Mild multi-progressive slow-release antibacterial composition and application thereof

Abstract

The invention provides a mild multi-progressive slow-release antibacterial composition and application thereof, and belongs to the field of medical, dental or cosmetic preparations. The components of the antibacterial gel mask comprise quaternary ammonium salt-chitosan antibacterial gel, a humectant, PEG-4000, juniper berry essential oil and Ami tree essential oil. The formula is simple, and raw material selection is advanced through a triple antibacterial system of organic quaternary ammonium salt, natural polysaccharide and essential oil slow release; good effects of resisting bacteria, diminishing inflammation and promoting wound repair are achieved; accidental risks such as allergy are reduced as much as possible; meanwhile, the dosage form is a freeze-dried dosage form; active ingredients (such as quaternary ammonium salt and gelatin network) of the gel can be retained for a long time, and microorganism breeding or chemical degradation in a water-phase environment is avoided. And whether deionized water needs to be added for redissolution or not can be selected according to an actual use scene.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medical, dental or cosmetic formulations, in particular to a mild multi-progressive sustained-release antibacterial composition and its application. BACKGROUND

[0002] As an important product of modern health protection, the core performance of the bactericidal gel depends on the bactericidal ingredients contained. The common bactericidal ingredients on the market have their own advantages and disadvantages, and some ingredients also endow the gel with multiple functions besides bactericidal function.

[0003] Chlorhexidine is a commonly used bactericidal ingredient, which has broad-spectrum antibacterial properties and can effectively inhibit Gram-positive bacteria, Gram-negative bacteria and fungi. It has significant inhibitory effect on common pathogenic bacteria such as Staphylococcus aureus and Escherichia coli, and has long-lasting effect. It is commonly used for skin disinfection before surgery and oral care. However, its disadvantage is that it has weak effect on spores and mycobacteria, and long-term use may cause staining of oral mucosa and may cause skin allergic reactions. In addition, chlorhexidine, besides bactericidal function, can form a protective film in the oral care scene, reduce the adhesion of dental plaque, and play a certain role in caries prevention.

[0004] Benzalkonium chloride has good water solubility and surface activity, broad antibacterial spectrum, and can kill most Gram-positive bacteria, Gram-negative bacteria and some fungi. It is relatively low in price and is commonly used in skin and mucosa disinfection and sanitary wipes. However, it has a certain irritant effect on the skin, and long-term contact may destroy the normal flora of the skin, leading to flora imbalance, and is prone to drug resistance. In actual use, benzalkonium chloride also has a certain decontamination ability, which can assist in cleaning oil and dirt on the surface of objects.

[0005] Silver ions, with their unique antibacterial mechanism, can bind to key enzymes in bacteria, disrupting their physiological activity, and have strong inhibitory effect on a variety of drug-resistant bacteria, and are safe, commonly used in wound dressings and medical instrument disinfection. However, silver ions are high in cost, and large-scale use may cause accumulation of silver ions in the body, causing argyria, making the skin and mucosa turn gray or brown. At the same time, silver ions can promote angiogenesis and cell proliferation in wound care, accelerating wound healing.

[0006] Povidone-iodine is a commonly used broad-spectrum bactericide for surgical and wound disinfection, which can quickly release iodine to effectively kill bacteria, viruses, fungi and spores, with fast killing speed and good effect, and is not prone to drug resistance. However, it is contraindicated in iodine allergy, and may cause yellow staining of the skin after use, and excessive accumulation of iodine in the body may affect thyroid function. In addition to bactericidal function, povidone-iodine can keep the wound moist during wound treatment, which is beneficial to the growth of new tissues.

[0007] Triclosan has been widely used in bactericidal products, and has a broad antibacterial spectrum, which can inhibit Gram-positive bacteria, Gram-negative bacteria and fungi, and has good stability. However, its use is limited because it may interfere with human endocrine and has great toxicity to aquatic organisms. In the past application, triclosan can also play a role in deodorization, reducing the odor caused by bacterial growth.

