KR102623910B1 - Microorganism strain having inhibitor for biofilm in animal intestines derived from Escherichia coli - Google Patents
Microorganism strain having inhibitor for biofilm in animal intestines derived from Escherichia coli Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
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Abstract
본 발명은 Escherichia coli 유래 가축 장내 바이오 필름 억제능을 가지는 미생물 균주를 개시한다.The present invention discloses a microbial strain derived from Escherichia coli that has the ability to inhibit intestinal biofilm in livestock.
Description
본 발명은 Escherichia coli 유래 가축 장내 바이오 필름 억제능을 가지는 미생물 균주에 대한 것으로서, 보다 구체적으로 고생산성 돼지 분변에서 분리된 바실러스 코아귤런스 GB-BC-2022 (Bacillus coagulans, GB-BC-2022, 수탁번호: KFCC11948P) 균주와 멧돼지 분변에서 분리된 엔테로코커스 페시움 GB-EF-2022 (Enterococcus faecium, GB-EF-2022, 수탁번호: KFCC11946P) 균주 2종에 관한 것으로서, 상기 균주 2종의 미생물의 경우 소화기 질병 유발 균주인 에스케리키아 콜라이(Escherichia coli; E.coli) 3종(KCCM 11234, E.coli K88, E.coli F18)의 바이오 필름 형성 억제 및 제거능을 갖는다. 본 발명에 따르면 바실러스 코아귤런스 GB-BC-2022 (Bacillus coagulans, GB-BC-2022, 수탁번호: KFCC11948P) 균주, 엔테로코커스 페시움 GB-EF-2022 (Enterococcus faecium, GB-EF-2022, 수탁번호: KFCC11946P) 균주는 소화기 질병 유발 균주인 에스케리키아 콜라이(Escherichia coli; E.coli) 3종(KCCM 11234, E.coli K88, E.coli F18)에 대한 바이오필름 억제 및 제거 능력과 이들 소화기 질병 유발 균주들에 항균활성을 가지고 있어 프로바이오틱스 등 사료 및 사료첨가제에 활용이 가능하다.The present invention relates to a microbial strain that has the ability to inhibit livestock intestinal biofilm derived from Escherichia coli , and more specifically, to Bacillus coagulans GB-BC-2022 ( Bacillus coagulans , GB-BC-2022, accession number) isolated from highly productive pig feces. : KFCC11948P) strain and Enterococcus faecium GB-EF-2022 ( Enterococcus faecium , GB-EF-2022, accession number: KFCC11946P) strain isolated from wild boar feces. In the case of the two strains of microorganisms, the digestive system It has the ability to inhibit biofilm formation and remove three types of disease-causing strains of Escherichia coli (E.coli) (KCCM 11234, E.coli K88, E.coli F18). According to the present invention, Bacillus coagulans GB-BC-2022 ( Bacillus coagulans , GB-BC-2022, accession number: KFCC11948P) strain, Enterococcus faecium GB-EF-2022 ( Enterococcus faecium , GB-EF-2022, accession number: KFCC11948P) strain Number: KFCC11946P) strain has the ability to inhibit and remove biofilms against three types of Escherichia coli (E.coli) (KCCM 11234, E.coli K88, E.coli F18), which are strains that cause digestive diseases, and their digestive properties. It has antibacterial activity against disease-causing strains and can be used in feed and feed additives such as probiotics.
바이오필름은 미생물들의 복합체로서, 스스로 만들어진 EPS(Extracelluar Polymeric Substances) 내에 함유된다. 따라서, 바이오필름은 그 매트릭스 안에 둘러 싸여져서 상호 간 또는 표면에 고착되거나 상호 교류하는 미생물군이라고 말할 수 있다.Biofilm is a complex of microorganisms contained within self-produced EPS (Extracelluar Polymeric Substances). Therefore, a biofilm can be said to be a group of microorganisms that are surrounded within the matrix and adhere to or interact with each other or on the surface.
미생물에 의하여 형성된 바이오필름은 다양한 표면에 강하게 부착되어 있어 제거하기가 힘들고, 부유생활을 할 때에 비하여, 가혹한 환경 (pH, 온도, 영양분의 고갈 등)과 항생제들의 공격 등에 대하여 훨씬 강한 저항력을 가지기 때문에 멸균, 소독이 힘들다. Biofilms formed by microorganisms are strongly attached to various surfaces and are difficult to remove, and have much stronger resistance to harsh environments (pH, temperature, depletion of nutrients, etc.) and attacks by antibiotics compared to when living in a floating environment. Sterilization and disinfection are difficult.
바이오필름은 세균, 효모, 곰팡이 등 미생물이 특정조건에서 동물(피부, 구강, 치아 등) 등의 표면에 필름처럼 부착되어 있는 미생물의 집합체라 할 수 있으며, 그 자체가 미생물의 서식처로도 제공되어 균의 서식을 가속화하기도 한다.Biofilm can be said to be a collection of microorganisms, such as bacteria, yeast, and mold, that adhere to the surface of animals (skin, mouth, teeth, etc.) like a film under certain conditions, and it itself serves as a habitat for microorganisms. It also accelerates the growth of fungi.
특히 질병 유발 균주들의 바이오필름은 축산업계 및 의학계 전반에서 심각한 문제를 일으킬 수 있으므로, 바이오필름 형성 억제용 조성물 및 바이오필름 형성을 억제하는 방법 등에 대한 연구가 요구되는 상황이다.In particular, biofilms from disease-causing strains can cause serious problems in the livestock industry and the medical community as a whole, so research on compositions for inhibiting biofilm formation and methods for inhibiting biofilm formation is required.
본 발명의 목적은 소화기 질병유발 균주 Escherichia coli 3종(KCCM 11234, E.coli K88, E.coli F18)에 대한 바이오필름 억제 및 제거 능력이 우수한 바실러스 코아귤런스 GB-BC-2022 (Bacillus coagulans, GB-BC-2022, 수탁번호: KFCC11948P) 균주, 엔테로코커스 페시움 GB-EF-2022 (Enterococcus faecium, GB-EF-2022, 수탁번호: KFCC11946P) 균주를 제공하고자 한다.The purpose of the present invention is to develop Bacillus coagulans GB - BC-2022 ( Bacillus coagulans , We would like to provide the strain GB-BC-2022, accession number: KFCC11948P) strain, Enterococcus faecium GB-EF-2022 ( Enterococcus faecium , GB-EF-2022, accession number: KFCC11946P) strain.
상기 과제를 해결하기 위하여, 본 발명에서는 하기와 같은 수단을 개시한다.In order to solve the above problems, the present invention discloses the following means.
일 양태에서, 본 발명은 소화기 질병 유발 균주 에스케리키아 콜라이(Escherichia coli; E.coli)에 대한 바이오필름 억제 및 제거 능력과 이들 소화기 질병 유발 균주들에 항균활성을 갖는 바실러스 코아귤런스 GB-BC-2022 (Bacillus coagulans, GB-BC-2022, 수탁번호: KFCC11948P) 균주를 개시한다.In one aspect, the present invention provides Bacillus coagulans GB-BC, which has biofilm inhibition and removal ability against digestive disease-causing strains Escherichia coli (E.coli) and antibacterial activity against these digestive disease-causing strains. -2022 ( Bacillus coagulans , GB-BC-2022, accession number: KFCC11948P) strain is disclosed.
