KR20050108110A - Novel bacillus strain and its use - Google Patents

Abstract

The present invention relates to Bacillus sp. 87 (KACC 91098) and its use, wherein the strain is recognized for acid resistance, bile resistance and biosafety, has pathogenic bacterial inhibitory activity, and antagonizes microorganisms as an independent intestinal flora (Niche) in the intestine. It is effective in preventing disease by reducing the growth of pathogenic flora through the action.

Description

Novel Bacillus Strain and Its Use

The present invention relates to a novel Bacillus microorganism and its industrial use, and more particularly, Bacillus sp 87 strain (KACC-91098) and a novel Bacillus sp. Microorganism isolated from fresh milk of healthy cows It relates to the probiotic composition, the use as a feed additive.

Probiotics are a group of microorganisms for living things, including Bacillus spp., Enterococcus spp., Streptococcus spp., Lactobacillus spp. And yeast. It is occupied by this master. Suitable functions as a probiotic are safe for the living body, and ingestion of the biochemical environment in vivo, such as acid resistance, bile resistance, intestinal adhesion, antagonism against harmful pathogens, the ability to synthesize useful ingredients such as vitamin B12, and the cells of the cell components themselves It presupposes the activating function of nonspecific immunological activity in the body.

Crude oil is a food of livestock, which replaces the function of breast milk, and is a natural food that is ingested by humans through various processing methods. Microorganisms in milk have been studied for a long time, and representative microorganisms include Bacillus spp., Enterococcus spp., Streptococcus spp. And Lactobacillus sp. Pathogenic microorganisms associated with the predominant species. In particular, microorganisms such as Bacillus spp, Enterococcus spp., Streptococcus spp., And Lactobacillus spp., Microorganisms, such as other harmful pathogens, are found in the intestines of animals including humans. And in fermentation of dairy products or crude milk. In fact, Lactobacillus microorganisms have been widely used as functional probiotics by maintaining the pH of the intestinal acid to inhibit the growth of harmful bacteria such as E. coli or Clostridium spp. . However, the antagonism and bacteriostatic ability against pathogenic microorganisms are not limited to the microorganisms of the genus Lactobacillus, but have been reported to have similar antimicrobial functions against pathogenic microorganisms in the genus Bacillus, enterococcus and yeast. .

In the livestock sector, in order to increase the productivity of livestock from an economic point of view, it is common to prevent the damage caused by pathogenic microorganisms by adding antibiotics to the feed, but problems such as the emergence of resistant bacteria to antibiotics and residual antibiotics in livestock products Is a serious problem. At this level, the use of functional probiotics that can replace antibiotic function is being actively studied.

The present inventors have completed the present invention by confirming that Bacillus sp. 87 strain, a strain useful as a probiotic that can be applied to animal feed, has excellent physicochemical properties, antagonism to harmful microorganisms, inhibitory activity, and nonspecific immune activity in animals. Was done.

Accordingly, an object of the present invention is to provide a novel Bacillus sp. Microorganism which has an immunoactive activity for preventing disease of an animal and can be used as a functional probiotic.

Another object of the present invention to provide a probiotic containing the novel Bacillus genus microorganisms.

Another object of the present invention to provide a feed additive containing a probiotic containing the novel microorganism of the genus Bacillus.

In order to achieve the above object, the present invention provides a novel Bacillus sp. 87 strain which is safe and has excellent immunological activity for preventing disease in animals.

The present invention provides a probiotic for preventing the development of pathogenic bacteria in animals comprising the novel Bacillus sp. 87 strain.

The present invention provides a feed additive for preventing diseases caused by pathogenic microorganisms containing probiotic, including the Bacillus sp. 87 strain.

Hereinafter, the content of the present invention will be described in detail.

The present invention provides a bacterium of the genus Bacillus isolated from the finest crude milk of healthy cows. Bacillus sp . Microorganism according to the present invention is characterized by excellent Bacillus ( Bacillus sp ) acid resistance, bile resistance, harmful microbial inhibitory activity and biosafety.

