CN114717286B - Preparation method of hazel mushroom antioxidant peptide - Google Patents
Preparation method of hazel mushroom antioxidant peptide Download PDFInfo
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- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K38/00—Medicinal preparations containing peptides
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Abstract
The preparation method of the hazel mushroom antioxidant peptide comprises the steps of sequentially extracting, precipitating, hydrolyzing, separating and purifying proteins by taking the hazel mushrooms as raw materials, wherein the protein extraction is carried out by firstly adopting alkali extraction, then carrying out ultrasonic alkali extraction on residues after alkali extraction, the precipitation is carried out by sequentially carrying out isoelectric point method and salting-out method, and finally drying to obtain the hazel mushroom protein powder. According to the invention, through the method of firstly carrying out alkali extraction on hazel mushrooms and then carrying out ultrasonic alkali extraction on protein, the extraction rate of the hazel mushrooms protein is effectively improved to 76.59%, the isoelectric point and salting-out method are combined in sequence in the precipitation process, the precipitation amount of the hazel mushrooms protein is effectively promoted to 66.2ug/mL, and the clearance of the prepared antioxidant polypeptide to DPPH free radicals is 86.37%. The clearance rate of the hydroxyl radical reaches 97.53 percent and approaches to that of a positive control. The total reducing power was equivalent to 0.71.
Description
Technical Field
The invention relates to the technical field of antioxidation peptide preparation, in particular to a preparation method of hazel mushroom antioxidation peptide.
Background
Oxidative damage of the body by oxidative radicals is one of the main causes of human diseases, including aging, coronary heart disease, inflammation, stroke, diabetes, cancer, etc., which pose a great threat to human health. The development of antioxidants is therefore of great importance for the treatment and prevention of these diseases.
Antioxidant peptide is favored by researchers as a safe and nontoxic natural antioxidant, and is one of the most popular research subjects in the international food industry and a functional factor with great development prospect. At present, the preparation of antioxidant peptides mainly adopts protease to hydrolyze peptide bonds in proteins, so that peptide substances with antioxidant activity are produced. However, at present, the antioxidant peptides are mainly derived from animal proteins and plant 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 hazel mushroom belongs to fungi, is a very important fungus used as both medicine and food special for Changbai mountain area, contains various amino acids, microelements and vitamins necessary for human body, and most obviously has protein content of more than 20% and is mostly high-quality protein. Modern pharmacological researches have shown that hazel mushroom polysaccharide has the effects of resisting radiation, promoting hematopoiesis, inhibiting tumor growth, regulating immunity, treating rickets, reducing cholesterol, preventing and treating arteriosclerosis, etc. Therefore, scholars at home and abroad mostly study the oxidation resistance of the hazel mushroom polysaccharide, and prove that the hazel mushroom polysaccharide has certain oxidation resistance. However, there are no reports on extraction and further development and utilization of hazel mushroom proteins for preparing antioxidant peptides, and thus, it is not known how to obtain antioxidant activity of the proteins in hazel mushrooms. Aiming at the hazel mushroom, how to extract protein with high efficiency, how to remarkably improve the antioxidant activity of the extracted protein in the process of preparing antioxidant peptide is to be studied.
Disclosure of Invention
The invention aims to provide a preparation method of hazel mushroom antioxidant peptide. The extraction rate of protein in the hazel mushrooms is effectively improved, and the prepared peptide has excellent oxidation resistance.
The invention aims at realizing the following technical scheme:
A preparation method of hazel mushroom antioxidant peptide is characterized by comprising the following steps: the preparation method comprises the steps of sequentially extracting, precipitating, hydrolyzing, separating and purifying the protein by taking hazel mushrooms as raw materials, wherein the protein is extracted by adopting alkali extraction firstly, then carrying out ultrasonic alkali extraction on residues after alkali extraction, the precipitation is sequentially carried out by an isoelectric point method and a salting-out method, and finally drying to prepare the hazel mushrooms protein powder.
Further, the alkali extraction is to crush hazel mushroom, add the crushed hazel mushroom into distilled water, stir the crushed hazel mushroom evenly, add NaOH with the concentration of 0.1mol/L to adjust the pH of the solution to 10, carry out water bath for 1.5 hours at the temperature of 75-85 ℃ and then carry out centrifugal treatment.
