KR102176078B1 - Solid culture method to culture with new microbial bacillus subtilis BS300 strains with resolution to carbohydrates and proteins and fibres among organic matter - Google Patents

Abstract

The present invention relates to a novel microorganism Bacillus subtilis BS300 having degradability to carbohydrates, proteins, and fibers among organic matters, and a solid culture method for culturing including a novel microorganism Bacillus subtilis BS300 strain. The novel microorganism Bacillus subtilis BS300 strain having degradability to carbohydrates, proteins, and fibers among organic matters has a nucleotide sequence represented by SEQ ID NO: 1, and is deposited as KCTC18263P. According to the present invention, Bacillus subtilis BS300, the novel microorganism having high degradability to carbohydrates, proteins, and fibers among organic matters even among homogeneous strains is provided, and at the same time, a solid medium composition for culturing Bacillus subtilis BS300 which can solid culture the novel microorganism Bacillus subtilis BS300 while exhibiting ease of manufacture and distribution stability is provided. In addition, by the same, a solid culture having degradability to carbohydrates, proteins, and fibers among organic matters, is provided together.

Description

Solid culture method to culture with new microbial bacillus subtilis BS300 strains with resolution to carbohydrates and proteins and fibers among organic matter organic matter}

The present invention relates to a solid culture method for culturing including new microorganisms Bacillus subtilis BS300 and Bacillus subtilis BS300 strains having decomposition power for carbohydrates, proteins, and fibrin among organic matter, and more specifically, organic matter even between homologous strains. It relates to a solid culture method for culturing including the new microorganism Bacillus subtilis BS300 strain while having high decomposition power for carbohydrates, proteins, and fiber, and ease of manufacture and distribution stability.

In recent years, the livestock sector is receiving demands from the times to enable eco-friendly breeding from the breeding stage by using microorganisms and eco-friendly resources of livestock manure and probiotics. Also, environmental pollution and soil salt accumulation problems due to excessive abuse of chemical fertilizers The use of compost and organic fertilizers is gradually increasing as an eco-friendly agricultural material that can be replaced by a large number of them.

However, since the organic matter contained in the organic fertilizer is composed of a large molecular weight, it is in a state that crops cannot be absorbed directly. Accordingly, it can be absorbed through the roots only when it is reduced by a separate microbial treatment.

Accordingly, as the interest in microbial agents capable of decomposing organic matters increases, several studies have been conducted.

However, these microbial preparations are usually commercialized and distributed in a liquid form, and in this case, there is a disadvantage in that the storage conditions are difficult to store at low temperatures and the concentration of the microorganisms is significantly reduced during distribution.

In addition, even if it is powdered, it is mainly manufactured in a form that is adsorbed to minerals or grains, so there is a limit to manufacturing a high-concentration microbial preparation.

1. Korean Patent Laid-Open Publication No. 10-2010-0045621'Macillus strain with excellent organic matter resolution' 2. Korean Patent Publication No. 10-2015-0101789'Bacillus subtilis BK418 strain having complex enzyme-producing ability and antibacterial activity and its use'

It is an object of the present invention to provide a new microorganism Bacillus subtilis strain BS300 having high decomposition ability for carbohydrates, proteins, and fibrin among organic matter even in the liver of homogeneous strains, and at the same time, the novel microorganism Bacillus subtilis BS300 can be solid cultured at high concentration. It is to provide a solid culture method for culturing, including Bacillus subtilis BS300 strain, which exhibits ease of manufacture and stability of distribution while still being.

The novel strain of the present invention for achieving the above object is a novel microorganism Bacillus subtilis BS300 strain having a content of 500 to 510 units of protease, 200 to 220 units of cellulase, and 400 to 420 units of amylase ( Bacillus subtilis BS300, accession number: KCTC 18263P).

