Bifidobacterium adolescentis, composition for inhibiting the generation and accumulation of lipofuscin, use and cosmetic

Abstract

The application belongs to the field of daily chemicals, and discloses a Bifidobacterium adolescentis, the classification name of the Bifidobacterium adolescentis BF23BA001 is Bifidobacterium adolescentis, the preservation number is CGMCC NO.26999, the preservation date is March 31, 2023, the preservation unit is China General Microbiological Culture Collection Center, and the preservation address is No.3, Xibahe Road, Chaoyang District, Beijing City. Compared with other similar bacteria on the market, the bacteria have unique advantages in the production of small molecule peptides and gamma-aminobutyric acid, according to the functional characteristics of the small molecule peptides and gamma-aminobutyric acid, the bacteria have significant anti-wrinkle advantages, and through subsequent functional verification, the bacteria have very important significance for further improving the whitening, brightening, repair and anti-aging effects of yeast fermentation products. In addition, the application also discloses a composition for inhibiting the generation and accumulation of lipofuscin, application based on the composition and cosmetics.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of daily chemicals, and particularly relates to a Bifidobacterium adolescentis, a composition for inhibiting the generation and accumulation of lipofuscin, application and cosmetics. BACKGROUND

[0002] The previous application of the present applicant, CN202310086320.2 Bifidobacterium adolescentis milk fermentation, product containing the same and preparation and application thereof, discloses that the Bifidobacterium adolescentis milk fermentation has the functions of free radical scavenging, antioxidation, skin barrier repair, improvement of skin elastin and soothing.

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

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

[0005] In the further in-depth research of the present applicant, it is found that in the whitening application of the fermentation product of the specific Bifidobacterium, the possible mechanism is not limited to this.

[0006] The technical problem to be solved by the present application is how to develop the potential whitening application of the Bifidobacterium adolescentis milk fermentation. SUMMARY

[0007] In view of the deficiencies of the prior art, the purpose of the present application is to provide a Bifidobacterium adolescentis, which has unique advantages in the production of small molecule peptides and gamma-aminobutyric acid compared with other similar bacteria on the market. According to the functional characteristics of small molecule peptides and gamma-aminobutyric acid, it has significant anti-wrinkle advantages. At the same time, through subsequent functional verification, it has very important significance for further improving the whitening effect of yeast fermentation.

[0008] In addition, the present application also discloses a composition for inhibiting the generation 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 gamma-aminobutyric acid in the milk fermentation products fermented by the Bifidobacterium adolescentis is more prominent, and the content of proteins, polysaccharides and various amino acids in the yeast fermentation products of the present application is more prominent. The combination of the two can effectively inhibit the generation and accumulation of lipofuscin.

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

[0010] In order to achieve the purpose of the present application, the following technical solutions are adopted:

[0011] A Bifidobacterium adolescentis, the taxonomic name of which is Bifidobacterium adolescentis, the preservation number of which is CGMCC NO. 26999, the preservation date of which is March 31, 2023, the preservation unit of which is China General Microbiological Culture Collection Center, and the preservation address of which is No. 1, Beichen West Road, Hua-yuan District, Beijing.

[0012] Meanwhile, the application also discloses a use of the Bifidobacterium adolescentis for fermenting milk to prepare a milk fermentation product.

[0013] Meanwhile, the application also discloses a milk fermentation product prepared by fermenting milk with the Bifidobacterium adolescentis.

[0014] In the milk fermentation product, the preparation method of the milk fermentation product is as follows: Bifidobacterium adolescentis bacterial liquid with a viable count of 10 6 ~ 10 8 CFU / mL is added into sterilized milk, and fermentation is carried out for 12-48 h; the volume ratio of the Bifidobacterium adolescentis bacterial liquid to the milk is 1-10:100.

[0015] In addition, the application also discloses a composition for inhibiting the generation and accumulation of lipofuscin, which comprises 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 strain of the milk fermentation product is Bifidobacterium adolescentis.

[0017] The conventional whitening idea is usually directed to the problem of melanin, and common ingredients include niacinamide, glabridin, resveratrol, etc., but in addition to the content and aggregation of melanin in the skin, yellow removal and improvement of dullness are also problems that are concerned in whitening. With the increasing in-depth and comprehensive research on skin whitening, healthy white or skin color management has become a trend, and pigments (melanin, lipofuscin, hemoglobin, etc.), inflammation (erythema, post-inflammatory hyperpigmentation) and structure (dryness, roughness, wrinkles) are all problems that need to be addressed in healthy whitening.

[0018] Wherein lipofuscin is a yellow-brown pigment particle accumulated in cells, and its deposition in skin cells can cause skin to appear yellow-brown spots, age spots, dull and rough skin, and wrinkles, etc. Ultraviolet rays and skin inflammation can increase the number of free radicals in the skin layer, accelerating the generation of lipofuscin; and mitochondrial-lysosome axis dysfunction can cause lipofuscin accumulation, lipofuscin promotes mitochondrial ROS production, and causes lysosome dysfunction through lysosome membrane permeabilization, resulting in a decrease in the number of lysosomes and impaired activity. Therefore, improving the relevant indicators can prevent the generation 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 exhibits photosensitization under visible light irradiation, producing singlet oxygen, which can cause DNA damage. DNA damage further promotes melanin production, deepening skin pigmentation; in addition, DNA damage can also affect the life cycle and renewal of skin cells. Therefore, improving the relevant indicators can improve the adverse effects of melanin deposition on the skin caused by lipofuscin accumulation, as well as improve skin laxity, wrinkles, and uneven pigmentation caused by DNA damage.

