KR100950113B1 - A new strain Enterobacter ludwigii KU201-3KACC91341P having cellulase and lignolytic activity and animal feed additive microbial agent comprising thereof - Google Patents

Abstract

The present invention is a novel strain Enterobacter Ludwig KU201-3 ( Enterobacter) having fibrinolytic and lignin degrading activity ludwigii KU201-3) (KACC91341P) and a microbial agent for animal feed addition comprising the same, comprising a fungal strain of fibrinolytic and lignin degrading from mushroom waste medium, Enterobacter ruud KU201-3 ( Enterobacter) ludwigii KU201-3) (KACC91341P). In addition, the present invention relates to a microbial preparation for adding livestock feed, comprising mixing an effective amount thereof and an additive thereof in a pure culture or pure culture.

Enterobacter lud crisis, livestock feed, microbial products

Description

Enterobacter lud crisis ＫＵ201-3 (ＫACC91341P), a new strain with fibrinolytic and lignin degrading activity, and a microbial agent for animal feed addition comprising the same additive microbial agent comprising

The present invention enterobacter rude crisis KU201-3 ( Enterobacter) ludwigii KU201-3) (KACC91341P).

The present invention the new strain Enterobacter Lud crisis KU201-3 ( Enterobacter It relates to a microbial agent for the addition of livestock feed using ludwigii KU201-3) (KACC91341P).

Mushroom is composed of moisture, carbohydrate, crude protein, crude fat, crude fiber, and ash, which not only has good protein and essential amino acid composition, but also has abundant trace elements, which plays an important role as a protein source. It is high. Mushrooms have a good taste and aroma and are rich in various nutrients. Also, polysaccharides are known to be anti-cancer and immunomodulatory and are used as health foods. Contains sheep's nutritional sources.

In particular, polysaccharides such as beta glucan (β-Glucan) contained in mushrooms and mushroom media are known to act to prevent the proliferation and metastasis of cancer cells by activating the immune function of cellular tissues. The medium used for mushroom cultivation contains many nutrients but contains a large amount of fiber, which makes it difficult for livestock to digest.However, during mushroom cultivation, the mushroom absorbs and decomposes the fiber, making it easier to digest. Higher nutrients can effectively use nutrients such as vitamins and minerals. Since the mushroom waste medium is high in fiber and contains a lot of hardly decomposable substances, microorganisms isolated from the mushroom waste medium are likely to have the ability to break down the fiber and the hardly decomposable substances. Therefore, the proper use of microorganisms isolated from mushroom waste media can produce microbial preparations that can be useful for livestock feeds that help beneficial microorganisms promote their activity and help feed animal feed degradation.

Korean Patent Application No. 10-2001-0006282 (application date 2001.02.09) relates to a microbial agent for feed addition and a method of manufacturing the same, and more particularly, to increase the livestock, improve feed efficiency and reduce the occurrence of odor due to livestock manure It relates to a microbial agent for the addition of livestock feed and a method for producing the same. More specifically, unlike the conventional muker microorganism that has been mainly used for food and alcohol production, such as cheese or soy sauce, a new muker strain KFCC-11239 has been identified as having an efficiency of suppressing odor by the increase of livestock and livestock manure. Is disclosed.

Korean Patent Application No. 10-2001-0047829 (application date 2001.08.09) relates to a microbial preparation for feed addition and a method of manufacturing the same. Lactobacillus amylophilus, yeast Saccharomyces cerevisiae, and Bacillus subtilis, Bacillus subtilis, were cultured, respectively, and then adsorbed and fixed in a zeolite and a degreasing steel mixture. After aging for 72 hours, lactobacillus amylophyllus adsorbate, Saccharomyces cerevisiae adsorbate, and Bacillus subtilis adsorbate made by drying the water content to 10-12% or less were 5: 3: 2. The present invention relates to a microbial preparation for feed addition, which is formulated at a ratio of and then crushed to a size of 250 to 300 mesh.

