KR101494230B1 - Novel strain of Bacillus amyloliquefaciens and animal feed additive containing it - Google Patents

Abstract

The present invention relates to a novel mutant of Bacillus amyloliquefaciens and an animal feed additive comprising the same. More specifically, the present invention relates to a novel mutant of Bacillus amyloliquefaciens , which is capable of inhibiting the growth of harmful bacteria in the intestinal tract, The present invention relates to a novel mutant of Bacillus amyloliquefaciens and an animal feed additive containing the mutant bacillus amyloliquefaciens. The present invention relates to a novel mutant of Bacillus amyloliquefaciens,
The mutant strain is Bacillus amyloliquefaciens strain BB1103 deposited on Feb. 6, 2013 under the deposit number KCCM 11372P of the Korean Society for Microbiology.

Description

[0001] The present invention relates to a novel mutant of Bacillus amyloliquefaciens and an animal feed additive containing the novel mutant,

The present invention relates to a novel mutant of Bacillus amyloliquefaciens and an animal feed additive comprising the same. More specifically, the present invention relates to a novel mutant of Bacillus amyloliquefaciens , which is capable of inhibiting the growth of harmful bacteria in the intestinal tract, The present invention relates to a novel mutant of Bacillus amyloliquefaciens and an animal feed additive containing the mutant bacillus amyloliquefaciens. The present invention relates to a novel mutant of Bacillus amyloliquefaciens,

All microorganisms in the natural ecosystem are carrying out various biochemical reactions themselves in order to carry out life activities. In the process, various metabolites are produced, and these metabolites are very important functions for man, animals and animals to carry out life activities .

Therefore, in the modern pharmaceutical field, efforts are being made to search for new microorganisms and to obtain more useful microorganism resources. However, microorganisms whose presence and function have been confirmed by humans so far are only a few.

Numerous microorganisms are parasitized in the body of animals including humans, and microorganisms inhabiting the intestines are collectively called intestinal bacteria. These intestinal bacteria can be classified into beneficial bacteria that harm the animal and harmful bacteria that harm them depending on their function.

For example, lactic acid bacterium is a kind of beneficial bacteria, and it has effects such as the action of form, suppression of intestinal decay products, inhibition of cancer, prevention of constipation and diarrhea, promotion of digestion and absorption of nutrients, It is known to help maintain. These lactic acid bacteria live not only in the digestive tract of humans and animals, but also in the vagina of women, and they also protect the invasion of harmful bacteria. Lactic acid bacteria are also contained in large quantities in traditional foods such as kimchi and yogurt, which is a health food.

On the other hand, bacteria such as Escherichia coli and Welshichiae are harmful bacteria which secrete harmful substances such as ammonia, indole, and hydrogen sulfide by changing a part of protein or amino acid, and in the process, they give a bad smell. In addition, the harmful substances affect the intestinal muscles and cause diarrhea and constipation, and promote headache, shoulder stiffness, rough skin, aging, and even nitrosoproteins may exhibit strong carcinogenic effects even in a trace amount .

Besides the like Salmonella (Sallmonella sp.), Shigella (Shigella sp.), Enterococcus (Enterococcus sp.) Belongs to the enteropathogenic bacteria. So when an animal is infected with Salmonella, it causes sepsis, chronic diarrhea, ileus colitis and chronic wasting disease. When pigs are infected with E. coli, premature colonic bacteria producing yellowish white or grayish white mucous spermatozoa are induced before 1 week of age, and fugitive coliform bacteria causing gray or white diarrhea occurs at 3 weeks of age. Also, infections of Enterococcus cause endocarditis, peritonitis or urinary tract infections.

On the other hand, animal, especially in order to suppress the harmful intestinal bacteria of livestock Bacillus subtilis (Bacillus subtilis), Bacillus core Tangerine Lance (Bacillus coagluans), Bacillus amyl Lowry query Pacific Enschede (Bacillus amyloliquefaciens), Bacillus poly flops mentee kusu (Bacillus polyfermenticus) and the efforts to develop probiotics and feed additives, such as using lactic acid bacteria has been attempted.

