Gastrodia elata polypeptide extraction process

Abstract

The invention discloses a gastrodia elata polypeptide extraction process, and belongs to the technical field of polypeptide extraction. According to the method, gastrodia elata cell membranes are broken through acetone pretreatment, so that protein and lipid are dissociated, trimethylsilyl ether passivates active groups, immobilized enzyme decomposes cellulose and adipose tissues through normal-temperature enzymolysis, a protein stabilizer prevents oxidation and inhibits enzyme activity, differential pressure extraction is combined with a sodium pyrophosphate buffer solution, polypeptide is promoted to be dissolved out, and activity is maintained; the high extraction rate of the gastrodia elata polypeptide and low-active group destructiveness are realized. Reaction sites and binding force are increased by epoxy groups on the surface of an immobilized enzyme carrier prepared by modifying corn starch, enzyme molecules are stable and appropriate due to good biocompatibility, enzyme molecule conformation is optimized through covalent binding, a rigid structure is enhanced, enzyme activity is kept at a high level for a long time, continuous and efficient enzymolysis is performed in gastrodia elata polypeptide extraction, and the gastrodia elata polypeptide extraction efficiency is improved. Compared with the traditional free enzyme, the gastrodia elata polypeptide enzyme has the advantages that the enzyme use cost is reduced, the enzyme consumption and waste generation are reduced, and an efficient and economical scheme is provided for gastrodia elata polypeptide extraction.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide extraction, and in particular relates to a process for extracting gastrodia elata polypeptide. Background Art

[0002] As a traditional precious Chinese medicinal material, Gastrodia elata has a long history of application in the field of traditional Chinese medicine. In the context of modern medical research, people are increasingly exploring natural bioactive substances, hoping to find ingredients with unique pharmacological effects and high safety from traditional Chinese medicinal materials to meet the growing demand for health care. Due to its biopharmacological activity, Gastrodia elata polypeptides have shown research value in multiple potential medicinal value fields such as neuroprotection, anti-oxidation, anti-inflammation, and immunomodulation. For example, in terms of neuroprotection, studies have found that Gastrodia elata polypeptides may have potential preventive and therapeutic effects on some nervous system diseases such as Alzheimer's disease and Parkinson's disease by regulating the metabolism of nerve cells and inhibiting the apoptosis of nerve cells; in terms of anti-oxidation, it can remove free radicals in the body and reduce the damage of oxidative stress to cells, thereby helping to prevent the occurrence and development of a variety of chronic diseases; in terms of anti-inflammatory and immunomodulation, Gastrodia elata polypeptides may regulate the expression of inflammatory factors and the activity of immune cells, and have also become a hot topic in the treatment of autoimmune diseases. However, there is currently little research on the extraction method of Gastrodia elata polypeptides, and a new process for efficient extraction of Gastrodia elata polypeptides is urgently needed. Summary of the invention

[0003] In view of the above situation, in order to overcome the defects of the prior art, the present invention realizes the efficient and high-activity extraction of Gastrodia elata polypeptides by performing multi-step fine treatment of Gastrodia elata raw materials and independently developing a special immobilized enzyme system, and effectively protects the active groups of the protein during the extraction process. At the same time, the prepared fixed enzyme is reusable, which significantly reduces production costs and resource consumption, and provides a highly promising new process for the industrial production of Gastrodia elata polypeptides.

[0004] In order to achieve the above object, the following technical scheme is adopted: The present invention provides a process for extracting Gastrodia elata polypeptide, comprising the following steps:

[0005] (1) washing, cutting, and crushing the gastrodia elata, adding the mixture to acetone, rapidly stirring the mixture at 0° C. to 2° C., filtering the mixture, and washing the mixture with trimethylsilyl ether. Then, adding an immobilized enzyme and a protein stabilizer, and subjecting the mixture to enzymolysis treatment at 30° C. to 35° C. for 1 to 4 hours to obtain a pretreated product;

[0006] (2) adding the pretreated product to a sodium pyrophosphate buffer solution at pH = 7.6, extracting three times at a pressure difference of 0.5 MPa-60 MPa, filtering, and obtaining a Gastrodia elata polypeptide extract;

[0007] (3) adjusting the pH value of the Gastrodia elata polypeptide extract to 4.5, standing the extract until precipitation occurs, and freeze-drying the precipitation to obtain the Gastrodia elata polypeptide.

