Process for the preparation of lysates of bifidobacterium fermented honey and extract of yarrowia lipolytica

Abstract

The present application relates to the preparation process of the lysate product of bifidobacterium fermentation honey and extract of yarrow, comprising the preparation of bifidobacterium activation medium; strain liquid propagation; composition preparation; anaerobic fermentation culture; finally, the fermentation broth of the above composition is inactivated by low-temperature micro-jet, centrifuged, and the supernatant is obtained, and the lysate product of bifidobacterium is obtained. Compared with the blank culture medium, the total number of viable bacteria and OD value can be obviously improved by adding glucose or honey as carbon source, and when the carbon source is further selected as honey, the total number of viable bacteria after fermentation culture by the culture medium is more, and the OD value is higher.

Description

Technical Field

[0001] This invention relates to the field of cosmetic processing technology, specifically to the preparation process of lysate products from Bifidobacterium fermentation of honey and yarrow extract. Background Technology

[0002] As we age, the number of dermal fibroblasts in human skin gradually decreases, reducing the body's ability to synthesize collagen. Furthermore, increased protease release promotes collagen degradation and the breakdown and breakage of elastic fibers, leading to skin laxity and wrinkles. On the other hand, ultraviolet and visible light significantly influence the induction of free radicals and reactive oxygen species (ROS), thereby activating inflammatory cytokines and matrix metalloproteinases. Inflammatory responses damage the skin barrier, and local metabolic disturbances and the aggregation of neutrophils and macrophages easily generate excessive free radicals, causing tissue damage. Excessive free radicals and reactive oxygen species generated by oxidative stress can easily lead to cell damage and collagen inactivation, accelerating the aging of the body and skin. When matrix metalloproteinases are overexpressed, they specifically degrade extracellular matrix components, disrupting the normal structure of collagen and elastic fibers, resulting in wrinkles and other signs of aging.

[0003] Honey's main components are glucose, fructose, and water, along with proteins, amino acids, polyphenols, organic acids, and minerals. It is a natural food with both medicinal and edible properties. The phenolic compounds and other antioxidants in honey can eliminate excess free radicals accumulated during human metabolism, promote skin cell metabolism, and delay skin aging. Honey can also regulate the levels of pro-inflammatory factors, C-reactive protein, and anti-inflammatory factors to exert its anti-inflammatory effect.

[0004] The active components in Bifidobacterium fermentation products can nourish the skin and replenish various nutrients needed by the skin, including small molecules such as amino acids, vitamins and minerals. They can penetrate deep into the skin to activate cells and enhance their biological functions; promote the expression of type I collagen factor, repair the skin barrier, inhibit the decomposition of collagen by MMP, and alleviate the damage of ultraviolet rays to the skin barrier function.

[0005] Yarrow contains a rich variety of chemical components, mainly flavonoids and their glycosides, phenolic acids, alkaloids, etc. Yarrow extract inhibits elastase and promotes the proliferation of epidermal keratinocytes, thus restoring the elasticity and balance of skin fibrous tissue and accelerating skin cell metabolism.

[0006] Technical issues:

[0007] (1) Honey is rich in protein. When used directly as an ingredient in cosmetics, the large protein molecules in honey are unstable in the cosmetic system, causing turbidity or precipitation. Figure 1As shown. Through bio-fermentation, large protein molecules are broken down into smaller peptides and other substances, making them more stable in the system and easier for the skin to absorb.

[0008] (2) Currently, most honey fermentation uses sake yeast. The alcohols produced during fermentation may irritate the skin, and long-term use may damage the skin barrier. Bifidobacteria are used to ferment honey. Honey contains abundant sugars, proteins, and amino acids, which provide carbon and nitrogen sources for Bifidobacteria during fermentation, thus promoting their proliferation. The fermentation product retains the characteristic flavor of honey, and the fermentation of Bifidobacteria and honey have a synergistic effect in terms of efficacy.

[0009] (3) Although honey, bifidobacteria products and yarrow extract all have corresponding effects in anti-aging, skin aging is caused by multiple factors, so there is a problem of single target pathway. Summary of the Invention

[0010] The purpose of this invention is to provide a process for preparing lysate products of Bifidobacterium fermented honey and yarrow extract, so as to solve the problems mentioned in the background art.

[0011] To achieve the above objectives, the present invention provides the following technical solution: a process for preparing lysate from Bifidobacterium-fermented honey and yarrow extract, comprising the following steps:

[0012] S1. Preparation of Bifidobacterium activation medium: Prepare the medium, dispense it into Erlenmeyer flasks, sterilize it at high temperature, and cool it to room temperature for use as a strain for activation.