또 다른 양태에서, 본 발명은 소화기 질병 유발 균주 에스케리키아 콜라이(Escherichia coli; E.coli)에 대한 바이오필름 억제 및 제거 능력과 이들 소화기 질병 유발 균주들에 항균활성을 갖는 바실러스 코아귤런스 GB-BC-2022 (Bacillus coagulans, GB-BC-2022, 수탁번호: KFCC11948P) 균주를 포함하는 사료용 조성물을 개시한다.In another aspect, the present invention provides Bacillus coagulans GB-, which has biofilm inhibition and removal ability against digestive disease-causing strains Escherichia coli (E.coli) and antibacterial activity against these digestive disease-causing strains. Disclosed is a composition for feed containing the strain BC-2022 ( Bacillus coagulans , GB-BC-2022, accession number: KFCC11948P).
또 다른 양태에서, 본 발명은 소화기 질병 유발 균주 에스케리키아 콜라이(Escherichia coli; E.coli)에 대한 바이오필름 억제 및 제거 능력과 이들 소화기 질병 유발 균주들에 항균활성을 갖는 엔테로코커스 페시움 GB-EF-2022 (Enterococcus faecium, GB-EF-2022, 수탁번호: KFCC11946P) 균주를 개시한다.In another aspect, the present invention provides Enterococcus faecium GB-, which has biofilm inhibition and removal ability against digestive disease-causing strains Escherichia coli (E.coli) and antibacterial activity against these digestive disease-causing strains. The strain EF-2022 ( Enterococcus faecium , GB-EF-2022, accession number: KFCC11946P) is disclosed.
또 다른 양태에서, 본 발명은 소화기 질병 유발 균주 에스케리키아 콜라이(Escherichia coli; E.coli)에 대한 바이오필름 억제 및 제거 능력과 이들 소화기 질병 유발 균주들에 항균활성을 갖는 엔테로코커스 페시움 GB-EF-2022 (Enterococcus faecium, GB-EF-2022, 수탁번호: KFCC11946P) 균주를 포함하는 사료용 조성물을 개시한다.In another aspect, the present invention provides Enterococcus faecium GB-, which has biofilm inhibition and removal ability against digestive disease-causing strains Escherichia coli (E.coli) and antibacterial activity against these digestive disease-causing strains. Disclosed is a composition for feed containing the strain EF-2022 ( Enterococcus faecium , GB-EF-2022, accession number: KFCC11946P).
본 발명에 따른 고생산성 돼지 및 멧돼지 분변에서 분리한 2종의 균주 중 바실러스 코아귤런스 GB-BC-2022 (Bacillus coagulans, GB-BC-2022, 수탁번호: KFCC11948P) 균주, 엔테로코커스 페시움 GB-EF-2022 (Enterococcus faecium, GB-EF-2022, 수탁번호: KFCC11946P) 균주 2종은 소화기 질병 유발 균주 에스케리키아 콜라이(Escherichia coli; E.coli) 3종(KCCM 11234, E.coli K88, E.coli F18)에 대한 바이오필름 억제 및 제거 능력과 이들 소화기 질병 유발 균주들에 항균활성을 가지고 있어 프로바이오틱스 등 사료 및 사료첨가제에 활용 가능한 이점이 있다.Among the two strains isolated from highly productive pig and wild boar feces according to the present invention, Bacillus coagulans GB-BC-2022 ( Bacillus coagulans , GB-BC-2022, Accession number: KFCC11948P) strain, Enterococcus faecium GB- EF-2022 ( Enterococcus faecium , GB-EF-2022, accession number: KFCC11946P), 2 strains of Escherichia coli ( E.coli ), which cause digestive diseases, and 3 types (KCCM 11234, E.coli K88, E It has the ability to suppress and remove biofilm against .coli F18) and has antibacterial activity against these strains that cause digestive diseases, so it has the advantage of being able to be used in feed and feed additives such as probiotics.
본 발명의 효과는 이상에서 언급한 효과들로 제한되지 않으며, 이하에서 설명할 내용으로부터 통상의 기술자에게 자명한 범위 내에서 다양한 효과들이 포함될 수 있다.The effects of the present invention are not limited to the effects mentioned above, and various effects may be included within the scope apparent to those skilled in the art from the contents described below.
도 1은 In-vivo 바이오필름 억제능 평가 결과를 400배 배율로 장 융모를 이루는 상피세포의 내강측 표면 부위를 나타낸 결과이다.
도 2은 In-vivo 바이오필름 억제능 평가 결과를 400배 배율로 장 융모를 이루는 상피세포의 내강측 표면 부위를 개체당 5개 단면에서 관찰하고 병변을 점수화 한 결과이다.
도 3은 본 발명에 따른 바실러스 코아귤런스 GB-BC-2022 (Bacillus coagulans, GB-BC-2022, 수탁번호: KFCC11948P) 균주, 엔테로코커스 페시움 GB-EF-2022 (Enterococcus faecium, GB-EF-2022, 수탁번호: KFCC11946P) 균주의 Escherichia coli (KCCM 11234)에 대한 항균활성 평가를 위한 최저생장저해농도(minimum inhibitory concentration; MIC) 시험 결과를 나타낸 것이다.
도 4는 본 발명에 따른 바실러스 코아귤런스 GB-BC-2022 (Bacillus coagulans, GB-BC-2022, 수탁번호: KFCC11948P) 균주, 엔테로코커스 페시움 GB-EF-2022 (Enterococcus faecium, GB-EF-2022, 수탁번호: KFCC11946P) 균주의 Escherichia coli(E.coli K88)에 대한 항균활성 평가를 위한 최저생장저해농도(minimum inhibitory concentration; MIC) 시험 결과를 나타낸 것이다.
도 5는 본 발명에 따른 바실러스 코아귤런스 GB-BC-2022 (Bacillus coagulans, GB-BC-2022, 수탁번호: KFCC11948P) 균주, 엔테로코커스 페시움 GB-EF-2022 (Enterococcus faecium, GB-EF-2022, 수탁번호: KFCC11946P) 균주의 Escherichia coli(E.coli F18)에 대한 항균활성 평가를 위한 최저생장저해농도(minimum inhibitory concentration; MIC) 시험 결과를 나타낸 것이다.Figure 1 shows the results of in-vivo biofilm inhibition ability evaluation showing the luminal surface area of the epithelial cells forming intestinal villi at 400 times magnification.
Figure 2 shows the results of in-vivo biofilm inhibition ability evaluation obtained by observing the luminal surface area of epithelial cells forming intestinal villi at 400x magnification in five sections per individual and scoring the lesions.
Figure 3 shows Bacillus coagulans GB-BC-2022 ( Bacillus coagulans , GB-BC-2022, accession number: KFCC11948P) strain, Enterococcus faecium GB-EF-2022 ( Enterococcus faecium , GB-EF-) according to the present invention. This shows the results of the minimum growth inhibitory concentration (MIC) test to evaluate the antibacterial activity of the strain 2022, accession number: KFCC11946P) against Escherichia coli (KCCM 11234).
Figure 4 shows Bacillus coagulans GB-BC-2022 ( Bacillus coagulans , GB-BC-2022, accession number: KFCC11948P) strain, Enterococcus faecium GB-EF-2022 ( Enterococcus faecium , GB-EF-) according to the present invention. This shows the results of the minimum growth inhibitory concentration (MIC) test for evaluating the antibacterial activity of the 2022, accession number: KFCC11946P) strain against Escherichia coli ( E.coli K88).
Figure 5 shows Bacillus coagulans GB-BC-2022 ( Bacillus coagulans , GB-BC-2022, accession number: KFCC11948P) strain, Enterococcus faecium GB-EF-2022 ( Enterococcus faecium , GB-EF-) according to the present invention. This shows the minimum growth inhibitory concentration (MIC) test results for evaluating the antibacterial activity of the strain 2022, accession number: KFCC11946P) against Escherichia coli ( E.coli F18).
이하, 본 명세서에 대하여 더욱 상세히 설명한다.Hereinafter, this specification will be described in more detail.
