CN113876934B - Lysozyme composition with antibacterial effect - Google Patents
Lysozyme composition with antibacterial effect Download PDFInfo
- Publication number
- CN113876934B CN113876934B CN202111336938.7A CN202111336938A CN113876934B CN 113876934 B CN113876934 B CN 113876934B CN 202111336938 A CN202111336938 A CN 202111336938A CN 113876934 B CN113876934 B CN 113876934B
- Authority
- CN
- China
- Prior art keywords
- lysozyme
- solution
- citral
- dialysis
- potassium phosphate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
-
- 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
-
- 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
- A61P31/10—Antimycotics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01017—Lysozyme (3.2.1.17)
-
- 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 relates to a lysozyme composition with a bacteriostatic effect, wherein an active ingredient is a lysozyme derivative modified by citral. The lysozyme derivative has excellent antibacterial performance, and the antibacterial effect and antibacterial spectrum breadth of the lysozyme derivative are superior to those of common lysozyme. The lysozyme has natural sources and no toxic or side effect, and the preparation method of the lysozyme composition can be industrialized and has popularization and application values.
Description
Technical Field
The invention relates to the field of medicines, in particular to a lysozyme composition.
Background
Lysozyme, also known as cell wall lytic enzyme, is an alkaline proteolytic enzyme that stabilizes cell wall hydrolysis. It acts mainly on bacterial cell wall, i.e. on beta-1, 4 glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid in its peptidoglycan, and it promotes the decomposition of polysaccharide in bacterial cell wall into glycopeptides by hydrolyzing it, reducing bacterial cell wall stability, destroying its structure, finally causing the content in cell wall-lysed cells to flow out of the cell, causing the bacteria to dissolve.
Lysozyme as a mononuclear, neutrophil and macrophage product has significant antimicrobial properties against gram-positive bacteria. In addition, lysozyme is an alkaline protease that is present in organisms and is also a nonspecific immune factor that does not generally have resistance to drugs when inhibiting and killing pathogens. The bacteriostatic mechanism of lysozyme, which is different from antibiotics, has been paid attention to and is applied to foods and medicines.
It was generally considered that the bactericidal activity of lysozyme was dependent on the enzymatic activity in the initial studies. As the research is advanced, it is found that when lysozyme enzyme activity is lost in a certain way, the bactericidal property is still present. Through a large number of experimental analyses in the early stage, a possible sterilization mode of lysozyme is obtained: depending on the type and mode of bacteria acting, lysozyme will adopt a lytic mode and a non-lytic mode. Both modes first generate electrostatic attraction by contact of positively charged lysozyme with negatively charged bacteria, then one may be that lysozyme-dependent enzyme activity hydrolyzes the peptidoglycan layer, or that the enzyme activity does not cause bacterial lysis death by bonding of a certain unknown specific region of lysozyme to phosphate groups on the bacterial cell wall or substitution of cations on the cell wall; it is also possible that bacterial death occurs by disruption of bacterial membrane function after action of lysozyme with bacteria, the first two being lysozyme death and the second being bacterial non-lytic death.
Lysozyme exists in a large amount in nature and has remarkable sterilization effect, but the lysozyme is a non-broad-spectrum antibacterial agent, so that in order to expand the antibacterial spectrum and the application range of the lysozyme, the scholars at home and abroad carry out lysozyme modification research, but the antibacterial spectrum of the derivatized lysozyme is still limited.
Citral (3, 7-dimethyl-2, 6-octadien-1-al) is a monoterpene compound with the molecular formula of C 10 H 16 O, colorless or pale yellow transparent liquid. Natural citral is mainly present in essential oils of plants such as litsea cubeba oil, lemon grass oil, verbena oil, and pendulous She Xiangmao oil. In China, the plant resources rich in natural citral are rich, wherein the citral content in the litsea cubeba plant essential oil reaches 65-80%. Citral has a strong lemon-like aroma and is commonly used as a flavoring agent, preservative and aromatic agent in foods and cosmetics, and is also a main raw material for synthesizing vitamin A, ionone and methyl ionone. The related literature data show that the citral also has the biological activities of insect disinfestation, bacteria inhibition, bacteria resistance, oxidation resistance and the like, and has the effects of treating cardiovascular diseases, leukemia resistance and the like. In recent years, citral has strong effects of relieving asthma, cough, allergy and the like.