The ratio of the hazel mushroom to distilled water is 1:45, the centrifugal speed is 10000rpmm, and the centrifugal time is 20min.
Further, the ultrasonic alkaline extraction is that the residue after alkaline extraction is added into distilled water, the ratio of the feed to the liquid is 1:45, after uniform mixing, naOH with the concentration of 0.1mol/L is used for adjusting the pH value to 9, and then ultrasonic treatment is carried out for 14-16min at room temperature with the power of 200W.
Further, the hydrolysis is to freeze-dry the precipitated protein and then sequentially hydrolyze the protein by ultrasonic treatment and flavourzyme to prepare the protein peptide.
Further, the ultrasonic treatment is to prepare hazel mushroom protein powder into a solution with the mass concentration of 3-4%, and the ultrasonic treatment is carried out for 15min at room temperature by adopting 200W.
Further, the flavourzyme hydrolysis is to hydrolyze the solution after ultrasonic treatment by flavourzyme, the hydrolysis temperature is 38-42 ℃ and the hydrolysis time is 2-3h.
In the enzymolysis process, if the hazel mushroom is improperly prepared, the proteolysis degree of the hazel mushroom is low, and the oxidation resistance of the prepared peptide is damaged, so that the hazel mushroom oxidation-resistant peptide has poor scavenging ability on DPPH free radicals, hydroxyl free radicals and the like. Therefore, the invention not only increases the hydrolysis degree of the flavor protease by carrying out ultrasonic treatment firstly and then carrying out hydrolysis of the flavor protease, but also further improves the antioxidant activity of the prepared peptide on the basis of the enzymolysis of the flavor protease, and the finally prepared peptide has excellent antioxidant activity by the synergistic effect of ultrasonic treatment and the hydrolysis of the flavor protease.
Further, the hydrolysis pH is 7, and the amount of the flavourzyme is 3.5% of the mass of the hazel mushroom protein powder.
And further, the separation and purification are to sequentially ultrafiltrate the protein peptide to obtain antioxidant peptides with different molecular weights, and then further purify by gel filtration chromatography to obtain antioxidant peptide 1 and antioxidant peptide 2.
The preparation method of the hazel mushroom antioxidant peptide is characterized by comprising the following steps of:
(one) protein extraction
(1) Washing, airing and crushing hazel mushrooms, sieving with a 60-mesh sieve, adding the crushed hazel mushrooms into distilled water according to a feed-liquid ratio of 1:45, uniformly mixing, adjusting the pH to 10 by using 0.1mol/L NaOH, and carrying out water bath at 80 ℃ for 1.5 hours;
(2) Filtering out residues after the alkali extraction is finished, adding distilled water according to the feed-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) Regulating pH of the supernatant after extraction to 3.6-3.7 with 0.1mol/L hydrochloric acid, standing at 4deg.C for 12 hr, centrifuging at 10000rpm for 20min at the temperature to obtain protein 1, collecting supernatant after isoelectric precipitation, adding saturated ammonium sulfate solution to make saturation 90%, standing at 4deg.C for 12 hr, and 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 hazel mushroom antioxidant peptide
(1) Preparing hazel mushroom protein powder into an aqueous solution with the mass concentration of 3-4%, performing ultrasonic treatment at normal temperature for 15min with the power of 200W, then adding flavourzyme, adjusting the pH to 7, and hydrolyzing at 38-42 ℃ for 2-3h, wherein the addition amount of the enzyme is 3.5% of the mass of the hazel mushroom protein powder;
(2) And after hydrolysis, inactivating the hydrolysis liquid in a boiling water bath for 15min, cooling, performing ultrafiltration treatment on the hydrolysis liquid by using ultrafiltration membranes of 3kD, 5kD and 10kD respectively to obtain polypeptide liquids with different molecular weights, and performing freeze drying to obtain antioxidant peptides with different molecular weights.
Further, the antioxidant peptide is prepared into a solution with the concentration of 30mg/L, the solution is subjected to filtration chromatography by using Sephadex (Sephadex G-25), then the solution is eluted by pure water at the rate of 2-3mL/5min, and the sample at the eluting peak is collected and separated into a component A1 and a component A2, and the solution is concentrated and freeze-dried.
