Combined leavening with whitening and anti-oxidation effects, preparation method and application in cosmetics

Abstract

The invention provides a combined leavening with whitening and anti-oxidation effects, a preparation method and an application of the combined leavening in cosmetics. The preparation method comprises the following steps: step 1, preparing a fermentation initial system in proportion and sterilizing, wherein the fermentation initial system comprises polygonum cuspidatum roots, grape seeds, cassia seed powder and deionized water; and step 2, inoculating the fermentation initial system with a zymophyte lactobacillus acidophilus, carrying out standing culture treatment, after the standing culture treatment is finished, carrying out sterilization and separation treatment in sequence, and taking a supernatant, so as to obtain the combined fermentation product. The lactobacillus acidophilus is adopted to conduct compound fermentation on the three raw materials including the polygonum cuspidatum roots, the grape seeds and the semen cassiae, active ingredients such as resveratrol in the raw materials can be fully released and enriched, the resource utilization rate is increased, the antioxidant and whitening effects of the lactobacillus acidophilus can be better and synergistically exerted, and the application of the polygonum cuspidatum roots, the grape seeds and the semen cassiae in the field of cosmetics is expanded.

Description

Technical Field

[0001] This invention belongs to the field of cosmetics, specifically relating to a combination fermentation product with whitening and antioxidant effects, its preparation method, and its application in cosmetics. Background Technology

[0002] In today's pursuit of healthy and ideal skin, the demand for whitening and antioxidant cosmetics is rising. Consumers desire products that effectively improve dull skin tone, fade dark spots, and protect against free radical damage, achieving radiant and youthful skin. They also demand higher standards for the products' greenness, environmental friendliness, and safety. Currently, while strictly adhering to relevant cosmetic laws and regulations, whitening and antioxidant cosmetics still largely utilize chemically synthesized ingredients. These ingredients offer advantages such as clear and controllable component structures, definite and significant whitening and antioxidant effects, and mature and stable quality control systems, allowing consumers to see skin improvements in a relatively short time. However, with accumulated user experience, people have gradually discovered that some chemically synthesized ingredients, while effective, can also have adverse effects on the skin, such as causing sensitivity, redness, stinging, burning sensations, and even damaging the skin barrier, leading to subsequent skin problems. Therefore, how to reduce adverse skin reactions caused by chemically synthesized ingredients while ensuring the whitening and antioxidant effects are not compromised has become one of the important directions for innovation and development in the whitening and antioxidant cosmetics industry.

[0003] Polygonum cuspidatum ( Polygonum cuspidatum Sieb. et Zucc. is a perennial herb belonging to the Polygonaceae family and the Polygonum genus. It is distributed in many parts of Northwest, East, and South China. Its core medicinal and skincare value comes from its dried rhizome. As a traditional Chinese medicine, it has been used for over a thousand years. Modern research has found that its rhizome is rich in various active ingredients such as resveratrol, polygalactoside (resveratrol glycoside), and emodin. Among these, resveratrol often exists in the more easily absorbed form of polygalactoside.

[0004] Grape seeds are from the grape plant (Vitis vinifera) of the Vitaceae family. Vitis vinifera The seeds of *L.*, usually a byproduct of winemaking, are extracted and processed into a high-value skincare ingredient. Its core active ingredient is proanthocyanidins, with a content reaching approximately 30%. It also contains polyphenolic compounds, vitamin E, and other nutrients, and is widely used in the food and cosmetic industries.

[0005] Cassia seeds are derived from the legume plant *Cassia tora* (Senna tora). Senna obtusifolia , formerly known as Cassia obtusifolia ) or small cassia seed ( Senna tora , formerly known as Cassia toraThe dried, mature seeds of Cassia tora, used as both food and medicine in traditional Chinese medicine, are widely applied in the field of healthcare. Cassia seeds are rich in active ingredients such as anthraquinones, naphthopyranones, and polysaccharides. Anthraquinones, such as rhein and emodin, are the material basis for its laxative effect and some antibacterial and anti-inflammatory effects; naphthopyranones, such as cassiaside, are key components for its heat-clearing and vision-improving effects, especially its antioxidant and neuroprotective effects; polysaccharides are generally associated with health-promoting functions such as immune regulation and lowering blood lipids.

[0006] Existing technologies suffer from low extraction efficiency for specific active ingredients, resulting in significant resource waste. Current techniques utilize organic solvents to extract active ingredients from single raw materials such as Polygonum cuspidatum root, grape seed, and cassia seed. This not only causes environmental pollution but also involves hazardous experimental procedures, strict production conditions, and is not conducive to industrialization. Furthermore, the extraction costs are high, and residual organic solvents may remain in the extracts, making them unsuitable for use as cosmetic raw materials. This application utilizes specific lactic acid bacteria to ferment Polygonum cuspidatum root, grape seed, and cassia seed, which can fully release the active ingredients in these materials and better exert their whitening and antioxidant effects. Summary of the Invention

[0007] A brief overview of this application is provided below to offer a basic understanding of certain aspects thereof. It should be understood that this overview is not an exhaustive summary of the application. It is not intended to identify key or essential parts of the application, nor is it intended to limit its scope. Its purpose is merely to present certain concepts in a simplified form as a prelude to the more detailed description that follows.

