JP6975701B2 - Bacillus subtilis isolation method, Bacillus subtilis, microbial preparation containing Bacillus subtilis, medium set for Bacillus subtilis isolation - Google Patents

Description

The present invention relates to a method for isolating Bacillus subtilis quickly, easily, at low cost and with high safety by using a microbial isolation source obtained from the environment such as activated sludge, soil, plant body, animal body and water body.

Traditionally, various chemicals have been used to treat wastewater. One such drug is a microbial preparation using a bacterium belonging to the genus Bacillus. Since Bacillus bacteria secrete large amounts of proteases (proteolytic enzymes) and lipases (oil-degrading enzymes), microbial preparations containing Bacillus bacteria are added when high-concentration proteins and fats flow into the aeration tank. By doing so, these loads are reduced.

In addition, Bacillus bacteria take the form of spores during environmental changes such as pH fluctuations, temperature changes, decreased dissolved oxygen, contamination with poisons, and depletion of nutrient sources, and can withstand environmental changes without dying. It is considered suitable for microbial preparations.

Patent Document 1 discloses a method described as a method for isolating a predetermined bacterium from many bacteria, although it is not a technique for isolating a bacterium belonging to the genus Bacillus. Patent Document 2 discloses a method of magnifying and observing a microorganism with a microscope, judging from a growth pattern, and isolating a microorganism such as Bacillus by a manipulator.

In addition, as a conventional technique for identifying microbial species, for example, a sample obtained from the environment is smeared on a standard agar medium to catch a single colony, and 16S ribosomal DNA is amplified by a PCR (Polymerase Chain Reaction) apparatus. There is a method of sequentially analyzing gene sequences with a sequencer and searching for homology in an international nucleotide sequence database, etc., which identifies microbial species (for example, Bacillus subtilis, Bacillus cereus, etc. in the case of Bacillus). Is known to be.

On the other hand, bacteria of the genus Bacillus are present in the environment such as activated sludge, soil, plants, animals, water bodies, urine, and fermented foods. About 300 species of Bacillus bacteria are known, and as a typical species, Bacillus is used in the production of fermented foods such as natto and has established safety in that it does not have pathogenicity. There is subtilis).

Japanese Unexamined Patent Publication No. 2008-271786 Japanese Unexamined Patent Publication No. 10-136973

In the above-mentioned method for identifying microbial species, equipment and reagents are extremely expensive, work procedures are complicated, and test days are long. Furthermore, various bacteria other than Bacillus bacteria occupy the overwhelming majority in the environment, and even if it is narrowed down to Bacillus bacteria by heat treatment etc., Bacillus subtilis after homology search in the presence of 300 species of Bacillus. The probability of hitting is not high.

On the other hand, there are some species of Bacillus bacteria having pathogenicity, for example, pathogenic Bacillus anthracis and Bacillus cereus. For this reason, not all of the 300 species belonging to the genus Bacillus are safe against pathogenicity.

In addition, anthrax and Bacillus cereus are species that have been proven to be pathogenic to humans, and it is hard to say that there is no possibility that new bacterial species that are pathogenic to animals and plants other than humans will be proposed in the future. It is not preferable to use a bacterial species that may carry such a risk as a microbial preparation in the water treatment process and release it into the environment.

If it becomes possible to develop a method for isolating Bacillus subtilis, which has been established to be safe against pathogenicity, from the environment, it can be widely applied not only to microbial preparations but also to fermented foods. ..

In the method of Patent Document 1 described above, a procedure for performing enrichment culture is required for isolation, but there is a problem that it takes time and effort to culture for a long period of time (3 to 4 weeks) in the enrichment culture. , Further simplification was required.

Since the method of Patent Document 2 described above requires a large number of expensive devices such as a microscope, a manipulator, a temperature control device, a computer, and an image processing device, capital investment is required for isolation, which is costly. rice field.

