Quadruple fermentation composition with acne removing and whitening effects as well as preparation method and application of quadruple fermentation composition

Abstract

The invention belongs to the technical field of cosmetics, and particularly relates to a quadruple fermentation composition with acne removing and whitening effects and application of the quadruple fermentation composition. The quadruple fermentation composition comprises the following components in parts by weight: 1-8 parts of a black tea fermentation product, 1-3 parts of a soybean seed extract fermentation product, 1-5 parts of a white birch juice fermentation product and 1-5 parts of a yeast / calcium fermentation product. According to the acne-removing and whitening composition provided by the invention, the black tea fermentation product, the soybean seed extract fermentation product, the white birch juice fermentation product and the yeast / calcium fermentation product oil are combined, and the four components are subjected to molecular weight screening, so that the acne-removing and whitening composition can be permeated and absorbed in each layer of the skin after being used on the skin; active ingredients effectively permeate the epidermal layer and the corium layer; and the four components interact in a preferable dosage range and can play an excellent synergistic effect, so that the water-oil balance is effectively improved, the oil is controlled, the acne is removed, and the whitening effect of inhibiting melanin is achieved.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, and in particular relates to a four-stage fermentation composition with acne-removing and whitening effects, its preparation method and application. Background Technology

[0002] With changing lifestyles and increasing environmental pollution in modern society, skin problems such as acne, blemishes, and dull skin have become health and beauty concerns for many. Currently, the market is flooded with skincare products targeting these issues, primarily focusing on the use of drugs, chemical ingredients, and plant extracts. However, existing skincare products often have limitations in terms of efficacy and safety. For example, many acne treatments rely heavily on antibacterial and anti-inflammatory chemicals, which may be effective in the short term, but long-term use can have negative effects on the skin, such as damaged skin barriers and sensitivity. Whitening products, while employing various whitening ingredients, often rely too heavily on high concentrations of chemicals, potentially causing skin allergies or irritation, and the effects are often short-lived. While some plant extracts are natural, their effects are relatively slow and require long-term use to see results.

[0003] Furthermore, existing skincare products typically address specific skin problems through single plant-based or synthetic chemical ingredients, often neglecting the multifaceted nature of skin issues and lacking products that can simultaneously provide multiple benefits. Therefore, there is an urgent need for a novel, effective, and safe skincare composition that can simultaneously resolve multiple skin problems such as acne, pigmentation, and uneven skin tone, while also offering good long-lasting effects and safety. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a four-stage fermentation composition with acne-removing and whitening effects, its preparation method and application, which significantly improves the acne-removing and whitening effects of the product through the synergistic effect between the components.

[0005] To achieve the above objectives, the technical solution adopted by the present invention includes:

[0006] In a first aspect, the present invention provides a quadruple fermentation composition with acne-removing and whitening effects, the composition comprising the following components in parts by weight: 1-8 parts of black tea fermentation product, 1-3 parts of soybean seed extract fermentation product, 0.5-5 parts of birch sap fermentation product, and 1-5 parts of yeast / calcium fermentation product.

[0007] Preferably, the black tea fermentation product is a yeast / acetobacterium xylinum / black tea fermentation product, that is, it is obtained by fermenting black tea with yeast and acetobacterium xylinum.

[0008] This invention scientifically combines fermented black tea products, fermented soybean seed extract, fermented birch sap, and yeast / calcium fermentation products. Within a preferred range, these four components work synergistically. The acne-removing and whitening effects are not simply the sum of individual ingredients, but rather the result of mutual promotion and complementarity between the components, which significantly enhances the overall efficacy of the composition and solves the problem of limited efficacy or insufficient effect of single fermentation products.

[0009] Among them, black tea fermentation products, fermented with a mixture of yeast and Acetobacter xylinum, produce rich antibacterial and anti-inflammatory active ingredients. When combined with the other three fermentation products, they can more effectively regulate sebum secretion and enhance acne treatment. Soybean seed extract fermentation products contain natural whitening active substances, such as small molecule peptides and amino acids. When combined with other components, they can more effectively inhibit tyrosinase activity and reduce melanin production and deposition, thereby significantly improving whitening efficiency. In addition, birch sap fermentation products are rich in natural soothing ingredients, such as minerals and amino acids. When combined with yeast / calcium fermentation products, they can better relieve skin sensitivity, redness and other problems, and enhance skin tolerance.

