Screening verification method of maltol as sweetening substance

Abstract

The invention relates to a screening verification method using maltol as a sweetening substance, which innovatively integrates three links of a molecular docking technology, an in-vitro cell function test and sensory evaluation, and constructs a comprehensive and efficient novel sweetener screening system. The system not only overcomes the limitations of a traditional screening method based on structures and ligands, such as complexity, time consumption, limited data sets, low model applicability and the like, but also makes up the sensory characteristic deviation possibly caused by prediction simply depending on a mathematical model. The operation process is simple and clear, the whole process from screening to verification can be completed only through three steps, the research and development cost and the time cost are greatly reduced, and a new way is opened up for rapid development and application of a novel sweetening agent.

Description

Technical Field

[0001] The present application relates to the field of compound analysis and identification, and in particular to a method for screening and verifying maltol as a sweetening substance. Background Art

[0002] Taste is an important part of human perception of the outside world. Human taste perception is mainly composed of sour, sweet, bitter, salty and other stimuli. Among them, sweetness often gives people the impression of happiness and energy. It is a flavor that is generally accepted by people and can bring consumers a good sensory experience. The intake of sugar can not only meet the body's demand for energy, but also activate the pleasure and reward system in the brain, causing positive emotions such as pleasure and satisfaction. With the rapid development of modern social economy, the intake of sugar in human diet has continued to increase, which has a certain degree of negative impact on health. Therefore, various types of non-caloric sweeteners such as acesulfame potassium and sucralose have been developed one after another, aiming to replace sugar substances to compensate for their sensory experience. However, these substances all have certain defects. For example, acesulfame potassium has obvious bitterness and metallic taste residues after consumption, and cannot fully reproduce the taste of sugar. In addition, many sweeteners also have problems such as delayed sweetness, lingering aftertaste, and limited maximum sweetness intensity. Therefore, finding novel and healthy non-nutritive sweeteners has become a hot issue that the world is paying attention to.

[0003] At present, the search for new sweeteners is mainly through accidental discovery or structure- and ligand-based methods. Although this method is widely used in the screening of new sweeteners, it is still a tedious and arduous task to screen sweet substances from a large number of natural and synthetic compounds based on structure-activity relationships. In addition, different sweeteners may have the same pharmacophore or the same type of characteristic group, but this does not completely determine their sensory properties. In recent years, there has been an increasing number of studies using mathematical models to explore new sweeteners. This type of research is based on the assumption that the sweetness of molecules is related to their molecular structure. It uses data from literature, patents, and databases to establish sweetness molecule data sets and sweetness molecule data sets to mine substances with sweetener potential in food. This method can reduce the time and money required for experiments, but these models also have some common problems, such as the generally small data sets, complete reliance on the physicochemical characteristics of the ligands, and limited scope of application of the models.

[0004] With the in-depth exploration of aroma substances, researchers have found that there is a delicate balance between smell and taste. The two can not only affect the flavor of food in an independent way, but also enhance or improve the flavor perception of food through cross-modal perceptual interaction. For example, odor-induced flavor enhancement, that is, certain odors can enhance the perceived intensity of taste, which is the result of cross-modal perceptual interaction between smell and taste. As an important aroma compound, maltol can be added to food to enhance the sensitivity of people's taste and olfactory receptor cells in the tongue and nose to the aroma and aftertaste of food, thereby enhancing the flavor, sweetening, deodorizing and removing fishy smell. It has a significant effect on enhancing and improving the aroma of food, and can also extend the shelf life of food. Summary of the invention

[0005] The invention purpose of this application is to innovatively integrate the three links of molecular docking technology, in vitro cell function test and sensory evaluation to construct a comprehensive and efficient new sweetener screening system. This system not only overcomes the limitations of traditional structure-based and ligand-based screening methods, such as cumbersome and time-consuming, limited data sets and limited model applicability, but also makes up for the sensory characteristics deviation that may be caused by relying solely on mathematical model predictions.

