Umami peptide, method for identifying same, and use thereof

Abstract

The application relates to the technical field of bioactive peptides, and provides umami peptides, an identification method and application thereof, the umami peptides comprising the following amino acid sequences: X1-X2-X3-X4-X5-X6-X7-X8 or Y1-Y2-Y3-Y4-Y5-Y6, wherein X1 comprises Ser, X2 comprises Gly, X3 comprises Glu, X4 comprises Thr, X5 comprises Leu, X6 comprises Thr, X7 comprises Phe, and X8 comprises Lys; Y1 comprises Leu, Y2 comprises Leu, Y3 comprises Asp, Y4 comprises Asp, Y5 comprises Leu, and Y6 comprises Arg. The umami peptides provided in the embodiments have good umami.

Description

Technical Field

[0001] This application relates to the field of bioactive peptide technology, and in particular to a umami peptide and its identification method and application. Background Technology

[0002] Umami is a pleasant taste that is essential for improving the flavor of food, such as enhancing the palatability of vegetables, broths, and sausages. Umami peptides are an important component of umami, not only imparting a delicious flavor to food, enhancing its umami taste and improving its smooth and mellow mouthfeel, but also synergistically enhancing umami with other substances and reducing the intake of NaCl and MSG.

[0003] However, the umami peptides currently available lack sufficient umami flavor. Therefore, reducing the salt, sugar, and fat content in food without compromising its taste and flavor while ensuring nutritional value is a pressing problem that the food industry needs to solve.

[0004] It should be noted that the above content is not necessarily prior art, nor is it intended to limit the scope of protection of this application. Summary of the Invention

[0005] This application provides an umami peptide, its identification method, and its application to solve or alleviate one or more of the technical problems mentioned above.

[0006] A first aspect of this application provides a umami peptide. The umami peptide comprises the following amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8 or Y1-Y2-Y3-Y4-Y5-Y6, wherein X1 includes Ser, X2 includes Gly, X3 includes Glu, X4 includes Thr, X5 includes Leu, X6 includes Thr, X7 includes Phe, and X8 includes Lys; Y1 includes Leu, Y2 includes Leu, Y3 includes Asp, Y4 includes Asp, Y5 includes Leu, and Y6 includes Arg.

[0007] The umami peptides in this application have good umami flavor. These umami peptides exhibit significant umami activity, with an umami threshold lower than that of the traditional umami substance monosodium glutamate (MSG), and possess a synergistic umami-enhancing effect, capable of improving the overall umami intensity of food without significantly increasing sodium intake.

[0008] A second aspect of this application provides a method for identifying umami peptides. The method includes the following steps: reacting Grifola frondosa sequentially with cellulase and flavor protease to obtain a mixed enzymatic hydrolysate containing umami peptides; purifying and identifying the mixed enzymatic hydrolysate to obtain candidate peptides; and screening the candidate peptides to obtain umami peptides, wherein the umami peptides include the following amino acid sequences: X1-X2-X3-X4-X5-X6-X7-X8 or Y1-Y2-Y3-Y4-Y5-Y6, wherein X1 includes Ser, X2 includes Gly, X3 includes Glu, X4 includes Thr, X5 includes Leu, X6 includes Thr, X7 includes Phe, and X8 includes Lys; Y1 includes Leu, Y2 includes Leu, Y3 includes Asp, Y4 includes Asp, Y5 includes Leu, and Y6 includes Arg.

[0009] The identification method described in this application is simple to operate and has good reproducibility. Computer-aided identification saves significant costs and improves identification efficiency. The umami peptides prepared by this method have the advantages of a low umami threshold, significant synergistic umami enhancement effect, non-toxicity, and good water solubility, and can be used for food umami enhancement and salt reduction seasoning.

[0010] A third aspect of this application provides the application of the umami peptide described in the first aspect or the umami peptide obtained by the identification method described in the second aspect in the preparation of food flavor enhancers. The umami peptide of this application has a pure umami flavor and a low umami threshold, and can be used to prepare the foods including seasonings, snack foods, soup bases, noodle products, meat products, or dairy products. The umami peptide can also be used alone. Attached Figure Description

[0011] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the various drawings denote the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings depict only some embodiments disclosed in this application and should not be construed as limiting the scope of this application.

[0012] Figure 1 This is the secondary mass spectrum of umami peptides; Figure 2 This is a molecular docking diagram of umami peptides; Figure 3 Sensory radar images for SGETLTFK and LLDDLR; Figure 4 Radar image of the electronic tongue for SGETLTFK and LLDDLR. Detailed Implementation

[0013] The embodiments of this application are described in detail below, examples of which are illustrated in the accompanying drawings. In the drawings, for clarity, the dimensions of layers, regions, and elements, as well as their relative dimensions, may be exaggerated. Throughout, the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain this application, and should not be construed as limiting this application. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other.

[0014] In this application, unless otherwise expressly specified and limited, the terms "installation," "connection," "joining," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components, unless otherwise expressly limited. Those skilled in the art can understand the specific meaning of the above terms in this application according to the specific circumstances.

[0015] It should be noted that the terms "first," "second," etc., used in this application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such terms can be used interchangeably where appropriate so that the embodiments of this application described herein can be implemented, for example, in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0016] In this application, when numerical intervals (i.e., numerical ranges) are involved, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed in this application should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include percentage intervals, ratio intervals, proportion intervals, etc.

[0017] A first aspect of this application provides a umami peptide. The umami peptide comprises the following amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8 or Y1-Y2-Y3-Y4-Y5-Y6, wherein X1 includes Ser, X2 includes Gly, X3 includes Glu, X4 includes Thr, X5 includes Leu, X6 includes Thr, X7 includes Phe, and X8 includes Lys; Y1 includes Leu, Y2 includes Leu, Y3 includes Asp, Y4 includes Asp, Y5 includes Leu, and Y6 includes Arg.

