Volume 74, Issue 7

The mechanistic understanding of sweet taste chemoreception has been advanced by the microscopic and macroscopic studies of sweetener­water interactions. This approach has led to the concept of water mobility as a key to interpreting sweetness. The apparent specific volume of a solution is a determinant of its taste quality, as sweetness is known to be confined to the range 0.51­0.71 cm 3 g -1 . Thus, the "ideal" quality of the sugars is presumed to be due to their occupancy of the center of this range (i.e., 0.618 cm 3 g -1 ). Most sweeteners elicit off-tastes and flavors and exhibit different apparent specific volumes. This leads to the conclusion that taste quality is broadly determined by the packing characteristics of sweet molecules among water molecules and the compactness of their hydration shells, expressed as their apparent specific isentropic compressibilities. The role of water can, therefore, be applied in modern attempts to optimize sweet taste quality, and different food salts can be explored as useful taste modifiers. Salts interact more strongly with water structure than do any other taste solutes, and it emerges that the ionic charge density is an important criterion. Such studies show how sweetener formulations are likely to improve within the next decade.

In this presentation, we describe an integrated approach for the molecular basis for sweet taste among dipeptide-based ligands. By comparing the results obtained from X-ray diffraction studies with the conformations from NMR analysis and molecular modeling, we have observed recurring topochemical motifs that agree with previous models for sweet taste. In our examination of the unexplored D zone of the Tinti­Nofre model, we have uncovered a sweet potency enhancing effect of a new set of aralkyl-substitutions on dipeptide ligands, which reveals the importance of aromatic­aromatic interactions in maintaining high potency.

The extracellular ligand binding domain of the sweet receptor T1R3 has been homology-modeled on the basis of the crystal structure of the metabotropic glutamate receptor (mGluR1). The region of the model that corresponds to the ligand binding site of mGluR1 has numerous polar and charged side-chains, consistent with expectations for a site that would respond to poly hydroxy compounds such as mono- and disaccharides. Docking studies show that proposed active conformations of the high-potency sweeteners neotame, superaspartame, and SC-45647 could interact favorably in this binding site, forming ion pairs or ionic hydrogen bonds with His-163, Glu-318, and His-407, in addition to hydrophobic interactions with numerous nonpolar side-chains.

Taste transduction is a specialized form of signal transduction by which taste receptor cells (TRCs) encode at the cellular level information about chemical substances encountered in the oral environment (so-called tastants). Bitter and sweet taste transduction pathways convert chemical information into a cellular second messenger code utilizing cyclic nucleotides, inositol trisphosphate, and/or diacyl glycerol. These messengers are components of signaling cascades that lead to TRC depolarization and Ca++ release. Bitter and sweet taste transduction pathways typically utilize taste-specific or taste-selective seven transmembrane-helix receptors, G proteins, effector enzymes, second messengers, and ion channels. The structural and chemical diversity of tastants has led to the need for multiple transduction mechanisms. Through molecular cloning and data mining, many of the receptors, G proteins, and effector enzymes involved in transducing responses to bitter and sweet compounds are now known. New insights into taste transduction and taste coding underlying sweet and bitter taste qualities have been gained from molecular cloning of the transduction elements, biochemical elucidation of the transduction pathways, electrophysiological analysis of the function of taste cell ion channels, and behavioral analysis of transgenic and knockout models.

Inbred mouse strains display marked differences in avidity for sweet solutions due in part to genetic differences among strains. Using several techniques, we have located a number of regions throughout the genome that influence sweetener acceptance. One prominent locus regulating differences in sweetener preferences among mouse strains is the saccharin preference ( Sac ) locus on distal chromosome 4. Afferent responses of gustatory nerves to sweeteners also vary as a function of allelic differences in the Sac locus, suggesting that this gene may encode a sweet taste receptor. Using a positional cloning approach, we identified a gene ( Tas1r3 ) encoding the third member of the T1R family of putative taste receptors, T1R3. Introgression by serial back-crossing of a chromosomal fragment containing the Tas1r3 allele from the high sweetener-preferring strain onto the genetic background of the low sweetener-preferring strain rescued its low sweetener-preference phenotype. Tas1r3 has two common haplotypes, one found in mouse strains with elevated sweetener preference and the other in strains relatively indifferent to sweeteners. This study, in conjunction with complimentary recent studies from other laboratories, provides compelling evidence that Tas1r3 is equivalent to the Sac locus and that the T1R3 receptor (when co-expressed with taste receptor T1R2) responds to sweeteners. However, other sweetness receptors may remain to be identified.

