KR20150066772A - A novel lactic acid bacteria and composition for preventing or treating diarrhea of an infant comprising the same - Google Patents

Abstract

The present invention relates to novel lactic acid bacteria and a composition for preventing or treating diarrhea of an infant including the same. The composition for preventing or treating the diarrhea of the infant according to the present invention includes the novel lactic acid having excellent antiviral activity among lactic acid bacteria separated from infant feces to express the antiviral activity against virus causing the diarrhea while being safe for the infant, thereby having an excellent effect of preventing and treating the diarrhea.

Description

TECHNICAL FIELD The present invention relates to a novel lactic acid bacterium and a composition for preventing or treating diarrheal diseases in infants and young children comprising the same. BACKGROUND ART [0002]

The present invention relates to a novel lactic acid bacterium and a composition for preventing or treating diarrhea in infants and young children comprising the same.

In general, Escherichia coli is a pathogenic E. coli that causes infectious diarrhea and acute enteritis in an infant, and Escherichia coli, a non-pathogenic Escherichia coli.

The pathogenic E. coli is classified into four types. Recently, Enterohemorrhageic E. coli producing Enterotoxigenic E (E. coli) and Enterotoxigen (E. coli) producing Enterotoxin such as Escherichia coli O157: H7 newly emerged all over the world such as Japan and USA, (Enteroinvasive E. coli), which causes intestinal infections by invading epithelial cells of the intestinal mucosa, and enteropathogenic E. coli, which causes acute gastroenteritis in adults.

Enteropathogenic viruses include Norovirus, Rotavirus, Sapovirus, Astrovirus, Adenovirus, Coxsackievirus, etc. Each year, More than 125 million infants under five years of age are infected more than once and more than 600,000 children are known to die. The transmission route of these viruses is mainly due to fecal-oral contact, and it is known that it is easily infectious in places where people are crowded such as nursing homes, nursing homes, and department stores because it is highly infectious.

In the case of such viruses, it affects the health of infants as well as the people seriously, and it causes social loss and economic loss. Therefore, it is necessary to develop research and product development for technology to reduce or eliminate viruses Although the development of expensive vaccines has been developed, there have been many side effects in the commercialization stage, and studies for the prevention and treatment of diarrhea in everyday dietary habits have been limited It is.

It is another object of the present invention to provide a novel lactic acid bacterium exhibiting antiviral activity and a composition for preventing or treating diarrhea in infants and young children comprising the same.

In order to solve the above-mentioned problems, the present invention provides a lactic acid bacterium of Bifidobacterium longum BG30 (Bifidobacterium longum BG30) deposited under the deposit number KCTC 12480BP in the Korean Collection for Type Culture (KCTC) do.

The present invention also provides a Lactobacillus acidophilus LA8 lactic acid bacterium deposited internationally as Accession No. KCTC 12481BP at the Center for Microbial Resources, Korea Research Institute of Bioscience and Biotechnology.

The present invention also relates to a method for the treatment of infants and young children comprising Bifidobacterium longum BG30 or Lactobacillus acidophilus LA8 or Bifidobacterium longum and Lactobacillus acidophilus LA8 A composition for preventing or treating diarrhea is provided.

In the composition for preventing or treating diarrheal diseases of infants and young children according to the present invention, the composition contains, as an active ingredient, a mixture of the two types of lactic acid bacteria strains in an amount of 10 ⁸ to 10 ¹² cfu / g, Characterized in that the culture medium contains an equivalent number of viable bacteria.

In the composition for preventing or treating diarrhea in infants and young children according to the present invention, the composition may be selected from the group consisting of Lactobacillus acidophilus, Lactobacillus plantarum, Bifidobacterium breve, Bifidobacterium lactis, Bifidobacterium longum, Lactobacillus salivarius, Lactobacillus brevis, Lactobacillus helveticus, Lactobacillus spp., Lactobacillus spp., Lactobacillus spp. Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus reuteri, Lactobacillus bochneri (Lactobacillus spp.), Lactobacillus spp. Lactobacillus buchneri), Lactobacillus gasseri, Lactobacillus But are not limited to, Lactobacillus johonsonii, Lactobacillus kefir, Lactococcus lactis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium pseudolongum, Bifidobacterium themophilum, Bifidobacterium adolescentis, Lactobacillus rhamnosus, Pediococcus spp. , Pediococcus spp. pentosaceus) . < / RTI >

In the composition for preventing or treating diarrhea in infants and young children according to the present invention, the composition is characterized by containing two kinds of lactic acid bacteria in the same ratio.