Another solid medium composition for cultivation of the novel microorganism Bacillus subtilis BS300 strain of the present invention is 25 to 30% by weight of skim steel, 45 to 55% by weight of protein, 3 to 5% by weight of glucose, 10 to alpha starch 15% by weight, yeast extract 3 to 7% by weight, peptone 1 to 2% by weight, soybean flour 1 to 3% by weight, potassium phosphate (K ₂ HPO ₄ ) 1 to 3% by weight, Magnesium sulfate heptahydrate (MgSO ₄ 7H ₂ 0) 0.01 to 0.1 wt%, manganese sulfate heptahydrate (MnSO ₄ 7H ₂ 0) 0.01 to 0.1 wt%, and calcium chloride (CaCl ₂ ) 0.01 to 0.1 wt% It is characterized by being constructed.

The solid culture method for culturing including a novel microorganism Bacillus subtilis BS300 strain having a decomposition ability for carbohydrates, proteins, and fibrin among organic matters of the present invention is the novel microorganism Bacillus subtilis BS300 strain of claim 1 ( Bacillus subtilis BS300, Accession No.: KCTC 18263P) inoculation to the liquid culture medium to prepare a liquid culture solution, skim steel 25-30% by weight, protein skin 45-55% by weight, glucose 3-5% by weight , Alpha starch 10~15% by weight, Yeast extract 3~7% by weight, Peptone 1~2% by weight, Soybean flour 1~3% by weight, Potassium phosphate (K ₂ HPO ₄ ) 1~3 weight%, Magnesium sulfate heptahydrate (MgSO ₄ 7H ₂ 0) 0.01 to 0.1 wt%, manganese sulfate heptahydrate (MnSO ₄ 7H ₂ 0) 0.01 to 0.1 wt%, and calcium chloride (CaCl ₂ ) 0.01 to 0.1 wt% Including a second step of preparing a solid medium composition by mixing, and a third step of adding the liquid culture solution of the first step to the solid medium composition of the second step, culturing, and then drying to prepare a solid culture in powder form. It is characterized by doing.

When preparing the liquid culture solution of the first step, the liquid culture medium includes glucose, starch, yeast extract, peptone, sodium chloride (NaCl), potassium phosphate (K ₂ HPO _{4 ).} ), It is characterized by containing magnesium sulfate·7 hydrate (MgSO _4· 7H ₂ 0), and ferrous sulfate·7 hydrate (FeSO _4· 7H ₂ 0).

The technical problems to be achieved by the present invention are not limited to the technical problems mentioned above, and other technical problems that are not mentioned can be clearly understood by those of ordinary skill in the technical field to which the present invention belongs from the following description. There will be.

According to the present invention, Bacillus subtilis BS300, a novel microorganism having high decomposition power for carbohydrates, proteins, and fibrin, among organic matter even in the liver of homogeneous strains is provided, and at the same time, the new microorganism Bacillus subtilis BS300 can be solid cultured at high concentration. It is to provide a solid culture method for culturing including Bacillus subtilis BS300 strain showing ease of manufacture and stability of distribution while still being.

1 is a view showing a similarity search result through a 16S rRNA nucleotide sequence and a gene bank of a novel microorganism Bacillus subtilis BS300 having resolution for carbohydrates, proteins, and fibers among organic substances of the present invention.
2 is a view showing a molecular schematic diagram of a novel microorganism Bacillus subtilis BS300 having decomposition power for carbohydrates, proteins and fibers among organic substances of the present invention.

Hereinafter, preferred embodiments and experimental examples of the present invention will be described in detail with reference to the accompanying drawings, and detailed descriptions of known functions and configurations that may unnecessarily obscure the subject matter of the present invention will be omitted.

The present invention relates to a new strain that forms spores well due to high growth rate due to high decomposition ability of carbohydrates, proteins, and fibres among organic matter decomposition capabilities, and more specifically, has a nucleotide sequence represented by SEQ ID NO: 1, It relates to a novel microorganism Bacillus subtilis BS300 having a high resolution and high enzyme production for carbohydrates, proteins, and fibers among organic substances compared to other homologous strains.

