KR101281973B1 - New antibacterial material with antagonistic effect against the food borne pathogenic microorganism such as Bacillus cereus and Listeria monocytogenes, and Bacullus substilis BY08 - Google Patents

Abstract

The present invention is a novel antimicrobial agent (UV254-B) having excellent antibacterial activity against Bacillus cereus and Listeria monocytogenes, which are food poisoning causing bacteria, and Bacillus subtilis as a strain producing the same. (B. substilis) relates to BY08. Specifically, according to the present invention, the bacterium Bacillus cereus (B. cereus) is a novel antibacterial substance (UV254-B) and a bactericidal bacterium by using Bacillus substilis BY08 as a strain to produce the same. And Listeria monocytogenes can be inhibited at the same time. In addition, the use of the antimicrobial agent according to the present invention provides antimicrobial and antagonistic effects specifically against Bacillus cereus and Listeria monocytognens without showing antimicrobial activity against other beneficial bacteria in the intestine. It is about showing.

Description

New antibacterial material with antagonistic effect against the food borne pathogenic microorganism such as Bacillus cereus and Listeria monocytogenes, and Bacullus substilis BY08}

The present invention relates to a novel antibacterial substance having a food poisoning antagonistic effect and B. subtilis BY08 producing the same.

Soybean fermented foods are foods that can be manufactured by anyone at home without any special knowledge. More specifically, they are microorganisms that exist around the living environment from soybeans such as soybeans. It is one of the representative Korean fermented foods aged through the fermentation process.

When soybean fermented food is manufactured by the traditional method, contamination of harmful bacteria scattered in the living environment remains a problem for food safety. Bacillus cereus is a genus such as Bacillus subtilis and Bacillus licheniformis, which are main fermenting fungi. It is distributed, and endogenous spores that can withstand heat treatment are not killed by the general sterilization method, and it can cause food poisoning by generating toxic substances. In 2006, the Food and Drug Administration (KFDA) enacted a law to add Bacillus cereus as a harmful contaminant in enteric foods and to limit the detection range to less than 10,000 / g per product.

In order to control various harmful bacteria including B. cereus, high concentrations of salts have been added or synthetic food preservatives have been used (Jang et al., 2003). However, according to the trend of preferring low salt foods or preservative-free foods to the well being trend, Bacillus subtilis, which has been recognized as a safe GRAS microorganism for use in the food and pharmaceutical industries to develop natural food preservatives ( Research into the use of antibacterial materials of the genus such as Bacillus subtilis and Bacillus licheniformis has been actively conducted.

Therefore, the necessity of research on new antimicrobial substances to the major harmful microorganisms of food poisoning bacteria, such as harm to the human body is emerging.

In addition, as an antibacterial substance against B. cereus, lipopeptide-based suctinin, penycin, fengycin, iturin, and lipopeptide produced by B. subtilis Bacillopeptins and the like are known, but many studies are being conducted to find new antimicrobial substances.

Accordingly, the present inventors completed the present invention by developing a novel antimicrobial agent and strain producing the same against antibacterial activity against food poisoning-inducing bacteria B. cereus and Listeria monocytogenes. It became.

Specifically, an object of the present invention is to provide a novel antibacterial substance (UV254-B) and B. subtilis BY08 strain producing the same.

Antimicrobial material (UV254-B) according to the present invention as described above is obtained by culturing Bacillus subtilis KCTC 18217P comprising SEQ ID NO: 1.

In addition, the culturing includes culturing, characterized in that the optimum temperature of 25 to 40 ℃, optimum pH 7.0 to 8.0, incubation time 12 hours to 48 hours.

According to another aspect of the present invention, there is provided a method for preparing an antimicrobial agent (UV254-B), comprising: separating Bacillus subtilis strain KCTC 18217P comprising SEQ ID NO: 1 and Bacillus subtilis after the separation. Bacillus subtilis Strain (Ba254us subtilis) Bacillus subtilis Strain (Bacillus subtilis) KCTC 18217P using an antimicrobial material (UV254-B) including the step of culturing and purifying the antimicrobial material (UV254-B) after the step of culturing ) Is a manufacturing method.

In addition, the culturing includes culturing, characterized in that the optimum temperature of 25 to 40 ℃, optimum pH 7.0 to 8.0, incubation time 12 hours to 48 hours.

