γ-Glutamyl Peptide, Its Screening Method and Application

Abstract

The present invention provides a γ-glutamyl peptide, a screening method thereof and applications. Among them, 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 nine γ-glutamyl peptides to be protected by this application have good taste, can be synthesized relatively simply and conveniently by biological enzyme methods, and are of great significance for improving the umami taste of food while reducing the amount of salt used.

Description

Technical Field

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

[0002] In contemporary society, with the increasing improvement of people's living standards, more and more attention is paid to healthy eating, and reducing salt and fat intake has become an increasingly popular lifestyle. However, the presence of seasonings and fats provides unique flavors to foods. Between the pursuit of deliciousness and health, people need to make a trade-off. 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 imparts a rich flavor to foods but also enhances the flavor intensity of basic flavor substances. Especially in the case of reducing the amount of salt used, it has the effects of increasing saltiness and enhancing umami, and has broad development prospects.

[0003] Therefore, how to effectively increase the content of γ-glutamyl peptide in foods has become a key problem to be solved urgently. Currently, it is mainly achieved by extracting from natural products or synthesizing using γ-glutamyl transpeptidase to obtain γ-glutamyl peptide, and by adding γ-glutamyl peptide to increase its content in foods. Therefore, how to provide γ-glutamyl peptide with better quality is of great significance for the food processing trend of reducing salt and fat. Summary of the Invention

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

[0005] To achieve the above object, according to the first aspect of the present invention, there is provided a γ-glutamyl peptide 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] To achieve the above object, according to the second aspect of the present invention, a screening method for γ-glutamyl peptides is provided. The screening method includes: S1, adding γ-glutamyl transpeptidase to amino acids and performing an enzyme synthesis reaction to obtain a synthesis mixture; S2, performing LC-MS / MS analysis on the synthesis mixture to obtain the amino acid sequences of the polypeptides in the synthesis mixture; S3, performing toxicity and water solubility analysis on the polypeptides obtained after LC-MS / MS analysis to obtain a first polypeptide set; S4, binding the polypeptides in the first polypeptide set to 5K5S receptor protein to perform affinity analysis to obtain the above-mentioned γ-glutamyl peptides; wherein the amino acids are 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] Further, in the enzyme synthesis reaction, the enzyme activity of γ-glutamyl transpeptidase is 0.6 - 1.2 U / g.

[0008] Further, S1 includes: mixing amino acids with deionized water, adding γ-glutamyl transpeptidase, and performing an enzyme synthesis reaction; preferably, the mass ratio of amino acids to deionized water is 1:4 - 5.

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

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

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

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

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

[0014] To achieve the above object, according to the third aspect of the present invention, there is provided an application of the above-mentioned γ-glutamyl peptide in food processing.

[0015] Applying the technical solution of the present invention, the nine γ-glutamyl peptides to be protected in this application have good taste, can be synthesized relatively simply and conveniently by the method of biological enzymes, and are of great significance for improving the umami taste of food while reducing the amount of salt used. Specific Embodiments

[0016] It should be noted that, without conflict, the embodiments in this application and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0017] As described in the background art, γ-glutamyl peptide has the property of increasing the saltiness and umami of food, and can be used as a main flavoring agent in the current food processing field where salt reduction and fat reduction are pursued. Therefore, the current market demand for γ-glutamyl peptide is gradually increasing. For this reason, this application aims to provide a γ-glutamyl peptide with good quality, that is, good taste. Among them, γ-glutamyl peptide, as a taste peptide, plays a role in enhancing the richness, fullness and persistence of taste in food.

[0018] In the first typical embodiment of this 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. Using the above nine γ-glutamyl peptides for food processing can make the food added with the polypeptide have good taste and meet the market demand for salt reduction and fat reduction.

