Extraction method of oat polypeptide
By combining modified graphene materials with oat processing byproducts, an efficient oat peptide extraction process was constructed, which solved the problems of insufficient selectivity and high cost in oat peptide separation, and achieved efficient extraction of high-purity peptides and preservation of their bioactivity.
Patent Information
- Application Number
- CN202511833917.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-08
- Publication Date
- 2026-02-06
AI Technical Summary
Existing technologies for separating oat peptides have limited selectivity and high costs. Graphene materials also lack specificity for peptide adsorption, making it difficult to achieve efficient extraction of oat peptides.
By combining modified graphene materials with oat processing byproducts, and employing oscillatory adsorption and reversed-phase C18 column gradient elution, a highly efficient and selective oat peptide extraction process was constructed. The large specific surface area and surface sulfonic acid groups of modified graphene were used to enrich peptides, which were then purified by chromatography.
This method enables the efficient and selective extraction of high-purity oat peptides from complex enzymatic hydrolysates, reducing costs while maintaining the bioactivity of the peptides, thus achieving resource recycling and green economic benefits.
Abstract
Description
Technical Field
[0001] This invention belongs to the field of polypeptide extraction technology, and particularly relates to a method for extracting oat polypeptides. Background Technology
[0002] Oats, a recognized all-around grain, boast nutritional value far exceeding that of traditional staple foods. Not only is it rich in soluble dietary fiber beta-glucan, but it is also renowned for its superior protein quality, with a protein content as high as 12%-20%, significantly better than wheat and rice. More importantly, oat protein has a balanced amino acid profile, especially rich in lysine, making it a high-quality plant protein source with immense development potential.
[0003] Plant peptides, small molecular fragments generated from proteins through enzymatic hydrolysis or fermentation, have become a research frontier in the fields of functional foods and biomedicine. Compared to intact proteins, these small peptide fragments are more easily absorbed by the human body and exhibit diverse and significant physiological regulatory activities. Oat peptides, obtained through targeted enzymatic hydrolysis of oat protein, are an important type of plant peptide. Studies have confirmed that oat peptides possess multiple biological benefits: their antioxidant activity helps scavenge free radicals and delay aging; their angiotensin-converting enzyme inhibitory activity has a positive effect on blood pressure regulation; simultaneously, they also show good potential in immune regulation, anti-inflammation, and improving lipid metabolism. This gives oat peptides a broad market prospect in the development of specific health functional foods, dietary supplements, and even lead drug ingredients.
[0004] However, efficiently and with high purity, obtaining target active peptides from complex oat hydrolysates remains a technological bottleneck for industrialization. Existing separation techniques, such as membrane separation, have limited selectivity, while precision methods like high-performance liquid chromatography (HPLC) are costly and susceptible to contamination if used to directly process crude extracts. Therefore, developing an integrated purification strategy that allows for both enrichment and purification is crucial. In recent years, novel adsorption materials such as graphene have attracted attention due to their enormous specific surface area; however, how to specifically modify them to selectively adsorb peptides remains a pressing technical problem. Summary of the Invention
[0005] To address the technical problems of limited separation selectivity, high cost, and insufficient specificity of graphene materials for peptide adsorption in the existing technologies, the present invention aims to provide a highly efficient, highly selective, and cost-effective method for extracting oat peptides. This method utilizes a specific adsorption material homologous to the raw material for pretreatment, combined with high-performance chromatographic purification, to achieve rapid enrichment and purification of target active peptides from complex enzymatic hydrolysates.
[0006] To achieve the above objectives, the following technical solution is adopted: This invention provides a method for extracting oat polypeptides, comprising the following steps:
[0007] S1. Mix oat protein raw materials with water, adjust the pH value, add protease to carry out enzymatic hydrolysis reaction, and inactivate the enzyme after the reaction is completed to obtain crude oat polypeptide extract;
[0008] S2. The crude extract of oat peptides obtained in step S1 is mixed with modified graphene material, and the mixture is subjected to oscillation and adsorption. Then, solid-liquid separation is performed to obtain modified graphene loaded with peptides.
[0009] S3. Use a desorption solvent to elute the modified graphene loaded with peptides described in step S2, collect the desorption solution, and obtain a preliminarily enriched peptide solution.
[0010] S4. Load the preliminarily enriched polypeptide solution obtained in step S3 onto a reversed-phase C18 column, and elute using a gradient elution program of aqueous and organic phases, collecting the elution fraction rich in the target polypeptide.
