WO2005007834A1 - Acid tolerant probiotic lactobacillus plantarum probio-38 that can suppress the growth of pathogenic microorganism and tge coronavirus - Google Patents

Acid tolerant probiotic lactobacillus plantarum probio-38 that can suppress the growth of pathogenic microorganism and tge coronavirus Download PDF

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Publication number
WO2005007834A1
WO2005007834A1 PCT/KR2003/001461 KR0301461W WO2005007834A1 WO 2005007834 A1 WO2005007834 A1 WO 2005007834A1 KR 0301461 W KR0301461 W KR 0301461W WO 2005007834 A1 WO2005007834 A1 WO 2005007834A1
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Prior art keywords
probio
coronavirus
lactobacillus plantarum
growth
present
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PCT/KR2003/001461
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French (fr)
Inventor
Yong-Ha Park
In-Seon Lee
Jung-Hoon Yoon
Chul-Joong Kim
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Probionic Corp.
M.D.Lab Corp.
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Priority to AU2003247193A priority Critical patent/AU2003247193A1/en
Priority to PCT/KR2003/001461 priority patent/WO2005007834A1/en
Publication of WO2005007834A1 publication Critical patent/WO2005007834A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/169—Plantarum
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/25—Lactobacillus plantarum

Definitions

  • the present invention relates to a novel Lactobacillus plantarum Probio-38 having the inhibition activity against porcine transmissible gastroenteritis (hereinafter, referred to as "TGE") Coronavirus and other pathogenic microbes and probiotics comprising the same. More particularly the present invention relates to Lactobacillus plantarum Probio-38 that is separated from pig's stomach, is tolerant of acid and bile acid, resistant to antibiotics and can suppress the growth of TGE Coronavirus and enteric pathogens effectively, probiotics comprising the same and methods for preventing and treating livestock' diarrhea by using the same.
  • TGE porcine transmissible gastroenteritis
  • TGE Transmissible gastroenteritis
  • TGE is so severe disease as a sort of diarrhea to kill 80 ⁇ 90% of lactivorous pigs and more than 20% of pigs, to decrease the appetite of hogs and boars and to suppress the growth of swine.
  • TGE is caused by Coronavirus that inhabits hog raising farm and natural environment and actually, becomes more harmful through the secondary infection of Escherichia coli.
  • this diarrhea against Coronavirus cannot be remedied easily by using an antibiotic since it is a viral disease.
  • the antibiotic is expected only to treat the secondary infection of microbes. On the other hand, it is a global trend to avoid the antibiotic abuse.
  • Coronavirus vaccine has been utilized to prevent the infection of porcine TGE Coronavirus, but there is also a problem that this vaccine may have some side effects.
  • various microbes exist and form normal intestinal flora.
  • some of microbes such as Lactobacillus sp. are known to be useful for host animals.
  • other microbes such as Esche chia coli, Salmonella sp., Staphylococcus sp. and the like are harmful directly or potentially for host cells.
  • the intestinal flora of normal microbes may be destructed if stress is increased, pathogenic bacteria are infected and outer environment is changed in human and animals. Therefore, the harmful microbes can proliferate rapidly, aggravates the health condition of host animal and become dangerous to cause diarrhea and even death.
  • the antibiotic is administered for therapeutic purpose, but it is not discharged completely after use and sustained within the body. Hence, if the antibiotic continued to be injected, the pathogenic microbes become resistant to drugs and will not be treated easily. In the meantime, the antibiotic content is strictly regulated in meat, milk, eggs and the like produced from livestock. Therefore, it is necessary to think over the use and abuse of antibiotics again.
  • Probiotic as a live vaccine is manufactured to pharmaceutical composition for commercial use, after useful microbes are separated from the intestine of human and animal.
  • aerobic bacteria, anaerobic bacteria, lactobacillus, yeast and the like can be exploited and especially, Lactobacillus sp. is widely adopted.
  • Lactobacillus sp. has been utilized to ferment and process traditional food and is proved to be safe for a long time.
  • several strains of Lactobacillus sp. have been already disclosed in GRAS (generally recognized as safe) list according to FDA standard.
  • the probiotic comprising Lactobacillus strain, causes no side effect, can suppress the abnormal fermentation of enteric pathogens, sustain the intestinal flora stably and reduce the infection of harmful microbe. Furthermore, it can increase the fodder efficiency as a live vaccine if continued to be administered, enhance the health condition and maximize the body weight of livestock.
  • the probiotic should be tolerant of acid and bile acid as well as suppress the growth of pathogenic microbes. Precisely, the live vaccine meets the strong acid of gastric juice after administered until reaching the intestine and afterward confronts bile acids.
  • the present inventors have attempted to screen novel live vaccine that can suppress the growth of pathogenic microbes more effectively than conventional live vaccines, be tolerant of acid and bile acid and specifically suppress the growth of porcine TGE Coronavirus.
  • an intestinal microbe that have the inhibition activity against pathogenic microbes, is highly resistant to acid and bile acids and suppresses the growth of porcine TGE Coronavirus effectively, from pig's stomach in an anaerobic condition and examined.
  • the present inventors have identified the microbe as Lactobacillus plantarum and completed the present invention successfully.
  • the object of the present invention is to provide a novel microbe that is harmless for human and animal and is tolerant of acid and bile acids remarkably.
  • Another object of the present invention is to provide compositions comprising the probiotic microbe, which can replace conventional antibiotics and suppress the growth of Coronavirus and pathogenic microbes in intestine, when it is administered to livestock and used as pharmaceuticals, fodder additive, veterinary drug or the like.
  • Another object of the present invention is to provide a method for suppressing Coronavirus or for preventing and/or treating disease caused by the virus, in which the microbe of the present invention or the compositions comprising the same is utilized.
  • the other object of the present invention is to provide a method for suppressing the growth of enteric pathogens or for preventing and/or treating disease caused by the pathogenic microbes, in which the microbe of the present invention is utilized.
  • FIG. 1 depicts the nucleotide sequence of 16S rRNA gene separated from Lactobacillus plantarum Probio-38 (accession number: KCCM-10329) of the present invention.
  • the present inventors have tried to provide a novel strain, Lactobacillus plantarum Probio-38 that can suppress the growth of porcine TGE Coronavirus and other enteric pathogens and is tolerant of acid and bile acid, probiotic compositions comprising the same such as food, pharmaceuticals, veterinary drug, fodder additive or the like and a method for suppressing the growth of porcine TGE Coronavirus and enteric pathogens in which Lactobacillus plantarum Probio-38 strain is used.
