KR20050025002A - Acid tolerant probiotic Lactobacillus reuteri Probio-054 that can suppresses the growth of pathogenic microorganisms - Google Patents
Acid tolerant probiotic Lactobacillus reuteri Probio-054 that can suppresses the growth of pathogenic microorganisms Download PDFInfo
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Abstract
Description
본 발명은 유해 미생물 억제 활성을 가지는 신규 내산성 락토바실러스 루테리 Probio-054 및 이를 함유하는 생균활성제에 관한 것으로, 보다 상세하게는 개의 분변으로부터 분리된 신규 미생물로서 내산성, 내담즙산성을 지니면서 동시에 유해 미생물의 생장을 효과적으로 억제할 수 있는 락토바실러스 루테리 Probio-054(Lactobacillus reuteri Probio-054), 상기 미생물을 함유하는 가축용 생균활성제 및 상기 미생물을 이용하여 장내 유해미생물의 생육을 억제하는 방법에 관한 것이다.The present invention relates to a novel acid resistant Lactobacillus luteri Probio-054 and a bioactive agent containing the same, and more particularly, a novel microorganism isolated from feces of a dog having acid resistance, bile acid resistance and at the same time harmful microorganisms. Lactobacillus luuteri Probio-054 ( Lactobacillus reuteri Probio-054) capable of effectively inhibiting the growth of the present invention, and a livestock bioactive agent containing the microorganism and a method for inhibiting the growth of harmful microorganisms in the gut using the microorganism.
국내·외적으로 개의 사육 시 발생하는 장염에 의한 설사, 구토, 탈수 등은 사육장 및 자연환경에 서식하는 파보바이러스와 대장균에 의한 2차 감염에 의해 일어나며 폐사율이 높은 심각한 질병이다. 개 파보바이러스 설사증은 질병발생 시 항생제를 투여하더라도 치료 효과를 볼 수 없으며, 단지 2차 감염을 일으키는 미생물에 대한 치료 효과를 기대할 수 있다. 파보바이러스 감염 예방을 위하여 백신이 사용되고 있으나 백신 사용에 의한 부작용 또한 발생할 수 있다는 문제점이 있다.Diarrhea, vomiting, and dehydration due to enteritis at home and abroad are serious diseases with high mortality due to secondary infection by parvovirus and Escherichia coli in the breeding ground and the natural environment. Canine parvovirus diarrhea does not have a therapeutic effect even if antibiotics are administered in the event of disease, and can only be expected to treat the microorganism causing the secondary infection. The vaccine is used to prevent parvovirus infection, but there is a problem that side effects due to the use of the vaccine may also occur.
인체 및 동물체의 장내에는 다양한 미생물들이 존재하면서 장내의 미생물 균총을 이루고 있다. 이러한 미생물 중에는 숙주 동물에 유용한 것으로 알려져 온 유산균 등의 유익한 미생물들이 있는가 하면 반대로 숙주에 직접적 또는 잠재적인 유해성을 가지는 대장균, 살모넬라, 포도상구균 등의 미생물들이 또한 포함된다. 인체 및 동물체의 경우 스트레스의 증가, 유해세균의 감염 등의 외부 환경의 변화에 의해서 장내 미생물의 균총이 파괴될 경우 유해 미생물들의 급격한 성장으로 이어져 숙주동물의 건강상태를 악화시키거나 설사에서부터 심지어는 폐사의 위험으로까지 진행될 수 있다. 이러한 일이 발생하였을 경우 치료의 목적으로 항생제를 투여하게 되나 이러한 항생제는 체외로 완전히 배설이 되지 않고 숙주 내에 잔존하게 되며 지속적인 투여시 유해미생물들이 내성을 가지게 되므로 효과적인 치료를 할 수 없게 되는 상황이 발생하게 된다. 이에 대한 대안으로 현재 생균활성제인 프로바이오틱스에 대한 관심이 증대되고 있다. 생균활성제인 프로바이오틱스는 인체나 동물의 장내에 서식하는 유익한 미생물을 분리하여 제제화한 것으로 호기성균군, 혐기성균군, 유산균, 효모균들이 사용되고 있으나 주로 유산균들이 주요 균주로 이용되고 있다. 이러한 유산균 프로바이오틱스는 항생제 남용으로 인해 발생할 수 있는 부작용을 일으키지 않고 장내 유해 세균의 이상발효를 억제하여 안정된 장내 균총을 유지하며 유해 미생물의 감염으로 인한 질병을 사전에 억제할 수 있다. 또한 생균활성제의 지속적인 투여는 사료의 이용효율을 높여주고 장내 유익한 미생물이 균형을 이루어 서식하게 되므로 건강 유지를 꾀할 수 있다. 생균활성제로서의 효과적인 사용을 위해서는 유해한 미생물에 대한 억제 활성뿐만 아니라 내산성, 내담즙산성을 지니는 것이 매우 중요하다. 동물체에 생균활성제를 투여할 후 경우 장에 도착할 때까지는 강산인 위액을 만나게 되고 그 다음 담즙산을 만나게 된다. 이들 위액과 담즙산은 일반적으로 세균을 죽이거나 활성을 떨어뜨리기 때문에 투여된 생균활성제가 무사히 장에 도달하여 그 기능을 발휘하기 위해서는 위액과 담즙산에 견딜 수 있는 성질을 가지고 있어야 한다.Various microorganisms exist in the intestine of the human body and the animal, forming a microbial flora in the intestine. Such microorganisms include beneficial microorganisms, such as lactic acid bacteria, which are known to be useful for host animals, and conversely, microorganisms such as E. coli, Salmonella, Staphylococcus, etc., which have direct or potential harmful effects on the host. In the case of the human body and animals, when the flora of the gut microorganism is destroyed by the change of external environment such as the increase of stress and the infection of harmful bacteria, it leads to the rapid growth of the harmful microorganisms, worsening the health of the host animal or even from diarrhea and even death. It can go up to risk. When this happens, antibiotics are administered for the purpose of treatment, but these antibiotics remain in the host without being completely excreted in vitro, and harmful microorganisms become resistant to continuous administration, resulting in a situation where effective treatment cannot be achieved. Done. As an alternative, there is a growing interest in probiotics, which are currently probiotic. Probiotics, a probiotic, are prepared by separating beneficial microorganisms that live in the intestines of humans or animals. Aerobic, anaerobic, and lactic acid bacteria and yeasts are used, but lactic acid bacteria are used as the main strains. These lactic acid bacteria probiotics can maintain the stable intestinal flora by inhibiting abnormal fermentation of harmful bacteria in the intestine without causing side effects that can occur due to antibiotic abuse, and can prevent diseases caused by infection of harmful microorganisms in advance. In addition, continuous administration of probiotic can increase the utilization efficiency of feed and intestinal beneficial microorganisms are inhabited in a balanced manner to maintain health. For effective use as probiotic, it is very important to have acid resistance and bile acid resistance as well as inhibitory activity against harmful microorganisms. After the probiotic is administered to the animal, it meets gastric juice, which is a strong acid, until it reaches the intestine, followed by bile acids. Since these gastric juices and bile acids generally kill bacteria or reduce their activity, the administered probiotic must reach the intestines and function properly to withstand the gastric juice and bile acids.
