WO2023177208A1 - Method for preparing lactic acid bacterium-derived extracellular vesicles with improved yield - Google Patents

Method for preparing lactic acid bacterium-derived extracellular vesicles with improved yield Download PDF

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WO2023177208A1
WO2023177208A1 PCT/KR2023/003468 KR2023003468W WO2023177208A1 WO 2023177208 A1 WO2023177208 A1 WO 2023177208A1 KR 2023003468 W KR2023003468 W KR 2023003468W WO 2023177208 A1 WO2023177208 A1 WO 2023177208A1
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lactobacillus
bifidobaterium
bifidobacterium
lactococcus
extracellular vesicles
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PCT/KR2023/003468
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French (fr)
Korean (ko)
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김윤근
신태섭
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주식회사 엠디헬스케어
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/02—Separating microorganisms from their culture media
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Definitions

  • the present invention relates to a method for producing extracellular vesicles secreted by lactic acid bacteria with improved yield.
  • Lactobacillus bacteria are Gram-positive bacilli that grow well not only in anaerobic environments but also in aerobic environments, and are known as beneficial bacteria that coexist in our bodies. Bacteria secrete double-membrane extracellular vesicles (EV) into the extracellular environment for the exchange of proteins, lipids, and genes between cells. Vesicles secreted by gram-positive bacteria such as Lactobacillus bacteria contain peptidoglycan and lipoteichoic acid, which are components of the bacterial cell wall, in addition to bacteria-derived proteins and nucleic acids.
  • EV extracellular vesicles
  • Lactobacillus secreted extracellular vesicles can be used as pharmaceutical ingredients, and a method for highly efficient mass production of such extracellular vesicles on an industrial scale is needed.
  • the present inventors have identified the optimal medium composition and fermentation conditions that can increase the yield of Lactobacillus-secreted extracellular vesicles. Based on this, the present inventors have The invention was completed.
  • the purpose of the present invention is to provide a method for producing extracellular vesicles derived from lactic acid bacteria with improved yield.
  • the present invention includes the steps of (a) preparing a culture medium for lactic acid bacteria that is free of toxic and animal-derived components and is edible; (b) cultivating lactic acid bacteria in the medium prepared in step (a); (c) centrifuging the culture medium of step (b) to separate the supernatant; (d) concentrating the supernatant of step (c) and re-concentrating it by diluting it with a buffer solution; and (e) filtering the concentrate of step (d) through a filter to obtain extracellular vesicles.
  • the medium may not contain ammonium citrate, which is an inedible ingredient, and beef extract, which is an animal-derived ingredient, but is not limited thereto.
  • the medium may contain glucose, soy peptone, yeast extract, sodium acetate, potassium dibasic phosphate, magnesium sulfate, manganese sulfate, citric acid and polysorbate 80, but is limited thereto. no.
  • step (b) may be performed in the order of seed culture, pre-culture, and main culture, respectively, for 8 to 16 hours, but is not limited thereto.
  • step (b) may be performed in the order of seed culture, pre-culture, and main culture, respectively, for 10 to 14 hours, but is not limited thereto.
  • step (d) may be concentration using an ultrafiltration membrane of 50 kDa to 150 kDa in size, but is not limited thereto.
  • the filter in step (e) may be a 0.1 to 1 ⁇ m size filter, but is not limited thereto.
  • the filter in step (e) may be a 0.1 ⁇ m to 0.5 ⁇ m size filter, but is not limited thereto.
  • the lactic acid bacteria may be bacteria of the genus Lactobacillus , Lactococcus , or Bifidobacterium , but are not limited thereto.
  • the bacteria of the Lactobacillus genus include Lactobacillus brevis , Lactobacillus acetolerans , Lactobacillus acidifrinae , and Lactobacillus acetate.
  • Lactobacillus acidipiscis Lactobacillus agilis, Lactobacillus algidus, Lactobacillus alimentarus , Lactobacillus amylolyticus , Lactobacillus amyl Lactobacillus amylotrophicus , Lactobacillus amylovorus, Lactobacillus animalis , Lactobacillus antri , Lactobacillus apodemi , Lactobacillus aviarius ( Lactobacillus aviarius , Lactobacillus bifermentans, Lactobacillus bombicola, Lactobacillus buchneri , Lactobacillus camelliae, Lactobacillus casei , lactobacillus Bacillus catenaformis, Lactobacillus ceti , Lactobacillus coleohominis, Lactobacillus collinoides , Lactobacillus composti , Lactobacillus concabus ( Lactobacillus
  • the bacteria of the Lactococcus genus are Lactococcus chungangensis , Lactococcus formosensis , Lactococcus fujiensis , and Lactococcus hirsi.
  • Lactococcus hircilactis Lactococcus garvieae , Lactococcus lactis , Lactococcus laudensis , Lactococcus nasutitermitis , Lactococcus piscium , Lactococcus plantarum , lacto It may be Lactococcus raffinolactis , or Lactococcus taiwanensis , but is not limited thereto.
  • the bacteria of the Bifidobacterium genus are Bifidobacterium actinocoloniiforme , Bifidobacterium adolescentis , and Bifidobacterium.
  • Bifidobacterium aemilianum Bifidobacterium aerophilum, Bifidobaterium aesculapii, Bifidobaterium amazonense , Bifidobacterium angulatum ( Bifidobaterium angulatum ), Bifidobacterium animalis , Bifidobacterium anseris , Bifidobaterium apousia , Bifidobaterium apri , Bifidobacterium apri Bifidobaterium aquikefiri , Bifidobaterium asteroides, Bifidobaterium avesanii , Bifidobaterium biavatii , Bifidobacterium bifidum ( Bifidobacterium bifidum ), Bifidobacterium bohemicum , Bifidobacterium bombi,
  • Bifidobaterium castoris Bifidobacterium catenulatum ( Bifidobaterium catulorum ), Bifidobacterium cebidarum ( Bifidobaterium cebidarum ) , Bifidobacterium coerinum ( Bifidobaterium choerinum ), Bifidobacterium choladohabitans , Bifidobaterium choloepi, Bifidobaterium colobi , Bifidobaterium commune , Bifidobacterium Bifidobaterium criceti , Bifidobaterium crudilactis , Bifidobaterium cuniculi, Bifidobaterium dentium , Bifidobacterium dolicotidis ( Bifidobaterium dolichotidis ), Bifidobaterium e
  • the extracellular vesicles may have an average diameter of 10 nm to 1000 nm, but are not limited thereto.
  • the present inventors identified the optimal medium composition and fermentation conditions that can increase the yield of Lactobacillus secreted extracellular vesicles. Specifically, when culturing with existing Lactobacillus culture medium, there were problems due to low yield of extracellular vesicles or toxicity due to inedible and animal-derived components of the medium, but the present inventors have found that there are no toxic or animal-derived components. An edible medium was prepared, and it was confirmed that it had no cytotoxicity and also increased the yield of extracellular vesicles during Lactobacillus culture. In addition, the optimal culturing time to increase yield was confirmed. Accordingly, the method for producing extracellular vesicles with improved yield according to the present invention is expected to be useful in the process of mass production on an industrial scale of extracellular vesicles secreted by lactic acid bacteria that can be used as pharmaceutical ingredients.
  • Figure 1 shows the results of evaluating the cytotoxicity of extracellular vesicles secreted by Lactobacillus paracasei in mouse macrophages (Raw264.7) depending on the type of culture medium.
  • Figure 2 shows the results of analyzing the growth pattern of Lactobacillus paracasei seed culture.
  • Figure 3 shows the results of analyzing the growth pattern of Lactobacillus paracasei pre-culture.
  • Figure 4 shows the results of analyzing the growth pattern of Lactobacillus paracasei main culture.
  • Figure 5 shows the results of confirming the ability of Lactobacillus paracasei secreted extracellular vesicles to inhibit TNF- ⁇ secretion according to the ultrafiltration membrane size in mouse macrophages (Raw264.7).
  • Figure 6 shows the results of SDS-PAGE analysis of the protein expression pattern of Lactobacillus paracasei secreted extracellular vesicles isolated by the method of the present invention.
  • the method for producing extracellular vesicles with improved yield of the present invention increases the yield of extracellular vesicles when culturing Lactobacillus. Therefore, the method for producing extracellular vesicles with improved yield of the present invention is expected to be useful in the process of mass production of Lactobacillus secreted extracellular vesicles that can be used as pharmaceutical ingredients on an industrial scale.
  • extracellular vesicles were isolated by culturing Lactobacillus paracasei in LB, MRS and EMP media, and as a result of comparative analysis of the yield and toxicity of the isolated extracellular vesicles, EMP It was confirmed that Lactobacillus paracasei cultured in medium had the highest yield of extracellular vesicles and had no toxicity (see Example 1).
  • a culture process optimization process was performed to increase the yield of Lactobacillus paracasei secreted extracellular vesicles, and the best growth was achieved at 12 hours in seed culture, pre-culture, and main culture, and extracellular It was confirmed that the yield of vesicles was also high (see Example 2).
  • a concentration process optimization process was performed to increase the yield of extracellular vesicles secreted by Lactobacillus paracasei.
  • the yield of extracellular vesicles was increased, and TNF- ⁇ It was confirmed that the secretion inhibition ability was also high (see Example 3).
  • a mass production process of Lactobacillus secreted extracellular vesicles was established by applying the optimal culture medium, culture time, and concentration process (see Example 4).
  • the characteristics of extracellular vesicles produced through the mass production process of extracellular vesicles of the present invention were confirmed.
  • the properties, pH, protein concentration, and major protein expression patterns of extravesicular vesicles were confirmed, and an average of 2.38 x 10 12 particles per 1 mg/mL with an average size of 116 nm were confirmed.
  • the isolated extracellular vesicles had an inhibitory effect on TNF- ⁇ secretion, and were confirmed to be non-toxic in a cytotoxicity test (see Example 5).
  • the present invention provides a method for producing extracellular vesicles derived from lactic acid bacteria with improved yield, comprising the following steps.
  • step (b) cultivating lactic acid bacteria in the medium prepared in step (a);
  • step (c) centrifuging the culture medium of step (b) to separate the supernatant
  • step (d) concentrating the supernatant of step (c) and re-concentrating it by diluting it with a buffer solution;
  • step (e) filtering the concentrate from step (d) through a filter to obtain extracellular vesicles.
  • the lactic acid bacteria may be bacteria of the genus Lactobacillus , Lactococcus , or Bifidobacterium , but are not limited thereto.
  • the bacteria of the Lactobacillus genus include Lactobacillus brevis , Lactobacillus acetolerans , Lactobacillus acidifarinae , and Lactobacillus acidifisis.
  • Lactobacillus agilis Lactobacillus algidus, Lactobacillus alimentarus , Lactobacillus amylolyticus , Lactobacillus amylotropicus ( Lactobacillus amylotrophicus , Lactobacillus amylovorus , Lactobacillus animalis , Lactobacillus antri , Lactobacillus apodemi, Lactobacillus aviarius , Lactobacillus bifermentans, Lactobacillus bombicola, Lactobacillus buchneri, Lactobacillus camelliae , Lactobacillus casei , Lactobacillus catnapomis ( Lactobacillus catenaformis ), Lactobacillus ceti , Lactobacillus coleohominis, Lactobacillus collinoides , Lactobacillus composti , Lactobacillus concavus , Lactobacill
  • Lactobacillus paracollinoides Lactobacillus parafarraginis, Lactobacillus parakefiri , Lactobacillus paralimentarius , Lactobacillus paraplantarum , Lactobacillus pentosus, Lactobacillus perolens, Lactobacillus pontis , Lactobacillus protectus , Lactobacillus psittaci , Lactobacillus rennini ( Lactobacillus rennin i), Lactobacillus reuteri, Lactobacillus rimae , Lactobacillus rogosae , Lactobacillus rossiae, Lactobacillus ruminis , Lactobacillus Lactobacillus saerimneri , Lactobacillus sakei , Lactobacillus salivarius, Lactobacillus sanfranciscensis , Lactobacillus satsumensis , Lactobac
  • the bacteria of the Lactococcus genus are Lactococcus chungangensis , Lactococcus formosensis , Lactococcus fujiensis , and Lactococcus hirsylactis. hircilactis ), Lactococcus garvieae , Lactococcus lactis , Lactococcus laudensis , Lactococcus nasutitermitis , Lactococcus piscium , Lactococcus plantarum , lacto It may be Lactococcus raffinolactis , or Lactococcus taiwanensis , but is not limited thereto.
  • the bacteria of the Bifidobacterium genus include Bifidobacterium actinocoloniiforme , Bifidobacterium adolescentis , and Bifidobacterium amyrianum ( Bifidobacterium aemilianum ), Bifidobacterium aerophilum , Bifidobaterium aesculapii, Bifidobacterium amazonense , Bifidobacterium angulatum , Bifidobacterium animalis , Bifidobacterium anseris, Bifidobaterium apousia , Bifidobaterium apri , Bifidobacterium aquiche Bifidobaterium aquikefiri , Bifidobacterium asteroides , Bifidobacterium avesanii , Bifidobaterium biavatii , Bifidobacterium aster
  • Murum Bifidobaterium lemurum ), Bifidobaterium leontopitheci , Bifidobacterium longum , Bifidobacterium magnum, Bifidobaterium margollesii , Bifidobacterium merycicum , Bifidobaterium miconis, Bifidobaterium miconisargentati , Bifidobacterium minimum , Bifidobacterium Mongoliense ( Bifidobaterium mongoliense ), Bifidobaterium moraviense , Bifidobaterium moukalabense , Bifidobaterium myosotis , Bifidobacterium oidipo Dis ( Bifidobaterium oedipodis ), Bifidobaterium olomucense ( Bifidobaterium olomucense ),
  • extracellular vesicle or “vesicle” refers to a nano-sized membrane structure secreted by various bacteria, and in the present invention, lactic acid bacteria including Lactobacillus paracasei It refers collectively to all structures made of membranes that are naturally secreted or artificially produced.
  • the extracellular vesicles have an average diameter of 10 nm to 1000 nm, 10 nm to 900 nm, 10 nm to 800 nm, 10 nm to 700 nm, 10 nm to 600 nm, 10 nm to 500 nm, 10 It may be from 10 nm to 400 nm, 10 nm to 300 nm, or 10 nm to 200 nm, but is not limited thereto.
  • “cultivation” refers to all actions performed to grow microorganisms in appropriately artificially controlled environmental conditions, and in the present invention, it is a concept that includes “fermentation.”
  • the “method for producing extracellular vesicles with improved yield” refers to producing extracellular vesicles with relatively high protein concentration and content among extracellular vesicles isolated from the same volume of lactic acid bacteria culture medium, and the present invention
  • the protein concentration of extracellular vesicles with improved yield may be 2 mg/mL to 5 mg/mL
  • the protein content may be 2 g/L to 5 g/L
  • the number of nanoparticles may be 100 nm to 150 nm.
  • Nanoparticles of nm size may be 1 x 10 12 particles/mL to 4 x 10 12 particles/mL, but are not limited thereto.
  • the medium in step (a) may not contain ammonium citrate, which is an inedible ingredient, and beef extract, which is an animal-derived ingredient, but is not limited thereto.
  • the medium may be EMP (Edible MRS), which is an edible Lactobacillus culture medium, and contains glucose, soy peptone, yeast extract, sodium acetate, potassium dibasic phosphate, magnesium sulfate, manganese sulfate, citric acid, and polysor. It may include bait 80, etc., but is not limited thereto.
  • EMP Edible MRS
  • the glucose is 5 g to 50 g, 10 g to 45 g, 10 g to 40 g, 10 g to 35 g, and 10 g to 30 g per 1 L of deionized water in the medium. g, 15 g to 40 g, 15 g to 35 g, 15 g to 30 g, or 20 g to 30 g of glucose, but is not limited thereto.
  • the soy peptone is used in the medium at an amount of 1 g to 10 g, 1 g to 9 g, 1 g to 8 g, 1 g to 7 g, and 2 g per 1 L of deionized water. to 10 g, 2 g to 9 g, 2 g to 8 g, 2 g to 7 g, 3 g to 10 g, 3 g to 9 g, 3 g to 8 g, 3 g to 7 g or 4 g to 6 g. It may include, but is not limited to, soy peptone.
  • the yeast extract is 1 g to 30 g, 3 g to 27 g, 3 g to 25 g, 3 g to 22 g, 3 g per 1 L of deionized water in the medium. to 20 g, 3 g to 18 g, 5 g to 30 g, 5 g to 27 g, 5 g to 25 g, 5 g to 22 g, 5 g to 20 g, 7 g to 30 g, 7 g to 27 g, 7 g to 25 g, 7 g to 22 g, 7 g to 20 g, 10 g to 30 g, 10 g to 27 g, 10 g to 25 g, 10 g to 22 g or 10 g to 20 g. It may include, but is not limited to, yeast extract.
  • the amount of sodium acetate is 1 g to 10 g, 1 g to 9 g, 1 g to 8 g, 1 g to 7 g, and 2 g per 1 L of deionized water in the medium. to 10 g, 2 g to 9 g, 2 g to 8 g, 2 g to 7 g, 3 g to 10 g, 3 g to 9 g, 3 g to 8 g, 3 g to 7 g or 4 g to 6 g. It may include, but is not limited to, sodium acetate.
  • the amount of dipotassium phosphate is 0.1 g to 5 g, 0.5 g to 5 g, 0.5 g to 4.5 g, 0.5 g to 4 g per 1 L of deionized water in the medium.
  • the magnesium sulfate (Magnesium sulfate) is 0.01 g to 1 g, 0.03 g to 1 g, 0.03 g to 0.7 g, 0.03 g to 0.5 g, 0.03 g per 1 L of pure water (Deionized water) in the medium. comprising from 0.3 g to 0.3 g, from 0.05 g to 1 g, from 0.05 g to 0.7 g, from 0.05 g to 0.5 g, from 0.07 g to 1 g, from 0.07 g to 0.7 g, from 0.07 g to 0.5 g or from 0.07 g to 0.3 g of magnesium sulfate. It can be done, but is not limited to this.
  • the amount of manganese sulfate is 0.01 g to 0.1 g, 0.02 g to 0.1 g, 0.02 g to 0.09 g, 0.02 g to 0.08 g, 0.02 g per 1 L of deionized water in the medium. It may include, but is not limited to, 0.07 g, 0.03 g to 0.1 g, 0.03 g to 0.09 g, 0.03 g to 0.08 g, 0.03 g to 0.07 g, or 0.04 g to 0.06 g of manganese sulfate.
  • the citric acid (Citric acid monohydrate) is 0.1 g to 5 g, 0.5 g to 5 g, 0.5 g to 4.5 g, 0.5 g to 4 g, 0.5 g per 1 L of pure water (Deionized water) in the medium. It may include, but is not limited to, citric acid, from 3.5 g to 3.5 g, 0.5 g to 3 g, 1 g to 5 g, 1 g to 4.5 g, 1 g to 4 g, 1 g to 3.5 g, or 1 g to 3 g. No.
  • polysorbate 80 is 0.1 g to 10 g, 0.3 g to 10 g, 0.3 g to 7 g, 0.3 g to 5 g, 0.3 g per 1 L of pure water (Deionized water) in the medium. g to 3 g, 0.5 g to 10 g, 0.5 g to 7 g, 0.5 g to 5 g, 0.7 g to 10 g, 0.7 g to 7 g, 0.7 g to 5 g or 0.7 g to 3 g of magnesium sulfate. It may include, but is not limited to this.
  • step (b) is performed in the order of seed culture, pre-culture, and main culture, respectively, for 5 hours to 20 hours, 5 hours to 18 hours, 5 hours to 16 hours, 5 hours to 14 hours, and 8 hours to 8 hours. You can incubate for 20 hours, 8 hours to 18 hours, 8 hours to 16 hours, 8 hours to 14 hours, 10 hours to 20 hours, 10 hours to 18 hours, 10 hours to 16 hours, or 10 hours to 14 hours, but now Not limited.
  • the culture medium in step (c), can be centrifuged to separate the supernatant and remove the bacterial cells.
  • step (d) is 30 to 180 kDa, 40 to 180 kDa, 50 to 180 kDa, 60 to 180 kDa, 70 to 180 kDa, 30 to 170 kDa, 40 to 170 kDa, 50 to 170 kDa.
  • the filter in step (e) is 0.01 ⁇ m to 1 ⁇ m, 0.05 ⁇ m to 1 ⁇ m, 0.05 ⁇ m to 0.8 ⁇ m, 0.05 ⁇ m to 0.5 ⁇ m, 0.05 ⁇ m to 0.3 ⁇ m, 0.1 ⁇ m to 1 ⁇ m, 0.1 ⁇ m to 1 ⁇ m. It may be a size filter of ⁇ m to 0.8 ⁇ m, 0.1 ⁇ m to 0.5 ⁇ m, or 0.1 ⁇ m to 0.3 ⁇ m, but is not limited thereto.
  • extracellular vesicles In the production of extracellular vesicles (EV) with improved yield of the present invention, in order to find the optimal culture conditions for the strain Lactobacillus paracasei , the basic medium, LB (Luria-Bertani) medium, Lactobacillus The yield and toxicity of isolated extracellular vesicles were compared and analyzed when cultured in MRS (De Man Rogosa and Sharpe) medium, a Bacillus culture medium, and EMP (Edible MRS), an edible Lactobacillus culture medium.
  • MRS De Man Rogosa and Sharpe
  • EMP Edible MRS
  • the LB, MRS and EMP media of the present invention were prepared and used according to the compositions and composition ratios shown in Tables 1 to 3 below.
  • ⁇ LB medium composition No.
  • Ingredient name g/L One Tryptone 10 2 Yeast extract 5 3 Sodium chloride 10 4 Agar 15
  • ⁇ EMP medium composition No. Ingredient name g/L One Glucose 25 2 Soy peptone 5 3 Yeast extract 15 4 Sodium acetate 5 5 Dipotassium phosphate 2 6 Magnesium sulfate 0.1 7 Manganese sulfate 0.05 8 Citric acid monohydrate 2 9 Tween 80 (polysorbate 80) One
  • Lactobacillus paracasei secreted extracellular vesicles
  • Lactobacillus paracasei was inoculated into LB, MRS and EMP media prepared with the composition of Example 1-1, respectively, and set at 37°C and 200 rpm. Cultured for 12 hours. Then, the culture medium containing the bacterial cells was recovered and continuously centrifuged at 10,000 x g at room temperature to obtain a supernatant from which the bacterial cells were removed.
  • the obtained supernatant was filtered again using a 0.22 ⁇ m filter, and the filtered supernatant was reduced to a volume of 50 mL or less using a 100 kDa Pellicon 2 Cassette filter membrane (Merck Millipore) and MasterFlex pump system (Cole-Parmer). Concentrated. The concentrated supernatant was filtered again using a 0.22 ⁇ m filter to separate Lactobacillus paracasei-derived vesicles. The amount of protein contained in the supernatant was measured using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific).
  • Mouse macrophages (Raw264.7) were cultured in LB, MRS, and EMP media and treated with Lactobacillus paracasei-derived extracellular vesicles (MDH-001) at concentrations of 0.1, 1, and 10 ⁇ g/mL, respectively. The viability was measured by OD (optical density).
  • the LB and EMP culture groups showed a cell viability of more than 80% based on the negative control that was not treated with the test substance, but at a concentration of 10 ⁇ g/mL in the MRS culture group, the cells Toxicity was confirmed.
  • a culture process optimization process was conducted for pilot production of Lactobacillus paracasei secreted extracellular vesicles (MDH-001) of the present invention.
  • a culture method using two subcultures was applied.
  • the sub-inoculation amount for each culture step was set at 1/100 of the total amount of target medium, and the growth pattern was analyzed by measuring colony forming unit (CFU) and optical density (OD) for each culture time at each culture step.
