CN112336677A

CN112336677A - Collagen moisturizing sunscreen isolation cream and preparation method thereof

Info

Publication number: CN112336677A
Application number: CN202011321716.3A
Authority: CN
Inventors: 肖暾
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-11-23
Filing date: 2020-11-23
Publication date: 2021-02-09

Abstract

The invention discloses a collagen moisturizing sunscreen isolation cream and a preparation method thereof. According to the invention, the recombinant gene TAT-Hlcollagen is transcribed into the pichia pastoris body by means of genetic engineering, so that the cell-penetrating peptide-human-like collagen complex is expressed in a large amount in the pichia pastoris at the same time, can be used in the fields of skin injury treatment, food additives, tissue engineering, cosmetics and the like, and has good social and economic values. The cell-penetrating peptide TAT can carry collagen to permeate cell membranes, so that the collagen directly reaches the interior of cells, and the effects of moisturizing and ageing resistance are really exerted. Tea leaves are added into pichia pastoris fermentation liquor, and low-temperature fermentation is carried out, so that pichia pastoris containing tea polyphenol can be obtained, the tea polyphenol fermentation liquor has super-strong oxidation resistance, and radiation protection and aging resistance, and the moisture-preserving sun-screening cream added into collagen can achieve a good sun-screening effect.

Description

Collagen moisturizing sunscreen isolation cream and preparation method thereof

Technical Field

The invention relates to the technical field of cosmetics, in particular to a collagen moisturizing sunscreen isolation cream and a preparation method thereof.

Background

The fermentation production of human-like collagen by using a yeast expression system is reported more at home and abroad, related researches have been started in the 80 s of the 20 th century, and the main expression bacteria of the yeast expression system are saccharomyces cerevisiae, hansenula, pichia pastoris and the like. In 1999, the dutch scholars expressed type I collagen by using Hansenula and Pichia pastoris and found that the Pichia pastoris can not secrete recombinant protein, and the Hansenula can express and secrete recombinant protein.

The collagen contains a large amount of polar groups, is a moisturizing factor and has the function of preventing tyrosine in the skin from being converted into melanin, so the collagen has the functions of purely natural moisturizing, whitening, crease resistance, freckle removal and the like, and can be widely applied to beauty products. The human-like collagen has the same root homology with human skin cells, has gene affinity, and has 108 times of the effect of promoting the growth of epithelial cells and fibroblasts compared with animal collagen. So that it is easily absorbed when the skin is smeared. The collagen fiber generated by the human-like collagen can maintain the elasticity and luster of the skin and delay the aging of the skin, and is one of ideal cosmetic raw materials.

However, in cosmetics, the molecular weight of pure nutritional skin care raw materials is generally required to be below 2KD, and the active collagen is generally above 3KD, so that a collagen moisturizing sunscreen cream which can keep the integrity of the collagen and can ensure the collagen to enter the skin and a preparation method thereof are necessary.

Disclosure of Invention

The invention aims to provide a collagen moisturizing sunscreen isolation cream and a preparation method thereof, and aims to solve the problems in the background technology.

In order to solve the technical problems, the invention provides the following technical scheme: a collagen moisturizing sunscreen cream comprises 5-10 parts of yeast fermentation extract, 2-6 parts of zinc oxide, 1-5 parts of rose essential oil, 0.5-2 parts of honey, 3-8 parts of glycerol, 5-10 parts of hyaluronic acid, 0.5-2 parts of thickening agent and 2-8 parts of water.

According to the technical scheme, the yeast fermentation extract comprises 8 parts by weight, 3 parts by weight of zinc oxide, 2 parts by weight of rose essential oil, 0.5 part by weight of honey, 8 parts by weight of glycerol, 6 parts by weight of hyaluronic acid, 1 part by weight of thickening agent and 5 parts by weight of water.

According to the technical scheme, the yeast fermentation extract is obtained by fermenting gene engineering yeast pichia pastoris.

