KR102688162B1 - Strain isolated from calf feces, and a preparation for preventing and alleviating diarrhea containing the same - Google Patents
Strain isolated from calf feces, and a preparation for preventing and alleviating diarrhea containing the same Download PDFInfo
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- 244000309466 calf Species 0.000 title claims abstract description 82
- 206010012735 Diarrhoea Diseases 0.000 title claims abstract description 56
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- 238000002360 preparation method Methods 0.000 title claims description 8
- 241000186713 Lactobacillus amylovorus Species 0.000 claims abstract description 51
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- 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
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/12—Antidiarrhoeals
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- 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
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Abstract
본 발명은 송아지 분변으로부터 분리된 균주와, 이를 포함하는 설사증 예방 및 완화 제제에 관한 것으로, 본 발명에 따르면, 락토바실러스 아밀로보루스(Lactobacillus amylovorus) 1394N20(기탁번호 KCCM 12999P)를 포함하는 송아지 분변으로부터 분리된 균주를 제공할 수 있다.
또한 송아지 분변으로부터 분리된 균주를 포함하는 설사증 예방 및 완화 제제를 제공할 수 있다.The present invention relates to a strain isolated from calf feces and a diarrhea prevention and alleviation agent containing the same. According to the present invention, from calf feces containing Lactobacillus amylovorus 1394N20 (accession number KCCM 12999P) Isolated strains can be provided.
Additionally, it is possible to provide a diarrhea prevention and alleviation agent containing a strain isolated from calf feces.
Description
본 발명은 송아지 분변으로부터 분리된 균주와, 이를 포함하는 설사증 예방 및 완화 제제에 관한 것으로, 더욱 자세하게는 건강한 한우 송아지, 즉 설사증에 걸리지 않은 한우 송아지의 분변으로부터 분리된 유익균주의 제제를 송아지에게 섭취시킴으로써, 장내 유익균을 증가시키고 설사증의 예방 및 완화에 기여할 수 있는 송아지 분변으로부터 분리된 균주와, 이를 포함하는 설사증 예방 및 완화 제제에 관한 것이다.The present invention relates to a strain isolated from calf feces and a diarrhea prevention and alleviation agent containing the same. More specifically, the present invention relates to a strain isolated from calf feces, and more specifically, to a calf by ingesting a preparation of beneficial bacteria isolated from the feces of a healthy Korean beef calf, that is, a Korean beef calf not suffering from diarrhea. , relates to a strain isolated from calf feces that can increase intestinal beneficial bacteria and contribute to the prevention and alleviation of diarrhea, and a diarrhea prevention and alleviation preparation containing the same.
락토바실러스 균은 체내에 들어가 장 건강에 좋은 효과를 주는 프로바이오틱스의 종류이다. 이 균은 당류를 발효하여 에너지를 획득하여 다량의 유산을 생성해 건강에 도움을 줄 수 있다.Lactobacillus is a type of probiotic that enters the body and has beneficial effects on intestinal health. This fungus ferments sugars to obtain energy and produces large amounts of lactic acid, which can be beneficial to health.
한편, 소를 키우는 축사 농가가 힘들어하는 부분 중 하나가 질병으로 인해 소나 한우 송아지가 폐사하는 경우이다. Meanwhile, one of the difficulties faced by cattle farms is when cattle or Korean beef calves die due to disease.
특히, 한우 송아지의 설사병은 소 질병 중 가장 많이 발생하며, 1개월 이내 사망률이 매우 높게 나타나, 이에 따른 경제적 피해가 큰 상황이다.In particular, diarrheal disease in Korean beef calves is the most common among cattle diseases, and the mortality rate within one month is very high, resulting in significant economic damage.
상기와 같은 문제를 해결하기 위해 항생제와 같은 약물 사용이 늘어나고 있는 추세이고, 이에 항생제 연구와 개발이 지속되고 있다.To solve the above problems, the use of drugs such as antibiotics is increasing, and antibiotic research and development continues.
그러나 항생제의 사용은 일시적일 수밖에 없으며, 가축의 장내에 서식하고 있는 미생물들 중 내성이 강한 경우에는 큰 효과를 기대하기 어렵고, 반대로 항생제의 성분이 가축 체내에 남는 문제와 그에 따라 사람에게도 영향을 미치게 되는 문제가 있었다. 이 때문에 선진국에서는 항생제의 남용을 적극적으로 차단하고 있고, 앞으로도 그 규제는 강화될 전망이다.However, the use of antibiotics is bound to be temporary, and it is difficult to expect a significant effect if the microorganisms living in the intestines of livestock are highly resistant, and on the contrary, the ingredients of antibiotics can cause problems that remain in the livestock's body and consequently affect humans. There was a problem. For this reason, developed countries are actively preventing the abuse of antibiotics, and regulations are expected to be strengthened in the future.
이 흐름에 따라 천연 항생제, 항생물질과 같은 항생제 대체제들이 떠오르고 있다. Following this trend, antibiotic alternatives such as natural antibiotics and antibiotics are emerging.
항생제 대체제 중 천연 항생제를 사용하게 될 경우, 다음과 같은 조건을 충족해야 그 효과를 제대로 볼 수 있다. 첫째, 정상 소화기관에 대해 비병원성일 것, 둘째, 장내에서 빠르게 성장할 수 있고, 대사활동을 해야하는 것, 셋째, 소화기관에서 집락을 잘 형성하고 유해미생물을 억제할 수 있는 항생물질을 충분히 분비할 수 있을 것이다. When using natural antibiotics as an alternative to antibiotics, the following conditions must be met for the effectiveness to be properly seen. First, it must be non-pathogenic to the normal digestive tract; second, it must be able to grow rapidly in the intestines and have metabolic activity; and third, it must be able to form colonies well in the digestive tract and secrete sufficient antibiotics to suppress harmful microorganisms. There will be.
상기 조건들을 종합해보았을 때, 가장 적합하고 많이 이용되고 있는 미생물이 바로 유산균이다.Considering the above conditions, the most suitable and widely used microorganism is lactic acid bacteria.
하지만 현재는 소의 특이성을 고려하지 않고, 통상적으로 인체나 다른 종들을 통해 분리된 유산균을 사용하고 있다. 이와 같은 유산균을 통해서는 제대로 된 효과를 보기 어려운 상황이다. 이에 소의 설사병에 맞춘 유산균 개발이 필요한 실정이다.However, currently, lactic acid bacteria isolated from humans or other species are usually used without considering the specificity of cows. It is difficult to see proper effects through such lactic acid bacteria. Accordingly, there is a need to develop lactic acid bacteria tailored to diarrheal diseases in cattle.
상기와 같은 문제를 해결하고자, 본 발명은 건강한 한우 송아지, 즉 설사증에 걸리지 않은 한우 송아지의 분변으로부터 분리된 유익균주의 제제를 송아지에게 섭취시킴으로써, 장내 유익균을 증가시키고 설사증의 예방 및 완화에 기여할 수 있는 송아지 분변으로부터 분리된 균주와, 이를 포함하는 설사증 예방 및 완화 제제를 제공하는데 목적이 있다.In order to solve the above problems, the present invention provides calves with a preparation of beneficial bacteria isolated from the feces of healthy Korean beef calves, that is, Korean cattle calves not suffering from diarrhea, which can increase intestinal beneficial bacteria and contribute to the prevention and alleviation of diarrhea. The purpose is to provide strains isolated from calf feces and preparations for preventing and alleviating diarrhea containing them.
상기와 같은 과제를 해결하기 위하여, 본 발명의 실시예에 따른 락토바실러스 아밀로보루스(Lactobacillus amylovorus) 1394N20(기탁번호 KCCM 12999P)를 포함하는 송아지 분변으로부터 분리된 균주를 제공할 수 있다.In order to solve the above problems, a strain isolated from calf feces containing Lactobacillus amylovorus 1394N20 (accession number KCCM 12999P) according to an embodiment of the present invention can be provided.
