CN111825748B - Antibacterial peptide of turbot and application thereof - Google Patents

Antibacterial peptide of turbot and application thereof Download PDF

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Publication number
CN111825748B
CN111825748B CN202010696832.7A CN202010696832A CN111825748B CN 111825748 B CN111825748 B CN 111825748B CN 202010696832 A CN202010696832 A CN 202010696832A CN 111825748 B CN111825748 B CN 111825748B
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turbot
antibacterial peptide
bacteria
antibacterial
food
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CN111825748A (en
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毕景然
姜景慧
侯红漫
张公亮
郝洪顺
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Dalian Polytechnic University
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    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/158—Fatty acids; Fats; Products containing oils or fats
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    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract

The invention discloses a turbot antibacterial peptide, the amino acid sequence of which is shown as SEQ ID No. 1. The antibacterial peptide is identified from the internal organs of the turbot, and is subjected to in vitro chemical synthesis, so that the turbot antibacterial peptide prepared in vitro is low in cytotoxicity and low in hemolysis, and has high-efficiency antibacterial activity on gram-positive bacteria, such as staphylococcus aureus, listeria monocytogenes, bacillus subtilis, gram-negative bacteria, such as escherichia coli, salmonella and hafnia alvei. Therefore, the antibacterial peptide of the turbot has wide application prospect in the aspects of preparing food additives, food packaging materials, anti-infective drugs, agriculture and animal husbandry development, human disease prevention and treatment, washing cosmetics and the like.

Description

Antibacterial peptide of turbot and application thereof
Technical Field
The invention belongs to the technical field of antibacterial peptide research, and particularly relates to antibacterial peptide of turbot and application thereof.
Background
The antibacterial peptide is a small molecular polypeptide with antibacterial activity, widely exists in organisms, is an important barrier for organism immunity, and can kill pathogenic microorganisms directly or indirectly by regulating the immune system of the organism. The antibacterial peptide is mostly composed of 10-50 amino acid residues, has net positive charges, and has good hydrophobicity and amphiphilicity. Unlike traditional antibiotics, it is generally accepted that the target of action of the antimicrobial peptide is the cell membrane of the bacteria. After the antimicrobial peptide is contacted with a microbial lipid membrane, a positively charged hydrophilic part of the antimicrobial peptide can be combined with a negatively charged phosphate group in the microbial lipid membrane through electrostatic interaction, and a hydrophobic part of the antimicrobial peptide can be inserted into a lipid bilayer through hydrophobic interaction, so that the permeability and barrier function of the microbial lipid membrane are damaged, the leakage or coagulation of cell contents is caused, and bacteria are killed. And the bacteria hardly change the components of the cell membranes of the bacteria in a short time, so that the bacteria are not easy to generate drug resistance to the antibacterial peptide.
At present, various types of antibacterial peptides are continuously isolated, purified and identified from animals, plants and microorganisms, and thousands of natural antibacterial peptides (ADP: The antibacterial Peptide Database, http:// APs. unmac. edu/AP/main. php) have been included in The antibacterial Peptide Database so far. Many antibacterial peptides have been prepared as drugs or food additives and widely used. Therefore, obtaining an antibacterial peptide with a simple structure, high antibacterial activity and easy preparation is an urgent need in the development and research of antibacterial peptides.
Turbot (Scophthalmus maximus) is a typical marine economic fish, and the visceral part of the turbot is mostly discarded as a processing byproduct, thereby causing resource waste and environmental pollution.
Disclosure of Invention
In order to improve the resource utilization rate, the turbot viscera is used as a raw material, and the antibacterial peptide is obtained according to the physicochemical characteristics of the antibacterial peptide, including total charge, hydrophobic distance, secondary structure, antibacterial activity, cytotoxicity and hemolysis rate.
The invention aims to provide a turbot antibacterial peptide and application thereof. The antibacterial peptide of the turbot has wide antibacterial spectrum, low cytotoxicity and hemolysis.
In order to achieve the purpose, the invention adopts the technical scheme that:
an antibacterial peptide of turbot contains 16 amino acid residues, and the amino acid sequence of the antibacterial peptide is shown as SEQ ID No. 1 and is GITDLRGMLKRLKKMK; the molecular weight is 1888.41Da, the charge amount is +5, and the hydrophobic distance is 0.480 muH.
The minimum inhibitory concentrations of the antibacterial peptide of the turbot to gram-positive bacteria Staphylococcus aureus (Staphylococcus aureus), Listeria monocytogenes (Listeria monocytogenes) and Bacillus subtilis (Bacillus subtilis) are respectively 2.5 mu M, 5 mu M and 5 mu M, and the minimum inhibitory concentrations to gram-negative bacteria Escherichia coli (Escherichia coli), Salmonella (Salmonella) and Hafnia alvei (Hafnia alvei) are respectively 2.5 mu M, 2.5 mu M and 5 mu M.
