CN110305920B

CN110305920B - Active fermentation product and preparation method and application thereof

Info

Publication number: CN110305920B
Application number: CN201910591583.2A
Authority: CN
Inventors: 刘露; 张娇; 韩志东
Original assignee: Quanhou (guangzhou) Research Institute Of Biotechnology Co Ltd
Current assignee: Quanhou (guangzhou) Research Institute Of Biotechnology Co Ltd
Priority date: 2019-07-02
Filing date: 2019-07-02
Publication date: 2020-01-24
Anticipated expiration: 2039-07-02
Also published as: CN110305920A

Abstract

The invention belongs to the technical field of biological fermentation, and particularly relates to an active fermentation product, a preparation method thereof and application thereof in cosmetics. The preparation method comprises the following steps: s1 mixing oat bran with water, performing enzyme treatment, and sterilizing to obtain oat bran culture solution; s2 transferring the oat bran culture solution to a fermentation tank, inoculating Schizophyllum commune strain subjected to three-stage culture, and fermenting to obtain Schizophyllum commune polysaccharide fermentation liquid; s3, filtering the schizophyllum commune polysaccharide fermentation liquor by using a plate-and-frame filter press to obtain a schizophyllum commune fermentation extracellular product, and then carrying out microfiltration water washing on the schizophyllum commune fermentation extracellular product by using a ceramic membrane until clear and bright active fermentation products are obtained. The method has the advantages of low production cost, simple and stable process, and the obtained fermentation product containing the active ingredients such as the oat beta-glucan, the schizophyllan and the like has obvious effects of repairing and moisturizing the skin, is high in stability and has good application prospect in the field of cosmetics.

Description

Active fermentation product and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological fermentation, and particularly relates to an active fermentation product, a preparation method thereof and application thereof in cosmetics.

Background

Glucans are natural polysaccharides found in oats, barley, yeast and fungi and include 1, 3-glucan, 1, 4-glucan (with branching) and 1, 6-glucan (without branching) linked by glucose, which may be alpha-and beta-forms. A large number of researches show that the beta-glucan has obvious effects of reducing blood fat and blood sugar, improving the immunity of organisms and the like, so that the beta-glucan has great application value in the aspects of biology, medicine, health care and food.

In recent years, β -glucan has been studied as a skin care ingredient. The beta-glucan high polysaccharide can stimulate Langerhans cells, promote proliferation of fibroblasts (dermal cells) and synthesis of skin tissue matrixes (such as collagen, elastin, proteoglycan and other components), increase skin elasticity, make the skin soft and tender, form a transparent, elastic and breathable film on the skin, effectively isolate damage of harmful substances in the environment to the skin, fully lock water in a macromolecular structure of the beta-glucan high polysaccharide, prevent water loss, and have high-efficiency moisture retention, so that the skin is moistened and smooth. The excellent skin care function enables the beta-glucan to have good development and application prospects.

Currently, there are three main ways to obtain β -glucan: the first is directly extracted from oat, barley and other plant fruits, the cost of raw materials is low, but the extraction process is complex and the efficiency is low, and the obtained beta-glucan has poor water solubility and structural stability; the second kind is extracted from mushroom directly, the extraction cost is high, the cycle is long, the production space is large, the yield is low, and the impurity is difficult to be removed; the third one is obtained by biological fermentation, the period is relatively short, and the product quality is controllable.

For example, the research on the extraction of oat beta-glucan by a fermentation method such as panyan and the like shows that the optimal fermentation conditions for extracting oat beta-glucan by a yeast fermentation method are as follows: adding protease, amylase and glucoamylase into a fermentation medium for treatment before sterilization, wherein the used strain is yellow wine yeast, the fermentation time is controlled to be 48h, the parameters of a shaking table are 170r/min and 28 ℃ (Panyan and the like, preliminary exploration for extracting oat beta-glucan by a fermentation method [ J ]. food and fermentation industry, 2009, 35 (4): 116-.

