KR20110035668A - Novel bacillus subtilis 5-b strain and composition for forage additive comprising the same - Google Patents

Abstract

PURPOSE: A novel Bacillus subtilis 5-B is provided to prevent reduction of activity in an intestine and to maintain viability. CONSTITUTION: A method for isolating a novel Bacillus subtilis 5-B comprises: a step of suspending 1g of soybean paste with 10 mL of saline solution; a step of smearing the suspend on a LB agar medium containing 10g/L of tryptone, 5 g/L of yeast extract, 5 g/L of sodium chloride, and 18 g/L of bactoagar and culturing at 37 degrees Celsius; a step of isolating different strains and inoculating the strains into a LB liquid medium and shaking at 37 degrees Celsius for 24 hours; and a step of mixing the medium with a corn powder. A feed additive composition contains the Bacillus subtilis 5-B(KCTC 11550BP) and phosphate or natural antioxidant.

Description

Novel Bacillus Subtilis 5-B Strain and Composition for Forage Additive Comprising the Same}

The present invention relates to a novel Bacillus subtilis 5-B strain (KCTC 11550BP) which is excellent in hydrolytic enzyme production capability of fibrous and semifibrous materials and has excellent long-term survival rate. The present invention also relates to a method for mass culturing the new strain and a composition for feed addition comprising the strain or a culture thereof.

In the context of increasing public health and safety interest in food at home and abroad, the persistent and resistant bacteria problem of antimicrobial feed additives added for the purpose of promoting livestock growth and improving feed efficiency has been the subject of debate. For this reason, it is urgent to develop useful resources to replace these antimicrobial feed additives, and for this, research on probiotics, prebiotics, enzymes, etc., is being actively conducted.

The intestinal flora of livestock is closely related to the productivity of livestock, and the maintenance of the intestinal flora is directly related to the prevention of livestock disease, the reduction of antibiotic use and the improvement of productivity. Is done.

Probiotics are microorganisms that act usefully in the digestive tract of livestock, and in the broad sense they also include microorganisms used for feeding livestock, such as silage additions and fermented feed additives. In the case of livestock probiotics, it means microorganisms that are fed directly to livestock. The term probiotics, meaning probiotics, was invented by Lilly and Stilwell in 1965 and has been generalized and used worldwide since 1975. Probiotics originate from the Greek meaning of symbiosis and, unlike antibiotics, mean a beneficial relationship between living organisms. Probiotic strains that have been developed and are commercially available include Bacillus, lactic acid bacteria, yeast, bifidus bacteria, enterococci and fungi. Among these probiotic strains, Bacillus produces enzymes that can degrade the refractory components of feed in the intestine and culture of livestock. In particular, Bacillus subtilis bacteria produce degrading enzymes of fibrous and semifibrous materials that are contained in large amounts in feed, and by hydrolyzing the cellulose and hemicellulose layers surrounding the grains, livestock can easily use the nutrients in the grains. As well as to improve the condition of the intestinal digest of livestock. Feeding with high hemicellulose content tends to lead to stool and swelling in livestock. Bacillus subtilis probiotics that produce xylanase, mannase and cellulase can prevent this and increase the value of feed. It is of great value as an enzyme for feed addition by improving the variation in the quality of wheat products so that they can be used as a stable feedstock. In addition, these Bacillus subtilis probiotics have the effect of improving the feed efficiency and gain rate of livestock, normalizing the intestinal microflora, suppressing the digestive diseases of harmful bacteria through the production of soft organic acids and replacing some antibiotics.

However, when using Bacillus subtilis bacteria as a probiotic, many of the strains that are currently used as probiotics have a problem that the survival rate is rapidly lowered after cultivation, so that the Bacillus subtilis bacteria have excellent characteristics. There is a problem that does not represent correctly.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have tried to identify and develop new Bacillus subtilis strains that can solve the problems that may occur when using the Bacillus strain as a probiotic for livestock feed. As a result, a novel Bacillus subtilis strain was isolated and identified to have excellent ability to produce degrading enzymes of fibrous and semi-fibrous materials and also have long-term viability.