In addition, this method is a method that cannot selectively isolate Bacillus subtilis among the bacteria of the genus Bacillus, does not require large-scale equipment, and selectively isolates Bacillus subtilis by a simple method. A method that can be used was desired.

Therefore, an object of the present invention is to provide a method for isolating Bacillus subtilis from the environment, which is highly safe against pathogenicity, by a simple, rapid and inexpensive method. Another object of the present invention is to provide a highly safe Bacillus subtilis isolated by the isolation method. Furthermore, an object of the present invention is to provide a highly safe microbial preparation using the Bacillus subtilis.

Furthermore, an object of the present invention is to provide a medium set for Bacillus subtilis isolation for isolating Bacillus subtilis.

In order to solve the above problems, the present invention can have the following configuration.
(1) A method for isolating the Bacillus subtilis from a sample containing Bacillus subtilis collected from the environment.
(A) A sterilization step of sterilizing the sample so that the spore-forming bacteria contained in the sample remain.
(B) Bacteria contained in the sterilized sample are cultured and cultured using a first medium that is substantially free of lecithin-containing substances, sugars that are decomposed by bacilli to produce acid, and blood. At least one of (C) lecithin-containing substances, sugars that are decomposed by bacilli to produce acid, and blood in the first culture step for obtaining Bacillus bacteria including bacilli. Bacteria of the genus Bacillus, including the bacillus obtained from the first culturing step, are cultivated using a second medium further containing a pH indicator when the bacterium is contained and is decomposed by the bacillus to produce acid. The second culture step, in which the bacillus is obtained from the cultivated bacteria,
including.
(2) The sterilization step is performed by heating the sample.
(3) The first medium contains 1 to 10 g of peptone, 1 to 5 g of yeast extract, 0.1 to 5 g of glucose, and 5 to 30 g of agar with respect to 1 L of water, and the second medium is water. Meat extract 0.1-5g, Peptone 1-20g, D-mannitol 0.5-20g, Sodium chloride 1-20g, Phenol red 0.01-0.1g, Agar 5-30g, Egg yolk solution per 1L (50%) Contains 1-100 mL, polymixin B 10,000-500,000 units.
(4) The sample containing Bacillus subtilis collected from the environment is activated sludge.
(5) The first culture step and the second culture step are performed by the plate culture method.
(6) Bacillus subtilis isolated by the above isolation method.
(7) A microbial preparation containing the Bacillus subtilis.
(8) A first medium for culturing Bacillus subtilis bacteria, which is substantially free of lecithin-containing substances, sugars that are decomposed by Bacillus subtilis to produce acid, and acid that is decomposed by Bacillus subtilis and lecithin-containing substances. A second medium for culturing Bacillus subtilis, which contains at least one of the sugars that produce Bacillus subtilis and, if contains a sugar that is degraded by the Bacillus subtilis to produce acid, further comprises a pH indicator. A set of media for Bacillus subtilis isolation, including.
(9) Bacillus subtilis B-1-2 strain Bacillus subtilis deposited under accession number NITE P-02795.
(10) Bacillus subtilis B-6-1 strain of Bacillus subtilis deposited under accession number NITE P-02796.
(11) Bacillus subtilis B-10-1 strain deposited under accession number NITE P-02797, Bacillus subtilis.
(12) Bacillus subtilis B-11-3 strain deposited under accession number NITE P-02798, Bacillus subtilis.
(13) A microbial preparation containing Bacillus subtilis according to any one of (9) to (12).

According to the present invention, Bacillus subtilis, which is highly safe against pathogenicity, can be isolated from the environment by a simple, rapid and inexpensive method.
In addition, Bacillus subtilis B-1-2 strain (accession number NITE P), which is a novel strain having high decomposition activity of organic substances (starch, protein, and fat) from Bacillus subtilis isolated from activated sludge using the method of the present invention. -02795), B-6-1 strain (accession number NITE P-02796), B-10-1 strain (accession number NITE P-02797), B-11-3 strain (accession number NITE P-02798), respectively. Isolated. These new strains were deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary on October 16, 2018.