[0018] The umami peptides in this application have good umami flavor. These umami peptides exhibit significant umami activity, with an umami threshold lower than that of the traditional umami substance monosodium glutamate (MSG), and possess a synergistic umami-enhancing effect, capable of improving the overall umami intensity of food without significantly increasing sodium intake.

[0019] In some embodiments, the amino acid sequence is Ser-Gly-Glu-Thr-Leu-Thr-Phe-Lys, the molecular weight is 881.4494 Da, the umami threshold is 0.0709 mmol / L, and the synergistic umami enhancement threshold is 0.0354 mmol / L.

[0020] In some embodiments, the amino acid sequence is Leu-Leu-Asp-Asp-Leu-Arg, the molecular weight is 743.4177 Da, the umami threshold is 0.1680 mmol / L, and the synergistic umami threshold is 0.0840 mmol / L.

[0021] The aforementioned umami threshold refers to the lowest concentration at which a human taste bud can perceive the presence of a certain umami substance (such as monosodium glutamate). The testing method involves preparing a 0.5 mg / ml solution of umami peptide (SGETLTKF) or (LLDDLR), diluting it at a 1:1 (v / v) ratio, and having sensory evaluators taste the solution in ascending order of concentration. Each evaluator holds 2 ml of the diluted solution in their mouth for 5 seconds and then spits it out. After tasting one sample, they rinse their mouth with water and wait 5 minutes before tasting the next sample. Each dilution level is evaluated using a triangular test until the evaluator can no longer distinguish between one sample solution and two ultrapure aqueous solutions; this dilution level is the umami threshold.

[0022] The aforementioned synergistic flavor-enhancing threshold refers to the lowest total concentration of umami substances that can be perceived by the human palate when two or more flavor compounds are mixed. The testing method involves preparing a 0.5 mg / ml solution of umami peptide (SGETLTKF) or (LLDDLR) with 1 mg / ml monosodium glutamate (MSG). This solution is then diluted 1:1 (v / v) and presented to sensory evaluators in ascending order of concentration. Each evaluator holds 2 ml of the diluted solution in their mouth for 5 seconds and then spits it out. After tasting one sample, the evaluator rinses their mouth with water and waits 5 minutes before tasting the next sample. Each dilution level is evaluated using a triangular test until the evaluator can no longer distinguish between one sample solution and two pure MSG solutions. The dilution level at this point is the synergistic flavor-enhancing threshold of the peptide.

[0023] The molecular weight mentioned above refers to the sum of the molecular weights of all amino acids that make up the peptide chain. The test method involves dissolving the sample in ultrapure water to prepare a 2 mg / mL solution, centrifuging at 10000 r / min for 10 min at 4℃, filtering through a 0.22 µm filter membrane, and then analyzing using ultra-high performance liquid chromatography-ion mobility-quadrupole time-of-flight mass spectrometry (UHPLC-IOM-QT-MS). The chromatographic column was a reversed-phase C18 column (2.1 mm × 100 mm, 2.2 µm); the flow rate was 0.3 mL / min; gradient elution was used with mobile phase A (containing 0.1 vol% formic acid aqueous solution) and mobile phase B (acetonitrile solution): 0 min–5 min: 98% A, 2% B; 5 min–8 min: 98%–95% A, 2%–5% B; 8 min–15 min: 95% A, 5% B; 15 min–20 min: 95%–85% A, 5%–15% B; 20%–25% B… min: 85%~80%A, 15%~20%B; 25min~30min: 80%~70%A, 20%~30%B; 30min~40min: 70%~15%A, 30%~85%B; 40min~45min: 15%A, 85%B; m / z: 50~1200; high-purity nitrogen is used for drying gas; flow rate is 8L / min; equipped with ESI-MS; atomizing gas pressure is 2bar.

[0024] Understandably, during gradient elution, the content gradient of mobile phase A decreases while the content gradient of mobile phase B increases.

[0025] Umami peptides can enhance the brain's perception of umami through interaction with umami receptors. T1R1 / T1R3, members of the G protein-coupled receptor family, are widely considered as heterodimers and are the primary receptors for umami sensation. In recent years, homology modeling and molecular docking have been primarily used in umami peptide research to calculate the binding sites and interactions between umami peptides and T1R1 / T1R3, explore the flavor-producing mechanisms of umami peptides, and provide an effective method for screening umami peptides. Molecular docking technology can shorten the time required for protein purification. This technology can be used to screen for desired umami peptides, predict and verify their biological activity, and obtain the umami peptides with the strongest umami flavor. Currently, the preparation of umami peptides is characterized by low efficiency, time-consuming and labor-intensive processes, and complex operations, making it difficult to efficiently screen for umami peptides with low threshold values.

[0026] Accordingly, a second aspect of this application provides a method for identifying umami peptides. The method includes the following steps: S100: reacting Grifola frondosa sequentially with cellulase and flavor protease to obtain a mixed enzymatic hydrolysate containing umami peptides; S200: purifying and identifying the mixed enzymatic hydrolysate to obtain candidate peptides; S300: screening the candidate peptides to obtain umami peptides, wherein the umami peptides include the following amino acid sequences: X1-X2-X3-X4-X5-X6-X7-X8 or Y1-Y2-Y3-Y4-Y5-Y6, wherein X1 includes Ser, X2 includes Gly, X3 includes Glu, X4 includes Thr, X5 includes Leu, X6 includes Thr, X7 includes Phe, and X8 includes Lys; Y1 includes Leu, Y2 includes Leu, Y3 includes Asp, Y4 includes Asp, Y5 includes Leu, and Y6 includes Arg.

