WO2004029082A1

WO2004029082A1 - Antibacterial substance produced by lactic acid bacterium

Info

Publication number: WO2004029082A1
Application number: PCT/JP2003/012123
Authority: WO
Inventors: Kyoko Kawano; Tomoko Higuchi; Kenji Sonomoto; Jiro Nakayama; Takeshi Zendo; Masanori Fukao
Original assignee: Fukuoka Prefectural Government; Omu Milk Products Co., Ltd.; Kumamoto Flour Milling Co., Ltd.
Priority date: 2002-09-25
Filing date: 2003-09-24
Publication date: 2004-04-08
Also published as: KR100662719B1; AU2003275532A1; KR20050057594A; JP3740499B2; CN1684976A; JPWO2004029082A1

Abstract

A novel antibacterial peptide (a bacteriocin) having a primary structure comprising the amino acid sequence Ile-Thr-Ser-Ile-Ser-Leu-Cys-Thr-Pro-Gly-Cys-Lys-Thr-Gly-Val-Leu-Met-Gly-Cys-Asn-Leu-Lys-Thr-Ala-Thr-Cys-Asn-Cys-Ser-Val-His-Val-Ser-Lys which is useful in preserving foods, etc. This substance is produced by a novel Lcatococcus lactis strain 61-14 (FERM BP-08492).

Description

Specification

Antibacterial substances produced by lactic acid bacteria

The present invention belongs to the technical field of antibacterial substances that can be used for purposes such as preservation of foods, and particularly relates to a novel lactic acid bacterium strain and a novel antibacterial peptide produced by the strain. Background art

As antibacterial substances produced by lactic acid bacteria, organic acids such as lactic acid, and low molecules such as hydrogen peroxide and diacetyl are already known. In addition, the existence of antibacterial substances composed of peptides is also known.For example, nisin A (J. Am. Chem. Soc. 1971 Sep 8; 93 (18)) produced by Lactococcus 1-actis : 4634-5), Nisin Z (Eur. J. Biochem. 1991 Nov 1; 201 (3): 581-4). However, only nisins A and Z have been reported to exist in nature.

The use of antibacterial substances produced by lactic acid bacteria to prevent food spoilage and spoilage is an important technology from the viewpoint of preserving food without deterioration in quality and providing safe food. Such antibacterial substances can be said to be a fight against resistant bacteria. In other words, there is a constant need for the development of new antibacterial substances that can effectively suppress the growth of a wide variety of bacterial species that appear in response to diversifying food types.

An object of the present invention is to provide a new antibacterial agent useful for preserving foods, etc., which has a different antibacterial spectrum (difference in antibacterial activity against multiple bacterial strains to be inhibited) from conventionally known antibacterial agents. To provide a substance. Ming disclosure

The present inventor focused on antibacterial substances produced by lactic acid bacteria, and as a result of repeated research, found that the genus Bacillus Bacillus, Micrococcus, Mici-ococcus, genus, Listeria. Enterococcus, Entei-ococcus, genus, genus Ratococcus (Lactococcus), genus Lactobacillus (Lactobacillus), and genus Leuconostoc (euconostoc), which have antimicrobial activity against microorganisms that cause food deterioration (that is, susceptible gram-positive bacteria) Lactococcus lactis, a novel lactic acid bacterium strain that produces a substance (having inhibitory activity against lactococcus), purification and identification of the antibacterial substance (peptide), elucidation of its primary structure, and higher-order structure Was estimated to derive the present invention.

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

1 5 10 1 5

li e Thr Ser He e Ser Leu Cys Thr Pro Gly Cys Lys Thr Gly Val Valu

20 25 30 34

Met Gly Cys Asn Leu Lys Thr Al a Thr Cys Asn Cys Ser Val Hi s Val Ser Lys

Further, according to the present invention, there is provided a precursor peptide of the above antibacterial peptide, which peptide has the following amino acid sequence (SEQ ID NO: 2): -20 -1 5-10

