CN112521444A - Extraction method of antibacterial peptide - Google Patents
Extraction method of antibacterial peptide Download PDFInfo
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- CN112521444A CN112521444A CN202011486210.8A CN202011486210A CN112521444A CN 112521444 A CN112521444 A CN 112521444A CN 202011486210 A CN202011486210 A CN 202011486210A CN 112521444 A CN112521444 A CN 112521444A
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- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 67
- 238000000605 extraction Methods 0.000 title claims abstract description 33
- 239000002994 raw material Substances 0.000 claims abstract description 37
- 239000000706 filtrate Substances 0.000 claims description 69
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 52
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 45
- 241000196324 Embryophyta Species 0.000 claims description 37
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 36
- 238000001914 filtration Methods 0.000 claims description 30
- 239000000047 product Substances 0.000 claims description 26
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- 238000010438 heat treatment Methods 0.000 claims description 19
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- BIVBRWYINDPWKA-VLQRKCJKSA-L Glycyrrhizinate dipotassium Chemical compound [K+].[K+].O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@@H]1O[C@H]1CC[C@]2(C)[C@H]3C(=O)C=C4[C@@H]5C[C@](C)(CC[C@@]5(CC[C@@]4(C)[C@]3(C)CC[C@H]2C1(C)C)C)C(O)=O)C([O-])=O)[C@@H]1O[C@H](C([O-])=O)[C@@H](O)[C@H](O)[C@H]1O BIVBRWYINDPWKA-VLQRKCJKSA-L 0.000 description 1
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- QCYLIQBVLZBPNK-UHFFFAOYSA-N asiaticoside A Natural products O1C(C(=O)C(C)C)=CC(C)C(C2(C(OC(C)=O)CC34C5)C)C1CC2(C)C3CCC(C1(C)C)C45CCC1OC1OCC(O)C(O)C1O QCYLIQBVLZBPNK-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- 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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/011—Hydrolysed proteins; Derivatives thereof from plants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/10—Anti-acne agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/005—Antimicrobial preparations
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/82—Preparation or application process involves sonication or ultrasonication
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/84—Products or compounds obtained by lyophilisation, freeze-drying
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- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
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- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention provides an extraction method of antibacterial peptide, which is characterized in that a plurality of plant raw materials are mixed and extracted to obtain the antibacterial peptide, the obtained antibacterial peptide has spectral applicability, the molecular weight of the whole active region is less than 5kDa, and the antibacterial peptide has a stable disulfide bond structure and good acid resistance, organic solvent resistance and thermal stability; the extraction method is simple to operate, improves the production efficiency, reduces the production cost and accords with industrial production; the extracted antibacterial peptide has a remarkable inhibition effect on propionibacterium acnes, can be effectively applied to acne-removing products, and has excellent antibacterial, anti-inflammatory and sedative effects. In addition, the extraction method adopts an online control system, so that the treatment conditions of the treatment kettle can be controlled more accurately, the impurity content is less, and the extraction quality is ensured.
Description
Technical Field
The invention relates to the field of extraction of chemical raw materials, and particularly relates to an extraction method of antibacterial peptide.
Background
The antibacterial peptide is a defensive polypeptide with broad-spectrum antibacterial property, and is composed of the antibacterial peptide. The antibacterial peptide in the prior art is generally used for medicines resisting antibiotic-resistant bacteria, shows activities of promoting tissue healing, regulating an in-vivo immune system and the like for an organism, is applied to the surface of the organism very few, and therefore has wide application space. However, obtaining a large amount of antibacterial peptides in the prior art is limited by the technical level of polypeptide synthesis, the cost of the antibacterial peptides obtained by a chemical synthesis method is too high, the extraction of the antibacterial peptides from animals and plants generally has the disadvantages of complex process, complex steps, low extraction rate, excessive impurity components of the extracted antibacterial peptides, low expression rate and poor effect.
In summary, the above problems still remain to be solved in the field of extraction of antimicrobial peptides.
