Method for identifying the effect of aroma substances on sweet perception based on sweet receptor cells
Patent Information
- Application Number
- CN202610693937.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-05-20
- Publication Date
- 2026-08-18
AI Technical Summary
[0004]然而，现有技术存在明显局限：首先，构建稳定表达双受体的细胞系过程繁琐、耗时长，且常面临细胞活力下降、受体表达量低、对甜味刺激响应弱等问题；其次，传统检测方法多依赖荧光探针长时间预孵育，背景噪声高，批次重复性差
[0024]1. 客观定量，克服主观偏差：本发明基于分子细胞水平的信号检测，完全避免了人工感官评价的主观性，能够提供精确、可重现的定量数据，直接反映香气物质对甜味受体通路的生物学效应。
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Figure CN122591627A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of sensory science and molecular biology, and in particular to a method for identifying the influence of aroma substances on sweetness perception based on sweetness receptor cells. Background Technology
[0002] With the global incidence of obesity, diabetes, and other metabolic diseases continuing to rise, public health agencies are strongly promoting sugar reduction initiatives in food and beverages. However, simply reducing sugar content often results in unpleasant tastes, making products difficult for consumers to accept. While artificial sweeteners provide sweetness, they are often accompanied by unpleasant flavors such as bitterness or metallic taste, and also raise certain health concerns. Therefore, developing "natural sugar reduction" technologies that can effectively enhance the perception of sweetness without increasing the amount of sugar or artificial sweeteners has become a critical issue that the food industry urgently needs to address.
[0003] The molecular basis of sweet taste perception lies in the sweet taste receptors expressed on the surface of taste bud cells on the tongue. These receptors belong to class C G protein-coupled receptors (GPCRs) and are heterodimers (T1R2 / T1R3) formed by the T1R2 and T1R3 subunits. After a sweet substance binds to the receptor, it causes an increase in intracellular calcium ion concentration via downstream signaling pathways, thereby generating nerve signals that transmit the sensation of sweetness. Based on this principle, using cell lines that stably express human T1R2 / T1R3 receptors (such as HEK293 cells) to detect calcium signals has become an objective method for evaluating the intensity of sweeteners in vitro.
[0004] However, existing technologies have significant limitations: First, constructing stable cell lines expressing dual receptors is a cumbersome and time-consuming process, often facing problems such as decreased cell viability, low receptor expression levels, and weak responses to sweet stimuli; second, traditional detection methods mostly rely on long-term pre-incubation of fluorescent probes, resulting in high background noise and poor batch-to-batch reproducibility. There is currently a lack of real-time, quantitative methods for studying how aroma substances affect sweet receptor activation at the cellular level, and no mature response evaluation system exists. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for identifying the influence of aroma substances on sweetness perception based on sweetness receptor cells. This method can objectively, rapidly, and quantitatively identify the enhancing or inhibiting effects of aroma substances on the activation of sweetness receptors.
[0006] To achieve the above objectives, the present invention adopts the following technical solution:
[0007] A method for identifying the influence of aroma compounds on sweetness perception based on sweetness receptor cells includes the following steps:
[0008] S1. Construction of a sweet taste receptor cell system: transient co-transfection of mammalian cells with human sweet taste receptor subunits T1R2 and T1R3 and chimeric G protein Gα. 16 The plasmid was used to obtain transfected cells;
[0009] S2. Cell seeding and incubation: The transfected cells are seeded into multi-well plates. After the cells adhere to the plate, calcium ion fluorescent probes are added for incubation.
[0010] S3. Preparation of test solutions: Prepare the solution of the sweet substance to be tested and the co-stimulation test solution of the aroma substance and sweet substance to be tested using buffer solution;
[0011] S4. Signal detection and data acquisition: The test solution prepared in step S3 is added to the cells incubated in step S2, and the changes in intracellular calcium ion fluorescence signal are monitored in real time using a fluorescence detection device.
[0012] S5. Data Analysis and Effect Identification: Based on the real-time fluorescence signal data collected in step S4, the effect of the aroma substance on sweetness perception is identified.
