CN111020598B - Preparation and antifouling evaluation method for traditional Chinese medicine extracted cyclic peptide modified metal surface - Google Patents
Preparation and antifouling evaluation method for traditional Chinese medicine extracted cyclic peptide modified metal surface Download PDFInfo
- Publication number
- CN111020598B CN111020598B CN201911412794.1A CN201911412794A CN111020598B CN 111020598 B CN111020598 B CN 111020598B CN 201911412794 A CN201911412794 A CN 201911412794A CN 111020598 B CN111020598 B CN 111020598B
- Authority
- CN
- China
- Prior art keywords
- cyclic peptide
- solution
- sample
- antibacterial
- bacteria
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C23—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; CHEMICAL SURFACE TREATMENT; DIFFUSION TREATMENT OF METALLIC MATERIAL; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL; INHIBITING CORROSION OF METALLIC MATERIAL OR INCRUSTATION IN GENERAL
- C23F—NON-MECHANICAL REMOVAL OF METALLIC MATERIAL FROM SURFACE; INHIBITING CORROSION OF METALLIC MATERIAL OR INCRUSTATION IN GENERAL; MULTI-STEP PROCESSES FOR SURFACE TREATMENT OF METALLIC MATERIAL INVOLVING AT LEAST ONE PROCESS PROVIDED FOR IN CLASS C23 AND AT LEAST ONE PROCESS COVERED BY SUBCLASS C21D OR C22F OR CLASS C25
- C23F11/00—Inhibiting corrosion of metallic material by applying inhibitors to the surface in danger of corrosion or adding them to the corrosive agent
- C23F11/08—Inhibiting corrosion of metallic material by applying inhibitors to the surface in danger of corrosion or adding them to the corrosive agent in other liquids
- C23F11/10—Inhibiting corrosion of metallic material by applying inhibitors to the surface in danger of corrosion or adding them to the corrosive agent in other liquids using organic inhibitors
- C23F11/14—Nitrogen-containing compounds
- C23F11/145—Amides; N-substituted amides
-
- 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
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/28—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Vibrionaceae (F)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/32—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Bacillus (G)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/405—Assays involving biological materials from specific organisms or of a specific nature from algae
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Materials Engineering (AREA)
- Mechanical Engineering (AREA)
- Metallurgy (AREA)
- Peptides Or Proteins (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a preparation and antifouling evaluation method of a metal surface modified by cyclic peptide extracted from traditional Chinese medicine, belonging to the technical field of metal material preparation, comprising the following steps: (1) drying herba Violae, and grinding into powder; (2) extracting antibacterial cyclic peptide in 1000 g of herba Violae powder with mixed solution of ethanol and water, and microwave-assisted; (3) carrying out solid-liquid separation on the solution containing the Chinese violet by high-speed centrifugation to obtain a solution containing the antibacterial cyclic peptide; (4) separating and purifying the antibacterial cyclic peptide by using a high performance liquid chromatograph; (5) reducing and digesting the cyclic peptide using tris (2-carboxyethyl) phosphine and trypsin; (6) selecting 304 stainless steel as a substrate, polishing the substrate to a mirror surface, cleaning, drying, and soaking for 24 hours by using a dopamine solution; (7) immersing the surface of the activated stainless steel into a solution of the antibacterial cyclic peptide for 24 hours, taking out a sample, and airing the sample in a nitrogen environment; the antifouling property can be improved by using the invention to modify the metal surface.
Description
Technical Field
The invention belongs to the technical field of metal material preparation, and particularly relates to a preparation and antifouling evaluation method of a metal surface modified by extracting cyclopeptide from traditional Chinese medicines.
Background
When a ship body is immersed in seawater, the surface of the ship body can be quickly attached by bacteria or other microorganisms to cause the fouling phenomenon of the surface of the ship body, the biofouling phenomenon is a serious problem in the global shipping industry, the ship maneuverability is reduced, the operation cost is improved, the conventional coating for the ship body is harmful to the surrounding environment, and the marine environment is not easily protected.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides a preparation method and an antifouling evaluation method of a metal surface modified by extracting cyclopeptide from traditional Chinese medicines, solves the technical problem of biofouling of the surface of a marine metal material in the prior art, and can improve antifouling performance by modifying the metal surface.