Bacillus microorganisms are widely present in nature and use carbohydrates aerobicly. In general, bacteria such as Bacillus subtilis, Bacillus rikenimoform, and Bacillus coagulus in Bacillus are known to be added directly or indirectly to feed to prevent damage caused by pathogenic microorganisms by antagonism or secretory antimicrobial substances in vivo. have. In addition, the added bacteria ingested through the feed enters the intestinal epithelial cells and enters the intestinal epithelial cells, which helps a lot of animals such as inhibition and antagonism of pathogenic microorganisms, and enhancement of immune activity.

Therefore, Bacillus bacteria have long been used as probiotics. However, in order to use the bacterium as a probiotic, the bacterium must be resistant to gastric acid or bile and have a high intestinal reach rate, and can be adsorbed and fixed on intestinal epithelial cells or mucous membranes. It should be able to stabilize the intestines, prevent intestinal colonization of harmful bacteria, and be safe and useable as an additive. Bacillus strain of the present invention ( Bacillus sp ) is a novel microorganism having all the above properties.

Isolation and identification of Bacillus sp. Microorganism according to the present invention was performed according to the procedure shown in Figure 1, which will be described in more detail below.

Crude milk collected over three months from cows ranches in 22 regions across the country was fed to Fossomatic series 4000 (Foss Electric Co., Denmark), a somatic cell counting device. Bacteria were cultured and isolated. Bacterial isolation was inoculated into blood agar using a sterile loop and incubated for 16-18 hours at 35-37 ° C. Independently selected by primary culture and gram staining of the formed independent colonies 116 strains were obtained. These selected strains were cultured in BHI (Brain Heart Infusion, Difco, USA) medium, and the identification of the selected microorganisms was based on the classification and criteria of the Burgy's manual based on morphological, cultural and physiological characteristics. Physiological characteristics such as gram staining and antibiotic resistance were examined, and bacteria were identified using the Vitek system (BioMerieux, USA).

In the present invention, the sugar availability patterns and 16S rRNA (ribosomal RNA) sequences of the isolated Bacillus sp. Microorganisms were analyzed and identified. As of April 8, 2004, the Korean Agricultural Culture Collection of the Korea Institute of Agricultural Biotechnology. Deposited under accession number KACC 91098.

Bacillus sp. 87 strain according to the present invention has the following biological properties.

Firstly, it is a Gram-positive aerobic bacillus, resistant to low pH (5.0), and capable of surviving 0.9% bile acid.

Second, the Bacillus sp. 87 strain of the present invention is active to inhibit harmful microorganisms. The harmful microorganisms include Gram-negative bacteria such as Salmonella spp. And E. coli , and Gram-positive bacteria such as Staphylococcus aureus .

Third, the Bacillus sp. 87 strain of the present invention is susceptible to various antibiotics.

Fourthly, the Bacillus sp. 87 strain of the present invention has a safe property in vivo.

Bacillus sp. 87 cells according to the present invention survived for 1 week even when 0.5ml injection into the mouse abdominal cavity at a concentration of 1.1 × 10 ⁸ CFU / ml cultured cells. In addition, it can be settled in the intestine without any abnormality when fed to the first-day herbivorous weight and can antagonize the pathogenic Salmonella spp.

The Bacillus sp. 87 strain of the present invention can be cultured in large quantities by conventional culture methods. As the culture medium, BHI broth and other broths can be used. Cultivation of microorganisms is possible under conventional aerobic culture conditions and can be incubated for 10 to 40 hours, for example, in a temperature range of 35 to 37 ° C. Centrifugation or filtration may be performed to remove the culture medium in the culture and recover only the cells, and this step may be performed depending on the intended use. The concentrated cells can be frozen or lyophilized with a protective agent such as 1 to 5% skim milk according to a conventional method so as not to lose their activity.

By using the microorganism of the present invention can be used as a feed additive. For example, a probiotic composition is provided for the prevention of animal intestinal diseases when a feed comprising Bacillus sp. 87 strain is administered to an animal. The probiotic composition of the present invention contains Bacillus sp. 87 strains, and 'animals' are mammals and birds except humans, and preferably include livestock such as cattle, pigs, and chickens.

