阅读说明：本技术 一种寡聚透明质酸钠的制备方法 (Preparation method of oligomeric sodium hyaluronate ) 是由 张建勇 俞超 王朋田 朱伟伟 刘守垒 田洪果 相茂功 王春朋 殷小崴 王海泉 秦 于 2021-01-13 设计创作，主要内容包括：本发明提供了一种寡聚透明质酸钠的制备方法：可发酵产生透明质酸的菌种接入发酵培养基中,适宜条件下搅拌发酵,发酵至动力粘度140000-250000 mPa·s时,添加透明质酸酶,至发酵终点获得发酵液；调节发酵液的pH,在适温下加入透明质酸酶,反应获得酶解液；酶解液中加入抗氧化剂,然后调节pH,升温灭活酶,获得寡聚透明质酸钠溶液；将寡聚透明质酸钠溶液纯化、干燥获得寡聚透明质酸钠。本发明提供的寡聚透明质酸钠的制备方法,在发酵过程中加入透明质酸水解酶,边发酵边酶解,提高了产量；高温灭活过程中加入抗氧化剂,提高了纯度。该方法获得的寡居透明质酸钠的收率高、工艺连续,直接获得目标分子量产物,所得产品活性高。(The invention provides a preparation method of oligomeric sodium hyaluronate, which comprises the following steps: inoculating a strain capable of fermenting to produce hyaluronic acid into a fermentation culture medium, stirring and fermenting under a proper condition, and adding hyaluronidase when the dynamic viscosity is 140000-250000 mPa & s until the fermentation end point to obtain fermentation liquor; adjusting the pH value of the fermentation liquor, adding hyaluronidase at a proper temperature, and reacting to obtain enzymatic hydrolysate; adding an antioxidant into the enzymolysis liquid, then adjusting the pH, heating and inactivating the enzyme to obtain an oligomeric sodium hyaluronate solution; and purifying and drying the oligomeric sodium hyaluronate solution to obtain the oligomeric sodium hyaluronate. According to the preparation method of oligomeric sodium hyaluronate, provided by the invention, hyaluronic acid hydrolase is added in the fermentation process, and enzymolysis is carried out while fermentation is carried out, so that the yield is improved; and an antioxidant is added in the high-temperature inactivation process, so that the purity is improved. The oligomeric sodium hyaluronate obtained by the method has high yield and continuous process, and can directly obtain a target molecular weight product with high activity.)

1. The preparation method of oligomeric sodium hyaluronate is characterized by comprising the following steps:

(1) inoculating a strain capable of fermenting to produce hyaluronic acid into a fermentation culture medium, stirring and fermenting under a proper condition, and adding hyaluronidase when the dynamic viscosity is 140000-250000 mPa & s until the fermentation end point to obtain fermentation liquor;

(2) adjusting the pH value of the fermentation liquor, adding hyaluronidase at a proper temperature, and reacting to obtain enzymatic hydrolysate;

(3) adding an antioxidant into the enzymolysis liquid, then adjusting the pH, heating and inactivating the enzyme to obtain an oligomeric sodium hyaluronate solution;

(4) and purifying and drying the oligomeric sodium hyaluronate solution to obtain the oligomeric sodium hyaluronate.

2. The method according to claim 1, wherein the hyaluronidase is added in an amount of 30-90AU/L fermentation broth in step (1); the fermentation end point is that the pH change is less than 0.05/min;

in the step (2), the addition amount of the hyaluronidase is as follows: the molecular weight section of the product is 5KDa-10KDa, 60000-120000 AU/kg; the molecular weight section of the product is 3KDa-5KDa, 120000-150000 AU/kg; the molecular weight section of the product is 800Da-3KDa, 150000-300000 AU/kg; the enzymolysis time of the hyaluronidase is 6-9 h.

3. The method of claim 1, wherein the hyaluronic acid producing bacteria is selected from Streptococcus zooepidemicus (Streptococcus zooepidemicus)Streptococcus zooepidemicus) Bacillus subtilis preparation (B)Bacillus subtilis)。

4. The method according to claim 1, wherein in the step (2), the pH is 6.0 to 7.5, and the suitable temperature is 30 to 45 ℃;

in the step (3), the pH is 4.5-5.6;

in the step (3), the temperature is increased to 80-120 ℃, and the inactivation time is 15-60 min.