[0006] In order to achieve the above-mentioned invention object and solve the above-mentioned technical problem, the present invention provides a method for screening and verifying maltol as a sweetening substance, characterized in that the method comprises the following steps:

[0007] Step 1) Homology modeling. According to the sequence similarity comparison results, metabotropic glutamate receptor 5 (mGlu5, PDB: 6N52) was finally selected as the template, and homology modeling was performed using the online SWISS-MODEL program. Tas1R2 (Genbank accession number: Q8TE23) and Tas1R3 (Q7RTX0) were multiple sequence aligned with the template by ClustalW and uploaded for modeling. The quality of the model was evaluated by ERRAT and PROCHECK programs, and the score met the requirements. The flexible docking mode of the molecular docking software Autodock was used to construct a docking model of maltol and the sweet taste receptor Tas1R2 / Tas1R3, and a visual analysis was performed based on Pymol. The interaction between maltol and the sweet taste receptor was determined by analyzing the docking binding energy and interaction mode of the two.

[0008] Step 2) Based on the in vitro functional test at the cell level, the functional detection of wild-type Tas1R2 / Tas1R3 was performed using a calcium excitation procedure. HEK293E cells were cultured, the cells were seeded on a 96-well plate, and then Tas1R2 / Tas1R3 and Ga16-gust44 vectors were transfected into the cells using Lipofectamine2000. After 48 hours of growth, the culture medium was replaced at regular intervals, the cells were washed, 50uL 3mM Fluo-4 AM was added, incubated for 1 hour, and then washed three times with HBSSH. After stimulation with the aroma solution, the calcium response changes were recorded on the FlexStation 3 multifunctional microplate reader using fluorescence changes 488nm excitation, 525nm emission, and 515nm cutoff. The calcium response intensity is expressed as a percentage change (peak fluorescence-baseline fluorescence level, expressed as △F) compared to the baseline fluorescence level (expressed as F). The values ​​represent the mean SEM of three independent experiments analyzed with GraphPad Prism 5 software. Statistical significance was analyzed using a two-tailed unpaired T test.

[0009] Furthermore, the method for culturing HEK293E cells is to culture them at 37° C. in Opti-MEM (Optimized medium) culture medium supplemented with 5% fetal bovine serum (FBS).

[0010] Furthermore, the cell washing method is to wash the cells with Hank's buffered salt solution (HBSSH) containing 20 mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES).

[0011] Step 3) Sensory evaluation, prepare sample solutions and control solutions. All solutions are prepared 24 hours before the test and stored at 4°C until use. Take out in advance when using and place at room temperature. Subjects should not eat or drink water for at least 1 hour before the test. Before the test, 10mL of sucrose solution containing aroma compounds and pure sucrose solution of equal concentration are injected into a 30mL transparent plastic cup. The test samples are coded with three random numbers and provided to the sensory evaluation team at room temperature of 25°C. The panelists are required to rinse their mouths before the test. For each sample, the panelists first pour all the liquid in the cup into their mouths without a nose clip, rotate it in the mouth for 3-5s, and spit it out, and ask the sensory personnel to choose the sweeter cup from each group of one cup with aroma substances added and one cup without adding. After each evaluation, the panelists rinse their mouths with water and have up to 1 minute to clear the taste in their mouths, then wear the nose clip and repeat the above steps. Data analysis uses non-parametric tests, and chi-square tests are used to determine the statistical significance of the data.

[0012] Furthermore, the sample solution is a mixed solution of 30 g / L sucrose and maltol, the concentration of maltol is 26.1 mg / kg, and the control group solution is a 30 g / L pure sucrose solution.

[0013] Furthermore, the sensory evaluation was conducted by 26 well-trained expert panel members from the Tobacco and Aromatic Plant Quality and Safety Risk Assessment Laboratory, a total of 13 males and 13 females, aged 18-26 years old.

[0014] Furthermore, the sweetening substance is a sweetener.

[0015] Beneficial effects of the present invention:

[0016] 1. Molecular docking technology can accurately and quickly screen out compounds with potential sweet taste receptor activation ability from a huge molecular library, greatly reducing the scope of subsequent verification and improving screening efficiency.

[0017] 2. Use in vitro cell function tests to verify the functionality of the screened candidate molecules to ensure that they can indeed trigger a sweet taste response at the cellular level. This step enhances the scientific nature and reliability of the screening results.

[0018] 3. Through the sensory evaluation process, the actual sweetness perception of the candidate molecules is directly evaluated, ensuring that the selected sweeteners not only meet theoretical predictions, but also meet the sensory needs of consumers, providing the food industry with a more market-oriented solution.

