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CN-121975674-A - Extraction method and application of Wemtman coagulans and extracellular vesicles thereof

CN121975674ACN 121975674 ACN121975674 ACN 121975674ACN-121975674-A

Abstract

The invention provides an extraction method and application of Weptmannia coagulans with a preservation number of CGMCC No.33498 and extracellular vesicles thereof, which takes the Weptmannia coagulans as a core, combines an improved MRS culture medium and a staged culture mode, can pertinently promote the Weptmannia coagulans to secrete EVs, not only solves the technical pain points that the yield of the existing EVs is low, the special instrument or the high-risk technology is needed and the cost is high, but also provides a safe and efficient new scheme for non-drug intervention of sleep dysfunction.

Inventors

  • SHANG NAN
  • WU RUIBIN
  • LI PINGLAN
  • GAO BOYA
  • ZHANG SHIQI

Assignees

  • 中国农业大学

Dates

Publication Date
20260505
Application Date
20251226

Claims (10)

  1. 1. The Wettman-coagulating bacteria is characterized in that the Wettman-coagulating bacteria is classified and named as Wettman-coagulating bacteria (Weizmannia coagulans), and the preservation number is CGMCC No. 33498.
  2. 2. The wizmann condensation bacterium according to claim 1, wherein the sequence of the 16S rDNA of the wizmann condensation bacterium is shown in SEQ ID No. 1.
  3. 3. A method for extracting extracellular vesicles from a condensation of a species of mannomyces wegener, the method comprising the steps of: S1, inoculating the condensation Wittman in an improved MRS culture medium, and performing first culture to obtain a fermentation broth; S2, taking the fermentation liquor for prefiltering, centrifuging and enriching to obtain extracellular vesicles; the Wettman coagulans is the Wettman coagulans of claim 1 and/or 2.
  4. 4. The extraction method according to claim 3, wherein the modified MRS medium contains 18-22 g/L glucose, 8-12 g/L peptone, 8-12 g/L yeast extract, 1-3 g/L dipotassium hydrogen phosphate, 4-6 g/L sodium acetate, 1-3 g/L ammonium citrate, 0.1-0.3 g/L magnesium sulfate, 0.04-0.06 g/L manganese sulfate, 0.5-2 mL/L tween 80, and 10-20 g/L calcium carbonate.
  5. 5. The extraction method according to claim 3, wherein the first culture comprises cell proliferation and EVs induction, wherein the conditions of cell proliferation comprise culturing under anaerobic conditions at 30-40℃, pH of 6-6.5 for 8-15 hours, and wherein the conditions of EVs induction comprise adding 0.1mM CaCl 2 to the modified MRS medium and culturing under pH 5-5.5 for 7.5-8.5 hours; The prefiltering comprises filtering the fermentation broth with a 0.4-0.5 μm filter membrane to remove thalli; The centrifugal enrichment treatment comprises centrifuging the fermentation liquor after the pre-filtration treatment at the temperature of (1-2) multiplied by 10 4 g and the temperature of 3-5 ℃ for 0.5-1.5h, collecting supernatant, transferring the supernatant into an ultrafiltration tube with the interception of 100kDa, centrifuging at the temperature of (3-6) multiplied by 10 3 g, carrying out ultrafiltration concentration, and continuously centrifuging the supernatant after the ultrafiltration concentration at the temperature of (1-2) multiplied by 10 5 g and the temperature of 3-5 ℃ for 1-2h.
  6. 6. A lyophilized formulation comprising extracellular vesicles extracted by the extraction method of any one of claims 3-5 and a lyoprotectant; The volume ratio of the extracellular vesicles to the lyoprotectant is 1 (0.5-1.5).
  7. 7. The lyophilized formulation of claim 6, wherein the lyoprotectant comprises mannitol and trehalose; The weight ratio of mannitol to trehalose is 1 (4-6).
  8. 8. Use of extracellular vesicles extracted by the extraction method of any one of claims 3-5 in the manufacture of a medicament for improving sleep dysfunction.
  9. 9. Use of a lyophilized formulation according to any one of claims 6-7 for the manufacture of a medicament for improving sleep dysfunction.
  10. 10. The use according to claim 8 or 9, wherein the effective dose of the medicament is 1-10 mg/kg body weight, calculated as the extracellular vesicle.