Specifically, in order to proceed with the separation of microorganisms of the genus Bacillus for completing the present invention and collecting soil samples from open field farmed lands using compost using corn and soybean residue (soybean meal) as main raw materials, first, carbohydrate decomposition ability is Bacillus genus strain of the present invention, except for Bacillus cereus microorganisms that can act as harmful factors on humans, by selecting microorganisms that have the ability to decompose proteins and fibrin using the selected microorganisms and using the selected microorganisms Separate

At this time, in order to select microorganisms capable of decomposing carbohydrates, a sample is diluted with physiological saline and inoculated in a nutrient agar medium (Nutrient Agar, Difco) containing 1.0% (w/w) of soluble starch, and inoculated at 30~32℃. It was first selected as a microorganism capable of decomposing carbohydrates in the order of cultivation for 16 to 24 hours in the order of formation of clear zones within the shortest time.

Then, in order to select microorganisms capable of decomposing proteins, the first selected microorganisms are inoculated into nutrient agar medium (Nutrient Agar, Difco) containing 1.0% (w/w) of skim milk powder (Skim Milk, Difco). And cultured at 32° C. for 16 to 24 hours, followed by secondary selection of microorganisms that formed a clear zone within the shortest time.

Then, in order to select microorganisms with fibrin decomposition ability, inoculate the secondly selected microorganisms in nutrient agar medium (Nutrient Agar, Difco) containing 1.0% (w/w) CMC (carboxymethyl cellilose), and By culturing for 16 to 24 hours, microorganisms that largely formed a clear zone within the shortest time were selected.

In order to exclude Bacillus cereus, which causes diarrhea in humans, among the selected microorganisms, streak smear on Bacillus cereus Agar (Difco) medium and incubate for 18 hours at 37°C. The prominent microorganism was determined as Bacillus cereus and excluded.

The microorganisms thus isolated were investigated with general bacteriological properties and API50CHB Kit (Biomerieux. Co. France) to confirm their characteristics.

As a result, it appeared as shown in Table 1 below.

Factors Characteristics Morphological
Characteristics Shape Rod Motility Motility Spore Oval terminal Sporangium Not swollen Gram Stain Positive Cultural
characteristics Colony of
Nutrient Agar
(37℃, 1~2 days) Form regular Surface Smoth Edge Erose Elevation Raised Opacity Opaque Color Creamy Brilliancy Not glistening Nutrient Broth(32℃, 1~2 days) Growth Physiological
characteristics Temperature range for growth 15~65℃ pH range for growth pH 3~9 NaCl tolerance for growth <5% Catalase + Lipase + Hydrolysis of starch + casein + cellulose - esculin + Levan formation from sucrose + NH ₃ production from peptone + NH ₃ production from arginine + Gelatin liquefaction + Utilization of citrate + Nitrate reduction + Denitrification - Action on milk; coagulation + peptonization + O-F test Fermentation Hydrolysis of tyrosine + Facultative Anaerobic growth +

As shown in Table 1 above, the characteristics of the novel microorganisms isolated in the present invention were Gram-positive bacteria, had motility and spore-forming ability, and as a bacillus, they were vigorous in aerobic conditions and capable of growing in microaerobic conditions.

In addition, it was confirmed that it had resolution such as starch and casein, and had ammonia reducing ability, and formed circular colonies in the solid medium (NA), and the color of the colonies was white.

Thus, as a result of analyzing the base sequence of 16s rRNA by applying the general bacterial identification method and molecular genetic method included in the Burergy's Manual of Derterminative Bacteriology for bacterial identification, Bacillus as shown in Figs. Subtilis strain is confirmed to have a similarity of 99%, and the above morphological, physiological, and molecular genetic characteristics were synthesized, and the isolate was identified as Bacillus subtilis and named Bacillus subtilis BS300. KCTC18263P was granted as of December 6, 2013 by depositing it with the gene bank of the Korea Research Institute of Bioscience and Biotechnology, which is a depository institution, and the Bacillus subtilis BS300 strain consists of the entire nucleotide sequence described in SEQ ID NO: 1 or a part thereof, and has an open reading of 821 bp. It consists of a frame (ORF).

In the present invention, a solid culture method including the novel microorganism Bacillus subtilis BS300 strain is provided so that it can be easily used industrially, at this time, a solid medium composition for cultivation of the new microorganism Bacillus subtilis BS300 strain in order to have ease of manufacture and distribution stability. Will also be provided.