At present, the sterilization effect and the antibacterial application range of lysozyme derivatives are limited, and a lysozyme composition with good sterilization effect and broad-spectrum antibacterial effect is needed.
Disclosure of Invention
The invention aims to solve the technical problem of providing the lysozyme composition with remarkable antibacterial effect, wide antibacterial spectrum and small toxic and side effects.
The invention solves the problems by adopting the technical scheme that a lysozyme derivative with an antibacterial effect is provided, and the preparation method is as follows:
(1) Dissolving a proper amount of citral in hexane to prepare a citral solution;
(2) Dissolving a proper amount of lysozyme in potassium phosphate buffer solution with the pH of 7.4 to prepare a lysozyme solution;
(3) Slowly dripping the citral solution in the step (1) into the lysozyme solution in the step (2), adding an equal volume of potassium phosphate buffer solution containing sodium borohydride and having the pH of 7.0 into the reaction mixture, and slowly stirring at the temperature of 0 ℃ for 0.5-2h;
(4) Adding 1-3 times of saturated sodium sulfate solution of the reaction solution to precipitate lysozyme, centrifuging at high speed, and re-dispersing the centrifuged precipitate into distilled water;
(5) And (3) putting the dispersion solution in the step (4) into a dialysis bag for dialysis, periodically replacing distilled water outside the dialysis bag, and freeze-drying the solution in the dialysis bag after dialysis to obtain the lysozyme derivative.
Preferably, the molar concentration ratio of citral in step (1) to lysozyme in step (2) is (1-10): 1, a step of; more preferably, the molar concentration ratio of citral to lysozyme is 10:1.
preferably, the concentration of citral in the citral solution of step (1) is 50-200. Mu.M; more preferably, the concentration of citral in the citral solution is 100. Mu.M.
Preferably, the concentration of lysozyme in the lysozyme solution of the step (2) is 5-20 mu M; more preferably, the concentration of lysozyme in the lysozyme solution is 10. Mu.M.
Preferably, the lysozyme in the step (2) is egg white lysozyme.
Preferably, the volume ratio of the citral solution in the step (1) to the lysozyme solution in the step (2) is 1: (1-10); more preferably, the volume ratio of citral solution to lysozyme solution is 1:5.
preferably, the citral solution in step (1) has a volume of 1 to 3mL, more preferably, the citral solution has a volume of 2mL.
Preferably, the volume of the lysozyme solution in the step (2) is 5-20mL, more preferably, the volume of the lysozyme solution is 10mL.
Preferably, the concentration of potassium phosphate in the potassium phosphate buffer in the step (2) is 30-80mM; more preferably, the concentration of potassium phosphate in the potassium phosphate buffer is 50mM.
Preferably, the concentration of potassium phosphate in the potassium phosphate buffer solution in the step (3) is 0.1-0.5M; more preferably, the concentration of potassium phosphate in the potassium phosphate buffer is 0.3M.
Preferably, the concentration of sodium borohydride in the potassium phosphate buffer solution in the step (3) is 1-5mg/mL; more preferably, the concentration of sodium borohydride in the potassium phosphate buffer is 3mg/mL.
Preferably, the stirring time of the reactants in the step (3) is 1h.
Preferably, in the step (4), a saturated sodium sulfate solution 2 times the volume of the reaction solution is added.
Preferably, 20000g is used for centrifugation in step (4) for 15 minutes.
Preferably, the precipitate in step (4) is redispersed in 30mL of distilled water.
Preferably, in the step (5), the dispersion solution is put into a dialysis bag for dialysis for 72 hours, and distilled water outside the dialysis bag is replaced every 24 hours.
In a second aspect the present invention provides a lysozyme composition comprising a lysozyme derivative as described above.
Preferably, the lysozyme composition further comprises pharmaceutically acceptable auxiliary materials.