Probiotics are microorganisms that have a beneficial effect on living organisms for life, that is, microbes that can survive in the gastrointestinal tract when ingested by an animal and can prevent certain pathological conditions. In general, probiotics have the effect of treating and ameliorating the symptoms caused by abnormal fermentation of the intestinal flora, and when administered to animals, the probiotics are concentrated and settled in the gut wall of the intestine, preventing harmful bacteria from settling and growing. In addition, the administered probiotics settle in the intestine of the animal to form a normal flora and produce enzymes, bacteriocins, which inhibit the growth of pathogens and help the absorption of nutrients. In addition, it produces a variety of decomposition products that help the absorption and utilization of nutrients, improve feed requirements in animals, and produce substances that neutralize the toxins produced by pathogens.

Probiotic compositions of the present invention can be prepared and administered in a variety of formulations and methods.

Hereinafter, the content of the present invention will be described in more detail with reference to Examples. However, these embodiments are merely illustrative of the contents of the present invention, it will be readily understood by those skilled in the art that the contents of the present invention are not limited thereto and may be comprehensively applied in similar cases.

Example 1 Isolation and Identification of Bacillus 87 Strains

1-1) Isolation of Bacillus 87 Strains from Crude Oil

Crude oil was collected from independent ranches nationwide, somatic cell count was measured on Fossomatic series 4000 (Foss Electric Co, Denmark). Selected crude oil was plated in agar blood medium (Blood agar) and incubated for 16 hours at 37 ℃. In order to separate the cultured cells first, different independent strains were selected by microscopic examination after colony characterization, hemagglutination ability, and Gram staining. There were 116 strains isolated primarily from high quality crude oil, and the selected strains were cultured in BHI medium and the identification of microorganisms was based on the morphological, cultural and physiological characteristics of the Burgy's manual. The physiological characteristics, such as gram staining and antibiotic resistance, were examined according to the standard, and the bacteria were identified using the Vitek system (BioMerieux, USA).

1-2) Identification of Isolated Strains

The strain isolated and screened in Example 1-1 was identified as Gram-positive bacillus, and was analyzed using the Vitek system (BioMerieux, USA) according to the supplier's experimental method. Is identified as a Bacillus (Bacillus sp) strain analysis result by the Vitek system Bacillus sp (Bacillus sp) was designated as 87 (Fig. 2).

1-3) Molecular Biology Identification of Isolated Strains

In order to more accurately identify the strains isolated and isolated in Examples 1-1 and 1-2, 16S rRNA genes known to be very effective for the identification of microorganisms in the genomic DNA of the bacterium were polymerized with primers. After amplification by a polymerase chain reaction, the nucleotide sequence was analyzed by an automatic base sequence determiner (see Table 1). The nucleotide sequence of 16S rRNA was determined by automatic base sequencer. The base sequence of the 16S rRNA is as shown in FIG. 3 and SEQ ID NO: 1. From the above results, the strain of the present invention confirmed a 99% concordance rate with the base sequence of 16S rRNA of Bacillus licheniformis (Af276309) registered in the GenBank.

TABLE 1

Strain primer Oligonucleotide Sequence (5 'to 3') Size of PCR Product B.sp 87 16S-F23 GGC GGC GTG CCT AAT ACA TGC AAG TCG 779 bp 16S-F310 CGG CCC AGA CTC CTA CGG GAG GCA GCA 481 bp 16S-R770 GCG TGG ACT ACC AGG GTA TCT AAT CC

Example 2 Characterization of Bacillus 87 Strains

2-1) Acid Resistance, Bile Resistance and Sugar Solubility Test

After adjusting the pH of the BHI liquid medium to pH 1-7 using HCl (Sigma), the isolated Bacillus 87 strain was suspended and treated at 37 ° C. for 18 hours, and the absorbance was measured at 620 nm. For comparison of acid resistance, commercial Bacillus species were disclosed under the same conditions. As a result, all of the test strains were grown at pH 5.0, and it was confirmed that Bacillus sp. 87 strain had acid resistance even under pH 5.0 conditions (Table 2).