According to the technical scheme, the pichia pastoris plasmid contains a recombinant gene of a cell-penetrating peptide TAT-human-like collagen (hlcollagen), histidine purification tags are respectively introduced into the 5 'end and the 3' end of the sequence, and the designed gene sequence has the full length of 828 bp. Membrane-penetrating peptide TAT gene and human-like collagen recombinant gene TAT-Hlcollagen are amplified by PCR technology, EcoRI enzyme-cutting sites and BamHI enzyme-cutting sites are respectively introduced into two ends of the membrane-penetrating peptide TAT gene and the human-like collagen recombinant gene TAT-Hlcollagen, and the membrane-penetrating peptide TAT gene and the human-like collagen recombinant gene TAT-Hlcollagen are connected to a pichia pastoris expression vector pGAPH alpha A after double enzyme cutting to obtain a recombinant expression vector pGAPH alpha A-TAT-H.

According to the technical scheme, PCR detection is carried out on the pichia pastoris: according to the designed pair of TAT-Hlcollagen gene primers, a band which is the same as the gene primers appears in electrophoresis detection, but the band is not amplified in the untransformed Pichia pastoris, so that the TAT-Hlcollagen recombinant gene is integrated into the genome of the Pichia pastoris, and the positive Pichia pastoris is obtained.

According to the technical scheme, the positive pichia pastoris strain is inoculated into a liquid culture medium containing 20 g of peptone, 10 g of yeast powder, 13.4 g of choline YNB, 10 ml of glycerol, 10 ml of methanol, 0.1 mol of potassium phosphate buffer solution and 0.4 mg of biotin per liter, shaken in a flask, cultured at the temperature of 28 ℃ and at the rpm of 250 for 18-24 hours, centrifuged and collected;

according to the technical scheme, the thalli is re-suspended and inoculated by using an amplification culture solution, and each liter of the amplification culture solution contains 20 g of peptone, 10 g of yeast powder, 13.4 g of choline YNB, 21.6 ml of methanol, 0.1 mol of potassium phosphate buffer solution and 0.4 mg of biotin; shaking-culturing at 25-35 deg.C and 100-300 rpm for 3-10D; during the fermentation, methanol is supplemented every 12h to induce and maintain the methanol concentration of 1.8-2.5% of the volume ratio.

According to the technical scheme, tea leaves are added into the pichia pastoris fermentation liquor for continuous fermentation, methanol is not added, and 2-3D fermentation is carried out at the low temperature of 20 ℃ to obtain the pichia pastoris containing tea polyphenol.

According to the technical scheme, the tea is one of oolong tea, Biluochun tea, Longjing tea, Tieguanyin tea and Wuyi rock tea.

A preparation method of a collagen moisturizing sunscreen cream comprises the following specific steps:

the method comprises the following steps: filtering the yeast fermentation liquid to remove tea, vacuum drying, and ultramicro grinding at 5-10 deg.C to obtain yeast fermentation extract;

step two: stirring yeast fermentation extract, rose essential oil, zinc oxide and water at 30 deg.C and 300rpm for 20min to obtain phase A

Step three: stirring honey, glycerol and hyaluronic acid at 80-90 ℃ and 200rpm for 60min to obtain phase B;

step four: and stirring the phase A and the phase B with a thickening agent at 35 ℃ and 200rpm for 2-3h to obtain the collagen moisturizing sun-screening cream.

TAT is a Cell Penetrating Peptide (CPPs), a short peptide that is positively charged under physiological conditions and has the ability to penetrate cell membranes. The CPPs have strong electropositivity so that the CPPs can form a physical complex with negatively charged biomacromolecules through electrostatic interaction, and the uptake mechanism of the CPPs for improving the cell-entry capacity of the CPPs relates to various cell-entry modes such as macropinocytosis and cell phagocytosis of clathrin. When the CPPs carry macromolecular substances, the CPPs are mainly inserted into cells in a macropinocytosis mode, and the CPPs with positive charges and the glycosaminoglycan with negative charges on the surface of cell membranes are electrically attracted to each other and then are inserted into the cells in an endocytosis mode.