[0008] In view of the above-mentioned deficiencies of the prior art, the purpose of this application is to provide a combination ferment with whitening and antioxidant effects, a preparation method thereof, and its application in cosmetics.

[0009] According to a first aspect of this application, a method for preparing a combined fermentation product with whitening and antioxidant effects is provided, comprising: Step 1: Prepare the initial fermentation system according to the specified ratio and sterilize it. The initial fermentation system includes Polygonum cuspidatum root, grape seeds, cassia seed powder and deionized water. Step 2: Inoculate the initial fermentation system with the fermenting bacterium Lactobacillus acidophilus (Lactobacillus acidophilus). Lactobacillus acidophilus The mixture was subjected to static culture, followed by sterilization and separation. The supernatant was then collected to obtain the combined fermentation product.

[0010] In the above-mentioned method for preparing a combination fermentation product with whitening and antioxidant effects, as a preferred embodiment, in step one, the mass ratio of Polygonum cuspidatum root, grape seed, and Cassia seed in the initial fermentation system is 1:1~3:1~3, more preferably 1:1:1; Preferably, the particle size of the powders of Polygonum cuspidatum root, grape seed, and cassia seed is 50-150 mesh; Preferably, in the initial fermentation system, the mass ratio of deionized water to the composition powder is 30~200:1, more preferably 50~150:1, and even more preferably 100:1.

[0011] In the above-mentioned method for preparing a combination fermentation product with whitening and antioxidant effects, as a preferred embodiment, in step one, after the initial fermentation system is prepared in proportion, it is subjected to high-temperature sterilization; wherein, the sterilization temperature is preferably 100~121℃, more preferably 100~110℃, and even more preferably 110℃; the sterilization time is 15~35min, more preferably 15~25min, and even more preferably 20min.

[0012] In the above-mentioned method for preparing a combination ferment with whitening and antioxidant effects, as a preferred embodiment, in step two, the Lactobacillus acidophilus is Lactobacillus acidophilus CICC20250 or Lactobacillus acidophilus CICC6083. Preferably, the Lactobacillus acidophilus is inoculated in the form of bacterial suspension; Preferably, the bacterial culture is cultured as follows: a single colony of *Lactobacillus acidophilus* is picked up using an inoculation loop and inoculated onto MRS medium, then shaken well and incubated statically in a culture flask at 42°C. The viable count of the *Lactobacillus acidophilus* culture is 10-1. 6 ~10 11 CFU / mL, more preferably 10 7 ~10 10 CFU / mL.

[0013] In the above-mentioned method for preparing a combination ferment with whitening and antioxidant effects, as a preferred embodiment, in step two, Lactobacillus acidophilus is inoculated into the initial fermentation system, and the inoculation amount is preferably 0.5% to 1.5% by volume fraction.

[0014] In the above-mentioned method for preparing a combination of fermented products with whitening and antioxidant effects, as a preferred embodiment, in step two, the fermentation temperature is 30~45℃, preferably 35~45℃; the fermentation time is 12~48h, preferably 24~48h.

[0015] In the above-mentioned method for preparing a combination fermentation product with whitening and antioxidant effects, as a preferred embodiment, in step two, after fermentation, high-temperature sterilization is performed at a temperature of 100~125℃, preferably 100~121℃, and more preferably 110℃; the sterilization time is 15~35min, preferably 15~25min, and more preferably 20min. Optionally, after sterilization, the fermentation broth is cooled to room temperature and centrifuged. The centrifugation speed is 3000~9000 rpm, preferably 4000~6000 rpm, and more preferably 5000 rpm. The centrifugation time is 20~50 min, preferably 20~40 min, and more preferably 35 min.

[0016] Preferably, after the separation process, a secondary sterilization operation is further included; the secondary sterilization method is preferably high-temperature sterilization; the temperature of the secondary sterilization is 100~125℃, more preferably 100~121℃; the time of the secondary sterilization is 20~40min, more preferably 25~35min; optionally, after the secondary sterilization, a preservative is added to the combined fermentation product, and during the mixing process of the combined fermentation product and the preservative, the mixing temperature is 50~80℃, preferably 70~80℃; the preservative includes p-Hydroxyacetophenone and / or 1,2-hexanediol; when the preservative includes p-hydroxyacetophenone and 1,2-hexanediol, the p-hydroxyacetophenone accounts for 0.5% to 1% of the mass of the material obtained after secondary sterilization, and the 1,2-hexanediol accounts for 0.5% to 1% of the mass of the material obtained after secondary sterilization; preferably, the p-hydroxyacetophenone accounts for 0.5% of the mass of the material obtained after secondary sterilization, and the 1,2-hexanediol accounts for 0.5% of the mass of the material obtained after secondary sterilization.

[0017] According to a second aspect of this application, a combined fermentation product with whitening and antioxidant effects is provided, which is a supernatant obtained by the above method, or a dry powder obtained from the supernatant.

[0018] According to a third aspect of this application, the above-mentioned combined fermentation product is also provided for use in the preparation of cosmetics; preferably, the cosmetics may be facial masks, serums, toners, lotions, etc.