Leptin, a hormone released from the adipose tissue, inhibits food intake and increases energy expenditure. We have found a novel function of leptin as a modulator of sweet taste sensitivity in mice. In lean normal mice, the gustatory nerve responses to sweet stimuli were selectively suppressed depending on plasma leptin level after an intraperitoneal injection of recombinant leptin. Patch-clamp studies using isolated taste cells of lean mice showed that extracellular leptin enhanced K + currents of sweet-responsive taste cells, which led to membrane hyperpolarization and a reduction of sweetener-induced depolarization. Reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization analyses demonstrated specific expression of mRNA of the long-form functional leptin receptor (Ob-Rb) in taste tissue and cells of lean mice. The genetically diabetic db / db mice, which have defects in Ob-Rb, demonstrated neither a suppression of gustatory neural responses to sweeteners nor an increment of whole-cell K + conductance of taste cells even with high doses of leptin. These results suggest that Ob-Rb is specifically expressed in sweet-responsive taste cells of lean mice and that leptin suppresses sweetener-induced depolarization via activation of K + channels, leading to a decrease in impulses of sweet-best fibers. The enhanced sweet responses of db / db mice may result from the lack of inhibitory modulation by leptin.

During the last decades, the comparison in various animal species of their gustatory responses to compounds eliciting a sweet taste in humans has extended our knowledge of the great biodiversity of the taste responses and evidenced some specialization and/or phyletic trends within species. Our interest was focused on responses to natural sugars, polyols, and naturally occurring sweeteners, but also on various artificial sweetening compounds, including the very powerful guanidine sweeteners. New results obtained with kangarooswhich originated about 130 MYAhave shown that their sweetness receptor is not designed to taste any of the artificial sweeteners tested. Therefore, the ability to taste complicated artificial sweeteners must have evolved later in higher developed mammals, about 100 million years ago.

Like the Sac locus controlling sugar sensitivity in mice, the taste gene Tre of the fruitfly Drosophila was discovered in wild populations as a genetic dimorphism controlling gustatory sensitivity to a sugar trehalose. By activating a P-element transposon near the gene locus we obtained induced Tre mutations and analyzed the associated changes in gene organizations and the mRNA expressions. The analysis showed that Tre is identical to Gr5a , a gene that belongs to a novel seven-transmembrane receptor family expressed in chemosensory neurons and predicted to encode chemosensory receptors. Thus, Gr5a is a candidate sweet taste receptor in the fly. An amino acid substitution in the second intracellular loop domain was identified to be functionally correlated with the genetic dimorphism of Tre . Since Tre controls sweet taste sensitivity to a limited subset of sugars, other Gr genes phylogenetically related to Tre may also encode sweet taste receptors. Those candidate sweet taste receptors, however, are phylogenetically distinct from vertebrate sweet taste receptors, suggesting that the sweet taste receptors in animals do not share a common origin.

Several plant-derived compounds of the terpenoid and phenolic types have commercial use as sweeteners. In our research program directed toward the discovery of additional sweet compounds of these chemical classes, candidate sweet plants for laboratory investigation may be selected after scrutiny of the available literature, as a result of making inquiries in the field, and/or from a limited amount of organoleptic testing. Sweet-tasting plants are extracted according to a standard protocol, and preliminary safety testing is conducted before crude extracts or pure compounds are tasted. The practicality of using Mongolian gerbil electrophysiological and behavioral assays to monitor plant extracts and pure isolates has been examined. A number of sweet-tasting, plant-derived terpenoids and phenolics have been isolated and characterized, including the bisabolane sesquiterpenoids, hernandulcin and 4β-hydroxyhernandulcin, the labdane diterpene glycoside, gaudichaudioside A, the oleanane triterpenoid glycoside, periandrin V, the cycloartane triterpene glycosides, abrusosides A­E, the 3,4- seco -dammarane triterpene glycosides, pterocaryosides A and B, the semisynthetic dihydroflavonol, dihydroquercetin 3-acetate (4'-methyl ether), and the proanthocyanidin, selligueain A. Most of these new compounds are prototype "high-intensity" sweeteners that may be worthy targets for chemical synthesis or for semi-synthetic modification to produce substances with enhanced sweetness properties.

Systematic modification of the structure of the sweet natural compound phyllodulcin, containing the isovanillyl glucophoric group, led to the synthesis of about 120 compounds. Features of the heterocyclic ring conferring high sweetness potency were identified. A strong increase in sweetness was obtained by the introduction of sulfur atoms in the ring and by separation of the enantiomers. Results of the quantitative structure­activity relationship (QSAR) studies on this series are reported. Application of the pseudoreceptor modeling approach afforded a three-dimensional binding site model for isovanillyl sweeteners. Extension of this methodology to a large group of structurally diverse compounds, including the commonly used sweeteners, gave a general pseudoreceptor for the sweet compounds, consisting of 16 amino acids. This pseudoreceptor, which has the peculiarity of giving a semiquantitative evaluation of the sweetness intensity, could be used as a valid tool to model the ligand­receptor interactions and to suggest some clues about the identification of a possible binding site once the receptor protein(s) are obtained.

