Kinetic method for rapidly sensing sweetness of sweetener by detection cells

Abstract

The invention provides a kinetic method for rapidly sensing the sweetness of a sweetening agent by detection cells. The method comprises the following steps: S1, determining a fluorescence signal of interaction of the sweetening agent and sweet recipient cells by adopting a staying fluorescence spectrophotometer; a Ca < 2 + > fluorescent probe is added into the sweet recipient cells; s2, performing data processing on the fluorescence signal to obtain an apparent rate constant kobs; s3, based on the apparent rate constant kobs, calculating a receptor isomerization rate constant k3, a receptor deisomerization rate constant k4 and the retention time tau of the sweetening agent in sweet recipient cells; and S4, judging the sensory result of the sweetening agent according to the calculation result of S3. According to the method for analyzing the sweetener activation cell signal by detecting the sweetness based on the stay spectrum, some sensory information of the sweetness can be rapidly obtained, and compared with traditional sensory evaluation, the method is more efficient and more economical.

Description

Technical Field

[0001] This invention relates to the field of food testing technology, and in particular to a kinetic method for detecting the rapid sweetness of sweeteners perceived by cells. Background Technology

[0002] Currently, sensory evaluation is the primary method used to determine the sweetness production process of sweeteners. For example, Bian's sensory experiments on erythritol, sucrose, redibutyrin A, redibutyrin M, and sucralose showed that the rate of sweetness reduction was sucrose > redibutyrin A > erythritol > redibutyrin M > sucralose. Alessandra's sensory experiments on sucrose and several high-intensity sweeteners found no significant difference in the overall time it took for the sweeteners to reach their maximum sweetness in the oral cavity, but they did show significant differences in sweetness intensity and the total time of sweetness perception in the oral cavity. Marcela studied the sensory time intensity of sweeteners in aqueous solutions and yogurt, and the results showed that the sweetness intensity perception time in stevioside aqueous solution was 13 s. MORAIS, through sensory evaluation, determined that the maximum sweetness duration for sucrose, sucralose, aspartame, stevioside, and neotame was 16.73 s, 16.43 s, 17.33 s, 16.33 s, and 17.80 s, respectively. Gwak's sensory study of 13 sweeteners revealed significant differences in duration of action between steviol, aspartame, sucralose, sucrose, fructose, tagatose, xylitol, xylose, erythritol, and maltitol. The duration of action for high-concentration sweeteners was approximately 9-17 seconds, while that for sugars and sugar alcohols was approximately 7-15 seconds. Other methods have also been used to study the interaction between sweeteners and sweet taste receptors. Qin used methods to determine the dose-dependent effect (∆F / F) and the half-maximal effective concentration (EC50) between sweeteners and sweet taste receptors. 50 The intensity of sweetness produced by lactose, sorbitol, xylose, arabinose, allulose, maltitol, xylitol, sucrose, and fructose was evaluated. In the HAO study, the interaction between sweeteners and sweet receptors was determined by radiolabeling hydrogen atoms onto the sweeteners.

[0003] However, sensory evaluation, one of the methods mentioned above, has drawbacks such as being labor-intensive, costly, and having a very long cycle. ∆F / F and EC 50 This method can only evaluate the interaction between sweeteners and sweet taste receptors, but cannot measure the perception of sweeteners and sweet taste receptors. While radioactive hydrogen atom labeling can measure the dynamics of sweeteners and sweet taste receptor cells, this method is expensive, lacks universality, and is environmentally unfriendly. In our previous research, the applicant's group used residence spectroscopy to collect signals generated by sweeteners stimulating sweet taste cells and calculated the sweetening rate (kJ) of different sugars and sugar alcohols. obsThere are differences. However, the results obtained by this study are not sufficient to establish a method to replace the sweet sensory evaluation. Therefore, it is of great significance to find a method for rapid detection of sweetener agonizing sweet signal to replace the traditional sensory evaluation. SUMMARY

[0004] In view of the above-mentioned disadvantages of the prior art, the purpose of the present application is to provide a method for detecting the rapid kinetics of cell perception of sweetener sweetness, which is used to solve the problems of traditional human sensory evaluation of sweetener, such as long period, high cost, and inability to determine the kinetics of sweetener and sweet receptor in an environmentally friendly manner.

