Sweetening agents of plant origin*

The most important sweet substance known is sucrose, which is obtained commercially from sugar cane and sugar beet. Because the intake of sucrose has been associated with a number of adverse effects on health, an intensive search has been undertaken to find alternative substances to satisfy the human craving for a sweet taste. Many other plant‐derived compounds are sweet, ranging in structural complexity from sugars and polyhydric alcohols through diterpene and triterpene glycosides to proteins; some of these compounds are intensely sweet, being hundreds or even thousand times sweeter than sucrose, and offer potential for commercial use in dietetic and diabetic foodstuffs. The present review examines the role of ethnobotany in the discovery of sweet‐tasting plants, the chemical isolation and elucidation of the sweet compounds, and some safety and sensory evaluation aspects of these compounds. A discussion on the future prospects of discovering and developing new plant‐derived sweeteners concludes the review.     

Whole nerve chorda tympani responses to sweeteners in C57BL/6ByJ and 129P3/J mice

The C57BL/6ByJ (B6) strain of mice exhibits higher preferences than does the 129P3/J (129) strain for a variety of sweet tasting compounds. We measured gustatory afferent responses of the whole chorda tympani nerve in these two strains using a broad array of sweeteners and other taste stimuli. Neural responses were greater in B6 than in 129 mice to the sugars sucrose and maltose, the polyol D-sorbitol and the non-caloric sweeteners Na saccharin, acesulfame-K, SC-45647 and sucralose. Lower neural response thresholds were also observed in the B6 strain for most of these stimuli. The strains did not differ in their neural responses to amino acids that are thought to taste sweet to mice, with the exception of L-proline, which evoked larger responses in the B6 strain. Aspartame and thaumatin, which taste sweet to humans but are not strongly preferred by B6 or 129 mice, did not evoke neural responses that exceeded threshold in either strain. The strains generally did not differ in their neural responses to NaCl, quinine and HCl. Thus, variation between the B6 and 129 strains in the peripheral gustatory system may contribute to differences in their consumption of many sweeteners.     

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.     

Inhibition of sweet taste in humans by methyl 4,6-dichloro-4,6-dideoxy-{alpha}-D-galactopyranoside

The glycoside methyl 4,6-dichloro–4,6-dideoxy--D-galactopyranoside,an inhibitor of electrophysiological responses to sweet tastein gerbils, was also found to suppress the perceived intensitiesof various sweeteners in human psychophysical experiments. Incontrast, this compound did not suppress the salty and sourtastes in either species.     

Chemical structure and sweet taste of isocoumarins and related compounds

The relationship between sweetness and chemical strucure of3,4-dihydroisocoumarins was studied because of the interestin the structure of phyllodulcin, the only sweet tasting compoundamong the naturally occurring 3,4-dihydroisocoumarins. It wasfound by structural modification of phyllodulcin and the synthesesof various derivatives that ß-(3-hydroxy-4-methoxyphenyl)ethylbenzene (XIII) is the essential part of sweet tasting 3,4-dihydroissocoumarins.Further studies on the structural modification of XIII wereundertaken to make the relationship between structure and sweettaste clearer. The application of the information obtained fromthe structure-sweetness relationship of 3,4-dihydroisocoumarinsto the design of other series of sweet compounds was attempted.The resulting chroman (LXXXVII), isochroman (LXXXVI), flavanone(LXXXIX), and dihydrochalcone (XCV) derivatives are potentlysweet, as was predicted. From these results, it may be concludedthat the relationship between sweetness and structure of 5-hydroxyflavanonesor dihydrochalcones lacking a glycoside moiety is similar tothat of the 3,4-dihydroisocoumarins.     

TIME-INTENSITY PROPERTIES OF SWEET AND BITTER STIMULI: IMPLICATIONS FOR SWEET AND BITTER TASTE CHEMORECEPTION

Interindividual differences in sweet and bitter taste sensitivity were investigated using time-intensity (TI) measurements and multivariate statistics. TI profiles were obtained in triplicate from 25 subjects for 23 sweet and/or bitter stimuli first matched to be approximately equi-intense to 200 mM NaCl. Sweet stimuli, except for the larger sweeteners, were less persistent, and required less time to reach maximum intensity than bitter stimuli. The results of principal component (PCA) and cluster (CA) analyses of the stimuli X subjects matrices for maximum intensity (Imax), time to maximum intensity (Tmax), total duration (Tdur), and area under the curve (Area) suggest that sweet and bitter stimuli do not share common receptors; and that there are at least two receptor mechanisms each for sweet taste (one for sugars and other small compounds, and the other for large sweeteners) and bitter taste (one for PTC/PROP and one for other bitter compounds).     

