CN114717286A - Preparation method of mellea armillaria sporophore antioxidant peptide - Google Patents
Preparation method of mellea armillaria sporophore antioxidant peptide Download PDFInfo
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Abstract
A method for preparing the mellea armillaria sporophore antioxidative peptide uses mellea as raw material and includes such steps as sequentially extracting protein by alkali extraction, ultrasonic alkali extraction of the residue after alkali extraction, isoelectric precipitation and salting out, and drying. According to the invention, the extraction rate of the hazel mushroom protein is effectively improved by the protein extraction mode of firstly carrying out alkali extraction on the hazel mushroom and then carrying out ultrasonic alkali extraction, the extraction rate reaches 76.59%, the precipitation amount of the hazel mushroom protein is effectively promoted to reach 66.2ug/mL by sequentially adopting isoelectric points and a salting-out method in the precipitation process, and the clearance rate of the prepared antioxidant polypeptide on DPPH free radicals reaches 86.37%. The clearance rate of hydroxyl free radicals reaches 97.53 percent, which is close to the clearance rate of a positive control. The total reducing power is equivalent and reaches 0.71.
Description
Technical Field
The invention relates to the technical field of antioxidant peptide preparation, and particularly relates to a preparation method of a mellea armillaria sporophore antioxidant peptide.
Background
Oxidative damage of oxidative free radicals to the body is one of the main causes of human diseases, including aging, coronary heart disease, inflammation, stroke, diabetes, cancer, etc., which pose great threats to human health. The development of antioxidants is therefore of great importance for the treatment and prevention of these diseases.
The antioxidant peptide is favored by researchers as a safe and nontoxic natural antioxidant, and is one of the most popular research subjects and functional factors with great development prospect in the international food industry at present. At present, the preparation of antioxidant peptides mainly uses protease to hydrolyze peptide bonds in proteins, thereby generating peptide substances with antioxidant activity. However, at present, the antioxidant peptides are mainly derived from animal proteins and vegetable proteins, and most of the antioxidant peptides belong to primary peptides which are not further separated and purified, so that the antioxidant activity of the antioxidant peptides is low.
The mellea armillaria sporophore belongs to the fungus world, is a very important medicine and food fungus special for Changbai mountain areas, contains various amino acids, trace elements and vitamins essential to human bodies, has the most considerable protein content of more than 20 percent, and is mostly high-quality protein. Modern pharmacological studies show that the mellea armillaria sporophore polysaccharide has the effects of resisting radiation, promoting hematopoiesis, inhibiting tumor growth, regulating immunity, treating rickets, reducing cholesterol, preventing and treating arteriosclerosis and the like. Therefore, scholars at home and abroad mostly study the oxidation resistance of the mellea armillaria sporophore polysaccharide, and prove that the mellea armillaria sporophore polysaccharide has certain oxidation resistance activity. However, no report on the extraction of the hazel mushroom protein and the further development and utilization of the hazel mushroom protein for preparing the antioxidant peptide exists at present, so that how to know the antioxidant activity of the hazel mushroom protein is not known. Aiming at how to extract proteins with high efficiency from hazel mushrooms, how to significantly improve the antioxidant activity of the extracted proteins in the process of preparing antioxidant peptides needs to be researched.
Disclosure of Invention
The invention aims to provide a preparation method of a mellea armillaria sporophore antioxidant peptide. The extraction rate of protein in the hazel mushroom is effectively improved, and the prepared peptide has excellent oxidation resistance.
The purpose of the invention is realized by the following technical scheme:
a preparation method of a mellea armillaria sporophore antioxidant peptide is characterized by comprising the following steps: the method comprises the steps of sequentially carrying out protein extraction, precipitation, hydrolysis, separation and purification on the hazel mushroom serving as a raw material, wherein the protein extraction is carried out by adopting alkali extraction, then carrying out ultrasonic alkali extraction on residues obtained after the alkali extraction, and the precipitation is carried out by sequentially carrying out isoelectric point method and salting-out method, and finally drying to prepare the hazel mushroom protein powder.
