CN116143876B - Carp scale antibacterial peptide, and preparation method and application thereof - Google Patents
Carp scale antibacterial peptide, and preparation method and application thereof Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- 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
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a carp scale antibacterial peptide, and a preparation method and application thereof, wherein the preparation method comprises the following steps: adding carp scales into citric acid solution for leaching, centrifuging, dialyzing, desalting, and freeze-drying to obtain carp scale collagen; preparing a carp scale collagen hydrolysate by ultrasonic-assisted protease hydrolysis; separating and purifying the carp scale collagen hydrolysate with the best antibacterial effect by ultrafiltration and semi-preparative liquid chromatograph; and carrying out LC-MS/MS mass spectrum analysis on the purified antibacterial peptide component and matching with a UniProt protein database to finally identify 3 novel carp fish scale antibacterial peptide sequences. The carp scale antibacterial peptide has good antibacterial effect on gram-negative bacteria and gram-positive bacteria, and meanwhile, safety evaluation experiment results show that the carp scale antibacterial peptide has low toxicity on liver cells LO 2. The invention provides a theoretical basis and an experimental basis for the comprehensive utilization of the fish scales of the later-stage carps and the research of novel antibiotic substitutes.
Description
Technical Field
The invention relates to the field of antibacterial peptide preparation, in particular to a carp scale antibacterial peptide, and a preparation method and application thereof.
Background
Antibacterial peptides are a class of small molecule polypeptides produced by the body in the defensive response against pathogenic microorganisms, and are also an important component of the innate immune system of the organism. The natural antibacterial peptide widely exists in bacteria, plants, mollusks, amphibians, fishes, birds, mammals and the like, and is expected to be a good substitute for antiseptic antibacterial drugs such as food preservatives, antibiotics and the like because the natural antibacterial peptide has the characteristics of strong antibacterial activity, good biocompatibility, wide sterilization range, difficult bacterial drug resistance and the like.
Fish are the major components of the aquatic animal population, in which various antimicrobial peptides are present. Many researches show that the antibacterial peptides in different fishes and different tissues and organs of the same fish are different, and the amino acid sequence homology is low, so that the antibacterial peptides from the fishes are various in variety, various in structure, wide in antibacterial spectrum and obvious in antibacterial activity difference. Meanwhile, the research shows that the fish antibacterial peptide has uniqueness, such as an antibacterial effect under extremely high salt concentration, and the fish antibacterial peptide has good development and application prospects.
China is a large country for freshwater fish culture, and yellow river carp is the most common variety in freshwater fish. The collagen content in the carp scales is rich, the carp scales are often discarded as processing byproducts, which not only results in the waste of protein resources, but also causes certain pollution to the environment due to unreasonable utilization of the carp scales, so that the improvement of the comprehensive utilization rate of the carp scales is particularly important. Based on the method, collagen is extracted from carp scales, and the carp scale antibacterial polypeptide is obtained through separation and purification by an enzymolysis method, so that not only is the waste scale resource fully utilized and the added value of fish products improved, but also the comprehensive utilization of freshwater fish byproducts is realized, and a theoretical basis and an experimental basis are provided for the application of the carp scale antibacterial peptide in the food or medicine industry in the later stage.
Disclosure of Invention
The invention aims to improve the comprehensive utilization rate of carp scales and simultaneously provides a simple and efficient preparation method of the carp scale antibacterial peptide. According to the invention, the carp scale antibacterial peptide is prepared by taking carp scales as raw materials for the first time through ultrasonic-assisted enzymolysis, ultrafiltration separation and semi-preparative liquid chromatography separation; and secondly, according to the invention, through mass spectrum identification of different peptide sequences and verification of antibacterial activity and cytotoxicity of the antibacterial peptide, three novel carp fish scale polypeptide sequences with biological safety and antibacterial potential are finally obtained.