  • CFU colony forming unit
  • OD optical density
  • the initial active culture time was 12 hours, as shown in Figure 2, and the seed culture time was set to 12 hours.
  • the active culture was passaged, and similarly, the culture was collected every two hours after inoculation and the growth pattern was analyzed by measuring CFU and OD.
  • the main culture stage which is the final stage of culture
  • 50 L culture was performed.
  • 500 mL of culture medium equivalent to 1/100 of the total amount of the target culture medium, was sub-inoculated, and the culture medium was collected every 3 hours to measure CFU and OD to analyze the growth pattern.
  • Ultrafiltration (UF) conditions were established to optimize the concentration process for pilot production of Lactobacillus paracasei secreted extracellular vesicles (MDH-001) of the present invention.
  • Example 1-2 Cultivation and sterilization processes were performed in the same manner as Example 1-2 using 8 L of EMP medium.
  • the supernatant from which the bacteria were removed was concentrated to a volume of 50 mL or less using ultrafiltration membranes of two sizes, 50 kDa and 100 kDa.
  • the concentrated supernatant was filtered again using a 0.22 ⁇ m filter to separate Lactobacillus paracasei-derived vesicles.
  • the amount of protein contained in the supernatant was measured using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific).
  • TNF- ⁇ secretion was performed to confirm the titer of extracellular vesicles according to the size of the ultrafiltration membrane.
  • the secretion amount of TNF- ⁇ (Tumor necrosis factor- ⁇ ), known as a representative inflammatory cytokine, was measured using enzyme-linked immunosorbent assay (ELISA) to evaluate its inflammation-modulating effect.
  • Mouse macrophages (Raw264.7) were treated with inflammation-inducing substances and extracellular vesicles concentrated in 50 kDa and 100 kDa sizes at concentrations of 0.1, 1, and 10 ⁇ g/mL, respectively, and the secretion amount of TNF- ⁇ was measured using enzyme-linked immunosorbent assay. It was measured by (ELISA).
  • the culture medium and culture time were optimized and applied to the mass production process of extracellular vesicles.
  • a large volume of EMP medium was prepared by the following method.
  • pure water was prepared in a pure water maker, received and stored in a container, and other ingredients except glucose were weighed on an electronic scale according to the composition and composition ratio in Table 3 below and placed in a 5 L plastic beaker. Pure water was added to a 10 L plastic beaker, the weighed ingredients were dissolved, and the ingredients dissolved in pure water were filled into the fermenter tank using a silicone tube using a pump. Pure water was discharged through the outlet of the container and received into an acrylic container in front of the outlet. Based on a 50 L culture, pure water was filled into the tank from an acrylic tank containing pure water to a volume of 42 L using a silicone tube using a pump. Press Heat Start in the sterilization window on the fermenter screen, and glucose was sterilized separately and added when the 50 L medium cooled and stabilized at 37°C.
  • Seed culture was performed 24 hours before inoculation into the fermenter (100 L fermenter, FE-101, BIOSYSTEMENG CO.,LTD).
  • EMP medium contained in a 60 mL tube was prepared, and the tube entrance was flame sterilized.
  • Lactobacillus paracasei was suspended in EMP medium and 200 ⁇ L was inoculated. The tube was completely capped and resuspended. Bacterial inoculation was performed on a clean bench. In order to maintain anaerobic conditions after inoculation, if there was empty space in the tube, EMP medium was filled and cultured in an incubator at a temperature of 37 ⁇ 2°C for 12 hours.
  • Pre-culture was performed 12 hours before inoculation into the fermenter.
  • 3 L EMP medium was prepared in a 5 L fermenter and sterilized.
  • a 5 L fermenter was installed and set at 37°C and 100 rpm, 30 mL of the seed culture medium of Example 2 was inoculated through the inoculation port, and cultured in an incubator at a temperature of 37 ⁇ 2°C for 12 hours.
  • Cotton soaked in 100% ethanol was placed in the flame hole groove of the fermenter, and silicone gloves were worn. The pressure was changed to 0.05, the flame bulb was installed appropriately in the inoculation hole, and the fire was ignited with a torch. Open the inoculation port cap, sterilize the mouth of the Duran bottle containing 500 mL of culture medium by flame, then quickly open the lid and pour in the culture medium to complete the inoculation.
  • the centrifuge rotation gravity acceleration was set to 10,000 xg and operated, and the speed of the metering pump (WT600-2J, LONGER Pump) was set to 100.
  • the supernatant from centrifugation was placed in a SUS-supernatant storage container.
  • a 0.22 ⁇ m bottle top filter (180C3, Sartorius) was sterilized with alcohol spray and then moved into the clean bench.
  • the nozzle was coupled to the pressure control hole of the 0.22 ⁇ m bottle top filter and the suction pipe was connected. Open the transparent plastic lid at the top of the 0.22 ⁇ m bottle top filter, add 150 mL of concentrated culture fluid inside, operate the suction tube, and filter the concentrated culture fluid using the 0.22 ⁇ m bottle top filter.
  • Bacterial cells were recovered from the culture medium after completion of incubation, and 16S rRNA analysis was performed to confirm whether the strain was the correct strain.
  • the extracellular vesicle MDH-001 of the present invention was confirmed to be a yellow-brown transparent liquid.
  • MDH-001 is an extracellular vesicle produced by lactic acid bacteria. During the culture process, lactic acid is produced and it is slightly acidic. It was washed with PBS at pH 7.4 to neutralize it, and the acidity was measured to determine whether it was cultured consistently.
  • the protein content was measured using the BCA asaay test method. 1 mL of the produced MDH-001 was serially diluted 10 times, then 25 ⁇ L was added to a 96 well plate, 200 ⁇ L of reagent was added, reaction was performed at 37°C for 30 minutes, and absorbance was measured at 562 nm. A standard graph was drawn using BSA, a standard reagent, and the concentration was calculated by comparing the graph.
  • the protein concentration was confirmed to be an average of 2 to 4 mg/mL.
  • SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis
  • a 12% acrylamide gel was prepared, SDS-PAGE analysis was performed, and the protein pattern was confirmed through silver stain. Analysis was performed using Bio-Rad's Image Lab program.
  • NTA Nanoparticle tracking analysis
  • MDH-001 MDH-001
  • NTA analysis is an analysis method that determines the size and distribution of nanoparticles using the Brownian motion speed and light scattering characteristics that vary depending on the size of the particle. It measures the nanoparticles (nano) present per 1 mg/mL of protein quantified through BCA analysis. -particle) was confirmed. This analysis method confirmed the number and distribution of particles using Malvern Panalytical's NS300 instrument.
  • TNF- ⁇ Tumor necrosis factor- ⁇
  • ELISA enzyme-linked immunosorbent assay
  • Mouse macrophages (Raw264.7) were treated with inflammation-inducing substances and MDH-001 at concentrations of 0.1, 1, and 10 ⁇ g/mL, and the secretion amount of TNF- ⁇ was measured by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the culture produced through the mass production process of the present invention contained extracellular vesicles, which consistently suppressed the secretion of TNF- ⁇ , an inflammatory mediator, without cytotoxicity.
  • the method for producing extracellular vesicles derived from lactic acid bacteria with improved yield according to the present invention is expected to be useful in the process of mass producing extracellular vesicles derived from lactic acid bacteria that can be used as pharmacological ingredients on an industrial scale. There is availability.

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Abstract

The present invention relates to a method for preparing lactic acid bacterium-derived extracellular vesicles with improved yield. The inventors of the present invention have identified the optimal medium composition and fermentation conditions that can increase the yield of extracellular vesicles secreted by lactic acid bacteria. Specifically, there were problems with the conventional Lactobacillus culture medium, such as a small yield of extracellular vesicle, or inedible components of the medium, and toxicity due to animal-derived components. However, the present inventors have developed a medium that is not toxic, contains no animal-derived components, and is edible, and confirming that the medium is free of cytotoxicity and increases the yield of extracellular vesicles when culturing Lactobacillus. Furthermore, the present inventors identified the optimal culture time that can increase the yield. Therefore, the method of preparing extracellular vesicles with improved yield according to the present invention is expected to be beneficially used in processes for mass production on an industrial scale of extracellular vesicles secreted by lactic acid bacteria, which can be utilized as pharmacological components.

Description

수율이 향상된 유산균 유래 세포외소포의 제조방법Method for manufacturing lactic acid bacteria-derived extracellular vesicles with improved yield
본 발명은 수율이 향상된 유산균이 분비하는 세포외소포의 제조방법에 관한 것이다.The present invention relates to a method for producing extracellular vesicles secreted by lactic acid bacteria with improved yield.
본 출원은 2022년 03월 18일에 출원된 한국특허출원 제10-2022-0034267호 및 2023년 03월 14일에 출원된 한국특허출원 제10-2023-0033499호에 기초한 우선권을 주장하며, 해당 출원의 명세서 및 도면에 개시된 모든 내용은 본 출원에 원용된다.This application claims priority based on Korean Patent Application No. 10-2022-0034267 filed on March 18, 2022 and Korean Patent Application No. 10-2023-0033499 filed on March 14, 2023. All contents disclosed in the specification and drawings of the application are incorporated into this application.
락토바실러스(Lactobacillus) 세균은 그람양성 간균으로서 혐기성 환경뿐만 아니라 호기성 환경에서도 잘 자라고, 우리 몸에 공생하는 유익한 세균으로 알려져 있다. 세균은 세포간 단백질, 지질, 유전자 등의 교환을 위해 세포 밖 환경으로 이중막 구조의 세포외소포(extracellular vesicle; EV)를 분비한다. 락토바실러스 세균과 같은 그람양성균(gram-positive bacteria)이 분비하는 소포는 세균 유래 단백질과 핵산 외에도 세균의 세포벽 구성성분인 펩티도글리칸(peptidoglycan)과 리포테이코산(lipoteichoic acid)도 가지고 있다. Lactobacillus bacteria are Gram-positive bacilli that grow well not only in anaerobic environments but also in aerobic environments, and are known as beneficial bacteria that coexist in our bodies. Bacteria secrete double-membrane extracellular vesicles (EV) into the extracellular environment for the exchange of proteins, lipids, and genes between cells. Vesicles secreted by gram-positive bacteria such as Lactobacillus bacteria contain peptidoglycan and lipoteichoic acid, which are components of the bacterial cell wall, in addition to bacteria-derived proteins and nucleic acids.
최근 락토바실러스 분비 세포외소포의 다양한 생리활성 효과에 대한 연구가 이루어지고 있으며, 특히, 락토바실러스 파라카제이의 세포외소포는 염증질환, 대사질환, 노화 및 암 등의 다양한 질환에서 예방 또는 치료효과가 있는 것으로 알려져 있다.Recently, research has been conducted on the various physiological activity effects of Lactobacillus secreted extracellular vesicles. In particular, the extracellular vesicles of Lactobacillus paracasei have preventive or therapeutic effects in various diseases such as inflammatory diseases, metabolic diseases, aging, and cancer. It is known that there is.
따라서, 락토바실러스 분비 세포외소포는 약리성분으로 활용될 수 있어, 이러한 세포외소포를 산업적 규모로 고효율 대량생산 하기 위한 방법이 필요한 실정이다. Therefore, Lactobacillus secreted extracellular vesicles can be used as pharmaceutical ingredients, and a method for highly efficient mass production of such extracellular vesicles on an industrial scale is needed.
본 발명자들은 락토바실러스 분비 세포외소포를 고효율로 대량생산 하기 위해 예의 연구한 결과, 락토바실러스 분비 세포외소포의 수율을 증가시킬 수 있는 최적의 배지조성 및 발효조건을 확인하였는 바, 이에 기초하여 본 발명을 완성하였다. As a result of intensive research to mass-produce Lactobacillus-secreted extracellular vesicles with high efficiency, the present inventors have identified the optimal medium composition and fermentation conditions that can increase the yield of Lactobacillus-secreted extracellular vesicles. Based on this, the present inventors have The invention was completed.
이에, 본 발명은 수율이 향상된 유산균 유래 세포외소포의 제조방법을 제공하는 것을 목적으로 한다. Accordingly, the purpose of the present invention is to provide a method for producing extracellular vesicles derived from lactic acid bacteria with improved yield.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.
상기와 같은 본 발명의 목적을 달성하기 위하여, 본 발명은 (a) 독성 및 동물 유래 성분이 없고 식용가능한 유산균 배양용 배지를 제조하는 단계; (b) 상기 (a) 단계에서 제조된 배지에 유산균을 배양하는 단계; (c) 상기 (b) 단계의 배양액을 원심분리하여 상층액을 분리하는 단계; (d) 상기 (c) 단계의 상층액을 농축하고, 완충용액으로 희석하여 재농축하는 단계; 및 (e) 상기 (d) 단계의 농축액을 필터로 여과하여 세포외소포를 수득하는 단계를 포함하는 수율이 향상된 유산균 유래 세포외소포의 제조방법을 제공한다.In order to achieve the object of the present invention as described above, the present invention includes the steps of (a) preparing a culture medium for lactic acid bacteria that is free of toxic and animal-derived components and is edible; (b) cultivating lactic acid bacteria in the medium prepared in step (a); (c) centrifuging the culture medium of step (b) to separate the supernatant; (d) concentrating the supernatant of step (c) and re-concentrating it by diluting it with a buffer solution; and (e) filtering the concentrate of step (d) through a filter to obtain extracellular vesicles.
본 발명의 일 구현예로서, 상기 배지는 식용 불가 성분인 구연산 암모늄 및 동물 유래 성분인 소고기 추출물을 포함하지 않는 것일 수 있으나, 이에 제한되는 것은 아니다.As an embodiment of the present invention, the medium may not contain ammonium citrate, which is an inedible ingredient, and beef extract, which is an animal-derived ingredient, but is not limited thereto.
본 발명의 다른 구현예로서, 상기 배지는 포도당, 대두 펩톤, 효모 추출물, 초산 나트륨, 제2인산칼륨, 황산 마그네슘, 황산 망간, 구연산 및 폴리소르베이트80을 포함하는 것일 수 있으나, 이에 제한되는 것은 아니다.In another embodiment of the present invention, the medium may contain glucose, soy peptone, yeast extract, sodium acetate, potassium dibasic phosphate, magnesium sulfate, manganese sulfate, citric acid and polysorbate 80, but is limited thereto. no.
본 발명의 또 다른 구현예로서, 상기 (b) 단계는 종균 배양, 전 배양 및 본 배양의 순서로 각각 8시간 내지 16시간 배양하는 것일 수 있으나, 이에 제한되는 것은 아니다.As another embodiment of the present invention, step (b) may be performed in the order of seed culture, pre-culture, and main culture, respectively, for 8 to 16 hours, but is not limited thereto.
본 발명의 또 다른 구현예로서, 상기 (b) 단계는 종균 배양, 전 배양 및 본 배양의 순서로 각각 10시간 내지 14시간 배양하는 것일 수 있으나, 이에 제한되는 것은 아니다.As another embodiment of the present invention, step (b) may be performed in the order of seed culture, pre-culture, and main culture, respectively, for 10 to 14 hours, but is not limited thereto.
본 발명의 또 다른 구현예로서, 상기 (d) 단계는 50 kDa 내지 150 kDa 사이즈 한외여과막을 사용하여 농축하는 것일 수 있으나, 이에 제한되는 것은 아니다.As another embodiment of the present invention, step (d) may be concentration using an ultrafiltration membrane of 50 kDa to 150 kDa in size, but is not limited thereto.
본 발명의 또 다른 구현예로서, 상기 (e) 단계의 필터는 0.1 내지 1 μm 사이즈 필터일 수 있으나, 이에 제한되는 것은 아니다.As another embodiment of the present invention, the filter in step (e) may be a 0.1 to 1 μm size filter, but is not limited thereto.
본 발명의 또 다른 구현예로서, 상기 (e) 단계의 필터는 0.1 μm 내지 0.5 μm 사이즈 필터일 수 있으나, 이에 제한되는 것은 아니다.As another embodiment of the present invention, the filter in step (e) may be a 0.1 μm to 0.5 μm size filter, but is not limited thereto.
본 발명의 또 다른 구현예로서, 상기 유산균은 락토바실러스(Lactobacillus), 락토코커스(Lactococcus), 또는 비피도박테리움(Bifidobacterium) 속 세균일 수 있으나, 이에 제한되는 것은 아니다.As another embodiment of the present invention, the lactic acid bacteria may be bacteria of the genus Lactobacillus , Lactococcus , or Bifidobacterium , but are not limited thereto.
본 발명의 또 다른 구현예로서, 상기 락토바실러스(Lactobacillus) 속 세균은 락토바실러스 브레비스(Lactobacillus brevis), 락토바실러스 아세톨레란스(Lactobacillus acetotolerans), 락토바실러스 아시디프리내(Lactobacillus acidifarinae), 락토바실러스 아시디피시스(Lactobacillus acidipiscis), 락토바실러스 아길리스(Lactobacillus agilis), 락토바실러스 알기두스(Lactobacillus algidus), 락토바실러스 알리먼타러스(Lactobacillus alimentarus), 락토바실러스 아밀로리티쿠스(Lactobacillus amylolyticus), 락토바실러스 아밀로트로피쿠스(Lactobacillus amylotrophicus), 락토바실러스 아밀로보러스(Lactobacillus amylovorus), 락토바실러스 아니말리스(Lactobacillus animalis), 락토바실러스 안트리(Lactobacillus antri), 락토바실러스 아포데미(Lactobacillus apodemi), 락토바실러스 아비아리우스(Lactobacillus aviarius), 락토바실러스 비퍼멘탄스(Lactobacillus bifermentans), 락토바실러스 봄피콜라(Lactobacillus bombicola), 락토바실러스 부흐너(Lactobacillus buchneri), 락토바실러스 카멜리애(Lactobacillus camelliae), 락토바실러스 카세이(Lactobacillus casei), 락토바실러스 카트나포미스(Lactobacillus catenaformis), 락토바실러스 세티(Lactobacillus ceti), 락토바실러스 콜레오호미니스(Lactobacillus coleohominis), 락토바실러스 콜리노이데스(Lactobacillus collinoides), 락토바실러스 콤포스티(Lactobacillus composti), 락토바실러스 콘카부스(Lactobacillus concavus), 락토바실러스 코리니포미스(Lactobacillus coryniformis), 락토바실러스 크리스파투스(Lactobacillus crispatus), 락토바실러스 크러스토룸(Lactobacillus crustorum), 락토바실러스 커바투스(Lactobacillus curvatus), 락토바실러스 델브루엑키(Lactobacillus delbruecki), 락토바실러스 덱스트리니쿠스(Lactobacillus dextrinicus), 락토바실러스 디올리보란스(Lactobacillus diolivorans), 락토바실러스 에퀴(Lactobacillus equi), 락토바실러스 에퀴제네로시(Lactobacillus equigenerosi), 락토바실러스 파라기니스(Lactobacillus farraginis), 락토바실러스 파르시미니스(Lactobacillus farciminis), 락토바실러스 퍼먼텀(Lactobacillus fermentum), 락토바실러스 포시니칼리스(Lactobacillus fornicalis), 락토바실러스 프룩티보란스(Lactobacillus fructivorans), 락토바실러스 프루먼티(Lactobacillus frumenti), 락토바실러스 푸흐엔시스(Lactobacillus fuchuensis), 락토바실러스 갈리나룸(Lactobacillus gallinarum), 락토바실러스 가세리(Lactobacillus gasseri), 락토바실러스 가스트리쿠스(Lactobacillus gastricus), 락토바실러스 강엔시스(Lactobacillus ghanensis), 락토바실러스 그라미니스(Lactobacillus graminis), 락토바실러스 하메시(Lactobacillus hammesii), 락토바실러스 하메스테리(Lactobacillus hamsteri), 락토바실러스 하비넨시스(Lactobacillus harbinensis), 락토바실러스 하야키텐시스(Lactobacillus hayakitensis), 락토바실러스 헬베티쿠스(Lactobacillus helveticus), 락토바실러스 힐가르디(Lactobacillus hilgardii), 락토바실러스 호모히오키(Lactobacillus homohiochii), 락토바실러스 이네르스(Lactobacillus iners), 락토바실러스 인글루비에이(Lactobacillus ingluviei), 락토바실러스 인테스티날리스(Lactobacillus intestinalis), 락토바실러스 젠세니(Lactobacillus jensenii), 락토바실러스 존소니(Lactobacillus johnsonii), 락토바실러스 칼릭센시스(Lactobacillus kalixensis), 락토바실러스 케피라노파시엔스(Lactobacillus kefiranofaciens), 락토바실러스 케피리(Lactobacillus kefiri), 락토바실러스 김치(Lactobacillus kimchii), 락토바실러스 키타사토니스(Lactobacillus kitasatonis), 락토바실러스 쿤키이(Lactobacillus kunkeei), 락토바실러스 레이크만니이(Lactobacillus leichmannii), 락토바실러스 린드네리(Lactobacillus lindneri), 락토바실러스 말레페르멘탄스(Lactobacillus malefermentans), 락토바실러스 말리(Lactobacillus mali), 락토바실러스 만니호티보란스(Lactobacillus manihotivorans), 락토바실러스 민덴시스(Lactobacillus mindensis), 락토바실러스 무코새(Lactobacillus mucosae), 락토바실러스 뮤리너스(Lactobacillus murinus), 락토바실러스 나겔리(Lactobacillus nagelii), 락토바실러스 나무렌시스(Lactobacillus namurensis), 락토바실러스 난텐시스(Lactobacillus nantensis), 락토바실러스 올리고퍼멘탄스(Lactobacillus oligofermentans), 락토바실러스 오리스(Lactobacillus oris), 락토바실러스 파니스(Lactobacillus panis), 락토바실러스 판테리스(Lactobacillus pantheris), 락토바실러스 파라브레비스(Lactobacillus parabrevis), 락토바실러스 파라부흐너(Lactobacillus parabuchneri), 락토바실러스 파라카제이(Lactobacillus paracasei), 락토바실러스 파라콜리노이데스(Lactobacillus paracollinoides), 락토바실러스 파라파라기니스(Lactobacillus parafarraginis), 락토바실러스 파라케피리(Lactobacillus parakefiri), 락토바실러스 파라리먼타리우스(Lactobacillus paralimentarius), 락토바실러스 파라플란타룸(Lactobacillus paraplantarum), 락토바실러스 펜토수스(Lactobacillus pentosus), 락토바실러스 페롤렌스(Lactobacillus perolens), 락토바실러스 폰티스(Lactobacillus pontis), 락토바실러스 프로텍투스(Lactobacillus protectus), 락토바실러스 시터씨(Lactobacillus psittaci), 락토바실러스 렌니니(Lactobacillus rennini), 락토바실러스 루테리(Lactobacillus reuteri), 락토바실러스 리매(Lactobacillus rimae), 락토바실러스 로고새(Lactobacillus rogosae), 락토바실러스 로지애(Lactobacillus rossiae), 락토바실러스 루미니스(Lactobacillus ruminis), 락토바실러스 새림네리(Lactobacillus saerimneri), 락토바실러스 사케이(Lactobacillus sakei), 락토바실러스 살리바리우스(Lactobacillus salivarius), 락토바실러스 샌프란시센시스(Lactobacillus sanfranciscensis), 락토바실러스 사츠멘시스(Lactobacillus satsumensis), 락토바실러스 세칼리필루스(Lactobacillus secaliphilus), 락토바실러스 사피애(Lactobacillus sharpeae), 락토바실러스 실리기니스(Lactobacillus siliginis), 락토바실러스 스피체리(Lactobacillus spicheri), 락토바실러스 수에비쿠스(Lactobacillus suebicus), 락토바실러스 타일랜덴시스(Lactobacillus thailandensis), 락토바실러스 울투넨시스(Lactobacillus ultunensis), 락토바실러스 백시노스터쿠스(Lactobacillus vaccinostercus), 락토바실러스 바기날리스(Lactobacillus vaginalis), 락토바실러스 버스몰덴시스(Lactobacillus versmoldensis), 락토바실러스 비니(Lactobacillus vini), 락토바실러스 비툴리누스(Lactobacillus vitulinus), 락토바실러스 지애(Lactobacillus zeae), 또는 락토바실러스 지매(Lactobacillus zymae)일 수 있으나, 이에 제한되는 것은 아니다.As another embodiment of the present invention, the bacteria of the Lactobacillus genus include Lactobacillus brevis , Lactobacillus acetolerans , Lactobacillus acidifrinae , and Lactobacillus acetate. Lactobacillus acidipiscis , Lactobacillus agilis, Lactobacillus algidus, Lactobacillus alimentarus , Lactobacillus amylolyticus , Lactobacillus amyl Lactobacillus amylotrophicus , Lactobacillus amylovorus, Lactobacillus animalis , Lactobacillus antri , Lactobacillus apodemi , Lactobacillus aviarius ( Lactobacillus aviarius , Lactobacillus bifermentans, Lactobacillus bombicola, Lactobacillus buchneri , Lactobacillus camelliae, Lactobacillus casei , lactobacillus Bacillus catenaformis, Lactobacillus ceti , Lactobacillus coleohominis, Lactobacillus collinoides , Lactobacillus composti , Lactobacillus concabus ( Lactobacillus concavus ), Lactobacillus coryniformis , Lactobacillus crispatus , Lactobacillus crustorum, Lactobacillus curvatus , Lactobacillus delbruecki ( Lactobacillus delbruecki ), Lactobacillus dextrinicus ( Lactobacillus dextrinicus ), Lactobacillus diolivorans ( Lactobacillus equi ), Lactobacillus equigenerosi ( Lactobacillus equigenerosi ), Lactobacillus paraginis ( Lactobacillus farraginis , Lactobacillus farciminis , Lactobacillus fermentum , Lactobacillus fornicalis, Lactobacillus fructivorans, Lactobacillus frumenti ), Lactobacillus fuchuensis , Lactobacillus gallinarum , Lactobacillus gasseri, Lactobacillus gastricus , Lactobacillus ghanensis , Lactobacillus Graminis ( Lactobacillus graminis ), Lactobacillus hammesii ( Lactobacillus hamsteri ), Lactobacillus harbinensis ( Lactobacillus harbinensis ) , Lactobacillus hayakitensis ( Lactobacillus hayakitensis ), Lactobacillus hell Lactobacillus helveticus , Lactobacillus hilgardii , Lactobacillus homohiochii , Lactobacillus iners , Lactobacillus ingluviei , Lactobacillus intes Lactobacillus intestinalis , Lactobacillus jensenii , Lactobacillus johnsonii , Lactobacillus kalixensis , Lactobacillus kefiranofaciens , Lactobacillus ke Piri ( Lactobacillus kefiri ), Lactobacillus kimchii ( Lactobacillus kimchii ), Lactobacillus kitasatonis ( Lactobacillus kunkeei ), Lactobacillus leichmannii ( Lactobacillus leichmannii ), Lactobacillus lindneri ), Lactobacillus malefermentans , Lactobacillus mali , Lactobacillus manihotivorans, Lactobacillus mindensis , Lactobacillus mucosae , Lactobacillus murinus, Lactobacillus nagelii, Lactobacillus namurensis , Lactobacillus nantensis , Lactobacillus oligofermentans , Lactobacillus oris ( Lactobacillus oris ), Lactobacillus panis , Lactobacillus pantheris , Lactobacillus parabrevis , Lactobacillus parabuchneri, Lactobacillus paracasei , Lactobacillus paracollinoides, Lactobacillus parafarraginis, Lactobacillus parakefiri , Lactobacillus paralimentarius , Lactobacillus paraplantarum ( Lactobacillus paraplantarum ), Lactobacillus pentosus, Lactobacillus perolens, Lactobacillus pontis , Lactobacillus protectus , Lactobacillus psittaci , lactobacillus Bacillus rennin i, Lactobacillus reuteri, Lactobacillus rimae, Lactobacillus rogosae , Lactobacillus rossiae, Lactobacillus ruminis ), Lactobacillus saerimneri , Lactobacillus sakei, Lactobacillus salivarius, Lactobacillus sanfranciscensis , Lactobacillus satsumensis , Lactobacillus secaliphilus , Lactobacillus sharpeae, Lactobacillus siliginis , Lactobacillus spicheri , Lactobacillus suebicus , Lactobacillus Lactobacillus thailandensis, Lactobacillus ultunensis , Lactobacillus vaccinostercus , Lactobacillus vaginalis , Lactobacillus versmoldensis , lactobacillus It may be Bacillus vini , Lactobacillus vitulinus, Lactobacillus zeae , or Lactobacillus zymae, but is not limited thereto.