The outline of the method of this invention is shown. Each step in the Example is shown. It is a photograph which shows the amorphous colony of the bacterium of the genus Bacillus cultured in the step (B). It is a photograph showing the colony of Bacillus subtilis cultivated in the step (C). It is a photograph which shows the colony of the bacterium other than Bacillus subtilis cultivated in the step (C).

Hereinafter, the present invention will be specifically described. FIG. 1 is a diagram showing an outline of the method of the present invention.
As described above, the isolation method of the present invention is a method for isolating the Bacillus subtilis from a sample containing Bacillus subtilis collected from the environment.
(A) A sterilization step of sterilizing the sample so that the spore-forming bacteria contained in the sample remain.
(B) Bacteria contained in the sterilized sample are cultured and cultured using a first medium that is substantially free of lecithin-containing substances, sugars that are decomposed by bacilli to produce acid, and blood. At least one of (C) lecithin-containing substances, sugars that are decomposed by bacilli to produce acid, and blood in the first culture step for obtaining Bacillus bacteria including bacilli. Bacteria of the genus Bacillus, including the bacillus obtained from the first culturing step, are cultivated using a second medium further containing a pH indicator when the bacterium is contained and is decomposed by the bacillus to produce acid. The second culture step, in which the bacillus is obtained from the cultivated bacteria,
including.

Bacillus subtilis forms spores and is resistant to various stresses, so it is universally present in all parts of the environment such as soil and plants. The present invention is for isolating Bacillus subtilis from a sample containing Bacillus subtilis collected from the environment. Hereinafter, each step will be described in detail.

[Step (A) (sterilization step)]
In the step (A), a sample collected from the environment is sterilized so that the spore-forming bacteria contained in the sample remain. Here, the environment includes both a natural environment and an artificial environment. Specifically, for example, natural soil (soil, leaf mold, gravel, compost, etc.), plant body (herbaceous, woody, flower, dead grass, dead leaf, moss, etc.), animal body (skin, organ, excrement, etc.) , Water areas (river water, lake water, seawater, etc.), and active sludge, fermented foods, slime (biofilm) for kitchens and pipes, human urine, etc. All environments in which can live are listed. In the present invention, a sample containing Bacillus subtilis collected from the environment (hereinafter, also referred to as "environmental sample") is used.

The environmental sample contains not only Bacillus subtilis but also various germs other than Bacillus subtilis. The method for sterilization may be any method as long as it can be sterilized so that the spore-forming bacteria contained in the environmental sample remain, but the preferred sterilization method is heating, acid treatment, base treatment, alcohol treatment, or hypochlorous acid. Examples include disinfectant treatment such as sodium chlorite, UV irradiation, dryness, and anaerobic conditions. Heating is particularly preferable.

The heating conditions may be any conditions as long as spore-forming bacteria remain and non-heat-resistant bacteria can be excluded. The heating temperature is, for example, 60 ° C to 100 ° C, preferably 70 ° C to 90 ° C. The heating time is, for example, 1 minute to 3 hours, preferably 20 minutes to 2 hours, and more preferably 30 minutes to 90 minutes. The heating pressure is preferably, for example, 0.8 to 1.2 atm, preferably under atmospheric pressure or a pressure close to this (for example, 0.9 to 1.1 atm).

Examples of the acid used for the acid treatment include inorganic acids such as hydrochloric acid and sulfuric acid, and organic acids. Examples of the base used for the base treatment include sodium hydroxide and potassium hydroxide. For the use of these acids and salts, those skilled in the art will appropriately select conditions under which spore-forming bacteria remain and other sterilization can be removed.

[Step (B) (First culture step)]
In step (B), a first medium that is substantially free of lecithin-containing substances, sugar that is decomposed by Bacillus subtilis to produce acid, and blood is included in the sample sterilized in step (A). Bacteria are cultivated, and Bacillus bacteria including Bacillus subtilis are obtained from the cultured bacteria.

