Extraction method of probiotic source extracellular vesicles

Abstract

The invention provides a probiotic source extracellular vesicle extraction method, and belongs to the technical field of extracellular vesicle extraction. The method comprises the following steps: adding an inducer into a culture medium, inoculating lactobacillus plantarum and lactobacillus rhamnosus, carrying out fermentation culture, respectively filtering, collecting a filtrate, adding an adsorbent, filtering, adding a solid into water, carrying out ultrasonic desorption, filtering, and freeze-drying the filtrate to prepare the probiotic-derived extracellular vesicles. The extraction method of the probiotic source extracellular vesicles is simple and low in cost, the high-purity extracellular vesicles can be obtained, the activity of the extracellular vesicles can be kept, and the extracellular vesicles have good anti-inflammatory, anti-oxidation and cell proliferation promoting effects and have wide application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of extracellular vesicle extraction, and in particular to a method for extracting probiotic-derived extracellular vesicles. Background Art

[0002] Extracellular vesicles (EVs) are biologically active, nanoscopic, spherical substances with a double-layer membrane structure that are actively secreted by cells. They are primarily composed of lipids, proteins, and genetic material. Their main types include exosomes (particle size 30-150 nm), microvesicles (particle size 100-1000 nm), and apoptotic bodies (particle size >1000 nm). EVs are characterized by their small molecular structure and high biocompatibility. They can be used to transport substances such as lipids, proteins, DNA, and RNA, acting as natural endogenous nanocarriers. EVs are widely present in organisms, including plants, mammals, and humans. In principle, all living cells secrete EVs. Currently, EVs are primarily extracted from cell culture media and body fluids such as blood, urine, cerebrospinal fluid, breast milk, amniotic fluid, and ascites.

[0003] EVs play an important role in the transmission of information between cells and are involved in a variety of physiological and pathological processes in the body, including inflammation, tissue homeostasis, neuronal communication, immune system regulation, tumorigenesis and metastasis. EVs have diverse functions and broad clinical application prospects, and can be used for disease diagnosis, nanodrug delivery, targeted therapy, immunotherapy, etc. As effective carriers for delivering drugs and functional RNA, EVs have obvious advantages: a broad and natural source, nanometer size, and membrane ligands that can be used for targeted therapy. Currently, there are many difficulties in the clinical translation of EVs, as follows: yield issues. Most current methods, such as ultracentrifugation, produce low yields of EVs; purity issues. Many methods will isolate biomolecules of similar size to EVs, causing potential contamination; source issues. Although EVs have a wide range of sources, it is difficult to obtain EVs with higher homogeneity; and economies of scale. Obtaining large quantities of EVs at a low cost is a key point in clinical translation.

[0004] Probiotic-derived EVs have unique advantages in some fields because they carry active ingredients (such as lipoteichoic acid and protein), but their extraction faces bottlenecks such as low yield, high cost, and complex operation. Summary of the Invention

[0005] The purpose of the present invention is to propose a method for extracting extracellular vesicles from probiotics. The method is simple, low-cost, can obtain high-purity extracellular vesicles, and the activity of the extracellular vesicles can be maintained. It has good anti-inflammatory, antioxidant and cell proliferation-promoting effects and has broad application prospects.

[0006] The technical solution of the present invention is achieved as follows: The invention provides a method for extracting probiotic-derived extracellular vesicles. The method comprises the following steps: adding an inducer to a culture medium, inoculating Lactobacillus plantarum and Lactobacillus rhamnosus, fermenting and culturing, filtering the culture media separately, collecting the filtrate, adding an adsorbent, filtering the filtrate, adding a solid to water, performing ultrasonic desorption, filtering, and freeze-drying the filtrate to obtain the probiotic-derived extracellular vesicles.

[0007] As a further improvement of the present invention, the following steps are included: S1. Mix serine and cholesterol to prepare an inducer; S2. The inducer, carbon source, nitrogen source, vitamins, and inorganic substances are added to water, stirred and mixed, and sterilized to prepare a culture medium; S3 Lactobacillus plantarum and Lactobacillus rhamnosus seed solution was inoculated into the culture medium to induce fermentation to obtain a mixed solution; S4. The mixture was frozen in liquid nitrogen, thawed at room temperature, and ultrasonically treated at low temperature to obtain a broken mixture; S5. Place a 0.45 μm centrifugal filter sleeve on the centrifuge tube and centrifuge once to separate debris >450 nm. Place a 0.22 μm centrifugal filter sleeve on the centrifuge tube and centrifuge again to separate bacteria >220 nm. Collect the filtrate. S6. Add an adsorbent to the filtrate, stir and adsorb, filter, add the solid to water, desorb under low-temperature ultrasonication, filter, dialyze the filtrate, and freeze-dry to produce probiotic-derived extracellular vesicles.

