阅读说明：本技术 牦牛奶发酵提取物的应用 (Application of yak milk fermentation extract ) 是由 李俊 章漳 黄�俊 于 2020-03-13 设计创作，主要内容包括：本发明属于天然提取物及日化领域,具体涉及牦牛奶发酵提取物的应用。牦牛奶经过植物乳杆菌的发酵,发酵液离心过滤后得到的牦牛奶发酵提取物,能够降低金黄色葡萄球菌/表皮葡萄球菌的比值、促进真皮成纤维细胞和表皮细胞增殖,从而实现调节皮肤菌群、维持和/或修复皮肤表面微生态,以及修复老化的皮肤及美白,因此,牦牛奶发酵提取物可用作制备皮肤外用剂,作为修复老化皮肤、皮肤美白、改善皮肤表面微生态的活性成分。(The invention belongs to the field of natural extracts and daily chemicals, and particularly relates to an application of a yak milk fermentation extract. The yak milk fermented extract obtained by fermenting yak milk with lactobacillus plantarum and centrifugally filtering fermentation liquor can reduce the ratio of staphylococcus aureus to staphylococcus epidermidis and promote the proliferation of dermal fibroblasts and epidermal cells, so that the purposes of regulating skin flora, maintaining and/or repairing skin surface micro-ecology, repairing aged skin and whitening are achieved, and therefore, the yak milk fermented extract can be used for preparing skin external preparations and used as active ingredients for repairing aged skin, whitening skin and improving skin surface micro-ecology.)

1. Application of Yak milk fermented extract in preparing skin external preparation is provided.

2. The use as claimed in claim 1, wherein said fermented extract of yak milk is used as an active ingredient for maintaining and/or restoring the micro-ecology of the skin surface; or as an anti-aging active ingredient; or as an active ingredient for whitening.

3. The use as claimed in claim 2, wherein said fermented extract of yak milk maintains and/or restores the skin surface micro-ecology by regulating the skin flora; the yak milk fermented extract realizes an anti-aging effect by promoting skin cell proliferation and repairing aged skin; the yak milk fermented extract realizes the whitening effect by inhibiting the tyrosinase activity of skin cells.

4. The use of claim 3, wherein said fermented Yak milk extract reduces the Staphylococcus aureus/Staphylococcus epidermidis ratio to modulate skin flora or promote proliferation of dermal fibroblasts and/or epidermal cells.

5. The application of claim 1, wherein the yak milk fermented extract is fermented filtrate of yak milk fermented by lactobacillus plantarum, or diluted solution, concentrated solution, extract or dried substance prepared from the fermented filtrate.

6. A skin external preparation is characterized by comprising fermented extract of yak milk; the yak milk fermented extract is used as an active ingredient for maintaining and/or repairing micro-ecology on the surface of the skin or used as an active ingredient for repairing aged skin; or as an active ingredient for whitening.

7. The external preparation for skin as claimed in claim 6, wherein the yak milk fermented extract is contained in an amount of 0.005wt% to 5wt% in terms of solid content.

8. An active ingredient raw material of a skin external agent is characterized in that yak milk fermentation extract is used as an active ingredient, and the active ingredient contains 4-10 wt% of butanediol, 0.45-0.72 wt% of phenoxyethanol and 0.05-0.08 wt% of ethylhexyl glycerol.

9. The method for preparing a raw material for an external preparation for skin according to claim 8, comprising the steps of: (1) centrifuging fermentation liquor obtained by fermenting yak milk with liquid at 4-10 ℃ and 1000-8000 rpm, and taking supernatant; (2) adjusting the pH of the supernatant to 5.0-7.0, heating at 80-90 ℃ for 30-60 min, centrifuging at 1000-8000 rpm, and taking the supernatant; (3) filtering the supernatant with a filter membrane with the pore diameter of 0.05-0.22 mu m under the pressure of 1-5 bar, and taking the fermentation filtrate; (4) adding butanediol, phenoxyethanol and ethylhexyl glycerol into the fermentation filtrate in sequence.