[0008] In summary, different bactericidal components have their own advantages and disadvantages in bactericidal gel, and also bring multiple functions such as promoting wound healing, cleaning and decontamination, preventing caries and deodorization. In the future, with the deepening of research, through reasonable compounding of components and development of new components, it is expected to further optimize the performance of bactericidal gel and expand its application boundary. SUMMARY

[0009] The purpose of the present application is to provide a mild multiple progressive sustained-release antibacterial composition, which comprises the following components by mass fraction:

[0010]

[0011] The juniper berry essential oil and the amboyna essential oil are embedded by casein.

[0012] Preferably, the preparation method of the quaternary ammonium salt-chitosan antibacterial gel comprises the following steps:

[0013] S1: Dissolve the gelatin in the PBS buffer, add the propargyl succinimidyl ester, stir and dialyze, and freeze-dry; obtain the alkyne-modified gelatin;

[0014] S2: Dissolve the chitosan and 2,3-epoxypropyltrimethylammonium chloride in water, heat and stir to react, then dialyze and freeze-dry; obtain the quaternary ammonium chitosan;

[0015] S3: Catalyze the reaction of benzyldimethylstearyl ammonium chloride and 3-azido-1-propylamine in acetonitrile using K2CO3, then purify by ether precipitation to obtain the azidated quaternary ammonium salt;

[0016] S4: Dissolve the alkyne-modified gelatin in deoxygenated PBS buffer, and dissolve the quaternary ammonium chitosan in deoxygenated acetic acid buffer; ultrasonically uniformize respectively, then mix the two solutions, add the azidated quaternary ammonium salt, and stir uniformly under nitrogen protection to obtain a mixed solution;

[0017] S5: Add the DBCO-PEG4-DBCO solution dropwise in the mixed solution, and stir to react in the dark; then add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide for further reaction;

[0018] S6: The reaction solution is left to gel, then dialyzed in PBS and freeze-dried.

[0019] Preferably, in the step S1, the molar ratio of gelatin and propargyl succinimidyl ester is 1: at least 1.2.

[0020] Preferably, in the step S2, the molar ratio of chitosan and 2,3-epoxypropyltrimethylammonium chloride is 1: at least 5.

[0021] Preferably, in the step S3, the molar ratio of benzyldimethyl dioctadecyl ammonium chloride and 3-azido-1-propanamine is 1: at least 1.2.

[0022] Preferably, in the step S4, the molar ratio of alkynylated gelatin and quaternary ammonium chitosan is 1: at least 1.5; the addition amount of azidated quaternary ammonium salt is at least 5% of the total mass of alkynylated gelatin and quaternary ammonium chitosan.

[0023] Preferably, the humectant is at least one of 1,3-propanediol, glycerol, and 1,3-butanediol.

[0024] Preferably, the preparation method of the juniper berry essential oil and the amboyna oil embedded by casein is as follows:

[0025] The casein is put into water and heated and stirred until completely dissolved, and the pH is adjusted to 9.0-10.0; a casein solution is obtained;

[0026] The emulsifier is mixed with the juniper berry essential oil and the amboyna oil, and then the pH is adjusted to 7.0 or below; the mixture is stirred uniformly to obtain a pre-emulsion;

[0027] The pre-emulsion is added dropwise to the casein solution, heated and stirred to form a coarse emulsion;

[0028] The coarse emulsion is homogenized, and then glutamine transaminase is added for reaction; then the mixture is cooled and allowed to stand to obtain a preliminary gel;

[0029] The preliminary gel is freeze-dried, washed, and dried again to obtain the essential oil embedded by casein.

[0030] The application also provides a preparation method of the aforementioned mild multiple progressive sustained-release anti-bacterial composition, comprising the following steps:

[0031] S1: PEG-4000 and a humectant are added to a mixture of tert-butyl alcohol and water to form a mixed solution, which is stirred until completely dissolved;

[0032] S2: Casein-embedded essential oil is added to the mixed solution of step S1, and homogenized to obtain a homogenized solution;

[0033] S3: The homogenized solution is frozen to ≤-40℃ at a cooling rate ≥5℃ / min, and then vacuum sublimation dried; then the temperature is increased for desorption drying under a vacuum degree ≤10Pa, and then broken to obtain a moisturizing freeze-dried agent;

[0034] S4: The moisturizing lyophilized agent is mixed with the quaternary ammonium salt-chitosan antibacterial gel uniformly to obtain a mild multiple progressive slow-release antibacterial composition.

