CN113730561A - Preparation method of phage endolysin-loaded cationic guar gum liposome - Google Patents
Preparation method of phage endolysin-loaded cationic guar gum liposome Download PDFInfo
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- CN113730561A CN113730561A CN202110936480.2A CN202110936480A CN113730561A CN 113730561 A CN113730561 A CN 113730561A CN 202110936480 A CN202110936480 A CN 202110936480A CN 113730561 A CN113730561 A CN 113730561A
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- Prior art keywords
- endolysin
- liposome
- cationic guar
- guar gum
- solution
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- KDXKERNSBIXSRK-UHFFFAOYSA-N lysine Chemical compound NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 title claims abstract description 114
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 title claims abstract description 110
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- 125000002091 cationic group Chemical group 0.000 title claims abstract description 58
- 229920002907 Guar gum Polymers 0.000 title claims abstract description 49
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- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 241000607272 Vibrio parahaemolyticus Species 0.000 claims abstract description 54
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- 239000003623 enhancer Substances 0.000 claims abstract description 12
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- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims abstract description 5
- 238000001704 evaporation Methods 0.000 claims abstract description 3
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 claims description 5
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- RUHCWQAFCGVQJX-RVWHZBQESA-N (3s,8s,9s,10r,13r,14s,17r)-3-hydroxy-10,13-dimethyl-17-[(2r)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-1-one Chemical compound C1C=C2C[C@H](O)CC(=O)[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 RUHCWQAFCGVQJX-RVWHZBQESA-N 0.000 claims description 3
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- NWGKJDSIEKMTRX-AAZCQSIUSA-N Sorbitan monooleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O NWGKJDSIEKMTRX-AAZCQSIUSA-N 0.000 claims description 3
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- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 claims description 2
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- NRHMKIHPTBHXPF-TUJRSCDTSA-M sodium cholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 NRHMKIHPTBHXPF-TUJRSCDTSA-M 0.000 claims description 2
- 239000001593 sorbitan monooleate Substances 0.000 claims description 2
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Images
Classifications
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- 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
- A61K38/51—Lyases (4)
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
-
- 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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
-
- 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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Zoology (AREA)
- Communicable Diseases (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Dispersion Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Agronomy & Crop Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Virology (AREA)
- Gastroenterology & Hepatology (AREA)
- Dentistry (AREA)
- Wood Science & Technology (AREA)
- Environmental Sciences (AREA)
- Inorganic Chemistry (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention relates to a preparation method of liposome, in particular to a preparation method of cationic guar gum liposome loaded with bacteriophage lysin, belonging to the technical field of biology. The method comprises the following steps: a) dissolving a phospholipid, cholesterol, and an elasticity enhancer in an organic solvent to form a premix; b) evaporating the organic solvent from the premix to form a phospholipid membrane; c) hydration of a system of blending of a phagolysin and cationic guar with a phospholipid membrane; d) homogenizing to form a liposome coating solution, wherein the weight ratio of the phage endolysin to the cationic guar gum in the coating solution is 1: 1 to 1: 15. the liposome prepared by the invention has effective bactericidal activity on vibrio parahaemolyticus, and still retains strong bactericidal activity after high-temperature and freeze-drying treatment.
Description
Technical Field
The invention relates to a preparation method of liposome, in particular to a preparation method of cationic guar gum liposome loaded with bacteriophage lysin, belonging to the technical field of biology.
Background
Vibrio parahaemolyticus (Vibrio parahaemolyticus) belongs to the genus Vibrio, is an important gram-negative zoonosis pathogenic bacterium, and is widely present in various marine products. When the bacterial gastroenteritis caused by the pathogen is eaten directly or after being eaten, the food polluted by the vibrio parahaemolyticus is easy to cause acute gastroenteritis and symptoms such as headache, diarrhea, nausea, vomiting, low fever and the like, and the proportion of the bacterial gastroenteritis caused by the pathogen is more than 45 percent all over the world. In recent decades, Vibrio parahaemolyticus has shown high resistance to a variety of antibiotics due to the abuse of antibiotics in aquaculture production. Vibrio parahaemolyticus isolated from marine products in parts of Asia and European countries (Korea, China, Italy, Poland, etc.) has high resistance to antibiotics such as ampicillin, rifampicin, streptomycin, etc.
