Wheat peptide as well as preparation method and application thereof in anti-oxidation and anti-inflammatory aspects

Abstract

The invention belongs to the technical field of plant peptides, and particularly relates to a wheat peptide as well as a preparation method and application thereof in the aspects of oxidation resistance and inflammation resistance. According to the present invention, the peptide fragment structure of the wheat peptide is determined, the wheat peptide contains at least 85 peptide fragments, and dipeptide, tripeptide and other small molecule peptides are mainly adopted; meanwhile, tests find that the wheat peptide has smaller molecular weight than wheat protein, is easily absorbed by human bodies, and has better thermal stability and a tighter structure. Based on the structural characteristics of the wheat peptide, the invention confirms the biological activities of the wheat peptide and the characteristic peptide fragments FQ, FV and FA in the wheat peptide in the aspects of oxidation resistance and inflammation resistance, so that the wheat peptide has a very good protection effect on alcohol-induced hepatocyte injury. Therefore, the novel wheat peptide and the characteristic peptide fragment with antioxidant and anti-inflammatory activity are developed, the types of the wheat peptide are enriched, and a technical support is provided for further application of the wheat peptide.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant peptides, and particularly relates to a wheat peptide, a preparation method thereof and application thereof in antioxidation and anti-inflammation. BACKGROUND

[0002] The wheat peptide is a small molecular polypeptide substance obtained by extracting and separating proteins extracted from wheat, and has the characteristics of good water solubility, dispersion stability, easy absorption and strong biological activity. The wheat peptide has multiple functions, such as nutritional supplement, immune enhancement, improvement of gastrointestinal function and regulation of blood sugar and blood lipid. Based on the functional activity and the characteristics of good water solubility and easy absorption, the wheat peptide is currently widely applied in drugs or health products.

[0003] After a large amount of research, the preparation methods of the wheat peptide currently mainly include biological extraction, protease hydrolysis, microbial fermentation, acid-base hydrolysis, enzymatic synthesis, recombinant DNA synthesis and chemical synthesis. Among them, the protease hydrolysis has become the main method for preparing peptide products due to its safety, easy control, high efficiency and low cost. However, the wheat peptide prepared by the existing protease hydrolysis is mostly a primary mixture, and the peptide segment structure of the wheat peptide cannot be accurately extracted, and the functional activity of the accurate peptide segment is also relatively lacking, so that the wheat peptide cannot be widely applied in practice. SUMMARY

[0004] The application aims to provide a wheat peptide, a preparation method thereof and application thereof in antioxidation and anti-inflammation. The wheat peptide is prepared, the peptide segment structure in the wheat peptide is determined, and the biological activity of the wheat peptide and the peptide segment structure in antioxidation and anti-inflammation is found.

[0005] The application provides a wheat peptide, which comprises one or more of the peptide segments shown in the following table:

[0006]

[0007] Preferably, the total protein content in the wheat peptide is 91.54±2.11%, the acid-soluble protein content is 87.23±1.76%, the ash content is 1.62±0.21%, and the water content is 1.97±0.09%.

[0008] The weight average molecular weight of the wheat peptide is 387.80 Da, and the molecular weight of the wheat peptide is less than 5000 Da.

[0009] Preferably, the wheat peptide comprises the amino acids shown in the following table, and the types and contents of the amino acids are shown in the following table:

[0010] Amino acid name Content (%) Amino acid name Content (%) Glutamic acid Arginine Proline 37.93±2.56 Alanine 2.66±0.15 Leucine 11.57±0.95 Isoleucine 2.64±0.23 Phenylalanine 5.68±0.28 Threonine 2.50±0.08 Serine 4.87±0.29 Histidine 1.81±0.09 Valine 4.32±0.41 Cysteine 1.69±0.11 Tyrosine 3.29±0.19 Methionine 1.23±0.06 Aspartic acid 2.83±0.10 Lysine 1.22±0.13 Glycine 2.72±0.18 Tryptophan 1.09±0.09 Figure 1 2.71±0.26 Figure 2 0.55±0.22

[0011] The application further provides a preparation method of the wheat peptide.

[0012] Mixing the gluten meal with deionized water to obtain a gluten meal suspension;

[0013] Mixing the gluten meal suspension with the alkaline protease to perform a first enzymolysis to obtain a first enzymolysis mixture;

[0014] Mixing the first enzymolysis mixture with the neutral protease to perform a second enzymolysis to obtain the second enzymolysis mixture;

[0015] Drying the second enzymolysis mixture to obtain the wheat peptide.

[0016] Preferably, the preparation method further comprises identifying the peptide segment composition of the wheat peptide, and the identification method comprises the following steps:

[0017] Mixing the wheat peptide with deionized water to obtain a wheat peptide aqueous solution;

[0018] Filtering the wheat peptide aqueous solution, and performing high performance liquid chromatography tandem mass spectrometry detection on the filtered wheat peptide solution to obtain the peptide segment structure in the wheat peptide;

[0019] The chromatographic conditions of the high performance liquid chromatography tandem mass spectrometry detection comprise: the chromatographic column is an Inertsil ODS-3 chromatographic column; the mobile phase comprises phase A and phase B, the phase A is formic acid and water, the volume concentration of formic acid in the phase A is 0.1%, the phase B is formic acid and acetonitrile, and the volume concentration of formic acid in the phase B is 0.1%; the chromatographic gradient is 0-15 min, 0%-40% B; 15-20 min, 40%-80% B; 20-25 min, 80% B; 25-35 min, 0% B; and the flow rate is 0.2 mL / min;

[0020] The mass spectrometry parameters of the high performance liquid chromatography tandem mass spectrometry detection comprise: the ionization mode of ESI, the atomization gas flow rate is 3.0 L / min, the heating gas flow rate is 10 L / min, the dry gas flow rate is 10 L / min, the ion spray voltage is 4.5 kV, the desolvation tube temperature is 250℃, the heating module temperature is 400℃, and the ion source temperature is 300℃.

[0021] Preferably, the mass to volume ratio of the alkaline protease to the gluten meal suspension is (0.10-0.14) g:100 mL, the enzyme activity of the alkaline protease is 2.4-4.0 AU-A / g, and the mass concentration of the gluten meal suspension is 11% w / w.

[0022] Preferably, the pH value of the first enzymolysis is 8-9; the temperature of the first enzymolysis is 55-65℃; and the time of the first enzymolysis is 2h.

[0023] Preferably, the mass / volume ratio of the neutral protease to the first enzymolysis mixture is (0.06-0.10) g:100 mL, and the enzyme activity of the neutral protease is 0.7-0.9 AU-N / g.

[0024] Preferably, the pH value of the second enzymolysis is 6.5-7.5; the temperature of the second enzymolysis is 45-55℃; and the time of the second enzymolysis is 3h.

[0025] The application also provides a use of the wheat peptide or a peptide segment in the wheat peptide in the preparation of an antioxidant and / or anti-inflammatory product; the wheat peptide is the wheat peptide described in the above technical solution or the wheat peptide prepared by the preparation method described in the above technical solution.

[0026] The peptide segment in the wheat peptide is one or more of FQ, FV and FA.

[0027] Beneficial effects:

[0028] The application aims to provide a wheat peptide, a preparation method thereof and an application thereof in antioxidant and anti-inflammatory aspects. The application determines the peptide segment structure of the wheat peptide, finds that the wheat peptide contains at least 85 peptide segments, and is mainly composed of small molecular peptides such as dipeptides and tripeptides. Meanwhile, the application determines through experiments that the molecular weight of the wheat peptide is smaller than that of wheat protein, and the wheat peptide has good thermal stability and a compact structure. Based on the above structural characteristics of the wheat peptide, the application confirms that the wheat peptide and the characteristic peptide segments FQ, FV and FA in the wheat peptide have biological activities in antioxidant and anti-inflammatory aspects, thereby having a good protective effect on alcohol-induced liver cell damage. It can be seen that the application develops new wheat peptides and characteristic peptide segments with antioxidant and anti-inflammatory effects, enriches the types of wheat peptides, and provides technical support for further application of the wheat peptides. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed to be used in the embodiments will be briefly introduced below.

[0030] Figure 3 Figure 2 is a molecular weight distribution graph of the wheat peptide in Example 2;

[0031] Figure 4 Figure 3 is a scanning electron microscope graph of the wheat peptide under 100 times and 6000 times in Example 3;

[0032] Figure 5 Figure 4 is a UV absorption spectrum graph of the wheat peptide in Example 3;

[0033] Figure 6 Fourier transform infrared spectroscopy of wheat peptide in Example 3;

[0034] Figure 7 Thermogravimetric analysis of wheat peptide in Example 3;

[0035] Figure 8 Results of the effect of different concentrations of wheat peptide on the viability of HepG2 cells in Example 4;

[0036] Figure 9 Results of the effect of different concentrations of wheat peptide on the viability of damaged HepG2 cells in Example 4;

[0037] Figure 10 SOD activity in HepG2 cells under the action of different concentrations of wheat peptide in Example 4;

[0038] Figure 11 MDA level in HepG2 cells under the action of different concentrations of wheat peptide in Example 4;

[0039] Figure 12 ROS level in HepG2 cells under the action of different concentrations of wheat peptide in Example 4;

[0040] Figure 13 GSH level in HepG2 cells under the action of different concentrations of wheat peptide in Example 4;

[0041] Figure 14 CAT activity in HepG2 cells under the action of different concentrations of wheat peptide in Example 4;

[0042] Figure 15 TNF-α level in HepG2 cells under the action of different concentrations of wheat peptide in Example 4;

[0043] Figure 16 IL-6 level in HepG2 cells under the action of different concentrations of wheat peptide in Example 4;

[0044] Figure 17 Expression of Nrf2 / Keap1 / HO-1 protein level in HepG2 cells under the action of different concentrations of wheat peptide in Example 4;

[0045] Figure 18 Effect of FQ, FV, and FA on the viability of damaged HepG2 cells in Example 4;

[0046] Figure 19 SOD level in HepG2 cells under the action of FQ, FV, and FA in Example 4;

[0047] Figure 20 MDA level in HepG2 cells under the action of FQ, FV, and FA in Example 4;

[0048] Figure 21 ROS level in HepG2 cells under the action of FQ, FV and FA in Example 4;

[0049] Figure 22 GSH level in HepG2 cells under the action of FQ, FV and FA in Example 4;

[0050] Figure 23 CAT activity in HepG2 cells under the action of FQ, FV and FA in Example 4;

[0051] Figure 24 TNF-α level in HepG2 cells under the action of FQ, FV and FA in Example 4;

[0052] Figures 6-24 IL-6 level in HepG2 cells under the action of FQ, FV and FA in Example 4;

[0053] Total protein content (%) Expression of Nrf2 / Keap1 / HO-1 protein level in HepG2 cells under the action of FQ, FV and FA in Example 4;

[0054] In Acid soluble protein content (%) In the table, different lowercase letters represent significant differences (P < 0.05). DETAILED DESCRIPTION

[0055] The present application provides a wheat peptide, which comprises one or more of the peptide segments shown in the following table:

[0056]

[0057] In the present application, "pE" in the N-terminal of some peptide segments in the above table is pGlu (pyroglutamic acid).