본 발명의 또 다른 구현예로서, 상기 락토코커스(Lactococcus) 속 세균은 락토코커스 중앙젠시스(Lactococcus chungangensis), 락토코커스 포모센시스(Lactococcus formosensis), 락토코커스 푸지엔시스(Lactococcus fujiensis) 락토코커스 히르시락티스(Lactococcus hircilactis), 락토코커스 갈비에(Lactococcus garvieae), 락토코커스 락티스(Lactococcus lactis), 락토코커스 라우덴시스(Lactococcus laudensis), 락토코커스 내슈티터미티스(Lactococcus nasutitermitis), 락토코커스 피시엄(Lactococcus piscium), 락토코커스 플란타룸(Lactococcus plantarum), 락토코커스 라피노락티스(Lactococcus raffinolactis), 또는 락토코커스 타이와네시스(Lactococcus taiwanensis)일 수 있으나, 이에 제한되는 것은 아니다.In another embodiment of the present invention, the bacteria of the Lactococcus genus are Lactococcus chungangensis , Lactococcus formosensis , Lactococcus fujiensis , and Lactococcus hirsi. Lactococcus hircilactis , Lactococcus garvieae , Lactococcus lactis , Lactococcus laudensis , Lactococcus nasutitermitis , Lactococcus piscium , Lactococcus plantarum , lacto It may be Lactococcus raffinolactis , or Lactococcus taiwanensis , but is not limited thereto.
본 발명의 또 다른 구현예로서, 상기 비피도박테리움(Bifidobacterium) 속 세균은 비피도박테리움 악티노코로니포르메(Bifidobacterium actinocoloniiforme), 비피도박테리움 아돌레신티스(Bifidobacterium adolescentis), 비피도박테리움 애미리아눔(Bifidobacterium aemilianum), 비피도박테리움 애로필럼(Bifidobaterium aerophilum), 비피도박테리움 애스쿨라피(Bifidobaterium aesculapii), 비피도박테리움 아마조넨스(Bifidobaterium amazonense), 비피도박테리움 안굴라툼(Bifidobaterium angulatum), 비피도박테리움 애니말리스(Bifidobacterium animalis), 비피도박테리움 안세리스(Bifidobaterium anseris), 비피도박테리움 아포우시아(Bifidobaterium apousia), 비피도박테리움 아프리(Bifidobaterium apri), 비피도박테리움 아퀴케피리(Bifidobaterium aquikefiri), 비피도박테리움 아스테로이데스(Bifidobaterium asteroides), 비피도박테리움 아베사니(Bifidobaterium avesanii), 비피도박테리움 비아바티(Bifidobaterium biavatii), 비피도박테리움 비피덤(Bifidobacterium bifidum), 비피도박테리움 보헤미쿰(Bifidobaterium bohemicum), 비피도박테리움 봄비(Bifidobaterium bombi), 비피도박테리움 보움(Bifidobaterium boum), 비피도박테리움 브레브(Bifidobacterium breve), 비피도박테리움 칼리미코니스(Bifidobaterium callimiconis), 비피도박테리움 칼리트리키다룸(Bifidobaterium callitrichidarum), 비피도박테리움 알리트리코스(Bifidobaterium allitrichos), 비피도박테리움 카니스(Bifidobaterium canis), 비피도박테리움 카스토리스(Bifidobaterium castoris), 비피도박테리움 카테눌라툼(Bifidobaterium catenulatum), 비피도박테리움 카툴로룸(Bifidobaterium catulorum), 비피도박테리움 세비다룸(Bifidobaterium cebidarum), 비피도박테리움 코에리눔(Bifidobaterium choerinum), 비피도박테리움 콜라도하비탄스(Bifidobaterium choladohabitans), 비피도박테리움 콜로에피(Bifidobaterium choloepi), 비피도박테리움 콜로비(Bifidobaterium colobi), 비피도박테리움 콤문(Bifidobaterium commune), 비피도박테리움 크리세티(Bifidobaterium criceti), 비피도박테리움 크루딜락티스(Bifidobaterium crudilactis), 비피도박테리움 쿠니쿨리(Bifidobaterium cuniculi), 비피도박테리움 덴티움(Bifidobaterium dentium), 비피도박테리움 돌리코티디스(Bifidobaterium dolichotidis), 비피도박테리움 에릭소니(Bifidobaterium eriksonii), 비피도박테리움 에리트로세비(Bifidobaterium erythrocebi), 비피도박테리움 을레무리스(Bifidobaterium eulemuris), 비피도박테리움 패칼리(Bifidobaterium faecale), 비피도박테리움 펠시네움(Bifidobaterium felsineum), 비피도박테리움 칼리쿰(Bifidobaterium gallicum), 비피도박테리움 갈리나룸(Bifidobaterium gallinarum), 비피도박테리움 글로보숨(Bifidobaterium globosum), 비피도박테리움 괼디(Bifidobaterium goeldii), 비피도박테리움 하팔리(Bifidobaterium hapali), 비피도박테리움 인디쿰(Bifidobaterium indicum), 비피도박테리움 이탈리쿰(Bifidobaterium italicum), 비피도박테리움 자치(Bifidobaterium jacchi), 비피도박테리움 레무룸(Bifidobaterium lemurum), 비피도박테리움 레오토피테치(Bifidobaterium leontopitheci), 비피도박테리움 롱검(Bifidobacterium longum), 비피도박테리움 마그눔(Bifidobaterium magnum), 비피도박테리움 마골레시(Bifidobaterium margollesii), 비피도박테리움 메리시쿰(Bifidobaterium merycicum), 비피도박테리움 미코니스(Bifidobaterium miconis), 비피도박테리움 미코니사젠카티(Bifidobaterium miconisargentati), 비피도박테리움 미니뭄(Bifidobaterium minimum), 비피도박테리움 몽골리엔스(Bifidobaterium mongoliense), 비피도박테리움 모라비엔스(Bifidobaterium moraviense), 비피도박테리움 모우칼라벤스(Bifidobaterium moukalabense), 비피도박테리움 미오소티스(Bifidobaterium myosotis), 비피도박테리움 오이디포디스(Bifidobaterium oedipodis), 비피도박테리움 올로무센스(Bifidobaterium olomucense), 비피도박테리움 파노스(Bifidobaterium panos), 비피도박테리움 파마에(Bifidobaterium parmae), 비피도박테리움 플라티리노룸(Bifidobaterium platyrrhinorum), 비피도박테리움 플러비알리실배(Bifidobaterium pluvialisilvae), 비피도박테리움 폴리사카롤리티쿰(Bifidobaterium polysaccharolyticum), 비피도박테리움 폰고니스(Bifidobaterium pongonis), 비피도박테리움 포르시눔(Bifidobaterium porcinum), 비피도박테리움 프리마티움(Bifidobaterium primatium), 비피도박테리움 슈도카테눌라툼(Bifidobaterium pseudocatenulatum), 비피도박테리움 슈도롱검(Bifidobacterium pseudolongum), 비피도박테리움 사이크래로필룸(Bifidobaterium psychraerophilum), 비피도박테리움 풀로룸(Bifidobaterium pullorum), 비피도박테리움 라모숨(Bifidobaterium ramosum), 비피도박테리움 루테리(Bifidobaterium reuteri), 비피도박테리움 루세티(Bifidobaterium rousetti), 비피도박테리움 루미날리(Bifidobaterium ruminale), 비피도박테리움 루미난티움(Bifidobaterium ruminantium), 비피도박테리움 산구이니(Bifidobaterium saguini), 비피도박테리움 산구이니비콜로리스(Bifidobaterium saguinibicoloris), 비피도박테리움 사이미리시우레(Bifidobaterium saimiriisciurei), 비피도박테리움 사미리(Bifidobaterium samirii), 비피도박테리움 산틸라넨스(Bifidobaterium santillanense), 비피도박테리움 스칼리제룸(Bifidobaterium scaligerum), 비피도박테리움 스카도비(Bifidobaterium scardovii), 비피도박테리움 시미아룸(Bifidobaterium simiarum), 비피도박테리움 시미벤트리스(Bifidobaterium simiiventris), 비피도박테리움 스텔렌보센스(Bifidobaterium stellenboschense), 비피도박테리움 서틸리(Bifidobaterium subtile), 비피도박테리움 써마시도필룸(Bifidobaterium thermacidophilum), 비피도박테리움 써모필룸(Bifidobaterium thermophilum), 비피도박테리움 티비그라눌리(Bifidobaterium tibiigranuli), 비피도박테리움 티시에리(Bifidobaterium tissieri), 비피도박테리움 쑤루미엔스(Bifidobaterium tsurumiense), 비피도박테리움 유리날리스(Bifidobaterium urinalis), 비피도박테리움 반신데레니(Bifidobaterium vansinderenii), 비피도박테리움 베스페틸리오니스(Bifidobaterium vespertilionis), 또는 비피도박테리움 자이로코패(Bifidobaterium xylocopae)일 수 있으나, 이제 제한되는 것은 아니다.As another embodiment of the present invention, the bacteria of the Bifidobacterium genus are Bifidobacterium actinocoloniiforme , Bifidobacterium adolescentis , and Bifidobacterium. Aemilianum ( Bifidobacterium aemilianum ), Bifidobacterium aerophilum, Bifidobaterium aesculapii, Bifidobaterium amazonense , Bifidobacterium angulatum ( Bifidobaterium angulatum ), Bifidobacterium animalis , Bifidobacterium anseris , Bifidobaterium apousia , Bifidobaterium apri , Bifidobacterium apri Bifidobaterium aquikefiri , Bifidobaterium asteroides, Bifidobaterium avesanii , Bifidobaterium biavatii , Bifidobacterium bifidum ( Bifidobacterium bifidum ), Bifidobacterium bohemicum , Bifidobacterium bombi, Bifidobaterium boum , Bifidobacterium breve , Bifidobacterium Bifidobaterium callimiconis , Bifidobaterium callitrichidarum, Bifidobaterium allitrichos , Bifidobaterium canis , Bifidobacterium cas. Toris ( Bifidobaterium castoris ), Bifidobacterium catenulatum ( Bifidobaterium catulorum ), Bifidobacterium cebidarum ( Bifidobaterium cebidarum ) , Bifidobacterium coerinum ( Bifidobaterium choerinum ), Bifidobacterium choladohabitans , Bifidobaterium choloepi, Bifidobaterium colobi , Bifidobaterium commune , Bifidobacterium Bifidobaterium criceti , Bifidobaterium crudilactis , Bifidobaterium cuniculi, Bifidobaterium dentium , Bifidobacterium dolicotidis ( Bifidobaterium dolichotidis ), Bifidobaterium eriksonii , Bifidobaterium erythrocebi , Bifidobaterium eulemuris , Bifidobaterium faecale , Bifidobacterium felsineum, Bifidobaterium gallicum, Bifidobacterium gallinarum , Bifidobaterium globosum , Bifidobacterium Goeldii ( Bifidobaterium goeldii ), Bifidobaterium hapali ( Bifidobaterium hapali ), Bifidobaterium indicum, Bifidobacterium italicum ( Bifidobaterium italicum ), Bifidobaterium jacchi , Bifidobacterium Bifidobaterium lemurum , Bifidobacterium leontopitheci, Bifidobacterium longum , Bifidobacterium magnum , Bifidobacterium margolesi ( Bifidobaterium margollesii ), Bifidobacterium merycicum , Bifidobaterium miconis , Bifidobacterium miconisargentati , Bifidobacterium minimum , Bifidobacterium mongoliense, Bifidobaterium moraviense, Bifidobaterium moukalabense , Bifidobaterium myosotis , Bifidobacterium Bifidobaterium oedipodis , Bifidobaterium olomucense, Bifidobaterium panos , Bifidobaterium parmae , Bifidobacterium platy Linorum ( Bifidobaterium platyrrhinorum ), Bifidobaterium pluvialisilvae , Bifidobaterium polysaccharolyticum , Bifidobaterium pongonis , Bifidobacterium porsi Bifidobaterium porcinum , Bifidobacterium primatium, Bifidobacterium pseudocatenulatum , Bifidobacterium pseudolongum , Bifidobacterium cyclophilum ( Bifidobaterium psychraerophilum ), Bifidobaterium pullorum ( Bifidobaterium ramosum ), Bifidobacterium reuteri ( Bifidobaterium reuteri ), Bifidobaterium rousetti , Bifidobacterium Bifidobaterium ruminale , Bifidobaterium ruminantium, Bifidobaterium saguini , Bifidobaterium saguinibicoloris , Bifidobacterium Bifidobaterium saimiriisciurei , Bifidobacterium samirii , Bifidobaterium santillanense , Bifidobaterium scaligerum , Bifidobacterium scadovi ( Bifidobaterium scardovii ), Bifidobaterium simiarum , Bifidobaterium simiiventris , Bifidobaterium stellenboschense , Bifidobaterium subtile , Bifidobaterium thermacidophilum , Bifidobaterium thermophilum , Bifidobaterium tibiigranuli , Bifidobaterium tissieri , Bifidobacterium Bifidobaterium tsurumiense, Bifidobaterium urinalis , Bifidobaterium vansinderenii , Bifidobaterium vespertilionis , or It may be, but is not limited to, Bifidobaterium xylocopae .
본 발명의 또 다른 구현예로서, 상기 세포외소포는 평균 직경이 10 nm 내지 1000 nm일 수 있으나, 이제 제한되는 것은 아니다.As another embodiment of the present invention, the extracellular vesicles may have an average diameter of 10 nm to 1000 nm, but are not limited thereto.
본 발명자들은 락토바실러스 분비 세포외소포의 수율을 증가시킬 수 있는 최적의 배지조성 및 발효조건을 확인하였다. 구체적으로, 기존의 락토바실러스 배양용 배지로 배양시 세포외소포의 적은 수득량 또는 배지의 식용 불가 성분 및 동물 유래 성분으로 인한 독성 등으로 인한 문제가 있었으나, 본 발명자들은 독성 및 동물 유래 성분이 없고 식용가능한 배지를 제조하였으며, 세포독성이 없고, 락토바실러스 배양시 세포외소포의 수율 또한 증가시키는 것을 확인하였다. 이에 더하여, 수율을 높일 수 있는 최적의 배양시간을 확인하였다. 이에, 본 발명에 따른 수율이 향상된 세포외소포의 제조방법은 약리성분으로 활용될 수 있는 유산균이 분비하는 세포외소포의 산업적 규모로 대량 생산하는 공정에 유용하게 사용될 수 있을 것으로 기대된다.The present inventors identified the optimal medium composition and fermentation conditions that can increase the yield of Lactobacillus secreted extracellular vesicles. Specifically, when culturing with existing Lactobacillus culture medium, there were problems due to low yield of extracellular vesicles or toxicity due to inedible and animal-derived components of the medium, but the present inventors have found that there are no toxic or animal-derived components. An edible medium was prepared, and it was confirmed that it had no cytotoxicity and also increased the yield of extracellular vesicles during Lactobacillus culture. In addition, the optimal culturing time to increase yield was confirmed. Accordingly, the method for producing extracellular vesicles with improved yield according to the present invention is expected to be useful in the process of mass production on an industrial scale of extracellular vesicles secreted by lactic acid bacteria that can be used as pharmaceutical ingredients.
도 1은 마우스 대식세포(Raw264.7)에서 배양 배지 종류에 따른 락토바실러스 파라카제이 분비 세포외소포의 세포 독성을 평가한 결과이다.Figure 1 shows the results of evaluating the cytotoxicity of extracellular vesicles secreted by Lactobacillus paracasei in mouse macrophages (Raw264.7) depending on the type of culture medium.
도 2는 락토바실러스 파라카제이 종균 배양 성장 패턴을 분석한 결과이다.Figure 2 shows the results of analyzing the growth pattern of Lactobacillus paracasei seed culture.
도 3은 락토바실러스 파라카제이 전 배양 성장 패턴을 분석한 결과이다.Figure 3 shows the results of analyzing the growth pattern of Lactobacillus paracasei pre-culture.
도 4는 락토바실러스 파라카제이 본 배양 성장 패턴을 분석한 결과이다.Figure 4 shows the results of analyzing the growth pattern of Lactobacillus paracasei main culture.
도 5는 마우스 대식세포(Raw264.7)에서 한외여과막 사이즈에 따른 락토바실러스 파라카제이 분비 세포외소포의 TNF-α 분비 저해능을 확인한 결과이다.Figure 5 shows the results of confirming the ability of Lactobacillus paracasei secreted extracellular vesicles to inhibit TNF-α secretion according to the ultrafiltration membrane size in mouse macrophages (Raw264.7).
도 6은 본 발명의 방법으로 분리된 락토바실러스 파라카제이 분비 세포외소포의 단백질 발현 패턴을 SDS-PAGE로 분석한 결과이다.Figure 6 shows the results of SDS-PAGE analysis of the protein expression pattern of Lactobacillus paracasei secreted extracellular vesicles isolated by the method of the present invention.
본 발명에서는 본 발명의 수율이 향상된 세포외소포의 제조방법이 락토바실러스 배양시 세포외소포의 수율을 증가시키는 것을 확인하였다. 따라서, 본 발명의 수율이 향상된 세포외소포의 제조방법은 약리성분으로 활용될 수 있는 락토바실러스 분비 세포외소포의 산업적 규모로 대량 생산하는 공정에 유용하게 사용될 수 있을 것으로 기대된다.In the present invention, it was confirmed that the method for producing extracellular vesicles with improved yield of the present invention increases the yield of extracellular vesicles when culturing Lactobacillus. Therefore, the method for producing extracellular vesicles with improved yield of the present invention is expected to be useful in the process of mass production of Lactobacillus secreted extracellular vesicles that can be used as pharmaceutical ingredients on an industrial scale.
본 발명의 일 실시예에서는, LB, MRS 및 EMP 배지에서 락토바실러스 파라카제이(Lactobacillus paracasei)를 배양하여 세포외소포를 분리하고, 분리된 세포외소포의 수율 및 독성을 비교 분석한 결과, EMP 배지에서 배양한 락토바실러스 파라카제이에서 세포외소포의 수율이 가장 높고, 독성이 없는 것을 확인하였다(실시예 1 참조).In one embodiment of the present invention, extracellular vesicles were isolated by culturing Lactobacillus paracasei in LB, MRS and EMP media, and as a result of comparative analysis of the yield and toxicity of the isolated extracellular vesicles, EMP It was confirmed that Lactobacillus paracasei cultured in medium had the highest yield of extracellular vesicles and had no toxicity (see Example 1).
본 발명의 다른 실시예에서는, 락토바실러스 파라카제이 분비 세포외소포의 수율을 높이기 위하여 배양 공정 최적화 과정을 진행하였으며, 종균 배양, 전 배양, 본 배양시 12시간에서 가장 생장성이 좋으며, 세포외소포의 수득률 또한 높은 것을 확인하였다(실시예 2 참조).In another embodiment of the present invention, a culture process optimization process was performed to increase the yield of Lactobacillus paracasei secreted extracellular vesicles, and the best growth was achieved at 12 hours in seed culture, pre-culture, and main culture, and extracellular It was confirmed that the yield of vesicles was also high (see Example 2).
