## 4 Mechanism for Synergism in Mixtures of Sweeteners  
Why can combinations of two or more sweeteners exert synergism? It is well known that sweet taste perception is mediated by a class C G protein-coupled receptor (GPCR)--the heterodimer T1R2 (taste type 1 receptor 2)/T1R3 (taste type 1 receptor 3) [42; 43]. Accordingly, this question certainly leads to elucidating the sweet taste elicitation process in which the sweeteners in mixtures cooperatively interact with and then activate the human sweet taste receptor T1R2/T1R3 [44; 45; 46]. However, due to the lack of structural information on the receptor, mechanical insights into the interplay between sweeteners and receptor are still elusive [47]. Nevertheless, molecular simulations and functional mutagenesis/chimera analysis can provide meaningful guidance for understanding the sweetener-receptor interaction as well as sweeteners' synergism [48; 49; 50]. In principle, different sweeteners have different weights, shapes and configurations, which definitely affect their specific interactions with the sweet taste receptor. In this regard, compared with the separate sweeteners, the mixing of sweeteners can result in a change or modification of their contact properties with the receptor, thus leading to activation of the receptor and eliciting their distinct sweetness [51]. Indeed, many previous studies have revealed that there are multiple binding sites in the sweet taste receptor for various sweeteners [47; 48]. For example, the artificial high-intensity sweeteners aspartate, neotame, saccharin and acesulfame K interact with the VFTM (Venus flytrap module) of human T1R2, while natural sugars (e.g., glucose and sucrose) and sucralose bind to the \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Sweetener 1** & **Sweetener 2** & **Concentration of 1** & **Concentration of 2** &
\begin{tabular}{c} **Evaluation** \\ **Method** \\ \end{tabular} & **References** \\ \hline Acesulfame-K & Sucrose & 1.0/4.0/6.0 mM & 0.1/0.25/0.5 M & II & [20] \\ Acesulfame-K & Glucose & 1.0/4.0/6.0 mM & 0.39/0.88/1.69 M & II & [20] \\ Acesulfame-K & Fructose & 1.0/4.0/6.0 mM & 0.15/0.39/0.74 M & II & [20] \\ Acesulfame-K & Xylitol & 1.0/4.0/6.0 mM & 0.2/0.6/1.0 M & II & [20] \\ Acesulfame-K & Aspartame & 1.0/4.0/6.0 mM & 0.7/2.7/4.9 mM & II & [20] \\ Acesulfame-K & Aspartame & Equal sweetness to 4%, 6% or 8% sucrose 4 & Same as sweetener 1 & III & [22] \\ Acesulfame-K & NHDC & Equal sweetness to 4%, 6% or 8% sucrose & Same as sweetener 1 & III & [22] \\ Acesulfame-K & Rebaudioside-A & Equal sweetness to 4%, 6% or 8% sucrose & Same as sweetener 1 & III & [22] \\ NHDC & Sucralose & Equal sweetness to 4%, 6% or 8% sucrose & Same as sweetener 1 & III & [22] \\ NHDC & Thaumat & Equal sweetness to 4%, 6% or 8% sucrose & Same as sweetener 1 & III & [22] \\ NHDC & Rebaudioside-A & Equal sweetness to 4%, 6% or 8% sucrose & Same as sweetener 1 & III & [22] \\ \hline \hline \end{tabular}  
* Each sweetener with specific concentrations in the mixtures displayed a sweetness intensity equal to that of 3% or 5% sucrose (\(w/w\)), respectively. 2 NHDC, rebaubergidin dihydrohalome. 3 Three molar concentrations divided by “/” were applied for each sweetener in the mixtures, respectively. 4 Each sweetener with specific concentrations in the mixtures displayed a sweetness intensity equal to that of 4%, 6% or 8% sucrose (\(w/w\)), respectively.  
\end{table} 
Table 1: \(Cont.\)VFTMs of both T1R2 and T1R3 [52; 53; 54]. However, cyclamate and NHDC act on the TMD (transmembrane domain) of human T1R3 [55; 56]. The sweet-tasting protein thaumatin was proposed to bind into the CRD (cysteine rich domain) of T1R3 [47; 57]. We have demonstrated that the intense sweeteners perillartine and saccharin (at high concentrations above 3 mM) can bind to the TMD of human T1R2 (Figure 1) [58; 59; 60]. According to these findings, it was postulated that the different sweeteners in the mixture could bind at different sites in the receptor to concertedly increase the efficiency of receptor activation, thus displaying synergism.