Keap1 inhibition type rice gluten antioxidative peptide as well as preparation method and application thereof

Abstract

The invention discloses a Keap1 inhibition type rice glutenin antioxidant peptide and a preparation method and application thereof, and relates to the technical field of functional foods.The preparation method of the Keap1 inhibition type rice glutenin antioxidant peptide comprises the following steps that rice glutenin powder and water are mixed to be uniform, pretreated through a physical modification method and then added with water to be properly diluted, pH is adjusted, and the Keap1 inhibition type rice glutenin antioxidant peptide is obtained; and adding protease with hydrophobic amino acid cleavage site specificity to carry out enzymolysis for 1-8 hours at 40-65 DEG C, then adding flavourzyme to carry out enzymolysis for 1-8 hours, carrying out enzyme deactivation and centrifugation, and drying the supernatant to obtain the rice gluten antioxidative peptide. Physical pretreatment is combined with a two-step enzymolysis method to carry out restrictive enzymolysis on the rice glutenin, and the obtained zymolyte is high in antioxidant activity, can inhibit oxidative stress by activating an Nrf2 / Keap1 signal channel, and can be used for preparing antioxidant and anti-aging foods, medicines, health care products and cosmetics.

Description

Technical Field

[0001] The present invention relates to the technical field of functional foods, and in particular to a Keap1-inhibiting rice gluten antioxidant peptide and a preparation method and application thereof. Background Art

[0002] With the accelerating pace of modern life and increasing stress, unhealthy lifestyles, chronic diseases, and aging can all induce oxidative stress in the body. Free radicals, such as reactive oxygen and reactive nitrogen species, accumulate within cells, damaging active components like proteins, lipids, and DNA. This can lead to pathological damage to organs and tissues, impacting normal physiological functions and increasing the risk of various chronic diseases, including cardiovascular disease, neurodegenerative disorders, and metabolic disorders, posing a threat to human health.

[0003] In recent years, food-derived antioxidant peptides, as a new type of antioxidant, have attracted much attention due to their wide range of sources, high safety, easy preparation, and multiple physiological activities. Antioxidant peptides are usually present in maternal proteins and can be released during food processing (such as fermentation, bio-enzymatic hydrolysis) or gastrointestinal digestion. Plant and animal antioxidant peptides have been widely proven to have free radical scavenging ability, metal ion chelating ability, and the ability to regulate redox signaling pathways. They have anti-aging effects, improve body function, and have good application prospects in the fields of food, medicine, and health products. However, most of the antioxidant peptides that have been developed so far act as free radical scavengers with a relatively simple mechanism of action. There is relatively little research on antioxidant peptides that specifically target and regulate signaling pathways.

[0004] Rice protein is a high-quality plant protein resource, boasting high nutritional value and low allergenicity. Gluten is the primary component of rice protein, accounting for approximately 80% of the total rice protein content. It has a rational amino acid composition, a high lysine content, and is easily digested and absorbed. However, gluten's low solubility limits its application in food processing. Bio-enzymatic hydrolysis technology can improve the structure and properties of gluten, enabling its high-value utilization. However, current bio-enzymatic hydrolysis technology still suffers from low enzymatic efficiency, low target peptide content in the product, and low gluten activity. Furthermore, the technology for preparing antioxidant peptides from gluten with well-defined functions, high activity, and specific effects is still immature. Summary of the Invention

[0005] The main purpose of the present invention is to propose a Keap1-inhibitory rice glutenin antioxidant peptide and its preparation method and application, aiming to solve the problems of low enzymatic hydrolysis efficiency, low content of target peptide segments in the product, unclear target site and low rice glutenin activity in the existing biological enzymatic hydrolysis technology.

[0006] To achieve the above objectives, the present invention proposes a method for preparing a Keap1-inhibitory rice gluten antioxidant peptide, wherein the Keap1-inhibitory peptide inhibits the expression of Keap1 protein, promotes the activation of Nrf2 protein and its target genes, and regulates the Nrf2 / Keap1 signaling pathway, thereby reducing intracellular oxidative stress.

