Preparation method and application of III-type recombinant collagen

Abstract

The invention provides III-type recombinant collagen, belongs to the technical field of biology, and comprises an amino acid sequence of the III-type recombinant collagen as shown in SEQ ID NO.1. The invention also provides a preparation method of the III-type recombinant collagen. According to the technical scheme provided by the invention, the purification process is simplified, the production cost is reduced, and the complex recombinant collagen repair gel provided by the invention can repair the skin barrier and relieve dry itching and stabbing pain of the skin.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for preparing type III recombinant collagen and its application. Background Technology

[0002] Collagen is a biological macromolecule, a major component of animal connective tissue, and the most abundant and widely distributed functional protein in mammals, accounting for 25%–30% of total protein, and even more than 80% in some organisms. There are many types of collagen, the most common being type I, II, III, V, and XI. Based on origin, they can be divided into animal-derived collagen and recombinant collagen. Compared with traditional animal-derived collagen, recombinant collagen has better biocompatibility and higher activity. Due to its good biocompatibility, biodegradability, and bioactivity, it has found wide application in food, medicine, tissue engineering, cosmetics, and other fields.

[0003] Chinese patent CN118909094A, entitled "A Recombinant Type III Collagen Yves-Col3 with Anti-aging Effects, Its Preparation Method and Application," discloses a recombinant collagen protein and its amino acid sequence with anti-aging effects. The protein is obtained by transfecting a recombinant expression plasmid into competent E. coli cells, followed by induced expression, lysis, and purification. It exhibits no cytotoxicity, high thermal stability, significant scavenging of hydroxyl and DPPH free radicals, and promotes cell proliferation, demonstrating significant anti-aging effects.

[0004] Chinese patent publication CN119552239A, entitled "An Anti-aging Composition Containing Recombinant Collagen and its Preparation Method," discloses that the main component of this composition is high-purity collagen obtained through gene recombination technology, combined with multiple anti-aging active ingredients such as sodium hyaluronate, vitamin C, coenzyme Q10, and vitamin E. This invention also provides a preparation method that ensures uniform distribution of each component and preserves its biological activity through low-temperature homogenization and precise proportioning. The composition of this invention can significantly promote the proliferation of human skin fibroblasts and enhance the expression of type I collagen genes, exhibiting significant anti-aging effects.

[0005] Chinese patent CN110934766A, entitled "Anti-wrinkle Essence Containing Recombinant Collagen," discloses that the anti-wrinkle essence comprises the following ingredients by mass percentage: 0.1%–1% recombinant collagen, 10%–30% glycerin, 1%–5% 1,2-hexanediol, 0.1%–0.5% sodium hyaluronate, 0.1%–0.5% polyglutamic acid, 0.05%–2% acetyl hexapeptide-8, 0.03%–2% elastin, 0.01%–1.5% fibronectin, and the balance being water. This invention uses recombinant collagen derived from microbial fermentation and other raw materials in combination, avoiding problems such as precipitation, discoloration, and separation. The anti-wrinkle essence of this invention combines recombinant collagen with sodium hyaluronate, sodium polyglutamate, etc., which can better exert the firming and wrinkle-reducing effects of recombinant collagen while moisturizing and locking in moisture.

[0006] However, the induced expression methods described above often result in the target protein existing in the form of inclusion bodies, requiring complex refolding steps and posing a high risk of endotoxin residue, thus affecting safety. For example, recombinant type III collagen expressed in *E. coli* requires multi-step purification through ultrafiltration and affinity chromatography, increasing process complexity and yield loss due to multiple purification steps; the prokaryotic system lacks key modifications such as hydroxyprolylation, resulting in an incomplete triple helix structure of collagen, affecting its mechanical strength and biological function; and type III collagen expressed in *E. coli* requires the artificial introduction of a hydroxylase system, increasing production costs.

[0007] On the other hand, the application of recombinant collagen in the above-mentioned technical solutions is for anti-aging, but the market still needs to further develop products that repair the skin barrier and relieve dry, itchy, and stinging skin. Summary of the Invention

[0008] In view of this, the present invention provides a method for preparing type III recombinant collagen and its application, thereby simplifying the purification process and reducing production costs.

[0009] To achieve the above objectives, the present invention provides a type III recombinant collagen, the amino acid sequence of which is shown in SEQ ID NO.1.

