Wheat gluten protein peptide with XOD inhibitory activity and application thereof

Abstract

The invention discloses a preparation process of wheat gluten protein peptide with XOD inhibitory activity, and belongs to the technical field of food-borne active peptide development. According to the method, the XOD inhibitory peptide is rapidly screened by utilizing a polypeptiomics and molecular docking technology, the defects of time consumption and labor consumption caused by step-by-step separation and purification are avoided, and the XOD inhibitory peptide can be used for assisting or replacing uric acid reducing medicines. The wheat gluten protein peptide disclosed by the invention has the characteristic of inhibiting XOD enzyme activity in vitro.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food-derived bioactive peptide development, and particularly relates to a wheat gluten protein peptide with XOD inhibitory activity and application thereof. BACKGROUND

[0002] Hyperuricemia has become the fourth health risk factor after hypertension, hyperglycemia and hyperlipidemia, and is a common metabolic disease in human body, mainly manifested as that the serum uric acid level of patients is significantly higher than that of normal people. When the serum uric acid level in the human body reaches the serum uric acid saturation, urate is easily deposited in tissues such as joints, cartilage and kidneys, forming urate crystals, and ultimately leading to gout. Uric acid (C5H4N4O3) is the final product of cell metabolism and metabolism of purines in food in the body. Under the condition of normal purine diet, if the fasting blood uric acid level of a male is >416 μmol / L and that of a female is >357 μmol / L within 2 days, the person is diagnosed as hyperuricemia. So far, the drugs for treating HUA or acute gout mainly include xanthine oxidase inhibitors (such as allopurinol and febuxostat) and uric acid excretion drugs (such as probenecid and benzbromarone). The clinical drugs have a significant effect on reducing uric acid, but can also cause different degrees of side effects and physiological toxicity, such as gastrointestinal intolerance, skin rash, respiratory tract infection, and influence on liver function. Therefore, it is an urgent need to find an active substance which is effective and safe for reducing the serum urate level to control hyperuricemia.

[0003] The occurrence of hyperuricemia is mainly related to excessive production of uric acid and insufficient excretion of uric acid. The generation of uric acid in the body is mainly controlled by purine metabolism-related enzymes, and adenosine deaminase (ADA) and xanthine oxidase (XOD) are the key enzymes for controlling the generation of uric acid. The excretion of uric acid is mainly participated by urate reabsorption proteins, including glucose transporter 9, urate anion transporter 1, OAT4 and OAT10 in the organic anion transporter family and sodium-coupled monocarboxylate transporter. If the expression of urate reabsorption proteins or secretory proteins in the body is abnormal, the reabsorption of uric acid increases or the excretion decreases, which will lead to hyperuricemia. Since the efficacy of uric acid excretion drugs is strongly regulated by drug genetics, targeting xanthine oxidase to regulate uric acid level is still the safest and most effective means.

[0004] Many food-derived proteins do not have uric acid-lowering activity by themselves, but through enzymolysis or fermentation, the proteins are decomposed to release peptide segments with uric acid-lowering activity. At present, a large number of highly active XOD inhibitory peptides have been isolated from a variety of food-derived proteins, showing good uric acid-lowering effect. For example, the patent with publication number CN118949001A discloses the production of uric acid-lowering peptides from potato protein. For another example, the patent with publication number CN118177233A discloses a method for producing uric acid-lowering peptides from rice protein. Such natural peptides can avoid the side effects of synthetic drugs, but the types of plant-derived uric acid-lowering peptides reported at present are limited, and new plant uric acid-lowering peptides need to be developed. SUMMARY

[0005] This section is intended to summarize some aspects of the embodiments of the present application and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of the specification of the present application in order to avoid obscuring the purpose of this section, the abstract and the title, and such simplifications or omissions cannot be used to limit the scope of the present application.

[0006] In view of the above and / or problems existing in the prior art, the present application is proposed.

[0007] Therefore, the purpose of the present application is to overcome the deficiencies in the prior art and provide a protein peptide with XOD inhibitory activity.

[0008] To solve the above technical problems, the present application provides the following technical solutions: a wheat gluten protein peptide with XOD inhibitory activity.

