EFFECT OF ACETYLATION AND METHYLATION ON THE SWEETNESS INTENSITY OF THAUMATIN I

The lysine residues in thaumatin I were chemically modifiedby acetylation with acetic anhydride and by reductive methylation,under various conditions. The acetylated and methylated thaumatinswere isolated by ion-exchange chromatography. The number ofremaining free amino groups was determined by trinitrophenylation. At least four acetylated thaumatins with either one, two, threeor four acetylated amino groups were obtained as well as onemethylated thaumatin with six dimethyl lysine residues and onemonomethyl lysine residue. The sweetness intensity of the acetylated thaumatins decreasedwith the increasing number of acetylated amino groups; the sweettaste had disappeared completely when four amino groups wereacetylated. The methylated thaumatin with seven modified lysineresidues had a sweetness intensity practically equal to thatof the original thaumatin. The total net change, i.e. the isoelectric point of thaumatin,might play a role in the physiological behaviour of thaumatincausing a sweet taste sensation.     

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.     

Highly Probable Active Site of the Sweet Protein Monellin

The sweet protein monellin consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. Synthetic monellin is 4000 times as sweet as sucrose on a weight basis, and the native conformation is essential for the sweet taste. Knowledge of the active site of monellin will provide important information on the mode of interaction between sweeteners and their receptors. If the replacement of a certain amino acid residue in monellin removes the sweet taste, while the native conformation is retained, it may be concluded that the position replaced is the active site. Our previous replacement studies on Asp residues in the A chain did not remove the sweet taste. The B chain contains two Asp residues at positions 7 and 21, which were replaced by Asn. [Asn^B21] Monellin and [Asn^B7]monellin were 7000 and 20 times sweeter than sucrose, respectively. The low potency of the [Asn^B7]monellin indicates that ASp^B7 participates in binding with the receptor. ASp^B7 was then replaced by Abu. [Abu^B7]Monellin was devoid of sweetness, and retained the native conformation. ASp^B7 is located at the surface of the molecule (Ogata et al.). These results suggest that Asp⁷ in the B chain is the highly probable active site of monellin.     

Cloning, expression and characterization of recombinant sweet-protein thaumatin II using the methylotrophic yeast Pichia pastoris

Thaumatin, an intensely sweet-tasting protein, was secreted by the methylotrophic yeast Pichia pastoris. The mature thaumatin II gene was directly cloned from Taq polymerase-amplified PCR products by using TA cloning methods and fused the pPIC9K expression vector that contains Saccharomyces cerevisiae prepro alpha-mating factor secretion signal. Several additional amino acid residues were introduced at both the N- and C-terminal ends by genetic modification to investigate the role of the terminal end region for elicitation of sweetness in the thaumatin molecule. The secondary and tertiary structures of purified recombinant thaumatin were almost identical to those of the plant thaumatin molecule. Recombinant thaumatin II elicited a sweet taste as native plant thaumatin II; its threshold value of sweetness to humans was around 50 nM, which is the same as that of plant thaumatin II. These results demonstrate that the functional expression of thaumatin II was attained by Pichia pastoris systems and that the N- and C-terminal regions of the thaumatin II molecule do not -play an important role in eliciting the sweet taste of thaumatin.     

Structure Activity Relationship amongst Some Fungitoxic α-Naphthoquinones of Angiosperm Origin

Rubusoside, the β-D-glucosyl ester of 13-O-D-glucosyl-steviol which was isolated from leaves of Rubus suavissimus collected in China as the major sweet principle (yield: 5.4%), was subjected to α-1 4 transglucosylation with the cyclodextrin glucosyltransferase produced by Bacillus megaterium Strain No. 5 using soluble starch as a donor. A significant improvement in the quality of sweetness was observed for the crude reaction mixture, which was separated into mono-, di-, tri-, tetra-, penta-, and hexa-glucosylated products. All isomers of the mono- and di-glucosylated products were further separated. Evaluation of the sweetness of these products compared with stevioside, rebaudioside A, etc. disclosed that the ratio of the number of glucose units at the 13-hydroxyl group to that at 19-carboxyl group seems to have a significant relationship with the sweetness as well as the quality of taste for glucosides of this type.     

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.     

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.     

不同类型特种稻种质营养及功能性成分含量的差异

本研究以具有有色种皮、巨胚、甜味、香味、糯性等单一特殊性状或2个以上特殊性状聚合于一体的新创制特种稻种质39份和1份白米种质(对照)为试验材料,进行了黑米、黑褐米、红米、香糯米、黑巨胚糯米、红巨胚糯米、巨胚糯米、黑甜米、红甜米、白甜米等不同类型特种稻种质的营养及功能性成分含量的差异评价。结果表明,供试特种稻类型在大部分氨基酸含量和矿质元素含量上与白米差异不显著,只在个别氨基酸和矿质元素含量上与白米呈显著或极显著差异。黑褐米、红米、黑巨胚糯米、红巨胚糯米、巨胚糯米、红甜米和白甜米等7种类型的赖氨酸含量显著或极显著高于白米,高2.91%~24.68%;黑米、黑褐米、红米、香糯米、黑巨胚糯米和红甜米等6种类型的铁含量显著或极显著高于白米,高17.62%~68.09%;黑褐米、红米、黑巨胚糯米、红巨胚糯米、黑甜米、红甜米、白甜米等7种类型的钙含量显著或极显著高于白米,高23.56%~49.46%;黑米、黑褐米、红米、黑甜米、红甜米、白甜米等6种类型的锌含量显著或极显著高于白米,高12.21%~55.87%。由此表明,具有有色种皮、巨胚、甜味、香味、糯性等单一特殊性状或2~3个特殊性状的聚合对赖氨酸含量与铁、钙和锌含量的提高方面具有一定的增加效应,认为在今后以赖氨酸、铁、钙和锌含量为目标性状的功能性水稻育种中,多个特殊性状的聚合将是增加上述功能性成分含量的有效途径之一。通过鉴定评价,从创新种质中还筛选出一些功能性成分含量相对较高的优异种质,白甜米1553和红巨胚糯米1476的赖氨酸含量较高,比白米分别高29.37%和23.42%;红米1439和红米1440的铁含量较高,比白米分别高99.05%和80.00%;黑甜米1511和黑甜米1515的硒含量较高,比白米分别高194.14%和136.48%;白甜米1551和香糯米1446的γ-氨基丁酸含量较高,比白米分别高14.56%和11.83%;黑巨胚糯米1464和黑米1432的花色苷含量较高,比供试18份有色稻米的平均值分别高253.23%和248.83%。这些新创制的功能性成分含量较高的水稻种质有待于今后在育种、生态适应性鉴定与产业化中进一步得到利用。