  • Lactobacillus plantarum Probio-38 of the present invention is an enteric microbe derived from pig's stomach and is elucidated to survive in an anaerobic condition, to suppress the growth of porcine TGE Coronavirus and other harmful microbes and as a result, to prevent and treat livestock' diarrhea efficiently.
  • the present inventors have already developed and modified the method for preventing or treating diarrhea caused by Coronavirus infection.
  • the attenuated vaccine of porcine epidemic diarrhea virus is commercially available to treat porcine diarrhea caused by TGE Coronavirus.
  • this vaccine is not utilized widely, since it is not promoted around domestic fields and due to the poor environment of hog raising farms and thus, TGE Coronavirus has not been eradicated yet.
  • the present invention provides an effective and potential probiotic that can treat diarrhea and/or gastroenteritis caused by the infection of porcine Coronavirus and other pathogenic microbes in intestine. Therefore, the probiotic microbe of the present invention can be
  • microbes that inhabit in food such as fodder and on livestock' intestine.
  • the microbe of the present invention can adsorb onto intestinal
  • Capacity to suppress the growth of porcine Coronavirus and other enteric pathogenic microbes Capacity to grow both in aerobic and an anaerobic condition and to grow in the wide range of pH; which can adjust the microbe to the physiological and pathological changes in stomach Tolerance to acid and bile acid Capacity to sustain the activity of live vaccine after lyophilized
  • novel microbe of the present invention has a
  • probiotic means "a living microbe which improves the enteric microbial
  • probiotic activity and manufactured to single or complex form can confer a beneficial effect upon the intestinal flora of host animal, when it is administered in the state of dried cell or fermented product for human or animal.
  • "porcine Coronavirus” defines "a major viral contagion derived from pigs", and includes porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and the like. It occurs even in domestic area every year and damages the hog raising industry enormously. Especially, the microbe of the present invention is outstanding to suppress the growth of TGEV. TGEV is highly transmissible, causes vomiting and severe diarrhea and is sensitive to all the swine regardless of age.
  • TGEV causes most severe diarrhea to newborn pigs under 2-weeked age and increases the fatality rate highly. It may infect and destruct cells on small intestine selectively so as to prevent the nutrient absorption and the regulation of body fluid (H. W. Moon, J. Am. Vet. Med. Assoc, 172, 443, 1978).
  • the present inventors have tried to screen the probiotic microbe that can suppress the growth of porcine TGE Coronavirus and enteric pathogenic microbes and is tolerant of acid and bile acids through the procedure as follows. That is to say, gastric remnant 30 g is collected from pigs, poured to an anaerobic bag (commercial name, Gas Pack pouch; purchased from BBL Co.
  • GAM semi-agar plate purchased from llsu
  • coli KCTC 2571 Klebsiella pneumoniae KCTC 2208, Staphylococcus aureus KCTC 1621 , Staphylococcus epidermidis KCTC 1917, Salmonella entehtidis, Shigella flexneri KCTC 2008, Salmonella gallinarum, Enterobacter cloacea KCTC 2361, Enterococcus lactis KCTC 1913, Salmonella typhimurium, Citrobacter freundii KCTC 2006 and Bacillus subtilis KCTC 1021.
  • NA nutrient agar
  • Lactobacillus plantarum of the present invention is cultivated anaerobically in MRS culture broth at
  • Lactobacillus plantarum Probio-38 one strain that can suppress the growth of pathogenic microbes most outstandingly are selected and named as Lactobacillus plantarum Probio-38.
  • Probio-38 strain is examined to elucidate morphological, physiological and biochemical characteristics, to determine the nucleotide sequence of 16S rRNA gene, and to analyze the sequence. Consequently, it is identified that Probio-38 strain is a Gram- positive bacterium, can proliferate both in aerobic and anaerobic conditions, does not make spores, has no mobility and has the bacillus
  • Probio-38 strain is preferable to proliferate at 30 ⁇ 37°C, does not
  • the nucleotide sequence of 16S rRNA gene from Probio-38 strain of the present invention contains the nucleotide sequence of SEQ ID NO: 1 as illustrated in FIG. 1.
  • Probio-38 strain is further investigated through the molecular phylogenic analysis based upon the nucleotide sequence of 16S rRNA. Therefore, Probio-38 strain of the present invention is identified to belong to Bacillus sp., to have 99.5% of 16S rDNA homology and to have the highest phylogenic relationship with the standard strain of Lactobacillus plantarum.
  • the Probio-38 strain of the present invention is named as Lactobacillus plantarum Probio-38 and has been deposited to Korean Culture Center of Microorganism, Korea Federation of Culture Collections (International Deposition Organization; Yoorim B/D FL 2, 361-221 Hongje-Dong, Seodaemoon-Gu, Seoul, Korea) as accession number KCCM-10329 on October 27, 2001.
  • Lactobacillus plantarum Probio-38 of the present invention is named as Lactobacillus plantarum Probio-38 and has been deposited to Korean Culture Center of Microorganism, Korea Federation of Culture Collections (International Deposition Organization; Yoorim B/D FL 2, 361-221 Hongje-Dong, Seodaemoon-Gu, Seoul, Korea) as accession number KCCM-10329 on October 27, 2001.
  • invention can be submerged in glycerol at -80°C or lyophilized after
  • the probiotic strain of the present invention can be improved or modified through conventional physiochemical methods, such as mutagenesis to enhance the stability or the antiviral activity.
  • the probiotic strain of the present invention is examined whether it can suppress the growth of TGE Coronavirus or not. As a result, it is confirmed to have the antiviral activity against Coronavirus since cytopathic effect is not observed.
  • the probiotic strain is investigated whether it can be sensitive to antibiotics or not. As a result, it is verified to have the inhibition activity against enteric pathogens in the stomach and to be resistant to various kinds of veterinary antibiotics.
  • the probiotic strain of the present invention is more advantageous to raise livestock since it is separated from pig's stomach and makes the normal flora of enteric microbes as demonstrated above.
  • the present invention provides food, pharmaceuticals, veterinary drug or fodder additive comprising Lactobacillus plantarum Probio-38 strain.
  • the probiotic microbe of the present invention can be prepared with pharmaceutically acceptable carrier, utilized independently or as a food additive and manufactured to compositions suitable for human and animal. Namely, the microbe of the present invention can be added to food without other probiotic microbe (conferring the probiotic activity, if added) and/or food with several probiotic microbes (enhancing or compensating the probiotic activity, if added) before use.
  • the pharmaceutical, veterinary or nutritional composition of the present invention can be composed of Lactobacillus plantarum Probio-38 strain as a probiotic microbe or additionally composed of more than 2 kinds of another useful strains and acceptable carriers.