[0020] Skin redness is a common symptom of skin inflammation, usually associated with vasodilation and release of inflammatory factors. Inflammatory factors, such as interleukins (IL), tumor necrosis factor (TNF-α), etc., can cause symptoms such as skin redness. Therefore, improving the relevant indicators (inhibiting the activity of inflammatory factors) can alleviate the irritation to the skin 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 luster and transparency of the skin, thereby indirectly affecting the skin color. Therefore, maintaining skin moisture and maintaining good skin barrier function are crucial for improving and maintaining skin color.

[0022] In summary, skin whitening has a complex pathway, in which reducing lipofuscin generation and accumulation, antioxidant, improving the activity of metabolism-related organelles, preventing DNA damage, inhibiting the activity of inflammatory factors, and repairing the skin barrier can help improve skin color to a certain extent.

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

[0024] The use of Bifidobacterium adolescentis can significantly increase the small molecule peptides and gamma-aminobutyric acid in the fermented product of milk; these two substances are closely related to the generation of collagen;

[0025] Meanwhile, the study shows that the increase of small molecule peptides and GABA can make the yeast fermentation product have better effect of inhibiting the generation and accumulation of lipofuscin. It is possible that B. adolescentis and milk fermentation product contain rich polysaccharides, small molecule peptides and GABA, and the yeast fermentation product contains rich amino acids, and the effective components of the two fermentation products complement and synergize with each other.

[0026] B. adolescentis and milk fermentation product can better improve lysosome activity, and the yeast fermentation product can better improve mitochondrion activity, and the combination of the two can better repair the damage of mitochondrion-lysosome axis, reduce the accumulation of lipofuscin, and improve the cell metabolism. Moreover, the two fermentation products can inhibit the activity of cell inflammatory factors, reduce the skin redness caused by skin inflammation, and better remove free radicals to avoid the generation of lipofuscin and skin aging caused by excessive free radicals. In addition, the combination of the two fermentation products can better repair skin irritation damage, repair skin barrier, maintain skin surface moisture, and thus maintain skin luster.

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

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

[0029] In the above composition for inhibiting the generation and accumulation of lipofuscin, the preparation method of the milk fermentation product is as follows:

[0030] B. adolescentis with a viable bacterial count of 10 6 ~ 10 8 CFU / mL is added to sterilized milk, and fermented for 12-48 h; the volume ratio of the B. adolescentis bacterial liquid and the milk is 1-10:100.

[0031] In the above composition for inhibiting the generation and accumulation of lipofuscin, the preparation method of the yeast fermentation product is as follows:

[0032] After the activated Saccharomyces cerevisiae is inoculated into a culture medium (1-3 wt% of glucose, 0.5-1.5 wt% of protein peptone, and 0.4-0.6 wt% of yeast extract powder), the culture medium is fermented at 28-32℃ for 12-24 h, centrifuged, preliminarily filtered, and then filtered after the impurities are adsorbed by activated carbon to obtain the yeast fermentation product.

[0033] In addition, the application also discloses the use of the above composition for preparing cosmetics.

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

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

[0036] and / or,

[0037] The cosmetic is a cosmetic having a multi-path whitening function;

[0038] and / or,

[0039] The cosmetic is a cosmetic having a function of repairing redness and antioxidant efficacy.

[0040] Finally, the present application also discloses a cosmetic containing the composition as described in any of the above.

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

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

[0043] The Bifidobacterium adolescentis / milk fermentation product can reduce the level of lipofuscin in nematodes, and can synergistically reduce the accumulation of lipofuscin with the yeast fermentation product. The fermentation liquor contains rich polysaccharides, small molecule proteins, gamma-aminobutyric acid and small molecule organic acids, and can comprehensively improve the skin color in terms of removing yellow spots, anti-inflammatory and redness, preventing and inhibiting blackening, moisturizing and lightening. Compared with traditional whitening agents, the Bifidobacterium adolescentis / milk fermentation liquor has high safety, has no toxicity to HaCat cells and the like at a concentration of ≤5%, and has no hemolytic effect on red blood cells, and can gently improve the skin color. The two kinds of fermentation liquor contain rich nutrients, which can supplement the nutrients for skin cells in a timely manner without damaging the cells, and are suitable as long-acting and mild skin whitening conditioning agents in skin care products. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 The nematode lipofuscin fluorescence photos of each sample, the control group and the blank group are taken;

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

[0046] The technical solutions of the present application are further illustrated by specific embodiments. Those skilled in the art should understand that the embodiments are only used to help understand the present application, and should not be regarded as specific limitations on the present application.