The phage lysosome is a peptidoglycan hydrolase encoded by the phage, has the advantages of high cracking speed, strong specificity, high safety, difficulty in generating drug resistance and the like, and is an ideal substitute of antibiotics. The endolysin Lysqdvp001 is derived from a vibrio parahaemolyticus phage qdvp001, has a wider lysis spectrum than a parent phage thereof, and shows peptidoglycan lysis activity to various vibrios. At present, the resistance of vibrio parahaemolyticus is becoming serious, endolysin Lysqdvp001 has certain advantages as a potential weapon for controlling multidrug-resistant vibrio parahaemolyticus. However, the vibrio parahaemolyticus has an outer membrane structure specific to gram-negative bacteria, and the bacterial outer membrane prevents endolysin from reaching a target peptidoglycan substrate, so that the vibrio parahaemolyticus is insensitive to the direct application of the endolysin, and the application of the endolysin in the aspect of controlling the harm of the vibrio parahaemolyticus is limited.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a preparation method of a cationic guar gum liposome loaded with a phage endolysin, and the liposome has a good bactericidal effect on vibrio parahaemolyticus.
In order to achieve the purpose, the invention specifically adopts the following scheme:
a method of preparing liposomes, the method comprising the steps of:
a) dissolving a phospholipid, cholesterol, and an elasticity enhancer in an organic solvent to form a premix;
b) evaporating the organic solvent from the premix to form a phospholipid membrane;
c) hydration of a system of blending of a phagolysin and cationic guar with a phospholipid membrane;
d) homogenizing to form a liposome coating solution, wherein the weight ratio of the phage endolysin to the cationic guar gum in the coating solution is 1: 1 to 1: 15. the amounts of phospholipid, cholesterol and elasticity enhancer constituting the phospholipid membrane are selected conventionally by those skilled in the art, and are not described in detail herein.
The cationic guar gum is cationic polysaccharide synthesized by quaternization of guar gum, and the research of the inventor finds that the cationic guar gum is positively charged, so that the cationic guar gum can effectively improve the surface positive charge of the liposome when added into the liposome, targets the surface environment with negative charge of bacteria, and improves the sterilization effect of the liposome.
The liposomes of the present invention comprise a phospholipid. The phospholipid may be selected from natural phospholipids, synthetic phospholipids, and combinations thereof. Lecithin is a phospholipid of one of its natural sources. Lecithin is a mixture of substances present in egg yolk and soybean. It contains a variety of phospholipids, including Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), and Phosphatidylinositol (PI). Natural phospholipids also include, for example, hydrogenated soy pc (hspc), sphingomyelin, and Phosphatidylglycerol (PG).
The liposomes of the present invention comprise an elasticity enhancer. Preferably, the elasticity enhancer is one or more selected from sodium cholate, sodium deoxycholate, polysorbate 80(Tween80), sorbitan monooleate (Span80), oleic acid, dipotassium glycyrrhizinate (KG), and cholesterol ether. Preferably, the elasticity enhancer is a surfactant, such as an anionic surfactant, a nonionic surfactant or a zwitterionic surfactant, preferably a nonionic surfactant. Further preferably, the nonionic surfactant is one or more of Tween80, Span-80 or polyoxyethylene cholesterol ether.
Preferably, the liposome coating solution comprises effective amounts of phospholipid, cholesterol and elasticity enhancer, and a formulated amount of cationic guar gum of 0.1-0.8mg/mL of bacteriophage lysin.
Preferably, the liposome coating solution in a 10mL system comprises: 80 mug of soybean lecithin, 20 mug of cholesterol, 2-8mg of vibrio parahaemolyticus phage endolysin, 10-30mg of cationic guar gum and 808-12 mug of Tween. The soybean lecithin, the cholesterol and the Tween80 are all commercial food-grade materials or additives, and are suitable for industrial production.
Preferably, the method comprises the steps of:
s1, pretreatment of endolysin: expressing recombinant phage endolysin by using escherichia coli, performing heat treatment to partially denature the recombinant phage endolysin, and centrifuging to remove precipitates to obtain an endolysin solution;
s2, preparation of phospholipid membrane: dissolving phospholipid, cholesterol and an elasticity enhancer in a mixed system of an organic solvent to obtain a premix, performing rotary evaporation on the premix to form a phospholipid membrane on the inner wall of a container, and drying;
s3, hydration of phospholipid membranes: adding cationic guar gum into the heat-denatured endolysin solution prepared in the step S1, wherein the weight ratio of the heat-denatured endolysin to the cationic guar gum is 1: 1 to 1: 15, adding the obtained mixed solution into the container in the step S2, and performing rotary evaporation to separate the phospholipid membrane from the wall of the container, so that the phospholipid membrane is fully hydrated and dissolved in the mixed solution;
s4, preparation of liposome coating solution: and carrying out water bath ultrasonic treatment on the mixed solution to obtain liposome coating solution.
