Selenium-rich cordyceps militaris antioxidant peptide as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a selenium-enriched cordyceps militaris antioxidant peptide and a preparation method and application thereof. The invention provides two selenium-enriched cordyceps militaris antioxidative peptides, the two selenium-enriched cordyceps militaris antioxidative peptides have obvious free radical scavenging effect, the activity presents concentration dependence, the effect of PHSeMSIIK is better, and the free radical scavenging rate at high concentration reaches 80% or above. Meanwhile, the preparation process of the selenium-rich cordyceps militaris antioxidant peptide is optimized by taking angkang selenium-rich cordyceps militaris as a raw material and adopting a single factor experiment and a response surface design, and the optimal preparation process of the selenium-rich cordyceps militaris antioxidant peptide is obtained through optimization of a response surface optimization result: the substrate concentration is 2.66 wt%, the enzymolysis temperature is 60 DEG C, the enzymolysis time is 2 hours, the pH value is 7.5, and the enzyme addition amount is 2 wt%. The theoretical calculation value of the DPPH clearance rate under the condition is 87.38%.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a selenium-rich cordyceps militaris antioxidant peptide as well as a preparation method and application thereof. BACKGROUND

[0002] Antioxidant peptides are a kind of active peptides that have been widely studied in recent years. As a natural antioxidant, antioxidant peptides have a relatively simple structure, are easy to absorb, have good stability and no immunoreactivity, and not only have strong antioxidant activity, but also have other health care effects such as blood pressure reduction and anticancer, and therefore are increasingly attracting people's attention in the fields of food and health care products.

[0003] Cordyceps militaris L. Link, also known as northern cordyceps, belongs to the subphylum Ascomycotina, order Mortal, and family Clavicipitaceae, and is a common edible fungus. Cordyceps militaris is rich in nutrients such as protein, fat, vitamins, polysaccharides and superoxide dismutase (SOD), and the content of cordyceps militaris protein is about 30%, and the content of fat is 8%. Meanwhile, the nucleoside compounds, cordyceps polysaccharides and cordyceps acid contained in cordyceps militaris have pharmacological effects such as antioxidant, antitumor, immune regulation, inflammation inhibition, bactericidal and virus replication inhibition, and are a traditional Chinese medicine with medicinal value.

[0004] At present, there are few selenium-rich cordyceps militaris and products thereof on the market, and more research and development are focused on ordinary cordyceps militaris. For example, medicinal diet, cordyceps militaris wine products and capsule health care products, but most of the above products are still in the stage of rough processing, and cannot obtain a large market. So far, there is no related report on selenium-containing polypeptides from cordyceps militaris at home and abroad, which is worthy of further research and development. SUMMARY

[0005] The present application aims to provide a selenium-rich cordyceps militaris antioxidant peptide as well as a preparation method and application thereof, so as to solve the problems in the prior art.

[0006] To achieve the above-mentioned purpose, the present application provides the following solutions.

[0007] The present application provides a selenium-rich cordyceps militaris antioxidant peptide, and the amino acid sequence of the selenium-rich cordyceps militaris antioxidant peptide is shown in SEQ ID NO. 1 or SEQ ID NO. 2.

[0008] The present application provides a preparation method of the above-mentioned selenium-rich cordyceps militaris antioxidant peptide, which comprises the following steps:

[0009] After the selenium-rich cordyceps militaris powder and water are mixed, ultrasonic treatment and centrifugal treatment are sequentially performed to obtain selenium-rich cordyceps militaris powder protein;

[0010] The selenium-rich Cordyceps militaris powder and papain are mixed, and after enzymolysis and ultrafiltration are sequentially performed, fragments with a molecular weight of 5-50 KDa are separated and identified to obtain the selenium-rich Cordyceps militaris powder antioxidant peptide.

[0011] Preferably, the amount of papain is 2% of the protein mass of the selenium-rich Cordyceps militaris powder.

[0012] Preferably, the enzymolysis time is 1.0-2.0 h, the temperature is 40-60 DEG C, and the pH value is 7.5.

[0013] Preferably, the mass-volume ratio of the selenium-rich Cordyceps militaris powder to water is 1 g:40 mL.

[0014] Preferably, the mass percentage of the selenium-rich Cordyceps militaris powder in the mixture obtained after mixing the selenium-rich Cordyceps militaris powder and water is 2.0-4.0%.

[0015] The application provides application of the selenium-rich Cordyceps militaris antioxidant peptide in preparation of an antioxidant.

[0016] The application provides an antioxidant, which comprises the selenium-rich Cordyceps militaris antioxidant peptide.

[0017] Preferably, the antioxidant further comprises a pharmaceutically acceptable adjuvant.

[0018] The application provides application of the selenium-rich Cordyceps militaris antioxidant peptide in preparation of an antioxidant product.

[0019] Preferably, the antioxidant product comprises a medicine.

[0020] The application discloses the following technical effects:

[0021] The application provides a selenium-rich Cordyceps militaris antioxidant peptide, wherein the amino acid sequence of the selenium-rich Cordyceps militaris antioxidant peptide is shown in SEQ ID NO. 1 or SEQ ID NO. 2. The two selenium-rich Cordyceps militaris antioxidant peptides have obvious free radical scavenging effects, and the activity is concentration-dependent, wherein the effect of PHSeMSIIK is better, and the free radical scavenging rate is more than 80% at a high concentration (0.4 mg / mL).

[0022] Meanwhile, the application takes An Kang selenium-rich Cordyceps militaris as raw material, adopts single factor experiment and response surface design to optimize the preparation process of selenium-rich Cordyceps militaris antioxidant peptides, and detects the flavor and taste, and the results show that: taking DPPH· clearance rate as an index, the best enzyme for preparing selenium-rich Cordyceps militaris antioxidant peptides is papain; the substrate concentration, temperature and time are closely related to the change of DPPH· clearance rate; the optimal preparation process of selenium-rich Cordyceps militaris antioxidant peptides is that the substrate concentration is 2.66 wt%, the enzymolysis temperature is 60 DEG C, the enzymolysis time is 2 h, the pH is 7.5, and the enzyme amount is 2 wt%, the DPPH· clearance rate under the condition is 87.38% in theory, and the result (87.26 ± 0.13%) is close to the theoretical value through verification experiment; through the methods of principal component analysis (PCA) and cluster analysis, the selenium-rich Cordyceps militaris protein and selenium-rich Cordyceps militaris antioxidant peptides are compared, and the results show that there is significant difference between the two in flavor and taste. It can be seen that the preparation method obtained by the application has strong operability, and the product has significant antioxidant effect. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0024] Figure 1 Effect of different proteases on DPPH· radical scavenging rate of selenium-rich Cordyceps militaris peptides

[0025] Figure 2 Effect of substrate concentration on DPPH· radical scavenging rate of selenium-rich Cordyceps militaris peptides