[0008] Furthermore, the immobilized enzyme is prepared by the following steps:

[0009] S1. Dissolve dry corn starch in ethyl acetate, add pyridine and stir to react, then add acryloyl chloride and stearyl chloride, stir and react at room temperature for 2 hours, after the reaction is completed, vacuum filter the product, wash with anhydrous ethanol, and dry in a vacuum oven at 40° C. to obtain esterified starch;

[0010] S2, adding esterified starch to deionized water, and stirring and gelatinizing at 80-90°C for 0.5h, after the starch gelatinization liquid becomes clear and transparent, lowering the reaction temperature to 70°C, slowly adding ethyl acrylate, reacting for 0.5h, then adding initiator KPS, continuing the reaction for 8h, adding allyl alcohol glycidyl ether by injection pump at a flow rate of 5mL / h, stirring and reacting for 3h after the addition is completed, breaking the emulsion by centrifugation, filtering, washing, and drying to obtain the modified esterified starch immobilized enzyme carrier;

[0011] S3. Immerse the modified esterified starch immobilized enzyme carrier in the auxiliary solution for 40-50 minutes, filter, dry the surface water, and then immerse it in the mixed enzyme solution. After stirring for 2-3 hours, refrigerate at 5°C for 12 hours, centrifuge to remove the solution, rinse with water, and freeze-dry to obtain the immobilized enzyme.

[0012] Furthermore, in the step (1), the mass ratio of Gastrodia elata, acetone, immobilized enzyme and protein stabilizer is 1:6-8:0.1-0.2:0.1-0.2.

[0013] Furthermore, in step (2), the mass ratio of the pretreated material to the sodium pyrophosphate buffer solution is 1:8-10.

[0014] Furthermore, the mixed enzyme solution is a mixed solution of papain, neutral protease and alkaline protease, wherein the mass fraction of papain is 1.0-2.5%, the mass fraction of neutral protease is 2.5-3%, and the mass fraction of alkaline protease is 0.5-1%.

[0015] Furthermore, the protein stabilizer is composed of high-ester pectin, sodium alginate and EDTA in a mass ratio of 2:1:1.

[0016] Furthermore, the auxiliary solution is a mixed solution of magnesium sulfate and calcium sulfate, wherein the concentration of magnesium sulfate is 0.5-1.0 mol / L, and the concentration of calcium sulfate is 0.07-0.15 mol / L.

[0017] Furthermore, in step S1, the mass ratio of corn starch, ethyl acetate, pyridine, acryloyl chloride and stearoyl chloride is 30-50:100-150:1-3:2-3:2-3.

[0018] Furthermore, in step S2, the mass ratio of esterified starch, deionized water, ethyl acrylate and allyl alcohol glycidyl ether is 4-6:25-35:8-12.

[0019] Furthermore, the amount of the initiator KPS added accounts for 0.5%-1% of the mass of the reaction system.

[0020] Furthermore, in step S3, the mass ratio of the modified esterified starch immobilized enzyme carrier, the auxiliary solution and the mixed enzyme solution is 1:5-6:8-10.

[0021] The beneficial effects of the present invention are:

[0022] (1) The present invention pre-treats Gastrodia elata with acetone, which can penetrate into the interior of Gastrodia elata to destroy the cell membrane and dissociate the binding of protein and lipid; then, it is treated with trimethylsilyl ether to passivate some of the active groups exposed by the protein, thereby reducing the risk of damage to the active groups during the extraction process; the Gastrodia elata is enzymatically hydrolyzed at room temperature using an immobilized enzyme, which can effectively decompose the cellulose and adipose tissue structure therein, thereby promoting the subsequent dissolution of polypeptides; the protein stabilizer can prevent polypeptide oxidation, inhibit the activity of proteases in Gastrodia elata, and ensure the activity of polypeptides; a differential pressure extraction method is used to promote the full dissolution of polypeptides in Gastrodia elata by means of differential pressure changes; the extraction conditions are mild, and the activity of polypeptides can be maintained; and the sodium pyrophosphate buffered extract can prevent metal ions from causing polypeptide denaturation, thereby ultimately achieving a high extraction rate of Gastrodia elata polypeptides and low destructiveness of active groups;