[0007] The preparation method of the Keap1-inhibiting rice gluten antioxidant peptide comprises the following steps:

[0008] S1. mixing water and rice gluten powder to obtain a mixture, and physically modifying the mixture to obtain a modified mixture;

[0009] S2, diluting the modified mixture with water, adjusting the pH of the system to 6.5-8.5, adding a protease having hydrophobic amino acid cleavage site specificity, and enzymolyzing at 40-60° C. for 1-8 hours to obtain a first enzymatic hydrolyzate;

[0010] S3, adding flavor protease to the first hydrolyzate, performing enzymatic hydrolysis at 40-60° C. for 1-8 hours to obtain a second hydrolyzate, inactivating the enzyme in the second hydrolyzate, centrifuging and separating the enzyme, and drying the supernatant to obtain the Keap1-inhibiting rice gluten antioxidant peptide;

[0011] Wherein, based on the protein content of rice gluten powder as a calculation basis, the added amount of the protease with hydrophobic amino acid cleavage site specificity is 0.1-2%, and the added amount of the flavor protease is 0.1-1%.

[0012] In one embodiment, in step S1, the mass ratio of the water to the rice gluten powder is 1:2-1:15.

[0013] In one embodiment, in step S1, the physical modification method includes one or more of ultrasonic treatment, air flow milling treatment, heating treatment, and high pressure treatment, wherein:

[0014] The power of the ultrasonic treatment is 100-600W, and the time of the ultrasonic treatment is 3-60min;

[0015] The pressure of the air flow milling treatment is 0.1-0.8 MPa;

[0016] The temperature of the heating treatment is 50-100°C, and the time of the heating treatment is 3-60 minutes;

[0017] The pressure of the high-pressure treatment is 50-500 MPa, and the time of the high-pressure treatment is 5-40 minutes.

[0018] In one embodiment, in step S2, in the step of diluting the modified mixture with water, the mass ratio of the modified mixture to the water is 1:1-1:15.

[0019] In one embodiment, in step S2, the protease having hydrophobic amino acid cleavage site specificity comprises one or more of pepsin, alkaline protease, trypsin, chymotrypsin, proteinase K, and papain;

[0020] Preferably, the protease having hydrophobic amino acid cleavage site specificity includes papain or alkaline protease.

[0021] In one embodiment, in step S3, the step of inactivating the enzyme in the second enzymatic hydrolysate comprises:

[0022] The second enzymatic hydrolysate is heated to 90-95° C. and kept warm for 15-30 minutes.

[0023] In one embodiment, in step S3, the centrifugal separation step includes: cooling the second enzymatic hydrolysate after enzyme inactivation to room temperature, then centrifuging at 6000-10000 rpm for 15-30 minutes, re-dissolving the precipitate with water, stirring evenly, and centrifuging again, and collecting the supernatants obtained twice.

[0024] The present invention also provides a Keap1-inhibiting rice gluten antioxidant peptide prepared by the aforementioned preparation method of the Keap1-inhibiting rice gluten antioxidant peptide, wherein the Keap1-inhibiting rice gluten antioxidant peptide comprises:

[0025] The hydrophobic amino acid content is 28-36 g / 100 g, the aromatic amino acid content is 7-12 g / 100 g, the leucine content is 5-9 g / 100 g, the arginine content is 8-13 g / 100 g, and the lysine content is 3-6 g / 100 g.

[0026] In one embodiment, the hydrophobic amino acids include alanine (Ala), phenylalanine (Phe), isoleucine (Ile), leucine (Leu), proline (Pro), valine (Val), tryptophan (Trp) and tyrosine (Tyr); the aromatic amino acids include phenylalanine (Phe), tryptophan (Trp) and tyrosine (Tyr).

[0027] The present invention also provides a use of the aforementioned Keap1-inhibiting rice gluten antioxidant peptide in the preparation of foods, medicines, health products or cosmetics with antioxidant and anti-aging effects, especially having antioxidant activity and alleviating cellular oxidative stress.

[0028] The antioxidant activity refers to the fact that the rice gluten antioxidant peptide has high ABTS free radical scavenging ability, DPPH free radical scavenging ability, etc.; alleviating the cellular oxidative stress level means that the rice gluten antioxidant peptide can increase the survival rate of cells and activate the Nrf2 / Keap1 signaling pathway.