This paper reviews our investigations on the chemical constituents of several kinds of botanically identified licorice roots, which led to the characterization of 13 then-new glucuronide-saponins named licorice-saponins (A­L), apioglycyrrhizin, and araboglycyrrhizin, together with glycyrrhizin and 18α-glycyrrhizin and also of 49 kinds of phenolic compounds and their glycosides (11 then-new). The restoration-promoting activity of licorice-saponin B2 for CCl 4 -intoxicated hepatocyte function and the structure­sweetness relationship of saponins were discussed. Biologically interesting, but isolable in minor quantities, several licorice-saponins were favorably synthesized from abundantly available glycyrrhizin. With 15 saponins and 49 phenolic compounds (including their glycosides) at hand, chemical evaluation of licorice root processings was undertaken. It was shown that the cortex contained a rich amount of phenolic compounds, whereas the xylem was rich in phenolic glycosides and the saponins contained were richer in the xylem than in the cortex. It was also found that roasted licorice root contained an increased amount of glycyrrhetic acid monoglucuronide, which was secondarily formed from glycyrrhizin through thermal hydrolysis and was known to taste 5 times sweeter than glycyrrhizin.

The genus Glycyrrhiza consists of about 30 species in which G. glabra , G. uralensis , G. inflata , G. aspera , G. korshinskyi , and G. eurycarpa are generally recognized as licorice because of their sweet taste. Except G. korshinskyi , we examined isoprenoid-substituted phenols of these licorices. Each plant could be characterized by some isoprenoid phenols. We also investigated the biological activities of the Glycyrrhiza phenols. In the course of screening phytoestrogen in medicinal plants, six Glycyrrhiza phenols exhibited the binding affinities for the bovine uterine estrogen receptor. The affinity of a dihydrostilbene with two 3-methyl-2-butenyl (prenyl) groups, gancaonin R, was higher than those of isoflavone phytoestrogens (genistein and daidzein) in dietary foods. The affinities of the other five phenols, a flavanone (liquiritigenin), two prenylflavanones (isobavachin and sigmoidin B), a prenylated coumestan (glycyrol), and a pyranoisoflav-3-ene (glabrene), were similar to that of the dietary isoflavone, genistein or daidzein. Cytotoxic activities of the Glycyrrhiza phenols against human oral tumor cell lines and HIV-infected MT-4 cells were also reviewed.

Quantitative structure­activity relationships (QSARs) are developed for two separate families of sweet-tasting molecules for which sweetness values relative to sucrose (RS) have been measured. For these two families of sucrose and guanidine derivatives, the molecules were divided into training and test sets. Linear multiple regression equations have been generated to relate separately log(RS) to two types of parameters, namely molecular descriptors and energies derived via molecular field analysis (MFA). The parameters used in the development of linear multiple regression equations were selected by the genetic algorithm. The equations obtained show high predictive quality, which is confirmed by statistical parameters obtained with the test sets. The data for these two families were then combined with data from two other families previously studied, namely the sulfamates and isovanillates, to make a set of 149 compounds. These molecules were also studied by QSAR methods. The generated equations show remarkable predictive power, and the quality of the results suggest that the mechanism of sweet taste receptor is similar and, therefore, that there could well be only one receptor site for sweet taste, particularly for the four sweet taste families considered in this work.

Sweet taste induction by alkyl 2,3-di- O -(l-aminoacyloxy)-α-d-glucopyranosides requires a combination of hydrophobic α-alkoxy and hydrophilic vicinal, diequatorially oriented, l-aminoacyloxy units. Pyranoside chair conformations afford the preferred stereochemical arrangements of these residues for optimum interaction with the receptor. For the design of new sweeteners based on sweetness inhibitors, the introduction of a di- O -aminoacyloxy unit as the hydrogen-bonding component was applied to effect their intertransformation. Thus, the known sweetness inhibitor, methyl 4,6-dichloro-4,6-dideoxy-α-d-galactopyranoside, was successfully transformed into sweet-tasting 2,3-di- O -(l-aminoacyl) derivatives. The inhibition of the 4,6-dichloro derivative is therefore competitive. Amongst the related amino-chloro-deoxysugars, methyl 6-chloro-6-deoxy-2,3-di- O -(l-alanyl)-α-d-gluco-pyrano side was found to be a full agonist. Our studies were then extended to disaccharide derivatives based on trehalose. This approach led to new highly intense sweeteners, as dimeric forms of the full agonist 2,3,2 ' ,3 ' -tetra- O -(l-alanyl)-6,6 ' -dichloro-6,6 ' -dideoxytrehalose. The derivatives with effective hydrophobic groups on the C-6 and C-6 ' positions, were found to be up to 800­1000 times sweeter than sucrose.

