Molecular Determinants of the Bittersweet Janus Head of Steviol

Chapter 15

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Molecular Determinants of the Bittersweet Janus Head of Steviol Glycosides from Stevia rebaudiana (Bert.) Bertoni C. Dawid,1 C. Well,1 A. Brockhoff,2 F. Stähler,2 W. Meyerhof,2 and T. Hofmann*,1 1Chair of Food Chemistry and Molecular Sensory Science, Technische Universität München, Lise-Meitner-Strasse 34, 85354 Freising, Germany 2Department of Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke (DIfE), Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany *E-mail: [email protected].

To identify the structural requirements for the organoleptic properties of steviol glycosides from Stevia rebaudiana, we correlated in vivo data obtained from human psychophysical experiments with in vitro data from cell-based taste receptor assays. While sensory evaluation demonstrated the structural features causing the sweet and bitter taste of these sweeteners, screening experiments with the 25 human bitter taste receptors revealed hTAS2R4 and hTAS2R14 to be the general sensors for bitter taste elicited by steviol glycosides. These results help to navigate breeding of Stevia rebaudiana and improve postharvest downstream processing toward the production of preferentially sweet and least bitter tasting Stevia extracts.

Introduction Due to a high number of undesirable health effects such as obesity, dental caries, type-2 diabetes or cardiovascular diseases and its risk factors which were associated with increasing sucrose consumptions (1–7), a new field of food research has emerged since the last decades - namely the investigation of low-calorie sweeteners. In this context, artificial sweeteners such as saccharine, © 2015 American Chemical Society In The Chemical Sensory Informatics of Food: Measurement, Analysis, Integration; Guthrie, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


aspartame, acesulfame K, sucralose or neotame found widespread use in food industry in the U.S.A. as well as in the European Union (8, 9). Since consumers were more and more aware of what ingredients go into their foods, special emphasis was given by producers to find taste active compounds of natural sources featuring functional, nutritional, dietary and tasty properties. Particularly, following the worldwide consumer demand for both non-nutritive high-potency and natural sweeteners, with no organoleptic drawbacks compared to sucrose, steviol glycosides the sweet principle of Stevia rebaudiana (Bert.) Bertoni, have recently approved as food additive in the EU (10). The leaves of Stevia rebaudiana, the so-called “sweet herb”, which are very popular, have a very long history as natural sweetener as they were already used by the native population in South America to sweeten and to mask the bitter off-taste of herbal teas (11, 12). Flavor research performed within the last years has shown that diterpenic ent-kaurene glycosides, all of which share steviol (1) as the common aglycone, are the sweet principle of Stevia (cf. Figure 1). Although, stevioside (2) was reported to be 210 to 300 times sweetener than sucrose, depending on the sensory protocol, especially, rebaudioside A (3) has the reputation for being the most potent sweetener with the most pleasant taste profile (13, 14). However, the data published on systematic and comparative sensory analysis of the purified individual steviol glycosides are rather fragmentary. Apart from their attractive sweetness the taste profile of steviol glycosides is hindered by a bitter off-taste and an unpleasant lingering aftertaste which is often the reason for consumer reactions and therefore a major problem for food makers (13, 15). Although several studies showed evidence that steviol glycosides play an important role in inducing the bitter taste of Stevia rebaudiana, it is still unclear which key structural requirements of the molecules do contribute to the overall bitterness of this non-nutritive sweetener. In general, sweet and bitter belong to the five basic taste modalities and are mediated by G protein-coupled receptors (GPCRs) expressed by taste receptor cells (bitter: hT2Rs and sweet: hT1R2/hT1R3) (16). In the last decade, functional expression studies have successfully enabled the identification of a broad range of cognate agonists for most of the 25 hT2R bitter taste receptors as well as for the heteromeric sweet taste receptor hTAS1R2/hTAS1R3 [e.g.: (17–23)]. Although, at atomic resolution the whole structure has still not been resolved, it is assumed that both sweet taste receptor subunits possess a large amino-terminal ectodomain, including a venus-flytrap binding domain that likely contains the orthosteric binding side for several sweet tasting activators like stevioside (23, 24). The objectives of the present work were, therefore, to investigate the structural requirements for sweet and bitter taste activities of the most important key steviol glycosides. Next to the characterisation of their psychophysical functions by means of different sensory experiments, the responses of the sweet taste receptor (hTAS1R2/hTAS1R3) to the most abundant steviol glycosides should be verified by functional expression studies in human embryonic kidney (HEK)-cells. In addition, to compare human psychophysical data with those obtained from cell-based taste receptor assays, the hTAS2 bitter taste receptors responding to the non-nutritive sweeteners ought to be identified by means of a similar functional receptor assay. 198 In The Chemical Sensory Informatics of Food: Measurement, Analysis, Integration; Guthrie, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Figure 1. Chemical structure of the aglycone steviol (1), steviol glycosides 2-11 and 16,17-dihydrostevioside (12).