First, the Bacillus poly flops mentee kusu (Bacillus Polyfermenticus is an endospore bacterium isolated from the air by Terakato in Japan in 1933. It secretes 20 kinds of digestive enzymes and synthesizes vitamins B1, B2, K and C to supply nutrition. And Syktyvkar seugyun, wave Latif seugyun, jeokrigyun, Vibrio cholerae, Shigella, Vibrio, Aspergillus way Russ (hwangguk station, black Aspergillus), the Staphylococcus aureus, E. coli 0157, tetanus (Clostridium), such that the effect of lysing or bacteriostatic .

In addition, Korean Patent Registration No. 10-0211529 (May 04, 1999) discloses " a novel acid-resistant Lactobacillus sp. Microorganism having harmful microorganism inhibitory activity and a livestock active agent for livestock containing the same. The microorganism is Lactobacillus reuterii anaerobically isolated from domestic feces feces. It is resistant to antibiotics with acid resistance and bile resistance, and inhibits the growth of harmful microorganisms such as pathogenic bacteria . ≪ / RTI > In addition, as a result of administration to pigs such as piglets, the lactic acid bacteria in the feces remained high while the coliform bacteria decreased with time, and thus it has been shown that lactic acid bacteria can be usefully used as live bacteria actives for livestock.

Korean Patent Registration No. 10-0427600 (April 06, 2004 ) discloses a strain of Bacillus amyloliquefaciens KBC1121 and a feed additive comprising the same. Since the strain has an excellent inhibitory activity against harmful bacteria in the intestines and has a high heat resistance, it has an advantage that the microorganisms do not die even when dried by hot air at a temperature of 80 ° C or higher in the production of feed. Therefore, diets can effectively inhibit harmful bacteria in the intestines Reported.

In addition, Korean Patent Registration No. 10-1104178 (Jan. 03, 2012) discloses a Bacillus amyloliquefaciens strain which secretes an acidic cellulase useful for increasing utilization efficiency of an indigestible carbohydrate of an animal feed, Feed additives containing cellulases have been introduced.

Patent Registration No. 10-0211529 (May 04, 1999) Patent Registration No. 10-0427600 (April 06, 2004) Patent Registration No. 10-1104178 (Jan. 03, 2012)

As described above, conventionally, various beneficial bacteria have been developed to suppress harmful bacteria in the intestines of animals. However, such conventional intestinal beneficial bacteria are expected to have a certain effect when they are put into livestock feeds in the past. However, When the conventional beneficial bacterial preparation is administered together with an antibiotic to treat an animal suffering from a disease or an infectious disease, all the bacterial flora in the intestines of an animal are killed by the antibiotic, resulting in a sequela there was.

Accordingly, a problem to be solved by the present invention is to provide a novel mutant of Bacillus amyloliquefaciens which has an activity to inhibit the growth of harmful bacteria in the intestines of animals and is highly resistant to various antibiotics for animals, and an animal feed additive .

The novel mutant of Bacillus amyloliquefaciens according to the present invention is characterized by having both an inhibitory activity against the harmful bacteria in the intestines of the animal and resistance against animal antibiotics.

The mutant strain is characterized by being a Bacillus amyloliquefaciens BB1103 strain deposited on February 02, 2013 (Accession No .: KCCM 11372P) with the Korean Society for Microbiological Care.

Further, the animal feed additive according to the present invention is characterized by comprising a novel mutant of Bacillus amyloliquefaciens or a culture thereof.

The novel mutant of Bacillus amyloliquefaciens according to the present invention has excellent antimicrobial resistance against various animal antimicrobial agents while retaining the inhibitory activity against the harmful bacteria in the intestines of the animal which is a characteristic of the parent strain.