[0029] The present invention has the following advantages and effects:

[0030] In the technical solution of the present invention, the purpose of physical modification in step S1 is to change the spatial structure or intermolecular interaction of rice gluten, increase its solubility and enzymolysis, improve the subsequent enzymolysis efficiency of rice gluten, and improve its functional properties; in step S2, the modified mixture is enzymatically hydrolyzed for the first time using a protease with hydrophobic amino acid cleavage site specificity, which can specifically recognize and cleave the peptide bonds of hydrophobic amino acids such as valine (Val), leucine (Leu), phenylalanine (Phe) or isoleucine (Ile), to generate a first enzymolyte containing a specific functional peptide segment, effectively improving the enrichment and content of the target peptide in the final enzymolyte; in step S3, the first enzymolyte is enzymatically hydrolyzed for the second time using a flavor protease to degrade the macromolecular peptide segments in the first enzymolyte, especially the endonuclease site reduces the bitterness of the enzymolyte and increases the content of hydrophobic amino acids and aromatic amino acids in the enzymolyte; the two-step enzymolysis technology can control the degree of enzymolysis while also increasing the content of the target amino acid or target polypeptide in the enzymolyte. The antioxidant peptides prepared by the present invention have good in vitro antioxidant capacity and can specifically target and regulate the Nrf2 / Keap1 signaling pathway, promote the dissociation of Nrf2 and Keap1, and prompt free Nrf2 to enter the cell nucleus, thereby activating the transcriptional expression of antioxidant-related proteins and enhancing the antioxidant defense ability of cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying any creative work.

[0032] Figure 1 The results of the effects of the Keap1 inhibitory rice gluten antioxidant peptide prepared in Example 1 of the present invention on Keap1-Nrf2 and related antioxidant enzymes are as follows: Figure 1 Middle (A) is the protein expression results of HO-1, NQO1, Keap1 and Nrf2. Figure 1 Middle (B) is the relative expression results of HO-1, NQO1, Keap1 and Nrf2.

[0033] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. Where the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes. Taking "A and / or B" as an example, it includes scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical solutions between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in this field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0035] The present invention provides a method for preparing a Keap1-inhibiting rice gluten antioxidant peptide, comprising the following steps:

[0036] S1. mixing water and rice gluten powder to obtain a mixture, and physically modifying the mixture to obtain a modified mixture;

[0037] S2, diluting the modified mixture with water, adjusting the pH of the system to 6.5-8.5, adding a protease having hydrophobic amino acid cleavage site specificity, and enzymolyzing at 40-60° C. for 1-8 hours to obtain a first enzymatic hydrolyzate;

[0038] S3, adding flavor protease to the first hydrolyzate, performing enzymatic hydrolysis at 40-60° C. for 1-8 hours to obtain a second hydrolyzate, inactivating the enzyme in the second hydrolyzate, centrifuging and separating the enzyme, and drying the supernatant to obtain the Keap1-inhibiting rice gluten antioxidant peptide;

[0039] Wherein, based on the protein content of rice gluten powder as a calculation basis, the added amount of the protease with hydrophobic amino acid cleavage site specificity is 0.1-2%, and the added amount of the flavor protease is 0.1-1%.

[0040] In the technical solution of the present invention, the purpose of physical modification in step S1 is to change the spatial structure or intermolecular interaction of rice gluten, increase its solubility and enzymolysis, improve the subsequent enzymolysis efficiency of rice gluten, and improve its functional properties; in step S2, the modified mixture is subjected to a first enzymolysis using a protease with hydrophobic amino acid cleavage site specificity, which can specifically recognize and cleave the peptide bonds of hydrophobic amino acids such as valine (Val), leucine (Leu), phenylalanine (Phe) or isoleucine (Ile) to generate a first enzymolyte containing specific functional peptide segments, effectively improving the enrichment and content of the target peptide in the final enzymolyte; in step S3, the first enzymolyte is subjected to a second enzymolysis using a flavor protease to degrade the macromolecular peptide segments in the first enzymolyte, especially the endonuclease site reduces the bitterness of the enzymolyte and increases the content of hydrophobic amino acids and aromatic amino acids in the enzymolyte; the two-step enzymolysis technology can control the degree of enzymolysis while also increasing the content of the target amino acid or target polypeptide in the enzymolyte. The antioxidant peptides (i.e., enzymatic hydrolysates) prepared by the present invention have good in vitro antioxidant capacity and can specifically target and regulate the Nrf2 / Keap1 signaling pathway, promote the dissociation of Nrf2 and Keap1, and encourage free Nrf2 to enter the cell nucleus, thereby activating the transcriptional expression of antioxidant-related proteins and enhancing the antioxidant defense ability of cells.

