NOVEL LACTOBACILLUS BREVIS BACTERIOPHAGE LAC-BRP-1 AND USE THEREOF FOR INHIBITING LACTOBACILLUS BREVIS PROLIFERATION

Abstract

The present invention relates to a Siphoviridae bacteriophage Lac-BRP-1 that is isolated from the nature and can kill Lactobacillus brevis cells specifically, which has a genome represented by the nucleotide sequence of SEQ. ID. NO: 1 (Accession NO: KCTC 12659BP), and a method for preventing and treating the contaminations of Lactobacillus brevis by using the composition comprising the bacteriophage as an active ingredient.

Claims

1. A Siphoviridae bacteriophage Lac-BRP-1 that is isolated from the nature and can kill Lactobacillus brevis cells specifically, which has the genome represented by the nucleotide sequence of SEQ. ID. NO: 1.

2. A composition for preventing and treating the contaminations of Lactobacillus brevis, which comprises the bacteriophage Lac-BRP-1 of claim 1 as an active ingredient.

3. The composition for preventing and treating the contaminations of Lactobacillus brevis according to claim 2, wherein said composition is used to prevent or treat the contaminations of Lactobacillus brevis in a process for producing bio-ethanol.

4. A method for preventing or treating the contaminations of Lactobacillus brevis, which comprises a step of adding the composition of claim 2 comprising the bacteriophage Lac-BRP-1 as an active ingredient.

5. The method for preventing or treating the contaminations of Lactobacillus brevis according to claim 4, wherein said composition is added to prevent or treat the contaminations of Lactobacillus brevis in a process for producing bio-ethanol.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

[0032] FIG. 1 is an electron micrograph showing the morphology of the bacteriophage Lac-BRP-1.

[0033] FIG. 2 is a photograph illustrating the capability of the bacteriophage Lac-BRP-1 to kill Lactobacillus brevis. The clear zone on the dish is the formation of plaque by lysis of bacteria cells.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

[0035] However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Isolation of Bacteriophage Capable of Killing Lactobacillus brevis

[0036] Samples were collected from the nature to screen the bacteriophage capable of killing Lactobacillus brevis. The Lactobacillus brevis used for the bacteriophage isolation herein were the one that had been isolated by the present inventors and identified as Lactobacillus brevis previously.

[0037] The isolation procedure of the bacteriophage is described in detail hereinafter. The collected sample was added to the MRS (deMan Rogosa and Sharpe Broth) medium (proteose peptone NO: 3, 10 g/L; beef extract, g/L; yeast extract, 5 g/L; dextrose, 20 g/L; polysorbate 80, 1 g/L; ammonium acetate, 2 g/L; sodium acetate, 5 g/L; magnesium sulfate, 0.1 g/L; manganese sulfate, 0.05 g/L; dipotassium phosphate, 2 g/L) inoculated with Lactobacillus brevis at the ratio of 1/100, followed by standing culture at 30° C. for 3˜4 hours. Upon completion of the culture, centrifugation was performed at 8,000 rpm for 20 minutes and supernatant was recovered. The recovered supernatant was inoculated with Lactobacillus brevis at the ratio of 1/100, followed by standing culture at 30° C. for 3˜4 hours. When the sample contained the bacteriophage, the above procedure was repeated total 5 times in order to increase the titer of the bacteriophage. After repeating the procedure 5 times, the culture solution proceeded to centrifugation at 8,000 rpm for minutes and the resulting supernatant was recovered. The recovered supernatant was filtrated by using a 0.45 μm filter. The obtained filtrate was used in spot assay for examining whether or not the bacteriophage capable of killing Lactobacillus brevis was included therein.

[0038] Spot assay was performed as follows; MRS medium was inoculated with Lactobacillus brevis at the ratio of 1/100, followed by standing culture at 30° C. for overnight. 3 ml (2.0 of OD.sub.600) of the culture broth of Lactobacillus brevis prepared above was spread on the MRS-A (deMan Rogosa and Sharpe Agar) medium (proteose peptone NO: 3, 10 g/L; beef extract, 10 g/L; yeast extract, 5 g/L; dextrose, 20 g/L; polysorbate 80, 1 g/L; ammonium acetate, 2 g/L; sodium acetate, 5 g/L; magnesium sulfate, 0.1 g/L; manganese sulfate, 0.05 g/L; dipotasium phosphate, 2 g/L; agar, 15 g/L) plate. The plate stood in a chamber for about 30 minutes to dry. After drying, 10 μl of the resulting filtrate was spotted directly onto the surface of the Lactobacillus brevis lawns and dried for about 30 minutes. Following drying, the plate was incubated at 30° C. for a day and then, examined for the formation of clear zones on the surface of the bacterial lawns. If a clear zone was generated where the filtrate was dropped, it could be judged that the bacteriophage capable of killing Lactobacillus brevis was included in the filtrate. Through the above procedure, the filtrate containing the bacteriophage having the killing ability of Lactobacillus brevis could be obtained.