The purpose of the invention is realized as follows: a preparation method of a traditional Chinese medicine extracted cyclic peptide modified metal surface comprises the following steps:
(1) drying herba Violae, and grinding into powder;
(2) extracting 1000 g of antibacterial cyclic peptide in the Chinese violet powder by using 10L of mixed solution of ethanol and water, and assisting by using microwaves;
(3) carrying out solid-liquid separation on the solution containing the Chinese violet by high-speed centrifugation to obtain a solution containing the antibacterial cyclic peptide;
(4) separating and purifying the antibacterial cyclic peptide by using a high performance liquid chromatograph;
(5) reducing and digesting the cyclic peptide using 0.1 μ M tris (2-carboxyethyl) phosphine and 0.5 μ g/μ L trypsin;
(6) selecting 304 stainless steel as a substrate, polishing the substrate to a mirror surface, cleaning, drying, and soaking in 2 mg/mL dopamine solution for 24h to obtain surface-activated stainless steel;
(7) and (3) immersing the activated stainless steel surface into the solution of the antibacterial cyclic peptide for 24h, taking out the sample, and airing the sample in a nitrogen environment.
In order to further realize the separation and purification of the antibacterial cyclic peptide, in the step (4), a solution A with trifluoroacetic acid (TFA) in a volume ratio of 0.1% as an eluent and a solution B with 80% acetonitrile containing TFA in a volume ratio of 0.1% as an eluent are used.
In order to further facilitate the analysis of the biochemical characteristics of the antibacterial cyclic peptide, in the step (5), after the cyclic peptide is reduced and digested, the amino acid sequence and the spatial structure of the antibacterial cyclic peptide are respectively obtained by using a liquid phase mass spectrometer and a dichroism spectrometer; in the design, the biochemical characteristics of the antibacterial cyclic peptide are analyzed by obtaining an amino acid sequence and a spatial structure, so that the structure and the performance of the cyclic peptide are improved subsequently, and the physicochemical performance of the prepared modified surface is improved.
As a further improvement of the invention, in the step (2), the volume ratio of the ethanol to the water is 3: 2.
The method for performing antifouling evaluation by using the prepared antibacterial cyclic peptide modified surface comprises the following steps:
(1) selecting dominant marine strains of vibrio natriegens, citrobacter fabarum and bacillus, dominant marine algae phaeodactylum tricornutum, chlorella vulgaris and navicula, and respectively culturing the bacteria and the marine algae;
(2) respectively immersing the prepared samples into bacteria and seaweed culture solution, respectively immersing the prepared samples into the bacteria and seaweed culture solution, taking out the samples after a period of time, fixing the samples by using glutaraldehyde after cleaning, taking out the samples after a period of time, cleaning the samples soaked in the bacteria solution, dyeing for 30min by using propidium iodide, then washing away residual solution, and taking care to avoid light in the process; fixing a sample soaked in the seaweed solution by using glutaraldehyde, observing the adhesion change of bacteria/seaweed on the surface of the sample by using a laser confocal fluorescence microscope, calculating and analyzing the adhesion rate change of the bacteria/seaweed on the surface of the sample, and analyzing the antibacterial/seaweed mechanism of the antibacterial cyclopeptide modified surface, thereby providing a method and a theoretical basis for obtaining a green antifouling surface with antibacterial/seaweed.
Compared with other antifouling material preparation methods, the preparation method takes hard metal as a research object, and the antibacterial cyclic peptide modified surface not only has the characteristics of green and environmental protection, but also has the advantages of easily obtained raw materials, simple operation and large-scale and large-area preparation.
Drawings
FIG. 1 is a flow chart of a method for preparing a metal antifouling surface modified by cyclic peptide extracted from Chinese herb Viola Yedoensis and evaluating antifouling property.