At this time, the novel microorganism Bacillus subtilis BS300 strain is prepared in a liquid culture medium and used as a seed for a solid medium.In order to ensure that the liquid culture solution containing the strain exhibits sufficient efficacy, the following optimal culture conditions It is good to introduce.

In other words, in the liquid culture medium used in the preparation of the liquid culture solution, glucose (Glucose), starch (starch), yeast extract (Yeast Extract), peptone (Peptone), sodium chloride (NaCl), potassium phosphate (K ₂ HPO ₄ ) , It is characterized in that it comprises magnesium sulfate 7 hydrate (MgSO ₄ 7H ₂ 0), and ferrous sulfate 7 hydrate (FeSO ₄ 7H ₂ 0), more preferably the total weight of the liquid culture medium As a standard, the glucose (Glucose) is 0.1 ~ 0.5% (W / V), the starch (starch) is 0.5 ~ 1% (W / V), the yeast extract (Yeast Extract) is 0.5 ~ 1% (W / V) ), the peptone (Peptone) is 0.1 ~ 0.5% (W / V), the sodium chloride (NaCl) is 0.01 ~ 0.1% (W / V), the potassium phosphate (K ₂ HPO ₄ ) is 0.05 ~ 0.3% (W /V), the magnesium sulfate 7 hydrate (MgSO ₄ 7H ₂ 0) is 0.01 to 0.05% (W/V), and the ferrous sulfate 7 hydrate (FeSO ₄ 7H ₂ 0) is 0.01 to 0.05 It is better to include it in% (W/V).

The liquid culture medium constructed in this way was prepared through sterilization and cooling before inoculation of bacteria, and then inoculated with the new microorganism Bacillus subtilis BS300 strain, and then cultured for 15 to 24 hours at 30 to 35°C under pH 6 to 7 conditions. Accordingly, a liquid culture solution having a cell concentration of 3.0~6.0×10 ⁸ cfu/g is prepared, which shows a faster cell growth rate of 30% or more than that of general bacteria, and this cell concentration is sufficient for use as a solid culture seed. It can be seen that it represents.

This liquid culture solution is to be used as a solid culture seed, which is another present invention, in which the solid medium composition includes skim steel, protein skin, alpha starch, and glucose as a carbon source, and yeast extract, peptone, and soybean meal as a nitrogen source. As trace elements, potassium phosphate, magnesium sulfate, manganese sulfate, and calcium chloride are further added to constitute a solid medium composition.

In other words, the solid medium composition for cultivating new strains of the present invention can compensate for the problems of existing microbial agents that are commercialized and distributed in a liquid form. At the same time, there is a disadvantage in that the concentration of microorganisms is significantly reduced during distribution, and there is a disadvantage in that the concentration of microorganisms is low and the ability to decompose organic matter is low, although there are currently commercially available products in the form of solid cultures. As the solution is solved, a solid medium composition capable of producing a new strain of high concentration while having ease of manufacture and stability of distribution is provided.

Thus, the solid medium composition of the present invention is skim steel, protein skin, glucose, alpha starch, yeast extract, peptone, soybean meal, potassium phosphate (K ₂ HPO ₄ ), Consisting of magnesium sulfate 7 hydrate (MgSO ₄ 7H ₂ 0), manganese sulfate 7 hydrate (MnSO ₄ 7H ₂ 0), and calcium chloride (CaCl ₂ ), more preferably 25 to 30% by weight of degreasing steel, protein Blood 45-55% by weight, glucose 3-5% by weight, alpha starch 10-15% by weight, yeast extract 3-7% by weight, peptone 1-2% by weight, soybean flour 1 ~3% by weight, potassium phosphate (K ₂ HPO ₄ ) 1 to 3% by weight, Magnesium sulfate heptahydrate (MgSO ₄ 7H ₂ 0) 0.01 to 0.1 wt%, manganese sulfate heptahydrate (MnSO ₄ 7H ₂ 0) 0.01 to 0.1 wt%, and calcium chloride (CaCl ₂ ) 0.01 to 0.1 wt% It is best to be organized.

