Method for evaluating the effect of aroma on sweetness based on dynamic response integral area and synergy index

Abstract

The application discloses a method for evaluating the sweetness of aroma based on dynamic response integral area and synergy index, so as to solve the problem that the response duration and combination synergy are easily neglected when the sweetness of aroma is evaluated by simply relying on the change rate of the fluorescence peak value. The method comprises the following steps: fluorescence kinetics detection, dynamic response integral area AUC calculation, and quantitative evaluation of the sweetness of aroma: taking the AUC of a sucrose reference stimulating solution as a reference value, comparing the AUC of a test solution of a to-be-detected aroma compound or an aroma-sucrose combination test solution with the reference value, and obtaining the relative response value of the sweetness of aroma; calculating the synergy index according to the normalized AUC corresponding to the sucrose single stimulation, the to-be-detected aroma compound single stimulation and the aroma-sucrose combination stimulation, and determining the synergistic enhancement effect between the to-be-detected aroma compound and the sucrose according to the synergy index.

Description

Technical Field

[0001] This invention relates to the field of food flavor analysis technology, and more specifically to a method for evaluating the influence of aroma on sweetness based on dynamic response integral area and synergistic index. Background Technology

[0002] Sweetness is a crucial component of food flavor quality. With the increasing demand for low-sugar foods, leveraging aroma-induced sweetness enhancement to improve perceived sweetness without significantly increasing sugar content has become an important direction in food formulation design. Some aroma compounds themselves do not possess typical sweetness but can enhance perceived sweetness in sucrose-containing systems. This phenomenon involves the interaction between aroma and sweetness, and relying solely on human sensory evaluation is easily influenced by individual differences, training levels, and psychological expectations.

[0003] Existing technologies have disclosed methods for quantifying sweetness using the intracellular calcium ion fluorescence response of sweetness receptor cells (see invention patent application publication number CN 109142298 A), and the cellular response to sweet substance stimulation can be characterized by the fluorescence intensity change rate ΔF / F0. These methods can evaluate the response of sweet substances at the cellular level, but their core analytical parameters are usually focused on single-point peak values ​​or peak value change rates. For the determination of the influence of aroma on sweetness, aroma compounds may not necessarily significantly increase the instantaneous peak value, but they may prolong the response duration, alter the response decay process, or cause the overall response of combined stimulation to exceed the independent additive level of individual stimuli.

[0004] Therefore, it is necessary to establish a completely new evaluation method so that the determination of the effect of aroma on sweetness can not only reflect the peak response, but also reflect the overall response load, synergistic relationship and concentration-dependent action mode within the complete time window. Summary of the Invention

[0005] To address the problem that existing technologies, which rely solely on the rate of change of fluorescence peaks to evaluate the influence of aroma on sweetness, often overlook response duration and synergistic relationships, this invention provides a method for evaluating the influence of aroma on sweetness based on the dynamic response integral area and synergistic index. This method establishes an evaluation system with the dynamic response integral area (AUC) as the core, further incorporating the synergistic index, and using the dose-response Hill equation fitting parameters as auxiliary judgment criteria, thereby more accurately evaluating the effect of aroma on sweetness.

[0006] To achieve the above objectives, the present invention provides a method for evaluating the influence of aroma on sweetness based on dynamic response integral area and synergistic index, comprising the following steps:

[0007] S1. Preparation of test cells and detection solutions: Sucrose reference stimulation solution, test solution of aroma compounds to be tested, and aroma-sucrose combination test solution were prepared using test cells expressing sweet taste receptors.

[0008] S2. Fluorescence kinetic detection: Load the calcium fluorescent probe onto the test cells described in step S1, and use a fluorescence kinetic detection system to continuously collect intracellular Ca2+ levels before and after stimulation with the detection solution. 2+ Fluorescence dynamic response curve;

[0009] S3. Calculation of the area under integration (AUC) of the dynamic response: Taking the stimulus addition time as the starting point of integration, the dynamic response curve after baseline subtraction is integrated within the preset detection time window to obtain the AUC.