FIG. 2 is a diagram showing the attachment of Vibrio natriegens to a sample before modification of cyclic peptides in the present invention.
FIG. 3 is a diagram showing the Vibrio natriegens attachment of the sample after modification of the cyclic peptide of the present invention.
FIG. 4 is a drawing showing the surface adherence of Citrobacter faberi to a sample before modification with cyclic peptide.
FIG. 5 is a drawing of the attachment of the sample surface F.citrobacter after cyclic peptide modification.
FIG. 6 is a sample surface bacillus attachment map before cyclic peptide modification.
FIG. 7 is a surface bacillus attachment map of a sample after cyclic peptide modification.
FIG. 8 is a drawing showing the attachment of Chlorella surfactin to a sample before modification of cyclic peptide.
FIG. 9 is a drawing showing the attachment of Chlorella surfactin after modification with cyclic peptide.
FIG. 10 is a drawing showing the adhesion of Phaeodactylum tricornutum on the surface of a sample before cyclic peptide modification.
FIG. 11 is a drawing of the attachment of Phaeodactylum tricornutum on the surface of a sample after cyclic peptide modification.
FIG. 12 is a drawing showing the attachment of navicula to the surface of a sample before modification with cyclic peptide.
FIG. 13 is a drawing showing the attachment of navicula on the surface of a sample after modification with cyclic peptide.
Detailed Description
The invention is further described below with reference to the accompanying drawings and methods of practice.
The preparation method of the traditional Chinese medicine extracted cyclic peptide modified metal surface shown in figure 1 comprises the following steps:
(1) drying herba Violae, and grinding into powder;
(2) according to the principle of 'polar similarity and intermiscibility' between chemical components of natural products and a solvent, 10L of mixed solution of ethanol and water is used for extracting antibacterial cyclic peptide in 1000 g of Chinese violet powder, and microwave assistance is adopted, wherein the volume ratio of the ethanol to the water is 3:2, the microwave assistance time is 0.5-3h, and the temperature is kept at 20 +/-2 ℃;
(3) carrying out solid-liquid separation on the solution containing the Chinese violet by high-speed centrifugation to obtain a solution containing the antibacterial cyclic peptide;
(4) separating and purifying the antibacterial cyclic peptide by using a high performance liquid chromatograph;
(5) reducing and digesting the cyclic peptide by using 0.1 mu M tri (2-carboxyethyl) phosphine and 0.5 mu g/mu L trypsin, and then respectively obtaining the amino acid sequence and the spatial structure of the antibacterial cyclic peptide by using a liquid phase mass spectrometer and a dichroism spectrometer;
(6) selecting 304 stainless steel as a substrate, polishing the substrate to a mirror surface, cleaning, drying, and soaking in 2 mg/mL dopamine solution for 24h to obtain surface-activated stainless steel;
(7) and (3) immersing the activated stainless steel surface into the solution of the antibacterial cyclic peptide for 24h, taking out the sample, and airing the sample in a nitrogen environment.
In the step (2), the temperature to be maintained is preferably 20 ℃, and in the step (4), an aqueous solution of 0.1% by volume of trifluoroacetic acid (TFA) is used as the solution a of the eluent, and an aqueous solution of 80% acetonitrile containing 0.1% by volume of TFA is used as the solution B of the eluent.
