CN106176277A - Gene recombinant human lysozyme facial film and preparation method thereof - Google Patents

Abstract

The invention provides a genetically recombinant human lysozyme facial mask, comprising: (1) lysozyme freeze-dried powder, including genetically recombinant human lysozyme and mannitol phosphate buffer, the ratio of the two is 0.5-0.8g: 1ml; (2) Solvent, each 1000ml solvent includes sodium hyaluronate 0.35g-0.5g, low molecular weight sodium hyaluronate 0.2g-0.6g, trimethylglycine 3g-7g, sodium lactate 40ml-80ml, sorbitol 30ml-50ml, Hydroxyethylcellulose 1.5g-3g, dipotassium glycyrrhizinate 1.5g-3g, EDTA-2Na 0.4g-0.7g and the rest of sodium phosphate buffer. The invention also provides a preparation method of the genetically recombined human lysozyme facial mask, including the steps of preparing lysozyme freeze-dried powder and the steps of preparing solvent.

Description

Gene recombinant human lysozyme facial mask and preparation method thereof

technical field

The invention belongs to the field of cosmetics. Specifically, the invention relates to a facial mask and a preparation method thereof, in particular to a gene recombinant human lysozyme facial mask and a preparation method thereof.

Background technique

Mask is a special beauty cosmetic, which is applied to the facial skin to form a thin film to isolate the skin from the outside world. The active ingredients in the mask can penetrate into the skin to moisturize the skin, increase nutrition, and promote skin function and metabolism. Make facial skin white, soft, comfortable and maintain youthful vitality. At present, commercially available facial mask products are divided into paste, liquid, gel and powder according to their form. From the perspective of composition, traditional Chinese medicine masks are mostly, while active protein masks are relatively rare.

Lysozyme (LYZ) is an alkaline enzyme that can hydrolyze mucopolysaccharides in pathogenic bacteria. This enzyme is also contained in liquids such as milk and milk, as well as microorganisms, among which egg white is the most abundant. Lysozyme is a very stable protein with strong heat resistance; it will not be inactivated by organic solvent treatment, and its activity can be fully restored when transferred to an aqueous solution; it can be frozen or dried, and its activity is stable; Wide antibacterial spectrum and high safety. When used on the skin surface, it can remove acne, reduce swelling, relieve pain, and eliminate bacteria.

Contents of the invention

The object of the present invention is to provide a gene recombinant human lysozyme facial mask and a preparation method thereof for the defects of the prior art.

On the one hand, the present invention provides a gene recombinant human lysozyme facial mask, comprising:

(1) Lysozyme freeze-dried powder, including genetically recombinant human lysozyme and mannitol phosphate buffer, the ratio of the two is 0.5-0.8g genetically recombinant human lysozyme: 1ml mannitol phosphate buffer;

(2) Solvent, wherein every 1000ml of solvent includes sodium hyaluronate 0.35g-0.5g, low molecular weight sodium hyaluronate 0.2g-0.6g, trimethylglycine 3g-7g, sodium lactate 40ml-80ml, sorbitol 30ml- 50ml, 1.5g-3g of hydroxyethyl cellulose, 1.5g-3g of dipotassium glycyrrhizinate, 0.4g-0.7g of EDTA-2Na and the rest of sodium phosphate buffer.

For the aforementioned recombinant human lysozyme facial mask, in the lysozyme freeze-dried powder, the ratio of genetic recombinant human lysozyme and mannitol phosphate buffer is 0.68g genetic recombinant human lysozyme: 1ml mannitol phosphate buffer.

For the aforementioned gene recombinant human lysozyme mask, every 1000ml solvent includes 0.45g sodium hyaluronate, 0.5g low molecular weight sodium hyaluronate, 5g trimethylglycine, 60ml sodium lactate, 40ml sorbitol, 2g hydroxyethyl cellulose, licorice Dipotassium acid 2g, EDTA-2Na 0.5g and the rest of the sodium phosphate buffer.

The aforementioned gene recombinant human lysozyme facial mask, said gene recombinant human lysozyme consists of 130 amino acids, and its amino acid sequence is: KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYNTRATNYNAGDRSTDYGIFQINSRYWCNDGKTPGAVNACHLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRNRCQNRDVRQYVQGCGV.

The aforementioned gene recombinant human lysozyme facial mask, the pH value of the mannitol phosphate buffer is 6.5-7.2 (preferably 7.0), and the concentration is 4.5-6.0mmol/L (preferably 5mmol/L); the mannitol The mass percent content of mannitol in the phosphate buffer solution is 2%-5% (preferably 3%).

In the aforementioned gene recombinant human lysozyme facial mask, the molecular weight of the sodium hyaluronate is 1.2-1.5 million daltons; the molecular weight of the low-molecular-weight sodium hyaluronate is 200,000-400,000 daltons.

For the aforementioned gene recombinant human lysozyme facial mask, the pH value of the sodium phosphate buffer is 6.5-7.2 (preferably 7.0), and the concentration is 4.5-6.0mmol/L (preferably 5mmol/L).

For the above-mentioned gene recombinant human lysozyme mask, when using it, first add 1ml of solvent to the lysozyme freeze-dried powder to dissolve evenly, then suck it out, return it to the solvent and mix evenly, and then inject it on the mask paper to soak it.

On the other hand, the present invention provides the preparation method of aforementioned gene recombinant human lysozyme mask, comprising the following steps:

(1) Preparation of lysozyme freeze-dried powder

(1.1) Dissolving 0.5g-0.8g of recombinant human lysozyme in sterile mannitol phosphate buffer, followed by filter sterilization to obtain a filtrate;

(1.2) Add aseptic mannitol phosphate buffer solution to the filtrate and set the volume to 100ml, mix evenly to obtain lysozyme solution;

(1.3) subpackage and freeze-dry the lysozyme solution to obtain lysozyme freeze-dried powder;

Wherein, the above-mentioned steps (1.1) to (1.3) are carried out under aseptic conditions;

(2) Preparation of solvent

(2.1) Dissolving 0.35g-0.5g (preferably 0.45g) sodium hyaluronate and 0.2g-0.6g (preferably 0.5g) low-molecular-weight sodium hyaluronate in sodium phosphate buffer to obtain a sodium hyaluronate solution;

(2.2) Dissolving 1.5g-3g (preferably 2g) hydroxyethyl cellulose in sodium phosphate buffer to obtain a hydroxyethyl cellulose solution;

(2.3) adding the sodium hyaluronate solution into the hydroxyethyl cellulose solution, and mixing evenly to obtain a mixed solution;

(2.4) Add 3g-7g (preferably 5g) trimethylglycine, 1.5g-3g (preferably 2g) dipotassium glycyrrhizinate, 0.4g-0.7g (preferably 0.5g) EDTA-2Na, 40ml- 80ml (preferably 60ml) of sodium lactate and 30ml-50ml (preferably 40ml) of sorbitol, then add sodium phosphate buffer to settle to 1000ml, mix well to obtain a solvent solution;

(2.5) Dispense and sterilize the solvent solution to obtain the solvent.

In the aforementioned preparation method, in step (1.1), a water phase filter with a pore size of 0.22 μm is used for filter sterilization.

In the aforementioned preparation method, steps (2.1) and (2.2) are carried out at 50-70°C (preferably 60°C).

In the aforementioned preparation method, in step (2.5), the sterilization is performed at 121° C. for 30 minutes.

According to the aforementioned preparation method, the recombinant human lysozyme consists of 130 amino acids, and its amino acid sequence is: KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYNTRATNYNAGDRSTDYGIFQINSRYWCNDGKTPGAVNACHLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRNRCQNRDVRQYVQGCGV.

In the aforementioned preparation method, the pH value of the mannitol phosphate buffer is 6.5-7.2 (preferably 7.0), and the concentration is 4.5-6.0mmol/L (preferably 5mmol/L); the mannitol phosphate buffer The mass percent content of mannitol in the medium is 2%-5% (preferably 3%).

In the aforementioned preparation method, the pH value of the sodium phosphate buffer solution is 6.5-7.2 (preferably 7.0), and the concentration is 4.5-6.0mmol/L (preferably 5mmol/L).