The solid medium composition thus constituted is sterilized with steam in a solid medium sterilizer, cooled, and inoculated with a liquid culture solution of Bacillus subtilis BS300 (KCTC18263P), a new microorganism prepared as a seed, at 20 to 25% by weight based on the weight of the solid medium composition, A solid culture method for preparing a solid culture, that is, a solid culture by culturing it at 30~35℃ for 24~36 hours, drying it at 40~45℃ for 30~40 hours, and then crushing it in 10~50μm in a roll mill. Will be provided.

At this time, the solid culture is characterized by having a cell concentration of 0.1×10 ⁹ to 6.0×10 ⁹ cfu/g and at the same time having a content of 500 to 510 units of protease, 200 to 220 units of cellulase, and 400 to 420 units of amylase, It can be seen that the value of use as a source of Bacillus bacillus powder will be very high considering that the minimum concentration of Bacillus bacillus is 1×10 ⁶ cfu/g under the permission of the soil microbial agent or feed probiotic.

Hereinafter, the present invention will be described in more detail with reference to experimental examples, but these experimental examples are for illustrative purposes only and are not intended to limit the protection scope of the present invention.

<Experimental Example 1> Optimization of liquid culture and confirmation of cell concentration of the novel strain of the present invention

1. Experiment method

The isolated new microorganism Bacillus subtilis BS300 was examined for optimal conditions for liquid culture in order to be used as a solid culture seed.

Optimum culture conditions were established by culturing under various conditions for carbon source, nitrogen source, trace element, growth factor, temperature, and pH. As a result, the optimized medium and culture conditions were shown in Table 2 below.

2. Experiment result

Ingredient Concentration (%,W/V) Glucose 0.25 Starch(Corn) 0.9 Yeast Extract 0.6 Peptone 0.2 NaCl 0.01 K ₂ HPO ₄ 0.1 MgSO ₄ 7H ₂ 0 0.02 FeSO ₄ 7H ₂ 0 0.01 pH 6.8 Temp. 32℃ Culture Time 16~24Hrs

As a result of liquid culture of Bacillus subtilis BS300 (KCTC18263P) of the present invention through the optimized medium shown in Table 2, the growth rate (μmax) of the Bacillus subtilis BS300 (KCTC18263P) strain of the present invention is a general Bacillus microorganism Compared to 0.25, it was confirmed that 0.32 was observed at 16 hours of cultivation.After 24 hours of cultivation, the cell concentration was 5.31×10 ⁸ cfu/g, and the growth rate appeared 30% faster than general bacteria, so it has sufficient utility as a seed for solid culture. It was confirmed that there is.

<Experimental Example 2> Optimization of solid culture and confirmation of cell concentration of the novel strain of the present invention

1. Experiment method

In order to optimize the solid culture medium, the amounts of degreasing steel, protein skin, glucose, starch as carbon sources, yeast extract, peptone, soybean meal as nitrogen sources, and potassium phosphate, magnesium sulfate, manganese sulfate, and calcium chloride as trace elements are adjusted to As shown in Table 3, a solid medium was formed, steamed, sterilized, cooled, and the Bacillus subtilis BS300 (KCTC18263P) culture solution cultured in Experimental Example 1 was inoculated with 20% as a seed, and in a solid incubator (For You Tech.) Temperature: 32°C, supply air volume: 30%. Incubate for 24 to 36 hours at an exhaust volume: 35%, and in the same facility, temperature: 42°C, supply air volume: 40%. Displacement: 45%, dried for 36 hours, crushed to 10-50 μm in a roll mill, and measured the cell concentration to establish optimal culture conditions.