5. The method according to claim 1, wherein in the step (3), the concentration of the antioxidant is 0.1% to 0.2% w/v; the antioxidant is selected from one or more of sodium bisulfite, potassium bisulfite, ascorbic acid and derivatives thereof.

6. The method according to claim 1, wherein in the step (4), the purification comprises the steps of:

(i) after filtering the oligomeric sodium hyaluronate solution, sequentially adding a bleaching agent and a metal ion chelating agent to adjust the pH value for reaction, then adjusting the pH value, and then filtering and concentrating to obtain a concentrated solution;

(ii) adding sodium chloride into the concentrated solution, adjusting pH, precipitating with ethanol solution, washing the precipitate, and vacuum filtering to obtain pure product.

7. The method according to claim 6, wherein in the step (i), the pore size of the filtration is 0.22 to 0.45 μm.

8. The method of claim 6, wherein in step (i), said bleaching agent is added in an amount of 0.1% to 0.2% w/v; the bleaching agent is one of sodium hypochlorite, hydrogen peroxide or activated carbon;

the addition amount of the metal ion chelating agent is 0.1-0.2% w/v; the metal ion chelating agent is EDTA, EDTA disodium or EDTA dipotassium;

in step (ii), the sodium chloride is added in an amount of 4-6% w/v.

9. The method according to claim 6, wherein in steps (i) and (ii), the pH is 6.0 to 7.5;

in step (i), the concentration is ultrafiltration concentration;

in step (ii), the concentration of the ethanol solution is 90-100% v/v; the solvent for washing is 90-100% v/v ethanol solution.

10. Oligomeric sodium hyaluronate obtainable by the process according to any of claims 1 to 9.

Technical Field

The invention belongs to the technical field of biomacromolecule fermentation production, relates to a preparation method of oligomeric sodium hyaluronate, and particularly relates to a method for preparing oligomeric sodium hyaluronate through degradation in a fermentation process.

Background

Compared with sodium hyaluronate with conventional molecular weight, the oligomeric sodium hyaluronate has good transdermal absorption property and bioactivity which macromolecular hyaluronic acid does not have on the basis of keeping good moisturizing property, and is widely used in daily necessities. Firstly, in the traditional sodium hyaluronate fermentation process, fermentation can only be stopped at the later stage of fermentation (10-12 g/L) due to the reasons of difficult mass and heat transfer caused by high viscosity and difficult stirring, so that the yield is low, and the cost for enzymolysis of hyaluronic acid is increased. Secondly, the conventional hyaluronic acid with the molecular weight is fed again for enzymolysis, so that the solution has large volume, small concentration and high production cost. Third, two common methods have difficulty obtaining a solid finished product: the precipitation method has large volume, wastes a large amount of raw material alcohol and has low yield; although the spray drying method has high yield, the finished sodium hyaluronate has poor color and lower content than the precipitation method.

Disclosure of Invention

In order to solve the problems, the invention provides a fermentation preparation method of high-yield high-activity oriented molecular weight oligomeric sodium hyaluronate. Adding hyaluronidase in the fermentation process of sodium hyaluronate, fermenting while performing enzymolysis, increasing the yield to about 14g/L, and performing secondary enzymolysis to obtain oligomeric sodium hyaluronate with fixed molecular weight. The method improves fermentation yield, has continuous process, high hyaluronic acid yield after enzymolysis, and high product activity.

In order to achieve the purpose, the invention adopts the following technical scheme.

The preparation method of oligomeric sodium hyaluronate comprises the following steps:

(1) inoculating a strain capable of fermenting to produce hyaluronic acid into a fermentation culture medium, stirring and fermenting under a proper condition, and adding hyaluronidase when the dynamic viscosity is 140000-250000 mPa & s until the fermentation end point to obtain fermentation liquor;

(2) adjusting the pH value of the fermentation liquor, adding hyaluronidase at a proper temperature, and reacting to obtain enzymatic hydrolysate;

(3) adding an antioxidant into the enzymolysis liquid, then adjusting the pH, heating and inactivating the enzyme to obtain an oligomeric sodium hyaluronate solution;

(4) and purifying and drying the oligomeric sodium hyaluronate solution to obtain the oligomeric sodium hyaluronate.

