CN112516283A - Quaternized abalone antibacterial peptide gel and preparation method and application thereof - Google Patents
Quaternized abalone antibacterial peptide gel and preparation method and application thereof Download PDFInfo
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- CN112516283A CN112516283A CN202011563767.7A CN202011563767A CN112516283A CN 112516283 A CN112516283 A CN 112516283A CN 202011563767 A CN202011563767 A CN 202011563767A CN 112516283 A CN112516283 A CN 112516283A
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Abstract
The invention provides a quaternized abalone antibacterial peptide gel and a preparation method and application thereof, wherein the gel comprises quaternized abalone antibacterial peptide powder, thyme essential oil, sodium alginate, propylene glycol alginate and water; the method adopts an enzymolysis technology and a membrane separation technology to extract abalone polypeptide, has higher degree and less impurities, obtains quaternized abalone antibacterial peptide powder through quaternary amination reaction, and then adds the quaternized abalone antibacterial peptide powder into a gel matrix to be mixed uniformly; the quaternized abalone antibacterial peptide gel disclosed by the invention is high in compatibility of each component, and a formed polymer network can effectively wrap quaternized abalone antibacterial peptide, is mild and non-irritant to skin and has a certain moisturizing effect; in addition, the quaternized abalone antibacterial polypeptide gel has a strong antibacterial effect, and is not easy to generate drug resistance, so that a method and a route are provided for the research and development of a new antibacterial agent, and the quaternized abalone antibacterial polypeptide gel has a good application prospect in the fields of medicines and cosmetics.
Description
Technical Field
The invention belongs to the technical field of deep processing of marine shellfish, and particularly relates to a quaternized abalone antibacterial peptide gel and a preparation method and application thereof.
Background
Abalone has abundant nutritive values such as protein, polysaccharide, fatty acid, etc., which are bioactive substances beneficial to the human body, wherein the protein accounts for more than 50% of the dry weight. Abalone polypeptide is a macromolecular substance with wide biological activity, and has been explored and applied in the field of health food by numerous scholars due to the functions of resisting tumors, resisting oxidation, regulating immunity and the like, but the research on the antibacterial action of the abalone polypeptide is less. Even though related researches show that the abalone polypeptide has a certain antibacterial function, the antibacterial effect is relatively weak and is not enough to be prepared into an antibacterial agent in practical application.
At present, quaternary ammonium salt is a cationic antibacterial agent, and the antibacterial mechanism of the quaternary ammonium salt is mainly that the quaternary ammonium salt is contacted with bacteria and permeates into cell walls of the bacteria, then reacts with the cell membranes of the bacteria to destroy the cell membranes, further causes substances among the cells to leak, and finally dissolves the cell walls, so that the antibacterial effect is realized. Quaternary ammonium based antibacterial agents do not generally develop bacterial resistance and are therefore of increasing interest to researchers and product engineers.
Antibiotics are added into the commercially available antibacterial gel to achieve the bacteriostatic action, and long-term use of the antibiotic-containing medicine can cause bacterial tolerance and possibly cause bacterial infection without medicine.
Disclosure of Invention
Aiming at the defects in the prior art, the invention mainly aims to provide a quaternized abalone antibacterial peptide gel.
The second purpose of the invention is to provide a preparation method of the quaternized abalone antibacterial peptide gel.
The third purpose of the invention is to provide the application of the quaternized abalone antibacterial peptide gel.
In order to achieve the above primary object, the solution of the present invention is:
a quaternized abalone antibacterial peptide gel comprises the following components:
further preferably, the preparation process of the quaternized abalone antibacterial peptide powder comprises the following steps: pulping abalone meat, preparing into solution, adding papain for enzymolysis for 4-5h, heating to 100 deg.C to inactivate enzyme, and centrifuging to obtain enzymolysis supernatant; separating the supernatant with nanofiltration membrane to obtain abalone polypeptide as the trapped liquid, and drying to obtain abalone polypeptide powder.
Dissolving dodecyl trimethyl ammonium chloride in isopropanol, continuously adding isopropanol solution of abalone polypeptide powder and dodecyl trimethyl ammonium chloride in a mass ratio of 1:20 under the action of magnetic stirring at 60-65 ℃, reacting for 6-8h, pouring the solution obtained by the reaction into excessive absolute ethyl alcohol for precipitation, performing suction filtration, washing, and drying to obtain the quaternized abalone antibacterial peptide powder.