[0058] As an embodiment, the total protein content in the wheat peptide is 91.54±2.11%, the acid-soluble protein content is 87.23±1.76%, the ash content is 1.62±0.21%, and the moisture content is 1.97±0.09%. As an embodiment, the weight average molecular weight of the wheat peptide is 387.80 Da; the molecular weight of the wheat peptide is <5000 Da, wherein the component with a molecular weight of <1000 Da accounts for 92.60%, the component with a molecular weight in the range of 189-1000 Da accounts for 49.60%, and the component with a molecular weight in the range of 132-189 Da accounts for 30.23%.

[0059] As an embodiment, the wheat peptide comprises the amino acids shown in the following table, and the types and contents of the amino acids are shown in the following table:

[0060]

[0061]

[0062] As an embodiment, the content of essential amino acids in the wheat peptide is 91.31±7.92%.

[0063] The present application also provides a preparation method of the wheat peptide according to the above technical solution, comprising the following steps:

[0064] Mixing gluten meal with deionized water to obtain a gluten meal suspension;

[0065] Mixing the gluten meal suspension with alkaline protease to perform first enzymolysis, to obtain a first enzymolysis mixture;

[0066] Mixing the first enzymolysis mixture with neutral protease to perform second enzymolysis, to obtain the second enzymolysis mixture; drying the second enzymolysis mixture to obtain the wheat peptide.

[0067] The present application mixes gluten meal with deionized water to obtain a gluten meal suspension. As an embodiment, the concentration of the gluten meal suspension is 11% w / w.

[0068] After obtaining the gluten meal suspension, the present application mixes the gluten meal suspension with alkaline protease to perform first enzymolysis, to obtain a first enzymolysis mixture. As an embodiment, the mass of alkaline protease to the volume of the gluten meal suspension is (0.10-0.14) g: 100 mL; as another embodiment, the mass of alkaline protease to the volume of the gluten meal suspension is 0.12 g: 100 mL. As an embodiment, the enzyme activity of the alkaline protease is 2.4-4.0 AU-A / g; as another embodiment, the enzyme activity of the alkaline protease is 2.4 AU-A / g. As an embodiment, the pH value of the first enzymolysis is 8-9; as another embodiment, the pH value of the first enzymolysis is 8.5. As an embodiment, the temperature of the first enzymolysis is 55-65℃; as another embodiment, the temperature of the first enzymolysis is 60℃. As an embodiment, the time of the first enzymolysis is 2 h.

[0069] After obtaining the first enzymatic hydrolysis mixture, the present application mixes the first enzymatic hydrolysis mixture with neutral protease to perform second enzymatic hydrolysis, and obtains the second enzymatic hydrolysis mixture. As an embodiment, the mass / volume ratio of the neutral protease to the first enzymatic hydrolysis mixture is (0.06-0.10) g:100 mL; as another embodiment, the mass / volume ratio of the neutral protease to the first enzymatic hydrolysis mixture is 0.08 g:100 mL. As an embodiment, the enzyme activity of the neutral protease is 0.7-0.9 AU-N / g; as another embodiment, the enzyme activity of the neutral protease is 0.8 AU-N / g. As an embodiment, the pH value of the second enzymatic hydrolysis is 6.5-7.5; as another embodiment, the pH value of the second enzymatic hydrolysis is 7.0. As an embodiment, the time of the second enzymatic hydrolysis is 3 h.

[0070] After obtaining the second enzymatic hydrolysis mixture, the present application dries the second enzymatic hydrolysis mixture to obtain the wheat peptide. The present application does not have special limitation on the drying method, and the conventional drying method in the art can be used.

[0071] As an embodiment, after obtaining the wheat peptide, the present application identifies the peptide segment structure of the wheat peptide, and the identification method comprises the following steps: mixing the wheat peptide with deionized water to obtain a wheat peptide aqueous solution;

[0072] The wheat peptide aqueous solution is filtered, and the filtered wheat peptide solution is detected by high performance liquid chromatography tandem mass spectrometry to obtain the peptide segment structure in the wheat peptide.

[0073] As an implementation form, the concentration of the aqueous solution of the wheat peptide is 20 pg / mL. As an implementation form, the filtration is nylon membrane filtration; as another implementation form, the pore size of the nylon membrane is 0.22 pm. As an implementation form, the chromatographic conditions of the high performance liquid chromatography-mass spectrometry detection include: the chromatographic column is an Inertsil ODS-3 chromatographic column; the mobile phase includes phase A and phase B, the phase A is formic acid and water, the volume concentration of formic acid in the phase A is 0.1%, the phase B is formic acid and acetonitrile, the volume concentration of formic acid in the phase B is 0.1%; the chromatographic gradient is 0-15 min, 0%-40% B; 15-20 min, 40%-80% B; 20-25 min, 80% B; 25-35 min, 0% B; the flow rate is 0.2 mL / min. As an implementation form, the mass spectrometry parameters of the high performance liquid chromatography-mass spectrometry detection include: the ionization mode of ESI, the flow rate of atomization gas is 3.0 L / min, the flow rate of heating gas is 10 L / min, the flow rate of dry gas is 10 L / min, the ion spray voltage is 4.5 kV, the desolvation tube temperature is 250°C, the heating module temperature is 400°C, and the ion source temperature is 300°C.

[0074] As an implementation form, the peptide segment structure in the wheat peptide has been defined in the above scheme and will not be repeated.

[0075] The application also provides an application of the wheat peptide or the peptide segment in the wheat peptide in the preparation of an antioxidant and / or anti-inflammatory product; the wheat peptide is the wheat peptide in the above technical solution or the wheat peptide prepared by the preparation method in the above technical solution; the peptide segment in the wheat peptide is one or more of FQ, FV and FA.

[0076] As an implementation form, the product includes a product having a protective effect on alcohol-induced hepatocyte damage; as another implementation form, the determination index of the protective effect on alcohol-induced hepatocyte damage includes the determination of the expression of one or more of Nrf2, Keap1 and HO-1 proteins. As an implementation form, the antioxidant index includes one or more of cell survival rate, superoxide dismutase (SOD) activity, catalase (CAT) activity, malondialdehyde (MDA) level, reactive oxygen species (ROS) level and glutathione (GSH) level. As an implementation form, the anti-inflammatory index includes tumor necrosis factor-α (TNF-α) level and / or interleukin-6 (IL-6) level.

[0077] As an implementation, the application is performed by applying a wheat peptide solution or a peptide segment solution, the concentration of the wheat peptide solution being 0.25-12 mg / mL; as another implementation, the concentration of the wheat peptide solution being 2-6 mg / mL; as an implementation, the concentration of the peptide segment solution being 50-300 μg / mL; as another implementation, the concentration of the peptide segment solution being 100-200 μg / mL.

[0078] In order to further illustrate the present application, the technical solutions provided by the present application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0079] Example 1

[0080] A preparation method of a wheat peptide, the steps being as follows:

[0081] A gluten suspension (concentration being 11% (w / w)) is mixed with an alkaline protease (enzyme activity being 2.4 AU-A / g, purchased from Novozymes (China) Biotechnology Co., Ltd., product name being Alcalase), the alkaline protease being added in an amount of 0.12 g / 100 mL, the pH being adjusted to 8.5, and the temperature being kept at 60°C for 2 h of enzymolysis; after the alkaline protease enzymolysis, a neutral protease (enzyme activity being 0.8 AU-N / g, purchased from Novozymes (China) Biotechnology Co., Ltd., product name being Neutrase) is added in the mixture in an amount of 0.08 g / 100 mL, the pH is adjusted to 7, and the temperature is kept at 50°C for 3 h of enzymolysis, the substrate concentration being 2.5 g / 100 mL, to obtain the wheat peptide.

[0082] Example 2

[0083] Detection of the physicochemical properties of the wheat peptide obtained in Example 1

[0084] 2.1 Experimental method

[0085] 2.1.1 Detection of basic physicochemical components

[0086] The physicochemical components are determined according to the national standard method, i.e., the protein content is determined according to the method in GB 5009.5-2016, the specific steps being that the nitrogen in the protein is converted into ammonium sulfate through sulfuric acid digestion, ammonia gas is released under alkaline conditions, and after being absorbed by boric acid, it is titrated with hydrochloric acid, the total nitrogen content is calculated and multiplied by the protein conversion coefficient to obtain the protein content. The acid-soluble protein content is determined according to the method in GB / T 22729-2008; the water content is determined according to the method in GB 5009.3-2016; and the ash content is determined according to the method in GB 5009.4-2016.

[0087] 2.1.2 Detection of relative molecular mass

[0088] The molecular weight distribution of wheat peptide was determined by high performance liquid chromatography (equipped with Empower workstation GPC software and 2487 ultraviolet detector), that is, the sample components were separated according to the difference in molecular volume, the macromolecules flowed out first due to short retention time, and the small molecules flowed out later due to long retention time. The relative molecular mass distribution range of wheat peptide was obtained by peak area normalization method, and the larger the peak area, the higher the content of the component. An appropriate amount of peptide standard was weighed and prepared into a 1 × 10 -3 g / mL standard solution, filtered through a 0.22 μm polytetrafluoroethylene filter membrane, and detected at a ultraviolet absorption wavelength of 220 nm. The chromatogram and its data were processed using the special data processing software (GPC software) for gel chromatography relative molecular mass distribution determination. The relative molecular mass calibration curve was drawn according to the molecular mass analysis results of the standard solution, and the relative molecular mass size and distribution range of the protein hydrolysate were calculated. The chromatographic detection conditions are shown in Table 1.

[0089] Table 1 Chromatographic detection conditions

[0090]

[0091]

[0092] 2.1.3 Amino acid composition determination

[0093] The amino acid composition of wheat peptide was determined according to the national standard GB 5009.124-2016 “Determination of amino acids in food” for food safety national standard. The specific determination steps are as follows: 0.1-0.5g sample is weighed, 6mol / L hydrochloric acid (containing phenol antioxidant) is added, vacuum is extracted and nitrogen is filled, and then hydrolysis is carried out at 110℃ for 22h. Sodium ion exchange column separation and gradient elution are carried out by using automatic amino acid analyzer. Combined with post-column ninhydrin color reaction (130℃), the content of each amino acid is quantitatively detected, and the detection wavelength is 570nm.

[0094] 2.1.4 Stability determination

[0095] (1) Wheat peptide acid-base stability determination

[0096] Wheat peptide was prepared into a 10mg / mL solution with Aesop ultrapure water, and evenly divided into 6 tubes, with 1 tube reserved as a control with pH 7. The pH of the remaining 5 tubes was adjusted to 2, 4, 6, 8 and 10 respectively with 1mol / L HCI and NaOH, and then cooled to room temperature after water bath at 37℃ for 2h. The relative molecular mass distribution was determined.