[0013] Preferably, in step S5, the method for identifying the effect of the aroma substance on sweetness perception based on the real-time fluorescence signal data collected in step S4 is as follows:
[0014] (1) Record the real-time fluorescence signal curve of the test solution, select the lowest fluorescence signal value within the first 30 seconds from the curve as the baseline value F0, and select the highest fluorescence signal value as the peak value F. p Calculate the response intensity value F pmax = F p / F0, if F pmax If the value is greater than 1, then the cell system is considered to be responsive to the stimulus;
[0015] (2) Select the highest fluorescence signal value within 30-150s as the peak value F1, and the initial response value as F2. If the test solution has a positive stimulating effect on sweet taste receptor cells, then the response intensity value is calculated. If F1 < F2, then the test solution is judged to have a negative stimulating effect on sweet taste receptor cells, and the response intensity value is calculated. ;
[0016] (3) When 1.1 < F max If the value is less than 1.5, the stimulus is considered to have a weak effect on the sweet taste sensory cell system. max A value >1.5 indicates that the stimulus has a strong effect on the sweet taste sensory cell system. max The value of the value is positively correlated with the strength of the influence.
[0017] Preferred options also include:
[0018] The response intensity value F based on the different concentrations of the sweetener solution to be tested. pmax Filter F pmax The concentration closest to 1.1 is used to prepare the aroma-sweetness co-stimulation test solution to be tested.
[0019] Preferably, in step S1, the mammalian cells are HEK293T cells; the transient co-transfection is performed using liposome transfection reagent, the culture medium is replaced after 6-8 hours of transfection, and the cells are cultured for another 12-18 hours.
[0020] Preferably, in step S2, cells are seeded into a 384-well plate at a density of 8,000-10,000 cells per well, with a seeding volume of 45-55 μL per well; after the cells adhere and grow overnight, they are incubated with a buffer containing a calcium ion fluorescent probe for 1-2 hours.
[0021] Preferably, in step S3, the sweetener is a sugar sweetener or a sugar alcohol sweetener; the buffer solution is an HBSS buffer containing 0.1%-1.0% DMSO, and the test solution is prepared using a buffer solution of the same concentration.
[0022] Preferably, in step S4, a high-throughput fluorescence imaging plate reader is used for signal detection; the total detection time is 250-350 seconds.
[0023] The beneficial effects of this invention are as follows:
[0024] 1. Objective quantification, overcoming subjective bias: This invention is based on signal detection at the molecular and cellular level, which completely avoids the subjectivity of human sensory evaluation and can provide accurate and reproducible quantitative data that directly reflects the biological effects of aroma substances on the sweet taste receptor pathway.
[0025] 2. High sensitivity and fast response: Employing transient co-transfection technology combined with Gα 16 Protein conjugation allows for the rapid (3-4 days) construction of a highly sensitive sweet taste sensory cell system that responds strongly to sweet and aroma stimuli with low background noise.
[0026] 3. Real-time dynamic analysis of sweetness and aroma interaction: This invention establishes a standardized method for detecting cells co-stimulated by "sweetness + aroma", which can record the changes in intracellular calcium signals in real time and dynamically. This not only allows us to determine the final effect of enhancement or inhibition, but also reveals the kinetic characteristics of its action.
[0027] 4. Significant application value: This method provides a powerful in vitro screening tool for the food and flavor industries, which can quickly and accurately identify aroma substances with the potential to "lower blood sugar and increase sweetness" or "regulate sweetness", and has important guiding significance for the development of a new generation of healthy low-sugar foods. Attached Figure Description
[0028] Figure 1 This is a graph showing the changes in the intensity of intracellular calcium ion fluorescence signal response in different concentrations of erythritol solutions described in Example 1 of the present invention.
[0029] Figure 2 This is a graph showing the change in the intracellular calcium ion fluorescence signal response intensity of the geraniol-erythritol test solution according to Example 1 of the present invention.
[0030] Figure 3 This is a graph showing the change in the intensity of intracellular calcium ion fluorescence signal response in the furanone-erythritol test solution according to Example 2 of the present invention. Detailed Implementation
[0031] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, cells and equipment used in the present invention are all commercially available products in the art.