The purpose of the endolysin heat treatment is: the heating denatures the endolysin part, so that the protein structure of the endolysin can be unfolded, the internal hydrophobic group is exposed, the hydrophobicity of the endolysin and the interaction capacity with a bacterial membrane are improved, and experiments prove that the bactericidal activity of the endolysin obtained by thermal denaturation is obviously improved compared with the unheated natural endolysin.
In S1, the heat treatment of endolysin has no significant influence on the enzymatic activity of endolysin, can improve the bactericidal activity of endolysin, has mild heat treatment conditions, and avoids the aggregation and denaturation inactivation of protein under extreme conditions. The temperature of the heat treatment of the endolysin is 70 plus or minus 2 ℃, and the heating time is 1-6 min; the optimal temperature is 70 deg.C for 6 min.
Preferably, in S2 and S3, the conditions of rotary evaporation are: rotary steaming at 70 + -10 RPM at 40 + -2 deg.C.
Preferably, in S1, the endolysin with higher purity is obtained by desalting treatment by nickel column purification and dialysis, and the obtained endolysin solution is adjusted so that the endolysin concentration in the endolysin solution is 200-400. mu.g/mL.
Preferably, in S2, the drying condition is 60 ℃ for 2 h.
Preferably, the organic solvent is a mixture of 10mL of chloroform and methanol 2: 1 in volume ratio.
Preferably, in S4, the conditions of the water bath ultrasonic treatment are: the ultrasonic time of the water bath is 2h under the condition of room temperature and the frequency of 40 Hz.
Preferably, in step S1, the recombinant phage lysin is prepared by a method comprising:
the engineering bacterium Rosetta (Lysqdvp001) capable of expressing endolysin is inoculated in 5mL of LB liquid medium containing 50 ug/mL of kanamycin and cultured overnight at 37 ℃ with shaking (150 r/min);
the next day, according to 1: 50 ratio overnight culture broth was added to fresh LB liquid medium (300mL, containing 50. mu.g/mL kanamycin) and cultured at 37 ℃ at 150r/min for 2-3h to OD600nmAbout 0.6; adding IPTG to the final concentration of 1mmol/L, and inducing at 25 ℃ for 4h at 150 r/min;
then centrifuging at 4000r/min and 4 deg.C for 20min, removing supernatant, and adding buffer (containing 20 mmol/LNa)2HPO3500mmol/L NaCl, pH 8.0), ultrasonic crushing after mixing uniformly, then centrifuging at 8000r/min for 30min to remove insoluble cell debris, and filtering with 0.22 μm filter membrane to obtain crude enzyme solution;
NiSepharose TM 6Fast Flow is adopted for purification, and a 10kD ultrafiltration tube is used for desalination to obtain the endolysin with higher purity.
Preferably, in step S3, the concentration of endolysin in the mixed solution system is 200-400 μ g/mL, and the concentration of cationic guar gum is 1-3 mg/mL. The optimal concentration is 1.5-2.5 mg/mL. The optimal concentration is 2.0 mg/mL.
The application of the liposome or the coating liquid thereof as a bactericide in aquaculture. Further, the sterilization refers to the killing of vibrio parahaemolyticus. Preferably, the liposomes are added to the body of aquaculture water at a final concentration of phage endolysin of 10 to 50 μ g/mL, most preferably 10 to 30 μ g/mL, most preferably 25 μ g/mL.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
1. the preparation method of the phage endolysin-loaded cationic guar gum liposome and the coating liquid thereof is simple and convenient to operate;
2. the phage endolysin-loaded cationic guar gum liposome and the coating liquid thereof have effective bactericidal activity on vibrio parahaemolyticus, still retain strong bactericidal activity after high-temperature and freeze-drying treatment, and can be used as a bactericide for vibrio parahaemolyticus in the aquaculture process.
