KR20120085451A - Novel bacteriocin from Bacillus cereus ATCC 13061 and antibiotic composition thereof - Google Patents

Abstract

PURPOSE: A composition containing bacteriocin cerein 13061 for killing germ is provided to replace antibiotics used in the food industry or livestock industry. CONSTITUTION: A polypeptide with a bacteriocin activity contains an amino acid sequence of sequence number 1 or 2. A polynucleotide encoding the amino acid of sequence number 1 has a nucleic acid sequence of sequence number 3. A polynucleotide encoding the amino acid of sequence number 2 has a nucleic acid sequence of sequence number 5. A composition for killing germ independently contains the polypeptide of sequence number 1 or 2 or a combination of the polypeptides. The composition contains a culture of Bacillus cereus ATCC 13061 as an active ingredient.

Description

Novel bacteriocin from Bacillus cereus ATCC 13061 and antibiotic composition according to Bacillus cereus ATC 13061 strain

The present invention Bacillus cereus ( Bacillus cereus ) It relates to a novel bacteriocin, and a composition for bacterial death comprising the same of the ATCC 13061 strain derived, and more particularly, to a new look for apoptotic function for the majority of Bacillus (Bacillus) and the L. monocytogenes Im sat jeneseu (Listeria monocytogenes) strain bacteriocin and It relates to a composition for killing bacteria comprising the same.

Bacillus (Bacillus) is primarily found in the soil aerobic or facultative aerobic bacteria. By secreting various kinds of hydrolase that decomposes various polymers, polysaccharides such as cellulose, starch, pectin, agar, nucleic acid, lipids, etc. can be used, so that foods can be transformed to produce acids and gases. Among them, Bacillus cereus is a causative agent of food poisoning that occurs mainly in starch foods, and it causes vomiting-type food poisoning that causes vomiting in a relatively short time (1-6 hours) after ingesting contaminated foods or 8 ~ after eating contaminated foods. It causes diarrhea-type food poisoning that causes abdominal pain and diarrhea after 16 hours. Other Bacillus Bacillus licheniformis ) is also contaminated with food, causing food poisoning, which causes diarrhea and vomiting. Bacillus coagulans is also known to be a decay bacterium in heated foods. However, since the spores made by these bacteria show high heat resistance and resistance to various chemical substances, it is urgent to develop effective control techniques.

On the other hand, bacteriocin is an antimicrobial peptide or protein produced by bacteria and is an antimicrobial substance that acts only on bacteria that have a close relationship with the producing strain. The bacteriocins identified so far are mainly related to lactic acid bacteria, particularly Bacillus genus Bacillus There has been little research on bacteriocin produced in cereus ).

On the other hand, substances that kill food poisoning bacteria when applied to foods should have a bactericidal effect specifically for harmful pathogens and should not affect the growth of beneficial bacteria, which is necessary because bacteriocins exhibit host-specific antimicrobial activity. Satisfies this, and loses its activity by proteolytic enzymes, so it can be used safely in food.

Accordingly, an object of the present invention is to develop and provide a new bacteriocin and a composition containing the same, which can control Bacillus bacteria, which are pathogenic microorganisms contaminated with food.

The present invention provides a polypeptide having the amino acid sequence of SEQ ID NO: 1 having bacteriocin activity. The present invention also provides a polypeptide having the amino acid sequence of SEQ ID NO: 2 having bacteriocin activity. The present invention also provides a polynucleotide having the nucleic acid sequence of SEQ ID NO: 3 by encoding the amino acid sequence of SEQ ID NO: 1 having bacteriocin activity. The present invention also provides a polynucleotide having the nucleic acid sequence of SEQ ID NO: 5 by encoding the amino acid sequence of SEQ ID NO: 2 having bacteriocin activity.

In the present invention, it is possible to control pathogenic microorganisms that are economically adversely affected in the food industry, and to isolate new antimicrobial agents that can control antibiotic resistant bacteria. As a result, a polypeptide having amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2 exhibiting a host specificity and a killing effect against a number of Bacillus and Listeria monocytogenes strains excluding the production strain from the culture of Bacillus cereus ATCC 13061, ie Bacteriocin was isolated.