[0047] Reagent sources:

[0048] Bifidobacterium adolescentis 1: Bifidobacterium adolescentis BF23BA001, classified as Bifidobacterium adolescentis, with a preservation number of CGMCC NO. 26999, a preservation date of March 31, 2023, and a preservation unit of China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Haidian District, Beijing;

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

[0050] Bifidobacterium adolescentis 3: Bifidobacterium adolescentis SF-B40, Shandong Xiangriwei Biological Engineering Co., Ltd.;

[0051] Bifidobacterium infantis: SHMCC D11206, Shanghai Microorganism Center;

[0052] Bifidobacterium longum: SHMCC D24500, Shanghai Microorganism Center;

[0053] Saccharomyces cerevisiae: SHMCC D11813, Shanghai Microorganism Center.

[0054] First part: preparation and identification of milk fermentation products

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

[0056] (1) sterilize the milk at a temperature of 95℃ for 30 min, and cool it to room temperature after sterilization to obtain a fermentation substrate;

[0057] (2) culture the Bifidobacterium to a viable bacterial count of 10 6 ~ 10 7 CFU / mL of Bifidobacterium bacterial solution;

[0058] (3) inoculate the Bifidobacterium bacterial solution into the fermentation substrate prepared in step (1), with a volume ratio of Bifidobacterium bacterial solution to milk of 5:100, and incubate at 37℃ for 24 h; after fermentation, sterilize the material at 95℃ for 30 min; cool it to room temperature after sterilization, centrifuge at a speed of 4800 rpm for 30 min, collect the supernatant, filter the supernatant with a 0.22μm filter membrane to remove Bifidobacterium and impurities, and obtain the Bifidobacterium milk fermentation product.

[0059] Through the above method, each milk fermentation product is prepared, and the relationship between the number of the milk fermentation product and the corresponding strain is shown in Table 3;

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

[0061] Small molecule peptide content test method:

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

[0063] (1) Gradient dilution of albumin (BSA) standard: open an ampoule containing BSA stock solution, and prepare A-I solution according to the contents of Table 1, and prepare BSA standard solution of each concentration in the standard curve according to Table 1.

[0064] Table 1 BSA standard dilution scheme in the standard curve

[0065]

[0066]

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

[0068] (3) Preparation of BCA working solution: take out A and B liquids in the kit, and prepare AB mixed solution of appropriate volume in the dark according to the ratio of A:B=50:1.

[0069] (4) Add 25 μL of diluted BSA standard, sample to be tested, ultrapure water to the 96-well transparent microplate, add 200 μL of BCA working solution, shake for 30 s to mix thoroughly, and seal in a 37℃ constant temperature incubator for incubation for 30 min.

[0070] (5) Take out the 96-well transparent microplate, and detect the absorbance value of each sample at 562 nm on the enzyme label instrument (the absorbance value of the sample should be controlled within the absorbance range of the standard).

[0071] (6) Draw the standard curve: set the standard concentration as the X axis, and the absorbance value corresponding to each concentration as the Y axis, and draw the standard curve Y=aX+b, and 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 measured absorbance value (background subtracted) of the sample to be tested into the formula to calculate the sample concentration X, and multiply by the corresponding dilution factor to obtain the sample protein concentration.

[0073] Gamma-aminobutyric acid test method:

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

[0075] Online derivatization: sample / standard 120 μL, add o-phthalaldehyde derivative solution 600 μL, and perform derivatization treatment. (Derivative reagent: o-phthalaldehyde derivative solution: dissolve 10 mg of o-phthalaldehyde in 0.5 mL of methanol, then add 2 mL of 0.4 mol / L boric acid buffer solution (pH-10.2) and 30 μL of 2-mercaptoethanol, and store at 4°C for 2 days.)

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

[0077] Table 2 Elution program table

[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 test method:

[0080] Test using HPLC-RID detector; ArtChrom H 8μ (300*7.8 mm), column temperature 30°C; sample injection volume 10 μL, mobile phase is 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 can be seen from Table 3, the use of B. adolescentis can effectively increase the concentrations of small molecule peptides, γ-aminobutyric acid and lactic acid. In particular, the use of B. adolescentis 1 selected and isolated has particularly good effects in this regard.

[0085] From the comparison of milk fermentation product 2 and milk fermentation product 5, it can be found that B. adolescentis is synchronous in improving the contents of small molecule peptides, γ-aminobutyric acid and lactic acid, and generally improves them synchronously. Other similar strains cannot exhibit similar rules, for example, the use of B. longum to ferment milk can significantly increase the content of small molecule peptides, but it does not help to increase the content of γ-aminobutyric acid.

