Application of mucoprotein in preparation of skin care product

Abstract

The invention belongs to the fields of biology and cosmetics, and particularly relates to application of mucoprotein in preparation of products for whitening, brightening, relieving, resisting inflammation, resisting oxidation, resisting aging and repairing cell damage. The yeast-source mucoprotein has the effects of whitening, brightening, relieving, resisting inflammation, resisting oxidation, resisting aging and repairing cell damage, is safe, mild and non-irritating, can be applied to products such as cosmetics and the like, and has application value.

Description

Technical Field

[0001] The invention belongs to the biological field and the cosmetic field, and specifically relates to the application of mucin in the preparation of skin care products. Background Art

[0002] With the increasing economic development and the continuous progress of society, people's pursuit of beauty will never stop; the fast-paced life, air pollution, air dust and other skin damage, as well as the increasingly serious aging of the population, make skin care particularly important. At present, people's demand for skin care products is no longer limited to moisturizing. Therefore, multi-functional skin care ingredients have emerged, including whitening, anti-aging, anti-allergy, anti-acne and other effects. Most of the functional ingredients on the market are single-effect, unstable and easy to fail. Not only that, the addition of functional ingredients may cause the instability of the system, and oil-water stratification is prone to occur, which will also affect the skin feel. In addition, most of the functional ingredients are relatively active and cannot be stably present in the system. They are easily oxidized or decomposed by light, resulting in the functional ingredients being ineffective before use and unable to exert their superior performance. Therefore, it is the unremitting goal of researchers in the field of daily chemicals to develop a multi-functional skin care ingredient with excellent performance, integrating moisturizing, whitening, anti-aging, soothing, etc., so that the functional ingredients can maintain high activity and be safe and non-irritating to the skin.

[0003] Mucin, also known as mucin, is a class of highly glycosylated high molecular weight proteins. From a molecular level, mucin is a copolymer of protein and sugar, which is formed by its unstructured polypeptide skeleton and dense serine (Ser) and threonine (Thr) connected O-sugar chain grafts. The glucan side chain can account for 50% or more of the molecular weight of glycoproteins, which is linear or branched. Because they are covered with sialic acid or sulfate groups, they are often negatively charged at neutral pH. The special structure of mucin gives it a variety of functional properties. The hydrophobic ends at both ends allow it to bind to hydrophobic surfaces, and the high-density polysaccharide region plays an important role in mucin hydration and lubrication. At the same time, mucins can form disulfide bonds through the cysteine-rich regions at both ends, thereby forming a mucin network. Studies have shown that the mucin network can form a protective barrier to reduce the infection of bacteria and external substances, thereby playing a bactericidal and anti-inflammatory role. At the same time, the mucin network can also prevent water evaporation and has the effect of locking water, moisturizing and lubricating.

[0004] Most of the mucin products on the market are extracted from animals. In addition to mucin, they also contain collagen, antimicrobial peptides and other ingredients. The claimed efficacy is produced by the synergistic effect of the mixed ingredients, and the specific efficacy of mucin alone cannot be determined. Glycoproteins, which are similar in structure to mucin, mainly serve as moisturizers, hair conditioners, and skin conditioners in products. Their efficacy in daily chemical products is mainly moisturizing and preventing hair loss. At present, mucin and glycoproteins with similar structures have a place in toiletries as the main moisturizing ingredients, but research on other efficacy aspects is still blank. Summary of the invention

[0005] In view of the gaps in the existing market and technical deficiencies, the present invention provides a new application of mucin in skin care, which, in addition to basic moisturizing, also has multiple functions of anti-aging, whitening and soothing, can meet the needs of the market, and has broad application prospects and huge market value.

[0006] The present invention provides an application of yeast-derived mucin in preparing a whitening and brightening product, wherein the amino acid sequence of the yeast-derived mucin is shown in SEQ ID NO: 1.

[0007] The present invention also provides a use of yeast-derived mucin in preparing a soothing and anti-inflammatory product, wherein the amino acid sequence of the yeast-derived mucin is shown in SEQ ID NO: 1.

[0008] The present invention also provides a use of yeast-derived mucin in preparing an antioxidant and anti-aging product, wherein the amino acid sequence of the yeast-derived mucin is shown in SEQ ID NO: 1.

[0009] The present invention further provides a use of yeast-derived mucin in preparing a product for repairing cell damage, wherein the amino acid sequence of the yeast-derived mucin is shown in SEQ ID NO:1.

[0010] Specifically, the product is a skin care product, a gel dressing, an ointment, a spray, a shampoo, a conditioner or an eye drop.

[0011] Preferably, the yeast-derived mucin is obtained by a recombinant expression method.

[0012] Specifically, yeast cells into which a recombinant expression vector containing a gene encoding the yeast-derived mucin is introduced are cultured to produce the yeast-derived mucin.

[0013] Preferably, the yeast cell is Pichia pastoris, specifically GS115 or KM71H strain.

[0014] More specifically, shake flask fermentation or fed-batch fermentation is adopted.

