KR100557397B1 - Acid tolerant probiotic Lactobacillus reuteri Probio-054 that can suppresses the growth of pathogenic microorganisms - Google Patents

Abstract

The present invention relates to a novel acid resistant Lactobacillus luteri probio-054 and a probiotic containing the same, wherein the bacterium of the present invention, Lactobacillus luteri probio-054, has harmful properties in the body and intestines of animals. Since it effectively inhibits the growth of various pathogenic microorganisms, and has acid resistance and bile acid function, it can be administered to humans and various livestock as a substitute for existing antibiotics, or used as medicines, feed additives, animal medicines, etc. to stabilize the intestinal microflora. It is possible to treat or prevent the symptoms that can be caused by abnormal fermentation of harmful microorganisms in the intestine, such as diarrhea.

Probiotics, Probiotic, Lactobacillus Luteri Probio-054, Acid Resistance, Bile Acid Resistance, Anticancer Effect, Immune Activity, Microorganism, Growth Inhibition

Description

Acid tolerant probiotic Lactobacillus reuteri Probio-054 that can suppresses the growth of pathogenic microorganisms}

The present invention relates to a novel acid resistant Lactobacillus luteri Probio-054 and a bioactive agent containing the same, and more particularly, a novel microorganism isolated from feces of a dog having acid resistance, bile acid resistance and at the same time harmful microorganisms. Lactobacillus luuteri Probio-054 ( Lactobacillus reuteri Probio-054) capable of effectively inhibiting the growth of the present invention, and a livestock bioactive agent containing the microorganism and a method for inhibiting the growth of harmful microorganisms in the gut using the microorganism.

Diarrhea, vomiting, and dehydration due to enteritis at home and abroad are serious diseases with high mortality due to secondary infection by parvovirus and Escherichia coli in the breeding ground and the natural environment. Canine parvovirus diarrhea does not have a therapeutic effect even if antibiotics are administered in the event of disease, and can only be expected to treat the microorganism causing the secondary infection. Vaccines are used to prevent parvovirus infection, but there is a problem that side effects may also occur due to vaccine use.

Various microorganisms exist in the intestine of the human body and the animal, forming a microbial flora in the intestine. Such microorganisms include beneficial microorganisms, such as lactic acid bacteria, which are known to be useful for host animals, and conversely, microorganisms such as E. coli, Salmonella, Staphylococcus, etc., which have direct or potential harmful effects on the host. In the case of the human body and animals, when the flora of the gut microorganism is destroyed by the change of external environment such as the increase of stress and the infection of harmful bacteria, it leads to the rapid growth of the harmful microorganisms, worsening the health of the host animal or even from diarrhea and even death. It can go up to risk. When this happens, antibiotics are administered for the purpose of treatment, but these antibiotics remain in the host without being completely excreted in vitro, and harmful microorganisms become resistant to continuous administration, resulting in a situation where effective treatment cannot be achieved. Done. As an alternative, there is a growing interest in probiotics, which are currently probiotic. Probiotics, a probiotic, are prepared by separating beneficial microorganisms that live in the intestines of humans or animals. Aerobic, anaerobic, and lactic acid bacteria and yeasts are used, but lactic acid bacteria are used as the main strains. These lactic acid bacteria probiotics can maintain the stable intestinal flora by inhibiting abnormal fermentation of harmful bacteria in the intestine without causing side effects that can occur due to antibiotic abuse, and can prevent diseases caused by infection of harmful microorganisms in advance. In addition, continuous administration of probiotic can increase the utilization efficiency of feed and intestinal beneficial microorganisms are inhabited in a balanced manner to maintain health. For effective use as a probiotic, it is very important to have acid and bile acid resistance as well as inhibitory activity against harmful microorganisms. After the probiotic is administered to the animal, it meets gastric juice, which is a strong acid, until it reaches the intestine, followed by bile acids. Since these gastric juices and bile acids generally kill bacteria or reduce their activity, the administered probiotic must reach the intestines and function properly to withstand the gastric juice and bile acids.