[0008] As a further improvement of the present invention, the mass ratio of serine to cholesterol in step S1 is 3-5:1-3; the mass ratio of the inducer, carbon source, nitrogen source, vitamins, inorganic matter, and water in step S2 is 2-4:15-20:8-12:2-3:3-5:200-300, the carbon source is selected from at least one of molasses, glucose, maltose, lactose, sucrose, fructose, and starch, the nitrogen source is selected from peptone, fish bone meal, ammonia water, urea, ammonium salt, nitrate, and amino acid, and the vitamins are selected from vitamin C, vitamin B1, vitamin B2, and vitamin B6. The present invention relates to a novel nanostructured carbon foam comprising at least one of vitamin A, vitamin B12, vitamin D3, and vitamin E; the inorganic salt is selected from at least one of sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride, zinc sulfate, copper sulfate, manganese sulfate, zinc chloride, copper chloride, and manganese chloride; the amino acid is selected from at least one of glycine, serine, threonine, valine, tryptophan, leucine, alanine, cysteine, methionine, lysine, isoleucine, and phenylalanine; the ammonium salt is selected from at least one of ammonium chloride and ammonium sulfate; and the nitrate is selected from at least one of sodium nitrate, potassium nitrate, and ammonium nitrate.

[0009] As a further improvement of the present invention, the bacterial content of the bacterial seed liquid in step S3 is 10 8 -10 9 cfu / mL, the inoculation amounts of the plant lactobacillus and rhamnosus lactobacillus seed liquids are 2-3v / v% and 1-3v / v%, respectively; the conditions for the induced fermentation culture are 36-38°C, 100-200r / min, 3-6v / v% CO2, and the induced fermentation culture is 36-48h; the conditions for the low-temperature ultrasonic treatment in step S4 are 200-300W ultrasound for 3-5s at a temperature of 2-4°C, and the ultrasonic treatment is performed again after stopping the treatment for 60-90s, and this is carried out for 10-15min.

[0010] As a further improvement of the present invention, the speed of the first centrifugation in step S5 is 2000-3000 r / min, the time is 10-15 min, the speed of the second centrifugation is 3000-4000 r / min, and the time is 5-10 min; the mass ratio of the filtrate to the adsorbent in step S6 is 10:2-3, the stirring adsorption time is 10-20 min, and the low-temperature ultrasonic desorption conditions are 200-400W ultrasonic treatment for 3-5 min at a temperature of 2-4 ° C.

[0011] As a further improvement of the present invention, the preparation method of the adsorbent is as follows: T1. Preparation of porous silica microspheres: A porogen and an alkyl orthosilicate were added to ethanol, concentrated hydrochloric acid and water were added, the reaction was stirred, filtered, washed, dried, and calcined to obtain porous silica microspheres; T2. Preparation of amino-modified porous silica microspheres: The porous silica microspheres were added to ethanol, an aminosilane coupling agent was added, the reaction was heated and stirred, filtered, washed, and dried to obtain amino-modified porous silica microspheres; T3. Preparation of adsorbent: Add lipopolysaccharide binding protein LBP and lipopolysaccharide-specific receptor CD14 to water, add EDC and NHS, stir to activate, add amino-modified porous silica microspheres, stir to react, filter, wash, and freeze-dry to prepare the adsorbent.

[0012] As a further improvement of the present invention, the mass ratio of the porogen, alkyl orthosilicate, ethanol, concentrated hydrochloric acid and water in step T1 is 1-2:10-12:80-100:5-7:4-8, the porogen is selected from at least one of D-glutamic acid, D-lysine, L-glycine, L-alanine, and L-valine; the alkyl orthosilicate is methyl orthosilicate or ethyl orthosilicate, the stirring reaction time is 10-12 hours, and the calcination temperature is 400-500° C. and the time is 1-3 hours.

[0013] As a further improvement of the present invention, the mass ratio of the porous silica microspheres to the aminosilane coupling agent in step T2 is 10:2-3, the temperature of the heating and stirring reaction is 45-55°C, and the time is 2-4 hours. The aminosilane coupling agent is selected from at least one of KH550, KH602, and KH792.

[0014] As a further improvement of the present invention, the mass ratio of the lipopolysaccharide binding protein LBP, the lipopolysaccharide-specific receptor CD14, EDC, NHS and amino-modified porous silica microspheres in step T3 is 3-5:2-3:1-2:1-2:10-12, the stirring activation time is 20-40 minutes, and the stirring reaction time is 8-10 hours.

[0015] The present invention further protects probiotic-derived extracellular vesicles obtained by the above extraction method.

