Preparation method of bifidobacterium-three flower wrinkle-removing fermentation liquor and skin-care fermentation liquor

Abstract

The invention relates to a preparation method of fermentation liquor of bifidobacterium-three-flower wrinkle-removing liquor and skin-care fermentation liquor. The method comprises the following steps: 1, activating a bifidobacterium strain; 2, preparing a seed solution; 3, bidirectional fermentation of the bifidobacterium-'three-flower wrinkle removal liquid '; according to the method, bifidobacterium adolescentis and the three-flower wrinkle removal liquid are subjected to bidirectional fermentation, so that the content and bioavailability of active ingredients in the three-flower wrinkle removal liquid are improved, and the skin care effects of oxidation resistance, whitening, inflammation resistance, aging resistance and the like of the three-flower wrinkle removal liquid are enhanced. The method is simple in technological process, high in controllability and high in production efficiency, and the prepared skin care fermentation liquor has the advantages of being safe, efficient and multifunctional and can be widely applied to the field of skin care products.

Description

Technical Field

[0001] The present invention relates to the technical field of preparation of skin care products, and more particularly to a preparation method of a Bifidobacterium-Trifolium wrinkle-removing fermentation liquid and the skin care fermentation liquid. Background Art

[0002] With the improvement of people's living standards and the change of aesthetic concepts, consumers have higher requirements for the efficacy and safety of skin care products. Traditional skin care products mostly use chemical synthetic ingredients. Although they have good efficacy, long-term use may cause irritation and damage to the skin, and even trigger adverse reactions such as allergies.

[0003] In recent years, with increasing interest in natural and safe skincare, Chinese herbal skincare products have become a research hotspot. Chinese herbal medicines are rich in bioactive ingredients, such as polyphenols, flavonoids, and saponins, offering antioxidant, whitening, anti-inflammatory, and anti-aging benefits. They are also highly safe and have minimal side effects. However, traditional Chinese herbal extraction methods suffer from low efficiency and significant loss of active ingredients, resulting in ineffective products.

[0004] "Three Flowers Wrinkle-Removing Liquid" is a traditional Chinese medicine formula made with peach, lotus, and hibiscus flowers. It promotes blood circulation, dissipates blood stasis, unclogs meridians, and improves complexion and wrinkle removal. Modern research shows that peach, lotus, and hibiscus flowers are rich in bioactive ingredients, such as polyphenols and flavonoids, which have antioxidant, whitening, anti-inflammatory, and anti-aging properties.

[0005] Bifidobacterium adolescentis is a probiotic that has antioxidant, whitening, and anti-inflammatory functions and can be used to improve skin health.

[0006] However, there are few reports on combining "Sanhua Wrinkle-Removing Liquid" with Bifidobacterium adolescentis to prepare skincare products, and there is a lack of systematic research. Therefore, the present invention aims to provide a method for preparing a fermentation broth of Bifidobacterium-Sanhua Wrinkle-Removing Liquid and a skincare fermentation broth, addressing the single efficacy and insufficient safety of existing skincare products and providing a skincare product with multiple benefits, safety, stability, and process controllability. Summary of the Invention

[0007] The present invention aims to provide a method for preparing a bifidobacterium-Sanhua wrinkle-removing liquid fermentation liquid and a skin care fermentation liquid. By combining traditional Chinese medicine ingredients with modern fermentation technology, bifidobacterium adolescentis and "Sanhua wrinkle-removing liquid" are subjected to bidirectional fermentation, and the enzyme system of microorganisms is used to structurally transform and modify the Chinese medicine ingredients to increase the content of active ingredients in the Chinese medicine, reduce toxicity, and enhance its biological activity, thereby solving the problems of single efficacy and insufficient safety of existing skin care products, and providing a new skin care fermentation liquid with multiple efficacy, safety, stability, and controllable process.

[0008] On the one hand, the technical solution adopted by the present invention to solve the technical problem is: a method for preparing a Bifidobacterium-Triphyllum wrinkle-removing liquid fermentation liquid, wherein the preparation method comprises the following steps:

[0009] Step 1: Inoculate Bifidobacterium adolescentis strains into a culture medium containing soy peptone, tryptone, yeast extract powder, glucose, L-cysteine, saline solution and deionized water, and culture under an anaerobic environment to activate the Bifidobacterium strains;

[0010] Step 2: Inoculate the activated bacteria in step 1, take a portion of the colony with an inoculating loop and inoculate it into a conical flask, and cultivate it under anaerobic conditions to prepare a seed solution;

[0011] Step 3: Mix the seed liquid prepared in step 2 with Sanhua wrinkle-removing liquid powder in a certain proportion, and carry out bi-directional fermentation of bifidobacterium and "Sanhua wrinkle-removing liquid" to form bifidobacterium-Sanhua wrinkle-removing liquid fermentation liquid.

[0012] The preparation method of the present invention, wherein, in step 2, the ratio of the soy peptone, the tryptone, the yeast extract powder, the glucose, the L-cysteine ​​and the saline solution is 1:1:2:2:0.1:8.

[0013] The preparation method of the present invention, wherein, in step 3, the fermentation conditions for the bifidobacterium-"Sanhua wrinkle removal liquid" bidirectional fermentation in step 3 are:

[0014] The mixture of Bifidobacterium and Sanhua wrinkle-removing liquid was sterilized in an autoclave at a high temperature of 121° C. for 20 minutes. The inoculation amount of the mixture was 2%, the initial pH was 5.7, and the mixture was anaerobically cultured at 37° C. for 24 hours.

[0015] The preparation method of the present invention, wherein the preparation method of the Sanhua wrinkle-removing liquid powder is as follows:

[0016] Peach blossoms, lotus flowers and hibiscus flowers are dried, crushed and sieved respectively, and then the three are evenly mixed in a certain proportion to obtain three-flower wrinkle-removing liquid powder.

[0017] On the other hand, the present invention also provides a skin care fermentation liquid, wherein the skin care fermentation liquid is produced by any of the above-mentioned preparation methods of the Bifidobacterium-Trifolium wrinkle-removing liquid fermentation liquid.

[0018] The beneficial effects of the present invention are as follows: the preparation method of the Bifidobacterium-Sanhua wrinkle-removing liquid fermentation liquid and the skin care fermentation liquid are simple. Through this method, the metabolites of Bifidobacterium adolescentis can synergize with the active ingredients in the "Sanhua wrinkle-removing liquid", such as polyphenols and flavonoids, thereby significantly improving the skin care effect. This method not only improves the bioavailability of the active ingredients in the "Sanhua wrinkle-removing liquid", but also reduces the toxicity of the skin care fermentation liquid, enhancing its multiple benefits such as antioxidant, whitening, anti-inflammatory, and anti-aging. In addition, this method has a simple process flow, strong controllability, and high production efficiency. The prepared skin care fermentation liquid is safe, efficient, and multifunctional, and can be widely used in the field of skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the present invention will be further described below with reference to the accompanying drawings and embodiments. The drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive efforts.

[0020] Figure 1 The present invention is a flowchart of a method for preparing a fermentation broth of Bifidobacterium-Trifolium wrinkle-removing liquid in Example 1.