[0010] Optional, the following steps may be included: S1. The bacterial strain is activated and cultured in a seed bottle, and then inoculated into a fermenter connected to an ammonia tank, a feed tank, and an antifoaming agent tank. Dissolved oxygen, pH, and foam levels in the fermenter are controlled. The OD value of the fermentation broth is to be determined. 600 Once the requirements are met, an inducing agent is added for low-temperature induction. Feed is added at the start of induction, and fermentation continues. After fermentation is completed, the obtained bacterial solution is centrifuged to collect the bacteria and obtain bacterial sludge. The bacterial sludge is dissolved in buffer solution and then homogenized. The homogenized bacterial solution is then filtered through a microfiltration membrane to obtain a protein solution. S2. The protein solution was purified by column chromatography, then detoxified and desalted to obtain type III recombinant collagen.

[0011] Optionally, the activation culture conditions are: temperature 37℃, rotation speed 220 rpm, and time 5-6 hours; the fermentation tank culture conditions are: initial parameters: temperature 37℃, dissolved oxygen 100%, pH 7.0±0.05, and air flow rate controlled at 0.8 m³ / min. 3 The initial rotation speed is 200 rpm. When the dissolved oxygen in the fermenter is below 30%, the speed is increased by 50 rpm to 500 rpm. The air flow is increased to control the dissolved oxygen. The dissolved oxygen is always kept above 30%, and the tank pressure is maintained at 0.05 MPa.

[0012] Optionally, the OD 600 The requirement is OD. 600 The temperature is 20-25℃; the low-temperature induction temperature is 25±0.5℃, and the total culture time of the strain in the fermenter is 24h.

[0013] Optionally, the centrifugation for bacterial collection is performed using a tubular centrifuge at 9000-11000 rpm for 1-1.5 hours; the mass ratio of buffer solution to bacterial sludge is 1:15 when the buffer solution is used to dissolve the bacterial sludge; the homogenization conditions are 4°C and 850-950 bar; and the microfiltration membrane is used at 15°C with a membrane thickness of 0.25 μm.

[0014] Optionally, the column chromatography purification is performed as nickel column chromatography. The preparation of the buffer solution for the column chromatography includes the following steps: Buffer A preparation: 3.12 g of sodium dihydrogen phosphate dihydrate and 2.84 g of anhydrous disodium hydrogen phosphate are added to 1 liter of water; 20 mm buffer A is mixed with 10 mm imidazole solution, 50 mm imidazole solution and 500 mm imidazole solution respectively to obtain three buffer solutions for later use.

[0015] Optionally, the endotoxin removal is performed by column chromatography, and the buffer solution includes buffer a and buffer b. Buffer a is a mixture of 20 mMPb and 0.1 M NaCl with a pH of 7.5; buffer b is a mixture of 20 mMPb and 1 M NaCl with a pH of 7.5; the desalting is performed using an 8 KD polyethersulfone membrane.

[0016] This invention also provides a recombinant collagen repair gel, which aims to repair the skin barrier and relieve dry, itchy, and stinging skin.

[0017] To achieve the above objectives, the present invention provides a recombinant collagen repair gel, comprising type III recombinant collagen as described in claim 1.

[0018] Optionally, it may also include the following components: water, butanediol, PEG / PPG-17 / 6 copolymer, glycerol, 1,2-hexanediol, erythritol, glyceryl glucoside, carbomer, arginine, p-hydroxyacetophenone, and sodium hyaluronate.

[0019] The above-described technical solution of the present invention has at least the following beneficial effects: The technical solution provided by this invention optimizes the purification process by combining nickel column and Q column affinity chromatography with an organic membrane (PES 8KD membrane). This allows for faster preparation of the target protein. After purification with a Ni column and endotoxin removal with a Q column, rapid desalting can be performed using the organic membrane. This ensures high purity of the target protein, simplifies the purification process, shortens the process cycle, and reduces production costs.