[0015] Furthermore, the method also includes the separation and purification step of yeast-derived mucin, and the specific purification steps are as follows:

[0016] 1) Using ethanol precipitation method to purify yeast fermentation broth, use 40% or more ethanol final concentration to directly precipitate the target protein; or first use 20%-35% ethanol final concentration, let it stand at 4°C for 1-2 hours to precipitate the impurity protein, and then use 40% ethanol final concentration, let it stand at 4°C for 1-2 hours to precipitate the target protein;

[0017] 2) further comprising, using chromatography to finely purify the target protein, specifically, the selected chromatographic medium is an anion exchange chromatographic medium, including but not limited to strong anion exchange, weak anion exchange, mixed mode ion exchange, the selected buffer is a buffer of pH 5.0-7.5, wherein the buffer pair can be a buffer such as citric acid-sodium citrate, Tris-HCl, Bis-Tris-HCl that can reach the target pH, and NaCl is used for gradient elution to obtain the target protein

[0018] Compared with the existing technology, the beneficial effects of the present invention are as follows: the mucin described in the present invention has multiple skin care effects. In addition to enhancing the skin's ability to lock in water and produce water, helping the skin to restore its moisture and elasticity, it can also remove free radicals and effectively resist oxidation for cells damaged by oxidation; mucin can also enhance cell repair, promote cell proliferation, and accelerate skin metabolism; in addition, mucin can inhibit the activity of tyrosinase and reduce the formation of melanin, thereby brightening the skin tone and having a whitening effect. Not only that, it also has a soothing effect, can enhance the skin's defense ability, resist skin allergies, and has low irritation and allergy, and has broad application prospects and huge market value in the field of skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Mucin purification results;

[0020] Figure 2 : Chicken embryo chorioallantoic membrane test results;

[0021] Figure 3 : Evaluation results of zebrafish moisturizing efficacy; a is the relative expression of aqp3a gene compared with the model control group, **p<0.01, ***p<0.001; b is the relative expression of has3 gene compared with the model control group, *p<0.05, ***p<0.001;

[0022] Figure 4 :Zebrafish neutrophil inhibition rate results;

[0023] Figure 5 : Cell migration rate results. DETAILED DESCRIPTION

[0024] The present invention is further described below through specific examples in order to better understand the present invention, but does not constitute a limitation of the present invention.

[0025] Example 1: Construction of recombinant strains of yeast-derived mucin and expression in shake flasks

[0026] 1. Mucin expression

[0027] In the early stage, our laboratory obtained the original mucin expression gene by amplifying the Pichia pastoris genome, and transformed it by truncating the sequence and replacing the signal peptide to obtain the mature mucin expression sequence (SEQ ID NO: 1):

[0028]

[0029] The gene was cloned into pPIC9K(+), linearized with SalI, and then electroporated into Pichia pastoris GS115 competent cells. High-copy single clones were screened using 2 mg / mL G418. The high-copy transformants obtained by screening were inoculated into BMGY medium and cultured at 30°C and 220 rpm until OD600 = 2, and the cells were collected. The cells were washed three times with BMMY medium and resuspended, and cultured at 30°C and 220 rpm for fermentation expression.

[0030] 2. Mucin Purification

[0031] The fermentation broth was centrifuged at 5000rpm for 10 minutes, and the fermentation supernatant was collected. The ethanol pre-cooled at 4°C was mixed with the fermentation supernatant in a volume ratio of 2:3, and then placed at 4°C for 2 hours until the precipitate automatically settled and became solid, and then centrifuged at 4°C and 10000rpm for 15 minutes, and the precipitate was retained as the cold ethanol precipitation product. The cold ethanol precipitation product was resuspended in pH 6.0, 20mM Bis-Tris-HCl buffer, and filtered through a 0.45μm microporous filter membrane to remove impurities. The treated sample was purified by anion mixing method, and the protein purification component was lyophilized to obtain protein lyophilized powder. The obtained sample was analyzed by SDS-PAGE, and the results are as follows Figure 1 As shown, the mucin is of high purity and can be used.

[0032] Example 2. Safety and toxicological evaluation

[0033] 1. Safety - Chicken Embryo Chorioallantoic Membrane Test

[0034] a. Test Principle

[0035] The chicken embryo chorioallantoic membrane test is an early in vitro eye irritation assessment method. The chorioallantoic membrane (CAM) is a respiratory membrane that surrounds the chicken embryo. This test uses the characteristics of the intact, clear and transparent vascular system of the chorioallantoic membrane of the mid-stage incubated chicken embryo. A certain amount of the test substance is directly exposed to the chicken embryo chorioallantoic membrane. After a period of action, the changes in the toxic effect indicators of the chorioallantoic membrane (such as bleeding, coagulation and vascular melting) are observed. These indicators reflect the changes in the morphological structure, color and permeability of the blood vessels and vascular networks, as well as the phenomena such as the denaturation of the chorioallantoic membrane proteins and the degree of damage. Then a score is obtained by combining them to evaluate the eye irritation of the test substance.

[0036] b. Test methods

[0037] The test is carried out in accordance with the entry-exit inspection and quarantine industry standard SN / T2329-2009 of the People's Republic of China.