[0041] It is understandable that the above-mentioned Nrf2 (nuclear factor E2-related factor) is a transcription factor that regulates the endogenous antioxidant system in cells. Under normal physiological conditions, Nrf2 binds to the Keap1 protein (Kelch-like ECH-related protein 1) in the cytoplasm and forms a complex. When the cell is stimulated by oxidative stress, Nrf2 dissociates from Keap1, prompting free Nrf2 to enter the cell nucleus, activating the transcriptional expression of antioxidant-related proteins and enhancing the cell's antioxidant defense ability. Therefore, activating the Nrf2 / Keap1 signaling pathway is an important way for the enzymatic hydrolysate in the present invention to inhibit oxidative stress-related diseases.

[0042] In some embodiments, in step S1, the mass ratio of water to rice gluten powder is 1:2-1:15. It is understood that the mass ratio of water to rice gluten powder can be 1:2, 1:6, or 1:15. When the mass ratio is within the above range, the physical modification effect on rice gluten is better.

[0043] In some embodiments, in step S1, the physical modification method includes one or more of ultrasonic treatment, air flow milling treatment, heat treatment, and high pressure treatment, wherein: the power of the ultrasonic treatment is 100-600W, and the ultrasonic treatment time is 3-60min; the pressure of the air flow milling treatment is 0.1-0.8MPa; the temperature of the heat treatment is 50-100°C, and the heat treatment time is 3-60min; the pressure of the high pressure treatment is 50-500MPa, and the high pressure treatment time is 5-40min. It is understood that the above-mentioned pretreatment method and conditions are controlled within an appropriate range to ensure that the enzymatic hydrolysis of rice glutenin is increased, thereby improving the subsequent enzymatic hydrolysis efficiency of rice glutenin and improving its functional properties.

[0044] In some embodiments, in step S2, during the step of diluting the modified mixture with water, the mass ratio of the modified mixture to water is 1:1-1:15. It is understood that the mass ratio of the modified mixture to water can be 1:1, 1:8, or 1:15. Within the above ranges, the mass ratio can ensure sufficient solvent to fully disperse the substrate during the subsequent enzymatic hydrolysis process while avoiding excessive dilution.

[0045] In some embodiments, in step S2, the protease with hydrophobic amino acid cleavage site specificity includes one or more of pepsin, alkaline protease, trypsin, chymotrypsin, proteinase K, and papain; preferably, the protease with hydrophobic amino acid cleavage site specificity includes papain or alkaline protease. Among them, pepsin tends to cut the peptide bond on the carboxyl side of aromatic amino acids such as phenylalanine, tyrosine (Tyr) and tryptophan (Trp); trypsin is a serine protease that can specifically recognize and cut the peptide bond on the carboxyl side of lysine and arginine; chymotrypsin also selectively acts on the peptide bond on the carboxyl side of hydrophobic amino acids with large side chains; alkaline protease, proteinase K and papain can act on the carboxyl side of various amino acids including hydrophobic amino acids. Selecting the above-mentioned enzyme with hydrophobic amino acid cleavage site specificity can ensure that the final enzymatic hydrolysis product (i.e., antioxidant peptide) has good in vitro antioxidant capacity, and can specifically target and regulate the Nrf2 / Keap1 signaling pathway, and can promote the dissociation of Nrf2 and Keap1.

[0046] In some embodiments, in step S3, the step of inactivating the second hydrolysate includes heating the second hydrolysate to 90-95°C for 15-30 minutes. It is understood that the inactivation temperature may be 90°C, 92°C, or 95°C, and the inactivation time may be 15 minutes, 20 minutes, or 30 minutes. Within the above ranges, the temperature and time can ensure that both the protease specific for the hydrophobic amino acid cleavage site and the flavor protease are effectively inactivated.