[0016] The present invention has the following beneficial effects: The present invention selects serine and cholesterol as inducers. Serine is an important raw material for phosphatidylserine, a membrane material of probiotic-derived extracellular vesicles. The probiotic-derived extracellular vesicles of the present invention also contain cholesterol. Cholesterol can regulate the fluidity, elasticity, and permeability of the membrane, enabling the vesicles to maintain their structural integrity in different physiological environments and facilitating the embedding and functioning of certain specific biomolecules. Therefore, the inducer can promote the secretion of extracellular vesicles by probiotics, thereby greatly increasing the secretion and production of extracellular vesicles.

[0017] The probiotics selected by the present invention include Lactobacillus plantarum and Lactobacillus rhamnosus, which have rapid proliferation and higher extracellular vesicles. The interaction between the two probiotics can promote them to quickly enter the logarithmic phase and promote each other's growth.

[0018] The logarithmic phase bacterial liquid of the present invention is frozen in liquid nitrogen and thawed at room temperature. Ice crystals are used to destroy the cell wall to prevent chemical reagents such as lysozyme from damaging the EVs membrane structure. At the same time, low-power intermittent ultrasonic treatment is used at low temperature to break the cell wall while avoiding excessive lysis of EVs, effectively protecting the intact structure of EVs.

[0019] In addition, the present invention uses a commercial 0.45μm centrifugal filter sleeve added to the centrifuge tube for centrifugation to intercept fragments larger than 450nm, and then uses a commercial 0.22μm centrifugal filter sleeve added to the centrifuge tube for centrifugation to intercept bacteria larger than 220nm. Centrifugal force is used to drive EVs through the membrane, avoiding manual squeezing and blocking the membrane, effectively separating the fragments and bacteria. At the same time, EVs with a pore size smaller than 0.22μm and a small amount of small-molecule glycophospholipid fragments pass through.

[0020] An adsorbent is added to the filtrate. This adsorbent, based on silica as a carrier and porogenized with a chiral amino acid, creates a rich surface porosity on the microspheres, increasing the specific surface area and pore size, and increasing the number of adsorption sites. Furthermore, the biocompatible porogen does not induce immune or toxic reactions in the organism, thereby maximizing the biological activity of EVs. After the porous silica microspheres are modified with an aminosilane coupling agent, the surface is then conjugated to lipopolysaccharide-binding protein (LBP) and the LPS-specific receptor CD14 through a condensation reaction between amino and carboxyl groups. LBP is an acute phase protein that recognizes and accumulates LPS. Lipopolysaccharide (LPS) is abundant on the surface of extracellular vesicles of Lactobacillus probiotics and is highly immunogenic, activating the host's innate immune response. LBP tightly binds to the lipid A portion of LPS, enhancing its biological activity and promoting its intercellular delivery. CD14 is a specific receptor for lipopolysaccharide. The LPS-LBP complex can bind to CD14 to form an LPS-LBP-CD14 triplet complex, thereby activating intracellular signaling pathways. Therefore, coupling LBP and CD14 on the adsorbent surface can specifically adsorb probiotic-derived extracellular vesicles without destroying their structure and function. Low-power, low-temperature ultrasonic desorption can promote the entry of extracellular vesicles into the solution, and dialysis removes inorganic salts and other small molecular impurities in the liquid, thereby obtaining high-purity extracellular vesicles. At the same time, the adsorbent can be reused after washing and freeze-drying, improving its resource utilization and reducing production costs.

[0021] The method for extracting probiotic-derived extracellular vesicles of the present invention is simple, low-cost, can obtain high-purity extracellular vesicles, and the activity of the extracellular vesicles can be maintained. It has good anti-inflammatory, antioxidant and cell proliferation-promoting effects and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 This is a TEM image of the probiotic-derived extracellular vesicles prepared in Example 1. DETAILED DESCRIPTION

[0024] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0025] Lipopolysaccharide-binding protein LBP, Huamei Biotechnology; lipopolysaccharide-specific receptor CD14, Huamei Biotechnology; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; NHS, N-hydroxysuccinimide.

[0026] Preparation method of Lactobacillus plantarum (20 billion cfu / g) and Lactobacillus rhamnosus (20 billion cfu / g) seed solution: inoculate the strain into Gao's medium, incubate at 37℃, 150r / min, activate and culture for 24-36h, and obtain a culture medium with a bacterial count of 10 8 -10 9 cfu / mL of bacterial seed liquid.

[0027] Preparation Example 1 Preparation of adsorbent Here’s how: T1. Preparation of porous silica microspheres: 1 g of L-alanine and 10 g of methyl orthosilicate were added to 80 g of ethanol, followed by 5 g of concentrated hydrochloric acid and 4 g of water. The mixture was stirred for 10 h, filtered, washed, dried, and calcined at 400°C for 1 h to produce porous silica microspheres. T2. Preparation of amino-modified porous silica microspheres: 10 g of porous silica microspheres were added to 150 mL of ethanol, along with 2 g of silane coupling agent KH602. The mixture was heated to 45°C and stirred for 2 h. The mixture was filtered, washed, and dried to obtain amino-modified porous silica microspheres. T3. Preparation of adsorbent: Add 3 g of lipopolysaccharide binding protein (LBP) and 2 g of the lipopolysaccharide-specific receptor CD14 to 150 mL of water, add 1 g of EDC and 1 g of NHS, and stir to activate for 20 min. Add 10 g of amino-modified porous silica microspheres and stir to react for 8 h. Filter, wash, and freeze-dry to prepare the adsorbent.