On the other hand, as a result of the analysis, the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2 were encoded in some of the polynucleotides having the nucleic acid sequences of SEQ ID NO: 3 and SEQ ID NO: 5, respectively. That is, as a result of analyzing the amino acid sequence of purely isolated cerein 13061 in the present invention, two peptide sequences of SEQ ID NO: 1 (serine 13061α) and SEQ ID NO: 2 (13061β) were identified. PCR was performed based on the results, and the entire amino acid sequence including these two peptide sequences was identified as SEQ ID NO: 4 and SEQ ID NO: 6, respectively, and the polynucleotide sequences encoding them were SEQ ID NO: 3 and SEQ ID NO: 5, respectively. Each was confirmed. However, in order to show the bacteriocin activity, the '↑' position (shown in FIG. 6) of the entire amino acid sequence of SEQ ID NO: 4 and SEQ ID NO: 6 is cut out to be SEQ ID NO: 1 and SEQ ID NO: 2.

On the other hand, the present invention provides a composition for killing bacteria, characterized in that it comprises a polypeptide of SEQ ID NO: 1 and a polypeptide of SEQ ID NO: 2 alone or in combination. Although only the polypeptides of SEQ ID NO: 1 or SEQ ID NO: 2 show bacteriocin activity, the bacteriocin activity is markedly increased when the two are present together.

On the other hand, the present invention provides a composition for killing bacteria characterized in that it comprises a culture solution of Bacillus cereus ATCC 13061 as an active ingredient. In addition, the present invention provides a composition for killing bacteria comprising the protein isolated from the culture medium of Bacillus cereus ATCC 13061 as an active ingredient. In this case, the protein is meant to include both hydrophilic and hydrophobic proteins. However, the composition for killing bacteria of the present invention may more preferably include only hydrophobic proteins among proteins isolated from the culture solution of Bacillus cereus ATCC 13061 as an active ingredient.

On the other hand, in the composition for killing bacteria of the present invention, the composition for killing bacteria may be more specifically a composition for killing strains of the genus Bacillus and Listeria monocytogenes.

Meanwhile, the bacteriocin of the present invention may be directly administered to a portion infected with a Bacillus or Listeria monocytogenes strain, or may be administered by preparing a composition in an appropriate form. The composition for killing bacteria of the present invention prepared in the form of a composition may be prepared in various forms such as liquid, semisolid, and powder. Liquid preparations include suspensions or injections, and semi-solid preparations include creams, lotions, gels and pastes, and solid preparations include powders, granules, and tablets. These formulations may be formulated by conventional methods for preparing protein formulations.

On the other hand, the composition for killing bacteria of the present invention may include a carrier, other adjuvants, etc., if necessary, the carrier may be physiological saline, distilled water and the like, the auxiliary agent is a buffer solution used for the purpose of pH correction and the like. .

On the other hand, the dosage of the composition for killing bacteria of the present invention means an amount required to exert the effect of killing Bacillus bacteria or Listeria monocytogenes bacteria in the administration target, the type and the age and weight of the dosage form Can be adjusted according to a variety of factors, including general health status, sex and diet, route of administration, time and duration of administration. In the case of livestock or fish, the route of administration may be formulated according to the veterinary allowance.

Bacterial killing composition comprising the bacteriocin of the present invention can kill Bacillus and Listeria monocytogenes bacteria regardless of resistance to existing antibiotics, and therefore, diseases caused by Bacillus and Listeria monocytogenes bacteria It can also be used for treatment, and especially when used in antibiotic resistant Bacillus and Listeria monocytogenes bacteria can kill antibiotic resistant bacteria that could not be removed by the existing antibiotics, it can be usefully used as a substitute for conventional antibiotics.

In particular, the development of a bacteriocin preparation that can control various bacterial diseases that are a problem in the livestock field, and the replacement of antibiotics can not only solve the problem of antibiotic resistance bacteria, but also guarantee domestic consumers' confidence in the safety of our livestock products. Increasing exports of livestock products could improve the added value of domestic livestock products and boost the overall domestic livestock industry.

In addition, the bacteriosin composition comprising the bacteriocin of the present invention can be used in food, by treating the bacteriocin during the processing and storage of food to prevent contamination of food caused by pathogenic bacteria, killing the bacteria causing rot, shelf life It can also bring many economic benefits, such as increasing the number of workers.

Bacteriocin serine 13061 newly identified in the present invention shows a specific lytic action against strains belonging to the genus Bacillus and Listeria monocytogenes, one of the causative agents of food poisoning, and shows a stable antibacterial activity at a wide temperature and pH range, It can be used for food, medicine and animal medicine industry.