또한, 락토바실러스 루테리(Lactobacillus reuteri) 1429C30(기탁번호 KCCM 13000P), 락토바실러스 존소니(Lactobacillus johnsonii) 7409N31(기탁번호 KCCM 13026P)를 더 포함할 수 있다.In addition, it may further include Lactobacillus reuteri 1429C30 (accession number KCCM 13000P) and Lactobacillus johnsonii 7409N31 (accession number KCCM 13026P).
또한 본 발명의 실시예에 따른 송아지 분변으로부터 분리된 균주를 포함하는 설사증 예방 및 완화 제제를 제공할 수 있다.In addition, it is possible to provide a diarrhea prevention and alleviation agent containing a strain isolated from calf feces according to an embodiment of the present invention.
또한 설사증 예방 및 완화 제제는 장내 유익균을 증가시키는 것을 특징으로 한다.In addition, diarrhea prevention and alleviation agents are characterized by increasing beneficial intestinal bacteria.
상기와 같은 본 발명의 실시예에 따른 송아지 분변으로부터 분리된 균주와, 이를 포함하는 설사증 예방 및 완화 제제는 건강한 한우 송아지, 즉 설사증에 걸리지 않은 한우 송아지의 분변으로부터 분리된 균주를 제제로 송아지에게 섭취시킴으로써, 장내 유익균을 증가시키고 설사증과 관련된 장내 미생물을 조절하도록 하여, 설사증의 예방 및 완화에 기여할 수 있다.The strain isolated from calf feces according to the embodiment of the present invention as described above, and the diarrhea prevention and alleviation agent containing the same, are administered to calves as a preparation made of strains isolated from the feces of healthy Korean beef calves, that is, Korean beef calves not suffering from diarrhea. By doing so, it can contribute to the prevention and alleviation of diarrhea by increasing beneficial intestinal bacteria and controlling intestinal microorganisms related to diarrhea.
또한 송아지의 분변으로부터 분리된 2종 이상의 균주를 혼합하여 사용함으로써, 단독으로 사용하는 경우 보다 장내 미생물의 다양성을 증가시키고, 유익균을 증가시킬 수 있다.In addition, by using a mixture of two or more strains isolated from calf feces, the diversity of intestinal microorganisms can be increased and the number of beneficial bacteria can be increased compared to when used alone.
또한 송아지의 분변으로부터 분리된 균주를 활용함으로써, 송아지에게 섭취 안전성이 우수할 수 있고 효과적 일 수 있다.Additionally, by using strains isolated from calf feces, the ingestion safety for calves can be excellent and it can be effective.
또한 항생제 대체제로 사용될 수 있으며, 설사증으로 인한 송아지의 폐사율을 줄일 수 있어, 결과적으로 축사 농가들의 경제적 피해를 줄이는 효과를 나타낼 수 있다.It can also be used as a replacement for antibiotics and can reduce the mortality rate of calves due to diarrhea, which can ultimately reduce the economic damage to livestock farms.
도 1은 정상 송아지와 설사증 송아지의 분변에 존재하는 DNA를 Alpha-diversity 분석한 결과를 나타낸 그래프.
도 2는 정상 송아지와 설사증 송아지의 분변에 존재하는 DNA를 문(Phylum) 수준에서 분석한 결과를 나타낸 그래프.
도 3은 정상 송아지와 설사증 송아지의 분변에 존재하는 DNA를 종(Species) 수준에서 분석한 결과를 나타낸 그래프.
도 4는 정상 송아지의 분변으로부터 분리된 균주를 colony PCR 검사한 결과 사진.
도 5는 선별된 L. amylovorus 1394N20, L. reuteri 1429C30, L. johnsonii 7409N31의 동정 분석 결과 데이터.
도 6a 내지 도 6c는 L. amylovorus 1394N20, L. reuteri 1429C30, L. johnsonii 7409N31의 16S rDNA 유전자 서열목록을 나타낸 데이터.
도 7a 내지 도 7c는 L. amylovorus 1394N20, L. reuteri 1429C30, L. johnsonii 7409N31의 항생제 내성 평가 결과 사진.
도 8a 및 도 8b는 L. amylovorus 1394N20, L. reuteri 1429C30, L. johnsonii 7409N31의 용혈 평가 결과 사진.
도 9는 L. amylovorus 1394N20, L. reuteri 1429C30, L. johnsonii 7409N31의 Urease 활성 및 젤라틴 액화 평가 결과 사진.
도 10은 Lactobacillus amylovorus 1394N20 투여 전/후에 따른 Alpha-diversity 분석 결과를 나타낸 그래프.
도 11은 Lactobacillus amylovorus 1394N20 투여 전/후에 따른 문(Phylum) 수준의장내미생물충 분석 결과 그래프.
도 12는 Lactobacillus amylovorus 1394N20 투여 전/후에 따른 종(Species) 수준의 장내미생물총 분석 결과 그래프.
도 13은 Lactobacillus amylovorus 1394N20 투여 후/투여종료 5일 후에 따른 종(Species) 수준의 장내미생물총 분석 결과 그래프.
도 14는 혼합균주 투여 전/후에 따른 Alpha-diversity 분석 결과를 나타낸 그래프.
도 15는 혼합균주 투여 전/후에 따른 문(Phylum) 수준의 장내미생물충 분석 결과 그래프.
도 16은 혼합균주 투여 전/후에 따른 속(Genus) 수준의 장내미생물충 분석 결과 그래프.
도 17은 혼합균주 투여 전/후에 따른 종(Species) 수준의 장내미생물총 분석 결과 그래프.
도 18은 실험군과 대조군의 병원체 검사 결과를 나타낸 표.
도 19a 내지 도 19d는 실험군과 대조군의 HCO3 -, BE(Base Excess), pH 및 SID를 측정한 일령별 평균 그래프.Figure 1 is a graph showing the results of alpha-diversity analysis of DNA present in the feces of normal calves and calves with diarrhea.
Figure 2 is a graph showing the results of analyzing DNA present in the feces of normal calves and calves with diarrhea at the phylum level.
Figure 3 is a graph showing the results of analyzing DNA present in the feces of normal calves and calves with diarrhea at the species level.
Figure 4 is a photograph of the colony PCR test results of a strain isolated from the feces of a normal calf.
Figure 5 shows the identification analysis result data of selected L. amylovorus 1394N20, L. reuteri 1429C30, and L. johnsonii 7409N31.
Figures 6a to 6c are data showing the 16S rDNA gene sequence list of L. amylovorus 1394N20, L. reuteri 1429C30, and L. johnsonii 7409N31.
Figures 7a to 7c are photographs of antibiotic resistance evaluation results of L. amylovorus 1394N20, L. reuteri 1429C30, and L. johnsonii 7409N31.
Figures 8a and 8b are pictures of the hemolysis evaluation results of L. amylovorus 1394N20, L. reuteri 1429C30, and L. johnsonii 7409N31.
Figure 9 is a photograph of the urease activity and gelatin liquefaction evaluation results of L. amylovorus 1394N20, L. reuteri 1429C30, and L. johnsonii 7409N31.
Figure 10 is a graph showing the results of alpha-diversity analysis before and after administration of Lactobacillus amylovorus 1394N20.
Figure 11 is a graph showing the results of intestinal microbial analysis at the phylum level before and after administration of Lactobacillus amylovorus 1394N20.
Figure 12 is a graph showing the results of intestinal microbiota analysis at the species level before and after administration of Lactobacillus amylovorus 1394N20.
Figure 13 is a graph showing the results of intestinal microbiota analysis at the species level after administration of Lactobacillus amylovorus 1394N20/5 days after completion of administration.
Figure 14 is a graph showing the results of alpha-diversity analysis before and after administration of mixed strains.
Figure 15 is a graph showing the results of intestinal microbial analysis at the phylum level before and after administration of mixed strains.
Figure 16 is a graph showing the results of intestinal microbial analysis at the genus level before and after administration of mixed strains.
Figure 17 is a graph showing the results of intestinal microbiota analysis at the species level before and after administration of mixed strains.
