阅读说明：本技术 一种柞蚕蛹虫草生物活性肽的制备方法 (Preparation method of tussah pupa cordyceps militaris bioactive peptide ) 是由 王美景 柳叶飞 王升厚 徐方旭 王泽� 赵晓云 王萍萍 辛意贝 余柳柳 于 2020-06-22 设计创作，主要内容包括：本发明公开了一种柞蚕蛹虫草生物活性肽的制备方法,该方法首次采用最为接近天然冬虫夏草的柞蚕蛹虫草为原料,以保证制得小分子生物活性肽的丰富度、含量和抗氧化活性；同时配合采用双蛋白酶联合酶解法,使酶解更彻底,小分子肽比例提高,活性更强；而且双蛋白酶联合酶解法中采用的是食品级碱性蛋白酶和胰蛋白酶,大大降低了活性肽的制备成本；整个活性肽的制备全程是在55℃以下,没有高温环节,因此,制得的活性肽抗氧化能力和清除超氧自由基的能力强,具有易溶解、耐酸、耐热、稳定性高的优点；此外,制备获得的活性肽经高效液相色谱法分离后,图谱清晰、物质分离度高、分散度好,达到优质水平。(The invention discloses a preparation method of biological active peptide of tussah silkworm chrysalis cordyceps, which adopts the tussah silkworm chrysalis cordyceps closest to natural cordyceps as a raw material for the first time to ensure the richness, content and antioxidant activity of the prepared small molecular biological active peptide; meanwhile, a double-protease combined enzymolysis method is adopted, so that enzymolysis is more thorough, the proportion of small molecular peptides is improved, and the activity is stronger; in addition, food-grade alkaline protease and trypsin are adopted in the double-protease combined enzymolysis method, so that the preparation cost of the active peptide is greatly reduced; the whole preparation process of the active peptide is below 55 ℃, and no high-temperature link exists, so that the prepared active peptide has strong oxidation resistance and superoxide radical removal capacity, and has the advantages of easy dissolution, acid resistance, heat resistance and high stability; in addition, after the prepared active peptide is separated by the high performance liquid chromatography, the spectrum is clear, the substance separation degree is high, the dispersion degree is good, and the high quality level is achieved.)

1. A preparation method of the biological active peptide of the tussah pupa cordyceps sinensis is characterized by comprising the following steps:

1) inoculating a functional cordyceps militaris 'Sugao No. 1' liquid strain by taking a living tussah pupa as a culture medium, and obtaining the cordyceps militaris with complete stroma through conventional culture;

2) freeze-drying the tussah cordyceps militaris stroma in the step 1), and then crushing to obtain dry powder;

3) adding deionized water into the dry powder obtained in the step 2), and adding two proteases, namely alkaline protease and trypsin, for enzymolysis to obtain an enzymolysis solution;

4) carrying out coarse filtration and centrifugation on the enzymolysis liquid in the step 3) to remove solid residues and leaving supernatant;

5) sequentially injecting the supernatant obtained in the step 4) into an ultrafiltration column with the molecular weight of 10 ten thousand daltons, an ultrafiltration column with the molecular weight of 1 ten thousand daltons and an ultrafiltration column with the molecular weight of 1000 daltons for filtering to obtain a mixed solution of micromolecular bioactive peptides;

6) under the aseptic condition, the mixed liquid of the micromolecule bioactive peptides in the step 5) is sterilized by a bacterial filter to obtain a finished product.

2. The method for preparing the biological active peptide of the cordyceps militaris of the tussah as claimed in claim 1, wherein the finished product is put into a vacuum freeze dryer after the step 6) to prepare peptide freeze-dried powder.

3. The preparation method of the bioactive peptide of the cordyceps militaris as claimed in claim 1, wherein in the step 1), the living tussah pupa is used as a culture medium, and after a functional cordyceps militaris 'Sugao No. 1' liquid strain is inoculated, the cordyceps militaris is cultured for 55 to 60 days at a temperature of 15 to 20 ℃ and a humidity of 70 to 90 percent, so that the cordyceps militaris with complete stroma is obtained.