The invention has the following technical effects:
according to the invention, through the protein extraction mode of carrying out alkali extraction on hazel mushrooms and then ultrasonic alkali extraction, the extraction rate of the hazel mushrooms protein is effectively improved to 76.59%, the isoelectric point and the salting-out method are combined in sequence in the precipitation process, the precipitation amount of the hazel mushrooms protein is effectively promoted to 66.2ug/mL, and the clearance of the prepared antioxidant polypeptide to DPPH free radicals is 86.37%. The clearance rate of the hydroxyl radical reaches 97.53 percent and approaches to that of a positive control. The total reducing power was equivalent to 0.71.
Drawings
Fig. 1: comparison of protein extraction rates from different methods.
Fig. 2: and (3) an absorbance value change curve of precipitation of hazel mushroom protein by an isoelectric point method under different pH values and a salting-out method under different saturation levels of ammonium sulfate solutions.
Fig. 3: ultrasound assisted effect of different protease hydrolysis on antioxidant activity of the prepared peptides.
Fig. 4: comparison of antioxidant activity of samples after ultrasonic assisted flavor protease and single flavor protease hydrolyzed hazel mushroom protein.
Fig. 5: antioxidant activity assay curves for each of the different molecular weight fractions after ultrafiltration.
Fig. 6: sephadex G-25 column chromatography and determination of antioxidant activity of each component.
Detailed Description
The present invention is described in detail below by way of examples, which are necessary to be pointed out herein for further illustration of the invention and are not to be construed as limiting the scope of the invention, since numerous insubstantial modifications and adaptations of the invention will be to those skilled in the art in light of the foregoing disclosure.
Example 1
A preparation method of hazel mushroom antioxidant peptide comprises the following steps of extracting protein:
(1) Washing, airing and crushing hazel mushrooms, sieving with a 60-mesh sieve, adding the crushed hazel mushrooms into distilled water according to a feed-liquid ratio of 1:45, uniformly mixing, adjusting the pH to 10 by using 0.1mol/L NaOH, and carrying out water bath at 80 ℃ for 1.5 hours;
(2) Filtering out residues after the alkali extraction is finished, adding distilled water according to the feed-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) Regulating pH of the supernatant after extraction to 3.7 with 0.1mol/L hydrochloric acid, standing at 4deg.C for 12 hr, centrifuging at 10000rpm for 20min at the temperature to obtain protein 1, collecting supernatant after isoelectric precipitation, adding saturated ammonium sulfate solution to make saturation 90%, standing at 4deg.C for 12 hr, and 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;
Comparative example 1
Scheme (a): unlike example 1, no separate alkaline extraction was performed in this scheme, but only a separate ultrasonic alkaline extraction was performed, and the rest of the procedure was the same as example 1.
Scheme (b): unlike example 1, only the same alkaline extraction treatment was performed during the alkaline extraction without further ultrasonic alkaline extraction, and the remaining steps were the same as example 1.
Scheme (c): the extraction sequence of the alkaline extraction and the ultrasonic alkaline extraction is exchanged, namely the ultrasonic alkaline extraction is carried out first, then the residue after the ultrasonic alkaline extraction is added into distilled water for alkaline extraction, and the rest steps are the same as those of the example 1.
By comparing the protein extraction schemes in comparative example 1 with the protein extraction scheme in example 1, the results are shown in fig. 1, the ultrasonic alkali extraction has the lowest protein extraction rate of 23.33%, the single alkali extraction method is 43.28%, the protein extraction rate of the alkali extraction scheme after ultrasonic alkali extraction is 66.72%, and the protein extraction rate of the ultrasonic alkali extraction is 76.59%. Therefore, the alkaline extraction followed by the ultrasonic method is selected to further optimize the protein extraction rate.
The proposal of alkali extraction and then ultrasonic alkali extraction with excellent protein extraction rate in the invention can not achieve good extraction effect when applied to other mushrooms such as tricholoma matsutake, but can achieve higher protein extraction efficiency when the protein extraction of the tricholoma matsutake is performed firstly and then the step of alkali extraction is performed.
In order to compare the effect of isoelectric point treatment and then salting-out on the protein yield of hazel mushrooms in the present invention, we conducted a corresponding comparative experiment.
Comparative example 2
Scheme (a): in comparison with example 1, the difference is that the isoelectric point treatment was not performed in step (3), and the protein precipitation was performed only by the salting-out method.
Scheme (b): the difference from example 1 is that in step (3), only isoelectric point treatment is performed for protein precipitation, and no other means are used for subsequent precipitation.