Experimental The following compounds were obtained commercially: sucralose (Merck, Darmstadt, Germany), colchicine, and aristolochic acid (Sigma-Aldrich, Steinheim, Germany). Lactisole was provided by Cargill (Minneapolis, U.S.A.). Dihydrostevioside (2H-Stev, 13), rebaudioside B (4) and steviolbioside (9) were synthesized as described before (25–27). While stevioside (2), rebaudioside A (3), rebaudioside C (5), rebaudioside D (6), and dulcoside A (10) were isolated and purified from commercial Stevia extracts (Cargill, Minneapolis, U.S.A.), rubusoside (11) was generated from a commercial extract of Rubus suavissimus (MedHerbs, Wiesbaden, Germany) following literature procedures (26–30). Prior to the psychophysical experiments and cell-culture assays, spectroscopic data and the purity (>98%) of each individual steviol derivatives 2−6 and 9−12 were 199 In The Chemical Sensory Informatics of Food: Measurement, Analysis, Integration; Guthrie, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

checked by means of 1H/13C NMR, LC-MS/MS, and LC-TOF-MS experiments. Thereby, spectroscopic data were in good agreement with those published in the literature. All psychophysical and functional expression experiments were performed as reported previously (27).


Results and Discussion Aimed at characterizing the alluring sweetness next to the undesirable bitter off-taste of steviol glycosides from Stevia rebaudiana data from human psychophysical experiments were combined with functional expressions of TAS1 sweet taste and TAS2 bitter taste receptors. Therefore, first, the individual steviol glycosides 2−6 and 9−11 and 12 were isolated or synthesized and their purities were confirmed by means of NMR, LC-MS and LC-TOF-MS experiments.

Human Sensory Studies on Steviol Glycosides To characterize the sensory activity of the selected chemosensates 2−6 and 9−12 their human sweet and bitter recognition thresholds were determined by means of triangle tests. While the human threshold concentrations determined for sweetness ranged from 5.3 to 32.9 µmol/L, the oral threshold concentration for bitterness ranged from 23 to 194 µmol/L and, was always above the recognition threshold determined for sweetness as given in Figure 2. Moreover, among the steviol glycosides, the lowest threshold concentration for sweetness was found for rebaudioside D (6), bearing the most β-glucose residues (5 β-glucose residues), followed by rebaudioside A (3, 4 residues) and stevioside (2, 3 residues), the highest thresholds were observed for the two rhamnose bearing glycosides no. 5 and 10, followed by the least hydrophilic glycosides no. 11 and 9. In general, the amount of glucose moieties influences the sweet recognition values significantly. For example, while 2 and 4, both bearing three β-glucose moieties, showed no significant differences in their threshold concentrations, the threshold of 4 significantly differed from that of compound 3, bearing four glucose moieties. In addition, the chemosensates decorated with rhamnose moieties induced higher sweet taste threshold concentrations. Besides the glycone chain length and pyranose substitution, additionally the exocyclic double bond plays an essential role for the orosensory impression of the highly appreciated low-calorie sweeteners. Hydrogenation of the double bond at position C(16), as found in 12, resulted in a significant increase of the threshold from 11.1 (1) to 28.1 µmol/L (12). Unlike their sweet recognition values, the bitter threshold concentrations of 2−6 and 9−12 could not be correlated to the amounts of β-glucose moieties, linked to the aglycone. But, interestingly, steviol glycosides evaluated with the highest sweet threshold values, exhibited the lowest bitter thresholds and, depending on their chemical structure, these showed low recognition thresholds between 49 and 200 In The Chemical Sensory Informatics of Food: Measurement, Analysis, Integration; Guthrie, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


