WO2017222295A1 - Composition for preventing and treating influenza virus, comprising kimchi- and soybean paste- derived lactic acid bacteria as active ingredient - Google Patents

Composition for preventing and treating influenza virus, comprising kimchi- and soybean paste- derived lactic acid bacteria as active ingredient Download PDF

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WO2017222295A1
WO2017222295A1 PCT/KR2017/006511 KR2017006511W WO2017222295A1 WO 2017222295 A1 WO2017222295 A1 WO 2017222295A1 KR 2017006511 W KR2017006511 W KR 2017006511W WO 2017222295 A1 WO2017222295 A1 WO 2017222295A1
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lactic acid
acid bacteria
influenza virus
composition
kimchi
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PCT/KR2017/006511
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French (fr)
Korean (ko)
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송창선
윤하나
김유진
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주식회사 카브
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • 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
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose

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  • the present invention relates to a composition for preventing and treating influenza virus comprising kimchi and miso-derived lactic acid bacteria as an active ingredient.
  • Influenza virus is a highly contagious cause of acute respiratory disease in humans. From the big and small seasonal flu, which is prevalent every year, and sometimes the pandemic, it costs a lot of social costs. Influenza viruses are currently known to have 144 serotypes, depending on the composition of the surface proteins. The most popular strains of the virus since the 20th century were the Spanish flu A / H1N1 in 1918, the Hong Kong flu A / H3N2 in 1968, and the A / H5N1 in Hong Kong in 1997. There is a case of human being infected.
  • amantadine is a substance that inhibits infection by blocking the hydrogen ion channel of influenza virus and preventing the genetic material of the virus from being exposed to the host cell.
  • Most influenza type A influenza is resistant to genetic mutations, and is ineffective against type B and C viruses, and may cause side effects such as nausea, dizziness, and insomnia.
  • Oseltamivir Phosphate and Janamivir are drugs that inhibit the function of the influenza virus surface glycoprotein neuraminidase, but are currently used most widely in clinical practice, but can be expected to be treated within 48 hours after the infection started. In addition, since a very deadly virus infection is accompanied by digestive disorders, drug absorption is difficult, and recently, a virus having resistance to the drug is found one after another.
  • influenza pandemic is unpredictable at that time and epidemic serotypes, but it is true.
  • the best way to prevent influenza outbreaks is to boost immunity through vaccination.
  • it is very difficult to provide the right vaccine at the right time because of the different kinds of viruses that are prevalent each time. .
  • problems such as delays and lack of vaccine supply.
  • the use of antiviral agents is effective in preventing the spread of infection when used immediately after infection, but prevention of infection is difficult due to preemptive administration.
  • the present invention has been made in view of the above necessity, and an object of the present invention is to provide a novel anti-influenza material which can effectively prevent or treat influenza and can be used safely.
  • the present invention provides a composition having a protective effect against influenza virus comprising Lactobacillus sakei strain or Pediococcus acidilactici strain as an active ingredient. .
  • the Lactobacillus sakei strain (Lactobacillus sakei) is Accession No. KCTC13031BP,
  • the Pediococcus strain is preferably No. KCTC13032BP is not limited thereto.
  • the two microbial strains of the present invention were deposited on June 1, 2016, at the Jeonbuk Branch Bioresource Center, Korea Biotechnology Research Institute, 181, Ipsin-gil, Jeongeup-si, Jeollabuk-do, Korea.
  • the composition is preferably for nasal or oral administration, but is not limited thereto.
  • the composition is preferably a pharmaceutical, food or feed composition, but is not limited thereto.
  • compositions, food compositions or feed compositions comprising Lactobacillus sakei strain [Accession No. KCTC13031BP] and Pediococcus acidilactici strain [Accession No. KCTC13032BP] or fragments thereof of the present invention.
  • the protection against antiviral was very good.
  • the herbal preparations of the pharmaceutical compositions according to the invention can be made in a general manner in the art.
  • suitable solid or liquid herbal formulations are, for example, granules, powders, dragees, tablets, (micro) capsules, suppositories, syrups, juices, suspensions or emulsions, and for their preparation, carrier materials, explosives, binders, coatings, expanding agents, lubricants Conventional means such as, flavors, sweeteners and solution mediators are used.
  • auxiliary substances animal and vegetable oils, such as magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk proteins, gelatin, starch, cellulose and derivatives, cod liver oil, sunflower oil, peanut oil or sesame oil, polyethylene glycol And solvents such as sterile water and monohydric or polyhydric alcohols such as glycerin.
  • the pharmaceutical composition according to the present invention is prepared by mixing the lactic acid bacterium according to the present invention in a predetermined amount with a pharmaceutically suitable and physiologically resistant carrier and optionally additional suitable activity, additions or auxiliary substances, and formulated in a desired dosage form. can do.
  • composition may contain or consist of 0.1 to 95% by weight carrier and 5 to 99.9% by weight lyophilized lactic acid bacteria relative to the total amount of cells and carriers.
  • the lactic acid bacteria of the present invention can be used as direct inoculation products.
  • bacteria can be added to foods, or pharmaceutical compositions, without any prior processing steps. It is preferred that the bacteria of the invention are cryopreserved in their own fermentation supernatants. This facilitates the production of the product and thus saves time and money.
  • “Fragment of the microorganism of the present invention” encompasses any part of the cells of the microorganism of the present invention.
  • the fragments are membrane fragments obtained from membrane preparations.
  • Membrane preparations of microorganisms belonging to the genus Lactobacillus are described by methods known in the art, for example in Rollan et al., Int. J. Food Microbiol. 70 (2001), 303-301, Matsuguchi et al., Clin. Diagn. Lab. Immunol. 10 (2003), 259-266 or Stentz et al., Appl. Environ. Microbiol. 66 (2000), 4272-4278 or Varmanen et al., J. Bacteriology 182 (2000), 146-154.
  • Kimchi-derived LABS defense against influenza virus A / NWS / 33 (H1N1) in mice was evaluated. After six intranasal administrations of lactic acid bacteria, they were challenged with the same route, and the weight change, clinical symptoms and survival rate were recorded daily in mice after infection. All mice in the positive control died and all mice in the negative control did not die.
  • L.plantarum-2, P.pentosaceus-2, and L. sakei showed high levels of defense efficiency, 80%, 70%, and 70%, respectively.
  • P.pentosaceus-1 showed 60%, L.plantarum-1, L.plantarum-3, and L.brevis-3 survival rates of 50%.
  • L.brevis-4, L. brevis-1 and L.brevis-2 showed low survival rates of 40%, 20% and 10%, respectively (FIG. 1).