[0035] The application also provides use of the aforementioned mild multiple progressive slow-release antibacterial composition in preparation of medical supplies.

[0036] The application is particularly suitable for a wet healing environment in terms of promoting repair and antibacterial requirements for various wound injuries.

[0037] The application has multiple antibacterial mechanisms:

[0038] Quaternized chitosan: Chitosan is modified by epoxypropyl trimethyl ammonium chloride to introduce permanent positive charges (quaternary ammonium groups), thereby enhancing electrostatic adsorption to the negatively charged bacterial cell membrane and destroying the membrane integrity. Compared with native chitosan, the water solubility is improved and the antibacterial spectrum is wider (covering gram-positive / negative bacteria).

[0039] Quaternary ammonium azide: After modification by azidation, the long alkyl chain of benzyl dimethyl octadecyl ammonium chloride enhances the hydrophobic penetration, and the quaternary ammonium group inserts into the lipid bilayer, resulting in intracellular solute leakage. The azide group (-N3) provides an active site for subsequent click chemistry reactions.

[0040] Quaternized chitosan is connected to the gelatin matrix through click chemistry reactions, and a three-dimensional network is constructed to achieve slow-release antibacterial effect.

[0041] Juniper berry essential oil and amboyna essential oil contain different volatile substances such as 3-carene, α-pinene, limonene, valerenol, elemol, and various eucalyptol, which have the effects of destroying microbial biofilms, inhibiting inflammatory factor TNF-α, and accelerating wound repair. The application finds that the two essential oils have good antibacterial and wound repair effects when used synergistically while reducing irritation.

[0042] The essential oil component of the application uses casein to solve the problems of essential oil volatilization, oxidation, and blending.

[0043] The polyhydric alcohol moisturizing agent in the application plays a role in maintaining a moist wound environment and promoting cell migration.

[0044] The application has at least the following beneficial technical effects:

[0045] The application has a simple formula, and through the "triple antibacterial system" of organic quaternary ammonium salt, natural polysaccharide and essential oil slow release, the raw material selection is advanced; good antibacterial and anti-inflammatory effects and promotion of wound repair are achieved; and allergic reactions and other unexpected risks are reduced as much as possible; and the dosage form is a freeze-dried dosage form; the gel active ingredients (such as quaternary ammonium salt and gel network) can be retained for a long time, avoiding microbial growth or chemical degradation in the aqueous environment. Whether to add deionized water for reconstitution can be selected according to the actual use scene; if it is used for body surface wound repair, pressure sores and the like, water can be added for reconstitution; if it is used for gynecological postoperative care, it can be pre-filled in a drug delivery device, and when used, it can be injected into the corresponding area without additional water reconstitution, and the body fluid can be used for natural reconstitution. Alternatively, it can be implanted in the body, relying on tissue fluid to slowly dissolve and release quaternary ammonium salt, and it can also be used for postoperative anti-infection without reconstitution. DETAILED DESCRIPTION

[0046] In order to better understand the present application, the present application will be further described below in combination with specific serial numbers, wherein the terms used in the serial numbers are used to describe specific embodiments and do not constitute a limitation on the protection scope of the present application.

[0047] In the detailed description, the experimental methods used are conventional methods, and the materials, reagents and the like used are commercially available unless otherwise specified.

[0048] Unless otherwise specified, the percentages, % and the like in the detailed description are mass percentages.

[0049] Some raw materials used in the present application are as follows

[0050] Amyris balsamifera essential oil refers to volatile aromatic substances obtained by distillation of the wood part of Amyris balsamifera.

[0051] Juniperus communis L. essential oil refers to volatile aromatic substances obtained by distillation of the fruits of Juniperus communis L.