Therefore, when the new mutant is administered in combination with an antibiotic to animals suffering from various diseases such as diarrhea, the antibiotic will kill all intestinal bacteria causing the disease, and at the same time, the mutant according to the present invention will prevent Thus, diarrhea and various diseases of an animal can be effectively treated without aftereffects.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a phylogenetic relationship of Bacillus amyloliquefaciens mutants according to the present invention; FIG.
2 is a 16S rDNA base sequence of Bacillus amyloliquefaciens BB1103 strain according to the present invention.

The present invention relates to a novel mutant of Bacillus amyloliquefaciens having animal growth inhibitory activity against intestinal harmful bacteria and tolerance to animal antibiotics, and an animal feed additive comprising the mutant.

The novel mutant of Bacillus amyloliquefaciens according to the present invention is a morphologically Gram-positive bacterium which is spore-forming from a pig house in a livestock farmhouse in Pyeongtaek, Gyeonggi-do.

In addition, the mutant was produced in a biochemical test (Bergg's manual of systematic bacteroiogy volum 2, 1986, Williams Wilkins), showed positive reaction in the Voges-Proskauer test, fermented sugar, And indole formation is negative, oxydase is negative, and urease is positive. It is characterized by hydrolysis of casein, gelatin and starch, growth at 30 to 50 ° C and reduction of nitrate, protein decay Thereby reducing the generation of odor.

Bacillus amyl Lowry query novel mutant strain of Pacific Enschede according to the present invention is Escherichia coli (Escherichia coli), Staphylococci (Staphylococcus aureus), gear group (Enterococcus), Shigella (Shigella), Serratia marcescens (Serratia), keurep when Ella (Klebsiella ) And salmonella (Salmonella) and the like. The mutant is also resistant to at least one animal antibiotic selected from among polyamicin, chloramphenicol, tetracycline, thiamulin, and aminobenzylpenicillin.

In the present invention, the Bacillus amyloliquefaciens mutant having such mycological characteristics was designated as 'BB1103 strain' and deposited with the Korean Microorganism Conservation Center (accession number: KCCM 11372P). The 16S rDNA sequence of the Bacillus amyloliquefaciens BB1103 strain is shown in FIG.

Meanwhile, the animal feed additive according to the present invention preferably contains 0.2 to 10% by weight of the Bacillus amyloliquefaciens mutant or its culture based on the total weight of the animal feed. If the content of the mutant strain or the culture medium is less than 0.2% by weight, there is a risk that the microorganism is killed due to the antibiotics contained in the animal feed itself, and if the amount exceeds 10% by weight, However, we can not expect a synergistic effect due to the increase of the content. For reference, the animal feed containing 50% by weight of the Bacillus amyloliquefaciens mutant according to the present invention or the culture thereof was fed to pigs for 7 days without any abnormality.

Hereinafter, the process of isolating and identifying new mutants according to the present invention will be described in detail with reference to the following examples. However, these examples are provided to aid understanding of the present invention, and the scope of protection of the present invention is not limited by the following examples.

[Example]

A) Microbial sample collection

(BHI), PDA and Luria (1% of tryptone, 0.5% of yeast extract, 0.5% of NaCl) were collected from the livestock wastewater that flowed from pig farms in Pyeongtaek City, Gyeonggi Province. ) Were plated on a plate culture medium (Difco Laboratories, USA) and cultured at 28 ° C for 5 days to observe colonies forming colonies. As a result, various bacterial and fungal colonies were observed. Among them, 12 kinds of bacteria and 6 kinds of fungi were separated and stored separately on a PDA plate medium.

B) Effective strain  Screening

Among these bacteria and fungi, 18 strains of the above strains were transplanted into BHI Broth and cultured at 32 ° C for 5 days in order to screen effective strains having an inhibitory activity against intestinal harmful bacteria.

Prepared Escherichia coli KCTC 1682 and Staphylococcus aureus KCTC 1916 culture medium were each plated on a culture medium and penicylinder for antimicrobial activity test was placed on the culture medium. 18 cultivars of the above-mentioned strains were placed in the culture medium and cultured at 32 ° C for 24 hours. And the results are shown in Table 1 below.

