Application of Actinidia arguta exosome-like nanoparticles in the preparation of products for improving intestinal flora

Abstract

The present invention provides an application of exosome-like nanoparticles of Actinidia arguta in the preparation of products for improving intestinal flora, belonging to the field of biological preparation technology, including centrifuging the juice of Actinidia arguta fruit at 3500-4500g for 5-15min to obtain supernatant a, centrifuging supernatant a at 6500-7500g for 15-25min to obtain supernatant b, centrifuging supernatant b at 9500-10500g for 25-35min to obtain supernatant c, centrifuging supernatant c1 at 19000-121000g for 65-75min to obtain precipitate 1, resuspending precipitate 1 with sterile PBS buffer to obtain resuspended liquid 1, centrifuging resuspended liquid 1 at 19000-121000g for 65-75min to obtain precipitate 2, and resuspending precipitate 2 with PBS buffer. The AAELNs prepared by the present invention have the effect of improving intestinal flora.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological preparation, and in particular relates to an application of Actinidia arguta exosome-like nanoparticles in the preparation of a product for improving intestinal flora. Background Art

[0002] Exosome-like nanoparticles (ELNs) are nano-sized vesicles secreted by cells. They are vesicle-like structures surrounded by a double layer of phospholipids and carry unique bioactive substances (such as lipids, proteins, miRNAs and secondary metabolites). Plant ELNs are rich in proteins, most of which are membrane proteins, which can maintain the stability of the ELN structure and play a role as bioactive ingredients. Plant ELNs are usually secreted by edible plants, and have the advantages of safety, reliability, and few side effects. They can also be used as drug carriers.

[0003] At present, a large number of plants with medicinal and edible properties are widely used to treat various diseases, and polysaccharides, polyphenols and other substances isolated from these plants are regarded as the main functional components for treating diseases. Studies in recent years have shown that in addition to the above chemical components, plants can secrete ELNs. Among them, Actinidia arguta, also known as soft date fruit and kiwi berry, is a perennial deciduous vine of the Actinidia family. Its fruit is small, thin and hairless. The fruit contains lutein, flavonoids, polyphenols, natural inositol and minerals (P, Ca, Fe, Zn, etc.), and has antioxidant, anti-tumor, anti-inflammatory, cholesterol-lowering, memory regulation, anti-diabetes, anti-allergy and anti-fatigue functions. Therefore, Actinidia arguta is a fruit with high value, wide application and broad development prospects, but there is no report on the preparation and functional effects of Actinidia arguta exosome-like nanoparticles (AAELNs). Summary of the invention

[0004] In view of this, the object of the present invention is to provide an application of Actinidia arguta exosome-like nanoparticles in the preparation of a product for improving intestinal flora.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides an application of exosome-like nanoparticles of Actinidia arguta in preparing a product for improving intestinal flora. The preparation method of the exosome-like nanoparticles of Actinidia arguta comprises the following steps:

[0007] The juice of Actinidia arguta fruit was centrifuged at 3500-4500g for 5-15min to obtain supernatant a, supernatant a was centrifuged at 6500-7500g for 15-25min to obtain supernatant b, supernatant b was centrifuged at 9500-10500g for 25-35min to obtain supernatant c, supernatant c was centrifuged at 119000-121000g for 65-75min to obtain precipitate 1, precipitate 1 was resuspended by blowing with sterile PBS buffer to obtain resuspension 1, resuspension 1 was centrifuged at 119000-121000g for 65-75min to obtain precipitate 2, precipitate 2 was resuspended with PBS buffer to obtain Actinidia arguta exosome-like nanoparticles.

[0008] Preferably, the centrifugation temperature is 2-6°C.

[0009] Preferably, the preparation of the juice of Actinidia arguta fruit comprises grinding the Actinidia arguta fruit.

[0010] Preferably, the Actinidia arguta exosome-like nanoparticles increase the abundance of Lactobacillus and Acinetobacter, and reduce the abundance of Proteus, Escherichia coli-Shigella and Clostridium sordellii.

[0011] The present invention also provides the use of the above-mentioned Actinidia arguta exosome-like nanoparticles in the preparation of a product that promotes the growth of probiotics.

[0012] Preferably, the probiotics include lactobacilli.

[0013] The present invention also provides the use of the above-mentioned Actinidia arguta exosome-like nanoparticles in the preparation of products for inhibiting the growth of harmful bacteria.

