KR101235561B1 - Lactobacillus plantarum clp-1 strain having anti-virus and anti-bacterial activity and direct-fed microorganisms comprising the same - Google Patents

Abstract

The present invention relates to a novel Lactobacillus plantarum CLP-1 strain having antiviral activity and antimicrobial activity and a probiotic comprising the same, wherein the strain of the present invention is antiviral to rotaviruses, E. coli and Salmonella and the like. Excellent antimicrobial activity can be effectively used for the prevention and treatment of diseases related thereto.

Description

LACTOBACILLUS PLANTARUM CLP-1 STRAIN HAVING ANTI-VIRUS AND ANTI-BACTERIAL ACTIVITY AND DIRECT-FED MICROORGANISMS COMPRISING THE SAME}

The present invention relates to a LactOBACILLUS PLANTARUM CLP-1 strain having a antiviral activity against diarrhea-induced viruses and an antimicrobial activity against Gram-negative pathogens or a probiotic comprising a culture of the strain as an active ingredient. .

In the livestock industry, it is now common to increase productivity by preventing diseases and adding antibiotics to feeds that have the effect of raising livestock. However, the abuse of these antibiotics leads to the development of resistant pathogens as well as the presence of antibiotics in the livestock products, so developed countries strictly regulate the use of antibiotics.

Due to these problems, efforts have been made to develop safe products while replacing antibiotics using organic acids, inorganic acids, plant-derived extracts, and probiotics. Among these, probiotics have various effects depending on the type of microorganism. In the case of lactic acid bacteria known to inhibit the growth of harmful bacteria in the intestine, recent studies on the antiviral and immunological effects of avian influenza virus have been reported. .

 On the other hand, in pig farms, mortality due to disease infections such as mortality and diarrhea caused by enteritis is about 20%, which is the biggest cause of productivity loss. The gastrointestinal disease of pigs occurs in all parts of the pigs (pigs, rearing pigs and sows), but among them, the frequency of infection is the most frequent and the damage is the most serious. The most problematic causes in piglets include rotavirus, coronavirus (Porcine epidemic diarrhea (PED) virus), Escherichia coli and Salmonella.

This rotavirus is a major cause of acute colitis and the induction of acute diarrhea and enteritis in young livestock. Various lactic acid bacteria having antiviral efficacy against such rotaviruses are disclosed. For example, Bifidobacterium infantis MAEIL-K9 (Patent No. 0813637), Lactobacillus reuteri Probio-16 ( Lactobacillus reuteri Probio-) 16; Patent No. 043 749 Ho), Bifidobacterium breve (Bifidobacterium breve ; Patent Registration No. 0232639), Bifidobacterium Rongum AR81 ( Bifidobacterium) longum AR81; Patent registration No. 0492455) and Lactobacillus casei HY7201 ( Lactobacillus casei HY7201; Patent Registration No. 0462192) is registered. However, these strains are strains included in dairy products, and it has not been reported that the Lactobacillus plantarum strain of the present invention has such efficacy.

In addition, coronavirus has been recognized mainly as an animal infection virus, such as dogs, pigs, cows, etc., but became known as the causative agent of acute respiratory syndrome (SARs) SARS is a new corona virus. When infected, coronavirus can cause fever, dyspnea, vomiting, diarrhea and dehydration, which can act as lethal pathogens, and are highly contagious and have a good ability to bind to other pathogens.

Accordingly, the present inventors found that the strains of the genus Lactobacillus plantarum isolated from kimchi have excellent antibacterial and antiviral activities, and named them as Lactobacillus plantarum CLP-1 strains.

Accordingly, it is an object of the present invention to provide a novel strain excellent in antimicrobial and antiviral activity.

Another object of the present invention to provide a probiotic comprising the strain as an active ingredient.

Still another object of the present invention is to provide a feed additive comprising the strain as an active ingredient.

In order to achieve the above object, the present invention provides Lactobacillus plantarum CLP-1 strain (Accession No. KACC91591P).

In order to achieve the above another object, the present invention provides a probiotic comprising the strain or its culture as an active ingredient.

In order to achieve the above another object, the present invention provides a feed additive comprising the strain or its culture as an active ingredient.

Since the Lactobacillus plantarum CLP-1 strain of the present invention shows antiviral activity against diarrheal causative agents rotavirus and coronavirus (PED virus), and excellent antibacterial activity against Escherichia coli and Salmonella, CLP-1 strain or the above Probiotics containing the culture of the strain is prepared and administered to the drinking water or added to the feed to provide feed, which can prevent livestock diseases and improve productivity, and unlike antibiotics, there is no risk of remaining in livestock or developing resistant bacteria. It can contribute to the production of safe livestock.

