KR101108472B1 - Bacillus subtilis cbs strain and direct-fed microorganisms comprising the same - Google Patents

Abstract

The present invention relates to a novel Bacillus subtilis CBS strain and a probiotic comprising the strain.

Bacillus subtilis, Probiotics

Description

BACILLUS SUBTILIS CBS STRAIN AND DIRECT-FED MICROORGANISMS COMPRISING THE SAME}

The present invention relates to a novel Bacillus subtilis CBS strain having excellent enzyme productivity isolated from barn recycled litter and a probiotic comprising the strain as an active ingredient.

The bottom of the barn where livestock cattle are raised is usually covered with chip-shaped pieces of wood (ie, litter), which are absorbers, to a certain thickness. Absorption keeps the inside environment of the barn comfortable and clean. However, such a litter not only becomes dirty by absorbed urination and the like after a certain time, but also the absorption rate is inhibited, and thus the function of the absorbent is lost, and the breeding environment is deteriorated due to odor due to manure. Therefore, a method of adding microbial agents has recently been studied as a method for improving and recycling the litter.

Bacillus subtilis ( Bacillus subtilis ) is a type of aerobic bacillus belonging to the diatoms and is a bacterium widely distributed in the natural world such as soil, dried grass and sewage, and is called Bacillus subtilis. In addition, it forms heat-resistant spores and belongs to Gram-positive bacteria, and spores are formed at the center or slightly away from the center of the cell to give an egg or cylinder shape. It is heat resistant and should be autoclaved if it is necessary to sterilize spores. Bacillus subtilis is known to have a variety of enzymes, including starch and proteolytic enzymes, and is therefore used in genetic engineering research such as industrial production or transformation, and genetic engineering.

Accordingly, the present inventors found a strain having excellent enzyme productivity from litter in barn, and identified the bacillus subtilis bacteria as a result of strain identification. As a result of comparing enzyme productivity, acid resistance and bile acid resistance with the existing strains, the bacterium was named Bacillus subtilis CBS strain. By using this property, it can be developed as a probiotic which can help the digestion of livestock and improve the growth ability than the existing probiotics.

Accordingly, it is an object of the present invention to provide a novel Bacillus subtilis CBS strain.

Still another object of the present invention is to provide a probiotic comprising the strain as an active ingredient.

In order to achieve the above object, the present invention provides a Bacillus subtilis CBS strain (Accession No. KACC91484P).

In order to achieve the above another object, the present invention provides a probiotic comprising the strain as an active ingredient.

Bacillus subtilis CBS strain of the present invention is excellent in growth and thermal stability, and excellent in enzyme productivity, acid resistance and bile acid resistance, when used as a livestock probiotic for feed, when used in feed, aids in digestion of livestock It is expected to improve livestock productivity by increasing growth.

Bacillus subtilis CBS strain according to the present invention exhibits the morphological and biochemical properties as shown in Table 1.

Therefore, according to Bergy's manual of determinative bacteriology, it was first identified as a strain of the genus Bacillus , and the result of 16S rRNA gene sequencing analysis as shown in SEQ ID NO: 1 (see FIG. 1), Bacillus subtilis ( Bacillus Subtilis ), but not exactly the same strains with excellent enzyme productivity was identified as Bacillus Subtilis new strain. The inventors named this new strain Bacillus subtilis CBS strain and deposited it with the Agricultural Genetic Resource Center under accession no. KACC91484P dated July 16, 2009. Bacillus subtilis has been shown in Table 2. The analysis of the availability of CBS per strain with API 50 CHB kit (Biomerieux ^®, France) (see Table 2). As shown in Table 2, it was confirmed that the Bacillus subtilis CBS strain of the present invention showed a positive availability to Lactose, compared to the majority of the existing Bacillus subtilis standard strain is confirmed that the availability to lactose is negative.