[0005] To achieve the above-mentioned purposes and other related purposes, the present application provides a rapid kinetics method for detecting sweetener agonizing sweet signal, comprising: S1: using a stopped fluorescence spectrometer to measure the fluorescence signal of the interaction between sweetener and sweet receptor cells; Ca 2+ fluorescent probe is added to the sweet receptor cells; S2: data processing of the fluorescence signal to obtain apparent rate constant k obs ; S3: based on the apparent rate constant k obs , the receptor isomerization rate constant k3, the receptor de-isomerization rate constant k4 and the retention time τ of the sweetener in the sweet receptor cells are calculated; S4: according to the calculation results of S3, the sensory results of the sweetener are determined: k3 is the rate of sweet perception of the sweetener; k4 is the rate of sweet perception of the sweetener; The retention time τ of the sweetener in the sweet receptor cells is the maximum sweetness duration of the sweetener in the oral cavity; When k3 is greater than k4, it is determined that the rate of sweet perception of the sweetener is faster than the rate of sweet perception of the sweetener; When k3 is equal to k4, it is determined that the rate of sweet perception of the sweetener is equal to the rate of sweet perception of the sweetener; When k3 is less than k4, it is determined that the rate of sweet perception of the sweetener is slower than the rate of sweet perception of the sweetener.

[0006] Based on the stopped spectrum technology, the present application collects the rapid kinetics of sweetener and sweet receptor cells at the level of milliseconds. Through formula fitting, the rising, maintaining and decaying processes of the whole sweet perception process are obtained. The stopped spectrum can be used to measure the apparent rate constant (k obs ) of sweetener activating sweet cell receptor signal at the level of milliseconds, which determines whether the whole process of sweet perception can be revealed.

[0007] The present application is based on the fact that the residence spectrum can collect the signal generated by the sweetener exciting the sweet cell in a very short time (millisecond level), and the apparent rate constant k obs After obtaining this key value, the isomerization rate constant of the sweet cell receptor after the sweetener activates the sweet receptor, the residence time of the sweetener on the receptor and the de-isomerization rate constant can be obtained according to the kinetic theory. In the macroscopic level, this represents the rise, persistence and decline of sweetness. Therefore, the residence spectrum of the present application for determining the signal generated by the sweetener exciting the sweet cell fills the technical gap in the kinetic part, and provides a basis for developing a rapid sweetness perception.

[0008] As a preferred embodiment, in S1, the sweet receptor cell is a HEK-293 cell transfected with a sweet receptor.

[0009] As a preferred embodiment, the concentration of the HEK-293 cell transfected with a sweet receptor is 12000 cells / mL.

[0010] As a preferred embodiment, the Ca 2+ The fluorescent probe is Fluo-4 AM.

[0011] As a preferred embodiment, S1 further comprises a control blank experiment, wherein the control blank experiment comprises: using the residence fluorescence spectrometer to determine the fluorescence signal of the interaction between the sweetener and the blank cell, wherein the blank cell is a parent HEK293 cell which is not transfected with a sweet receptor; and S2 further comprises subtracting the fluorescence signal collected in the control blank experiment before the data processing of the fluorescence signal.

[0012] As a preferred embodiment, in S1, the instrument parameters of the residence fluorescence spectrometer are as follows: the test temperature is 36.5-37.5℃, the dead time is 3 ms, the excitation wavelength is 488 nm, the slit length is 2.5 nm, the 515 nm cutoff filter is used to collect data, 1000 points are used in each experiment, and the kinetic curve within 10 s of reaction is obtained.

[0013] As a preferred embodiment, the method for obtaining the apparent rate constant k obs in S2 is as follows: the Pro-Data software is used to perform kinetic fitting on the fluorescence signal, wherein the interaction process between the sweetener and the sweet receptor cell conforms to the exponential function as shown in formula (1): (1); Wherein, Y is the voltage value in the instrument test process, the unit is V, which represents the fluorescence intensity generated after the interaction between the sweetener and the sweet receptor cell; c is the intercept, A i is the amplitude, k obs is the apparent rate constant, and t is the reaction time.

[0014] As preferred, in S4, the receptor isomerization rate constant k3 and de-isomerization rate constant k4 after the sweetener activates the sweet receptor cell are obtained by fitting formula (2) after the interaction: (2) ; Wherein, K d1 is the first step dissociation equilibrium constant; C is the sweetener concentration, mmol·L -1 .

[0015] As preferred, the retention time τ of the sweetener in the sweet receptor cell is calculated according to formula (3): (3).

[0016] As preferred, the sweetener is selected from one of neotame, sucralose, rebaudioside, aspartame, acesulfame, aspartame and cyclamate.

[0017] As preferred, the sweetener is added to the stopped fluorescence spectrometer in the form of an aqueous solution, and a concentration gradient of the sweetener is set.

