A method for extracting extracellular vesicles from probiotic source cells

Abstract

This invention proposes a method for extracting probiotic-derived extracellular vesicles, belonging to the field of extracellular vesicle extraction technology. The method includes: adding an inducer to a culture medium, inoculating with *Lactobacillus plantarum* and *Lactobacillus rhamnosus*, fermenting and culturing, filtering separately, collecting the filtrate, adding an adsorbent, filtering again, adding the solid to water, ultrasonically desorbing, filtering again, and freeze-drying the filtrate to obtain probiotic-derived extracellular vesicles. This invention's method for extracting probiotic-derived extracellular vesicles is simple, low-cost, and can obtain high-purity extracellular vesicles while maintaining their activity. These vesicles exhibit good anti-inflammatory, antioxidant, and cell proliferation-promoting effects, and have broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of extracellular vesicle extraction technology, specifically to a method for extracting probiotic-derived extracellular vesicles. Background Technology

[0002] Extracellular vesicles (EVs) are bioactive nanospheres with a double-membrane structure, actively secreted by cells. They are primarily composed of lipids, proteins, and genetic material. The main types include exosomes (30-150 nm), microvesicles (100-1000 nm), and apoptotic bodies (>1000 nm). EVs are characterized by their small molecular size and high biocompatibility, making them suitable for transporting lipids, proteins, DNA, and RNA, and thus serving as natural endogenous nanocarriers. EVs are widely distributed in organisms, including plants, mammals, and humans. In principle, all living cells secrete EVs. Currently, EVs are mainly extracted from cell culture media and bodily fluids such as blood, urine, cerebrospinal fluid, breast milk, amniotic fluid, and ascites.

[0003] EVs play a crucial role in intercellular communication and participate in various physiological and pathological processes in vivo, including inflammation, tissue homeostasis, neuronal communication, immune system regulation, and tumor development and metastasis. EVs possess diverse functions and broad clinical application prospects, applicable to disease diagnosis, nanomedicine delivery, targeted therapy, and immunotherapy. As effective carriers for delivering drugs and functional RNA, EVs offer significant advantages: abundant and natural sources, nanoscale size, and the presence of membrane ligands suitable for targeted therapy. Currently, there are several challenges in the clinical translation of EVs, including: yield issues (most current methods, such as ultracentrifugation, yield low amounts of EVs); purity issues (many methods isolate biomolecules similar in size to EVs, causing potential contamination); source issues (although EVs are widely available, obtaining EVs with higher homogeneity is difficult); and economies of scale (obtaining large quantities of EVs at a lower cost is a key aspect of clinical translation).

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

[0005] The purpose of this invention is to propose a method for extracting probiotic-derived extracellular vesicles. The method is simple, low-cost, and can obtain high-purity extracellular vesicles while maintaining their activity. These vesicles exhibit good anti-inflammatory, antioxidant, and cell proliferation-promoting effects, and have broad application prospects.

[0006] The technical solution of this invention is implemented as follows:

[0007] This invention provides a method for extracting probiotic-derived extracellular vesicles. An inducer is added to a culture medium, and *Lactobacillus plantarum* and *Lactobacillus rhamnosus* are inoculated, fermented, filtered, and the filtrate is collected. An adsorbent is added, filtered again, the solid is added to water, ultrasonically desorbed, filtered again, and the filtrate is freeze-dried to obtain probiotic-derived extracellular vesicles.

[0008] As a further improvement to the present invention, the following steps are included:

[0009] S1. Serine and cholesterol are mixed evenly to prepare an inducer;

[0010] S2. Add the inducer, carbon source, nitrogen source, vitamins, and inorganic substances to water, stir and mix evenly, sterilize, and prepare the culture medium;

[0011] S3. Inoculate the seed liquids of Lactobacillus plantarum and Lactobacillus rhamnosus into the culture medium, induce fermentation culture, and obtain a mixed liquid;

[0012] S4. Freeze the mixture in liquid nitrogen, thaw it at room temperature, and treat it with low-temperature ultrasound to obtain a cell-wall-broken mixture;

[0013] S5. Add a 0.45μm centrifugal filter sleeve to the centrifuge tube, centrifuge for the first time to separate fragments >450nm, then add a 0.22μm centrifugal filter sleeve to the centrifuge tube, centrifuge for the second time to separate bacterial cells >220nm, and collect the filtrate.

[0014] S6. Add adsorbent to the filtrate, stir to adsorb, filter, add solid to water, desorb by low-temperature ultrasonication, filter, dialyze the filtrate, freeze dry, and obtain probiotic-derived extracellular vesicles.

