Inhibitor-assisted cellular level sweetness quantitative determination method

Abstract

The invention discloses an inhibitor-assisted cellular level sweetness quantitative determination method, and belongs to the technical field of molecular biology. A large number of experiments find that in the process of detecting the sweetness at the cellular level, cell stress caused by operation steps of exogenously adding a probe and the like can interfere with the sweetness detection result and influence the accurate quantification of the sweetness detection result. According to the invention, an inhibitor group containing a sweetness receptor inhibitor is arranged in the detection process so as to eliminate the change of the concentration of calcium ions in cells caused by cell stress and further cause a false positive result in the detection process, so that the aim of more accurately and quantitatively detecting the sweetness of the to-be-detected compound is fulfilled. Furthermore, the detection method disclosed by the invention can be used for various sweet feeling cells, is simple and convenient to operate, and can effectively reduce the false positive rate of a final detection result.

Description

TECHNICAL FIELD

[0001] The present application relates to an inhibitor-assisted cell level sweetness quantitative determination method, belonging to the technical field of molecular biology. BACKGROUND

[0002] Taste sense is one of the important physiological senses of human body, which largely determines the choice of animals on diet, so that they can supplement the nutrients beneficial to survival in time according to their own needs, and plays an important role in feeding regulation, body nutrition and metabolism. The taste receptor is taste bud, which is mainly distributed on the surface of tongue and lingual margin, and is also scattered on the surface of oral and pharyngeal mucosa. The taste bud is composed of taste cells, which express taste receptors and can detect and distinguish various tastes.

[0003] The taste system can sense and distinguish a variety of tastes, which are currently believed to be composed of five basic tastes of acid, sweet, bitter, salty and umami. Among them, sweet taste is a taste like sugar and honey, which symbolizes a good feeling in many cultures around the world, and is the most popular taste for humans. Sweet compounds are diverse, which can be divided into natural sweet substances and artificial sweet substances. Natural sweet substances include some natural sugars, sugar alcohols, proteins and amino acids; artificial sweet substances include some sulfonamide lipids, dipeptides, halogenated sugars and sulfonyl amides. These sweet compounds can activate the sweet taste receptor and the sweet taste perception pathway to produce sweet taste, so the sweet taste receptor is a key link to sense sweet taste. The sweet taste receptor belongs to the member of the C class GPCR family, which forms a heterodimer protein complex (T1R2 / T1R3) with two subunits T1R2 and T1R3. The activated sweet taste receptor can regulate the intracellular calcium ion concentration through two pathways of GPCR-Gs-cNMP and GPCR-Gα / Gβγ-IP3, thereby changing the cell membrane potential and releasing neurotransmitters to transmit sweet taste information.

[0004] Sweetness is in a sense the evaluation index of human sensory to food taste, simply speaking, it represents the sweetness degree of compound or food. Since sweetness can be used to improve the palatability and certain edible properties of food (such as sweeteners), the detection of compound sweetness is crucial. There are various methods for sweetness determination, mainly divided into analytical chemistry detection method, biological detection method and cell level detection. Among them, the analytical chemistry detection method mainly includes high performance liquid chromatography, electronic tongue detection method, sweetness meter detection method, spectrophotometer detection method and the like, which can accurately measure the content of sugar, but cannot quantitatively analyze the physiological level of sweetness. The biological detection method is to find a sample, control the variable, and compare the quantitative value by professional sweetness evaluators, including descriptive analysis, scale and difference test. Although the quantitative results of this method have value, they are highly subjective and have poor accuracy in quantifying sweetness. Therefore, it is urgent to establish a new method that can accurately quantify the sweetness at the physiological level.

[0005] The Chinese invention patent with publication date of April 6, 2021 and publication number CN109142298B discloses a method for quantitatively determining sweetness at the cell level. By constructing a cell line expressing T1R2 and T1R3 sweet receptors, and using calcium ion imaging technology, the perception intensity of sweet substances can be simulated and quantified at the cell level. This method can quantitatively determine the perception degree of sweetness of unknown samples in vitro, effectively avoiding the influence of human subjective factors. However, the molecules and reaction networks in cells are very complex, and may be disturbed by various factors during the quantitative detection of sweetness. It is unknown whether the interference will affect the stress response of cells and thus affect the final quantitative detection results. SUMMARY

[0006] The purpose of the present application is to provide an inhibitor-assisted cell level sweetness quantitative determination method, and to provide a cell level sweetness quantitative determination method with more accurate detection results.