[0010] S4. Quantitative analysis of the effect of aroma on sweetness: Using the AUC of the sucrose reference stimulation solution as the reference value, the AUC of the test solution of the aroma compound to be tested or the aroma-sucrose combination test solution is compared with the reference value to obtain the relative response value of the effect of aroma on sweetness.

[0011] S5. Synergistic Effect Determination: The synergistic index ΔE is calculated based on the normalized AUC corresponding to sucrose stimulation alone, aroma compound stimulation alone, and aroma-sucrose combination stimulation. Bliss And based on ΔE Bliss Determine the synergistic enhancement effect between the aroma compound and sucrose; synergistic index ΔE Bliss Calculate ΔE using the following formula: Bliss =E Observed -E Predicted ; where E Observed E represents the normalized AUC measured value of the aroma-sucrose combination stimulus. Predicted The theoretical predictions for sucrose stimulation alone and the aroma compound test alone under the Bliss independent model; when ΔE Bliss When ΔE > 0, it is determined that the aroma compound being tested has a synergistic enhancing effect with sucrose; when ΔE Bliss When ΔE ≈ 0, it is determined to be an approximately independent additive effect; when ΔE Bliss When <0, it is determined to be an antagonistic or inhibitory effect.

[0012] The method uses the dynamic response integral area (AUC) as the core parameter for determining the effect of aroma on sweetness, which can simultaneously reflect the response intensity and response duration. Furthermore, by combining the Bliss synergy index, the measured response to the aroma-sucrose combination stimulus is compared with the theoretical independent additive response, which can distinguish between synergistic enhancement, approximate additive response and antagonistic inhibition, and avoid misjudging single-point peak fluctuations as true synergy.

[0013] Furthermore, the method also includes step S6, dose-response auxiliary analysis: Under a sucrose-based stimulation background, test cells are stimulated with test solutions of different concentrations of the aroma compounds to be tested. Based on the fluorescence response data, the Hill equation is fitted to obtain the half-maximum effect concentration (EC5). 50 The Hill coefficient (n), maximum response value (Emax), and coefficient of determination (R²) were used as auxiliary parameters to evaluate the influence of aroma on sweetness. Thus, the Hill equation was used for fitting, and EC... 50 Dose-response parameters such as Hill coefficient n, Emax, and R2 can help identify potent, weakly potent, and concentration-window aroma compounds, providing a more complete dose-dependent interpretation of the AUC and synergistic index evaluation results.

[0014] Further, in step S6, the Hill equation is:

[0015] Where Y is the response value, X is the logarithm of the concentration of the aroma compound to be measured, Emin is the minimum response value, Emax is the maximum response value, and EC is the maximum response value. 50 is the half-maximal effect concentration, and n is the Hill coefficient.

[0016] Further, in step S1, the test cells are host cells expressing human sweet taste receptor subunits T1R2 and T1R3; more preferably, HEK293T cells expressing T1R2 / T1R3 and the chimeric G protein Gα16gust44.

[0017] Furthermore, in step S1, the concentration of the sucrose reference stimulus solution is determined through preliminary experiments, and the non-saturated response range that can induce a stable response and has not reached the plateau period is selected as the reference stimulus concentration.

[0018] Further, in step S1, the aroma compound to be tested is methyl salicylate, phenylethanol, β-damascone, hexanal, γ-octyl lactone, γ-heptyl lactone, or a combination thereof.

[0019] Furthermore, in step S1, the concentration of the sucrose reference stimulant is 80 mM; the concentration of the aroma compound to be tested is independently selected from 0.125 mM, 0.25 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, 8 mM, 16 mM or 32 mM.

[0020] Further, in step S3, the dynamic response integral area AUC is calculated according to the following formula:

[0021]

[0022] Where t0 is the time of stimulus addition, t1 is the detection endpoint, F(t) is the fluorescence intensity at time t, and F0 is the average baseline fluorescence value before stimulation.

