KR100662719B1 - Antibacterial substance produced by lactic acid bacterium - Google Patents

Abstract

Ile-Thr-Ser-Ile-Ser-Leu-Cys-Thr-Pro-Gly-Cys-Lys-Thr-Gly-Val-Leu-Met-Gly-Cys-Asn-Leu A novel antimicrobial peptide (bacteriocin) having a primary structure consisting of the amino acid sequence of -Lys-Thr-Ala-Thr-Cys-Asn-Cys-Ser-Val-His-Val-Ser-Lys. The antimicrobial material of the present invention is produced by the novel Lactococcus lactis strain 61-14 (FERM BP-08492).

Antimicrobial peptide

Description

Antibacterial substance produced from lactic acid bacteria {ANTIBACTERIAL SUBSTANCE PRODUCED BY LACTIC ACID BACTERIUM}

The present invention relates to an antimicrobial substance that can be used for the purpose of food preservation and the like, and more particularly to a novel lactic acid strain and a novel antimicrobial peptide produced by the strain.

As an antimicrobial substance which lactic acid bacteria produce, low molecules, such as organic acids, such as lactic acid, hydrogen peroxide, and diacetyl, are known. In addition, the presence of an antimicrobial substance consisting of a peptide is also known, for example, Niacin A produced by Lactococcus lactis (J. Am. Chem. Soc. 1971 Sep 8; 93 (18): 4634-5) , Nisin Z (Eur. J. Biochem. 1991 Nov 1; 201 (3): 581-4) and the like. However, the nisin are reported to have only Nisin A and Z present in nature.

The use of antimicrobial substances produced by lactic acid bacteria to prevent deterioration and decay of food is an important technique in that it provides safe food by preserving food without deteriorating its quality. The antimicrobial substance can be said to be a fight against resistant bacteria. In other words, the development of novel antimicrobial substances capable of effectively suppressing the proliferation of a wide variety of species appearing according to various kinds of foods has been constantly required.

An object of the present invention is to provide a new antimicrobial substance useful for food preservation and the like, which has a different antimicrobial spectrum (difference in antimicrobial activity in a plurality of species which are multiplicatively inhibited) from conventionally known antimicrobial agents.

Disclosure of the Invention

The present inventor result of extensive research in view of the anti-microbial substance for lactic acid production, Bacillus (Bacillus), A micro Rhodococcus (Micrococcus) genus Listeria (Listeria), A Phedi O Rhodococcus (Pediococcus) genus Enterococcus (Enterococcus) , a (with other words, inhibitory activity against susceptible gram-positive bacteria) Lactococcus (Lactococcus) genus Lactobacillus bacteria (Lactobacillus), a flow Pocono stock having, antibacterial with respect to the cause of microorganisms in the food deterioration belonging to the genus (Leuconostoc) substance Lactococcus lactis , a novel lactic acid strain producing Lactococcus lactis , was discovered, and the present invention was completed by performing purification and identification of an antimicrobial substance (peptide), primary structure analysis and higher order structure estimation.

Accordingly, the present invention provides an antimicrobial peptide having a primary structure of the following amino acid sequence (SEQ ID NO: 1):

In addition, the present invention provides a peptide consisting of the following amino acid sequence (SEQ ID NO: 2) as a propeptide of the antimicrobial peptide:

The present invention also provides a DNA encoding each of the peptides. In particular, a preferred example of the DNA encoding the precursor peptide consists of the following nucleotide sequence (SEQ ID NO: 4):

The present invention also provides Lactococcus lactis , strain 61-14 (FERM BP-08492), which produces the antimicrobial peptide.

The present invention also provides a food preservative comprising the antimicrobial peptide as an active ingredient.

To practice the invention  Best form for

The antimicrobial agent according to the present invention is a novel peptide (bacteriocin) produced by Lactococcus lactis strains newly isolated from river water, and is a new type of amino acid sequence composed of different amino acid sequences from known Nisin A or Nisin Z. It's nisin.

Fig. 1 shows the primary amino acid sequence of the antimicrobial peptide of the present invention (nicin Q) in comparison with the conventional amino acid sequence of Nisin Z.