Figure 1 shows the growth inhibition of Bacillus cereus ATCC 10876 (indicator) by the Bacillus cereus ATCC 13061 culture and the decrease in the size of the inhibitory ring with dilution fold of the culture.
Figure 2 shows the results of high performance liquid chromatography (A) and lysis activity of two fractions (B) for Bacillus cereus ATCC 13061 culture.
FIG. 3 shows the change in absorbance when the bacteriocin serine 13061 of the present invention is treated with a stagnant stage indicator strain, Bacillus cereus ATCC 10876 (A) and Listeria monocytogenes ScottA (B).
Figure 4 shows the antimicrobial effect of the present invention bacteriocin cerane 13061 at various temperatures (A), pH range (B), organic solvent environment (C).
Figure 5 shows the antimicrobial effect of the present invention bacteriocin sere 13061 in reduction conditions (reduction conditions).
Figure 6 shows the entire DNA, the total amino acid sequence of the two peptides constituting the bacteriocin serein 13061 of the present invention (ceramic 13061α, cerane 13061β) and the part cut off to have lytic activity (indicated by '↑').

Hereinafter, the content of the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples, and includes modifications of equivalent technical spirits.

Example  1: present invention bacteriocin Serein  Activity Measurement and Isolation of 13061

(1) bacteriocin Serein  Active measurement

Bacillus cereus ATCC 13061, a bacteriocin producing strain, was inoculated in TSB liquid medium and shaken at 37 ° C. for 8 hours at 220 rpm. After centrifugation, the strain was removed by filtration and only pure culture was obtained.

Subsequently, Bacillus cereus ATCC 10876, an indicator strain, was incubated for 8 hours in TSB liquid medium, and then 100 μl of this culture was mixed with 5 ml of PA semi-solid medium (0.7%) and poured onto TSB solid medium to harden.

Meanwhile, 10 μl of the Bacillus cereus ATCC 13061 culture solution obtained above was dropped on a solid medium containing the above-mentioned indicator bacteria and incubated at 37 ° C. for 5 hours, and the susceptibility of antimicrobial activity was measured by observing growth inhibition rings. It was.

As a result, as shown in Figure 1 it was confirmed that the inhibition of the growth of the indicator bacteria by the Bacillus cereus ATCC 13061 culture medium and the decrease in the size of the inhibitory ring according to the dilution factor of the culture medium. Figure 1 is the number of dilution samples, '0' is not diluted, '1' is diluted 1 / 2-fold, '2' is 1 / 4-fold diluted, '3' is 1 / 8-fold diluted.

(2) Antibacterial activity range of bacteriocin

On the other hand, as shown in Table 1 below, as a result of measuring the antimicrobial activity of various strains of the genus Bacillus, all the Bacillus strains except the production strains ( B. thuringiensis, B. mycoides , B. weihenstephanensis , B. circulans , B. . licheniformis, megaterium B., B. pumilus, was B. produce a growth inhibition ring in sphaericus), showed a growth inhibition effect for the L. monocytogenes Im jeneseu it sat.

In addition, more than 140 Bacillus cereus and 38 Listeria monocytogenes isolates from various dry food groups showed the same activity. However, Staphylococcus epi deolmi display (Staphylococcus Other Gram-positive bacteria, such as epidermidis ) and Streptococcus pyogenes , or Gram-negative bacteria such as Salmonella and Escherichia coli, had no growth inhibitory effect.

Therefore, the antibacterial activity of the bacteriocin of the present invention was confirmed to be limited to Bacillus and Listeria monocytogenes.

Antimicrobial range of the present invention bacteriocin cerane 13061 Indicator Antimicrobial activity Gram-positive bacteria Bacillus cereus ATCC 13061 - ATCC 10876 + ATCC 27348 + ATCC 21768 + ATCC 14579 + ATCC 21772 + Bacillus thuringiensis ATCC 10792 + ATCC 29730 + ATCC 35646 + ATCC 35866 + KCCM 11428 + Bacillus mycoides ATCC 21929 + ATCC 6462 + Bacillus weihenstephanensis CCM 4872 + Bacillus circulans ATCC 4513 + Bacillus licheniformis ATCC 14580 + Bacillus megaterium ATCC 14581 + Bacillus pumilus ATCC 7061 + Bacillus sphaericus ATCC 14577 + Listeria monocytogens LO28 + ScottA + Staphylococcus epidermidis ATCC 35983 - Streptococcus pyogenes ATCC 19615 - Gram-negative bacteria Salmonella Typhimurium SL1344 - LT2 - Esherichia coli MG1655 - Enterobacter sakazakii ATCC 29544 - Enterococcus faecalis ATCC 29212 - Pseudomonas aeruginosa ATCC 27835 - Klebsiella pneumoniae ATCC 10031 - Isolated strain Bacillus cereus (140)
+ Listeria monocytogens (38)
+