[0013] S2. Propagation of bacterial culture: Bifidobacterium strains are inoculated into the culture medium and anaerobic fermented in a constant temperature shaking incubator to obtain Bifidobacterium strain propagation solution.

[0014] S3. Preparation of the composition: The yarrow herb is pulverized and mixed with a solvent to obtain a mixture. The mixture is extracted by a microfluidic extractor. The yarrow extract pulp and honey are mixed in proportion and sterilized at high temperature in a fermenter.

[0015] S4. Anaerobic fermentation culture: The Bifidobacterium inoculum propagation liquid obtained in S2 is inoculated into the mixed liquid obtained in S3 and anaerobic fermentation culture is carried out to obtain the combined fermentation broth.

[0016] S5. The fermentation broth of the above composition is inactivated by low-temperature microfluidic disruption, centrifuged, and the supernatant is collected to obtain the Bifidobacterium lysate.

[0017] In some embodiments, the Bifidobacterium activation culture medium in S1 comprises the following components in parts by weight: yeast extract 2-6 g / L, bacteriological peptone 4-10 g / L, honey 30-120 g / L, beef extract powder 2-10 g / L, magnesium sulfate 0.05-0.5 g / L, sodium acetate 3-8 g / L, diammonium citrate 1-5 g / L, dipotassium hydrogen phosphate 0.5-5 g / L, manganese sulfate 0.01-0-0.06 g / L, Tween 80 0.5-2 g / L, with the remainder made up with pure water.

[0018] In some embodiments, the Bifidobacterium activation culture medium comprises the following components in parts by weight: yeast extract 3-4 g / L, bacteriological peptone 6-8 g / L, honey 50-100 g / L, beef extract powder 2-10 g / L, magnesium sulfate 0.1-0.3 g / L, sodium acetate 4-6 g / L, diammonium citrate 1-3 g / L, dipotassium hydrogen phosphate 1-3 g / L, manganese sulfate 0.02-0.04 g / L, Tween 80 1.0-1.5 g / L, with the remainder made up with pure water.

[0019] In some embodiments, the solvent in S3 is water and / or a lower alcohol, and the mass ratio of the solvent to yarrow is 10 to 40:1; preferably, the lower alcohol is propylene glycol, glycerol, or butylene glycol, and the preferred mass ratio of the solvent to yarrow is 15 to 30:1.

[0020] In some embodiments, propylene glycol is 1,2-propanediol, butanediol is 1,3-butanediol, and the mass ratio of solvent to yarrow is 20:1.

[0021] In some embodiments, the composition preparation in step S3 includes the following components in parts by weight: 5-20 parts honey, 0.5-2 parts yarrow extract, and water to make up the mass; preferably, the composition includes the following components in parts by weight: 10-15 parts honey, 1-2 parts yarrow extract, and water to make up the mass.

[0022] In some embodiments, the micropore diameter of the low-temperature microfluidic extractor is 800 μm, and the extraction conditions of the low-temperature microfluidic extractor are: feeding rate of 15-25 L / min, temperature of 20-35 °C, stirring speed of 300-700 rpm, and stirring time of 20-40 min.

[0023] In some embodiments, the high-temperature sterilization temperature in S3 is 100-120°C and the time is 10-25 min, with the preferred high-temperature sterilization temperature being 115°C and the time being 15 min.

[0024] In some embodiments, the amount of Bifidobacterium strain in S2 is 3-15% of the volume ratio of the activation medium, and preferably the amount of Bifidobacterium strain inoculated is 3-6% of the volume ratio of the activation medium.

[0025] In some embodiments, the amount of Bifidobacterium strain propagation solution in S4 is 4-20% of the volume ratio of the mixed solution; preferably, the amount of Bifidobacterium strain propagation solution in S4 is 7-12% of the volume ratio of the mixed solution.

[0026] The anaerobic fermentation culture conditions are: rotation speed 120-240 rpm, temperature 30-40℃, and time 16-30 h. Preferably, the anaerobic fermentation culture conditions are: rotation speed 160 rpm, temperature 37℃, and time 24 h.

[0027] In some embodiments, the centrifugation device in S5 is a high-speed centrifuge with a rotation speed of 8000-10000 rpm and a time of 10-30 min.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] (1) Compared with the blank culture medium, the addition of glucose or honey as a carbon source can significantly increase the total number of viable bacteria and the OD value. When honey is further selected as the carbon source, the total number of viable bacteria and the OD value are higher after fermentation in this culture medium.