Bacillus subtilis KCTC 18217P strain according to another feature of the present invention is a strain comprising SEQ ID NO: 1 and produces an antibacterial substance (UV254-B).

In addition, the production of the antimicrobial material (UV254-B) includes the one produced by culturing the characteristics of the optimum temperature 25 to 40 ℃, optimum pH 7.0 to 8.0, incubation time 12 hours to 48 hours.

The novel antibacterial substance (UV254-B) and B. subtilis BY08 strains producing the same according to the present invention are food poisoning-inducing bacteria B. cereus, Listeria monocytogenes (L. monocytogenes). New antimicrobial agents and strains that have antimicrobial activity simultaneously. In addition, the novel antibacterial substance (UV254-B) and B. subtilis BY08 strains producing the same according to the present invention without affecting other strains in the gut, Bacillus cereus (B. cereus) and Listeria monocytogenes (L. monocytogenes) is only a novel antibacterial substance and strain that does not affect the beneficial strains in the gut, because it shows only antibacterial activity.

1 is a diagram showing a phylogenetic relationship between B. subtilis BY08 and other bacteria according to an embodiment of the present invention.
FIG. 2 is agarose gel electrophoresis diagram of PCR-generated amplified B. cereus HblA fraction (Lane M: molecular size marker; Lane 1: B. subtilis BY08 (A); B. cereus (KCTC 3624) (B): Lane 2: enterotoxin-positive control; Lane 3: enterotoxin-negative control.
3 is a graph illustrating the effect of the carbon source on the cell growth and antimicrobial activity of the culture medium of B. subtilis BY08.
4 is a graph illustrating the effect of metal ions on the cell growth and antimicrobial activity of the culture medium of B. subtilis BY08 (A: KCl; B: CaCl2; C: FeSO4; D: MgSO4; E: CuSO 4; F: ZnSO 4; G: NaCl).
5 shows thin film chromatography results of antimicrobial material produced from B. subtilis BY08 under UV 254 nm.
6 is a diagram showing the antimicrobial activity of the antimicrobial substance eluted from TLC silica gel with methanol.
7 shows TLC chromatography results of the antimicrobial material produced from B. subtilis BY08.
FIG. 8 is a plot of the minimum inhibitory concentrations of antimicrobial material (UV254-B) purified from B. subtilis BY08 for Bacillus cereus by microelution.
9 is a chromatogram of antimicrobial material (UV254-B) scanned by HPLC at 254 nm.
10 is a chromatogram of the fractional ion spectrum obtained from a full scan of antimicrobial material (UV-254-B) by ESI-MS detection, positive ion mode, TIC.
FIG. 11 is the product ion spectrum of the molecule at retention time from 15.186 minutes to 15.387 minutes.

The present inventors have made intensive research efforts to develop antimicrobial substances and strains. As a result, the inventors have found novel antimicrobial substances and strains that produce them. Specifically, a novel antibacterial substance (UV254-B) having antimicrobial activity against food poisoning bacteria B. cereus and Listeria monocytogenes and B. subtilis producing the same The present invention was completed by discovering BY08 strain.

Hereinafter, the present invention will be described in detail.

The present invention is a novel antibacterial substance (UV254-B) having antimicrobial activity against food poisoning bacteria B. cereus and Listeria monocytogenes and Bacillus subtilis BY08 to produce the same. It is about a strain. The Bacillus subtilis BY08 was deposited on August 10, 2011 to the Korea Institute of Bioscience and Biotechnology, 125, Gwahak-ro, Yuseong-gu, Daejeon, Korea, and received the accession number KCTC 18217P on August 17, 2011. , The nucleotide sequence according to SEQ ID NO: 1. In addition, the novel antimicrobial material (UV254-B) according to the present invention and Bacillus subtilis BY08 to produce the same is the bacterium Bacillus cereus (B. cereus) and Listeria monocytogenes (Listeria monocytogenes) The invention relates to novel antimicrobial substances and strains that produce them, while having specific antimicrobial activity only, while not affecting other intestinal strains.

In addition, in order to increase the production of the antimicrobial substance (UV254-B) according to the present invention, the culture conditions relating to the temperature, pH, incubation time of the Bacillus subtilis BY08, temperature 25 ~ 40 ℃, pH 7.0 to 8.0, the incubation time is 12 hours to 48 hours when the production is the maximum value shows the maximum antimicrobial activity.

