Bifidobacterium adolescentis, composition for inhibiting generation and accumulation of lipofuscin, application and cosmetics

Abstract

The invention belongs to the field of daily chemicals, and discloses a strain of bifidobacterium adolescentis, the classification name of the bifidobacterium adolescentis BF23BA001 is bifidobacterium adolescentis, the preservation number is CGMCC NO.26999, and the strain of bifidobacterium adolescentis BF23BA001 is named as bifidobacterium adolescentis BF23BA001. The preservation date is March 31, 2023, and the preservation unit is China General Microbiological Culture Collection Center (CGMCC); the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Compared with other commercially available similar bacteria, the strain has unique advantages in the production of small molecule peptides and gamma-aminobutyric acid, has a remarkable anti-wrinkle advantage according to the functional characteristics of the small molecule peptides and gamma-aminobutyric acid, and has a remarkable anti-wrinkle effect through subsequent functional verification. The method is of great significance to further improvement of whitening, brightening, repairing and anti-aging effects of yeast fermentation products. In addition, the invention further discloses a composition for inhibiting generation and accumulation of lipofuscin, application based on the composition and cosmetics.

Description

Technical Field

[0001] The invention belongs to the field of daily chemicals, and specifically relates to a strain of Bifidobacterium adolescentis, a composition for inhibiting the generation and accumulation of lipofuscin, an application thereof, and cosmetics. Background Art

[0002] The applicant's prior application CN202310086320.2 Bifidobacterium adolescentis milk fermentation, including its products and their preparation and application, discloses the functions of Bifidobacterium adolescentis milk fermentation in free radical scavenging, anti-oxidation, repairing skin barrier, increasing skin elastin, soothing, etc.

[0003] The document does not mention whether it has any effect on whitening.

[0004] CN117180132A discloses a bifidobacterium longum exopolysaccharide and its use, which discloses the use of bifidobacterium exopolysaccharide in anti-glycation or inhibiting the formation of one or more advanced glycation end products or anti-skin aging or inhibiting the formation of age spots and wrinkles.

[0005] After further in-depth research by the applicant, it was found that in the whitening application of the fermentation product of specific bifidobacteria, its possible mechanism is not limited to this.

[0006] The technical problem to be solved in this case is: how to develop the potential whitening use of Bifidobacterium adolescentis milk fermentation product. Summary of the invention

[0007] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a strain of Bifidobacterium adolescentis, which has unique advantages in the production of small molecule peptides and γ-aminobutyric acid compared to other similar bacteria on the market. According to the functional characteristics of small molecule peptides and γ-aminobutyric acid themselves, it has significant anti-wrinkle advantages. At the same time, through subsequent functional verification, it is of great significance to further improve the whitening effect of yeast fermentation.

[0008] In addition, the present invention also discloses a composition for inhibiting the production and accumulation of lipofuscin, which contains milk fermentation products and yeast fermentation products. It has been verified that the content of small molecule peptides and γ-aminobutyric acid in the milk fermentation product fermented by Bifidobacterium adolescentis relatively prominent, and the content of protein, polysaccharide, and various amino acids in the yeast fermentation product of the present invention is relatively prominent. The combination of the two can effectively inhibit the production and accumulation of lipofuscin.

[0009] At the same time, the invention also provides applications and cosmetics based on the composition.

[0010] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0011] A strain of Bifidobacterium adolescentis, the classification name of the Bifidobacterium adolescentis is Bifidobacterium adolescentis, the preservation number is: CGMCC NO.26999; preservation date: March 31, 2023, preservation unit: General Microbiology Center of China Microorganism Culture Collection Administration; preservation address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0012] At the same time, the present invention also discloses the use of fermenting milk with the Bifidobacterium adolescentis to prepare milk fermentation products.

[0013] Meanwhile, the invention also discloses a milk fermentation product, which is prepared by fermenting milk with the Bifidobacterium adolescentis as described above.

[0014] In the above-mentioned milk fermentation product, the preparation method of the milk fermentation product is: 6 ~10 8 CFU / mL of Bifidobacterium adolescentis bacterial liquid is added into sterilized milk and fermented for 12 to 48 hours; the volume ratio of Bifidobacterium adolescentis bacterial liquid to milk is 1 to 10:100.

[0015] In addition, the present invention also discloses a composition for inhibiting the generation and accumulation of lipofuscin, comprising a milk fermentation product and a yeast fermentation product; the mass ratio of the milk fermentation product to the yeast fermentation product is 0.5-10:1-10;

[0016] The fermentation bacteria of the milk fermentation product is Bifidobacterium adolescentis.

[0017] Conventional whitening ideas usually target melanin issues, with common ingredients such as niacinamide, glabridin, and resveratrol. However, in addition to the content and accumulation of melanin in the skin, removing yellowness and improving dullness are also issues of concern when whitening. As people's understanding and research on skin whitening become more in-depth and comprehensive, healthy white or skin color management has become a trend. Pigment (melanin, lipofuscin, hemoglobin, etc.), inflammation (erythema, post-inflammatory pigmentation), and structure (dryness, roughness, wrinkles) are all issues that need to be addressed for healthy whitening.

[0018] Lipofuscin is a yellow-brown pigment particle that accumulates in cells. Its deposition in skin cells may cause skin problems such as chloasma, age spots, dull skin, roughness, and wrinkles. Ultraviolet rays and skin inflammation can lead to an increase in free radicals in the skin layer, accelerating the production of lipofuscin; while dysfunction of the mitochondrial-lysosomal axis can lead to lipofuscin accumulation. Lipofuscin promotes the production of mitochondrial ROS and causes lysosomal dysfunction through lysosomal membrane permeabilization, resulting in a decrease in the number of lysosomes and impaired activity. Therefore, improving relevant indicators can prevent the production and accumulation of lipofuscin, as well as the accumulation and aging of skin pigments caused by ultraviolet rays and free radicals.

[0019] The accumulation of lipofuscin in cells shows photosensitization under visible light, producing singlet oxygen, which may cause DNA damage. DNA damage further promotes melanin production and deepens skin pigmentation; in addition, DNA damage may also affect the life cycle and renewal of skin cells. Therefore, improving relevant indicators can improve the adverse effects of lipofuscin accumulation on the skin, such as melanin deposition, as well as improve skin sagging, wrinkles and uneven pigmentation caused by DNA damage.

