JP7601957B2 - Recombinant collagen for skin soothing and repair and uses thereof - Google Patents

Description

The present invention relates to the field of skin care, specifically to recombinant collagen and its uses for skin soothing and repair.

When the environment inside and outside the skin cells changes, the skin's defense and repair mechanisms cannot adapt to the changes, causing skin damage and aging due to the accumulation of damage. Studies have shown that radicals are one of the important factors that cause aging in the human body. Various radicals, such as superoxide anion radicals, hydroxyl radicals, and DPPH radicals, exist in the human body, and radicals may cause cell and tissue decomposition and a decline in metabolic function. In addition to normal metabolism, the concentration of radicals in the human body is also affected by factors such as drought, pollution, exercise level, and mental state. Therefore, skin care products containing anti-oxidant ingredients can be used to remove excess radicals, delay body degeneration, and inhibit skin aging.

Schizophyllan is a naturally occurring β-D-glucan from Schizophyllum commune, which consists of a main chain of (1-3)-linked β-D-pyran type glucose residues and a side chain of (1-6)-linked pyran type glucose residues, and has the ability to resist oxidation and radiation. Schizophyllan can suppress the generation of radicals by inhibiting the cellular oxidative enzyme system and inducing the complexation of oxidized transition metal ions, and exerts a radiation resistance effect by means of radical scavenging, regulating gene expression, and protein synthesis, and can extend the apoptosis time of cells damaged by radiation and inhibit lipid peroxidation.

Collagen is a biopolymer and the main component of animal connective tissues. It is also the functional protein with the highest content and widest distribution in mammalian bodies, accounting for 25-30% of the total protein. Collagen has good biocompatibility, biodegradability and bioactivity, and is therefore widely used in the fields of food, medicine, tissue engineering and other fields. Collagen includes type I collagen molecules, type III collagen molecules, type IV collagen molecules, type XI collagen molecules, etc., and has the effects of moisturizing and hydrating the skin, promoting cell proliferation and reinforcing the cell barrier.

CN113057932A discloses a collagen skin care product that improves skin wrinkles. This collagen skin care product that improves skin wrinkles contains, by weight, 5-15 parts of active plants, 10-15 parts of glycerin, 1-2 parts of butylene glycol, 7-13 parts of active additives, 5-10 parts of collagen, and 30-40 parts of deionized water as raw materials, and is prepared by grinding, mixing, foaming stirring, secondary mixing, sterilization, adding collagen, and final mixing. The invention adopts skin care product production equipment during preparation, which improves production and makes collection convenient, and the final skin care product has collagen activity and activity to promote collagen production, and can effectively improve the problem of wrinkles appearing due to skin aging with age.

CN102670477A discloses a collagen skin care product that has good therapeutic effects, no side effects, and improves skin defects. The skin care product contains, by weight, 10-30 parts of seaweed (high in plant collagen), 10-30 parts of honey, and 1-5 parts of vitamin E.

Therefore, it has become very meaningful to explore skin care product compositions with multiple effects such as anti-oxidation, anti-aging, repair and moisturizing activity, and to reinforce the repair of skin damage from multiple angles.

In response to the deficiencies of the prior art, the present invention aims to provide recombinant collagen and uses thereof, in particular for soothing and repairing the skin, and uses thereof.

To achieve this objective, the present invention employs the following technical solutions:

In a first aspect, the present invention provides a recombinant collagen for soothing and repairing skin, the nucleotide sequence encoding the recombinant collagen for soothing and repairing skin comprising the sequence shown in SEQ ID NO:1.

The nucleotide sequence shown in SEQ ID NO:1 is as follows:

GGGCCTCAAGGTATTGCTGGACAGCGTGGTGTGGTCGGCCTGCCTGGTCAGAGAGGAGAGAGAGGCTTCCCTGGTCTTCCTGGCCCCTCTGGTGAACCTGG CAAACAAGGTCCCTCTGGAGCAAGTGGTGAACGTGGTCCCCCTGGTCCCATGGGCCCCCTGGATTGGCTGGACCCCCTGGTGAATCTGGACGTGAGGGGG CTCCTGGTGCCGAAGGTTCCCCTGGACGAGACGGTTCTCCTGGCGCCAAGGGTGACCGTGGTGAGACCGGCCCCGCTGGACCCCCTGGTGCTCCTGGTGCT CCTGGTGCCCCTGGCCCCGTTGGCCCTGCTGGCAAGAGTGGTGATCGTGGTGAGACTGGTCCTGCTGGTCCCGCCGGAGAACGAGGTGGCCCTGGAGGACCT GGCCCTCAGGGTCCTCCTGGAAAGAATGGTGAAAACTGGACCTCAGGGACCCCAGGGCCCTACTGGGCCTGGTGGTGACAAAGGAGACACAGGACCCCCTGG TCCACAAGGATTACAAGGCTTGCCTGGTACAGGTGGTCCTCCAGGAGAAATGGAAAACCTGGGGAACCAGGTCCAAAGGGTGATGCCGGTGGCACCTGGAGC TCCAGGAGGCAAGGGTGATGCTGGTGCCCCTGGTGAACGTGGACCTCCTGGATTGGCAGGGGCCCCAGGACTTAGAGGTGGAGCTGGTCCCCCTGGTCCCG AAGGAGGAAAGGGTGCTGCTGGTCCTCCTGGGCCACCTGGTGCTGCTGGTACTCCTGGTCTGCAAGGAATGCCTGGACCTGGTCCTTGCTGTGGTGGTTAA