The invention also provides the application of the antibacterial peptide of the turbot;
the application of the antibacterial peptide of the turbot in preparing the bacteriostatic agent for inhibiting bacteria, wherein the bacteriostatic agent takes the antibacterial peptide of the turbot as an active ingredient and comprises a food bacteriostatic agent, a feed bacteriostatic agent for animals or a medicine bacteriostatic agent.
Under the preferred mode, the food bacteriostatic agent is a fresh food bacteriostatic agent, and the antibacterial peptide aqueous solution of the turbot with the concentration of 5-50 mu M is sprayed on the surface of the fresh food for keeping the fresh of the fresh food.
Preferably, the raw fresh food is salmon.
In a preferred mode, the food bacteriostatic agent is a health-care product bacteriostatic agent; when the health-care product is prepared, the antibacterial peptide of the turbot is added into the health-care product by 0.01-1 per mill of weight fraction to inhibit bacteria.
Preferably, the health-care product is a vitamin C effervescent tablet.
In a preferable mode, the feed is silage alfalfa feed, and 5-50 mu M of a turbot antibacterial peptide aqueous solution is sprayed on the surface of the silage and is used for bacteriostasis of the silage.
Preferably, the feed is a granular feed; when preparing granular feed, the antibacterial peptide of the turbot is added into the granular animal feed by 0.01-1 per mill of weight fraction for bacteriostasis.
Preferably, the granular feed is dog food.
In a preferred mode, the dog food is prepared from the following raw materials in percentage by weight: 20% of corn flour, 17% of milk powder, 10% of potato, 10% of soybean flour, 2.995% of salt, 10% of chicken powder, 20% of chicken bone powder, 10% of lard oil and 0.005% of antibacterial peptide of turbot.
Preferably, the bacteria comprise: staphylococcus aureus (Staphylococcus aureus), Listeria monocytogenes (Listeria monocytogenes), Bacillus subtilis (Bacillus subtilis), Escherichia coli (Escherichia coli), Salmonella (Salmonella), Hafnia alvei (Hafnia alvei).
The application of the antibacterial peptide of the turbot in preparing food, medicines and/or health-care products is disclosed, and the antibacterial peptide of the turbot is used for inhibiting bacteria in the food, medicines and/or health-care products.
Preferably, the medicine is hydrogel for bacteriostatic dressing.
In a preferred mode, the hydrogel for the bacteriostatic dressing is prepared from the following components: the antibacterial peptide of the turbot, sodium alginate, gelatin and water; the weight ratio of the antibacterial peptide of the turbot, the sodium alginate and the gelatin is 0.125-1: 1: 1; the weight volume ratio of the gelatin to the water is 1:1 mg/L; the amino acid sequence of the antibacterial peptide of the turbot is shown as SEQ ID No. 1. The antibacterial dressing hydrogel can be used for wound healing. The invention also provides a preparation method of the hydrogel for the antibacterial dressing, which comprises the following steps: dissolving the antibacterial peptide of the turbot, sodium alginate and gelatin in ultrapure water respectively, carrying out ultrasonic treatment at 200-1500W for 5-120 min, and standing for 5-120 min to obtain the hydrogel for the antibacterial peptide dressing of the turbot.
The application of the antibacterial peptide of the turbot in preparing a packaging material for inhibiting the growth of bacteria can be used for packaging food, medicines and cosmetics.
In a preferred mode, the antibacterial peptide of the turbot is applied to the preparation of a packaging material for inhibiting the growth of bacteria, and the bacteria comprise: staphylococcus aureus (Staphylococcus aureus), Listeria monocytogenes (Listeria monocytogenes), Bacillus subtilis (Bacillus subtilis), Escherichia coli (Escherichia coli), Salmonella (Salmonella), Hafnia alvei (Hafnia alvei).
Preferably, the packaging material is prepared from the following components: the antibacterial peptide comprises turbot antibacterial peptide, 1g/mL polyvinyl alcohol aqueous solution and 1g/mL chitosan acetic acid solution; the preparation method of the 1g/mL chitosan acetic acid solution comprises the following steps: dissolving chitosan in 2% volume fraction acetic acid solution; the weight ratio of the antibacterial peptide of the turbot, the chitosan and the polyvinyl alcohol is 1 per mill-2 percent to 1: 1-30; the amino acid sequence of the antibacterial peptide of the turbot is shown as SEQ ID No. 1; and (2) blending the chitosan acetic acid solution, the polyvinyl alcohol aqueous solution and the antibacterial peptide of the turbot, pouring the mixture into a polyvinyl chloride mould, freezing the mixture for 4 to 24 hours at a temperature of between 80 ℃ below zero and 20 ℃ below zero, melting the mixture for 4 to 24 hours at a temperature of between 4 and 37 ℃ to form 1 cycle, and performing 3 to 10 cycles of co-circulation to obtain the antibacterial peptide-chitosan-polyvinyl alcohol packaging material of the turbot.
The invention has the beneficial effects that:
the antibacterial peptide extracted and screened by the invention has the advantages of small molecular weight, strong antibacterial action and the like, and also has the characteristics of small toxicity to normal human cells and low hemolysis.