[0011] S5. The eluted fraction collected in step S4 is concentrated and dried to obtain a high-purity oat polypeptide product.
[0012] Further, in step S1, the protease is one or more of alkaline protease, neutral protease or trypsin; the pH value of the enzymatic hydrolysis reaction is 7.0-10.0, the reaction temperature is 45-60℃, the enzymatic hydrolysis time is 2-6h, and the amount of enzyme added is 1%-4% of the mass of the substrate protein.
[0013] Furthermore, the modified graphene is prepared through the following steps:
[0014] P1. Oat bran and oat protein hydrolysis residue are washed, dried and pulverized to obtain biomass powder;
[0015] P2. Mix the biomass powder obtained in step P1 with concentrated sulfuric acid at a mass-to-volume ratio of 1g:(5-15)mL, and carry out a hydrothermal reaction at 180-220℃ for 6-12h under nitrogen protection. After the reaction is completed, cool the product, dilute it with deionized water, filter it, and then purify it by dialysis or ultrafiltration to obtain an aqueous solution of carbon dots with sulfonic acid groups on the surface, and then concentrate it.
[0016] P3. Disperse graphene oxide in water to prepare a dispersion of 0.5-2.0 mg / mL;
[0017] P4. Mix the sulfonic acid carbon dot solution obtained in step P2 with the graphene oxide dispersion obtained in step P3 at a carbon dot to graphene oxide mass ratio of 1:(2-5). Under acidic conditions of pH=3-5, stir at 40-60℃ for 2-4 hours. Then transfer the mixture to a hydrothermal reactor and react at 120-160℃ for 4-8 hours.
[0018] P5. After the reaction is complete, the product is naturally cooled, collected by centrifugation, and washed with ethanol and deionized water until neutral. Finally, the obtained solid is vacuum dried at 60-80℃ and ground to obtain the modified graphene.
[0019] Further, in step S2, the amount of modified graphene added is 0.1%-1.0% of the mass of the crude oat polypeptide extract; the conditions for oscillation adsorption are: temperature 20-40℃, rotation speed 100-200rpm, and adsorption time 30-90min.
[0020] Further, in step S3, the desorption solvent is an acidic aqueous solution with a pH of 2.0-4.0, or an ethanol aqueous solution with a volume concentration of 50%-80%, or an alcohol-water solution containing 0.1%-1.0% ammonia; the desorption method is ultrasonic-assisted desorption or static extraction, and the desorption time is 10-30 min.
[0021] Further, in step S4, the particle size of the packing material of the reversed-phase C18 column is 5-15 μm, the column size is 10-50 mm inner diameter and 150-250 mm column length; before loading the sample, the preliminarily enriched polypeptide solution needs to be filtered through a 0.22 μm filter membrane.
[0022] Further, in step S4, the gradient elution program is as follows: using a 0.1% trifluoroacetic acid aqueous solution as mobile phase A, and a 0.1% trifluoroacetic acid acetonitrile solution as mobile phase B; the elution gradient is as follows: 0-10 min, phase B linearly increases from 5% to 25%; 10-25 min, phase B linearly increases from 25% to 45%; 25-30 min, phase B is maintained at 45% for rinsing; 30-35 min, phase B rapidly decreases from 45% to 5% for column equilibration; the flow rate is 1-5 mL / min, and the detection wavelength is 214 nm.
[0023] Furthermore, in step S5, the concentration is carried out using a rotary evaporator at a temperature not exceeding 50°C; the drying is carried out by vacuum freeze drying or spray drying.
[0024] Furthermore, in step P2, the hydrothermal reaction is carried out in a high-pressure reactor with a polytetrafluoroethylene liner; the purification is performed using a dialysis bag with a molecular weight cutoff of 500-1000 Da.
[0025] Furthermore, in step P4, the acidic condition is achieved by adding citrate-sodium citrate buffer or acetic acid-sodium acetate buffer.
