POTENCY OF SWEETNESS OF ASPARTAME, D-TRYPTOPHAN AND THAUMATIN EVALUATED BY SINGLE VALUE AND TIME-INTENSITY MEASUREMENTS

Perceived sweetness of sucrose, aspartame, D-tryptophan and thaumatin in a sour, citric acid background was analyzed in terms of the potency of these compounds relative to sucrose-water combinations. Potencies of the sweeteners were determined from (1) maximum intensity using single value and time-intensity (T-I) measurements and (2) average intensity calculated as the ratio of area under the T-I curve and total perceived time. Stevens' law was applied to sweet responses, either in static or dynamic conditions. It was found that the exponent of the concentration-response function reflected the relative capacity of a compound to sweeten a given food and stressed differences of potency among sweeteners. Aspartame, D-tryptophan and thaumatin exhibited a decrease in sweetness potency relative to sucrose as sweetness increased from 10 to 100% of the full scale of response. Across the entire sweetness range, thaumatin showed the greatest potency but its long persistence time led to differentiate this intense sweetener from the other sweeteners evaluated.     

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.     

植物甜蛋白Thaumatin研究进展

甜蛋白自 2 0世纪 70年代发现以来 ,一直倍受人们关注 ,而源于自然的Thaumatin是植物甜蛋白中的一种 ,它具有低热量、高甜度、安全无毒 ,并可降解为人体所需的氨基酸等多种优点 ,是一种新型甜味剂。在物质文化生活日益丰富的今天 ,人们越来越重视饮食的科学性 ,吃饱的同时更加关注所摄入食品的品质 ,无疑具多功能的非糖类物质 Thaumatin就是人们所需求的理想食品。因此 ,Thaumatin成为热门研究领域之一也就不足为怪了。1 　植物甜蛋白研究概况迄今为止 ,人们从多种植物中发现并分离出 7种甜味蛋白 [1 ]。更确切地说 ,其中 5种( Thaumatin,…     

Photoaffinity labeling of thaumatin-binding protein in monkey circumvallate papillae

Thaumatin I is an intensely sweet-tasting protein. It was photo-crosslinked with taste papillae of crab-eating monkey by using a conjugated photo-affinity reagent [3H]azidobenzoylthaumatin I. Serial sections of SDS-polyacrylamide gel electrophoresis of the 0.1 M sodium phosphate buffer-soluble fraction from taste papillae had a large peak of radioactivity at the Mr region of approx. 70,000; fractions from non-taste papillae did not. Excess unlabeled thaumatin I reduced the photo-crosslinking at the 70 kDa region; acetylated thaumatin I (which is not sweet) did not. The results show that taste papillae of the monkey contain a protein of Mr approx. 50,000, which binds to thaumatin I (Mr 22,209) but not to completely acetylated thaumatin I. The possibility that the thaumatin-binding protein is a sweet receptor protein is discussed.     

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.     

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.     

Crystal structure of the sweet-tasting protein thaumatin II at 1.27Å

Thaumatin, an intensely sweet-tasting protein, elicits a sweet taste sensation at 50 nM. Here the X-ray crystallographic structure of one of its variants, thaumatin II, was determined at a resolution of 1.27 ?. Overall structure of thaumatin II is similar to thaumatin I, but a slight shift of the Cα atom of G96 in thaumatin II was observed. Furthermore, the side chain of residue 67 in thaumatin II is highly disordered. Since residue 67 is one of two residues critical to the sweetness of thaumatin, the present results suggested that the critical positive charges at positions 67 and 82 are disordered and the flexibility and fluctuation of these side chains would be suitable for interaction of thaumatin molecules with sweet receptors.     