Accordingly, an object of the present invention is to provide a Bacillus subtilis 5-B strain (KCTC 11550BP) having excellent hydrolytic enzyme production ability of fibrous and semifibrous materials and excellent long-term survival rate.

Another object of the present invention is to provide a culture method for mass production of the Bacillus subtilis 5-B strain (KCTC 11550BP).

Another object of the present invention to provide a composition for feed addition comprising the Bacillus subtilis (Bcillus subtilis ) 5-B strain (KCTC 11550BP) or a culture thereof as an active ingredient.

The 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 provides a Bacillus subtilis 5-B strain (KCTC 11550BP) which has excellent hydrolytic enzyme production ability of fibrous and semifibrous materials and has excellent survival even in long-term storage. To provide.

According to another aspect of the present invention, the present invention provides a Bacillus subtilis 5-B strain (KCTC 11550BP) comprising TSB comprising tryptone, soyton, dextrose, sodium chloride (NaCl) and potassium phosphate. It provides a culture method for mass production of Bacillus subtilis 5-B strain (KCTC 11550BP) comprising the step of shaking culture in (tryptic soy broth) medium.

According to another aspect of the invention, the present invention provides a composition for feed addition comprising Bacillus subtilis 5-B strain (KCTC 11550BP) or a culture thereof as an active ingredient.

Bacillus subtilis (Bcillus subtilis ) 5-B strain of the present invention (KCTC 11550BP) is obtained by separating a strain having excellent viability in Doenjang, a traditional Korean food.

The strain of the present invention was identified as the final Bacillus subtilis 5-B strain by investigating its morphological, biochemical and genetic characteristics, and deposited on the Bank of Korea Biotechnology Research Institute on August 24, 2009. Deposited as number KCTC 11550BP.

The Bacillus subtilis 5-B strain (KCTC 11550BP) of the present invention is characterized in that the production and secretion ability of cellulase, xylanase, and mannalas with excellent resolution of fibrous and semifibrous materials are excellent. . Cellulase, xylanase and mannase produced by the strains of the present invention do not significantly reduce activity even in the intestinal environment of livestock. The enzyme contained in the culture of the strain of the present invention as a main component of the feed to effectively break down the fibrous and semi-fiber-based materials that are difficult to break down in the body of the livestock to increase the feed utilization efficiency of livestock animals and livestock It is suitable for maintaining the intestinal flora and preventing the digestive diseases.

Another characteristic of the Bacillus subtilis 5-B strain (KCTC 11550BP) of the present invention is excellent in long-term survival when stored in dry conditions.

The strain of the present invention shows a very good survival rate even after long-term storage at room temperature mixed with excipients for livestock feed. Therefore, the Bacillus subtilis strain of the present invention can be maintained for a long time as a probiotic even if formulated and stored as a feed additive after cultivation, the utilization is very high as a probiotic for feed.

Bacillus subtilis (Bcillus subtilis ) 5-B strain (KCTC 11550BP) of the present invention can be cultured through a known method for culturing Bacillus subtilis strains. The medium may be natural or synthetic medium. As the carbon source of the medium, for example, glucose, sucrose, dextrin, glycerol, starch and the like can be used, and as the nitrogen source, peptone, meat extract, yeast extract, dried yeast, soy cake, urea, thiourea, ammonium salt, knight Rates and other organic or inorganic nitrogen-containing compounds may be used, but are not limited to these components. Inorganic salts included in the medium may be phosphate such as magnesium, manganese, potassium, calcium, iron, nitrate, carbonate, chloride, etc., but are not limited thereto. In addition to the components of the carbon source, nitrogen source and inorganic salts, amino acids, vitamins, nucleic acids and related compounds may be added to the medium.

The strain of the present invention can be cultured according to the culture conditions of known Bacillus subtilis strains. For example, it can be incubated for 12 hours-5 days in the temperature range of 20-50 ℃.

The present invention provides a culture method for the mass production of the new strain of the present invention. Bacillus subtilis 5-B strain (KCTC 11550BP) of the present invention was cultured in shake media of tryptic soy broth (TSB) containing tryptone, soyton, dextrose, sodium chloride (NaCl) and potassium phosphate When spores of the strains are induced, it is possible to mass culture bacteria having excellent viability. In order to induce sporulation of the strain to maximize viability, the culture is preferably at least 20 hours, more preferably at least 24 hours in the temperature range of 30-40 ℃, pH 6.0-8.0.