Description

Extraction method and application of Wemtman coagulans and extracellular vesicles thereof Technical Field The application relates to the technical field of microorganisms, in particular to a method for extracting Wemtschia coagulans and extracellular vesicles thereof and application thereof. Background Probiotics (e.g. lactobacillus, bifidobacterium) have been widely studied for their functions of regulating intestinal flora, enhancing immunity, etc., by means of secretion of active substances such as short chain fatty acids, antimicrobial peptides, etc., or interaction with hosts via surface molecules of the bacteria. Bacterial outer vesicles (EVs) as emerging bioactive carriers have been studied for improving EVs yield or function (e.g., over-expressing outer membrane proteins or loading anti-inflammatory molecules) by genetically engineering probiotics for intervention in intestinal inflammation or metabolic diseases, because of their nanoscale structure, low immunogenicity and ability to cross biological barriers. However, the prior art focuses on local effects of EVs (e.g., intestinal or immune regulation), lacks a targeting design for the central nervous system (e.g., sleep disorders), and does not address the problem of EVs penetrating the blood brain barrier, limiting its application in neuromodulation. Currently, related studies for improving the yield of EVs by modifying probiotics mainly comprise two types, namely, one type of engineering probiotics (such as escherichia coli Nissle 1917) to overexpress specific proteins to enhance the yield of EVs and deliver anti-inflammatory factors (such as IL-10) to treat intestinal diseases by using the EVs, but the technology does not involve the regulation and control of sleep-related neurotransmitters (such as 5-hydroxytryptamine and GABA) by the EVs and does not carry out brain-targeted modification on the EVs, so that the intervention effect on a central system is limited, and the other type of technology improves the sleep by screening probiotics (such as lactobacillus plantarum) with high yield of GABA to directly secrete GABA, but the GABA is easy to be degraded by intestinal enzymes and difficult to penetrate through blood brain barrier and has low bioavailability. In addition, the prior art generally relies on a single pathway (such as GABA or anti-inflammatory pathway), does not integrate a multi-pathway synergistic mechanism (such as e.g. a synchronous regulation neurotransmitter, melatonin synthesis and oxidative stress pathway), and part of genetically engineered bacteria has potential safety risks, which restricts practical application value. Therefore, it is needed to provide a natural probiotic strain capable of efficiently secreting EVs of probiotics, which solves the problems of low EVs yield, high preparation cost and the like in the prior art. Disclosure of Invention The purpose of the present disclosure is to provide a natural probiotic strain capable of efficiently secreting EVs of probiotics, which solves the problems of low EVs yield, high preparation cost and the like in the prior art. To achieve the above object, a first aspect of the present disclosure provides a condensation of Wittman's bacteria, classified as Wittman's bacteria condensation (Weizmannia coagulans), with a collection number of CGMCC No. 33498. Alternatively, the sequence of the 16S rDNA of the M.Weizhindicum is shown as SEQ ID NO. 1. In another aspect, the present disclosure provides a method of extracting extracellular vesicles in a condensation of a species of mannzia, the method comprising the steps of: S1, inoculating the condensation Wittman in an improved MRS culture medium, and performing first culture to obtain a fermentation broth; S2, taking the fermentation liquor for prefiltering, centrifuging and enriching to obtain extracellular vesicles; The Wettman-coagulating bacteria are Wettman-coagulating bacteria with a preservation number of CGMCC No. 33498, and/or the sequence of 16S rDNA of the Wettman-coagulating bacteria is shown as SEQ ID No. 1. Optionally, the modified MRS medium contains 18-22 g/L glucose, 8-12 g/L peptone, 8-12 g/L yeast extract, 1-3 g/L dipotassium hydrogen phosphate, 4-6 g/L sodium acetate, 1-3 g/L ammonium citrate, 0.1-0.3 g/L magnesium sulfate, 0.04-0.06 g/L manganese sulfate, 0.5-2 mL/L tween 80, and 10-20 g/L calcium carbonate. Optionally, the first culturing comprises thallus proliferation and EVs induction, wherein the thallus proliferation conditions comprise culturing for 8-15 hours under anaerobic conditions at 30-40℃, pH of 6-6.5, and the EVs induction conditions comprise adding 0.1mM CaCl 2 into the modified MRS culture medium, and culturing for 7.5-8.5 hours under pH 5-5.5. Optionally, the prefiltering comprises filtering the fermentation broth with a 0.4-0.5 μm filter membrane to remove thalli; The centrifugal enrichment treatment comprises centrifuging the fermentation liquor after the pre-filtration treatment at the