Collagen microcapsule as well as preparation method and application thereof

Abstract

The invention discloses a collagen microcapsule as well as a preparation method and application thereof, and belongs to the technical field of biological material preparation. The collagen microcapsule comprises a collagen core material and a capsule material coated on the surface of the collagen core material, and the capsule material comprises a mitochondrial membrane. According to the collagen microcapsule and the preparation method thereof, the mitochondrial membrane is adopted as a wrapping material, accurate wrapping of the collagen can be achieved, the biocompatibility of the collagen and the fusion of the collagen and the skin are improved, the collagen microcapsule has a good anti-oxidation effect and can be used for resisting aging, and the prepared collagen microcapsule has good stability when dispersed in water at different temperatures and can be used for preparing the collagen microcapsule. And the application stability requirement in a solution state can be met. When the collagen microcapsule is applied to the skin care product, beneficial elements in the collagen can be better absorbed by the skin, and the use effect of the skin care product is improved.

Description

Technical Field

[0001] The present application relates to the technical field of biomaterial preparation, and in particular to a collagen microcapsule and a preparation method and application thereof. Background Art

[0002] Collagen is a biological macromolecular substance, accounting for 25% to 35% of the total protein in the human body. It is one of the most abundant proteins in the human body. It has been proven that collagen hydrolysate has significant biological activity, such as antioxidant properties, antihypertensive activity, lipid-lowering activity, and the ability to repair damaged skin. Collagen can supplement the nutrients needed by each layer of the skin, enhance the activity of collagen in the skin, and has the effects of repairing the skin, delaying aging, beautifying, and anti-wrinkle. It is widely used in skin care products. However, when using traditional collagen skin care products on the market, the collagen has poor transdermal properties and cannot be well integrated with human skin, resulting in the inability of the beneficial elements in it to be absorbed by the skin, thus affecting the use effect of the skin care products.

[0003] In order to improve the transdermal absorption of collagen, chemical permeation enhancers or biological enhancers can be used to encapsulate collagen. However, traditional encapsulation materials may cause immune responses in the human body, and it is difficult to achieve accurate encapsulation of collagen. The product is not stable and its functions are limited. Summary of the invention

[0004] Based on this, it is necessary to provide a collagen microcapsule and a preparation method and application thereof. By using mitochondrial membrane as a wrapping material, the collagen can be precisely wrapped, the biocompatibility of the collagen and the fusion with the skin are improved, and the prepared collagen microcapsule has good stability. When the collagen microcapsule is applied to skin care products, the beneficial elements in the collagen can be better absorbed by the skin, thereby improving the use effect of the skin care products.

[0005] In a first aspect of the present application, a collagen microcapsule is provided, comprising: a collagen core material, and a capsule material coated on the surface of the collagen core material;

[0006] The material of the capsule material includes mitochondrial membrane.

[0007] In some embodiments, the mass ratio of the collagen core material to the capsule material is 1-5:1.

[0008] In some embodiments, the collagen comprises recombinant collagen.

[0009] In some embodiments, the recombinant collagen includes at least one of recombinant human collagen, recombinant humanized collagen and recombinant collagen-like protein.

[0010] In some embodiments, the recombinant collagen includes at least one of type I recombinant collagen, type II recombinant collagen, type III recombinant collagen, type IV recombinant collagen, and type XVII recombinant collagen.

[0011] In some embodiments, the mitochondrial membrane comprises at least one of a yeast mitochondrial membrane, a bacterial mitochondrial membrane, a plant cell line mitochondrial membrane, and an animal cell mitochondrial membrane.

[0012] In some embodiments, the mitochondrial membrane preparation process comprises the following steps:

[0013] The raw material containing mitochondria is dispersed in a first extraction solvent, and the wall is broken to extract the mitochondria; the mitochondria are dispersed in a second extraction solvent, and the membrane is broken to extract the mitochondrial membrane.

[0014] In some embodiments, the first extraction solvent comprises a buffer solution of yeast cell wall-breaking enzyme and / or a buffer solution of snail enzyme;

[0015] The second extraction solvent includes water and sucrose;

[0016] And / or the wall breaking treatment and the membrane breaking treatment independently include physical breaking and / or chemical breaking.

[0017] The second aspect of the present application provides a method for preparing collagen microcapsules, comprising the following steps:

[0018] The collagen microcapsules are prepared by injecting the mitochondrial membrane solution and the collagen solution into different channels of the microfluidic device for mixing.

[0019] In some embodiments, the concentration of the mitochondrial membrane solution is 0.1-5 mg / mL;

[0020] And / or the concentration of the collagen solution is 0.1-20wt%.

[0021] In some embodiments, the solvent of the mitochondrial membrane solution and the solvent of the collagen solution are each independently selected from at least one of a buffer solution and water.

[0022] In some embodiments, the flow rate ratio of the mitochondrial membrane solution to the collagen solution is 1:1-5.

[0023] In some embodiments, the step of injecting the mitochondrial membrane solution and the collagen solution into different channels of the microfluidic device for mixing further includes a freeze-drying step.

[0024] In some embodiments, in the freeze-drying step, a protective agent is added before freeze-drying.

[0025] In some embodiments, the protective agent includes at least one of lecithin, mannitol, and cyclodextrin.

[0026] The third aspect of the present application provides the use of the aforementioned collagen microcapsules in skin care products.

[0027] The fourth aspect of the present application provides a skin care product, the raw materials of which include the aforementioned collagen microcapsules.

[0028] Beneficial effects of this application:

[0029] 1. This application uses yeast mitochondrial membrane as a collagen encapsulation material for the first time, achieving precise encapsulation of collagen, improving the biocompatibility of collagen and its integration with the skin, and has good antioxidant effects, which can be used for anti-aging. The prepared collagen microcapsules are dispersed in water and have good stability at different temperatures, which can meet the application stability requirements in the solution state.

[0030] 2. The collagen microcapsules prepared in this application have improved biocompatibility and safety, and reduce the risk of human immune response when used in medical cosmetology, skin repair and other fields.

[0031] 3. When the collagen microcapsules prepared in the present application are used in skin care products, due to their unique structure and biocompatibility, they help to improve the integration of collagen with the skin, so that the beneficial elements in the collagen can be better absorbed by the skin, thereby improving the use effect of the skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art description. The drawings are only used to illustrate the preferred implementation methods and are not considered to be limitations of the present application. The same reference symbols are used throughout the drawings to represent the same components. In the drawings:

[0033] Figure 1 is a SEM image of the collagen microcapsule powder of Example 1;

[0034] Figure 2 This is the Dio staining image of the mitochondrial membrane in Preparation Example 1. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of this application clearer and to provide a more thorough and comprehensive understanding of the disclosed content of this application, the technical solution of this application will be clearly and completely described below in conjunction with the specific embodiments of this application and the corresponding drawings. The described embodiments are only part of the embodiments of this application, not all of them.

[0036] The implementation of the present application is described in detail below in conjunction with the accompanying drawings. This embodiment is implemented based on the technical solution of the present application, and a detailed implementation method and specific operation process are given, but the protection scope of the present application is not limited to the following embodiments.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0038] the term

[0039] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0040] In the present application, "plurality", "multiple" and the like, unless otherwise specified, refer to a number greater than or equal to 2. For example, "one or more" or "at least one" means one or greater than or equal to two.

[0041] In the present application, the terms "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0042] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0043] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0044] In the present application, the term "room temperature" generally refers to 4-35° C., preferably 20±5° C. In the embodiments of the present application, room temperature refers to 20-30° C.

[0045] In this application, unless otherwise specified, the temperature parameter is allowed to be either a constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C and ±1°C are allowed.

[0046] In this application, when referring to the unit of a data range, if there is a unit only after the right endpoint, it means that the units of the left and right endpoints are the same. For example, 2-5h means that the units of the left endpoint "2" and the right endpoint "5" are both h (hours).

[0047] In a first aspect of the present application, a collagen microcapsule is provided, comprising: a collagen core material, and a capsule material coated on the surface of the collagen core material;

[0048] The material of the capsule material includes mitochondrial membrane.

[0049] Mitochondria are the bioenergetics center of eukaryotic cells and are the organelles that play a primary role in energy generation and oxidative stress. Mitochondria not only provide more than 90% of the energy source for cells, but also deeply regulate energy metabolism, ROS and free radicals, oxidative stress, inflammation, wound healing, autophagy, calcium homeostasis, stem cells, tissue reconstruction, pigmentation, biological rhythms and hair growth. In addition, mitochondria are considered to be the main cause of cell or organism aging. Mitochondrial dysfunction leads to skin aging and damage, and targeting mitochondria helps restore skin vitality and accurately solves the problem of deep repair and anti-aging.

[0050] As a natural material, mitochondrial membrane has excellent biocompatibility and can be used as a carrier for mitochondrial targeted delivery. Using mitochondrial membrane to wrap collagen can promote its absorption in the skin, give full play to the efficacy of collagen, and improve the biocompatibility and safety of collagen. When used in medical beauty, skin repair and other fields, it reduces the risk of human immune response.

[0051] Specifically, the mass ratio of the collagen core material to the capsule material is 1-5:1, for example, 1:1, 2:1, 3:1, 4:1, 5:1, etc.

[0052] In some embodiments, the collagen comprises recombinant collagen. Optionally, the recombinant collagen comprises at least one of recombinant human collagen, recombinant humanized collagen and recombinant collagen-like protein.

[0053] It should be noted that there is no special requirement for the type of collagen, and it can be a single collagen or a composite of multiple collagens. Among them, recombinant collagen has better biocompatibility with the human body and is more preferred. In addition, the recombinant collagen can be divided into type I, type II, type III, type IV and type XVII.

[0054] In some embodiments, the mitochondrial membrane comprises at least one of a yeast mitochondrial membrane, a bacterial mitochondrial membrane, a plant cell line mitochondrial membrane, and an animal cell mitochondrial membrane.

[0055] It should be noted that the source of the mitochondrial membrane is not particularly limited, and any mitochondrial membrane can be provided. Among them, yeast mitochondrial membrane is a more common source and is more preferred.

[0056] In some embodiments, the mitochondrial membrane preparation process comprises the following steps:

[0057] The raw material containing mitochondria is dispersed in a first extraction solvent, and the wall is broken to extract the mitochondria; the mitochondria are dispersed in a second extraction solvent, and the membrane is broken to extract the mitochondrial membrane.

[0058] In some embodiments, the first extraction solvent includes a buffer solution of yeast cell wall-breaking enzyme and / or a buffer solution of snail enzyme; the second extraction solvent includes water and sucrose; the cell wall-breaking treatment and the membrane-breaking treatment each independently include physical disruption and / or chemical disruption.

[0059] It should be noted that in the preparation process of mitochondrial membrane, the methods of wall breaking and membrane disruption are not particularly limited, as long as they can play the role of wall breaking and membrane disruption. The physical disruption includes at least one of ultrasound, homogenization, homogenization and vibration; the chemical disruption includes enzymatic hydrolysis.

[0060] Mitochondria are separated by differential centrifugation, that is, low-speed centrifugation is first performed to separate mitochondria, at which time the supernatant is obtained, and then the supernatant is centrifuged at high speed to enrich mitochondria.

[0061] Optionally, the step of separating mitochondria includes: centrifuging at 300×g-700×g for 3min-8min, taking the supernatant, centrifuging at 10000×g-20000×g for 15min-30min, and taking the precipitate. Specifically, during low-speed centrifugation, the centrifugal force can be 300×g, 400×g, 500×g, 600×g, 700×g, etc.; the centrifugal time can be 3min, 4min, 5min, 6min, 7min, 8min, etc.; during high-speed centrifugation, the centrifugal force can be 10000×g, 12000×g, 14000×g, 16000×g, 18000×g, 20000×g, etc.; the centrifugal time can be 15min, 16min, 17min, 18min, 19min, 20min, 25min, 30min, etc.

[0062] The separation conditions of mitochondrial membrane include: centrifugation at 10000×g-20000×g for 5min-20min, taking the supernatant, centrifuging at 50000×g-150000×g for 20-40min, and collecting the precipitate; specifically, during low-speed centrifugation, the centrifugal force can be 10000×g, 12000×g, 14000×g, 16000×g, 18000×g, 20000×g, etc.; the centrifugation time can be 5min, 1 0min, 15min, 20min, etc.; during high-speed centrifugation, the centrifugal force can be 50000×g, 60000×g, 70000×g, 80000×g, 90000×g, 100000×g, 110000×g, 120000×g, 130000×g, 140000×g, 150000×g, etc.; the centrifugation time can be 20min, 25min, 30min, 35min, 40min, etc.

[0063] The first extraction solvent is used to extract and disperse mitochondria, and the second solvent is used to extract and disperse mitochondrial membranes. The composition is not particularly limited. Specifically, in the first extraction solvent, the concentration of the enzyme is 0.5-5 mg / mL; in the second extraction solvent, the mass ratio of water to sucrose is 1:0.5-2.

[0064] The second aspect of the present application provides a method for preparing collagen microcapsules, comprising the following steps:

[0065] The collagen microcapsules are prepared by injecting the mitochondrial membrane solution and the collagen solution into different channels of the microfluidic device for mixing.

[0066] Understandably, the use of microfluidics technology can achieve precise encapsulation of collagen by mitochondrial membrane, improve the stability and encapsulation efficiency of collagen microcapsules, and further improve the efficiency of collagen use. By regulating the concentration, pressure, flow rate ratio and other parameters of the mitochondrial membrane solution and the collagen solution, the uniform mixing and encapsulation of the mitochondrial membrane and collagen in the microchannel can be optimized. Among them, the flow rate ratio is adjusted according to the concentration of collagen and mitochondrial membrane, and the pressure is based on ensuring uniform mixing without damaging the membrane structure.

[0067] In some embodiments, the concentration of the mitochondrial membrane solution is 0.1-5 mg / mL, for example, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, etc.

[0068] In some embodiments, the concentration of the collagen solution is 0.5-20wt%, for example, it can be 0.5wt%, 1wt%, 1.25wt%, 2.5wt%, 5wt%, 7.5wt%, 10wt%, 12.5wt%, 15wt%, 17.5wt%, 20wt%, etc.

[0069] In some embodiments, the solvent of the mitochondrial membrane solution and the solvent of the collagen solution are each independently selected from a buffer solution and water;

[0070] Optionally, the buffer comprises PBS buffer and / or HBSS buffer.

[0071] In some embodiments, the flow rate ratio of the mitochondrial membrane solution to the collagen solution is 1:1-5, specifically 1:1, 1:2, 1:3, 1:4, 1:5, etc. During microchannel mixing, the pressure can be adjusted to 1-10Pa, such as 1Pa, 2Pa, 3Pa, 4Pa, 5Pa, 6Pa, 7Pa, 8Pa, 9Pa, 10Pa, etc.

[0072] In some embodiments, the step of injecting the mitochondrial membrane solution and the collagen solution into different channels of the microfluidic device for mixing further includes a freeze-drying step.

[0073] In some embodiments, in the freeze-drying step, a protective agent is added before freeze-drying.

[0074] In some embodiments, the protective agent includes at least one of lecithin, mannitol, and cyclodextrin.

[0075] It is understandable that the collagen microcapsules collected from the microfluidic device before freeze drying are solutions. Adding a protective agent to the solution and then freeze drying can prevent the structure of the collagen microcapsules from being damaged by freeze drying. There is no special limitation on the conditions of freeze drying. The specific process of freeze drying may include: pre-freezing, vacuum drying (sublimation drying, analytical drying) and exhaust after freeze drying. The vacuum degree of each stage can be 0-20, the temperature can be -45℃-25℃, and the time is 1-30h. The pressure is gradually reduced at low temperature to remove the water in the microcapsules by sublimation. The freeze drying temperature is determined according to the thermal stability of the microcapsules, and the pressure is based on ensuring the smooth sublimation of the water.

[0076] The third aspect of the present application provides the use of the aforementioned collagen microcapsules in skin care products.

[0077] It can be understood that the collagen microcapsules prepared in the present application, when used in skin care products, due to their unique structure and biocompatibility, help to improve the integration of collagen with the skin, so that the beneficial elements in the collagen can be better absorbed by the skin, thereby improving the use effect of the skin care products; at the same time, the prepared collagen microcapsules have good antioxidant effects and can be used for anti-aging.

[0078] Specifically, the skin care products may include toner, essence water, lotion or cream, and their functions may be anti-wrinkle, anti-aging, anti-oxidation, etc.

[0079] The fourth aspect of the present application provides a skin care product, the raw materials of which include the aforementioned collagen microcapsules.

[0080] Unless otherwise specified, the raw materials used in the following experiments can be purchased from the market.

[0081] The following is an exemplary description of some raw materials:

[0082] Type I recombinant collagen powder and type III recombinant collagen powder were purchased from Zhejiang Zhuji Juyuan Biotechnology Co., Ltd.

[0083] Type XVII recombinant collagen powder: purchased from Jiangsu Chuangjian Medical Technology Co., Ltd.

[0084] The following are specific embodiments.

[0085] Preparation Example 1

[0086] Yeast mitochondrial membrane extraction:

[0087] Step 1: Yeast mitochondrial extraction

[0088] 1) Take the yeast culture and centrifuge it at 4°C, 1000×g for 5 minutes. Carefully aspirate the culture medium and dry it as much as possible to collect the yeast precipitate.

[0089] 2) Wash the yeast pellet twice with PBS, and absorb the supernatant as much as possible after each wash;

[0090] 3) Take 1 mL of pre-cooled extraction solvent (1 mg / mL snail enzyme / sorbitol solution), resuspend the yeast pellet (about 1000 mg), mix thoroughly, shake on an oscillator in a 30°C water bath for 50 minutes at 300 rpm, centrifuge at 4°C, 500 × g for 5 minutes, discard the pellet, collect the supernatant, and transfer it to another clean centrifuge tube;

[0091] 4) Centrifuge at 4°C, 12,000 × g for 20 minutes, discard the supernatant, and collect the precipitate, which is the mitochondria;

[0092] 5) Resuspend the pellet with 100 μL of PBS and store at -80°C to obtain the mitochondrial solution.

[0093] Step 2: Mitochondrial membrane extraction

[0094] 1) Centrifuge the extracted mitochondrial solution at 4°C, 12,000 × g for 20 minutes, collect the precipitate, resuspend it in 500 μL ultrapure water, and shake it in an ice box at 4°C for 20 minutes at 300 rpm;

[0095] 2) Add 500 μL of sucrose solution (1.4 M) to the above 1), mix at 4°C, shake at 300 rpm for 30 min in an ice bath, then centrifuge at 4°C, 12,000 × g for 10 min, and collect the supernatant;

[0096] 3) Centrifuge at 4°C, 100,000 × g for 30 min, collect the precipitate, add appropriate amount of PBS to resuspend, adjust the mitochondrial membrane concentration to 1 mg / mL, store at -80°C, and thaw in a 4°C refrigerator before use.

[0097] Mitochondrial membrane extraction rate (%) = mitochondrial membrane protein concentration / mitochondrial protein concentration = 180.3 mg / mL / (1120.5 mg / mL) = 16.09%.

[0098] Example 1

[0099] Preparation of collagen microcapsules:

[0100] 1) Prepare collagen solution: dissolve 0.25 g of type I recombinant collagen, 0.25 g of type XVII recombinant collagen and 2.5 g of type III recombinant collagen in 20 g of water, heat and stir (≤40°C) until completely dissolved to obtain a collagen solution;

[0101] 2) Microfluidic technology encapsulation of collagen:

[0102] a. First, clean the microfluidic chip with deionized water to remove impurities and contaminants on the surface;

[0103] b. Inject the collagen solution and the mitochondrial membrane solution of Preparation Example 1 into the corresponding channels of the microfluidic chip through micro-injectors, control the flow rate ratio to 1:1.5 and the pressure to 3Pa to achieve uniform mixing and encapsulation of the two in the microchannel; collect the collagen microcapsule solution flowing out of the outlet of the microfluidic chip.

[0104] 3) Freeze-drying process and product preservation:

[0105] a. Pre-freeze-drying treatment: Add protective agent to the collagen microcapsule solution. Specifically, add 2g of lecithin, 7g of mannitol, and 25g of cyclodextrin to 100g of collagen microcapsule solution, stir well to dissolve, place the mixed solution in a freeze-drying dish, and cover with film.

[0106] b. Freeze-drying: The freeze-drying process is: -45°C, 6 hours, vacuum degree 0; -15°C, 24 hours, vacuum degree 20; -10°C, 16 hours, vacuum degree 20; 0°C, 3.5 hours, vacuum degree 20; 25°C, 3 hours, vacuum degree 20.

[0107] c. Storage: The freeze-dried collagen microcapsules were quickly placed in a sample bottle and stored at room temperature in a dark environment. The SEM image is shown in Figure 1 .

[0108] Example 2

[0109] The method is basically the same as Example 1, except that: the collagen solution is prepared as follows: 0.25 g of type III recombinant collagen powder is dissolved in 20 g of water, and heated and stirred (≤40° C.) until completely dissolved to obtain a collagen solution.

[0110] Example 3

[0111] The method is basically the same as Example 1, except that: the collagen solution is prepared as follows: 2.5 g of X VII recombinant collagen powder is dissolved in 20 g of water, and heated and stirred (≤40° C.) until completely dissolved to obtain a collagen solution.

[0112] Test Example 1

[0113] Yeast mitochondrial membrane characterization:

[0114] The steps for extracting yeast mitochondrial membranes are shown in Preparation Example 1. The extracted mitochondrial membranes were stained and characterized as follows: Dio staining solution of appropriate concentration was added, incubated at 4°C for 30 min, mitochondrial membranes were collected by centrifugation at 12000×g for 5 min, washed twice with PBS, 100 μL PBS was added to resuspend the precipitate, an appropriate amount of the suspension was dropped on a glass slide, a slide was prepared, and observation and photography were performed under an inverted fluorescence microscope. The results are shown in FIG. Figure 2 As shown, it can be seen that the extracted mitochondrial membrane is intact.

[0115] Test Example 2

[0116] The antioxidant capacity of collagen microcapsules was studied to reflect its ability to scavenge free radicals and further verify its anti-aging effect.

[0117] 1. Evaluation of the antioxidant capacity of the collagen microcapsules of Example 1 - Study on the scavenging effect on ABTS free radicals.

[0118] Detection method: Mix 7 mM ABTS solution and 2.45 mM potassium persulfate in equal volumes, react at room temperature in the dark for 16 hours to obtain ABTS+·free radical stock solution, which is stored at 4°C for later use. Then dilute the ABTS+·free radical stock solution with PBS until the absorbance of the solution at 734 nm is 0.7 ± 0.1 to prepare the ABTS working solution. Prepare a series of collagen microcapsule solutions with different addition amounts, each with 5 mL for later use. Mix 2 mL of collagen microcapsule solutions with different addition amounts with 2 mL of ABTS working solution, and react the mixture in the dark for 10 minutes.

[0119] Transfer the reaction solution to a cuvette and use a UV-visible spectrophotometer to measure its absorbance at 734 nm, which is the absorbance of the material reaction group. Mix 2 mL of ABTS working solution with an equal volume of ultrapure water to obtain the absorbance of the ABTS control group. Mix 2 mL of collagen microcapsule solution with different addition amounts with an equal volume of control solution (40 mL PBS + 1 mL ultrapure water) to obtain the absorbance of the material control group.

[0120] ABTS clearance rate = [A ABTS 对照组 - (A 反应组 -A 材料对照组 )] / A ABTS 对照组 ×100%

[0121] Principle: 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS for short) can generate stable ABTS+ free radicals under the oxidation of potassium persulfate (K2S2O8), which is dark green in aqueous solution and has strong absorption near 734nm. When there is a free radical scavenger (antioxidant), the ABTS+ free radical solution fades due to single electron pairing with it, and the absorption value at 734nm decreases. This can evaluate the ability of the test sample to scavenge ABTS free radicals.

[0122] Table 1 Evaluation of the antioxidant capacity of collagen microcapsules to scavenge ABTS free radicals

[0123]

[0124] 2. Evaluation of the antioxidant capacity of the collagen microcapsules in Example 1 - Study on the scavenging effect on OH free radicals.

[0125] Detection method: Prepare 1 mL of 1 mM TMB, 40 mM H2O2, 4 mM FeSO4, HAc / NaAc buffer (pH=4), and different amounts of collagen microcapsule solution for later use.

[0126] OH control group: add 150μL TMB, 150μL H2O2 and 150μL ddH2O to a 1.5mL centrifuge tube, and finally add 150μL FeSO4. Start timing immediately after addition and react for 5 minutes in the dark. Transfer to a 96-well plate and measure the absorbance at 650nm in a microplate reader, which is the absorbance of the OH control group.

[0127] Material reaction group: add 150 μL TMB, 150 μL H2O2 and 150 μL material solution to a 1.5 mL centrifuge tube, and finally add 150 μL FeSO4, start timing immediately, and react for 5 minutes in the dark. Measure the absorbance at 650 nm in an ELISA instrument, which is the absorbance of the material reaction group.

[0128] Material control group: 150 μL of the above material solution + 450 μL of ddH2O, mix well and measure the absorbance value, which is the absorbance value of the material control group.

[0129] OH clearance rate = [A OH 对照组 - (A 反应组 -A 材料对照组 )] / A OH 对照组 ×100%

[0130] Principle: Hydrogen peroxide (H2O2) reacts with ferrous ions to generate hydroxyl radicals (·OH). Under acidic conditions, ·OH reacts with tetramethylbenzidine (TMB) to produce a color reaction (from light yellow to blue-green or yellow), causing the absorbance of TMB at 650nm to change. When there is a free radical scavenger (antioxidant), the solution turns yellow-green due to single electron pairing with it, and the absorbance at 650nm decreases, thereby evaluating the ability of the test sample to scavenge hydroxyl radicals.

[0131] Table 2 Hydroxyl radical scavenging ability of collagen microcapsules in Example 1

[0132]

[0133] Test Example 3

[0134] Storage stability of the aqueous solution of collagen microcapsules of Example 1

[0135] The collagen microcapsules of Example 1 were prepared into a 2% aqueous solution and stored for 3 months under the following conditions: low temperature: -20°C, 4°C; room temperature; high temperature 40°C, 48°C; high and low temperature alternation 48°C × 24h + 4°C × 24h, and the solution state was observed regularly. It was found that the aqueous solution of the collagen microcapsules of Example 1 was unstable at 48°C, and protein denaturation and precipitation occurred after 24h, while it was relatively stable under the other conditions. It can be seen that the aqueous solution of the collagen microcapsules of Example 1 has good stability at both high and low temperatures.

[0136] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0137] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.

Claims

1. A collagen microcapsule, characterized in that: include: A collagen core material and a capsule material coated on the surface of the collagen core material; The material of the capsule material includes mitochondrial membrane.

2. The collagen microcapsule according to claim 1, characterized in that The mass ratio of the collagen core material to the capsule material is 1-5:1; and / or the collagen comprises recombinant collagen; And / or the mitochondrial membrane includes at least one of a yeast mitochondrial membrane, a bacterial mitochondrial membrane, a plant cell line mitochondrial membrane and an animal cell mitochondrial membrane.

3. The collagen microcapsule according to claim 2, characterized in that: One or more of the following conditions are met: (1) The recombinant collagen includes at least one of type I recombinant collagen, type II recombinant collagen, type III recombinant collagen, type IV recombinant collagen and type XVII recombinant collagen; (2) The recombinant collagen includes at least one of recombinant human collagen, recombinant humanized collagen and recombinant collagen-like protein; (3) The mitochondrial membrane preparation process comprises the following steps: The raw material containing mitochondria is dispersed in a first extraction solvent, and the wall is broken to extract the mitochondria; the mitochondria are dispersed in a second extraction solvent, and the membrane is broken to extract the mitochondrial membrane.

4. The collagen microcapsule according to claim 3, characterized in that The first extraction solvent comprises a buffer solution of yeast cell wall-breaking enzyme and / or a buffer solution of snail enzyme; The second extraction solvent includes water and sucrose; And / or the wall breaking treatment and the membrane breaking treatment independently include physical breaking and / or chemical breaking.

5. A method for preparing collagen microcapsules, characterized in that: The steps include: The collagen microcapsules are prepared by injecting the mitochondrial membrane solution and the collagen solution into different channels of the microfluidic device for mixing.

6. The preparation method according to claim 5, characterized in that: One or more of the following conditions are met: (1) The concentration of the mitochondrial membrane solution is 0.1-5 mg / mL; (2) The concentration of the collagen solution is 0.1-20wt%; (3) The solvent of the mitochondrial membrane solution and the solvent of the collagen solution are independently selected from at least one of a buffer solution and water; (4) The flow rate ratio of the mitochondrial membrane solution to the collagen solution is 1:1-5.

7. The preparation method according to claim 5 or 6, characterized in that: The step of injecting the mitochondrial membrane solution and the collagen solution into different channels of the microfluidic device for mixing also includes a freeze-drying step.

8. The preparation method according to claim 7, characterized in that: In the freeze-drying step, a protective agent is added before freeze-drying; Optionally, the protective agent includes at least one of lecithin, mannitol and cyclodextrin.

9. Use of the collagen microcapsules according to any one of claims 1 to 4 or the collagen microcapsules prepared by the preparation method according to any one of claims 5 to 8 in skin care products.

10. A skin care product, characterized in that: The raw materials include the collagen microcapsules described in any one of claims 1 to 4 or the collagen microcapsules prepared by the preparation method described in any one of claims 5 to 8.

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