[0018] As described above, the present application has the following beneficial effects: Based on the stopped spectrum, the present application can collect the signal generated by the sweetener stimulating the sweet cell in a very short time (millisecond level), and after processing the collected fluorescence signal, the key values are obtained, and the isomerization rate constant of the sweet receptor cell after the sweetener activates the sweet receptor, the retention time of the sweetener on the receptor and the de-isomerization rate constant can be obtained according to the kinetic theory. In the macroscopic level, it shows the rise, duration and decline of sweetness. Therefore, the stopped spectrum of the present application for determining the signal of the sweetener activating the sweet cell fills the technical gap in the kinetic part, and provides a basis for developing a fast sweet perception. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The figure shows the fluorescence signal generated by the interaction of neotame and sweet receptor cells.

[0020] Figure 2 The figure shows the fluorescence signal generated by the interaction of sucralose and sweet receptor cells.

[0021] Figure 3 The figure shows the fluorescence signal generated by the interaction of rebaudioside and sweet receptor cells.

[0022] Figure 4 The figure shows the fluorescence signal generated by the interaction of aspartame and sweet receptor cells.

[0023] Figure 5A plot of the fluorescence signal produced by the interaction of aspartame with sweet receptor cells.

[0024] Figure 6 A plot of the fluorescence signal produced by the interaction of aspartame with sweet receptor cells.

[0025] Figure 7 A plot of k obs vs. concentration data for the six high-potency sweeteners at 37°C.

[0026] Figure 8 A plot of k obs vs. concentration data for the six high-potency sweeteners at 37°C.

[0027] Figure 9 A plot of k obs vs. concentration data for the six high-potency sweeteners at 37°C.

[0028] Figure 10 A plot of k obs vs. concentration data for the six high-potency sweeteners at 37°C.

[0029] Figure 11 A plot of k obs vs. concentration data for the six high-potency sweeteners at 37°C.

[0030] Figure 12 A plot of k obs vs. concentration data for the six high-potency sweeteners at 37°C.

[0031] Figure 13 A plot of k obs vs. concentration data for the six high-potency sweeteners at 37°C.

[0032] Figure 14 A plot of T-I curves for the six high-potency sweeteners at the same sweetness level. DETAILED DESCRIPTION

[0033] The present application is herein described, by way of example only, with the comprehension that the advantages and utility thereof are not limited thereto. It will be clear that the application can be practiced otherwise than as specifically described herein without departing from the spirit and scope of the application.

[0034] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a component" or "the component" is a reference to one or more components and includes the possibilities of one or more components. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The materials, methods, and examples provided herein are illustrative only and in no way limiting. Other embodiments of the present application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein.

[0035] The reagents used in the following examples of the present application are as follows: acesulfame (>99%), cyclamate (>99%) and phosphate buffer (DPBS, containing calcium and magnesium ions) were purchased from Shanghai Aladdin Biochem Technology Co., Ltd. Steviol glycoside (>90%), sucralose (>99.9%) and aspartame (>99%) were purchased from Shanghai McLean Biotechnology Co., Ltd. Fluo-4AM (increased fluorescence intensity when combined Ca 2+ :100-fold) were purchased from Thermo Fisher Scientific Corporation.

[0036] The examples of the present application provide a method for detecting the rapid perception of sweetener sweetness by cells, comprising the following steps: Preparation of high-intensity sweetener samples: Six high-intensity sweeteners, neotame, sucralose, steviol glycoside, aspartame, acesulfame, aspartame and cyclamate, were selected, with the sweetness of sucrose being 1 as a reference, and the same sweetness of 6 kinds of high-intensity sweetener pure solution system was prepared, in turn 3.03 mmol·L -1 , 2.42 mmol·L -1 , 1.94 mmol·L -1 , 1.55 mmol·L -1 , 1.24 mmol·L -1 , 0.99 mmol·L -1 ; the concentration of sucralose was 50 mmol·L -1 , 40 mmol·L -1 , 32 mmol·L -1 , 25.6 mmol·L -1 , 20 mmol·L -1 , 16.4 mmol·L -1 ; the concentration of steviol glycoside was 100 mmol·L -1 , 80 mmol·L -1 , 62.5 mmol·L -1 , 51.2 mmol·L -1 , 41.0 mmol·L -1 , 32.8 mmol·L -1 ; the concentration of acesulfame was 122 mmol·L -1 , 98 mmol·L -1 , 78.1 mmol·L -1 , 62.5 mmol·L -1 , 50 mmol·L -1 , 40 mmol·L -1; aspartame concentrations were 1.56 mmol·L -1 , 1.25 mmol·L -1 , 1.0 mmol·L -1 , 0.8 mmol·L -1 , 0.64 mmol·L -1 , 0.511 mmol·L -1 ; cyclamate concentrations were 750 mmol·L -1 , 600 mmol·L -1 , 480 mmol·L -1 , 384 mmol·L -1 , 307 mmol·L -1 , 245 mmol·L -1 .