Further, the alkali extraction is that the hazel mushroom is crushed and then added into distilled water, the mixture is stirred evenly, NaOH with the concentration of 0.1mol/L is added to adjust the pH value of the solution to 10, the solution is bathed for 1.5h at the temperature of 75-85 ℃, and then the centrifugal treatment is carried out.
The feed-liquid ratio of the hazel mushroom to the distilled water is 1:45, the centrifugal rotation speed is 10000rpmm, and the centrifugal time is 20 min.
Further, the ultrasonic alkali extraction is to add distilled water into the residue after the alkali extraction at a material-liquid ratio of 1:45, uniformly mix the mixture, adjust the pH to 9 by using NaOH with the concentration of 0.1mol/L, and then perform ultrasonic treatment at room temperature for 14-16min at the power of 200W.
Further, the hydrolysis is to prepare the protein peptide by freeze-drying the precipitated protein and then sequentially adopting ultrasonic treatment and flavourzyme hydrolysis.
Further, the ultrasonic treatment is to prepare the hazel mushroom protein powder into a solution with the mass concentration of 3-4%, and ultrasonic treatment is carried out for 15min at 200W at room temperature.
Further, the flavourzyme hydrolysis is to hydrolyze the solution after ultrasonic treatment by using flavourzyme, wherein the hydrolysis temperature is 38-42 ℃, and the hydrolysis time is 2-3 h.
During the enzymolysis process, if the preparation is improper, the protein hydrolysis degree of the mellea armillaria sporophore is low, and the inoxidizability of the prepared peptide is damaged, so that the removing capability of the mellea armillaria sporophore antioxidant peptide on DPPH free radicals, hydroxyl free radicals and the like is poor. Therefore, according to the invention, the ultrasonic treatment is carried out firstly, and then the hydrolysis of the flavourzyme is carried out, so that the hydrolysis degree of the flavourzyme is increased through the ultrasonic treatment, the antioxidant activity of the prepared peptide is further improved on the basis of the enzymolysis of the flavourzyme, and the finally prepared peptide has excellent antioxidant activity through the synergistic effect of the ultrasonic treatment and the hydrolysis of the flavourzyme.
Further, the hydrolysis pH is 7, and the amount of the flavourzyme is 3.5% of the hazel mushroom protein powder by mass.
Further, the separation and purification are to sequentially carry out ultrafiltration on the protein peptide to obtain antioxidant peptides with different molecular weights, and then further purify the antioxidant peptides by adopting gel filtration chromatography to obtain antioxidant peptide 1 and antioxidant peptide 2.
Most specifically, the preparation method of the mellea armillaria sporophore antioxidant peptide is characterized by comprising the following steps of:
(I) extracting proteins
(1) Cleaning and airing the hazel mushroom, crushing the hazel mushroom, sieving the hazel mushroom by a 60-mesh sieve, adding the hazel mushroom into distilled water according to the feed-liquid ratio of 1:45, uniformly mixing, adjusting the pH value to 10 by using 0.1mol/L NaOH, and carrying out water bath at 80 ℃ for 1.5 hours;
(2) filtering out residues after alkali extraction is finished, adding distilled water according to the material-liquid ratio of 1:45, uniformly mixing, adding 0.1mol/L NaOH to adjust the pH value to 9, and carrying out ultrasonic treatment for 14-16min at normal temperature by using 200W power;
(3) adjusting the pH of the extracted supernatant to 3.6-3.7 by using 0.1mol/L hydrochloric acid, standing for 12h at 4 ℃, then centrifuging at 10000rpm for 20min at the temperature to obtain protein 1, taking the supernatant after isoelectric point precipitation, adding a saturated ammonium sulfate solution to ensure that the saturation is 90%, standing for 12h at 4 ℃, and then centrifuging at 10000rpm for 20min at the temperature to obtain protein 2;
(4) mixing the protein 1 and the protein 2, and freeze-drying to obtain hazel mushroom protein powder;
preparation of (II) mellea armillaria anti-oxidation peptide
(1) Preparing 3-4% aqueous solution of Corylus armillaria sporophore protein powder, treating with 200W ultrasonic wave at normal temperature for 15min, adding flavourzyme, adjusting pH to 7, hydrolyzing at 38-42 deg.C for 2-3h, wherein the enzyme addition amount is 3.5% of Corylus armillaria sporophore protein powder;
(2) and (3) inactivating the hydrolysate in boiling water bath for 15min after the hydrolysis is finished, respectively carrying out ultrafiltration treatment on the hydrolysate with ultrafiltration membranes of 3kD, 5kD and 10kD after cooling to obtain polypeptide solutions with different molecular weights, and carrying out freeze drying to obtain the antioxidant peptides with different molecular weights.