Figure 1 is a screening of lactic acid bacteria having antimicrobial activity against hemolytic E. coli.
Figure 2 shows the rotavirus and coronavirus content according to the CLP-1 strain treatment through agarose gel electrophoresis after PCR.
3 and 4 show the biochemical properties and identification results of the CLP-1 strain.
Figure 5 shows the growth of the CLP-1 strain according to the concentration of bile added.
Figure 6 shows the results of cell adhesion to MA104, Vero and Caco-2 cells of the CLP-1 strain.
Figure 7 shows the IFN-β expression level of MA104 cells corresponding to rotavirus infection, with or without the addition of CLP-1 strain.
Figure 8 shows the growth inhibition rate of CLP-1 strain culture against disease-causing bacteria.
9 and 10 show the pH and thermal stability of the antimicrobial active material produced by the CLP-1 strain, respectively.
11 and 12 show the antimicrobial activity against hemolytic E. coli according to the culture period and culture temperature of the CLP-1 strain.
Figure 13 confirms the antiviral activity against rotavirus of CLP-1 strain according to the culture temperature by gel electrophoresis.

The present invention is a novel Lactobacillus plantarum CLP-1 strain having antiviral activity against rotavirus, a causative agent of diarrheal disease, and coronavirus, a causative agent of swine pandemic diarrheal disease (PED), and antibacterial activity against Escherichia coli and Salmonella. To provide.

The Lactobacillus plantarum CLP-1 strain of the present invention has the 16S rRNA sequence represented by SEQ ID NO: 1, and most of the existing Lactobacillus standard strains are available for galactose, esculin, melibiose and genthiobiose. Compared to this positive, the strain of the present invention is characterized by a negative availability to galactose, esculin, melibiose and genthiobiose.

The present inventors named this new strain Lactobacillus plantarum CLP-1 strain and deposited it with the Agricultural Genetic Resource Center on October 20, 2010 and deposited it as accession number KACC91591P.

CPL-1 strain according to the present invention is pH 4.5 to 7.5, preferably 6.0 to medium in a medium such as MRS, ATP, Elliker, Tomato Juice Logo SL, Todd Hewitt or crude oil. It grows well at temperatures of 7.0 and 20 ° C to 40 ° C, preferably at 25 ° C to 37 ° C, more preferably at 30 ° C.

According to one embodiment of the present invention, the strain of the present invention comprises 10 g to 120 g of glucose, preferably 50 g, 20 g to 80 g of molasses, preferably 50 g of sugar, 2 g to 30 g, preferably 10 g, skimmed soy flour per L. Medium containing 5 g to 50 g, preferably 20 g, 20 g to 70 g corn steep liquor, preferably 40 g, ₂ g to 7 g K ₂ HPO ₄ , preferably 3 g, and 0.2 g to 3 g sodium acetate, preferably 1 g Mass can be produced by inoculating 0.05 to 10% by weight, preferably 0.1% by weight, followed by incubation at 20 ° C to 37 ° C, preferably 30 ° C for 16 to 48 hours, preferably 24 hours.

The present invention also provides a probiotic comprising the Lactobacillus plantarum CLP-1 strain as an active ingredient.

Specifically, the probiotic of the present invention alone or an active ingredient such as Lactobacillus plantarum CLP-1 strain or its culture, or a group consisting of lactose, glucose, skim milk, zeolite and mixtures thereof. Together with excipients selected from. When the excipient is included, the probiotic may include 10 to 95 wt% of the active ingredient, and 5 to 90 wt% of the excipient based on the total weight of the probiotic.

The probiotic of the present invention may be supplied in liquid form, and may be formulated in the form of powder, granules or pellets through lyophilization and vacuum drying, and the form of powder is preferred.

Probiotics prepared in this way can be administered to the drinking water or feed to suppress the occurrence of disease in the livestock.

In addition, the present invention provides a feed additive comprising a Lactobacillus plantarum CLP-1 strain or a culture thereof. Specifically, the feed additive may include 10 ⁶ to 10 ⁷ cfu / g of live bacteria or 1 to 30% by weight of the culture solution dried, it may be prepared by further mixing Bacillus, yeast, oligosaccharides, vitamins and minerals.