To date, various types of lactic acid bacteria have been published as probiotic probiotic agents, and bioactive agents identified as Lactobacillus luteri, which have the characteristics of acid and bile acids and inhibit harmful microorganisms, have also been reported (Jang, Young-Hyo et al., Kor. J. Appl. Microbiol.Biotechnol. 27, 23-27) (Korean Patent 10-211529-0000).

The inventors of the present invention have attempted to search for new bioactive agents that can more effectively inhibit harmful pathogenic microorganisms than existing published probiotic agents and have acid resistance and bile acid resistance, and are harmful microorganisms for one strain anaerobicly isolated from dog feces. The results of the present invention by confirming that the strain is Lactobacillus ruteri strain as a strain having excellent acid resistance and bile acid resistance while effectively inhibiting harmful microorganisms as a result of growth inhibition ability test, acid resistance, bile acid resistance test and microbial identification test Completed.

Accordingly, an object of the present invention is to provide a novel probiotic Lactobacillus luterie Probio-054 ( Lactobacillus reuteri Probio-054), which is harmless to humans and animals, and is excellent in acid and bile resistance.

Another object of the present invention is to inhibit harmful microorganisms in the intestine and high immune activity by using as a fermented food spawning agent, including a formal drug, lactic acid bacteria, health supplements, etc., which are administered to the human body as a substitute for the existing antibiotics, It is to provide a probiotic-containing composition excellent in acid resistance and bile resistance.

Still another object of the present invention is to provide a method for inhibiting the growth of harmful enteropathogenic microorganisms in the intestine or preventing or treating diarrhea caused by the microorganisms using the present invention Lactobacillus luteri Probio-054.

Hereinafter, the configuration and operation of the present invention will be described in detail. Other objects and advantages of the present invention will be more clearly understood through the following configuration.

The object of the present invention is to inhibit harmful microorganisms and inhibit the growth of harmful microorganisms intestinal inhabitants including Lactobacillus reuteri Probio-054 ( Lactobacillus reuteri Probio-054), the probiotic microorganism having acid resistance, anti-acid resistance By providing food or pharmaceutical compositions such as food, pharmaceuticals, animal drugs, feed additives, and the like.

Lactobacillus reuteri Probio-054 ( Lactobacillus reuteri Probio-054), a novel microorganism of the present invention, is an anaerobic probiotic bacterium that has been anaerobicly isolated from dog feces and has been shown to have excellent antimicrobial inhibitory activity, acid resistance and bile acid resistance. Therefore, the probiotic microorganism of the present invention and the probiotic containing the same can be used in food or pharmacological composition, that is, pharmaceuticals, lactic acid bacteria and health supplements containing the microorganisms for the purpose of inhibiting the growth of intestinal harmful bacteria and enhancing immune activity. Can be.

The novel probiotic microorganism of the present invention, Lactobacillus luteri Probio-054, can not only adsorb to enteric cells but also possess the following probiotic properties.

-Growth inhibition of rotavirus and intestinal harmful microorganisms

-Immune activity enhancer

Growth ability at aerobic, anaerobic conditions and various pHs; This property confers the ability to adapt well to various physiological and pathological conditions that change within the gastrointestinal tract.

-Stomach acid and bile acid resistance

-Probiotic retention in lyophilization

In addition, the novel microorganism of the present invention is characterized by no side effects such as antibiotic resistance or biotoxicity as in other known prevention or treatment methods.

In the present invention, 'probiotic' is understood to mean 'living microorganisms which have a beneficial effect on the host by improving the intestinal microbial balance of the host in the gastrointestinal tract of animals including humans'. Probiotic microorganisms are living microorganisms with probiotic activity that can have a beneficial effect on the intestinal flora of the host when fed in the form of dried cells or fermented products to humans or animals in the form of single or complex strains.                     

Specifically, the results of examining the morphology and physiological and biochemical characteristics of the probiotic Probio-054 strain isolated from dog feces are as follows: Probio-054 is a Gram-positive bacterium under aerobic and anaerobic conditions. It grows at the same time, does not form spores, has no motility, and the cells are bacilli. As growth conditions of the said strain, 30-37 degreeC is preferable. It does not produce gases and indole, does not show hemolysis and does not reduce nitric acid. The microorganism of the present invention is preferably stored at −80 ° C. including a glycerol component or suspended in sterile 10% skim milk to freeze-dried for long-term stable storage.