International Patent Application No. PCT / AU2000 / 00021 (filed Jan. 14, 2000) is a microbial agent having increased growth / harvestability or increased survival / recovery rate in a product, which is based on or contains indigestible starch. Preparations comprising microorganisms grown or cultured in a medium; It provides a method for producing the same and a product containing the microbial agent.

Although described with respect to the microbial agent for the addition of various feeds as described above, Enterobacter Lud crisis KU201-3 ( Enterobacter isolated and used in the present invention) There is no reference to ludwigii KU201-3) (KACC91341P).

Therefore, the present inventors have isolated and identified the microorganisms in the relevant mushroom waste medium for the development of a fermentation microorganism dedicated to mushroom waste medium with the goal of developing the microorganisms most suitable for the characteristics of the mushroom waste medium.

The present invention enterobacter rude crisis KU201-3 ( Enterobacter) It is an object to provide ludwigii KU201-3) (KACC91341P).

The present invention the new strain Enterobacter Lud crisis KU201-3 ( Enterobacter ludwigii KU201-3) (KACC91341P) is a pure culture of an object of the invention to provide a microbial agent for animal feed is added and a method containing as an active ingredient.

In order to achieve the above object, the present invention provides novel strain Enterobacter Ruud crisis (Enterobater with cellulolytic and lignin-decomposing activity ludwigii ) KU201-3 (KACC91341P).

The present invention enterobacter lude crisis ( Enterobater ludwigii ) It provides a microbial agent for the addition of livestock feed containing the pure culture of KU201-3 (KACC91341P) as an active ingredient.

The present invention comprises the steps of preparing a culture medium; Culturing Enterobater ludwigii KU201-3 (KACC91341P); Collecting the culture solution; Mixing the collected culture solution with an additive; And it provides a method for producing a microbial preparation for livestock feed comprising the step of drying the mixture.

Embodiment of the first aspect of the present invention are novel strain Enterobacter Ruud crisis (Enterobater with cellulolytic and lignin-decomposing activity ludwigii ) KU201-3 (KACC91341P).

The strain according to the present invention was 1% soybean meal as nitrogen source and 2% sucrose as carbon source for optimal growth. Spores do not form when the fungus grows, and when the colonies are formed, the brightness is dark white like rice. Growth is vigorous at moderate temperature (36 ° C), pH is in the range of 6-7. In addition, Enterobater ludwigii KU201-3 strain according to the present invention was deposited with the strain KAKA91341P on December 5, 2007 to the Institute of Agricultural Biotechnology.

As used herein, "fibrinolytic activity" is protease, cellulase (CMCase), amylase or xylanase activity. Proteases are proteolytic enzymes, cellulases are cellulose degrading enzymes, amylases are starch degrading enzymes, and xylanase is xylan degrading enzymes.

As used herein, "lignin degrading activity" is laccase activity. Lactase, also called polyphenol oxidase, breaks down complex lignin molecules by oxidizing phenol residues in lignin to quinones.

A second aspect of the invention is Enterobater ludwigii ) It provides a microbial agent for the addition of livestock feed containing the pure culture of KU201-3 (KACC91341P) as an active ingredient.

A third aspect of the invention comprises the steps of preparing a culture medium; Enterobacter Ruud crisis (Enterobater ludwigii ) culturing KU201-3 (KACC91341P); Collecting the culture solution; Mixing the collected culture solution with zeolite; And it provides a method for producing a microbial preparation for livestock feed comprising the step of drying the mixture.

Enterobacter lud crisis of the present invention ( Enterobater ludwigii ) KU201-3 (KACC91341P) strain can be used as a microbial preparation for livestock feed addition by formulating it as a powder, pellets, granules or solutions by mixing with the carrier itself, or its culture, its extract or its spores alone. As the carrier, water, white carbon, kaolin, zeolite, or the like can be used.

Formulated microbial formulations can be usefully used in livestock feed to help feed degradation of livestock by adding to livestock feed.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are merely to illustrate the present invention is not limited to the contents of the present invention.