[0037] First, the present application will stay the temperature of the spectrum at 37℃, which is in line with the normal temperature of the human body. The signal of the interaction between sweet taste receptor and sweet taste cells is collected by using the spectrum of residence. The concentration of cells is 12000 cells / mL, and finally the concentration of cells after mixing with sweeteners is 6000 cells / mL. The same experiment sets a blank cell group as a control, deducts the signal of the blank group, and ensures that the final signal is the signal of the interaction between sweeteners and sweet taste receptor cells.

[0038] The sweet taste receptor cells in the present application are HEK-293 cells transfected with sweet taste receptors, which are obtained by the following method: By using plasmids (pLVX-Puro) encoding TAS1R2, TAS1R3 and Gα16-gust44, HEK293 cells stably expressing human taste receptor 1 type 2 and 3 (TAS1R2 and TAS1R3) and Gα16-gust44 were generated by slow virus packaging. After transfection, the cells were screened by treatment with neomycin and hygromycin B for 2-3 weeks. HEK-293 cells with stable expression of human TAS1R2, TAS1R3 and Gα16-gust44 are referred to as "sweet cells". The cells were cultured in 25 cm 2 of gas-permeable culture dishes containing 5% South American fetal bovine serum (BSA) (Gibco) in DMEM (1x) + GlutaMAX-I medium (Invitrogen, USA). When the cell confluence reached 90%, 1.5 mL trypsin-EDTA was used for digestion, and after PBS washing, 12.5 mM Ca 2+Fluo-4 AM, incubate for 60 min at room temperature; wash with PBS for three times, resuspend with 5 mL DPBS and adjust the density to 12000 cells / mL, stand for 15 min at room temperature before use. Before the experiment, dilute to 12000 cells / mL with DPBS.

[0039] The control blank experiment in the present application is the parent HEK293 cells without transfection of sweet taste receptor as negative control, called "blank cells", which are cultured under the same conditions as the sweet taste receptor cells as described above, and will not be repeated here.

[0040] After the sweeteners bind to the sweet taste receptor cells on the receptor, due to the different binding sites of the sweeteners, they activate the cell signal from the following two paths respectively. First, after sucrose binds to the sweet taste receptor cells, α-gustducin (a heterotrimeric G protein similar to transducin) is activated, which activates intracellular adenylyl cyclase (AC) to produce cMAP, resulting in an increase in the intracellular cMAP concentration. The phosphorylation of protein kinase A (PKA) closes the K + channel, at the same time, L voltage-sensitive Ca 2+ channels (L-VSCCs) are opened, resulting in the influx of extracellular Ca 2+ and increasing the intracellular free Ca 2+ concentration; second, after artificial sweeteners bind to sweet taste receptor cells, α-gustducin-Gβ3-Gγ13 trimer is activated, releasing gustducin-βγ subunit dimer, activating phospholipase Cβ2 (PLCβ2), and PLCβ2 hydrolyzes phosphatidylinositol diphosphate (PIP2) to inositol triphosphate (IP3) and diacylglycerol (DAG). The increase of IP3 leads to the opening of intracellular IP3-gated Ca 2+ ion channels, Ca 2+ release, transient receptor potential melastatin 5 (TRPM5) activation, and DAG promotes the phosphorylation of PKC and PKD, which closes the K + channel, increases the intracellular K + concentration. Mainly by using Fluo-4 AM to label Ca 2+ , collect signals through residence spectrum.

[0041] The rapid kinetics characterization of sweeteners was determined using the fluorescence of a stopped-flow fluorimeter (SX 20, Applied Photophysics, Surrey, U.K.) to determine the interaction between sweeteners and sweet receptor cells. The instrument dead time was 3 ms. The excitation wavelength was 488 nm, with a slit length of 2.5 nm, and data were collected using a 515 nm cutoff filter. 1000 points were taken for each experiment, giving a kinetic curve over 10 s of reaction. Each experiment was repeated at least three times for each concentration, with 15 instrument repeats for each mix, and the average was taken after fitting. The resulting signals were fitted for kinetics using the Pro-Data software that comes with the instrument. The interaction process between sweet molecules and receptors was fitted to an exponential function: (1) ; where Y is the voltage value during the instrument testing process, in V, representing the fluorescence intensity produced after the interaction between sweeteners and sweet receptor cells; c is the intercept, A i is the amplitude, k obs is the apparent rate constant, and t is the reaction time.

[0042] The present application found that the main binding region of the six high-potency sweeteners is the extracellular VFT region and the transmembrane region. Then according to the research of GCPRs receptor shows that the extracellular flytrap structure region (VFT) oscillates back and forth between the two states of opening and closing, and the sweetener binding induces a change in the conformation of the receptor in the interaction of the sweetener with the living cell sweet receptor. And the research shows that the transmembrane region changes from the TMD 5-6 interface to the TMD 6 interface, and the conformation of the receptor also changes. Therefore, according to the induced conformational kinetics binding step, the process of mutual combination of sweeteners and sweet receptor cells may be the following process: .

