JP2003339377A - New bacteriocin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new bacteriocin derived from Lactobacillus, being an antibacterial substance readily decomposed in the body, having a high inhibitory selectivity to microorganisms and providing high safety, a bacterial strain producing the same, a gene thereof, a relating gene thereof and use of the bacteriocin and the bacterial strain to foods. <P>SOLUTION: A new Lactobacillus, Enterococcus mundtii 7393 strain is separated using an antibacterial activity to Enterococcus faecium as an index and a new bacteriocin, mundticin KS is separated from the bacterial strain. The gene of the new bacteriocin, the resistance gene thereof and the extracellular exporter gene thereof are cloned from the genome gene of the bacterial strain. The new bacteriocin, the mundticin KS and the Enterococcus mundtii 7393 strain are used for production of conservable foods. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel bacteriocin which is an antibacterial peptide, a method for producing the same, its use and the like. More specifically, the novel bacteriocin Munditicin KS, a method for producing the same, and the bacteriocin The present invention relates to a coding gene, a bacteriocin-related gene and a protein coded by the gene, a method for producing a food product having excellent storability using the bacteriocin, a producing microorganism thereof and a culture thereof.

[0002]

2. Description of the Related Art Food preservatives have been added to various foods for the purpose of preventing spoilage and deterioration of quality of foods. As such food preservatives, synthetic preservatives that have been chemically synthesized have been mainly used so far. However, ingestion of a large amount of a synthetic preservative is not preferable for the human body from the viewpoint of health, and one of the causes is that the chemically synthesized preservative is not easily decomposed in the human body. In order to solve such problems, it is necessary to develop an antibacterial substance that is easily decomposed in the human body.

By the way, lactic acid bacteria have been one of the useful microorganisms that have been used for the production of various fermented foods and beverages such as soy sauce, miso, pickles and sake since ancient times. Lactic acid fermentation is carried out by using lactic acid bacteria in the production process of fermented foods, etc., the pH of the system is lowered by the produced lactic acid, and the lactic acid bacteria produce antibacterial substances, thereby producing processes and products. It is possible to inhibit the growth of various bacteria in the inside, and prevent the product from spoiling and deterioration of quality.

There are some reports on bacteriocin produced by lactic acid bacteria. Japanese Food Science and Engineering Magazine No. 47
Volume 10 (October 2000), pp. 752-759, describes the use of nisin produced from Lactococcus lactis for brewing miso. Biochimica et Biophysica Acta
1373 (1998) 47-58, a bacteriocin produced by Enterococcus munditi is disclosed in JP-A-7-5.
No. 1055 discloses a novel microorganism belonging to the genus Enterococcus having a growth inhibitory effect on Listeria monocytogenes. Also, the patent No.
2618148 discloses a gene sequence encoding a bacteriocin derived from a plasmid in Pediococcus acidilactici.

[0005]

The object of the present invention is to provide a highly safe novel bacteriocin produced by lactic acid bacteria which has been used for food and food processing since ancient times, a method for producing the same, a method for using the same and the bacteriocin. It is to provide a gene or the like encoding the gene. Specifically, it is an antibacterial substance derived from lactic acid bacteria, such as nisin and pediocin, which is easily decomposed in the human body, and is widely used as bacteriocin produced by lactic acid bacteria, and can inhibit the growth of many microorganisms. Compared to known bacteriocin known
A novel bacteriocin having high selectivity of a microorganism to be inhibited and having higher safety, a gene encoding the novel bacteriocin, a gene related to the gene and a protein encoded by the gene, and storage using the novel bacteriocin It is to provide a method for producing a food having excellent properties.

[0006]

[Means for Solving the Problems] The inventors of the present invention have conducted diligent research in order to achieve the above-mentioned object, isolated lactic acid bacteria present in fermented foods and silage, and searched for a strain having a bacteriocin-producing ability. As a result, they succeeded in isolating a novel lactic acid bacterium strain capable of producing bacteriocin, and further confirmed that the bacteriocin and its gene produced by the bacterium are novel substances, thus completing the present invention.

That is, the present invention is a DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 or its complementary sequence or a sequence containing a part or all of these sequences (claim 1).
Or a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2 or a complementary sequence thereof (claim 2), a protein consisting of the amino acid sequence shown in SEQ ID NO: 5, or 1 in the amino acid sequence shown in SEQ ID NO: 5 DNA consisting of an amino acid sequence in which several amino acids have been deleted, substituted or added, and encoding a protein having bacteriocin extracellular efflux activity (claim 3), the nucleotide sequence shown in SEQ ID NO: 4 or its complement 1 or a number in the DNA consisting of a specific sequence or a sequence containing a part or all of these sequences (claim 4), a protein consisting of the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence shown in SEQ ID NO: 7 Consisting of an amino acid sequence in which one amino acid has been deleted, substituted or added, and has a bacteriocin resistance function. DNA encoding a protein that
(Claim 5), or a DNA comprising the nucleotide sequence shown in SEQ ID NO: 6 or its complementary sequence, or a sequence containing a part or all of these sequences (Claim 6).

The present invention also relates to a munditicin KS having the bacteriocin activity consisting of the amino acid sequence shown in SEQ ID NO: 3 (claim 7) and a protein consisting of the amino acid sequence shown in SEQ ID NO: 5 (claim 8). Or a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5 and having bacteriocin extracellular efflux activity (claim 9), or a sequence A protein consisting of the amino acid sequence shown in No. 7 (claim 10), or an amino acid sequence obtained by deleting, substituting or adding one or several amino acids in the amino acid sequence shown in SEQ ID No. 7, and comprising bacteriocin It relates to a protein having a resistance function (claim 11).

Further, the present invention provides a method for producing munditicin KS (claim 12), which comprises culturing a munditicin KS-producing bacterium belonging to the genus Enterococcus and collecting munditicin KS from the culture. The Munditicin KS-producing bacterium is Enterococcus munditi 7393 strain (FERM-P18300).
The method for producing Munditishin KS according to claim 2 (claim 1
3) and the Enterococcus munditi 7393 strain (FERM-P1) that has the ability to produce Munditicin KS.
8300) (claim 14), Munditishin KS,
A method for producing a food having excellent preservability, which comprises adding a microorganism having a munditicin KS-producing ability or a culture solution thereof to the food (claim 15), and munditicin KS
Microbes that have the ability to produce Listeria
A lactic acid bacterium capable of killing or reducing monocytogenes, the method for producing a food product having excellent preservability according to claim 15 (claim 16), and the lactic acid bacterium being Enterococcus munditi 7393 strain. (FE
RM-P18300).
The method for producing a food product having excellent shelf life according to item 6 (claim 17).
Or the food is non-heated meat.
The present invention relates to a method for producing a food having excellent preservability according to any one of 5 to 17 (claim 18).

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The DNA which is the subject of the present invention includes DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 in the Sequence Listing or its complementary sequence, or a sequence containing a part or all of these sequences, In the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2 or its complementary sequence, the protein consisting of the amino acid sequence shown in SEQ ID NO: 5 or the amino acid sequence shown in SEQ ID NO: 5, one or several amino acids are deleted, A DNA consisting of a substituted or added amino acid sequence and coding for a protein having bacteriocin extracellular efflux activity, the nucleotide sequence shown in SEQ ID NO: 4 or its complementary sequence, or part or all of these sequences DNA consisting of the sequence, protein consisting of the amino acid sequence shown in SEQ ID NO: 7 or shown in SEQ ID NO: 7 A DNA consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, and coding for a protein having a bacteriocin resistance function, the base sequence shown in SEQ ID NO: 6 or its complementary sequence. There is no particular limitation as long as it is a DNA consisting of a sequence or a sequence containing a part or all of these sequences, and among them, munditicin KS which is a novel bacteriocin, a protein having bacteriocin extracellular efflux activity, and , A gene D encoding a protein having a bacteriocin resistance function, respectively
NA can be preferably exemplified, and the origin of these DNAs is not particularly limited. The DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2 which encodes munditicin KS imparts bacteriocin-producing ability to bacteriocin-resistant microorganisms that are usually used in mass production of recombinant munditicin KS and food production. And its complementary sequence can be used for detection of the bacteriocin gene at the genetic level. Further, the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 4 and the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 6 elucidate the extracellular secretion mechanism of bacteriocin at the gene level and elucidate the action mechanism of bacteriocin. It is useful for doing. Furthermore, the nucleotide sequence shown in SEQ ID NO: 1 or a sequence containing a part or all of these sequences can be used for imparting a bacteriocin-producing ability to a microorganism usually used in food production. Then, the transformed microorganism imparted with the bacteriocin-producing ability can be advantageously used for foods such as raw ham that are severe in pH.