Strain No. Size of transparent ring (mm) Escherichia coli Staphylococcus aureus One 10 13 2 - - 3 - - 4 17 15 5 - - 6 10 11 7 - - 8 12 14 9 10 10 10 - - 11 - - 12 - - 13 - - 14 11 12 15 - - 16 - - 17 - - 18 - -

As shown in Table 1, a transparent ring having the largest diameter appeared around the fourth strain. Therefore, the 4th strain showed the best activity to inhibit the growth of harmful bacteria compared with other strains.

C) Antibiotic resistance induction

Next, as a procedure for inducing tolerance to various antibiotics, antibiotics aminobenzylpenicillin was added to 10 μg / ml, 30 μg / ml, 60 μg / ml, 90 μg / ml , And 120 μg / ml. The strain No. 4 was inoculated therein and grown at 37 ° C. for 24 hours. Effective strains were collected in a 30 μg / ml flask having the most stable growth of bacteria, Resistant strains were continuously subcultured by the incremental method to induce tolerance to various antibiotics.

That is, 20 times on the polymyxin B-gradient plate, 25 times on the chloramphenicol-gradient plate, 30 times on the tetracycline-gradient plate, 16 times on the thiamulin-gradient plate and 10 times on the aminobenzylpenicillin- Antibiotic resistance was induced and the mutant strain was given the name 'BB1103'.

D) Minimum development inhibition concentration ( MIC )

The strain of BB1103 was suspended in a Luria broth medium and the absorbance was adjusted to 0.3 to be used as a test bacterium for measuring the minimum growth inhibitory concentration (MIC). First, the antibiotic concentration was gradually diluted so that the expected MIC concentration was maximized in the first test tube, and the dilution was doubled in the first test tube.

The antibiotics diluted in each step were added to a previously prepared Luria agar medium (maintained at 45 캜), poured into a Petri dish and hardened, and 0.1 ml of the prepared test broth was plated on the Luria agar plate.

The cultures were incubated at 37 ° C. for 24 hours using the strain No. 4, which was not resistant to induction, and the concentration of antibiotics on the last plate without growth of the bacteria was regarded as the minimum growth inhibitory concentration (MIC) Respectively.

Antibiotic type  MIC ([mu] g / ml)  No. 4 strain  BB1103 strain  Polymyxin B  > 5 100 Chloramphenicol 5 100 Tetracycline 5 5 Colistin > 5 100 Aminobenzylpenicillin 5 100 Thia mellin 5 40 Penicillin G 5 150 Lincomycin 5 50 Kanamycin 50 1,000 Erythromycin 10 > 1,000

As shown in the following Table 2, it can be confirmed that the antibiotic resistance-induced strain BB1103 according to the present invention is significantly more resistant to various antibiotics than the parent strain No. 4.

E) BB1103  Identification of Strain ( Identification )

In order to identify the BB1103 strain, morphological and biochemical investigations were performed first, and 16S rDNA sequencing was finally performed. As a morphological investigation, Gram staining revealed that the BB1103 strain was a Gram-positive bacterium, and it was confirmed that spores were formed by observation using a phase contrast microscope.

In addition, a biochemical test (Bergg's manual of systematic bacteroiogy volum 2, 1986, Williams Wilkins) was performed, and the result was positive for Vates-Proskauer test. , H2S and indole formation were negative, oxydase was negative, and urease was positive. And it was confirmed that casein, gelatin and starch were hydrolyzed, grown at 30 ~ 50 ℃ and nitrate was most similar to Bacillus subtilis.

For more accurate identification, the 16S rDNA nucleotide sequence of the above isolate was determined and compared with the nucleotide sequence of other strains registered in GenBank, and 16S rDNA sequence analysis was performed. Based on this, 1 < / RTI >

As a result of the analysis, the 16S rDNA sequence of the BB1103 strain, which was isolated from the 4th strain and induced resistance to various animal antibiotics, was determined as shown in FIG. Then, the result of comparing the base sequences of the other strains registered in GenBank, Bacillus amyl Lowry query Pacific Enschede (Bacillus amyloliquefaciens ) with a similarity of 99.0%.