3. Taking clam culture as an example, the cationic guar gum liposome loaded with the phage endolysin and the coating liquid thereof disclosed by the invention are applied in the clam culture process, so that vibrio parahaemolyticus in a water environment can be effectively killed, the sterilization rate is up to more than 99%, and the pollution of the vibrio parahaemolyticus of the clam is reduced; the sterilization rate of the vibrio parahaemolyticus in the clam is over 90 percent when the liposome is added to the thermally denatured endolysin with the final concentration of 25 mug/mL.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention.
FIG. 1 is a process flow diagram of the process of the present invention;
FIG. 2 is a graph comparing the heating time to the endolysin enzyme activity;
FIG. 3 is a graph comparing the time of heating to the bactericidal activity of endolysin;
FIG. 4 is a graph comparing cationic guar gum addition concentration to liposomal antimicrobial activity;
FIG. 5 is a schematic diagram of the measurement of the thermostability of a cationic guar gum liposome loaded with heat-denatured phage endolysin;
fig. 6 is a schematic diagram of bactericidal activity determination before and after lyophilization of heat-denatured phage endolysin-loaded cationic guar gum liposomes.
Detailed Description
The technical solution of the present invention will be further specifically described below by way of specific examples. It is to be understood that the practice of the invention is not limited to the following examples, and that various changes and modifications may be made without departing from the scope of the invention.
The equipment and materials used in the present invention are commercially available or commonly used in the art, unless otherwise specified, all parts and percentages are by weight.
The strains involved in the test of the invention: vibrio parahaemolyticus VP17802 (accession number ATCC17802) was purchased from American ATCC center, and an engineering bacterium Rosetta (Lysqdvp001) capable of expressing endolysin was deposited in the food safety laboratory of China university of oceans.
LB liquid medium, 2216E medium, qingdao haibo biotechnology ltd.c.;
NiSepharose TM6Fast Flow packing, GE USA;
isopropyl-beta-D-l thiogalactoside (IPTG), cholesterol (Chol), Beijing Soilebao Tech Co., Ltd.;
soybean lecithin (PC), tween80 (T-80), shanghai aladine reagents ltd;
cationic guar gum, guangzhou republic of chemical technology ltd.
Example 1A method for preparing Heat-denatured endolysin
1. Preparation of heat-denatured endolysins
The engineered bacterium Rosetta (Lysqdvp001) expressing endolysin was inoculated into 5mL of LB liquid medium containing 50. mu.g/mL of kanamycin and cultured overnight at 37 ℃ with shaking (150 r/min). The next day, according to 1: 50 ratio overnight-cultured broth was added to fresh LB liquid medium (300)mL, containing 50. mu.g/mL kanamycin), cultured at 37 ℃ for 2-3h at 150r/min to OD600nmAbout 0.6. IPTG was added to a final concentration of 1mmol/L and induction was carried out at 25 ℃ at 150r/min for 4 h. Then centrifuging at 4000r/min and 4 deg.C for 20min, removing supernatant, and adding buffer (containing 20 mmol/LNa)2HPO3500mmol/L NaCl, pH 8.0), ultrasonic crushing after mixing, then centrifuging at 8000r/min for 30min to remove insoluble cell debris, and filtering with 0.22 μm filter membrane to obtain crude enzyme solution. By using NiSepharoseTMPurifying 6Fast Flow, and desalting with 10kD ultrafiltering tube to obtain high purity endolysin. Heating the endolysin solution (200-.
2. Effect of heating time on Heat-denatured endoplasmin Activity
Selecting Vibrio parahaemolyticus VP17802 to 300mL2216E liquid culture medium, culturing overnight, centrifuging thallus, thawing with 100mmol/L EDTA for 5min, centrifuging at 4 deg.C and 8000r/min for 10min, thawing with pure water, centrifuging twice to obtain bacterial precipitate, and storing at-80 deg.C. Before the activity was measured, the Vibrio parahaemolyticus bacterial pellet was treated with 50mmol/LTris buffer (pH 8.2 containing 0.1% Triton X-100). mu.L of the bacterial reconstitution solution was added to a 96-well plate, 100. mu.L of heat-denatured endolysin Lysqdvp001 (final concentration: 200. mu.g/mL) was added thereto, and after 30min of treatment at 20 ℃ the OD was measured450nmThe value is obtained. Tris buffer was used as negative control, endolysin with heat treatment time of 0 was used as positive control, OD450nmThe decrease in value reflects endolysin activity. The relative enzyme activity is calculated as: relative enzyme activity ═ Δ assay set OD450nm(decrease value of heat-denatured endolysin experimental group)/Delta positive control group OD450nm(reduction of the untreated endolysin treatment group).