[0027] The identification method described in this application is simple to operate and has good reproducibility. Computer-aided identification saves significant costs and improves identification efficiency. The umami peptides prepared by this method have the advantages of a low umami threshold, significant synergistic umami enhancement effect, non-toxicity, and good water solubility, and can be used for food umami enhancement and salt reduction seasoning.

[0028] According to an embodiment of this application, in step S100, Grifola frondosa is reacted sequentially with cellulase and flavor protease to obtain a mixed enzymatic hydrolysate containing umami peptides.

[0029] In some embodiments, the reaction includes: mixing Grifola frondosa with water and placing the mixture in a flash extractor to obtain a flash extract; reacting the flash extract with cellulase at pH 3.5-5.5 to carry out a first reaction; reacting the product of the first reaction with a flavor protease at pH 5.0-7.0 to carry out a second reaction; and subjecting the product of the second reaction to enzyme inactivation treatment, centrifugation treatment, and concentration treatment to obtain a mixed enzymatic hydrolysate containing umami peptides.

[0030] Furthermore, the conditions for obtaining the flash extract include a extraction time of 20-120 seconds, a voltage of 160V-210V, at least one extraction cycle, and a mass ratio of Grifola frondosa to water of 1:(8-30). This allows for the rapid and efficient release of proteins from Grifola frondosa, providing a foundation for subsequent first and second reactions. The flash extractor allows for cell disruption in a short time, and the flash extraction can be performed at room temperature.

[0031] Optionally, the number of extractions is 1 to 5 times, for example, 1 time, 2 times, 3 times, 4 times, or 5 times.

[0032] In some embodiments, the first reaction is an enzymatic hydrolysis reaction, in which cellulase is used to directionally break down substances in Grifola frondosa. The mass ratio of cellulase to Grifola frondosa is (0.12-0.2):1, for example, 0.012:1, 0.015:1, 0.017:1, 0.019:1, 0.02:1, etc. This improves the efficiency of enzymatic hydrolysis in the first reaction process.

[0033] Optionally, the temperature of the first reaction is 35℃-55℃, such as 35℃, 40℃, 45℃, 50℃, 55℃, etc., and the time is 1.5h-2.5h, such as 1.5h, 2.0h, 2.5h, etc. Under the aforementioned temperature and time conditions, cellulase can maintain high activity, promoting the enzymatic hydrolysis of Grifola frondosa.

[0034] In some embodiments, the second reaction is also an enzymatic hydrolysis reaction, using a flavor protease to hydrolyze the cellulase product. The mass ratio of the flavor protease to maitake mushroom is (0.03-0.05):1, for example, 0.03:1, 0.035:1, 0.04:1, 0.045:1, 0.05:1, etc. This allows for the efficient and targeted conversion of maitake mushroom protein into small-molecule umami peptides. Optionally, the temperature of the second reaction is 40℃-60℃, such as 40℃, 45℃, 50℃, 55℃, 60℃, etc., and the time is 1.5h-2.5h, such as 1.5h, 2.0h, 2.5h, etc. Under the aforementioned temperature and time conditions, the yield of candidate peptides can be optimized, and the inactivation of flavor proteases can be reduced during enzymatic hydrolysis.

[0035] In some embodiments, the enzyme inactivation treatment involves heating the product from the second reaction to 70°C-100°C and inactivating the enzyme for 5-15 minutes. This further increases the content of the candidate peptide. In some embodiments, the centrifugation conditions include centrifuging the enzyme-inactivating product at 5000 r / min-9000 r / min for 10 min-20 min at 1 °C-10 °C.

[0036] As a specific example, dried Grifola frondosa powder was added to ultrapure water at a material-to-liquid ratio of 1:10 g / mL to 1:30 g / mL and mixed thoroughly. The mixture was then placed in a flash extractor, with an extraction time of 20-120 seconds, an extraction voltage of 160V-210V, and 1-5 extraction cycles. After flash pretreatment, the solution pH was adjusted to 3.5-5.5, and 0.12%-0.2% cellulase (based on the mass of Grifola frondosa powder) was added. Enzymatic hydrolysis was performed at 35℃-55℃ for 1.5-2.5 hours. The solution pH was then adjusted to 5.0-7.0, and 0.03wt%-0.05wt% flavor protease (based on the mass of Grifola frondosa powder) was added. Enzymatic hydrolysis was performed at 40℃-60℃ for 1.5-2.5 hours. After hydrolysis, the temperature was rapidly increased to 90℃ for 10 minutes to inactivate the enzyme. After the enzymatic hydrolysate cools, centrifuge at 7000 r / min for 15 min at 4℃, collect the supernatant, concentrate it and use it for later use.

[0037] According to an embodiment of this application, step S200: the mixed enzymatic hydrolysate is purified and identified to obtain candidate peptides.

[0038] In some embodiments, the mixed enzymatic hydrolysate is purified, the purification process including ethanol fractionation, enrichment, and purification. Optionally, ethanol fractionation includes adding anhydrous ethanol to the mixed enzymatic hydrolysate to achieve ethanol volume concentrations of 20%, 40%, 60%, and 80% sequentially, stirring at 1500 rpm for 20 min at room temperature, and then centrifuging at 8000 rpm for 20 min. The precipitate obtained by centrifugation is reconstituted with water, residual ethanol is removed by rotary evaporation, and then it is freeze-dried or spray-dried to obtain a powder, thus obtaining a first powder.