CBS strain according to the present invention is well in the culture medium for bacterial growth such as Lubria-Bertani agar (LB medium), Bennett agar plate (nutrient agar), nutrient agar medium (nutrient agar) It grows and shows good productivity even in feed medium. The feed medium may be a pig feed medium, cattle feed medium or pig fecal medium prepared by adding the respective feed ingredients to a predetermined amount of sterile distilled water.

In addition, the CBS strain of the present invention grows well at a temperature of 25 ℃ to 65 ℃, preferably, 30 ℃ to 37 ℃, about 37 ℃ is the optimum growth temperature. It also grows relatively well in medium at pH 6.5 to pH 7.5 and best at pH 6.8.

In addition, the present invention provides a probiotic comprising a Bacillus subtilis CBS strain as an active ingredient.

Specifically, the probiotic of the present invention may include Bacillus subtilis CBS strain, culture or extract thereof.

In order to facilitate the mixing of the probiotics when added to the feed, the probiotic of the present invention is preferably dried and granulated. After mixing the excipients with the probiotic dry matter, the number of the viable bacteria is 10 ⁷ to 10 ⁸ cfu / g, and then the binder The solution can be used to carry out granulation of the probiotics in a conventional fluid bed granulator. The excipient may be a component selected from the group consisting of lactose, starch, glucose, and mixtures thereof. As a binder constituting the binder solution, a component selected from the group consisting of hydroxypropyl cellulose (HPC), polyvinyl propylene (PVP), and mixtures thereof may be used, and the concentration of the binder solution may be 1.0. To 3.0% by volume, preferably 1.0% by volume is suitable.

Probiotics prepared in the present invention can be added to the feed, which is expected to have the effect of helping the digestion of livestock and improve growth.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1: Production of Bacillus subtilis CBS strain

More than 30 litter samples were collected from Hanwoo farm barns in Daegu Metropolitan City, and sterile distilled water was added to the collected samples and plated on a Bennett agar plate for 2 days. Subsequently, the colonies of the agar medium cultured for 2 days were inoculated into a medium containing 2% skim milk and 3% NaCl, and cultured at 37 ° C. for 2 days, and the cells were transparent by the protease produced by the strain. Colonies forming a ring were selected. The strains selected above were first identified as Bacillus genus strains according to the manual of the virgin, and the strains having excellent enzyme productivity were identified as Bacillus subtilis new strains and named as Bacillus subtilis CBS strains. On July 16, 2009, it was deposited with KACC91484P. 16S rRNA nucleotide sequence of the Bacillus subtilis CBS strain is shown in SEQ ID NO: 1.

To test the productivity of the CBS strain, the CBS strain was precultured aerobicly at 37 ° C. for 24 hours using TS broth (10 g / L casein pancreatic degradation product, 5 g / L yeast extract and 10 g / L NaCl). For the main culture, 50 ml of Bacillus subtilis CBS seed culture medium was inoculated using 3 liters of LB medium (casein pancreatic degradation product 10 g / L, yeast extract 5 g / L and NaCl 10 g / L), and a 5 liter fermenter (Fermentor). Air supply amount using 0.5), agitation speed 350rpm and incubation temperature of 37 ℃ for the same time as shown in Table 3, and then measured by taking a sample every hour after the fermentor operation using the absorbance measurement method In one way, absorbance and viable cell count at 660 nm were measured and the results are shown in Table 3 below. As shown in Table 3, the number of viable cells of 1.1 × 10 ¹⁰ was obtained at 12 hours after spawn inoculation, and the result of 24 hours of incubation time was 1.2 × 10 ¹⁰ . I could see.