[0023] (2) The present invention prepares an immobilized enzyme carrier with epoxy groups on the surface by modifying corn starch. The abundant epoxy groups on the surface greatly increase the surface reaction sites, providing more possibilities and stronger binding force for the binding of enzyme molecules. The good biocompatibility enables the enzyme molecules to be in a relatively stable and suitable state in the carrier environment, effectively avoiding damage to the enzyme molecules caused by external extreme environmental factors, greatly prolonging the service life of the enzyme. Through its covalent binding mode with the enzyme, the conformation of the enzyme molecule is further optimized and stabilized, and the rigid structure is significantly enhanced, so that a high enzyme activity level can be maintained for a long time, so that the enzymatic hydrolysis effect can be continuously and efficiently exerted during the extraction of Gastrodia elata polypeptide. After multiple uses, the immobilized enzyme can still maintain a considerable proportion of activity. Compared with traditional free enzymes, the use cost of the enzyme is greatly reduced, and the consumption of the enzyme and the generation of waste are reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1The results of polypeptide extraction rate and reuse performance evaluation of various embodiments and comparative examples of the present invention are shown;

[0025] Figure 2 The enzyme activities of the enzymes of various embodiments and comparative examples of the present invention at different temperatures;

[0026] Figure 3 The following are the enzyme activities of the enzymes of various embodiments and comparative examples of the present invention at different pH values.

[0027] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0029] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.

[0030] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0031] Example 1

[0032] A gastrodia elata polypeptide extraction process comprises the following steps:

[0033] (1) cleaning, cutting, and crushing Gastrodia elata, adding the mixture to acetone, rapidly stirring the mixture at 2°C, filtering the mixture, and washing the mixture with trimethylsilyl ether, and then adding an immobilized enzyme and a protein stabilizer, and subjecting the mixture to enzymatic hydrolysis at 35°C for 4 hours to obtain a pretreated product; wherein the mass ratio of Gastrodia elata, acetone, immobilized enzyme, and protein stabilizer is 1:8:0.2:0.2;

[0034] (2) adding the pretreated product to a sodium pyrophosphate buffer solution at pH = 7.6, extracting three times at a pressure difference of 60 MPa, filtering, and obtaining a Gastrodia elata polypeptide extract; wherein the mass ratio of the pretreated product to the sodium pyrophosphate buffer solution is 1:10;

[0035] (3) adjusting the pH value of the Gastrodia elata polypeptide extract to 4.5, standing the extract until precipitation occurs, and freeze-drying the precipitation to obtain the Gastrodia elata polypeptide.

[0036] The immobilized enzyme is prepared by the following steps:

[0037] S1. Dissolve dry corn starch in ethyl acetate, add pyridine and stir to react, then add acryloyl chloride and stearoyl chloride, stir and react at room temperature for 2 hours, after the reaction, vacuum filter the product, wash with anhydrous ethanol, and dry in a vacuum oven at 40°C to obtain esterified starch; wherein the mass ratio of corn starch, ethyl acetate, pyridine, acryloyl chloride and stearoyl chloride is 50:150:3:3:3;

[0038] S2, adding esterified starch to deionized water, and stirring and gelatinizing at 90°C for 0.5h, after the starch gelatinization liquid becomes clear and transparent, lowering the reaction temperature to 70°C, slowly dropping ethyl acrylate, reacting for 0.5h, then adding initiator KPS, continuing the reaction for 8h, dropping allyl alcohol glycidyl ether with a syringe pump at a flow rate of 5mL / h, stirring and reacting for 3h after the addition is completed, after the reaction is completed, the emulsion is demulsified by centrifugation, filtered, washed, and dried to obtain a modified esterified starch immobilized enzyme carrier; wherein the mass ratio of esterified starch, deionized water, ethyl acrylate and allyl alcohol glycidyl ether is 6:35:12;

[0039] S3. Immerse the modified esterified starch immobilized enzyme carrier in the auxiliary solution for 50 minutes, filter, dry the surface moisture, and then immerse it in the mixed enzyme solution. After stirring for 3 hours, refrigerate it at 5°C for 12 hours, centrifuge to remove the solution, rinse with water, and freeze-dry to obtain the immobilized enzyme; the mass ratio of the modified esterified starch immobilized enzyme carrier, the auxiliary solution and the mixed enzyme solution is 1:6:10.