In the composition for preventing or treating diarrheal diseases of infants and young children according to the present invention, the two kinds of lactic acid bacterial strains are double-coated with proteins and polysaccharides or triple-coated with proteins, polysaccharides and nanoparticles.

In the composition for preventing or treating diarrheal diseases of infants and young children according to the present invention, the composition further comprises a pharmaceutically acceptable carrier or excipient.

In the composition for preventing or treating diarrheal diseases of infants and young children according to the present invention, the composition is prepared by lyophilizing lactic acid bacteria strains in the form of a probiotic agent.

The composition for the prevention or treatment of diarrheal diseases of infants and young children according to the present invention contains a novel lactic acid bacterium having excellent antiviral activity among the lactic acid bacteria isolated from the infant feces of infants and has a strong antiviral activity against viruses that cause diarrhea in infants and young children, And treatment.

FIGS. 1 and 2 show the results of performing plaque reduction analysis on lactic acid bacteria isolated from infants.
FIG. 3 shows the result of performing 16S rRNA sequencing on lactic acid bacteria isolated according to an embodiment of the present invention.
FIG. 4 shows the result of Random Amplification of Polymorphic DNA (RAPD) performed on lactic acid bacteria isolated according to an embodiment of the present invention.
FIG. 5 shows the results of evaluating the hemolytic activity of lactic acid bacteria isolated according to an embodiment of the present invention.
FIGS. 6 to 8 show the results of observing changes in body weight, changes in the amount of feed intake, and changes in the amount of insulin when the lactic acid bacteria isolated according to an embodiment of the present invention are once orally administered to rats.

Example 1: Isolation of lactic acid bacteria

Feces were collected from 18 infants with normal eating habits. The cultures were incubated for 48 h in anaerobic condition (90% N ₂ , 5% H ₂ , 5% CO ₂ ) at 37 ° C with a Bactron Anaerobic Chamber (Sheldon Manufacturing Inc, USA) using General Anaerobic Medium Branches were separated.

Example 2: Isolation of lactic acid bacteria showing antiviral activity against rotavirus

Plaque reduction assay was performed to select lactic acid bacteria showing 18 antiviral effects against rotavirus of 18 kinds of lactic acid bacteria isolated in Example 1.

The plaque reduction assay was performed according to the method described by Edgar et al. (See Manual of clinical microbiology-5th ed. A. Balows (chief ed.) Amer. Soc. For Microbiology, Washington DC (1991), p.1184-1191) Respectively.

The results of plaque reduction assay for the 18 strains isolated are shown in FIGS. 1 and 2. FIG. In FIGS. 1 and 2, ALR2 and CLA2 exhibit antiviral activity of 50% or more against rotavirus.

Example 3 Identification of Isolated Lactic Acid Bacteria

≪ Example 3-1 > 16S rRNA sequencing

16S rRNA sequencing was performed for identification of LA8 and BG30 lactic acid bacteria showing antiviral activity against rotavirus in Example 3 above.

Genomic DNA was extracted from 1 ml of pure culture broth of infant feces using Accuprep gemomic extraction kit (Bioneer, Korea). PCR (MyCycler, BIO-RAD, USA) was performed using the extracted DNA as a template using a primer F (5'-AGAGTTTGATCCTGGCTCAG-3 ') and a primer R (5'-AAGGAGGTGATCCAGCC-3' The product was ligation in pGEM-Teasy vector (Promega, USA), transformed into E. coli DH5a, plated on LB plate containing X-gal and ampicillin and incubated at 37 ° C for about 16 hours. The recombinant plasmid containing the PCR product was separated from the colony grown on the LB plate, followed by DNA sequencing and the results are shown in FIG.