Drawings
FIG. 1 is a projection of a Schiff-Edmonton helical wheel of antibacterial peptide of turbot according to example 2 of the present invention (the direction of the arrow is the hydrophobic side).
FIG. 2 is the MIC of antibacterial peptide of scophthalmus maximus to Escherichia coli in example 3 of the present invention.
FIG. 3 is the MIC of antibacterial peptide of scophthalmus maximus for Salmonella in example 3 of the present invention.
FIG. 4 is the MIC of antibacterial peptide of scophthalmus maximus to Hafnia alvei in example 3 of the present invention.
FIG. 5 shows MIC of antibacterial peptide of scophthalmus maximus against Staphylococcus aureus in example 3 of the present invention.
FIG. 6 shows MIC of antibacterial peptide of Scophthalmus maximus to Listeria monocytogenes in example 3 of the present invention.
FIG. 7 shows MIC of antibacterial peptide of Scophthalmus maximus to Bacillus subtilis in example 3 of the present invention.
FIG. 8 shows the cytotoxicity of antibacterial peptide of Scophthalmus maximus of example 4 of the present invention.
FIG. 9 shows the hemolysis rate of antibacterial peptide from Scophthalmus maximus in example 5 of the present invention.
FIG. 10 shows the effect of antibacterial peptide content in Scophthalmus maximus on the clearance of Escherichia coli biofilm according to example 6 of the present invention.
FIG. 11 is a graph showing the effect of concentration of antibacterial peptide of scophthalmus maximus on the total number of colonies during the storage of salmon sides in accordance with example 7 of the present invention.
FIG. 12 shows the effect of the antibacterial peptide content of Scophthalmus maximus on the growth of Escherichia coli in example 9 of the present invention.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.
An antibacterial peptide of Scophthalmus maximus contains 16 amino acid residues, the amino acid sequence of the antibacterial peptide is shown in SEQ ID No. 1, the molecular weight of the antibacterial peptide is 1888.41Da, the charge capacity is +5, and the hydrophobic distance is 0.480 mu H.
The minimum inhibitory concentrations of the antibacterial peptide of the turbot to gram-positive bacteria Staphylococcus aureus (Staphylococcus aureus), Listeria monocytogenes (Listeria monocytogenes) and Bacillus subtilis (Bacillus subtilis) are respectively 2.5 mu M, 5 mu M and 5 mu M, and the minimum inhibitory concentrations to gram-negative bacteria Escherichia coli (Escherichia coli), Salmonella (Salmonella) and Hafnia alvei (Hafnia alvei) are respectively 2.5 mu M, 2.5 mu M and 5 mu M.
The invention also provides the application of the antibacterial peptide of the turbot;
an application of antibacterial peptide of turbot in preparing antibacterial agent, wherein the amino acid sequence of the antibacterial peptide of turbot is shown in SEQ ID No. 1.
An application of antibacterial peptide of Scophthalmus maximus in preparing antibacterial drugs, foods and/or health products and animal feeds, wherein the amino acid sequence of the antibacterial peptide of Scophthalmus maximus is shown in SEQ ID No. 1.
An application of antibacterial peptide of Scophthalmus maximus in preparing food additive, food packaging material, animal feed additive, antibacterial drug or cosmetic additive, wherein the amino acid sequence of the antibacterial peptide of Scophthalmus maximus is shown as SEQ ID No. 1.
In a preferred mode, the antibacterial medicine is hydrogel for bacteriostatic dressings, and is prepared from the following components: the antibacterial peptide of the turbot, sodium alginate, gelatin and water; the weight ratio of the antibacterial peptide of the turbot, the sodium alginate and the gelatin is 0.125-1: 1: 1; the weight volume ratio of the gelatin to the water is 1:1 mg/L; the amino acid sequence of the antibacterial peptide of the turbot is shown as SEQ ID No. 1. The antibacterial dressing hydrogel can be used for wound healing.
The invention also provides a preparation method of the hydrogel for the antibacterial dressing, which comprises the following steps: dissolving the antibacterial peptide of the turbot, sodium alginate and gelatin in ultrapure water respectively, carrying out ultrasonic treatment at 200-1500W for 5-120 min, and standing for 5-120 min to obtain the hydrogel for the antibacterial peptide dressing of the turbot.
The application of antibacterial peptide of turbot in fresh-keeping of fresh food can be used as preservative to keep fresh of fresh food.
Preferably, the food is salmon.
The application of the antibacterial peptide of the turbot in the preservation of health-care food can be used as a preservative to preserve the health-care food.
Preferably, the health food is vitamin C effervescent tablets.
The application of antibacterial peptide of turbot in food packaging can be used for packaging food, medicines and cosmetics to inhibit the growth of bacteria.