[0009] As a preferred scheme of the wheat gluten protein peptide according to the present application, wherein: the wheat gluten protein peptide with XOD inhibitory activity is characterized in that: the wheat gluten protein peptide is obtained by rapid screening of polypeptidomics and molecular docking technology, and the wheat gluten protein peptide comprises a peptide segment as shown in SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 or SEQ ID No. 4 or SEQ ID No. 5 or SEQ ID No. 6.

[0010] As a preferred scheme of the wheat gluten protein peptide according to the present application, wherein: the molecular docking binding energy of the peptide segment is less than -5 kcal / mol.

[0011] Another purpose of the present application is to overcome the deficiencies in the prior art and provide an application of the wheat gluten protein peptide in preparing an XOD inhibitor.

[0012] Another purpose of the present application is to overcome the deficiencies in the prior art and provide an application of the wheat gluten protein peptide in preparing a uric acid-lowering drug.

[0013] Still another object of the present application is to overcome the deficiencies in the prior art and provide a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the wheat gluten peptide as described above.

[0014] As a preferred embodiment of the pharmaceutical composition of the present application, the pharmaceutical composition is in the form of an oral dosage or a pharmaceutically acceptable excipient.

[0015] Still another object of the present application is to overcome the deficiencies in the prior art and provide a method for screening and identifying a protein peptide having XOD inhibitory activity, characterized in that the method comprises,

[0016] hydrolyzing wheat gluten protein with a protease to produce a wheat gluten peptide;

[0017] separating and purifying by ultrafiltration;

[0018] sequencing the <3 kDa wheat gluten peptide by polypeptidomics;

[0019] screening a potential high-activity XOD inhibitory peptide;

[0020] verifying the screened peptide segment by molecular docking technology;

[0021] computer simulation of gastrointestinal digestion.

[0022] As a preferred embodiment of the method of the present application, the hydrolysis of wheat gluten protein with a protease to produce a wheat gluten peptide is carried out at a temperature of 35-55°C and pH = 6.5-8.5.

[0023] As a preferred embodiment of the method of the present application, the screening of a potential high-activity XOD inhibitory peptide comprises:

[0024] biological activity prediction and scoring of the peptide segment in PeptideRanker;

[0025] querying the protein source of the peptide segment in a protein database;

[0026] toxicity prediction by Toxinpred;

[0027] allergenicity prediction by AllerTOP;

[0028] selecting from the sequenced peptide segment a peptide segment that is derived from a protein, has not been modified by a functional group, has a peak area greater than 1.00 x 10 7 , has a sequence of 3-10 amino acids, has no toxicity and allergenicity, and has not been disclosed.

[0029] As a preferred scheme of the method, wherein: the verification of the screened peptide segments by using the molecular docking technology comprises:

[0030] Preparing the XOD enzyme PDB file: retrieving the protein crystal structure of the XOD enzyme from the protein database, removing the excess ligands and H2O from 1N5X for subsequent docking experiments with the peptide segments;

[0031] Evaluating the XOD inhibitory activity: obtaining the binding energy after docking the screened peptide sequence with 1M5X, and evaluating the potential XOD inhibitory activity according to the size of the binding energy.

[0032] As a preferred scheme of the method, wherein: the evaluation of the XOD inhibitory activity, wherein the lower the molecular docking binding energy, the stronger the potential XOD inhibitory activity.

[0033] As a preferred scheme of the method, wherein: the computer simulation of gastrointestinal digestion comprises:

[0034] Enzymatic digestion of the peptide segments by BIOPEP-UWM: ANALYSIS function in the BIOPEP database;

[0035] Selecting rennet trypsin A, trypsin, pepsin for simulated enzymatic digestion.

[0036] The present application has the following beneficial effects:

[0037] (1) The present application uses polypeptidomics and molecular docking technology to quickly screen XOD inhibitory peptides, avoiding the time-consuming and laborious shortcomings caused by step-by-step separation and purification, and can be used to assist or replace uric acid-lowering drugs.