[0020] The recombinant collagen repair gel provided by this invention repairs the skin barrier and relieves dry, itchy, and stinging skin. Attached Figure Description

[0021] Figure 1 These are electrophoretic images of nickel column chromatography with different flow-through solutions in Example 1 of the present invention; Figure 2 This is a molecular weight diagram of mark in Example 1 of the present invention; Figure 3 Electrophoretic images of the three elution solutions in Example 1 of this invention; Figure 4 This is an electrophoresis image of the Q column after endotoxin removal in Example 1 of the present invention; Figure 5 This is a graph showing the liquid phase detection results of the freeze-dried type III recombinant collagen in Example 1 of this invention; Figure 6 Graph showing the transdermal water loss (TEWL) values ​​of the recombinant collagen repair gel. Figure 7 A graph showing the difference in transdermal water loss (TEWL) values ​​for recombinant collagen repair gel. Figure 8 The image shows the skin a-value test results for the recombinant collagen repair gel. Figure 9 A graph showing the difference in skin a-value relative to the mean initial value for the recombinant collagen repair gel. Figure 10 The image shows the self-evaluation results of subjects after using the recombinant collagen repair gel. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will be described in conjunction with the accompanying drawings of the embodiments of the present invention. Figures 1-8The technical solutions of the embodiments of the present invention will be clearly and completely described herein. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention are within the scope of protection of the present invention.

[0023] Example 1 This invention provides a method for preparing type III recombinant collagen, comprising the following steps: 1. Preparations before inoculation and fermentation: Preparation of culture media and feed reagents: Seed culture medium preparation: Table 1. Seed Culture Medium Composition Table

[0024] Table 2 Composition of Ampicillin Mother Solution

[0025] Table 3. Fermentation tank culture medium composition table

[0026] Table 4. Composition of Feed Additives

[0027] Use the pH electrode and dissolved oxygen electrode after calibration.

[0028] Cleaning and sterilization: If the tank is not used for a long time or after the previous batch of fermentation is completed, clean it by first applying acid and then alkali and heating it to 60°C to ensure that the fermentation tank and auxiliary equipment are thoroughly cleaned and sterilized.

[0029] Weigh out 30L of fermentation culture medium (calcium chloride dissolved separately) according to the ingredient list, add feed and deionized water to dissolve, and adjust the pH of the fermentation culture medium to 7.0. Add defoamer and feed to the feed bottle. Place the feed, defoamer, empty ammonia bottle, and two bottles of pure water into an autoclave for sterilization.

[0030] High-temperature sterilization program parameters: 121℃, 1.10MPa, 20min, end temperature 37℃.

[0031] 2. Inoculation and fermentation: Strain preparation: Select suitable engineered strains for target product expression. The amino acid sequence is SEQ ID NO.1: MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLD ANLAGSGSGHMHHHHHHLEVLFQGYGSGSPGERGSPGPPGPAGSPGAPGPPGEPGEKGERGSPGEKGSPGPPGEPGPPGSPGPAGPPGPPGEKGERGSPGSPGER GSPGPPGPAGSPGAPGPPGEPGEKGERGSPGEKGSPGPPGEPGPPGSPGPAGPPGPPGEKGERGSPGSPGERGSPGPPGPAGSPGAPGPPGEPGEKGERGSPGEKGSPGPPGEPGPPGSPGPAGPPGPPGEKGERGSPGSPGERGSPGPPGPAGSPGAPGPPGEPGEKGERGSPGEKGSPGPPGEPGPPGSPGPAGPPGPPGEKGERGSP.

[0032] Inoculum preparation: Inoculate one frozen bacterial culture into 1 liter of LB liquid medium, co-culture to 2 liters, and dispense into two bottles. Add 100 μl (100 mg / ml) of ampicillin to each bottle. Activation conditions for the inoculum: 37℃, 220 r / min (incubate for 5-6 hours).

[0033] Connect the ammonia tank, feed tank, and defoamer tank to the fermenter. Pour 100 μl / ml ampicillin solution into the fermentation medium through the feed inlet with a flame ring. When the OD value of the seed bottle reaches 2-3, inoculate the activated inoculum from the seed bottle into the fermenter. Once the OD600 of the fermentation broth reaches the required level, add an inducer for low-temperature induction. Add feed at the start of induction and continue fermentation. After fermentation, centrifuge the resulting bacterial solution to collect the bacteria, obtaining bacterial sludge. Dissolve the sludge in buffer solution, then homogenize. Filter the homogenized bacterial solution through a microfiltration membrane to obtain a protein solution.

