KR101970439B1 - A novel Komagataeibacter rhaeticus Strain and Method for Producing Cellulose Sheet Using the Same - Google Patents

Abstract

The present invention provides a Komagataeibacter rhaeticus KOSS-15 strain, a composition for producing a cellulose sheet comprising the strain or a culture, and a method for producing a cellulose sheet comprising a step of culturing the strain. The cellulose sheet produced by the Komagataeibacter rhaeticus KOSS-15 strain of the present invention exhibits high tensile strength and burst strength as compared with the cellulose sheet of Acetobacter xylinum, which is conventionally known as the most excellent microorganism in the production yield of cellulose, and it is possible to reduce, by 5-6 times, a content of the saccharide source necessary for producing a cellulose sheet of the same weight.

Description

[0001] The present invention relates to a novel coma strain, and more particularly, to a method for producing a cellulosic sheet using the same,

The present invention relates to a novel coma strain, and a method for producing a cellulose sheet using the same.

Sheet-type masks used in the cosmetics field mainly include (i) nonwoven fabrics made of vegetable cellulose fibers such as cotton and pulp, or synthetic fibers such as nylon and DuPont, (Ii) hydrogel masks made from natural polysaccharides such as natural agar, carrageenan, locust bean gum, and (iii) bacterial cellulose (bacterial cellulose, biocellulose) There are three types of bacterial cellulosic masks that are immersed in fresh water bodies and used as mask-shaped supports.

The sheet-type mask pack used in the cosmetics field has a great advantage in the cosmetics market because it is possible to obtain a moisturizing and cleaning effect and the like, by attaching a face-shaped sheet without needing to be hand-worn.

Cellulose (aka, bacterial cellulose) made by culturing microorganisms has recently been used as a material for healing drugs for medicines and cosmetic mask packs. Cellulose is the most abundant biomaterial resource in the natural world. In particular, the cellulose produced by the acetic acid bacteria is attracting much attention as a high value-added industrial new material as well as a food additive. In addition, the cellulose produced by the bacteria is superior in physicochemical properties to the cellulose produced by the plant.

Strains producing bacterial celluloses include Acetobacter Agrobacterium sp., Rhizobium sp., Pseudomonas sp., and Sarcina sp., among others. Among the acetobacterium strains, A. xylinum , A. pasteurinanus and A. hansenii are well known, and among them, the microorganism having the highest yield of cellulose is Acetobacter xylinum (KIC News, 2013 16 (4) 37-45).

Microorganisms producing such bacterial celluloses are known to have low production efficiency, and therefore, there is an urgent need to develop new microorganisms having high efficiency of cellulosic production.

Korea Patent Publication 2014-0130569 Korea Patent Publication 2018-0056929

 Satoshi Masaokaet al. Journal of fermentation and bioengineering 1993 75 (1): 18-22  Rachel Machado et al. Carbohydrate polymers 2016 152: 841-849

The present inventors have searched for new strains and changed the quality and production efficiency of bacterial cellulose according to the source of the culture in order to develop a method of producing the bacterial cellulase with improved quality and improved production efficiency. As a result, a novel coma has been obtained from Komagataeibacter The inventors of the present invention have completed the present invention by identifying a KOSS-15 strain and confirming that the above-mentioned strain has high cellulosic production ability by using honey as a sugar source and that the quality of the produced bacterial cellulose is excellent.

Accordingly, it is an object of the present invention to provide a strain of T. bacteriatis KOSS-15.

Another object of the present invention is to provide a composition for producing a bacterial cellulose sheet.

It is still another object of the present invention to provide a method for producing bacterial cellulose.

According to one aspect of the present invention, the present invention provides a strain of Komagataeibacter rhaeticus KOSS-15, which is Deposit No. KCCM12270P.

The present inventors have searched for new strains and changed the quality and production efficiency of bacterial cellulose according to the source of the culture in order to develop a method of producing the bacterial cellulase with improved quality and improved production efficiency. As a result, a novel coma has been obtained from Komagataeibacter The inventors of the present invention have completed the present invention by identifying a KOSS-15 strain and confirming that the above-mentioned strain has high cellulosic production ability by using honey as a sugar source and that the quality of the produced bacterial cellulose is excellent.