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

[0087] Table 4 Effective components of the milk fermentation product 1

[0088] Ingredient mg / mL Unit Test method Small molecule peptide 341.1 mg / L BCA protein quantitative kit Gamma-aminobutyric acid 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 quantitative kit

[0089] Preparation and identification of the second part of the yeast fermentation product

[0090] The preparation method of the yeast fermentation product is as follows: glucose 2wt%, peptone 1wt%, yeast extract powder 0.5wt%, and deionized water 96.5wt% are dissolved and sterilized, and then cooled to room temperature. Saccharomyces cerevisiae is inoculated and activated, and then fermented in a shaker for 20h. After centrifugation at 5000rpm for 30min, the crude filtrate is obtained by preliminary filtration. 1% activated carbon is added to the crude filtrate, stirred for about 10min to remove impurities, and then filtered through a 0.45μm filter membrane to obtain the yeast fermentation product.

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

[0092] Table 5 Effective components of the yeast fermentation product

[0093] Ingredient 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 Automatic amino acid analyzer Glutamine mg / L 39.2 HPLC Proline mg / L 2.26 Automatic amino acid analyzer

[0094] Preparation of the third part 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 the milk fermentation product of Bifidobacterium adolescentis. Through previous tests, we found that the milk fermentation product of Bifidobacterium adolescentis alone cannot achieve particularly excellent whitening effect. Therefore, further verification is needed to determine whether it can be compounded with other whitening active ingredients to achieve better whitening effect.

[0097] The milk fermentation product and other whitening active ingredients are compounded to evaluate the performance of lipofuscin inhibition and deposition.

[0098] According to existing data, 0.001-0.005% resveratrol has good lipofuscin inhibition effect; glycyrrhizin and niacinamide are classic whitening ingredients commonly used in cosmetics. Glycyrrhizin has a high cost, and the addition amount is generally not more than 0.05%. Niacinamide has a regulatory requirement for the addition amount of <5%.

[0099] Pre-experiments found that the nematodes in the 0.05% glycyrrhizin and 5% niacinamide concentration groups were smaller in size after a certain period of cultivation compared with the blank group, which may be due to the toxic effect of high concentration on nematodes; after reducing the concentration by 5 times, the nematodes were normal in shape, so this concentration was used for formal lipofuscin test.

[0100] In the preliminary 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 Table 6; in Table 6, the balance is water.

[0101] Table 5 Formula Table Unit: wt%

[0102]

[0103]

[0104] 3.2 Performance test

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

[0106] Nematode lipofuscin test:

[0107] (1) Nematode synchronization (hypochlorous acid lysis method): collect Caenorhabditis elegans, and lyse the nematodes with nematode lysis solution (5N NaOH:

[0108] 5% NaClO = 1:2), and then culture the nematodes to L4 stage for subsequent experiments (culture temperature is 20°C, using NGM plate containing E. coli OP50).

[0109] (2) Nematode in vivo lipofuscin level determination

[0110] Prepare the culture medium containing the sample, and the final concentration of the sample in the culture medium is shown in Table 5, and the buffer is blank.

[0111] Transfer L4 nematodes to the prepared sample-containing culture plate, each plate containing 500 nematodes, and place the treated nematode plate in a 20°C incubator for culture.

[0112] After 5 days of culture at 20°C, collect the nematodes, and then wash the nematodes 3 times. Transfer the nematodes to an agarose plate, add 1% tetramisole hydrochloride, and stand for 1 h for anesthesia.

[0113] (3) Nematode in vivo lipofuscin fluorescence observation and analysis

[0114] Observe the fluorescence intensity under a fluorescence microscope and take pictures. When taking pictures, randomly take 30 different nematodes from the same plate, and the exposure parameters and the like should be consistent with those used in the same experiment. Save all the pictures for subsequent analysis.

[0115] (4) Image J software analysis: Image J quantitatively analyzes the fluorescence intensity of nematodes, and analyzes the autofluorescence content of nematode lipofuscin.

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

[0117] The test results can refer to 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] In combination Figure 1 With Table 7, compared with the blank group, the effect of 5% milk fermentation product 1 alone is not obvious, and the lipofuscin is only reduced by about 3%. The use of 5% yeast fermentation product alone only reduces the lipofuscin by about 6%, and the effect is not obvious; after adding 5% milk fermentation product 1, the effect is obviously enhanced, and the lipofuscin is reduced by 25.52%, which shows that the two have good synergistic effect in reducing lipofuscin.

[0122] 0.01% glabridin has no obvious effect on the reduction of lipofuscin, and the effect is slightly enhanced after adding milk fermentation product 1, but it is not obvious. 0.005% resveratrol and 1% nicotinamide can reduce lipofuscin to a certain extent, but there is no obvious synergistic effect when used with milk fermentation product 1, and the effect on reducing lipofuscin in nematodes is not as good as sample 1.

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

[0124] Through the above screening, it can be known that the performance of sample 1 in reducing lipofuscin in nematodes is the best. In order to further clarify the root of its good performance and explore the other effects of sample 1 in whitening and brightening, we further test the antioxidant and anti-inflammatory properties of samples 1-8.