[0023] Furthermore, in step S4, the relative response value of the aroma to sweetness is AUC. sample / AUC sucrose AUC sample Here is the AUC corresponding to the sample to be tested. sucrose The value is the AUC corresponding to the sucrose-based stimulus solution.

[0024] Furthermore, the E Predicted Calculate E according to the following formula: Predicted =E S +E A -E S ×E A ; where E S Normalized AUC for sucrose-only stimulation, E A The normalized AUC is the value of the aroma alone.

[0025] The above technical solution is only one feasible technical solution of the present invention. The scope of protection of the present invention is not limited thereto. Those skilled in the art can reasonably adjust the specific design according to actual needs.

[0026] The above invention has the following advantages or beneficial effects:

[0027] (1) This invention uses the dynamic response integral area AUC as the core parameter for the determination of the effect of aroma on sweetness. It can simultaneously reflect the response intensity and response duration. Furthermore, by combining the Bliss synergy index, the measured response of the aroma-sucrose combination stimulus is compared with the theoretical independent additive response. This can distinguish between synergistic enhancement, approximate additive response and antagonistic inhibition, and avoid misjudging single-point peak fluctuations as true synergy. Compared with the method of using only the fluorescence peak change rate, it can better evaluate the overall regulatory effect of aroma on the sweetness response process.

[0028] (2) This invention uses Hill's equation fitting and EC 50 Dose-response parameters such as Hill coefficient n, Emax, and R2 can help identify potent, weakly potent, and concentration-window aroma compounds, providing a more complete dose-dependent interpretation of the AUC and synergistic index evaluation results.

[0029] (3) This invention verifies the results by comparing proportions, AUC examples, AUC combined with synergistic index examples and Hill equation fitting examples, demonstrating that the appropriate selection of dynamic response integral area AUC and synergistic index will significantly affect the evaluation results of aroma affecting sweetness, thus providing an objective and quantifiable methodological basis for screening sweetening aromas in low-sugar food formulations. Attached Figure Description

[0030] The invention, its features and advantages will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings.

[0031] Figure 1 These are real-time calcium flow trajectory diagrams of all aroma compounds in the embodiments of the present invention after stimulating cells individually and in combination with sucrose; wherein diagram a corresponds to the test diagram of aroma compound methyl salicylate (MS), diagram b corresponds to the test diagram of aroma compound β-damascone (DM), diagram c corresponds to the test diagram of aroma compound phenylethanol (PA), diagram d corresponds to the test diagram of aroma compound γ-octanolide (OT), diagram e corresponds to the test diagram of aroma compound hexanal (HX), diagram f corresponds to the test diagram of aroma compound γ-heptanol (HT), and diagram g corresponds to the test diagram of aroma compound benzaldehyde (BZ); sucrose is labeled in the diagrams.

[0032] Figure 2 This is a comparison of the area under the curve (AUC) within a specified time window after cells are stimulated by different aroma compounds and sucrose mixtures in this embodiment of the invention; in the figure, S marks sucrose, A marks aroma compounds, and S+A marks a mixture of sucrose and aroma compounds.

[0033] Figure 3 This is a semi-log dose-response curve based on Hill's equation fitting in an embodiment of the present invention;

[0034] Figure 4 This is a real-time calcium flow trajectory diagram of the T1R2 / T1R3 calcium response induced by 28 mM methyl salicylate (MS) in response to different concentrations of sucrose (S) in an embodiment of the present invention;

[0035] Figure 5 This is an AUC plot showing the base-dependent regulatory effect of methyl salicylate (MS) on the T1R2 / T1R3 calcium response induced by different concentrations of sucrose (S) in embodiments of the present invention. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0037] The reaction apparatus and reaction materials involved in the following embodiments and examples are all commercially available.

[0038] The detection instruments and reagents used in the following examples are all commercially available, and the detection methods used are existing technologies that can be found online.