As shown in FIG. 2, the relative enzyme activities of endolysin after heating at 70 ℃ for 2-6min were not significantly reduced (both close to 1.0), indicating that the enzyme activities of endolysin were not significantly affected within 6min, and the relative enzyme activities of endolysin after heating at 70 ℃ for 8 and 10min were reduced to 0.75 and 0.74, indicating that the enzyme activities of endolysin were significantly reduced after heating for more than 8min, compared to the heat-untreated endolysin.
3. Effect of heating time on the Bactericidal Activity of Heat-denatured endolysin
The vibrio parahaemolyticus VP17802 to 300mL2216E liquid culture medium is picked up, cultured overnight, the thalli is centrifuged, and the Tris buffer solution is used for thawing. mu.L of the bacterial reconstituted solution was added to an EP tube, and then 500. mu.L of heat-denatured endolysin Lysqdvp001 (final concentration: 200. mu.g/mL) was added thereto, and the mixture was treated at 37 ℃ and 150r/min with Tris buffer as a control for 2 hours, and then the number of culturable bacteria in each group was measured by plate counting method using a TCBS plate.
As shown in FIG. 3, the numbers of cultured Vibrio parahaemolyticus in the control group were 7.70log10 CFU/mL, and the numbers of cultured Vibrio parahaemolyticus treated with endolysin after heating at 70 ℃ for 0-6min were 6.90, 6.02, 6.25 and 5.37log10CFU/mL, respectively. The numbers of culturable bacteria of endolysin-treated Vibrio parahaemolyticus after heating at 70 ℃ for 8 and 10min were 7.54 and 7.32log10 CFU/mL, respectively. These data indicate that the endolysin bactericidal activity tends to increase with increasing heat treatment time when heated at 70 ℃ for 0 to 6min, and that the endolysin bactericidal activity decreases significantly when heated at 70 ℃ for more than 8 min.
Example 2 preparation method of cation guar gum liposome loaded with heat-denatured endolysin
1. Preparation of heat-denatured endolysin-loaded cationic guar gum liposome
The process flow diagram of the method is shown in FIG. 1, and 80. mu.g of soybean lecithin (PC), 20. mu.g of cholesterol (Chol) and 10. mu.L of Tween80 (T-80) are dissolved in a mixed system containing 10mL of chloroform and methanol (2: 1 v/v). Transferring the mixed solution into a round-bottom flask, introducing into a rotary evaporator, performing vacuum rotary evaporation at 75rpm and 40 ℃, and performing rotary evaporation for 1.5-2h to form unilamellar liposomes on the inner wall of the flask. And (3) putting the round-bottom flask with the formed unilamellar liposome into a constant-temperature oven at normal pressure, setting the temperature to be 60 ℃, and drying for 2 hours. The resulting heat-denatured endolysin solution heated at 70 ℃ for 6min was adjusted to a heat-denatured endolysin concentration of 0.2mg/mL with PBS buffer (pH 7.4), and cationic guar gum was added to 0-3 mg/mL. 10mL of the mixture of the heat-denatured endolysin and the guar gum is added into a round-bottom flask, and the mixture is rotationally steamed for 10min at the rotating speed of 70rpm under the condition of 40 ℃ so as to separate the film from the glass wall, so that the mixture is fully dissolved in the mixture after hydration. And placing the obtained dispersion mixed solution in an ultrasonic cleaner, setting the temperature to be 25 ℃ and the frequency to be 40Hz, and carrying out water bath ultrasonic treatment for 2h to obtain the liposome coating solution. And (4) freeze-drying the obtained liposome coating solution to obtain liposome dry powder.
2. Effect of cationic guar concentration on liposome particle size, PDI and ZETA potential
The particle size, PDI and ZETA potential of the liposomes were determined using a laser particle sizer (Nanotrac wave II, marvens instruments, uk).
Table 1 mean particle size, PdI and ZETA analysis of liposomes with different concentrations of cationic guar
The results are shown in table 1, with increasing cationic guar concentration, increasing liposome mean particle size and ZETA potential. The PdIs of the liposome are between 0.34 and 0.53, and are all less than 0.6, which shows that the liposome has good uniformity.
