A lyophilized powder composition containing camellia flower exosomes, and a preparation method and application thereof

Abstract

This invention belongs to the field of cosmetic preparation technology, specifically disclosing a freeze-dried powder composition containing camellia exosomes, its preparation method, and its application. The freeze-dried powder composition comprises the following components in parts by weight: 2.5-3.5 parts camellia exosomes, 6-10 parts grapefruit peel extract, 8-10 parts compound microbial fermentation supernatant, 6-10 parts trehalose, 4-6 parts mannitol, 0.5-1.5 parts glycine, 0.1-0.2 parts Tween 80, 0.1-0.2 parts disodium hydrogen phosphate, 0.05-0.1 parts sodium dihydrogen phosphate, 0.1-0.5 parts vitamin E, 0.2-0.5 parts soybean lecithin, and 0.1-0.5 parts sodium chloride. This freeze-dried powder composition combines active functional components to exert a synergistic effect of antioxidation and anti-inflammation, effectively scavenging DPPH free radicals, exhibiting high total antioxidant capacity and activity, and can be used to prepare beauty and skincare products.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic preparation technology, specifically relating to a freeze-dried powder composition containing camellia exosomes, its preparation method, and its application. Background Technology

[0002] In recent years, with the deepening research on intercellular communication mechanisms, exosomes, as 30-150 nm nanoscale vesicles secreted by cells, have become a research hotspot in the fields of cosmetics and regenerative medicine due to their unique functions in mediating intercellular signal transduction and transporting bioactive molecules. Compared with animal-derived exosomes, plant-derived exosome-like nanovesicles (PENs) have attracted much attention in recent years due to their high safety, low immunogenicity, abundant sources, and unique antioxidant, anti-inflammatory, anti-aging, and skin repair-promoting bioactivities. Plant exosomes can penetrate the skin barrier, reduce fine lines, improve skin texture and moisturizing ability, and enhance skin elasticity, making them a highly promising natural active ingredient in cosmetic formulations. Camellia, a traditional ornamental and medicinal plant in my country, has had its flower and leaf extracts reported to have various anti-aging effects, including promoting collagen synthesis, inhibiting matrix metalloproteinase expression, scavenging reactive oxygen species, and inhibiting skin glycation. However, research on the application of camellia-derived exosomes in the cosmetics field is still limited, and there is a lack of systematic development and application reports. Exosomes have poor stability in liquid environments and degrade within a few days at room temperature, a characteristic that severely restricts their application in skincare products. Summary of the Invention

[0003] The present invention aims to provide a freeze-dried powder composition containing camellia exosomes, its preparation method and application. The freeze-dried powder composition combines active functional components to exert a synergistic effect of antioxidation and anti-inflammation, effectively scavenging DPPH free radicals, with high total antioxidant capacity and activity, and can be used to prepare beauty and skin care products.

[0004] A freeze-dried powder composition containing camellia exosomes comprises the following components in parts by weight: 2.5-3.5 parts camellia exosomes, 6-10 parts grapefruit peel extract, 8-10 parts compound microbial fermentation supernatant, 6-10 parts trehalose, 4-6 parts mannitol, 0.5-1.5 parts glycine, 0.1-0.2 parts Tween 80, 0.1-0.2 parts disodium hydrogen phosphate, 0.05-0.1 parts sodium dihydrogen phosphate, 0.1-0.5 parts vitamin E, 0.2-0.5 parts soybean lecithin, and 0.1-0.5 parts sodium chloride.

[0005] The present invention also provides a method for preparing the freeze-dried powder composition containing camellia exosomes, comprising the following steps: T1. Grapefruit peel is mixed with 60-70% ethanol by mass, heated under reflux and extracted three times. The three extracts are combined, filtered, concentrated, and freeze-dried under vacuum to obtain grapefruit peel extract. T2. Inoculate the compound microbial seed liquid into the fermentation medium, ferment and culture, centrifuge to collect the supernatant, filter through a filter membrane to obtain the compound microbial fermentation supernatant. T3. Preparation of camellia exosomes; T4. Camellia exosomes obtained in T3, grapefruit peel extract prepared in T1, supernatant of compound microbial fermentation prepared in T2, trehalose, mannitol, glycine, Tween 80, disodium hydrogen phosphate, sodium dihydrogen phosphate, vitamin E, soybean lecithin, sodium chloride and deionized water are mixed in proportion and the pH is adjusted to 6.5-7.5 to obtain the pre-lyophilized solution. T5. The pre-freeze liquid obtained in T4 is subjected to pre-freezing treatment and vacuum drying to obtain freeze-dried powder.

