KR102076089B1 - A new Lactococcus lactis subsp. lactis LDTM 6804 having advanced nisin Z production(KCTC 13564BP) - Google Patents

Abstract

The present invention relates to a new Lactococcus lactis subsp. lactis LDTM 6804 strain having excellent antibacterial activity, and isolated from raw milk from Gangwon-do, South Korea. The present invention, particularly, relates to a new strain which can be utilized as a natural preservative in various processing methods of processed foods, such as food processing and preservation, and for a starter for fermentation, and the like, by producing nisin Z among bacteriocins which are antibacterial materials unique to microorganisms, thereby having strong antibacterial activity against various Gram-positive and Gram-negative bacteria and spoilage bacteria.

Description

Lactococcus lactis subspesis Lactose LDTM 6804 new strain (KCTC 13564BP) having excellent nisin Z production capacity {A new Lactococcus lactis subsp. lactis LDTM 6804 having advanced nisin Z production (KCTC 13564BP)}

The present invention is Lactococcus Lactococcus Lactococcus Lactococcus excellent antibacterial activity isolated from crude oil in Gangwon-do, Korea lactis subsp. lactis) LDTM 6804 new strains, especially nisin Z (bacterial bacteriocin-specific bacteriocin) to produce a strong antimicrobial action against a variety of Gram-positive and Gram-negative pathogens and decay bacteria, such as spawn for food processing, preservation and fermentation It relates to a new strain that can be used as a natural preservative during the processing of various processed foods.

As consumers' interest in health has increased recently, there has been a growing demand for consumers to prefer no-added and unprocessed foods without food additives. Accordingly, manufacturers have studied biological preservation methods to replace conventional chemical preservation methods. Lactic acid bacteria are attracting attention. This is because lactic acid bacteria naturally secrete the antibacterial peptide bacteriocin, including organic acids, and because of this, they are antibacterial against pathogens and rot bacteria.

Bacteriocin is active at high temperatures, stable at a wide range of pH, non-toxic, colorless and odorless, whereas antibiotics are secondary metabolites, while bacteriocin is biosynthesized from its genes, maximizing production at the molecular level through genetic analysis and manipulation. Not only is it easy to do, but also it is advantageous to synthesize better bacteriocin through molecular variation. In addition, antibiotics have side effects when administered, but bacteriocin is decomposed by proteolytic enzymes when ingested into the human body. Therefore, it is recognized as a good means to ensure stability even with minimal heat treatment and low temperature distribution in food because it is non-toxic and persistent. have.

Bacteriocin improves the shelf life of fermented milk and fermented alcoholic beverages, improves the shelf life of canned products, improves the shelf life of refrigerated and frozen products, improves the shelf life of fermented products such as red pepper paste, soybean paste, tofu, and lactic acid bacteria, and traditional foods (kimchi, medicinal herbs, Takju, etc.). It can be applied to preventing rancidity and deterioration, maintaining freshness of fish and shellfish, improving the shelf life of fruits and vegetables, and supporting high quality products such as productivity improvement by improving shelf life of fresh food, reducing consumer anxiety about chemical preservatives, and preventing livestock diseases.

Lactococcus is widely used as a cheese and dairy starter in the dairy industry, especially because of its rapid growth in the commercialization stage, and in the United States, some of the selected Lactococcus microorganisms are being introduced into the dairy process.

However, there are not many microorganisms in the lactococcus known in terms of production of bacteriocin, and most of them are developed in foreign countries, which are not suitable for the domestic dairy industry, and they have to pay a fee to foreign countries. It was one of the biggest challenges in the dairy industry.

Published Patent Publication No. 1993-00008697 (published on Jan. 1995) Registered Patent Publication No. 10-1521123 (August 19, 2015)

In order to solve this problem, the present invention is one of bacteriocins and is intended to select domestic dairy strains derived from dairy products excellent in nisin production ability is known as a very powerful biological preservatives to utilize in the production of dairy products.