Figure 18 is a table showing the pathogen test results of the experimental and control groups.
Figures 19a to 19d are average graphs by day measuring HCO 3 - , BE (Base Excess), pH, and SID of the experimental group and the control group.
본 발명에서 사용되는 단수의 표현은 문맥상 명백하게 다르게 뜻하지 않는 한, 복수의 표현을 포함한다. 또한, 이하에서 기재되는 "포함하다", "구비하다" 또는 "가지다" 등의 용어는 명세서상에 기재된 특징, 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것이 존재함을 지정하려는 것으로 해석되어야 하며, 하나 또는 그 이상의 다른 특징들이나, 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것들의 존재 또는 부가 가능성을 미리 배제하지 않는 것으로 이해되어야 한다.As used herein, singular expressions include plural expressions, unless the context clearly dictates otherwise. In addition, terms such as “comprise,” “provide,” or “have” used below are intended to designate the presence of features, numbers, steps, operations, components, parts, or a combination thereof described in the specification. It should be construed and understood as not precluding the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts or combinations thereof.
여기서, 반복되는 설명, 본 발명의 요지를 불필요하게 흐릴 수 있는 공지 기능, 및 구성에 대한 상세한 설명은 생략한다. 본 발명의 실시형태는 당 업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해서 제공되는 것이다.Here, repeated descriptions, known functions that may unnecessarily obscure the gist of the present invention, and detailed descriptions of configurations are omitted. Embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.
본 발명은 건강한 한우 송아지로부터 분리되어 장내 유익균 증가 효과를 가지고 설사증 예방 및 치료가 가능한 락토바실러스 아밀로보루스(Lactobacillus amylovorus) 1394N20(기탁번호 KCCM 12999P), 락토바실러스 루테리(Lactobacillus reuteri) 1429C30(기탁번호 KCCM 13000P), 락토바실러스 존소니(Lactobacillus johnsonii) 7409N31(기탁번호 KCCM 13026P) 균주 중 1종 이상을 포함하는 송아지 분변으로부터 분리된 균주 및 이를 포함한 설사증 예방 및 완화 제제를 제공하고자 한다.The present invention relates to Lactobacillus amylovorus 1394N20 (Accession number KCCM 12999P) and Lactobacillus reuteri 1429C30 (Accession number KCCM), which are isolated from healthy Korean beef calves and have the effect of increasing intestinal beneficial bacteria and can prevent and treat diarrhea. 13000P), Lactobacillus johnsonii ( Lactobacillus johnsonii ) 7409N31 (accession number KCCM 13026P) strains isolated from calf feces, and preparations for preventing and alleviating diarrhea containing the same.
이러한 본 발명의 락토바실러스 아밀로보루스(Lactobacillus amylovorus) 1394N20(기탁번호 KCCM 12999P), 락토바실러스 루테리(Lactobacillus reuteri) 1429C30(기탁번호 KCCM 13000P) 및 락토바실러스 존소니(Lactobacillus johnsonii) 7409N31(기탁번호 KCCM 13026P)는 본 발명에서 최초로 분리 동정된 신규한 균주로, 송아지 분변에서 내생하고 있는 미생물을 배양법에 기초하여 처음으로 분리 동정하였다.The present invention includes Lactobacillus amylovorus 1394N20 (accession number KCCM 12999P), Lactobacillus reuteri 1429C30 (accession number KCCM 13000P), and Lactobacillus johnsonii 7409N31 (accession number KCCM 13026P ) is a novel strain isolated and identified for the first time in the present invention, and was the first to isolate and identify endogenous microorganisms in calf feces based on a culture method.
이하, 한우송아지의 분변으로부터 신규주를 동정하기 위한 방법 및 신균주의 설사증 예방 및 치료를 위한 장내 유익균 증가 효과, 설사증 관련 장내 미생물 조절 효과에 대하여 실시예를 중심으로 설명하다.Hereinafter, the method for identifying a new strain from the feces of Korean beef calves, the effect of the new strain on increasing intestinal beneficial bacteria for preventing and treating diarrhea, and the effect of controlling intestinal microorganisms related to diarrhea will be explained, focusing on examples.
1. 장내 유용 미생물 후보군 선정1. Selection of beneficial intestinal microorganism candidates
설사증 개선을 위한 유용 균주를 분리하기 위하여, 정상 송아지와 설사증 송아지의 장내 미생물 군집 분석을 통해 설사증 유무에 따른 장내 미생물 군집 변화를 분석하여, 장내 유용 미생물 후보군을 선정하였다. 설사증에 따라 송아지의 장내 미생물총에 변화가 있음을 확인한 바 있어, 이를 기반으로 진행하였다(한국등록특허 제10-2282884호).In order to isolate useful strains for improving diarrhea, we analyzed the intestinal microbial communities of normal calves and calves with diarrhea and analyzed changes in the intestinal microbial communities according to the presence or absence of diarrhea, and selected useful intestinal microorganism candidates. It has been confirmed that there are changes in the intestinal microflora of calves due to diarrhea, so the process was conducted based on this (Korean Patent No. 10-2282884).
정상 송아지와 설사증 송아지로부터 각각 분변을 수집하고, 수집된 분변에서 DNA를 추출한 후, Alpha-diversity 및 Taxonomic composition를 진행하였다.Feces were collected from normal calves and calves with diarrhea, DNA was extracted from the collected feces, and alpha-diversity and taxonomic composition were performed.
그 결과는 도 1 내지 도 3과 같다.The results are as shown in Figures 1 to 3.
1) Alpha-diversity1) Alpha-diversity
도 1은 정상 송아지와 설사증 송아지의 분변에 존재하는 DNA를 Alpha-diversity 분석한 결과를 나타낸 그래프이다.Figure 1 is a graph showing the results of alpha-diversity analysis of DNA present in the feces of normal calves and calves with diarrhea.
Richness 지수인 Ace, Chao1, JakKnife는 정상 송아지 대비 설사증 송아지에서 유의적으로 감소하였고, evenness 지수인 Shannon는 정상 송아지 대비 설사증 송아지에서 유의적으로 감소, Simpson는 증가하는 것을 확인할 수 있었다.The richness indexes Ace, Chao1, and JakKnife were significantly decreased in diarrheal calves compared to normal calves, and the evenness index Shannon was significantly decreased in diarrheal calves compared to normal calves, and Simpson was confirmed to be increased.
2) Taxonomic composition2) Taxonomic composition
도 2는 정상 송아지와 설사증 송아지의 분변에 존재하는 DNA를 문(Phylum) 수준에서 분석한 결과를 나타낸 그래프이며, 도 3은 정상 송아지와 설사증 송아지의 분변에 존재하는 DNA를 종(Species) 수준에서 분석한 결과를 나타낸 그래프이다.Figure 2 is a graph showing the results of analyzing the DNA present in the feces of normal calves and calves with diarrhea at the phylum level, and Figure 3 is a graph showing the results of analyzing the DNA present in the feces of normal calves and calves with diarrhea at the species level. This is a graph showing the analysis results.
Taxonomic composition 분석을 통해 문(phylum) 수준에서 정상 송아지와 설사증 송아지 간의 relative abundance를 비교 분석한 결과, 정상 송아지 대비 설사증 송아지에서 Firmicutes, Fusobacteria의 증가 및 Bacteroidetes의 감소를 확인할 수 있었다.As a result of comparing the relative abundance between normal calves and diarrheal calves at the phylum level through taxonomic composition analysis, it was confirmed that Firmicutes and Fusobacteria increased and Bacteroidetes decreased in diarrheal calves compared to normal calves.
Taxonomic composition 분석을 통해 종(Species) 수준에서 정상 송아지와 설사증 송아지 간의 relative abundance를 비교 분석한 결과, 정상 송아지 대비 설사증 송아지에서 Faecalibacterium prausnitzii group, Lactobacillus reuteri group, Lactobaillus gasseri group, Lactobacillus plantarum group 의 감소, Clostridium perfringens, Escherichia coli의 증가를 확인할 수 있었다.As a result of comparing the relative abundance between normal calves and diarrheal calves at the species level through taxonomic composition analysis, there was a decrease in Faecalibacterium prausnitzii group, Lactobacillus reuteri group, Lactobaillus gasseri group, and Lactobacillus plantarum group in diarrheal calves compared to normal calves, and Clostridium An increase in perfringens and Escherichia coli was confirmed.