The human-like collagen has the same root homology with human skin cells, has gene affinity, and has 108 times of the effect of promoting the growth of epithelial cells and fibroblasts compared with animal collagen. The recombinant gene TAT-Hlcollagen is transcribed into a pichia pastoris body through a genetic engineering means, so that a large amount of cell-penetrating peptide-human-like collagen complexes are simultaneously expressed in the pichia pastoris, a large amount of cell-penetrating peptide composite collagen can be obtained, the cost for obtaining the collagen is much lower than that for extracting the collagen from an animal body, the animal is protected, a large amount of collagen can be obtained, the recombinant gene TAT-Hlcollagen can be used for the fields of skin injury treatment, food additives, tissue engineering, cosmetics and the like, and the recombinant gene TAT-Hlcollagen has good social and economic values.

The cell-penetrating peptide TAT can carry collagen to permeate cell membranes and reach the inside of cells directly, so that the skin permeability of macromolecular collagen is solved, the macromolecular collagen can permeate epidermal cells, and the effects of moisturizing and ageing resistance are really exerted.

Tea leaves are added into pichia pastoris fermentation liquor, and 2-3D fermentation is carried out at low temperature, so that tea polyphenol-containing pichia pastoris can be obtained, wherein the tea polyphenol has oxidation resistance which is higher than that of vitamin E10, and specifically, the tea polyphenol-containing pichia pastoris has various physiological activities of radiation protection, aging resistance, bacteriostasis, enzyme inhibition and the like, and the collagen moisturizing sunscreen cream can achieve good sunscreen effect when added into the collagen moisturizing sunscreen cream.

Compared with the prior art, the invention has the following beneficial effects: in the invention, the raw materials are mixed,

(1) by means of genetic engineering, the recombinant gene TAT-Hlcollagen is transcribed into a pichia pastoris body, so that a large amount of cell-penetrating peptide-human-like collagen complexes are simultaneously expressed in the pichia pastoris, a large amount of cell-penetrating peptide composite collagen can be obtained, and the recombinant gene TAT-Hlcollagen can be used in the fields of skin injury treatment, food additives, tissue engineering, cosmetics and the like and has good social and economic values;

(2) the cell-penetrating peptide TAT can carry collagen to permeate cell membranes, so that the collagen directly reaches the inside of cells, the problem that the collagen with the KD of more than 2KD can not enter the inside of skin is solved, the obtained human-like collagen can permeate the epidermis to the inside of the cells even if being coated on the skin simply, and the effects of water replenishing, moisture preserving and anti-aging are really exerted;

(3) tea leaves are added into pichia pastoris fermentation liquor, and low-temperature fermentation is carried out to obtain pichia pastoris containing tea polyphenol, wherein the tea polyphenol has super oxidation resistance, radiation protection and aging resistance, and the moisturizing sun-screening cream added into collagen can achieve a good sun-screening effect.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides the technical scheme that: a collagen moisturizing sunscreen cream comprises 5-10 parts of yeast fermentation extract, 2-6 parts of zinc oxide, 1-5 parts of rose essential oil, 0.5-2 parts of honey, 3-8 parts of glycerol, 5-10 parts of hyaluronic acid, 0.5-2 parts of thickening agent and 2-8 parts of water.

Example 1

A collagen moisturizing sunscreen cream comprises 9 parts of yeast fermentation extract, 5 parts of zinc oxide, 3 parts of rose essential oil, 1.5 parts of honey, 5 parts of glycerol, 6 parts of hyaluronic acid, 1.2 parts of thickening agent and 7 parts of water.

according to the technical scheme, the thalli is re-suspended and inoculated by using an amplification culture solution, and each liter of the amplification culture solution contains 20 g of peptone, 10 g of yeast powder, 13.4 g of choline YNB, 21.6 ml of methanol, 0.1 mol of potassium phosphate buffer solution and 0.4 mg of biotin; shaking-culturing at 28 deg.C and 180rpm for 3-10D; methanol was supplemented every 12h during the fermentation to induce maintenance of a 2.0% methanol concentration by volume.