3. Effect of cationic guar concentration on Liposome Bactericidal Activity
The vibrio parahaemolyticus VP17802 to 300mL2216E liquid culture medium is picked up, cultured overnight, the thalli is centrifuged, and the Tris buffer solution is used for thawing. The number of culturable bacteria in each group was determined by plate counting using a TCBS plate after adding 500. mu.L of a bacterial reconstituted solution to an EP tube, 500. mu.L of PBS buffer containing a cationic guar liposome loaded with heat-denatured endolysin (final concentration of heat-denatured endolysin: 25. mu.g/mL) and treating the mixture at 37 ℃ and 150r/min for 2 hours using Tris buffer as a control.
As shown in FIG. 4, as the concentration of cationic guar added in the preparation of liposome increases, the number of culturable bacteria of Vibrio parahaemolyticus after liposome treatment increases and then decreases and reaches equilibrium, and when the concentration of cationic guar increases to more than 2.0mg/mL, the number of culturable bacteria of Vibrio parahaemolyticus after liposome treatment decreases to about 5.0log10CFU/mL. These results indicate that as the concentration of cationic guar used to prepare the liposomes increases, the antimicrobial activity of the liposomes decreases and then increases, and that the cationic guar concentration increases above 2.0mg/mL, there is no significant increase in the antimicrobial activity of the liposomes.
Example 3 measurement of the thermostability and antibacterial Activity of Heat-denatured endolysin-loaded cationic guar Liposome and before and after lyophilization
1. Determination of thermal stability of cationic guar gum liposome loaded with heat-denatured endolysin
The cationic guar gum liposome Z2 and Z3 loaded with heat-denatured endolysin, wherein the concentration of the cationic guar gum is added in an amount of 2.0 and 3.0mg/mL, are respectively heated at 60-90 ℃ for 10min for later use. The vibrio parahaemolyticus VP17802 to 300mL2216E liquid culture medium is picked up, cultured overnight, the thalli is centrifuged, and the cells are thawed by Tris buffer solution. Adding 500 mu L of bacterial re-melting solution into an EP tube, then respectively adding 500 mu L of PBS buffer solution (the final concentration of heat-denatured endolysin is 25 mu g/mL) containing Z2 and Z3 liposomes, taking Tris buffer solution as a negative control, taking the liposomes without heat treatment as a positive control, treating for 2h under the conditions of 37 ℃ and 150r/min, using a TCBS plate, determining the number of culturable bacteria of each group by a plate counting method, and reacting the bactericidal activity of the liposomes by using the reduction value of the number of culturable bacteria of vibrio parahaemolyticus. The formula for calculating the relative bactericidal activity is: relative bactericidal activity ═ Δ assay viable count/Δ positive control viable count.
As shown in FIG. 5, the results show that the Z2 and Z3 liposomes have good thermal stability and can maintain at least 80% of bactericidal activity after being heated at 60-90 ℃ for 10 min.
2. Determination of antibacterial activity of heat-denatured endolysin-loaded cationic guar gum liposome before and after freeze-drying
Z2 and Z3 liposomes (cationic guar concentration added in 2.0 and 3mg/mL) were lyophilized and reconstituted with an equal volume of sterile water for use. The vibrio parahaemolyticus VP17802 to 300mL2216E liquid culture medium is picked up, cultured overnight, the thalli is centrifuged, and the Tris buffer solution is used for thawing. Adding 500 mu L of bacterial re-melting liquid into an EP tube, then adding 500 mu L of PBS buffer solution (the final concentration of the heat-denatured endolysin is 25 mu g/mL) containing cation guar gum liposome loaded with the heat-denatured endolysin, taking Tris buffer solution as a negative control, taking liposome without freeze-drying treatment as a positive control, treating for 2h under the conditions of 37 ℃ and 150r/min, using a TCBS plate, determining the number of culturable bacteria of each group by a plate counting method, and reacting the sterilization activity of the liposome by using the reduction value of the number of culturable bacteria of vibrio parahaemolyticus. The formula for calculating the relative bactericidal activity is: relative bactericidal activity ═ Δ assay viable count/Δ positive control viable count.