[0020] Skin redness is a common symptom of skin inflammation, which is usually related to vasodilation and the release of inflammatory factors. Inflammatory factors, such as interleukin (IL) and tumor necrosis factor (TNF-α), can cause symptoms such as skin redness. Therefore, improving related indicators (inhibiting the activity of inflammatory factors) can relieve skin irritation to a certain extent and reduce skin redness.

[0021] Damage to the skin barrier function can affect the moisture content of the skin, leading to dryness, scaling, roughness, and even fine lines and dry lines. These conditions can affect the radiance and transparency of the skin, which can indirectly affect the skin tone. Therefore, keeping the skin hydrated and maintaining a good skin barrier function are essential for improving and maintaining skin tone.

[0022] In summary, skin whitening has a complex pathway, among which reducing the production and accumulation of lipofuscin, anti-oxidation, increasing the activity of metabolism-related organelles, preventing DNA damage, inhibiting the activity of inflammatory factors, and repairing the skin barrier are all helpful in improving skin color.

[0023] The core mechanism of the present invention is:

[0024] The use of Bifidobacterium adolescentis can significantly increase the small molecule peptides and γ-aminobutyric acid in milk fermentation products; these two substances are closely related to the production of collagen;

[0025] At the same time, we found in our research that the increase of small molecule peptides and γ-aminobutyric acid will make the yeast fermentation products more effective in inhibiting the production and accumulation of lipofuscin. This may be because the bifidobacterium adolescentis and milk fermentation products are rich in polysaccharides, small molecule peptides, and γ-aminobutyric acid, and the yeast fermentation products are rich in amino acids, and the effective ingredients of the two fermentation broths complement and synergize.

[0026] Bifidobacterium adolescentis and milk fermentation products can effectively improve lysosomal activity, and yeast fermentation products can effectively improve mitochondrial activity. The combination of the two can better repair mitochondrial-lysosomal axis damage, reduce lipofuscin accumulation, and improve cell metabolism. Both fermentation products inhibit the activity of cellular inflammatory factors and reduce skin redness caused by skin inflammation. At the same time, they can effectively remove free radicals and avoid excessive free radicals causing lipofuscin production and skin aging. In addition, the use of the two fermentation products together can better repair skin irritation damage, repair the skin barrier, and maintain the moisture of the skin surface to maintain skin luster.

[0027] In the above composition for inhibiting the production and accumulation of lipofuscin, the Bifidobacterium adolescentis is one or a combination of the above-mentioned Bifidobacterium adolescentis, Bifidobacterium adolescentis BBF-06, and Bifidobacterium adolescentis SF-B40.

[0028] In the above-mentioned composition for inhibiting the production and accumulation of lipofuscin, the mass ratio of the milk fermentation product to the yeast fermentation product is 0.5-8:2-9.5, preferably the mass ratio of the milk fermentation product to the yeast fermentation product is 2-8:2-8.

[0029] In the above-mentioned composition for inhibiting the production and accumulation of lipofuscin, the preparation method of the milk fermentation product is:

[0030] The viable count was 10 6 ~10 8 CFU / mL of Bifidobacterium adolescentis bacterial liquid is added into sterilized milk and fermented for 12 to 48 hours; the volume ratio of Bifidobacterium adolescentis bacterial liquid to milk is 1 to 10:100.

[0031] In the above-mentioned composition for inhibiting the production and accumulation of lipofuscin, the preparation method of the yeast fermentation product is:

[0032] After the activation of brewer's yeast, the culture medium (1-3wt% glucose, 0.5-1.5wt% peptone, 0.4-0.6wt% yeast extract powder) is inoculated and fermented at 28-32°C for 12-24h, centrifuged and initially filtered, and then the impurities are adsorbed with activated carbon and then filtered to obtain the yeast fermentation product.

[0033] In addition, the present invention also discloses the use of any of the above compositions in preparing cosmetics.

[0034] In the above-mentioned use, the cosmetic is a cosmetic for skin care use.

[0035] In the above-mentioned use, the cosmetic is a cosmetic having the function of inhibiting the generation and accumulation of lipofuscin;

[0036] and / or,

[0037] The cosmetics are cosmetics with multi-pathway whitening and brightening functions;

[0038] and / or,

[0039] The cosmetics have the functions of repairing, removing redness and resisting oxidation.

[0040] Finally, the present invention also discloses a cosmetic comprising any one of the above compositions.

[0041] In the above-mentioned cosmetics, the concentration of the composition in the cosmetics is 5 to 20 wt %.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The adolescent bifidobacterium / milk fermentation product of the present invention can reduce the level of lipofuscin in nematodes, and can cooperate with yeast fermentation products to reduce lipofuscin accumulation. The fermentation broth of the present invention is rich in polysaccharides, small molecule proteins, γ-aminobutyric acid and small molecule organic acids, which can comprehensively improve skin color from the aspects of removing yellow spots, anti-inflammatory and redness, protecting and suppressing blackness, moisturizing and brightening. Compared with traditional whitening agents, the adolescent bifidobacterium / milk fermentation broth of the present invention has high safety, and a concentration of ≤5% is non-toxic to cells such as HaCat, and has no hemolytic effect on red blood cells, and can gently improve skin color. The two fermentation broths are rich in nutrients, which can replenish nutrition for skin cells in time without damaging cells, and are suitable as long-lasting and mild whitening skin conditioners in skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Take pictures of the lipofuscin fluorescence of nematodes for each sample, control group, and blank group;

[0045] Figure 2 This is the gel electrophoresis diagram of the cell DNA damage test of sample 1 and sample 5. DETAILED DESCRIPTION

[0046] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0047] Source of reagents:

[0048] Bifidobacterium adolescentis 1: Bifidobacterium adolescentis BF23BA001, classified as Bifidobacterium adolescentis (Bifidobacterium adolescentis), deposit number: CGMCC NO.26999; deposit date: March 31, 2023, deposit unit: General Microbiology Center of China Microbiological Culture Collection Administration; deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing;

[0049] Bifidobacterium adolescentis 2: Bifidobacterium adolescentis BBF-06, Shandong Zhongke Jiayi Bioengineering Co., Ltd.;

[0050] Bifidobacterium adolescentis 3: Bifidobacterium adolescentis SF-B40, Shandong Sunflower Bioengineering Co., Ltd.;

[0051] Bifidobacterium infantis: SHMCC D11206, Shanghai Collection of Microorganisms;

[0052] Bifidobacterium longum: SHMCC D24500, Shanghai Collection of Microorganisms;

[0053] Saccharomyces cerevisiae: SHMCC D11813, Shanghai Collection of Microorganisms.