According to the technical scheme, tea leaves are added into the pichia pastoris fermentation liquor for continuous fermentation, methanol is not added, and 3D fermentation is carried out at the low temperature of 20 ℃ to obtain the pichia pastoris containing tea polyphenol.

step two: stirring yeast fermentation extract, rose essential oil, zinc oxide and water at 30 deg.C and 150rpm for 20min to obtain phase A

Step three: stirring honey, glycerol and hyaluronic acid at 90 deg.C and 200rpm for 60min to obtain phase B;

step four: and stirring the phase A and the phase B with a thickening agent at 35 ℃ and 200rpm for 2.5h to obtain the collagen moisturizing sun-screening cream.

Example 2

A collagen moisturizing sunscreen cream comprises 6 parts of yeast fermentation extract, 4 parts of zinc oxide, 3 parts of rose essential oil, 0.9 part of honey, 6 parts of glycerol, 8 parts of hyaluronic acid, 0.8 part of thickening agent and 6 parts of water.

according to the technical scheme, the thalli is re-suspended and inoculated by using an amplification culture solution, and each liter of the amplification culture solution contains 20 g of peptone, 10 g of yeast powder, 13.4 g of choline YNB, 21.6 ml of methanol, 0.1 mol of potassium phosphate buffer solution and 0.4 mg of biotin; shaking-culturing at 30 deg.C and 250rpm for 3-10D; methanol was supplemented every 12h during the fermentation to induce maintenance of a 2.1% methanol concentration by volume.

step two: stirring yeast fermentation extract, rose essential oil, zinc oxide and water at 30 deg.C and 250rpm for 20min to obtain phase A

Step three: stirring honey, glycerol and hyaluronic acid at 90 deg.C and 180rpm for 60min to obtain phase B;

step four: and stirring the phase A and the phase B with a thickening agent at 35 ℃ and 150rpm for 3h to obtain the collagen moisturizing sun-screening cream.

Test 1 measurement of the content of the cell-penetrating peptide-human-like collagen Complex

Taking the freeze-dried pichia pastoris fermentation extract, and carrying out redissolution precipitation by using an ammonium sulfate solution to obtain crude protein. The purity of the final human-like collagen is about 92% by SDS-PAGE and QuantityOne software analysis.

The expression quantity of the human-like collagen is about 3.19g/L and the molecular weight of the target protein is about 34.94kD through protein content calculation according to SDS-PAGE and Westernblotting verification.

Experiment 2, human clinical moisturizing model test moisturizing ability

2.1 test Instrument

Skin elasticity tester, skin moisture content test probe and percutaneous water loss test probe

2.2 test principles and Primary parameters

The test is divided into two parts, one part is used for calibrating the skin hydration rate of the moisture content of the stratum corneum of the skin, the test principle adopts the Corneometer method according to the moisture content of the skin, the subtle change of the capacitance of the skin is sensed and recorded by a sensitive capacitor, and the moisture content of the skin is changed into a detection value through the change of an electric signal and the conversion of a computer chip. The measurement of the moisture content of the skin by capacitance is a stable and reliable method, because the skin of the tested part is hardly in abnormal contact with the test probe, basically no current flows through the skin of the tested part, and the detection result is basically not influenced by the polarization effect and the ionic conductivity. Inertia cannot be shown in the process of establishing balance between the probe for measurement and water in the skin, so that the rapid measurement can be realized, and the interference of skin activity on the test result can be solved.

Skin hydration rate (MMV) involves the main technical parameters:

1) area measurement: 49mm²；

2) And (3) testing pressure: 1.1-1.5N;

3) precision: plus or minus 3 percent;

4) numerical ranges: 0 to 130;

5) weight: 41 g.

One part is the percutaneous water loss (TEWL) test, which is based on the following basic principle:

fick's law of diffusion: dm/dt ═ DAdp/dx

In the formula: a-area (m)²) (ii) a M-diffusion amount of moisture (g); t-time (h); d-diffusion constant (0.0877 g/mgmmHg); p-vapor pressure (mmHg); x-the distance (m) of the skin surface measurement point.