[0010] Therefore, the quadruple fermentation composition of the present invention has significant acne-removing, whitening, and soothing effects, and is natural, safe, gentle and non-irritating, suitable for a variety of skin types including sensitive skin, and has a wider range of applications.

[0011] Preferably, the composition comprises the following components in parts by weight: 2-7 parts of black tea fermentation product, 1.5-2.5 parts of soybean seed extract fermentation product, 1.5-4 parts of birch sap fermentation product, and 2-4 parts of yeast / calcium fermentation product.

[0012] Preferably, the composition comprises the following components in parts by weight: 6 parts of black tea fermentation product, 2 parts of soybean seed extract fermentation product, 3 parts of birch sap fermentation product, and 3 parts of yeast / calcium fermentation product.

[0013] Experimental research has shown that when the four components are combined in the optimal dosage ratio specified above, their respective effects can be maximized, thereby ensuring the excellent effects of the prepared composition in multiple aspects such as oil control, whitening and acne removal, and the best cost performance.

[0014] Preferably, the preparation method of the soybean seed extract fermentation product includes the following steps:

[0015] S1. After crushing soybeans, water is added for extraction. The solid-liquid separation is performed and the filtrate is collected to obtain soybean seed extract.

[0016] S2. Inoculate Bacillus subtilis into a liquid culture medium containing 5-15% soybean seed extract for activation culture;

[0017] S3. The activated Bacillus subtilis from step S2 is inoculated into a sterilized fermentation medium containing soybean seed extract for fermentation culture to obtain the fermentation product.

[0018] S4. Inactivate the fermentation product from step S3, centrifuge, filter to obtain a coarse filtrate, add β-cyclodextrin to the coarse filtrate, evaporate and concentrate to obtain the soybean seed extract fermentation product.

[0019] Preferably, in step S1, the soybeans are pulverized to 80-100 mesh to effectively ensure complete fermentation of the substrate, thereby ensuring the efficacy of the final product; the ratio of soybeans to water is 1:(5-15). If there are too few soybeans, the efficacy of the composition will be affected, while too many soybeans will cause dissolution and caking, which will also weaken the final efficacy; the specific extraction conditions are: extraction at 45-55℃ for 1.5-2.5h.

[0020] Preferably, in step S2, the activation culture is performed 2-3 times to ensure that the bacterial strain reaches its optimal viability.

[0021] Preferably, in step S3, the fermentation medium comprises the following components: 90-110 g / L soybean seed extract, 0.3-0.7 g / L glucose, 0.1-0.5 g / L yeast extract, 0.03-0.07 g / L potassium dihydrogen phosphate, and 0.01-0.05 g / L magnesium sulfate.

[0022] Experimental research revealed that the fermentation process of soybean seed extract requires precise control of conditions; otherwise, the activity and quality of the product may be affected. Furthermore, compared to non-fermented soybean extract, fermented soybean seed extract exhibits more active ingredients and more definitive efficacy, and demonstrates excellent compatibility with the other three components. Therefore, this invention provides a specific preparation process for fermented soybean seed extract, effectively ensuring that no active interactions occur when the four components are combined, thereby achieving an ideal synergistic effect to further enhance the oil-controlling and whitening effects of the composition, and making it more suitable for cosmetic applications.

[0023] Preferably, in step S3, the degree of protein hydrolysis in the obtained fermentation product reaches 25-30%; the spore formation rate is ≥90%; in step S1, the soybean is a high-quality non-GMO soybean with a protein content of ≥35% and free from mold and insect infestation.

[0024] Preferably, the molecular weight range of the main components of the black tea fermentation product is above 10,000 Da; the molecular weight range of the main components of the soybean seed extract fermentation product is 5,000-10,000 Da; the molecular weight range of the main components of the birch sap fermentation product is 1,000-5,000 Da; and the molecular weight range of the main components of the yeast / calcium fermentation product is below 1,000 Da.

[0025] This invention employs molecular weight gradient screening of the four components to ensure effective penetration and retention of each active ingredient at different skin layers. This avoids the limitations of single-molecular-weight components acting only at localized levels, achieving full-layer active ingredient coverage across the epidermis and dermis. Furthermore, the molecular weight screening of each component allows it to exert targeted effects at different skin layers, creating a complementary synergistic effect. Through superficial regulation of the stratum corneum, mid-layer barrier repair, and deep anti-inflammatory and antioxidant action, it avoids the shortcomings of single-layer effects. The molecular weight differences also prevent the components from losing activity due to intermolecular interactions during penetration and action, ensuring that each ingredient can independently function at its target layer, thereby significantly enhancing the acne-reducing and whitening efficacy of the composition.