In addition, the novel antimicrobial material (UV254-B) according to the present invention exhibited antibacterial activity specifically against Bacillus cereus and Listeria monocytogenes. At this time, the minimum inhibitory concentration value for B. cereus was 64 μg / mL or more.

In addition, the analysis of the antimicrobial substance (UV254-B) using LC / ESI-MS / MS showed peaks at m / z 1133.6 and 1700.5. Thus, the antibacterial substance (UV254-B) produced from the Bacillus subtilis BY08 strain according to the present invention is completely different from the conventional antimicrobial substance in molecular weight, and the antibacterial substance (UV254-B) according to the present invention is It is identified as a novel antimicrobial.

Therefore, the present invention provides a novel antimicrobial agent (UV254-B) having an antagonistic effect and an excellent antimicrobial activity against food poisoning bacteria such as Bacillus cereus and Listeria monocytogenes, and a novel method for producing the same. Strain B. subtilis BY08.

Hereinafter, the present invention will be described in detail with reference to the preferred embodiments, which will be readily apparent to those skilled in the art to which the present invention pertains. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Example :

Disclosure strain

The strains used for the antimicrobial activity test were Bacillus subtilis KCTC1998, Bacillus cereus KCTC 3624, Bacillus amyloliquefaciens, KCTC 1660 from Daejeon, Korea. ), Bacillus licheniformis KCTC 3559, Salmonellaenteric subsp. Enterica (KCTC 2514) Listeria monocytogenes KCTC 3710 and Staphylococcus aureus KCTC 1621 were used.

< Example  1> Bacillus Subtilis (B. subtilis  ) BY08  Isolation and Identification of Strain

Isolation of strain

To isolate the B. subtilis BY08 strain, 2 g of Cheonggukjang was first added to 18 mL of sterile saline, followed by bathing at 80 ° C. for 15 minutes. Subsequently, the supernatant was spread on TSB (tryptic soy broth, Bacto, MD, USA) solid medium and incubated at 37 ° C. for 12 hours to isolate bacterial colonies. Selected strains were inoculated in TSB medium (Bacto Co, MD, USA), shaken for 36 hours at 37 ° C, and the supernatant obtained by centrifugation (15,000 rpm, 15 min) was used as a sample for antibacterial activity test. . The assay was performed by injecting 100 microliters of each microbial culture into wells on a nutrient cell of 107-8 CFU / mL, and incubating at 37 ° C for 6 hours to inhibit the growth of harmful microorganisms. The diameter (mm) of the clear zone indicated was measured.

Bacillus In B. cereus  Antagonistic strain screening for

In this example, each isolated strain against Bacillus cereus (B. cereus KCTC 3624) for the development of a biological control method to effectively block the B. cereus KCTC 3624, a major harmful microorganism of soybean fermented foods. The antimicrobial activity of was compared and verified (see Table 1 below).

Specifically, strains showing antimicrobial activity against B. cereus were primarily isolated and tested for antimicrobial activity on B. cereus KCTC 3624 lawn cell medium. As a result, the diameters of the growth-lowering rings were 8 mm and 11 mm, respectively, and the antibacterial activity against Bacillus cereus (B. cereus KCTC 3624) was isolated from the experimental group to which the cultures of the isolates BY03 and BY08 were added. It was. Among them, the BY08 strain was selected, particularly excellent in antibacterial activity.

Identification of the strain

Gyrase B gene forward primer (5`-CCC AAG CTT AAC TGC ACT GGG AAA TYG THG AYA AYA G-3`) GyrB135 and reverse primer (5`-CGG AAT TCG GAT CCA CRT CGG CRT CBG TCA TRA T-3 ' PCR was carried out using a polymerase chain reaction (PCR) reaction.

Specific PCR reaction conditions were 1 cycle of thermal denaturation at 94 ° C. for 4 minutes, thermal denaturation at 94 ° C. for 1 minute, and annealing at 58 ° C. for 1 minute, followed by 1 at 72 ° C. After 30 minutes of elongation, the reaction was amplified by final extension at 72 ° C. for 10 minutes. The amplified DNA bands were purified using Agarose Gel Extraction Kit (MEGA-spinTM, Intron Co, Seongnam, Korea), and commissioned to sequence analysis at Cosmogenetech Genetic Analysis Center (Seoul, Korea).