奇甜蛋白基因在园艺作物中的表达

奇甜蛋白（thaumatin）是从非洲西部植物katemfe（Thaumatococcus daniellii Benth）中提取得到的几种关系相近的甜味蛋白的统称,其中最主要的为奇甜蛋白Ⅰ和奇甜蛋白Ⅱ。奇甜蛋白不仅甜度高,而且具有低热量、安全无毒以及不易诱发糖尿病等优点。因此,将奇甜蛋白基因转入园艺作物中并使之表达,用以提高可食部分的甜味,有其特别的研究意义。奇甜蛋白基因已先后在马铃薯、梨树、黄瓜、番茄等园艺作物得到表达,但仍有一些问题需要解决。现从奇甜蛋白基因的克隆、测序与表达,转基因果实的安全性检测,甜度的感官评价,甜味遗传特点以及奇甜蛋白抗真菌病害检验等几个方面综述了国内外研究进展,并对今后的研究提出了建议。     

Crystal structure of the sweet-tasting protein thaumatin II at 1.27 Å

Thaumatin, an intensely sweet-tasting protein, elicits a sweet taste sensation at 50 nM. Here the X-ray crystallographic structure of one of its variants, thaumatin II, was determined at a resolution of 1.27 Å. Overall structure of thaumatin II is similar to thaumatin I, but a slight shift of the Cα atom of G96 in thaumatin II was observed. Furthermore, the side chain of residue 67 in thaumatin II is highly disordered. Since residue 67 is one of two residues critical to the sweetness of thaumatin, the present results suggested that the critical positive charges at positions 67 and 82 are disordered and the flexibility and fluctuation of these side chains would be suitable for interaction of thaumatin molecules with sweet receptors.     

Transgenic tomato plants as supersweet protein thaumatin II producers

Yalf tomato plants have been transformed with a gene for thaumatin II from Thaumatococcus daniellii Benth. The nucleotide sequence for thaumatin II cDNA was cloned in the pBI121 vector under the control of the CaMV 35S promoter of cauliflower mosaic virus. Expression of the thaumatin II gene was detected in all of the studied transgenic lines. A quantitative estimation of the thaumatin II accumulation in fruits was performed by ELISA. The highest content of thaumatin in transgenic tomato fruits (line 91) was 46.4 ± 10.5 μg/mg of total soluble protein (4.6%). In the other studied lines, the thaumatin content ranged from 17.6 ± 6.1 to 41.3 ± 12.3 μg/mg of total soluble protein (1.8–4.1%). The fruits of transgenic plants had a well-defined sweet taste with a long aftertaste typical of thaumatin II. Transgenic tomato lines with high expression levels can be potentially used as producers of thaumatin for the food and pharmaceutical industries.     

Enhancement of the perceived sucrose sweetness in the rat by thaumatin

After conditioned aversion to a 0.18 mol/l sucrose solutionrats do not reject a solution of 0.03 mol sucrose per l (nearthreshold) or a solution of thaumatin (0.2 g/l), which elicitsno response in the rats' taste nerves. However, a mixture ofthe latter two solutions is rejected just as the 0.18 mol/lsucrose solution. This means that for the rat the mixture hasa sucrose-like taste. So, thaumatin enhances sucrose-like sweetnessin this animal. Electrophysiological measurements in the chordatympani indeed show an enhancement of the sucrose response ofalmost 20 percent by the addition of thaumatin (0.2 g/l) toa sucrose solution of 0.5 mol/l.     

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.     

An intensity/time study of the taste of amino acids

An intensity/time study of the taste of selected amino acidswas carried out. Intensity, persistence and total gustatoryresponse were assessed at five concentrations. Ten amino acidswere assessed for sweetness and eleven amino acids were assessedfor bitterness, four amino acids being assessed for both sweetnessand bitterness. Both a linear function and a power function,I = Kcⁿ (where I is taste intensity, c is concentration, K isa constant and n is the exponent of taste intensity), were fittedto the data. The accession efficiencies for taste recognitionand taste detection were found. Kinetic equations were usedto find K_m, the affinity of the receptor site for the sapidmolecule. Limited relationships between chemical structure ofthe amino acids and their temporal properties were found.     

The taste responses in primates to the proteins thaumatin and monellin and their phylogenetic implications.