[0026] Preferably, the method for preparing the birch sap fermentation product includes the following steps:

[0027] S1. Mix birch sap powder with water, stir until dissolved, filter, and obtain a liquid matrix;

[0028] S2. The activated Lactobacillus plantarum culture solution is inoculated into a liquid substrate for fermentation culture, inactivated, filtered and collected to obtain the birch sap fermentation product.

[0029] Preferably, in step S1, the mass ratio of birch sap powder to water is 1:(5-10); in step S2, the specific conditions for fermentation culture are: fermentation at 35-37℃ for 24-48 hours; and the inoculation amount of Lactobacillus plantarum solution is 5-10%.

[0030] The present invention uses the above-mentioned specific preparation process to prepare birch sap fermentation products, which can further increase the content of active ingredients in the products, thereby significantly enhancing the multiple effects of the final composition.

[0031] Secondly, the present invention provides the use of the described composition in cosmetics.

[0032] Thirdly, the present invention provides a toner with acne-removing and whitening effects, the toner comprising the aforementioned composition.

[0033] Preferably, the cosmetics include serums, creams, sunscreens, face masks, and makeup products.

[0034] Preferably, the toner further includes a thickener and a preservative; the thickener includes at least one of carbomer; and / or, the preservative includes at least one of phenoxyethanol.

[0035] Fourthly, the present invention provides a method for preparing the aforementioned toner, comprising the following steps:

[0036] S1. The black tea fermentation product is filtered through a hollow fiber ultrafiltration membrane with a molecular weight cutoff of 10,000 Da, and the retentate is collected to obtain the macromolecular-level black tea fermentation product.

[0037] S2. The fermented product of soybean seed extract is first passed through an ultrafiltration membrane with a molecular weight cutoff of 10,000 Da to obtain permeate. The permeate is then passed through an ultrafiltration membrane with a molecular weight cutoff of 5,000 Da, and the retentate is collected to obtain the large molecular weight soybean seed extract fermented product.

[0038] S3. Pass the birch sap fermentation product through a nanofiltration membrane with a molecular weight cutoff of 5000 Da to obtain permeate. Pass the permeate through a nanofiltration membrane with a molecular weight cutoff of 1000 Da and collect the retentate to obtain medium molecular weight birch sap fermentation product.

[0039] S4. Place the yeast / calcium fermentation product into a dialysis bag with a molecular weight cutoff of 1000 Da and dialyze for 17-19 hours. Collect the dialysate to obtain small molecular-level yeast / calcium fermentation product.

[0040] S5. Add water to the thickener, heat and stir until gel-like, cool down, add the product obtained in steps S1-S4 and the preservative, stir and mix well, adjust the pH to 5.5-6.5, filter, and cool to obtain the toner.

[0041] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0042] (1) This invention provides an acne-removing and whitening composition combining yeast / Acetobacter xylinum / black tea fermentation product, soybean seed extract fermentation product, birch sap fermentation product and yeast / calcium fermentation product. By screening the molecular weight of the four components, it is possible to achieve absorption in all layers of the skin after application, with effective penetration of active ingredients in both the epidermis and dermis. Moreover, the four components interact with each other within the preferred dosage range, which can exert excellent synergistic effects, effectively improving the water-oil balance, controlling oil and removing acne, and achieving a whitening effect by inhibiting melanin.

[0043] (2) The present invention provides a specific preparation process for soybean seed extract fermentation products, which can effectively ensure that no active interaction occurs when the four components are compounded, thereby exerting an ideal synergistic effect to further enhance the oil control and whitening effects of the composition, making it more suitable for cosmetic applications. Attached Figure Description

[0044] Figure 1 The graph shows the antibacterial effect of the toners in different embodiments and comparative examples of Example 1 on Propionibacterium acnes. Detailed Implementation

[0045] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0046] Unless otherwise specified, all raw materials used in the following examples and comparative examples are commercially available general-purpose materials.