Bio-protective effects of homologous disaccharides on biological macromolecules

In this contribution the effects of the homologous disaccharides trehalose and sucrose on both water and hydrated lysozyme dynamics are considered by determining the mean square displacement (MSD) from elastic incoherent neutron scattering (EINS) experiments. The self-distribution function (SDF) procedure is applied to the data collected, by use of IN13 and IN10 spectrometers (Institute Laue Langevin, France), on trehalose and sucrose aqueous mixtures (at a concentration corresponding to 19 water molecules per disaccharide molecule), and on dry and hydrated (H₂O and D₂O) lysozyme also in the presence of the disaccharides. As a result, above the glass transition temperature of water, the MSD of the water–trehalose system is lower than that of the water–sucrose system. This result suggests that the hydrogen-bond network of the water–trehalose system is stronger than that of the water–sucrose system. Furthermore, by taking into account instrumental resolution effects it was found that the system relaxation time of the water–trehalose system is longer than that of the water–sucrose system, and the system relaxation time of the protein in a hydrated environment in the presence of disaccharides increases sensitively. These results explain the higher bioprotectant effectiveness of trehalose. Finally, the partial MSDs of sucrose/water and trehalose/water have been evaluated. It clearly emerges from the analysis that these are almost equivalent in the low-Q domain (0–1.7 ?⁻¹) but differ substantially in the high-Q range (1.7–4 ?⁻¹). These findings reveal that the lower structural sensitivity of trehalose to thermal changes is connected with the local spatial scale.     

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.     

The history of sweet taste: not exactly a piece of cake

Understanding the molecular bases of sweet taste is of crucial importance not only in biotechnology but also for its medical implications, since an increasing number of people is affected by food-related diseases like, diabetes, hyperlipemia, caries, that are more or less directly linked to the secondary effects of sugar intake. Despite the interest paid to the field, it is only through the recent identification and functional expression of the receptor for sweet taste that new perspectives have been opened, drastically changing our approach to the development of new sweeteners. We shall give an overview of the field starting from the early days up to discussing the newest developments. After a review of early models of the active site, the mechanisms of interaction of small and macromolecular sweet molecules will be examined in the light of accurate modeling of the sweet taste receptor. The analysis of the homology models of all possible dimers allowed by combinations of the human T1R2 and T1R3 sequences of the sweet receptor and the closed (A) and open (B) conformations of the mGluR1 glutamate receptor shows that only 'type B' sites, either T1R2(B) and T1R3(B), can host the majority of small molecular weight sweeteners. Simultaneous binding to the A and B sites is not possible with two large sweeteners but is possible with a small molecule in site A and a large one in site B. This observation accounted for the first time for the peculiar phenomenon of synergy between some sweeteners. In addition to these two sites, the models showed an external binding site that can host sweet proteins.     

Study of the effect of gymnemic acid on taste in hamster

Behavioral and electrophysiological experiments with 10 sweetenershave been made to test if gymnemic acid (GA) is able to blockthe response to sweet stimuli in single taste fibres of thechorda tympani proper nerve in hamsters. The hamster was chosenbecause earlier studies show that it is more sensitive to GAthan any other non-human species. Since GA has been shown toaffect the sweetness of many different substances, its effectswere studied on an array of sweeteners. To avoid, however, theinclusion of sweeteners unpalatable to the hamster, the hamsters'liking of this array was tested with two-bottle preference techniquefor –24 h. It was found that acesulfam-K, fructose, glucose,sucrose and xylitol were strongly liked, while the animals showedno preference for aspartame, D-tryptophane, sodium cyclamate,sodium saccharin and thaumatin over water. The summated nerveresponse of these stimuli was then recorded. It was found thatneither thaumatin nor aspartame elicited a response, while theother stimuli gave a good response. Finally, the sweetenerswhich were both preferred in the two-bottle tests and gave anerve response were used as taste stimuli in single fibre experimentstogether with sodium chloride, quinine hydrochloride, citricacid and saccharin. The single fibre recordings were made beforeand after application of 5 mg GA for 3 min on the tongue. Itwas found that GA did not cause any dramatic decrease or disappearanceof the responses to either the sweet or the non-sweet substances.The responses to the sweeteners, however, were more depressedthan those to the non-sweet stimuli.     