[0039] The test cells used in this invention are cells expressing human sweetness receptors T1R2 / T1R3, preferably HEK293T cells co-expressing T1R2, T1R3, and Gα16gust44. The construction, culture, and loading of the calcium fluorescent probe into the test cells can be performed using methods known in the art. The improvement of this invention lies in performing AUC integral analysis on the continuous fluorescence dynamic response curve and further combining it with the synergistic index to determine the influence of aroma on sweetness, while retaining the Hill equation fitting parameters for auxiliary classification of concentration-dependent response patterns. The detection system used in this invention is a fluorescence dynamics detection system capable of continuously reading changes in cell fluorescence, preferably a FLIPR system; the calcium fluorescent probe used is preferably Fluo-4 AM.

[0040] This invention discloses a method for evaluating the influence of aroma on sweetness based on dynamic response integral area and synergistic index, comprising the following steps:

[0041] S1. Preparation of test cells and detection solutions: Sucrose reference stimulation solution, test solution of aroma compounds to be tested, and aroma-sucrose combination test solution were prepared using test cells expressing sweet taste receptors.

[0042] S2. Fluorescence kinetic detection: Load the calcium fluorescent probe onto the test cells described in step S1, and use a fluorescence kinetic detection system to continuously collect intracellular Ca2+ levels before and after stimulation with the detection solution. 2+ Fluorescence dynamic response curve; the fluorescence kinetic detection system is a FLIPR system or a detection system capable of continuously recording dynamic changes in cell fluorescence;

[0043] S3. Calculation of the area under integration (AUC) of the dynamic response: Taking the stimulus addition time as the starting point of integration, the dynamic response curve after baseline subtraction is integrated within the preset detection time window to obtain the AUC.

[0044] S4. Quantitative analysis of the effect of aroma on sweetness: Using the AUC of the sucrose reference stimulation solution as the reference value, the AUC of the test solution of the aroma compound to be tested or the aroma-sucrose combination test solution is compared with the reference value to obtain the relative response value of the effect of aroma on sweetness.

[0045] S5. Synergistic Effect Determination: The synergistic index ΔE is calculated based on the normalized AUC corresponding to sucrose stimulation alone, aroma compound stimulation alone, and aroma-sucrose combination stimulation. Bliss And based on ΔE Bliss Determine the synergistic enhancement effect between the aroma compound and sucrose; synergistic index ΔE Bliss Calculate ΔE using the following formula: Bliss =E Observed -E Predicted ; where E ObservedE represents the normalized AUC measured value of the aroma-sucrose combination stimulus. Predicted The theoretical predictions for sucrose stimulation alone and the aroma compound test alone under the Bliss independent model; when ΔE Bliss When ΔE > 0, it is determined that the aroma compound being tested has a synergistic enhancing effect with sucrose; when ΔE Bliss When ΔE ≈ 0, it is determined to be an approximately independent additive effect; when ΔE Bliss When <0, it is determined to be an antagonistic or inhibitory effect.

[0046] Furthermore, the method also includes step S6, dose-response auxiliary analysis: Under a sucrose-based stimulation background, test cells are stimulated with test solutions of different concentrations of the aroma compounds to be tested. Based on the fluorescence response data, the Hill equation is fitted to obtain the half-maximum effect concentration (EC5). 50 The Hill coefficient (n), maximum response value (Emax), and coefficient of determination (R²) were used as auxiliary parameters to evaluate the influence of aroma on sweetness. Thus, the Hill equation was used for fitting, and EC... 50 Dose-response parameters such as Hill coefficient n, Emax, and R2 can help identify potent, weakly potent, and concentration-window aroma compounds, providing a more complete dose-dependent interpretation of the AUC and synergistic index evaluation results.

[0047] The following section uses methyl salicylate (MS), phenylethanol (PA), β-damascone (DM), hexanal (HX), γ-octyl lactone (OT), and γ-heptyl lactone (HT) as examples, combined with comparative examples, to explain in detail the technical effects produced by the above technical solutions.