4. The method for preparing the bioactive peptide of the cordyceps militaris of the tussah as claimed in claim 1, wherein the freeze-drying environment in the step 2) is that the temperature is-25 ℃, the vacuum degree is 10-12Pa, and the high-pressure homogenizing pressure is 1.2 × 10⁵KPa。

5. The method for preparing the biological active peptide of the cordyceps militaris of the tussah as claimed in claim 1, wherein the temperature of the dry powder obtained by crushing in the step 2) is-10 ℃ to-20 ℃, and the fineness of the dry powder is 120 meshes.

6. The method for preparing the biological active peptide of the cordyceps militaris of the tussah as claimed in claim 1, wherein the adding amount of the protease in the step 3) is 6% of the dry powder by weight, wherein the adding amount of the alkaline protease and the trypsin is 1:1, and the alkaline protease is added first and then the trypsin is added.

7. The method for preparing the biological active peptide of the cordyceps militaris of the tussah as claimed in claim 6, wherein the enzymolysis temperature of the alkaline protease and the enzymolysis temperature of the trypsin are both 55 ℃, the enzymolysis time is both 2 hours according to the specified sequence, and the substrate concentration is 10%.

8. The method for preparing the tussah pupa cordyceps militaris bioactive peptide according to any one of claims 1, 6 or 7, wherein the alkaline protease is food grade 20 ten thousand U, and the trypsin is food grade 10 ten thousand U.

9. The method for preparing the bioactive peptide of the cordyceps militaris of the tussah as recited in claim 1, wherein the bacterial filter adopted in the step 6) is a 0.22 micron bacterial filter.

Technical Field

The invention relates to the technical field of active peptide preparation, in particular to a preparation method of a biological active peptide of tussah pupa cordyceps.

Background

Cordyceps militaris, also known as Cordyceps militaris and Cordyceps militaris, is a species of the same genus as Cordyceps militaris. Research shows that the cordyceps militaris has active ingredients and medicinal effects which are extremely similar to those of cordyceps sinensis, such as the effects of benefiting vital energy, tonifying deficiency, inhibiting bacteria, reducing blood pressure, calming, reducing blood sugar, regulating immunity and the like. Compared with expensive wild cordyceps sinensis, the artificially cultured cordyceps militaris ingredient system has no loss, and particularly has more advantages in the content of anticancer ingredients cordycepin and pentostatin.

Bioactive peptides are important substances essential for the body to perform various physiological activities, and play an irreplaceable role in the metabolic activities of organisms and the treatment of diseases. At present, the bioactive peptide is mostly prepared from cordyceps sinensis, and with the improvement of the technology of artificially cultivating cordyceps militaris, whether the artificially cultivated cordyceps militaris can be used as a raw material for preparing the bioactive peptide becomes a research object of people.

Disclosure of Invention

In view of the above, the invention discloses a preparation method of a biological active peptide of tussah pupa cordyceps sinensis, which is used for preparing and obtaining the biological active peptide with high activity by taking artificially cultured cordyceps militaris as a raw material.

The technical scheme provided by the invention is specifically a preparation method of the biological active peptide of the tussah pupa cordyceps sinensis, which is characterized by comprising the following steps:

1) inoculating a functional cordyceps militaris 'Sugao No. 1' liquid strain by taking a living tussah pupa as a culture medium, and obtaining the cordyceps militaris with complete stroma through conventional culture;

2) freeze-drying the tussah cordyceps militaris stroma in the step 1), and then crushing to obtain dry powder;

3) adding deionized water into the dry powder obtained in the step 2), and adding two proteases, namely alkaline protease and trypsin, for enzymolysis to obtain an enzymolysis solution;

4) carrying out coarse filtration and centrifugation on the enzymolysis liquid in the step 3) to remove solid residues and leaving supernatant;

5) sequentially injecting the supernatant obtained in the step 4) into an ultrafiltration column with the molecular weight of 10 ten thousand daltons, an ultrafiltration column with the molecular weight of 1 ten thousand daltons and an ultrafiltration column with the molecular weight of 1000 daltons for filtering to obtain a mixed solution of micromolecular bioactive peptides;

6) under the aseptic condition, the mixed liquid of the micromolecule bioactive peptides in the step 5) is sterilized by a bacterial filter to obtain a finished product.