[0043] where L is a sweetener; R is a sweet receptor cell; LR is the initial complex formed after the interaction between the sweetener and the sweet receptor cell; LR * is the complex after the sweetener induces a change in the conformation of the sweet receptor cell; k1 is the forward rate of the interaction between the sweetener and the sweet receptor cell, and k2 is the reverse rate of the interaction between the sweetener and the sweet receptor cell. K d1 is the dissociation equilibrium constant k2 / k1 of the first step, and the dissociation constant K d = K d1 x k4 / k3 in the interaction between the sweetener and the sweet receptor cell.

[0044] According to formula (2), k3, k4 and K d1 can be fitted.

[0045] (2) ; where K d1 is the dissociation equilibrium constant of the first step; C is the concentration of the sweetener, mmol L -1 , the rate constant of the receptor isomerization k3and the rate constant of the receptor de-isomerization k4.

[0046] The retention time τ of the sweetener in the sweet receptor cell was calculated according to equation (3): (3).

[0047] Figures 1-6 Figure 6 is a graph of the fluorescence signal of six high-potency sweeteners with sweet cells at 37 °C, with three batches of samples repeated. During the collection of the residence spectrum, one of the sample injectors was filled with a sweetener solution, and the other sample injector was filled with sweet receptor cells or "blank cells". When the instrument started to work, the two injectors injected samples into the detector at the same time. When the Ca 2+ in the sweet cells was activated, the instrument automatically collected the signal. It can be seen that after the sweet receptor cells interacted with the sweetener, the residence spectrum could collect the increase of the fluorescence signal. In the interaction of the sweetener and the blank cells, the signal collected by the residence spectrum decreased. This also shows that the instrument can accurately collect the changes in the cell signal when the sweetener interacts with the sweet receptor cells. Similarly, the signal of the sweetener and the blank cells collected by the present application represents the control blank in the entire experiment. Therefore, the signal of the blank experiment is deducted in the final data processing stage, and the final data obtained is the signal generated by the interaction of the sweetener and the sweet receptor on the sweet cells. Next, the present application analyzes this signal.

[0048] According to the above data, the present application uses equation (1) to fit the signals of the six high-potency sweeteners at the same sweetness, and the apparent rate constant (k obs ) of the interaction of different sweeteners with sweet cells is obtained by fitting, which represents the speed of the sweetener sweetening rate, and the results are shown in Figure 7 : the k obs of the six high-potency sweeteners is cyclamate > aspartame > sucralose > neotame > acesulfame > stevioside; but the increasing speed of k obs is sucralose > aspartame > acesulfame > stevioside > neotame > cyclamate. k obs is a key indicator in the determination of kinetics, because the non-linear fitting of k obs and concentration can obtain the change of the sweet receptor conformation in the process of the binding of the sweetener and the sweet receptor.

[0049] As shown in Figures 8-13As shown, the present application obtained k obs The fitting graph of the concentration data, further calculated the interaction kinetics parameters of the 6 sweeteners and the sweet receptor cells, the results are shown in Table 1: Table 1. Kinetics parameters of 6 high-fold sweeteners at 37℃

[0050] In Table 1, k3 is the receptor isomerization rate constant, k4 is the receptor deisomerization rate constant, K d1 is the dissociation equilibrium constant of the first step; K d is the dissociation constant in the process of sweetener and sweet receptor cell interaction; τ is the residence time of sweetener on the sweet receptor cell; G bind is the binding energy; RSI is the relative sweetness intensity of the sweetener. a,b,c,d,e : Significant difference analysis (P < 0.05).