[0026] The beneficial effects of this invention are as follows: This invention utilizes sulfonic acid-modified graphene prepared from oat processing byproducts as an adsorbent, combined with reversed-phase C18 chromatography, to construct a highly efficient and selective oat peptide extraction and purification process. Firstly, by leveraging the large specific surface area and abundant sulfonic acid groups on the surface of the modified graphene, peptides can be efficiently and efficiently enriched from complex enzymatic hydrolysates through strong electrostatic interactions, significantly removing impurities and solving the problems of load and interference in subsequent chromatographic separation. Secondly, this process uses oat byproducts to prepare the modified material, which not only achieves resource recycling and cost reduction but may also enhance the specific adsorption of oat peptides due to homology. The entire process of this invention is characterized by mild conditions and recyclable solvents, demonstrating green economic efficiency. Finally, through fine gradient separation and mild post-processing using reversed-phase C18 chromatography, high-purity and highly active target oat peptide products can be obtained, providing a reliable technical solution for the development of functional peptide products. Detailed Implementation
[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.
[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.
[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the experimental materials used in the following examples are all purchased from commercial channels.
[0030] Example 1:
[0031] A method for extracting oat polypeptides includes the following steps:
[0032] S1. Mix oat protein raw materials with water, adjust the pH value, add protease to carry out enzymatic hydrolysis reaction, and inactivate the enzyme after the reaction is completed to obtain crude oat polypeptide extract;
[0033] The protease is an alkaline protease; the pH value of the enzymatic hydrolysis reaction is 7.0, the reaction temperature is 45℃, the hydrolysis time is 2h, and the amount of enzyme added is 1% of the mass of the substrate protein;
[0034] S2. The crude extract of oat peptides obtained in step S1 is mixed with modified graphene material, and the mixture is subjected to oscillation and adsorption. Then, solid-liquid separation is performed to obtain modified graphene loaded with peptides.
[0035] The amount of modified graphene added was 0.1% of the mass of the crude oat polypeptide extract; the conditions for oscillation adsorption were: temperature 20℃, rotation speed 100rpm, and adsorption time 30min.
[0036] S3. Use a desorption solvent to elute the modified graphene loaded with peptides described in step S2, collect the desorption solution, and obtain a preliminarily enriched peptide solution.
[0037] The desorption solvent is an acidic aqueous solution with a pH of 2.0; the desorption method is static extraction, and the desorption time is 10 min.
[0038] S4. Load the preliminarily enriched polypeptide solution obtained in step S3 onto a reversed-phase C18 column, and elute using a gradient elution program of aqueous and organic phases, collecting the elution fraction rich in the target polypeptide.
[0039] The reversed-phase C18 column has a packing particle size of 5 μm, a column size of 10 mm inner diameter and 150 mm length; before loading the sample, the preliminarily enriched polypeptide solution needs to be filtered through a 0.22 μm filter membrane.
[0040] The gradient elution program was as follows: 0.1% trifluoroacetic acid aqueous solution was used as mobile phase A, and 0.1% trifluoroacetic acid in acetonitrile solution was used as mobile phase B; the elution gradient was as follows: 0-10 min, phase B linearly increased from 5% to 25%; 10-25 min, phase B linearly increased from 25% to 45%; 25-30 min, phase B was maintained at 45% for rinsing; 30-35 min, phase B was rapidly reduced from 45% to 5% for column equilibration; the flow rate was 1 mL / min, and the detection wavelength was 214 nm.
[0041] S5. The elution fraction collected in step S4 is concentrated and dried to obtain a high-purity oat polypeptide product; the concentration is carried out by rotary evaporation at a temperature not exceeding 50°C; the drying is vacuum freeze drying.
[0042] The modified graphene is prepared through the following steps:
[0043] P1. Oat bran and oat protein hydrolysis residue are washed, dried and pulverized to obtain biomass powder;
[0044] P2. The biomass powder obtained in step P1 was mixed with concentrated sulfuric acid at a mass-volume ratio of 1g:5mL. Under nitrogen protection, the mixture was subjected to a hydrothermal reaction at 180℃ for 6h. After the reaction was completed, the mixture was cooled, the product was diluted with deionized water, filtered, and then purified by dialysis to obtain an aqueous solution of carbon dots with sulfonic acid groups on the surface, which was then concentrated.
[0045] The hydrothermal reaction was carried out in a high-pressure reactor with a polytetrafluoroethylene liner; the purification was performed by dialysis using a dialysis bag with a molecular weight cutoff of 500 Da.