[0026] Figure 3 Effect of enzyme amount on DPPH· radical scavenging rate of selenium-rich Cordyceps militaris peptides

[0027] Figure 4 Effect of temperature on DPPH· radical scavenging rate of selenium-rich Cordyceps militaris peptides

[0028] Figure 5 Effect of enzymolysis time on DPPH· radical scavenging rate of selenium-rich Cordyceps militaris peptides

[0029] Figure 6 Effect of pH on DPPH· radical scavenging rate of selenium-rich Cordyceps militaris peptides

[0030] Figure 7 Residual normal chart

[0031] Figure 8 Residual and predicted value

[0032] Figure 9 isotherm plot of temperature and substrate concentration;

[0033] Figure 10 response surface plot of temperature and substrate concentration;

[0034] Figure 11 is principal component analysis plot of selenium-rich Cordyceps militaris protein and polypeptide electronic tongue;

[0035] Figure 12 is cluster analysis plot of selenium-rich Cordyceps militaris protein and polypeptide electronic tongue detection;

[0036] Figure 13 is DPPH· scavenging rate of each component after ultrafiltration separation of Cordyceps militaris selenium peptide; wherein, F0 is polypeptide without ultrafiltration separation, F1 is a component greater than 50 kDa, F2 is a component of 5-50 kDa, and F3 is a component less than 5 kDa;

[0037] Figure 14 is separation plot of F2 component reverse phase high performance liquid chromatography;

[0038] Figure 15 is DPPH· scavenging rate of each component separated by reverse phase high performance liquid chromatography;

[0039] Figure 16 is secondary mass spectrum (A) of polypeptide I and polypeptide structure diagram (B) thereof;

[0040] Figure 17 is secondary mass spectrum (A) of polypeptide II and polypeptide structure diagram (B) thereof;

[0041] Figure 18 is selenium-rich Cordyceps militaris polypeptide gastrointestinal simulated digestion enzyme hydrolysis site; wherein, Pn is pepsin enzyme cleavage site, and Ts is trypsin enzyme cleavage site;

[0042] Figure 19 is molecular docking model of interaction of Keap1 and PHSeMSIIK; wherein, A is three-dimensional structure plot of docking of PHSeMSIIK and Keap1 protein, and B is theoretical binding mode of PHSeMSIIK and Keap1 protein;

[0043] Figure 20 is molecular docking model of interaction of Keap1 and KVVVALGDYSeMEITSLA; wherein, A is three-dimensional structure plot of docking of KVVVALGDYSeMEITSLA and Keap1 protein, and B is theoretical binding mode of KVVVALGDYSeMEITSLA and Keap1 protein

[0044] Figure 21The clearance rate of selenium-enriched cordyceps selenium peptide on DPPH·;

[0045] Figure 22 The clearance rate of selenium-enriched cordyceps selenium peptide on ABTS + free radicals;

[0046] Figure 23 The relief effect of selenium-enriched cordyceps selenium peptide on cell oxidative damage. DETAILED DESCRIPTION

[0047] The detailed description set forth below is intended as a description of various example embodiments of the application and is not intended to represent the only embodiments in which the application can be practiced. The detailed description is intended only to afford those skilled in the art the broadest possible understanding of the present application as it exists presently.

[0048] It should be understood that the terms used herein are merely descriptive, but are not intended to limit the application. In addition, for numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is specifically disclosed. Each intermediate value within any stated value or stated range, as well as any other stated value or intermediate value within the stated range, is also included in the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the content of the present specification will control.

[0050] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative only.

[0051] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended terms that are intended to mean "including but not limited to".

[0052] Example 1

[0053] 1. Materials and Methods

[0054] 1.1 Materials and reagents

[0055] Experimental materials: selenium-enriched cordyceps was provided by Ankang Agricultural Science Institute.

[0056] Experimental reagents: papain, neutral protease, alkaline protease, complex protease, pepsin, trypsin were purchased from Shanghai Yuan Ye Technology Co., Ltd.

[0057] The main chemical reagents were all of analytical purity: sodium hydroxide, anhydrous ethanol were purchased from Tianjin Tianli Chemical Reagent Co., Ltd.; hydrochloric acid was purchased from Chengdu Kolon Chemicals Co., Ltd.; 1,1-diphenyl-2-picrylhydrazyl (DPPH) was purchased from Fuzhou Feijing Biological Technology Co., Ltd.

[0058] 1.2 Instruments and equipment

[0059] The instruments and equipment used in this experiment are shown in Table 1.

[0060] Table 1 Instruments and equipment

[0061]

[0062] 1.3 Experimental method

[0063] 1.3.1 Raw material pretreatment

[0064] After the selenium-enriched cordyceps militaris was washed with deionized water, it was dried at 60℃ until the weight was constant, crushed by a pulverizer and then passed through an 80 mesh sieve to obtain selenium-enriched cordyceps militaris powder with uniform texture, which was sealed for later use.

[0065] 1.3.2 Extraction of selenium-enriched cordyceps militaris protein

[0066] 40.0 g of selenium-enriched cordyceps militaris powder was weighed, deionized water was added according to the solid-liquid ratio of 1:40, and ultrasonic extraction (600W) was performed for 10 min. The pH value was adjusted to 8.5 with 1 mol / L NaOH, and extraction was performed in a constant temperature water bath with stirring at 60℃ for 2 h. After the extraction was completed, the above solution was centrifuged (8000 r / min, 15 min) to remove the precipitate, and the supernatant was adjusted to pH 4.0 with 1 mol / L HCl. After standing for 2 h, the precipitate was centrifuged and placed in a culture dish. After vacuum freeze-drying for 24 h, the precipitate was weighed, and the obtained precipitate was selenium-enriched cordyceps militaris protein.

[0067] 1.3.3 Preparation of selenium-enriched cordyceps militaris polypeptide

[0068] Take 10.0 g of selenium-rich Cordyceps protein, and prepare a 5wt% concentration of selenium-rich Cordyceps protein solution with deionized water, stir well, and ultrasonic (600 W) for 10 min. Select the corresponding protease, and adjust the temperature and pH according to the optimum conditions of different proteases (see Table 2). Add 0.2 g of protease according to the enzyme / substrate protein mass ratio of 2%. Perform enzymolysis in a water bath, and detect the pH change of the solution every 15 min, and adjust the pH in time. After 3 h of enzymolysis, boil the water bath to inactivate the enzyme for 10 min. After cooling, centrifuge the solution (4000 r / min, 15 min), transfer the supernatant to a petri dish, and vacuum freeze-dry for 24 h. Collect the enzymolysis product, which is selenium-rich Cordyceps polypeptide.