Preferably, the packaging material is prepared from the following components: antibacterial peptide of turbot, chitosan and polyvinyl alcohol; the mass ratio of the antibacterial peptide to the chitosan of the turbot is 1 per mill-2% to 1; the mass ratio of the chitosan to the polyvinyl alcohol is 1: 1-30; the amino acid sequence of the antibacterial peptide of the turbot is shown as SEQ ID No. 1. Mixing a chitosan acetic acid solution, a polyvinyl alcohol aqueous solution and the antibacterial peptide of the turbot, pouring the mixture into a polyvinyl chloride mould, and circulating for 3-10 times through freezing (-80 ℃ to-20 ℃, 4-24 h) -thawing (4 ℃ to 37 ℃, 4-24 h) to prepare the antibacterial peptide-chitosan-polyvinyl alcohol packaging material of the turbot
The application of the antibacterial peptide of the turbot in bacteriostasis of animal feed can be used as a bacteriostat to carry out bacteriostasis on the animal feed.
Preferably, the animal feed is silage alfalfa feed and dog food.
Example 1: preparation and identification of turbot polypeptide
Firstly, preparing the turbot viscera hydrolysate polypeptide: mixing 100g of the washed turbot viscera with 900mL of deionized water, and stirring. Keeping the temperature in a water bath at 95 ℃ for 5min to inactivate endogenous enzymes in the internal organs of the turbot. Adding pepsin into the aqueous solution of the internal organs of the turbot according to the proportion of 2000U/g protein, adjusting the pH to 2.0 by adopting 2mol/L hydrochloric acid solution, and carrying out enzymolysis for 4 hours in water bath at 37 ℃. Then, the temperature of the water bath was adjusted to 50 ℃, 2000U/g protein trypsin was added to the sample, pH was adjusted to 8.0 with 2mol/L sodium hydroxide solution, and enzymatic hydrolysis was carried out for 4 hours. And after enzyme deactivation at 90 ℃ for 10min, centrifuging the sample for 10min at 4000r/min to obtain a turbot viscera hydrolysate polypeptide sample.
II, ultrafiltration: and (3) performing ultrafiltration on the turbot viscera hydrolysate by adopting a membrane with the molecular weight cutoff of 3KDa to obtain the filtered turbot viscera enzymolysis solution.
Thirdly, desalting: the C18-SPE column was washed with 3mL of methanol, and 3mL of 0.1% FA-H was added2O activates the C18-SPE column. Then adding 1.5mL of ultrafiltered turbot viscera enzymatic hydrolysate sample for desalting treatment, and finally collecting the sample by adopting 1.5mL of 0.1% formic acid-water solution.
And fourthly, identifying the turbot viscera hydrolysate polypeptide: passing the sample through a 0.22 mu m sterilization membrane, and then carrying out UPLC-QTOF-MS analysis, wherein the model of UPLC equipment is Ultimate 3000, Dionex, Thermo Fisher Scientific, a chromatographic column is a Phenomenex Luna C18 chromatographic column (150 multiplied by 3.0mm, 3.0 mu m), a mobile phase A is a formic acid aqueous solution with the mass fraction of 0.1%, a mobile phase B is formic acid acetonitrile with the mass fraction of 0.1%, the elution procedure is gradient elution from 99% of phase A to 50% of phase A, the flow rate is 0.5mL/min, the column temperature is 45 ℃, and the sample feeding amount is 10 mu L; the QTOF-MS is a time-of-flight mass spectrometer, an electrospray ionization source is adopted, the positive ion mode is adopted, the mass scanning range is 50-3500 m/z, and the capillary voltage is 4.5 kv. The obtained polypeptide sequence results were compared with data in the national center for Biotechnology information (http:// www.ncbi.nlm.nih.gov) and identified.
Example 2: physical and chemical property and structure screening of turbot polypeptide
Firstly, analyzing molecular weight and total charge number: turbot polypeptide sequences were entered into the protein analysis website (ProtParam resource in the protein analysis site EXPASY) https:// www.expasy.org/and analyzed for molecular weight and total charge number, the results are shown in Table 1.
As shown in Table 1, the molecular weight of the separated and identified turbot polypeptide is distributed in the range of 1489.76-2969.54 Da, and the total charge number is distributed in the range of + 1- + 7. Generally, the larger the number of positive charges of the antimicrobial peptide, the better the bacteriostatic effect. Therefore, polypeptides having a total charge amount of +5 to +7 were selected for further structural screening.
TABLE 1 amino acid sequence, molecular weight, sequence length and total charge of turbot polypeptide
Figure GDA0003193819550000061
Figure GDA0003193819550000071
Secondly, structure screening: inputting the turbot polypeptide sequence subjected to total charge number screening in a HeliQuest server website (https:// helix.ipmc.cnrs.fr /) and a protein sequence analysis tool NPS @ (https:// NPSA-prabi.ibcp.fr/cgi-bin/NPSA _ Automat.pl.
In general, the alpha-helix structure and the hydrophobic distance in the secondary structure of the antibacterial peptide are positively correlated with the bacteriostatic effect. Therefore, obtaining the antibacterial peptide sequence GITDLRGMLKRLKKMK (shown as SEQ ID No: 1) with the highest alpha-helix proportion content (up to 75%) and the largest hydrophobic distance (0.480 muH) in the secondary structure is the antibacterial peptide of the turbot obtained by the final screening, and the Schiff-Edmunson helix projection of the antibacterial peptide of the turbot is shown as the graph 1.