[0021] Figure 2 This is a comparison chart of the DPPH clearance rates of different samples in Example 1 of the present invention.

[0022] Figure 3 This is a graph showing the DPPH clearance rate at different BTBF concentrations in Example 1 of the present invention.

[0023] Figure 4 This is a comparison chart of the ABTS clearance rates of different samples in Example 1 of the present invention.

[0024] Figure 5 This is a graph showing the effect of different BTBF concentrations on ABTS clearance in Example 1 of the present invention.

[0025] Figure 6 This is a standard curve diagram of FeSO4 in Example 1 of the present invention.

[0026] Figure 7 is the Fe of different samples in Example 1 of the present invention 2+ Comparison chart of equivalents.

[0027] Figure 8 The Fe content of BTBF of different concentrations in Example 1 of the present invention is 2+ Equivalent curve diagram.

[0028] Figure 9This is a comparison chart of the inhibition rates of different samples on AAPH-induced erythrocyte hemolysis in Example 1 of the present invention.

[0029] Figure 10 This is a comparison chart of the AAPH-induced hemolysis of red blood cells in different samples in Example 1 of the present invention.

[0030] Figure 11 This is a curve diagram showing the inhibition rate of AAPH-induced erythrocyte hemolysis at different concentrations of BTBF in Example 1 of the present invention.

[0031] Figure 12 This is a comparison chart of the inhibition rates of BTBF at different concentrations on AAPH-induced erythrocyte hemolysis in Example 1 of the present invention.

[0032] Figure 13 This is a comparison chart of the red blood cell states observed under an optical microscope in Example 1 of the present invention.

[0033] Figure 14 This is a comparison chart showing the effects of different samples on AAPH-induced ROS generation in erythrocytes in Example 1 of the present invention.

[0034] Figure 15 This is a graph showing the effects of different concentrations of BTBF on AAPH-induced ROS generation in erythrocytes in Example 1 of the present invention.

[0035] Figure 16 This is a comparison chart showing the effects of different samples on the tyrosinase inhibition rate in Example 1 of the present invention.

[0036] Figure 17 This is a graph showing the effects of different concentrations of BTBF on tyrosinase inhibition in Example 1 of the present invention.

[0037] Figure 18 This is a comparison chart showing the effects of different samples on the inhibition rate of elastase in Example 1 of the present invention.

[0038] Figure 19 This is a curve diagram showing the effects of different concentrations of BTBF on the inhibition rate of elastase in Example 1 of the present invention.

[0039] Figure 20 This is a comparison chart showing the effects of different samples on the hyaluronidase inhibition rate in Example 1 of the present invention.

[0040] Figure 21 This is a comparison chart showing the effects of different samples on the AGEs inhibition rate in Example 1 of the present invention.

[0041] Figure 22 This is a curve diagram showing the effects of different concentrations of BTBF on the AGEs inhibition rate in Example 1 of the present invention.

[0042] Figure 23 This is a comparison chart showing the effects of different samples on the α-glucosidase inhibition rate in Example 1 of the present invention.

[0043] Figure 24 This is a curve diagram showing the effects of different concentrations of BTBF on the α-glucosidase inhibition rate in Example 1 of the present invention.

[0044] Figure 25 This is a graph showing wavelength scanning of different samples in Example 1 of the present invention.

[0045] Figure 26 This is a comparison chart of the average ultraviolet absorption rate (%) of the samples in Example 1 of the present invention in the ultraviolet light region.

[0046] Figure 27 This is a curve diagram of wavelength scanning of different BTBF concentrations in Example 1 of the present invention. DETAILED DESCRIPTION

[0047] The terms "first," "second," "third," and "fourth," etc., in the specification, claims, and accompanying drawings of the present invention are used to distinguish between different items, not to describe a specific order. Furthermore, the terms "including," "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, system, product, or apparatus comprising a series of steps or elements is not limited to the listed steps or elements, but may optionally include steps or elements not listed, or may optionally include other steps or elements inherent to the process, method, product, or apparatus.

[0048] References herein to "embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments may be included in at least one embodiment of the present invention. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it constitute a separate or alternative embodiment that is mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0049] "Multiple" refers to two or more. "And / or" describes the relationship between associated objects, indicating that three possible relationships exist. For example, "A and / or B" can mean: A exists alone, A and B exist simultaneously, or B exists alone. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0050] Moreover, the terms "up, down, front, back, left, right, upper end, lower end, longitudinal" and the like indicating directions are all based on the posture and position of the device or apparatus described in this solution during normal use.

[0051] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the following will be a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work shall fall within the scope of protection of the present invention.

[0052] Bidirectional fermentation technology, first proposed by Zhuang Yi, involves Chinese medicinal herbs providing nutrients to microorganisms, while the enzymes produced by these microorganisms catalyze the structural transformation of the herbs' natural components. Microbial enzymes can modify and transform the active ingredients in these herbs, enhancing their effectiveness. For potentially toxic or irritating herbs, microbial fermentation can decompose or transform these toxic and irritating components, thereby reducing or eliminating adverse reactions.

[0053] "Three Flower Wrinkle Remover" is recorded in the "Compendium of Secret Prescriptions," and its composition is "peach blossoms, lotus flowers, and hibiscus flowers in any quantity." This formula has the effects of promoting blood circulation, dispersing blood stasis, unblocking meridians, and rejuvenating the complexion and removing wrinkles.

[0054] Peach blossoms (Prunus persica) are the buds of the peach tree, a member of the Rosaceae family. The Shennong Bencao Jing (Classic of Materia Medica) records that peach blossoms have the effect of improving complexion. Traditional Chinese Medicine believes that peach blossoms, with their mild and bitter nature, have benefits such as promoting blood circulation, soothing the complexion, and moisturizing the skin. Modern research indicates that peach blossoms possess antioxidant, whitening, anti-inflammatory, and antibacterial properties. Lotus (Nelumbonucifera), also known as lotus, is a medicinal and edible plant. Lotus anthocyanins and extracts have excellent scavenging properties against DPPH, ABTS, and hydroxyl free radicals. Both aqueous and alcoholic extracts of lotus flowers can inhibit lipid peroxidation products, advanced oxidation protein products, and glycation end products, demonstrating their excellent antioxidant properties. Hibiscus (Hibiscus rosea) is the flower of the Hibiscus mutabilis. Its chemical components primarily include flavonoids, organic acids, stigmasterols, anthraquinones, coumarins, and volatile components. The total flavonoids in Hibiscus mutabilis flowers can scavenge hydroxyl free radicals and superoxide anion free radicals.

[0055] Raw material preparation

[0056] Chinese medicinal materials: peach blossoms, lotus flowers, hibiscus flowers (dried or fresh, the proportion can be adjusted according to actual needs).

[0057] Bacterial species: Bifidobacterium adolescentis.