지금까지 프로바이오틱 생균활성제로 다양한 종류의 유산균이 발표되어 왔으며 내산, 내담즙산 특성을 가지면서 유해 미생물을 억제하는 락토바실러스 루테리로 동정된 생균활성제 또한 보고된 바 있다(장영효 등, Kor. J. Appl. Microbiol. Biotechnol. 27, 23-27) (대한민국 특허 10-211529-0000).To date, various types of lactic acid bacteria have been published as probiotic probiotic agents, and bioactive agents identified as Lactobacillus luteri, which have the characteristics of acid and bile acids and inhibit harmful microorganisms, have also been reported (Jang, Young-Hyo et al., Kor. J. Appl. Microbiol.Biotechnol. 27, 23-27) (Korean Patent 10-211529-0000).
본 발명자들은 기존의 발표된 생균활성제 보다 유해 병원성 미생물들을 더욱 효과적으로 억제할 수 있고 내산성, 내담즙산성을 지니는 신규 생균활성제의 탐색을 시도하였으며, 개의 분변으로부터 혐기적으로 분리한 한 균주에 대해 유해 미생물에 대한 생육억제 능력 실험과 내산성, 내담즙산성 실험 및 미생물 동정 실험을 한 결과, 이 균주가 유해 미생물을 효과적으로 억제하면서도 내산성, 내담즙산성이 우수한 균주로 락토바실러스 루테리 균주라는 것을 확인함으로써 본 발명을 완성하였다.The inventors of the present invention have attempted to search for new bioactive agents that can more effectively inhibit harmful pathogenic microorganisms than existing published probiotic agents and have acid resistance and bile acid resistance, and are harmful microorganisms for one strain anaerobicly isolated from dog feces. The results of the present invention by confirming that the strain is Lactobacillus ruteri strain as a strain having excellent acid resistance and bile acid resistance while effectively inhibiting harmful microorganisms as a result of growth inhibition ability test, acid resistance, bile acid resistance test and microbial identification test Completed.
따라서, 본 발명의 목적은 인간 및 동물에는 해가 없고, 내 산성 및 내 담즙성이 뛰어난 신규 프로바이오틱 락토바실러스 루테리 Probio-054(Lactobacillus reuteri Probio-054)를 제공하는 것이다.Accordingly, an object of the present invention is to provide a novel probiotic Lactobacillus luterie Probio-054 ( Lactobacillus reuteri Probio-054), which is harmless to humans and animals, and is excellent in acid and bile resistance.
본 발명의 다른 목적은 기존 항생제의 대체용으로 인체에게 투여하거나 의약품용으로 사용되는 정장제, 유산균제제, 건강보조식품를 포함하는 발효식품 종균제 등으로 이용하여 장내 유해한 미생물을 억제하고 면역 활성이 높고, 내산성과 내담즙성이 우수한 생균활성제 함유 조성물을 제공하는 것이다.Another object of the present invention is to inhibit harmful microorganisms in the intestine and high immune activity by using as a fermented food spawning agent, including a formal drug, lactic acid bacteria, health supplements, etc., which are administered to the human body as a substitute for the existing antibiotics, It is to provide a probiotic-containing composition excellent in acid resistance and bile resistance.
본 발명의 또 다른 목적은 본 발명 락토바실러스 루테리 Probio-054를 이용하여 동물에서 장내 유해 병원성미생물의 생육을 억제하거나 상기 미생물에 의한 설사를 예방 또는 치료하는 방법을 제공하는 것이다.Still another object of the present invention is to provide a method for inhibiting the growth of harmful enteropathogenic microorganisms in the intestine or preventing or treating diarrhea caused by the microorganisms using the present invention Lactobacillus luteri Probio-054.
이하, 본 발명의 구성 및 작용을 상세히 설명한다. 본 발명의 다른 목적들 및 잇점들은 하기 구성을 통해 보다 명백히 이해될 것이다.Hereinafter, the configuration and operation of the present invention will be described in detail. Other objects and advantages of the present invention will be more clearly understood through the following configuration.
본 발명의 상기 목적은 유해 미생물을 억제하며 내산성, 내담증산성을 가지는 신규 미생물 락토바실러스 루테리 Probio-054(Lactobacillus reuteri Probio-054), 상기 프로바이오틱 미생물을 포함하여 장내 서식 유해 미생물의 생육을 억제할 수 있는 식품, 의약품, 동물약품, 사료첨가제 등의 식품학적 또는 약리학적 조성물을 제공함으로써 달성되었다.The object of the present invention is to inhibit harmful microorganisms and inhibit the growth of harmful microorganisms intestinal inhabitants including Lactobacillus reuteri Probio-054 ( Lactobacillus reuteri Probio-054), the probiotic microorganism having acid resistance, anti-acid resistance By providing food or pharmaceutical compositions such as food, pharmaceuticals, animal drugs, feed additives, and the like.
본 발명 신규 미생물인 락토바실러스 루테리 Probio-054(Lactobacillus reuteri Probio-054)는 개의 분변으로부터 혐기적으로 분리한 프로바이오틱 세균으로 유해 미생물 억제활성과 내산성, 내담즙산성 능력이 뛰어난 것으로 확인되었다. 그러므로, 본 발명 프로바이오틱 미생물 및 이를 함유한 생균활성제는 장내 유해세균들의 생장 억제 및 면역활성 증강 목적으로 식품학적 또는 약리학적 조성물, 즉 상기 미생물을 함유하는 의약품, 유산균제제 및 건강보조식품 등에 사용할 수 있다. Lactobacillus reuteri Probio-054 ( Lactobacillus reuteri Probio-054), a novel microorganism of the present invention, is an anaerobic probiotic bacterium that has been anaerobicly isolated from dog feces and has been shown to have excellent antimicrobial inhibitory activity, acid resistance and bile acid resistance. Therefore, the probiotic microorganism of the present invention and the probiotic containing the same can be used in food or pharmacological composition, that is, pharmaceuticals, lactic acid bacteria and health supplements containing the microorganisms for the purpose of inhibiting the growth of intestinal harmful bacteria and enhancing immune activity. Can be.