[0014] Preferably, the harmful bacteria include one or more of Proteus, Escherichia coli-Shigella and Clostridium sordellii.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention provides an application of exosome-like nanoparticles of actinidia arguta in preparing a product for improving intestinal flora. The present invention adopts an ultrahigh-speed gradient centrifugation method to prepare exosome-like nanoparticles of actinidia arguta. The method is simple and rapid to operate. The prepared exosome-like nanoparticles of actinidia arguta have good structural integrity and stability, and have the effect of improving intestinal flora. Moreover, the product is derived from edible actinidia arguta and has high safety, thus providing a safer and more reliable new option for the screening of products for preparing products for improving intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1The morphology of AAELNs is characterized. A is a picture of AAELNs precipitated at the bottom of the centrifuge tube after ultra-high-speed centrifugation; B: A picture of AAELNs observed by transmission electron microscopy;

[0018] Figure 2 Characterization of particle size and Zeta potential of AAELNs, A is particle size distribution; B is Zeta potential;

[0019] Figure 3 The results of RNA and protein characterization of AAELNs, A: agarose gel electrophoresis; B: SDS-PAGE results;

[0020] Figure 4 The particle size change results of AAELNs at different storage times and temperatures, A is the change results of the particle size of AAELNs at different storage temperatures over time; B is the change results of the particle size of AAELNs at different storage times under -80℃;

[0021] Figure 5 The effect of AAELNs in different pH solutions (1.2, 6.8 and 7.8) on their particle size;

[0022] Figure 6 The particle size results of AAELNs in simulated gastric fluid (0 and 4 h), small intestinal fluid (0 and 24 h), and colonic fluid (0 and 72 h);

[0023] Figure 7 The uptake and distribution of AAELNs in C57BL / 6 mice. A is the result of in vivo fluorescence imaging; B is the result of organ imaging.

[0024] Figure 8 The results are the comparison of pH values ​​of the fermentation broth during the fermentation process under different treatment conditions;

[0025] Fig. 9 is the sequence length distribution result;

[0026] Fig.10 This is the β-diversity analysis of mouse intestinal flora. A is the principal component analysis; B is the principal coordinate analysis;

[0027] Fig.11 The taxonomic composition of the bacterial community at the phylum level and the abundance distribution of taxa with higher content among groups;

[0028] Fig.12 The taxonomic composition of the bacterial community at the genus level and the abundance distribution of taxa with higher content among groups. DETAILED DESCRIPTION

[0029] The present invention provides an application of exosome-like nanoparticles of Actinidia arguta in preparing a product for improving intestinal flora. The preparation method of the exosome-like nanoparticles of Actinidia arguta comprises the following steps:

[0030] The juice of Actinidia arguta fruit was centrifuged at 3500-4500g for 5-15min to obtain supernatant a, supernatant a was centrifuged at 6500-7500g for 15-25min to obtain supernatant b, supernatant b was centrifuged at 9500-10500g for 25-35min to obtain supernatant c, supernatant c was centrifuged at 119000-121000g for 65-75min to obtain precipitate 1, precipitate 1 was resuspended by blowing with sterile PBS buffer to obtain resuspension 1, resuspension 1 was centrifuged at 119000-121000g for 65-75min to obtain precipitate 2, precipitate 2 was resuspended with PBS buffer to obtain Actinidia arguta exosome-like nanoparticles.

[0031] In the present invention, the preparation of the juice of the kiwifruit fruit preferably includes grinding the kiwifruit fruit, and the present invention adopts a grinding process to ensure the structural integrity of the kiwifruit exosome-like nanoparticles. The present invention adopts an ultra-high-speed gradient centrifugation method to remove pulp tissue, miscellaneous cells, cell fragments, organelles and other substances in the plant juice, thereby preparing purified kiwifruit exosome-like nanoparticles. As a preferred embodiment, supernatant a is obtained by centrifugation at 3700-4200g for 7-13min, supernatant b is obtained by centrifugation at 6600-7300g for 17-23min, supernatant c is obtained by centrifugation at 9800-10300g for 27-34min, supernatant c is obtained by centrifugation at 119500-120500g for 68-72min, precipitate 1 is obtained by blowing and resuspending precipitate 1 with sterile PBS buffer, resuspended precipitate 1 is obtained by centrifugation at 119500-120500g for 68-72min, and precipitate 2 is resuspended with PBS buffer. The centrifugation temperature is preferably 2-6°C, and more preferably 3-5°C.