<Table 2> Acid Resistance Comparison of B. 87 and Commercial Bacillus spp.

Strain pH Cell growth (Cell growth, 620nm) One 2 3 4 5 6 7  B. sp 87 0.0110 0.0030 0.0000 0.0060 0.3150 0.4110 0.3100  B. subtilis (A) 0.0110 0.0000 0.0040 0.0020 0.1160 0.1700 0.1140  B. subtilis (B) 0.0060 0.0030 0.0100 0.0030 0.0900 0.1160 0.1880  B. coagulans (C) 0.0130 0.0050 0.0000 0.0020 0.1030 0.1430 0.2010  B. mesentericus (D) 0.0080 0.0160 0.0120 0.0000 0.1920 0.2500 0.2340

* A: A company, B: B company, C: C company, D: D company

Bile resistance test was performed by adding Bile salt (Difco) at 0.3%, 0.6%, and 0.9% to BHI broth, and then suspending Bacillus strains and incubating for 16 hours at 37 ° C. It was confirmed using. As a result of the experiment, all strains grew even at 0.6% or more of Bile salt (Table 3).

Table 3 Comparison of Bile Resistance between B.sp 87 and Commercial Bacillus spp.

Strains Cell growth (bile salts, 620nm) 0.3% 0.6% 0.9%  B.sp 87 0.040 0.032 0.041  B. subtilis (A) 0.012 0.007 0.005  B. subtilis (B) 0.055 0.048 0.025  B. coagulans (C) 0.056 0.055 0.039  B. mesentericus (D) 0.009 0.029 0.025

* A: A company, B: B company, C: C company, D: D company

2-2) Biochemical Sugar Availability of Bacillus 87 strains

The sugar availability of Bacillus sp. 87 strain was investigated. As shown in Table 4, biochemical glycolysis properties of the typical Bacillus genus could be confirmed.

Table 4 Glucose Solubility of Bacillus sp. 87 Strains and Commercial Bacillus Strains

No. carbohydrate B.sp 87 B. subtilis (A) B. subtilis (B) B. coagulans (C) B. mesentericus (D) One Maltose + + ± + ± 2 D-(+)-glucose + ± + + + 3 Rhamnoose - - - ± - 4 Dextrose + + + + + 5 D-lactose + - - ± - 6 D-(+)-trehaloise + ± + + + 7 D-(+)-Mannose + ± + + + 8 Sucrose + ± + + + 9 D-sorbitol - - - + + 10 Inulin - - - - - 11 D-mannitol + + + + + 12 D-(+)-Xiloose - - - - - 13 D-(-)-Arabinose - - - ± - 14 D-(+)-cellobiose + + + + + 15 Methyl α-D-glucopyranoside ± + - ± ± 16 L-arginine - - - - - 17 D-(+)-rapinose + - - ± -

* A: A company, B: B company, C: C company, D: D company

2-3) Inhibitory Activity of Bacillus 87 Strains on Harmful Microorganisms

The harmful microbial inhibitory activity of Bacillus sp. 87 strain was measured using Staphylococcus aureus 305.

Staphylococcus aureus (Staphylococcus aureus, ATCC 305) is by enrichment in the (Difco, USA) BHI broth was used for the test. After inoculating the harmful microorganisms, the initial bacterial count was measured, and then the Bacillus sp. 87 strain according to the present invention was added to 1% (v / v), and the culture medium was diluted in 1, 3, 6, and 24 hours at 37 ° C. in a decimal manner. After colonization, the colony counts were calculated and the ratio to the initial bacterial count was investigated.

As a result of the experiment, as shown in Table 5, the control group was also able to observe almost equal or superior bacteriostatic effect compared to other probiotics used in the past.