And entrusting a related polypeptide synthesis company, synthesizing and preparing an GITDLRGMLKRLKKMK antibacterial peptide sample with an amino acid sequence shown in SEQ ID No. 1 by adopting a solid phase synthesis method, and detecting that the purity is up to 98%.
Example 3 measurement of bacteriostatic Effect of antibacterial peptide from Scophthalmus maximus
Minimum Inhibitory Concentration (MIC) determination of antibacterial peptide of turbot: the tested microorganism strains are gram-positive bacteria Staphylococcus aureus (Staphylococcus aureus), Listeria monocytogenes (Listeria monocytogenes) and Bacillus subtilis (Bacillus subtilis); and gram-negative bacteria, Escherichia coli (Escherichia coli), Salmonella (Salmonella), Hafnia alvei (H)afnia alvei). The above strains were purchased from Nanjing feces Biotech Ltd. Adding Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Hafnia alvei into LB culture medium, adding Salmonella into beef extract peptone liquid culture medium, adding Listeria monocytogenes into tryptone soybean broth culture medium, respectively performing amplification culture, and measuring OD of the bacteria liquid600The value is obtained. When OD is reached600When the value reaches 0.6-0.8, centrifuging the bacterial liquid at 6000r/min, discarding the supernatant, collecting the bacterial precipitate, re-suspending the bacterial precipitate with 0.01M PBS buffer solution with the same volume, and diluting the bacterial suspension to 1 × 10 with the culture medium6CFU/mL. The turbot antibacterial peptide (the amino acid sequence of which is shown in SEQ ID No: 1) sample synthesized in example 2 is diluted twice by deionized water to make the concentration of the turbot antibacterial peptide 0.15625-20 mu M. Adding 50 μ L antibacterial peptide of Scophthalmus maximus and 50 μ L bacterial solution into 96-well cell culture plate, repeating each concentration for three times, culturing at 37 deg.C for 12 hr, mixing the solutions, and determining OD600And measuring the OD600 value of the bacterial liquid by using a full-wavelength microplate reader, determining the MIC value of the antibacterial peptide of the turbot, wherein the result is shown in figures 2-7, respectively calculating the Geometric Mean (GM) of the antibacterial peptide of the turbot to the MIC of gram-positive bacteria and gram-negative bacteria, and the result is shown in table 2.
TABLE 2 MIC and GM of antibacterial peptides of turbot
Figure GDA0003193819550000081
Figure GDA0003193819550000091
As shown in FIGS. 2-7 and Table 2, the minimum inhibitory concentrations of the antibacterial peptide of turbot to Staphylococcus aureus, Listeria monocytogenes and Bacillus subtilis are respectively 2.5 μ M, 5 μ M and 5 μ M, and the minimum inhibitory concentrations to Escherichia coli, Salmonella and Hafnia alvei are respectively 2.5 μ M, 2.5 μ M and 5 μ M. The GM of the antibacterial peptide of the turbot is 3.97 mu M for gram-positive bacteria and 3.15 mu M for gram-negative bacteria, so that the antibacterial peptide of the turbot has extremely strong bacteriostatic activity.
Example 4 cytotoxicity assay of antibacterial peptide of Scophthalmus maximus
The antibacterial peptide of the turbot is synthesized in the embodiment 2, and the amino acid sequence is shown in SEQ ID No. 1.
The tested cells are hepatocyte L-02 cells, which are purchased from cell resource center of Shanghai Biotech institute of Chinese academy of sciences. L-02 cells were incubated to reach logarithmic growth phase and transferred to 96-well plates at a cell concentration of 5.0X 103Putting the mixture into an incubator, and introducing CO2(content: 5%, relative humidity: 90%), incubating at 37 ℃ for 24h until the cells adhere to the wall. And (3) sucking and removing the supernatant, adding the antibacterial peptide sample of the turbot which is twice diluted by deionized water into each group, wherein the concentration is 3.125-200 mu M, and taking the deionized water as a control group. Incubating at 37 deg.C for 24 hr, adding 5mg/mL MTT 20 μ L, incubating for 3 hr, removing supernatant, adding 100 μ L DMSO solution, oscillating for 10min, mixing, and measuring absorbance (OD) with fluorescence enzyme-labeling instrument490Value), cell activity (%) ═ assay OD was calculated490Value/control OD490The value is 100%, and the results are shown in FIG. 8.
As shown in FIG. 8, when the concentration of the antibacterial peptide of turbot reaches 200. mu.M, the cell activity of L-02 cells is kept above 95%, which indicates that the cytotoxicity of the antibacterial peptide of turbot is weak.
Example 5 hemolytic assay of antibacterial peptide of turbot
The antibacterial peptide of the turbot is synthesized in the embodiment 2, and the amino acid sequence is shown in SEQ ID No. 1.