  • the strain that is compatible with Lactobacillus plantarum Probio-38 to prepare the compositions of the present invention should be edible for animal, suppress the proliferation of virus or pathogenic microbes and improve the microbial balance of mammal's intestine, in addition to the probiotic activity.
  • yeast such as Saccharomyces, Candida, Pichia, Torulopsis and the like
  • fungi such as Aspergillus, Rhizopus, Mucor, Penicillium and the like
  • bacteria such as Lactobacillus, Bifidobacte um, Clostridium, Leuconostoc, Bacteroides, Staphylococcus, Lactococcus, Bacillus, Streptococcus, Fusobactehum, Propionibacterium, Enterococcus, Pediococcus, and Micrococcus sp.
  • yeast such as Saccharomyces, Candida, Pichia, Torulopsis and the like
  • fungi such as Aspergillus, Rhizopus, Mucor, Penicillium and the like
  • bacteria such as Lactobacillus, Bifidobacte um, Clostridium, Leuconostoc, Bacteroides, Staphylococcus, Lactococcus, Bacillus,
  • the strain can be selected among Saccharomyces cereviseae, Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis, Bifidobacterium bifidum, Bifidobactehum infantis, Bifidobacterium longum, Enterococcus faecium, Enterococcus faecalis, Lactobacillus acidophilus, Lactobacillus alimentarius, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus delbruckii, Lactobacillus johnsonii, Lactobacillus farciminus, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus sake, Lactococcus lactis, Micrococcus varians, Pediococcus acidilactici, Staphylococcus, Bac
  • Lactobacillus reuteri Probio-16 (accession number: KCCM 10214) that can suppress the growth of Rotavirus causing porcine diarrhea as well as has the probiotic activity and Lactobacillus salibarius Probio-37 (accession number: KCCM 10328) that can suppress the growth of Coronavirus or their microbial mixture, can be added to the compositions of the present invention.
  • the composition of the present invention can include one more acceptable carrier selected among weight-increase agent, polymer additive, capsules, lipid and the like. These carriers are disclosed clearly to those skilled in the art.
  • Lactobacillus plantarum Probio-38 of the present invention can be lyophilized or made to a capsule, culture suspension or dry powder.
  • the microbe and the compositions of the present invention can be administered to animals directly or coordinately with food, pharmaceuticals, veterinary drug or fodder additive as a probiotic agent.
  • the probiotic agent aims to replace the existed antibiotics, to suppress the growth of enteric pathogens in human and various livestock, to sustain the intestinal flora stably, to make the animal body healthier, to increase the weight of livestock, to improve the quality of meat, to increase the productivity of milk and to enhance the immunity and the like.
  • the probiotic composition of the present invention can be treated even after the antibiotic is already injected.
  • the microbe of the present invention also has the antiviral activity and is used to alleviate and treat most symptoms caused by Coronavirus infection, when other treatment is not effective and has the side effect on clinical use. Therefore, the present invention provides the method for preventing the Coronavirus infection or for treating gastroenteritis, diarrhea and the like caused by Coronavirus, by using the probiotic strain and the compositions comprising the same. Also, the present invention provides the method for suppressing the growth of enteric pathogenic microbes or for treating gastroenteritis, diarrhea and the like caused by the pathogens.
  • Gastric remnant from normal pig was collected to an anaerobic bag (commercial name, Gas Pack pouch; a product from BBL Co. Ltd.), moved in the anaerobic condition and examined. 30 g of the resulting remnant collected above was diluted to 1 : 10 ratio by using 270 ml of
  • horse serum 50m£ were dissolved in distilled water 1,000ml. and then
  • agar 15 g was added; solution A, 10% KH 2 P0 4 and 10% K 2 HP0 4 and solution B, magnesium sulfate 10 g, iron sulfate 0.5 g, salt 0.5 g and
  • GAM semi-agar plate purchased from llsu Pharmaceutics, Japan
  • peptone 10 g Soya peptone 3 g, proteose peptone No.3 10 g, peptonized whale serum 13.5 g
  • yeast extract 5 g beef extract 2.2 g
  • liver extract 1.2 g KH 2 P0 4 2.5 g
  • NaCI 3 g L-cystein 0.3 g
  • sodium thioglycolate 0.3 g and glucose 2 g were dissolved in
  • Probio-38 strain separated in Example 1 was cultivated on MRS
  • Probio-38 strain the morphological and physiological properties were examined through the method of Yoon et al. and by using API 32A kit and CHL kit (purchased from Biomerieux Co. Ltd.)(Yoon et al., Int. J. Syst Bactehol., 47, 904, 1997).
  • the nucleotide sequence of 16S rRNA gene was determined and analyzed by performing the method of Yoon et al. (Yoon et al., Int. J. Syst. Bactehol., 47, 933, 1997).
  • the morphological, physiological and biochemical characteristics of Probio-38 strain are illustrated in Table 1. ⁇ Table 1 > Morphological, physiological and biochemical characteristics of Probio-38 strain
  • Probio-38 strain is a Gram-positive bacterium, can proliferate both in aerobic and anaerobic conditions, does not make spores, have no mobility and be a bacillus in the shape.
  • Probio-38 strain is preferable to grow at 30 ⁇ 37°C, does not generate gas and indoles, does not induce hemolysis, not reduce nitric acid and is tolerant of bile acid 0.5%.
  • the nucleotide sequence of 16S rRNA gene purified from Probio- 38 strain of the present invention is illustrated in FIG. 1. Probio-38 strain was investigated through the molecular phylogenic analysis, based upon the nucleotide sequence of 16S rRNA.
  • Probio-38 strain of the present invention is verified to belong to Bacillus sp., to have 99.5% sequence homology of 16S rDNA and to have the highest phylogenic relationship with the standard strain of Lactobacillus plantarum.
  • Probio-38 strain of the present invention was named as Lactobacillus plantarum Probio-38 and has been deposited to Korean Culture Center of Microorganism, International Deposition Organization on October 27, 2001 (accession number KCCM-10329).
  • TGE virus strain and ST cell were prepared in the Example.
  • Virus culture TGE virus strain was inoculated onto ST cell monolayer, adsorbed on cell for about 30 minutes, mixed with fresh Eagle's minimum essential medium (MEM) containing 5% bovine serum in a
  • CPE cytopathic effect
  • Virus titration ST cell monolayer was prepared on 96-well culture plate and TGE virus was diluted through 10-fold serial dilution. Then, each virus aliquot was poured to 10 wells of culture plate respectively and
  • the virus titer of the culture medium was estimated according to Reed and Muench method.
  • ST cell monolayer was prepared onto 96-well culture plate and 90 ⁇ l of virus titer 10.0 TCID 5 o/0.1m£, 100 TCID 50 /0J ⁇ and 1000 TCID 5 o/OJm£ were poured to the first, the second and the third wells independently.