Preferably, the lysozyme composition is in the form of an oral preparation or an injection.
The third aspect of the invention also provides the use of a lysozyme derivative or lysozyme composition as described above in the manufacture of a medicament for inhibiting bacterial and/or fungal growth.
Preferably, the bacteria are selected from one or more of escherichia coli, bacillus subtilis and staphylococcus aureus, and the fungi are aspergillus niger; more preferably, the bacteria are E.coli, B.subtilis and Staphylococcus aureus.
Most preferably, the present invention provides the use of the above-described lysozyme derivative or lysozyme composition in the manufacture of a medicament for simultaneously inhibiting the growth of E.coli, B.subtilis, staphylococcus aureus and Aspergillus niger.
The invention has the positive beneficial effects that: surprisingly, through repeated experiments, the lysozyme derivative obtained by using citral modification has excellent antibacterial performance, and the antibacterial effect and antibacterial spectrum breadth of the lysozyme derivative are far superior to those of common egg white lysozyme in the prior art. In addition, the lysozyme has natural sources and no toxic or side effect, and the preparation method of the lysozyme composition can be industrialized and has popularization and application values.
Detailed Description
The present invention will be further described with reference to examples, but the embodiments of the present invention are not limited thereto. The experimental methods used in the following examples are conventional methods unless otherwise specified.
EXAMPLE 1 preparation of citral-modified lysozyme derivative L1
2mL of a hexane solution containing 100. Mu.M citral was gradually added to 10mL of a potassium phosphate buffer (pH 7.4) containing 10. Mu.M egg white lysozyme at a concentration of 50mM, and an equal volume of a potassium phosphate buffer (pH 7.0) containing 3mg/mL sodium borohydride at a concentration of 0.3M was added to the above reaction mixture. The mixture was stirred slowly at 0℃for 1h. Then adding 2 times volume of saturated sodium sulfate to precipitate lysozyme, centrifuging 20000g for 15 minutes, and re-dispersing the centrifuged precipitate into 30mL of distilled water. And (3) putting the dispersion solution into a dialysis bag for dialysis for 72 hours, replacing distilled water outside the dialysis bag every 24 hours, and freeze-drying the solution in the dialysis bag after 72 hours to obtain the citral modified lysozyme derivative L1.
EXAMPLE 2 preparation of citral-modified lysozyme derivative L2
2mL of hexane solution containing 50. Mu.M citral was gradually added to 10mL of potassium phosphate buffer (pH 7.4) containing 10. Mu.M egg white lysozyme at a concentration of 50mM, and an equal volume of potassium phosphate buffer (pH 7.0) containing 3mg/mL sodium borohydride at a concentration of 0.3M was added to the above reaction mixture. The mixture was stirred slowly at 0℃for 1h. Then adding 2 times volume of saturated sodium sulfate to precipitate lysozyme, centrifuging 20000g for 15 minutes, and re-dispersing the centrifuged precipitate into 30mL of distilled water. And (3) putting the dispersion solution into a dialysis bag for dialysis for 72 hours, replacing distilled water outside the dialysis bag every 24 hours, and freeze-drying the solution in the dialysis bag after 72 hours to obtain the citral modified lysozyme derivative L2.
EXAMPLE 3 preparation of citral-modified lysozyme derivative L3
2mL of hexane solution containing 200. Mu.M citral was gradually added to 10mL of potassium phosphate buffer (pH 7.4) containing 10. Mu.M egg white lysozyme at a concentration of 50mM, and an equal volume of potassium phosphate buffer (pH 7.0) containing 3mg/mL sodium borohydride at a concentration of 0.3M was added to the above reaction mixture. The mixture was stirred slowly at 0℃for 1h. Then adding 2 times volume of saturated sodium sulfate to precipitate lysozyme, centrifuging 20000g for 15 minutes, and re-dispersing the centrifuged precipitate into 30mL of distilled water. And (3) putting the dispersion solution into a dialysis bag for dialysis for 72 hours, replacing distilled water outside the dialysis bag every 24 hours, and freeze-drying the solution in the dialysis bag after 72 hours to obtain the citral modified lysozyme derivative L3.