  • mice The group of non-vaccinated mice (negative control) showed little change in body weight, but the weights of the positive and experimental groups decreased after viral challenge. Body weight decreased from 1 day after challenge, mostly down to 8 days, and then recovered to or remained close to its original condition after 9 days. Mean onset time of clinical symptoms was 3 days in all groups. Clinical symptoms observed (fever), eyelid abnormalities and hair abnormalities were better in the LAB treated group than in the positive control group (FIG. 2).
  • mice-derived LABS defense against influenza virus A / Korea / 01/2009 (H1N1) in mice was evaluated. All mice in the positive control died and all mice in the negative control did not die. 30 P. acidilactici or P. lolii and 23 E. faecium showed the highest level of protection with a survival rate of 90%, followed by 80%, 21 B. coagulans, 17 S with 35 B.linchenformis and 22 L.mesenteroides. .nakayamae, 18 B.coagulans, 46 B.coagulans, 28 L.alimentarius showed survival rates of 60%, 55%, 50%, 40% and 25%, respectively (FIG. 3).
  • mice The group of non-vaccinated mice (negative control) showed little change in body weight, but the weights of the positive and experimental groups decreased after viral challenge. Body weight decreased from 1 day after challenge, mostly down to 8 days, and then recovered or no longer returned to its original condition after 8 days. Mean onset time of clinical symptoms was 3 days in all groups. Clinical symptoms observed (fever), eyelid abnormalities and hair abnormalities were better in the LAB treated group than in the positive control group (FIG. 4).
  • the lactic acid bacteria of the present invention were mixed with dextrin additives and fed, and observed the ERA, E. faecium strains, etc. were completely killed as a result of the challenge control group, but P. acidilactici was finally survived 6 out of 10 Results were shown (data not shown).
  • strains with good protective effect against influenza virus infection were selected (L. plantarum-2, L. brevis-5 and L. sakei). Influenza virus A / Korea / 01/2009 after oral and nasal administration of this strain The protective effect on (H1N1) was evaluated.
  • the control group was challenged simultaneously with the experimental group after PBS administration. Body weight changes, clinical symptoms, and death inhibition effects were then observed for two weeks. Nasal treated Lactobacillus had mild clinical symptoms and significant weight loss was inhibited (FIG. 5).
  • the mean onset time of clinical symptoms was 3 days in all groups except the negative control.
  • the observed clinical symptoms body temperature (fever), eyelid abnormalities and hair abnormalities were better in the LAB treated group than in the positive control.
  • H1N1 Three candidate strains P.acidilactici, E.faecium, and B.linchenformis with excellent antiviral effects against influenza virus A / Korea / 01/2009 (H1N1) upon nasal administration were selected and influenza virus A / The protective effect against Korea / 01/2009 (H1N1) was evaluated. Mice in the positive control group and the oral administration of E. faecium, B.linchenformis died completely within 12 days after challenge, but the group receiving P. acidilactici showed a protective effect of 40% (Fig. 7).
  • the mean onset time of clinical symptoms was 3 days in all groups except the negative control.
  • P. acidilactici was shown to recover near the normal body weight in surviving subjects (FIG. 8).
  • L. sakei in the kimchi-derived lactic acid bacteria of the present invention and P. acidilactici in the miso-derived lactic acid bacteria showed a high defense efficiency against influenza in both nasal and oral administration. There is a possibility to prevent influenza infection through the use of the lactic acid bacteria.
  • 1 is a survival rate of influenza virus challenge vaccination after kimchi-derived lactobacillus nasal administration
  • Figure 2 shows the weight change during influenza virus challenge after nasal administration of kimchi-derived lactic acid bacteria
  • Figure 3 is a figure of weight change during influenza virus challenge vaccination after doenjang-derived lactic acid bacteria nasal
  • Figure 4 is a survival rate when influenza virus challenge vaccination after doenjang-derived lactobacillus nasal administration
  • Figure 5 shows the weight change during influenza virus challenge after lactic acid bacteria and oral administration of kimchi derived
  • Figure 6 is a survival rate of influenza virus challenge vaccination after oral administration of lactic acid bacteria and kimchi derived
  • Figure 8 shows the weight change during influenza virus challenge after oral administration of miso-derived lactic acid bacteria
  • 9 and 10 are 16s rRNA gene sequence information of Kimchi-derived L. sakei and miso-derived lactic acid bacteria P. acidilactici of the present invention, respectively.
  • Lactic acid bacteria were isolated from Kimchi (Chinese cabbage, Korean cabbage, and Dongchimi) and Doenjang collected from various parts of Korea.
  • 1 ml fermentation soup was first added to 30 ml MRS liquid medium and incubated at 37 ° C. for 24 hours.
  • One loop of cultured liquid medium was streaked in Rogosa solid medium and incubated at 37 ° C. for 48 hours.
  • Each grown colony was further streaked in MRS solid medium and incubated at 37 ° C. for 24 hours.
  • For the identification of lactic acid bacteria strains single colonies isolated from each sample were sequenced and LABs were identified by 16s rRNA gene sequencing.
  • the identified lactic acid bacteria were again cultured in MRS liquid medium and mixed with cryopreservative and stored at -70 ° C.
  • the isolated lactic acid bacteria are shown in Table 1 (kimchi derived) and Table 2 (miso derived).
  • Influenza virus A / NWS / 33 (H1N1), influenza virus A / Korea / 01/2009 (H1N1) were grown in the ureteral cavity of 11-day-old fetuses at 37 ° C. for 3 days. Allantoic fluid was collected and stored at minus 70 ° C. until use. The titer of virus obtained from the urea was continuously injected with 10-fold diluted urea into embryonated eggs, and the presence of the virus in the urea of each egg was determined based on the hemagglutination ability of the virus. To make the virus adapted to the mouse, the virus was first isolated from the mouse lung, propagated in SPF eggs and re-inoculated into the mouse. In the challenge study, mice were anesthetized by injecting Avertin (375 mg / kg) by the intraperitoneal injection route and challenged by intranasal route with 90 ⁇ L of 10 ⁇ 4.5 EID50 influenza virus.
  • Avertin 375 mg /
  • mice were challenged and inoculated with the virus after administration of lactic acid bacteria. Ten animals were grouped, one lactic acid bacterium per group was administered six times, respectively, by intranasal route (1 ⁇ 10 8 cfu / horse) prior to virus challenge. Positive and negative control groups were inoculated with PBS in the same format as the test group.
  • Kimchi-derived lactic acid bacteria and miso-derived lactic acid bacteria were inoculated with influenza virus A / NWS / 33 (10 ⁇ 4.5 / 90 ⁇ L / dose) and A / Korea / 01/2009 (H1N1) (10 ⁇ 4.5 / 90 ⁇ L / dose), respectively.
  • the clinical effects, mortality and body weight change were observed for 2 weeks to assess the protective effect of influenza virus in nasal-administered lactobacilli.
  • Strains resistant to influenza virus infection were selected and oral (300ul) was administered to mice for 3 weeks at a concentration of 1x10 ⁇ 8 cfu / horse to determine whether there was a difference in the protective effect according to the administration route of lactic acid bacteria.
