Journal of Agricultural and Food Chemistry signals to aspartame (Figure 2B,C). In contrast, BCECF did not produce nonspeciﬁc signals at the same concentrations in the aequorin assay (Figure 2D F). This result indicates not only that the luminescence assay is useful for ﬂuorescent ligands but also that BCECF is an ideal model ﬂuorescent substance. Riboﬂavin was selected as an example of a ﬂuorescent sub- stance that is commonly contained in food materials because it is known to possess ﬂuorescent characteristics and is contained at high levels in various kinds of food, including milk, meat, ﬁsh, and leafy green vegetables.36 When we measured the sweet taste signals developed in response to the addition of riboﬂavin at concentrations relevant to food-derived samples (0.25 1.0 μg/mL; e.g., leafy vegetables contain several μg/g edible portion), the response mediated by the sweet taste receptor could be detected using the mitochondrial clytin-II lumines- cence-based assay. We concluded that riboﬂavin did not modulate activation of the sweet taste receptor within the range tested (Figure 5E G). However, we were unable to evaluate this phenomenon using the conventional ﬂuores- cence-based assay system (Figure 5B D). Recently, artiﬁcial chemical compounds that have sweet taste- enhancing eﬀects have been identiﬁed using both cell-based assays and human taste tests.37 This result suggests that cell- based assays could function together with the human sensory test as a taste evaluation system. In our data, the EC50 values of aspartame, saccharin, and sucrose were measured as 1.0, 0.3, and 104 mM, respectively (Figure 5G and Figure S1 in the Support- ing Information). This result is well correlated with the data from human taste tests showing that aspartame and saccharin were about 100 200 and 200 400 times as sweet as sucrose, respectively.32 Furthermore, cell-based assays are very useful not only for screening taste modulators but also for analyzing the mechanism of taste modulation.38 41 In this study, we used the luminescence-based assay only for evaluating the sweet taste receptor, but the assay can also be applied to evaluate other taste GPCRs, such as the umami and bitter taste receptors, and ion channels, such as TRPV1, which senses hot taste.23 Many classes of taste modulators are likely to be contained in food materials, and this novel taste evaluation system would be a powerful tool to elucidate these food-derived modulators even at an industrial level. Further investigation is in progress to characterize new taste modulators in foods. ’ ASSOCIATED CONTENT bS Supporting Information. Figure of the dose response curves to sweeteners using the luminescence assay. This material is available free of charge via the Internet at http://pubs.acs.org. ’ AUTHOR INFORMATION Corresponding Author *Tel: +81-3-5841-8100. Fax: +81-3-5841-8118. E-mail: amisaka@ mail.ecc.u-tokyo.ac.jp. Funding Sources The work was partially supported by the Funding Program for Next Generation World-Leading Researchers from the Japan Society for the Promotion of Science (LS037 to T.M.) and by a grant (E0111501) from the Korea Food Research Institute (to T.M.). ARTICLE ’ ACKNOWLEDGMENT We thank Dr. Keiko Abe (The University of Tokyo) for critically reading the manuscript. ’ ABBREVIATIONS USED AUC, area under the curve; BCECF, 20,70-bis-(2-carboxyethyl)- 5-(and-6)-carboxyﬂuorescein; mt-, mitochondrial; ΔRFU, Δ relative ﬂuorescence units; RLU, relative light units ’ REFERENCES (1) Chandrashekar, J.; Hoon, M. A.; Ryba, N. J.; Zuker, C. S. The receptors and cells for mammalian taste. 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