< Example  1> Mushroom From waste media  Isolation of Strains with Fibrin and Lignin Degradation Activity

1. Sample Preparation

Waste medium samples for microbial separation were collected in the early summer according to the mushroom type 5 waste medium. Enoki mushroom waste medium was collected from Chungju farm located in Gaeum-myeon, Chungju. The temperature of waste medium was 41 ℃. Sage mushroom waste badges were collected from a farm located in Noeun-myeon, Chungju, and the samples were collected from the hot part (57 ℃) and the medium temperature (40 ℃) as the central temperature of the waste medium rose high. Pleurotus erythrocytes were collected from a farm located in Gaeum-myeon, Chungju.

2. Microbial Separation

10 g of each sample collected was put into 190 ml of sterile water, ground (Homogenizer, Heidalph diax 900) and then diluted appropriately by serial dilution. The dilutions were plate count agar ( PCA , casein peptone 5g, yeast extract 2.5g, dextrose 1.0g, agar 15g), MRS agar, brain heart infusion ( BHI ) agar, yeast and malt. Yeast & malt extract ( YM ) agar, potato dextrose Agar (potatoes dextrose Agar; PDA ) were plated in each medium and incubated for 48 hours at 30 ℃ ( Table 1 ).

According to the colony form formed after the culture, bacteria were purified by PCA, yeast and molds (yeast, molds) in PDA medium. A total of 367 isolates were isolated from 290 bacteria and 77 fungi.

<Composition of Culture Medium> badge Furtherance g / L  PCA Casein peptone 5.0 Yeast extract 2.5 Dextrose 1.0 Agar 15.0     MRS Proteos peptone No.3 10.0 Beef extract 10.0 Yeast extract 5.0 Dextrose 20.0 Polysorbate 80 1.0 Ammonium citrate 2.0 Sodium acetate 5.0 Magnesium sulfate 0.1 Manganese sulfate 0.05 Dipotassium phosphate 2.0 Agar 15.0    BHI Calf brain infusion (200 g) 7.7 Beef heart infusion (250 g) 9.8 Proteose peptone 10.0 Dextrose 2.0 Sodium chloride 5.0 Disodium phosphate 2.5 Agar 15.0   YM Yeast extract 3.0 Malt extract 3.0 Pepton 5.0 Dextrose 10.0 Agar 15.0  PDA Potatoes 4.0 Dextrose 20.0 Agar 15.0

3. Microbial Selection

(1) Selection of fibrinase producing strains

Since the waste medium is high in fiber and contains a lot of hardly decomposable substances, the strains which are the enzymes of xylanase or cellulase (CMCase) as fibrinase among the strains isolated from the waste medium are selected first. It was. The isolated strains were selected from strains showing a clear zone after incubation at 30 ° C. using a screening medium containing 1% of cellulose and xylan as substrates. The xylanase immediately evaluated the clear zone formed after incubation, and in the case of cellulase, after clearing, the medium was stained with Congo red staining solution and washed with 1M NaCl. Clear zones were evaluated.

As a result of the first strain selection, 25 strains were selected, and the selected strains were measured for cellulase, xylanase, amylase and protease enzyme activities using the media shown in Table 1 . . In consideration of the high temperature conditions during fermentation, the enzyme activity and growth at 50 ° C high temperature conditions were also analyzed simultaneously. Clear zone was stained as shown in Table 2, and evaluated according to the size as '+++', '++', '+', '-' ( Table 3 ).

