Gamma-glutamyl peptide as well as screening method and application thereof

Abstract

The invention provides gamma-glutamyl peptide as well as a screening method and application thereof. Wherein the gamma-glutamyl peptide is selected from one or more of amino acid sequences as shown in SEQ ID NO: 92, SEQ ID NO: 91, SEQ ID NO: 83, SEQ ID NO: 130, SEQ ID NO: 150, SEQ ID NO: 60, SEQ ID NO: 82, SEQ ID NO: 90 or SEQ ID NO: 93. The nine kinds of gamma-glutamyl peptides to be protected have good taste, can be simply and conveniently synthesized through a biological enzyme method, and have important significance for improving the delicate flavor of food under the condition that the use amount of salt is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, and in particular to a gamma-glutamyl peptide, a screening method and an application thereof. Background Art

[0002] In contemporary society, people's living standards are improving day by day, and they pay more and more attention to healthy diet. Reducing salt and fat has become a lifestyle that people increasingly admire. However, the presence of seasonings and fats provides food with a unique flavor. People need to make a trade-off between pursuing deliciousness and health. γ-Glutamyl peptide (kokumi) has unique taste characteristics and can effectively solve this contradiction. As a new type of flavor peptide, γ-glutamyl peptide not only gives food a strong taste, but also enhances the taste intensity of basic flavoring substances. Especially when the amount of salt is reduced, it has the effect of increasing saltiness and freshness, and has broad development prospects.

[0003] Therefore, how to effectively increase the content of γ-glutamyl peptides in food has become a key issue that needs to be solved urgently. At present, γ-glutamyl peptides are mainly obtained by extracting them from natural products or synthesizing them using γ-glutamyl transpeptidase, and their content in food is increased by adding γ-glutamyl peptides. Therefore, how to provide γ-glutamyl peptides with better quality is of great significance for the food processing trend of reducing salt and fat. Summary of the invention

[0004] The main purpose of the present invention is to provide a γ-glutamyl peptide, a screening method and application thereof, so as to provide a new γ-glutamyl peptide with better taste.

[0005] In order to achieve the above object, according to the first aspect of the present invention, a γ-glutamyl peptide is provided, which is selected from one or more of the amino acid sequences shown in SEQ ID NO:92, SEQ ID NO:91, SEQ ID NO:83, SEQ ID NO:130, SEQ ID NO:150, SEQ ID NO:60, SEQ ID NO:82, SEQ ID NO:90 or SEQ ID NO:93.

[0006] In order to achieve the above-mentioned purpose, according to the second aspect of the present invention, a screening method for γ-glutamyl peptides is provided, and the screening method comprises: S1, adding γ-glutamyl transpeptidase to amino acids, performing enzyme synthesis reaction, and obtaining a synthetic mixture; S2, performing LC-MS / MS analysis on the synthetic mixture to obtain the amino acid sequence of the polypeptide in the synthetic mixture; S3, performing toxicity and water solubility analysis on the polypeptides obtained after LC-MS / MS analysis to obtain a first polypeptide set; S4, allowing the polypeptides in the first polypeptide set to bind to the 5K5S receptor protein for affinity analysis to obtain the above-mentioned γ-glutamyl peptide; wherein the amino acid is selected from one or more of the following: glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, asparagine, glutamic acid, lysine, glutamine, methionine, serine, threonine, cysteine, proline, histidine or arginine.

[0007] Furthermore, in the enzyme synthesis reaction, the enzyme activity of γ-glutamyl transpeptidase was 0.6-1.2 U / g.

[0008] Furthermore, S1 comprises: after mixing the amino acid with deionized water, adding γ-glutamyl transpeptidase to carry out an enzyme synthesis reaction; preferably, the mass ratio of the amino acid to the deionized water is 1:4-5.

[0009] Furthermore, the temperature of the enzyme synthesis reaction is 30-37° C.; preferably, the time of the enzyme synthesis reaction is 2-3 h.

[0010] Furthermore, S2 includes: before performing LC-MS / MS analysis, freeze-drying the synthetic mixed solution to obtain a synthetic mixed powder, and performing LC-MS / MS analysis on the synthetic mixed powder.