In the present invention, a creatively novel recombinant collagen sequence is discovered. The nucleotide sequence encoding the recombinant collagen for skin soothing and repair comprises the sequence shown in SEQ ID NO:1, and the amino acid sequence encoded by SEQ ID NO:1 comprises amino acids 950-1075 of type I collagen, and amino acids 594-729 and 1193-1199 of type III collagen. The recombinant collagen has effects such as moisturizing, replenishing collagen in the skin, promoting cell proliferation, anti-oxidation, and anti-aging, and also has good tissue compatibility, skin permeability, and safety.

In a second aspect, the present invention provides a method for producing a composition comprising:
The present invention provides a composition having a soothing and repairing effect, comprising the recombinant collagen for soothing and repairing skin according to the first aspect and schizophyllan.

The composition having a soothing and repairing effect according to the present invention contains the recombinant collagen for soothing and repairing the skin described in the first aspect and schizophyllan, and the two components are used in combination to have a synergistic effect in terms of moisturizing, oxidation resistance, promotion of cell proliferation, and improved repair of skin damage.

Preferably, the soothing and restorative composition comprises, in parts by weight:
It contains 0.01 to 0.5 parts of recombinant collagen for skin soothing and repair and 0.1 to 5 parts of schizophyllan.

The mass parts of the skin soothing and repairing recombinant collagen can be selected from 0.01 parts, 0.1 parts, 0.2 parts, 0.3 parts, 0.4 parts, 0.5 parts, etc.

The mass parts of schizophyllan can be selected from 0.1 parts, 1 parts, 2 parts, 3 parts, 4 parts, 5 parts, etc.

All specific point values within each of the above numerical ranges can be selected, and so we will not explain them here.

When the composition having a soothing and repairing effect according to the present invention contains, by mass, 0.01 to 0.5 parts of recombinant collagen for soothing and repairing the skin and 0.1 to 5 parts of schizophyllan, the use of both in combination provides an even more excellent repairing effect against skin damage.

Preferably, the composition having the soothing and restorative effect further contains a moisturizer.

Preferably, the moisturizer includes one or a combination of at least two of glycerin, 1,3-butanediol, sodium hyaluronate, sodium acetylated hyaluronate, hydrolyzed sodium hyaluronate, or ceramide 3. The combination of at least two includes a combination of glycerin and 1,3-butanediol, a combination of 1,3-butanediol and sodium hyaluronate, or a combination of sodium hyaluronate and sodium acetylated hyaluronate, and any other combination form can be selected, and the description thereof will be omitted here.

Preferably, the moisturizer is glycerin.

When the moisturizer of the present invention specifically selects glycerin, the combination of the recombinant collagen and schizophyllan for skin soothing and repair can be further used in combination with glycerin to exert a stronger synergistic effect in terms of moisturization.

Preferably, the soothing and restorative composition comprises, in parts by weight:
It comprises 0.01 to 0.5 parts of the recombinant collagen for skin soothing and repair according to the first aspect, 0.1 to 5 parts of schizophyllan and 5 to 50 parts of glycerin.

The mass parts of the recombinant collagen for skin soothing and repair can be selected from 0.01 parts, 0.1 parts, 0.2 parts, 0.3 parts, 0.4 parts, 0.5 parts, etc.

The mass parts of schizophyllan can be selected from 0.1 parts, 1 parts, 2 parts, 3 parts, 4 parts, 5 parts, etc.

The mass parts of glycerin can be selected from 5 parts, 10 parts, 15 parts, 20 parts, 25 parts, 30 parts, 35 parts, 40 parts, 45 parts, or 50 parts.

All specific point values within each of the above numerical ranges can be selected, and so we will not explain them here.

Preferably, the method for preparing schizophyllan comprises:
The method includes fermenting and culturing Schizophyllan in a medium containing kudzu root, removing proteins from the fermentation culture liquid by the Sevag method, and then adding an ethanol solution to precipitate polysaccharides in the fermentation culture liquid, thereby preparing the schizophyllan.

The schizophyllan of the present invention is prepared by fermenting Schizophyllum commune in a medium containing kudzu root. The prepared schizophyllan has more significant effects in terms of oxidation resistance, cell repair, moisturizing, and immune regulation. Therefore, when schizophyllan prepared by adding kudzu root to a medium is used to prepare a composition having the above-mentioned sedative and repairing effects, it further reinforces the oxidation resistance, repair, and moisturizing effects of the composition.