Further, the antioxidant peptide is prepared into a solution with the concentration of 30mg/L, filtration chromatography is carried out by using Sephadex G-25, then pure water is used for eluting at the speed of 2-3mL/5min, samples at the eluting peak are collected and are divided into a component A1 and a component A2, and freeze-drying is carried out after concentration.
The invention has the following technical effects:
according to the invention, the extraction rate of the hazel mushroom protein is effectively improved by a protein extraction mode of firstly carrying out alkali extraction and then carrying out ultrasonic alkali extraction on the hazel mushroom, the extraction rate reaches 76.59%, the isoelectric point and the salting-out method are sequentially adopted in the precipitation process, the precipitation amount of the hazel mushroom protein is effectively promoted to reach 66.2ug/mL, and the clearance rate of the prepared antioxidant polypeptide to DPPH free radicals reaches 86.37%. The clearance rate of hydroxyl free radicals reaches 97.53 percent, which is close to the clearance rate of a positive control. The total reducing power is equivalent to 0.71.
Drawings
FIG. 1: and (3) comparing the extraction rate of the extracted protein by different methods.
FIG. 2: the absorbance value change curve of the precipitation of the mellea armillaria sporophore protein by the isoelectric point method under different pH values and the salting-out method under different ammonium sulfate solution saturation values.
FIG. 3: the effect of ultrasound-assisted hydrolysis of different proteases on the antioxidant activity of the peptides prepared.
FIG. 4 is a schematic view of: and (3) comparing the antioxidant activity of the sample after hydrolysis of the hazel mushroom protein by the ultrasonic-assisted flavourzyme and the single flavourzyme.
FIG. 5: and (3) measuring the antioxidant activity of the components with different molecular weights after ultrafiltration.
FIG. 6: sephadex G-25 column chromatography and determination of antioxidant activity of each component thereof.
Detailed Description
The present invention is described in detail below by way of examples, it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and those skilled in the art can make some insubstantial modifications and adaptations of the present invention based on the above-mentioned disclosure.
Example 1
A preparation method of a mellea armillaria sporophore antioxidant peptide comprises the following steps of extracting protein:
(1) cleaning and airing the hazel mushroom, crushing the hazel mushroom, sieving the hazel mushroom by a 60-mesh sieve, adding the hazel mushroom into distilled water according to the feed-liquid ratio of 1:45, uniformly mixing, adjusting the pH value to 10 by using 0.1mol/L NaOH, and carrying out water bath at 80 ℃ for 1.5 hours;
(2) filtering out residues after alkali extraction is finished, adding distilled water according to the material-liquid ratio of 1:45, uniformly mixing, adding 0.1mol/L NaOH to adjust the pH value to 9, and carrying out ultrasonic treatment for 14-16min at normal temperature by using 200W power;
(3) adjusting the pH of the extracted supernatant to 3.7 by using 0.1mol/L hydrochloric acid, standing for 12h at 4 ℃, then centrifuging for 20min at 10000rpm at the temperature to obtain protein 1, taking the supernatant after isoelectric point precipitation, adding a saturated ammonium sulfate solution to ensure that the saturation is 90%, standing for 12h at 4 ℃, and then centrifuging for 20min at 10000rpm at the temperature to obtain protein 2;
(4) mixing the protein 1 and the protein 2, and freeze-drying to obtain hazel mushroom protein powder;
comparative example 1
Scheme (a): unlike example 1, the present scheme does not perform a separate alkali extraction, but performs a separate ultrasonic alkali extraction, and the rest of the steps are the same as example 1.
Scheme (b): unlike example 1, only the same alkali extraction treatment was performed during the alkali extraction without further ultrasonic alkali extraction, and the remaining steps were the same as example 1.