[0047] The yeast / Acetobacter xylinum / black tea fermentation products described in the following examples and comparative examples were purchased from Bozhou Mingqi Biotechnology Co., Ltd.; soybeans were purchased from Taobao Maicai (a grocery delivery platform), directly from the farm; Bacillus subtilis was purchased from Shandong Dehe Mingxing Biotechnology Co., Ltd.; yeast / calcium fermentation products were purchased from Guangzhou Huayang Biotechnology Co., Ltd.; birch sap powder was purchased from Peptide Biotechnology (Xi'an) Co., Ltd.; and Lactobacillus plantarum was purchased from Xi'an Meihe Biotechnology Co., Ltd.

[0048] Examples 1-5

[0049] Examples 1-5 provide a toner with acne-removing and whitening effects, the formula of which is shown in Table 1 (total weight parts: 100 parts). The preparation method of the toner includes the following steps:

[0050] (1) Preparation of fermentation products of soybean seed extract:

[0051] S1. Select high-quality non-GMO soybeans with a protein content ≥35% and free from mold and insect infestation. Grind the soybeans to 100 mesh and add deionized water at a material-to-liquid ratio of 1:10 (g / mL). Stir and extract at 50℃ for 2 hours. Then, separate the solid and liquid using a plate and frame filter press, collect the filtrate, and obtain soybean seed extract. Add 1M sodium hydroxide solution to adjust the pH of the extract to 7.

[0052] S2. Select Bacillus subtilis strains and inoculate them into LB liquid medium containing 10% soybean seed extract. Incubate at 37°C and 200 rpm with shaking for 14 hours until the OD600nm value of the bacterial solution reaches 0.8-1.0, indicating that it has entered the logarithmic growth phase. Transfer the inoculum to fresh medium at a rate of 10% and repeat the activation process twice. Examine the activated strains under a microscope to confirm that they are free from contamination before use.

[0053] S3. Add 10g soybean seed extract, 0.053g glucose, 0.027g yeast powder, 0.0053g potassium dihydrogen phosphate, 0.0027g magnesium sulfate, and the remaining water to a final volume of 100ml. Dissolve all ingredients thoroughly, adjust the pH to 7.0±0.2 with 1M hydrochloric acid, transfer to a 50mL Erlenmeyer flask, autoclave at 121℃ for 30 minutes, and cool to 35-37℃ to complete the preparation of the fermentation medium.

[0054] S4. Inoculate the activated strain from step S2 into the fermentation medium from step S3 at a 9% (v / v) inoculation rate, place in a 100 mL shake flask, and ferment in a constant temperature shaker at 37°C and 200 rpm. Measure the OD600nm, protein hydrolysis degree using the ninhydrin colorimetric method, and spore formation rate using a microscope every 4 hours. Terminate fermentation within 48 hours when the protein hydrolysis degree reaches 25-30% and the spore formation rate is ≥90%. If the pH is below 6.5, adjust by adding 5% sodium carbonate solution using a micropipette.

[0055] S5. Transfer the fermentation broth from step S4 into a 15mL centrifuge tube, inactivate the enzyme in an 85℃ water bath for 25 minutes, centrifuge at 8000rpm for 20 minutes in a small centrifuge, and collect the supernatant to obtain the coarse filtrate; then filter the coarse filtrate through a 0.45μm syringe-type membrane filter, add 0.02g of β-cyclodextrin, stir at 45℃ for 40 minutes, then filter through a 0.22μm syringe-type sterile membrane filter, and finally concentrate to 1 / 3 of the original volume using a micro rotary evaporator at a temperature ≤50℃ and a pressure of -0.06MPa to obtain the fermentation product of soybean seed extract.

[0056] (2) Preparation of birch sap fermentation products:

[0057] S1. Add birch sap powder to deionized water at a ratio of 1:8, stir until completely dissolved, filter once with 200-mesh gauze to obtain a liquid matrix.

[0058] S2. Using a sterile pipette, take 0.1g of Lactobacillus plantarum powder, add it to 10mL of activated liquid culture medium, shake gently, and incubate at 37℃ for 12-16h until the liquid is slightly turbid to obtain the activated strain.

[0059] S3. After activation, inoculate the bacterial solution at an 8% rate (100 mL of substrate plus 8 mL of bacterial solution) and stir well. Place the mixed liquid substrate into a clean, capped glass bottle, seal tightly, and store at 35-37℃. Ferment for 24-48 hours, observing during this time. Stop fermentation when the liquid becomes cloudy and has a slightly sour taste (pH drops to around 4.0). Pour the fermentation liquid into a pot, heat over low heat to 60℃, maintain for 10 minutes, let cool, and then filter once through 200-mesh gauze to remove a small amount of bacterial precipitate, obtaining a clear birch sap fermentation product.

