Efficient polypeptide raw material production method based on biological fermentation technology

Abstract

The invention belongs to the technical field of polypeptide raw materials, and discloses an efficient polypeptide raw material production method based on a biological fermentation technology, which comprises the following steps: 1, screening and culturing microorganisms: selecting appropriate microorganisms (bacteria, yeast and fungi) as a leavening agent, the microorganisms having efficient polypeptide synthesis ability; 2, performing microbial fermentation and liquid fermentation, wherein the liquid fermentation is mainly performed in a liquid culture medium; 3, solid-phase synthesis: optimizing the environmental conditions of polypeptide production, and controlling the temperature, the pH value, the dissolved oxygen content, the nutrient substances, the stirring rate and the fermentation time; cells are broken through an ultrasonic method, the method can be carried out at a low temperature, chemical reagents do not need to be added during ultrasonic breaking, possible pollution and influence on polypeptide are avoided, and therefore the total yield of polypeptide extracted from the cells can be remarkably increased, and the polypeptide can be produced through microorganisms on the basis of a biological fermentation technology.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide raw materials, and specifically relates to a method for producing high-efficiency polypeptide raw materials based on biological fermentation technology. Background Art

[0002] High-efficiency peptide raw materials are usually used in the fields of pharmaceuticals, food and research. The functions of peptide raw materials mainly include: as a drug ingredient, it has multiple biological activities such as antibacterial, antiviral and anti-inflammatory. It can be used as a nutritional supplement to support physical health and enhance immunity. It can be used to develop functional foods and enhance the health value of food. In biomedical research, it is used to explore the mechanisms of signal transduction and cell function. It is used in skin care products and has moisturizing and anti-aging effects.

[0003] However, the existing methods for producing high-efficiency peptide raw materials based on biological fermentation technology are not perfect and still have certain defects:

[0004] In the production of peptide raw materials, if the cells are not broken uniformly, it will lead to incomplete release of the peptides, affecting the final yield. Failure to effectively separate impurities may result in low purity of the target peptide, affecting subsequent applications. Traditional crushing methods may introduce chemical substances and increase the risk of peptide degradation. Subsequent processing and separation processes may be more complicated and time-consuming, reducing the quality of peptide raw material production. Summary of the invention

[0005] The purpose of the present invention is to provide a method for producing an efficient polypeptide raw material based on biological fermentation technology to solve the problems raised in the above background technology.

[0006] In order to achieve the above object, the present invention provides the following technical solution: a method for producing a high-efficiency polypeptide raw material based on biological fermentation technology, comprising the following steps:

[0007] Step 1: Microbial screening and cultivation: Select appropriate microorganisms (bacteria, yeast and fungi) as fermentation agents, which have the ability to efficiently synthesize peptides;

[0008] The second step: microbial fermentation and liquid fermentation. Liquid fermentation is mainly carried out in liquid culture medium;

[0009] Step 3: Solid phase synthesis: Select a suitable solid phase carrier (resin) whose surface can effectively bind amino acids, optimize the environmental conditions for peptide production, and control temperature, pH value, dissolved oxygen, nutrients, stirring rate and fermentation time;

[0010] Step 4: Genetic engineering: After the target gene is amplified by PCR, it is cloned into an appropriate expression vector for subsequent transformation. The recombinant expression vector is introduced into the host cell by electroporation, the expressed polypeptide is harvested, and subsequent extraction and purification are performed;

[0011] Step 5: The cells are broken by ultrasonic method to release the peptides, and the peptides are extracted from the broken cells using a solvent;

[0012] Step 6: Centrifugation and filtration: remove insoluble substances, separate the supernatant, and obtain the liquid containing polypeptides;

[0013] Step 7: Concentration: Use evaporation technology to concentrate the extract to increase the concentration of the polypeptide. Purification: Use ion exchange chromatography to separate and remove impurities according to the properties of the polypeptide.

[0014] Step 8: Quality testing: The purity and activity of the peptide are tested by mass spectrometry and amino acid analysis.