본 발명의 또 다른 실시예에서는, 락토바실러스 파라카제이 분비 세포외소포의 수율을 높이기 위하여 농축 공정 최적화 과정을 진행하였으며, 한외여과막 100 kDa 사이즈로 농축시 세포외소포의 수득률이 높아지고, TNF-α 분비 저해능 또한 높게 나타나는 것을 확인하였다(실시예 3 참조).In another embodiment of the present invention, a concentration process optimization process was performed to increase the yield of extracellular vesicles secreted by Lactobacillus paracasei. When concentrated with an ultrafiltration membrane of 100 kDa size, the yield of extracellular vesicles was increased, and TNF-α It was confirmed that the secretion inhibition ability was also high (see Example 3).
본 발명의 또 다른 실시예에서는, 최적의 배양 배지, 배양 시간 및 농축 공정을 적용하여 락토바실러스 분비 세포외소포의 대량생산 공정을 수립하였다(실시예 4 참조).In another example of the present invention, a mass production process of Lactobacillus secreted extracellular vesicles was established by applying the optimal culture medium, culture time, and concentration process (see Example 4).
본 발명의 또 다른 실시예에서는, 본 발명의 세포외소포 대량생산 공정을 통해 생산된 세포외소포의 특성을 확인하였다. 그 결과, 소포외소포의 성상, pH, 단백질 농도 및 주요 단백질 발현 패턴이 확인되었으며, 평균 116 nm 크기의 1 mg/mL당 평균 2.38 x 1012개의 입자가 확인 되었다. 또한, TNF-α 분비 저해능 시험을 통하여 분리된 세포외소포가 TNF-α 분비 저해 효과가 있는 것을 확인하였으며, 세포 독성 시험에서 독성이 없는 것으로 확인되었다(실시예 5 참조).In another example of the present invention, the characteristics of extracellular vesicles produced through the mass production process of extracellular vesicles of the present invention were confirmed. As a result, the properties, pH, protein concentration, and major protein expression patterns of extravesicular vesicles were confirmed, and an average of 2.38 x 10 12 particles per 1 mg/mL with an average size of 116 nm were confirmed. In addition, through a test for inhibition of TNF-α secretion, it was confirmed that the isolated extracellular vesicles had an inhibitory effect on TNF-α secretion, and were confirmed to be non-toxic in a cytotoxicity test (see Example 5).
이에, 본 발명은 하기 단계를 포함하는 수율이 향상된 유산균 유래 세포외소포의 제조방법을 제공한다. Accordingly, the present invention provides a method for producing extracellular vesicles derived from lactic acid bacteria with improved yield, comprising the following steps.
(a) 독성 및 동물 유래 성분이 없고 식용가능한 유산균 배양용 배지를 제조하는 단계; (a) preparing a culture medium for lactic acid bacteria that is free of toxic and animal-derived ingredients and is edible;
(b) 상기 (a) 단계에서 제조된 배지에 유산균을 배양하는 단계; (b) cultivating lactic acid bacteria in the medium prepared in step (a);
(c) 상기 (b) 단계의 배양액을 원심분리하여 상층액을 분리하는 단계; (c) centrifuging the culture medium of step (b) to separate the supernatant;
(d) 상기 (c) 단계의 상층액을 농축하고, 완충용액으로 희석하여 재농축하는 단계; 및 (d) concentrating the supernatant of step (c) and re-concentrating it by diluting it with a buffer solution; and
(e) 상기 (d) 단계의 농축액을 필터로 여과하여 세포외소포를 수득하는 단계.(e) filtering the concentrate from step (d) through a filter to obtain extracellular vesicles.
본 발명에 있어서, 상기 유산균은 락토바실러스(Lactobacillus), 락토코커스(Lactococcus), 또는 비피도박테리움(Bifidobacterium) 속 세균일 수 있으나, 이에 제한되지 않는다.In the present invention, the lactic acid bacteria may be bacteria of the genus Lactobacillus , Lactococcus , or Bifidobacterium , but are not limited thereto.
본 발명에 있어서, 상기 락토바실러스(Lactobacillus) 속 세균은 락토바실러스 브레비스(Lactobacillus brevis), 락토바실러스 아세톨레란스(Lactobacillus acetotolerans), 락토바실러스 아시디프리내(Lactobacillus acidifarinae), 락토바실러스 아시디피시스(Lactobacillus acidipiscis), 락토바실러스 아길리스(Lactobacillus agilis), 락토바실러스 알기두스(Lactobacillus algidus), 락토바실러스 알리먼타러스(Lactobacillus alimentarus), 락토바실러스 아밀로리티쿠스(Lactobacillus amylolyticus), 락토바실러스 아밀로트로피쿠스(Lactobacillus amylotrophicus), 락토바실러스 아밀로보러스(Lactobacillus amylovorus), 락토바실러스 아니말리스(Lactobacillus animalis), 락토바실러스 안트리(Lactobacillus antri), 락토바실러스 아포데미(Lactobacillus apodemi), 락토바실러스 아비아리우스(Lactobacillus aviarius), 락토바실러스 비퍼멘탄스(Lactobacillus bifermentans), 락토바실러스 봄피콜라(Lactobacillus bombicola), 락토바실러스 부흐너(Lactobacillus buchneri), 락토바실러스 카멜리애(Lactobacillus camelliae), 락토바실러스 카세이(Lactobacillus casei), 락토바실러스 카트나포미스(Lactobacillus catenaformis), 락토바실러스 세티(Lactobacillus ceti), 락토바실러스 콜레오호미니스(Lactobacillus coleohominis), 락토바실러스 콜리노이데스(Lactobacillus collinoides), 락토바실러스 콤포스티(Lactobacillus composti), 락토바실러스 콘카부스(Lactobacillus concavus), 락토바실러스 코리니포미스(Lactobacillus coryniformis), 락토바실러스 크리스파투스(Lactobacillus crispatus), 락토바실러스 크러스토룸(Lactobacillus crustorum), 락토바실러스 커바투스(Lactobacillus curvatus), 락토바실러스 델브루엑키(Lactobacillus delbruecki), 락토바실러스 덱스트리니쿠스(Lactobacillus dextrinicus), 락토바실러스 디올리보란스(Lactobacillus diolivorans), 락토바실러스 에퀴(Lactobacillus equi), 락토바실러스 에퀴제네로시(Lactobacillus equigenerosi), 락토바실러스 파라기니스(Lactobacillus farraginis), 락토바실러스 파르시미니스(Lactobacillus farciminis), 락토바실러스 퍼먼텀(Lactobacillus fermentum), 락토바실러스 포시니칼리스(Lactobacillus fornicalis), 락토바실러스 프룩티보란스(Lactobacillus fructivorans), 락토바실러스 프루먼티(Lactobacillus frumenti), 락토바실러스 푸흐엔시스(Lactobacillus fuchuensis), 락토바실러스 갈리나룸(Lactobacillus gallinarum), 락토바실러스 가세리(Lactobacillus gasseri), 락토바실러스 가스트리쿠스(Lactobacillus gastricus), 락토바실러스 강엔시스(Lactobacillus ghanensis), 락토바실러스 그라미니스(Lactobacillus graminis), 락토바실러스 하메시(Lactobacillus hammesii), 락토바실러스 하메스테리(Lactobacillus hamsteri), 락토바실러스 하비넨시스(Lactobacillus harbinensis), 락토바실러스 하야키텐시스(Lactobacillus hayakitensis), 락토바실러스 헬베티쿠스(Lactobacillus helveticus), 락토바실러스 힐가르디(Lactobacillus hilgardii), 락토바실러스 호모히오키(Lactobacillus homohiochii), 락토바실러스 이네르스(Lactobacillus iners), 락토바실러스 인글루비에이(Lactobacillus ingluviei), 락토바실러스 인테스티날리스(Lactobacillus intestinalis), 락토바실러스 젠세니(Lactobacillus jensenii), 락토바실러스 존소니(Lactobacillus johnsonii), 락토바실러스 칼릭센시스(Lactobacillus kalixensis), 락토바실러스 케피라노파시엔스(Lactobacillus kefiranofaciens), 락토바실러스 케피리(Lactobacillus kefiri), 락토바실러스 김치(Lactobacillus kimchii), 락토바실러스 키타사토니스(Lactobacillus kitasatonis), 락토바실러스 쿤키이(Lactobacillus kunkeei), 락토바실러스 레이크만니이(Lactobacillus leichmannii), 락토바실러스 린드네리(Lactobacillus lindneri), 락토바실러스 말레페르멘탄스(Lactobacillus malefermentans), 락토바실러스 말리(Lactobacillus mali), 락토바실러스 만니호티보란스(Lactobacillus manihotivorans), 락토바실러스 민덴시스(Lactobacillus mindensis), 락토바실러스 무코새(Lactobacillus mucosae), 락토바실러스 뮤리너스(Lactobacillus murinus), 락토바실러스 나겔리(Lactobacillus nagelii), 락토바실러스 나무렌시스(Lactobacillus namurensis), 락토바실러스 난텐시스(Lactobacillus nantensis), 락토바실러스 올리고퍼멘탄스(Lactobacillus oligofermentans), 락토바실러스 오리스(Lactobacillus oris), 락토바실러스 파니스(Lactobacillus panis), 락토바실러스 판테리스(Lactobacillus pantheris), 락토바실러스 파라브레비스(Lactobacillus parabrevis), 락토바실러스 파라부흐너(Lactobacillus parabuchneri), 락토바실러스 파라카제이(Lactobacillus paracasei), 락토바실러스 파라콜리노이데스(Lactobacillus paracollinoides), 락토바실러스 파라파라기니스(Lactobacillus parafarraginis), 락토바실러스 파라케피리(Lactobacillus parakefiri), 락토바실러스 파라리먼타리우스(Lactobacillus paralimentarius), 락토바실러스 파라플란타룸(Lactobacillus paraplantarum), 락토바실러스 펜토수스(Lactobacillus pentosus), 락토바실러스 페롤렌스(Lactobacillus perolens), 락토바실러스 폰티스(Lactobacillus pontis), 락토바실러스 프로텍투스(Lactobacillus protectus), 락토바실러스 시터씨(Lactobacillus psittaci), 락토바실러스 렌니니(Lactobacillus rennini), 락토바실러스 루테리(Lactobacillus reuteri), 락토바실러스 리매(Lactobacillus rimae), 락토바실러스 로고새(Lactobacillus rogosae), 락토바실러스 로지애(Lactobacillus rossiae), 락토바실러스 루미니스(Lactobacillus ruminis), 락토바실러스 새림네리(Lactobacillus saerimneri), 락토바실러스 사케이(Lactobacillus sakei), 락토바실러스 살리바리우스(Lactobacillus salivarius), 락토바실러스 샌프란시센시스(Lactobacillus sanfranciscensis), 락토바실러스 사츠멘시스(Lactobacillus satsumensis), 락토바실러스 세칼리필루스(Lactobacillus secaliphilus), 락토바실러스 사피애(Lactobacillus sharpeae), 락토바실러스 실리기니스(Lactobacillus siliginis), 락토바실러스 스피체리(Lactobacillus spicheri), 락토바실러스 수에비쿠스(Lactobacillus suebicus), 락토바실러스 타일랜덴시스(Lactobacillus thailandensis), 락토바실러스 울투넨시스(Lactobacillus ultunensis), 락토바실러스 백시노스터쿠스(Lactobacillus vaccinostercus), 락토바실러스 바기날리스(Lactobacillus vaginalis), 락토바실러스 버스몰덴시스(Lactobacillus versmoldensis), 락토바실러스 비니(Lactobacillus vini), 락토바실러스 비툴리누스(Lactobacillus vitulinus), 락토바실러스 지애(Lactobacillus zeae), 또는 락토바실러스 지매(Lactobacillus zymae)일 수 있으나, 이에 제한되지 않는다.In the present invention, the bacteria of the Lactobacillus genus include Lactobacillus brevis , Lactobacillus acetolerans , Lactobacillus acidifarinae , and Lactobacillus acidifisis. acidipiscis ), Lactobacillus agilis, Lactobacillus algidus, Lactobacillus alimentarus , Lactobacillus amylolyticus , Lactobacillus amylotropicus ( Lactobacillus amylotrophicus , Lactobacillus amylovorus , Lactobacillus animalis , Lactobacillus antri , Lactobacillus apodemi, Lactobacillus aviarius , Lactobacillus bifermentans, Lactobacillus bombicola, Lactobacillus buchneri, Lactobacillus camelliae , Lactobacillus casei , Lactobacillus catnapomis ( Lactobacillus catenaformis ), Lactobacillus ceti , Lactobacillus coleohominis, Lactobacillus collinoides , Lactobacillus composti , Lactobacillus concavus , Lactobacillus coryniformis , Lactobacillus crispatus , Lactobacillus crustorum, Lactobacillus curvatus , Lactobacillus delbruecki , Lactobacillus dextrinicus , Lactobacillus diolivorans , Lactobacillus equi , Lactobacillus equigenerosi , Lactobacillus farraginis , Lactobacillus farciminis, Lactobacillus fermentum , Lactobacillus fornicalis , Lactobacillus fructivorans , Lactobacillus frumenti , Lactobacillus Lactobacillus fuchuensis , Lactobacillus gallinarum , Lactobacillus gasseri , Lactobacillus gastricus , Lactobacillus ghanensis , Lactobacillus graminis ( Lactobacillus graminis , Lactobacillus hammesii, Lactobacillus hamsteri , Lactobacillus harbinensis , Lactobacillus hayakitensis, Lactobacillus helveticus helveticus ), Lactobacillus hilgardii , Lactobacillus homohiochii , Lactobacillus iners, Lactobacillus ingluviei , Lactobacillus internalis ( Lactobacillus ) intestinalis ), Lactobacillus jensenii , Lactobacillus johnsonii , Lactobacillus kalixensis, Lactobacillus kefiranofaciens , Lactobacillus kefiri ), Lactobacillus kimchii , Lactobacillus kitasatonis, Lactobacillus kunkeei, Lactobacillus leichmannii , Lactobacillus lindneri , Lactobacillus Lactobacillus malefermentans, Lactobacillus mali , Lactobacillus manihotivorans, Lactobacillus mindensis , Lactobacillus mucosae, Lactobacillus murinus ( Lactobacillus murinus ), Lactobacillus nagelii , Lactobacillus namurensis, Lactobacillus nantensis , Lactobacillus oligofermentans , Lactobacillus oris , Lactobacillus panis , Lactobacillus pantheris, Lactobacillus parabrevis, Lactobacillus parabuchneri, Lactobacillus paracasei , Lactobacillus para. Lactobacillus paracollinoides , Lactobacillus parafarraginis, Lactobacillus parakefiri , Lactobacillus paralimentarius , Lactobacillus paraplantarum , Lactobacillus pentosus, Lactobacillus perolens, Lactobacillus pontis , Lactobacillus protectus , Lactobacillus psittaci , Lactobacillus rennini ( Lactobacillus rennin i), Lactobacillus reuteri, Lactobacillus rimae , Lactobacillus rogosae , Lactobacillus rossiae, Lactobacillus ruminis , Lactobacillus Lactobacillus saerimneri , Lactobacillus sakei , Lactobacillus salivarius, Lactobacillus sanfranciscensis , Lactobacillus satsumensis , Lactobacillus secaliphile Lactobacillus secaliphilus , Lactobacillus sharpeae , Lactobacillus siliginis , Lactobacillus spicheri, Lactobacillus suebicus , Lactobacillus tylandensis ( Lactobacillus thailandensis , Lactobacillus ultunensis , Lactobacillus vaccinostercus , Lactobacillus vaginalis , Lactobacillus versmoldensis, Lactobacillus vini ), Lactobacillus vitulinus , Lactobacillus zeae , or Lactobacillus zymae , but is not limited thereto.
본 발명에 있어서, 상기 락토코커스(Lactococcus) 속 세균은 락토코커스 중앙젠시스(Lactococcus chungangensis), 락토코커스 포모센시스(Lactococcus formosensis), 락토코커스 푸지엔시스(Lactococcus fujiensis) 락토코커스 히르시락티스(Lactococcus hircilactis), 락토코커스 갈비에(Lactococcus garvieae), 락토코커스 락티스(Lactococcus lactis), 락토코커스 라우덴시스(Lactococcus laudensis), 락토코커스 내슈티터미티스(Lactococcus nasutitermitis), 락토코커스 피시엄(Lactococcus piscium), 락토코커스 플란타룸(Lactococcus plantarum), 락토코커스 라피노락티스(Lactococcus raffinolactis), 또는 락토코커스 타이와네시스(Lactococcus taiwanensis)일 수 있으나, 이에 제한되지 않는다.In the present invention, the bacteria of the Lactococcus genus are Lactococcus chungangensis , Lactococcus formosensis , Lactococcus fujiensis , and Lactococcus hirsylactis. hircilactis ), Lactococcus garvieae , Lactococcus lactis , Lactococcus laudensis , Lactococcus nasutitermitis , Lactococcus piscium , Lactococcus plantarum , lacto It may be Lactococcus raffinolactis , or Lactococcus taiwanensis , but is not limited thereto.
본 발명에 있어서, 상기 비피도박테리움(Bifidobacterium) 속 세균은 비피도박테리움 악티노코로니포르메(Bifidobacterium actinocoloniiforme), 비피도박테리움 아돌레신티스(Bifidobacterium adolescentis), 비피도박테리움 애미리아눔(Bifidobacterium aemilianum), 비피도박테리움 애로필럼(Bifidobaterium aerophilum), 비피도박테리움 애스쿨라피(Bifidobaterium aesculapii), 비피도박테리움 아마조넨스(Bifidobaterium amazonense), 비피도박테리움 안굴라툼(Bifidobaterium angulatum), 비피도박테리움 애니말리스(Bifidobacterium animalis), 비피도박테리움 안세리스(Bifidobaterium anseris), 비피도박테리움 아포우시아(Bifidobaterium apousia), 비피도박테리움 아프리(Bifidobaterium apri), 비피도박테리움 아퀴케피리(Bifidobaterium aquikefiri), 비피도박테리움 아스테로이데스(Bifidobaterium asteroides), 비피도박테리움 아베사니(Bifidobaterium avesanii), 비피도박테리움 비아바티(Bifidobaterium biavatii), 비피도박테리움 비피덤(Bifidobacterium bifidum), 비피도박테리움 보헤미쿰(Bifidobaterium bohemicum), 비피도박테리움 봄비(Bifidobaterium bombi), 비피도박테리움 보움(Bifidobaterium boum), 비피도박테리움 브레브(Bifidobacterium breve), 비피도박테리움 칼리미코니스(Bifidobaterium callimiconis), 비피도박테리움 칼리트리키다룸(Bifidobaterium callitrichidarum), 비피도박테리움 알리트리코스(Bifidobaterium allitrichos), 비피도박테리움 카니스(Bifidobaterium canis), 비피도박테리움 카스토리스(Bifidobaterium castoris), 비피도박테리움 카테눌라툼(Bifidobaterium catenulatum), 비피도박테리움 카툴로룸(Bifidobaterium catulorum), 비피도박테리움 세비다룸(Bifidobaterium cebidarum), 비피도박테리움 코에리눔(Bifidobaterium choerinum), 비피도박테리움 콜라도하비탄스(Bifidobaterium choladohabitans), 비피도박테리움 콜로에피(Bifidobaterium choloepi), 비피도박테리움 콜로비(Bifidobaterium colobi), 비피도박테리움 콤문(Bifidobaterium commune), 비피도박테리움 크리세티(Bifidobaterium criceti), 비피도박테리움 크루딜락티스(Bifidobaterium crudilactis), 비피도박테리움 쿠니쿨리(Bifidobaterium cuniculi), 비피도박테리움 덴티움(Bifidobaterium dentium), 비피도박테리움 돌리코티디스(Bifidobaterium dolichotidis), 비피도박테리움 에릭소니(Bifidobaterium eriksonii), 비피도박테리움 에리트로세비(Bifidobaterium erythrocebi), 비피도박테리움 을레무리스(Bifidobaterium eulemuris), 비피도박테리움 패칼리(Bifidobaterium faecale), 비피도박테리움 펠시네움(Bifidobaterium felsineum), 비피도박테리움 칼리쿰(Bifidobaterium gallicum), 비피도박테리움 갈리나룸(Bifidobaterium gallinarum), 비피도박테리움 글로보숨(Bifidobaterium globosum), 비피도박테리움 괼디(Bifidobaterium goeldii), 비피도박테리움 하팔리(Bifidobaterium hapali), 비피도박테리움 인디쿰(Bifidobaterium indicum), 비피도박테리움 이탈리쿰(Bifidobaterium italicum), 비피도박테리움 자치(Bifidobaterium jacchi), 비피도박테리움 레무룸(Bifidobaterium lemurum), 비피도박테리움 레오토피테치(Bifidobaterium leontopitheci), 비피도박테리움 롱검(Bifidobacterium longum), 비피도박테리움 마그눔(Bifidobaterium magnum), 비피도박테리움 마골레시(Bifidobaterium margollesii), 비피도박테리움 메리시쿰(Bifidobaterium merycicum), 비피도박테리움 미코니스(Bifidobaterium miconis), 비피도박테리움 미코니사젠카티(Bifidobaterium miconisargentati), 비피도박테리움 미니뭄(Bifidobaterium minimum), 비피도박테리움 몽골리엔스(Bifidobaterium mongoliense), 비피도박테리움 모라비엔스(Bifidobaterium moraviense), 비피도박테리움 모우칼라벤스(Bifidobaterium moukalabense), 비피도박테리움 미오소티스(Bifidobaterium myosotis), 비피도박테리움 오이디포디스(Bifidobaterium oedipodis), 비피도박테리움 올로무센스(Bifidobaterium olomucense), 비피도박테리움 파노스(Bifidobaterium panos), 비피도박테리움 파마에(Bifidobaterium parmae), 비피도박테리움 플라티리노룸(Bifidobaterium platyrrhinorum), 비피도박테리움 플러비알리실배(Bifidobaterium pluvialisilvae), 비피도박테리움 폴리사카롤리티쿰(Bifidobaterium polysaccharolyticum), 비피도박테리움 폰고니스(Bifidobaterium pongonis), 비피도박테리움 포르시눔(Bifidobaterium porcinum), 비피도박테리움 프리마티움(Bifidobaterium primatium), 비피도박테리움 슈도카테눌라툼(Bifidobaterium pseudocatenulatum), 비피도박테리움 슈도롱검(Bifidobacterium pseudolongum), 비피도박테리움 사이크래로필룸(Bifidobaterium psychraerophilum), 비피도박테리움 풀로룸(Bifidobaterium pullorum), 비피도박테리움 라모숨(Bifidobaterium ramosum), 비피도박테리움 루테리(Bifidobaterium reuteri), 비피도박테리움 루세티(Bifidobaterium rousetti), 비피도박테리움 루미날리(Bifidobaterium ruminale), 비피도박테리움 루미난티움(Bifidobaterium ruminantium), 비피도박테리움 산구이니(Bifidobaterium saguini), 비피도박테리움 산구이니비콜로리스(Bifidobaterium saguinibicoloris), 비피도박테리움 사이미리시우레(Bifidobaterium saimiriisciurei), 비피도박테리움 사미리(Bifidobaterium samirii), 비피도박테리움 산틸라넨스(Bifidobaterium santillanense), 비피도박테리움 스칼리제룸(Bifidobaterium scaligerum), 비피도박테리움 스카도비(Bifidobaterium scardovii), 비피도박테리움 시미아룸(Bifidobaterium simiarum), 비피도박테리움 시미벤트리스(Bifidobaterium simiiventris), 비피도박테리움 스텔렌보센스(Bifidobaterium stellenboschense), 비피도박테리움 서틸리(Bifidobaterium subtile), 비피도박테리움 써마시도필룸(Bifidobaterium thermacidophilum), 비피도박테리움 써모필룸(Bifidobaterium thermophilum), 비피도박테리움 티비그라눌리(Bifidobaterium tibiigranuli), 비피도박테리움 티시에리(Bifidobaterium tissieri), 비피도박테리움 쑤루미엔스(Bifidobaterium tsurumiense), 비피도박테리움 유리날리스(Bifidobaterium urinalis), 비피도박테리움 반신데레니(Bifidobaterium vansinderenii), 비피도박테리움 베스페틸리오니스(Bifidobaterium vespertilionis), 또는 비피도박테리움 자이로코패(Bifidobaterium xylocopae)일 수 있으나, 이에 제한되지 않는다.In the present invention, the bacteria of the Bifidobacterium genus include Bifidobacterium actinocoloniiforme , Bifidobacterium adolescentis , and Bifidobacterium amyrianum ( Bifidobacterium aemilianum ), Bifidobacterium aerophilum , Bifidobaterium aesculapii, Bifidobacterium amazonense , Bifidobacterium angulatum , Bifidobacterium animalis , Bifidobacterium anseris, Bifidobaterium apousia , Bifidobaterium apri , Bifidobacterium aquiche Bifidobaterium aquikefiri , Bifidobacterium asteroides , Bifidobacterium avesanii , Bifidobaterium biavatii , Bifidobacterium bifidum , Bifidobacterium bohemicum, Bifidobacterium bombi, Bifidobaterium boum , Bifidobacterium breve , Bifidobacterium calimico Nice ( Bifidobaterium callimiconis ), Bifidobaterium callitrichidarum , Bifidobaterium allitrichos, Bifidobaterium canis , Bifidobaterium castoris ), Bifidobaterium catenulatum , Bifidobaterium catulorum , Bifidobacterium cebidarum , Bifidobacterium choerinum , Bifidobacterium Bifidobaterium choladohabitans , Bifidobacterium choloepi, Bifidobaterium colobi, Bifidobaterium commune , Bifidobacterium chryseti ( Bifidobaterium criceti ), Bifidobaterium crudilactis , Bifidobaterium cuniculi , Bifidobaterium dentium , Bifidobaterium dolichotidis , Bifidobacterium eriksonii , Bifidobaterium erythrocebi, Bifidobaterium eulemuris , Bifidobaterium faecale , Bifidobacterium Bifidobaterium felsineum , Bifidobaterium gallicum , Bifidobaterium gallinarum , Bifidobaterium globosum , Bifidobaterium goeldii ), Bifidobaterium hapali , Bifidobaterium indicum, Bifidobacterium italicum, Bifidobacterium jacchi , Bifidobacterium re. Murum ( Bifidobaterium lemurum ), Bifidobaterium leontopitheci , Bifidobacterium longum , Bifidobacterium magnum, Bifidobaterium margollesii , Bifidobacterium merycicum , Bifidobaterium miconis, Bifidobaterium miconisargentati , Bifidobacterium minimum , Bifidobacterium Mongoliense ( Bifidobaterium mongoliense ), Bifidobaterium moraviense , Bifidobaterium moukalabense , Bifidobaterium myosotis , Bifidobacterium oidipo Dis ( Bifidobaterium oedipodis ), Bifidobaterium olomucense ( Bifidobaterium olomucense ), Bifidobaterium panos , Bifidobaterium parmae ( Bifidobaterium parmae ), Bifidobacterium platirinorum ( Bifidobaterium platyrrhinorum ), Bifidobaterium pluvialisilvae , Bifidobaterium polysaccharolyticum, Bifidobaterium pongonis , Bifidobaterium porcinum ), Bifidobaterium primatium , Bifidobaterium pseudocatenulatum , Bifidobacterium pseudolongum , Bifidobacterium psychraerophilum , Bifidobacterium pullorum , Bifidobacterium ramosum, Bifidobaterium reuteri , Bifidobaterium rousetti , Bifidobacterium luminali ( Bifidobaterium ruminale ), Bifidobaterium ruminantium ( Bifidobaterium ruminantium ), Bifidobacterium saguini ( Bifidobaterium saguini ), Bifidobaterium saguinibicoloris ( Bifidobaterium saguinibicoloris ), Bifidobacterium cymyriciure ( Bifidobaterium saimiriisciurei ), Bifidobacterium samirii ( Bifidobaterium samirii ), Bifidobaterium santillanense ( Bifidobaterium santillanense ), Bifidobaterium scaligerum ( Bifidobaterium scaligerum ), Bifidobacterium scardovii ( Bifidobaterium scardovii ) , Bifidobacterium simiarum, Bifidobaterium simiiventris , Bifidobaterium stellenboschense , Bifidobaterium subtile , Bifidobacterium Bifidobaterium thermacidophilum , Bifidobaterium thermophilum , Bifidobaterium tibiigranuli , Bifidobaterium tissieri , Bifidobacterium su Bifidobaterium tsurumiense, Bifidobaterium urinalis , Bifidobaterium vansinderenii , Bifidobaterium vespertilionis , or Bifidobacterium It may be, but is not limited to, Bifidobaterium xylocopae .