According to one embodiment of the present invention, the Komai gabi Lacticus KOSS-15 strain is derived from black mushroom kombucha.

The above-mentioned black tea mushroom seeds are a kind of organic acid-producing bacteria (lactic acid bacteria), which are called as bear bamboo, black tea mushroom yeast, red mushroom and longevity mushroom. The black mushroom strain is a bacterium that increases acidity by fermenting alcohol produced by yeast such as A. xylinum with acetic acid and other acids, and bacteria such as Brettanomyces, Zygosaccharomyces ( Zygosaccharomyces). Is a symbiotic yeast such as Candida, Schizosaccharomyces, and Saccharomyces.

According to one embodiment of the present invention, the Coma tachyta lacticus KOSS-15 strain has cellulose production ability.

As used herein, the term " cellulose " refers to a polysaccharide in which the polymer forms a polymer through a glucosidic bond, the formula is (C ₆ H ₁₀ O ₅ ) n, and the unit is a cellobiose do.

According to another embodiment of the present invention, the Coma teichteracticus KOSS-15 strain produces honey by using honey as a source of cellulose.

As used herein, the term " honey " refers to a viscous substance collected by bees from a plant's nectar and includes a high content of glucose, fructose, as well as various vitamins, proteins, and minerals.

According to another aspect of the present invention, the present invention relates to a composition for preparing a cellulose sheet, which comprises a strain of KOMAGA T. lactis var. KOSS-15, or a culture of said strain, having the accession number KCCM12270P.

The term " culture product " of the present invention refers to a cultured product obtained by culturing the strain for a certain period in a medium capable of supplying nutrients so that the strain of the present invention, Teabactaracticus KOSS-15, can grow and survive in vitro Means a medium containing a strain, its metabolites, extra nutrients, and the like. Since the T. bacterium Laticus strain KOSS-15 is a strain having cellulase production ability, the T. bacterium Laticus strain KOSS-15 or its culture is used as a composition for producing cellulose sheet from cellulose to produce cellulose .

According to still another aspect of the present invention, the present invention relates to a method for producing a cellulose sheet, which comprises culturing a strain of Kombagativa lacticus KOSS-15, which has a deposit number KCCM12270P.

The above-mentioned T. lactis strain KOSS-15 can produce cellulose by using honey, fructose, glucose or a combination thereof as a sugar source.

According to one embodiment of the present invention, the Coma japonicus Lacticus KOSS-15 strain comprises (a) honey or (b) glucose and fructose; And proline. ≪ / RTI >

The culture medium contains honey in an amount of 0.1 to 2.0 vol%, 0.2 to 2.0 vol%, 0.3 to 2.0 vol%, 0.4 to 2.0 vol%, 0.5 to 2.0 vol%, 0.6 to 2.0 vol%, 0.7 to 2.0 vol% 0.1 to 1.4 volume%, 0.1 to 1.9 volume%, 0.1 to 1.8 volume%, 0.1 to 1.7 volume%, 0.1 to 1.6 volume%, 0.1 to 1.5 volume%, 0.1 to 1.4 volume%, 0.1 to 1.3 volume% , 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 contains 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% of glucose and fructose, 0.1 to 1.6 wt%, 0.1 to 1.5 wt%, 0.1 to 1.4 wt%, 0.1 to 1.2 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 1.2% by weight, 0.1 to 1.1% by weight, 0.2 to 1.8% by weight, 0.2 to 1.6% by weight, 0.2 to 1.4% by weight or 0.2 to 1.2% by weight.

The culture medium contains 0.01 to 1.0% by weight, 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, 0.01 to 0.6% by weight, 0.01 to 0.4% by weight, 0.01 to 0.2% by weight, 0.02 to 0.6% by weight, 0.03 to 0.6% by weight, 0.04 to 0.6% by weight, 0.05 to 0.4% by weight %, 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 major sugars and amino acids that constitute royal jelly. In the cultivation of the Koma gigantei Lacticus KOSS-15 strain, the main sugars and amino acids constituting royal jelly instead of honey are added to produce bacterial cellulose can do.

