WO2020060164A1 - Souche de komagateibacter rhaeticus et procédé de production de feuille cellulosique l'utilisant - Google Patents

Souche de komagateibacter rhaeticus et procédé de production de feuille cellulosique l'utilisant Download PDF

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WO2020060164A1
WO2020060164A1 PCT/KR2019/012009 KR2019012009W WO2020060164A1 WO 2020060164 A1 WO2020060164 A1 WO 2020060164A1 KR 2019012009 W KR2019012009 W KR 2019012009W WO 2020060164 A1 WO2020060164 A1 WO 2020060164A1
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weight
strain
culture
koss15
cellulose
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PCT/KR2019/012009
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Korean (ko)
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홍두성
박지호
권희수
김기수
이재우
이여준
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에스케이바이오랜드 주식회사
에스케바이오랜드 해문지사
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Priority to CN201980007631.7A priority Critical patent/CN111684057B/zh
Publication of WO2020060164A1 publication Critical patent/WO2020060164A1/fr

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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
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A61K8/731—Cellulose; Quaternized cellulose derivatives
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J5/00—Manufacture of articles or shaped materials containing macromolecular substances
    • C08J5/18—Manufacture of films or sheets
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L1/00—Compositions of cellulose, modified cellulose or cellulose derivatives
    • C08L1/08—Cellulose derivatives
    • C08L1/10—Esters of organic acids, i.e. acylates
    • C08L1/12—Cellulose acetate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • 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

Definitions

  • the present invention relates to a novel Comaga bacterial bacterium Laticus strain and a method of manufacturing a cellulose sheet using the same.
  • the sheet-type mask used in the cosmetic field is largely (i) a non-woven fabric made of vegetable cellulose fibers such as cotton and pulp or synthetic fibers such as nylon and dupont is soaked in a fresh water body of a cosmetic fluid and used as a mask-shaped support.
  • Cellulose masks used (ii) hydrogel masks made from natural polysaccharides such as natural agar, carrageenan, locust bean gum, and (iii) bacterial cellulose (biocellulose) made through microbial culture
  • bacterial cellulose biocellulose
  • the sheet-type mask pack used in the cosmetic field is attracting attention in the cosmetics market because it is possible to obtain a moisturizing and clean effect as the most important advantage as attaching a sheet made in the form of a face without having to apply by hand.
  • Cellulose (aka bacterial cellulose) made through microbial culture has recently been used as a material for burn healers for cosmetics or cosmetic mask packs.
  • Cellulose is the most abundant biomaterial resource in nature.
  • cellulose produced by acetic acid bacteria has attracted much attention as a high-value industrial new material as well as food additives.
  • cellulose produced by bacteria has excellent physical and chemical properties, unlike cellulose produced by plants.
  • Acetobacter is a strain that produces bacterial cellulose. sp.), Agrobacterium sp., Rhizobium sp., Pseudomonas sp. and Sarcina sp., among the acetobacter strains Xylinum ( A. xylinum ), Acetobacter Pasteurianus ( A. pasteurinanus ) and Acetobacter Hansenii ( A. hansenii ) are widely known. Among them, the microorganism with the highest yield of cellulose is acetobacter xylinum . (Korean Industrial Chemistry Society KIC News, 2013 16 (4) 37-45).
  • Microorganisms producing such bacterial cellulose are known to have low production efficiency, and thus, there is an urgent need for development of new microorganisms having high cellulose production efficiency.
  • the present inventors have investigated the changes in the quality and production efficiency of bacterial cellulose according to the discovery of new strains and the sugar content of the culture medium.
  • a novel Komaga-Tate bacterium Lacticus ( Komagataeibacter rhaeticus) KOSS15 strain was identified, and the strain was found to have high cellulosic production capacity with honey as a sugar source, and to demonstrate that the quality of the produced bacterial cellulose is excellent. It was completed.
  • Another object of the present invention is to provide a composition for preparing a bacterial cellulose sheet.