[0032] Example 1
[0033] S1. Constructing a sweet taste sensory cell system:
[0034] HEK293T cells were cultured in DMEM high glucose medium according to standard conditions and passaged every 24 hours until the third passage.
[0035] When the cells reached 70%-80% confluence in the culture dish, Lipo3000 and p3000 transfection reagents were used to transfect the cells with the human sweet taste receptors T1R2 and T1R3 subunits and the chimeric G protein Gα. 16 The eukaryotic expression plasmids were co-transfected into the cells at a mass ratio of 1:1:1. Six hours after transfection, the culture medium was replaced with complete medium.
[0036] S2. Cell seeding and incubation:
[0037] Continue culturing for 12 hours, then seed the cells into 384-well plates at a density of approximately 10,000 cells per well, with a volume of 50 μL per well, and incubate in an incubator for 18 hours to allow the cells to fully adhere to the plate.
[0038] Discard the culture medium in each well, add 25 μL of the calcium ion fluorescent probe from the Fluo-4 Direct™ Calcium Assay Kit to each well, and incubate in a cell culture incubator at 5% carbon dioxide concentration and 37°C in the dark for 1 hour.
[0039] S3. Prepare the test solution:
[0040] Preparation of erythritol test solutions of different concentrations: Erythritol test solutions with concentration gradients of 5 mM, 10 mM, 20 mM, 40 mM, 80 mM, 160 mM, and 320 mM were prepared using HBSS buffer containing 0.5% DMSO.
[0041] The negative control was HBSS buffer containing 0.5% DMSO.
[0042] S4. Signal Detection and Data Acquisition:
[0043] Each test solution was added to the corresponding well of the prepared cell plate through the sample loading module of the FLIPR-PENTA system (3 replicates per concentration). The total monitoring time was 300 seconds, with the sample loading point set at the 6th second. The fluorescence intensity value F of each test solution was recorded once per second.
[0044] S5. Data Analysis and Effect Assessment:
[0045] The response intensity values for each mixed solution group and the control group were calculated using the following method:
[0046] For each fluorescence signal curve, the lowest fluorescence signal value within the first 30 seconds is taken as the baseline value F0, and the highest fluorescence signal value is taken as the peak value F. p Calculate the response intensity value F pmax = F p / F0.
[0047] like Figure 1 As shown, the intracellular calcium signaling response intensity value F pmax It showed a concentration dependence on erythritol, generally exhibiting a trend of increasing stimulus response with increasing concentration. When the concentration was below 40 mM, F pmax When the value is less than 1.1, the cellular response is not significant; when the concentration reaches 80 mM, F pmaxThe value rose to approximately 1.15, indicating a strong stimulus; the peak response was reached at 160 mM, and while the response slightly decreased at 320 mM, it still remained at a strong level. This result demonstrates that the sweetness-sensing cell system of this invention can effectively detect the activation effect of sweet substances on sweetness receptors and has a suitable sensitivity range. Simultaneously, a 40 mM erythritol solution was selected as the baseline concentration to prepare a geraniol-erythritol co-stimulation test solution: using 40 mM erythritol (when stimulated alone, 1 < F...). pmax Based on a response of <1.1 (weak response), a mixed solution containing 40 mM erythritol and different concentrations (8, 16, 32, 64, 128 mM) of geraniol was prepared using HBSS buffer containing 0.5% DMSO. A 40 mM erythritol solution was prepared as a control.
[0048] Repeat steps S4 and S5.
[0049] Geraniol is a natural monoterpene compound with a typical rose aroma. It is widely found in the essential oils of plants such as rose, geranium, and lemongrass. It is one of the core raw materials in the daily fragrance industry. It is also used as a food additive to provide fruity flavors to various beverages and candies.