Compared with the prior art, the gene recombinant human lysozyme facial mask provided by the invention and its preparation method have the following advantages:

(1) The lysozyme raw material used is genetically recombinant human lysozyme, which is prepared by transforming Pichia pastoris with bioengineering technology, and is prepared through large-scale fermentation, expression and purification, with high purity, high activity, good safety, and low cost .

(2) To overcome the shortcoming that the active protein is easy to degrade and inactivate when stored in an aqueous solution for a long time, it is prepared into a freeze-dried powder to ensure the activity of lysozyme, and does not contain preservatives.

detailed description

In order to fully understand the purpose, features and effects of the present invention, the present invention will be described in detail through the following specific implementation modes.

Aiming at the problem that most of the facial masks currently on the market are traditional Chinese medicine facial masks, less active protein facial masks, and unsatisfactory effects, in the first aspect, the present invention provides a genetically recombinant human lysozyme facial mask, which comprises The following components:

(1) lysozyme freeze-dried powder, including genetically recombinant human lysozyme and mannitol phosphate buffer, the ratio of the two is 0.5-0.8g genetically recombinant human lysozyme: 1ml mannitol phosphate buffer, preferably, two The ratio is 0.68g recombinant human lysozyme: 1ml mannitol phosphate buffer.

Among them, the recombinant human lysozyme is produced by genetic engineering and is homologous to human lysozyme. It consists of 130 amino acids and its amino acid sequence is: KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYNTRATNYNAGDRSTDYGIFQINSRYWCNDGKTPGAVNACHLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRNRCQNRDVRQYVQGCGV. The lysozyme is obtained by screening Pichia pastoris genetically engineered bacteria expressing lysozyme, and then producing through fermentation. The recombinant human lysozyme used in the present invention is consistent with the lysozyme in the human body. First, its biohomology, biocompatibility, and biosafety are all good; secondly, the recombinant human lysozyme has acne-removing, Reducing swelling, relieving pain, and degerming. It is itself a kind of bacteriolytic substance, which can play the role of antisepsis and sterilization, and avoid the harm of adding chemical preservatives to the human body.

Wherein, the pH value of mannitol phosphate buffer is 6.5-7.2 (preferably 7.0), and concentration is 4.5-6.0mmol/L (preferably 5mmol/L); In mannitol phosphate buffer, the quality of mannitol The percentage is 2%-5% (preferably 3%). During the freeze-drying process of genetically recombinant human lysozyme, the pH value and concentration of mannitol phosphate buffer used in the present invention can protect the activity of lysozyme, and after freeze-drying, it can lyophilize the bacteria The enzyme is uniformly dispersed and has high uniformity.

(2) Solvent, including sodium hyaluronate, low molecular weight sodium hyaluronate, trimethylglycine, sodium lactate, sorbitol, hydroxyethyl cellulose, dipotassium glycyrrhizinate, EDTA-2Na and sodium phosphate buffer, wherein, Each 1000ml vehicle includes sodium hyaluronate 0.35g-0.5g, low molecular weight sodium hyaluronate 0.2g-0.6g, trimethylglycine 3g-7g, sodium lactate 40ml-80ml, sorbitol 30ml-50ml, hydroxyethyl cellulose 1.5g-3g, dipotassium glycyrrhizinate 1.5g-3g, EDTA-2Na 0.4g-0.7g and the rest of the sodium phosphate buffer; preferably, every 1000ml solvent includes sodium hyaluronate 0.45g, low molecular weight hyaluronic acid Sodium 0.5g, trimethylglycine 5g, sodium lactate 60ml, sorbitol 40ml, hydroxyethyl cellulose 2g, dipotassium glycyrrhizinate 2g, EDTA-2Na 0.5g and the rest of sodium phosphate buffer.

Wherein, the pH value of the sodium phosphate buffer solution is 6.5-7.2 (preferably 7.0), and the concentration is 4.5-6.0mmol/L (preferably 5mmol/L). The molecular weight of sodium hyaluronate is 1.2-1.5 million daltons, and the molecular weight of low-molecular-weight sodium hyaluronate is 200,000-400,000 daltons.

After adopting the above-mentioned specific components and specific ratios, there is a synergistic effect between the components of the solvent, which is more conducive to dispersion and dissolution when dissolving the lysozyme freeze-dried powder. After making a mask, it can promote the skin’s nutrition when used Absorption, and can maintain the activity of lysozyme for a longer time.