Table 5 Bacillus Comparison of In Vitro Antimicrobial Effects of 87 and Commercial Bacillus Probiotics

Strain 0 hr 1 hr 3 hr 6 hr 24 hr 48 hr 72 hr S. aureus 305 (control) <10 <10 <10 1.9 × 10 ² 9.2 × 10 ⁷ 6.5 × 10 ⁷ 7.3 × 10 ⁷ S. aureus 305 & B. subtilis ¹ <10 <10 <10 <10 4.3 × 10 ⁷ 7.0 × 10 ⁷ 3.4 × 10 ⁷ S. aureus 305 & B. subtilis ² <10 2.1 × 10 ² <10 <10 3.7 × 10 ⁶ 2.4 × 10 ⁷ 4.9 × 10 ⁷ S. aureus 305 & B. sp 87 <10 <10 <10 <10 5.1 × 10 ⁷ 7.8 × 10 ⁷ 2.6 × 10 ⁷

* ¹ Company A, ² Company B

2-4) Resistance and Susceptibility Testing to Antibiotics

The resistance and susceptibility to antibiotics of the Bacillus sp. 87 strain according to the present invention were tested. Antibiotic resistance tests were performed according to the standard method of NCCLS (2002).

Pure cultured 87 Bacillus strains were inoculated in the enrichment medium and incubated at 37 ℃. Turbidity of the culture was determined by MacFarland No. It was adjusted to 0.5 and used as test bacteria. The test bacteria were plated on a Mueller Hinton agar plate medium and placed on an antibiotic sensitivity test disc and incubated at 37 ° C. for 16 hours. When the culture was completed, it was checked visually to determine the antibiotic susceptibility or resistance by measuring the growth inhibition diameter including the marginal diameter.

As a result of the experiment, the Bacillus sp. 87 strain according to the present invention showed susceptibility to various antibiotics when read by the resistance standard according to NCCLS (2002). Table 6

Table 6. Antibiotic Resistance of B.sp 87 and Bacillus Probiotics

      Antibiotic strains  Antibiotic Susceptibility (mm) AN N S L K TE P E ENR VA GM OX AM CF CX B. sp 87 21 21 24 - 24 28 18 30 28 22 22 - 19 21 - B. subtilis ^a 27 25 20 - 28 22 13 12 ND 25 32 11 16 25 15 B. subtilis ^b 30 23 20 - 11 25 33 30 ND 28 30 24 32 42 15 B. coagulans ^c 26 21 18 - 24 19 30 25 ND 20 26 23 30 40 22 B. mesentericus ^d 22 23 20 - 30 19 27 27 ND 20 26 17 25 40 17

VA: 30 μg of vancomycin, CF: 30 μg of cephalosin, P: penicillin G 10U, CX: 1 μg of clocksacillin, K: 30 μg of kanamycin, N: 30 μg of neomycin, E: 15 μg of irisromycin, OX: 1 μg oxacillin, AM: 10 μg ampicillin, TE: 30 μg tetracycline,

a: A company, b: B company, c: C company, d: D company, e: E company

* ND: Not detected.

Example 3 Safety Test in Mice of Bacillus sp. 87 Strains

To test the in vivo safety of Bacillus sp. 87 strains, incubated for 18 hours at 37 ° C in BHI liquid medium, adjusted to 1.4 × 10 ⁸ CFU / ml with saline solution, and then intraperitoneally with 40 ml of 22 g of 40-week-old ICR mice. Observations were made for 1 week after inoculation. 40 water under the same conditions was observed as a control group after inoculation with saline. Survival was measured for 1 week after 24 hours of inoculation and all survived (Table 7).

Table 7 In Vivo Safety Investigation of Mice of Bacillus Esp 87 Strains

division Inoculation amount Inoculation site Dosage Experiment head Period (% mortality) 1 day 5 days 7 days Control Saline Abdominal cavity 0.5ml 40 0 0 0 B.sp 87 1.4 × 10 ⁸ cfu / ml Abdominal cavity 0.5ml 40 0 0 0

<Example 4> Antibiotic resistance against antibiotic-resistant standard pathogenic Staphylococcus aureus and Escherichia coli strains of 87 strains of Bacillus sp.