The invention provides three novel antibacterial peptides derived from carp scales, which have inhibition effects on gram-negative bacteria and gram-positive bacteria, and the amino acid sequence of the antibacterial peptides is as follows:
SEQ ID NO.1,I-G-F-I-S-Q-P-Q-E-K-A-P-D-P-F;
SEQ ID NO.2,F-A-M-P-L-A-E-K-G-P-D-P-L-R-G-G-Y;
SEQ ID NO.3,I-G-F-I-S-Q-P-Q-E-K-A-P-D-P-F-R-S-Y。
the invention provides a preparation method of carp scale antibacterial peptide, which comprises the following steps:
1) Cutting carp scales, adding the cut carp scales into citric acid solution, placing the solution into a water bath with the temperature of 45-60 ℃ for leaching for 13-18 hours, centrifuging to obtain supernatant, dialyzing for desalination, concentrating and freeze-drying to obtain carp scale collagen;
in the step 1), the mass percentage of the citric acid solution is 5-15%, and more preferably 10%.
The ratio of the carp scales to the citric acid solution is 1 g:10-30 mL, and more preferably 1g:15mL.
Leaching in water bath at 50-55 deg.c for 15-16 hr.
The centrifugal conditions are as follows: the centrifugation is carried out at a temperature of 2 to 6℃and a rotational speed of 7000 to 9000rpm for 5 to 15 minutes, and more preferably at a temperature of 4℃and a rotational speed of 8000rpm for 10 to 15 minutes.
The dialysis desalination is performed for 40 to 56 hours, and more preferably, the dialysis desalination is performed for 48 hours.
2) Adding carp scale collagen into water to prepare a protein water solution, adding protease, carrying out enzymolysis for 2-3 hours at 37-55 ℃ by using an ultrasonic auxiliary protease hydrolysis method, inactivating enzyme at high temperature for 10-15 minutes after the reaction is finished, centrifuging the enzymolysis solution, taking supernatant, and freeze-drying and collecting to obtain the carp scale collagen hydrolysate;
in the step 2), the mass percentage concentration of the protein water solution is 1-10%, and more preferably 5%;
adding protease according to 1-4% (more preferably 2%) of the mass of the carp scale collagen;
3) Sequentially carrying out ultrafiltration and semi-preparative liquid chromatography separation on the carp scale collagen hydrolysate, collecting each antibacterial peptide component, and carrying out dialysis and desalination to obtain a carp scale antibacterial peptide purification component.
The preparation method of the carp scale antibacterial peptide comprises the following specific steps:
1) Extracting carp scale collagen: adding the sheared carp scales into 10% citric acid solution according to the ratio of 1g to 15mL, leaching for 150-16h in a water bath at 50-55 ℃, centrifuging for 10-15min at 4 ℃ and 8000rpm, taking supernatant, dialyzing for 48h, concentrating and freeze-drying to obtain the carp scale collagen;
2) Enzymolysis of carp scale collagen: adding carp scale collagen into ultrapure water to prepare an aqueous solution with the concentration of 5%, adding 2% protease (w/w), performing enzymolysis for 2-3 hours at 37-55 ℃ by using an ultrasonic auxiliary protease hydrolysis method, performing high-temperature enzyme deactivation treatment for 10-15 minutes, cooling to room temperature, centrifuging the enzymolysis solution, taking supernatant, and freeze-drying to obtain the carp scale collagen hydrolysate;
3) Separation and purification of carp scale antibacterial peptide: separating and purifying the carp scale collagen hydrolysate sequentially through ultrafiltration and semi-preparative liquid chromatography, collecting each antibacterial peptide purification component, and obtaining the carp scale antibacterial peptide purification component after dialysis and desalination;
4) Sequence identification of carp scale antibacterial peptide: and (3) selecting an antibacterial peptide component with optimal antibacterial activity for LC-MS/MS mass spectrometry analysis and identifying the amino acid sequence of the antibacterial peptide component through a matching pair UniProt database.
Preferably, the concentration of the citric acid solution in the step 1) is 10%, and the water extraction temperature is 50-55 ℃.