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 operation sequence of the isoelectric point method and the salting-out method in step (3) is changed, that is, the same salting-out as in example 1 is performed first, and then the same isoelectric point treatment as in example 1 is performed.
The results of comparing the isoelectric point-first salting-out and isoelectric point-later-salting-out method with the isoelectric point-first-salting-out method of example 1 by the isoelectric point-first-salting-out method, the salting-out method and the isoelectric point-synchronous method of comparative example 2 are shown in Table 1.
Table 1: comparison of protein precipitation amount by different precipitation methods
It is seen that the protein obtained by isoelectric point first and then salting out was highest in quality by 1.81 times as compared with the isoelectric point alone, and the precipitation amount achieved by the isoelectric point treatment scheme (d) and the isoelectric point treatment scheme (a) by salting out first and then salting out alone was the same, i.e., the isoelectric point treatment after salting out did not exert any effect, whereas the protein obtained in example 1 was 34.28% higher in quality than the two schemes and 22.14% higher in quality than the scheme (b) in which salting out and isoelectric point were performed simultaneously.
The yield of the protein powder obtained after freeze-drying of the hazel mushroom protein extracted in example 1 was 19.9%.
Example 2
A preparation method of hazel mushroom antioxidant peptide comprises the following steps:
(one) protein extraction
(1) Washing, airing and crushing hazel mushrooms, sieving with a 60-mesh sieve, adding the crushed hazel mushrooms into distilled water according to a feed-liquid ratio of 1:45, uniformly mixing, adjusting the pH to 10 by using 0.1mol/L NaOH, and carrying out water bath at 80 ℃ for 1.5 hours;
(2) Filtering out residues after the alkali extraction is finished, adding distilled water according to the feed-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) Regulating pH of the supernatant after extraction to 3.6 with 0.1mol/L hydrochloric acid, standing at 4deg.C for 12 hr, centrifuging at 10000rpm for 20min at the temperature to obtain protein 1, collecting supernatant after isoelectric precipitation, adding saturated ammonium sulfate solution to make saturation 90%, standing at 4deg.C for 12 hr, and 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 hazel mushroom antioxidant peptide
(1) Preparing hazel mushroom protein powder into an aqueous solution with the mass concentration of 3%, performing ultrasonic treatment at normal temperature for 15min with the power of 200W, then adding flavourzyme, adjusting the 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 after hydrolysis, inactivating the hydrolysis liquid in a boiling water bath for 15min, cooling, performing ultrafiltration treatment on the hydrolysis liquid by using ultrafiltration membranes of 3kD, 5kD and 10kD respectively to obtain polypeptide liquids with different molecular weights, and performing freeze drying to obtain antioxidant peptides with different molecular weights.
Comparative example 3
In order to verify the superiority of the scheme of the invention that ultrasonic treatment is firstly carried out, then the flavourzyme is adopted for further hydrolysis, then alkaline protease, neutral protease and trypsin are respectively adopted for carrying out ultrasonic treatment, and the hazel mushroom protein powder is hydrolyzed under the respective optimal conditions, and the optimal reaction conditions of the enzymes are shown in the table 2.
Table 2: optimal reaction conditions for hydrolyzing hazel mushroom protein by different enzymes
| Protease enzyme | pH | Reaction time (h) | Temperature (. Degree. C.) | Enzyme addition amount (%) | Substrate concentration (%) |
| Trypsin, trypsin and its preparation method | 8.0 | 2 | 37 | 3.5 | 4 |
| Flavoured 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 |
When trypsin hydrolysis is carried out for 2 hours after ultrasonic treatment, the degree of hydrolysis of the hazel mushroom protein is improved to the greatest extent and is improved to 7.06% from 5.94%; when the ultrasonic treatment is carried out for 3 hours by adopting flavourzyme for hydrolysis, the hydrolysis degree of the hazel mushroom protein is improved from 13.51% to 16.32%; when alkaline protease is adopted for hydrolysis for 3 hours after ultrasonic treatment, the hydrolysis degree is improved from 14.92% to 15.47%; when neutral protease is adopted for hydrolysis for 4 hours after ultrasonic treatment, the hydrolysis degree is improved from 9.65 percent to 10.31 percent; the hazel mushroom protein is hydrolyzed by adopting the combination of ultrasonic treatment and flavor protease hydrolysis, and the improvement of the degree of hydrolysis by ultrasonic treatment is more remarkable.