[0054] Part I. Preparation and Identification of Milk Fermentation Products

[0055] The preparation method of milk fermentation product is as follows:

[0056] (1) sterilizing milk at 95° C. for 30 minutes, and cooling to room temperature after sterilization to obtain a fermentation substrate;

[0057] (2) Cultivate the bifidobacterium until the viable count is 10 6 ~10 7 CFU / mL of Bifidobacterium liquid;

[0058] (3) inoculating the fermentation substrate prepared in step (1) with bifidobacterium liquid, the volume ratio of bifidobacterium liquid to milk being 5:100, and fermenting and culturing at 37°C for 24 hours; after the fermentation, sterilizing the material at 95°C for 30 minutes; cooling to room temperature after sterilization, centrifuging at 4800 rpm for 30 minutes, collecting the supernatant, and filtering the supernatant with a 0.22 μm filter membrane to remove bifidobacteria and impurities, thereby obtaining bifidobacterium milk fermentation product.

[0059] By the above method, various milk fermentation products were prepared. The relationship between the numbers of the milk fermentation products and the corresponding bacterial species is shown in Table 3;

[0060] The contents of small molecule peptides, γ-aminobutyric acid and lactic acid in milk fermentation products were detected. The contents of small molecule peptides, γ-aminobutyric acid and lactic acid in milk and milk fermentation products can be seen in Table 3;

[0061] Small molecule peptide content test method:

[0062] The fermentation product samples were passed through a 10 kDa ultrafiltration tube, and the polypeptide content in the filtrate <10 kDa was tested according to the BCA kit method of Thermo.

[0063] (1) Gradient dilution of albumin (BSA) standard: Open an ampoule containing BSA stock solution and prepare solutions A to I in sequence according to the contents in Table 1. Refer to Table 1 to prepare the BSA standard solutions of various concentrations in the standard curve.

[0064] Table 1 Dilution scheme of BSA standard at various concentrations in the standard curve

[0065]

[0066]

[0067] (2) Preparation of the sample to be tested: The sample was diluted 10 times for testing (100 μL sample stock solution + 900 μL ultrapure water).

[0068] (3) Preparation of BCA working solution: Take out solution A and solution B from the kit and prepare an appropriate volume of AB mixed solution in a ratio of A:B = 50:1 in the dark.

[0069] (4) Add 25 μL of diluted BSA standard, test sample, and ultrapure water to a 96-well transparent microplate, add 200 μL of BCA working solution, shake for 30 seconds to mix thoroughly, seal and incubate in a 37°C constant temperature incubator for 30 minutes.

[0070] (5) Take out the 96-well transparent microplate and measure the absorbance value of each sample at 562 nm using an ELISA reader (the absorbance value of the sample should be controlled within the absorbance range of the standard).

[0071] (6) Draw a standard curve: Set the concentration of the standard as the X-axis and the absorbance value corresponding to each concentration as the Y-axis, and draw a standard curve Y=aX+b. The standard curve requires R 2 ≥0.99.

[0072] (7) Calculate the final protein concentration of the sample according to the standard curve formula: Substitute the absorbance value of the sample to be tested (minus the background) into the formula to calculate the sample concentration X, and multiply it by the corresponding dilution factor to obtain the sample protein concentration.

[0073] GABA test method:

[0074] Extraction: Take an appropriate amount of sample, add 25mL 60% ethanol, shake in a water bath at 30℃ for 3h, transfer the supernatant, add another 25mL, shake in a water bath at 30℃ for 3h, centrifuge at 5000rpm for 8min, transfer the supernatant and combine it with the first one. Take 25mL, evaporate at 40℃ until almost dry, transfer to a 5mL volumetric flask with 60% ethanol and make up to volume.

[0075] Online derivatization: 120μL of sample / standard product, add 600μL of o-phthalaldehyde derivatization solution, and derivatize. (Derivatization reagent: o-phthalaldehyde derivatization solution: Dissolve 10mg o-phthalaldehyde in 0.5mL methanol, add 2mL of 0.4mol / L boric acid buffer solution (pH-10.2) and 30μL of 2-mercaptoethanol, and store at 4℃ for 2 days).

[0076] Aglient C 18 (4.6×250mm×5μm); column temperature 40℃; detection wavelength 332nm; injection volume 10μL. Mobile phase A is 25mmol / L sodium acetate buffer solution (adjusted to pH 5.90 with 4% acetic acid), mobile phase B is acetonitrile; elution program is shown in Table 2;

[0077] Table 2 Elution schedule

[0078] Time min Flow rate (mL / min) Mobile phase A (%) Mobile phase B (%) 0 1.0 90 10 17.0 1.0 40 60 17.1 1.0 90 10 20.0 1.0 90 10

[0079] Lactic acid testing method:

[0080] The test was conducted using an HPLC-RID detector; ArtChrom H 8μ (300*7.8 mm), column temperature 30° C.; sample injection volume 10 μL, mobile phase 0.1% TFA, flow rate 0.8 mL / min.

[0081] Table 3 Results of marker components in milk and milk fermentation products

[0082]

[0083] Result analysis:

[0084] As shown in Table 3, the use of Bifidobacterium adolescentis can effectively increase the concentrations of small molecule peptides, γ-aminobutyric acid, and lactic acid. In particular, the use of Bifidobacterium adolescentis 1 that has been screened and separated has a particularly good effect in this regard.

[0085] By comparing milk fermentation product 2 and milk fermentation product 5, it can be found that Bifidobacterium adolescentis synchronously increases the content of small molecule peptides, γ-aminobutyric acid, and lactic acid, and generally increases synchronously. Other similar bacteria species cannot show similar patterns. For example, fermenting milk with Bifidobacterium longum can significantly increase the content of small molecule peptides, but it does not significantly help to increase γ-aminobutyric acid.