Probiotics and feed additives prepared in the present invention exhibits antiviral activity against diarrheal causative agents rotavirus, coronavirus (PED virus), and excellent antibacterial activity against Escherichia coli and Salmonella. In addition to preventing disease and improving productivity in livestock, unlike antibiotics, there is no risk of remaining in livestock or developing resistant bacteria, which can contribute to the production of safe livestock.

[Example]

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1 Isolation and Identification of New Lactobacillus Planta Room CLP-1

About 300 samples from kimchi, salted fish, pig intestine, livestock manure and dairy products were diluted properly in sterile saline solution, and then MRS added with 0.0001% (w / v) bromocresol purple reagent. Plated in agar medium and incubated for 24 to 48 hours at 37 ℃ and anaerobic conditions. Among them, about 800 strains of yellow color were selected due to the low pH around the strain by the organic acid produced by the organic acid producing strain. Subsequently, in order to select lactic acid bacteria having antimicrobial activity against Escherichia coli, the strains were cultured at 37 ° C. and anaerobic conditions in MRS agar (p-MRS agar) medium containing 0.1 N concentration of phosphate buffer (pH 7.0). . Overlaid on E. coli culture and incubated at 37 ° C. for 24 hours, 29 strains were first screened to generate growth halo of E. coli (FIG. 1).

In order to select strains having antiviral activity against rotavirus, the first selected strains were inoculated in MRS medium, and cultured at 37 ° C. for 24 hours, and then the culture solution was recovered, and the pH was recovered using 6N NaOH. Adjusted to about 6.0. The culture solution and the Rotavirus culture solution at a concentration of 10 ⁵ TCID ₅₀ were mixed at a ratio of 9: 1 and left in a 5% CO ₂ incubator at 37 ° C. for 1 hour, followed by 40% (v / v) of MA104 cells (African green monkey kidney cells). ) And left in a 5% CO ₂ incubator at 37 ° C. for 1 hour. The medium was then removed and washed twice with phosphate buffered saline (PBS). Subsequently, DMEM maintenance medium with 1% fetal bovine serum (FBS) was added and incubated for 24 hours in a 5% CO ₂ incubator at 37 ° C. After incubation, the cytopathic effect (CPE) was observed, 0.2 mL of the culture was added, 1 mL of Trizol and 0.2 mL of chloroform were added and mixed well, followed by centrifugation at 15,000 rpm for 10 minutes. It was. After centrifugation, 0.65 mL of the supernatant was collected, transferred to a new microtube, 0.5 mL of isopropanol was added thereto, mixed, and left at −70 ° C. for 30 minutes. After 30 minutes, the microtubes were centrifuged at 12,000 rpm for 10 minutes and all of the supernatant was removed. After 1 ml of 70% alcohol was added to the microtube and centrifuged at 12,000 rpm for 5 minutes, the supernatant was discarded and dried in a 60 ° C. drying oven for 5 minutes, and then 0.02 mL of sterilized distilled water was added to extract rotavirus RNA. The extracted RNA was mixed with 10 μl of RNA and 10 μl of sterilized distilled water using an RT premix kit and reacted at 37 ° C. for 60 minutes and 95 ° C. for 5 minutes to synthesize cDNA. The synthesized cDNA was mixed with 3 μl of cDNA, 1 μl of forward primer (SEQ ID NO: 2), 1 μl of reverse primer (SEQ ID NO: 3) and 15 μl of sterilized distilled water using a PCR premix kit for 1 minute at 95 ° C. Rotavirus DNA was synthesized by reacting 35 times at 53 ° C. for 1 minute and at 72 ° C. for 1 minute. Synthesized DNA was loaded into the wells of 1.2% agarose gel (agarose gel) by 5µl, followed by electrophoresis for 20 minutes and the virus content was confirmed by DNA band identification using a UV spectrophotometer.

Subsequently, the three strains that were found to have reduced or completely eliminated rotavirus RNA from the culture-added sample were selected as secondary strains by determining that the rotavirus had antiviral activity. Among the secondary screened strains, in order to select strains having antiviral activity against coronavirus (PEDVirus), coronaviruses and Vero cells (African green monkey kidney cells) were used for the antiviral activity-bearing strains of the rotavirus. In the same manner as the selection method, the Lactobacillus plantarum CLP-1 strain derived from kimchi was finally selected. The results of confirming the antiviral activity against the rotavirus and coronavirus of the CLP-1 strain are shown in FIG.

The results of confirming the morphological and biochemical properties of the selected strains are shown in Table 1 below. As shown in Table 1 below, the strains of the present invention are catalase negative as gram positive bacilli and do not produce CO ₂ using glucose.