In addition, Probio-054 is a strain belonging to the genus Lactobacillus , which is 99.5% homologous to the 16S rDNA homology, which is the highest in the standard strain of Lactobacillus reuteri . It was identified as a strain showing a mathematical relationship. Therefore, based on the above results, Probio-054 was identified as a Lactobacillus reuteri strain, and was deposited on July 21, 2003 to the Korea Biotechnology Research Institute Gene Bank (Accession Number: KCTC 10499BP).

Specifically, the process of searching for acid-resistant and bile acid-resistant microorganisms having harmful microbial inhibitory activity may be performed as follows: After collecting 1 g of dog feces using an anaerobic bag (trade name Gas Pack pouch; manufactured by BBL) The mixture was diluted with an anaerobic dilution adjusted to pH 2 at a ratio of 1:10, and shaken for 120 minutes using a shaker. 1 ml of the shake solution was spread on a BL solid medium (manufactured by Young Yeon Chemical Co., Ltd.) to which 0.2% of oxal (made by Diffco Co., Ltd.) was added and incubated at 37 ° C for 48 hours. After observing the colonies appearing after a certain time under a microscope, 150 microorganisms were selected and transferred to GAM semi-agar medium (Japan Daihan Pharmaceutical Co., Ltd.) culture, and then stored at minus 80 ℃. Inhibition experiments of harmful microorganisms on isolated microorganisms were conducted in Kuroiwa et al. (Kuroiwa et al., Journal of Infectious Diseases, 64, 257, 1990) and five microorganisms used for antibiotic evaluation. . Escherichia coli KCTC 2441, Shigella flexneri KCTC 2008, Enterobacter cloacea KCTC 2361, Citrobacter freundii KCTC 2006, Klebsiella pneumoniae KCTC 2208. Smeared. Paper disks with a diameter of 8 mm were placed on solid medium coated with harmful microorganisms, and inoculated with 30 ul of each isolated microbial culture medium anaerobicly cultured in an MRS liquid medium for 18 hours. Growth inhibitory rings appearing after a certain period of time was measured and compared with the control group, the strains with the highest inhibitory activity of harmful microorganisms were selected and named as Probio-054.

On the other hand, the strain Lactobacillus luteri Probio-054 of the present invention is antibiotic-resistant, so that it can be administered to humans or animals as well as antibiotics or to replace existing antibiotics in order to suppress the growth of harmful microorganisms in the intestine.

In one advantageous aspect of the present invention, the present invention provides foods, pharmaceuticals, animal drugs, animal feed, lactic acid bacteria preparations and dietary supplements containing the novel probiotic microorganisms, Lactobacillus luteri Probio-054. The microorganisms of the present invention may be used alone or in combination with an acceptable carrier or in the form of a composition suitable for ingestion by humans or animals. That is, the microorganism of the present invention is added to foods that do not contain other probiotic bacteria (added to give probiotic activity) and foods that already contain some probiotic bacteria (added to enhance probiotic activity. Or complementary).

The pharmaceutical, food or nutritional composition of the present invention may be an isolated probiotic bacterium and include Lactobacillus luteri Probio-054 or two or more other beneficial strains together with a suitable carrier.