[0011] Furthermore, the toxicity analysis in S3 includes: analyzing the aerobic biodegradability, rodent carcinogenicity, rat oral LD50 and mutagenicity of peptides less than 1000 Da obtained after LC-MS / MS analysis; preferably, using Discovery Studio TOPKAT to perform toxicity analysis on the peptides obtained after LC-MS / MS analysis.

[0012] Furthermore, the water solubility analysis in S3 includes: using proteomics to perform water solubility analysis on the peptides obtained after LC-MS / MS analysis.

[0013] Further, S4 includes: using Discovery Studio 2017 to perform affinity analysis, and the polypeptide with affinity >86.891 kcal / mol is the screened γ-glutamyl peptide.

[0014] In order to achieve the above object, according to the third aspect of the present invention, there is provided a use of the above γ-glutamyl peptide in food processing.

[0015] By applying the technical solution of the present invention, the nine γ-glutamyl peptides to be protected in the present application have good taste and can be synthesized relatively simply and conveniently by a biological enzyme method, which is of great significance for improving the umami taste of food while reducing the amount of salt used. DETAILED DESCRIPTION

[0016] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0017] As described in the background technology, γ-glutamyl peptide has the property of increasing the saltiness and freshness of food, and can be used as the main seasoning agent in the current pursuit of reducing salt and fat in the field of food processing. Therefore, the current market demand for γ-glutamyl peptide is gradually increasing. For this reason, the present application intends to provide a γ-glutamyl peptide with better quality, i.e., better flavor. Among them, γ-glutamyl peptide, as a flavor peptide, plays a role in enhancing the richness, full mouthfeel and sustainability of the taste in food.

[0018] In a first typical embodiment of the present application, a γ-glutamyl peptide is provided, and the γ-glutamyl peptide is selected from one or more of the amino acid sequences shown in SEQ ID NO: 92, SEQ ID NO: 91, SEQ ID NO: 83, SEQ ID NO: 130, SEQ ID NO: 150, SEQ ID NO: 60, SEQ ID NO: 82, SEQ ID NO: 90 or SEQ ID NO: 93. The use of the above nine γ-glutamyl peptides for food processing can make the food added with the polypeptide have better taste and meet the market demand for reducing salt and fat.

[0019] In a second typical embodiment of the present application, a method for screening γ-glutamyl peptides is provided, which screening method comprises: S1, adding γ-glutamyl transpeptidase to amino acids, performing enzyme synthesis reaction, and obtaining a synthetic mixture; S2, performing LC-MS / MS analysis on the synthetic mixture to obtain the amino acid sequence of the polypeptide in the synthetic mixture; S3, performing toxicity and water solubility analysis on the polypeptides obtained after LC-MS / MS analysis to obtain a first polypeptide set; S4, allowing the polypeptides in the first polypeptide set to bind to the 5K5S receptor protein for affinity analysis to obtain the above-mentioned γ-glutamyl peptide; wherein the amino acid is selected from one or more of the following: glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, asparagine, glutamic acid, lysine, glutamine, methionine, serine, threonine, cysteine, proline, histidine or arginine.

[0020] Among the existing methods for obtaining γ-glutamyl peptides, the method of extracting from natural products cannot meet the requirements of industrial production due to its low yield and high cost; and the method of synthesizing using γ-glutamyl transpeptidase, that is, using glutamine, free amino acids or polypeptides in protein hydrolysates as receptors for artificial synthesis, requires screening of the synthetic products, and the screening mainly relies on sensory evaluation and calcium sensitivity, which is time-consuming and costly overall. Using the screening method of the present application, it is possible to screen and obtain suitable γ-glutamyl peptides more accurately and efficiently.

[0021] Any γ-glutamyl transpeptidase (GGT, EC: 2.3.2.2) suitable for synthesizing and screening γ-glutamyl peptides is suitable for the present application. In a preferred embodiment, in the enzyme synthesis reaction, the enzyme activity of the γ-glutamyl transpeptidase is 0.6-1.2 U / g.

[0022] In order to further perform a more efficient enzyme synthesis reaction, in a preferred embodiment, S1 includes: after mixing the amino acid with the buffer, adding γ-glutamyl transpeptidase to perform the enzyme synthesis reaction; preferably, the mass ratio of the amino acid to deionized water is 1:4-5.