[0069] 1.3.4 Preferred protease preparation

[0070] According to the conditions of 1.3.3, six proteases were used to prepare selenium-rich Cordyceps polypeptide, and the DPPH· clearance rate was used as an index. The amount of enzyme added was 10000 U / g protein, and the DPPH· clearance rate of each polypeptide was evaluated to determine the optimal protease for preparing selenium-rich Cordyceps polypeptide. The optimum temperature, pH and enzyme activity of each enzyme are shown in Table 2.

[0071] Table 2 Optimum temperature, pH and enzyme activity of each enzyme

[0072]

[0073] 1.3.5 Determination of DPPH· clearance rate

[0074] Transfer 2 mL of 2 mg / mL selenium-rich Cordyceps polypeptide solution to a test tube, add 1 mL of 0.2 mmol / L DPPH solution, shake well, and place in the dark for 30 min. Measure the absorbance of the selenium-rich Cordyceps polypeptide solution at 517 nm using a UV spectrophotometer and record it as A1. Mix 1 mL of anhydrous ethanol and 2 mL of selenium-rich Cordyceps polypeptide solution, shake well, and measure the absorbance as A2. Mix 2 mL of anhydrous ethanol and 1 mL of DPPH evenly, and measure the absorbance as A3. Mix 2 mL of anhydrous ethanol and 2 mL of deionized water in a test tube, mix well, and adjust to zero as a blank control.

[0075] Calculate the DPPH· clearance rate (K) according to formula (1):

[0076] Formula (1).

[0077] 1.3.6 Single factor experiment

[0078] 1.3.6.1 Effect of substrate concentration on preparation of selenium-rich Cordyceps polypeptide

[0079] Under the conditions of enzyme dosage 2.0 wt% (based on the mass of the substrate), temperature 50℃, pH 7.5, and enzymolysis time 2 h, the effect of different substrate concentrations (2.0 wt%, 3.0 wt%, 4.0 wt%, 5.0 wt%, 6.0 wt%, 7.0 wt%) on the DPPH· clearance rate of selenium-rich Cordyceps militaris peptides was explored.

[0080] 1.3.6.2 Effect of enzyme dosage on preparation of selenium-rich Cordyceps militaris peptides

[0081] Under the conditions of substrate (selenium-rich Cordyceps militaris protein) concentration 3 wt%, temperature 50℃, pH 7.5, and enzymolysis time 2 h, the effect of different enzyme dosages (0.5 wt%, 1.0 wt%, 1.5 wt%, 2.0 wt%, 2.5 wt%, 3.0 wt%, based on the mass of the substrate) on the DPPH· clearance rate of selenium-rich Cordyceps militaris peptides was explored.

[0082] 1.3.6.3 Effect of temperature on preparation of selenium-rich Cordyceps militaris peptides

[0083] Under the conditions of substrate concentration 3.0 wt%, enzyme dosage 2.0 wt% (based on the mass of the substrate), pH 7.5, and enzymolysis time 2 h, the effect of different enzymolysis temperatures (30℃, 40℃, 50℃, 60℃, 65℃, 70℃) on the DPPH· clearance rate of selenium-rich Cordyceps militaris peptides was explored.

[0084] 1.3.6.4 Effect of pH on preparation of selenium-rich Cordyceps militaris peptides

[0085] Under the conditions of substrate concentration 3.0 wt%, enzyme dosage 2.0 wt% (based on the mass of the substrate), temperature 50℃, and enzymolysis time 2 h, the effect of different enzymolysis pH (6.0, 6.5, 7.0, 7.5, 8.0, 8.5) on the DPPH· clearance rate of selenium-rich Cordyceps militaris peptides was explored.

[0086] 1.3.6.5 Effect of time on preparation of selenium-rich Cordyceps militaris peptides

[0087] Under the conditions of substrate concentration 3.0 wt%, enzyme dosage 2.0 wt% (based on the mass of the substrate), temperature 50℃, and pH 7.5, the effect of different enzymolysis times (0.5 h, 1.0 h, 1.5 h, 2.0 h, 2.5 h, 3.0 h, 3.5 h) on the DPPH· clearance rate of selenium-rich Cordyceps militaris peptides was explored.

[0088] 1.3.7 Response surface design

[0089] According to the single factor experiment results, the substrate concentration, enzymolysis temperature, and time were selected as the factors, and the DPPH· clearance rate was used as the index to optimize the preparation conditions of the selenium-rich Cordyceps militaris antioxidant peptide by the response surface experiment with 3 factors and 3 levels.

[0090] The specific experimental design factors and levels are shown in Table 3.

[0091] Table 3 Factor and level table of Box-Behnken experimental design

[0092]

[0093] 1.3.8 Electronic tongue detection

[0094] 0.5 g of each sample was weighed, deionized water was added at a ratio of 1 g:40 mL, and stirring was performed for rehydration. Ultrasonic treatment (50 W) was performed for 10 min to ensure complete dissolution. The samples were detected by the electronic tongue, and the sample collection time was 140 s. The samples were detected repeatedly, and principal component analysis (PCA) and cluster analysis were used for data processing.

[0095] 1.3.9 DPPH· clearance rate detection of different Cordyceps militaris polypeptides

[0096] The selenium-rich Cordyceps militaris polypeptide was prepared according to the optimal process optimized by the response surface in 1.3.6. The DPPH· clearance rates of the two polypeptides at different concentrations (2 mg / mL, 1.5 mg / mL, 1.0 mg / mL, and 0.5 mg / mL) were explored according to the conditions in 1.3.4.

[0097] 1.3.10 Data processing

[0098] Three groups of experiments were performed under the same experimental conditions, and the experimental data were recorded. After preliminary sorting of the experimental data, the data were analyzed and processed by SPSS software, and the data graph was drawn by GraphPad Prism.

[0099] 2. Experimental results and analysis

[0100] 2.1 Selection of protease preparation

[0101] The effects of different proteases on the DPPH· clearance rate of the selenium-rich Cordyceps militaris polypeptide are shown in Figure 1 . From Figure 1It was found that the type of protease significantly affected the antioxidant activity of selenium-enriched Cordyceps militaris peptides. The DPPH· scavenging rates of papain and the neutral protease group were 81.93 ± 1.09% and 81.51 ± 0.96%, respectively, significantly higher than other groups (p < 0.05). Different proteases recognize specific amino acid sequences, resulting in different cleavage sites and thus differences in DPPH· scavenging rates. Although there was no significant difference in DPPH· scavenging rates between papain and the neutral protease group, the data showed that papain was more effective. Therefore, papain was selected as the optimal protease for the preparation of selenium-enriched Cordyceps militaris peptides.