[0032] In the present invention, the average particle size of the Actinidia arguta exosome-like nanoparticles is 157.8 nm, and the Zeta potential is -23.07 mV, indicating that the AAELNs have good stability and the number of vesicles is 6.5×10 12 Particle / mL, the ratio of the number of vesicles (Particles) to the protein or RNA concentration reflects the purity of the sample, and the ratios of the two for AAELNs were 1.08×10 12 Particles / μg RNA and 7.56×10 11Particles / μg protein, indicating that the AAELNs of the present invention have high purity. At the same time, the AAELNs prepared by the method of the present invention have good structural integrity and acid resistance.

[0033] In the present invention, the Actinidia arguta exosome-like nanoparticles increase the abundance of Lactobacillus and Acinetobacter, and reduce the abundance of Proteus, Escherichia coli-Shigella and Clostridium sordellii.

[0034] The present invention also provides the use of the above-mentioned Actinidia arguta exosome-like nanoparticles in the preparation of a product that promotes the growth of probiotics.

[0035] In the present invention, the probiotics preferably include lactobacillus. Adding the exosome-like nanoparticles of Actinidia arguta to products containing lactobacillus, such as yogurt, can ensure the number of live lactobacillus in the yogurt and improve the quality of products such as yogurt.

[0036] The present invention also provides the use of the above-mentioned Actinidia arguta exosome-like nanoparticles in the preparation of products for inhibiting the growth of harmful bacteria.

[0037] In the present invention, the harmful bacteria preferably include one or more of Proteus, Escherichia coli-Shigella and Clostridium sordellii. When the Actinidia arguta exosome-like nanoparticles are used in food, reagents or drugs, the contamination of harmful bacteria can be prevented.

[0038] In the present invention, the product includes reagents, foods or medicines. The product also preferably includes pharmaceutically acceptable excipients, such as one or more of adhesives, excipients, diluents, dispersants, wetting agents, cosolvents, buffers and surfactants. The dosage form of the medicine preferably includes tablets, pills, solutions, injections, granules, capsules or sprays. The medicine described in the present invention can be administered orally, rectally, intraperitoneally, subcutaneously, intramuscularly, intravenously or nasally.

[0039] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0040] In the following examples, the materials, reagent names and manufacturers used in the experiments are shown in Table 1.

[0041] Materials and Reagents:

[0042] Table 1 Names and manufacturers of reagents used in the test

[0043]

[0044]

[0045] In the following examples, the statistical analysis used in the present invention: the experimental data were analyzed and plotted using GraphPad Prism8 software. SPSS 22.0 software was used for statistical analysis, using one-way ANOVA, and the least significant difference (LSD) method was used for pairwise comparisons, and p<0.05 was considered statistically significant.

[0046] Example 1

[0047] Effects of Actinidia arguta exosome-like nanoparticles (AAELNs) on intestinal microbiota

[0048] 1.1 Preparation of Actinidia arguta exosome-like nanoparticles (AAELNs)

[0049] A method for preparing Actinidia arguta exosome-like nanoparticles (AAELNs) comprises the following steps:

[0050] Select fresh mature fruits of Actinidia arguta fruit, clean them and grind them into juice, take the juice in a 50mL centrifuge tube, centrifuge at 4000g for 10min at 4℃ to take supernatant a, supernatant a at 7000g for 20min to take supernatant b, supernatant b at 10000g for 30min to take supernatant c, supernatant c at 20000g for 70min to take precipitate 1, resuspend precipitate 1 with sterile PBS buffer to obtain resuspension 1, resuspension 11 at 20000g for 70min, carefully discard the supernatant to obtain precipitate 2, dissolve precipitate 2 with PBS buffer, and mix by blowing to obtain AAELNs. All the above operations were performed under sterile conditions.

[0051] 1.2 Characterization of AAELNs prepared in this example

[0052] 1.2.1 The AAELNs precipitated at the bottom of the centrifuge tube after ultra-high speed centrifugation in Example 1 were photographed.

[0053] 1.2.2 Transmission electron microscopy characterization

[0054] 10 μL AAELNs were added to the carbon-coated grid, left to stand for 20 min, washed with PBS, treated with 50 μL 1% glutaraldehyde solution for 5 min, washed with double distilled water for 2 min (repeated 8 times), treated with 50 μL uranyl acetate solution for 5 min, and treated with 50 μL methylcellulose-uranyl acetate solution for 10 min. Excess liquid was removed with filter paper, dried in air for 10 min, and electron microscope photos were taken at 80 kV.

[0055] Depend on Figure 1The results showed that the precipitate was clearly seen at the bottom of the ultracentrifuge tube, showing off-white color (black is the marker pen mark) ( Figure 1 A). Subsequently, the morphology of the precipitate was observed using a transmission electron microscope ( Figure 1 B), AAELNs present a teacup-shaped structure (indicated by the arrow), indicating that the AAELNs prepared by the present invention have the appearance characteristics of exosome-like nanoparticles.

[0056] 1.2.3 Particle size and Zeta potential characterization

[0057] The sample pool was washed with ultrapure water for 3 to 5 times, and the AAELNs were diluted with PBS at 1:1000 (v / v), and the particle size and Zeta potential were detected at room temperature.

[0058] like Figure 2 As shown in A, the sample particle size ranges from 78 to 220 nm, the average particle size is 157.8 nm, the PDI index is 0.211, and the number of vesicles is 6.5×10 12 Particle / mL. This indicates that AAELNs have good stability and are consistent with the particle size distribution of ELNs. The higher the absolute value of the Zeta potential, the more stable the system is and the less likely it is to agglomerate. The Zeta potential of AAELNs is -23.07mV ( Figure 2 B), AAELNs are negatively charged and the solution system is relatively stable.

[0059] 1.2.4 RNAs characterization

[0060] 1.2.4.1 RNA extraction

[0061] RNAs from AAELNs were extracted according to the instructions of Trizol Universal Total RNA Extraction Kit.

[0062] 1.2.4.2 Agarose gel electrophoresis

[0063] The RNA concentration was measured on an ultra-micro UV spectrophotometer. The loading amount was 1 μg, and the AAELNs group and the RNA-free group after adding an equal volume of RNase were set up, the agarose gel concentration was 1%, the electrophoresis voltage was 80 V, the electrophoresis time was 30 min, and the imaging was developed.

[0064] 1.2.5 Protein characterization

[0065] 1.2.5.1 Protein extraction

[0066] All operations were performed on ice. AAELNs were re-dissolved at 4°C, centrifuged at 10,000 g for 5 min at 4°C, and washed with pre-cooled PBS. An equal volume of RIPA lysis buffer (RIPA: PMSF: phosphatase inhibitor = 100:1:1) was added, pipetted and mixed, and placed on ice for 30 min, vortexed on a vortex mixer for 30 s every 10 min. Centrifuged at 12,000 g for 10 min at 4°C, and the supernatant was transferred to a new EP tube to obtain the AAELNs total protein product.

[0067] 1.2.5.2 Protein concentration determination

[0068] The protein concentration was determined by the BCA method.

[0069] 1.1.5.3SDS-PAGE

[0070] (1) Glue preparation

[0071] Separation gel and stacking gel were prepared in sequence according to the SDS-PAGE gel kit.

[0072] (2) SDS-PAGE electrophoresis

[0073] The samples were denatured by boiling in a boiling water bath for 3 to 5 minutes, and electrophoresis was started at a constant voltage of 80 V. When the protein band moved to the separation gel, the voltage was adjusted to a constant voltage of 120 V, and the electrophoresis was stopped after the protein left the separation gel and entered the electrophoresis solution.

[0074] (3) Fixed

[0075] Fixative solution: 30 mL of anhydrous ethanol and 0.5 mL of glutaraldehyde, dilute to 100 mL with double distilled water. Place the film in the fixative for 30 minutes.

[0076] (4) Dyeing

[0077] Preparation of staining solution: Accurately weigh 1.25 g of Coomassie Brilliant Blue R-250, add 225 mL of methanol and 50 mL of glacial acetic acid, and dilute to 500 mL with double distilled water. Place the film in the staining solution and stain for 4 hours.

[0078] (5) Decolorization

[0079] Destaining solution: 8 mL acetic acid and 25 mL methanol, dilute to 100 mL with double distilled water. Place the film in the destaining solution, decolorize and diffuse on a destaining shaker, and take a photo when the background is clear.