[0039] After that, the bacteriophage was isolated from the filtrate confirmed above to have the bacteriophage capable of killing Lactobacillus brevis. The conventional plaque assay was used for the isolation of pure bacteriophages. In detail, a plaque formed in the course of the plaque assay was picked up by using a sterilized tip, which was then added to the culture solution of Lactobacillus brevis, followed by standing culture at 30° C. for 4˜5 hours. Upon completion of the culture, centrifugation was performed at 8,000 rpm for 20 minutes to obtain supernatant. The recovered supernatant was inoculated with Lactobacillus brevis culture at the ratio of 1/50, followed by standing culture again at 30° C. for 4˜5 hours. To increase the titer of the bacteriophage, the above procedure was repeated at least 5 times. Then, centrifugation was performed at 8,000 rpm for 20 minutes to obtain supernatant. Plaque assay was performed with the obtained supernatant. In general, the pure bacteriophage isolation is not completed by one-time procedure, so the above procedure was repeated by using the plaque formed above. After at least 5 times of repeated procedure, the solution containing the pure bacteriophage was obtained. The procedure for the isolation of the pure bacteriophage was generally repeated until the generated plaques became similar in sizes and morphologies. And the final pure bacteriophage isolation was confirmed by the observation under electron microscope. Until the pure bacteriophage isolation was confirmed under electron microscope, the above procedure was repeated. The observation under electron microscope was performed by the conventional method. Briefly, the solution containing the pure bacteriophage was loaded on copper grid, followed by negative staining with 2% uranyl acetate. After drying thereof, the morphology was observed under transmission electron microscope. The electron micrograph of the bacteriophage isolated in the present invention is presented in FIG. 1. From the morphological observation, the bacteriophage isolated above was identified as belonging to the family Siphoviridae.

[0040] The solution containing the pure bacteriophage confirmed above proceeded to purification. The culture broth of Lactobacillus brevis was added to the solution containing the pure bacteriophage at the volume of 1/50 of the total volume of the bacteriophage solution, followed by culturing again for 4˜5 hours. Upon completion of the culture, centrifugation was performed at 8,000 rpm for 20 minutes to obtain supernatant. This procedure was repeated 5 times to obtain a solution containing enough numbers of the bacteriophage. The supernatant obtained from the final centrifugation was filtered by a 0.45 μm filter, followed by the conventional polyethylene glycol (PEG) precipitation. Particularly, PEG and NaCl were added to 100 ml of the filtrate until reaching 10% PEG 8000/0.5 M NaCl, which stood at 4° C. for 2˜3 hours. Then, centrifugation was performed at 8,000 rpm for 30 minutes to obtain the bacteriophage precipitate. The resulting bacteriophage precipitate was resuspended in 5 ml of buffer (10 mM Tris-HCl, 10 mM MgSO.sub.4, 0.1% Gelatin, pH 8.0). This solution was called as the bacteriophage suspension or bacteriophage solution.

[0041] As a result, the pure bacteriophage purified above was collected, which was named as the bacteriophage Lac-BRP-1 and then deposited at Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology in Aug. 21, 2014 (Accession NO: KCTC 12659BP).

Example 2: Separation and Sequence Analysis of the Bacteriophage Lac-BRP-1 Genome