[0097] (2) Wheat peptide thermal stability determination

[0098] The wheat peptide powder sample was prepared into a solution with a concentration of 10 mg / mL using Aesop ultrapure water, and evenly distributed into 6 tubes, respectively, and placed in a water bath at 25°C (control group), 20°C, 40°C, 60°C, 80°C, and 100°C for 2 hours. After the water bath, the temperature was balanced at room temperature, and the relative molecular mass distribution was measured.

[0099] (3) In vitro digestion stability determination of wheat peptides

[0100] ① Pepsin digestion

[0101] The wheat peptide sample was dissolved in Aesop ultrapure water to 2 mg / mL, and the pH value of the sample solution was adjusted to 2 using HCl (1 mol / L). The sample was placed in a water bath at 37°C for 5 minutes, and pepsin was added at 2% (w / w) of the substrate and mixed uniformly. The sample was then placed in a water bath at 37°C for 3 hours, and enzyme inactivation was performed at 100°C for 10 minutes. After the temperature was stabilized at room temperature, the relative molecular mass distribution was measured.

[0102] ② Trypsin digestion

[0103] The pH value of the wheat peptide solution was adjusted to 6.8 using NaOH (1 mol / L), and the solution was placed in a water bath at 37°C for 5 minutes. Then, trypsin was added at 2% (w / w) of the substrate and mixed uniformly. The solution was then placed in a water bath at 37°C for 3 hours, and enzyme inactivation was performed at 100°C for 10 minutes. After the temperature was stabilized at room temperature, the relative molecular mass distribution was measured.

[0104] ③ Pepsin digestion followed by trypsin digestion

[0105] After digestion according to the aforementioned pepsin digestion method, the pH value of the reaction solution was immediately adjusted to 6.8 using NaOH (1 mol / L), and 2% (w / w) trypsin was added and mixed uniformly. The solution was then placed in a water bath at 37°C for 3 hours, and enzyme inactivation was performed at 100°C for 10 minutes. After the temperature was stabilized at room temperature, the relative molecular mass distribution was measured.

[0106] 2.2 Experimental results

[0107] 2.2.1 Composition of wheat peptides

[0108] As shown in Table 2, the moisture content of the wheat peptides was (1.97 ± 0.09)%, the ash content was (1.62 ± 0.21)%, the total protein content was (91.54 ± 2.11)%, and the acid-soluble protein content was (87.23 ± 1.76)%. Acid-soluble protein refers to protein molecules that can be dissolved under specific pH conditions. This type of biological macromolecule typically contains a large number of acidic residues, such as glutamic acid and aspartic acid, in its amino acid composition. Due to the dissociation characteristics of the side chain groups, this type of protein exhibits significant solubility in low-pH environments.

[0109] Composition of wheat peptides

[0110] Ash content (%) Moisture content (%) Figure 1 Figure 2 91.54±2.11 87.23±1.76 1.62±0.21 1.97±0.09

[0111] 2.2.2 Relative molecular mass of wheat peptides

[0112] In this example, the molecular weight of the smallest dipeptide (Gly-Gly) is 132 Da, and the molecular weight of the smallest tripeptide (Gly-Gly-Gly) is 189 Da, which are used as the molecular weight range boundaries. From Table 2 and Table 3, it can be seen that the weight average molecular weight of the wheat peptides is 387.8 Da, and the molecular weight of the wheat peptides is less than 5000 Da, of which the component with a molecular weight of less than 1000 Da accounts for as high as 92.60%. Half of the molecular weight of the wheat peptides is concentrated in the range of 189-1000 Da, accounting for 49.60%, and the molecular weight in the range of 132-189 Da is 30.23%, which can indicate that most of the peptide segments in the wheat peptides are dipeptides and tripeptides. The size of the molecular mass is closely related to the absorption and utilization rate of the human body, and the digestion and transport speed of small peptides in the gastrointestinal tract can be stronger than that of macromolecular peptides or proteins. Figure 3 Table 3 Molecular weight distribution of wheat peptides

[0113]

[0114]

[0115] 2.2.3 Amino acid composition of wheat peptides

[0116] ​The amino acid species, quantity and arrangement order are important factors to determine the function of wheat peptide. The content of essential amino acids in wheat peptide is very rich, which is (22.15±3.23)%. Amino acids are the units that constitute proteins, and components rich in amino acids can play an important role in regulating human physiological functions. As can be seen from Table 4, the amino acids with the highest content in wheat peptide are glutamic acid (36.23±1.86)% and proline (11.57±0.95)%, and the amino acid with the lowest content is lysine, which accounts for only (1.25±0.13)%. In addition, wheat peptide is rich in leucine, phenylalanine, serine, arginine, isoleucine and alanine, with a total mass fraction of 22.67%. These amino acids may synergistically enhance their biological activity. Aromatic amino acids (AAA) are an important indicator of liver metabolism, and the content of AAA is the sum of phenylalanine, tyrosine and tryptophan. The content of AAA in wheat peptide is only (8.25±0.38)%. Studies have found that an increase in AAA levels is a potential marker of abnormal liver function, especially in hepatic encephalopathy or advanced liver disease. In addition, the content of non-essential amino acids is (68.61±1.02)%, and part of the amino acids can also be converted into essential amino acids through chemical changes and modifications under normal physiological metabolism of the body, such as tyrosine can be converted into phenylalanine. The content of hydrophobic amino acids is (31.77±0.43)%. Studies have shown that hydrophobic amino acid residues usually have hydrophobic groups, so the presence of hydrophobic amino acids in polypeptides helps to enhance the solubility of the peptides in lipids, thereby removing free radicals in lipids. Therefore, the presence of hydrophobic amino acids is a key point for polypeptides to remove free radicals and play an antioxidant role.

[0117] Table 4 Distribution of amino acid species and content of wheat peptide

[0118]

[0119] Note: Glutamic acid a is glutamic acid + glutamine, aspartic acid b is aspartic acid + asparagine.

[0120] 2.2.4 Acid-base stability of wheat peptide

[0121] The relative molecular weight distribution of wheat peptide under different pH conditions is shown in Table 5. When the pH of the solution is adjusted to 2, 4, 6, 8, and 10, respectively, the mass percentage of low molecular weight components below 1000 Da is 90.08%, 90.45%, 91.80%, 90.93%, 90.99%, and 90.75%, respectively, with a difference range of 1.72% between each experimental group, indicating that the pH fluctuation has less effect on the molecular weight distribution. The molecular weight of the smallest dipeptide (Gly-Gly) is 132 Da and the molecular weight of the smallest tripeptide (Gly-Gly-Gly) is 189 Da, which are used as the range of molecular weight. The content change of the molecular weight in the range of 132 Da, 132-189 Da, 189-1000 Da, etc. before and after the enzymatic hydrolysis of the wheat peptide sample is analyzed, and the change amplitude is used as an important indicator to judge the stability of the sample. The data shows that the wheat peptide has high stability under acidic and alkaline conditions, and no significant degradation of high molecular weight components to low molecular weight is observed. It is worth noting that when the pH is adjusted to 2, the content of the 189-1000 Da component is slightly lower than that of other experimental groups, and the proportion of ultra-small molecular weight substances below 132 Da increases accordingly. This phenomenon may be related to the hydrolysis and fragmentation of some peptide chains under extreme acidic conditions.

[0122] Table 5 Molecular weight distribution of wheat peptide under different pH conditions

[0123]

[0124] 2.2.5 Thermal stability of wheat peptide

[0125] The molecular weight distribution of wheat peptide after treatment at different temperatures is shown in Table 6. The content below 1000 Da is 91.08%, 91.08%, 91.22%, 91.12%, 91.15%, and 93.92%, respectively. With the increase of temperature, the content of 2000-5000 Da part gradually decreases, and the content of 2000-5000 Da part is the least and the content of 132 Da part is the most under the condition of 100°C, indicating that a small amount of degradation of wheat peptide occurs with the increase of temperature. Compared with high temperature conditions, wheat peptide is more stable under low temperature conditions. Except for 100°C, the molecular weight distribution changes slightly under each temperature, with a maximum change of 2.24%, indicating that wheat peptide should be stored in high temperature environment as much as possible.

[0126] Table 6 Molecular weight distribution of wheat peptide under different temperatures

[0127]

[0128] 2.2.6 In vitro digestion stability of wheat peptide

[0129] As shown in Table 7, the proportion of the components with molecular weight below 1000 Da in the wheat peptide control group and under different digestion modes was 73.9%, 92.72%, 92.19%, and 92.49%. Compared with the control group, after digestion, the content of wheat peptides with molecular weight above 2000 Da decreased, and the content of 1000 Da components increased, indicating that after digestion, the large molecular weight part became small molecular weight part, and the small molecular weight peptide was more easily absorbed and utilized by humans, and could produce bioactive peptide effect in the human body. And under the action of the three digestion modes, although decomposition occurred, the content of the part with molecular weight less than 132 Da was less than 10%, indicating that the wheat peptide was not over-digested, but concentrated in the range of 1000-132 Da suitable for human utilization. The change under the digestion of pepsin followed by trypsin was slightly larger than the other two digestion modes, and the reason for analyzing this phenomenon may be due to the acidic environment. It can be known from the above analysis that the wheat peptide is not easy to be digested and decomposed, and most of them can maintain their original structure to play a good bioactive function in the body.

[0130] Table 7 Molecular weight distribution of wheat peptides under different digestion modes

[0131]

[0132] Example 3

[0133] Structure characterization and structure identification of the wheat peptide prepared in Example 1

[0134] 3.1 Experimental method

[0135] 3.1.1 Scanning electron microscope morphology observation

[0136] The morphology and structure of the wheat peptide were observed by scanning electron microscope: the powder sample was fixed on an aluminum rod using double-sided tape, the surface was sprayed with gold, the acceleration voltage was 5 kV, and the magnification was 100 times and 6000 times to take pictures.

[0137] 3.1.2 Ultraviolet spectrum scanning

[0138] Turn on the ultraviolet-visible spectrophotometer and preheat to stable state, set the scanning wavelength to 240-400 nm, the slit width to 1.0 mm, and the sampling interval to 0.5 nm. Prepare a 2.0 mg / mL wheat peptide solution with pure water, place it in a cuvette, and place the cuvette in the sample slot of the spectrophotometer. Observe the absorption peaks, which can reflect the molecular structure and optical properties of the wheat peptide.

[0139] 3.1.3 Fourier transform infrared spectrum scanning

[0140] Take 5 mg of wheat peptide powder and 500 mg of dry potassium bromide (KBr) respectively, mix them well, put them in a marble mortar, and turn on the infrared lamp to irradiate. Grind the mixture until it becomes a powder with a particle size of 2.5 μm or less. Put the treated powder under the tablet press to press it into a transparent sheet. The wavelength range is 4000 to 500 cm -1 , the spectral resolution is 4 cm -1 , the scanning times are 32, the sample is scanned for the full wavelength, and the results are expressed in transmittance (T%).