[0007] In order to achieve the above purpose, the technical scheme adopted by the inhibitor-assisted cell level sweetness quantitative determination method in the present application is as follows:

[0008] An inhibitor-assisted cell level sweetness quantitative determination method, comprising the following steps:

[0009] (1) Set up an inhibitor group in which a sweet receptor inhibitor and a calcium ion fluorescent probe are co-incubated with sweet receptor cells, and a non-inhibitor group in which a calcium ion fluorescent probe is incubated with sweet receptor cells, add different concentrations of standard sugars to each group respectively, and continuously detect the fluorescence, determine the fluorescence intensity characteristic value under each standard sugar concentration according to the fluorescence intensity change of each treatment group to exclude the interference caused by cell stress;

[0010] (2) constructing a correlation between the fluorescence intensity characteristic value and the standard sugar concentration in step (1) ;

[0011] (3) determining the fluorescence intensity characteristic value of the sample to be tested according to step (1), and obtaining the sweetness of the sample to be tested relative to the standard sugar according to the correlation in step (2).

[0012] The above-mentioned scheme has the beneficial effect that the present application is an improved method for inhibitor-assisted cell-level sweetness quantitative determination. Through a large number of experiments, the present application finds that during the detection of sweetness at the cell level, the cell stress caused by the exogenous addition of probes and other operation steps will interfere with the detection results of sweetness and affect the accurate quantification of the sweetness detection results. The present application sets an inhibitor group containing a sweet receptor inhibitor in the detection process to eliminate the changes in intracellular calcium ion concentration caused by cell stress and the false positive results caused by the detection process, so as to achieve the purpose of more accurate sweetness quantitative detection of the test compound.

[0013] Further, the detection method of the present application can be used for a variety of sweet taste sensing cells, and the detection method is simple to operate and can effectively reduce the false positive rate of the final detection results.

[0014] As a further improvement, the fluorescence intensity characteristic value is the difference between the maximum fluorescence intensity change value of the non-inhibitor group and the maximum fluorescence intensity change value of the inhibitor group.

[0015] As a further improvement, the fluorescence intensity change value = (F t -F0), wherein F t is the average fluorescence intensity value of the sweet taste sensing cell at a certain detection time point, and F0 is the average fluorescence intensity value of the sweet taste sensing cell at the initial time point.

[0016] As a further improvement, the continuous fluorescence detection is recording the fluorescence intensity every 5-7 seconds, a total of 12-15 times.

[0017] As a further improvement, the sweet taste sensing cell is a cell transiently or stably co-expressing T1R2 and T1R3.

[0018] As a further improvement, the sweet receptor inhibitor is lactitol.

[0019] As a further improvement, the concentration of the lactitol is 5-10 mM.

[0020] As a further improvement, the calcium ion fluorescence probe is Calbryte 520 AM.

[0021] As a further improvement, the concentration of the Calbryte 520AM is 2-3 μM.

[0022] As a further improvement, the incubation time of step (1) is 25-30 min. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The characterization results of the transient cells and the stable cells in Example 1 of the present application (wherein A is the detection result of WB, and B is the detection result of fluorescent quantitative PCR);

[0024] Figure 2 The morphological changes of the cells incubated with different concentrations of inhibitors in Example 2 of the present application;

[0025] Figure 3 The fluorescence intensity change graph of the sweet taste receptor cells before and after adding the inhibitors in Example 3 of the present application;

[0026] Figure 4 The fluorescence intensity change curve of the experimental groups and the control groups at different time points in Example 4 of the present application;

[0027] Figure 5 The correlation between the different sucrose concentrations and the fluorescence intensity change difference in Example 4 of the present application;

[0028] Figure 6 The fluorescence graph of the sweet taste receptor cells stimulated by 4 μM thaumatin at different time points in Example 5 of the present application;

[0029] Figure 7 The fluorescence graph of the sweet taste receptor cells stimulated by 0.5 μM advantame at different time points in Example 5 of the present application;

[0030] Figure 8 The fluorescence graph of the sweet taste receptor cells stimulated by 1.4 μM neotame at different time points in Example 5 of the present application. DETAILED DESCRIPTION

[0031] The prior art does not consider the possible cell stress reaction in the cell detection process when detecting the sweetness of a compound at the cell level. However, the present application proves through experiments that when cells are used to detect sweetness, the intracellular calcium ion concentration changes when the cells are stressed, which easily causes false positive results. Based on this, the present application provides an inhibitor-assisted cell-level sweetness quantitative determination method.