84 µmol/L (5, 9-11). Thereby, the threshold values of Dulc A (10) and Reb C (5), both containing rhamnose residues, are only ~1.5 times higher compared to their sweet thresholds. Moreover, the lowest bitter threshold concentration was determined for compound 12, the hydrogenated analogon of stevioside (1). In conclusion, saturation of the double bond in compound 12 compared to 1 induced an archaic increase in bitterness and is a prerequisite for its bitterness.

Figure 2. Correlation of taste threshold concentrations (bars) measured for sweetness (A) (error bars indicate standard deviation) and bitterness (B) and the amount of β-glycosylic residues (○) of different steviol glycosides and of 12. The structures of the individual compounds are given in Figure 1.

Sweet Taste Receptor Responses to Steviol Glycosides In order to assess the structure/activity relationships of steviol glycosides on the human sweet taste receptor, functional experiments were carried out using the human embryonic kidney cell line HEK293 expressing the human sweet taste receptor subunits hTAS1R2 and hTAS1R3 and the chimeric G protein subunit Gα15Gαi3 following the protocol reported recently (23, 27). Thereby, the functional sweet taste receptor heteromer is implemented by stable expression of the subunit hTAS1R2, and inducible expression of the second subunit, hTAS1R3, through a tetracycline-responsive element (27, 31, 32). Especially, the G protein subunit couples the sweet taste receptor to the release of calcium from intracellular stores that can be monitored by means of a calcium-indicator fluorescent dye (cf. Figure 3). 201 In The Chemical Sensory Informatics of Food: Measurement, Analysis, Integration; Guthrie, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Figure 3. Scheme of the functionally expressed human sweet taste receptor hTAS1R2/hTAS1R3 assay according to Behrens et al., 2011.

Using this strategy, we observed that cells expressing hTAS1R2/hTAS1R3 responded with a transient increase of calcium fluorescence to application of all tested steviol glycosides (2−6 and 9−12). Being well in agreement with the findings of the psychophysical experiments, the onset of responses from the sweet receptor-expressing cells were in the same range as the sensory data observed in vivo (cf. Figure 2 and Table 1). For example, substance no. 6 was found to be the most sweet potent steviol glycoside in vitro as well as in vivo (threshold concentration in vivo: 5.3 µmol/L; in vitro: 2.2 µmol/L). Intriguingly, also in the cell assay steviol glycosides, exhibiting a high number of β-glycosyl residues, such as Reb A (3) and Reb D (6), revealed the lowest threshold concentrations, while high threshold values could be observed for stevia compounds which contain rhamnose residues (cf. Figure 2 and Table 1). Therefore, the recently identified Rebaudiside M, bearing six β-glucose moieties, could be a highly promising stevia sweetener (33). 202 In The Chemical Sensory Informatics of Food: Measurement, Analysis, Integration; Guthrie, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Table 1. Threshold Concentration for Activation of Bitter Receptors TAS2R4 and TAS2R14 and the TAS1R2/TAS1R3 Sweet Taste Receptor by Steviol Glycosides Threshold concentration1 (µmol/L) in cells expressing


Compound2

TAS1R2/TAS1R3

TAS2R4

TAS2R14

Stev (2)

4.3

200

600

RebA (3)

4.3

200

600

RebB (4)

12.9

200

1000

RebC (5)

38.8

400

400

RebD (6)

2.2

n.s.3

n.s.3

Stbs (9)

12.9

400

n.s.4

Dulc A (10)

38.8

200

50

Rub (11)

25.9

50

400

2H-Stev (12)

38.8

n.d.

n.d.