The method for performing antifouling evaluation by using the prepared antibacterial cyclic peptide modified surface comprises the following steps:
(1) selecting dominant marine strains of vibrio natriegens, citrobacter fabarum and bacillus, dominant marine algae phaeodactylum tricornutum, chlorella vulgaris and navicula, and respectively culturing the bacteria and the marine algae;
(2) respectively immersing the prepared samples into bacteria and seaweed culture solution, taking out the samples after a period of time (the bacteria solution is immersed for 24 hours, and the seaweed solution is immersed for 7 days), cleaning the samples immersed in the bacteria solution, dyeing for 30min by using propidium iodide (PI, 50 microgram/mL, and the solvent is artificial seawater), and then washing away the residual solution, wherein the process needs to be protected from light; fixing a sample soaked in the seaweed solution by using glutaraldehyde (2.5%), observing the adhesion change of bacteria/seaweed on the surface of the sample by using a laser confocal fluorescence microscope (CLSM), calculating and analyzing the adhesion change of the bacteria/seaweed on the surface of the sample by using Image J, and analyzing the antibacterial/seaweed mechanism of the antibacterial cyclic peptide modified surface to provide a method and a theoretical basis for obtaining a green antifouling surface with antibacterial/seaweed (for example, if the microbial biofilm attached to the surface of the sample is observed to be broken, the cyclic peptide residue on the modified surface is considered to have the effect on the cell wall of the microorganism to break the cell wall, so that the aim of resisting the bacteria/seaweed is fulfilled).
In specific implementation, the antibacterial cyclic peptide is extracted from Chinese herbal medicine herba violae (collected from Chinese herbal medicine herba violae in Yongchuan county of Sichuan province), the antibacterial cyclic peptide is used for modifying the surface of 304 stainless steel to obtain the surface of the stainless steel modified by the antibacterial cyclic peptide, and the antifouling property of the modified surface is evaluated through a static adhesion experiment of marine fouling organisms, and the specific process steps are as follows:
(1) selecting Chinese violet as a raw material, airing and crushing the Chinese violet, soaking 800 g of Chinese violet powder in 3.5L of dichloromethane for 24 hours, filtering out large-particle powder, repeating the operation for three times within 96 hours, evaporating the dichloromethane to obtain a Chinese violet crude extract, soaking the Chinese violet crude extract in 3.5L of ethanol/water (volume ratio of 3: 2) for 48 hours, and then performing high-speed centrifugation to realize solid-liquid separation on a dissolving solution containing the Chinese violet to obtain a solution containing the antibacterial cyclic peptide;
(2) the epoxide was separated from the n-butanol fraction using gel column chromatography (3 cm inner diameter × 42 cm long column packed with 70-230 mesh silica gel) and using CHCl3And absolute ethanol, increasing the proportion of ethanol to 100% at a rate of 70 drops/min;
(3) further fractionating the purified sample by HPLC, wherein the solution A is 0.1% trifluoroacetic acid (TFA) in water and the solution B is 80% acetonitrile in water containing 0.1% TFA, the time is set to 25 min, the percentage of the solution B is linearly increased from 10% to 70%, and the flow rate of the eluent in HPLC is 4 mL/min;
(4) treating each fraction with a low temperature vacuum drier to obtain antibacterial cyclic peptide powder;
(5) reducing and digesting the antibacterial cyclic peptide by using 0.1 mu M tri (2-carboxyethyl) phosphine and 0.5 mu g/mu L trypsin, and then respectively obtaining the amino acid sequence and the spatial structure of the antibacterial cyclic peptide by using a liquid phase mass spectrometer and a dichroism spectrograph;
(6) using dopamine as a coupling agent (the pH of a dopamine solution is =8.5, the concentration is 2 mg/mL), and combining the antibacterial cyclic peptide on the surface of stainless steel (the sample is soaked for 24 h), wherein the surface of the stainless steel is dark black;
(7) respectively using an XPS (X-ray diffraction), a contact angle measuring instrument and a surface profiler to represent chemical components, hydrophilicity and hydrophobicity and surface morphology of the surface of a sample, and analyzing the acquired data to obtain the roughness, surface energy and stability of the surface of the sample;
(8) selecting vibrio natriegens, citrobacter fabarum, bacillus, alga phaeodactylum tricornutum, chlorella and navicula algae, respectively expanding and culturing bacteria and alga to logarithmic phase, soaking the metal surface modified by the antibacterial cyclic peptide in the diluted bacterial liquid, observing the adhesion condition of the bacteria every 12 h, soaking the metal surface modified by the antibacterial cyclic peptide in the diluted alga solution, and observing the adhesion condition of the alga every 24h (the adhesion experiment of the alga requires 12 h darkness and 12 h illumination alternation conditions);