Morphology and Biochemical Features reaction Colony shape Small round shape Colony color Pale ivory Cell morphology Short bacilli Gram staining Gram positive Expiratory condition Facultative anaerobic Spore Formability (Endospore) voice CO ₂ production from glucose voice Catalase test voice

In addition, the enzyme activity of the strain using the API ZYM kit (biomerieux, France) using the results of the range of the enzyme reaction expressed in 0 (no reaction) to 5 points (maximum reaction) is shown in Table 2 below. As a result, the strains of the present invention had very high activity of leucine arylamidase and β-galactosidase, and β-glucuronidase, a carcinase that converts benzopyrene into a carcinogenic substance, has no activity and is safe. It was found that the strain. In addition, α-galactosidase generally shows strong activity in Bifidobacterium strains, and is known to be very weak or inactive in Lactobacillus plantarum (applied animal microorganism 2006), but the CLP-1 strain of the present invention. Shows a different activity than the existing Lactobacillus plantarum standard strain in that it shows a strong activity.

enzyme Reaction degree Alkaline phosphatase One Esterase C4 One Esterase Lipase C8 One Lipase C14 0 Leucine arylamidase 4 Valine arylamidase 3 Cystine arylamidase 2 Trypsine 0 α-chimotrypsine 0 Acid phosphatase 2 Naphthol-AS-BI-phosphohydrolase
(Naphtol-AS-BI-phosphohydrolase) 2 α-galactosidase 2 β-galactosidase 4 β-glucuronidase 0 α-glucosidase 3 β-glucosidase 2 N-ethyl-β-glucosaminidase 3 α-mannosidase 0 α-fucosidase 0

In addition, the results of analyzing the sugar availability of the strain using the API 50 CHL kit (bioMerieux, France) is shown in Table 3 and FIG.

In addition, the 16S rRNA gene sequence of the Lactobacillus plantarum CLP-1 strain was the same as SEQ ID NO: 1. As a result of analyzing the 16S rRNA gene sequence, the strain of the present invention is OC-2 of the genus Lactobacillus. It could be seen that the new strain exhibits about 98% homology with the strain (see FIG. 4).

Strip 0-25
Tube / substrate +/- Strip 26-49
Tube / substrate +/-  1 glycerol -  26 salincin +  2 erythritol -  27 cellobiose +  3 d-arabinose -  28 maltose +  4l-arabinos +  29 lactose +  5 ribose +  30 melibiose -  6 d-xylose -  31 sucrose +  7 l-xylose -  32 trehalose +  8 adonitol -  33 Inulin -  9 β-methyl-d-xylose -  34 melezitose +  10 galactose -  35 Raffinose -  11 glucose +  36 starches -  12 fructose +  37 glycogen -  13 manose +  38 xylitol -  14 sorbose -  39 gentiobiose -  15 rhamnose -  40 d-turanose +  16 Dulcitol -  41 d-lyxose -  17 inositol -  42 d-tagatose -  18 mannitol +  43 d-fucose -  19 Sorbitol +  44 l-fucose -  20 α-methyl-d-mannoside -  45 d-arabitol -  21 α-methyl-d-glucoside -  46 l-arabitol -  22 n-acetyl-glucosamine +  47 Gluconate -  23 amygdalin +  48 2-keto-gluconate -  24 arbutin +  49 5-keto-gluconate -  25 esculin -

Based on these results, the selected strain was identified as Lactobacillus plantarum new strain, and it was named as Lactobacillus plantarum CLP-1 strain and deposited with the Agricultural Genetic Resource Center on October 20, 2010. Deposited as No. KACC91591P.

One) Lactobacillus Flora Room CLP Of -1 strain Acid resistance  And Bile Acid Resistance  Confirm

In order to determine the intestinal reachability of the CLP-1 strain, acid resistance and bile acid resistance were confirmed.

First, in order to determine the acid resistance of the CLP-1 strain of the present invention, CLP-1 strains were inoculated in MRS medium and cultured at 37 ° C. for 24 hours, followed by centrifugation to recover only the cells. As a result of confirming the survival rate of the strain after the addition of fresh MRS medium having a pH adjusted to 2.03 to the recovered cells and suspended cells at 37 ℃ for 2 hours, the CLP-1 strain of the present invention is 82.5% survival resistance It was found to be excellent.