이에 설사증에 대한 장내 유용 미생물 후보군으로 락토바실러스(Lactobacillus) 속을 선정하였고, 다양한 종류의 락토바실러스(Lactobacillus)를 분리하기 위하여, 정상 송아지에서 상대빈도가 10% 이상인 Lactobacillus amylovorus, Lactobaillus gasseri, Lactobacillus reuteri를 주요 분리대상으로 선정하였다.Accordingly, the genus Lactobacillus was selected as a candidate group of beneficial intestinal microorganisms for diarrhea, and in order to isolate various types of Lactobacillus , Lactobacillus amylovorus, Lactobaillus gasseri, and Lactobacillus reuteri, which have a relative frequency of more than 10% in normal calves, were selected. It was selected as the main separation target.
2. 후보군 선정에 따른 배양조건 설정2. Setting culture conditions according to candidate selection
장내 유용 미생물 후보군으로 락토바실러스(Lactobacillus) 속을 선정함에 따라, 균 분리를 위햐여 주요 분리대상의 배양 조건을 확인하였다. 주요 분리대상의 배양 조건은 하기 표 1과 같다.As the genus Lactobacillus was selected as a candidate group of useful intestinal microorganisms, the culture conditions for the main isolation target were confirmed for bacterial isolation. The culture conditions for the main isolation targets are shown in Table 1 below.
상기 표 1의 배양 조건을 참고하여, 균 분리 시 배양 조건을 37℃에서 CO2 gas pack 또는 5% CO2 인큐베이터 또는 CO2 없이 배양하는 것으로 설정하였다.Referring to the culture conditions in Table 1 above, the culture conditions for bacterial isolation were set to culture at 37°C in a CO 2 gas pack or 5% CO 2 incubator or without CO 2 .
3. 균주 분리 3. Strain isolation
장내 유용 미생물 후보군으로 선정된 락토바실러스(Lactobacillus) 속에 해당하는 균주를 분리하기 위하여, 하기 표 2와 같은 설사 증세가 없는 정상 송아지 5마리(일령 30일 이내)로부터 분변을 채취하고 당일 배양을 진행하여 균주를 분리하였다.In order to isolate strains belonging to the Lactobacillus genus, which were selected as useful intestinal microorganism candidates, feces were collected from five normal calves (less than 30 days old) without diarrhea as shown in Table 2 below, and culture was performed on the same day. The strain was isolated.
이때, 호기적 조건에서 다양한 장내 미생물을 분리하기 위하여 37℃ CO2 조건(CO2 gas pack, 5% CO2 인큐베이터, CO2 없음)을 달리하여 각각 배양하고 균주를 분리하였다. At this time, in order to isolate various intestinal microorganisms under aerobic conditions, they were cultured under different CO 2 conditions at 37°C (CO 2 gas pack, 5% CO 2 incubator, no CO 2 ) and the strains were isolated.
또한 순수한 균주를 분리하기 위해, 콜로니(colony)가 단일 콜로니로 순수 분리될 때까지 콜로니(colony)를 분리한 후 배양하는 과정을 반복하여 수행하였다.Additionally, in order to isolate pure strains, the process of separating colonies and then culturing them was repeated until the colonies were purely isolated into single colonies.
균주를 분리한 결과, 채취한 분변에서 총 99 균주가 분리되었다.As a result of strain isolation, a total of 99 strains were isolated from the collected feces.
4. 균주 동정4. Strain identification
정상 송아지의 분변으로부터 분리된 균주를 colony PCR을 통해 락토바실러스가 검출되는지 1차적으로 확인한 다음, 16S rRNA 염기서열 분석을 통해 균주 동정을 실시하였다. 그 결과는 도 4 및 표 3과 같다.The strain isolated from the feces of a normal calf was first confirmed to be Lactobacillus through colony PCR, and then the strain was identified through 16S rRNA base sequence analysis. The results are shown in Figure 4 and Table 3.
도 4는 정상 송아지의 분변으로부터 분리된 균주를 colony PCR 검사한 결과 사진이다.Figure 4 is a photograph of the colony PCR test results of a strain isolated from the feces of a normal calf.
도 4를 보면 알 수 있듯이, 정상 송아지의 분변으로부터 분리된 균주는 락토바실러스(Lactobacillus) 속에 속하는 것을 확인할 수 있었다. 구체적으로, Lactobacillus amylovorus(A), Lactobaillus gasseri(G), Lactobacillus reuteri(R)가 확인되는 것을 확인할 수 있었다.As can be seen in Figure 4, it was confirmed that the strain isolated from the feces of a normal calf belonged to the genus Lactobacillus . Specifically, it was confirmed that Lactobacillus amylovorus (A), Lactobaillus gasseri (G), and Lactobacillus reuteri (R) were identified.
상기 표 3에 나타난 바와 같이, 동정된 총 99균주 중 중복되는 균주를 제외하면, 균주는 총 8종으로 확인되었다.As shown in Table 3, a total of 8 strains were identified among the 99 strains identified, excluding overlapping strains.
구체적으로, L. reuteri, L. johnsonii, L. amylovorus, L. oris, L. vaginalis, L. mucosae, L. salivarius, L. faecis의 8 종의 Lactobaillus 균주를 확보하였다.Specifically, eight Lactobaillus strains, including L. reuteri , L. johnsonii , L. amylovorus, L. oris, L. vaginalis, L. mucosae, L. salivarius, and L. faecis, were obtained.
여기서, L. faecis의 경우 코로나 바이러스 감염에 따라 증가하기 때문에 제외하였다.Here, L. faecis was excluded because it increases with coronavirus infection.
한편, 주요 분리대상으로 선정하였던 L. gasseri는 1차적으로 colony PCR을 통해 확인되었지만, 실제 16s rRNA 시퀀싱으로 최종 확인하였을 때 L. johnsonii로 확인되었다.Meanwhile, L. gasseri , which was selected as the main isolation target, was initially confirmed through colony PCR, but was confirmed to be L. johnsonii when finally confirmed by actual 16s rRNA sequencing.
이는 현재 Microbiota 분석시 사용되는 MTP Taxonomy 상에서 Lactobacillus의 경우 하기 표 4와 같이 대다수 group으로 표기되고 있는데, Lactobacillus gasseri group에는 L. johnsonii가 포함되어 있는 것으로 확인되어 정상 송아지에 존재하는 Lactobacillus gasseri group의 다수는 L. johnsonii인 것으로 추정된다.In the MTP Taxonomy currently used for microbiota analysis, Lactobacillus is indicated as the majority group as shown in Table 4 below. It was confirmed that the Lactobacillus gasseri group includes L. johnsonii, so the majority of the Lactobacillus gasseri group present in normal calves is It is believed to be L. johnsonii .
5. 최종 균주 선별5. Final strain selection
상기와 같은 동정 결과에 따라, 분리 동정된 L. reuteri, L. johnsonii, L. amylovorus, L. oris, L. vaginalis, L. mucosae, L. salivarius 균주 중 주요 분리대상으로 선정했던 L. amylovorus, L. reuteri를 최종 균주로 선별하였고, 정상 송아지에서 주요 분리대상으로 선정하였던 L. gasseri가 L. johnsonii로 확인됨에 따라 L. johnsonii를 최종 균주로 선별하였다.According to the above identification results, among the isolated and identified L. reuteri , L. johnsonii , L. amylovorus, L. oris, L. vaginalis, L. mucosae, and L. salivarius strains, L. amylovorus was selected as the main isolation target, L. reuteri was selected as the final strain, and as L. gasseri, which had been selected as the main target of isolation from normal calves, was confirmed to be L. johnsonii, L. johnsonii was selected as the final strain.