본 발명 신규 프로바이오틱 미생물인 락토바실러스 루테리 Probio-054는 장세포에 흡착할 수 있을 뿐만 아니라, 다음과 같은 프로바이오틱 특성을 보유한다.The novel probiotic microorganism of the present invention, Lactobacillus luteri Probio-054, can not only adsorb to enteric cells but also possess the following probiotic properties.
- 로타바이러스 및 장내 유해미생물의 생장 억제능-Growth inhibition of rotavirus and intestinal harmful microorganisms
- 면역 활성 증강능-Immune activity enhancer
- 호기적, 혐기적 조건 및 다양한 pH에서의 생장능; 이러한 성질은 위장관 내에서 변화되는 다양한 생리적 및 병리적 조건에 잘 적응할 수 있는 능력을 부여한다.Growth ability at aerobic, anaerobic conditions and various pHs; This property confers the ability to adapt well to various physiological and pathological conditions that change within the gastrointestinal tract.
- 위산 및 담즙산 내성-Stomach acid and bile acid resistance
- 동결건조에서의 생균활성 보유능-Probiotic retention in lyophilization
또한, 본 발명 신규 미생물은 공지의 다른 예방 또는 치료방법과 같이 항생제 내성이나 생물 독성 등의 부작용이 없는 것이 특징이다.In addition, the novel microorganism of the present invention is characterized by no side effects such as antibiotic resistance or biotoxicity as in other known prevention or treatment methods.
본 발명에 있어서, '프로바이오틱(probiotic)' 은 ' 사람을 포함한 동물의 위장관내에서 숙주의 장내 미생물 균형을 개선하여 숙주에 유익한 영향을 미치는 살아있는 미생물' 의 의미로 이해된다. 프로바이오틱 미생물은 프로바이오틱 활성을 갖는 살아 있는 미생물로 단일 또는 복합균주 형태로 사람이나 동물에 건조된 세포 형태나 발효산물 형태로 급여될 경우, 숙주의 장내 균총에 유익한 영향을 미칠 수 있다.In the present invention, 'probiotic' is understood to mean 'living microorganisms which have a beneficial effect on the host by improving the intestinal microbial balance of the host in the gastrointestinal tract of animals including humans'. Probiotic microorganisms are living microorganisms with probiotic activity that can have a beneficial effect on the intestinal flora of the host when fed in the form of dried cells or fermented products to humans or animals in the form of single or complex strains.
구체적으로, 개의 분변으로부터 분리한 본 발명 프로바이오틱 Probio-054 균주의 형태 및 생리·생화학적 특성을 조사한 결과는 다음과 같다 : Probio-054는 그람양성의 세균으로 호기적인 조건과 혐기적인 조건에서 동시에 성장하며 포자를 형성하지 않고 운동성이 없으며, 세포의 형태는 간균이다. 상기 균주의 성장 조건으로는 30∼37℃이 바람직하다. 가스와 인돌을 생성하지 않으며 용혈성을 나타내지 않고 질산을 환원하지 않는다. 본 발명의 미생물은 장기간 안정적으로 보존하기 위해 글리세롤 성분을 포함하여 -80℃에 보관하거나 멸균된 10% 탈지유에 현탁하여 동결건조하는 것이 바람직하다.Specifically, the results of examining the morphology and physiological and biochemical characteristics of the probiotic Probio-054 strain isolated from dog feces are as follows: Probio-054 is a Gram-positive bacterium under aerobic and anaerobic conditions. It grows at the same time, does not form spores, has no motility, and the cells are bacilli. As growth conditions of the said strain, 30-37 degreeC is preferable. It does not produce gases and indole, does not show hemolysis and does not reduce nitric acid. The microorganism of the present invention is preferably stored at −80 ° C. including a glycerol component or suspended in sterile 10% skim milk to freeze-dried for long-term stable storage.
또한, 16S rDNA 염기서열에 기초한 분자계통분류학적 분석에서 Probio-054는 락토바실러스 (Lactobacillus) 속에 속하는 균주로서 99.5%의 16S rDNA 상동성으로 락토바실러스 루테리(Lactobacillus reuteri)의 표준균주에 가장 높은 분자계통학적 유연관계를 보여주는 균주로 동정되었다. 따라서 이상의 결과에 기초하여 Probio-054를 락토바실러스 루테리(Lactobacillus reuteri) 균주로 동정하고, 한국생명공학연구원 유전자은행에 2003년 7월 21일자로 기탁하였다(수탁번호: KCTC 10499BP).In addition, Probio-054 is a strain belonging to the genus Lactobacillus , which is 99.5% homologous to the 16S rDNA homology, which is the highest in the standard strain of Lactobacillus reuteri . It was identified as a strain showing a mathematical relationship. Therefore, based on the above results, Probio-054 was identified as a Lactobacillus reuteri strain, and was deposited on July 21, 2003 to the Korea Biotechnology Research Institute Gene Bank (Accession Number: KCTC 10499BP).