[0058] Example 1:

[0059] The first embodiment of the present invention provides a method for preparing a fermentation broth of Bifidobacterium-Sanhua wrinkle-removing liquid, such as Figure 1As shown, the preparation method comprises the following steps:

[0060] Step 1: Under sterile conditions, inoculate Bifidobacterium adolescentis strains into a culture medium containing soy peptone, tryptone, yeast extract powder, glucose, L-cysteine, saline solution and deionized water, and culture at 37°C under anaerobic conditions for 72 hours to activate the Bifidobacterium strains;

[0061] The ratio of the soy peptone, the tryptone, the yeast extract powder, the glucose, the L-cysteine, and the saline solution is 1:1:2:2:0.1:8. For example, in this embodiment, 5 g of soy peptone, 5 g of tryptone, 10 g of yeast extract powder, 10.1 g of glucose, 0.5 g of L-cysteine, 40 mL of saline solution, and 1 L of deionized water are used. It is worth noting that the actual amount of each component used may differ slightly from the above ratio, for example, ±0.1.

[0062] Step 2: Inoculate the activated bacteria in step 1, take a portion of the colony with an inoculating loop and inoculate it into a conical flask, and culture it at 37°C under anaerobic conditions for 48 hours to prepare a seed solution;

[0063] Step 3: Mix the seed solution prepared in Step 2 with a mixture containing 1.2g / L of Sanhua Wrinkle-Removing Liquid powder in a specific ratio (the specific ratio can be flexibly adjusted based on the actual dosage and potency) to perform a bidirectional fermentation of Bifidobacterium and Sanhua Wrinkle-Removing Liquid to form a Bifidobacterium-Sanhua Wrinkle-Removing Liquid fermentation broth (BTBF). During the fermentation process, regularly monitor the pH value, colony count, and fermentation product formation.

[0064] Depending on the equipment conditions, low-speed stirring (100-150rpm) can be used to maintain a suitable anaerobic environment and promote bacterial growth and metabolism.

[0065] Step 4: After fermentation is complete, the fermentation broth is centrifuged to remove the bacteria and unfermented Chinese medicinal material residues. The supernatant is filtered and sterilized to obtain the final fermentation broth product.

[0066] Furthermore, the fermentation conditions for the bifidobacterium-"Sanhua wrinkle removal liquid" bidirectional fermentation in step 3 are:

[0067] The mixture of Bifidobacterium and Sanhua wrinkle-removing liquid was sterilized in an autoclave at a high temperature of 121° C. for 20 minutes. The inoculation amount of the mixture was 2%, the initial pH was 5.7, and the mixture was anaerobically cultured at 37° C. for 24 hours.

[0068] Furthermore, the preparation method of the three-flower wrinkle-removing liquid powder is as follows: peach blossoms, lotus flowers, and hibiscus flowers are dried and crushed separately, sieved (for example, 80-mesh sieve, or other mesh sizes, which can be selected according to actual conditions), and then the three are evenly mixed in a certain proportion to obtain the three-flower wrinkle-removing liquid powder.

[0069] In this example, in order to test the various functions of the prepared Bifidobacterium-Sanhua wrinkle removal liquid fermentation liquid (BTBF), three control groups were prepared: Bifidobacterium fermentation liquid (BFL), "Sanhua wrinkle removal liquid" blank culture medium (TBC), and culture medium blank control (MBC) for comparative analysis of efficacy experiments;

[0070] Among them, Bifidobacterium fermentation broth (BFL): fermentation was carried out using the same fermentation medium, treatment conditions, and culture conditions, but without adding "Sanhua Wrinkle Remover Liquid";

[0071] "Sanhua Wrinkle-Removing Liquid" blank culture medium (TBC): Add 1.2 g / L Sanhua Wrinkle-Removing Liquid powder to the same fermentation medium. The same treatment and culture conditions were used, but without the inoculation of Bifidobacterium seed liquid.

[0072] Medium blank control (MBC): The same medium, treatment conditions, and culture conditions were used, but no "Sanhua Wrinkle Removal Liquid" was added, and no Bifidobacterium seed liquid was inoculated.

[0073] 1. The experimental method for testing the following seven major effects of Sanhua Wrinkle-Removing Liquid - Bifidobacterium adolescentis fermented liquid is as follows:

[0074] 1. Efficacy 1: Antioxidant activity analysis

[0075] 1.1 The experimental method for detecting DPPH scavenging ability is as follows:

[0076] Experimental group (As): 2 mL sample solution and 2 mL DPPH solution; sample blank group (Ar): 2 mL sample solution and 2 mL solvent; solvent blank group (A0): 2 mL solvent and 2 mL DPPH solution; react at room temperature in the dark for 30 min and measure the absorbance (wavelength 517 nm), using Vc as the positive control.

[0077] DPPH scavenging rate (%) = [(A0 + Ar) - As] / A0 * 100% (1)

[0078] Where A0 represents the absorbance value of the solvent blank group; Ar represents the absorbance value of the sample blank group; As represents the absorbance value of the experimental group.

[0079] 1.2 The experimental method for detecting the free radical scavenging ability of ABTS is as follows:

[0080] ABTS and K₂S₂O₄ were prepared in deionized water to 7 mM and 2.45 mM aqueous solutions, respectively. Equal volumes were mixed and allowed to react in the dark for 14 hours. This was the ABTS experimental stock solution. Prior to the experiment, the stock solution was diluted to an absorbance of 0.7 ± 0.02 at 734 nm. This was the ABTS working solution. The experiment was divided into experimental group (A): 0.7 mL sample solution + 2.8 mL ABTS working solution; blank group (A0): 0.7 mL deionized water + 2.8 mL ABTS working solution. Vc was used as a positive control. After reacting in the dark for 30 minutes at room temperature, the absorbance was measured at 734 nm.

[0081] ABTS free radical scavenging rate (%) = [(A0-A) / A0]*100% (2)

[0082] Where A0 represents the absorbance value of the blank group; A represents the absorbance value of the experimental group.

[0083] 1.3 The experimental method of ferric reducing ability (FRAP) detection is as follows:

[0084] Take 105 μL of ferric sulfite at various concentrations, add 3.15 mL of FRAP working solution, then 315 μL of distilled water, mix thoroughly, and use water as a blank control. Incubate at 37°C in the dark for 15 minutes. Measure the absorbance at 593 nm to construct a standard curve. Dilute the sample to a 5% volume fraction and perform the same procedure. The antioxidant capacity is expressed as the concentration of the ferric sulfite solution.

[0085] 1.4 The experimental method for the effects of AAPH-induced erythrocyte hemolysis and cell morphology is as follows:

[0086] 10 mL of sterile sheep blood containing the anticoagulant sodium heparin was placed in a 50 mL centrifuge tube. 20 mL of PBS buffer (pH 7.4) (3.63 g Na2HPO4·12H2O, 0.24 g KH2PO4, 8 g NaCl, 0.2 g KCl, dissolved in 1 L deionized water, sterilized at 121°C for 20 min, and refrigerated at 4°C) was added. The tube was centrifuged at 2600 × g for 10 min. The supernatant and buffy coat were aspirated with a pipette, and the red blood cells were resuspended in 20 mL of PBS buffer. This process was repeated three to four times until the supernatant was clear. Four volumes of PBS buffer were added to the red blood cell volume to prepare a 20% red blood cell suspension for later use. The sample was diluted with PBS buffer and set aside. AAPH was prepared in deionized water to a 200 mM aqueous solution. The experiment was divided into: A: PBS blank control group; B: sample protection group; C: AAPH oxidative damage group; D: complete hemolysis control group. The experimental steps are shown in Table 1.