Bacillus subtilis 5-B strain (KCTC 11550BP) of the present invention generates enzymes that have high resolution of fibrous and semifibrous materials, and has excellent viability even when stored at room temperature for a long time. It may be included as an active ingredient in the composition.

The feed composition of the present invention includes organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate and polyphosphate (polyphosphate), polyphenols, and catechins (catechin). It may further include any one or more of natural antioxidants, such as alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid.

The feed composition of the present invention includes auxiliaries such as amino acids, inorganic salts, vitamins, antibiotics, antibacterial substances, antioxidants, antifungal enzymes, microbial preparations of other types of live bacteria; Grains such as milled or crushed wheat, oats, barley, corn and rice; Vegetable protein feed, such as rape, soybeans and sunflower; Animal protein feeds such as blood meal, meat meal, bone meal and fish meal; Dry ingredients consisting of sugars and dairy products such as various powdered milk and whey powders; Main components such as lipids, for example animal fats and vegetable fats, optionally liquefied by heating; Additives such as nutritional supplements, digestion and absorption enhancers, growth promoters and disease prevention agents may be included.

The feed composition of the present invention may be in the form of a dry or liquid formulation, and may include excipients for feed addition. Excipients for feed addition may include, but are not limited to, for example, zeolite, jade powder or rice bran.

Feed composition of the present invention may further comprise one or more enzyme preparation. For example, alpha-1,4-glycosidic bonds (α-1) contained in lipases such as lipases, phytases that break down phytic acid to form phosphates and inositol phosphates, and starch and glycogen. Two molecules are amylase, an enzyme that hydrolyzes and 4-glycoside bonds, phosphatase, an enzyme that hydrolyzes organic phosphate esters, carboxymethylcellulose that breaks down cellulose, xylanase that breaks down xylose, and maltose. Maltase hydrolyzed to glucose and saccharose may be converted to a glucose-fructose mixture to form a glucose-fructose mixture.

The composition for adding livestock feed of the present invention may be administered to an animal alone or in combination with other feed additives in an edible carrier. In addition, the composition for adding livestock feed can be easily administered as a top dressing or mixed directly with the livestock feed or separately from the feed, in a separate oral dosage form, or in combination with other ingredients. Typically, a single daily intake or divided daily intake may be used, as is well known in the art.

Animals that can be used in the feed composition of the present invention can be used for example cattle, chicks, chickens, domestic chickens, such as edible cattle, cows, calves, pigs, piglets, sheep, goats, horses, rabbits, dogs, cats, etc. Poultry such as roosters, ducks, geese, turkeys, quails, birds, and the like.

The amount of the Bacillus subtilis 5-B strain of the present invention included in the feed composition of the present invention is not particularly limited, but in order to decompose fibrous and semi-fibrous substances during long-term survival in the digestive tract of livestock. Included in suitable amounts. Preferably feed composition for additives is feed 1 ㎏ per Bacillus subtilis (Bacillus subtilis) 5-B strain ^{(KCTC 11550BP) 10 2 - 10} 15 to the cells, more preferably 10 ⁴ - 10 ¹² cells, more preferably Contains 10 ⁷ -10 ¹¹ cells.

As described in detail above, the present invention relates to Bacillus subtilis 5-B (KCTC 11550BP) strain which is a renal strain. Bacillus subtilis new strain of the present invention produces and secretes cellulase, xylanase and mannase, which are not significantly reduced in the intestine of the livestock, thereby providing excellent resolution of the fibrous and semifibrous materials that are the main components of livestock feed. It has characteristics, and long-term survival rate is maintained under dry conditions at room temperature, so it is very excellent in storage. When the strain of the present invention and the culture thereof are used in the additive composition for feed, the main component of the feed effectively decomposes fibrous and semi-fibrous components, which are difficult to decompose in the body of the livestock, thereby increasing feed efficiency of livestock animals and raising livestock. It is effective in maintaining the intestinal flora of the intestine and preventing digestive diseases. In addition, the viability is maintained even if stored for a long time as an additive for feed is very useful as a probiotic for feed.