[0042] The genome of the bacteriophage Lac-BRP-1 was separated as follows. The genome was separated from the bacteriophage suspension obtained in Example 1. First, in order to eliminate DNA and RNA of Lactobacillus brevis included in the suspension, DNase I and RNase A were added 200 U each to 10 ml of the bacteriophage suspension, which was incubated at 37° C. for 30 minutes. 30 minutes later, to remove the DNase I and RNase A activity, 500 μl of 0.5 M ethylenediaminetetraacetic acid (EDTA) was added thereto, which was incubated for 10 minutes. The suspension was further incubated at 65° C. for 10 minutes and then added with 100 μl of proteinase K (20 mg/ml) to break the outer wall of the bacteriophage, followed by incubation at 37° C. for 20 minutes. After that, 500 μl of 10% sodium dodecyl sulfate (SDS) solution was added thereto, followed by incubation at 65° C. for 1 hour. 10 ml of the mixture of phenol:chloroform:isoamylalcohol in a ratio of 25:24:1 was added thereto, followed by mixing well. The mixture was centrifuged at 13,000 rpm for 15 minutes to separate each layer. The upper layer was obtained, to which isopropyl alcohol was added at the volume of 1.5 times the volume of the upper layer, followed by centrifugation at 13,000 rpm for 10 minutes to precipitate the genome of the bacteriophage. After collecting the precipitate, 70% ethanol was added to the precipitate, followed by centrifugation at 13,000 rpm for 10 minutes to wash the precipitate. The washed precipitate was recovered, vacuum-dried and then dissolved in 100 μl of water. This procedure was repeated to obtain a sufficient amount of the bacteriophage Lac-BRP-1 genome.

[0043] The nucleotide sequence of the genome of the bacteriophage Lac-BRP-1 obtained above was analyzed by Next Generation Sequencing (NGS) using illumina Mi-Seq device at National Instrumentation Center for Environmental Management, Seoul National University. As a result, it is suggested that the final genome of bacteriophage Lac-BRP-1 have 136,174 bp of size and the nucleotide sequence of the whole genome has SEQ. ID. NO: 1.

[0044] Similarity of the genomic sequence of the bacteriophage Lac-BRP-1 obtained above with the previously reported bacteriophage genome sequences was investigated by using BLAST on Web (http://www.ncbi.nlm.nih.gov/BLAST/). From the BLAST result, it is noted that genomic sequences having more than 50% homology with the genomic sequence of bacteriophage Lac-BRP-1 were not found.

[0045] Based upon this result, it is concluded that the bacteriophage Lac-BRP-1 should be a novel bacteriophage not reported previously.

Example 3: Investigation of Killing Ability of the Bacteriophage Lac-BRP-1 Against Lactobacillus brevis

[0046] The killing ability of the isolated bacteriophage Lac-BRP-1 against Lactobacillus brevis was investigated. To do so, the formation of clear zone was observed by the spot assay by the same manner as described in Example 1. The Lactobacillus brevis used for this investigation were total 15 strains which had been isolated and identified as Lactobacillus brevis previously by the present inventors. The bacteriophage Lac-BRP-1 demonstrated the killing ability against 11 strains of the Lactobacillus brevis used in this experiment. The representative result of the killing ability test is shown in FIG. 2. In the meantime, the activity of the bacteriophage Lac-BRP-1 to kill Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Streptococcus agalactiae, Streptococcus uberis, Haemophilus parasuis, Bordetella bronchiseptica, and Escherichia coli was also investigated. As a result, it is decided that the bacteriophage Lac-BRP-1 did not have the killing activity against these microorganisms.

[0047] Therefore, it is confirmed that the bacteriophage Lac-BRP-1 had the specific killing ability against Lactobacillus brevis and a broad antibacterial spectrum against Lactobacillus brevis, suggesting that the bacteriophage Lac-BRP-1 of the present invention could be used as an active ingredient for the composition for preventing and treating the contaminations of Lactobacillus brevis.

Example 4: Preventive Effect of Bacteriophage Lac-BRP-1 on the Contaminations of Lactobacillus brevis

[0048] The bacteriophage Lac-BRP-1 was investigated under a similar bio-ethanol-producing condition whether it could be used to prevent the contaminations of Lactobacillus brevis or not. 100 mL of molasses medium (16% total sugars, 0.046% KH.sub.2PO.sub.4, 0.225% urea) was added to 4 of 300 mL erlenmeyer flasks respectively. Then, the bacteriophage Lac-BRP-1 suspension prepared by the same manner as described in Example 1 was added to only 2 flasks to adjust the concentration of bacteriophage at 1×10.sup.6 pfu/mL, while the other 2 flasks remained intact. After that, Lactobacillus brevis cells were added to all flasks to reach 1×10.sup.4 cfu/mL. The resulting solutions were cultivated at 30° C. for 16 hours. Then, 100 μL of the solution was collected from each flask, ten-fold serially diluted in physiological saline solution, spread onto MRS-A medium plates respectively and then cultivated at 30° C. in a plate incubator for overnight. Upon completion of overnight culture, the number of colonies formed was counted. Then, based upon the count of colonies, the concentration of Lactobacillus brevis was calculated in each flask. The results are as follows.