[0019] According to a fourth aspect of this application, a topical skin agent is also provided, comprising the supernatant; preferably, the supernatant accounts for 1% to 50% of the topical skin agent by mass, more preferably 10% to 30%.

[0020] Compared with the prior art, this application has the following advantages: 1. The proposed method in this application adopts microbial fermentation treatment, especially the compound fermentation of three raw materials, namely Polygonum cuspidatum root, grape seed and cassia seed, by Lactobacillus acidophilus. This method can fully release and enrich the active ingredients in the raw materials, such as resveratrol, and improve the resource utilization rate.

[0021] 2. The method proposed in this application adopts a microbial fermentation extraction method, which does not require organic reagents and is environmentally friendly; the resulting product has no chemical reagent residues and has better safety; at the same time, the fermentation conditions are mild, the fermentation cycle is short, and the production cost is low, which is conducive to large-scale production.

[0022] 3. The proposed solution improves the problem of poor efficacy of fermentation broth obtained from single-component fermentation. By using a combination of Polygonum cuspidatum root, grape seed, and cassia seed as a substrate for fermentation, the antioxidant and whitening effects can be better synergistically exerted, thus expanding the application of Polygonum cuspidatum root, grape seed, and cassia seed in the cosmetics field.

[0023] 4. The fermentation product prepared by the method of this application is enriched with more active ingredient resveratrol. Its function is mainly based on its chemical properties (such as the antioxidant properties of polyphenols) and its regulation of skin physiological mechanisms, and it shows outstanding performance in anti-oxidation and improving skin dullness. Attached Figure Description

[0024] This application can be better understood by referring to the description given below in conjunction with the accompanying drawings. These drawings, together with the detailed description below, are included in and form part of this specification, and are used to further illustrate preferred embodiments of the application and explain the principles and advantages of the application. Wherein: Figure 1 The experimental results of DPPH free radical scavenging in Example 1 of this application are shown; Figure 2 The results of the hydroxyl radical scavenging experiment in Example 2 of this application are shown; Figure 3 The results of the tyrosinase inhibition experiment in Example 3 of this application are shown; Figure 4 The results of resveratrol content determination in Example 4 of this application are shown; Figure 5 The results of the human skin fibroblast toxicity experiment of Example 5 of this application are shown; Figure 6 These are photographs of the combined ferments obtained in Examples 1-5 of this application; Figure 7 Photographs of the combined ferments prepared by Comparative Examples 1-8 of this application. Detailed Implementation

[0025] This application provides a method for preparing a combination fermentation product with whitening and antioxidant effects. The method uses Lactobacillus acidophilus to ferment a combination of Polygonum cuspidatum root, grape seed, and cassia seed, which reduces the loss of active ingredients in the combination. The resulting fermentation liquid has good whitening and antioxidant effects. No chemical components are added during the preparation of the fermentation liquid, so it is gentle on the skin and has good safety. It is a pure natural raw material and has a good application prospect in the cosmetics field.

[0026] A specific embodiment of the method for preparing a combined fermentation product with whitening and antioxidant effects provided in this application includes the following steps: First, the initial fermentation system is prepared in proportion and sterilized. The initial fermentation system includes Polygonum cuspidatum root, grape seeds, cassia seeds and deionized water. Secondly, the fermentation bacteria Lactobacillus acidophilus (L. acidophilus) were inoculated into the initial fermentation system. Lactobacillus acidophilus The mixture was subjected to static culture, followed by sterilization and separation. The supernatant was then collected to obtain the combined fermentation product.

[0027] The initial fermentation system of this application includes a powdered composition and deionized water. The powdered composition allows for more complete fermentation. The preferred mass ratio of deionized water to the powdered composition is 30-200:1 (e.g., 50:1, 80:1, 100:1, 120:1, 150:1, 180:1, 200:1, etc.), more preferably 50-150:1, and even more preferably 100:1. In the composition, the preferred mass ratio of Polygonum cuspidatum root, grape seeds, and cassia seeds is 1:1-3:1-3, more preferably 1:1:1. The powdered Polygonum cuspidatum root, grape seeds, and cassia seeds can be obtained by pulverizing and sieving dried Polygonum cuspidatum root, grape seeds, and cassia seeds, preferably with a particle size of 50-150 mesh (e.g., sieve size below 50-150 mesh, such as 150 mesh, 120 mesh, 100 mesh, 75 mesh, below 50 mesh, etc.). Polygonum cuspidatum root, grape seeds, and cassia seeds can be purchased from the market.

[0028] After the initial fermentation system is prepared according to the specified ratio, it is preferable to sterilize the initial fermentation system at high temperature. The sterilization temperature is preferably 100-121℃ (e.g., 105℃, 110℃, 115℃, 118℃, etc.), more preferably 100-110℃, and even more preferably 110℃; the sterilization time is 15-35 min (e.g., 18 min, 20 min, 25 min, 30 min, etc.), preferably 15-25 min, and even more preferably 20 min. Sterilizing the initial fermentation system can prevent the subsequent fermentation process from being affected by other microorganisms.