Preferably, the ultrasound-assisted conditions described in step 2) are: the ultrasonic power is 100-200W, and the ultrasonic time is 10-30min; further preferred, ultrasound-assisted conditions: the ultrasonic power is 100W and 200W, the ultrasonic temperature is 20-25 ℃, and the ultrasonic time is 10min, 20min and 30min.
Preferably, the protease in step 2) is: pepsin, alkaline protease, flavourzyme or papain, wherein pepsin is the optimal enzyme.
The enzymolysis conditions are as follows:
pepsin at 36-38 ℃ and pH 1.5-2.5, more preferably, pepsin at 37 ℃ and pH 2.0;
alkaline protease 53-57 ℃, pH 8.5-9.5, more preferably, alkaline protease 55 ℃, pH 9.0;
the flavourzyme is 48-52 ℃, the pH is 7.5-8.5, more preferably, the flavourzyme is 50 ℃, and the pH is 8.0;
papain 48-52 deg.c, pH 7.5-8.5, and preferably papain 50 deg.c and pH 8.0.
Preferably, the enzyme deactivation temperature of the hydrolysate in the step 2) is 95-100 ℃ and the time is 10-15min; the centrifugation conditions are as follows: centrifugal force is applied at 9000-11000 rpm at 2-6deg.C for 10-30min, more preferably at 10000rpm at 4deg.C for 15-20min.
Preferably, the ultrafiltration in the step 3) adopts PES ultrafiltration membranes with molecular weights of 3kDa and 10kDa, and the specific process is that the carp scale collagen hydrolysate is subjected to ultrafiltration treatment by adopting different ultrafiltration membranes, and the hydrolysate components with the retention amounts of <3,3-10 and >10kDa are collected.
Preferably, the semi-preparative liquid chromatography separation elution procedure in step 3) is as follows: 0-5min,5% mobile phase B,5-15min,40% mobile phase B,15-25min,95% mobile phase B; the flow rate is 5-6mg/mL, the loading volume is 1-2mL, and the sample concentration is 10-20mg/mL. Wherein mobile phase A is ultrapure water and mobile phase B is acetonitrile.
Preferably, the dialysis time in step 3) is 24-48 hours and the dialysis bag size is 100Da.
Preferably, the amino acid sequence of the polypeptide identified in step 4) having antibacterial potential is:
SEQ ID NO.1,I-G-F-I-S-Q-P-Q-E-K-A-P-D-P-F;
SEQ ID NO.2,F-A-M-P-L-A-E-K-G-P-D-P-L-R-G-G-Y;
SEQ ID NO.3,I-G-F-I-S-Q-P-Q-E-K-A-P-D-P-F-R-S-Y。
the invention also provides application of the carp scale antibacterial peptide identified according to the preparation method in inhibiting various gram-positive bacteria (staphylococcus aureus, bacillus subtilis and listeria monocytogenes) and gram-negative bacteria (escherichia coli, salmonella and pseudomonas aeruginosa). Verification of human hepatocyte LO2 cytotoxicity by carp scale antibacterial peptide identified by mass spectrometry. The bacteria are one or more of escherichia coli, salmonella typhimurium, pseudomonas aeruginosa, staphylococcus aureus, listeria monocytogenes and bacillus subtilis.
Compared with the prior art, the invention has the advantages that:
(1) According to the invention, three novel carp scale antibacterial peptides with antibacterial potential are obtained by taking carp scales as raw materials for the first time through identification;
(2) According to the invention, the carp scale antibacterial peptide is prepared by combining a plurality of separation and purification technologies through an ultrasonic-assisted enzymolysis method, and the preparation method is simple and feasible, low in cost and high in safety;
(3) The invention can effectively expand the application range of the carp byproducts, improve the comprehensive utilization rate of the carp processing byproduct resources, reduce the problems of unreasonable utilization of the resources and environmental pollution, and has important significance for the development of industries such as food, medicine, health care products and the like.
Drawings
FIG. 1 is a semi-preparative liquid chromatographic separation of <3kDa antimicrobial peptide component;
FIG. 2 is an amino acid sequence and a secondary mass spectrum of the carp scale antibacterial peptide;
FIG. 3 shows cytotoxicity of carp scale antibacterial peptide on human liver cell LO2 at different concentrations.