[0038] (2) The wheat gluten protein peptides extracted by the present application have higher in vitro XOD inhibitory activity compared with food-derived XOD inhibitory peptides. DETAILED DESCRIPTION

[0039] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiment description. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor. Among them:

[0040] Figure 1 Comparison chart of food-derived XOD inhibitory peptide activity;

[0041] Figure 2 Effect of protease species on XOD inhibition rate and degree of hydrolysis;

[0042] Figure 3Effect of different substrate concentrations on XOD inhibition rate and hydrolysis degree;

[0043] Figure 4 Effect of different enzymatic hydrolysis time on XOD inhibition rate and hydrolysis degree;

[0044] Figure 5 Protein crystal structure of XOD;

[0045] Figure 6 Molecular docking of APF and XOD and interaction force analysis diagram;

[0046] Figure 7 Molecular docking of LNL and XOD and interaction force analysis diagram;

[0047] Figure 8 Molecular docking of APFA and XOD and interaction force analysis diagram;

[0048] Figure 9 Molecular docking of TLPL and XOD and interaction force analysis diagram;

[0049] Figure 10 Molecular docking of TIPL and XOD and interaction force analysis diagram;

[0050] Figure 11 Molecular docking of GIFGTN and XOD and interaction force analysis diagram. DETAILED DESCRIPTION

[0051] In order to make the above objectives, features and advantages of the present application more apparent and comprehensible, the specific embodiments of the present application will be described in detail below with reference to the description and examples.

[0052] In the following description, a large number of specific details are set forth in order to facilitate a thorough understanding of the present application, but the present application can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without departing from the connotation of the present application, therefore the present application is not limited by the specific examples disclosed below.

[0053] Secondly, the "one embodiment" or "embodiment" referred to herein means that the specific features, structures or characteristics can be included in at least one implementation of the present application. "In one embodiment" appearing in different places in the specification does not mean the same embodiment, nor is it an independent or alternative embodiment that excludes other embodiments.

[0054] Experimental materials and reagents used in the embodiments of the present application: raw wheat gluten powder, Binzhou Zhongyu Food Co., Ltd.; trypsin, National Pharmaceutical Group Chemical Reagent Co., Ltd.; xanthine oxidase (XOD), Sigma-Aldrich Company, USA;

[0055] The instruments used in the embodiments of the present application: SW22 type electric heating constant temperature water bath, Germany JULABO company; LXJ-IIB type centrifuge, Shanghai Anting Instrument Factory; FE20K type precision pH meter, AL104 type electronic analytical balance, Shanghai Mettler Instrument Co., Ltd.; LGJ-10E type freeze dryer, Ningbo Scents Biological Technology Co., Ltd.; Muliskan GO full-wavelength enzyme label instrument, China Thermo Fisher Scientific Co., Ltd.

[0056] Example 1

[0057] This embodiment is the activity comparison of XOD inhibitory peptides from various food sources. By consulting domestic and foreign literatures, several common food source XOD inhibitory peptides IC 50 values are summarized.

[0058] The results are shown as Figure 1 follows. It can be found through comparison that the in vitro XOD inhibitory activity of wheat gluten peptide is in the upper middle level, the IC 50 value is relatively low, and it has great research value. After water absorption, wheat gluten protein can form wet gluten with network structure, which has excellent viscoelasticity, extensibility and film forming property, and is widely used in food industry. Moreover, wheat gluten protein is a byproduct in the process of wheat starch processing, and its protein content is as high as more than 75%. Therefore, the wheat gluten XOD inhibitory peptide is further studied.

[0059] Example 2

[0060] The embodiment provides a method for obtaining wheat gluten protein hydrolysate from wheat gluten protein, which comprises the following steps:

[0061] An appropriate amount of wheat gluten protein powder is weighed to prepare a protein solution with a substrate concentration of 4-12%, which is subjected to alkaline, neutral, trypsin and complex protease hydrolysis. The temperature is adjusted to 35-65℃ and the pH is adjusted to 4.5-9.5 to achieve the optimal hydrolysis conditions of the protease. The protease is added according to the enzyme substrate ratio (6000-12000 U / g protein), and the reaction is continuously stirred. The pH of the reaction system is maintained by NaOH until it is constant. After the hydrolysis is completed, the heating and stirring are stopped, and the enzyme is inactivated by placing in a boiling water bath for 10-30 min. The pH is adjusted to 7.0 by HCl, and then centrifuged at 8000-10000 r / min for 10-30 min. The supernatant is collected into a dialysis bag and dialyzed at 4℃ for 2-3 days. After freeze-drying, the wheat gluten peptide is obtained and stored at -20℃ for standby.