[0030] (2) This invention achieves highly efficient cell wall disruption of yarrow herb through specific low-temperature microfluidic process parameters. After cell wall disruption, the solvent fully penetrates the herb, allowing its active ingredients to be better dissolved, resulting in a high extraction rate and strong efficacy. The Bifidobacterium lysate is extracted via low-temperature microfluidic cell wall disruption, achieving inactivation of Bifidobacterium and further decomposing it into smaller molecular weight substances, which are better absorbed by the skin.

[0031] (3) The present invention uses Bifidobacterium activated by the above culture medium as the fermentation strain of honey and yarrow composition. It is non-irritating to the skin and can effectively increase the effect of the composition in scavenging DPPH free radicals and scavenging reactive oxygen species, slow down the damage of free radicals to collagen and inhibit the decomposition of elastin by MMPs-1 and elastase. The fermentation product of this combination can reduce the production of inflammation by promoting the regeneration and expression of collagen and elastin, inhibiting the expression of inflammatory factors, inhibiting the activity of hyaluronidase, repairing the skin epidermal barrier, maintaining skin elasticity, and reducing the production of wrinkles, thus achieving multi-target and multi-faceted anti-aging of the skin.

[0032] The above description is merely an overview of the technical solutions of the embodiments of this application. In order to better understand the technical means of the embodiments of this application and to implement them in accordance with the contents of the specification, and to make the above and other objects, features and advantages of the embodiments of this application more obvious and understandable, specific implementation methods of this application are described below. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 This is a diagram illustrating the stability of the product when unfermented honey and fermented honey are added respectively.

[0035] Figure 2 This is a schematic diagram of the chicken embryo chorioallantoic membrane irritation test.

[0036] Figure 3 This is a schematic diagram of the results of the zebrafish embryo reactive oxygen species (ROS) scavenging test.

[0037] Figure 4 This is a schematic diagram of the neutrophil inhibition test results in zebrafish embryos.

[0038] Figure 5 This is a schematic diagram of the melanin inhibition test results in zebrafish embryos. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to limit the application; the terms “comprising” and “having”, and any variations thereof, in the specification, claims and drawings of this application are intended to cover non-exclusive inclusion.

[0041] The term "embodiment" as used herein means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of the phrase "embodiment" in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0042] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings.

[0043] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," and "circumferential" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and simplifying the description, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0044] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0045] Example 1

[0046] A lysate of honey and yarrow extract fermented by Bifidobacterium is prepared as follows:

[0047] (1) Prepare the culture medium, which includes the following components by mass: yeast extract 4 g / L, bacteriological peptone 6 g / L, honey 100 g / L, beef extract powder 6 g / L, magnesium sulfate 0.2 g / L, sodium acetate 5 g / L, diammonium citrate 2 g / L, dipotassium hydrogen phosphate 2 g / L, manganese sulfate 0.04 g / L, Tween 80 1 g / L, and make up the remainder with pure water.

[0048] (2) After preparing the Bifidobacterium culture medium in step 1, dispense it into Erlenmeyer flasks, place them in a high-temperature steam sterilizer, sterilize at 115°C for 15 minutes, and place them at room temperature for activation of the inoculum.

[0049] (3) The amount of Bifidobacterium in the sterilized culture medium was 5%, and it was placed in a constant temperature shaking incubator and anaerobic cultured at 37°C for 24 hours. The OD value was then measured.

[0050] (4) Extract yarrow herb by low temperature microfluidic extraction. The mass ratio of solvent to yarrow is 20:1. Mix 1 part of yarrow extract concentrate with 13 parts of honey and make up the mass with pure water.

[0051] (5) Place the above mixture in a fermenter and sterilize at 115°C for 15 minutes;

[0052] (6) Inoculate the activated Bifidobacterium from step 3 into the mixture at an inoculation rate of 8% of the mixture mass and anaerobic culture at 37°C for 24 hours.

[0053] (7) The above fermentation product was inactivated by low temperature microfluidic disruption, centrifuged at 10,000 rpm for 10 min, and the supernatant was collected to obtain the cell lysate.

[0054] Example 2

[0055] A lysate of honey and yarrow extract fermented by Bifidobacterium, which differs from Example 1 in that the honey content in the culture medium is 50 g / L;

[0056] Example 3

[0057] A lysate of honey and yarrow extract fermented by Bifidobacterium, differing from Example 1 in that the yarrow herb is extracted by low-temperature microfluidic extraction, the mass ratio of solvent to yarrow is 30:1, and 0.5 parts of yarrow extract concentrate and 5 parts of honey are mixed.