For example, other microorganisms that can be used with the strains of the present invention in the compositions of the present invention are suitable for ingestion by humans or animals and have probiotic activity that can inhibit harmful pathogenic harmful bacteria or improve microbial balance in the mammalian intestine. Those having are preferred. Examples of such probiotic microorganisms include yeasts including Saccharomyces, Candida, Pichia and Torulopsis , molds such as Aspergillus , Rhizopus, Mucor, Penicillium and Lactobacillus, Bifidobacterium, Clostridium, Leuconostoc, Bacteroides, Staactococcus, Lactusoccus, Lactobacillus Streptococcus, Fusobacterium, Propionibacterium, Enterococcus, Pediococcus, and Micrococcus are bacteria. Specific examples of suitable probiotic microorganisms include Saccharomyces cereviseae, Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Enterococcus faecium, Enterococcus faecalis, Lactobacillcillus caseus ruciusus lucus alitususus Lactobacillus johnsonii, Lactobacillus farciminus, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus sake, Lactococcus lactis, Micrococcus varians, Pediococcus acidilactici, and Staphylococcus x . Preferably, a probiotic microbial mixed bacterium having excellent probiotic activity and excellent immune activity enhancement and anticancer activity may be further included in the composition of the present invention to further enhance the effect of the composition of the present invention. Examples of carriers that may be contained in the compositions of the present invention may be extenders, high fiber additives, encapsulating agents, lipids, and the like and examples of such carriers are well known in the art. The Lactobacillus luteri Probio-054 strain of the present invention may be in lyophilized or encapsulated form or in culture suspension or dry powder form.

Instead of conventional antibiotics, the novel microorganisms and compositions of the present invention may be administered to humans or animals as probiotic or to humans or animals with foods, pharmaceuticals, animal drugs, feed additives, or dietary supplements. The probiotic microorganism of the present invention and the probiotic in the form of a composition containing the same inhibit the harmful microorganisms in the gut as a substitute for the existing antibiotics and improve the health of the animal body by maintaining a stable intestinal flora, and increase the weight gain of the livestock, improve the meat quality, and produce the milk It can be used for the purpose of increasing, enhancing immune activity. The composition of the present invention may also be administered after antibiotic administration to maintain the intestinal microflora.

In particular, the novel microorganisms of the present invention having antibiotic resistance as well as the ability to inhibit the growth of harmful enteric microorganisms are particularly effective for alleviating or treating most symptoms caused by infection of enteric harmful microbial infections in which other therapies are ineffective or have clinical side effects. useful. Thus, the present invention provides a method for preventing infection of intestinal harmful microorganisms or the occurrence of intestinal diseases or treating gastroenteritis and diarrhea caused by intestinal harmful microbial infections using the novel microorganisms or compositions containing an effective amount thereof. As the optimum temperature condition for inhibiting the harmful microorganisms Lactobacillus luterie Probio-054 is 30-37 ℃ is preferred.

Hereinafter, the configuration and the effect of the present invention through the embodiment will be described in more detail. However, the following examples are not intended to limit the scope of the invention for the purpose of illustrating the present invention is limited to the following examples.

Example 1 Isolation of Bacteria with Acid and Bile Acid Properties

The feces of healthy Jindo dogs were collected in an anaerobic bag (trade name Gas Pack pouch; manufactured by BBL). 1 g of the collected feces was anaerobic diluent adjusted to pH 2 (0.78% K ₂ HPO ₄ , salt mixture 37.5ml, L-cystein 0.5g, 25% L-ascorbic acid 2ml, 8% Na ₂ CO ₃ 50ml, 0.1% resazurin 1 ml was dissolved in 860 ml of distilled water, 0.5 g of agar was added), and the mixture was diluted at a ratio of 1:10 and shaken for 120 minutes using a shaker. BL solid medium (made by Young Yeon Chemical Co., Ltd.) with 0.2 ml of oxal (made by Diffco Co., Ltd.) [1 ml of shaking liquid] [Lab-lemco powder 2.4 g, Proteose peptone No. 3 10 g, Trypticase 5 g, Phytone peptone 3 g, 5g yeast extract, 150ml liver extract, 10g glucose, soluble starch 0.5g, sol.A 10ml, sol.B 5ml, 10% FS antifoam F-20 5ml, Tween 80 1g, L-cystein 0.5g, horse serum 50ml Dissolve in 1 L and add 15 g of agar; a composition of sol.A, 10% KH ₂ PO ₄ and 10% K ₂ HPO ₄ ; The composition of sol.B, 10 g of magnesium sulfate, 0.5 g of iron sulfate, 0.5 g of salt, and 0.337 g of manganese sulfate were dissolved in 250 ml of distilled water) and incubated at 37 ° C for 48 hours. 150 colonies formed after incubation were isolated by pure separation and selected by GAM semi-agar medium (manufactured by Japan Pharmaceutics Co., Ltd.) 2.2g, liver extract 1.2g, KH2PO4 2.5g, NaCl 3g, L-cystein 0.3g, Nathioglycolate 0.3g, glucose 2g are dissolved in 1L of distilled water, and 1.5g of agar is added to incubate. Preserved.