In the step (1), the addition amount of the hyaluronidase is 30-90AU/L fermentation liquor.

The fermentation end point is that the pH change is less than 0.05/min.

Preferably, the species that can ferment to produce hyaluronic acid is selected from the group consisting of streptococcus zooepidemicus (streptococcus: (a))Streptococcus zooepidemicus) Bacillus subtilis preparation (B)Bacillus subtilis)。

The fermentation medium is fermentation liquor which is suitable for fermentation of hyaluronic acid and at least contains a carbon source, a nitrogen source and inorganic salt.

In the step (2), the pH is 6.0-7.5, and the proper temperature is 30-45 ℃, preferably 37-40 ℃.

In the step (2), the addition amount of the hyaluronidase is adjusted according to the molecular weight of the target product and the enzymolysis time. Preferably, the molecular weight section is between 5KDa and 10KDa, and the enzyme 60000-120000AU is added per kg of HA; the molecular weight section is 3KDa-5KDa, and 120000-150000AU is added into each kg of HA; the molecular weight section is 800Da-3KDa, and enzyme 150000-300000AU is added per kg HA.

In the step (2), the enzymolysis time of the hyaluronidase is 6-9 h.

In steps (1) and (2), the source of the hyaluronic acid is not limited, and the hyaluronic acid can be of leech origin, animal origin, such as leech and bovine testis, and can also be of microbial origin, such as obtained by fermentation of recombinant engineering bacteria.

In the step (3), the concentration of the antioxidant is 0.1-0.2% w/v. The antioxidant is selected from one or more of sodium bisulfite, potassium bisulfite, ascorbic acid and derivatives thereof; sodium bisulfite is preferred.

In the step (3), the pH is 4.5-5.6.

In the step (3), the temperature is increased to 80-120 ℃, and the inactivation time is 15-60 min.

Preferably, in the step (4), the purification comprises the following steps:

(i) after filtering the oligomeric sodium hyaluronate solution, sequentially adding a bleaching agent and a metal ion chelating agent to adjust the pH value for reaction, then adjusting the pH value, and then filtering and concentrating to obtain a concentrated solution;

(ii) adding sodium chloride into the concentrated solution, adjusting pH, precipitating with ethanol solution, washing the precipitate, and vacuum filtering to obtain pure product.

In step (i), the pore size of the filtration is 0.22-0.45 μm.

In step (i), the bleaching agent is added in an amount of 0.1% to 0.2% w/v. The addition amount of the metal ion chelating agent is 0.1-0.2% w/v.

In the step (i), the bleaching agent is one of sodium hypochlorite, hydrogen peroxide or activated carbon; preferably sodium hypochlorite. The metal ion chelating agent is EDTA, EDTA disodium or EDTA dipotassium.

In steps (i) and (ii), the pH is in the range of 6.0 to 7.5.

In step (i), the concentration is preferably ultrafiltration concentration, the pore size of ultrafiltration is selected according to different target products, for example, the molecular weight range is 5KDa-10KDa, and 500Da ultrafiltration membrane can be used; the molecular weight range is 3KDa-5KDa, and 300Da ultrafiltration membrane can be used; the molecular weight range is 800Da-3KDa, and a reverse osmosis membrane of 300Da can be used.

In step (ii), the sodium chloride is added in an amount of 4-6% w/v.

In step (ii), the concentration of the ethanol solution is 90-100% v/v.

In step (ii), the washing solvent is a 90-100% v/v ethanol solution.

Oligomeric sodium hyaluronate obtained by the preparation method.

The invention has the following advantages:

according to the preparation method of oligomeric sodium hyaluronate, provided by the invention, hyaluronic acid hydrolase is added in the fermentation process, enzymolysis is carried out while fermentation is carried out, the yield is increased to 14g/L, and then secondary enzymolysis is carried out to obtain oligomeric sodium hyaluronate with a fixed molecular weight; adding antioxidant to protect sodium hyaluronate during high temperature inactivation. The method improves fermentation yield, has high hyaluronic acid yield after enzymolysis, and continuous process, and can directly obtain target molecular weight product with high activity.

