CN115607656B

CN115607656B - Application of chitosan enzyme and composite gel thereof in preparation of products for preventing or treating skin diseases caused by malassezia

Info

Publication number: CN115607656B
Application number: CN202211634508.8A
Authority: CN
Inventors: 张晓云; 张晓立; 王楠; 张慧泽
Original assignee: Baikuiri Tianjin Biotechnology Co ltd
Current assignee: Baikuiri Tianjin Biotechnology Co ltd
Priority date: 2022-12-19
Filing date: 2022-12-19
Publication date: 2023-04-21
Anticipated expiration: 2042-12-19
Also published as: CN115607656A

Abstract

The invention discloses an inhibition effect of chitosanase on malassezia, a preparation method and application of chitosan enzyme composite gel. The composite gel comprises chitosanase, hyaluronic acid and deionized water. The invention discovers that the chitosanase has good killing effect on malassezia, can be used for treating diseases caused by malassezia, and can form composite gel by combining with hyaluronic acid without influencing the sterilizing effect, thereby having great application value.

Description

Application of chitosan enzyme and composite gel thereof in preparation of products for preventing or treating skin diseases caused by malassezia

Technical Field

The invention belongs to the field of applied biological materials, and relates to a series of composite gel containing chitosanase, and application and a preparation method thereof in the aspect of killing malassezia.

Background

Malasezia (Malasezia) is a lipophilic yeast-like fungus, which is one of the resident bacteria on the surface of human skin. Because of their physiological properties (except for Piemala thick) of lipid, they are mainly distributed in the sebum-rich parts of the human body, such as scalp, face and chest back. There are data statistics that malassezia accounts for about 50-80% of the skin colonization fungi of the keykang population. Malachite bacteria are conditional pathogenic bacteria, are related to various skin diseases of human and animals, can directly infect skin and appendages thereof to cause pityriasis versicolor and Malachite folliculitis, and are involved in the occurrence and development of certain diseases such as seborrheic dermatitis, atopic dermatitis, onychomycosis, external auditory meatus inflammation, glandular prepuce balanitis and the like under certain conditions, wherein, spherical, furfur-pityrosis, syncytial and blunt malassezia bacteria are main pathogenic bacteria causing the diseases. The malassezia causes multiple skin infection diseases, has high incidence rate, is easy to repeatedly attack, and brings great influence to the health of patients. At present, systemic treatment or local treatment is adopted when malassezia-related skin diseases are clinically treated. Systemic treatment is with oral antifungal agents such as ketoconazole, itraconazole, fluconazole, and the like; topical treatment is applied by antifungal topical ointment, such as Caile, kangwang. Glucocorticoids and immunosuppressants can also be used, but are easy to generate drug resistance and have large side effects, and itch and edema symptoms frequently appear when the medicine is used limit the application of the medicines. In addition, polypeptides can be used for sterilization, but they have limitations in application due to the high cost of polypeptide synthesis and the problem of skin penetration and absorption.

The chitosan enzyme is a specific enzyme with a decomposition effect on linear chitosan, and can selectively and specifically cut off beta- (1-4) -glycosidic bonds of the chitosan to obtain chitosan oligosaccharide with a specific molecular weight range. The chitosan enzyme is mainly used for preparing various chitosan oligosaccharides with different polymerization degrees in industry, and researches show that the chitosan oligosaccharides with specific polymerization degrees have an inhibition effect on bacteria or fungi. And since the main component of the cell wall of most fungi is chitosan, there have been researchers to prepare protoplasts using chitosan enzymes or for the prevention of plant pathogens such as the fungi f. However, no inhibiting effect of chitosanase on malassezia has been reported at present.

Disclosure of Invention

The invention discloses an effect of chitosanase in killing malassezia, and develops and provides a chitosan enzyme composite gel capable of effectively killing malassezia as a substitute for the existing malassezia diseases.