[0058] Example 4

[0059] A lysate of honey and yarrow extract fermented by Bifidobacterium, which differs from Example 1 in that the activated Bifidobacterium is inoculated into the mixture at an inoculum amount of 5% by weight of the mixture.

[0060] Comparative Example 1

[0061] A lysate of honey and yarrow extract fermented by Bifidobacterium, which differs from Example 1 in that no honey is added to the culture medium;

[0062] Comparative Example 2

[0063] A Bifidobacterium lysate, the preparation process of which is as follows:

[0064] (1) Prepare the culture medium, which includes the following components by mass: yeast extract 4 g / L, bacteriological peptone 6 g / L, glucose 20 g / L, beef extract powder 6 g / L, magnesium sulfate 0.2 g / L, sodium acetate 5 g / L, diammonium citrate 2 g / L, dipotassium hydrogen phosphate 2 g / L, manganese sulfate 0.04 g / L, Tween 80 1 g / L, and make up the remainder with pure water.

[0065] (2) After preparing the Bifidobacterium culture medium in step 1, dispense it into Erlenmeyer flasks, place them in a high-temperature steam sterilizer, sterilize at 115°C for 15 minutes, and place them at room temperature for activation of the inoculum.

[0066] (3) The amount of Bifidobacterium in the sterilized culture medium was 5%, and it was placed in a constant temperature shaking incubator and anaerobic cultured at 37°C for 24 hours. The OD value was then measured.

[0067] (4) The fermentation broth of Bifidobacterium was inactivated by low temperature micro-jet disruption, centrifuged at 10,000 rpm for 10 min, and the supernatant was collected to obtain the Bifidobacterium lysate.

[0068] Comparative Example 3

[0069] A lysate of bifidobacterium-fermented honey and yarrow extract, which differs from Example 1 in that step (4) does not include yarrow extract.

[0070] Comparative Example 4

[0071] A lysate of honey and yarrow extract fermented by Bifidobacterium, which differs from Example 1 in that honey is not added in step (4);

[0072] Comparative Example 5

[0073] A lysate of honey and yarrow extract fermented with sake yeast, differing from Example 1 in that the inoculated strain is sake yeast, and the culture medium comprises the following components by weight: 10 g / L yeast extract, 20 g / L bacteriological peptone, 100 g / L honey, with the remainder made up with pure water.

[0074] Test Example 1: OD value of the test sample

[0075] This test case was used to determine the OD values ​​of Bifidobacterium in different culture media of Examples 1 and 2 and Comparative Examples 1 and 2. The specific determination method is as follows:

[0076] (1) Shake the bacterial culture thoroughly, transfer 200 μL of the bacterial culture into a 96-well plate using a pipette, and measure the absorbance of the bacterial culture at 600 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0077] (2) Experimental results:

[0078] Table 1 OD values ​​of Bifidobacterium inoculum

[0079] Example 1 Example 2 Comparative Example 1 Comparative Example 2 OD value 1.959 1.589 0.348 0.935

[0080] Test Example 2: Test Sample Bacterial Quantity

[0081] This test case was used to determine the bacterial count of Bifidobacterium in different culture media of Examples 1 and 2 and Comparative Examples 1 and 2. The specific determination method is as follows:

[0082] (1) Shake the fermentation broth thoroughly, take an equal mass of the fermentation broth into a centrifuge bottle, and centrifuge at 10000 rpm for 10 min; (2) Take the evaporating dish and record its mass as M0;

[0083] (3) Pour off the supernatant from the centrifuge, transfer the bottom precipitate to an evaporating dish, and dry it in a 60°C forced-air oven. Once the evaporating dish containing the sample has reached a constant weight, record the final mass as M. 1；

[0084] (4) Formula for calculating the total mass of microorganisms M: M = M1 - M0

[0085] (5) Experimental results:

[0086] Table 2 Total weight of Bifidobacterium strains

[0087] Example 1 Example 2 Comparative Example 1 Comparative Example 2 M (unit: grams) 7.12 5.61 1.20 3.36

[0088] The data in Tables 1 and 2 show that when 50 g / L or 100 g / L of honey is added to the culture medium as a carbon source, compared with glucose as a carbon source or a blank carbon source, the total number of Bifidobacterium strains and the OD value can be significantly increased. When 100 g / L of honey is further selected as the carbon source for the culture medium, the total number of viable bacteria and the OD value are even higher after fermentation in this medium.

