WO2020060164A1 - Komagateibacter rhaeticus strain and cellulose sheet production method using same - Google Patents

Abstract

The present invention provides: a Komagataeibacter rhaeticus KOSS15 strain; a composition comprising the strain or a culture thereof for production of a cellulose sheet; and a method for production of a cellulose sheet, the method comprising a step of culturing the strain. The cellulose sheet produced by the Komagataeibacter rhaeticus KOSS15 strain of the present invention exhibits higher tensile strength and rupture strength than the cellulose sheet of Acetobacter xylinum, which is known as a microorganism having the highest cellulose production yield, and enables a 5-6 fold reduction in the amount of saccharide source necessary for producing a cellulose sheet having the same weight, compared to that of Acetobacter xylinum.

Description

Comagatabacter laticus strain and method of manufacturing cellulose sheet using same

This patent application claims priority to Korean Patent Application No. 10-2018-0111099 filed with the Korean Intellectual Property Office on September 17, 2018, and the disclosures of the patent application are incorporated herein by reference.

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 There are three types of bacterial cellulose masks that are soaked in fresh water and used as a mask-shaped support.

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. In particular, cellulose produced by acetic acid bacteria has attracted much attention as a high-value industrial new material as well as food additives. In addition, 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.

In order to develop a method for producing a bacterial cellulose with improved quality and improved 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. As a result, 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.

Accordingly, it is an object of the present invention to provide a strain of Komagatabacter laticus KOSS15.

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.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to an aspect of the present invention, the present invention is deposited with the accession number KCCM12270P, Komagatabacter laticus ( Komagataeibacter rhaeticus ) KOSS15 strain.

In order to develop a method for producing a bacterial cellulose with improved quality and improved 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. As a result, 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.

According to an embodiment of the present invention, 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.

According to one embodiment of the present invention, the Komagatabacter laticus KOSS15 strain has the ability to produce cellulose.

The term "cellulose" as used herein refers to a polysaccharide in which β-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.

According to another embodiment of the present invention, the Komagatabacter laticus KOSS15 strain produces cellulose with honey as a sugar source.

As used herein, 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.

According to another aspect of the present invention, 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.

The term "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.

According to another aspect of the present invention, 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.

According to one embodiment of the present invention, 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.

The 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.

The 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.

According to another aspect of the present invention, 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:

(A) pre-culturing step of stirring the strain in a pre-culture medium containing sugar and cellulase;

(B) the main culture step of adding the pre-culture solution of the above step to the main culture medium containing sugar and proline and stirring culture; And

(C) sheet conversion culture step of adding the main culture solution of the above step to a culture medium containing sugar and proline and stationary culture.

The method of manufacturing the bacterial cellulose sheet of the present invention will be described step by step.

Step (a): Preculture  step

First, 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.

According to one embodiment of the present invention, the pre-culture medium includes sugar.

In one embodiment of the present invention, 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).

According to a specific embodiment of the present invention, 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.

In another embodiment of the present invention, 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% or 0.2 to 1.2% by volume, but is not limited thereto.

According to another specific embodiment of the present invention, 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.

In another embodiment of the present invention, 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 wt%, 0.1-1.3 wt%, 0.1-1.2 wt%, 0.1-1.1 wt%, 0.2-1.8 wt%, 0.2-1.6 wt% , 0.2 to 1.4 wt% or 0.2 to 1.2 wt%, but is not limited thereto.

According to another embodiment of the present invention, 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.

According to one embodiment of the invention, 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.

As used herein, the term, “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.

According to one embodiment of the present invention, 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.

According to another embodiment of the present invention, the pre-incubation step is stirred and cultured at 160 rpm for 48 hours.

Step (b): Main culture  step

Next, 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.

As used herein, the term “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.

(c) step: sheet conversion culture step

Finally, 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.

In this specification, the term “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).

Since 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.

Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Throughout this specification, "%" 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)%.

1. Strain selection

After brewing 3 packs of brown rice green tea in 1 L of water, 30% (w / v) of sugar was added, and 500 ml was dispensed into the flask, followed by sterilization and cooling at 121 ° C for 30 minutes. Five kinds of obtained black tea mushroom spawn solution (Vietnam black tea mushroom; Jiri green tea black tea mushroom; Boseong green tea black tea mushroom; Chinese Yunnan province black tea mushroom; Tibetan black tea mushroom) were inoculated in 10% (v / v), respectively, and incubated at 28 ℃ Cultured. After confirming that cellulose was formed from 2 weeks of culture, 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. After adjusting the pH of the medium to 4.0 with acetic acid, 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. After separating the colonies having the same shape and fungus into six groups, 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.

After culturing at 28 ° C for 3 days, 40 ml of acetic acid pre-culture medium was prepared, and 3 ml of cultured 3 days was added to acetic acid pre-culture medium, and then cultured at 28 ° C. to observe cellulose formation after 4 days. . Finally, colonies with good cellulose morphology were displayed and fungal stock was prepared. The colonies that were most well formed among the six experimental groups were designated as KOSS15 and deposited with the Korean Microbiological Conservation Center for patent accession number KCCM12270P.