Fig. 2 shows an example of the experimental results conducted to investigate the applicability of the antimicrobial peptide (Nicine Q) of the present invention as a food preservative.

Hereinafter, embodiments of the present invention will be described in detail according to isolation of novel strains, characterization, production of antimicrobial peptides from the strains, determination of DNA sequences encoding the peptides, and preservation tests for foods.

(1) How to separate lactic acid bacteria

In order to investigate the wild type of lactic acid bacteria, various river water was used as a separation source and inoculated in GYP white agar medium to plate culture. Thereafter, screening was performed using the antibacterial activity as an index. That is, after growth of colonies in MRS medium, Bacillus subtilis IAM12118 was stacked on the upper layer, and the strain showing inhibition of growth inhibited in the upper layer was isolated.

(2) analysis of strain properties

As a result of screening, strains having excellent antimicrobial properties were found. The strain is isolated from river water recovered from the East Bridge of the Shikosan River basin in Tagawa, Fukuoka Prefecture. Gram staining, catalase test, motility test, cell morphology observation, sugar fermentability test, production lactic acid yield, production lactic acid photosensitivity, gas evolution test from glucose were examined. Characteristics of the strains included familial anaerobic, gram positive, catalase negative, motility, and the cell morphology was elliptic, L-lactic acid-producing bacteria and homo-fermenting bacteria. D-xylose magnetization, lactose magnetization, and sucrose magnetization were also present. As a result of 59 kinds of sugar magnetization test (API system) including these, Lactococcus lactis was identified and named 61-14 strain. The 61-14 strains were deposited in the Patent Biological Deposit Center and received FERM BP-08492 (FERM P-18994: September 4, 2002).

(3) antimicrobial test

In order to examine whether the antimicrobial material was produced in liquid medium by the 61-14 strain, the cells were cultured in an MRS liquid medium for 16 hours, centrifuged, the supernatant was collected, adjusted to pH 6, and filtered and sterilized. Two-fold dilutions were used after testing. 10 μL of the step dilutions were added to the agar medium inoculated with the indicator bacteria. The antimicrobial activity was X x 1000/10 (A.U./ml), where X was the magnification at which clear inhibitory rings were observed. The test results are shown in Table 1 compared to the case with Nisin Z. As shown in Table 1, the antimicrobial activity pattern of the antimicrobial material produced by the strain 61-14 is clearly different from that of Nisin Z. In addition, the antimicrobial substance (antibacterial peptide) produced by the 61-14 strain (FERM BP-08492) of this invention is called niacin Q hereafter.

Table 1

Antibacterial activity measurement

Surface bacteria Nisin Q (A.U./ml) Nisin Z (A.U./ml) Bacillus coagullans JCM2257 ^T 102,400 102,400 Bacillus subtilis JCM2504 ^T 3,200 6,400 Bacillus supulans JCM2154 ^T 3,200 3,200 Microcaucus Luteus IF012708 200 400 Listeria Inokua ATCC33090 ^T 400 1,600 Pediococcus pentosaceus JCM5885 200 400 Enterococcus faecalis JCM5803 ^T 200 400 Lactococcus lactis subs. Lactis ATCC19435 ^T 400 800 Lactococcus lactis subs.cremoris TUA1344L 400 400 Lactobacillus plantarum atcc14917 ^t 200 800 Lactobacillus sakei cec.Sakkei JCM1157 ^T 25,600 ≥204,800 Lucono Stock Mesenteroides subs.Mesenteroides JCM6124 ^T 12,800 3,200

(4) Production and Purification Methods of Antimicrobial Substances

The strain 61-14 was precultured, and 8 ml of the preculture was inoculated into 1 L of MRS (oxoid) liquid medium, and cultured at 30 ° C. for 18 hours, and the supernatant was recovered by centrifugation. 1 L of culture supernatant could be recovered and its activity was 1.28 × 10 ⁴ (AU / ml). 20 g of Amberlite XAD-16 (Sigma) sufficiently immersed in 2-propanol was added to the supernatant, followed by shaking for 3 hours to adsorb the supernatant.