  • Another object of the present invention is to provide a method for producing bacterial cellulose.
  • the present invention is deposited with the accession number KCCM12270P, Komagatabacter laticus ( Komagataeibacter rhaeticus ) KOSS15 strain.
  • the present inventors have investigated the changes in the quality and production efficiency of bacterial cellulose according to the discovery of new strains and the sugar content of the culture medium.
  • the new Komagataeibacter rhaeticus ) KOSS15 strains were identified, and the present invention was completed by identifying the strains having high cellulosic production capacity with honey as a sugar and excellent quality of bacterial cellulose produced.
  • the strain of Komygabacteractus latiscus KOSS15 is derived from black tea mushroom strain (kombucha).
  • the black tea mushroom spp. Is called Kombucha, black tea mushroom yeast, red mushroom, longevity mushroom, etc. and is a type of organic acid-producing bacteria (lactic acid bacteria) in which bacteria and yeast coexist with hyphae like threads.
  • Black tea mushrooms are bacteria and Bretanomyces that increase the acidity by fermenting alcohol produced by yeast such as acetobacter xylinum with acetic acid and other acids, and Zygosaccharomyces ( Zygosaccharomyces). It is a symbiosis of yeasts such as Candida, Skizosaccharomyces and Saccharomyces.
  • the Komagatabacter laticus KOSS15 strain has the ability to produce cellulose.
  • cellulose refers to a polysaccharide in which ⁇ -D-glucose forms a polymer through a glucoside bond, the formula is (C 6 H 10 O 5 ) n, and the unit is cellobiose. do.
  • the Komagatabacter laticus KOSS15 strain produces cellulose with honey as a sugar source.
  • the term "honey” is a viscous substance collected by bees collected from wheat plants of plants, and includes a high content of glucose and fructose, as well as a substance containing various vitamin, protein, and mineral components.
  • the present invention relates to a composition for producing a cellulosic sheet comprising a strain of Komagatabacter laticus KOSS15 or a culture of the strain having accession number KCCM12270P.
  • culture of the present invention is a cultured strain obtained by culturing the strain for a period of time in a medium capable of supplying nutrients so that the Komaga bacterial lacticus KOSS15 strain of the present invention can grow and survive in vitro. It means a medium containing its metabolites, extra nutrients, and the like. Since the Komaga tate bacterium KOSS15 strain is a strain having cellulosic production ability, the Komaga bacterial bacterium KOSS15 strain or a culture thereof can be used as a composition for producing a cellulose sheet for producing cellulose from sugar.
  • the present invention relates to a method for producing a cellulose sheet comprising culturing a strain of Komaga tate bacterium Laticus KOSS15 having accession number KCCM12270P.
  • the Komaga bacterial bacterium KOSS15 strain can produce cellulose using honey, fructose, glucose or a combination thereof as a sugar source.
  • the Komaga bacterial bacterium KOSS15 strain comprises: (a) honey or (b) glucose and fructose; And cultured in a culture medium containing proline (proline).
  • the culture medium is 0.1 to 2.0% by volume, 0.2 to 2.0% by volume, 0.3 to 2.0% by volume, 0.4 to 2.0% by volume, 0.5 to 2.0% by volume, 0.6 to 2.0% by volume, 0.7 to 2.0% by volume, 0.8 to 2.0 vol%, 0.9-2.0 vol%, 0.1-1.9 vol%, 0.1-1.8 vol%, 0.1-1.7 vol%, 0.1-1.6 vol%, 0.1-1.5 vol%, 0.1-1.4 vol%, 0.1-1.3 vol %, 0.1 to 1.2% by volume, 0.1 to 1.1% by volume, 0.2 to 1.8% by volume, 0.2 to 1.6% by volume, 0.2 to 1.4% by volume, or 0.2 to 1.2% by volume.