[0060] (3) Preparation of toner:

[0061] S1. The yeast / Acetobacter xylinum / black tea fermentation product is filtered through a hollow fiber ultrafiltration membrane with a molecular weight cutoff of 10000 Da. The membrane is pumped into the ultrafiltration device at a pressure of 0.2 MPa and the flow rate is controlled at 25 L / h. Large molecular weight bacterial cellulose, some polysaccharides and proteins are retained, while small molecular weight impurities flow out with the filtrate. The retentate is collected, which is the large molecular weight yeast / Acetobacter xylinum / black tea fermentation product.

[0062] S2. The fermented product of soybean seed extract is first passed through a 10000Da ultrafiltration membrane to retain large molecular proteins and some polysaccharides; then the permeate is passed through a 5000Da ultrafiltration membrane at a pressure of 0.15MPa, and the 5000Da retentate containing active peptides, isoflavones, etc. is collected to obtain the filtrate of soybean seed extract fermentation product of medium and large molecular weight.

[0063] S3. The birch sap fermentation product is first passed through a 5000 Da nanofiltration membrane to retain large molecular weight polysaccharides and proteins; the permeate is then passed through a 1000 Da nanofiltration membrane to retain 1000-5000 Da organic acids, oligopeptides, etc. The retentate from the 1000 Da nanofiltration membrane is collected to obtain the medium molecular weight birch sap fermentation product filtrate;

[0064] S4. The yeast / calcium fermentation product is placed in a dialysis bag with a molecular weight cutoff of 1000 Da. It is then placed in deionized water dialysis solution and dialyzed for 18 hours at 4°C and 100 rpm on a shaker. The dialysis solution is changed 4 times during the dialysis process. Small molecule amino acid chelates, calcium, and small peptides diffuse into the dialysis solution, while large molecule proteins remain in the dialysis bag. The dialysis solution is collected, which is the small molecule-level yeast / calcium fermentation product.

[0065] S5. Heat deionized water to 70°C, add carbomer to form a gel, cool to 40°C, then add the products obtained in steps S1-S4 and phenoxyethanol, stir until dissolved and homogeneous, adjust the pH to 5.5-6.5, filter, and cool to obtain the toner.

[0066] Table 1. Formulation of the toners described in Examples 1-5 (parts by weight)

[0067]

[0068]

[0069] Example 6

[0070] This embodiment provides a toner with acne-removing and whitening effects. The only difference between this toner and Example 1 is the preparation method of the soybean seed extract fermentation product. The difference is as follows:

[0071] In step S1, soybeans are pulverized to 80 mesh, and deionized water is added at a material-to-liquid ratio of 1:5 (g / mL). The mixture is then stirred and extracted at 45°C for 2.5 hours.

[0072] In step S2, the bacterial strain is activated three times;

[0073] In step S3, the fermentation medium comprises 90 g / L soybean seed extract, 0.3 g / L glucose, 0.1 g / L yeast extract, 0.07 g / L potassium dihydrogen phosphate, and 0.05 g / L magnesium sulfate.

[0074] Example 7

[0075] This embodiment provides a toner with acne-removing and whitening effects. The only difference between this toner and Example 1 is the preparation method of the soybean seed extract fermentation product. The difference is as follows:

[0076] In step S1, soybeans are pulverized to 100 mesh, and deionized water is added at a material-to-liquid ratio of 1:15 (g / mL). The mixture is then stirred and extracted at 55°C for 1.5 hours.

[0077] In step S3, the fermentation medium comprises 110 g / L soybean seed extract, 0.7 g / L glucose, 0.1 g / L yeast extract, 0.07 g / L potassium dihydrogen phosphate, and 0.05 g / L magnesium sulfate.

[0078] Example 8

[0079] This embodiment provides a toner with acne-removing and whitening effects. The only difference between this toner and Example 1 is the preparation method of the soybean seed extract fermentation product. The difference is as follows:

[0080] In step S1, the soybeans are not crushed, and deionized water is added at a material-to-liquid ratio of 1:20 (g / mL) for extraction.

[0081] In step S2, the bacterial strain is activated once.