As shown in FIG. 6, the Z2 (cationic guar gum concentration added 2.0mg/mL) liposome has good reconstitution effect after freeze-drying, and the antibacterial activity is not reduced, while the Z3 (cationic guar gum concentration added 3.0mg/mL) liposome has poor reconstitution effect after freeze-drying, has obvious flocculent precipitate, and has obviously reduced antibacterial activity.
Example 4 application of cationic guar gum liposome loaded with heat-denatured endolysin in prevention and treatment of Vibrio parahaemolyticus contamination of clams
1. Application of heat-denatured endolysin-loaded cationic guar gum liposome in prevention of clam vibrio parahaemolyticus pollution
Selecting fresh clams, and standing in sterile seawater for 48h, wherein water is continuously changed in the period. Then inoculating VP17802 (about 1.13X 10) in the aquaculture water body5CFU/mL), treatment groups added Z2 and Z3 liposomes to a final concentration of heat denatured endolysin of 25 and 50. mu.g/mL for 4 h. And after the treatment is finished, measuring the number of the vibrio parahaemolyticus in the water body, and averaging the results of three parallel tests in each group. Opening the shell of the treated clams, taking meat, measuring the number of vibrio parahaemolyticus, and averaging the results of six parallel tests in each group.
Table 2 analysis of bactericidal effect of heat-denatured endolysin-loaded cationic guar gum liposome in clam culture water body
Table 2 shows the bactericidal effect of the heat-denatured endolysin-loaded cationic guar gum liposome in the clam culture water body, and Z2 and Z3 liposomes can effectively kill vibrio parahaemolyticus in the clam culture water body. The Z2 liposome (heat denatured endolysin 25 and 50 ug/mL) treatment killed more than 99% of Vibrio parahaemolyticus in water, and the Z3 liposome (heat denatured endolysin 25 and 50 ug/mL) treatment killed more than about 82% -95% of Vibrio parahaemolyticus in water.
TABLE 3 analysis of the Effect of the cationic guar gum liposome loaded with heat-denatured endolysin on the prevention of Vibrio parahaemolyticus contamination of clams
Table 3 shows the application effect of the heat-denatured endolysin-loaded cationic guar gum liposome in preventing the contamination of the clam vibrio parahaemolyticus. The Z2 and Z3 liposomes added in the clam culture water body can effectively prevent the enrichment of vibrio parahaemolyticus in the clam, and the number of the vibrio parahaemolyticus in the clam in the liposome treatment group is obviously lower than that of the control group, and is reduced by about 0.83-1.34log10CFU/g。
2. Application of cation guar gum liposome loaded with heat-denatured endolysin in clam purification
Selecting fresh clams, and standing in sterile seawater for 48h, wherein water is continuously changed in the period. Then inoculating VP17802 (about 2.92X 10) in the aquaculture water body7CFU/mL), culturing at 16 deg.C for 4h to enrich the clams with Vibrio parahaemolyticus, and transferring to sterile seawater. Treatment groups were treated with Z2 and Z3 liposomes to a final concentration of heat denatured endolysin of 25 and 50. mu.g/mL for 4 h. Opening the shell of the treated clams, taking meat, measuring the number of vibrio parahaemolyticus, and averaging the results of six parallel tests in each group.
Table 4 analysis of the effect of heat denatured endolysin loaded cationic guar gum liposomes on clam cleansing
As shown in Table 4, the numbers of Vibrio parahaemolyticus in clams were significantly reduced after treatment with Z2 and Z3 liposomes, and when Z2 and Z3 liposomes were added to a final concentration of heat-denatured endolysin of 25. mu.g/mL, more than 90% of Vibrio parahaemolyticus were killed, and the Vibrio parahaemolyticus was killed when the liposomes were added to the heat-denatured endolysinThe clam has certain antibacterial effect and can reduce Vibrio parahaemolyticus by about 1.4log at most10CFU/g. However, the bactericidal effect is rather reduced with the increase of the addition amount of the liposome, and when the Z2 and Z3 liposome are added to the heat-denatured endolysin with the final concentration of 50 mug/mL, only about 50% -60% of vibrio parahaemolyticus in the clam is killed.