  • the culture medium is 0.1 to 2.0% by weight of glucose and fructose, 0.2 to 2.0% by weight, 0.3 to 2.0% by weight, 0.4 to 2.0% by weight, 0.5 to 2.0% by weight, 0.6 to 2.0% by weight, 0.7 to 2.0% by weight, 0.8 to 2.0 wt%, 0.9 to 2.0 wt%, 0.1 to 1.9 wt%, 0.1 to 1.8 wt%, 0.1 to 1.7 wt%, 0.1 to 1.6 wt%, 0.1 to 1.5 wt%, 0.1 to 1.4 wt%, 0.1 to It may include 1.3 wt%, 0.1 to 1.2 wt%, 0.1 to 1.1 wt%, 0.2 to 1.8 wt%, 0.2 to 1.6 wt%, 0.2 to 1.4 wt% or 0.2 to 1.2 wt%.
  • the culture medium is 0.01 to 1.0 wt% of proline, 0.02 to 1.0 wt%, 0.03 to 1.0 wt%, 0.04 to 1.0 wt%, 0.05 to 1.0 wt%, 0.06 to 1.0 wt%, 0.07 to 1.0 wt%, 0.08 to 1.0 wt%, 0.01 to 0.8 wt%, 0.01 to 0.6 wt%, 0.01 to 0.4 wt%, 0.01 to 0.2 wt%, 0.02 to 0.6 wt%, 0.03 to 0.6 wt%, 0.04 to 0.6 wt%, 0.05 to 0.4 wt% %, 0.06 to 0.3 wt%, 0.04 to 0.3 wt%, 0.04 to 0.2 wt% or 0.05 to 0.15 wt%, but is not limited thereto.
  • the fructose, glucose and proline are the main sugars and amino acids constituting the royal jelly, and in the cultivation of the Komaga bacterial bacterium KOSS15 strain, bacterial cellulose can be prepared by adding the main sugars and amino acids constituting the royal jelly instead of honey. have.
  • composition for preparing a cellulose sheet of the present invention may additionally include a carbon source, a nitrogen source, and a phosphorus source for cultivation of a strain of Komagatabacter laticus KOSS15.
  • the carbon source is one selected from the group consisting of ethanol, glucose, sucrose, fructose, galactose, soluble stark, inositol, glycerol, xylose, dextrose, lactose, dextrin, adonitol, mannitol, mannose, maltose, and raffinose.
  • the above carbon sources include, but are not limited to.
  • the nitrogen source is at least one organic nitrogen source selected from the group comprising yeast extract, peptone, soytone, casamino acid, tryptone and malt extract, NH 4 Cl, (COONH 4 ) 2 , NH 4 H 2 PO 4 , (NH 4 ) 2 HPO 4 , NH 4 NO 3 , NaNO 3 and (NH 4 ) 2 SO 4 includes at least one inorganic nitrogen source selected from the group comprising, but is not limited to.
  • the personnel includes, but is not limited to, one or more personnel selected from the group comprising potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate.
  • composition for preparing the cellulose sheet is, MgSO 4 , CaCl 2 , KCl, BaCl 2 , Li 2 SO 4 , MnSO, MnSO 4 , ZnSO 4 , FeSO 4 , Na 2 MoO 4 , KH 2 PO 4 and FeCl 3 may be further included, but is not limited thereto.
  • the present invention relates to a method for producing a bacterial cellulose sheet using a strain of Komaga bacterial lacticus KOSS15 having accession number KCCM12270P, comprising the following steps:
  • the method of manufacturing the bacterial cellulose sheet of the present invention will be described step by step.
  • the pre-culture by stirring culture in a pre-culture medium containing glycogen and cellulase, a strain of Komaga bacterial Lactus KOSS15 having accession number KCCM12270P.
  • the pre-culture medium includes sugar.
  • the type and concentration of the sugar source is (a) 0.1 to 5 volumes (v / v) of honey or (b) glucose and fructose 0.1 to 5% by weight (w / v).
