Preparation process of lysate of bifidobacterium fermented honey and achillea millefolium extract

Abstract

The invention relates to a preparation process of lysate of bifidobacterium fermented honey and achillea millefolium extract. The preparation process comprises the following steps: preparing a bifidobacterium activation culture medium; propagating the strain liquid; preparing the composition; carrying out anaerobic fermentation culture; and finally, carrying out low-temperature microjet wall-breaking inactivation and centrifugation on the fermentation liquor of the composition, and taking supernate to obtain a bifidobacterium lysate. Compared with a blank culture medium, the total number of viable bacteria and the OD value can be obviously increased by adding glucose or honey as the carbon source, and when the honey is further selected as the carbon source, the total number of viable bacteria obtained after fermentation culture of the culture medium is larger, and the OD value is higher.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetic processing, and specifically to a preparation process for the lysate of bifidobacterium-fermented honey and yarrow extract. Background Art

[0002] As people age, the number of dermal fibroblasts in the human skin gradually decreases, and the ability to synthesize collagen is reduced. Moreover, due to the increased release of proteases, collagen degradation is promoted, and elastic fibers will degrade and break, resulting in skin relaxation and thus the formation of wrinkles. On the other hand, ultraviolet and visible light have a significant impact on inducing free radicals and ROS, and thus activating inflammatory cytokines and matrix metalloproteinases, etc. Inflammatory reactions will damage the skin barrier. Due to local metabolic disorders and the aggregation of neutrophils, macrophages, etc., excessive free radicals are easily generated, causing tissue damage. Excessive free radicals in the body and reactive oxygen species generated by oxidative stress easily lead to cell damage and collagen inactivation, accelerating the aging of the body and skin. When matrix metalloproteinases are overexpressed, they specifically degrade the components of the extracellular matrix, destroying the normal structure of collagen fibers and elastic fibers, resulting in aging manifestations such as wrinkles on the skin.

[0003] The main components of honey are glucose, fructose and water, as well as proteins, amino acids, polyphenols, organic acids and minerals, etc. It is a natural food with both medicinal and edible properties. Antioxidant factors such as phenolic compounds in honey can scavenge excessive free radicals accumulated in the human metabolic process, promote the metabolism of skin cells, and delay skin aging. Honey can play an anti-inflammatory role by regulating the levels of pro-inflammatory factors, C-reactive protein and anti-inflammatory factors.

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

[0005] The yarrow herb contains a rich variety of chemical components, mainly flavonoid compounds and their glycosides, phenolic acids, alkaloids, etc. The inhibition of elastase by the yarrow extract and the proliferation effect on epidermal keratinocytes have the functions of restoring the elasticity of skin fibrous tissue, balancing and accelerating the metabolism of skin cells.

[0006] Technical Problem:

[0007] (1) Honey contains rich proteins. When directly used as an ingredient added to cosmetics, due to the characteristics of macromolecular proteins in honey, it is unstable in the cosmetic system and will produce turbidity or precipitation phenomena, such as Figure 1As shown, through the biological fermentation process, macromolecular proteins are decomposed into small molecule peptides and other substances, which are more stable in the system and easier to be absorbed by the skin.

[0008] (2) At present, the most commonly used strain for honey fermentation is Saccharomyces sake. The alcohol substances produced during the fermentation process may not only irritate the skin, but also cause damage to the skin barrier if used for a long time. Bifidobacterium is used as the strain to ferment honey. Honey contains rich nutrients such as sugars, proteins, and amino acids, which can provide carbon and nitrogen sources for Bifidobacterium during the fermentation process, more favorably promoting the proliferation of Bifidobacterium. The fermentation product still retains the characteristic flavor of honey, and the fermentation of Bifidobacterium and honey can exert a synergistic effect in terms of efficacy.

[0009] (3) Although honey, Bifidobacterium products, and Achillea millefolium extracts all have corresponding anti-aging effects, due to the fact that skin aging is the result of the combined action of multiple factors, there is a problem of a single target pathway. Summary of the Invention

[0010] The purpose of the present invention is to provide a preparation process for the lysate of Bifidobacterium-fermented honey and Achillea millefolium extract to solve the problems raised in the above background technology.