[0028] Preparation Example 2 Preparation of adsorbent Here’s how: T1. Preparation of porous silica microspheres: 2 g of L-valine and 12 g of ethyl orthosilicate were added to 100 g of ethanol, followed by 7 g of concentrated hydrochloric acid and 8 g of water. The mixture was stirred for 12 h, filtered, washed, dried, and calcined at 500°C for 3 h to produce porous silica microspheres. T2. Preparation of amino-modified porous silica microspheres: 10 g of porous silica microspheres were added to 150 mL of ethanol, along with 3 g of silane coupling agent KH792. The mixture was heated to 55°C and stirred for 4 h. The mixture was filtered, washed, and dried to obtain amino-modified porous silica microspheres. T3. Preparation of adsorbent: Add 5 g of lipopolysaccharide binding protein (LBP) and 3 g of the lipopolysaccharide-specific receptor CD14 to 150 mL of water, add 2 g of EDC and 2 g of NHS, and stir to activate for 40 min. Then add 12 g of amino-modified porous silica microspheres and stir to react for 10 h. Filter, wash, and freeze-dry to prepare the adsorbent.

[0029] Preparation Example 3 Preparation of adsorbent Here’s how: T1. Preparation of porous silica microspheres: 1.5 g of D-glutamic acid and 11 g of tetraethyl orthosilicate were added to 90 g of ethanol, followed by 6 g of concentrated hydrochloric acid and 6 g of water. The mixture was stirred for 11 h, filtered, washed, dried, and calcined at 450°C for 2 h to produce porous silica microspheres. T2. Preparation of amino-modified porous silica microspheres: 10 g of porous silica microspheres were added to 150 mL of ethanol, along with 2.5 g of silane coupling agent KH550. The mixture was heated to 50°C and stirred for 3 h. The mixture was then filtered, washed, and dried to obtain amino-modified porous silica microspheres. T3. Preparation of adsorbent: Add 4 g of lipopolysaccharide binding protein (LBP) and 2.5 g of the lipopolysaccharide-specific receptor CD14 to 150 mL of water, add 1.5 g of EDC and 1.5 g of NHS, and stir to activate for 30 min. Add 11 g of amino-modified porous silica microspheres, stir to react for 9 h, filter, wash, and freeze-dry to prepare the adsorbent.

[0030] Comparative Preparation Example 1 The difference compared with Preparation Example 3 is that D-glutamic acid is not added in step T1.

[0031] The details are as follows: T1. Preparation of silica microspheres: 11 g of ethyl orthosilicate was added to 90 g of ethanol, followed by 6 g of concentrated hydrochloric acid and 6 g of water. The mixture was stirred for 11 h, filtered, washed, dried, and calcined at 450°C for 2 h to obtain silica microspheres.

[0032] Comparative Preparation Example 2 Compared with Preparation Example 3, the difference is that lipopolysaccharide binding protein LBP is not added in step T3.

[0033] The details are as follows: T3. Preparation of adsorbent: Add 6.5 g of CD14, a specific receptor for lipopolysaccharide, to 150 mL of water, followed by 1.5 g of EDC and 1.5 g of NHS. Stir and activate for 30 min. Add 11 g of amino-modified porous silica microspheres and stir for 9 h. Filter, wash, and freeze-dry to obtain the adsorbent.

[0034] Comparative Preparation Example 3 Compared with Preparation Example 3, the difference is that the specific receptor CD14 for lipopolysaccharide is not added in step T3.

[0035] The details are as follows: T3. Preparation of adsorbent: Add 6.5 g of lipopolysaccharide binding protein (LBP) to 150 mL of water, add 1.5 g of EDC and 1.5 g of NHS, and stir to activate for 30 min. Then add 11 g of amino-modified porous silica microspheres and stir to react for 9 h. The adsorbent is filtered, washed, and freeze-dried.

[0036] Comparative Preparation Example 4 Compared with Preparation Example 3, the difference is that step T3 is not performed.

[0037] The details are as follows: T1. Preparation of porous silica microspheres: 1.5 g of D-glutamic acid and 11 g of tetraethyl orthosilicate were added to 90 g of ethanol, followed by 6 g of concentrated hydrochloric acid and 6 g of water. The mixture was stirred for 11 h, filtered, washed, dried, and calcined at 450°C for 2 h to produce porous silica microspheres. T2. Preparation of amino-modified porous silica microspheres: 10 g of porous silica microspheres were added to 150 mL of ethanol, along with 2.5 g of silane coupling agent KH550. The mixture was heated to 50°C and stirred for 3 h. The mixture was filtered, washed, and dried to obtain amino-modified porous silica microspheres, which served as the adsorbent.