[0089] Test Example 3: DPPH Free Radical Scavenging Experiment

[0090] This test example is used to determine the antioxidant properties of Examples 1-4 and Comparative Examples 1-4, specifically the DPPH free radical scavenging experiment. The determination method is as follows:

[0091] (1) Add 4.0 ml of DPPH solution and 1.0 ml of 95% ethanol to a 10 ml test tube, mix and shake well, react in the dark for 30 min, and after stabilization, use 95% ethanol as a reference to measure the absorbance at 517 nm and record it as A0.

[0092] (2) Add 4.0 ml of DPPH solution and 1.0 ml of the test sample solution to a 10 ml test tube, mix and shake well, react in the dark for 30 min, and after stabilization, use 95% ethanol as a reference to measure the absorbance at 517 nm and record it as Ar.

[0093] (3) Add 4.0 ml of 95% ethanol solution and 1.0 ml of the test sample solution to a 10 ml test tube, mix and shake well, react in the dark for 30 min, and after stabilization, use 95% ethanol as a reference to measure the absorbance at 517 nm and record it as As.

[0094] (4) Calculation formula:

[0095] Scavenging rate of DPPH free radicals by the sample

[0096] Where: Ar -- absorbance of the DPPH solution after reaction with the test solution;

[0097] As -- The absorbance of a solution without DPPH, containing only the test solution and 95% ethanol;

[0098] A0 -- Absorbance of a solution containing only DPPH and 95% ethanol without test solution.

[0099] (5) Experimental results:

[0100] Table 3 Experimental Results

[0101]

[0102]

[0103] According to the comparison of experimental data, the lysate obtained by fermenting honey and yarrow extract with Bifidobacterium is more effective at scavenging DPPH free radicals than the single component, and is superior to the lysate obtained by fermenting sake yeast, thus exhibiting antioxidant effects.

[0104] Test Example 4: Hydroxyl Radical Scavenging Experiment

[0105] This test example is used to determine the antioxidant properties of Examples 1-4 and Comparative Examples 1-4, specifically the hydroxyl radical scavenging experiment. The determination method is as follows:

[0106] (1) Accurately weigh 1 mL of methyl violet solution, 1 mL of buffer solution, 1 mL of sample solution, and 1 mL of ferrous sulfate solution. Finally, add 1 mL of H2O2, mix well, and record as AS.

[0107] (2) Accurately weigh 1 mL of methyl violet solution, 1 mL of buffer solution, 1 mL of distilled water, and 1 mL of ferrous sulfate solution. Finally, add 1 mL of H2O2, mix well, and record as A.

[0108] (3) Accurately weigh 1 mL of methyl violet solution, 1 mL of buffer solution and 3 mL of distilled water, mix well and record as A0;

[0109] (4) After mixing, let stand at room temperature for 5 minutes, using pure water as a control, measure the change in absorbance ΔA of each system on a spectrophotometer at an absorption wavelength of 582 nm with a 1 cm cuvette, and calculate the clearance rate CR.

[0110] (5) Calculation formula:

[0111]

[0112] Where: AS -- absorbance after reaction with sample solution;

[0113] A -- Absorbance measured without sample;

[0114] A0 -- Absorbance of the colorimetric reagent;

[0115] (6) Experimental Results

[0116] Table 4 Experimental Results

[0117] Group Clearance rate (%) 5% Example 1 62.52 5% Example 2 53.22 5% Example 3 38.51 5% Example 4 42.76 5% Comparative Example 1 15.44 5% Comparative Example 2 21.97 5% Comparative Example 3 25.17 5% Comparative Example 4 28.55 5% vs. 5 36.49 1% Vitamin C Ethyl Ether 78.70

[0118] According to the comparison of experimental data, the lysate obtained by fermenting honey and yarrow extract with Bifidobacterium is more effective at scavenging hydroxyl radicals than the single component, and is superior to the lysate obtained by fermenting sake yeast, thus exhibiting antioxidant effects.

[0119] Test Example 5: Irritation Test of Chicken Embryo Villi Allantoic Membrane

[0120] (1) Select SPF fertilized chicken embryos. When the chicken embryos are 9 days old, perform egg candling inspection and discard unfertilized and inactive chicken embryos. Select chicken embryos with well-developed blood vessels and mark the location of the air cell on the eggshell surface.

[0121] (2) Carefully remove the eggshell and shell membrane with tweezers, ensuring the exposed allantoic membrane remains intact and undamaged. Apply the test substance to the CAM. After the treatment period, observe the degree of change in each toxic effect under a stereomicroscope and assign a score (ES).