Specifically, sodium alginate is derived from brown algae, is a natural polysaccharide, has high viscosity of water solution, is a main raw material of the gel matrix, and is safer and nontoxic compared with other high-molecular gel matrix raw materials; the propylene glycol alginate is also derived from seaweed and used as a stabilizer, which can enhance the stability of the sodium alginate gel matrix.
In order to achieve the second objective, the solution of the invention is:
a preparation method of the quaternized abalone antibacterial peptide gel comprises the following steps:
(1) and preparing the quaternized abalone antibacterial peptide powder:
preparing abalone polypeptide: pulping Carnis Haliotidis meat, adding distilled water to obtain solution, adding papain for enzymolysis for 4-5 hr, heating to 100 deg.C to inactivate enzyme, centrifuging to obtain enzymolysis supernatant; separating the supernatant by adopting a 500-plus-1000 Da nanofiltration membrane to obtain trapped fluid which is the abalone polypeptide, and performing spray drying to obtain abalone polypeptide powder;
polypeptide quaternization: dissolving dodecyl trimethyl ammonium chloride in isopropanol, continuously adding isopropanol solution of abalone polypeptide powder and dodecyl trimethyl ammonium chloride in a mass ratio of 1:20 under the action of magnetic stirring at 60-65 ℃, reacting for 6-8h, pouring the solution obtained by the reaction into absolute ethyl alcohol for precipitation, performing suction filtration, washing, and drying to obtain quaternized abalone antibacterial peptide powder;
(2) and preparing a gel matrix:
weighing 0.5-1 part of sodium alginate, adding the sodium alginate into 55-105 parts of water at the speed of one drop per 30s while stirring, heating to 65 ℃ for accelerating swelling, and adding 0.2-0.4 part of propylene glycol alginate into a gel matrix at the speed of one drop per 30s for later use;
(3) and preparing polysaccharide gel:
dissolving the quaternized antibacterial peptide: weighing 1-2 parts of quaternized abalone antibacterial peptide powder, dissolving in 5-10 parts of pure water, and adding the quaternized abalone antibacterial peptide solution into 0.3-0.6 part of thyme essential oil for later use;
compounding: and adding the quaternary ammonium abalone antibacterial peptide dissolved solution into the gel matrix in a sterile environment, and uniformly mixing to obtain the quaternary ammonium abalone antibacterial peptide gel.
In order to achieve the third object, the solution of the invention is:
an application of the quaternized abalone antibacterial peptide gel in preparation of skin wounds.
The application of the quaternized abalone antibacterial peptide gel in the fields of medicines and cosmetics.
Due to the adoption of the scheme, the invention has the beneficial effects that:
firstly, the quaternary ammonium abalone antibacterial polypeptide is combined with the surface of bacteria with negative electricity, so that the positive charge outside the bacterial cell membrane is increased, the membrane potential on two sides is increased, when the concentration of the antibacterial polypeptide reaches a critical value, the bacterial cell membrane is damaged in a blanket type, barrel plate type or annular hole type mode, and the bacteria die due to imbalance of internal and external osmotic pressure. The physical action mode ensures that the quaternized abalone antibacterial polypeptide is not easy to generate drug resistance, thereby providing a method and a route for researching and developing a new antibacterial agent and having wide market prospect; therefore, the quaternized abalone antibacterial polypeptide has a unique antibacterial mechanism, and the quaternized abalone antibacterial peptide gel has strong antibacterial activity, so that the quaternized abalone antibacterial peptide gel has a good application prospect in the fields of medicines and cosmetics.
Secondly, the abalone polypeptide has a certain antibacterial property, and the antibacterial effect of the quaternized abalone antibacterial peptide obtained by performing quaternization reaction on the abalone polypeptide is stronger; in addition, as the abalone polypeptide is difficult to dissolve in an organic solvent, and the organic solvent is added into the gel matrix, the quaternized abalone antibacterial peptide is more easily dissolved in the gel matrix and is easily made into the gel.