The present invention first provides the use of a chitosan enzyme in the preparation of a product for the prevention or treatment of skin disorders caused by malassezia, such as excessive dandruff, itching, rash, folliculitis, etc.

Specifically, the chitosan enzyme is derived from bacillus, and more specifically adopts the specification: 10 ten thousand U/g.

The invention provides a chitosan enzyme composite gel for killing malassezia, which is formed by chitosan enzyme and hyaluronic acid in a buffer solution.

Preferably, the content of the chitosan enzyme is 2-4mg/ml, the content of the hyaluronic acid is 0.1-0.3%, and the buffer solution is acetic acid-sodium acetate buffer solution; more preferably, the chitosan enzyme content is 3.1mg/ml, the hyaluronic acid content is 0.2%, and the rest is 20mM acetic acid-sodium acetate buffer solution with pH of 5.8; further preferably, the molecular weight of hyaluronic acid is from 1000 to 1800kDa, preferably the molecular weight of hyaluronic acid is from 1200 to 1600kDa, more preferably the molecular weight of hyaluronic acid is 1400 kDa.

The invention also provides a preparation method of the composite gel, which comprises the following steps: 1) Dissolving chitosan enzyme in acetic acid-sodium acetate buffer solution; 2) Dissolving hyaluronic acid in acetic acid-sodium acetate buffer solution; 3) And slowly adding the chitosan enzyme solution into the hyaluronic acid solution under the condition of stirring, and uniformly mixing to obtain a mixed solution, namely the chitosan enzyme/hyaluronic acid composite gel.

The invention further provides the chitosan enzyme composite gel obtained by the preparation method and used for killing malassezia.

Further provides the application of the chitosan enzyme composite gel for killing malassezia in preparing products for preventing or treating skin diseases caused by malassezia, such as excessive dandruff, pruritus, rash, folliculitis and the like. The product is a cosmetic, health product or pharmaceutical product, in particular a gel is coated on the surface of skin.

Compared with the prior art, the invention has the following beneficial effects: the invention discloses a new application of chitosanase, which can inhibit malassezia, and compared with other fungicidal drugs, the chitosanase has the advantages of thorough sterilization, difficult generation of drug resistance and the like; furthermore, the invention also provides a chitosan enzyme composite gel, the formula comprises chitosan enzyme, hyaluronic acid, acetic acid and water, the stability is good, the cytotoxicity is low, the bactericidal activity of the chitosan enzyme is maintained, and the chitosan enzyme composite gel also plays a role in skin moisture preservation, and can be used for treating diseases caused by malassezia, such as seborrheic dermatitis, tinea versicolor, malassezia folliculitis and the like.

The invention discovers that the chitosanase has good killing effect on malassezia, can be used for treating diseases caused by malassezia, and can form composite gel by combining with hyaluronic acid without influencing the sterilizing effect, thereby having great application value.

Drawings

Fig. 1: antibacterial effect of chitosanase of 100mg/ml on malassezia.

Fig. 2: the chitosan enzyme and the hyaluronic acid with different concentrations are compounded to realize the antibacterial effect on malassezia.

Fig. 3: hyaluronic acid-chitosan enzyme composite gel with different molecular weights has antibacterial effect.

Detailed Description

The following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.

Example 1: preliminary antibacterial experiment of chitosan enzyme

And preliminarily verifying the inhibition effect of chitosanase on malassezia according to a W_ST650 standard method.

Experimental strains: limiting malassezia (M. Furdur, CICC 31847);

preparation of malassezia culture medium: 20g/L Malt Extract Broth (MEB) medium, 15g/L agar powder, purified water, and sterilizing at 121deg.C for 20min;

preparation of malassezia suspension: culturing the strain to be tested on MEB fixed culture plate at 30deg.C for 48 hr, inoculating single colony into 5ml liquid MEB test tube culture mediumCulturing for 48h at 30 ℃ and 220rmp. Centrifuging 1ml of bacterial liquid, and carrying out gradient dilution with acetic acid-sodium acetate buffer solution with pH of 5.8-20 mM to obtain colony number of 1×10 ⁶ cfu/ml of bacterial suspension, which is used on the same day.