The antioxidant property of the hydrolyzed polypeptide sample after the completion of the enzymatic hydrolysis is tested, and the result is shown in fig. 3: the clearance rate of the polypeptide sample subjected to ultrasonic treatment combined with neutral protease hydrolysis on DPPH free radicals is 87.49%, the clearance rate of hydroxyl free radicals and the total reducing force are both highest when the polypeptide sample is subjected to ultrasonic auxiliary treatment combined with the protease hydrolysis and are 86.12% and 0.72 respectively, the clearance rate of the polypeptide sample subjected to ultrasonic treatment combined with neutral protease hydrolysis also reaches 79.61%, and the integral oxidation resistance of the polypeptide sample prepared by ultrasonic treatment combined with alkaline protease hydrolysis is worst.
Whereas for the effect of sonication on the hydrolysis process, the antioxidant comparison of the polypeptide samples prepared by hydrolysis with flavourzyme alone found: the polypeptide sample obtained by combining the ultrasonic treatment and the hydrolysis of the flavourzyme has a certain improvement on the DPPH free radical scavenging capacity, the hydroxyl free radical scavenging capacity and the total reducing power compared with the polypeptide prepared by the hydrolysis of the single flavourzyme, and is particularly shown in figure 4. It can be seen that the ultrasonic treatment increases the degree of hydrolysis of the protein by the flavourzyme and also increases the antioxidant activity of the hydrolysate during hydrolysis.
The components of different molecular weights prepared in example 2 were respectively: the components <3kD,3-5kD,5-10kD and >10kD are rich in polysaccharides with antioxidant activity, and the prepared components possibly contain polysaccharide components to form polysaccharide proteins, so that the polysaccharide proteins have antioxidant activity, DPPH free radical scavenging is respectively carried out, hydroxyl free radical scavenging and total reducing power are measured as indexes, and the antioxidant active components are screened for further purification.
Test results: the antioxidant activity of the <3kD component clearly exhibits a dose-dependent effect, presumably contributing mainly. The DPPH radical scavenging rate of the <3kD fraction reached a maximum of 86.37% when the concentration reached 10 mg/mL. When the concentration reaches 2.5mg/mL, the hydroxyl radical clearance of the <3kD component reaches the highest, 97.53%, approaching that of the positive control. At 10mg/mL, the total reducing power of each component was comparable, with the total reducing power of the <3kD component reaching 0.71. In comprehensive consideration, the components with the molecular weight of less than 3kD are selected for further experiments, and the components with the molecular weight of more than 3kD are selected, so that the components with the smaller molecular weight contain less polysaccharide, and the measured antioxidant activity can more accurately represent the antioxidant activity of the prepared protein peptide, and the antioxidant activity is shown in a figure 5.
Example 3
Gel filtration chromatography:
Taking a proper amount of gel Sephadex G-25, adding excessive distilled water at room temperature for soaking for 24 hours, continuously stirring until the gel is fully swelled, and pouring out the upper layer floating matters. The well-expanded 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 stationary. The antioxidant peptide of <3kD was prepared as a solution at a concentration of 30mg/mL, sterilized by filtration through a sterile filter, and loaded with 2mL. Eluting with pure water, controlling flow rate at 2-3mL/5min, measuring OD254nm value, collecting sample at eluting 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 have antioxidant activity and are dose dependent. The DPPH radical clearance of the A2 component reaches the highest at the concentration of 5mg/mL, is 87.33 percent and approaches to the clearance of the positive control. At a concentration of 5mg/mL, the hydroxyl radical scavenging rate of the A2 component reached a maximum of 100%. But the total reduction of the A1 component is higher than that of the A2 component. When the concentration was 5mg/mL, the total reducing power of the A1 component was 0.57 at the highest, and at this time, the total reducing power of A2 was 0.32 at the highest. Therefore, the antioxidant peptide components with different antioxidant activities are formed through further filtration chromatography, which lays a foundation for the subsequent further research and development of hazel mushroom antioxidant peptide.