[0006] Preferably, in T1, the heating reflux temperature is 50-90℃, and the extraction time is 2-4 hours each time; the concentration to 1 / 5-1 / 10 of the original volume is carried out at a temperature of 50-60℃; the vacuum freeze-drying is specifically carried out at -80℃ and 10-100Pa for 48 hours.

[0007] Preferably, in T2, the composite microorganism is composed of *Lactobacillus plantarum* and *Saccharomyces cerevisiae* in a 1:1 ratio of live bacteria, wherein *Lactobacillus plantarum* is *Lactobacillus plantarum* ATCC 14917 and *Saccharomyces cerevisiae* is *Saccharomyces cerevisiae* CGMCC 2.6673.

[0008] Preferably, in T2, the fermentation culture temperature is 28-32℃ and the time is 48-96h.

[0009] Preferably, in T3, the method for preparing the camellia exosomes includes the following steps: S1. Select fresh camellia flowers, wash and drain them to obtain pre-treated camellia flowers; S2. Place the pretreated camellia flowers obtained in S1 in PBS buffer, add the complex enzyme, and obtain the enzymatic hydrolysate; S3. Homogenize the enzymatic hydrolysate obtained in S2, centrifuge at differential speed, collect the supernatant, ultrafilter it through a 100kDa ultrafiltration membrane, collect the retentate, let it stand, centrifuge at ultraspeed, collect the precipitate, and obtain camellia exosomes. Resuspend them in PBS solution containing 5% trehalose and store them in a -80℃ freezer.

[0010] Preferably, in S2, the complex enzyme is composed of cellulase, pectinase and hemicellulase in a mass ratio of (3-5):(2-3):(1-2); the enzymatic hydrolysis temperature is 35-40℃ and the time is 2-4h.

[0011] Preferably, in S3, the homogenization conditions are: rotation speed of 5000-8000 rpm, time of 15-20 min, and temperature of 4℃.

[0012] Preferably, in S3, the differential centrifugation includes: centrifugation at 300-500g for 10-15 min at 4°C; centrifugation at 2000-3000g for 15-20 min at 4°C; and centrifugation at 10000-12000g for 20-30 min at 4°C.

[0013] Preferably, in S3, the ultracentrifugation includes: centrifugation at 100,000-120,000g for 80-90 minutes at a temperature of 4°C.

[0014] Preferably, in T5, the pre-freezing treatment specifically involves cooling to -40°C at a rate of 0.5-1.0°C / min and holding at that temperature for 2-4 hours.

[0015] Preferably, in T5, the vacuum drying specifically involves: raising the temperature from -40℃ to -20℃ at a rate of 1-2℃ / h under 10-50Pa, drying for 24-48h, then raising the temperature to 25-30℃ at a rate of 1-2℃ / h, drying for 8-12h, until the moisture content of the freeze-dried powder is ≤3%.

[0016] The present invention also provides the application of the freeze-dried powder composition containing camellia exosomes in the preparation of beauty and skin care products.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects: This invention discloses a freeze-dried powder composition containing camellia exosomes, its preparation method, and its applications. Camellia exosomes, as core signaling molecules, can penetrate the skin barrier and directly enter the cell layer. By delivering plant-derived miRNAs and active lipids, they activate fibroblast proliferation and collagen synthesis at the gene level, achieving deep repair and anti-aging. Grapefruit peel extract, rich in flavonoids such as naringin, constructs an antioxidant protective network on the skin surface, effectively scavenging UV and pollution-induced free radicals. Simultaneously, it activates TRPV4 channels to promote tight junction protein expression, strengthening the skin barrier. The two form a complementary combination of "external antioxidant + deep repair." The lipid vesicle structure of the exosomes also helps carry water-soluble active ingredients from the grapefruit peel across layers for delivery. The supernatant from the complex microbial fermentation provides organic acids, bacteriocins, and B vitamins, gently exfoliating dead skin cells, opening microchannels to promote the penetration of exosomes and plant extracts, regulating the skin microecology, inhibiting the release of inflammatory factors, and achieving metabolic renewal. Antioxidant tests showed that the DPPH scavenging rate of the example group exceeded 61%, and the total antioxidant capacity (T-AOC) and T-SOD activity were high. The freeze-dried powder composition provided by this invention achieves efficient, stable, and safe multi-target whitening and anti-aging skincare effects, and has good prospects for industrialization.