[0019] 4. The operation process of the present invention is concise and clear, and the whole process from screening to verification can be completed in only three steps, which greatly reduces the R&D cost and time cost, and opens up a new way for the rapid development and application of new sweeteners. The present invention has demonstrated unprecedented comprehensive advantages in the field of new sweetener screening, and has injected new vitality into food science research and industrial development.

[0020] 5. The present invention involves multiple fields such as computational biology, cell biology, sensory science, etc., promotes the cross-integration of food science and biotechnology, helps to promote scientific and technological progress and industrial upgrading in related fields, and provides more innovative solutions for food safety, nutrition and health. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Detailed information on the maltol binding site in hTas1R2 / hTas1R3

[0022] Figure 2 Dose-response curves of maltol stimulation at different concentrations DETAILED DESCRIPTION

[0023] The present invention is described in more detail hereinafter to facilitate understanding of the present invention.

[0024] The present invention provides a method for screening and verifying maltol as a sweetening substance, characterized in that the method comprises the following steps:

[0025] Step 1), homology modeling, based on the sequence similarity comparison results, metabotropic glutamate receptor 5 (mGlu5, PDB: 6N52) was finally selected as the template, and homology modeling was performed using the online SWISS-MODEL program. Tas1R2 (Genbank accession number: Q8TE23) and Tas1R3 (Q7RTX0) were uploaded for modeling after multiple sequence alignment with the template using ClustalW. The quality of the model was evaluated by the ERRAT and PROCHECK programs, and the score met the requirements. The flexible docking mode of the molecular docking software Autodock was used to construct a docking model of maltol and the sweet taste receptor Tas1R2 / Tas1R3, and a visual analysis was performed based on Pymol. The interaction between maltol and the sweet taste receptor was determined by analyzing the docking binding energy and interaction mode of the two.

[0026] The experimental results show that the minimum docking binding free energy of maltol and sweet taste receptor Tas1R2 / Tas1R3 is -4.97kcal / mol, which is less than -1.2kcal / mol, indicating that the conformation obtained by docking is reasonable. Through Autodock prediction, it is believed that maltol has the ability to activate sweet taste receptors and induce sweetness perception. Figure 1The binding site of maltol in the receptor is shown (Note: the carbon atoms of maltol are represented by pink, and their AH, B and residues that interact with the ligand are represented by orange-red, dark blue and light blue respectively). This site is located in the extracellular VFTM domain of Tas1R2. The hydroxyl group on maltol can act as a hydrogen bond donor to form a hydrogen bond with glutamine Q435 at position 435 in Tas1R2. In addition, phenylalanine F437 at position 437 and phenylalanine F438 at position 438 of Tas1R2 can act as hydrogen bond acceptors to form hydrogen bonds with oxygen atoms in maltol. The oxygen atom with strong electronegativity on the maltol ring structure can be used as a hydrogen bond acceptor B to bind to the adjacent hydroxyl group, imino group and other groups that can donate hydrogen atoms on the sweet receptor to form hydrogen bonds, and vice versa. The formation of hydrogen bonds allows maltol and the sweet receptor to form an activated complex together. The sweet receptor is activated, and the nerve fibers transmit the generated signals to the brain, thereby producing sweet stimulation. In summary, the hydroxyl and carbonyl groups in maltol are AH and B donors necessary for the formation of sweet taste perception. In addition, the pyran ring of maltol can also produce hydrophobic interactions with the benzene ring in phenylalanine. As a weak interaction, hydrophobic interaction is also an important factor affecting the stability of the complex. The above results show that, theoretically, maltol can activate the human sweet taste receptor Tas1R2 / Tas1R3 through hydrogen bonds and hydrophobic interactions, causing sweet taste perception, so it has the potential to become a new sweetener.