In a third aspect, the present invention provides a soothing and restorative coating, the soothing and restorative coating comprising:
The composition includes a microencapsulated material and the soothing and restorative composition according to the second aspect coated inside the microencapsulated material.

The coating having a soothing and repairing effect according to the present invention is a coating of a microcapsule material on the outside of the composition having a soothing and repairing effect described in the second embodiment. After coating, the composition having a soothing and repairing effect has a stronger penetrating effect, and when used on skin tissue, it further reinforces the moisturizing, oxidation resistance, soothing and repairing effects that have already been exerted.

Preferably, the raw material for preparing the microcapsule material contains a polyglyceryl nonionic surfactant.

Preferably, the polyglyceryl nonionic surfactant comprises one or a combination of at least two of polyglycerin-10 diisostearate, polyglycerin-10 laurate, or polyglycerin-10 oleate. The combination of at least two includes a combination of polyglycerin-10 diisostearate and polyglycerin-10 laurate, a combination of polyglycerin-10 laurate or polyglycerin-10 oleate, or a combination of polyglycerin-10 diisostearate and polyglycerin-10 oleate, and any other combination form can be selected, and a description thereof will be omitted here.

In a fourth aspect, the present invention provides a method for preparing a soothing and restorative coating according to the third aspect, said method comprising the steps of:
(1) mixing a polyglyceryl nonionic surfactant with the soothing and restorative composition according to the second aspect;
and step (2) of subjecting the mixture prepared in step (1) to a coating-assisted process to prepare the coating having the soothing and repairing effect, the coating-assisted process including a high-voltage pulsed electric field-assisted process and/or an ultrasonic-assisted process.

The method for preparing a coating having a soothing and repairing effect according to the present invention can prepare the coating under the action of a coating-assisted treatment by simply mixing the active ingredient with the microcapsule material. This method has a simple preparation process, facilitating industrial expansion.

Preferably, the coating-assisted process in step (2) is a high-voltage pulsed electric field-assisted process.

Preferably, the high-voltage pulse electric field assisted treatment has an electric field strength of 0.5 to 2 kV/cm, a number of pulse treatments of 10 to 60, and a material conductivity of 2000 to 6000 μS/cm.

Specific values from the range of 0.5 to 2 kV/cm can be selected as 0.5 kV/cm, 0.7 kV/cm, 0.9 kV/cm, 1.1 kV/cm, 1.3 kV/cm, 1.5 kV/cm, 1.7 kV/cm, 1.9 kV/cm, or 2 kV/cm, etc.

Specific values from the 10 to 60 range can be selected as 10, 20, 30, 40, 50, or 60, etc.

Specific values within the range of 2000 to 6000 μS/cm can be selected as 2000 μS/cm, 3000 μS/cm, 4000 μS/cm, 5000 μS/cm, or 6000 μS/cm, etc.

All specific point values within each of the above numerical ranges can be selected, and so we will not explain them here.

In a fifth aspect, the present invention provides the use of the recombinant collagen according to the first aspect, the composition having a soothing and restorative effect according to the second aspect, or the coating having a soothing and restorative effect according to the third aspect in the preparation of a skin care product.

Compared to conventional technology, the present invention has the following beneficial effects:

1. In the present invention, the nucleotide sequence encoding the recombinant collagen for skin soothing and repairing comprises the sequence shown in SEQ ID NO:1, and the amino acid sequence encoded by SEQ ID NO:1 comprises amino acids 950-1075 of type I collagen, and amino acids 594-729 and 1193-1199 of type III collagen. The recombinant collagen has effects such as moisturizing, supplementing collagen in the skin, promoting cell proliferation, anti-oxidation, and anti-aging, and also has good tissue compatibility, skin permeability, and safety.

2. The composition having a soothing and repairing effect according to the present invention contains the recombinant collagen for soothing and repairing the skin described in the first aspect and schizophyllan, and the two components are used in combination to have a synergistic effect in terms of moisturizing, oxidation resistance, promotion of cell proliferation, and repair of skin damage.

3. The coating having a soothing and repairing effect according to the present invention is a coating of a microcapsule material on the outside of the composition having a soothing and repairing effect described in the second aspect. After coating, the composition having a soothing and repairing effect has a stronger penetrating effect, and when used on skin tissue, it further reinforces the moisturizing, oxidation resistance, soothing and repairing effects that have already been exerted.

4. The method for preparing a coating having a soothing and restorative effect according to the present invention can prepare the coating under the action of a coating-assisted treatment by simply mixing the active ingredient with the microcapsule material. This method has a simple preparation process, facilitating industrial expansion.

The technical solution of the present invention will be further described below by using specific embodiments. Those skilled in the art should understand that such examples are merely for understanding the present invention and should not be regarded as specifically limiting the present invention.