[0122] (3) Calculation formula:

[0123]

[0124] (4) Data analysis: After taking photos, the subjects were scored according to the degree of bleeding, vascular dissolution and coagulation, and the ES value was calculated. The irritation classification was determined according to Table 5.

[0125] Table 5 Irritation Classification

[0126] Finish line score Finish line score Irritant Classification Irritant Classification ES≤4 ES≤4 Non-irritating Non-irritating 4 < ES ≤ 12 4 < ES ≤ 12

[0127] Judgment criteria: If the reaction results of the negative control and positive control in the experiment fall within the classification ranges of non-irritating and strongly irritating, respectively, the test results are considered acceptable. A negative control (0.9% physiological saline) IS = 0.00 indicates that the sample is non-irritating and meets the standard for a negative control sample; a positive control (0.1 mol / L sodium hydroxide solution) IS = 10–19 indicates that the sample is strongly irritating / corrosive and meets the standard for a positive control sample. The irritant content of the sample is determined based on the ES test results.

[0128] (5) Experimental Results: The experimental results are shown in Table 6 and Figure 2 As shown.

[0129] Table 6 ES Values ​​and Irritation Classification

[0130]

[0131] Provided that the negative and positive controls of the test system meet the standards of the reference standard (negative control IS = 0.00, positive control IS = 15.63), when the sample concentration is 10%, the sample ES = 0.00, indicating that the sample is non-irritating and has mild efficacy.

[0132] Test Example 6: Inhibition of hyaluronidase

[0133] (1) Four groups (A, B, C, and D) were set up, with three replicates in each group. Calcium chloride, buffer solution, sample solution, sodium hydroxide, and acetylacetone were added to the test tubes in sequence, and the mixtures were then placed in boiling water, ice water, and room temperature environments for reaction. P-DAB colorimetric reagent was then added to the four tubes. After the solutions had reacted completely, the absorbance values ​​of each group were measured at a wavelength of 530 nm.

[0134] (2) Calculation formula:

[0135]

[0136] In the formula: A-- is the absorbance of the reaction solution containing the sample and enzyme;

[0137] B-- represents the absorbance of the reaction solution containing the sample and the reaction solution without the enzyme;

[0138] C -- is the absorbance of the reaction solution containing the enzyme but not the sample;

[0139] D -- Absorbance of the reaction solution without sample and enzyme.

[0140] (3) Results analysis: Under the condition of ensuring the effectiveness of the test system (the positive control hyaluronidase inhibition rate is >50%), if the hyaluronidase inhibition rate of the test sample is higher than that of the negative control hyaluronidase inhibition rate and there is a significant difference between the two (P<0.05), it can be determined that the test sample has a soothing effect.

[0141] (4) Experimental results:

[0142] Table 7 Experimental Results

[0143]

[0144] Under the condition that the experimental system is effective (the positive control hyaluronidase inhibition rate is >50%), when the sample is the original solution, the hyaluronidase inhibition rate is 70.257%, which is significantly different from the negative control (P<0.05), indicating that the sample has a soothing effect.

[0145] Test Example 7: Moisturizing Performance Test

[0146] The moisturizing rate of the sample and the positive control was calculated by measuring the ratio of the sample amount to the initial sample amount after 1h, 2h and 4h of storage.

[0147] (1) Weigh the glass plate with 3cm adhesive tape attached using an analytical balance (M0);

[0148] (2) Use a glass rod to spread the sample evenly on a glass plate covered with 3cm tape, and weigh the mass (M1) using an analytical balance;

[0149] (3) Then place it in a constant temperature and humidity chamber (temperature: 20℃, relative humidity 50%±5%).

[0150] (4) Calculation formula:

[0151]

[0152] In the formula: Mt -- the mass of the sample (positive control) and the plate after 1h, 2h, and 4h;

[0153] M0 -- The mass of the empty board;

[0154] M -- Mass of the sample (positive control) taken.

[0155] Table 8 Experimental Results

[0156] Moisturizing rate (%) 1h 2h 4h Example 1 51.78±2.276 28.56±1.466 26.95±0.008 3% glycerin 46.80±0.278 14.86±0.926 7.91±0.689

[0157] The moisturizing rates of the sample at 1h, 2h, and 4h were 51.78%, 28.56%, and 26.95%, respectively. It can be seen that the moisturizing rates of the tested sample at 1h, 2h, and 4h were all higher than those of the positive control, indicating that the sample has moisturizing effect.