TABLE-US-00001 TABLE 1 Suppresion of Lactobacillus brevis contamination Concentration of Item Lactobacillus brevis cells Flask 1 (− bacteriophage approximately 10.sup.6 cfu/mL solution) Flask 2 (− bacteriophage approximately 10.sup.8 cfu/mL solution) Flask 1 (+ bacteriophage approximately 10.sup.2 cfu/mL solution) Flask 2 (+ bacteriophage approximately 10.sup.2 cfu/mL solution)

[0049] The above results indicate that the bacteriophage Lac-BRP-1 not only inhibited the growth of Lactobacillus brevis but also could kill them. Therefore, it is concluded that the bacteriophage Lac-BRP-1 could be used as an active ingredient of the composition in order to prevent the contaminations of Lactobacillus brevis.

Example 5: Effect of the Treatment with Bacteriophage Lac-BRP-1 on the Contaminations of Lactobacillus brevis

[0050] The bacteriophage Lac-BRP-1 was investigated under a similar bio-ethanol-producing condition whether it could be used to treat the contaminations of Lactobacillus brevis or not. 100 mL of molasses medium was added to 4 of 300 mL erlenmeyer flasks respectively and inoculated with yeast cells to reach 5×10.sup.7 cfu/mL. Then, Lactobacillus brevis (lx 10.sup.4 cfu/mL) was innocuated to all erlenmeyer flasks. After innoculation, the bacteriophage Lac-BRP-1 suspension prepared by the same manner as described in Example 1 was added to only 2 flasks to adjust the concentration of bacteriophage at 1×10.sup.7 pfu/mL, while the other 2 flasks remained intact. The resulting solutions were cultivated at 30° C. for 24 hours. Then, 100 μL of the solution was collected from each flask, ten-fold serially diluted in physiological saline solution and spread onto MRS-A medium plates respectively. The resulting plates were cultivated at 30° C. in a plate incubator for 24 hours. Upon completion of overnight culture, the number of colonies formed was counted. Then, based upon the count of colonies, the concentration of Lactobacillus brevis was calculated in each flask. The results are as follows.

TABLE-US-00002 TABLE 2 Treatment of Lactobacillus brevis contamination Concentration of Item Lactobacillus brevis Flask 1 (− bacteriophage approximately 10.sup.8 cfu/mL solution) Flask 2 (− bacteriophage approximately 10.sup.8 cfu/mL solution) Flask 1 (+ bacteriophage approximately 10.sup.1 cfu/mL solution) Flask 2 (+ bacteriophage Not detected solution)

[0051] From the above results, it is concluded that the bacteriophage Lac-BRP-1 of the present invention could be used as an active ingredient of the composition in order to treat the contaminations of Lactobacillus brevis.

Example 6: Application Tests

[0052] The bacteriophage Lac-BRP-1 was investigated practically in the process for producing bio-ethanol whether it could be applied to improve the productive yield of bio-ethanol or not. For this application, the bacteriophage Lac-BRP-1 suspension prepared by the same manner as described in Example 1 was utilized. The application tests were performed by adding bacteriophage suspension to a yeast cream to be the bacteriophage concentration of 1×10.sup.7 pfu/mL (test {circle around (1)}), putting bacteriophage suspension into a fermentation tank to be the bacteriophage concentration of 1×10.sup.6 pfu/mL (test {circle around (2)}), putting bacteriophage suspension into a fermentation tank to be the bacteriophage concentration of 1×10.sup.6 pfu/mL along with the bacteriophage suspension added to a yeast cream to be the bacteriophage concentration of 1×10.sup.7 pfu/mL (test {circle around (3)}), and without any treatment (test {circle around (4)}). Test {circle around (4)} was included as a control group. These application tests were conducted total 10 times and the results are as follows. In Table 3, the bio-ethanol productivity is average values obtained after measuring 10 times and considering that of test {circle around (4)} as 100%.

TABLE-US-00003 TABLE 3 Results of application tests Item Productivity of bio-ethanol Test {circle around (1)} 104% Test {circle around (2)} 106% Test {circle around (3)} 107% Test {circle around (4)} 100%

[0053] From the above results, it is confirmed that the composition of the present invention comprising the bacteriophage Lac-BRP-1 could be effective to improve the productive yield of bio-ethanol.

[0054] Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended Claims.