선발된 L. amylovorus, L. reuteri, L. johnsonii를 16S rRNA 염기서열 분석을 통해 다시 한번 검증하였고, 그 결과는 도 5 및 도 6a 내지 도 6c와 같다.The selected L. amylovorus , L. reuteri , and L. johnsonii were verified again through 16S rRNA base sequence analysis, and the results are shown in Figures 5 and 6a to 6c.
선별된 L. amylovorus는 Lactobacillus amylovorus 1394N20 균주로 명명되었고, 한국미생물보존센터에 2021년 06월 01일자로 기탁하였으며, 기탁번호는 KCCM 12999P이다.The selected L. amylovorus was named Lactobacillus amylovorus 1394N20 strain and was deposited at the Korea Microbiological Conservation Center on June 1, 2021, and the deposit number is KCCM 12999P.
선별된 L. reuteri는 Lactobacillus reuteri 1429C30 균주로 명명되었고, 한국미생물보존센터에 2021년 06월 01일자로 기탁하였으며, 기탁번호는 KCCM 13000P이다.The selected L. reuteri was named Lactobacillus reuteri 1429C30 strain and was deposited at the Korea Microorganism Conservation Center on June 1, 2021, and the deposit number is KCCM 13000P.
선별된 L. johnsonii는 Lactobacillus johnsonii 7409N31 균주로 명명되었고, 한국미생물보존센터에 2021년 08월 03일자로 기탁하였으며, 기탁번호는 KCCM 13026P이다.The selected L. johnsonii was named Lactobacillus johnsonii 7409N31 strain and was deposited with the Korea Microbial Conservation Center on August 3, 2021, and the deposit number is KCCM 13026P.
6. 최종 균주 안전성 평가6. Final strain safety evaluation
최종적으로 선발된 Lactobacillus amylovorus 1394N20, Lactobacillus reuteri 1429C30, Lactobacillus johnsonii 7409N31 균주의 안전성을 확인하기 위하여, 항생제내성, 용혈성, Urease 활성 및 젤라틴 액화성을 평가하였다.To confirm the safety of the finally selected strains of Lactobacillus amylovorus 1394N20, Lactobacillus reuteri 1429C30, and Lactobacillus johnsonii 7409N31, antibiotic resistance, hemolysis, urease activity, and gelatin liquefaction were evaluated.
1) 항생제내성 평가1) Antibiotic resistance evaluation
Lactobacillus amylovorus 1394N20, Lactobacillus reuteri 1429C30, Lactobacillus johnsonii 7409N31 균주에 대하여 항생제내성 시험을 MRS 배지를 이용하여 E-test method에 의해 수행하였다. 이때, ampicillin (AM, 0.016-256 μg), chloramphenicol (C, 0.016-256 μg), ciprofloxacin (CIP, 0.016-32 μg), erythromycin (E, 0.016-256 μg), gentamicin (GM, 0.016-256 μg), kanamycin (K, 0.016-256 μg), penicillin (P, 0.016-256 μg), rifampin (RA, 0.016-32 μg), tetracycline (TE, 0.016-256 μg), vancomycin (VA, 0.016-256 μg)에 대하여 평가하였다.Antibiotic resistance testing was performed on Lactobacillus amylovorus 1394N20, Lactobacillus reuteri 1429C30, and Lactobacillus johnsonii 7409N31 using MRS medium using the E-test method. At this time, ampicillin (AM, 0.016-256 μg), chloramphenicol (C, 0.016-256 μg), ciprofloxacin (CIP, 0.016-32 μg), erythromycin (E, 0.016-256 μg), gentamicin (GM, 0.016-256 μg) ), kanamycin (K, 0.016-256 μg), penicillin (P, 0.016-256 μg), rifampin (RA, 0.016-32 μg), tetracycline (TE, 0.016-256 μg), vancomycin (VA, 0.016-256 μg) ) was evaluated.
그 결과는 도 7a 내지 도 7c 및 하기 표 5와 같다.The results are shown in Figures 7A to 7C and Table 5 below.
도 7a 내지 도 7c 및 표 5를 보면 알 수 있듯이, L. amylovorus 1394N20, L. reuteri 1429C30, L. johnsonii 7409N31 모두 대부분의 항생제에 대해 내성이 없는 것을 확인할 수 있었다.As can be seen from Figures 7a to 7c and Table 5, it was confirmed that L. amylovorus 1394N20, L. reuteri 1429C30, and L. johnsonii 7409N31 were all non-resistant to most antibiotics.
2) 용혈성 평가2) Hemolytic evaluation
용혈성 시험은 Lactobacillus amylovorus 1394N20, Lactobacillus reuteri 1429C30, Lactobacillus johnsonii 7409N31 균주를 blood가 첨가된 용혈성 배지에 streaking 한 후 37℃, 24시간 동안 배양하여 균체 주위의 투명환 생성여부로 용혈성을 판단하였다.For the hemolytic test, the strains Lactobacillus amylovorus 1394N20, Lactobacillus reuteri 1429C30, and Lactobacillus johnsonii 7409N31 were streaked on a hemolytic medium supplemented with blood, then cultured at 37°C for 24 hours, and hemolyticity was judged by whether or not a transparent ring was formed around the bacterial cells.
적혈구의 완전한 용혈을 일으키는 β-hemolysis 검사를 수행하였고, 그 결과는 도 8a 및 도 8b와 같다.A β-hemolysis test that causes complete hemolysis of red blood cells was performed, and the results are shown in Figures 8a and 8b.
도 8a 및 도 8b를 보면 알 수 있듯이, Lactobacillus amylovorus 1394N20, Lactobacillus reuteri 1429C30, Lactobacillus johnsonii 7409N31 균주는 용혈성이 나타나지 않는 것을 확인하였다.As can be seen in FIGS. 8A and 8B, it was confirmed that Lactobacillus amylovorus 1394N20, Lactobacillus reuteri 1429C30, and Lactobacillus johnsonii 7409N31 strains did not exhibit hemolysis.
3) Urease 활성 및 젤라틴 액화성 평가3) Evaluation of urease activity and gelatin liquefaction
Lactobacillus amylovorus 1394N20, Lactobacillus reuteri 1429C30, Lactobacillus johnsonii 7409N31 균주에 대한 Urease 활성 및 젤라틴 액화성 평가를 수행하였고, 그 결과는 도 9와 같다.Urease activity and gelatin liquefaction were evaluated for Lactobacillus amylovorus 1394N20, Lactobacillus reuteri 1429C30, and Lactobacillus johnsonii 7409N31 strains, and the results are shown in Figure 9.
도 9를 보면 알 수 있듯이, Lactobacillus amylovorus 1394N20, Lactobacillus reuteri 1429C30, Lactobacillus johnsonii 7409N31 균주 모두 Urease 활성 및 젤라틴 액화 특성을 갖지 않는 것을 확인할 수 있었다.As can be seen in Figure 9, it was confirmed that Lactobacillus amylovorus 1394N20, Lactobacillus reuteri 1429C30, and Lactobacillus johnsonii 7409N31 strains did not have urease activity and gelatin liquefaction characteristics.
상기에서 설명한 바와 같이, 본 발명의 송아지 분변으로부터 분리한 Lactobacillus amylovorus 1394N20(기탁번호 KCCM 12999P), Lactobacillus reuteri 1429C30(기탁번호 KCCM 13000P), Lactobacillus johnsonii 7409N31(기탁번호 KCCM 13026P)은 장내 유익균을 증가시키는 것으로 송아지의 설사증에 대한 치료 및 예방 효과가 있으며, 항생제 내성이 없고, 용혈성이 없으며, 섭취 안전성이 우수할 수 있다.As described above, Lactobacillus amylovorus 1394N20 (accession number KCCM 12999P), Lactobacillus reuteri 1429C30 (accession number KCCM 13000P), and Lactobacillus johnsonii 7409N31 (accession number KCCM 13026P) isolated from calf feces of the present invention are known to increase beneficial intestinal bacteria. It is effective in treating and preventing diarrhea in calves, has no antibiotic resistance, is not hemolytic, and has excellent safety for ingestion.