[0046] P3. Disperse graphene oxide in water to prepare a dispersion of 0.5 mg / mL;
[0047] P4. The sulfonic acid-based carbon dot solution obtained in step P2 and the graphene oxide dispersion obtained in step P3 are mixed at a carbon dot to graphene oxide mass ratio of 1:2. The mixture is stirred at 40°C for 2 hours under acidic conditions of pH=3. The mixture is then transferred to a hydrothermal reactor and reacted at 120°C for 4 hours. The acidic conditions are achieved by adding a citric acid-sodium citrate buffer solution.
[0048] P5. After the reaction is complete, the product is naturally cooled, collected by centrifugation, and washed with ethanol and deionized water until neutral. Finally, the obtained solid is vacuum dried at 60°C and ground to obtain the modified graphene.
[0049] Example 2:
[0050] A method for extracting oat polypeptides includes the following steps:
[0051] S1. Mix oat protein raw materials with water, adjust the pH value, add protease to carry out enzymatic hydrolysis reaction, and inactivate the enzyme after the reaction is completed to obtain crude oat polypeptide extract;
[0052] The protease is a neutral protease; the pH value of the enzymatic hydrolysis reaction is 10.0, the reaction temperature is 60℃, the enzymatic hydrolysis time is 6h, and the amount of enzyme added is 4% of the mass of the substrate protein;
[0053] S2. The crude extract of oat peptides obtained in step S1 is mixed with modified graphene material, and the mixture is subjected to oscillation and adsorption. Then, solid-liquid separation is performed to obtain modified graphene loaded with peptides.
[0054] The amount of modified graphene added was 1.0% of the mass of the crude oat polypeptide extract; the conditions for oscillation adsorption were: temperature 40℃, rotation speed 200rpm, and adsorption time 90min.
[0055] S3. Use a desorption solvent to elute the modified graphene loaded with peptides described in step S2, collect the desorption solution, and obtain a preliminarily enriched peptide solution.
[0056] The desorption solvent is an alcohol-water solution containing 1.0% ammonia; the desorption method is ultrasonic-assisted desorption, and the desorption time is 30 min.
[0057] S4. Load the preliminarily enriched polypeptide solution obtained in step S3 onto a reversed-phase C18 column, and elute using a gradient elution program of aqueous and organic phases, collecting the elution fraction rich in the target polypeptide.
[0058] The reversed-phase C18 column has a packing particle size of 15 μm, a column size of 50 mm inner diameter and 250 mm length; before loading the sample, the preliminarily enriched polypeptide solution needs to be filtered through a 0.22 μm filter membrane.
[0059] The gradient elution program was as follows: 0.1% trifluoroacetic acid aqueous solution was used as mobile phase A, and 0.1% trifluoroacetic acid in acetonitrile solution was used as mobile phase B; the elution gradient was as follows: 0-10 min, phase B linearly increased from 5% to 25%; 10-25 min, phase B linearly increased from 25% to 45%; 25-30 min, phase B was maintained at 45% for rinsing; 30-35 min, phase B was rapidly reduced from 45% to 5% for column equilibration; the flow rate was 5 mL / min, and the detection wavelength was 214 nm.
[0060] S5. The elution fraction collected in step S4 is concentrated and dried to obtain a high-purity oat polypeptide product; the concentration is carried out using a rotary evaporator at a temperature not exceeding 50°C; the drying is spray drying.
[0061] The modified graphene is prepared through the following steps:
[0062] P1. Oat bran and oat protein hydrolysis residue are washed, dried and pulverized to obtain biomass powder;
[0063] P2. The biomass powder obtained in step P1 was mixed with concentrated sulfuric acid at a mass-volume ratio of 1g:15mL. Under nitrogen protection, the mixture was subjected to a hydrothermal reaction at 220℃ for 12h. After the reaction was completed, the mixture was cooled, the product was diluted with deionized water, filtered, and then purified by ultrafiltration to obtain an aqueous solution of carbon dots with sulfonic acid groups on the surface, which was then concentrated.
[0064] The hydrothermal reaction was carried out in a high-pressure reactor with a polytetrafluoroethylene liner; the purification was performed by dialysis using a dialysis bag with a molecular weight cutoff of 1000 Da.
[0065] P3. Disperse graphene oxide in water to prepare a dispersion of 2.0 mg / mL;
[0066] P4. The sulfonic acid carbon dot solution obtained in step P2 and the graphene oxide dispersion obtained in step P3 are mixed at a carbon dot to graphene oxide mass ratio of 1:5. Under acidic conditions of pH=5, the mixture is stirred at 60°C for 4 hours. Then, the mixture is transferred to a hydrothermal reactor and reacted at 160°C for 8 hours. The acidic conditions are achieved by adding an acetic acid-sodium acetate buffer solution.