[0052] Tea tree essential oil refers to volatile aromatic substances obtained by distillation of the branches and leaves of Melaleuca alternifolia.

[0053] Origanum Vulgare essential oil refers to volatile aromatic substances obtained by distillation of the stems and leaves of Origanum Vulgare.

[0054] PEG-4000 refers to polyethylene glycol-4000, i.e., a polyethylene glycol molecule with an average molecular weight of about 4000.

[0055] Example 1

[0056] The preparation of casein-embedded essential oil includes the following steps:

[0057] S1: The casein powder was put into 50℃ deionized water, the concentration was controlled to be 5% (w / v), mechanically stirred until completely dissolved, NaOH was added dropwise, the pH was adjusted to 9.0, and stirred for 15 min; a casein solution was obtained.

[0058] S2: The required essential oils were prepared, mixed according to the proportions in Table 1, and 5% of the mass of the essential oils was added to glycerol monooleate; then 0.1 mol / L HCl was added dropwise to adjust the pH to 7; homogenized for 5 min to form a pre-emulsion.

[0059] S3: The pre-emulsion was slowly added dropwise into the casein solution, kept at a constant temperature of 50℃ and stirred (300 rpm), and after the addition was completed, the stirring was continued for 30 min to allow the casein to adsorb the essential oils by hydrophobic interaction; a coarse emulsion was obtained.

[0060] S4: The coarse emulsion was homogenized twice at 50 MPa, then glutamine transaminase was added (the amount was 0.5% of the weight of the casein), and the reaction was carried out at 40℃ for 1 hour to form a three-dimensional network gel structure; cooled to 4℃ and stood for 12 hours to form a preliminary gel.

[0061] S5: The preliminary gel was cut into slices, pre-frozen at -40℃, and freeze-dried for 24 hours (vacuum degree ≤ 30 Pa); the freeze-dried product was washed with anhydrous ethanol 3 times, and vacuum dried at 40℃ to a constant weight to obtain casein-embedded essential oils.

[0062] Table 1

[0063]

[0064]

[0065] Example Two

[0066] The preparation method of the quaternary ammonium salt-chitosan antibacterial gel comprises the following steps:

[0067] S1: The gelatin was dissolved in a PBS buffer, stirred at 50–60℃ until completely dissolved; propargyl succinimidyl ester was added, and the reaction was carried out at room temperature under dark conditions for 12 hours, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 8–14 kDa, dialyzed with deionized water for 3 days (water was changed 3 times a day), and freeze-dried to obtain white sponge-like alkyne-modified gelatin.

[0068] The molar ratio of gelatin to propargyl succinimidyl ester was 1:1.2.

[0069] S2: Chitosan and 2,3-epoxypropyl trimethyl ammonium chloride were dissolved in 2% acetic acid aqueous solution, and ultrasonic dispersion was performed for 10 minutes; magnetic stirring was performed in a 70-80 °C water bath for 24 hours, and the pH was maintained at 8.5-9.0 (0.5 mol / L NaOH was added dropwise for adjustment); the reaction solution was dialyzed (molecular weight cut-off 3.5 kDa) for 4 days, and freeze-drying was performed to obtain quaternary ammonium chitosan;

[0070] The molar ratio of chitosan to 2,3-epoxypropyl trimethyl ammonium chloride was 1:5.

[0071] S3: Benzyl dimethyl octadecyl ammonium chloride and 3-azido-1-propanamine were dissolved in acetonitrile, and K2CO3 (catalytic amount, 0.1 eq) was added; under nitrogen protection, reflux reaction was performed at 60 °C for 6 hours;

[0072] After the reaction solution was cooled, it was precipitated with ethyl ether for 3 times, centrifuged (8000 rpm, 10 minutes), and vacuum dried to obtain white azidated quaternary ammonium salt;

[0073] The molar ratio of benzyl dimethyl octadecyl ammonium chloride to 3-azido-1-propanamine was 1:1.2.