Ingredient name content(%) Skim steel 21 Protein blood 41 Glucose 2.5 Alpha starch 8.5 Yeast Extract 3.5 Peptone 1.0 Soybean flour 1.5 K ₂ HPO ₄ 1.0 MgSO ₄ 7H ₂ 0 0.01 MnSO ₄ 7H ₂ 0 0.01 CaCl ₂ 0.01

Through this optimized medium, a solid culture cell powder containing Bacillus subtilis BS300 (KCTC18263P) of the present invention was prepared, and 20 g of the cell powder was added to the sterilized cell line solution in order to measure the cell concentration thereof, and then finally After adjusting to 400ml with a magnetic stirrer, eluting for 1 hour using a magnetic stirrer, diluting at an appropriate ratio, spreading on Nutrient Agar, incubating for 24 hours at 32℃ in an incubator, and dilution with the number of colonies. The ratio was converted to measure the cell concentration of Bacillus subtilis BS300 (KCTC18263P).

2. Experiment result

As a result of the above experiment, the bacterial cell concentration of the novel microorganism Bacillus subtilis BS300 (KCTC18263P) of the present invention was confirmed to be 5.6×10 ⁹ cfu/g, and the minimum concentration of Bacillus bacillus under the permission of the soil microorganism or feed probiotic was 1×10 ⁶ cfu/g. Considering the matters, it was confirmed that there is a value of use as a source of Bacillus bacillus powder for microbial production by diluting 100 to 500 times.

<Experimental Example 3> Confirmation of the amount of enzyme produced in the novel strain of the present invention

1. Experiment method

To measure the enzyme activity, 1 ml of the eluate from Experimental Example 2 was taken and centrifuged at 10,000 g for 10 minutes, and then the supernatant was sterilized with a membrane syringe filter having a pore size of 0.45 μm, and used as a coenzyme.

Enzymatic activity of protease is determined by dissolving casein to 1% in 100 mM Tris-HCI buffer (pH 7.3) and used as a substrate solution. 0.5 ml of the coenzyme was added to 0.5 ml of the substrate solution and reacted at 32°C for 1 hour. The reaction was stopped by adding 40% (w/v) TCA (trichloroacetic acid) solution, and then allowed to stand at room temperature for 15 minutes. After centrifugation at 12,000 g for 15 minutes, the supernatant was taken and the absorbance was measured at 280 nm to measure the amount of soluble peptide. The standard curve was L-tyrosine solution, and the enzyme activity unit was defined as 1 unit as the amount of enzyme that produced tyrosine equivalent to 1 μg for 1 minute.

Amylase enzyme activity was reacted at 30° C. for 1 hour by adding 0.5 ml of 1% (w/v) soluble starch coenzyme. After stopping the reaction by adding 1 ml of cold 1N HCl, 0.1 ml of Lugol's iodine solution was added to develop color. Distilled water was added and the absorbance was measured at 620 nm in water determined to be 25 ml. The standard curve was a maltose solution, and the enzyme activity unit was defined as 1 unit, which is equivalent to reducing soluble starch by 0.01% for 1 minute.

Cellulase enzyme activity was reacted at 50° C. for 10 minutes by mixing 0.1 ml of crude enzyme solution and 0.5 ml of 1% (W/V) CMC (Carboxy methyl cellulose) substrate solution dissolved in 0.05M glycin-NaOH buffer solution. Reducing sugar produced by CMC hydrolysis was colorimetrically quantified by measuring absorbance at 550 nm according to the method using 3,5-dinitrosalicylic acid (DNS), and the amount of enzyme that generates 1 μmol of reducing sugar for 1 minute was 1 unit.

In addition, in order to confirm the excellence of the enzyme activity of the novel microorganism Bacillus subtilis BS300 (KCTC18263P) of the present invention, the present invention is obtained by comparing the enzyme production capacity and the cell concentration during solid culture by receiving general Bacillus subtilis , which has an enzyme production ability. The superiority of KCTC18263P was confirmed.

In other words, Bacillus subtilis (KCTC No. 3042) capable of producing Protease and Cellulase enzymes and Bacillus subtilis (KCTC No. 1028) capable of producing Amylase enzymes were sold and compared.

Bacillus subtilis (KCTC No. 3042) is known as KCTC Media No. With a composition of 108, Bacillus subtilis (KCTC No. 1028) was obtained from KCTC Media No. It was cultured in a liquid phase with the composition of 1 and used as a solid culture seed. Bacillus subtilis BS300 (KCTC18263P) of the present invention was used as a solid culture seed by liquid culture by the above method.