Disclosure of Invention
Based on this, in order to solve the problems that in the prior art, the acquisition of a large amount of antibacterial peptide is limited by the technical level of polypeptide synthesis, the antibacterial cost obtained by a chemical synthesis method is too high, the extraction of the antibacterial peptide from animals and plants generally has the defects of complex process, complicated steps, low extraction rate and excessive impurity components of the extracted antibacterial peptide, so that the expression rate is low and the effect is poor, the invention provides an extraction method of the antibacterial peptide, and the specific technical scheme is as follows:
an extraction method of antibacterial peptide, comprising the following steps:
preparing plant raw materials according to the weight part ratio;
adding plant raw materials into a treatment kettle, and adding acetic acid into the treatment kettle according to the weight ratio of the material liquid of 1: 15-30;
controlling the treatment conditions in the treatment kettle through an online control system, and after the first-stage treatment is finished, performing first-stage filtration to obtain filtrate A;
adding complex enzyme into the filtrate A according to the proportion of 0.01g/mL-0.09g/mL, heating to inactivate enzyme after enzymolysis is finished, cooling to room temperature, dropwise adding a pH regulator to regulate the pH to 7.0, and performing secondary filtration to obtain filtrate B;
dialyzing and concentrating the filtrate B to obtain filtrate C;
and (3) carrying out reversed-phase high performance liquid chromatography separation on the filtrate C, freezing the separated product at the temperature of-12 to-5 ℃, and then transferring the product to a vacuum freeze drying device for drying to obtain the antibacterial peptide.
Preferably, the plant raw materials are obtained by mixing barley, alfalfa, peas and sesame according to the weight ratio of 1:1-5:2-7: 1-4.
Preferably, a temperature adjusting device, a pH adjusting device, a pressure adjusting device, an ultrasonic treatment device and a filtering device are arranged in the treatment kettle, and a feeding device is arranged outside the treatment kettle.
Preferably, the treatment conditions include temperature conditions, pH conditions, pressure conditions, and sonication conditions.
Preferably, the temperature condition is 40 ℃ to 85 ℃; the pressure condition is 20MPa-40 MPa; the power of the ultrasonic treatment condition is 150W-300W, the frequency is 30KHz-45KHz, and the time is 20min-45 min.
Preferably, a ceramic membrane with the pore diameter of 15nm-35nm is adopted in the primary filtration.
Preferably, the complex enzyme is obtained by mixing papain, cellulase and mannanase according to the weight ratio of 1-10:5-9: 0.2-1.8.
Preferably, the pH regulator is an alkali solution.
Preferably, the dialysis time is 15h to 22 h.
Preferably, the reversed-phase high performance liquid chromatography is to load the filtrate C onto a reversed-phase C4 silica gel column and carry out elution treatment on the C4 silica gel column for 20min to 30min by using double distilled water containing trifluoroacetic acid and acetonitrile containing trifluoroacetic acid in sequence.
In the scheme, the antibacterial peptide is obtained by mixing and extracting a plurality of plant raw materials, the obtained antibacterial peptide has spectral applicability, the molecular weight of the whole active region is less than 5kDa, and the antibacterial peptide has a stable disulfide bond structure and good acid resistance, organic solvent resistance and thermal stability; the extraction method is simple to operate, improves the production efficiency, reduces the production cost and accords with industrial production; the extracted antibacterial peptide has a remarkable inhibition effect on propionibacterium acnes, can be effectively applied to acne-removing products, and has excellent antibacterial, anti-inflammatory and sedative effects. In addition, the extraction method adopts an online control system, so that the treatment conditions of the treatment kettle can be controlled more accurately, the impurity content is less, and the extraction quality is ensured.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to embodiments thereof. It should be understood that the detailed description and specific examples, while indicating the scope of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The extraction method of the antibacterial peptide in one embodiment of the invention comprises the following steps:
preparing plant raw materials according to the weight part ratio;
adding plant raw materials into a treatment kettle, and adding acetic acid into the treatment kettle according to the weight ratio of the material liquid of 1: 15-30;
controlling the treatment conditions in the treatment kettle through an online control system, and after the first-stage treatment is finished, performing first-stage filtration to obtain filtrate A;
adding complex enzyme into the filtrate A according to the proportion of 0.01g/mL-0.09g/mL, heating to inactivate enzyme after enzymolysis is finished, cooling to room temperature, dropwise adding a pH regulator to regulate the pH to 7.0, and performing secondary filtration to obtain filtrate B;
dialyzing and concentrating the filtrate B to obtain filtrate C;
and (3) carrying out reversed-phase high performance liquid chromatography separation on the filtrate C, freezing the separated product at the temperature of between 12 ℃ below zero and 5 ℃ below zero, and then transferring the product to a vacuum freeze drying device for drying to obtain the antibacterial peptide.
In the scheme, the antibacterial peptide is obtained by mixing and extracting a plurality of plant raw materials, the obtained antibacterial peptide has spectral applicability, the molecular weight of the whole active region is less than 5kDa, and the antibacterial peptide has a stable disulfide bond structure and good acid resistance, organic solvent resistance and thermal stability; the extraction method is simple to operate, improves the production efficiency, reduces the production cost and accords with industrial production; the extracted antibacterial peptide has a remarkable inhibition effect on propionibacterium acnes, can be effectively applied to acne-removing products, and has excellent antibacterial, anti-inflammatory and sedative effects. In addition, the extraction method adopts an online control system, so that the treatment conditions of the treatment kettle can be controlled more accurately, the impurity content is less, and the extraction quality is ensured.