The present invention is to provide a functional live bacteria that can be applied quickly and safely to the dairy industry, which is widely used industrially by selecting and providing lactococcus strains that are recognized as safety and have excellent nisin production ability as GRAS (generally recognized as safe) strains.

The present invention also provides a reliable native strain excellent in nisin production capacity, which has been genetically verified to include nisin expression genes.

Therefore, in order to solve the above problems, the present invention has been deposited with the Korea Research Institute of Bioscience and Biotechnology as accession number KCTC13564BP, (a) has a 16s-rRNA sequence of SEQ ID NO: 1. (b) ampicillin, vancomycin, gentamycin, kanamycin, kanamycin, streptomycin, erythromycin, tetratracycline, clindamycin and clindamycin (chloramphenicol) Not antibiotic resistant. (d) The strain contains eleven genes of nis Z, nis B, nis T, nis C, nis I, nis P, nis R, nis K, nis F, nis E, and nis G. (e) Has antibacterial effects against Listeria monocytogenes , Staphylococcus aureus and Escherichia coli . Features of Lactococcus lactis lactis sub Spanish sheath representing (Lactococcus lactis subsp. Lactis) may be achieved by providing a novel strain LDTM 6804.
The present invention can also be achieved by providing a food preservative using the LDTM 6804 new strain.

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Lactococcus lactis new strain according to the present invention was isolated from crude oil products produced in Gangwon-do, South Korea, and particularly to provide Lactococcus lactis subsp. Lactis LDTM 6804 new strain having excellent antibacterial activity. Can be.

That is, Lactococcus lactis subsp. Lactis LDTM 6804 according to the present invention can be effectively used for the prevention of decay bacteria and pathogens.

Further, the present invention Lactococcus lactis (Lactococcus lactis subsp. Lactis LDTM 6804 ) novel strain is the strain that in addition to its own, and dairy and food across a variety of forms such as nisin purified water purification the culture, the culture using a strain of the It can be used as a safe preservative.

In addition, the present invention can be utilized as feed additives, colostrum added lactic acid bacteria for pups, silage mold production inhibitory lactic acid bacteria, probiotics excellent antibacterial activity.

Figure 1 shows Lactococcus lactis subsp.lactis Lactococcus lactis subsp.lactis Lactobacillus LDTM 6804 strain and control group Lactobacillus plantarum MR-14, Lactobacillus paracaze THM-1, Pediococcus axix Antimicrobial activity evaluation results, such as dilactic TWE-4 and Lactobacillus casei DH-12. As a result, +++ is> 10.6mm, ++ is> 9.3mm, and + is> 8mm. (Please check whether it is correct.) The new strain LDTM 6804 of the present invention, especially B. circulans, B It was confirmed that .licheniformis, B. cereus and L. innocua strains exhibited very strong antibacterial effects.
Figure 2 shows biochemical properties, unlike the control lactococcus lactis subspis lactis IL1403 control LDTM 6804 new strain of the present invention is D-arabinose, inositol, N-acetylglucosamine, D- maltose, esterase -Differences in using lipase, beta-galactosidase, beta-glucosidase, and the like more effectively can be seen.
Figure 3 shows the growth of the LDTM 6804 strain of the present invention according to the medium composition and the change in pH accordingly, in graph (a) it can be seen that the growth of up to 5 times the number of bacteria within 24 hours of incubation time of viable cells, In graph (b), it can be seen that the pH is lowered to 4.5.
Figure 4 confirmed the control strain IL1403 and SO strain and the whole genome sequence (Whole genome sequence) for the new strain of the present invention was able to identify the nisin expression gene of LDTM 6804 new strain of the present invention, Figure 5 is Nisin Z Representing the gene cluster schematics related to biosynthesis, it can be seen that consists of a total of 11 genes.
6, 7 and 8 are (a) 4 ° C. against Pseudomonas fluorescens KCTC42821, Burkholderia cepacia KCTC2966, and pathogen Listeria monocytogenes KVCC-BA0001449, respectively, in 10% skim milk; as a result of inhibiting pathogens of the inventive bacterial strains tested at (b) 10 ° C and (c) 15 ° C. (●) is cultured the low-temperature rot bacteria or pathogens alone; (■) is the lactic acid bacteria IL1403 addition group in addition to a single bacterium; (▲) was investigated in comparison with the addition of a single bacterium, the present invention strain LDTM 6804, through which the present invention Lactococcus lactis subspis lactis compared to Lactococcus lactis subspis lactis IL1403 of the same genus ( Lactococcus lactis subsp.lactis ) LDTM 6804 shows an excellent pathogen inhibitory effect.
9 performed 55 cycles of 20 seconds at 95 ° C, 10 seconds at 58 ° C, and 30 seconds at 72 ° C. The melting curve was confirmed by increasing the temperature by 0.5 ° C per cycle from 45 ° C to 95 ° C.
FIG. 10 shows that the indicator bacteria P. fluorescence, B. cepacia, and L. monocytogenes in 10% skim milk were effectively reduced by the nisZ gene nisZ of the LDTM6804 strain.
11 is a flavor component determination function analysis table between the experimental groups, the cheese added with the control and pathogenic microorganisms after 25 days of cheese aging can be seen that the flavor component of the cheese containing LDTM6804 is similar.
12 was confirmed that effectively reduced the nisin gene nisZ of LDTM6804 strain against the indicator bacteria L. monocytogenes, S. aureus, E. coli in cheese.