Example 2: Production of Bacillus subtilis CBS strains in feed medium

In order to confirm the productivity in the feed medium of the Bacillus subtilis CBS strain selected in Example 1, Bacillus subtilis CBS strains were TS broth (10 g / L casein pancreatic degradation products, yeast extract 5 g / L and NaCl 10 g / L) was preincubated for 24 hours at 37 ℃. Pig feed medium (containing 10% pig feed), cattle feed medium (containing 10% cattle feed) and pig stool medium (containing 10% pig stool), prepared by adding each feed ingredient to a certain amount of sterile distilled water. 1% of Bacillus subtilis CBS culture medium was inoculated and incubated at 37 ° C. for 24 hours, followed by aerobic and anaerobic tests. In addition, in order to compare the productivity with other known Bacillus subtilis species Bacillus subtilis ATCC 6633 and Bacillus subtilis KCTC 1331 using the experiment in the same manner as described above the results shown in the feed medium Table 4 Shown in

As shown in Table 4, the Bacillus subtilis CBS strain was able to confirm the productivity of more than 10 ⁷ in all three kinds of feed medium, it can be confirmed that compared to other Bacillus subtilis species.

Example 3: Thermal Stability of Bacillus Subtilis CBS Strains

In order to confirm the thermal stability of the Bacillus subtilis CBS strain selected in Example 1, the spray drying after the end of the culture The Bacillus subtilis CBS strain was heat-treated in an incubator maintained at 80 ° C. for 30 minutes, and the change in viable cell count was measured. Also, in order to compare thermal stability with other known Bacillus subtilis species, the change of viable cell number after heat treatment was measured in the same manner as above using Bacillus subtilis ATCC 6633 and Bacillus subtilis KCTC 1331. Table 5 shows.

As shown in Table 5 below, other existing Bacillus subtilis species was confirmed that the maximum number of viable bacteria of about 10 ¹ after heat treatment. On the other hand, the CBS strain of the present invention was found to maintain a 10 ⁹ level of viable count even after heat treatment. As such, it was confirmed that the Bacillus subtilis CBS strain of the present invention had higher thermal stability than the existing Bacillus subtilis species.

Example 4 Enzyme Production Capacity of Bacillus Subtilis CBS Strains

The enzyme production capacity of the Bacillus subtilis CBS strain selected in Example 1 was measured and compared with the known Bacillus subtilis species.

Specifically, Bacillus subtilis ATCC 6633, Bacillus subtilis ATCC 33068, Bacillus subtilis as CBS strain and comparative group in TS broth (casein pancreatic degradation product 10g / L, yeast extract 5g / L and NaCl 10g / L) ATCC 9372 and Bacillus subtilis KCTC 1331 were inoculated at 1% and incubated at 37 ° C. for 24 hours. Each Bacillus species culture medium was centrifuged (3000 RPM, 10 minutes), and the supernatant was filtered using a 0.45 μm filter. Starch, carboxymethyl cellulose (Carboxy-methyl) was added to the medium to which 5 g / L of peptone, 3 g / L of yeast extract (Yeast-extract) and 5 g / L of NaCl (except for NaCl in the case of protease) were added to determine the enzyme activity. A method of inoculating 20 μl of each Bacillus culture medium filtered on a sterilized paper disc (radius 5 mm) on a plate medium for enzyme activity measurement prepared by adding different substrates for each enzyme for measuring enzymes such as cellulose and CMC. The enzyme activity of amylase (Amylase), protease (Protease), lipase (Lipase), xylanase (Xylanase), cellulase (Cellulase) and phytase (Phytase) was measured and the results are shown in Table 6 below.

As shown in Table 6, the Bacillus subtilis CBS strain of the present invention was found to be very excellent in the enzymatic activity of amylase, protease, xylanase, phytase, etc. when cultured aerobicly compared to the existing strain and cellulase Was found to be nearly similar. In the case of lipase activity, activity was not confirmed in other Bacillus species except KCTC 1331 strain, but it was confirmed that the activity appeared when aerobic culture of the CBS strain of the present invention. In addition, when the CBS strain of the present invention is anaerobicly cultured, the CBS strain may contribute to improving digestion and absorption by increasing feed rate in the intestine of the livestock because the enzyme activity is superior to the existing strain.