The composition for preparing a cellulose sheet of the present invention may further comprise a carbon source, a nitrogen source, and a phosphorus source for culturing a thalassemperticus KOSS-15 strain.

Wherein the carbon source is selected from the group consisting of ethanol, glucose, sucrose, proctose, galactose, solubilast, inositol, glycerol, xylose, dextrose, lactose, dextrin, adonitol, mannitol, mannose, maltose and raffinose Carbon sources such as, but not limited to, carbon sources.

Wherein the nitrogen source comprises at least one organic nitrogen source selected from the group consisting of yeast extract, peptone, soybean, casamino acid, tryptone and malt extract, NH ₄ Cl, (COONH ₄ ) ₂ , NH ₄ H ₂ PO ₄ , ₄ ) ₂ HPO ₄ , NH ₄ NO ₃ , NaNO ₃ and (NH ₄ ) ₂ SO ₄ .

Such personnel include, but are not limited to, one or more personnel selected from the group comprising potassium dihydrogenphosphate, dipotassium hydrogenphosphate, sodium dihydrogenphosphate and disodium hydrogenphosphate.

The composition for preparing a cellulose sheet may further contain at least one selected from the group consisting of MgSO ₄ , CaCl ₂ , KCl, BaCl ₂ , Li ₂ SO ₄ , MnSO ₄ , MnSO ₄ , ZnSO ₄ , FeSO ₄ , Na ₂ MoO ₄ , KH ₂ PO ₄ , But is not limited to, FeCl ₃ .

According to another aspect of the present invention, the present invention relates to a method for producing a bacterial cellulosic sheet using a Kombageti bacteraracticus KOSS-15 strain, accession number KCCM12270P, comprising the steps of:

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

(b) a main culture step in which the pre-culture solution of the above step is added to the present culture medium containing glycogen and proline and stirred and cultured; And

(c) a sheet conversion culture step in which the present culture medium in the above step is added to a culture medium containing glycogen and proline, followed by constant culture.

The process for producing the bacterial cellulose sheet of the present invention will be described step by step.

(a) Pre-culture  step

First, pre-culture is carried out in which the strain KCMM12270P KOMAGAI Bacterial Lacticus KOSS-15 is stirred and cultured in a preculture medium containing a sugar source and a cellulase agent.

According to one embodiment of the present invention, the preculture medium comprises a saccharide.

In one embodiment of the present invention, the species and concentration of the glycogen are (a) 0.1 to 5 volume (v / v) of honey or (b) 0.1 to 5 weight% (w / v) glucose and fructose.

According to a specific embodiment of the present invention, the culture medium of the present invention contains 0.1 to 5% by volume, 0.1 to 4% by volume, 0.1 to 3% by volume, 0.1 to 2.5% by volume, 0.1 to 2% by volume, By volume, or 0.1 to 1% by volume.

In another embodiment of the present invention, the culture medium of the present invention contains honey in an amount of 0.1 to 3.0% by volume, 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% From 0.3 to 2.0 volume%, from 0.4 to 2.0 volume%, from 0.7 to 3.0 volume%, from 0.8 to 3.0 volume%, from 0.9 to 3.0 volume%, from 1 to 3.0 volume%, from 0.1 to 2.0 volume%, from 0.2 to 2.0 volume% 0.1 to 1.8% by volume, 0.1 to 1.7% by volume, 0.1 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% by volume, 0.9 to 2.0% 0.1 to 1.4 volume%, 0.1 to 1.3 volume%, 0.1 to 1.2 volume%, 0.1 to 1.1 volume%, 0.2 to 1.8 volume%, 0.2 to 1.6 volume%, 0.2 to 1.4 volume% Vol%, or 0.2 to 1.2% by volume.

According to another specific embodiment of the present invention, the culture medium composition of the present invention comprises 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 wt%, or 0.1 to 1 wt%.