Example 2 Experiment of Inhibiting Harmful Microorganism Growth of Isolated Microorganisms

Inhibitory experiments on the growth of harmful microorganisms on isolated microorganisms were carried out on the following five types of microorganisms used for antibiotic evaluation using Kuroiwa et al. (Kuroiwa et al., Infectious Disease Journal, 64, 257, 1990). It was. Escherichia coli KCTC 2441, Shigella flexneri KCTC 2008, Enterobacter cloacea KCTC 2361, Citrobacter freundii KCTC 2006, Klebsiella pneumoniae KCTC 2208. Smeared. Paper disks with a diameter of 8 mm were placed on solid medium coated with harmful microorganisms, and inoculated with 30 ul of each isolated microbial culture medium anaerobicly cultured in an MRS liquid medium for 18 hours. Growth inhibitory rings appearing after a certain period of time was measured and compared with the control group, the strains with the highest inhibitory activity of harmful microorganisms were selected and named as Probio-054. Inhibitory activity of probio-054 against harmful microorganisms is shown in Table 1 below. As shown in Table 1, Probio-054 was found to be a very useful microorganism with mostly similar inhibitory activity against the five harmful microorganisms used. In addition, Probio-054 is more effective against harmful microorganisms than the existing Lactobacillus lusteri strain (BSA-131) (Kang, Young-Hyo et al., Kor. J. Appl. Microbiol. Biotechnol. 27, 23-27) (Korean Patent 10-211529-0000). The inhibitory activity was found to be slightly better, and the survival experiment was also confirmed to be superior.

Example 4 Identification of Isolated Microorganism Probio-054

Probio-054 strain isolated in Example 1 was incubated in 37 ° C conditions in MRS medium (Difco). To identify Probio-054 strains, the morphology and physiological properties of Probio-054 were determined by Yoon et al., Int. J. Syst. Bacteriol ., 47, 904, 1997 and the API 32A and CHL kits. It determined using (Biomerio company make). The sequencing and analysis of the 16S rRNA gene was performed using the method of Yoon et al., Int. J. Syst. Bacterill ., 47, 933, 1997.

Probio-054 is a Gram-positive bacterium that grows simultaneously in both aerobic and anaerobic conditions. It does not form spores, has no motility, and has a cell type. As growth conditions of the said strain, 30-37 degreeC is preferable. It does not produce gases and indole, does not show hemolysis and does not reduce nitric acid.

The base sequence of the 16S rRNA gene of Probio-054 is set forth in SEQ ID NO: 1 . In biomolecular taxonomic analysis based on 16S rDNA sequencing, Probio-054 is a strain belonging to the genus Lactobacillus , with 99.5% homology of 16S rDNA, which is the highest in the molecular strain of Lactobacillus reuteri . Strains showing relationships were identified. In addition, Probio-054 is a probiotic (BSA-131) identified by Lactobacillus luteri, which has the characteristics of acid and bile acids, which have been previously announced, and Jang, Young-Hyo et al., Kor J. Appl.Microbiol.Biotechnol. 27, 23-27) (Korean Patent 10-211529-0000) compared to the two nucleotide sequence was found to be different strains from the other. Therefore, based on the above results, Probio-054 was identified as a strain of Lactobacillus reuteri , which is different from the existing probiotic BSA-131, and was deposited on July 21, 2003 with the Korea Biotechnology Research Institute Gene Bank. Accession number: KCTC 10499BP).

As discussed above, Lactobacillus luteri Probio-054, a new microorganism of the genus Lactobacillus of the present invention, is a probiotic that is a probiotic activator that inhibits rotavirus and pathogenic bacteria, and rotavirus and various species that exhibit harmful properties in animals and intestines. It effectively inhibits pathogenic bacteria in the body and has acid-resistant and bile-acid function, so it is stabilized in the intestinal microflora when administered to humans and various livestocks as a substitute for existing antibiotics to treat symptoms that may be caused by abnormal fermentation of harmful microorganisms in the intestines. It can be prevented in advance, improve the health and improve the livestock weight gain, improve the quality of meat, increase the output of milk, increase immunity, etc.