[0032] The present application will be further described in detail below in conjunction with specific embodiments. Unless otherwise specified, the equipment and reagents used in each embodiment, experimental example and comparative example can be obtained from commercial channels.

[0033] The experimental methods in the following examples are conventional methods, such as those described in Sambrook et al. (Sambrook J & Russell DW. Molecular cloning: a laboratory manual. 2001) or the instructions provided by the product manufacturers, unless otherwise specified.

[0034] An embodiment of the present application is a method for determining the sweetness of a cell at the level of an inhibitor:

[0035] Example 1 Characterization of stable cells of transient cells

[0036] In this example, HEK-293 cells transiently co-expressing T1R2 and T1R3 receptors and stably co-expressing T1R2 and T1R3 receptors were detected by fluorescent quantitative PCR and WB, and the specific implementation steps are as follows:

[0037] 1. Construction of transient cells and stable cells

[0038] T1R2 (as shown in SEQ ID NO. 1) and T1R3 genes (as shown in SEQ ID NO. 2) were artificially synthesized by gene synthesis, and the gene fragments were inserted into plasmid vectors (pcDNA3.1 and pEGFP-C1 plasmid vectors) by double enzyme digestion reaction (enzyme digestion sites were BamH I and Xho I, respectively). After transformation and monoclonal screening, T1R2 and T1R3 recombinant vectors were obtained. The constructed recombinant plasmids pcDNA3.1-T1R2 and pEGFP-C1-T1R3 were added to HEK293 cells, and transiently transfected sweet taste receptor cells were obtained by transient transfection. The cells were treated with G418 for two weeks, and then single clone cells were selected. Stable sweet taste receptor cells were obtained by fluorescent quantitative PCR and WB detection.

[0039] 2. Fluorescent quantitative PCR and WB detection

[0040] The mRNA in the cells was detected and quantitatively analyzed by using specific primers for T1R2 and T1R3 (T1R2: Forward primer: 5'-TCCAGCCGGTGCTCTACTT-3' (shown in SEQ ID NO. 3); Reverse primer: 5'-CCACCACACGGGAAATGTAGT-3' (shown in SEQ ID NO. 4); T1R3: Forward primer: 5'-CCGCCTACTGCAACTACACG-3' (shown in SEQ ID NO. 5); Reverse primer: 5'-CTAGCACCGTAGCTGACCTG-3' (shown in SEQ ID NO. 6)), and the sweet receptor protein expression in the monoclonal cells was detected by using specific antibodies for T1R2 and T1R3 (Anti-T1R3 antibody (abcam, ab229015); TAS1R2 Polyclonal antibody (proteintech, 29344-1-AP)). The specific detection results are shown in Table 1. Figure 1

[0041] As shown in Table 1B, after transfection, the relative expression amount of T1R2 and T1R3 mRNA of both the HEK-293 cells transiently transfected with the sweet receptor and the HEK-293 cells stably transfected with the sweet receptor was significantly increased (p<0.05). Figure 1 As shown in Table 1A, after transfection, the relative expression amount of T1R2 and T1R3 protein of both the HEK-293 cells transiently transfected with the sweet receptor and the HEK-293 cells stably transfected with the sweet receptor was significantly increased, which was consistent with the result of real-time quantitative PCR. The above indicated that the cell line expressing the recombinant human sweet receptor was successfully constructed in the embodiment, which can be used for subsequent cell level sweetness quantitative determination. Figure 1

[0042] Example 2 Optimization of the amount of inhibitor

[0043] In order to ensure that the amount of inhibitor can inhibit the activity of sweet receptor to the maximum extent without significantly affecting the survival of cells, the amount of the inhibitor lactisole was optimized by using the HEK293 cells transiently transfected constructed in Example 1, and the specific implementation operation was as follows:

[0044] ​​HEK293 cells (HEK293 cells transiently transfected with recombinant plasmids pcDNA3.1-T1R2 and pEGFP-C1-T1R3 obtained in Example 1) were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S) in a cell incubator at 37°C, 5% CO2 and 95% humidity. 30000 transiently transfected cells were seeded into a 96-well plate one day in advance and incubated in the cell incubator for 24h. The cells were washed once with PBS solution, and different concentrations of inhibitors (diluted with culture medium) were added to each well, a total of 100μL, and incubated in the cell incubator at 37°C for 30min.

