Preparation and application of Chuzhou chrysanthemum exosome
By combining mixed bacterial fermentation and plasma-activated water treatment, the problems of low extraction rate and reduced activity of Chrysanthemum in Chuzhou were solved, enabling the efficient extraction of highly active exosomes for use in daily chemical products.
Patent Information
- Application Number
- CN202511592612.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-03
- Publication Date
- 2026-03-03
AI Technical Summary
How to improve the extraction rate of exosomes from Chrysanthemum indicum while maintaining their activity, and addressing the problem of physical damage and reduced activity of exosomes caused by existing plasma treatment methods.
A method combining mixed bacterial fermentation with plasma-activated water treatment was adopted. Probiotic fermentation secreted polysaccharides coated the surface of exosome vesicles to form a protective film, and plasma-activated water was used to destroy the cell wall of Chrysanthemum indicum, thereby improving the extraction rate and maintaining biological activity.
It significantly improved the extraction rate of Chrysanthemum indicum exosomes while maintaining high bioactivity, making it suitable for preparing daily chemical products with free radical scavenging and anti-aging activities.
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Figure CN121592576A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for preparing plant-derived exosomes, and more particularly to the preparation and application of Chrysanthemum indicum exosomes. Background Technology
[0002] Chrysanthemum morifolium cv. Chuju is rich in active ingredients such as flavonoids (e.g., luteolin and apigenin), polyphenols, amino acids, and vitamins, and has potential skin care value in terms of anti-oxidation, anti-inflammation, and skin soothing.
[0003] Exosomes are spherical lipid bilayer vesicles secreted by cells. They are a subset of vesicles involved in intercellular communication, with a particle size of approximately 30-150 nm. They contain bioactive components such as lipids, proteins, and nucleic acids used for intercellular communication, and have significant effects on scar improvement, pigmentation correction, skin rejuvenation, and hair loss recovery. Chrysanthemum morifolium, due to its superior antioxidant activity, can be used as a high-quality raw material for extracting plant exosomes.
[0004] Generally, animal, plant, and microbial cells can produce exosomes, and Chuzhou chrysanthemum is no exception. However, compared to other types of chrysanthemum, Chuzhou chrysanthemum has a "relatively compact" structure. The core reason is that the compact cell arrangement enhances the toughness and morphological stability of the petals, preventing the flowers from being easily damaged by external forces (such as wind); the compact intercellular spaces reduce water evaporation, helping the chrysanthemum retain moisture in dry environments. For Chuzhou chrysanthemum, the compact cell structure further increases the difficulty of exosome extraction.
[0005] Plant exosomes are distributed in the intercellular spaces and intercellular matrix. High-energy particles in plasma-activated water collide with the cell walls and intercellular matrix of *Chrysanthemum indicum*, forming micropores and fissures on the surface. This loosens the arrangement of cellulose microfibrils and breaks the lignin cross-linking structure, reducing solvent permeation resistance and increasing solvent contact area. The ROS oxidation effect and microbubble shock waves of plasma-activated water further disrupt the enzymatically hydrolyzed cell walls, promoting exosome release. While plasma can theoretically increase exosome extraction rate by disrupting the cell walls of *Chrysanthemum indicum*, it also causes physical damage to the exosomes, leading to leakage of contents and lipid peroxidation. This not only makes it difficult to improve exosome yield but also significantly reduces the bioactivity of the exosome contents, resulting in a substantial weakening of the exosome's antioxidant activity. Therefore, how to improve the yield while maintaining the activity of *Chrysanthemum indicum* exosomes is a pressing technical problem that needs to be solved. Summary of the Invention Purpose of the Invention: The purpose of this invention is to provide a method for preparing Chrysanthemum indicum exosomes, solving the problem of how to efficiently extract highly active Chrysanthemum indicum exosomes. Another purpose of this invention is to propose the application of Chrysanthemum indicum exosomes in the preparation of daily chemical products with free radical scavenging and anti-aging activities, solving the problem of how to prepare daily chemical products with free radical scavenging and anti-aging activities.