[0086] The effective components of milk fermentation product 1 are shown in Table 4 below:

[0087] Table 4 Effective ingredients of milk fermentation product 1

[0088] Element mg / mL unit Test Method Small molecule peptides 341.1 mg / L BCA Protein Quantitation Kit GABA 16.24 mg / L HPLC lactic acid 3.024 mg / mL HPLC Acetic acid 2.60 mg / mL HPLC glucose 3.08 mg / mL HPLC Galactose 1.67 mg / mL HPLC lactose 4.81 mg / mL HPLC Total Sugar 22.35 mg / mL Phenol-sulfuric acid colorimetry Total Protein 2.02 mg / mL BCA Protein Quantitation Kit

[0089] Part II Preparation and identification of yeast fermentation products

[0090] The preparation method of yeast fermentation product is as follows: 2wt% of glucose, 1wt% of peptone, 0.5wt% of yeast extract powder and 96.5wt% of deionized water are added into a conical flask, dissolved and sterilized, and cooled to room temperature, and brewer's yeast is inoculated and activated, and then fermented on a shaker for 20 hours, centrifuged at 5000rpm for 30 minutes, and then initially filtered to obtain a coarse filtrate, 1% activated carbon is added to the coarse filtrate, and stirred for about 10 minutes to remove impurities, and then filtered through a 0.45μm filter membrane to obtain a yeast fermentation product.

[0091] The effective ingredient content of the yeast fermentation product prepared by the above method is shown in Table 5 below;

[0092] Table 5 Effective ingredients of yeast fermentation products

[0093] Element unit content Test Method Total Sugar mg / mL 7 Phenol-sulfuric acid colorimetry Total Protein mg / mL 0.7 Kjeldahl nitrogen determination Asparagine mg / L 71.30 HPLC Threonine mg / L 2.26 Amino Acid Automatic Analyzer Glutamine mg / L 39.2 HPLC Proline mg / L 2.26 Amino Acid Automatic Analyzer

[0094] Part III Preparation of the composition

[0095] 3.1 Screening of whitening active ingredients

[0096] One of the main purposes of this project is to explore the whitening potential of milk fermentation products of Bifidobacterium adolescentis. After preliminary testing, we found that milk fermentation products of Bifidobacterium adolescentis alone cannot achieve particularly excellent whitening effects. Therefore, we further verify whether it can be combined with other whitening active ingredients to achieve better whitening effects.

[0097] Milk fermentation products and other whitening active ingredients were compounded to evaluate the lipofuscin inhibition and deposition performance.

[0098] According to existing data, 0.001-0.005% resveratrol has a good inhibitory effect on lipofuscin; glabridin and niacinamide are classic whitening ingredients commonly used in cosmetics. Glabridin is generally added in an amount not exceeding 0.05% due to its high cost. Nicotinamide is added in an amount less than 5% due to regulatory requirements.

[0099] Preliminary experiments found that after a certain period of culture, the nematodes in the 0.05% glabridin and 5% nicotinamide concentration groups were smaller in size than those in the blank group. This may be because too high a concentration is toxic to the nematodes. After reducing the concentration by 5 times, the nematodes had a more normal morphology, so this concentration was used for formal lipofuscin testing.

[0100] In the early screening process, we screened the compounding performance of active ingredients with obvious whitening function and milk fermentation product 1. The specific compounding can be seen in the following Table 6; in Table 6, the balance is water.

[0101] Table 5 Formulation Unit: wt%

[0102]

[0103]

[0104] 3.2 Performance Testing

[0105] The performance test package is for nematode lipofuscin test, and the test method is:

[0106] Nematode lipofuscin test:

[0107] (1) Synchronization of nematodes (hypochlorous acid lysis method): Collect Caenorhabditis elegans and use nematode lysis solution (5N NaOH:

[0108] 5% NaClO=1:2) to lyse the nematodes, and then cultured them to the L4 stage for subsequent experiments (culture temperature was 20°C, using NGM plates containing Escherichia coli OP50).

[0109] (2) Determination of lipofuscin levels in nematodes

[0110] The culture medium containing the sample was prepared. The final concentration of the sample in the culture medium was as shown in Table 5, and the buffer solution was used as blank.

[0111] The L4 stage nematodes were transferred to the prepared sample culture plates, each plate containing 500 nematodes, and the treated nematode plates were placed in a 20°C incubator for culture.

[0112] After culturing at 20°C for 5 days, the nematodes were collected and washed 3 times. The nematodes were transferred to an agarose plate, 1% tetramisole hydrochloride was added, and the plate was left to stand for 1 hour for anesthesia.

[0113] (3) Observation and analysis of lipofuscin fluorescence in nematodes

[0114] Observe the fluorescence intensity and take photos under a fluorescence microscope. When taking photos, randomly select 30 different nematodes from the same plate for photography, and the parameters such as exposure should be consistent in the same experiment. All photos should be saved for subsequent analysis.

[0115] (4) Image J software analysis: Image J was used to quantitatively analyze the fluorescence intensity of the nematodes and the autofluorescence content of lipofuscin in the nematodes.

[0116] (5) Lipofuscin relative fluorescence intensity = sample fluorescence intensity / blank group fluorescence intensity * 100%

[0117] The test results can be found in Table 7 and Figure 1-2 ;

[0118] Table 7 Performance test results

[0119] Lipofuscin relative fluorescence intensity / % Sample 1 74.48 Sample 2 80.07 Sample 3 96.24 Sample 4 85.38 Sample 5 94.23 Sample 6 83.10 Sample 7 101.65 Sample 8 88.26 Control group 96.83 Blank Group 100.00

[0120] Result analysis:

[0121] Combination Figure 1 As shown in Table 7, compared with the blank group, the effect of using 5% milk fermentation product 1 alone was not obvious, and lipofuscin only decreased by about 3%. The effect of using 5% yeast fermentation product alone was only about 6% lower than that of the blank group, and the effect was not obvious; after adding 5% milk fermentation product 1, the effect was significantly enhanced, and lipofuscin decreased by 25.52%, indicating that the two together had a good synergistic effect in reducing lipofuscin.

[0122] 0.01% glabridin had no significant effect on reducing lipofuscin, and the effect was slightly enhanced after adding milk fermentation product 1, but not obvious. 0.005% resveratrol and 1% nicotinamide could reduce lipofuscin to a certain extent, but there was no obvious enhancement effect when used together with milk fermentation product 1, and the effect of reducing lipofuscin in nematodes was not as good as sample 1.

[0123] This indicates that the combination of Bifidobacterium adolescentis / milk fermentation products and yeast fermentation products has a good synergistic effect on reducing the level of lipofuscin in nematodes, but has no obvious synergistic effect on other classic whitening ingredients.