[0019] In the second typical embodiment of the present application, a screening method for γ-glutamyl peptides is provided. The screening method includes: S1, adding γ-glutamyl transpeptidase to amino acids to perform an enzymatic synthesis reaction to obtain a synthesis mixture; S2, performing LC-MS / MS analysis on the synthesis mixture to obtain the amino acid sequences of the polypeptides in the synthesis mixture; S3, performing toxicity and water solubility analysis on the polypeptides obtained after LC-MS / MS analysis to obtain a first polypeptide set; S4, binding the polypeptides in the first polypeptide set to 5K5S receptor protein for affinity analysis to obtain the above-mentioned γ-glutamyl peptides; wherein the amino acids are 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] In the existing methods for obtaining γ-glutamyl peptides, the method of extracting from natural products cannot meet industrial production due to its low yield and high cost; while 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 synthesized products, and the screening mainly relies on sensory evaluation method and calcium sensitivity method, with a long overall time consumption and high cost. However, using the screening method of the present application, it is possible to more accurately and efficiently screen and obtain suitable γ-glutamyl peptides.

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

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

[0023] In order to further perform a more efficient enzymatic synthesis reaction, in a preferred embodiment, the temperature of the enzymatic synthesis reaction is 30 - 37 °C; preferably, the time of the enzymatic 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, performing freeze-drying treatment on the synthesis mixture to obtain a synthesis mixed powder, and performing LC-MS / MS analysis on the synthesis mixed powder.

[0025] In order to further accurately screen for γ-glutamyl peptides with suitable quality, in a preferred embodiment, the toxicity analysis in S3 includes: analyzing the aerobic biodegradability of the polypeptides obtained after LC-MS / MS analysis, rodent carcinogenicity (male mice), oral LD50 of rats, and mutagenicity of polypeptides with a molecular weight less than 1000 Da. Among them, when the oral LD50 of rats > 5 g / kg and the values of aerobic biodegradability, rodent carcinogenicity (male mice), and mutagenicity of polypeptides with a molecular weight less than 1000 Da are all < 0.3, it is determined to be non-toxic. Preferably, Discovery Studio TOPKAT is used to perform toxicity analysis on the polypeptides obtained after LC-MS / MS analysis.

[0026] In order to further efficiently and accurately screen for γ-glutamyl peptides with better quality, 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 the calcium-sensing receptor (5K5S receptor protein) is the main receptor for humans to perceive kokumi and is mostly used for detecting the content and quality control of taste peptides (i.e., γ-glutamyl peptides in this application) in food. In a preferred embodiment, S4 includes: performing affinity analysis using Discovery Studio 2017, and the polypeptides with an affinity > 86.891 kcal / mol are the screened γ-glutamyl peptides.

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

[0029] The following further describes this application in detail with specific embodiments, and these embodiments should not be construed as limiting the scope claimed in this application.

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

[0031] 1. Experimental method

[0032] Mix 20 kinds of amino acids with deionized water at a mass ratio of 1:4 and adjust the pH to 9. Add γ-glutamyl transpeptidase with an enzyme concentration of 1 U / g, and use this as the starting point of the reaction. React at a constant temperature of 37°C for 3 h. After the reaction is completed, inactivate the enzyme in a boiling water bath for 15 min. Freeze-dry the synthesis mixture obtained from the enzyme synthesis reaction into a powder sample, named S2, and use LC-MS / MS to identify and analyze S2 to obtain 255 γ-glutamyl peptide sequences. Among them, γ-glutamyl transpeptidase is obtained by fermenting Bacillus subtilis strain BZT1 in the patent application document with the published application number 2023113386095.

[0033] 2. Experimental Results

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

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

[0036] Table 1 Polypeptide Identification Results

[0037]

[0038] Table 2 Polypeptide Identification Results

[0039]

[0040] Table 3 Polypeptide Identification Results

[0041]

[0042] Table 4 Polypeptide Identification Results

[0043]

[0044] Example 2 Prediction of Toxicity and Solubility of γ - Glutamyl Peptides

[0045] 1. Experimental Method

[0046] The 3D structures of the polypeptides identified by LC - MS / MS were drawn using ChemDraw 20.0 and Chem3D 20.0. The mutagenicity (Ames mutagenicity), aerobic biodegradability, carcinogenicity in rodents (male mice) (NTP carcinogenicityCall (Male Mouse)), and oral LD50 in rats (Rat Oral LD50) of the polypeptides were predicted with the help of the Discovery Studio 2017 TOPKAT process. The water solubility of the polypeptides was evaluated using proteomics.