[0038] Purchase 0-day-old chicken embryos, cultivate them in an incubator at a temperature of 37.5°C ± 0.5°C and a relative humidity of 50%-60%, and check the chicken embryos with an egg candling device when they are 9 days old, and discard the chicken embryos that have not hatched alive or have not been fertilized. Take out the available chorioallantoic membrane according to the correct operation method. Take 0.3mL of the 0.5% mucin described in the present invention and directly drip it on the surface of the chorioallantoic membrane, observe the reaction of the chorioallantoic membrane under a microscope, and record the time when each toxic effect appears within 5 minutes of action. In order to exclude false positives, verify that a positive control group is set up, and a 0.1mol / L sodium hydroxide solution is selected as the positive control group.

[0039] c. Result evaluation

[0040] This test uses a liquid sample and the reaction time method to evaluate irritation.

[0041] Bleeding: refers to the outflow of blood from the blood vessels or capillaries of the chorioallantoic membrane. It can appear in various forms, such as cauliflower-like, smooth, diffuse gauze-like or punctate bleeding.

[0042] Coagulation: It is divided into intravascular coagulation, which appears as dark coagulation spots inside the blood vessels; extravascular coagulation, which appears as dark coagulation spots outside the blood vessels. It can also appear as turbidity (opacity), appearing in all or part of the membrane, which may be similar to milky white gauze or milky.

[0043] Vascular melting: refers to the disappearance of blood vessels on the chorioallantoic membrane, which may be caused by multiple factors such as bleeding and changes in vascular wall tension.

[0044] The stimulation score (IS) was calculated using the following formula:

[0045]

[0046] Where:

[0047] SecH: (bleeding time, the average time to observe the onset of bleeding on the chorioallantoic membrane, in seconds)

[0048] SecL: (Vascular melting time, the average time when vascular melting begins to occur on the chorioallantoic membrane, in seconds)

[0049] SecC: (Coagulation time, the average time for coagulation to begin to occur on the chorioallantoic membrane, in seconds)

[0050] Note: If no corresponding vascular effect occurs, the time is recorded as 301 seconds.

[0051] The eye irritation of the test substances is classified according to the calculated IS values ​​as shown in Table 1:

[0052] Table 1 Irritation classification table

[0053] Stimulus score Irritation classification IS<1 Non-irritating 1≤IS<5 Mild irritant 5≤IS<9 Moderate irritation IS ≥ 10 Strong irritation

[0054] d. Experimental results

[0055] The test results are detailed in Figure 2 And Table 2

[0056] Table 2 Comparison test results

[0057] 0.1mol / L sodium hydroxide 0.5% mucin HkDJ 35s 301s sec 108s 301s secC 170s 301s IS 12.83 0 Irritation classification Strong irritation Non-irritating

[0058] It can be seen from the pictures and IS scores that the mucin of the present invention did not cause vasoconstriction or other abnormal reactions after contacting the chicken embryo chorioallantoic membrane, which indicates that the mucin is non-irritating. Therefore, it can be seen that the mucin is relatively safe and non-irritating to the skin.

[0059] 2. Toxicity-Cytotoxicity Test

[0060] a. Experimental Principle

[0061] The full name of CCK8 is Cell Counting Kit-8. The principle of the kit is that WST-8 can be detected by NAD in the presence of electron coupling reagent (that is, the cells are alive, breathing, and have energy metabolism). + The redox reaction becomes a water-soluble yellow formazan product (Formazan). The more living cells there are, the more formazan is produced and the darker the color. CCK8 can be used to evaluate the cytotoxicity of the sample and its effect on the proliferation ability of cells.

[0062] b. Experimental procedures

[0063] Take cells in logarithmic growth phase and inoculate the cell suspension into 96-well plates at 100 μL per well to make the cell density 5000-10000 per well. After the cells adhere to the wall for about 24 hours, add protein samples with concentrations of 0.10%, 0.25%, 0.50%, and 1%, respectively, and place in 5% CO 2 Continue to culture at 37°C in the incubator for 24 hours. After the culture is completed, add 10 μL of CCK8 reagent to each well. Incubate the 96-well plate in the incubator for about 45-90 minutes, and measure the absorbance at 450nm using an enzyme reader. Calculate the cell proliferation rate according to the following formula

[0064] Cell proliferation rate (CRS) = (OD value of experimental group - blank group) / (control group - blank group) × 100%

[0065] Evaluate according to the following criteria

[0066] When the viability is low, the test sample has a higher potential cytotoxicity. If the viability drops to <70% of the blank, it has potential cytotoxicity.

[0067] c. Experimental results

[0068] The experimental results are shown in the table below.

[0069] Table 3: CCK8 cytotoxicity test results

[0070] CRS Cytotoxicity Comparison 100 - 0.1％ 100.7 No cytotoxicity 0.25％ 99.1 No cytotoxicity 0.5％ 134.5 No cytotoxicity 1％ 138.2 No cytotoxicity

[0071] From the results in the above table, it can be seen that mucin at a concentration of 0.1%-1% is non-cytotoxic, and 0.5% and 1% mucin have a cell proliferation effect, indicating that mucin can promote cell division and proliferation, thereby enhancing cell activity, repairing cell damage, accelerating cell renewal, thereby improving skin condition, and has great potential in wrinkle removal and anti-aging.

[0072] 3. Toxicology--Acute eye irritation

[0073] a. Experimental Principle

[0074] The acute eye irritation and corrosion test is an acute test to detect the primary irritation and corrosion effects of foreign compounds on the eyes and mucous membranes of experimental animals, and to provide information on the potential hazards of human eyes and mucous membranes coming into contact with the test substances.