본 발명에 있어서, “세포외소포(extracellular vesicle)” 또는 “소포(vesicle)”란 다양한 세균에서 분비되는 나노 크기의 막으로 된 구조물을 의미하며, 본 발명에서는 락토바실러스 파라카제이를 포함하는 유산균으로부터 자연적으로 분비되거나, 인공적으로 생산된 막으로 된 모든 구조물을 총칭한다. In the present invention, “extracellular vesicle” or “vesicle” refers to a nano-sized membrane structure secreted by various bacteria, and in the present invention, lactic acid bacteria including Lactobacillus paracasei It refers collectively to all structures made of membranes that are naturally secreted or artificially produced.
본 발명에 있어서, 상기 세포외소포는 평균 직경이 10 nm 내지 1000 nm, 10 nm 내지 900 nm, 10 nm 내지 800 nm, 10 nm 내지 700 nm, 10 nm 내지 600 nm, 10 nm 내지 500 nm, 10 nm 내지 400 nm, 10 nm 내지 300 nm, 또는 10 nm 내지 200 nm일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the extracellular vesicles have an average diameter of 10 nm to 1000 nm, 10 nm to 900 nm, 10 nm to 800 nm, 10 nm to 700 nm, 10 nm to 600 nm, 10 nm to 500 nm, 10 It may be from 10 nm to 400 nm, 10 nm to 300 nm, or 10 nm to 200 nm, but is not limited thereto.
본 발명에 있어서, “배양”이란 미생물을 적당히 인공적으로 조절한 환경 조건에서 생육시키기 위하여 수행하는 모든 행위를 의미하며, 본 발명에서는 “발효”를 포함하는 개념이다.In the present invention, “cultivation” refers to all actions performed to grow microorganisms in appropriately artificially controlled environmental conditions, and in the present invention, it is a concept that includes “fermentation.”
본 발명에 있어서, 상기 “수율이 향상된 세포외소포의 제조방법”이란 동일한 용량의 유산균 배양액으로부터 분리된 세포외소포 중 단백질 농도 및 함량이 상대적으로 높은 세포외소포를 제조하는 것을 의미하며, 본 발명에서 수율이 향상된 세포외소포의 단백질의 농도는 2 mg/mL 내지 5 mg/mL일 수 있으며, 단백질의 함량은 2 g/L 내지 5 g/L 일 수 있고, 나노파티클 개수는 100 nm 내지 150 nm 크기의 나노파티클 1 x 1012 particles/mL 내지 4 x 1012 particles/mL일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the “method for producing extracellular vesicles with improved yield” refers to producing extracellular vesicles with relatively high protein concentration and content among extracellular vesicles isolated from the same volume of lactic acid bacteria culture medium, and the present invention The protein concentration of extracellular vesicles with improved yield may be 2 mg/mL to 5 mg/mL, the protein content may be 2 g/L to 5 g/L, and the number of nanoparticles may be 100 nm to 150 nm. Nanoparticles of nm size may be 1 x 10 12 particles/mL to 4 x 10 12 particles/mL, but are not limited thereto.
본 발명에 있어서, 상기 (a) 단계의 배지는 식용 불가 성분인 구연산 암모늄 및 동물 유래 성분인 소고기 추출물을 포함하지 않을 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the medium in step (a) may not contain ammonium citrate, which is an inedible ingredient, and beef extract, which is an animal-derived ingredient, but is not limited thereto.
본 발명에 있어서, 상기 배지는 식용가능한 락토바실러스 배양 배지인 EMP(Edible MRS) 일 수 있으며, 포도당, 대두 펩톤, 효모 추출물, 초산 나트륨, 제2인산칼륨, 황산 마그네슘, 황산 망간, 구연산, 폴리소르베이트80 등을 포함 할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the medium may be EMP (Edible MRS), which is an edible Lactobacillus culture medium, and contains glucose, soy peptone, yeast extract, sodium acetate, potassium dibasic phosphate, magnesium sulfate, manganese sulfate, citric acid, and polysor. It may include bait 80, etc., but is not limited thereto.
본 발명에 있어서, 상기 포도당(Glucose)은 상기 배지에서 순수(Deionized water) 1 L 당 5 g 내지 50 g, 10 g 내지 45 g, 10 g 내지 40 g, 10 g 내지 35 g, 10 g 내지 30 g, 15 g 내지 40 g, 15 g 내지 35 g, 15 g 내지 30 g 또는 20 g 내지 30 g의 포도당을 포함할 수 있으나, 이에 제한되지 않는다.In the present invention, the glucose is 5 g to 50 g, 10 g to 45 g, 10 g to 40 g, 10 g to 35 g, and 10 g to 30 g per 1 L of deionized water in the medium. g, 15 g to 40 g, 15 g to 35 g, 15 g to 30 g, or 20 g to 30 g of glucose, but is not limited thereto.
본 발명에 있어서, 상기 대두 펩톤(Soy peptone)은 상기 배지에서 순수(Deionized water) 1 L 당 1 g 내지 10 g, 1 g 내지 9 g, 1 g 내지 8 g, 1 g 내지 7 g, 2 g 내지 10g, 2 g 내지 9 g, 2 g 내지 8 g, 2 g 내지 7 g, 3 g 내지 10 g, 3 g 내지 9 g, 3 g 내지 8 g, 3 g 내지 7 g 또는 4 g 내지 6 g의 대두 펩톤을 포함할 수 있으나, 이에 제한되지 않는다.In the present invention, the soy peptone is used in the medium at an amount of 1 g to 10 g, 1 g to 9 g, 1 g to 8 g, 1 g to 7 g, and 2 g per 1 L of deionized water. to 10 g, 2 g to 9 g, 2 g to 8 g, 2 g to 7 g, 3 g to 10 g, 3 g to 9 g, 3 g to 8 g, 3 g to 7 g or 4 g to 6 g. It may include, but is not limited to, soy peptone.
본 발명에 있어서, 상기 효모 추출물(Yeast extract)은 상기 배지에서 순수(Deionized water) 1 L 당 1 g 내지 30 g, 3 g 내지 27 g, 3 g 내지 25 g, 3 g 내지 22 g, 3 g 내지 20 g, 3 g 내지 18 g, 5 g 내지 30 g, 5 g 내지 27 g, 5 g 내지 25 g, 5 g 내지 22 g, 5 g 내지 20 g, 7 g 내지 30 g, 7 g 내지 27 g, 7 g 내지 25 g, 7 g 내지 22 g, 7 g 내지 20 g, 10 g 내지 30 g, 10 g 내지 27 g, 10 g 내지 25 g, 10 g 내지 22 g 또는 10 g 내지 20 g의 효모 추출물을 포함할 수 있으나, 이에 제한되지 않는다.In the present invention, the yeast extract is 1 g to 30 g, 3 g to 27 g, 3 g to 25 g, 3 g to 22 g, 3 g per 1 L of deionized water in the medium. to 20 g, 3 g to 18 g, 5 g to 30 g, 5 g to 27 g, 5 g to 25 g, 5 g to 22 g, 5 g to 20 g, 7 g to 30 g, 7 g to 27 g, 7 g to 25 g, 7 g to 22 g, 7 g to 20 g, 10 g to 30 g, 10 g to 27 g, 10 g to 25 g, 10 g to 22 g or 10 g to 20 g. It may include, but is not limited to, yeast extract.
본 발명에 있어서, 상기 초산 나트륨(Sodium acetate)은 상기 배지에서 순수(Deionized water) 1 L 당 1 g 내지 10 g, 1 g 내지 9 g, 1 g 내지 8 g, 1 g 내지 7 g, 2 g 내지 10g, 2 g 내지 9 g, 2 g 내지 8 g, 2 g 내지 7 g, 3 g 내지 10 g, 3 g 내지 9 g, 3 g 내지 8 g, 3 g 내지 7 g 또는 4 g 내지 6 g의 초산 나트륨을 포함할 수 있으나, 이에 제한되지 않는다.In the present invention, the amount of sodium acetate is 1 g to 10 g, 1 g to 9 g, 1 g to 8 g, 1 g to 7 g, and 2 g per 1 L of deionized water in the medium. to 10 g, 2 g to 9 g, 2 g to 8 g, 2 g to 7 g, 3 g to 10 g, 3 g to 9 g, 3 g to 8 g, 3 g to 7 g or 4 g to 6 g. It may include, but is not limited to, sodium acetate.
본 발명에 있어서, 상기 제2인산칼륨(Dipotassium phosphate)은 상기 배지에서 순수(Deionized water) 1 L 당 0.1 g 내지 5 g, 0.5 g 내지 5 g, 0.5 g 내지 4.5 g, 0.5 g 내지 4 g, 0.5 g 내지 3.5 g, 0.5 g 내지 3 g, 1 g 내지 5 g, 1 g 내지 4.5 g, 1 g 내지 4 g, 1 g 내지 3.5 g 또는 1 g 내지 3 g의 제2인산칼륨을 포함할 수 있으나, 이에 제한되지 않는다.In the present invention, the amount of dipotassium phosphate is 0.1 g to 5 g, 0.5 g to 5 g, 0.5 g to 4.5 g, 0.5 g to 4 g per 1 L of deionized water in the medium. 0.5 g to 3.5 g, 0.5 g to 3 g, 1 g to 5 g, 1 g to 4.5 g, 1 g to 4 g, 1 g to 3.5 g or 1 g to 3 g of potassium phosphate dibasic. However, it is not limited to this.
본 발명에 있어서, 상기 황산 마그네슘(Magnesium sulfate)은 상기 배지에서 순수(Deionized water) 1 L 당 0.01 g 내지 1 g, 0.03 g 내지 1 g, 0.03 g 내지 0.7 g, 0.03 g 내지 0.5 g, 0.03 g 내지 0.3 g, 0.05 g 내지 1 g, 0.05 g 내지 0.7 g, 0.05 g 내지 0.5 g, 0.07 g 내지 1 g, 0.07 g 내지 0.7 g, 0.07 g 내지 0.5 g 또는 0.07 g 내지 0.3 g의 황산 마그네슘을 포함할 수 있으나, 이에 제한되지 않는다.In the present invention, the magnesium sulfate (Magnesium sulfate) is 0.01 g to 1 g, 0.03 g to 1 g, 0.03 g to 0.7 g, 0.03 g to 0.5 g, 0.03 g per 1 L of pure water (Deionized water) in the medium. comprising from 0.3 g to 0.3 g, from 0.05 g to 1 g, from 0.05 g to 0.7 g, from 0.05 g to 0.5 g, from 0.07 g to 1 g, from 0.07 g to 0.7 g, from 0.07 g to 0.5 g or from 0.07 g to 0.3 g of magnesium sulfate. It can be done, but is not limited to this.
본 발명에 있어서, 상기 황산 망간(Manganese sulfate)은 상기 배지에서 순수(Deionized water) 1 L 당 0.01 g 내지 0.1 g, 0.02 g 내지 0.1 g, 0.02 g 내지 0.09 g, 0.02 g 내지 0.08 g, 0.02 g 내지 0.07 g, 0.03 g 내지 0.1 g, 0.03 g 내지 0.09 g, 0.03 g 내지 0.08 g, 0.03 g 내지 0.07 g 또는 0.04 g 내지 0.06 g 의 황산 망간을 포함할 수 있으나, 이에 제한되지 않는다.In the present invention, the amount of manganese sulfate is 0.01 g to 0.1 g, 0.02 g to 0.1 g, 0.02 g to 0.09 g, 0.02 g to 0.08 g, 0.02 g per 1 L of deionized water in the medium. It may include, but is not limited to, 0.07 g, 0.03 g to 0.1 g, 0.03 g to 0.09 g, 0.03 g to 0.08 g, 0.03 g to 0.07 g, or 0.04 g to 0.06 g of manganese sulfate.
본 발명에 있어서, 상기 구연산(Citric acid monohydrate)은 상기 배지에서 순수(Deionized water) 1 L 당 0.1 g 내지 5 g, 0.5 g 내지 5 g, 0.5 g 내지 4.5 g, 0.5 g 내지 4 g, 0.5 g 내지 3.5 g, 0.5 g 내지 3 g, 1 g 내지 5 g, 1 g 내지 4.5 g, 1 g 내지 4 g, 1 g 내지 3.5 g 또는 1 g 내지 3 g의 구연산을 포함할 수 있으나, 이에 제한되지 않는다.In the present invention, the citric acid (Citric acid monohydrate) is 0.1 g to 5 g, 0.5 g to 5 g, 0.5 g to 4.5 g, 0.5 g to 4 g, 0.5 g per 1 L of pure water (Deionized water) in the medium. It may include, but is not limited to, citric acid, from 3.5 g to 3.5 g, 0.5 g to 3 g, 1 g to 5 g, 1 g to 4.5 g, 1 g to 4 g, 1 g to 3.5 g, or 1 g to 3 g. No.
본 발명에 있어서, 폴리소르베이트80(Tween 80)은 상기 배지에서 순수(Deionized water) 1 L 당 0.1 g 내지 10 g, 0.3 g 내지 10 g, 0.3 g 내지 7 g, 0.3 g 내지 5 g, 0.3 g 내지 3 g, 0.5 g 내지 10 g, 0.5 g 내지 7 g, 0.5 g 내지 5 g, 0.7 g 내지 10 g, 0.7 g 내지 7 g, 0.7 g 내지 5 g 또는 0.7 g 내지 3 g의 황산 마그네슘을 포함할 수 있으나, 이에 제한되지 않는다.In the present invention, polysorbate 80 (Tween 80) is 0.1 g to 10 g, 0.3 g to 10 g, 0.3 g to 7 g, 0.3 g to 5 g, 0.3 g per 1 L of pure water (Deionized water) in the medium. g to 3 g, 0.5 g to 10 g, 0.5 g to 7 g, 0.5 g to 5 g, 0.7 g to 10 g, 0.7 g to 7 g, 0.7 g to 5 g or 0.7 g to 3 g of magnesium sulfate. It may include, but is not limited to this.
본 발명에 있어서, 상기 (b) 단계는 종균 배양, 전 배양 및 본 배양의 순서로 각각 5시간 내지 20시간, 5시간 내지 18시간, 5시간 내지 16시간, 5시간 내지 14시간, 8시간 내지 20시간, 8시간 내지 18시간, 8시간 내지 16시간, 8시간 내지 14시간, 10시간 내지 20시간, 10시간 내지 18시간, 10시간 내지 16시간 또는 10시간 내지 14시간 배양할 수 있으나, 이제 제한되지 않는다.In the present invention, step (b) is performed in the order of seed culture, pre-culture, and main culture, respectively, for 5 hours to 20 hours, 5 hours to 18 hours, 5 hours to 16 hours, 5 hours to 14 hours, and 8 hours to 8 hours. You can incubate for 20 hours, 8 hours to 18 hours, 8 hours to 16 hours, 8 hours to 14 hours, 10 hours to 20 hours, 10 hours to 18 hours, 10 hours to 16 hours, or 10 hours to 14 hours, but now Not limited.
본 발명에 있어서, 상기 (c) 단계에서는 배양액을 원심분리하여 상층액을 분리하고 균체를 제거하는 과정을 수행할 수 있다.In the present invention, in step (c), the culture medium can be centrifuged to separate the supernatant and remove the bacterial cells.
본 발명에 있어서, 상기 (d) 단계는 30 내지 180 kDa, 40 내지 180 kDa, 50 내지 180 kDa, 60 내지 180 kDa, 70 내지 180 kDa, 30 내지 170 kDa, 40 내지 170 kDa, 50 내지 170 kDa, 60 내지 170 kDa, 70 내지 170 kDa, 30 내지 160 kDa, 40 내지 160 kDa, 50 내지 160 kDa, 60 내지 160 kDa, 70 내지 160 kDa, 30 내지 150 kDa, 40 내지 150 kDa, 50 내지 150 kDa, 60 내지 150 kDa, 70 내지 150 kDa, 30 내지 140 kDa, 40 내지 140 kDa, 50 내지 140 kDa, 60 내지 140 kDa, 70 내지 140 kDa, 30 내지 130 kDa, 40 내지 130 kDa, 50 내지 130 kDa, 60 내지 130 kDa 또는 70 내지 130 kDa 사이즈 한외여과막을 사용하여 농축 할 수 있으나, 이에 제한되지 않는다.In the present invention, step (d) is 30 to 180 kDa, 40 to 180 kDa, 50 to 180 kDa, 60 to 180 kDa, 70 to 180 kDa, 30 to 170 kDa, 40 to 170 kDa, 50 to 170 kDa. , 60 to 170 kDa, 70 to 170 kDa, 30 to 160 kDa, 40 to 160 kDa, 50 to 160 kDa, 60 to 160 kDa, 70 to 160 kDa, 30 to 150 kDa, 40 to 150 kDa, 50 to 150 kDa , 60 to 150 kDa, 70 to 150 kDa, 30 to 140 kDa, 40 to 140 kDa, 50 to 140 kDa, 60 to 140 kDa, 70 to 140 kDa, 30 to 130 kDa, 40 to 130 kDa, 50 to 130 kDa , it can be concentrated using a 60 to 130 kDa or 70 to 130 kDa size ultrafiltration membrane, but is not limited thereto.
본 발명에 있어서, 상기 (e) 단계의 필터는 0.01 μm 내지 1 μm, 0.05 μm 내지 1 μm, 0.05 μm 내지 0.8 μm, 0.05 μm 내지 0.5 μm, 0.05 μm 내지 0.3 μm, 0.1 μm 내지 1 μm, 0.1 μm 내지 0.8 μm, 0.1 μm 내지 0.5 μm 또는 0.1 μm 내지 0.3 μm 사이즈 필터일 수 있으나, 이에 제한되지 않는다. In the present invention, the filter in step (e) is 0.01 μm to 1 μm, 0.05 μm to 1 μm, 0.05 μm to 0.8 μm, 0.05 μm to 0.5 μm, 0.05 μm to 0.3 μm, 0.1 μm to 1 μm, 0.1 μm to 1 μm. It may be a size filter of μm to 0.8 μm, 0.1 μm to 0.5 μm, or 0.1 μm to 0.3 μm, but is not limited thereto.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.
[실시예][Example]
실시예 1. 배양 배지 선정Example 1. Selection of culture medium
본 발명의 수율이 향상된 세포외소포(extracellular vesicle; EV) 생산에 있어, 균주인 락토바실러스 파라카제이(Lactobacillus paracasei)의 배양 최적 조건을 찾기 위하여, 기본 배지인 LB(Luria-Bertani) 배지, 락토바실러스 배양 배지인 MRS(De Man Rogosa and Sharpe) 배지 및 식용가능한 락토바실러스 배양 배지인 EMP(Edible MRS)에서 배양 시, 분리된 세포외소포의 수율 및 독성을 비교 분석 하였다.In the production of extracellular vesicles (EV) with improved yield of the present invention, in order to find the optimal culture conditions for the strain Lactobacillus paracasei , the basic medium, LB (Luria-Bertani) medium, Lactobacillus The yield and toxicity of isolated extracellular vesicles were compared and analyzed when cultured in MRS (De Man Rogosa and Sharpe) medium, a Bacillus culture medium, and EMP (Edible MRS), an edible Lactobacillus culture medium.