  • the culture medium of the present invention is 0.1-5% by volume of honey, 0.1-4% by volume, 0.1-3% by volume, 0.1-2.5% by volume, 0.1-2% by volume, 0.1-1.5% It is included in a concentration of volume% or 0.1 to 1% by volume.
  • the culture medium of the present invention contains 0.1 to 3.0% by volume of honey, 0.2 to 3.0% by volume, 0.3 to 3.0% by volume, 0.4 to 3.0% by volume, 0.5 to 3.0% by volume, 0.6 to 3.0% Volume%, 0.7-3.0 volume%, 0.8-3.0 volume%, 0.9-3.0 volume%, 1-3.0 volume%, 0.1-2.0 volume%, 0.2-2.0 volume%, 0.3-2.0 volume%, 0.4-2.0 volume% , 0.5 to 2.0 vol%, 0.6 to 2.0 vol%, 0.7 to 2.0 vol%, 0.8 to 2.0 vol%, 0.9 to 2.0 vol%, 0.1 to 1.9 vol%, 0.1 to 1.8 vol%, 0.1 to 1.7 vol%, 0.1 To 1.6 vol%, 0.1 to 1.5 vol%, 0.1 to 1.4 vol%, 0.1 to 1.3 vol%, 0.1 to 1.2 vol%, 0.1 to 1.1 vol%, 0.2 to 1.8 vol%, 0.2 to 1.6 vol%, 0.2 to 1.4 Volume%, 0.1 to 1.3 vol
  • the medium composition of the present invention is a combination of glucose and fructose 0.1 to 5% by weight, 0.1 to 4% by weight, 0.1 to 3% by weight, 0.1 to 2.5% by weight, 0.1 to 2% by weight , 0.1 to 1.5% by weight, or 0.1 to 1% by weight.
  • the culture medium of the present invention adds glucose and fructose 0.1 to 3.0 wt%, 0.2 to 3.0 wt%, 0.3 to 3.0 wt%, 0.4 to 3.0 wt%, 0.5 to 3.0 wt% , 0.6 to 3.0 wt%, 0.7 to 3.0 wt%, 0.8 to 3.0 wt%, 0.9 to 3.0 wt%, 1 to 3.0 wt%, 0.1 to 2.0 wt%, 0.2 to 2.0 wt%, 0.3 to 2.0 wt%, 0.4 To 2.0 wt%, 0.5 to 2.0 wt%, 0.6 to 2.0 wt%, 0.7 to 2.0 wt%, 0.8 to 2.0 wt%, 0.9 to 2.0 wt%, 0.1 to 1.9 wt%, 0.1 to 1.8 wt%, 0.1 to 1.7 Wt%, 0.1-1.6 wt%, 0.1-1.5 wt%, 0.1-1.4
  • the glucose and fructose are included in a weight ratio of 2: 3.
  • the 2: 3 ratio is derived from the ratio of glucose and fructose contained in royal jelly.
  • the pre-culture medium is 0.1 to 10.0% by weight of yeast extract, MgSO 4 0.002 to 0.2% by weight, CaCl 2 0.002 to 0.2% by weight, ethanol 0.2 to 20.0% by weight, and cellulase 0.0005 to 0.05% by weight % Or combinations thereof.
  • pre-cultivation refers to a step of culturing a cellulose-producing strain in a relatively small scale (eg, 100 L or less) to activate or increase the cellulosic-producing strain.
  • the pre-culture medium is 0.1 to 10.0% by weight of yeast extract, 0.5 to 10.0% by weight, 0.7 to 10.0% by weight, 0.9 to 10.0% by weight, 0.1 to 8.0% by weight, 0.1 to 6.0% by weight, 0.1 to 4.0% by weight, 0.1 to 2.0 wt%, 0.5 to 10.0 wt%, 0.5 to 8.0 wt%, 0.5 to 6.0 wt%, 0.5 to 4.0 wt%, 0.5 to 2.0 wt%, 0.9 to 10.0 wt%, 0.9 to 8.0 wt%, 0.9 to 6.0 wt%, 0.9 to 4.0 wt%, 0.9 to 2.0 wt% or 0.9 to 1.5 wt%.