Drawings

FIG. 1 is a graph of different treatment group model tissue morphologies.

Detailed Description

The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.

Example 15 preparation of sodium hyaluronate with a KDa-10KDa

(1) Inoculating streptococcus zooepidemicus into 30L of fermentation medium in a 40L fermentation tank, stirring and fermenting under proper conditions, adding hyaluronidase 1200AU when fermenting to reach dynamic viscosity of 210000 mPa & s, fermenting until pH changes to be less than 0.05/min, stopping fermenting to obtain fermentation liquor, and detecting the content of sodium hyaluronate to be 13.5g/L by a carbazole sulfate method;

(2) adjusting the pH value of the fermentation liquor to 6.5, adding hyaluronidase 30000AU at 37 ℃, and reacting for 7.5h to obtain enzymatic hydrolysate;

(3) cooling the enzymolysis solution to room temperature, adding 60g of sodium bisulfite, adjusting the pH to 4.5, heating to 80 ℃, keeping for 30min, and inactivating the enzyme to obtain oligomeric sodium hyaluronate solution;

(4) filtering oligomeric sodium hyaluronate solution with a 0.45-micron filter plate, sequentially adding 150g of sodium hypochlorite solution (30% w/v) and 40g of EDTA disodium, adding water to 40L, adjusting the pH to 8.0, stirring at 30 ℃ for 30min, adjusting the pH to 6.5, filtering with a 0.22-micron filter plate, and performing ultrafiltration concentration with a 500Da ultrafiltration membrane to obtain 4L of concentrated solution;

(5) adding 160g of sodium chloride into the concentrated solution, adjusting the pH value to 6.5, pouring 16L of ethanol solution with the concentration of 95% v/v, stirring to generate precipitation, standing for 30min, removing supernatant, adding 4L of ethanol solution with the concentration of 95% v/v, stirring and washing for 30min, repeating until the alcoholic strength of supernatant is more than 90, then carrying out suction filtration, and carrying out vacuum drying at 65 ℃ to obtain 345g of pure white product with the average molecular weight of 8KDa, the yield of 85% and the content of 98%.

Example 23 preparation of sodium hyaluronate with kDa of 5kDa

(1) Inoculating streptococcus zooepidemicus into 30L of fermentation medium in a 40L fermentation tank, stirring and fermenting under proper conditions, adding 1350AU hyaluronidase when fermenting to 195000 mPa & s dynamic viscosity, fermenting until pH changes to be less than 0.05/min, stopping fermenting to obtain fermentation liquor, and detecting the content of sodium hyaluronate to be 13.7g/L by a carbazole sulfate method;

(2) adjusting the pH value of the fermentation liquor to 6.5, adding 48000AU of hyaluronidase at 40 ℃, and reacting for 8h to obtain enzymatic hydrolysate;

(3) cooling the enzymolysis solution to room temperature, adding 40g of ascorbic acid, adjusting the pH to 5.0, heating to 100 ℃, keeping for 20min, and inactivating the enzyme to obtain an oligomeric sodium hyaluronate solution;

(4) filtering oligomeric sodium hyaluronate solution with a 0.45-micron filter plate, sequentially adding 80g of active carbon and 40g of EDTA, adding water to 40L, adjusting the pH to 8.0, stirring at 30 ℃ for 30min, adjusting the pH to 6.8, filtering with a 0.22-micron filter plate, and performing ultrafiltration concentration with a 300Da ultrafiltration membrane to obtain 2L of concentrated solution;

(5) adding 160g of sodium chloride into the concentrated solution, adjusting the pH value to 6.5, pouring 24L of ethanol solution with the concentration of 90% v/v, precipitating and stirring to generate precipitate, standing for 30min, removing supernatant, adding 4L of ethanol solution with the concentration of 95% v/v, stirring and washing for 30min, repeating until the alcoholic strength of supernatant is about 90, then carrying out suction filtration, and carrying out vacuum drying at 65 ℃ to obtain 332g of pure white product, wherein the average molecular weight is 4.5KDa, the yield is 80%, and the content is 99%.