1-1. LB, MRS 및 EMP 배지 조성1-1. LB, MRS and EMP medium composition
본 발명의 LB, MRS 및 EMP 배지는 하기의 표 1 내지 표 3의 조성 및 조성비로 제조하여 사용하였다. The LB, MRS and EMP media of the present invention were prepared and used according to the compositions and composition ratios shown in Tables 1 to 3 below.
<LB 배지 조성><LB medium composition>
No.No. 성분명Ingredient name g/Lg/L
1One
Tryptone (트립톤)Tryptone 1010
22 Yeast extract (효모 추출물) Yeast extract 55
33 Sodium chloride (염화 나트륨) Sodium chloride 1010
44 Agar (한천)Agar 1515
<MRS 배지 조성><MRS medium composition>
No.No. 성분명Ingredient name g/Lg/L
1 One Proteose peptone (프로테오스 펩톤)Proteose peptone 1010
22 Beef extract (소고기 추출물) Beef extract 1010
33 Yeast extract (효모 추출물) Yeast extract 55
44 Dextrose (덱스트로즈)Dextrose 2020
55 Sorbitan monooleate (소르비탄 모노올레이트)Sorbitan monooleate 1One
66 Ammonium citrate (구연산 암모늄) Ammonium citrate 22
77 Sodium acetate (초산 나트륨) Sodium acetate 55
88 Manganese sulfate (황산 망간)Manganese sulfate 0.050.05
99 Disodium phosphate (제2인산나트륨) Disodium phosphate 22
<EMP 배지 조성><EMP medium composition>
No.No. 성분명Ingredient name g/Lg/L
1One Glucose (포도당)Glucose 2525
22 Soy peptone (대두 펩톤) Soy peptone 55
33 Yeast extract (효모 추출물)Yeast extract 1515
44 Sodium acetate (초산 나트륨) Sodium acetate 55
55 Dipotassium phosphate (제2인산칼륨) Dipotassium phosphate 22
66 Magnesium sulfate (황산 마그네슘)Magnesium sulfate 0.10.1
77 Manganese sulfate (황산 망간)Manganese sulfate 0.050.05
88 Citric acid monohydrate (구연산) Citric acid monohydrate 22
99 Tween 80 (폴리소르베이트80)Tween 80 (polysorbate 80) 1One
1-2. 수율 확인1-2. Check yield
락토바실러스 파라카제이 분비 세포외소포를 분리하기 위하여, 락토바실러스 파라카제이를 상기 실시예 1-1의 조성으로 제조된 LB, MRS 및 EMP 배지에 각각 접종하고, 37 ℃, 200 rpm으로 설정하여 12시간 배양하였다. 그리고 균체가 포함되어 있는 배양액을 회수하여 10,000 x g로 상온에서 연속원심분리하여 균체가 제거된 상층액을 획득하였다. 획득된 상층액은 다시 0.22 μm의 필터를 이용하여 여과시키고, 여과시킨 상층액은 100 kDa Pellicon 2 Cassette 필터 멤브레인(Merck Millipore)과 MasterFlex pump system(Cole-Parmer)을 이용하여 50 mL 이하의 부피로 농축하였다. 농축시킨 상층액은 다시 0.22 μm의 필터를 이용하여 여과시켜, 락토바실러스 파라카제이 유래 소포를 분리하였다. 상층액에 포함되어 있는 단백질의 양은 Pierce BCA Protein Assay kit(Thermo Fisher Scientific)를 이용하여 측정하였다.In order to isolate Lactobacillus paracasei secreted extracellular vesicles, Lactobacillus paracasei was inoculated into LB, MRS and EMP media prepared with the composition of Example 1-1, respectively, and set at 37°C and 200 rpm. Cultured for 12 hours. Then, the culture medium containing the bacterial cells was recovered and continuously centrifuged at 10,000 x g at room temperature to obtain a supernatant from which the bacterial cells were removed. The obtained supernatant was filtered again using a 0.22 μm filter, and the filtered supernatant was reduced to a volume of 50 mL or less using a 100 kDa Pellicon 2 Cassette filter membrane (Merck Millipore) and MasterFlex pump system (Cole-Parmer). Concentrated. The concentrated supernatant was filtered again using a 0.22 μm filter to separate Lactobacillus paracasei-derived vesicles. The amount of protein contained in the supernatant was measured using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific).
그 결과, 하기 표 4에 나타난 바와 같이, 4 L 배양 기준 EMP 배지에서 배양한 락토바실러스 파라카제이에서 세포외소포의 수득량 및 총 단백질량이 가장 높은 것으로 확인되었다.As a result, as shown in Table 4 below, it was confirmed that the yield of extracellular vesicles and the total amount of protein were the highest in Lactobacillus paracasei cultured in EMP medium based on 4 L culture.
배지 종류Badge Type 단백질 농도 (mg/ml)Protein concentration (mg/ml) 수득량 (ml)Yield (ml) 총 단백질 (mg)Total protein (mg)
LBLB 0.770.77 5151 39.2739.27
MRSM.R.S. 2.42.4 4242 100.8100.8
EMPEMP 2.062.06 5454 111.24111.24
1-3. 세포 독성 평가1-3. Cytotoxicity evaluation
상기 실시예 1-2의 방법으로 분리된 락토바실러스 파라카제이 분비 세포외소포의 세포 독성 유무를 판단하기 위한 실험을 진행하였다.An experiment was conducted to determine the cytotoxicity of Lactobacillus paracasei secreted extracellular vesicles isolated by the method of Example 1-2.
마우스 대식세포(Raw264.7)에 LB, MRS 및 EMP 배지에서 배양하여 분리된 락토바실러스 파라카제이 유래 세포외소포(MDH-001)를 각각 0.1, 1, 10 μg/mL 농도로 처리한 후 세포의 생존능을 OD(optical density)로 측정하였다.Mouse macrophages (Raw264.7) were cultured in LB, MRS, and EMP media and treated with Lactobacillus paracasei-derived extracellular vesicles (MDH-001) at concentrations of 0.1, 1, and 10 μg/mL, respectively. The viability was measured by OD (optical density).
그 결과, 도 1에서 나타난 바와 같이, 시험물질을 처리하지 않은 음성대조군 (negative control)을 기준으로 LB 및 EMP 배양군에서는 80% 이상의 세포 생존능을 보였으나, MRS 배양군 10 μg/mL 농도에서는 세포독성이 확인되었다.As a result, as shown in Figure 1, the LB and EMP culture groups showed a cell viability of more than 80% based on the negative control that was not treated with the test substance, but at a concentration of 10 μg/mL in the MRS culture group, the cells Toxicity was confirmed.
실시예 2. 배양 공정 최적화Example 2. Culture process optimization
본 발명의 락토바실러스 파라카제이 분비 세포외소포(MDH-001)의 파일럿 생산을 위한 배양 공정 최적화 과정을 진행하였다. A culture process optimization process was conducted for pilot production of Lactobacillus paracasei secreted extracellular vesicles (MDH-001) of the present invention.
최대 용량 100 L의 발효조를 이용하여 대량 생산을 진행하기 위하여 2번의 계대 배양을 통한 배양법이 적용되었다. 각 배양 단계의 계대 접종량은 대상 배지 총량의 1/100으로 정하였고, 각 배양 단계별 배양 시간별 Colony froming unit (CFU), Optical Density(OD)를 측정하여 성장 패턴을 분석하였다. In order to proceed with mass production using a fermenter with a maximum capacity of 100 L, a culture method using two subcultures was applied. The sub-inoculation amount for each culture step was set at 1/100 of the total amount of target medium, and the growth pattern was analyzed by measuring colony forming unit (CFU) and optical density (OD) for each culture time at each culture step.
성장 패턴 분석 결과, 도 2에 나타난 바와 같이 초기 활성 배양 시간이 12시간인 것으로 확인되어, 종균 배양 시간을 12시간으로 설정하였다. As a result of growth pattern analysis, it was confirmed that the initial active culture time was 12 hours, as shown in Figure 2, and the seed culture time was set to 12 hours.
이후 전배양 단계에서는 활성 배양액을 계대하여 진행하였고, 마찬가지로 접종 후 두시간 마다 배양액을 채취하여 CFU와 OD를 측정하여 성장 패턴을 분석하였다.Afterwards, in the pre-culture stage, the active culture was passaged, and similarly, the culture was collected every two hours after inoculation and the growth pattern was analyzed by measuring CFU and OD.
그 결과, 도 3에 나타난 바와 같이 두 번의 반복시험을 통하여 생장성이 가장 좋다고 판단되는 시간인 12시간을 전배양 시간으로 설정하였다.As a result, as shown in Figure 3, 12 hours, which was the time judged to have the best growth potential through two repeated tests, was set as the pre-culture time.
배양의 최종 단계인 본배양 단계에서는 50 L 배양을 진행하였다. 상기와 같이 대상 배양액 총량의 1/100에 해당하는 500 mL의 배양액을 계대 접종하였고, 3시간 마다 배양액을 체취하여 CFU와 OD를 측정하여 성장 패턴을 분석하였다. In the main culture stage, which is the final stage of culture, 50 L culture was performed. As described above, 500 mL of culture medium, equivalent to 1/100 of the total amount of the target culture medium, was sub-inoculated, and the culture medium was collected every 3 hours to measure CFU and OD to analyze the growth pattern.
그 결과, 도 4에 나타난 바와 같이 두 번의 반복시험을 통하여 CFU와 OD를 종합하여 볼 때, 접종 후 12시간에서 가장 높은 성장이 확인되었고, 이를 바탕으로 본배양 시간을 12시간으로 설정하였다.As a result, as shown in Figure 4, when looking at CFU and OD through two repeated tests, the highest growth was confirmed at 12 hours after inoculation, and based on this, the main culture time was set at 12 hours.
또한, 각 배양 시간별 배양액 내에 존재하는 세포외소포를 추출하여 시간대별 세포외소포의 수득량을 확인한 결과, 하기 표 5에 나타난 바와 같이, 12시간에서 2.67 mg/ml, 단백질 총량으로는 98.79 mg 으로 가장 높은 수득률을 보임을 확인하였다.In addition, as a result of extracting extracellular vesicles present in the culture medium for each culture time and confirming the yield of extracellular vesicles by time period, as shown in Table 5 below, the amount was 2.67 mg/ml at 12 hours, and the total amount of protein was 98.79 mg. It was confirmed that it showed the highest yield.
Lot No.Lot No. 단백질 농도protein concentration
(mg/mL)(mg/mL) 		수득량yield
(mL)(mL) 		총 단백질 양total amount of protein
(mg)(mg)
MDH-001-CM-200808_Z (0 hr)MDH-001-CM-200808_Z (0 hr) 1.481.48 3737 54.7654.76
MDH-001-CM-200808_Z (3 hr)MDH-001-CM-200808_Z (3 hrs) 1.781.78 3737 65.8665.86
MDH-001-CM-200808_Z (6 hr)MDH-001-CM-200808_Z (6 hrs) 2.042.04 3737 75.4875.48
MDH-001-CM-200808_Z (9 hr)MDH-001-CM-200808_Z (9 hrs) 2.592.59 3737 95.8395.83
MDH-001-CM-200808_Z (12 hr)MDH-001-CM-200808_Z (12 hrs) 2.672.67 3737 98.7998.79
MDH-001-CM-200808_Z (15 hr)MDH-001-CM-200808_Z (15 hrs) 2.082.08 3737 76.9676.96
실시예 3. 농축 공정 최적화Example 3. Concentration process optimization
본 발명의 락토바실러스 파라카제이 분비 세포외소포(MDH-001)의 파일럿 생산을 위한 농축 공정을 최적화하기 위하여 한외여과(UF, Ultrafiltration) 조건을 확립하였다.Ultrafiltration (UF) conditions were established to optimize the concentration process for pilot production of Lactobacillus paracasei secreted extracellular vesicles (MDH-001) of the present invention.
8 L의 EMP 배지를 사용하여 상기 실시예 1-2와 동일한 방법으로 배양 및 제균 공정을 수행하였다. 균체가 제거된 상층액은 두 가지 사이즈 50 kDa 및 100 kDa의 한외여과막(Ultrafiltration membrane)을 이용하여 50 mL 이하의 부피로 농축하였다. 농축시킨 상층액은 다시 0.22 μm의 필터를 이용하여 여과시켜, 락토바실러스 파라카제이 유래 소포를 분리하였다. 상층액에 포함되어 있는 단백질의 양은 Pierce BCA Protein Assay kit(Thermo Fisher Scientific)를 이용하여 측정하였다.Cultivation and sterilization processes were performed in the same manner as Example 1-2 using 8 L of EMP medium. The supernatant from which the bacteria were removed was concentrated to a volume of 50 mL or less using ultrafiltration membranes of two sizes, 50 kDa and 100 kDa. The concentrated supernatant was filtered again using a 0.22 μm filter to separate Lactobacillus paracasei-derived vesicles. The amount of protein contained in the supernatant was measured using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific).
한외여과막 사이즈에 따른 세포외소포의 수득량을 확인한 결과, 하기 표 6에 나타난 바와 같이, 100 kDa이 50 kDa에서보다 평균 수득률(총 단백질량)이 높은 것으로 확인되었다. As a result of checking the yield of extracellular vesicles according to the ultrafiltration membrane size, it was confirmed that the average yield (total protein amount) of 100 kDa was higher than that of 50 kDa, as shown in Table 6 below.
UF membrane sizeUF membrane size Lot No.Lot No. 단백질 농도protein concentration
(mg/mL)(mg/mL) 		수득량yield
(mL)(mL) 		총 단백질 양total amount of protein
(mg)(mg)
50 kDa50 kDa MDH-001-CM-200214fMDH-001-CM-200214f 2.602.60 3636 93.693.6
MDH-001-CM-200219fMDH-001-CM-200219f 2.702.70 4242 113.4113.4
MDH-001-CM-200221fMDH-001-CM-200221f 2.762.76 3636 99.499.4
100 kDa100 kDa MDH-001-CM-200108fMDH-001-CM-200108f 2.172.17 57.557.5 124.8124.8
MDH-001-CM-200205fMDH-001-CM-200205f 2.882.88 4040 115.2115.2
MDH-001-CM-200212fMDH-001-CM-200212f 2.872.87 3434 97.697.6
한외여과막 사이즈에 따른 세포외소포의 역가를 확인하기 위해 TNF-α 분비 저해능 시험을 진행하였다. 대표적인 염증성 사이토카인으로 알려진 TNF-α(Tumor necrosis factor-α)의 분비량을 효소면역측정법(enzyme-linked immunosorbent assay, ELISA)으로 측정하여, 염증 조절 효력을 평가하였다. A test for inhibition of TNF-α secretion was performed to confirm the titer of extracellular vesicles according to the size of the ultrafiltration membrane. The secretion amount of TNF-α (Tumor necrosis factor-α), known as a representative inflammatory cytokine, was measured using enzyme-linked immunosorbent assay (ELISA) to evaluate its inflammation-modulating effect.
마우스 대식세포(Raw264.7)에 염증유도물질과 50 kDa 사이즈 및 100 kDa 사이즈로 농축한 세포외소포를 각각 0.1, 1, 10 μg/mL 농도로 처리하고, TNF-α의 분비량을 효소면역측정법 (ELISA)으로 측정하였다. Mouse macrophages (Raw264.7) were treated with inflammation-inducing substances and extracellular vesicles concentrated in 50 kDa and 100 kDa sizes at concentrations of 0.1, 1, and 10 μg/mL, respectively, and the secretion amount of TNF-α was measured using enzyme-linked immunosorbent assay. It was measured by (ELISA).
그 결과, 도 5에 나타난 바와 같이, 50 kDa 사이즈로 농축한 세포외소포에 비하여 100 kDa 사이즈로 농축한 세포외소포에서 TNF-α 분비 저해능이 더 높게 나타난 것으로 확인되었다.As a result, as shown in Figure 5, it was confirmed that the ability to inhibit TNF-α secretion was higher in the extracellular vesicles concentrated in the 100 kDa size compared to the extracellular vesicles concentrated in the 50 kDa size.
실시예 4. 세포외소포 대량생산 공정Example 4. Extracellular vesicle mass production process
본 발명에서는 상기 실시예 1 내지 실시예 3의 결과에 따라, 배양 배지 및 배양 시간을 최적화하여 세포외소포의 대량생산 공정에 적용하였다.In the present invention, according to the results of Examples 1 to 3, the culture medium and culture time were optimized and applied to the mass production process of extracellular vesicles.
4-1. 대용량 EMP 배지 제조4-1. Manufacturing of large-capacity EMP badges
대용량의 EMP 배지는 하기의 방법으로 제조하였다.A large volume of EMP medium was prepared by the following method.
먼저, 순수 제조기에 순수를 제조하여 컨테이너에 받아 저장하고, 하기 표 3의조성물 및 조성비에 따라 글루코스를 제외한 다른 성분을 전자저울로 무게를 재어 5 L 플라스틱 비커에 넣었다. 10 L 플라스틱 비커에 순수를 넣고 무게를 잰 성분들을 넣어 녹이고, 순수에 녹인 성분들을 펌프를 이용하여 실리콘 튜브로 발효조 탱크에 채워 넣었다. 컨테이너의 배출구로 순수를 내보내고 배출구 앞에 아크릴 통으로 받아주었다. 50 L 배양 기준 42 L까지 펌프를 이용해 순수가 들어가는 아크릴 통에서 실리콘 튜브로 순수를 탱크 안으로 채워주었다. 발효조 화면의 멸균(Sterilization) 창의 가열 시작(Heat Start)을 누르고, 글루코스는 따로 멸균하여 50 L 배지가 식어 37℃로 안정화되면 투입하였다.First, pure water was prepared in a pure water maker, received and stored in a container, and other ingredients except glucose were weighed on an electronic scale according to the composition and composition ratio in Table 3 below and placed in a 5 L plastic beaker. Pure water was added to a 10 L plastic beaker, the weighed ingredients were dissolved, and the ingredients dissolved in pure water were filled into the fermenter tank using a silicone tube using a pump. Pure water was discharged through the outlet of the container and received into an acrylic container in front of the outlet. Based on a 50 L culture, pure water was filled into the tank from an acrylic tank containing pure water to a volume of 42 L using a silicone tube using a pump. Press Heat Start in the sterilization window on the fermenter screen, and glucose was sterilized separately and added when the 50 L medium cooled and stabilized at 37°C.
4-2. 종균 배양(Activation culture)4-2. Activation culture
발효조(100 L fermenter, FE-101, BIOSYSTEMENG CO.,LTD)에 접종하기 24시간 전에 종균 배양을 시행하였다. Seed culture was performed 24 hours before inoculation into the fermenter (100 L fermenter, FE-101, BIOSYSTEMENG CO.,LTD).
60 mL의 EMP는 접종 2시간 전에 37℃ 인큐베이터에서 보관하였다. -80℃ 초저온 냉장고(deep freezer)에서 락토바실러스 파라카제이 균을 꺼내서 실온 온도로 맞춰주고, 60 mL 튜브에 담긴 EMP 배지를 준비하여, 튜브 입구를 화염 멸균하였다.60 mL of EMP was stored in an incubator at 37°C 2 hours before inoculation. Lactobacillus paracasei was taken out of the -80°C deep freezer, brought to room temperature, EMP medium contained in a 60 mL tube was prepared, and the tube entrance was flame sterilized.
EMP 배지에 락토바실러스 파라카제이 균을 현탁시켜 200 μL를 접종하고, 튜브의 뚜껑을 완전히 잠그고 다시 현탁하였다. 균 접종은 클린벤치(Clean bench)에서 시행하였다. 접종 후 혐기 조건을 유지하기 위하여 튜브에 빈 공간이 있을 경우, EMP 배지를 채워주고, 37±2℃ 온도의 인큐베이터에서 12시간 정치 배양하였다.Lactobacillus paracasei was suspended in EMP medium and 200 μL was inoculated. The tube was completely capped and resuspended. Bacterial inoculation was performed on a clean bench. In order to maintain anaerobic conditions after inoculation, if there was empty space in the tube, EMP medium was filled and cultured in an incubator at a temperature of 37 ± 2°C for 12 hours.
4-3. 전 배양(Seed culture)4-3. Seed culture
발효조에 접종하기 12시간 전에 전 배양을 시행하였다. Pre-culture was performed 12 hours before inoculation into the fermenter.
5 L 발효조에서 3 L EMP 배지를 제조하여 멸균하였다. 5 L 발효조를 설치하여 37℃, 100 rpm으로 설정하고, 상기 실시예 2의 종균 배양 배지 30 mL을 접종구를 통하여 접종하였고, 37±2℃ 온도의 인큐베이터에서 12시간 배양하였다.3 L EMP medium was prepared in a 5 L fermenter and sterilized. A 5 L fermenter was installed and set at 37°C and 100 rpm, 30 mL of the seed culture medium of Example 2 was inoculated through the inoculation port, and cultured in an incubator at a temperature of 37±2°C for 12 hours.
4-4. 본배양(Main culture)4-4. Main culture
클린벤치에서 상기 실시예 3의 전 배양 배양액 2,400 mL을 준비하였고, 500 mL만 듀란 보틀(duran bottle)로 옮겼다.2,400 mL of the pre-culture medium of Example 3 was prepared on a clean bench, and only 500 mL was transferred to a Duran bottle.
100% 에탄올에 적신 솜을 발효조 화염구 홈에 넣고, 실리콘 장갑을 착용하였다. 압력을 0.05로 바꾸고, 화염구를 접종구에 알맞게 설치한 뒤 토치로 불을 점화하였다. 접종구 마개를 열고, 500 mL 배양액이 담긴 듀란 보틀 입구 부분을 화염 멸균 후 신속히 뚜껑을 열어 배양액을 부어 접종을 완료하였다.Cotton soaked in 100% ethanol was placed in the flame hole groove of the fermenter, and silicone gloves were worn. The pressure was changed to 0.05, the flame bulb was installed appropriately in the inoculation hole, and the fire was ignited with a torch. Open the inoculation port cap, sterilize the mouth of the Duran bottle containing 500 mL of culture medium by flame, then quickly open the lid and pour in the culture medium to complete the inoculation.
4-5. 제균 공정4-5. Sterilization process
상기 배양액에서 균체를 제거하기 위하여 원심분리를 이용하여 제균 공정을 시행하였다.To remove bacteria from the culture medium, a sterilization process was performed using centrifugation.
원심분리기 회전 중력가속도를 10,000 xg로 설정하여 가동하고, 정량 펌프(WT600-2J, LONGER Pump)의 속도를 100으로 하여 투입하였다. 원심분리의 상층액은 SUS-상층액 보관통에 받았다.The centrifuge rotation gravity acceleration was set to 10,000 xg and operated, and the speed of the metering pump (WT600-2J, LONGER Pump) was set to 100. The supernatant from centrifugation was placed in a SUS-supernatant storage container.
4-6. 농축 공정4-6. concentration process
연속원심분리의 상층액을 받아 놓은 SUS-상층액 보관통 하단부에 장착되어 있는 퇴수 노즐에 튜브로 농축기(MAXFLOW10, BTR) 탱크 주입구 부분을 연결하고, 농축프로그램(CymonX) P4.1 매뉴얼 모드를 가동하여 탱크에 상층액을 주입하였다. 필터는 100 kDa 사이즈의 한외여과막(Ultrafiltration membrane)을 이용하였다. 농축 공정은 최종 용량(volume) 2,400 mL 까지 농축을 진행하였다. Connect the concentrator (MAXFLOW10, BTR) tank inlet with a tube to the drain nozzle mounted at the bottom of the SUS-supernatant storage tank that received the supernatant from continuous centrifugation, and run the concentration program (CymonX) P4.1 manual mode. The supernatant was injected into the tank. The filter used was an ultrafiltration membrane with a size of 100 kDa. The concentration process was carried out to a final volume of 2,400 mL.