[0074] S4: Alkynylated gelatin was dissolved in deoxygenated PBS buffer, and the concentration was 5 w / v%; ultrasonic dispersion was performed at 50 °C for 15 min;

[0075] Quaternary ammonium chitosan was dissolved in deoxygenated acetic acid buffer; the concentration was 4 w / v%, and ultrasonic dispersion was performed at room temperature for 15 min;

[0076] Then, the two solutions were mixed, and azidated quaternary ammonium salt was added; catalyst copper sulfate pentahydrate (0.4 eq) and sodium ascorbate (1 eq) were added, and stirring reaction was performed at 40 °C for 12 hours to obtain a mixed solution;

[0077] The molar ratio of alkynylated gelatin to quaternary ammonium chitosan was 1:1.5; the addition amount of azidated quaternary ammonium salt was 5% of the total mass of alkynylated gelatin and quaternary ammonium chitosan.

[0078] S5: A DMSO solution of 10 mM DBCO-PEG4-DBCO (molecular weight 2-5 kDa) was prepared, and the addition amount was 1.2 times the molar amount of alkynyl groups in alkynylated gelatin;

[0079] Stirring reaction was performed at 25 °C under light protection and nitrogen protection for 4 hours, and the pH was maintained at 7.0-7.5. Copper-free click reaction occurred between DBCO and azidated quaternary ammonium salt to form a triazole bond crosslinking network;

[0080] Then 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC-HCl) and N-hydroxysuccinimide (NHS) were added with a molar ratio of EDC:NHS:total carboxyl groups = 5:2:1 (carboxyl groups from gelatin and quaternary ammonium salt); avoid light, adjust pH to 5.0, stir the reaction at 25℃ for 2-4 hours;

[0081] S6: Transfer the reaction solution to a mold, place it at 37℃ in the dark for 24 hours to form a three-dimensional network gel; place the gel in a dialysis bag with a molecular weight cut-off of 8-14 kDa, dialyze it against 4℃ pre-cooled PBS buffer (pH 7.4) for 3 days, change the buffer 3 times a day to remove unreacted monomers and catalysts;

[0082] After dialysis, the gel was quickly frozen with liquid nitrogen, vacuum freeze-dried at -50℃ and 0.05mBar for 48 hours to obtain a porous sponge-like solid quaternary ammonium salt-chitosan antibacterial gel, which was stored at -20℃ in the dark.

[0083] Example Three

[0084] The preparation method of the gel composition is as follows:

[0085] S1: Add PEG-4000 and humectant to a mixture of tert-butyl alcohol and water to form a mixed solution, stir until completely dissolved;

[0086] S2: Add the embedded essential oil from Example One to the mixed solution of Step S1, homogenize to obtain a homogeneous solution;

[0087] S3: Freeze the homogeneous solution to ≤-40℃ at a cooling rate ≥5℃ / min, then vacuum sublimation dry; then warm up and desorb dry at a vacuum degree ≤10Pa, then crush to obtain a humectant lyophilizate;

[0088] S4: Mix the humectant lyophilizate with the quaternary ammonium salt-chitosan antibacterial gel uniformly to obtain a gel composition.

[0089] The addition amount of the embedded essential oil and other ingredients of each serial number in Example Three is shown in Table 2 below.

[0090] Table 2

[0091]

[0092]

[0093] The "embedded essential oil serial number" in Table 2 is the serial number corresponding to Table 1 in Example 1.

[0094] The gel composition prepared in Example Three was tested for performance, as follows:

[0095] Antibacterial performance test:

[0096] The gel composition is reconstituted by adding 9 times the mass of water before testing to form a hydrogel.

[0097] The antibacterial performance is tested according to the Disinfection Technical Specification (2002 edition) item 2.2.3.2.1, and the results are shown in Table 3 below.