DNA sequencing was performed using the Cluster V program of the DNA star program. The LA8 and BG30 sequences were compared with those of the L. acidophilus (GenBank accession number: CP000033) and SEQ ID NO: 2 And 99.87% and 99.50% homology with B. longum (GenBank accession number; AP010888).

≪ Example 3-2 > Random Amplification of Polymorphic DNA (RAPD)

Random Amplified Polymorphic DNA (RAPD) -fingerprinting was performed to investigate the genetic relationship between isolated lactic acid bacteria and existing standard strains.

PCR-RAPD (MyCycler, BIO-RAD, USA) was performed using GTG ₅ (5'-GTGGTGGTGGTGGTG-3 ') as a template after extracting genomic DNA from isolated lactic acid bacteria. The PCR products were electrophoresed on 0.8% agarose gel, stained with EtBr, and observed with G: BOX (SYNGENE, UK). The results are shown in FIG.

As shown in FIG. 4, when the RAPD band pattern of LA8 was compared with the type strain, the band patterns of the two strains were similar to each other, so that the identification was correctly performed. In addition, the RAPD pattern of B. longum was known to be different in the same species, so it was not possible to identify it by comparing the similarity with the type strain. This fact was confirmed in the RAPD pattern of the different B. longum strains.

The present inventors have named ALR2 and CLA2, which are excellent in antiviral activity, as BG30 and LA8, respectively, and deposited them as KCTC 12480BP and KCTC 12481BP, respectively, in the Korean Collection for Type Culture (KCTC) .

<Example 4> Safety evaluation of isolated strains

&Lt; Example 4-1 > Evaluation of hemolytic activity

Since the protozoan microorganism shows a hemolysis phenomenon which destroys red blood cells of blood, the hemolytic activity evaluation was performed in this embodiment to check whether the lactic acid bacteria isolated from the feces were pathogenic.

Lactobacillus acidophilus LA8 and Bifidobacterium longum BG30 strains cultured in liquid medium were inoculated on a medium containing 5% sheep or horse blood using sterilized rods after containing 5% sheep or horse blood in MRS agar or BL agar. After the incubation for the time, the hemolysis reaction was confirmed, and the results are shown in Fig.

As shown in FIG. 5, since no hemolysis was observed at the cultures of LA8 and BG30, the two strains were confirmed as safe lactic acid bacteria free from hemolytic activity.

&Lt; Example 4-2 > Antimicrobial susceptibility test [

Ten antibiotics (ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, chloramphenicol, The minimum inhibitory concentration (MIC) was measured in the quinupristin + dalfopristin (QU + DA)) and the measured MIC was compared with the breakpoint suggested by the Eurean Food Safety Authority to assess whether the antibiotic was susceptible Respectively. If the measured MIC was higher than the EFSA breakpoint, it was considered to be resistant to the antibiotic.

MIC was performed using a micro-dilution test (Clinical and Laboratory Standards Institute, 2006) and E-test (BD, USA).

The microdilution test was performed by stepwise dilution of 9 antibiotics from 256 μg / ml to 0.5 μg / ml, followed by stepwise dilution of LA8 and BG30 strains to 10 ⁶ to 10 ⁷ CFU / ml in lactic acid susceptible medium (LSM, BD) After inoculation in all wells of stepwise diluted antibiotics, MICs were determined after incubation for 37 to 48 hours.

In the E-test, LA8 and BG30 were inoculated into MRS agar or BL agar at a concentration of 10 ⁶ to 10 ⁷ CFU / mL, and then the medium was cultured. Seven antibiotics (STM, ERM, CP, TET, And QU + DA) was placed on a culture medium, and after incubation at 37 ° C for 48 hours, it was confirmed whether or not it inhibited, and the results are shown in Table 1 below.