4-7. 최종 제균 공정4-7. Final sterilization process
0.22 μm 바틀탑 필터(Bottle Top Filter, 180C3, Sartorius)를 알코올 스프레이로 살균 후 클린벤치 내부로 이동시켰다. 0.22 μm 바틀탑 필터의 압력 조절구멍에 노즐을 결합하고 흡입관을 연결하였다. 0.22 μm 바틀탑 필터 상단 부의 플라스틱 투명 뚜껑을 열어 내부에 농축된 배양액 150 mL를 넣고, 흡입관을 가동시켜 농축된 배양액을 0.22 μm 바틀탑 필터를 이용하여 여과하였다.A 0.22 μm bottle top filter (180C3, Sartorius) was sterilized with alcohol spray and then moved into the clean bench. The nozzle was coupled to the pressure control hole of the 0.22 μm bottle top filter and the suction pipe was connected. Open the transparent plastic lid at the top of the 0.22 μm bottle top filter, add 150 mL of concentrated culture fluid inside, operate the suction tube, and filter the concentrated culture fluid using the 0.22 μm bottle top filter.
상기 과정을 500 mL의 배양액이 전부 여과될 때까지 반복하고, 0.22 μm 바틀탑 필터의 상단부를 제거 후 바틀의 뚜껑을 닫았다.The above process was repeated until all 500 mL of culture fluid was filtered, the upper part of the 0.22 μm bottle top filter was removed, and the lid of the bottle was closed.
실시예 5. 배양물의 특성 분석Example 5. Characterization of cultures
본 발명의 세포외소포 대량생산 공정을 통해 생산된 세포외소포의 특성을 확인하였다.The characteristics of extracellular vesicles produced through the extracellular vesicle mass production process of the present invention were confirmed.
5-1. 배양물의 동정(Identification) 및 확인5-1. Identification and confirmation of cultures
배양이 완료된 배양액에서 균체를 회수하여 16S rRNA 분석을 진행하여 해당 균주가 맞는지 확인하였으며, 육안으로 성상을 관찰시 본 발명의 세포외소포 MDH-001은 황갈색의 투명한 액체인 것으로 확인되었다.Bacterial cells were recovered from the culture medium after completion of incubation, and 16S rRNA analysis was performed to confirm whether the strain was the correct strain. When observing the properties with the naked eye, the extracellular vesicle MDH-001 of the present invention was confirmed to be a yellow-brown transparent liquid.
5-2. pH 측정5-2. pH measurement
MDH-001은 유산균이 생산한 세포외소포로 배양 되는 과정에서 젖산이 생성되어 약간의 산성을 나타내며, 이를 pH 7.4의 PBS로 세척하여 중화과정을 거치고, 산도를 측정하여 일관되게 배양 되었는지 측정하였다.MDH-001 is an extracellular vesicle produced by lactic acid bacteria. During the culture process, lactic acid is produced and it is slightly acidic. It was washed with PBS at pH 7.4 to neutralize it, and the acidity was measured to determine whether it was cultured consistently.
그 결과, MDH-001을 대한민국약전 일반시험법 pH 측정법 항목에 따라 측정했을 때 pH 7.0±1.0 이내로 확인되었다.As a result, when MDH-001 was measured according to the pH measurement method of the General Test Method in the Korean Pharmacopoeia, the pH was confirmed to be within 7.0 ± 1.0.
5-3. 단백질 정량 분석 (BCA assay)5-3. Protein quantitative analysis (BCA assay)
MDH-001의 함량을 확인하기 위하여 BCA asaay 시험법을 이용하여 단백질 함량을 측정하였다. 생산된 MDH-001 1 mL을 10배 단계 희석한 후 96 well plate에 25 μL를 넣고 반응, 시약 200 μL를 넣고 37℃에서 30분간 반응시킨 후 562 nm에서 흡광도를 측정하였다. 표준 시약인 BSA로 Standard graph를 그려 그래프와 비교하여 농도를 계산하였다. To confirm the content of MDH-001, the protein content was measured using the BCA asaay test method. 1 mL of the produced MDH-001 was serially diluted 10 times, then 25 μL was added to a 96 well plate, 200 μL of reagent was added, reaction was performed at 37°C for 30 minutes, and absorbance was measured at 562 nm. A standard graph was drawn using BSA, a standard reagent, and the concentration was calculated by comparing the graph.
하기 표 7에 나타난 바와 같이, 49개의 배치 시료 분석 결과, 단백질의 농도는 평균 2 내지 4 mg/mL로 확인되었다.As shown in Table 7 below, as a result of analyzing 49 batch samples, the protein concentration was confirmed to be an average of 2 to 4 mg/mL.
No.No. B/T No.B/T No. 단백질 농도 (mg/mL)Protein concentration (mg/mL)
1One MDH-001-CM-210106_ZMDH-001-CM-210106_Z 3.543.54
22 MDH-001-CM-210107_ZMDH-001-CM-210107_Z 3.573.57
33 MDH-001-CM-210108_ZMDH-001-CM-210108_Z 3.863.86
44 MDH-001-CM-210112_ZMDH-001-CM-210112_Z 3.773.77
55 MDH-001-CM-210116_ZMDH-001-CM-210116_Z 3.173.17
66 MDH-001-CM-210117_ZMDH-001-CM-210117_Z 3.323.32
77 MDH-001-CM-210118_ZMDH-001-CM-210118_Z 3.83.8
88 MDH-001-CM-210121_ZMDH-001-CM-210121_Z 3.863.86
99 MDH-001-CM-210126_ZMDH-001-CM-210126_Z 3.953.95
1010 MDH-001-CM-210127_ZMDH-001-CM-210127_Z 3.643.64
1111 MDH-001-CM-210128_ZMDH-001-CM-210128_Z 3.673.67
1212 MDH-001-CM-210129_ZMDH-001-CM-210129_Z 3.043.04
1313 MDH-001-CM-210130_ZMDH-001-CM-210130_Z 3.533.53
1414 MDH-001-CM-210131_ZMDH-001-CM-210131_Z 3.873.87
1515 MDH-001-CM-210201_ZMDH-001-CM-210201_Z 3.973.97
1616 MDH-001-CM-210202_ZMDH-001-CM-210202_Z 3.003.00
1717 MDH-001-CM-210203_ZMDH-001-CM-210203_Z 3.633.63
1818 MDH-001-CM-210204_ZMDH-001-CM-210204_Z 3.943.94
1919 MDH-001-CM-210205_ZMDH-001-CM-210205_Z 3.003.00
2020 MDH-001-CM-210206_ZMDH-001-CM-210206_Z 3.703.70
2121 MDH-001-CM-210209_ZMDH-001-CM-210209_Z 3.853.85
2222 MDH-001-CM-210217_ZMDH-001-CM-210217_Z 3.293.29
2323 MDH-001-CM-210224_ZMDH-001-CM-210224_Z 3.663.66
2424 MDH-001-CM-210304_ZMDH-001-CM-210304_Z 3.433.43
2525 MDH-001-CM-210310_ZMDH-001-CM-210310_Z 3.313.31
2626 MDH-001-CM-210311_ZMDH-001-CM-210311_Z 3.613.61
2727 MDH-001-CM-210315_ZMDH-001-CM-210315_Z 3.223.22
2828 MDH-001-CM-210317_ZMDH-001-CM-210317_Z 3.353.35
2929 MDH-001-CM-210318_ZMDH-001-CM-210318_Z 3.213.21
3030 MDH-001-CM-210324_ZMDH-001-CM-210324_Z 3.233.23
3131 MDH-001-CM-210325_ZMDH-001-CM-210325_Z 3.323.32
3232 MDH-001-CM-210401_ZMDH-001-CM-210401_Z 3.753.75
3333 MDH-001-CM-210407_ZMDH-001-CM-210407_Z 3.693.69
3434 MDH-001-CM-210408_ZMDH-001-CM-210408_Z 3.193.19
3535 MDH-001-CM-210414_ZMDH-001-CM-210414_Z 3.53.5
3636 MDH-001-CM-210415_ZMDH-001-CM-210415_Z 3.843.84
3737 MDH-001-CM-210421_ZMDH-001-CM-210421_Z 3.563.56
3838 MDH-001-CM-210423_ZMDH-001-CM-210423_Z 3.793.79
3939 MDH-001-CM-210428_ZMDH-001-CM-210428_Z 3.743.74
4040 MDH-001-CM-210429_ZMDH-001-CM-210429_Z 3.383.38
4141 MDH-001-CM-210430_ZMDH-001-CM-210430_Z 3.933.93
4242 MDH-001-CM-210505_ZMDH-001-CM-210505_Z 3.543.54
4343 MDH-001-CM-210506_ZMDH-001-CM-210506_Z 3.473.47
4444 MDH-001-CM-210514_ZMDH-001-CM-210514_Z 3.353.35
4545 MDH-001-CM-210519_ZMDH-001-CM-210519_Z 3.553.55
4646 MDH-001-CM-210520_ZMDH-001-CM-210520_Z 3.483.48
4747 MDH-001-CM-210602_ZMDH-001-CM-210602_Z 2.902.90
4848 MDH-001-CM-210603_ZMDH-001-CM-210603_Z 2.702.70
4949 MDH-001-CM-210604_ZMDH-001-CM-210604_Z 2.942.94
5050 MDH-001-CM-210707_ZMDH-001-CM-210707_Z 2.502.50
5151 MDH-001-CM-210805_ZMDH-001-CM-210805_Z 2.552.55
5252 MDH-001-CM-210812_ZMDH-001-CM-210812_Z 2.762.76
5-4. 단백질 발현 패턴 분석 (SDS-PAGE)5-4. Protein expression pattern analysis (SDS-PAGE)
MDH-001의 단백질 발현 패턴을 확인하기 위하여 SDS-PAGE (Sodium dodecyl sulphate polyacrylamide gel electrophoresis) 분석을 진행하였다. 12%의 acrylamide gel을 제조하여 SDS-PAGE 분석을 진행하고 Silver stain을 통해 단백질 패턴을 확인하였다. Bio-Rad 사의 Image Lab 프로그램을 이용해 분석하였다.To confirm the protein expression pattern of MDH-001, SDS-PAGE (Sodium dodecyl sulphate polyacrylamide gel electrophoresis) analysis was performed. A 12% acrylamide gel was prepared, SDS-PAGE analysis was performed, and the protein pattern was confirmed through silver stain. Analysis was performed using Bio-Rad's Image Lab program.
그 결과, 도 6에 나타난 바와 같이, 특성 분석을 통해 관찰된 MDH-001의 주요 다섯 개의 밴드(131 kDa, 94 kDa, 86 kDa, 69 kDa, 45 kDa 부근)가 모두 확인되었다.As a result, as shown in Figure 6, all five major bands (around 131 kDa, 94 kDa, 86 kDa, 69 kDa, and 45 kDa) of MDH-001 observed through characterization were confirmed.
5-5. 입자의 크기 및 개수 확인 (NTA)5-5. Determination of particle size and number (NTA)
MDH-001 내 세포외소포를 확인하기 위해 NTA (Nanoparticle tracking analysis) 분석을 수행하였다. NTA 분석은 입자의 크기에 따라 달라지는 브라운 운동 속도와 광 산란의 특성을 이용하여 나노 분자의 크기 및 분포를 확인하는 분석법으로, BCA 분석을 통해 정량한 단백질 1 mg/mL당 존재하는 나노입자(nano-particle)의 개수를 확인하였다. 본 분석법은 Malvern Panalytical 사의 NS300 기기를 이용하여 particle 개수와 분포를 확인하였다. NTA (Nanoparticle tracking analysis) analysis was performed to identify extracellular vesicles in MDH-001. NTA analysis is an analysis method that determines the size and distribution of nanoparticles using the Brownian motion speed and light scattering characteristics that vary depending on the size of the particle. It measures the nanoparticles (nano) present per 1 mg/mL of protein quantified through BCA analysis. -particle) was confirmed. This analysis method confirmed the number and distribution of particles using Malvern Panalytical's NS300 instrument.
세 배치의 분석 결과, 하기의 표 8에 나타난 바와 같이, 평균 116 nm 크기의 입자(particle)가 제일 많았으며, 1 mg/mL당 평균 2.38 x 1012개의 입자가 확인되었다.As a result of the analysis of three batches, as shown in Table 8 below, particles with an average size of 116 nm were the most frequent, and an average of 2.38 x 10 12 particles were confirmed per 1 mg/mL.
No.No. B/T No.B/T No. NTA 결과NTA Results
mode(nm)mode(nm) Particles(mg/mL)Particles(mg/mL)
1One MDH-001-CM-210602_ZMDH-001-CM-210602_Z 113.5±4.1113.5±4.1 2.82E+122.82E+12
22 MDH-001-CM-210603_ZMDH-001-CM-210603_Z 123.8±6.4123.8±6.4 2.18E+122.18E+12
33 MDH-001-CM-210604_ZMDH-001-CM-210604_Z 113.3±2.1113.3±2.1 2.15E+122.15E+12
5-6. 역가 측정 (TNF-α 분비 저해능 시험)5-6. Potency measurement (TNF-α secretion inhibition ability test)
MDH-001의 생물학적 특성을 평가하기 위해 TNF-α 분비 저해능 시험을 진행하였다. 대표적인 염증성 사이토카인으로 알려진 TNF-α(Tumor necrosis factor-α)의 분비량을 효소면역측정법(enzyme-linked immunosorbent assay, ELISA)으로 측정하여, 염증 억제 능을 평가하였다. To evaluate the biological properties of MDH-001, a test for inhibition of TNF-α secretion was performed. The secretion amount of TNF-α (Tumor necrosis factor-α), known as a representative inflammatory cytokine, was measured using enzyme-linked immunosorbent assay (ELISA) to evaluate the ability to suppress inflammation.
마우스 대식세포(Raw264.7)에 염증유도물질과 MDH-001을 0.1, 1, 10 μg/mL 농도로 처리하고, TNF-α의 분비량을 효소면역측정법 (ELISA)으로 측정하였다. 본 발명에서는 병원성 세포외소포인 E. coli 유래 EV에 의해 분비되는 TNF-α를 억제하는 정도를 측정하여 평가하였다.Mouse macrophages (Raw264.7) were treated with inflammation-inducing substances and MDH-001 at concentrations of 0.1, 1, and 10 μg/mL, and the secretion amount of TNF-α was measured by enzyme-linked immunosorbent assay (ELISA). In the present invention, the degree of inhibition of TNF-α secreted by E. coli- derived EVs, which are pathogenic extracellular vesicles, was measured and evaluated.
그 결과, 하기의 표 9에 나타난 바와 같이, 양성 대조군(positive control)을 기준으로 MDH-001 10 μg/mL 처리군 모두에서 E. coli 유래 EV에 의한 TNF-α의 분비량을 60% 이하로 억제하는 것을 확인하였다.As a result, as shown in Table 9 below, the secretion amount of TNF-α by E. coli- derived EV was suppressed by less than 60% in all MDH-001 10 μg/mL treatment groups based on the positive control. It was confirmed that
No.No. B/T No.B/T No. TNF-α 분비량 (대조군 대비)TNF-α secretion amount (compared to control group)
1One MDH-001-CM-210602_ZMDH-001-CM-210602_Z 19.0%19.0%
22 MDH-001-CM-210603_ZMDH-001-CM-210603_Z 13.8%13.8%
33 MDH-001-CM-210604_ZMDH-001-CM-210604_Z 15.8%15.8%
5-7. 세포독성 평가5-7. Cytotoxicity evaluation
MDH-001의 세포독성 유무를 판단하기 위한 실험을 진행하였다. 세포독성시험을 통해서 시료가 세포에 독성을 나타내는지 여부를 확인할 수 있으며, 본 시험에서 적합성을 보여야, 대식세포의 감소 없이 TNF-α의 분비가 감소하였음을 확인할 수 있다. An experiment was conducted to determine the cytotoxicity of MDH-001. Through a cytotoxicity test, it can be confirmed whether the sample is toxic to cells, and if it shows compatibility in this test, it can be confirmed that the secretion of TNF-α is reduced without a decrease in macrophages.
마우스 대식세포 (Raw264.7)에 MDH-001을 0.1, 1, 10 μg/mL 농도로 처리한 후 세포의 생존능을 OD(optical density)값으로 측정하였다.After treating mouse macrophages (Raw264.7) with MDH-001 at concentrations of 0.1, 1, and 10 μg/mL, cell viability was measured using OD (optical density) values.
그 결과, 하기 표 10에 나타난 바와 같이, 시험물질을 처리하지 않은 음성대조군(negative control)을 기준으로 MDH-001의 처리군에서 80% 이상의 세포 생존능을 보이는 것으로 확인되었다.As a result, as shown in Table 10 below, it was confirmed that the cell viability was greater than 80% in the group treated with MDH-001, based on the negative control group that was not treated with the test substance.
No.No. B/T No.B/T No. 세포 독성cytotoxicity
1One MDH-001-CM-210602_ZMDH-001-CM-210602_Z 적합fitness
22 MDH-001-CM-210603_ZMDH-001-CM-210603_Z 적합fitness
33 MDH-001-CM-210604_ZMDH-001-CM-210604_Z 적합fitness
상기와 같은 결과를 통해, 본 발명의 대량생산 공정으로 생산된 배양물이 세포외소포를 함유하고 있으며, 이는 세포독성이 없이 염증 매개체인 TNF-α 분비를 일관적으로 억제함을 확인하였다.Through the above results, it was confirmed that the culture produced through the mass production process of the present invention contained extracellular vesicles, which consistently suppressed the secretion of TNF-α, an inflammatory mediator, without cytotoxicity.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.
본 발명에 따른 수율이 향상된 유산균 유래 세포외소포의 제조방법은 약리성분으로 활용될 수 있는 유산균 유래 세포외소포를 산업적 규모로 대량 생산하는 공정에 유용하게 이용될 수 있을 것으로 기대되는 바, 산업상 이용가능성이 있다.The method for producing extracellular vesicles derived from lactic acid bacteria with improved yield according to the present invention is expected to be useful in the process of mass producing extracellular vesicles derived from lactic acid bacteria that can be used as pharmacological ingredients on an industrial scale. There is availability.

Claims (13)

  1. 하기 단계를 포함하는 수율이 향상된 유산균 유래 세포외소포의 제조방법:Method for producing extracellular vesicles derived from lactic acid bacteria with improved yield comprising the following steps:
    (a) 독성 및 동물 유래 성분이 없고 식용가능한 유산균 배양용 배지를 제조하는 단계;(a) preparing a culture medium for lactic acid bacteria that is free of toxic and animal-derived ingredients and is edible;
    (b) 상기 (a) 단계에서 제조된 배지에 유산균을 배양하는 단계;(b) cultivating lactic acid bacteria in the medium prepared in step (a);
    (c) 상기 (b) 단계의 배양액을 원심분리하여 상층액을 분리하는 단계;(c) centrifuging the culture medium of step (b) to separate the supernatant;
    (d) 상기 (c) 단계의 상층액을 농축하고, 완충용액으로 희석하여 재농축하는 단계; 및(d) concentrating the supernatant of step (c) and re-concentrating it by diluting it with a buffer solution; and
    (e) 상기 (d) 단계의 농축액을 필터로 여과하여 세포외소포를 수득하는 단계.(e) filtering the concentrate from step (d) through a filter to obtain extracellular vesicles.
  2. 제1항에 있어서,According to paragraph 1,
    상기 배지는 식용 불가 성분인 구연산 암모늄 및 동물 유래 성분인 소고기 추출물을 포함하지 않는 것을 특징으로 하는, 제조방법.The production method is characterized in that the medium does not contain ammonium citrate, which is an inedible ingredient, and beef extract, which is an animal-derived ingredient.
  3. 제1항에 있어서, According to paragraph 1,
    상기 배지는 포도당, 대두 펩톤, 효모 추출물, 초산 나트륨, 제2인산칼륨, 황산 마그네슘, 황산 망간, 구연산 및 폴리소르베이트80을 포함하는 것을 특징으로 하는, 제조방법.The production method is characterized in that the medium contains glucose, soy peptone, yeast extract, sodium acetate, potassium dibasic phosphate, magnesium sulfate, manganese sulfate, citric acid and polysorbate 80.
  4. 제1항에 있어서,According to paragraph 1,
    상기 (b) 단계는 종균 배양, 전 배양 및 본 배양의 순서로 각각 8시간 내지 16시간 배양하는 것을 특징으로 하는, 제조방법.The manufacturing method is characterized in that step (b) is cultured for 8 to 16 hours in the order of seed culture, pre-culture, and main culture, respectively.
  5. 제4항에 있어서,According to paragraph 4,
    상기 (b) 단계는 종균 배양, 전 배양 및 본 배양의 순서로 각각 10시간 내지 14시간 배양하는 것을 특징으로 하는, 제조방법.The manufacturing method is characterized in that step (b) is cultured for 10 to 14 hours each in the order of seed culture, pre-culture, and main culture.
  6. 제1항에 있어서,According to paragraph 1,
    상기 (d) 단계는 50 kDa 내지 150 kDa 사이즈 한외여과막을 사용하여 농축하는 것을 특징으로 하는, 제조방법.The preparation method is characterized in that step (d) is concentrated using an ultrafiltration membrane with a size of 50 kDa to 150 kDa.
  7. 제1항에 있어서,According to paragraph 1,
    상기 (e) 단계의 필터는 0.1 μm 내지 1 μm 사이즈 필터인 것을 특징으로 하는, 제조방법.The manufacturing method, wherein the filter in step (e) is a 0.1 μm to 1 μm size filter.
  8. 제7항에 있어서,In clause 7,
    상기 (e) 단계의 필터는 0.1 μm 내지 0.5 μm 사이즈 필터인 것을 특징으로 하는, 제조방법.The manufacturing method, wherein the filter in step (e) is a 0.1 μm to 0.5 μm size filter.
  9. 제1항에 있어서,According to paragraph 1,
    상기 유산균은 락토바실러스(Lactobacillus), 락토코커스(Lactococcus), 또는 비피도박테리움(Bifidobacterium) 속 세균인 것을 특징으로 하는, 제조방법.A manufacturing method, characterized in that the lactic acid bacteria are bacteria of the genus Lactobacillus , Lactococcus , or Bifidobacterium .