  • the pre-culture medium is 0.002 to 0.2% by weight of MgSO 4 , 0.005 to 0.2% by weight, 0.008 to 0.2% by weight, 0.01 to 0.2% by weight, 0.013 to 0.2% by weight, 0.015 to 0.2% by weight, 0.01 to 0.15% by weight, It may include 0.01 to 0.1% by weight, 0.01 to 0.05% by weight, 0.01 to 0.03% by weight, or 0.015 to 0.03% by weight.
  • the pre-culture medium is 0.002 to 0.2% by weight of CaCl 2 , 0.005 to 0.2% by weight, 0.008 to 0.2% by weight, 0.01 to 0.2% by weight, 0.013 to 0.2% by weight, 0.015 to 0.2% by weight, 0.01 to 0.15% by weight, It may include 0.01 to 0.1% by weight, 0.01 to 0.05% by weight, 0.01 to 0.03% by weight, or 0.015 to 0.03% by weight.
  • the pre-culture medium is 0.2 to 20.0% by volume of ethanol, 0.5 to 20.0% by volume, 1.0 to 20.0% by volume, 1.5 to 20.0% by volume, 1.0 to 20.0% by volume, 1.0 to 17.0% by volume, 1.0 to 14.0% by volume, 1.0 To 11.0% by volume, 1.0 to 8.0% by volume, 1.0 to 5.0% by volume, or 1.0 to 3.0% by volume.
  • the pre-culture medium may include 0.0005 to 0.05% by weight, 0.001 to 0.05% by weight, 0.003 to 0.05% by weight, 0.001 to 0.03% by weight, 0.001 to 0.02% by weight, or 0.001 to 0.01% by weight of cellulase.
  • the pre-incubation step is agitated and cultured at 100 to 200 rpm for 24 to 72 hours.
  • the pre-culture step is 30 to 72 hours, 36 to 72 hours, 42 to 72 hours, 36 to 72 hours, 36 to 66 hours, 36 to 60 hours, 36 to 54 hours, 42 to 66 hours, 42 to 60 hours, Stirring can be carried out for 42 to 54 hours or 42 to 52 hours.
  • the pre-incubation step is stirred and cultured at 160 rpm for 48 hours.
  • the pre-culture solution of the above step is added to the main culture medium containing the above-described sugars and proline and cultured with stirring.
  • main culture refers to a large scale (eg, 500 L or more and 50,000 L or less) of bacterial cellulose-producing strains up to the stage immediately before “sheet conversion culture” in which the “pre-cultured” culture medium is actually producing a bacterial cellulose sheet. Means to incubate with.
  • the main culture medium includes 0.01 to 1.0% by weight of proline, 0.002 to 0.2% by weight of MgSO 4, 0.002 to 0.2% by weight of CaCl 2 , 0.01 to 1.0% by weight of sodium acetate, and 0.02 to 2.0% by weight of acetic acid, or a combination thereof. .
  • the main culture medium is 0.01 to 1.0 wt% of proline, 0.02 to 1.0 wt%, 0.03 to 1.0 wt%, 0.04 to 1.0 wt%, 0.05 to 1.0 wt%, 0.06 to 1.0 wt%, 0.07 to 1.0 wt%, 0.08 To 1.0 wt%, 0.01 to 0.8 wt%, 0.01 to 0.6 wt%, 0.01 to 0.4 wt%, 0.01 to 0.2 wt%, 0.02 to 0.6 wt%, 0.03 to 0.6 wt%, 0.04 to 0.6 wt%, 0.05 to 0.4 Weight percent, 0.06 to 0.3 weight percent, 0.04 to 0.3 weight percent, 0.04 to 0.2 weight percent or 0.05 to 0.15 weight percent.