2. Culture of strain

(1) Cultivation of Acetobactor xylinum IFO13693 strain

1) Pre-cultivation (1 passage)

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.

2) Main culture (2 passages)

Subsequently, 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).

3) Sheet conversion culture (main culture mixture mixing and tray dispensing) and washing

Finally, the main culture of 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). After the cultivation was over, the prepared cellulose sheet was dehydrated, and 0.1% of NaOH was treated to remove and wash the excess strain. Then, 5% of sodium percarbonate was treated to remove proteins and impurities and washed to obtain a sheet (FIG. 4).

(2) Cultivation of Komagatabacter laticus KOSS15 (KCCM12270P)

1) Pre-cultivation (1 passage)

Comagatabacter laticus in a culture medium containing sugar (0.6% fructose and 0.4% glucose; or 1% honey), 1.0% yeast extract, 0.02% MgSO _4, 0.02% CaCl ₂ , 2% ethanol, and 0.005% cellulase. 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).

2) Main culture (2 passages)

Next, 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.

3) Sheet conversion culture (main culture mixture mixing and tray dispensing) and washing

Finally, 5-10% of the main culture of Komaga bacterial bacterium Lacticus KOSS15 was inoculated and mixed with the culture medium, dispensed into trays, and incubated for 2 days at 0 rpm and 0.3 vvm conditions to induce cellulose sheet conversion (FIG. 2 and 3). After the incubation was over, 0.1% of NaOH was treated overnight to remove excess strain, dehydrated and washed. Subsequently, 5% of sodium percarbonate was treated to remove proteins and impurities and washed to obtain a sheet (FIG. 4). The cellulose sheet produced by the Komagateibacter laticus KOSS15, unlike the cellulose sheet produced by Acetobacter xylinum IFO13693, 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.

3. Preparation of cellulose sheet

3.1. Depending on the type and content of sugar in the medium Cellulose sheet  Dry weight

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.

Species	Content of sugar in the medium	Dry weight of cellulose sheet (18.15cm x 18.15cm g)
K. rhaeticus KOSS15	0.3% honey (0.18% fructose + 0.12% glucose)	0.22
	0.5% honey (0.3% fructose + 0.2% glucose)	0.35
	1.0% honey (0.6% fructose + 0.4% glucose)	0.55
A. xylinum IFO13693	Glucose 2.0%	0.25
	Glucose 3.0%	0.30
	Glucose 5.0%	0.53

Therefore, when manufacturing the sheet of the same weight, it was confirmed that the comagatabacter laticus KOSS15 can reduce the content of sugar in the medium by about 6 times compared to acetobacter xylinum IFO13693. In addition, it was confirmed that even when fructose and glucose contained in a ratio of 3: 2 instead of honey were used as sugar sources, an equivalent effect was exhibited.

3.2. Composition of cellulose sheet according to sugar content and content in the medium

The content of the constituent sugars of the cellulose sheet was confirmed.

Strains and sugar	Party member	glucose(%)	Cellobiose (%)	Etc(%)	Sheet formation state
A. xylinum IFO13693	Glucose2.5%	18.9	54.7	26.4	Good
	Fructose 2.5%	16.4	58.9	24.7	Good
	Sugar 2.5%	-	-	-	Bad
	Honey 2.5%	-	-	-	Bad
K. rhaeticus KOSS15	Glucose2.5%	22.6	65.0	12.4	Bad
	Fructose 2.5%	27.7	67.1	5.2	Good
	Sugar 2.5%	-	-	-	Bad
	Honey 2.5%	32.3	59.4	8.3	Good

3.3. Properties of cellulose sheet according to sugar content in the medium

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).

In addition, the wet burst strength of the sheet was measured by increasing the fluid pressure (KS M ISO 2758).

Strains and sugar	Wet tensile strength-vertical (kN / m)	Wet elongation-vertical (%)	Wet burst strength kPa	Sheet formation state
A.xylinum IFO13693 (glucose)	0.32	7.9	149	Good
A.xylinum IFO13693 (fructose)	0.49	12.5	104	Good
A.xylinum IFO13693 (sugar)	-	-	-	Bad
A.xylinum IFO13693 (honey)	-	-	-	Bad
K. rhaeticus KOSS15 (glucose)	1.30	9.0	193	Bad
K. rhaeticus KOSS15 (fructose)	0.41	6.2	68	Good
K. rhaeticus KOSS15 (sugar)	-	-	-	Bad
K. rhaeticus KOSS15 (honey)	1.03	9.1	264	Good

3.4. Comparison of physical properties of constituent sugars and cellulose sheets according to the concentration of honey in the medium

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).