  • Positive and negative controls were inoculated with PBS in the same format as the test group.
  • Kimchi-derived lactic acid bacteria and miso-derived lactic acid bacteria were inoculated with influenza virus A / NWS / 33 (10 ⁇ 4.5 / 90 ⁇ L / dose) and A / Korea / 01/2009 (H1N1) (10 ⁇ 4.5 / 90 ⁇ L / dose), respectively.
  • the clinical effects, mortality and body weight change were observed for 2 weeks to assess the protective effect of influenza virus in nasal-administered lactobacilli.

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Abstract

The present invention relates to a composition for preventing and treating an influenza virus, comprising kimchi- and soybean paste-derived lactic acid bacteria as an active ingredient and, more specifically, to a composition having a protective effect against an influenza virus, comprising a Lactobacillus sakei strain or a Pediococcus acidilactici strain as an active ingredient. L.sakei among the kimchi-derived lactic acid bacteria and P.acidilactici among the soybean paste-derived lactic acid bacteria of the present invention have shown high protective efficiency against influenza in both nasal administration and oral administration. There is a possibility that an influenza infection can be prevented by using the lactic acid bacteria.

Description

김치 및 된장 유래 젖산균을 유효성분으로 포함하는 인플루엔자 바이러스 예방 및 치료용 조성물Influenza virus prevention and treatment composition comprising kimchi and miso-derived lactic acid bacteria as an active ingredient
본 발명은 김치 및 된장 유래 젖산균을 유효성분으로 포함하는 인플루엔자 바이러스 예방 및 치료용 조성물에 관한 것이다.The present invention relates to a composition for preventing and treating influenza virus comprising kimchi and miso-derived lactic acid bacteria as an active ingredient.
인플루엔자 바이러스는 사람에서 전염성이 매우 높은 급성 호흡기 질환의 원인체이다. 매년 유행하는 크고 작은 계절성 독감부터 때로는 범세계적 유행으로 많은 사회적 비용을 발생시킨다. 인플루엔자 바이러스는 표면 단백질의 구성에 따라 현재 144가지의 혈청형을 가진다고 알려져 있다. 20세기 이후 대유행한 바이러스주는 1918년 스페인독감 A/H1N1, 1968년 홍콩독감 A/H3N2, 1997년 홍콩조류독감 A/H5N1 등으로 다양한 혈청형이 유행하였으며, 최근에도 홍콩에서 A/H9N2 등의 바이러스가 사람에 감염된 사례가 있다.Influenza virus is a highly contagious cause of acute respiratory disease in humans. From the big and small seasonal flu, which is prevalent every year, and sometimes the pandemic, it costs a lot of social costs. Influenza viruses are currently known to have 144 serotypes, depending on the composition of the surface proteins. The most popular strains of the virus since the 20th century were the Spanish flu A / H1N1 in 1918, the Hong Kong flu A / H3N2 in 1968, and the A / H5N1 in Hong Kong in 1997. There is a case of human being infected.
현재 인플루엔자 바이러스에 감염될 경우 몇 가지 치료제가 사용되고 있다. 이 치료제들은 바이러스가 생물체 내에서 증식하는 기전 중 일부를 차단하는 방식으로 바이러스 감염을 억제한다. 아만타딘, 오셀타미비르(타미플루, Tamiflu), 자나미비어(리렌자, Relenza) 등이 주로 사용된다. 이 중 아만타딘은 인플루엔자바이러스의 수소이온통로를 차단하여 바이러스의 유전물질이 숙주세포 안으로 노출되는 것을 막아 감염을 억제하는 물질이다. 최근 유행하는 대부분의 A형 인플루엔자는 내성 유전자변이를 가지고 있고, B형과 C형 바이러스에는 효과가 없으며, 오심, 현기증, 불면증 등의 부작용을 유발할 가능성이 있다. 인산오셀타미비르, 자나미비어는 인플루엔자 바이러스 표면 당단백질인 뉴라미니데이즈의 기능을 억제하는 약물로, 현재 임상에서 가장 많이 사용되고 있으나, 바이러스에 감염이 시작된 지 48시간 이내에 투여하여야 치료 효과를 기대할 수 있으며, 아주 치명적인 바이러스 감염시 소화기 장애를 동반하기 때문에 약물 흡수가 어렵고, 최근 이 약물에 저항성을 가지는 바이러스가 속속 발견되는 등의 문제점을 보이고 있다.Currently, several treatments are used when infected with influenza virus. These agents inhibit viral infection in a way that blocks some of the mechanisms by which viruses multiply in organisms. Amantadine, oseltamivir (Tamiflu) and zanamivir (Relenza) are mainly used. Among them, amantadine is a substance that inhibits infection by blocking the hydrogen ion channel of influenza virus and preventing the genetic material of the virus from being exposed to the host cell. Most influenza type A influenza is resistant to genetic mutations, and is ineffective against type B and C viruses, and may cause side effects such as nausea, dizziness, and insomnia. Oseltamivir Phosphate and Janamivir are drugs that inhibit the function of the influenza virus surface glycoprotein neuraminidase, but are currently used most widely in clinical practice, but can be expected to be treated within 48 hours after the infection started. In addition, since a very deadly virus infection is accompanied by digestive disorders, drug absorption is difficult, and recently, a virus having resistance to the drug is found one after another.
인플루엔자 대유행은 그 시기와 유행하는 혈청형을 예측할 수 없지만 반복해서 발생하고 있는 것이 사실이다. 인플루엔자 발생을 막는 가장 좋은 방책으로 생각되는 것은 백신 접종을 통한 면역력의 증진이다. 그러나 매번 유행하는 바이러스의 종류가 다르기 때문에 적절한 시기에 정확한 백신을 공급하는 것이 매우 어렵다. . 또 대유행 상황에서는 백신 공급의 지연 및 부족 등의 문제가 나타날 수 있다. 항바이러스제의 사용은 감염 이후 즉시 사용할 경우 감염의 확산을 막는데 효율적이지만, 선제적 투여로 감염의 예방은 어렵다.The influenza pandemic is unpredictable at that time and epidemic serotypes, but it is true. The best way to prevent influenza outbreaks is to boost immunity through vaccination. However, it is very difficult to provide the right vaccine at the right time because of the different kinds of viruses that are prevalent each time. . In the pandemic, there may be problems such as delays and lack of vaccine supply. The use of antiviral agents is effective in preventing the spread of infection when used immediately after infection, but prevention of infection is difficult due to preemptive administration.
이에 인플루엔자를 효과적으로 예방 또는 치료하고, 일상적으로 안전하게 이용될 수 있는 항인플루엔자 물질의 개발이 필요하다.Accordingly, there is a need for the development of anti-influenza substances that can effectively prevent or treat influenza and be used safely on a daily basis.