Met Ser Thr Lys Asp Phe Asn Leu Asp Leu Val Ser Val Ser Lys Thr

-5 -1 1 5

Asp Ser Gly y Al a Ser Thr Arg l i e Thr Ser lie Ser Leu Cys Thr Pro

10 15 20 25

Gly Cys Lys Thr Gly Val Leu Met Gly Cys Asn Leu Lys Thr Al a Thr

30

Cys Asn Cys Ser Val His Val Ser Lys According to the present invention, there is provided DNA encoding each of the above peptides. In particular, a preferred example of a DNA encoding the above-mentioned precursor peptide consists of the following nucleotide sequence (SEQ ID NO: 4):

atg agt aca aaa gat ttc aac tta gat ttg gta tct gtt tea aaa aca gat tct ggc get 60 tea aca cgt att acc age att tcg ctt tgt aca cca ggt tgt aaa aca ggt gtt ctg atg 120 gga tgt aac ctg aaa aca gca tgt aat tgt age gtt cac gta age aaa taa 174

Further, according to the present invention, there is provided a novel strain of Lactococcus lactis producing the above-mentioned antimicrobial peptide, ie, a 61-14 strain (FERM BP-08492).

The present invention further provides a food preservative containing the above antibacterial peptide as an active ingredient. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the primary amino acid sequence of the antimicrobial peptide (Nisin Q) of the present invention. This is shown in comparison with the known primary amino acid sequence of nisin Z.

FIG. 2 shows an example of the results of an experiment conducted to investigate that the antimicrobial peptide (Nisin Q) of the present invention can be used as a food preservative. BEST MODE FOR CARRYING OUT THE INVENTION

The antibacterial substance obtained by the present invention is a novel peptide (bacteriocin) produced by a ratatococcus ratatis strain newly isolated from river water. Is considered to be a new type of nisin composed of different amino acid sequences.

The practice of the present invention will now be described in accordance with isolation of a new strain and analysis of its characteristics, production of an antibacterial peptide from the strain, measurement of a DNA sequence encoding the peptide, and a preservation test for food. The form will be described in detail.

(1) Lactic acid bacteria isolation method

In order to examine natural wild strains of lactic acid bacteria, pour plate culture was carried out on GYP chalk agar medium using various river waters as isolation sources. Next, screening was performed using the antibacterial activity as an index. That is, after growing the colony in the MRS medium, the upper layer was covered with Bacillus subtilis (Bacillus subtilis) IAM12118, and a strain in which the inhibition zone in which growth was suppressed was observed in the upper layer was isolated.

(2) Characterization of isolated strains

As a result of screening, a strain having excellent antibacterial properties was discovered. This strain was isolated from the river water collected at Higashi-Ohashi Bridge in the Hikoyama River basin of Tagawa 巿, Fukuoka Prefecture. Gram stain, catalase test, motility test, cell morphology observation, sugar fermentation test, lactic acid production, lactic optical rotation, and gas generation test from glucose were investigated. The characteristics of the strain were facultative aerobic, gram-positive, catalase-negative, non-motile, and the cell morphology was elliptical, L-lactic acid-producing bacteria, and homozygous fermenting bacteria. Also, D-xylo There were utilization of sucrose, utilization of ratatose, and utilization of sucrose. As a result of 59 kinds of sugar utilization tests (API system) including these, ratatococcus.

It was identified as {Lactococcus lac tis) and named 61-14 strain. The 61-14 strain has been deposited with the Patent Organism Depositary, and has the accession number FERM BP-08492 (FERM P-18994: original date of deposit: September 4, 2002).

(3) Antibacterial test

To investigate whether the antimicrobial substance was produced in the liquid medium by the strain 61-14, the cells were cultured in MRS liquid medium for 16 hours, centrifuged, the supernatant was collected, and the pH was adjusted to 6. After that, it was sterilized by filtration. Two-fold serial dilutions were performed and used for testing. A serial dilution of 10 μ 希釈 was placed on the agar medium inoculated with the indicator bacteria. The antimicrobial activity was defined as XX 1000/10 (AU / ml), where X is the magnification at which a clear inhibition circle was observed. The test results are shown in Table 1 in comparison with the case using Nisin Z. As shown in Table 1, the pattern of antimicrobial activity of the antimicrobial substances produced by the 61-14 strain is clearly different from that of Nisin Z. In addition, the antibacterial substance (antibacterial peptide) produced by the 61-14 strain (FERM BP-08492) according to the present invention is referred to herein as nisin Q.