[0038] Example 1

[0039] This embodiment provides a method for extracting probiotic-derived extracellular vesicles, comprising the following steps: S1. Mix 3g of serine and 1g of cholesterol to prepare an inducer; S2 2g inducer, 15g molasses, 8g ammonium nitrate, 1g vitamin C, 1g vitamin A, 3g inorganic matter were added to 200g of water, stirred and mixed, and sterilized to obtain a culture medium; The inorganic substance is a mixture of sodium chloride, calcium chloride, copper chloride, manganese chloride and magnesium chloride in a mass ratio of 10:3:0.5:0.2:1; S3. Lactobacillus plantarum and Lactobacillus rhamnosus seed suspensions were inoculated into the culture medium at inoculum levels of 2 v / v% and 1 v / v%, respectively, and fermented at 36°C, 100 rpm, and 3 v / v% CO2 for 36 h to produce a mixed solution. S4. The mixture was frozen in liquid nitrogen, thawed at room temperature, and ultrasonically treated at 200W for 3 seconds at 2°C. After 60 seconds of treatment, ultrasonic treatment was repeated for 10 minutes to obtain a broken cell mixture. S5. Place a 0.45 μm centrifugal filter cannula on the centrifuge tube and centrifuge at 2000 rpm for 10 minutes to separate debris >450 nm. Place a 0.22 μm centrifugal filter cannula on the centrifuge tube and centrifuge at 3000 rpm for 5 minutes to separate bacteria >220 nm. Collect the filtrate. S6. To 100 g of the filtrate, 20 g of the adsorbent prepared in Preparation Example 1 was added, and the mixture was stirred and adsorbed for 10 min. The mixture was filtered, and the solid was added to 100 mL of water. The mixture was ultrasonically treated at 200 W for 3 min at 2°C, filtered, and the filtrate was dialyzed using a 5000 Da dialysis bag for 3 days. The mixture was freeze-dried to obtain probiotic-derived extracellular vesicles. Figure 1 This is a TEM image of the prepared probiotic-derived extracellular vesicles. As can be seen from the image, the probiotic-derived extracellular vesicles are spherical structures with a particle size of about 200 nm.

[0040] Example 2

[0041] This embodiment provides a method for extracting probiotic-derived extracellular vesicles, comprising the following steps: S1. 5g of serine and 3g of cholesterol were mixed to prepare an inducer; S2 4g inducer, 20g glucose, 12g fish bone meal, 1g vitamin C, 1g vitamin A, 1g vitamin B1, 5g inorganic matter were added to 300g of water, stirred and mixed, and sterilized to obtain a culture medium; The inorganic substance is a mixture of sodium chloride, calcium chloride, copper chloride, manganese chloride and magnesium chloride in a mass ratio of 10:3:0.5:0.2:1; S3. Lactobacillus plantarum and Lactobacillus rhamnosus seed suspensions were inoculated into the culture medium at inoculum levels of 3 v / v% and 3 v / v%, respectively, and fermented at 38°C, 200 rpm, and 6 v / v% CO2 for 48 h to produce a mixed solution. S4. The mixture was frozen in liquid nitrogen, thawed at room temperature, and ultrasonically treated at 300W for 5 seconds at 4°C. After 90 seconds, ultrasonic treatment was repeated for 10-15 minutes to obtain a broken cell mixture. S5. Place a 0.45 μm centrifugal filter cannula on the centrifuge tube and centrifuge at 3000 rpm for 15 minutes to separate debris >450 nm. Place a 0.22 μm centrifugal filter cannula on the centrifuge tube and centrifuge at 4000 rpm for 10 minutes to separate bacteria >220 nm. Collect the filtrate. S6. To 100 g of the filtrate, 30 g of the adsorbent prepared in Preparation Example 2 was added. The mixture was stirred and adsorbed for 20 min. The mixture was filtered. The solid was added to 100 mL of water and sonicated at 400 W for 5 min at 4°C. The mixture was filtered. The filtrate was dialyzed using a 5000 Da dialysis bag for 3 days and freeze-dried to obtain probiotic-derived extracellular vesicles.