[0023] In order to further perform a more efficient enzyme synthesis reaction, in a preferred embodiment, the temperature of the enzyme synthesis reaction is 30-37° C.; preferably, the time of the enzyme synthesis reaction is 2-3 h.

[0024] In order to obtain a sample suitable for LC-MS / MS analysis, in a preferred embodiment, S2 includes: before performing LC-MS / MS analysis, freeze-drying the synthetic mixed solution to obtain a synthetic mixed powder, and performing LC-MS / MS analysis on the synthetic mixed powder.

[0025] In order to further accurately screen and obtain γ-glutamyl peptides of suitable quality, in a preferred embodiment, the toxicity analysis in S3 includes: analyzing the aerobic biodegradability, rodent carcinogenicity (male mice), rat oral LD50 and mutagenicity of polypeptides less than 1000 Da obtained after LC-MS / MS analysis, wherein when the rat oral LD50>5g / kg and the values ​​of aerobic biodegradability, rodent carcinogenicity (male mice) and mutagenicity of polypeptides less than 1000 Da are all <0.3, it is judged to be non-toxic. Preferably, the toxicity analysis of the polypeptide obtained after LC-MS / MS analysis is performed using Discovery Studio TOPKAT.

[0026] In order to further screen out γ-glutamyl peptides with better quality efficiently and accurately, in a preferred embodiment, the water solubility analysis in S3 includes: using proteomics to perform water solubility analysis on the polypeptides obtained after LC-MS / MS analysis.

[0027] Since calcium-sensitive receptor (5K5S receptor protein) is the main receptor for human perception of kokumi, it is mostly used to detect the content and quality control of flavor peptides (i.e., γ-glutamyl peptides in this application) in food. In a preferred embodiment, S4 includes: using Discovery Studio 2017 to perform affinity analysis, and the peptide with affinity > 86.891 kcal / mol is the γ-glutamyl peptide screened.

[0028] In a third typical embodiment of the present application, a use of the above-mentioned γ-glutamyl peptide in food processing is provided.

[0029] The present application is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.

[0030] Example 1 Synthesis of γ-glutamyl peptides and LC-MS / MS analysis

[0031] 1. Experimental methods

[0032] The 20 amino acids were mixed with deionized water in a mass ratio of 1:4 and the pH was adjusted to 9. γ-glutamyl transpeptidase with an enzyme concentration of 1 U / g was added as the starting point of the reaction and reacted at a constant temperature of 37°C for 3 h. After the reaction, the enzyme was inactivated in a boiling water bath for 15 min. The synthetic mixture obtained by the enzyme synthesis reaction was freeze-dried into a powder sample, named S2. S2 was identified and analyzed by LC-MS / MS to obtain 255 γ-glutamyl peptide sequences. Among them, γ-glutamyl transpeptidase was fermented by Bacillus subtilis BZT1 strain in the patent application document with the published application number of 2023113386095.

[0033] 2. Experimental results

[0034] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) has the advantages of high resolution, high selectivity, high sensitivity, and wide dynamic range, and is widely used in biochemistry, drug metabolism, protein structure analysis, etc.

[0035] Analysis of the LC-MS / MS test results showed that after the enzyme-catalyzed reaction, a total of 255 new γ-glutamyl peptides were generated (see Table 1-4 for details). These peptides were mainly small molecule peptides with a length of 5 to 10 amino acids, and most of them contained receptor amino acids such as phenylalanine, leucine, proline, tyrosine, valine, isoleucine and glycine.

[0036] Table 1 Peptide identification results

[0037]

[0038] Table 2 Peptide identification results

[0039]

[0040] Table 3 Peptide identification results

[0041]

[0042] Table 4 Peptide identification results

[0043]

[0044] Example 2 Prediction of toxicity and solubility of γ-glutamyl peptides

[0045] 1. Experimental methods

[0046] The 3D structures of peptides identified by LC-MS / MS were drawn using ChemDraw 20.0 and Chem3D 20.0, and the Ames mutagenicity, aerobic biodegradability, NTP carcinogenicityCall (Male Mouse), and rat oral LD50 of peptides were predicted using the Discovery Studio 2017 TOPKAT process. Proteomics was used to evaluate the water solubility of peptides.