In the present invention, the implementation process, conditions, reagents, test methods, etc., are all common knowledge and common general knowledge in this field, except for those intentionally mentioned below, and there are no particular limitations. Test methods for which specific conditions are not specified in each example are usually based on conventional conditions or conditions recommended by the manufacturer.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present invention, including definitions, shall control.

The pET-28a(+) vector in the following studies was purchased from MACKLIN. Schizophyllum commune is Schizophyllum commune GIM 5.43 strain purchased from Guangdong Provincial Institute of Microbiology. BL21(DE3) chemically competent cells were purchased from MERCK, model number CMC0014-4X40UL.

[Preparation Example 1]
This preparation example provides a recombinant collagen freeze-dried powder for skin soothing and repair. The preparation method of the recombinant collagen freeze-dried powder for skin soothing and repair includes the following steps:

(1) Construction of recombinant plasmid: According to the nucleotide sequence shown in SEQ ID NO: 1 above, the pET-28a(+) vector to be applied to the E. coli host as the expression carrier was selected, and sent to a sequencing company for synthesis to prepare the recombinant collagen expression vector pET-28a-1.

(2) Construction of recombinant fermentation strain: The pET-28a-1 expression vector was transformed into E. coli (BL21(DE3)) competent cells by chemical conversion. After transformation, positive single clones from the plate colonies were selected by agarose gel electrophoresis, and the selected positive single clones were transferred and inoculated into 2 mL of LB medium and cultured at 37°C and 200 rpm for 12 hours. After culture, the bacterial solution was diluted 1-fold with 50% volumetric glycerin, dispensed into cryopreservation tubes, and cryopreserved at -80°C to prepare a recombinant collagen fermentation strain.

(3) Fermentation culture of recombinant fermentation strain: 10 μL of LB recombinant collagen fermentation culture was applied to a solid LB plate and cultured in an incubator at 37°C for 12 hours, after which a monoclone was selected and inoculated into 50 mL of LB liquid medium containing 50 μg/mL kanamycin, placed on a shaker and cultured at 37°C and 200 rpm for 14 hours. Then, it was inoculated into 200 mL of LB liquid medium containing 50 μg/mL kanamycin at a ratio of 1:100, cultured at 37°C until the culture OD was 0.6, IPTG was added to the final concentration of 1 mM, and cultured for 4 hours. Then, the cells were collected by centrifugation at 4°C and 4000 rpm for 15 minutes.

(4) Purification of recombinant collagen: The bacterial cells were ultrasonically crushed, centrifuged, the precipitate was washed with TritonX-100 washing solution, and centrifuged again to remove the supernatant. 6M guanidine hydrochloride was added to the precipitate, the pH was adjusted to 8, and the precipitate was fully dissolved. The solution was then passed through a nickel affinity chromatography resin and eluted sequentially using imidazole solutions with a concentration gradient of 20mM, 50mM, 100mM, 200mM, and 500mM. The eluate was concentrated using a protein concentration column, and the concentrate was collected and dialyzed overnight (dialysis bag, RC membrane, 14KD, flat width 44mm (Seiko F600112)). The protein concentration was then detected using a BCA kit, and the mixture was uniformly mixed with an aqueous mannitol solution at a ratio of 0.5mL of a 5% volumetric concentration aqueous mannitol solution per 5mg of protein. Thereafter, the mixture was pre-frozen at −40° C. for 10 min using a freeze dryer, and finally freeze-dried at −35° C. to prepare a freeze-dried powder of recombinant collagen.

[Preparation Example 2]
This preparation example provides schizophyllan. The method for preparing the schizophyllan is as follows.

(1) Preparation of Schizophyllum commune fermentation product: Schizophyllum commune was inoculated into the fermentation medium with a bacterial inoculum amount of 5%, and fermented under the conditions of 28°C and 160 rpm. After fermentation for 120 h, the fermentation product was centrifuged to remove the fungus body and kudzu root powder to obtain the fermentation product. The components of the fermentation medium were 0.6% kudzu root powder, 0.3% yeast extract, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, 3% glucose, and the remaining amount was water.

(2) Preparation of schizophyllan: The fermentation product was diluted by adding an equal volume of distilled water. After dilution, 0.5% volume fraction of activated carbon was added, and the mixture was decolorized in a water bath at 80°C for 40 minutes, centrifuged, and the filtrate was sieved through a 400-mesh sieve to remove the activated carbon, yielding a clear fermentation liquid. Proteins were eluted from the fermentation liquid five times using the Sevag method. Polysaccharides in the fermentation liquid were then precipitated with three volumes of 95% ethanol. After washing, distilled water was added to redissolve the product, and the mixture was dialyzed against running water and distilled water for 24 hours. The dialyzed liquid was concentrated and freeze-dried to prepare the schizophyllan.

[Preparation Example 3]
This preparation example provides schizophyllan. The preparation method of schizophyllan is different from that of Preparation Example 2 only in that the fermentation medium does not contain pueraria root powder, and the other operations are all the same as those of Preparation Example 2.