[0102] 2.2 Analysis of Single-Factor Experiment Results

[0103] 2.2.1 Effect of substrate concentration on the scavenging rate of selenium-enriched Cordyceps militaris peptide DPPH·

[0104] The effect of substrate concentration on the scavenging rate of selenium-enriched Cordyceps militaris peptide DPPH· is shown in the figure. Figure 2 .Depend on Figure 2 It was found that substrate concentration significantly affected the DPPH· scavenging rate of selenium-enriched Cordyceps militaris peptides. With increasing substrate concentration, the DPPH· scavenging rate initially increased and then decreased. The highest DPPH· scavenging rate (91.50 ± 0.58%) was observed at a substrate concentration of 3.0 wt%, significantly higher than other groups (P < 0.05). At lower substrate concentrations, the enzyme had a lower contact probability with the selenium-enriched Cordyceps militaris protein, resulting in limited hydrolysis. Conversely, at excessively high substrate concentrations, the protease became oversaturated, further reducing its hydrolytic effect and leading to a lower DPPH· scavenging rate. Therefore, substrate concentrations of 2.0–4.0 wt% were selected for subsequent response surface methodology optimization experiments.

[0105] 2.2.2 Effect of enzyme dosage on DPPH scavenging rate of selenium-enriched Cordyceps militaris peptides

[0106] The effect of enzyme dosage on the DPPH· scavenging rate of selenium-enriched Cordyceps militaris polypeptide is shown in the figure. Figure 3 .Depend on Figure 3 It was found that the enzyme dosage significantly affected the DPPH· scavenging rate of selenium-enriched Cordyceps militaris peptides. With increasing enzyme dosage, the DPPH· scavenging rate initially increased slowly and then decreased. The highest DPPH· scavenging rate (86.08 ± 0.71%) was observed at an enzyme dosage of 2.0 wt%, significantly higher than other groups (p < 0.05). Further increases in enzyme dosage led to a decrease in the DPPH· scavenging rate of selenium-enriched Cordyceps militaris peptides. Therefore, an enzyme dosage of 2.0 wt% was selected for subsequent response surface methodology optimization experiments.

[0107] 2.2.3 Effect of temperature on the scavenging rate of selenium-enriched Cordyceps militaris polypeptide DPPH·

[0108] Effect of temperature on DPPH· scavenging rate of selenium-rich Cordyceps militaris polypeptide Figure 4 It can be seen from Figure 4 that the DPPH· scavenging rate of selenium-rich Cordyceps militaris polypeptide increased first and then decreased with the increase of temperature, and the DPPH· scavenging rate (87.93 ± 0.57%) at 50℃ was significantly higher than that of other groups (p < 0.05). This is because the optimum temperature of papain is 50℃, and when the temperature is too high or too low, it will affect the activity of papain, thereby affecting the enzymolysis efficiency and the generation of antioxidant peptides. Therefore, the temperature of 40-60℃ was selected for the subsequent response surface optimization experiment.

[0109] 2.2.4 Effect of pH on DPPH· scavenging rate of selenium-rich Cordyceps militaris polypeptide

[0110] Effect of pH on DPPH· scavenging rate of selenium-rich Cordyceps militaris polypeptide Figure 5 It can be seen from Figure 5 that the DPPH· scavenging rate of selenium-rich Cordyceps militaris polypeptide increased first and then decreased with the increase of pH. The DPPH· scavenging rate reached the highest at pH 7.5, which was 88.00 ± 0.26%. Further increasing the pH, the DPPH· scavenging rate of selenium-rich Cordyceps militaris polypeptide decreased rapidly. This may be due to the fact that the optimum pH of papain is 7.5, and too much acid or too much alkali will destroy the spatial conformation of the enzyme molecule, affecting the hydrolysis efficiency of the enzyme, and thus affecting the generation of antioxidant peptides. Some studies have shown that the catalytic reaction is affected by the external pH environment, and too high or too low pH will affect the dissociation state of enzyme molecules and substrate molecules, as well as the combination and catalysis of enzyme and substrate, which will have adverse effects. Therefore, the pH of 7.5 was selected for the subsequent response surface optimization experiment.

[0111] 2.2.5 Effect of enzymolysis time on DPPH scavenging rate of selenium-rich Cordyceps militaris polypeptide

[0112] Effect of enzymolysis time on DPPH· scavenging rate of selenium-rich Cordyceps militaris polypeptide Figure 6 It can be seen from Figure 6 that under the premise of other factors being constant, the DPPH· scavenging rate increased first and then gently with the increase of enzymolysis time. The DPPH· scavenging rate at 2.0 h was 87.73 ± 0.85%, which was significantly higher than that of other time points (P < 0.05). The above research results show that the hydrolysis effect of protease on polypeptide mainly concentrates in 1.0-2.0 h, and the hydrolysis effect is weakened when the hydrolysis time is further prolonged, so there is no obvious promoting effect on the antioxidant activity. Therefore, the enzymolysis time of 1.0-2.0 h was selected for the subsequent response surface optimization experiment.

[0113] 2.3 Analysis of response surface results

[0114] 2.3.1 Establishment of regression model and significance analysis

[0115] According to the Box-Behnken center design principle, the DPPH· clearance rate was selected as the index, and 3 factors including substrate concentration, enzymolysis temperature and enzymolysis time were selected for 17 groups of experiments. The response surface experiment results are shown in Table 4.

[0116] Table 4 Box-Behnken experimental design scheme and experimental results

[0117]

[0118] The experimental data were preliminarily evaluated by residual normal graph Figure 7 , Figure 8 and other indicators, and the results are shown in Figure 7 and Figure 8 . The results show that all the residuals and normal probability basically present a smooth straight line, indicating that the data presents a good normal distribution, and the residual values and predicted values of the experimental results present a regular distribution. These results show that the experimental data provide a favorable basis for the subsequent model construction.

[0119] On the basis of the above theoretical analysis, Design-Expert software was used for multiple regression analysis, and variance analysis was performed on the regression model. As shown in Table 5, the F value of the model is 2.08, and the P value is 0.0447 < 0.05, which is significantly different, and the P value of the loss of fit is 0.9671 > 0.05, indicating that the regression model has certain practicality and reliability, and can be used for accurate analysis of the DPPH· clearance rate of selenium-rich Cordyceps militaris antioxidant peptides and guidance for process optimization.

[0120] Table 5 Variance analysis table of response surface design regression model

[0121]

[0122] 2.3.2 Analysis of interaction of each factor of response surface

[0123] According to the establishment of the model in 2.3.1, the interaction of each influencing factor was further clarified. The interaction between A (substrate concentration) and B (enzymolysis temperature) is shown in Figure 9 and Figure 10 . From Figure 9 and Figure 10 , it can be seen that the interaction between A and B is not significant.It can be seen that the DPPH· scavenging rate continuously increases with increasing temperature. With increasing substrate concentration, the DPPH· scavenging rate shows a trend of first increasing and then decreasing within the substrate concentration range of 2.0–4.0 wt%, and this trend is more pronounced within the 2.5–3.5 wt% range. Therefore, substrate concentration and temperature have a certain influence on the DPPH· scavenging rate of selenium-enriched Cordyceps militaris peptides, and there is an interaction between the two, resulting in a significant difference (P = 0.0447 < 0.05).