[0050] like Figure 2 As shown, the group of geraniol + erythritol mixed solutions with a concentration of 32 mM or higher has F max The values were all above 1.2, indicating that after adding an appropriate amount of geraniol, the stimulation of the sweet taste receptor cell system by the 40 mM erythritol mixed solution reached or exceeded that of the sweet taste receptor cell system by the high concentration of erythritol monosol. Among them, 64 mM geraniol, while producing a strong enhancing effect, allowed the response signal to return to near the baseline level within 300 s, with a complete signal dynamics curve and good recovery, indicating that this concentration of geraniol-erythritol stimulation system had a high degree of stimulation on the sweet taste receptor cells and caused only minor or almost no damage to the cells.
[0051] Example 2
[0052] The test solution was changed to: a mixed solution containing 40 mM erythritol and different concentrations (8, 16, 32, 64, 128 mM) of furanone was prepared using HBSS buffer containing 0.5% DMSO; other steps were the same as in Example 1.
[0053] Furanones play multiple roles in the food industry. They are not only the core components in the formulation of flavorings such as strawberry, pineapple, and caramel, but also synergists and harmonizers. Even a small amount can enhance the richness and mellowness of dairy products and baked goods, and harmonize various flavors.
[0054] like Figure 3As shown, compared with stimulation alone by 40 mM erythritol, the addition of a certain concentration of furanone exhibited a negative stimulating effect, and the addition of furanone above 16 mM led to F max A value > 1.1 indicates a strong inhibitory effect. The inhibitory effect exhibits a certain concentration-dependent characteristic: the addition of a high concentration of 128 mM furanone ultimately has the weakest inhibitory effect on the sweet taste perception of 40 mM erythritol. However, it can also be observed that the higher the concentration of furanone solution, the stronger the initial inhibitory effect on the sweet taste perception system, but the inhibitory effect gradually weakens as the stimulation occurs and the calcium ion concentration increases.
[0055] Example 3
[0056] The test solution was changed to: glucose solutions of different concentrations (5, 10, 20, 40, 80, 160, 320 mM) were prepared and mixed solutions containing 40 mM glucose and eugenol of different concentrations (8, 16, 32, 64, 128 mM) were prepared using HBSS buffer containing 0.5% DMSO; other steps were the same as in Example 1.
[0057] Example 4
[0058] The test solution was changed to: a mixed solution containing 40 mM glucose and different concentrations (8, 16, 32, 64, 128 mM) of benzyl acetate was prepared using HBSS buffer containing 0.5% DMSO; other steps were the same as in Example 3.
[0059] Example 5
[0060] The test solution was changed to: a mixed solution containing 40 mM glucose and different concentrations (8, 16, 32, 64, 128 mM) of phenylacetaldehyde was prepared using HBSS buffer containing 0.5% DMSO; other steps were the same as in Example 3.
[0061] Example 6
[0062] The test solutions were changed as follows: fructose solutions of different concentrations (5, 10, 20, 40, 80, 160, 320 mM) were prepared and mixed with HBSS buffer containing 0.5% DMSO to prepare a mixed solution containing 40 mM fructose and different concentrations (8, 16, 32, 64, 128 mM) of eugenol. The 40 mM fructose solution alone was used as a control. Other steps were the same as in Example 1.
[0063] Example 7
[0064] The test solution was changed to: a mixed solution containing 40 mM fructose and different concentrations (8, 16, 32, 64, 128 mM) of benzyl acetate was prepared using HBSS buffer containing 0.5% DMSO; other steps were the same as in Example 6.
[0065] Example 8
[0066] The test solution was changed to: a mixed solution containing 40 mM fructose and different concentrations (8, 16, 32, 64, 128 mM) of phenylacetaldehyde was prepared using HBSS buffer containing 0.5% DMSO; other steps were the same as in Example 6.
[0067] Table 1
[0068]
[0069] Table 1 shows the single-sample measurement data of some test solutions in the examples. The experimental results show that the identification method of this application can determine the influence of aroma substances on sweetness perception, and there are significant differences in sweetness perception among different aroma substances. Among them, the response intensity value F of the co-stimulation group of 40mM fructose and 128mM benzyl acetate is... MAX The response intensity reached 1.9414, showing a significant positive enhancement effect. When the same concentration of fructose was co-stimulated with 64 mM eugenol and 128 mM phenylacetaldehyde, the response intensity values were 1.4455 and 1.4296, respectively, exhibiting a weak enhancement effect. These results systematically verify that the sweet taste sensory cell model constructed in this invention can accurately quantify the calcium signal response intensity F0. MAX This enables an objective evaluation of the interaction between aroma compounds and sweetness, providing a standardized in vitro research tool for the field of food sensory science.