[0067] P5. After the reaction is complete, the product is naturally cooled, centrifuged and collected, and washed with ethanol and deionized water until neutral. Finally, the obtained solid is vacuum dried at 80°C and ground to obtain the modified graphene.
[0068] Example 3:
[0069] A method for extracting oat polypeptides includes the following steps:
[0070] S1. Mix oat protein raw materials with water, adjust the pH value, add protease to carry out enzymatic hydrolysis reaction, and inactivate the enzyme after the reaction is completed to obtain crude oat polypeptide extract;
[0071] The protease was obtained by mixing neutral protease and trypsin in a 1:1 mass ratio; the pH value of the enzymatic hydrolysis reaction was 8.5, the reaction temperature was 52℃, the hydrolysis time was 4h, and the amount of enzyme added was 2.5% of the mass of the substrate protein;
[0072] S2. The crude extract of oat peptides obtained in step S1 is mixed with modified graphene material, and the mixture is subjected to oscillation and adsorption. Then, solid-liquid separation is performed to obtain modified graphene loaded with peptides.
[0073] The amount of modified graphene added was 0.55% of the mass of the crude oat polypeptide extract; the conditions for oscillation adsorption were: temperature 30℃, rotation speed 150rpm, and adsorption time 60min.
[0074] S3. Use a desorption solvent to elute the modified graphene loaded with peptides described in step S2, collect the desorption solution, and obtain a preliminarily enriched peptide solution.
[0075] The desorption solvent is an aqueous ethanol solution with a volume concentration of 65%; the desorption method is ultrasonic-assisted desorption, and the desorption time is 20 min.
[0076] S4. Load the preliminarily enriched polypeptide solution obtained in step S3 onto a reversed-phase C18 column, and elute using a gradient elution program of aqueous and organic phases, collecting the elution fraction rich in the target polypeptide.
[0077] The reversed-phase C18 column has a packing particle size of 10 μm, a column size of 30 mm inner diameter and 200 mm length; before loading the sample, the preliminarily enriched polypeptide solution needs to be filtered through a 0.22 μm filter membrane.
[0078] The gradient elution program is as follows: 0.1% trifluoroacetic acid aqueous solution is used as mobile phase A, and 0.1% trifluoroacetic acid in acetonitrile solution is used as mobile phase B; the elution gradient is as follows: 0-10 min, phase B increases linearly from 5% to 25%; 10-25 min, phase B increases linearly from 25% to 45%; 25-30 min, phase B is maintained at 45% for rinsing; 30-35 min, phase B is rapidly reduced from 45% to 5% for column equilibration; the flow rate is 3 mL / min, and the detection wavelength is 214 nm.
[0079] S5. The elution fraction collected in step S4 is concentrated and dried to obtain a high-purity oat polypeptide product; the concentration is carried out using a rotary evaporator at a temperature not exceeding 50°C; the drying is carried out by vacuum freeze drying.
[0080] The modified graphene is prepared through the following steps:
[0081] P1. Oat bran and oat protein hydrolysis residue are washed, dried and pulverized to obtain biomass powder;
[0082] P2. The biomass powder obtained in step P1 was mixed with concentrated sulfuric acid at a mass-to-volume ratio of 1 g:10 mL, and subjected to a hydrothermal reaction at 200 °C for 9 h under nitrogen protection. After the reaction was completed, the mixture was cooled, diluted with deionized water, filtered, and then purified by dialysis to obtain an aqueous solution of carbon dots rich in sulfonic acid groups on the surface, which was then concentrated. The hydrothermal reaction was carried out in a high-pressure reactor with a polytetrafluoroethylene liner. The purification was performed using a dialysis bag with a molecular weight cutoff of 750 Da.
[0083] P3. Disperse graphene oxide in water to prepare a dispersion of 1.2 mg / mL;
[0084] P4. The sulfonic acid-based carbon dot solution obtained in step P2 and the graphene oxide dispersion obtained in step P3 are mixed at a carbon dot to graphene oxide mass ratio of 1:3.5. The mixture is stirred at 50°C for 3 hours under acidic conditions of pH=4. The mixture is then transferred to a hydrothermal reactor and reacted at 140°C for 6 hours. The acidic conditions are achieved by adding a citric acid-sodium citrate buffer solution.