[0015] 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, vitamin, inorganic substance, and water in step S2 is 2-4:15-20:8-12:2-3:3-5:200-300, wherein 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, urea, ammonium salts, nitrates, and amino acids; and the vitamin is selected from vitamin C, vitamin B1, vitamin B2, and vitamin D. The inorganic salt is selected from 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.

[0016] As a further improvement of the present invention, the bacterial count of the spore solution in step S3 is 10. 8 -10 9 The inoculum concentrations of *Lactobacillus plantarum* and *Lactobacillus rhamnosus* seed culture were 2-3 v / v% and 1-3 v / v%, respectively. The conditions for induced fermentation culture were 36-38℃, 100-200 r / min, 3-6 v / v% CO2, and induced fermentation culture for 36-48 h. The conditions for low-temperature ultrasonic treatment in step S4 were: a temperature of 2-4℃, ultrasonic treatment at 200-300 W for 3-5 s, stopping treatment for 60-90 s, and then ultrasonic treatment again, repeating this process for 10-15 min.

[0017] As a further improvement of the present invention, in step S5, the rotation speed of the first centrifugation is 2000-3000 r / min and the time is 10-15 min, and the rotation speed of the second centrifugation is 3000-4000 r / min and the time is 5-10 min; in step S6, the mass ratio of the filtrate to the adsorbent is 10:2-3, the stirring adsorption time is 10-20 min, and the conditions for low-temperature ultrasonic desorption are ultrasonic treatment at 200-400 W for 3-5 min at a temperature of 2-4℃.

[0018] As a further improvement of the present invention, the preparation method of the adsorbent is as follows:

[0019] T1. Preparation of porous silica microspheres: Porositrifying agent and alkyl ester of orthosilicate are added to ethanol, concentrated hydrochloric acid and water are added, the mixture is stirred and reacted, filtered, washed, dried and calcined to obtain porous silica microspheres;

[0020] T2. Preparation of amino-modified porous silica microspheres: Porous silica microspheres were added to ethanol, aminosilane coupling agent was added, the mixture was heated and stirred to react, filtered, washed, and dried to obtain amino-modified porous silica microspheres;

[0021] T3. Preparation of the adsorbent: Lipopolysaccharide-binding protein LBP and lipopolysaccharide-specific receptor CD14 were added to water, along with EDC and NHS. The mixture was stirred and activated, then amino-modified porous silica microspheres were added. The mixture was stirred and reacted, filtered, washed, and freeze-dried to obtain the adsorbent.

[0022] 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, wherein 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 h, and the calcination temperature is 400-500 °C for 1-3 h.

[0023] As a further improvement of the present invention, the mass ratio of the porous silica microspheres and the aminosilane coupling agent in step T2 is 10:2-3, the temperature of the heating and stirring reaction is 45-55℃, and the time is 2-4h. The aminosilane coupling agent is selected from at least one of KH550, KH602, and KH792.

[0024] As a further improvement of the present invention, the mass ratio of lipopolysaccharide binding protein LBP, 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 min, and the stirring reaction time is 8-10 h.

[0025] This invention further protects a probiotic-derived extracellular vesicle obtained by the above extraction method.

[0026] The present invention has the following beneficial effects:

[0027] This invention selects serine and cholesterol as inducers. Serine is an important raw material for phosphatidylserine, a probiotic-derived extracellular vesicle membrane material. The probiotic-derived extracellular vesicles of this invention also contain cholesterol. Cholesterol can regulate membrane fluidity, elasticity, and permeability, enabling vesicles to maintain their structural integrity in different physiological environments and facilitating the embedding and function of certain biomolecules. Therefore, the inducers can promote the secretion of extracellular vesicles by probiotics, thereby significantly increasing the secretion amount and yield of extracellular vesicles.

[0028] The probiotics selected in this invention include Lactobacillus plantarum and Lactobacillus rhamnosus, which have rapid proliferation and high levels of extracellular vesicles. The interaction between the two probiotics can promote their rapid entry into the logarithmic phase and promote each other's growth.

[0029] In this invention, the bacterial culture in the logarithmic phase is frozen in liquid nitrogen and thawed at room temperature. The ice crystals disrupt the cell wall, avoiding damage to the EV membrane structure by chemical reagents such as lysozyme. At the same time, low-power intermittent ultrasonic treatment is used at low temperature to break the cell wall while avoiding excessive EV lysis, effectively protecting the integrity of the EV structure.