구체적으로, 유해 미생물 억제 활성을 가지는 내산성, 내담즙산성 미생물을 탐색하는 과정은 하기와 같이 수행할 수 있다 : 혐기백(상품명 Gas Pack pouch; BBL사 제품)을 이용하여 개 분변 1g을 채취한 후 pH 2로 조정한 혐기 희석액으로 1:10의 비율로 희석하고, 진탕기를 이용하여 120분간 진탕하였다. 진탕액 1ml을 옥스갈(디프코사 제품, 상품명) 0.2%를 첨가한 BL 고체배지 (일본 영연화학사 제품)에 도말하고, 37℃에서 48시간 배양하였다. 일정시간이 지난 후 나타나는 콜로니를 현미경으로 관찰한 후 미생물 150개를 선발하여 GAM semi-agar 배지 (일본 일수제약사 제품)에 옮겨 배양한 뒤, 영하 80℃에서 보존하였다. 분리된 미생물들을 대상으로 한 유해미생물의 생장억제실험은 구로이와 등의 문헌(Kuroiwa et al., 감염증학지, 64, 257, 1990)과 항생제 평가시 사용되는 미생물 5종을 대상으로 하였으며 다음과 같다. Escherichia coli KCTC 2441, Shigella flexneri KCTC 2008, Enterobacter cloacea KCTC 2361, Citrobacter freundii KCTC 2006, Klebsiella pneumoniae KCTC 2208. 이러한 유해 미생물은 MRS 액체배지에서 18시간 혐기적으로 배양한 후 배양액 0.1ml을 취하여 MRS 고체배지에 도말 하였다. 지름 8 mm의 페이퍼디스크를 유해미생물이 도말된 고체배지에 올려놓고, MRS 액체배지에서 18시간 혐기적으로 배양된 각각의 분리 미생물 배양액 30ul를 접종하였다. 일정 시간 배양 후 나타나는 생육 억제환을 측정하여 대조군과 비교하고 유해미생물의 억제 활성이 가장 우수한 균주를 선별하여 Probio-054로 명명하였다.Specifically, the process of searching for acid-resistant and bile acid-resistant microorganisms having harmful microbial inhibitory activity may be performed as follows: After collecting 1 g of dog feces using an anaerobic bag (trade name Gas Pack pouch; manufactured by BBL) The mixture was diluted with an anaerobic dilution adjusted to pH 2 at a ratio of 1:10, and shaken for 120 minutes using a shaker. 1 ml of the shake solution was spread on a BL solid medium (manufactured by Young Yeon Chemical Co., Ltd.) to which 0.2% of Oxgal (manufactured by Diffco Co., Ltd.) was added and incubated at 37 ° C for 48 hours. After observing the colonies appearing after a certain time under a microscope, 150 microorganisms were selected and transferred to GAM semi-agar medium (Japan Daihan Pharmaceutical Co., Ltd.) culture, and then stored at minus 80 ℃. Inhibition experiments of harmful microorganisms on isolated microorganisms were conducted in Kuroiwa et al. (Kuroiwa et al., Journal of Infectious Diseases, 64, 257, 1990) and five microorganisms used for antibiotic evaluation. . Escherichia coli KCTC 2441, Shigella flexneri KCTC 2008, Enterobacter cloacea KCTC 2361, Citrobacter freundii KCTC 2006, Klebsiella pneumoniae KCTC 2208. Smeared. Paper disks with a diameter of 8 mm were placed on solid medium coated with harmful microorganisms, and inoculated with 30 ul of each isolated microbial culture medium anaerobicly cultured in an MRS liquid medium for 18 hours. Growth inhibitory rings appearing after a certain period of time was measured and compared with the control group, the strains with the highest inhibitory activity of harmful microorganisms were selected and named as Probio-054.
한편, 본 발명 균주 락토바실러스 루테리 Probio-054는 항생제 내성이 있어, 장내 유해 미생물의 생육을 억제하기 위해서 항생제와 같이 또는 기존 항생제의 대체용으로도 인체 또는 가축에게 투여할 수 있는 것으로 나타났다.On the other hand, the strain Lactobacillus luteri Probio-054 of the present invention is antibiotic-resistant, so that it can be administered to humans or animals as well as antibiotics or to replace existing antibiotics in order to suppress the growth of harmful microorganisms in the intestine.
본 발명의 한 유익한 측면에 있어서, 본 발명은 신규의 프로바이오틱 미생물인 락토바실러스 루테리 Probio-054를 함유하는 식품, 의약품, 동물약품, 가축용 사료, 유산균제제 및 건강보조식품을 제공한다. 본 발명 미생물은 허용 가능한 담체와 함께 독립적으로, 또는 첨가되어 사용되거나 사람 또는 동물이 섭취하기에 적합한 조성물 형태로 사용될 수 있다. 즉, 본 발명 미생물은 다른 프로바이오틱 세균을 함유하지 않은 식품(첨가되어 프로바이오틱 활성을 부여함) 및 이미 몇 가지의 프로바이오틱 세균을 함유한 식품(첨가되어 프로바이오틱 활성을 증강시키거나 보완함)에 첨가되어 사용될 수 있다.In one advantageous aspect of the present invention, the present invention provides foods, pharmaceuticals, animal drugs, animal feed, lactic acid bacteria preparations and dietary supplements containing the novel probiotic microorganisms, Lactobacillus luteri Probio-054. The microorganisms of the present invention may be used alone or in addition to an acceptable carrier or in the form of a composition suitable for ingestion by humans or animals. That is, the microorganism of the present invention is added to foods that do not contain other probiotic bacteria (added to give probiotic activity) and foods that already contain some probiotic bacteria (added to enhance probiotic activity. Or complementary).
본 발명의 의약학적, 식품학적 또는 영양학적 조성물은 분리된 프로바이오틱 세균으로 락토바실러스 루테리 Probio-054 또는 이와 함께 2종 이상의 다른 유익한 균주와 적당한 담체를 함께 포함할 수 있다.The pharmaceutical, food or nutritional composition of the present invention may be an isolated probiotic bacterium and include Lactobacillus luteri Probio-054 or two or more other beneficial strains together with a suitable carrier.