[0158] Test Example 8: Elastase Inhibition Experiment

[0159] (1) Set up four groups, A, B, C, and D, with three replicates in each group. Add buffer solution, sample solution, enzyme solution, and substrate to the 96-well plate in sequence. Place the 96-well plate in a low-temperature shaking water bath at a certain temperature and turn on the shaking mode. After the solution inside has reacted completely, measure the absorbance value of each group at a wavelength of 410 nm.

[0160] (2) Calculation formula:

[0161]

[0162] In the formula: A-- is the absorbance of the reaction solution containing the sample and enzyme;

[0163] B-- represents the absorbance of the reaction solution containing the sample and the reaction solution without the enzyme;

[0164] C -- is the absorbance of the reaction solution containing the enzyme but not the sample;

[0165] D -- Absorbance of the reaction solution without sample and enzyme.

[0166] (3) Experimental results:

[0167] Table 9 Experimental Results

[0168] name Experimental results unit p-value Example 1 35.766±0.403 ％ <0.05 5% Example 1 24.769±1.322 ％ <0.05 buffer solution -1.801±0.566 ％ / 1% EGCG solution 88.603±0.299 ％ <0.05

[0169] The test results showed that, under the premise of ensuring the effectiveness of the experimental reaction system (positive control elastase inhibition rate > 50%), when the sample concentration was 5%, the elastase inhibition rate was 24.769%, and when the sample concentration was 100%, the elastase inhibition rate was 35.766%, which was significantly different from the negative control (P < 0.05), indicating that the sample concentrations of 5% and 100% had anti-wrinkle effects.

[0170] Test Example 9: Removal of Reactive Oxygen Species (ROS) from Zebrafish Embryos

[0171] (1) Twenty-four 48-h zebrafish embryos were exposed to sample solutions of Example 1 with formulations of 0.5%, 1%, 3%, 5%, and 10% respectively. A blank control group was set up at the same time. After 24 hours of exposure, the fish embryos were stained with H2DCFDA, and the ROS signal intensity was measured by fluorescence imaging and statistical analysis was performed.

[0172] (2) The test results are shown in Table 10 and Figure 3 As shown:

[0173] Table 10 Experimental Results

[0174]

[0175] Test results show that the formulations of the sample solutions in Example 1 with addition amounts of 0.5%, 1%, 3%, 5%, and 10% achieved ROS scavenging rates of 29%, 48%, 47%, 59%, and 62% respectively in zebrafish embryos. The samples can significantly remove ROS from zebrafish embryos, demonstrating antioxidant effects and supporting claims of anti-wrinkle and firming efficacy.

[0176] Test Example 10: Zebrafish Embryo Neutrophil Test Method

[0177] (1) A model of neutrophil aggregation induced by damage to neurothalamic cells in the lateral line region of zebrafish embryos using copper sulfate was tested. Twenty-four fish embryos were exposed to 10 μM anhydrous copper sulfate and the sample solution from Example 1. A blank control group, a positive control group, and a model control group were set up. After 40 min of exposure, the fish embryos were fixed and stained with Sudan Black. The number of neutrophils in the lateral line region was counted and statistically analyzed.

[0178] (2) The test results are shown in Table 11 and Figure 4 As shown:

[0179] Table 11 Experimental Results

[0180]

[0181] Test results showed that the formulations of the sample solutions in Example 1 with added concentrations of 0.5%, 1%, 3%, 5%, and 10% inhibited the aggregation of neutrophils in zebrafish embryos by 13%, 12%, 11%, 20%, and 28%, respectively. The samples significantly inhibited the aggregation of neutrophils in zebrafish embryos and had a soothing effect, supporting the claim of soothing efficacy.

[0182] Test Example 11: Zebrafish Embryo Melanin Inhibition Test

[0183] (1) Twenty 8-hour zebrafish embryos were exposed to sample solutions of Example 1 with formulations of 0.5%, 1%, 3%, 5%, and 10% concentration, respectively. A blank control group, a 100% melanin inhibition model group (30 mg / L phenylthiourea), and a positive control group (2.5 g / L kojic acid) were set up. After 48 hours of exposure, the fish embryos were photographed under a microscope to measure the melanin signal intensity and perform statistical analysis.