[0062] The XOD inhibition rate and the hydrolysis degree were used as indexes to screen the types of proteases commonly used for preparing XOD inhibitory peptides, and the substrate concentration and enzymolysis time were optimized to determine the optimal enzymolysis conditions. The effects of the types of proteases on the XOD inhibition rate and the hydrolysis degree are shown in Figure 2 The effects of different substrate concentrations on the XOD inhibition rate and the hydrolysis degree are shown in Figure 3 The effects of different enzymolysis times on the XOD inhibition rate and the hydrolysis degree are shown in Figure 4

[0063] Therefore, the optimal enzymolysis conditions are determined as follows: the substrate concentration is 6%, the trypsin addition amount is 10000 U / g, and the enzymolysis time is 150 min.

[0064] Example 3

[0065] This example is a method for detecting the XOD inhibitory activity of wheat gluten protein hydrolysate in vitro, which comprises the following steps:

[0066] 50 μL of the hydrolysate sample and 50 μL of xanthine oxidase (XOD) solution (0.05 U / mL) are added to a 96-well plate, and then incubated at 37°C for 15 min, followed by adding 150 μL of xanthine solution to start the reaction, and then incubating for 10 min before measuring the absorbance at 290 nm. Allopurinol is selected as the positive control.

[0067] The calculation method of the XOD inhibition rate is as follows:

[0068]

[0069] A1 is the absorbance value of XOD, xanthine and the sample solution; A2 is the absorbance value of xanthine and the sample solution; A3 is the absorbance value of the buffer, XOD and xanthine solution; and A4 is the absorbance value of the buffer and xanthine solution.

[0070] Example 4

[0071] This example provides a method for separating and identifying XOD inhibitory peptides from wheat gluten protein hydrolysate, which comprises the following steps:

[0072] (1) The wheat gluten protein hydrolysate is separated and purified by ultrafiltration, and the component with a molecular weight of <3 kDa is selected for sequencing in the next step:

[0073] A 50 mL ultrafiltration tube with a molecular weight of 3 kDa is used for ultrafiltration operation. The wheat gluten peptide in step (1) is dissolved in water and placed in the ultrafiltration tube, and then centrifuged at 3000-4000 r / min for 5-10 min. The operation is repeated, and the component with a molecular weight of <3 kDa in the lower layer of the ultrafiltration tube is taken for freeze-drying.

[0074] (2) The wheat gluten peptide with a molecular weight of <3 kDa is sequenced by polypeptidomics:​

[0075] The <3 KDa wheat gluten protein peptide sample obtained in step (1) was analyzed by LC-MS / MS equipped with an online nanospray ion source.

[0076] The whole system was an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, MA, USA) connected with an EASY-nanoLC 1200.

[0077] 5 μL of wheat gluten proteolyzate was co-loaded (analytical column: Acclaim PepMap C18, 75 μm x 25 cm) to separate the sample in a 60 min gradient, with a column flow rate controlled at 300 nL / min, a column temperature of 40 °C, an electrospray voltage of 2 kV, a gradient starting from 4% of phase B, increasing to 50% in a non-linear gradient in 53 min 40 s, and increasing to 95% in 40 s, maintaining for 5 min 40 s.

[0078] The mass spectrometer was operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition.

[0079] The mass spectrometer parameters were set as follows:

[0080] ① MS: scan range (m / z): 100-1500; resolution: 120,000; AGC target: 200%; maximum injection time: 100 ms;

[0081] ② HCD-MS / MS: resolution: 50,000; AGC target: 200%; maximum injection time: 86 ms; collision energy: 25%, 30%, 35%; dynamic exclusion time: 30 s. The tandem mass spectra were analyzed by PEAKS Studio version 10.6 (Bioinformatics Solutions Inc., Waterloo, Canada). The database was searched against the uniprot Cicer arietinum (version 202112, 24812 entries) database by PEAKS DB, with None enzyme set. The search parameters were as follows: fragment ion mass tolerance: 0.02 Da, parent ion mass tolerance: 10 ppm, variable modification: Oxidation (M) 15.99, Deamidation (NQ) 0.98. The protein score was at least 1 unique peptide; the peptide score was -10 lgP≥20.