10. The toning lotion is characterized by comprising the following components: 0.01wt% -3 wt% of yak milk fermented extract by solid content, and: 0-5 wt% of glycerol, 2-5 wt% of butanediol, 0-4 wt% of hydroxyethyl urea, 0-2 wt% of nicotinamide, 0-0.02 wt% of sodium hyaluronate or potassium hyaluronate, 0-0.2 wt% of dipotassium glycyrrhizinate, 0-0.2 wt% of allantoin, 0-0.2 wt% of methylparaben, 0-0.1 wt% of EDTA disodium, 0-0.5 wt% of phenoxyethanol, 0-0.1 wt% of ethylhexyl glycerol, 0-acceptable amount of essence, and the balance of deionized water.

Technical Field

The invention belongs to the field of plant extraction and the field of daily chemicals, and particularly relates to an application of a yak milk fermentation extract.

Background

It is not uncommon for humans to record cosmetic skin care with milk and dairy products, as gulomains believe that a soaked bread wipe in milk may be white, tender, and smooth in skin. The milk protein and fat in the milk can be attached to the surface of the skin, so that the effect of moistening the skin can be achieved; the enzyme in milk can promote decomposition of cutin on skin surface. The yak milk contains high content of vitamins, proteins, amino acids, trace elements, etc. Compared with milk, the milk has more abundant nutrient substances and higher content, for example, the content of amino acid and vitamin A is respectively 15 percent and 6 percent higher than that of the milk; the calcium content is 1.13 times of common milk, and the zinc content is 3 times of milk. It has been reported that casein in milk can be used as a humectant, a nutrient and a conditioner in cosmetics, and yak milk protein can be used as an additive in hair conditioner and shampoo.

Most of the lactic acid bacteria are non-toxic and harmless, and the lactic acid bacteria and metabolites thereof have strong antioxidant activity. Its antioxidant activity is mainly directed at superoxide anion free radical (O)₂-), hydroxyl radical (OH-), and 1, 1-diphenyl-2-bitter hydrazino radical (DPPH), and can be used for preventing and treating lipid peroxidation. Lactobacillus plantarum (A)Lactobacillus plantarum; L. plantarum) The lactobacillus acidophilus milk is commonly used for preparing traditional dairy products such as yoghourt and the like, can regulate the intestinal microbial balance of a human body, has good effects of reducing blood fat, reducing blood sugar and the like, is common probiotic food and dairy product producing bacteria, and has a cell-dissolving supernatant which also has an inhibiting effect on melanin generation. Meanwhile, the research that the lactobacillus has an inhibiting effect on main pathogenic bacteria (staphylococcus aureus and streptococcus pyogenes) of bacterial skin infection can be seen.

Human skin fibroblasts are the most important cells in the dermal reticulum of the skin and are one of the major repair cells after skin aging and cell damage. It can promote the migration, proliferation and differentiation of epidermal cells, secrete great amount of collagen, elastic fiber protein and cell repairing factors, and has powerful self-renewing capacity to repair aged skin.

The surface of human skin and the intestinal tract store a great amount of microorganisms, which accompany us from birth and are in symbiotic relationship with human beings. Wherein, the skin surface flora or surface microecology is used as an important biological barrier on the outer layer of the physical barrier of the human skin, and has an important effect on the health state of the skin. The surface flora has obvious individual difference according to different sexes, ages, external environments and the like, and the unbalance of the surface flora, particularly the proportion of some conditional pathogenic bacteria in the surface flora is abnormal, can cause various skin diseases. Staphylococcus epidermidisS. e（Staphylococcus epidermidis; S. epidermidis) Is one of the resident bacteria of human skin, staphylococcus aureusS. a（Staphylococcus aureus; S. aureus) When the ratio is abnormal, skin surface barrier is broken or skin problems such as inflammation are caused.