In another embodiment of the present invention, the culture medium of the present invention comprises 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% of glucose and fructose, 0.1 to 2.0% by weight, 0.2 to 2.0% by weight, 0.3 to 2.0% by weight, 0.4 to 3.0% by weight, 0.6 to 3.0% by weight, 0.7 to 3.0% by weight, 0.8 to 3.0% by weight, 0.9 to 3.0% 0.9 to 2.0 wt%, 0.1 to 1.9 wt%, 0.1 to 1.8 wt%, 0.1 to 1.7 wt%, and 0.1 wt% 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% 0.1 to 1.2 weight%, 0.1 to 1.1 weight%, 0.2 to 1.8 weight%, 0.2 to 1.6 weight%, 0.1 to 1.6 weight%, 0.1 to 1.5 weight%, 0.1 to 1.4 weight%, 0.1 to 1.3 weight% , 0.2 to 1.4 wt%, or 0.2 to 1.2 wt%.

According to another embodiment of the present invention, the glucose and fructose are contained in a weight ratio of 2: 3. The ratio of 2: 3 is derived from the ratio of glucose and fructose contained in royal jelly.

According to one embodiment of the present invention, the preculture medium comprises 0.1 to 10.0 wt% of yeast extract, 0.002 to 0.2 wt% of MgSO _4, 0.002 to 0.2 wt% of CaCl ₂ , 0.2 to 20.0 wt% of ethanol, and 0.0005 to 0.05 wt% of cellulase %, Or a combination thereof.

In the present specification, pre-culture refers to a step of culturing the stored cellulosic producing strain to a relatively small scale (for example, 100 L or less) in order to activate or boost the cellulosic producing strain.

The pre-culture medium may contain 0.1 to 10.0 wt%, 0.5 to 10.0 wt%, 0.7 to 10.0 wt%, 0.9 to 10.0 wt%, 0.1 to 8.0 wt%, 0.1 to 6.0 wt%, 0.1 to 4.0 wt% 0.5 to 2.0% by weight, 0.5 to 10.0% by weight, 0.5 to 8.0% by weight, 0.5 to 6.0% by weight, 0.5 to 4.0% by weight, 0.5 to 2.0% by weight, 0.9 to 10.0% by weight, 0.9 to 8.0% 6.0% by weight, 0.9% by weight to 4.0% by weight, 0.9% by 2.0% by weight or 0.9% by weight to 1.5% by weight.

Wherein the preculture medium comprises 0.002-0.2 wt%, 0.005-0.2 wt%, 0.008-0.2 wt%, 0.01-0.2 wt%, 0.013-0.2 wt%, 0.015-0.2 wt%, 0.01-0.15 wt% of MgSO ₄ , 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 preculture medium comprises 0.002 to 0.2%, 0.005 to 0.2%, 0.008 to 0.2%, 0.01 to 0.2%, 0.013 to 0.2%, 0.015 to 0.2%, 0.01 to 0.15% by weight of CaCl ₂ , 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 preculture medium comprises ethanol in an amount of 0.2 to 20.0% by volume, 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% To 11.0 vol%, 1.0 to 8.0 vol%, 1.0 to 5.0 vol%, or 1.0 to 3.0 vol%.

The pre-culture medium may contain 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 a cellulase.

According to an embodiment of the present invention, the pre-culturing step is agitated at 100 to 200 rpm for 24 to 72 hours.

The pre-culturing step may be performed for 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, 42 to 54 hours or 42 to 52 hours.

According to another embodiment of the present invention, the pre-culturing step is agitated at 160 rpm for 48 hours.

(b) Cultivation  step

Next, the pre-culture solution of the above step is added to the present culture medium containing the above-mentioned saccharide and proline, followed by agitation.

The present culture medium comprises 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, .

The present culture medium contains proline in an amount of 0.01 to 1.0 wt%, 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 0.01 to 0.6% by weight, 0.01 to 0.4% by weight, 0.01 to 0.2% by weight, 0.02 to 0.6% by weight, 0.03 to 0.6% by weight, 0.04 to 0.6% by weight, 0.05 to 0.4% by weight, 0.06 to 0.3% by weight, 0.04 to 0.3% by weight, 0.04 to 0.2% by weight or 0.05 to 0.15% by weight.