Solid culture was carried out according to Experimental Example 2 of the present invention, and enzyme activity was measured according to the above method.

2. Experiment result

The experimental results were shown in Table 4 below.

division The present invention
New microorganism Common microbes
(Korea Research Institute of Bioscience and Biotechnology Gene Bank) Bacillus subtilis BS300(KCTC18263P) Bacillus subtilis
(KCTC No.3042) Bacillus subtilis
(KCTC No.1028) Protease 502 unit 45 unit 9 unit Cellulase 212 unit 7 unit 11 unit Amylase 412 unit 12 unit 36 unit Solid cultured cell concentration 5.6x10 ⁹ cfu/g 3.2x10 ⁸ cfu/g 5.1x10 ⁸ cfu/g

As shown in Table 4, in the eluate of Experimental Example 2 containing the novel microorganism Bacillus subtilis BS300 (KCTC18263P) of the present invention, the enzyme activity of Protease was 502 unit, the enzyme activity of Amylase was 412 unit, and the enzyme activity of Cellulase was 212 unit.

Thus, it was found that even with the same homogeneous strain, there was a significant difference in its enzyme activity. In other words, Bacillus subtilis BS300 (KCTC18263P) of the present invention showed remarkably excellent results, which confirmed that the strain of the present invention had sufficient value as a new microorganism.

<Experimental Example 4> Confirmation of the storage ability of the novel strain of the present invention

1. Experiment method

Bacillus subtilis BS300 (KCTC18263P) solid culture is the key to confirming the stability of the cells according to the storage conditions in order to provide ease of manufacture of the powdered microbial agent and prevent the decrease in the number of cells even during distribution, so the cells obtained in Experimental Example 2 are placed in a plastic bag. Each 100g was dispensed and sealed to check storage at room temperature (a place where direct sunlight is avoided and well ventilated). The cell concentration over time was performed in the same manner as in Experimental Example 2.

2. Experiment result

Storage months Room temperature storage
- Bacillus subtilis BS300(KCTC18263P) Room temperature storage
- Bacillus subtilis (KCTC No. 1028) Cell concentration (10 ^x cfu/g) Cell concentration (10 ^x cfu/g) 0 5.31E+09 5.1E+08 3 5.29E+09 1.30E+08 6 5.27E+09 5.29E+07 9 5.21E+09 2.22E+07 12 5.01E+09 5.11E+06 15 4.81E+09 5.03E+05

As shown in Table 5, Bacillus subtilis (KCTC No. 1028) is 3 months after the elapse of 3 months, considering that the longest shelf life of the microbial agent is usually 1 year and the preservation of this result is 1 year or more. As the cell storage rate is rapidly falling, it is judged that it is difficult to store at room temperature, whereas the Bacillus subtilis BS300 (KCTC18263P) solid culture of the present invention has a reduction rate of 0.38% even when stored at room temperature for 3 months, 0.75% when stored for 6 months, and 9 months. Storage is 1.88%, storage for 12 months, 5.65%, storage for 15 months, 9.42%, so it can be safely distributed even at room temperature, so it is considered to have sufficient effectiveness as the raw material (raw material) for producing microbial agents.

As described above, the present invention provides Bacillus subtilis BS300, a novel microorganism with high resolution for carbohydrates, proteins, and fibrin, among organic matter even in the liver of homogeneous strains, and at the same time, solid culture of Bacillus subtilis BS300, a new microorganism It provides a solid culture method including Bacillus subtilis BS300 strain, which is capable of and exhibits ease of manufacture and stability of distribution.