[0029] After sterilization of the initial fermentation system of this application, it is allowed to cool to room temperature before being inoculated with fermentation bacteria. The preferred fermentation bacteria are *Lactobacillus acidophilus* (…). Lactobacillus acidophilus The *Lactobacillus acidophilus* were all inoculated in the form of a bacterial suspension. The bacterial suspension can be cultured as follows: a single colony of *Lactobacillus acidophilus* is picked up using an inoculation loop and inoculated onto MRS medium, then shaken well and incubated statically in a culture flask at 42°C. The viable count of the *Lactobacillus acidophilus* bacterial suspension is 10-1. 6 ~10 11 CFU / mL, more preferably 10 7 ~10 10 CFU / mL. The fermentation strain was further selected as Lactobacillus acidophilus CICC20250, purchased from the China Industrial Microbial Culture Collection Center.

[0030] In this application, *Lactobacillus acidophilus* is inoculated into the initial fermentation system, and the inoculation amount, calculated by volume fraction, is preferably 0.5% to 1.5% (e.g., 0.5%, 0.8%, 1.0%, 1.2%, 1.5%, etc.). For example, in the bacterial solution used for inoculation in this application, the viable count of the fermenting bacteria is 10. 6 ~10 11 CFU / mL, more preferably 10 7 ~10 10 CFU / mL; if the inoculation amount is 1%, that is, based on the volume of deionized water in the initial fermentation system, the viable count in the initial fermentation system after inoculation is 10-1. 4 ~10 9 CFU / mL, more preferably 10 5 ~10 8 CFU / mL.

[0031] In this application, the fermentation culture is carried out in a constant temperature incubator under static conditions. In this invention, the fermentation temperature is preferably 30~45℃ (e.g., 32℃, 35℃, 38℃, 40℃, 42℃, etc.), more preferably 35~45℃; the fermentation time is 12~48h (e.g., 15h, 20h, 24h, 30h, 36h, 40h, 45h, etc.), more preferably 24~48h.

[0032] This application uses *Lactobacillus acidophilus* for fermentation, which requires mild fermentation conditions and effectively improves the bioactivity of the composition. After fermentation, the fermentation broth is preferably sterilized at high temperature to inactivate the fermenting bacteria and terminate fermentation. The sterilization temperature is preferably 100-125℃, more preferably 100-121℃, and even more preferably 110℃; the sterilization time is 15-35 min, more preferably 15-25 min, and even more preferably 20 min. After sterilization, the fermentation broth is cooled to room temperature and centrifuged to obtain the fermentation product of the composition. The centrifugation speed is 3000-9000 rpm, more preferably 4000-6000 rpm, and even more preferably 5000 rpm; the centrifugation time is 20-50 min, more preferably 20-40 min, and even more preferably 35 min.

[0033] After centrifugation, a secondary sterilization process is further included to prevent the introduction of other microorganisms during the centrifugation process; the secondary sterilization method is preferably high-temperature sterilization; the temperature of the secondary sterilization is preferably 100~125℃, more preferably 100~121℃, and even more preferably 110℃; the time of the secondary sterilization is preferably 20~40min, more preferably 25~35min, and even more preferably 30min.

[0034] After secondary sterilization, a preservative needs to be added to the combined fermentation product for preservation. During mixing with the preservative, the mixing temperature is 50-80℃, preferably 70-80℃. The preservative includes p-hydroxyacetophenone and / or 1,2-hexanediol. When the preservative includes p-hydroxyacetophenone and 1,2-hexanediol, the mass percentage of p-hydroxyacetophenone in the sterilized material is 0.5%-1%, and the mass percentage of 1,2-hexanediol in the sterilized material is 0.5%-1%. Preferably, the mass percentage of p-hydroxyacetophenone in the sterilized material is 0.5%, and the mass percentage of 1,2-hexanediol in the sterilized material is 0.5%. The combined fermentation product can also be further processed into a dry powder for preservation using conventional processes such as freeze-drying or spray drying.

[0035] The combined fermentation product obtained in this application can be used directly as a cosmetic or added to a basic cosmetic formulation. When preparing a topical skin agent, the combined fermentation product preferably accounts for 1% to 50% of the agent's mass, more preferably 10% to 30%.

[0036] Exemplary embodiments of this application will be described below with reference to the accompanying drawings.

[0037] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods.

[0038] Unless otherwise specified, all materials and reagents used in the following examples and comparative examples are commercially available.

[0039] In the following examples and comparative examples, the suppliers of Polygonum cuspidatum root and Cassia tora seed were Anhui Changfu Pharmaceutical Co., Ltd.; the supplier of grape seed was Bozhou Yuanshengtang Pharmaceutical Co., Ltd.; and the Lactobacillus acidophilus, including Lactobacillus acidophilus CICC20250, Lactobacillus acidophilus CICC6083, and other fermentation strains in the comparative examples, were all purchased from the China Industrial Microbial Culture Collection Center.

[0040] Example 1: The dried Polygonum cuspidatum root, grape seeds, and cassia seeds were crushed and passed through a 100-mesh sieve. 300 mL of deionized water was mixed with 1 g of Polygonum cuspidatum root powder, 1 g of grape seed powder, and 1 g of cassia seed powder. The mixture was then sterilized in an autoclave at 110 °C for 20 min. After sterilization, the mixture was cooled to room temperature to obtain the initial fermentation system.