[0087] Table 1 Experimental operation of inhibition rate of sample hemolysis

[0088]

[0089] After the reaction, 3.2 mL of PBS buffer was added to all experimental groups, and the mixture was centrifuged at 2600 × g for 5 min. The supernatant was aspirated and the absorbance at 540 nm was measured, which were Aa, Ab, Ac, and Ad, respectively. The hemolysis inhibition rate of each experimental group was calculated according to formulas (3), (4), and (5).

[0090] Hemolysis inhibition rate of PBS blank control group = (Ad-Aa) / Ad*100% (3)

[0091] Hemolysis inhibition rate of sample protection group = (Ad-Ab) / Ad*100% (4)

[0092] Hemolysis inhibition rate of AAPH oxidative damage group = (Ad-Ac) / Ad*100% (5)

[0093] Wherein, Aa is the absorbance value of the control group; Ab is the absorbance value of the sample protection group; Ac is the absorbance value of the AAPH oxidative damage group; Ad is the absorbance value of the toxicity control group;

[0094] After the reaction, add 3.2 mL of PBS buffer to each experimental group, shake well, pipette a small amount of the suspension onto a glass slide, and dry it over fire to create a thin coating. Cover with a coverslip and observe the cell morphology under an optical microscope.

[0095] 1.5 The experimental method for determining the intracellular ROS content of erythrocytes is as follows:

[0096] Resuspend the red blood cells in 100 μL of PBS buffer. Add 200 μL of 10 mM DCFH-DA and mix thoroughly. Incubate at 37°C in the dark for 30 minutes, then wash three times with PBS buffer (see 1.4 for details on the composition) and resuspend in 1 mL of PBS buffer. Experimental group A, treated with PBS, serves as the blank control. Measure the fluorescence intensity using a fluorescence spectrophotometer at EX: 485 nm, EM: 535 nm. Calculate the relative fluorescence intensity of the sample according to formula (6).

[0097] Relative fluorescence intensity (%) = fluorescence intensity (sample) / fluorescence intensity (experimental group A) * 100 (6)

[0098] 2. Efficacy 2: Tyrosinase inhibition

[0099] The experimental method is as follows: The experimental group (B) accurately aspirates 20 μL of the extract solution, 170 μL of the L-tyrosine solution, and 10 μL of the tyrosinase solution; the sample blank group (C) consists of 170 μL of the L-tyrosine solution, 20 μL of the sample, and 10 μL of PBS; the sample solvent blank group (A) consists of 20 μL of the solvent, 170 μL of the L-tyrosine solution, and 10 μL of the tyrosinase solution. After incubation in a 37°C incubator for 30 minutes, the absorbance is measured at 475 nm.

[0100] Taking Vc as the control, tyrosinase inhibition rate (%) = A-(BC) / A*100% (7)

[0101] Where A is the absorbance value of the sample solvent blank group; B is the absorbance value of the experimental group; C is the absorbance value of the sample blank group.

[0102] 3. Efficacy three: Inhibition of elastase

[0103] The experimental method is as follows: Elastase and the reaction substrate (N-(methoxysuccinyl)-L-alanyl-L-alanyl-L-prolyl-L-valine-4-nitroaniline) were prepared in 0.1 mol / L Tris-Cl buffer (pH 8.0) to concentrations of 0.5 U / mL and 1.015 M, respectively. Samples were diluted to the specified concentrations in the same buffer. The experiment was divided into A: sample reaction group; B: blank control group; and C: sample blank group. The experimental procedures are shown in Table 2.

[0104] Table 2 Experimental procedures for the inhibition of elastase by samples

[0105]

[0106] After incubation, the absorbance was measured at 405 nm.

[0107] Elastase inhibition rate (%) = Ab-(Aa-Ac) / Ab*100% (8)

[0108] Where Aa represents the absorbance value of the experimental group, Ab represents the absorbance value of the sample blank group, and Ac represents the absorbance value of the solvent blank group.

[0109] 4. Effect 4: Inhibition of hyaluronidase

[0110] The experimental method is as follows: Samples were diluted with deionized water to the specified concentration and set aside. 5 mg / mL dipotassium glycyrrhizate was used as a positive control. The experiment was divided into A: sample experimental group; B: sample blank group; C: reaction group; and D: solvent blank group. The experimental procedures are shown in Table 3.

[0111] Table 3 Experimental procedures for the inhibition of hyaluronidase by samples

[0112]

[0113]

[0114] After each group returned to room temperature, it was left to stand for 30 minutes to allow for color development, and the absorbance was measured at 530 nm.

[0115] Hyaluronidase inhibition rate = [(CD)-(AB)] / (CD)*100% (9)

[0116] 5. Efficacy 5: Inhibition of advanced glycation end products (AGEs)

[0117] The experimental method was as follows: blank glycated group (A0): 1.5 mL deionized water, 3 mL bovine serum albumin, and 3 mL glucose; blank non-glycated group (A1): 1.5 mL deionized water, 3 mL bovine serum albumin, and 3 mL deionized water; sample experimental group (B0): 1.5 mL sample, 3 mL bovine serum albumin, and 3 mL glucose; non-glycated sample group (B1): 1.5 mL sample, 3 mL bovine serum albumin, and 3 mL deionized water. These were added sequentially and shaken evenly. The mixture was incubated in a 60°C constant temperature and humidity incubator for 40 h. Fluorescence absorption was recorded using a fluorescence spectrophotometer with excitation / emission wavelengths of 370 / 440 nm.

[0118] AGEs inhibition rate (%) = 1-[(B0-B1) / (A0-A1)]*100% (10)

[0119] Wherein, A0 is the absorbance value of the blank glycation group; A1 is the absorbance value of the blank non-glycated group; B0 is the absorbance value of the sample experimental group; B1 is the absorbance value of the sample non-glycated group.

[0120] 6. Efficacy 6: Inhibition of α-glucosidase

[0121] The experimental method was as follows: α-glucosidase and pNPG solutions were prepared in PBS buffer (pH 6.8) at concentrations of 1 U / mL and 10 mM, respectively. Samples were diluted to the specified concentrations in the same PBS buffer and set aside. A 1 M Na₂CO₃ solution was prepared in deionized water. Acarbose (1 mg / mL) was used as a positive control. The experiment was divided into A: sample experimental group; B: reaction group; and C: sample blank group. The experimental procedures are shown in Table 4.

[0122] Table 4 Experimental procedures for the inhibition of α-glucosidase by samples

[0123]

[0124] The absorbance of each group was measured at 405 nm.

[0125] α-glucosidase inhibition rate = [B-(AC) / ]B*100 (11)

[0126] 7. Efficacy 7. Ultraviolet absorption capacity test

[0127] The experimental method is as follows: Dilute the sample with deionized water and scan the sample wavelengths under UVA (200-280), UVB (280-320), and UVC (320-400) light using a UV spectrophotometer, with each scan interval of 1 nm. Deionized water is used as the reference solution. 1 mg / mL ethylhexyl methoxycinnamate is used as the control (omc), and ethylhexyl methoxycinnamate is compared with anhydrous ethanol as the reference solution. Calculate the average absorbance of the sample in the UVA, UVB, and UVC light regions according to formulas (3-12), (3-13), and (3-14);

[0128]

[0129]

[0130] Among them, the wavelength range of the UVA light zone is 320-400. The denominator 81 in the formula represents that each wavelength interval is 1nm. The UVA light zone has a total of 400-320+1=81 wavelength points.