  • the main culture medium is 0.002 to 0.2% by weight of MgSO 4 , 0.005 to 0.2% by weight, 0.008 to 0.2% by weight, 0.01 to 0.2% by weight, 0.013 to 0.2% by weight, 0.015 to 0.2% by weight, 0.01 to 0.15% by weight, It may include 0.01 to 0.1% by weight, 0.01 to 0.05% by weight, 0.01 to 0.03% by weight, or 0.015 to 0.03% by weight.
  • the main culture medium is CaCl 2 0.002 to 0.2% by weight, 0.005 to 0.2% by weight, 0.008 to 0.2% by weight, 0.01 to 0.2% by weight, 0.013 to 0.2% by weight, 0.015 to 0.2% by weight, 0.01 to 0.15% by weight, It may include 0.01 to 0.1% by weight, 0.01 to 0.05% by weight, 0.01 to 0.03% by weight, or 0.015 to 0.03% by weight.
  • the main culture medium is 0.01 to 1.0% by weight of sodium acetate, 0.02 to 1.0% by weight, 0.03 to 1.0% by weight, 0.04 to 1.0% by weight, 0.05 to 1.0% by weight, 0.06 to 1.0% by weight, 0.07 to 1.0% by weight, 0.08 to 1.0 wt%, 0.01 to 0.8 wt%, 0.01 to 0.6 wt%, 0.01 to 0.4 wt%, 0.01 to 0.2 wt%, 0.02 to 0.6 wt%, 0.03 to 0.6 wt%, 0.04 to 0.6 wt%, 0.05 to 0.4 wt%, 0.06 to 0.3 wt%, 0.04 to 0.3 wt%, 0.04 to 0.2 wt%, or 0.05 to 0.15 wt%.
  • the main culture medium may include 0.02 to 2.0 wt%, 0.05 to 2.0 wt%, 0.1 to 2.0 wt%, 0.15 to wt%, 0.1 to 1.5 wt%, 0.1 to 1.0 wt% or 0.1 to 0.5 wt% acetic acid have.
  • the main culture solution of the above step is added to a culture medium containing sugar and proline, and subjected to stationary culture to perform sheet conversion culture.
  • sheet conversion culture refers to a step in which the bacterial cellulose producing strain produces a bacterial cellulose sheet by statically culturing the main culture solution containing the bacterial cellulose producing strain.
  • the culture medium has the same composition as the main culture medium of step (b).
  • the method of manufacturing the bacterial cellulose sheet of the present invention is similar to the composition for preparing the Komagatebacter laticus KOSS15 strain described above, the culture of the strain and the cellulosic sheet, the common content between the two is to avoid excessive complexity in this specification In order to avoid this, the description is omitted.
  • the present invention is a Komagata bacterium Laticus ( Komagataeibacter rhaeticus ) KOSS15 strain, a composition for preparing a cellulosic sheet comprising the strain or culture, and a method for producing a cellulosic sheet comprising culturing the strain.
  • Cellulose sheet produced by the Komaga bacterial bacterium Lacticus KOSS15 strain of the present invention exhibits high tensile strength and bursting strength compared to the cellulose sheet of Acetobacter xylinum, which is known as a microorganism having the best production yield of cellulose, and has the same weight. It is possible to reduce the content of sugars required to manufacture the cellulose sheet of 5-6 times.
  • Figure 1 shows the phylogenetic tree of the Komaga bacterial Lactus KOSS15 strain.
  • Figure 2 shows the production process of cellulose using a strain of Komaga bacterial Lactus KOSS15.
  • Figure 3 schematically shows the manufacturing process of the cellulosic sheet through passage culture of the Komaga bacterial bacterium Lacusis KOSS15 strain.
  • Figure 4 shows a cellulose sheet prepared through the cultivation of the Komaga bacterial Bacillus KOSS15 strain.
  • FIG. 5 shows the cellulose sheet of Comagathebacter laticus KOSS15 and Acetobacter xylinum IFO13693 according to cellulase treatment.
  • Figure 6 shows an image of a cellulose sheet prepared by Komaga tate bacterium Laticus KOSS15 according to the honey concentration in the medium.