[0051] As can be seen from Table 1, different sweeteners cause different rates of receptor isomerization (k3), which macroscopically manifests as the speed of sweetness rising. The data obtained from the stopped-flow spectrum is in the range of 0.80-1.91. The rate of sweetness rising of aspartame is the fastest, followed by acesulfame, stevioside and sucralose have no significant difference, followed by neotame, and the k3 of cyclamate is the smallest, which indicates that acesulfame is the slowest in the sweetness rising among the several sweeteners. The greater the rate of inducing receptor conformation, the faster the rate of sweetness perception. The table shows that the degree of de-isomerization of sweet receptor protein (k4) is also different, which macroscopically manifests as the speed of sweetness decline. The de-isomerization rate (k4) of the six high-potency sweeteners ranges from 0.12 to 0.59, with the smallest k4 of stevioside, followed by sucralose, no significant difference between stevioside and sucralose; the third is the dissociation rate constant of acesulfame, which has no significant difference with the dissociation rate constant of sucralose, but there is a significant difference between the dissociation rate of stevioside and acesulfame; then the dissociation rate constant of aspartame, and finally the k4 of neotame and cyclamate is the largest, and there is no significant difference between the two, indicating that neotame and cyclamate have the fastest dissociation rate from the sweet receptor. This also indicates that the sweetness decay of the six high-potency sweeteners is similar, so the weakening of the sweetness of several sugar sweeteners should also be similar. The binding of sweeteners to sweet receptors causes the conformation of sweet receptor protein, and the greater the residence time (τ) of sweeteners on the receptor, the longer the isomerization time of the receptor, which should macroscopically manifest as the duration of sweetness. Table 1 shows that among the six sweeteners, the residence time of stevioside on the receptor is 6.8 s, sucralose is 5.44 s, acesulfame is 3.8 s, aspartame is 2.7 s, cyclamate is 2.1 s, and neotame is 1.7 s. Therefore, the sweetness duration of the six sweeteners calculated by the present application should be in the range of 1-7 s. And the present application found that in the data of the six high-potency sweeteners, the de-conformation rate is smaller than the conformation rate constant of the receptor protein, which indicates that the sweetness rising time of the six sweeteners is less than the sweetness decline time. Table 1 shows the dissociation equilibrium constant of the six high-potency sweeteners, except that the dissociation equilibrium constant of cyclamate is large and the affinity is small, the other five sweeteners have no significant difference, but there is a trend of aspartame < sucralose < neotame < stevioside < acesulfame < cyclamate. The greater the equilibrium dissociation constant indicates the smaller the affinity of the sweetener to the sweet receptor, and vice versa. The present application found that except aspartame, the affinity changes of the other five high-potency sweeteners are almost consistent with their sweetness changes, so the present application speculates that the stronger the sweetness of the sweetener, the greater the affinity between the sweetener and the sweet receptor. The change trend of the free energy of the combination of the five different sweeteners with the receptor is the same as the change trend of their sweetness, so the present application speculates that there may be a higher sweetness, a lower binding free energy, a more stable complex, and a stronger sweetness.In general, the reason for the difference in sweetness of the six high-intensity sweeteners is that the sweeteners cause the sweet cell receptors to have different rates of isomerization and de-isomerization, resulting in different degrees of conformational change in the sweet receptors in response to sweetness, and thus different lengths of time spent on the receptors, which determines the difference in sweetness of the sweeteners. Thus, the data collected by these methods can provide key data on the rate of onset of sweetness, the rate of rise of sweetness, the duration of sweetness, and the rate of decline of sweetness for different sweeteners.

[0052] In traditional experiments, the rate of onset of sweetness, the rate of rise of sweetness, the duration of sweetness, and the rate of decline of sweetness for a sweetener can only be obtained through sensory experiments. Thus, the present application greatly reduces the cost, manpower, and time required for experiments by using the method of collecting signals by residence spectroscopy, and can more efficiently obtain the process of activation of sweet signals.

[0053] To verify the consistency of the results of the method of measuring the rapid kinetics of sweetener activation of sweet signals based on residence spectroscopy and the results of traditional sensory evaluation experiments, the present application next performed sensory evaluation experiments on six high-intensity sweeteners as a comparative example.

[0054] The comparative example used six high-intensity sweeteners identical to those of the examples, namely, neotame with a purity of 99%, acesulfame potassium, cyclamate, aspartame, stevioside with a purity of 90% (Jiangsu Vido Co., Ltd., Jiangsu, China), sucralose (Anhui Jinhe Industry Co., Ltd., Anhui, China), and sucrose (Angel Yeast Co., Ltd., Hubei, China). Considering the sensitivity of the trained evaluators, the comparative example used a 10 cm linear scale, and selected pure water as 0 (not felt), a 0.75% sucrose solution as 5 (felt moderately warm), and a 1.5% sucrose solution as 10 (felt very strong). The sweetness equivalent concentrations of the sweeteners were determined accordingly.

[0055] In the experiments, 10 trained evaluators scored a series of concentrations of each sweetener, and selected a concentration of sucralose of 0.013‰ in the range. The evaluation process was as follows: the evaluator held the sample in the mouth for 5 s, and then continued to evaluate for 5 s after spitting it out, for a total evaluation time of 10 s. Next, the concentrations of the other five sweeteners that were not significantly different from sucralose were selected, and the concentrations were as follows: neotame at 0.017 , stevioside at 0.071‰, acesulfame potassium at 0.061‰, aspartame at 0.046‰, and cyclamate at 0.38‰.