[0047] In some embodiments, in step S3, the centrifugal separation step includes: cooling the second enzymatic hydrolysate after enzyme inactivation to room temperature, then centrifuging at 6,000 to 10,000 rpm for 15 to 30 minutes, re-dissolving the precipitate in water, stirring evenly, and centrifuging again, and collecting the supernatants obtained twice. It is understood that the purpose of centrifugation is to precipitate insoluble substances in the enzymatic hydrolysate, such as incompletely digested macromolecules, thereby separating them from soluble components and improving the purity of the final product.

[0048] The present invention also provides a Keap1-inhibiting rice gluten antioxidant peptide prepared by the preparation method of the aforementioned Keap1-inhibiting rice gluten antioxidant peptide, wherein the Keap1-inhibiting rice gluten antioxidant peptide comprises: a hydrophobic amino acid content of 28 to 36 g / 100 g, an aromatic amino acid content of 7 to 12 g / 100 g, a leucine content of 5 to 9 g / 100 g, an arginine content of 8 to 13 g / 100 g, and a lysine content of 3 to 6 g / 100 g. The contents of hydrophobic amino acids, aromatic amino acids, leucine, arginine, and lysine in the antioxidant peptide prepared by the present invention are within the above-mentioned specific ranges, and the peptide has good in vitro antioxidant capacity, and can specifically target and regulate the Nrf2 / Keap1 signaling pathway, promote the dissociation of Nrf2 and Keap1, and promote the entry of free Nrf2 into the cell nucleus, thereby activating the transcriptional expression of antioxidant-related proteins and enhancing the antioxidant defense capacity of the cell.

[0049] In addition, since the Keap1-inhibiting rice gluten antioxidant peptide is prepared by the aforementioned preparation method, it has all the beneficial effects of the aforementioned preparation method of the Keap1-inhibiting rice gluten antioxidant peptide, which will not be described in detail here.

[0050] In some embodiments, the hydrophobic amino acids include alanine (Ala), phenylalanine (Phe), isoleucine (Ile), leucine (Leu), proline (Pro), valine (Val), tryptophan (Trp) and tyrosine (Tyr); the aromatic amino acids include phenylalanine (Phe), tryptophan (Trp) and tyrosine (Tyr).

[0051] The present invention also provides a use of the aforementioned Keap1-inhibiting rice gluten antioxidant peptide in the preparation of food, medicine, health product or cosmetic with antioxidant and anti-aging effects. Therefore, all the beneficial effects of the aforementioned Keap1-inhibiting rice gluten antioxidant peptide are achieved, which will not be described in detail here.

[0052] The technical solutions of the present invention are further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0053] Example 1

[0054] A method for preparing a Keap1-inhibiting rice gluten antioxidant peptide comprises the following steps:

[0055] (1) The rice gluten powder was ultrafinely pulverized using a jet mill with a pulverization pressure of 0.6 MPa. Subsequently, the pulverized rice gluten powder was dispersed in water at a ratio of 1:10 and subjected to ultrasonic treatment with an ultrasonic power of 400 W and an ultrasonic time of 10 min. The sonicated solution was freeze-dried to obtain physically modified rice gluten.

[0056] (2) The physically modified rice gluten powder was mixed evenly with water in a ratio of 1:8 (w / w), the pH of the solution was adjusted to 7.0, and then 1% papain (purchased from Nanning Pangbo Bioengineering Co., Ltd.) was added for enzymatic hydrolysis at 55°C for 8 h, and then 0.2% flavor protease (purchased from Novozymes) was added and the enzymatic hydrolysis was continued for 4 h. The enzyme was inactivated, the enzymatic solution was cooled to room temperature and centrifuged at 8000×g for 20 min, the supernatant was collected, and freeze-dried to obtain Keap1 inhibitory rice gluten antioxidant peptide.

[0057] Example 2

[0058] A method for preparing a Keap1-inhibiting rice gluten antioxidant peptide comprises the following steps:

[0059] (1) The rice gluten powder was pretreated under high pressure (250 MPa) for 20 min. Subsequently, the treated rice gluten powder was dispersed in water and ultrasonically treated at an ultrasonic power of 400 W for 10 min. The ultrasonicated powder was freeze-dried to obtain physically modified rice gluten.