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 .

Example

Example 1: Isolation of Bacillus Strain with High Viability from Doenjang

Suspend 1g of traditional soybean paste sample in 10 ml of physiological saline solution, take an appropriate amount of suspension, tryptone 10 g / L, yeast extract 5 g / L, sodium chloride (NaCl) 5 g / L, Bactoagar 18 Plated in LB agar medium containing g / L and incubated at 37 ℃. Strains of different shapes and sizes were isolated from colonies formed on solid LB agar medium, inoculated into LB liquid medium, shaken for 24 hours at 37 ° C, and the cultured cells were mixed with jade to be 5% (v / w). It was. After leaving the mixed jade powder for 1 week at room temperature, 1 g of the mixture was suspended in 10 ml of physiological saline, the suspension was taken in an appropriate amount, plated on LB agar medium, and cultured at 37 ° C. Strains were finally selected. The selected strain was named Bacillus subtilis 5-B strain.

Example 2: Identification and Characterization of Bacillus subtilis 5-B Strains

Morphological and biochemical properties and the nucleotide sequence of 16S ribosomal RNA (16S rRNA) were examined to identify the strains isolated from Example 1 with excellent survival rates.

Morphological and biochemical characteristics of the strain isolated in Example 1 are shown in Tables 1 and 2, respectively. Morphological and biochemical characteristics of the isolated strains were compared to those of the strains described in the Manual of Buggy's manuals of determinative bacteriology.

In addition, as a result of investigating the biochemical characteristics of the sugar fermentation of the isolated strain, the bacterium had the highest similarity with Bacillus subtilis, and analyzed the base sequence of 16S ribosomal RNA (see FIG. 1, sequence listing No. 1 sequence). ) Showed 100% homology with Bacillus subtilis. Therefore, the strain isolated in the present invention was finally identified as Bacillus subtilis 5-B strain. The isolated and identified new Bacillus subtilis 5-B strain was deposited in Korean Collection for Type Cultures of Korea Research Institute of Bioscience and Biotechnology on August 24, 2009 under accession number KCTC 11550BP.

division reaction  Shape Bacillus  Spore Formation Capacity (Endospore) +  Spore shape Oval  Spore position center  Motility +  Gram staining +  Catalase Activity +  Xylan hydrolysis +  Galactomannan hydrolysis +  Cellulose hydrolysis +  Kim milk hydrolysis +  Starch hydrolysis +

Acid Production by Sugar Fermentation Responsive Glycerol, L-Arabinose, D-Ribose,
D-Xylose, Glucose, Fructose,
Mannose, Mannitol, Sorbitol,
Methyl-α-D-glucopyranoside,
Acetylglucosamine (N-Acetylglucosamine), amigdalin,
Arbutin, Esculin, Salicin,
Cellobiose, Maltose, Saccharose,
Trehalose, Inulin, Amidon,
Glycogen, Gentiobiose,
D-Turanose, D-Tagatose, Rhamnose,
Gluconate, Galactose, Inositol,
Erythritol, arabinose, lactose,
L-Xylose, adonitol, Melibiose,
Methyl-β-D-xylopyranoside,
L-Sorbose, Dulcitol, Raffinose,
Methyl-α-D-mannopyranoside,
Melezitose (D-Melezitose), Xylitol, Resource (D-Lyxose),
D-Fucose, L-Fucose, Arabitol,
Arabitol (L-Arabitol),
Potassium 2-ketogluconate,
Potassium 5-ketogluconate +
+
+
+
+
+
+
+
+
-
-
-
-
-
-
-
-
-
-
-
-

Example 3: Reaction Characteristics of Hydrolytic Enzymes of Extracellular Fibrous and Semifibrous Materials Produced by Bacillus Subtilis 5-B Strains

Among the enzymes secreted from the strain Bacillus subtilis 5-B isolated in the present invention, the activity of the enzymes capable of degrading the fibrous and semifibrous materials mainly included in the feed of livestock was evaluated. In the activity evaluation of the enzyme, the hydrolytic activity of carboxymethylcellulose, oat xylan, and acacia fruit resin galactomannan was examined for the enzymes of cellulase, xylanase and mannase according to reaction temperature and pH.