A number of sweeteners contain the sulfur atom and a sulfonyl group. In our current search for new glucophores, several new compounds containing a sulfonyl group were obtained. Synthetic targets have been found both by intuitive modifications of the structural motifs of the known sweet compounds and by the use of molecular modeling techniques. We developed the efficient synthetic routes yielding these compounds and obtained a number of these analogs. We have evaluated the taste quality of the amides and sulfonylated amino acids, finding out that almost all of them are bitter actives. We are now performing basic toxicological tests preceding the systematic taste study within the tetrazoles obtained.

Structure­taste relationship studies were carried out by chemically synthesizing monellin and its analogs. Replacement of the Asp B7 by l-2-aminobutyric acid, Gly and d-Asp resulted in complete loss of sweetness. Replacement of Ile B6 and Ile B8 by different amino acids resulted in a significant decrease of sweetness, or complete loss of sweetness. Comparison of short- and long-range nuclear Overhauser effects (NOEs) and chemical shifts between monellin and [Abu B7 ]monellin showed no marked differences except for the region of the Asp B7 . Thus, the complete loss of sweetness in [Abu B7 ]monellin is caused by the lack of free β-carboxyl group in the Asp B7 and not by a result of major disruption in the overall 3-dimensional structure. These results suggested that the free β-carboxyl group of the Asp B7 would possibly bind to the receptor site through ionic bonding and trigger the sensation of intense sweet taste, and Ile B6 and/or Ile B8 would be involved in the hydrophobic interaction with the receptor site. Selectively labeled monellin was synthesized by the solid-phase method by incorporating 15 N-labeled amino acids into 10 key residues including Asp B7 . Relaxation analysis shows that Asp B7 is the most flexible of these 10 residues. The flexibility of the active site may be important for receptor binding.

Several oligosaccharides such as glycosylsucrose, fructo-oligosaccharides, malto-oligosaccharides, isomalto-oligosaccharides (branched-oligosaccharides), galacto-oligosaccharides, xylo-oligosaccharides, isomaltulose (palatinose), and lactosucrose have been produced on an industrial scale. Recent developments in industrial enzymology have made possible a series of new starch oligosaccharides such as β-1,6 linked gentio-oligosaccharides, α,α-1,1 linked trehalose, α-1,3 linked nigero-oligosaccharides, and branched-cyclodextrins. Some new sweeteners, including trehalose and nigero-oligosaccharides, are being developed as food ingredients with physiologically unique functions such as superoxide dismutase-like activity and immunological activity. Also, soybean oligosaccharides containing raffinose, stachyose, and other oligosaccharides mentioned above are now used in beverages, confectionery, bakery products, yogurts, daily products, and infant milk. In 1991, the Japanese government legislated for Foods for Specified Health Use (FOSHU). FOSHU increased the total to 223 items of which more than 50 % incorporate oligosaccharides as the functional components. Furthermore, the Ministry of Health, Labor, and Welfare published the proposal for a new system named Foods with Health Claims (FHC), which was carried out in April 2002.

Many kinds of oligosaccharides and sugar alcohols have been newly developed as bulking sugar substitutes that have beneficial health effects. A sugar substitute that is not digested and absorbed in the small intestine reaches the large intestine, where it is completely fermented by intestinal bacteria and produces short-chain fatty acids, which are converted to energy. The available energy of a nondigestible sugar substitute, which is completely fermented by intestinal microbes, is estimated as approximately 2 kcal/g. On the other hand, a sufficiently high ingestion of nondigestible sugar substitutes regularly causes overt diarrhea in human and animals. However, the diarrhea disappears within a few days because intestinal microbes, which readily utilize these saccharides, increase during ingestion. The maximum permissible dose, by which transitory diarrhea is not caused by the ingestion of nondigestible sugar substitutes, is estimated as approximately 0.3 g per kg body weight in single ingestion for Japanese adults. The value is varied by some factors, such as separate ingestion, repeat ingestion, test substance, and body condition, etc.