[0039] In some embodiments, the first powder is enriched. A macroporous resin is used in the enrichment process. The first powder is prepared into a first aqueous solution, which is placed in a chromatography column. Ultrapure water is used as the eluent. The flow rates of the eluent and the first loading solution are controlled, and the chromatography column is irradiated with ultraviolet light to obtain the first enriched component.

[0040] The ratio of the mass of the macroporous resin to the volume of the first aqueous solution is 1:(2-6)g / mL, which results in a higher yield of candidate peptides and a higher utilization rate of the macroporous resin.

[0041] Furthermore, in the first aqueous solution, the concentration of the first powder is controlled between 10 mg / ml and 30 mg / ml. The first loading solution formed by the first aqueous solution and the macroporous resin has a flow rate that is 1 to 1.5 times that of the eluent flow rate.

[0042] Furthermore, the flow rate of the eluent is 0.5 mL / min to 2 mL / min.

[0043] As a specific example, macroporous resin enrichment was performed using Amberlite XAD 16N, with a first aqueous solution concentration of 20 mg / mL, ultrapure water as the eluent, an eluent flow rate of 1 mL / min, a loading volume (the first loading solution formed by the first aqueous solution and the macroporous resin) of 10 mL, a glass chromatography column of 2.6 cm × 60 cm, and a UV detection wavelength of 220 nm. The first enriched fraction was obtained after irradiation.

[0044] Furthermore, it also includes rotary evaporation, concentration, and drying of the first enriched component to form a second powder. The conditions for rotary evaporation, concentration, and drying are not specifically limited.

[0045] In some embodiments, purification is performed using a gel. The second powder is prepared into a second aqueous solution, which is placed in a chromatography column. Ultrapure water is used as the eluent. The flow rate of the eluent and the flow rate of the second loading solution are controlled, and the chromatography column is irradiated with ultraviolet light to obtain a second enriched fraction. The second enriched fraction is then subjected to rotary evaporation, concentration, and drying to form a third powder.

[0046] The ratio of the mass of the dextran gel particles to the volume of the second aqueous solution is 1:(2-6) g / mL, which results in a higher yield of candidate peptides and a higher utilization rate of the dextran gel particles.

[0047] Furthermore, in the second aqueous solution, the concentration of the second powder is controlled between 10 mg / ml and 30 mg / ml. The second aqueous solution forms a second loading solution with the dextran gel particles, and the flow rate of the second loading solution is 1-1.5 times the flow rate of the elution solution.

[0048] Furthermore, the flow rate of the eluent is 0.5 mL / min to 2 mL / min.

[0049] As a specific example, the enrichment of dextran gel particles was performed using Sephadex G-15, with a second aqueous solution concentration of 20 mg / mL, ultrapure water as the eluent, an eluent flow rate of 0.8 mL / min, a loading volume (the second loading solution formed by the second aqueous solution and the dextran gel) of 8 mL, a glass chromatography column of 2.6 cm × 60 cm, and a UV detection wavelength of 220 nm. After irradiation, the second enriched fraction was obtained.

[0050] In some embodiments, the third powder is identified to obtain candidate peptides. The identification is performed using liquid chromatography-tandem mass spectrometry. Specifically, the mass spectrometer is connected to an EASY-nanoLC system, set to data-dependent acquisition (DDA) mode, and automatically switches between MS and MS / MS modes. Chromatographic conditions: Poroshell 120 EC-C18 column (4.6 mm × 150 mm, 4 μm); column temperature 30 °C; injection volume 2 μL (0.1 vol% formic acid-water solution); flow rate 500 nL / min; mobile phase A: 0.1% (v / v) formic acid-water solution; mobile phase B: 0.1% (v / v) formic acid-acetonitrile solution (84%), column equilibrated with 94% of solution A; analysis time 60 min. Gradient elution: 0~38 min: 6%~23% (v / v) B; 38 min~50 min: 23%~32% (v / v) B; 50~55 min: 32%~80% (v / v) B; 55 min~60 min: 80% (v / v) B.

[0051] Mass spectrometry conditions: analysis time 60 min; capillary temperature 320℃; ion source spray voltage 2.3 kV; capillary voltage 45 V; normalized collision energy 27 eV; ion source ESI; detection mode positive ion; precursor ion scan range 200 m / z ~ 1500 m / z; scan mode Full MS, dd-MS2; resolution Full MS: 70000 m / z, dd-MS2: 17500 m / z.

[0052] According to an embodiment of this application, step S300: the candidate peptides are screened to obtain umami peptides, wherein the umami peptides include the following amino acid sequences: X1-X2-X3-X4-X5-X6-X7-X8 or Y1-Y2-Y3-Y4-Y5-Y6, wherein X1 includes Ser, X2 includes Gly, X3 includes Glu, X4 includes Thr, X5 includes Leu, X6 includes Thr, X7 includes Phe, and X8 includes Lys; Y1 includes Leu, Y2 includes Leu, Y3 includes Asp, Y4 includes Asp, Y5 includes Leu, and Y6 includes Arg.

[0053] In some embodiments, the screening includes: obtaining characteristic conditions of the umami peptide, the characteristic conditions including ALC greater than 95%, peptide length of 3-9, molecular weight less than 1000 Da, and the peptide containing both basic and acidic amino acids, the basic amino acids including Lys, His, or Arg, and the acidic amino acids including Glu, Asp, Asn, or Gln; performing flexible molecular docking on candidate peptides according to the characteristic conditions, and selecting candidate peptides with a first threshold greater than 6; predicting the candidate peptides with the first threshold greater than 6, the prediction conditions including umami activity, toxicity, and water solubility, to obtain candidate peptides with umami activity greater than 588; and performing semi-flexible docking on the candidate peptides with umami activity greater than 588 to obtain the umami peptide.