In one embodiment, the plant raw materials are obtained by mixing barley, alfalfa, peas and sesame in a weight ratio of 1:1-5:2-7: 1-4.
In one embodiment, the concentration of acetic acid is 0.5% to 8%.
In one embodiment, the online control system comprises a temperature adjusting module, a pH adjusting module, a pressure adjusting module, an ultrasonic processing adjusting module, a time adjusting module, a mobile terminal and a master controller, wherein the temperature adjusting module, the pH adjusting module, the pressure adjusting module, the ultrasonic processing adjusting module, the time adjusting module and the mobile terminal are respectively connected with the master controller.
In one embodiment, the temperature adjusting module, the pH adjusting module, the pressure adjusting module, the ultrasonic treatment adjusting module and the time adjusting module are respectively connected with the mobile terminal.
In one embodiment, the temperature adjustment module stores a preset temperature value.
In one embodiment, the preset temperature value includes a first preset temperature value and a second preset temperature value.
In one embodiment, the first preset temperature value is 40 ℃.
In one embodiment, the second preset temperature value is 85 ℃.
In one embodiment, the pH adjusting module stores a preset pH value.
In one embodiment, the predetermined pH is 7.0.
In one embodiment, the pressure adjustment module stores a preset pressure value.
In one embodiment, the preset pressure values include a first preset pressure value and a second preset pressure value.
In one embodiment, the first preset pressure value is 20 MPa.
In one embodiment, the second preset pressure value is 40 MPa.
In one embodiment, the preset temperature value, the preset pH value and the preset pressure value can be set by the mobile terminal.
In one embodiment, the sonication conditions are set by the mobile terminal.
In one embodiment, a temperature adjusting device, a pH adjusting device, a pressure adjusting device, an ultrasonic treatment device and a filtering device are arranged in the treatment kettle, and a feeding device is arranged outside the treatment kettle.
In one embodiment, the feeding device comprises a liquid storage tank for containing the pH regulator.
In one embodiment, the liquid storage tank is connected with the treatment kettle through a liquid pipe, and a control valve is arranged on the liquid pipe.
In one embodiment, the temperature adjusting device, the pH adjusting device, the pressure adjusting device, the ultrasonic processing device, the filtering device and the control valve are respectively connected with the overall controller.
In one embodiment, the temperature regulating device comprises a temperature sensor for acquiring a temperature value in the processing kettle and sending the temperature value.
In one embodiment, the temperature adjusting module receives the temperature value, compares the temperature value with the preset temperature value, and when the temperature value is smaller than the first preset temperature value, the temperature adjusting module sends a heating control instruction, and the master controller receives the heating control instruction and controls the temperature adjusting device to start heating; the temperature value is equal to the second preset temperature value, the temperature adjusting module sends a heating stop control instruction, and the master controller receives the heating stop control instruction and controls the temperature adjusting device to stop heating.
In one embodiment, the pH adjusting device comprises a pH sensor for acquiring the pH value in the treatment kettle and sending the pH value.
In one embodiment, the pH adjusting module receives the pH value and compares the pH value with the preset pH value, when the pH value is smaller than the preset pH value, the pH adjusting module sends a command of adding a pH adjusting agent, and the general controller receives the command of adding the pH adjusting agent and controls the control valve to perform an action of opening the control valve until the pH value is equal to the preset pH value.
In one embodiment, the pressure regulating device comprises a pressure sensor for acquiring a pressure value in the treatment kettle and sending the pressure value.
In one embodiment, the pressure adjusting module receives the pressure value, compares the pressure value with the preset pressure value, and when the pressure value is smaller than the first preset pressure value, the pressure adjusting module sends a pressurization control command, and the master controller receives the pressurization control command and controls the pressure adjusting device to perform a pressure increasing action; the pressure value is equal to or larger than the second preset pressure value, the pressure adjusting module sends a decompression control instruction, and the master controller receives the decompression control instruction and controls the pressure adjusting device to perform decompression.
In one embodiment, the ultrasonic processing module converts power, frequency and time of ultrasonic processing conditions set by a user through the mobile terminal into a power value, a frequency value and a time value, and sends the power value, the frequency value and the time value to the general controller, and the general controller receives the power value, the frequency value and the time value and controls the ultrasonic processing device to perform power control action, frequency control action and time control action.
In one embodiment, the treatment conditions include temperature conditions, pH conditions, pressure conditions, and sonication conditions.
In one embodiment, the temperature conditions are 40 ℃ to 85 ℃; the pressure condition is 20MPa-40 MPa; the power of the ultrasonic treatment condition is 150W-300W, the frequency is 30KHz-45KHz, and the time is 20min-45 min.