EXAMPLE 4 preparation of citral-modified lysozyme derivative L4
2mL of a hexane solution containing 100. Mu.M citral was gradually added to 10mL of a potassium phosphate buffer (pH 7.4) containing 10. Mu.M egg white lysozyme at a concentration of 50mM, and an equal volume of a potassium phosphate buffer (pH 7.0) containing 1mg/mL sodium borohydride at a concentration of 0.3M was added to the above reaction mixture. The mixture was stirred slowly at 0℃for 1h. Then adding 2 times volume of saturated sodium sulfate to precipitate lysozyme, centrifuging 20000g for 15 minutes, and re-dispersing the centrifuged precipitate into 30mL of distilled water. And (3) putting the dispersion solution into a dialysis bag for dialysis for 72 hours, replacing distilled water outside the dialysis bag every 24 hours, and freeze-drying the solution in the dialysis bag after 72 hours to obtain the citral modified lysozyme derivative L4.
EXAMPLE 5 preparation of citral-modified lysozyme derivative L5
2mL of a hexane solution containing 100. Mu.M citral was gradually added to 10mL of a potassium phosphate buffer (pH 7.4) containing 10. Mu.M egg white lysozyme at a concentration of 50mM, and an equal volume of a potassium phosphate buffer (pH 7.0) containing 5mg/mL sodium borohydride at a concentration of 0.3M was added to the above reaction mixture. The mixture was stirred slowly at 0℃for 1h. Then adding 2 times volume of saturated sodium sulfate to precipitate lysozyme, centrifuging 20000g for 15 minutes, and re-dispersing the centrifuged precipitate into 30mL of distilled water. And (3) putting the dispersion solution into a dialysis bag for dialysis for 72 hours, replacing distilled water outside the dialysis bag every 24 hours, and freeze-drying the solution in the dialysis bag after 72 hours to obtain the citral modified lysozyme derivative L5.
Test example 1 test of the inhibitory Effect of citral-modified lysozyme derivatives of the present invention on bacterial growth
1. Test subjects
(1) The lysozyme derivatives prepared in examples 1-5 were dissolved with deionized water to prepare lysozyme derivative solutions having concentrations of 50mg/mL, 100mg/mL and 150mg/mL, and the supernatant was collected after low-speed centrifugation to obtain the lysozyme derivative bacteriostat (L1-L5) of test examples 1-5 having three concentrations of low, medium and high.
(2) And (3) dissolving egg white lysozyme with deionized water to prepare an egg white lysozyme solution with the concentration of 50mg/mL, 100mg/mL and 150mg/mL, centrifuging at a low speed, and taking supernatant to obtain the egg white lysozyme bacteriostat (C1) of the comparative example 1 with the concentration of low, medium and high.
2. Test method
(1) LB solid medium (containing yeast extract 1g, tryptone 0.5g, naCl 0.5g, agar powder 2g, per 100 mL) was prepared, sterilized by high-pressure steam at 121℃for 20min, and poured into a petri dish until it solidified. E.coli, bacillus subtilis and staphylococcus aureus are respectively streaked and inoculated on LB solid culture medium, and are cultured for 24 hours at 37 ℃ in a constant temperature incubator.
(2) Preparing LB liquid culture medium (containing yeast extract powder 1g, tryptone 0.5g, naCl 0.5g per 100 mL), sterilizing with high pressure steam at 121deg.C for 20min, and coolingRespectively picking 1 loop of strain from the culture medium of activated strain with sterile inoculating loop, inoculating into LB liquid culture medium, shaking (37deg.C, 130 rpm) in shaking table for 12 hr, centrifuging at 10000rpm/min for 15min, removing supernatant, washing with sterile water to obtain precipitate (i.e. adding sterile water, mixing, centrifuging, removing supernatant), repeatedly washing for 3 times, suspending thallus precipitate with a certain amount of sterile water, and measuring and regulating OD of bacterial suspension 600 =0.1-0.2 to produce a bacterial content of about 1 x 10 7 -2*10 7 CFU/mL of bacterial suspension.