Detailed Description
The present invention will be described in detail with reference to specific embodiments thereof for the purpose of more clearly describing the objects and advantages of the present invention. The scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Meanwhile, the embodiments described herein are only for explaining the present invention, and do not represent the scope of the present invention as defined by the claims.
The following is a further explanation of the present invention by way of specific examples.
Example 1
The method is used for screening the optimal process for preparing the carp scale collagen hydrolysate by pepsin hydrolysis. The method comprises the following specific steps:
1) Extracting carp scale collagen: weighing 100g of treated carp scales (shearing treatment), adding the carp scales into 1500mL of citric acid aqueous solution with mass concentration of 10% according to the ratio of 1g to 15mL, leaching for 15.5h in a water bath at 53 ℃, centrifuging the solution for 15min at 4 ℃ and 8000rpm, taking supernatant, dialyzing and desalting for 48h, concentrating, collecting, freeze-drying, collecting to obtain carp scale collagen, and storing at-80 ℃ for later use;
2) Preparation of carp scale collagen hydrolysate: weighing 10g of carp scale collagen, adding into ultrapure water to prepare a protein water solution with the mass percentage concentration of 5%, adding pepsin (0.2 g) according to 2% of the mass of the carp scale collagen, pretreating the protein solution by using different ultrasonic conditions, and then performing enzymolysis under the optimum condition of pepsin (pepsin enzymolysis condition: 37 ℃ and pH of 2.0), wherein the ultrasonic treatment conditions are as follows: ultrasonic power 100W, 200W; the ultrasonic time is 10min, 20min and 30min. Stirring for 3h at 500rpm, and after the reaction is finished, carrying out boiling water bath on the enzymolysis liquid for 15min, and rapidly cooling to room temperature of 25 ℃. Centrifuging at 10000rpm at 4deg.C for 15min, collecting supernatant, dialyzing, and lyophilizing to obtain carp scale collagen hydrolysate.
Different processing condition groupings: h0- -no sonication
H1-Ult-A---200W,10min(H1-A);H1-Ult-B---200W,20min(H1-B);
H1-Ult-C---200W,30min(H1-C);H1-Ult-D---100W,10min(H1-D);
H1-Ult-E---100W,20min(H1-E);H1-Ult-F---100W,30min(H1-F);
3) Antibacterial activity assay: the antimicrobial activity of the different carp scale collagen hydrolysates was evaluated by determining their Minimum Inhibitory Concentration (MIC). The specific operation is as follows: selecting Escherichia coli and Staphylococcus aureus as strains to be detected, inoculating the bacteria into NB in a shaking table at 37deg.C and rotation speed of 180rpmThe broth is cultured overnight to logarithmic phase, and the broth is diluted to about 1×10 with fresh NB broth 6 CFU/mL for use. Adding 100 μl of the prepared bacterial suspension into sterile 96-well plate, adding 100 μl of enzymolysis solution with different concentrations into 96-well plate, standing 96-well plate at 37deg.C in constant temperature incubator for 24 hr, and detecting absorbance (OD) of each well at 600nm with enzyme marker 600 ) The minimum inhibitory concentration was determined as absorbance value (OD 600 )<Minimum antimicrobial peptide concentration of 0.1.
As can be seen from table 1, the MIC results of the carp scale crude antimicrobial peptides are significantly affected by different ultrasound-assisted conditions. Compared with other ultrasonic treatment conditions, at ultrasonic power of 200W and ultrasonic time of 20min, the crude antibacterial peptide prepared by pepsin hydrolysis of carp squamous collagen has the lowest MIC value to escherichia coli (ATCC 8749) and staphylococcus aureus (ATCC 25923), and the MIC values are 5mg/mL and 3.5mg/mL respectively. Therefore, the optimal ultrasonic treatment condition for preparing the carp scale collagen hydrolysate by gastric proteolysis in the embodiment is that ultrasonic power: 200W, ultrasonic time: 20min.