[0048] Comparative Example 1: The effect of fluorescence peak change rate ΔF / F0 on sweetness was quantified solely using the effect of aroma on sweetness.

[0049] Test cells expressing T1R2 / T1R3 were used. Cells were stimulated with 80 mM sucrose, the target aroma compound, and an aroma-sucrose combination, and fluorescence dynamic response curves were continuously acquired. The response was calculated as ΔF / F0 = (F... max The fluorescence peak change rate is calculated as -F0) / F0, where F max F0 represents the highest fluorescence intensity after stimulation, while F0 represents the baseline fluorescence average before stimulation.

[0050] Using 80 mM sucrose as the baseline stimulus, the peak responses of some samples were enhanced after adding different aroma compounds at concentrations of 0.125 mM, 0.25 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, 8 mM, 16 mM, or 32 mM against the 80 mM sucrose background. For example, methyl salicylate and β-damascone produced higher peak responses, while the peak enhancement of some aroma compounds was weaker or exhibited atypical dose responses.

[0051] The results show that the peak change rate ΔF / F0 can only reflect the maximum response at a certain time point, and cannot reflect the response duration, decay process, and whether the combined effect exceeds the theoretical additive effect. Therefore, it is difficult to accurately determine the overall influence of aroma on sweetness response using ΔF / F0, especially since aroma-sucrose combinations with insignificant peak increase but significantly prolonged response duration are easily overlooked.

[0052] Example 1: Quantifying the effects of sweetness and aroma on sweetness using dynamic response integral area (AUC).

[0053] Under the same test conditions as Comparative Example 1, AUC analysis was performed on the acquired fluorescence dynamic response curves. The area under integration (AUC) of the dynamic response after baseline subtraction was calculated within a specified time window, using the stimulus introduction time as the starting point for integration.

[0054] The AUC is calculated according to the following formula:

[0055]

[0056] Where F(t) is the fluorescence intensity at time t, F0 is the average baseline fluorescence value before stimulation, t0 is the time of stimulation addition, and t1 is the detection endpoint.

[0057] Using the AUC of the 80 mM sucrose group as the baseline, the ratio of the AUC of the aroma-sucrose combination group to the AUC of the sucrose group was calculated to obtain the relative response value of aroma to sweetness. The detection time window was the period from the moment the stimulus was added until the response basically recovered to the baseline level, or a fixed detection time window. The results are shown in Table 1. Figure 1 and Figure 2 .

[0058] Table 1 Results of quantitative analysis of the effect of aroma on sweetness based on AUC

[0059]

[0060] Table 1 shows that the AUC of the 80 mM sucrose group was 48.9, while the AUC of the aroma-sucrose combination group reached 205.0 after the addition of methyl salicylate, with a relative response value of 4.19. The AUC of the β-damascone and phenylethyl alcohol combination group was also significantly higher than that of the sucrose-only stimulation group. These results indicate that AUC can reflect the overall amplification effect of aroma compounds on the entire sweetness response process, and is more suitable for determining the influence of aroma on sweetness than using only the peak change rate.

[0061] Example 2: Quantifying the Influence of Sweetness and Aroma on Sweetness Using Dynamic Response Integral Area (AUC) and Synergy Index

[0062] Building upon Example 1, a Bliss independent model was further introduced to calculate the synergistic index. The AUCs for sucrose alone, aroma alone, and the aroma-sucrose combination were normalized, and the measured effect E of the aroma-sucrose combination was calculated. Observed Theoretical independent additive effect E Predicted and with ΔE Bliss =E Observed -E Predicted As a synergy index.

[0063] Among them, E Predicted =E S +E A -E S ×E A E S Normalized AUC for sucrose-only stimulation, E A The normalized AUC is the value for aroma stimulation alone. When ΔE Bliss When ΔE > 0, it is determined to be positive synergistic enhancement; when ΔE Bliss When ΔE ≈ 0, it is determined to be an approximately independent sum; when ΔE Bliss When <0, it is determined to be antagonistic or inhibitory.