[0125] The test method is as follows:

[0126] Hydroxyl radical scavenging rate test:

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

[0128] H2O2 mixed with divalent iron ions to produce hydroxyl radicals, salicylic acid and hydroxyl radical reaction to produce colored products 2, 3-dihydroxybenzoic acid at 510 nm has a strong absorption peak. If the system is added with the measured substance with the function of scavenging hydroxyl radicals, and the effect of the measured substance on capturing hydroxyl radicals is greater than that of salicylic acid, hydroxyl radicals can be removed in time, so that the generation of colored products is reduced, resulting in the decrease of absorbance. Therefore, by using the fixed reaction time method, the absorbance of the reaction solution containing the measured substance at 510 nm is measured, and compared with the blank group, the scavenging effect of the measured substance on hydroxyl radicals can be determined.

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

[0130] Table 8 Hydroxyl radical test steps

[0131]

[0132]

[0133] Hydroxyl radical scavenging rate (%) = [A2-(A1-A3)] / A2x100%; the greater the scavenging rate under the same test concentration indicates the better antioxidant effect.

[0134] Cell inflammation factor TNF-a, IL-1β test

[0135] The skin inflammation site is usually accompanied by redness phenomenon, and improving skin inflammation can improve the skin redness phenomenon. The expression of cell inflammation factors TNF-a, IL-1β is tested to evaluate the relief effect of the sample on skin inflammation. The test steps are as follows:

[0136] (1) Cell preparation: the THP-1 cell suspension stably passaged and cultured in the culture bottle was transferred to a 15 mL centrifuge tube, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, 8 mL of complete culture medium was used to resuspend the cells, and the cell counter was used for counting.

[0137] (2) Plate seeding: 5x10^5 THP-1 cell suspension was inoculated into each well of a 24-well plate in turn, and complete culture medium was added to the well to a total volume of 0.5 mL.

[0138] (3) Sample adding and sample pre-incubation: Set up blank control group, model group and sample group, add 0.5 mL complete medium to the corresponding wells of the blank control group and the model group, and add 0.5 mL sample-containing working solution to the corresponding wells of the sample group, shake to mix, and incubate in an incubator for 18-24 h. Among them, the test concentration of sample 1 is reduced to half of the original because the system contains about 10% of the fermentation broth, which has a slight effect on the survival rate of cells. The sample in the sample 1 test system is "2.5% milk fermentation product 1 + 2.5% yeast fermentation product"; according to the pre-experiment, the cell safety concentration of the classic whitening ingredients (see Table 9 below) is much smaller than the test concentration of the sample setting group, so the group containing the classic whitening ingredients will not be formally tested. The final test concentration of other groups is shown in Table 5.

[0139] Table 9 Cell safety concentration table of classic whitening ingredients

[0140] Classic whitening ingredients Cell safety concentration / % Resveratrol 0.0002 Glycyrrhizin 0.0002 Nicotinamide 0.00001

[0141] (4) LPS stimulation: add 10 μL LPS working solution (100 μg / mL) to the corresponding wells of the model group and the sample group, so that the final concentration of LPS in the wells is about 1 μg / mL, shake to mix, and incubate in an incubator for 24 h.

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

[0143] (6) Reverse transcription: use a reverse transcription kit to reverse transcribe the total RNA of each group of cells into cDNA.

[0144] (7) qPCR detection: use GAPDH as an internal reference to detect the relative expression of TNF-a and IL-1β in each group.

[0145] (8) Comparison method: the model group is only the stimulation source group, without adding samples or other stimulation relieving ingredients; the sample group is the sample added and stimulation applied group; the blank control group is the normal cell group without stimulation and sample. Therefore, the expression of inflammatory factors TNF-a and IL-1β is less than the model group, indicating that the sample has the effect of reducing the expression of inflammatory factors, and the closer the data is to the blank control group, the stronger the effect.

[0146] Lysosome activity test:

[0147] Lysosomes contain a variety of hydrolytic enzymes that can degrade macromolecules. Lysosome activity and number can indirectly reflect the metabolic renewal capacity of cells. The higher the lysosome activity, the stronger the metabolic capacity of cells.

[0148] At physiological pH, the net charge of neutral red dye is almost zero, so it can penetrate the cell membrane into the cell by non-ionic passive diffusion. The proton gradient in lysosomes makes the pH in lysosomes lower than that in the cytoplasm, so that the neutral red can carry an electric charge and accumulate in lysosomes, thereby detecting lysosomal activity.

[0149] Test method: skin fibroblasts were plated in a 96-well plate at 8000 cells per well. After the cells adhered, a blank control group and a sample group were set up, and the blank control group was added with serum-free culture medium, and the sample group was added with serum-free culture medium containing the sample. The final test concentration of the sample is shown in Table 5. After 24 hours of treatment, the cell culture medium was discarded, and 200 μL of fresh serum-free culture medium was added, 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 lysate was added, lysed on a shaking table at room temperature for 10 minutes, and the A540 of each well was measured.

[0150] The lysosomal activity of the sample group was calculated with the blank control as the reference.

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

[0152] Mitochondrial activity test:

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

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

[0155] The mitochondrial activity of the sample group was calculated with the blank control as the reference. 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 molecular size and configuration. When DNA molecules are damaged, they will be broken into small fragments, which will show a tailing phenomenon during electrophoresis. The longer the tailing, the stronger the damage, so this method can be used to detect the strength of DNA damage in cells.