[0054] Among them, short peptides with umami activity usually have common characteristics. Umami peptides are mostly small molecule peptides produced by enzymatic hydrolysis. The selection criteria include ALC of more than 95%, peptide length of 3-9, molecular weight of less than 1000 Da, and the presence of both basic and acidic amino acids in the peptide. The basic amino acids include Lys, His or Arg, and the acidic amino acids include Glu, Asp, Asn or Gln. These characteristics can be used to quickly narrow down the candidate peptide range and then select umami peptides.

[0055] In this study, SYBYL-X 2.0 software was used to perform flexible molecular docking on the screened candidate peptides, identifying those with a first threshold (T-Score) greater than 6. This method preserves candidate peptides with potential umami activity. A first threshold greater than 6 comprehensively considers the effects of ligand-receptor binding polarity, hydrophobicity, enthalpy, and solvation, converting these factors into a numerical value. This value represents the binding strength and affinity of the umami peptide to T1R1 / T1R3. When the first threshold is greater than 6, the docking results are stable.

[0056] In this process, candidate peptides with a first threshold greater than 6 are predicted, with prediction conditions including umami activity, toxicity, and water solubility, resulting in candidate peptides with an umami activity greater than 588. This evaluation of umami peptides from multiple dimensions, including umami activity, toxicity, and water solubility, improves the safety of the final umami peptides. Setting an umami activity greater than 588 further ensures that the screened candidate peptides have strong umami potential. An umami activity greater than 588 is determined by obtaining umami scores for peptides from the iUmami-SCM database; peptides with scores higher than 588 are considered umami peptides.

[0057] Furthermore, prediction tools include BIOPEP-UWM, ToxinPred, Proteomics-Tools, or iUmami-SCM.

[0058] In some embodiments, the umami receptors in the semi-flexible docking process include T1R1 or T1R3. The umami heterodimer T1R1 / T1R3 receptors are homologous and modeled. 99.8% of the results in the Laplace plot are in the allowable region, which meets the 90% criticality principle. The model is reasonable in terms of dihedral distribution and three-dimensional collision.

[0059] Furthermore, the semi-flexible docking is performed using Autodock Vina software. Based on the software's built-in scoring criteria, the conformation with the lowest docking score is selected as the optimal binding model for the umami peptide and T1R1 / T1R3, thereby screening out the umami peptide.

[0060] In some embodiments, after performing the semi-flexible docking but before obtaining the umami peptide, the process further includes selecting candidate peptides with a second threshold greater than 9. Defining a second threshold (T-Score) greater than 9 results in higher reliability of the semi-flexible docking results.

[0061] In some embodiments, the umami characteristics of the umami peptides are verified using a human sensory rating and an electronic tongue system. The methods of human sensory rating and electronic tongue system analysis are not specifically limited.

[0062] A third aspect of this application provides the application of the umami peptide described in the first aspect or the umami peptide obtained by the identification method described in the second aspect in the preparation of food flavor enhancers. The umami peptide of this application has a pure umami flavor and a low umami threshold, and can be used to prepare the foods including seasonings, snack foods, soup bases, noodle products, meat products, or dairy products. The umami peptide can also be used alone.

[0063] Exemplary embodiments according to this application will now be described in more detail with reference to the accompanying drawings. It should be understood that these exemplary embodiments may be implemented in many different forms and should not be construed as being limited to the embodiments set forth herein.

[0064] Example 1 (1) Preparation of mixed enzyme hydrolysate.

[0065] Dried maitake mushrooms were pulverized using a ball mill and passed through a 60-mesh sieve to obtain dried maitake mushroom powder. Ultrapure water was added at a material-to-liquid ratio of 1:23 g / mL and mixed thoroughly. The mixture was then placed in a flash extractor for pretreatment. The flash extraction parameters were set as follows: extraction time 82 s, extraction voltage 200 V, and extraction once. After flash pretreatment, the pH was adjusted to 4.5, and 0.16 wt% cellulase (relative to the mass of maitake mushrooms) was added. Enzymatic hydrolysis was carried out at 45℃ for 1.5 h. The pH was then adjusted to 6.0, and 0.05 wt% flavor protease (relative to the mass of maitake mushrooms) was added. Enzymatic hydrolysis was carried out at 50℃ for 2 h. After hydrolysis, the temperature was rapidly increased to 90℃ for 10 min to inactivate the enzyme. After cooling, the hydrolysate was centrifuged at 7000 rpm for 15 min at 4℃. The supernatant was collected, concentrated, and used for later use.

[0066] (2) Isolation and purification of Grifola frondosa umami peptides.

[0067] ① Ethanol fractionation.

[0068] Add 25 mL of anhydrous ethanol to the concentrated enzymatic hydrolysate and stir at 25°C for 30 min. Centrifuge at 8000 r / min for 20 min at 4°C to obtain precipitate A and supernatant A. Dissolve precipitate A in ultrapure water to obtain the 20%-F fraction. Add 42 mL of anhydrous ethanol to supernatant A and stir and centrifuge under the same conditions to obtain precipitate B and supernatant B. Dissolve precipitate B in ultrapure water to obtain the 40%-F fraction. Similarly, add 83 mL of anhydrous ethanol to supernatant B to obtain the 60%-F fraction, and add 250 mL of anhydrous ethanol to supernatant C to obtain the 80%-F fraction. Stir at 1500 r / min for 20 min at room temperature and centrifuge at 8000 r / min for 20 min at 4°C. Redissolve the centrifuged precipitate in water, remove residual ethanol by rotary evaporation, and then freeze-dry or spray-dry to obtain the first powder.

[0069] ② Enrichment of macroporous resin.