[0124] 2.4 Verification of Experimental Results

[0125] The constructed model was differentiated and the optimal value was calculated using Design-Expert. The optimal process conditions for selenium-enriched Cordyceps militaris antioxidant peptides were obtained as follows: substrate concentration 2.66 wt%, enzymatic hydrolysis temperature 60℃, enzymatic hydrolysis time 2 h, pH 7.5, and enzyme dosage 2.0 wt%. Under these conditions, the theoretically calculated DPPH· scavenging rate was 87.38%.

[0126] To further verify the optimized process, three verification experiments were conducted using these conditions. The DPPH· scavenging rate was 87.26 ± 0.13%, which is in good agreement with the theoretical model prediction. This indicates that the results are reliable for the analysis and prediction of the extraction and preparation process optimization of selenium-enriched Cordyceps militaris antioxidant peptides, and the obtained optimal process has high accuracy and precision. This demonstrates the successful development of this product and its certain antioxidant effects.

[0127] 2.5 Detection Results and Analysis of Electronic Tongue

[0128] 2.5.1 Principal Component Analysis (PCA) Results of Electronic Tongue

[0129] Depend on Figure 11 As can be seen, there are two principal components: the proportions of selenium-enriched Cordyceps militaris protein (red) in Sample 1 and selenium-enriched Cordyceps militaris polypeptide (blue) are 62.3834% and 21.1978%, respectively. The cumulative contribution of principal components 1 and 2 is 83.58%, indicating that most flavor compounds and sample information can be reflected, and the results can be used to evaluate the overall quality of the samples. According to the gravel map distribution sites, in the PC1 direction, Sample 1 and Sample 2 do not overlap and are relatively far apart, showing clear distinction. The closer the distance between different points, the smaller the difference; the farther the distance, the greater the difference. However, in the PC2 direction, there is partial overlap. The PCA results indicate that the flavor components of selenium-enriched Cordyceps militaris protein and selenium-enriched Cordyceps militaris polypeptide are different, with significant differences in taste (p > 0.05).

[0130] 2.5.2 Results of electronic tongue clustering analysis

[0131] The results of cluster analysis of selenium-enriched Cordyceps militaris protein and polypeptide electronic tongue are shown below. Figure 12 .Depend on Figure 12 As can be seen, at a Euclidean distance of 0.1, the samples were divided into two clusters: Cluster I consisted of selenium-enriched Cordyceps militaris peptides, and Cluster II consisted of selenium-enriched Cordyceps militaris protein. The significant distance between the two clusters indicates a clear difference in the volatile components of selenium-enriched Cordyceps militaris protein and peptides, resulting in a certain difference in taste, which is consistent with the results of principal component analysis (PCA). The results show that the flavor compounds of selenium-enriched Cordyceps militaris protein undergo significant changes after enzymatic hydrolysis. Electronic tongue technology can effectively distinguish between selenium-enriched Cordyceps militaris protein and selenium-enriched Cordyceps militaris antioxidant peptides, and the results accurately reflect their volatile components, overall quality, and taste, thus promoting future product development and utilization.

[0132] 2.6 Results and Analysis of DPPH Scavenging Rate of Different Cordyceps militaris Polypeptides

[0133] To investigate the effect of selenium content on the antioxidant activity of Cordyceps militaris peptides, the DPPH· scavenging rates of ordinary Cordyceps militaris peptides (prepared using the same method as the optimized selenium-enriched Cordyceps militaris peptides, the only difference being that the raw material was ordinary Cordyceps militaris peptides) and selenium-enriched Cordyceps militaris peptides were compared, as shown in Table 6. The results showed that the antioxidant activity of both peptides increased with increasing peptide concentration, reaching its highest value at 2.0 mg / mL, at 83.98 ± 0.66% and 87.26 ± 0.13%, respectively, indicating that the antioxidant activity of these peptides is concentration-dependent. Furthermore, at the same concentration, the DPPH· scavenging rate of selenium-enriched Cordyceps militaris peptides was significantly higher than that of ordinary Cordyceps militaris peptides (p < 0.05). Therefore, the antioxidant effect of selenium-enriched Cordyceps militaris peptides is more prominent than that of ordinary Cordyceps militaris peptides, and its antioxidant effect is relatively stable within a certain range. These results not only contribute to a deeper understanding of the antioxidant mechanism of the active ingredients in Cordyceps militaris but also demonstrate that selenium can promote the antioxidant efficacy of Cordyceps militaris peptides.

[0134] Table 6. Scavenging rate of different Cordyceps militaris polypeptides DPPH

[0135]

[0136] In summary, in this embodiment, An Kang selenium-rich Cordyceps militaris was used as raw material, single factor experiment and response surface design were used to optimize the preparation process of selenium-rich Cordyceps militaris antioxidant peptides, and the flavor and taste were detected. The results showed that: taking DPPH· clearance rate as the index, papain was selected as the best enzyme for preparing selenium-rich Cordyceps militaris antioxidant peptides; substrate concentration, enzyme hydrolysis temperature and enzyme hydrolysis time were closely related to the change of DPPH· clearance rate; the optimal preparation process of selenium-rich Cordyceps militaris antioxidant peptides was substrate concentration 2.66 wt%, enzyme hydrolysis temperature 60℃, enzyme hydrolysis time 2 h, pH 7.5, enzyme addition amount 2 wt%, the theoretical calculation value of DPPH· clearance rate under this condition was 87.38%, and the result (87.26 ± 0.13%) was close to the theoretical value after verification experiment. Electronic tongue detection showed that: by comparing selenium-rich Cordyceps militaris protein and selenium-rich Cordyceps militaris antioxidant peptides through principal component analysis (PCA) and cluster analysis, the results showed that there were significant differences in flavor and taste. It can be seen that the process optimized in this embodiment has strong operability, and the product has significant antioxidant effect.

[0137] Example 2

[0138] 1.1 Experimental materials and reagents

[0139] Selenium-rich Cordyceps militaris (dried product) was taken from Shaanxi Ankang Agricultural Science Institute;

[0140] Papain was purchased from Shanghai Yuan Ye Technology Co., Ltd;

[0141] Anhydrous ethanol, hydrochloric acid, nitric acid, sodium hydroxide, acetonitrile, and hydrogen peroxide were all analytical pure;

[0142] 1,1-diphenyl-2-trinitrobenzene hydrazine (DPPH), Fuzhou Feijing Biological Technology Co., Ltd.

[0143] 1.2 Main instruments and equipment

[0144] The main instruments and equipment are shown in Table 7.