In one embodiment, a ceramic membrane with a pore size of 15nm-35nm is used in the first stage of filtration.
In one embodiment, the complex enzyme is prepared by mixing papain, cellulase and mannanase according to the weight ratio of 1-10:5-9: 0.2-1.8. The components have synergistic effect, so that more effective enzymolysis is achieved, various plant raw materials have better enzymolysis effect, and the extraction rate of the antibacterial peptide is further promoted.
In one embodiment, the pH adjusting agent is a lye.
In one embodiment, the lye is NaOH.
In one embodiment, the enzymolysis time is 25min-40 min.
In one embodiment, the temperature for heating to inactivate enzyme is 65-100 ℃; the heating enzyme deactivation time is 10min-30 min.
In one embodiment, the secondary filtration is performed by using an ultrafiltration membrane with a pore size capable of isolating molecular weight of 1000-.
In one embodiment, the dialysis time is 15h to 22 h.
In one embodiment, the reversed-phase high performance liquid chromatography separation is to load the filtrate C onto a reversed-phase C4 silica gel column and perform elution treatment on the C4 silica gel column for 20min to 30min by using double distilled water containing trifluoroacetic acid and acetonitrile containing trifluoroacetic acid in sequence.
In one embodiment, the trifluoroacetic acid content in the double distilled water containing trifluoroacetic acid is 0.1-0.5% by mass.
In one embodiment, the trifluoroacetic acid-containing acetonitrile contains 0.1-0.5% by mass of trifluoroacetic acid and 70-85% by mass of acetonitrile.
In one embodiment, the antimicrobial peptide is used in cosmetic preparation.
In one embodiment, the cosmetic is an anti-acne cosmetic.
Embodiments of the present invention will be described in detail below with reference to specific examples.
Example 1:
an extraction method of antibacterial peptide, comprising the following steps:
mixing barley, alfalfa, peas and sesame in a weight ratio of 1:1:2:4 to prepare 8kg of plant raw materials, wherein the weight of the plant raw materials is 1kg of barley, 1kg of alfalfa, 2kg of peas and 4kg of sesame;
adding plant raw materials into a treatment kettle, and adding acetic acid with the concentration of 8% into the treatment kettle according to the weight ratio of the material liquid to the treatment kettle of 1: 15;
controlling the temperature condition in the treatment kettle to be 40 ℃ through an online control system; the pressure condition is 20 MPa; the power of ultrasonic treatment conditions is 150W, the frequency is 45KHz, the time is 20min, and after the first-stage treatment is finished, a ceramic membrane with the aperture of 15nm is adopted for filtration to obtain filtrate A;
adding 0.01g/mL (relative to the addition amount of the filtrate A) of papain, cellulase and mannase in a weight ratio of 1:5:0.2 into the filtrate A, mixing to obtain a complex enzyme, after enzymolysis is finished, heating to 65 ℃, treating for 10min, cooling to room temperature, dropwise adding NaOH to adjust the pH to 7.0, and filtering by adopting an ultrafiltration membrane with an isolatable pore size of 1000 daltons to obtain a filtrate B;
dialyzing the filtrate B for 15h and concentrating to obtain filtrate C;
loading the sample onto a reversed phase C4 silica gel column, sequentially eluting the C4 silica gel column by using double distilled water containing 0.5% of trifluoroacetic acid by mass and aqueous solution containing 0.5% of trifluoroacetic acid by mass and acetonitrile by mass of 80% of acetonitrile by mass for 30min, freezing the separated product at-12 ℃, and then transferring the product into a vacuum freeze drying device for drying to obtain the antibacterial peptide.
Example 2:
an extraction method of antibacterial peptide, comprising the following steps:
mixing barley, alfalfa, peas and sesame in a weight ratio of 1:5:7:1 to prepare 14kg of plant raw materials, wherein the weight of the plant raw materials is 1kg of barley, 5kg of alfalfa, 7kg of peas and 1kg of sesame;
adding plant raw materials into a treatment kettle, and adding 0.5% acetic acid into the treatment kettle according to the weight ratio of the material liquid to the treatment kettle of 1: 30;
controlling the temperature condition in the treatment kettle to be 85 ℃ through an online control system; the pressure condition is 40 MPa; the power of ultrasonic treatment conditions is 300W, the frequency is 45KHz, the time is 45min, and after the first-stage treatment is finished, a ceramic membrane with the aperture of 35nm is adopted for filtration to obtain filtrate A;
adding 0.09g/mL (relative to the addition amount of the filtrate A) of papain, cellulase and mannase in a weight ratio of 10:9:1.8 into the filtrate A, mixing to obtain a complex enzyme, heating to 100 ℃ after enzymolysis is finished, treating for 30min, cooling to room temperature, dropwise adding NaOH to adjust the pH to 7.0, and filtering by adopting an ultrafiltration membrane with an isolatable pore size of 5000 daltons to obtain a filtrate B;
dialyzing the filtrate B for 22h and concentrating to obtain filtrate C;
loading the sample onto a reversed phase C4 silica gel column, sequentially eluting the C4 silica gel column by using double distilled water containing 0.5% of trifluoroacetic acid by mass and aqueous solution containing 0.5% of trifluoroacetic acid by mass and acetonitrile by mass of 80% of acetonitrile by mass for 30min, freezing the separated product at-5 ℃, and then transferring the product into a vacuum freeze drying device for drying to obtain the antibacterial peptide.