Microorganisms are important members of maintaining the skin's microecological balance. Skin microorganisms are mainly bacteria, and can be divided into resident bacteria and transient bacteria. The resident flora is closely related to skin health, and the resident flora is planted on the surface of the skin to form a biological film, so that the resident flora has an occupation protection effect and can be used as a physical barrier to block the invasion of exogenous pathogenic bacteria, and the resident flora can also inhibit or kill pathogenic bacteria by generating antibacterial peptide, wherein the pathogenic bacteria mainly comprise staphylococcus epidermidis, micrococcus, corynebacterium, propionibacterium, acinetobacter, malassezia and the like. The transient bacteria include Staphylococcus aureusS. aureusStreptococcus, escherichia coli, candida albicans, and the like, which are major pathogenic bacteria causing skin infections.S. aureusIs one of common skin infection bacteria. Studies have shown that atopic dermatitis patients (AD) have skin lesionsS. aureusThe planting rate reaches 78 percent. The existing research is conducted to find that the microecology on the surface of the AD skin is in an unbalanced state, which not only shows that the AD skin is in an unbalanced stateNumber of aureusThe quantity is obviously increased, and the relative abundance, the diversity and the abundance of other skin resident bacteria are reflectedThe reduction of the degree.

In the report of the determination of skin flora of patients with eczema dermatitis and the clinical research thereof (menghua, electronic journal of clinical medicine literature, 2018), the determination of skin flora shows that: the staphylococcus aureus (57.50%) and staphylococcus epidermidis (32.50%) in the observation group (40 cases of various dermatitis eczema patients) are significantly higher than those in staphylococcus aureus (0%) and staphylococcus epidermidis (7.50%) in the control group (40 cases of healthy people). According to the calculation of the report, the skin of the patientS. a/S. eHas a ratio of 1.77, and is found in healthy skinS. a/S. eThe ratio of (A) to (B) is 0%.

Human skin fibroblasts are the most important cells in the dermal reticulum of the skin and are one of the major repair cells after skin aging and cell damage. It can promote the migration, proliferation and differentiation of epidermal cells, secrete great amount of collagen, elastic fiber protein and cell repairing factors, and has powerful self-renewing capacity to repair aged skin.

Thus, if the proliferation of human skin fibroblasts can be promoted, the skin aging can be delayed and repaired, or the ratio of staphylococcus aureus/staphylococcus epidermidis is reduced, namelyS. a/S. eThe skin surface flora and surface micro-ecology can be improved, and the skin is healthier. If a single active substance can solve the above problems simultaneously, the skin external preparation can have multiple effects on the basis of simplification of the ingredients.

At the same time, the color of the skin comes from melanin stored in keratinocytes. Generally, people who store melanin many have darker and more protected skin color from solar radiation. Melanin is an important factor in determining skin color in terms of quantity and quality of melanin. Tyrosinase is a copper-containing oxidoreductase with a complex structure, and is widely present in microorganisms, animals, plants, and human bodies. Thus, tyrosinase is a key enzyme in the synthesis of melanin by the skin in humans.

However, at present, there is no report that an extract obtained by fermenting yak milk is used as a skin external preparation, and there is no report that the yak milk fermented extract is used for improving skin surface micro-ecology or repairing aged skin, and is used as an active ingredient for whitening by inhibiting tyrosinase activity.

Disclosure of Invention

The invention aims to provide application of a yak milk fermentation extract in preparation of a skin external preparation.

The invention also provides an external skin preparation which contains the yak milk fermentation extract.

The technical scheme is as follows: a fermented extract of yak milk is used for preparing skin external preparation. Specifically, the yak milk fermented extract is used as an active ingredient for maintaining and/or repairing skin surface micro-ecology; or as an active ingredient for repairing aged skin; or as an active ingredient for whitening.

The yak milk fermented extract maintains and/or restores the skin surface micro-ecology by regulating skin flora; the yak milk fermented extract can repair aged skin by promoting skin cell proliferation; the yak milk fermented extract realizes the whitening effect by inhibiting the tyrosinase activity of skin cells.