Honey concentration	step	fruit sugar(%)	glucose(%)	Sugar(%)	Dry weight (g)	Sheet properties
0.3%	Before culture	0.14	0.00	0.03	-	Good tactile feel, slipperiness
	After culture	0.11	0.00	0.06	0.22
0.5%	Before culture	0.23	0.07	0.05	-	Good tactile feel, slipperiness
	After culture	0.13	0.00	0.06	0.35
1.0%	Before culture	0.45	0.21	0.08	-	Good touch, thick feel
	After culture	0.17	0.00	0.10	0.55

As shown in Table 4, it was confirmed that the proportion of constituent sugar and the dry weight of the sheet increased in proportion to the concentration of honey contained in the medium.

3.5. Comparison of dry weight of constituent sugars and cellulose sheets according to the type of honey in the medium

In order to confirm the production efficiency of the cellulose sheet according to the type of honey, 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.

Table 5 shows the results.

Honey types	Sample status	fruit sugar(%)	glucose(%)	Sugar(%)	Dry weight (g)
Specification honey	Before sterilization	0.27	0.18	0.00	0.31
	After sterilization	0.22	0.13	0.00
	After culture	0.08	0.00	0.18
Acacia honey	Before sterilization	0.27	0.14	0.00	0.42
	After sterilization	0.24	0.13	0.00
	After culture	0.10	0.00	0.17
Wildflower Honey	Before sterilization	0.28	0.14	0.00	0.44
	After sterilization	0.24	0.13	0.00
	After culture	0.08	0.00	0.16

As shown in Table 5, the dry weight (production efficiency) according to the type of honey used for cellulose production was all confirmed to be excellent, among them, acacia honey and wildflower honey (honey) were more excellent, and wildflower honey (honey) It was confirmed that this is the best (Fig. 6 and 7).

4. K. rhaeticus KOSS15's  Cellulose Gene Analysis

4.1. Identification of new cellulosic gene

IFO13693 and KOSS15 were sequenced through Pacbio sequence analysis, a type of next generation sequencing (NGS). For the IFO13693 strain, chromosome 3,539,432 bp was identified. In the case of the 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

Since the specific parts of the present invention have been described in detail above, it is obvious that for those skilled in the art, these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto.

[Accession Number]

Depository name: Korea Microbial Conservation Center (Overseas)

Accession number: KCCM12270P

Date of Deposit: 20180530

Claims (15)

1. Komagataeibacter rhaeticus KOSS15 strain, deposit number KCCM12270P.
2. The strain of claim 1, wherein the strain of Komagateibacter latiscus KOSS15 has a cellulosic capacity.
3. Composition for the production of cellulose sheets comprising a strain of Komagataeibacter rhaeticus KOSS15 or a culture of the strain, with accession number KCCM12270P.
4. Method for producing a cellulose sheet comprising the step of culturing the Komagataeibacter rhaeticus KOSS15 strain having accession number KCCM12270P.
5. The method of claim 4, wherein the Komaga bacterial bacterium KOSS15 strain is (a) honey or (b) glucose and fructose; And culturing in a culture medium comprising proline.
6. According to claim 5, wherein the sugar is (a) 0.1 to 5% by volume of honey; Or (b) 0.1 to 5% by weight of glucose and fructose.
7. The method of claim 5 or 6, wherein the glucose and fructose are included in a weight ratio of 2: 3.
8. A method for preparing a bacterial cellulose sheet using Komagataeibacter rhaeticus , comprising the following steps:

   (A) pre-culturing step of stirring the strain in a pre-culture medium containing sugar and cellulase;

   (B) the main culture step of adding the pre-culture solution of the above step to the main culture medium containing sugar and proline and stirring culture; And

   (C) sheet conversion culture step of adding the main culture solution of the above step to a culture medium containing sugar and proline and stationary culture.
9. The method of claim 8, wherein the pre-culture medium is 0.1 to 10.0% by weight of yeast extract, 0.002 to 0.2% by weight of MgSO _4, 0.002 to 0.2% by weight of CaCl ₂ , 0.2 to 20.0% by weight of ethanol, and 0.0005 to 0.05% by weight of cellulase or these. The method comprising a combination of.
10. The method of claim 8, wherein the culture medium of the main culture medium and step (c) is 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 A method comprising 0.02 to 2.0% by weight of acetic acid, or a combination thereof.
11. The method according to claim 8, wherein the sugar is honey or (b) glucose and fructose.
12. The method according to any one of claims 8 to 11, wherein the sugar is (a) 0.1 to 5% by volume of honey; Or (b) 0.1 to 5% by weight of glucose and fructose.
13. The method of claim 11 or 12, wherein the glucose and fructose are included in a weight ratio of 2: 3.
14. The method according to any one of claims 8 to 13, wherein the pre-incubation step is agitated and cultured at 100 to 200 rpm for 24 to 72 hours.
15. 15. The method according to any one of claims 8 to 14, wherein the comagatabacter laticus is a strain of Komagataeibacter rhaeticus KOSS15 with accession number KCCM12270P.

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