[선행 특허 문헌][Previous Patent Document]
대한민국 특허공개번호 1020040080078Republic of Korea Patent Publication No. 1020040080078
본 발명은 상기의 필요성에 의하여 안출된 것으로서 본 발명의 목적은 인플루엔자를 효과적으로 예방 또는 치료하고, 안전하게 이용될 수 있는 신규한 항인플루엔자 물질을 제공하는 것이다.   The present invention has been made in view of the above necessity, and an object of the present invention is to provide a novel anti-influenza material which can effectively prevent or treat influenza and can be used safely.
상기의 목적을 달성하기 위하여 본 발명은 락토바실러스 사케이(Lactobacillus sakei) 균주 또는 페디오코커스 애시디락티시(Pediococcus acidilactici) 균주를 유효성분으로 포함하는 인플루엔자 바이러스에 대한 보호 효과를 가지는 조성물을 제공한다.In order to achieve the above object, the present invention provides a composition having a protective effect against influenza virus comprising Lactobacillus sakei strain or Pediococcus acidilactici strain as an active ingredient. .
본 발명의 일 구현예에 있어서, 상기 락토바실러스 사케이(Lactobacillus sakei) 균주는 기탁번호 KCTC13031BP이고, 상기 페디오코커스(Pediococcus) 균주는 기탁번호 KCTC13032BP인 것이 바람직하나 이에 한정되지 아니한다.In one embodiment of the present invention, the Lactobacillus sakei strain (Lactobacillus sakei) is Accession No. KCTC13031BP, The Pediococcus strain is preferably No. KCTC13032BP is not limited thereto.
본 발명의 두 미생물 균주는 대한민국 전라북도 정읍시 입신길 181 (신정동) 한국생명공학연구원 전북분원 생물자원센터에 2016년 6월1일자로 기탁하였다.The two microbial strains of the present invention were deposited on June 1, 2016, at the Jeonbuk Branch Bioresource Center, Korea Biotechnology Research Institute, 181, Ipsin-gil, Jeongeup-si, Jeollabuk-do, Korea.
본 발명의 다른 구현예에 있어서, 상기 조성물은 비강 또는 경구 투여용인 것이 바람직하나 이에 한정되지 아니한다.In another embodiment of the present invention, the composition is preferably for nasal or oral administration, but is not limited thereto.
본 발명의 또 다른 구현예에 있어서, 상기 조성물은 약학, 식품 또는 사료 조성물인 것이 바람직하나 이에 한정되지 아니한다.In another embodiment of the present invention, the composition is preferably a pharmaceutical, food or feed composition, but is not limited thereto.
본 발명의 락토바실러스 사케이(Lactobacillus sakei) 균주[기탁번호 KCTC13031BP] 및 페디오코커스 애시디락티시(Pediococcus acidilactici) 균주[기탁번호 KCTC13032BP] 또는 그들의 단편을 포함하는 약학적 조성물, 식품 조성물 또는 사료 조성물의 항바이러스로부터 보호효과는 매우 우수하였다.Pharmaceutical compositions, food compositions or feed compositions comprising Lactobacillus sakei strain [Accession No. KCTC13031BP] and Pediococcus acidilactici strain [Accession No. KCTC13032BP] or fragments thereof of the present invention. The protection against antiviral was very good.
본 발명에 따른 약학적 조성물의 생약 제제는 이 기술 분야에서 일반적인 방식으로 만들어질 수 있다. 적합한 고체 또는 액체 생약 제형은 예를 들어 과립, 분말, 당의정, 태블릿, (마이크로)캡슐, 좌약, 시럽, 즙, 현탁액 또는 유화액이고, 그 제조를 위해 담체 물질, 폭약, 결합제, 코팅제, 팽창제, 윤활제, 미각제,감미료 및 용액 매개제와 같은 통상의 수단이 사용된다. 보조 물질로 여기에서 탄산 마그네슘, 이산화 티타늄,락토오스, 만니톨 및 다른 당, 활석, 우유 단백질, 젤라틴, 녹말, 셀룰로오스 및 유도체, 대구 간유, 해바라기유, 땅콩 기름 또는 참기름과 같은 동물성 및 식물성 기름, 폴리에틸렌 글리콜 및 멸균수와 같은 용매 및 일가 또는 다가 알콜, 예를 들어 글리세린이 나열된다. 본 발명에 따른 약학적 조성물은 본 발명에 따른 젖산 박테리아가 약학적으로 적합하고 생리학적으로 내성 좋은 담체 및 임의로 추가적인 적합한 활성, 첨가 또는 보조 물질과 정해진 양으로 혼합되고, 원하는 투여형으로 제제됨으로써 제조할 수 있다. The herbal preparations of the pharmaceutical compositions according to the invention can be made in a general manner in the art. Suitable solid or liquid herbal formulations are, for example, granules, powders, dragees, tablets, (micro) capsules, suppositories, syrups, juices, suspensions or emulsions, and for their preparation, carrier materials, explosives, binders, coatings, expanding agents, lubricants Conventional means such as, flavors, sweeteners and solution mediators are used. As auxiliary substances here animal and vegetable oils, such as magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk proteins, gelatin, starch, cellulose and derivatives, cod liver oil, sunflower oil, peanut oil or sesame oil, polyethylene glycol And solvents such as sterile water and monohydric or polyhydric alcohols such as glycerin. The pharmaceutical composition according to the present invention is prepared by mixing the lactic acid bacterium according to the present invention in a predetermined amount with a pharmaceutically suitable and physiologically resistant carrier and optionally additional suitable activity, additions or auxiliary substances, and formulated in a desired dosage form. can do.
조성물은 세포 및 담체의 총량 대비 0.1 내지 95 중량% 담체 및 5 내지 99.9 중량% 동결건조된 젖산 박테리아를 함유하거나 이로 이루어질 수 있다.The composition may contain or consist of 0.1 to 95% by weight carrier and 5 to 99.9% by weight lyophilized lactic acid bacteria relative to the total amount of cells and carriers.
본 발명의 젖산 박테리아는 직접 접종 제품으로 사용될 수 있다. 따라서 박테리아는 임의의 선행 가공 단계 없이 식품, 또는 약학적 조성물에 첨가될 수 있다. 본 발명의 박테리아가 그들 자신의 발효 상청액에 저온 보존되는 것이 바람직하다. 이는 제품의 생산을 용이하게 하고 따라서 시간 및 비용 절약에 도움이 된다.The lactic acid bacteria of the present invention can be used as direct inoculation products. Thus, bacteria can be added to foods, or pharmaceutical compositions, without any prior processing steps. It is preferred that the bacteria of the invention are cryopreserved in their own fermentation supernatants. This facilitates the production of the product and thus saves time and money.