[0124] Through the above screening, we can know that sample 1 has the best performance in reducing lipofuscin in nematodes. In order to further clarify the source of its better performance and explore other effects of sample 1 in whitening and brightening, we further tested the antioxidant, anti-inflammatory and other properties of samples 1-8.

[0125] The test method is as follows:

[0126] Hydroxyl free radical scavenging rate test:

[0127] Hydroxyl free radicals act on biological molecules such as proteins, nucleic acids, and lipids in the body, causing damage to cell structure and function. Hydroxyl free radical scavenging ability is one of the important indicators of the sample's antioxidant capacity. The test method is as follows:

[0128] After H2O2 is mixed with divalent iron ions, hydroxyl radicals are generated. Salicylic acid reacts with hydroxyl radicals to produce a colored product, 2,3-dihydroxybenzoic acid, which has a strong absorption peak at 510nm. If a test substance with the function of scavenging hydroxyl radicals is added to the system, and the effect of the test substance in capturing hydroxyl radicals is greater than that of salicylic acid, the hydroxyl radicals can be scavenged in time, thereby reducing the amount of colored products generated and causing a decrease in absorbance. Therefore, the fixed reaction time method is used to measure the absorbance of the reaction solution containing the test substance at 510nm, and compare it with the blank group, so that the scavenging effect of the test substance on hydroxyl radicals can be determined.

[0129] Prepare the test sample according to Table 8, add the reagents in Table 7 into a 25 mL test tube in sequence and react.

[0130] Table 8 Hydroxyl radical test steps

[0131]

[0132]

[0133] Hydroxyl free radical scavenging rate (%) = [A2-(A1-A3)] / A2×100%; at the same test concentration, the greater the scavenging rate, the better the antioxidant effect.

[0134] Cellular inflammatory factor TNF-α, IL-1β test

[0135] Skin inflammation is usually accompanied by redness and swelling. Improving skin inflammation can improve skin redness. Test the expression of inflammatory factors TNF-a and IL-1β to evaluate the sample's effect on alleviating skin inflammation. The test steps are as follows:

[0136] (1) Cell preparation: Harvest the THP-1 cell suspension of stable subculture in the culture flask, transfer it to a 15 mL centrifuge tube, centrifuge it at 1000 rpm for 5 min, discard the supernatant, resuspend the cells in 8 mL of complete medium, and count them using a cell counter.

[0137] (2) Seed plate: Inoculate each well of a 24-well plate with a cell suspension containing 5×10^5 THP-1 cells, and add complete culture medium until the total volume in the well is 0.5 mL.

[0138] (3) Sample addition and sample pre-incubation: Set up a blank control group, a model group, and a sample group, add 0.5 mL of complete culture medium to the corresponding wells of the blank control group and the model group, add 0.5 mL of working solution containing the sample to the corresponding wells of the sample group, shake and mix, and culture in an incubator for 18-24 hours. When testing sample 1, because the system contains about 10% fermentation liquid, it has a slight effect on cell survival rate, so the test concentration is reduced to half of the original. The sample in the sample 1 test system is "2.5% milk fermentation product 1 + 2.5% yeast fermentation product"; according to the preliminary experiment, the cell safety concentration of the classic whitening ingredient (see Table 9 below) is much lower than the nematode test concentration in the sample setting group, so the group containing the classic whitening ingredient is no longer subject to formal cell testing. The final test concentrations of other groups are shown in Table 5.

[0139] Table 9 Classic whitening ingredient cell safety concentration table

[0140] Classic whitening ingredients Cell safety concentration / % Resveratrol 0.0002 Glabridin 0.0002 Niacinamide 0.00001

[0141] (4) LPS stimulation: Add 10 μL of LPS working solution (100 μg / mL) to each well of the model group and sample group to make the final concentration of LPS in the well about 1 μg / mL, shake to mix, and culture in the incubator for 24 h.

[0142] (5) RNA extraction: Collect cells from each group of wells and use an RNA extraction kit to extract total RNA from each group of cells.

[0143] (6) Reverse transcription: The total RNA of each group of cells was reverse transcribed into cDNA using a reverse transcription kit.

[0144] (7) qPCR detection: Using GAPDH as the internal reference, the relative expression levels of TNF-a and IL-1β in each group were detected.

[0145] (8) Comparison method: The model group is a group that only applies the stimulus source without adding samples or other components to relieve stimulation; the sample group is a group that adds samples and applies stimulation; the blank control group is a normal cell group that does not stimulate or contain samples. Therefore, the expression of inflammatory factors TNF-a and IL-1β is lower than that of the model group, indicating that the sample has the effect of reducing the expression of inflammatory factors. The closer the data is to the blank control group, the stronger the effect.

[0146] Lysosomal activity test:

[0147] Lysosomes contain a variety of hydrolases that can degrade macromolecules. The activity and number of lysosomes can indirectly reflect the metabolic renewal capacity of cells. The higher the lysosome activity, the stronger the cell's metabolic capacity.

[0148] Under physiological pH conditions, the net charge of neutral red dye is almost zero, allowing it to penetrate the cell membrane and enter the cell by non-ionic passive diffusion. The proton gradient in the lysosome makes the pH value in the lysosome lower than that in the cytoplasm, which can make neutral red charged and accumulate in the lysosome, thereby detecting lysosomal activity.

[0149] Test method: Skin fibroblasts were plated in a 96-well plate, with 8,000 cells per well. After the cells adhered to the wall, a blank control group and a sample group were set up. Serum-free culture medium was added to the blank control group, and serum-free culture medium containing the sample was added to the sample group. The final test concentration of the sample is shown in Table 5. After 24 hours of treatment, the cell culture medium was discarded, replaced with 200 μL of fresh serum-free culture medium, 20 μL of neutral red was added and incubated for 2 hours, then the culture medium containing neutral red was removed, washed twice with PBS, and then 200 μL of neutral red detection lysis solution was added, lysed on a shaker at room temperature for 10 minutes, and the A540 of each well was measured.

[0150] The blank control was used as a reference to calculate the lysosomal activity of the sample groups.

[0151] Lysosomal activity %=(A540 sample group / A540 blank)*100%.