[0011] To achieve the above purpose, the present invention provides the following technical solution: A preparation process for the lysate of Bifidobacterium-fermented honey and Achillea millefolium extract, including the following steps:

[0012] S1. Prepare the Bifidobacterium activation medium: Prepare the medium, dispense it into Erlenmeyer flasks, and then perform high-temperature sterilization. Cool it to room temperature for standby as the strain activation.

[0013] S2. Expand the strain liquid: Inoculate the Bifidobacterium strain into the medium and perform anaerobic fermentation culture in a constant temperature shaker to obtain the Bifidobacterium strain expansion liquid.

[0014] S3. Prepare the composition: Crush the Achillea millefolium medicinal material and mix it with a solvent to obtain a mixed liquid. The mixed liquid is extracted by a microfluidic extractor. Mix the original extract of Achillea millefolium and honey according to the number of parts, and perform high-temperature sterilization in a fermentation tank.

[0015] S4. Anaerobic fermentation culture: Inoculate the Bifidobacterium strain expansion liquid obtained in S2 into the mixed liquid obtained in S3, and perform anaerobic fermentation culture to obtain the composition fermentation liquid.

[0016] S5. Break the wall and inactivate the above composition fermentation liquid through low-temperature microfluidics, centrifuge, and take the supernatant to obtain the Bifidobacterium lysate.

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

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

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

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

[0021] In some embodiments, the composition preparation in step S3 comprises the following components in parts by mass: 5 - 20 parts of honey, 0.5 - 2 parts of Achillea millefolium extract, and the remaining is made up to the mass with pure water; preferably, the composition comprises the following components in parts by mass: 10 - 15 parts of honey, 1 - 2 parts of Achillea millefolium extract, and the remaining is made up to the mass with pure water.

[0022] In some embodiments, the micropore aperture of the low - temperature micro - jet extractor is 800 μm, and the conditions for extraction by the low - temperature micro - jet extractor are: the feeding rate is 15 - 25 L / min, the temperature is 20 - 35 °C, the stirring speed is 300 - 700 rpm, and the stirring time is 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. Preferably, the high - temperature sterilization temperature is 115 °C, and the time is 15 min.

[0024] In some embodiments, the inoculation amount of the Bifidobacterium strain in S2 is 3 - 15% by volume of the activation medium. Preferably, the inoculation amount of the Bifidobacterium strain is 3 - 6% by volume of the activation medium.

[0025] In some embodiments, the inoculation amount of the Bifidobacterium strain propagation liquid in S4 is 4-20% by volume ratio of the mixed liquid. Preferably, the inoculation amount of the Bifidobacterium strain propagation liquid is 7-12% by volume ratio of the mixed liquid.

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

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

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

[0029] (1) Compared with the blank medium, adding glucose or honey as a carbon source in the present invention 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 after fermentation culture with this medium is more and the OD value is higher.

[0030] (2) Through the specific process parameters of low-temperature microfluidics, the present invention can achieve the effect of highly efficient cell wall breaking of Achillea millefolium herbs. After cell wall breaking, the solvent fully penetrates into the herbs, enabling its active ingredients to be better dissolved, achieving high extraction rate and strong efficacy of the extract. The Bifidobacterium lysate is extracted by low-temperature microfluidics cell wall breaking, achieving cell wall breaking and inactivation of Bifidobacterium, and at the same time being further decomposed into substances with smaller molecular weights, which can be better absorbed by the skin.

[0031] (3) Using the Bifidobacterium activated by the above medium as the fermentation strain of the honey and Achillea millefolium composition in the present invention has no irritation to the skin, 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 MMPs-1 and elastase to elastin; the combined fermentation product can inhibit the expression of inflammatory factors, inhibit the activity of hyaluronidase to reduce inflammation, repair the skin epidermal barrier, maintain skin elasticity, and reduce the generation of wrinkles by promoting the regenerative expression of collagen and elastin, achieving multi-target and multi-faceted skin anti-aging.

[0032] The above description is only an overview of the technical solutions of the embodiments of the present application. In order to be able to understand the technical means of the embodiments of the present application more clearly, it can be implemented according to the content of the description. And in order to make the above and other purposes, features and advantages of the embodiments of the present application more obvious and understandable, the specific embodiments of the present application are hereinafter specifically exemplified. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] To more clearly illustrate the technical solutions of the embodiments of the present application, the accompanying drawings required for the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on these drawings.

[0034] Figure 1 It is a schematic diagram of the stability of adding unfermented honey and fermented honey in the product respectively.