Among them, the above-mentioned sodium hyaluronate, low-molecular-weight sodium hyaluronate, trimethylglycine, sodium lactate, sorbitol, hydroxyethyl cellulose, dipotassium glycyrrhizinate, EDTA-2Na (that is, disodium ethylenediaminetetraacetate) All are commercially available.

When using the above-mentioned recombinant human lysozyme mask, first take a small amount of solvent (such as 1ml) and add it to the lysozyme freeze-dried powder to dissolve it evenly, suck it out, and return it to the solvent and mix it evenly to obtain the genetic recombinant human lysozyme mask solution. Aspirate the solution with a syringe and inject it onto the sheet mask, ready to use. There is no special requirement for the ratio of lysozyme freeze-dried powder to solvent, as long as the solvent can fully dissolve the lysozyme freeze-dried powder; The volume ratio is preferably 1:10-1:15, and more preferably 1:13.5.

The lysozyme component of the above-mentioned gene recombinant human lysozyme mask is prepared into freeze-dried powder, and is stored separately from the solvent during storage, which can ensure the activity of lysozyme and avoid the degradation and loss of active protein in aqueous solution for a long time. live problem.

In a second aspect, the present invention provides a method for preparing the above-mentioned gene recombinant human lysozyme facial mask, which method includes the steps of preparing lysozyme freeze-dried powder and the step of preparing solvent, specifically as follows:

The first step is to prepare lysozyme freeze-dried powder

Firstly, 0.5g-0.8g of recombinant human lysozyme is dissolved in sterile mannitol phosphate buffer (for example, 5ml) until it is completely dissolved, and then sterilized by filtration to obtain a filtrate. Wherein, the filtration sterilization can be performed by using a water phase filter, for example, a water phase filter with a pore size of 0.22 μm can be used for filtration sterilization.

Then add aseptic mannitol phosphate buffer solution to the above filtrate and adjust the volume to 100ml, and mix evenly to obtain a lysozyme solution.

Then the lysozyme solution is subpackaged and freeze-dried with a freeze dryer to obtain a lysozyme freeze-dried powder. According to actual needs, those skilled in the art can determine the volume of subpackaging, for example, it may be 1 ml.

The above-mentioned steps of preparing lysozyme freeze-dried powder are carried out under aseptic conditions, for example, can be aseptically completed in an ultra-clean workbench.

The mannitol phosphate buffer used in this step can be prepared by conventional methods, its pH value is 6.5-7.2 (preferably 7.0), and the concentration is 4.5-6.0mmol/L (preferably 5mmol/L); In the salt buffer solution, the mass percentage of mannitol is 2%-5% (preferably 3%). According to the properties of the mannitol phosphate buffer, those skilled in the art can select a suitable sterilization method, for example, the mannitol phosphate buffer prepared can be filtered with a pore size of 0.45 μm, followed by 121 °C for 30 minutes.

The second step is to prepare the solvent

Firstly, 0.35g-0.5g (preferably 0.45g) sodium hyaluronate and 0.2g-0.6g (preferably 0.5g) low-molecular-weight sodium hyaluronate are dissolved in sodium phosphate buffer (for example, 200ml) to obtain sodium hyaluronate solution , at a temperature of 50-70°C, preferably 60°C, and mix well.

Subsequently, 1.5g-3g of hydroxyethyl cellulose was dissolved in sodium phosphate buffer (for example, 300ml) to obtain a hydroxyethyl cellulose solution, and mixed uniformly at a temperature of 50-70°C, preferably 60°C.

Then add the sodium hyaluronate solution into the hydroxyethyl cellulose solution, and mix evenly to obtain a mixed solution. Then add 3g-7g trimethylglycine, 1.5g-3g dipotassium glycyrrhizinate, 0.4g-0.7g EDTA-2Na, 40ml-80ml sodium lactate and 30ml-50ml sorbitol to the mixed solution, then add sodium phosphate buffer Make up to 1000ml, mix evenly to obtain a solvent solution; preferably, add 5g trimethylglycine, 2g dipotassium glycyrrhizinate, O.5g EDTA-2Na, 60ml sodium lactate and 40ml sorbitol to the mixed solution, and then add sodium phosphate buffer Dilute to 1000ml, mix evenly to obtain a solvent solution.