The first medium is one capable of culturing Bacillus bacteria including Bacillus subtilis. Therefore, the first medium usually contains at least a gelling agent and an amino acid source.
Examples of the gelling agent include, but are not limited to, agar, gellan gum, xanthan gum, locust bean gum, polyvinyl alcohol, alkyl cellulose, carboxyalkyl cellulose, hydroxyalkyl cellulose and the like. Agar is particularly preferable.

Examples of the amino acid source include peptone, tryptone, and extracts. Examples of the peptone include casein peptone and soybean peptone. Examples of the extracts include yeast extract, meat extract, malt extract, potato extract and the like.

The first medium may contain other components. Other components include sugars, extracts (for the supply of vitamins, minerals, etc.), sodium chloride and the like. Examples of saccharides include glucose, sucrose and the like. Examples of the extracts include yeast extract, meat extract, malt extract, potato extract and the like.

The content of various components of the first medium is not particularly limited as long as it can culture Bacillus bacteria, but for example, 1 to 30 g of a gelling agent, 1 to 10 g of an amino acid source, and 1 to 5 g of extracts. The amount of saccharides is 1 to 5 g and the amount of sodium chloride is 1 to 10 g (these contents are for 1 L of purified water).

The pH of the first medium is preferably 7.0 ± 0.1.
The specific composition of the preferred first medium is 1-10 g of casein-pancreatic peptone, preferably 3-7 g, 1-5 g of yeast extract, preferably 1.5-4 g, per 1 L of water (such as purified water). , 0.1 to 5 g of glucose, preferably 0.5 to 3 g, and 5 to 30 g of agar, preferably 10 to 20 g, and the pH is 7.0 ± 0.1.

As a particularly preferable first medium, for example, a standard agar medium can be used. The standard agar medium is a non-selective medium that can be used for culturing general bacteria, and is defined and used by, for example, the Ministry of Waterworks Law-related ordinance, the Japanese Pharmacopoeia, and the like.

As mentioned above, the first medium is substantially free of lecithin-containing substances, sugars that are degraded by Bacillus subtilis to produce acid, and blood. "Substantially free" means that in the case of lecithin-containing substances or blood, the content of the lecithin-containing substances or blood in the first medium is less than 0.5 mL with respect to 1 L of purified water. In the case of sugar that is decomposed by lecithin to produce acid, the content of sugar that is decomposed by lecithin to produce acid in the first medium is less than 0.5 g with respect to 1 L of purified water. Means that.

It is preferred that the first medium is substantially free of antibiotics. The term "substantially free of antibiotics" means that the content of antibiotics in the first medium is less than 1 unit with respect to 1 L of purified water.

The culture in step (B) is usually carried out by a plate culture method under aerobic conditions. The culture temperature is, for example, 20 to 40 ° C. The culture time is, for example, 12 to 72 hours.
When the first culture step is performed in the step (B), in addition to the colonies of Bacillus bacteria, a certain number of colonies of spore-forming bacteria other than Bacillus and colonies of miscellaneous bacteria surviving in the sterilization step of step (A) are cultured. NS. From these, Bacillus bacteria (including Bacillus subtilis) can be obtained by fishing for coarse and amorphous colonies peculiar to Bacillus bacteria.

[Step (C) (second culture step)]
In step (C), a lecithin-containing substance, a sugar that is decomposed by Bacillus subtilis to produce acid, and at least one of blood, and pH if a sugar that is decomposed by Bacillus subtilis to produce acid is contained. Bacillus bacteria including Bacillus subtilis obtained from the first culture step are cultured using a second medium further containing an indicator, and Bacillus subtilis is obtained from the cultured bacteria.

Examples of the lecithin-containing substance include egg yolk, soybean lecithin, and fish lecithin. Egg yolk is preferred. Examples of sugars that are decomposed by Bacillus subtilis to produce acid include mannitol, arabinose, and xylose. Mannitol is preferred. Examples of blood include sheep blood, horse blood, human blood and the like. Blood is preferably in the form of defibrillated. Examples of pH indicators include phenol red and bromcresol purple.