Example 3800 Da-3kDa preparation of sodium hyaluronate

(1) Inoculating streptococcus zooepidemicus into 30L of fermentation medium in a 40L fermentation tank, stirring and fermenting under proper conditions, adding hyaluronidase 1800AU when fermenting to reach the dynamic viscosity of 180000 mPa & s, fermenting until the pH change is less than 0.05/min, stopping fermenting to obtain fermentation liquor, and detecting the content of sodium hyaluronate to be 13.4g/L by a carbazole sulfate method;

(2) adjusting the pH value of the fermentation liquor to 6.5, adding hyaluronidase 60000AU at 37 ℃, and reacting for 7h to obtain enzymatic hydrolysate;

(3) cooling the enzymolysis solution to room temperature, adding 70g of potassium bisulfite, adjusting the pH to 5.6, heating to 120 ℃, keeping the temperature for 15min, and inactivating the enzyme to obtain an oligomeric sodium hyaluronate solution;

(4) after filtering an oligomeric sodium hyaluronate solution by using a 0.45 mu m filter plate, sequentially adding 120g of hydrogen peroxide (50% w/w) and 60g of EDTA dipotassium, adding water to 40L, adjusting the pH to 8.0, stirring at 30 ℃ for 30min, adjusting the pH to 6.5, filtering by using a 0.22 mu m filter plate, and concentrating by using a 300Da reverse osmosis membrane to obtain 2L of a concentrated solution;

(5) adding 160g of sodium chloride into the concentrated solution, adjusting the pH value to 6.5, pouring 24L of ethanol solution with the concentration of 90% v/v, precipitating and stirring to generate precipitate, standing for 30min, removing supernatant, adding 4L of ethanol solution with the concentration of 95% v/v, stirring and washing for 30min, repeating until the alcoholic strength of supernatant is more than 90, then carrying out suction filtration, and carrying out vacuum drying at 65 ℃ to obtain 302g of pure white product, wherein the average molecular weight is 1.2KDa, the yield is 75%, and the content is 98%.

Comparative example 1

Performing hyaluronic acid fermentation in a 40L fermentation tank, wherein the volume of the fermentation liquid is 30L, the dynamic viscosity is 140000 mPa & s, 150000mPa & s, 160000 mPa & s and 170000-. When the kinetic viscosity is higher than 250000 pas, the viscosity becomes too high, and the stirring fermentation cannot be continued.

It can be seen that when the viscosity is lower than 150000 mPas, the addition of hyaluronidase will affect the normal fermentation process, resulting in too low yield; when the viscosity is higher than 250000 Pa.s, stirring cannot be continued to cause difficulty in mass and heat transfer, and the enzymolysis effect of the hyaluronic acid is affected.

Comparative example 2

Fermentation, enzymatic hydrolysis and purification were carried out according to the raw material ratios and procedures of example 1, except that no antioxidant was added in the step (3), and 347g of a product was finally obtained, having an average molecular weight of 8kDa, yield of 85.5%, content of 91.5% and dark yellow color. This shows that the addition of antioxidant during the purification of oligomeric sodium hyaluronate helps to inhibit the degradation of the product during the purification process, which is beneficial to increase the yield and product content.

Comparative example 3 preparation of 5-10 kDa sodium hyaluronate from macromolecular hyaluronic acid

Taking 400g of sodium hyaluronate with the average molecular weight of 80kDa produced by fermentation in the same fermentation medium as the strain in the example 1, dissolving the sodium hyaluronate in 40L of water, adding 30000AU hyaluronidase after complete dissolution, adjusting the pH to 6.5, stirring and carrying out enzymolysis for 8h at 37 ℃, inactivating the sodium hyaluronate for 30min at 80 ℃ to obtain an enzymolysis solution, then purifying, concentrating and drying the enzymolysis solution according to the purification method in the example 1 to obtain 288g of oligomeric sodium hyaluronate with the average molecular weight of 8KDa, wherein the precipitate is 200L of alcohol, the yield is 72%, the color is white, and the content is determined by a carbazole sulfate method to be 92%. The method for degrading by adding hyaluronidase in the fermentation process has the advantages of high yield, high purity and less alcohol consumption compared with the traditional method for preparing macromolecular hyaluronic acid and then carrying out enzymolysis.