[0080] The RNA and protein concentrations extracted from the samples were 6 ng / μL and 8.60 μg / μL, respectively. The ratio of the number of vesicles (Particles) to the protein or RNA concentration can reflect the purity of the sample. The ratios of AAELNs were 1.08×10 12Particles / μg RNA and 7.56×10 11 Particles / μg protein. The value of mammalian cell ELNs is usually around 10 9 The higher particle-to-protein ratios in the range of 2.1% to 1.7% suggest that the AAELNs of the present invention may contain more RNAs and proteins per vesicle than mammalian cell-derived ELNs.

[0081] Agarose gel electrophoresis results showed that there were small molecular weight RNAs in AAELNs ( Figure 3 A). SDS-PAGE results showed that the molecular weight of the sample protein was below 35 kDa ( Figure 3 B). In summary, the sample extracted in Example 1 has the basic characteristics of exosomes and can be determined to be AAELNs.

[0082] 1.3 Storage stability of AAELNs prepared in this example

[0083] 1.3.1 Effect of time on the stability of AAELNs

[0084] 10 μL of AAELNs was dispensed into multiple EP tubes and stored at -80°C. AAELNs were diluted with PBS at 1:1000 (v / v), and the changes in particle size were measured at 0, 1, 3, 5, 7, 9, 11, 13, 18, 24, and 30 days.

[0085] 1.3.2 Effect of temperature on the stability of AAELNs

[0086] 10 μL of AAELNs was dispensed into multiple EP tubes and stored at 4°C and -80°C. AAELNs were diluted with PBS at 1:1000 (v / v), and the particle sizes were measured at different temperatures on days 0, 3, 6, 9, 12, and 15.

[0087] The results are as follows Figure 4 As shown in the figure, at -80℃, the particle size of AAELNs did not change significantly after 30 days; at 4℃, the particle size did not change significantly within 0-6 days, and with the increase of time, the particle size increased to varying degrees, and there may be aggregation, indicating that AAELNs are relatively stable when stored at -80℃. In summary, AAELNs can be stored at 4℃ for short-term (such as within 6 days), and at -80℃ for long-term storage (such as more than 30 days).

[0088] 1.3.3 Effect of pH on the stability of AAELNs

[0089] An appropriate amount of AAELNs was dispensed into EP tubes, and three different pH solutions (1.2, 6.8 and 7.8) prepared with distilled water, HCl and NaOH were added respectively. The tubes were placed in a constant temperature shaking water bath (37°C), and the particle size was measured at 0 and 2 h respectively.

[0090] The pH values ​​of gastric juice, small intestinal juice and colonic juice are approximately 1.2, 6.8 and 7.8, respectively. The particle size stability of AAELNs at different pH values ​​was investigated to provide reference data for the oral delivery of AAELNs.

[0091] like Figure 5 As shown in the figure, within 0 to 2 hours, the particle size range of AAELNs increased to varying degrees in three different environments. Under the acidic condition of pH 1.2, the particle size basically did not change; in the solutions of pH 6.8 and pH 7.8, the particle size span was more obvious. This shows that AAELNs are relatively stable in acidic solutions for a short time.

[0092] 1.4 In vitro simulated digestion of AAELNs prepared in this example

[0093] The stability of AAELNs in the gastrointestinal tract was evaluated by measuring the changes in particle size in simulated gastric fluid, small intestinal fluid, and colonic fluid at different time points. The formulas of gastric simulated fluid, small intestinal simulated fluid, and colonic simulated fluid are as follows:

[0094] Table 2 Name and formula of simulated digestive fluid

[0095]

[0096] When administering orally, it is necessary to consider the effects of the gastrointestinal environment on the stability of plant ELNs, such as the low pH of gastric juice and various digestive enzymes.

[0097] Table 3 Particle size of AAELNs in simulated gastric fluid, small intestinal fluid and colonic fluid Simulated gastric fluid

[0098]

[0099]

[0100] Simulated intestinal fluid

[0101]

[0102] Simulated colon fluid

[0103]

[0104] The results showed that in the three simulated fluids, the particle size of AAELNs showed an increasing trend (Table 3). Among them, in the simulated gastric fluid, the particle size of AAELNs increased from 170nm to 179nm within 0-4h; in the simulated small intestinal fluid, from the initial 184.8±8.9nm, after 12h of incubation, the particle size gradually stabilized at 209nm; in the simulated colonic fluid, the initial particle size was 143.4±8.5nm, and the particle sizes at the 24th, 48th and 72nd hours of incubation were 211.5±4.9nm, 215.8±2.1nm and 217.4±7.9nm, respectively, and the increase gradually decreased. Compared with simulated gastric fluid and small intestinal fluid, the particle size of AAELNs in colonic fluid changed the most ( Figure 6 ). Therefore, AAELNs may maintain their integrity when passing through simulated gastric fluid and reach the intestine.