[0131] Similarly, 41 also refers to 41 wavelength points.

[0132] Calculate the average ultraviolet absorption rate of the sample in the UVA, UVB, and UVC light regions according to formulas (3-15), (3-16), (3-17), and (3-18), respectively.

[0133] Ultraviolet absorption (A%) = 1-transmittance (T)% (15)

[0134]

[0135] 2. The Bifidobacterium-Sanhua wrinkle removal liquid fermentation liquid (BTBF), Bifidobacterium fermentation liquid (BFL), "Sanhua wrinkle removal liquid" blank culture medium (TBC) and culture medium blank control (MBC) were compared and analyzed according to the experimental method above.

[0136] 2.1 Analysis of chemical antioxidant activity

[0137] 2.1.1 Detection of DPPH scavenging ability

[0138] The four samples (BTBF, BFL, TBC and MBC) were diluted to 1% by volume and DPPH clearance test was performed. The specific experimental steps are shown in 1.1 above. Figure 2As shown, it shows the schematic diagram of the clearance rate of DPPH by different samples. Figure 2 In the table, ** indicates P < 0.01 compared with the BFL group; ## indicates P < 0.01 compared with the TBC group. Analysis showed that among the four samples, the DPPH clearance rate of the BTBF group was the highest, at 66.72 ± 1.77%, the DPPH clearance rate of the BFL group was 45.85 ± 2.76%, the DPPH clearance rate of the TBL group was 55.63 ± 2.1%, and the DPPH clearance rate of the MBC group was 39.74 ± 1.09%.

[0139] It can be seen that all four groups of samples have good antioxidant effects. The antioxidant effect of the MBC group is mainly due to the L-cysteine ​​in the culture medium.

[0140] Further, the filtrate of the bifidobacterium-Sanhua wrinkle-removing liquid bidirectional fermentation product (BTBF) was diluted in a concentration gradient. Figure 3 The figure shows the effect of different BTBF concentrations on DPPH clearance. It can be clearly seen that BTBF has a significant dose-effect relationship on DPPH clearance. Specifically, it can be seen that when the volume fraction is 5%, the DPPH clearance rate reaches 90.3±1.18%. The IC 50 is 0.97%; among which IC 50 Refers to the half-maximal inhibitory concentration, which can quantify the scavenging ability of antioxidants to compare the oxidative activity of different samples and can intuitively express the scavenging ability of each sample.

[0141] When the volume fraction of BTBF is in the range of 0% to 2%, its DPPH clearance rate also increases significantly; when the volume fraction of BTBF is in the range of 2% to 3%, its DPPH clearance rate increases slowly; when the volume fraction of BTBF is in the range of 3% to 5%, its DPPH clearance rate increases gently; when the volume fraction of BTBF reaches 5%, the DPPH clearance rate reaches its maximum value and is in a saturated clearance state.

[0142] 2.1.2 Detection of ABTS free radical scavenging ability

[0143] The four samples (BTBF, BFL, TBC and MBC) were diluted to 1% by volume and the ABTS clearance test was performed. For details, please refer to the experimental method in 1.2 above. Figure 4 Shown are the ABTS clearance rates of the four samples. Figure 4In the middle: ** indicates P < 0.01 compared with the BFL group; ## indicates P < 0.01 compared with the TBC group. The analysis showed that the ABTS free radical scavenging rate in the BTBF group was 60.89 ± 2.4%, in the BFL group was 14.92 ± 0.82%, in the TBC group was 51.48 ± 1.15%, and in the MBC group was 17.16 ± 2.09%.

[0144] This suggests that the ABTS free radical scavenging capacity of the BFL group was lower than that of the MBC group, possibly due to the consumption of L-cysteine ​​in the culture medium. Compared with the BFL and TBC blank groups, the ABTS free radical scavenging capacity of BTBF was significantly improved (P<0.01).

[0145] Further, the filtrate of the bifidobacterium-Sanhua wrinkle-removing liquid bidirectional fermentation product (BFBT) was diluted in a concentration gradient. Figure 5 As shown, the effect of different BTBF concentrations on ABTS clearance is shown; Figure 5 It can be seen that BTBF shows a significant dose-effect relationship on the ABTS free radical scavenging rate. It can be seen that when the volume fraction is 2%, the ABTS free radical scavenging rate reaches 87.97±0.13%. Its IC 50 It is 0.5%.

[0146] When the volume fraction of BTBF is in the growth range of 0%-2%, its ABTS clearance rate also increases significantly; when the volume fraction of BTBF reaches 2%, the ABTS clearance rate reaches the maximum value and is in a clearance saturation state; when the volume fraction of BTBF is in the growth range of 2%-10%, its ABTS clearance rate remains basically unchanged and is in a flat state.

[0147] 2.1.3 Ferric Reducing Ability (FRAP) Assay

[0148] Ferric reduction assay (FRAP) is a method that uses the sample to reduce Fe 3+ Reduced to Fe 2+ , Fe 2+ The sample reacts with TPTZ to form a colored compound, and the change in absorbance is used to measure the antioxidant activity of the sample. For detailed experimental procedures, see 1.3 above. Figure 6 To detect Fe 2+ The standard curve corresponding to the content, where the linear regression equation is y = 0.5111x + 0.216, R 2 is 0.9997, which has a good linear correlation, so Fe can be calculated using this equation 2+ The content of Fe 2+ The concentration represents the antioxidant capacity of the sample.

[0149] like Figure 7As shown, the Fe 2+ Equivalent, where ** indicates P < 0.01 compared with the BFL group; ## indicates P < 0.01 compared with the TBC group. Figure 7 As shown, BTBF group Fe 2+ The equivalent was 4.396±0.242mM; TBC group Fe 2+ The equivalent was 4.244±0.234mM; Fe 2+ The equivalent was 0.667±0.043mM; Fe 2+ The equivalent is 0.599±0.04mM; the reducing ability of the bifidobacterium fermentation broth alone is slightly lower than that of the culture medium blank control, indicating that bifidobacteria utilize L-cysteine ​​in the culture medium during the fermentation process. The concentration of L-cysteine ​​in the blank culture medium is higher than that in the bifidobacterium fermentation group, so the reducing ability of the blank culture medium is higher than that of the bifidobacterium fermentation group alone.

[0150] The reducing ability of the bifidobacterium-Sanhua wrinkle-removing liquid bidirectional fermentation liquid was significantly increased compared with the bifidobacterium fermentation liquid and the "Sanhua" blank control group, and the reducing ability of the bifidobacterium fermentation liquid alone was significantly lower than that of the "Sanhua" blank control group, while the reducing ability of the bifidobacterium-Sanhua wrinkle-removing liquid bidirectional fermentation liquid and the "Sanhua" blank control group was slightly increased, indicating that in the Bifidobacterium-Sanhua wrinkle-removing liquid bidirectional fermentation liquid group, it was mainly "Sanhua" that played the main reducing role.