[0082] After sequencing the <3 kDa wheat gluten protein hydrolyzate by polypeptidomics, a total of 2393 polypeptides were identified.

[0083] (3)Screening potential high-activity XOD inhibitory peptides from the identified polypeptides using multiple screening conditions, mainly including the following steps:

[0084] ①Inquiring the protein source of the peptide segment in the protein database (https: / / www.uniprot.org / );

[0085] ②Toxicity prediction by Toxinpred (https: / / crdd.osdd.net / raghava / toxinpred / );

[0086] ③Allergenicity prediction by AllerTOP (https: / / www.ddg-pharmfac.net / AllerTOP / );

[0087] ④Selecting the peptide segment from the sequenced peptide segment, which is derived from a protein, not modified by a functional group, has a peak area greater than 1.00 x 10 7 , 3-10 amino acid sequences, has no toxicity and allergenicity, and has not been disclosed.

[0088] (4)Using molecular docking technology to verify the peptide segment obtained from step (3), mainly including the following steps:

[0089] ①Preparation of XOD enzyme PDB file: retrieving the protein crystal structure 1N5X of XOD enzyme from the protein database, removing excess ligands and H2O for subsequent docking experiments with peptide segments;

[0090] ②After docking the screened peptide sequence with 1N5X, the binding energy is obtained, and the potential XOD inhibitory activity is evaluated according to the size of the binding energy. The lower the molecular docking binding energy, the stronger the potential XOD inhibitory activity.

[0091] (5)Computer simulation of gastrointestinal digestion

[0092] Computer simulation of gastrointestinal digestion of the peptide segment with low molecular docking binding energy screened in step (4) to see if the screened peptide segment has the potential to resist gastrointestinal digestion. The specific steps are as follows:

[0093] ①Enzymatic digestion of the peptide segment by BIOPEP-UWM: ANALYSIS function in the BIOPEP database (https: / / biochemia.uwm.edu.pl);

[0094] ②Selecting chymotrypsin A, trysin, and pepsin (pH 1.3) for simulated enzyme digestion, and selecting peptide segments that can resist digestion or have potential activity after digestion.

[0095] Example 5

[0096] This example is an analysis of the XOD inhibitory peptides screened in Example 4, specifically:

[0097] (1) Sequence number, peptide sequence, molecular weight and binding energy

[0098] Table 1 Sequence number, peptide sequence, molecular weight and binding energy

[0099] Sequence No. Peptide segment sequence Molecular weight (Da) Binding energy (kcal / mol) SEQ ID No. 1 APF 333.1688 -6.8 SEQ ID No. 2 LNL 358.2216 -6.53 SEQ ID No. 3 APFA 404.2059 -6.12 SEQ ID No. 4 TLPL 430.2427 -5.31 SEQ ID No. 5 TIPL 430.2427 -5.04 SEQ ID No. 6 GIFGTN 607.2966 -5.06

[0100] (2) XOD structure

[0101] As shown in Figure 1, it is the protein crystal structure of XOD (1N5X). Figure 5

[0102] (3) APF and XOD molecular docking

[0103] As shown in Figure 2, APF forms six hydrogen bonds with GLU-802, SER-876, LEU-873, and ASP-872 amino acid residues, with a binding energy of -6.8 kcal / mol, showing good XOD inhibitory properties at the molecular docking level. Figure 6

[0104] (4) LNL and XOD molecular docking

[0105] As shown in Figure 3, LNL forms three hydrogen bonds with LYS-771, GLU-802, and ASN-768, with a binding energy of -6.53 kcal / mol, showing good XOD inhibitory properties at the molecular docking level. Figure 7