The nucleotide sequence of the strain obtained through the sequencing analysis was analyzed by the Blast Network Service using the Blast Network Service and compared with the nucleotide sequence of the NCBI GenBank database. Flexible relationships were analyzed using the Multiple Sequence Alignment program. Biochemical test was conducted using the API 50CHB kit (Bio Merieux, France) to examine the sugar availability of the strain, and the results of sugar fermentation test were analyzed using apiweb (apiweb, apiweb.biomerieux.com).

Results of comparing homology with gyrB gene base sequences of selected BY08 strains and phylogenic trees showing phylogenetic relationships are shown in Table 2 and FIG. 1.

As shown in Table 2, BY08 showed 98% similarity to Bacillus subtilis and Bacillus amyloliquefaciens, but in Bacillus subtilis (phylogenic tree) showing the phylogenetic relationship of FIG. Bacillus subtilis) was found to be the most flexible.

In addition, in Table 3 below, which summarizes the biochemical test results using the API 50 CHB kit, BY08 showed a similarity rate of 99.8% with B. subtilis, so the isolated strain BY08 was replaced with B. subtilis. ) BY08.

< Example  2> optimal Badge composition

The medium composition test for the nitrogen source for the optimal medium composition of the isolated strains was carried out on the base TSB medium (glucose 0.25%, Na2HPO4 0.5%, NaCl 0.25%), tryptone, yeast extract, soytone, peptone, beef extract, skim milk and Soybean extract was added 3% each, incubated for 24 hours at 30 ℃, 180 rpm and then the supernatant was tested for the antimicrobial activity against B. cereus (B. cereus).

The medium composition test for carbon source was performed by adding 2% of monosaccharides (glucose and fructose), disaccharides (lactose and sucrose), polysaccharides (starch), and polyols (mannitol and glycerol), respectively. The same was done. The media composition test for inorganic salts was examined in the same manner as the effect of nitrogen source by adding KCl, CaCl 2, FeSO 4, MgSO 4, CuSO 4, ZnSO 4, NaCl, etc. at 1 mM concentration.

Carbon source With nitrogen sources With inorganic salts  Of antimicrobial activity

Effects of nitrogen source on the antibacterial activity of B. subtilis BY08 by adding 3.0% of nitrogen source in basic TSB medium (glucose 0.25%, Na2HPO4 0.5%, NaCl 0.25%) except TS It was investigated (Table 4).

As a result, as shown in Table 4, the antimicrobial activity against B. cereus was higher in the yeast extract, soytone, and soybean extract, and the nitrogen source was the highest. It was found in the test group to which the extract was added, and the base TSB medium test group to which tryptone, peptone, beef extract and skim milk was added did not inhibit the growth of Bacillus cereus KCTC 3624.

In addition, the effects of carbon source on the growth and antimicrobial activity of B. subtilis BY08 by adding 2.0% of each carbon source to basal TSB medium (soybean extract 3.0%, Na2HPO4 0.5%, NaCl 0.25%) were investigated. As shown in Figure 3, the growth of bacteria was promoted in the order of soluble starch, sucrose, glucose, but appeared to be irrelevant in the case of addition of fructose, lactose, mannitol and glycerol. In addition, the antibacterial activity against B. cereus was high in the test group containing glucose, sucrose and glycerol.

From the above results, it was confirmed that the antimicrobial activity of B. subtilis BY08 strain had the synergistic effect of promoting the growth of bacteria and the antimicrobial activity when glucose was added as a carbon source.

Furthermore, in order to test the change of the antimicrobial activity according to the addition of the inorganic salt, medium containing various inorganic salts in 1 mM concentration (3% soy extract, 2% glucose and 1% NaCl and 1% Na2HPO4), respectively The change of antimicrobial activity was tested using and the results are summarized in FIG. 4.

As shown in Figure 4, the control group without the addition of the inorganic salt and the test group with the addition of KCl, CaCl2, FeSO4, MgSO4, ZnSO4 and NaCl inorganic salts did not show a significant difference as a result of the observation. However, the test group containing CuSO4 had no antimicrobial activity, and the result of losing the antimicrobial activity in the test group containing CuSO4 was probably Bacillus subtilis BY08 at the concentration of 1 mM CuSO4. It is inferred from the result of ingrowth.