[0082] Example 9

[0083] This embodiment provides a toner with acne-removing and whitening effects. The only difference between this toner and Example 1 is the preparation method of the soybean seed extract fermentation product. The difference is as follows:

[0084] In step S1, soybeans are pulverized to 100 mesh and extracted with deionized water at a material-to-liquid ratio of 1:1 (g / mL).

[0085] Comparative Examples 1-10

[0086] Comparative Examples 1-10 provide a toner with acne-reducing and whitening effects, the formula of which is shown in Table 2 (total weight parts: 100 parts). The preparation method of the toner is the same as that of Example 1, the only difference being the formula.

[0087] Table 2. Formulation of the toners described in Comparative Examples 1-10 (parts by weight)

[0088]

[0089] Comparative Example 11

[0090] This comparative example provides a toner that differs from Example 1 only in that an equal amount of soybean seed extract prepared in Example 1 is used to directly replace the soybean seed extract fermentation product, i.e., no fermentation treatment is performed. All other components and amounts are the same as in Example 1.

[0091] Comparative Example 12

[0092] This comparative example provides a toner that differs from Example 1 only in that: an equal amount of birch sap liquid matrix as described in Example 1 is used to replace the birch sap fermentation product, i.e., no fermentation treatment is performed, while the remaining components and amounts are the same as in Example 1.

[0093] Comparative Example 13

[0094] This comparative example provides a toner, which differs from Example 1 only in that the preparation process of the toner is different. The preparation method of the toner in this comparative example includes the following steps:

[0095] Deionized water was heated to 70°C, and carbomer was added to form a gel. The temperature was then lowered to 40°C, and then purchased black tea fermentation product, yeast / calcium fermentation product, soybean seed extract fermentation product and birch sap fermentation product prepared by the method described in Example 1 were added. Phenoxyethanol was then added and stirred until dissolved and homogeneous. The pH was adjusted to 5.5-6.5, filtered, and cooled to obtain the toner.

[0096] Example 1

[0097] This efficacy example uses the toners prepared in Examples 1-9 and Comparative Examples 1-13 as test samples. The acne-removing efficacy of the samples was tested according to the in vitro testing method for cosmetic acne-removing effects (antibacterial rate method). Since acne is mainly caused by the accumulation of Propionibacterium acnes, the acne-removing effect of the samples can be evaluated by testing their antibacterial effect against Propionibacterium acnes. The specific method is as follows:

[0098] Negative control group: sterile standard hard water.

[0099] 1. Main reagents: Clostridium enrichment medium, phosphate buffered saline (PBS), standard strain: Propionibacterium acnes ATCC6919.

[0100] 2. Main equipment: Biosafety cabinet (Lubo brand, model LB-9100), electronic balance (Sartiorius brand, model BSA-224S), biochemical incubator (Thermo brand, model RI-150).

[0101] 3. Experimental Procedure (3.1) Dilute the test bacterial suspension appropriately with PBS solution. The required concentration is: take 0.1 mL and drop it into 5.0 mL of control sample solution (PBS). The recovered bacterial count is 1 × 10⁻⁶. 4 -9×10 4 CFU / mL; Dilute the test sample with sterile standard hard water to the specified concentration; (3.2) Take 5 mL of the original sample solution or its dilution into a sterile test tube, take 0.1 mL of the above test bacterial solution into a test tube containing 5 mL of sample, mix quickly, and start timing immediately; (3.3) After acting for 20 min, take 0.5 mL of the above mixture and add it into a test tube containing 4.5 mL of sterilized PBS, and mix thoroughly; (3.4) After standing for 10 min, take 1 mL of sample solution into a sterile Petri dish, and inoculate two sterile Petri dishes for each sample solution or dilution; (3.5) Pour 15 mL of Clostridium enrichment medium cooled to 40℃-45℃ into the Petri dish, rotate the Petri dish to mix thoroughly, and after the medium solidifies, turn the Petri dish over and anaerobic incubate at 37℃ for 72 h, and then count the viable colonies; (3.6) Use PBS instead of the test sample and follow the above steps as a blank control group. The experiment was repeated 3 times and the average value was calculated. (3.7) A negative control group (bacterial suspension, culture medium, sterile standard hard water) needs to be prepared separately for the experiment.

[0102] 4. Calculate the antibacterial rate using the following formula:

[0103]

[0104] In the above formula: A—average colony count of blank control sample; B—average colony count of experimental group.