[0040] The mixed enzyme solution is a mixed solution of papain, neutral protease and alkaline protease, wherein the mass fraction of papain is 2.5%, the mass fraction of neutral protease is 3%, and the mass fraction of alkaline protease is 1%.

[0041] The protein stabilizer is composed of high-ester pectin, sodium alginate and EDTA in a mass ratio of 2:1:1.

[0042] The auxiliary solution is a mixed solution of magnesium sulfate and calcium sulfate, wherein the concentration of magnesium sulfate is 1.0 mol / L and the concentration of calcium sulfate is 0.15 mol / L.

[0043] The added amount of the initiator KPS accounts for 1% of the mass of the reaction system.

[0044] Example 2

[0045] A gastrodia elata polypeptide extraction process comprises the following steps:

[0046] (1) cleaning, cutting, and crushing Gastrodia elata, adding the mixture to acetone, rapidly stirring the mixture at 0°C, filtering the mixture, and washing the mixture with trimethylsilyl ether, and then adding an immobilized enzyme and a protein stabilizer, and subjecting the mixture to enzymolysis at 30°C for 1 hour to obtain a pretreated product; wherein the mass ratio of Gastrodia elata, acetone, immobilized enzyme, and protein stabilizer is 1:6:0.1:0.1;

[0047] (2) adding the pretreated product to a sodium pyrophosphate buffer solution at pH = 7.6, extracting three times at a pressure difference of 0.5 MPa, filtering, and obtaining a Gastrodia elata polypeptide extract; wherein the mass ratio of the pretreated product to the sodium pyrophosphate buffer solution is 1:8;

[0048] (3) adjusting the pH value of the Gastrodia elata polypeptide extract to 4.5, standing the extract until precipitation occurs, and freeze-drying the precipitation to obtain the Gastrodia elata polypeptide.

[0049] The immobilized enzyme is prepared by the following steps:

[0050] S1. Dissolve dry corn starch in ethyl acetate, add pyridine and stir to react, then add acryloyl chloride and stearoyl chloride, stir and react at room temperature for 2 hours, after the reaction, vacuum filter the product, wash with anhydrous ethanol, and dry in a vacuum oven at 40°C to obtain esterified starch; wherein the mass ratio of corn starch, ethyl acetate, pyridine, acryloyl chloride and stearoyl chloride is 30:100:1:2:2;

[0051] S2, adding esterified starch to deionized water, and stirring and gelatinizing at 80°C for 0.5h, after the starch gelatinization liquid becomes clear and transparent, lowering the reaction temperature to 70°C, slowly adding ethyl acrylate, reacting for 0.5h, then adding initiator KPS, continuing the reaction for 8h, using a syringe pump to add allyl alcohol glycidyl ether at a flow rate of 5mL / h, stirring and reacting for 3h after the addition is completed, after the reaction is completed, the emulsion is demulsified by centrifugation, filtered, washed, and dried to obtain a modified esterified starch immobilized enzyme carrier; wherein the mass ratio of esterified starch, deionized water, ethyl acrylate and allyl alcohol glycidyl ether is 4:25:8;

[0052] S3. Immerse the modified esterified starch immobilized enzyme carrier in the auxiliary solution for 40 minutes, filter, dry the surface moisture, and then immerse it in the mixed enzyme solution. After stirring for 2 hours, refrigerate it at 5°C for 12 hours, centrifuge to remove the solution, rinse with water, and freeze-dry to obtain the immobilized enzyme; the mass ratio of the modified esterified starch immobilized enzyme carrier, the auxiliary solution and the mixed enzyme solution is 1:5:8.

[0053] The mixed enzyme solution is a mixed solution of papain, neutral protease and alkaline protease, wherein the mass fraction of papain is 1.0%, the mass fraction of neutral protease is 2.5% and the mass fraction of alkaline protease is 0.5%.

[0054] The protein stabilizer is composed of high-ester pectin, sodium alginate and EDTA in a mass ratio of 2:1:1.

[0055] The auxiliary solution is a mixed solution of magnesium sulfate and calcium sulfate, wherein the concentration of magnesium sulfate is 0.5 mol / L and the concentration of calcium sulfate is 0.07 mol / L.

[0056] The added amount of the initiator KPS accounts for 0.5% of the mass of the reaction system.