Screening media and dyes used to detect various enzyme activities enzyme temperament dyes Confirm Protease 2% (w / v) skimmed milk Clear zone Cellulase (CMCase) 1% (w / v) CMC ¹ 0.2% (w / v) Congo Red Solution Clear zone Amylase 1% (w / v) soluble starch 0.2% (w / v) I ₂ + 2% (w / v) KI solution Clear zone Xylanase 1% (w / v) Haute Consumption Xylan (oat spelt xylan) Clear zone

^* Basic medium: 5 g of casein peptone, 2.5 g of yeast extract, 1 g of dextrose, 15 g / l of agar

¹ Carboxymethyl cellulose-medium viscosity

<Enzyme Activity of Selected 25 Strains> ¹ Strain number Enzyme activity ² 30 ℃ 50 ℃ Xylanase Cellulase Amylase Protease Xylanase Cellulase Amylase Protease 7 ++ ++ ++ ++ - +++ ++ +++ 11 ++ ++ ++ ++ - +++ +++ ++ 12 ++ + ++ ++ - +++ +++ ++ 89 ++ ++ +++ ++ - +++ ++ ++ 95 + ++ ++ +++ - +++ ++ ++ 96 + ++ ++ +++ - +++ ++ ++ 97 ++ ++ + +++ - ++ +++ ++ 101 + +++ +++ ++ - +++ ++ ++ 104 ++ +++ ++ ++ - +++ +++ ++ 127 ++ ++ +++ ++ - ++ +++ +++ 135 ++ + + - - - + - 148 - ++ + - + +++ +++ ++ 153 - ++ + - + - ++ - 161 + + ++ - + ++ ++ + 162 + + ++ - + +++ ++ - 169 - ++ + - + + - - 196 + ++ +++ +++ - +++ +++ ++ 198 + ++ +++ +++ - - +++ ++ 201 + + ++ +++ - + ++ ++ 203 + + ++ + - ++ + + 205 + + +++ +++ - +++ + + 206 - ++ +++ +++ + +++ + + 207 - ++ + +++ + +++ ++ + 208 - + + - + ++ ++ + 216 +++ +++ + - - +++ + +

¹ growth rate was measured after incubation for 24 hours in plate count agar.

² +++ shows good clear zone formation in screening medium; ++ good; + Medium; - voice

(2) Lignin Degradation Active Microorganism Selection

The selected strains showing fibrinase activity were selected strains showing lignin degradation activity through lignin degradation activity through the discoloration ring generation and strain growth in a liquid broth using lignin as a carbon source.

0.05% remazol brilliant blue R ( RBBR ), 0.01% guiacol ( GU ), 0.1% galic acid GA were added to each of the six strains selected on the agar plate (agar plate) was incubated for 48 hours in a 30 ℃ incubator. In the medium inoculated with the lignin degrading bacteria, the color change ring was formed red around the bacteria. The strain was selected by measuring the size of the color change ring in the same manner as the clear zone measurement method.

As shown in Table 4 , there were no strains showing discoloration in the medium containing RBBR and guaiacol, but four strains (NO. 196, 201) in the medium containing galic acid. , 206, 208) showed a discoloration ring, indicating that it has lignin-degrading activity. In particular, No. 206 showed the best enzyme activity. 196, 201, and 208 showed the same enzymatic activity, but the results of growth analysis and fibrinase activity in a medium supplemented with lignosufonic acid as a carbon source were comprehensively examined. Strain 1 was selected and 201 strain was named KU201-3.

<Lignin Degradation Activity of Selected Six Microorganisms> ¹  Strain number Lignolytic activity ² at 30 ° C Remazol brilliant blue R Guaiacol Gallic acid 96 - - - 162 - - - 196 - - ++ 201 - - ++ 206 - - +++ 208 - - ++

¹ growth rate was measured after 48 hours of incubation in plate count agar.

² +++ shows good clear zone formation in screening medium; ++ good; + Medium; - voice

(3) Determination of Lignin Degradability by Enzyme Activity Measurement

① Laccase activity

As a result of measuring the laccase activity of the selected strains KU201-3 and 206, it was 0.716 unit / min at 8 hours of culture in KU201-3 ( FIG. 1 ).

In order to measure laccase activity, 1.9 ml of coenzyme solution and PBS (pH 7.0) were mixed, and absorbance was measured at 525 ml immediately after adding 0.3 ml of 5 mM p-phenylenediamine, and then absorbance was measured every minute. The activity was calculated by the following formula (Morohoshi, N. et al. 1985. Tokyo Univ. Of Agri. And Tech. 21: 101-105).