[0064] The results are shown in Table 2. When the AUC of the aroma-sucrose combination stimulus was used as the normalization benchmark, E... Observed The value is 1. The synergy index in the table has been verified to be calculated according to the Bliss independent model, and the calculation formula is ΔE. Bliss =1−E Predicted E Predicted =E S +E A -E S ×E A E S =AUC(S) / AUC(S+A), E A =AUC(A) / AUC(S+A).

[0065] Table 2. Results of the aroma-sucrose synergistic effect evaluated based on AUC and synergistic index.

[0066]

[0067] Table 2 shows that the AUC of the combined stimulation of methyl salicylate and sucrose was 4.19 times that of sucrose alone, and ΔE Bliss The value was 0.694, indicating that the combined response exceeded the predicted value of the Bliss independent model, demonstrating a significant synergistic enhancement effect. Although β-damascone had a low AUC when stimulated by aroma alone, its AUC increased significantly after stimulation with sucrose, with ΔE... Bliss The value of 0.602 indicates that it has a synergistic amplification effect on the sucrose response.

[0068] Compared to Example 1, this example not only evaluates whether the aroma-sucrose combined response is enhanced, but also determines whether the enhancement exceeds the theoretical independent additive level. Therefore, the AUC-based synergistic index can further improve the accuracy and interpretability of aroma-influenced sweetness determination.

[0069] Example 3: Dose-response characteristics of key aroma compounds in peach and Hill equation fitting

[0070] To preserve the concentration-dependent information of aroma compounds, concentration gradients of the target aroma compounds were set under an 80 mM sucrose baseline stimulation background to stimulate test cells and collect fluorescence response data. The target aroma compounds included key peach aroma compounds such as methyl salicylate, phenethyl alcohol, β-damascone, hexanal, γ-octyl lactone, γ-heptyl lactone, and benzaldehyde. Dose-response fitting results are shown below. Figure 3 .

[0071] The dose-response data were nonlinearly fitted using the Hill equation, which is as follows: Where Y is the response value, X is the logarithm of the concentration of the aroma compound to be measured, Emin is the minimum response value, Emax is the maximum response value, and EC is the maximum response value. 50 is the half-maximal effect concentration, and n is the Hill coefficient.

[0072] The EC values ​​of each aroma compound were obtained by fitting the Hill equation. 50 Hill coefficient n, Emax, and R² were used. This step is not the sole basis for evaluating the influence of aroma on sweetness in this invention, but rather serves as an auxiliary parameter for the AUC and synergy index evaluation results, used to identify potent, weakly potent, and concentration-window aroma compounds. The results are shown in Table 3.

[0073] Table 3. Kinetic parameters of aroma compounds enhancing sweet taste receptor activity

[0074]

[0075] Table 3 shows that phenylethanol, β-damascone, and methyl salicylate exhibit relatively clear dose-response relationships, with methyl salicylate reaching a maximum response value (Emax) of 1.24 and β-damascone at 0.90. Hexanal, although possessing a lower EC50, shows a different response. 50 However, its response decreases at higher concentrations, exhibiting a concentration window effect. γ-Heprolactone and benzaldehyde did not form a reliable fitting curve.

[0076] This example illustrates that Hill equation fitting can provide the concentration sensitivity and maximum response capability of the aroma compound being tested, but it still mainly reflects the peak or dose-response characteristics and cannot replace AUC in characterizing the complete dynamic response process, nor can it determine whether the combined effect exceeds the theoretical independent summation.

[0077] Example 4: Evaluation of the base dependence of aroma on sweetness

[0078] To verify whether the effect of aroma on sweetness is influenced by the sucrose base concentration, sucrose solutions of 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, and 80 mM were prepared, and a fixed concentration of methyl salicylate (28 mM) was added under the same sucrose gradient. Real-time calcium flow trajectories were collected and AUC was calculated according to the method in Example 1. The results are shown in Table 4. Figure 4 and Figure 5 .