[0034] The initial parameters for the fermenter were: temperature 37℃, dissolved oxygen 100%, pH 7.0 (±0.05), and air flow rate controlled at 0.8 m³ / h. 3 The initial rotation speed is 200 rpm. Whenever the dissolved oxygen level drops below 30%, the speed is increased by 50 rpm. When the rotation speed reaches 500 rpm, the air flow rate is increased to control the dissolved oxygen level, which is always maintained above 30%. The tank pressure is maintained at 0.05 MPa.

[0035] Monitor the fermentation process: (1) Record the fermentation-related data displayed on the fermenter every hour. (2) Aseptically sample and measure OD every 2 hours. 600 (3) When dissolved oxygen and pH rise, start adding feed at a rate of 120ml / h-540ml / h. (4) pH: control between 7.0±0.05. (5) When a large amount of foam appears, add an appropriate amount of defoamer until the large amount of foam disappears.

[0036] Low-temperature induction includes the following steps: approximately 5-6 hours after fermentation begins, the OD of the fermentation broth... 600 Between 20 and 25 minutes, add the inducing agent and begin low-temperature induction at 25 ± 0.5℃. The fermentation process is terminated after 24 hours. The inducing agent is 2.38 g IPTG dissolved in 100 ml of sterile water. Feeding should continue at the start of induction at a rate of 240 ml / h.

[0037] Centrifugation was performed using a tube centrifuge at 10,000 rpm for 1.5 hours.

[0038] To dissolve the bacterial sludge in buffer solution, prepare a 20mM PB buffer solution and dissolve the bacterial sludge at a mass ratio of 1:15, and use a stirrer to accelerate the dissolution.

[0039] Homogenization involves passing the dissolved bacterial solution through a high-pressure homogenizer to homogenize both sides under the following conditions: 4°C and 900 bar.

[0040] Microfiltration involves passing the homogenized bacterial solution through a 0.25μm membrane at 15℃ and collecting the permeate, which is the protein solution.

[0041] 3. Purification process The protein solution was purified by column chromatography, then detoxified, and finally desalted to obtain type III recombinant collagen. The specific steps included: The column chromatography was nickel column chromatography, with a Ni column volume of 3000 ml and a sample loading volume of 50 liters. The buffer solution was prepared as follows: Buffer A: 20 mm Pb (pH 7.5). 1 L Pb solution was prepared by adding 3.12 g of sodium dihydrogen phosphate dihydrate and 2.84 g of anhydrous disodium hydrogen phosphate to 1 liter of water.

[0042] Buffer B: Take 20 mm Pb and mix it with 10 mm imidazole solution, 50 mm imidazole solution, and 500 mm imidazole solution respectively to obtain three buffer solutions for later use: Buffer A + 10 mm imidazole, Buffer A + 50 mm imidazole, and Buffer A + 10 mm imidazole. 0.5 mol / L imidazole preparation: Add 34 g imidazole to 1 L of water.

[0043] Nickel column chromatography elution conditions: Sample loading flow rate: 450 ml / min. Electrophoresis was performed with flow-through buffers of 15, 30, 40, and 50 liters respectively. Figure 1 The molecular weight of the mark is shown in [reference needed]. Figure 2 .

[0044] Equilibrium: 3 cv (i.e., elution volume equal to 3 column volumes) Elution flow rate: 450 ml / min, and the three buffer solutions obtained were used for elution.

[0045] 20mm pb + 10mm imidazole washed 3cv.

[0046] 20mm pb + 50mm imidazole washed 3cv.

[0047] 20mm pb + 500mm imidazole washed 3cv.

[0048] Collect the three eluents and perform electrophoresis separately to obtain... Figure 3 .

[0049] Wash with 0.5mm NaOH for 1.5 cv, wash with 500mm imidazole for 1 cv, and wash with pure water for 2 cv.

[0050] Nickel column preservation solution: 20% ethanol 1.5 cv.