예컨대, 본 발명 조성물에 본 발명 균주와 함께 사용 가능한 다른 미생물들은 사람이나 동물이 섭취하기에 적합하고 섭취시 유해 병원성 유해세균을 억제하거나 포유동물 장관 내의 미생물 균형을 개선시킬 수 있는 프로바이오틱 활성을 갖는 것들이 바람직하다. 그러한 프로바이오틱 미생물의 예로는 Saccharomyces, Candida, Pichia 및 Torulopsis를 포함하는 효모(yeasts), Aspergillus, Rhizopus, Mucor, Penicillium 등과 같은 곰팡이 및 Lactobacillus, Bifidobacterium, Clostridium, Leuconostoc, Bacteroides, Staphylococcus, Lactococcus, Bacillus, Streptococcus, Fusobacterium, Propionibacterium, Enterococcus, Pediococcus, 그리고 Micrococcus 속 세균들이 있다. 적당한 프로바이오틱 미생물의 구체적인 예로는 Saccharomyces cereviseae, Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis, Bifidobacterium bifidum, Bifidobacterium 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, 및 Staphylococcus xylosus 등을 들 수 있다. 바람직하게는 우수한 프로바이오틱 활성을 가지면서 면역활성 증강 및 항암작용이 뛰어난 프로바이오틱 미생물 혼합균을 본 발명 조성물에 추가로 포함하여 본 발명 조성물의 효과를 더욱 증진시킬 수도 있다. 본 발명 조성물에 함유될 수 있는 담체의 예로는 증량제, 고섬유 첨가제, 캡슐화제, 지질 등일 수 있으며 이러한 담체들의 예는 당업계에 충분히 공지되어 있다. 본 발명 락토바실러스 루테리 Probio-054 균주는 동결건조되거나 캡슐화된 형태 또는 배양현탁액이나 건조분말 형태일 수 있다.For example, other microorganisms that can be used with the strains of the present invention in the compositions of the present invention are probiotic activities that are suitable for human or animal ingestion and that can inhibit harmful pathogenic harmful bacteria or improve microbial balance in the mammalian intestine upon ingestion. Those having are preferred. Examples of such probiotic microorganisms include yeasts including Saccharomyces, Candida, Pichia and Torulopsis , molds such as Aspergillus , Rhizopus, Mucor, Penicillium and Lactobacillus, Bifidobacterium, Clostridium, Leuconostoc, Bacteroides, Staactococcus, Lactusoccus, Lactobacillus Streptococcus, Fusobacterium, Propionibacterium, Enterococcus, Pediococcus, and Micrococcus are bacteria. Specific examples of suitable probiotic microorganisms include Saccharomyces cereviseae, Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Enterococcus faecium, Enterococcus faecalis, Lactobacillcillus caseus ruciusus lucus alitususus Lactobacillus johnsonii, Lactobacillus farciminus, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus sake, Lactococcus lactis, Micrococcus varians, Pediococcus acidilactici, and Staphylococcus x . Preferably, a probiotic microbial mixed bacterium having excellent probiotic activity and excellent immune activity enhancement and anticancer activity may be further included in the composition of the present invention to further enhance the effect of the composition of the present invention. Examples of carriers that may be contained in the compositions of the present invention may be extenders, high fiber additives, encapsulating agents, lipids, and the like and examples of such carriers are well known in the art. The Lactobacillus luteri Probio-054 strain of the present invention may be in lyophilized or encapsulated form or in culture suspension or dry powder form.
통상적인 항생제 대신에, 본 발명 신규 미생물 및 조성물은 생균활성제로 사람 또는 동물에 투여되거나 식품, 의약품, 동물약품, 사료첨가제, 또는 건강보조식품과 함께 사람이나 동물에 투여될 수 있다. 본 발명 프로바이오닉 미생물 및 이를 포함한 조성물 형태의 생균활성제는 기존 항생제의 대체용으로 장내 유해한 미생물을 억제하며 안정된 장내 균총의 유지를 통해 동물체의 건강상태를 양호하게 하고 가축의 증체량 증가, 육질 개선, 산유량 증가, 면역활성을 증강시키는 등의 목적으로 사용할 수 있다. 본 발명 조성물은 또한 장내 미생물 균총을 유지하기 위해 항생제 투여 후에도 투여될 수 있다.Instead of conventional antibiotics, the novel microorganisms and compositions of the present invention may be administered to humans or animals as probiotic or to humans or animals with foods, pharmaceuticals, animal drugs, feed additives, or dietary supplements. The probiotic microorganism of the present invention and the probiotic in the form of a composition containing the same inhibit the harmful microorganisms in the gut as a substitute for the existing antibiotics and improve the health of the animal body by maintaining a stable intestinal flora, and increase the weight gain of the livestock, improve the meat quality, and produce the milk It can be used for the purpose of increasing, enhancing immune activity. The composition of the present invention may also be administered after antibiotic administration to maintain the intestinal microflora.
특히, 항생제 내성뿐만 아니라 유해 장내미생물 생육 억제능을 갖는 본 발명 신규 미생물은 특히, 다른 치료법이 비효과적이거나 임상적으로 부작용이 있는 장내 유해미생물 감염의 감염에 의해 유발된 대부분의 증상 완화나 치료에 매우 유용하다. 그리하여, 본 발명은 신규 미생물 또는 이를 유효량 함유한 조성물을 사용하여 장내 유해 미생물의 감염이나 장내 질환의 발생을 저지하거나 장내 유해 미생물 감염에 의한 위장염 및 설사 등을 치료하는 방법을 제공한다. 락토바실러스 루테리 Probio-054가 유해 미생물을 억제하기 위한 최적 온도조건으로는 30-37℃가 바람직하다.In particular, the novel microorganisms of the present invention having antibiotic resistance as well as the ability to inhibit the growth of harmful enteric microorganisms are particularly effective for alleviating or treating most symptoms caused by infection of enteric harmful microbial infections in which other therapies are ineffective or have clinical side effects. useful. Thus, the present invention provides a method for preventing infection of intestinal harmful microorganisms or the occurrence of intestinal diseases or treating gastroenteritis and diarrhea caused by intestinal harmful microbial infections using the novel microorganisms or compositions containing an effective amount thereof. As the optimum temperature condition for inhibiting the harmful microorganisms Lactobacillus luterie Probio-054 is 30-37 ℃ is preferred.
이하, 실시 예를 통하여 본 발명의 구성 및 작용효과를 보다 구체적으로 설명한다. 단, 하기 실시 예는 본 발명을 예시하기 위한 목적으로 발명의 권리범위가 하기 실시 예에 한정되는 것은 아니다.Hereinafter, the configuration and the effect of the present invention through the embodiment will be described in more detail. However, the following examples are not intended to limit the scope of the invention for the purpose of illustrating the present invention is limited to the following examples.