In addition, in order to determine the bile acid resistance of the CLP-1 strain of the present invention, CLP-1 strains were inoculated into MRS agar medium containing bile extract (Sigma) at concentrations of 0%, 0.5% and 1.0%, respectively. The growth of the strain was confirmed after standing for 24 to 48 hours at ℃. As shown in Figure 5, it was found that the strain of the present invention grows well in the medium to which the bile extract is added at 1.0% concentration. That is, the CLP-1 strain is capable of growing in a medium in which bile is added at least 0.5% and has a high bile acid resistance, and thus is considered to be an excellent strain.

2) Lactobacillus Flora Room CLP Of -1 strain Intestinal attachment  Confirm

To confirm the intestinal adhesion of the CLP-1 strain of the present invention, Caco-2 cells (human epithelial colorectal adenocarcinoma cell line), MA104 cells and Vero cells were used as follows. As described above, cell adhesion was confirmed.

First, CLP-1 strains were inoculated in MRS liquid medium and incubated anaerobicly at 37 ° C. for 24 hours, and then the pH of the culture medium was adjusted to 6.0 ± 0.2 using 6N NaOH, followed by centrifugation at 3,000 rpm for 10 minutes. Only recovered. The recovered CLP-1 cells were washed twice with phosphate buffered saline (PBS) and suspended in 1 mL of DMEM medium. Sterile cover glass was placed on a 6-well plate, and then Caco-2, MA104, and Vero cells, which were prepared to be 10 ⁶ cells / mL, were divided into monolayers and incubated at 37 ° C. and 5% CO ₂ conditions for 24 hours. The supernatant was then removed and washed twice with 0.5 mL PBS and the PBS was completely removed. 0.5 mL of CLP-1 cell suspension was added to each prepared cell and reacted for 2 hours at 37 ° C. and 5% CO ₂ , followed by washing 6 times with 1 mL PBS. After carefully lifting the cover glass, gram staining was performed, and then observed under a microscope. The results are shown in FIG. 6. In FIG. 6, A and B are photographs before and after MA104 cell attachment, C and D are photographs before and after Vero cell adhesion, and E and F are photographs before and after attachment to Caco-2 cells.

As shown in Figure 6, the strain of the present invention was attached to the cell surface on the cell surface irrespective of the type of the cell was shown around the purple. Therefore, the CLP-1 strain of the present invention was found to be excellent in intestinal adhesion capacity.

3) Lactobacillus Flora Room CLP -Immunoassay of Strains

In general, due to virus infection, organisms try to maintain antiviral status by secreting proteins that regulate immune responses such as intracellular interferon (IFN). Interferon occurs in the initial immune response and when IFN-stimulated gene (ISG) factor 3 (IRF3) is activated through phosphorylation, it moves into the nucleus to stimulate ISG and IFN-α and IFN-β. Promotes the production of This initial immune activity has a great influence on protecting cells from the outside.

However, nonstructural protein 1 (NSP1), one of the nonstructural proteins of rotavirus, binds to IRF3 and inhibits dimerization and intranuclear migration of IRF3, thereby inhibiting IFN production. Thus, to determine whether the CLP-1 strain of the present invention has immunological characteristics against virus infection, pIF-Luc plasmid with IRF3-stimulated IFN-β promoter element (acquired from Hallym University Ilsong Life Science Research Institute) And expression of IFN-β were confirmed using MA104 cells.

Specifically, the polyethyleneimine reagent was mixed with the pIF-Luc plasmid at a ratio of 1: 1, and reacted at room temperature for 30 minutes. The medium of MA104 cells, which had been grown in advance, was removed, and the mixed solution was treated with 1 mL. After 1 hour of reaction at 37 ° C. incubator, the mixture was removed and cultured for 24 hours with the addition of a medium without any addition, a medium treated with rotavirus, and a medium treated with a culture solution of rotavirus and CLP-1 strains, respectively. I was. After 24 hours, the cultures were removed and washed twice with PBS, and then 0.2 mL of luciferase cell lysis reagent was added to recover the cells with a scraper. The recovered cells were completely lysed using a 1 ml syringe, centrifuged at 15,000 rpm, and 0.15 mL of the supernatant was recovered. The recovered supernatant was measured for expression of IFN-β present in the supernatant through a Luciferase repoter assay machine.

As shown in FIG. 7, the expression level of IFN-β was small in the group treated with rotavirus alone (V), and increased in the group treated with culture medium of CLP-1 strain (CS), and the rotavirus. The group treated with the culture medium of CLP-1 strain (V + CS) was slightly less than the CS group. Therefore, it was confirmed that the substances in the culture medium of the CLP-1 strain inhibit the influx of viruses and increase the cell immunity.