[0047] 2. Experimental results

[0048] The toxicity and water solubility of peptides are crucial in the development and application of peptide-based functional foods and drugs, and are the main issues in the development of new peptides. With the help of Discovery Studio TOPKAT, the mutagenicity, aerobic biodegradability, rodent carcinogenicity (male mice), and rat oral LD50 of newly generated γ-glutamyl peptides less than 1000 Da were predicted. On this basis, proteomics was used to predict the water solubility of peptides, and a total of 93 peptide sequences with good water solubility and non-toxicity were screened out, as shown in Tables 5-13. Among them, the amino acids with unmeasurable toxicity prediction results in Table 13 are all sequences with a molecular weight greater than 1000Da. Since only peptides less than 1000Da have flavor, sequences with a molecular weight greater than 1000Da have not been tested for toxicological prediction.

[0049] Table 5 Toxicity and water solubility prediction results

[0050]

[0051] Table 6 Toxicity and water solubility prediction results

[0052]

[0053] Table 7 Toxicity and water solubility prediction results

[0054]

[0055] Table 8 Toxicity and water solubility prediction results

[0056]

[0057] Table 9 Toxicity and water solubility prediction results

[0058]

[0059] Table 10 Toxicity and water solubility prediction results

[0060]

[0061] Table 11 Toxicity and water solubility prediction results

[0062]

[0063] Table 12 Toxicity and water solubility prediction results

[0064]

[0065] Table 13 Toxicity and water solubility prediction results

[0066]

[0067] Example 3 Molecular docking

[0068] 1. Experimental methods

[0069] Discovery Studio 2017 software was used to perform molecular docking between the γ-glutamyl peptides that had passed the toxicity prediction and solubility prediction screening and the calcium-sensing receptor CaSR (PDB 5K5S).

[0070] 2. Experimental results

[0071] The molecular docking results are shown in Tables 14-15. Among the 93 γ-glutamyl peptides with good water solubility and non-toxic properties, 88 γ-glutamyl peptides were able to dock successfully with 5K5S (the 5 γ-glutamyl peptides that could not be successfully docked are recorded in Table 15). The molecular docking results are arranged according to the CDocker Enegry value, among which the lowest CDocker Enegry value is ELDDQFID (SEQ ID NO: 22) and the highest CDocker Enegry value is ELSEI (SEQ ID NO: 92), which are -420.341 kcal / mol and 108.438 kcal / mol, respectively.

[0072] The CDocker Enegry value of the reported thick-flavored peptide glutathione is 86.891 kcal / mol (detected by Discovery Studio 2017 software). Through screening, a total of 9 peptides (ELSEI (SEQ ID NO:92), EISEI (SEQ ID NO:91), ELADL (SEQ ID NO:83), ETEDTF (SEQ ID NO:130), EVASF (SEQ ID NO:150), EFEGV (SEQ ID NO:60), ELADI (SEQ ID NO:82), ELSEL (SEQ ID NO:90) and EISEL (SEQID NO:93)) are higher than the CDocker Enegry value of glutathione reported above (86.891 kcal / mol), indicating that these 9 peptides have a strong affinity with the calcium-sensitive receptor CaSR (PDB 5K5S). These 9 peptides are more likely to bind to the 5K5S receptor, and thus are more likely to provide a better flavoring effect and provide a higher thick flavor for food.

[0073] Table 14 Molecular docking results

[0074]

[0075] Table 15 Molecular docking results

[0076]

[0077] Example 4 Electronic tongue verification experiment

[0078] The three γ-glutamyl peptides with the highest scores among the nine γ-glutamyl peptides screened above were subjected to electronic tongue analysis.

[0079] 1. Experimental equipment and raw materials

[0080] ELSEI (SEQ ID NO:92), EISEI (SEQ ID NO:91), ELADL (SEQ ID NO:83), Japanese insent 5000Z electronic tongue, glutathione (GSH), monosodium glutamate (MSG), and NaCl.

[0081] 2. Experimental Methods

[0082] Weigh 100 mg of target peptides (ELSEI (SEQ ID NO: 92), EISEI (SEQ ID NO: 91), ELADL (SEQID NO: 83)) and glutathione, dilute to 100 mL with a mixed solution of monosodium glutamate (30 mmol / L) and NaCl (10 mmol / L), shake well and set aside. Two cups of each sample were used, and each sample was measured 4 times using the Japanese insent 5000Z electronic tongue. The first data was used for differential correction, and the data of the last three times were retained.