[0030] In addition, this invention uses a commercially available 0.45μm centrifugal filter sleeve attached to a centrifuge tube for centrifugation to intercept fragments >450nm, and then uses a commercially available 0.22μm centrifugal filter sleeve attached to a centrifuge tube for centrifugation to intercept bacterial cells >220nm. Centrifugal force is used to drive EVs through the membrane, avoiding manual squeezing and clogging of the membrane, effectively separating fragments and bacterial cells. At the same time, EVs with a pore size smaller than 0.22μm and a small amount of small molecule glycophospholipid fragments pass through.

[0031] An adsorbent, using silica as a carrier, is added to the filtrate. This adsorbent is pore-forming via a chiral amino acid porogen, resulting in a highly porous microsphere surface. This increases the specific surface area and pore size of the microspheres, thus increasing the number of adsorption sites. Furthermore, the use of a biocompatible porogen avoids triggering immune or toxic responses in the organism, thereby maximizing the physiological activity of EVs. After modifying the surface of the porous silica microspheres with an aminosilane coupling agent, a condensation reaction between amino and carboxyl groups is used to prepare a microsphere with a surface coupled to lipopolysaccharide-binding protein (LBP) and the specific receptor CD14 for LBP. LBP is an acute-phase protein that recognizes and accumulates LBP. LBP is abundant on the surface of extracellular vesicles of Lactobacillus probiotics and possesses strong immunogenicity, capable of activating the host's innate immune response. LBP binds tightly to the lipid A portion of LBP, enhancing its biological activity and promoting its intercellular transport. CD14 is a specific receptor for lipopolysaccharide (LPS). The LPS-LBP complex can bind to CD14, forming an LPS-LBP-CD14 triplet complex, which in turn activates intracellular signaling pathways. Therefore, coupling LBP and CD14 to the surface of an adsorbent can specifically adsorb probiotic-derived extracellular vesicles without damaging their structure and function. Low-power, low-temperature ultrasonic desorption promotes the entry of extracellular vesicles into the solution, and dialysis removes inorganic salts and other small molecule impurities from the liquid, thus obtaining high-purity extracellular vesicles. Furthermore, the adsorbent can be reused after washing and freeze-drying, improving its resource utilization rate and reducing production costs.

[0032] The present invention provides a method for extracting probiotic-derived extracellular vesicles. The method is simple, low-cost, and can obtain high-purity extracellular vesicles while maintaining their activity. These vesicles exhibit good anti-inflammatory, antioxidant, and cell proliferation-promoting effects, and have broad application prospects. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 This is a TEM image of the probiotic-derived extracellular vesicles prepared in Example 1. Detailed Implementation

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Lipopolysaccharide-binding protein LBP, Huamei Biotechnology; lipopolysaccharide-specific receptor CD14, Huamei Biotechnology; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; NHS, N-hydroxysuccinimide.

[0037] Preparation method of seed culture of *Lactobacillus plantarum* (20 billion CFU / g) and *Lactobacillus rhamnosus* (20 billion CFU / g): Inoculate the inoculum into Gao's medium and activate it at 37°C and 150 rpm for 24-36 h to obtain a culture with a bacterial count of 10... 8 -10 9 CFU / mL bacterial seed solution.

[0038] Preparation Example 1: Preparation of Adsorbent

[0039] The method is as follows:

[0040] T1. Preparation of porous silica microspheres: 1g L-alanine and 10g methyl orthosilicate were added to 80g ethanol, 5g concentrated hydrochloric acid and 4g water were added, the mixture was stirred and reacted for 10h, filtered, washed, dried and calcined at 400℃ for 1h to obtain porous silica microspheres.

[0041] T2. Preparation of amino-modified porous silica microspheres: 10g of porous silica microspheres were added to 150mL of ethanol, 2g of silane coupling agent KH602 was added, the mixture was heated to 45℃, stirred for 2h, filtered, washed and dried to obtain amino-modified porous silica microspheres.

[0042] T3. Preparation of the adsorbent: 3g of lipopolysaccharide-binding protein LBP and 2g of lipopolysaccharide specific receptor CD14 were added to 150mL of water, along with 1g of EDC and 1g of NHS. The mixture was stirred and activated for 20min. Then, 10g of amino-modified porous silica microspheres were added and the mixture was stirred and reacted for 8h. The mixture was then filtered, washed, and freeze-dried to obtain the adsorbent.