Example 3:
an extraction method of antibacterial peptide, comprising the following steps:
mixing barley, alfalfa, peas and sesame in a weight ratio of 1:3:5:3 to prepare 12kg of plant raw materials, wherein the weight of the plant raw materials is 1kg of barley, 3kg of alfalfa, 5kg of peas and 3kg of sesame;
adding plant raw materials into a treatment kettle, and adding acetic acid with the concentration of 5% into the treatment kettle according to the weight ratio of the material liquid to the treatment kettle of 1: 20;
controlling the temperature condition in the treatment kettle to be 50 ℃ through an online control system; the pressure condition is 30 MPa; the power of ultrasonic treatment conditions is 250W, the frequency is 40KHz, the time is 35min, and after the first-stage treatment is finished, a ceramic membrane with the aperture of 25nm is adopted for filtration to obtain filtrate A;
adding 0.08g/mL (relative to the addition amount of the filtrate A) of papain, cellulase and mannase in a weight ratio of 5:6:1.2 into the filtrate A, mixing to obtain a complex enzyme, after enzymolysis is finished, heating to 85 ℃, treating for 20min, cooling to room temperature, dropwise adding NaOH to adjust the pH value to 7.0, and filtering by adopting an ultrafiltration membrane with an isolatable pore size of 3000 daltons to obtain a filtrate B;
dialyzing the filtrate B for 20h and concentrating to obtain filtrate C;
loading the sample onto a reversed phase C4 silica gel column, sequentially eluting the C4 silica gel column by using double distilled water containing 0.4 mass percent of trifluoroacetic acid and aqueous solution containing 0.4 mass percent of trifluoroacetic acid and 78 mass percent of acetonitrile for 25min, freezing the separated product at-10 ℃, and then transferring the product into a vacuum freeze drying device for drying to obtain the antibacterial peptide.
Example 4:
an extraction method of antibacterial peptide, comprising the following steps:
mixing barley, alfalfa, peas and sesame in a weight ratio of 1:2:3:2 to prepare 8kg of plant raw materials, wherein the weight of the plant raw materials is 1kg of barley, 2kg of alfalfa, 3kg of peas and 2kg of sesame;
adding plant raw materials into a treatment kettle, and adding 2% acetic acid into the treatment kettle according to the weight ratio of the material liquid to the treatment kettle of 1: 18;
controlling the temperature condition in the treatment kettle to be 50 ℃ through an online control system; the pressure condition is 33 MPa; the power of ultrasonic treatment conditions is 260W, the frequency is 40KHz, the time is 33min, and after the first-stage treatment is finished, a ceramic membrane with the aperture of 33nm is adopted for filtration to obtain filtrate A;
adding 0.03g/mL (relative to the addition amount of the filtrate A) of papain, cellulase and mannase in a weight ratio of 3:6:0.8 into the filtrate A, mixing to obtain a complex enzyme, after enzymolysis is finished, heating to 85 ℃, treating for 22min, cooling to room temperature, dropwise adding NaOH to adjust the pH value to 7.0, and filtering by adopting an ultrafiltration membrane with an isolatable pore size of 2000 daltons to obtain a filtrate B;
dialyzing the filtrate B for 18h and concentrating to obtain filtrate C;
loading the sample onto a reversed phase C4 silica gel column, sequentially eluting the C4 silica gel column by using double distilled water containing 0.4 mass percent of trifluoroacetic acid and aqueous solution containing 0.2 mass percent of trifluoroacetic acid and 72 mass percent of acetonitrile for 23min, freezing the separated product at-5 ℃, and then transferring the product into a vacuum freeze drying device for drying to obtain the antibacterial peptide.