  10. 제9항에 있어서,According to clause 9,
    상기 락토바실러스(Lactobacillus) 속 세균은 락토바실러스 브레비스(Lactobacillus brevis), 락토바실러스 아세톨레란스(Lactobacillus acetotolerans), 락토바실러스 아시디프리내(Lactobacillus acidifarinae), 락토바실러스 아시디피시스(Lactobacillus acidipiscis), 락토바실러스 아길리스(Lactobacillus agilis), 락토바실러스 알기두스(Lactobacillus algidus), 락토바실러스 알리먼타러스(Lactobacillus alimentarus), 락토바실러스 아밀로리티쿠스(Lactobacillus amylolyticus), 락토바실러스 아밀로트로피쿠스(Lactobacillus amylotrophicus), 락토바실러스 아밀로보러스(Lactobacillus amylovorus), 락토바실러스 아니말리스(Lactobacillus animalis), 락토바실러스 안트리(Lactobacillus antri), 락토바실러스 아포데미(Lactobacillus apodemi), 락토바실러스 아비아리우스(Lactobacillus aviarius), 락토바실러스 비퍼멘탄스(Lactobacillus bifermentans), 락토바실러스 봄피콜라(Lactobacillus bombicola), 락토바실러스 부흐너(Lactobacillus buchneri), 락토바실러스 카멜리애(Lactobacillus camelliae), 락토바실러스 카세이(Lactobacillus casei), 락토바실러스 카트나포미스(Lactobacillus catenaformis), 락토바실러스 세티(Lactobacillus ceti), 락토바실러스 콜레오호미니스(Lactobacillus coleohominis), 락토바실러스 콜리노이데스(Lactobacillus collinoides), 락토바실러스 콤포스티(Lactobacillus composti), 락토바실러스 콘카부스(Lactobacillus concavus), 락토바실러스 코리니포미스(Lactobacillus coryniformis), 락토바실러스 크리스파투스(Lactobacillus crispatus), 락토바실러스 크러스토룸(Lactobacillus crustorum), 락토바실러스 커바투스(Lactobacillus curvatus), 락토바실러스 델브루엑키(Lactobacillus delbruecki), 락토바실러스 덱스트리니쿠스(Lactobacillus dextrinicus), 락토바실러스 디올리보란스(Lactobacillus diolivorans), 락토바실러스 에퀴(Lactobacillus equi), 락토바실러스 에퀴제네로시(Lactobacillus equigenerosi), 락토바실러스 파라기니스(Lactobacillus farraginis), 락토바실러스 파르시미니스(Lactobacillus farciminis), 락토바실러스 퍼먼텀(Lactobacillus fermentum), 락토바실러스 포시니칼리스(Lactobacillus fornicalis), 락토바실러스 프룩티보란스(Lactobacillus fructivorans), 락토바실러스 프루먼티(Lactobacillus frumenti), 락토바실러스 푸흐엔시스(Lactobacillus fuchuensis), 락토바실러스 갈리나룸(Lactobacillus gallinarum), 락토바실러스 가세리(Lactobacillus gasseri), 락토바실러스 가스트리쿠스(Lactobacillus gastricus), 락토바실러스 강엔시스(Lactobacillus ghanensis), 락토바실러스 그라미니스(Lactobacillus graminis), 락토바실러스 하메시(Lactobacillus hammesii), 락토바실러스 하메스테리(Lactobacillus hamsteri), 락토바실러스 하비넨시스(Lactobacillus harbinensis), 락토바실러스 하야키텐시스(Lactobacillus hayakitensis), 락토바실러스 헬베티쿠스(Lactobacillus helveticus), 락토바실러스 힐가르디(Lactobacillus hilgardii), 락토바실러스 호모히오키(Lactobacillus homohiochii), 락토바실러스 이네르스(Lactobacillus iners), 락토바실러스 인글루비에이(Lactobacillus ingluviei), 락토바실러스 인테스티날리스(Lactobacillus intestinalis), 락토바실러스 젠세니(Lactobacillus jensenii), 락토바실러스 존소니(Lactobacillus johnsonii), 락토바실러스 칼릭센시스(Lactobacillus kalixensis), 락토바실러스 케피라노파시엔스(Lactobacillus kefiranofaciens), 락토바실러스 케피리(Lactobacillus kefiri), 락토바실러스 김치(Lactobacillus kimchii), 락토바실러스 키타사토니스(Lactobacillus kitasatonis), 락토바실러스 쿤키이(Lactobacillus kunkeei), 락토바실러스 레이크만니이(Lactobacillus leichmannii), 락토바실러스 린드네리(Lactobacillus lindneri), 락토바실러스 말레페르멘탄스(Lactobacillus malefermentans), 락토바실러스 말리(Lactobacillus mali), 락토바실러스 만니호티보란스(Lactobacillus manihotivorans), 락토바실러스 민덴시스(Lactobacillus mindensis), 락토바실러스 무코새(Lactobacillus mucosae), 락토바실러스 뮤리너스(Lactobacillus murinus), 락토바실러스 나겔리(Lactobacillus nagelii), 락토바실러스 나무렌시스(Lactobacillus namurensis), 락토바실러스 난텐시스(Lactobacillus nantensis), 락토바실러스 올리고퍼멘탄스(Lactobacillus oligofermentans), 락토바실러스 오리스(Lactobacillus oris), 락토바실러스 파니스(Lactobacillus panis), 락토바실러스 판테리스(Lactobacillus pantheris), 락토바실러스 파라브레비스(Lactobacillus parabrevis), 락토바실러스 파라부흐너(Lactobacillus parabuchneri), 락토바실러스 파라카제이(Lactobacillus paracasei), 락토바실러스 파라콜리노이데스(Lactobacillus paracollinoides), 락토바실러스 파라파라기니스(Lactobacillus parafarraginis), 락토바실러스 파라케피리(Lactobacillus parakefiri), 락토바실러스 파라리먼타리우스(Lactobacillus paralimentarius), 락토바실러스 파라플란타룸(Lactobacillus paraplantarum), 락토바실러스 펜토수스(Lactobacillus pentosus), 락토바실러스 페롤렌스(Lactobacillus perolens), 락토바실러스 폰티스(Lactobacillus pontis), 락토바실러스 프로텍투스(Lactobacillus protectus), 락토바실러스 시터씨(Lactobacillus psittaci), 락토바실러스 렌니니(Lactobacillus rennini), 락토바실러스 루테리(Lactobacillus reuteri), 락토바실러스 리매(Lactobacillus rimae), 락토바실러스 로고새(Lactobacillus rogosae), 락토바실러스 로지애(Lactobacillus rossiae), 락토바실러스 루미니스(Lactobacillus ruminis), 락토바실러스 새림네리(Lactobacillus saerimneri), 락토바실러스 사케이(Lactobacillus sakei), 락토바실러스 살리바리우스(Lactobacillus salivarius), 락토바실러스 샌프란시센시스(Lactobacillus sanfranciscensis), 락토바실러스 사츠멘시스(Lactobacillus satsumensis), 락토바실러스 세칼리필루스(Lactobacillus secaliphilus), 락토바실러스 사피애(Lactobacillus sharpeae), 락토바실러스 실리기니스(Lactobacillus siliginis), 락토바실러스 스피체리(Lactobacillus spicheri), 락토바실러스 수에비쿠스(Lactobacillus suebicus), 락토바실러스 타일랜덴시스(Lactobacillus thailandensis), 락토바실러스 울투넨시스(Lactobacillus ultunensis), 락토바실러스 백시노스터쿠스(Lactobacillus vaccinostercus), 락토바실러스 바기날리스(Lactobacillus vaginalis), 락토바실러스 버스몰덴시스(Lactobacillus versmoldensis), 락토바실러스 비니(Lactobacillus vini), 락토바실러스 비툴리누스(Lactobacillus vitulinus), 락토바실러스 지애(Lactobacillus zeae), 또는 락토바실러스 지매(Lactobacillus zymae)인 것을 특징으로 하는, 제조방법.The bacteria of the Lactobacillus genus include Lactobacillus brevis , Lactobacillus acetolerans, Lactobacillus acidifrinae , Lactobacillus acidipiscis , and Lactobacillus. Lactobacillus agilis, Lactobacillus algidus, Lactobacillus alimentarus , Lactobacillus amylolyticus , Lactobacillus amylotrophicus, lactobacillus amylotrophicus Bacillus amylovorus , Lactobacillus animalis , Lactobacillus antri, Lactobacillus apodemi , Lactobacillus aviarius , Lactobacillus vifermentans ( Lactobacillus bifermentans ), Lactobacillus bombicola, Lactobacillus buchneri, Lactobacillus camelliae , Lactobacillus casei , Lactobacillus catenaformis , Lactobacillus ceti , Lactobacillus coleohominis, Lactobacillus collinoides, Lactobacillus composti , Lactobacillus concavus , Lactobacillus colini Lactobacillus coryniformis , Lactobacillus crispatus, Lactobacillus crustorum , Lactobacillus curvatus , Lactobacillus delbruecki , Lactobacillus dextrini Lactobacillus dextrinicus , Lactobacillus diolivorans , Lactobacillus equi , Lactobacillus equigenerosi, Lactobacillus farraginis , Lactobacillus parsiminis ( Lactobacillus farciminis ), Lactobacillus fermentum , Lactobacillus fornicalis, Lactobacillus fructivorans , Lactobacillus frumenti , Lactobacillus fuchuensis ), Lactobacillus gallinarum , Lactobacillus gasseri , Lactobacillus gastricus, Lactobacillus ghanensis , Lactobacillus graminis , lactobacillus Bacillus hammesii, Lactobacillus hamsteri, Lactobacillus harbinensis , Lactobacillus hayakitensis , Lactobacillus helveticus , Lactobacillus Lactobacillus hilgardii , Lactobacillus homohiochii , Lactobacillus iners, Lactobacillus ingluviei , Lactobacillus intestinalis , Lactobacillus Lactobacillus jensenii , Lactobacillus johnsonii , Lactobacillus kalixensis, Lactobacillus kefiranofaciens , Lactobacillus kefiri , Lactobacillus kimchi ( Lactobacillus kimchii ), Lactobacillus kitasatonis ( Lactobacillus kunkeei ), Lactobacillus leichmannii ( Lactobacillus leichmannii ) , Lactobacillus lindneri ( Lactobacillus lindneri ), Lactobacillus malefermentans ( Lactobacillus malefermentans , Lactobacillus mali , Lactobacillus manihotivorans , Lactobacillus mindensis , Lactobacillus mucosae, Lactobacillus murinus , Lactobacillus nagelii, Lactobacillus namurensis , Lactobacillus nantensis, Lactobacillus oligofermentans , Lactobacillus oris , Lactobacillus panis ( Lactobacillus panis , Lactobacillus pantheris , Lactobacillus parabrevis , Lactobacillus parabuchneri , Lactobacillus paracasei, Lactobacillus paracholinoides. paracollinoides ), Lactobacillus parafarraginis, Lactobacillus parakefiri , Lactobacillus paralimentarius , Lactobacillus paraplantarum , Lactobacillus pentosus ( Lactobacillus pentosus , Lactobacillus perolens, Lactobacillus pontis , Lactobacillus protectus , Lactobacillus psittaci , Lactobacillus rennin i, Lactobacillus reuteri, Lactobacillus rimae , Lactobacillus rogosae, Lactobacillus rossiae , Lactobacillus ruminis , Lactobacillus saerimneri ), Lactobacillus sakei , Lactobacillus salivarius, Lactobacillus sanfranciscensis , Lactobacillus satsumensis, Lactobacillus secaliphilus , Lactobacillus sharpeae , Lactobacillus siliginis , Lactobacillus spicheri, Lactobacillus suebicus , Lactobacillus thailandensis , lactobacillus Bacillus ultunensis, Lactobacillus vaccinostercus, Lactobacillus vaginalis , Lactobacillus versmoldensis , Lactobacillus vini , Lactobacillus A production method, characterized in that it is Lactobacillus vitulinus, Lactobacillus zeae , or Lactobacillus zymae .
  11. 제9항에 있어서,According to clause 9,
    상기 락토코커스(Lactococcus) 속 세균은 락토코커스 중앙젠시스(Lactococcus chungangensis), 락토코커스 포모센시스(Lactococcus formosensis), 락토코커스 푸지엔시스(Lactococcus fujiensis) 락토코커스 히르시락티스(Lactococcus hircilactis), 락토코커스 갈비에(Lactococcus garvieae), 락토코커스 락티스(Lactococcus lactis), 락토코커스 라우덴시스(Lactococcus laudensis), 락토코커스 내슈티터미티스(Lactococcus nasutitermitis), 락토코커스 피시엄(Lactococcus piscium), 락토코커스 플란타룸(Lactococcus plantarum), 락토코커스 라피노락티스(Lactococcus raffinolactis), 또는 락토코커스 타이와네시스(Lactococcus taiwanensis)인 것을 특징으로 하는, 제조방법.The bacteria of the Lactococcus genus include Lactococcus chungangensis , Lactococcus formosensis , Lactococcus fujiensis, Lactococcus hircilactis , and Lactococcus. Lactococcus garvieae , Lactococcus lactis , Lactococcus laudensis , Lactococcus nasutitermitis , Lactococcus piscium , Lactococcus plantarum , lacto A manufacturing method, characterized in that Lactococcus raffinolactis , or Lactococcus taiwanensis .
  12. 제9항에 있어서,According to clause 9,
    상기 비피도박테리움(Bifidobacterium) 속 세균은 비피도박테리움 악티노코로니포르메(Bifidobacterium actinocoloniiforme), 비피도박테리움 아돌레신티스(Bifidobacterium adolescentis), 비피도박테리움 애미리아눔(Bifidobacterium aemilianum), 비피도박테리움 애로필럼(Bifidobaterium aerophilum), 비피도박테리움 애스쿨라피(Bifidobaterium aesculapii), 비피도박테리움 아마조넨스(Bifidobaterium amazonense), 비피도박테리움 안굴라툼(Bifidobaterium angulatum), 비피도박테리움 애니말리스(Bifidobacterium animalis), 비피도박테리움 안세리스(Bifidobaterium anseris), 비피도박테리움 아포우시아(Bifidobaterium apousia), 비피도박테리움 아프리(Bifidobaterium apri), 비피도박테리움 아퀴케피리(Bifidobaterium aquikefiri), 비피도박테리움 아스테로이데스(Bifidobaterium asteroides), 비피도박테리움 아베사니(Bifidobaterium avesanii), 비피도박테리움 비아바티(Bifidobaterium biavatii), 비피도박테리움 비피덤(Bifidobacterium bifidum), 비피도박테리움 보헤미쿰(Bifidobaterium bohemicum), 비피도박테리움 봄비(Bifidobaterium bombi), 비피도박테리움 보움(Bifidobaterium boum), 비피도박테리움 브레브(Bifidobacterium breve), 비피도박테리움 칼리미코니스(Bifidobaterium callimiconis), 비피도박테리움 칼리트리키다룸(Bifidobaterium callitrichidarum), 비피도박테리움 알리트리코스(Bifidobaterium allitrichos), 비피도박테리움 카니스(Bifidobaterium canis), 비피도박테리움 카스토리스(Bifidobaterium castoris), 비피도박테리움 카테눌라툼(Bifidobaterium catenulatum), 비피도박테리움 카툴로룸(Bifidobaterium catulorum), 비피도박테리움 세비다룸(Bifidobaterium cebidarum), 비피도박테리움 코에리눔(Bifidobaterium choerinum), 비피도박테리움 콜라도하비탄스(Bifidobaterium choladohabitans), 비피도박테리움 콜로에피(Bifidobaterium choloepi), 비피도박테리움 콜로비(Bifidobaterium colobi), 비피도박테리움 콤문(Bifidobaterium commune), 비피도박테리움 크리세티(Bifidobaterium criceti), 비피도박테리움 크루딜락티스(Bifidobaterium crudilactis), 비피도박테리움 쿠니쿨리(Bifidobaterium cuniculi), 비피도박테리움 덴티움(Bifidobaterium dentium), 비피도박테리움 돌리코티디스(Bifidobaterium dolichotidis), 비피도박테리움 에릭소니(Bifidobaterium eriksonii), 비피도박테리움 에리트로세비(Bifidobaterium erythrocebi), 비피도박테리움 을레무리스(Bifidobaterium eulemuris), 비피도박테리움 패칼리(Bifidobaterium faecale), 비피도박테리움 펠시네움(Bifidobaterium felsineum), 비피도박테리움 칼리쿰(Bifidobaterium gallicum), 비피도박테리움 갈리나룸(Bifidobaterium gallinarum), 비피도박테리움 글로보숨(Bifidobaterium globosum), 비피도박테리움 괼디(Bifidobaterium goeldii), 비피도박테리움 하팔리(Bifidobaterium hapali), 비피도박테리움 인디쿰(Bifidobaterium indicum), 비피도박테리움 이탈리쿰(Bifidobaterium italicum), 비피도박테리움 자치(Bifidobaterium jacchi), 비피도박테리움 레무룸(Bifidobaterium lemurum), 비피도박테리움 레오토피테치(Bifidobaterium leontopitheci), 비피도박테리움 롱검(Bifidobacterium longum), 비피도박테리움 마그눔(Bifidobaterium magnum), 비피도박테리움 마골레시(Bifidobaterium margollesii), 비피도박테리움 메리시쿰(Bifidobaterium merycicum), 비피도박테리움 미코니스(Bifidobaterium miconis), 비피도박테리움 미코니사젠카티(Bifidobaterium miconisargentati), 비피도박테리움 미니뭄(Bifidobaterium minimum), 비피도박테리움 몽골리엔스(Bifidobaterium mongoliense), 비피도박테리움 모라비엔스(Bifidobaterium moraviense), 비피도박테리움 모우칼라벤스(Bifidobaterium moukalabense), 비피도박테리움 미오소티스(Bifidobaterium myosotis), 비피도박테리움 오이디포디스(Bifidobaterium oedipodis), 비피도박테리움 올로무센스(Bifidobaterium olomucense), 비피도박테리움 파노스(Bifidobaterium panos), 비피도박테리움 파마에(Bifidobaterium parmae), 비피도박테리움 플라티리노룸(Bifidobaterium platyrrhinorum), 비피도박테리움 플러비알리실배(Bifidobaterium pluvialisilvae), 비피도박테리움 폴리사카롤리티쿰(Bifidobaterium polysaccharolyticum), 비피도박테리움 폰고니스(Bifidobaterium pongonis), 비피도박테리움 포르시눔(Bifidobaterium porcinum), 비피도박테리움 프리마티움(Bifidobaterium primatium), 비피도박테리움 슈도카테눌라툼(Bifidobaterium pseudocatenulatum), 비피도박테리움 슈도롱검(Bifidobacterium pseudolongum), 비피도박테리움 사이크래로필룸(Bifidobaterium psychraerophilum), 비피도박테리움 풀로룸(Bifidobaterium pullorum), 비피도박테리움 라모숨(Bifidobaterium ramosum), 비피도박테리움 루테리(Bifidobaterium reuteri), 비피도박테리움 루세티(Bifidobaterium rousetti), 비피도박테리움 루미날리(Bifidobaterium ruminale), 비피도박테리움 루미난티움(Bifidobaterium ruminantium), 비피도박테리움 산구이니(Bifidobaterium saguini), 비피도박테리움 산구이니비콜로리스(Bifidobaterium saguinibicoloris), 비피도박테리움 사이미리시우레(Bifidobaterium saimiriisciurei), 비피도박테리움 사미리(Bifidobaterium samirii), 비피도박테리움 산틸라넨스(Bifidobaterium santillanense), 비피도박테리움 스칼리제룸(Bifidobaterium scaligerum), 비피도박테리움 스카도비(Bifidobaterium scardovii), 비피도박테리움 시미아룸(Bifidobaterium simiarum), 비피도박테리움 시미벤트리스(Bifidobaterium simiiventris), 비피도박테리움 스텔렌보센스(Bifidobaterium stellenboschense), 비피도박테리움 서틸리(Bifidobaterium subtile), 비피도박테리움 써마시도필룸(Bifidobaterium thermacidophilum), 비피도박테리움 써모필룸(Bifidobaterium thermophilum), 비피도박테리움 티비그라눌리(Bifidobaterium tibiigranuli), 비피도박테리움 티시에리(Bifidobaterium tissieri), 비피도박테리움 쑤루미엔스(Bifidobaterium tsurumiense), 비피도박테리움 유리날리스(Bifidobaterium urinalis), 비피도박테리움 반신데레니(Bifidobaterium vansinderenii), 비피도박테리움 베스페틸리오니스(Bifidobaterium vespertilionis), 또는 비피도박테리움 자이로코패(Bifidobaterium xylocopae)인 것을 특징으로 하는, 제조방법.The bacteria in the Bifidobacterium genus include Bifidobacterium actinocoloniiforme , Bifidobacterium adolescentis , Bifidobacterium aemilianum , and Bifidobacterium aemilianum. Bifidobaterium aerophilum , Bifidobaterium aesculapii, Bifidobaterium amazonense , Bifidobaterium angulatum , Bifidobacterium ani Malis ( Bifidobacterium animalis ), Bifidobacterium anseris ( Bifidobaterium anseris ), Bifidobaterium apousia , Bifidobaterium apri , Bifidobaterium aquikefiri , Bifidobaterium asteroides , Bifidobaterium avesanii , Bifidobaterium biavatii , Bifidobacterium bifidum , Bifidobacterium Bohemicum ( Bifidobaterium bohemicum ), Bifidobaterium bombi , Bifidobaterium boum , Bifidobacterium breve , Bifidobacterium callimiconis , Bifidobacterium callitrichidarum , Bifidobaterium allitrichos, Bifidobaterium canis , Bifidobaterium castoris , Bifidobacterium Bifidobaterium catenulatum , Bifidobaterium catulorum , Bifidobaterium cebidarum , Bifidobacterium choerinum , Bifidobacterium coladoha Bifidobaterium choladohabitans , Bifidobaterium choloepi , Bifidobaterium colobi, Bifidobaterium commune , Bifidobaterium criceti , Bifidobacterium crudilactis , Bifidobaterium cuniculi, Bifidobaterium dentium , Bifidobaterium dolichotidis , Bifidobacterium Ericksonii ( Bifidobaterium eriksonii ), Bifidobaterium erythrocebi , Bifidobaterium eulemuris, Bifidobaterium faecale , Bifidobacterium felsineum ( Bifidobaterium ) fesineum ), Bifidobaterium gallicum, Bifidobaterium gallinarum , Bifidobaterium globosum , Bifidobaterium goeldii , Bifidobacterium Bifidobaterium hapali , Bifidobaterium indicum, Bifidobaterium italicum, Bifidobaterium jacchi , Bifidobaterium lemurum , Bifidobacterium leontopitheci , Bifidobacterium longum , Bifidobaterium magnum , Bifidobaterium margollesii , Bifidobacterium Mary. Bifidobaterium merycicum , Bifidobaterium miconis , Bifidobaterium miconisargentati , Bifidobaterium minimum , Bifidobacterium mongoliens mongoliense ), Bifidobaterium moraviense , Bifidobaterium moukalabense , Bifidobaterium myosotis , Bifidobaterium oedipodis , Bifidobaterium olomucense , Bifidobaterium panos , Bifidobaterium parmae , Bifidobaterium platyrrhinorum , Bifidobacterium Bifidobaterium pluvialisilvae , Bifidobaterium polysaccharolyticum , Bifidobaterium pongonis , Bifidobaterium porcinum , Bifidobacterium Bifidobaterium primatium , Bifidobaterium pseudocatenulatum , Bifidobacterium pseudolongum , Bifidobacterium psychraerophilum , Bifidobacterium Pullorum ( Bifidobaterium pullorum ), Bifidobaterium ramosum ( Bifidobaterium ramosum ), Bifidobaterium reuteri , Bifidobaterium rousetti , Bifidobaterium ruminale , Bifidobacterium ruminantium , Bifidobaterium saguini, Bifidobaterium saguinibicoloris , Bifidobaterium saimiriisciurei , Bifidobacterium samirii , Bifidobaterium santillanense, Bifidobaterium scaligerum , Bifidobaterium scardovii , Bifidobacterium Bifidobaterium simiarum , Bifidobacterium simiiventris , Bifidobacterium stellenboschense , Bifidobaterium subtile , Bifidobacterium thermosidophilum ( Bifidobaterium thermacidophilum ), Bifidobaterium thermophilum , Bifidobaterium tibiigranuli , Bifidobaterium tissieri , Bifidobaterium tsurumiense ), Bifidobaterium urinalis , Bifidobaterium vansinderenii, Bifidobaterium vespertilionis , or Bifidobaterium xylocopae ), a manufacturing method characterized in that.
  13. 제1항에 있어서,According to paragraph 1,
    상기 세포외소포는 평균 직경이 10 nm 내지 1000 nm인 것을 특징으로 하는, 제조방법.The production method, wherein the extracellular vesicles have an average diameter of 10 nm to 1000 nm.
PCT/KR2023/003468 2022-03-18 2023-03-15 Method for preparing lactic acid bacterium-derived extracellular vesicles with improved yield WO2023177208A1 (en)

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CN117243885B (en) * 2023-11-15 2024-01-26 北京青藤谷禧干细胞科技研究院有限公司 Stem cell exosome composition for improving skin and preparation method thereof

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