The test method comprises the following steps: 100mg/ml chitosanase was formulated using 20mM acetic acid-sodium acetate pH5.8 and sterilized by passing through the membrane. A reaction system was prepared from 450ul of chitosanase and 50ul of malassezia suspension, placed in a 30℃incubator for 1h of incubation, and 100ul of plates (MEB plates) were then applied. The control group is incubated by replacing the chitosan enzyme solution with acetic acid-sodium acetate buffer solution, plating culture is carried out, and the growth condition of malassezia is observed and compared.

Results: the malassezia after the treatment of the chitosan enzyme is basically sterile in colony growth, which shows that the chitosan enzyme has strong malassezia inhibition effect, the colony numbers of the control group and the experimental group are shown in table 1, and the corresponding plate inhibition effect is shown in fig. 1.

Table 1: antibacterial rate of 100mg/ml chitosan enzyme

Group of	Colony count	Antibacterial rate
			Control group	>10 ⁴	0%
100mg/ml chitosan enzyme	32	>99%

Example 2: minimum inhibitory concentration test of chitosan enzyme

The experimental method comprises the following steps: the minimal inhibitory concentration (minimum inhibitory concentration, MIC) of chitosanase was determined using a microdilution method (Wiegand Nature Protocol 2008) in 96-well plates. The chitosanase was diluted with acetic acid-sodium acetate buffer and mixed with the malassezia suspension of example 1 according to 9:1 to prepare a 400ul system, wherein the final concentration of the protein is 100mg/ml, 50mg/ml, 25mg/ml, 12.5mg/ml, 6.2mg/ml, 3.1mg/ml, 1.5mg/ml and 0.7mg/ml respectively. The optical density was measured at 600nm wavelength after incubation of the well plate at 30℃for 24 hours, and the MIC value of the protein was recorded as the lowest protein concentration at which no bacterial growth occurred. The positive control replaced the chitosan enzyme with acetic acid-sodium acetate buffer, the negative control replaced the bacterial solution with acetic acid-sodium acetate buffer, and the experimental results are shown in table 2.

Results: table 2 shows that the minimum inhibitory concentration of chitosanase was 3.1mg/ml using the method of measuring absorbance values in 96-well plates.

Table 2: measurement of minimum inhibitory concentration of chitosan enzyme

Example 3: preparation of chitosanase-hyaluronic acid composite gel and bacteriostasis experiment

Preparation of the chitosanase gel: 0.62g of chitosan enzyme was dissolved in 100ml of 20mM acetic acid-sodium acetate buffer solution of pH5.8, and 2%, 1%, 0.4% and 0.2% of a hyaluronic acid solution having a molecular weight of 1400kDa was prepared in the same manner, and the above solutions were mixed in a volume ratio of 1:1, and obtaining the chitosanase/hyaluronic acid gel. The prepared chitosanase/hyaluronic acid gel was subjected to membrane sterilization, and the antibacterial rate was determined as in example 1. Blank control group 1:20mM acetic acid-sodium acetate buffer pH 5.8; blank control group 2: acetic acid-sodium acetate solution containing 0.2% hyaluronic acid; control experimental group: acetic acid-sodium acetate solution containing 3.1mg/ml chitosanase; experimental groups 1-4: hyaluronic acid was added at final concentrations of 1%, 0.5%, 0.2% and 0.1% based on the control experimental group, respectively. The specific colony numbers are shown in the following table 3, and the corresponding plate bacteriostasis effects are shown in fig. 2.

Results: under the minimum inhibitory concentration of the chitosan enzyme (3.1 mg/ml), the chitosan enzyme and the hyaluronic acid are compounded into a series of hyaluronic acid gel with the concentration, and the result shows that the hyaluronic acid and the chitosan enzyme have the best compound antibacterial effect when the final concentration is 0.2 percent.