[0041] Lactobacillus acidophilus was inoculated into the initial fermentation system. Lactobacillus acidophilus Fermentation was carried out using CICC20250 bacterial culture, in which the viable count of Lactobacillus acidophilus in the culture was 10. 9 The bacterial culture concentration was 3 mL, and after inoculation, the culture was shaken well and incubated at 42℃ for 24 hours.

[0042] After fermentation, the resulting fermentation product was sterilized in an autoclave at 110°C for 20 minutes. After sterilization, the fermentation product was cooled to room temperature and centrifuged at 5000 rpm for 35 minutes. After centrifugation, the supernatant was sterilized a second time at 110°C for 30 minutes. The resulting fermentation product was then obtained.

[0043] A portion of the fermentation broth was tested (in both the examples and comparative examples of this application, fermentation broth without preservatives was used for testing), and the remaining portion was preserved with preservatives. Specifically, based on the mass of the original fermentation broth, 0.5% p-hydroxyacetophenone and 0.5% 1,2-hexanediol were added and mixed thoroughly at 75°C.

[0044] Example 2: The preparation method is the same as in Example 1, except that the fermentation time is 48 hours, and other conditions and parameters are the same as in Example 1.

[0045] Example 3: The preparation method is the same as in Example 1, except that the fermentation strain is Lactobacillus acidophilus CICC6083, and other conditions and parameters are the same as in Example 1.

[0046] Example 4: The preparation method is the same as in Example 1, except that the fermentation temperature is 30℃, and other conditions and parameters are the same as in Example 1.

[0047] Example 5: The preparation method is the same as in Example 1, except that the fermentation time is 45℃, and other conditions and parameters are the same as in Example 1.

[0048] Comparative Example 1: The preparation method is the same as in Example 1, except that fermentation is not performed. After sterilization, centrifugation is performed directly. Other conditions and parameters are the same as in Example 1.

[0049] Comparative Example 2: The preparation method is the same as in Example 1, except that 0.2g of Polygonum cuspidatum root powder, 1.4g of grape seed powder and 1.4g of cassia seed powder are added respectively, and other conditions and parameters are the same as in Example 1.

[0050] Comparative Example 3: The preparation method is the same as in Example 1, except that brewer's yeast (Saccharomyces cerevisiae) is used. Saccharomyces cerevisiae Fermentation was carried out using CICC32880, with other conditions and parameters the same as in Example 1.

[0051] Comparative Example 4: The preparation method is the same as in Example 1, except that Lactobacillus plantarum ( Lactiplantibacillus plantarum Fermentation was carried out using CICC23180, with other conditions and parameters the same as in Example 1.

[0052] Comparative Example 5: The preparation method is the same as in Example 1, except that Lactobacillus helveticus (…) is used. Lactobacillus helveticus CICC6261 was used for fermentation, and other conditions and parameters were the same as in Example 1.

[0053] Comparative Example 6: The preparation method is the same as in Example 1, except that 0 g of Polygonum cuspidatum root powder is added, and 1.5 g of grape seed powder and cassia seed powder are added respectively. Other conditions and parameters are the same as in Example 1.

[0054] Comparative Example 7: The preparation method is the same as in Example 1, except that 0 g of grape seed powder is added, and 1.5 g each of Polygonum cuspidatum root powder and Cassia seed powder are added. Other conditions and parameters are the same as in Example 1.

[0055] Comparative Example 8: The preparation method is the same as in Example 1, except that 0 g of cassia seed powder is added, and 1.5 g each of grape seed powder and Polygonum cuspidatum root powder are added. Other conditions and parameters are the same as in Example 1.

[0056] The raw material ratios and process conditions for the embodiments and comparative examples of this application are shown in Table 1 below, and photographs of the obtained fermentation broth are shown in [reference needed]. Figure 6 and Figure 7 .

[0057] Table 1. Raw material ratios and process conditions for the examples and comparative examples.

[0058] Example 1: DPPH free radical scavenging experiment DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) is an early synthesized organic free radical often used to evaluate the hydrogen-donating capacity of antioxidants. It is very stable in organic solvents, exhibits a purple color, and has a characteristic absorption peak at 517 nm. When it encounters a free radical scavenger, the lone pair electrons of DPPH are paired, causing it to fade, meaning the absorbance at the maximum absorption wavelength decreases. Therefore, the scavenging effect of a sample on DPPH free radicals can be evaluated by measuring the change in absorbance.

[0059] The test solution was obtained by diluting the fermentation broth prepared in the examples and comparative examples by ten times.

[0060] The specific experimental steps of the DPPH free radical scavenging experiment are as follows: (1) Take an equal volume (1 mL) of the test solution and 2 × 10 -4 Mix the mol / L DPPH solution thoroughly (tube A1). (2) Take an equal volume (1 mL) of anhydrous ethanol (the solvent for the analyte) and 2 × 10 -4 Mix the mol / L DPPH solution thoroughly (tube A2). (3) Take an equal volume (1 mL) of anhydrous ethanol and mix it with the test solution (A3 tube). (4) After the reaction in the dark for 30 minutes, the absorbance values ​​of tubes A1, A2 and A3 were measured at 517 nm. The formula for calculating the clearance rate is: clearance rate = [(A2+A3)-A1] / A2×100%.