[0027] Step 2) Based on the in vitro functional test at the cell level, the functional detection of wild-type Tas1R2 / Tas1R3 was performed using a calcium stimulation procedure. HEK293E cells were cultured in Opti-MEM (Opti-mized medium) medium supplemented with 5% fetal bovine serum (FBS) at 37°C. The cells were seeded on a 96-well plate, and then Tas1R2 / Tas1R3 and Ga16-gust44 vectors were transfected into the cells using Lipofectamine 2000. After 48 hours of growth, the culture medium was replaced at regular intervals, and then the cells were washed with Hank's buffered saline solution (HBSSH) containing 20mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), and then 50uL 3mM Fluo-4 AM was added, incubated for 1 hour, and then washed three times with HBSSH. After stimulation with the aroma solution, the calcium response changes were recorded on the FlexStation 3 multifunctional microplate reader using fluorescence changes 488nm excitation, 525nm emission, and 515nm cutoff. The intensity of calcium response is expressed as the percentage change (peak fluorescence - baseline fluorescence level, expressed as ΔF) compared with the baseline fluorescence level (expressed as F). Values ​​represent the mean SEM of three independent experiments analyzed by GraphPad Prism 5 software. Statistical significance was determined by two-tailed unpaired T test.

[0028] Sweet taste is caused by the binding of sweet molecules to sweet receptors. When the sweet receptor Tas1R2 / Tas1R3 binds to the sweet molecules, hydrogen bonds are formed between the sweet molecules and the receptors, which causes the ion bonds and hydrogen bonds between the extracellular domains of the sweet receptor proteins to break, and eventually changes the contracted conformation of the receptor proteins to form an open conformation, which is called the activated state of the receptor proteins. When the sweet receptors are activated, they induce the intracellular Ca 2+ Increase, monitor Ca 2+ The fluctuation of can characterize the binding effect between sweet substances and receptors. This application uses Tas1R2, Tas1R3 and G protein plasmids with good functionality for transfection. By stimulating HEK293 cells that stably express Tas1R2, Tas1R3 and G protein with different concentrations of maltol solution, we obtained the concentration-response intensity curve of aroma compounds, which showed obvious S-shaped characteristics, such as Figure 2 As shown. The curves show that maltol solutions at different concentrations can cause an increase in the intracellular response signal, and the intracellular signal increases with the increase in concentration. The above results prove that maltol can cause changes in the intensity of the response in HEK293 cells, that is, maltol can effectively activate sweet taste receptor cells and transmit sweet taste perception signals to the brain. Combined with the molecular docking results, we believe that maltol has the potential to become a new sweetener both at the theoretical level and at the cellular level.

[0029] Step 3) sensory evaluation, the sample solution is a mixed solution of 30g / L sucrose and maltol, the maltol concentration is 26.1mg / kg, the control group is a 30g / L pure sucrose solution, all solutions are made 24h before the test and stored at 4°C until use. Take it out in advance and place it at room temperature when used. The sensory evaluation was conducted by 26 well-trained expert panel members from the Tobacco and Aromatic Plant Quality and Safety Risk Assessment Laboratory, a total of 13 males and 13 females, aged 18-26 years old. The subjects were not allowed to eat or drink water for at least 1 hour before the test. Before the test, 10mL of sucrose solution containing aroma compounds and pure sucrose solution of equal concentration were injected into a 30mL transparent plastic cup. The test samples were coded with three random numbers and provided to the sensory evaluation team at room temperature of 25°C. The panel members were required to rinse their mouths before the test. For each sample, the panelists first poured all the liquid in the cup into their mouths without a nose clip, spit it out after rotating it in their mouths for 3-5 seconds, and asked the sensory personnel to choose the sweeter cup from each group, one with added aroma substances and one without. After each evaluation, the panelists rinsed their mouths with water and had up to 1 minute to clear the taste in their mouths, then put on the nose clip and repeated the above steps. Data analysis used non-parametric tests, and the chi-square test was used to determine the statistical significance of the data.

[0030] The experimental results are shown in Table 1. The results show that when there is no nose clip, maltol can enhance the sweetness perceived by sensory evaluators in both pure water solution and 30g / L sucrose solution. When the sensory evaluators wear the nose clip, the olfactory pathway is blocked. At this time, maltol can still significantly enhance the perception of sweetness, and this enhancement effect is even more significant in 30g / L sucrose solution. From this, we can infer that even without the participation of the olfactory system, maltol can cause an enhancement in human sweetness perception by activating sweet taste receptors. Therefore, we believe that maltol can significantly enhance its sweetness perception in the presence or absence of sweet substances. In summary, maltol can not only be used as a sweetener to enhance the sweetness perception caused by sweet substances, but it can also be used as a sweetener to increase the sweetness in sugar-free products.