[0043] Preparation Example 2: Preparation of Adsorbent

[0044] The method is as follows:

[0045] T1. Preparation of porous silica microspheres: 2g L-valine and 12g tetraethyl orthosilicate were added to 100g ethanol, 7g concentrated hydrochloric acid and 8g water were added, the mixture was stirred and reacted for 12h, filtered, washed, dried and calcined at 500℃ for 3h to obtain porous silica microspheres.

[0046] T2. Preparation of amino-modified porous silica microspheres: 10g of porous silica microspheres were added to 150mL of ethanol, 3g of silane coupling agent KH792 was added, the mixture was heated to 55℃, stirred for 4h, filtered, washed, and dried to obtain amino-modified porous silica microspheres.

[0047] T3. Preparation of the adsorbent: 5g of lipopolysaccharide-binding protein LBP and 3g of lipopolysaccharide specific receptor CD14 were added to 150mL of water, along with 2g of EDC and 2g of NHS. The mixture was stirred and activated for 40min. Then, 12g of amino-modified porous silica microspheres were added and stirred for 10h. The mixture was then filtered, washed, and freeze-dried to obtain the adsorbent.

[0048] Preparation Example 3: Preparation of Adsorbent

[0049] The method is as follows:

[0050] T1. Preparation of porous silica microspheres: 1.5g D-glutamic acid and 11g tetraethyl orthosilicate were added to 90g ethanol, 6g concentrated hydrochloric acid and 6g water were added, the mixture was stirred and reacted for 11h, filtered, washed, dried and calcined at 450℃ for 2h to obtain porous silica microspheres.

[0051] T2. Preparation of amino-modified porous silica microspheres: 10g of porous silica microspheres were added to 150mL of ethanol, 2.5g of silane coupling agent KH550 was added, the mixture was heated to 50℃, stirred for 3h, filtered, washed and dried to obtain amino-modified porous silica microspheres.

[0052] T3. Preparation of the adsorbent: 4g of lipopolysaccharide binding protein LBP and 2.5g of lipopolysaccharide specific receptor CD14 were added to 150mL of water, along with 1.5g of EDC and 1.5g of NHS. The mixture was stirred and activated for 30min. Then, 11g of amino-modified porous silica microspheres were added, and the mixture was stirred and reacted for 9h. The mixture was then filtered, washed, and freeze-dried to obtain the adsorbent.

[0053] Comparative Preparation Example 1

[0054] The difference from Preparation Example 3 is that D-glutamic acid was not added in step T1.

[0055] Specifically as follows:

[0056] T1. Preparation of silica microspheres: 11g of tetraethyl orthosilicate was added to 90g of ethanol, along with 6g of concentrated hydrochloric acid and 6g of water. The mixture was stirred and reacted for 11h, then filtered, washed, dried, and calcined at 450℃ for 2h to obtain silica microspheres.

[0057] Comparative Preparation Example 2

[0058] The difference from Preparation Example 3 is that lipopolysaccharide-binding protein (LBP) was not added in step T3.

[0059] Specifically as follows:

[0060] T3. Preparation of the adsorbent: 6.5g of the specific receptor CD14 of lipopolysaccharide was added to 150mL of water, along with 1.5g of EDC and 1.5g of NHS. The mixture was stirred and activated for 30min. Then, 11g of amino-modified porous silica microspheres were added and the mixture was stirred and reacted for 9h. The mixture was then filtered, washed, and freeze-dried to obtain the adsorbent.

[0061] Comparative preparation example 3

[0062] The difference from Preparation Example 3 is that the lipopolysaccharide-specific receptor CD14 was not added in step T3.

[0063] Specifically as follows:

[0064] T3. Preparation of the adsorbent: 6.5g of lipopolysaccharide-binding protein (LBP) was added to 150mL of water, along with 1.5g of EDC and 1.5g of NHS. The mixture was stirred and activated for 30min. Then, 11g of amino-modified porous silica microspheres were added, and the mixture was stirred and reacted for 9h. The mixture was then filtered, washed, and freeze-dried to obtain the adsorbent.

[0065] Comparative preparation example 4

[0066] The difference from preparation example 3 is that step T3 was not performed.

[0067] Specifically as follows:

[0068] T1. Preparation of porous silica microspheres: 1.5g D-glutamic acid and 11g tetraethyl orthosilicate were added to 90g ethanol, 6g concentrated hydrochloric acid and 6g water were added, the mixture was stirred and reacted for 11h, filtered, washed, dried and calcined at 450℃ for 2h to obtain porous silica microspheres.

