KR102443494B1 - Novel Strain of Bifidobacterium animalis CACC 789, and feed composition using thereof - Google Patents

Novel Strain of Bifidobacterium animalis CACC 789, and feed composition using thereof Download PDF

Info

Publication number
KR102443494B1
KR102443494B1 KR1020210099278A KR20210099278A KR102443494B1 KR 102443494 B1 KR102443494 B1 KR 102443494B1 KR 1020210099278 A KR1020210099278 A KR 1020210099278A KR 20210099278 A KR20210099278 A KR 20210099278A KR 102443494 B1 KR102443494 B1 KR 102443494B1
Authority
KR
South Korea
Prior art keywords
strain
cacc
present
bifidobacterium animalis
activity
Prior art date
Application number
KR1020210099278A
Other languages
Korean (ko)
Inventor
김양선
장현준
김정애
송정섭
김대혁
Original Assignee
재단법인 농축산용미생물산업육성지원센터
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 재단법인 농축산용미생물산업육성지원센터 filed Critical 재단법인 농축산용미생물산업육성지원센터
Priority to KR1020210099278A priority Critical patent/KR102443494B1/en
Application granted granted Critical
Publication of KR102443494B1 publication Critical patent/KR102443494B1/en

Links

Images

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, 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
    • C12N1/20Bacteria; Culture media therefor
    • C12N1/205Bacterial isolates
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23KFODDER
    • A23K10/00Animal feeding-stuffs
    • A23K10/10Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12RINDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00Microorganisms ; Processes using microorganisms
    • C12R2001/01Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Zoology (AREA)
  • Polymers & Plastics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Animal Husbandry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Physiology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention relates to a novel strain Bifidobacterium animalis CACC 789, and a feed composition for companion animals comprising the same as an active component. Since a Bifidobacterium animalis CACC 789 strain, and a culture medium thereof exhibit antibacterial activity against livestock pathogens, and exhibit excellent effect of enhancing immunity and suppressing inflammation, and thus can be usefully used for antibacterial, immune enhancement, and anti-inflammatory purposes of companion animals.

Description

신균주 비피도박테리움 애니멀리스 CACC 789 및 이를 유효성분으로 함유하는 반려동물용 사료 조성물{Novel Strain of Bifidobacterium animalis CACC 789, and feed composition using thereof}New strain Bifidobacterium animalis CACC 789 and a feed composition for companion animals containing the same as an active ingredient {Novel Strain of Bifidobacterium animalis CACC 789, and feed composition using thereof}

본 발명은 비피도박테리움 애니멀리스 CACC 789 및 이를 유효성분으로 함유하는 반려동물용 사료 조성물에 관한 것으로, 더욱 구체적으로 가축 병원균에 대한 항균 활성을 나타내고, 면역증진 및 염증 억제에 우수한 효과가 있는 비피도박테리움 애니멀리스 CACC 789 및 이를 이용한 반려동물용 사료 조성물에 관한 것이다.The present invention relates to Bifidobacterium animallis CACC 789 and a feed composition for companion animals containing the same as an active ingredient. It relates to a gamblerium animalis CACC 789 and a feed composition for companion animals using the same.

현재 우리나라는 핵가족화로 인하여 1인 및 2인 가구가 급격하게 증가하고 있다. 이로 인해, 정서적 안정을 찾기 위해 반려견, 반려묘 등 반려동물을 키우고 있는 인구도 증가하고 있다. Currently, the number of single-person and two-person households is rapidly increasing in Korea due to the nuclear family. As a result, the number of people raising companion animals such as dogs and cats to find emotional stability is increasing.

그러나, 우리나라의 반려동물은 대부분 주택 내에서 생활하기 때문에 운동이 부족하고 스트레스를 많이 받아 손쉽게 소화 및 대사 활성이 낮아지게 되고, 면역력이 낮아지는 경향이 있다. 이로 인해 사람과 유사하게 대사성 질환이나 면역 관련 질환이 발생하고, 향후 더욱 치명적인 내외과적 질환으로 악화되어 반려동물과 보호자의 삶의 질이 떨어지는 문제가 발생하고 있다.However, since most companion animals in Korea live in houses, they lack exercise and are under a lot of stress, so their digestive and metabolic activities are easily lowered, and their immunity tends to be lowered. Due to this, metabolic diseases or immune-related diseases similar to humans occur, and in the future, it will worsen into more fatal internal and external diseases, resulting in a decrease in the quality of life of companion animals and their guardians.

현재 우리나라 동물 의료 및 복지에 대한 관심이 증가하고 있으나, 현실적인 치료 비용이 높아 의료비 부담이 크므로, 반려동물의 운동 부족으로 인한 대사성 질환이나 면역 관련 질환의 발병을 손쉽게 예방할 수 있는 제제의 개발이 시급한 실정이다.Currently, interest in animal health and welfare in Korea is increasing, but realistic treatment costs are high and medical expenses are high. the current situation.

한편, 살아있는 미생물인 프로바이오틱스(probiotics)는 장내에서 미생물의 기능을 활성화하여 동물의 건강을 증진한다(Fuller, 1989). 많은 연구자들이 락토바실러스와 비피도박테리움과 같은 프로바이오틱스(probiotics) 균주가 설사 등과 같은 증상을 감소시키거나, 방어한다는 사실을 보고하였다(Gilliland, 1990). On the other hand, living microorganisms, probiotics, promote the health of animals by activating the functions of microorganisms in the intestine (Fuller, 1989). Many researchers have reported that probiotic strains such as Lactobacillus and Bifidobacterium reduce or protect symptoms such as diarrhea (Gilliland, 1990).

이들 프로바이오틱스 균주는 장내로 유입된 후 장내 상피세포에 착생하게 되어 유해 미생물의 장 정착을 방지하고 항균 물질을 분비함으로써 유해 미생물의 생육을 억제하고 설사와 변비를 개선할 뿐만 아니라, 면역활성 증진, 항암 작용 등의 효능을 가지고 있다. 프로바이오틱스 균주가 장내에 유입되어 자체적인 대사활동을 통해 분비하는 물질은 숙주에 많은 이로움을 주기 때문에 프로바이오틱스 균주를 이용한 많은 연구가 진행되고 있다. 프로바이오틱스 균주는 대부분 동물의 장내에 정상적으로 존재하며 장내 미생물의 균형을 유지하고 숙주의 건강에 유익한 영향을 끼친다. These probiotic strains, after being introduced into the intestine, become epithelial cells in the intestine to prevent the colonization of harmful microorganisms and secrete antibacterial substances to suppress the growth of harmful microorganisms and improve diarrhea and constipation, as well as enhance immune activity, anticancer It has effects such as action. Since probiotic strains are introduced into the intestine and secreted through their own metabolic activities, many studies are being conducted using probiotic strains because they provide many benefits to the host. Probiotic strains are normally present in the intestinal tract of most animals, maintain the balance of the intestinal microflora and have a beneficial effect on the health of the host.

따라서 장내 유용한 프로바이오틱스 균주를 선정하기 위해서는 다음의 몇 가지 중요한 성상을 확인하며 선정한다(Fuller, 1989; Garriga et al., 1998; Martin et al., 2006; Mishra and Prasad, 2005). 즉, 프로바이오틱스 균주가 소화관 내에서도 생존해야 하므로, 내산성이나 내담즙성을 가져야 하고, 장에 도달할 때까지 소화관에서 콜로니를 형성해야 한다. 또한, 식품이나 사료 또는 임상적으로 사용 시 안전해야 하며, 동시에 숙주에 유익한 영향을 줄 수 있어야 한다. 최근에는 많은 기능적인 특성이 있는 프로바이오틱스 균주들이 개발되고 있는데, 병원체의 증식을 억제하는 기능을 갖는 프로바이오틱스 균주가 있고, BSH 활성(Begley et al., 2006; Pereira et al., 2003) 또는 전분가수분해 활성(amylolytic activity, Lee et al., 2001; Vizoso Pinto et al., 2006)을 갖는 프로바이오틱스 균주 등이 있다.Therefore, in order to select a useful intestinal probiotic strain, several important characteristics are identified and selected (Fuller, 1989; Garriga et al., 1998; Martin et al., 2006; Mishra and Prasad, 2005). That is, since the probiotic strain must survive in the digestive tract, it must have acid resistance or bile resistance, and must form colonies in the digestive tract until it reaches the intestine. In addition, it must be safe for food, feed, or clinical use, while at the same time having a beneficial effect on the host. Recently, probiotic strains with many functional properties have been developed, and there are probiotic strains having the function of inhibiting the proliferation of pathogens, BSH activity (Begley et al., 2006; Pereira et al., 2003) or starch hydrolysis. There are probiotic strains having amylolytic activity, Lee et al., 2001; Vizoso Pinto et al., 2006).