(3) Isolation of Bacteriocin

  1) Strain Culture

Bacillus cereus ATCC 13061 한 strain inoculated in sterile TSB liquid medium at 1% and incubated for 9 hours at 37 ℃.

  2) Hydrophobic Chromatography

400 ml of the supernatant from which the strain was removed by centrifugation and filtration were mixed with 20 g of Amberlite XAD-16 resin at 23 ° C. for 6 hours. The mixed solution was filled in an open column, washed with 100 ml of distilled water and 100 ml of 40% ethanol, and the bacteriocin was eluted with 400 ml of 70% 2-propanol containing 0.1% trifluoroacetic acid. The 2-propanol was then evaporated in a vacuum at 40 ° C. and then dissolved in 20 mM Tris buffer (pH 8.0).

  3) High speed liquid chromatography

Bacteriocin active fraction eluted from hydrophobic chromatography was dissolved in 0.1% trifluoroacetic acid solution and injected into the C18 column equilibrated with the same solution to a linear gradient of 5% to 100% acetonitrile using high performance liquid chromatography. Separated.

As shown in FIG. 2, bacteriocin activity was shown in two of the eluted fractions. However, when the two fractions were combined, the bacteriocin activity was about 8 times higher than that of each fraction.

Therefore, bacteriocin cerane 13061 of the present invention is composed of two substances having lytic activity, and it can be confirmed that there is a synergistic effect when the two substances work together.

Example  2: bacteriocin Serein  Characterization of 13061

(1) bacteriocin Serein  13061 hosts Solvability

The bacteriocin 13061 isolated in Example 1 was treated with the Bacillus cereus ATCC 10876 culture in the standing phase stage, and the absorbance was measured for about 7 hours. As a result, the absorbance decreased to about 1 hour (A in FIG. 3).

On the other hand, Listeria monocytogenes, a susceptible bacterium of serine 13061 belonging to another genus, also gradually reduced the absorbance of the strain culture solution in the stagnant state by treatment with serine 13061 (B in FIG. 3).

Therefore, it could be judged that the action of serine 13061 was not to inhibit the growth of susceptible strains but to lyse the strains.

(2) bacteriocins Serein  Biochemical Properties of 13061

Bacteriocin sere 13061 of the present invention has no change in activity by α-amylase, lipase, lysozyme, but loses lytic activity by proteinase K. It can be seen that it is an antibacterial substance derived from protein.

The antimicrobial activity was stable in a wide range of temperature and pH range (A, B in Fig. 4), and exhibited about half the original antimicrobial activity even in the presence of organic solvents such as butanol, chloroform and ethanol (Fig. 4 C).

On the other hand, it was intended to find out whether the activity is maintained even if the bacteriocin of the present invention is reduced. First, double-electrophoresis of styrene 13061 on an acrylamide gel was followed by dyeing one part and fixing the other part. The fixed gel was placed in an empty petri dish, poured with 20 ml of a semi-solid medium mixed with the bacterium Bacillus cereus ATCC 10876, incubated at 37 ° C. for 5 hours.

As a result, a growth inhibitory ring was generated at the same size (2-3 kDa) as the band of the dyed gel, and when the band was cut and placed in a liquid medium inoculated with the indicator bacterium, the absorbance was remarkably different and the absorbance decreased. .

As a result, it was confirmed that even in the reducing conditions induced with 2-mercaptoethanol, cerine 13061 lysates the target strain without losing its activity (FIG. 5).

Example  3: bacteriocin Serein  13061 amino acids and DNA  Sequencing

In Example 1 the amino acid sequence of purely isolated Serene 13061 was analyzed.

As a result, two peptide sequences were identified, each named serine 13061α (SEQ ID NO: 1) and 13061β (SEQ ID NO: 2).