이를 구체적으로 설명하면 다음과 같다. 본 명세서에서 사용되는 용어는 본 발명에서의 기능을 고려하면서 가능한 현재 널리 사용되는 일반적인 용어들을 선택하였으나, 이는 당 분야에 종사하는 기술자의 의도 또는 판례, 새로운 기술의 출현 등에 따라 달라질 수 있다. 또한, 특정한 경우는 출원인이 임의로 선정한 용어도 있으며, 이 경우 해당되는 발명의 설명 부분에서 상세히 그 의미를 기재할 것이다. 따라서 본 발명에서 사용되는 용어는 단순한 용어의 명칭이 아닌, 그 용어가 가지는 의미와 본 발명의 전반에 걸친 내용을 토대로 정의되어야 한다.This is explained in detail as follows. The terms used in this specification are general terms that are currently widely used as much as possible while considering the function in the present invention, but this may vary depending on the intention or precedent of a person skilled in the art, the emergence of new technology, etc. In addition, in certain cases, there are terms arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the relevant invention. Therefore, the terms used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, rather than simply the name of the term.
다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 관련 기술의 문맥상 가지는 의미와 일치하는 의미를 가지는 것으로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한, 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다.Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the technical field to which the present invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and unless clearly defined in the present application, should not be interpreted in an ideal or excessively formal sense. No.
수치 범위는 상기 범위에 정의된 수치를 포함한다. 본 명세서에 걸쳐 주어진 모든 최대의 수치 제한은 낮은 수치 제한이 명확히 쓰여져 있는 것처럼 모든 더 낮은 수치 제한을 포함한다. 본 명세서에 걸쳐 주어진 모든 최소의 수치 제한은 더 높은 수치 제한이 명확히 쓰여져 있는 것처럼 모든 더 높은 수치 제한을 포함한다. 본 명세서에 걸쳐 주어진 모든 수치 제한은 더 좁은 수치 제한이 명확히 쓰여져 있는 것처럼, 더 넓은 수치 범위 내의 더 좋은 모든 수치 범위를 포함할 것이다.The numerical range includes the values defined in the range above. Every maximum numerical limit given throughout this specification includes all lower numerical limits as if the lower numerical limit were explicitly written out. Every minimum numerical limit given throughout this specification includes every higher numerical limit as if such higher numerical limit was clearly written. All numerical limits given throughout this specification will include all better numerical ranges within the broader numerical range, as if the narrower numerical limits were clearly written.
이하, 본 발명에서 개시된 각각의 설명 및 실시형태는 각각에 대한 다른 설명 및 실시형태에도 적용될 수 있다. 즉, 본 발명에 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기에 기술된 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 볼 수 없다.Hereinafter, each description and embodiment disclosed in the present invention may also be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Additionally, the scope of the present invention cannot be considered limited by the specific description set forth below.
본 명세서에서 사용되는 「포함하는」과 같은 표현은, 해당 표현이 포함되는 문구 또는 문장에서 특별히 다르게 언급되지 않는 한, 다른 실시예를 포함할 가능성을 내포하는 개방형 용어(open-ended terms)로 이해되어야 한다.Expressions such as “comprising” used in this specification are understood as open-ended terms that imply the possibility of including other embodiments, unless specifically stated otherwise in the phrase or sentence containing the expression. It has to be.
본 발명의 발명자들은, 돼지 질병 예방을 위한 유용 미생물을 선발하기 위해 연구 개발하던 중, 사료 등의 기준 및 규격'의 제 5조 '보조사료의 범위'에 명시된 균주의 허용 범위에 따라 분류하여 축산 사료로 이용할 수 있는 22종의 후보 미생물을 선발하였고, 이 중에서 소화기 질병유발 균주 Escherichia coli 3종(KCCM 11234, E.coli K88, E.coli F18)에 대한 바이오필름 억제 및 제거 능력이 우수한 바실러스 코아귤런스 GB-BC-2022 (Bacillus coagulans, GB-BC-2022, 수탁번호: KFCC11948P) 균주, 엔테로코커스 페시움 GB-EF-2022 (Enterococcus faecium, GB-EF-2022, 수탁번호: KFCC11946P) 균주 2종을 발견하여 본 발명을 완성하게 되었다.While conducting research and development to select useful microorganisms for preventing swine diseases, the inventors of the present invention classified them according to the allowable range of strains specified in Article 5, 'Scope of Supplementary Feed' of 'Standards and Specifications for Feed, etc.' and used them in livestock farming. We selected 22 types of candidate microorganisms that can be used as feed, and among them, Bacillus Core has excellent biofilm inhibition and removal ability against 3 types of Escherichia coli (KCCM 11234, E.coli K88, E.coli F18) that cause digestive diseases. Gyulens GB-BC-2022 ( Bacillus coagulans , GB-BC-2022, accession number: KFCC11948P) strain, Enterococcus faecium GB-EF-2022 ( Enterococcus faecium , GB-EF-2022, accession number: KFCC11946P) strain 2 The discovery of the species led to the completion of this invention.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
소화기 질병 유발 균주 에스케리키아 콜라이(Escherichia coli, a strain that causes digestive diseases ( Escherichia coliEscherichia coli ; E.coli)에 대한 바이오필름 억제 및 제거 능력과 이들 소화기 질병 유발 균주들에 항균활성을 갖는 균주; A strain that has the ability to inhibit and remove biofilm against E. coli and has antibacterial activity against these strains that cause digestive diseases
본 발명은 소화기 질병 유발 균주 에스케리키아 콜라이(Escherichia coli; E.coli)에 대한 바이오필름 억제 및 제거 능력과 이들 소화기 질병 유발 균주들에 항균활성을 갖는 바실러스 코아귤런스 GB-BC-2022 (Bacillus coagulans, GB-BC-2022, 수탁번호: KFCC11948P) 균주를 제공한다.The present invention relates to Bacillus coagulans GB-BC-2022 ( Bacillus ), which has the ability to inhibit and remove biofilms against Escherichia coli (E.coli), a strain causing digestive diseases, and has antibacterial activity against these strains. coagulans , GB-BC-2022, accession number: KFCC11948P) strain is provided.
본 발명에서 용어, 「바실러스 코아귤런스(Bacillus coagulans)」란, 충남지역 양돈장의 고생산성 육성돈(3원 교잡([LxY]xD)) 분변에서 채취한 균주로, 에스케리키아 콜라이(Escherichia coli; E.coli)에 대한 바이오필름 억제 및 제거 능력과 이들 소화기 질병 유발 균주들에 항균활성을 갖는 바실러스 코아귤런스 GB-BC-2022 (Bacillus coagulans, GB-BC-2022, 수탁번호: KFCC11948P)를 의미한다.In the present invention, the term " Bacillus coagulans " refers to a strain collected from the feces of high-productivity raised pigs (three-way hybridization ([LxY]xD)) from a pig farm in the Chungnam region. Escherichia coli ( Escherichia coli) ; refers to Bacillus coagulans GB-BC-2022 ( Bacillus coagulans , GB-BC-2022, accession number: KFCC11948P), which has the ability to suppress and remove biofilm against E.coli) and has antibacterial activity against these digestive disease-causing strains. do.
또한, 본 발명은 소화기 질병 유발 균주 에스케리키아 콜라이(Escherichia coli; E.coli)에 대한 바이오필름 억제 및 제거 능력과 이들 소화기 질병 유발 균주들에 항균활성을 갖는 엔테로코커스 페시움 GB-EF-2022 (Enterococcus faecium, GB-EF-2022, 수탁번호: KFCC11946P) 균주를 제공한다.In addition, the present invention provides Enterococcus faecium GB-EF-2022, which has biofilm inhibition and removal ability against Escherichia coli (E.coli), a strain causing digestive diseases, and antibacterial activity against these strains causing digestive diseases. ( Enterococcus faecium , GB-EF-2022, accession number: KFCC11946P) strain is provided.