(9) soaking the sample after the experiment by using glutaraldehyde fixing solution, dyeing the sample soaked by the bacteria solution by using PI, placing the sample soaked by the seaweed solution into a refrigerator at the temperature of-4 ℃ for standby, collecting an attached fluorescence Image by using a laser confocal fluorescence microscope, observing the change of the bacteria/seaweed adhesion rate on the surface of the sample, and calculating the adsorption capacity by using Image J. The experimental results show that: the attachment rates of vibrio natriegens, citrobacter faecium and bacillus on the surface of the original sample are 4.505%, 6.13% and 6.347% respectively, the attachment rates of the three marine bacteria on the surface of the modified sample are 0.796%, 0.894% and 0.618% respectively, and compared with the original sample, the attachment rates of the three marine bacteria after cyclic peptide modification are reduced by 82.33%, 85.42% and 90.26% respectively (fig. 2-7); the attachment rates of chlorella, phaeodactylum tricornutum and navicula on the surface of the original sample are 8.51%, 8.60% and 2.23% respectively, the attachment rates of three marine bacteria on the surface of the modified sample are 0.697%, 0.468% and 0.358% respectively, and compared with the original sample, the attachment rates of the surfaces of the three seaweeds after cyclic peptide modification are reduced by 92.02%, 94.56% and 83.95% respectively (fig. 8-13), which proves that the cyclic peptide modified surface has obvious antibacterial/seaweed adsorption resistance; wherein, the white spots in FIGS. 2 to 7 are the attachment amounts of bacteria, and the white spots in FIGS. 8 to 13 are the attachment amounts of seaweeds.
Compared with other antifouling material preparation methods, the preparation method takes hard metal as a research object, and the antibacterial cyclic peptide modified surface not only has the characteristics of green and environmental protection, but also has the advantages of easily obtained raw materials, simple operation and large-scale and large-area preparation.
The present invention is not limited to the above-mentioned embodiments, and based on the technical solutions disclosed in the present invention, those skilled in the art can make some substitutions and modifications to some technical features without creative efforts according to the disclosed technical contents, and these substitutions and modifications are all within the protection scope of the present invention.
Claims (5)
1. A preparation method of a traditional Chinese medicine extracted cyclic peptide modified metal surface comprises the following steps:
(1) drying herba Violae, and grinding into powder;
(2) extracting 1000 g of antibacterial cyclic peptide in the Chinese violet powder by using 10L of mixed solution of ethanol and water, and assisting by using microwaves;
(3) carrying out solid-liquid separation on the solution containing the Chinese violet by high-speed centrifugation to obtain a solution containing the antibacterial cyclic peptide;
(4) separating and purifying the antibacterial cyclic peptide by using a high performance liquid chromatograph;
(5) reducing and digesting the cyclic peptide using 0.1 μ M tris (2-carboxyethyl) phosphine and 0.5 μ g/μ L trypsin;
(6) selecting 304 stainless steel as a substrate, polishing the substrate to a mirror surface, cleaning, drying, and soaking in 2 mg/mL dopamine solution for 24h to obtain surface-activated stainless steel;
(7) immersing the surface of the activated stainless steel into a solution of the antibacterial cyclic peptide for 24 hours, taking out a sample, and airing the sample in a nitrogen environment;
(8) respectively using an XPS (X-ray diffraction), a contact angle measuring instrument and a surface profiler to represent chemical components, hydrophilicity and hydrophobicity and surface morphology of the surface of a sample, and analyzing the acquired data to obtain the roughness, surface energy and stability of the surface of the sample;
(9) selecting vibrio natriegens, citrobacter fabarum, bacillus, alga phaeodactylum tricornutum, chlorella and navicula algae, respectively expanding and culturing bacteria and alga to logarithmic phase, soaking the metal surface modified by the antibacterial cyclic peptide in the diluted bacterial liquid, observing the adhesion condition of the bacteria every 12 h, soaking the metal surface modified by the antibacterial cyclic peptide in the diluted alga solution, and observing the adhesion condition of the alga every 24h, wherein the adhesion experiment of the alga requires the alternate conditions of 12 h darkness and 12 h illumination;
(10) soaking the sample after the experiment by using glutaraldehyde fixing solution, dyeing the sample soaked by the bacteria solution by using PI, placing the sample soaked by the seaweed solution into a refrigerator at the temperature of-4 ℃ for standby, collecting an attached fluorescence Image by using a laser confocal fluorescence microscope, observing the change of the bacteria/seaweed adhesion rate on the surface of the sample, and calculating the adsorption capacity by using Image J.