The second medium is one in which Bacillus subtilis can be cultivated. Therefore, the second medium usually contains at least a gelling agent and an amino acid source, in addition to one of the above lecithin-containing substances, sugars that are degraded by Bacillus subtilis to produce acid, and blood.

As the gelling agent and the amino acid source, the same ones as described above for the first medium can be used.
The second medium may further contain other components. Other components include sugars, extracts, sodium chloride, antibiotics and the like. As the saccharides and extracts, the same ones as described above can be used for the first medium. Examples of the antibiotic include an antibiotic insensitive to Bacillus subtilis, for example, polymyxin B. In the present invention, in the first medium and the second medium, one of the listed components may be used alone, or two or more of them may be combined.

The contents of various components of the second medium are, for example, 0.5 to 50 mL for lecithin-containing substances, 0.5 to 20 g for sugars that are decomposed by Bacillus subtilis to produce acid, and 0.5 to 100 mL for blood. The gelling agent is 1 to 30 g, the amino acid source is 1 to 10 g, the extracts are 1 to 5 g, the saccharide is 1 to 5 g, and the sodium chloride is 1 to 10 g (these contents are for 1 L of purified water). ..

The pH of the second medium is preferably in the range of 7.0 to 7.4, particularly preferably in the range of 7.1 to 7.3.
The specific composition of the preferred second medium is 0.1 to 5 g of meat extract, preferably 0.5 to 2 g, 1 to 20 g of peptone, preferably 5 to 15 g, relative to 1 L of water (such as purified water). D-mannitol 0.5 to 20 g, preferably 5 to 15 g, sodium chloride 1 to 20 g, preferably 5 to 15 g, phenol red 0.01 to 0.1 g, preferably 0.02 to 0.05 g, agar 5- 30 g, preferably 10 to 20 g, egg yolk solution (50%) 1 to 100 mL, preferably 1 to 80 mL, more preferably 10 mL to 70 mL, polymixin B 10,000 to 500,000 units, preferably 50,000 to 200,000. It is a unit and has a pH of 7.0 to 7.4.

The culture in step (C) is usually carried out by a plate culture method under aerobic conditions. The culture temperature is, for example, 20 to 40 ° C. The culture time is, for example, 12 to 72 hours.
If the second medium contains a sugar that is broken down by the bacillus to produce an acid and phenol red as a pH indicator, the bacillus breaks down the sugar to generate an acid and the pH indicator is acidic (ie). , The colony is yellow). On the other hand, other Bacillus bacteria (Bacillus cereus, Bacillus cereus, etc.) do not decompose the sugar and the colonies do not turn yellow. Therefore, after the culture is completed in the step (C), it can be determined that the colony exhibiting yellow is a colony of Bacillus subtilis.

On the other hand, when the second medium contains a lecithin-containing substance, Bacillus subtilis does not react with the lecithin-containing substance, while other Bacillus bacteria, especially pathogenic Bacillus bacteria (Bacillus cereus, anthrax, etc.) contain lecithin. Causes a recitinase reaction with a substance. Since the colonies that have undergone the recitinase reaction exhibit an opaque zone around them, it is determined that the colonies having the opaque zone are Bacillus bacteria other than Bacillus subtilis and the colonies without the opaque zone are the colonies of Bacillus subtilis. Can be done.

The second medium may contain both sugars (and pH indicators) that are degraded by Bacillus subtilis to produce acid and lecithin-containing substances, in which case yellow colonies without opaque bands are Bacillus subtilis colonies. It can be determined that there is. Although Bacillus subtilis can be isolated from environmental samples with an extremely high probability by this method, it is presumed that the possibility of isolating a strain that is not Bacillus subtilis is not zero. It is desirable to perform a cross check of.

When blood is contained in the second medium (blood agar medium), Bacillus subtilis can be discriminated from the cultured bacteria from the viewpoint of the presence or absence of hemolytic property. Since Bacillus subtilis is hemolytic, the cultured colonies are colonies with concentric hemolytic zones.