The protein to which the present invention is applied includes
Munditicin KS having the bacteriocin activity consisting of the amino acid sequence shown in SEQ ID NO: 3 and SEQ ID NO: 5
A protein consisting of the amino acid sequence shown in SEQ ID NO: 5 or a protein consisting of the amino acid sequence shown in SEQ ID NO: 5 with one or several amino acids deleted, substituted or added, and having bacteriocin extracellular efflux activity Or a protein consisting of the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence obtained by deleting, substituting or adding one or several amino acids in the amino acid sequence shown in SEQ ID NO: 7, and having a bacteriocin resistance function. It is not particularly limited as long as it is a protein having a bacteriocin, and the bacteriocin extracellular efflux activity means the activity of bacteriocin produced in the bacterium to the extracellular part, and the bacteriocin resistance function. , It refers to the function of protecting the bacteria themselves from the bacteriocin generated in the cells. Munditicin KS consisting of the amino acid sequence shown in SEQ ID NO: 3, or the protein consisting of the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 7,
It can be obtained by expressing a DNA having the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a DNA having the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 6 using a known host vector system. it can. The above-mentioned Munditicin KS can be advantageously used for killing or reducing Listeria monocytogenes present in foods such as non-heated meat, and for producing foods excellent in preservability, and is shown in SEQ ID NO: 5. The protein having the bacteriocin extracellular efflux activity consisting of the amino acid sequence and the protein having the bacteriocin resistance function consisting of the amino acid sequence shown in SEQ ID NO: 7 are useful for elucidating the extracellular secretory mechanism of bacteriocin and the mechanism of action of bacteriocin. It is useful in clarifying

The above Munditicin KS is a Munditicin KS-producing bacterium belonging to the genus Enterococcus, preferably Enterococcus munditi 7393 strain (FE
It can also be advantageously produced by culturing RM-P18300) and collecting munditicin KS from the culture. The culture medium for such a munditicin KS-producing bacterium is not particularly limited, but TG
E medium (composition: 6 g of beef extract (manufactured by Difco), 10 g tryptone (manufactured by Difco), 2 g glucose (manufactured by Wako Pure Chemical Industries) per liter) is preferable as a medium for mass production, and this medium is used. Anaerobically 10 at 37 ° C
It is preferable to culture for 12 hours. The munditicin KS thus obtained is more effective than nisin (Journal of Food Science and Technology, Vol. 47, No. 10, October 2000), which is the only bacteriocin currently used as a food preservative. Strong antibacterial activity against bacteria,
Nisin exhibits antibacterial activity against Bacillus and the like, but Munditicin KS of the present invention has no antibacterial activity against Bacillus and the like, and is considered to be more specific to Listeria monocytogenes and safe.

[0013] Wild type microorganisms and transformed microorganisms having the ability to produce munditicin KS, preferably lactic acid bacteria capable of killing or reducing Listeria monocytogenes present in foods, more preferably Enterococcus munditi of the present invention. 7393 strain (FERM-P
18300) or by adding these culture solutions to foods, foods having excellent preservability can be produced. When Munditicin KS-producing microorganisms or a culture solution containing the microorganisms are added to food, the Munditicin KS-producing microorganisms grow on the surface or inside of the food, and Munditicin KS
Since it secretes, it is possible to easily produce a food having excellent preservability. Such foods include Listeria
A non-heated food that cannot sterilize contaminated microorganisms such as monocytogenes by heating, for example, non-heated meat such as raw ham can be preferably exemplified. When using lactic acid bacteria as a starter in non-heated meat products such as raw ham,
Although there is concern that taste may be affected by a decrease in pH, the above-mentioned Enterococcus munditi strain 7393 strain (FE
RM-P18300) has the characteristic of not lowering the pH in salted meat compared to other starter strains,
It can be used particularly advantageously as a starter strain that has little influence on flavor.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.

EXAMPLES Example 1 [Production of Munditicin KS] (Separation of Lactic Acid Bacteria) The present inventors separated a large number of lactic acid bacteria using fermented foods and silage as a separation source. From the separated lactic acid bacteria, lactic acid bacteria capable of producing an antibacterial substance were selected using the antibacterial activity against Enterococcus phaseum as an index. The lactic acid bacteria were separated by the following method. 0.5% for samples collected from various fermented foods and silage
Lactobacilli MRS medium containing calcium carbonate (composition:
Proteose peptone 1%, beef extract 1%, yeast extract 0.5%, glucose 2%, Tween 80 0.1%, ammonium citrate 0.5%, magnesium sulfate 0.0.
1%, manganese sulfate 0.005%, dipotassium phosphate 0.2%, manufactured by Difco), and after subculture several times as necessary, Lactobacilli MRS agar medium (composition) : 2% agar added to the above medium composition)
Then, 550 strains of lactic acid bacteria were isolated from the resulting colonies.

(Identification of isolated lactic acid bacteria) Next, from the thus isolated lactic acid bacteria 550 strain, the paper disk method (Hoover, DG & Harlander, SK, In Bacte) was used with the antibacterial activity against Enterococcus phaseum as an index.
Forty-five strains of lactic acid bacteria capable of producing antibacterial substances were selected by riocins of lactic acid bacteria, Academic Press, 23-39, 1993). Among these, a strain having a particularly strong antibacterial activity was selected, and the mycological properties of the selected lactic acid bacterium were examined. As a result, 16S ribosomal DNA (rDNA)
Sequence homology (Mori, K. et al .: Int.J.Syst.Bact)
eriol., 47, 54-57, 1997).
Due to the fact that it showed 100% homology with the Munditi JCM8731 strain, and that the fermentability of sugars was the same as that of the Enterococcus munditi JCM8731 strain,
It was found that this bacterium belongs to Enterococcus munditi. Therefore, this bacterium was named Enterococcus munditi 7393. Table 1 shows the findings regarding the identification of this bacterium.

[0016]

[Table 1]

(Comparison with conventional strains) The antibacterial activity of Enterococcus munditi JCM 8731 strain (Type Strain) culture supernatant and Enterococcus munditi 7393 culture supernatant against Listeria monocytogenes was determined by the Agarwell method. , Trypto soya agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.). It was evaluated as having antibacterial activity when a blocking circle of 7 mm or more was formed. The results are shown in Table 2. In Table 2, "+" indicates that a blocking circle of 7 to 12 mm was formed, and "++" indicates that a blocking circle of 13 to 18 mm was formed. From Table 2, it was revealed that Enterococcus munditi 7393 strain produces Listeria antibacterial active substance, while Enterococcus munditi JCM8731 strain does not produce antibacterial active substance. From this, enterococcus
Munditi 7393 strain was identified as a new strain,
The 7393 strains have been deposited at the Patent Organism Depositary Center, National Institute of Advanced Industrial Science and Technology, Ministry of Economy, Trade and Industry. Its accession number is FERM P-18300.

[0018]

[Table 2]

(Enterococcus munditi 739
Production of Antibacterial Substances by 3 Strains) Next, the isolated Enterococcus munditi 7393 strain (FERM P-1
8300) was inoculated into a Lactobacilli MRS medium (composition: manufactured by Difco as described above) and cultured at 37 ° C. for 0 to 10 hours to examine the growth state of 7393 strain and the productivity of antibacterial substances. The results are shown in Fig. 1. In Figure 1,
The growth amount of the 7393 strain when cultured at 37 ° C was measured by OD _600.
The results show that the activity of the antibacterial substance was measured by the Agarwell method using the antibacterial activity against Enterococcus phaseum as an index. As is clear from FIG. 1, when cultured at 37 ° C., sufficient bacterial growth was observed in 4 hours, and it was found that a large amount of antibacterial substance was produced. In the figure, ■ indicates the growth of 7393 strains, and ● indicates the productivity of antibacterial substances. In addition, the influence of the culture temperature on the productivity of antibacterial substances was investigated. The results of culturing at 15 ° C to 45 ° C are shown in Fig. 2. Figure 2 shows the productivity of antibacterial substances when cultured at 15 ° C to 45 ° C.
The results obtained by the Agarwell method using the antibacterial activity against phaseum as an index are shown. As is clear from FIG. 2, it was found that the antibacterial activity was strong when cultured at 20 ° C. to 37 ° C.