Those of ordinary skill in the art to which the present invention pertains will be able to implement embodiments of different forms from the detailed description of the present invention within the essential technical scope of the present invention. Here, the essential technical scope of the present invention is shown in the claims, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Name of donated institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC18263P

Consignment Date: 20131202

<110> LEE, Jeong-Bok <120> New microorganism Bacillus subtilis BS300 having an excellent decomposition effect of organic matter and solid medium composition for culture of Bacillus subtilis BS300 and the microbial agent comprising the same <130> 01-000001 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 821 <212> DNA <213> Bacillus subtilis <400> 1 gagtaacacg tgggtaacct gcctgtaaga ctgggataac tccgggaaac cggggctaat 60 accggatggt tgtttgaacc gcatggttca aacataaaag gtggcttcgg ctaccactta 120 cagatggacc cgcggcgcat tagctagttg gtgaggtaac ggctcaccaa ggcaacgatg 180 cgtagccgac ctgagagggt gatcggccac actgggactg agacacggcc cagactccta 240 cgggaggcag cagtagggaa tcttccgcaa tggacgaaag tctgacggag caacgccgcg 300 tgagtgatga aggttttcgg atcgtaaagc tctgttgtta gggaagaaca agtaccgttc 360 gaatagggcg gtaccttgac ggtacctaac cagaaagcca cggctaacta cgtgccagca 420 gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta ttgggcgtaa agggctcgca 480 ggcggtttct taagtctgat gtgaaagccc ccggctcaac cggggagggt cattggaaac 540 tggggaactt gagtgcagaa gaggagagtg gaattccacg tgtagcggtg aaatgcgtag 600 agatgtggag gaacaccagt ggcgaaggcg actctctggt ctgtaactga cgctgaggag 660 cgaaagcgtg gggagcgaac aggattagat accctggtag tccacgccgt aaacgatgag 720 tgctaagtgg ttagggggtt tccgcccctt agtgctgcag ctaacgcatt aagcactccg 780 cctgggggag tacggtcgca agactgaaac tcaaaggaat t 821

Claims (5)

Protease 500-510 unit, cellulase 200-220 unit and amylase 400-420 unit content,
Degradation of carbohydrates, proteins, and fibrin among organic matter and possible to survive for 15 months at room temperature in solid culture.
New microorganism Bacillus subtilis BS300 strain ( Bacillus subtilis BS300, accession number: KCTC 18263P)
delete The Bacillus subtilis BS300 strain (Bacillus subtilis BS300, accession number: KCTC 18263P) selected to have resolution for carbohydrates, proteins, and fibres among organic matter was isolated,
Based on the total weight of the liquid culture medium for the isolated strain, glucose (Glucose) is 0.1 ~ 0.5% (W / V), starch (starch) is 0.5 ~ 1% (W / V), yeast extract (Yeast Extract) 0.5~1% (W/V), Peptone 0.1~0.5% (W/V), sodium chloride (NaCl) 0.01~0.1% (W/V), potassium phosphate (K ₂ HPO ₄ ) 0.05 ~0.3% (W/V), magnesium sulfate 7 hydrate (MgSO ₄ 7H ₂ 0) is 0.01 to 0.05% (W/V), and ferrous sulfate 7 hydrate (FeSO ₄ 7H ₂ 0) is A first step of preparing a liquid culture solution by inoculating a liquid culture medium consisting of a composition containing 0.01 to 0.05% (W/V);
Skim steel 25 to 30% by weight, protein skin 45 to 55% by weight, glucose 3 to 5% by weight, alpha starch 10 to 15% by weight, yeast extract 3 to 7% by weight, Peptone 1 to 2 wt%, soy flour 1 to 3 wt%, potassium phosphate (K ₂ HPO ₄ ) 1 to 3 wt%, Magnesium sulfate heptahydrate (MgSO ₄ 7H ₂ 0) 0.01 to 0.1 wt%, manganese sulfate heptahydrate (MnSO ₄ 7H ₂ 0) 0.01 to 0.1 wt%, and calcium chloride (CaCl ₂ ) 0.01 to 0.1 wt% A second step of preparing a solid medium composition by mixing;
Including; a third step of adding the liquid culture solution of the first step to the solid medium composition of the second step, followed by culturing and then drying to prepare a solid culture in the form of a powder;
A solid culture method comprising cultivating a new microorganism Bacillus subtilis BS300 (Bacillus subtilis BS300, accession number: KCTC 18263P), which can survive for 15 months at room temperature when culturing carbohydrates, proteins, and fibers among organic matter. delete delete

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