2. The method for preparing a metal surface modified by a cyclopeptide extracted from Chinese herbs according to claim 1, wherein in the step (4), an aqueous solution of 0.1% by volume of trifluoroacetic acid (TFA) is used as the A solution of the eluent, and an aqueous solution of 80% acetonitrile containing 0.1% by volume of TFA is used as the B solution of the eluent.
3. The method for preparing the metal surface modified by the cyclopeptide extracted from the traditional Chinese medicine according to claim 1, wherein in the step (5), after the cyclopeptide is reduced and digested, the amino acid sequence and the spatial structure of the antibacterial cyclopeptide are obtained by using a liquid phase mass spectrometer and a dichroism spectrometer respectively.
4. The method for preparing a metal surface modified by cyclopeptide extracted from traditional Chinese medicines according to any one of claims 1 to 3, wherein in the step (2), the volume ratio of ethanol to water is 3: 2.
5. The method for evaluating stain resistance using the cyclic peptide-modified metal surface according to any one of 1 to 4,
(1) selecting dominant marine strains of vibrio natriegens, citrobacter fabarum and bacillus, dominant marine algae phaeodactylum tricornutum, chlorella vulgaris and navicula, and respectively culturing the bacteria and the marine algae;
(2) respectively immersing the prepared samples into bacteria and seaweed culture solution, taking out the samples after a period of time, cleaning, fixing by using glutaraldehyde, taking out the samples after a period of time, cleaning the samples soaked in the bacteria solution, dyeing for 30min by using propidium iodide, and then washing away residual solution, wherein the process needs to be protected from light; fixing a sample soaked in the seaweed solution by using glutaraldehyde, observing the adhesion change of bacteria/seaweed on the surface of the sample by using a laser confocal fluorescence microscope, calculating and analyzing the adhesion rate change of the bacteria/seaweed on the surface of the sample, and analyzing the antibacterial/seaweed mechanism of the antibacterial cyclopeptide modified surface, thereby providing a method and a theoretical basis for obtaining a green antifouling surface with antibacterial/seaweed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911412794.1A CN111020598B (en) | 2019-12-31 | 2019-12-31 | Preparation and antifouling evaluation method for traditional Chinese medicine extracted cyclic peptide modified metal surface |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911412794.1A CN111020598B (en) | 2019-12-31 | 2019-12-31 | Preparation and antifouling evaluation method for traditional Chinese medicine extracted cyclic peptide modified metal surface |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111020598A CN111020598A (en) | 2020-04-17 |
| CN111020598B true CN111020598B (en) | 2022-01-11 |
Family
ID=70201121
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911412794.1A Active CN111020598B (en) | 2019-12-31 | 2019-12-31 | Preparation and antifouling evaluation method for traditional Chinese medicine extracted cyclic peptide modified metal surface |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111020598B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112626525B (en) * | 2020-10-16 | 2022-12-27 | 辽宁师范大学 | Soybean extract hydrolysate corrosion inhibitor and preparation method and application thereof |
| CN115739565B (en) * | 2022-10-28 | 2023-12-08 | 扬州大学 | Method for multiple modification of metal surface |
| CN117169393B (en) * | 2023-11-03 | 2024-03-19 | 杭州湃肽生化科技有限公司 | Method for detecting cyclic peptide in plant tissue |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108658923A (en) * | 2018-05-07 | 2018-10-16 | 山西纳安生物科技有限公司 | A method of extracting purple sweet potato cyanidin with acidic oxidized electric potential water |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7090856B2 (en) * | 2003-06-19 | 2006-08-15 | Hong Kong University Of Science And Technology | Anti-fouling exopolysaccharides isolated from cultures of Vibrio alginolyticus and Vibrio proteolyticus |