식품 조성물, 동물 사료, 약학적 조성물이 절대값으로, 또는 세포를 함유하는 100 g의 식품, 동물 사료, 또는 약학적 조성물에 대해 10^2 내지 10^15, 바람직하게는 10^6 또는 10^8 내지 10^12, 젖산 박테리아 세포 또는 그의 단편을 함유하는 것이 바람직하다. 10 ^ 2 to 10 ^ 15, preferably 10 ^ 6 or 10 ^ for food compositions, animal feed, pharmaceutical compositions in absolute value or for 100 g of food, animal feed, or pharmaceutical compositions containing cells It is preferred to contain 8 to 10 ^ 12, lactic acid bacterial cells or fragments thereof.
"본 발명의 미생물의 단편"은 본 발명의 미생물의 세포의 임의의 부분을 포괄한다. 바람직하게는, 상기 단편은 막 제제로부터 얻어지는 막 단편이다. 락토바실러스 속에 속하는 미생물의 막 제제는 기술 분야에서 공지된 방법에 의해, 예를 들어, 문헌 [Rollan et al., Int. J. Food Microbiol. 70 (2001), 303-301], 문헌 [Matsuguchi et al., Clin. Diagn. Lab. Immunol. 10 (2003), 259-266] 또는 문헌 [Stentz et al., Appl.Environ. Microbiol. 66 (2000), 4272-4278] 또는 문헌 [Varmanen et al., J. Bacteriology 182 (2000), 146-154]에 기술된 방법을 사용함으로써 얻을 수 있다."Fragment of the microorganism of the present invention" encompasses any part of the cells of the microorganism of the present invention. Preferably, the fragments are membrane fragments obtained from membrane preparations. Membrane preparations of microorganisms belonging to the genus Lactobacillus are described by methods known in the art, for example in Rollan et al., Int. J. Food Microbiol. 70 (2001), 303-301, Matsuguchi et al., Clin. Diagn. Lab. Immunol. 10 (2003), 259-266 or Stentz et al., Appl. Environ. Microbiol. 66 (2000), 4272-4278 or Varmanen et al., J. Bacteriology 182 (2000), 146-154.
이하 본 발명을 설명한다.Hereinafter, the present invention will be described.
전통 식품 유래 유산균의 비강 투여시 인플루엔자 방어 효과Influenza Defense Effects of Nasal Administration of Lactic Acid Bacteria Derived from Traditional Foods
마우스에서 인플루엔자 바이러스 A/NWS/33 (H1N1)에 대한 김치 유래 LABS 방어 효과를 평가하였다. 6번의 유산균 비강내 투여 후 같은 경로로 공격접종하였고, 감염 후 마우스에서 매일 체중 변화, 임상증상 및 생존율를 기록하였다. 양성 대조군의 모든 마우스는 죽고 음성 대조군의 모든 마우스는 죽지 않았다. L.plantarum- 2, P.pentosaceus-2, L. sakei가 각각 80%, 70%, 70%로 높은 수준의 방어 효율을 나타내었다. 다음으로 P.pentosaceus-1이 60%, L.plantarum -1, L.plantarum -3, L.brevis-3이 50%의 생존율을 보였다. L.brevis-4, L. brevis-1, L.brevis-2는 각각 40%, 20%, 10%로 낮은 생존율을 보였다.(도 1)Kimchi-derived LABS defense against influenza virus A / NWS / 33 (H1N1) in mice was evaluated. After six intranasal administrations of lactic acid bacteria, they were challenged with the same route, and the weight change, clinical symptoms and survival rate were recorded daily in mice after infection. All mice in the positive control died and all mice in the negative control did not die. L.plantarum-2, P.pentosaceus-2, and L. sakei showed high levels of defense efficiency, 80%, 70%, and 70%, respectively. Next, P.pentosaceus-1 showed 60%, L.plantarum-1, L.plantarum-3, and L.brevis-3 survival rates of 50%. L.brevis-4, L. brevis-1 and L.brevis-2 showed low survival rates of 40%, 20% and 10%, respectively (FIG. 1).
공격접종하지 않은 마우스 그룹(음성 대조군)은 체중의 변화가 거의 없지만, 양성 대조군 및 실험군의 체중은 바이러스 공격접종 후 감소하였다. 체중은 공격접종 후 1일부터 감소하여 대부분 8일까지 감소한 다음 9일 이후 원래 상태와 가깝게 회복하거나 더 이상 감소하지 않았다. 임상 증상의 평균 시작 시간은 모든 군에서 3일이었다. 관찰된 임상적 증상 체온(열), 눈꺼풀 이상 및 털 이상이 양성대조군에서 보다 LAB 처리된 군에서 더 나았다.(도 2)The group of non-vaccinated mice (negative control) showed little change in body weight, but the weights of the positive and experimental groups decreased after viral challenge. Body weight decreased from 1 day after challenge, mostly down to 8 days, and then recovered to or remained close to its original condition after 9 days. Mean onset time of clinical symptoms was 3 days in all groups. Clinical symptoms observed (fever), eyelid abnormalities and hair abnormalities were better in the LAB treated group than in the positive control group (FIG. 2).
마우스에서 인플루엔자 바이러스 A/Korea/01/2009 (H1N1)에 대한 된장 유래 LABS 방어 효과를 평가하였다. 양성 대조군의 모든 마우스는 죽고 음성 대조군의 모든 마우스는 죽지 않았다. 30 P.acidilactici or P.lolii와 23 E.faecium은 90%의 생존율로 가장 높은 보호 수준을 나타내었고, 그 다음이 35 B.linchenformis와 22 L.mesenteroides로 80%, 21 B.coagulans, 17 S.nakayamae, 18 B.coagulans, 46 B.coagulans, 28 L.alimentarius이 각각 60%, 55%, 50%, 40% 및 25%의 생존율을 나타내었다.(도 3)Mice-derived LABS defense against influenza virus A / Korea / 01/2009 (H1N1) in mice was evaluated. All mice in the positive control died and all mice in the negative control did not die. 30 P. acidilactici or P. lolii and 23 E. faecium showed the highest level of protection with a survival rate of 90%, followed by 80%, 21 B. coagulans, 17 S with 35 B.linchenformis and 22 L.mesenteroides. .nakayamae, 18 B.coagulans, 46 B.coagulans, 28 L.alimentarius showed survival rates of 60%, 55%, 50%, 40% and 25%, respectively (FIG. 3).