In order to select the new strain of the present invention, domestically produced crude oil was used, and specific experimental methods are as follows.

Experimental Example 1

1. Isolation of Lactic Acid Bacteria from Domestic Crude Oil

In order to separate the desired lactic acid bacteria from domestic crude oil, 20 ranches of Pyeongchang-gun and Hoengseong-gun, Gangwon-do, Korea were used.The crude oil was diluted 10 times with 0.85% NaCl and added vancomycin (20mg / L). Plated in De Man Rogosa, Sharpe agar (MRS broth; Difco, USA) and bromocresol purple agar (BCP; Eiken chemical Co., Ltd, Japan), titrated to pH 5.0, for 48 hours at temperatures of 30 ° C. and 37 ° C. Aerobic culture.

After colonies different colonies (colony) formed according to the culture was selected by 16s rRNA sequencing to identify the strain, after passage in MRS broth and Tryptic Soy broth (TSB; Difco, USA) for the selection strain MRS broth and TSB containing 20% glycerol were stored frozen at -80 ° C.

Experimental Example 2

2. Antibacterial activity test against low temperature rot bacteria and pathogens

Agar plate disc assay was used to test the antibacterial activity against 7 strains of low-temperature decaying bacteria and 6 pathogens. The stored lactic acid strain and the indicator strain were passaged in broth. 100 μl of the indicator bacteria prepared in accordance with O.D600 1.0 on the indicator bacteria medium using a sterile swab, and then placed on a sterilized paper disc, inoculated 20 ul of the lactic acid strain culture medium onto the paper disc, and then at a temperature of 30 ° C. and 37 ° C. When aerobic incubation for 24 to 48 hours and a clear zone occurred, the size was measured horizontally and vertically and used as an average value.

Lactococcus than other strains lactis subsp. The highest antimicrobial activity of lactis LDTM 6804 (LDTM 6804) was confirmed by agar well diffusion assay and colony-based top agar assay in addition to the paper disc assay as shown in Table 1. This ensured reproducible results and reliability.

That is, the low temperature decay bacteria as shown in Table 1 (psychrotrophic spoilage bacteria) and Lactococcus for pathogens (pathogenic bacteria) lactis sub Spanish cis lactis (Lactococcus lactis subsp. Lactis) LDTM strong antimicrobial activity of 6804 new strain cells, supernatant And agar-diffusion (paper disc assay), agar well diffusion assay, colony-based top agar assay, which were performed with pH-adjusted supernatant.