[0015] Preferably, in step three, the first amino acid is linked to a solid support via a covalent bond, and the protecting group on the amino acid is usually removed by a reaction of the amino group, so that it can react with the next amino acid.

[0016] Preferably, in step three, the optimal growth temperature of the microorganisms is between 25-37° C. to promote metabolic activities.

[0017] Preferably, in step three, the pH value is controlled within a suitable range of 5.5-7.5, and the nutrients are appropriate amounts of carbon sources, nitrogen sources and trace elements.

[0018] Preferably, in step five, the solvent is a salt solution.

[0019] Preferably, in step three, protected amino acids are added sequentially, and the deprotection and connection processes are repeated to form a polypeptide chain. After each amino acid is added, the carrier needs to be cleaned to remove unreacted amino acids and impurities.

[0020] Preferably, in step three, the stirring speed is between 200-1000 rpm, and the fermentation time is 24-72 hours.

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

[0022] 1. The present invention uses an ultrasonic method to break cells, which can be performed at a lower temperature, reducing the risk of degradation of heat-sensitive polypeptides, and can quickly and effectively destroy cell membranes to release polypeptides and other biological molecules in cells. Ultrasonic disruption does not require the addition of chemical reagents, avoiding possible pollution and effects on polypeptides, thereby significantly increasing the total yield of polypeptides extracted from cells. Microorganisms can be used to produce polypeptides based on biofermentation technology.

[0023] 2. The present invention removes impurities through the ion exchange chromatography, which can effectively separate charged molecules, has strong pertinence, removes specific impurities, and significantly improves the purity of the target polypeptide. The ion exchange material is renewable, which reduces the cost of long-term use. Through high-efficiency polypeptide raw material quality testing, the quality and purity of each batch of products can be verified to ensure consistency, and potential impurities can be discovered and eliminated in a timely manner, reducing product quality risks. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0025] Figure 1 It is a production flow chart of the present invention. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] like Figure 1 As shown, the embodiment of the present invention provides a technical solution for a method for producing an efficient polypeptide raw material based on biological fermentation technology, which includes the following steps:

[0028] Step 1: Microbial screening and cultivation: Select appropriate microorganisms (bacteria, yeast and fungi) as fermentation agents, which have the ability to efficiently synthesize peptides;

[0029] The second step: microbial fermentation and liquid fermentation. Liquid fermentation is mainly carried out in liquid culture medium;

[0030] Step 3: Solid phase synthesis: Select a suitable solid phase carrier (resin) whose surface can effectively bind amino acids, optimize the environmental conditions for peptide production, and control temperature, pH value, dissolved oxygen, nutrients, stirring rate and fermentation time;

[0031] Step 4: Genetic engineering: After the target gene is amplified by PCR, it is cloned into an appropriate expression vector for subsequent transformation. The recombinant expression vector is introduced into the host cell by electroporation, the expressed polypeptide is harvested, and subsequent extraction and purification are performed;

[0032] Step 5: The cells are broken by ultrasonic method to release the peptides, and the peptides are extracted from the broken cells using a solvent;

[0033] Step 6: Centrifugation and filtration: remove insoluble substances, separate the supernatant, and obtain the liquid containing polypeptides;

[0034] Step 7: Concentration: Use evaporation technology to concentrate the extract to increase the concentration of the polypeptide. Purification: Use ion exchange chromatography to separate and remove impurities according to the properties of the polypeptide.

[0035] Step 8: Quality testing: The purity and activity of the peptide are tested by mass spectrometry and amino acid analysis.

[0036] Among them, in step three, the first amino acid is connected to the solid phase carrier through a covalent bond, and the protecting group on the amino acid is usually removed by the reaction of the amino group to enable it to react with the next amino acid. In step three, the optimal growth temperature of the microorganism is between 25-37°C to promote metabolic activity. In step three, the pH value is controlled within the appropriate range of 5.5-7.5, and the nutrients are appropriate amounts of carbon sources, nitrogen sources and trace elements. The cells are broken by the set ultrasonic method, which can be carried out at a lower temperature to reduce the degradation risk of heat-sensitive polypeptides, and can quickly and effectively destroy the cell membrane and release polypeptides and other biological molecules in the cell. Ultrasonic disruption does not require the addition of chemical reagents, avoiding possible pollution and effects on polypeptides, thereby significantly increasing the total yield of polypeptides extracted from cells. Microorganisms can be used to produce polypeptides based on biofermentation technology.