First, the culture supernatant of the strain was concentrated and the concentration of ammonium sulfate was fractionated in the range of 25-75% to obtain a precipitate, which was then suspended in 20 mM sodium phosphate buffer (pH 6.0). Dialysis with buffer. Cellulase, xylanase and mannase activity were measured by varying the reaction temperature and pH using the dialyzed enzyme solution.

As shown in Figure 2, the cellulase produced by the Bacillus subtilis 5-B strain of the present invention showed the highest enzymatic activity at 60 ℃ and pH 5.0, the intestinal pH of the animal pH 6.5 and 37 The activities of 80% and 50% of the highest enzymatic activity were shown at ℃. Therefore, it was confirmed that the cellulase produced by the strain of the present invention had excellent activity at pH and bio temperature in the vicinity of neutral physiological conditions.

As shown in the results of Figure 3, the xylanase produced by the Bacillus subtilis 5-B strain of the present invention showed the highest enzymatic activity around 55 ℃ and pH 6.0, pH 6.5 and the intestinal pH of the animal The highest enzyme activity was 80% and 50%, respectively, at the intestinal temperature at 37 ° C. Therefore, it was confirmed that the xylanase produced by the strain of the present invention has excellent activity at pH and bio temperature in the vicinity of neutral physiological conditions.

As shown in the results of FIG. 4, the mannase produced by the Bacillus subtilis 5-B strain of the present invention showed the highest enzymatic activity near 60 ° C. and pH 5.5, and the pH 6.5 of the intestinal pH of the animal. It showed reactivity of 80% and 50% of the highest enzyme activity at 37 ° C., the intestinal temperature, and showed excellent reactivity at pH and biological temperature near the neutral physiological condition.

Example 4: Investigation of Spore Rate of Bacillus Subtilis 5-B Strains

In order to maintain viability of liquid cultured Bacillus subtilis, it is important that the cultured cells are converted to spores which are resting cells. Therefore, Bacillus subtilis 5-B strain (KCTC 11550BP) was prepared using tryptone 17g / L, soyton 3g / L, dextrose 2.5g / L, sodium chloride (NaCl) 5g / L, potassium phosphate 2.5g / L, and inoculated in TSB (tryptic soy broth) medium with a pH of 7.0 was shaken at 37 ℃. When the culture time was 16 hours, 20 hours, and 24 hours, the culture medium was recovered, and then heat-treated at 80 ° C. for 30 minutes to kill nutrient cells and measure the number of spores with high heat resistance. The number of spores was determined by taking an appropriate amount of the heat-treated bacteria and spreading them on an LB plate medium and measuring the number of colonies formed after incubation at 37 ° C. for 20 hours. The viable cell number of the culture solution obtained by the same method was taken as a control group by taking an appropriate amount of the non-heat treated bacteria solution.

As shown in Table 3, spore efficiency of Bacillus subtilis 5-B strains, the number of cells survived after heat treatment was 1% or less in the culture solution cultured for 16 hours. Was 66%, and in the bacterial cultures cultured for 24 hours, the number of viable cells after heat treatment and the number of viable cells before heat treatment were the same, indicating that most of them were spores. Spores have a strong characteristic of drying or heat, the high Bacillus subtilis 5-B strain of the present invention may be a property that can maintain a high survival rate when used as a feed agent probiotic.

Incubation time (hours) Before heat treatment After heat treatment Spore yield (%) 16h 3.110 ⁸ / ml <1.010 ⁶ / ml <1 20h 3.010 ⁸ / ml 2.010 ⁸ / ml 66.7 24h 7.010 ⁸ / ml 7.110 ⁸ / ml 100

Example  5: Of the present invention Bacillus Subtilis  Survival Investigation of 5-B Strains

Bacillus subtilis 5-B strain of the present invention (KCTC 11550BP) can be directly fed in liquid form when used as a probiotic for livestock feed, but is usually mixed with it by using jade powder, zeolite, rice bran, etc. Keep it in the feed and mix it with feed if necessary. Most Bacillus strains have a problem that the survival rate is sharply lowered when the water activity is mixed with the excipient. Therefore, in order to increase the probiotic efficacy of the Bacillus subtilis 5-B strain, the survival rate should be kept high when mixed with excipients.