[0051] Endotoxin removal is performed using column chromatography, and specifically includes the following steps: Q-Column - Detoxification (100ml) Buffer:A: 20mMPb+0.1M NaCl (pH 7.5); B: 20mMPb+1M NaCl (pH 7.5) Flow rate: 20 ml / min Steps: (1) Treat with 1.5 cv alkali (0.5M NaOH) for 30 min (2) Rinse with 2cv B solution (3) Rinse with 3cv A solution (4) Sample loading volume: 800ml (5) When liquid A reaches equilibrium at 3 cv, flush until absorbance and conductivity are in equilibrium. (6) Elute 2 cv with solution B (7) Preserve the Q column with 1.5 cv of 20% ethanol. Collect endotoxin-free samples and perform electrophoresis tests. Figure 4 Then, desalting was performed using an 8KD PES (polyethersulfone) membrane to obtain purified type III recombinant collagen.

[0052] The purified type III recombinant collagen was desalted and concentrated using an organic membrane. Water was added during the concentration process to ensure complete desalting. The concentration was stopped when 7-9 liters were reached, and the retentate was collected for freeze-drying. The parameters for freeze-drying are shown in Table 5 below. Table 5. Parameters for Freeze-drying Process

[0053] The lyophilized type III recombinant collagen was analyzed by liquid chromatography to obtain... Figure 5 .

[0054] Depend on Figure 5 It can be seen that the recombinant collagen prepared by the technical solution provided by the present invention has high purification degree, and the purity of the recombinant collagen is greater than 90%.

[0055] Example 2 This invention provides a recombinant collagen repair gel, comprising type III recombinant collagen prepared according to this invention, and other components, the specific components of which are shown in Table 6.

[0056] Table 6. Ingredients of Reconstituted Collagen Repair Gel

[0057] The following results were obtained from the testing of the recombinant collagen repair gel: Human efficacy testing of cosmetics: Refer to T / CAB 0152-2022 "Test Methods for Seven Efficacy Items of Cosmetics: Anti-wrinkle, Firming, Moisturizing, Oil Control, Repairing, Nourishing and Soothing" and "Cosmetic Safety Technical Specifications" (2015 edition).

[0058] Asian adult subjects used the recombinant collagen repair gel sample for 14 and 28 days. Transepidermal water loss (TEWL) values ​​and skin a-values ​​were measured at the test sites using specialized equipment. Before and after use, the skin repair efficacy of the test sample was evaluated. Subject self-assessment was also used to evaluate the repair efficacy of the survey sample.

[0059] Instrument and equipment test results: see Table 7 Table 7. Results of Skin Repair Test by Instrument and Equipment

[0060] a: Comparison before and after sample use.

[0061] Subject self-report results 100.00% of the test subjects agreed that the product was gentle and non-irritating. 93.33% of the participants agreed that the product was easily absorbed. 100.00% of the test subjects agreed that the product could make the skin moisturized, soft, and elastic. 100.00% of the test subjects agreed that the product could relieve dry, itchy, and stinging skin. 100.00% of the test subjects agreed that the product could relieve skin discomfort. 100.00% of the test subjects agreed that the product improved rough and dehydrated skin. 100.00% of the test subjects agreed that the product relieved skin redness. 96.67% of the respondents agreed that the product had a moisturizing effect. 96.67% of the respondents agreed that the product could repair the skin barrier and stabilize the skin environment. 96.67% of the respondents agreed that they were generally satisfied with the product.

[0062] Test Conclusion Based on the results of instrument testing, 30 Asian subjects (aged 25-42 years) showed a significant decrease in transepidermal water loss (TEWL) and skin a value after using the recombinant collagen repair gel for 14 and 28 days compared with baseline values, indicating that the recombinant collagen repair gel has the effect of repairing the skin barrier.

[0063] In this self-evaluation by the participants, after 28 consecutive days of use of the sample recombinant collagen repair gel, 30 Asian participants (aged 25-42) reported that over 93.33% of the participants found the product easily absorbed; over 96.67% believed the product could repair the skin barrier and stabilize the skin environment; 100.00% of the participants found the product could relieve dry, itchy, and stinging skin; and 96.67% of the participants were generally satisfied with the product after use.

[0064] The recombinant collagen repair gel prepared in this invention is a leave-on cosmetic product suitable for the face. In accordance with the relevant provisions of the "Technical Guidelines for Cosmetic Safety Assessment," 12 raw materials used in the formula, including water, butylene glycol, PEG / PPG-17 / 6 copolymer, glycerin, 1,2-hexanediol, erythritol, glyceryl glucoside, carbomer, arginine, p-hydroxyacetophenone, soluble collagen, and sodium hyaluronate, were evaluated. The product was tested for harmful substances and microorganisms, and four potentially hazardous substances—dioxane, phenol, diethylene glycol, and benzene—were assessed. The results show that under normal, reasonable, and foreseeable use, this product will not pose a threat to human health.