[0037] Among them, in step five, the solvent is a salt solution, in step three, protected amino acids are added in sequence, and the process of deprotection and connection is repeated to form a polypeptide chain. After each amino acid is added, the carrier needs to be cleaned to remove unreacted amino acids and impurities. In step three, the stirring speed is between 200-1000rpm, and the fermentation time is 24-72 hours. The impurities are removed by the set ion exchange chromatography, which can effectively separate charged molecules, has strong pertinence, removes specific impurities, and significantly improves the purity of the target polypeptide. The ion exchange material is renewable, which reduces the cost of long-term use. Through the high-efficiency polypeptide raw material quality detection, the quality and purity of each batch of products can be verified to ensure consistency, and potential impurities can be discovered and eliminated in time to reduce product quality risks.

[0038] 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.

Claims

1. A method for producing high-efficiency polypeptide raw materials based on biological fermentation technology, characterized in that: The following steps are involved: Step 1: Microbial screening and cultivation: Select appropriate microorganisms (bacteria, yeast and fungi) as fermentation agents, which have the ability to efficiently synthesize peptides; The second step: microbial fermentation and liquid fermentation. Liquid fermentation is mainly carried out in liquid culture medium; Step 3: Solid phase synthesis: Select a suitable solid phase carrier (resin) whose surface can effectively bind amino acids, optimize the environmental conditions for peptide production, and control temperature, pH value, dissolved oxygen, nutrients, stirring rate and fermentation time; Step 4: Genetic engineering: After the target gene is amplified by PCR, it is cloned into an appropriate expression vector for subsequent transformation. The recombinant expression vector is introduced into the host cell by electroporation, the expressed polypeptide is harvested, and subsequent extraction and purification are performed; Step 5: The cells are broken by ultrasonic method to release the peptides, and the peptides are extracted from the broken cells using a solvent; Step 6: Centrifugation and filtration: remove insoluble substances, separate the supernatant, and obtain the liquid containing polypeptides; Step 7: Concentration: Use evaporation technology to concentrate the extract to increase the concentration of the polypeptide. Purification: Use ion exchange chromatography to separate and remove impurities according to the properties of the polypeptide. Step 8: Quality testing: The purity and activity of the peptide are tested by mass spectrometry and amino acid analysis.

2. The method for producing high-efficiency polypeptide raw materials based on biological fermentation technology according to claim 1, characterized in that: In step three, the first amino acid is covalently linked to a solid support, and the protecting group on the amino acid is usually removed by a reaction of the amino group, so that it can react with the next amino acid.

3. The method for producing high-efficiency polypeptide raw materials based on biological fermentation technology according to claim 1, characterized in that: In step three, the optimal growth temperature of microorganisms is between 25-37°C to promote metabolic activities.

4. The method for producing high-efficiency polypeptide raw materials based on biological fermentation technology according to claim 3, characterized in that: In step three, the pH value is controlled within the appropriate range of 5.5-7.5, and the nutrients are appropriate amounts of carbon sources, nitrogen sources and trace elements.

5. The method for producing high-efficiency polypeptide raw materials based on biological fermentation technology according to claim 1, characterized in that: In step 5, the solvent is a salt solution.

6. The method for producing high-efficiency polypeptide raw materials based on biological fermentation technology according to claim 4, characterized in that: In step three, the protected amino acids are added in sequence, and the deprotection and connection processes are repeated to form a polypeptide chain. After each amino acid is added, the carrier needs to be cleaned to remove unreacted amino acids and impurities.

7. The method for producing high-efficiency polypeptide raw materials based on biological fermentation technology according to claim 6, characterized in that: In step 3, the stirring speed is between 200-1000 rpm, and the fermentation time is 24-72 hours.

Images