공격접종하지 않은 마우스 그룹(음성 대조군)은 체중의 변화가 거의 없지만, 양성 대조군 및 실험군의 체중은 바이러스 공격접종 후 감소하였다. 체중은 공격접종 후 1일부터 감소하여 대부분 8일까지 감소한 다음 8일 이후 원래 상태와 가깝게 회복하거나 더 이상 감소하지 않았다. 임상 증상의 평균 시작 시간은 모든 군에서 3일이었다. 관찰된 임상적 증상 체온(열), 눈꺼풀 이상 및 털 이상이 양성대조군에서 보다 LAB 처리된 군에서 더 나았다.(도 4)The group of non-vaccinated mice (negative control) showed little change in body weight, but the weights of the positive and experimental groups decreased after viral challenge. Body weight decreased from 1 day after challenge, mostly down to 8 days, and then recovered or no longer returned to its original condition after 8 days. Mean onset time of clinical symptoms was 3 days in all groups. Clinical symptoms observed (fever), eyelid abnormalities and hair abnormalities were better in the LAB treated group than in the positive control group (FIG. 4).
또한 하기 본 발명의 유산균을 덱스트린 첨가제와 섞어서 먹이고 방어율을 관찰하였더니, E.faecium은 등의 균주는 공격접종 대조군의 결과와 같이 전수폐사하였으나, P.acidilactici는 10마리 중 최종적으로 6마리가 살아남는 결과를 보였다(자료 미첨부).In addition, the lactic acid bacteria of the present invention were mixed with dextrin additives and fed, and observed the ERA, E. faecium strains, etc. were completely killed as a result of the challenge control group, but P. acidilactici was finally survived 6 out of 10 Results were shown (data not shown).
투여 경로에 따른 유산균의 인플루엔자 방어 효과Influenza Defense Effect of Lactic Acid Bacteria with Different Administration Routes
비강 투여 결과 인플루엔자 바이러스 감염에 대한 방어 효과가 좋은 균주를 선발하여 (L. plantarum-2, L. brevis-5 및 L. sakei) 이 균주의 경구 및 비강 투여 후 인플루엔자 바이러스 A/Korea/01/2009 (H1N1)에 대한 보호 효과를 평가하였다. 대조군은 PBS 투여 후 실험군과 동시에 공격접종하였다. 그 후 체중 변화, 임상 증상 및 사망 저해 효과를 2 주 동안 관찰하였다. 비강 처리된 Lactobacillus는 완화된 임상 증상을 가지고 현저한 체중 감소가 억제되었다.(도 5) As a result of nasal administration, strains with good protective effect against influenza virus infection were selected (L. plantarum-2, L. brevis-5 and L. sakei). Influenza virus A / Korea / 01/2009 after oral and nasal administration of this strain The protective effect on (H1N1) was evaluated. The control group was challenged simultaneously with the experimental group after PBS administration. Body weight changes, clinical symptoms, and death inhibition effects were then observed for two weeks. Nasal treated Lactobacillus had mild clinical symptoms and significant weight loss was inhibited (FIG. 5).
감소된 체중은 비강 투여된 L.plantarum-2 및 L. brevis-5 에서 공격접종 10일 후 증가하였고, L. sakei에서 9일부터 증가하였다. 경구 투여된 Lactobacillus와 비교하여 비강 처리된 Lactobacillus 는 인플루엔자 바이러스 A/H1N1에 대한 더 높은 보호 효과를 나타내었다.(도 6) Reduced body weight increased 10 days after challenge in L. plantarum-2 and L. brevis-5 administered nasal, and increased from day 9 in L. sakei. Nasal treated Lactobacillus showed a higher protective effect against influenza virus A / H1N1 compared to orally administered Lactobacillus (FIG. 6).
임상 증상의 평균 시작 시간은 음성 대조군을 제외하고 모든 군에서 3일이었다. 이것 외에도, 관찰된 임상적 증상 체온(열), 눈꺼풀 이상 및 털 이상이 양성 대조군에서 보다 LAB 처리된 군에서 더 나아졌다.The mean onset time of clinical symptoms was 3 days in all groups except the negative control. In addition to this, the observed clinical symptoms body temperature (fever), eyelid abnormalities and hair abnormalities were better in the LAB treated group than in the positive control.
비강 투여시 인플루엔자 바이러스 A/Korea/01/2009 (H1N1)에 대한 항바이러스 효과가 우수한 세 후보 균주 P.acidilactici, E.faecium, B.linchenformis를 선택하고 균주의 경구 투여 후 마우스에서 인플루엔자 바이러스 A/Korea/01/2009 (H1N1)에 대한 보호 효과를 평가하였다. 양성 대조군 및 E.faecium, B.linchenformis를 경구 투여한 그룹의 마우스는 공격접종 후 12일 이내에 전수 폐사하였으나 P.acidilactici를 투여한 그룹은 40%의 보호 효과를 보였다.(도 7)Three candidate strains P.acidilactici, E.faecium, and B.linchenformis with excellent antiviral effects against influenza virus A / Korea / 01/2009 (H1N1) upon nasal administration were selected and influenza virus A / The protective effect against Korea / 01/2009 (H1N1) was evaluated. Mice in the positive control group and the oral administration of E. faecium, B.linchenformis died completely within 12 days after challenge, but the group receiving P. acidilactici showed a protective effect of 40% (Fig. 7).
임상 증상의 평균 시작 시간은 음성 대조군을 제외하고 모든 군에서 3일이었다. 특히 P.acidilactici의 경우 생존 개체에서 정상 체중 가까이 회복하는 것을 보였다.(도 8) The mean onset time of clinical symptoms was 3 days in all groups except the negative control. In particular, P. acidilactici was shown to recover near the normal body weight in surviving subjects (FIG. 8).
본 발명을 통하여 알 수 있는 바와 같이 본 발명의 김치 유래 유산균 중 L.sakei와 된장 유래 유산균 중 P.acidilactici는 비강 투여와 경구 투여에서 모두 높은 인플루엔자에 대한 방어 효율을 보였다. 상기 유산균의 사용을 통하여 인플루엔자 감염을 예방할 수 있는 가능성이 있다.As can be seen through the present invention, L. sakei in the kimchi-derived lactic acid bacteria of the present invention and P. acidilactici in the miso-derived lactic acid bacteria showed a high defense efficiency against influenza in both nasal and oral administration. There is a possibility to prevent influenza infection through the use of the lactic acid bacteria.