(3) 0.1mL of the bacterial suspension is aseptically sucked onto the surface of a solid medium, the surface of the solid medium is uniformly coated by an aseptic coating rod, then an aseptic oxford cup is placed on the surface of the bacteria-containing medium, 0.2mL of the bacteriostat (L1-L5) of the lysozyme derivative of test examples 1-5 and the bacteriostat (C1) of the egg white lysozyme of comparative example 1 with different concentrations are added into the sterile oxford cup, and the medium is slowly transferred into a constant temperature incubator for culturing for 24 hours at 37 ℃. The growth of the colony is observed, and the diameter (mm) of the inhibition zone is measured.
3. Test results
The antibacterial effects of the lysozyme derivative and the unmodified egg white lysozyme on escherichia coli, bacillus subtilis and staphylococcus aureus under different concentration conditions are shown in tables 1-3.
TABLE 1 antibacterial effect of lysozyme derivatives of the present invention at different concentrations on E.coli
TABLE 2 bacteriostatic effects of lysozyme derivatives of the present invention at different concentrations on Bacillus subtilis
TABLE 3 antibacterial effect of lysozyme derivatives of the present invention at different concentrations on Staphylococcus aureus
As shown in the test results of tables 1-3 above, the diameters of the bacteriostasis circles of the lysozyme derivatives L1-L5 of the invention at three concentrations are obviously larger than those of egg white lysozyme C1 serving as a comparison example, which shows that the lysozyme derivatives modified by citral obviously improve the bacteriostasis effects on escherichia coli, bacillus subtilis and staphylococcus aureus. In addition, the lysozyme derivatives L1-L5 of the invention have remarkable antibacterial effects on both gram-positive bacteria and gram-negative bacteria, and also prove that the lysozyme derivatives have broad-spectrum antibacterial activity. It is particularly notable that the lysozyme derivative L1 prepared in example 1 obtained a significantly superior bacteriostatic effect on E.coli, B.subtilis and Staphylococcus aureus to other examples (L2-L5) at the same time, resulting in an unexpectedly superior bacteriostatic effect.
Test example 2 test of the effect of citral-modified lysozyme derivatives of the invention on inhibiting fungal growth
1. Test subjects
(1) The lysozyme derivatives prepared in examples 1-5 were dissolved with deionized water to prepare lysozyme derivative solutions having concentrations of 50mg/mL, 100mg/mL and 150mg/mL, and the supernatant was collected after low-speed centrifugation to obtain the lysozyme derivative bacteriostat (L1-L5) of test examples 1-5 having three concentrations of low, medium and high.
(2) And (3) dissolving egg white lysozyme with deionized water to prepare an egg white lysozyme solution with the concentration of 50mg/mL, 100mg/mL and 150mg/mL, centrifuging at a low speed, and taking supernatant to obtain the egg white lysozyme bacteriostat (C1) of the comparative example 1 with the concentration of low, medium and high.
2. Test method
(1) PDA solid culture medium is prepared, sterilized by high pressure steam at 121 ℃ for 20min, and poured into a culture dish for solidification. Aspergillus niger was streaked onto PDA solid medium and incubated for 2d at 28℃in a thermostated incubator.
(2) Aspergillus niger is removed from a plant by sterile waterWashing the culture medium, centrifuging at 10000rpm/min for 15min, removing supernatant, washing with sterile water to obtain precipitate (i.e. adding sterile water, mixing, centrifuging, removing supernatant), repeatedly washing for 3 times, suspending thallus precipitate with a certain amount of sterile water, and measuring and adjusting bacterial suspension OD 600 =0.1-0.2 to produce a bacterial content of about 1 x 10 7 -2*10 7 CFU/mL of bacterial suspension.
(3) 0.1mL of the bacterial suspension is aseptically sucked onto the surface of a solid medium, the surface of the solid medium is uniformly coated by an aseptic coating rod, then an aseptic oxford cup is placed on the surface of the bacteria-containing medium, 0.2mL of the bacteriostat (L1-L5) of the lysozyme derivative of test examples 1-5 and the bacteriostat (C1) of the egg white lysozyme of comparative example 1 with different concentrations are added into the sterile oxford cup, and the medium is slowly transferred into a constant temperature incubator at 28 ℃ for culturing for 24 hours. The growth of the colony is observed, and the diameter (mm) of the inhibition zone is measured.