[Example 1]
This example provides a coating with soothing and restorative effects. The method for preparing said coating is as follows:

(1) 1 part of polyglycerin-10 diisostearate, 0.5 parts of polyglycerin-10 laurate, 0.2 parts of the recombinant collagen prepared in Preparation Example 1, 0.3 parts of the schizophyllan prepared in Preparation Example 2, and 15 parts of glycerin were mixed and water was added until the total volume reached 100 parts.

(2) The mixed solution was subjected to high-voltage pulse electric field assisted treatment. The parameters were an electric field strength of 0.5 kV/cm, the number of pulse treatments was 10, and the material conductivity was 2000 μS/cm. After the treatment, a coating having the above-mentioned calming and repairing effects was obtained.

[Example 2]
This example provides a coating with soothing and restorative effects. The method for preparing said coating is as follows:

(1) 1 part of polyglycerin-10 diisostearate, 0.5 parts of polyglycerin-10 laurate, 0.01 parts of recombinant collagen prepared in Preparation Example 1, 5 parts of schizophyllan prepared in Preparation Example 2, and 50 parts of glycerin were mixed and water was added until the total volume reached 100 parts.

(2) The mixed solution was subjected to high-voltage pulse electric field assisted treatment. The parameters were electric field strength of 0.5 kV/cm, the number of pulse treatments was 20, and the conductivity of the material was 600.
After the treatment, a coating having the above-mentioned soothing and repairing effect was obtained.

[Example 3]
This example provides a coating with soothing and restorative effects. The method for preparing said coating is as follows:

(1) 1 part of polyglycerin-10 diisostearate, 0.5 parts of polyglycerin-10 laurate, 0.5 parts of recombinant collagen prepared in Preparation Example 1, 0.1 parts of schizophyllan prepared in Preparation Example 2, and 5 parts of glycerin were mixed and water was added until the total volume reached 100 parts.

(2) A high-voltage pulsed electric field assisted treatment was performed on the mixed solution. The parameters were an electric field strength of 2 kV/cm, the number of pulse treatments was 60, and the material conductivity was 2000 μS/cm. After the treatment, a coating having the above-mentioned calming and repairing effects was obtained.

[Example 4]
This embodiment provides a coating having soothing and restorative effects. The preparation method of the coating is different from that of Example 1 only in that the raw materials do not contain glycerin, and the mass parts of glycerin are allocated to recombinant collagen and schizophyllan in a proportion, but the other operations are all the same as those of Example 1.

[Example 5]
This example provides a coating having soothing and restorative effects. The preparation method of the coating is different from that of Example 1 only in that the schizophyllan prepared in Preparation Example 2 is replaced with the schizophyllan prepared in Preparation Example 3, and the other operations are all the same as those of Example 1.

[Example 6]
This example provides a coating with soothing and repairing effects, the preparation method of said coating is different from that of Example 1 only in that 0.2 parts of the recombinant collagen prepared in Preparation Example 1 and 0.3 parts of the schizophyllan prepared in Preparation Example 2 are replaced with 0.5 parts of a mixture of recombinant collagen and schizophyllan (the ratio of recombinant collagen to schizophyllan in the mixture is 0.01:5.1), and all other operations are the same as those of Example 1.

[Example 7]
This example provides a coating with soothing and repairing effects, the preparation method of said coating is different from that of Example 1 only in that 0.2 parts of the recombinant collagen prepared in Preparation Example 1 and 0.3 parts of the schizophyllan prepared in Preparation Example 2 are replaced with 0.5 parts of a mixture of recombinant collagen and schizophyllan (the ratio of recombinant collagen to schizophyllan in the mixture is 0.6:0.05), and all other operations are the same as those of Example 1.

[Comparative Example 1]
This comparative example provides a coating having soothing and restorative effects. The preparation method of the coating is different from that of Example 1 only in that the raw materials do not contain recombinant collagen and the mass part of recombinant collagen is allocated to schizophyllan, but the other operations are all the same as those of Example 1.

[Comparative Example 2]
This comparative example provides a coating having soothing and repairing effects. The preparation method of the coating is different from that of Example 1 only in that the preparation raw material does not contain schizophyllan, and the mass part of schizophyllan is allocated to recombinant collagen, but the other operations are all the same as those of Example 1.

[Comparative Example 3]
This comparative example provides a coating having a soothing and restorative effect. The preparation method of the coating is different from that of Example 1 only in that the preparation raw materials do not contain schizophyllan and recombinant collagen, and the parts by mass of schizophyllan and recombinant collagen are allocated to glycerin, but the other operations are all the same as those of Example 1.

[Test Example 1]
In this test example, the oxidation resistance of the coating having the soothing and repairing effect of each of the Examples and Comparative Examples was evaluated. The specific evaluation method was as follows.