[0061] The results are shown in Table 2 and Figure 1 It can be seen that the DPPH free radical scavenging rate of the products prepared in Examples 1-5 is much higher than that of the products prepared in Comparative Examples 1-8, indicating that the products prepared in the examples of this application have a more ideal antioxidant effect.

[0062] Table 2. Experimental results of DPPH free radical scavenging

[0063] Example 2: Hydroxyl radical scavenging experiment Hydroxyl radicals (·OH) are generated using the Fenton reaction. Salicylic acid is added to the reaction system, and the ·OH reacts with the salicylic acid to form a colored compound, 2,3-dihydroxybenzoic acid, which exhibits characteristic absorption at 510 nm. Using a fixed reaction time method, the absorbance of the reaction solution containing the analyte is measured at 510 nm and compared with a blank solution to determine the analyte's scavenging effect on ·OH.

[0064] Add reagents to the test tube according to Table 3, including 9 mmol / L FeSO4, 9 mmol / L salicylic acid ethanol solution, the test solution (diluted four times with the fermentation broth prepared in the above examples and comparative examples), an appropriate amount of deionized water, and 8.8 mmol / L H2O2 solution. Shake well, heat in a 37°C water bath for 15 min, and then measure the absorbance A0 of the blank control and the absorbance A of the sample group. x Background absorbance A of the solution without added H2O2 x0 When determining A0, the reference solution was a system without hydrogen peroxide. Three parallel experiments were performed for each group, and the absorbance values ​​were measured and averaged. The hydroxyl radical scavenging rate for each group was calculated using the following formula: Hydroxyl radical scavenging rate = (A0 - (A...)) / (A...) x -A x0 )) / A0×100%.

[0065] Table 3. Reagent dosage for each group in the hydroxyl radical scavenging experiment.

[0066] Table 4. Results of hydroxyl radical scavenging experiments

[0067] The results are shown in Table 4 and Figure 2 It can be seen that the hydroxyl radical scavenging rates of the products prepared in Examples 1-5 are much higher than those of the products prepared in Comparative Examples 1-8, indicating that the products prepared in the examples of this application have relatively ideal antioxidant effects.

[0068] Example 3: Tyrosinase Inhibition Experiment 1. Experimental instruments and reagents Experimental instruments: microplate reader; analytical balance; volumetric flasks (10mL, 50mL, 100mL, 250mL); digital display constant temperature water bath; pipette; beaker; pH meter; graduated cylinders; EP tubes.

[0069] Experimental reagents: disodium hydrogen phosphate dodecahydrate (AR); sodium dihydrogen phosphate (AR); L-tyrosine; tyrosinase; hydrochloric acid, etc.

[0070] 2. Reagent preparation (1) Buffer preparation: Preparation of PBS (pH=6.5): Weigh 3.5g of disodium hydrogen phosphate dodecahydrate and 1.56g of sodium dihydrogen phosphate dihydrate, add deionized water to make up to 200mL, measure the pH value with a pH meter, and adjust the pH value by adding a small amount of sodium dihydrogen phosphate dihydrate; Preparation of PBS (pH=6.8): Weigh 3.58g of disodium hydrogen phosphate dodecahydrate and 1.56g of sodium dihydrogen phosphate dihydrate, add deionized water to make up to 200mL, measure the pH value with a pH meter, and adjust the pH value by adding a small amount of sodium dihydrogen phosphate dihydrate. (2) Preparation of tyrosinase solution (activity 80-200 U / ml): Dissolve 50 mg of tyrosinase in 250 mL (pH=6.5) PBS buffer and shake well. This operation should be performed on ice. (3) Preparation of 0.1% hydrochloric acid solution: Take 0.865 mL of 36% hydrochloric acid solution, dilute to 100 mL of deionized water in a volumetric flask, and mix well; (4) Preparation of 0.05% L-tyrosine solution: Weigh 50.7 mg of L-tyrosine, dissolve it in 35 mL of 0.1 mol / L hydrochloric acid solution, then add 65 mL of PBS (pH=6.8) buffer and mix well; (5) Preparation of test samples: Take 100µl of the test sample (fermentation broth obtained in the examples and comparative examples of this application) and dilute it with PBS (pH=6.8) to 2, 4, 8, 16 times to prepare 5 gradient concentrations of 0 (original solution), 2, 4, 8, 16 times, and determine its ability to inhibit tyrosinase activity respectively. (6) Blank group: PBS buffer with pH=6.8.

[0071] Each sample was tested in triplicate.

[0072] 3. Determination of tyrosinase activity The tyrosinase activity was determined according to the steps shown in Table 5 below. The prepared reaction solution was quickly transferred to an ELISA plate, and the absorbance of each solution was measured at 475 nm and recorded. Substituting these values ​​into the formula for calculating the tyrosinase activity inhibition rate (inhibition rate (%) = [(BD) - (AC)] / (BD)%), the inhibition rate of the extract on tyrosinase activity at a given concentration can be obtained.