[0070] The first powder was prepared into a first aqueous solution with a concentration of 20 mg / mL. An Amberlite XAD 16N macroporous resin column (2.6 cm × 60 cm) was used for chromatography. The sample loading volume (first aqueous solution and macroporous resin) was 10 mL. After the resin adsorption reached equilibrium, ultrapure water was used for desorption at a flow rate of 1 mL / min. The solution was collected using an automatic collector for 3 min per tube. The absorbance was measured at a wavelength of 220 nm to obtain the first enriched fraction. The first enriched fraction was then subjected to rotary evaporation, concentration, and drying to form the second powder.

[0071] ③ Gel chromatography separation.

[0072] The second powder was prepared into a second aqueous solution with a concentration of 25 mg / mL. A Sephadex G-15 gel chromatography column (2.6 cm × 60 cm) was used, with a loading volume (second aqueous solution and macroporous resin) of 8 mL. After gel adsorption equilibrium was reached, ultrapure water was used for elution at a flow rate of 0.8 mL / min. The samples were collected using an automatic collector for 3.75 min per tube, and the absorbance was measured at 220 nm. The F2 and F3 fractions, which exhibited the strongest umami flavor, were collected and freeze-dried separately. The freeze-dried sample powders were stored at -20℃ to obtain the third powder.

[0073] ④ Structural identification of polypeptide sequences in F2 and F3.

[0074] The third powder was identified to obtain candidate peptides. Specifically, the third powder was dissolved in a 0.1 wt% formic acid solution, filtered through a 0.22 μm filter membrane, and then placed in a chromatographic vial for later use.

[0075] The UHPLC-Q-Orbitrap-MS / MS determination conditions are as follows: the mass spectrometer is connected to the EASY-nano LC system, the mass spectrometer is set to data-dependent acquisition (DDA) mode, and it automatically switches between MS and MS / MS modes.

[0076] Chromatographic conditions: Poroshell 120 EC-C18 column (4.6 mm × 150 mm, 4 μm); column temperature 30 °C; injection volume 2 μL; flow rate 500 nL / min; mobile phase A: 0.1% (v / v) formic acid-water solution; mobile phase B: 0.1% (v / v) formic acid-acetonitrile solution (84%), column equilibrated with 94% of solution A; analysis time 60 min. Gradient elution: 0–38 min: 6%–23% (v / v) B; 38–50 min: 23%–32% (v / v) B; 50–55 min: 32%–80% (v / v) B; 55–60 min: 80% (v / v) B.

[0077] Mass spectrometry conditions: analysis time 60 min; capillary temperature 320℃; ion source spray voltage 2.3 kV; capillary voltage 45 V; normalized collision energy 27 eV; ion source ESI; detection mode positive ion; precursor ion scan range 200 m / z ~ 1500 m / z; scan mode Full MS, dd-MS2; resolution Full MS: 70000 m / z, dd-MS2: 17500 m / z.

[0078] (3) Identification of umami peptides.

[0079] After analyzing the mass spectrometry data collected in step (2) using De novo software and searching the corresponding species database, the obtained umami peptides were screened according to the following conditions: ALC greater than 95%, peptide length 3-9, molecular weight less than 1000 Da, and the peptide containing both basic and acidic amino acids. The basic amino acids included Lys, His, or Arg, and the acidic amino acids included Glu, Asp, Asn, or Gln. The screened peptides were subjected to flexible molecular docking using SYBYL-X 2.0 software, and candidate peptides with a total docking score (T-Score) greater than 6.0 were entered into the BIOPEP-UWM, ToxinPred, Proteomics-Tools, and iUmami-SCM websites for prediction. Tables 1 and 2 show that the umami activity scores of all 7 identified peptides were greater than 588, and all contained umami-active fragments, indicating that all peptides were umami peptides, non-toxic, and had good water solubility. Figure 1 As shown, the molecular weight range of umami peptides is 700.3755~942.4334 Da, and the peptide length is 6 to 8. The peptides with a total docking score greater than 6 are Ser-Gly-Glu-Thr-Leu-Thr-Phe-Lys (SGETLTFK) and Leu-Glu-Leu-Asn-Val-Asp-Ala-Lys (LELNVDAK) in F2, and Leu-Leu-Asp-Asp-Leu-Arg (LLDDLR), Leu-Ala-Glu-Leu-Asn-Arg (LAELNR), and Thr-Leu-Glu-Asn-Arg (Thr-Leu-Glu-Asn-Arg) in F3. The umami peptides n-Glu-Phe-Lys (TLENEFK), Ser-Val-Glu-Glu-Leu-Leu-Gly-Lys (SVEELLGK), and Pro-Gln-Glu-Leu-Ser-Lys (PQELSK) have molecular weights of 881.4494 Da, 900.4916 Da, 743.4177 Da, 714.4024 Da, 879.4338 Da, 873.4807 Da, and 700.3755 Da, respectively, indicating that the umami peptides of Grifola frondosa are mainly composed of short peptides. Umami peptides with a total docking score (T-Score) greater than 6.0 were selected for semi-flexible docking with AutoDock vina.

[0080] Table 1. Umami activity score, toxicity, and water solubility prediction of umami peptides.

[0081] Table 2. Frequency of taste-active fragments in identified peptides

[0082] Note: "-" indicates that the corresponding identification peptide does not have a taste-active fragment.