TABLE 1 MIC results of carp Scale collagen hydrolysate
Example 2
The embodiment is used for explaining the separation and purification method of the carp fish scale antibacterial peptide. The method comprises the following specific steps:
1) Ultrafiltration separation: the carp scale collagen hydrolysate component with higher antibacterial activity in the embodiment 1 sequentially passes through ultrafiltration membranes with the cutoff molecular weight of 3kDa and 10kDa, the hydrolysate components with cutoff amounts of <3kDa,3-10kDa and >10kDa are collected, and antibacterial peptide cutoff filtrates with different molecular weights are concentrated and dialyzed and then freeze-dried and collected at the temperature of minus 80 ℃ for storage.
2) Semi-preparative liquid chromatography separation: and (3) further purifying the antibacterial peptide component with better antibacterial activity obtained by ultrafiltration separation in the step (1). Dissolving the lyophilized antibacterial peptide component with ultrapure water to prepare a preparation solution of 10mg/mL, loading the solution to a 0.22 mu m filter membrane, loading the solution to a 1mL semi-prepared column by using Zorbax SB-300C 18, taking the mobile phase A as ultrapure water, taking the mobile phase B as acetonitrile at a flow rate of 6mL/min, detecting the wavelength of 220nm, and collecting the components after gradient elution of the sample, wherein the elution procedure is as follows: 0-5min,5% mobile phase B,5-15min,40% mobile phase B,15-25min,95% mobile phase B. The 3 peak fractions with higher response values were collected and named F1, F2 and F3, respectively (fig. 1). And (3) after multiple collection, dialyzing and desalting, and freeze-drying to obtain the purified component of the carp scale antibacterial peptide.
3) Identification of antibacterial peptide sequences: and (3) carrying out LC-MS/MS identification on the antibacterial peptide component with optimal antibacterial activity in the step (2) and analyzing the amino acid sequence of the antibacterial peptide component, and finally identifying by matching with a UniProt protein database to obtain the carp scale antibacterial peptide with antibacterial potential.
In this example, 3 molecular weight ranges of <3kDa,3-10kDa, and >10kDa were isolated by ultrafiltration. As can be seen from Table 2, the MIC of the <10kDa antimicrobial peptide component was significantly reduced compared to that of the carp collagen hydrolysate and the >10kDa component, wherein the <3kDa antimicrobial peptide component has the lowest MIC value of 3mg/mL, 4mg/mL, 2.5mg/mL and 2.5mg/mL for E.coli (ATCC 8739), salmonella typhimurium (ATCC 14028), staphylococcus aureus (ATCC 25923) and Bacillus subtilis (ATCC 6633), respectively, indicating that the <3kDa antimicrobial peptide component contains more polypeptide having antimicrobial activity. Therefore, the invention selects the antibacterial peptide component with the molecular weight of <3kDa to separate and purify by semi-preparative liquid chromatography.
TABLE 2 MIC results of Cyprinus Carpio Scale collagen hydrolysate and Ultrafiltration fractions of different antimicrobial peptides
The MIC values of the three purified components F1-F3 obtained by separating the carp fish scale antibacterial peptide <3kDa component through semi-preparative liquid chromatography are shown in Table 3, and the antibacterial peptide component purified through semi-preparative liquid chromatography has good antibacterial activity on all of escherichia coli (ATCC 8739), salmonella typhimurium (ATCC 14028), staphylococcus aureus (ATCC 25923) and bacillus subtilis (ATCC 6633). The F3 component has better inhibiting effect relative to other antibacterial peptide separation components, the MIC value of the F3 component is obviously lower than that of other purification components, the MIC of the F3 component is 2.5mg/mL for escherichia coli, the MIC of the F3 component for salmonella typhimurium is 3mg/mL, the MIC of the F3 component for staphylococcus aureus is 1.5mg/mL, and the MIC of the F3 component for bacillus subtilis is 1.75mg/mL, which shows that the F3 component has huge antibacterial potential and possibly contains high-efficiency antibacterial active peptide, so that the F3 component is selected for LC-MS/MS mass spectrum analysis and identification of antibacterial peptide sequences. The mass spectrum identification result is shown in figure 2, and three novel carp scale antibacterial peptide sequences are mainly obtained through identification.