[0141] 3.1.4 Thermogravimetric analysis

[0142] Use a TG-DSC thermal analyzer to scan. Place 5 mg of wheat peptide powder on an aluminum crucible, and take an empty crucible as a reference. Put the wheat peptide sample and the reference into the sample cell of the thermal gravimetric analyzer. Set the TG-DSC thermal analyzer scanning temperature to 25-900℃, the heating rate to 10 K / min, and the N2 flow rate to 30 mL / min. Record the real-time mass change of the sample, and obtain the temperature-mass relationship curve and the temperature-DTA relationship curve during the heating process of the sample.

[0143] 3.1.5 Identification of wheat peptide segment composition

[0144] Prepare a 2 mg / mL aqueous solution of wheat peptide, then dilute the sample aqueous solution to 20 μg / mL with distilled water, and filter it with a nylon membrane with a pore size of 0.22 μm. After filtration, proceed with the subsequent determination work.

[0145] During the determination, first connect the Inertsil ODS-3 column to the liquid chromatograph, and take 5 μL of the sample to be tested. In positive ion mode, perform Q3-Scan scanning of the sample, and set the mass-to-charge ratio scanning range to 100-600 Da. The liquid chromatography parameters are as follows: mobile phase A (containing 0.1% formic acid by volume in water), mobile phase B (containing 0.1% formic acid by volume in acetonitrile), flow rate set to 0.2 mL / min. Use a gradient elution program of 0-15 min, B 0-40%; 15-20 min, B 40-80%; 20-25 min, B 80%; 25-35 min, B 0%, and set the column oven temperature to 40℃. The mass spectrometry conditions are shown in Table 8.

[0146] Perform product ion scanning of the sample to be tested, and take 5 μL of the sample. The parent ion is input according to the results of Q3 scanning analysis, the collision energy is set to -25 V and -35 V, the liquid chromatography and mass spectrometry conditions are the same as those of Q3 scanning analysis, and then the product ions generated after collision are further analyzed.

[0147] The obtained precursor ion information and product ion information are compared with ion information of 2-3 peptides contained in a self-built database of functional peptides of China Institute of Food Fermentation, and if the characteristic ions corresponding to the target peptide segment are contained in the product ion results, it is determined that the sample contains the peptide segment.

[0148] Table 8 Mass spectrometer determination conditions

[0149]

[0150] 3.2 Experimental results

[0151] 3.2.1 Structure identification of wheat peptide segments

[0152] The arrangement order, quantity and type of amino acids are closely related to the biological activity of the peptide segment. It is reported that dipeptides and tripeptides in components with a molecular weight of less than 1000 Da may have higher nutritional value and physiological function than free amino acids, therefore, the components with a molecular weight of less than 1000 Da are intercepted for identification. The wheat peptide is subjected to PIS precursor ion scanning to obtain mass-to-charge ratio (m / z) information of the sample to be tested, i.e. precursor ion information, then the precursor ion is cracked by PIS, and fragment ion information of the peptide segment is collected to obtain product ion information. Subsequently, the ion information in the existing original peptide segment database of China Institute of Food Fermentation is compared with the obtained precursor ion information and product ion information one by one for analysis, and the peptide segment sequence can be determined. A total of 85 short peptides are detected (as shown in Table 9), all of which are peptide segments with an amino acid quantity of 2-3, indicating that most of the wheat peptide components are composed of small molecule peptides.

[0153] Table 9 Identification results of composition of wheat peptide segments

[0154]

[0155] 3.2.2 Structure characterization analysis of wheat peptides

[0156] (1) Morphology analysis of wheat peptides by scanning electron microscope

[0157] As shown in Figure 4 , the wheat peptides are mixed peptides with different molecular weights, so they present different sizes. When magnified by 6000 times, it can be observed that the wheat peptides are single, complete and smooth globular bodies, or wrinkled globular bodies. The wrinkling of the globular bodies is mainly caused by dehydration and drying, and there are some irregular wrinkles and larger gaps on the molecular surface, and the arrangement between molecules is relatively sparse. When magnified by 100 times, more broken bodies can be seen. The reason for containing broken bodies may be that the structure of the wheat peptides is relatively soft and fragile, and after being broken, the molecular weight is smaller, which is easy to be digested and absorbed by the human body.

[0158] (2) Analysis of wheat peptide by ultraviolet spectrum scanning

[0159] Ultraviolet spectral analysis of wheat peptides, such as Figure 5 As shown, the absorption characteristics of wheat peptides in the ultraviolet spectrum are mainly concentrated in the 200-300 nm range. The spectral characteristics of wheat peptides show obvious absorption peaks at 212 nm and 274 nm. The appearance of these absorption peaks is related to specific chemical groups in the peptide chain, especially the contributions of aromatic amino acids and peptide bonds. The absorption peak at 212 nm: This absorption peak is mainly related to the n→π* transition of peptide bonds (amide bonds). Peptide bonds typically show absorption in the 200-220 nm range in ultraviolet spectra; the presence of this peak indicates that the sample contains abundant peptide bond structures, which is a typical characteristic of proteins and polypeptides. The absorption peak at 274 nm is mainly related to the π→π* transition of aromatic amino acids. This suggests that the presence of several specific chemical groups in wheat peptides, including aromatic amino acids and hydrophobic amino acids, can enhance the antioxidant properties of the substance.

[0160] (3) Fourier transform infrared spectroscopy analysis of wheat peptides

[0161] The absorbable functional groups differ across different wavelength ranges, so the functional groups and structural characteristics of a substance can be detected based on the different features of its spectral pattern. For example... Figure 6 As shown, Fourier transform infrared spectroscopy analysis provides important information on the molecular structural characteristics of wheat peptides. The spectrum at 3294 cm⁻¹... 1 The broad absorption peak at 3077 cm⁻¹ can be attributed to the stretching vibration modes of NH (amide A band) and OH, indicating the presence of amino and hydroxyl functional groups in the molecular structure. This is consistent with the structural characteristics of the amide group and carboxylic acid terminus in the peptide chain. -1 and 2962cm -1 Unsaturated CH4(sp) was observed in the regions respectively. 2 Hybridized) and saturated CH (sp) 3 The characteristic peaks of stretching vibrations (hybridization) suggest that the sample may contain aromatic amino acid residues, while the latter is closely related to aliphatic alkyl structures (including -CH3, -CH2-, and -CH groups). Regarding the characteristic amide absorption band: 1665 cm⁻¹ -1 The strong absorption peak at 1544 cm⁻¹ corresponds to the amide I band (C=O stretching vibration). -1 The characteristic peak at 1247 cm⁻¹ belongs to the amide II band (a coupled mode of NH in-plane bending vibration and CN stretching vibration), while the peak at 1247 cm⁻¹... -1 The absorption at 1317 cm⁻¹ corresponds to the amide III band (a combination of CN stretching and NH bending vibrations). The synergistic appearance of these three characteristic peaks confirms the presence of a peptide bond (-CONH-). Notably, the absorption at 1317 cm⁻¹... -1The C-N stretching vibration absorption peaks at 1451 cm -1 and 1396 cm -1 are attributed to the bending vibration modes of methylene (-CH2-) and methyl (-CH3), while the characteristic peak at 1080 cm -1 is related to the stretching vibration of C-O bond. The weak absorption at 611 cm -1 may be due to the bending vibration of C-C in the aliphatic chain or the out-of-plane deformation vibration of the aromatic ring. The regular appearance of the amide characteristic absorption bands (1665, 1544, 1247 cm -1 ) in the spectrum confirms the presence of typical peptide bond structures in the wheat peptide.

[0162] (4) Thermal gravimetric analysis of wheat peptide

[0163] By observing the process of sample mass change with temperature through thermal gravimetric analysis, the degradation rate at any temperature, the starting point, the termination point and the residual amount, etc. can be obtained to reflect the thermal stability of the sample. Through analysis, the thermal stability, decomposition temperature and other temperature-related mass change characteristics of the wheat peptide can be understood to evaluate its stability and durability in actual processing applications. The thermal gravimetric analysis results of the wheat peptide are shown in Figure 7 . In the range of room temperature to 200°C, the mass of the wheat peptide fluctuates slightly and remains basically unchanged, indicating that in this temperature range, the wheat peptide is relatively stable and does not undergo significant volatilization or decomposition, only a small amount of adsorbed water is released, resulting in a small fluctuation in the mass of the wheat peptide. When the temperature reaches 252°C, the high temperature breaks the hydrogen bonds between the amino, carboxyl groups in the wheat peptide molecules and water molecules, and the crystal water inside the protein that is not easy to separate also releases due to the breaking of the bonds, resulting in a rapid decrease in sample mass. When the temperature exceeds 252°C, the molecular motion of the peptide bond is intensified, and the weight loss rate is accelerated. This may be due to the volatilization of peptide segments or free amino acids into the gas phase after the breakage. After more than 409°C, the overall mass is nearly constant, because the early-decomposing peptide chains have been decomposed, leaving some more stable peptide segments that have little effect on the mass, thus greatly slowing down the mass decrease rate. This may be due to the fact that the structure of the small molecular weight peptide is mainly composed of primary structure and a small amount of secondary peptide chain, and the wheat peptide does not have a high-level structure, resulting in its low sensitivity to heat treatment, indicating that the wheat peptide has good thermal stability and a relatively tight structure.

[0164] Example 4

[0165] Detection of antioxidant and anti-inflammatory functions of the wheat peptide prepared in Example 1

[0166] 4.1 Experimental method

[0167] 4.1.1 Protective effect of different concentrations of wheat peptides on alcohol-induced injury of HepG2 cells

[0168] 4.1.1.1 Culture of HepG2 cells

[0169] In this example, the HepG2 cell line was cultured: after the thawed and recovered cell suspension was mixed with 5 mL of fresh culture medium, it was inoculated into a culture bottle and placed in a 37°C, 5% CO2 incubator for primary culture. The cell adhesion process was monitored daily by an inverted microscope and the morphological characteristics were recorded. The culture system was maintained by replacing the fresh culture medium every 48-72 h, and after 2-3 passages, the formal experiment could be started.

[0170] 4.1.1.2 Screening of the optimal concentration and time of alcohol by CCK-8 method

[0171] The CCK-8 method was used to determine the activity of HepG2 cells. The logarithmic growth phase of HepG2 cells was inoculated into a 96-well plate at a density of 1×10 4 After adhesion, the original culture medium was discarded, 100 μL of culture medium with alcohol concentrations of 0.05 mol / L, 0.1 mol / L, 0.2 mol / L, 0.4 mol / L, 0.6 mol / L, 0.8 mol / L, 1.0 mol / L, and 1.2 mol / L was added to the experimental group, and 100 μL of fresh culture medium was added to the control group. At the same time, each group was repeated, and after 12, 24, and 48 h, the culture medium was aspirated, and 90 μL and 10 μL of base medium and CCK-8 reagent were added to each well, respectively. Incubate at 37°C in the dark for 1 h. The absorbance value at 450 nm was measured by a microplate reader, and the cell survival rate was calculated by the following formula:

[0172]

[0173] A sample - Experimental group (cells, culture medium containing different concentrations of wheat peptides, CCK-8)

[0174] A blank - Blank group (culture medium, CCK-8);

[0175] A control - Control group (cells, culture medium, CCK-8).