[0105] 1.4.2 In vivo fluorescence tracing

[0106] DIR-labeled AAELNs: Add DIR dye at a final concentration of 5 μM to 2.2 mg / mL AAELNs, incubate at 37°C on a shaker for 20 min, centrifuge for 60 min (120,000 g, 4°C), discard the supernatant, and resuspend in PBS. The above experiments were performed under light-protected conditions.

[0107] In vivo imaging: C57BL / 6 mice were intragastrically administered with DIR-AAELNs at 40 mg / mouse / 200 μL. The mice were anesthetized at 3, 6, 9, and 12 h for in vivo imaging.

[0108] Organ imaging: Mice were sacrificed and the heart, liver, spleen, lung, kidney, colon, and adipose tissue were removed for imaging.

[0109] To further determine whether AAELNs can reach the intestine, the distribution of AAELNs in vivo was tracked by fluorescence.

[0110] like Figure 7 As shown, by X-Ray live imaging of mice, fluorescence signals appeared at 3h, gradually accumulated at 6h, and gradually weakened at 9h and 12h. Mice were selected for organ imaging after 6h of AAELNs digestion in vivo (when the fluorescence intensity was the highest). The results showed that there were fluorescence signals in the liver, kidneys and colon of mice, among which the fluorescence signal intensity in the colon was the highest. In summary, AAELNs can be absorbed by C57BL / 6 mice and are mainly distributed in colon tissue.

[0111] 1.4.3 Pretreatment of mouse feces

[0112] SPF female C57BL / 6 mice (18-20 g) aged 6-8 weeks were selected and raised in an IVC system at a temperature of 21°C-25°C, relative humidity (50±10%), and a 12-h light / dark cycle. After one week of adaptive feeding, the feces in the colon were collected in a sterile tube (4°C). In an anaerobic workstation, the mouse colon feces sample was diluted with sterile PBS at 1:10 (w / v), vortexed for 5 minutes, and the intestinal bacteria solution was obtained by filtration with sterile gauze, stored at 4°C and used within 30 minutes.

[0113] 1.4.4 Fermentation of fecal samples

[0114] Prepare fecal culture medium (1L): 2.0g peptone, 2.0g yeast extract, 0.1g NaCl, 0.04g K2HPO4, 0.04g KH2PO4, 0.01g MgSO4·7H2O, 0.01g CaCl2·6H2O, 2g NaHCO3, 0.005g hemin, 0.5g L-cysteine ​​hydrochloride, 0.5g porcine bile salt, 1mg resazurin (1%, m / V), 2.0mL Tween-80, 1.0mL vitamin K1, and distilled water.

[0115] The pH of the culture medium was adjusted to 7.0 and sterilized by high pressure (115°C, 20min), and placed in an anaerobic gas-generating bag overnight (37.8°C). The pH of the culture medium was adjusted to 6.8 to simulate the colon environment. The culture medium (13.5mL) was quantitatively divided into fermentation tubes, and a 200μg / mL AAELNs sample and 1.5mL intestinal bacteria solution were added. An equal volume of PBS was added to the control tube, and each group was paralleled for 3 times. The culture was cultured in an anaerobic incubator at a constant temperature of 37°C and a gas composition of 85% N2, 10% H2, and 5% CO2 for 48h.

[0116] 1.4.5 pH determination

[0117] At 0, 4, 8, 16, 24, 36, and 48 hours of anaerobic fermentation, samples were taken and placed at room temperature for 10 minutes and measured with a pH meter in three replicates.

[0118] The colonic feces of mice were collected for in vitro fermentation, and the pH of the fermentation broth was measured during the fermentation process. Figure 8 As shown in the figure, the pH of the two groups showed an overall downward trend, and the pH of the AAELNs group was always lower than that of the PBS group. After 48 hours of fermentation, the pH of the two groups decreased from the initial 7.13 and 7.19 to 6.74 and 6.58, respectively. In summary, AAELNs affected the pH environment of the colon microecology.

[0119] 1.4.6 Intestinal flora determination

[0120] The total genomic DNA of microorganisms in the cecal contents was extracted using a rapid DNA spin extraction kit. The 16S rRNA gene V3-V4 region was amplified using primers 338F (5'-ACTCCTACGGGAGGCAGCA-3' (SEQ ID No.1)) and 806R (5'-GGACTACHVGGGTWTCTAAT-3' (SEQ ID No.2)). The sequencing library was prepared after PCR amplification of the product and sequenced on the Illumina MiSeq platform. QIIME (v1.8.0, http: / / qiime.org / ) was used to identify and eliminate problematic sequences. Sequences were merged based on 97% sequence similarity, and operational taxonomic units (OTUs) were given. Based on α-diversity and β-diversity, the fecal microecology was analyzed for colony composition, and the effects of AAELNs treatment on microorganisms at the phylum and genus levels were compared.

[0121] 1.4.6.1 Effect of AAELNs of this Example on α-diversity of intestinal flora

[0122] The Illumina MiSeq platform was used to analyze the effects of AAELNs on intestinal flora. A total of 1,027,752 sequences were identified, with sequence lengths ranging from 400 to 500 bp ( Fig. 9 ). The sequencing depth of the samples was determined by using the QIIME software through Chao1 index, Simpson index, Shannon index, Faith-pd and Observed-species index analysis (Table 4).

[0123] Table 4 Analysis of α-diversity of mouse intestinal flora

[0124]

[0125] The results showed that Chao1 index and observed-species in AAELNs group were significantly higher than those in PBS group (p<0.05). The analysis of α-diversity of colonic fecal microecology showed that AAELNs may be a potential nutrient to change the diversity of intestinal flora.

[0126] 1.4.6.2 Effects of AAELNs on β-diversity of intestinal flora

[0127] The results of principal component analysis based on species level are as follows Fig.10 As shown in A, the community contributes 79.7% to the detection results in the first principal component, and 17.5% in the second principal component, and the sum of the two is greater than 50%. Principal coordinate analysis is performed based on the Unweighted Unifrac distance. Fig.10B. The contribution rate of the community to the test results in the first principal component was 29%, and the contribution rate of the second principal component was 21.6%. The intestinal microbial community of the mouse fecal fermentation liquid treated with AAELNs can be well distinguished from that of the PBS group, indicating that the composition of the two groups of flora is different. In summary, it is shown that AAELNs can affect the community structure of colonic microorganisms.

[0128] 1.4.6.3 Effects of AAELNs on the composition of intestinal flora

[0129] like Fig.11 As shown in the figure, after 48 hours of fecal microbial fermentation, at the phylum level, the main dominant bacteria in the two groups were Proteobacteria and Firmicutes. Compared with the PBS group, the abundance of Proteobacteria in the AAELNs group was significantly reduced (p<0.01), while the abundance of Firmicutes was significantly increased (p<0.01). This shows that the bacterial community structure is different at different pH values. At the genus level ( Fig.12 ), the main dominant flora in both groups included Proteus and Lactobacillus. After AAELNs intervention, the relative abundance of Acinetobacter and Lactobacillus increased, and the abundance of Proteus, Escherichia-Shigella, and Paeniclostridium decreased. Proteus, Escherichia-Shigella, and Paeniclostridium, whose abundance decreased, are all intestinal pathogens, and their abundance is higher in patients with intestinal diseases. Acinetobacter, whose abundance increased, has the effect of producing short-chain fatty acids to improve colitis. In summary, AAELNs changed the composition of the colonic intestinal flora, promoted the abundance of beneficial bacteria, and inhibited the abundance of harmful bacteria.

[0130] Example 2

[0131] A method for preparing Actinidia arguta exosome-like nanoparticles (AAELNs) comprises the following steps:

[0132] Select fresh mature fruits of Actinidia arguta fruit, clean them and grind them into juice, take the juice in a 50mL centrifuge tube, centrifuge at 3500g for 15min at 3℃, take supernatant a, supernatant a 6500g centrifuge for 35min, take supernatant b, supernatant b 9500g centrifuge for 35min, take supernatant c, supernatant c119000g centrifuge for 75min, take precipitate 1, resuspend precipitate 1 with sterile PBS buffer, centrifuge resuspension 1119000g for 75min, discard the supernatant carefully to get precipitate 2, dissolve precipitate 2 with PBS buffer, and mix by blowing to get AAELNs. All the above operations were performed under sterile conditions.

[0133] Example 3

[0134] A method for preparing Actinidia arguta exosome-like nanoparticles (AAELNs) comprises the following steps:

[0135] Select fresh mature fruits of Actinidia arguta fruit, clean them and grind them into juice, take the juice in a 50mL centrifuge tube, centrifuge at 4500g for 5min at 6℃, take supernatant a, supernatant b, supernatant c, supernatant c121000g for 65min, take precipitate 1, resuspend precipitate 1 with sterile PBS buffer, resuspend 1121000g for 65min, discard the supernatant carefully to get precipitate 2, dissolve precipitate 2 with PBS buffer, and mix well to get AAELNs. All the above operations were performed under sterile conditions.

[0136] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An application of Actinidia arguta exosome-like nanoparticles in the preparation of a product for improving intestinal flora, characterized in that: The method for preparing the Actinidia arguta exosome-like nanoparticles comprises the following steps: The juice of Actinidia arguta fruit was centrifuged at 3500-4500 g for 5-15 min to obtain supernatant a, supernatant a was centrifuged at 6500-7500 g for 15-25 min to obtain supernatant b, supernatant b was centrifuged at 9500-10500 g for 25-35 min to obtain supernatant c, supernatant c1 was centrifuged at 19000-121000 g for 65-75 min to obtain precipitate 1, precipitate 1 was resuspended by blowing with sterile PBS buffer to obtain resuspension 1, resuspension 1 was centrifuged at 119000-121000 g for 65-75 min to obtain precipitate 2, precipitate 2 was resuspended with PBS buffer to obtain Actinidia arguta exosome-like nanoparticles; The Actinidia arguta exosome-like nanoparticles increase the abundance of Lactobacillus and Acinetobacter, and reduce the abundance of Proteus, Escherichia coli-Shigella and Clostridium sordellii.

2. The use according to claim 1, characterized in that: The centrifugal temperature is 2-6°C.

3. The use according to claim 1, characterized in that: The juice of the Actinidia arguta fruit is obtained by grinding the Actinidia arguta fruit.

4. An application of Actinidia arguta exosome-like nanoparticles in the preparation of a product for improving intestinal flora, characterized in that: The method for preparing the Actinidia arguta exosome-like nanoparticles comprises the following steps: The juice of Actinidia arguta fruit was centrifuged at 3500-4500 g for 5-15 min to obtain supernatant a, supernatant a was centrifuged at 6500-7500 g for 15-25 min to obtain supernatant b, supernatant b was centrifuged at 9500-10500 g for 25-35 min to obtain supernatant c, supernatant c1 was centrifuged at 19000-121000 g for 65-75 min to obtain precipitate 1, precipitate 1 was resuspended by blowing with sterile PBS buffer to obtain resuspension 1, resuspension 1 was centrifuged at 119000-121000 g for 65-75 min to obtain precipitate 2, precipitate 2 was resuspended with PBS buffer to obtain Actinidia arguta exosome-like nanoparticles; The Actinidia arguta exosome-like nanoparticles promote the growth of lactobacilli.

5. An application of Actinidia arguta exosome-like nanoparticles in the preparation of a product for improving intestinal flora, characterized in that: The method for preparing the Actinidia arguta exosome-like nanoparticles comprises the following steps: The juice of Actinidia arguta fruit was centrifuged at 3500-4500 g for 5-15 min to obtain supernatant a, supernatant a was centrifuged at 6500-7500 g for 15-25 min to obtain supernatant b, supernatant b was centrifuged at 9500-10500 g for 25-35 min to obtain supernatant c, supernatant c1 was centrifuged at 19000-121000 g for 65-75 min to obtain precipitate 1, precipitate 1 was resuspended by blowing with sterile PBS buffer to obtain resuspension 1, resuspension 1 was centrifuged at 119000-121000 g for 65-75 min to obtain precipitate 2, precipitate 2 was resuspended with PBS buffer to obtain Actinidia arguta exosome-like nanoparticles; The Actinidia arguta exosome-like nanoparticles inhibit the growth of harmful bacteria; The harmful bacteria are one or more of Proteus, Escherichia coli-Shigella and Clostridium sordellii.

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