그러나, 프로바이오틱스 균주에 대한 연구는 사람의 건강이나 가축의 생산성 향상에 주로 초점을 맞추어 진행되고 있는 측면이 있었으며, 아직까지 반려동물, 특히 반려묘의 면역 관련 질환의 발병을 효과적으로 억제할 수 있는 프로바이오틱스 균주의 개발은 부진한 실정이다. However, research on probiotic strains has been mainly focused on improving human health or productivity of livestock, and there are still probiotic strains that can effectively suppress the onset of immune-related diseases in companion animals, especially cats. Development is sluggish.

이에, 본 발명자들은 반려묘의 면역 관련 질환에 효과적인 프로바이오틱스 균주를 찾기 위해 예의 연구노력한 결과, 반려묘의 분변에서 분리된 신균주 비피도박테리움 애니멀리스 CACC 789 균주가 내산성, 내담즙성, 및 장내부착능이 우수하여 프로바이오틱스 균주로 사용하기에 적합한 동시에 가축 병원균에 대한 항균 활성이 있으며, 면역증진, 염증 억제에 우수한 효과가 있을 확인하고, 본 발명을 완성하게 되었다.Accordingly, the present inventors made intensive research efforts to find a probiotic strain effective for immune-related diseases in cats, and as a result, the new strain Bifidobacterium animalis CACC 789 isolated from the feces of cats exhibited acid resistance, bile resistance, and intestinal adhesion. It is excellent and suitable for use as a probiotic strain, and at the same time has antibacterial activity against livestock pathogens, and has an excellent effect on immune enhancement and inflammation suppression, thereby completing the present invention.

따라서, 본 발명의 주된 목적은 내산성, 내담즙성, 및 장내부착능이 우수한 프로바이오틱스 신균주인 신균주 비피도박테리움 애니멀리스 CACC 789 균주를 제공하는데 있다. Therefore, the main object of the present invention is to provide a new strain Bifidobacterium animalis CACC 789 strain, which is a new probiotic strain excellent in acid resistance, bile resistance, and intestinal adhesion.

또한, 본 발명의 다른 목적은 상기 신균주 비피도박테리움 애니멀리스 CACC 789 균주를 유효성분으로 함유한 반려동물용 사료 조성물을 제공하는 데 있다.Another object of the present invention is to provide a feed composition for companion animals containing the new strain Bifidobacterium animalis CACC 789 as an active ingredient.

본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

본 발명의 한 양태에 따르면, 본 발명은 프로바이오틱스 신균주인 비피도박테리움 애니멀리스 CACC 789(수탁번호: KACC 92349P) 균주를 제공한다.According to one aspect of the present invention, the present invention provides a new probiotic strain, Bifidobacterium animalis CACC 789 (accession number: KACC 92349P) strain.

본 발명의 용어 "비피도박테리움 애니멀리스(Bifidobacterium animalis) CACC 789(수탁번호: KACC 92349P)" 균주는 본 발명자들에 의해 반려묘의 분변에서 새롭게 분리 동정된 신규한 비피도박테리움 애니멀리스 균주로서, "CACC 789 균주" 또는 "789 균주"와 혼용하여 사용할 수 있다. The term " Bifidobacterium animalis CACC 789 (Accession No.: KACC 92349P)" strain of the present invention is a novel Bifidobacterium animalis strain newly isolated and identified from the feces of cats by the present inventors. , "CACC 789 strain" or "789 strain" can be used interchangeably.

본 발명의 신균주 비피도박테리움 애니멀리스 CACC 789(수탁번호: KACC 92349P) 균주는 항균 활성, 면역 증진 활성 및 항염증 활성을 갖는 것을 특징으로 한다. The novel strain Bifidobacterium animalis CACC 789 (accession number: KACC 92349P) strain of the present invention is characterized in that it has antibacterial activity, immune enhancing activity and anti-inflammatory activity.

본 발명자들은 반려동물에 사용할 수 있는 프로바이오틱스의 개발을 위해 반려동물에 유용한 미생물에 대해 수년간 조사 분석하였고, 그 결과 반려묘의 분변에서 유래한 비피도박테리움 애니멀리스 CACC 789 균주가 내산성, 내담즙성, 및 장내부착능이 우수하여 프로바이오틱스 균주로 사용하기에 적합한 동시에 가축 병원균에 대한 항균 활성이 있으며, 면역증진, 염증 억제에도 우수한 효과가 있음을 확인하였고, 저장시 안전성에도 문제가 없음을 확인하였다.The present inventors investigated and analyzed microorganisms useful for companion animals for many years for the development of probiotics that can be used for companion animals. And it was confirmed that it was suitable for use as a probiotic strain due to its excellent intestinal adhesion, and had antibacterial activity against livestock pathogens, and had excellent effects on immunity enhancement and inflammation suppression, and there was no problem with safety during storage.

본 발명의 신균주 비피도박테리움 애니멀리스 CACC 789(수탁번호: KACC 92349P)는 서열번호 1의 16s rRNA 서열을 갖는 것을 특징으로 한다.The novel strain Bifidobacterium animalis CACC 789 (accession number: KACC 92349P) of the present invention is characterized in that it has the 16s rRNA sequence of SEQ ID NO: 1.

본 발명자들은 상기 유전자 서열을 종래 공지된 균주들의 유전자 서열들과 비교 분석한 결과, 본 발명의 균주가 비피도박테리움 애니멀리스에 속하는 새로운 균주임을 확인하여 비피도박테리움 애니멀리스 CACC 789로 명명하였고, 국립농업과학원 농업유전자원정보센터에 2021년 06월 01일자로 기탁하였고, 수탁번호 KACC 92349P를 부여받았다.As a result of comparative analysis of the gene sequence with the gene sequences of previously known strains, the present inventors confirmed that the strain of the present invention was a new strain belonging to Bifidobacterium animalis, and named it Bifidobacterium animallis CACC 789. , was deposited with the Agricultural Genetic Resources Information Center of the National Academy of Agricultural Sciences on June 1, 2021, and was given an accession number KACC 92349P.

본 발명의 다른 양태에 따르면, 본 발명은 신균주 신균주 비피도박테리움 애니멀리스 CACC 789(수탁번호: KACC 92349P), 또는 그 배양액을 유효성분으로 하는 반려동물용 사료 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a feed composition for companion animals comprising the new strain Bifidobacterium animalis CACC 789 (Accession No.: KACC 92349P) or a culture solution thereof as an active ingredient.

본 발명의 상기 반려동물용 사료 조성물은 내산성, 내담즙성, 및 장내부착 활성이 우수하여 프로바이오틱 균주로 사용되기에 적합하다. 본 발명의 바람직한 실시예에서는 본 발명의 CACC 789 균주가 pH 2.5의 강산성 조건에서도 생존율이 82.9%로 측정되어 매우 우수한 내산성을 갖는 것을 확인할 수 있었고, 0.3% 담즙 조건에서도 86.3% 이상의 생존율을 보여 매우 우수한 내담즙성을 갖는 것을 확인할 수 있었다. 또한, 본 발명의 CACC 789 균주는 78.43%의 높은 장내부착 활성을 나타내는 것을 확인할 수 있었다(도 1 참조). The feed composition for companion animals of the present invention is suitable for use as a probiotic strain because of its excellent acid resistance, bile resistance, and intestinal adhesion activity. In a preferred embodiment of the present invention, it was confirmed that the CACC 789 strain of the present invention had a very good acid resistance as the survival rate was measured to be 82.9% even under a strongly acidic condition of pH 2.5, and showed a survival rate of 86.3% or more even under a 0.3% bile condition. It was confirmed that it has bile resistance. In addition, it was confirmed that the CACC 789 strain of the present invention exhibited a high intestinal adhesion activity of 78.43% (see FIG. 1).