Preferably, the finished product is placed into a vacuum freeze dryer after the step 6) to prepare peptide freeze-dried powder.

Further preferably, in the step 1), living tussah pupas are used as a culture medium, liquid strains of 'Sugao No. 1' of functional cordyceps militaris are inoculated, and the culture is carried out for 55 to 60 days under the conditions that the temperature is 15 to 20 ℃ and the humidity is 70 to 90 percent, so that the cordyceps militaris with complete stroma is obtained by culture.

Further preferably, the freeze drying in step 2) is carried out at-25 deg.C under vacuum degree of 10-12Pa and under high-pressure homogenizing pressure of 1.2 × 10⁵KPa。

Further preferably, the temperature of the dry powder obtained by crushing in the step 2) is-10 ℃ to-20 ℃, and the fineness of the dry powder is 120 meshes.

Further preferably, the adding amount of the protease in the step 3) is 6% of the dry powder by weight, wherein the adding amount of the alkaline protease and the trypsin is 1:1, and the alkaline protease is added first, and then the trypsin is added.

More preferably, the enzymolysis temperature of the alkaline protease and the enzymolysis temperature of the trypsin are both 55 ℃, the enzymolysis time is both 2h according to the specified sequence, and the substrate concentration is 10%.

Further preferably, the alkaline protease is food grade 20 ten thousand U, and the trypsin is food grade 10 ten thousand U.

Further preferably, the bacterial filter used in step 6) is a 0.22 micron bacterial filter.

The method for preparing the biological active peptide of the tussah pupa cordyceps provided by the invention adopts the tussah pupa cordyceps which is most similar to natural cordyceps sinensis as a raw material for the first time so as to ensure the richness, the content and the antioxidant activity of the prepared small molecular biological active peptide; meanwhile, a double-protease combined enzymolysis method is adopted, so that enzymolysis is more thorough, the proportion of small molecular peptides is improved, and the activity is stronger; in addition, alkaline protease and trypsin are adopted in the double-protease combined enzymolysis method, so that the preparation method of the active peptide is greatly reduced; the whole preparation process of the active peptide is below 55 ℃, and no high-temperature link exists, so that the prepared active peptide has strong oxidation resistance and superoxide radical removal capacity, and has the advantages of easy dissolution, acid resistance, heat resistance and high stability; in addition, after the prepared active peptide is separated by the high performance liquid chromatography, the spectrum is clear, the substance separation degree is high, the dispersion degree is good, and the high quality level is achieved.

The preparation method of the tussah pupa cordyceps militaris bioactive peptide provided by the invention has the advantages of simple preparation method, easiness in operation and the like, and the prepared bioactive peptide has the advantages of high proportion of small-molecular peptides, strong activity and the like.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a standard curve of the protein prepared in example 1;

FIG. 2 is a graph showing the time-dependent change of trypsin in example 2;

FIG. 3 is a graph showing the variation of trypsin enzymolysis according to the amount of added enzyme in example 2;

FIG. 4 is a graph showing the variation of the trypsin concentration factor in example 2;

FIG. 5 is a graph showing the time-dependent change of the alkaline protease in example 2;

FIG. 6 is a graph showing the variation of the alkaline protease in accordance with the amount of enzyme added in the case of enzymolysis with alkaline protease in example 2;

FIG. 7 is a graph showing the change in the alkaline protease hydrolysis according to the substrate concentration in example 2.

Detailed Description

The invention is further illustrated by the following specific embodiments, which are not intended to limit the scope of the invention.