Experimental Example 3

3. Characterization and Identification of Strains

The biochemical properties of the strains were examined using API CH50 kit and API ZYM kit (BioMetrieux Co. France) and once again, 16s-rRNA sequencing was obtained from Macrogen, a sequencing company, for accurate identification. Lactococcus by identifying the 16s-rRNA sequence of SEQ ID NO: 1 lactis subsp. It was identified as one of lactis , and it was deposited with Korea Research Institute of Bioscience and Biotechnology (KCTC 13564BP).

In addition, in order to confirm the usability in dairy products, the growth of the medium was designed in various ways such as M17, MRS, and 10% skim milk to confirm the growth of LDTM 6804. As shown in FIG. 3, the bacterial count increased up to 5 times in about 24 hours. It was confirmed that the pH is also lowered up to 4.5.

Experimental Example 4

4. Safety Assessment

The microdilution method (CLSI, M07-A9) was used to confirm the antibiotic resistance of the present invention LDTM 6804.

The strain was washed twice and the cells were prepared by diluting in 2 × LSM broth to 0.3 in O.D600. 100 µl each of the prepared microbial dilution solution is placed on a microplate, and 100 µl each of the 2 × antibiotic dilution solution subjected to 2-fold dilution. The antibiotics were prepared by diluting ampicillin, vancomycin, gentamycin, kanamycin, streptomycin, erythromycin, tetracycline, clindamycin and chloramphenicol in steps from 1mg / L to 128mg / L. After 48 hours of incubation, the Minimal inhibitory concentration was confirmed.

As a result of the investigation, it was confirmed that according to the standards of EFSA and EUCAST, LDTM 6804 is not antibiotic resistant.

Hemolytic activity evaluation was performed by adding 5% (v / v) sheep blood to the TSA medium to stretch the highly active strains to confirm clear zone after 24 hours of culture. As a result, the LDTM 6804 strain as shown in Table 2 below was confirmed that there is no hemolytic ability.

5. Investigating nisin-expressing genes in the whole genome sequence

The LDTM 6804 strain of the present invention identified the nisin expression gene by examining the entire genome sequence.

That is, gDNA extraction of LDTM 6804 was followed by sequencing using PacBio RSII and Illumina Hiseq 4000 platform. First, the device was sequenced with PacBio RSII and Hiseq, followed by precise de novo assembly using HGAP3 software and Pilon tool, and finally two contigs were obtained from the LDTM 6804 strain. As a result of comparing and analyzing the tRNA, rRNA, coding sequence information and annotation based on NCBI database, homology of the reference strain IL1403 with 98.31% was confirmed.

Meanwhile, Lactococcus lactis subsp. lactis IL1403 strain has become already decode the entire genome sequence (whole genome sequencing) a strain widely used in cheese starter as a cheese-derived strains, and often used as a reference strain when analyzed dielectric because easy to obtain the genetic information, Lactococcus lactis subsp. Since lactis S0 is a dairy-derived strain, such as LDTM 6804, which has been found to have nisin Z biosynthesis genes, genome analysis of the LDTM 6804 new strain of the present invention compared to the two strains has shown that LDTM 6804 is an IL1403. The gene clusters related to nisin Z biosynthesis, which have been absent, were clearly identified.

That is, the LDTM 6804 of the present invention has 11 genes ( nis Z, nis B, nis T, nis C, nis I, nis P, nis R, nis K, nis F, and nis E, as shown in FIGS. 4 and 5). , nis G ), and it was confirmed that LDTM 6804 is a new strain that has excellent genetic ability to produce nisin.

Experimental Example 5

6. Measurement of Crude Oil Reduction Effect in Skim Milk Model

Coculture was carried out to measure the reduction effect by lactic acid bacteria selected from three crude microorganisms in 10% skim milk.