[0085] P5. After the reaction is complete, the product is naturally cooled, centrifuged and collected, and washed with ethanol and deionized water until neutral. Finally, the obtained solid is vacuum dried at 70°C and ground to obtain the modified graphene.
[0086] Comparative Example 1:
[0087] This comparative example differs from Example 3 in that the adsorbent material is replaced with an equal amount of unmodified graphene oxide.
[0088] Comparative Example 2:
[0089] Except for the preparation method of the modified graphene, the steps in this comparative example are exactly the same as those in Example 3. The modified graphene in this comparative example is prepared by conventional chemical sulfonation: graphene oxide is dispersed in concentrated sulfuric acid, stirred and reacted at 80°C for 6 hours, and then washed and dried to obtain sulfonated graphene oxide.
[0090] Comparative Example 3:
[0091] The difference between this comparative example and Example 3 is that steps S2 and S3 are omitted, that is, no adsorption material is used for enrichment, the crude extract of oat peptides obtained in step S1 is directly filtered, and then reversed-phase C18 chromatography separation is performed in step S4.
[0092] Results Analysis
[0093] The indicators of the various embodiments and comparative examples of the present invention were evaluated using the following methods:
[0094] The peptide concentration in the oat peptide solution before and after adsorption was determined by the BCA method. The peptide adsorption rate (%) was calculated as (peptide content in crude extract before adsorption - peptide content in supernatant after adsorption) / peptide content in crude extract before adsorption × 100%.
[0095] The impurity removal rate (%) of each set of examples was estimated by comparing the changes in the UV absorbance of the solution at 400 nm (for impurities such as pigments) before and after adsorption.
[0096] The peptide purity (%) of the final products obtained from each set of examples and comparative examples was calculated using the high performance liquid chromatography area normalization method.
[0097] The ACE inhibitory activity (IC50) of the peptides obtained in each group of examples and comparative examples was determined by in vitro angiotensin-converting enzyme inhibition assay. 50 (mg / mL), IC 50 The lower the value, the stronger the peptide activity.
[0098] The results of the above measurements are shown in Table 1.
[0099] Table 1. Comparison of evaluation results of indicators between each embodiment and the comparative example.
[0100] Group Peptide adsorption rate (%) Impurity removal rate (%) Peptide purity (%) IC50 (mg / mL) Example 1 87.2 65 92.1 0.78 Example 2 91.5 70 94.3 0.75 Example 3 89.8 68 93.7 0.80 Comparative Example 1 41.3 24 88.5 1.12 Comparative Example 2 82.3 60 91.0 0.92 Comparative Example 3 / / 90.2 1.05
[0101] Examples 1-3 show that the modified graphene prepared using this invention exhibits an adsorption rate of over 87% for oat peptides, while the impurity removal rate exceeds 65%. This demonstrates that the method described in this invention, which utilizes homologous byproducts to prepare sulfonic acid carbon dots for modification and then modifies the graphene, can effectively introduce abundant specific adsorption sites of sulfonic acid groups, thereby significantly improving its adsorption capacity and selectivity for target peptides. This invention overcomes the bottlenecks of poor adsorption selectivity and difficulty in maintaining product activity in existing technologies, achieving not only efficient and highly selective extraction and purification of oat peptides but also maximizing the preservation of their biological activity.
[0102] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
[0103] The present invention and its embodiments have been described above. This description is not restrictive, and the embodiments shown are only one of the embodiments of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.
Claims
1. A method for extracting oat polypeptides, characterized in that: Includes the following steps: S1. Mix oat protein raw materials with water, adjust the pH value, add protease to carry out enzymatic hydrolysis reaction, and inactivate the enzyme after the reaction is completed to obtain crude oat polypeptide extract; S2. The crude extract of oat peptides obtained in step S1 is mixed with modified graphene material, and the mixture is subjected to oscillation and adsorption. Then, solid-liquid separation is performed to obtain modified graphene loaded with peptides. S3. Use a desorption solvent to elute the modified graphene loaded with peptides described in step S2, collect the desorption solution, and obtain a preliminarily enriched peptide solution. S4. Load the preliminarily enriched polypeptide solution obtained in step S3 onto a reversed-phase C18 column, and elute using a gradient elution program of aqueous and organic phases, collecting the elution fraction rich in the target polypeptide. S5. The eluted fraction collected in step S4 is concentrated and dried to obtain a high-purity oat polypeptide product.