EXAMPLE 4 product Effect

The anti-allergic, anti-inflammatory and barrier injury repairing activities of the products of the above examples and comparative examples were compared, and oligomeric sodium hyaluronate prepared by a commercially available acidolysis method was used as a control (98% content, average molecular weight 8.1KDa, abbreviated as acidolysis HA, manufactured by shandong shangshan biotechnology limited). The concentration of each sample is calculated according to the content of the oligomeric sodium hyaluronate.

1. Anti-allergy efficacy testing

The desensitization efficacy of the samples was evaluated by morphological observation of mast cell degranulation and degranulation rate using the C48/80 stimulated mast cell model, grouped according to the following table:

the determination was carried out according to the following procedure: by 1.5X 10⁵The mast cells were seeded at a density of one well per well in 24 well plates at 37 ℃ with 5% CO₂Incubating under 95% RH conditions; and (3) when the cell plating rate in the 24-well plate reaches 40% -60%, the administration amount of the blank control group is 1mL, the administration amount of each well of other groups is 900 muL, and each group is provided with 3 multiple wells. Continuously culturing for 2h under the original condition; then, 100 mu L of corresponding 100 mu g/ml C48/80 mother solution prepared by the test object working solution, the negative control working solution and the positive control working solution is added into each hole, the hole plate is shaken to uniformly mix the liquid in the holes, and then the hole plate is put under the original condition to be continuously cultured for 45 min; placing the 24-pore plate in ice water bath at 0 ℃ for cold soaking for 10min to terminate the reactionCell morphology was observed under an inverted microscope and photographed (20 ×); the results were calculated according to the following formula: degranulation = degranulation cell number/total cell number × 100%.

The results are shown in table 1: compared with the blank group, the mast cell degranulation rate of the negative group is obviously increased, which indicates that the stimulation condition of C48/80 in the experiment is effective. Compared with the negative group, the mast cell degranulation rate phenomenon of the positive group is obviously inhibited, which indicates that the experiment is effective. The four groups of example 1, comparative example 2, comparative example 3 and acid hydrolysis HA showed a significant decrease in mast cell degranulation at a concentration of 7.81. mu.g/mL, and the most significant decrease in example 1, compared to the negative group, indicating that the anti-allergic activity of hyaluronic acid prepared according to the present invention was the best at the same molecular weight. Example 1, example 2 and example 3 have lower and lower degranulation rate, which shows that the smaller the molecular weight of the oligomeric hyaluronic acid prepared by the method, the higher the anti-sensitization activity.

TABLE 1 allergy relief effect of different treatments

2. Anti-inflammatory efficacy testing

UVB is selected to stimulate keratinocytes to establish an in-vitro model of skin inflammatory injury, the anti-inflammatory efficacy of a sample is evaluated by detecting the expression conditions of inflammatory factors TNF alpha and IL-alpha, and experimental grouping is carried out according to the following table:

the determination was carried out according to the following procedure: by 1.5X 10⁵The cells were seeded at a density of one cell per well in 24-well plates at 37 ℃ with 5% CO₂Incubating under 95% RH conditions; and (3) administering when the cell plating rate in the 24-well plate reaches 40% -60%, wherein each well is loaded with 2mL, and each group is provided with 3 multiple wells. Continuously culturing for 24h under the original condition; then, after washing the cells with PBS, the group irradiated with UVB was subjected to 300mJ/cm according to the experimental groups²The hole plate is placed under the original condition to be continuously cultured for 24 hours; collecting the supernatant in a 24-well plateIn an EP tube, the tube was stored in a refrigerator at-80 ℃. And (3) detecting the contents of TNF alpha and IL-l alpha according to the operation instruction of the ELISA detection kit of each inflammatory factor.