[0151] Further, the bifidobacterium-Sanhua wrinkle-removing liquid bidirectional fermentation liquid was diluted to different concentrations. Figure 8 As shown, the Fe content in BTBF with different concentrations is shown. 2+ The reducing ability showed dose-dependence.

[0152] 2.1.4 Effects on AAPH-induced erythrocyte hemolysis and cell morphology

[0153] 2.1.4.1 Determination of hemolysis inhibition rate

[0154] AAPH is a free radical initiator that can cause cell membrane lipid peroxidation and cell hemolysis. Adding antioxidants to the reaction system can reduce cell rupture and reduce the degree of hemolysis. For specific experimental steps, please refer to 1.4 above. The protective effect on red blood cells is demonstrated by measuring the degree of hemolysis after the sample is added. Figure 9 The figure shows the inhibition rates of AAPH-induced erythrocyte hemolysis by the four samples, where ## indicates that the AAPH oxidative damage group was compared with the PBS blank control group, P < 0.01; ** indicates that the BTBF group was compared with the AAPH oxidative damage group, BFL group, and TBC group, P < 0.01.

[0155] Depend on Figure 9 The results showed that the hemolysis rate of red blood cells caused by AAPH reached 46.17±0.67%, with nearly half of the red blood cells experiencing hemolysis. The hemolysis inhibition rate in the BTBF group was significantly increased, reaching 66.92±3.06%, while the hemolysis inhibition rates in the BFL and TBC groups were 55.72±0.25% and 55.2±3.13%, respectively.

[0156] This indicates that all four groups of samples can clear AAPH and protect cells. Figure 10 The image shows the effects of four samples on AAPH-induced erythrocyte hemolysis. From left to right, the image shows the full hemolysis control group, the AAPH oxidative damage group, and the experimental groups containing 10% of each of the four samples (BTBF, BFL, TBC, and MBC). Hemolysis was reduced in the BTBF group. This also demonstrates that the dual fermentation of Bifidobacterium and Sanhua Wrinkle Removal Solution effectively scavenges free radicals.

[0157] Furthermore, the BTBF group was diluted in a concentration gradient, e.g. Figure 11 The graph shows the inhibition rate of AAPH-induced red blood cell hemolysis by different concentrations of BTBF. Figure 12 Shown are photos of AAPH-induced erythrocyte hemolysis induced by different concentrations of BTBF; Figure 12 In the figure, from left to right are the full hemolysis control group, the AAPH oxidative damage group, and multiple experimental groups with different BTBF sample concentrations (1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%).

[0158] Depend on Figure 11 、 12 As can be seen, the hemolysis inhibition rate increases with increasing volume fraction and then tends to stabilize. At a volume fraction of 20%, the hemolysis inhibition rate reaches 94.33%. At a volume fraction of 100%, the hemolysis inhibition rate decreases. This may be because the osmotic pressure in the fermentation broth is lower than the osmotic pressure within the cells, causing the cells to absorb water and rupture. At other concentrations, the osmotic pressure is balanced with the intracellular osmotic pressure by dilution with PBS.

[0159] Free radical initiators can cause damage to the red blood cell membrane, leading to hemolysis. In order to verify that BTBF can reduce the hemolysis of red blood cells induced by AAPH, the morphological changes of red blood cells were observed under a microscope, such as Figure 13 , showing the state of red blood cells observed under an optical microscope. The results showed that normal red blood cells (PBS blank control group) had a smooth surface, while the red blood cells in the AAPH oxidative damage group ruptured into fragments, indicating that the red blood cell membrane was oxidatively damaged by the AAPH free radical initiator. The red blood cells in the BTBF protection group showed normal morphology, indicating that BTBF can scavenge AAPH free radicals, has an antioxidant effect, and has a good protective effect against red blood cell oxidative damage.

[0160] 2.1.4.2 Determination of intracellular ROS content in erythrocytes

[0161] On the basis of the above red blood cell experiment (for specific experimental steps, see 1.5 above), the fluorescent probe DCFH-DA (2'7'-fluorescein diacetate) was added to detect the content of intracellular reactive oxygen species (ROS) in red blood cells, which was expressed as the fluorescence intensity of each group. Figure 14 The figure shows the effects of different samples on the generation of ROS in erythrocytes induced by AAPH, where ** indicates that the BTBF group was compared with the AAPH oxidative damage group, P < 0.01; ## indicates that the BTBF group was compared with the BFL and TBC groups, P < 0.01. Figure 14 It can be seen that the ROS content in the erythrocytes of the AAPH oxidative damage group increased significantly due to the addition of AAPH oxidant. In the sample group, the ROS content in the erythrocytes decreased significantly due to the effective removal of AAPH, and the ROS content in the erythrocytes of the BTBF group was the lowest. The BTBF group was diluted to different concentrations, such as Figure 15 As shown, it shows the effect of different concentrations of BTBF on the ROS generation in erythrocytes induced by AAPH. It can be seen from the figure that with the increase of concentration, the ROS content in erythrocytes decreases sharply and tends to be stable when the volume fraction is 20%.

[0162] 2.2 Inhibition of tyrosinase

[0163] Tyrosinase is an oxidoreductase that plays an important role in the synthesis of melanin. For the specific experimental steps, please refer to the second point above. By inhibiting the content of tyrosinase, the whitening effect is demonstrated. Figure 16 The figure below shows the effects of different samples on tyrosinase inhibition. In the figure, ** indicates P < 0.01 compared to the BFL group; ## indicates P < 0.01 compared to the TBC group. As can be seen, the BTBF group had a tyrosinase inhibition rate of 72.21 ± 2%, the BFL group had an inhibition rate of 22.82 ± 4.52%, the TBC group had an inhibition rate of 33.08 ± 2.4%, and the MBC group had an inhibition rate of 39.26 ± 2.01%. The study showed that the BTBF group significantly increased the whitening effect compared to the BFL and TBC control groups.

[0164] like Figure 17 The effect of different concentrations of BTBF on tyrosinase inhibition is shown. The IC values ​​were obtained by Origin nonlinear curve fitting. 50 It is 78.83%.

[0165] 2.3 Inhibition of elastase

[0166] Elastase degrades proteins by hydrolyzing the peptide bonds of collagen, elastin and other proteins. Elastin accounts for 90% of the elastic fiber components. The degradation and destruction of elastic fibers will cause the skin to lose elasticity and cause wrinkles. Elastase is closely related to skin aging, so inhibiting the activity of elastase is one of the important ways to delay skin aging. For specific experimental steps, please refer to the third point above, efficacy three. The inhibitory effect of BTBF and BFL, TBC, and MBC blank control groups on elastase activity was tested through experiments. Studies have shown that Figure 18 The figure shows the effect of different samples on the inhibition rate of elastase. In the figure, ** indicates P < 0.01 compared with the BFL group; ## indicates P < 0.01 compared with the TBC group. It can be seen that compared with BFL and TBL, the inhibitory activity of BTBF on elastase was significantly increased, which was 63.33 ± 1.9%. As the volume fraction of BTBF increased, the inhibition rate of elastase increased, showing a dose-dependent inhibition.