[0152] Mitochondrial activity test:

[0153] The dye can be reduced by dehydrogenase in mitochondria to generate a highly water-soluble orange-yellow formazan product. The depth of its color is proportional to cell proliferation and inversely proportional to cytotoxicity. For the same cells, the depth of the color is linearly related to the number of cells.

[0154] 7500 cells / 100 μL were inoculated into a 96-well plate and cultured for 24 h, and the culture medium was discarded; serum-free DMEM containing the sample was added for 24 h, and the same dose of water was added as the blank group. The final test concentration of the sample was shown in Table 5; then 10 μL of CCK-8 reagent was added to each well, and the incubation was continued at 37°C for 2 h, and OD450nm was measured.

[0155] The blank control was used as a reference to calculate the mitochondrial activity of the sample group: Mitochondrial activity % = (A450 sample group / A450 blank)*100%.

[0156] DNA damage test:

[0157] DNA molecules are negatively charged during electrophoresis, and their migration speed is related to their own molecular size and configuration. When DNA molecules are damaged, they will break and form small molecular fragments, which will show a tailing phenomenon during electrophoresis. The longer the tail, the greater the degree of damage. Therefore, this method can be used to detect the degree of damage to DNA molecules in cells.

[0158] (1) According to the sample addition settings, the samples were divided into blank group, model group, and sample group.

[0159] Blank group: sample solvent control group without UV irradiation;

[0160] Model group: 3J / cm 2 UVA-irradiated sample solvent control group;

[0161] Sample group: Use 3J / cm 2 Sample loading group for UVA illumination.

[0162] (2) Experimental operation

[0163] Cell preparation: Prepare Bal b / c3T3 cell suspension in a 24-well plate to a concentration of 1.0×10 5 cells / well and placed in a CO2 incubator for 24 h.

[0164] Sample pretreatment: cells were pretreated with samples for 24 h, and the blank group and model group were added with equal amounts of solvent control (three replicates were set for each well), and the final test concentration of the samples was consistent with Table 5.

[0165] UVA irradiation treatment: After 24 hours of sample pretreatment, the cells were washed with PBS. After washing, the model group and the sample group were irradiated with UVA at 3J / cm 2 The blank group was treated with light protection for the same period of time.

[0166] Cell collection and DNA extraction: Cells were collected after UVA irradiation treatment, and cell DNA was extracted using a DNA extraction kit. The extracted DNA was tested for DNA concentration and purity using a micro-spectrophotometer.

[0167] Gel electrophoresis of cell DNA: Prepare 1% agarose gel for cell DNA electrophoresis.

[0168] Photographic analysis of results: After electrophoresis, the electrophoretic bands were photographed using a gel imaging analyzer.

[0169] Skin repair test:

[0170] After the skin is stimulated by SLS, the epidermal permeability barrier function is damaged and the transepidermal water loss rate increases. The repair effect of the sample on the skin is evaluated by comparing the recovery rate before and after the use of the sample. Test the transepidermal water loss rate T0 of the inner arm skin; apply 1% sodium dodecyl sulfate (SLS) aqueous solution to the inner arm skin, remove the patch after 24 hours, and test the transepidermal water loss rate Timm after washing with clean water. The increase of Timm after modeling by 2-4 times compared with the T0 value before modeling indicates that the modeling is successful; apply sample 1, sample 5, and control group samples to the test area for 3 days and measure the transepidermal water loss rate T3d, leaving one test area without applying the sample as the model group control. Recovery rate = (Tn-Timm) / (T0-Timm)×100%. The number of testers was 15, and the recovery rate was calculated by taking the average value after eliminating abnormal data.

[0171] Red blood cell hemolysis test

[0172] SLS compounds that can cause irritation to human skin generally have the characteristics of rupturing cell membranes or denaturing proteins. Based on this characteristic, mammalian red blood cells are used to simulate in vivo reactions in vitro. According to the rupture of red blood cells by the compound, the hemolysis rate is calculated and compared with the autolysis group (negative group) and the full lysis group (positive group) to determine the irritation of the compound. Generally, a hemolysis rate below 5% can be considered non-irritating, but the stability of in vitro cell experiments is not good, and the experimental results are for reference only. Hemolysis rate = (sample absorbance - negative control absorbance) / (positive control absorbance - negative control absorbance) × 100%. The lower the hemolysis rate, the better the blood compatibility of the material. The test method is as follows:

[0173] (1) Mix normal saline with 4% sheep red blood cells in a 1:1 ratio to prepare 2% sheep red blood cells for later use.

[0174] (2) Sodium dodecyl sulfate (SDS) diluent was prepared, with PBS as the blank group and 100 μg / mL SDS as the positive group.

[0175] (3) The sample was mixed with 2% sheep red blood cells in a ratio of 1:1 to form a reaction system, and then placed in a 37°C constant temperature incubator for 3 hours. The final test concentration of the sample was consistent with that in Table 2.

[0176] (4) After removal, centrifuge at 2000 rpm for 15 min and measure the absorbance with an enzyme-labeled instrument.

[0177] (5) Calculate the hemolysis rate of each sample concentration, and use software fitting to calculate the sample concentration HC50 at which the red blood cell hemolysis rate is 50%. The larger the HC50 value, the less irritating the sample is.

[0178] The test results can be found in Table 10-12;

[0179] Table 10 Hydroxyl radical scavenging performance test results

[0180] Hydroxyl free radical scavenging rate / % Sample 1 57.67 Sample 2 24.32 Sample 3 26.25 Sample 4 48.31 Sample 5 30.05 Sample 6 4.63 Sample 7 2.49 Sample 8 47.73 Control group 56.69

[0181] The scavenging rate of milk fermentation product 1 on hydroxyl free radicals was about 57%. The scavenging rate of 5% yeast fermentation product on hydroxyl free radicals was about 30%, and the scavenging rate was about 58% after adding 5% milk fermentation product 1. The scavenging rate of 1% niacinamide was 48%, and the scavenging rate did not increase significantly after adding 5% milk fermentation product 1, and was slightly lower than the group adding 5% milk fermentation product 1 alone. The scavenging rates of 0.005% resveratrol and 0.01% glabridin on hydroxyl free radicals were low, and the scavenging rates increased after adding 5% milk fermentation product 1, but were significantly lower than the group adding 5% milk fermentation product 1 alone. This shows that the scavenging effect of Bifidobacterium adolescentis / milk fermentation product and yeast fermentation product on hydroxyl free radicals is the best, which can better prevent the formation of lipofuscin caused by hydroxyl free radicals.