[0145] Table 7 Main instruments and equipment

[0146]

[0147] 1.3 Experimental method

[0148] 1.3.1 Preparation of selenium-rich Cordyceps militaris protein

[0149] The extraction of selenium-rich Cordyceps militaris protein was based on the alkali dissolution and acid precipitation method. 20.0 g of selenium-rich Cordyceps militaris powder was dissolved in ultrapure water at a solid-liquid ratio of 1 g:40 mL. After ultrasonic-assisted dissolution for 10 min, the pH value was adjusted to 8.5 with 2 mol / L NaOH. The extraction was carried out at 60°C for 2 h. The precipitate was removed by centrifugation at 4000 r / min for 15 min. The supernatant was adjusted to pH 4.0 with 1 mol / L HCl. After standing for 4 h, the precipitate was collected by centrifugation. Finally, the vacuum freeze-drying was performed to obtain the Cordyceps militaris protein.

[0150] 1.3.2 Preparation of selenium-rich Cordyceps militaris polypeptide

[0151] A Cordyceps militaris protein solution with a mass concentration of 2.66% was prepared with deionized water. After thorough stirring, ultrasonic treatment was performed for 10 min. The pH value of the solution was adjusted to 7.5. Papain was added according to an enzyme / substrate protein mass ratio of 2%. After enzymolysis at 60°C for 2 h, the enzyme was inactivated in a boiling water bath for 10 min. The solution was centrifuged at 4000 r / min for 15 min, and the supernatant was collected. Vacuum freeze-drying was performed for 24 h to obtain selenium-rich Cordyceps militaris polypeptide.

[0152] 1.3.3 Ultrafiltration separation of selenium-rich Cordyceps militaris antioxidant peptide

[0153] 4 g of selenium-rich Cordyceps militaris polypeptide was dissolved in deionized water at a solid-liquid ratio of 1 g:50 mL. Under a pressure of 0.3 MPa, 50 kD and 5 kD ultrafiltration membranes were used for step-by-step separation of the selenium-rich Cordyceps militaris polypeptide. The > 50 kDa, 5-50 kDa, and < 5 kDa components were collected, freeze-dried, and the DPPH· scavenging rates of the components were determined.

[0154] 1.3.4 Reverse-phase high-performance liquid chromatography separation of selenium-rich Cordyceps militaris antioxidant peptide

[0155] The optimal antioxidant polypeptide component selected in 2.3.3 was prepared into a solution with a concentration of 30 mg / mL. After filtration through a 0.25 µm filter membrane, reverse-phase high-performance liquid chromatography separation was performed. The chromatographic column was Nucifera-C 18 A chromatographic column (10 mm×250 mm, 5 μm). The mobile phase A was ultrapure water, and the mobile phase B was acetonitrile. After equilibration with 95% A+5% B mixed mobile phase for 2 column volumes, 1 mL of sample was loaded. The gradient elution program was 95% A+5% B for 14 min at a flow rate of 3 mL / min; 90% A+10% B for 14-30 min at a flow rate of 3 mL / min. The detection wavelength was 214 nm. After collection of the components, low-temperature vacuum concentration and freeze-drying were performed, and then the DPPH· scavenging rates of the components were determined.

[0156] 1.3.5 Determination of selenium content in selenium-rich Cordyceps militaris antioxidant peptide

[0157] Weigh the solid sample (> 50 kDa, 5-50 kDa, < 5 kDa, and 0.02 g of each component separated by liquid phase separation) (accurate to 0.001 g), and determine the selenium content of each component according to GB5009.93—2017 "National Food Safety Standard Determination of Selenium in Food".

[0158] 1.3.6 Amino acid sequence identification of selenium-rich Cordyceps militaris antioxidant peptide

[0159] The amino acid sequence of selenium-rich Cordyceps militaris antioxidant peptide was identified using Agilent 1290 liquid chromatography-quadrupole time-of-flight tandem mass spectrometry technology. The chromatographic conditions were as follows: the chromatographic column was C 18 18 column (2.1 x 100 mm); the mobile phase was A: 0.1% formic acid water, B: acetonitrile; the column temperature was 30°C; the injection volume was 5 μL, the flow rate was 0.3 mL / min; the elution gradient was 0-5 min, 5% B; 5-20 min, 5-10% B; 20-35 min, 10-30% B; 35-40 min, 30-5% B; 40-45 min, 5% B. The mass spectrometry conditions were as follows: electrospray ionization; positive ion mode; the ion source parameters were set as follows: capillary voltage 4500 V, nebulizer pressure 30 psi; gas flow temperature 325°C, fragment pressure 125 V, secondary fragmentation energy 10-40 ev, scan mass-to-charge ratio range between 100 and 2000 (m / z). The parent ion was selected by using the intensity of the primary mass spectrum signal for collision-induced dissociation (CID) to obtain secondary mass spectrum data, and the Mascot database was searched and analyzed. Alternatively, PEAKS Studio 10.0 s software was used for Denovo analysis to obtain the amino acid sequence of the polypeptide.

[0160] 1.3.7 Online analysis of the physicochemical properties of selenium-rich Cordyceps militaris antioxidant peptide

[0161] The present embodiment refers to the method used in the reference "Preparation and physicochemical property analysis of mung bean antioxidant peptides" (Jiang Y J, Feng Y C, Zhang S, et al. Preparation and physicochemical property analysis of mung bean antioxidant peptides [J]. Chinese Journal of Oils and Fats, 2023, 38(03): 75-85.) Online tools are used to analyze the physicochemical properties of polypeptides such as residue number, molecular weight, isoelectric point, stability, water solubility, and allergenicity, providing a reference for predicting peptide function. The method in the reference "Effect of simulated gastrointestinal digestion on the antioxidant activity of sheepskin collagen peptides and its digestion protection analysis" (Wang B B, Yu Z, Li Q, et al. Effect of simulated gastrointestinal digestion on the antioxidant activity of sheepskin collagen peptides and its digestion protection analysis [J]. Food Science, 2022, 43(07): 128-138.) is used for online gastrointestinal simulation digestion to evaluate the gastrointestinal stability of polypeptides.

[0162] 1.3.8 Potential mechanism of selenium-rich cordyceps antioxidant peptides

[0163] The crystal structure of the receptor molecule Keap1 protein (PBD ID: 4l7B) was downloaded from the protein database (https: / / www.rcsb.org / structure / 4L7B). The water molecules and original ligands in the receptor molecule were removed using pyMol software and stored as a PDB file. On this basis, the receptor molecule after pyMol processing was catalytically hydrogenated by Autodock Tools1.5.6 and saved as a PDBQT file. The two-dimensional structure of the ligand was drawn using MarvinSketch software and optimized by energy minimization. The 3D structure was output as a mol file. The Autodock tool was used to open the mol2 format file, add hydrogen, calculate Gasteiger, set the rotation key, and save the file in PDBQT format. The center position and size of the grid box were set to (X:−2.636, Y:3.267, Z:−27.433), and the box size was 15×15×15. Molecular docking was performed using AutodockVina1.1.2, with default values for all parameters except special instruction parameters. Finally, the molecular docking results were visualized and analyzed using Discovery Studio 4.5 software.