Scheme (c): the extraction sequence of the alkali extraction and the ultrasonic alkali extraction is changed, namely, the ultrasonic alkali extraction is firstly carried out, then the residue after the ultrasonic alkali extraction is added into distilled water for the alkali extraction, and the rest steps are the same as the steps in the embodiment 1.
As a result of comparing each protein extraction scheme of comparative example 1 with the protein extraction scheme of example 1, as shown in fig. 1, the protein extraction rate of the ultrasonic alkali extraction was the lowest, 23.33%, the alkali extraction method alone was 43.28%, the protein extraction rate of the ultrasonic alkali extraction followed by the alkali extraction scheme was 66.72%, and the protein extraction rate of the ultrasonic alkali extraction followed by the alkali extraction in the present invention was 76.59%. Therefore, the alkali extraction method is selected first, and then the ultrasonic method is selected to further optimize the protein extraction rate.
The scheme of firstly carrying out alkali extraction and then further carrying out ultrasonic alkali extraction with excellent protein extraction rate in the invention can not achieve good extraction effect when being applied to other mushrooms such as tricholoma matsutake, and for the protein extraction of the tricholoma matsutake, the steps of firstly carrying out ultrasonic alkali extraction and then carrying out alkali extraction can realize higher protein extraction efficiency.
In order to compare the effect of isoelectric point treatment and salting-out on the yield of hazel mushroom protein in the present invention, we performed corresponding comparative experiments.
Comparative example 2
Scheme (a): the difference from example 1 is that no isoelectric point treatment was performed in step (3), and only salting-out was used for protein precipitation.
Scheme (b): compared with the example 1, the difference is that only isoelectric point treatment is carried out in the step (3) to precipitate the protein, and other means are not adopted for precipitation subsequently.
Scheme (c): the difference from example 1 is that the isoelectric point method and the salting-out method in step (3) are performed simultaneously.
Scheme (d): the difference from example 1 is that the isoelectric point method and the salting-out method are performed in step (3) in the same order, that is, the same salting-out as in example 1 is performed first, and then the isoelectric point treatment as in example 1 is performed.
The results of comparing the precipitation of proteins by the single isoelectric point method, the single salting-out method, the salting-out method and the isoelectric point synchronization method in comparative example 2 with the first isoelectric point and then salting-out method in example 1 are shown in Table 1.
Table 1: comparison of protein precipitation amounts by different precipitation methods
It can be seen that the highest quality of protein obtained by first isoelectric point and then salting out is 1.81 times higher than that obtained by single isoelectric point method, while the same precipitation amount is achieved by the scheme (d) of first salting out and then performing isoelectric point treatment and the scheme (a) of single salting out, i.e. no effect is achieved by performing isoelectric point treatment after salting out, while the quality of protein obtained in example 1 is 34.28% higher than those of the two schemes, and is 22.14% higher than that of the scheme (b) in which salting out and isoelectric point are performed simultaneously.
The yield of the protein powder obtained by freeze-drying the hazel mushroom protein extracted in example 1 was 19.9%.
Example 2
A preparation method of a mellea armillaria sporophore antioxidant peptide comprises the following steps:
(I) extracting proteins
(1) Cleaning and airing the hazel mushroom, crushing the hazel mushroom, sieving the hazel mushroom by a 60-mesh sieve, adding the hazel mushroom into distilled water according to the feed-liquid ratio of 1:45, uniformly mixing, adjusting the pH value to 10 by using 0.1mol/L NaOH, and carrying out water bath at 80 ℃ for 1.5 hours;
(2) filtering out residues after alkali extraction is finished, adding distilled water according to the material-liquid ratio of 1:45, uniformly mixing, adding 0.1mol/L NaOH to adjust the pH value to 9, and carrying out ultrasonic treatment for 14-16min at normal temperature by using 200W power;
(3) adjusting the pH of the extracted supernatant to 3.6 by using 0.1mol/L hydrochloric acid, standing for 12h at 4 ℃, then centrifuging for 20min at 10000rpm at the temperature to obtain protein 1, taking the supernatant after isoelectric point precipitation, adding a saturated ammonium sulfate solution to ensure that the saturation is 90%, standing for 12h at 4 ℃, and then centrifuging for 20min at 10000rpm at the temperature to obtain protein 2;
(4) mixing the protein 1 and the protein 2, and freeze-drying to obtain hazel mushroom protein powder;
preparation of (II) mellea armillaria anti-oxidation peptide
(1) Preparing 3% aqueous solution of the hazel mushroom protein powder, performing ultrasonic treatment at normal temperature with power of 200W for 15min, adding flavourzyme, adjusting pH to 7, and hydrolyzing at 41 ℃ for 2-3h, wherein the addition amount of the enzyme is 3.5% of the mass of the hazel mushroom protein powder;
(2) and (3) inactivating the hydrolysate in boiling water bath for 15min after the hydrolysis is finished, respectively carrying out ultrafiltration treatment on the hydrolysate with ultrafiltration membranes of 3kD, 5kD and 10kD after cooling to obtain polypeptide solutions with different molecular weights, and carrying out freeze drying to obtain the antioxidant peptides with different molecular weights.