[0098] Table 3

[0099] No. / Bacteriostatic rate (%) Escherichia coli Staphylococcus aureus Candida albicans 1 ＞99 ＞99 ＞99 2 ＞99 ＞99 ＞99 3 ＞99 ＞99 ＞99 4 94 ＞99 96 5 98 ＞99 97 6 96 ＞99 ＞99 7 ＞99 ＞99 ＞99 8 94 98 98 9 ＞99 ＞99 ＞99 10 ＞99 ＞99 ＞99 11 ＞99 ＞99 ＞99 12 80 76 84 13 90 96 94

[0100] As can be seen from the results in Table 3, the quaternary ammonium salt-chitosan antibacterial gel of the present application has very strong bactericidal effect, but it may not be able to achieve an effect of > 99% due to concentration problems, and after adding the combination of juniper berry essential oil and amyris essential oil, it has excellent bacteriostatic effect. The results of serial numbers 4, 5, 10, and 11 show that the bacteriostatic effect of tea tree essential oil and oregano essential oil is better than that of juniper berry essential oil and amyris essential oil; however, the present application does not use tea tree essential oil and oregano essential oil due to other considerations, and the bactericidal performance of the limited essential oil combination in the present application is sufficient.

[0101] Skin water content test:

[0102] The gel composition is reconstituted by adding 9 times the mass of water before testing to form a hydrogel.

[0103] Fifteen volunteers are selected and divided into three groups; the skin surface water content is detected and averaged for each group, the gel composition is applied to the skin for 30 min, then removed, and the skin water content is detected again after 30 min of standing and averaged, and the skin water content before and after using the gel composition is recorded, and the results are shown in Table 4.

[0104] Table 4

[0105]

[0106] Wound healing animal experiment:

[0107] In order to verify whether the gel composition prepared by the present application has the function of promoting wound healing, an animal modeling test is performed.

[0108] (1) A total of 70 SD rats with similar growth states are selected, with a male to female ratio of 1:1, and randomly divided into 14 groups, one group as a control group and the other 13 groups as experimental groups; the back is depilated with a depilatory agent, and the depilation area is about 16 cm 2 After anesthesia, paraffin liquid is dropped onto the depilation site to form a burn and scald wound;

[0109] (2) In each experimental group, the wound site was uniformly coated with the gel composition of each serial number of Example 3 (the serial number is carried forward from Example 3), with a coating thickness of 1 mm, and then sterile gauze was used for bandaging, and the sterile gauze was replaced every 2 days for re-bandaging; for 14 consecutive days, the wound was cleaned once on the 7th day, and each serial number of gel composition was re-coated, with a coating thickness of 1 mm; the scabbed area of the wound on the 7th day and the 14th day was counted, and the results are shown in Table 5.

[0110] (3) The control group was bandaged using sterile gauze, which was replaced every 2 days for re-bandaging; for 14 consecutive days; the scabbed area of the wound on the 7th day and the 14th day was counted, and the results are shown in Table 5.

[0111] Table 5

[0112]

[0113] According to the experimental results in Table 5, serial numbers 4-11 are different non-inventive essential oil combinations, and their effects on promoting wound healing are relatively poor compared to the combination of juniper berry essential oil and amica essential oil selected by the present application; and serial number 13 does not add essential oil, but the presence of the gel composition provides a sterile low-oxygen environment for moist healing, which also has a certain auxiliary effect on wound healing.

[0114] Serial number 12 uses gelatin instead of quaternary ammonium salt-chitosan antibacterial gel to make a gel composition, which relies only on the essential oil composition for corresponding bacteriostasis and wound promotion, but the essential oil composition has a low concentration and weak bacteriostatic ability, which may cause partial infection of the wound; its auxiliary wound healing effect is even lower than that of serial number 13.

[0115] The above detailed description is a specific description of one of the feasible embodiments of the present application, and this embodiment is not used to limit the patent scope of the present application. Any equivalent implementation or change that does not deviate from the present application should be included in the scope of the technical solutions of the present application.

Claims

1. A mild, multiple progressive, sustained-release, anti-bacterial composition, characterized in that, The following ingredients are included by mass: The juniper berry essential oil and the amboyna essential oil are encapsulated by casein.