[Bacillus subtilis, microbial preparation, medium set]
The present invention also provides Bacillus subtilis obtained by the isolation method of the present invention. The present invention also provides a microbial preparation containing the Bacillus subtilis of the present invention. Furthermore, the present invention provides a medium set for Bacillus subtilis isolation containing the above-mentioned first medium and second medium.

As described above, according to the present invention, Bacillus subtilis, which is highly pathogenic and safe, can be isolated from the environment by a simple, rapid and inexpensive method. The isolated Bacillus subtilis can be used not only for microbial preparations used in water treatment processes, but also for soil conditioners, compost fermentation accelerators, microbial pesticides, deodorants, fermented foods such as natto, enzyme production, supplements, laboratory tests, etc. It can be appropriately used for strain collection and the like. Hereinafter, the present invention will be described with reference to examples.

[1] Example 1
Example 1 was performed in the order shown in FIG. First, 90 mg of a sample collected from the environment (soil, dead leaves, dead grass, moss, activated sludge) was suspended in 9 ml of sterile water. The suspended sample is first serially diluted, followed by standard agar medium (peptone 5 g / L, yeast extract 2.5 g / L, glucose 1 g / L, agar 15 g / L, pH 7.0) according to the sewage test method. Dilution was performed according to ± 0.1).

Next, after standing still culturing at 35 ° C. for 24 hours, the number of general bacteria was calculated (reference value, Table 1 below). This general bacterium count indicates the general bacterium contained in the sample (all mesophilic aerobic bacteria growing on a standard agar medium), for example, 125,000 CFU / ml of soil and 1 ml of sterile water after suspension. It shows that there are 125,000 live bacteria in it.

Then, the suspension was immersed in a water bath at 80 ° C. together with the container and heat-treated for 1 hour (step (A)). As a result, germs other than spore-forming bacteria (such as Bacillus bacteria) were sterilized.
Next, after serially diluting the sterilized suspension, a standard agar medium (peptone 5 g / L, yeast extract 2.5 g / L, glucose 1 g / L, agar 15 g / L, pH 7. It was smeared to 0 ± 0.1) and allowed to stand at 30 ° C. for 24 hours for plate culture. After culturing, coarse and amorphous colonies (colonyes indicated by arrows in FIG. 3) peculiar to Bacillus bacteria were obtained from the spore-forming bacteria and the microorganisms that survived the heat sterilization in step (A) (step (B)).

As shown in Table 1, 2 strains of Bacillus bacteria were obtained from soil, 1 strain from dead leaves, 3 strains from dead grass, 5 strains from moss, and 16 strains from activated sludge.
For each of the acquired colonies of Bacillus subtilis, MYP agar medium (meat extract 1 g / L, peptone 10 g / L, D-mannitol 10 g / L, sodium chloride 10 g / L, phenol red 0.025 g) as a second medium. / L, agar 15 g / L, egg yolk solution (50%) 50 ml / L, polymixin B100,000 units / L, pH 7.2 ± 0.1), smeared with a stroke, and statically cultured at 30 ° C for 24 hours. Yellow colonies without opaque bands (FIG. 4) were discriminated as Bacillus subtilis from the cultured Bacillus spp. (Step (C)).

As shown in Table 1, 1 strain of Bacillus subtilis was obtained from soil, 1 strain of Bacillus subtilis was obtained from dead grass, and 4 strains of Bacillus subtilis were obtained from activated sludge. A homology search was performed on these 6 strains by gene analysis, and all of them were 100% consistent with the known Bacillus subtilis sequence. Of the soil, dead leaves, dead grass, moss, and activated sludge, it was possible to isolate them most efficiently from activated sludge. As shown in FIG. 5, the colonies that do not exhibit yellow color and have an opaque zone are Bacillus bacteria other than Bacillus subtilis.