[0069] T2. Preparation of amino-modified porous silica microspheres: 10g of porous silica microspheres were added to 150mL of ethanol, 2.5g of silane coupling agent KH550 was added, the mixture was heated to 50℃, stirred for 3h, filtered, washed and dried to obtain amino-modified porous silica microspheres, which are the adsorbent.

[0070] Example 1

[0071] This embodiment provides a method for extracting probiotic-derived extracellular vesicles, including the following steps:

[0072] S1. Mix 3g of serine and 1g of cholesterol evenly to prepare an inducer;

[0073] S2. Add 2g of inducer, 15g of molasses, 8g of ammonium nitrate, 1g of vitamin C, 1g of vitamin A, and 3g of inorganic matter to 200g of water, stir and mix evenly, sterilize, and prepare the culture medium;

[0074] 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;

[0075] S3. Inoculate the seed liquids of Lactobacillus plantarum and Lactobacillus rhamnosus into the culture medium at inoculation amounts of 2 v / v% and 1 v / v%, respectively. Induce fermentation at 36℃, 100 r / min, and 3 v / v% CO2 for 36 h to obtain a mixed solution.

[0076] S4. Freeze the mixture in liquid nitrogen, thaw it at room temperature, and then ultrasonically treat it at 200W for 3 seconds at 2℃. Stop the treatment for 60 seconds and then ultrasonically treat it again. Repeat this process for 10 minutes to obtain the cell-wall broken mixture.

[0077] S5. Add a 0.45μm centrifugal filter sleeve to the centrifuge tube, centrifuge at 2000r / min for 10min to separate fragments >450nm, then add a 0.22μm centrifugal filter sleeve to the centrifuge tube, centrifuge at 3000r / min for 5min to separate bacterial cells >220nm, and collect the filtrate.

[0078] S6. Add 20g of the adsorbent prepared in Preparation Example 1 to 100g of filtrate, stir and adsorb for 10min, filter, add the solid to 100mL of water, sonicate at 200W for 3min at 2℃, filter, dialyze the filtrate through a dialysis bag with a pore size of 5000Da for 3 days, freeze-dry to obtain probiotic-derived extracellular vesicles. Figure 1 The image shows a TEM image of the obtained 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.

[0079] Example 2

[0080] This embodiment provides a method for extracting probiotic-derived extracellular vesicles, including the following steps:

[0081] S1. Mix 5g of serine and 3g of cholesterol evenly to prepare an inducer;

[0082] S2. Add 4g inducer, 20g glucose, 12g fish bone powder, 1g vitamin C, 1g vitamin A, 1g vitamin B1, and 5g inorganic matter to 300g water, stir and mix evenly, sterilize, and prepare the culture medium.

[0083] 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;

[0084] S3. Inoculate the seed liquids of Lactobacillus plantarum and Lactobacillus rhamnosus into the culture medium at inoculation amounts of 3v / v% and 3v / v%, respectively. Induce fermentation at 38℃, 200r / min, and 6v / v% CO2 for 48h to obtain a mixed solution.

[0085] S4. Freeze the mixture in liquid nitrogen, thaw it at room temperature, and then ultrasonically treat it at 4°C with 300W for 5 seconds. Stop the treatment for 90 seconds and then ultrasonically treat it again. Repeat this process for 10-15 minutes to obtain the cell-wall broken mixture.

[0086] S5. Add a 0.45μm centrifugal filter sleeve to the centrifuge tube, centrifuge at 3000r / min for 15min to separate fragments >450nm, then add a 0.22μm centrifugal filter sleeve to the centrifuge tube, centrifuge at 4000r / min for 10min to separate bacterial cells >220nm, and collect the filtrate.

[0087] S6. Add 30g of the adsorbent prepared in Preparation Example 2 to 100g of filtrate, stir and adsorb for 20min, filter, add the solid to 100mL of water, sonicate at 400W for 5min at 4℃, filter, dialyze the filtrate through a dialysis bag with a pore size of 5000Da for 3 days, freeze dry, and obtain probiotic-derived extracellular vesicles.

[0088] Example 3

[0089] This embodiment provides a method for extracting probiotic-derived extracellular vesicles, including the following steps:

[0090] S1. Mix 4g of serine and 2g of cholesterol evenly to prepare an inducer;

[0091] S2. Add 3g inducer, 10g molasses, 7g glucose, 10g peptone, 1g vitamin C, 1.5g vitamin A, and 4g inorganic matter to 250g water, stir and mix evenly, sterilize, and prepare the culture medium;

[0092] 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;

[0093] S3. Inoculate the seed liquids of Lactobacillus plantarum and Lactobacillus rhamnosus into the culture medium at inoculation amounts of 2.5 v / v% and 2 v / v%, respectively. Induce fermentation at 37℃, 150 r / min, and 5 v / v% CO2 for 42 h to obtain a mixed solution.