Since the bacteria cultured in TSB medium used in Example 4 for 24 hours reached about 100% spore, 5% (v / w) of the culture solution of Bacillus subtilis 5-B strain cultured for 24 hours After mixing with zeolite, dried at room temperature, sealed and stored for 180 days, the viable cell count of Bacillus subtilis 5-B strain was measured in the mixture at regular intervals. As a result, as shown in FIG. 5, 100% survival rate was maintained until 60 days based on the viable cell count of Bacillus subtilis 5-B strain immediately after mixing with zeolite, and 95% and 180 days were stored when stored for 90 days. When the survival rate of about 75% was maintained, the Bacillus subtilis 5-B strain was found to have a very high survival rate when mixed with a feed additive. In fact, considering that the livestock probiotic is consumed within 3 months, the Bacillus subtilis 5-B strain, which is mixed with excipients, survives over 95% of the shelf life. .

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

1 is a Bacillus subtilis of the present invention ( Bacillus) subtilis ) shows the nucleotide sequence of 16S ribosomal RNA (16S ribosomal RNA) of 5-B strain.

Figure 2 is a graph showing the activity of the cellulase produced by the Bacillus subtilis ( Bcillus subtilis ) 5-B strain according to the temperature and acidity (pH).

Figure 3 is a graph showing the activity according to the temperature and acidity (pH) of the xylanase produced by the Bacillus subtilis 5-B strain of the present invention.

Figure 4 is a graph showing the activity according to the temperature and acidity (pH) of the mannase produced by the Bacillus subtilis ( Bcillus subtilis ) 5-B strain.

5 is a graph showing the survival rate of the subtilis ( Bcillus subtilis ) 5-B strain of the present invention by mixing with zeolite and leaving at room temperature.

<110> YOON, Ki-Hong          Yecheongun agriculture technology center <120> Novel Bacillus Subtilis 5-B Strain and Composition for Forage          Additive Comprising the Same <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1422 <212> DNA <213> Bacillus subtilis <400> 1 tgcaagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga cgggtgagta 60 acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg ctaataccgg 120 atggttgttt gaaccgcatg gttcaaacat aaaaggtggc ttcggctacc acttacagat 180 ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcaa cgatgcgtag 240 ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga 300 ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt 360 gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtac cgttcgaata 420 gggcggtacc ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc 480 ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc tcgcaggcgg 540 tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg gaaactgggg 600 aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg 660 tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg aggagcgaaa 720 gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta 780 agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac tccgcctggg 840 gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca agcggtggag 900 catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgacaa 960 tcctagagat aggacgtccc cttcgggggc agagtgacag gtggtgcatg gttgtcgtca 1020 gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg atcttagttg 1080 ccagcattca gttgggcact ctaaggtgac tgccggtgac aaaccggagg aaggtgggga 1140 tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggacagaa 1200 caaagggcag cgaaaccgcg aggttaagcc aatcccacaa atctgttctc agttcggatc 1260 gcagtctgca actcgactgc gtgaagctgg aatcgctagt aatcgcggat cagcatgccg 1320 cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccacgaga gtttgtaaca 1380 cccgaagtcg gtgaggtaac cttttaggag ccagccgccg aa 1422  

Claims (3)

Fiber-based and semi-fiber-based material of the hydrolase producing capability at the same time, excellent long-term survival is superior Bacillus subtilis (Bacillus subtilis ) 5-B strain (KCTC 11550BP). The first term Bacillus subtilis (Bacillus subtilis ) Bacillus subtilis comprising shaking culture of 5-B strain (KCTC 11550BP) in Tryptic Soy Broth (TSB) medium containing tryptone, soyton, dextrose, sodium chloride (NaCl) and potassium phosphate ( Bacillus subtilis ) A culture method for mass production of 5-B strain (KCTC 11550BP). Bacillus subtilis subtilis ) feed composition comprising a 5-B strain (KCTC 11550BP) or a culture thereof as an active ingredient.

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