Enzyme activity (unit / min) = ΔE / Δt x 15.3846

                            ΔE = absorbance

                     Δt = reaction time (min)

② Manganase peroxidase activity

Manganase peroxidase was measured for each culture time for KU201-3 and 206, which were selected strains, but the enzyme activity was not found.

(4) Measurement of growth curve of selected strains

KU201-3 and 206, the first strains, measured the total growth in PCA medium and measured the growth curve. The two strains showed excellent growth rate, and the KU201-3 strain had a shorter induction period than the 206 strain. At 12 hours the log phase was over and reached the steady state ( FIG. 2 ).

< Example  2> Mushroom From waste media  Identification of Strains with Isolated Fibrinolytic and Lignin Degrading Activity

(1) Identification of isolated strain KU201-3

Identification of selection strains was identified according to the Bergey's Mannual of Systematic Bacteriology. To investigate the physiological and biochemical characteristics of the isolates, sugar fermentation / availability was investigated using API 50 kit (Bio-Merieux, France), and cellular fatty acid profiles and 16S rDNA sequencing were performed. .

Sugar fermentation / availability using the API 50 kit of the isolated strain KU201-3 is shown in Table 5 . This strain is ribose, glucose, fructose, N-acetyl glucosamine, esculine, salicycin, cellobiose, maltose maltose, sacchorose, trehalose, amidone and glycogen have been shown to ferment / use to produce acids. As shown in Table 6 and FIG. 3 , the intracellular fatty acid content was highest in C15: 0 ISO and high in C13: 0 ISO. The result of genetic analysis of 16S rDNA is shown in SEQ ID NO: 1 . Based on this, the% similarity (similarity) was determined by comparison with the control strain. As a result, the strain KU201-3 was found to be a bacterium belonging to the genus Enterobacter , and 98% similarity with the standard strain of Enterobacter ludwigii . It can be called the nearest bacteria. In addition, a phylogenetic tree was created by a neighbor joining method, and the results are shown in FIG. 4 . In this case, the scale bar of the tree refers to 0.01 substitution per site.

As a result of examining the characteristics of the KU201-3 strain, the KU201-3 strain is Enterobacter It was classified as yuyeongyun of ludwigii), and finally Enterobacter Ruud crisis (Enterobacter ludwigii ) identified as KU201-3. In addition, Enterobacter Ruud crisis (Enterobacter ludwigii ) KU201-3 was deposited on December 5, 2007 with the deposit number KACC91341P to the Institute of Agricultural Biotechnology.

<Biochemical Properties of Carbohydrate KU201-3 (Carbohydrates)> ¹ Characteristics KU201-3 Characteristics KU201-3 Glycerol - Esculline + Ertythritol - Salicine + D-arabinose - Cellobiose + L-arabinose - Maltose + Ribose + Lactose + D-Xylose - Melibiose - L-Xylose - Saccharose + Adonitol - Trehalose + β methyl-xyloside  -  Inuline  - Galactose - Melezitose - D-Glucose + D-Raffinose - D-Fructose + Amidon + D-Mannose - Glycogen + L-sorbose - Xylitol - Rhamnose - β Genentiobiose - Dulcitol - D-Turanose - Inositol - D-Resource (Lyxose) - Mannitol - D-Tagatose - Sorbitol - D-Fucose -  α Methyl-D-mannoside  -  L-Fucose  -  α Methyl-D-glucoside  -  D-arabitol  - N acetyl glucosamine  +  L-Arabitol  - Amygdaline - Gluconate -  Arbutine  - 2 aceto-gluconate  - 5 aceto-gluconate  -