Antibacterial activity measurement

(4) Production and purification method of antibacterial substance

Preculture the isolates 61-14, inoculate 8 ml of the preculture into 1 L of MRS (oxoid) liquid medium, culture at 30 ° C for 18 hours, centrifuge, Qing was recovered. 1 L of the culture supernatant was recovered, and the 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, and the mixture was shaken for 3 hours to adsorb the supernatant.

This was loaded onto a column (200 ml _{volume), H 2 0 100 ml,} 40% ethanol 100 ml, 70% 2-propanol 100 ml (TFA 0. 1%) , and eluted with 100% 2-propanol 50 ml. The fraction in which the activity was most strongly detected was 100% of 70% 2-propanol (0.1% of TFA), and the activity of the eluted fraction was 1.64 × 10 ⁶ (AU / ml). This fraction was concentrated on a speedback evaporator, and the final total volume was adjusted to 50 ml with 20 mM sodium phosphate buffer. SP—Sepharose cation exchange chromatography resin (Pharmacia) The column (ltol volume) was packed, 50 ml of the concentrated sample was added, and equilibrated with 20 mM sodium phosphate buffer. Elution was performed at a flow rate of 100 ml I hour using a sodium phosphate buffer containing 0.25, 0.5, 0.75, and 0.1 M NaCl.

The antibacterial activity of each eluted fraction was measured, and the activity of the 0.25 M NaCl eluted fraction, which was the strongest, was 2.62 × 10 ⁵ (AU / ml). 10 ml of the eluted total volume of 50 ml was subjected to reverse phase HPLC (column: Pharmacia P-marked RPC HR 5/5). Acetonitrile was used as the mobile phase, and fractionation was performed at a concentration gradient (linear gradient of 0 to 100%) at a flow rate of 1 ml / min at a flow rate of 1 ml / min. UV (UV 220, 280 nm) was used for the detector. The antibacterial activities of the 30 fraction samples were measured, and the fractions with elution times of 12 to 13 minutes having strong antibacterial activities were concentrated and subjected to reverse-phase HPLC again under the same conditions. A single peak and a fraction with an elution time of 9-11 minutes with strong antibacterial activity were obtained. It should be noted that the indicator bacteria of antibacterial activity, using the ^{Bacillus cos "/? S JCM2257 T} .

(5) N-terminal amino acid sequencing

The antibacterial substance obtained as described above was subjected to N-terminal analysis using an amino acid sequencer (SHMADZU PSQ-1U protein sequener) utilizing the Edman degradation method. As a result, the N-terminus of the peptide sequence was identified as lie, but the next amino acid could not be identified. The amino acid second from the N-terminus was Thr in the primary structure, but was shown to be modified to a special amino acid.

(6) DNA sequencing

The whole genome DNA was extracted using MagExtractor-Genome- (T0Y0B0) from the isolate FERM BP-08 ^ 92 (Lactococcus 1 act is). The concentration was 60.5 ng / μ1. Next, a polymerase chain reaction (PCR) reaction was performed. The PCR primers used at this time were designed based on the nucleotide sequences around the nisin A and nisin Z precursor structural genes. It consists of an oligonucleotide sequence. cgt tcg aag aaa cta caa aat aaa tt (Rooster column number 5) and cca tgt ctg aac taa caa aat act at (SEQ ID NO: 6). PCR was performed using Premix TaqONA polymerase (Ex-Taq TAKARA). The extension reaction was performed at 94 ° C for 30 seconds, 51 ° C for 30 seconds, and 72 ° C for 60 seconds for 30 cycles.

Thereafter, TA cloning was performed using a pUC18-derived T vector. DNA Ligation Kit Ver. 2 (TAKARA) was used for ligation of the PCR amplified fragment and the T vector. Using this ligation product, E. coli JM109 was transformed by electroporation. After transformants were cultured in LB medium for 8 hours, plasmids were extracted using MagExtractor-Plasmid- (T0Y0B0). This plasmid was designated as type II of the sequence reaction. For the sequencing reaction, a Thrmo Sequense fluorescent label led primer cycle sequencing kit was used. The sequence reaction was incubated at 95 ° C for 5 minutes, followed by 15 cycles of 95 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 1 minute, followed by 15 cycles of 95 ° C for 30 seconds and 72 ° C for 1 minute I went.