도 1은 김치 유래 유산균 비강 투여 후 인플루엔자 바이러스 공격접종시 생존율 그림,1 is a survival rate of influenza virus challenge vaccination after kimchi-derived lactobacillus nasal administration,
도 2는 김치 유래 유산균 비강 투여 후 인플루엔자 바이러스 공격접종시 체중 변화 그림,Figure 2 shows the weight change during influenza virus challenge after nasal administration of kimchi-derived lactic acid bacteria,
도 3은 된장 유래 유산균 비강 투여 후 인플루엔자 바이러스 공격접종시 체중 변화 그림,Figure 3 is a figure of weight change during influenza virus challenge vaccination after doenjang-derived lactic acid bacteria nasal
도 4는 된장 유래 유산균 비강 투여 후 인플루엔자 바이러스 공격접종시 생존율 그림,Figure 4 is a survival rate when influenza virus challenge vaccination after doenjang-derived lactobacillus nasal administration,
도 5는 김치 유래 유산균 비강 및 경구 투여 후 인플루엔자 바이러스 공격접종시 체중 변화 그림,Figure 5 shows the weight change during influenza virus challenge after lactic acid bacteria and oral administration of kimchi derived,
도 6은 김치 유래 유산균 비강 및 경구 투여 후 인플루엔자 바이러스 공격접종시 생존율 그림,Figure 6 is a survival rate of influenza virus challenge vaccination after oral administration of lactic acid bacteria and kimchi derived,
도 7은 된장 유래 유산균 경구 투여 후 인플루엔자 바이러스 공격접종시 생존율 그림,7 is a survival rate of influenza virus challenge after oral administration of miso-derived lactic acid bacteria,
도 8은 된장 유래 유산균 경구 투여 후 인플루엔자 바이러스 공격접종시 체중변화 그림,Figure 8 shows the weight change during influenza virus challenge after oral administration of miso-derived lactic acid bacteria,
도 9 및 도 10은 각각 본 발명의 김치 유래 L.sakei와 된장 유래 유산균 P.acidilactici의 16s rRNA 유전자 서열 정보이다.9 and 10 are 16s rRNA gene sequence information of Kimchi-derived L. sakei and miso-derived lactic acid bacteria P. acidilactici of the present invention, respectively.
이하 비한정적인 실시예를 통하여 본 발명을 더욱 상세하게 설명한다. 단 하기 실시예는 본 발명을 예시하기 위한 의도로 기재된 것으로서 본 발명의 범위는 하기 실시예에 의하여 제한되는 것으로 해석되지 아니한다.Hereinafter, the present invention will be described in more detail with reference to non-limiting examples. However, the following examples are intended to illustrate the invention and the scope of the present invention is not to be construed as limited by the following examples.
실시예Example 1:사용1: use 동물 animal
6주령 SPF BALB/c 마우스 암컷(Orient Bio Laboratories, Seoul, Korea)으로 체중 18-20 g을 사용하였다. 모든 실험을 건국대학교 동물실험윤리위원회(IACUC)의 승인을 얻어 프로토콜에 따라 수행하였다.A 6-week-old SPF BALB / c mouse female (Orient Bio Laboratories, Seoul, Korea) used a body weight of 18-20 g. All experiments were performed in accordance with the protocol with the approval of the Animal Experimental Ethics Committee of Konkuk University.
실시예Example 2: 유산균의 분리 2: Isolation of Lactic Acid Bacteria
한국의 여러 지역에서 수집한 김치(배추, 깍두기 및 동치미)와 된장으로부터 유산균을 분리하였다. 유산균 분리를 위하여, 먼저 1 ml 발효 수프를 30 ml MRS 액체 배지에 첨가하고 24 시간 동안 37℃에서 배양하였다. 1 루프의 배양된 액체배지를 Rogosa 고체배지에 스트리킹하고 37℃에서 48 시간 동안 배양하였다. 각 성장한 집락들을 MRS 고체배지에서 더 스트리킹하고 37℃에서 24시간 동안 배양하였다. 젖산균 균주의 동정을 위하여, 각 시료로부터 분리된 단일 집락을 시퀀싱을 하고 16s rRNA 유전자 서열 분석에 의하여 LAB를 동정하였다. 동정된 유산균은 다시 MRS 액체배지에서 배양하여 동결보존제와 혼합하여 영하 70℃에서 보관하였다. 분리한 유산균은 표 1(김치유래), 표 2(된장유래)와 같다.Lactic acid bacteria were isolated from Kimchi (Chinese cabbage, Korean cabbage, and Dongchimi) and Doenjang collected from various parts of Korea. For lactic acid bacteria isolation, 1 ml fermentation soup was first added to 30 ml MRS liquid medium and incubated at 37 ° C. for 24 hours. One loop of cultured liquid medium was streaked in Rogosa solid medium and incubated at 37 ° C. for 48 hours. Each grown colony was further streaked in MRS solid medium and incubated at 37 ° C. for 24 hours. For the identification of lactic acid bacteria strains, single colonies isolated from each sample were sequenced and LABs were identified by 16s rRNA gene sequencing. The identified lactic acid bacteria were again cultured in MRS liquid medium and mixed with cryopreservative and stored at -70 ° C. The isolated lactic acid bacteria are shown in Table 1 (kimchi derived) and Table 2 (miso derived).
그룹  group 유산균  Lactobacillus
G1  G1 L.L. plantarumplantarum - 1- One
G2  G2 L.L. plantarumplantarum - 2- 2
G3  G3 L.L. plantarumplantarum - 3-3
G4  G4 L.L. brevisbrevis -1-One
G5  G5 L.L. brevisbrevis -2-2
G6  G6 L.L. brevisbrevis -3-3
G7  G7 L.L. brevisbrevis -4-4
G8  G8 P.P. pentosaceuspentosaceus -1-One
G9  G9 P.P. pentosaceuspentosaceus -2-2
G10  G10 L. L. sakeisakei
G11  G11 공격접종 양성대조군  Attack control positive control group
G12  G12 공격접종 음성대조군  Attack Vaccine Voice Control
그룹  group 유산균  Lactobacillus
G1  G1 18 B. coagulans 18 B. coagulans
G2  G2 37 S. nakayamae 37 S. nakayamae
G3  G3 17 S. nakayamae 17 S. nakayamae
G4  G4 46 B. coagulans 46 B. coagulans
G5  G5 21 B. coagulans 21 B. coagulans
G6  G6 35 B. linchenformis 35 B. linchenformis
G7  G7 30 P . acidilactici 30 P. acidilactici
G8  G8 28 L. alimentarius 28 L. alimentarius
G9  G9 23 E. faecium 23 E. faecium
G10  G10 22 L. mesenteroides 22 L. mesenteroides
G11  G11 공격접종 양성대조군  Attack control positive control group
G12  G12 공격접종 음성대조군  Attack Vaccine Voice Control
실시예Example 3: 바이러스  3: virus
인플루엔자 바이러스 A/NWS/33 (H1N1), 인플루엔자 바이러스 A/Korea/01/2009 (H1N1)를 3일 동안 37℃에서 11일령 계태아의 요막강에서 성장시켰다. 요막액(allantoic fluid)을 모아서 사용시까지 영하 70℃에 저장하였다. 요막액에서 얻은 바이러스의 역가는 연속적으로 10-배 희석된 요막액을 발육계란(embryonated eggs)에 주사하고, 각 계란의 요막액에서 바이러스의 존재를 바이러스의 혈구응집 능력에 기초하여 결정하였다. 마우스에 적응된 바이러스를 만들기 위해, 바이러스를 먼저 마우스 폐에서 분리하여 SPF 계란에서 증식시키고 다시 마우스에 재접종하였다. 공격접종 연구에서, Avertin (375 mg/kg)을 복강내 주사 경로로 주입하여 마우스를 마취한 뒤 90μL의 10^4.5 EID50 인플루엔자 바이러스로 비강내 경로로 공격접종하였다.Influenza virus A / NWS / 33 (H1N1), influenza virus A / Korea / 01/2009 (H1N1) were grown in the ureteral cavity of 11-day-old fetuses at 37 ° C. for 3 days. Allantoic fluid was collected and stored at minus 70 ° C. until use. The titer of virus obtained from the urea was continuously injected with 10-fold diluted urea into embryonated eggs, and the presence of the virus in the urea of each egg was determined based on the hemagglutination ability of the virus. To make the virus adapted to the mouse, the virus was first isolated from the mouse lung, propagated in SPF eggs and re-inoculated into the mouse. In the challenge study, mice were anesthetized by injecting Avertin (375 mg / kg) by the intraperitoneal injection route and challenged by intranasal route with 90 μL of 10 ^ 4.5 EID50 influenza virus.