[0158] (1) According to the sample setting, it is divided into blank group, model group and sample group.

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

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

[0161] Sample group: sample addition group irradiated by 3J / cm 2 UVA light.

[0162] (2) Experimental operation

[0163] Preparation of cells: prepare Bal b / c3T3 cell suspension in 24-well plates, with a concentration of 1.0x10 5 cells / well, and place in a CO2 incubator for 24h.

[0164] Sample pretreatment: add sample to pretreat cells for 24h, and add equal amount of solvent control to blank group and model group (three replicate wells are set for each well), and the final test concentration of sample is consistent with Table 5.

[0165] UVA irradiation treatment: after sample pretreatment for 24h, wash the cells with PBS once, and then perform UVA irradiation on the model group and sample group, with irradiation conditions of UVA 3J / cm 2 ; the blank group is treated with light shielding for the same time.

[0166] Cell collection and DNA extraction: after UVA irradiation treatment, collect the cells, extract the cell DNA according to the DNA extraction kit, and detect the DNA concentration and purity by microspectrophotometer.

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

[0168] Result analysis: after electrophoresis, take a photo of the electrophoretic bands by gel imaging analyzer.

[0169] Skin care test:

[0170] Skin barrier function is impaired after SLS stimulation, and the trans-epidermal water loss rate increases. The recovery rate before and after the use of the sample is compared to evaluate the repair effect of the sample on the skin. The trans-epidermal water loss rate of the inner arm skin T0 is tested; the inner arm skin is pasted with a 1% sodium dodecyl sulfate (SLS) aqueous solution, and after 24 hours, the patch is removed and washed with water, and the trans-epidermal water loss rate Timm is tested. The Timm value after modeling is 2-4 times higher than the T0 value before modeling, indicating that the modeling is successful. The trans-epidermal water loss rate T3d is measured after 3 days of applying sample 1, sample 5, and control sample 3 to the test area, respectively. Leave one test area without sample as a model group control. Recovery rate = (Tn-Timm) / (T0-Timm) x 100%. The number of test persons is 15, and the average recovery rate is calculated after excluding abnormal data.

[0171] Red blood cell hemolysis test

[0172] SLS can cause irritation to human skin. Generally, compounds with such characteristics as rupturing cell membranes or denaturing proteins, etc. are used to simulate in vitro reactions in vivo based on this characteristic. According to the rupturing of red blood cells by the compound, compared with the autolysis group (negative group) and the total dissolution group (positive group), the hemolysis rate is calculated to judge the irritability of the compound. Generally, a hemolysis rate of less than 5% can be considered non-irritating, but the in vitro cell experiment has poor stability, and the experimental results are only for reference. Hemolysis rate = (sample absorbance-negative control absorbance) / (positive control absorbance-negative control absorbance) x 100%. The lower the hemolysis rate, the better the blood compatibility of the material. The test method is as follows:

[0173] (1) Mix physiological saline with 4% sheep red blood cells 1:1 to prepare 2% sheep red blood cells for standby.

[0174] (2) Prepare a dilute solution of sodium dodecyl sulfate (SDS), with PBS as the blank group and 100 μg / mL SDS as the positive group.

[0175] (3) Mix the sample with 2% sheep red blood cells 1:1 to prepare the reaction system, and incubate in a 37°C constant temperature incubator for 3h. The final test concentration of the sample is consistent with Table 2.

[0176] (4) After removal, centrifuge at 2000 rpm for 15 min, and measure the absorbance value on the enzyme marker.

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

[0178] The test results can be referred to Tables 10-12;

[0179] Table 10 Hydroxyl radical scavenging rate performance test results

[0180] Hydroxyl radical removal 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 hydroxyl radical scavenging rate of milk fermentation product 1 was about 57%. The hydroxyl radical scavenging rate of 5% yeast fermentation product was about 30%, and the hydroxyl radical scavenging rate after adding 5% milk fermentation product 1 was about 58%. The scavenging rate of 1% nicotinamide was 48%, and the scavenging rate after adding 5% milk fermentation product 1 was not significantly improved and was slightly lower than that of the group of adding 5% milk fermentation product 1 alone. The hydroxyl radical scavenging rates of 0.005% resveratrol and 0.01% glabridin were low, and the hydroxyl radical scavenging rate after adding 5% milk fermentation product 1 was improved, but was significantly lower than that of the group of adding 5% milk fermentation product 1 alone. It can be seen that the co-use of Bifidobacterium adolescentis / milk fermentation product and yeast fermentation product has the best effect on hydroxyl radical scavenging, and can better prevent the generation of lipofuscin caused by hydroxyl radicals.

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

[0183]

[0184] As can be seen from the test results in Table 11, compared with the model group, 5% milk fermentation product 1, 5% yeast fermentation product alone and co-use all have a reducing effect on the expression amount of inflammatory factors TNF-α and IL-β. Among them, 5% milk fermentation product 1 has a more obvious reducing effect on TNF-α, 5% yeast fermentation product has a more obvious reducing effect on IL-β, and the expression amount of inflammatory factors of sample 1 (the test concentration is 2.5% milk fermentation product 1+2.5% yeast fermentation product) of the co-use group is slightly lower than that of the single use. It is indicated that the co-use of Bifidobacterium adolescentis / milk fermentation product and yeast fermentation product can simultaneously reduce the expression amount of two kinds of 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 pigment precipitation caused after inflammation.