Comparative example 3
In order to verify the superiority of the scheme of carrying out ultrasonic treatment and then adopting flavourzyme for further hydrolysis, alkaline protease, neutral protease and trypsin are respectively adopted after the ultrasonic treatment, the hazel mushroom protein powder is hydrolyzed under respective optimal conditions, and the optimal reaction conditions of various enzymes are specifically shown in table 2.
Table 2: optimal reaction conditions for hydrolyzing mellea armillaria sporophore protein by different enzymes
| Protease enzyme | pH | Reaction time (h) | Temperature (. degree.C.) | Amount of enzyme added (%) | Substrate concentration (%) |
| Trypsin | 8.0 | 2 | 37 | 3.5 | 4 |
| Flavourzyme protease | 7.0 | 3 | 41 | 3.5 | 4 |
| Alkaline protease | 9.0 | 3 | 50 | 3.5 | 4 |
| Neutral protease | 7.0 | 4 | 48 | 3.5 | 4 |
After ultrasonic treatment, trypsin hydrolysis is carried out for 2 hours, the degree of hydrolysis of the hazel mushroom protein is improved to the maximum extent from 5.94% to 7.06%; after ultrasonic treatment, when flavourzyme is adopted for hydrolysis for 3 hours, the degree of hydrolysis of the hazel mushroom protein is improved from 13.51% to 16.32%; after ultrasonic treatment, when alkaline protease is adopted for hydrolysis for 3 hours, the hydrolysis degree is improved from 14.92 percent to 15.47 percent; after ultrasonic treatment, when neutral protease is adopted for hydrolysis for 4 hours, the hydrolysis degree is improved from 9.65% to 10.31%; the hazel mushroom protein is hydrolyzed by adopting the combination of ultrasonic treatment and flavourzyme hydrolysis, and the ultrasonic treatment can obviously improve the hydrolysis degree.
The hydrolyzed polypeptide sample after the enzymatic hydrolysis is subjected to an antioxidant performance test, and the result is shown in fig. 3: the clearance rate of DPPH free radicals of the polypeptide sample prepared by combining ultrasonic treatment with neutral protease hydrolysis is the highest and is 87.49%, the clearance rate of hydroxyl free radicals and the total reducing force of the polypeptide sample prepared by combining ultrasonic auxiliary treatment with gustatory protease hydrolysis are the highest and are 86.12% and 0.72 respectively, the clearance rate of DPPH free radicals also reaches 79.61%, and the whole oxidation resistance of the polypeptide sample prepared by combining ultrasonic treatment with alkaline protease hydrolysis is the worst.
And for the effect of the ultrasonic treatment in the hydrolysis process, the oxidation resistance ratio of the polypeptide sample prepared by hydrolysis with the flavourzyme alone finds that: the polypeptide sample obtained by combining the hydrolysis of the flavourzyme after the ultrasonic treatment has a certain improvement on the polypeptide prepared by the hydrolysis of the flavourzyme, wherein the DPPH free radical scavenging capacity, the hydroxyl free radical scavenging capacity and the total reducing power of the polypeptide sample are all single, and the specific scheme is shown in figure 4. Therefore, the ultrasonic treatment not only increases the hydrolysis degree of the flavourzyme to protein during hydrolysis, but also improves the antioxidant activity of the hydrolysate.