The oxidation resistance of the examples and comparative examples was evaluated using the DPPH elimination efficiency. The specific evaluation method is as follows. 0.5 g of the coated body was immersed in 4 mL of DPPH (100 μM, in 99 wt % ethanol) solution (a solution containing only DPPH was used as the control group) and incubated in the dark for 60 min. The absorbance value of the mixed solution at 517 nm was then measured using a UV-Vis spectrophotometer. The DPPH elimination rate was calculated using the formula: DPPH elimination rate = (absorbance of control group - absorbance of test group) / absorbance of control group x 100%, and the results are shown in Table 1.

As can be seen from the data in Table 1, in Comparative Example 1 and Comparative Example 2, compared with Example 1, the preparation raw materials lack one of recombinant collagen and schizophyllan, respectively, and the DPPH elimination rate is lower than that of Example 1, so it was found that the two components, schizophyllan and recombinant collagen, work together to have an unexpected synergistic effect in elimination of DPPH. In Example 5, compared with Example 1, the fermentation medium contains kudzu root powder, and the DPPH elimination rate is lower than that of Example 1, so it was found that the addition of kudzu root powder reinforces the oxidation resistance of the schizophyllan product. In comparison with Example 1, in Examples 6 and 7, the ratio of mass parts of recombinant collagen to schizophyllan is not within the range of (0.01-0.5):(0.1-5), and the DPPH elimination rate is lower than that of Example 1. This indicates that when the ratio of mass parts of recombinant collagen to schizophyllan is within the range of (0.01-0.5):(0.1-5), the oxidation resistance performance is stronger when these two components are used in combination.

[Test Example 2]
In this test example, the moisturizing effect of the covering having soothing and repairing effects of each of the Examples and Comparative Examples was evaluated. The specific evaluation method was as follows.

One hundred volunteers with healthy skin aged 18-40 years were selected, half of whom were male and half were randomly divided into 10 groups, and the coverings having soothing and repairing effects according to the examples and comparative examples were used. After the volunteers washed their arms, they evenly applied the coverings to a test area of 2 cm in diameter on the inside of their arms at a dose of 2 mg/cm, and used them once a day for 30 consecutive days. The skin moisture content of the volunteers was measured before and after use using a skin test device (MC750, CK, Germany).

The skin moisture content of each subject was measured before use and after 30 days of continuous use, with each data point measured three times, and the average value for each group was calculated. The rate of change in skin moisture content was calculated according to the formula: rate of change in skin moisture content = (skin moisture content after 30 days of use - skin moisture content before use) / skin moisture content before use x 100%, and the measurement results are shown in Table 2.

As can be seen from the data in Table 2, in Comparative Example 1 and Comparative Example 2, the preparation raw materials lack recombinant collagen and one type of schizophyllan, respectively, compared with Example 1, and the change rate of the skin moisture content is lower than that of Example 1, so it was found that the two components of schizophyllan and recombinant collagen work together to reinforce the moisturizing effect of the cover. In Example 4 and Comparative Example 3, the preparation raw materials of the cover having the soothing and repairing effect lack glycerin or a combination of recombinant collagen and schizophyllan, respectively, compared with Example 1, and the change rate of the skin moisture content is both lower than that of Example 1, so it was found that the combination of glycerin, recombinant collagen, and schizophyllan has an unexpected synergistic effect in improving the moisturizing effect of the skin.

[Test Example 3]
In this test example, the cell proliferation effect of the recombinant collagen of Preparation Example 1 and the coatings having soothing and repairing effects of the Examples and Comparative Examples was evaluated. The specific evaluation method was as follows.

HaCaT cells (human immortalized keratinocytes) in good growth condition were taken, seeded on a 96-well plate, and cultured for 17 hours, then replaced with serum-free basic medium. After 7 hours of culture, the old medium was discarded and a medium containing 1% recombinant collagen aqueous solution or the coated body solution prepared in each Example or Comparative Example (the coated body concentration in the medium was 2 μg/mL) was added. The blank was the same as the test group except that the medium did not contain the coated body. After 48 hours of culture, the cell viability was detected by the CCK8 method (630 nm was used as the reference wavelength, and the absorbance value was measured at a wavelength of 450 nm), and measurements were made three times per data point, and the average value of each group was calculated. The relative cell proliferation rate was calculated according to the formula: relative cell proliferation rate = (absorbance of test group - absorbance of blank) / blank absorbance x 100%.

As can be seen from the data in Table 3, the recombinant collagen of Preparation Example 1 has an excellent cell proliferation promoting effect. In addition, in Comparative Examples 1 and 2, compared to Example 1, the preparation raw materials lack one of recombinant collagen and schizophyllan, respectively, and the relative cell proliferation rate is lower than that of Example 1, so it was found that the two components schizophyllan and recombinant collagen work together to have an unexpected synergistic effect in terms of promoting cell proliferation. In Example 5, compared to Example 1, the fermentation medium contains kudzu root powder, and the relative cell proliferation rate is lower than that of Example 1, so it was found that the addition of kudzu root powder reinforces the cell proliferation promoting ability of schizophyllan.

[Test Example 4]
In this test example, the skin soothing and repair properties of the coverings having soothing and repair effects of each of the Examples and Comparative Examples were evaluated. The specific evaluation method was as follows.