(Enterococcus munditi 739
Cultivation of 3 strains) For the purpose of obtaining Munditicin KS which is the bacteriocin of the present invention, when culturing a large amount of 7393 strain, TGE medium (composition: beef extract per liter (manufactured by Difco)) is used as a medium. 6 g, tryptone (manufactured by Difco) 10 g, glucose (manufactured by Wako Pure Chemical Industries)
2g) is preferably used and 37
It is preferable to anaerobically cultivate at 10 ° C. for 10 to 12 hours.

(Detection of antibacterial active substance) 60% of culture filtrate
Precipitated with ammonium sulphate, and eluted stepwise with acetonitrile using a C-18 reverse phase column.
Electrophoresis was performed by cin-SDS PAGE, and activity staining for Enterococcus phaseum was performed.
FIG. 3 is a photograph of SDS-PAGE (A) and activity staining (B). As is clear from FIG. 3, the molecular weight is about 5 kD.
Antibacterial activity was detected at position a.

Example 2 [Munditicin KS Gene and Related Genes] (Cloning of Munditicin KS Gene) Cloning of a DNA fragment containing the Munditicin KS gene (DNA described in SEQ ID NO: 1 in the Sequence Listing) was performed as follows. I went like. The lactic acid bacterium-E. Coli shuttle vector pRH100 (selection marker; ampicillin resistance <Escherichia coli>, erythromycin resistance <lactic acid bacterium>) was used as a reference (Maniatis, T., EF Fr.
itsch, and J. Sambrook. 1982. Molecular cloning: a
laboratory manual.Cold Spring Harbor Laboratory, C
old Spring Harbor) and constructed by the restriction enzyme Sau3AI, and then partially digested with strain 7393 strain Genome DN.
A was inserted to create a genomic library. Using the bacteriocin-susceptible bacterium Enterococcus phaseum as a host bacterium, the prepared genomic library was introduced by electroporation, and the bacteriocin-containing culture solution (5% v / v) from which the 7393 strain was removed and erythromycin (5 μg / ml) ) Added Lactobacilli MRS
Transformants were selected on agar medium.

Next, the plasmid DNA extracted from this transformant was partially digested with the restriction enzymes BamHI and HindIII and inserted into the vector pBluescript II KS (+) (manufactured by STRATAGENE, selection marker; ampicillin resistance <Escherichia coli>). , Escherichia coli was introduced by electroporation, and transformants were selected on an agar medium supplemented with ampicillin. The base sequence of this transformant was determined by a DNA sequencer. As a result, a DNA fragment having the sequence shown in SEQ ID NO: 1 was obtained. At least three open reading frames (ORFs) are present in this DNA fragment, and the amino acid sequence encoded by ORF1 (SEQ ID NO: 2) present at positions 1776 to 1949 in the sequence of SEQ ID NO: 1 and other bacteriocins are present. Was compared with the amino acid sequence of. The results are shown in Fig. 4. As is clear from FIG. 4, this amino acid sequence was highly similar to known bacteriocin and was found to represent the bacteriocin amino acid sequence. ORF1 was PCR-amplified by a conventional method, this PCR product was expressed in Escherichia coli by a conventional method using the expression vector pET-3, and bacteriocin was purified from Escherichia coli. The purified product was confirmed to have antibacterial activity against Enterococcus phaseum. did. Therefore, it was also revealed that ORF1 contains a bacteriocin biosynthesis gene. In addition, analysis of the purified preparation of the bacteriocin using a protein sequencer revealed that the lysine residue, which is the 16th residue of the amino acid sequence encoded by ORF1, and the lysine residues are the bacteriocin amino acid sequence ( SEQ ID NO: 3).

(Munditicin KS) SEQ ID NO: 3 above
Since the amino acid sequence shown in 1 was found to have a novel structure, this bacteriocin was named munditicin KS. From the conserved amino acids possessed by this bacteriocin, this bacteriocin is classified as Klaenhammer (Klaenhammer, TR: FEMS).
Microbiol. Rev., Volume 12, 39-86, 1993), it was also found to belong to the low molecular weight and heat resistance class (class IIa).

(Determination of bacteriocin-related gene region) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 above.
The fragment contains at least two ORs in addition to the ORF1 described above.
F, that is, in the nucleotide sequence shown in SEQ ID NO: 1,
There are ORF2 (SEQ ID NO: 4) existing from 2094th to 4118th and ORF3 (SEQ ID NO: 6) existing from 4143rd to 4439th. The properties of the proteins encoded by these ORF2 and ORF3 were investigated. As a result of the homology search, the protein consisting of the amino acid sequence (SEQ ID NO: 5) encoded by ORF2 (SEQ ID NO: 4) showed high homology with some of the existing bacterial ATP-dependent membrane transport proteins over the entire amino acid sequence. From this, it was found that the protein has bacteriocin extracellular efflux activity. Therefore, ORF2
It was also clarified that (SEQ ID NO: 4) is a gene involved in extracellular bacteriocin export. Also, OR
Only F3 (SEQ ID NO: 6) was PCR-amplified by a conventional method, and this PCR product was used as the shuttle vector pRH1 for lactic acid bacteria-E. Coli.
When it was expressed in Enterococcus phaseum using 00 in a conventional manner, it was confirmed that it showed resistance to Munditicin KS. From this, it was also clarified that ORF3 (SEQ ID NO: 6) is a gene involved in imparting resistance to bacteriocin encoding a protein having bacteriocin resistance imparting activity.

Example 3 [Physical properties of munditicin KS] (pH stability of munditicin KS) The purified munditicin KS obtained in Example 1 had a final concentration of 0.
Dissolve it in buffer solutions of various pHs so that it becomes 01% by weight, and
After incubating at 7 ° C for 30 minutes, the pH was returned to 6.0 and the pH stability of Munditicin KS was examined. As the buffer solution, 10 mM MES (pH 4 to 6), 10 mM TE
S (pH 6-8), 10 mM Tris.HCl (pH
8-11) to adjust the pH to 6.0.
It was performed by adding mM MES (pH 6.0). The antibacterial activity against Listeria monocytogenes was measured by the Agarwell method using the trypto soya agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.). As a result, it was revealed that Munditicin KS maintained 80% to 100% activity at pH 4 to 11 and had stability in a wide pH range.

(Temperature stability of Munditicin KS) The purified Munditicin KS obtained in Example 1 was treated with 2- (N-morpholino) ethanesulfonic acid (so that the final concentration was 0.01% by weight). MES) buffer (pH 6.
0) and add this bacteriocin solution to 0-100
The temperature stability of Munditicin KS was examined by measuring the residual antibacterial activity after heating or cooling at 1 ° C for 1 hour. The residual antibacterial activity against Listeria monocytogenes was measured by the Agarwell method using the trypto soya agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.). Results are shown in FIG. From FIG. 5, 100% antibacterial activity is maintained at 0 ° C to 70 ° C, about 80% at 80 ° C, about 50% at 100 ° C.
It was found to maintain the antibacterial activity of%.

Example 4 [Antibacterial property of Munditicin KS] (Antibacterial spectrum of Munditicin KS) The purified Munditicin KS obtained in Example 1 was adjusted to a final concentration of 0.01% by weight. MES buffer (pH 6.
0), and using this bacteriocin solution, the antibacterial spectrum of Munditicin KS against various pathogens was examined by the Agarwell method. When a blocking circle of 7 mm or more was formed, it was evaluated as having an antibacterial effect. Table 3 shows the measurement results and the culture conditions of the test bacteria. In Table 3, "+" is 7
"++" means that a stop circle of ~ 12mm was formed.
Each shows the formation of an inhibition circle of ~ 18 mm. As is clear from Table 3, Listeria monocytogenes and Clostridium, which cause food poisoning,
It was revealed that Munditicin KS has a growth inhibitory effect on perfringens. The composition of the medium used for the test is as follows. Tryptosome Agar Medium: Peptone 1.5%, Soybean Peptone 0.5%, Sodium Chloride 0.5%, Agar 1.5
% (Nissui Pharmaceutical Co., Ltd.) GAM agar medium: peptone 1%, soybean peptone 0.3
%, Proteose peptone 1%, digested serum powder 1.35
%, Yeast extract 0.5%, meat extract 0.22%, liver extract 0.12%, glucose 0.3%, potassium dihydrogen phosphate 0.25%, sodium chloride 0.3%, soluble starch 0. 5%, L-cysteine hydrochloride 0.03%, sodium thioglycolate 0.03%, agar 1.5% (Nissui Pharmaceutical Co., Ltd.)