[0045] The morphology of the cells was observed under a microscope, and the survival rate of the cells was detected using a CCK8 kit. The cells were seeded and the inhibitors were incubated as described above. After incubation, 10μL of CCK-8 solution was added to each well, and the 96-well plate was placed in the incubator for 1h. The absorbance of each well at 450nm was measured using a microplate reader, and the cell survival rate was calculated. The results are shown in Figure 2 As shown in Table 1, there was no obvious change in cell morphology and the cell survival rate remained above 90% at 5-10mM, so 5-10mM inhibitors were selected for subsequent experiments. In order to further inhibit the activity of sweet receptors, 10mM inhibitors were preferred for subsequent experiments.

[0046] Table 1 Cell survival rate after adding different concentrations of inhibitors

[0047]

[0048] Example 3 Change in fluorescence of sweet taste receptor cells before and after inhibitor treatment

[0049] The addition of fluorescent probes and other substances during detection may cause cell stress, and the stress response of cells causes changes in intracellular calcium ion concentration, which may cause false positive results. In order to verify whether it will affect the results, this example was detected, and the specific implementation operation is as follows:

[0050] HEK293 cells (HEK293 cells transiently transfected with recombinant plasmids pcDNA3.1-T1R2 and pEGFP-C1-T1R3 obtained in Example 1) were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S) in a cell incubator at 37°C, 5% CO2 and 95% humidity. 30000 transiently transfected cells were seeded into a 96-well plate one day in advance and incubated in the cell incubator for 24 hours. The cells were washed once with 100 μL of PBS solution, 2 μM of Calbryte520 AM calcium ion fluorescent probe and 10 mM of inhibitor lactisole were added to each well, a total of 100 μL, and incubated in the cell incubator at 37°C for 30 minutes. The plate was taken out, the calcium ion probe solution was discarded, and the cells were washed once with PBS. 30 μL of PBS solution was added and incubated at room temperature for 20 minutes until the AM ester in the cells was completely hydrolyzed. The staining effect was observed under a fluorescence microscope (Ex / Em = 490 / 525 nm), and a field of view with good cell morphology and uniform dispersion was photographed. The liquid in the well was removed, and an initial image was taken. 30 μL of the sweetener solution to be tested was added to stimulate the cells, and the fluorescence state of the cells was continuously observed. An image was taken every 5 seconds. The specific results are shown in Figure 2 (this figure is the time point with the strongest fluorescence intensity). Figure 3

[0051] As can be seen from the figure, when sucrose is directly used without adding an inhibitor, the fluorescence intensity of the cells is found to increase. When the same concentration of sucrose is used after adding an inhibitor, the fluorescence intensity of the cells is found to change slightly. This fully demonstrates that the increase in fluorescence intensity of the cells is mainly due to sucrose stimulation, and the inhibitor can inhibit the sweet taste of the cells.

[0052] Example 4 Construction of standard curve

[0053] In this example, the correlation between sucrose concentration and fluorescence intensity change was constructed, and the specific implementation operation was as follows:

[0054] 1. Cell culture:

[0055] Sweet taste receptor cells (HEK293 cells transiently transfected with recombinant plasmids pcDNA3.1-T1R2 and pEGFP-C1-T1R3 obtained in Example 1) were inoculated into a 96-well plate, and the number of cells inoculated in each well was 30000. After 48 hours of incubation in the incubator, they were ready for use.

[0056] 2. Co-incubation of inhibitor and fluorescent probe:

[0057] ​HEK293 cells (HEK293 cells transiently transfected with recombinant plasmids pcDNA3.1-T1R2 and pEGFP-C1-T1R3 obtained in Example 1) were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S) in a cell culture incubator at 37°C, 5% CO2, and 95% humidity. 30,000 transiently or stably transfected cells were seeded into a 96-well plate one day in advance and cultured in an incubator for 24 hours. The cells were washed once with 100 μL of PBS solution, and 2 μM Calbryte520AM calcium ion fluorescent probe and 10 mM inhibitor lactitol (Lactisole) were added to each well for a total of 100 μL and incubated in a cell culture incubator at 37°C for 30 minutes. The well plate was removed, the staining solution was discarded, the cells were washed once with PBS, 30 μL of PBS solution was added, and incubated at room temperature for 20 minutes until the intracellular AM ester was completely hydrolyzed.