¹ '+' = positive; '-' = Voice

<Cellular fatty acid composition of isolate KU201-3> fatty acid % C9: 0 0.19 C11: 0 ISO 0.51 C12: 0 ISO 1.30 C12: 0 0.77 C13: 0 ISO 20.97 C13: 0 ANTEISO 1.57 C14: 0 ISO 3.82 C14: 0 3.54 C15: 0 ISO 35.37 C15: 0 ANTEISO 2.91 C15: 0 - C16: 1 w7c alcohol 0.97 C16: 0 ISO 3.12 C16: 0 2.28 C15: 0 2OH 0.89 ISO C17: 1 w10c 1.86 ISO C17: 1 w5c 4.06 C17: 1 ANTEISO A - C17: 0 ISO 4.71 C17: 0 ANTEISO -

(2) Identification of Separated Strain 206

Sugar fermentation / availability using the API 50 kit of the isolated strain 206 is shown in Table 7 . The strains are glycerol, ribose, glucose, fructose, mannitol, mannitol, N acetyl glucosamine, amygdaline, arbutine, and arsine. Eculine, salicine, cellobiose, maltose, lactose, saccharose, trehalose, amidodon, glycogen ( fermented or used glycogene) to produce acids.

As shown in FIG. 5 and Table 8 , the intracellular fatty acid content was highest in 15: 0 ISO and high in C13: 0 ISO. The result of gene analysis of 16S rDNA is shown in SEQ ID NO: 2 . Based on this, the% similarity was obtained from the control strain. As a result, the strain 206 was found to be a bacterium belonging to the genus Bacillus and showed 99% similarity to the standard strain of Bacillus cereus . It can be called a fungus. In addition, a phylogenetic tree was created by a neighbor joining method and the results are shown in FIG. 6 . In this case, the scale bar of the tree refers to 0.01 substitution per site.

Results 206 strains have reviewed the properties of the 206 strain as described above was divided into yuyeongyun of Bacillus cereus (Bacillus cereus), and finally Bacillus cereus (Bacillus cereus ) 206.

<Biochemical Properties (Carbohydrates) of Isolated Strain 206> ¹ Characteristics 206 Characteristics 206 Glycerol + Esculline + Ertythritol - Salicine + D-arabinose - Cellobiose + L-arabinose - Maltose + Ribose + Lactose + D-Xylose - Melibiose - L-Xylose - Saccharose + Adonitol - Trehalose + β methyl-xyloside  -  Inuline  - Galactose - Melezitose - D-Glucose + D-Raffinose - D-Fructose + Amidon + D-Mannose - Glycogen + L-sorbose - Xylitol - Rhamnose - β Genentiobiose - Dulcitol - D-Turanose - Inositol - D-Resource (Lyxose) - Mannitol + D-Tagatose - Sorbitol - D-Fucose -  α Methyl-D-mannoside  -  L-Fucose  -  α Methyl-D-glucoside  -  D-arabitol  - N acetyl glucosamine  +  L-Arabitol  - Amygdaline + Gluconate -  Arbutine  + 2 aceto-gluconate  - 5 aceto-gluconate  -

¹ '+' = positive; '-' = Voice

<Cellular fatty acid composition of isolate 206> fatty acid % C12: 0 ISO 2.02 C12: 0 1.20 C13: 0 ISO 20.07 C13: 0 ANTEISO 2.14 C14: 0 ISO 5.33 C14: 0 4.71 C15: 0 ISO 27.27 C15: 0 ANTEISO 3.55 C15: 0 0.61 C16: 1 0.85 C16: 0 ISO 4.54 C16: 0 5.46 C15: 0 2OH 0.59 ISO C17: 1 w10c 1.86 ISO C17: 1 w5c 3.03 C17: 1 ANTEISO A 0.76 C17: 0 ISO 4.60 C17: 0 ANTEISO 0.73

< Example  3> Enterobacter Ruud crisis ( Enterobacter ludwigii ) KU201 -3 Preparation of microbial agent containing [KACC91341P]

1. Prototype of fermenter for mushroom waste medium

(1) Liquid culture of spawn

-Incubate the liquid spawn incubated at 36 ℃ for 12 hours using PCB (Plate count broth).