The present culture medium contains 0.002 to 0.2 wt%, 0.005 to 0.2 wt%, 0.008 to 0.2 wt%, 0.01 to 0.2 wt%, 0.013 to 0.2 wt%, 0.015 to 0.2 wt%, 0.01 to 0.15 wt% of MgSO ₄ , 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 present culture medium contains 0.002 to 0.2 wt%, 0.005 to 0.2 wt%, 0.008 to 0.2 wt%, 0.01 to 0.2 wt%, 0.013 to 0.2 wt%, 0.015 to 0.2 wt%, 0.01 to 0.15 wt% of CaCl ₂ , 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 present culture medium comprises 0.01 to 1.0 wt%, 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% of sodium acetate, 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% 0.4 wt%, 0.06-0.3 wt%, 0.04-0.3 wt%, 0.04-0.2 wt%, or 0.05-0.15 wt%.

The present culture medium may contain 0.02 to 2.0 wt%, 0.05 to 2.0 wt%, 0.1 to 2.0 wt%, 0.15 to 0.1 wt%, 0.1 to 1.5 wt%, 0.1 to 1.0 wt%, or 0.1 to 0.5 wt% of acetic acid have.

(c) step: sheet conversion culture step

Finally, the present culture in the above step is added to a culture medium containing glycogen and proline, followed by stationary culture to transform the sheet.

The culture medium has the same composition as the present culture medium of step (b).

The method of producing the bacterial cellulose sheet of the present invention is similar to that of the above-mentioned Coma teabactaracticus KOSS-15 strain, the culture of the strain and the composition for producing a cellulose sheet, The description thereof will be omitted.

The present invention is based on the finding that the coma is a Komagataeibacter rhaeticus KOSS-15 strain, a composition for preparing a cellulose sheet comprising the strain or the culture, and a method for culturing the strain. The cellulosic sheet produced by the Coma gutta lacticus KOSS-15 strain of the present invention exhibits high tensile strength and tear strength as compared with the cellulose sheet of Acetobacter xylinum, which is known as the most excellent microorganism in the production yield of the conventional cellulose, It is possible to reduce the content of the saccharide source necessary for producing the cellulose sheet having the same weight by 5-6 times.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the phylogenetic number of a strain of T. bacteriatis KOSS-15.
FIG. 2 shows a process for producing cellulose using a strain of T. bacteriaceticus KOSS-15.
Fig. 3 schematically shows a process for producing a cellulose sheet through subculture of a strain of T. bacteriatis KOSS-15.
Fig. 4 shows a cellulose sheet prepared by culturing a strain of T. bacteriatis KOSS-15.
Fig. 5 shows the cellulose sheet of Coma followed by cellulase treatment with Teabacter lactis KOSS-15 and Acetobacter xylenes IFO13693.
Fig. 6 shows an image of a cellulose sheet produced by Teabactor Laticus KOSS-15 according to the concentration of honey in the culture medium.
Fig. 7 shows an image of a cellulose sheet produced by Teabactor Laticus KOSS-15 according to the kind of honey in the culture medium.
Fig. 8 shows the results of the genome analysis of the cellulase-producing gene cluster of Coma japonica Lacticus KOSS-15 and Acetobacter xylenol IFO13693.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Throughout this specification, "%" used to denote the concentration of a particular substance is intended to include solids / solids (wt / wt), solid / liquid (wt / The liquid / liquid is (vol / vol)%.