[0079] Figure 4 The real-time calcium flow trajectories shown indicate that sucrose alone induces a weak dynamic response in the range of 10 mM to 80 mM. However, the addition of 28 mM methyl salicylate to the same sucrose concentration gradient significantly enhances the real-time calcium flow response for different sucrose substrates, and prolongs the response duration. These results demonstrate that 28 mM methyl salicylate can amplify the sucrose-induced T1R2 / T1R3 calcium response and provide dynamic curve evidence for subsequent AUC integral analysis.

[0080] Table 4. Results of AUC evaluation of the dynamic response of methyl salicylate to different concentrations of sucrose.

[0081]

[0082] Table 4 shows that the AUC induced by sucrose alone was generally low, ranging from 4.02 to 6.98. The AUC stimulated by the sucrose-methyl salicylate combination significantly increased after the addition of 28 mM methyl salicylate, ranging from 109.54 to 264.92. Specifically, the AUC stimulated by the combination of 80 mM sucrose and 28 mM methyl salicylate was 264.92, which was 38.07 times that of the 80 mM sucrose alone group, ΔE Bliss The value was 0.892, indicating that methyl salicylate significantly enhanced the dynamic response of sweet receptors under different sucrose bases. This result demonstrates that AUC evaluation can reveal the base-dependent relationship between aroma and sweetness.

[0083] Example 5 Evaluation of aroma compounds with concentration window effect

[0084] Using hexanal as a representative aroma compound, different concentration gradients were set against an 80 mM sucrose background, and the AUC and ΔE were calculated according to the methods of Examples 1, 2, and 3.Bliss And perform Hill equation fitting.

[0085] The results showed that hexanal could increase the AUC of the aroma-sucrose combination at lower concentrations, but as the concentration increased further, the AUC no longer increased and even decreased, exhibiting a concentration window effect. AUC and ΔE were used as the modulus parameters. Bliss The combined evaluation of Hill equation fitting can identify the effective concentration range of this type of aroma compound, avoiding over-judgment of its sweetening effect based solely on a single peak response.

[0086] The above results collectively demonstrate that, through progressive verification using comparative examples, AUC examples, AUC combined with synergistic index examples, and Hill equation fitting examples, it is proven that the selection of dynamic response integral area (AUC) and synergistic index significantly affects the evaluation results of aroma's influence on sweetness. This provides an objective and quantifiable methodological basis for screening sweetening aromas in low-sugar food formulations. Hill equation fitting and EC... 50 Dose-response parameters such as Hill coefficient n, Emax, and R2 can help identify potent, weakly potent, and concentration-window aroma compounds, providing a more complete dose-dependent interpretation of the AUC and synergistic index evaluation results.

[0087] Those skilled in the art should understand that variations can be implemented by combining existing technology with the above embodiments, which will not be elaborated here. Such variations do not affect the essence of the present invention, and will not be elaborated here either.

[0088] The preferred embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and the devices and structures not described in detail should be understood as being implemented in a conventional manner in the art. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the scope of the present invention. This does not affect the essential content of the present invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the present invention's technical solutions still fall within the protection scope of the present invention.