[0056] All samples were dissolved in purified water before use. Each sample was 15 mL, and 6 samples were placed in plastic cups with numbers 1-6 written on them. To clean the mouth, fresh lemonade and purified water were provided as taste cleansers. All subjects used lemonade and purified water to rinse their mouths between tasting each sample and the samples, and the amount of use of the two cleansers was kept relatively consistent.

[0057] Dynamic sensory experiment procedure: TI test was performed using Fizz Network software with the following settings: initial waiting time 3 s; sample flow time in the mouth 5 s; post-spit duration 90 s; at the start of the evaluation, the maximum sweetness was set, and then the evaluator recorded the perceived intensity according to the instructions using the mouse software, and the evaluation was repeated three times. The TI curve of different sweeteners was obtained by sensory analysis software, including: IMAX (maximum observed intensity), TStart (test start time), Tend (test end time), SIMInc (slope of first reaching maximum observed intensity), SlMDec (slope of first maximum observed intensity decrease), TSPI (time of first reaching maximum observed intensity), TEPl (maximum observed intensity end time), DurPl (maximum observed intensity duration), DurInc (duration of first rising to maximum observed intensity), DurDec (duration of first maximum observed intensity decrease), etc.

[0058] The results of the three repetitions of each sample during sensory evaluation are shown in Table 1 and Table 2: Figure 14 Table 2. Sensory evaluation parameters of 6 high-fold sweeteners at 37°C

[0059] ​In Table 2, Imax (maximum intensity observed for the curve) is the maximum observed intensity of the curve; TStart (computed start time) is the start time, and Tend (computed end time) is the end time; SlMInc (maximum slope measured in the increasing phase) is the maximum slope measured in the ascending phase; SlMDec (maximum slope measured in the decreasing phase) is the maximum slope measured in the descending phase; TSPl (plateau start time) is the plateau start time, and TEPl (plateau end time) is the plateau end time; DurPl (duration of the plateau) is the plateau duration; DurInc (duration of the increasing phase) is the ascending duration; and DurDec (duration of the decreasing phase) is the descending duration. a: Significant difference analysis (P<0.05).

[0060] Sensory data results of 6 high-intensity sweeteners are as follows Figure 14 As shown, from Figure 14 The data in Table 2 show that the sensory sweetening rate of the six high-intensity sweeteners is very rapid. The data indicate that their sweetening time points (TStart) are within 1 second, in the order of cyclamate > aspartame > sucralose > neotame > steviol glycoside > acesulfame potassium. This aligns with our previous analysis of the kinetics. obsThe order of the data of the six high-potency sweeteners is highly similar. The process from the beginning to the highest point of sweetness is also very fast. The rate of the rise of the sensory (SlMInc) is sucralose > aspartame > neotame > acesulfame > rebaudioside > thaumatin, but there is no significant difference in the range of 0.91-4.13, which is highly consistent with the range of 0.80-1.91 of the residence assay. The time of the six high-potency sweeteners to remain at the highest point (DurPI) is 3-8 s, neotame is 7.5 s, aspartame is 6.7 s, sucralose is 3.6 s, rebaudioside is 4.3 s, acesulfame is 5.7 s, and thaumatin is 6.1 s. The residence time obtained by the residence assay is 1-7 s, and the data of the two are highly similar. The dissociation rate constant of the six high-potency sweeteners (SIMDec) falls in a range of approximately 0.14-0.85, sucralose > aspartame > acesulfame > rebaudioside > neotame > thaumatin, but there is no significant difference between the dissociation rates of several high-potency sweeteners. This may be because the human mouth is less sensitive to similar sweetness, so it is difficult to distinguish. This is similar to the residence data, which is more sensitive to data collection, so it can better distinguish the significance. Because there is no significant difference between the dissociation rates, the order of the dissociation time of the six high-potency sweeteners (DurDec) is sucralose > aspartame > acesulfame > rebaudioside > neotame > thaumatin. The results obtained by the residence fall in the range of 0.11-0.58, and the dissociation rate results obtained by the two experimental analyses are highly cross. The time of the sensory experiment (TEnd) is 90 s, and the human sensory needs a reaction time to determine the sweetness, and the residence is relatively fast to collect the reaction signal. The sensory evaluation of the high-potency sweetener is actually 90 s and cannot completely be without sweetness, so this may cause a large error in the data, and the residence measurement has a relatively small error. At the same time, the sensory data shows that the difference between the dissociation time and the binding time of the six sweeteners is approximately one order of magnitude, and the residence also measures that the difference between the two is approximately one order of magnitude. This shows that the sensory evaluation is completely consistent with the sweetener kinetic parameters mentioned above. The data above shows that the data measured by the residence is highly consistent with the sensory data.