  • 10m (10%) culture medium of Lactobacillus plantarum Probio-38 was poured to each well and cultivated at 37°C with a incubator containing 5% C0 2 so as to measure CPE after 24, 48 and 72 hours lapsed.
  • the culture medium of Lactobacillus plantarum Probio-38 was verified not to have the antiviral activity when the cytopathic effect (CPE) was observed.
  • the inhibition activity against Coronavirus in Lactobacillus plantarum Probio- 38 is illustrated in Table 2.
  • coli KCTC 2571 Klebsiella pneumoniae KCTC 2208, Staphylococcus aureus KCTC 1621 , Staphylococcus epidermidis KCTC 1917, Salmonella ente tidis, Shigella flexneri KCTC 2008, Salmonella gallinarum, Enterobacter cloacea KCTC 2361 , Enterococcus lactis KCTC 1913, Salmonella typhimuhum, Citrobacter freundii KCTC 2006 and Bacillus subtilis KCTC 1021.
  • the pathogenic microbes described above were cultivated in NB (nutrient broth) medium at 37°C for 18 hours. Then 10 mi of the culture medium was smeared on
  • Lactobacillus plantarum of the present invention was cultivated in an anaerobic condition by using MRS culture broth at 37°C for 18 hours. Again, 30 ⁇ l of culture medium was inoculated on 8 mm paper disc placed on the above-mentioned NA plate and cultivated at 37°C for 24 hours. Afterward, the clear zone was measured to compare that of standard group. As a result, one strain was selected to suppress the growth of pathogenic microbes most outstandingly and named as Lactobacillus plantarum Probio-38. The inhibition activity against pathogenic microbes is illustrated in Table 3. ⁇ Table 3> Inhibition activity against pathogenic microbes by Lactobacillus plantarum Probio-38
  • Lactobacillus plantarum Probio-38 suppress the growth of almost 13 pathogenic microbes and be a useful probiotic microbe.
  • Lactobacillus plantarum Probio-38 was utilized according to the protocol of Becton Dickinson Microbiology Systems.
  • 11 kinds of antibiotics were adopted as follows: tetracycline, ampicillin, gentamicin, cephalothin, sulfisoxazole, carbenicillin, streptomycin, lincomycin, neomycin, penicillin and erythromycin.
  • Lactobacillus plantarum Probio-38 of the present invention was cultivated in MRS culture broth at 37°C for 18 hours and smeared homogeneously on MRS culture plate.
  • Lactobacillus plantarum Probio-38 is highly sensitive to ampicillin, carbenicillin and erythromycin.
  • the novel microbe of the present invention separated from pig's intestine, Lactobacillus plantarum Probio-38 can suppress the growth of TGE Coronavirus and other pathogenic microbes effectively and is tolerant of acid and bile acid. Therefore instead of conventional antibiotics, the microbe and the compositions of the present invention can be administered for human and animals to make the animal body healthier, to increase the weight of livestock, to improve the quality of meat, to increase the productivity of milk and to enhance the immunity and the like as well as to prevent and treat the symptoms by the abnormal fermentation of enteric pathogens since it sustains the intestinal flora stably.

Abstract

The present invention relates to a Lactobacillus prantarum Probio-38 from animal sources that inhibits TGE coronavirus infection and other pathogenic microorganisms, as well as being tolerant of gastric and bile acids. Also, this invention relates to a prophylactic and therapeutic composition comprising the same for contributing in many probiotic ways to the host's general health and preventing and treating diseases or conditions associated with coronavirus and other enteric pathogens.

Description

ACID TOLERANT PROBIOTIC LACTOBACILLUS PLANTARUM PROBIO-38 THAT CAN SUPPRESS THE GROWTH OF PATHOGENIC MICROORGANISM AND TGE CORONAVIRUS
Technical Field The present invention relates to a novel Lactobacillus plantarum Probio-38 having the inhibition activity against porcine transmissible gastroenteritis (hereinafter, referred to as "TGE") Coronavirus and other pathogenic microbes and probiotics comprising the same. More particularly the present invention relates to Lactobacillus plantarum Probio-38 that is separated from pig's stomach, is tolerant of acid and bile acid, resistant to antibiotics and can suppress the growth of TGE Coronavirus and enteric pathogens effectively, probiotics comprising the same and methods for preventing and treating livestock' diarrhea by using the same.
Background Art Transmissible gastroenteritis (TGE) occurs in hog raising farms around domestic and oversea fields. TGE is so severe disease as a sort of diarrhea to kill 80 ~ 90% of lactivorous pigs and more than 20% of pigs, to decrease the appetite of hogs and boars and to suppress the growth of swine. TGE is caused by Coronavirus that inhabits hog raising farm and natural environment and actually, becomes more harmful through the secondary infection of Escherichia coli. In addition, this diarrhea against Coronavirus cannot be remedied easily by using an antibiotic since it is a viral disease. The antibiotic is expected only to treat the secondary infection of microbes. On the other hand, it is a global trend to avoid the antibiotic abuse. Also, there is a problem that the antibiotic may be sustained within meat and move into human body. Therefore, it is required to develop a novel substance instead of antibiotic. Presently, Coronavirus vaccine has been utilized to prevent the infection of porcine TGE Coronavirus, but there is also a problem that this vaccine may have some side effects. On intestines of human and animal, various microbes exist and form normal intestinal flora. Especially, some of microbes such as Lactobacillus sp. are known to be useful for host animals. On the contrary, other microbes such as Esche chia coli, Salmonella sp., Staphylococcus sp. and the like are harmful directly or potentially for host cells. The intestinal flora of normal microbes may be destructed if stress is increased, pathogenic bacteria are infected and outer environment is changed in human and animals. Therefore, the harmful microbes can proliferate rapidly, aggravates the health condition of host animal and become dangerous to cause diarrhea and even death. At this moment, the antibiotic is administered for therapeutic purpose, but it is not discharged completely after use and sustained within the body. Hence, if the antibiotic continued to be injected, the pathogenic microbes become resistant to drugs and will not be treated easily. In the meantime, the antibiotic content is strictly regulated in meat, milk, eggs and the like produced from livestock. Therefore, it is necessary to think over the use and abuse of antibiotics again. In order to settle this problem, many researchers are paying more attention to probiotics and tried to develop an effective live vaccine. Probiotic as a live vaccine is manufactured to pharmaceutical composition for commercial use, after useful microbes are separated from the intestine of human and animal. For this purpose, aerobic bacteria, anaerobic bacteria, lactobacillus, yeast and the like can be exploited and especially, Lactobacillus sp. is widely adopted. Lactobacillus sp. has been utilized to ferment and process traditional food and is proved to be safe for a long time. Also, several strains of Lactobacillus sp. have been already disclosed in GRAS (generally recognized as safe) list according to FDA standard. Advantageously, the probiotic comprising Lactobacillus strain, causes no side effect, can suppress the abnormal fermentation of enteric pathogens, sustain the intestinal flora stably and reduce the infection of harmful microbe. Furthermore, it can increase the fodder efficiency as a live vaccine if continued to be administered, enhance the health condition and maximize the body weight of livestock. In order to become an effective live vaccine, the probiotic should be tolerant of acid and bile acid as well as suppress the growth of pathogenic microbes. Precisely, the live vaccine meets the strong acid of gastric juice after administered until reaching the intestine and afterward confronts bile acids. Since gastric juice and bile acids usually remove external microbes and decrease the activity of live vaccine, the probiotic should be tolerant of gastric juice and bile acids to reach the intestine stably and to be functional in the body. Up to now, various strains of Lactobacillus sp. have been disclosed to be used for live vaccines. Also, Lactobacillus plantarum has been identified to be tolerant of acid and bile acid and to suppress the growth of pathogenic microbes and reported as a live vaccine (Handbook
of Probiotics / Yuan-Kun Lee... [et al.).John Wiley & Sons, Inc., New
York, 1999, pp.4-6).