Thirdly, the quaternized abalone antibacterial peptide gel disclosed by the invention is simple and safe in components and high in compatibility, and a formed polymer network can effectively wrap quaternized abalone antibacterial peptides, is mild and non-irritant to skin, has a certain moisturizing effect, and can be used for adjuvant therapy of skin wounds. In addition, the thyme essential oil added into the gel has the functions of clearing away heat and toxic materials, and the fishy smell of the quaternized abalone antibacterial peptide can be well covered by the medicinal fragrance of the thyme essential oil and the medicinal fragrance of the thyme essential oil, so that the thyme essential oil and the medicinal fragrance supplement each other.
Fourthly, the quaternized abalone antibacterial polypeptide is made into a product in the form of a gel, so that the application field of abalone polypeptide is widened, the additional value of abalone-related industries is promoted, and the development of the deep processing industry of marine shellfish is promoted.
Drawings
FIG. 1 is a bacteriostatic performance diagram of the quaternized abalone antibacterial peptide gel in the embodiment of the invention ((a) is escherichia coli bacteriostatic effect, (b) is staphylococcus aureus bacteriostatic effect; -is a negative control inoculated with sterile water; + is a positive control inoculated with streptomycin at 1mg/mL and kanamycin).
Detailed Description
The invention provides a quaternized abalone antibacterial peptide gel and a preparation method and application thereof.
The present invention will be further described with reference to the following examples.
Example (b):
the preparation method of the quaternized abalone antibacterial peptide gel comprises the following steps:
(1) and preparing the quaternized abalone antibacterial peptide powder:
preparing abalone polypeptide: pulping Carnis Haliotidis meat, adding distilled water to obtain solution, adding papain for enzymolysis for 4.5 hr, heating to 100 deg.C to inactivate enzyme, centrifuging to obtain enzymolysis supernatant; separating the supernatant by adopting a 800Da nanofiltration membrane to obtain trapped fluid, namely abalone polypeptide, and performing spray drying to obtain abalone polypeptide powder;
polypeptide quaternization: dissolving dodecyl trimethyl ammonium chloride in isopropanol, continuously adding isopropanol solution of abalone polypeptide powder and dodecyl trimethyl ammonium chloride in a mass ratio of 1:20 under the action of magnetic stirring at 60-65 ℃, reacting for 7 hours, pouring the solution obtained by the reaction into absolute ethyl alcohol for precipitation, performing suction filtration, washing and drying to obtain quaternized abalone antibacterial peptide powder;
(2) and preparing a gel matrix:
weighing 0.5 part of sodium alginate, adding the sodium alginate into 100 parts of water at the speed of one drop per 30s while stirring, heating to 65 ℃ to accelerate swelling, and adding 0.2 part of propylene glycol alginate into a gel matrix at the speed of one drop per 30s for later use;
(3) and preparing polysaccharide gel:
dissolving the quaternized antibacterial peptide: weighing 2 parts of quaternized abalone antibacterial peptide powder, dissolving the quaternized abalone antibacterial peptide powder in 5 parts of pure water, and adding the quaternized abalone antibacterial peptide solution into 0.4 part of thyme essential oil for later use;
compounding: and adding the quaternary ammonium abalone antibacterial peptide dissolved solution into the gel matrix in a sterile environment, and uniformly mixing to obtain the quaternary ammonium abalone antibacterial peptide gel.
The prepared quaternized abalone antibacterial peptide gel is subjected to the following antibacterial performance experiments:
and determining the bacteriostatic performance of the quaternized abalone antibacterial peptide gel by measuring the size of a bacteriostatic zone by an agar diffusion method. Respectively streaking Escherichia coli and Staphylococcus aureus which are frozen and stored at-80 deg.C on LB solid medium, and culturing at 30 deg.C for 24 h. Single colonies were picked from the activated plates, inoculated into 25mL of LB liquid medium, and shaken at 30 ℃ and 200r/min for 24 hours. Taking an LB culture medium containing 1% of agar as a lower-layer culture medium, inoculating an experimental strain into the LB culture medium containing 0.7% of agar, shaking uniformly to prepare an upper-layer culture medium, punching a hole on a flat plate by using a puncher, wherein the diameter of the hole is 0.8cm, punching three holes on each flat plate, inoculating 1 piece of quaternized abalone antibacterial peptide bacteriostatic gel, inoculating 1 piece of sterile water negative control and inoculating 1 piece of positive control (a mixed solution of streptomycin and kanamycin at 1 mg/mL) and culturing at the constant temperature of 30 ℃ for 24 hours, and observing and measuring the size of a bacteriostatic ring, wherein the quaternized abalone antibacterial peptide bacteriostatic gel disclosed by the invention has an obvious inhibitory effect on the growth of escherichia coli and staphylococcus aureus as shown in figure 1 and table 1.