Mice inoculated with Bacillus sp. 87 cells of Example 3 were adjusted to 2.0 × 10 ⁸ CFU / ml of pathogenic Staphylococcus aureus ( S. aureus ) and inoculated 1 ml intraperitoneally at 1, 2, and 4 weeks. Survival was compared from 24 hours after inoculation. The control group showed survival rates of 10% at 1 and 2 weeks and 40% at 4 weeks, whereas 80% and 2 weeks were given to the group inoculated with Bacillus sp. 87 cells. The survival rate was 70% for the main group and 90% for the 4 week group (Table 8).

<Table 8> Effect of mouse Bacillus sp. 87 strains on the survival of mice by the inoculation of S. aureus

^※ Inoculate the selected strain to the abdominal cavity and inoculate 2.0 × 10 ⁸ cfu / ml / mouse intraperitoneally with S. aureus after 1, 2, 3, 4 weeks.

In the case of Escherichia coli ( E. coli ) 3 days after the same experiment for 10 water was adjusted to 2.5 × 10 ⁹ CFU / ml was inoculated 1ml intraperitoneally by 10 water. The survival rate was compared with the control group after 72 hours of inoculation. The control group showed a survival rate of 10% after inoculation, but the Bacillus sp. 87 cell inoculation group showed a survival rate of 80% (see Table 9).

<Table 9> Protective effect of E. coli 055 vaccination against Escherichia coli of Bacillus sp. 87 strains in mice

division Inoculation amount Experiment head Number of deaths by date after E. coli inoculation Survival rate (%) 0 days 1 day 3 days 4 days Control Saline 10 2 7 0 0 10 B.sp 87 1.4 × 10 ⁸ cfu / ml 10 0 2 0 0 80

The Bacillus sp. 87 strain of the present invention was confirmed to be excellent in acid resistance, bile resistance and biosafety, has a pathogenic bacterial inhibitory activity and has a prophylactic effect of reducing the growth of pathogenic flora by antagonism during preoccupation in the intestine.

1 is a flow chart for identification and characterization of Bacillus sp. 87 strain according to the present invention.

Figure 2 is a micrograph after gram staining of Bacillus sp. 87 strain according to the present invention

3 is a nucleotide sequence of 16S rRNA of Bacillus sp. 87 strain according to the present invention

<110> Republic of Korea <120> Novel Bacillus Strain and Its Use <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 782 <212> DNA <213> Bacillus sp. <220> <221> gene (222) (1) .. (782) <223> gene coding 16SrRNA <400> 1 ggcggcgtgc ctaatacatg caagtcgagc ggaccgacgg gagcttgctc ccttaggtca 60 gcggcggacg ggtgagtaac acgtgggtaa cctgcctgta agactgggat aactccggga 120 aaccggggct aataccggat gcttgattga accgcatggt tccaatcata aaaggtggct 180 ttcagctacc acttacagat ggacccgcgg cgcattagct agttggtgag gtaacggctc 240 accaaggcga cgatgcgtag ccgacctgag agggtgatcg gccacactgg gactgagaca 300 cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga 360 cggagcaacg ccgcgtgagt gatgaaggtt ttcggatcgt aaaactctgt tgttagggaa 420 gaacaagtac cgttcgaata gggcggtacc ttgacggtac ctaaccagaa agccacggct 480 aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg aattattggg 540 cgtaaagcgc gcgcaggcgg tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg 600 agggtcattg gaaactgggg aacttgagtg cagaagagga gagtggaatt ccacgtgtag 660 cggtgaaatg cgtagagatg tggaggaaca ccagtggcga aggcgactct ctggtctgta 720 actgacgctg aggcgcgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac 780 gc 782

Claims (3)

Bacillus sp. 87 strain (KACC 91098) excellent in the inhibition of pathogenic bacteria. Probiotics for the prevention of pathogenic bacteria in animals, including Bacillus sp. 87 strain (KACC 91098) Feed additive containing probiotic containing Bacillus sp. 87 strain (KACC 91098)