[0029]

[Table 3]

(Antibacterial Effect of Muditycin KS against Food Spoilage / Degrading Bacteria) The antibacterial effect of Munditicin KS against Enterococcus faecalis which causes spoilage / deterioration of various foods was examined. The antibacterial effect is measured by a liquid medium system (medium composition: tryptone 1.25%, yeast extract 0.75%, glucose 1.0%, sodium citrate 0.5%, thiamine hydrochloride 0.0001%, sodium chloride. 1.5%, dipotassium hydrogen phosphate 0.5%, magnesium sulfate 0.08%, manganese chloride 0.014%, iron (II) sulfate 0.004%, Tween 80 0.02%,
The culture supernatant of Enterococcus munditi 7393 strain was concentrated 10-fold at pH 6.0), and 0% (control; ◆), 1% (●) and 10% (Δ) were added thereto. Results are shown in FIG. As is clear from FIG. 6, it was revealed that Munditicin KS has a growth inhibitory effect on Enterococcus faecalis.

(Comparison between Munditicin KS and Nisin) The antibacterial effects of Munditicin KS and nisin, a known bacteriocin, against Listeria monocytogenes were compared. The antibacterial effect was measured in the above liquid medium system, and the culture supernatant of the Enterococcus munditi strain 7393 was concentrated 10-fold to 0% (control;
◆), 1% (∘) and 10% (x) were added respectively. Nisin is a 1% solution of 400 IU / ml.
(4 IU / ml; △) and 10% (40 IU / ml;
■) was added respectively. The results are shown in Fig. 7. As is clear from FIG. 7, Munditicin KS was found to have a stronger growth inhibitory effect than nisin against Listeria monocytogenes that causes food poisoning.

(Munditishin KS and enterocin S
Comparison with E-K4) Munditicin KS and enterocin SE-K4 (Biosci.Bio), which is a known bacteriocin.
technol.Biochem. Vol.65, No.2, 2001, 247-253) was compared for the antibacterial effect against Listeria monocytogenes. The antibacterial effect was measured by the Agarwell method using trypto soya agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.).
The results are shown in Table 4. In Table 4, "+" indicates that a blocking circle of 7 to 12 mm was formed, and "++" indicates that a blocking circle of 13 to 18 mm was formed. From Table 4,
It was found that Munditicin KS has a higher antibacterial effect on Listeria than enterocin SE-K4.

[0033]

[Table 4]

Example 5 [Application of Munditicin KS to food] Enterococcus munditi strain 7393 strain was used as a starter in the production of raw ham. Salted meat modeled on raw ham (200g ground meat, 4.13 salt)
%, Sodium nitrite 300 ppm, sodium ascorbate 0.06%, saccharide 2.0%) were inoculated with 10 ⁴ / g of Listeria monocytogenes. Next, Enterococcus munditi 7 as starter stock
393 strains and Lactobacillus salmon 10 ⁷ / g-10 ⁸
10 / g each after inoculation and air-filled packaging
It was stored at 9 ° C, 15 ° C and 20 ° C for 9 days, respectively. Figure 8 shows changes in the viable cell count of Listeria monocytogenes when Enterococcus munditi strain 7393 was used as a starter, and changes in the viable cell count of Listeria monocytogenes when Lactobacillus salmon was used as a starter. 9 respectively. As a result, if Enterococcus munditi 7393 strain is used as a starter, 2
The bactericidal effect of Listeria was observed at 0 ° C storage, but when Lactobacillus salmon was used as the starter, the bactericidal effect of Listeria was not observed. Moreover, when Enterococcus munditi 7393 strain is used as a starter,
The pH of the product on the 9th day of storage at 20 ° C is 10 ° C;
H5.73, 15 ° C; pH 5.55, 20 ° C; pH 5.
While it was 37, when Lactobacillus salmon was used as the starter, the p
H is 10 ° C; pH 4.85, 15 ° C; pH 4.80,
It was 20 ° C .; pH was 4.75, and it was found that when Enterococcus munditi 7393 strain was used as a starter, the decrease in pH was small and the influence on the taste of meat products was small. The pH of the product was determined by adding a 5-fold amount of deionized water and then measuring the homogenized supernatant with a pH meter.

Example 6 [Expression of recombinant munditicin KS and munditicin ATO6 in Escherichia coli] In order to verify the difference in biological activity between munditicin KS and munditicin ATO6, a combination in E. coli was performed. Recombinant munditicin KS and ATO6 were expressed. Histidine tag sequence and rTEV (recomb
Munditicin with a protease cleavage recognition sequence was expressed in Escherichia coli in a large amount and purified by affinity chromatography using the tag sequence. Then, an excess N-terminal additional sequence was removed by cleaving rTEV protease to obtain recombinant munditicin rMunKS and rMunATO6. rMun
Both KS and rMunATO6 peptides had an extra N-terminal glycine residue that does not exist in the natural form. However, these recombinant munditicins were all against E. faecium. hand,
Antibacterial specific activity equivalent to natural Munditicin KS (4.
1 × 10 ⁶ AU / mg protein), but for other munditicin-sensitive lactic acid bacteria, rMun
There is a difference in antibacterial specific activity between KS and rMunATO6,
The degree varied depending on the susceptible bacteria. That is,
RMunATO6 is about 10% of rMunKS against L. plantarum and rMun against L. carvatus
ATO6 showed only about 50% antibacterial specific activity of rMunKS (see Figure 10). from these things,
Munditicin KS has both primary structure and antibacterial activity,
It was revealed that it is a novel bacteriocin different from Munditicin ATO6. Furthermore, it was revealed that a specific amino acid sequence at the C-terminal of bacteriocin is involved in the specificity of target cells.

[0036]

The munditicin KS of the present invention is a bacteriocin having excellent heat resistance and exhibits an antibacterial spectrum against specific bacteria such as Listeria monocytogenes and Clostridium. Therefore, by adding this bacteriocin to foods and the like, it is possible to inhibit specific growth such as food poisoning bacteria and some spoilage and spoilage bacteria, and effectively prevent spoilage and deterioration of quality of foods. In addition, since this bacteriocin is derived from lactic acid bacteria and is easily decomposed in the human body, compared to conventional chemically synthesized food preservatives, it is safe to consume even in large amounts. It is preferable from the viewpoint of health. Moreover, since it has a specific antibacterial spectrum as compared with conventional bacteriocin, it has less influence on intestinal bacteria and the like, and is more safe.

The munditicin KS-producing novel lactic acid bacterium strain of the present invention has specific antibacterial activity against Listeria which causes food poisoning, and even lactic acid bacterium has a characteristic that the pH is not lowered so much. For example, in the production of non-heated meat, it can be advantageously used in food production, such as imparting effective antibacterial activity without changing the taste of the product.

Further, the DNA encoding the munditicin KS of the present invention can be used for mass production of recombinant munditicin KS and the like. Further, the bacteriocin-related gene of the present invention and the protein encoded by the gene can be expected to be applied to mass production of the recombinant munditicin KS and the like, and the elucidation of the mechanism of extracellular bacteriocin excretion and , It is useful for elucidating the action mechanism of bacteriocin.

[0039]

[Sequence list]                                SEQUENCE LISTING <110> Prima Meat Packers, Ltd. <120> A novel Bacteriocin <130> 2001P865 <140> <141> <160> 7 <170> PatentIn Ver. 2.1 <210> 1 <211> 5334 <212> DNA <213> Enterococcus mundtii <400> 1 ttttggctgg actgcacaag gcgtagagat cattcgtatc gatgaagata aagaagaatt 60 tggatttgaa cgattggcag aactggtcaa acagtacgaa gtagatccca caccacctga 120 aagcatcaaa gaagcaccaa tcactgaaat ggccaaacca gaaggcgcat tgcccaagac 180 taatacgatc ccccggtgac tgttgctttc ggatcaattc gttttacggg cacttacgtg 240 ccaatcgggg ccccagatca atcaatttca ttaatgatga aagaccgttt tagttcgatt 300 cgataccgca gacattctat gaataatgtt tatgatttag cacatgccct tgttttacta 360 gaaacaattg gtcacttcgg taaactctaa acgaaagaat caggaaagga gacatacgaa 420 agggtttcat aaccctttcg tatcaatcaa aagatgaaga taaatgaagg aaagaagtat 480 aaatgcaaaa attcttttgt ctttcaagaa gaagaaatta ggcaagggga tatgattggt 540 atcaatcata tcgagtcgcc agtattagtc gattgcgaag tgtatcgtga agatgggcga 600 ggcatgaaac gattcagaac gattgaaatc aacctggcac gcttagaaca atcgatcgat 660 ttttcgagtg aagcgacttt ttacggcgac tgtgaagaat gccaccgact acttgattat 720 ttaccaaggg gggcttgggg cgtaggttat gtttgcgctc cttgtgcaaa aagactaaga 780 ggagagtatg agtaacgtcg ttagtgagat tagtagaaca atgattcaat tcattttatt 840 ctttaaggta aatcagcaca tatattccga aaagcgaata tatatggcaa cttaaattac 900 aaataaaaag ttaataggag atgacattat gaaattaatc gatatttacg ataataatag 960 cttaaagtat aaggggtctt ttgaacacac cgatgaaaat attatcaatt atgtagcagc 1020 aatcccacag ttcaagttta tacgattagt tgaattatct agtgatgaaa tagttctaac 1080 gacaatcggc aatttccttg actatgtacc tgatcaaaat tggttagaaa aaattcgtcc 1140 tagcttgatt gctaaacaga tgaaagaaga tgttatagat gaagtgctaa taattgatcg 1200 atataaagaa gatggagatt tctaaaaaaa ttaaacatac aaaggaaaaa gataaatgag 1260 gtgaccattt taataaaaca acaaatcatg aaatgattgt tttgcagaag aagaacaagt 1320 gaacactgaa gccctagagt agtaggatac tctaaggctt ttctttttgg ctaatctcgc 1380 cgtgtttgtt ttacttcttg ttgcgagtat ttagccagta ctcgtatatg gttactagca 1440 aaccgataag aatgtgggat taagcataca cgaatacttt ttcttgcttg taagactata 1500 ggatagacaa ctattcactt gtttaaaata tttttttaga tttaggcaaa tgaatcaaag 1560 taagtatatt tacgattatg acattaaaaa taacttttat gacatcaaaa gctacgttta 1620 tgaaatccat atttataata aaataaaata tgtttttaga tgacaaaatt gtaagttaaa 1680 ataacgtaat tttaagtaaa aaaatataaa taatatgcta tcttttaaat aaggacaatt 1740 atttattgtt caaaaaatga gagaaggttt aagttttgaa gaaattaaca gcaaaagaaa 1800 tgtcacaagt agtaggtgga aaatactacg gtaatggagt ctcatgtaat aaaaaagggt 1860 gcagtgttga ttggggaaaa gctattggca ttattggaaa taattctgct gcgaatttag 1920 ctactggtgg agcagctggt tggaaaagtt aatttgagcc ttttatttta gtatattata 1980 tttttagaat actcattcaa aaattctatg gatgagtatt cttatttata ggtgattctt 2040 tgagacaaaa attaataaaa cagtataatg aagttgattg tggtatagcc tgtatgcaga 2100 tgattttaaa taattttcat tcatggattt cagtggaagt tttaagagac ttaactgaaa 2160 ccgattctga aggtacctgt gcattaggta tagttaacgg atttgctaaa ttaggaataa 2220 attgtgaagc ctataaagct aatagtgatg tatggaaaga gaatgagttc aattatcccg 2280 taattgctaa tatagtaacg aataatcaat ttcttcatta ttgtattgta tatggtgtga 2340 aaaaagagaa attgttaata gctgaccctg cgattggaaa atacaaagaa tcaatagaaa 2400 agttcaacaa caattggact ggtgttattt tagttgctga aaagacgcct gatttccaac 2460 ccataaataa tacaaaaaaa agtttttttt cttcaataag tttattaaaa gatcaatata 2520 aaaaaatttt attggtgata ttatcttcat taataataac aattatagga atactatcaa 2580 gttactattt tagaatttta atagactggt tacttcctga aaaagacttt ttaaatctat 2640 ttatgatatc aattagctat atcataggca tttttataac aagtatattt gaaattacaa 2700 gaagatataa tttagaaaag ctaggacaag atgtaggtag aagcatttta tttaaatatt 2760 tagatcatat tttcatttta ccagcttcct ttttttctaa aagaaaaact ggagatattg 2820 tctctagatt ttctgatgct aataaaatta tagaagcttt agctagcttt actatatcta 2880 tttttttaga tttaagttca gtcattgttg tggggattat attgatcaat attaataaac 2940 aattattttt aataacgtta agttctattc cattttatat actaattata ttaggatcaa 3000 ataaaaaaat gagtcgatta aacggagaag agatgcaaac aaattcaata gttgattcta 3060 attttattga aggattaaac ggaatatata ctataaaagc actttgtagt gagaataaga 3120 ttgtaaatca aatatataga agtttaaatg aattttttga tgtatcacta aagagaaata 3180 tgtatgattc tataattcaa aatttaaaaa ttttggtttc tcttttaacc tcggcttttg 3240 tattatggtt tggttcgtat tatgttatca atggagaaat tacaatagga gaactaataa 3300 ctttcaattc attatctata tttttttcta cacctctaca aaatataata aatctacaag 3360 aaaaattcca aaaagcacaa gttgcaaata atcggcttaa cgatgtattt tctataaata 3420 atgaaaataa agacaagttt attcatttgg ctaaattaac tgaaaaagca acgattacat 3480 ttgaaaatgt atattttagt tattctacta aatatcctaa tgtgttagat aatatgagtt 3540 tttctctacc tgtgagtaaa aatataggag taaaaggtga tagtggtgct gggaaatcaa 3600 ctttagcaca acttctagct ggattttact ctccagataa tggaagaatt tgtataaatg 3660 agcaaaatat tgaaaatatt aatagaaaag atttacgtaa gttgattacc tatgtgcctc 3720 aagaatcttt tattatgagt ggaactatta aagacaattt atttttaggt ttagaaagta 3780 ttcctgatga acaagaactc gaaaaagtac tgaaagatac ttgtttatcg agttatatta 3840 ctgcgcttcc tctaggactt gatacgtatc tagaggaaaa tggtgcgaat ttatcaggtg 3900 gtcaaaagca aagaattgct ttagcaagag ttttattatc aggaagtaaa attttattat 3960 tagatgaagc tacgagtgct ctagattcta aaactgaaat gctgattttt gaaaaaatat 4020 taaagtaccc taataagtca atcattatta tatctcataa tgataaatta atagacaagt 4080 gtgacataat cattgattta gacgaaaggg attcgtaaaa aggaatcaag gaggatgaat 4140 aaatgagtaa tttaaagtgg ttttctggtg gagacgatcg acgtaaaaaa gcagaagtga 4200 ttattactga attattagat gatttagaga tggatcttgg aaatgaatct cttcggaaag 4260 tattaggctc ctatcttaaa aagttgaaaa atgaagggac ttcagttccg ttagttttaa 4320 gtcgtatgaa tatagagata tctaatgcaa taaaaaaaga cggtgtatcg ttaaatgaaa 4380 atcaatctaa aaaattaaaa gaactaatgt ctatatctaa tattagatat ggttactagt 4440 gatttattta aattagatat agttaatttt tactattgga tcataaaatt attaattttt 4500 gttggtatct ttaaaaatgc tgataaatag ttattggttg tggggacaat gtataacgat 4560 tttaattgaa tccctattgt taattctaaa atcctgaata agttactaaa agttacaaac 4620 gagaaataca attaataaaa gggatcagaa gcttttaagt ttttgatcct ttttaacttg 4680 cttatttgat aattctttca gttgctcact taattaggtt tagtaagata tttacctaga 4740 gtccaccaga gtataccctg ttggactttc tatttcgctt ttaaacactt aaattagaaa 4800 atgacatcga agttgagcaa gaaaaataaa agataagaat caaaaaactt aaatattttt 4860 gattcttacc ttttattttt ttttgagcac taattatttt ctcccattca ttctgatgaa 4920 tcaatgattt catttgtttt tttgggttat tttctagata tgcataacga ggtgaaaatt 4980 gtgattttca accttatata aattgttata agataatgaa tataaggaag gctaaaggtt 5040 aagccttgtt tattcactaa aacgtattag aaaccatcgt tttttagaat aacgaatact 5100 tatgtatgct aagtcatcga aacgccaatt taacaacatt actttagaca tctgacaagt 5160 tcagatgtct attttgctgt acacccaaaa acgatagttt ttgagataga tgttaaagga 