[0105] The specific test results are shown in Table 3.

[0106] Table 3

[0107] Sample source Antibacterial rate (%) Sample source Antibacterial rate (%) Sample source Antibacterial rate (%) Example 1 76.2 Example 9 64.2 Comparative Example 8 16.2 Example 2 65.3 Comparative Example 1 43.1 Comparative Example 9 58.3 Example 3 70.7 Comparative Example 2 46.4 Comparative Example 10 55.7 Example 4 72.2 Comparative Example 3 47.7 Comparative Example 11 53.2 Example 5 73.8 Comparative Example 4 48.3 Comparative Example 12 54.8 Example 6 74.5 Comparative Example 5 23.4 Comparative Example 13 45.6 Example 7 74.1 Comparative Example 6 20.5 Positive control group 7.52 Example 8 62.1 Comparative Example 7 18.5

[0108] Table 3 shows that the toner containing the quadruple fermentation composition prepared by the technical solution of the present invention has a good antibacterial effect against Propionibacterium acnes, thus achieving a better acne-removing effect. Among them, the toner described in Example 1 has the best antibacterial effect, i.e., the best acne-removing effect. However, the acne-removing effect of the toners described in Examples 8-9 is somewhat lower than that of Example 1, indicating that the preparation process of the soybean seed extract fermentation product has a certain impact on the efficacy of the product.

[0109] In Comparative Examples 1-4, when any one of the four active components (black tea fermentation product, soybean seed extract fermentation product, birch sap fermentation product, and yeast / calcium fermentation product) was missing, or in Comparative Examples 4-8, when only one component was used, the antibacterial effect of the resulting toner was significantly reduced compared to the examples. This indicates that the four components selected in this invention work synergistically to effectively enhance the acne-removing efficacy of the product, and all four components are indispensable. In Comparative Examples 9-10, when the ratio of the four components exceeded the limits specified in this invention, the acne-removing effect of the toner also decreased, further confirming that the four active components selected in this invention... The active components can exert a better synergistic effect only within a specific ratio range. In Comparative Examples 11-12, the fermented soybean seed extract and birch sap fermented product were replaced with the corresponding unfermented liquids, and their antibacterial effects were significantly lower than those in the Example. This indicates that fermentation treatment can effectively enhance the antibacterial activity of the components, thereby synergistically enhancing the acne-removing effect of the product when combined with the other three fermented products. In Comparative Example 13, the four components were not subjected to appropriate molecular weight screening, and their antibacterial effect was also reduced compared with the Example. This indicates that molecular weight screening of the components can effectively increase the content of active substances in the components, thereby further enhancing the antibacterial effect.

[0110] Example 2

[0111] This effect example uses the toners prepared in Examples 1-9 and Comparative Examples 1-13 as test samples to test their whitening effect. The specific methods are as follows:

[0112] Mouse melanoma B16 cells were used as the research subject. B16 cells were cultured to restore them to a melanin-synthesizing state. The test sample was dissolved in PBS to obtain the experimental solution (80 mg / mL), which was added to the B16 cell culture medium and incubated for 24 h. An equal volume of PBS was used as a control group. After incubation, the cells were centrifuged, and the supernatant was added to a 96-well plate. 5 mL of Dopa solution was added, mixed well, and incubated in a 37°C water bath for 10 min. The absorbance of each well was measured at 475 nm. Intracellular tyrosinase activity (%) was calculated as A0 / A1 × 100%, where A0 was the average absorbance of the experimental solution group, and A1 was the average absorbance of the control group. Nicotinamide was used as a positive control. Specific test results are shown in Table 4. The lower the cellular tyrosinase activity, the better the whitening effect.

[0113] Table 4

[0114]

[0115] Table 4 shows that the toner containing the quadruple fermentation composition prepared in the examples can effectively inhibit tyrosinase activity, thereby achieving a better whitening effect. In contrast, the whitening effect of the toners prepared in the comparative examples was significantly lower than that of the examples due to the absence or proportions of the four key components selected in this invention exceeding the preferred range. These results indicate that when the soybean seed extract fermentation product, birch sap fermentation product, black tea fermentation product, and yeast / calcium fermentation product prepared using the specific preparation process of this invention are combined within the preferred proportions, the four components interact and synergistically enhance the whitening effect of the product.