Comparative example 1:
an extraction method of antibacterial peptide, comprising the following steps:
adding 8kg of barley into a treatment kettle, and adding 5% acetic acid into the treatment kettle according to the weight ratio of the material liquid to the treatment kettle of 1: 20;
controlling the temperature condition in the treatment kettle to be 50 ℃ through an online control system; the pressure condition is 30 MPa; the power of the ultrasonic treatment condition is 250W, the frequency is 40KHz, the time is 35min, and after the first-stage treatment is finished, a ceramic membrane with the aperture of 25nm is adopted for filtering to obtain a filtrate A;
adding 0.08g/mL (relative to the addition amount of the filtrate A) of papain, cellulase and mannase in a weight ratio of 5:6:1.2 into the filtrate A, mixing to obtain a complex enzyme, after enzymolysis is finished, heating to 85 ℃, treating for 20min, cooling to room temperature, dropwise adding NaOH to adjust the pH value to 7.0, and filtering by adopting an ultrafiltration membrane with an isolatable pore size of 3000 daltons to obtain a filtrate B;
dialyzing the filtrate B for 20h and concentrating to obtain filtrate C;
loading the sample onto a reversed phase C4 silica gel column, sequentially eluting the C4 silica gel column by using double distilled water containing 0.4 mass percent of trifluoroacetic acid and aqueous solution containing 0.4 mass percent of trifluoroacetic acid and 78 mass percent of acetonitrile for 25min, freezing the separated product at-10 ℃, and then transferring the product into a vacuum freeze drying device for drying to obtain the antibacterial peptide.
Comparative example 2:
an extraction method of antibacterial peptide, comprising the following steps:
mixing barley, alfalfa, peas and sesame in a weight ratio of 10:3:5:4 to prepare 22kg of plant raw materials, wherein the weight of the plant raw materials is 10kg of barley, 3kg of alfalfa, 5kg of peas and 4kg of sesame;
adding plant raw materials into a treatment kettle, and adding acetic acid with the concentration of 5% into the treatment kettle according to the weight ratio of the material liquid to the treatment kettle of 1: 20;
controlling the temperature condition in the treatment kettle to be 50 ℃ through an online control system; the pressure condition is 30 MPa; the power of ultrasonic treatment conditions is 250W, the frequency is 40KHz, the time is 35min, and after the first-stage treatment is finished, a ceramic membrane with the aperture of 25nm is adopted for filtration to obtain filtrate A;
adding 0.08g/mL (relative to the addition amount of the filtrate A) of papain, cellulase and mannase in a weight ratio of 5:6:1.2 into the filtrate A, mixing to obtain a complex enzyme, after enzymolysis is finished, heating to 85 ℃, treating for 20min, cooling to room temperature, dropwise adding NaOH to adjust the pH value to 7.0, and filtering by adopting an ultrafiltration membrane with an isolatable pore size of 3000 daltons to obtain a filtrate B;
dialyzing the filtrate B for 20h and concentrating to obtain filtrate C;
loading the sample onto a reversed phase C4 silica gel column, sequentially eluting the C4 silica gel column by using double distilled water containing 0.4 mass percent of trifluoroacetic acid and aqueous solution containing 0.4 mass percent of trifluoroacetic acid and 78 mass percent of acetonitrile for 25min, freezing the separated product at-10 ℃, and then transferring the product into a vacuum freeze drying device for drying to obtain the antibacterial peptide.
Comparative example 3:
an extraction method of antibacterial peptide, comprising the following steps:
mixing barley, alfalfa, peas and sesame in a weight ratio of 1:3:5:3 to prepare 12kg of plant raw materials, wherein the weight of the plant raw materials is 1kg of barley, 3kg of alfalfa, 5kg of peas and 3kg of sesame;
adding plant raw materials into a treatment kettle, and adding acetic acid with the concentration of 5% into the treatment kettle according to the weight ratio of the material liquid to the treatment kettle of 1: 20;
setting the temperature condition in the treatment kettle to be 50 ℃; the pressure condition is 30 MPa; the power of ultrasonic treatment conditions is 250W, the frequency is 40KHz, the time is 35min, and after the first-stage treatment is finished, a ceramic membrane with the aperture of 25nm is adopted for filtration to obtain filtrate A;
adding 0.08g/mL (relative to the addition amount of the filtrate A) of papain, cellulase and mannase in a weight ratio of 5:6:1.2 into the filtrate A, mixing to obtain a complex enzyme, after enzymolysis is finished, heating to 85 ℃, treating for 20min, cooling to room temperature, dropwise adding NaOH to adjust the pH value to 7.0, and filtering by adopting an ultrafiltration membrane with an isolatable pore size of 3000 daltons to obtain a filtrate B;
dialyzing the filtrate B for 20h and concentrating to obtain filtrate C;
loading the sample onto a reversed phase C4 silica gel column, sequentially eluting the C4 silica gel column by using double distilled water containing 0.4 mass percent of trifluoroacetic acid and aqueous solution containing 0.4 mass percent of trifluoroacetic acid and 78 mass percent of acetonitrile for 25min, freezing the separated product at-10 ℃, and then transferring the product into a vacuum freeze drying device for drying to obtain the antibacterial peptide.