[0167] like Figure 19 The effect of different concentrations of BTBF on the inhibition rate of elastase is shown. The IC 50 It is 76.96%.

[0168] 2.4 Inhibition of hyaluronidase

[0169] Hyaluronidase is an endogenous glycosidase that degrades hyaluronic acid. Most hyaluronidases can not only degrade hyaluronic acid but also other glycosaminoglycan substrates such as dermatan sulfate. Studies have shown that hyaluronidases can significantly promote cell migration and proliferation, and are strongly correlated with inflammation and allergies. Furthermore, most type I allergic reactions are associated with hyaluronidase activity in vivo, making hyaluronidase inhibition testing a rapid and effective method for screening anti-allergic cosmetic ingredients. Hyaluronidase inhibitors are particularly effective in reducing skin damage caused by oxidative stress and inflammation due to their potent antioxidant and anti-inflammatory properties. This mechanism not only helps slow the aging process but also effectively maintains skin elasticity and smoothness, effectively regulates skin pigmentation, and achieves a synergistic whitening effect. For specific experimental procedures, please refer to the fourth point above, "Benefit 4."

[0170] like Figure 20The figure shows the effect of different samples on hyaluronidase inhibition. In the figure, ** indicates P < 0.01 for the BTBF group compared to the BFL group; * indicates P < 0.05 for the BTBF group compared to the TBC group. The study found that the inhibition rates of BFL and MBC on hyaluronidase were -138.41 ± 2.17% and -55.8 ± 3.62%, respectively, indicating that Bifidobacterium fermentation alone and blank culture medium failed to inhibit hyaluronidase activity. BTBF effectively inhibited hyaluronidase activity compared to the TBC group (24.62 ± 6.15%).

[0171] 2.5 Inhibitory effect on advanced glycation end products (AGEs)

[0172] Non-enzymatic glycation (NEG) is a process in which proteins, lipids, and reducing sugars undergo a series of complex biochemical reactions without the catalysis of enzymes, generating irreversible AGEs. AGEs alter protein structure, causing color changes, brittleness, and yellowing, leading to changes in the biomechanical properties and biological changes of the skin. For specific experimental steps, please refer to point 5 above, efficacy 5. Figure 21 The figure shows the effects of different samples on AGE inhibition. In the figure, ** indicates P < 0.01 compared to the BFL group. The experimental results showed that the BTBF group had an AGE inhibition rate of 82.26 ± 3.82%, higher than the BFL and TBC groups, and P < 0.01 compared to the TBC group.

[0173] The filtrate of the bifidobacterium-Sanhua wrinkle-removing liquid fermentation product was diluted in a concentration gradient. Figure 22 The figure shows the effect of different concentrations of BTBF on the inhibition rate of AGEs. It can be seen that the inhibition rate of BTBF on AGEs shows an obvious dose-effect relationship. The IC 50 It is 54.31%.

[0174] 2.6 Inhibition of α-glucosidase

[0175] Non-enzymatic glycosylation reactions require the participation of glucose or other reducing monosaccharides to generate stable covalent adducts. α-glucosidase is an enzyme that can hydrolyze polysaccharides into glucose. I Yamamoto et al. detected relatively high α-glucosidase activity in the lysates of fibroblasts cultured in vitro, indicating the possibility that α-glucosidase exists in the fibroblasts of the dermis. The sample reduces the glucose content in the skin by inhibiting the activity of α-glucosidase, thereby reducing the amount of substrate for non-enzymatic glycosylation reactions and thus reducing the amount of AGEs produced. For specific experimental steps, please refer to the sixth point above, Efficacy 6. Figure 23The figure shows the effect of different samples on the inhibition rate of α-glucosidase. In the figure, ** indicates that compared with the BFL group, P < 0.01; ## indicates that compared with the TBC group, P < 0.01. The experimental results show that the inhibition rate of the BTBF group on α-glucosidase is 68.02 ± 0.75%, which is higher than that of the BFL group: -62.49 ± 3.11% and the TBC group: 44.97 ± 1.80%. The inhibition rate and significance are improved (P < 0.01). The filtrate of the product of the bifidobacterium-Sanhua wrinkle removal liquid was diluted in a concentration gradient. Figure 24 The effect curve of different concentrations of BTBF on the inhibition rate of α-glucosidase shows that BTBF shows a significant dose-effect relationship in the inhibition rate of α-glucosidase. The IC 50 It is 64.95%.

[0176] 2.7 UV absorption capacity test

[0177] Aging is caused by both intrinsic genetic factors and exogenous factors, with ultraviolet rays being the primary exogenous factor contributing to skin aging. Ultraviolet radiation (UVR) primarily includes three categories: long-wave ultraviolet (UVA), medium-wave ultraviolet (UVB), and short-wave ultraviolet (UVC). UVA, with a wavelength of 320 to 400 nm, has high penetrating power and can penetrate deep into the dermis, damaging fibroblasts, the main cells of the dermis, leading to skin aging. It also stimulates melanocytes, increasing melanin levels through the α-MSH-MC1R pathway and darkening the skin. UVB, with a wavelength of 280 to 320 nm, can reach the basal layer of the epidermis. The photodamage caused by UVB is 800 to 1000 times greater than that of the same dose of UVA and is the primary cause of cellular DNA damage. UVC, with a wavelength of 200 to 280 nm, has shorter wavelengths and higher energy. Normally, the Earth's ozone layer effectively absorbs this UVC radiation, preventing it from reaching the Earth's surface. However, due to environmental pollution, the ozone layer has been damaged and its ability to absorb ultraviolet rays has decreased significantly, which has led to a significant increase in the risk of humans being exposed to excessive ultraviolet radiation.

[0178] For specific experimental steps, please refer to the seventh point above. Figure 25The figure shows wavelength scans of different samples. Plotting the absorbance values ​​obtained by wavelength scans reveals that among the four samples with a volume fraction of 2.5%, BTBF exhibits higher absorbance between 200 and 400 nm than the other three samples, demonstrating superior sun protection. In the UVC region, BTBF's absorbance ranges from 1 to 3.5, BFL's from 0.4 to 3.0, TBC's from 1 to 3.4, and MBC's from 0.4 to 3.0. All four samples exhibit maximum absorption peaks around 220 nm, with values ​​of 3.501, 3.063, 3.417, and 3.095, respectively. From 220 to 400 nm, the absorbance decreases with increasing wavelength.

[0179] The UV absorption values ​​obtained by scanning are averaged in each light zone. Figure 26 The figure shows the average UV absorbance (%) of various samples in the UV region. ** in the figure indicates P < 0.01 compared to the BFL group. Table 5 shows that the average absorbance values ​​for the four samples in the UVA, UVB, and UVC regions follow the order BTBF > TBC > MBC > BFL. UV absorbance exceeding 60% is considered to be a strong sunscreen.