[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. The mutagenicity, aerobic biodegradability, rodent carcinogenicity (male mice), and oral LD50 of rats of newly generated γ-glutamyl peptides with a molecular weight less than 1000 Da were predicted using Discovery Studio TOPKAT. On this basis, proteomics was used to predict the water solubility of polypeptides, and a total of 93 peptide sequences with good water solubility and no toxicity were screened out, as shown in Table 5-13. Among them, the amino acids with unpredictable toxicity prediction results in Table 13 are all sequences with a molecular weight greater than 1000 Da. Since only polypeptides with a molecular weight less than 1000 Da have taste properties, sequences with a molecular weight greater than 1000 Da were not subjected to toxicological prediction tests.

[0049] Table 5 Prediction Results of Toxicity and Water Solubility

[0050]

[0051] Table 6 Prediction Results of Toxicity and Water Solubility

[0052]

[0053] Table 7 Prediction Results of Toxicity and Water Solubility

[0054]

[0055] Table 8 Prediction Results of Toxicity and Water Solubility

[0056]

[0057] Table 9 Prediction Results of Toxicity and Water Solubility

[0058]

[0059] Table 10 Prediction Results of Toxicity and Water Solubility

[0060]

[0061] Table 11 Prediction Results of Toxicity and Water Solubility

[0062]

[0063] Table 12 Prediction Results of Toxicity and Water Solubility

[0064]

[0065] Table 13 Prediction Results of Toxicity and Water Solubility

[0066]

[0067] Example 3 Molecular Docking

[0068] 1. Experimental Method

[0069] The γ-glutamyl peptides that have passed the toxicity prediction and solubility prediction screening and the calcium-sensing receptor CaSR (PDB 5K5S) were subjected to molecular docking using Discovery Studio 2017 software.

[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 successfully docked with 5K5S (the 5 γ-glutamyl peptides that could not be successfully docked are listed in Table 15). The molecular docking results are arranged according to the CDocker Enegry value. 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 umami peptide glutathione that has been reported is 86.891 kcal / mol (detected using 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 the above-reported glutathione (86.891 kcal / mol), indicating that these 9 peptides have a strong affinity for the calcium-sensing 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 umami effect and a higher umami taste for food.

[0073] Table 14 Molecular Docking Results

[0074]

[0075] Table 15 Molecular Docking Results

[0076]

[0077] Example 4 Electronic Tongue Verification Experiment

[0078] Perform electronic tongue analysis on the 3 types of γ-glutamyl peptides with the highest scores among the 9 types of γ-glutamyl peptides screened above.

[0079] I. Experimental Equipment and Raw Materials

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

[0081] II. Experimental Methods

[0082] Weigh 100 mg of the target peptides (ELSEI (SEQ ID NO:92), EISEI (SEQ ID NO:91), ELADL (SEQ ID NO:83)) and glutathione respectively, and make up the volume to 100 mL with a mixed solution of monosodium glutamate (30 mmol / L) and NaCl (10 mmol / L). Shake well for later use. There are two cups for each sample. Use the insent 5000Z electronic tongue from Japan to measure each sample 4 times repeatedly. Perform differential correction with the data of the first time and retain the data of the last three times.

[0083] III. Experimental Results

[0084] Table 16:

[0085]

[0086] Among them, glutathione is the most common γ-glutamyl peptide. As a standard product, the aftertaste of umami can be expressed as richness, which coincides with the thickness and persistence in the γ-glutamyl peptide (kokumi) property. Therefore, it can be used as an index to evaluate kokumi. The saltiness of the above three polypeptides in this detection 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 previous simulation analysis results. It can be seen from Table 16 that the saltiness and kokumi property (richness) of the MSG / NaCl model solution added with the above polypeptides are both enhanced to a certain extent, which will have a certain significance in the application of enhancing 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: using the screening method of the present application can obtain γ-glutamyl peptides with better properties more accurately and efficiently, and the operation is simpler and the cost is lower. Moreover, the 9 kinds of γ-glutamyl peptides obtained by screening have good taste and can be synthesized relatively simply and conveniently by biological enzyme methods, which is of great significance for improving the umami of food while reducing the amount of salt used.

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

Claims

1. A γ-glutamyl peptide, characterized in that, The γ-glutamyl peptide is selected from the amino acid sequences shown in SEQ ID NO:92, SEQ ID NO:91 or SEQ ID NO:

83.

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

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