The low temperature rot bacteria known to be a problem during cold storage of crude oil are Pseudomonas fluorescens KCTC42821, Burkholderia cepacia KCTC2966 with pathogens Listeria monocytogenes KVCC-BA0001449 such strains selected by the instruction, and three groups, that is, instructions strains (P. fluorescens, B. cepacia or L. monocytogenes) to a single culture, LDTM 6804 strain and the strain indicated 1: 1 ratio ( CFU / ml) was added and cultured by adding the IL1403 strain in a 1: 1 ratio with the indicator strain (control group) and compared with the control strain, and compared with the control strain. In FIGS. 6 to 8, indicators were cultured alone (marked), LDTM 6804 added (marked), and IL1403 added (marked).

6 (a) (b) (c) to 8 (a) (b) (c) of the three groups are 4 ° C., 10 ° C., and 15 for P. fluorescens, B. cepacia, and L. monocytogenes, respectively. It shows the result of incubation at ℃, it was made by counting the number of indicator strains while sampling over 0,1,3,5,7,10,14 days,

As a result, the inoculation of P. fluorescens was 6.69 logCFU / ml as shown in FIG. 6, and (b) the LDTM 6804 group was 2 (logCFU / ml) at 14 days of culture compared to the control strain added with reference strain IL1403 at 10 ° C culture. The extent was reduced (c) and completely inhibited on day 10 at 15 ° C. incubation.

In addition, as shown in FIG. 7, the initial inoculation amount of B. cepacia was 6.46 logCFU / ml, and (b) the LDTM +6804 group was 2 (logCFU / ml), and (c) was completely inhibited on day 10 at 15 ° C.

L. As for the monocytogenes bacteria, the initial inoculation amount was 6.57 logCFU / ml as shown in FIG. 8, and (b) the LDTM 6804 group showed a log of 0.6 (logCFU / ml) at 14 days of culture compared to the control group to which the IL1403 strain, a reference strain, was added at 10 ° C. Units were decreased, and (c) at 15 ° C. was decreased by 4.93 (logCFU / ml) on day 14.

On the other hand, at 4 ℃ of refrigeration temperature, the growth rate of all strains was low, and the effect of reduction was small.However, at 10 ℃ and 15 ℃, which are room temperature, all indicator strains decreased very effectively in the group to which LDTM 6804 was added. At temperature, it confirmed that all disappeared after about 10 days. This confirms that the antimicrobial activity of LDTM 6804 at room temperature is very superior to that of IL 1403.

Experimental Example 6

7. Confirmation of Nisin Gene Expression Using qRT-PCR in Skim Milk Model

As shown in Table 3, the primers specific for the LDTM 6804 nisin genes nisZ, nisB, and nisP were used as shown in Table 3 in order to confirm that the nisin gene of the LDTM 6804 new strain of the present invention was expressed in 10% skim milk. The preparation was confirmed the mRNA expression of nisin. Samples incubated with LDTM6804, P. fluorescens, B. cepacia, and L. monocytogenes in 10% skim milk were collected on days 0, 5, 10, and 14, respectively. The collected samples were collected by centrifugation and vortexed after 1 ml of AccuZolTM (Bioneer, USA) solution. After 1-2 hours storage at -80 ℃, they were dissolved once and stored again at -80 ℃. RNA extraction according to the reagent protocol followed by cDNA synthesis using the ReverTra Ace qPCR RT Master Mix with gDNA Remover kit (TOYOBO, Japan) and quantitative real using TOPrealTMqPCR2Ⅹ PreMIX (SYBR Green with low ROX) kit (Enzynomics, Korea) -time PCR (qRT-PCR) was performed. The qRT-PCR condition is shown in FIG. As a result, it was confirmed that the expression rate of gene nisZ was high, although there were differences by temperature and culture time.

In this study , it was confirmed that nisZ of LDTM6804 mycobacteria was effectively inhibited by P. fluorescens, B. cepacia, and L. monocytogenes in skim milk.

Experimental Example 7

8. Determination of Pathogen Reduction Effects in Laboratory Unit Cheese Models

Listeria , widely known as a pathogen during the ripening and distribution of cheese The pathogenic effect of selected lactic acid bacteria including LDTM 6804 against monocytogenes KVCC-BA0001449, Staphylococcus aureus KCTC1621, and E. coli KCTC1682 was tested.

In other words, after dividing by Test 1, 2, 3 according to the type of pathogen, L. lactis subsp. lactis LDTM6402 and L. lactis subsp. cremoris LDTM6403 Two Control 1 cheeses were prepared and LDTM 6804 was combined with Starter strains at different ratios of Test 1-2, Test 1-3, Test 2-2, Test 2-3, Test 3-2, The experiment was planned as Test 3-3.

The experiment was performed by dispensing 400 ml of crude oil into 500 ml centrifuge tubes before sterilization and sterilizing at 68 ° C. for 25 minutes, and after standing for about 30 minutes in a 30 ° C. constant temperature water bath, preparing lactic acid bacteria in the combination of Table 4 below. Inoculate 1% (v / v) and inoculate pathogens after standing for 10 minutes, and then incubate for 1 hour in a constant temperature water bath at 30 ° C and inoculate STD PLUS 290 Lennet (Chr.Hansen, Denmark) at the recommended dose It was left in a 30 ° C. constant temperature water bath for about two hours until it reached.

After cutting the curd with a sterilized disposable stick and stirring 20 minutes at 10rpm using Laboshaker R100 (Labogene, South Korea) to remove about 40% of whey, curd washing by adding the same amount of sterilized 30 ℃ distilled water and again at 10rpm After stirring for a minute, the liquid was removed by centrifugation (380 g, 10 min, 30 ° C.).

Transfer the curd to a new sterile centrifuge pail and centrifuge again (1,400 g, 1 h, 30 ° C.), finally remove the whey and centrifuge it by inverting the curd (1,400 g, 30 min, 30 ° C.) and catch the cheese. After transfer to the aging vessel and immersed in 66% NaCl solution for 5 minutes to remove the solution. It was sampled at regular intervals while ripening at 10 ° C. for 25 days, and then the pathogens were counted using the selective medium.

The data in Tables 5 to 7 below are calculated as mean log CFU / g ± standard deviation. The L. monocytogenes bacteria in Table 5 had an initial inoculation of 5.04 logCFU / ml and were aged compared to the control group without the LDTM 6804 strain. On the day, about 0.15 and 0.42 logCFU / ml decreased according to the amount of LDTM 6804 strain added. Through this, it was confirmed that the effect of reducing the pathogen of LDTM 6804 is much stronger as the amount added.

The initial inoculation of S. aureus in Table 6 was 4.58 logCFU / ml, and 0.2 and 0.57 logCFU / ml were decreased by the addition of LDTM 6804 strain at 25 days of aging compared to the control group without addition of LDTM 6804 strain. Through this, it was confirmed that the effect of reducing the pathogen of LDTM 6804 is much stronger as the amount added.

The initial inoculation of E. coli in Table 7 was 5.86 logCFU / ml, and 0.74 and 0.26 log units decreased with the addition of LDTM 6804 strain at 25 days of aging compared to the control without LDTM 6804 strain .

Although Lactococcus lactis LDTM 6802 and 6803 were homogenized or increased in the L. monocytogenes and E. coli compared to the initial inoculation during the maturation period, the Lactococcus lactis LDTM 6804 group was added only to LDTM 6802 and 6803. Unlike the addition, it was clearly confirmed that significantly reducing the pathogens, it was clearly confirmed that the strong antibacterial effect of LDTM 6804 is not the usual expected from all Lactococcus lactis spawn.

Experimental Example 8

9. Analysis of Flavor Components in Cheese Model Using Electronic Nose

Flavor components of the LDTM 6804 strains of the present invention were analyzed by Heracles II Analyzer (Alpha MOS, France) according to the type and age of inoculation. Heracles II Analyzer has two columns: DB-5 and DB-WAX. ) Is a gas chromatography-type electronic nose instrument equipped with Combi PAL Auto-Sampler (CTC Analytics AG, Switzerland), and each chromatogram generated by two columns is AroChemBase provided by Alpha MOS. The flavor component of cheese aged at 10 ° C., analyzed by software, was analyzed by Principal Component Analysis and Discriminant Function Analysis.

According to the analysis, the flavor components differed according to the ripening period, the type of indicator, and the addition of LDTM 6804. E. coli has a doubling time of 20 minutes, which is superior to other strains used in this study. In the other two groups except test 3 group inoculated with E. coli , the non-LDTM 6804 group had a change in flavor due to the pathogen, but the group inoculated with LDTM 6804 suppressed the change of flavor by the pathogen. From the grouping of the control group and the control group, it can be assumed to have a similar profile to the control group.

Most of the flavor components increased during the ripening period, and common representative components were ethanol, 3-Buten-2-one, acetoin, isopropyl isothiocyanate, and formic acid. Commonly increased components in Test 1-1, Test 2-1, Test 3-1, and Test 3-2 in Table 8 include 2-nitropropane, which can suppress flavor changes due to pathogen contamination when LDTM 6804 strain is added. The effect was clearly confirmed.

Taken together, the above investigation, in vitro experiments (in vitro) Antibacterial ability is superior and the LDTM 6804 strain evaluation exact test (in situ) a difference degree, but was clear that there is antibacterial activity confirmed in in, in 10% skim milk model that Antibacterial activity was more clearly shown. In addition, LDTM 6804 suppressed the flavor change due to pathogen contamination when LDTM 6804 was added to the cheese using electronic noses. LDTM 6804 suppresses the number of pathogens and has the potential to be used as a biopreservative in the food industry where flavor is important. It can be confirmed for sure.

Experimental Example 9

10. Confirmation of Nisin Gene Expression Using qRT-PCR in Cheese Models

In order to confirm whether the L. monocytogenes , S. aureus , and E. coli were reduced in cheese by the LDTM 6804 new strain of the present invention, the mRNA expression level of the nisin gene was confirmed.

Cheese Test 1-3, Test 2-3, Test 3-3 samples were sampled for 0, 14, and 25 days respectively. After 1 ml of AccuZolTM (Bioneer, USA) solution was vortexed, it was vortexed at -80 ℃. After time storage, dissolve once and store again at -80 ° C. RNA extraction according to the reagent protocol followed by cDNA synthesis using the ReverTra Ace qPCR RT Master Mix with gDNA Remover kit (TOYOBO, Japan) and quantitative real using TOPrealTMqPCR2Ⅹ PreMIX (SYBR Green with low ROX) kit (Enzynomics, Korea) -time PCR (qRT-PCR) was performed. qRT-PCR was performed under the conditions of FIG. 9 using the primers of Table 3. As a result, as shown in FIG. 12, it was confirmed that the expression rate of the gene nisZ was significantly higher at 14 days of cheese aging. In addition, it was confirmed that nisZ was highly expressed on day 25 in E. coli -inoculated cheese. The inside of the cheese by nisZ LDTM6804 new strains were able to confirm that the indicated bacteria L. monocytogenes, S. aureus, E. coli is effectively suppressed.

Meanwhile, the depositing institution, accession number and date for the LDTM 6804 new strain of the present invention are as follows.

Depositary Name: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC13564BP

Deposit date: 2018627

<110> Seoul National University R & DB Foundation <120> A new Lactococcus lactis subsp. lactis LDTM 6804 having          advanced nisin Z production (KCTC 13564BP) <130> P18-006 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 971 <212> DNA <213> Unknown <220> Lactococcus lactis subsp. lactis LDTM 6804 <400> 1 acaggtcggc tctccagcgg agtgcttatt gcgttagctg cgatacagag aacttatagc 60 tccctacatc tagcactcat cgtttacggc gtggactacc agggtatcta atcctgtttg 120 ctccccacgc tttcgagcct cagtgtcagt tacaggccag agagccgctt tcgccaccgg 180 tgttcctcca tatatctacg catttcaccg ctacacatgg aattccactc tcctctcctg 240 cactcaagtc taccagtttc caatgcatac aatggttgag ccactgcctt ttacaccaga 300 cttaataaac cacctgcgct cgctttacgc ccaataaatc cggacaacgc tcgggaccta 360 cgtattaccg cggctgctgg cacgtagtta gccgtccctt tctgggtagt taccgtcact 420 tgatgagctt tccactctca ccaacgttct tctctaccaa cagagtttta cgatccgaaa 480 accttcttca ctcacgcggc gttgctcggt cagactttcg tccattgccg aagattccct 540 actgctgcct cccgtaggag tttgggccgt gtctcagtcc caatgtggcc gatcaccctc 600 tcaggtcggc tatgtatcat cgccttggtg agcctttacc tcaccaacta gctaatacaa 660 cgcgggatca tctttgagtg atgcaattgc atctttcaaa cttaaaactt gtgtttaaag 720 tttttatgcg gtattagcat tcgtttccaa atgttgtccc ccgctcaaag gcagattccc 780 cacgcgttac tcacccgttc gctgctcatc cagtcggtac aagtaccaac cttcagcgct 840 caacttgcat gtattaggca cgccgccagc gttcgtctga gggggggaaa gaaaatataa 900 tatataaaat tttttttttt tttcccccct tttttgtatt attttctttc ctgcgggaaa 960 aatctctatt g 971 <210> 2 <211> 722 <212> DNA <213> Unknown <220> Lactococcus lactis subsp. lactis strain LDTM 6804 <400> 2 tgaggtcgat gagtgctaga tgtagggagc tataagttct ctgtatcgca gctaacgcaa 60 taagcactcc gcctggggag tacgaccgca aggttgaaac tcaaaggaat tgacgggggc 120 ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc 180 ttgacatact cgtgctattc ctagagatag gaagttcctt cgggacacgg gatacaggtg 240 gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca 300 acccctattg ttagttgcca tcattaagtt gggcactcta acgagactgc cggtgataaa 360 ccggaggaag gtggggatga cgtcaaatca tcatgcccct tatgacctgg gctacacacg 420 tgctacaatg gatggtacaa cgagtcgcga gacagtgatg tttagctaat ctcttaaaac 480 cattctcagt tcggattgta ggctgcaact cgcctacatg aagtcggaat cgctagtaat 540 cgcggatcag cacgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac 600 cacgggagtt gggagtaccc gaagtaggtt gcctaaccgc aaggagggcg cttcctaagg 660 taagaccgat gactggggtg aatcaaaagg gggggggcaa aaaaaaaatt ttttattttt 720 tg 722

Claims (2)

Lactococcus lactis sub Legaspi system lactis (Lactococcus lactis subsp. Lactis) LDTM 6804 novel strain showing the characteristics of the deposit to the Korea Research Institute of Bioscience and Biotechnology as Accession No. KCTC13564BP.
(a) has the 16s-rRNA nucleotide sequence of SEQ ID NO: 1.
(b) ampicillin, vancomycin, gentamycin, kanamycin, kanamycin, streptomycin, erythromycin, tetratracycline, clindamycin and clindamycin (chloramphenicol) Not antibiotic resistant.
(c) No hemolytic activity.
(d) The strain contains eleven genes of nis Z, nis B, nis T, nis C, nis I, nis P, nis R, nis K, nis F, nis E, and nis G.
(e) Has antibacterial effects against Listeria monocytogenes , Staphylococcus aureus and Escherichia coli . Food preservative using LDTM 6804 new strain of claim 1

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