본 발명에서 용어, 「엔테로코커스 페시움(Enterococcus faecium)」란, 경남 및 전남지역 야생 멧돼지 분변에서 채취한 균주로, 에스케리키아 콜라이(Escherichia coli; E.coli)에 대한 바이오필름 억제 및 제거 능력과 이들 소화기 질병 유발 균주들에 항균활성을 갖는 엔테로코커스 페시움 GB-EF-2022 (Enterococcus faecium, GB-EF-2022, 수탁번호: KFCC11946P)을 의미한다.In the present invention, the term " Enterococcus faecium " refers to a strain collected from wild boar feces in the Gyeongnam and Jeonnam regions, and has the ability to inhibit and remove biofilm against Escherichia coli (E.coli). and Enterococcus faecium GB-EF-2022 ( Enterococcus faecium , GB-EF-2022, accession number: KFCC11946P), which has antibacterial activity against these digestive disease-causing strains.
본 발명에 있어서, 상기 소화기 질병 유발 균주 에스케리키아 콜라이(Escherichia coli; E.coli)는 KCCM 11234, E.coli K88 또는 E.coli F18 중에서 선택된 1 이상일 수 있으며, 이에 한정되는 것은 아니다.In the present invention, the digestive disease-causing strain of Escherichia coli (E.coli) may be one or more selected from KCCM 11234, E.coli K88, or E.coli F18, but is not limited thereto.
소화기 질병 유발 균주 에스케리키아 콜라이(Escherichia coli, a strain that causes digestive diseases ( Escherichia coliEscherichia coli ; E.coli)에 대한 바이오필름 억제 및 제거 능력과 이들 소화기 질병 유발 균주들에 항균활성을 갖는 균주를 포함하는 사료용 조성물; A feed composition containing a strain that has biofilm inhibition and removal ability against E. coli and antibacterial activity against these digestive disease-causing strains.
본 발명은 소화기 질병 유발 균주 에스케리키아 콜라이(Escherichia coli; E.coli)에 대한 바이오필름 억제 및 제거 능력과 이들 소화기 질병 유발 균주들에 항균활성을 갖는 바실러스 코아귤런스 GB-BC-2022 (Bacillus coagulans, GB-BC-2022, 수탁번호: KFCC11948P) 균주를 포함하는 사료용 조성물을 제공한다.The present invention relates to Bacillus coagulans GB-BC-2022 ( Bacillus ), which has the ability to inhibit and remove biofilms against Escherichia coli (E.coli), a strain causing digestive diseases, and has antibacterial activity against these strains. coagulans , GB-BC-2022, accession number: KFCC11948P) A feed composition containing the strain is provided.
또한, 본 발명은 소화기 질병 유발 균주 에스케리키아 콜라이(Escherichia coli; E.coli)에 대한 바이오필름 억제 및 제거 능력과 이들 소화기 질병 유발 균주들에 항균활성을 갖는 엔테로코커스 페시움 GB-EF-2022 (Enterococcus faecium, GB-EF-2022, 수탁번호: KFCC11946P) 균주를 포함하는 사료용 조성물을 제공한다.In addition, the present invention provides Enterococcus faecium GB-EF-2022, which has biofilm inhibition and removal ability against Escherichia coli (E.coli), a strain causing digestive diseases, and antibacterial activity against these strains causing digestive diseases. ( Enterococcus faecium , GB-EF-2022, accession number: KFCC11946P) A feed composition containing the strain is provided.
본 발명에 있어서, 상기 사료용 조성물은 사료 또는 사료 첨가제일 수 있고, 이에 한정되는 것은 아니다.In the present invention, the feed composition may be feed or a feed additive, but is not limited thereto.
본 발명에 있어서, 용어 「사료」란, 동물이 먹고, 섭취하며, 소화시키기 위한 또는 이에 적당한 임의의 천연 또는 인공 규정식, 한끼식 등 또는 상기 한끼식의 성분을 의미한다.In the present invention, the term “feed” means any natural or artificial diet, meal, etc., or a component of the meal, for or suitable for eating, ingestion, and digestion by animals.
본 발명에 있어서, 용어 「사료첨가제」란, 배합사료에 첨가되어 소화력 향상, 병원균 저감, 지방 대사 개선, 항산화 효과, 독성 물질 제거 등의 효과를 나타내며, 종류에는 보존제, 아미노산제, 비타민제, 생균제, 효소제 등이 있다.In the present invention, the term "feed additive" refers to effects such as improvement of digestion, reduction of pathogens, improvement of fat metabolism, antioxidant effect, removal of toxic substances, etc. when added to compound feed. Types include preservatives, amino acids, vitamins, probiotics, etc. There are enzyme preparations, etc.
상기 사료의 종류는 특별히 제한되지 아니하며, 당해 기술 분야에서 통상적으로 사용되는 사료를 사용할 수 있다. 상기 사료의 비제한적인 예로는, 곡물류, 근과류, 식품 가공 부산물류, 조류, 섬유질류, 제약 부산물류, 유지류, 전분류, 박류 또는 곡물 부산물류 등과 같은 식물성 사료; 단백질류, 무기물류, 유지류, 광물성류, 유지류, 단세포 단백질류, 동물성 플랑크톤류 또는 음식물 등과 같은 동물성 사료를 들 수 있다. 이들은 단독으로 사용되거나 2종 이상을 혼합하여 사용될 수 있다.The type of feed is not particularly limited, and feed commonly used in the art can be used. Non-limiting examples of the feed include plant feeds such as grains, roots and fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, cucurbits or grain by-products; Examples include animal feeds such as proteins, inorganic substances, fats and oils, minerals, oils and fats, single-cell proteins, zooplanktons or foods. These may be used alone or in combination of two or more types.
본 발명에 있어서, 본 발명의 신규 균주인 바실러스 코아귤런스 GB-BC-2022 (Bacillus coagulans, GB-BC-2022, 수탁번호: KFCC11948P) 균주와 엔테로코커스 페시움 GB-EF-2022 (Enterococcus faecium, GB-EF-2022, 수탁번호: KFCC11946P) 균주는 본 발명에 따른 사료용 조성물의 총 중량에 대해 0.001중량% 내지 50중량% 포함될 수 있으며, 보다 구체적으로는 0.01중량% 내지 20중량%로 포함될 수 있으나, 이에 제한되지 않고 당업자의 사용 목적에 따라 용이하게 변경할 수 있다.In the present invention, the new strain of the present invention, Bacillus coagulans GB-BC-2022 ( Bacillus coagulans , GB-BC-2022, accession number: KFCC11948P) strain and Enterococcus faecium GB-EF-2022 ( Enterococcus faecium , GB-EF-2022, accession number: KFCC11946P) strains may be included in an amount of 0.001% by weight to 50% by weight, more specifically, in an amount of 0.01% by weight to 20% by weight, based on the total weight of the composition for feed according to the present invention. , but is not limited to this and can be easily changed depending on the purpose of use by those skilled in the art.
본 발명에 있어서, 상기 조성물은 소, 돼지, 양, 염소, 사슴 등 발굽이 둘로 갈라진 동물(우제류), 닭, 오리, 거위, 꿩 등 조류, 장어, 광어 등 어류, 또는 개, 고양이 등의 반려동물 중 어느 하나 이상의 사료용일 수 있으며, 구체적으로는 육계 사료, 양돈 사료 또는 축우 사료일 수 있고, 이에 한정되는 것은 아니다.In the present invention, the composition is used for animals with cloven hooves such as cows, pigs, sheep, goats, and deer (artiodactyls), birds such as chickens, ducks, geese, and pheasants, fish such as eels and flounders, or companion animals such as dogs and cats. It may be used as feed for one or more animals, and specifically may be broiler feed, pig feed, or cattle feed, but is not limited thereto.