Example

1. Selection of strains

Three liters of brown rice green tea were poured into 1 L of water, 30% (w / v) of sugar was added, and 500 ml was added to each flask. The mixture was sterilized and cooled at 121 캜 for 30 minutes. (V / v) were inoculated in the incubator at 28 ° C in the incubator at the temperature of 28 ° C, respectively, and 5 kinds of the obtained mushroom extracts (Vietnamese mushroom, Jiri green tea mushroom, Bosung tea mushroom, Chinese Yunnan mushroom and Tibet tea mushroom) Lt; / RTI > After confirming that cellulose was formed from 2 weeks of culture, the bacteria were taken in one experiment and 2% of glucose, 1% of yeast extract, 0.02% of magnesium sulfate, 0.02% of calcium chloride and 1.8% of agar were added. After adjusting the pH of the medium to 4.0 with acetic acid, agar medium was prepared. One of the five solutions of agar medium (Jiri green tea mushroom) was plated in 0.1 ml and cultured at 28 ° C for 7 days. Colonies having the same shape and bacterium shape were separated into six groups, and then the culture medium containing 60 ml of glucose 2%, yeast extract 1%, magnesium sulfate 0.02% and calcium chloride 0.02% was sterilized. Thereafter, 10 ml of the sterilized medium was dispensed, and 6 colonies were inoculated, respectively.

After culturing at 28 ° C for 3 days, 40 ml of acetic acid pre-culture medium was prepared, and 4 ml of the culture solution was incubated for 3 days in an acetic acid pre-culture medium and fixed at 28 ° C to observe the formation of cellulose after 4 days . Finally, colonies with a good cellulose form were labeled and a fungusstock was prepared. The most well-formed colonies were named KOSS-15 and KCCM12270P, respectively.

2. Culture of strain

(1) Culture of Acetobactorxylinum IFO13693 strain (ATCC 53582)

1) Pre-cultivation (1 strain)

Acetobactor xylinum IFO13693 (ATCC 53582) was used as a control to compare the cell biotransformation efficiency of the coma teabactaracticus KOSS-15. The Acetobacter xylenol is a representative cellulose production strain.

The culture medium containing 2% of glucose, 1.0% of yeast extract, 0.02% of MgSO _4, 0.02% of CaCl ₂ , and 2% of ethanol was inoculated with 2.0 × 10 ⁷ CFU / ml of Acetobacterium xylenol IFO 13693, And the cells were pre-cultured for 48 hours.

2) Main culture (2 passages)

Subsequently, the slurry produced in the pre-culture was filtered through a sieve of 80 mesh and inoculated with 10% of the culture. The 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% vvm (aeration volume / medium volume / minute) for 48 hours.

3) Sheet conversion culture (mixing of culture and tray dispensing) and washing

Finally, the present cultures of acetobacterium xylenol IFO13693 were inoculated with 5-10% of the culture medium and mixed with each other, dispensed into trays and incubated for 3 days at 0 rpm and 0.3 vvm to induce cellulosic sheet conversion 2 and 3). After the culture was completed, the prepared cellulose sheet was dewatered and treated with 0.1% NaOH to remove excess strains and washed. Then 5% sodium carbonate was treated to remove proteins and impurities and wash to obtain sheets (Figure 4).

(2) Culture of Coma tei Bacteria Lacticus KOSS-15 (KCCM12270P)

1) Pre-cultivation (1 strain)

To the culture medium containing the glycogen (0.6% fructose and 0.4% glucose or 1% honey), yeast extract 1.0%, MgSO ₄ 0.02%, CaCl ₂ 0.02%, ethanol 2% and cellulase 0.005% KOSS-15 was inoculated at 1.0 x ¹⁰ < ⁸ > CFU / ml and preincubated in Flask at 30 DEG C with stirring at 80-160 rpm for 48 hours. The cellulase was added to prevent the development of pellicle during pre-culture, and when the cellulase was added to the pre-culture medium of the acetobacterium xylenol IFO13693 of the above (1), defective sheets were formed and were not applied 5).

2) Main culture (2 passages)

Next, the pre-culture was inoculated 10% of the culture medium. The above culture medium was prepared by adding a sugar source (0.6% fructose, 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 culture was agitated at 80 rpm and 0.3 vvm for 48 hours.