<110> Probionic Copr. <120> Acid tolerant probiotic Lactobacillus reuteri Probio-054 that can          suppresses the growth of pathogenic microorganisms <130> probio-54 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1481 <212> DNA <213> Lactobacillus reuteri Probio-054 <220> <221> rRNA (222) (1) .. (1481) <223> 16 rRNA (cDNA) sequence of Lactobacillus reuteri Probio-054 <400> 1 cgccggcggt gtgcctaata catgcaagtc gtacgcactg gcccaactga ttgatggtgc 60 ttgcacctga ttgacgatgg atcaccattg agtggcggac gggtgagtaa cacgtaggta 120 acctgccccg gagcggggga taacatttgg aaacagatgc taataccgca taacaacaaa 180 agccacatgg cttttgtttg aaagatggct ttggctatca ctctgggatg gacctgcggt 240 gcattagcta gttggtaagg taacggctta ccaaggcgat gatgcatatc cgagttgaga 300 gactgatcgg ccacaatgga actgagacac ggtccatact cctacgggag gcagcagtag 360 ggaatcttcc acaatgggcg caagcctgat ggagcaacac cgcgtgagtg aagaagggtt 420 tcggctcgta aagctctgtt gttggagaag aacgtgcgtg agagtaactg ttcacgcagt 480 gacggtatcc aaccagaaag tcacggctaa ctacgtgcca gcagccgcgg taatacgtag 540 gtggcaagcg ttatccggat ttattgggcg taaagcgagc gcaggcggtt gcttaggtct 600 gatgtgaaag ccttcggctt aaccgaagaa gtgcatcgga aaccgggcga cttgagtgca 660 gaagaggaca gtggaactcc atgtgtagcg gtggaatgcg tagatatatg gaagaacacc 720 agtggcgaag gcggctgtct ggtctgcaac tgacgctgag gctcgaaagc atgggtagcg 780 aacaggatta gataccctgg tagtccatgc cgtaaacgat gagtgctagg tgttggaggg 840 tttccgccct tcagtgccgg agctaacgca ttaagcactc cgcctgggga gtacgaccgc 900 aaggttgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 960 ttcgaagcta cgcgaagaac cttaccaggt cttgacatct tgcgctaacc ttagagataa 1020 ggcgttccct tcggggacgc aatgacaggt ggtgcatggt cgtcgtcagc tcgtgtcgtg 1080 agatgttggg ttaagtccyg caacgagcgc aacccttgtt actagttgcc agcattaagt 1140 tgggcactct agtgagactg ccggtgacaa accggaggaa ggtggggacg acgtcagatc 1200 atcatgcccc ttatgacctg ggctacacac gtgctacaat ggacggtaca acgagtcgca 1260 agctcgcgag agtaagctaa tctcttaaag ccgttctcag ttcggactgt aggctgcaac 1320 tcgcctacac gaagtcggaa tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt 1380 tcccgggcct tgtacacacc gcccgtcaca ccatgggagt ttgtaacgcc caaagtcggt 1440 ggcctaacct ttatggaggg agccgcctaa ggcgggacag a 1481

Claims (4)

Lactobacillus reuteri Probio-054 (Accession No .: KCTC 10499BP) with acid resistance, bile acid resistance and ability to inhibit harmful microbial growth. A veterinary composition comprising an effective amount of the probiotic Lactobacillus luterie Probio-054 (Accession No .: KCTC 10499BP) and an acceptable carrier and having probiotic activity. The composition of claim 2, wherein the composition is useful for preventing or treating diarrhea in a mammal upon oral ingestion. A method for preventing the growth of harmful intestinal pathogenic microorganisms in pigs or preventing or treating diarrhea caused by the microorganisms using the probiotic Lactobacillus luteri Probio-054 of claim 1 or the probiotic composition of claim 2.