실시예 1 : 내산, 내담즙산 성질을 가지는 세균의 분리Example 1 Isolation of Bacteria with Acid and Bile Acid Properties
건강한 진돗개의 분변을 혐기백(상품명 Gas Pack pouch; BBL사 제품)에 채취하였다. 채취한 분변 1g은 pH 2로 조정한 혐기희석액 (0.78% K2HPO4, 염혼합액 37.5ml, L-cystein 0.5g, 25% L-ascorbic acid 2ml, 8% Na2CO3 50ml, 0.1% resazurin 1ml을 증류수 860ml에 녹이고, agar 0.5g을 첨가) 을 이용하여 1:10의 비율로 희석하고, 진탕기를 이용하여 120분간 진탕하였다. 진탕액 1ml을 옥스갈 (디프코사 제품, 상품명) 0.2%를 첨가한 BL 고체배지(일본 영연화학사 제품)[ Lab-lemco powder 2.4g, Proteose peptone No.3 10g, Trypticase 5g, Phytone peptone 3g, yeast extract 5g, liver extract 150ml, glucose 10g, soluble starch 0.5g, sol.A 10ml, sol.B 5ml, 10% FS antifoam F-20 5ml, Tween 80 1g, L-cystein 0.5g, horse serum 50ml을 증류수 1 L에 녹이고, agar 15g을 첨가; sol.A 의 조성, 10% KH2PO4과 10% K2HPO4을 혼합한 것이며; sol.B의 조성, 황산마그네슘 10g, 황산철 0.5g, 소금 0.5g, 황산망간 0.337g을 증류수 250ml에 녹임)에 도말하고, 37℃에서 48시간 배양하였다. 배양 후 형성된 콜로니 150개를 순수분리하여 선발하고 GAM semi-agar 배지(일본 일수제약사 제품) [peptone 100g, Soya peptone 3g, Proteose peptone No.3 10g, Peptonized whale serum 13.5g, yeast extract 5g, Beef extract 2.2g, liver extract 1.2g, KH2PO4 2.5g, NaCl 3g, L-cystein 0.3g, Nathioglycolate 0.3g, glucose 2g를 증류수 1 L에 녹이고, agar 1.5g을 첨가]에 옮겨 배양한 뒤, 영하 80℃에서 보존하였다.The feces of healthy Jindo dogs were collected in an anaerobic bag (trade name Gas Pack pouch; manufactured by BBL). 1 g of the collected feces was anaerobic diluent adjusted to pH 2 (0.78% K 2 HPO 4 , salt mixture 37.5ml, L-cystein 0.5g, 25% L-ascorbic acid 2ml, 8% Na 2 CO 3 50ml, 0.1% resazurin 1 ml was dissolved in 860 ml of distilled water, 0.5 g of agar was added), and the mixture was diluted at a ratio of 1:10 and shaken for 120 minutes using a shaker. BL solid medium (made by Young Yeon Chemical Co., Ltd.) with 0.2 ml of oxal (made by Diffco Co., Ltd.) [1 ml of shaking liquid] [Lab-lemco powder 2.4 g, Proteose peptone No. 3 10 g, Trypticase 5 g, Phytone peptone 3 g, 5g yeast extract, 150ml liver extract, 10g glucose, soluble starch 0.5g, sol.A 10ml, sol.B 5ml, 10% FS antifoam F-20 5ml, Tween 80 1g, L-cystein 0.5g, horse serum 50ml Dissolve in 1 L and add 15 g of agar; a composition of sol.A, 10% KH 2 PO 4 and 10% K 2 HPO 4 ; The composition of sol.B, 10 g of magnesium sulfate, 0.5 g of iron sulfate, 0.5 g of salt, and 0.337 g of manganese sulfate were dissolved in 250 ml of distilled water) and incubated at 37 ° C for 48 hours. 150 colonies formed after incubation were isolated by pure separation and selected by GAM semi-agar medium (manufactured by Japan Pharmaceutics Co., Ltd.) 2.2g, liver extract 1.2g, KH2PO4 2.5g, NaCl 3g, L-cystein 0.3g, Nathioglycolate 0.3g, glucose 2g are dissolved in 1L of distilled water, and 1.5g of agar is added to incubate. Preserved.
실시예 2 : 분리된 미생물의 유해 미생물 생장 억제 실험Example 2 Experiment of Inhibiting Harmful Microorganism Growth of Isolated Microorganisms
분리된 미생물들을 대상으로 한 유해미생물의 생장억제실험은 구로이와 등(Kuroiwa et al., 감염증학지, 64, 257, 1990)의 방법을 이용하여 항생제 평가시 사용되는 다음의 미생물 5종을 대상으로 하였다. Escherichia coli KCTC 2441 , Shigella flexneri KCTC 2008 , Enterobacter cloacea KCTC 2361 , Citrobacter freundii KCTC 2006 , Klebsiella pneumoniae KCTC 2208. 이러한 유해 미생물은 MRS 액체배지에서 18시간 혐기적으로 배양한 후 배양액 0.1ml을 취하여 MRS 고체배지에 도말 하였다. 지름 8 mm의 페이퍼디스크를 유해미생물이 도말된 고체배지에 올려놓고, MRS 액체배지에서 18시간 혐기적으로 배양된 각각의 분리 미생물 배양액 30ul를 접종하였다. 일정 시간 배양 후 나타나는 생육 억제환을 측정하여 대조군과 비교하고 유해미생물의 억제 활성이 가장 우수한 균주를 선별하여 Probio-054로 명명하였다. Probio-054의 유해 미생물에 대한 억제 활성은 다음 표1과 같다. 표 1에서 보는 바와 같이 Probio-054는 사용된 유해미생물 5종에 대해서 대부분 유사한 억제 활성을 지니는 매우 유용한 미생물인 것으로 확인되었다. 또한 Probio-054는 기존의 락토바실러스 루테리 균주 (BSA-131) (장영효 등, Kor. J. Appl. Microbiol. Biotechnol. 27, 23-27) (대한민국 특허 10-211529-0000) 보다 유해 미생물에 대한 억제 활성이 약간 더 뛰어남을 확인할 수 있었고 생존력 실험 또한 더 우수함을 확인하였다.Inhibitory experiments on the growth of harmful microorganisms on isolated microorganisms were carried out on the following five microorganisms used for antibiotic evaluation using the method of Kuroiwa et al. (Kuroiwa et al., Journal of Infectious Diseases, 64, 257, 1990). It was. Escherichia coli KCTC 2441, Shigella flexneri KCTC 2008, Enterobacter cloacea KCTC 2361, Citrobacter freundii KCTC 2006, Klebsiella pneumoniae KCTC 2208. Smeared. Paper disks with a diameter of 8 mm were placed on solid medium coated with harmful microorganisms, and inoculated with 30 ul of each isolated microbial culture medium anaerobicly cultured in an MRS liquid medium for 18 hours. Growth inhibitory rings appearing after a certain period of time was measured and compared with the control group, the strains with the highest inhibitory activity of harmful microorganisms were selected and named as Probio-054. Inhibitory activity of probio-054 against harmful microorganisms is shown in Table 1 below. As shown in Table 1, Probio-054 was found to be a very useful microorganism with mostly similar inhibitory activity against the five harmful microorganisms used. In addition, Probio-054 is more effective against harmful microorganisms than the existing Lactobacillus lusteri strain (BSA-131) (Kang, Young-Hyo et al., Kor. J. Appl. Microbiol. Biotechnol. 27, 23-27) (Korean Patent 10-211529-0000). The inhibitory activity was found to be slightly better, and the survival experiment was also confirmed to be superior.