Fresh blood of healthy adult Kunming mouse is obtained by taking blood from eyeball, sodium citrate is added for anticoagulation, centrifugation is carried out at 4 ℃ and 3000rpm/min for 5min, serum is removed, and PBS solution is adopted to dilute lower layer red blood cell sediment. And (3) diluting the antibacterial peptide sample of the turbot by deionized water twice, wherein the concentration is 0.3125-160 mu M. Respectively adding 400 mu L of turbot antibacterial peptide sample and 1600 mu L of red blood cell suspension into a 5mL EP tube, and taking PBS buffer solution as a positive control and Tritonx-100 as a negative control. Incubating at 37 deg.C for 0.5h, centrifuging at 4 deg.C and 3000rpm/min for 5min, collecting supernatant 500 μ L, and adding into 96 wellsIn the plate, OD was measured540Value, hemolysis rate (%) (test group OD)540Value-negative control OD540Value)/(positive control OD540Value-negative control OD540Value) × 100%, the structure is shown in fig. 9. The Minimum Hemolysis Concentration (MHC) was defined as the concentration of the antimicrobial peptide at which the hemolysis rate was 10%.
As shown in FIG. 9, when the concentration of the antibacterial peptide of turbot reaches 160 μ M, the haemolysis rate of the red blood cells is still lower than 20%, and the MHC of the antibacterial peptide of turbot is only 80 μ M, which indicates that the antibacterial peptide of turbot has good safety.
Example 6 application of antibacterial peptide of turbot in antibacterial dressing hydrogel
A hydrogel for bacteriostatic dressing is prepared from the following components: the antibacterial peptide of the turbot, sodium alginate, gelatin and water; the mass ratio of the antibacterial peptide of the turbot, the sodium alginate and the gelatin is 0.125-1: 1: 1; the mass-volume ratio of the gelatin to the water is 1:1 mg/L; the amino acid sequence of the antibacterial peptide of the turbot is shown as SEQ ID No. 1. The antibacterial dressing hydrogel can be used for wound healing.
Dissolving 1mg, 0.5mg, 0.25mg and 0.125mg of antibacterial peptide of turbot, 1mg of sodium alginate and 1mg of gelatin in 1mL of ultrapure water respectively, carrying out ultrasonic treatment (800W for 10min), standing for 20min to self-assemble antibacterial peptide dressing hydrogel of turbot, and measuring the removing capacity of the dressing to in-vitro bacterial biofilms. Add 200. mu.L of E.coli suspension (1.5X 10) to each well of 6-well plate8CFU/ml) and 1800. mu.L of LB medium, the 6-well plate was incubated in a 37 ℃ incubator for 24 hours at constant temperature. Adding 200 mu L of antibacterial dressing hydrogel of the antibacterial peptide of the turbot into the bacterial liquid, incubating for 24h together, discarding the suspension, and washing each hole for 3 times by 2000 mu L of PBS (pH 7.4, 0.01M) to remove loosely adhered cells; then fixing each hole with 2000 μ L anhydrous methanol for 15min, pouring off methanol, and drying at 60 deg.C for 10 min; then dip-dyeing with 2000 μ L of 0.1% crystal violet solution for 15 min; the well plate was washed 3 times with double distilled water to remove residual dye solution and dried at 60 ℃. Finally, the biofilm was eluted by dropping 2000. mu.L of 33% glacial acetic acid solution in each well and incubated at room temperature for 20min with shaking. The absorbance was measured at 590nm using a microplate reader, and the biofilm clearance rate, i.e., (vs.) was calculatedPhoto group OD590nmExperimental group OD590nm) Control group OD590nmX 100%. The result is shown in fig. 10, when the content of the antibacterial peptide in the antibacterial dressing hydrogel is 1mg, the clearance rate of the escherichia coli biofilm can reach 93.6%.
Example 7 application of antibacterial peptide of turbot in fresh food preservation
Dissolving antibacterial peptide of turbot in water to prepare an antibacterial peptide aqueous solution with the concentration of 5-50 mu M, and spraying the antibacterial peptide aqueous solution on the surface of the fresh food for keeping the fresh of the fresh food; the amino acid sequence of the antibacterial peptide of the turbot is shown as SEQ ID No. 1.
Uniformly spraying 50 mu M, 20 mu M, 10 mu M and 5 mu M aqueous solution (2mL) of the antibacterial peptide of the turbot on the surface of a fresh salmon block (3 multiplied by 3cm), placing the salmon block in a refrigerator at 4 ℃ for storage, detecting the total colony number according to GB4789.2-2016, and taking the salmon block which is not sprayed with the aqueous solution of the antibacterial peptide of the turbot as a control group. As shown in FIG. 11, the total number of colonies in the control group was 10 on day 1 of storage4.11The total number of colonies of the CFU/mL group with the concentration of 50 mu M of the antibacterial peptide aqueous solution of the turbot is 101.74CFU/mL; at day 14 of storage, the total number of colonies in the control group was 106.00The total number of colonies of the CFU/mL group with the concentration of 50 mu M of the antibacterial peptide aqueous solution of the turbot is 103.75CFU/mL indicates that the antibacterial peptide of the turbot can effectively inhibit the growth of colonies in the salmon sample block.