Claims (9)
1. A preparation method of hazel mushroom antioxidant peptide is characterized by comprising the following steps: the preparation method comprises the steps of sequentially extracting, precipitating, hydrolyzing and separating and purifying the protein by taking hazel mushrooms as raw materials, wherein the protein is extracted by adopting alkali, then carrying out ultrasonic alkali extraction on residues after alkali extraction, the precipitation is carried out by sequentially carrying out isoelectric point method and salting-out method for precipitation, finally drying to obtain hazel mushrooms protein powder, and the hydrolysis is carried out by freeze-drying the precipitated protein, preparing a solution, and sequentially carrying out ultrasonic treatment and flavor protease hydrolysis to prepare the protein peptide.
2. The method for preparing the hazel mushroom antioxidant peptide according to claim 1, wherein the method comprises the steps of: the alkali extraction is to crush hazel mushroom, add the crushed hazel mushroom into distilled water, stir the distilled water uniformly, add NaOH with the concentration of 0.1mol/L to adjust the pH of the solution to 10, carry out water bath for 1.5h at 80 ℃, and then carry out centrifugal treatment.
3. The method for preparing the hazel mushroom antioxidant peptide according to claim 2, wherein: the feed liquid ratio of the hazel mushrooms to the distilled water is 1:45, the centrifugal speed is 10000rpm, and the centrifugal time is 20min.
4. A method for preparing an antioxidant peptide of hazel mushroom according to any one of claims 1 to 3, wherein: the ultrasonic alkaline extraction is to add the residue after alkaline extraction into distilled water, the feed-liquid ratio is 1:45, adjust the pH to 9 by NaOH with the concentration of 0.1mol/L after uniform mixing, and then carry out ultrasonic treatment for 14-16min at room temperature with the power of 200W.
5. The method for preparing the hazel mushroom antioxidant peptide according to claim 4, wherein the method comprises the steps of: the ultrasonic treatment is to prepare 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 adopting 200W.
6. The method for preparing the hazel mushroom antioxidant peptide according to claim 5, wherein the method comprises the steps of: the flavourzyme hydrolysis is to hydrolyze the solution after ultrasonic treatment by flavourzyme, the hydrolysis temperature is 38-42 ℃ and the hydrolysis time is 2-3h.
7. The method for preparing the hazel mushroom antioxidant peptide according to claim 6, wherein the method comprises the steps of: the hydrolysis pH is 7, and the amount of the flavourzyme is 3.5% of the mass of the hazel mushroom protein powder.
8. The preparation method of the hazel mushroom antioxidant peptide is characterized by comprising the following steps of:
(one) protein extraction
(1) Washing, airing and crushing hazel mushrooms, sieving with a 60-mesh sieve, adding the crushed hazel mushrooms into distilled water according to a feed-liquid ratio of 1:45, uniformly mixing, adjusting the pH to 10 by using 0.1mol/L NaOH, and carrying out water bath at 80 ℃ for 1.5 hours;
(2) Filtering out residues after the alkali extraction is finished, adding distilled water according to the feed-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) Regulating pH of the supernatant after extraction to 3.6-3.7 with 0.1mol/L hydrochloric acid, standing at 4deg.C for 12 hr, centrifuging at 10000rpm for 20min at the temperature to obtain protein 1, collecting supernatant after isoelectric precipitation, adding saturated ammonium sulfate solution to make saturation 90%, standing at 4deg.C for 12 hr, and 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 hazel mushroom antioxidant peptide
(1) Preparing hazel mushroom protein powder into an aqueous solution with the mass concentration of 3-4%, performing ultrasonic treatment at normal temperature for 15min with the power of 200W, then adding flavourzyme, adjusting the pH to 7, and hydrolyzing at 38-42 ℃ for 2-3h, wherein the addition amount of the enzyme is 4.5% of the mass of the hazel mushroom protein powder;
(2) And after hydrolysis, inactivating the hydrolysis liquid in a boiling water bath for 15min, cooling, performing ultrafiltration treatment on the hydrolysis liquid by using ultrafiltration membranes of 3kD, 5kD and 10kD respectively to obtain polypeptide liquids with different molecular weights, and performing freeze drying to obtain antioxidant peptides with different molecular weights.
9. The method for preparing the hazel mushroom antioxidant peptide according to claim 8, wherein: preparing the antioxidant peptide into a solution with the concentration of 30mg/L, filtering and chromatography by adopting sephadex Sephadex G-25, eluting by using pure water at the rate of 2-3mL/5min, collecting a sample at the eluting peak, separating the sample into antioxidant peptide 1 and antioxidant peptide 2, concentrating and freeze-drying.
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