[0058] 3. Fluorescence detection:

[0059] Before detection, the liquid in the wells was removed and 30 μL of sucrose solution of different concentrations (0, 2, 5, 10, 20, 30, 50, 100, 200 mM) was added. The changes in cell fluorescence intensity were continuously observed under a fluorescence microscope, and the fluorescence intensity was recorded every 5 s for a total of 180 s.

[0060] 4. Data processing:

[0061] ImageJ software was used to process the fluorescence images, with time as the horizontal axis and the fluorescence intensity change value as the vertical axis, to obtain the fluorescence intensity change curve of the experimental group and the control group cells under different concentrations of sucrose stimulation over time, as shown in the figure. Figure 4 As shown in the figure, △F=F-F0; F t is the average fluorescence intensity value of sweet taste receptor cells at different time points, and the average fluorescence intensity value of sweet taste receptor cells at the initial time point F0.

[0062] As can be seen from the figure, without adding inhibitors, the cell fluorescence intensity increased significantly, and after adding inhibitors, the cell fluorescence intensity did not change significantly.

[0063] Under the same sucrose concentration, the fluorescence value of the highest point of the fluorescence intensity of the experimental group was subtracted from the fluorescence value of the highest point of the fluorescence intensity of the control group, and the difference was used as the vertical axis. The standard curve was established with different sucrose concentrations as the horizontal axis, such as Figure 5 As shown in the figure, △F max =ΔF / F0 (the highest point of fluorescence value in the group without inhibitor) - ΔF / F0 (the highest point of fluorescence value in the group with inhibitor).

[0064] It can be seen from the figure that in the range of 0-20 mM sucrose concentration, the sucrose concentration and the change value of fluorescence intensity are linearly related, and the standard curve can be used to accurately quantify the sweetness intensity and sweetness value of different sweeteners within a certain range.

[0065] Example 5 Detection of actual samples

[0066] This example uses the standard curve constructed in Example 4 to quantitatively detect the sweetness of thaumatin, advantame and neotame, and the specific implementation operation is as follows:

[0067] 1. Thaumatin

[0068] (1) Cell culture:

[0069] The sweet taste receptor cells (transient transfection) were inoculated into a 96-well plate at a cell number of 30,000 per well, and cultured in an incubator for 48 hours before use.

[0070] (2) Co-incubation of inhibitors and fluorescent probes:

[0071] The sweet taste receptor cells in the experimental and control groups were removed from the culture medium and washed with 100 μL of PBS buffer. Among them, the sweet taste receptor cells in the experimental group were added with 2 μM calcium ion fluorescent probe (model: Calbryte 520AM, manufacturer: AATBioquest) for incubation at room temperature for 30 min in the dark, and then the probe solution was removed and the cells were washed once with PBS, and then 30 μL of PBS was added and the cells were placed in the PBS solution for detection. The control group cells were operated similarly, and 10 mM of the inhibitor lactisole was added at the same time as the calcium ion fluorescent probe.

[0072] (3) Fluorescence detection:

[0073] Before detection, remove the PBS in the hole, and add 4 μM of thaumatin solution, and continuously observe the change of cell fluorescence intensity under the fluorescence microscope, and record once every 5 s.

[0074] (4) Data processing:

[0075] The change of cell fluorescence intensity was continuously observed under the fluorescence microscope, and recorded once every 5 s. The detection results are as follows: Figure 6The fluorescence intensity values of each cell at different time points were counted, the average fluorescence intensity was calculated and brought into the sweetness quantification standard curve, and it was calculated that the sweetness value of 4 μM thaumatin at the cell level was approximately equal to 18.78 mM sucrose, so it was deduced that the sweetness of thaumatin was 4694 times that of sucrose, which was close to the reported value. If the inhibitor was not added during the detection, the sweetness was directly calculated, and the sweetness value of 4 μM thaumatin was approximately equal to 22.72 mM sucrose, so it was deduced that the sweetness of thaumatin was 5680 times that of sucrose, which was quite different from the reported value.