Further, the yak milk fermented extract can reduce the ratio of staphylococcus aureus to staphylococcus epidermidis to regulate skin flora, or promote proliferation of dermal fibroblasts and/or epidermal cells.

The fermented extract of yak milk can be obtained by liquid fermentation method, mixing yak milk with water, and fermenting. Preferably, the lactobacillus plantarum is used. Centrifuging the fermentation liquor to obtain supernatant, and filtering to obtain filtrate to obtain yak milk fermented extract. Or diluting, concentrating, or making into extract or dry powder.

The preparation method of the yak milk fermentation extract comprises the following steps: (1) mixing yak milk or dry yak milk powder with water to obtain culture medium, inoculating Lactobacillus plantarumLactobacillus plantarum; L. plantarum) Fermenting, namely fermenting for 16-48 hours at constant temperature of 30-40 ℃ and 150-250rpm to carry out liquid fermentation; (2) centrifuging the fermentation liquor for 10-60 min at the temperature of 4-10 ℃ and the rpm of 1000-8000, and taking supernatant; (3) adjusting the pH of the supernatant to 5.0-7.0, heating at 80-90 deg.C for 30-60 min, and centrifuging at 1000-8000 rpm for 10 ℃Taking supernatant after 30 min; (4) filtering the supernatant with a filter membrane with the pore diameter of 0.05-0.22 mu m under the pressure of 1-5 bar, and taking the fermentation filtrate. The content of yak milk solid in the culture medium is 1wt% -5 wt%, and the balance is water. The preferable fermentation temperature is 35-40 ℃.

The yak milk fermentation extract is yak milk fermentation filtrate, or a diluent, a concentrated solution, an extract or a dried substance prepared from the yak milk fermentation filtrate.

Preferably, the yak milk is produced in the pan-himalayas region.

A skin external preparation contains fermented extract of yak milk; the yak milk fermented extract is used as an active ingredient for maintaining and/or repairing micro-ecology on the surface of the skin or used as an active ingredient for repairing aged skin; or as an active ingredient for whitening.

The content of the yak milk fermented extract is 0.001-5 wt% based on the dry matter content; preferably, the content of the yak milk fermented extract is 0.005wt% -5 wt% based on the dry matter content. More preferably, the content of the yak milk fermented extract is 0.01wt% -2 wt% based on the dry matter content.

Preferably, the external preparation for skin further contains at least one of the following ingredients: a moisturizing active ingredient, a whitening active ingredient, a moisturizing active ingredient, an antioxidant active ingredient, an anti-wrinkle active ingredient, a spot-removing active ingredient, an acne-eliminating active ingredient, a sunscreen active ingredient, an acne/acne-removing active ingredient, a dandruff-removing active ingredient, an antiallergic active ingredient, or a sebaceous gland-inhibiting active ingredient.

The skin external agent is a product which is spread on any part of the surface of a human body by smearing, spraying or other similar methods so as to clean, maintain, beautify, modify, change the appearance or correct the smell of the human body and keep a good state. The skin external preparation can be basic cosmetics, facial cosmetics, head care products, and body care products for cleaning, caring, beautifying or caring skin.

Preferably, the external preparation for skin is a cosmetic.

A skin external preparation raw material takes yak milk fermentation extract as an active ingredient, and contains 4-10 wt% of butanediol, 0.45-0.72 wt% of phenoxyethanol and 0.05-0.08 wt% of ethylhexyl glycerin.

The preparation method of the raw materials of the skin external agent comprises the following steps: (1) centrifuging fermentation liquor obtained by fermenting yak milk or dry yak milk powder in liquid for 10-60 min at the temperature of 4-10 ℃ and the rpm of 1000-8000, and taking supernatant; (2) adjusting the pH of the supernatant to 5.0-7.0, heating at 80-90 ℃ for 30-60 min, centrifuging at 1000-8000 rpm for 10-30 min, and taking the supernatant; (3) filtering the supernatant with a filter membrane with the pore diameter of 0.05-0.22 mu m under the pressure of 1-5 bar, and taking the fermentation filtrate; (4) adding butanediol, phenoxyethanol and ethylhexyl glycerol into the fermentation filtrate in sequence.