[0182] Table 11 Test results of inflammatory factors TNF-α, IL-1β, lysosomal activity and mitochondrial activity

[0183]

[0184] According to the test results in Table 11, compared with the model group, 5% milk fermentation product 1 and 5% yeast fermentation product alone and together have a reducing effect on the expression of inflammatory factors TNF-α and IL-β. Among them, 5% milk fermentation product 1 has a more obvious reducing effect on TNF-α, and 5% yeast fermentation product has a more obvious reducing effect on IL-β. The expression of inflammatory factors in sample 1 of the shared group of the two (this test concentration is 2.5% milk fermentation product 1 + 2.5% yeast fermentation product) is slightly lower than that of single use. This shows that the use of Bifidobacterium adolescentis / milk fermentation product and yeast fermentation product can simultaneously reduce the expression of two inflammatory factors, more comprehensively prevent the formation of lipofuscin caused by skin inflammation, and has the potential to reduce erythema caused by skin inflammation and pigmentation caused by inflammation.

[0185] 5% milk fermentation product 1 has a more prominent effect on improving lysosomal activity, while yeast fermentation product sample 5 has a more prominent effect on improving mitochondrial activity. The combination of the two groups, sample 1, has a more obvious effect on improving both lysosomal and mitochondrial activity. This shows that the use of Bifidobacterium adolescentis / milk fermentation products and yeast fermentation products can better alleviate the dysfunction of the mitochondrial-lysosomal axis, restore organelle activity, and jointly prevent the accumulation of skin lipofuscin.

[0186] In summary, sample 1 can reduce the inflammatory factors produced by cells due to stimulation, reduce the production of free radicals by stimulated cells in the skin, and can better remove free radicals, thereby preventing the production of lipofuscin and melanin; at the same time, it can increase lysosomal activity and mitochondrial activity, and reduce lipofuscin accumulation. In addition, analysis Figure 2 The test results show that sample 1 is more effective in reducing DNA damage caused by UV radiation, and can reduce the photosensitive damage caused by the accumulation of lipofuscin in the skin and the resulting melanin production.

[0187] Table 12 Skin repair test results

[0188] T0 Timm T3d Recovery rate / % Sample 1 7.06 23.14 18.06 31.59 Sample 5 6.6 20.84 17.86 20.93 Control group 7.02 21.6 18.3 22.63 Model Group 6.94 18.88 17.46 11.89

[0189] Comparing the data in Table 12, it can be seen that the transepidermal water loss rate after SLS stimulation modeling is significantly higher than that before modeling, and the skin barrier is damaged. The recovery rate of sample 1 is the highest after three days of use, indicating that it has the best repair effect on damaged skin barrier. Sample 5 and the control group alone also have a certain repair effect on damaged skin barrier.

[0190] Table 13 Cell hemolysis rate test results

[0191] Cell hemolysis rate HC50 / % Sample 1 No hemolysis Sample 2 38.42 Sample 3 23.39 Sample 4 52.13 Sample 5 No hemolysis Sample 6 39.01 Sample 7 23.33 Sample 8 51.6 Control group No hemolysis Blank Group No hemolysis

[0192] According to the test results in Table 13, samples 1, 5, the control group and the test blank group will not cause hemolysis of red blood cells, indicating that 5% milk fermentation product 1 and 5% yeast fermentation product alone or together have almost no irritation to cells. Samples 2 / 3 / 4 / 6 / 7 / 8 containing classic whitening ingredients are irritating to a certain extent, among which resveratrol and glabridin are irritating to a certain extent at lower concentrations, while the safe concentration of milk fermentation product 1 and yeast fermentation product is more than 500 times that of resveratrol and glabridin.

[0193] Cell DNA damage test results can be referenced Figure 2 ,pass Figure 2 It can be seen that sample 1 and sample 5 have no obvious tailing phenomenon, and both will not cause damage to cell DNA.

[0194] In summary, the products of Bifidobacterium adolescentis / milk fermentation and yeast fermentation have low irritation to the skin and are relatively safe; while the above-mentioned classic whitening ingredients will cause hemolysis of red blood cells to a certain extent. Classic whitening ingredients such as resveratrol and glabridin may be more effective in inhibiting tyrosinase, but due to their irritation, long-term use may cause skin irritation, sensitivity and other problems. The filtrate of Bifidobacterium adolescentis / milk fermentation and yeast fermentation products is not only highly safe, but also can reduce inflammatory factors, repair skin barrier damage, and has certain advantages in reducing lipofuscin production and accumulation; it can be used as a mild ingredient for long-term improvement and brightening of skin color.

[0195] 3.2 Screening and performance testing of Bifidobacterium

[0196] By performing performance tests on different bifidobacterium fermentation products, the correlation between the factors that affect the function of whitening ingredients can be determined.

[0197] The specific formula can be referred to Table 14; in Table 14, the balance component is water;

[0198] Table 14 Formulation Unit: wt%

[0199]

[0200] Refer to the performance test in Section 3.1 for testing;

[0201] The test results can be seen in Table 15;

[0202] Table 15 Performance test results

[0203] Lipofuscin relative fluorescence intensity / % Sample 1 74.48 Sample 9 83.22 Sample 10 81.13 Sample 11 93.61 Sample 12 89.35 Blank Group 100

[0204] It can be seen from the test results in Table 15 that compared with other Bifidobacterium adolescentis, Bifidobacterium infantis and Bifidobacterium longum, the Bifidobacterium adolescentis obtained by the present invention can be compounded with yeast fermentation products after fermenting milk to obtain the product with the best lipofuscin inhibition effect.

[0205] 3.3 Study on the compound performance of Bifidobacterium adolescentis milk fermentation products and yeast fermentation products

[0206] This section mainly discusses the compounding performance of Bifidobacterium adolescentis milk fermentation products and yeast fermentation products. By compounding Bifidobacterium adolescentis milk fermentation products and yeast fermentation products in different proportions, the performance change trend is determined.