이에 설사증 예방 및 완화 제제로 활용될 수 있다.Therefore, it can be used as an agent to prevent and relieve diarrhea.
이하, 상기에서 설명한 실시예에 대하여 실험예를 들어 본 발명을 더욱 구체적으로 설명하지만, 본 발명이 반드시 이들 실시예 및 실험예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the above-described examples and experimental examples, but the present invention is not necessarily limited to these examples and experimental examples.
[실험예 1] 단독 균주 투여에 따른 장내미생물총 확인[Experimental Example 1] Confirmation of intestinal microflora following administration of a single strain
최종 균주 중 Lactobacillus amylovorus 1394N20(기탁번호 KCCM 12999P)를 단독으로 사용하는 경우의 설사증 예방 효과를 평가하기 위하여, 한국의 한 농장에서 생후 10일 내의 정상 송아지를 무작위로 선정하여 Lactobacillus amylovorus 1394N20를 투여하였고, 분변을 수집하여 Lactobacillus amylovorus 1394N20 투여 전/투여 후/투여종료 5일 후에 따른 장내미생물총 차이를 분석하였다.To evaluate the effectiveness of preventing diarrhea when using Lactobacillus amylovorus 1394N20 (accession number KCCM 12999P) alone among the final strains, normal calves within 10 days of age were randomly selected from a farm in Korea and administered Lactobacillus amylovorus 1394N20. Feces were collected and the differences in intestinal microflora were analyzed before/after administration of Lactobacillus amylovorus 1394N20 and 5 days after the end of administration.
● Lactobacillus amylovorus 1394N20 투여 전/투여 후에 따른 장내미생물총 분석 ● Intestinal microbiota analysis before/after administration of Lactobacillus amylovorus 1394N20
Lactobacillus amylovorus 1394N20 투여 전/투여 후 수집된 분변으로부터 DNA를 추출한 후, Alpha-diversity 분석, 시퀀싱 분석을 수행하였고, 그 결과는 도 10 내지 도 12와 같다.After extracting DNA from feces collected before and after administration of Lactobacillus amylovorus 1394N20, alpha-diversity analysis and sequencing analysis were performed, and the results are shown in Figures 10 to 12.
도 10은 Lactobacillus amylovorus 1394N20 투여 전/후에 따른 Alpha-diversity 분석 결과를 나타낸 그래프이다. 여기서 NA는 투여 전, LA treat는 투여 후이다.Figure 10 is a graph showing the results of alpha-diversity analysis before and after administration of Lactobacillus amylovorus 1394N20. Here, NA is before administration and LA treat is after administration.
도 10을 보면 알 수 있듯이, Lactobacillus amylovorus 1394N20 투여 후 군에서 richness 지수인 ACE, Chao1, JakKnife와 evenness 지수인 Shannon에서 유의적 증가를 보이는 것을 확인할 수 있었다.As can be seen in Figure 10, it was confirmed that the group after administration of Lactobacillus amylovorus 1394N20 showed a significant increase in the richness indexes of ACE, Chao1, and JakKnife and the evenness index of Shannon.
도 11은 Lactobacillus amylovorus 1394N20 투여 전/후에 따른 문(Phylum) 수준의 장내미생물충 분석 결과 그래프이다.Figure 11 is a graph showing the results of intestinal microbial analysis at the phylum level before and after administration of Lactobacillus amylovorus 1394N20.
도 11을 보면 알 수 있듯이, Taxonomic composition 분석을 통해 문(Phylum) 수준에서 투여 전/후 군 간의 relative abundance를 비교 분석한 결과, Lactobacillus amylovorus 1394N20 투여 군에서 투여 전 군 보다 Proteobacteria, Actinobacteria, Firmicutes가 감소하고, Bacteroidetes가 증가하는 것을 확인할 수 있었다.As can be seen in Figure 11, as a result of comparing the relative abundance between groups before and after administration at the phylum level through taxonomic composition analysis, Proteobacteria, Actinobacteria, and Firmicutes were reduced in the group administered Lactobacillus amylovorus 1394N20 compared to the group before administration. And it was confirmed that Bacteroidetes increased.
여기서 Firmicutes는 설사증에 의해 증가하고, Bacteroidetes는 설사증에 의해 감소하는 장내미생물로서(1. 장내 유용 미생물 후보군 선정 참조), Lactobacillus amylovorus 1394N20 투여에 따라 설사증 예방 효과를 나타내는 것으로 사료된다.Here, Firmicutes is an intestinal microorganism that increases due to diarrhea, and Bacteroidetes decreases due to diarrhea (see 1. Selection of useful intestinal microorganism candidates), and it is believed that administration of Lactobacillus amylovorus 1394N20 has a diarrhea prevention effect.
도 12는 Lactobacillus amylovorus 1394N20 투여 전/후에 따른 종(Species) 수준의 장내미생물총 분석 결과 그래프이다.Figure 12 is a graph showing the results of intestinal microbiota analysis at the species level before and after administration of Lactobacillus amylovorus 1394N20.
도 12를 보면 알 수 있듯이, Taxonomic composition 분석을 통해 종(Species) 수준에서 투여 전/후 군 간의 relative abundance를 비교 분석한 결과, Lactobacillus amylovorus 1394N20 투여 군에서 Faecalibacterium prausnitzii group, Bacteroides vulgatus, Lactobacillus gasseri group의 비율이 증가하고, Clostridium perfringens, Escherichia coli의 비율이 감소된 것을 확인할 수 있었다.As can be seen in Figure 12, as a result of comparing the relative abundance between groups before and after administration at the species level through taxonomic composition analysis, in the group administered Lactobacillus amylovorus 1394N20 , Faecalibacterium prausnitzii group, Bacteroides vulgatus, and Lactobacillus gasseri group It was confirmed that the ratio increased, and the ratio of Clostridium perfringens and Escherichia coli decreased.
여기서, Faecalibacterium prausnitzii group, Lactobaillus gasseri group은 설사증에 의해 감소하고, Clostridium perfringens, Escherichia coli은 설사증에 의해 증가하는 장내미생물로서(1. 장내 유용 미생물 후보군 선정 참조), Lactobacillus amylovorus 1394N20 투여에 따라 설사증 예방 효과가 나타나는 것으로 사료된다.Here, Faecalibacterium prausnitzii group and Lactobaillus gasseri group are intestinal microorganisms that decrease due to diarrhea, and Clostridium perfringens and Escherichia coli are intestinal microorganisms that increase due to diarrhea (see 1. Selection of useful intestinal microorganism candidates). The administration of Lactobacillus amylovorus 1394N20 has a diarrhea prevention effect. It is believed that appears.
● Lactobacillus amylovorus 1394N20 투여 후/투여종료 5일 후에 따른 장내미생물총 분석 ● Intestinal microbiota analysis after administration of Lactobacillus amylovorus 1394N20/5 days after completion of administration
Lactobacillus amylovorus 1394N20 투여 후/투여종료 5일 후 수집된 분변으로부터 DNA를 추출한 후, 시퀀싱 분석을 수행하였고, 그 결과는 도 13과 같다.DNA was extracted from feces collected after administration of Lactobacillus amylovorus 1394N20/5 days after completion of administration, and then sequencing analysis was performed, and the results are shown in FIG. 13.
도 13은 Lactobacillus amylovorus 1394N20 투여 후/투여종료 5일 후에 따른 종(Species) 수준의 장내미생물총 분석 결과 그래프이다.Figure 13 is a graph showing the results of intestinal microbiota analysis at the species level after administration of Lactobacillus amylovorus 1394N20/5 days after completion of administration.