A lotion comprises the following components: 0.01wt% -3 wt% of yak milk fermented extract by solid content, and: 0-5 wt% of glycerol, 2-5 wt% of butanediol, 0-4 wt% of hydroxyethyl urea, 0-2 wt% of nicotinamide, 0-0.02 wt% of sodium hyaluronate or potassium hyaluronate, 0-0.2 wt% of dipotassium glycyrrhizinate, 0-0.2 wt% of allantoin, 0-0.2 wt% of methylparaben, 0-0.1 wt% of EDTA disodium, 0-0.5 wt% of phenoxyethanol, 0-0.1 wt% of ethylhexyl glycerol, 0-acceptable amount of essence, and the balance of deionized water.

The invention has the beneficial effects that the yak milk fermented extract obtained by fermenting yak milk with lactobacillus plantarum and centrifugally filtering fermentation liquor can reduce the ratio of staphylococcus aureus to staphylococcus epidermidis and promote the proliferation of dermal fibroblasts and epidermal cells, thereby realizing the regulation of skin flora, the maintenance and/or restoration of skin surface micro-ecology and the restoration of aged skin. Therefore, the yak milk fermented extract can be used as an active ingredient of a skin external agent, especially a cosmetic; can be used for preparing skin external preparations, in particular to skin external preparations with the functions of regulating skin flora, maintaining and repairing skin surface micro-ecology, repairing aged skin and whitening skin, and has good application prospect.

Drawings

FIG. 1 shows the relationship between filtrate of fermented product of yak milk and surface flora,S. a/S. einfluence of the ratio

Fig. 2 shows the effect of yak milk fermentation product filtrate on the growth of fibroplast.

FIG. 3 shows the effect of filtrate of yak milk fermentation product on HaCaT growth as shown in FIG. 3.

Detailed Description

EXAMPLE 1 preparation of Yak milk fermentation product filtrate

The matrix raw material of the fermentation product is yak milk or dry yak milk powder, and is produced in pan-xi-Mara areas. Mixing yak milk with deionized water, wherein the content of yak milk solid in a fermentation medium/culture solution is 4 wt%; lactobacillus plantarum for cooling after high-temperature and high-pressure sterilizationLactobacillus plantarum) Fermenting at 35-40 deg.C and 150-250rpm for 36 hr.

Preparing yak milk fermentation filtrate:

(1) centrifuging the fermentation product for 10-30 min at 4-10 ℃ and 6000rpm, and taking supernatant;

(2) adjusting the pH value to 5.0-7.0, carrying out water bath at 80-90 ℃ for 30-60 min, centrifuging at 1000-4000 rpm for 10-30 min, and taking supernatant;

(3) selecting a filter membrane with the aperture of 0.05, 0.10 or 0.22 mu m, and pre-soaking the filter membrane in 75% (v/v) ethanol; the filter and the equipment are cleaned by 75% (v/v) ethanol and dried by a 70 ℃ oven for use;

(5) the membrane filtration device is firstly used for circulating and soaking 0.5 percent NaOH aqueous solution for 5-10 min, and then 0.5 percent NaHSO is used₃Circulating or soaking the water solution for 5-10 min, and then cleaning with deionized water. After the membrane is arranged in equipment, sterilizing water is added for cleaning, and then the water is emptied;

(6) inoculating the supernatant in the step (2) for filtration, controlling the filtration speed (1.0-2.0L/h) by adjusting the pressure (less than or equal to 5 bar), and storing the fermentation filtrate after filtration in a sterile container.

The dry matter content of the obtained yak milk fermentation filtrate was 1.5 wt%.