Example 5: Acid and Bile Acid Resistance of Bacillus subtilis CBS Strains

In order to confirm the acid resistance and bile acid resistance of the Bacillus subtilis CBS strain selected in Example 1, in the case of the acid resistance test was inoculated 1% of the culture solution of Bacillus subtilis CBS strain in TS broth 24 hours at 37 ℃ After aerobic incubation, the culture was adjusted to pH 2 with 10% HCl and treated for 2 hours. After that, the ratio of viable cells after pH 2 treatment to initial viable cells was shown. In the case of the bile acid resistance test, 1.0% bile was added to the Bacillus subtilis CBS strain and growth was confirmed. In addition, Bacillus subtilis ATCC 6633, Bacillus subtilis ATCC 33068, Bacillus subtilis ATCC 9372, and Bacillus subtilis KCTC 1331 were treated under the same conditions as the comparison group, and the comparison results are shown in Table 7 below.

As shown in Table 7, in the case of bile acid resistance, all other existing Bacillus species including the Bacillus subtilis CBS strain of the present invention was confirmed that growth was possible even when 1.0% bile was added, but in the case of acid resistance, Bacillus subtilis CBS strain and Bacillus subtilis KCTC 1331 strain was confirmed that there is no significant change in the number of viable cells even after pH 2 treatment. In particular, Bacillus subtilis ATCC 9372 was found to be vulnerable to acidic conditions. As confirmed in the above results, the Bacillus subtilis CBS strain of the present invention was found to be similar or superior to other existing Bacillus species in bile acid resistance and acid resistance.

Preparation Example 1 Preparation of Probiotic

After the culture medium in which the Bacillus subtilis CBS strain prepared in Example 1 was incubated for 1 day was dried using a spray dryer, a probiotic was prepared in which the number of cells was 1.2 × 10 ¹⁰ / g.

Figure 1 shows the sequence homology analysis of the Bacillus subtilis CBS strain.

<110> CHEIL BIO CO., LTD. <120> BACILLUS SUBTILIS CBS STRAIN AND DIRECT-FED MICROORGANISMS          COMPRISING THE SAME <130> FPD200906-0031 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 850 <212> DNA <213> BACILLUS SUBTILIS CBS STRAIN <400> 1 nnnggggagc agctgctatc atgcagtgag cggacgatgg gagcttgctc cctgatgtta 60 gcggcggacg ggtgagtaac acgtgggtaa cctgcctgta agactgggat aactccggga 120 aaccggggct aataccggat ggttgtctga accgcatggt tcagacataa aaggtggctt 180 cggctaccac ttacagatgg acccgcggcg cattagctag ttggtgaggt aacggctcac 240 caaggcgacg atgcgtagcc gacctgagag ggtgatcggc cacactggga ctgagacacg 300 gcccagactc ctacgggagg cagcagtagg gaatcttccg caatggacga aagtctgacg 360 gagcaacgcc gcgtgagtga tgaaggtttt cggatcgtaa agctctgttg ttagggaaga 420 acaagtgccg ttcaaatagg gcggcacctt gacggtacct aaccagaaag ccacggctaa 480 ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttgtccggaa ttattgggcg 540 taaagggctc gcaggcggtt tcttaagtct gatgtgaaag cccccggctc aaccggggag 600 ggtcattgga aactggggaa cttgagtgca gaagaggaga gtggaattcc acgtgtagcg 660 gtgaaatgcg tagagatgtg gaggaacacc agtggcgaag gcgactctct ggtctgtaac 720 tgacgctgag gagcgaaagc gtggggagcg aacaggatta gataccctgg tagtccacgc 780 cgtanacgat gagtgctaag tgttaggggg tttccgcccc ttagtgctgc agctaacgca 840 ttaagcactc 850  

Claims (2)

Bacillus subtilis CBS strain (Accession No. KACC91484P). Bacillus subtilis (Bcillus subtilis ) CBS strain (Accession No. KACC91484P), a probiotic comprising a culture solution or a culture extract thereof as an active ingredient.

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