[0065] The specific testing protocol and data for the recombinant collagen repair gel are as follows: The experimental protocol is shown in Table 8: Table 8. Detection scheme for transepidermal water loss (TEWL) value and skin a value.

[0066] The criteria for the subject survey are shown in Table 9.

[0067] Table 9. Subject Survey Standards

[0068] Requirements for participants: Inclusion criteria: 1. Healthy Asian adult subjects aged 18-55, regardless of gender; 2. No allergic diseases, and no history of allergies to cosmetics or other topical preparations; 3. Those who can understand the testing process, are willing to participate in the trial, and sign a written informed consent form; 4. Be able to cooperate with and participate in the follow-up visits of the experimental research, maintain the regularity of daily life during the research period, do not stay up late, do not smoke, do not drink excessively, do not overeat, and promptly report any changes in one's own health status or medication, and any adverse reaction symptoms; 5. The test results meet the testing requirements after screening by the inspectors; 6. The skin at the test site should be free of pigmentation, inflammation, scars, moles, hirsutism, etc.

[0069] Exclusion criteria: 1. Women who are currently pregnant or breastfeeding, or women who plan to become pregnant within the next two months; 2. History of skin cancer within the past 5 years; 3. Individuals with bacterial, viral, or fungal infections at the test site; 4. The test site has obvious sunburn, scars, excessive hair, or other conditions that may affect the test measurement results; 5. Individuals with skin diseases (such as psoriasis, rosacea, eczema, lupus erythematosus, seborrheic dermatitis, psoriasis, severe epidermal exfoliation, etc.); 6. History of immunosuppression or immunodeficiency (including HIV or AIDS), or current use of immunosuppressive drugs or radiation therapy; 7. Have undergone cosmetic medical treatment on the test area within the past month prior to participating in the test; 8. Those who have participated in other clinical trials (such as hormone replacement therapy HRT) or taken oral hormones that may affect the test results within 3 months prior to participating in the test, or those who plan to undergo HRT treatment during the trial period; 9. Have used oral vitamin A or steroids within the past 6 months prior to participating in the test; 10. Currently using any medications that may affect skin condition or reaction, such as antihistamines, antibiotics, insulin, anti-inflammatory drugs, steroids, corticosteroids, aspirin, thyroid medications, etc. 11. Have undergone chemotherapy or other radiation therapy, or received any medication or treatment that may affect skin condition; 12. Other diseases or conditions that, in the researcher's judgment, may reduce the likelihood of enrollment or complicate enrollment, such as frequent changes in work environment or unstable living environment, which may lead to loss to follow-up: alcoholism or psychoactive substances, drug abuse and dependence.

[0070] Exit criteria: 1. Subjects may withdraw from this study if they experience complications, adverse reactions, or other reasons (specific details must be provided) during the testing period; 2. The subjects did not follow up within the agreed time; 3. The client terminates the test.

[0071] Subject Valid Data Standards 1. The subject's testing protocol has completed the testing process, and the subject's test data is acceptable; 2. If a subject fails to meet any of the withdrawal criteria, the subject's data will be considered invalid.

[0072] Subject inclusion information Number of participants recruited: 33 Actual number of participants: 30 Gender ratio of participants: 20 women and 10 men Participant age range: 25-42 years old, average age 36 years old Number of people who withdrew from the test: 3 Data Statistics and Analysis Descriptive statistics: Calculate the mean, standard deviation, maximum, median, and minimum values ​​of each skin parameter at each time point.

[0073] Normality test: If a normality test is performed on the above means and the asymptotic significance (two-tailed) value of the normality test is >0.05, then the data series follows a normal distribution.

[0074] Difference analysis: To analyze the differences between the above means, when both sets of data are normally distributed, a t-test is used to analyze the differences between the two sets of data; when the two sets of data are not normally distributed, a rank-sum test is used to analyze the differences between the two sets of data.

[0075] Evaluation criteria: If the statistical significance level p < 0.05, it indicates that there is a statistically significant difference in skin parameters before and after the subjects used the sample; if the statistical significance level p ≥ 0.05, it indicates that there is no statistically significant difference in skin parameters before and after the subjects used the sample.