[0042] Example 3

[0043] This embodiment provides a method for extracting probiotic-derived extracellular vesicles, comprising the following steps: S1. 4 g of serine and 2 g of cholesterol were mixed to prepare an inducer; S2 3g inducer, 10g molasses, 7g glucose, 10g peptone, 1g vitamin C, 1.5g vitamin A, 4g inorganic matter were added to 250g of water, stirred and mixed, and sterilized to obtain a culture medium; The inorganic substance is a mixture of sodium chloride, calcium chloride, copper chloride, manganese chloride and magnesium chloride in a mass ratio of 10:3:0.5:0.2:1; S3. Lactobacillus plantarum and Lactobacillus rhamnosus seed suspensions were inoculated into the culture medium at inoculum levels of 2.5 v / v% and 2 v / v%, respectively, and fermented at 37°C, 150 rpm, and 5 v / v% CO2 for 42 h to produce a mixed solution. S4. The mixture was frozen in liquid nitrogen, thawed at room temperature, and ultrasonically treated at 250W for 4 seconds at 3°C. After 75 seconds, ultrasonic treatment was repeated for 12 minutes to obtain a cell-broken mixture. S5. Place a 0.45 μm centrifugal filter cannula on the centrifuge tube and centrifuge at 2500 rpm for 12 minutes to separate debris >450 nm. Place a 0.22 μm centrifugal filter cannula on the centrifuge tube and centrifuge at 3500 rpm for 7 minutes to separate bacteria >220 nm. Collect the filtrate. S6. To 100 g of the filtrate, 25 g of the adsorbent prepared in Preparation Example 3 was added. The mixture was stirred and adsorbed for 15 min. The mixture was filtered. The solid was added to 100 mL of water and sonicated at 300 W for 4 min at 3°C. The mixture was filtered. The filtrate was dialyzed using a 5000 Da dialysis bag for 3 d and freeze-dried to obtain probiotic-derived extracellular vesicles.

[0044] Comparative Example 1 Compared with Example 3, the difference is that the adsorbent is prepared by Comparative Preparation Example 1. The purity of the probiotic-derived extracellular vesicles in the product is significantly reduced.

[0045] Comparative Example 2 Compared with Example 3, the difference is that the adsorbent is prepared by Comparative Preparation Example 2.

[0046] Comparative Example 3 Compared with Example 3, the difference is that the adsorbent is prepared by Comparative Preparation Example 3.

[0047] Comparative Example 4 Compared with Example 3, the difference is that the adsorbent is prepared by Comparative Preparation Example 4.

[0048] Comparative Example 5 Compared with Example 3, the difference is that no inducer is added in step S2.

[0049] The details are as follows: S2 10g molasses, 7g glucose, 10g peptone, 1g vitamin C, 1.5g vitamin A, 4g inorganic matter were added to 250g of water, stirred and mixed, and sterilized to obtain a culture medium; The inorganic substance is a mixture of sodium chloride, calcium chloride, copper chloride, manganese chloride and magnesium chloride in a mass ratio of 10:3:0.5:0.2:1.

[0050] Comparative Example 6 Compared with Example 3, the difference is that no ultrasonic treatment is performed in step S4.

[0051] The details are as follows: S4. Freeze the mixture in liquid nitrogen and thaw it at room temperature to obtain a cell wall-broken mixture.

[0052] Comparative Example 7 Compared with Example 3, the difference is that no adsorbent is added in step S6, and the filtrate is dialyzed and then freeze-dried.

[0053] The details are as follows: S6. 100 g of the filtrate was dialyzed using a dialysis bag with a pore size of 5000 Da for 3 days and freeze-dried to obtain probiotic-derived extracellular vesicles.

[0054] Test Example 1 Anti-inflammatory effect SPF grade NIH male mice were selected as experimental mice and divided into 12 groups, with 10 mice in each group. Distilled water was used as the negative control group, fluocinonide ointment was used as the positive control group, and the probiotic-derived extracellular vesicle aqueous suspension prepared in Examples 1-3 and Comparative Examples 1-7 was used as the experimental group at a concentration of 2×108 Particles / mL.

[0055] Each mouse's right auricle was inflamed by applying xylene evenly to both the inner and outer surfaces at a dose of 100 μL / mouse. The left ear remained untreated and served as a blank control. 30 minutes after xylene induction, all animals in the negative control group received the corresponding test substance in their right ear at a dose of 50 μL / mouse, ensuring that the substance was evenly applied to both the inner and outer surfaces of the right auricle. Animals in the negative control group received distilled water at a dose of 50 μL / mouse. One hour after test substance administration, the mice were sacrificed by cervical dislocation, and both auricles were removed. The test substance on the right auricle was washed clean with saline and dried. The auricles were overlapped, and the left and right ear pieces were punched out using an 8 mm diameter punch. The pieces were weighed, and the swelling value was calculated. The results are shown in Table 1.

[0056] Swelling value = m 右耳耳片 -m 左耳耳片 Table 1 Group Swelling value (mg) Negative control group 9.4±2.2 Positive control group <![CDATA[6.3±2.7 * ]]> Example 1 <![CDATA[3.7±1.9 * ]]> Example 2 <![CDATA[3.8±2.1 * ]]> Example 3 <![CDATA[3.5±1.6 * ]]> Comparative Example 1 5.2±2.9 Comparative Example 2 4.8±3.1 Comparative Example 3 4.9±3.0 Comparative Example 4 5.6±3.5 Comparative Example 5 6.1±3.8 Comparative Example 6 5.0±2.8 Comparative Example 7 7.9±3.9 Note: *P<0.05 compared with the negative control group.