[0094] S4. Freeze the mixture in liquid nitrogen, thaw it at room temperature, and then sonicate it at 3°C ​​for 4 seconds at 250W. Stop the treatment for 75 seconds and then sonicate it again. Repeat this process for 12 minutes to obtain the cell-wall-broken mixture.

[0095] S5. Add a 0.45μm centrifugal filter sleeve to the centrifuge tube, centrifuge at 2500r / min for 12min to separate fragments >450nm, then add a 0.22μm centrifugal filter sleeve to the centrifuge tube, centrifuge at 3500r / min for 7min to separate bacterial cells >220nm, and collect the filtrate.

[0096] S6. Add 25g of the adsorbent prepared in Preparation Example 3 to 100g of filtrate, stir and adsorb for 15min, filter, add the solid to 100mL of water, sonicate at 300W for 4min at 3℃, filter, dialyze the filtrate through a dialysis bag with a pore size of 5000Da for 3 days, freeze dry, and obtain probiotic-derived extracellular vesicles.

[0097] Comparative Example 1

[0098] The difference compared to Example 3 is that the adsorbent was prepared using Comparative Preparation Example 1. The purity of the probiotic-derived extracellular vesicles in the product was significantly reduced.

[0099] Comparative Example 2

[0100] The difference from Example 3 is that the adsorbent was prepared from Comparative Preparation Example 2.

[0101] Comparative Example 3

[0102] The difference from Example 3 is that the adsorbent was prepared by Comparative Preparation Example 3.

[0103] Comparative Example 4

[0104] The difference from Example 3 is that the adsorbent was prepared by Comparative Preparation Example 4.

[0105] Comparative Example 5

[0106] The difference from Example 3 is that no inducing agent was added in step S2.

[0107] Specifically as follows:

[0108] S2. Add 10g molasses, 7g glucose, 10g peptone, 1g vitamin C, 1.5g vitamin A, and 4g inorganic matter to 250g water, stir and mix evenly, sterilize, and prepare the culture medium;

[0109] 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.

[0110] Comparative Example 6

[0111] The difference from Example 3 is that no ultrasonic treatment was performed in step S4.

[0112] Specifically as follows:

[0113] S4. Freeze the mixture in liquid nitrogen and thaw it at room temperature to obtain a cell wall-broken mixture.

[0114] Comparative Example 7

[0115] The difference from Example 3 is that no adsorbent was added in step S6, and the filtrate was freeze-dried after dialyzing.

[0116] Specifically as follows:

[0117] S6. Dialyze 100g of filtrate through a dialysis bag with a pore size of 5000Da for 3 days, then freeze-dry to obtain probiotic-derived extracellular vesicles.

[0118] Test Example 1: Anti-inflammatory effect

[0119] SPF-grade NIH male mice were selected as experimental mice and divided into 12 groups of 10 mice each. Distilled water was used as the negative control group, fluocinolone acetonide ointment was used as the positive control group, and the probiotic-derived extracellular vesicle aqueous suspensions prepared in Examples 1-3 and Comparative Examples 1-7 were used as experimental groups at a concentration of 2×10⁻⁶. 8 Particles / mL.

[0120] Xylene was applied evenly to both the inner and outer surfaces of the right auricle of each mouse to induce inflammation at a dose of 100 μL / mouse. The left ear was left untreated as a blank control group. Thirty minutes after xylene induction, except for the negative control group, each group of animals received the corresponding test substance in their right ear at a dose of 50 μL / mouse, ensuring even application to both the inner and outer surfaces of the right auricle. The negative control group received distilled water in their right auricle at a dose of 50 μL / mouse. One hour after administration of the test substance, the mice were euthanized by cervical dislocation. Both auricles were removed, and the test substance on the right auricle was washed off with physiological saline and dried. The two auricles were overlapped, and ear pieces were punched out using an 8 mm diameter punch. The ear pieces were weighed separately, and the swelling value was calculated. The results are shown in Table 1.

[0121] Swelling value = m右耳耳片 -m 左耳耳片

[0122] Table 1

[0123] 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

[0124] Note: * indicates P<0.05 compared to the negative control group.

[0125] The lower the swelling value, the better the anti-inflammatory effect. As shown in the table above, the probiotic-derived extracellular vesicles prepared in Examples 1-3 of this invention have good anti-inflammatory effects.

[0126] Test Example 2: Antioxidant Test

[0127] 20 μL of the probiotic-derived extracellular vesicle aqueous suspensions prepared in Examples 1-3 and Comparative Examples 1-7 were added to each well of a 96-well plate, with a concentration of 2 × 10⁻⁶. 8 Particles / mL were added, followed by DPPH solution (0.12 mg / mL), mixed thoroughly, and incubated at 37°C for 30 min. The absorbance value A was then 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.

[0128] Calculate the free radical scavenging rate of the test solution using the formula:

[0129] Clearance rate (%) = [1 - (A - A0) / (C - C0)] × 100%.

[0130] Table 2

[0131] 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

[0132] 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) is a stable nitrogen-centered chromogenic free radical that produces a characteristic absorption peak at 517 nm. In the presence of free radical scavengers, the absorbance of DPPH decreases until it disappears. The change in absorbance shows a linear relationship with the content of antioxidants within a certain range, which can be used to evaluate the sample's ability to scavenge free radicals, i.e., its antioxidant efficacy. As shown in the table above, the probiotic-derived extracellular vesicles prepared in Examples 1-3 of this invention have good antioxidant efficacy.

[0133] Test Example 3: Cell Proliferation Test

[0134] Select healthy mouse embryonic fibroblasts (NIH-3T3) and seed them into 96-well plates. Discard the old culture medium, wash twice with PBS, digest with trypsin, resuspend and count the cells, and seed at a density of 4000 cells / well. Incubate at 37°C, 5v / v% CO2 for 24 hours. Discard the old culture medium from each well, wash the cells once with PBS at pH 7.4, and add 100 μL of culture medium from the negative control group or experimental group to each well.

[0135] The negative control group was prepared using DMEM high-glucose basal medium. The experimental groups were prepared using DMEM high-glucose basal medium supplemented with probiotic-derived extracellular vesicle aqueous suspensions prepared in Examples 1-3 and Comparative Examples 1-7, respectively, to a final concentration of 1×10⁻⁶. 8 Particles / mL. After culturing for another 48 hours, 10 μL / well of CCK8 reagent was added directly, and the cells were incubated at 37°C in the dark for 2 hours. The absorbance was measured on a microplate reader (450 nm), and the relative cell viability (relative to the negative control group) was calculated. The results are shown in Table 3.

[0136] Table 3

[0137] 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

[0138] As shown in the table above, the probiotic-derived extracellular vesicles prepared in Examples 1-3 of this invention have a good effect on promoting fibroblast proliferation.

[0139] 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 within the protection scope of the present invention.

Claims

1. A method for extracting extracellular vesicles from probiotic source cells, characterized in that, The inducing agent is added into the culture medium, Lactobacillus plantarum and Lactobacillus rhamnosus are inoculated, fermentation culture is carried out, the filtrate is collected by filtering respectively, the adsorbent is added, filtration is carried out, the solid is added into water, ultrasonic desorption is carried out, filtration is carried out, the filtrate is freeze-dried, and the probiotic source extracellular vesicle is prepared, The extraction method comprises the following steps: S1. Serine and cholesterol are uniformly mixed to prepare an inducing agent; S2. The inducing agent, carbon source, nitrogen source, vitamin, inorganic substance are added into water, and stirred and uniformly mixed to prepare a culture medium; S3. Lactobacillus plantarum and Lactobacillus rhamnosus seed liquid are inoculated into the culture medium to induce fermentation culture to prepare a mixed liquid; S4. The mixed liquid is frozen in liquid nitrogen, thawed at room temperature, and treated with low-temperature ultrasonic waves to prepare a broken-wall mixed liquid; S5. A 0.45 μm centrifugal filter sleeve is added to a centrifugal tube, and first centrifugation is carried out to separate fragments greater than 450 nm; then a 0.22 μm centrifugal filter sleeve is added to the centrifugal tube, and second centrifugation is carried out to separate bacteria greater than 220 nm, and the filtrate is collected; S6. The adsorbent is added to the filtrate, stirred and adsorbed, filtered, the solid is added into water, low-temperature ultrasonic desorption is carried out, filtration is carried out, the filtrate is dialyzed, and freeze-drying is carried out to prepare the probiotic source extracellular vesicle, The preparation method of the adsorbent is as follows: T1. Preparation of porous silica microspheres: a porogen and tetraalkyl orthosilicate are added into ethanol, concentrated hydrochloric acid and water are added, stirring reaction is carried out, filtration is carried out, washing is carried out, drying is carried out, and calcination is carried out to prepare the porous silica microspheres; T2. Preparation of amino-modified porous silica microspheres: the porous silica microspheres are added into ethanol, and amino silane coupling agent is added, heating stirring reaction is carried out, filtration is carried out, washing is carried out, and drying is carried out to prepare the amino-modified porous silica microspheres; T3. Preparation of the adsorbent: lipopolysaccharide binding protein LBP and the specific receptor CD14 of lipopolysaccharide are added into water, EDC and NHS are added, stirring activation is carried out, the amino-modified porous silica microspheres are added, stirring reaction is carried out, filtration is carried out, washing is carried out, and freeze-drying is carried out to prepare the adsorbent.