TABLE 2 TNF alpha, IL-l alpha content in supernatants of differently treated cells

The results are shown in table 2: compared with the blank group, the content of TNF alpha and IL-l alpha in the negative group is obviously increased, which shows that the experimental stimulation condition is effective. Compared with the negative group, the positive group has obviously reduced contents of TNF alpha and IL-l alpha, which indicates that the experiment is effective. In example 1, comparative example 2, comparative example 3 and acid hydrolysis HA four groups compared with the negative group, the contents of TNF alpha and IL-l alpha are obviously reduced under the concentration of 1000 mu g/mL, and the example 1 is most obviously reduced, which shows that the anti-inflammatory activity of the hyaluronic acid prepared by the invention is best under the same molecular weight. Example 1, example 2 and example 3 have lower and lower contents of TNF alpha and IL-l alpha, which shows that the lower the molecular weight of the oligomeric hyaluronic acid prepared by the method, the higher the anti-inflammatory activity.

3. Barrier damage repair function detection

Stimulation of 3D skin model (EpiKutis) with SLS (sodium dodecyl sulfate)^®) And simulating a human body patch experiment to construct an SLS-EpiKutis skin irritation injury model. A surface administration mode is adopted, a sample is used for simulating the use process of a human body and is uniformly coated on the surface of an SLS-EpiKutis skin irritation injury model constructed by an experiment, the effect of promoting the repair of the SLS-Epikutis irritation injury barrier by the sample is evaluated by detecting the structural change of the model tissue morphology and barrier related protein, and the experiment grouping is carried out according to the following table:

the determination was carried out according to the following procedure: according to the experimental design above, the model was transferred to a 6-well plate (0.9 mL of model culture solution was added in advance), and the 6-well plate was marked with the test group number. Get configuredAdding 25 μ L of different groups of sample liquid on the surface of the model, slightly shaking the model to uniformly distribute the sample on the surface of the model, and placing in CO₂Incubator (37 ℃, 5% CO)₂95% RH) for 24 h. After the incubation is finished, the test object remained on the surface of the model is washed by a wash bottle filled with sterile PBS solution, and the residual liquid inside and outside the model is lightly wiped off by a sterile cotton swab. Fixing the model for tissue morphology observation with 4% paraformaldehyde for 24 hr, cutting off the model ring, and performing H&E, staining treatment, photographing and observing under a microscope, and collecting pictures to observe the tissue morphology (figure 1). The method comprises the steps of sequentially embedding a model for FLG, LOR and CD44 immunohistochemical detection in paraffin, pre-treating (dewaxing, hydrating, antigen repairing and peroxidase blocking), sealing serum, respectively dropwise adding a primary antibody and a secondary antibody for incubation, incubating an ABC compound, staining (DAB staining and hematoxylin counterstaining), dehydrating to obtain a section, and measuring an Integrated Optical Density (IOD) value of the section by using an image analysis system.

TABLE 3 FLG, LOR and CD44 Integrated Optical Density (IOD) values for different treatments

The results are shown in FIG. 1 and Table 3: compared with the blank group, the negative group epidermal model has loose and thickened horny layer, damaged living cell layer and air bubbles, which indicates that the SLS stimulation condition is effective. Compared with a negative group, the positive (WY14643) group model has clear four-layer structure boundary, compact arrangement of living cell layer cells and obvious improvement of stratum corneum loosening and thickening phenomena, and the detection system is effective. Compared with a negative group, the four-layer structure boundary of the HA group model in the example 1 group and the acidolysis group is clear, the damage condition of a living cell layer is obviously improved, and the phenomenon of stratum corneum looseness and thickening is also obviously relieved; therefore, the example 1 group and the acid hydrolysis HA group have certain repairing efficacy on the 3D epidermis model morphological damage caused by SLS under the concentration of 10 mg/mL.

Compared with the blank group, the content of FLG, LOR and CD44 proteins in the negative group is obviously reduced, which indicates that the experimental stimulation condition is effective. Compared with the negative group, the positive group has obviously improved FLG, LOR and CD44 protein contents, which indicates that the experiment is effective. In the example 1 group, the FLG, LOR and CD44 protein contents are obviously increased compared with the negative control group and the acidolysis HA group respectively. The micromolecule hyaluronic acid prepared by the method has better barrier repair and repair activity.