The MIC of the two strains was measured by the micro-dilution method. As shown in Table 1, the ESIA breakpoint was measured for all 10 antibiotics. E-test of 7 antibiotics showed that both strains had lower MIC than micro-dilution method. Therefore, it can be confirmed that LA8 and BG30 according to the present invention are safe strains having no antibiotic resistance.

&Lt; Example 5 >

When LA8 and BG30 according to the present invention were administered to rats at a concentration of 10 ¹¹ CFU / kg or more, mortality, clinical symptoms, body weight, change in body water volume, change in body temperature and body weight were observed, Are shown in Tables 2 to 5 and Figs. 6 to 8.

As shown in Tables 2 and 3, no 14-day mortality was observed, and no clinical symptoms were observed when the administration group and the control group were compared.

In FIGS. 6 to 8, no significant difference was observed in body weight, feed quantity, water volume, body temperature and organ weight as compared with the control group, and thus LA8 and BG30 according to the present invention were confirmed as safe strains having no toxicity.

&Lt; Example 6 > Preparation of composition for prevention or treatment of diarrhea in infants and young children and clinical experiment

&Lt; Example 6-1 & gt; Preparation of composition for preventing or treating diarrhea in infants and young children

( Lactobacillus plantarum LP3, L. rhamnosus LR5, Bifidobacterium lactis BL3, and Pediococcus pentosaceus SL4) in addition to LA8 and BG30 according to the present invention at the composition ratios shown in Table 6 below to prepare compositions for preventing or treating diarrhea in infants and young children .

Clinical trials in patients with irritable bowel syndrome and atopic patients have shown that four types of lactic acid bacteria ( Lactobacillus plantarum LP3 KCTC 10782BP, L. rhamnosus LR5 KCTC 12202BP, Bifidobacterium lactis BL3 KCTC 11904BP and Pediococcus pentosaceus SL4 KCTC 11870BP) Which have already proven efficacy and safety in the treatment of cancer, are already commercially available.

Each lactic acid bacterium was mixed with 6.0 × ^{10 8} cells containing maltose and polydextrose as excipients. The placebo group produces placebo containing only maltose and polydextrose without live bacteria. The placebo was prepared so that its color, flavor, and taste were almost identical to those of the prototype.

&Lt; Example 6-2 > Clinical trial for infants and young children

50 infants were recruited for 6 months from the date of IRB approval for patients who visited the Department of Pediatrics and Adolescent Clinic of Chungbuk National University Hospital for symptoms of viral enteritis such as diarrhea, vomiting, fever and abdominal pain among infants younger than 5 years old. Fifty infants were divided into test groups and dosage groups, and the dosage groups were administered twice per day in units of 1 g of the composition for preventing or treating diarrhea in the infant and child prepared in Example 5-1.

The Mann-Whitney test was used to compare the pre-dose and post-dose ages, diarrhea, fever, abdominal pain, and duration of vomiting between the test and placebo groups and to compare pre- and post- The t-test was used. In addition, paired t-test was used to compare changes in blood test results between pre- and post-doses in each group, and the comparison of clinical characteristics, blood test results, and blood test values before and after dosing were compared 6 and Table 7, respectively.

As shown in Table 6 and Table 7, the duration of symptoms such as fever, abdominal pain and vomiting did not differ between the two groups, but the duration of diarrhea after the treatment was 3.38 ± 0.87 days in the test group and 4.56 ± 0.20 days, and hemoglobin was significantly lower in the test group (11.69 ± 0.35 g / dl) than the placebo group (12.64 ± 0.80 g / dl, p value = 0.021).