본 발명에 있어서, 상기 조성물은 부형제를 더 포함할 수 있다. 구체적으로, 본 발명에 있어서, 「부형제」는 약제에 적당한 굳기나 형상을 주기 위해서, 또는 주제(主劑)의 양이 적은 경우에 일정 용량, 중량을 주어 취급하기 쉬운 크기로 할 목적으로 첨가되는 성분을 의미한다. 상기 부형제는 사료용 조성물의 일 예인 사료 또는 사료첨가제의 제조에 있어서 통상적으로 사용되는 것을 포함할 수 있고, 이에 한정되지는 않으나, 밀기울(wheat bran), 옥수수 분말, 곡류 전분, 실리카분말, 규조토 중 어느 하나 이상을 포함할 수 있으며, 사료 등 기준 및 규격(농림축산식품부 고시 제2014-106호)에서 인정하고 있는 부형제 등을 포함할 수 있다. 부형제는 전체 사료용 조성물 100 중량부에 대해 50 내지 99 중량부로 포함될 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the composition may further include an excipient. Specifically, in the present invention, “excipient” is added to give the drug an appropriate hardness or shape, or to give it a certain volume and weight when the amount of the main drug is small to make it easy to handle. It means ingredients. The excipients may include those commonly used in the production of feed or feed additives, which are examples of feed compositions, but are not limited thereto, and may include any of wheat bran, corn powder, grain starch, silica powder, and diatomaceous earth. It may contain one or more excipients recognized in the standards and specifications for feed, etc. (Ministry of Agriculture, Food and Rural Affairs Notice No. 2014-106). The excipient may be included in an amount of 50 to 99 parts by weight based on 100 parts by weight of the total feed composition, but is not limited thereto.
본 발명에 있어서, 상기 사료용 조성물은 당업계의 통상적인 제형으로 제조될 수 있으며, 분말 또는 소정의 형태를 가진 펠릿 형상으로 구현될 수 있으나 이에 한정되는 것은 아니다. 또한, 본 발명에 따른 사료용 조성물은 체내 흡수를 증진시키기 위해 나노화되거나, 소장에서의 항균 활성을 강화하기 위해 지방코팅 제형으로 제조될 수 있고, 이에 한정되는 것은 아니다.In the present invention, the composition for feed may be manufactured in a conventional formulation in the art, and may be implemented in the form of powder or pellets having a predetermined shape, but is not limited thereto. In addition, the composition for feed according to the present invention may be nano-sized to improve absorption in the body, or may be manufactured in a fat-coated formulation to enhance antibacterial activity in the small intestine, but is not limited thereto.
위에서 기술된 균주에 관한 내용은 서로 모순되지 않는 한 상기 사료용 조성물에 모두 적용될 수 있다.The information regarding the strains described above can be applied to the feed composition as long as they do not contradict each other.
이하, 본 발명을 실시예로서 더욱 상세하게 설명하나, 본 발명이 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited by the examples.
실시예.Example.
이하 실시예에서 사용하는 바실러스 코아귤런스 GB-BC-2022 (Bacillus coagulans, GB-BC-2022, 수탁번호: KFCC11948P) 균주는 2022년 11월 01일 자로 한국미생물보존센터(국내)에 기탁하여 수탁번호(KFCC11948P)를 받은 균주이고, 엔테로코커스 페시움 GB-EF-2022 (Enterococcus faecium, GB-EF-2022, 수탁번호: KFCC11946P) 균주는 2022년 11월 01일 자로 한국미생물보존센터(국내)에 기탁하여 수탁번호(KFCC11946P)를 받은 균주이다.The Bacillus coagulans GB-BC-2022 ( Bacillus coagulans , GB-BC-2022, accession number: KFCC11948P) strain used in the examples below was deposited and entrusted to the Korea Microorganism Conservation Center (Korea) on November 1, 2022. It is a strain that received a number (KFCC11948P), and the Enterococcus faecium GB-EF-2022 ( Enterococcus faecium , GB-EF-2022, accession number: KFCC11946P) strain was registered with the Korea Microbiological Conservation Center (Korea) as of November 1, 2022. This strain was deposited and received an accession number (KFCC11946P).
실시예 1. 미생물 스크리닝을 통한 최종 후보 미생물 선발Example 1. Selection of final candidate microorganisms through microbial screening
1. 미생물 스크리닝1. Microbial screening
(1) 멧돼지 분변, 돈사 주변 환경 등에서 미생물 분리(1) Isolation of microorganisms from wild boar feces and the environment around pig farms, etc.
본 발명에서는 돼지 질병 예방을 위한 유용 미생물을 선발하기 위해 충남 및 경기도 지역의 양돈장 분변, 경남 및 전남 지역의 야생 멧돼지 분변 및 음식물 쓰레기를 비롯한 식품 등에서 샘플을 채취하였고, 채취한 샘플로부터 150여종의 미생물을 분리하였다. 미생물의 분리는 LB 배지(Tryptone, 10 g/L; Yeast extract, 5 g/L; Sodium chloride, 10 g/L; Agar, 15 g/L), MRS 배지(Proteose Peptone No.3 10 g/L; Beef Extract 10 g/L; Yeast Extract 5 g/L; Dextrose 20 g/L; Polysorbate80 1 g/L; Ammonium Citrate 2 g/L; Sodium Acetate 5 g/L; Magnesium Sulfate 0.1 g/L; Manganese Sulfate 0.05 g/L; Dipotassium Phosphate 2 g/L; Agar 15 g/L) 및 PDA 배지(Potato starch, 4 g/L, Dextrose, 20 g/L, Agar, 15 g/L)를 이용하였다. 순수 분리된 미생물은 각각의 증식 배지를 이용하여 33℃ 에서 24시간 이상 배양 후 15% glycerol stock 과 1:1 비율로 혼합하여 -70℃ depp freezer에 보관하였다. In the present invention, in order to select useful microorganisms for preventing pig diseases, samples were collected from pig farm feces in Chungnam and Gyeonggi-do regions, wild boar feces and food waste in Gyeongnam and Jeollanam-do regions, and food, and about 150 types of microorganisms were collected from the collected samples. was separated. Isolation of microorganisms was performed using LB medium (Tryptone, 10 g/L; Yeast extract, 5 g/L; Sodium chloride, 10 g/L; Agar, 15 g/L), MRS medium (Proteose Peptone No.3 10 g/L) ; Beef Extract 10 g/L; Yeast Extract 5 g/L; Dextrose 20 g/L; Polysorbate80 1 g/L; Ammonium Citrate 2 g/L; Sodium Acetate 5 g/L; Magnesium Sulfate 0.1 g/L; Manganese Sulfate 0.05 g/L; Dipotassium Phosphate 2 g/L; Agar 15 g/L) and PDA medium (Potato starch, 4 g/L, Dextrose, 20 g/L, Agar, 15 g/L) were used. Purely isolated microorganisms were cultured at 33°C for more than 24 hours using each growth medium, then mixed with 15% glycerol stock in a 1:1 ratio and stored in a depp freezer at -70°C.