[0083] Candidate peptides with umami activity greater than 588 were molecularly docked with the umami receptors T1R1 / T1R3 to screen for umami peptides. Specifically, the structures of the umami receptors T1R1 / T1R3 were obtained through homology modeling, and semi-flexible docking with AutoDock vina was used to screen for peptides with low docking binding energies to the umami receptors T1R1 / T1R3. The docking results of seven peptides are shown in Table 3. Figure 2 As shown in Table 3, the ease of ligand-receptor docking is represented by the docking binding energy. The larger the negative value of the energy, the stronger the interaction between the ligand and the receptor, and the greater the docking affinity. Table 3 shows that the T-Score values ​​of the seven umami peptides with T1R1 / T1R3 are all greater than 6.0. When the T-Score > 9.0, the docking results are more reliable. Among them, the hexapeptide LLDDLR has the highest total docking score of 11.7 and the lowest docking binding energy of -9.1 kcal / mol, proving that this umami peptide has the strongest binding affinity. Next, the octapeptide SGETLTFK has the highest total docking score of 9.1 and the lowest docking binding energy of -8.7 kcal / mol. The peptides SGETLTFK and LLDDLR were synthesized and subjected to artificial sensory evaluation and electronic tongue system analysis to verify the umami flavor.

[0084] Table 3. Total molecular docking score and docking binding energy of Grifola frondosa umami peptides with T1R1 / T1R3

[0085] Test case Umami rating (1) Solid-phase synthesis of target peptides. The two target peptides, SGETLTFK and LLDDLR, were synthesized by Shanghai Jier Biochemical Co., Ltd. using solid-phase synthesis, with a purity of not less than 95%.

[0086] (2) The taste characteristics of synthetic peptides were determined by using artificial sensory evaluation and electronic tongue system analysis.

[0087] ① Artificial sensory rating method.

[0088] The sensory evaluation panel consisted of 5 men and 5 women, aged 22-30 years, and was conducted in a sensory evaluation room at a temperature of 23±2℃. Panel members underwent screening and sensory training to ensure accurate evaluation results. Standard control solutions were prepared using 0.8 mg / mL citric acid solution, 10 mg / mL sucrose solution, 5 mg / mL L-isoleucine solution, 3.5 mg / mL NaCl solution, 3.5 mg / mL MSG solution, and 1 mg / mL glutathione solution, respectively, for sour, sweet, bitter, salty, umami, and strong taste. Sample solutions at 0.5 mg / mL were prepared, and scores were calculated using a 10-point linear scaling method: 0–3 points (weak); 4–7 points (moderate); 8–10 points (strong). All standard solutions were scored out of 5. Sensory evaluators were required to rinse their mouths with water before evaluation and between evaluations of every two samples. Group members tasted the samples randomly without discussion or communication throughout the process. The average score of the 10 members was used as the taste intensity value, and a radar chart was plotted to determine the sensory evaluation value of different samples.

[0089] ②Analysis of the electronic tongue system.

[0090] To ensure the stability and reliability of the electronic tongue detection data, the electronic tongue was first activated, calibrated, and diagnosed. An Insert SA402B electronic tongue system was used, employing five taste sensors: AAE (umami), CTO (salty), CA0 (sour), C00 (bitter), and AE1 (aftertaste). Standard taste solutions were prepared, including umami solution (3.5 mg / mL MSG solution), salty solution (3.5 mg / mL NaCl solution), sour solution (0.8 mg / mL citric acid solution), bitter solution (5 mg / mL L-isoleucine solution), and aftertaste solution (1 mg / mL glutathione solution). The synthetic peptides were prepared into a 0.5 mg / mL solution using ultrapure water. An Ag / AgCl electrode was used as the reference electrode, and the temperature was 25°C. Each test was repeated three times.

[0091] (3) Method for determining the umami threshold.

[0092] The synthetic peptide was prepared into a 0.5 mg / mL solution and serially diluted at a 1:1 (v / v) ratio. Sensory evaluators tasted the solution in ascending order of concentration. Each evaluator held 2 ml of the diluted solution in their mouth for 5 seconds and then spat it out. After tasting one sample, they rinsed their mouth with water and waited 5 minutes before tasting the next sample. Each dilution level was evaluated using a triangulation method until the evaluator could no longer distinguish between one sample solution and two ultrapure aqueous solutions. This dilution level was recorded as the taste dilution value (TDA value). Alternatively, the blank in the taste dilution analysis was replaced with a 1 mg / mL MSG solution, and the other steps were the same. The recorded dilution level was the synergistic flavor enhancement threshold.

[0093] Table 4. Umami Threshold and Synergistic Umami Threshold of Synthetic Peptides

[0094] Combination Figure 3 , Figure 4 The results of electronic tongue and sensory evaluation in Table 4 show that these two peptides have different intensities of umami. Ser-Gly-Glu-Thr-Leu-Thr-Phe-Lys (SGETLTFK) exhibits distinct umami, saltiness, and sweetness, with the lowest umami threshold of 0.0709 mmol / L, a rich umami flavor, strong flavor extension, and a synergistic umami enhancement threshold of 0.0354 mmol / L. Leu-Leu-Asp-Asp-Leu-Arg (LLDDLR) exhibits distinct umami, saltiness, and a strong flavor profile, with an umami threshold of 0.1680 mmol / L and a synergistic umami enhancement threshold of 0.0840 mmol / L. Both peptide thresholds are lower than the umami recognition threshold (1.635 mmol / L) of the traditional umami substance MSG. Therefore, the application of umami peptides in the development of MSG flavor base materials can effectively reduce sodium... + This invention reduces salt intake and enhances flavor, demonstrating that the computer-aided prediction and screening method for umami peptides can efficiently select the most flavorful umami peptides, and has broad application prospects.