Table 3: antibacterial rate of chitosanase/hyaluronic acid gel on malassezia

Example 4: compounding hyaluronic acid and chitosan enzyme with different molecular weights and antibacterial verification

Gel dressing containing 0.2% hyaluronic acid and 3.1mg/ml chitosanase was prepared in the manner of experimental group 3 in example 3, wherein the hyaluronic acid was selected from the group consisting of different molecular weights of 50kDa, 200kDa and 1400kDa, respectively, and the control group was 20mM acetic acid-sodium acetate buffer pH 5.8; it was verified whether hyaluronic acid of different molecular weights affected the bacteriostatic activity of the gel dressing.

The results are shown in Table 4 and FIG. 3.

Table 4: antibacterial rate of malassezia by compounding hyaluronic acid with different components and chitosan enzyme

Hyaluronic acid molecular weight	Antibacterial rate
		Control group	0
50kDa	57%
		200kDa	80%
1400kDa	>99%

From the results, the antibacterial effect of the hyaluronic acid compounded chitosan enzyme gel with the molecular weight of 1400kDa is optimal, the antibacterial rate is more than 99%, and the antibacterial effect of the hyaluronic acid gel with the molecular weight of 200kDa is worst, which indicates that the molecular weight of the hyaluronic acid gel is positively correlated with the antibacterial rate within a certain range.

Claims

1. The application of the chitosan enzyme composite gel in preparing a malassezia-killing product is characterized in that the chitosan enzyme and hyaluronic acid form the composite gel in a buffer solution, the minimum inhibition concentration of the chitosan enzyme is 3.1mg/ml, the content of the hyaluronic acid is 0.1-0.2%, the molecular weight of the hyaluronic acid is 1000-1800 kDa, and the buffer solution is acetic acid-sodium acetate buffer solution.

2. The use according to claim 1, wherein the chitosan enzyme content is 3.1-4 mg/ml, the hyaluronic acid content is 0.2%, the remainder is 20mM acetic acid-sodium acetate buffer at pH 5.8.

3. The use according to claim 1 or 2, wherein the chitosan enzyme is derived from bacillus.

4. A preparation method for preparing a chitosan enzyme composite gel, which comprises the following steps: 1) Dissolving chitosan enzyme in acetic acid-sodium acetate buffer solution; 2) Dissolving hyaluronic acid in acetic acid-sodium acetate buffer solution; 3) Slowly adding the chitosan enzyme solution into the hyaluronic acid solution under the condition of stirring, and uniformly mixing to obtain a mixed solution, namely the composite gel; wherein the concentration of chitosanase is at least 3.1mg/ml, the content of hyaluronic acid is 0.1-0.2%, and the molecular weight of hyaluronic acid is 1000-1800 kDa.

5. The method according to claim 4, wherein the chitosan enzyme content is 3.1-4 mg/ml, the hyaluronic acid content is 0.2%, and the balance is 20mM acetic acid-sodium acetate buffer solution with pH 5.8.

6. Use of a chitosan enzyme complex gel prepared by a method comprising the steps of: 1) Dissolving chitosan enzyme in acetic acid-sodium acetate buffer solution; 2) Dissolving hyaluronic acid in acetic acid-sodium acetate buffer solution; 3) Slowly adding the chitosan enzyme solution into the hyaluronic acid solution under the condition of stirring, and uniformly mixing to obtain a mixed solution, namely the composite gel; wherein the concentration of chitosanase is at least 3.1mg/ml, the content of hyaluronic acid is 0.2%, and the molecular weight of hyaluronic acid is 1000-1800 kDa.

7. The use according to claim 6, wherein the condition is excessive dandruff, itching, rash or folliculitis.

8. The use according to claim 7, wherein the product is a cosmetic or pharmaceutical product.