[0018] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0019] Figure 1 Statistical graph showing the DPPH free radical scavenging rate results for different treatment groups; Figure 2 Statistical graph of total antioxidant capacity (T-AOC) results for different treatment groups; Figure 3 Statistical chart showing the total superoxide dismutase (T-SOD) activity results for different treatment groups. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0022] Source of experimental materials: In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0023] Fermentation medium: glucose 20 g / L, peptone 10 g / L, yeast extract 5 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 2 g / L, pH 6.5.

[0024] Example 1 The freeze-dried powder composition containing camellia exosomes is composed of the following raw materials in parts by weight: The composition consisted of 3 parts camellia exosomes, 8 parts grapefruit peel extract, 9 parts compound microbial fermentation supernatant, 8 parts trehalose, 5 parts mannitol, 1 part glycine, 0.15 parts Tween 80, 0.15 parts disodium hydrogen phosphate, 0.08 parts sodium dihydrogen phosphate, 0.3 parts vitamin E, 0.35 parts soybean lecithin, 0.3 parts sodium chloride, and the remaining components were deionized water.

[0025] T1. Wash and chop 200g of fresh grapefruit peel. Add 65% ethanol at a ratio of 1:10g / mL. Heat and reflux at 70℃ for 3 times, 3 hours each time. Combine the three extracts, filter, and concentrate under reduced pressure at 55℃ to 1 / 8 of the original volume. Freeze-dry the concentrate at -80℃ and 50Pa for 48 hours. Crush and sieve to obtain grapefruit peel extract. T2. Activate *Lactobacillus plantarum* (ATCC 14917) and *Saccharomyces cerevisiae* (CGMCC 2.6673) separately, and adjust the bacterial concentration to 10. 8 CFU / mL, mixed at a viable count of 1:1 to obtain a composite seed culture, the composite seed culture was inoculated into the fermentation medium at a 5% inoculation rate, and incubated at 30℃ for 72h. The fermentation broth was centrifuged at 8000rpm for 15min to obtain the supernatant, and then filtered through a 0.22μm filter membrane to obtain the composite microbial fermentation supernatant. T3. Preparation of camellia exosomes, including the following steps: S1. Clean, wash, and drain fresh camellia flowers; S2. Take 100g of S1 pretreated camellia flowers, add 500mL of PBS buffer (pH 7.2), add compound enzyme (cellulase: pectinase: hemicellulase = 4:2.5:1.5, total enzyme amount 1.5g), and enzymatically hydrolyze at 37℃ for 3h. S3. Homogenize the enzymatic hydrolysate obtained in S2 at 4℃ and 6000rpm for 18min. Centrifuge the homogenate sequentially at 4℃ using the following differential speeds: 400g for 12min, 2500g for 18min, and 11000g for 25min. Collect the supernatant, filter it through a 100kDa ultrafiltration membrane, collect the retentate, and centrifuge the retentate at 4℃ and 110000g for 85min. Discard the supernatant, and resuspend the precipitate in PBS containing 5% trehalose to obtain a camellia exosome suspension, which is stored at -80℃ for later use. T4. Weigh the grapefruit peel extract obtained from T1, the fermentation supernatant obtained from T2, the camellia exosomes obtained from T3, trehalose, mannitol, glycine, Tween 80, disodium hydrogen phosphate, sodium dihydrogen phosphate, vitamin E, soybean lecithin, and sodium chloride according to the formula. Add deionized water to a total weight of 100 parts, stir until completely dissolved, adjust the pH to 7.0, and obtain the pre-lyophilized solution. T5. The pre-lyophilized liquid obtained in T4 is cooled to -40℃ at a rate of 0.8℃ / min and kept at that temperature for 3 hours. Under a vacuum of 30Pa, the temperature is increased from -40℃ to -20℃ at a rate of 1.5℃ / h and dried for 36 hours. Then, the temperature is increased from -20℃ to 28℃ at a rate of 1.5℃ / h and dried for 10 hours until the moisture content is ≤3%, thus obtaining a lyophilized powder composition containing camellia exosomes.

[0026] Example 2 The freeze-dried powder composition containing camellia exosomes is composed of the following raw materials in parts by weight: The composition consisted of 2.5 parts camellia exosomes, 10 parts grapefruit peel extract, 8 parts compound microbial fermentation supernatant, 10 parts trehalose, 4 parts mannitol, 1.5 parts glycine, 0.1 parts Tween 80, 0.2 parts disodium hydrogen phosphate, 0.05 parts sodium dihydrogen phosphate, 0.5 parts vitamin E, 0.2 parts soybean lecithin, 0.5 parts sodium chloride, and the remaining components were deionized water.

[0027] T1. Take 200g of fresh grapefruit peel, wash and chop it. Add 70% ethanol at a material-to-liquid ratio of 1:10 (g / mL). Heat and reflux at 90℃ for 2 hours. Extract three times in total. Combine the three extracts, filter, and concentrate under reduced pressure at 60℃ to 1 / 10 of the original volume. Freeze-dry the concentrate at -80℃ and 30Pa for 48 hours. Crush and sieve to obtain grapefruit peel extract. T2, *Lactobacillus plantarum* (ATCC 14917), and *Saccharomyces cerevisiae* (CGMCC 2.6673) were activated separately, and the bacterial concentration was adjusted to 10. 8 CFU / mL, mixed at a viable count of 1:1 to obtain a composite seed culture, the composite seed culture was inoculated into the fermentation medium at a 5% inoculation rate, and incubated at 30℃ for 72h. The fermentation broth was centrifuged at 8000rpm for 15min to obtain the supernatant, and then filtered through a 0.22μm filter membrane to obtain the composite microbial fermentation supernatant. T3. Preparation of camellia exosomes, including the following steps: S1. Clean, wash, and drain fresh camellia flowers; S2. Take 100g of pretreated camellia flowers, add 500mL of PBS buffer (pH 7.2), add a compound enzyme (cellulase: pectinase: hemicellulase = 5:2:1, total enzyme amount 1.2g), and enzymatically hydrolyze at 35℃ for 4h. S3. Homogenize the enzymatic hydrolysate obtained in S2 at 4℃ and 5000rpm for 20min. Centrifuge the homogenate sequentially at 4℃ using the following differential speeds: 300g for 15min, 2000g for 20min, and 10000g for 30min. Collect the supernatant and filter it through a 100kDa ultrafiltration membrane. Collect the retentate and centrifuge it at 4℃ and 100000g for 90min. Discard the supernatant and resuspend the precipitate in PBS containing 5% trehalose to obtain a camellia exosome suspension. T4. Weigh the grapefruit peel extract obtained from T1, the fermentation supernatant obtained from T2, the camellia exosomes obtained from T3, trehalose, mannitol, glycine, Tween 80, disodium hydrogen phosphate, sodium dihydrogen phosphate, vitamin E, soybean lecithin, and sodium chloride according to the formula. Add deionized water to a total weight of 100 parts, stir to dissolve, and adjust the pH to 6.5 to obtain the pre-freeze-drying solution. T5. The pre-lyophilized liquid obtained in T4 is cooled to -40℃ at a rate of 0.5℃ / min and kept at that temperature for 4 hours. Under a vacuum of 10Pa, it is heated to -20℃ at a rate of 1.0℃ / h and dried for 48 hours. Then, it is heated to 25℃ at a rate of 1.0℃ / h and dried for 12 hours until the moisture content is ≤3%, thus obtaining a lyophilized powder composition containing camellia exosomes.

[0028] Example 3 The freeze-dried powder composition containing camellia exosomes is composed of the following raw materials in parts by weight: The composition consisted of 3.5 parts camellia exosomes, 6 parts grapefruit peel extract, 10 parts compound microbial fermentation supernatant, 6 parts trehalose, 6 parts mannitol, 0.5 parts glycine, 0.2 parts Tween 80, 0.1 parts disodium hydrogen phosphate, 0.1 parts sodium dihydrogen phosphate, 0.1 parts vitamin E, 0.5 parts soybean lecithin, 0.1 parts sodium chloride, and the remaining components were deionized water.

[0029] T1. Take 200g of fresh grapefruit peel, wash and chop it. Add 60% ethanol at a material-to-liquid ratio of 1:10 (g / mL). Heat and reflux at 50℃ for 4 hours. Extract three times in total. Combine the three extracts, filter, and concentrate under reduced pressure at 50℃ to 1 / 5 of the original volume. Freeze-dry the concentrate at -80℃ and 80Pa for 48 hours. Crush and sieve to obtain grapefruit peel extract. T2. Preparation of supernatant from compound microbial fermentation Lactobacillus plantarum (ATCC 14917) and Saccharomyces cerevisiae (CGMCC 2.6673) were activated separately, and the bacterial concentration was adjusted to 10. 8 CFU / mL was mixed at a 1:1 ratio and inoculated into the fermentation medium at a 5% inoculum. The mixture was then incubated at 30°C for 72 hours. The supernatant was collected by centrifuging the fermentation broth at 8000 rpm for 15 minutes and filtered through a 0.22 μm filter membrane to obtain the supernatant of the composite microbial fermentation. T3. Preparation of camellia exosomes, including the following steps: S1. Clean, wash, and drain fresh camellia flowers; S2. Take 100g of S1 pretreated camellia flowers, add 500mL PBS (pH 7.2), add compound enzyme (cellulase: pectinase: hemicellulase = 3:3:2, total enzyme amount 1.8g), and enzymatically hydrolyze at 40℃ for 2h. S3. Homogenize the enzymatic hydrolysate obtained in S2 at 4℃ and 8000rpm for 15min. Centrifuge the homogenate sequentially at 4℃: 500g for 10min, 3000g for 15min, and 12000g for 20min. Collect the supernatant and filter it through a 100kDa ultrafiltration membrane. Collect the retentate and centrifuge it at 4℃ and 120000g for 80min. Discard the supernatant and resuspend the precipitate in PBS containing 5% trehalose to obtain a camellia exosome suspension. Store it at -80℃ for later use. T4. Weigh the grapefruit peel extract obtained from T1, the fermentation supernatant obtained from T2, the camellia exosomes obtained from T3, trehalose, mannitol, glycine, Tween 80, disodium hydrogen phosphate, sodium dihydrogen phosphate, vitamin E, soybean lecithin, and sodium chloride according to the formula. Add deionized water to a total weight of 100 parts, stir to dissolve, and adjust the pH to 6.5 to obtain the pre-lyophilized solution. T5. The pre-lyophilized liquid obtained in T4 is cooled to -40℃ at a rate of 1.0℃ / min and kept at that temperature for 2 hours. Then, under a vacuum of 50Pa, the temperature is increased to -20℃ at a rate of 2.0℃ / h and dried for 24 hours. Next, the temperature is increased to 30℃ at a rate of 2.0℃ / h and dried for 8 hours until the moisture content is ≤3%, thus obtaining a lyophilized powder composition containing camellia exosomes.

[0030] Comparative Example 1 The freeze-dried powder composition comprises the following raw materials in parts by weight: The composition consisted of 8 parts grapefruit peel extract, 9 parts compound microbial fermentation supernatant, 8 parts trehalose, 5 parts mannitol, 1 part glycine, 0.15 parts Tween 80, 0.15 parts disodium hydrogen phosphate, 0.08 parts sodium dihydrogen phosphate, 0.3 parts vitamin E, 0.35 parts soybean lecithin, 0.3 parts sodium chloride, and the remaining components were deionized water.

[0031] T1. Wash and chop 200g of fresh grapefruit peel. Add 65% ethanol at a ratio of 1:10 (g / mL) and extract by reflux at 70℃ three times for 3 hours each time. Combine the three extracts, filter, and concentrate under reduced pressure at 55℃ to 1 / 8 of the original volume. Freeze-dry the concentrate at -80℃ and 50Pa for 48 hours, pulverize and sieve to obtain grapefruit peel extract. T2. Activate *Lactobacillus plantarum* (ATCC 14917) and *Saccharomyces cerevisiae* (CGMCC 2.6673) separately, and adjust the bacterial concentration to 10.8 CFU / mL, mixed at a viable count of 1:1 to obtain a composite seed culture, the composite seed culture was inoculated into the fermentation medium at a 5% inoculation rate, and incubated at 30℃ for 72h. The fermentation broth was centrifuged at 8000rpm for 15min to obtain the supernatant, and then filtered through a 0.22μm filter membrane to obtain the composite microbial fermentation supernatant. T3. Weigh the grapefruit peel extract obtained from T1, the fermentation supernatant obtained from T2, trehalose, mannitol, glycine, Tween 80, disodium hydrogen phosphate, sodium dihydrogen phosphate, vitamin E, soybean lecithin, and sodium chloride according to the formula. Add deionized water to a total weight of 100 parts, stir until completely dissolved, adjust the pH to 7.0, and obtain the pre-freeze-drying solution. T4. The pre-lyophilized liquid obtained in T3 is cooled to -40℃ at a rate of 0.8℃ / min and kept at that temperature for 3 hours. Under a vacuum of 30Pa, the temperature is increased from -40℃ to -20℃ at a rate of 1.5℃ / h and dried for 36 hours. Then, the temperature is increased from -20℃ to 28℃ at a rate of 1.5℃ / h and dried for 10 hours until the moisture content is ≤3%, thus obtaining the lyophilized powder composition.

[0032] Comparative Example 2 The freeze-dried powder composition containing camellia exosomes is composed of the following raw materials in parts by weight: The composition consisted of 3 parts camellia exosomes, 9 parts compound microbial fermentation supernatant, 8 parts trehalose, 5 parts mannitol, 1 part glycine, 0.15 parts Tween 80, 0.15 parts disodium hydrogen phosphate, 0.08 parts sodium dihydrogen phosphate, 0.3 parts vitamin E, 0.35 parts soybean lecithin, 0.3 parts sodium chloride, and the remaining components were deionized water.

[0033] T1. Activate *Lactobacillus plantarum* (ATCC 14917) and *Saccharomyces cerevisiae* (CGMCC 2.6673) separately, and adjust the bacterial concentration to 10. 8 CFU / mL, mixed at a viable count of 1:1 to obtain a composite seed culture, the composite seed culture was inoculated into the fermentation medium at a 5% inoculation rate, and incubated at 30℃ for 72h. The fermentation broth was centrifuged at 8000rpm for 15min to obtain the supernatant, and then filtered through a 0.22μm filter membrane to obtain the composite microbial fermentation supernatant. T2. Preparation of camellia exosomes, including the following steps: S1. Clean, wash, and drain fresh camellia flowers; S2. Take 100g of S1 pretreated camellia flowers, add 500mL of PBS buffer (pH 7.2), add compound enzyme (cellulase: pectinase: hemicellulase = 4:2.5:1.5, total enzyme amount 1.5g), and enzymatically hydrolyze at 37℃ for 3h. S3. Homogenize the enzymatic hydrolysate obtained in S2 at 4℃ and 6000rpm for 18min. Centrifuge the homogenate sequentially at 4℃ using the following differential speeds: 400g for 12min, 2500g for 18min, and 11000g for 25min. Collect the supernatant, filter it through a 100kDa ultrafiltration membrane, collect the retentate, and centrifuge the retentate at 4℃ and 110000g for 85min. Discard the supernatant, and resuspend the precipitate in PBS containing 5% trehalose to obtain a camellia exosome suspension, which is stored at -80℃ for later use. T3. Weigh the fermentation supernatant obtained from T1, the camellia exosomes obtained from T2, trehalose, mannitol, glycine, Tween 80, disodium hydrogen phosphate, sodium dihydrogen phosphate, vitamin E, soybean lecithin, and sodium chloride according to the formula. Add deionized water to a total weight of 100 parts, stir until completely dissolved, adjust the pH to 7.0, and obtain the liquid before freeze-drying. T4. The pre-lyophilized liquid obtained in T3 is cooled to -40℃ at a rate of 0.8℃ / min and kept at that temperature for 3 hours. Under a vacuum of 30Pa, the temperature is increased from -40℃ to -20℃ at a rate of 1.5℃ / h and dried for 36 hours. Then, the temperature is increased from -20℃ to 28℃ at a rate of 1.5℃ / h and dried for 10 hours until the moisture content is ≤3%, thus obtaining a lyophilized powder composition containing camellia exosomes.

[0034] Comparative Example 3 The freeze-dried powder composition containing camellia exosomes is composed of the following raw materials in parts by weight: The composition consisted of 3 parts camellia exosomes, 8 parts grapefruit peel extract, 8 parts trehalose, 5 parts mannitol, 1 part glycine, 0.15 parts Tween 80, 0.15 parts disodium hydrogen phosphate, 0.08 parts sodium dihydrogen phosphate, 0.3 parts vitamin E, 0.35 parts soybean lecithin, 0.3 parts sodium chloride, and the remaining components were deionized water.

[0035] T1. Wash and chop 200g of fresh grapefruit peel. Add 65% ethanol at a ratio of 1:10g / mL. Heat and reflux at 70℃ for 3 times, 3 hours each time. Combine the three extracts, filter, and concentrate under reduced pressure at 55℃ to 1 / 8 of the original volume. Freeze-dry the concentrate at -80℃ and 50Pa for 48 hours. Crush and sieve to obtain grapefruit peel extract. T2. Preparation of camellia exosomes, including the following steps: S1. Clean, wash, and drain fresh camellia flowers; S2. Take 100g of S1 pretreated camellia flowers, add 500mL of PBS buffer (pH 7.2), add compound enzyme (cellulase: pectinase: hemicellulase = 4:2.5:1.5, total enzyme amount 1.5g), and enzymatically hydrolyze at 37℃ for 3h. S3. Homogenize the enzymatic hydrolysate obtained in S2 at 4℃ and 6000rpm for 18min. Centrifuge the homogenate sequentially at 4℃ using the following differential speeds: 400g for 12min, 2500g for 18min, and 11000g for 25min. Collect the supernatant, filter it through a 100kDa ultrafiltration membrane, collect the retentate, and centrifuge the retentate at 4℃ and 110000g for 85min. Discard the supernatant, and resuspend the precipitate in PBS containing 5% trehalose to obtain a camellia exosome suspension, which is stored at -80℃ for later use. T3. Weigh the grapefruit peel extract obtained in T1, the camellia exosomes obtained in T2, trehalose, mannitol, glycine, Tween 80, disodium hydrogen phosphate, sodium dihydrogen phosphate, vitamin E, soybean lecithin, and sodium chloride according to the formula. Add deionized water to a total weight of 100 parts, stir until completely dissolved, adjust the pH to 7.0, and obtain the pre-lyophilized solution. T4. The pre-lyophilized liquid obtained in T3 is cooled to -40℃ at a rate of 0.8℃ / min and kept at that temperature for 3 hours. Under a vacuum of 30Pa, the temperature is increased from -40℃ to -20℃ at a rate of 1.5℃ / h and dried for 36 hours. Then, the temperature is increased from -20℃ to 28℃ at a rate of 1.5℃ / h and dried for 10 hours until the moisture content is ≤3%, thus obtaining a lyophilized powder composition containing camellia exosomes.

[0036] The effects of the freeze-dried powder compositions provided in Examples 1-3 and Comparative Examples 1-3 were verified.

[0037] The antioxidant activity test (DPPH free radical scavenging method) is conducted using the following specific experimental protocol: Take the lyophilized powders from Examples 1-3 and Comparative Examples 1-3 respectively, and set them as Group A, Group B, Group C, Group D, Group E and Group F respectively, and prepare 200 μg / mL with deionized water.

[0038] Weigh out DPPH and prepare a 0.1 mmol / L solution with anhydrous ethanol. Store in the dark. Before testing, dilute with anhydrous ethanol to a absorbance of 0.70 ± 0.02 at 517 nm.

[0039] Reaction system (96-well plate, total volume per well 200 μL): Sample well: 100 μL DPPH working solution + 100 μL sample solution; Blank well: 100μL DPPH working solution + 100μL deionized water; Control well: 100 μL anhydrous ethanol + 100 μL sample solution; Positive control: Take 100 μL of 50 μg / mL vitamin C solution to replace the sample solution and operate according to the sample well.

[0040] The positive control group was set as Group G.

[0041] After standing at room temperature in the dark for 30 minutes, the absorbance at 517 nm was measured using an ELISA reader and recorded as A. 样品 A 对照 A 空白 The clearance rate is calculated using the following formula: .

[0042] The results are as follows Figure 1 As shown.

[0043] Depend on Figure 1 It can be seen that the DPPH scavenging rates of Examples 1-3 were significantly higher than those of Comparative Examples 1-3, demonstrating excellent free radical scavenging ability, indicating that the combined use of camellia exosomes, grapefruit peel extract and compound microbial fermentation supernatant has a significant synergistic effect.

[0044] The total antioxidant capacity (T-AOC) assay kit (purchased from Tongwei Biotechnology) was used to determine the T-AOC content according to the kit instructions. The results are as follows: Figure 2 As shown.

[0045] Depend on Figure 2 It can be seen that the T-AOC of Examples 1, 2 and 3 are all at a high level; while the T-AOC of Comparative Examples 1, 2 and 3 are significantly lower than those of the respective example groups, indicating that the freeze-dried powder composition of the present invention has excellent total antioxidant capacity.

[0046] The total superoxide dismutase (T-SOD) assay kit (purchased from Nanjing Jiancheng) was used to determine the T-SOD content according to the kit instructions. The results are as follows: Figure 3 As shown.