[0184] (2) The test results are shown in Table 12 and Figure 5 As shown:

[0185] Table 12 Experimental Results

[0186]

[0187] Test results showed that the formulations of the sample solutions in Example 1 with added concentrations of 0.5%, 1%, 3%, 5%, and 10% exhibited melanin inhibition rates of 7.96%, 6.52%, 9.41%, 10.36%, and 16.80%, respectively. The samples significantly inhibited melanin production in zebrafish embryos and had a whitening effect, which can serve as supporting evidence for melanin inhibition efficacy, but does not replace human efficacy testing.

[0188] In this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components, unless otherwise explicitly limited. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0189] In this invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can mean that the first feature is in direct contact with the second feature, or that the first feature is in indirect contact with the second feature through an intermediate medium. Furthermore, "above," "over," and "on top" of the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply that the first feature is at a lower horizontal level than the second feature.

[0190] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0191] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing lysate from Bifidobacterium-fermented honey and yarrow extract, characterized in that: Includes the following steps: S1. Preparation of Bifidobacterium activation medium: Prepare the medium, dispense it into Erlenmeyer flasks, sterilize it at high temperature, and cool it to room temperature for use as a strain for activation. S2. Propagation of bacterial culture: Bifidobacterium strains are inoculated into the culture medium and anaerobic fermented in a constant temperature shaking incubator to obtain Bifidobacterium strain propagation solution. S3. Preparation of the composition: The yarrow herb is pulverized and mixed with a solvent to obtain a mixture. The mixture is extracted by a microfluidic extractor. The yarrow extract pulp and honey are mixed in proportion and sterilized at high temperature in a fermenter. S4. Anaerobic fermentation culture: The Bifidobacterium inoculum propagation liquid obtained in S2 is inoculated into the mixture obtained in S3 and anaerobic fermentation culture is carried out to obtain the composite fermentation broth. S5. The fermentation broth of the above composition is inactivated by low-temperature microfluidic disruption, centrifuged, and the supernatant is collected to obtain the Bifidobacterium lysate. The Bifidobacterium activation culture medium in S1 comprises the following components in parts by weight: yeast extract 3-4 g / L, bacteriological peptone 6-8 g / L, honey 50-100 g / L, beef extract powder 2-10 g / L, magnesium sulfate 0.1-0.3 g / L, sodium acetate 4-6 g / L, diammonium citrate 1-3 g / L, dipotassium hydrogen phosphate 1-3 g / L, manganese sulfate 0.02-0.04 g / L, Tween 80 1.0-1.5 g / L, with the remainder made up with pure water.

2. The method for preparing the lysate of Bifidobacterium-fermented honey and yarrow extract according to claim 1, characterized in that: The solvent in S3 is water and / or a lower alcohol, wherein the lower alcohol is propylene glycol, glycerol, or butylene glycol, and the mass ratio of the solvent to yarrow is 15-30:

1.

3. The method for preparing the lysate of Bifidobacterium-fermented honey and yarrow extract according to claim 2, characterized in that: The propylene glycol is 1,2-propanediol, the butanediol is 1,3-butanediol, and the mass ratio of the solvent to yarrow is 20:

1.

4. The method for preparing the lysate of Bifidobacterium-fermented honey and yarrow extract according to claim 1, characterized in that: The preparation of the composition in step S3 includes the following components in parts by weight: 10-15 parts honey, 1-2 parts yarrow extract, and the remainder made up with pure water.

5. The method for preparing the lysate of Bifidobacterium-fermented honey and yarrow extract according to claim 1, characterized in that: The microfluidic extractor has a pore size of 800µm, and the extraction conditions are as follows: feeding rate of 15-25L / min, temperature of 20-35℃, stirring speed of 300-700rpm, and stirring time of 20-40min.

6. The method for preparing the lysate of Bifidobacterium-fermented honey and yarrow extract according to claim 1, characterized in that: The high-temperature sterilization temperature in S3 is 115℃, and the time is 15 minutes.

7. The method for preparing the lysate of Bifidobacterium-fermented honey and yarrow extract according to claim 1, characterized in that: The amount of Bifidobacterium strain introduced into S2 is 3-6% of the volume of the activated culture medium.

8. The method for preparing the lysate of Bifidobacterium-fermented honey and yarrow extract according to claim 1, characterized in that: The amount of Bifidobacterium bacterial propagation solution in S4 is 7-12% by volume of the mixture; The anaerobic fermentation culture conditions are: 160 rpm, 37°C, and 24 h.

9. The method for preparing the lysate of Bifidobacterium-fermented honey and yarrow extract according to claim 1, characterized in that: The centrifugation equipment in S5 is a high-speed centrifuge with a rotation speed of 8000-10000 rpm and a time of 10-30 min.

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