(1) 66 guinea pigs were taken, half of which were male and half of which were female. One day before the test, the hair on the right hind leg was shaved, leaving a hairless area of approximately 2 ^cm2 . The guinea pigs were randomly divided into 11 groups, each of which contained 6 guinea pigs, and were designated as Example groups 1 to 7, Comparative examples 1 to 3, and blank, respectively.

(2) For the guinea pigs in the Example 1-7 groups, the Comparative Example 1-3 groups, and the blank, 0.05 g of the coating solution prepared in each Example or Comparative Example was applied to the hair-removed area once per day for 2 consecutive days, and an equal amount of deionized water was applied to the blank.

(3) On the third day of the test, 0.01% (w/v), 0.02% (w/v), 0.03% (w/v), 0.04% (w/v), 0.05% (w/v), 0.06% (w/v), 0.07% (w/v), 0.08% (w/v), 0.09% (w/v) and 0.1% (w/v) histamine phosphate solutions were prepared in saline. The depilated area of the right hind paw of the guinea pig was scratched with coarse sandpaper, and the scratch area was about 1 ^cm2 . In addition, the corresponding coating solution was applied locally once, with an application amount of 0.05 g. For the blank, an equal amount of deionized water was applied, and 10 min later, 0.05 mL of 0.01% (w/v) histamine phosphate solution was dripped onto the scratched area. Observation was performed to see whether the guinea pig turned around and licked its right hind paw within 3 minutes. If not, 0.05 mL of 0.02% (w/v) histamine phosphate solution was continuously dripped onto the scratched area, and observation was performed to see whether the guinea pig turned around and licked its right hind paw within 3 minutes. In this way, the concentration was increased according to the above gradient every 3 minutes, and 0.05 mL of histamine phosphate solution was dripped every time until the guinea pig licked its right hind paw within 3 minutes. The cumulative total amount of histamine phosphate dripped at this time was taken as the itch threshold (μg), and the average value of the itch threshold and the difference rate of the itch threshold of the guinea pig in the same group were calculated, and the difference rate of the itch threshold=(itch threshold of the test group-itch threshold of the blank)/itch threshold of the blank×100%, and the results are shown in Table 4.

As can be seen from the data in Table 4, the higher the itch threshold, the stronger the corresponding skin barrier action, i.e., the soothing and repairing ability of the covering solution. In Example 4 and Comparative Example 3, compared to Example 1, the preparation raw materials for the covering having the soothing and repairing effect lacked glycerin or a combination of recombinant collagen and schizophyllan, respectively, and the difference rates of the itch threshold were all lower than those of Example 1, so it was found that the combination of glycerin, recombinant collagen, and schizophyllan had an unexpected synergistic effect in terms of soothing and repairing the skin. In Comparative Example 1 and Comparative Example 2, compared to Example 1, the preparation raw materials lacked one of recombinant collagen and schizophyllan, respectively, and the difference rates of the itch threshold were all lower than those of Example 1, so it was found that the combination of the two components, schizophyllan and recombinant collagen, could improve the anti-itching and repairing action of the covering. In Example 5, compared to Example 1, the fermentation medium contains kudzu root powder, and the difference rate of the itching threshold is lower than that of Example 1, so it was found that the addition of kudzu root powder can improve the oxidation resistance of the schizophyllan product and improve the barrier function of the skin. In Examples 6 and 7, compared to Example 1, the ratio of mass parts of recombinant collagen to schizophyllan is not in the range of (0.01-0.5):(0.1-5), and the difference rate of the itching threshold is lower than that of Example 1, so it was found that when the ratio of mass parts of recombinant collagen to schizophyllan is in the range of (0.01-0.5):(0.1-5), the use of the two components in combination can enhance the soothing and repairing function of the skin.

[Test Example 5]
The safety evaluation was carried out for the coating with soothing and repairing effects prepared in the examples. The specific evaluation method is as follows. The test was carried out according to the human skin patch test method in the human safety detection method in Chapter 7 of the "Cosmetics Safety Technical Standards 2015 Edition". (1) Female volunteers aged 18-60 years were selected who met the study requirements, with the exclusion criteria being: (a) those who had used antihistamines in the last week or immunosuppressants within the last month; (b) those who had used any anti-inflammatory drugs on the test site within the last two months; (c) subjects with inflammatory skin diseases that were not clinically cured; (d) insulin-dependent diabetes mellitus patients; (e) treated asthma or other chronic respiratory disease patients; (f) those who had undergone anti-cancer chemotherapy within the last six months; (g) immunodeficiency or autoimmune disease patients; (h) lactating or pregnant women; (i) those who had undergone bilateral mastectomy and bilateral axillary lymph node removal; (n) those who judged that the test results would be affected by scarring, pigmentation, atrophy, erythema or other defects on the skin test site; (o) those who had participated in other clinical trial studies; (p) those with extremely sensitive constitutions; and (q) non-volunteers or those who were unable to complete the prescribed content according to the study requirements. (2) Each example corresponds to one group, and there are 10 volunteers in each group. A qualified patch test device with an area not exceeding 50 ^mm2 and a depth of about 1 mm is selected, and the coating solution prepared in each example is put into the cell of the patch test device, and the amount used is about 0.025 g. Nothing is put into the control well as a blank. The low sensitizing tape for the patch test device containing the test sample is attached to the back or forearm of the subject, and is lightly pressed with the palm of the hand to make it evenly attached to the skin, and is maintained for 24 hours. (3) After removing the test sample patch test device, the skin reaction is observed 30 minutes (after the indentation disappears), 24 hours and 48 hours according to the standards shown in Table 5. Evaluation is performed according to the standards shown in Table 5.

As can be seen from the results, the product prepared according to the present invention was safe, non-irritating, and had higher usage value.

The present invention describes the preparation process of the present invention through the above examples, but the applicant declares that the present invention is not limited to the above examples, that is, the present invention does not have to be carried out according to the above examples. It is clear to those skilled in the art that any improvements to the present invention, equivalent replacement of each raw material of the product of the present invention, addition of auxiliary ingredients, selection of specific embodiments, etc., are all within the protection scope and disclosure scope of the present invention.

Although the preferred embodiments of the present invention have been described in detail above, the present invention is not limited to the specific details of the above embodiments. Within the technical concept of the present invention, a number of simple modifications can be made to the technical solution of the present invention, and all of these simple modifications fall within the protection scope of the present invention.

The specific technical features described in the above specific embodiments can be combined in any suitable manner if not contradictory. In order to avoid unnecessary repetition, the present invention does not separately describe various possible combination forms.

Claims (15)

Recombinant collagen for skin soothing and repair; and schizophyllan.
The nucleotide sequence encoding the recombinant collagen for skin soothing and repair is SEQ ID NO:
The sequence shown in ID NO: 1
A composition having a soothing and restorative effect. The composition having a soothing and repairing effect comprises, by weight, 0.01 to 0.5 parts of recombinant collagen for soothing and repairing the skin, and 0.1 to 5 parts of schizophyllan.
A composition having a soothing and restorative effect according to claim 1 . The soothing and restorative composition further comprises a moisturizer.
A composition having a soothing and restorative effect according to claim 1 . The moisturizing agent includes any one or a combination of at least two of glycerin, 1,3-butanediol, sodium hyaluronate, sodium acetylated hyaluronate, hydrolyzed sodium hyaluronate, or ceramide 3.
A composition having a soothing and restorative effect according to claim 3 . The moisturizer is glycerin.
A composition having a soothing and restorative effect as claimed in claim 4 . The soothing and restorative composition comprises, in parts by weight:
A skin soothing and repairing composition, comprising 0.01 to 0.5 parts of recombinant collagen, 0.1 to 5 parts of schizophyllan, and 5 to 50 parts of glycerin.
A composition having a soothing and restorative effect according to claim 1 . The method for preparing schizophyllan comprises the steps of:
The method includes fermenting Schizophyllum commune in a medium containing kudzu root, removing proteins from the fermentation culture liquid by the Sevag method, adding an ethanol solution to precipitate polysaccharides in the fermentation culture liquid, and centrifuging the mixture to prepare the schizophyllan.
A composition having a soothing and restorative effect according to claim 1 . A composition having a soothing and restorative effect according to any one of claims 1 to 7 , comprising a microcapsule material and the composition is coated on the inside of the microcapsule material.
A coating having a soothing and restorative effect. The preparation raw material of the microencapsulation material includes a polyglyceryl nonionic surfactant;
9. The soothing and restorative covering according to claim 8 . The polyglyceryl nonionic surfactant includes any one or a combination of at least two of polyglycerin-10 diisostearate, polyglycerin-10 laurate, or polyglycerin-10 oleate.
10. The soothing and restorative covering according to claim 9 . (1) mixing a polyglyceryl nonionic surfactant with a soothing and restorative composition according to any one of claims 1 to 7 ;
Step (2) of subjecting the mixture prepared in step (1) to a coating-assisted treatment to prepare the coating having the soothing and repairing effect, the coating-assisted treatment including a high-voltage pulsed electric field-assisted treatment and/or an ultrasonic-assisted treatment;
9. A method for preparing a coating having a soothing and restorative effect according to claim 8 . The coating assisted process according to step (2) is a high voltage pulse electric field assisted process;
A method for preparing a coating having a soothing and restorative effect according to claim 11 . The high voltage pulse electric field assisted treatment has an electric field strength of 0.5-2 kV/cm, a pulse treatment number of 10-60, and a material conductivity of 2000-6000 μS/cm;
A method for preparing a coating having a soothing and restorative effect according to claim 11 . 13. Use of the soothing and restorative composition according to claim 1 in the preparation of a skin care product. 9. Use of the soothing and restorative coating according to claim 8 in the preparation of a skin care product.