Comparative example 4:
an extraction method of antibacterial peptide, comprising the following steps:
mixing barley, alfalfa, peas and sesame in a weight ratio of 1:3:5:3 to prepare 12kg of plant raw materials, wherein the weight of the plant raw materials is 1kg of barley, 3kg of alfalfa, 5kg of peas and 3kg of sesame;
adding plant raw materials into a treatment kettle, and adding acetic acid with the concentration of 5% into the treatment kettle according to the weight ratio of the material liquid to the treatment kettle of 1: 20;
controlling the temperature condition in the treatment kettle to be 50 ℃ through an online control system; the pressure condition is 30 MPa; the power of the ultrasonic treatment condition is 250W, the frequency is 40KHz, the time is 35min, and after the first-stage treatment is finished, a ceramic membrane with the aperture of 25nm is adopted for filtering to obtain a filtrate A;
adding 0.08g/mL (relative to the addition amount of the filtrate A) of papain into the filtrate A, heating to 85 ℃ after enzymolysis is finished, treating for 20min, cooling to room temperature, dropwise adding NaOH to adjust the pH value to 7.0, and filtering by adopting an ultrafiltration membrane with an isolatable pore size of 3000 daltons to obtain a filtrate B;
dialyzing the filtrate B for 20h and concentrating to obtain filtrate C;
loading the sample onto a reversed phase C4 silica gel column, sequentially eluting the C4 silica gel column by using double distilled water containing 0.4 mass percent of trifluoroacetic acid and aqueous solution containing 0.4 mass percent of trifluoroacetic acid and 78 mass percent of acetonitrile for 25min, freezing the separated product at-10 ℃, and then transferring the product into a vacuum freeze drying device for drying to obtain the antibacterial peptide.
The first test example: bacteriostasis test
The antimicrobial peptides extracted in examples 1 to 4 and the antimicrobial peptides extracted in comparative examples 1 to 4 were added with distilled water at a volume ratio of 1:8, and 1mL each was used as a sample solution.
The determination method comprises the following steps: antibacterial activity determination is performed by adopting filter paper determination method, 0.5ml of bacterial suspension containing propionibacterium acnes is inoculated on an NA plate by using coating method, filter paper sheets of determination test solution soaked respectively are taken by using sterilized forceps and are flatly placed in a culture dish containing test bacteria, each group is repeated for 3 times, and sterile water is used as blank control. The prepared culture dish is placed in a constant temperature incubator at 30 ℃, observed after 48 hours, and the diameter of the inhibition zone is measured by a cross method. In addition, the test solutions of comparative examples 1 to 4 and the test solutions of examples 1 to 4 were placed in a 40 ℃ incubator and a 50 ℃ incubator, respectively, using the same measurement method as described above, and the results of measurement of the zone diameters of inhibition by 5 repetitions were shown in Table 1 below, using the same measurement method as described above.
Table 1:
as can be seen from the data analysis in Table 1, the antibacterial peptides extracted in examples 1-4 have excellent inhibitory effect on Propionibacterium acnes, and the inhibitory diameter is greater than 18mm in the inhibitory test. In the comparative example 1, a single plant raw material is adopted for extraction, and the bacteriostatic effect of the antibacterial peptide is obviously poorer than that of the antibacterial peptide extracted in the examples 1-4; the proportion of the barley, the alfalfa, the pea and the sesame in the comparative example 2 is different from that in the examples 1 to 4, but the bacteriostatic effect is obviously poorer than that in the examples; in comparative example 2, an online control system is not adopted to control the treatment conditions of the treatment kettle, although the antibacterial effect reaches 14mm, the antibacterial effect is better than that of comparative example 1, comparative example 2 and comparative example 3, but the antibacterial effect is poorer than that of the antibacterial peptide in examples 1-4; the comparative example 4 adopts single enzyme for enzymolysis, so that the bacteriostatic effect of the composition is obviously poorer than that of the examples 1-4, and shows that the components, the component proportion, the online control system and the enzymolyzed compound enzyme component of the plant raw materials can influence the bacteriostatic effect of extracting the antibacterial peptide. In addition, the extracted antibacterial peptide still keeps good antibacterial effect at 50 ℃, which shows that the extracted antibacterial peptide is not easy to denature under the action of heat, and has better heat resistance and thermal stability.
Test example two: recovery rate and purity of antibacterial peptide
The recovery rate and purity of the antibacterial peptides extracted in examples 1 to 4 and comparative examples 1 to 4 were measured by the conventional kjeldahl method, and the results are shown in table 2.