실시예Example 4: 실험 설계 4: experimental design
인플루엔자 바이러스 type A, Influenza virus type A, H1N1에On H1N1 대한 LABS 방어 효과 LABS defense against
김치와 된장에서 분리한 여러 유산균의 인플루엔자 바이러스에 대한 방어 효과를 확인하기 위하여 마우스에 유산균 투여 후 바이러스를 공격 접종하여 관찰하였다. 10마리를 한 그룹으로 하여, 그룹당 한 가지 유산균을 각각 바이러스 공격접종 전 비강 내 경로(1x 10^8 cfu/마리)로 6회 투여하였다. 양성 대조군 및 음성 대조군 그룹은 테스트 그룹과 동일한 형식으로 PBS를 접종하였다. 김치 유래 유산균과 된장 유래 유산균은 각각 인플루엔자 바이러스 A/NWS/33 (10^4.5/ 90μL/ dose)와 A/Korea/01/2009 (H1N1) (10^4.5/ 90μL/ dose)으로 공격 접종한 후, 임상 증상, 사망률 및 체중 변화를 2주 동안 관찰하여 비강 투여된 유산균에서 인플루엔자 바이러스의 보호 효과를 평가하였다.In order to confirm the protective effect of various lactic acid bacteria isolated from kimchi and doenjang against influenza virus, mice were challenged and inoculated with the virus after administration of lactic acid bacteria. Ten animals were grouped, one lactic acid bacterium per group was administered six times, respectively, by intranasal route (1 × 10 8 cfu / horse) prior to virus challenge. Positive and negative control groups were inoculated with PBS in the same format as the test group. Kimchi-derived lactic acid bacteria and miso-derived lactic acid bacteria were inoculated with influenza virus A / NWS / 33 (10 ^ 4.5 / 90μL / dose) and A / Korea / 01/2009 (H1N1) (10 ^ 4.5 / 90μL / dose), respectively. The clinical effects, mortality and body weight change were observed for 2 weeks to assess the protective effect of influenza virus in nasal-administered lactobacilli.
투여 경로에 따른 인플루엔자 바이러스 type A, H1N1에 대한 Lactobacillus의 보호 효과Protective effect of Lactobacillus against influenza virus type A and H1N1 according to the route of administration
인플루엔자 바이러스 감염에 저항이 있는 균주를 선택하여 유산균의 투여 경로에 따른 방어 효과에 차이가 있는지 확인하기 위하여 마우스에 1x 10^8cfu/마리의 농도로 3주간 경구(300ul) 투여를 수행하였다. 양성 대조군 및 음성 대조군을 테스트 군과 동일한 형식으로 PBS로 접종하였다. 김치 유래 유산균과 된장 유래 유산균은 각각 인플루엔자 바이러스 A/NWS/33 (10^4.5/ 90μL/ dose)와 A/Korea/01/2009 (H1N1) (10^4.5/ 90μL/ dose)으로 공격 접종한 후, 임상 증상, 사망률 및 체중 변화를 2주 동안 관찰하여 비강 투여된 유산균에서 인플루엔자 바이러스의 보호 효과를 평가하였다.Strains resistant to influenza virus infection were selected and oral (300ul) was administered to mice for 3 weeks at a concentration of 1x10 ^ 8 cfu / horse to determine whether there was a difference in the protective effect according to the administration route of lactic acid bacteria. Positive and negative controls were inoculated with PBS in the same format as the test group. Kimchi-derived lactic acid bacteria and miso-derived lactic acid bacteria were inoculated with influenza virus A / NWS / 33 (10 ^ 4.5 / 90μL / dose) and A / Korea / 01/2009 (H1N1) (10 ^ 4.5 / 90μL / dose), respectively. The clinical effects, mortality and body weight change were observed for 2 weeks to assess the protective effect of influenza virus in nasal-administered lactobacilli.
Figure PCTKR2017006511-appb-T000001
Figure PCTKR2017006511-appb-T000001
Figure PCTKR2017006511-appb-T000002
Figure PCTKR2017006511-appb-T000002

Claims (4)

  1. 락토바실러스 사케이(Lactobacillus sakei) 균주 또는 페디오코커스 애시디락티시(Pediococcus acidilactici) 균주를 유효성분으로 포함하는 인플루엔자 바이러스에 대한 보호 효과를 가지는 조성물.A composition having a protective effect against influenza virus comprising Lactobacillus sakei strain or Pediococcus acidilactici strain as an active ingredient.
  2. 제 1항에 있어서, 상기 락토바실러스 사케이(Lactobacillus sakei) 균주는 기탁번호 KCTC13031BP이고, 상기 페디오코커스 애시디락티시(Pediococcus acidilactici) 균주는 기탁번호 KCTC13032BP인 것을 특징으로 하는 인플루엔자 바이러스에 대한 보호 효과를 가지는 조성물.According to claim 1, The Lactobacillus sakei (Lactobacillus sakei) strain is Accession No. KCTC13031BP, The Pediococcus acididiactici strain (Pediococcus acidilactici) The protective effect against influenza virus, characterized in that the Accession No. KCTC13032BP. Composition having a.
  3. 제 1항 또는 제2항에 있어서, 상기 조성물은 비강 또는 경구 투여용인 것을 특징으로 하는 인플루엔자 바이러스에 대한 보호 효과를 가지는 조성물.The composition according to claim 1 or 2, wherein the composition has a protective effect against influenza virus, which is for nasal or oral administration.
  4. 제 1항 또는 제2항에 있어서, 상기 조성물은 약학, 식품 또는 사료 조성물인 것을 특징으로 하는 인플루엔자 바이러스에 대한 보호 효과를 가지는 조성물.According to claim 1 or 2, wherein the composition is a pharmaceutical, food or feed composition having a protective effect against influenza virus, characterized in that the composition.
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