[0031] Table 1 Maltol sweetness evaluation results

[0032]

[0033] Note: The threshold is the threshold in water. Non-parametric chi-square test was used for significance analysis. * and ** indicate that the data are significantly or extremely significantly different from those in the control group.

[0034] The preferred embodiments of the present invention are described above, but they are not intended to limit the present invention. Those skilled in the art may make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the present invention.

Claims

1. A method for screening and verifying maltol as a sweetening substance, characterized in that: The method comprises the following steps: Step 1) Homology modeling: According to the sequence similarity comparison results, metabolic glutamate receptor 5 was finally selected as the template. Homology modeling was performed using the online SWISS-MODEL program. Tas1R2 and Tas1R3 were multiple sequence aligned with the template using ClustalW and uploaded for modeling. The quality of the model was evaluated by ERRAT and PROCHECK programs. The score met the requirements. The flexible docking mode of the molecular docking software Autodock was used to construct a docking model of maltol and the sweet taste receptor Tas1R2 / Tas1R3. Visual analysis was performed based on Pymol. The interaction between maltol and the sweet taste receptor was determined by analyzing the docking binding energy and interaction mode of the two. Step 2) In vitro functional testing based on cell level, the functional detection of wild-type Tas1R2 / Tas1R3 was performed using a calcium stimulation procedure. HEK293E cells were cultured and seeded on 96-well plates. Tas1R2 / Tas1R3 and Ga16-gust44 vectors were then transfected into the cells using Lipofectamine2000. After 48 hours of growth, the culture medium was replaced at regular intervals, and the cells were washed and then added with 50 uL 3 mM Fluo-4 AM. The cells were incubated for 1 hour and then washed three times with HBSSH. After stimulation with aroma solution, the calcium response changes were recorded on a FlexStation 3 multi-function microplate reader using fluorescence changes at 488 nm excitation, 525 nm emission, and 515 nm cutoff. The calcium response intensity was expressed as a percentage of the baseline fluorescence level. The values ​​represent the mean SEM of three independent experiments analyzed by GraphPad Prism 5 software. The statistical significance was analyzed using a two-tailed unpaired T test. Step 3) Sensory evaluation: prepare sample solutions and control group solutions. All solutions were prepared 24 h before the test and stored at 4°C until use. They were taken out in advance and placed at room temperature. The subjects were not allowed to eat or drink water for at least 1 hour before the test. Before the test, 10 mL of sucrose solution containing aroma compounds and pure sucrose solution of equal concentration were injected into a 30 mL transparent plastic cup. The test samples were coded with three-digit random numbers and provided to the sensory evaluation team at room temperature of 25°C. The team members rinsed their mouths before the test. For each sample, the team members first poured all the liquid in the cup into their mouths without a nose clip, rotated it in the mouth for 3-5 s, and spit it out. The sensory personnel were asked to select the sweeter cup from each group, one with added aroma substances and one without. After each evaluation, the team members rinsed their mouths with water and had up to 1 minute to clear the taste in their mouths. They then wore the nose clip and repeated the above steps. Non-parametric tests were used for data analysis, and chi-square tests were used to determine the statistical significance of the data.

2. The method for screening and verifying maltol as a sweetening substance according to claim 1, characterized in that: The method for culturing HEK293E cells is to culture them at 37° C. in Opti-MEM medium supplemented with 5% fetal bovine serum.

3. The method for screening and verifying maltol as a sweetening substance according to claim 1, characterized in that: The method for washing cells is to wash the cells with Hank's buffered saline solution containing 20 mM 4-hydroxyethylpiperazineethanesulfonic acid.

4. The method for screening and verifying maltol as a sweetening substance according to claim 1, characterized in that: The sample solution is a mixed solution of 30 g / L sucrose and maltol, the concentration of maltol is 26.1 mg / kg, and the control group solution is a 30 g / L pure sucrose solution.

5. The method for screening and verifying maltol as a sweetening substance according to claim 1, characterized in that: The sensory evaluation was conducted by 26 well-trained expert panel members from the Tobacco and Aromatic Plant Quality and Safety Risk Assessment Laboratory, a total of 13 males and 13 females, aged 18-26 years old.

6. The method for screening and verifying maltol as a sweetening substance according to claim 1, characterized in that: The sweetening substance is a sweetener.

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