실시예 4 : 분리한 미생물 Probio-054의 동정Example 4 Identification of Isolated Microorganism Probio-054
실시예 1에서 분리된 Probio-054 균주를 MRS 배지(Difco사 제품)에서 37℃ 조건으로 배양하였다. Probio-054 균주를 동정하기 위하여, Probio-054의 형태 및 생리학적 특성은 윤 등(Yoon et al., Int. J. Syst. Bacteriol., 47, 904, 1997)의 방법과 API 32A 및 CHL 키트 (바이오메리오사 제품)를 사용하여 결정하였다. 16S rRNA 유전자의 염기 서열 결정 및 분석은 윤 등 (Yoon et. al., Int. J. Syst. Bacterill., 47, 933, 1997)의 방법을 사용하였다.Probio-054 strain isolated in Example 1 was incubated in 37 ° C conditions in MRS medium (Difco). To identify Probio-054 strains, the morphology and physiological properties of Probio-054 were determined by Yoon et al., Int. J. Syst. Bacteriol ., 47, 904, 1997 and the API 32A and CHL kits. It determined using (Biomerio company make). The sequencing and analysis of the 16S rRNA gene was performed using the method of Yoon et al., Int. J. Syst. Bacterill ., 47, 933, 1997.
Probio-054는 그람양성의 세균으로 호기적인 조건과 혐기적인 조건에서 동시에 성장하며 포자를 형성하지 않고 운동성이 없으며, 세포의 형태는 간균이다. 상기 균주의 성장 조건으로는 30∼37℃이 바람직하다. 가스와 인돌을 생성하지 않으며 용혈성을 나타내지 않고 질산을 환원하지 않는다.Probio-054 is a Gram-positive bacterium that grows simultaneously in both aerobic and anaerobic conditions. It does not form spores, has no motility, and has a cell type. As growth conditions of the said strain, 30-37 degreeC is preferable. It does not produce gases and indole, does not show hemolysis and does not reduce nitric acid.
Probio-054의 16S rRNA 유전자의 염기 서열은 서열번호 1로 기재된다. 16S rDNA 염기서열에 기초한 분자계통분류학적 분석에서 Probio-054는 락토바실러스(Lactobacillus) 속에 속하는 균주로서 99.5%의 16S rDNA 상동성으로 락토바실러스 루테리 (Lactobacillus reuteri)의 표준균주에 가장 높은 분자계통학적 유연관계를 보여주는 균주로 동정되었다. 또한 Probio-054는 기존에 발표된 내산, 내담즙산 특성을 가지면서 유해 미생물을 억제하는 락토바실러스 루테리로 동정된 생균활성제(BSA-131)(장영효 등, Kor J. Appl. Microbiol. Biotechnol. 27, 23-27) (대한민국 특허 10-211529-0000)와 비교시 2 개의 염기서열이 다른 것으로부터 서로 다른 균주인 것으로 확인할 수 있었다. 따라서 이상의 결과에 기초하여 Probio-054는 기존의 생균활성제 BSA-131과는 다른 락토바실러스 루테리(Lactobacillus reuteri)의 균주로 동정하고, 한국생명공학연구원 유전자은행에 2003년 7월 21일자로 기탁하였다 (수탁번호: KCTC 10499BP).The base sequence of the 16S rRNA gene of Probio-054 is set forth in SEQ ID NO: 1 . In biomolecular taxonomic analysis based on 16S rDNA sequencing, Probio-054 is a strain belonging to the genus Lactobacillus , with 99.5% homology of 16S rDNA, which is the highest in the molecular strain of Lactobacillus reuteri . Strains showing relationships were identified. In addition, Probio-054 is a probiotic (BSA-131) identified by Lactobacillus luteri, which has the characteristics of acid and bile acids, which have been previously announced, and Jang, Young-Hyo et al., Kor J. Appl.Microbiol.Biotechnol. 27, 23-27) (Korean Patent 10-211529-0000) compared to the two nucleotide sequence was found to be different strains from the other. Therefore, based on the above results, Probio-054 was identified as a strain of Lactobacillus reuteri , which is different from the existing probiotic BSA-131, and was deposited on July 21, 2003 with the Korea Biotechnology Research Institute Gene Bank. Accession number: KCTC 10499BP).
상기에서 살펴본 바와 같이, 본 발명의 락토바실러스 속의 새로운 미생물인 락토바실러스 루테리 Probio-054은 로타바이러스 및 병원성 세균을 억제하는 생균활성제인 프로바이오틱스로 동물의 체내 및 장내에서 유해한 성질을 나타내는 로타바이러스 및 여러 종의 병원성 세균을 효과적으로 억제하고 내산성, 내담즙산 기능을 가지고 있어 기존 항생제의 대체용으로 인체 및 각종 가축에게 투여 시 장내 미생물균총의 안정화를 이루어 장내 유해한 미생물의 이상발효에 의하여 발생할 수 있는 증상들을 치료하거나 사전에 예방할 수 있으며 건강상태를 양호하게 하고 가축의 증체량증가, 육질 개선, 산유량증가, 면역력증가 등의 효과를 얻을 수 있다.As described above, Lactobacillus luteri Probio-054, a novel microorganism of the genus Lactobacillus of the present invention, is a probiotic that is a probiotic that inhibits rotavirus and pathogenic bacteria, and rotavirus and various species that exhibit harmful properties in the body and intestines of animals. It effectively inhibits pathogenic bacteria in the body and has acid-resistant and bile-acid function, so it is stabilized in the intestinal microflora when administered to humans and various livestocks as a substitute for existing antibiotics to treat symptoms that may be caused by abnormal fermentation of harmful microorganisms in the intestines. It can be prevented in advance, improve the health and improve the livestock weight gain, improve the quality of meat, increase the output of milk, increase immunity, etc.