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

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

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

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

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are some, but not all, of the embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present application without creative efforts fall within the scope of protection of the present application.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs; the terms used in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification, claims, and drawings of this application are intended to cover non-exclusive inclusion.

[0041] Reference to "embodiments" herein means that the specific features, structures, or characteristics described in connection with the embodiments can be included in at least one embodiment of this application. The phrase "embodiments" appearing in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. Those skilled in the art will explicitly and implicitly understand that the embodiments described herein can be combined with other embodiments.

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

[0043] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential", etc. indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present invention.

[0044] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include at least one of such features. In the description of the present invention, "a plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0045] Example 1

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

[0047] (1) Prepare a culture medium, which includes the following components in parts 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 the remaining is made up to the mass with pure water.

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

[0049] (3) Inoculate 5% of Bifidobacterium in the sterilized culture medium above, place it in a constant temperature shaker and anaerobically culture at 37 °C for 24 h, and measure the OD value;

[0050] (4) Extract Achillea millefolium herbs by low-temperature microfluidics. The mass ratio of the solvent to Achillea millefolium is 20:1. Mix 1 part of the original Achillea millefolium extract and 13 parts of honey, and make up to the mass with pure water;

[0051] (5) Place the above-mentioned mixed solution in a fermenter and sterilize it at 115 °C for 15 min;

[0052] (6) Inoculate the activated Bifidobacterium in step 3 into the mixed solution at an inoculation amount of 8% of the mass of the mixed solution, and anaerobically culture it at 37 °C for 24 h;

[0053] (7) Inactivate the above-mentioned fermentation product by low-temperature microfluidic cell disruption, centrifuge it at 10000 rpm for 10 min, take the supernatant, and obtain the cell lysate.

[0054] Example 2

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

[0056] Example 3

[0057] A cell lysate of Bifidobacterium fermented honey and yarrow extract, which is different from Example 1 in that the yarrow medicinal material is extracted by low-temperature microfluidics, the mass ratio of the solvent to yarrow is 30:1, and 0.5 parts of the yarrow extract original pulp and 5 parts of honey are mixed evenly;

[0058] Example 4

[0059] A cell lysate of Bifidobacterium fermented honey and yarrow extract, which is different from Example 1 in that the activated Bifidobacterium is inoculated into the mixed solution at an inoculation amount of 5% of the mass of the mixed solution;

[0060] Comparative Example 1

[0061] A cell lysate of Bifidobacterium fermented honey and yarrow extract, which is different from Example 1 in that honey is not added to the culture medium;

[0062] Comparative Example 2

[0063] A Bifidobacterium cell lysate, and its preparation process is as follows:

[0064] (1) Prepare a culture medium, which includes the following components in parts 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 the remaining is made up to the mass with pure water.

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

[0066] (3) Inoculate the sterilized culture medium with Bifidobacterium at an inoculum size of 5%, and place it in a constant temperature shaker for anaerobic culture at 37°C for 24 h, then measure the OD value.

[0067] (4) Inactivate the Bifidobacterium fermentation broth by low-temperature microfluidic cell disruption, centrifuge at 10000 rpm for 10 min, take the supernatant to obtain the Bifidobacterium lysate.

[0068] Comparative Example 3

[0069] A lysate of Bifidobacterium-fermented honey and Achillea millefolium extract, which is different from Example 1 in that Achillea millefolium extract is not added in step (4);

[0070] Comparative Example 4

[0071] A lysate of Bifidobacterium-fermented honey and Achillea millefolium extract, which is different from Example 1 in that honey is not added in step (4);

[0072] Comparative Example 5

[0073] A lysate of sake yeast-fermented honey and Achillea millefolium extract, which is different from Example 1 in that the inoculated strain is sake yeast, and the culture medium includes the following components in parts by mass: yeast extract 10 g / L, bacteriological peptone 20 g / L, honey 100 g / L, and the remaining is made up to the mass with pure water;

[0074] Test Example 1 OD value of test samples

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

[0076] (1) Shake the strain solution well, use a pipette to transfer 200 μL of the strain solution into a 96-well plate, and use a microplate reader to measure the absorbance value of the strain solution at 600 nm.

[0077] (2) Experimental results:

[0078] Table 1 OD values of Bifidobacterium strain solutions

[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 Strain quantity of test samples

[0081] This test example is used to determine the strain quantities of different culture media of Bifidobacterium in Examples 1, 2 and Comparative Examples 1, 2. The specific determination method is as follows:

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

[0083] (3) Pour out the centrifuged supernatant, transfer the bottom precipitate to an evaporating dish, and dry it in a blast drying oven at 60 °C. Wait until the mass of the evaporating dish containing the sample is constant, record the final mass, denoted as M. 1；

[0084] (4) The formula for calculating the total mass M of the bacteria: M = M1 - M0

[0085] (5) Experimental results:

[0086] Table 2 Total mass of Bifidobacterium strains

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

[0088] From the relevant data in Table 1 and Table 2, it is found that when adding 50 g / L and 100 g / L of honey as carbon sources to the medium, compared with using glucose as the carbon source and the blank carbon source in the medium, it can significantly increase the total number of Bifidobacterium strains and the OD value; when further selecting 100 g / L of honey as the carbon source of the medium, the total number of viable bacteria after fermentation and culture with this medium is more and the OD value is higher.

[0089] Test Example 3 DPPH free radical scavenging experiment

[0090] This test example is used to conduct antioxidant tests on Examples 1-4 and Comparative Examples 1-4, specifically for DPPH free radical scavenging experiment tests. The measurement method is as follows:

[0091] (1) In a 10 ml test tube, add 4.0 ml of DPPH solution and 1.0 ml of 95% ethanol in sequence, mix well, react in the dark for 30 min. After stabilization, using 95% ethanol as the reference, measure the absorbance at 517 nm, denoted as A0.

[0092] (2) In a 10 ml test tube, add 4.0 ml of DPPH solution and 1.0 ml of the test sample solution in sequence, mix well, react in the dark for 30 min. After stabilization, using 95% ethanol as the reference, measure the absorbance at 517 nm, denoted as Ar.

[0093] (3) In a 10 ml test tube, add 4.0 ml of 95% ethanol solution and 1.0 ml of the test sample solution in sequence, mix well, react in the dark for 30 min. After stabilization, using 95% ethanol as the reference, measure the absorbance at 517 nm, denoted as As.

[0094] (4) Calculation formula:

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

[0096] In the formula: Ar--Absorbance of the DPPH solution after reacting with the test solution;

[0097] As - Absorbance of the solution with only the test solution and 95% ethanol added, without DPPH.

[0098] A0 - Absorbance of the solution with only DPPH and 95% ethanol added, without the test solution.

[0099] (5) Experimental results:

[0100] Table 3 Experimental results table

[0101]

[0102]

[0103] According to the comparison of experimental data, the cell lysate obtained by fermenting honey and yarrow extract with Bifidobacterium has a better effect on scavenging DPPH free radicals than single components, and is superior to the cell lysate fermented by Saccharomyces sake, showing antioxidant efficacy.

[0104] Test Example 4 Hydroxyl radical scavenging experiment

[0105] This test example is used to conduct antioxidant tests on Examples 1 - 4 and Comparative Examples 1 - 4, specifically for the hydroxyl radical scavenging experiment test. The measurement 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 and mix well, denoted as AS;

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

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

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

[0110] (5) Calculation formula:

[0111]

[0112] Where: AS - Absorbance after reacting with the sample solution;

[0113] A - Absorbance measured without the sample;

[0114] A0--Absorbance of the chromogenic agent;

[0115] (6) Experimental results

[0116] Table 4 Experimental result table

[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% Comparative Example 5 36.49 1% Vitamin C Ethyl Ether 78.70

[0118] According to the comparison of experimental data, the lytic products obtained by fermenting honey and yarrow extract with Bifidobacterium have a better effect on scavenging hydroxyl radicals than single components, and are superior to the lytic products of Saccharomyces sake fermentation, with antioxidant effects.

[0119] Test Example 5 Chicken embryo chorioallantoic membrane irritation experiment

[0120] (1) Select SPF fertilized chicken embryos. When the chicken embryos are at 9 days old, candling inspection is carried out to discard unfertilized and inactive chicken embryos, and chicken embryos with well-developed blood vessels are selected, and the position of the air chamber is marked on the eggshell surface.

[0121] (2) Carefully remove the eggshell and eggshell membrane with forceps to ensure that the exposed chorioallantoic membrane is intact without any damage. Apply the test substance to the CAM. After the action time ends, observe the degree of change of each toxic effect under a stereomicroscope and give a score (ES).

[0122] (3) Calculation formula:

[0123]

[0124] (4) Data analysis: After taking pictures, score according to the degree of bleeding, blood vessel lysis and blood coagulation of the test chicken embryos, and calculate the ES value. Judge the irritation classification according to Table 5.

[0125] Table 5 Irritation classification

[0126] Final score Final score Irritation classification Irritation classification ES ≤ 4 ES ≤ 4 Non-irritating Non-irritating 4 < ES ≤ 12 4 < ES ≤ 12

[0127] Judgment criteria: If the reaction results produced by the negative control and positive control set in the test are exactly within the non-irritating and strongly irritating classification ranges respectively, the test results are considered acceptable. The negative control (0.9% normal saline) IS = 0.00, indicating that the sample is non-irritating, then it meets the standard of the negative control sample; the positive control (0.1 mol / L sodium hydroxide solution) IS = 10 - 19, indicating that the sample is strongly irritating / corrosive and meets the standard of the positive control sample; judge the irritation contained in the sample according to the ES test result of the sample.

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

[0129] Table 6 ES value and irritation classification

[0130]

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

[0132] Test Example 6: Inhibition of Hyaluronidase

[0133] (1) Set up four groups A, B, C, and D, with 3 parallels in each group. Add calcium chloride, buffer solution, sample solution, sodium hydroxide, acetylacetone and other solutions into the test tubes in sequence. After mixing, place them in boiling water, ice water, and room temperature environments for reaction respectively, and then add P-DAB color reagent to the four tubes. After the solutions inside react completely, measure the absorbance values of each group at a wavelength of 530 nm.

[0134] (2) Calculation formula:

[0135]

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

[0137] B--is the absorbance of the reaction solution containing the sample but without the enzyme;

[0138] C--is the absorbance of the reaction solution containing the enzyme but without the sample;

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

[0140] (3) Result analysis: When the test system is effective (the inhibition rate of the positive control hyaluronidase > 50%), if the inhibition rate of the test sample's hyaluronidase is higher than that of the negative control sample's hyaluronidase, 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 Table

[0143]

[0144] When the test system is effective (the inhibition rate of the positive control hyaluronidase > 50%), when the sample is the stock solution, the inhibition rate of hyaluronidase is 70.257%, and there is a significant difference compared with the negative control (P < 0.05), indicating that the sample has a moisturizing effect.

[0145] Test Example 7: Moisture Retention Performance Test

[0146] The moisturizing rate of the sample and the positive control was calculated by measuring the ratio of the sample amount after the aqueous sample and the positive control were placed for 1 h, 2 h, and 4 h to the initially weighed sample amount.

[0147] (1) Weigh the mass (M0) of the glass plate with a 3-cm adhesive tape using an analytical balance.

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

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

[0150] (4) Calculation formula:

[0151]

[0152] Where: Mt—the mass of the sample (positive control) and the plate after being placed for 1 h, 2 h, and 4 h;

[0153] M0—the mass of the empty plate;

[0154] M—the mass of the weighed sample (positive control).

[0155] Table 8 Experimental result table

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

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

[0158] Test example 8 Elastase inhibition experiment

[0159] (1) Set four groups A, B, C, and D respectively, with 3 parallels 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] Where: A—the absorbance of the reaction solution containing the sample and the enzyme;

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

[0164] C is the absorbance of the reaction solution containing the enzyme but no sample;

[0165] D is the absorbance of the reaction solution without sample and enzyme.

[0166] (3) Experimental results:

[0167] Table 9 Experimental results table

[0168] Name Experimental result 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 show that: on the premise of ensuring the effectiveness of the test reaction system (the elastase inhibition rate of the positive control > 50%), when the sample concentration is 5%, the elastase inhibition rate is 24.769%, and when the sample concentration is 100%, the elastase inhibition rate is 35.766%, showing a significant difference compared with the negative control (P < 0.05), indicating that when the sample concentration is 5% and 100%, it has anti-wrinkle effects.

[0170] Test Example 9 Scavenging of reactive oxygen species (ROS) in zebrafish embryos

[0171] (1) Twenty-four 48-hour-old zebrafish embryos were respectively exposed to the sample solutions of Example 1 with formulation addition concentrations of 0.5%, 1%, 3%, 5%, and 10%. At the same time, a blank control group was set up. After 24 hours of exposure, the fish embryos were stained with H2DCFDA, and fluorescence photography was used to measure the ROS signal intensity and statistical analysis was carried out.

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

[0173] Table 10 Experimental results table

[0174]

[0175] The test results show that: the formulations of the sample solutions of Example 1 with addition amounts of 0.5%, 1%, 3%, 5%, and 10% have ROS scavenging rates for zebrafish embryos of 29%, 48%, 47%, 59%, and 62% respectively. The sample can significantly scavenge ROS in zebrafish embryos, has antioxidant effects, and supports the claims of anti-wrinkle and firming effects.

[0176] Test Example 10 Test method for neutrophils in zebrafish embryos

[0177] (1) The model of neutrophil aggregation induced by copper sulfate-induced injury of neuromast cells in the lateral line region of zebrafish embryos was used for testing. Twenty-four fish embryos were exposed to 10 μM anhydrous copper sulfate and the sample solution of Example 1. At the same time, 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 statistical analysis was performed.

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

[0179] Table 11 Experimental Results Table

[0180]

[0181] The test results show that the formulas of the sample solution of Example 1 with addition amounts of 0.5%, 1%, 3%, 5%, and 10% concentrations have neutrophil aggregation inhibition rates for zebrafish embryos of 13%, 12%, 11%, 20%, and 28% respectively. The sample can significantly inhibit neutrophil aggregation in zebrafish embryos, has a soothing effect, and supports the claim of soothing efficacy.

[0182] Test Example 11 Melanin Inhibition Test on Zebrafish Embryos

[0183] (1) Twenty 8-hour-old zebrafish embryos were respectively exposed to the sample solution of Example 1 with formulation addition concentrations of 0.5%, 1%, 3%, 5%, and 10%. At the same time, 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 h of exposure, the fish embryos were photographed under a microscope to measure the melanin signal intensity and statistical analysis was performed.

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

[0185] Table 12 Experimental Results Table

[0186]

[0187] The test results show that the formulas of the sample solution of Example 1 with addition amounts of 0.5%, 1%, 3%, 5%, and 10% concentrations have melanin inhibition rates for zebrafish embryos of 7.96%, 6.52%, 9.41%, 10.36%, and 16.80% respectively. The sample can significantly inhibit melanin production in zebrafish embryos, has a whitening effect, and can be used as supporting evidence for the melanin inhibition efficacy, but does not replace human efficacy testing.

[0188] In the present invention, unless otherwise clearly defined or limited, terms such as "installed", "connected", "coupled", "fixed", etc. shall be construed in a broad sense. For example, it may be a fixed connection, a detachable connection, or an integral one; it may be a mechanical connection or an electrical connection; it may be directly connected or indirectly connected through an intermediate medium, and it may be the internal communication of two components or the interaction relationship between two components, unless otherwise clearly defined. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0189] In the present invention, unless otherwise clearly defined or limited, the first feature being "on" or "under" the second feature may be that the first and second features are in direct contact, or the first and second features are indirectly in contact through an intermediate medium. Moreover, the first feature being "above", "over" and "on top of" the second feature may be that the first feature is directly above or obliquely above the second feature, or merely indicates that the first feature has a higher horizontal height than the second feature. The first feature being "under", "beneath" and "underneath" the second feature may be that the first feature is directly below or obliquely below the second feature, or merely indicates that the first feature has a lower horizontal height than the second feature.

[0190] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic descriptions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

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

Claims

1. Preparation process of lysate of honey and yarrow extract fermented by Bifidobacterium, characterized in that: The following steps are involved: S1. Preparation of Bifidobacterium activation culture medium: prepare the culture medium, dispense it into triangular bottles, sterilize it at high temperature, and cool it to room temperature for use as bacterial activation; S2, bacterial culture liquid expansion: inoculate the bifidobacterium strain into the culture medium and perform anaerobic fermentation in a constant temperature shaking incubator to obtain the bifidobacterium bacterial culture liquid; S3, preparation of the composition: grinding the Achillea millefolium material and mixing it with a solvent to obtain a mixed solution, extracting the mixed solution through a microfluidizer, mixing the Achillea millefolium extract puree and honey according to the number of portions, and sterilizing them at high temperature in a fermentation tank; S4, anaerobic fermentation culture: the bifidobacterium strain expansion liquid obtained in S2 is added to the mixed liquid obtained in S3, and anaerobic fermentation culture is performed to obtain a composite fermentation liquid; S5. Inactivate the fermentation broth of the above-mentioned composition by microfluidization, centrifuge, and take the supernatant to obtain a bifidobacterium lysate.

2. The preparation process of the lysate of bifidobacterium-fermented honey and yarrow extract according to claim 1, characterized in that: The bifidobacterium activation medium in S1 includes the following components in parts by weight: 2-6 g / L yeast extract, 4-10 g / L bacteriological peptone, 30-120 g / L honey, 2-10 g / L beef extract powder, 0.05-0.5 g / L magnesium sulfate, 3-8 g / L sodium acetate, 1-5 g / L diammonium citrate, 0.5-5 g / L dipotassium hydrogen phosphate, 0.01-0-0.06 g / L manganese sulfate, 0.5-2 g / L Tween 800, and the rest is supplemented with pure water; Preferably, the bifidobacterium activation medium comprises the following components in parts by mass: 3-4 g / L yeast extract, 6-8 g / L bacteriological peptone, 50-100 g / L honey, 2-10 g / L beef extract powder, 0.1-0.3 g / L magnesium sulfate, 4-6 g / L sodium acetate, 1-3 g / L diammonium citrate, 1-3 g / L dipotassium hydrogen phosphate, 0.02-0.04 g / L manganese sulfate, 1.0-1.5 g / L Tween 80, and the remainder is made up to the mass with pure water.

3. The preparation process of the lysate of bifidobacterium-fermented honey and yarrow extract according to claim 1, characterized in that: The solvent in S3 is water and / or lower alcohol, and the mass ratio of the solvent to the yarrow is 10-40:1; preferably, the lower alcohol is propylene glycol or glycerol or butylene glycol, and the mass ratio of the solvent to the yarrow is 15-30:

1.

4. The preparation process of the lysate of Bifidobacterium-fermented honey and yarrow extract according to claim 3, characterized in that: The propylene glycol is 1,2-propylene glycol, the butylene glycol is 1,3-butylene glycol, and the mass ratio of the solvent to the yarrow is 20:

1.

5. The preparation process of the lysate of bifidobacterium-fermented honey and yarrow extract according to claim 1, characterized in that: The composition preparation in step S3 includes the following components in parts by mass: 5-20 parts of honey, 0.5-2 parts of yarrow extract, and the mass is supplemented with pure water; preferably, the composition includes the following components in parts by mass: 10-15 parts of honey, 1-2 parts of yarrow extract, and the mass is supplemented with pure water.

6. The preparation process of the lysate of bifidobacterium-fermented honey and yarrow extract according to claim 1, characterized in that: The micropore diameter of the low-temperature microfluidic extractor is 800 μm. The extraction conditions of the low-temperature microfluidic extractor are: feeding speed of 15-25 L / min, temperature of 20-35° C., stirring speed of 300-700 rpm, and stirring time of 20-40 min.

7. The preparation process of the lysate of bifidobacterium-fermented honey and yarrow extract according to claim 1, characterized in that: The high temperature sterilization temperature in S3 is 100-120° C., and the time is 10-25 min. The preferred high temperature sterilization temperature is 115° C., and the time is 15 min.

8. The preparation process of the lysate of bifidobacterium-fermented honey and yarrow extract according to claim 1, characterized in that: The inoculation amount of the Bifidobacterium strain in S2 is 3-15% based on the volume ratio of the activation medium, and preferably, the inoculation amount of the Bifidobacterium strain is 3-6% based on the volume ratio of the activation medium.

9. The preparation process of the lysate of bifidobacterium-fermented honey and yarrow extract according to claim 1, characterized in that: The inoculation amount of the Bifidobacterium species propagation liquid in S4 is 4-20% based on the volume ratio of the mixed liquid. Preferably, the inoculation amount of the Bifidobacterium species propagation liquid is 7-12% based on the volume ratio of the mixed liquid. The conditions for anaerobic fermentation culture are: rotation speed 120-240 rpm, temperature 30-40 °C, and time 16-30 h. Preferably, the conditions for anaerobic fermentation culture are: rotation speed 160 rpm, temperature 37 °C, and time 24 h.

10. The preparation process of the lysate of bifidobacterium-fermented honey and yarrow extract according to claim 1, characterized in that: In S5, a high-speed centrifuge is used as the centrifugation equipment. The rotation speed of the high-speed separator is 8000-10000 rpm, and the time is 10-30 min.

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