본 발명의 반려동물용 사료 조성물은 가축 병원균에 대한 항균 활성을 나타내는 것을 특징으로 한다. 본 발명의 CACC 789 균주 또는 그 배양액은 그 자체로 항균 활성을 나타내며, 다양한 가축 병원균에 항균 활성이 있다. 본 발명의 일 실시예에서는 본 발명에 따른 CACC 789 균주 배양액이 다양한 가축 병원균에 대해 생장 억제 효능이 있는 것을 확인하였다. 구체적으로는 상기 가축 병원균은 살모넬라 엔테리티디스(Salmonella Enteritidis) 및 살모넬라 타이피뮤리움(Salmonella Typhimurium)에 대해 항균 활성이 있으나, 이에 한정되는 것은 아니다.The feed composition for companion animals of the present invention is characterized in that it exhibits antibacterial activity against livestock pathogens. The CACC 789 strain of the present invention or its culture medium exhibits antibacterial activity by itself, and has antibacterial activity against various livestock pathogens. In one embodiment of the present invention, it was confirmed that the CACC 789 strain culture solution according to the present invention has a growth inhibitory effect against various livestock pathogens. Specifically, the livestock pathogen has antibacterial activity against Salmonella Enteritidis and Salmonella Typhimurium, but is not limited thereto.

본 발명의 상기 반려동물용 사료 조성물은 저장 안정성이 있는 것을 특징으로 한다. 본 발명의 바람직한 실시예에서는 상기 CACC 789 균주를 저장하는 경우에 3개월의 기간 동안 생균 수에 크게 변화가 없음을 확인할 수 있었으며, 이를 통해 저장 안정성이 우수한 균주임을 확인할 수 있었다(도 2 참조). The feed composition for companion animals of the present invention is characterized in that it has storage stability. In a preferred embodiment of the present invention, it was confirmed that there was no significant change in the number of viable cells for a period of 3 months when the CACC 789 strain was stored, thereby confirming that the strain had excellent storage stability (see FIG. 2 ).

본 발명의 반려동물용 사료 조성물에서, 상기 균주, 또는 그 배양액은 염증 관련 유전자인 IFN-γ 및 TLR3 유전자의 발현을 억제하여 항염증 활성을 나타내는 것을 특징으로 한다. 본 발명의 바람직한 실시예에서는 상기 CACC 789 균주의 배양액을 처리한 군에서 poly(I:C)에 의해서 유도된 염증 반응 관련 유전자(IFN-γ 및 TLR3 유전자)들의 발현이 유의적으로 감소한 것을 확인할 수 있었다(도 4 참조). In the feed composition for companion animals of the present invention, the strain, or its culture medium, is characterized in that it exhibits anti-inflammatory activity by suppressing the expression of IFN-γ and TLR3 genes, which are inflammation-related genes. In a preferred embodiment of the present invention, it can be confirmed that the expression of inflammatory response-related genes (IFN-γ and TLR3 genes) induced by poly(I:C) was significantly reduced in the group treated with the culture medium of the CACC 789 strain. There was (see Fig. 4).

본 발명의 반려동물용 사료 조성물에서, 상기 균주, 또는 그 배양액은 면역세포를 활성화하고, 단핵구(monocytes) 및 과립성백혈구(WBC-granulocyte)의 수치를 정상범위 내에서 증가시켜 면역증진 활성을 갖는 것을 특징으로 한다. 본 발명의 바람직한 실시예에서는 상기 CACC 789 균주의 배양액이 고양이 면역세포주인 Fcwf-4 세포를 유의적으로 활성화시키고, 백혈구(WBC) 내 호산구(eosinophils)의 비율을 정상범위 내에서 증가시켜 면역증진 활성을 갖는 것을 확인한 바 있으며, 이러한 결과를 통해 본 발명의 CACC 789 균주가 세포성 면역반응에 주요 역할을 담당하는 백혈구를 정상범위에서 증가시켜 면역기능을 증가시키는 효과가 있음을 확인할 수 있었다(도 3a 및 도 5 참조). In the feed composition for companion animals of the present invention, the strain, or its culture medium, activates immune cells and increases the levels of monocytes and granulocytes (WBC-granulocyte) within a normal range to have immune enhancing activity. characterized in that In a preferred embodiment of the present invention, the culture medium of the CACC 789 strain significantly activates Fcwf-4 cells, a cat immune cell line, and increases the ratio of eosinophils in leukocytes (WBC) within a normal range, thereby enhancing immunity , and through these results, it was confirmed that the CACC 789 strain of the present invention had the effect of increasing the immune function by increasing the leukocytes that play a major role in the cellular immune response in the normal range (FIG. 3a). and Figure 5).

본 발명의 상기 사료 조성물은 반려동물이 먹고, 섭취하며, 소화시키기 위한 또는 이에 적당한 임의의 천연 또는 인공 규정식, 한끼식, 간식 또는 상기 한끼식의 성분을 의미할 수 있으며, 당업계의 공지된 다양한 형태로 제조가능하다.The feed composition of the present invention may mean any natural or artificial diet, meal, snack, or ingredient of the meal for companion animals to eat, ingest, and digest or suitable for, and is known in the art. It can be manufactured in various forms.

상기 사료의 종류는 특별히 제한되지 아니하며, 당해 기술 분야에서 통상적으로 사용되는 사료를 더 추가하여 사용할 수 있으며, 이 경우 본 발명의 사료 조성물은 사료 첨가제로서 역할을 한다. 상기 추가될 수 있는 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 the feed is not particularly limited, and a feed commonly used in the art may be further used, and in this case, the feed composition of the present invention serves as a feed additive. Non-limiting examples of the feed that can be added include plant feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, gourds or grain by-products; and animal feeds such as proteins, inorganic materials, oils and fats, minerals, oils and fats, single cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.

다른 일 예로, 상기 사료 조성물은 투여될 반려동물의 종류, 나이 및 기타 사료 성분의 종류에 따라 적절하게 사용될 수 있다.As another example, the feed composition may be appropriately used depending on the type of companion animal to be administered, age, and other types of feed components.

본 발명의 사료 조성물은 영양소 보충 및 체중감소 예방, 사료 내 섬유소의 소화 이용성 증진, 유질 개선, 번식장애 예방 및 수태율 향상, 하절기 고온 스트레스 예방 등 다양한 효과를 나타내는 물질을 추가로 포함할 수 있다. 예를 들어 탄산수소나트륨, 벤토나이트(bentonite), 산화마그네슘, 복합광물질 등의 광물질제제, 아연, 구리, 코발트, 셀레늄 등의 미량 광물질인 미네랄제제, 케로틴, 비타민 E, 비타민 A, D, E, 니코틴산, 비타민 B 복합체 등의 비타민제, 메티오닌, 리이산 등의 보호아미노산제, 지방산 칼슘염 등의 보호지방산제, 효모배양물, 곰팡이 발효물 등의 생균, 효모제 등이 추가로 포함될 수 있다. The feed composition of the present invention may further contain substances exhibiting various effects such as nutrient supplementation and weight loss prevention, enhancement of digestibility of fiber in feed, improvement of oil quality, prevention of reproductive disorders and improvement of fertility rate, prevention of high temperature stress in summer. For example, mineral preparations such as sodium bicarbonate, bentonite, magnesium oxide, and complex minerals, trace minerals such as zinc, copper, cobalt, selenium, kerotene, vitamin E, vitamins A, D, E, nicotinic acid , vitamins such as vitamin B complex, protective amino acids such as methionine and lyic acid, protective fatty acids such as fatty acid calcium salts, live bacteria such as yeast cultures and mold fermented products, yeast agents, etc. may be further included.