[0057] The smaller the swelling value, the better the anti-inflammatory effect. As can be seen from the above table, the probiotic-derived extracellular vesicles prepared in Examples 1-3 of the present invention have a good anti-inflammatory effect.

[0058] Test Example 2 Antioxidant Test 20 μL of the probiotic-derived extracellular vesicle aqueous suspensions prepared in Examples 1-3 and Comparative Examples 1-7 were added to a 96-well plate at a concentration of 2×10 8 Particles / mL, followed by the addition of DPPH solution (0.12 mg / mL), mixing thoroughly, and incubating at 37°C for 30 min. The absorbance A was measured. The absorbance of the sample background was A0, the absorbance of the DPPH well was C, and the absorbance of the solvent background was C0. The results are shown in Table 2.

[0059] Calculate the free radical scavenging rate of the test solution according to the formula: Clearance rate (%) = [1-(A-A0) / (C-C0)] 100%.

[0060] Table 2 Group Free radical scavenging rate (%) Example 1 82.5 Example 2 82.1 Example 3 83.2 Comparative Example 1 74.5 Comparative Example 2 77.9 Comparative Example 3 78.2 Comparative Example 4 70.1 Comparative Example 5 64.4 Comparative Example 6 76.3 Comparative Example 7 54.2 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) is a stable nitrogen-centered chromogenic free radical with a characteristic absorption peak at a wavelength of 517 nm. In the presence of a free radical scavenger, the absorbance of DPPH decreases until it disappears. The change in absorbance is linearly correlated with the antioxidant content within a certain range, allowing the sample's free radical scavenging ability, i.e., antioxidant efficacy, to be evaluated. As shown in the table above, the probiotic-derived extracellular vesicles prepared in Examples 1-3 of the present invention exhibited good antioxidant efficacy.

[0061] Test Example 3 Cell Proliferation Test Select healthy mouse embryonic fibroblasts (NIH-3T3) and seed them into 96-well plates. Discard the old culture medium, wash twice with PBS, trypsinize, resuspend, count, and seed at a density of 4,000 cells per well. Incubate in a 37°C, 5% CO2 incubator for 24 hours. Discard the old culture medium from each well, wash the cells once with PBS (pH 7.4), and add the culture medium from the negative control or experimental group, with 100 μL of system per well.

[0062] The negative control group was a DMEM high-glucose basal medium, and the experimental group was a DMEM high-glucose basal medium supplemented with the probiotic-derived extracellular vesicle aqueous suspensions prepared in Examples 1-3 and Comparative Examples 1-7, respectively, to a final concentration of 1×10 8 Particles / mL. After 48 hours of continued culture, 10 μL / well of CCK8 reagent was added and incubated at 37°C in the dark for 2 hours. The absorbance (450 nm) was measured on a microplate reader, and the relative cell viability (relative to the negative control group) was calculated. The results are shown in Table 3.

[0063] Table 3 Group Relative cell viability (%) Example 1 138 Example 2 137 Example 3 140 Comparative Example 1 122 Comparative Example 2 125 Comparative Example 3 127 Comparative Example 4 118 Comparative Example 5 115 Comparative Example 6 123 Comparative Example 7 103 As can be seen from the above table, the probiotic-derived extracellular vesicles prepared in Examples 1-3 of the present invention have a good effect of promoting fibroblast proliferation.

[0064] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for extracting extracellular vesicles from probiotics, characterized in that: The inducer is added to the culture medium, and Lactobacillus plantarum and Lactobacillus rhamnosus are inoculated, fermented and cultured, filtered separately, the filtrate is collected, an adsorbent is added, filtered, the solid is added to water, ultrasonically desorbed, filtered, and the filtrate is freeze-dried to prepare probiotic-derived extracellular vesicles.

2. The extraction method according to claim 1, wherein The following steps are involved: S1. Mix serine and cholesterol to prepare an inducer; S2. The inducer, carbon source, nitrogen source, vitamins, and inorganic substances are added to water, stirred and mixed, and sterilized to prepare a culture medium; S3 Lactobacillus plantarum and Lactobacillus rhamnosus seed solution was inoculated into the culture medium to induce fermentation to obtain a mixed solution; S4. The mixture was frozen in liquid nitrogen, thawed at room temperature, and ultrasonically treated at low temperature to obtain a broken mixture; S5. Place a 0.45 μm centrifugal filter sleeve on the centrifuge tube and centrifuge once to separate debris >450 nm. Place a 0.22 μm centrifugal filter sleeve on the centrifuge tube and centrifuge again to separate bacteria >220 nm. Collect the filtrate. S6. Add an adsorbent to the filtrate, stir and adsorb, filter, add the solid to water, desorb under low-temperature ultrasonic wave, filter, dialyze the filtrate, and freeze-dry to obtain probiotic-derived extracellular vesicles.