Example 8 application of antibacterial peptide of Scophthalmus maximus in health food
Adding the antibacterial peptide of the turbot into the health food by weight fraction of 0.01-1 per mill for preserving the health food; the amino acid sequence of the antibacterial peptide of the turbot is shown as SEQ ID No. 1.
Firstly, preparing acid granules: respectively crushing 25g of tartaric acid, 10g of sodium bicarbonate, 10g of sucrose and 15g of lactose, sieving by a 100-mesh nylon sieve, adding 100mL of 10% polyvinylpyrrolidone absolute ethanol solution by weight fraction, granulating by a 20-mesh nylon sieve, and drying at 40 ℃ to obtain 18-mesh whole granules;
secondly, preparing alkali particles: 25g of sodium bicarbonate, 20g of sucrose and 2g of aspartame are respectively crushed, and after the materials are sieved by a 100-mesh nylon sieve, 100mL of 10% polyvinylpyrrolidone absolute ethanol solution by weight is added, and after the materials are granulated by a 20-mesh nylon sieve, the granules are dried at 40 ℃ and are granulated by a 18-mesh sieve.
Thirdly, mixing acid particles and alkali particles: mixing the acid granules and the alkali granules, adding 100mg of sodium ascorbate, 10mg of antibacterial peptide of turbot, 5g of polyethylene glycol 6000 and 3g of potassium chloride, mixing, and tabletting to obtain effervescent tablet of vitamin C.
Example 9 application of antibacterial peptide of Scophthalmus maximus in packaging material
A packaging material is made from the following components: antibacterial peptide of turbot, chitosan and polyvinyl alcohol; the weight ratio of the antibacterial peptide of the turbot, the chitosan and the polyvinyl alcohol is 1 per mill-2 percent to 1: 1-30; the amino acid sequence of the antibacterial peptide of the turbot is shown as SEQ ID No. 1. The packaging material can be used for packaging food, medicine and cosmetics to inhibit the growth of bacteria.
Dissolving 100g of polyvinyl alcohol in 100mL of deionized water to obtain 1g/mL of polyvinyl alcohol aqueous solution; 10g of chitosan was dissolved in 100mL of 2% volume fraction acetic acid solution to obtain 1g/mL of chitosan acetic acid solution. Mixing 10mL of chitosan acetic acid solution (1g/mL) with 50mL of polyvinyl alcohol aqueous solution (1g/mL), adding 0.09375g, 0.1875g and 0.375g of antibacterial peptide of turbot respectively, stirring uniformly to prepare a mixed solution of antibacterial peptide of turbot, chitosan and polyvinyl alcohol with the mass ratio of 1.875 per mill, 3.75 per mill and 7.5 per mill: 1:5, pouring the mixed solution into a polyvinyl chloride mould with the length of 25cm multiplied by the width of 20cm (the thickness of the mixed solution is 0.1cm), and circulating for 5 times through freezing (-80 ℃, 8h) -melting (25 ℃, 8h) to prepare the antibacterial peptide-chitosan-polyvinyl alcohol packaging material of turbot, and determining the influence of the antibacterial peptide-chitosan-polyvinyl alcohol packaging material on the growth of bacteria. Adding Escherichia coli into LB culture medium, performing amplification culture, and measuring OD of bacteria liquid600The value is obtained. When OD is reached600When the value reaches 0.6-0.8, centrifuging the bacterial liquid at 6000r/min, discarding the supernatant, collecting the bacterial precipitate, re-suspending the bacterial precipitate with 0.01M PBS buffer solution with the same volume, and diluting the bacterial suspension to 1 × 10 with the culture medium6CFU/mL. The antibacterial peptide-chitosan-polyvinyl alcohol packaging material of the turbot is made into a sheet shape of 1 multiplied by 1cm by a puncher. Adding 1 tablet into the triangular pyramid bottle respectivelyCulturing the turbot antibacterial peptide-chitosan-polyvinyl alcohol packaging material and 50mL of bacterial solution (taking the turbot antibacterial peptide-chitosan-polyvinyl alcohol packaging material as a control group without being added) at 37 ℃, and measuring the OD600 value of the bacterial solution when culturing for 0h, 4h, 8h, 12h, 24h and 48h respectively. As shown in figure 12, the antibacterial peptide-chitosan-polyvinyl alcohol packaging material for turbot has an obvious inhibition effect on the growth of escherichia coli, and the inhibition effect is increased along with the increase of the content of the antibacterial peptide in the turbot.
Example 10 application of antibacterial peptide of turbot in bacteriostasis of animal feed
Dissolving antibacterial peptide of turbot in water to prepare an antibacterial peptide aqueous solution with the concentration of 5-50 mu M, and spraying the antibacterial peptide aqueous solution on the surface of silage for bacteriostasis of the silage; the amino acid sequence of the antibacterial peptide of the turbot is shown as SEQ ID No. 1.