3. Test results
The antibacterial effect of the lysozyme derivatives L1-L5 and unmodified egg white lysozyme C1 on aspergillus niger under different concentration conditions is shown in table 4.
TABLE 4 bacteriostatic effects of lysozyme derivatives of the invention at different concentrations on Aspergillus niger
As shown in the test results of the table 4, the diameters of the bacteriostasis rings of the lysozyme derivatives L1-L5 of the invention at three concentrations are obviously larger than that of the egg white lysozyme C1 serving as a comparison example, which shows that the lysozyme derivatives modified by citral obviously improve the bacteriostasis effect on aspergillus niger. In addition, the lysozyme derivatives L1-L5 of the present invention have remarkable antibacterial effects on both the above fungi and the bacteria in tables 1-3, and also demonstrate broad-spectrum antibacterial activity. It is particularly notable that lysozyme derivative L1 prepared in example 1 gave significantly better bacteriostatic effects on Aspergillus niger than the other examples (L2-L5), resulting in unexpectedly superior fungicidal effects.
Claims (6)
1. The lysozyme derivative with the antibacterial effect is characterized by comprising the following steps of:
(1) Dissolving a proper amount of citral in hexane to prepare a citral solution;
(2) Dissolving a proper amount of lysozyme in potassium phosphate buffer solution with the pH of 7.4 to prepare a lysozyme solution;
(3) Slowly dripping the citral solution in the step (1) into the lysozyme solution in the step (2), adding an equal volume of potassium phosphate buffer solution containing sodium borohydride and having the pH of 7.0 into the reaction mixture, and slowly stirring at the temperature of 0 ℃ for 0.5-2h;
(4) Adding 1-3 times of saturated sodium sulfate solution of the reaction solution to precipitate lysozyme, centrifuging at high speed, and re-dispersing the centrifuged precipitate into distilled water;
(5) Putting the dispersion solution in the step (4) into a dialysis bag for dialysis, periodically replacing distilled water outside the dialysis bag, and freeze-drying the solution in the dialysis bag after dialysis to obtain lysozyme derivatives;
the molar concentration ratio of citral in the step (1) to lysozyme in the step (2) is 10:1, a step of;
the volume ratio of the citral solution in the step (1) to the lysozyme solution in the step (2) is 1:5, a step of;
the concentration of sodium borohydride in the potassium phosphate buffer solution in the step (3) is 3mg/mL.
2. The lysozyme derivative according to claim 1, characterized in that the concentration of sodium borohydride in the potassium phosphate buffer in step (3) is 1-5mg/mL.
3. The lysozyme derivative according to claim 1, characterized in that in step (4) centrifugation is carried out for 15 minutes with 20000 g.
4. The lysozyme derivative according to claim 1, wherein in the step (5), the dispersion solution is put into a dialysis bag for dialysis for 72 hours, and distilled water outside the dialysis bag is replaced every 24 hours.