3. The extraction method according to claim 2, characterized in that The mass ratio of serine to cholesterol in step S1 is 3-5:1-3; the mass ratio of the inducer, carbon source, nitrogen source, vitamins, inorganic matter, and water in step S2 is 2-4:15-20:8-12:2-3:3-5:200-300, the carbon source is selected from at least one of molasses, glucose, maltose, lactose, sucrose, fructose, and starch, the nitrogen source is selected from peptone, fish bone meal, ammonia water, urea, ammonium salt, nitrate, and amino acid, and the vitamins are selected from vitamin C, vitamin B1, vitamin B2, vitamin A, and vitamin B6. The inorganic salt is selected from at least one of sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride, zinc sulfate, copper sulfate, manganese sulfate, zinc chloride, copper chloride, and manganese chloride; the amino acid is selected from at least one of glycine, serine, threonine, valine, tryptophan, leucine, alanine, cysteine, methionine, lysine, isoleucine, and phenylalanine; the ammonium salt is selected from at least one of ammonium chloride and ammonium sulfate; and the nitrate is selected from at least one of sodium nitrate, potassium nitrate, and ammonium nitrate.

4. The extraction method according to claim 2, wherein The bacterial content of the bacterial seed liquid in step S3 is 10 8 -10 9 cfu / mL, the inoculation amounts of the plant lactobacillus and rhamnosus lactobacillus seed liquids are 2-3v / v% and 1-3v / v%, respectively; the conditions for the induced fermentation culture are 36-38°C, 100-200r / min, 3-6v / v% CO2, and the induced fermentation culture is 36-48h; the conditions for the low-temperature ultrasonic treatment in step S4 are 200-300W ultrasound for 3-5s at a temperature of 2-4°C, and the ultrasonic treatment is performed again after stopping the treatment for 60-90s, and this is carried out for 10-15min.

5. The extraction method according to claim 2, characterized in that The speed of the first centrifugation in step S5 is 2000-3000 r / min, and the time is 10-15 min. The speed of the second centrifugation is 3000-4000 r / min, and the time is 5-10 min. The mass ratio of the filtrate to the adsorbent in step S6 is 10:2-3, the stirring adsorption time is 10-20 min, and the low-temperature ultrasonic desorption conditions are 200-400W ultrasonic treatment for 3-5 min at a temperature of 2-4°C.

6. The extraction method according to claim 5, characterized in that The preparation method of the adsorbent is as follows: T1. Preparation of porous silica microspheres: A porogen and an alkyl orthosilicate were added to ethanol, concentrated hydrochloric acid and water were added, the reaction was stirred, filtered, washed, dried, and calcined to obtain porous silica microspheres; T2. Preparation of amino-modified porous silica microspheres: The porous silica microspheres were added to ethanol, an aminosilane coupling agent was added, the reaction was heated and stirred, filtered, washed, and dried to obtain amino-modified porous silica microspheres; T3. Preparation of adsorbent: Add lipopolysaccharide binding protein LBP and lipopolysaccharide-specific receptor CD14 to water, add EDC and NHS, stir to activate, add amino-modified porous silica microspheres, stir to react, filter, wash, and freeze-dry to prepare the adsorbent.

7. The extraction method according to claim 6, characterized in that In step T1, the mass ratio of the porogen, alkyl orthosilicate, ethanol, concentrated hydrochloric acid, and water is 1-2:10-12:80-100:5-7:4-8, the porogen is selected from at least one of D-glutamic acid, D-lysine, L-glycine, L-alanine, and L-valine; the alkyl orthosilicate is methyl orthosilicate or ethyl orthosilicate, the stirring reaction time is 10-12 hours, and the calcination temperature is 400-500° C. and the time is 1-3 hours.

8. The extraction method according to claim 6, characterized in that The mass ratio of the porous silica microspheres to the aminosilane coupling agent in step T2 is 10:2-3, the heating and stirring reaction temperature is 45-55° C., and the time is 2-4 hours. The aminosilane coupling agent is selected from at least one of KH550, KH602, and KH792.

9. The extraction method according to claim 6, characterized in that In step T3, the mass ratio of lipopolysaccharide binding protein LBP, lipopolysaccharide specific receptor CD14, EDC, NHS and amino-modified porous silica microspheres is 3-5:2-3:1-2:1-2:10-12, the stirring activation time is 20-40 minutes, and the stirring reaction time is 8-10 hours.

10. Probiotic-derived extracellular vesicles obtained by the extraction method according to any one of claims 1 to 9.

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