[0060] (2) The physically modified rice gluten powder was evenly mixed with water in a ratio of 1:10 (w / w), the pH of the solution was adjusted to 7.0, and then 0.75% alkaline protease (purchased from Novozymes) was added for enzymatic hydrolysis at 55°C for 8 h, and then 0.2% flavor protease (purchased from Novozymes) was added for further enzymatic hydrolysis for 2 h. The enzyme was inactivated, the enzymatic solution was cooled to room temperature and centrifuged at 8000×g for 20 min, the supernatant was collected, and freeze-dried to obtain Keap1 inhibitory rice gluten antioxidant peptide.

[0061] Example 3 Effect of Keap1-inhibiting rice gluten hydrolysate on the Keap1 / Nrf2 signaling pathway in HepG2 cells

[0062] (1) HepG2 cell culture

[0063] HepG2 cells were cultured in MEM medium containing 10% fetal bovine serum (FBS) and 1% double antibiotics (penicillin and streptomycin). The cells were cultured in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity. The medium was replaced every 2-3 days, and the cells were passaged according to their growth.

[0064] (2) Exploring the protective effect of Keap1 inhibitory rice gluten antioxidant peptides on H2O2-damaged HepG2 cells in Example 1

[0065] HepG2 cells in the logarithmic growth phase were cultured at a rate of 1×10 5 Cells were seeded at a density of 100 μg / mL in culture flasks. After 24 hours of cell attachment, the cells were divided into control, sample, and model groups for the following treatments. The culture medium used in the following experiments was serum-free medium. Sample group: After removing the original culture medium, the cells were replaced with culture medium containing different concentrations of Keap1 inhibitory rice gluten antioxidant peptide (low concentration: 0.5 mg / mL, high concentration: 1.0 mg / mL) and continued to be cultured. After 24 hours, the original culture medium was discarded and replaced with 500 μM H2O2 and cultured for 2 hours. The cells were then collected for lysis. Control group: The original culture medium was replaced with fresh culture medium. After 24 hours, the culture medium was discarded and replaced with fresh culture medium. After 2 hours of culture, the cells were collected for lysis. Model group: The original culture medium was replaced with fresh culture medium. After 24 hours, the culture medium was discarded and replaced with culture medium containing 500 μM H2O2. After 2 hours of culture, the cells were collected for lysis. The lysed cells were quantified by BCA method for protein. Subsequently, the protein expression levels of Keap1, Nrf2 and its target genes heme oxygenase-1 (HO-1) and quinone oxidoreductase 1 (NQO1) in the cells collected from the four groups were determined by Western blot, and semi-quantitative analysis was performed using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a benchmark. Figure 1 shown.

[0066] Figure 1 The four columns of strips from left to right in (A) are the results of the control group, model group, low-concentration sample group 1, and high-concentration sample group 1; Figure 1 (B) The white bar graphs from left to right are the relative expression results of HO-1 in the control group, model group, low-concentration sample group and high-concentration sample group, the green bar graphs from left to right are the relative expression results of NQO1 in the control group, model group, low-concentration sample group and high-concentration sample group, the blue bar graphs from left to right are the relative expression results of Keap1 in the control group, model group, low-concentration sample group and high-concentration sample group, and the purple bar graphs from left to right are the relative expression results of Nrf2 in the control group, model group, low-concentration sample group and high-concentration sample group.

[0067] Depend on Figure 1 It can be seen that compared with the control group, the protein expression level of Keap1 in the cells of the model group was significantly increased, the protein expression level of Nrf2 was significantly decreased, and the protein expression of its target genes HO-1 and NQO1 was also significantly decreased, indicating that H2O2 damage caused the redox state of the cells to be unbalanced. In comparison, the pretreatment with the rice gluten antioxidant peptide in Example 1 activated the Nrf2 signaling pathway and enhanced the protein expression levels of the target genes HO-1 and NQO1. At a sample concentration of 1 mg / mL, the protein expression levels of HO-1 and NQO1 in the high-concentration sample group were 2.86 times and 2.11 times that of the model group. Compared with the model group, the protein expression of Keap1 in the high-concentration sample group decreased by 43.44%, and the protein expression level of Nrf2 increased by 7.29% (p<0.05), indicating that the rice gluten antioxidant peptide can regulate the Keap1 / Nrf2 signaling pathway and promote the expression of its target genes to reduce cellular oxidative damage.