Disclosure of the Invention The present inventors have attempted to screen novel live vaccine that can suppress the growth of pathogenic microbes more effectively than conventional live vaccines, be tolerant of acid and bile acid and specifically suppress the growth of porcine TGE Coronavirus. As a result, we have separated an intestinal microbe that have the inhibition activity against pathogenic microbes, is highly resistant to acid and bile acids and suppresses the growth of porcine TGE Coronavirus effectively, from pig's stomach in an anaerobic condition and examined. The present inventors have identified the microbe as Lactobacillus plantarum and completed the present invention successfully. The object of the present invention is to provide a novel microbe that is harmless for human and animal and is tolerant of acid and bile acids remarkably. Another object of the present invention is to provide compositions comprising the probiotic microbe, which can replace conventional antibiotics and suppress the growth of Coronavirus and pathogenic microbes in intestine, when it is administered to livestock and used as pharmaceuticals, fodder additive, veterinary drug or the like. Another object of the present invention is to provide a method for suppressing Coronavirus or for preventing and/or treating disease caused by the virus, in which the microbe of the present invention or the compositions comprising the same is utilized. The other object of the present invention is to provide a method for suppressing the growth of enteric pathogens or for preventing and/or treating disease caused by the pathogenic microbes, in which the microbe of the present invention is utilized.
Brief description of the drawing The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which; FIG. 1 depicts the nucleotide sequence of 16S rRNA gene separated from Lactobacillus plantarum Probio-38 (accession number: KCCM-10329) of the present invention. Best mode for carrying out the Invention In order to attain the above-mentioned objects of the present invention, the present inventors have tried to provide a novel strain, Lactobacillus plantarum Probio-38 that can suppress the growth of porcine TGE Coronavirus and other enteric pathogens and is tolerant of acid and bile acid, probiotic compositions comprising the same such as food, pharmaceuticals, veterinary drug, fodder additive or the like and a method for suppressing the growth of porcine TGE Coronavirus and enteric pathogens in which Lactobacillus plantarum Probio-38 strain is used. Lactobacillus plantarum Probio-38 of the present invention is an enteric microbe derived from pig's stomach and is elucidated to survive in an anaerobic condition, to suppress the growth of porcine TGE Coronavirus and other harmful microbes and as a result, to prevent and treat livestock' diarrhea efficiently. The present inventors have already developed and modified the method for preventing or treating diarrhea caused by Coronavirus infection. Presently, the attenuated vaccine of porcine epidemic diarrhea virus is commercially available to treat porcine diarrhea caused by TGE Coronavirus. However, this vaccine is not utilized widely, since it is not promoted around domestic fields and due to the poor environment of hog raising farms and thus, TGE Coronavirus has not been eradicated yet. The present invention provides an effective and potential probiotic that can treat diarrhea and/or gastroenteritis caused by the infection of porcine Coronavirus and other pathogenic microbes in intestine. Therefore, the probiotic microbe of the present invention can be
utilized to reduce the number of Coronavirus and other pathogenic
microbes that inhabit in food such as fodder and on livestock' intestine. The microbe of the present invention can adsorb onto intestinal
cells and has probiotic characteristics as follows.
Capacity to suppress the growth of porcine Coronavirus and other enteric pathogenic microbes. Capacity to grow both in aerobic and an anaerobic condition and to grow in the wide range of pH; which can adjust the microbe to the physiological and pathological changes in stomach Tolerance to acid and bile acid Capacity to sustain the activity of live vaccine after lyophilized
In addition, the novel microbe of the present invention has a
feature to exclude side effect such as antibiotic resistance, biological
toxicity or the like, compared with the prior arts for the prevention and the
treatment. In the present invention, several terms are defined as follows,
"probiotic" means "a living microbe which improves the enteric microbial
balance in the stomach and gives a beneficial effect on host animal
including human", "probiotic microbe" denotes "a microbe left alive with
the probiotic activity and manufactured to single or complex form" and can confer a beneficial effect upon the intestinal flora of host animal, when it is administered in the state of dried cell or fermented product for human or animal. In the present invention, "porcine Coronavirus" defines "a major viral contagion derived from pigs", and includes porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and the like. It occurs even in domestic area every year and damages the hog raising industry enormously. Especially, the microbe of the present invention is outstanding to suppress the growth of TGEV. TGEV is highly transmissible, causes vomiting and severe diarrhea and is sensitive to all the swine regardless of age. Furthermore, TGEV causes most severe diarrhea to newborn pigs under 2-weeked age and increases the fatality rate highly. It may infect and destruct cells on small intestine selectively so as to prevent the nutrient absorption and the regulation of body fluid (H. W. Moon, J. Am. Vet. Med. Assoc, 172, 443, 1978). Concretely, the present inventors have tried to screen the probiotic microbe that can suppress the growth of porcine TGE Coronavirus and enteric pathogenic microbes and is tolerant of acid and bile acids through the procedure as follows. That is to say, gastric remnant 30 g is collected from pigs, poured to an anaerobic bag (commercial name, Gas Pack pouch; purchased from BBL Co. Ltd.) accurately, diluted to 1 : 10 ratio by using 270 ml of anaerobic dilution buffer adjusted to pH 2 and blended for 120 minutes with a shaker. 1 ml of aliquot is smeared onto BL culture plate containing 0.5% Oxgal (commercial name, purchased from Dipco Co. Ltd.) and cultivated at
37°C for 48 hours with an anaerobic incubator. Afterward, 310 colonies
are selected, moved onto GAM semi-agar plate (purchased from llsu
Pharmaceutics, Japan), cultivated and stored at -80°C for the next