By configuring the standard agar medium and MYP agar medium used in this example as a medium set and including a platinum loop for smearing and sterile diluted water as accessories, even if there is no special large-scale equipment, the place can be used. Anyone can easily and quickly isolate Bacillus subtilis.

[2] Comparative Example 1
As Comparative Example 1, after preparing a suspension of an environmental sample in the same manner as in Example 1, 1. 2. When the sample sterilized in the above MYP agar medium is cultured (at 30 ° C. for 24 hours) after the step of sterilizing the environmental sample by heating (same conditions as in the example). 3. When the environmental sample is cultured in the above standard agar medium (at 30 ° C. for 24 hours) from the beginning without performing the sterilization step. Tables 2 to 4 show the results when the environmental sample was cultured (at 30 ° C. for 24 hours) in the above-mentioned MYP agar medium from the beginning without performing the sterilization step. In either case, germs proliferated vigorously, and the next step could not be proceeded, and Bacillus subtilis could not be isolated.

[3] Example 2
In the same manner as in Example 1, about 40 strains of Bacillus subtilis were isolated from a plurality of activated sludge sources. For these Bacillus subtilis strains, each strain is cultured in a nutrient medium (polypeptone 10 g / L, yeast extract 2 g / L, magnesium sulfate heptahydrate 1 g / L, pH 7.0), and the cells are washed and centrifuged. After recovery, it was pulverized with a holding carrier in a freeze-dryer to produce a microbial preparation. Using this microbial preparation, the degrading activity of organic substances (starch, protein, and fats and oils) was evaluated. Also. In addition to the acquired strain, another company's Bacillus subtilis-containing microbial preparation (other company's product A) was also used for the same test as a control group. Incidentally, Bacillus subtilis bacterial count of microorganisms formulations and Competitor A was produced, were both 10 8 ^CFU / g.

The starch degrading activity was evaluated by the following method. A 200 mL triangular flask was filled with 100 mL of simulated drainage (yeast extract 0.5 g / L, diammonium hydrogen phosphate 0.5 g / L, pH 7.0) and sterilized by autoclave. After autoclaving, another sterilized starch aqueous solution was added with a 0.22 μm filter to adjust the final starch concentration to 0.2 g / L. 100 mg / L of the microbial preparation was added to the adjusted medium, and the mixture was shaken back and forth at 150 rpm and 30 ° C. Sampling was performed in cultures 0h and 48h, starch was quantified using a starch measurement kit (K-TSTA-100A, manufactured by Megazyme), and the starch decomposition rate was calculated by the following formula:
(Culture 0h-Culture 48h) ÷ Culture 0h x 100 = Starch decomposition rate (%).

The evaluation of proteolytic activity was carried out by the following method. A 200 mL triangular flask was filled with 100 mL of the above-mentioned simulated drainage and sterilized by autoclave. After autoclaving, another sterilized casein Na aqueous solution was added with a 0.22 μm filter to adjust the final concentration of casein Na to 2 g / L. 100 mg / L of the microbial preparation was added to the adjusted medium, and the mixture was shaken back and forth at 150 rpm and 30 ° C. Sampling was performed at cultures 0h and 6h, protein was quantified using a protein measurement kit (Protein Assay Rapid Kit Wako II manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.), and the proteolysis rate was calculated by the following formula:
(Culture 0h-Culture 6h) ÷ Culture 0h x 100 = Proteolysis rate (%).