Changes in Taste Perception Following Mental or Physical Stress

Taste perception depends not only on the chemical and physicalproperties of tastants, but may also depend on the physiologicaland psychological conditions of those who do the tasting. Inthis study, the effects of mood state on taste sensitive wasevaluated in humans who were exposed to conditions of mentalor physical fatigue and tension. Taste responses to quininesulfate (bitter), citric acid (sour) and sucrose (sweet) weretested. The intensity of the taste sensations were recordedby a computerized time-intensity (TI) on-line system. Subjectsperformed mental tasks by personal computer or physical tasksby ergometer for 10–40 min. Before and after these sessions,the duration of the after-taste and the intensity of the sensationof taste were recorded by the TI system, and in addition, psychologicalmood states were evaluated with POMS (Profile of Mood State).TI evaluation showed that after the mental tasks, the perceivedduration of bitter, sour and sweet taste sensations was shortenedrelative to the control. Total amount of bitterness, sournessand sweetness was also significantly reduced. Furthermore, themaximum intensity of bitterness was significantly reduced. Therewere no significant differences in bitterness and sweetnesssensations following physical tasks. However, relative to beforethe physical task, the duration of the after-taste of sournesswas significantly shortened by the physical task. After thephysical task, the buffering capacity of saliva was significantlyincreased. Thus mental and physical tasks alter taste perceptionin different ways; the mechanisms underlying these changes remainto be determined. Chem. Senses 21: 195–200, 1996.     

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.     

Drosophila melanogaster prefers compounds perceived sweet by humans

To understand the functional similarities of fly and mammalian taste receptors, we used a top-down approach that first established the fly sweetener-response profile. We employed the fruit fly Drosophila melanogaster, an omnivorous human commensal, and determined its sensitivity to an extended set of stimuli that humans find sweet. Flies were tested with all sweeteners in 2 assays that measured their taste reactivity (proboscis extension assay) and their ingestive preferences (free roaming ingestion choice test). A total of 21 sweeteners, comprised of 11 high-potency sweeteners, 2 amino acids, 5 sugars, 2 sugar alcohols, and a sweet salt (PbCl2), were tested in both assays. We found that wild-type Drosophila responded appetitively to most high-potency sweeteners preferred by humans, even those not considered sweet by rodents or new world monkeys. The similarities in taste preferences for sweeteners suggest that frugivorous/omnivorous apes and flies have evolved promiscuous carbohydrate taste detectors with similar affinities for myriad high-potency sweeteners. Whether these perceptual parallels are the result of convergent evolution of saccharide receptor-binding mechanisms remains to be determined.     

Changes in Outward K+ Currents in Response to Two Types of Sweeteners in Sweet Taste Transduction of Gerbil Taste Cells

Using the whole cell patch clamp technique, we measured changesin outward K⁺ currents of gerbil taste cells in response todifferent kinds of sweeteners. Outward K⁺ currents of the tastecell induced by depolarizing pulses were suppressed by sweetstimuli such as 10 mM Na-saccharin. The membrane-permeable analogof cAMP, cpt-cAMP, also decreased outward K⁺ currents. On theother hand, the K+ currents were enhanced by amino acid sweetenerssuch as 10 mM D-tryptophan. The outward K⁺ current was enhancedby external application of Ca²⁺-transporting ionophore, 5 µMionomycin, and intracellular application of 5 µM inositol-1,4,5-trisphosphate(IP₃). The outward K⁺ currents were no longer suppressed by10 mM Na-saccharin containing 20 µM gurmarin, but werestill enhanced by 10 mM D-tryptophan containing 20 µMgurmarin. These results suggest that sweet taste transductionfor one group of sweeteners such as Na-saccharin in gerbilsis concerned with an increase of the intracellular cAMP level,and that the transduction for the other group of sweetenerssuch as D-tryptophan is concerned with an increase of the intracellularIP₃ level which releases Ca²⁺ from the internal stores. Chem.Senses 22: 163–169, 1997.     

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.     

Selective inhibition of sweetness by the sodium salt of +/-2-(4-methoxyphenoxy)propanoic acid.