3) Sheet conversion culture (mixing of culture and tray dispensing) and washing

Finally, the coma was fed to a tray at 5-10% of the culture medium of the Teabactoracticus KOSS-15 and mixed with the culture medium and cultured at 0 rpm and 0.3 vvm for 2 days to induce cellulosic sheet conversion (Figs. 2 and 3). After the incubation, 0.1% NaOH was added overnight to remove excess strains, dehydrated and washed. Subsequently, 5% sodium percarbonate was treated to remove proteins and impurities and wash to obtain a sheet (FIG. 4). Unlike the cellulose sheet produced by Acerobacterium xylenol IFO 13693, the cellulosic sheet produced by Teema Bacteriacticus KOSS-15 is superior in strength when the sheet is first immersed in the NaOH aqueous solution before dehydrating the sheet .

3. Preparation of cellulose sheet

3.1. Depending on the type and content of the source in the medium Of cellulose sheet  Dry weight

The washed cellulose sheet was flattened and cut to 18.15 x 18.15 cm, and the weight was measured. After the measurement, the resultant was dried in a drying oven at 95 DEG C for 1 hour and weighed to determine the weight before and after drying.

The content of the glycogen in the medium of Table 1 below means the content of glycogen in the culture medium at the time of cellulosic sheet conversion culture.

Fungi Content of the party members in the medium Dry weight of cellulose sheet
(G per 18.15 cm x 18.15 cm) K. rhaeticus KOSS-15 Honey 0.3%
(0.18% fructose + 0.12% glucose) 0.22 Honey 0.5%
(0.3% fructose + 0.2% glucose) 0.35 Honey 1.0%
(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

Thus, when producing sheets of the same weight, it was confirmed that Coma gutta lacticus KOSS-15 can reduce the content of glycogen in the medium by about 6 times as compared with Acetobacter xylenol IFO13693. In addition, it was confirmed that the same effect was obtained when fructose and glucose contained in a ratio of 3: 2 instead of honey were used as a sugar source.

3.2. Composition of cellulose sheet according to content and content in media

The constituent sugar content of the cellulose sheet was confirmed.

Strain and member Party member glucose(%) Cellobiose (%) Other(%) Sheet formation state A. xylinum IFO13693 glucose
2.5% 18.9 54.7 26.4 Good fruit sugar
2.5% 16.4 58.9 24.7 Good Sugar
2.5% - - - Bad Honey 2.5% - - - Bad K. rhaeticus KOSS-15 glucose
2.5% 22.6 65.0 12.4 Bad fruit sugar
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. Physical Properties of Cellulose Sheet According to the Party Member in Media

The tensile strength of the sheet was measured (KS M ISO 1924-2) using a test machine to which a constant elongation at break (20 mm / min) was applied.

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

Strain and member Wet Tensile Strength - Length
(kN / m) Wet Elongation - Vertical
(%) Wet rupture strength
kPa Sheet formation state A. xylinum IFO13693
(glucose) 0.32 7.9 149 Good A. xylinum IFO13693
(fruit sugar) 0.49 12.5 104 Good A. xylinum IFO13693 (sugar) - - - Bad A. xylinum IFO13693
(honey) - - - Bad K. rhaeticus KOSS-15
(glucose) 1.30 9.0 193 Bad K. rhaeticus KOSS-15
(fruit sugar) 0.41 6.2 68 Good K. rhaeticus KOSS-15
(Sugar) - - - Bad K. rhaeticus KOSS-15
(honey) 1.03 9.1 264 Good

3.4. Comparison of properties of constituent sugar and cellulose sheet according to honey concentration in medium

Cellulose sheet was prepared in the same manner as in Example 2 except that the kind and content of the sugar source of the medium used in the sheet conversion culture step were respectively 0.3% of honey, 0.5% of honey, and 1.0% of honey, . The types and contents of sugar sources in the medium were measured according to the concentration of honey before and after culturing, and the properties of the cellulose sheet prepared according to 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 touch, slippery After incubation 0.11 0.00 0.06 0.22 0.5% Before culture 0.23 0.07 0.05 - Good touch, slippery After incubation 0.13 0.00 0.06 0.35 1.0% Before culture 0.45 0.21 0.08 - Feeling good, thick feeling After incubation 0.17 0.00 0.10 0.55

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

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

In order to confirm the production efficiency of the cellulose sheet according to the kinds of honey, the sugar sources of the medium used in the sheet conversion culture step were respectively 0.5% of the specimen honey, 0.5% of the acacia honey, 0.5% of the wild flower honey %, The cellulose sheet was prepared in the same manner as in Example 2 above. The type and content of the glycoproteins in the medium were measured according to the type of honey before and after the culture, and the dry weight of the cellulosic sheet prepared for each type of honey contained in the medium was compared.