도 13을 보면 알 수 있듯이, Taxonomic composition 분석을 통해 종(Species) 수준에서 투여 후/투여종료 5일 후 군 간의 relative abundance를 비교 분석한 결과, Lactobacillus amylovorus 1394N20 투여 종료 5일 후 군에서 Bacteroides vulgatus, Lactobacillus gasseri group, Lactobacillus reuteri group, Lactobacillus helveticus group, Bacteroides xylanisolvens group의 비율이 증가하고, Clostridium perfringens, Escherichia coli의 비율이 감소되는 것을 확인할 수 있었다.As can be seen in Figure 13, as a result of comparing the relative abundance between groups at the species level through taxonomic composition analysis / 5 days after the end of administration, Lactobacillus amylovorus 1394N20 group 5 days after the end of administration , Bacteroides vulgatus, It was confirmed that the proportions of Lactobacillus gasseri group, Lactobacillus reuteri group, Lactobacillus helveticus group, and Bacteroides xylanisolvens group increased, and the proportions of Clostridium perfringens and Escherichia coli decreased.
이에 따라, Lactobacillus amylovorus 1394N20 투여에 의해 설사증이 예방된 것으로 판단되며, 이를 통해 Lactobacillus amylovorus 1394N20는 설사증 예방 효과를 나타내는 것으로 판단된다.Accordingly, it is judged that diarrhea was prevented by administration of Lactobacillus amylovorus 1394N20, and through this , Lactobacillus amylovorus 1394N20 is judged to have a diarrhea prevention effect.
[실험예 2] 혼합 균주 투여에 따른 장내미생물총 확인[Experimental Example 2] Confirmation of intestinal microflora following administration of mixed strains
최종 균주인 Lactobacillus amylovorus 1394N20(기탁번호 KCCM 12999P), Lactobacillus reuteri 1429C30(기탁번호 KCCM 13000P), Lactobacillus johnsonii 7409N31(기탁번호 KCCM 13026P)를 혼합(이하 '혼합균주'라 함)하여 사용할 경우의 설사증 예방 효과를 평가하기 위하여, 한국의 한 농장에서 생후 10일 내의 정상 송아지를 무작위로 선정하여 혼합균주를 투여하였고, 분변을 수집하여 혼합균주 투여 전/투여 후에 따른 장내미생물총 차이를 분석하였다. Lactobacillus amylovorus 1394N20 (deposit number kccm 1299p), lactobacillus reuteri 1429c30 Mixing m 13026p (hereinafter referred to as 'mixed strain') To evaluate this, normal calves within 10 days of age were randomly selected from a farm in Korea and administered a mixed strain, and feces were collected to analyze the differences in intestinal microflora before and after administration of the mixed strain.
혼합균주 투여 전/투여 후 수집된 분변으로부터 DNA를 추출한 후, Alpha-diversity 분석, 시퀀싱 분석을 수행하였고, 그 결과는 도 14 내지 도 17과 같다.After extracting DNA from feces collected before and after administration of the mixed strain, alpha-diversity analysis and sequencing analysis were performed, and the results are shown in Figures 14 to 17.
도 14는 혼합균주 투여 전/후에 따른 Alpha-diversity 분석 결과를 나타낸 그래프이다. 여기서 NA는 투여 전, LM treat는 투여 후이다.Figure 14 is a graph showing the results of alpha-diversity analysis before and after administration of mixed strains. Here, NA is before administration and LM treatment is after administration.
도 14를 보면 알 수 있듯이, 혼합균주 투여 후 군(LM treat)에서 richness 지수인 ACE, Chao1, JakKnife와 evenness 지수인 Shannon에서 유의적 증가를 보이는 것을 확인할 수 있었다.As can be seen in Figure 14, it was confirmed that in the group (LM treat) after administration of mixed strains, there was a significant increase in the richness indexes of ACE, Chao1, and JakKnife and the evenness index of Shannon.
도 15는 혼합균주 투여 전/후에 따른 문(Phylum) 수준의 장내미생물충 분석 결과 그래프이다.Figure 15 is a graph showing the results of intestinal microbial analysis at the phylum level before and after administration of mixed strains.
도 15를 보면 알 수 있듯이, Taxonomic composition 분석을 통해 문(Phylum) 수준에서 투여 전/후(NA, LM treat) 군 간의 relative abundance를 비교 분석한 결과, 혼합균주 투여 군(LM treat)에서 투여 전 군(NA) 보다 Proteobacteria가 감소하고, Bacteroidetes가 증가하는 것을 확인할 수 있었다.As can be seen in Figure 15, as a result of comparative analysis of the relative abundance between groups before and after administration (NA, LM treat) at the phylum level through taxonomic composition analysis, before administration in the mixed strain administration group (LM treat) It was confirmed that Proteobacteria decreased and Bacteroidetes increased compared to group (NA).
도 16은 혼합균주 투여 전/후에 따른 속(Genus) 수준의 장내미생물충 분석 결과 그래프이다.Figure 16 is a graph showing the results of intestinal microbial analysis at the genus level before and after administration of mixed strains.
도 16을 보면 알 수 있듯이, Taxonomic composition 분석을 통해 속(Genus) 수준에서 투여 전/후(NA, LM treat) 군 간의 relative abundance를 비교 분석한 결과, 혼합균주 투여 군(LM treat)에서 투여 전 군(NA) 보다 Lactobacillus, Faecalibacterium, Bacteroides의 비율이 증가하고, Escherichia의 비율이 감소하는 것을 확인할 수 있었다.As can be seen in Figure 16, as a result of comparative analysis of the relative abundance between groups before and after administration (NA, LM treat) at the genus level through taxonomic composition analysis, before administration in the mixed strain administration group (LM treat) It was confirmed that the proportion of Lactobacillus, Faecalibacterium, and Bacteroides increased, and the proportion of Escherichia decreased compared to group (NA).
도 17은 혼합균주 투여 전/후에 따른 종(Species) 수준의 장내미생물총 분석 결과 그래프이다.Figure 17 is a graph showing the results of intestinal microbiota analysis at the species level before and after administration of mixed strains.
도 17을 보면 알 수 있듯이, Taxonomic composition 분석을 통해 속(genus) 수준에서 투여 전/후(NA, LM treat) 군 간의 relative abundance를 비교 분석한 결과, 혼합균주 투여 군(LM treat)에서 투여 전 군(NA) 보다 Escherichia coli group, Clostridium celatum group, Bacteroides fragilis의 비율이 감소하고, Faecalibacterium prausnitzii group, Bacteroides vulgatus, Lactobacillus gasseri group의 비율이 증가하는 것을 확인할 수 있었다.As can be seen in Figure 17, as a result of comparative analysis of the relative abundance between the groups before and after administration (NA, LM treat) at the genus level through taxonomic composition analysis, before administration in the mixed strain administration group (LM treat) It was confirmed that the proportions of Escherichia coli group, Clostridium celatum group, and Bacteroides fragilis decreased, and the proportions of Faecalibacterium prausnitzii group, Bacteroides vulgatus, and Lactobacillus gasseri group increased compared to group (NA).
이와 같이, 혼합균주를 투여한 군(LM treat)에서 혼합균주를 투여하지 않은 군(NA) 보다 장내 미생물의 다양성이 증가하였을 뿐만 아니라, Lactobacillus, Faecalibacterium 등과 같은 유익균의 비율은 높아지는 반면 Escherichia, Bacteroides 등과 같은 유해균의 비율은 낮아지는 경향을 보이는 것을 확인할 수 있었다.In this way, not only did the diversity of intestinal microorganisms increase in the group administered mixed strains (LM treat) compared to the group not administered mixed strains (NA), but the proportion of beneficial bacteria such as Lactobacillus and Faecalibacterium increased, while Escherichia, Bacteroides , etc. It was confirmed that the proportion of the same harmful bacteria tended to decrease.
따라서, 혼합균주 투여에 따라 유익균이 우점할 수 있는 장내 환경을 유도하며, 질병을 일으키는 유해균의 증식을 억제하는 효과를 나타내어, 설사증 예방이 가능할 것으로 판단된다.Therefore, it is believed that the administration of mixed strains induces an intestinal environment in which beneficial bacteria can dominate and has the effect of suppressing the growth of harmful bacteria that cause disease, thereby preventing diarrhea.