The vehicle solution is then aliquoted and sterilized to obtain a vehicle. According to actual needs, those skilled in the art can determine the volume of dispensing, for example, it may be 13.5ml. Conventional methods can be used for sterilization, for example, at 121° C. for 30 minutes.

The sodium phosphate buffer used in this step can be prepared by conventional methods, its pH value is 6.5-7.2 (preferably 7.0), and its concentration is 4.5-6.0mmol/L (preferably 5mmol/L). Before use, the sodium phosphate buffer solution is filtered, for example, using a 0.45 μm aqueous filter.

Example

The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions. All materials used in the examples are commercially available.

Example 1

The composition of the recombinant human lysozyme facial mask of the present embodiment is as follows:

(1) Lysozyme freeze-dried powder, including genetically recombinant human lysozyme and mannitol phosphate buffer solution with a pH value of 7.0 and a concentration of 5 mmol/L, the ratio of the two is 0.68g genetically recombinant human lysozyme: 1ml mannitol Phosphate buffer.

(2) Solvent, wherein every 1000ml of solvent includes sodium hyaluronate 0.45g, low molecular weight sodium hyaluronate 0.5g, trimethylglycine 5g, sodium lactate 60ml, sorbitol 40ml, hydroxyethyl cellulose 2g, glycyrrhizic acid di Potassium 2g, EDTA-2Na 0.5g, and the remainder have a pH value of 7.0 and a sodium phosphate buffer solution with a concentration of 5mmol/L.

The preparation method of the gene recombinant human lysozyme facial mask of the present embodiment is as follows:

(1) Preparation of lysozyme freeze-dried powder: first prepare 200ml of 5mmol/L pH7.0 phosphate buffer containing 3% mannitol (i.e. mannitol phosphate buffer) and filter through an aqueous filter with a pore size of 0.45 μm. Then sterilize at 121°C for 30 minutes. Then weigh 0.68g of recombinant human lysozyme dry powder, add 5ml of sterilized mannitol phosphate buffer to dissolve, after the dissolution is complete, use a water phase filter with a pore size of 0.22μm to filter and sterilize, and then absorb a small amount of mannitol phosphate buffer Filter and rinse, then add mannitol phosphate buffer to make up to 100ml, and finally divide it into sterile vials, 1ml per bottle. The whole operation process is aseptically completed in the ultra-clean bench. Dry.

(2) Solvent preparation: prepare 1200 ml of 5 mmol/L sodium phosphate buffer solution with pH 7.0, and filter with an aqueous phase filter with a pore size of 0.45 μm. Then weigh 0.45 g of sodium hyaluronate and 0.5 g of low-molecular-weight sodium hyaluronate, dissolve them in 200 ml of 5 mmol/L sodium phosphate buffer at pH 7.0, and stir evenly at a temperature of 60° C. for later use. Weigh 2g of hydroxyethyl cellulose, dissolve it in 300ml of 5mmol/L sodium phosphate buffer solution with pH7.0, stir evenly at a temperature of 60°C, then slowly add the evenly dissolved hyaluronic acid solution into the hydroxyethyl cellulose solution Continue to stir until uniform, add 5g of trimethylglycine, 2g of dipotassium glycyrrhizinate, 0.5g of EDTA-2Na, 60ml of sodium lactate, and 40ml of sorbitol one after another. to 1000ml, stir again evenly. Take 15ml vials and divide into 13.5ml vials, sterilize at 121°C for 30min.

Before use, take a piece of lysozyme freeze-dried powder, use a syringe to draw 1ml of solvent, add it to the freeze-dried powder, dissolve it evenly, suck it out, infuse it back into the solvent and mix it evenly, then suck it all out with a syringe, inject it on the mask paper to soak it, and get Gene recombinant human lysozyme mask.

Example 2

The composition of the recombinant human lysozyme facial mask of the present embodiment is as follows:

(1) Lysozyme freeze-dried powder, including recombinant human lysozyme and mannitol phosphate buffer with a pH value of 6.5 and a concentration of 4.5mmol/L, the ratio of the two is 0.5g recombinant human lysozyme: 1ml manna alcohol phosphate buffer.