The structures of the individual compounds are given in Figure 1. Threshold concentration is defined as the lowest concentration which was used and led to a cellular response which is significant higher than that obtained by applying buffer solutions to the cell. 3 No response to the test compound up to the maximal soluble concentration of 400 µmol/L. 4 No response to the test compound up to the maximal soluble concentration of 800 µmol/L. n.d. Not determined due to receptor-independent fluorescence signal in control cells. 1

2

Although most of the sweet receptor responses to the steviol glycosides were rather similar to the data observed in the psycophysical experiments, there are noteworthy differences in potency between compound no. 2 and its hydrated derivative 12, which are much more pronounced in vitro than in vivo and which are apparent by the 9-fold increased threshold value of 12 compared to 2.

Identification of the hTAS2 Bitter Taste Receptors Responding to Bitter Steviol Glycosides To analyze the bitter off-taste of steviol glycosides in more detail, we selected stevioside (2) as a representative to identify the responding bitter taste receptors (cf. Figure 4). Therefore, we used HEK293T Gα16gust44 cells, which transiently expressed each of the 25 hTAS2Rs individually. As already described for the sweet receptor assay also the activation of hTAS2R receptors was coupled to the release of Ca2+ from intracellular stores, which could be measured using a calciumsensitive fluorescence dye (27). Intriguingly, two of the 25 bitter taste receptors were activated by compound no. 2, namely hTAS2R4 and hTAS2R14 (cf. Figure 4). 203 In The Chemical Sensory Informatics of Food: Measurement, Analysis, Integration; Guthrie, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Figure 4. Calcium responses of HEK-293T-Gα16gust44 cells expressing one of the 25 hTAS2Rs or empty vector (mock, M) elicited by bath application of stevioside (2) (1mM). Arrows point to positive fluorescence signals in TAS2R4and TAS2R14-expressing cells. As positive control calcium responses of hTAS2R46 to 10 µmol/L strychnine were recorded. Due to their structural similarities, we conclude that these two bitter receptors are selective sensors for all steviol glycosides. In order to compare the potency of the steviol glycosides, the cells expressing hTAS2R4 and hTAS2R14, respectively, were challenged with the tastants 2−6 and 9−12 and their threshold concentration were determined. Therefore, increasing concentrations (up to 1.2 mmol/L) of each compound were tested in our cell-assay and the lowest concentration leading to a significant fluorescence signal was determined. All steviol glycosides that have been used in this assay were capable of activating hTAS2R4 and/or the hTAS2R14 (cf. Table 1), except of rebaudioside D (6). Due to its limited solubility, rebaudioside D was measured at a lower concentration compared to other steviol glycosides. Comparison of the threshold data revealed, that the two bitter receptors showed neither the same rank order of potency nor were equally sensitive to the steviol glycosides. But among the test compounds, lower threshold concentrations were found for the activation of hTAS2R4, than for that of hTAS2R14. Interestingly, hTAS2R4 seems to be more sensitive to short short-chained and rhamnose-containing steviol glycosides since comparatively low threshold concentrations were observed for 10, 5 and 11 (cf. Table 1). In addition, the high threshold values for Reb A and Reb B indicated that hTAS2R14 is less sensitive to steviol glycosides with a high number of β-glycosyl moieties. In sum, the bitter taste thresholds determined in vitro were well in line with those observed in vivo.

Conclusion Based on the findings of the present study, we conclude that, glycone chain length, pyranose substitution, and the C16 double bond play an essential role for the orosensory impression of the highly appreciated low-calorie steviol 204 In The Chemical Sensory Informatics of Food: Measurement, Analysis, Integration; Guthrie, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

glycosides (2-11). Sensory and cell-based studies revealed and characterized the sweet profiles of individual steviol glycosides. For the first time, comprehensive screening experiments with the 25 human bitter taste receptors demonstrated that two members, hTAS2R4 and hTAS2R14, act as general sensors for the bitter off-taste elicited by steviol glycosides. These results might help to develop commercially available preferentially sweet and least bitter tasting Stevia extracts.

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Molecular Determinants of the Bittersweet Janus Head of Steviol

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