And for example, Wudi and the like screen out 3 strains, namely pholiota adiposa, clitocybe maxima and grifola frondosa, which can increase the content of the oat beta-glucan from 12 medicinal fungi, take oat bran of China as a culture medium, extract the beta-glucan in the oat bran by a two-way fermentation method, and perform the research of process optimization, separation and purification and physicochemical properties (Wudi and the like, research on the extraction of the oat beta-glucan by two-way fermentation and the physicochemical properties thereof [ J ] food research and development, 2019,40(1): 184-.

Schizophyllum commune (Schizophyllum commune), also known as white ginseng, flowers of trees, Kadsura coccinea, belongs to the phylum Eumycophyta, Basidiomycetes, Agaricales, Schizophyllaceae, Schizophyllum (Schizophyllum commune), Schizophyllum (Shizophyllum). Schizophyllum commune polysaccharide (SPG for short), also called schizophyllan, is one of the extracellular main active ingredients of schizophyllum commune fermentation, and is glucan with a repeating unit of 3 beta- (1, 3) glucosides as a main chain and 1 beta- (1, 6) glucoside as a side chain, and has various physiological activities of inhibiting tumor, resisting bacteria, diminishing inflammation, resisting radiation, improving the immunity of the organism and the like, and researches indicate that the moisturizing effect of the schizophyllum commune polysaccharide is superior to that of oat beta-glucan.

Oat bran contains rich beta-glucan, and if the oat bran which is a byproduct in the grain and oil processing process is used as a substrate and inoculated with schizophyllum commune for fermentation, an active fermented product with high skin-beautifying activity is expected to be obtained.

Therefore, there is a need for improvements in the prior art to better utilize oat bran and Schizophyllum commune and to obtain an active ferment with high skin-beautifying activity.

Disclosure of Invention

In order to solve the problem that the content of beta-glucan is obviously reduced in the oat bran fermented by schizophyllum commune in the prior art, the invention firstly provides a preparation method of an active fermented product containing the beta-glucan, which specifically comprises the following steps:

s1, mixing oat bran and water according to a feed-liquid ratio of 1: 10-25 (W/V, g/mL), then sequentially performing enzyme treatment by using protease, saccharifying enzyme and amylase, and sterilizing after the enzyme treatment to obtain an oat bran culture solution;

s2, transferring the oat bran culture solution into a 50L fermentation tank, filling the fermentation tank with liquid in 30-35L, adding the Schizophyllum commune strain subjected to three-stage culture in an inoculum size of 10%, fermenting for 3-4 d under the conditions that the temperature is 25-28 ℃, the ventilation rate is 0.25-1.50 vvm and the rotating speed is 100-400 r/min, and the pH is natural to obtain Schizophyllum commune polysaccharide fermentation liquid;

s3, filtering the schizophyllum commune polysaccharide fermentation liquor by using a plate-and-frame filter press to obtain a schizophyllum commune fermentation extracellular product, and then carrying out microfiltration circulating water washing on the schizophyllum commune fermentation extracellular product by using a ceramic membrane until clear and bright active fermentation products are obtained.

Further, the steps of the enzyme treatment are as follows:

a1, protease treatment: adjusting the pH value to 6-7, then adding thiol protease accounting for 1-5% of the weight of the oat bran culture solution, and carrying out water bath at 60-65 ℃ for 90-120 min;

a2, saccharifying enzyme treatment: adjusting the pH value to 4.0-4.5, adding saccharifying enzyme accounting for 1-3% of the weight of the oat bran culture solution, and carrying out water bath at 55-60 ℃ for 2-3 h;

a3, amylase treatment: adding amylase accounting for 1-5% of the weight of the oat bran culture solution, and carrying out water bath at 70-75 ℃ for 30-40 min.

Further, the thiol protease is papain or ficin.

Further, the amylase is alpha-amylase, and the weight percentage of the amylase is 10%.

Further, the three-stage culture comprises the following steps:

b1, tube bevel: adopting agar to fix a culture medium, inoculating ampoule bottle strains, and culturing at 28 ℃ for 7 days until the ampoule bottle strains are full of tubes;

b2, primary culture: taking two-ring hyphae from a slant test tube by using an inoculating ring, transferring the two-ring hyphae into a first-class seed bottle with the liquid loading capacity of 100mL, and culturing for 3d at 28 ℃ at 170 r/min;

b3, secondary culture: transferring the first-stage seed culture solution to a second-stage seed culture bottle with the inoculation amount of 10%, and culturing at 28 deg.C and 170r/min for 3d to obtain third-stage cultured Schizophyllum commune strain.

Further, the culture solution prepared from the following raw materials is adopted in the primary culture process and the secondary culture process: 35.0g of glucose, 3.0g of yeast extract powder, 1.0g of ammonium chloride, 1.0g of monopotassium phosphate, 0.5g of magnesium sulfate and 1000ml of deionized water, wherein the pH value is 6.5 +/-0.2.

Furthermore, the aperture of the ceramic membrane is 0.1-0.5 μm.

And further, mixing the extracellular products of the Schizophyllum commune fermentation with pure water according to the volume ratio of 1 (2-5) to carry out microfiltration circulating water washing.

Further, the microfiltration speed is 1-5L/min.

Correspondingly, the invention also provides the active fermentation product prepared by the preparation method, the active fermentation product contains active ingredients such as oat beta-glucan, schizophyllan and the like, and has obvious effects of repairing and moisturizing the skin, so the invention also provides the application of the active fermentation product in cosmetics, and the cosmetics with obvious skin beautifying effect can be brought to consumers.

Therefore, compared with the prior art, the invention has the following advantages:

(1) the beta-glucan is prepared by fermenting the schizophyllum commune, which is a byproduct in the grain and oil processing process, serving as a substrate, and utilizing the schizophyllum commune, the production cost is low, the process is simple and stable, and the obtained fermentation product containing the active ingredients such as the oat beta-glucan, the schizophyllum commune polysaccharide and the like has remarkable repairing and moisturizing effects on the skin, is high in stability, and has good application prospects in the field of cosmetics.

(2) The preparation method of the active fermentation product comprises the steps of firstly carrying out enzymolysis treatment on oat bran, particularly adopting specific thiol protease to carry out treatment, then fermenting an oat bran culture solution by using schizophyllum commune, preventing beta-glucan from being decomposed and utilized by the schizophyllum commune by using free oat polypeptide generated by enzymolysis, generating schizophyllum commune polysaccharide while propagating a large amount of schizophyllum commune, and enabling the schizophyllum commune to utilize nutrient components such as starch, protein and the like in the oat bran culture solution, thereby improving the yield and the purity of the beta-glucan in the oat bran fermentation solution.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

In the following examples, the agar fixed medium was a PDA medium manufactured by Kyork, Guangdong, Microscience and technology Co.Ltd; the first-stage culture and the second-stage culture both adopt culture solutions prepared from the following raw materials: 35.0g of glucose, 3.0g of yeast extract powder, 1.0g of ammonium chloride, 1.0g of monopotassium phosphate, 0.5g of magnesium sulfate and 1000ml of deionized water, wherein the pH value is 6.5 +/-0.2.

Example 1 active fermentates of the invention and methods for their preparation

S1, mixing oat bran and water according to a feed-liquid ratio of 1:10(W/V, g/mL), then sequentially performing enzyme treatment by using protease, saccharifying enzyme and amylase, and sterilizing after the enzyme treatment to obtain an oat bran culture solution;

the steps of the enzyme treatment are as follows:

a1, protease treatment: adjusting the pH value to 6-7, then adding papain with the weight of 1% of the oat bran culture solution, and carrying out water bath at 60 ℃ for 120 min;

a2, saccharifying enzyme treatment: adjusting the pH value to 4.0-4.5, adding saccharifying enzyme accounting for 1% of the weight of the oat bran culture solution, and carrying out water bath at 55 ℃ for 3 hours;

a3, amylase treatment: adding alpha-amylase (lyophilized powder of alpha-amylase, 10 wt%) 1 wt% of oat bran culture solution, and water bath at 70 deg.C for 40 min.

S2, transferring the oat bran culture solution into a 50L fermentation tank, wherein the liquid loading amount is 35L, then adding the Schizophyllum commune strain subjected to three-stage culture in an inoculum size of 10%, fermenting for 4d under the conditions that the temperature is 25 ℃, the ventilation volume is 0.25vvm and the rotating speed is 100r/min, and the pH is natural to obtain Schizophyllum commune polysaccharide fermentation liquid;

the three-stage culture comprises the following steps:

S3, filtering the schizophyllum commune polysaccharide fermentation liquor by using a plate-and-frame filter press to obtain a schizophyllum commune fermentation extracellular product, wherein the product contains components such as pigments, saccharides, polypeptides, amino acids and the like, then carrying out microfiltration circulating water washing on the schizophyllum commune fermentation extracellular product by using a ceramic membrane with the pore diameter of 0.1 mu m, wherein small molecular impurities such as pigments, polypeptides, amino acids and the like can flow out along with pure water in the microfiltration process to achieve the effect of impurity removal, and finally concentrating to 1/3 of the initial volume to obtain a clear and bright active fermentation product. Wherein the Schizophyllum commune fermentation extracellular product is mixed with pure water according to the volume ratio of 1:2, and the microfiltration speed is 1L/min.

Example 2 active fermentates of the invention and methods for their preparation

S1, mixing oat bran and water according to a feed-liquid ratio of 1:25(W/V, g/mL), then sequentially performing enzyme treatment by using protease, saccharifying enzyme and amylase, and sterilizing after the enzyme treatment to obtain an oat bran culture solution;

the steps of the enzyme treatment are as follows:

a1, protease treatment: adjusting the pH value to 6-7, then adding ficin accounting for 5% of the weight of the oat bran culture solution, and carrying out water bath at 65 ℃ for 90 min;

a2, saccharifying enzyme treatment: adjusting the pH value to 4.0-4.5, adding saccharifying enzyme accounting for 3% of the weight of the oat bran culture solution, and carrying out water bath at 60 ℃ for 2 h;

a3, amylase treatment: adding alpha-amylase (lyophilized powder of alpha-amylase, 10 wt%) 5 wt% of oat bran culture solution, and water bath at 75 deg.C for 30 min.

S2, transferring the oat bran culture solution into a 50L fermentation tank, wherein the liquid loading amount is 35L, then adding the Schizophyllum commune strain subjected to three-stage culture in an inoculum size of 10%, fermenting for 3d under the conditions that the temperature is 28 ℃, the ventilation volume is 1.50vvm and the rotating speed is 400r/min, and the pH is natural to obtain Schizophyllum commune polysaccharide fermentation liquid;

the three-stage culture comprises the following steps:

S3, filtering the schizophyllum commune polysaccharide fermentation liquor by using a plate-and-frame filter press to obtain a schizophyllum commune fermentation extracellular product, wherein the product contains components such as pigments, saccharides, polypeptides, amino acids and the like, then carrying out microfiltration circulating water washing on the schizophyllum commune fermentation extracellular product by using a ceramic membrane with the pore diameter of 0.5 mu m, wherein small molecular impurities such as pigments, polypeptides, amino acids and the like can flow out along with pure water in the microfiltration process to achieve the effect of impurity removal, and finally concentrating to 1/3 of the initial volume to obtain a clear and bright active fermentation product. Wherein the Schizophyllum commune fermentation extracellular product is mixed with pure water according to the volume ratio of 1:5, and the microfiltration speed is 5L/min.

Example 3 active fermentates of the invention and methods of making the same

S1, mixing oat bran and water according to a feed-liquid ratio of 1:15(W/V, g/mL), then sequentially performing enzyme treatment by using protease, saccharifying enzyme and amylase, and sterilizing after the enzyme treatment to obtain an oat bran culture solution;

the steps of the enzyme treatment are as follows:

a1, protease treatment: adjusting the pH value to 6-7, then adding papain with the weight being 3% of that of the oat bran culture solution, and carrying out water bath at 60 ℃ for 100 min;

a2, saccharifying enzyme treatment: adjusting the pH value to 4.0-4.5, adding saccharifying enzyme accounting for 2% of the weight of the oat bran culture solution, and carrying out water bath at 60 ℃ for 2.5 h;

a3, amylase treatment: adding alpha-amylase (lyophilized powder of alpha-amylase, 10 wt%) 3 wt% of oat bran culture solution, and water bath at 75 deg.C for 30 min.

S2, transferring the oat bran culture solution into a 50L fermentation tank, wherein the liquid loading amount is 35L, adding the Schizophyllum commune strain subjected to three-stage culture in an inoculum size of 10%, fermenting for 3d under the conditions that the temperature is 26 ℃, the ventilation volume is 1.00vvm and the rotating speed is 200r/min, and the pH is natural to obtain Schizophyllum commune polysaccharide fermentation liquid;

the three-stage culture comprises the following steps:

S3, filtering the schizophyllum commune polysaccharide fermentation liquor by using a plate-and-frame filter press to obtain a schizophyllum commune fermentation extracellular product, wherein the product contains components such as pigments, saccharides, polypeptides, amino acids and the like, then carrying out microfiltration circulating water washing on the schizophyllum commune fermentation extracellular product by using a ceramic membrane with the pore diameter of 0.3 mu m, wherein small molecular impurities such as pigments, polypeptides, amino acids and the like can flow out along with pure water in the microfiltration process to achieve the effect of impurity removal, and finally concentrating to 1/3 of the initial volume to obtain a clear and bright active fermentation product. Wherein the Schizophyllum commune fermentation extracellular product is mixed with pure water according to the volume ratio of 1:3, and the microfiltration speed is 2L/min.

Comparative example 1

The comparative example differs from example 3 only in that: trypsin is used to replace papain, and the specific treatment process is as follows:

a1, protease treatment: adjusting the pH value to 8.0-8.5, then adding trypsin which accounts for 3% of the weight of the oat bran culture solution, and carrying out water bath at 37 ℃ for 100 min.

Comparative example 2

The comparative example differs from example 3 only in that: the method uses alkaline protease (the main enzyme component is bacillus licheniformis protease) to replace papain, and comprises the following specific treatment processes:

a1, protease treatment: adjusting the pH value to 10-11, then adding alkaline protease accounting for 3% of the oat bran culture solution by weight, and carrying out water bath at 50 ℃ for 100 min.

Comparative example 3

The comparative example differs from example 3 only in that: the enzyme treatment was not carried out with protease.

Test example one measurement of the content of beta-glucan in the active fermented product of the present invention

Beta-glucan can be specifically combined with Congo red (Congo red) to increase the light absorption intensity, and the absorption value can be measured at the wavelength of 550 nm.

Preparing the beta-glucan standard solution into 20 mu g/ml, 40 mu g/m, 60 mu g/ml, 80 mu g/ml and 100 mu g/ml solutions, respectively adding 4ml Congo red solution, reacting in a water bath at 25 ℃ for 10min, and measuring the absorbance value at the wavelength of 550nm to obtain a standard curve.

Samples were taken from the active fermentates prepared in examples 1-3 and comparative examples 1-3 and the oat bran culture solution of example 3, and the content of beta-glucan in the samples was determined by the following specific method:

(1) preparation of a reference solution: 4ml of the prepared Congo red solution is added with 2ml of distilled water and mixed evenly.

(2) 1.9ml of water is added into a test tube of the sample, then 0.1ml of the sample is added, finally 4ml of Congo red solution is added, and the reaction is carried out in water bath at 25 ℃ for 10 min.

(3) And (3) measuring the absorbance value at the wavelength of 550nm, and converting the content of the beta-glucan in the sample according to the standard curve.

The results of the measurements are shown in Table 1 below.

TABLE 1 measurement results of the content of beta-glucan in each sample

From table 1 above, it can be seen that: compared with the oat bran culture solution which is not subjected to fermentation treatment, the content of beta-glucan in the product of the comparative example 3 is reduced; compared with the scheme without protease treatment (comparative example 3), the content of beta-glucan in the products of examples 1-3 is obviously increased, and the content of beta-glucan in the products of comparative examples 1-2 is also increased, but the obvious difference exists compared with the products of examples 1-3, and the specific reasons are as follows:

according to the enzyme activity center, the protein can be divided into serine protein, thiol protease and metal protein, and the enzymatic hydrolysis produces polypeptide and other small molecules with different properties, but the invention finds that the free oat polypeptide obtained by hydrolysis of thiol protease such as papain, ficin and the like can prevent beta-glucan from being utilized and decomposed, and the prevention effect may be the protection effect of the free oat polypeptide on oat beta-glucan and the inhibition effect of the free oat polypeptide on the decomposition of oat beta-glucan.

Test example two evaluation of efficacy of the active fermented product of the present invention

repair-Effect on fibroblast proliferation

The active fermentation products prepared in examples 1-3 were prepared into stock solutions with beta-glucan content of 5 mg/ml. The stock solution was diluted to 6 final concentrations (5. mu.g/ml, 10. mu.g/ml, 20. mu.g/ml, 50. mu.g/ml, 100. mu.g/ml, 200. mu.g/ml) in serum-free medium just prior to each spotting.

Selecting logarithmic phase growing fibroblast, digesting, centrifuging, counting, diluting with DMEM medium containing 10% fetal calf serum to 3 × 10⁴Each ml^-1The concentration of (4) was inoculated in a 96-well plate using a line gun, 100. mu.l per well. At 5% CO₂Culturing for 4-5 h in a constant-temperature incubator at 37 ℃ (ensuring that cells adhere to the wall), sucking out the culture medium in a 96-well plate, washing twice with PBS, and adding culture solution containing samples with different concentrations. And 6 multiple wells of each sample are cultured for 48h, and then the light absorption values of the control and different treatment concentrations are determined by a methylene blue method.

After 48h incubation, the medium in the 96-well plate was aspirated, washed twice with PBS, replaced with 50. mu.l of methylene blue stain, and placed on 5% CO₂And continuously culturing for 60min in a constant-temperature incubator at 37 ℃. The methylene blue dye solution is sucked away, and the washing is carried out for six times by using distilled water, and the washing times of each hole are ensured to be the same. After draining the distilled water (or naturally drying for several minutes) in the well plate, 100. mu.l of methylene blue washing solution was added to each well, and the well plate was placed at room temperature and shaken on a shaking plate to completely dissolve the blue crystals. The absorbance (OD) at 570nm of each well was measured using an enzyme linked immunosorbent assay. The fibroblast proliferation rate was calculated according to the following formula:

the test results are shown in table 2 below.

(II) moisturizing effect

(1) Principle for measuring moisture content in skin by MMV method

The CM825 model skin oil-water acid-base tester is used for testing the hydration of the skin, and the change of the electrical conductivity reflects the hydration state of the skin after the test probe is contacted with the skin.

Evaluation formula of moisturizing effect evaluation:

in the formula: p-sample t ═ x: conductivity of the product after a certain period of use; blank P: the electrical conductivity of the uncoated sample; p denotes t ═ x: conductivity 2 of the reference after a certain time of use.

(2) Principle of measuring moisturizing effect by TEWL method

The test principle is as follows: the TEWL value does not directly indicate the water content of the stratum corneum, but indicates the loss of water from the stratum corneum, which indicates the function of the stratum corneum barrier and is an important parameter for evaluating the state of the stratum corneum.

The test method comprises the following steps: the testing time is selected, the cylindrical probe is vertically placed on the skin to be tested for 20s, the data can be read after the measured value is stable, and the TEWL data are automatically collected by the instrument and displayed according to the time sequence to form a TEWL time curve. The instrument automatically displays the TEWL values, curves, mean and standard deviation of skin moisture loss, taking the average of several different test time values as the final result. The measured TEWL value is high, which indicates that the water lost through the skin is much, the barrier effect of the horny layer is not good, after the cosmetics are used, the TEWL value is obviously reduced, the larger the difference value is, the better the moisturizing effect of the cosmetics is, and the barrier effect of the horny layer is obviously enhanced after the cosmetics are used. Can be used for comparing the moisture retention performance of different cosmetics and monitoring the moisture loss condition of skin for a long time.

(3) The test steps are as follows:

1) 30 subjects aged between 16 and 65 years (except pregnant or lactating women). The basic value of a capacitance-method skin moisture tester of a forearm testing area is required to be 15-40, and the tester has no serious system diseases, immunodeficiency or autoimmune diseases, active allergic diseases, severe allergic history to skin care cosmetics, hormone medicines and immunosuppressant which are not used in the last month and other clinical testers.

2) Any product (cosmetics or external medicine) cannot be used 2-3 days before the tested part. ) Before the test, the subject had to agree to clean the forearm of both hands, wipe clean with dry facial tissue. Measurement zone markings were made on the forearm of the subject after cleaning. Before formal test, the patient should sit still in a room (with the test environment temperature: 22 + -1 deg.C, humidity: 50 + -5%, and real-time dynamic monitoring) meeting the standard for at least 30min, and the patient should not drink water, and the forearm should be exposed and kept in the test state to be relaxed.

3) The inner side of the left and right arms in the experiment is marked with 3 multiplied by 3cm²In the test area, the same arm can mark a plurality of areas at the same time, and the intervals of the areas are 1 cm. The test product (the test sample is the active fermentation product of examples 1-3, prepared into a 1% aqueous solution) and the blank control were randomly distributed on the left and right arms. The MMV and TEWL values of the test area were tested and recorded. Each area was assayed 5 times in parallel. The blank value of each test area was measured and then measured at 2.0. + -. 0.1mg sample/cm²The test was evenly spread into the test area using a latex finger cot. The skin moisture content was measured at 1 hour, 2 hours, 4 hours, test area and blank control area after application (measured at this time at the time of verification). The test of the same volunteer was performed by the same measurement staff.

(4) The test results are shown in table 2 below.

Table 2 evaluation of repairing and moisturizing effects of the active fermented product of the present invention

Item	Example 1	Example 2	Example 3
				Fibroblast proliferation Rate (%)	129	132	139
1h MMV(％)	15.32	18.25	21.12
				2h MMV(％)	22.41	26.56	28.35
4h MMV(％)	26.18	29.45	32.17
				1h TEWL	8.17	8.12	7.14
2h TEWL	6.78	6.47	5.4
				4h TEWL	6.01	5.78	4.15

As can be seen from table 2 above, the active fermentation products of examples 1 to 3 of the present invention have significant skin repairing and moisturizing effects, and thus have high skin-beautifying activity, and can be applied to cosmetics. In examples 1 to 3, the effect of the active fermentation product in example 3 was most remarkable, and thus example 3 is the most preferable example of the present invention.

Test example III, application of the active fermentation product of the invention in cosmetics and evaluation of efficacy

(1) The active ferment of example 3 was applied to a toner formulation as follows:

10.0g of butanediol, 3.0g of dipropylene glycol, 2.0g of 1.2-pentanediol, 1.5g of the active fermentation product of example 3 and 0.5g of phenoxyethanol are added with water to 100g, and the moisturizing and repairing toner is prepared according to a conventional method;

(2) the active fermentate of example 3 was applied in an eye cream formulation as follows:

5.0g of glycerin, 5.0g of jojoba esters, 3.0g of caprylic/capric triglyceride, 3.0g of tocopherol glucoside, 3.0g of sucrose stearate, 2.0g of cyclomethicone, 2.0g of batyl alcohol, 2.0g of caprylic/capric triglyceride, 1.5g of sucrose distearate, 2.0g of the active fermentation product of example 3, 1.0g of bisabolol, 0.5g of carbomer, 0.05g of triethanolamine, 0.05g of disodium EDTA, 1.0g of dextran sulfate, 2.0g of 1.2-pentanediol, 0.5g of phenoxyethanol, and water is added to 100g to prepare the repair eye cream according to a conventional method.

(3) The active fermentate of example 3 was applied to the following emulsion formulation, as follows:

sweet almond oil 8.0g, squalane 3.0g, glycerin 5.0g, dipropylene glycol 4.0g, polysorbate-802.0 g, active fermented product 1.0g of example 3, carbomer 0.1g, triethanolamine 0.1g, and water to 100g, and emulsion 1 was prepared according to a conventional method.

Setting blank emulsion without adding active leavening, wherein the formula of the blank emulsion is as follows: sweet almond oil 8.0g, squalane 3.0g, glycerin 5.0g, dipropylene glycol 4.0g, polysorbate-802.0 g, carbomer 0.1g, triethanolamine 0.1g, adding water to 100g, and making into emulsion 2 by conventional method.

30 volunteer subjects were invited and 4 pieces of 2X 2cm were selected on the inside of the arms of their hands²The size of the area is 0.2g of emulsion 1 or emulsion 2 coated on each area, and the left hand is coated with one piece of emulsion 1 and one piece of emulsion 2 respectively, and the right hand is the same. After the application time of 4h, 8h, 12h and 24h, the moisture content of the applied area is measured by a percutaneous moisture loss instrument. The test results are given in table 3 below.

TABLE 3 moisturizing test results for each emulsion

Sample (I)	Water content in 4 h%	Water content of 8 h%	Water content in 12 h%	Water content of 24 h%
					Emulsion 1	25.16	35.02	35.33	34.28
Emulsion 2	20.04	24.13	20.10	19.21

From table 3 above, it can be seen that: compared with the emulsion 2, the emulsion 1 added with the active leavening of the embodiment 3 of the invention still maintains higher moisture content after being used for 24 hours, shows obvious moisturizing effect on skin, and has good application prospect in the field of cosmetics.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Claims

1. A method for preparing an active fermentation product is characterized by comprising the following steps:

s1, mixing the oat bran with water according to the mass volume ratio of 1g (10-25) mL of the feed liquid, then sequentially performing enzyme treatment by adopting thiol protease, saccharifying enzyme and amylase, and sterilizing after the enzyme treatment to prepare the oat bran culture solution;

s3, filtering the schizophyllum commune polysaccharide fermentation liquor by using a plate-and-frame filter press to obtain a schizophyllum commune fermentation extracellular product, and then carrying out microfiltration circulating water washing on the schizophyllum commune fermentation extracellular product by using a ceramic membrane until clear and bright active fermentation products are obtained;

the thiol protease is papain or ficin.

2. The method for producing an active fermentation product according to claim 1, wherein the enzyme treatment comprises the steps of:

a1, thiol protease treatment: adjusting the pH value to 6-7, then adding thiol protease accounting for 1-5% of the weight of the oat bran culture solution, and carrying out water bath at 60-65 ℃ for 90-120 min;

a2, saccharifying enzyme treatment, namely adjusting the pH value to 4.0 ~ 4.5.5, adding saccharifying enzyme accounting for 1-3% of the weight of the oat bran culture solution, and carrying out water bath at 55-60 ℃ for 2-3 h;

3. The method for preparing an active fermentation product according to claim 1, wherein the amylase is alpha-amylase, the alpha-amylase is lyophilized powder, and the alpha-amylase accounts for 10% of the lyophilized powder by weight.

4. The method for producing an active fermentation product according to claim 1, wherein the three-stage cultivation comprises the steps of:

5. The method for producing an active fermented product according to claim 1, wherein the ceramic membrane has a pore size of 0.1 to 0.5 μm.

6. The preparation method of the active fermentation product according to claim 1, wherein the Schizophyllum commune fermentation extracellular product and pure water are mixed according to a volume ratio of 1 (2-5) to be subjected to microfiltration circulating water washing.

7. The method for preparing active fermentation product according to claim 1, wherein the microfiltration rate is 1-5L/min.

8. An active fermentation product prepared by the method according to any one of claims 1 to 7.

9. Use of an active ferment according to claim 8 in cosmetics.