[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.
Claims
1. A method for identifying the influence of aroma substances on sweetness perception based on sweetness receptor cells, characterized in that, Includes the following steps: S1, constructing sweet taste receptor cell system: co-transfecting plasmids encoding human sweet taste receptor subunits T1R2, T1R3 and chimeric G protein Gα 16 in mammalian cells to obtain transfected cells; S2. Cell seeding and incubation: The transfected cells are seeded into multi-well plates. After the cells adhere to the plate, calcium ion fluorescent probes are added for incubation. S3. Preparation of test solutions: Prepare the solution of the sweet substance to be tested and the co-stimulation test solution of the aroma substance and sweet substance to be tested using buffer solution; S4. Signal detection and data acquisition: The test solution prepared in step S3 is added to the cells incubated in step S2, and the changes in intracellular calcium ion fluorescence signal are monitored in real time using a fluorescence detection device. S5. Data Analysis and Effect Identification: Based on the real-time fluorescence signal data collected in step S4, the effect of the aroma substance on sweetness perception is identified.
2. The method for identifying the influence of aroma substances on sweetness perception based on sweetness receptor cells according to claim 1, characterized in that, In step S5, the method for identifying the effect of the aroma substance on sweetness perception based on the real-time fluorescence signal data collected in step S4 is as follows: (1) Record the real-time fluorescence signal curve of the test solution, select the lowest fluorescence signal value within the first 30 seconds from the curve as the baseline value F0, and select the highest fluorescence signal value as the peak value F. p Calculate the response intensity value If F pmax If the value is greater than 1, then the cell system is considered to be responsive to the stimulus; (2) Select the highest fluorescence signal value within 30-150s as the peak value. The initial response value is ,like If the test solution has a positive stimulating effect on sweet taste receptor cells, then the response intensity value is calculated. ;like If the test solution has a negative stimulating effect on sweet taste receptor cells, then the response intensity value is calculated. ; (3) When This indicates that the stimulus has a weak effect on the sweet taste sensory cell system. This indicates that the stimulus has a strong effect on the sweet taste sensory cell system, F. max The value of the value is positively correlated with the strength of the influence.
3. The method for identifying the influence of aroma substances on sweetness perception based on sweetness receptor cells according to claim 2, characterized in that, Also includes: response intensity value F based on different concentrations of the solution of the sweet substance to be tested pmax , screening F pmax value closest to 1.1, and the sweet substance-aroma substance co-stimulation test solution to be tested is prepared at this concentration.
4. The method for identifying the influence of aroma substances on sweetness perception based on sweetness receptor cells according to claim 1, characterized in that, In step S1, the mammalian cells are HEK293T cells; the transient co-transfection is performed using liposome transfection reagent, and the culture medium is replaced 6-8 hours after transfection, and the cells are cultured for another 12-18 hours.
5. The method for identifying the influence of aroma substances on sweetness perception based on sweetness receptor cells according to claim 1, characterized in that, In step S2, cells are seeded into 384-well plates at a density of 8,000-10,000 cells per well, with a seeding volume of 45-55 μL per well. After the cells adhere and grow overnight, they are incubated with a buffer containing a calcium ion fluorescent probe for 1-2 hours.
6. The method for identifying the influence of aroma substances on sweetness perception based on sweetness receptor cells according to claim 1, characterized in that, In step S3, the sweetener is a sugar sweetener or a sugar alcohol sweetener; the buffer solution is an HBSS buffer containing 0.1%-1.0% DMSO, and the test solution is prepared using a buffer solution of the same concentration.
7. The method for identifying the influence of aroma substances on sweetness perception based on sweetness receptor cells according to claim 1, characterized in that, In step S4, a high-throughput fluorescence imaging plate reader is used for signal detection; the total detection time is 250-350 seconds.