2. The extraction method according to claim 1, characterized in that, The mass ratio of serine and cholesterol in step S1 is 3-5:1-3; the mass ratio of the inducing agent, carbon source, nitrogen source, vitamin, inorganic substance and water in step S2 is 2-4:15-20:8-12:2-3:3-5:200-300, the carbon source is at least one selected from molasses, glucose, maltose, lactose, sucrose, fructose and starch, the nitrogen source is at least one selected from proteose peptone, fish bone powder, ammonia water, urea, ammonium salt, nitrate and amino acid, the vitamin is at least one selected from vitamin C, vitamin B1, vitamin B2, vitamin A, vitamin B12, vitamin D3 and vitamin E, and the inorganic substance is at least one selected from sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, iron chloride, zinc sulfate, copper sulfate, manganese sulfate, zinc chloride, copper chloride and manganese chloride.

3. The extraction method according to claim 2, characterized in that, The amino acid is at least one selected from glycine, serine, threonine, valine, tryptophan, leucine, alanine, cysteine, methionine, lysine, isoleucine, phenylalanine, the ammonium salt is at least one selected from ammonium chloride, ammonium sulfate, and the nitrate salt is at least one selected from sodium nitrate, potassium nitrate, and ammonium nitrate.

4. The extraction method according to claim 1, characterized in that, The bacterial content of the seed liquid of the strain in step S3 is 10 8 -10 9 cfu / mL, the inoculation amount of the seed liquid of the Lactobacillus plantarum and Lactobacillus rhamnosus is 2-3 v / v% and 1-3 v / v% respectively, the condition of the induction fermentation culture is 36-38℃, 100-200 r / min, 3-6 v / v% CO2, and the induction fermentation culture is performed for 36-48 h; the condition of the low-temperature ultrasonic treatment in step S4 is that the ultrasonic treatment is performed at 200-300 W for 3-5 s at a temperature of 2-4℃, and after stopping the treatment for 60-90 s, the ultrasonic treatment is performed again, and the process is repeated for 10-15 min.

5. The extraction method of claim 1, wherein, The first centrifugation in step S5 has a speed of 2000-3000 r / min and a time of 10-15 min, and the second centrifugation has a speed of 3000-4000 r / min and a time of 5-10 min; in step S6, the mass ratio of the filtrate to the adsorbent is 10:2-3, the stirring and adsorption time is 10-20 min, and the low-temperature ultrasonic desorption conditions are 200-400 W ultrasonic treatment for 3-5 min at a temperature of 2-4℃.

6. The extraction method of claim 1, wherein, In step T1, the mass ratio of the pore-forming agent, alkyl orthosilicate, ethanol, concentrated hydrochloric acid, and water is 1-2:10-12:80-100:5-7:4-8, the pore-forming agent is at least one selected from 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 h, and the calcination temperature is 400-500℃ for 1-3 h.

7. The extraction method of claim 1, wherein, In step T2, the mass ratio of the porous silica microspheres and the amino silane coupling agent is 10:2-3, the heating and stirring reaction temperature is 45-55℃, and the time is 2-4 h, and the amino silane coupling agent is at least one selected from KH550, KH602, and KH792.

8. The extraction method of claim 1, wherein, In step T3, the mass ratio of the lipopolysaccharide binding protein LBP, the specific receptor CD14 of lipopolysaccharide, EDC, NHS, and the amino-modified porous silica microspheres is 3-5:2-3:1-2:1-2:10-12, the stirring activation time is 20-40 min, and the stirring reaction time is 8-10 h.

9. A source of extracellular vesicles of probiotics prepared by the extraction method of any one of claims 1-8.

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