[0076] 2, Advantame

[0077] The cells were stimulated with 0.5 μM Advantame, and the detection process was as shown in Example 5 thaumatin detection. The change in fluorescence intensity of the cells was continuously observed under a fluorescence microscope, and was recorded every 5 s. The detection results are shown in Table 2. Figure 7 The fluorescence intensity values of each cell at different time points were counted, the average fluorescence intensity was calculated and brought into the sweetness quantification standard curve, and it was calculated that the sweetness value of 4 μM thaumatin at the cell level was approximately equal to 18.78 mM sucrose, so it was deduced that the sweetness of thaumatin was 4694 times that of sucrose, which was close to the reported value. If the inhibitor was not added during the detection, the sweetness was directly calculated, and the sweetness value of 4 μM thaumatin was approximately equal to 22.72 mM sucrose, so it was deduced that the sweetness of thaumatin was 5680 times that of sucrose, which was quite different from the reported value.

[0078] 3, Neotame

[0079] The cells were stimulated with 1.4 μM neotame, and the detection process was as shown in Example 5 thaumatin detection. The change in fluorescence intensity of the cells was continuously observed under a fluorescence microscope, and was recorded every 5 s. The detection results are shown in Table 3. Figure 8 The fluorescence intensity values of each cell at different time points were counted, the average fluorescence intensity was calculated and brought into the sweetness quantification standard curve, and it was calculated that the sweetness value of 4 μM thaumatin at the cell level was approximately equal to 18.78 mM sucrose, so it was deduced that the sweetness of thaumatin was 4694 times that of sucrose, which was close to the reported value. If the inhibitor was not added during the detection, the sweetness was directly calculated, and the sweetness value of 4 μM thaumatin was approximately equal to 22.72 mM sucrose, so it was deduced that the sweetness of thaumatin was 5680 times that of sucrose, which was quite different from the reported value.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can still be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. An inhibitor-assisted cell-level sweetness quantification method, characterized by: The method comprises the following steps: (1) setting an inhibitor group for co-incubating a sweet receptor inhibitor and a calcium ion fluorescent probe with sweet receptor cells and a non-inhibitor group for incubating a calcium ion fluorescent probe with sweet receptor cells, adding different concentrations of standard sugars respectively, continuously detecting fluorescence, determining fluorescence intensity characteristic values at different concentrations of standard sugars according to the fluorescence intensity changes of different treatment groups to exclude interference caused by cell stress; (2) constructing a correlation between the fluorescence intensity characteristic values and the concentrations of standard sugars according to step (1); (3) determining the fluorescence intensity characteristic values of the sample to be tested according to step (1), and obtaining the sweetness of the sample to be tested relative to standard sugars in combination with the correlation of step (2).

2. The inhibitor-assisted cell-level sweetness quantification assay method according to claim 1, characterized in that: The fluorescence intensity characteristic value is the difference between the maximum fluorescence intensity change value of the non-inhibitor group and the maximum fluorescence intensity change value of the inhibitor group.

3. The inhibitor-assisted cell-level sweetness quantification assay method of claim 2, wherein: The fluorescence intensity change value = (F t - F0), wherein F t is the average fluorescence intensity value of the sweet taste receptor cell at a certain detection time point, and F0is the average fluorescence intensity value of the sweet taste receptor cell at an initial time point.

4. The inhibitor-assisted cell-level sweetness quantification assay method according to any one of claims 1 to 3, characterized in that: The continuous fluorescence detection is recording the fluorescence intensity once every 5-7 seconds, and a total of 12-15 times.

5. The inhibitor-assisted cell-level sweetness quantification assay method of claim 4, wherein: The sweet receptor cells are cells transiently or stably co-expressing T1R2 and T1R3.

6. The inhibitor-assisted cell-level sweetness quantification assay method according to any one of claims 1 to 3, characterized in that: The sweet receptor inhibitor is lactitol.

7. The inhibitor-assisted cell-level sweetness quantification assay method of claim 6, wherein: The concentration of the lactitol is 5-10 mM.

8. The inhibitor-assisted cell-level sweetness quantification assay method of claim 7, wherein: The calcium ion fluorescent probe is Calbryte 520AM.

9. The inhibitor-assisted cell-level sweetness quantification assay method of claim 8, wherein: The concentration of the Calbryte 520AM is 2-3 μM.

10. The inhibitor-assisted cell-level sweetness quantification assay method of claim 9, wherein: The incubation time of step (1) is 25-30 min.

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