4) Lactobacillus Flora Room CLP -1 strain of organic acid Production capacity  Confirm

In order to determine the amount of organic acid produced during the cultivation of the CLP-1 strain of the present invention, the CLP-1 strain was inoculated in an MRS liquid medium and incubated anaerobicly at 37 ° C. for 24 hours. The amount of organic acid was determined by lactic acid quantification method. As a result, the strain culture of the present invention based on lactic acid contained 3.5% of the organic acid, it can be seen that the strain of the present invention produces a large amount of organic acid.

Example 2: Antimicrobial Activity of Lactobacillus Planta Room CLP-1 Strains

In order to investigate the antimicrobial activity of the CLP-1 strain of the present invention, Swine disease-inducing strains Staphylococcus aureus ( Staphylococcus aureus ) , Salmonella choleraesuis , Salmonella typhimurium and Salmonella enteritimurium , Salmolella enteritidis , which are polio- causing strains, and The antimicrobial spectrum of Salmonella gallinarium was investigated.

First, inoculate CLP-1 strain in MRS medium and incubate at 37 ° C. for 20 hours, remove the cells, adjust the pH to 6.0 ± 0.2 using 6N NaOH, filter with 0.45 um sterile filter, and test the filtrate. It was used as a sample. The prepared sample was mixed with tryptic soy medium (BD) in a ratio of 2: 3, and then inoculated with 0.5% (v / v) of the indicators (disease causing bacteria) activated in tryptic soy medium. Incubated for 24 hours at 37 ℃. Subsequently, the absorbance value was measured at 660 nm using a spectrophotometer (1650PC; Simadzu Co., Ltd.).

The strain of the present invention showed more than 50% antimicrobial activity against the five kinds of pathogenic strains as shown in Figure 8, in particular, the antimicrobial activity against Salmonella gallinarium was found to be very high as 92% or more.

Example 3: Properties of Antimicrobial Activity of Lactobacillus Planta Room CLP-1 Strains

The characteristics of the antimicrobial active material produced by the CLP-1 strain of the present invention was confirmed as follows.

One) PH  stability

CLP-1 strain of the present invention was inoculated in MRS medium and cultured at 37 ° C. for 20 to 24 hours, and the pH of the culture medium after culture was 4.2 ± 0.2. The pH was adjusted to 2, 4, 6, 9 and 11 with 18% HCl and 6N NaOH. Subsequently, the cells were removed and left at 4 ° C. for 2 hours, after which the pH was adjusted to 6.0 ± 0.2 and the antibacterial activity (%) against E. coli was shown in FIG. 9. When the antimicrobial activity of the sample was not adjusted to 100% after incubation, the antimicrobial activity was stable to about 100% until pH 2 to 6, but when the pH was increased to 8 or more, the antimicrobial activity was rapidly reduced.

2) thermal stability

The CLP-1 strain culture solution of the present invention was adjusted to pH 6.0 using 6N NaOH and then sterilized using a 0.45 um filter, and samples were taken at intervals of 5 minutes, 15 minutes, 30 minutes and 60 minutes while standing at 80 ° C. The results of confirming the antimicrobial activity (%) against E. coli is shown in FIG. 10. As a result, when the heat treatment for 60 minutes at 80 ℃ was reduced to about 92%, it was found that it is very stable to heat.

Example 4: Culture conditions of Lactobacillus plantarum CLP-1 strain

1) Growth and Antimicrobial Activity of CLP-1 Strains According to Culture Period

CLP-1 strains were inoculated in MRS medium and cultured anaerobicly at 37 ° C. for 72 hours, followed by culture at 24, 48 and 72 hours after incubation to measure the number of viable cells and the pH of the culture. Shown in

Incubation time (hours) 24 48 72 Viable Count (cfu / mL) 1.5 X 10 ⁹ 1.4 X 10 ⁹ 8.7 X 10 ⁸ Culture pH 4.15 4.09 4.10

As shown in Table 4, the pH after 24 hours of cultivation appeared similar regardless of the culturing time, and the number of viable cells did not increase any more, but it was found to decrease slightly at 72 hours.

In addition, after correcting the pH of the culture medium to 6.0 ± 0.2, the antimicrobial activity against hemolytic E. coli was confirmed in the same manner as described in Example 2, the highest antibacterial activity in the culture cultured for 24 hours was 65.2% It could be seen (Fig. 11). Therefore, the antimicrobial active material present in the CLP-1 culture is produced in a growth-associated form, such as bacteriocin, and the antimicrobial activity is reduced when the culture time is 48 hours or more. After this, it is assumed that the antimicrobial active substance is decomposed.