TABLE 3 MIC results for different semi-preparative liquid chromatography separation and purification fractions F1-F3
Example 3
This example was used to synthesize antimicrobial peptides and determine the Minimum Inhibitory Concentration (MIC) of the antimicrobial peptides against bacteria. Peptide sequences identified by mass spectrometry were synthesized and the purity of the synthesized peptides was analyzed by HPLC (> 95%). Wherein the antibacterial peptide sequence is as follows:
SEQ ID NO.1, I-G-F-I-S-Q-P-Q-E-K-A-P-D-P-F, abbreviated CSC-1;
SEQ ID NO.2, F-A-M-P-L-A-E-K-G-P-D-P-L-R-G-G-Y, abbreviated CSC-2
SEQ ID NO.3, I-G-F-I-S-Q-P-Q-E-K-A-P-D-P-F-R-S-Y, abbreviated CSC-3.
The antibacterial activity of this example was evaluated by determining the Minimum Inhibitory Concentration (MIC) of three carp scale antibacterial peptides against escherichia coli, salmonella, pseudomonas aeruginosa, staphylococcus aureus, bacillus subtilis and listeria monocytogenes.
Determining the Minimum Inhibitory Concentration (MIC) of the carp scale antibacterial peptide by using a microdilution method: firstly, respectively inoculating the above strains into NB broth liquid culture medium for activation culture, shake culturing at 37deg.C in a constant temperature shaking table with rotation speed of 180rpmDuring several growth periods, the bacterial liquid is diluted to about 1X 10 by fresh NB liquid culture medium 6 CFU/mL for use. Next, 100. Mu.L of the above prepared bacterial suspension was added to a sterile 96-well plate, 100. Mu.L of NB liquid medium containing antimicrobial peptides of different concentrations was added to the 96-well plate one by one, and finally the 96-well plate was placed in a constant temperature incubator at 37℃for stationary culture for 24 hours. Detection of absorbance values (OD) of each well at 600nm wavelength by using a microplate reader 600 ) The minimum inhibitory concentration was determined as absorbance value (OD 600 )<Minimum antimicrobial peptide concentration of 0.1.
As shown in Table 4, the three novel carp scale antibacterial peptides (CSC-1-3) obtained by the invention have good inhibition effect on various bacteria. Wherein, compared to the other two antimicrobial peptides, CSC-3 has the lowest MIC value for a variety of bacteria, indicating that CSC-3 has the optimal bacteriostatic activity, with a MIC value for escherichia coli (ATCC 8739) of 2mg/mL, for salmonella typhimurium of 2.5mg/mL, for pseudomonas aeruginosa of 1.5mg/mL, for staphylococcus aureus of 1mg/mL, for bacillus subtilis of 1mg/mL, for listeria monocytogenes of 0.5mg/mL. The antibacterial experimental result shows that the invention successfully identifies the novel carp fish scale antibacterial peptide with broad-spectrum antibacterial activity.
TABLE 4 MIC results of different carp Scale antibacterial peptides CSC-1-CSC-3
Example 4
This example was used to determine the cytotoxicity of carp scale antibacterial peptide to human hepatocytes LO 2. The invention adopts MTT method to measure the cytotoxicity of the antibacterial peptide, and comprises the following specific steps:
cell resuscitation: taking frozen liver cells LO2 in liquid nitrogen, rapidly placing in a constant-temperature water bath kettle at 37 ℃, shaking to melt, blowing uniformly, transferring into a 10mL centrifuge tube, adding 3mL DMEM culture medium, mixing uniformly, centrifuging at 1000rpm for 3min, discarding supernatant, adding 8mL DMEM culture medium, blowing uniformly, transferring into a cell culture bottle, placing in 37 ℃ and 5% CO 2 Is cultured overnight in a cell incubator.