[0076] Testing process: Before using the sample: 1. Two days before the test, the subject should stop using cosmetics or topical medications on the area to be tested; 2. The night before the test, the participants washed their faces at home and did not apply any cosmetics. 3. On the morning of the test, the participants washed their faces with water at home and did not apply any cosmetics. 4. Upon the first visit, explain the test to the participants and obtain their informed consent. 5. Subjects cleansed their faces using facial cleansing products provided by the laboratory. 6. Subjects waited for 30 minutes in a constant temperature and humidity environment of 21±1℃ and 50±10%; 7. The tester used a VAPOSCAN AS-VT100 transdermal water loss meter to measure the transdermal water loss (TEWL) value in this area; 8. The tester took photos using a VISIA-7; 9. The tester distributes test samples and instructions for use.

[0077] 14 days after using the sample: 1. Subjects cleansed their faces using facial cleansing products provided by the laboratory. 2. Subjects waited for 30 minutes in a constant temperature and humidity environment of 21±1℃ and 50±10%; 3. The tester used a VAPOSCAN AS-VT100 transdermal water loss meter to measure the transdermal water loss (TEWL) value in this area; 4. The tester took photos using a VISIA-7.

[0078] 28 days after using the sample: 1. Subjects cleansed their faces using facial cleansing products provided by the laboratory. 2. Subjects waited for 30 minutes in a constant temperature and humidity environment of 21±1℃ and 50±10%; 3. The tester used a VAPOSCAN AS-VT100 transdermal water loss meter to measure the transdermal water loss (TEWL) value in this area; 4. The tester took photos using a VISIA-7; 5. Subject self-report.

[0079] Test Results 1. Description of Transepidermal Water Loss (TEWL) Values The transdermal water loss (TEWL) values ​​before sample use, 14 days after sample use, and 28 days after sample use are shown in Table 10 below. Figure 6 .

[0080] Table 10 Descriptive statistics of transepidermal water loss (TEWL) values ​​(n=30) Unit: g / cm³ 2 / h

[0081] 2. Difference in transepidermal water loss (TEWL) values The transdermal water loss (TEWL) value difference refers to the difference between the TEWL values ​​of the calibrated area before and 28 days after sample application. The calculation formula is as follows: Difference in transdermal water loss (TEWL) values ​​= Transdermal water loss (TEWL) value before sample application - Transdermal water loss (TEWL) value after sample application The results of the transepidermal water loss (TEWL) value difference test are shown in Tables 11 and 12 below. Figure 7 .

[0082] Table 11 Descriptive statistics of the difference in transepidermal water loss (TEWL) values ​​(n=30) Unit: g / cm³ 2 / h

[0083] Note: a. The difference rate represents the difference between the percentage improvement in the sample area before and after the comparison and the percentage improvement in the negative area before and after the comparison.

[0084] 3. Analysis of the differences in transepidermal water loss (TEWL) values Table 12 Results of the analysis of differences in transepidermal water loss (TEWL) values ​​(n=30)

[0085] Note: "ns" means p ≥ 0.05, "*" means 0.01 ≤ p < 0.05, "**" means 0.001 ≤ p < 0.01, "***" means p < 0.001, and "****" means p < 0.0001.

[0086] 4. Description of skin a-value Table 13 shows the skin a-value test results before sample use, 14 days after sample use, and 28 days after sample use. Figure 8 .

[0087] Table 13 Descriptive statistics of skin a-value (n=30)

[0088] Note: a. Difference rate = (Mean after use - Mean before use) / (Mean before use) 5. Analysis of differences in skin a-values Table 14 Results of the analysis of differences in skin a-values ​​(n=30)

[0089] Note: "ns" means p ≥ 0.05, "*" means 0.01 ≤ p < 0.05, "**" means 0.001 ≤ p < 0.01, "***" means p < 0.001, and "****" means p < 0.0001.

[0090] Figure 9 This is a graph showing the difference rate of skin a value relative to the initial mean value.