2) Growth and Antimicrobial Activity of CLP-1 Strains According to Culture Temperature

The CLP-1 strains were first activated in MRS liquid medium, and the activated bacteria were inoculated at 1% in fresh MRS liquid medium and anaerobicly for 24 hours at 10 ° C, 25 ° C, 30 ° C, 37 ° C and 40 ° C. The culture was performed, and the results of measuring the number of viable bacteria and the culture solution thereof are shown in Table 5 below.

Culture temperature (℃) 10 25 30 37 40 Viable Count (cfu / mL) 5.0 X 10 ⁶ 2.9 X 10 ⁹ 2.5 X 10 ⁹ 1.5 X 10 ⁹ 1.1 X 10 ⁹ Culture pH 6.23 4.05 3.96 4.18 4.33

As shown in Table 5, 2.9 X 10 ⁹ cfu / mL when incubated at 25 ℃ and 30 ℃ And 2.5 X 10 ⁹ cfu / mL of viable cells. This is considered to be excellent in low temperature growth because CLP-1 strain is lactic acid bacteria isolated from kimchi.

In addition, the antimicrobial activity against hemolytic E. coli of the strain cultured in the same manner as described above was confirmed by the method of Example 2, the highest antimicrobial activity appeared at 30 ℃ (Fig. 12).

In addition, the results of measuring the antiviral activity against rotavirus according to the culture temperature of the CLP-1 strain in the same manner as in Example 1 is shown in FIG. As a result, antiviral activity was not significantly different in cultures at 30 ° C and 37 ° C.

Example 5: Mass Production of Lactobacillus Planta Room CLP-1 Strains

In order to mass-produce Lactobacillus plantarum CLP-1 strains, firstly inoculate the strain stored freeze at -70 ℃ to 200% MRS medium to 0.1% and incubated anaerobicly at 30 ℃ for 24 hours Activated. Activated fungi were inoculated at a concentration of 0.1% in a medium containing 50 g of glucose, 50 g of molasses, 10 g of yeast extract, 20 g of skim soy flour, 40 g of corn steep liquor, 3 g of K ₂ HPO ₄ , and 1 g of sodium acetate, and then 30 The cells were left incubated at 24 ° C. for mass production of CLP-1 bacteria.

After completion of the culture, the pH of the culture solution was 3.9 ± 0.2, the viable cell count was 1.7 × 10 ⁹ cfu / mL, and the organic acid content (lactic acid basis) contained in the culture was 3.4%.

Example 6: Formulation of Lactobacillus Planta Room CLP-1 Strains

After filtering the viable culture medium produced in Example 5 with a 1 mm sieve, the solution was passed through a sieve, glucose, lactose and skim milk were added or mixed with excipients to 15% (w / v), and then lyophilized (Hanil, Probiotics in the dry form were prepared by lyophilization for 24 hours using Clean Vac 12).

In addition, after correcting the pH of the live cell culture produced in Example 5 to 6.0 ± 0.2 using 6N NaOH, and lyophilized for 24 hours using a freeze dryer (Hanil, Clean Vac 12) to obtain a dry probiotic in the form of a dry product Prepared.

In addition, glucose, lactose, skimmed milk and zeolite were added to the viable culture medium produced in Example 5 or 10 times the concentration of the culture solution in an amount of 5 to 15%, preferably 10% (w / v), respectively. Thereafter, a probiotic in the form of a dry product was prepared by drying the vacuum dryer (Dong-A, DA-VO-64) at 20 ° C. to 25 ° C. under 70 cmHg for 4 hours to 6 hours.

Experimental Example 1: Pig specification test

A 40-day-old piglet infected with rotavirus and toxin-type Escherichia coli in a pig farm in Paju, Gyeonggi-do, Korea was prepared with a bacterial culture sample as shown in Table 6, mixed in negative water to 10 mL / du / day, and 8 Specimen experiment was carried out with daily salary, and the results are shown in Table 7 below.

Experimental group Non-administered group A B C Fungus - Bacillus
Subtilis Lactobacillus
Lactis
ATCC 2013 Lactobacillus
Planta Room CLP-1 Viable Count (cfu / mL) - 1.7 X 10 ⁹ 1.2 X 10 ⁹ 1.5 X 10 ⁹

Experimental group Non-administered group A B C % Mortality 30 30 20 0 Diarrhea Occur Occur Occur Occur

As shown in Table 7, lactic acid bacteria non-administered group, Bacillus subtilis And 20% or more mortality occurred in the feed group of coliform bacteria ( Lactobacillus lactis ), while the water-borne diarrhea occurred in the Lactobacillus plantarum CLP-1 strain fed group, but mortality was 0%. The rotavirus and toxin-type E. coli showed excellent therapeutic effects.