Item g / ℓ Pancreatic digest of casein 5 Yeast extract 2.5 Dextrose One

(2) Mass cultivation of spawn using agricultural processing by-products

Enterobacter ludwigii ) For mass production of KU201-3, strains are produced through mass culture using soybean meal and rice bran.

-The mixing ratio of soybean meal and rice bran is 8: 2, and molasses is added at the level of 2% of the building to promote the growth of the strain.

-Inoculate liquid cultured spawn in solid medium using agricultural processing by-product at 5% level.

Incubate for 48 hours in a 36 ° C incubator.

(3) Enterobacter ludwigii ) drying of 201-3 cultures

-Dry at 40 ° C for 48 hours using a ventilated drying oven to ensure optimum bacterial count.

(4) Utilization of prototype

-Prepared prototypes are packaged in 25kg units and used as fermentation accelerator between mushroom waste medium fermented feed.

New strain Enterobacter ruud crisis KU201-3 (KACC91341P) according to the present invention is a strain having fibrinolytic and lignin degrading activity isolated from mushroom waste medium. Since the mushroom waste medium contains a lot of hardly decomposable substances, the microorganisms isolated from the mushroom waste medium are excellent in the degrading activity of the hardly decomposable substance. Therefore, if appropriately used enterobacter lud crisis KU201-3 (KACC91341P) used in the present invention to produce a microbial agent that can be useful in livestock feed by helping the strain to feed feed of livestock with high fiber content Can be.

1 shows laccase activity and cell growth of KU201-3 strain.

2 shows cell growth of KU201-3 and 206 strains.

3 shows a cell fatty acid gas chromatogram of the isolate strain KU201-3.

4 shows a phylogenetic tree of isolated strain KU201-3.

5 shows the cell fatty acid gas chromatogram of the isolate strain 206.

6 shows a phylogenetic tree of isolated strain 206.