[0068] Comparative Example 1

[0069] Comparative Example 1 is different from Example 1 in that:

[0070] In step (2), alkaline protease was not used for enzymatic hydrolysis. Flavor protease was used for one-step enzymatic hydrolysis. The amount of flavor protease added was 1% and the enzymatic hydrolysis time was 10 h. The remaining steps were the same as those in Example 1.

[0071] Comparative Example 2

[0072] Comparative Example 2 is different from Example 1 in that:

[0073] In step (2), flavor protease was not used for enzymatic hydrolysis, and papain was used for one-step enzymatic hydrolysis. The amount of papain added was 1%, and the enzymatic hydrolysis time was 10 h. The remaining steps were the same as in Example 1.

[0074] Performance Testing

[0075] The amino acid composition of the Keap1 inhibitory rice gluten antioxidant peptide prepared in Example 1 was analyzed according to the national standard GB / T 5009.124-2016 “National Food Safety Standard - Determination of Amino Acids in Foods”. The results are shown in Table 1.

[0076] Table 1 Amino acid composition of Keap1 inhibitory rice gluten antioxidant peptide prepared in Example 1

[0077]

[0078]

[0079] As can be seen from Table 1, the amino acid composition of the Keap1 inhibitory rice gluten antioxidant peptide prepared in Example 1 is: the content of hydrophobic amino acids is 34.38 g / 100 g, the content of aromatic amino acids is 9.21 g / 100 g, the content of leucine is 7.14 g / 100 g, the content of arginine is 10.65 g / 100 g, and the content of lysine is 4.53 g / 100 g.

[0080] The antioxidant activity of the antioxidant peptides prepared in Example 1-2 and Comparative Example 1-2 was determined by the following method:

[0081] (1) Determination of DPPH free radical scavenging ability: 2 mL of DPPH solution (0.2 mmol / L, dissolved in 95% ethanol) was placed in a test tube, 2 mL of rice gluten antioxidant peptide was added, and the mixture was shaken and mixed. After standing at room temperature for 30 min, the absorbance was measured at 517 nm. The absorbance was adjusted to zero by adding 2 mL of 95% ethanol to 2 mL of distilled water. A control group was also set. The DPPH scavenging ability of the sample was expressed as the inhibition rate.

[0082] (2) Determination of ABTS free radical scavenging ability: Equal volumes of ABTS solution (concentration of 7 mmol / L) and potassium persulfate solution (concentration of 2.45 mmol / L) were mixed and placed in the dark at room temperature for 12-16 hours to generate ABTS free radical cations (ABTS·+). Before use, the solution was diluted to a working solution with an absorbance of 0.7±0.02 at 734 nm after mixing with phosphate buffer (50 mmol / L, pH 7.4). Subsequently, 50 μL of rice gluten antioxidant peptide solution was mixed with 150 μL of ABTS working solution and incubated at 30°C for 30 minutes. The absorbance at 734 nm was measured using a microplate reader to calculate the ABTS free radical scavenging rate (%). The results are shown in Table 2.

[0083] Table 2 Antioxidant activity of rice gluten antioxidant peptides from different examples

[0084]

[0085]

[0086] As shown in Table 2, the rice gluten antioxidant peptides prepared by the preparation method of the present invention have high antioxidant activity.

[0087] In summary, the present invention provides a method for preparing an antioxidant peptide that can target and regulate the Keap1 / Nrf2 signaling pathway, which opens up a way for deep processing of rice resources and high-value utilization of rice gluten resources; the rice gluten antioxidant peptide prepared by the present invention not only has high in vitro antioxidant capacity, but also can target and regulate the Nrf2 / Keap1 signaling pathway, and has a small molecular weight and is easy to digest and absorb, and can be used to prepare functional foods, medicines, health products and cosmetics that improve oxidative stress damage in the body, and can also be used in combination with other health products or food excipients.

[0088] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of the present invention.