[0116] Finally, it should be noted that the above embodiments are used to illustrate the technical solutions of the present invention and not to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A four-stage fermentation composition with acne-removing and whitening effects, characterized in that, The composition comprises the following components in parts by weight: 1-8 parts of black tea fermentation product, 1-3 parts of soybean seed extract fermentation product, 0.5-5 parts of birch sap fermentation product, and 1-5 parts of yeast / calcium fermentation product.

2. The composition according to claim 1, characterized in that, The composition comprises the following components in parts by weight: 2-7 parts of black tea fermentation product, 1.5-2.5 parts of soybean seed extract fermentation product, 1.5-4 parts of birch sap fermentation product, and 2-4 parts of yeast / calcium fermentation product.

3. The composition according to claim 2, characterized in that, The composition comprises the following components in parts by weight: 6 parts of black tea fermentation product, 2 parts of soybean seed extract fermentation product, 3 parts of birch sap fermentation product, and 3 parts of yeast / calcium fermentation product.

4. The composition according to any one of claims 1-3, characterized in that, The preparation method of the soybean seed extract fermentation product includes the following steps: S1. After crushing soybeans, water is added for extraction. The filtrate is collected by solid-liquid separation to obtain soybean seed extract. S2. Inoculate Bacillus subtilis into a liquid culture medium containing 5-15% soybean seed extract for activation culture; S3. The activated Bacillus subtilis from step S2 is inoculated into a sterilized fermentation medium containing soybean seed extract for fermentation culture to obtain the fermentation product. S4. Inactivate the fermentation product from step S3, centrifuge, filter to obtain a coarse filtrate, add β-cyclodextrin to the coarse filtrate, evaporate and concentrate to obtain the soybean seed extract fermentation product.

5. The composition according to claim 4, characterized in that, In step S1, the soybeans are pulverized to 80-100 mesh, the ratio of soybeans to water is 1:(5-15), and the specific extraction conditions are: extraction at 45-55℃ for 1.5-2.5 hours. In step S2, the activation culture is performed 2-3 times; In step S3, the fermentation medium comprises the following components: 90-110 g / L soybean seed extract, 0.3-0.7 g / L glucose, 0.1-0.5 g / L yeast extract, 0.03-0.07 g / L potassium dihydrogen phosphate, and 0.01-0.05 g / L magnesium sulfate.

6. The composition according to any one of claims 1-3, characterized in that, The main components of the black tea fermentation product have a molecular weight range of 10,000 Da or higher; the main components of the soybean seed extract fermentation product have a molecular weight range of 5,000-10,000 Da; the main components of the birch sap fermentation product have a molecular weight range of 1,000-5,000 Da; and the main components of the yeast / calcium fermentation product have a molecular weight range of less than 1,000 Da.

7. The use of the composition according to any one of claims 1-6 in cosmetics.

8. A toner with acne-removing and whitening effects, characterized in that, The toner comprises the composition according to any one of claims 1-6.

9. The toner as described in claim 8, characterized in that, The toner further includes a thickener and a preservative; the thickener includes at least one of carbomer; and / or, the preservative includes at least one of phenoxyethanol.

10. The method for preparing the toner as described in claim 9, characterized in that, Includes the following steps: S1. The black tea fermentation product is filtered through a hollow fiber ultrafiltration membrane with a molecular weight cutoff of 10,000 Da, and the retentate is collected to obtain the macromolecular-level black tea fermentation product. S2. The fermented product of soybean seed extract is first passed through an ultrafiltration membrane with a molecular weight cutoff of 10,000 Da to obtain permeate. The permeate is then passed through an ultrafiltration membrane with a molecular weight cutoff of 5,000 Da, and the retentate is collected to obtain the large molecular weight soybean seed extract fermented product. S3. Pass the birch sap fermentation product through a nanofiltration membrane with a molecular weight cutoff of 5000 Da to obtain permeate. Pass the permeate through a nanofiltration membrane with a molecular weight cutoff of 1000 Da and collect the retentate to obtain medium molecular weight birch sap fermentation product. S4. Place the yeast / calcium fermentation product into a dialysis bag with a molecular weight cutoff of 1000 Da and dialyze for 17-19 hours. Collect the dialysate to obtain small molecular-level yeast / calcium fermentation product. S5. Add water to the thickener, heat and stir until gel-like, cool down, add the product obtained in steps S1-S4 and the preservative, stir and mix well, adjust the pH to 5.5-6.5, filter, and cool to obtain the toner.

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