[0180] Table 5 Average absorbance values ​​of samples in the ultraviolet region

[0181] sample UVA light zone UVB light zone UVC light zone BTBF 0.327±0.001 0.781±0.002 2.083±0.012 BFL 0.061±0.002 0.204±0.012 1.385±0.023 TBC 0.310±0.015 0.738±0.032 2.015±0.013 MBC 0.072±0.009 0.229±0.012 1.455±0.034 1mg / mLomc 1.231±0.023 3.539±0.019 3.497±0.034

[0182] Table 6 shows that the average UVC absorbance of all four samples is greater than 60%, indicating that all four samples have good UVC absorption. The average UVB absorbance of BTBF and TBC is greater than 60%, while that of BFL and MBC is less than 60%. Meanwhile, the average UVA absorbance of all four samples is less than 60%, indicating that at a volume fraction of 2.5%, the four samples have low UVA absorption capacity and weaker UVA protection. However, the BTBF group still outperforms the other control groups.

[0183] Table 6 Average UV absorption rate of samples in the UV region (%)

[0184]

[0185]

[0186] BTBF is diluted to different volume fractions, Figure 27 The wavelength scanning curves of different concentrations of BTBF can be seen. As the concentration increases, the absorbance value increases accordingly. The absorbance value of BTBF with a volume fraction of 20% at 200-400nm is higher than that of the positive control (1 mg·mL-1 Ethylhexyl methoxycinnamate solution, omc).

[0187] Table 7 shows that as BTBF concentration increases, the average absorbance values ​​in the UVC, UVB, and UVA regions also increase. In the UVA and UVC regions, the average absorbance values ​​of 10% and 20% BTBF are superior to those of OMC. In the UVB region, the average absorbance value of 20% BTBF is superior to that of OMC.

[0188] Table 7 Average absorbance values ​​of different BTBF concentrations in the ultraviolet region

[0189]

[0190] It can be seen from Table 8 that the average UV absorption rate of BTBF with a concentration of more than 5% is greater than 60%, indicating that BTBF has a good sun protection effect only when the concentration is above 5%.

[0191] Table 8 Average UV absorption rate of different BTBF concentrations in the UV region (%)

[0192] BTBF concentration (%) UVA light zone UVB light zone UVC light zone 0.5 15.29±0.12 33.78±0.02 68.27±0.23 1 24.8±0.02 50.68±0.03 81.73±0.33 2.5 49.79±0.03 82.54±0.02 97.08±0.12 5 71.82±0.01 96.66±0.01 99.83±0.12 10 88.93±0.11 99.84±0.08 99.99±0.02 20 97.38±0.12 99.99±0.01 99.99±0.02 1mg / mLomc 49.66±0.34 99.97±0.13 99.70±0.13

[0193] 3. Conclusion

[0194] A comprehensive study of the antioxidant properties, whitening effects, anti-aging properties, and sunscreen effects of BTBF, BFL, TBC, and MBC was conducted. In antioxidant experiments, BTBF demonstrated significantly higher DPPH scavenging rates, ABTS scavenging rates, and total reducing power compared to the other control groups, demonstrating excellent antioxidant capacity. The antioxidant effect of BTBF increased in a dose-dependent manner with increasing volume fraction. Furthermore, BTBF demonstrated significant protection against AAPH-induced erythrocyte oxidative damage, as evidenced by lower erythrocyte hemolysis rates and ROS levels, demonstrating its antioxidant and cytoprotective properties. Regarding whitening effects, the BTBF group showed the highest activity for tyrosinase, reaching 72.21±2%, indicating its regulatory effect on melanin synthesis. Furthermore, BTBF exhibited significant inhibitory effects on elastase, AGEs, and α-glucosidase, demonstrating its superior anti-aging properties. Regarding sunscreen effects, BTBF exhibited the highest absorbance in the UV region, demonstrating excellent UV absorptivity and excellent sunscreen properties. In addition, the sun protection effect of BTBF also showed an enhanced trend with the increase of concentration. In summary, BTBF showed significant advantages in anti-oxidation, whitening, anti-aging and sun protection.

[0195] IV. Application Prospects and Advantages

[0196] 4.1 Skin Care Products

[0197] The fermentation liquid can be used to develop skin care products with antioxidant, whitening, anti-aging, anti-inflammatory, anti-allergic, anti-glycation and other effects.

[0198] 4.2 Functional Foods

[0199] The fermentation liquid can also be used to develop foods or health foods with functions such as anti-oxidation, whitening, and anti-aging.

[0200] 4.3 Pharmaceutical field

[0201] The fermentation liquid can be used as an external medicine with anti-inflammatory, anti-allergic, anti-glycation and other effects.

[0202] Implementation 2:

[0203] The present invention also provides a skin care fermentation liquid, wherein the skin care fermentation liquid is prepared by the preparation method of the Bifidobacterium-Trifolium wrinkle-removing liquid fermentation liquid described in Example 1.

[0204] The skin care fermentation liquid prepared by the method in Example 1 not only retains the traditional efficacy of Sanhua Chinese medicine, but also gives it stronger activity and stability through microbial fermentation technology. It is a new type of bio-fermentation skin care product raw material with both safety and efficacy, and has broad application prospects.

[0205] It should be understood that those skilled in the art can make improvements or changes based on the above description, and all such improvements and changes should fall within the scope of protection of the appended claims of the present invention.

Claims

1. A method for preparing a Bifidobacterium-Sanhua wrinkle-removing liquid fermentation liquid, characterized in that: The preparation method comprises the following steps: Step 1: Inoculate Bifidobacterium adolescentis strains into a culture medium containing soy peptone, tryptone, yeast extract powder, glucose, L-cysteine, saline solution and deionized water, and culture under an anaerobic environment to activate the Bifidobacterium strains; Step 2: Inoculate the activated bacteria in step 1, take a portion of the colony with an inoculating loop and inoculate it into a conical flask, and cultivate it under anaerobic conditions to prepare a seed solution; Step 3: Mix the seed liquid prepared in step 2 with the Sanhua wrinkle-removing liquid powder in a certain proportion, and carry out a bi-directional fermentation of Bifidobacterium-"Sanhua wrinkle-removing liquid" to form a Bifidobacterium-Sanhua wrinkle-removing liquid fermentation liquid.

2. The preparation method according to claim 1, characterized in that In step 2, the ratio of the soy peptone, the tryptone, the yeast extract powder, the glucose, the L-cysteine ​​and the saline solution is 1:1:2:2:0.1:

8.

3. The preparation method according to claim 1 or 2, characterized in that In step 3, the fermentation conditions for the bifidobacterium-"Sanhua wrinkle removal liquid" bidirectional fermentation in step 3 are: The mixture of Bifidobacterium and Sanhua wrinkle-removing liquid was sterilized in an autoclave at a high temperature of 121° C. for 20 minutes. The inoculation amount of the mixture was 2%, the initial pH was 5.7, and the mixture was anaerobically cultured at 37° C. for 24 hours.

4. The preparation method according to claim 1, characterized in that The preparation method of the three-flower wrinkle-removing liquid powder is as follows: Peach blossoms, lotus flowers and hibiscus flowers are dried, crushed and sieved respectively, and then the three are evenly mixed in a certain proportion to obtain three-flower wrinkle-removing liquid powder.

5. A skin care fermentation liquid, characterized in that: The skin care fermentation liquid is prepared by the preparation method of the Bifidobacterium-Trifolium wrinkle-removing liquid fermentation liquid according to any one of claims 1 to 4.

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