< Example  3> antibacterial substance ( UV254 -B) separation

Preparation of Antimicrobial Substances

Selected BY 08 strains were inoculated into optimal medium and shaken at 180 rpm for 24 hours at 30 ° C (VS-245MTi, Vision Scientific, Korea). 300 mL of the culture was centrifuged (8000 rpm, 15 min), and then the supernatant was used for antimicrobial extraction. Extraction of the antimicrobial material was performed according to the method of Huang et al. (2007). That is, by adjusting the pH (2.0) of the supernatant using 6N hydrochloric acid, and left for 24 hours at 4 ℃ and centrifuged (8,000 rpm, 15 min) to separate the resulting precipitate. The precipitate was dissolved in 10 mL of 80% methanol, adjusted to pH (7.0) with 6 N NaOH, and left at 4 ° C. for 12 hours, followed by centrifugation (10,000 rpm, 15 min) to purify the antimicrobial material. The supernatant was concentrated under reduced pressure and then lyophilized (Model FD5508; ilshin, Netherlands) to prepare a powder sample.

TLC Purification of antimicrobial substances by

Antibacterial material was purified using TLC plate (silica gel 60 F254, Merck Co, Darmstadt, Germany) (FIG. 5). In other words, a solution obtained by dissolving a lyophilized powder cultured with 90% methanol in 100% methanol was instilled and then developed with a mixture of chloroform / methanol / water (13: 5: 0.9, v / v / v), followed by UV Bands were observed at a wavelength of 254 nm using a lamp (77202 Vilber lourmat, Marne La Vallee Cedex1, France). The antimicrobial material was separated and extracted with methanol at 4 ° C. for 12 hours (Lee et al., 2010) and centrifuged again (15,000 rpm, 15 min) to dry the supernatant with nitrogen gas, and then stored in a -20 ° C. freezer.

Separation and purification of antimicrobial substances

The results of measuring the antimicrobial activity using the antimicrobial material are shown in FIG. 6.

Antimicrobial activity was measured by paper-disc method against B. cereus by scraping silica gel of A, B and C bands shown in TLC, eluting with methanol, and using concentrated material. As a result, the antimicrobial effect could be confirmed in the second band (B) (see FIG. 6), and was emitted at 254 nm. The antimicrobial material was named UV254-B based on the emitted wavelength band.

The antimicrobial material identified by the UV lamp was colored using ninhydrin and iodine staining as qualitative analysis.

Lane A of FIG. 7 was stained with a ninhydrin reagent, but was light reddish purple, but not clear. Lane B was light yellow when stained with iodine reagent. For reference, the ninhydrin staining reagent is colored in reddish purple by combining with the amine of the peptide, and the iodine dyeing reagent is colored in yellow by binding to unsaturated fatty acids. UV254-B) can be interpreted as a substance containing unsaturated fatty acids, and the result of weak reddish purple color developed by ninhydrin indicates that the antimicrobial substance (UV254-B) is a peptide substance.

In addition, the antimicrobial substance (UV254-B) is estimated to be a lipopeptide-based substance when the antimicrobial substance (UV254-B) is fluoresced by a 254 nm UV lamp.

Experimental Example :

< Experimental Example  1> Toxin  Generation test

Detection of the presence or absence of enterotoxin production in Bacillus sp. Was carried out using a hemolysin BL (HblA) gene for polymerase chain reaction (PCR) and immunoreactive toxin detection. Reaction (reversed passive latex agglutination, RPLA kit, Oxoid) method was confirmed.

As primer for HblA gene identification, the sequence of forward primer is 5'-CGG GCG TTC TAG GGC ATA TTG AG-3 ', and the sequence of reverse primer is 5'-GCG AGT AGT TTA TTA GGG ATT TTT TTC A-3') Was used.

The test bacteria were inoculated in Mueller-Hinton agar (Difco co, france), and colonies incubated overnight at 37 ° C were suspended in 50 µL of distilled water and heated at 98 ° C for 20 minutes to extract DNA. 2 μL of DNA extract, 2 μL of mixed primer, 4 μL of PCR mixture (deoxy-nucleotide triphosphate, dNTP; Taq polymerase, 10 x buffer), 12 μL of 8-MOP (methoxypsoralene), or 20 μL total )

The reaction using the HblAl primer pair with a GeneAmp PCR system 9600, Perkin-Elmer Cetus Corp., Norwalk, CT, USA was carried out for 5 minutes of predenaturation at 94 ° C and for 45 seconds at 94 ° C. PCR was carried out for 30 cycles of annealing at 65 ° C. for 45 seconds, extension at 72 ° C. for 45 seconds, and last extension at 72 ° C. for 5 minutes. 5 μL of the amplified product by PCR was placed in the groove of 2% agarose gel (promega, Madision, WI, USA) and electrophoresed for 20 minutes to confirm the presence or absence of bands (FIG. 2).