procedure. In order to examine the growth inhibition of Lactobacillus plantarum Probio-38 strain separated in the present invention, Kuroiwa' method (Kuroiwa et al., Journal of Infectious disease, 64, 257, 1990) is exploited. In this experiment, 13 kinds of target microbes that are used to estimate the antibiotic activity are selected as follows: Esche chia coli KCTC 2441 , E. coli KCTC 2571 , Klebsiella pneumoniae KCTC 2208, Staphylococcus aureus KCTC 1621 , Staphylococcus epidermidis KCTC 1917, Salmonella entehtidis, Shigella flexneri KCTC 2008, Salmonella gallinarum, Enterobacter cloacea KCTC 2361, Enterococcus lactis KCTC 1913, Salmonella typhimurium, Citrobacter freundii KCTC 2006 and Bacillus subtilis KCTC 1021. The enteric pathogenic microbes described
above are cultivated in NB (nutrient broth) medium at 37°C for 18 hours
and 10 mi of culture broth is smeared on NA (nutrient agar) plate, dried
and covered with 8 mm radius of paper disc. Lactobacillus plantarum of the present invention is cultivated anaerobically in MRS culture broth at
37°C for 18 hours. 30 μl of culture medium is inoculated to 8 mm radius
of paper disc placed on the above NA plate and then cultivated at 37°C for 24 hours. Afterward, the clear zone is measured to compare that of standard group. As a result, one strain that can suppress the growth of pathogenic microbes most outstandingly are selected and named as Lactobacillus plantarum Probio-38. In order to identify the probiotic microbe of the present invention,
Lactobacillus plantarum Probio-38 strain is examined to elucidate morphological, physiological and biochemical characteristics, to determine the nucleotide sequence of 16S rRNA gene, and to analyze the sequence. Consequently, it is identified that Probio-38 strain is a Gram- positive bacterium, can proliferate both in aerobic and anaerobic conditions, does not make spores, has no mobility and has the bacillus
shape. Probio-38 strain is preferable to proliferate at 30 ~ 37°C, does not
generate gas and indoles, does not induce hemolysis and does not reduce nitric acid. The nucleotide sequence of 16S rRNA gene from Probio-38 strain of the present invention contains the nucleotide sequence of SEQ ID NO: 1 as illustrated in FIG. 1. Probio-38 strain is further investigated through the molecular phylogenic analysis based upon the nucleotide sequence of 16S rRNA. Therefore, Probio-38 strain of the present invention is identified to belong to Bacillus sp., to have 99.5% of 16S rDNA homology and to have the highest phylogenic relationship with the standard strain of Lactobacillus plantarum. Depending upon the experimental data, the Probio-38 strain of the present invention is named as Lactobacillus plantarum Probio-38 and has been deposited to Korean Culture Center of Microorganism, Korea Federation of Culture Collections (International Deposition Organization; Yoorim B/D FL 2, 361-221 Hongje-Dong, Seodaemoon-Gu, Seoul, Korea) as accession number KCCM-10329 on October 27, 2001. Preferably, Lactobacillus plantarum Probio-38 of the present
invention can be submerged in glycerol at -80°C or lyophilized after
suspended in 10% of sterilized skim milk in order to be stored for a long time.
It is clear to those skilled in the art that the probiotic strain of the present invention can be improved or modified through conventional physiochemical methods, such as mutagenesis to enhance the stability or the antiviral activity. The probiotic strain of the present invention is examined whether it can suppress the growth of TGE Coronavirus or not. As a result, it is confirmed to have the antiviral activity against Coronavirus since cytopathic effect is not observed. In addition, the probiotic strain is investigated whether it can be sensitive to antibiotics or not. As a result, it is verified to have the inhibition activity against enteric pathogens in the stomach and to be resistant to various kinds of veterinary antibiotics. The probiotic strain of the present invention is more advantageous to raise livestock since it is separated from pig's stomach and makes the normal flora of enteric microbes as demonstrated above. Preferably, the present invention provides food, pharmaceuticals, veterinary drug or fodder additive comprising Lactobacillus plantarum Probio-38 strain. The probiotic microbe of the present invention can be prepared with pharmaceutically acceptable carrier, utilized independently or as a food additive and manufactured to compositions suitable for human and animal. Namely, the microbe of the present invention can be added to food without other probiotic microbe (conferring the probiotic activity, if added) and/or food with several probiotic microbes (enhancing or compensating the probiotic activity, if added) before use. The pharmaceutical, veterinary or nutritional composition of the present invention can be composed of Lactobacillus plantarum Probio-38 strain as a probiotic microbe or additionally composed of more than 2 kinds of another useful strains and acceptable carriers. Preferably, the strain that is compatible with Lactobacillus plantarum Probio-38 to prepare the compositions of the present invention, should be edible for animal, suppress the proliferation of virus or pathogenic microbes and improve the microbial balance of mammal's intestine, in addition to the probiotic activity. For example, yeast such as Saccharomyces, Candida, Pichia, Torulopsis and the like; fungi such as Aspergillus, Rhizopus, Mucor, Penicillium and the like; and bacteria such as Lactobacillus, Bifidobacte um, Clostridium, Leuconostoc, Bacteroides, Staphylococcus, Lactococcus, Bacillus, Streptococcus, Fusobactehum, Propionibacterium, Enterococcus, Pediococcus, and Micrococcus sp. can be adopted for this purpose. Preferably, the strain can be selected among Saccharomyces cereviseae, Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis, Bifidobacterium bifidum, Bifidobactehum infantis, Bifidobacterium longum, Enterococcus faecium, Enterococcus faecalis, Lactobacillus acidophilus, Lactobacillus alimentarius, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus delbruckii, Lactobacillus johnsonii, Lactobacillus farciminus, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus sake, Lactococcus lactis, Micrococcus varians, Pediococcus acidilactici, Staphylococcus xylosus and the like. More preferably, Lactobacillus reuteri Probio-16 (accession number: KCCM 10214) that can suppress the growth of Rotavirus causing porcine diarrhea as well as has the probiotic activity and Lactobacillus salibarius Probio-37 (accession number: KCCM 10328) that can suppress the growth of Coronavirus or their microbial mixture, can be added to the compositions of the present invention. The composition of the present invention can include one more acceptable carrier selected among weight-increase agent, polymer additive, capsules, lipid and the like. These carriers are disclosed clearly to those skilled in the art. Lactobacillus plantarum Probio-38 of the present invention can be lyophilized or made to a capsule, culture suspension or dry powder.