[0057] Example 3

[0058] A gastrodia elata polypeptide extraction process comprises the following steps:

[0059] (1) cleaning, cutting, and crushing Gastrodia elata, adding the mixture to acetone, rapidly stirring the mixture at 1°C, filtering the mixture, and washing the mixture with trimethylsilyl ether, and then adding an immobilized enzyme and a protein stabilizer, and subjecting the mixture to enzymatic hydrolysis at 32°C for 2 hours to obtain a pretreated product; wherein the mass ratio of Gastrodia elata, acetone, immobilized enzyme, and protein stabilizer is 1:7:0.15:0.15;

[0060] (2) adding the pretreated product to a sodium pyrophosphate buffer solution at pH = 7.6, extracting three times at a pressure difference of 30 MPa, filtering, and obtaining a Gastrodia elata polypeptide extract; wherein the mass ratio of the pretreated product to the sodium pyrophosphate buffer solution is 1:9;

[0061] (3) adjusting the pH value of the Gastrodia elata polypeptide extract to 4.5, standing the extract until precipitation occurs, and freeze-drying the precipitation to obtain the Gastrodia elata polypeptide.

[0062] The immobilized enzyme is prepared by the following steps:

[0063] S1. Dissolve dry corn starch in ethyl acetate, add pyridine and stir to react, then add acryloyl chloride and stearoyl chloride, stir and react at room temperature for 2 hours, after the reaction, vacuum filter the product, wash with anhydrous ethanol, and dry in a vacuum oven at 40°C to obtain esterified starch; wherein the mass ratio of corn starch, ethyl acetate, pyridine, acryloyl chloride and stearoyl chloride is 40:120:2:2.5:2.5;

[0064] S2, adding esterified starch to deionized water, and stirring and gelatinizing at 85°C for 0.5h, after the starch gelatinization liquid becomes clear and transparent, lowering the reaction temperature to 70°C, slowly dropping ethyl acrylate, reacting for 0.5h, then adding initiator KPS, continuing the reaction for 8h, dropping allyl alcohol glycidyl ether with a syringe pump at a flow rate of 5mL / h, stirring and reacting for 3h after the addition is completed, after the reaction is completed, the emulsion is demulsified by centrifugation, filtered, washed, and dried to obtain a modified esterified starch immobilized enzyme carrier; wherein the mass ratio of esterified starch, deionized water, ethyl acrylate and allyl alcohol glycidyl ether is 5:30:10;

[0065] S3. Immerse the modified esterified starch immobilized enzyme carrier in the auxiliary solution for 45 minutes, filter, dry the surface moisture, and then immerse it in the mixed enzyme solution. After stirring for 2.5 hours, refrigerate it at 5°C for 12 hours, centrifuge to remove the solution, rinse with water, and freeze-dry to obtain the immobilized enzyme; the mass ratio of the modified esterified starch immobilized enzyme carrier, the auxiliary solution and the mixed enzyme solution is 1:5.5:9.

[0066] The mixed enzyme solution is a mixed solution of papain, neutral protease and alkaline protease, wherein the mass fraction of papain is 1.5%, the mass fraction of neutral protease is 2.8% and the mass fraction of alkaline protease is 0.8%.

[0067] The protein stabilizer is composed of high-ester pectin, sodium alginate and EDTA in a mass ratio of 2:1:1.

[0068] The auxiliary solution is a mixed solution of magnesium sulfate and calcium sulfate, wherein the concentration of magnesium sulfate is 0.8 mol / L and the concentration of calcium sulfate is 0.1 mol / L.

[0069] The added amount of the initiator KPS accounts for 0.8% of the mass of the reaction system.

[0070] Comparative Example 1

[0071] The difference between this comparative example and Example 3 is that corn starch is used instead of modified esterified starch to immobilize the enzyme carrier, and the rest is the same as Example 3.

[0072] Comparative Example 2

[0073] The difference between this comparative example and Example 3 is that the fixed enzyme is replaced by free enzyme with the same content as the fixed enzyme in Example 3, and the rest is the same as Example 3.

[0074] Results Analysis

[0075] Test Example 1

[0076] Evaluation of peptide extraction rate and reusability

[0077] The polypeptide extraction was repeated three times using the methods of Examples 1-3 and Comparative Examples 1-2. The polypeptide extraction rate of Gastrodia elata was calculated for each extraction. The polypeptide extraction rate of Gastrodia elata = the mass of extracted polypeptide / the mass of polypeptide in Gastrodia elata raw material × 100%. The results are shown in Figure 1 .