Example 2 Effect of Yak milk fermentation extract on surface flora

(I) test materials

1. Experimental strains

EpidermisStaphylococcus aureusS. e（Staphylococcus epidermidis) Staphylococcus aureusS. a（Staphylococcus aureus）。

2. Culture medium

LB culture medium: 0.5 percent of powder, 1.0 percent of tryptone, 1.0 percent of NaCl and the balance of water, wherein the percentages are mass percentages. Sterilizing at 121 deg.C for 20min under high temperature and high pressure, and cooling.

3. Sample information

Blank sample (Control): sterile physiological saline: 0.9 percent of NaCl, and the balance of water, wherein the percentages are mass percentages. Sterilizing at 121 deg.C for 20min under high temperature and high pressure, and cooling.

Experimental samples: the filtrate of the fermented yak milk obtained in example 1 had a solid content of 1.5 wt%.

(II) Experimental procedure

1. To be preservedS. aAndS. einoculating (single colony or frozen tube bacterial liquid) into a sterile LB liquid culture medium for activation recovery (1 CFU/4 mL or 50 mu L/mL); culturing for 8-24 hours at 37 ℃ and 250rpm of a shaking table; LB Medium sustainableS. a AndS. eafter culturing for 16h, both can reach the growth stable period;

2.2000-6000 rpm, removing the culture medium by centrifugation, and gradually diluting the resuspended bacteria solution with physiological saline (C)₀) To 1/2C₀、1/4C₀And 1/8C₀Determining the OD of two kinds of resuspended bacteria solutions at each concentration₆₀₀Determining the ratio of the concentration of the two bacteria, and determining the OD of each₆₀₀The values are diluted proportionally and the equal OD is regained₆₀₀And (4) bacterial suspension.

3. By usingS. aAndS. eadding yak milk fermentation product filtrates with different concentrations into a sterile LB culture medium respectively to obtain the yak milk fermentation filtrate (dry weight) culture medium with the mass percentages of 0.015%, 0.15%, 0.3% and 0.45%, testing the total volume of the system to be 4mL, and inoculating according to the volume ratio of 0.2-2%. Culturing for 8-24 hr, and measuring light absorption OD by turbidimetry₆₀₀Denotes the biomass of the microorganism. Significant differences were considered as p < 0.05, p < 0.01, p < 0.001.

Filtrate (dry weight content) pair of yak milk fermentation products with different concentrationsS. a/S. eThe regulating action of (2) is shown in fig. 1. The experimental result shows that the filtrate of the yak milk fermentation product can influenceS. a/S. eRatio, selectivity influenceS. aAndS. egrow, can reduceS. a/S. eThe value helps to maintain the steady state of the microbial flora on the surface of the human body or adjust and restore the unbalanced skin surface micro-ecology.

As shown in fig. 1, at four concentrations,S. a/S. ethe values of (a) were reduced by 5.24%, 33.31%, 49.64 and 65.84%, respectively, compared to 1.660 for the blank. This indicates that the yak milk fermentation product can significantly reduce the biomass ratio and gradually decrease with increasing concentration; the yak milk fermented product has the function of promoting the growth of skin surface microorganismsS. aAndS. eis adjusted to the potential for a more healthy state.

Example 3 cell proliferation of fermented product of Yak milk

(I) test materials

Cell: dermal Fibroblast, human immortalized epidermal cell HaCaT.

Experimental samples: preparing yak milk fermentation product filtrate into solutions with different concentrations by using deionized water, and adding yak milk fermentation product filtrate with each concentration into serum-free DMEM or MEM culture media required by different cell growth, so that the dry matter content of the yak milk fermentation product filtrate in the culture media is 0.015%, 0.075% and 0.15%, wherein the percentages are mass percentages.