[0083] 3. Experimental Results

[0084] Table 16:

[0085]

[0086] Among them, glutathione is the most common γ-glutamyl peptide. As a standard, the aftertaste of umami can be expressed as richness. This indicator coincides with the thickness and persistence of the properties of γ-glutamyl peptide (kokumi), so it can be used as an indicator to evaluate kokumi. The saltiness of the above three peptides in this test is significantly higher than that of the blank solution and glutathione. The richness of the three peptides (0.24, 0.22333, 0.24667) is higher than that of the blank solution and glutathione (0.10667), which is consistent with the results of the previous simulation analysis. As can be seen from Table 16, the saltiness and kokumi properties (richness) of the MSG / NaCl model solution with the addition of the above peptides are enhanced to a certain extent, which will be of certain significance in the application of increasing freshness and reducing salt.

[0087] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the screening method of the present application can more accurately and efficiently obtain γ-glutamyl peptides with better properties, and the operation is simpler and the cost is lower. The 9 γ-glutamyl peptides screened have good taste and can be synthesized more simply and conveniently by a biological enzyme method, which is of great significance for improving the umami taste of food while reducing the amount of salt.

[0088] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A γ-glutamyl peptide, characterized in that The γ-glutamyl peptide is selected from one or more of the amino acid sequences shown in SEQ ID NO:92, SEQ ID NO:91, SEQ ID NO:83, SEQ ID NO:130, SEQ ID NO:150, SEQ ID NO:60, SEQ ID NO:82, SEQ ID NO:90 or SEQ ID NO:

93.

2. A method for screening γ-glutamyl peptides, characterized in that: The screening method comprises: S1, adding γ-glutamyl transpeptidase to amino acids to perform enzyme synthesis reaction to obtain a synthetic mixture; S2, performing LC-MS / MS analysis on the synthetic mixture to obtain the amino acid sequence of the polypeptide in the synthetic mixture; S3, performing toxicity and water solubility analysis on the polypeptides obtained after the LC-MS / MS analysis to obtain a first polypeptide set; S4, allowing the polypeptides in the first polypeptide set to bind to the 5K5S receptor protein to perform affinity analysis to obtain the γ-glutamyl peptide described in claim 1; Wherein, the amino acid is selected from one or more of the following: glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, asparagine, glutamic acid, lysine, glutamine, methionine, serine, threonine, cysteine, proline, histidine or arginine.

3. The screening method according to claim 2, characterized in that In the enzyme synthesis reaction, the enzyme activity of the γ-glutamyl transpeptidase is 0.6-1.2 U / g.

4. The screening method according to claim 2, characterized in that The S1 includes: After the amino acid is mixed with deionized water, the γ-glutamyl transpeptidase is added to carry out the enzyme synthesis reaction.

5. The screening method according to claim 4, characterized in that The mass ratio of the amino acid to the deionized water is 1:4-5.

6. The screening method according to claim 2, characterized in that The temperature of the enzyme synthesis reaction is 30-37°C.

7. The screening method according to claim 2, characterized in that The time of the enzyme synthesis reaction is 2-3h.

8. The screening method according to claim 2, characterized in that The S2 comprises: before the LC-MS / MS analysis, freeze-drying the synthetic mixed solution to obtain a synthetic mixed powder, and performing LC-MS / MS analysis on the synthetic mixed powder.

9. The screening method according to claim 2, characterized in that The toxicity analysis in S3 includes: The aerobic biodegradability, rodent carcinogenicity, rat oral LD50 and mutagenicity of peptides less than 1000 Da obtained by the LC-MS / MS analysis were analyzed.

10. The screening method according to claim 9, characterized in that The toxicity analysis was performed on the peptides obtained after the LC-MS / MS analysis using Discovery Studio TOPKAT.

11. The screening method according to claim 2, characterized in that The water solubility analysis in S3 includes: using proteomics to perform the water solubility analysis on the polypeptide obtained after the LC-MS / MS analysis.

12. The screening method according to claim 2, characterized in that The S4 includes: The affinity analysis was performed using Discovery Studio 2017, and the polypeptide with affinity > 86.891 kcal / mol was the γ-glutamyl peptide obtained by screening.

13. Use of the γ-glutamyl peptide according to claim 1 in food processing.

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