5220 ggagggaaga tgcgaaaaac tattggtctt atgattggtc taatgtccct aataatagcg 5280 atacaatttc cgctacaggt gtatggtaca caggtccaat ccgtcgagtc agaa 5334 <210> 2 <211> 150 <212> DNA <213> Enterococcus mundtii <220> <221> CDS <222> (1) .. (150) <400> 2 ttg aag aaa tta aca gca aaa gaa atg tca caa gta gta ggt gga aaa 48 Met Lys Lys Leu Thr Ala Lys Glu Met Ser Gln Val Val Gly Gly Lys   1 5 10 15 tac tac ggt aat gga gtc tca tgt aat aaa aaa ggg tgc agt gtt gat 96 Tyr Tyr Gly Asn Gly Val Ser Cys Asn Lys Lys Gly Cys Ser Val Asp              20 25 30 tgg gga aaa gct att ggc att att gga aat aat tct gct gcg aat tta 144 Trp Gly Lys Ala Ile Gly Ile Ile Gly Asn Asn Ser Ala Ala Asn Leu          35 40 45 gct act ggt gga gca gct ggt tgg aaa agt 174 Ala Thr Gly Gly Ala Ala Gly Trp Lys Ser          50 55 <210> 3 <211> 50 <212> PRT <213> Enterococcus mundtii <400> 3 Lys Tyr Tyr Gly Asn Gly Val Ser Cys Asn Lys Lys Gly Cys Ser Val   1 5 10 15 Asp Trp Gly Lys Ala Ile Gly Ile Ile Gly Asn Asn Ser Ala Ala Asn              20 25 30 Leu Ala Thr Gly Gly Ala Ala Gly Trp Lys Ser          35 40 <210> 4 <211> 2025 <212> DNA <213> Enterococcus mundtii <220> <221> CDS <222> (1) .. (2025) <400> 4 atg cag atg att tta aat aat ttt cat tca tgg att tca gtg gaa gtt 48 Met Gln Met Ile Leu Asn Asn Phe His Ser Trp Ile Ser Val Glu Val   1 5 10 15 tta aga gac tta act gaa acc gat tct gaa ggt acc tgt gca tta ggt 96 Leu Arg Asp Leu Thr Glu Thr Asp Ser Glu Gly Thr Cys Ala Leu Gly              20 25 30 ata gtt aac gga ttt gct aaa tta gga ata aat tgt gaa gcc tat aaa 144 Ile Val Asn Gly Phe Ala Lys Leu Gly Ile Asn Cys Glu Ala Tyr Lys          35 40 45 gct aat agt gat gta tgg aaa gag aat gag ttc aat tat ccc gta att 192 Ala Asn Ser Asp Val Trp Lys Glu Asn Glu Phe Asn Tyr Pro Val Ile      50 55 60 gct aat ata gta acg aat aat caa ttt ctt cat tat tgt att gta tat 240 Ala Asn Ile Val Thr Asn Asn Gln Phe Leu His Tyr Cys Ile Val Tyr  65 70 75 80 ggt gtg aaa aaa gag aaa ttg tta ata gct gac cct gcg att gga aaa 288 Gly Val Lys Lys Glu Lys Leu Leu Ile Ala Asp Pro Ala Ile Gly Lys                  85 90 95 tac aaa gaa tca ata gaa aag ttc aac aac aat tgg act ggt gtt att 336 Tyr Lys Glu Ser Ile Glu Lys Phe Asn Asn Asn Trp Thr Gly Val Ile             100 105 110 tta gtt gct gaa aag acg cct gat ttc caa ccc ata aat aat aca aaa 384 Leu Val Ala Glu Lys Thr Pro Asp Phe Gln Pro Ile Asn Asn Thr Lys         115 120 125 aaa agt ttt ttt tct tca ata agt tta tta aaa gat caa tat aaa aaa 432 Lys Ser Phe Phe Ser Ser Ile Ser Leu Leu Lys Asp Gln Tyr Lys Lys     130 135 140 att tta ttg gtg ata tta tct tca tta ata ata aca att ata gga ata 480 Ile Leu Leu Val Ile Leu Ser Ser Leu Ile Ile Thr Ile Ile Gly Ile 145 150 155 160 cta tca agt tac tat ttt aga att tta ata gac tgg tta ctt cct gaa 528 Leu Ser Ser Tyr Tyr Phe Arg Ile Leu Ile Asp Trp Leu Leu Pro Glu                 165 170 175 aaa gac ttt tta aat cta ttt atg ata tca att agc tat atc ata ggc 576 Lys Asp Phe Leu Asn Leu Phe Met Ile Ser Ile Ser Tyr Ile Ile Gly             180 185 190 att ttt ata aca agt ata ttt gaa att aca aga aga tat aat tta gaa 624 Ile Phe Ile Thr Ser Ile Phe Glu Ile Thr Arg Arg Tyr Asn Leu Glu         195 200 205 aag cta gga caa gat gta ggt aga agc att tta ttt aaa tat tta gat 672 Lys Leu Gly Gln Asp Val Gly Arg Ser Ile Leu Phe Lys Tyr Leu Asp     210 215 220 cat att ttc att tta cca gct tcc ttt ttt tct aaa aga aaa act gga 720 His Ile Phe Ile Leu Pro Ala Ser Phe Phe Ser Lys Arg Lys Thr Gly 225 230 235 240 gat att gtc tct aga ttt tct gat gct aat aaa att ata gaa gct tta 768 Asp Ile Val Ser Arg Phe Ser Asp Ala Asn Lys Ile Ile Glu Ala Leu                 245 250 255 gct agc ttt act ata tct att ttt tta gat tta agt tca gtc att gtt 816 Ala Ser Phe Thr Ile Ser Ile Phe Leu Asp Leu Ser Ser Val Ile Val             260 265 270 gtg ggg att ata ttg atc aat att aat aaa caa tta ttt tta ata acg 864 Val Gly Ile Ile Leu Ile Asn Ile Asn Lys Gln Leu Phe Leu Ile Thr         275 280 285 tta agt tct att cca ttt tat ata cta att ata tta gga tca aat aaa 912 Leu Ser Ser Ile Pro Phe Tyr Ile Leu Ile Ile Leu Gly Ser Asn Lys     290 295 300 aaa atg agt cga tta aac gga gaa gag atg caa aca aat tca ata gtt 960 Lys Met Ser Arg Leu Asn Gly Glu Glu Met Gln Thr Asn Ser Ile Val 305 310 315 320 gat tct aat ttt att gaa gga tta aac gga ata tat act ata aaa gca 1008 Asp Ser Asn Phe Ile Glu Gly Leu Asn Gly Ile Tyr Thr Ile Lys Ala                 325 330 335 ctt tgt agt gag aat aag att gta aat caa ata tat aga agt tta aat 1056 Leu Cys Ser Glu Asn Lys Ile Val Asn Gln Ile Tyr Arg Ser Leu Asn             340 345 350 gaa ttt ttt gat gta tca cta aag aga aat atg tat gat tct ata att 1104 Glu Phe Phe Asp Val Ser Leu Lys Arg Asn Met Tyr Asp Ser Ile Ile         355 360 365 caa aat tta aaa att ttg gtt tct ctt tta acc tcg gct ttt gta tta 1152 Gln Asn Leu Lys Ile Leu Val Ser Leu Leu Thr Ser Ala Phe Val Leu     370 375 380 tgg ttt ggt tcg tat tat gtt atc aat gga gaa att aca ata gga gaa 1200 Trp Phe Gly Ser Tyr Tyr Val Ile Asn Gly Glu Ile Thr Ile Gly Glu 385 390 395 400 cta ata act ttc aat tca tta tct ata ttt ttt tct aca cct cta caa 1248 Leu Ile Thr Phe Asn Ser Leu Ser Ile Phe Phe Ser Thr Pro Leu Gln                 405 410 415 aat ata ata aat cta caa gaa aaa ttc caa aaa gca caa gtt gca aat 1296 Asn Ile Ile Asn Leu Gln Glu Lys Phe Gln Lys Ala Gln Val Ala Asn             420 425 430 aat cgg ctt aac gat gta ttt tct ata aat aat gaa aat aaa gac aag 1344 Asn Arg Leu Asn Asp Val Phe Ser Ile Asn Asn Glu Asn Lys Asp Lys         435 440 445 ttt att cat ttg gct aaa tta act gaa aaa gca acg att aca ttt gaa 1392 Phe Ile His Leu Ala Lys Leu Thr Glu Lys Ala Thr Ile Thr Phe Glu     450 455 460 aat gta tat ttt agt tat tct act aaa tat cct aat gtg tta gat aat 1440 Asn Val Tyr Phe Ser Tyr Ser Thr Lys Tyr Pro Asn Val Leu Asp Asn 465 470 475 480 atg agt ttt tct cta cct gtg agt aaa aat ata gga gta aaa ggt gat 1488 Met Ser Phe Ser Leu Pro Val Ser Lys Asn Ile Gly Val Lys Gly Asp                 485 490 495 agt ggt gct ggg aaa tca act tta gca caa ctt cta gct gga ttt tac 1536 Ser Gly Ala Gly Lys Ser Thr Leu Ala Gln Leu Leu Ala Gly Phe Tyr             500 505 510 tct cca gat aat gga aga att tgt ata aat gag caa aat att gaa aat 1584 Ser Pro Asp Asn Gly Arg Ile Cys Ile Asn Glu Gln Asn Ile Glu Asn         515 520 525 att aat aga aaa gat tta cgt aag ttg att acc tat gtg cct caa gaa 1632 Ile Asn Arg Lys Asp Leu Arg Lys Leu Ile Thr Tyr Val Pro Gln Glu     530 535 540 tct ttt att atg agt gga act att aaa gac aat tta ttt tta ggt tta 1680 Ser Phe Ile Met Ser Gly Thr Ile Lys Asp Asn Leu Phe Leu Gly Leu 545 550 555 560 gaa agt att cct gat gaa caa gaa ctc gaa aaa gta ctg aaa gat act 1728 Glu Ser Ile Pro Asp Glu Gln Glu Leu Glu Lys Val Leu Lys Asp Thr                 565 570 575 tgt tta tcg agt tat att act gcg ctt cct cta gga ctt gat acg tat 1776 Cys Leu Ser Ser Tyr Ile Thr Ala Leu Pro Leu Gly Leu Asp Thr Tyr             580 585 590 cta gag gaa aat ggt gcg aat tta tca ggt ggt caa aag caa aga att 1824 Leu Glu Glu Asn Gly Ala Asn Leu Ser Gly Gly Gln Lys Gln Arg Ile         595 600 605 gct tta gca aga gtt tta tta tca gga agt aaa att tta tta tta gat 1872 Ala Leu Ala Arg Val Leu Leu Ser Gly Ser Lys Ile Leu Leu Leu Asp     610 615 620 gaa gct acg agt gct cta gat tct aaa act gaa atg ctg att ttt gaa 1920 Glu Ala Thr Ser Ala Leu Asp Ser Lys Thr Glu Met Leu Ile Phe Glu 625 630 635 640 aaa ata tta aag tac cct aat aag tca atc att att ata tct cat aat 1968 Lys Ile Leu Lys Tyr Pro Asn Lys Ser Ile Ile Ile Ile Ser His Asn                 645 650 655 gat aaa tta ata gac aag tgt gac ata atc att gat tta gac gaa agg 2016 Asp Lys Leu Ile Asp Lys Cys Asp Ile Ile Ile Asp Leu Asp Glu Arg             660 665 670 gat tcg taa 2025 Asp Ser         675 <210> 5 <211> 674 <212> PRT <213> Enterococcus mundtii <400> 5 Met Gln Met Ile Leu Asn Asn Phe His Ser Trp Ile Ser Val Glu Val   1 5 10 15 Leu Arg Asp Leu Thr Glu Thr Asp Ser Glu Gly Thr Cys Ala Leu Gly              20 25 30 Ile Val Asn Gly Phe Ala Lys Leu Gly Ile Asn Cys Glu Ala Tyr Lys          35 40 45 Ala Asn Ser Asp Val Trp Lys Glu Asn Glu Phe Asn Tyr Pro Val Ile      50 55 60 Ala Asn Ile Val Thr Asn Asn Gln Phe Leu His Tyr Cys Ile Val Tyr  65 70 75 80 Gly Val Lys Lys Glu Lys Leu Leu Ile Ala Asp Pro Ala Ile Gly Lys                  85 90 95 Tyr Lys Glu Ser Ile Glu Lys Phe Asn Asn Asn Trp Thr Gly Val Ile             100 105 110 Leu Val Ala Glu Lys Thr Pro Asp Phe Gln Pro Ile Asn Asn Thr Lys         115 120 125 Lys Ser Phe Phe Ser Ser Ile Ser Leu Leu Lys Asp Gln Tyr Lys Lys     130 135 140 Ile Leu Leu Val Ile Leu Ser Ser Leu Ile Ile Thr Ile Ile Gly Ile 145 150 155 160 Leu Ser Ser Tyr Tyr Phe Arg Ile Leu Ile Asp Trp Leu Leu Pro Glu                 165 170 175 Lys Asp Phe Leu Asn Leu Phe Met Ile Ser Ile Ser Tyr Ile Ile Gly             180 185 190 Ile Phe Ile Thr Ser Ile Phe Glu Ile Thr Arg Arg Tyr Asn Leu Glu         195 200 205 Lys Leu Gly Gln Asp Val Gly Arg Ser Ile Leu Phe Lys Tyr Leu Asp     210 215 220 His Ile Phe Ile Leu Pro Ala Ser Phe Phe Ser Lys Arg Lys Thr Gly 225 230 235 240 Asp Ile Val Ser Arg Phe Ser Asp Ala Asn Lys Ile Ile Glu Ala Leu                 245 250 255 Ala Ser Phe Thr Ile Ser Ile Phe Leu Asp Leu Ser Ser Val Ile Val             260 265 270 Val Gly Ile Ile Leu Ile Asn Ile Asn Lys Gln Leu Phe Leu Ile Thr         275 280 285 Leu Ser Ser Ile Pro Phe Tyr Ile Leu Ile Ile Leu Gly Ser Asn Lys     290 295 300 Lys Met Ser Arg Leu Asn Gly Glu Glu Met Gln Thr Asn Ser Ile Val 305 310 315 320 Asp Ser Asn Phe Ile Glu Gly Leu Asn Gly Ile Tyr Thr Ile Lys Ala                 325 330 335 Leu Cys Ser Glu Asn Lys Ile Val Asn Gln Ile Tyr Arg Ser Leu Asn             340 345 350 Glu Phe Phe Asp Val Ser Leu Lys Arg Asn Met Tyr Asp Ser Ile Ile         355 360 365 Gln Asn Leu Lys Ile Leu Val Ser Leu Leu Thr Ser Ala Phe Val Leu     370 375 380 Trp Phe Gly Ser Tyr Tyr Val Ile Asn Gly Glu Ile Thr Ile Gly Glu 385 390 395 400 Leu Ile Thr Phe Asn Ser Leu Ser Ile Phe Phe Ser Thr Pro Leu Gln                 405 410 415 Asn Ile Ile Asn Leu Gln Glu Lys Phe Gln Lys Ala Gln Val Ala Asn             420 425 430 Asn Arg Leu Asn Asp Val Phe Ser Ile Asn Asn Glu Asn Lys Asp Lys         435 440 445 Phe Ile His Leu Ala Lys Leu Thr Glu Lys Ala Thr Ile Thr Phe Glu     450 455 460 Asn Val Tyr Phe Ser Tyr Ser Thr Lys Tyr Pro Asn Val Leu Asp Asn 465 470 475 480 Met Ser Phe Ser Leu Pro Val Ser Lys Asn Ile Gly Val Lys Gly Asp                 485 490 495 Ser Gly Ala Gly Lys Ser Thr Leu Ala Gln Leu Leu Ala Gly Phe Tyr             500 505 510 Ser Pro Asp Asn Gly Arg Ile Cys Ile Asn Glu Gln Asn Ile Glu Asn         515 520 525 Ile Asn Arg Lys Asp Leu Arg Lys Leu Ile Thr Tyr Val Pro Gln Glu     530 535 540 Ser Phe Ile Met Ser Gly Thr Ile Lys Asp Asn Leu Phe Leu Gly Leu 545 550 555 560 Glu Ser Ile Pro Asp Glu Gln Glu Leu Glu Lys Val Leu Lys Asp Thr                 565 570 575 Cys Leu Ser Ser Tyr Ile Thr Ala Leu Pro Leu Gly Leu Asp Thr Tyr             580 585 590 Leu Glu Glu Asn Gly Ala Asn Leu Ser Gly Gly Gln Lys Gln Arg Ile         595 600 605 Ala Leu Ala Arg Val Leu Leu Ser Gly Ser Lys Ile Leu Leu Leu Asp     610 615 620 Glu Ala Thr Ser Ala Leu Asp Ser Lys Thr Glu Met Leu Ile Phe Glu 625 630 635 640 Lys Ile Leu Lys Tyr Pro Asn Lys Ser Ile Ile Ile Ile Ser His Asn                 645 650 655 Asp Lys Leu Ile Asp Lys Cys Asp Ile Ile Ile Asp Leu Asp Glu Arg             660 665 670 Asp Ser <210> 6 <211> 297 <212> DNA <213> Enterococcus mundtii <220> <221> CDS <222> (1) .. (297) <400> 6 atg agt aat tta aag tgg ttt tct ggt gga gac gat cga cgt aaa aaa 48 Met Ser Asn Leu Lys Trp Phe Ser Gly Gly Asp Asp Arg Arg Lys Lys   1 5 10 15 gca gaa gtg att att act gaa tta tta gat gat tta gag atg gat ctt 96 Ala Glu Val Ile Ile Thr Glu Leu Leu Asp Asp Leu Glu Met Asp Leu              20 25 30 gga aat gaa tct ctt cgg aaa gta tta ggc tcc tat ctt aaa aag ttg 144 Gly Asn Glu Ser Leu Arg Lys Val Leu Gly Ser Tyr Leu Lys Lys Leu          35 40 45 aaa aat gaa ggg act tca gtt ccg tta gtt tta agt cgt atg aat ata 192 Lys Asn Glu Gly Thr Ser Val Pro Leu Val Leu Ser Arg Met Asn Ile      50 55 60 gag ata tct aat gca ata aaa aaa gac ggt gta tcg tta aat gaa aat 240 Glu Ile Ser Asn Ala Ile Lys Lys Asp Gly Val Ser Leu Asn Glu Asn  65 70 75 80 caa tct aaa aaa tta aaa gaa cta atg tct ata tct aat att aga tat 288 Gln Ser Lys Lys Leu Lys Glu Leu Met Ser Ile Ser Asn Ile Arg Tyr                  85 90 95 ggt tac tag 297 Gly Tyr <210> 7 <211> 98 <212> PRT <213> Enterococcus mundtii <400> 7 Met Ser Asn Leu Lys Trp Phe Ser Gly Gly Asp Asp Arg Arg Lys Lys   1 5 10 15 Ala Glu Val Ile Ile Thr Glu Leu Leu Asp Asp Leu Glu Met Asp Leu              20 25 30 Gly Asn Glu Ser Leu Arg Lys Val Leu Gly Ser Tyr Leu Lys Lys Leu          35 40 45 Lys Asn Glu Gly Thr Ser Val Pro Leu Val Leu Ser Arg Met Asn Ile      50 55 60 Glu Ile Ser Asn Ala Ile Lys Lys Asp Gly Val Ser Leu Asn Glu Asn  65 70 75 80 Gln Ser Lys Lys Leu Lys Glu Leu Met Ser Ile Ser Asn Ile Arg Tyr                  85 90 95 Gly Tyr

[Brief description of drawings]

FIG. 1 shows the growth of Enterococcus munditi strain 7393 at 37 ° C. and the amount of munditicin KS produced.

FIG. 2 is a diagram showing the effect of culture temperature on the amount of munditicin KS produced.

FIG. 3 SDS-PAGE of Munditishin KS
It is a figure which shows the result of (A) and activity dyeing (B).

FIG. 4 is a diagram showing a comparison between the amino acid sequence of munditicin KS and other bacteriocins.

FIG. 5 is a diagram showing the thermal stability of Munditicin KS.

FIG. 6 is a graph showing the growth inhibitory effect of Munditicin KS on food spoilage and spoilage bacteria.

FIG. 7 is a graph showing the growth inhibitory effect of Munditicin KS on Listeria monocytogenes.

FIG. 8 is a graph showing the bactericidal effect of Enterococcus munditi strain 7393 against Listeria.

FIG. 9 shows the bactericidal effect of Lactobacillus salmon against Listeria.

FIG. 10 is a diagram showing a difference in antibacterial spectrum between recombinant munditicin KS and ATO6.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12P 21/02 A23B 4/00 J (72) Inventor Chise Suzuki 667-7 Ino, Toride-shi, Ibaraki (72) Inventor Sadahiro Ohmo 953-502 off Arakawa, Ami-cho, Inashiki-gun, Ibaraki Prefecture (72) Inventor Nahoko Horikoshi 635 Nakamuhara, Tsuchiura City, Ibaraki Prefecture Primaham Co., Ltd. (72) Junko Shito 635 Nakamukaihara, Tsuchiura City, Ibaraki Prefecture Prima Ham (72) Inventor Kazuko Takeshita 635 Nakamuhara, Tsuchiura City, Ibaraki Prefecture Primaham Co., Ltd. (72) Inventor Takashi Samejima 635 Nakamuhara, Tsuchiura City, Ibaraki Prefecture F Term (reference) 4B021 MC01 MK06 MK23 4B024 AA05 CA02 CA04 GA19 HA03 4B064 AG30 CA02 DA10 4B065 AA01 AC14 CA34 CA42 4H045 AA10 AA20 AA30 BA10 CA11 DA83 EA01 FA73

Claims (18)

[Claims] 1. A DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 or its complementary sequence, or a sequence containing a part or all of these sequences. 2. A DNA comprising the nucleotide sequence shown in SEQ ID NO: 2 or its complementary sequence. 3. A DNA encoding the following protein (a) or (b). (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 5 (b) consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5, and comprising bacteriocin Protein with extracellular efflux activity 4. A DNA consisting of the nucleotide sequence shown in SEQ ID NO: 4 or its complementary sequence, or a sequence containing a part or all of these sequences. 5. A DNA encoding the following protein (a) or (b): (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 7 (b) an amino acid sequence represented by SEQ ID NO: 7 in which one or several amino acids are deleted, substituted or added, and a bacteriocin Protein with resistance function 6. A DNA comprising the nucleotide sequence shown in SEQ ID NO: 6 or its complementary sequence, or a sequence containing a part or all of these sequences. 7. Munditicin K having bacteriocin activity, which comprises the amino acid sequence shown in SEQ ID NO: 3.
S. 8. A protein comprising the amino acid sequence shown in SEQ ID NO: 5. 9. A protein having an amino acid sequence represented by SEQ ID NO: 5 in which one or several amino acids have been deleted, substituted or added, and which has bacteriocin extracellular efflux activity. 10. A protein comprising the amino acid sequence shown in SEQ ID NO: 7. 11. A protein having an amino acid sequence represented by SEQ ID NO: 7 in which one or several amino acids are deleted, substituted or added, and having a bacteriocin resistance function. 12. A method for producing munditicin KS, which comprises culturing a munditicin KS-producing bacterium belonging to the genus Enterococcus and collecting munditicin KS from the culture. 13. A Munditicin KS-producing strain is Enterococcus munditi strain 7393 (FERM-P1).
8300), The method for producing munditicin KS according to claim 12. 14. Enterococcus munditi strain 7393 strain (FER which has the ability to produce Munditicin KS)
M-P18300). 15. A method for producing a food product having excellent preservability, which comprises adding munditicin KS, a microorganism having munditicin KS-producing ability, or a culture solution thereof to a food product. 16. The preservative according to claim 15, wherein the microorganism having the ability to produce munditicin KS is a lactic acid bacterium capable of killing or reducing Listeria monocytogenes present in foods. Excellent food manufacturing method. 17. The method for producing a food product having excellent preservability according to claim 16, wherein the lactic acid bacterium is Enterococcus munditi strain 7393 strain (FERM-P18300). 18. The method for producing a food having excellent preservability according to claim 15, wherein the food is non-heated meat.