The diffusion flow dm/dt refers to the amount of water delivered per square meter of area over a period of time, the flow of delivered water being proportional to the area over which it is delivered and the change in concentration dc/dx per unit length.

D is the diffusion coefficient of water vapor when air is the transport medium.

The main technical parameters of the percutaneous water loss (TEWL) are:

1) the size of the probe is as follows: phi 1 is multiplied by 2 cm;

2) weight of the probe: 90g of the total weight of the mixture;

3) measurement range: temperature. + -. 0.01 ℃ and humidity. + -. 0.01% RH.

2.3 Experimental environmental requirements

Indoor, no strong light or direct sunlight exists, the room temperature is 20 +/-2 ℃, and the indoor relative humidity is 40-60%.

2.4 Subjects claim

The age is 18-60 years, female is preferred. There is no immune disease. Has no allergic skin diseases. There is also no history of allergy to skin care products. There is no systemic application of hormonal drugs or immunotherapeutic drugs for one month. Other skin clinical trials must not be attended at the same time. The number of subjects should not be less than 30.

Before the test, the test part of the volunteer is required not to use any cosmetics, and before the test, the test part is wiped clean, and the test part is required to relax and rest for more than 30min in a room with the temperature of 18-22 ℃ and the relative humidity of 40-60%.

2.5 test procedure

The tested part can not use cosmetics or external medicines within 3 days. Before testing, the tested person must clean the inner sides of the forearms of both hands with clear water and then wipe the forearms clean with dry and clean non-woven fabrics. After cleaning, marking the measuring parts on the inner sides of the forearms of the two hands of the tested person. The forearm should be exposed to rest and relaxed in a conditioned environment for at least 30min before testing.

When an immediate moisturizing effect evaluation experiment is carried out, a plurality of 4 multiplied by 4cm are marked on the inner side of the forearm of a subject²Test areas of size, each area being separated by a distance of at least 2 cm. The test areas of the samples were randomly and irregularly selected. Firstly, measuring blank values 30min after cleaning of each test area, detecting each test part by using an instrument, repeating the detection for 5 times respectively, and obtaining an average value which is a basic value. Then according to per cm²The sample dosage is 2.0 +/-0.1 mg sample/cm²The latex finger cot is worn to uniformly coat the sample in the selected tested part (the non-woven fabric mask is directly and softly pasted with the membrane cloth soaked with liquid with the same size). After smearing, the water content of the horny layer and the water loss of the epidermis in each test area are respectively measured for 0.5h, 1h, 2h, 3h, 4h, 5h and 6h, the detection should be repeated for more than 5 times, and the results are averaged.

The skin moisture content increase rate calculation formula is as follows:

skin moisture content increase rate%

In the formula: MMV0 — skin MMV before application; MMVt — time period t after application skin MMV.

The water content increase rate of skin%	0.5h	1h	2h	3h	4h	5h	6h
								Blank control	100	100	100	100	100	100	100
Cream without yeast fermentation extract	104	110	114	50	108	105	103
								Cream containing yeast fermentation extract	110	121	128	129	130	128	125

TABLE 2 skin moisture content growth rate

According to the test results, the collagen moisturizing sunscreen isolation cream has a good moisturizing effect.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A collagen moisturizing sunscreen cream comprises 5-10 parts of yeast fermentation extract, 2-6 parts of zinc oxide, 1-5 parts of rose essential oil, 0.5-2 parts of honey, 3-8 parts of glycerol, 5-10 parts of hyaluronic acid, 0.5-2 parts of thickening agent and 2-8 parts of water.

2. The collagen moisturizing and sun-blocking moisturizer as claimed in claim 1, wherein the collagen moisturizing and sun-blocking moisturizer is characterized in that: 8 parts of yeast fermentation extract, 3 parts of zinc oxide, 2 parts of rose essential oil, 0.5 part of honey, 8 parts of glycerol, 6 parts of hyaluronic acid, 1 part of thickening agent and 5 parts of water.