[0075] b. Experimental procedures

[0076] New Zealand rabbits were adapted to the laboratory animal room environment for 3 days. Both eyes of the New Zealand rabbits were examined within 24 hours before the start of the test (including the use of sodium fluorescein for examination), and no obvious eye irritation symptoms, corneal defects and conjunctival damage were found. Gently pull open the left lower eyelid, drop 0.1mL of the test substance into the conjunctival sac, and passively close the upper and lower eyelids for 1 second to prevent the loss of the test substance. The eyes were not rinsed within 24 hours after the test substance was administered, and the right eye was not treated as a self-control. The damage to the conjunctiva, cornea and iris was examined and recorded 1, 24, 48 and 72 hours after the administration of the test substance. The scoring was carried out in accordance with the "Table 1 Scoring Standards for Eye Damage" in Chapter 6, 5 of the "Technical Specifications for Safety of Cosmetics" (2015 Edition), and the eyes of all animals were further examined with sodium fluorescein 24 hours later. No irritation reaction occurred 72 hours after exposure, and the test was terminated. The intensity of eye irritation reaction is determined by evaluating the mean value of the irritation response scores of the cornea, iris and conjunctiva of the animals at 24h, 48h and 72h after administration of the test substance and the recovery time, and judging the intensity of eye irritation reaction in accordance with “Table 2 Classification of Eye Irritation Reaction of Raw Materials” in Chapter 6.5 of “Technical Specifications for Safety of Cosmetics” (2015 edition).

[0077] c. Experimental results

[0078] Table 4 Acute eye irritation results

[0079] Results of acute eye irritation test on New Zealand rabbits

[0080] (Do not rinse)

[0081]

[0082] Note: The integral mean is rounded to 2 decimal places.

[0083] Acute eye irritation of 0.5% mucin to New Zealand rabbits: No irritation under (no flushing) conditions. Furthermore, it is less irritating to human eyes.

[0084] This example verifies the safety of mucin from many aspects. The results show that mucin has the characteristics of low sensitivity and low toxicity, is a highly safe ingredient, complies with the "Cosmetic Hygiene Standards", and can be used in daily chemical products to ensure the safety of consumers.

[0085] Example 3: Animal experiment to evaluate moisturizing effect

[0086] The moisturizing effect of mucin has been previously evaluated by in vitro weighing. Here, animal experiments were performed to further confirm the moisturizing effect of mucin at the genetic level.

[0087] a. Experimental Principle

[0088] When zebrafish are treated with sodium chloride, the skin surface will lose water and shrink due to osmotic pressure. Hyaluronic acid (HA) exists in the dermis of the skin and has a moisturizing function. Hyaluronan synthase 3 (HAS3) is an enzyme molecule involved in the synthesis of hyaluronic acid, responsible for the synthesis of low molecular weight hyaluronic acid (100-1000ku). HAS3 is expressed in many tissues of late embryos and adults, can spontaneously act on the matrix around cells, and can bind to HS receptors on the cell surface to stimulate signal cascade reactions and produce a variety of physiological effects in cells. Aquaporins (AQPs) are proteins located on the cell membrane, forming "pores" on the cell membrane to control the entry and exit of water in the cell. AQP-3, or aquaporin 3, is mainly an epidermal aquaporin, a membrane transporter of water and glycerol, expressed in the plasma membrane of keratinocytes in the epidermal basal layer of normal skin, promoting water permeability and hydration of the stratum corneum. Therefore, detecting the relative expression levels of has3 and aqp3a genes can indicate whether the sample has moisturizing effect.

[0089] b. Experimental methods

[0090] The experiment was conducted according to the Standard Operating Procedures for the Evaluation of Moisturizing Efficacy of Zebrafish

[0091] Zebrafish were randomly selected in a 6-well plate, with 30 fish in each well. Water-soluble samples were given, and normal control group and model control group were set up at the same time, with a capacity of 3 mL in each well. Three biological replicates were performed. At the same time, sodium chloride was given in water-soluble solution to establish a zebrafish skin dehydration model. Incubate in the dark at 28°C for 22 hours. Total RNA of zebrafish in each experimental group was extracted, cDNA was synthesized, and q-PCR was used to detect the expression of β-actin and target genes. β-actin was used as an internal reference for gene expression to calculate the relative RNA expression of the target gene. Statistical analysis p<0.05 was judged to be significantly different.

[0092] c. Experimental results

[0093] Sample efficacy test bar graph Figure 3 As shown:

[0094] Compared with the control group, the relative expression of aqp3a gene in the 0.5% mucin sample was significantly increased, indicating that it promoted the synthesis of aquaporins, thereby promoting water permeability and hydration of the stratum corneum, and achieving a moisturizing effect.

[0095] Compared with the control group, the relative expression of has3 gene in 0.5% mucin sample was significantly increased, indicating that the sample can promote the synthesis of hyaluronidase, thereby increasing the low molecular weight hyaluronic acid in the skin, which can reach the dermis of the skin and play a deep moisturizing effect. At the same time, mucin can form a protective film on the surface of the skin and reduce water loss.

[0096] Overall, mucin can directly or indirectly target different layers of the skin, locking in moisture in the outer layer and replenishing moisture in the inner layer, making the skin hydrated and radiant from the inside out.