Table 2:
as can be seen from the data analysis in Table 2, the recovery rate and purity of the antimicrobial peptides in examples 1-4 are above 90% and the purity is above 95%, while the recovery rate and purity of the antimicrobial peptides in comparative example 1, comparative example 3 and comparative example 4 are obviously inferior to those of the antimicrobial peptides in examples 1-4; the results show that the treatment conditions in the treatment kettle and the treatment by adopting the complex enzyme through the online control system are beneficial to improving the recovery rate and the purity of the antibacterial peptide.
Application example:
the antibacterial peptide extracted in example 4 was applied in an anti-acne lotion.
An acne-removing emulsion comprises the following components in parts by weight: 5 parts of plant oil control factors, 3 parts of carboxymethyl yeast glucan, 5 parts of asiaticoside, 4 parts of dipotassium glycyrrhizinate, 7 parts of millettia extract and 6 parts of antibacterial peptide. Prepared by a conventional emulsion preparation method.
Human body patch test: randomly selecting 50 volunteers with healthy skin, pasting the acne-removing lotion prepared by the method on arms, tearing the volunteers after 12 hours of pasting, and after 50 volunteers complete a skin spot pasting test, no allergic phenomena such as redness and the like are found at the place pasted with the acne-removing lotion, so that the acne-removing lotion is not easy to cause allergic problems, and then entering a functional test.
And (4) functional test: randomly selecting 50 volunteers with age of 18-25 years and face with acne skin, using the acne removing lotion for one month by 50 volunteers, not using other skin care products within a one-month test period, and evaluating the effect after one month. The result shows that the acne skin of 50 volunteers is obviously improved, wherein 94% of the acne skin is in a healing state, which indicates that the antibacterial peptide applied in the acne-removing product can play an excellent inhibition role of propionibacterium acnes, and has good effects of diminishing inflammation, calming and improving the acne-removing skin.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. An extraction method of antibacterial peptide, which is characterized by comprising the following steps:
preparing plant raw materials according to the weight part ratio;
adding plant raw materials into a treatment kettle, and adding acetic acid into the treatment kettle according to the weight ratio of the material liquid of 1: 15-30;
controlling the treatment conditions in the treatment kettle through an online control system, and after the first-stage treatment is finished, performing first-stage filtration to obtain filtrate A;
adding complex enzyme into the filtrate A according to the proportion of 0.01g/mL-0.09g/mL, heating to inactivate enzyme after enzymolysis is finished, cooling to room temperature, dropwise adding a pH regulator to regulate the pH to 7.0, and performing secondary filtration to obtain filtrate B;
dialyzing and concentrating the filtrate B to obtain filtrate C;
and (3) carrying out reversed-phase high performance liquid chromatography separation on the filtrate C, freezing the separated product at the temperature of-12 to-5 ℃, and then transferring the product to a vacuum freeze drying device for drying to obtain the antibacterial peptide.
2. The method for extracting the antibacterial peptide according to claim 1, wherein the plant raw materials are obtained by mixing barley, alfalfa, pea and sesame in a weight ratio of 1:1-5:2-7: 1-4.
3. The method for extracting antimicrobial peptide according to claim 1, wherein a temperature adjusting device, a pH adjusting device, a pressure adjusting device, an ultrasonic processing device and a filtering device are arranged in the processing kettle, and a feeding device is arranged outside the processing kettle.
4. The method for extracting antimicrobial peptide according to claim 3, wherein said treatment conditions include temperature conditions, pH conditions, pressure conditions, and sonication conditions.
5. The method for extracting antibacterial peptide according to claim 4, wherein the temperature condition is 40-85 ℃; the pressure condition is 20MPa-40 MPa; the power of the ultrasonic treatment condition is 150W-300W, the frequency is 30KHz-45KHz, and the time is 20min-45 min.
6. The method for extracting antibacterial peptide according to claim 1, wherein a ceramic membrane with a pore size of 15nm-35nm is used in the primary filtration.
7. The method for extracting antibacterial peptide according to claim 1, wherein the complex enzyme is obtained by mixing papain, cellulase and mannanase according to a weight ratio of 1-10:5-9: 0.2-1.8.
8. The method for extracting antimicrobial peptide according to claim 1, wherein the pH adjusting agent is an alkaline solution.
9. The method for extracting antibacterial peptide according to claim 1, wherein the dialysis time is 15h-22 h.
10. The method for extracting antimicrobial peptide according to claim 9, wherein the reversed-phase high performance liquid chromatography is performed by loading the filtrate C onto a reversed-phase C4 silica gel column, and sequentially eluting the C4 silica gel column with double distilled water containing trifluoroacetic acid and acetonitrile containing trifluoroacetic acid for 20min to 30 min.
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