(II) Experimental method

Preparing solutions with different concentrations from yak milk fermentation product filtrate by using deionized water, adding the solutions into human fibroblast culture solution, taking deionized water without a sample as a blank control, culturing for 48 hours, dyeing cells by using an MTT method, measuring absorbance at 550nm by using an enzyme labeling instrument, and evaluating the proliferation effect on human fibroblasts by referring to the blank control, wherein the proliferation rate of the blank control is 100%. Significant differences were considered as p < 0.05, p < 0.01. The dry matter content of the yak milk fermentation product filtrate in the serum-free medium is respectively 0.015%, 0.075% and 0.15%, and the percentages are mass percentages.

(III) results of the experiment

The effect of yak milk fermentation product filtrate on fibroplast growth is shown in fig. 2, and the effect on HaCaT growth is shown in fig. 3.

As shown in the results of figures 2 and 3, yak milk fermentation product filtrates (dry weight content) with different concentrations have proliferation effects on fibroblasts and epidermal cells, and with the increase of the concentrations, the growth promotion effects on the fibroblasts and the epidermal cells are gradually enhanced, and the yak milk fermentation product filtrate with the concentration of 0.15 wt% can enable the highest cell activities of the fibroblasts and the HaCaT to reach 143.72% and 140.03% respectively.

Therefore, the yak milk fermentation product filtrate, namely the yak milk fermentation extract, can promote the proliferation of human skin cells, so that aged skin is repaired, and the anti-aging effect is achieved.

Example 4 cell proliferation of fermented product of Yak milk

(I) test materials

Cell: mouse melanoma cell B16

Experimental samples: and adding the sterile yak milk fermentation product filtrate stock solution into a B16 cell culture medium, wherein the dry matter content of the yak milk fermentation product filtrate is 0.015 percent, and the percentages are mass percentages.

(II) Experimental method

The inhibition effect of the sample on tyrosinase was determined by the L-Dopa oxidation method. B16 melanocyte is cultured in 96-well plate at 1 × 105 density, after 24h, sterile yak milk fermentation product filtrate is added into the cells at 0.015% for culturing for 48h, culture solution is removed, 100 μ L of PBS buffer solution containing 1% TritonX-100 is added into each well, 50 μ L of 0.2 mg/mL L-DOPA is added, and light absorption value at 490 nm is measured after treatment at 37 ℃ for 3 h. The enzyme activity was calculated as follows: tyrosinase inhibition rate = [1- (experimental OD value/experimental cell density)/(control OD value/control cell density) ] × 100%. Meanwhile, the MTT method is used for measuring the influence of the sample on the vitality of the B16 cells.

(III) results of the experiment

The experimental results show that 0.015% of yak milk fermentation product filtrate has no significant influence on the activity of B16 cells and can obviously inhibit the activity of tyrosinase.

TABLE 1 mouse B16 melanoma cell tyrosinase inhibition results

	Test concentration	B16 cell viability (%)	B16 tyrosinase inhibition (%)
				Control	-	100	0
Arbutin (positive control)	0.1mg/mL	99	25
				Filtrate of yak milk fermentation product	0.015%	98	35

Example 5 use of Yak milk fermentation product filtrate for preparation of cosmetic/skin external preparation raw Material

The treatment process comprises the following steps: butanediol, phenoxyethanol and ethylhexyl glycerol are added into the filtrate obtained in the example 1 in sequence, and are fully stirred to be uniformly mixed, wherein the content of butanediol, phenoxyethanol and ethylhexyl glycerol in the mixed sample is 4-10 wt%, 0.45-0.72 wt% and 0.05-0.08 wt%. Finally, the samples are stored at room temperature or 4 ℃ in the dark. When in production, the cosmetic water/essence/emulsion/cream is added according to the preparation method of the cosmetic water/essence/emulsion/cream conventional in the field. The above formula product can also be directly used as a cosmetic lotion.

Example 6 cosmetic lotion containing fermented extract of Yak milk

Taking the yak milk fermentation product filtrate of example 1 to prepare the toning lotion with the functions of regulating the skin microecological flora and repairing, and according to the preparation method of the toning lotion which is conventional in the field, the components contained in the toning lotion are shown in the table 1:

TABLE 2

。