[0164] 1.3.9 Data processing

[0165] Three groups of experiments were conducted under the same experimental conditions, and experimental data were recorded. SPSS software was used to analyze and process the data, GraphPad Prism was used to plot the data graph, and ChemDraw was used to draw the polypeptide structure graph.

[0166] 2 Results and analysis

[0167] 2.1 Ultrafiltration separation of selenium-rich Cordyceps militaris antioxidant peptides

[0168] Ultrafiltration was used to separate selenium-rich Cordyceps militaris antioxidant peptides, and polypeptides of different molecular weights were separated and collected. Three ultrafiltration components greater than 50 kDa (F1), 5-50 kDa (F2), and less than 5 kDa (F3) were obtained, and the DPPH· clearance rates of each component were determined, as shown in Table 1. Figure 13

[0169] According to Figure 13 , the DPPH· clearance rate of the polypeptide F0 component without ultrafiltration separation was significantly lower than that of each component after separation (p < 0.01), and among the separated components, the DPPH· clearance rate of the smaller molecular weight F2 component was the highest, at 81.61 ± 0.18%. In summary, polypeptides with smaller molecular weights have higher DPPH· clearance capacity, so the 5-50 kDa F2 component was selected for subsequent separation and purification in this example.

[0170] 2.2 Reverse phase high performance liquid chromatography separation of selenium-rich Cordyceps militaris antioxidant peptides

[0171] Further separation of the 5-50 kDa selenium-rich Cordyceps militaris polypeptide component was performed using reverse phase high performance liquid chromatography, and the elution diagram is shown in Figure 2. Figure 14 As can be seen from Figure 14 , four components were obtained after further separation, namely F 2-1 , F 2-2 , F 2-3 , and F 2-4 . The DPPH· clearance rates of the four components were detected, and the results are shown in Table 2. Figure 15 As can be seen from Figure 15 , the DPPH· clearance rates of the four separated components were significantly different, with the DPPH· clearance rate of the F 2-3 component (91.67 ± 1.24%) being significantly higher than that of the other three components (p < 0.05). The hydrophobicity of polypeptides is closely related to their antioxidant activity, and the higher the degree of hydrophobicity, the better the antioxidant activity of polypeptides. In reverse phase high performance liquid chromatography separation, the components that come out later have higher hydrophobicity than those that come out earlier, but in this study, it was found that the antioxidant activity of the last component F 2-4 was significantly lower than that of F 2-3 ​This may be related to the presence of other antioxidant substances in the components. Selenium is an important component of glutathione peroxidase (GSH-Px), which can catalyze the redox reaction of reducing glutathione (GSH) and peroxide (GSH), thereby exerting significant antioxidant function. Studies have reported that selenium plays a crucial role in the antioxidant capacity of polypeptides, and selenium and polypeptides have synergistic effects on antioxidant activity. Therefore, the selenium content of the four components was further analyzed, and the results are shown in Table 8. As shown in Table 8, the selenium content of the four components was significantly different (p < 0.05). Among them, F 2-3 The component with the highest selenium content was 25.94 ± 0.37 µg / g.

[0172] Table 8. Separation of selenium content of each component by reverse phase high performance liquid chromatography

[0173]

[0174] Note: Different lowercase letters represent significant differences.

[0175] 2.3 Identification of Amino Acid Sequence of Selenium-rich Cordyceps militaris Antioxidant Peptide

[0176] It was found that different amino acid compositions have a greater impact on their antioxidant activity, and appropriate combinations can improve their antioxidant activity and stability. Therefore, analyzing the amino acid sequence is of great significance to its structure and function. In order to determine the amino acid composition and primary structure of the F 2-3 component with the highest selenium content, LC MS / MS was used to identify the cordyceps selenium peptide components after liquid separation and purification, and two selenium-rich cordyceps active polypeptides were obtained: (I) PHSeMSIIK (SEQ ID NO. 1) and (II) KVVVALGDYSeMEITSLA (SEQ ID NO. 2), and their secondary mass spectrometry, polypeptide structure prediction is as follows Figure 16 and Figure 17 The parent proteins of these two polypeptides are protein-tyrosine-phosphatase and ATP-dependent RNAhelicase eIF4A, respectively, and the two polypeptides identified in this example have antioxidant structural advantages in amino acid composition.

[0177] 2.4 Physicochemical properties of selenium-rich Cordyceps militaris antioxidant peptide

[0178] The molecular weights of the two polypeptides in this embodiment are all below 2000 Da, and the hydrophobic residues of the two selenium-containing polypeptides are all higher than 44%, which are consistent with the structure characteristics of antioxidant peptides. In terms of water solubility and allergen stability, the two selenium-containing polypeptides both perform well. However, in terms of intracellular stability, KVVVALGDYSeMEITSLA performs better (Table 9).

[0179] In terms of gastrointestinal stability, it is found through online simulation of enzyme digestion analysis that PHSeMSIIK is stable to pepsin and trypsin and has no potential enzyme cutting site, but KVVVALGDYSeMEITSLA has a pepsin and trypsin enzyme cutting site, indicating that the gastrointestinal stability is not high, and this problem needs to be paid attention to in subsequent research and development Figure 18 ).

[0180] Table 9 Physicochemical property prediction of selenium-rich Cordyceps militaris antioxidant peptides

[0181]

[0182] 2.5 Mechanism of activity of selenium-rich Cordyceps militaris antioxidant peptides

[0183] An important defense pathway of human cells to reduce exogenous oxidative damage is the Keap1-Nrf2-ARE signaling pathway. Inhibition of the Keap1-Nrf2 interaction can activate the expression of downstream antioxidant and cell protection protein-related genes of the pathway, thereby improving the antioxidant capacity of the body. In order to clarify the intrinsic molecular mechanism of the antioxidant activity of the two selenium peptides identified in this embodiment, the binding of the two polypeptides to Keap1 was explored by bioinformatics means. According to the detection results, the affinity of PHSeMSIIK and KVVVALGDYSeMEITSLA to Keap1 protein is -6.2 and -5.6 Kcal / mol, respectively, and the binding energy is below 0, indicating that the two selenium-rich Cordyceps militaris antioxidant peptides can spontaneously bind to the Keap1 protein. Figure 19 and Figure 20 It can be seen that the two selenium-rich Cordyceps militaris antioxidant peptides can be embedded into the active cavity of Keap1 to form a stable docking conformation. In terms of specific binding sites, PHSeMSIIK can bind to residues such as Pro384 and Ser602 in Keap1 through various forces; similarly, KVVVALGDYSeMEITSLA can bind to residues such as His432, Gly433, Ser431, Arg336, and Asn382 in Keap1 (Table 10). The above results show that the two selenium-rich Cordyceps militaris peptides isolated and identified can spontaneously enter the active site of Keap1, thereby activating the antioxidant signaling pathway Keap1-Nrf2-ARE, which also explains the mechanism of antioxidant activity of the two polypeptides at the molecular level.