Korea Biotechnology Research Institute KCTC12480BP 20130903 Korea Biotechnology Research Institute KCTC12481BP 20130903

<110> CELL BIOTECH CO., LTD. <120> A NOVEL LACTIC ACID BACTERIA AND COMPOSITION FOR PREVENTING OR          TREATING DIARRHEA OF AN INFANT COMPRISING THE SAME <130> DPP-2013-0193 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 1556 <212> DNA <213> Lactobacillus acidophilus <400> 1 cagatcgagc gagctgaacc aacagattca cttcggtgat gacgttggga acgcgagcgg cggatgggtg 120 agtaacacgt ggggaacctg ccccatagtc tgggatacca cttggaaaca ggtgctaata 180 ccggataaga aagcagatcg catgatcagc ttataaaagg cggcgtaagc tgtcgctatg 240 ggatggcccc gcggtgcatt agctagttgg tagggtaacg gcctaccaag gcaatgatgc 300 atagccgagt tgagagactg atcggccaca ttgggactga gacacggccc aaactcctac 360 gggaggcagc agtagggaat cttccacaat ggacgaaagt ctgatggagc aacgccgcgt 420 gagtgaagaa ggttttcgga tcgtaaagct ctgttgttgg tgaagaagga tagaggtagt 480 aactggcctt tatttgacgg taatcaacca gaaagtcacg gctaactacg tgccagcagc 540 cgcggtaata cgtaggtggc aagcgttgtc cggatttatt gggcgtaaag cgagcgcagg 600 cggaagaata agtctgatgt gaaagccctc ggcttaaccg aggaactgca tcggaaactg 660 tttttcttga gtgcagaaga ggagagtgga actccatgtg tagcggtgga atgcgtagat 720 atatggaaga acaccagtgg cgaaggcggc tctctggtct gcaactgacg ctgaggcccg 780 aaagcatggg tagcgaacag gattagatac cctggtagtc catgccgtaa acgatgagtg 840 ctaagtgttg ggaggtttcc gcctctcagt gctgcagcta acgcattaag cactccgcct 900 ggggagtacg accgcaaggt tgaaactcaa aggaattgac gggggcccgc acaagcggtg 960 gagcatgtgg tttaattcga agcaacgcga agaaccttac caggtcttga catctagtgc 1020 aatccgtaga gatacggagt tcccttcggg gacactaaga caggtggtgc atggctgtcg 1080 tcagctcgtg tcgtgaggtg ttgggttaag tcccgcaacg agcgcaaccc ttgtcattag 1140 ttgccagcat taagttgggc actctaatga gactgccggt gacaaaccgg aggaaggtgg 1200 ggatgacgtc aagtcatcat gccccttatg acctgggcta cacacgtgct acaatggaca 1260 gtacaacgag gagcaagcct gcgaaggcaa gcgaatctct taaagctgtt ctcagttcgg 1320 actgcagtct gcaactcgac tgcacgaagc tggaatcgct agtaatcgcg gatcagcacg 1380 ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccatg ggagtctgca 1440 atgcccaaag ccggtggcct aaccttcggg aaggagccgt ctaaggcagg gcagatgact 1500 ggggtgaagt cgtaacaagg tagccgtagg agaacctgcg gctggatcac ctcctt 1556 <210> 2 <211> 1403 <212> DNA <213> Bifidobacterium longum BG30 <400> 2 catgcagtcg acgggatcca tcaggctttg cttggtggtg agagtggcga acgggtgagt 60 aatgcgtgac cgacctgccc catacaccgg aatagctcct ggaaacgggt ggtaatgccg 120 gatgctccag ttgatcgcat ggtcttctgg gaaagctttc gcggtatggg atggggtcgc 180 gtcctatcag cttgacggcg gggtaacggc ccaccgtggc ttcgacgggt agccggcctg 240 agagggcgac cggccacatt gggactgaga tacggcccag actcctacgg gaggcagcag 300 tggggaatat tgcacaatgg gcgcaagcct gatgcagcga cgccgcgtga gggatggagg 360 ccttcgggtt gtaaacctct tttatcgggg agcaagcgag agtgagttta cccgttgaat 420 aagcaccggc taactacgtg ccagcagccg cggtaatacg tagggtgcaa gcgttatccg 480 gaattattgg gcgtaaaggg ctcgtaggcg gttcgtcgcg tccggtgtga aagtccatcg 540 cttaacggtg gatccgcgcc gggtacgggc gggcttgagt gcggtagggg agactggaat 600 tcccggtgta acggtggaat gtgtagatat cgggaagaac accaatggcg aaggcaggtc 660 tctgggccgt tactgacgct gaggagcgaa agcgtgggga gcgaacagga ttagataccc 720 tggtagtcca cgccgtaaac ggtggatgct ggatgtgggg cccgttccac gggttccgtg 780 tcggagctaa cgcgttaagc atcccgcctg gggagtacgg ccgcaaggct aaaactcaaa 840 gaaattgacg ggggcccgca caagcggcgg agcatgcgga ttaattcgat gcaacgcgaa 900 gaaccttacc tgggcttgac atgttcccga cggtcgtaga gatacggctt cccttcgggg 960 cgggttcaca ggtggtgcat ggtcgtcgtc agctcgtgtc gtgagatgtt gggttaagtc 1020 ccgcaacgag cgcaaccctc gccccgtgtt gccagcggat tatgccggga actcacgggg 1080 gaccgccggg gttaactcgg aggaaggtgg ggatgacgtc agatcatcat gccccttacg 1140 tccagggctt cacgcatgct acaatggccg gtacaacggg atgcgacgcg gcgacgcgga 1200 gcggatccct gaaaaccggt ctcagttcgg atcgcagtct gcaactcgac tgcgtgaagg 1260 cggagtcgct agtaatcgcg aatcagcaac gtcgcggtga atgcgttccc gggccttgta 1320 cacaccgccc gtcaagtcat gaaagtgggc agcacccgaa gccggtggcc taaccccttg 1380 tgggatgagc cgtctaatat agt 1403