5. A lysozyme composition comprising a lysozyme derivative according to any one of claims 1 to 4.
6. Use of a lysozyme derivative according to any one of claims 1 to 4 or a lysozyme composition according to claim 5 for the preparation of a medicament for inhibiting bacterial and/or fungal growth.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111336938.7A CN113876934B (en) | 2021-11-12 | 2021-11-12 | Lysozyme composition with antibacterial effect |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111336938.7A CN113876934B (en) | 2021-11-12 | 2021-11-12 | Lysozyme composition with antibacterial effect |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113876934A CN113876934A (en) | 2022-01-04 |
| CN113876934B true CN113876934B (en) | 2023-10-13 |
Family
ID=79017373
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111336938.7A Active CN113876934B (en) | 2021-11-12 | 2021-11-12 | Lysozyme composition with antibacterial effect |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113876934B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112159802A (en) * | 2020-09-18 | 2021-01-01 | 北京安佰特科技发展有限公司 | Lysozyme modified by cinnamyl aldehyde coupling and modification method thereof |
-
2021
- 2021-11-12 CN CN202111336938.7A patent/CN113876934B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112159802A (en) * | 2020-09-18 | 2021-01-01 | 北京安佰特科技发展有限公司 | Lysozyme modified by cinnamyl aldehyde coupling and modification method thereof |
Non-Patent Citations (5)
| Title |
|---|
| Covalently immobilized chemically modified lysozyme as a sorbent for bacterial endotoxins (lipopolysaccharides);Pavel A. Levashov等;Biotechnology Reports;第24卷;e00381 * |
| Enhanced Antimicrobial Action of Lysozyme against Gram-Negative and Gram-positive Bacteria Due to Modification with Perillaldehyde;Hisham R. Ibrahim等;J. Agric. Food Chem.;第42卷(第8期);第1813-1817页 * |
| 壳聚糖与柠檬醛缩合反应产席夫碱及其抗菌活性;金晓晓;王江涛;白洁;;化工进展(第11期);摘要，第2015页左栏第2段 * |
| 天然柠檬醛衍生物对食品腐败细菌的抑制活性;李兆双;王喜男;王鹏;陈尚;范国荣;王宗德;;浙江农业学报(第11期);全文 * |
| 植物精油对大菱鲆弧菌的体外和体内抗菌活性;崔惠敬;孟玉霞;徐永平;赵前程;姜龙;马永生;;微生物学通报(第02期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113876934A (en) | 2022-01-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jeon et al. | Antimicrobial effect of chitooligosaccharides produced by bioreactor | |
| Kamil et al. | Isolation and identification of rhizosphere soil chitinolytic bacteria and their potential in antifungal biocontrol | |
| Wang et al. | Antimicrobial effect of chitooligosaccharides produced by chitosanase from Pseudomonas CUY8. | |
| US5057542A (en) | Cosmetic preparation containing chitosan | |
| Wang et al. | Production of antifungal materials by bioconversion of shellfish chitin wastes fermented by Pseudomonas fluorescens K-188 | |
| CN113876934B (en) | Lysozyme composition with antibacterial effect | |
| CN109452283A (en) | A kind of application of mannosylerythritol lipid in suppressing growth of microorganism | |
| WO1991010435A1 (en) | Chitosan pyrithione as antimicrobial agent useful in personal care products | |
| KR102450693B1 (en) | A lactobacillus paracasei uos1 strain and antibacterial composition comprising the same | |
| Seo et al. | Development of a natural preservative system using the mixture of chitosan‐Inula helenium L. extract | |
| CN101507703B (en) | High-efficiency sterilization hands washing liquid | |
| CN101239034B (en) | Sterilization shampoo | |
| EP1481666B1 (en) | A composition for re-establishment of the vaginal ecosystem | |
| Debnath et al. | Fungal exopolysaccharides: types, production and application | |
| JPH0156755B2 (en) | ||
| Upadhyay | Growth and bacteriolytic activity of a soil amoeba, Hartmannella glebae | |
| KR20200027482A (en) | Novel chitinase-producing Salinivibrio sp. Kostichola BAO1801 strain and use thereof | |
| KR20140123131A (en) | Antibiotic composition containing low molecular water soluble chitosan or derivative thereof | |
| KR101113729B1 (en) | Process for Preparation of Middle Molecular Chitosan Having Antibacterial Activity against Antibiotic Resistance Bacteria and Uses thereof | |
| JPH08333213A (en) | Antiviral and antimicrobial agent | |
| CN114874619B (en) | Polyethyleneimine/oxidized cellulose nanogel, preparation method and application | |
| JPH07258015A (en) | Insecticidal composition and method for killing insect pest using the same | |
| JPH0124121B2 (en) | ||
| CN114159337B (en) | Facial antibacterial care solution containing recombinant human lysozyme | |
| KR20190130355A (en) | Novel chitinase-producing Salinivibrio sp. Kostichola BAO1801 strain and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |