An oil-controlling, acne-removing and pore-improving composition obtained by fermentation of bifid yeast and a preparation method thereof

Abstract

The present invention belongs to the technical field of probiotic fermentation, and discloses a preparation method and application of a bifid yeast fermentation product, the preparation method of the bifid yeast fermentation product, comprising the following steps: S1. preparing a medicinal porphyra extract A; S2. preparing a composite extract B of Rhizoma Anemarrhenae, Radix Puerariae, bud of Sophora japonica, and Fructus Sapindi; S3. preparing a bifid yeast fermentation product: inoculating a bifidobacterium seed solution in a fermentation medium, culturing and fermenting under anaerobic conditions, and obtaining a primary fermentation liquid C; adding A, B and bifidobacterium seed solution to C for secondary fermentation, and obtaining the bifid yeast fermentation product after post-treatment. The bifid yeast fermentation product obtained by the present invention can reduce skin oxidative stress, inhibit inflammatory factors IL-6 and 5α-reductase, is externally beneficial to skin absorption, has a good transdermal effect on the skin, has an excellent oil control effect, can promote skin water and oil balance, and achieves the technical effects of oil control and acne removal and pore improvement.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotic fermentation, and more specifically to an oil-controlling, acne-removing and pore-improving composition obtained by fermenting bifid yeast and a preparation method thereof. Background Art

[0002] The skin is the largest organ in the human body. Fine and even skin texture is one of the important criteria for measuring healthy skin. Excessive secretion of sebum in the skin and imbalance of water and oil in the skin cause the proliferation of lipophilic microorganisms, resulting in blocked hair follicles, enlarged skin pores, acne and blackheads, affecting the health and beauty of the skin. Therefore, it is necessary to control excessive oil secretion in the skin.

[0003] There are several main reasons for excessive sebum secretion: (1) Oxidative stress. When the skin is exposed to ultraviolet rays or reactive oxygen species are formed, it stimulates the release of α-melanocyte-stimulating hormone, causing androgen effects and sebaceous gland cell differentiation, leading to lipid synthesis. (2) Inflammatory response. When the skin is stimulated by arachidonic acid, inflammatory factors (IL-6 and TNF-α) are expressed, causing lipid synthesis. (3) Activation of 5α-reductase. 5α-reductase can convert testosterone into dihydrotestosterone, which binds to androgen receptors in cells to stimulate sebaceous glands to secrete oil.

[0004] Regarding oil control products, the existing technology mainly combines plant extracts such as Anemarrhena asphodeloides, Pueraria lobata, and Sophora japonica buds with other components to form an oil control composition. The saponin components in these plants can have a certain oil control effect, and the polysaccharide components have a certain moisturizing effect. However, some natural active substances in the above-mentioned plant extracts have a large molecular weight, are not easily absorbed by the skin, and have a poor transdermal effect, so the overall oil control effect is limited. Summary of the invention

[0005] The invention provides a method for preparing a bifid yeast fermentation product. The obtained bifid yeast fermentation product has a technical effect on skin external oil control, acne removal and pore improvement that is better than the prior art.

[0006] Another object of the present invention is to provide a bifid yeast fermentation product.

[0007] Another object of the present invention is to provide the use of the bifid yeast fermentation product in the preparation of oil-controlling, acne-removing and pore-improving external products.

[0008] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0009] A method for preparing a bifid yeast fermentation product comprises the following steps:

[0010] S1. Preparation of medicinal porphyra extract A;

[0011] S2. Extracting a plant composite extract: crushing, heating, extracting, filtering and concentrating the Anemarrhena asphodeloides, Puerariae lobata, Sophora japonica buds and Sapindus mukorossi to obtain a plant composite extract B; the mass ratio of the Anemarrhena asphodeloides, Puerariae lobata, Sophora japonica buds and Sapindus mukorossi is (1-15): (1-15): (0.4-8): (0.2-3);

[0012] S3. Preparing a bifid yeast fermentation product: inoculating a bifidobacterium seed solution into a fermentation medium, culturing and fermenting under anaerobic conditions to obtain a primary fermentation solution C; adding A, B and bifidobacterium seed solution to C for secondary fermentation, and obtaining the bifid yeast fermentation product after post-treatment; the mass ratio of A, B and C is (1-5): (7-35): (15-80).

[0013] Active substances such as polysaccharides, sterols and amino acids in medicinal porphyra extract A can reduce skin oxidative stress and inhibit inflammatory factors IL-6 and 5α-reductase.

[0014] Among the plant complex extracts in S2: Anemarrhena asphodeloides and Pueraria lobata extracts have a relatively strong oil-control effect, but their solubility and stability are poor, while Sapindus mukorossi saponins have the effect of natural solubilization and promoting dissolution; Sophora japonica buds contain total flavonoids, which can scavenge free radicals and resist oxidation, reduce skin oxidative stress and thus avoid increased oil secretion.

[0015] In step S3, bifidobacteria are fermented in two steps. The first fermentation is the primary fermentation of bifidobacteria, which is mainly the process of culturing the reproduction and growth of bifidobacterium seed liquid. In the second fermentation, A obtained in S1 and B obtained in S2 are added to the primary fermentation liquid C, which has the following main effects: (1) The bifidobacteria fermentation process synergizes with the polyphenols or phenolic reducing substances in the plants in B to increase the ability of the product to scavenge hydroxyl free radicals and oxygen free radicals; (2) Bifidobacteria can synthesize antioxidant enzymes such as SOD superoxide dismutase in the secondary fermentation process and the lactic acid bacteria fermentation process in A, which can also scavenge free radicals. The antioxidant properties of the resulting fermentation product are enhanced and can effectively reduce skin oxidative stress; (3) Bifidobacteria express a variety of glycosidases such as α / β-glucosidase and amylase in the secondary fermentation process, which can degrade the complex polysaccharides introduced from A and B into small molecular sugars, and degrade some saponin components containing multiple glycosides to reduce the number of sugar molecules, making the polarity smaller and easier to absorb by the skin, thereby improving the transdermal effect of the fermentation product of bifidobacterium.

[0016] In summary, the bifid yeast fermentation product obtained by the present invention is beneficial to skin absorption when applied externally, has good skin transdermal effect, excellent oil control effect, can promote the water-oil balance of the skin, and achieve the technical effects of oil control, acne removal, and pore improvement.

[0017] In the method for preparing the bifid yeast fermentation product, step S1 is to prepare the medicinal porphyrin extract A and step S2 is to prepare the plant composite extract. There is no order for these two steps. In the process of preparing the bifid yeast fermentation product, either the medicinal porphyrin extract A or the plant composite extract B can be prepared first.

[0018] Preferably, the preparation method of the medicinal pore fungus extract A in S1 comprises the following steps: adding medicinal pore fungus to MRS culture medium to obtain a medicinal pore fungus culture medium, inoculating lactic acid bacteria seed liquid into the medicinal pore fungus culture medium for culturing and fermenting to obtain the medicinal pore fungus extract A, wherein the mass ratio of the MRS culture medium to the medicinal pore fungus is (1~7):(0.5~5).

[0019] In the process of preparing the medicinal pore fungus extract A, the medicinal pore fungus is fermented by lactic acid bacteria, which can increase the extraction amount of active substances such as polysaccharides, sterols and amino acids in the medicinal pore fungus.

[0020] Preferably, the inoculation amount of the lactic acid bacteria seed solution in S1 is 10% to 25% of the volume of the culture medium, and the pH of the culture medium is 6 to 8.

[0021] Preferably, the culture and fermentation temperature in S1 is 30-40° C., and the time is 30-70 hours. The fermented liquid is sterilized and filtered to obtain the medicinal porphyra extract A.

[0022] Preferably, the inoculation amount of the bifidobacterium seed solution in S3 is 4-10% of the volume of the culture medium, and the pH of the culture medium is 6-8.

[0023] Preferably, the primary fermentation temperature in S3 is 35-40°C, and the time is 45-70 hours; the secondary fermentation temperature in S3 is 35-40°C, and the time is 8-15 hours.

[0024] Preferably, the mass of the bifidobacterium seed liquid added in the secondary fermentation in S3 is (2% to 7%) of the mass of C.

[0025] Preferably, the post-treatment process in S3 is to first separate the feed liquid obtained from the secondary fermentation and perform cell lysis to obtain a bifid yeast fermentation filtrate and cell wall fragments; then add an enzyme to the obtained cell wall fragments for enzymatic hydrolysis, inactivation, centrifugation, and filtration to obtain a cell fragment hydrolyzate; finally, the bifid yeast fermentation filtrate and the cell fragment hydrolyzate are compounded to obtain the bifid yeast fermentation product.

[0026] Preferably, the enzyme added in the enzymatic treatment in S3 is glycoside hydrolase and one of the following enzymes: Alcalase alkaline protease, animal protease, plant protease and microbial protease.

[0027] The mass ratio of cell wall fragments to enzyme is (5-15): (10-25).

[0028] The enzymatic hydrolysis process is to enzymatically hydrolyze the fermented cell fragments to obtain soluble polysaccharides and small molecule peptides to form water-soluble components, which is beneficial to the absorption and utilization of active ingredients in the cell fragments. There is no precipitation in the product, which is beneficial to its use in various dosage forms of cosmetics.

[0029] Preferably, the bifidobacterium is selected from one of Bifidobacterium longum, Bifidobacterium breve and Bifidobacterium adolescentis.

[0030] The present invention also provides the use of the bifid yeast fermentation product prepared by the bifid yeast fermentation product preparation method in the preparation of oil-controlling, acne-removing and pore-improving products.

[0031] Preferably, the oil-controlling, acne-removing and pore-improving product of the present invention is selected from toner, essence and cream. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The inhibitory effect of the bifid yeast obtained in Example 1 on inflammatory factors, wherein: (a) is blank (55% butanediol), model (LPS+), benign (LPS+, dexamethasone), Examples 1 to 4; (b) is blank (55% butanediol), model (LPS+), benign (LPS+, dexamethasone), Example 1, Comparative Example 1; (c) is blank (55% butanediol), model (LPS+), benign (LPS+, dexamethasone), Example 1, Comparative Examples 2 to 4; (d) is blank (55% butanediol), model (LPS+), benign (LPS+, dexamethasone), Example 1, Comparative Example 5. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below in conjunction with specific implementation methods.

[0034] The lactic acid bacteria (Lactococcus lactis) used in the relevant embodiments and comparative examples have a strain number of CICC20408; the bifidobacterium is Bifidobacterium longum (Bifidobacterium longum), which is deposited in the China General Microbiological Culture Collection Center with a number of CGMCC No.6273.

[0035] The method for preparing the lactic acid bacteria seed solution used in the relevant embodiments and comparative examples comprises the following steps:

[0036] Place 5wt% MRS culture medium in water, stir and dissolve at room temperature, sterilize under high pressure at 125°C for 10 min, and cool to room temperature to use as lactic acid bacteria seed culture medium; pick lactic acid bacteria and place them in the seed culture medium, culture at a constant temperature of 38°C for 48 h to obtain lactic acid bacteria seed solution, and the lactic acid bacteria inoculation amount accounts for 0.5% of the volume of the seed culture medium.

[0037] The method for preparing the bifidobacterium seed solution used in the relevant embodiments and comparative examples comprises the following steps:

[0038] Bifidobacterium longum was inoculated into a seed culture medium at a volume ratio of 3%, and cultured at a culture temperature of 37° C. under anaerobic conditions for 40 hours to obtain a Bifidobacterium longum seed solution.

[0039] The seed liquid culture medium comprises the following components in parts by weight: 20 parts of glucose, 3 parts of tryptone, 3 parts of beef peptone, 3 parts of yeast extract powder, 3 parts of sodium acetate, 1 part of diammonium hydrogen citrate, 1 part of dipotassium hydrogen phosphate, and 15 parts of agar, and water is added to make up to 1000 parts.

[0040] Related Examples and Comparative Examples The culture medium for the primary and secondary fermentation of Bifidobacterium longum includes the following components in parts by weight: 25 parts of glucose, 3 parts of tryptone, 2 parts of beef peptone, 2 parts of yeast extract powder, 2 parts of sodium acetate, 1 part of diammonium hydrogen citrate, 1 part of dipotassium hydrogen phosphate and 1000 parts of water.

[0041] Example 1

[0042] A method for preparing a bifid yeast fermentation product comprises the following steps:

[0043] S1. Preparation of medicinal porphyra extract A: First, MRS medium and medicinal porphyra powder are placed in water and stirred at room temperature to dissolve, the pH is adjusted to 7.0, and high-pressure sterilization is performed at 125°C for 10 minutes to obtain a fermentation medium containing medicinal porphyra, and the fermentation medium is high-pressure sterilized at 120°C for 15 minutes, wherein the mass ratio of MRS medium: medicinal porphyra: water is 5:2:100; secondly, lactic acid bacteria seed liquid is inoculated into the fermentation medium at an inoculation amount of 13%, and fermented at a fermentation temperature of 35°C for 48 hours; finally, the fermented liquid is taken out, high-pressure sterilized at 120°C for 15 minutes, and filtered to obtain medicinal porphyra extract A;

[0044] S2. Plant compound extract: Anemarrhena, Pueraria, Sophora japonica, and Sophora japonica buds are weighed in proportion and put into a wall breaking machine, and water 1 times the total mass of the input plants is added to break the wall to obtain a composite slurry; the composite slurry is transferred to an extraction device, water 8 times the total mass of the input plants is added, and the mixture is heated to 85°C for extraction, filtered, and the residue is discarded to obtain a filtrate; the filtrate is concentrated to a mass of 1.5 times the total mass of the input plants to obtain a plant compound extract, which is recorded as B; the input mass ratio of Anemarrhena, Pueraria, Sophora japonica, and Sophora japonica is 10:10:3:2;

[0045] S3. Preparation of bifid yeast fermentation:

[0046] S31. Primary fermentation: The cultured Bifidobacterium longum seed solution was inoculated into a sterilized fermentation medium at 7% of the volume of the medium, the pH was adjusted to 7.0, and the culture temperature was 37°C and cultured under anaerobic conditions for 55h to obtain a fermentation broth, denoted as C;

[0047] S32 secondary fermentation: A, B and long bifidobacterium seed solution were added to C into the medium for secondary fermentation, cultured at a temperature of 37 ° C, anaerobic conditions for 12h to obtain a secondary fermentation product; the long bifidobacterium seed solution was added in an amount of 5% of the mass of C; the mass ratio of A, B and C was 3:21:50;

[0048] S33. Enzymatic dissolution of secondary fermentation products:

[0049] First, the secondary fermentation product obtained by S32 is subjected to high-speed centrifugation and filtration, and the supernatant is separated from the lower layer of cells. The lower layer of cells obtained after filtration is subjected to high-pressure homogenization for 2 to 3 times. Through microscopic examination, the cell lysis degree reaches more than 95%, and the upper layer of lysate and the lower layer of cell wall fragments are separated; All the lysates after high-pressure homogenization are combined with the supernatant separated by centrifugation to obtain Bifidobacterium longum lysate;

[0050] Secondly, it is calculated based on the mass ratio of each substance to the cell fragment enzymatic hydrolysate. Mix 9wt% of the lower layer of cell wall fragments with deionized water, add 5-6wt% Alcalase alkaline protease (enzyme activity>600,000 U / g, enzyme substrate ratio>5000U / g), 10-12wt% glycoside hydrolase (enzyme activity>300,000 U / g, enzyme substrate ratio>5000U / g), add the above substances into a reaction container, the balance is deionized water, use a water bath oscillator to fully disperse and mix the substances, adjust the pH value to 8-8.5, the water bath temperature is 55°C, the heating time is 3-4 hours, and then place the reaction container in a 90-95°C water bath for 15-20min to inactivate Alcalase alkaline protease and glycoside hydrolase, after inactivation, place in a refrigerator at 0-5°C for refrigeration and centrifugation, the centrifugation condition is 6000rmp / min, 8min, separate the supernatant after centrifugation, and obtain a cell fragment enzymatic solution; the cell wall fragment: enzyme mass ratio is 10:20;

[0051] Finally, the lysate of Bifidobacterium longum and the enzymatic hydrolysate of cell fragments were mixed, and 55% butanediol was added to the mixed liquid at a ratio of 55 wt % of the total amount, and the mixture was mixed evenly to obtain the bifid yeast fermentation product.

[0052] Example 2

[0053] This embodiment is the second embodiment of the present invention, which is different from the embodiment 1 in that S1. Preparation of medicinal porphyra extract A: First, MRS culture medium and medicinal porphyra powder are placed in water and stirred to dissolve at room temperature, the pH is adjusted to 6.0, and high-pressure sterilization is performed at 125°C for 10 minutes to obtain a fermentation medium containing medicinal porphyra, and the fermentation medium is high-pressure sterilized at 120°C for 15 minutes, wherein the mass ratio of MRS culture medium: medicinal porphyra: water is 1:0.5:100; secondly, lactic acid bacteria seed liquid is inoculated into the fermentation medium at an inoculation amount of 10%, and fermented at a fermentation temperature of 30°C for 70 hours; finally, the fermented liquid is taken out, high-pressure sterilized at 120°C for 15 minutes, and filtered to obtain medicinal porphyra extract A;

[0054] S2. Plant compound extract: Anemarrhena, Pueraria, Sophora japonica, and Sophora japonica buds are weighed in proportion and put into a wall breaking machine, and water of 1 times the total mass of the input plants is added to break the wall to obtain a composite slurry; the composite slurry is transferred to an extraction device, water of 8 times the total mass of the input plants is added, and the mixture is heated to 85°C for extraction, filtered, and the residue is discarded to obtain a filtrate; the filtrate is concentrated to a mass of 1.5 times the total mass of the input plants to obtain a plant compound extract, which is recorded as B; the input mass ratio of Anemarrhena, Pueraria, Sophora japonica, and Sophora japonica is 1:1:0.4:0.2;

[0055] S3. Preparation of bifid yeast fermentation:

[0056] S31. Primary fermentation: The cultured Bifidobacterium longum seed solution was inoculated into a sterilized fermentation medium at 4% of the volume of the medium, the pH was adjusted to 6.0, and the culture temperature was 35 ° C. Under anaerobic conditions, the culture was cultured for 70 h to obtain a fermentation broth, denoted as C;

[0057] S32. Secondary fermentation: A, B and Bifidobacterium longum seed solution were added to C and placed in a culture medium for secondary fermentation, and cultured at a temperature of 35 ° C under anaerobic conditions for 15 h to obtain a secondary fermentation product; the amount of Bifidobacterium longum seed solution added was 2% of the mass of C; the mass ratio of A, B and C was 1:7:15;

[0058] S33. Enzymatic dissolution of secondary fermentation products: the mass ratio of cell wall fragments to enzyme is 5:10.

[0059] Example 3

[0060] This embodiment is the third embodiment of the present invention, which is different from the embodiment 1 in that S1. Preparation of medicinal porphyra extract A: First, MRS culture medium and medicinal porphyra powder are placed in water and stirred to dissolve at room temperature, the pH is adjusted to 8.0, and high-pressure sterilization is performed at 125°C for 10 minutes to obtain a fermentation medium containing medicinal porphyra, and the fermentation medium is high-pressure sterilized at 120°C for 15 minutes, wherein the mass ratio of MRS culture medium: medicinal porphyra: water is 7:5:100; secondly, lactic acid bacteria seed liquid is inoculated into the fermentation medium at an inoculation amount of 25%, and fermented at a fermentation temperature of 40°C for 30 hours; finally, the fermented liquid is taken out, high-pressure sterilized at 120°C for 15 minutes, and filtered to obtain medicinal porphyra extract A;

[0061] S2. Plant compound extract: Anemarrhena, Pueraria, Sophora japonica, and Sophora japonica buds are weighed in proportion and put into a wall breaking machine, and water of 1 times the total mass of the input plants is added to break the wall to obtain a composite slurry; the composite slurry is transferred to an extraction device, water of 8 times the total mass of the input plants is added, and the mixture is heated to 85°C for extraction, filtered, and the residue is discarded to obtain a filtrate; the filtrate is concentrated to a mass of 1.5 times the total mass of the input plants to obtain a plant compound extract, which is recorded as B; the input mass ratio of Anemarrhena, Pueraria, Sophora japonica, and Sophora japonica is 15:15:8:3;

[0062] S3. Preparation of bifid yeast fermentation:

[0063] S31. Primary fermentation: The cultured Bifidobacterium longum seed solution was inoculated into a sterilized fermentation medium at 10% of the volume of the medium, the pH was adjusted to 8.0, and the culture temperature was 40 ° C. Under anaerobic conditions, the culture was cultured for 45 h to obtain a fermentation broth, denoted as C;

[0064] S32. Secondary fermentation: A, B and Bifidobacterium longum seed solution were added to C and placed in a culture medium for secondary fermentation, and cultured at a temperature of 40 ° C under anaerobic conditions for 8 h to obtain a secondary fermentation product; the amount of Bifidobacterium longum seed solution added was 7% of the mass of C; the mass ratio of A, B and C was 3:21:50;

[0065] S33. Enzymatic dissolution of secondary fermentation products: the mass ratio of cell wall fragments to enzyme is 15:25.

[0066] Example 4

[0067] This embodiment is the fourth embodiment of the present invention. Different from Embodiment 1, the secondary fermentation product obtained in S32 is not enzymatically dissolved, and the secondary fermentation product obtained in S32 is directly added to anhydrous ethanol at a volume ratio of 1:5, and ultrasonically treated at 500W for 10 to 15min, and then centrifuged at 2000 to 3000rmp / min for 15min, the filtrate is collected, and then inactivated at 60°C for 30min; filtered at 1.0μm, and pasteurized, butanediol is added to the mixed liquid at a ratio of 55wt% of the total amount, and mixed evenly to obtain the bifid yeast fermentation product.

[0068] Comparative Example 1

[0069] This comparative example is the first comparative example of the present invention. Different from Example 1, the medicinal pore fungus in S1 is not fermented with lactic acid bacteria, but is directly crushed, soaked, and extracted with ethanol to obtain the medicinal pore fungus extract A.

[0070] Comparative Example 2

[0071] This comparative example is the second comparative example of the present invention. Different from Example 1, S3 is fermented only once, and A and B are directly added to the culture medium during the fermentation of Bifidobacterium longum.

[0072] Comparative Example 3

[0073] This comparative example is the third comparative example of the present invention. Different from Example 1, only A is directly added to the culture medium during the secondary fermentation of Bifidobacterium longum S32.

[0074] Comparative Example 4

[0075] This comparative example is the fourth comparative example of the present invention. Different from Example 1, in the secondary fermentation process of Bifidobacterium longum S32, only B is directly added to the culture medium.

[0076] Comparative Example 5

[0077] This comparative example is the fifth comparative example of the present invention. Different from Example 1, in the secondary fermentation process of Bifidobacterium longum S32, no Bifidobacterium longum seed liquid is directly added to the culture medium.

[0078] Performance testing:

[0079] Inhibition of inflammatory factors:

[0080] The test method steps are as follows:

[0081] (1) Inoculation: Macrophages were inoculated into 24-well plates and incubated at 37°C in a 5% CO2 incubator for 18 to 24 h;

[0082] (2) Sampling: After 18 to 24 hours of cell plating in the 24-well plate, the cells were sampled in groups, with three replicate wells set up for each treatment group. The cells were cultured in a 37°C, 5% CO2 incubator for another 18 to 24 hours.

[0083] (3) LPS induction: After 18 to 24 hours of culture, the culture medium in the plate was aspirated and the plates were washed once with PBS:

[0084] Blank control group: only 55% butanediol was added to the cell culture medium;

[0085] Model control group: only LPS was added;

[0086] Benign control group: dexamethasone and LPS were added;

[0087] Experimental group: 3.5% concentration of the bifid yeast fermentation product and LPS obtained in any of the examples and comparative examples were added to the cell culture medium;

[0088] The above cell culture medium was cultured in a 37°C, 5% CO2 incubator for 18-24 hours and the supernatant was collected;

[0089] (4) Detection of inflammatory factor content: Take the cell supernatant from each well and detect the cell inflammatory factor content according to the ELISA kit operating instructions;

[0090] (5) Data processing: All data obtained in the experiment were processed by Excel software. GraphPadPrism_9.5 was used for statistical analysis and drawing. One-way analysis of variance (ANOVA) was used for component comparison. When P < 0.05, the difference was considered significant.

[0091] The inhibition of inflammatory factor IL-6 by the bifid yeast fermentation products obtained in Examples 1 to 4 and Comparative Examples 1 to 5 is shown in Figure 1, wherein (a) is the inhibition of IL-6 by the products obtained in Examples 1 to 4; (b) is the inhibition of IL-6 by the products obtained in Example 1 and Comparative Example 1; (c) is the inhibition of IL-6 by the products obtained in Example 1 and Comparative Examples 2 to 4; (d) is the inhibition of IL-6 by the products obtained in Example 1 and Comparative Example 5, and in the figure, an asterisk (*) usually indicates that the p value is less than 0.05, that is, the result is statistically significant; two asterisks (**) usually indicate that the p value is less than 0.01, that is, the result is highly statistically significant; three asterisks: (***) usually indicate that the p value is less than 0.001, that is, the result is statistically extremely significant; four asterisks (****) usually indicate that the p value is less than 0.0001, that is, the result is statistically particularly significant. The final detection of IL-6 content in the embodiments and comparative examples as test examples is shown in Table 1.

[0092] Table 1 Inhibitory effect of the bifid yeast fermentation products obtained in the examples and comparative examples on the inflammatory factor IL-6

[0093]

[0094] The inhibition of IL-6 inflammatory factor by the obtained bifid yeast fermentation product is shown in Tables 1 and Figure 1 The final IL-6 inflammatory factor concentration of the bifid yeast fermentation product obtained in the example was as low as 14 μg / mL, which was basically consistent with the positive control concentration of dexamethasone, and was better than the inhibitory effect of the bifid yeast fermentation product obtained in the comparative example on the IL-6 inflammatory factor.

[0095] Figure 1 From Example 1 and Example 4 in (a), it can be seen that after the secondary fermentation product obtained by S32 is not subjected to the enzyme dissolution process, the inhibitory effect of the obtained bifid yeast fermentation product on the IL-6 inflammatory factor is reduced, and the concentration is 35 μg / mL; Figure 1 (b) It can be seen from Example 1 and Comparative Example 1 that the medicinal pore fungus is not fermented with lactic acid bacteria, and the final IL-6 inflammatory factor concentration of the obtained bifid yeast fermentation product is greatly increased, up to 39.6 μg / mL; Figure 1 (c) It can be seen from Example 1 and Comparative Example 2 that the bifidobacterium was fermented only once, and the final IL-6 inflammatory factor concentration of the obtained bifid yeast fermentation product increased significantly, reaching 57.6 μg / mL; Figure 1 (c) It can be seen from Example 1 and Comparative Example 3 that during the secondary fermentation of S32 Bifidobacterium longum, only A was directly added to the culture medium without adding B, and the final IL-6 inflammatory factor concentration of the obtained bifid yeast fermentation product was greatly increased, reaching 43.6 μg / mL; Figure 1(c) It can be seen from Example 1 and Comparative Example 4 that during the secondary fermentation of S32 Bifidobacterium longum, only B was directly added to the culture medium without A. The final IL-6 inflammatory factor concentration of the obtained bifidobacterium fermentation product was greatly increased, up to 50 μg / mL; Figure 1 (d) From Example 1 and Comparative Example 5, it can be seen that during the secondary fermentation of S32 Bifidobacterium longum, the final IL-6 inflammatory factor concentration of the obtained bifid yeast fermentation product was significantly increased, reaching 40.6 μg / mL, without adding Bifidobacterium longum seed liquid to the culture medium.

[0096] 5α-Reductase Inhibition:

[0097] Test method:

[0098] (1) Female SD rats were fasted overnight and then killed by cervical dislocation. The liver was removed aseptically, weighed, and repeatedly rinsed with pre-cooled buffer. The connective tissue was removed and the liver was chopped up. The pre-cooled homogenate solution 4 times the amount of the tissue was added and homogenized in a glass homogenizer. The homogenate was transferred to a centrifuge tube and centrifuged at 12000r·min-1 at 4℃ for 30-60min. The supernatant was taken and the operation was repeated until the supernatant was clear. The mixture was aliquoted and stored at -80℃ for later use.

[0099] (2) Enzyme extraction buffer: 0.32 mol / L sucrose, 1.0 mmol / L disitol, 1.0 mmol / L EDTA, 20.0 mmol / L sodium phosphate;

[0100] Reaction buffer (0.2 mM, pH 6.5): Dissolve 1.0687 g of NaH2PO4·2H2O and 1.1281 g of Na2HPO4·2H2O in 50 ml of deionized water and dilute 10 times before use;

[0101] NADPH solution: weigh 8.3 mg of NADPH and dissolve it in 10 mL of reaction buffer to obtain a concentration of 1.0 mmol / L;

[0102] Testosterone solution: Weigh 28.84 mg of testosterone and dilute to 100 mL with anhydrous ethanol to obtain a concentration of 1.0 mmol / L.

[0103] (3) Add 2 mL of reaction buffer, 50 μL of 5α-reductase solution, and 100 μL of the sample solution to the cuvette, incubate at 37°C for 10 min, then add 200 μL of NADPH solution and 50 μL of substrate testosterone (T) solution in sequence, then immediately use a UV spectrophotometer to measure the absorbance value A0 at 340 nm, incubate at 37°C for 30 min and measure the absorbance value A30 again at 340 nm, calculate the change value A of the absorbance, and repeat twice. Set up a blank group (phosphate buffer + 5α-reductase + 55% butanediol + NADPH + T), a positive control group (phosphate buffer + 5α-reductase + finasteride + NADPH + T), and a sample group (phosphate buffer + 5α-reductase + sample to be tested + NADPH + T), and the sample to be tested is the bifid yeast fermentation product obtained in the embodiment and the comparative example; then determine the inhibition rate of the sample on 5α-reductase.

[0104] The inhibition rate of 5α-reductase was calculated as follows:

[0105]

[0106] The inhibition of 5α-reductase by the obtained bifid yeast fermentation products in the examples and comparative examples is shown in Table 3. The inhibition rate of 5α-reductase by the bifid yeast fermentation product obtained in the examples was up to 55.8%, which was better than the 50.2% of the positive control group using finasteride, and better than the inhibition rate of 5α-reductase by the bifid yeast fermentation product obtained in the comparative example.

[0107] It can be seen from Examples 1 and 4 that after the secondary fermentation product obtained by S32 is not subjected to the enzyme dissolution process, the inhibition rate of the obtained bifid yeast fermentation product on 5α-reductase is reduced; it can be seen from Example 1 and Comparative Example 1 that the medicinal pore fungus is not fermented with lactic acid bacteria, and the inhibition rate of the obtained bifid yeast fermentation product on 5α-reductase is greatly reduced to only 35.7%; it can be seen from Example 1 and Comparative Example 2 that the bifidobacterium is fermented only once, and the inhibition rate of the obtained bifid yeast fermentation product on 5α-reductase is greatly reduced to only 32.1%; it can be seen from Example 1 and Comparative Example 3 that the secondary fermentation of Bifidobacterium longum in S32 In the process, only A is directly added to the culture medium without adding B, and the inhibition rate of the obtained bifid yeast fermentation product on 5α-reductase is greatly reduced to only 37.1%; it can be seen from Example 1 and Comparative Example 4 that in the secondary fermentation process of S32 Bifidobacterium longum, only B is directly added to the culture medium without adding A, and the inhibition rate of the obtained bifid yeast fermentation product on 5α-reductase is greatly reduced to only 29.9%; it can be seen from Example 1 and Comparative Example 5 that in the secondary fermentation process of S32 Bifidobacterium longum, Bifidobacterium longum seed liquid is directly added to the culture medium, and the inhibition rate of the obtained bifid yeast fermentation product on 5α-reductase is greatly reduced to only 37.7%.

[0108] Oxidative Stress Inhibition:

[0109] Test method: Dilute the sample to a concentration of 5% and vortex to mix. Add each reagent according to the reaction system in Table 2, shake well, and let stand for 30 minutes. Read the absorbance at 517nm on a UV spectrophotometer, measure three times in parallel, take the average value, and calculate the inhibition rate according to the clearance formula. The different clearance rates obtained from different samples are used to calculate IC50 using graphpad prism8 software.

[0110] Table 2 Reaction system of scavenging DPPH free radicals by bifid yeast products obtained in test examples and comparative examples

[0111]

[0112] DPPH free radical scavenging rate (SR) = (1-(Ai-Aj) / Ac) × 100%

[0113] Wherein, Ai: reaction system with the test sample and DPPH solution added; Aj: control with the test sample and anhydrous ethanol added; Ac: blank control with DPPH solution and 55% butanediol added. The test sample is the bifid yeast fermentation product obtained in the embodiment and comparative example.

[0114] The scavenging effects of the bifid yeast fermentation products obtained in the examples and comparative examples on DPPH free radicals are shown in Table 3. The inhibition rate of the bifid yeast fermentation product obtained in the examples on DPPH is up to 55.8%, which is better than the inhibition rate of the bifid yeast fermentation product obtained in the comparative example on DPPH.

[0115] It can be seen from Examples 1 and 4 that after the secondary fermentation product obtained by S32 is not subjected to the enzyme dissolution process, the inhibition rate of the obtained bifid yeast fermentation product on DPPH is reduced; it can be seen from Example 1 and Comparative Example 1 that the medicinal pore fungus is not fermented with lactic acid bacteria, and the inhibition rate of the obtained bifid yeast fermentation product on DPPH is greatly reduced to only 60.3%; it can be seen from Example 1 and Comparative Example 2 that the bifidobacterium is only fermented once, and the inhibition rate of the obtained bifid yeast fermentation product on DPPH is greatly reduced to only 57.2%; it can be seen from Example 1 and Comparative Example 3 that the secondary fermentation of Bifidobacterium longum in S32 In the process, only A is directly added to the culture medium without adding B, and the inhibition rate of the obtained bifid yeast fermentation product on DPPH is greatly reduced to only 52.4%; from Example 1 and Comparative Example 4, it can be seen that in the secondary fermentation process of S32 Bifidobacterium longum, only B is directly added to the culture medium without adding A, and the inhibition rate of the obtained bifid yeast fermentation product on DPPH is greatly reduced to only 40.7%; from Example 1 and Comparative Example 5, it can be seen that in the secondary fermentation process of S32 Bifidobacterium longum, Bifidobacterium longum seed liquid is directly added to the culture medium, and the inhibition rate of the obtained bifid yeast fermentation product on DPPH is greatly reduced to only 54.7%.

[0116] Oil control effect test:

[0117] Test instrument: cuotometer MPA580 grease test probe Sebumeter manufactured by Courage+Khazaka, Germany (CK, Germany).

[0118] Test method: Follow up and test once before use and 7 and 14 days after use. Select the test area of ​​the forehead of the subject's face. 50 subjects were randomly divided into 10 groups. The first 9 groups used the samples prepared by the embodiments and comparative examples, respectively. The 10th group also used a commercially available product (a cosmetic or skin care product on the market that claims to have a good oil control effect) as a control group. The technician used a skin oil test probe to test the test site 3 times, took the average value, and collected a blank value for the first time. Use it twice a day, morning and evening. After the subjects used the sample continuously for 14 and 28 days, the technician used the skin oil probe to measure 3 times, and took the average test site forehead. The measured values ​​were statistically analyzed, and the mean method was used to analyze the changing rules and persistence of skin oil secretion. The experimental results are shown in Table 3.

[0119] Subjects: All subjects have signed informed consent and voluntarily participated in this test.

[0120] Informed consent was obtained verbally and in paper. Inclusion criteria: ① Chinese ② Age 20-55 years old (at the start of the trial) ③ Subjects should be insensitive to commonly used cosmetics ④ Subjects have not participated in other clinical studies in the past three months 3.3. Exclusion criteria ① Those who have travel plans during the experiment ②) Have used antihistamines in the past week or immunosuppressants in the past month Yujia Skin Research Institute 2 ③ Have used any anti-inflammatory drugs in the test area in the past two months ④ Insulin-dependent diabetic patients ⑤ Patients with respiratory diseases being treated ⑥ Women in lactation or pregnancy ⑦ People with allergic constitution, allergic dermatitis, etc., with skin diseases or disease history ⑧ People who are receiving dermatological treatment, or subjects who have taken hydroxy acids, whitening and anti-aging drugs within one month ⑨ The test area has large birthmarks, scratches, white spots, pigmented moles, keloids and other skin manifestations that affect the test. 3.4. Restrictions ① During the test, it is prohibited to perform treatments that may affect the test on the test area. ② During the test, it is prohibited to use other skin care products and other products that may affect the test on the test area. ③ During the test, daily necessities such as shower gel, soap, shampoo, etc. must be the same as those used before the start of the test and are prohibited from being replaced.

[0121] The target number of eligible subjects is 50, and the final number of subjects is 50, and the sebum content of the subjects is ensured to be above 100 μg / cm.

[0122] Skin oil secretion: Tested using Sebumeter. The amount of skin sebum before use is recorded as Se0, and the amount of skin sebum on the nth day is recorded as Sen. Parameter explanation: The lower the test value, the less sebum the skin produces. Result judgment: After using the product, the amount of sebum produced in the test area is significantly reduced, indicating that the test sample has an oil control effect.

[0123] Skin oil secretion reduction rate = (Se0-Se n ) / Se0*100%, where Se0, Se n The unit is μg / cm 2 .

[0124] The oil control effects of the obtained bifid yeast fermentation products of the embodiments and comparative examples are shown in Table 3. When the oil control test experiment was 14 days, the bifid yeast fermentation product obtained in the embodiment had a maximum oil secretion reduction rate of 23.1%, which was 8.9% better than the control group using a commercially available oil control product; when the experiment was 28 days, the bifid yeast fermentation product obtained in the embodiment had a maximum oil secretion reduction rate of 31.7%, which was 14.3% better than the control group using a commercially available oil control product, and better than the bifid yeast fermentation product obtained in the comparative example.

[0125] At 28 days of the oil control test experiment, it can be seen from Examples 1 and 4 that after the secondary fermentation product obtained by S32 is not subjected to the enzyme dissolution process, the oil secretion reduction rate of the obtained bifid yeast fermentation product is reduced; it can be seen from Example 1 and Comparative Example 1 that the medicinal pore fungus is not fermented with lactic acid bacteria, and the oil secretion reduction rate of the obtained bifid yeast fermentation product is greatly reduced, which is only 18.5%; it can be seen from Example 1 and Comparative Example 2 that the bifidobacterium is only fermented once, and the oil secretion reduction rate of the obtained bifid yeast fermentation product is greatly reduced, which is only 16.8%; it can be seen from Example 1 and Comparative Example 3 that during the secondary fermentation of Bifidobacterium longum S32, only A is directly added to the culture medium without adding B, and the oil secretion reduction rate of the obtained bifid yeast fermentation product is greatly reduced, which is only 15.3%; it can be seen from Example 1 and Comparative Example 4 that during the secondary fermentation of Bifidobacterium longum S32, only B is directly added to the culture medium without adding A, and the oil secretion reduction rate of the obtained bifid yeast fermentation product is greatly reduced, which is only 14.8 %; From Example 1 and Comparative Example 5, it can be seen that the secondary fermentation process of S32 Bifidobacterium longum directly does not add Bifidobacterium longum seed liquid to the culture medium, and the reduction rate of oil secretion by the obtained bifid yeast fermentation product is greatly reduced, which is only 16.5%. The oil control test experiment was carried out for 14 days. The rules of reducing oil secretion by the bifid yeast fermentation products obtained in each embodiment and comparative example were similar to 28 days, as shown in Table 3 for details.

[0126] Table 3 Performance test of bifid yeast fermentation products obtained in Examples and Comparative Examples

[0127]

[0128] Water and oil balance effect test:

[0129] Mice were randomly divided into 12 groups, 5 mice in each group, weighed, and after 5 days of adaptive feeding, all mice were depilated using an electric pusher and depilatory cream, with an abdominal depilatory area of ​​1*2cm2 and a back depilatory area of ​​2cm2. The mice were fed with modeling feed (2.5g fish oil and 48g linoleic acid were added to every 100g mouse feed) for 7 weeks. In the first 15 days, passive fish oil / linoleic acid (1:20) was gavaged (gavage scheme: 0.5mL / mouse in the morning and evening for 1-10 days; 0.35mL / mouse in the morning and evening for 11-15 days); drug treatment was carried out simultaneously, once a day. After that, only oily feed was fed for 30 days, and the rest remained unchanged. The blank group was fed with normal maintenance feed. The model group, positive control group, and treatment group were sandpapered, and sensitized and stimulated with DNCB.

[0130] The model is constructed as follows:

[0131] Blank control: only fed with normal feed;

[0132] Model control: modeling feed + DNCB;

[0133] Positive control: modeling feed + DNCB + commercially available tacrolimus ointment, 0.2 mL / animal / day;

[0134] Test group: Modeling feed + DNCB + bifid yeast fermentation products obtained in the examples and comparative examples

[0135] On the first day of modeling, 7% DNCB acetone solution was applied to the depilated area of ​​the abdomen for sensitization, and the second sensitization was performed at the same location 2 days later; on the sixth day of modeling, 0.5% DNCB acetone solution was applied to the back for stimulation, and then stimulated every 3 days until the end of the experiment. On the 15th day, the second sensitization was performed on the abdomen to observe the recurrence of dermatitis on the back. Samples were collected on the 15th, 30th, and 45th days of the experiment, and the differences between the groups were compared. The skin moisture and skin oil content were measured with a skin meter on the 15th, 30th, and 45th days, and the ratio of water content to oil content (water content / oil content) was calculated to evaluate the level of water and oil content in the mouse skin.

[0136] The test of the obtained bifid yeast fermentation product on the water-oil balance regulation of the skin in the embodiment and the comparative example is shown in Table 4. After 45 days of testing, the bifid yeast fermentation product obtained in the embodiment was used for the mouse epidermis, and the final skin water content / oil content reached 1.45, which was slightly better than the skin water content / oil content value of 1.30 obtained by using the positive control group, and was better than the bifid yeast fermentation product obtained in the comparative example for the water-oil regulation ability of the mouse epidermis.

[0137] After 45 days of testing, it can be seen from Examples 1 and 4 that the secondary fermentation product obtained by S32 does not undergo an enzyme dissolution process, and the final skin water content / oil content of the obtained bifid yeast fermentation product reaches 1.27; it can be seen from Example 1 and Comparative Example 1 that the medicinal pore fungus is not fermented with lactic acid bacteria, and the final skin water content / oil content of the obtained bifid yeast fermentation product reaches 1.19; it can be seen from Example 1 and Comparative Example 2 that the bifidobacterium is fermented only once, and the final skin water content / oil content of the obtained bifid yeast fermentation product reaches 1.17; it can be seen from Example 1 and Comparative Example 3 that S32 has long bifidobacteria. In the secondary fermentation process of Bifidobacterium longum, only A was directly added to the culture medium without adding B, and the final skin water content / oil content of the obtained bifid yeast fermentation product reached 1.10; it can be seen from Example 1 and Comparative Example 4 that in the secondary fermentation process of S32 Bifidobacterium longum, only B was directly added to the culture medium without adding A, and the final skin water content / oil content of the obtained bifid yeast fermentation product reached 1.07; it can be seen from Example 1 and Comparative Example 5 that in the secondary fermentation process of S32 Bifidobacterium longum, Bifidobacterium longum seed liquid was not added directly to the culture medium, and the final skin water content / oil content of the obtained bifid yeast fermentation product reached 1.12. The specific changes in the skin water content / oil content of mice tested for 15 days and 30 days are shown in Table 4.

[0138] Table 4 Water-oil balance test results of the bifid yeast fermentation products obtained in the examples and comparative examples

[0139]

[0140] In summary, the bifid yeast fermentation product obtained by the present invention can reduce skin oxidative stress, inhibit inflammatory factors IL-6 and 5α-reductase, is conducive to skin absorption when applied externally, has good skin transdermal effect, excellent oil control effect, can promote skin water and oil balance, and achieve the technical effects of oil control, acne removal and pore improvement.

[0141] The above is only an implementation method of the invention, and does not limit the scope of patent protection. Those skilled in the art can make non-substantial changes or substitutions based on the invention, which still fall within the scope of patent protection.

Claims

1. A method for preparing a bifid yeast fermentation product, characterized in that: The following steps are involved: S1. preparing medicinal porphyra extract A; S2. Extracting a plant composite extract: crushing, heating, extracting, filtering and concentrating the Anemarrhena asphodeloides, Puerariae lobata, Sophora japonica buds and Sapindus mukorossi to obtain a plant composite extract B; the mass ratio of the Anemarrhena asphodeloides, Puerariae lobata, Sophora japonica buds and Sapindus mukorossi is (1-15): (1-15): (0.4-8): (0.2-3); S3. Preparing a bifid yeast fermentation product: inoculating a long bifidobacterium seed solution into a fermentation medium, culturing and fermenting under anaerobic conditions to obtain a primary fermentation solution C; adding A, B and long bifidobacterium seed solution to C for secondary fermentation, and post-treating to obtain the bifid yeast fermentation product; the mass ratio of A, B and C is (1~5): (7~35): (15~80); The post-treatment process in S3 is to first separate and lyse the feed liquid obtained from the secondary fermentation to obtain a bifid yeast fermentation filtrate and cell wall fragments; then add an enzyme to the obtained cell wall fragments for enzymatic hydrolysis, inactivation, centrifugation, and filtration to obtain a cell wall fragment hydrolyzate; and finally, compound the bifid yeast fermentation filtrate and the cell wall fragment hydrolyzate to obtain the bifid yeast fermentation product. The enzymes added in the enzymatic treatment in S3 are glycoside hydrolase and Alcalase alkaline protease; The preparation method of the medicinal fusilli extract A in S1 comprises the following steps: adding medicinal fusilli to an MRS culture medium to obtain a medicinal fusilli culture medium, inoculating a lactic acid bacteria seed solution into the medicinal fusilli culture medium for culturing, fermenting, and extracting to obtain the medicinal fusilli extract A, wherein the mass ratio of the MRS culture medium to the medicinal fusilli is (1-7):(0.5-5).

2. The method for preparing the bifid yeast fermentation product according to claim 1, characterized in that: The inoculation amount of the lactic acid bacteria seed liquid in S1 is 10% to 25% of the volume of the culture medium, and the pH of the culture medium is 6 to 8.

3. The method for preparing the bifid yeast fermentation product according to claim 1, characterized in that: In the S1, the culture and fermentation temperature is 30-40° C. and the time is 30-70 hours. The fermented liquid is sterilized and filtered to obtain the medicinal porphyra extract A.

4. The method for preparing the bifid yeast fermentation product according to claim 1, characterized in that: The inoculation amount of the Bifidobacterium longum seed solution in S3 is 4-10% of the volume of the culture medium, and the pH of the culture medium is 6-8.

5. The method for preparing the bifid yeast fermentation product according to claim 1, characterized in that: The temperature of the first fermentation in S3 is 35-40°C, and the time is 45-70 hours; the temperature of the second fermentation in S3 is 35-40°C, and the time is 8-15 hours.

6. The method for preparing the bifid yeast fermentation product according to claim 1, characterized in that: The mass of the Bifidobacterium longum seed liquid added to the secondary fermentation in S3 is (2% to 7%) of the mass of C.

7. Use of a bifid yeast fermentation product prepared by the method for preparing a bifid yeast fermentation product according to any one of claims 1 to 6 in preparing an oil control product.

Images