The 20 mu M aqueous solution of the antibacterial peptide of the turbot is uniformly sprayed on the silage alfalfa feed for cattle, so that the growth of bacteria in the storage alfalfa feed in the storage process can be effectively inhibited.
Secondly, adding the antibacterial peptide of the turbot into the animal granular feed by 0.01-1 per mill of weight fraction for bacteriostasis of the animal feed; the amino acid sequence of the antibacterial peptide of the turbot is shown as SEQ ID No. 1.
The dog food is prepared from the following raw materials in percentage by weight: 20% of corn flour, 17% of milk powder, 10% of potato, 10% of soybean flour, 2.995% of salt, 10% of chicken powder, 20% of chicken bone powder, 10% of lard oil and 0.005% of antibacterial peptide of turbot.
In conclusion, the antibacterial peptide of the turbot is wide in antibacterial spectrum, low in cytotoxicity and hemolytic property, and has efficient antibacterial effect on gram-negative bacteria and gram-positive bacteria. Therefore, the antibacterial peptide of the turbot can be better applied to preparing antibacterial agents resisting gram-positive bacteria and gram-negative bacteria, and can also be used for preparing food additives, food packaging materials, animal feed additives, antibacterial drugs or cosmetic additives.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Figure GDA0003193819550000131
Sequence listing
<110> university of Dalian Industrial university
<120> antibacterial peptide of turbot and application thereof
<130> ZR201186LQ
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 16
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 1
Gly Ile Thr Asp Leu Arg Gly Met Leu Lys Arg Leu Lys Lys Met Lys
1 5 10 15

Claims (10)

1. The antibacterial peptide of turbot is characterized in that the amino acid sequence is shown as SEQ ID No. 1.
2. The use of the antibacterial peptide of turbot according to claim 1 for the preparation of bacteriostatic agents for inhibiting bacteria, wherein the bacteriostatic agents comprise the antibacterial peptide of turbot as an active ingredient, including food bacteriostatic agents, animal feed bacteriostatic agents or pharmaceutical bacteriostatic agents.
3. The application of the antibacterial peptide of turbot in preparing the bacteriostatic agent for inhibiting bacteria according to claim 2, wherein the food bacteriostatic agent is a fresh food bacteriostatic agent, and the aqueous solution of the antibacterial peptide of turbot with the concentration of 5-50 μ M is sprayed on the surface of the fresh food for keeping the fresh of the fresh food.
4. The use of antibacterial peptide of turbot according to claim 2, wherein the food bacteriostatic agent is a health product bacteriostatic agent; when the health-care product is prepared, the antibacterial peptide of the turbot is added into the health-care product by 0.01-1 per mill of weight fraction to inhibit bacteria.
5. The application of the antibacterial peptide of turbot in preparing the bacteriostatic agent for inhibiting bacteria according to claim 2, wherein the feed is silage alfalfa feed, and 5-50 μ M of the aqueous solution of the antibacterial peptide of turbot is sprayed on the surface of the silage for bacteriostasis of the silage.
6. The use of antibacterial peptide of turbot according to claim 2, wherein the feed is a granulated feed; when preparing the granular animal feed, the antibacterial peptide of the turbot is added into the granular animal feed by 0.01-1 per mill of weight fraction for bacteriostasis.
7. The use of antibacterial peptide of turbot according to any one of claims 2 to 6, for the preparation of bacteriostatic agents for inhibiting bacteria, wherein said bacteria comprise: staphylococcus aureus (Staphylococcus aureus), Listeria monocytogenes (Listeria monocytogenes), Bacillus subtilis (Bacillus subtilis), Escherichia coli (Escherichia coli), Salmonella (Salmonella), Hafnia alvei (Hafnia alvei).
8. The use of the antibacterial peptide of scophthalmus maximus according to claim 1 for preparing food, medicine and/or health care products, wherein the antibacterial peptide of scophthalmus maximus is used for inhibiting bacteria in the food, medicine and/or health care products.
9. Use of the antibacterial peptide of turbot according to claim 1 for the preparation of a packaging material for inhibiting the growth of bacteria, which is used for the packaging of foods, pharmaceuticals or cosmetics.
10. The use of antibacterial peptide of turbot according to claim 9, for the preparation of a packaging material for inhibiting the growth of bacteria, wherein said bacteria comprise: staphylococcus aureus (Staphylococcus aureus), Listeria monocytogenes (Listeria monocytogenes), Bacillus subtilis (Bacillus subtilis), Escherichia coli (Escherichia coli), Salmonella (Salmonella), Hafnia alvei (Hafnia alvei).
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Citations (3)

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CN1778920A (en) * 2005-09-27 2006-05-31 中国水产科学研究院黄海水产研究所 Antibiotic peptide gene and its yeast expression carrier
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WO2006089121A2 (en) * 2005-02-18 2006-08-24 Kent Seatech Corporation Streptococcus iniae phosphoglucomutase is a virulence factor and target for vaccine development
CN1778920A (en) * 2005-09-27 2006-05-31 中国水产科学研究院黄海水产研究所 Antibiotic peptide gene and its yeast expression carrier
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