[0091] 6. Subjects' self-assessment results Table 15 Self-Assessment Results

[0092] Note: 1. 5 points indicates complete agreement, 4 points indicates partial agreement, 3 points indicates both agreement and disagreement (i.e., neutral), 2 points indicates partial disagreement, and 1 point indicates complete disagreement. 2. Statistics: Satisfaction rate = (Number of valid respondents with a score > 3) ÷ (Total number of valid respondents) × 100% Figure 10 This is a graph showing the self-evaluation results of the test subjects after using the product.

[0093] Table 16 Subject Information Table

[0094] Table 17 Transepidermal Water Loss (TEWL) Value Data

[0095] Table 18 Skin a-value data table

[0096] The above are preferred embodiments of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type III recombinant collagen, characterized in that, The amino acid sequence of the type III recombinant collagen is shown in SEQ ID NO.

1.

2. A method for preparing type III recombinant collagen as described in claim 1, characterized in that, Includes the following steps: S1. The bacterial strain is activated and cultured in a seed bottle, and then inoculated into a fermenter connected to an ammonia tank, a feed tank, and an antifoaming agent tank. Dissolved oxygen, pH, and foam levels in the fermenter are controlled. The OD value of the fermentation broth is to be determined. 600 Once the requirements are met, an inducing agent is added for low-temperature induction. Feed is added at the start of induction, and fermentation continues. After fermentation is completed, the obtained bacterial solution is centrifuged to collect the bacteria and obtain bacterial sludge. The bacterial sludge is dissolved in buffer solution and then homogenized. The homogenized bacterial solution is then filtered through a microfiltration membrane to obtain a protein solution. S2. The protein solution was purified by column chromatography, then detoxified and desalted to obtain type III recombinant collagen.

3. The method for preparing type III recombinant collagen according to claim 2, characterized in that, The activation culture conditions were: temperature 37℃, rotation speed 220 rpm, and time 5-6 hours; the fermentation tank culture conditions were as follows: initial parameters: temperature 37℃, dissolved oxygen 100%, pH 7.0±0.05, and air flow rate controlled at 0.8 m³ / min. 3 The initial rotation speed is 200 rpm. When the dissolved oxygen in the fermenter is below 30%, the speed is increased by 50 rpm to 500 rpm. The air flow is increased to control the dissolved oxygen. The dissolved oxygen is always kept above 30%, and the tank pressure is maintained at 0.05 MPa.

4. The method for preparing type III recombinant collagen according to claim 2, characterized in that, The OD 600 The requirement is OD. 600 The temperature is 20-25℃; the low-temperature induction temperature is 25±0.5℃, and the total culture time of the strain in the fermenter is 24h.

5. The method for preparing type III recombinant collagen according to claim 2, characterized in that, The centrifugation for bacterial collection is performed using a tubular centrifuge at 9000-11000 rpm for 1-1.5 hours. The mass ratio of buffer solution to bacterial sludge is 1:15 when the buffer solution is used to dissolve the bacterial sludge. The homogenization conditions are 4°C and 850-950 bar. The microfiltration membrane is used at 15°C with a membrane thickness of 0.25 μm.

6. The method for preparing type III recombinant collagen according to claim 2, characterized in that, The column chromatography purification is to nickel column chromatography. The preparation of the buffer for the column chromatography includes the following steps: Buffer A preparation: 3.12 g of sodium dihydrogen phosphate dihydrate and 2.84 g of anhydrous disodium hydrogen phosphate are added to 1 liter of water; 20 mm buffer A is mixed with 10 mm imidazole solution, 50 mm imidazole solution and 500 mm imidazole solution respectively to obtain three buffers for later use.

7. The method for preparing type III recombinant collagen according to claim 2, characterized in that, The endotoxin removal is performed by column chromatography. The buffer solution includes buffer a and buffer b. Buffer a is a mixture of 20 mMPb and 0.1 M NaCl with a pH of 7.

5. Buffer b is a mixture of 20 mMPb and 1 M NaCl with a pH of 7.

5. The desalting is performed using an 8 KD polyethersulfone membrane.

8. A recombinant collagen repair gel, characterized in that, Includes the type III recombinant collagen as described in claim 1.

9. The recombinant collagen repair gel according to claim 8, characterized in that, It also includes the following components: water, butanediol, PEG / PPG-17 / 6 copolymer, glycerol, 1,2-hexanediol, erythritol, glyceryl glucoside, carbomer, arginine, p-hydroxyacetophenone, and sodium hyaluronate.

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