Claims

1. A method for preparing a Keap1-inhibiting rice gluten antioxidant peptide, characterized in that: The following steps are involved: S1. mixing water and rice gluten powder to obtain a mixture, and physically modifying the mixture to obtain a modified mixture; S2, diluting the modified mixture with water, adjusting the pH of the system to 6.5-8.5, adding a protease having hydrophobic amino acid cleavage site specificity, and enzymolyzing at 40-60° C. for 1-8 hours to obtain a first enzymatic hydrolyzate; S3, adding flavor protease to the first hydrolyzate, performing enzymatic hydrolysis at 40-60° C. for 1-8 hours to obtain a second hydrolyzate, inactivating the enzyme in the second hydrolyzate, centrifuging and separating the enzyme, and drying the supernatant to obtain the Keap1-inhibiting rice gluten antioxidant peptide; Wherein, based on the protein content of rice gluten powder as a calculation basis, the added amount of the protease with hydrophobic amino acid cleavage site specificity is 0.1-2%, and the added amount of the flavor protease is 0.1-1%.

2. The method for preparing the Keap1-inhibiting rice gluten antioxidant peptide according to claim 1, wherein: In step S1, the mass ratio of the water to the rice gluten powder is 1:2-1:

15.

3. The method for preparing the Keap1-inhibiting rice gluten antioxidant peptide according to claim 1, wherein: In step S1, the physical modification method includes one or more of ultrasonic treatment, air flow milling treatment, heating treatment, and high pressure treatment, wherein: The power of the ultrasonic treatment is 100-600W, and the time of the ultrasonic treatment is 3-60min; The pressure of the air flow milling treatment is 0.1-0.8 MPa; The temperature of the heating treatment is 50-100°C, and the time of the heating treatment is 3-60 minutes; The pressure of the high-pressure treatment is 50-500 MPa, and the time of the high-pressure treatment is 5-40 minutes.

4. The method for preparing the Keap1-inhibiting rice gluten antioxidant peptide according to claim 1, wherein: In step S2, in the step of diluting the modified mixture with water, the mass ratio of the modified mixture to the water is 1:1-1:

15.

5. The method for preparing the Keap1-inhibiting rice gluten antioxidant peptide according to claim 1, wherein: In step S2, the protease having hydrophobic amino acid cleavage site specificity includes one or more of pepsin, alkaline protease, trypsin, chymotrypsin, proteinase K, and papain; Preferably, the protease having hydrophobic amino acid cleavage site specificity includes papain or alkaline protease.

6. The method for preparing the Keap1-inhibiting rice gluten antioxidant peptide according to claim 1, wherein: In step S3, the step of inactivating the enzyme in the second enzymatic hydrolysate comprises: The second enzymatic hydrolysate is heated to 90-95° C. and kept warm for 15-30 minutes.

7. The method for preparing the Keap1-inhibiting rice gluten antioxidant peptide according to claim 1, wherein: In step S3, the centrifugal separation step includes: cooling the second enzymatic hydrolysate after enzyme inactivation to room temperature, then centrifuging at 6000-10000 rpm for 15-30 minutes, re-dissolving the precipitate with water, stirring evenly and centrifuging again, and collecting the supernatant obtained twice.

8. A Keap1-inhibiting rice gluten antioxidant peptide prepared by the method for preparing a Keap1-inhibiting rice gluten antioxidant peptide according to any one of claims 1 to 7, characterized in that: The Keap1-inhibiting rice gluten antioxidant peptide: The hydrophobic amino acid content is 28-36 g / 100 g, the aromatic amino acid content is 7-12 g / 100 g, the leucine content is 5-9 g / 100 g, the arginine content is 8-13 g / 100 g, and the lysine content is 3-6 g / 100 g.

9. The Keap1-inhibiting rice gluten antioxidant peptide according to claim 8, wherein The hydrophobic amino acids include Ala, Phe, Ile, Leu, Pro, Val, Trp and Tyr; and the aromatic amino acids include Phe, Trp and Tyr.

10. Use of the Keap1-inhibiting rice gluten antioxidant peptide according to any one of claims 8 to 9 in the preparation of foods, medicines, health products or cosmetics with antioxidant and anti-aging effects.

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