[0061] In summary, the method for detecting sweet taste and analyzing sweetener activation of cell signals based on the residence spectrum can obtain some information on the sweet sensory more quickly, which is more efficient and more economical than the traditional sensory evaluation. This provides a new method and technology for quickly perceiving sweetness. Therefore, the present application effectively overcomes the various shortcomings in the prior art and has a high industrial utilization value.

[0062] The above embodiments are only illustrative of the principles of the present application and its efficacy, and are not intended to limit the present application. Any modification or change made by any person skilled in the art without departing from the spirit and scope of the present application shall be covered by the claims of the present application.

Claims

1. A method of detecting the kinetics of rapid gustatory perception of sweetener sweetness by cells, comprising, The method comprises the following steps: S1: using a stopped fluorescence spectrometer to measure the fluorescence signal of the interaction between the sweetener and the sweet receptor cell; The sweet receptor cell is added with Ca 2+ Fluorescent probe; S2: data processing on the fluorescence signal to obtain apparent rate constant k obs ; S3: apparent rate constant k obs , the rate constant of receptor isomerization k 3 , the rate constant of receptor de-isomerization k 4 and the retention time τ of the sweetener in the sweet receptor cell; S4: determining the sensory result of the sweetener according to the calculation result of S3: k 3 the rate of rise of sweet sensation for a sweetener; k 4 the rate of sweet taste perception reduction for the sweetener; The retention time τ of the sweetener in the sweet receptor cell is the maximum sweetness duration of the sweetener in the oral cavity; When k 3 greater than k 4 determining that the rate of rise in the perception of sweetness is faster than the rate of fall in the perception of sweetness. When k 3 is equal to k 4 determines that the rate of rise of the sweetness perception of the sweetener is equal to the rate of fall of the sweetness perception. When k 3 Less than k 4 The rate of increase in the sweetness perception of the sweetener is determined to be slower than the rate of decrease in the sweetness perception.

2. The method of claim 1, wherein the cell rapidly senses the sweetness of the sweetener. In S1, the sweet receptor cell is a HEK-293 cell transfected with a sweet receptor.

3. The method of claim 2, wherein the cell rapidly senses the sweetness of the sweetener. The concentration of the HEK-293 cell transfected with the sweet receptor is 12000 cells / mL.

4. The method of claim 1, wherein the cell rapidly senses the sweetness of the sweetener. The Ca 2+ The fluorescent probe was Fluo-4 AM.

5. The method of claim 1, wherein the method is for detecting the kinetics of rapid gustatory perception of sweetener sweetness by cells. In S1, a control blank experiment is further included, which comprises: using a stopped fluorescence spectrometer to measure the fluorescence signal of the interaction between the sweetener and a blank cell, wherein the blank cell is a parent HEK293 cell which is not transfected with a sweet receptor; and before the data processing of the fluorescence signal in S2, the fluorescence signal collected in the control blank experiment is further deducted.

6. The method of claim 1, wherein the method is for detecting the kinetics of rapid gustatory perception of sweetener sweetness by cells. In S1: the instrument parameters of the stopped fluorescence spectrometer are as follows: the test temperature is 36.5-37.5℃, the dead time is 3 ms, the excitation wavelength is 488 nm, the slit length is 2.5 nm, the 515 nm cutoff filter is used to collect data, 1000 points are used in each experiment, and the kinetic curve within 10 s of reaction is obtained.

7. The method of claim 1, wherein the cell rapidly senses the sweetness of the sweetener. Method for obtaining apparent rate constant in S2 k obs The method is: using Pro-Data software to carry out kinetic fitting on the fluorescence signal, wherein the interaction process of the sweetener and the sweet taste receptor cell conforms to the exponential function as shown in formula (1): （1）； wherein Y is the voltage value during the instrument test process, unit is V, representing the fluorescence intensity generated after the sweetener interacts with the sweet receptor cells; c is the intercept, A i is the amplitude, k obs is the apparent rate constant, and t is the reaction time.

8. The method of claim 1, wherein the cell rapidly senses the sweetness of the sweetener. In S4, the receptor isomerization rate constant is fitted by formula (2) k 3 and de-isomerization rate constant k 4 : （2）； wherein, K d1 is the first step dissociation equilibrium constant; C is the sweetener concentration, mmol L -1 .

9. The method of claim 8, wherein the cell rapidly senses the kinetics of sweetness of the sweetener. The retention time τ of the sweetener in the sweet receptor cell is calculated according to formula (3): （3）。 10. The method of claim 1, wherein the method is for detecting the kinetics of rapid gustatory perception of sweetener sweetness by cells. The sweetener is selected from one of neotame, sucralose, rebaudioside, aspartame, acesulfame, aspartame and cyclamate; the sweetener is added to the stopped fluorescence spectrometer in the form of an aqueous solution, and the concentration gradient of the sweetener is set.

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