  • Figure 7 shows an image of a cellulose sheet prepared by Komaga tate bacterium Laticus KOSS15 according to the type of honey in the medium.
  • FIG. 8 shows the results of genomic analysis of the cellulose-producing gene group of Komagateibacter laticus KOSS15 and Acetobacter xylinum IFO13693.
  • % used to indicate the concentration of a specific substance, unless otherwise specified, solids / solids (weight / weight)%, solids / liquids (weight / volume)%, and The liquid / liquid is (volume / volume)%.
  • bacteria were taken from one experimental group, and 2% glucose, 1% yeast extract, 0.02% magnesium sulfate, 0.02% calcium chloride, and 1.8% agar were added.
  • an agar medium was prepared.
  • One of the five liquids (agar green tea mushrooms of Jirisan) selected from 5 liquids of agar medium was spread on 0.1 ml and cultured at 28 ° C for 7 days.
  • a medium solution containing 60 ml of glucose 2%, yeast extract 1%, magnesium sulfate 0.02%, and calcium chloride 0.02% was sterilized. Then, 10 ml of the sterilized medium solution was dispensed, and 6 colonies were inoculated therein.
  • Acetobactor xylinum (IFo13693) was used as a control for comparing the cellulose bioconversion efficiency of Komagateibacter laticus KOSS15.
  • the acetobacter xylinum is a representative cellulose production strain.
  • Acetobacter xylinum IFO13693 was inoculated with 2.0x10 7 CFU / ml in a culture medium containing 2% glucose, 1.0% yeast extract, 0.02% MgSO 4, 0.02% CaCl 2 and 2% ethanol, and 160 rpm under 30 ° C at Flask. It was stirred and pre-incubated for 48 hours.
  • the slurry (residue) produced from the pre-culture was filtered through an 80 mesh sieve and inoculated with 10% of the main culture.
  • the main culture medium was prepared by adding glucose 3%, MSG ((monosodium glutamate) 0.5%, MgSO 4 0.02%, CaCl 2 0.02%, sodium acetate 0.1% and acetic acid 0.2%. Incubated for 48 hours under the condition of vvm (aeration volume / medium volume / minute).
  • acetobacter xylinum IFO13693 was inoculated and mixed with 5-10% of the culture medium and dispensed into a tray, and incubated for 3 days at 0 rpm and 0.3 vvm conditions to induce cellulose sheet conversion (FIG. 2 and 3).
  • the prepared cellulose sheet was dehydrated, and 0.1% of NaOH was treated to remove and wash the excess strain.
  • 5% of sodium percarbonate was treated to remove proteins and impurities and washed to obtain a sheet (FIG. 4).
  • KOSS15 was inoculated at 1.0x10 8 CFU / ml and pre-incubated for 48 hours at 80-160 rpm under stirring at 30 ° C in Flask. The cellulase was added to prevent the phenomenon of pellicle during pre-cultivation, and when the cellulase was added to the pre-culture medium of acetobacter xylinum IFO13693 in (1), a bad sheet was generated and was not applied ( Fig. 5).
  • the whole culture was inoculated 10% compared to the main culture medium.
  • the main culture medium was prepared by adding sugar (0.6% fructose and 0.4% glucose; or 1% honey), 0.1% proline, 0.02% MgSO 4, 0.02% CaCl 2 , 0.1% sodium acetate, and 0.2% acetic acid.
  • the main culture was incubated with stirring at 48 rpm and 0.3 vvm for 48 hours.
  • the cellulose sheet produced by the Komagateibacter laticus KOSS15 is characterized in that when the sheet is first immersed in an aqueous NaOH solution before dehydration, the sheet becomes hard and the strength is excellent. There was.
  • the washed cellulose sheets were flattened, cut to 18.15x18.15 cm, and weighed. After the measurement, the dried oven was dried at 95 ° C for 1 hour, and the weight was measured to obtain the weight before and after drying.