The components of different molecular weights prepared in example 2 were: the fractions <3kD, 3-5kD, 5-10kD and >10kD are rich in polysaccharides with antioxidant activity, so that the prepared fractions possibly contain polysaccharide fractions to form polysaccharide protein, so that the polysaccharide protein has antioxidant activity, DPPH free radical scavenging, hydroxyl free radical scavenging and total reducing power measurement are respectively carried out as indexes, and the antioxidant active fractions are screened for further purification.
And (3) testing results: the antioxidant activity of the <3kD component showed a significant dose-dependent effect, which is presumed to play a major role. The DPPH radical clearance of the <3kD fraction reached a maximum of 86.37% when the concentration reached 10 mg/mL. The clearance of hydroxyl radicals of the <3kD component reached a maximum of 97.53% near the clearance of the positive control when the concentration reached 2.5 mg/mL. At 10mg/mL, the total reducing power of each component is comparable, with the total reducing power of the <3kD component reaching 0.71. Comprehensively considering, selecting the component with the molecular weight of less than 3kD for further test, wherein the component with the smaller molecular weight contains less polysaccharide, and the measured antioxidant activity can more accurately represent the antioxidant activity of the prepared protein peptide, as shown in fig. 5.
Example 3
Gel filtration chromatography:
taking a proper amount of gel Sephadex G-25, adding excessive distilled water at room temperature, soaking for 24h, continuously stirring until the gel is fully swelled, and pouring out upper-layer floating substances. The swollen gel is poured into the column along the inner wall of the column at one time, and bubbles and delamination cannot be generated. The bed was equilibrated with pure water until the baseline was stable. Antioxidant peptide with <3kD is prepared into a solution with the concentration of 30mg/mL, and the solution is filtered and sterilized by a sterile filter membrane and loaded with 2 mL. Eluting with pure water, controlling flow rate at 2-3mL/5min, measuring OD254nm value, collecting sample at elution peak to obtain component A1 and component A2, concentrating, and lyophilizing.
The antioxidant activity was measured. The results are shown in FIG. 6: both component a1 and component a2 had antioxidant activity and were dose dependent. At a concentration of 5mg/mL, the DPPH free radical clearance of the A2 fraction reached a maximum of 87.33%, approaching the clearance of the positive control. The hydroxyl radical clearance of the A2 component was the highest at a concentration of 5mg/mL, and was 100%. However, the total reducing power of the A1 component was higher than that of the A2 component. When the concentration is 5mg/mL, the total reducing power of the A1 component is the highest and is 0.57, and the total reducing power of the A2 component is also the highest and is 0.32. Therefore, antioxidant peptide components with different antioxidant activities are formed through further filtration chromatography, and a foundation is laid for further research and development of the mellea armillaria sporophore antioxidant peptide.
Claims (10)
1. A preparation method of a mellea armillaria sporophore antioxidant peptide is characterized by comprising the following steps: the method comprises the steps of sequentially carrying out protein extraction, precipitation, hydrolysis, separation and purification on the hazel mushroom serving as a raw material, wherein the protein extraction is carried out by adopting alkali extraction, then carrying out ultrasonic alkali extraction on residues obtained after the alkali extraction, and the precipitation is carried out by sequentially carrying out isoelectric point method and salting-out method, and finally drying to prepare the hazel mushroom protein powder.
2. The method for preparing the mellea armillaria sporophore antioxidant peptide according to claim 1, wherein the steps of: the alkali extraction is to crush the hazel mushroom, add the crushed hazel mushroom into distilled water, stir the solution evenly, add NaOH with the concentration of 0.1mol/L to adjust the pH of the solution to 10, perform water bath for 1.5h at the temperature of 80 ℃, and then perform centrifugal treatment.
3. The method for preparing the mellea armillaria sporophore antioxidant peptide as claimed in claim 1 or 2, wherein the steps of: the feed-liquid ratio of the hazel mushrooms to the distilled water is 1:45, the centrifugal rotation speed is 10000rpmm, and the centrifugal time is 20 min.