Based on these results, PCR was performed to confirm the entire DNA sequence (SEQ ID NO: 3 and SEQ ID NO: 5) and the entire amino acid sequence (SEQ ID NO: 4 and SEQ ID NO: 6, respectively), and the bacteriocins were cut off to have activity. We could check up to the place (marked with '↑'). (Fig. 6)

<110> SNU R & DB FOUNDATION <120> Novel bacteriocin from Bacillus cereus ATCC 13061 and antibiotic          composition <130> AP-2010-0183 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 30 <212> PRT <213> Bacillus cereus ATCC 13061 <400> 1 Gly Asn Ala Ala Cys Val Ile Gly Cys Ile Gly Ser Cys Val Ile Ser   1 5 10 15 Glu Gly Ile Gly Ser Leu Val Gly Thr Ala Phe Thr Leu Gly              20 25 30 <210> 2 <211> 30 <212> PRT <213> Bacillus cereus ATCC 13061 <400> 2 Gly Trp Val Ala Cys Val Gly Ala Cys Gly Thr Val Cys Leu Ala Ser   1 5 10 15 Gly Gly Val Gly Thr Glu Phe Ala Ala Ala Ser Tyr Phe Leu              20 25 30 <210> 3 <211> 144 <212> DNA <213> Bacillus cereus ATCC 13061 <400> 3 atggaagtta tgaacaatgc tttaattaca aaagtagatg aggagattgg aggaaacgct 60 gcttgtgtaa ttggttgtat tggcagttgc gtaattagtg aaggaattgg ttcacttgta 120 ggaacagcat ttactttagg ttaa 144 <210> 4 <211> 47 <212> PRT <213> Bacillus cereus ATCC 13061 <400> 4 Met Glu Val Met Asn Asn Ala Leu Ile Thr Lys Val Asp Glu Glu Ile   1 5 10 15 Gly Gly Asn Ala Ala Cys Val Ile Gly Cys Ile Gly Ser Cys Val Ile              20 25 30 Ser Glu Gly Ile Gly Ser Leu Val Gly Thr Ala Phe Thr Leu Gly          35 40 45 <210> 5 <211> 150 <212> DNA <213> Bacillus cereus ATCC 13061 <400> 5 atggaagttt taaacaaaca aaatgtaaat attattccag aatctgaaga agtaggtgga 60 tgggtagcat gtgttggagc atgtggtaca gtatgtcttg ctagtggtgg tgttggaaca 120 gagtttgcag ctgcatctta tttcctataa 150 <210> 6 <211> 49 <212> PRT <213> Bacillus cereus ATCC 13061 <400> 6 Met Glu Val Leu Asn Lys Gln Asn Val Asn Ile Pro Glu Ser Glu   1 5 10 15 Glu Val Gly Gly Trp Val Ala Cys Val Gly Ala Cys Gly Thr Val Cys              20 25 30 Leu Ala Ser Gly Gly Val Gly Thr Glu Phe Ala Ala Ala Ser Tyr Phe          35 40 45 Leu    

Claims (9)

A polypeptide having the amino acid sequence of SEQ ID NO: 1 having bacteriocin activity
A polypeptide having an amino acid sequence of SEQ ID NO: 2 having bacteriocin activity
A polynucleotide having a nucleic acid sequence of SEQ ID NO: 3 encoding and containing an amino acid sequence of SEQ ID NO: 1 having bacteriocin activity
A polynucleotide having a nucleic acid sequence of SEQ ID NO: 5 encoding and containing an amino acid sequence of SEQ ID NO: 2 having bacteriocin activity
Bacterial killing composition comprising a polypeptide of SEQ ID NO: 1 and a polypeptide of SEQ ID NO: 2 alone or in combination
Bacillus ssere mouse (Bacillus cereus ) Bacterial killing composition comprising the culture medium of ATCC 13061 as an active ingredient
Bacillus ssere mouse (Bacillus cereus ) Bacterial killing composition comprising the protein isolated from the culture medium of ATCC 13061 as an active ingredient
The method of claim 7, wherein
The protein is,
Bacterial killing composition, characterized in that the hydrophobic protein
The method according to any one of claims 5 to 8,
The bacteria,
Bacillus (Bacillus) sp and Listeria monocytogenes jeneseu (Listeria monocytogenes) in the composition for killing bacteria, characterized

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