(2) 미생물의 유전학적 동정(2) Genetic identification of microorganisms
선발한 미생물들의 명확한 동정을 위하여 16S rRNA sequencing을 진행하였다. 순수 분리된 균주는 ㈜마크로젠(Seoul, Korea)에 염기서열 분석을 의뢰하여 16S rRNA sequencing을 통해 동정하였으며, NCBI GenBank database의 염기 서열 비교를 통해 미생물을 확인하였다.16S rRNA sequencing was performed to clearly identify the selected microorganisms. The pure isolated strain was identified through 16S rRNA sequencing by requesting base sequence analysis from Macrogen Co., Ltd. (Seoul, Korea), and the microorganism was confirmed through base sequence comparison in the NCBI GenBank database.
확인된 150여종의 미생물들은 행정규칙 ‘사료 등의 기준 및 규격‘의 제 5조 ‘보조사료의 범위' 에 명시된 균주의 허용 범위에 따라 분류하여 축산 사료로 이용할 수 있는 22종의 후보 미생물을 선발하였다. 이들은 혐기성 미생물 17종, 호기성 미생물 5종으로 구성되어있다 (표 1 참조).About 150 types of microorganisms identified were classified according to the allowable range of strains specified in Article 5, ‘Scope of Supplementary Feed’ of the Administrative Regulations ‘Standards and Specifications for Feed, etc.’, and 22 types of candidate microorganisms that can be used as livestock feed were selected. did. They consist of 17 types of anaerobic microorganisms and 5 types of aerobic microorganisms (see Table 1).
(Bacillus coagulans GB-BC-2022, KFCC11948P) GBD1
( Bacillus coagulans GB-BC-2022, KFCC11948P)
(Enterococcus faecium GB-EF-2022, KFCC11946P)GM9
( Enterococcus faecium GB-EF-2022, KFCC11946P )
2. 미생물의 특성2. Characteristics of microorganisms
(1) (One) In-vitroIn-vitro 바이오필름 억제능 평가 Evaluation of biofilm inhibition ability
선발된 22종의 미생물들의 소화기 질병유발 균주 Escherichia coli 3종(KCCM 11234, E.coli K88, E.coli F18)에 대한 바이오필름 억제 및 제거 능력을 확인하기 위하여 Biofilm Formation Assay Kit(Dojindo)를 이용하였다. Biofilm Formation Assay Kit (Dojindo) was used to confirm the biofilm inhibition and removal ability of 22 selected microorganisms against 3 types of Escherichia coli (KCCM 11234, E.coli K88, E.coli F18) that cause digestive diseases. did.
- 바이오필름 억제능 평가(Biofilm inhibition test) 방법- Biofilm inhibition test method
i) 바이오필름 생성 균주(KCCM 11234, E.coli K88, E.coli F18) 각각을 루리아-버타니(Luria-Bertani) broth (LB broth)를 이용하여 33℃, 24~48시간 배양 후 O.D 0.2(600 nm)로 희석한다.i) Each of the biofilm-producing strains (KCCM 11234, E.coli K88, E.coli F18) was cultured at 33°C for 24 to 48 hours using Luria-Bertani broth (LB broth), and then the OD was 0.2. (600 nm).
ii) 희석한 바이오필름 생성 균주 배양액을 96-well plate에 180 μl를 분주한 후 48시간 배양하여 바이오필름을 생성시킨다.ii) Dispense 180 μl of the diluted biofilm-producing strain culture into a 96-well plate and incubate for 48 hours to generate a biofilm.
iii) 바이오필름 생성 균주 배양액에 담겨있던 lid를 멸균 식염수로 3번 세척한다.iii) Wash the lid contained in the biofilm-producing strain culture medium three times with sterile saline solution.
iv) 선발된 22종의 미생물의 균주 배양액(O.D 0.2(600 nm))을 96-well plate에 180 μl를 분주한 후 세척된 lid를 넣고 24~48시간 배양한다. 이 때, 균주 배양액은 유산균의 경우 MRS broth, 바실러스 균의 경우 루리아-버타니(Luria-Bertani) broth (LB broth)를 이용하여 33℃, 24~48시간 배양하여 준비한다.iv) Dispense 180 μl of the selected 22 types of microorganism strain culture (O.D 0.2 (600 nm)) into a 96-well plate, add a washed lid, and incubate for 24 to 48 hours. At this time, the strain culture medium is prepared by culturing at 33°C for 24 to 48 hours using MRS broth for lactic acid bacteria and Luria-Bertani broth (LB broth) for Bacillus bacteria.
v) 선발된 22종의 미생물의 균주 배양액에 담겨있던 lid를 멸균 식염수로 3번 세척 후 crystal violet solution을 이용하여 30분간 염색시킨다.v) The lid contained in the strain culture medium of the selected 22 types of microorganisms was washed three times with sterile saline solution and then stained for 30 minutes using crystal violet solution.
vi) 염색된 lid를 멸균식염수로 3번 세척한다.vi) Wash the stained lid three times with sterile saline solution.
vii) 바이오필름만 남은 세척된 lid를 에탄올(ethanol)을 이용하여 15분간 녹인 후 590 nm에서 흡광도를 측정하여 바이오필름 생성 정도를 확인한다.vii) Dissolve the washed lid with only the biofilm remaining using ethanol for 15 minutes and then measure the absorbance at 590 nm to check the degree of biofilm formation.
- 바이오필름 억제능 평가 결과- Biofilm inhibition ability evaluation results
상기 방법을 통해 실험한 결과, 본 발명에 따른 신규한 균주인 바실러스 코아귤런스 GB-BC-2022 (Bacillus coagulans, GB-BC-2022, 수탁번호: KFCC11948P) 균주, 엔테로코커스 페시움 GB-EF-2022 (Enterococcus faecium, GB-EF-2022, 수탁번호: KFCC11946P) 균주 2종의 미생물이 Escherichia coli 3종(KCCM 11234, E.coli K88, E.coli F18)에 대하여 바이오필름 억제 및 제거에 70% 이상 효과가 있는 것으로 확인되었다 (표 2 참조). As a result of the experiment using the above method, the new strain according to the present invention, Bacillus coagulans GB-BC-2022 ( Bacillus coagulans , GB-BC-2022, accession number: KFCC11948P) strain, Enterococcus faecium GB-EF- 2022 ( Enterococcus faecium , GB-EF-2022, accession number: KFCC11946P) Two types of microorganisms inhibit and remove biofilms by 70% against three types of Escherichia coli (KCCM 11234, E.coli K88, E.coli F18) It was confirmed to have the above effect (see Table 2).
특히, 선발된 22종의 미생물 균주 중에서, 본 발명에 따른 신규한 균주인 바실러스 코아귤런스 GB-BC-2022 (Bacillus coagulans, GB-BC-2022, 수탁번호: KFCC11948P) 균주, 엔테로코커스 페시움 GB-EF-2022 (Enterococcus faecium, GB-EF-2022, 수탁번호: KFCC11946P) 균주 2종의 미생물이 바이오필름 억제 및 제거에 있어서 탁월한 효과가 있음을 확인하였다.In particular, among the 22 selected microbial strains, the new strain according to the present invention, Bacillus coagulans GB-BC-2022 ( Bacillus coagulans , GB-BC-2022, accession number: KFCC11948P) strain, Enterococcus faecium GB -EF-2022 ( Enterococcus faecium , GB-EF-2022, accession number: KFCC11946P) It was confirmed that two types of microorganisms were excellent in suppressing and removing biofilm.