4. The method for preparing the mellea armillaria sporophore antioxidant peptide according to any one of claims 1 to 3, wherein the steps of: the ultrasonic alkali extraction is to add distilled water into residues after the alkali extraction, the material-liquid ratio is 1:45, the mixture is uniformly mixed, the pH value is adjusted to 9 by NaOH with the concentration of 0.1mol/L, and then ultrasonic treatment is carried out for 14-16min at room temperature with the power of 200W.
5. The method for preparing the mellea armillaria sporophore antioxidant peptide as claimed in any one of claims 1-4, wherein the steps of: the hydrolysis is to prepare protein peptide by freeze-drying the precipitated protein and then sequentially adopting ultrasonic treatment and flavourzyme hydrolysis.
6. The method for preparing the mellea armillaria sporophore antioxidant peptide according to claim 5, wherein the steps of: the ultrasonic treatment is to prepare the hazel mushroom protein powder into a solution with the mass concentration of 3-4%, and carry out ultrasonic treatment for 15min at room temperature by 200W.
7. The method for preparing the mellea armillaria sporophore antioxidant peptide as claimed in claim 5 or 6, wherein the steps of: the flavourzyme hydrolysis is to hydrolyze the solution after ultrasonic treatment by using flavourzyme, wherein the hydrolysis temperature is 38-42 ℃, and the hydrolysis time is 2-3 h.
8. The method for preparing the mellea armillaria sporophore antioxidant peptide as claimed in any one of claims 5-7, wherein the steps of: the hydrolysis pH is 7, and the using amount of the flavourzyme is 3.5% of the hazel mushroom protein powder by mass.
9. The preparation method of the mellea armillaria sporophore antioxidant peptide is characterized by comprising the following steps of:
(I) extracting proteins
(1) Cleaning and airing the hazel mushroom, crushing the hazel mushroom, sieving the hazel mushroom by a 60-mesh sieve, adding the hazel mushroom into distilled water according to the feed-liquid ratio of 1:45, uniformly mixing, adjusting the pH value to 10 by using 0.1mol/L NaOH, and carrying out water bath at 80 ℃ for 1.5 hours;
(2) filtering out residues after alkali extraction is finished, adding distilled water according to the material-liquid ratio of 1:45, uniformly mixing, adding 0.1mol/L NaOH to adjust the pH value to 9, and carrying out ultrasonic treatment for 14-16min at normal temperature by using 200W power;
(3) adjusting the pH of the extracted supernatant to 3.6-3.7 by using 0.1mol/L hydrochloric acid, standing for 12h at 4 ℃, then centrifuging at 10000rpm for 20min at the temperature to obtain protein 1, taking the supernatant after isoelectric point precipitation, adding a saturated ammonium sulfate solution to ensure that the saturation is 90%, standing for 12h at 4 ℃, and then centrifuging at 10000rpm for 20min at the temperature to obtain protein 2;
(4) mixing the protein 1 and the protein 2, and freeze-drying to obtain hazel mushroom protein powder;
preparation of (II) mellea armillaria anti-oxidation peptide
(1) Preparing 3-4% aqueous solution of Armillariella mellea protein powder, treating with 200W ultrasonic wave at normal temperature for 15min, adding flavourzyme, adjusting pH to 7, hydrolyzing at 38-42 deg.C for 2-3 hr, wherein the enzyme content is 4.5% of Armillariella mellea protein powder;
(2) and (3) inactivating the hydrolysate in boiling water bath for 15min after the hydrolysis is finished, respectively carrying out ultrafiltration treatment on the hydrolysate with ultrafiltration membranes of 3kD, 5kD and 10kD after cooling to obtain polypeptide liquids with different molecular weights, and carrying out freeze drying to obtain antioxidant peptides with different molecular weights.
10. The method for preparing the mellea armillaria sporophore antioxidant peptide according to claim 9, wherein the steps of: preparing the antioxidant peptide into a solution with the concentration of 30mg/L, performing filtration chromatography by using Sephadex (Sephadex G-25), eluting at the rate of 2-3mL/5min by using pure water, collecting a sample at an elution peak, dividing the sample into the antioxidant peptide 1 and the antioxidant peptide 2, and freeze-drying after concentration.
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