[0006] Technical solution: The present invention provides a method for preparing Chrysanthemum morifolium exosomes, comprising the following steps: (1) Add water or buffer solution to Chuzhou chrysanthemum, homogenize to obtain Chuzhou chrysanthemum homogenate; (2) A mixed bacterial solution containing lactobacillus, yeast and Bacillus was inoculated into the Chuzhou chrysanthemum homogenate, and fermented to obtain Chuzhou chrysanthemum fermentation broth; (3) Add plasma-activated water to the fermentation liquid of Chuzhou chrysanthemum, mix well and react to obtain raw material liquid; (4) Centrifuge the raw material solution and collect the supernatant; (5) After the supernatant is filtered through two stages, the retentate is collected to obtain Chrysanthemum exosomes.
[0007] Preferably, in step (1), the chrysanthemum is dried or fresh chrysanthemum, the buffer solution is phosphate buffer, and the ratio of chrysanthemum to water or buffer solution is 5-15g:20-40mL.
[0008] Preferably, in step (2), the method for preparing the mixed bacterial solution is as follows: Lactobacillus, yeast and Bacillus are cultured in the corresponding culture medium respectively, and the culture is shaken until the logarithmic growth phase of the cells. The cells are collected separately, and Lactobacillus, yeast and Bacillus are mixed and resuspended in buffer solution according to the ratio of live bacteria 1-3:1-3:1-5 to obtain the mixed bacterial solution.
[0009] Preferably, in step (2), the volume ratio of the mixed bacterial solution to the Chuzhou chrysanthemum homogenate is 1-5:100, and the total number of viable bacteria in the mixed bacterial solution is not less than 1×10⁻⁶. 5 per mL.
[0010] Preferably, in step (2), the fermentation conditions are 25-35℃ for 24-72h, during which the pH of the fermentation system is controlled at 4.0-6.5.
[0011] In some embodiments, fermentation is divided into two stages: the initial stage is cultured in a constant temperature shaker at 35°C for 12-32 hours; the subsequent stage is cultured statically at 35°C for 14-40 hours. The aerobic conditions in the initial stage promote the rapid proliferation of Bacillus and yeast and the secretion of metabolites, while the static culture in the later stage creates a facultative anaerobic environment, promoting the proliferation of yeast and lactobacillus and the formation of metabolites.
[0012] Preferably, in step (3), the method for preparing plasma-activated water is as follows: placing the jet plasma nozzle in water, and treating the water with plasma for 0.5-10 minutes under the conditions of input voltage 150-250V and discharge power 25-40W to obtain plasma-activated water.
[0013] Preferably, in step (3), the volume ratio of Chuzhou chrysanthemum fermentation liquid to plasma-activated water is 1-3:1-3, and the reaction conditions are constant temperature water bath shaking at 30~35℃ for 30~45min.
[0014] Preferably, in step (4), the centrifugation method is as follows: at 2-4℃, the raw material liquid is centrifuged at 5000-8000×g for 20-30min, and the supernatant is taken to obtain the first supernatant; the first supernatant is centrifuged at 10000-12000×g for 30-40min to obtain the second supernatant.
[0015] Preferably, in step (5), the two-stage filtration method is as follows: the supernatant is filtered through a 0.22-0.45μm microporous membrane for primary filtration, the filtrate is collected, and then filtered through a 10-200nm asymmetric nanomembrane for secondary filtration, and the retentate is collected.
[0016] Another aspect of this invention discloses the application of the *Chrysanthemum indicum* exosomes prepared by the above-described method in the preparation of daily chemical products with free radical scavenging and anti-aging activities. These daily chemical products include cosmetics or skincare products such as serums, creams, lotions, and masks, as well as washing products such as hand sanitizers, shower gels, laundry detergents, and soaps.
[0017] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: This invention utilizes plasma activation technology to effectively improve exosome extraction efficiency. Simultaneously, it leverages polysaccharides secreted by microbial fermentation to coat the exosome vesicle surface, forming a protective film and reducing the risk of rupture and activity reduction caused by plasma treatment. Furthermore, probiotic fermentation of Chuzhou chrysanthemum enhances antioxidant activity, strengthening the exosomes' resistance to ROS. In addition, enzymes secreted by probiotic fermentation can loosen the cell wall structure, further improving exosome yield. This invention, employing a specific combination of mixed bacterial fermentation and plasma activation, not only significantly improves the extraction rate of Chuzhou chrysanthemum exosomes but also effectively maintains their high bioactivity, demonstrating promising application prospects. Attached Figure Description
[0018] Figure 1 Microscopic morphology of Chrysanthemum indicum without plasma activation treatment; Figure 2 The image shows the microscopic morphology of Chrysanthemum indicum after plasma activation treatment. Detailed Implementation The technical solution of the present invention will be further described below.