[0097] Example 4: Evaluation of Antioxidant Efficacy by In Vitro and Cell Methods

[0098] 1. In vitro DPPH free radical scavenging experiment

[0099] a. Experimental Principle

[0100] 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH for short) is a stable long-lived free radical. Its ethanol solution is dark purple and has strong absorption near 517nm. When there is a free radical scavenger, the light absorption of DPPH ethanol solution is weakened due to pairing with its single electron. The degree of fading of DPPH ethanol solution is linearly related to the number of electrons it accepts, which can be used to evaluate the ability of the test sample to scavenge free radicals, that is, the size of the antioxidant activity.

[0101] b. Experimental methods

[0102] This experiment was conducted in accordance with the Shanghai Daily Cosmetics Industry Association Group Standard T / SHRH 006-2018 "Cosmetics-Free Radical (DPPH) Scavenging Experiment Method".

[0103] 1) Test substance treatment: Water-soluble test substances were diluted with water to form samples of multiple concentrations, and oil-soluble test substances were diluted with 95% ethanol to form samples of multiple concentrations.

[0104] 2) According to Table 5, use 10mL test tubes to set up sample tubes (T), sample background (T0), DPPH tube (C) and solvent background (C0). Three parallel tubes should be set up for each sample sample tube (T), and three parallel tubes should be set up for DPPH (C)

[0105] 3) Add the sample solution, water or 95% ethanol solvent, 95% ethanol and DPPH ethanol solution in the order of Table 5, mix well, and stand at room temperature in the dark for 5 minutes;

[0106] 4) Transfer each reaction solution into a 1 cm cuvette or well plate and measure the absorbance at 517 nm.

[0107] To exclude false positive results, 0.3 mg / ml vitamin C was used as a positive control.

[0108] The data is processed and calculated according to the following figure:

[0109] Calculate the DPPH free radical scavenging rate:

[0110]

[0111] (1) Where:

[0112] T-sample tube absorbance, that is, the absorbance of the solution after the sample reacts with DPPH:

[0113] T 0 -Sample background absorbance:

[0114] The average value of the absorbance of the C-DPPH tube for 3 times. That is, the absorbance of the DPPH solution without adding a sample:

[0115] C 0 -Solvent background absorbance.

[0116] Table 5: Sample loading requirements

[0117] T-sample tube <![CDATA[T 0 -Sample Background]]> C-DPPH tube <![CDATA[C 0 -Solvent Background]]> Sample solution (mL) 1 1 - - Water or 95% ethanol solvent (mL) 2 2 3 3 DPPH ethanol solution (mL) 1 - 1 - 95% ethanol (mL) - 1 - 1 Parallel times 3 / Sample 1 / sample 3 / Experimental 1 / Experiment

[0118] c. Experimental results

[0119] The experimental results are shown in the following table.

[0120] Table 6 Mucin DPPH free radical scavenging rate

[0121] DPPH free radical scavenging rate 0.5% mucin 21.80％ Vc 98.26％

[0122] From the data in the table above, we can see that 0.5% mucin has a free radical scavenging rate of 21.8%, which has a certain antioxidant effect.

[0123] 2. Cell-based ROS assay

[0124] a. Experimental Principle

[0125] Malondialdehyde (MDA) is an important product of lipid peroxidation. Reactive oxygen species (ROS) are also produced in large quantities when damaged. The accumulation of these substances causes degradation of the extracellular matrix, and inflammation leads to skin aging. Moreover, MDA can also cause a large number of abnormal cross-linking of proteins, which is also one of the most important causes of skin aging. Peroxidase in cells plays a role in removing ROS and has an antioxidant effect. Among them, superoxide dismutase (SOD) and catalase (CAT) play a preventive role in the initial stage of free radical generation, while glutathione peroxidase (GSH-Px) plays an anti-lipid peroxidation role. The anti-aging effect of the sample can be preliminarily evaluated by detecting these indicators.

[0126] b. Experimental procedures

[0127] (1) 4 groups: NC, Model, 0.25% mucin

[0128] (2) Cell plating was performed with a density of 1.2*10^5 cells / ml (photographs were taken on 6-well plates and data were detected by ELISA reader on 12-well plates / 96-well plates, 5% CO 2 Incubate the cells in a stable incubator for 24 hours.

[0129] (3) Add AAPH to NC, Model, and 0.25% mucin in the plate and place in 5% CO 2 Incubate for 2 h (without Nc treatment).

[0130] (4) Prepare the drug in complete culture medium, add 0.25% mucin, M, NC and replace the medium (complete culture medium), 5% CO 2 Culture in incubator for 24 hours.

[0131] (5) Dilute the purified DCFH with serum-free medium at a ratio of 1:1000 to obtain a probe concentration of 10 μm

[0132] (6) Discard the culture medium and add 100ul (96-well plate), 600ul (12-well plate), and 1ml (6-well plate) of DCFH mixed solution and incubate for 20 min.