<110> Konkuk university industrial cooperation agency <120> A new strain Enterobacter ludwigii KU201-3 (KACC91341P) having          cellulase and lignolytic activity and animal feed additive          microbial agent comprising <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 1449 <212> DNA <213> Enterobacter ludwigii KU201-3 <400> 1 ggggactgcg gcaggctacc atgcaagtcg aacggtagca cagagagctt gctctcgggt 60 gacgagtggc ggacgggtga gtaatgtctg ggaaactgcc tgatggaggg ggataactac 120 tggaaacggt agctaatacc gcataacgtc gcaagaccaa agagggggac cttcgggcct 180 cttgccatca gatgtgccca gatgggatta gctagtaggt ggggtaacgg ctcacctagg 240 cgacgatccc tagctggtct gagaggatga ccagccacac tggaactgag acacggtcca 300 gactcctacg ggaggcagca gtggggaata ttgcacaatg ggcgcaagcc tgatgcagcc 360 atgccgcgtg tatgaagaag gccttcgggt tgtaaagtac tttcagcggg gaggaaggtg 420 ttgtggttaa taaccgcagc aattgacgtt acccgcagaa gaagcaccgg ctaactccgt 480 gccagcagcc gcggtaatac ggagggtgca agcgttaatc ggaattactg ggcgtaaagc 540 gcacgcaggc ggtctgtcaa gtcggatgtg aaatccccgg gctcaacctg ggaactgcat 600 tcgaaactgg caggctagag tcttgtagag gggggtagaa ttccaggtgt agcggtgaaa 660 tgcgtagaga tctggaggaa taccggtggc gaaggcggcc ccctggacaa agactgacgc 720 tcaggtgcga aagcgtgggg agcaaacagg attagatacc ctggtagtcc acgccgtaaa 780 cgatgtcgac ttggaggttg tgcccttgag gcgtggcttc cggagctaac gcgttaagtc 840 gaccgcctgg ggagtacggc cgcaaggtta aaactcaaat gaattgacgg gggcccgcac 900 aagcggtgga gcatgtggtt taattcgatg caacgcgaag aaccttacct actcttgaca 960 tccagagaac ttagcagaga tgctttggtg ccttcgggaa ctctgagaca ggtgctgcat 1020 ggctgtcgtc agctcgtgtt gtgaaatgtt gggttaagtc ccgcaacgag cgcaaccctt 1080 atcctttgtt gccagcggtc cggccgggaa ctcaaaggag actgccagtg ataaactgga 1140 ggaaggtggg gatgacgtca agtcatcatg gcccttacga gtagggctac acacgtgcta 1200 caatggcgca tacaaagaga agcgaactcg cgagagcaag cggacctcat aaagtgcgtc 1260 gtagtccgga ttggagtctg caactcgact ccatgaagtc ggaatcgcta gtaatcgtag 1320 atcagaatgc tacggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccatgg 1380 gagtgggtgc aaaagaagta ggtagcttaa ccttcgggag ggcgcttacc acttgtgatn 1440 cagaacggg 1449 <210> 2 <211> 1429 <212> DNA <213> Bacillus cereus 206 <400> 2 agcagggggc tatctgcagt cggcgatgga ttagagcttg ctcttatgaa gttagcggcg 60 gacgggtgag taacacgtgg gtaacctgcc cataagactg ggataactcc gggaaaccgg 120 ggctaatacc ggataacatt ttgaaccgca tggttcgaaa ttgaaaggcg gcttcggctg 180 tcacttatgg atggacccgc gtcgcattag ctagttggtg aggtaacggc tcaccaaggc 240 aacgatgcgt agccgacctg agagggtgat cggccacact gggactgaga cacggcccag 300 actcctacgg gaggcagcag tagggaatct tccgcaatgg acgaaagtct gacggagcaa 360 cgccgcgtga gtgatgaagg ctttcgggtc gtaaaactct gttgttaggg aagaacaagt 420 gctagttgaa taagctggca ccttgacggt acctaaccag aaagccacgg ctaactacgt 480 gccagcagcc gcggtaatac gtaggtggca agcgttatcc ggaattattg ggcgtaaagc 540 gcgcgcaggt ggtttcttaa gtctgatgtg aaagcccacg gctcaaccgt ggagggtcat 600 tggaaactgg gagacttgag tgcagaagag gaaagtggaa ttccatgtgt agcggtgaaa 660 tgcgtagaga tatggaggaa caccagtggc gaaggcgact ttctggtctg taactgacac 720 tgaggcgcga aagcgtgggg agcaaacagg attagatacc ctggtagtcc acgccgtaaa 780 cgatgagtgc taagtgttag agggtttccg ccctttagtg ctgaagttaa cgcattaagc 840 actccgcctg gggagtacgg ccgcaaggct gaaactcaaa ggaattgacg ggggcccgca 900 caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc aggtcttgac 960 atcctctgaa aaccctagag atagggcttc tccttcggga gcagagtgac aggtggtgca 1020 tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct 1080 tgatcttagt tgccatcatt aagttgggca ctctaaggtg actgccggtg acaaaccgga 1140 ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac acacgtgcta 1200 caatggacgg tacaaagagc tgcaagaccg cgaggtggag ctaatctcat aaaaccgttc 1260 tcagttcgga ttgtaggctg caactcgcct acatgaagct ggaatcgcta gtaatcgcgg 1320 atcagcatgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccacga 1380 gagtttgtaa cacccgaagt cggtggggta accttttgga gccagccgc 1429  

Claims (3)

New strains with fibrinolysis and lignin-degrading activity Enterobacter Ruud crisis (Enterobater ludwigii ) KU201-3 (KACC91341P). Fermentation promoter of feed containing cellulose and / or lignin, characterized in that it contains a pure culture of Enterobater ludwigii KU201-3 (KACC91341P) of claim 1 as an active ingredient. Preparing a culture medium; Culturing the Enterobater ludwigii KU201-3 (KACC91341P) of claim 1; Collecting the culture solution; And Fermentation promoter manufacturing method of the feed containing fiber and / or lignin, characterized in that it comprises the step of drying the culture.

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