2. The mild, multiple progressive, slow release, anti-bacterial composition according to claim 1, wherein, The preparation method of the quaternary ammonium salt-chitosan antibacterial gel comprises the following steps: S1: dissolving gelatin in PBS buffer, adding propargyl succinimidyl ester, stirring and dialyzing, and freeze-drying; obtaining alkyne-modified gelatin; S2: dissolving chitosan and 2,3-epoxypropyltrimethylammonium chloride in water, heating and stirring to react, dialyzing, and then freeze-drying; obtaining quaternary ammonium chitosan; S3: benzyl dimethyl octadecyl ammonium chloride and 3-azido-1-propanamine are catalyzed by K2CO3 in acetonitrile, then purified by ether precipitation, and then freeze-drying; obtaining azido quaternary ammonium salt; S4: dissolving alkyne-modified gelatin in deoxygenated PBS buffer and dissolving quaternary ammonium chitosan in deoxygenated acetic acid buffer; ultrasonic treatment respectively until uniform, then mixing the two solutions, adding azido quaternary ammonium salt, stirring uniformly under nitrogen protection, and obtaining a mixed solution; S5: adding DBCO-PEG4-DBCO solution dropwise in the mixed solution, stirring in the dark to react; then adding 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide for further reaction; S6: the reaction solution is left to gel, then dialyzed in PBS and freeze-dried.

3. The mild multiple progressive delayed release anti-bacterial composition of claim 2, wherein, In the step S1, the molar ratio of gelatin and propargyl succinimidyl ester is 1: at least 1.

2.

4. The mild multiple progressive delayed release anti-bacterial composition of claim 2, wherein, In the step S2, the molar ratio of chitosan and 2,3-epoxypropyltrimethylammonium chloride is 1: at least 5.

5. The mild multiple progressive delayed release anti-bacterial composition of claim 2, wherein, In the step S3, the molar ratio of benzyl dimethyl octadecyl ammonium chloride and 3-azido-1-propanamine is 1: at least 1.

2.

6. The mild multiple progressive delayed release anti-bacterial composition of claim 2, wherein, In the step S4, the molar ratio of alkyne-modified gelatin and quaternary ammonium chitosan is 1: at least 1.5; and the addition amount of azido quaternary ammonium salt is at least 5% of the total mass of alkyne-modified gelatin and quaternary ammonium chitosan.

7. The mild multiple progressive release antibacterial composition according to claim 1, wherein The humectant is at least one of 1,3-propanediol, glycerol, and 1,3-butanediol.

8. The mild multiple progressive release antibacterial composition according to claim 1, wherein The preparation method of the juniper berry essential oil and the amboyna essential oil encapsulated by casein is as follows: The casein is put into water, heated and stirred until completely dissolved, and the pH is adjusted to 9.0-10.0; obtaining a casein solution; The emulsifier is mixed with the juniper berry essential oil and the amboyna essential oil, and then the pH is adjusted to 7.0 or below; stirring uniformly to obtain a pre-emulsion; The pre-emulsion is added dropwise into the casein solution, heated and stirred to form a coarse emulsion; The coarse emulsion is homogenized, then reacted with glutamine transaminase; then cooled and left to stand, obtaining a primary gel; The primary gel is freeze-dried, washed, and dried again, obtaining the essential oil encapsulated by casein.

9. A process for the preparation of a mild multiple progressive release antibacterial composition as claimed in any one of claims 1 to 8, characterized in that, The following steps are included: S1: adding PEG-4000 and a humectant to a mixture of tert-butyl alcohol and water to form a mixed solution, and stirring until completely dissolved; S2: adding the essential oil encapsulated by casein to the mixed solution of step S1, and homogenizing to obtain a homogenized solution; S3: freezing the homogenized solution to ≤-40℃ under the condition of a cooling rate ≥5℃ / min, and then vacuum sublimation drying; Then warming and desorption drying under the condition of a vacuum degree ≤10Pa, and then crushing to obtain a moisturizing freeze-dried agent; S4: mixing the moisturizing freeze-dried agent with the quaternary ammonium salt-chitosan antibacterial gel uniformly, and obtaining a mild multiple progressive slow-release antibacterial composition.

10. Use of a mild multiple progressive release antibacterial composition according to any one of claims 1 to 8 for the manufacture of a medical article.

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