[0133] (7) Fluorescence microscope detection (qualitative) (six-well plate): The ROS kit uses blue light to excite green light. Adjust to the corresponding filter, focus, dim the background and take pictures (1 picture with a 10X objective lens, 5 pictures with a 20X objective lens, upper left, upper right, lower left, lower right, and middle, the above is one with a ruler and one with a ruler)

[0134] (8) Detection (quantification) by using an enzyme-labeled instrument to measure absorbance at 450 nm

[0135] (9) Result calculation

[0136] ROS clearance rate (%) = (experimental group - model group) / (model group - blank NC) * 100

[0137] c. Experimental results

[0138] Table 7 Mucin ROS clearance rate

[0139] NC Model 0.25% mucin Cell viability 100.00 80.09 80.33 ROS clearance rate (%) - - 27.49

[0140] Compared with the model group, the protein activity of 0.25% mucin increased, and the ROS clearance rate was 27.49%. This result proves that mucin has a certain degree of antioxidant activity and has application prospects in anti-aging.

[0141] Example 5: In vitro evaluation of whitening efficacy

[0142] a. Experimental Principle

[0143] Melanin synthesis is affected by tyrosinase, which can catalyze the hydroxylation of L-tyrosine into dopa and oxidize dopa into dopaquinone, which forms the final reaction product, melanin. Test substances with tyrosinase activity inhibition can slow down the tyrosinase-catalyzed conversion of L-tyrosine into dopaquinone. The absorbance of dopaquinone at 475nm is measured, and the inhibitory effect of the test substance on tyrosinase activity is evaluated based on the change in absorbance.

[0144] b. Experimental methods

[0145] The operation is carried out in accordance with the Guangdong Cosmetics Association group standard T / GDCA 006-2021 "Test Method for Inhibition of Tyrosinase Activity by Cosmetic Raw Materials".

[0146] Refer to the reagent addition amount in Table 9, add L-tyrosine solution, sample solution / solvent, and PBS buffer to each well in turn, mix thoroughly, incubate at 37°C for 10 minutes, then add 20 μL of tyrosinase solution to each well in turn, mix at 37°C for 5 minutes ± 5 seconds, then put into the microplate reader immediately, measure at 475nm wavelength, and calculate DPPH clearance. In order to exclude false positives in the experiment, 1 mg / mL kojic acid was set as a positive control.

[0147] Table 8 Tyrosinase activity loading table

[0148]

[0149]

[0150] Where:

[0151] Y-tyrosinase activity inhibition rate;

[0152] A d - Absorbance of sample reaction wells;

[0153] A c -Sample background well absorbance;

[0154] A b - Average absorbance of solvent reaction wells;

[0155] A e - Average absorbance of solvent background wells.

[0156] c. Experimental results

[0157] The experimental results are shown in the following table.

[0158] Table 9 Mucin tyrosinase inhibition rate

[0159] Tyrosinase inhibition rate 0.5% mucin 38.77％ Kojic acid (positive control) 91.33％

[0160] The inhibition rate of 0.5% mucin on tyrosinase is 38.77%. Through this experiment, it is proved that mucin has inhibitory activity on tyrosinase, which can inhibit the metabolic process of melanocytes and reduce the formation of melanin, thereby playing a role in whitening and brightening.

[0161] Example 6: Evaluation of soothing and repairing effects by in vitro, cell and animal experiments

[0162] 1. In vitro method - hyaluronidase inhibition test

[0163] a. Experimental Principle

[0164] When the body develops allergic diseases or inflammation, histamine in mast cells plays an important role as a chemical transmitter. By measuring the concentration of histamine, the anti-allergic effect of the sample can be evaluated. Hyaluronidase is a participant in allergic reactions. Studies have shown that hyaluronidase is strongly correlated with inflammation and allergies, and many anti-allergic drugs have a strong inhibitory effect on hyaluronidase activity. The anti-allergic activity is indexed by the hyaluronidase inhibition rate. The greater the hyaluronidase inhibition rate, the stronger the anti-allergic activity.

[0165] b. Test methods

[0166] Take 0.1mL 0.25mmol / L CaCl 2 The solution and 0.5 mL of hyaluronidase solution were incubated at 37°C for 20 min; 0.5 mL of 0.5% mucin was added, and the incubation was continued at 37°C for 20 min; 0.5 mL of sodium hyaluronate solution was added and incubated at 37°C for 30 min, and then placed at room temperature for 5 min; 0.1 mL of 0.4 mol / L NaOH solution and 0.5 mL of acetylacetone solution were added, and the mixture was heated in a boiling water bath for 15 min, and then immediately cooled with ice water for 5 min; 1.0 mL of Ehrlich reagent was added and diluted with 3.0 mL of anhydrous ethanol, and the mixture was placed for 30 min to develop color, and the absorbance at 530 nm was determined by a spectrophotometer.

[0167] In order to exclude false positives, a positive control of 6 mg / ml dipotassium glycyrrhizinate was set up.

[0168] Calculation of hyaluronidase inhibition rate

[0169]

[0170] Where:

[0171] A: OD value of control solution (acetic acid buffer solution is used instead of sample solution);

[0172] B: OD value of the control blank solution (using acetate buffer solution instead of sample solution and enzyme solution);

[0173] C: OD value of sample solution;

[0174] D: OD value of the sample blank solution (using acetate buffer solution instead of enzyme solution).

[0175] c. Experimental results

[0176] The specific experimental results are shown in the table below.

[0177] Table 10 Mucin hyaluronidase inhibition rate

[0178] Hyaluronidase inhibition rate 0.5% mucin 26.54％ Dipotassium glycyrrhizinate 88.53％

[0179] According to the above results, 0.5% mucin has a certain inhibitory effect on hyaluronidase, and can alleviate the inflammation and allergy problems caused by hyaluronidase.

[0180] 2. Cell method - detection of inflammatory factor IL-6

[0181] a. Experimental Principle

[0182] Lipopolysaccharide (LPS) stimulates macrophages through TOLL-like receptor 4 (TLR4), activates inflammatory signaling pathways such as NF-KB, thereby inducing the release of inflammatory factors such as IL-6 and aggravating the inflammatory response. The classic cell model of lipopolysaccharide-induced inflammatory response in mouse macrophage RAW264.7 was used to evaluate the inhibition of IL-6 secretion by the samples by comparing the difference in IL-6 content between the model control group and the sample group, thereby determining whether the samples have a soothing effect.

[0183] b. Experimental procedures

[0184] Operate in accordance with the China Association of Non-Public Medical Institutions Standard T / CNMIA 0013-2020 "Safety / Efficacy Evaluation Standards for Allergy-Relief Functional Skin Care Products".

[0185] Take cells with good morphology and prepare cell suspension with complete medium, inoculate them in 24-well plates and incubate in CO 2 After incubation in the incubator for 24 h, 0.25% mucin sample was added, and a blank control group and a positive control group (20 ng / mL dexamethasone, etc.) were set up in parallel, with 3 replicates in each group. 2 The cells were incubated in an incubator for 2 h. LPS was added to each group except the blank control group at a final concentration of 1 μg / ml. 2 After incubation in the incubator for 24 h, the supernatant was collected by low-temperature centrifugation, and the IL-6 content was determined according to the instructions of the ELISA kit, and the experimental results were analyzed by data processing software.

[0186] c. Experimental results

[0187] The experimental results are shown in the following table

[0188] Table 11 Mucin anti-inflammatory results

[0189]

[0190] Compared with the model control group, the IL-6 content in the sample group was reduced, proving that 0.25% mucin can reduce inflammatory response and play a soothing role.

[0191] 3. Animal experiment-zebrafish neutrophil inhibition rate

[0192] a. Experimental Principle

[0193] Sodium dodecyl sulfate (SLS) may cause irritation in the body after acting on it. The irritant enters the zebrafish body, induces an inflammatory response, and neutrophils have an immune response, migrating to the skin epidermis and gathering. The changes in the number of neutrophils in the skin of transgenic neutrophil green fluorescent strain zebrafish (MPX) before and after treatment were used to detect whether the sample has a soothing effect.

[0194] b. Test methods

[0195] According to the Zhejiang Health Products and Cosmetics Industry Association T / ZHCA 016-2022 "Evaluation of the Soothing Efficacy of Cosmetics by Neutrophil Inhibition Rate in Zebrafish Fry"

[0196] Randomly select zebrafish in a 6-well plate, 15 per well. Water-soluble SLS was given to establish a zebrafish skin inflammation model. Water-soluble samples were given, and a normal control group and a model control group were set up at the same time, with a capacity of 3 mL per well. Incubate at 28°C in the dark for 18 hours. Ten zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. Advanced image processing software was used to analyze and collect data, analyze the number of neutrophils in zebrafish skin (N), and calculate the soothing effect of the sample according to the formula to determine whether it has a soothing effect.

[0197]

[0198] c. Experimental results

[0199] The experimental results are shown in Figure 4 It was observed that the number of neutrophils in the sample group was significantly reduced compared with the model control group, indicating that mucin can relieve inflammatory response.

[0200] Example 7: Cell experiment to evaluate the repair efficacy

[0201] 1. Cell Migration Assay

[0202] a. Experimental Principle

[0203] The wound healing assay is a simple, inexpensive method and one of the earliest methods developed to study directed cell migration in vitro. The method mimics the process of cell migration during healing in vivo. The basic steps include creating a "wound" in a cell monolayer, capturing images at the beginning and periodically during cell migration to close the wound, and comparing the images to determine the rate of cell migration.

[0204] b. Experimental procedures

[0205] 1) Marking: First, use a marker pen to draw horizontal lines evenly on the back of the 6-well plate, using a ruler to measure, approximately one line every 0.5 to 1 cm, across the holes, and at least 5 lines in each hole;

[0206] 2) Plating: Cells in the logarithmic growth phase are trypsinized into single cell suspension and inoculated into 6-well culture plates; 6 cells are plated / well, and the inoculation principle is that the fusion rate reaches 100% after overnight, and the final total volume of culture medium per well is 2 mL;

[0207] 3) The cells were cultured in a 37°C, 5% CO2 incubator for 24 h;

[0208] 4) Scratching: On the second day, use a 200 μL pipette tip to scratch the 6-well plate cover or ruler parallel or perpendicular to the horizontal line on the back. The pipette tip should be vertical and not tilted.

[0209] 5) Washing: Wash the cells 3 times with PBS, remove the scratched cells, and add serum-free culture medium;

[0210] 6) Take photos: Wipe off the horizontal scratch marks on the back of the 6-well plate. Take photos under a 4x microscope, make sure the scratch marks are centered and vertical, and pay attention to the consistency of the background. Add the sample to be tested, set the concentration gradient, and take samples at 4, 6, or 24 hours and take photos.

[0211] 7) Result analysis: After opening the image using Image J software, 6 to 8 horizontal lines were randomly drawn to calculate the mean of the distance between cells or the mean of the scratch area.

[0212] 8) Data processing:

[0213] 9) Cell migration rate (wound healing rate) = (initial scratch area - scratch area at time t) / initial scratch area

[0214] c. Experimental results

[0215] The experimental results are shown in the following table and Figure 5 shown.

[0216] Table 12 Cell scratch repair results

[0217] 4h 6h 24h 0.25％ 1.23 4.18 88.26 0.5％ -1.45 2.01 81.04

[0218] From the above table, we can see that the 0.25% and 0.5% concentrations of mucin have significant effects on cell repair, and the cell repair rate can reach up to 88% in 24 hours, which reveals that mucin has great potential in wound repair.

[0219] 2. Oxidative damage repair

[0220] a. Experimental Principle

[0221] Malondialdehyde (MDA) is an important product of lipid peroxidation. Reactive oxygen species (ROS) are also produced in large quantities when damaged. The accumulation of these substances causes degradation of the extracellular matrix and inflammation leading to skin aging. AAPH (azobisisobutylamidine hydrochloride) can induce the production and accumulation of cellular ROS. By using CCK8 to detect the effect of raw materials on the proliferation activity of AAPH-treated skin keratinocytes (HaCaT), the safety of the raw materials can be determined and the repair effect of the raw materials on damaged skin keratinocytes can be evaluated.

[0222] b. Experimental procedures

[0223] First, take cells in the logarithmic growth phase and inoculate the cell suspension in a 96-well plate with 100 μL per well to make the cell density 7000 / well. After 24 days of culture, perform the drug addition operation (AAPH) and place it in a 5% CO2 incubator at 37°C for 2 hours; then, after the culture is completed, discard the culture medium and add complete culture medium containing samples of different concentrations to continue culturing for 24 hours; finally, after the culture is completed, add 10 μL of CCK8 reagent to each well, set up a blank group, and exclude the influence of the color of the working solution. Incubate the 96-well plate in the incubator for about 45-90 minutes, and measure the absorbance at 450nm using an enzyme reader.

[0224] The cell proliferation rate was calculated according to the following formula:

[0225] Cell repair rate % = (OD value of experimental group - blank group) / (control group - blank group) × 100.

[0226] c. Experimental results

[0227] The experimental results are shown in the following table:

[0228] Table 13 Cell oxidative damage repair results

[0229] blank Model Group 0.5% mucin Cell viability 100.00 80.09 92.14 Repair rate - - 60.51

[0230] Compared with the model group, the cell activity in the sample group was significantly increased, proving that it has a repairing effect on oxidatively damaged cells and has potential applications in anti-aging skin care products.

Claims

1. A use of yeast-derived mucin in preparing a whitening and brightening product, wherein the amino acid sequence of the yeast-derived mucin is as shown in SEQ ID NO:

1.

2. Use of yeast-derived mucin in preparing a soothing and anti-inflammatory product, wherein the amino acid sequence of the yeast-derived mucin is as shown in SEQ ID NO:

1.

3. Use of yeast-derived mucin in the preparation of antioxidant and anti-aging products, wherein the amino acid sequence of the yeast-derived mucin is as shown in SEQ ID NO:

1.

4. Use of yeast-derived mucin in preparing a product for repairing cell damage, wherein the amino acid sequence of the yeast-derived mucin is as shown in SEQ ID NO:

1.

5. The use according to any one of claims 1 to 4, It is characterized in that The product is a skin care product, gel dressing, ointment, spray, shampoo, conditioner or eye drops.

6. The use according to any one of claims 1 to 4, It is characterized in that The yeast-derived mucin is obtained by a recombinant expression method.

7. The use according to claim 6, It is characterized in that The yeast cells into which the recombinant expression vector containing the gene encoding the yeast-derived mucin is introduced are cultured to produce the yeast-derived mucin.

8. The use according to claim 7, It is characterized in that The yeast cell is Pichia pastoris, specifically GS115 or KM71H strain.

9. The use according to claim 8, It is characterized in that The fermentation was carried out by shake flask fermentation or fed-batch fermentation.

10. The use according to claim 9, It is characterized in that The method also includes the separation and purification steps of yeast-derived mucin, and the specific purification steps are as follows: 1) Use ethanol precipitation method to purify yeast fermentation broth, use 40% or more ethanol final concentration to directly precipitate the target protein; or first use 20%-35% ethanol final concentration, let it stand at 4°C for 1-2 hours to precipitate the impurities, and then use 40% ethanol final concentration, let it stand at 4°C for 1-2 hours to precipitate the target protein; 2) Further comprising: using chromatography to perform fine purification of the target protein, specifically, the selected chromatographic medium is an anion exchange chromatographic medium, including but not limited to strong anion exchange, weak anion exchange, and mixed mode ion exchange, and the selected buffer is a buffer of pH 5.0-7.5, wherein the buffer pair can be a buffer that can reach the target pH, such as citric acid-sodium citrate, Tris-HCl, Bis-Tris-HCl, etc., and NaCl is used for gradient elution to obtain the target protein.

Images