Trehalose is a disaccharide with important functional properties. Although these properties have been recognized for many years, trehalose was not produced on an industrial scale. Recently, a novel enzymatic system for the production of trehalose was developed, and the cost has been dramatically reduced enough to permit widespread application. In the present study, the stabilization mechanism of unsaturated fatty acids by trehalose was found. Furthermore, possible suppressive effects of trehalose on osteoporosis development emerged from our experiments. Trehalose is also expected to be used for various applications in the fields of cosmetics and pharmaceuticals.

This paper reviews the evidence for novel physiological functions of oligosaccharides. A history of the research and development and the types of health benefits are discussed. A key property of the physiological functions lies in the indigestibility of oligosaccharides, which gives rise to fermentation in the large intestine, followed by an increase of Bifidobacteria and short-chain fatty acid (SCFA) production. This property leads to several kinds of physiological functions, classified into three types of health benefits. The first is the improvement of gastrointestinal conditions, including a normal stool frequency, less constipation, and healthy intestinal microflora. The second is the promotion of mineral absorption, including an increase of bone density and relief of anemia. The third is an immunomodulation effect, such as allergy and cancer prevention.

Noncaloric functional beverages are a key segment in the rapidly growing functional food market. Erythritol, an all-natural bulk sweetener, offers sensorial as well as functional benefits for use in such beverages. It can improve taste quality by adding body and mouthfeel, and it is capable of masking unwanted off-flavors often associated with intense sweeteners. In addition, erythritol can strengthen the functional concept since it is noncaloric, noncariogenic, and nonglycemic, and it has antioxidant properties.

Dental caries is a multifactorial disease that is caused by an interplay of three major factors, i.e., teeth, cariogenic bacteria, and fermentable sugars. Streptococcus mutans and S. sobrinus , collectively termed mutans streptococci (MS), are principal causative agents of dental caries. Initial MS-tooth surface attachment is followed by firm and irreversible adhesion of MS to the tooth surface, accompanied by the synthesis of water-insoluble glucan from sucrose via enzymatic action of glucosyltransferases (GTases). MS induce severe dental caries in rats fed on a high-sucrose diet. Epidemiological surveys indicate that frequent sucrose intakes are associated with high prevalence of dental caries in humans. In contrast, dietary sucrose restrictions and/or use of nonfermentable sucrose substitutes clearly influence the GTase activities of MS, resulting in decreased caries development. Structural isomers of sucrose (i.e., disaccharides composed of glucose and fructose with different linkages) will not function as substrates for GTases of MS, nor be utilized as energy sources by MS. Palatinose and trehalulose are included in this category, and are produced in commercial scales in Japan. Glucose oligomers containing α-1, 6 and/or α-1, 4 linkages are found to inhibit glucan synthesis by MS from sucrose, although these oligomers are hydrolyzed by MS to release acids. Lastly, sugar alcohols, including maltitol and palatinit, are useful as noncaries-inducing sweeteners.

In the course of our studies on antidiabetogenic and antidiabetic principles of natural medicines and medicinal foodstuffs, we have isolated salacinol and kotalanol with unique thiosugar sulfonium sulfate inner salt structures from the antidiabetic Ayurvedic traditional medicines, Salacia reticulata and S. oblonga . Salacinol and kotalanol showed potent inhibitory activities against intestinal α-glucosidase, and also inhibitory effects of salacinol on the increase in serum glucose levels in maltose- and sucrose-loaded rats were found to be more potent than those of acarbose. In addition, various flavonoids with potent inhibitory activities against rat lens aldose reductase such as quercitrin desmanthin-1 and guaijaverin were isolated from Myrcia multiflora and several natural medicines, and some structural requirements of flavonoids for aldose reductase inhibitory activity were clarified.

To search for possible cancer-chemopreventive agents from natural resources, several natural sweeteners were screened by the in vitro assay indicated by the inhibitory effects of Epstein-Barr virus early antigen (EBV-EA) induction. Of active compounds that showed the remarkable inhibitory effects on the EBV-EA induction, stevioside, from the leaves of Stevia rebaudiana , and mogroside V, from the fruits of Momordica grosvenori , exhibited significant inhibitory effects on the two-stage mouse skin carcinogenesis in vivo induced by 7,12-dimethylbenz[a]anthracene (DMBA) and 12- O -tetradecanoylphorbol-13-acetate (TPA). The inhibitory effect of stevioside is stronger than that of glycyrrhizin, which had been known as an antitumor-promoter in chemical carcinogenesis. Furthermore, stevioside also inhibited mouse skin carcinogenesis initiated by peroxynitrite. These results suggest that stevioside and mogroside V might be valuable as chemopreventive agents for chemical carcinogenesis.