Instead of conventional antibiotics, the microbe and the compositions of the present invention can be administered to animals directly or coordinately with food, pharmaceuticals, veterinary drug or fodder additive as a probiotic agent. The probiotic agent aims to replace the existed antibiotics, to suppress the growth of enteric pathogens in human and various livestock, to sustain the intestinal flora stably, to make the animal body healthier, to increase the weight of livestock, to improve the quality of meat, to increase the productivity of milk and to enhance the immunity and the like. In order to sustain the normal flora of enteric microbes, the probiotic composition of the present invention can be treated even after the antibiotic is already injected. Especially, the microbe of the present invention also has the antiviral activity and is used to alleviate and treat most symptoms caused by Coronavirus infection, when other treatment is not effective and has the side effect on clinical use. Therefore, the present invention provides the method for preventing the Coronavirus infection or for treating gastroenteritis, diarrhea and the like caused by Coronavirus, by using the probiotic strain and the compositions comprising the same. Also, the present invention provides the method for suppressing the growth of enteric pathogenic microbes or for treating gastroenteritis, diarrhea and the like caused by the pathogens.
EXAMPLES
Practical and presently preferred embodiments of the present invention are illustrated as shown in the following Examples. 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> Separation of microbes tolerant of acid and bile acid
Gastric remnant from normal pig was collected to an anaerobic bag (commercial name, Gas Pack pouch; a product from BBL Co. Ltd.), moved in the anaerobic condition and examined. 30 g of the resulting remnant collected above was diluted to 1 : 10 ratio by using 270 ml of
anaerobic dilution buffer (0.78% K2HP0 , salt mixture 37.5ml.. L-cystein
0.5 g, 25% L-ascorbic acid 2m£, 8% Na2C03 50mϋ. and 0.1% resazurin _ral were dissolved in distilled water 860ιn£ and finally agar 0.5g was
added) adjusted to pH 2 and blended for 120 minutes with a shaker. 1 ml of aliquot was smeared onto BL plate (Lab-lemco powder 2.4 g, proteose peptone No. 3 10 g, trypticase 5 g, phytone peptone 3 g, yeast extract 5
g, liver extract 150 ml, glucose 10 g, soluble starch 0.5 g, solution A 10m£,
solution B 5mβ, 10% FS antifoam F-20 5ml., Tween 80 1 g, L-cystein 0.5 g,
horse serum 50m£ were dissolved in distilled water 1,000ml. and then
agar 15 g was added; solution A, 10% KH2P04 and 10% K2HP04 and solution B, magnesium sulfate 10 g, iron sulfate 0.5 g, salt 0.5 g and
manganese sulfate 0.337 g dissolved in distilled water 250m£) containing
- --.z (commercial name, purchased from Dipco Co. Ltd.) and then cultivated at 37°C for 48 hours. Afterward, 310 colonies were selected,
transferred onto GAM semi-agar plate (purchased from llsu Pharmaceutics, Japan) [peptone 10 g, Soya peptone 3 g, proteose peptone No.3 10 g, peptonized whale serum 13.5 g, yeast extract 5 g, beef extract 2.2 g, liver extract 1.2 g, KH2P04 2.5 g, NaCI 3 g, L-cystein 0.3 g, sodium thioglycolate 0.3 g and glucose 2 g were dissolved in
distilled water 1000ml!. and 1.5g of agar was added], cultivated at 37°C
and stored at -80°C.
<Example 2> Identification of Probio-38 strain
Probio-38 strain separated in Example 1 was cultivated on MRS
medium (purchased from Dipco Co. Ltd.) at 37°C. In order to identify
Probio-38 strain, the morphological and physiological properties were examined through the method of Yoon et al. and by using API 32A kit and CHL kit (purchased from Biomerieux Co. Ltd.)(Yoon et al., Int. J. Syst Bactehol., 47, 904, 1997). The nucleotide sequence of 16S rRNA gene was determined and analyzed by performing the method of Yoon et al. (Yoon et al., Int. J. Syst. Bactehol., 47, 933, 1997). The morphological, physiological and biochemical characteristics of Probio-38 strain are illustrated in Table 1. <Table 1 > Morphological, physiological and biochemical characteristics of Probio-38 strain
Figure imgf000019_0001
(+): positive; (-): negative
As described in Table 1 , it is confirmed that Probio-38 strain is a Gram-positive bacterium, can proliferate both in aerobic and anaerobic conditions, does not make spores, have no mobility and be a bacillus in the shape. Probio-38 strain is preferable to grow at 30 ~ 37°C, does not generate gas and indoles, does not induce hemolysis, not reduce nitric acid and is tolerant of bile acid 0.5%. The nucleotide sequence of 16S rRNA gene purified from Probio- 38 strain of the present invention is illustrated in FIG. 1. Probio-38 strain was investigated through the molecular phylogenic analysis, based upon the nucleotide sequence of 16S rRNA. As a result, Probio-38 strain of the present invention is verified to belong to Bacillus sp., to have 99.5% sequence homology of 16S rDNA and to have the highest phylogenic relationship with the standard strain of Lactobacillus plantarum. Depending upon the experimental data, Probio-38 strain of the present invention was named as Lactobacillus plantarum Probio-38 and has been deposited to Korean Culture Center of Microorganism, International Deposition Organization on October 27, 2001 (accession number KCCM-10329).
<Example 3> Examination of growth inhibition of Coronavirus by Lactobacillus plantarum Probio-38
In order to examine the growth inhibition of Coronavirus by Probio-38 strain separated in the present invention, TGE virus strain and ST cell were prepared in the Example.
(1) Virus culture TGE virus strain was inoculated onto ST cell monolayer, adsorbed on cell for about 30 minutes, mixed with fresh Eagle's minimum essential medium (MEM) containing 5% bovine serum in a
proper ratio and cultivated at 37°C with a incubator containing 5% C02.
Then, the cytopathic effect (CPE) was observed. When CPE reached 70% approximately, the monolayer cells infected above were
submerged in fresh MEM medium, freezed and thawed twice at -70°C
repeatedly, harvested and centrifuged so as to remove cell debris. As a
result, the cell supernatant was stored at -70°C for the next procedure
of the experiment.
(2) Virus titration ST cell monolayer was prepared on 96-well culture plate and TGE virus was diluted through 10-fold serial dilution. Then, each virus aliquot was poured to 10 wells of culture plate respectively and
cultivated for 72 hours at 37°C with a cell incubator containing 5% C02
so as to measure CPE values. Based upon the CPE data, the virus titer of the culture medium was estimated according to Reed and Muench method.
(3) Antiviral activity test The culture medium of TGE virus was diluted by using MEM medium containing 10% bovine serum and adjusted to the virus titer,
10.0 TCID50/0Jm , 100 TCID50/OJm£ and 1000 TCID5o/OJm£
respectively. ST cell monolayer was prepared onto 96-well culture plate and 90 μl of virus titer 10.0 TCID5o/0.1m£, 100 TCID50/0Jιιι^ and 1000 TCID5o/OJm£ were poured to the first, the second and the third wells independently. Immediately, 10m (10%) culture medium of Lactobacillus plantarum Probio-38 was poured to each well and cultivated at 37°C with a incubator containing 5% C02 so as to measure CPE after 24, 48 and 72 hours lapsed. Through the above-mentioned procedure, the culture medium of Lactobacillus plantarum Probio-38 was verified not to have the antiviral activity when the cytopathic effect (CPE) was observed. The inhibition activity against Coronavirus in Lactobacillus plantarum Probio- 38 is illustrated in Table 2.
<Table 2> Cytopathic effect (CPE) against TGE Coronavirus of Lactobacillus plantarum Probio-38
Figure imgf000022_0001
Figure imgf000023_0001
<Example 4> Examination of growth inhibition of enteric pathogens by Lactobacillus plantarum Probio-38
In order to examine the growth inhibition of pathogenic microbes by Lactobacillus plantarum Probio-38 strain separated in the present invention, Kuroiwa' method (Kuroiwa et al., Journal of Infectious disease, 64, 257, 1990) was exploited. In this experiment, 13 kinds of microbes were adopted to estimate the antibiotic activity as follows: Escherichia coli KCTC 2441 , E. coli KCTC 2571 , Klebsiella pneumoniae KCTC 2208, Staphylococcus aureus KCTC 1621 , Staphylococcus epidermidis KCTC 1917, Salmonella ente tidis, Shigella flexneri KCTC 2008, Salmonella gallinarum, Enterobacter cloacea KCTC 2361 , Enterococcus lactis KCTC 1913, Salmonella typhimuhum, Citrobacter freundii KCTC 2006 and Bacillus subtilis KCTC 1021. The pathogenic microbes described above were cultivated in NB (nutrient broth) medium at 37°C for 18 hours. Then 10 mi of the culture medium was smeared on
NA (nutrient agar) plate, dried and covered with 8 mm radius paper disc. Lactobacillus plantarum of the present invention was cultivated in an anaerobic condition by using MRS culture broth at 37°C for 18 hours. Again, 30 μl of culture medium was inoculated on 8 mm paper disc placed on the above-mentioned NA plate and cultivated at 37°C for 24 hours. Afterward, the clear zone was measured to compare that of standard group. As a result, one strain was selected to suppress the growth of pathogenic microbes most outstandingly and named as Lactobacillus plantarum Probio-38. The inhibition activity against pathogenic microbes is illustrated in Table 3. <Table 3> Inhibition activity against pathogenic microbes by Lactobacillus plantarum Probio-38
Figure imgf000024_0001
Figure imgf000025_0001
As demonstrated in Table 3, it is confirmed that Lactobacillus plantarum Probio-38 suppress the growth of almost 13 pathogenic microbes and be a useful probiotic microbe.
<Example 5> Examination of antibiotic sensitivity of Lactobacillus plantarum Probio-38
In order to examine the sensitivity to antibiotics of Lactobacillus plantarum Probio-38, BBL Sensi-Disc was utilized according to the protocol of Becton Dickinson Microbiology Systems. For this experiment, 11 kinds of antibiotics were adopted as follows: tetracycline, ampicillin, gentamicin, cephalothin, sulfisoxazole, carbenicillin, streptomycin, lincomycin, neomycin, penicillin and erythromycin. Lactobacillus plantarum Probio-38 of the present invention was cultivated in MRS culture broth at 37°C for 18 hours and smeared homogeneously on MRS culture plate. Then, BBL Sensi-Disc with 8 mm radius was placed on the above plate and further cultivated at 37°C for 24 hours. Afterward, the radius at clear zone was measured and the sensitivity to antibiotics of Lactobacillus plantarum Probio-38 is illustrated in Table 4. <Table 4> Sensitivity to antibiotics of Lactobacillus plantarum Probio-38
Figure imgf000026_0001
As illustrated in Table 4, it is confirmed that Lactobacillus plantarum Probio-38 is highly sensitive to ampicillin, carbenicillin and erythromycin.
As described above, the novel microbe of the present invention separated from pig's intestine, Lactobacillus plantarum Probio-38 can suppress the growth of TGE Coronavirus and other pathogenic microbes effectively and is tolerant of acid and bile acid. Therefore instead of conventional antibiotics, the microbe and the compositions of the present invention can be administered for human and animals to make the animal body healthier, to increase the weight of livestock, to improve the quality of meat, to increase the productivity of milk and to enhance the immunity and the like as well as to prevent and treat the symptoms by the abnormal fermentation of enteric pathogens since it sustains the intestinal flora stably.
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.

Claims

What is claimed is:
1. Lactobacillus plantarum Probio-38 that can suppress the growth of Coronavirus and pathogenic microbes in intestine (accession number: KCCM-10329).
2. A pharmaceutical, veterinary or nutritional composition, which comprises Lactobacillus plantarum Probio-38 of claim 1 and acceptable carriers and has probiotic activity.
3. The pharmaceutical, veterinary or nutritional composition according to claim 2, in which Lactobacillus Probio-38 (accession number: KCCM 10214), Lactobacillus salibarius Probio-37 (accession number: KCCM 10328) or their microbial mixture is added.
4. The pharmaceutical, veterinary or nutritional composition according to claim 2 or claim 3, which can be used for preventing or treating diarrhea of mammals when it is administered orally.
5. Food, pharmaceutical, veterinary drug or fodder additive, which comprises Lactobacillus plantarum Probio-38 (accession number: KCCM-10329) of claim 1 or the composition of claim 2 or claim 3 in effective amounts.
6. A method for suppressing Coronavirus or for preventing or treating diarrhea caused by Coronavirus, in which Lactobacillus plantarum Probio-38 (accession number: KCCM-10329) of claim 1 or the composition of claim 2 or claim 3 are used.
6. A method for suppressing the growth of pathogenic microbes in intestine or for preventing or treating diarrhea caused by the microbes, in which Lactobacillus plantarum Probio-38 (accession number: KCCM- 10329) of claim 1 or the composition of claim 2 or claim 3 are used.
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