Claims (9)

Bifidobacterium longum BG30 (Bifidobacterium longum BG30) deposited with the Korean Collection for Type Culture (KCTC) under the accession number KCTC 12480BP
Lactobacillus acidophilus LA8 (Lactobacillus acidophilus LA8), deposited with the Korea Research Institute of Bioscience and Biotechnology as Accession No. KCTC 12481BP,
Prevention or treatment of diarrhea in infants and young children, including Bifidobacterium longum BG30 or Lactobacillus acidophilus LA8, or Bifidobacterium longum BG30 and Lactobacillus acidophilus LA8 Composition
The method of claim 3,
Characterized in that the composition comprises, as an active ingredient, a mixture of the two types of lactic acid bacterium strains in an amount of 10 ⁸ to 10 ¹² cfu / g, or an equivalent number of viable bacteria, relative to the total weight of the composition A composition for preventing or treating diarrhea in infants and young children.
5. The method of claim 4,
Wherein the composition is selected from the group consisting of Lactobacillus acidophilus, Lactobacillus plantarum, Bifidobacterium breve, Bifidobacterium lactis, Bifidobacterium, Bacillus thuringiensis, Bifidobacterium longum, Lactobacillus salivarius, Lactobacillus brevis, Lactobacillus helveticus, Lactobacillus fermentum, Lactobacillus paracasei, ), Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus reuteri, Lactobacillus buchneri, Lactobacillus gasseri, Lactobacillus gasseri, Lactobacillus spp., Lactobacillus spp. Lactobacillus johonsonii, Lactobacillus kefir, (Lactococcus lactis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium pseudolongum, Bifidobacterium thrombophilus (Bifidobacterium pseudolongum, Bifidobacterium pseudolongum, The present invention further comprises at least one lactic acid bacterium selected from the group consisting of Bifidobacterium themophilum, Bifidobacterium adolescentis, Lactobacillus rhamnosus, and Pediococcus pentosaceus. A composition for preventing or treating diarrhea in infants and young children
5. The method of claim 4,
Wherein the composition comprises two kinds of lactic acid bacteria in the same ratio. The composition for preventing or treating diarrhea in infants and young children
5. The method of claim 4,
Wherein the two kinds of lactic acid bacterial strains are double-coated with proteins and polysaccharides or are triple-coated with proteins, polysaccharides and nanoparticles.
5. The method of claim 4,
Wherein the composition further comprises a pharmaceutically acceptable carrier or excipient. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;
5. The method of claim 4,
Wherein the composition is prepared by lyophilizing lactic acid bacterial strains in the form of a probiotic agent.

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