Claims

1. A method for evaluating the influence of aroma on sweetness based on dynamic response integral area and synergistic index, characterized in that: Includes the following steps: S1. Preparation of test cells and detection solutions: Sucrose reference stimulation solution, test solution of aroma compounds to be tested, and aroma-sucrose combination test solution were prepared using test cells expressing sweet taste receptors. S2. Fluorescence kinetic detection: Load the calcium fluorescent probe onto the test cells described in step S1, and use a fluorescence kinetic detection system to continuously collect intracellular Ca2+ levels before and after stimulation with the detection solution. 2+ Fluorescence dynamic response curve; S3. Calculation of the area under integration (AUC) of the dynamic response: Taking the stimulus addition time as the starting point of integration, the dynamic response curve after baseline subtraction is integrated within the preset detection time window to obtain the AUC. S4. Quantitative analysis of the effect of aroma on sweetness: Using the AUC of the sucrose reference stimulation solution as the reference value, the AUC of the test solution of the aroma compound to be tested or the aroma-sucrose combination test solution is compared with the reference value to obtain the relative response value of the effect of aroma on sweetness. S5. Synergistic Effect Determination: The synergistic index ΔE is calculated based on the normalized AUC corresponding to sucrose stimulation alone, aroma compound stimulation alone, and aroma-sucrose combination stimulation. Bliss And based on ΔE Bliss Determine the synergistic enhancement effect between the aroma compound and sucrose; synergistic index ΔE Bliss Calculate ΔE using the following formula: Bliss =E Observed -E Predicted ; where E Observed E represents the normalized AUC measured value of the aroma-sucrose combination stimulus. Predicted The theoretical predictions for sucrose stimulation alone and the aroma compound test alone under the Bliss independent model; when ΔE Bliss When ΔE > 0, it is determined that the aroma compound being tested has a synergistic enhancing effect with sucrose; when ΔE Bliss When ΔE ≈ 0, it is determined to be an approximately independent additive effect; when ΔE Bliss When <0, it is determined to be an antagonistic or inhibitory effect.

2. The method according to claim 1, characterized in that, The procedure also includes step S6, dose-response auxiliary analysis: Under a sucrose-based stimulation background, test cells are stimulated with different concentrations of the test solution containing the analyte aroma compounds. The Hill equation is fitted based on the fluorescence response data to obtain the half-maximum effect concentration (EC5). 50 The Hill coefficient n, maximum response value Emax, and coefficient of determination R2 were used as auxiliary parameters for evaluating the influence of aroma on sweetness.

3. The method according to claim 2, characterized in that, In step S6, the Hill equation is: Where Y is the response value, X is the logarithm of the concentration of the aroma compound to be measured, Emin is the minimum response value, Emax is the maximum response value, and EC is the maximum response value. 50 is the half-maximal effect concentration, and n is the Hill coefficient.

4. The method according to claim 1, characterized in that, In step S1, the test cells are host cells that express human sweet taste receptor subunits T1R2 and T1R3.

5. The method according to claim 1, characterized in that, In step S1, the concentration of the sucrose reference stimulus solution is determined through preliminary experiments, and the non-saturated response range that can induce a stable response and has not reached the plateau period is selected as the reference stimulus concentration.

6. The method according to claim 1, characterized in that, In step S1, the aroma compound to be tested is methyl salicylate, phenylethanol, β-damascone, hexanal, γ-octyl lactone, γ-heptyl lactone, or a combination thereof.

7. The method according to claim 6, characterized in that, In step S1, the concentration of the sucrose reference stimulant is 80 mM; the concentrations of the aroma compounds to be tested are independently selected from 0.125 mM, 0.25 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, 8 mM, 16 mM or 32 mM.

8. The method according to claim 1, characterized in that, In step S3, the dynamic response integral area AUC is calculated according to the following formula: Where t0 is the time of stimulus addition, t1 is the detection endpoint, F(t) is the fluorescence intensity at time t, and F0 is the average baseline fluorescence value before stimulation.

9. The method according to claim 1, characterized in that, In step S4, the relative response value of aroma to sweetness is AUC. sample / AUC sucrose AUC sample Here is the AUC corresponding to the sample to be tested. sucrose The value is the AUC corresponding to the sucrose-based stimulus solution.

10. The method according to claim 1, characterized in that, The E Predicted Calculate E according to the following formula: Predicted =E S +E A -E S ×E A ; where E S Normalized AUC for sucrose-only stimulation, E A Normalized AUC for aroma stimulation alone.

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