[0185] 5% milk fermentation product 1 has a more prominent effect on improving lysosome activity, while yeast fermentation product sample 5 has a more prominent effect on improving mitochondrial activity, and sample 1 of the co-use group has a more obvious improving effect on lysosome and mitochondrial activity. It is indicated that the co-use of Bifidobacterium adolescentis / milk fermentation product and yeast fermentation product can better alleviate mitochondrial-lysosome axis dysfunction, restore organelle activity, and jointly prevent skin lipofuscin accumulation.

[0186] In summary, sample 1 can reduce the inflammatory factors produced by cell stimulation, reduce the free radicals produced by stimulated cells in the skin, and can better scavenge free radicals, thereby preventing lipofuscin and melanin production; at the same time, it can improve lysosome activity and mitochondrial activity, and reduce lipofuscin accumulation. In addition, the analysis Figure 2 The test results show that sample 1 has a more obvious effect on reducing DNA damage caused by UV irradiation, 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] As can be seen from the data in Table 12, the transdermal water loss rate after sls stimulation is significantly higher than before stimulation, and the skin barrier is damaged. The recovery rate is highest after using sample 1 for three days, 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 Non-hemolysis Sample 2 38.42 Sample 3 23.39 Sample 4 52.13 Sample 5 Non-hemolysis Sample 6 39.01 Sample 7 23.33 Sample 8 51.6 Control group Non-hemolysis Blank group Non-hemolysis

[0192] As can be seen from the test results in Table 13, samples 1, 5, the control group and the test blank group do not cause red blood cell hemolysis, indicating that 5% milk fermentation product 1 and 5% yeast fermentation product alone and together have little stimulation to cells. Samples 2 / 3 / 4 / 6 / 7 / 8 containing classic whitening ingredients have some stimulation, among which resveratrol and glabridin have some stimulation in samples with low concentration, and the safe concentration of milk fermentation product 1 and yeast fermentation product is more than 500 times that of resveratrol and glabridin.

[0193] The cell DNA damage test results can be referred to Figure 2 , by Figure 2 It can be seen that sample 1 and sample 5 have no obvious tailing phenomenon, and both of them will not cause damage to cell DNA.

[0194] In summary, Bifidobacterium adolescentis / milk fermentation product and yeast fermentation product have little skin irritation and high safety; while the above-mentioned classic whitening ingredients will cause red blood cell hemolysis to some extent. Classic whitening ingredients such as resveratrol and glabridin may have a more prominent effect on inhibiting tyrosinase, but long-term use of them may cause skin irritation, sensitivity and other problems. Bifidobacterium adolescentis / milk fermentation product and yeast fermentation product filtrate not only have high safety, but also can reduce inflammatory factors, repair skin barrier damage, and have certain advantages in reducing lipofuscin production and accumulation; they can be used as mild ingredients for long-term improvement of skin lightening.

[0195] 3.2 Screening and performance testing of Bifidobacterium

[0196] By testing the performance of different Bifidobacterium fermentation products, the correlation between the influencing factors affecting the function of whitening ingredients is determined.

[0197] The specific formula can refer to Table 14; in Table 14, the balance ingredient is water;

[0198] Table 14 Formula Table Unit: wt%

[0199]

[0200] Test according to the performance test in Section 3.1;

[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] From the test results in Table 15, it can be seen that compared with other Bifidobacterium adolescentis and Bifidobacterium infantis and Bifidobacterium longum, the Bifidobacterium adolescentis screened in the present application can obtain the best product with the best lipofuscin inhibition effect after fermenting milk and compounding with yeast fermentation products.

[0205] 3.3 Exploration of the compounding performance of Bifidobacterium adolescentis milk fermentation product and yeast fermentation product

[0206] This part mainly explores the compounding performance of Bifidobacterium adolescentis milk fermentation product and yeast fermentation product. By compounding different proportions of Bifidobacterium adolescentis milk fermentation product and yeast fermentation product, the performance change trend is determined.

[0207] The above mainly evaluates from nematode lipofuscin test, and the specific formula is as follows Table 16; in Table 16, the balance ingredient is water;

[0208] Table 16 Formula Table Unit: wt%

[0209]

[0210]

[0211] The nematode lipofuscin test results can be referred to Table 17;

[0212] Table 17 Performance Test Results

[0213]

[0214]

[0215] The test results in Table 17 show that a higher concentration of yeast fermentation products can exhibit a more obvious lipofuscin inhibition effect.

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

[0217] When yeast fermentation products and milk fermentation products 1 are combined, they exhibit significant synergistic effects.

[0218] 3.4 Formulation Safety Testing

[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. Participants: A total of 31 participants, including 14 males and 17 females, aged 25 to 55 years, who met the criteria for voluntary participation.