Toxin  To check the existence PCR Result of

In FIG. 2, PCR amplification products of HblA gene fragments of B. subtilis BY08 and B. cereu KCTC 3624 were electrophoresed on agarose gels, and then the presence of enterotoxin was produced through band presence. As a result, the test strain Bacillus cereus (B. cereus KCTC 3624) was found to produce harmful toxins to the human body from the results of the clear band band at 586 bp, Bacillus subtilis (B of the present invention) subtilis) BY08 has not been shown to produce harmful toxins.

< Experimental Example  2> Bacillus To Cereus (B. cereus KCTC 3624)  Antimicrobial Activity Measurement

In order to show the best antimicrobial activity of the isolated B. subtilis BY08 strain, the change of antimicrobial activity with medium pH and culture time was as follows.

The optimum pH was set in a 20mL TSB medium in a 50mL Erlenmeyer plush and sterilized at 121 ° C. for 15 minutes by adjusting the initial pH of the medium to a range of 5.5-8.5 with 0.1 N HCl and 0.1 N NaOH solution. Inoculated with 1% of the pre-culture solution of the selected strains in the sterilized medium and incubated at 30 ℃, 180rpm for 24 hours and examined the antimicrobial activity. The optimum culture temperature was examined in the same manner as the setting test of the optimum pH in the temperature range of 25 ~ 50 ℃. The optimum incubation time was also inoculated in TSB medium and incubated at 30 ° C and 180 rpm for 60 hours while taking samples for 0, 6, 12, 24, 36, 48, and 60 hours for antimicrobial activity against various types of harmful microorganisms. Measured.

Incubation temperature and pH Change of antimicrobial activity

The results of experiments on the effects of incubation temperature and pH on the change of antibacterial activity of B. subtilis BY08 are shown in Table 5.

As shown in Table 5, the antimicrobial activity against Bacillus cereus (B. cereus KCTC 3624) was high at 25 to 40 ℃ as a result of culturing while changing the culture temperature in the range of 25 ~ 50 ℃.

From the above results, the expression level of the antimicrobial substance (UV254-B) varies according to the culture temperature of B. subtilis BY08 strain, and a large amount of antimicrobial substance (UV254-B) is expressed at 25 to 40 ° C. It can be seen that the activity is excellent. In addition, it can be seen that the most preferable culture temperature is 30 ℃ from the results. Thus, the optimum incubation temperature range for the maximum antimicrobial (UV254-B) expression was found to be 25 to 40 ℃.

In addition, after adjusting the initial pH in the range of 5.5 ~ 8.0 and incubated at 30 ℃ for 24 hours to examine the effect of the initial pH, the production of antimicrobial material (UV254-B) changes depending on the pH of the medium, pH is 7.0 to 8.0 It showed a strong antimicrobial activity in the range, it was found that the most potent antimicrobial activity in the test zone which is most preferably 7.5. From these results, it was found that the B. subtilis BY08 strain had a difference in the expression level of the antimicrobial substance (UV254-B), which had the highest antimicrobial activity depending on pH, and the maximum antimicrobial substance (UV254-B). B) The optimal pH for expression was found to be 7.0 to 8.0.

Antimicrobial Activity According to Incubation Time

After inoculating B. subtilis BY08 strain to TSB medium and incubating at 30 rpm at 180 rpm, the change of the antimicrobial activity according to the culture time is shown in FIGS. 5 and 6. The antimicrobial activity against B. cereus was very high from 12 to 48 hours. However, at 60 hours of culture, the antimicrobial activity was almost disappeared.

From the above results, it can be seen that there is a difference in the production of the antimicrobial material (UV254-B) according to the incubation time, and the optimal culture for the production of the antimicrobial material (UV254-B) derived from B. subtilis BY08 12 ~ 48 hours was considered appropriate.