[0073] Table 5. Procedures and conditions for determining tyrosinase activity.

[0074] Table 6 Results of Tyrosinase Inhibition Experiment

[0075] The results are shown in Table 6 and Figure 3It can be seen that the tyrosinase inhibition rates of the products prepared in Examples 1-5 are all much higher than those of the products prepared in Comparative Examples 1-8. Therefore, the products prepared in the examples of this application have excellent whitening effects.

[0076] Example 4: Determination of Resveratrol Content The determination was performed according to the method in DB35 / T1514-2015 "Determination of Resveratrol in Agricultural Products by High Performance Liquid Chromatography".

[0077] Chromatographic conditions: A Shim-pack GIST C(18) column (4.6 mm × 250 mm, 5 μm) was used; mobile phase A was 0.1% acetic acid solution, and mobile phase B was acetonitrile, with a ratio of A:B = 74:26; flow rate: 1 mL / min. -1 The detection wavelength was 306 nm, the injection volume was 10 μL, the column temperature was 40℃, and the time was set to 35 min.

[0078] Accurately weigh 5 mg of resveratrol standard reference, dissolve it in methanol and dilute to 10 ml to prepare a standard solution with a concentration of 500 μg / mL. Dilute the standard solutions with methanol to a series of concentrations of 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL and 10 μg / mL, filter through a 0.45 μm filter membrane and perform HPLC analysis to plot a standard curve.

[0079] The samples (fermentation broths obtained in the examples and comparative examples of this application) were prepared into a 50% concentration test solution with 85% ethanol, filtered through a 0.45 μm filter membrane, and then subjected to HPLC analysis.

[0080] The resveratrol content in the fermentation supernatant is shown in Table 7 and 8. Figure 4 It can be seen that the resveratrol content in the products obtained in Examples 1-5 is much higher than that in the products obtained in Comparative Examples 1-8, indicating that the resveratrol content extracted from the products obtained in the examples of this application is higher.

[0081] Table 7 Results of resveratrol content determination

[0082] Example 5: Human skin fibroblast toxicity experiment This experiment used human skin fibroblasts (from the Chinese Scientific Cell Bank) to verify the cytotoxicity of the products prepared in the above examples and comparative examples.

[0083] Reagents: 0.25% (EDTA-containing) trypsin was manufactured by GIBCO, USA; DMEM medium was manufactured by GIBCO, USA; penicillin-dextrose antibody was manufactured by Corning, USA; CCK-8 was manufactured by Beijing Bairui Biotechnology Co., Ltd.; fetal bovine serum was manufactured by GIBCO, USA; phosphate buffer was manufactured by Beijing Bairui Biotechnology Co., Ltd.

[0084] Equipment: The WJ-80A-Ⅱ CO2 constant temperature incubator is manufactured by Shanghai Shengke Instrument Equipment Co., Ltd.; the Sunrise microplate reader is manufactured by Diken Trading Co., Ltd.; the TL80-2 medical centrifuge is manufactured by Jiangsu Tianli Medical Equipment Co., Ltd.; and the NUNC 96-well cell culture plate is manufactured by Thermo Fisher Scientific.

[0085] Experimental Procedure: The products (fermentation broth) obtained in the above examples and comparative examples were prepared into experimental test solutions with a volume percentage of 1% using serum-free DMEM medium. Human skin fibroblasts were cultured in a solution containing 10% fetal bovine serum and 1% penicillin-streptomycin (1×10⁻⁶) antibiotics. 5 Cells were cultured in DMEM medium containing 100 mg / L penicillin and 100 mg / L streptomycin. Cells were grown in a 37°C, 5% CO2 saturated humidity incubator. When cell confluence reached 85% or higher, logarithmic growth phase cells were digested with 0.05% trypsin, and the digestion was terminated with serum-containing DMEM. Cells were counted using a cell counting chamber, and the cell suspension concentration was adjusted to 7 × 10⁻⁶ cells / mL. 4 Cells were seeded at a rate of 100 μL per well in a 96-well plate and incubated at 37°C and 5% CO2 for 12 h. The old culture medium was removed, and the cells were washed twice with phosphate-buffered saline (PBS). In the experimental groups, 100 μL of the filtered and sterilized test solution prepared above was added to each well, with six replicates per test solution. The control group contained cells and was inoculated with serum-free DMEM medium; the blank control group contained no cells and was inoculated with 100 μL of PBS. The cells were then incubated at 37°C and 5% CO2 for 24 h. Then, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for another 3 h. The absorbance was measured at 450 nm, and the cell viability was calculated. The cell viability was calculated using the following formula: Cell viability (%) = (A experimental group - A blank control group) / (A control group - A blank control group) × 100%.

[0086] Table 8 Results of human skin fibroblast toxicity test

[0087] The results are shown in Table 8 and Figure 5It can be seen that the cell viability of the products prepared in Examples 1 to 5 is significantly higher than that of the products prepared in Comparative Examples 1 to 8, indicating that the products prepared in this application are safer.