[0095] It should also be noted that the terms "some embodiments," "other embodiments," and "embodiments" used in this application refer to specific features, structures, or characteristics described in connection with those embodiments, which are included in at least one embodiment described in the general description of this application. The appearance of the same expression in multiple places in the specification does not necessarily refer to the same embodiment. Furthermore, when a specific feature, structure, or characteristic is described in connection with any embodiment, the intention is to suggest that implementing such a feature, structure, or characteristic in conjunction with other embodiments also falls within the scope of this application.

[0096] In the above embodiments, the descriptions of each embodiment have different focuses. For parts not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0097] It should also be noted that the above are merely preferred embodiments of this application and do not limit the scope of protection of this application. Any equivalent structural or procedural transformations made using the content of this application specification, or direct or indirect applications in other related technical fields, are similarly included within the scope of protection of this application.

Claims

1. A umami peptide, characterized in that, It contains the following amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8 or Y1-Y2-Y3-Y4-Y5-Y6. Among them, X1 includes Ser, X2 includes Gly, X3 includes Glu, X4 includes Thr, X5 includes Leu, X6 includes Thr, X7 includes Phe, and X8 includes Lys. Y1 includes Leu, Y2 includes Leu, Y3 includes Asp, Y4 includes Asp, Y5 includes Leu, and Y6 includes Arg.

2. The umami peptide according to claim 1, characterized in that, The amino acid sequence is Ser-Gly-Glu-Thr-Leu-Thr-Phe-Lys, the molecular weight is 881.4494 Da, the umami threshold is 0.0709 mmol / L, and the synergistic umami enhancement threshold is 0.0354 mmol / L. Alternatively, the amino acid sequence is Leu-Leu-Asp-Asp-Leu-Arg, the molecular weight is 743.4177 Da, the umami threshold is 0.1680 mmol / L, and the synergistic umami threshold is 0.0840 mmol / L.

3. A method for identifying umami-rich titanium, characterized in that, Includes the following steps: Grifola frondosa was reacted sequentially with cellulase and flavor protease to obtain a mixed enzymatic hydrolysate containing umami peptides. The mixed enzymatic hydrolysate was purified and identified to obtain candidate peptides; The candidate peptides are screened to obtain umami peptides, which include the following amino acid sequences: X1-X2-X3-X4-X5-X6-X7-X8 or Y1-Y2-Y3-Y4-Y5-Y6. Among them, X1 includes Ser, X2 includes Gly, X3 includes Glu, X4 includes Thr, X5 includes Leu, X6 includes Thr, X7 includes Phe, and X8 includes Lys. Y1 includes Leu, Y2 includes Leu, Y3 includes Asp, Y4 includes Asp, Y5 includes Leu, and Y6 includes Arg.

4. The identification method according to claim 3, characterized in that, The reaction includes: Grifola frondosa was mixed with water and placed in a flash extractor to obtain a flash extract; The flash extract was reacted with cellulase at a pH of 3.5-5.5 in the first reaction. The product obtained from the first reaction was subjected to a second reaction with flavor protease at a pH of 5.0-7.0; The product obtained from the second reaction is subjected to enzyme inactivation treatment, centrifugation treatment, and concentration treatment to obtain a mixed enzymatic hydrolysate containing umami peptides.

5. The identification method according to claim 4, characterized in that, The conditions for obtaining the flash extract include a time of 20s-120s, a voltage of 160V-210V, an extraction frequency of at least one time, and a mass ratio of Grifola frondosa to water of 1:(8-30). And / or, the conditions for the first reaction include: the mass ratio of the cellulase to the maitake mushroom is (0.12-0.2):1, the temperature is 35℃-55℃, and the time is 1.5h-2.5h; And / or, the conditions for the second reaction include: the mass ratio of the flavor protease to the maitake mushroom is (0.03-0.05):1, the temperature is 40℃-60℃, and the time is 1.5h-2.5h; And / or, the conditions for the enzyme inactivation treatment include: heating the product obtained from the second reaction to 70°C-100°C and inactivating the enzyme for 5 min-15 min; And / or, the centrifugation conditions include: centrifuging the enzyme-inactivating product at 5000r / min-9000r / min for 10min-20min at 1℃-10℃.

6. The identification method according to claim 3, characterized in that, The identification was performed using liquid chromatography-tandem mass spectrometry.

7. The identification method according to claim 3, characterized in that, The filtering includes: The characteristic conditions for obtaining the umami peptide include ALC exceeding 95%, peptide length of 3-9, molecular weight less than 1000 Da, and the peptide containing both basic and acidic amino acids. The basic amino acids include Lys, His, or Arg, and the acidic amino acids include Glu, Asp, Asn, or Gln. Based on the aforementioned characteristic conditions, the candidate peptides are subjected to flexible molecular docking, and candidate peptides with a first threshold greater than 6 are selected. Candidate peptides with a first threshold greater than 6 are predicted, and the prediction conditions include umami activity, toxicity and water solubility, to obtain candidate peptides with an umami activity greater than 588. The candidate peptides with umami activity greater than 588 were subjected to semi-flexible docking to obtain umami peptides.

8. The identification method according to claim 7, characterized in that, The prediction tools include BIOPEP-UWM, ToxinPred, Proteomics-Tools, or iUmami-SCM; And / or, the umami receptors during the semi-flexible docking process include T1R1 or T1R3.

9. The identification method according to claim 7, characterized in that, After performing the semi-flexible docking and before obtaining the umami peptide, the process also includes selecting candidate peptides with a second threshold greater than 9. And / or, the semi-flexible docking is performed using Autodockvina software.

10. The use of the umami peptide according to claim 1 or 2, or the umami peptide obtained by the identification method according to any one of claims 3-9, in the preparation of food flavor enhancers; The food products include condiments, snack foods, soup bases, noodle products, meat products, or dairy products.

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