< Experimental Example  3> Stability test for antibacterial substance

pH  Stability test of antimicrobial material by adjustment and heat treatment

In order to investigate the effect of the antimicrobial substance (UV254-B) on the pH, it was dissolved in a buffer solution adjusted to pH 3.0, pH 5.0, pH 7.0, pH 9.0, pH 11.0, treated at 37 ° C. for 2 hours, and then the paper disk ( The diameter (mm) of the clear zone in which growth inhibition appeared was measured by the paper-disc method. In addition, to determine the thermal stability of the antimicrobial substance (UV254-B), the lyophilized antimicrobial substance was dissolved in sterile water and heat-treated at 30, 40, 60, 80, and 100 ° C for 1 hour, and then the antibacterial activity by the paper disc method. Was measured.

The results of examining the stability of pH and temperature of the antimicrobial substance (UV254-B) are shown in Table 7.

Change of Antimicrobial Activity by Enzyme Treatment of TLC Isolating Antibiotic (UV254-B)

In order to investigate the effect of the antimicrobial substance (UV254-B) on the change of antimicrobial activity after enzyme treatment, proteinase K, lipase and esterase (UV) were applied to the antimicrobial substance (UV254-B). The change of antimicrobial activity after esterase) was also shown in Table 7.

As shown in Table 7, the antimicrobial material (UV254-B) did not show antimicrobial activity after heating for 1 hour at a high temperature of 100 ℃, but maintained a relatively strong antibacterial even after heating for 1 hour at 80 ℃.

In addition, the antimicrobial substance (UV254-B) was reduced in the acidic side of pH 3 and 5, but maintained a very stable antimicrobial activity in the alkaline range up to pH 7-11, especially in the alkaline pH 9 Appeared strongly. It was found that the antimicrobial substance (UV254-B), which is a purified substance using TLC, was not significantly affected by the pH change. Therefore, when passing through the stomach is expected to be able to exert its efficacy by the acidic pH of the stomach.

Furthermore, proteinase K, lipase and estera were applied to the antimicrobial substance (UV254-B) in order to determine the effect of the antimicrobial substance (UV254-B) after the enzyme treatment. After the reaction of esterase, the antibacterial activity (UV254-B) was completely lost after treatment with proteinase K and lipase, but after treatment with esterase Not reduced.

From these results, the antimicrobial substance (UV254-B) is a substance of fatty acid having a structure having a protein or a long peptide chain, and is assumed to be a lipopeptide-based substance.

< Experimental Example  4> Minimum Antimicrobial Activity Concentration Test

The minimum inhibition concentration (MIC) of B. cereus was measured by the liquid microdilution method (Bhatta and Kapadnis, 2010). That is, put sterile TSB medium (100 μL) into each well of a 96-well microplate, add 256 μg / mL (100 μL) of the antimicrobial substance (UV254-B), and apply the antimicrobial substance (UV254-B) twice by step. Diluted. B. cereus KCTC 3624 cultured at 1.5 × 108 cells / mL (McFarland; 0.5) was diluted 10-fold (1.5 × 107 cells / mL) and inoculated in each well by 10 μL at 37 ° C. After growing for 24 hours at, the extent of growth was read. At this time, the antimicrobial activity was expressed as MIC, and MIC was determined as the minimum concentration of the sample without 100% growth of Bacillus cereus (B. cereus KCTC 3624). In addition, the control group was compared with the group containing no B. cereus KCTC 3624 culture.

Minimum Antimicrobial Activity Concentration of Antimicrobial Substances

The results of testing the minimum inhibitory concentration of bacteria by performing a broad microdilution method (Baker et al., 1991) on the antimicrobial substance (UV254-B) are shown in FIG. 8. The antimicrobial activity (UV254-B) showed a low antimicrobial activity with a minimum antimicrobial activity concentration value of ≧ 64 μg / mL against B. cereus (1.5 × 10 5 CFU / mL).

< Experimental Example  5> LC Of ESI - MS Characterization experiment such as molecular weight measurement of antimicrobial substance through analysis

Antimicrobial LC Of ESI - MS analysis

Antimicrobial (UV254-B) analysis isolated from TLC selected electrospray ionization (ESI) ionization as the MS / MS analysis condition and used MRM (multiple reaction monitoring) in positive ion mode. A: water, 0.1% formic acid, B: acetonitrile, 0.1% formic acid was used as a mobile phase, and the column was Capcell Pak C18 (2.0 × 100 mm, 5 μm, Shiseido, Japan).

The flow rate was 0.23 mL / min, and the analysis was carried out at a flow rate of 0.23 mL / min while maintaining the mobile phase B from 10% to 20 minutes at 100% from 0% in a linear gradient of analysis time. From 21 to 25 minutes, the linear gradient allowed the mobile phase B to be 10%.

The temperature of the analytical column was kept constant at 40 ℃. Both the nebulizing gas and the collision gas used nitrogen gas, and other MS / MS analysis parameters were set to spray voltage 110 V and ion spray temperature to 300 ° C. The scan range was m / z 100-1,800.

LC Of ESI - MS Of MS Analysis of Antimicrobial Molecular Weight Using

Chromatograms obtained by LC / ESI-MS / MS analysis of the antimicrobial substance (UV254-B) are shown in FIGS. 9, 10 and 11. The peak peak retention time was about 15 minutes, and the molecular weight of the peaks in 15 minutes was detected by protonated molecular ion / product ion pairs when scanned using LC / ESI-MS / MS.

In other words, peaks were observed at m / z 1133.6 and 1700.5 (FIG. 11), and these results and the results of color reaction in TLC showed that the antimicrobial compounds isolated in this study were relatively cyclic lipopeptides based on molecular weight. As a compound of the present invention, suspectin (surfactin [1044.6005-1074.5780 (m / z)]), iturine (ituline [1061.5812, 1075.5810 (m / z)]), pengisin ( fengycin [1449.5587-1515.5127 (m / z)]) has been identified as a novel antimicrobial material that is completely different from the material of the family.

< Experimental Example  6> Bacillus Cereus (B. cereus ) And Listeria  Monocytogenes (L. monocytogenes)  Experiment showing specific antimicrobial and antagonistic effects

B. cereus, pathogenic microorganisms from various foods, Listeria monocytogenes, S. enterica and S. aureus, and B. subtilis used for fermentation of feed. , B. licheniformis (B. licheniformis) and B. amyloliqufaciens (B. amyloliqufaciens) and the antimicrobial range of the antimicrobial material (UV254-B) according to the present invention was shown in Table 8 the results of the investigation.

As a result, the antibacterial substance (UV254-B) according to the present invention showed antimicrobial activity against gram positive strains Bacillus cereus and Listeria monocytogenes, whereas Bacillus subtilis (B. subtilis), B. licheniformis, B. amyloliqufaciens, S. enterica and S. aureus did not show antimicrobial effects (Table 9). Antimicrobial agents isolated from Bacillus spp. Were found to be iturin, bacillomycin, prepastatin and surfactin. These antimicrobial agents were Gram-negative bacteria, benign bacteria and fungi. (Shelburne et al., 2007) Isolation and identification of microorganisms showing antimicrobial activity against B. cereus and L. monocytogenes specifically This is the first result. The antimicrobial substance (UV254-B) isolated from this study has high antibacterial activity against B. cereus and L. monocytogenes strains and is commonly applied to Bacillus (B. cereus). Bacillus) did not show antimicrobial activity against beneficial bacteria.

As described above, the novel antibacterial substance (UV254-B) and B. subtilis BY08 producing the same according to the present invention are Bacillus cereus (B. cereus) and Listeria monocytogenes (L. monocytogenes). It can be seen that the antimicrobial activity and antagonistic effect are excellent.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. It is natural.

Korea Biotechnology Research Institute KCTC18217P 20110810

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Claims (8)

delete delete delete delete Bacillus subtilis having antimicrobial activity against B. cereus and Listeria monocytogenes at the same time, producing antilipides based on lipopeptides, and comprising the nucleotide sequence of SEQ ID NO: 1 ) KCTC 18217P strain.
6. The method of claim 5,
The Bacillus subtilis KCTC 18217P strain is cultured under the conditions of the following (a) to (c) Bacillus subtilis KCTC 18217P strain.
(a) a temperature of 25 to 40 ° C;
(b) pH 7.0 to 8.0;
(c) incubation time 12 hours to 48 hours;
The method of claim 5,
The Bacillus subtilis KCTC 18217P strain is a Bacillus subtilis (Bacillus) characterized in that cultured in a medium containing any one or more nitrogen sources selected from the group consisting of yeast extract, soytone and soybean extract subtilis) KCTC 18217P strain.
The method of claim 5,
The Bacillus subtilis KCTC 18217P strain is cultured in a medium comprising any one or more carbon sources selected from the group consisting of glucose, sucrose and glycerol. Bacillus subtilis KCTC 18217P strain.

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