It was packed into a column (200 ml capacity) and eluted with 100 ml of H ₂ O, 100 ml of 40% ethanol, 100 ml of 70% 2-propanol (0.1% TFA) and 50 ml of 100% 2-propanol. The strongest detected fraction was 100 ml of 70% 2-propanol (0.1% TFA) and the eluting fraction had an activity of 1.64 × 10 ⁶ (AU / ml). The fractions were concentrated on a speed vac evaporator and adjusted to a final volume of 50 ml with 20 mM sodium phosphate buffer. SP-Sepharose Cation Exchange Chromatography Resin (Pharmacia) was charged to a column (16 ml volume), 50 ml of concentrated sample was injected and equilibrated with 20 mM sodium phosphate buffer. Elution was carried out at a flow rate of 100 ml / hour using sodium phosphate buffer containing 0.25, 0.5, 0.75, 0.1 M NaCl.

The antimicrobial activity of each eluting fraction was measured and the activity of the strongest 0.25 M NaCl eluting fraction was 2.62 × 10 ⁵ (AU / ml). 10 ml in 50 ml total elution was injected into reverse phase HPLC (column: Pharmacia Pep RPC HR 5/5). As the mobile phase, acetonitrile was used at a concentration gradient (straight gradient 0-100%) at a flow rate of 1 ml / min, and 30 fractions were obtained at 1 ml per fraction. UV (UV 220, 280 nm) was used as a detector. The antimicrobial activity of the 30 fraction samples was measured, and the fractions with elution time of 12 minutes to 13 minutes with strong antimicrobial activity were concentrated, and reverse phase HPLC was performed again under the same conditions. A fraction with a dissolution time of 9-11 minutes, which appeared as a single peak and had strong antibacterial activity, was obtained. In addition, Bacillus coagulans JCM2257 ^T was used as an indicator of antimicrobial activity.

(5) N-terminal amino acid sequence determination

The antimicrobial substance obtained as described above was analyzed for the N terminus on an amino acid sequencer (SHIMADZU PSQ-1U protein sequencer) using Adman degradation. As a result, Ile was identified as the N terminus of the peptide sequence, but no amino acid was analyzed. The second amino acid at the N-terminal was Thr as the primary structure, but was modified with a special amino acid.

(6) DNA sequencing

Isolate FERM BP-08492 ( Lactococcus lactis ) was used to extract whole genomic DNA using MagExtractor-Genome- (TOYOBO). The concentration was 60.5 ng / μL. Subsequently, polymerase chain reaction (PCR) was performed, wherein the PCR primers used were designed based on the surrounding nucleotide sequences of the Nisin A and Nisin Z precursor structural genes, and consisted of the following deoxyoligonucleotide sequences: cgt tcg aag aaa cta caa aat aaa tt (SEQ ID NO: 5) and cca tgt ctg aac taa caa aat act at (SEQ ID NO: 6). PCR was performed using Premix TaqDNA polymerase (Ex-Taq TAKARA). The stretch reaction was performed 30 times at 94 ° C 30 seconds, 51 ° C 30 seconds, and 72 ° C 60 seconds.

Thereafter, TA cloning was performed using a pUC 18 derived T vector. NA ligation kit Ver. 2 (TAKARA) was used for ligation of the PCR amplification fragment and the T vector. The ligated product was used to transform E. coli JM109 by electroporation. The transformants were incubated for 8 hours in LB medium and plasmids were extracted using MagExtractor-Plasmid- (TOYOBO). The plasmid was used as a template for the sequencing reaction. A sequencing thermo sequence Fluorescent Label labeled primer cycle sequencing kit (Pharmacia) was used. The sequencing reaction was performed at 95 ° C. for 5 minutes and then 15 times at 95 ° C., 30 seconds, 55 ° C., 30 seconds, and 72 ° C. for 1 minute, and 15 times at 95 ° C., 30 seconds, 72 ° C., and 1 minute. It was.

As a result, the nucleotide sequence (nucleotide sequence) of SEQ ID NO: 4 was confirmed. Compared with the gene encoding the nisin Z precursor peptide, T of C18 base, G of A base of 24, G of A of base 42, G of A of base 45, 47 Base A is C, Base 54 is A T, Base 57 is T C, Base 60 is A T, Base 64 is C A, Base 69 is C To T, A to C of base 75, T to C of base 78, A to T of base 87, C to A of base 96, A to T of base 111, 113 Base C was T, base 123 T was A, base 130 A was C, base 156 T was C, and base 157 A was G.

Base difference that affects peptide sequence compared to Nisin Z, with amino acid residues -8th Lys due to base 47 difference and Thrs and amino acid residues -2nd Pro due to difference 64 bases Thr Thus, Ala of the 15th amino acid residue due to the difference of base 113 is Val, 21st Met of the amino acid residue 21 due to the difference of base 130 is Leu, and Ile of the 30th amino acid residue of the amino acid residue due to the difference of base 157 Val (see FIG. 1). In Fig. 1, it is indicated by Nisin Q but it is the amino acid sequence of the propeptide of the antimicrobial peptide produced from FERM P-18994 of the present invention (SEQ ID NO: 2), among which the C terminus starts from I (isoleucine: Ile). The part consisting of 34 amino acid residues of the side is the amino acid sequence of the primary structure of the peptide which finally functions as an antimicrobial peptide (SEQ ID NO: 1), and becomes the amino acid sequence of the leader peptide which is the N-terminal squared part. As described above, the antimicrobial peptide (nicin Q) of the present invention is a substance completely different from the existing nicin Z.

Incidentally, the encoding of nisin Q (nisin Q precursor peptide) is not limited to the DNA sequence of SEQ ID NO: 4, and considering the degeneracy of the codon, the nisin Q consisting of the amino acid sequence of SEQ ID NO: 2 is selected. Includes all DNA sequences that can be encoded.

(7) Estimation of higher order structure by MALDI - TOF MS analysis

Compared with Nisin Z, since the DNA sequence of Nisin Q is similar and the special structure is near the N terminus, Nisin Q is likely to be similar to the higher order structure of Nisin Z. Thus, the higher order structure of nisin Q was estimated as follows. That is, the following amino acid sequence:

It has a primary structure consisting of, threonine in the second position is modified to be 2, 3-didehydrobutyrin, serine in the 5, 33rd position is modified to be 2, 3-didehydroalanine, respectively, 3rd position Serine is modified to be alanine, and the 8th, 13, 23, and 25th threonine are modified to be 2-aminobutyric acid (Abu), and at the same time, the 3rd and 7th bases are Ala-S-Cys Lanthionine is formed by ether bonding, 8th and 11th bases are thioether bonded with Abu-S-Cys to form 3-methyllanthionine, and 13th and 19th bases are Abu-S-Cys. Thioether bonds to form 3-methyllanthionine, the 23rd and 26th bases are thioether bonded to Abu-S-Cys to form 3-methyllanthionine, and the 25th and 28th bases are Abu Thioether bonds with -S-Cys to form 3-methyllanthionine (SEQ ID NO: 3).

Theoretically calculating the molecular weight from the amino acid sequence of SEQ ID NO: 3 estimated as described above is 3327.43. Thus, the molecular weight was measured using MALDI-TOF MS (PE Biosystems Voyager System 4025) that can measure the exact molecular weight. Since a fluoroton is added to a measurement sample and it measures, it becomes a measured value which increased 1 Da more than an actual molecular weight. As a result of the measurement, two peaks consisting of a peak of 3345.890 Da corresponding to the substance in which methionine contained in Nisin Q was oxidized and a peak of 3328.50 Da considered to correspond to the molecular weight measurement value of Nisin Q were observed. The said molecular weight measured value was substantially in agreement with the theoretical value within 0.1% of theoretical value 3327.43 Da + 1 Da = 3328.43 Da. The results suggest that Nisin Q is an estimated structure.

(8) Applicability Test as Food Preservative

In order to investigate the applicability of the antimicrobial peptide (nicin Q) of the present invention as a food preservative, the antimicrobial activity when retort sterilization treatment (110 ° C., 10 minutes) was performed at pH 4-7, which can be assumed as a pH range in food. It was confirmed.

Nisin Q Adjusting Agent

Lactococcus lactis 61-14 strain (FERM BP-08492) was incubated for 24 hours at 30 ℃ in 10ml of MRS (Difco) medium. The culture solution was inoculated in 100 ml of MRS medium (containing 2% calcium carbonate) and incubated at 30 ° C for 24 hours. The culture solution was inoculated in 1 L of MRS medium (containing 2% calcium carbonate), incubated for 16 hours, and then centrifuged to recover the supernatant. 20 g of Amberlite XAD-16 (Sigma) (which was swollen sufficiently with 2-propanol and then washed with distilled water) was added to the supernatant, followed by deposition for 16 hours to adsorb. It was packed into a column, washed with 700 ml of distilled water and 500 ml of 40% ethanol and then eluted with 100% 2-propanol. The eluted fractions were dried in a rotary evaporator. The dried material was placed in 50 ml of Britton-Robinson wide area buffer (pH 3.94) and dissolved at 4 ° C. for 16 hours. This was used as a stock solution.

<Antibacterial Test Method>

Lactobacillus four K seopseu as indicator bacteria. Kay Inc. JCM1157 ^T (Lactobacillus sakei subsp. Sakei JCM1157 ^T ) was used. Samples were added to the indicator bacteria and then inoculated in MRS medium at a final concentration of 4 × 10 ⁵ cells / ml, and cultured for 24 hours at 30 ° C. on 96 well plates (nunc), and then absorbance was measured at 630 nm. When the growth of the indicator bacteria was observed, the absorbance increased.

Nisin Q adjuster solution was adjusted to each pH and autosterilized at 110 ° C for 10 minutes. Each pH sample was diluted twice in fold and then tested for antimicrobial activity at each dilution step.

<Result>

The test results are shown in FIG. At each pH, it was examined under the same conditions as the retort sterilization conditions. Since dilution had an anti-proliferative effect, it was confirmed that it could be used in food materials accompanied with a retort-like heat treatment and also in non-heated food materials.

(9) Food Preservation Test

The effect of the antimicrobial peptide (Nisin Q) of this invention was examined about rice. MRS medium (manufactured by DIFCO Co., Ltd.) was sterilized at 121 ° C. for 15 minutes, inoculated with 61-14 weeks (FERM BP-08492), incubated at 37 ° C. for 15 hours, and prepared by removing and lyophilizing the cells. As a comparative group, an improving agent, a vinegar preparation, and a lactic acid for commercially available oils and fats, which are widely used at low cost, were used.

After washing with water and absorbing rice, rice seeded Bacillus subtilis spores were inoculated at 100 / g and cooked by adding nisin Q, vinegar preparation and lactic acid according to Table 2. After cooking, the mixture was cooled and then placed in a sterile cup and subjected to a preservation test at 30 ° C. for 48 hours. For each sample, normal viable counts were measured using a standard agar medium (produced by Youngyeon Chemical Co., Ltd.). The results are shown in Table 3.

TABLE 2

Sample 1 Sample 2 Sample 3 Nisin Q 0.3% Heritage 0.5% Vinegar 0.5%

TABLE 3

Sample 1 Sample 2 Sample 3 No addition 100 500 240000 1000000

(Pcs / g)

By adding nisin Q, there was an improvement effect of maintaining good freshness. In addition, as a result of sensory evaluation, the nisin Q-added group had better flavor and taste than the lactic acid and vinegar added groups. From this, it became clear that Nisin Q has an effect of improving shelf life without affecting food quality.

The present invention provides novel antimicrobial materials useful as food preservatives and the like.

Attach sequence list electronic file  

Claims (7)

An antimicrobial peptide characterized by having a primary structure of the following amino acid sequence (SEQ ID NO: 1):

The peptide of claim 1, wherein the propeptide of the antimicrobial peptide consists of an amino acid sequence (SEQ ID NO: 2):

DNA encoding the peptide of claim 1. DNA encoding the peptide of claim 2. The DNA of claim 4, wherein the DNA consists of the following nucleotide sequence (SEQ ID NO: 3):

The Lactococcus lactis to produce antimicrobial peptides paragraph 1 (Lactococcus lactis ) 61-14 strain (FERM BP-08492). A food preservative comprising the antimicrobial peptide of claim 1 as an active ingredient.

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