[0047] Depend on Figure 3 It can be seen that the T-SOD activities of Examples 1, 2, and 3 are significantly higher than those of Comparative Examples 1, 2, and 3. The T-SOD activities of the Example groups are on average about 70% to 80% higher than those of the Comparative Examples, indicating that the composition of the present invention can effectively enhance superoxide dismutase-like activity, thereby efficiently scavenging superoxide anion free radicals.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A lyophilized powder composition comprising Camellia japonica exosome, characterized by, It includes the following components by weight: 2.5-3.5 parts camellia exosomes, 6-10 parts grapefruit peel extract, 8-10 parts compound microbial fermentation supernatant, 6-10 parts trehalose, 4-6 parts mannitol, 0.5-1.5 parts glycine, 0.1-0.2 parts Tween 80, 0.1-0.2 parts disodium hydrogen phosphate, 0.05-0.1 parts sodium dihydrogen phosphate, 0.1-0.5 parts vitamin E, 0.2-0.5 parts soybean lecithin, and 0.1-0.5 parts sodium chloride.

2. The method for preparing the freeze-dried powder composition containing camellia exosomes as described in claim 1, characterized in that, Includes the following steps: T1. Grapefruit peel is mixed with 60-70% ethanol by mass, heated under reflux and extracted three times. The three extracts are combined, filtered, concentrated, and freeze-dried under vacuum to obtain grapefruit peel extract. T2. Inoculate the compound microbial seed liquid into the fermentation medium, ferment and culture, centrifuge to collect the supernatant, filter through a filter membrane to obtain the compound microbial fermentation supernatant. T3. Preparation of camellia exosomes; T4. Camellia exosomes obtained in T3, grapefruit peel extract prepared in T1, supernatant of compound microbial fermentation prepared in T2, trehalose, mannitol, glycine, Tween 80, disodium hydrogen phosphate, sodium dihydrogen phosphate, vitamin E, soybean lecithin, sodium chloride and deionized water are mixed in proportion and the pH is adjusted to 6.5-7.5 to obtain the pre-lyophilized solution. T5. The pre-freeze liquid obtained in T4 is subjected to pre-freezing treatment and vacuum drying to obtain freeze-dried powder.

3. The preparation method according to claim 2, characterized in that, In T1, the heating reflux temperature is 50-90℃, and the extraction time is 2-4h each time; the concentration to 1 / 5-1 / 10 of the original volume is carried out at a temperature of 50-60℃; the vacuum freeze drying is specifically carried out at -80℃ and 10-100Pa for 48h.

4. The preparation method according to claim 2, characterized in that, In T2, the composite microorganism is composed of *Lactobacillus plantarum* and *Saccharomyces cerevisiae* in a 1:1 ratio of live bacteria. The *Lactobacillus plantarum* is *Lactobacillus plantarum* ATCC 14917, and the *Saccharomyces cerevisiae* is *Saccharomyces cerevisiae* CGMCC 2.6673.

5. The preparation method according to claim 2, characterized in that, In T2, the fermentation culture temperature is 28-32℃ and the time is 48-96h.

6. The preparation method according to claim 2, characterized in that, In T3, the method for preparing the camellia exosomes includes the following steps: S1. Select fresh camellia flowers, wash and drain them to obtain pre-treated camellia flowers; S2. Place the pretreated camellia flowers obtained in S1 in PBS buffer, add the complex enzyme, and obtain the enzymatic hydrolysate; S3. Homogenize the enzymatic hydrolysate obtained in S2, centrifuge at differential speed, collect the supernatant, ultrafilter it through a 100kDa ultrafiltration membrane, collect the retentate, let it stand, centrifuge at ultraspeed, collect the precipitate, and obtain camellia exosomes. Resuspend them in PBS solution containing 5% trehalose and store them in a -80℃ freezer.

7. The preparation method according to claim 6, characterized in that, In S3, the homogenization conditions are: rotation speed of 5000-8000 rpm, time of 15-20 min, and temperature of 4℃.

8. The preparation method according to claim 2, characterized in that, In T5, the pre-freezing treatment specifically involves cooling to -40°C at a rate of 0.5-1.0°C / min and holding at that temperature for 2-4 hours.

9. The preparation method according to claim 2, characterized in that, In T5, the vacuum drying process specifically involves: raising the temperature from -40℃ to -20℃ at a rate of 1-2℃ / h under 10-50Pa, drying for 24-48h, then raising the temperature to 25-30℃ at a rate of 1-2℃ / h, drying for 8-12h, until the moisture content of the freeze-dried powder is ≤3%.

10. The use of the freeze-dried powder composition containing camellia exosomes as described in claim 1 in the preparation of beauty and skin care products.

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