또 다른 양태로서, 본 발명은 상기 사료 조성물을 반려동물에 투여하는 단계를 포함하는, 반려동물의 면역증진 및 염증 억제 방법을 제공한다.In another aspect, the present invention provides a method for enhancing immunity and inhibiting inflammation of a companion animal, comprising administering the feed composition to the companion animal.

상기 투여는 어떠한 적절한 방법으로 반려동물에 본 발명의 조성물을 도입하는 것을 의미하며, 본 발명의 조성물 투여 경로는 목적 조직에 도달할 수 있는 한 경구 또는 비경구의 다양한 경로를 통하여 투여될 수 있다. 본 발명자들은 본 발명의 사료 조성물을 경구 투여하더라도 급성 독성이 나타나지 않는다.The administration means introducing the composition of the present invention to the companion animal by any suitable method, and the composition administration route of the present invention may be administered through various routes, either oral or parenteral, as long as it can reach the target tissue. The present inventors do not show acute toxicity even when the feed composition of the present invention is orally administered.

본 발명의 특징 및 이점을 요약하면 다음과 같다:The features and advantages of the present invention are summarized as follows:

(1) 본 발명은 반려묘의 분변에서 유래한 신균주 비피도박테리움 애니멀리스 CACC 789 및 이를 유효성분으로 함유하는 반려동물용 사료 조성물을 제공한다.(1) The present invention provides a new strain Bifidobacterium animalis CACC 789 derived from cat feces and a feed composition for companion animals containing the same as an active ingredient.

(2) 본 발명의 비피도박테리움 애니멀리스 CACC 789 균주는 내산성, 내담즙성 및 장내부착능이 우수하여 프로바이오틱스 균주로 사용하기에 적합하며, 가축 병원균에 대한 우수한 항균 활성을 나타내고, 면역증진 및 염증 억제에 우수한 효과가 있다.(2) The Bifidobacterium animalis CACC 789 strain of the present invention has excellent acid resistance, bile resistance and intestinal adhesion, so it is suitable for use as a probiotic strain, and exhibits excellent antibacterial activity against livestock pathogens, immunity enhancement and inflammation It has an excellent effect on suppression.

도 1은 본 발명의 비피도박테리움 애니멀리스 CACC 789 균주의 내산성, 내담즙성 및 장내부착능을 측정하여 나타낸 그래프이다.
도 2는 본 발명의 비피도박테리움 애니멀리스 CACC 789 균주의 장기 보관시 생균 수의 변화를 측정하여 나타낸 그래프이다.
도 3은 본 발명의 비피도박테리움 애니멀리스 CACC 789 균주의 면역세포 활성 효과를 측정하여 나타낸 그래프이다[도 3a: (-) poly(I:C), 도 3b: (+) poly(I:C)].
도 4는 본 발명의 비피도박테리움 애니멀리스 CACC 789 균주의 배양 상등액 처리에 따른 염증 반응 관련 유전자(IFN-γ, TRL3)의 유전자 발현 변화를 측정하여 나타낸 그래프이다.
도 5는 본 발명의 비피도박테리움 애니멀리스 CACC 789 균주의 배양 상등액 처리에 따른 임상시험 전후 52종 혈액지표 값에 대한 부분최소자승 판별분석(Partial least squares- discriminant analysis, PLS-DA) 결과 그래프(A)와 백혈구(WBC) 내 단핵구(monocytes) 및 과립성백혈구(WBC-granulocyte)의 비율변화(B) 그래프이다.
도 6은 본 발명의 비피도박테리움 애니멀리스 CACC 789 균주의 계통도를 모식화하여 나타낸 모식도이다.
1 is a graph showing the measurement of acid resistance, bile resistance and intestinal adhesion of the Bifidobacterium animalis CACC 789 strain of the present invention.
2 is a graph showing the change in the number of live cells during long-term storage of the Bifidobacterium animalis CACC 789 strain of the present invention.
3 is a graph showing the effect of measuring the immune cell activation effect of the Bifidobacterium animalis CACC 789 strain of the present invention [FIG. 3a: (-) poly(I:C), FIG. 3b: (+) poly(I: C)].
4 is a graph showing changes in gene expression of inflammatory response-related genes (IFN-γ, TRL3) according to the treatment of the culture supernatant of the Bifidobacterium animalis CACC 789 strain of the present invention.
5 is a partial least squares-discriminant analysis (PLS-DA) result graph for the values of 52 blood indicators before and after clinical trials according to the treatment of the culture supernatant of the Bifidobacterium animalis CACC 789 strain of the present invention; (A) and white blood cells (WBC) in monocytes (monocytes) and granular leukocytes (WBC-granulocyte) ratio change (B) is a graph.
6 is a schematic diagram schematically showing the phylogenetic diagram of the Bifidobacterium animalis CACC 789 strain of the present invention.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention by these examples.

실시예 1. 반려묘 분변 유래 미생물의 내산성, 내담즙성 및 장내부착 활성 검증Example 1. Verification of acid resistance, bile resistance and intestinal adhesion activity of microorganisms derived from cat feces

가축 병원균에 대한 항균 및 면역증진 활성을 갖는 프로바이오틱스(probiotics)의 후보군을 선별하기 위해서 반려묘의 분변 시료에서 혐기적 조건으로 채취된 미생물들의 생균제 특성을 검증하였다. In order to select a candidate group of probiotics having antibacterial and immune-enhancing activity against livestock pathogens, the probiotic properties of microorganisms collected under anaerobic conditions from cat fecal samples were verified.

1-1. 내산성 및 내담즙성1-1. acid and bile resistance

반려묘 분변으로부터 분리한 균주의 내산성 및 내담즙 특성 연구를 위해 분리 균주들을 혐기챔버(Coy Laboratories, Grass Lake, MI)를 이용하여 혐기적 조건(90% N2, 5% CO2, 5% H2)에서 BL broth(BD Difco™, USA)를 이용하여 37℃에서 24시간 배양하여 실험에 사용하였다.For the study of acid resistance and bile resistance characteristics of strains isolated from cat feces, the isolated strains were subjected to anaerobic conditions (90% N 2 , 5% CO 2 , 5% H 2 ) using an anaerobic chamber (Coy Laboratories, Grass Lake, MI). ) in BL broth (BD Difco™, USA) was used in the experiment by culturing at 37° C. for 24 hours.

내산성은 1N HCl을 이용하여 pH 2.5로 조정된 BL 배지, 내담즙성은 0.3%, 1%(w/v) Oxgall(BD Difco™, USA)이 첨가된 BL 배지에 접종하여 2시간 동안 배양한 후, 0시간과 2시간 후의 생균수를 측정하여 생존율(%) 하기 [표 1] 나타내었다.Acid resistance is inoculated into BL medium adjusted to pH 2.5 using 1N HCl, and biliary resistance is 0.3%, 1% (w/v) Oxgall (BD Difco™, USA) is added to BL medium and cultured for 2 hours. , the number of viable cells after 0 and 2 hours was measured and the survival rate (%) is shown in [Table 1] below.