[0078] From the comparison between Example 1-3 and Comparative Example 1-2, Example 1-3 shows a higher extraction rate of Gastrodia elata polypeptide, which is due to the unique process adopted by the present invention. In the pretreatment stage, Gastrodia elata is treated with acetone. Acetone can penetrate into the interior of Gastrodia elata to destroy the cell membrane, so that the protein originally bound to lipid is dissociated, preparing for subsequent enzymatic hydrolysis and extraction. It is rinsed with trimethylsilyl ether to passivate some active groups exposed by the protein, reducing the risk of damage to the active groups in the subsequent extraction process. Self-made fixed enzymes are used for enzymatic hydrolysis. The immobilized enzyme carrier prepared by multi-step modification of corn starch is combined with the corresponding mixed enzyme solution, which can effectively decompose the cellulose and adipose tissue structure in Gastrodia elata and promote protein dissolution. In the subsequent extraction step, the differential pressure extraction method is adopted, and the pressure difference change is used to promote the full dissolution of the protein in Gastrodia elata, and the sodium pyrophosphate buffered extract can prevent metal ions from causing protein denaturation. These links cooperate with each other to jointly ensure a higher extraction rate.

[0079] In Comparative Example 1, corn starch was used instead of modified esterified starch as an immobilized enzyme carrier. However, due to the lack of many advantages of the modified immobilized enzyme carrier, such as abundant surface reaction sites, good biocompatibility, and optimization and stabilization of the enzyme molecular conformation, the enzymatic hydrolysis effect was poor, which in turn affected the extraction rate of the polypeptide, resulting in its extraction rate being lower than that of Examples 1-3.

[0080] Comparative Example 2 uses free enzyme instead of immobilized enzyme. The free enzyme lacks the protection and stabilization effect of the immobilized carrier in the reaction system and is easily affected by external environmental factors. The activity of the enzyme cannot be well maintained. It is not as effective as the fixed enzyme in decomposing the related tissue structure in Gastrodia elata and promoting protein dissolution. Therefore, the extraction rate is also lower than that of Examples 1-3.

[0081] And the immobilized enzyme prepared by the present invention has the remarkable advantage of reusability, the reason is that the surface of the immobilized enzyme carrier prepared by modifying corn starch has epoxy groups, these epoxy groups not only greatly increase the surface reaction sites, provide stronger binding force for the binding of enzyme molecules, but also make the enzyme molecules in a relatively stable and suitable state in the carrier environment, effectively avoid the damage to the enzyme molecules caused by external extreme environmental factors, greatly extend the service life of the enzyme, after multiple uses, the fixed enzyme can still maintain a considerable proportion of activity, so when the extraction of gastrodia polypeptide is repeated three times, it can still maintain a good extraction effect, reflecting good reusability.

[0082] Test Example 2

[0083] Evaluation of enzyme thermal stability and pH stability

[0084] The enzyme activities of the enzymes of Examples 1-3 and Comparative Examples 1-2 at different temperatures and pH values ​​were measured. Figure 2 and Figure 3 .

[0085] The immobilized enzymes in Examples 1-3 all exhibited high enzyme activity at different temperatures, especially reaching the highest at 40°C. This is because the immobilized enzymes are covalently bound to the modified esterified starch immobilized enzyme carrier, so that the conformation of the enzyme molecules is further optimized and stabilized, and the rigid structure is significantly enhanced. This stable structure enables the enzyme molecules to maintain a good activity state under temperature changes, and are not easily denatured and inactivated due to temperature increases.

[0086] Moreover, the immobilized enzymes of Examples 1-3 also have high enzyme activity under different pH conditions, with the highest activity at pH = 7. This is because the good biocompatibility of the immobilized enzyme carrier makes the microenvironment in which the enzyme molecules are located relatively stable, and has a certain buffering and tolerance capacity to pH changes, enabling it to effectively exert its enzymatic effect on Gastrodia elata within a suitable pH range and promote the extraction of polypeptides.