And (3) passage: taking out the cell culture flask, sucking the DMEM medium, adding 1mL of PBS, washing 2 times, adding 1mL of 0.25% pancreatin, standing at 37deg.C, and 5% CO 2 Digestion for 60-90s, adding 3mL fresh DMEM culture medium to stop digestion, purging bottle wall to completely drop cells, transferring into 4mL centrifuge tube, centrifuging at 1000rpm for 3min, discarding supernatant, adding 3mL DMEM culture medium into the centrifuge tube, blowing uniformly, transferring 1mL into cell culture bottle containing 6mL DMEM culture medium, placing at 37deg.C, and placing in 5% CO 2 The culture is carried out for 48 hours in a cell incubator, and the liquid can be changed every other day according to the cell state, so that the activity of the resuscitated cells is ensured.
And (3) paving: taking out cell culture flask, sucking DMEM medium, adding 1mL PBS, washing for 2 times (washing serum), digesting liver cells LO2 with 1mL 0.25% pancreatin, centrifuging at 1000rpm for 3min, collecting cells, adjusting cell suspension concentration, inoculating into 96-well plate, and 100 μL per well to maintain cell number at 5000 cells/well, placing at 37deg.C and 5% CO 2 Is cultured overnight in a cell box of (2) to allow cells to grow on the wall.
Sample adding: taking out the 96-well plate inoculated with cells, sucking the waste DMEM culture medium, adding fresh DMEM culture medium containing different concentrations (0.5-4 mg/mL) of antibacterial peptide, setting 3-5 parallel concentrations each, 100 mu L of each pore volume, adding the same volume of fresh DMEM culture medium without antibacterial peptide into a control group, and placing at 37 ℃ and 5% CO 2 The cell culture was continued for 24 hours in the incubator.
Adding MTT for dyeing: after 24h of incubation, the 96-well plates were removed, 20. Mu.L of 5mg/mL MTT solution was added to each well, and the incubation was continued in a cell incubator for 4h under dark conditions.
Dissolving and crystallizing, and measuring absorbance: the DMEM medium in the wells was aspirated, 100. Mu.L of dimethyl sulfoxide (DMSO) was added to each well, and the wells were placed in a shaking table at 37℃for 10min with low-speed shaking, after which crystals were sufficiently dissolved, absorbance at 490nm was measured using an ELISA.
As can be seen from the data in fig. 3, the survival rate of LO2 cells gradually decreased with increasing concentration of antimicrobial peptide. The proliferation and activity of the carp scale antibacterial peptide on LO2 cells can not be obviously inhibited in the concentration range of 0.5-1mg/mL, namely, the cell survival rate is more than 80%, and in the concentration range of 1-4mg/mL, the LO2 cell survival rate after CSC-3 treatment is still more than 80%, which indicates that the CSC-3 has lower cytotoxicity in the concentration range of 0.5-4mg/mL, and can be considered to not cause toxic threat to normal cells, and the biological safety is reliable.
In conclusion, the carp scale source polypeptide with broad-spectrum antibacterial activity and low biotoxicity is successfully prepared, can be widely applied to the fields of food, agriculture and medicine, improves the comprehensive utilization rate of carp resources, and provides a reference basis for recycling freshwater fish processing byproducts.
The foregoing is merely illustrative of the preferred embodiments of this invention, and it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the principles of the invention.