[0088] Finally, it should be noted that in this application, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0089] Although this application has been disclosed above through the description of specific embodiments, it should be understood that those skilled in the art can devise various modifications, improvements, or equivalents to this application within the spirit and scope of the appended solutions. Such modifications, improvements, or equivalents should also be considered to be included within the scope of protection claimed in this application.

Claims

1. A method for preparing a combined fermentation product with whitening and antioxidant effects, characterized in that, include: Step 1: Prepare the initial fermentation system according to the specified ratio and sterilize it. The initial fermentation system includes Polygonum cuspidatum root, grape seeds, cassia seed powder and deionized water. Step 2: Inoculate the initial fermentation system with the fermenting bacterium Lactobacillus acidophilus (Lactobacillus acidophilus). Lactobacillus acidophilus The mixture was subjected to static culture, followed by sterilization and separation. The supernatant was then collected to obtain the combined fermentation product.

2. The method for preparing the combined fermentation product according to claim 1, characterized in that, In step one, the mass ratio of Polygonum cuspidatum root, grape seed, and cassia seed in the initial fermentation system is 1:1~3:1~3, more preferably 1:1:1; Preferably, the particle size of the powders of Polygonum cuspidatum root, grape seed, and cassia seed is 50-150 mesh; Preferably, in the initial fermentation system, the mass ratio of deionized water to the composition powder is 30~200:1, more preferably 50~150:1, and even more preferably 100:

1.

3. The method for preparing the combined fermentation product according to claim 1 or 2, characterized in that, In step one, after the initial fermentation system is prepared in proportion, it is sterilized at high temperature; wherein, the sterilization temperature is preferably 100~121℃, more preferably 100~110℃, and even more preferably 110℃; the sterilization time is 15~35min, more preferably 15~25min, and even more preferably 20min.

4. The method for preparing the combined fermentation product according to any one of claims 1-3, characterized in that, In step two, the Lactobacillus acidophilus is Lactobacillus acidophilus CICC20250 or Lactobacillus acidophilus CICC6083.

5. The method for preparing the combined fermentation product according to claim 4, characterized in that, In step two, the Lactobacillus acidophilus were all inoculated in the form of bacterial suspension; Preferably, the bacterial culture is cultured as follows: a single colony of *Lactobacillus acidophilus* is picked up using an inoculation loop and inoculated onto MRS medium, then shaken well and incubated statically in a culture flask at 42°C. The viable count of the *Lactobacillus acidophilus* culture is 10-1. 6 ~10 11 CFU / mL, more preferably 10 7 ~10 10 CFU / mL; Preferably, the initial fermentation system is inoculated with Lactobacillus acidophilus culture, and the inoculation amount is preferably 0.5% to 1.5% by volume.

6. The method for preparing the combined fermentation product according to any one of claims 1-5, characterized in that, In step two, the fermentation temperature is 30~45℃, preferably 35~45℃; the fermentation time is 12~48h, preferably 24~48h.

7. The method for preparing the combined fermentation product according to any one of claims 1-6, characterized in that, In step two, after fermentation, high-temperature sterilization is carried out. The sterilization temperature is 100~125℃, preferably 100~121℃, and more preferably 110℃; the sterilization time is 15~35min, preferably 15~25min, and more preferably 20min. Optionally, after sterilization, the fermentation broth is cooled to room temperature and centrifuged. The centrifugation speed is 3000~9000 rpm, preferably 4000~6000 rpm, and more preferably 5000 rpm. The centrifugation time is 20~50 min, preferably 20~40 min, and more preferably 35 min. Preferably, after the separation process, a secondary sterilization operation is further included; the secondary sterilization method is preferably high-temperature sterilization; the temperature of the secondary sterilization is 100~125℃, more preferably 100~121℃; the time of the secondary sterilization is 20~40min, more preferably 25~35min. Optionally, after the secondary sterilization, a preservative is added to the combined fermentation product. During the mixing process of the combined fermentation product and the preservative, the mixing temperature is 50~80℃, preferably 70~80℃. The preservative includes p-hydroxyacetophenone and / or 1,2-hexanediol. When the preservative includes p-hydroxyacetophenone and 1,2-hexanediol, the mass percentage of p-hydroxyacetophenone in the material obtained after secondary sterilization is 0.5%~1%, and the mass percentage of 1,2-hexanediol in the material obtained after secondary sterilization is 0.5%~1%. Preferably, the mass percentage of p-hydroxyacetophenone in the material obtained after secondary sterilization is 0.5%, and the mass percentage of 1,2-hexanediol in the material obtained after secondary sterilization is 0.5%.

8. A combined fermentation product with soothing and antioxidant effects, which is a supernatant prepared by any one of the preparation methods of claims 1-7, or a dry powder prepared from the supernatant.

9. The application of the combined fermentation product as described in claim 8 in the preparation of cosmetics; preferably, the cosmetic is any one of a face mask, serum, toner, or lotion.

10. A topical skin agent, characterized in that, The topical skin agent comprises the supernatant as described in claim 8; preferably, the supernatant accounts for 1% to 50% of the mass percentage of the topical skin agent, more preferably 10% to 30%.

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