[0222] 4. Spot test method: Select an area not exceeding 50mm. 2 A qualified spot test apparatus with a depth of approximately 1 mm is used. The test substance is placed in the small chamber of the spot test apparatus, with a volume of approximately 0.020 mL–0.025 mL. The spot test apparatus containing the test substance is applied to the flexor side of the subject's forearm using hypoallergenic adhesive tape. Gently press with the palm of the hand to ensure even application to the skin, and leave for 24 hours. Skin reactions are observed 30 minutes (after the indentation disappears), 24 hours, and 48 hours after removing the spot test apparatus. The results are recorded according to the skin reaction grading standards (Table 18) in the *Cosmetic Safety Technical Specifications* (2015 edition).

[0223] Table 18 Criteria for Evaluating Skin Reactions in Closed Patch Tests

[0224]

[0225]

[0226] Result determination criteria: If more than 5 out of 30 subjects have a grade 1 skin adverse reaction, or more than 2 out of 30 subjects have a grade 2 skin adverse reaction (more than 5 out of 30 subjects have a grade 2 reaction in the patch test for deodorant products), or if any one out of 3 subjects has a grade 3 or higher skin adverse reaction, the test substance is determined to have an adverse skin reaction on the human body. The test results are shown in Table 19.

[0227] Table 19 Criteria for Evaluating Skin Reactions in Closed Patch Tests

[0228]

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

[0230] 31 cases of subjects showed negative reactions (score level 0) in the skin, according to the judgment standard of human patch test in the Cosmetics Safety Technical Specifications, sample 1 has no adverse reactions on human skin.

[0231] Four, application formula

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

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

[0234]

[0235]

[0236] Application formula 2: emulsion

[0237] Table 21 Formula table of emulsion

[0238]

[0239]

[0240] Application formula 3: cream

[0241] Table 22 Formula table of cream

[0242]

[0243]

[0244] The applicant declares that the process of the present application is illustrated by the above examples, but the present application is not limited to the above process steps, nor does it mean that the present application must rely on the above process steps to be implemented. It should be understood by those skilled in the art that any improvement on the present application, equivalent replacement of the materials selected by the present application, addition of auxiliary ingredients, selection of specific methods, etc. fall within the scope of protection and disclosure of the present application.

Claims

1. A strain of Bifidobacterium adolescentis, characterized in that, The Bifidobacterium adolescentis is Bifidobacterium adolescentis BF23BA001, the classification name is Bifidobacterium adolescentis (Bifidobacterium adolescentis) Bifidobacterium adolescentis ), the preservation number is: CGMCC NO: 26999; the preservation date is: March 31, 2023, the preservation unit is: China General Microbiological Culture Collection Center, and the preservation address is: No. 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the Bifidobacterium adolescentis for fermenting milk according to claim 1 to prepare a milk ferment.

3. A milk ferment, characterized in that, The Bifidobacterium adolescentis is prepared according to the method of claim 1, and the preparation method of the milk fermentation product is as follows: Bifidobacterium adolescentis liquid with a viable count of 10 6 ~ 10 8 CFU / mL is added into sterilized milk, and then fermented for 12-48 hours, sterilized at 95°C for 30 minutes, cooled to room temperature, centrifuged at 4800 rpm for 30 minutes, collected supernatant, filtered through a 0.22 μm filter to remove Bifidobacterium adolescentis and impurities, and thus a Bifidobacterium adolescentis milk fermentation product is prepared; the volume ratio of Bifidobacterium adolescentis liquid to milk is 1-10:

100.

4. A composition for inhibiting lipofuscin production and accumulation, comprising, The milk ferment and the yeast ferment according to claim 3; the mass ratio of the milk ferment and the yeast ferment is 0.5-10:1-10; the yeast used in the yeast ferment is Saccharomyces cerevisiae, and the Saccharomyces cerevisiae is derived from Saccharomyces cerevisiae with the accession number SHMCC D11813 of Shanghai Collection Center of Microorganisms; the mass ratio of the milk ferment and the yeast ferment is 0.5-8:2-9.5, and the preparation method of the yeast ferment is as follows: after the Saccharomyces cerevisiae is activated, it is inoculated into a culture medium and fermented at 28-34 ℃ for 12-24 h, then centrifuged, preliminarily filtered, and then filtered after the impurities are adsorbed by activated carbon to obtain the yeast ferment; The culture medium contains 1-3 wt% of glucose, 0.5-1.5 wt% of proteose peptone, and 0.4-0.6 wt% of yeast extract powder.

5. Use of the composition according to claim 4 to prepare a cosmetic; the cosmetic is a cosmetic for skin care.

6. Use according to claim 5, characterized in that, The cosmetic is a cosmetic with the function of inhibiting the generation and accumulation of lipofuscin; and / or, The cosmetic is a cosmetic with the function of whitening and lightening; and / or, The cosmetic is a cosmetic with the function of repairing redness and antioxidation.

7. A cosmetic product, characterized by The cosmetic contains the composition according to claim 4.

8. The cosmetic product according to claim 7, characterized in that, In the cosmetic, the concentration of the composition is 5-20 wt%.

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