[0184] Table 10 Ligand, binding energy and binding site prediction of selenium-rich Cordyceps militaris antioxidant polypeptide binding with Keap1

[0185]

[0186] Table 11 Ligand interaction report

[0187]

[0188] In summary, in this embodiment, selenium-rich Cordyceps militaris was used as raw material, and selenium peptides with strong antioxidant activity were prepared by gradually separating and purifying through ultrafiltration, reverse phase high performance liquid chromatography and other technologies. Finally, two selenium-containing polypeptide segments were identified: (I) PHSeMSIIK and (II) KVVVALGDYSeMEITSLA. The molecular docking results show that the two antioxidant peptides have the potential to activate the Keap1-Nrf2-ARE signaling pathway, thereby reflecting the antioxidant activity.

[0189] Example 3

[0190] 1. Determination of DPPH free radical scavenging rate

[0191] Selenium peptide samples (PHSeMSIIK and KVVVALGDYSeMEITSLA) were prepared into solutions of different concentrations (0.1, 0.2, 0.4 mg / mL), respectively. 1 mL of selenium peptide solution was removed into a test tube and dissolved in 2 mL of 95% ethanol DPPH solution (0.1 mmol / L, prepared by accurately weighing 4 mg of DPPH powder and dissolving it in 95% ethanol to a final volume of 100 mL to obtain a 0.1 mmol / L DPPH solution, which was placed in a brown bottle for later use.). The mixture was thoroughly mixed and allowed to stand in the dark for 30 min. The absorbance (A1) was measured at 517 nm using a UV spectrophotometer. Each sample was repeated three times. The DPPH· scavenging rate (R1) was calculated according to formula (2).

[0192] Formula (2);

[0193] Wherein, R1 = DPPH free radical scavenging rate, %; A0 = blank group absorbance value (distilled water instead of selenium peptide solution); A1 = sample group absorbance value; A2 = control group absorbance value (95% ethanol instead of DPPH solution).

[0194] 2. Determination of ABTS scavenging rate

[0195] ABTS working solution was prepared: 7 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate (1:1, V / V) were reacted in the dark for 12-16 h to generate ABTS free radical cation. The ABTS working solution was diluted with deionized water, and used when the absorbance value at 734 nm was 0.70 ± 0.02.

[0196] Selenium peptide samples were prepared into solutions of different concentrations (0.1, 0.2, 0.4 mg / mL), respectively. After 1.0 mL of selenium peptide solution was dissolved in 4.0 mL of ABTS solution, it was reacted in the dark for 6 min, and the absorbance value of the sample was measured at 734 nm. Each sample was tested in triplicate. The ABTS clearance rate (R2) was calculated according to formula (3).

[0197] , formula (3);

[0198] Wherein, R2 = ABTS clearance rate, %; A0 = blank group absorbance value (distilled water instead of selenium peptide solution); A1 = sample group absorbance value; A2 = control group absorbance value (distilled water instead of ABTS solution).

[0199] 3. Alleviating oxidative stress effect

[0200] The H2O2-induced Caco-2 cell damage model was used to evaluate the alleviating effect of selenium peptides on oxidative damage. The experiment was divided into blank control, model group (oxidative damage group), GSH group (glutathione GSH, positive control group), PHSeMSIIK group and KVVVALGDYSeMEITSLA group.

[0201] 100 μL of Caco-2 cell solution diluted to a density of 5.0 × 10 5 cells / mL was added to a 96-well plate and placed in a 37°C, 5% CO2 incubator for cell adhesion. The blank control was added with 100 μL of DMEM complete medium, the GSH group was added with H2O2-containing medium solution, the concentration was 2.0 mmol / L, and the PHSeMSIIK group and the KVVVALGDYSeMEITSLA group were added with 0.1 mg / mL of PHSeMSIIK or KVVVALGDYSeMEITSLA, respectively, before H2O2 treatment. The cells were incubated in a 37°C, 5% CO2 environment for 6 h, and then the cell viability rate was determined by a CCK-8 kit.

[0202] 4. Experimental results

[0203] The DPPH· and ABTS+· clearance rates of selenium peptides were as follows: Figure 21 and Figure 22The results showed that the two identified selenium-enriched Cordyceps militaris antioxidant peptides had obvious free radical scavenging effect, and the activity was concentration-dependent, and the effect of PHSeMSIIK was better, and the free radical scavenging rate reached more than 80% at high concentration (0.4 mg / mL).

[0204] The selenium peptide alleviates the oxidative stress damage to cells, and the effect is as shown in the following table: Figure 23 The results showed that the two selenium-enriched Cordyceps militaris antioxidant peptides could effectively alleviate the oxidative damage of cells induced by hydrogen peroxide, and the effect of PHSeMSIIK was equivalent to that of the positive control (GSH).

[0205] The above-described examples only describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A selenium-enriched Cordyceps militaris antioxidant peptide, characterized in that, The amino acid sequence of the selenium-enriched Cordyceps militaris antioxidant peptide is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The method for preparing selenium-enriched Cordyceps militaris antioxidant peptides according to claim 1, characterized in that, Includes the following steps: After mixing selenium-enriched Cordyceps militaris powder with water, the mixture was subjected to ultrasonication and centrifugation to obtain selenium-enriched Cordyceps militaris powder protein. The selenium-enriched Cordyceps militaris powder and papain were mixed, and then subjected to enzymatic hydrolysis and ultrafiltration. Fragments with a molecular weight of 5-50 kDa were separated and identified to obtain the selenium-enriched Cordyceps militaris powder antioxidant peptides.

3. The preparation method according to claim 2, characterized in that, The amount of papain used is 2% of the protein content of the selenium-enriched Cordyceps militaris powder.

4. The preparation method according to claim 2, characterized in that, The enzymatic hydrolysis time is 1.0-2.0 h, the temperature is 40-60℃, and the pH value is 7.

5.

5. The preparation method according to claim 2, characterized in that, The mass-to-volume ratio of the selenium-enriched Cordyceps militaris powder to the water is 1 g: 40 mL. The mass percentage of selenium-enriched Cordyceps militaris powder in the mixture obtained by mixing selenium-enriched Cordyceps militaris powder and water is 2.0-4.0%.

6. The application of the selenium-enriched Cordyceps militaris antioxidant peptide according to claim 1 in the preparation of antioxidants.

7. An antioxidant, characterized in that, The antioxidant includes the selenium-enriched Cordyceps militaris antioxidant peptide as described in claim 1.

8. The antioxidant according to claim 7, characterized in that, The antioxidants also include pharmaceutically acceptable excipients.

9. The application of the selenium-enriched Cordyceps militaris antioxidant peptide according to claim 1 in the preparation of antioxidant products.

10. The application according to claim 9, characterized in that, The antioxidant products include pharmaceuticals.

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