The purpose of this study was to determine the degree to which the sodium salt of +/-2-(4-methoxyphenoxy)propanoic acid (Na-PMP) reduced sweet intensity ratings of 15 sweeteners in mixtures. Na-PMP has been approved for use in confectionary/frostings, soft candy and snack products in the USA at concentrations up to 150 p.p.m. A trained panel evaluated the effect of Na-PMP on the intensity of the following 15 sweeteners: three sugars (fructose, glucose, sucrose), three terpenoid glycosides (monoammonium glycyrrhizinate, rebaudioside-A, stevioside), two dipeptide derivatives (alitame, aspartame), two N-sulfonylamides (acesulfame-K, sodium saccharin), two polyhydric alcohols (mannitol, sorbitol), 1 dihydrochalcone (neohesperidin dihydrochalcone), one protein (thaumatin) and one sulfamate (sodium cyclamate). Sweeteners were tested at concentrations isosweet with 2.5, 5, 7.5 and 10% sucrose in mixtures with two levels of Na-PMP: 250 and 500 p.p.m. In addition, the 15 sweeteners were tested either immediately or 30 s after a pre-rinse with 500 p.p.m. Na-PMP. In mixtures, Na-PMP at both the 250 and 500 p.p.m. levels significantly blocked sweetness intensity for 12 of the 15 sweeteners. However, when Na-PMP was mixed with three of the 15 sweeteners (monoammonium glycyrrhizinate, neohesperidin dihydrochalcone and thaumatin), there was little reduction in sweetness intensity. Pre-rinsing with Na-PMP both inhibited and enhanced sweetness with the greatest enhancements found for monoammonium glycyrrhizinate, neohesperidin dihydrochalcone and thaumatin, which were not suppressed by Na-PMP in mixtures. The mixture data suggest that Na-PMP is a selective competitive inhibitor of sweet taste. The finding that pre-treatment can produce enhancement may be due to sensitization of sweetener receptors by Na-PMP.     

Sweet taste involves several distinct receptor mechanisms

Measures of human sensitivities to various sweet compounds conductedat threshold (91 subjects, 7 sweeteners) and at suprathresholdlevels (9 subjects, 12 sweeteners) show interindividual differences.Multidimensional analysis indicates that sweet taste can berepresented in a tridimensional continuum if 12 compounds areconsidered. Results are speculatively interpreted as indirectevidence for the existence of several receptor sites cooperatingin sweet taste chemoreception.     

甜味分子与G蛋白偶联受体Tas1R2/3的相互作用及激活机制

甜味分子与C家族G蛋白偶联受体(G protein-coupled receptor,GPCR)的成员之一甜味受体相互作用,从而激活受体并引起甜味觉的感知。本文简要总结了甜味受体(taste receptor 2 and 3,Tas1R2/3)的结构与功能、甜味分子与受体相互作用并激活受体的机制,并对甜味受体研究领域的发展前景进行了展望。甜味分子与受体相互作用机制的阐明对于理解甜味觉的产生与GPCR的结构与功能具有重要的意义。此外,甜味受体结构与功能的研究可为有针对性地设计新型甜味化合物提供理论基础。     

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.     

Taste modifiers: parotid reflexes can differ from taste judgements

Sweetness-depressing gymnemic acid (G) and sweetness-inducingmiraculin (M) helped determine the extent to which parotid salivaryresponses match behavioral and neural gustatory responses. Parotidflow rates and tastes intensities were obtained from four subjectsfor four sweeteners (before and after G) and for citric acid(CA) before and after M and G. A mixture of 20 mM CA and 10%sucrose was also tested. Although G depressed sweetness forglucose, sucrose, fructose and aspartame, G generally failedto alter parotid responses or depress post-G ratings for bitterintensity. In fact, G markedly elevated parotid responses forthe acid–sucrose mixture. Residual sweetness after G,detected mainly from the posterior tongue, probably contributedonly partially to sustaining post–G parotid responses.We speculate that side tastes from the sweeteners and oral irritationfrom CA in the mixture contributed to elevated flow rates afterG. Sucrose- and M-induced sweetness generally elevated parotidresponses for 20 mM CA. This result agrees with human chordatympani responses after M but differs for sucrose–acidmixtures in that parotid responses approached the calculatedsum of the components. We speculate that non-gustatory inputsmay also affect sweet–sour responses and advise cautionin relating parotid reflexes only to taste judgements. ¹Present address: Department of Biological Sciences, San JoseState University, San Jose, CA 95192, USA