Claims (7)
1. The carp scale antibacterial peptide is characterized in that the amino acid sequence of the antibacterial peptide is specifically as follows:
SEQ ID NO.1:I-G-F-I-S-Q-P-Q-E-K-A-P-D-P-F;
SEQ ID NO.2:F-A-M-P-L-A-E-K-G-P-D-P-L-R-G-G-Y;
SEQ ID NO.3:I-G-F-I-S-Q-P-Q-E-K-A-P-D-P-F-R-S-Y。
2. the method for preparing the carp scale antibacterial peptide according to claim 1, which comprises the following steps:
1) Cutting carp scales, adding the cut carp scales into citric acid solution, placing the solution into a water bath with the temperature of 45-60 ℃ for leaching for 13-18 hours, centrifuging to obtain supernatant, dialyzing for desalination, concentrating and freeze-drying to obtain carp scale collagen;
2) Adding carp scale collagen into water to prepare a protein water solution with the mass percentage concentration of 5%, adding pepsin according to the mass of 2% of the carp scale collagen, and utilizing an ultrasonic-assisted pepsin hydrolysis method, wherein the pepsin enzymolysis condition is as follows: the ultrasonic auxiliary conditions are that the temperature is 37 ℃, the pH is 2.0: ultrasonic power is 200W, ultrasonic time is 20min, stirring is carried out for 3h at a rotating speed of 500rpm, after the reaction is finished, the enzymolysis liquid is subjected to boiling water bath for 15min, the temperature is quickly cooled to room temperature of 25 ℃, the supernatant is taken after the enzymolysis liquid is centrifuged, and the carp scale collagen hydrolysate is obtained through dialysis, freeze-drying and collection;
3) Sequentially carrying out ultrafiltration and semi-preparative liquid chromatography separation on the carp scale collagen hydrolysate, ultrafiltering, separating and collecting the hydrolysate component with the retention of less than 3kDa, then carrying out semi-preparative liquid chromatography separation, collecting the peak component with the highest response value, and obtaining the purified component of the carp scale antibacterial peptide after dialysis and desalination.
3. The method for preparing the carp scale antibacterial peptide according to claim 2, wherein in the step 1), the mass percentage of the citric acid solution is 5-15%;
the ratio of the carp scales to the citric acid solution is 1 g:10-30 mL.
4. The method for preparing the carp scale antibacterial peptide according to claim 2, wherein in the step 1), the carp scale antibacterial peptide is placed in a water bath with the temperature of 50-55 ℃ for leaching for 15-16 hours;
the centrifugal conditions are as follows: centrifuging for 5-15min at 2-6 ℃ and rotating speed of 7000-9000 rpm;
and (5) dialyzing and desalting for 40-56 h.
5. The method for producing an antibacterial peptide against carp scales according to claim 2, wherein in step 2), the centrifugation conditions are: centrifuging at the temperature of 2-6 ℃ and the rotating speed of 9000-11000 rpm for 10-30 min.
6. The method for preparing the carp scale antibacterial peptide according to claim 2, wherein in the step 3), a PES ultrafiltration membrane with molecular weight of 3kDa and 10kDa is adopted for ultrafiltration;
elution conditions for semi-preparative liquid chromatography separation: mobile phase a was ultrapure water and mobile phase B was acetonitrile.
7. The use of a carp scale antibacterial peptide according to claim 1, in the preparation of a medicament for inhibiting gram-negative bacteria or gram-positive bacteria, wherein the gram-positive bacteria is staphylococcus aureus, bacillus subtilis or listeria monocytogenes, and the gram-negative bacteria is escherichia coli, salmonella typhimurium or pseudomonas aeruginosa.
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CN110590908A (en) * | 2019-08-26 | 2019-12-20 | 舟山市常青海洋食品有限公司 | Micropterus-derived antibacterial peptide additive and preparation method thereof |
CN111647043A (en) * | 2019-08-07 | 2020-09-11 | 中国农业大学 | Oligopeptide with platelet resisting and antithrombotic functions containing Hyp-Gly sequence |
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WO2009149554A1 (en) * | 2008-06-11 | 2009-12-17 | National Research Council Of Canada | Antimicrobial components of the mucus and extruded slime of hagfish (myxine glutinosa) |
CN111647043A (en) * | 2019-08-07 | 2020-09-11 | 中国农业大学 | Oligopeptide with platelet resisting and antithrombotic functions containing Hyp-Gly sequence |
CN110590908A (en) * | 2019-08-26 | 2019-12-20 | 舟山市常青海洋食品有限公司 | Micropterus-derived antibacterial peptide additive and preparation method thereof |
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