[0176] 4.1.1.3 Effect of wheat peptides on the activity of HepG2 cells

[0177] The HepG2 cell suspension in the exponential growth phase was collected and inoculated into a 96-well plate at a density of 1×10 4Cell density of 1 x 104cells / well was moved to 96-well plates. The plates were placed in a 37°C, 5% CO2 environment for 24 h of adherent culture, and then the culture medium was aspirated. The experimental groups were respectively added with 100 μL of 0.25 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 4.0 mg / mL, 6.0 mg / mL, 8.0 mg / mL, 10.0 mg / mL and 12.0 mg / mL wheat peptide aqueous solution in gradient, and the control group was supplemented with an equal amount of fresh culture medium. After 24 h of continuous culture, the old liquid was removed, and 90 μL and 10 μL of the base medium and CCK-8 reagent were added to the cells, which were incubated at 37°C in the dark for 1 h. The absorbance value at 450 nm was determined by an enzyme marker, and the cell survival rate was calculated by the formula.

[0178] 4.1.1.4 Effect of wheat peptide on the viability of damaged HepG2 cells

[0179] Different concentrations of wheat peptide were added to the experimental groups for 24 h, and the same amount of medium was added to the MC group for 24 h. After the culture medium was aspirated, 0.6 mol / L alcohol-containing base medium was added to each well for 24 h, and the NC group was cultured with fresh medium for 48 h, with the medium being changed every 24 h. After the treatment was completed, CCK-8 reagent was added for 1 h of incubation, the absorbance value at 450 nm was determined, and the cell viability was calculated.

[0180] 4.1.1.5 Determination of antioxidant and oxidative stress indicators of HepG2 cells

[0181] According to the requirements of the Nanjing Jiancheng and Biyun Tian kit instructions, the SOD activity, CAT activity, MDA level, ROS level and GSH level in the samples were determined and calculated by an enzyme marker.

[0182] 4.1.1.6 Determination of anti-inflammatory factor indicators of HepG2 cells

[0183] According to the detailed requirements of the kit instructions, the TNF-α level and IL-6 level in the samples were determined and calculated by an enzyme marker.

[0184] 4.1.1.7 Western-Blot detection and analysis of Nrf2 / Keap1 / HO-1 protein expression level

[0185] A small amount of protein in the test sample (NC, MC) group and (2 mg / mL, 4 mg / mL, 6 mg / mL) group was determined by BCA kit. The remaining samples were mixed with the loading buffer and boiled in a water bath for 15 min. Vertical electrophoresis was performed using polyacrylamide gel (SDS-PAGE). The appropriate concentration of polyacrylamide gel was prepared according to the molecular weight of the protein, and the samples in each group were loaded into the gel hole. The bands were leveled at 80V constant voltage for 20 min, and the electrophoresis was continued at 120V. Wet transfer method was used to transfer the protein to PVDF membrane, and then the membrane was blocked with 5% skim milk in TBS solution at room temperature for 1 h. The membrane was incubated with primary antibody at 4°C overnight, including Nrf2, Keap1, HO-1 and β-actin. Then incubated with horseradish peroxidase (HRP) labeled secondary antibody, washed the membrane with TBST for 3 times, and developed with hypersensitive ECL luminescent reagent. Image Lab and Photo-shop were used to observe and measure the gray value, and the experimental results were quantified according to the internal reference protein.

[0186] 4.1.2 Study on the protective effect of different concentrations of characteristic peptide segments on alcohol-induced HepG2 cell damage

[0187] 4.1.2.1 Culture of HepG2 cells

[0188] The culture method of HepG2 cells is the same as 4.1.1.1

[0189] 4.1.2.2 Effect of short peptides FQ, FV and FA on the viability of HepG2 cells

[0190] The cells were cultured in a 96-well plate at a density of 1×10 4 The old culture medium was discarded and washed with PBS, and the NC group (culture medium), MC control group (cells + culture medium) and different concentrations of peptide groups (FQ, FV, FA: 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL) were set up, with 6 replicate wells in each group. After 24 h of culture, the old culture medium was removed and washed with PBS, and 90 μL and 10 μL of basal medium and CCK-8 solution were added to each well, respectively, after 10 min of gentle shaking, incubated at 37°C for 1 h, and the absorbance at 450 nm was measured. The sample concentration without toxicity to cells was selected for subsequent experiments.

[0191] 4.1.2.3 Protective effect of FQ, FV and FA on HepG2 cell damage

[0192] The method for determining the viability of HepG2 cells damaged by FQ, FV and FA is the same as 4.1.1.4.

[0193] 4.1.2.4 Measurement of antioxidant and oxidative stress markers in HepG2 cells

[0194] The method for measuring antioxidant and oxidative stress markers in HepG2 cells is the same as 4.1.1.5

[0195] 4.1.2.5 Measurement of anti-inflammatory factor markers in HepG2 cells

[0196] The method for measuring anti-inflammatory factor markers in HepG2 cells is the same as 4.1.16

[0197] 4.1.2.6 Western-Blot detection of Nrf2 / Keap1 / HO-1 protein expression levels

[0198] A small amount of sample (NC, MC) group and (FQ 150 μg / mL, FV 150 μg / mL, FA 150 μg / mL) group protein was taken to determine its protein concentration using a BCA kit. The remaining samples were mixed with the loading buffer, and then boiled in a water bath for 15 min before use. Vertical electrophoresis was performed using polyacrylamide gel (SDS-PAGE). The appropriate concentration of polyacrylamide gel was prepared according to the molecular weight of the protein, and the samples of each group were loaded into the gel holes in equal amounts. The electrophoresis was carried out at 80 V for 20 min until the bands were aligned, and then the electrophoresis was continued at 120 V. Wet transfer method was used to transfer the protein to PVDF membrane, and then the membrane was blocked with 5% skim milk in TBS solution at room temperature for 1 h. The membrane was incubated with the first antibody at 4°C overnight, including Nrf2, Keap1, HO-1 and β-actin. Then incubated with horseradish peroxidase (HRP) labeled secondary antibody, washed the membrane with TBST for 3 times, and added hypersensitive ECL luminescent reagent for development. Image Lab and Photo-shop were used for observation and determination of gray value, and the experimental results were quantified according to the internal reference protein.

[0199] 4.2 Experimental results

[0200] 4.2.1 Protective effect of different concentrations of wheat peptide on alcohol-induced HepG2 cell damage

[0201] 4.2.1.1 Optimal modeling alcohol concentration and action time screening results

[0202] The results of cell survival rate detected by CCK-8 method are shown in Table 10. When the alcohol acts for 12 h, the cell proliferation of 0.05, 0.1, 0.2 mol / L concentration groups is good, and there is a significant difference (P<0.05) compared with the NC group. The cell survival rate of 0.4 mol / L group is reduced to (84.50±5.08)%, and there is a significant difference (P<0.05) compared with the NC group. It is shown that the alcohol has caused damage to HepG2 cells from this concentration condition, and the alcohol concentration is low when the culture is 12 h, which does not have damage to HepG2 cells. When the culture is 24 h, the final concentration is 0.05, 0.1, 0.2 mol / L, and there is a significant difference (P<0.05) compared with the NC group, and the cell proliferation state is good. When the final concentration of alcohol reaches 0.4 mol / L, the survival of HepG2 cells is significantly reduced to (75.02±3.38)%(P<0.05), and there is a significant difference compared with the NC group. With the increase of the final concentration of alcohol to 0.6 mol / L, the cell survival rate is further reduced to about 50% threshold level. Among them, when the alcohol continuously acts for 48 h, the final concentration is only 0.1 mol / L, and the cell survival rate is still reduced to (89.65±5.60)%(P<0.05), which confirms that the alcohol-induced cell damage has time-dose dependent characteristics. The results show that the extension of the alcohol action time can amplify its cytotoxic effect, and even a lower concentration (0.1 mol / L) can still cause irreversible cell damage under long-term action.

[0203] When the alcohol action time is 12, 24 h, and the final concentration is 0.05, 0.1, 0.2 mol / L, the cell survival rate is improved, which shows that the low concentration alcohol does not cause damage to the cells. When the final concentration of alcohol is 0.4 mol / L, the cell survival rate begins to decrease, which shows that the alcohol has begun to cause damage to the cells. When the alcohol action time is 24 h, and the final concentration is 0.6 mol / L, it has caused nearly 50% damage to HepG2 cells. When the final concentration of alcohol is 1.2 mol / L, the cell survival rate is only (10.53±3.05)%.

[0204] At the same time of implementing CCK-8 cell activity evaluation, cell morphology is observed by using inverted microscope. HepG2 cells in NC group maintain typical spindle morphology, showing clear cell boundary, good plasma membrane integrity and stable adherent growth phenotype. Under the action of 0.05, 0.1, 0.2 mol / L alcohol concentration, no obvious change in cell morphology is observed; under the action of 0.4 mol / L alcohol concentration, the overall number of cells shows a significant decreasing trend; when the alcohol concentration increases to 0.6 mol / L, HepG2 cells show typical stress morphological changes, showing cell volume shrinkage and accompanying decrease in adhesion ability, and cells are easy to fall off. When the alcohol concentration further increases to 0.8 mol / L, cell density is observed to decrease under the microscope, and the edge is blurred. When the alcohol concentration reaches 1 mol / L, the cell survival rate drops sharply, and most of the cells are found to have fallen off and died under the microscope. Combined with the CCK-8 and cell morphology observation results, the optimal alcohol damage model condition is determined to be 0.6 mol / L alcohol for 24 h.

[0205] Table 10 HepG2 cell survival rate after treatment with different concentrations of alcohol for different times

[0206]

[0207] Note: Different lowercase letters in the same column represent significant difference (P < 0.05).

[0208] 4.2.1.2 Effect of different concentrations of wheat peptide on the activity of HepG2 cells

[0209] Before studying the protective effect of wheat peptide, it is necessary to confirm that it has no toxic effect on cells. Before the subsequent experiments, the effect of different concentrations of wheat peptide on cell proliferation ability is determined by CCK-8 method after the cells are cultured for 24 h. The effect of different concentrations of wheat peptide on cell survival rate is shown in Table 10 and Figure 8 The experimental data show that, compared with the NC group, the wheat peptide (0.25, 1, 2, 4, 6, 10 mg / mL) group has no significant difference in the survival rate of HepG2 cells (P > 0.05), neither showing a proliferation activity nor showing a cytotoxicity. This result can rule out the interference effect of itself on the experimental results, and the protective mechanism of wheat peptide on alcohol-damaged HepG2 cells is not dependent on the cell proliferation effect. Based on this, when studying the protective effect of alcohol-damaged cells in the subsequent experiments, the proliferation interference factor of wheat peptide does not need to be considered. Combined with cell morphology observation, the cell morphology is complete, the cell wall is clear, and the adhesion is good when the peptide concentration is (2, 4, 6 mg / mL), and there is no cell debris. At the same time, high-concentration peptides can affect the osmotic pressure balance of cells. Therefore, 2, 4, 6 mg / mL wheat peptides are selected for subsequent experiments in this embodiment.

[0210] 4.2.1.3 Effect of different concentrations of wheat peptides on the viability of damaged HepG2 cells

[0211] Since wheat peptides had no toxic effect on cells, different concentrations of wheat peptides were applied to the alcohol-induced damage model of HepG2 cells to discuss their effects on the proliferation ability of damaged cells. The effects of different concentrations of wheat peptides on the survival rate of model cells are shown in Figure 9 The survival rates of the NC group, the MC group, and the 2, 4, and 6 mg / mL groups were (100.28 ± 6.04)%, (48.59 ± 6.32)%, (64.48 ± 11.42)%, (76.06 ± 11.01)%, and (80.72 ± 3.57)%, respectively, indicating that the alcohol-induced damage model of HepG2 cells was successfully constructed, and the cell survival rates were improved after intervention with wheat peptides. Compared with the MC group, the cell survival rates increased by 15%, 28%, and 32% after treatment with 2, 4, and 6 mg / mL wheat peptides, respectively, showing significant differences in cell survival rates between the MC group and the 2, 4, and 6 mg / mL wheat peptide groups (P < 0.05). There was no significant difference in survival rates between the 4 mg / mL and 6 mg / mL wheat peptide groups (P > 0.05).

[0212] 4.2.1.4 Effect of different concentrations of wheat peptides on the activity of superoxide dismutase in HepG2 cells

[0213] SOD mediates the decomposition and metabolism of superoxide anion free radicals to generate molecular oxygen (O2) and hydrogen peroxide (H2O2), thereby achieving the biological detoxification of free radicals. This enzyme-catalyzed reaction plays an important regulatory role in maintaining the redox homeostasis of the body. The SOD activity in HepG2 cells in different treatment groups is shown in Figure 10 As can be seen from the figure, the SOD activity in the MC group (11.89 ± 0.33 U / mg prot) was significantly lower than that in the NC group (35.86 ± 0.92 U / mg prot) (P < 0.05), and the SOD activity in different treatment groups was significantly reversed to different levels after treatment with 2, 4, and 6 mg / mL wheat peptides (P < 0.05). The 6 mg / mL wheat peptide group had the best intervention effect, and its SOD activity increased to (31.70 ± 0.78 U / mg prot). Compared with the MC group, the intervention of 2, 4, and 6 mg / mL wheat peptides significantly improved the trend of decreased SOD levels in alcohol-induced HepG2 cells, and showed a dose-dependent effect (P < 0.05).

[0214] 4.2.1.5 Effect of different concentrations of wheat peptides on the level of malondialdehyde in HepG2 cells

[0215] The MDA levels in HepG2 cells in different treatment groups are shown in Figure 11As shown in the figure, MDA, a key product of lipid peroxidation metabolism, can cause damage to biological membranes when expressed in excess. It alters cell membrane permeability and consequently affects a series of abnormal physiological responses in cells. As can be seen from the figure, the MDA level in the MC group (16.81±0.49 nmol / mgprot) was significantly higher than that in the NC group (2.71±0.50 nmol / mgprot) (P<0.05). After treatment with 2, 4, and 6 mg / mL wheat peptide, the MDA levels in each treatment group significantly decreased to different levels (P<0.05), showing a dose-dependent effect. Compared to the 2 and 4 mg / mL wheat peptide groups, the 6 mg / mL group better mitigated the trend of increased MDA levels in alcohol-induced HepG2 cells, reducing the MDA level to (4.78±0.16 nmol / mgprot), which is consistent with the findings of Pareek et al.

[127] The findings are consistent, indicating that wheat peptides have the potential to reduce MDA production.

[0216] 4.2.1.6 Effects of different concentrations of wheat peptides on reactive oxygen species levels in HepG2 cells

[0217] ROS levels in HepG2 cells from different treatment groups are as follows: Figure 12 As shown in the figure, the fluorescence intensity ratio of the MC group (1.28±0.13) was significantly higher than that of the NC group (0.75±0.10) (P<0.05). After treatment with 2, 4, and 6 mg / mL wheat peptide, the fluorescence intensity ratios in different treatment groups were significantly reduced to different levels compared with the MC group (P<0.05), and showed a dose-dependent decrease. Among them, there was no significant difference in fluorescence intensity ratio among the 4 mg / mL wheat peptide group, the 6 mg / mL wheat peptide group, and the NC group (P>0.05), indicating that the 4 mg / mL and 6 mg / mL wheat peptide groups could restore the ROS level of alcohol-induced HepG2 cells to a normal state. The fluorescence intensity ratios of the 4 mg / mL and 6 mg / mL wheat peptide groups were (0.85±0.06) and (0.82±0.04), respectively.

[0218] 4.2.1.7 Effects of different concentrations of wheat peptides on glutathione levels in HepG2 cells

[0219] GSH levels in HepG2 cells of different treatment groups as follows Figure 13As shown in the figure, compared with the NC group (6.07±0.13 μmol / gprot), the GSH level in the MC group (4.05±0.16 μmol / gprot) was significantly decreased (P<0.05). Compared with the MC group, after treatment with 2, 4, and 6 mg / mL wheat peptide, the GSH levels in different treatment groups were significantly reversed to different levels (P<0.05). Compared with the 2 mg / mL and 4 mg / mL wheat peptide groups, the 6 mg / mL wheat peptide group better improved the trend of decreased GSH level in alcohol-induced HepG2 cells, with a GSH level of (5.24±0.16 μmol / gprot), which was dose-dependent (P<0.05).

[0220] 4.2.1.8 Effects of different concentrations of wheat peptides on catalase activity in HepG2 cells

[0221] CAT activity in HepG2 cells from different treatment groups, such as Figure 14 As shown in the figure, the CAT activity in the MC group (16.46±1.10 U / mgprot) was significantly lower than that in the NC group (35.40±1.57 U / mgprot) (P<0.05). Compared with the MC group, after treatment with 2, 4, and 6 mg / mL wheat peptide, the CAT activity in different treatment groups was significantly reversed to different levels (P<0.05). Compared with the 2 mg / mL and 4 mg / mL wheat peptide groups, the 6 mg / mL wheat peptide group better improved the trend of decreased CAT activity in alcohol-induced HepG2 cells, with a CAT activity of (32.63±1.31 U / mgprot), which was dose-dependent (P<0.05).

[0222] 4.2.1.9 Effects of different concentrations of wheat peptides on the level of tumor necrosis factor-α in HepG2 cells

[0223] TNF-α levels in HepG2 cells from different treatment groups were as follows: Figure 15 As shown in the figure, compared with the NC group (52.24±1.96 pg / mL), the TNF-α level in the MC group (83.49±1.41 pg / mL) was significantly increased (P<0.05). After treatment with 2, 4, and 6 mg / mL wheat peptide, the TNF-α levels in different treatment groups were significantly reversed to different levels (P<0.05). Compared with the 2 mg / mL and 4 mg / mL wheat peptide groups, the 6 mg / mL wheat peptide group better improved the trend of alcohol-induced increase in TNF-α levels in HepG2 cells, with a TNF-α level of (61.87±3.76 pg / mL), which was dose-dependent (P<0.05).

[0224] 4.2.1.10 Effect of different concentrations of wheat peptides on the level of interleukin-6 in HepG2 cells

[0225] The changes of IL-6 levels in HepG2 cells in different treatment groups are shown in Figure 16 The results show that the IL-6 level in the MC group (74.85 ± 3.17 pg / mL) was significantly higher than that in the NC group (21.23 ± 2.21 pg / mL) (P < 0.05). After intervention with 2, 4, and 6 mg / mL wheat peptides, the IL-6 level decreased in a dose-dependent manner, reaching (52.17 ± 1.21 pg / mL), (44.41 ± 1.82 pg / mL), and (32.45 ± 1.98 pg / mL), respectively, which was significantly different from the MC group (P < 0.05). Among them, the IL-6 level in the 6 mg / mL wheat peptide group was significantly lower than that in the 2 mg / mL wheat peptide group and the 4 mg / mL wheat peptide group (P < 0.05), and the 6 mg / mL wheat peptide group could better improve the trend of increased IL-6 level in alcohol-induced HepG2 cells.

[0226] 4.2.1.11 Western-Blot method for detecting Nrf2 / Keap1 / HO-1 protein expression level analysis

[0227] The relative protein expression levels of Nrf2, Keap1, and HO-1 in HepG2 cells in each group were detected by Western-blot method. The results are shown in Figure 17 Compared with the NC group, the relative protein expression levels of Nrf2 and HO-1 in the MC group were significantly reduced (P < 0.05), while the relative protein expression level of Keap1 in the MC group was significantly increased (P < 0.05). After treatment with 2, 4, and 6 mg / mL wheat peptides, the protein expression levels of Nrf2, HO-1, and Keap1 in different treatment groups were significantly reversed to varying degrees (P < 0.05). Among them, the intervention effect of the 6 mg / mL group was more obvious than that of the 2 and 4 mg / mL groups. The protein expression level of Nrf2 increased in a dose-dependent manner with increasing concentration of wheat peptides, but the protein expression level of HO-1 in the 4 mg / mL wheat peptide group was higher than that in the 6 mg / mL wheat peptide group. The protein expression level of Keap1 decreased in a dose-dependent manner (P < 0.05). This indicates that the intervention effect of wheat peptides is effective, and can regulate protein expression levels by promoting the activation of the Nrf2 signaling pathway to protect HepG2 cells.

[0228] 4.2.2 Study on the protective effect of different concentrations of characteristic peptide segments on alcohol-induced HepG2 cell damage

[0229] 4.2.2.1 Effect of different concentrations of FQ, FV, and FA peptide segments on the viability of HepG2 cells

[0230] First, the CCK-8 method was used to detect whether different concentrations of the three peptide segments FQ, FV and FA would produce toxicity to HepG2 cells after intervention, and the most suitable peptide concentration for the growth of HepG2 cells was selected. The results are shown in Table 11. There was no significant difference between each concentration of peptide and the NC group after 24h of intervention (P>0.05). Combined with cell morphology observation, the cell morphology was complete, the cell wall was clear, the adhesion was good, and there was no cell debris when the peptide segment concentration was (200, 150, 100 μg / mL). FQ, FV and FA were all selected at 200, 150 and 100 μg / mL for subsequent experiments, and the cell survival rates were FQ (200, 150, 100 μg / mL): (107.59±4.47)%, (106.56±8.65)%, (106.42±7.04)%; FV (200, 150, 100 μg / mL): (102.29±6.60)%, (100.67±2.53)%, (101.06±7.48)%; FA (200, 150, 100 μg / mL): (101.99±10.26)%, (101.93±7.91)%, (102.89±6.04)%. These concentrations had no effect on cell growth, indicating that the three peptide segments had no toxicity to HepG2 cells.

[0231] Table 11 Effect of FQ, FV and FA on the viability of HepG2 cells

[0232]

[0233] Note: Different lowercase letters represent significant differences (P<0.05).

[0234] 4.2.2.2 Effect of FQ, FV and FA peptide segments at different concentrations on the viability of damaged HepG2 cells

[0235] As Figure 18The effects of wheat peptides FQ, FV and FA on the viability of HepG2 cells induced by alcohol were detected. Compared with the MC group (48.08±4.79)%, the cell survival rates of the groups treated with different concentrations of the three peptides FQ, FV and FA were increased (P<0.05). There was no significant difference between the 200 μg / mL and 150 μg / mL FQ treatment groups (P>0.05), and there was no significant difference between the 100 μg / mL and 150 μg / mL FQ treatment groups (P>0.05); there were significant differences between the three FV and FA treatment groups (P<0.05). The 200 μg / mL FA treatment group had the best improvement effect on cell damage, and the cell survival rate was increased by about 39% compared with the MC group. The cell survival rates of the FQ and FV treatment groups increased with the increase of the concentration, indicating that the three peptides could inhibit the decrease of the survival rate of HepG2 cells damaged by alcohol and protect the cells.

[0236] 4.2.2.3 Effects of FQ, FV and FA Peptides at Different Concentrations on the Activity of Superoxide Dismutase in HepG2 Cells

[0237] The SOD activities in the HepG2 cells of different treatment groups were as follows Figure 19The SOD activity of the MC group was significantly lower than that of the NC group (P < 0.05). After the treatment of the three peptide segments FQ, FV and FA at different concentrations, the SOD activity of each peptide segment treatment group was significantly increased (P < 0.05). There was no significant difference between the 150 μg / mL and 200 μg / mL FQ treatment groups (P > 0.05), and there was no significant difference between the 150 μg / mL and 100 μg / mL FV treatment groups (P > 0.05). The SOD activity of the 200 μg / mL FV treatment group was not significantly different from that of the NC group (P > 0.05), and the SOD activity of the 150 μg / mL FA treatment group was significantly higher than that of the 100 μg / mL and 200 μg / mL FA treatment groups (P < 0.05), and there was no significant difference between the 150 μg / mL FA treatment group and the NC group (P > 0.05). The SOD activity of the three peptide segment treatment groups showed an upward trend with the increase of the concentration. The results showed that the three peptide segments effectively improved the antioxidant level.

[0238] 4.2.2.4 Effect of FQ, FV and FA peptide segments at different concentrations on the level of malondialdehyde in HepG2 cells

[0239] The MDA levels in HepG2 cells of different treatment groups were as follows: Figure 20The MDA levels of the different concentrations of FQ, FV and FA peptide segments were significantly lower than those of the MC group (P < 0.05). Compared with the NC group (2.79 ± 0.37 nmol / mgprot), the MDA level of the MC group (17.16 ± 0.35 nmol / mgprot) was significantly higher (P < 0.05). There were significant differences in the MDA levels among the different concentrations of FQ and FA peptide segments (P < 0.05). The MDA level of the 150 μg / mL FA group was significantly lower than those of the 100 μg / mL and 200 μg / mL FA groups (P < 0.05), and the contents were (4.49 ± 0.76 nmol / mgprot), (12.00 ± 0.79 nmol / mgprot) and (7.53 ± 0.69 nmol / mgprot), respectively. The MDA levels of the FV, FQ and FA groups showed a downward trend with the increase of the concentration. There was no significant difference in the MDA levels between the 100 μg / mL and 150 μg / mL FV groups (P > 0.05), and the contents were (10.31 ± 0.70 nmol / mgprot) and (9.79 ± 0.52 nmol / mgprot), respectively. Notably, the 200 μg / mL FQ group had the best effect on improving the MDA level, and the MDA level was (3.67 ± 0.40 nmol / mgprot). The results showed that the three peptide segments could effectively improve the MDA level. The 150 μg / mL FA could significantly reduce the MDA level, which indicated that, compared with the FQ and FV peptide segments, the FA had a stronger effect on inhibiting lipid peroxidation and relieving oxidative stress.

[0240] 4.2.2.5 Effect of different concentrations of FQ, FV and FA peptide segments on the level of reactive oxygen species in HepG2 cells

[0241] The results of the fluorescence intensity ratio of the HepG2 cells in the different treatment groups were as follows: Figure 21The fluorescence intensity ratio of each group was shown in Fig. 6. Compared with the MC group, the fluorescence intensity ratio of each group treated with different concentrations of FQ, FV and FA was significantly reduced to different levels (P < 0.05). Compared with the NC group, the fluorescence intensity ratio of the MC group was significantly increased (P < 0.05). After treatment with different concentrations of FQ, FV and FA, the fluorescence intensity ratio of the 150 μg / mL FA treatment group was lower than that of the 200 μg / mL FA treatment group, but the difference was not significant (P > 0.05), and the ratios were (0.82 ± 0.04) and (0.89 ± 0.08), respectively. The ROS levels of the FV and FQ treatment groups showed a downward trend with the concentration gradient. In addition, there was no significant difference in the fluorescence intensity ratio between the FQ and FV treatment groups (P > 0.05). Compared with the NC group, there was no significant difference in the fluorescence intensity ratio of the 150 μg / mL FA treatment group (P > 0.05), and the ratio was (0.82 ± 0.04). At the same time, there was no significant difference in the fluorescence intensity ratio of the 200 μg / mL FV treatment group (P > 0.05), and the ratio was (0.81 ± 0.08). This indicated that the three peptide segments effectively improved the ROS level of the damaged HepG2 cells. In oxidative stress, ROS is the core driving factor, and its dynamic balance is directly related to cell damage and disease occurrence. ROS activates NF-κB in the body, releases TNF-α, IL-6 and other pro-inflammatory factors, causes inflammation in the body, and links oxidative stress and inflammation pathways. The three peptide segments can improve the increase of ROS, and it can be speculated that each peptide segment also has strong anti-inflammatory ability to inhibit TNF-α, IL-6 and other pro-inflammatory factors, which will be verified in the subsequent inflammation level experiment.

[0242] 4.2.2.6 Effect of different concentrations of FQ, FV and FA peptide segments on the level of glutathione in HepG2 cells

[0243] The GSH levels in HepG2 cells of different treatment groups were as shown in Fig. 7. Compared with the MC group, the GSH level of each group treated with different concentrations of FQ, FV and FA was significantly increased to different levels (P < 0.05). Compared with the NC group, the GSH level of the MC group was significantly decreased (P < 0.05). After treatment with different concentrations of FQ, FV and FA, the GSH level of the 150 μg / mL FA treatment group was lower than that of the 200 μg / mL FA treatment group, but the difference was not significant (P > 0.05), and the ratios were (0.82 ± 0.04) and (0.89 ± 0.08), respectively. The GSH levels of the FV and FQ treatment groups showed a downward trend with the concentration gradient. In addition, there was no significant difference in the GSH level between the FQ and FV treatment groups (P > 0.05). Compared with the NC group, there was no significant difference in the GSH level of the 150 μg / mL FA treatment group (P > 0.05), and the ratio was (0.82 ± 0.04). At the same time, there was no significant difference in the GSH level of the 200 μg / mL FV treatment group (P > 0.05), and the ratio was (0.81 ± 0.08). This indicated that the three peptide segments effectively improved the ROS level of the damaged HepG2 cells. In oxidative stress, ROS is the core driving factor, and its dynamic balance is directly related to cell damage and disease occurrence. ROS activates NF-κB in the body, releases TNF-α, IL-6 and other pro-inflammatory factors, causes inflammation in the body, and links oxidative stress and inflammation pathways. The three peptide segments can improve the increase of ROS, and it can be speculated that each peptide segment also has strong anti-inflammatory ability to inhibit TNF-α, IL-6 and other pro-inflammatory factors, which will be verified in the subsequent inflammation level experiment. Figure 22The GSH levels of the different concentrations of FQ, FV and FA peptide segments were significantly reversed to different levels compared with the MC group (P < 0.05). Compared with the NC group (6.07 ± 0.13 μmol / gprot), the GSH level of the MC group (4.05 ± 0.16 μmol / gprot) was significantly reduced (P < 0.05). There was no significant difference between the 150 μg / mL and 200 μg / mL treatment groups of FQ (P > 0.05). Their contents were (5.10 ± 0.06 μmol / gprot) and (5.28 ± 0.13 μmol / gprot), respectively. There was also no significant difference between the 150 μg / mL and 100 μg / mL treatment groups of FQ (P > 0.05), and their contents were (5.10 ± 0.06 μmol / gprot) and (4.95 ± 0.31 μmol / gprot), respectively. There was no significant difference between the 150 μg / mL and 100 μg / mL treatment groups of FV (P > 0.05), and their contents were (4.94 ± 0.14 μmol / gprot) and (4.53 ± 0.08 μmol / gprot), respectively. There were significant differences between the different treatment groups of FA (P < 0.05). The 200 μg / mL treatment group of FA had the best effect on improving the GSH level compared with the 100 μg / mL and 150 μg / mL treatment groups of FA, and their contents were (6.25 ± 0.12 μmol / gprot), (5.40 ± 0.18 μmol / gprot) and (5.96 ± 0.12 μmol / gprot), respectively. The GSH levels of the different concentrations of FQ, FV and FA peptide segments were all increased, indicating that the three peptide segments effectively improved the GSH level. GSH is the most critical endogenous antioxidant in cells, which plays a core protective role by directly neutralizing free radicals, repairing oxidative damage and maintaining redox homeostasis. At the same time, by activating the Nrf2 pathway, it can up-regulate GSH synthetase, increase the intracellular GSH level and enhance the antioxidant capacity. We can speculate that the three peptide segments can activate the Nrf2 pathway to increase the intracellular GSH level.

[0244] 4.2.2.7 Effect of different concentrations of FQ, FV and FA peptide segments on the activity of catalase in HepG2 cells

[0245] The CAT activities in the HepG2 cells of the different treatment groups were as follows: Figures 4-18The CAT activity of the MC group (17.81 ± 1.27 U / mgprot) was significantly lower than that of the NC group (36.73 ± 1.45 U / mgprot) (P < 0.05). After treatment with different concentrations of the three peptide segments, the CAT activity of the FQ 150 μg / mL treatment group was not significantly different from that of the FQ 200 μg / mL treatment group (P > 0.05), and the CAT activity of the FQ 150 μg / mL treatment group was higher than that of the FQ 200 μg / mL treatment group, with values of (34.42 ± 0.46 U / mgprot) and (33.71 ± 0.85 U / mgprot), respectively. The CAT activity of the FV and FA treatment groups showed an upward trend with increasing concentration. The CAT activity of the FA 150 and 200 μg / mL treatment groups was not significantly different (P > 0.05), with values of (34.06 ± 2.39 U / mgprot) and (35.67 ± 0.86 U / mgprot), respectively. Meanwhile, the CAT activity of the NC and FA 200 μg / mL treatment groups was not significantly different (P > 0.05), and the CAT activity of the FA 200 μg / mL treatment group was (35.67 ± 0.86 U / mgprot), which may have returned to the normal cell level, indicating that the FA 200 μg / mL treatment group had the best ability to improve CAT activity. The CAT activity of each FV treatment group was significantly different from that of the other groups, and the CAT activity of the FV 200, 150, and 100 μg / mL treatment groups was (34.87 ± 0.26 U / mgprot), (32.5 ± 0.43 U / mgprot), and (21.78 ± 0.64 U / mgprot), respectively. The CAT activity of each group of HepG2 damaged cells treated with different concentrations of the three peptide segments FQ, FV, and FA showed an upward trend, indicating that the three peptide segments effectively improved CAT activity.

[0246] 4.2.2.8 Effect of different concentrations of FQ, FV, and FA peptide segments on the level of tumor necrosis factor-α in HepG2 cells

[0247] The TNF-α levels in HepG2 cells in different treatment groups were as follows: Figure 24The levels of TNF-a in the MC group were significantly higher than those in the NC group (83.71 ± 2.75 pg / mL vs. 54.93 ± 1.34 pg / mL, P < 0.05). After treatment with different concentrations of the three peptide segments, the levels of TNF-a in the FV and FQ groups showed a downward trend with increasing concentration, while the level of TNF-a in the 150 pg / mL FA group was significantly different from that in the 100 pg / mL and 200 pg / mL FA groups (P < 0.05), and it was better at reducing the level of TNF-a (56.57 ± 2.79 pg / mL, 77.46 ± 1.41 pg / mL, and 62.48 ± 1.02 pg / mL, respectively). The level of TNF-a in the 150 pg / mL FA group was not significantly different from that in the NC group (P > 0.05), indicating that the level of TNF-a might have returned to normal. The 150 pg / mL FA group had the best ability to inhibit the level of TNF-a. The levels of TNF-a in the 200, 150, and 100 pg / mL FQ groups were significantly different from each other (58.61 ± 1.46 pg / mL, 66.97 ± 1.54 pg / mL, and 75.53 ± 1.35 pg / mL, respectively). The levels of TNF-a in the 200, 150, and 100 pg / mL FV groups were also significantly different from each other (61.63 ± 1.77 pg / mL, 67.83 ± 0.83 pg / mL, and 76.18 ± 1.00 pg / mL, respectively). The levels of TNF-a in the HepG2 damaged cells treated with different concentrations of the three peptide segments showed a downward trend, indicating that the three peptide segments effectively improved the anti-inflammatory level. TNF-a is mainly secreted by activated macrophages, T cells, and other immune cells, and is a key mediator of inflammatory response. It can activate the NF-kB pathway and induce the expression of pro-inflammatory factors such as IL-1β, IL-6, and chemokines.

[0248] 4.2.2.9 Effect of different concentrations of FQ, FV, and FA peptide segments on the level of interleukin-6 in HepG2 cells

[0249] IL-6 is a pleiotropic cytokine that plays a core pro-inflammatory role in inflammatory response and also has partial immune regulatory function. It has important pathological significance in inflammation, infection, autoimmune diseases, and cancer. The levels of IL-6 in HepG2 cells in different treatment groups were as follows: ​The IL-6 levels of the MC group were significantly higher than those of the NC group (P < 0.05), and the IL-6 level of the MC group was (75.52 ± 5.49 pg / mL), while the IL-6 level of the NC group was (23.80 ± 2.87 pg / mL). The IL-6 levels of the FV and FQ groups showed a downward trend with the increase of the concentration. The IL-6 level of the 150 μg / mL FA group was lower than that of the 200 μg / mL FA group, but there was no significant difference between the 150 μg / mL FA group and the 200 μg / mL FA group (P > 0.05), and the results were (45.06 ± 3.21 pg / mL) and (47.11 ± 3.18 pg / mL), respectively. FA did not show a concentration gradient decrease in regulating the expression of IL-6, and the linear dose effect of FQ and FV might be due to direct pathway intervention. The IL-6 levels of the 200, 150, and 100 μg / mL FQ groups were significantly different, and the IL-6 levels were (37.11 ± 2.38 pg / mL), (44.50 ± 3.60 pg / mL), and (53.72 ± 2.63 pg / mL), respectively. The IL-6 levels of the 200, 150, and 100 μg / mL FV groups were significantly different, and the IL-6 levels were (34.44 ± 3.33 pg / mL), (46.07 ± 3.35 pg / mL), and (55.13 ± 2.60 pg / mL), respectively. The IL-6 levels of the HepG2 damaged cells treated with different concentrations of the three peptide segments FQ, FV, and FA showed a downward trend, indicating that the three peptide segments effectively improved the anti-inflammatory level.

[0250] 4.2.2.10 Western-Blot method for detecting Nrf2 / Keap1 / HO-1 protein expression level analysis

[0251] The Nrf2 signaling pathway is an important regulatory pathway for cellular antioxidant defense and detoxification function, and plays a key role in improving oxidative stress and inflammation. Alcohol metabolism induces ROS explosion, and the activation of the Nrf2 / HO-1 pathway can alleviate oxidative stress, in which the bilirubin generated by HO-1 neutralizes free radicals and can inhibit lipid peroxidation (such as reducing MDA). In terms of anti-inflammatory, Nrf2 can inhibit the release of pro-inflammatory factors (TNF-α, IL-6) by reducing ROS accumulation, and can improve the anti-inflammatory ability. The relative expression of Nrf2, Keap1, and HO-1 proteins in HepG2 cells was detected by Western-blot method. The results are as follows ​As shown, the damage caused by alcohol was improved after treatment with the three peptide segments, and the expression of Nrf2 in each group showed an upward trend, and the FQ group had the best effect. Compared with the MC group, the Nrf2 in the NC group had a significant statistical difference (P<0.05), and the FQ group, the FA group and the FQ group significantly improved the expression level of Nrf2 protein (P<0.05). After treatment with different peptide segments, the expression of Keap1 in each group showed a downward trend, and compared with the MC group, the FQ group, the FV group and the FA group had a significant statistical difference (P<0.05). After treatment with different peptide segments, the expression of HO-1 in each group showed an upward trend, and compared with the MC group, the FV, FQ and FA groups had a statistical difference (P<0.05). It is proved that after the different peptide segments act on the alcohol-damaged HepG2 cells, the expression of Nrf2, Keap1 and HO-1 proteins in the cells can be effectively improved, and the expression of the three proteins can be effectively regulated, which shows that the FV, FA and FQ peptide segments activate the antioxidant proteins in the Nrf2 signal pathway in HepG2 cells to reduce the damage to HepG2 cells caused by alcohol, and the effect of FA activation is better.

[0252] From the above examples, it can be concluded that the wheat peptide is prepared, the peptide segment structure in the wheat peptide is determined, and the biological activity of the wheat peptide and the peptide segment structure in antioxidant and anti-inflammatory aspects is found.

[0253] Although the above examples make a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which all belong to the protection scope of the present application.

Claims

1. A wheat peptide, characterized in that, The wheat peptide comprises one or more of the peptide segments shown in the following table:

2. The wheat peptide according to claim 1, characterized in that, The total protein content of the wheat peptide is 91.54±2.11%, the acid-soluble protein content is 87.23±1.76%, the ash content is 1.62±0.21%, and the moisture content is 1.97±0.09%; The weight average molecular weight of the wheat peptide is 387.80 Da; and the molecular weight of the wheat peptide is less than 5000 Da.

3. The wheat peptide according to claim 1, characterized in that, The wheat peptide comprises the amino acids shown in the following table, and the types and contents of the amino acids are shown in the following table:

4. Process for the preparation of the wheat peptide according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: Mixing the gluten meal with deionized water to obtain a gluten meal suspension; Mixing the gluten meal suspension with alkaline protease to perform first enzymolysis to obtain a first enzymolysis mixture; Mixing the first enzymolysis mixture with neutral protease to perform second enzymolysis to obtain the second enzymolysis mixture; Drying the second enzymolysis mixture to obtain the wheat peptide.

5. The preparation method according to claim 4, characterized in that, The preparation method further comprises identifying the peptide segment composition of the wheat peptide, and the identification method comprises the following steps: Mixing the wheat peptide with deionized water to obtain a wheat peptide aqueous solution; Filtering the wheat peptide aqueous solution, and performing high-performance liquid chromatography tandem mass spectrometry on the filtered wheat peptide solution to obtain the peptide segment structure in the wheat peptide; The chromatographic conditions of the high-performance liquid chromatography tandem mass spectrometry comprise: the chromatographic column is an Inertsil ODS-3 chromatographic column; the mobile phase comprises phase A and phase B, the phase A is formic acid and water, the volume concentration of formic acid in the phase A is 0.1%, the phase B is formic acid and acetonitrile, and the volume concentration of formic acid in the phase B is 0.1%; the chromatographic gradient is 0-15 min, 0%-40% B; 15-20 min, 40%-80% B; 20-25 min, 80% B; 25-35 min, 0% B; and the flow rate is 0.2 mL / min; The mass spectrometry parameters of the high-performance liquid chromatography tandem mass spectrometry comprise: the ionization mode of ESI, the atomization gas flow rate is 3.0 L / min, the heating gas flow rate is 10 L / min, the dry gas flow rate is 10 L / min, the ion spray voltage is 4.5 kV, the desolvation tube temperature is 250℃, the heating module temperature is 400℃, and the ion source temperature is 300℃.

6. The preparation method according to claim 4, characterized in that, The mass of the alkaline protease to the volume of the gluten meal suspension is (0.10-0.14) g:100 mL, the enzyme activity of the alkaline protease is 2.4-4.0 AU-A / g, and the mass concentration of the gluten meal suspension is 11% w / w.

7. The production method according to claim 4 or 6, characterized by, The pH value of the first enzymolysis is 8-9, the temperature of the first enzymolysis is 55-65℃, and the time of the first enzymolysis is 2 h.

8. The preparation method according to claim 4, characterized in that, The mass of the neutral protease to the volume of the first enzymolysis mixture is (0.06-0.10) g:100 mL, and the enzyme activity of the neutral protease is 0.7-0.9 AU-N / g.

9. The production method according to claim 4 or 8, characterized by, The pH value of the second enzymolysis is 6.5-7.5, the temperature of the second enzymolysis is 45-55℃, and the time of the second enzymolysis is 3 h.

10. Use of a wheat peptide or a peptide segment in a wheat peptide in the preparation of an antioxidant and / or anti-inflammatory product; the wheat peptide being a wheat peptide as defined in any one of claims 1 to 3 or a wheat peptide prepared by the method of any one of claims 4 to 9; the peptide segment in the wheat peptide being one or more of FQ, FV and FA.

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