(Bacillus coagulans GB-BC-2022, KFCC11948P)GBD1
( Bacillus coagulans GB-BC-2022, KFCC11948P)
(Enterococcus faecium)GBD5
( Enterococcus faecium )
(Lactobacillus plantarum)GBD8
( Lactobacillus plantarum )
(Lactobacillus reuteri)GBD11
( Lactobacillus reuteri )
(Lactobacillus paracasei)GBD27
( Lactobacillus paracasei )
(Lactobacillus plantarum)GBD28
( Lactobacillus plantarum )
(Lactobacillus reuteri)GBD31
( Lactobacillus reuteri )
(Bacillus coagulans)GBD32
( Bacillus coagulans )
(Pediococcus pentosaceus)GM3
( Pediococcus pentosaceus )
(Enterococcus faecium GB-EF-2022, KFCC11946P)GM9
( Enterococcus faecium GB-EF-2022, KFCC11946P)
(Enteroccocus faecium)GM11
( Enteroccocus faecium )
(Enterococcus faecium)GM13
( Enterococcus faecium )
(Enteroccocus faecium)GM15
( Enteroccocus faecium )
(Enterococcus faecium)GM21
( Enterococcus faecium )
(Lactoplantbacillus plantarum)GM22
( Lactoplantbacillus plantarum )
(Enterococcus faecium)GM24
( Enterococcus faecium )
(Bacillus subtilis)GL26
( Bacillus subtilis )
(Bacillus subtilis)GL27
( Bacillus subtilis )
(Bacillus subtilis)GL28
( Bacillus subtilis )
(Bacillus licheniformis)GL29
( Bacillus licheniformis )
(Bacillus subtilis)GL30
( Bacillus subtilis )
(2) (2) In-vivoIn-vivo 바이오필름 억제능 평가 Evaluation of biofilm inhibition ability
바이오필름을 유도하지 않고 유용미생물도 투여하지 않은 음성대조군(negative control; NC)과, 바이오필름을 유도하고 유용미생물은 투여하지 않은 양성대조군(positive control; PC), 바이오필름을 유도하고 바실러스 코아귤런스 GB-BC-2022 (Bacillus coagulans, GB-BC-2022, 수탁번호: KFCC11948P) 균주, 엔테로코커스 페시움 GB-EF-2022 (Enterococcus faecium, GB-EF-2022, 수탁번호: KFCC11946P) 균주를 각각 투여한 2개 군(G3 및 G4)으로 총 4개의 군에 군당 8마리의 C57BL/6(수컷, 6주령) 마우스로 실험하였다 (표 3 참조). 여기서 유용미생물이라 함은 본 발명에 따른 바실러스 코아귤런스 GB-BC-2022 (Bacillus coagulans, GB-BC-2022, 수탁번호: KFCC11948P) 균주, 엔테로코커스 페시움 GB-EF-2022 (Enterococcus faecium, GB-EF-2022, 수탁번호: KFCC11946P) 균주를 의미한다.A negative control (NC) group in which biofilm was not induced and beneficial microorganisms were not administered, a positive control group (PC) in which biofilm was induced and beneficial microorganisms were not administered, and a biofilm induced and beneficial microorganisms were not administered (positive control (PC)). lance GB-BC-2022 ( Bacillus coagulans , GB-BC-2022, accession number: KFCC11948P) strain, and Enterococcus faecium GB-EF-2022 ( Enterococcus faecium , GB-EF-2022, accession number: KFCC11946P) strain, respectively. The experiment was conducted with 8 C57BL/6 (male, 6-week-old) mice per group in a total of 4 groups (G3 and G4) (see Table 3). Here, useful microorganisms include Bacillus coagulans GB-BC-2022 ( Bacillus coagulans , GB-BC-2022, Accession number: KFCC11948P) strain, Enterococcus faecium GB-EF-2022 ( Enterococcus faecium , GB) according to the present invention. -EF-2022, accession number: KFCC11946P) refers to the strain.
- - In-vivoIn-vivo 바이오필름 억제능 평가 방법 Method for evaluating biofilm inhibition ability
i) C57BL/6 마우스에 5x10^9 cfu/ml 농도의 Escherichia coli(KCCM 11234) 200ul씩 D0와 D6에 각 1회씩, 총 2회 bid 투여하여 바이오필름을 유도한다.i) Induce biofilm by administering 200ul of Escherichia coli (KCCM 11234) at a concentration of 5x10^9 cfu/ml to C57BL/6 mice bid twice, once each on D0 and D6.
ii) 바이오필름이 유도된 C57BL/6 마우스에 주 1회 2주간 유용미생물을 5x10^9 cfu/ml 농도로 준비하여 마리당 200ul씩 D8부터 D23까지 오전 10시와 4시에 약 6시간 간격을 두고 1일 2회 bid 투여하여 바이오필름 억제 및 제거 효능을 확인한다.ii) Prepare useful microorganisms to biofilm-induced C57BL/6 mice once a week for 2 weeks at a concentration of 5x10^9 cfu/ml, and administer 200ul per mouse at 10:00 am and 4:00 pm from D8 to D23 at approximately 6-hour intervals. Confirm biofilm inhibition and removal efficacy by administering bid twice a day.
- - In-vivoIn-vivo 바이오필름 억제능 평가 결과 Biofilm inhibition ability evaluation results
바이오필름 억제 효과는 조직병리학적 분석을 통해 평가하였으며 평가 체계는 400배 배율로 장 융모를 이루는 상피세포의 내강측 표면 부위를 개체당 5개 단면에서 관찰하고 병변을 점수화하여 그 결과를 도 1에 나타내었다. 점수화 기준으로는, 바이오필름이 관찰되지 않는 경우 0점, 바이오필름이 세균체의 형태를 보이는 경우 1점, 바이오필름이 선형으로 관찰되는 경우 2점, 바이오필름과 병변이 동반되는 경우 3점으로 처리하였다. 도 1을 참조하면, 상기 실험 결과 본 발명에 따른 신규한 균주인 바실러스 코아귤런스 GB-BC-2022 (Bacillus coagulans, GB-BC-2022, 수탁번호: KFCC11948P) 균주, 엔테로코커스 페시움 GB-EF-2022 (Enterococcus faecium, GB-EF-2022, 수탁번호: KFCC11946P) 균주 2종의 미생물 모두 양성대조군(G2, PC)과 비교하여 바이오필름 억제 측면에서 통계적으로 유의한 결과가 관찰되어 Escherichia coli에 대하여 바이오필름 억제 및 제거에 유의적인 효과가 있는 것으로 확인되었다(도 1 참조, 도 1에서, P- value < 0.05인 경우 유의성이 있다고 판단했을 때 양성대조군(PC) 처리구, 그리고 음성대조군(NC) 처리구 간 유의적 차이를 확인할 수 있으며, 각 처리구 간 유의성은 superscript (*, + or none)으로 표기하였음).The biofilm inhibition effect was evaluated through histopathological analysis, and the evaluation system observed the luminal surface area of the epithelial cells forming intestinal villi at 400x magnification in five sections per individual and scored the lesions. The results are shown in Figure 1. indicated. The scoring criteria is 0 points if no biofilm is observed, 1 point if the biofilm appears in the form of a bacterial body, 2 points if the biofilm is observed in a linear form, and 3 points if the biofilm and lesion are accompanied. Processed. Referring to Figure 1, as a result of the above experiment, the new strain according to the present invention, Bacillus coagulans GB-BC-2022 ( Bacillus coagulans , GB-BC-2022, accession number: KFCC11948P) strain, Enterococcus faecium GB-EF -2022 ( Enterococcus faecium , GB-EF-2022, accession number: KFCC11946P) statistically significant results were observed in terms of biofilm inhibition compared to the positive control group (G2, PC) for both types of microorganisms, indicating that it was effective against Escherichia coli. It was confirmed that there was a significant effect on biofilm inhibition and removal (see Figure 1, when P-value < 0.05 was judged to be significant, the positive control (PC) treatment group, and the negative control (NC) treatment group Significant differences between treatments can be confirmed, and the significance between each treatment group is indicated with superscript (*, + or none).
[표 3][Table 3]
(표 3에서, a와 b는 다음과 같다. a: negative control, b: positive control, 상기 표 3에서 투여량(μl)이라 함은, 바이오필름 유도물질 및 미생물 투여량은 마우스의 권장 투여 용량을 참고하여 선정한 것으로서, 본 실험에서는 미생물의 효과만 확인한 것임.)(In Table 3, a and b are as follows. a: negative control, b: positive control. In Table 3, the dosage (μl) refers to the dosage of biofilm-inducing substances and microorganisms, which is the recommended dosage for mice. It was selected with reference to , and only the effect of microorganisms was confirmed in this experiment.)
(3) 항균 활성 평가(3) Evaluation of antibacterial activity
항균 활성 평가 방법Antibacterial activity evaluation method
바이오필름 억제 및 제거 능력이 있는 2종의 미생물의 Escherichia coli 3종(KCCM 11234, E.coli K88, E.coli F18)에 대한 항균활성을 최저생장저해농도(minimum inhibitory concentration; MIC) 시험을 통하여 분석하였다. 최저생장저해농도 시험은 병원성 균주인 E. coli 3종 (KCCM 11234, E.coli K88, E.coli F18) 각각을 LB 배지에 접종하고 37℃ 배양기에서 24시간 배양한 다음 분광광도계를 이용하여 630 nm에서 흡광도 값이 0.2±0.03이 되도록 희석하여 준비하였다. 그리고 나서, 본 발명에 따른 신규한 균주인 바실러스 코아귤런스 GB-BC-2022 (Bacillus coagulans, GB-BC-2022, 수탁번호: KFCC11948P) 균주, 엔테로코커스 페시움 GB-EF-2022 (Enterococcus faecium, GB-EF-2022, 수탁번호: KFCC11946P) 균주 2종 각각을 MRS 및 LB배지 50 mL에 각각 1%씩 접종한 다음 35℃, 130 rpm 조건으로 24시간 동안 진탕배양하고 원심분리기를 이용하여 4000 rpm에서 10분 간 원심분리 하였다. 피펫을 이용하여 상등액 5 mL을 분리한 다음 0.2 μm 시린지 필터(syringe filter)에 여과하여 샘플을 준비하였다. 96 well plate에 여과된 배양액과 병원성 균주 배양액을 1:1 비율로 혼합하여 분주하고 37℃ 진탕배양기에서 48시간 동안 발효를 진행하였다. 발효 24시간, 48시간 차에 흡광도값을 측정하여 결과를 도 3 내지 도 5에 나타내었으며, 양성 대조군(Postive control; PC)는 병원성 균주 배양액과 미생물이 들어있지 않은 LB 배지를 1:1로 혼합하여 분주하였고, 음성 대조군(Negative control; NC)은 멸균 증류수와 미생물이 들어있지 않은 LB배지를 1:1로 혼합하여 분주하였다. The antibacterial activity of two types of microorganisms capable of inhibiting and removing biofilms against three types of Escherichia coli (KCCM 11234, E.coli K88, E.coli F18) was tested through minimum inhibitory concentration (MIC) test. analyzed. In the minimum growth inhibition concentration test, each of the three pathogenic strains of E. coli (KCCM 11234, E.coli K88, E.coli F18) was inoculated into LB medium, cultured in an incubator at 37°C for 24 hours, and then measured at 630 degrees Celsius using a spectrophotometer. It was prepared by diluting the absorbance value to 0.2±0.03 nm. Then, the new strain according to the present invention, Bacillus coagulans GB-BC-2022 ( Bacillus coagulans , GB-BC-2022, accession number: KFCC11948P) strain, Enterococcus faecium GB-EF-2022 ( Enterococcus faecium , GB-EF-2022, accession number: KFCC11946P) Inoculate 1% of each of the two strains into 50 mL of MRS and LB media, then culture with shaking at 35°C and 130 rpm for 24 hours, and centrifuge at 4000 rpm. It was centrifuged for 10 minutes. A sample was prepared by separating 5 mL of supernatant using a pipette and filtering it through a 0.2 μm syringe filter. The filtered culture medium and the pathogenic strain culture medium were mixed and distributed in a 1:1 ratio in a 96 well plate, and fermentation was carried out in a shaking incubator at 37°C for 48 hours. Absorbance values were measured 24 and 48 hours after fermentation, and the results are shown in Figures 3 to 5. The positive control (PC) was a 1:1 mixture of pathogenic strain culture medium and LB medium without microorganisms. The negative control (NC) was dispensed by mixing sterilized distilled water and LB medium without microorganisms in a ratio of 1:1.
항균 활성 평가 결과Antibacterial activity evaluation results
상기 방법을 통해 진행된 실험 결과를 참조하면, 본 발명에 따른 신규한 균주인 바실러스 코아귤런스 GB-BC-2022 (Bacillus coagulans, GB-BC-2022, 수탁번호: KFCC11948P) 균주, 엔테로코커스 페시움 GB-EF-2022 (Enterococcus faecium, GB-EF-2022, 수탁번호: KFCC11946P) 균주 2종의 미생물이 Escherichia coli 3종(KCCM 11234, E.coli K88, E.coli F18)에 대하여 항균활성을 나타내었다 (도 3 내지 도 5 참조).Referring to the experimental results conducted through the above method, the new strain according to the present invention, Bacillus coagulans GB-BC-2022 ( Bacillus coagulans , GB-BC-2022, accession number: KFCC11948P) strain, Enterococcus faecium GB -EF-2022 ( Enterococcus faecium , GB-EF-2022, accession number: KFCC11946P) Two types of microorganisms showed antibacterial activity against three types of Escherichia coli (KCCM 11234, E.coli K88, E.coli F18) (See Figures 3 to 5).
그러나, 양성 대조군(G2)의 경우, 24시간, 48시간에서 O.D값이 증가하여 Escherichia coli 3종(KCCM 11234, E.coli K88, E.coli F18)이 성장한 것을 확인할 수 있었고 이는 항균활성이 없는 것을 나타내었다. However, for the positive control (G2), The OD value increased at 24 and 48 hours, confirming the growth of three types of Escherichia coli (KCCM 11234, E.coli K88, E.coli F18), indicating no antibacterial activity.
3. 결론3. Conclusion
본 발명은 환경 및 멧돼지 분변에서 분리한 균주 중 바실러스 코아귤런스 GB-BC-2022 (Bacillus coagulans, GB-BC-2022, 수탁번호: KFCC11948P) 균주, 엔테로코커스 페시움 GB-EF-2022 (Enterococcus faecium, GB-EF-2022, 수탁번호: KFCC11946P) 2종은 소화기 질병 유발 균주 Escherichia coli 3종(KCCM 11234, E.coli K88, E.coli F18)에 대한 바이오필름 억제 및 제거 능력과 이들 소화기 질병 유발 균주들에 항균활성을 가지고 있어 프로바이오틱스 등 사료 및 사료첨가제에 활용 가능할 것으로 판단된다.The present invention relates to Bacillus coagulans GB-BC-2022 ( Bacillus coagulans , GB-BC-2022, accession number: KFCC11948P) strain, Enterococcus faecium GB-EF-2022 ( Enterococcus faecium ) among strains isolated from the environment and wild boar feces. , GB-EF-2022, accession number: KFCC11946P), two of which have the ability to inhibit and remove biofilms against three types of Escherichia coli (KCCM 11234, E.coli K88, E.coli F18) that cause digestive diseases and cause these digestive diseases. Since the strains have antibacterial activity, it is believed that they can be used in feed and feed additives such as probiotics.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 관련 기술 분야의 통상의 지식을 가진 자에게 있어 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구범위와 그의 등가물에 의하여 정의될 것이다.As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
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