As a result, the nucleotide sequence (nucleotide sequence) of SEQ ID NO: 4 was obtained. Compared to the gene encoding the Nisin Z precursor peptide, T at base 18 is C, G at base 24 is A, G at base 42 is A, G at base 45 is A, A of base 47 is C, A of base 54 is T, T of base 57 is C, A of base 60 is T, C of base 64 is A, C of base 69 is A At base T, base 75 A is base C, base 78 base T is base C, base 87 base A is base T, base 96 base C is base A, base 111 base A is base T, base 113 The base C was different from T, the base 123 was different from A, the base 130 was different from A, the base 156 was different from T, and the base 157 was different from G.

The difference in the nucleotide sequence that affects the peptide sequence compared to Nisin Z is that the amino acid residue _8th Lys is Thr due to the difference in base 47 and the amino acid residue-Thr 2 , 113 base difference, amino acid residue 15 Ala is Val, 130 base difference is amino acid residue 21 Met is Leu, 157 base difference is amino acid residue The 30th lie is Val (see Figure 1). In FIG. 1, it is shown as nisin Q, which is the amino acid sequence (SEQ ID NO: 2) of the precursor peptide of the antibacterial peptide produced from FERM P-18994 according to the present invention, and I (isoleucine: lie) ) Is the amino acid sequence of the primary structure of the peptide (SEQ ID NO: 1), which finally functions as an antibacterial peptide. The attached portion becomes the amino acid sequence of the leader peptide. As described above, the antimicrobial peptide (Nisin Q) of the present invention is a substance completely different from known Nisin Z.

The coding of nisin Q (nisin Q precursor peptide) is not limited to the DNA sequence of SEQ ID NO: 4, and DNA that can encode nisin Q consisting of the amino acid sequence of SEQ ID NO: 2 in consideration of codon degeneracy. All sequences are included.

(7) Estimation of higher-order structure by MALDI-T0F MS analysis

Compared with nisin Z, the similar DNA sequence of nisin Q and the special structure around the N-terminal suggest that nisin Q may be similar to the higher-order structure of nisin Z Is done. Therefore, the higher-order structure of nisin Q was estimated as follows. That is, the following amino acid sequence:

1 5 10 1 5

li e Thr Ser li e Ser Leu Cys Thr Pro Gly Cys Lys Thr 61 y Val Leu

20 25 30 34

Met Gly Cys Asn Leu Lys Thr Ala Thr Cys Asn Cys Ser ValHis Has a primary structure consisting of Val Ser Lys. Serine is modified to 2,3-didehydroalanine, respectively, and serine at position 3 is modified to alanine, and threonine at positions 8, 13, 23, 25 is modified. Are modified to 2-aminobutyric acid (Abu), respectively. Cys thioether bond to form 3-methyllantonin, position 13 and position 19 form Abu-S-Cys thioether bond to form 3-methyllantonin, position ²³ and position 26 The thioether bond of Abu-S-Cys forms 3-methyllantonin, and positions 25 and 28 form the thioether bond of Abu-S-Cys to form 3-methyllantonin (SEQ ID NO: 3).

The theoretical molecular weight was calculated from the amino acid sequence of SEQ ID NO: 3 estimated as described above, and the result was 33327.43. Therefore, the molecular weight was measured using MALDI-TOF MS (PE Biosystems Voyager System 4025), which can accurately measure the molecular weight. Since the measured sample is measured by adding a proton, the measured value is 1 Da. Higher than the actual molecular weight. The measurement results, from the peak of Mechio two emissions corresponding to the oxidant 3345. 890 Da, with the peak of 33 ² 8. 50 Da considered to correspond to definite measuring the molecular weight of nisin Q contained in the nisin Q Two peaks were observed. The measured value of the molecular weight was within the range of 0.1% to the theoretical value of 3327.43 Da + 1 Da = 3328.43 Da, which was almost consistent with the theoretical value. These results suggested that nisin Q had the predicted structure.

(8) Applicability test as food preservative

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

Kunisin Q crude purification>

Ratatococcus lactis 61-14 strain (FERM BP-08492) was cultured in 10 ml of MRS (D co) medium at 30 ° C. for 24 hours. The culture solution was inoculated into 100 ml of MRS medium (containing 2% calcium carbonate) and cultured at 30 ° C for 24 hours. This culture solution is added to MRS medium (2% 1 L was inoculated, cultured for 16 hours, centrifuged, and the supernatant was collected. To the supernatant was added 20 g of Amberley 1, XAD-16 (Sigma) (a substance sufficiently swollen with 2-propanol and washed with distilled water), immersed for 16 hours, and adsorbed. This was packed in a column, washed 700 ml with distilled water and 500 ml with 40% ethanol, and eluted with 100% 2-propanol. The eluted fraction was dried using a rotary evaporator. The dried product was dissolved in 50 ml of Britton-Robinson wide area buffer (pH3.94) at 4 ° C for 16 hours. This was used as a stock solution.

Antibacterial test method>

^ Cto aciZ / i / s sakei subsp. Sakei JCM1157 ^T was used as the indicator bacterium. After adding the sample to the sample, inoculate the MRS medium to a final concentration of 4 x 10 ⁵ cells / ml, incubate in a 96-well plate (manufactured by Nunc) at 30 ° C for 24 hours, and then absorb at 630 nm. Was measured. When the growth of the indicator bacteria was observed, the absorbance increased.

After adjusting the pH of the Nisin Q crudely purified solution to each pH, autoclaving was performed at 110 ° C for 10 minutes. Each pH sample was serially diluted two-fold, and an antibacterial test was performed at each dilution step.

Figure 2 shows the test results. At each pH, we examined the conditions under the same conditions as the retort sterilization conditions.Since dilution had an inhibitory effect on growth, food materials that are expected to undergo heat treatment at about the retort level, and unheated food materials are also considered. Has also been confirmed to be available.

(9) Food preservability test

The effect of the antimicrobial peptide (Nisin Q) of the present invention on cooked rice was examined. MRS medium (manufactured by DIFCO) was sterilized at 121 ° C for 15 minutes, inoculated with strain 61-14 (FERM BP-08492), cultured at 37 ° C for 15 hours, and the cells were removed and freeze-dried. In addition, in comparison, commercially available inexpensive and widely used date keeping improvers, vinegar preparations and lactic acid were used. Was.

After washing the rice with water and absorbing water, 100 spores / g of rice-derived ssw ^ iZz's spores were inoculated, and nisin Q, a dietary preparation, and lactic acid were added as in Table 2 and rice was cooked. After cooking, the rice was cooled, put in a sterilized cup, and stored at 30 ° C for 48 hours. For each sample, the standard viable cell count was measured using a standard agar medium (manufactured by Eiken Chemical Co., Ltd.). The results are shown in Table 3. Table 2

Table 3

(Pcs / g) By adding Nisin Q, a good day keeping and improving effect was obtained. In addition, as a result of the sensory test, the group added with nisin Q had better flavor and taste than the group added with lactic acid and vinegar. From this, it was clarified that Nisin Q can give a day keeping and improving effect without affecting the quality of food. Industrial applicability

According to the present invention, a novel antibacterial substance useful as a food preservative or the like is provided,

Claims

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CZllT0 / f00Zdf / X3d J806i0 / O0Z OAV atg agt aca aaa gat ttc aac tta gat ttg gta tct gtt tea aaa aca gat tct ggc get 60 tea aca cgt att acc age att tcg ctt tgt aca cca ggt tgt aaa aca ggt gtt ctg atg 120 gga tgt aac ctg aaa aca gca The DNA according to claim 4, comprising tgt aat tgt age gtt cac gta age aaa taa 174.

6. Lactococcus lactis which produces the antimicrobial peptide of claim 1 ■ Lactococcus lactis 61-14 strain (FERM BP-08492).

7. A food preservative comprising the antibacterial peptide of claim 1 as an active ingredient.