TABLE 1 determination of antibacterial zones of different strains by quaternized abalone antibacterial peptide gel
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments and the generic principles defined herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments. Those skilled in the art should appreciate that many modifications and variations are possible in light of the above teaching without departing from the scope of the invention.
Claims (6)
1. A quaternized abalone antibacterial peptide gel is characterized in that: the paint comprises the following components:
2. the quaternized abalone antimicrobial peptide gel of claim 1, wherein: the preparation process of the quaternized abalone antibacterial peptide powder comprises the following steps: pulping abalone meat, preparing into solution, adding papain for enzymolysis for 4-5h, heating to 100 deg.C to inactivate enzyme, and centrifuging to obtain enzymolysis supernatant; separating the supernatant by using a nanofiltration membrane to obtain a retentate which is abalone polypeptide, and drying to obtain abalone polypeptide powder;
dissolving dodecyl trimethyl ammonium chloride in isopropanol, continuously adding isopropanol solution of abalone polypeptide powder and dodecyl trimethyl ammonium chloride in a mass ratio of 1:20 under the action of magnetic stirring at 60-65 ℃, reacting for 6-8h, pouring the solution obtained by the reaction into absolute ethyl alcohol for precipitation, carrying out suction filtration, washing and drying to obtain the quaternized abalone antibacterial peptide powder.
3. A method of preparing a quaternized abalone antibacterial peptide gel of claim 1, characterized in that: which comprises the following steps:
(1) preparing quaternized abalone antibacterial peptide powder;
(2) and preparing a gel matrix:
weighing 0.5-1 part of sodium alginate, adding the sodium alginate into 55-105 parts of water at the speed of one drop per 30s while stirring, heating to 65 ℃ for accelerating swelling, and adding 0.2-0.4 part of propylene glycol alginate into a gel matrix at the speed of one drop per 30s for later use;
(3) and preparing polysaccharide gel:
dissolving the quaternized antibacterial peptide: weighing 1-2 parts of quaternized abalone antibacterial peptide powder, dissolving in 5-10 parts of pure water, and adding the quaternized abalone antibacterial peptide solution into 0.3-0.6 part of thyme essential oil for later use;
compounding: and adding the quaternary ammonium abalone antibacterial peptide dissolved solution into the gel matrix in a sterile environment, and uniformly mixing to obtain the quaternary ammonium abalone antibacterial peptide gel.
4. The production method according to claim 3, characterized in that: in the step (1), preparing the quaternized abalone antibacterial peptide powder:
preparing abalone polypeptide: pulping Carnis Haliotidis meat, preparing into solution, adding papain for enzymolysis for 4-5 hr, heating to 100 deg.C to inactivate enzyme, centrifuging to obtain enzymolysis supernatant; separating the supernatant by using a nanofiltration membrane to obtain a trapped fluid, namely abalone polypeptide, and performing spray drying to obtain abalone polypeptide powder;
polypeptide quaternization: dissolving dodecyl trimethyl ammonium chloride in isopropanol, continuously adding isopropanol solution of abalone polypeptide powder and dodecyl trimethyl ammonium chloride in a mass ratio of 1:20 under the action of magnetic stirring at 60-65 ℃, reacting for 6-8h, pouring the solution obtained by the reaction into absolute ethyl alcohol for precipitation, performing suction filtration, washing, and drying to obtain the quaternized abalone antibacterial peptide powder.
5. Use of a quaternized abalone antimicrobial peptide gel according to claim 1 in the preparation of a medicament for the treatment of skin wounds.
6. The use of a quaternized abalone antimicrobial peptide gel of claim 1 in the fields of medicine and cosmetics.
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