The results are shown in Table 5.

Honey type 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 incubation 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 incubation 0.10 0.00 0.17 Wildflower honey
(Honey) Before sterilization 0.28 0.14 0.00 0.44 After sterilization 0.24 0.13 0.00 After incubation 0.08 0.00 0.16

As shown in Table 5, the dry weight (production efficiency) according to the type of honey used in the cellulose production was found to be excellent. Among them, acacia honey and wild honey (honey) (Figs. 6 and 7).

4. K. rhaeticus KOSS -15 Cellulose Generation Analysis

4.1. Identification of New Cellulose Genes

Sequencing of IFO13693 and KOSS15 was performed through PacBio sequencing, a type of next generation sequencing (NGS). In the case of IFO13693 strain, chromosome 3,539,432 bp was confirmed. In the case of KOSS15 strain, 3,275,555 bp of chromosome and 3 plasmids were confirmed. On the basis of NGS, the production gene of each bio-cellulose was confirmed in two kinds of production strains (Fig. 8).

- gene A. xylinum IFO13693 K. rhaeticus KOSS-15 Type I BcsA I 2950 1826 BcsB I 2951 1824, 1825 BcsC I 2952 1823 BcsD I 2953 1822 Type II BcsBII 717, 1148 3004, 1007 BcsX 718 3007 BcsY 719 3008 BcsCII 720 3009

Korea Microorganism Conservation Center (overseas) KCCM12270P 20180530

Claims (11)

Accession No. KCCM12270P, Komagataeibacter rhaeticus strain KOSS15.
2. The method according to claim 1, wherein the Coma gibbsite Lacticus KOSS15 strain has a cellulose production ability.
Accession No. KCCM12270P Comagataeibacter rhaeticus KOSS15 strain or a culture of said strain.
A method for producing a cellulosic sheet, comprising the step of culturing a strain of Komagataeibacter rhaeticus KOSS15 with accession number KCCM12270P.
5. The method of claim 4, wherein the Coma germicidal KOSS15 strain is selected from the group consisting of (a) honey or (b) glucose and fructose; And proline. ≪ / RTI >
A method for producing a bacterial cellulose sheet using the Komagataeibacter rhaeticus KOSS15 strain, accession number KCCM12270P, comprising the steps of:
(a) a step of pre-culturing the strain in a pre-culture medium containing a sugar source and a cellulase agent;
(b) a main culture step in which the pre-culture solution of the above step is added to the present culture medium containing glycogen and proline and stirred and cultured; And
(c) a sheet conversion culture step in which the present culture medium in the above step is added to a culture medium containing glycogen and proline, followed by constant culture.
7. The method of claim 6, wherein the preculture medium comprises 0.1 to 10.0 wt.% Of yeast extract, 0.002 to 0.2 wt.% Of MgSO _4, 0.002 to 0.2 wt.% Of CaCl ₂ , 0.2 to 20.0 wt.% Of ethanol and 0.0005 to 0.05 wt.% Of cellulase, ≪ / RTI >
7. The method of claim 6, wherein the culture medium and the culture medium of step (c) comprise 0.01 to 1.0 wt.% Of proline, 0.002 to 0.2 wt.% Of MgSO _4, 0.002 to 0.2 wt.% Of CaCl ₂ , 0.01 to 1.0 wt. 0.02 to 2.0 wt% of acetic acid, or a combination thereof.
7. The method of claim 6, wherein the source comprises: (a) from 0.1 to 5% by volume of honey; Or (b) 0.1 to 5% by weight of glucose and fructose.
10. The method of claim 9, wherein the glucose and fructose are included in a weight ratio of 2: 3.
The method according to claim 6, wherein (a) the pre-culturing step is agitated at 100 to 200 rpm for 24 to 72 hours.

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