The cationic guar gum liposome loaded with heat-denaturation vibrio parahaemolyticus phage endolysin, the preparation method and the application thereof are described in detail. The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its core concepts. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
Claims (10)
1. A preparation method of liposome is characterized by comprising the following steps:
a) dissolving a phospholipid, cholesterol, and an elasticity enhancer in an organic solvent to form a premix;
b) evaporating the organic solvent from the premix to form a phospholipid membrane;
c) hydration of a system of blending of a phagolysin and cationic guar with a phospholipid membrane;
d) homogenizing to form a liposome coating solution, wherein the weight ratio of the phage endolysin to the cationic guar gum in the coating solution is 1: 1 to 1: 15.
2. the method of claim 1, wherein: the liposome coating solution comprises effective dose of phospholipid, cholesterol and elasticity enhancer, and phage endolysin 0.1-0.8mg/mL, and formula dose of cationic guar gum.
3. The method of claim 1, wherein: the elasticity enhancer is selected from one or more of sodium cholate, sodium deoxycholate, polysorbate 80(Tween80), sorbitan monooleate (Span80), oleic acid, dipotassium glycyrrhizinate (KG) and cholesterol ether.
4. The method of claim 1, wherein: the 10mL system of the liposome coating solution contains: 80 mug of soybean lecithin, 20 mug of cholesterol, 2-8mg of vibrio parahaemolyticus phage endolysin, 10-30mg of cationic guar gum and 808-12 mug L of Tween.
5. The method of claim 1, wherein the method comprises the steps of:
s1, pretreatment of endolysin: expressing recombinant phage endolysin by using escherichia coli, performing heat treatment to partially denature the recombinant phage endolysin, and centrifuging to remove precipitates to obtain an endolysin solution;
s2, preparation of phospholipid membrane: dissolving phospholipid, cholesterol and an elasticity enhancer in a mixed system of an organic solvent to obtain a premix, performing rotary evaporation on the premix to form a phospholipid membrane on the inner wall of a container, and drying;
s3, hydration of phospholipid membranes: adding cationic guar gum into the endolysin solution prepared in the step S1, wherein the weight ratio of the endolysin to the cationic guar gum is 1: 1 to 1: 15, adding the obtained mixed solution into the container in the step S2, and performing rotary evaporation to separate the phospholipid membrane from the wall of the container, so that the phospholipid membrane is fully hydrated and dissolved in the mixed solution;
s4, preparation of liposome coating solution: and carrying out water bath ultrasonic treatment on the mixed solution to obtain liposome coating solution.
6. The method of claim 1, further comprising: s5, freeze-drying the liposome coating solution to obtain the dry powder liposome.
7. The method of claim 1, wherein: in step S1, the method for preparing recombinant phage lysin comprises:
inoculating the engineering bacterium Rosetta capable of expressing endolysin into 5mL LB liquid culture medium containing 50 ug/mL kanamycin, and shaking at 37 ℃ for overnight culture;
the next day, according to 1: adding the overnight cultured bacterial liquid into a fresh LB liquid culture medium according to the proportion of 50, and culturing for 2-3h at the temperature of 37 ℃ and at the speed of 150 r/min; adding IPTG to the final concentration of 1mmol/L, and inducing at 25 ℃ for 4h at 150 r/min;
centrifuging at 4000r/min at 4 deg.C for 20min, removing supernatant, and adding Na containing 20mmol/L2HPO3Re-melting bacterial precipitation by using 500mmol/L NaCl and a buffer solution with pH of 8.0, uniformly mixing, performing ultrasonic crushing, centrifuging at 8000r/min for 30min to remove insoluble cell fragments, and filtering with a filter membrane of 0.22 mu m to obtain a crude enzyme solution;
NiSepharose TM 6Fast Flow is adopted for purification, and a 10kD ultrafiltration tube is used for desalination to obtain the endolysin with higher purity.
8. The method of claim 1, wherein: in the step S3, in the mixed solution system, the endolysin concentration is 200-400 mug/mL, and the cationic guar gum addition concentration is 1-3 mg/mL.
9. The method of claim 1, wherein: in S2 and S3, the conditions of rotary evaporation are: rotary steaming at 70 + -10 RPM at 40 + -2 deg.C.
10. The method of claim 1, wherein: in S1, carrying out desalination treatment by using nickel column purification and dialysis to obtain endolysin with higher purity, and adjusting the obtained endolysin solution to enable the concentration of the endolysin in the endolysin solution to be 200-; the heat treatment temperature of endolysin is 70 + -2 deg.C, and the heating time is 1-6 min.
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