[실험예 3] 설사증 예방 효과 평가 1[Experimental Example 3] Evaluation of diarrhea prevention effect 1
송아지 분변으로부터 분리된 균주의 설사증 예방 효과를 확인하기 위하여, 한국의 한 농장에서 생후 10일 내의 정상 송아지를 무작위로 선정하여, 혼합균주를 투여하였다. 실험군의 투여 4일차/투여 8일차/투여종료 10일 후 분변을 수집하여 병원체 검사를 수행하였다. 대조군으로 혼합균주를 투여하지 않은 송아지에 대해 동일하게 병원체 검사를 수행하였다.To confirm the effectiveness of strains isolated from calf feces in preventing diarrhea, normal calves within 10 days of age were randomly selected from a farm in Korea and administered a mixed strain. On the 4th day of administration/8th day of administration/10 days after the end of administration, feces from the experimental group were collected and tested for pathogens. As a control group, the same pathogen test was performed on calves that were not administered the mixed strains.
그 결과는 도 18과 같다.The results are as shown in Figure 18.
도 18을 보면 알 수 있듯이, 혼합균주를 섭취한 실험군에서 대조군 보다 병원체가 검출되는 두수가 적은 것을 확인할 수 있었으며, 검출되는 병원체 총수 또한 감소되는 것을 확인할 수 있었다.As can be seen in Figure 18, it was confirmed that the number of pathogens detected was lower in the experimental group that consumed the mixed strains than the control group, and the total number of detected pathogens was also reduced.
[실험예 4] 설사증 예방 효과 평가 2[Experimental Example 4] Evaluation of diarrhea prevention effect 2
송아지 분변으로부터 분리된 균주의 설사증 예방 효과를 확인하기 위하여, 한국의 두개 농장에서 생후 10일 내의 송아지를 무작위로 선정하여, 혼합균주를 투여하였다. 투여 5일차/투여 10일차/투여종료 후 20일차에 각각 실험군의 혈액을 수집하여 혈액 가스 분석을 수행하여, 일령 변화에 따른 생체정보 변화를 분석하여 대조군과 비교하였다. 대조군으로 혼합균주를 투여하지 않은 송아지에 대해 동일하게 혈액 가스 분석을 수행하였다. 설사증에 따라 송아지의 생체정보에 변화가 있음을 확인한 바 있어, 이를 기반으로 진행하였다(한국등록특허 제10-2302739호).To confirm the effectiveness of strains isolated from calf feces in preventing diarrhea, calves within 10 days of age were randomly selected from two farms in Korea and administered a mixed strain. On the 5th day of administration, the 10th day of administration, and the 20th day after the end of administration, the blood of the experimental group was collected and blood gas analysis was performed. Changes in biometric information according to age were analyzed and compared with the control group. As a control group, the same blood gas analysis was performed on calves that were not administered the mixed strains. It has been confirmed that there are changes in the calf's biometric information depending on diarrhea, so we proceeded based on this (Korean Patent No. 10-2302739).
그 결과는 도 19a 내지 도 19d와 같다.The results are as shown in Figures 19A to 19D.
도 19a 내지 도 19d는 실험군과 대조군의 HCO3 -, BE(Base Excess), pH 및 SID를 측정한 일령별 평균 그래프이다.Figures 19a to 19d are average graphs by day measuring HCO 3 - , BE (Base Excess), pH, and SID of the experimental group and the control group.
도 19a를 참조하면, HCO3 -은 투여 5일차에 대조군 보다 실험군에서 낮게 나타났으나, 투여 10일차에 실험군에서 대조군 보다 높게 나타나는 것을 확인할 수 있었고, 투여종료 후 20일차에서도 실험군에서 대조군 보다 높게 유지하고 있는 것을 확인할 수 있었다.Referring to Figure 19a, HCO 3 - appeared lower in the experimental group than the control group on the 5th day of administration, but was confirmed to be higher in the experimental group than the control group on the 10th day of administration, and remained higher in the experimental group than the control group even on the 20th day after the end of administration. I was able to confirm what was being done.
도 19b를 참조하면, BE(Base Excess)는 투여 5일차에서 실험군과 대조군에서 유사하게 나타났으나, 투여 10일차에 실험군에서 대조군 보다 높게 나타나는 것을 확인할 수 있었다. 또한 투여종료 후 20일차에서도 실험군이 대조군 보다 높게 유지하고 있는 것을 확인할 수 있었다.Referring to Figure 19b, BE (Base Excess) appeared similarly in the experimental and control groups on the 5th day of administration, but was confirmed to be higher in the experimental group than the control group on the 10th day of administration. In addition, it was confirmed that the experimental group maintained a higher level than the control group even on the 20th day after the end of administration.
도 19c를 참조하면, pH는 투여 5일차, 10일차에 대조군 보다 실험군에서 높게 나타나는 것을 확인할 수 있었다. Referring to Figure 19c, it was confirmed that pH was higher in the experimental group than in the control group on the 5th and 10th days of administration.
도 19d를 참조하면, SID는 투여 5일차에서 실험군과 대조군에서 유사하게 나타났으나, 투여 10일차에 실험군에서 대조군 보다 높게 나타나는 것을 확인할 수 있었다. 또한 투여종료 후 20일차에서도 실험군이 대조군 보다 높게 유지하고 있는 것을 확인할 수 있었다.Referring to Figure 19d, SID appeared similar in the experimental and control groups on the 5th day of administration, but was confirmed to be higher in the experimental group than the control group on the 10th day of administration. In addition, it was confirmed that the experimental group maintained a higher level than the control group even on the 20th day after the end of administration.
이상에서 본 발명은 기재된 특정한 실시형태 및 실시예에 대해서만 상세히 기술되었지만, 본 발명의 기술사상범위 내에서 다양한 변형 및 수정이 가능함은 당업자에게 명백한 것이며, 이러한 변형 및 수정이 첨부된 특허청구범위에 속함은 당연한 것이다.Although the present invention has been described in detail only with respect to the specific embodiments and examples described above, it is clear to those skilled in the art that various changes and modifications are possible within the technical scope of the present invention, and such changes and modifications fall within the scope of the appended patent claims. is natural.
Claims (4)
A diarrhea prevention and alleviation agent containing a strain of Lactobacillus amylovorus 1394N20 (accession number KCCM 12999P) isolated from calf feces and characterized by increasing the intestinal beneficial bacteria Faecalibacterium prausnitzii group and Lactobaillus gasseri group. .
Lactobacillus amylovorus 1394N20 (accession number KCCM 12999P), Lactobacillus reuteri 1429C30 (accession number KCCM 13000P), Lactobacillus johnsonii 7409N31 (accession number KCCM 13000P) isolated from calf feces Tak number KCCM A preparation for preventing and alleviating diarrhea, comprising a strain consisting of (13026P) and increasing the intestinal beneficial bacteria Faecalibacterium prausnitzii group and Lactobaillus gasseri group .
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| KR102282884B1 (en) | 2019-10-11 | 2021-07-28 | 전북대학교산학협력단 | A Method for diagnosing rotavirus diarrhea using Neonatal Calves by Using an Index of Microbiome Data |
| KR102302739B1 (en) | 2019-11-15 | 2021-09-14 | 전북대학교산학협력단 | A Method for Determining of Health Status of Neonatal Calves by using an Index of Biologic Information |
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| KR102282884B1 (en) | 2019-10-11 | 2021-07-28 | 전북대학교산학협력단 | A Method for diagnosing rotavirus diarrhea using Neonatal Calves by Using an Index of Microbiome Data |
| KR102302739B1 (en) | 2019-11-15 | 2021-09-14 | 전북대학교산학협력단 | A Method for Determining of Health Status of Neonatal Calves by using an Index of Biologic Information |
Non-Patent Citations (3)
| Title |
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| Front. Nutr., Vol.9, Article No.846991, pp.1-15(2022.03.29.) |
| Frontiers in Microbiology, Vol.12, Article No.772863(2021.10.)* |
| JARQ, Vol.35, pp.189-194(2001.) |
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