[0019] Example 1: A method for preparing Chrysanthemum chrysanthemum exosomes is as follows: (1) Add 1×PBS to the dried flowers of Chrysanthemum chuchun at a material-to-liquid ratio of 10g:30mL, homogenize at 2500rpm for 20min to obtain Chrysanthemum chuchun homogenate; (2) Lactobacillus plantarum ATCC 8014 was inoculated into MRS medium, Saccharomyces cerevisiae CGMCC 2.10 was inoculated into YPD medium, and Bacillus subtilis ATCC 23857 was inoculated into LB medium. The cultures were shaken and cultured until the logarithmic growth phase. The cells were collected by centrifugation, and the Lactobacillus, yeast, and Bacillus were mixed and resuspended in PBS at a viable cell ratio of 1:1:1 to obtain a total viable cell count of 1×10⁻⁶. 7 Mixed bacterial culture with cells / mL.
[0020] (3) Inoculate the mixed bacterial solution into the Chuzhou chrysanthemum homogenate. The volume ratio of the mixed bacterial solution to the Chuzhou chrysanthemum homogenate is 3:100. Under aerobic conditions, the mixture is cultured in a constant temperature shaker at 30℃ for 24 hours in the early stage and then in a static culture at 30℃ for 36 hours in the later stage to obtain the Chuzhou chrysanthemum fermentation broth. During this period, lactic acid can be used to control the pH of the fermentation system to 4.5. (4) Place the jet plasma nozzle in deionized water, with the nozzle submerged 0.5 cm in the water. Treat the water with plasma for 7 min under the conditions of input voltage 200 V and discharge power 35 W to obtain plasma-activated water. Add plasma-activated water to the Chuzhou chrysanthemum fermentation broth at a volume ratio of 2:1, and shake in a constant temperature water bath at 32℃ for 40 min to obtain the raw material broth; (5) At 4℃, the raw material liquid was centrifuged at 6500×g for 25min, and the supernatant was taken to obtain the first supernatant; the first supernatant was centrifuged at 11000×g for 35min to obtain the second supernatant; (6) The second supernatant was filtered through a 0.45 μm microporous membrane for primary filtration, and the filtrate was then filtered through a 100 nm asymmetric nanomembrane for secondary filtration. The retentate was collected to obtain Chrysanthemum exosomes.
[0021] Example 2: A method for preparing Chrysanthemum chrysanthemum exosomes is as follows: (1) Add 1×PBS to fresh Chuzhou chrysanthemum at a material-to-liquid ratio of 5g:20mL, homogenize at 2500rpm for 20min to obtain Chuzhou chrysanthemum homogenate; (2) Lactobacillus rhamnosus ATCC 7469 was inoculated into MRS medium, Saccharomyces cerevisiae CGMCC2.1000 into YPD medium, and Bacillus licheniformis CGMCC1.198 into LB medium. The cultures were shaken and cultured until the logarithmic growth phase. The cells were collected by centrifugation, and the lactobacillus, yeast, and Bacillus were mixed and resuspended in PBS at a viable cell ratio of 3:3:5 to obtain a total viable cell count of 1×10⁻⁶. 9 Mixed bacterial culture with cells / mL.
[0022] (3) Inoculate the mixed bacterial solution into the Chuzhou chrysanthemum homogenate. The volume ratio of the mixed bacterial solution to the Chuzhou chrysanthemum homogenate is 1:100. Under aerobic conditions, the mixture is cultured in a constant temperature shaker at 35℃ for 12 hours in the early stage and then in a static culture at 35℃ for 14 hours in the later stage to obtain the Chuzhou chrysanthemum fermentation broth. During this period, lactic acid can be used to control the pH of the fermentation system to 4.0.
[0023] (4) Place the jet plasma nozzle in deionized water, submerging the nozzle 0.5 cm in the water. Treat the water with plasma for 10 min under the conditions of input voltage 150 V and discharge power 25 W to obtain plasma-activated water. Add plasma-activated water to the Chuzhou chrysanthemum fermentation broth at a volume ratio of 1:3, and shake in a constant temperature water bath at 35℃ for 30 min to obtain the raw material broth; (5) At 2℃, the raw material liquid is centrifuged at 5000×g for 30min, and the supernatant is taken to obtain the first supernatant; the first supernatant is centrifuged at 10000×g for 40min to obtain the second supernatant.
[0024] (6) The second supernatant was filtered through a 0.22 μm microporous membrane for primary filtration, and the filtrate was then filtered through a 10 nm asymmetric nanomembrane for secondary filtration. The retentate was collected to obtain Chrysanthemum exosomes.
[0025] Example 3: A method for preparing Chrysanthemum chrysanthemum exosomes is as follows: (1) Add water to the dried flowers of Chuzhou chrysanthemum at a material-to-liquid ratio of 15g:40mL, homogenize at 2500rpm for 20min to obtain Chuzhou chrysanthemum homogenate; (2) Lactobacillus reuteri BNCC192190 was inoculated into MRS medium, Kluyveromyces martensii CGMCC2.1440 into YPD medium, and Bacillus coagulans CGMCC1.10 into LB medium. The cultures were shaken and cultured until the logarithmic growth phase. The cells were collected by centrifugation, and the lactobacillus, yeast, and Bacillus were mixed and resuspended in PBS at a viable cell ratio of 2:2:3, yielding a total viable cell count of 1×10⁻⁶. 5 Mixed bacterial culture with cells / mL.
[0026] (3) Inoculate the mixed bacterial solution into the Chuzhou chrysanthemum homogenate. The volume ratio of the mixed bacterial solution to the Chuzhou chrysanthemum homogenate is 5:100. Under aerobic conditions, the mixture is cultured in a constant temperature shaker at 25℃ for 32 hours in the early stage and then in a static culture at 25℃ for 40 hours in the later stage to obtain the Chuzhou chrysanthemum fermentation broth. During this period, lactic acid can be used to control the pH of the fermentation system to 6.5.
[0027] (4) Place the jet plasma nozzle in deionized water, submerging the nozzle 0.5 cm in the water. Treat the water with plasma for 0.5 min under the conditions of input voltage 250 V and discharge power 40 W to obtain plasma-activated water. Add plasma-activated water to the Chuzhou chrysanthemum fermentation broth at a volume ratio of 1:1, and shake in a constant temperature water bath at 30 °C for 45 min to obtain the raw material broth; (5) At 3℃, the raw material liquid is centrifuged at 8000×g for 20min, and the supernatant is taken to obtain the first supernatant; the first supernatant is centrifuged at 12000×g for 30min to obtain the second supernatant.
[0028] (6) The second supernatant was filtered through a 0.45 μm microporous membrane for primary filtration, and the filtrate was then filtered through a 200 nm asymmetric nanomembrane for secondary filtration. The retentate was collected to obtain Chrysanthemum exosomes.
[0029] Comparative Example 1: Everything else is the same as in Example 1, except that: Replace the plasma-activated water with deionized water.
[0030] Comparative Example 2: Everything else is the same as in Example 1, except that: Replace the mixed bacterial culture with PBS buffer.
[0031] Comparative Example 3: Everything else is the same as in Example 1, except that: No lactobacillus added.
[0032] Comparative Example 4: Everything else is the same as in Example 1, except that: Replace Lactobacillus with Acetic Acid Bacillus.
[0033] Comparative Example 5: Everything else is the same as in Example 1, except that: No yeast is added.
[0034] Comparative Example 6: Everything else is the same as in Example 1, except that: Replace the yeast with Trichoderma viride.
[0035] Comparative Example 7: Everything else is the same as in Example 1, except that: No Bacillus subtilis added.
[0036] Comparative Example 8: Everything else is the same as in Example 1, except that: Replace Bacillus with Actinomycetes fibrinolyticus.
[0037] Comparative Example 9: Everything else is the same as in Example 1, except that: Exosomes were extracted directly from the homogenate of Chrysanthemum in Chuzhou by centrifugation and filtration.
[0038] The extraction amounts of Chrysanthemum indicum exosomes in Examples 1-3 and Comparative Examples 1-9 were determined using the following methods: Nanoparticle tracing (NTA) technology was used to characterize particle concentration. The original *Chrysanthemum indicum* exosome sample was diluted to a suitable factor, and the number of particles (30-150 nm) in the extracted product was counted. Combined with the sample dilution factor, the particle concentration of the original sample (unit: particles / mL) was finally calculated. Simultaneous transmission electron microscopy (TEM) observation verified that the particles exhibited typical exosome vesicle structural characteristics. The particle concentration reflects the extraction efficiency of different methods for *Chrysanthemum indicum* exosomes.
[0039] The free radical scavenging ability of Chrysanthemum indicum exosomes extracted in Examples 1-3 and Comparative Examples 1-9 was tested using the following methods: Take 2 mL of exosomes from the above examples and 2 mL of 0.1 mmol / L DPPH anhydrous ethanol solution, add them to the same test tube, mix thoroughly, and place in the dark at room temperature for 30 min. Measure the absorbance at 517 nm and record it as A1. Add 2 mL of anhydrous ethanol and 2 mL of DPPH solution to the same test tube, mix thoroughly, and place in the dark at room temperature for 30 min. Measure the absorbance at 517 nm and record it as A0. Add 2 mL of exosomes and 2 mL of anhydrous ethanol to the same test tube, mix thoroughly, and place in the dark at room temperature for 30 min. Measure the absorbance at 517 nm and record it as A2. Each group is repeated three times. Calculate the DPPH free radical scavenging rate using the following formula: DPPH free radical scavenging rate (%) = (A0 - (A1 - A2)) / A0 × 100% The anti-aging activity of Chrysanthemum indicum exosomes in Examples 1-3 and Comparative Examples 1-9 was detected by measuring elastase inhibition rate, as follows: Using N-succinyl-alanine-alanine-alanine-p-nitroaniline (AAAPNA) as a substrate, elastase can catalyze its hydrolysis. After the addition of the test substance, if the Chrysanthemum chrysanthemum exosome sample inhibits enzyme activity, the amount of substrate hydrolyzed decreases, and the change in absorbance at 380 nm decreases. The inhibition rate is calculated by the difference in absorbance, and the anti-wrinkle and firming efficacy is indirectly evaluated.
[0040] 1. Add samples in groups according to the reagent volumes in the table below: Table 1. Reagent ratio for elastase activity assay
[0041] Enzyme-catalyzed reaction and absorbance detection steps: (1) Referring to Table 1, use a 96-well plate to set up test sample wells (T), test sample control wells (T0), model wells (C), and model control wells (C). 0) .
[0042] (2) Add 100 μL of the same concentration (diluted to 1×10⁻⁶) to each of the test sample well (T) and the test sample control well (T0). 10 The test solution (particles / mL) was added to the model well (C) and the model control well (C0), and 100 μL of the test solution (i.e., water) was added to each well.
[0043] (3) Add 50 μL of elastase working solution (final elastase concentration 5 IU / mL, diluted with Tris-HCl buffer (250 mM, pH 7.5)) to each of the test sample well (T), test sample control well (T0), model well (C) and model control well (C0), and incubate at 25 °C for 15 min.
[0044] (4) Add 50 μL of 1 mmol / L AAAPNA working solution to each of the test sample well (T) and the model well (C). Replace the test sample control well (T0) and the model control well (C0) with 50 μL Tris-HCl (250 mM, pH 7.5) buffer.
[0045] (5) After placing the 96-well plate at room temperature for 20 min, measure the absorbance at 380 nm.
[0046] in, ODc - Absorbance value of the model group; ODc0 - Absorbance value of the model control group; OD T -Absorbance of the test group; OD T0 - Absorbance of the test sample control group.
[0047] The test results are as follows: Table 2. Effects of different preparation methods on the extraction rate and bioactivity of Chrysanthemum indicum exosomes.
[0048] As shown in Table 2, in Comparative Example 1, the lack of plasma treatment of the Chuzhou chrysanthemum fermentation broth resulted in the inability to effectively disrupt the dense cell walls of the Chuzhou chrysanthemum, leading to insufficient exosome release and a significantly lower extraction rate compared to Example 1. However, the bioactivity of the Chuzhou chrysanthemum exosomes was not significantly different from that of Example 1, indicating that fermentation with mixed bacteria alone does not affect exosome activity. Similarly, in Comparative Example 9, the absence of fermentation and plasma treatment resulted in a lower exosome extraction rate due to the dense cell walls of the Chuzhou chrysanthemum, but the exosome activity was higher. This demonstrates that the extraction rate of exosomes is not correlated with their activity. While some common cell wall disruption methods can effectively disrupt cell walls and release exosomes, thereby increasing the exosome yield, these methods also significantly reduce exosome activity. Conservative cell wall disruption extraction methods, although effectively preserving exosome activity, result in excessively low extraction rates, hindering industrial application.
[0049] In Comparative Example 2, only plasma-activated water treatment of Chrysanthemum morifolium was used. Although it could effectively destroy the dense cell wall of Chrysanthemum morifolium, the high-energy plasma also destroyed exosome vesicles. As a result, the extraction rate of Comparative Example 2 did not increase significantly and was significantly lower than that of Example 1. In addition, the exosome activity extracted in Comparative Example 2 was also relatively low, indicating that plasma can also cause the exosome contents to become inactive.
[0050] In Comparative Examples 3-8, when the composition of the mixed bacteria was changed to a dual-species mixture or replaced with other bacterial combinations, the extraction rate of exosomes decreased significantly, much lower than that of Example 1 but close to that of Comparative Example 2. This indicates that the polysaccharide complexes produced by the mixed bacterial fermentation in these comparative examples could not effectively protect the Chuzhou chrysanthemum exosomes from plasma damage, and the problems of exosome rupture and decreased biological activity also occurred.
[0051] In Examples 1-3, fermentation with appropriate bacterial strains resulted in a polysaccharide protective layer that coats the surface of exosome vesicles, protecting *Chrysanthemum indicum* exosomes from plasma damage. Therefore, this invention can both utilize plasma to disrupt the dense cell walls of *Chrysanthemum indicum* to increase exosome yield and prevent plasma damage to exosome vesicles and loss of exosome biological activity through the polysaccharide complex produced by mixed fermentation.
[0052] To further confirm that the surface of Chrysanthemum indicum is coated with polysaccharides produced during fermentation, the following experiment was conducted: The Chrysanthemum chrysanthemum exosomes extracted in Examples 1-3 and Comparative Examples 1-9 were centrifuged at 15000g for 60 min. After separating the precipitate and supernatant, the polysaccharide content in the precipitate and supernatant was determined by the 3,5-dinitrosalicylic acid method (DNS method). The results are as follows: Table 3 Effects of different preparation methods on polysaccharide coating on the surface of Chrysanthemum indicum exosomes
[0053] As shown in Table 3, in the *Chrysanthemum indicum* exosomes prepared in Examples 1-3, most of the polysaccharides coated the surface of the exosome vesicles, with relatively little free polysaccharide in the supernatant. Similarly, in Comparative Example 1, most of the polysaccharides produced by fermentation were also present in the exosome precipitate, i.e., coated on the surface of the exosome vesicles, thus protecting the structure and activity of the exosomes. However, the polysaccharide content in the *Chrysanthemum indicum* exosomes extracted in Comparative Example 2 was very low. In Comparative Examples 3, 5, and 7, due to the use of dual-strain fermentation, the total amount of polysaccharides in the *Chrysanthemum indicum* exosomes was insufficient, failing to effectively protect the exosomes. In Comparative Examples 4, 6, and 8, although the total amount of polysaccharides was not significantly different from that in Example 1 after the composition of the mixed strains was changed, the polysaccharide complexes produced by the combined fermentation of these strains could not effectively attach to the exosome vesicles, remaining mostly in the supernatant, and thus failing to effectively protect the exosomes from plasma damage.
[0054] After separating the residual Chuzhou chrysanthemum tissue (i.e., centrifuged precipitate) from the Chuzhou chrysanthemum fermentation broth in Example 1, the morphological image of the Chuzhou chrysanthemum tissue was examined using a scanning electron microscope. The results are as follows: Figure 1 As shown. The centrifuged precipitate of the raw material solution in Example 1 was examined using scanning electron microscopy, and the results are as follows. Figure 2 As shown. By Figure 1 and Figure 2 visible, Figure 1 The surface of plant tissues is smooth and evenly distributed, while Figure 2 The structure appears relatively coarse, indicating that plasma-activated water can make the surface of plant tissues more porous, thereby promoting the efficient release of exosomes.
Claims
1. A method for preparing Chrysanthemum indicum exosomes, characterized in that, Includes the following steps: (1) Add water or buffer solution to Chuzhou chrysanthemum, homogenize to obtain Chuzhou chrysanthemum homogenate; (2) A mixed bacterial solution containing lactobacillus, yeast and Bacillus was inoculated into the Chuzhou chrysanthemum homogenate, and fermented to obtain Chuzhou chrysanthemum fermentation broth; (3) Add plasma-activated water to the fermentation liquid of Chuzhou chrysanthemum, mix well and react to obtain raw material liquid; (4) Centrifuge the raw material solution and collect the supernatant; (5) After the supernatant is filtered through two stages, the retentate is collected to obtain Chrysanthemum exosomes.
2. The method for preparing Chrysanthemum exosomes according to claim 1, characterized in that, In step (1), the chrysanthemum is dried or fresh chrysanthemum, the buffer solution is phosphate buffer, and the ratio of chrysanthemum to water or buffer solution is 5-15g:20-40mL.
3. The method for preparing Chrysanthemum exosomes according to claim 1, characterized in that, In step (2), the method for preparing the mixed bacterial solution is as follows: Lactobacillus, yeast and Bacillus are cultured in the corresponding culture medium respectively, and the culture is shaken until the logarithmic growth phase of the cells. The cells are collected separately, and Lactobacillus, yeast and Bacillus are mixed and resuspended in buffer solution according to the ratio of live bacteria 1-3:1-3:1-5 to obtain the mixed bacterial solution.
4. The method for preparing Chrysanthemum exosomes according to claim 3, characterized in that, In step (2), the volume ratio of the mixed bacterial solution to the Chuzhou chrysanthemum homogenate is 1-5:100, and the total number of viable bacteria in the mixed bacterial solution is not less than 1×10⁻⁶. 5 per mL.
5. The method for preparing Chrysanthemum exosomes according to claim 3, characterized in that, In step (2), the fermentation conditions are 25-35℃ for 24-72h, during which the pH of the fermentation system is controlled at 4.0-6.
5.
6. The method for preparing Chrysanthemum exosomes according to claim 1, characterized in that, In step (3), the method for preparing plasma-activated water is as follows: place the jet plasma nozzle in water, and treat the water with plasma for 0.5-10 minutes under the conditions of input voltage 150-250V and discharge power 25-40W to obtain plasma-activated water.
7. The method for preparing Chrysanthemum exosomes according to claim 1, characterized in that, In step (3), the volume ratio of Chuzhou chrysanthemum fermentation liquid to plasma-activated water is 1-3:1-3, and the reaction conditions are constant temperature water bath shaking at 30~35℃ for 30~45min.
8. The method for preparing Chrysanthemum exosomes according to claim 1, characterized in that, In step (4), the centrifugation method is as follows: at 2-4℃, the raw material liquid is centrifuged at 5000-8000×g for 20-30min, and the supernatant is taken to obtain the first supernatant; the first supernatant is centrifuged at 10000-12000×g for 30-40min to obtain the second supernatant.
9. The method for preparing Chrysanthemum exosomes according to claim 1, characterized in that, In step (5), the two-stage filtration method is as follows: the supernatant is filtered through a 0.22-0.45μm microporous membrane for primary filtration, the filtrate is collected, and then filtered through a 10-200nm asymmetric nanomembrane for secondary filtration, and the retentate is collected.
10. The use of Chrysanthemum exosomes prepared by the preparation method according to any one of claims 1-9 in the preparation of daily chemical products with free radical scavenging and anti-aging activities.