3. The collagen moisturizing and sun-blocking moisturizer as claimed in claim 2, wherein the collagen moisturizing and sun-blocking moisturizer comprises the following components in percentage by weight: the yeast fermentation extract is obtained by fermenting gene engineering yeast pichia pastoris.

4. The collagen moisturizing and sun-blocking moisturizer as claimed in claim 3, wherein the collagen moisturizing and sun-blocking moisturizer comprises the following components in percentage by weight: the pichia pastoris plasmid contains a cell-penetrating peptide TAT-human-like collagen (Hlcollagen) recombinant gene, histidine purification labels are respectively introduced into the 5 'end and the 3' end of the sequence, the designed gene sequence has the full length of 828bp, the cell-penetrating peptide TAT gene and the human-like collagen recombinant gene TAT-Hlcollagen are amplified through a PCR technology, Eco RI enzyme cutting sites and Bam HI enzyme cutting sites are respectively introduced into the two ends of the cell-penetrating peptide TAT gene and the human-like collagen recombinant gene TAT-Hlcollagen, and the cell-penetrating peptide TAT gene and the human-like collagen recombinant gene pGAPH alpha A are connected to a pichia pastoris expression vector pGAPH alpha A after double enzyme cutting, so that the recombinant expression vector pG.

5. The collagen moisturizing and sun-blocking moisturizer as claimed in claim 4, wherein the collagen moisturizing and sun-blocking moisturizer comprises the following components in percentage by weight: carrying out PCR detection on the pichia pastoris: according to the designed pair of TAT-Hlcollagen gene primers, a band which is the same as the gene primers appears in electrophoresis detection, but the band is not amplified in the untransformed Pichia pastoris, so that the TAT-Hlcollagen recombinant gene is integrated into the genome of the Pichia pastoris, and the positive Pichia pastoris is obtained.

6. The collagen moisturizing and sun-blocking moisturizer as claimed in claim 5, wherein the collagen moisturizing and sun-blocking moisturizer comprises the following components in percentage by weight: the positive pichia pastoris strain is inoculated into a liquid culture medium containing 20 g of peptone, 10 g of yeast powder, 13.4 g of choline YNB, 10 ml of glycerol, 10 ml of methanol, 0.1 mol of potassium phosphate buffer solution and 0.4 mg of biotin per liter, shaken in a flask, cultured at the temperature of 28 ℃ and 250rpm for 18-24h, centrifuged and collected.

7. The collagen moisturizing and sun-blocking moisturizer as claimed in claim 6, wherein the collagen moisturizing and sun-blocking moisturizer comprises the following components in percentage by weight: the thalli is re-suspended and inoculated by an amplification culture solution, and each liter of the amplification culture solution contains 20 g of peptone, 10 g of yeast powder, 13.4 g of yeast choline YNB, 21.6 ml of methanol, 0.1 mol of potassium phosphate buffer solution and 0.4 mg of biotin; shaking-culturing at 25-35 deg.C and 100-300 rpm for 3-10D; during the fermentation, methanol is supplemented every 12h to induce and maintain the methanol concentration of 1.8-2.5% of the volume ratio.

8. The collagen moisturizing and sun-blocking moisturizer as claimed in claim 7, wherein the collagen moisturizing and sun-blocking moisturizer comprises the following components in percentage by weight: and adding tea leaves into the pichia pastoris fermentation liquor for continuous fermentation, and fermenting at the low temperature of 20 ℃ for 2-3D without adding methanol to obtain the pichia pastoris containing tea polyphenol.

9. The collagen moisturizing and sun-blocking moisturizer as claimed in claim 8, wherein the collagen moisturizing and sun-blocking moisturizer comprises the following components in percentage by weight: the tea is one of oolong tea, Biluochun tea, Longjing tea, TIEGUANYIN, and WUYIYAN tea.

10. A preparation method of a collagen moisturizing sunscreen cream comprises the following specific steps: