Preparation method of chlorella exosome as well as product and application of chlorella exosome

Abstract

The invention relates to a preparation method of a chlorella exosome as well as a product and application of the chlorella exosome, the preparation method comprises the following steps: (1) mixing extremely micro chlorella with a separation buffer solution, and then carrying out wall breaking treatment to obtain a cell breaking solution; (2) centrifuging the cell disruption liquid, and reserving supernate; and (3) filtering the supernate to obtain the chlorella exosome. The preparation method of the chlorella exosome, provided by the invention, is simple in steps and easy to operate, the chlorella raw material and the specific separation buffer solution are subjected to wall breaking treatment, so that active ingredients in the prepared chlorella can be better extracted, the content of protein and polysaccharide in the obtained exosome is higher, and the purity of the exosome is improved. Meanwhile, gradient centrifugation, filter membrane filtration and the like are assisted in the preparation process, so that the prepared exosome is high in purity and less in impurities.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant exosome extraction, and specifically relates to a preparation method of Chlorella exosomes and its products and applications. Background Art

[0002] Chlorella is a common plant in the Chromococcales order, belonging to the Chlorophyta. It contains a variety of nutrients and can be taken as a nutritional supplement. It has anti-aging and beauty-enhancing effects. It is rich in vitamins, minerals, amino acids and other substances. It can completely repair damaged cell tissues and improve the regeneration capacity of normal cells by more than 25%. It helps the skin promote collagen whitening and elastin synthesis, plays a role in repair and anti-aging, and also has powerful antioxidant and free radical scavenging functions, restoring the skin to moisture and luster. Therefore, it is increasingly used in skin care products.

[0003] CN114805485A discloses an anti-aging cosmetic containing chlorella active peptides, which comprises dissolving chlorella powder in hot water, performing high-pressure extraction after dissolution, enzymolyzing the extract with papain, ultrafiltration separation of the enzymolyzed solution through an ultrafiltration tube, collecting and freeze-drying after purification, and preparing chlorella active peptide extract freeze-dried powder. The chlorella active peptides provided by the invention can delay the natural aging of the skin caused by excessive oxidation and the photoaging of the skin caused by UV irradiation, and at the same time promote cell proliferation, enhance skin metabolism, and delay skin aging.

[0004] CN110801413A discloses a chlorella fermentation extract and a preparation method thereof. The invention utilizes the culture fluid of bacterial species such as plant lactobacillus to carry out liquid fermentation culture on chlorella powder to obtain a fermentation product, and the fermentation product is centrifuged, filtered, and sterilized to obtain a chlorella powder fermentation extract. The method has mild reaction conditions, a simple preparation process, and does not add any chemical components. Experiments have shown that the chlorella powder fermentation extract prepared by the above method is rich in functional ingredients such as protein, crude polysaccharides, total amino acids and lutein, has good anti-free radical, skin irritation and anti-aging effects, and has high biological safety, and can be widely used in various cosmetics.

[0005] More and more Chlorella is used in cosmetics in the prior art, but few use Chlorella exosomes. Plant exosomes contain 90% of the active ingredients in plants and show significant advantages in biocompatibility, stability, distribution in the body, extended half-life and cellular internalization. Therefore, in order to better exert the efficacy of Chlorella, it is urgently necessary to provide a method for preparing Chlorella exosomes to meet the needs of application. Summary of the invention

[0006] In view of the deficiencies in the prior art, the object of the present invention is to provide a method for preparing Chlorella exosomes and its products and applications.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing Chlorella exosomes, the preparation method comprising:

[0009] (1) mixing Chlorella microsporum with a separation buffer and performing cell wall breaking treatment to obtain a cell breaking solution;

[0010] (2) centrifuging the cell lysate and retaining the supernatant;

[0011] (3) Filter the supernatant to obtain the Chlorella exosomes.

[0012] The method provided by the present invention uses microscopic chlorella as a raw material, and then mixes the microscopic chlorella with a separation buffer and processes the mixture through wall breaking, centrifugation and filtration, thereby solving the problem of low purity in the preparation process of chlorella exosomes. The final product contains high levels of active ingredients such as protein, polysaccharides, amino acids, etc., which not only has excellent moisturizing and anti-aging effects, but also has anti-inflammatory and anti-allergic effects, and can be used for sensitive skin to help reduce redness, swelling and irritation.

[0013] Preferably, the separation buffer comprises Tris, Na2HPO4, NaCl, Tween 80, sorbitol and water.

[0014] Preferably, the separation buffer comprises, in molar concentration, Tris 15-20 mM, Na2HPO4 20-40 mM, NaCl 1-3 mM, Tween 80 0.1-2 mM, and sorbitol 0.1-1 mM.

[0015] For example, the molar concentration of Tris in the separation buffer is 15-20 mM, for example, 16 mM, 17 mM, 18 mM or 19 mM, etc.;

[0016] The molar concentration of the Na2HPO4 is 20-40 mM, for example, 22 mM, 24 mM, 26 mM, 28 mM, 30 mM, 32 mM, 34 mM, 36 mM or 38 mM, etc.;

[0017] The molar concentration of NaCl is 1-3 mM, for example, 1.1 mM, 1.3 mM, 1.5 mM, 1.7 mM, 2 mM, 2.2 mM, 2.5 mM, 2.7 mM or 2.9 mM, etc.;

[0018] The molar concentration of Tween 80 is 0.1-2 mM, for example, 0.3 mM, 0.5 mM, 0.7 mM, 1 mM, 1.1 mM, 1.3 mM, 1.5 mM, 1.7 mM or 1.9 mM, etc.;

[0019] The molar concentration of sorbitol is 0.1-1 mM, for example, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM or 0.9 mM, etc.

[0020] Preferably, the pH of the separation buffer is 6-7, for example, it can be 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8 or 6.9.

[0021] Preferably, the solid-liquid ratio of the microalgae to the separation buffer is 0.1-0.8 g / mL, for example, 0.2 g / mL, 0.3 g / mL, 0.4 g / mL, 0.5 g / mL, 0.6 g / mL or 0.7 g / mL.

[0022] Preferably, the rotation speed during the wall breaking treatment is 2000-5000 rpm, the time is 10-20 min, and the temperature is 25-35°C.

[0023] For example, the rotation speed can be 2500rpm, 3000rpm, 3500rpm, 4000rpm or 4500rpm, etc., the time can be 11min, 13min, 15min, 17min or 19min, etc., and the temperature can be 26℃, 28℃, 30℃, 32℃ or 34℃, etc.

[0024] Preferably, the centrifugation in step (2) is gradient centrifugation.

[0025] Preferably, the specific steps of the gradient centrifugation are: 300-500g (for example, 320g, 350g, 380g, 400g, 420g, 450g or 480g, etc.) centrifugation for 5-15min (for example, 6min, 8min, 10min, 12min or 14min, etc.), 1000-2000g (for example, 1100g, 1300g, 1500g, 1700g or 1900g, etc.) centrifugation for 10-30min (for example, 12min, 14min, 16min, 18min, 20min, 22min, 24min, 26min or 28min, etc.), 400 0-5000g (for example, 4100g, 4300g, 4500g, 4700g or 4900g, etc.) for centrifugation for 10-30min (for example, 12min, 14min, 16min, 18min, 20min, 22min, 24min, 26min or 28min, etc.), 6000-7000g (for example, 6100g, 6300g, 6500g, 6700g or 6900g, etc.) for centrifugation for 10-30min (for example, 12min, 14min, 16min, 18min, 20min, 22min, 24min, 26min or 28min, etc.).

[0026] Preferably, the filtration in step (3) is membrane filtration.

[0027] Preferably, the pore size of the filter membrane is 0.5-1 μm, for example, 0.6 μm, 0.7 μm, 0.8 μm or 0.9 μm.

[0028] Preferably, step (3) further includes concentration treatment after the filtration.

[0029] Preferably, the concentration is to 15-30% of the original volume of the filtrate, for example, 16%, 18%, 20%, 22%, 24%, 26% or 28%.

[0030] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0031] In a second aspect, the present invention provides a Chlorella exosome, wherein the Chlorella exosome is prepared by the method described in the first aspect.

[0032] In a third aspect, the present invention provides a use of the Chlorella exosomes as described in the second aspect in the preparation of skin care products.

[0033] Preferably, the skin care product comprises any one of a gel, an essence, a lotion, a cream, an ointment or a mask.

[0034] Preferably, the amount of the Chlorella exosomes added to the skin care product is 0.1-10%, for example, 0.3%, 0.5%, 0.8%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% or 9%, etc.

[0035] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) The preparation method of Chlorella exosomes provided by the present invention has simple steps and is easy to operate. By breaking the wall of Chlorella raw materials with a specific separation buffer, the active ingredients in the prepared Chlorella can be better extracted, thereby making the protein and polysaccharide content in the obtained exosomes higher. At the same time, gradient centrifugation, membrane filtration, etc. are used in the preparation process to make the prepared exosomes high in purity and less in impurities.

[0038] (2) The separation buffer of the present invention contains Tris, Na2HPO4, NaCl, Tween 80, sorbitol and water, which not only provides a stable environment for the preparation of Chlorella exosomes, but also prevents the active ingredients of Chlorella from being damaged during the separation process. DETAILED DESCRIPTION

[0039] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0040] The raw materials involved in the following examples and comparative examples can be used as long as they are purchased from regular dealers.

[0041] Example 1

[0042] This embodiment provides a method for preparing Chlorella exosomes, the preparation method comprising:

[0043] (1) mixing Chlorella microsporum with a separation buffer (the separation buffer comprises Tris, Na2HPO4, NaCl, Tween 80, sorbitol and water, with a pH of 6.5, wherein the molar concentration of Tris is 18 mM, the molar concentration of Na2HPO4 is 30 mM, the molar concentration of NaCl is 1.5 mM, the molar concentration of Tween 80 is 1 mM, and the molar concentration of sorbitol is 0.5 mM) at a solid-liquid ratio of 0.5 g / mL, and then performing a cell wall breaking treatment, wherein the temperature during the cell wall breaking treatment is 30° C., the rotation speed is 4000 rpm, and the time is 15 min to obtain a cell disruption solution;

[0044] (2) subjecting the cell lysate to gradient centrifugation and retaining the supernatant, wherein the specific steps of the gradient centrifugation are: centrifugation at 450 g for 10 min, centrifugation at 1800 g for 25 min, centrifugation at 4800 g for 25 min, and centrifugation at 6300 g for 20 min;

[0045] (3) The supernatant was filtered using a filter membrane with a pore size of 0.8 μm, and the filtrate was concentrated to 20% of the original volume, and the filtrate was allowed to stand to obtain the Chlorella exosomes.

[0046] Example 2

[0047] This embodiment provides a method for preparing Chlorella exosomes, the preparation method comprising:

[0048] (1) mixing Chlorella microsporum with a separation buffer (the separation buffer comprises Tris, Na2HPO4, NaCl, Tween 80, sorbitol and water, with a pH of 6, wherein the molar concentration of Tris is 20 mM, the molar concentration of Na2HPO4 is 25 mM, the molar concentration of NaCl is 2.5 mM, the molar concentration of Tween 80 is 0.5 mM, and the molar concentration of sorbitol is 1 mM) at a solid-liquid ratio of 0.8 g / mL, and then performing a cell wall breaking treatment, wherein the temperature during the cell wall breaking treatment is 25° C., the rotation speed is 5000 rpm, and the time is 10 min to obtain a cell disruption solution;

[0049] (2) subjecting the cell lysate to gradient centrifugation and retaining the supernatant, wherein the specific steps of the gradient centrifugation are: centrifugation at 350 g for 15 min, centrifugation at 1500 g for 15 min, centrifugation at 4500 g for 20 min, and centrifugation at 6500 g for 25 min;

[0050] (3) The supernatant was filtered using a filter membrane with a pore size of 0.7 μm, and the filtrate was concentrated to 18% of the original volume, and the filtrate was allowed to stand to obtain the Chlorella exosomes.

[0051] Example 3

[0052] This embodiment provides a method for preparing Chlorella exosomes, the preparation method comprising:

[0053] (1) mixing Chlorella microsporum with a separation buffer (the separation buffer comprises Tris, Na2HPO4, NaCl, Tween 80, sorbitol and water, with a pH of 7, wherein the molar concentration of Tris is 15 mM, the molar concentration of Na2HPO4 is 40 mM, the molar concentration of NaCl is 1 mM, the molar concentration of Tween 80 is 2 mM, and the molar concentration of sorbitol is 0.8 mM) at a solid-liquid ratio of 0.4 g / mL, and then performing a cell wall breaking treatment, wherein the temperature during the cell wall breaking treatment is 35° C., the rotation speed is 3000 rpm, and the time is 20 min to obtain a cell disruption solution;

[0054] (2) subjecting the cell lysate to gradient centrifugation and retaining the supernatant, wherein the specific steps of the gradient centrifugation are: centrifugation at 400 g for 5 min, centrifugation at 2000 g for 10 min, centrifugation at 5000 g for 10 min, and centrifugation at 7000 g for 10 min;

[0055] (3) The supernatant was filtered using a filter membrane with a pore size of 0.5 μm, and the filtrate was concentrated to 25% of the original volume, and the filtrate was allowed to stand to obtain the Chlorella exosomes.

[0056] Example 4

[0057] This embodiment provides a method for preparing Chlorella exosomes, the preparation method differing from that in Example 1 only in that the separation buffer is Na2HPO4, NaCl, Tween 80, sorbitol and water, with a pH of 6.5, wherein the molar concentration of Na2HPO4 is 30 mM, the molar concentration of NaCl is 1.5 mM, the molar concentration of Tween 80 is 1 mM, and the molar concentration of sorbitol is 0.5 mM, and the other raw materials, steps and parameters remain unchanged.

[0058] Example 5

[0059] This embodiment provides a method for preparing Chlorella exosomes, the preparation method being different from that in Example 1 only in that the separation buffer is Tris, NaCl, Tween 80, sorbitol and water, with a pH of 6.5, wherein the molar concentration of Tris is 18 mM, the molar concentration of NaCl is 1.5 mM, the molar concentration of Tween 80 is 1 mM, and the molar concentration of sorbitol is 0.5 mM, and the other raw materials, steps and parameters remain unchanged.

[0060] Example 6

[0061] This embodiment provides a method for preparing Chlorella exosomes, the preparation method differing from that in Example 1 only in that the separation buffer is Tris, Na2HPO4, Tween 80, sorbitol and water, with a pH of 6.5, wherein the molar concentration of Tris is 18 mM, the molar concentration of Na2HPO4 is 30 mM, the molar concentration of Tween 80 is 1 mM, and the molar concentration of sorbitol is 0.5 mM, and the other raw materials, steps and parameters remain unchanged.

[0062] Example 7

[0063] This embodiment provides a method for preparing Chlorella exosomes, the preparation method differing from that in Example 1 only in that the separation buffer is Tris, Na2HPO4, NaCl, sorbitol and water, with a pH of 6.5, wherein the molar concentration of Tris is 18 mM, the molar concentration of Na2HPO4 is 30 mM, the molar concentration of NaCl is 1.5 mM, and the molar concentration of sorbitol is 0.5 mM, and the other raw materials, steps and parameters remain unchanged.

[0064] Example 8

[0065] This embodiment provides a method for preparing Chlorella exosomes, the preparation method differing from that in Example 1 only in that the separation buffer is Tris, Na2HPO4, NaCl, Tween 80 and water, with a pH of 6.5, wherein the molar concentration of Tris is 18 mM, the molar concentration of Na2HPO4 is 30 mM, the molar concentration of NaCl is 1.5 mM, and the molar concentration of Tween 80 is 1 mM, and the other raw materials, steps and parameters remain unchanged.

[0066] Example 9

[0067] This embodiment provides a method for preparing Chlorella exosomes. The difference between the preparation method and Example 1 is that the centrifugation step in step (2) is 450g for 80 minutes, and the other raw materials, steps and parameters remain unchanged.

[0068] Example 10

[0069] This embodiment provides a method for preparing Chlorella exosomes. The difference between the preparation method and Example 1 is that the centrifugation step in step (2) is 6300g for 80 minutes, and the other raw materials, steps and parameters remain unchanged.

[0070] Comparative Example 1

[0071] This comparative example provides a method for preparing Chlorella exosomes, and the preparation method is:

[0072] (1) performing cell disruption treatment on Chlorella microsporum, wherein the temperature during the cell disruption treatment is 30° C., the rotation speed is 4000 rpm, and the time is 15 min to obtain a cell disruption solution;

[0073] (2) subjecting the cell lysate to gradient centrifugation and retaining the supernatant, wherein the specific steps of the gradient centrifugation are: centrifugation at 450 g for 10 min, centrifugation at 1800 g for 25 min, centrifugation at 4800 g for 25 min, and centrifugation at 6300 g for 20 min;

[0074] (3) The supernatant was filtered using a filter membrane with a pore size of 0.8 μm, and the filtrate was concentrated to 20% of the original volume, and the filtrate was allowed to stand to obtain the Chlorella exosomes.

[0075] Comparative Example 2

[0076] The present invention provides a chlorella extract, and the preparation method of the chlorella extract is as follows:

[0077] The microscopic Chlorella is broken and crushed to obtain algae powder, and the algae powder is mixed with 10 times the mass of an ethanol aqueous solution with a volume fraction of 70%, heated to boiling, and refluxed for 3 times, each time for 30 minutes, and the extract is concentrated to 20% of the original volume to obtain the Chlorella extract.

[0078] Test Example 1: Moisturizing performance test

[0079] The samples obtained in Examples 1-10 and Comparative Examples 1-2 (configured as aqueous solutions with a concentration of 1 g / mL) were tested for their moisturizing properties, and the changes in the skin moisture content of the test subjects were compared before and after use (the use operation was kept consistent). A total of 120 volunteers, 60 men and 60 women, aged 20-40 years old, with an average age of 28 years old, were randomly divided into 12 groups, with 5 women and 5 men in each group.

[0080] The subjects washed their forearms and sat quietly for 30 minutes in a set humidity environment. A 3×3 cm 2 The left arm was used as the test area for the test sample, and the symmetrical area on the right arm was used as the blank control. The moisture content of each experimental part was detected by Corneometer CM825. The test was repeated 3 times and the average value was recorded.

[0081] The formula for calculating skin hydration rate is:

[0082] The average skin hydration rate of the subjects after using the samples for 2 hours is shown in Table 1:

[0083] Table 1

[0084]

[0085]

[0086] It can be seen from the test data in Table 1 that compared with the Chlorella extract prepared by the conventional method, the Chlorella exosomes prepared by the method of the present invention in Examples 1-3 had a higher skin hydration rate after use by volunteers, which was above 44%, indicating that the exosomes obtained by the method contained more active ingredients, and thus had a better moisturizing effect.

[0087] Test Example 2 Antioxidant Efficacy Study - DPPH Free Radical Scavenging Experiment

[0088] Test samples: samples provided in Examples 1-10 and Comparative Examples 1-2;

[0089] Test method:

[0090] Accurately weigh 47.7 mg of DPPH reagent, dilute to 100 ml brown volumetric flask, take 5 ml of it and dilute to 50 ml brown volumetric flask with anhydrous ethanol to obtain DPPH test solution (47.7 mg / L). Pipette 2 mL of DPPH test solution respectively, add 0.1 mL of Chlorella exosomes prepared in Examples 1-7 and Comparative Examples 1-4 (configured as an aqueous solution with a concentration of 1 g / mL) as shown in Table 2, and then add an appropriate amount of anhydrous ethanol to make the total mass equal. After fully mixing, let it stand in the dark for 30 minutes, use anhydrous ethanol as a blank control, measure its absorbance at 517 nm, and set vitamin C as a control, and calculate the scavenging rate of each extract for DPPH according to the following formula:

[0091] DPPH removal rate (%) = [1-(As-Ar) / A0] × 100%

[0092] Where: As is the absorbance of the extract and DPPH reaction system; Ar is the absorbance of the extract-anhydrous ethanol; A0 is the absorbance of the blank control anhydrous ethanol.

[0093] Table 2 Reaction solution composition

[0094]

[0095] The DPPH free radical scavenging rate reflects its antioxidant capacity to a certain extent. The higher the scavenging rate, the better the antioxidant degree and the better the anti-aging effect. Therefore, the anti-aging effect of the essence can be judged by studying its ability to scavenge DPPH free radicals. Vitamin C is a highly effective antioxidant recognized by the cosmetics industry. This experiment used vitamin C as a control to examine its antioxidant capacity. The test results are shown in Table 3.

[0096] Table 3

[0097]

[0098]

[0099] As can be seen from Table 3, compared with the Chlorella extract prepared by the conventional method of Comparative Example 2, the exosomes prepared by the specific method of the present invention in Examples 1-3 have better scavenging effects on DPPH, and the scavenging rates are all above 89%. Among them, the scavenging rates of Examples 1-3 are higher than those of Comparative Examples 1-4, indicating that the combination of the algae raw materials of the present invention can make the antioxidant capacity of the final product better, verifying the rationality of the raw material selection. And compared with the positive control vitamin C, the scavenging rates of Examples 1-3 are only slightly lower and very close, indicating that the Chlorella exosomes of the present invention have an antioxidant capacity similar to that of vitamin C, and have a good antioxidant effect.

[0100] Test Example 3 Anti-inflammatory efficacy test

[0101] Test sample: samples provided in Examples 1-10 and Comparative Examples 1-2 (configured as aqueous solutions with a concentration of 1 g / mL);

[0102] The test method is as follows:

[0103] Inhibition of TNF-α expression: Fresh blood was treated with EDTA as anticoagulant, blood cells were separated by Ficoll, and cells were resuspended in RIMP 1640 medium containing 10% fetal bovine serum. 100 mL of 1×10 5 cells / mL Freshly isolated cells, total number of cells per well: 10 4 3 wells for each sample; weigh the test sample, add sterile PBS solution to prepare the test sample stock solution (10 mg / mL), and then use sterile PBS to dilute the stock solution to the corresponding working concentration for use; add 10 mL of the test sample solution of the corresponding working concentration to the well of the culture plate. The culture plate is placed in an incubator at 37°C and 5% CO2 for 15 minutes; add 10 mL of LPS with a concentration of 100 mg / mL and incubate in an incubator at 37°C and 5% CO2 for 16 hours; the culture plate is centrifuged at 1000 rpm for 15 minutes, the supernatant is transferred to a new culture plate, and the TNF-α concentration in the culture supernatant is determined using an enzyme marker; or frozen at -20°C to avoid repeated freezing and thawing, and the inhibition rate is calculated.

[0104] The specific test results are shown in Table 4 below:

[0105] Table 4

[0106] Test samples Inhibition rate of TNF-a release / % Blank control 8.0 Example 1 90.5 Example 2 89.7 Example 3 88.6 Example 4 82.2 Example 5 83.7 Example 6 83.8 Example 7 84.7 Example 8 84.5 Example 9 80.9 Example 10 83.4 Comparative Example 1 62.6 Comparative Example 2 85.7

[0107] It can be seen from the data in Table 4 that compared with the conventional Chlorella extract preparation method in Comparative Example 2, the Chlorella exosomes provided in Examples 1-3 of the present invention in the culture medium have a higher inhibition rate on TNF-a release, reaching more than 88%, which fully demonstrates that the product of the present invention can play an anti-inflammatory role, thereby effectively repairing inflamed skin.

[0108] Test Example 4 Safety Evaluation:

[0109] The specific method is as follows: 40 females aged 18-35 years old with no history of allergies are selected, and each subject is given the sample prepared in Examples 1-10 (configured as an aqueous solution with a concentration of 1g / mL). After cleaning the back of the subject, apply the spot tester with the sample to the selected position on the back with a non-irritating tape, and after pasting, press it lightly with your fingers to make it evenly attached to the skin for 48 hours. The subject keeps the patch area dry within 48 hours, avoids intense exercise, scratching the patch area, and long-term sunlight exposure. After 48 hours, remove the tester and mark it. After 30 minutes, wait for the indentation to disappear and make a judgment under sufficient light.

[0110] The grading standards for adverse skin reactions are shown in Table 5:

[0111] Table 5

[0112]

[0113] The results are shown in Table 6:

[0114] Table 6

[0115]

[0116]

[0117] It can be seen from the data in Table 6 that after removing the spot tester, no adverse reactions were observed on the skin of the volunteers, which shows that the raw materials used for the Chlorella exosomes of the present invention are safe and non-irritating, have no adverse reactions to the human body, and are highly safe.

[0118] The applicant declares that the present invention illustrates the preparation method of a chlorella exosome and its product and application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0119] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0120] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for preparing Chlorella exosomes, characterized in that: The preparation method comprises: (1) mixing Chlorella microsporum with a separation buffer and performing cell wall breaking treatment to obtain a cell breaking solution; (2) centrifuging the cell lysate and retaining the supernatant; (3) Filter the supernatant to obtain the Chlorella exosomes.

2. The preparation method according to claim 1, characterized in that The separation buffer includes Tris, Na2HPO4, NaCl, Tween 80, sorbitol and water.

3. The preparation method according to claim 2, characterized in that: The separation buffer comprises, in molar concentration, Tris 15-20 mM, Na2HPO4 20-40 mM, NaCl 1-3 mM, Tween 80 0.1-2 mM, and sorbitol 0.1-1 mM; Preferably, the pH of the separation buffer is 6-7.

4. The preparation method according to any one of claims 1 to 3, characterized in that The solid-liquid ratio of the microalgae to the separation buffer is 0.1-0.8 g / mL; Preferably, the rotation speed during the wall breaking treatment is 2000-5000 rpm, the time is 10-20 min, and the temperature is 25-35°C.

5. The preparation method according to any one of claims 1 to 4, characterized in that The centrifugation in step (2) is gradient centrifugation; Preferably, the specific steps of the gradient centrifugation are: 300-500g centrifugation for 5-15min, 1000-2000g centrifugation for 10-30min, 4000-5000g centrifugation for 10-30min, and 6000-7000g centrifugation for 10-30min.

6. The preparation method according to any one of claims 1 to 5, characterized in that The filtration in step (3) is membrane filtration; Preferably, the pore size of the filter membrane is 0.5-1 μm.

7. The preparation method according to any one of claims 1 to 7, characterized in that Step (3) further includes concentration treatment after the filtration; Preferably, the concentration is to 15-30% of the original volume of the filtrate.

8. A Chlorella exosome, characterized in that The Chlorella exosomes are prepared by the method according to any one of claims 1 to 7.

9. Use of the Chlorella exosomes according to claim 8 in preparing skin care products.

10. The use according to claim 9, characterized in that The skin care product includes any one of a gel, an essence, a lotion, a cream, an ointment or a mask; Preferably, the amount of the Chlorella exosomes added to the skin care product is 0.1-10%.

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