  • the content of sugar in the medium of Table 1 below refers to the content of sugar in the culture medium during cellulosic sheet conversion culture.
  • the comagatabacter laticus KOSS15 can reduce the content of sugar in the medium by about 6 times compared to acetobacter xylinum IFO13693.
  • fructose and glucose contained in a ratio of 3: 2 instead of honey were used as sugar sources, an equivalent effect was exhibited.
  • the content of the constituent sugars of the cellulose sheet was confirmed.
  • the tensile strength of the sheet was measured using a tester applying a constant speed elongation method (20 mm / min) to the sheet (KS M ISO 1924-2).
  • wet burst strength of the sheet was measured by increasing the fluid pressure (KS M ISO 2758).
  • Cellulose sheets were prepared in the same manner as in Example 2, except that 0.3% of honey, 0.5% of honey, and 1.0% of honey were used as shown in Table 4 below, respectively, as shown in Table 4 below. .
  • the types and contents of sugar in the medium were measured according to the concentration of honey before and after cultivation, and the physical properties of the cellulose sheets produced by the concentration of honey contained in the medium were compared (Table 4).
  • the sugar source of the medium used in the sheet conversion culture step is 0.5% of specification honey, 0.5% of acacia honey, and 0.5% of wild flower honey (hybrid honey) as shown in Table 5 below.
  • a cellulose sheet was produced in the same manner as in Example 2, except that one was used. The type and content of sugar in the medium were measured according to the type of honey before and after cultivation, and the dry weight of the cellulose sheet produced by the type of honey contained in the medium was compared.
  • IFO13693 and KOSS15 were sequenced through Pacbio sequence analysis, a type of next generation sequencing (NGS).
  • NGS next generation sequencing
  • chromosome 3,539,432 bp was identified.
  • KOSS15 strain it was confirmed to have 3,275,555 bp of chromosome and 3 plasmids.
  • the production genes of each bio-cellulose were identified from two production strains based on NGS (FIG. 8).
  • Type gene A xylinum IFO13693 K. rhaeticus KOSS15 Type I BcsAI 2950 1826 BcsBI 2951 1824, 1825 BcsCI 2952 1823 BcsDI 2953 1822 Type II BcsB II 717, 1148 3004, 1007 BcsX 718 3007 BcsY 719 3008 BcsC II 720 3009

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Abstract

La présente invention concerne : une souche de Komagataeibacter rhaeticus KOSS15; une composition comprenant la souche ou une culture de celle-ci pour la production d'une feuille de cellulose; et un procédé de production d'une feuille de cellulose, le procédé comprenant une étape de culture de la souche. La feuille de cellulose produite par la souche de Komagataeibacter rhaeticus KOSS15 de la présente invention présente une résistance à la traction et une résistance à la rupture supérieures à celles de la feuille de cellulose produite à partir d'Acetobacter xylinum, qui est connu en tant que micro-organisme ayant le rendement de production de cellulose le plus élevé, et permet une réduction de facteur 5 à 6 de la quantité de source de saccharide nécessaire pour produire une feuille de cellulose ayant le même poids que celle produite à partir d'Acetobacter xylinum.
PCT/KR2019/012009 2018-09-17 2019-09-17 Souche de komagateibacter rhaeticus et procédé de production de feuille cellulosique l'utilisant WO2020060164A1 (fr)

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KR102262644B1 (ko) 2020-03-03 2021-06-09 주식회사 현대바이오랜드 피부 상재균총 조절효과를 갖는 하이브리드 박테리얼 셀룰로오스 마스크 시트 및 이의 제조방법
TWI776240B (zh) * 2020-09-22 2022-09-01 財團法人食品工業發展研究所 微生物纖維之製備方法
KR102499330B1 (ko) 2022-04-26 2023-02-10 주식회사 현대바이오랜드 콤부차로부터 분리된 균주의 복합 배양물을 함유하는 피부장벽 강화용 화장료 조성물
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