The evaluation of fat decomposition activity was carried out by the following method. A 200 mL triangular flask was filled with 100 mL of the above-mentioned simulated drainage and sterilized by autoclave. After autoclaving, another sterilized lard (manufactured by Bell Shokuhin, genuine lard) was added with a 0.22 μm filter to adjust the final lard concentration to 1 g / L. 100 mg / L of the microbial preparation was added to the adjusted medium, and the mixture was shaken back and forth at 150 rpm and 30 ° C. Sampling was performed at 0h and 48h of the culture, and 30 mg of a lipase preparation (Swing 120, manufactured by Swing Corporation) was added to 4 mL of the sample, followed by an enzymatic reaction at 40 ° C. for 60 minutes to release glycerin. Then, the amount of fat and oil was measured by measuring the residual glycerin with an F-kit (F-kit glycerol manufactured by JK International Co., Ltd.). It has been confirmed in advance that glycerin, which was present before the enzymatic reaction with the lipase preparation, is rapidly assimilated by Bacillus bacteria. For the obtained amount of fat and oil, the fat and oil decomposition rate was calculated by the following formula:
(Culture 0h-Culture 48h) ÷ Culture 0h x 100 = Fat decomposition rate (%).

The test results are shown in Table 5. Tests were carried out on multiple strains obtained from activated sludge, and the results of 4 strains with particularly high decomposition activity are shown. These four strains tended to have a significantly higher decomposition activity of organic substances than the other company's product A.

The base sequence (about 1,500 bp) of 16SrDNA was analyzed for each strain named B-1-2 strain, B-6-1 strain, B-10-1 strain, and B-11-3 strain, and internationally. nucleotide sequence databases result of homology search by (BLAST), both strains were also consistent sequence 99.9% of the known Bacillus subtilis (Ba c illus subtilis). The results of homology search and morphology observation, these strains were confirmed to belong to Bacillus subtilis (Ba c illus subtilis). These strains are novel strains independently isolated from the natural world (activated sludge). Compared with known strains (competitor's product A), these novel strains produce high amylase, protease, and lipase, and have significantly higher starch, protein, and fat-decomposing activity. Each of these strains was deposited with the National Institute of Technology and Evaluation Patent Microorganisms Depositary on October 16, 2018 (the access numbers are NITE P-02795, NITE P-02796, and NITE P-02797, respectively). , NITE P-02798).

The characteristics of these new strains are as follows.
Separation source: Separation from used activated sludge storage tank (collection site: Japan)
Isolation method: Method according to Example 1 Cell: Rod-shaped, with peripheral hair, having motility Colony: Cream-colored, coarse, amorphous Culturing method: Culturing by the same method as that for normal Bacillus subtilis. Possible characteristics: Highly produce amylase, protease, and lipase, and as shown in Table 5, have significantly higher starch, protein, and fat-decomposing activity compared to existing Bacillus subtilis strains.

The isolated Bacillus subtilis is formulated, for example, as it is, or mixed with an arbitrary conventional carrier or nutritional component, or powdered or granulated by an arbitrary conventional method. It can be used as a microbial preparation.

Claims (4)

A method for isolating the Bacillus subtilis from a sample containing Bacillus subtilis collected from activated sludge.
(A) A sterilization step of heating the sample so that the spore-forming bacteria contained in the sample remain.
(B) The sterilized sample contains a first medium containing 1 to 10 g of peptone, 1 to 5 g of yeast extract, 0.1 to 5 g of glucose, and 5 to 30 g of agar per 1 L of water. bacteria were plated smear culture is, first culturing step to obtain a Bacillus bacterium comprising Bacillus subtilis from the cultured bacteria, and with respect to (C) water 1L, meat extract 0.1-5 g, peptone 1 ~ 20 g, D-mannitol 0.5-20 g, sodium chloride 1-20 g, phenol red 0.01-0.1 g, agar 5-30 g, egg yolk solution (50%) 1-100 mL, polymixin B 10,000-500, Using a second medium containing 000 units, a plate smear culture of Bacillus subtilis containing the bacillus obtained from the first culture step is performed, and the bacillus is obtained from the cultured bacteria in the second culture step. ,
Methods for isolating Bacillus subtilis, including. The method according to claim 1, further comprising a confirmation step of confirming that the bacterium acquired in the second culture step belongs to Bacillus subtilis by performing a genetic analysis and performing a homology search . Bacillus subtilis B-11-3 strain deposited under accession number NITE P-02798, Bacillus subtilis. The microbial preparation containing Bacillus subtilis according to claim 3.

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