Electrophysiological and behavioural methods have been applied to 34 species of the primates and, for comparison, to the Madagascan hedgehog to determine their responses to the proteins thaumatin and monellin. These substances elicit an intensely sweet taste sensation in man. All Catarrhina prefer monellin to water. The responses of the Prosimii as well as those of the South American primates to monellin are different, some species show a reaction, other species are not sensitive. In the case of thaumatin neither the Prosimii--including Tupaia and Tarsius--nor the South American primates show any response to this protein. Only the Cercopithecidae, the Hylobatidae and the Pongidae respond to this protein like man and prefer this substance to water. This physiological aspect of taste constitutes a clear dichotomy within the order Primates. This capability to taste thaumatin probably developed as long as 38 million years ago.     

A structure-activity study of thaumatin using pyridoxal 5'-phosphate (PLP) as a probe

The structural features responsible for the sensory propertiesof the sweet protein, thaumatin, have been investigated by sidechain modification of amino acid residues using pyridoxal 5'-phosphate(PLP). PLP molecules bind covalently to proteins by reactingwith the -amino group and the -amino group of lysine residues.Spectral and sensory studies have been performed on thaumatin-PLPderivatives prepared at various molar ratios. The incorporationof one mole of PLP into thaumatin causes substantial modificationof the sensory properties which include generation of astringency,an unpleasant taste and the loss of sweetness intensity. Theintroduction of more than one mole of PLP has no further effecton the gustatory properties of thaumatin. Removal by alkalinephosphatase of the phosphate group of PLP bound to thaumatinhas no influence on the ability of PLP to modify the sensorycharacteristics of thaumatin. This suggests that the sensoryalteration caused by PLP cannot be ascribed to the changes inthe net charge of the protein, but is likely to be due to themodification of specific lysine residue(s) which are thus implicatedin the sweet site.     

Use of the "SMURF" in taste analysis

An improved moving chart recording of intensity/time of tasteresponse has been achieved using a potentiometer ‘dialbox’ linked by a cable to a Telsec recorder. The deviceallows rates of taste response to be determined and is describedas a Sensory Measuring Unit for Recording Flux (SMURF) on theassumption that the flux of stimuli at the taste receptor isresponsible for the time course of response. Fourteen trained and sixteen untrained panellists evaluatedone standard and four unknown sucrose solutions using the SMURFand determined their intensity and persistence time of responsefor each of these same solutions by conventional interval scalingand use of a stop-clock. The SMURF gave results which were higher(but not significantly so) than the conventional method. Trainedpanellists tended to prefer the SMURF and found it quicker andeasier to use than the conventional method. Untrained panelliststended to prefer the conventional method but these results weregenerally not significant. The SMURF is therefore an extremelyuseful device in reducing time and effort whilst still maintainingaccuracy in the measurement of intensity and time of taste response. The SMURF was also used to obtain intensity/time data for threeother sugars so that a comparison between the sugars could bemade.     

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).     

Is multiple sclerosis an infectious disease? Inference about an input process based on the output

The output process of an infinite-server queue with a Poisson process input is observed starting at time 0 with an empty queue. It is assumed that the service time distribution is known. This article discusses statistical inference about the input intensity. A controversial issue in the study of multiple sclerosis is addressed as a motivation for the model and methods developed.     

Oral zinc sulfate solutions inhibit sweet taste perception

We investigated the ability of zinc sulfate (5, 25, 50 mM) to inhibit the sweetness of 12 chemically diverse sweeteners, which were all intensity matched to 300 mM sucrose [800 mM glucose, 475 mM fructose, 3.25 mM aspartame, 3.5 mM saccharin, 12 mM sodium cyclamate, 14 mM acesulfame-K, 1.04 M sorbitol, 0.629 mM sucralose, 0.375 mM neohesperidin dihydrochalcone (NHDC), 1.5 mM stevioside and 0.0163 mM thaumatin]. Zinc sulfate inhibited the sweetness of most compounds in a concentration dependent manner, peaking with 80% inhibition by 50 mM. Curiously, zinc sulfate never inhibited the sweetness of Na-cyclamate. This suggests that Na-cyclamate may access a sweet taste mechanism that is different from the other sweeteners, which were inhibited uniformly (except thaumatin) at every concentration of zinc sulfate. We hypothesize that this set of compounds either accesses a single receptor or multiple receptors that are inhibited equally by zinc sulfate at each concentration.