2. The method for extracting oat polypeptides according to claim 1, characterized in that: In step S1, the protease is one or more of alkaline protease, neutral protease, or trypsin; the pH value of the enzymatic hydrolysis reaction is 7.0-10.0, the reaction temperature is 45-60℃, the enzymatic hydrolysis time is 2-6h, and the amount of enzyme added is 1%-4% of the mass of the substrate protein.
3. The method for extracting oat polypeptides according to claim 1, characterized in that: The modified graphene is prepared through the following steps: P1. Oat bran and oat protein hydrolysis residue are washed, dried and pulverized to obtain biomass powder; P2. Mix the biomass powder obtained in step P1 with concentrated sulfuric acid at a mass-to-volume ratio of 1g:(5-15)mL, and carry out a hydrothermal reaction at 180-220℃ for 6-12h under nitrogen protection. After the reaction is completed, cool the product, dilute it with deionized water, filter it, and then purify it by dialysis or ultrafiltration to obtain an aqueous solution of carbon dots with sulfonic acid groups on the surface, and then concentrate it. P3. Disperse graphene oxide in water to prepare a dispersion of 0.5-2.0 mg / mL; P4. Mix the sulfonic acid carbon dot solution obtained in step P2 with the graphene oxide dispersion obtained in step P3 at a carbon dot to graphene oxide mass ratio of 1:(2-5). Under acidic conditions of pH=3-5, stir at 40-60℃ for 2-4 hours. Then transfer the mixture to a hydrothermal reactor and react at 120-160℃ for 4-8 hours. P5. After the reaction is complete, the product is naturally cooled, collected by centrifugation, and washed with ethanol and deionized water until neutral. Finally, the obtained solid is vacuum dried at 60-80℃ and ground to obtain the modified graphene.
4. The method for extracting oat polypeptides according to any one of claims 1 or 2, characterized in that: In step S2, the amount of modified graphene added is 0.1%-1.0% of the mass of the crude oat polypeptide extract; the conditions for oscillation adsorption are: temperature 20-40℃, rotation speed 100-200rpm, and adsorption time 30-90min.
5. The method for extracting oat polypeptides according to claim 4, characterized in that: In step S3, the desorption solvent is an acidic aqueous solution with a pH of 2.0-4.0, or an ethanol aqueous solution with a volume concentration of 50%-80%, or an alcohol-water solution containing 0.1%-1.0% ammonia; the desorption method is ultrasonic-assisted desorption or static extraction, and the desorption time is 10-30 min.
6. The method for extracting oat polypeptides according to claim 4, characterized in that: In step S4, the particle size of the packing material of the reversed-phase C18 column is 5-15 μm, the column size is 10-50 mm inner diameter and 150-250 mm column length; before loading the sample, the preliminarily enriched polypeptide solution needs to be filtered through a 0.22 μm filter membrane.
7. The method for extracting oat polypeptides according to claim 4, characterized in that: In step S4, the gradient elution program is as follows: using a 0.1% trifluoroacetic acid aqueous solution as mobile phase A and a 0.1% trifluoroacetic acid acetonitrile solution as mobile phase B; the elution gradient is as follows: 0-10 min, phase B linearly increases from 5% to 25%; 10-25 min, phase B linearly increases from 25% to 45%; 25-30 min, phase B is maintained at 45% for rinsing; 30-35 min, phase B is rapidly reduced from 45% to 5% for column equilibration; the flow rate is 1-5 mL / min, and the detection wavelength is 214 nm.
8. The method for extracting oat polypeptides according to claim 4, characterized in that: In step S5, the concentration is carried out using a rotary evaporator at a temperature not exceeding 50°C; the drying is carried out by vacuum freeze drying or spray drying.
9. The method for extracting oat polypeptides according to claim 3, characterized in that: In step P2, the hydrothermal reaction is carried out in a high-pressure reactor with a polytetrafluoroethylene liner; the purification is performed by dialysis using a dialysis bag with a molecular weight cutoff of 500-1000 Da.
10. The method for extracting oat polypeptides according to claim 3, characterized in that: In step P4, the acidic conditions are achieved by adding citrate-sodium citrate buffer or acetic acid-sodium acetate buffer.