<110> Probionic Copr. <120> Acid tolerant probiotic Lactobacillus reuteri Probio-054 that can suppresses the growth of pathogenic microorganisms <130> probio-54 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1481 <212> DNA <213> Lactobacillus reuteri Probio-054 <220> <221> rRNA <222> (1)..(1481) <223> 16rRNA(cDNA) sequence of Lactobacillus reuteri Probio-054 <400> 1 cgccggcggt gtgcctaata catgcaagtc gtacgcactg gcccaactga ttgatggtgc 60 ttgcacctga ttgacgatgg atcaccattg agtggcggac gggtgagtaa cacgtaggta 120 acctgccccg gagcggggga taacatttgg aaacagatgc taataccgca taacaacaaa 180 agccacatgg cttttgtttg aaagatggct ttggctatca ctctgggatg gacctgcggt 240 gcattagcta gttggtaagg taacggctta ccaaggcgat gatgcatatc cgagttgaga 300 gactgatcgg ccacaatgga actgagacac ggtccatact cctacgggag gcagcagtag 360 ggaatcttcc acaatgggcg caagcctgat ggagcaacac cgcgtgagtg aagaagggtt 420 tcggctcgta aagctctgtt gttggagaag aacgtgcgtg agagtaactg ttcacgcagt 480 gacggtatcc aaccagaaag tcacggctaa ctacgtgcca gcagccgcgg taatacgtag 540 gtggcaagcg ttatccggat ttattgggcg taaagcgagc gcaggcggtt gcttaggtct 600 gatgtgaaag ccttcggctt aaccgaagaa gtgcatcgga aaccgggcga cttgagtgca 660 gaagaggaca gtggaactcc atgtgtagcg gtggaatgcg tagatatatg gaagaacacc 720 agtggcgaag gcggctgtct ggtctgcaac tgacgctgag gctcgaaagc atgggtagcg 780 aacaggatta gataccctgg tagtccatgc cgtaaacgat gagtgctagg tgttggaggg 840 tttccgccct tcagtgccgg agctaacgca ttaagcactc cgcctgggga gtacgaccgc 900 aaggttgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 960 ttcgaagcta cgcgaagaac cttaccaggt cttgacatct tgcgctaacc ttagagataa 1020 ggcgttccct tcggggacgc aatgacaggt ggtgcatggt cgtcgtcagc tcgtgtcgtg 1080 agatgttggg ttaagtccyg caacgagcgc aacccttgtt actagttgcc agcattaagt 1140 tgggcactct agtgagactg ccggtgacaa accggaggaa ggtggggacg acgtcagatc 1200 atcatgcccc ttatgacctg ggctacacac gtgctacaat ggacggtaca acgagtcgca 1260 agctcgcgag agtaagctaa tctcttaaag ccgttctcag ttcggactgt aggctgcaac 1320 tcgcctacac gaagtcggaa tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt 1380 tcccgggcct tgtacacacc gcccgtcaca ccatgggagt ttgtaacgcc caaagtcggt 1440 ggcctaacct ttatggaggg agccgcctaa ggcgggacag a 1481<110> Probionic Copr. <120> Acid tolerant probiotic Lactobacillus reuteri Probio-054 that can suppresses the growth of pathogenic microorganisms <130> probio-54 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1481 <212> DNA <213> Lactobacillus reuteri Probio-054 <220> <221> rRNA (222) (1) .. (1481) <223> 16 rRNA (cDNA) sequence of Lactobacillus reuteri Probio-054 <400> 1 cgccggcggt gtgcctaata catgcaagtc gtacgcactg gcccaactga ttgatggtgc 60 ttgcacctga ttgacgatgg atcaccattg agtggcggac gggtgagtaa cacgtaggta 120 acctgccccg gagcggggga taacatttgg aaacagatgc taataccgca taacaacaaa 180 agccacatgg cttttgtttg aaagatggct ttggctatca ctctgggatg gacctgcggt 240 gcattagcta gttggtaagg taacggctta ccaaggcgat gatgcatatc cgagttgaga 300 gactgatcgg ccacaatgga actgagacac ggtccatact cctacgggag gcagcagtag 360 ggaatcttcc acaatgggcg caagcctgat ggagcaacac cgcgtgagtg aagaagggtt 420 tcggctcgta aagctctgtt gttggagaag aacgtgcgtg agagtaactg ttcacgcagt 480 gacggtatcc aaccagaaag tcacggctaa ctacgtgcca gcagccgcgg taatacgtag 540 gtggcaagcg ttatccggat ttattgggcg taaagcgagc gcaggcggtt gcttaggtct 600 gatgtgaaag ccttcggctt aaccgaagaa gtgcatcgga aaccgggcga cttgagtgca 660 gaagaggaca gtggaactcc atgtgtagcg gtggaatgcg tagatatatg gaagaacacc 720 agtggcgaag gcggctgtct ggtctgcaac tgacgctgag gctcgaaagc atgggtagcg 780 aacaggatta gataccctgg tagtccatgc cgtaaacgat gagtgctagg tgttggaggg 840 tttccgccct tcagtgccgg agctaacgca ttaagcactc cgcctgggga gtacgaccgc 900 aaggttgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 960 ttcgaagcta cgcgaagaac cttaccaggt cttgacatct tgcgctaacc ttagagataa 1020 ggcgttccct tcggggacgc aatgacaggt ggtgcatggt cgtcgtcagc tcgtgtcgtg 1080 agatgttggg ttaagtccyg caacgagcgc aacccttgtt actagttgcc agcattaagt 1140 tgggcactct agtgagactg ccggtgacaa accggaggaa ggtggggacg acgtcagatc 1200 atcatgcccc ttatgacctg ggctacacac gtgctacaat ggacggtaca acgagtcgca 1260 agctcgcgag agtaagctaa tctcttaaag ccgttctcag ttcggactgt aggctgcaac 1320 tcgcctacac gaagtcggaa tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt 1380 tcccgggcct tgtacacacc gcccgtcaca ccatgggagt ttgtaacgcc caaagtcggt 1440 ggcctaacct ttatggaggg agccgcctaa ggcgggacag a 1481
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| KR20110092178A (en) * | 2010-02-08 | 2011-08-17 | 영남대학교 산학협력단 | Pediococcus parvulus yml 002 against pathogen of american foulbrood, microbial agent comprising the same, and method for preventing pathogen using the same |
| KR20110092177A (en) * | 2010-02-08 | 2011-08-17 | 영남대학교 산학협력단 | Lactobacillus plantarum yml 001 against pathogen of american foulbrood, microbial agent comprising the same, and method for preventing pathogen using the same |
| KR101675035B1 (en) * | 2015-07-17 | 2016-11-22 | 주식회사 빙그레 | Probiotics comprising Lactobacillus sp., additives for animal feedstuff and animal feedstuff |
| WO2018050650A1 (en) * | 2016-09-16 | 2018-03-22 | Dupont Nutrition Biosciences Aps | Bacteria |
| KR101851297B1 (en) * | 2017-02-13 | 2018-04-23 | 강원대학교 산학협력단 | A novel Lactobacillus reuteri and anti-bacterial use of the same |
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| CN115210360A (en) * | 2021-02-09 | 2022-10-18 | 乐土美森有限公司 | Novel lactobacillus reuteri LM1071 strain derived from breast milk, and composition for alleviating premenstrual syndrome comprising the same or culture thereof |
| CN112940970B (en) * | 2021-02-09 | 2023-01-24 | 中国科学院近代物理研究所 | Intestinal probiotics and application thereof in treating tumors and replacing antibiotics |
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