The above-mentioned Bacillus amyloliquefaciens BB1103 strain was deposited on February 02, 2013 with the Korean Society for Microbiological Care (Accession No .: KCCM 11372P).

[Activity test]

A) Test Bacterial  Produce

The test strain was prepared to confirm the antimicrobial activity and the antibiotic resistance of the Bacillus amyloliquefaciens BB1103 strain. Namely, the strain of BB1103 was cultured by inoculating it into a test tube, and 1% of tryptone, 0.5% of yeast extract and 0.5% of NaCl were put into a 500 ml Erlenmeyer flask, dissolved with 100 ml of purified water, and then cultured at 37 ° C for 72 hours.

The seed culture was added to 20 L of Luria broth preculture prepared beforehand and cultured at 37 ° C for 48 hours. The culture was added to a culture tank containing 250 L of Luria broth medium, For 48 hours, and fermentation was stopped. At this time, the number of Bacillus amyloliquefaciens was more than 1x109 / ml.

B) Growth inhibition activity and antibiotic resistance test for harmful bacteria

Gram-negative bacteria that induce swine diarrhea were isolated and cultured using a MACCONKEY agar medium. Gram-negative bacteria were plated on a Luria agar medium prepared in advance. The penicillin strain was placed on the medium, and the BB1103 strain, which is a test strain, Amyloliquefaciens KCTC 1660 were added to each well and cultured at 37 ° C for 24 hours.

As a result, the Bacillus amyloliquefaciens KCTC 1660 strain, which is a standard strain, had a size of 8 mm and a size of 12 mm of the BB1103 strain. Therefore, it was confirmed that the strain of BB1103 according to the present invention is more effective in inhibiting growth of intestinal harmful bacteria than the standard strain.

C) Production of animal feed

An animal feed according to the present invention was prepared by adding 10 ml of BB1103 strain to 1 kg of animal feed supplemented with 23 mg of antibiotic thiamulin.

74 pigs were divided into a control group and a test group. The control group was fed with a conventional feed containing an antibiotic thiamulin, and the test group was fed with the feed of the present invention containing antibiotics thiamine and BB1103, The average weight gain (Kg), daily average weight gain (g / day), feed efficiency (total weight / feed consumption) and fat thickness (mm) .

As a result, in the control group, the intestinal flora was killed, diarrhea and bacterial diarrhea occurred, and intestinal disease occurred. However, in the test group, intestinal disease or bacterial diarrhea did not occur, and meat quality was improved with weight gain.

Item Control group Test group Deviation Average weight gain (Kg / 3months) 63.4 66.6 + 3.2kg Daily average weight gain (g / day) 701.0 740.4 + 39.4 g Feed efficiency (%) 32.4 37.3 + 4.9% Back fat thickness (mm) 18.8 16.1 -2.7 mm

Thus, when the Bacillus amyloliquefaciens strain BB1103 according to the present invention is added to an animal feed together with an antibiotic, the antibiotic treats harmful bacteria in the intestines, and the BB1103 strain inhibits bacterial growth caused by the death of the intestinal bacteria As a result, it is possible to obtain an effect that the meat quality is improved together with an increase in the body weight of the animal.

Korea Microorganism Conservation Center (overseas) KCCM11372P 20130206

Claims (5)

Has an activity of inhibiting the growth of Escherichia coli and Staphylococcus aureus, which are harmful bacteria in the intestines of animals; (Bacillus amyloliquefaciens) BB1103 strain (accession number: KCCM 11372P ) , which is resistant to any one of animal antibiotics selected from polymyxin, chloramphenicol, tetracycline, thiamulin and aminobenzylpenicillin ).
delete delete An animal feed additive characterized by comprising the Bacillus amyloliquefaciens strain BB1103 of claim 1 or a culture thereof.
The animal feed additive according to claim 4, wherein the strain of BB1103 or the culture thereof is contained in an amount of 0.2 to 10% by weight based on the animal feed.

Images