[표 1][Table 1]

Figure 112021087360180-pat00001
Figure 112021087360180-pat00001

실험 결과, 분리된 후보군 중에서 CACC 789 균주가 pH 2.5의 강산성 조건에서도 생존율이 82.92%로 측정되어 매우 우수한 내산성을 갖는 것을 확인할 수 있었고, 담즙 조건에서도 80% 이상의 생존율을 보여 매우 우수한 내담즙성을 갖는 것을 확인할 수 있었다.As a result of the experiment, it was confirmed that the CACC 789 strain from the isolated candidate group had very good acid resistance as the survival rate was measured to be 82.92% even under the strongly acidic condition of pH 2.5, and it was confirmed that the CACC 789 strain had very good acid resistance even in the bile condition, showing a survival rate of 80% or more. could confirm that

1-2. 장내부착 활성1-2. intestinal adhesion activity

장상피세포 HT-29 세포주는 10% fecal calf serum(Gibdo-BRL, USA)과 1% 페니실린/스트렙토마이신(10,000U/ml, Gibdo-BRL, USA)이 첨가된 DMEM(Gibdo-BRL, USA) 배지를 사용하여 37℃, 5% CO2 조건에서 배양하였다. 장내부착 활성 시험도 혐기적 조건(90% N2, 5% CO2, 5% H2)에서 수행되었다. The intestinal epithelial HT-29 cell line is DMEM (Gibdo-BRL, USA) supplemented with 10% fecal calf serum (Gibdo-BRL, USA) and 1% penicillin/streptomycin (10,000U/ml, Gibdo-BRL, USA). The culture medium was used at 37° C., 5% CO 2 conditions. The intestinal adhesion activity test was also performed under anaerobic conditions (90% N 2 , 5% CO 2 , 5% H 2 ).

HT-29 세포 단일층(monolayer)이 형성된 24-well plate에 1.0×108 CFU/mL 이상의 후보 균주들을 접종하였고, 2시간 배양 후, 0.1% triton X 100을 사용하여 HT-29 세포와 상기 균주를 떼어낸 후 생균수를 측정하였다. 비교 실험을 위해 종래 장내부착 활성이 우수한 것으로 알려진 락토바실러스 람노서스(Lactobacillus rhamnosus) GG 균주를 대조군 균주로 사용하였고, 실험 결과를 하기 [표 2]에 정리하였다. Candidate strains of 1.0×10 8 CFU/mL or more were inoculated into a 24-well plate in which a HT-29 cell monolayer was formed, and after 2 hours of incubation, HT-29 cells and the strain were used using 0.1% triton X 100. After removing the , the number of viable cells was measured. For comparative experiments, Lactobacillus rhamnosus GG strain, which is known to have excellent intestinal adhesion activity, was used as a control strain, and the experimental results are summarized in [Table 2] below.

[표 2][Table 2]

Figure 112021087360180-pat00002
Figure 112021087360180-pat00002

실험 결과, 분리된 후보군 중에서 CACC 789 균주의 장내 부착능은 78.43%로 측정되었으며, 이러한 CACC 789 균주의 장내부착 활성은 종래 프로바이오틱스 균주 중 장내 부착능이 우수한 것으로 알려진 락토바실러스 람노서스(Lactobacillus rhamnosus) GG 균주와 비교하여도 더욱 우수한 것을 확인할 수 있었다. As a result of the experiment, the intestinal adhesion capacity of the CACC 789 strain among the isolated candidate groups was measured to be 78.43%, and the intestinal adhesion activity of the CACC 789 strain was Lactobacillus rhamnosus GG strain, which is known to have excellent intestinal adhesion among conventional probiotic strains. It was also confirmed that it was even better compared with .

실시예 2. 가축 병원균에 대한 항균 활성Example 2. Antimicrobial activity against livestock pathogens

가축 병원균에 대한 항균 활성을 검증하기 위하여 Disk diffusion method 방법으로 항균 활성을 검증하였다. To verify the antimicrobial activity against livestock pathogens, the antimicrobial activity was verified by the disk diffusion method.

병원균은 Salmonella Enteritidis NCCP 14546 및 Salmonella Typhimurium NCCP 10328 균주를 국가병원자원은행과 한국생명공학연구원 생물자원센터에서 분양받아 사용하였다. 상기 2종 병원균을 LB(BD Difco™, USA) 배지를 사용하여 37℃에서 24시간 배양하였다. As pathogens, Salmonella Enteritidis NCCP 14546 and Salmonella Typhimurium NCCP 10328 strains were purchased from the National Hospital Resources Bank and the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center and used. The two pathogens were cultured at 37° C. for 24 hours using LB (BD Difco™, USA) medium.

반려묘 분변으로부터 분리한 균주들을 BL broth에 접종한 후, 37℃에서 48시간 동안 배양하여 0.22㎛ pore size 막 필터로 제균한 배양 상등액 80㎕를 8mm paper disc에 접종하고 clear zone의 지름을 재어 항균력을 평가하고, 하기 [표 3]에 정리하였다. After inoculating the strains isolated from cat feces in BL broth, incubate at 37°C for 48 hours, inoculate 80 μl of the culture supernatant sterilized with a 0.22㎛ pore size membrane filter on an 8mm paper disc, and measure the diameter of the clear zone to determine the antibacterial activity. It was evaluated and summarized in [Table 3] below.

[표 3][Table 3]

Figure 112021087360180-pat00003
Figure 112021087360180-pat00003

실험 결과, 분리된 후보군 중에서 CACC 789 균주는 가축 병원균인 Salmonella Typhimurium NCCP 10328 균주에 대해 9-10mm의 clear zone을 형성하였고, Salmonella Enteritidis NCCP 14546 균주에 대해 11-12mm의 clear zone을 형성하여 가축병원균에 대하여 매우 우수한 항균 활성을 갖는 것으로 확인되었다. As a result of the experiment, among the separated candidate groups, the CACC 789 strain formed a clear zone of 9-10 mm against the livestock pathogen Salmonella Typhimurium NCCP 10328 strain, and formed a clear zone of 11-12 mm against the Salmonella Enteritidis NCCP 14546 strain to protect the livestock pathogens. It was confirmed to have very good antibacterial activity against

실시예 3. 저정 안정성Example 3. Storage stability

상기 CACC 789 균주를 BL broth를 이용하여 혐기적 조건으로 37℃에서 48시간 배양하고, 13,000rpm에서 5분 동안 원심분리한 후, 균체만을 수집하였다. The CACC 789 strain was cultured anaerobically at 37° C. for 48 hours using BL broth, centrifuged at 13,000 rpm for 5 minutes, and only the cells were collected.

수집된 균체는 유산균 생균제 분말 동결건조보호제(5% fructo-oligosaccharide, 10% skim milk, 15% trehalose, 0.5% glycerin, 1% NaCl)를 첨가하여 동결건조하였다. 동결건조된 균체를 분쇄하여 분말화한 후, 1포에 0.2g씩 소포장을 하고, 5℃에서 3개월 저장하였다. 저장 기간 중 생균 수를 측정하여 제형 안정성을 확인하였다.The collected cells were freeze-dried by adding lactic acid bacteria probiotic powder freeze-drying protectant (5% fructo-oligosaccharide, 10% skim milk, 15% trehalose, 0.5% glycerin, 1% NaCl). After the freeze-dried cells were pulverized and powdered, 0.2 g each was packaged in small packages, and stored at 5° C. for 3 months. Formulation stability was confirmed by measuring the number of viable cells during storage.

실험 결과, 상기 CACC 789 균주는 초기 4.6×108 CFU(1포 0.2g 소포장)의 생균 수가 3개월 저장 후에도 98% 수준으로 유지되어 저장기간 동안 생균 수의 변화가 없는 것으로 관찰되었고, 저장 안정성이 매우 우수한 균주임을 확인할 수 있었다(도 2 참조). As a result of the experiment, the CACC 789 strain was observed to have no change in the number of viable cells during the storage period as the initial number of viable cells of 4.6×10 8 CFU (1 package, 0.2g small package) was maintained at 98% even after 3 months of storage, and storage stability was improved. It was confirmed that it was a very good strain (see FIG. 2).

실시예 4. 면역세포 활성 증가 및 염증 반응 억제Example 4. Increased immune cell activity and inhibition of inflammatory response

4-1. 면역세포의 활성화4-1. activation of immune cells

상기 CACC 789 균주가 면역세포의 활성에 미치는 효과를 평가하기 위하여 고양이 대식세포주인 Fcwf-4 세포를 세포배양배지(10% FBS, 1% anitibiotics in DMEM)에서 37℃, 5% CO2 조건으로 배양하였다.In order to evaluate the effect of the CACC 789 strain on the activity of immune cells, Fcwf-4 cells, a cat macrophage cell line, were cultured in a cell culture medium (10% FBS, 1% anitibiotics in DMEM) at 37° C. and 5% CO 2 conditions. did.

CACC 789 균주를 BL broth를 이용하여 혐기적 조건에서 37℃, 24시간 배양하고, 13,000rpm에서 5분 원심분리 후, 상등액만을 수집하였다.The CACC 789 strain was cultured anaerobically at 37° C. for 24 hours using BL broth, and after centrifugation at 13,000 rpm for 5 minutes, only the supernatant was collected.

배양 중인 Fcwf-4 세포에 세포배양액 부피의 10% 수준으로 CACC 789 균주의 배양 상층액을 넣어주고, 37℃, 5% CO2 조건에서 24시간 동안 배양한 후, WST-1 assay를 이용하여 Fcwf-4 세포의 면역세포 활성을 배양액 무첨가 조건인 음성대조군과 비교하여 평가하였다.The culture supernatant of the CACC 789 strain was added to the Fcwf-4 cells in culture at a level of 10% of the cell culture volume, and after culturing for 24 hours at 37° C. and 5% CO 2 conditions, Fcwf using WST-1 assay. The immune cell activity of -4 cells was evaluated in comparison with the negative control group, which was a condition in which the culture medium was not added.

실험 결과 [도 3a]을 참조하면, CACC 789 균주의 배양 상등액은 고양이 대식세포주인 Fcwf-4 세포를 유의적으로 활성화시키는 것을 확인할 수 있었다. Referring to the experimental results [Fig. 3a], it was confirmed that the culture supernatant of the CACC 789 strain significantly activated Fcwf-4 cells, a cat macrophage cell line.

한편, 배양 중인 Fcwf-4 세포에 면역유도물질인 poly(I:C)를 24시간 동안 처리하여 염증 반응을 유도한 후, 세포배양액 부피의 10% 수준으로 CACC 789 균주의 배양 상층액을 넣어주었다. 37℃, 5% CO2 조건에서 24시간 동안 배양한 후, WST-1 assay를 이용하여 Fcwf-4 세포 염증 억제 활성을 배양액 무첨가 조건인 음성대조군과 비교하여 평가하였다. On the other hand, after inducing an inflammatory response by treating the Fcwf-4 cells in culture with poly(I:C), an immune-inducing material, for 24 hours, the culture supernatant of the CACC 789 strain was added at a level of 10% of the cell culture solution volume. . After culturing for 24 hours at 37 ° C., 5% CO 2 condition, the Fcwf-4 cell inflammation inhibitory activity was evaluated by using WST-1 assay compared to the negative control, which is a condition in which the culture medium is not added.

실험 결과 [도 3b]을 참조하면, 염증 반응에 의해 저하된 고양이 대식세포주의 활성을 CACC 789 균주의 배양 상등액이 일부 회복시키는 것을 확인할 수 있었다(P < 0.05).Referring to the experimental results [Fig. 3b], it was confirmed that the culture supernatant of the CACC 789 strain partially restored the activity of the cat macrophage line decreased by the inflammatory reaction (P < 0.05).

4-2. 염증 반응 관련 유전자 발현량 분석4-2. Inflammatory response-related gene expression analysis

추가적으로 Fcwf-4 세포의 RNA를 추출하여 염증 반응과 관련된 유전자 발현 양상을 확인하였다.Additionally, by extracting RNA from Fcwf-4 cells, gene expression patterns related to the inflammatory response were confirmed.

실험은 배양 중인 Fcwf-4 세포에 면역유도물질인 poly(I:C)를 24시간 동안 처리하여 염증 반응을 유도한 후, 세포배양액 부피의 10% 수준으로 CACC 789 균주의 배양 상층액을 넣어주고. 37℃, 5% CO2 조건에서 24시간 동안 배양한 후, Fcwf-4 세포의 RNA를 추출하여 염증 반응 관련 유전자 발현 양상을 분석하였다. 유전자 발현 분석은 하우스키핑 유전자(housekeeping gene)인 GAPDH 유전자로 정규화(normalization)하였고, 하기 [표 4]의 프라이머를 이용하였다. In the experiment, after inducing an inflammatory response by treating Fcwf-4 cells in culture with poly(I:C), an immune-inducing substance, for 24 hours, the culture supernatant of the CACC 789 strain was added at a level of 10% of the cell culture medium volume. . After culturing for 24 hours at 37° C., 5% CO 2 conditions, RNA of Fcwf-4 cells was extracted and the inflammatory response-related gene expression pattern was analyzed. Gene expression analysis was normalized to the GAPDH gene, which is a housekeeping gene, and the primers in Table 4 below were used.

[표 4][Table 4]

Figure 112021087360180-pat00004
Figure 112021087360180-pat00004

실험 결과 [도 4]를 참조하면, CACC 789 균주의 배양 상등액이 poly(I:C)에 의해서 유도된 염증 반응 관련 유전자(IFN-γ, TLR3)들의 유전자 발현을 유의적으로 억제하는 것으로 관찰되었다(P < 0.05).As a result of the experiment [Fig. 4], the culture supernatant of the CACC 789 strain was observed to significantly inhibit the gene expression of inflammatory response-related genes (IFN-γ, TLR3) induced by poly(I:C). (P < 0.05).

상기와 같은 결과를 통해 CACC 789 균주 또는 그 배양액은 면역세포를 활성화시키고, 염증 관련 유전자의 발현을 억제하여 면역기능 강화 또는 항염증 용도로 유용하게 활용될 수 있음을 확인할 수 있었다.Through the above results, it was confirmed that the CACC 789 strain or its culture medium can be usefully used for enhancing immune function or for anti-inflammatory purposes by activating immune cells and suppressing the expression of inflammation-related genes.

실시예 5. 면역 증진 활성에 대한 임상시험Example 5. Clinical trial on immune enhancing activity

보호자 동의 아래 반려묘 7마리를 대상으로 하여 실시예 1 및 2를 통해 생균제 활성이 검증된 CACC 789 균주의 면역증진 효과에 대한 임상시험을 실시하였다. A clinical test was conducted on the immune-enhancing effect of the CACC 789 strain whose probiotic activity was verified through Examples 1 and 2 on 7 cats under the consent of the guardian.

실험은 CACC 789 균주를 실시예 3과 같이 분말 형태로 제제화하여 45일 동안 하루 1회 0.2g(4.6 x 108 CFU/g 이상)씩 급여한 후, 미생물제제 급여 전후의 혈액을 채취하여 혈액 검사를 실시하였다. 혈액 검사는 52종의 혈액 지표값을 측정하고 이들 지표값을 이용하여 부분최소자승 판별분석(Partial least squares- discriminant analysis, PLS-DA)을 수행하였고, CACC 789 균주의 급여 전후로 혈액 지표값들이 정상범위 안에서 변화하는 것으로 확인되었다. For the experiment, the CACC 789 strain was formulated in powder form as in Example 3, and 0.2 g (4.6 x 10 8 CFU/g or more) was fed once a day for 45 days, and then blood was collected before and after feeding the microorganism and blood test was carried out. For the blood test, 52 types of blood index values were measured and partial least squares-discriminant analysis (PLS-DA) was performed using these index values, and blood index values were normal before and after feeding the CACC 789 strain. It was found to change within the range.

또한, 혈액 검사 결과를 정리한 [도 5]를 참조하면, 백혈구(WBC) 내 단핵구(monocytes)와 과립성백혈구(WBC-granulocyte)의 비율이 정상범위 내에서 각각 7.2%와 4.9% 증가하는 경향을 보였다.In addition, referring to [Fig. 5], which summarizes the blood test results, the ratio of monocytes and WBC-granulocytes in white blood cells (WBC) tends to increase by 7.2% and 4.9%, respectively, within the normal range. showed

상기와 같은 결과를 통해 CACC 789 균주가 세포성 면역반응에 주요 역할을 담당하는 백혈구를 정상범위에서 증가시켜 건강한 고양이의 면역력을 강화시키는데 효과가 있음을 확인할 수 있었다. Through the above results, it was confirmed that the CACC 789 strain was effective in strengthening the immunity of healthy cats by increasing the leukocytes that play a major role in the cellular immune response in the normal range.

실시예 6. 균주 동정Example 6. Identification of strains

상기 실시예 1 내지 5에서 생균제 활성이 검증된 CACC 789 균주를 동정하기 위해 16S rRNA gene sequencing 분석을 실시하였다. 16S rRNA gene sequencing analysis was performed to identify the CACC 789 strain whose probiotic activity was verified in Examples 1 to 5 above.

프라이머는 27F 및 1492R 프라이머를 사용하였고, PCR 기기를 이용하여 16S rRNA 유전자를 증폭시킨 후 inter-primer인 785F, 907R 프라이머로 약 1,400bp 이상의 염기서열을 확보하였다. 27F and 1492R primers were used as primers, and after amplifying the 16S rRNA gene using a PCR device, a nucleotide sequence of about 1,400 bp or more was secured with inter-primer 785F and 907R primers.

[표 5][Table 5]

Figure 112021087360180-pat00005
Figure 112021087360180-pat00005

확보된 염기서열은 Ezbiocloud(https://www.ezbiocloud.net)의 16S-based ID를 통해 16S rRNA 분석을 수행하였다.The obtained nucleotide sequence was analyzed by 16S rRNA through the 16S-based ID of Ezbiocloud (https://www.ezbiocloud.net).

분석 결과, CACC 789 균주는 Bifidobacterium animalis subsp. lactis (accession number, DSM 10140) 균주와 99.93%의 상동성을 보이는 신규한 균주로 동정되었다. Phylogenetic tree는 Mega 7 program을 사용하여 neighbor joining method를 기반으로 [도 6]에 나타내었다.As a result of the analysis, the CACC 789 strain was Bifidobacterium animalis subsp. lactis (accession number, DSM 10140) strain was identified as a novel strain showing 99.93% homology. The phylogenetic tree is shown in [Fig. 6] based on the neighbor joining method using the Mega 7 program.

상기와 같이 동정된 우수한 생균제 활성을 갖는 본 발명의 균주를 비피도박테리움 애니멀리스(Bifidobacterium animalis) CACC 789로 명명하고, 2021년 06월 01일 국립농업과학원 농업유전자원정보센터에 기탁번호(KACC 92349P)로 기탁하였다. The strain of the present invention having excellent probiotic activity identified as described above was named Bifidobacterium animalis CACC 789, and on June 01, 2021, the Agricultural Genetic Resources Information Center of the National Academy of Agricultural Sciences was deposited with the deposit number (KACC). 92349P).

이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

농업생명공학연구원Agricultural Biotechnology Research Institute KACC92349KACC92349 2021060120210601

<110> Center for Industrialization of Agricultural and Livestock Microorganisms <120> Novel Strain of Bifidobacterium animalis CACC 789, and feed composition using thereof <130> PN210041AN <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1499 <212> DNA <213> Bifidobacterium animalis <400> 1 tctgctcagg atgaacgctg gcggcgtgct taacacatgc aagtcgaacg ggatccctgg 60 cagcttgctg tcggggtgag agtggcgaac gggtgagtaa tgcgtgacca acctgccctg 120 tgcaccggaa tagctcctgg aaacgggtgg taataccgga tgctccgctc catcgcatgg 180 tggggtggga aatgcttttg cggcatggga tggggtcgcg tcctatcagc ttgttggcgg 240 ggtgatggcc caccaaggcg ttgacgggta gccggcctga gagggtgacc ggccacattg 300 ggactgagat acggcccaga ctcctacggg aggcagcagt ggggaatatt gcacaatggg 360 cgcaagcctg atgcagcgac gccgcgtgcg ggatggaggc cttcgggttg taaaccgctt 420 ttgttcaagg gcaaggcacg gtttcggccg tgttgagtgg attgttcgaa taagcaccgg 480 ctaactacgt gccagcagcc gcggtaatac gtagggtgcg agcgttatcc ggatttattg 540 ggcgtaaagg gctcgtaggc ggttcgtcgc gtccggtgtg aaagtccatc gcctaacggt 600 ggatctgcgc cgggtacggg cgggctggag tgcggtaggg gagactggaa ttcccggtgt 660 aacggtggaa tgtgtagata tcgggaagaa caccaatggc gaaggcaggt ctctgggccg 720 tcactgacgc tgaggagcga aagcgtgggg agcgaacagg attagatacc ctggtagtcc 780 acgccgtaaa cggtggatgc tggatgtggg gccctttcca cgggtcccgt gtcggagcca 840 acgcgttaag catcccgcct ggggagtacg gccgcaaggc taaaactcaa agaaattgac 900 gggggcccgc acaagcggcg gagcatgcgg attaattcga tgcaacgcga agaaccttac 960 ctgggcttga catgtgccgg atcgccgtgg agacacggtt tcccttcggg gccggttcac 1020 aggtggtgca tggtcgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1080 gcgcaaccct cgccgcatgt tgccagcggg tgatgccggg aactcatgtg ggaccgccgg 1140 ggtcaactcg gaggaaggtg gggatgacgt cagatcatca tgccccttac gtccagggct 1200 tcacgcatgc tacaatggcc ggtacaacgc ggtgcgacac ggtgacgtgg ggcggatcgc 1260 tgaaaaccgg tctcagttcg gatcgcagtc tgcaactcga ctgcgtgaag gcggagtcgc 1320 tagtaatcgc ggatcagcaa cgccgcggtg aatgcgttcc cgggccttgt acacaccgcc 1380 cgtcaagtca tgaaagtggg tagcacccga agccggtggc ccgacccttg tggggggagc 1440 cgtctaagtg agaatcgtga ttgggactaa gtcgtaacaa ggtagcgtac cggaagtgc 1499 <110> Center for Industrialization of Agricultural and Livestock Microorganisms <120> Novel Strain of Bifidobacterium animalis CACC 789, and feed composition using <130> PN210041AN <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1499 <212> DNA <213> Bifidobacterium animalis <400> 1 tctgctcagg atgaacgctg gcggcgtgct taacacatgc aagtcgaacg ggatccctgg 60 cagcttgctg tcggggtgag agtggcgaac gggtgagtaa tgcgtgacca acctgccctg 120 tgcaccggaa tagctcctgg aaacgggtgg taataccgga tgctccgctc catcgcatgg 180 tggggtggga aatgcttttg cggcatggga tggggtcgcg tcctatcagc ttgttggcgg 240 ggtgatggcc caccaaggcg ttgacgggta gccggcctga gagggtgacc ggccacattg 300 ggactgagat acggcccaga ctcctacggg aggcagcagt ggggaatatt gcacaatggg 360 cgcaagcctg atgcagcgac gccgcgtgcg ggatggaggc cttcgggttg taaaccgctt 420 ttgttcaagg gcaaggcacg gtttcggccg tgttgagtgg attgttcgaa taagcaccgg 480 ctaactacgt gccagcagcc gcggtaatac gtagggtgcg agcgttatcc ggatttattg 540 ggcgtaaagg gctcgtaggc ggttcgtcgc gtccggtgtg aaagtccatc gcctaacggt 600 ggatctgcgc cgggtacggg cgggctggag tgcggtaggg gagactggaa ttcccggtgt 660 aacggtggaa tgtgtagata tcgggaagaa caccaatggc gaaggcaggt ctctgggccg 720 tcactgacgc tgaggagcga aagcgtgggg agcgaacagg attagatacc ctggtagtcc 780 acgccgtaaa cggtggatgc tggatgtggg gccctttcca cgggtcccgt gtcggagcca 840 acgcgttaag catcccgcct ggggagtacg gccgcaaggc taaaactcaa agaaattgac 900 gggggcccgc acaagcggcg gagcatgcgg attaattcga tgcaacgcga agaaccttac 960 ctgggcttga catgtgccgg atcgccgtgg agacacggtt tcccttcggg gccggttcac 1020 aggtggtgca tggtcgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1080 gcgcaaccct cgccgcatgt tgccagcggg tgatgccggg aactcatgtg ggaccgccgg 1140 ggtcaactcg gaggaaggtg gggatgacgt cagatcatca tgccccttac gtccagggct 1200 tcacgcatgc tacaatggcc ggtacaacgc ggtgcgacac ggtgacgtgg ggcggatcgc 1260 tgaaaaccgg tctcagttcg gatcgcagtc tgcaactcga ctgcgtgaag gcggagtcgc 1320 tagtaatcgc ggatcagcaa cgccgcggtg aatgcgttcc cgggccttgt acacaccgcc 1380 cgtcaagtca tgaaagtggg tagcacccga agccggtggc ccgacccttg tggggggagc 1440 cgtctaagtg agaatcgtga ttgggactaa gtcgtaacaa ggtagcgtac cggaagtgc 1499

Claims (8)

pH 2.5 조건에서 2시간 배양시 82.92% 생존률을 나타내는 우수한 내산성; 1.0% 옥스갈(Oxgall) 조건에서 2시간 배양시 84.16%의 생존율을 나타내는 우수한 내담즙성; 및 장상피세포(HT-29)에 78.43% 부착되는 우수한 장내부착 활성;을 나타내고,
3개월 저장 조건에서 98%의 생균수를 유지하는 우수한 저장 안정성을 갖고,
IFN-γ 및 TLR3의 염증 반응 관련 유전자 발현을 억제하여 항염증 활성을 나타내며,
가축 병원균인 살모넬라 엔테리티디스(Salmonella Enteritidis) 및 살모넬라 타이피뮤리움(Salmonella Typhimurium)에 대한 항균 활성을 갖는 것을 특징으로 하고,
백혈구(WBC) 내 단핵구(monocytes)와 과립성백혈구(WBC-granulocyte)의 증식을 유도하여 세포성 면역 증진 활성을 나타내는 것을 특징으로 하는,
신균주 비피도박테리움 애니멀리스 CACC 789(수탁번호: KACC 92349P).
Excellent acid resistance showing 82.92% viability when cultured for 2 hours at pH 2.5; Excellent bile resistance showing a survival rate of 84.16% when cultured for 2 hours in 1.0% Oxgall condition; and excellent intestinal adhesion activity with 78.43% adhesion to intestinal epithelial cells (HT-29);
It has excellent storage stability that maintains 98% of the number of viable cells under storage conditions for 3 months,
It exhibits anti-inflammatory activity by suppressing the expression of inflammatory response-related genes of IFN-γ and TLR3,
Characterized in that it has antibacterial activity against livestock pathogens Salmonella Enteritidis and Salmonella Typhimurium,
Characterized in that it induces proliferation of monocytes and granulocytes (WBC-granulocyte) in white blood cells (WBC) to exhibit cellular immunity enhancing activity,
New strain Bifidobacterium animalless CACC 789 (accession number: KACC 92349P).
삭제delete 제1항에 있어서,
상기 균주는 서열번호 1의 16s rRNA 서열을 갖는 것을 특징으로 하는 신균주 비피도박테리움 애니멀리스 CACC 789(수탁번호: KACC 92349P).
According to claim 1,
The strain is a new strain Bifidobacterium animalis CACC 789 (accession number: KACC 92349P), characterized in that it has the 16s rRNA sequence of SEQ ID NO: 1.
제1항의 균주, 또는 그 배양액을 유효성분으로 포함하는 반려동물용 사료 조성물. A feed composition for companion animals comprising the strain of claim 1 or a culture solution thereof as an active ingredient. 삭제delete 삭제delete 삭제delete 삭제delete
KR1020210099278A 2021-07-28 2021-07-28 Novel Strain of Bifidobacterium animalis CACC 789, and feed composition using thereof KR102443494B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020210099278A KR102443494B1 (en) 2021-07-28 2021-07-28 Novel Strain of Bifidobacterium animalis CACC 789, and feed composition using thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
KR1020210099278A KR102443494B1 (en) 2021-07-28 2021-07-28 Novel Strain of Bifidobacterium animalis CACC 789, and feed composition using thereof

Publications (1)

Publication Number Publication Date
KR102443494B1 true KR102443494B1 (en) 2022-09-15

Family

ID=83281615

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1020210099278A KR102443494B1 (en) 2021-07-28 2021-07-28 Novel Strain of Bifidobacterium animalis CACC 789, and feed composition using thereof

Country Status (1)

Country Link
KR (1) KR102443494B1 (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20140008563A (en) * 2012-07-06 2014-01-22 (주)케비젠 Bifidobacterium animalis strain producing conjugated linoleic acid and uses thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20140008563A (en) * 2012-07-06 2014-01-22 (주)케비젠 Bifidobacterium animalis strain producing conjugated linoleic acid and uses thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
J Microbiol Biotechnol. 2020, 30(12):1793-1800.* *

Similar Documents

Publication Publication Date Title
JP5718916B2 (en) Novel Lactobacillus plantarum and composition containing the same
CN102597214B (en) Novel lactobacillus plantarum and composition comprising same
DK2360237T3 (en) NEW Lactobacillus plantarum AND COMPOSITION CONTAINING SAME
US20110269165A1 (en) Lactic acid bacteria and their cellular components inducing immunoregulatory function, and method of obtaining the same
CN112011481B (en) Lactobacillus reuteri for preventing and treating bacterial diarrhea of livestock and poultry and application thereof
KR101724601B1 (en) Composition for preventing or treating atopic dermatitis
AU2006253006A1 (en) Feline probiotic Lactobacilli
CN110878267A (en) Lactobacillus salivarius and application thereof
KR102064134B1 (en) Novel Strain of Pediococcus acidilactici CACC 537, and feed composition using thereof
KR20200034169A (en) Lactobacillus plantarum JDFM LP11 strain having antimicrobial activity, anti-aging activity and probiotics properties and uses thereof
KR101075558B1 (en) Novel Lactobacillus plantarum and compositions comprising the same
KR101098946B1 (en) A compound for feed additive comprising novel Lactobacillus salivarius G1-1
KR100557397B1 (en) Acid tolerant probiotic Lactobacillus reuteri Probio-054 that can suppresses the growth of pathogenic microorganisms
KR102525173B1 (en) Bacillus bacteria, interleukin-22 production inducer, skin barrier function enhancer
KR102443494B1 (en) Novel Strain of Bifidobacterium animalis CACC 789, and feed composition using thereof
KR102140405B1 (en) Novel Strain of Bifidobacterium longum CACC 517, and feed composition using thereof
KR102443495B1 (en) Novel Strain of Lactobacillus rhamnosus CACC 612, and feed composition using thereof
Qin et al. Identification and Characterisation of Potential Probiotic Lactic Acid Bacteria Extracted from Pig Faeces.
KR100513167B1 (en) Acid tolerant probiotic Enterococcus faecalis Probio-053 that can suppresses the growth of pathogenic microorganisms and Salmonella gallinarum
KR100523255B1 (en) Acid tolerant probiotic Enterococcus faecium Probio-048 that can suppresses the growth of pathogenic microorganisms and Salmonella gallinarum
JP5158986B2 (en) Method for selecting microbial strain / component having immunoregulatory function
Chandra et al. Development and assessment of a fish feed to assist in aquaculture nutrition management
CN111212902B (en) Growth-stimulating lactobacillus strains
KR20100076263A (en) Microorganisms for prevention of calves with diarrhea and use thereof
Karami et al. Survey of Probiotic Bacillus in Poultry Farm of Arak, Iran

Legal Events

Date Code Title Description
E701 Decision to grant or registration of patent right
GRNT Written decision to grant