[0106] (5) APFA and XOD molecular docking

[0107] As shown in Figure 4, APFA forms two hydrogen bonds with GLU-802 and SER-876 amino acid residues in the MOS site, with a binding energy of -6.12 kcal / mol. Figure 8

[0108] (6) TLPL and XOD molecular docking

[0109] As shown in Figure 5, TLPL forms one hydrogen bond with GLU-802 amino acid residues in the MOS site, with a binding energy of -5.31 kcal / mol. Figure 9

[0110] (7) TIPL and XOD molecular docking

[0111] As shown in Figure 6, TIPL forms one hydrogen bond with GLU-802 amino acid residues in the MOS site, with a binding energy of -5.31 kcal / mol. Figure 10 ​​​​​As shown, TLPL forms two hydrogen bonds with the GLU-802 amino acid residue at the MOS site, with a binding energy of -5.04 kcal / mol.

[0112] (8) Molecular docking of GIFGTN and XOD

[0113] like Figure 11 As shown, GIFGTN forms eight hydrogen bonds with amino acid residues SER-710, ARG-871, HIS-875, and GLU-879, with a binding energy of -5.06 kcal / mol.

[0114] This invention utilizes peptidomics and molecular docking technology to rapidly screen XOD-inhibiting peptides, avoiding the time-consuming and labor-intensive drawbacks of stepwise separation and purification. These peptides can be used as adjuncts or alternatives to uric acid-lowering drugs. This invention effectively identified highly active XOD-inhibiting peptides from wheat protein peptides, with the peptide sequences being APF (SEQ ID No. 1), LNL (SEQ ID No. 2), APFA (SEQ ID No. 3), TLPL (SEQ ID No. 4), TIPL (SEQ ID No. 5), and GIFGTN (SEQ ID No. 6).

[0115] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A wheat gluten protein peptide with XOD inhibitory activity, characterized in that: The wheat gluten protein peptide is derived from the hydrolysis of wheat gluten protein, and the wheat gluten protein peptide includes peptide segments as shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, or SEQ ID No.

6.

2. The wheat gluten protein peptide as described in claim 1, characterized in that: The molecular docking binding energies of the peptides are all less than -5 kcal / mol.

3. The application of the wheat gluten peptide as described in claim 1 or 2 in the preparation of XOD inhibitors.

4. The use of wheat gluten peptide as described in claim 1 or 2 in the preparation of uric acid-lowering drugs.

5. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the wheat gluten peptide as described in claim 1 or 2.

6. The pharmaceutical composition according to claim 5, characterized in that: The pharmaceutical composition is an oral dosage form or a pharmaceutically acceptable excipient.

7. The method for screening and identifying protein peptides with XOD inhibitory activity as described in claim 1, characterized in that: include, Wheat gluten protein peptides were produced by hydrolyzing wheat gluten protein with proteases. Ultrafiltration is used for separation and purification; Sequencing of wheat gluten peptides <3kDa using peptidomics; Screening for potentially highly active XOD-inhibiting peptides; The selected peptides were validated using molecular docking technology. Computer simulation of gastrointestinal digestion.

8. The method as described in claim 7, characterized in that: The method involves using protease to hydrolyze wheat gluten protein to produce wheat gluten peptides, wherein the hydrolysis temperature is 35–55°C and the pH is 6.5–8.

5.

9. The method as described in claim 7, characterized in that: The screening of potentially highly active XOD-inhibiting peptides includes: PeptideRanker is used to predict and score the bioactivity of peptides. Search for the protein source of a peptide in a protein database; Toxicity prediction using Toxinpred; Allergenicity prediction using AllerTOP; Select protein-derived peptides from the sequenced peptides that are not modified by functional groups and have a peak area greater than 1.00 × 10⁻⁶. 7 A peptide with a sequence of 3 to 10 amino acids that is non-toxic and non-allergenic and has not yet been disclosed.

10. The method as described in claim 7, characterized in that: The computer simulation of gastrointestinal digestion includes: Peptides were digested using the BIOPEP-UWM:ANALYSIS function in the BIOPEP database; Simulated enzyme digestion was performed using chymotrypsin A, trypsin, and pepsin.

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