[0087] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0088] The present invention and its implementation methods are described above, which is not restrictive. The drawings are only one of the implementation methods of the present invention, and the actual application is not limited thereto. In short, if ordinary technicians in the field are inspired by it and design methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. A process for extracting Gastrodia elata polypeptide, characterized in that: The steps include: (1) washing, cutting, and crushing the gastrodia elata, adding the mixture to acetone, rapidly stirring the mixture at 0° C. to 2° C., filtering the mixture, and washing the mixture with trimethylsilyl ether. Then, adding an immobilized enzyme and a protein stabilizer, and subjecting the mixture to enzymolysis treatment at 30° C. to 35° C. for 1 to 4 hours to obtain a pretreated product; (2) adding the pretreated product to a sodium pyrophosphate buffer solution at pH = 7.6, extracting three times at a pressure difference of 0.5 MPa-60 MPa, filtering, and obtaining a Gastrodia elata polypeptide extract; (3) adjusting the pH value of the Gastrodia elata polypeptide extract to 4.5, standing until precipitation occurs, and freeze-drying the precipitation to obtain the Gastrodia elata polypeptide; The immobilized enzyme is prepared by the following steps: S1. Dissolve dry corn starch in ethyl acetate, add pyridine and stir to react, then add acryloyl chloride and stearyl chloride, stir and react at room temperature for 2 hours, after the reaction is completed, vacuum filter the product, wash with anhydrous ethanol, and dry in a vacuum oven at 40° C. to obtain esterified starch; S2, adding esterified starch to deionized water, and stirring and gelatinizing at 80-90°C for 0.5h, after the starch gelatinization liquid becomes clear and transparent, lowering the reaction temperature to 70°C, slowly adding ethyl acrylate, reacting for 0.5h, then adding initiator KPS, continuing the reaction for 8h, adding allyl alcohol glycidyl ether by injection pump at a flow rate of 5mL / h, stirring and reacting for 3h after the addition is completed, breaking the emulsion by centrifugation, filtering, washing, and drying to obtain the modified esterified starch immobilized enzyme carrier; S3. Immerse the modified esterified starch immobilized enzyme carrier in the auxiliary solution for 40-50 minutes, filter, dry the surface water, and then immerse it in the mixed enzyme solution. After stirring for 2-3 hours, refrigerate at 5°C for 12 hours, centrifuge to remove the solution, rinse with water, and freeze-dry to obtain the immobilized enzyme.

2. The process for extracting Gastrodia elata polypeptide according to claim 1, characterized in that: In the step (1), the mass ratio of Gastrodia elata, acetone, immobilized enzyme and protein stabilizer is 1:6-8:0.1-0.2:0.1-0.

2.

3. The process for extracting Gastrodia elata polypeptide according to claim 1, characterized in that: In the step (2), the mass ratio of the pretreated material to the sodium pyrophosphate buffer solution is 1:8-10.

4. The process for extracting Gastrodia elata polypeptide according to claim 1, characterized in that: The mixed enzyme solution is a mixed solution of papain, neutral protease and alkaline protease, wherein the mass fraction of papain is 1.0-2.5%, the mass fraction of neutral protease is 2.5-3%, and the mass fraction of alkaline protease is 0.5-1%.

5. The process for extracting Gastrodia elata polypeptide according to claim 1, characterized in that: The protein stabilizer is composed of high-ester pectin, sodium alginate and EDTA in a mass ratio of 2:1:

1.

6. The process for extracting Gastrodia elata polypeptide according to claim 1, characterized in that: The auxiliary solution is a mixed solution of magnesium sulfate and calcium sulfate, wherein the concentration of magnesium sulfate is 0.5-1.0 mol / L, and the concentration of calcium sulfate is 0.07-0.15 mol / L.

7. The process for extracting Gastrodia elata polypeptide according to claim 1, characterized in that: In the step S1, the mass ratio of corn starch, ethyl acetate, pyridine, acryloyl chloride and stearoyl chloride is 30-50:100-150:1-3:2-3:2-3.

8. The process for extracting Gastrodia elata polypeptide according to claim 1, characterized in that: In the step S2, the mass ratio of esterified starch, deionized water, ethyl acrylate and allyl alcohol glycidyl ether is 4-6:25-35:8-12.

9. The process for extracting Gastrodia elata polypeptide according to claim 1, characterized in that: The added amount of the initiator KPS accounts for 0.5%-1% of the mass of the reaction system.

10. The process for extracting Gastrodia elata polypeptide according to claim 1, characterized in that: In the step S3, the mass ratio of the modified esterified starch immobilized enzyme carrier, the auxiliary solution and the mixed enzyme solution is 1:5-6:8-10.

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