[0207] The above is mainly evaluated from the nematode lipofuscin test, and the specific formula is as shown in Table 16; in Table 16, the balance component is water;

[0208] Table 16 Formulation Unit: wt%

[0209]

[0210]

[0211] The results of the nematode lipofuscin test can be found in Table 17;

[0212] Table 17 Performance test results

[0213]

[0214]

[0215] According to the test results in Table 17, when the concentration of yeast fermentation products is high, a more obvious lipofuscin inhibition effect can be shown;

[0216] From the results of samples 15 to 18 and the control group, even if the concentration of milk fermentation product 1 reaches 20wt%, it cannot achieve a very excellent lipofuscin inhibition effect; when the amount of milk fermentation product 1 reaches 15wt%, the best lipofuscin inhibition effect can be achieved;

[0217] When yeast fermentation products and milk fermentation products 1 are compounded, they can show obvious synergy.

[0218] 3.4 Formula safety test

[0219] 1. Test substance: Sample 1 (5% milk fermentation product 1 + 5% yeast fermentation product + 90% water)

[0220] 2. Negative control: deionized water

[0221] 3. Subjects: A total of 31 people, including 14 males and 17 females, aged 25 to 55 years old, met the voluntary inclusion criteria.

[0222] 4. Spot test method: the selected area should not exceed 50mm 2 , a qualified spot test device with a depth of about 1mm. Place the test substance in the small chamber of the spot tester, with a dosage of about 0.020mL-0.025mL. Apply the spot tester with the test substance to the curved side of the subject's forearm with hypoallergenic tape, and gently press it with the palm of your hand to evenly apply it to the skin for 24 hours. Observe the skin reaction 30 minutes (after the indentation disappears), 24 hours and 48 hours after removing the test substance spot tester, and record the results according to the skin reaction grading standard (Table 18) in the "Technical Specifications for Safety of Cosmetics" (2015 Edition).

[0223] Table 18 Evaluation criteria for skin reactions to closed patch test

[0224]

[0225]

[0226] Result judgment criteria: When more than 5 cases of Grade 1 adverse skin reactions occur among 30 subjects, or more than 2 cases of Grade 2 adverse skin reactions occur (more than 5 cases of Grade 2 reactions in the deodorant product patch test), or any case of Grade 3 or above adverse skin reaction occurs, the test substance is judged to have adverse skin reactions on humans. The test results can be seen in Table 19.

[0227] Table 19 Evaluation criteria for skin reactions to closed patch test

[0228]

[0229] Conclusion: The human patch test results of sample 1 showed:

[0230] 31 out of 31 test subjects showed negative reactions on their skin (scoring level 0). According to the human patch test judgment standard of the "Technical Specifications for Safety of Cosmetics", sample 1 had no adverse reaction to human skin.

[0231] 4. Application formula

[0232] The application formula of the composition of the present invention can refer to Table 20 to Table 22; Application formula 1: semi-permeable essence

[0233] Table 20: Formula of semi-transparent essence

[0234]

[0235]

[0236] Application formula 2: lotion

[0237] Table 21 Emulsion formula

[0238]

[0239]

[0240] Application formula 3: facial cream

[0241] Table 22 Formulation of facial cream

[0242]

[0243]

[0244] The applicant declares that the present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, and it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A strain of Bifidobacterium adolescentis, characterized in that The Bifidobacterium adolescentis is Bifidobacterium adolescentis BF23BA001, classified as Bifidobacterium adolescentis (Bifidobacterium adolescentis), with a preservation number of CGMCC NO.26999; preservation date: March 31, 2023, preservation unit: General Microbiology Center of China National Microbiological Culture Collection Administration; preservation address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the Bifidobacterium adolescentis as claimed in claim 1 to ferment milk to prepare milk fermentation products.

3. A milk fermentation product, characterized in that: The method is prepared by fermenting milk with the Bifidobacterium adolescentis as claimed in claim 1.

4. The milk fermentation product according to claim 3, characterized in that: The preparation method of the milk fermentation product is: 6 ~10 8 CFU / mL of Bifidobacterium adolescentis bacterial liquid is added into sterilized milk and fermented for 12 to 48 hours; the volume ratio of Bifidobacterium adolescentis bacterial liquid to milk is 1 to 10:

100.

5. A composition for inhibiting the production and accumulation of lipofuscin, characterized in that: It includes milk fermentation products and yeast fermentation products; the mass ratio of the milk fermentation products to the yeast fermentation products is 0.5-10:1-10; The fermentation bacteria of the milk fermentation product is Bifidobacterium adolescentis.

6. The composition for inhibiting the production and accumulation of lipofuscin according to claim 5, characterized in that: The Bifidobacterium adolescentis is one or more combinations of the Bifidobacterium adolescentis, Bifidobacterium adolescentis BBF-06, and Bifidobacterium adolescentis SF-B40 as described in claim 1.

7. The composition for inhibiting the production and accumulation of lipofuscin according to claim 5, characterized in that: The mass ratio of the milk fermentation product to the yeast fermentation product is 0.5-8:2-9.

5.

8. The composition for inhibiting the production and accumulation of lipofuscin according to claim 5, characterized in that: The preparation method of the milk fermentation product is: The viable count was 10 6 ~10 8 CFU / mL of Bifidobacterium adolescentis bacterial liquid is added into sterilized milk and fermented for 12 to 48 hours; the volume ratio of Bifidobacterium adolescentis bacterial liquid to milk is 1 to 10:

100.

9. The composition for inhibiting the production and accumulation of lipofuscin according to claim 5, characterized in that: The preparation method of the yeast fermentation product is as follows: after activating the brewer's yeast, inoculate the culture medium and ferment at 28-34°C for 12-24 hours, centrifuge and perform preliminary filtration, then use activated carbon to absorb impurities and then filter to obtain the yeast fermentation product; The culture medium contains 1-3 wt% glucose, 0.5-1.5 wt% peptone and 0.4-0.6 wt% yeast extract powder.

10. Use of the composition according to any one of claims 5 to 9 in preparing cosmetics.

11. The use according to claim 10, characterized in that The cosmetics are cosmetics for skin care purposes.

12. The use according to claim 11, characterized in that The cosmetics are cosmetics that have the function of inhibiting the generation and accumulation of lipofuscin; and / or, The cosmetics are cosmetics with whitening and brightening functions; and / or, The cosmetics are cosmetics with the functions of repairing, removing redness and anti-oxidation.

13. A cosmetic, characterized in that: Contains the composition as described in any one of 5 to 9.

14. The cosmetic according to claim 13, characterized in that In the cosmetic, the concentration of the composition is 5 to 20 wt %.

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