Experimental Example 2: Poultry Specification

The in vivo efficacy of CLP-1 strains confirmed in vitro antibacterial activity against Salmonella enteritidis , one of the causes of diarrheal diseases of herbaceous herb in Example 2, was as follows. Confirmed.

After hatching and incubating SPF eggs (Spafas), 1% (v / v) of CLP-1 strain culture and Lactobacillus lactis ATCC 2013 strain culture as a control group were negatively added at 1 day of age. After the addition was allowed for 8 days autonomous benefits. Two days after the start of feeding, Salmonella enteritidis was activated and orally administered at a concentration of 10 ⁹ cfu / mL / horse, and lesions were observed after challenge. As a result, weak diarrhea occurred after 3 days in the non-inoculated group, but improved in all groups (non-administered group, Lactobacillus lactis group, and CLP-1 strain group). Later diarrhea occurred.

Since the mortality rate of the mortality of each group was shown in Table 8 below.

Experimental Section Blank Non-administered group Lactobacillus lactis group CLP-1 group % Mortality 0 66 62 32

As shown in Table 8, the non-lactated group not fed the lactic acid bacteria showed the highest mortality rate of 66%, and the group fed the Lactobacillus lactis ATCC 2013 strain culture showed 62% mortality, and the CLP-1 of the present invention. The group fed the strain culture showed a mortality rate of 32%, which showed an improvement in mortality rate of about 34% compared to the non-administered group.

Therefore, the CLP-1 strain of the present invention can prevent infection by Salmonella enteritidis and inhibit mortality.

Agricultural Genetic Resource Center KACC91591P 20101020

<110> Cheil Bio. Co., Ltd. <120> LACTOBACILLUS PLANTARUM CLP-1 STRAIN HAVING ANTI-VIRUS AND          ANTI-BACTERIAL ACTIVITY AND DIRECT-FED MICROORGANISMS COMPRISING          THE SAME <130> FPD / 201007-0030 <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 950 <212> DNA <213> Lactobacillus plantarum <400> 1 atnngnancg gtgngntgcn atnatgcagt cgaacgaact ctggtattga ttggtgcttg 60 catcatgntt tacatttgag tgagtggcga actggtgagt aacacgtggg aaacctgccc 120 agaagcgggg gataacacct ggaaacagat gctaataccg cataacaact tggaccgcat 180 ggtccgagyt tgaaagatgg cttcggctat cacttttgga tggtcccgcg gcgtattagc 240 tagatggtgr ggtaacggct caccatggca atgatacgta gccgacctga gagggtaatc 300 ggccacattg ggactgagac acggcccaaa ctcctacggg aggcagcagt agggaatctt 360 ccacaatgga cgaaagtctg atggagcaac gccgcgtgag tgaagaaggg tttcggctcg 420 taaaactctg ttgttaaaga agaacatatc tgagagtaac tgttcaggta ttgacggtat 480 ttaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag 540 cgttgtccgg atttattggg cgtaaagcga gcgcaggcgg ttttttaagt ctgatgtgaa 600 agccttcggc tcaaccgaag aagtgcatcg gaaactggga aacttgagtg cagaagagga 660 cagtggaact ccatgtgtag cggtgaaatg cgtagatata tggaagaaca ccagtggcga 720 aggcggctgt ctggtctgta actgacgctg aggctcgaaa gtatgggtag caaacaggat 780 tagataccct ggtagtccat accgtaaacg atgaatgcta agtgttggag ggtttccgcc 840 cttcagtgct gcagctaacg cattaagcat tccgcctggg gagtacggcc gcaaggctga 900 actcaaggaa ttgacggggg cccgcacagc ggtggagcat gtggtttaat 950 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR forward primer <400> 2 ttcagattgt ggagctattc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> PCR reverse primer <400> 3 aaagatgcta gggacaaaat 20

Claims (3)

Lactobacillus ( Lactobacillus) plantarum ) CLP-1 strain (Accession No. KACC91591P). A probiotic comprising Lactobacillus plantarum CLP-1 strain (Accession No. KACC91591P) or a culture thereof as an active ingredient. A feed additive comprising Lactobacillus plantarum CLP-1 strain (Accession No. KACC91591P) or a culture thereof as an active ingredient.

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