(2) Solvent, wherein every 1000ml of solvent includes sodium hyaluronate 0.35g, low molecular weight sodium hyaluronate 0.6g, trimethylglycine 3g, sodium lactate 80ml, sorbitol 30ml, hydroxyethyl cellulose 1.5g, glycyrrhizic acid Dipotassium 1.5g, EDTA-2Na0.4g and the pH value of the remainder are 6.5 and the sodium phosphate buffer solution whose concentration is 4.5mmol/L.

The preparation method of the gene recombinant human lysozyme facial mask of the present embodiment is as follows:

(1) Preparation of lysozyme freeze-dried powder: first prepare 200ml of 4.5mmol/L pH6.5 phosphate buffer containing 2% mannitol (i.e. mannitol phosphate buffer), and filter with an aqueous phase filter with a pore size of 0.45 μm , and then sterilized at 121°C for 30 minutes. Then weigh 0.5g of recombinant human lysozyme dry powder, add 5ml of sterilized mannitol phosphate buffer to dissolve, after the dissolution is complete, use a water phase filter with a pore size of 0.22μm to filter and sterilize, and then absorb a small amount of mannitol phosphate buffer Filter and rinse, then add mannitol phosphate buffer to make up to 100ml, and finally divide it into sterile vials, 1ml per bottle. The whole operation process is aseptically completed in the ultra-clean bench. Dry.

(2) Solvent preparation: Prepare 1200ml of 4.5mmol/L sodium phosphate buffer solution with a pH of 6.5, and filter through an aqueous phase filter with a pore size of 0.45μm. Then weigh 0.35 g of sodium hyaluronate and 0.6 g of low-molecular-weight sodium hyaluronate, dissolve them in 200 ml of 4.5 mmol/L sodium phosphate buffer solution with a pH of 6.5, and stir them evenly at a temperature of 60° C. for later use. Weigh 1.5g of hydroxyethyl cellulose, dissolve it in 300ml 4.5mmol/L sodium phosphate buffer solution of pH 6.5, stir well at a temperature of 60°C, then slowly add hydroxyethyl cellulose to the homogeneously dissolved hyaluronic acid solution Continue to stir in the cellulose solution until it is uniform, add 3g of trimethylglycine, 1.5g of dipotassium glycyrrhizinate, 0.4g of EDTA-2Na, 80ml of sodium lactate, and 30ml of sorbitol one after another. The buffer solution was adjusted to 1000ml, and stirred evenly again. Take 15ml vials and divide into 13.5ml vials, sterilize at 121°C for 30min.

Before use, take a piece of lysozyme freeze-dried powder, use a syringe to draw 1ml of solvent, add it to the freeze-dried powder, dissolve it evenly, suck it out, infuse it back into the solvent and mix it evenly, then suck it all out with a syringe, inject it on the mask paper to soak it, and get Gene recombinant human lysozyme mask.

Example 3

The composition of the recombinant human lysozyme facial mask of the present embodiment is as follows:

(1) Lysozyme freeze-dried powder, including recombinant human lysozyme and mannitol phosphate buffer solution with a pH value of 7.2 and a concentration of 6mmol/L, the ratio of the two is 0.5g recombinant human lysozyme: 1ml mannitol Phosphate buffer.

(2) Solvent, wherein every 1000ml of solvent includes sodium hyaluronate 0.5g, low molecular weight sodium hyaluronate 0.2g, trimethylglycine 7g, sodium lactate 40ml, sorbitol 50ml, hydroxyethyl cellulose 3g, glycyrrhizic acid di Potassium 3g, EDTA-2Na 0.7g, and the remainder have a pH value of 7.2 and a sodium phosphate buffer solution with a concentration of 6mmol/L.

The preparation method of the gene recombinant human lysozyme facial mask of the present embodiment is as follows:

(1) Preparation of lysozyme freeze-dried powder: first prepare 200ml of 6mmol/L pH7.2 phosphate buffer (i.e. mannitol phosphate buffer) containing 5% mannitol, filter through an aqueous filter with a pore size of 0.45 μm, Then sterilize at 121°C for 30 minutes. Then weigh 0.5g of recombinant human lysozyme dry powder, add 5ml of sterilized mannitol phosphate buffer to dissolve, after the dissolution is complete, use a water phase filter with a pore size of 0.22μm to filter and sterilize, and then absorb a small amount of mannitol phosphate buffer Filter and rinse, then add mannitol phosphate buffer to make up to 100ml, and finally divide it into sterile vials, 1ml per bottle. The whole operation process is aseptically completed in the ultra-clean bench. Dry.

(2) Solvent preparation: prepare 1200ml of 6mmol/L sodium phosphate buffer solution with pH7.2, and filter it with an aqueous phase filter with a pore size of 0.45μm. Then weigh 0.5 g of sodium hyaluronate and 0.2 g of low-molecular-weight sodium hyaluronate, dissolve them in 200 ml of 6 mmol/L sodium phosphate buffer at pH 7.2, and stir evenly at a temperature of 60° C. for later use. Weigh 3g of hydroxyethyl cellulose, dissolve it in 300ml 6mmol/L sodium phosphate buffer solution with pH7.2, stir evenly at a temperature of 60°C, then slowly add the evenly dissolved hyaluronic acid solution into the hydroxyethyl cellulose solution Continue to stir until uniform, then add 7g of trimethylglycine, 3g of dipotassium glycyrrhizinate, 0.7g of EDTA-2Na, 40ml of sodium lactate, and 50ml of sorbitol. to 1000ml, stir again evenly. Take 15ml vials and divide into 13.5ml vials, sterilize at 121°C for 30min.

Before use, take a piece of lysozyme freeze-dried powder, use a syringe to draw 1ml of solvent, add it to the freeze-dried powder, dissolve it evenly, suck it out, infuse it back into the solvent and mix it evenly, then suck it all out with a syringe, inject it on the mask paper to soak it, and get Gene recombinant human lysozyme mask.

Claims (15)

1. a gene recombinant human lysozyme facial film, it is characterised in that comprise:

(1) lysozyme lyophilized powder, including gene recombinant human lysozyme and mannitol phosphate buffer, the ratio of the two is 0.5- 0.8g gene recombinant human lysozyme: 1ml mannitol phosphate buffer；

(2) solvent, wherein, every 1000ml solvent includes hyaluronate sodium 0.35g-0.5g, low-numerator sodium hyaluronate 0.2g- 0.6g, trimethyl glycine 3g-7g, sodium lactate 40ml-80ml, sorbitol 30ml-50ml, hydroxyethyl cellulose 1.5g-3g, sweet Oxalic acid dipotassium 1.5g-3g, EDTA-2Na 0.4g-0.7g and the sodium phosphate buffer of surplus.

Gene recombinant human lysozyme facial film the most according to claim 1, it is characterised in that in lysozyme lyophilized powder, base Because the proportioning of recombinant human lysozyme and mannitol phosphate buffer is 0.68g gene recombinant human lysozyme: 1ml mannitol phosphoric acid Salt buffer.

Gene recombinant human lysozyme facial film the most according to claim 1, it is characterised in that every 1000ml solvent includes transparent Matter acid sodium 0.45g, low-numerator sodium hyaluronate 0.5g, trimethyl glycine 5g, sodium lactate 60ml, sorbitol 40ml, ethoxy Cellulose 2g, glycyrrhizic acid dipotassium 2g, EDTA-2Na 0.5g and the sodium phosphate buffer of surplus.

4. according to the gene recombinant human lysozyme facial film described in any one of claim 1-3, it is characterised in that described gene recombinaton Human lysozyme is made up of 130 aminoacid, and its aminoacid sequence is: KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYNTRATNYNAGDRSTDYGIFQINSRYWCNDGKTPGAVNAC HLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRNRCQNRDVRQYVQGCGV。

5. according to the gene recombinant human lysozyme facial film described in any one of claim 1-3, it is characterised in that described mannitol phosphorus The pH value of phthalate buffer is 6.5-7.2 (preferably 7.0), and concentration is 4.5-6.0mmol/L (preferably 5mmol/L)；Described In mannitol phosphate buffer, the weight/mass percentage composition of mannitol is 2%-5% (preferably 3%).

6. according to the gene recombinant human lysozyme facial film described in any one of claim 1-3, it is characterised in that described hyaluronic acid The molecular weight of sodium is 120-150 ten thousand dalton；The molecular weight of described low-numerator sodium hyaluronate is 20-40 ten thousand dalton.

7. according to the gene recombinant human lysozyme facial film described in any one of claim 1-3, it is characterised in that described sodium phosphate delays The pH value rushing liquid is 6.5-7.2 (preferably 7.0), and concentration is 4.5-6.0mmol/L (preferably 5mmol/L).

8. according to the gene recombinant human lysozyme facial film described in any one of claim 1-3, it is characterised in that in use, first By 1ml solvent addition lysozyme lyophilized powder being uniformly dissolved rear sucking-off, being fed back to mix homogeneously in solvent, then it is injected to facial film Soak on paper.

9. the preparation method of the gene recombinant human lysozyme facial film described in any one of claim 1-8, it is characterised in that include as Lower step:

(1) lysozyme lyophilized powder is prepared

(1.1) 0.5g-0.8g gene recombinant human lysozyme is dissolved in aseptic mannitol phosphate buffer, filters subsequently Degerming, obtain filtrate；

(1.2) adding aseptic mannitol phosphate buffer in filtrate and be settled to 100ml, it is molten that mix homogeneously obtains lysozyme Liquid；

(1.3) by lysozyme soln subpackage, lyophilizing, lysozyme lyophilized powder is obtained；

Wherein, above-mentioned steps (1.1) is aseptically carried out to (1.3)；

(2) solvent is prepared

(2.1) by 0.35g-0.5g (preferably 0.45g) hyaluronate sodium and 0.2g-0.6g (preferably 0.5g) low molecular weight hyaluronic acid Sodium is dissolved in sodium phosphate buffer and obtains sodium hyaluronate solution；

(2.2) 1.5g-3g (preferably 2g) hydroxyethyl cellulose is dissolved in sodium phosphate buffer obtains hydroxyethyl cellulose solution；

(2.3) adding in hydroxyethyl cellulose solution by sodium hyaluronate solution, mix homogeneously obtains mixed solution；

(2.4) in mixed solution, 3g-7g (preferably 5g) trimethyl glycine, 1.5g-3g (preferably 2g) glycyrrhizic acid are added successively Dipotassium, 0.4g-0.7g (preferably 0.5g) EDTA-2Na, 40ml-80ml (preferably 60ml) sodium lactate and 30ml-50ml are (preferably 40ml) sorbitol, is subsequently added sodium phosphate buffer and is settled to 1000ml, and mix homogeneously obtains solvent solutions；

(2.5) by solvent solutions subpackage, sterilizing, solvent is obtained.

Preparation method the most according to claim 9, it is characterised in that in step (1.1), using aperture is 0.22 μm Aqueous phase filter carry out filtration sterilization.

11. preparation methoies according to claim 9, it is characterised in that step (2.1) and (2.2) are at 50-70 DEG C (preferably 60 DEG C) carry out.

12. preparation methoies according to claim 9, it is characterised in that in step (2.5), described sterilizing is at 121 DEG C Sterilizing 30 minutes.

13. according to the preparation method described in claim 9-12, it is characterised in that described gene recombinant human lysozyme is by 130 Aminoacid forms, and its aminoacid sequence is: KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYNTRATNYNAGDRS TDYGIFQINSRYWCNDGKTPGAVNACHLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRNRCQNRDVRQYVQGC GV。

14. according to the preparation method described in claim 9-12, it is characterised in that the pH value of described mannitol phosphate buffer Being 6.5-7.2 (preferably 7.0), concentration is 4.5-6.0mmol/L (preferably 5mmol/L)；Described mannitol phosphate-buffered In liquid, the weight/mass percentage composition of mannitol is 2%-5% (preferably 3%).

15. according to the preparation method described in claim 9-12, it is characterised in that the pH value of described sodium phosphate buffer is 6.5- 7.2 (preferably 7.0), concentration is 4.5-6.0mmol/L (preferably 5mmol/L).