| CN105696333A (en) * | 2016-03-17 | 2016-06-22 | 袁春华 | Preparation method of compound antibacterial fibers |
-
2019
- 2019-12-31 CN CN201911412794.1A patent/CN111020598B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108658923A (en) * | 2018-05-07 | 2018-10-16 | 山西纳安生物科技有限公司 | A method of extracting purple sweet potato cyanidin with acidic oxidized electric potential water |
Non-Patent Citations (2)
| Title |
|---|
| 基于生物修饰的304不锈钢表面抗菌性能;曹攀等;《中国表面工程》;20170630;第30卷(第3期);第1-7页 * |
| 紫花地丁中抗甲型H1N1 流感病毒的环肽;刘忞之等;《药学学报》;20141231;第49卷(第6期);第905-912页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111020598A (en) | 2020-04-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111020598B (en) | Preparation and antifouling evaluation method for traditional Chinese medicine extracted cyclic peptide modified metal surface | |
| Amador et al. | Extraction of chromophoric humic substances from seawater | |
| Hendey | The diagnostic value of diatoms in cases of drowning | |
| Esteves et al. | Comparative characterization of humic substances from the open ocean, estuarine water and fresh water | |
| Shan et al. | Incubation habitats and aging treatments affect the formation of biofilms on polypropylene microplastics | |
| CN101041486A (en) | Method for treatment and comprehensive utilization of silk production waste water | |
| Zigah et al. | An investigation of size-fractionated organic matter from Lake Superior and a tributary stream using radiocarbon, stable isotopes and NMR | |
| CN104782626B (en) | A kind of allelochemical algae-inhibiting agent and method for extraction and purification thereof | |
| EP3067324A1 (en) | Method for the preparation of electrodes of boron-doped nanocrystalline diamond, an electrode prepared by this method and a use thereof | |
| CN114606134B (en) | Sponge coanda fungus and application thereof in preparation of oxaanthraquinone compounds | |
| KR101827777B1 (en) | Method for recovering non-destructive soil algae from soil | |
| Medwed et al. | Ecophysiological, morphological, and biochemical traits of free-living Diplosphaera chodatii (Trebouxiophyceae) reveal adaptation to harsh environmental conditions | |
| CN112725230A (en) | Selenium-resistant strain Enterobacter ludwigii GX-C3 and application thereof | |
| CN109053720B (en) | Alkaloid compound and preparation method and application thereof | |
| CN115161232A (en) | Pseudoalteromonas strain SCSIO43740 capable of producing quorum sensing inhibitor from coral and application thereof | |
| Burnison et al. | Isolation of colloidal fibrils from lake water by physical separation techniques | |
| Sun et al. | New insight in algal cell adhesion and cake layer evolution in algal-related membrane processes: Size-fractioned particles, initial foulant seeds and EDEM simulation | |
| CN110241111A (en) | A kind of method of modified activated carbon immobilized cell enhancing bacterium chromium reducing power | |
| CN111909881B (en) | Bacillus pumilus capable of producing feruloyl esterase and application thereof | |
| CN109503535B (en) | Bicyclic ring nerolidine type sesquiterpene derivative and preparation and application thereof | |
| Minami et al. | Chemical techniques to extract organic fractions from fossil bones for accurate 14C dating | |
| Šabanović et al. | Sorption of metals on pulverized pumpkin (Cucurbita pepo L.) peels | |
| CN102604868A (en) | Alteromonas and application thereof in inhibiting growth of red-tide algae | |
| CN111285937B (en) | Method for producing transparent extracellular polymeric particles based on cyanobacterial bloom water body | |
| CN112831431B (en) | Selenium-resistant strain Comamonas testosteroni GX-A1 and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |