KR102529206B1 - Novel Lactobacillus reuteri DB2 strain and use of the same - Google Patents
Novel Lactobacillus reuteri DB2 strain and use of the same Download PDFInfo
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- KR102529206B1 KR102529206B1 KR1020200179713A KR20200179713A KR102529206B1 KR 102529206 B1 KR102529206 B1 KR 102529206B1 KR 1020200179713 A KR1020200179713 A KR 1020200179713A KR 20200179713 A KR20200179713 A KR 20200179713A KR 102529206 B1 KR102529206 B1 KR 102529206B1
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- strain
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- lactobacillus reuteri
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Abstract
본 발명은 락토바실러스 루테리 DB2 신균주(KACC 92293P) 및 이의 용도에 관한 것으로, 본 발명의 락토바실러스 루테리 DB2 신균주(KACC 92293P)는 장내 균총을 개선하는 활성이 있고 동시에 NK(Natural killer) 세포 활성 및 면역반응 관련 사이토카인의 생성 증진 활성을 가지고 있어, 인간뿐만 아니라 반려동물의 장 기능 개선 및 면역증진 개선을 위한 기능성 식품 및 의약품의 제조에 사용될 수 있으며, 나아가 정장용, 생균제, 사료용 조성물 및 반려동물 간식의 제조에도 유용하게 사용될 수 있다.The present invention relates to a new Lactobacillus reuteri DB2 strain (KACC 92293P) and a use thereof, and the new Lactobacillus reuteri DB2 strain (KACC 92293P) of the present invention has an activity to improve intestinal flora and at the same time activates NK (Natural killer) cells and immune response-related cytokine production enhancement activity, so it can be used in the manufacture of functional foods and pharmaceuticals for improving intestinal function and immunity enhancement of humans as well as companion animals, and furthermore, for formal wear, probiotics, feed compositions and companion animals It can also be usefully used in the manufacture of snacks.
Description
본 발명은 신규한 락토바실러스 루테리 DB2 균주 및 이의 용도에 관한 것이다.The present invention relates to a novel Lactobacillus reuteri DB2 strain and uses thereof.
생균제는 인간과 동물 건강에 이로운 효과를 주는 살아있는 미생물 첨가제로서 대표적으로 유산균(Lactic acid bacteria)이 있다. 생균제로 가장 많이 사용되는 유산균으로는 비피도박테리움(Bifidobacterium)과 락토바실러스(Lactobacillus)가 있고, 이들 유산균은 장내 유익균으로 알려져 있다.Probiotics are live microbial additives that have beneficial effects on human and animal health, and representatively include lactic acid bacteria. Lactic acid bacteria most commonly used as probiotics include Bifidobacterium and Lactobacillus, and these lactic acid bacteria are known as intestinal beneficial bacteria.
유산균은 장내 pH를 산성으로 유지시켜 대장균이나 크로스트리디움(Clostridium sp.)과 같은 유해균의 번식을 억제하고 설사와 변비를 개선할 뿐 아니라, 비타민 합성, 항암작용, 혈청 콜레스테롤 저하 등의 역할을 한다. 특히, 유산균은 장의 점막과 상피세포에 강하게 결합할 수 있는 특정 단백질을 가지고 있어 유해 세균의 성장을 막는 정장 작용에 많은 도움을 준다. 또한, 유산균은 대식세포의 증식을 촉진하여 대식세포(macrophage)의 장내 유해 세균에 대한 인지능력, 살균능력 등을 강화시키고, 면역 관련 물질의 분비를 촉진하여 면역증강 효과를 나타내는 것으로 알려져 있다(Gabriela perdigon et al., J. of food Protection 53:404-410, 1990; Katsumasa sato et al., Microbiol. Immunol., 32(7):689-698, 1988).Lactic acid bacteria keep the intestinal pH acidic, suppress the growth of harmful bacteria such as Escherichia coli or Clostridium sp., improve diarrhea and constipation, and also play a role in vitamin synthesis, anti-cancer activity, and lowering serum cholesterol. . In particular, lactic acid bacteria have a specific protein that can strongly bind to intestinal mucosa and epithelial cells, so it is very helpful in the intestinal function to prevent the growth of harmful bacteria. In addition, lactic acid bacteria are known to promote the proliferation of macrophages, enhance the ability of macrophages to recognize harmful bacteria in the intestine, bactericidal ability, etc., and promote the secretion of immune-related substances to exhibit an immune-enhancing effect (Gabriela perdigon et al., J. of food protection 53:404-410, 1990; Katsumasa sato et al., Microbiol. Immunol., 32(7):689-698, 1988).
이에 최근에는 유산균을 장내에 정착시키기 위해, 포유동물의 장내에서 안정적으로 정착할 수 있는 유산균을 분리하고, 이를 생균제, 정장제, 식품첨가제 등으로 개발하고자 하는 연구가 활발히 진행되고 있으나, 이러한 목적을 달성하기 위한 효과적인 균주가 아직 개발되지 못하고 있는 실정이다.In recent years, in order to settle lactic acid bacteria in the intestine, studies are being actively conducted to isolate lactic acid bacteria that can stably settle in the intestines of mammals and develop them as probiotics, intestinal agents, food additives, etc. An effective strain for this has not yet been developed.
한편, 현대의 생활은 인간이 반려동물과 더불어 살아가는 환경에 조성되고 있고, 반려 동물은 인간에게 심리적으로 안정감과 친밀감을 주는 친구나 가족과 같은 존재가 되어 가고 있다. 반려 동물은 전통적인 반려 동물인 개와 고양이뿐만 아니라 앵무새, 고슴도치, 토끼 및 햄스터 등으로 그 종류도 다양해지고 있으며, 반려동물을 보살피기 위한 관련 산업도 점차 각광을 받고 있다.On the other hand, modern life is being created in an environment where humans live together with companion animals, and companion animals are becoming like friends or family that give humans psychological stability and intimacy. Companion animals are diversifying into not only traditional companion animals such as dogs and cats, but also parrots, hedgehogs, rabbits and hamsters.
이에 반려동물의 사료 및 간식과 관련된 산업은 반려동물의 건강과 직결된 산업에 해당하기 때문에 더욱 관심이 집중되고 있고, 반려동물의 사료도 사람이 섭취하는 음식과 비슷한 종류의 것까지 접근해 있는 추세이며, 특히 반려동물 장 내에 유산균을 증식시켜 장 건강과 면역력을 개선하는 효과를 갖는 반려동물용 기능성 제품의 개발도 필요한 실정이다.Accordingly, the industry related to companion animal food and snacks is receiving more attention because it corresponds to the industry directly related to the health of companion animals, and the trend is that pet food is also approaching similar types of food consumed by humans. In particular, it is necessary to develop functional products for companion animals that have the effect of improving intestinal health and immunity by proliferating lactic acid bacteria in the intestines of companion animals.
이에 본 발명자들은 인간을 비롯한 반려동물의 장기능 개선 효과뿐만 아니라 면역증진 효과를 모두 갖는 새로운 유산균을 동정하기 위해 연구하던 중, 신규한 락토바실러스 루테리 DB2 균주를 분리 및 동정하였고 상기 균주가 장기능 개선 및 면역증진에 대해 우수한 효능이 있음을 규명함으로써 본 발명을 완성하였다.Accordingly, the present inventors isolated and identified a novel Lactobacillus reuteri DB2 strain while studying to identify a new lactic acid bacterium having both the effect of improving the intestinal function of companion animals, including humans, as well as the effect of enhancing immunity, and the strain improved the intestinal function. And the present invention was completed by identifying that there is an excellent efficacy for immune enhancement.
따라서 본 발명의 목적은 락토바실러스 루테리 DB2 신균주(KACC 92293P)를 제공하는 것이다.Accordingly, an object of the present invention is to provide a new strain of Lactobacillus reuteri DB2 (KACC 92293P).
본 발명의 다른 목적은 락토바실러스 루테리 DB2 신균주(KACC 92293P), 이의 파쇄물 또는 이의 배양물을 유효성분으로 포함하는, 장기능 개선 또는 면역증진용 식품 조성물; 장 질환 또는 면역질환의 예방 또는 치료용 약학적 조성물; 정장용 조성물; 생균제 조성물; 또는 사료용 조성물을 제공하는 것이다.Another object of the present invention is a food composition for improving intestinal function or enhancing immunity, comprising a new strain of Lactobacillus reuteri DB2 (KACC 92293P), a lysate thereof or a culture thereof as an active ingredient; A pharmaceutical composition for preventing or treating intestinal or immune diseases; compositions for formal wear; probiotic composition; Or to provide a composition for feed.
상기와 같은 본 발명의 목적을 달성하기 위해서, 본 발명은 락토바실러스 루테리 DB2 신균주(KACC 92293P)을 제공한다.In order to achieve the object of the present invention as described above, the present invention provides a new strain of Lactobacillus reuteri DB2 (KACC 92293P).
본 발명의 일실시예에 있어서, 상기 균주는 장내 균총 개선 또는 면역증진 활성을 갖는 것일 수 있다.In one embodiment of the present invention, the strain may have intestinal microflora improvement or immunity enhancing activity.
또한 본 발명은 락토바실러스 루테리 DB2 신균주(KACC 92293P), 이의 파쇄물 또는 이의 배양물을 유효성분으로 포함하는, 장기능 개선 또는 면역증진용 식품 조성물을 제공한다.In addition, the present invention provides a food composition for improving intestinal function or enhancing immunity, comprising a new strain of Lactobacillus reuteri DB2 (KACC 92293P), a lysate thereof, or a culture thereof as an active ingredient.
본 발명의 일실시예에 있어서, 상기 장기능 개선은 장내 균총을 개선하는 것이고, 상기 면역증진은 NK(Natural killer) 세포활성 증진; TNF-α, IFN-γ 또는 IL-6의 사이토카인 생성 증진; 또는 NF-kB 활성 증진을 통해 이루어지는 것일 수 있다.In one embodiment of the present invention, the improvement of intestinal function is to improve the intestinal flora, and the immunity enhancement is to enhance NK (Natural killer) cell activity; enhancing cytokine production of TNF-α, IFN-γ or IL-6; Alternatively, it may be achieved through enhancement of NF-kB activity.
본 발명의 일실시예에 있어서, 상기 조성물은 비피도박테리움 롱검 유산균을 추가로 더 포함하는 것일 수 있다. In one embodiment of the present invention, the composition may further contain Bifidobacterium longum lactic acid bacteria.
본 발명의 일실시예에 있어서, 상기 조성물은 반려 동물용일 수 있다.In one embodiment of the present invention, the composition may be for companion animals.
또한 본 발명은 락토바실러스 루테리 DB2 신균주(KACC 92293P), 이의 파쇄물 또는 이의 배양물을 유효성분으로 포함하는, 장 질환 또는 면역질환의 예방 또는 치료용 약학적 조성물을 제공한다. In addition, the present invention provides a pharmaceutical composition for preventing or treating intestinal diseases or immune diseases, comprising a new strain of Lactobacillus reuteri DB2 (KACC 92293P), a lysate thereof, or a culture thereof as an active ingredient.
또한 본 발명은 락토바실러스 루테리 DB2 신균주(KACC 92293P), 이의 파쇄물 또는 이의 배양물을 유효성분으로 포함하는, 정장용 조성물을 제공한다.In addition, the present invention provides a composition for intestinal disorders comprising a new strain of Lactobacillus reuteri DB2 (KACC 92293P), a lysate thereof, or a culture thereof as an active ingredient.
또한 본 발명은 락토바실러스 루테리 DB2 신균주(KACC 92293P), 이의 파쇄물 또는 이의 배양물을 유효성분으로 포함하는, 생균제 조성물을 제공한다.In addition, the present invention provides a probiotic composition comprising a new strain of Lactobacillus reuteri DB2 (KACC 92293P), a lysate thereof, or a culture thereof as an active ingredient.
또한 본 발명은 락토바실러스 루테리 DB2 신균주(KACC 92293P), 이의 파쇄물 또는 이의 배양물을 유효성분으로 포함하는, 사료용 조성물을 제공한다.In addition, the present invention provides a feed composition comprising a new strain of Lactobacillus reuteri DB2 (KACC 92293P), a lysate thereof, or a culture thereof as an active ingredient.
본 발명은 락토바실러스 루테리 DB2 신균주(KACC 92293P) 및 이의 용도에 관한 것으로, 본 발명의 락토바실러스 루테리 DB2 신균주(KACC 92293P)는 장내 균총을 개선하는 활성이 있고 동시에 NK(Natural killer) 세포 활성 및 면역반응 관련 사이토카인의 생성 증진 활성을 가지고 있어, 인간뿐만 아니라 반려동물의 장 기능 개선 및 면역증진 개선을 위한 기능성 식품 및 의약품의 제조에 사용될 수 있으며, 나아가 정장용, 생균제, 사료용 조성물의 제조에도 유용하게 사용될 수 있다.The present invention relates to a new Lactobacillus reuteri DB2 strain (KACC 92293P) and a use thereof, and the new Lactobacillus reuteri DB2 strain (KACC 92293P) of the present invention has an activity to improve intestinal flora and at the same time activates NK (Natural killer) cells and immune response-related cytokine production enhancement activity, so it can be used in the manufacture of functional foods and pharmaceuticals for improvement of intestinal function and immunity enhancement of humans as well as companion animals, and furthermore, it is also used in the manufacture of compositions for formal wear, probiotics, and feed. can be useful
도 1은 본 발명의 일실시예에서 본 발명의 락토바실러스 루테리 DB2 신균주에 대한 NO 생성능을 분석한 결과를 나타낸 것으로, 본 발명의 도면에서 LGG는 Lactobacillus rhamnosus GG 균주를, BIF4는 비피도박테리움 롱검 BIF4를, KACC91563는 비피도박테리움 롱검 KACC91563 균주를 나타낸 것이다.
도 2는 본 발명의 일실시예에서 본 발명의 락토바실러스 루테리 DB2 신균주와 다른 균주들에 대한 NF-kB의 활성화를 분석한 결과를 나타낸 것이다.
도 3은 본 발명의 일실시예에서 본 발명의 락토바실러스 루테리 DB2 신균주와 다른 균주들에 대한 IL-6 생성능을 분석한 결과를 나타낸 것이다.
도 4는 본 발명의 일실시예에서 본 발명의 락토바실러스 루테리 DB2 신균주와 다른 균주들에 대한 TNF-a 생성능을 분석한 결과를 나타낸 것이다.
도 5는 본 발명의 일실시예에서 본 발명의 락토바실러스 루테리 DB2 신균주와 비피도박테리움 롱검 KACC91563 균주의 혼합균주를 첨가하여 제조한 퀘소블랑코 치즈를 섭취시킨 비글 견(QCBL) 및 균주를 첨가하지 않고 제조한 퀘소블랑코 치즈를 섭취시킨 비글 견(QC)에 대한 혈청 내 TNF-α의 함량을 측정한 결과를 나타낸 것이다.
도 6은 본 발명의 일실시예에서 본 발명의 락토바실러스 루테리 DB2 신균주와 비피도박테리움 롱검 KACC91563 균주의 혼합균주를 첨가하여 제조한 퀘소블랑코 치즈를 섭취시킨 비글 견(QCBL) 및 균주를 첨가하지 않고 제조한 퀘소블랑코 치즈를 섭취시킨 비글 견(QC)에 대한 혈청 내 IFN-γ의 함량을 측정한 결과를 나타낸 것이다.1 shows the results of analyzing the NO production ability of the new Lactobacillus reuteri DB2 strain of the present invention in one embodiment of the present invention. Longum BIF4, KACC91563 indicates the Bifidobacterium longum KACC91563 strain.
Figure 2 shows the results of analyzing the activation of NF-kB for the new Lactobacillus reuteri DB2 strain of the present invention and other strains in one embodiment of the present invention.
Figure 3 shows the results of analyzing the IL-6 producing ability of the new Lactobacillus reuteri DB2 strain of the present invention and other strains in one embodiment of the present invention.
Figure 4 shows the results of analyzing the TNF-a producing ability of the new Lactobacillus reuteri DB2 strain of the present invention and other strains in one embodiment of the present invention.
5 is a beagle dog (QCBL) ingested with Queso Blanco cheese prepared by adding a mixed strain of the new strain of Lactobacillus reuteri DB2 and the strain of Bifidobacterium longum KACC91563 of the present invention in one embodiment of the present invention and adding the strain. It shows the results of measuring the content of TNF-α in the serum of beagle dogs (QC) fed with Queso Blanco cheese prepared without processing.
6 is a beagle dog (QCBL) fed with Queso Blanco cheese prepared by adding a mixed strain of the new strain of Lactobacillus reuteri DB2 and the strain of Bifidobacterium longum KACC91563 of the present invention in one embodiment of the present invention and adding the strain. It shows the results of measuring the content of IFN-γ in the serum of beagle dogs (QC) fed with Queso Blanco cheese prepared without processing.
본 발명은 락토바실러스 루테리 DB2 신균주(KACC 92293P) 및 상기 균주의 용도를 제공하는 것으로, 장기능 개선 및 면역증진 활성을 모두 가지고 있는 락토바실러스 루테리 DB2 신균주를 최초로 분리 및 동정하였다.The present invention provides a new strain of Lactobacillus reuteri DB2 (KACC 92293P) and a use of the strain, and a new strain of Lactobacillus reuteri DB2 having both intestinal function improvement and immunity enhancing activity was isolated and identified for the first time.
본 발명자들은 장기능 개선활성을 가지면서 동시에 면역증진 활성을 갖는 신규한 유산균을 동정하기 위해 연구하던 중, 건강한 반려견의 장관으로부터 락토바실러스 루테리 DB2 신균주(KACC 92293P)를 분리 및 동정하였고, 상기 동정한 균주가 종래 알려지지 않은 신균주임을 확인함에 따라, 상기 균주를 락토바실러스 루테리 DB2로 명명하였고, 2019년 11월 22일에 국립농업과학원 미생물은행에 기탁하여 기탁번호 KACC 92293P를 부여받았다.The present inventors isolated and identified a new Lactobacillus reuteri DB2 strain (KACC 92293P) from the intestinal tract of a healthy dog while conducting research to identify a novel lactic acid bacterium having intestinal function-improving activity and immunity-enhancing activity at the same time, and the identification As one strain was confirmed to be a previously unknown new strain, the strain was named Lactobacillus reuteri DB2, and on November 22, 2019, it was deposited with the National Institute of Agricultural Sciences Microorganism Bank and was given accession number KACC 92293P.
한편, 본 발명자들은 본 발명의 신균주에 대한 특성을 확인하기 위해 연구한 결과, 락토바실러스 루테리 DB2 신균주(KACC 92293P)를 함유한 치즈를 섭취한 실험동물군이 락토바실러스 루테리 DB2 신균주를 함유하지 않은 치즈를 섭취한 대조군에 비해 장내 유익균의 총균수가 증가되어 있는 것으로 나타났고, 반면 유해균의 총균수는 감소되어 있는 것으로 나타났다.On the other hand, the inventors of the present invention studied to confirm the characteristics of the new strain of the present invention, and as a result, the experimental animal group consuming cheese containing the new Lactobacillus reuteri DB2 strain (KACC 92293P) contained the new strain of Lactobacillus reuteri DB2. It was found that the total number of beneficial bacteria in the intestine was increased compared to the control group who consumed cheese without cheese, while the total number of harmful bacteria was decreased.
따라서 이러한 결과를 통해 본 발명자들은 본 발명의 신균주가 장내 균총을 개선할 수 있는 활성이 있어 인간뿐만 아니라 반려동물의 장 기능을 증진 또는 개선할 수 있고, 장질환을 예방, 개선 또는 치료할 수 있음을 알 수 있었다.Therefore, through these results, the present inventors found that the new strain of the present invention has an activity to improve the intestinal flora, so that the intestinal function of humans as well as companion animals can be promoted or improved, and intestinal diseases can be prevented, improved, or treated And it was found.
나아가 본 발명의 락토바실러스 루테리 DB2 신균주(KACC 92293P) 함유 치즈를 섭취한 실험동물군의 경우, 혈청 내 면역반응 관련 사이토카인인 TNF-α, IFN-γ 또는 IL-6의 생성량이 대조군에 비해 월등히 증진되어 있는 것으로 나타났고, NK(Natural killer) 세포 활성 또한 증가되어 있는 것으로 나타났다.Furthermore, in the case of the experimental animal group consuming the cheese containing the new Lactobacillus reuteri DB2 strain (KACC 92293P) of the present invention, the production of TNF-α, IFN-γ or IL-6, which are immune response-related cytokines, in serum was higher than that of the control group. It was shown to be significantly enhanced, and NK (Natural killer) cell activity was also increased.
특히 자연살해세포(natural killer cell; NK 세포)는 항원에 의한 사전 감작없이도 종양세포, 박테리아, 세포 내 기생생물 또는 바이러스에 감염된 숙주세포를 제거하는 기능을 수행하며, 부적절한 골수 이식을 거부하고, T세포의 면역반응을 조절하는 등 생체 내 면역계 방어 기작의 제 1선에서 작용하는 행동세포이다.In particular, natural killer cells (NK cells) perform the function of removing host cells infected with tumor cells, bacteria, intracellular parasites or viruses without prior sensitization by antigens, rejecting inappropriate bone marrow transplantation, and T It is a behavioral cell that acts in the first line of the immune system defense mechanism in vivo, such as regulating the cell's immune response.
또한, NK 세포는 숙주에 감염된 병원균 또는 암세포에 대항하기 위한 선천성 면역 반응과 사이토카인 분비를 통한 후천성 면역 반응에 중요한 역할을 하는데, NK 세포가 표적 세포를 살상하는데 사용하는 주된 메커니즘은 퍼포린(perforin) 및 그랜자임(granzyme) B와 같은 세포 용해성 과립을 면역 시냅스를 통하여 표적 세포에 분비하는 것으로, 분비된 퍼포린은 표적 세포벽에 구멍을 만들고, 구멍을 통하여 표적 세포 내로 들어간 그랜자임이 표적 세포의 세포사멸(apoptosis)을 유도한다. 이와 같이, NK 세포는 세포가 악성으로 변형되는 발암과정이나, 바이러스 감염 초기의 방어기전에서 결정적인 역할을 하는 것으로 확인되었으며, 이러한 연구들을 바탕으로 암 또는 면역질환 치료를 위해 NK 세포 치료법이 대두되고 있다.In addition, NK cells play an important role in the innate immune response against pathogens or cancer cells infected by the host and the acquired immune response through cytokine secretion. The main mechanism used by NK cells to kill target cells is perforin ) and granzyme B are secreted into the target cell through the immune synapse. induces apoptosis. As such, NK cells have been confirmed to play a crucial role in the carcinogenesis process in which cells are transformed into malignant cells or in the defense mechanism in the early stages of viral infection. Based on these studies, NK cell therapy is emerging for the treatment of cancer or immune diseases. .
이러한 점에서 본원발명의 신균주는 NK 세포를 활성화 수 있고, 면역반응을 유도하는 사이토카인인 TNF-α, IFN-γ 또는 IL-6의 생성을 증가시킬 수 있어 면역질환을 예방, 개선 또는 치료할 수 있다.In this regard, the new strain of the present invention can activate NK cells and increase the production of TNF-α, IFN-γ or IL-6, which are cytokines that induce immune responses, so that immune diseases can be prevented, improved, or treated. can
또한 본 발명의 락토바실러스 루테리 DB2 신균주(KACC 92293P)가 갖는 장 기능 개선, 즉 장내 균총 개선 활성 및 면역증진 활성은 다른 종류의 유산균들에 비해서도 더 우수한 효과가 있는 것으로 확인되었다.In addition, it was confirmed that the intestinal function improvement of the new Lactobacillus reuteri DB2 strain (KACC 92293P) of the present invention, that is, the activity of improving intestinal flora and the activity of enhancing immunity, is more effective than other types of lactic acid bacteria.
뿐만 아니라 본 발명자들은 본 발명의 락토바실러스 루테리 DB2 신균주(KACC 92293P)와 함께 혼합균주로서 면역증진 활성을 더욱 증진시킬 수 있는 균주들을 스크리닝한 결과, 다른 유산균에 비해 비피도박테리움 롱검 균주를 본 발명의 신균주와 함께 혼합하여 사용할 경우, 우수한 면역증진 활성을 도출할 수 있음을 알 수 있었다.In addition, the present inventors screened strains capable of further enhancing the immune-enhancing activity as a mixed strain together with the new Lactobacillus reuteri DB2 strain (KACC 92293P) of the present invention, and as a result, compared to other lactic acid bacteria, the Bifidobacterium longum strain was found. It was found that when mixed with the new strain of the invention and used, excellent immune enhancing activity can be derived.
그러므로 본 발명은 락토바실러스 루테리 DB2 신균주(KACC 92293P)를 제공할 수 있으며, 나아가 락토바실러스 루테리 DB2 신균주(KACC 92293P), 이의 파쇄물 또는 이의 배양물을 유효성분으로 포함하는, 장기능 개선 또는 면역증진용 식품 조성물을 제공할 수 있다.Therefore, the present invention can provide a new strain of Lactobacillus reuteri DB2 (KACC 92293P), and furthermore, improve intestinal function or immunity, including a new strain of Lactobacillus reuteri DB2 (KACC 92293P), a lysate thereof, or a culture thereof as an active ingredient. A food composition for enhancement can be provided.
본 발명의 식품 조성물은 기능성 식품(functional food), 영양 보조제(nutritional supplement), 건강식품(health food) 및 식품 첨가제(food additives) 등의 모든 형태를 포함한다. 상기 유형의 식품 조성물은 당 업계에 공지된 통상적인 방법에 따라 다양한 형태로 제조할 수 있다. 예를 들면, 건강식품으로는 본 발명의 락토바실러스 루테리 DB2 신균주(KACC 92293P) 자체, 이의 파쇄물 및 상기 균주 배양물 군 중 하나 이상을 차, 쥬스 및 드링크의 형태로 제조하여 음용하도록 하거나, 과립화, 캡슐화 및 분말화 하여 섭취할 수 있다. 또한, 본 발명의 락토바실러스 루테리 DB2 신균주(KACC 92293P), 이의 파쇄물 및 배양물 군 중 하나 이상을 장기능 개선능 또는 면역증진능이 있다고 알려진 공지의 물질 또는 활성 성분과 함께 혼합하여 조성물의 형태로 제조할 수 있다. 또한, 기능성 식품으로는 음료(알콜성 음료 포함), 과실 및 그의 가공식품(예: 과일통조림, 병조림, 잼, 마아말 레이드 등), 어류, 육류 및 그 가공식품(예: 햄, 소시지콘비이프 등), 빵류 및 면류(예: 우동, 메밀국수, 라면, 스파게티, 마카로니 등), 과즙, 각종 드링크, 쿠키, 엿, 유제품(예: 버터, 치이즈 등), 식용식물유지, 마아가린, 식물성 단백질, 레토르트 식품, 냉동식품, 각종 조미료(예: 된장, 간장, 소스 등) 등에 본 발명의 락토바실러스 루테리 DB2 신균주(KACC 92293P), 이의 파쇄물 및 배양물 군 중 하나 이상을 첨가하여 제조할 수 있다. The food composition of the present invention includes all forms such as functional food, nutritional supplement, health food and food additives. Food compositions of this type can be prepared in various forms according to conventional methods known in the art. For example, as a health food, the new Lactobacillus reuteri DB2 strain (KACC 92293P) itself, its lysate, and at least one of the strain culture groups of the present invention are prepared in the form of tea, juice, and drink for drinking, or granules It can be consumed as capsules, encapsulates, and powders. In addition, the new strain of Lactobacillus reuteri DB2 (KACC 92293P) of the present invention, its lysate and culture group is mixed with a known substance or active ingredient known to have an ability to improve intestinal function or enhance immunity to form a composition. can be manufactured In addition, functional foods include beverages (including alcoholic beverages), fruits and their processed foods (e.g. canned fruit, bottled fruit, jam, marmalade, etc.), fish, meat and their processed foods (e.g. ham, sausage corned beef). etc.), breads and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein , Retort food, frozen food, various seasonings (e.g., soybean paste, soy sauce, sauce, etc.) can be prepared by adding at least one of the group of the new strain of Lactobacillus reuteri DB2 (KACC 92293P), its lysates and cultures of the present invention. .
본 발명의 식품 조성물 중 상기 본 발명의 신균주, 이의 파쇄물 및 배양물 등의 바람직한 함유량으로는 이에 한정되지 않지만 바람직하게는 최종적으로 제조된 식품 중 0.01 내지 50 중량%이다. 또한, 본 발명의 신균주, 이의 파쇄물 및 배양물로 이루어진 군에서 선택된 하나 이상을 유효성분으로서 식품 첨가제의 형태로 사용하기 위해서는 분말 또는 농축액 형태로 제조하여 사용할 수 있다.In the food composition of the present invention, the preferred content of the new strain of the present invention, its lysate and culture is not limited thereto, but is preferably 0.01 to 50% by weight of the final food product. In addition, in order to use at least one selected from the group consisting of the new strain of the present invention, its lysate and culture as an active ingredient in the form of a food additive, it can be prepared and used in the form of a powder or concentrate.
또한 본 발명의 락토바실러스 루테리 DB2 신균주(KACC 92293P)는 건강한 반려견의 장관에서 분리된 균주라는 점에서 개와 고양이 등 반려동물의 장내 균총 개선 또는 면역증진 활성을 효과적으로 증진시킬 수 있으므로, 반려동물용 장기능 개선 또는 면역증진용 간식의 제조에도 사용될 수 있다.In addition, since the new Lactobacillus reuteri DB2 strain (KACC 92293P) of the present invention is a strain isolated from the intestinal tract of a healthy companion dog, it can effectively improve the intestinal flora or enhance immunity in companion animals such as dogs and cats. It can also be used in the manufacture of snacks for function improvement or immunity enhancement.
따라서 본 발명은 락토바실러스 루테리 DB2 신균주(KACC 92293P), 이의 파쇄물 또는 이의 배양물을 유효성분으로 포함하는, 반려동물용 간식 조성물을 제공할 수 있다. Therefore, the present invention can provide a snack composition for companion animals, comprising the new strain of Lactobacillus reuteri DB2 (KACC 92293P), its lysate or its culture as an active ingredient.
상기 반려동물용 간식 조성물은 반려동물에게 섭취시킬 수 있는 다양한 간식, 예컨대 비스켓, 껌, 육포, 음료, 치즈 및 유제품 등의 제조에 첨가하여 장기능 개선 또는 면역증진 활성을 갖는 반려동물용 간식을 제조할 수 있다.The companion animal snack composition is added to the manufacture of various snacks that can be consumed by companion animals, such as biscuits, chewing gum, jerky, beverages, cheese and dairy products, etc. can do.
또한 본 발명은 락토바실러스 루테리 DB2 신균주(KACC 92293P), 이의 파쇄물 또는 이의 배양물을 유효성분으로 포함하는, 장 질환 또는 면역질환의 예방 또는 치료용 약학적 조성물을 제공할 수 있다.In addition, the present invention can provide a pharmaceutical composition for preventing or treating intestinal diseases or immune diseases, comprising a new strain of Lactobacillus reuteri DB2 (KACC 92293P), a lysate thereof, or a culture thereof as an active ingredient.
상기 장 질환으로는 이에 제한되지는 않으나, 크론병, 베체트병에 수반되는 장 병변, 궤양성 대장염, 출혈성 직장 궤양, 회장 낭염 또는 변비일 수 있고, 또한 반려동물에게 발생하는 장 질환을 포함할 수 있다.The intestinal disease may be, but is not limited to, Crohn's disease, intestinal lesions accompanying Behcet's disease, ulcerative colitis, bleeding rectal ulcer, ileum pouchitis or constipation, and may also include intestinal diseases that occur in companion animals. there is.
상기 면역질환은 면역기능 저하로 인해 유발되는 질환으로, 이에 제한되지는 않으나, 류마티스 관절염, 타입 I 당뇨병, 다발성 경화증 및 전신성 홍반 루푸스와 같은 자가면역질환, 천식, 엔세필리티스(encephilitis), 염증성 장염, 만성 폐쇄성 폐질환, 알러지, 폐혈병성 쇼크증, 폐섬유증, 미분화 척추관절증, 미분화 관절병증, 관절염, 염증성 골용해, 및 만성 바이러스 또는 박테리아 감염에 의한 만성 염증과 같은 염증성 질환, 각종 암, 및 감기와 같은 바이러스 질환을 포함한다. 또한 상기 면역질환은 반려동물에게서 발생하는 면역질환을 포함할 수 있다.The immune disease is a disease caused by a decrease in immune function, but is not limited thereto, autoimmune diseases such as rheumatoid arthritis, type I diabetes, multiple sclerosis and systemic lupus erythematosus, asthma, encephilitis, inflammatory enteritis , inflammatory diseases such as chronic obstructive pulmonary disease, allergy, septic shock, pulmonary fibrosis, undifferentiated spondyloarthrosis, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation caused by chronic viral or bacterial infections, various cancers, and Including viral diseases such as the common cold. In addition, the immune disease may include an immune disease occurring in companion animals.
상기 약학적 조성물에는 약학적으로 허용 가능한 염을 단독으로 함유하거나 또는 하나 이상의 약학적으로 허용되는 담체, 부형제 또는 희석제를 추가로 함유할 수 있다. 약학적으로 허용되는 담체로는 예컨대, 경구 투여용 담체 또는 비경구 투여용 담체를 추가로 포함할 수 있다. 경구 투여용 담체는 락토스, 전분, 셀룰로스 유도체, 마그네슘 스테아레이트, 스테아르산 등을 포함할 수 있다. 또한, 비경구 투여용 담체는 물, 적합한 오일, 식염수, 수성 글루코스 및 글리콜 등을 포함할 수 있으며, 안정화제 및 보존제를 추가로 포함할 수 있다. 적합한 안정화제로는 아황산수소나트륨, 아황산나트륨 또는 아스코르브산과 같은 항산화제가 있다. 적합한 보존제로는 벤즈알코늄 클로라이드, 메틸- 또는 프로필-파라벤 및 클로로부탄올이 있다. 그 밖의 약학적으로 허용되는 담체로는 다음의 문헌에 기재되어 있는 것을 참고로 할 수 있다(Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995). The pharmaceutical composition may contain a pharmaceutically acceptable salt alone or may further contain one or more pharmaceutically acceptable carriers, excipients or diluents. A pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like. In addition, the carrier for parenteral administration may include water, suitable oil, saline, aqueous glucose, glycol, and the like, and may further include a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. As other pharmaceutically acceptable carriers, reference may be made to those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).
본 발명의 약학적 조성물은 인간을 비롯한 포유동물에 어떠한 방법으로도 투여할 수 있다. 예를 들면, 경구 또는 비경구적으로 투여할 수 있다. 비경구적인 투여방법으로는 이에 한정되지는 않으나, 정맥내, 근육내, 동맥내, 골수내, 경막내, 심장내, 경피, 피하, 복강내, 비강내, 장관, 국소, 설하 또는 직장내 투여일 수 있다. 바람직하게는 본 발명의 약학적 조성물은 경구 또는 경피 투여될 수 있다.The pharmaceutical composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal administration can be Preferably, the pharmaceutical composition of the present invention can be administered orally or transdermally.
본 발명의 약학적 조성물은 상술한 바와 같은 투여 경로에 따라 경구 투여용 또는 비경구 투여용 제제로 제형화 할 수 있다. The pharmaceutical composition of the present invention may be formulated into a preparation for oral administration or parenteral administration according to the administration route as described above.
경구 투여용 제제의 경우에 본 발명의 조성물은 분말, 과립, 정제, 환제, 당의정제, 캡슐제, 액제, 겔제, 시럽제, 슬러리제, 현탁액 등으로 당업계에 공지된 방법을 이용하여 제형화될 수 있다. 예를 들어, 경구용 제제는 활성성분을 고체 부형제와 배합한 다음 이를 분쇄하고 적합한 보조제를 첨가한 후 과립 혼합물로 가공함으로써 정제 또는 당의정제를 수득할 수 있다. 적합한 부형제의 예로는 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨 및 말티톨 등을 포함하는 당류와 옥수수 전분, 밀 전분, 쌀 전분 및 감자 전분 등을 포함하는 전분류, 셀룰로즈, 메틸 셀룰로즈, 나트륨 카르복시메틸셀룰로오즈 및 하이드록시프로필메틸-셀룰로즈 등을 포함하는 셀룰로즈류, 젤라틴, 폴리비닐피롤리돈 등과 같은 충전제가 포함될 수 있다. 또한, 경우에 따라 가교결합 폴리비닐피롤리돈, 한천, 알긴산 또는 나트륨 알기네이트 등을 붕해제로 첨가할 수 있다. 나아가, 본 발명의 약학적 조성물은 항응집제, 윤활제, 습윤제, 향료, 유화제 및 방부제 등을 추가로 포함할 수 있다. In the case of preparations for oral administration, the composition of the present invention may be formulated into powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions, etc. using a method known in the art. can For example, preparations for oral use may be obtained by combining the active ingredient with a solid excipient, which is then milled and, after adding suitable auxiliaries, processed into a mixture of granules to obtain tablets or dragees. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, starches including corn starch, wheat starch, rice starch and potato starch, cellulose, Celluloses including methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose, and the like, fillers such as gelatin, polyvinylpyrrolidone, and the like may be included. In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant, if desired. Furthermore, the pharmaceutical composition of the present invention may further include an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, and a preservative.
비경구 투여용 제제의 경우에는 주사제, 크림제, 로션제, 외용연고제, 오일제, 보습제, 겔제, 에어로졸 및 비강 흡입제의 형태로 당업계에 공지된 방법으로 제형화할 수 있다. 이들 제형은 모든 제약 화학에 일반적으로 공지된 처방서인 문헌(Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour)에 기재되어 있다. In the case of preparations for parenteral administration, they may be formulated in the form of injections, creams, lotions, external ointments, oils, moisturizers, gels, aerosols, and nasal inhalants by methods known in the art. These formulations are described in Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, which is a generally known formula for all pharmaceutical chemistry.
본 발명의 균주 자체, 이의 파쇄물 및 상기 균주 배양물 등의 총 유효량은 단일 투여량(single dose)으로 이를 필요로 하는 개체에게 투여될 수 있으며, 다중 투여량(multiple dose)으로 장기간 투여되는 분할 치료 방법(fractionated treatment protocol)에 의해 투여될 수 있다. 본 발명의 약학적 조성물은 질환의 정도에 따라 유효성분의 함량을 달리할 수 있다. 바람직하게는 본 발명의 균주, 이의 파쇄물 및 배양물의 바람직한 전체 용량은 1일당 개체 체중 1 ㎏ 당 약 0.01ug 내지 1,000 mg, 가장 바람직하게는 0.1 ug 내지 100 mg일 수 있다. 그러나 상기 본 발명의 균주 자체, 이의 파쇄물 및 상기 균주 배양물 등의 용량은 약학적 조성물의 투여 경로 및 치료 횟수뿐만 아니라 개체의 연령, 체중, 건강 상태, 성별, 질환의 중증도, 식이 및 배설율 등 다양한 요인들을 고려하여 개체에 대한 유효 투여량이 결정되는 것이므로, 이러한 점을 고려할 때 당 분야의 통상적인 지식을 가진 자라면 상기 본 발명의 균주, 이의 파쇄물 및 배양물을 면역기능 증진제로서의 특정한 용도에 따른 적절한 유효 투여량을 결정할 수 있을 것이다. 본 발명에 따른 약학적 조성물은 본 발명의 효과를 보이는 한 그 제형, 투여 경로 및 투여 방법에 특별히 제한되지 아니한다. The total effective amount of the strain itself, its lysate, and the culture of the strain of the present invention can be administered to a subject in need thereof with a single dose, and divided treatment administered for a long period of time with multiple doses. It may be administered by a fractionated treatment protocol. The pharmaceutical composition of the present invention may vary the content of the active ingredient according to the severity of the disease. Preferably, the preferred total dose of the strain of the present invention, its lysate and culture may be about 0.01 ug to 1,000 mg, most preferably 0.1 ug to 100 mg per 1 kg of body weight per day. However, the dose of the strain itself, its lysate and the strain culture of the present invention depends on the route of administration of the pharmaceutical composition and the number of treatments, as well as the age, weight, health condition, gender, severity of disease, diet and excretion rate, etc. Since the effective dosage for the subject is determined in consideration of various factors, considering this point, those with ordinary knowledge in the art can use the strain of the present invention, its lysate and culture according to specific uses as an immune function enhancer. Appropriate effective dosages will be determined. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route and administration method as long as it exhibits the effects of the present invention.
또한, 본 발명은 락토바실러스 루테리 DB2 신균주(KACC 92293P), 이의 파쇄물 및 배양물로 이루어진 군에서 선택된 하나 이상을 유효성분으로 포함하는 정장용 조성물, 생균제 조성물 또는 사료용 조성물을 제공할 수 있다.In addition, the present invention can provide a composition for intestinal disorders, a probiotic composition, or a composition for feed containing at least one selected from the group consisting of a new strain of Lactobacillus reuteri DB2 (KACC 92293P), a lysate and a culture thereof as an active ingredient.
또한 상기 조성물은 반려동물용으로 사용될 수 있다.In addition, the composition may be used for companion animals.
본 발명의 락토바실러스 루테리 DB2 신균주(KACC 92293P)는 장기능 개선 및 면역증진 활성을 갖는 균주로 인간 및 동물의 건강 증진을 위한 용도, 즉, 정장용, 생균제 또는 사료용 조성물로 사용될 수 있다. 상기 조성물은 상기 균주 자체, 이의 파쇄물 및 상기 균주 배양물 등을 유효성분으로 포함할 수 있으며, 부형제 또는 담체를 추가로 포함할 수 있다. 상기 조성물 내에 함유된 본 발명의 균주 함량은 조성물의 용도 및 제형에 따라 달라질 수 있다. The new Lactobacillus reuteri DB2 strain (KACC 92293P) of the present invention is a strain having improved intestinal function and immune-enhancing activity, and can be used for improving the health of humans and animals, that is, as a composition for intestinal regulation, probiotics, or feed. The composition may include the strain itself, its lysate, and the culture of the strain as active ingredients, and may further include an excipient or carrier. The content of the strain of the present invention contained in the composition may vary depending on the use and formulation of the composition.
본 발명에 따른 정장용 또는 생균제 조성물은 다양한 제형과 방법으로 제조 및 투여될 수 있다. 예를 들어, 락토바실러스 루테리 DB2 신균주(KACC 92293P), 이의 파쇄물 및 이의 배양물을 약제학적 분야에서 통상적으로 사용하는 담체 및 향료와 혼합하여 정제 (tablet), 트로키(troche), 캡슐(capsule), 엘릭실(elixir), 시럽(syrup), 산제(powder), 현탁제(suspension) 또는 과립제(granule) 등의 형태로 제조 및 투여될 수 있다. The intestinal or probiotic composition according to the present invention can be prepared and administered in various formulations and methods. For example, Lactobacillus reuteri DB2 new strain (KACC 92293P), its lysate and its culture are mixed with carriers and flavorings commonly used in the pharmaceutical field to form tablets, troches, and capsules ), elixir, syrup, powder, suspension, or granules.
담체로는 결합제, 활탁제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제 등을 사용할 수 있다. 투여방식은 경구, 비경구 또는 도포법을 사용할 수 있으나, 바람직하게는 경구투여하는 것이 바람직하다. 또한, 투여용량은 개체의 체내에서 활성성분의 흡수도, 불활성율 및 배설속도, 피투여자의 연령, 성별, 상태 등에 따 라 적절히 선택할 수 있다.As the carrier, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, and the like may be used. The administration method may use oral, parenteral or application methods, but oral administration is preferred. In addition, the dosage can be appropriately selected depending on the degree of absorption, inactivity rate and excretion rate of the active ingredient in the body of the individual, age, sex, condition of the recipient, and the like.
또한, 본 발명에 따른 사료용 조성물은 발효사료, 배합 사료, 펠렛 형태 및 사일레지(silage) 등 다양한 형태로 제조될 수 있다.In addition, the composition for feed according to the present invention may be prepared in various forms such as fermented feed, formulated feed, pellet form and silage.
상기 발효사료는 본 발명의 신균주와 여러 가지 미생물균 또는 효소들을 첨가함으로써 유기물을 발효시켜 제조될 수 있으며, 상기 배합사료는 여러 종류의 일반 사료와 본 발명의 균주를 혼합하여 제조할 수 있다. 펠렛 형태의 사료는 상기 발효사료 또는 배합사료를 펠렛기로 제형화하여 제조될 수 있으며, 사일레지는 청예사료를 본 발명에 따른 균주를 발효시킴으로서 제조될 수 있다.The fermented feed may be prepared by fermenting organic matter by adding the new strain of the present invention and various microorganisms or enzymes, and the formulated feed may be prepared by mixing various types of general feed with the strain of the present invention. Feed in the form of pellets may be prepared by formulating the fermented feed or formulated feed into a pellet machine, and silage may be prepared by fermenting green feed with the strain according to the present invention.
본 발명의 사료용 조성물은 당업계에 공지된 방법에 따라 건조 사료, 반-습윤(semimoist) 사료, 습윤 사료 등의 다양한 형태로 제작이 가능하며, 예를 들어, 분말, 과립, 환, 펠릿, 젤리, 캔 사료(canned feed), 비스켓, 크로켓, 너겟, 플레이크, 스낵 등의 형태 중 어느 하나의 형태로 제조될 수 있다.The composition for feed of the present invention can be produced in various forms such as dry feed, semi-moist feed, wet feed, etc. according to a method known in the art, for example, powder, granule, pill, pellet, jelly , Canned feed (canned feed), biscuits, croquettes, nuggets, flakes, can be manufactured in any one of the forms of snacks.
또한, 본 발명의 사료 조성물은 하나 이상의 담체, 부형제 또는 희석제를 포함할 수 있으며, 상기 담체, 부형제 및 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.In addition, the feed composition of the present invention may include one or more carriers, excipients, or diluents. Examples of the carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol. , starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. can
또한, 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제 및 방부제 등을 추가로 포함할 수 있다.In addition, fillers, anti-coagulants, lubricants, wetting agents, flavoring agents, emulsifiers and preservatives may be further included.
특히 본 발명의 신균주는 개로부터 분리된 균주이며 본 발명의 일실시예를 통해 본 발명의 균주를 첨가하여 제조된 치즈를 섭취하게 한 비글견에서 장내 균총이 개선되고 면역관련 인자들의 발현 및 활성이 증진되어 면역 기능을 향상시킬 수 있음을 확인하였다.In particular, the new strain of the present invention is a strain isolated from dogs, and through an embodiment of the present invention, intestinal flora is improved in beagle dogs fed cheese prepared by adding the strain of the present invention, and expression and activity of immune-related factors It was confirmed that this can be enhanced to improve immune function.
따라서 본 발명의 신균주, 이의 파쇄물 또는 이의 배양물을 포함하는 상기 조성물은 반려동물의 영양 상태를 개선시키고, 기초 체력을 강화시켜 초기 면역체계를 빨리 구축하고, 면역력을 증진시켜 내병성을 증진시킬 수 있다.Therefore, the composition comprising the new strain of the present invention, its lysate or its culture can improve the nutritional status of companion animals, strengthen basic physical strength to quickly build the initial immune system, and improve immunity by enhancing disease resistance. there is.
상기 면역력 증진은 반려동물의 건강에 중요한 특정 면역 기능을 자극하거나, 잠재적으로 염증, 알러지 등과 같은 면역 장애에 관여할 수 있는 다른 면역 기능을 조정하는 능력을 갖는다는 것을 의미하며, 장내 균총의 안정화, 사망률의 감소를 포함하고, 세균 및 바이러스 감염과 같은 질병 및 염증의 회피 및 억제를 모두 포함한다.The enhancement of immunity means that it has the ability to stimulate specific immune functions important to the health of companion animals or modulate other immune functions that can potentially be involved in immune disorders such as inflammation, allergy, etc., stabilization of intestinal flora, This includes reduction of mortality and includes both avoidance and inhibition of inflammation and diseases such as bacterial and viral infections.
이하, 실시예를 통하여 본 발명을 보다 상세히 설명하고자 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. These examples are intended to explain the present invention in more detail, and the scope of the present invention is not limited to these examples.
<실시예 1><Example 1>
락토바실러스 루테리 DB2 균주의 분리 및 동정Isolation and identification of Lactobacillus reuteri DB2 strains
건강한 반려견의 장관에서 채취한 신선한 분변 1g을 희석액 A(KH2PO4 4.5 g, Na2HPO4 6.0 g, L-cysteinㆍHCl 0.5 g, Tween 80 0.5 g, Agar 1.0 g, 증류수 1000 ㎖) 9 ㎖을 사용하여 10배 단계로 희석한 다음, 유산균 분리용 배지인 TATAC, LBS, BL 배지를 사용하여 37℃에서 24시간 동안 Steel wool 방법으로 혐기배양을 통해 유산균을 분리하였다.1 g of fresh feces collected from the intestinal tract of a healthy dog was diluted 10 times using 9 ml of diluent A (4.5 g of KH2PO4, 6.0 g of Na2HPO4, 0.5 g of L-cysteinㆍHCl, 0.5 g of
이후 분리된 유산균의 분자생물학적 동정을 위하여, 분리된 유산균의 16S rRNA 염기서열을 분석하였으며, 분석한 16S rRNA 유전자 염기서열을 NCBI의 BLAST program을 이용하여 기존에 알려진 미생물과 상동성 여부를 분석하였다.Then, for molecular biological identification of the isolated lactic acid bacteria, the 16S rRNA sequence of the isolated lactic acid bacteria was analyzed, and the analyzed 16S rRNA gene sequence was analyzed for homology with previously known microorganisms using NCBI's BLAST program.
그 결과, 본 발명에서 분리한 유산균은 락토바실러스 루테리(Lactobacillus reuteri)와 99%의 상동성을 나타내었으며 기존에 존재하지 않는 신규한 균주임을 확인하였다. 본 발명에서 동정한 락토바실러스 루테리 DB2의 16S rRNA 염기서열은 서열번호 1에 나타내었고, 본 발명자들은 본 발명에서 분리한 신규한 유산균을 락토바실러스 루테리 DB2로 명명하였으며, 2019년 11월 22일에 국립농업과학원 미생물은행에 기탁하여 기탁번호 KACC 92293P를 부여받았다.As a result, it was confirmed that the lactic acid bacteria isolated in the present invention showed 99% homology with Lactobacillus reuteri and were a novel strain that did not exist previously. The 16S rRNA base sequence of Lactobacillus reuteri DB2 identified in the present invention is shown in SEQ ID NO: 1, and the present inventors named the novel lactic acid bacteria isolated in the present invention as Lactobacillus reuteri DB2, and on November 22, 2019, the National It was deposited with the Microbial Bank of the Academy of Agricultural Sciences and was given the deposit number KACC 92293P.
<실시예 2><Example 2>
본 발명의 락토바실러스 루테리 DB2 균주의 특성 분석Characterization of the Lactobacillus reuteri DB2 strain of the present invention
본 발명에서 분리한 신균주인 락토바실러스 루테리 KACC 92293P 균주를 Bergey's Manual of Determinative bacteriology의 방법에 따라 형태학적 특성 및 생화학적 특성을 조사하였고, API 50 CHL kit(Biomerieux, Lyan, France)를 사용하여 탄수화물 이용성을 분석하였으며, 그 결과를 하기 표 1에 나타내었다.The morphological and biochemical characteristics of the Lactobacillus reuteri KACC 92293P strain, a new strain isolated in the present invention, were investigated according to the method of Bergey's Manual of Determinative bacteriology, and carbohydrates were analyzed using the
<실시예 3><Example 3>
본 발명의 락토바실러스 루테리 DB2 균주의 장기능 개선능 분석Analysis of long-term function improvement of the Lactobacillus reuteri DB2 strain of the present invention
본 발명에서 분리한 신균주인 락토바실러스 루테리 DB2 균주의 장기능 개선능을 확인하기 위해, 상기 락토바실러스 루테리 DB2 균주를 퀘소 블랑코 치즈의 제조에 첨가하여, 상기 균주가 함유된 퀘소 블랑코 치즈를 제조하였다. 상기 치즈의 제조방법은 당업계에 공지된 치즈 제조 공정에 따라 제조하였으며, 다만 치즈 원료에 본 발명의 락토바실러스 루테리 DB2 균주를 첨가하여 수행하였다.In order to confirm the long-term function improvement ability of the Lactobacillus reuteri DB2 strain, a new strain isolated in the present invention, the Lactobacillus reuteri DB2 strain was added to the production of Queso Blanco cheese to prepare Queso Blanco cheese containing the strain. . The cheese manufacturing method was prepared according to a cheese manufacturing process known in the art, but was performed by adding the Lactobacillus reuteri DB2 strain of the present invention to cheese raw materials.
이후 제조된 본 발명의 신균주를 함유한 치즈를 비글견에서 8주 동안 섭취하게 한 다음, 장내 세균총 분석을 수행하였다.Thereafter, the cheese containing the new strain of the present invention prepared was fed to beagle dogs for 8 weeks, and then the intestinal flora was analyzed.
그 결과, 본 발명의 신균주를 함유한 치즈를 섭취하게 한 군이 본 발명의 신균주를 함유하지 않은 퀘소 블랑코 치즈 섭취 군에 비해 장내 유익균의 미생물 수가 증가한 것으로 나타났고, 반면 유해균은 감소한 것으로 나타났다.As a result, the group ingesting the cheese containing the new strain of the present invention showed an increase in the number of beneficial bacteria in the intestine, while the number of harmful bacteria decreased compared to the group consuming Queso Blanco cheese that did not contain the new strain of the present invention. .
따라서 이러한 결과를 통해 본 발명자들은 본 발명의 락토바실러스 루테리 DB2 균주는 장내 균총을 개선할 수 있는 효과가 있는 것으로 나타났고, 따라서 장기능을 개선할 수 있으며 나아가 장질환을 예방 또는 치료할 수 있음을 알 수 있었다.Therefore, through these results, the present inventors have shown that the Lactobacillus reuteri DB2 strain of the present invention has an effect of improving intestinal flora, and thus can improve intestinal function and further prevent or treat intestinal diseases. could
<실시예 4><Example 4>
본 발명의 락토바실러스 루테리 DB2 균주의 면역증진 활성 분석Analysis of immune enhancing activity of the Lactobacillus reuteri DB2 strain of the present invention
본 발명의 락토바실러스 루테리 DB2 신균주가 면역증진 활성이 있는지를 확인하기 위해, 마우스 대식세포주인 RAW 264.7 세포를 6-웰에 분주한 후, 균주의 배양물을 처리 후 48시간 배양한 다음 각 실험군에서의 NO, NF-κB, IL-6 및 TNF-α의 생성량을 ELISA kit 사용하여 각각 분석하였다. 이때 비교 균주로서, 락토바실러스 람노서스 LGG(Lactobacillus rhamnosus GG), 비피도박테리움 롱검 BIF4(Bifidobacteium longum) 및 비피도박테리움 롱검 KACC91563(Bifidobacteium longum) 균주를 사용하였다. 또한 상기 균주의 배양물은 각 균주를 배양 후, 세포를 용해버퍼로 용해한 세포 용해물을 원심분리하여, 세포 펠릿을 제거하고 세포 용해물을 수득하여 이를 사용하였다.In order to confirm whether the new strain of Lactobacillus reuteri DB2 of the present invention has immunostimulating activity, RAW 264.7 cells, a mouse macrophage cell line, were dispensed into 6-wells, and the culture of the strain was cultured for 48 hours after treatment, and each experimental group The production amounts of NO, NF-κB, IL-6, and TNF-α were analyzed using an ELISA kit, respectively. At this time, as comparative strains, Lactobacillus rhamnosus LGG (Lactobacillus rhamnosus GG), Bifidobacterium longum BIF4 (Bifidobacteium longum) and Bifidobacterium longum KACC91563 (Bifidobacteium longum) strains were used. In addition, after culturing each strain, the cell lysate obtained by dissolving the cells in a lysis buffer was centrifuged to remove the cell pellet, and the cell lysate was obtained and used for the culture of the strain.
그 결과, 도 1 내지 도 4에 나타낸 바와 같이, 본 발명의 신균주 락토바실러스 루테리 DB2 균주가 면역활성이 있다고 알려진 상업용 균주인 Lactobacillus rhamnosus GG (LGG)와 비교하여 NO, NF-κB, IL-6 및 TNF-α 생성이 더 증가되어 있는 것으로 나타났다. 또한, 본 발명의 DB2 균주는 Bifidobacteium longum BIF4 및 KACC 91563 균주에 비해서도 NO, NF-κB, IL-6 및 TNF-α 생성을 더 증가시키는 활성이 있음을 확인하였다.As a result, as shown in FIGS. 1 to 4, the new strain Lactobacillus reuteri DB2 strain of the present invention has NO, NF-κB, and IL-6 compared to Lactobacillus rhamnosus GG (LGG), a commercial strain known to have immunological activity. And TNF-α production was found to be further increased. In addition, it was confirmed that the DB2 strain of the present invention has an activity to further increase production of NO, NF-κB, IL-6, and TNF-α compared to Bifidobacteium longum BIF4 and KACC 91563 strains.
따라서 이러한 결과를 통해 본 발명자들은 본 발명의 락토바실러스 루테리 DB2 균주가 종래 알려진 유산균들에 비해 대식세포를 효과적으로 활성화시켜 NO, NF-κB 및 TNF-α의 생성을 더 우수하게 유도할 수 있고 이로 인해 면역활성을 더 증진시킬 수 있음을 확인하였다.Therefore, through these results, the present inventors found that the Lactobacillus reuteri DB2 strain of the present invention can effectively activate macrophages and induce the production of NO, NF-κB and TNF-α better than conventionally known lactic acid bacteria, thereby It was confirmed that the immune activity can be further enhanced.
그러므로 in vitro 실험을 통해서도 본 발명의 락토바실러스 루테리 DB2 균주가 우수한 면역증강 효과를 갖는다는 것을 알 수 있었다.Therefore, it was found through in vitro experiments that the Lactobacillus reuteri DB2 strain of the present invention had an excellent immune enhancing effect.
<실시예 5><Example 5>
본 발명의 락토바실러스 루테리 DB2 균주를 포함하는 혼합균주에 대한 면역증진 활성 분석Analysis of immune enhancing activity for mixed strains including the Lactobacillus reuteri DB2 strain of the present invention
나아가 본 발명자들은 본 발명의 루테리 DB2 균주와 함께 혼합 사용할 경우, 더 효과적인 면역증진 활성을 유도할 수 있는 최적의 혼합 균주를 선별하기 위해, 상기 실시예 4에서 비교 균주로 사용한 LGG(Lactobacillus rhamnosus GG), 비피도박테리움 롱검 BIF4(Bifidobacteium longum) 및 비피도박테리움 롱검 KACC91563(Bifidobacteium longum) 균주를 본 발명의 루테리 DB2 균주와 각각 1:1의 동일한 세포수 비율로 혼합하여 혼합 균주를 제조한 후, 상기 실시예 3에 기재된 바와 같이 퀘소 블랑코 치즈의 제조에 첨가하여 각 혼합균주가 균주가 함유된 퀘소 블랑코 치즈를 제조하였다. 이후 각 제조된 치즈를 비글견에 8주 동안 섭취하게 한 다음, 4주째 및 8주째에 각각 비글견으로부터 혈액을 체취한 후, TNF-α, IFN-γ 및 NK 세포 활성도를 측정하였다. Furthermore, the inventors of the present invention LGG (Lactobacillus rhamnosus GG) used as a comparative strain in Example 4 in order to select an optimal mixed strain capable of inducing more effective immune-enhancing activity when mixed and used together with the Reuteri DB2 strain of the present invention , Bifidobacterium longum BIF4 (Bifidobacteium longum) and Bifidobacterium longum KACC91563 (Bifidobacteium longum) strains were mixed with the Reuteri DB2 strain of the present invention at the same cell number ratio of 1: 1, respectively, to prepare a mixed strain, As described in Example 3, each mixed strain was added to the production of Queso Blanco cheese to prepare strain-containing Queso Blanco cheese. Thereafter, each prepared cheese was fed to a beagle dog for 8 weeks, and then blood was collected from the beagle dog at 4 weeks and 8 weeks, respectively, and then TNF-α, IFN-γ and NK cell activity were measured.
상기 표에서 QC는 균주를 첨가하지 않고 제조된 퀘소 블랑코 치즈를 섭취시킨 군이고, QCLB는 본 발명의 루테리 DB2 균주 및 비피도박테리움 롱검 KACC91563 균주의 혼합균주를 첨가하여 제조된 퀘소 블랑코 치즈를 섭취시킨 군이고, QCLL은 본 발명의 루테리 DB2 균주 및 LGG 균주의 혼합균주를 첨가하여 제조된 퀘소 블랑코 치즈를 섭취시킨 군이다.In the table above, QC is a group that consumes Queso Blanco cheese prepared without adding strain, and QCLB is a group that consumes Queso Blanco cheese prepared by adding a mixed strain of Reuteri DB2 strain and Bifidobacterium longum KACC91563 strain according to the present invention. QCLL was a group that consumed Queso Blanco cheese prepared by adding a mixed strain of the Reuteri DB2 strain and the LGG strain of the present invention.
그 결과, 상기 표 2 및 도 5~6에 나타낸 바와 같이, 본 발명의 루테리 DB2 균주를 비피도박테리움 롱검 KACC91563 균주와 혼합하여 사용할 경우, NK 세포 활성을 더 증진시킬 수 있는 것으로 나타났고, 또한 혈청 내 TNF-α, IFN-γ의 사이토카인 함량도 더 높은 것으로 나타났다. 한편, 본 발명의 루테리 DB2 균주를 다른 균주와 혼합하여 사용할 경우에는 비피도박테리움 롱검 KACC91563 균주와 함께 사용한 경우에 비해 그 효과가 낮은 것으로 나타났다.As a result, as shown in Table 2 and FIGS. 5 and 6, when the Reuteri DB2 strain of the present invention is mixed with the Bifidobacterium longum KACC91563 strain, it was found that NK cell activity can be further enhanced, and The cytokine content of TNF-α and IFN-γ in serum was also higher. On the other hand, when the Reuteri DB2 strain of the present invention is mixed with other strains, the effect is lower than that when used together with the Bifidobacterium longum KACC91563 strain.
그러므로 이러한 실험결과들을 통해, 본 발명자들은 본 발명의 신균주인 락토바실러스 루테리 ＤＢ２ 균주는 장 건강 및 면역활성 증진을 위한 기능성식품, 사료첨가제, 정장제 제조 및 의약품 등 다양한 산업분야에 유용하게 활용할 수 있으며, 반려동물의 장 기능 개선 및 면역력을 증진시키는 효과가 우수하므로 펫푸드 관련 산업에도 유용하게 사용될 수 있다.Therefore, through these experimental results, the present inventors found that the new strain of the present invention, Lactobacillus reuteri DB2 strain, can be usefully used in various industrial fields such as functional foods, feed additives, intestinal medicine manufacturing and pharmaceuticals for improving intestinal health and immune activity, , It can be usefully used in pet food-related industries because it has excellent effects on improving intestinal function and enhancing immunity of companion animals.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far, the present invention has been looked at with respect to its preferred embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.
<110> Industry University Cooperation Foundation of Chungbuk National University <120> Novel Lactobacillus reuteri DB2 strain and use of the same <130> NPDC83550 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1163 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus reuteri DB2 strain 16S rRNA sequence <400> 1 ctccctccat aaaggttagg ccaccgactt tgggcgttac aaactcccat ggtgtgacgg 60 gcggtgtgta caaggcccgg gaacgtattc accgcggcat gctgatccgc gattactagc 120 gattccgact tcgtgtaggc gagttgcagc ctacagtccg aactgagaac ggctttaaga 180 gattagctta ctctcgcgag cttgcgactc gttgtaccgt ccattgtagc acgtgtgtag 240 cccaggtcat aaggggcatg atgatctgac gtcgtcccca ccttcctccg gtttgtcacc 300 ggcagtctca ctagagtgcc caacttaatg ctggcaacta gtaacaaggg ttgcgctcgt 360 tgcgggactt aacccaacat ctcacgacac gagctgacga cgaccatgca ccacctgtca 420 ttgcgtcccc gaagggaacg ccttatctct aaggttagcg caagatgtca agacctggta 480 aggttcttcg cgtagcttcg aattaaacca catgctccac cgcttgtgcg ggcccccgtc 540 aattcctttg agtttcaacc ttgcggtcgt actccccagg cggagtgctt aatgcgttag 600 ctccggcact gaagggcgga aaccctccaa cacctagcac tcatcgttta cggcatggac 660 taccagggta tctaatcctg ttcgctaccc atgctttcga gcctcagcgt cagttgcaga 720 ccagacagcc gccttcgcca ctggtgttct tccatatatc tacgcattcc accgctacac 780 atggagttcc actgtcctct tctgcactca agtcgcccgg tttccgatgc acttcttcgg 840 ttaagccgaa ggctttcaca tcagacctaa gcaaccgcct gcgctcgctt tacgcccaat 900 aaatccggat aacgcttgcc acctacgtat taccgcggct gctggcacgt agttagccgt 960 gactttctgg ttggataccg tcactgcgtg aacagttact ctcacgcacg ttcttctcca 1020 acaacagagc tttacgagcc gaaacccttc ttcactcacg cggtgttgct ccatcaggct 1080 tgcgcccatt gtggaagatt ccctactgct gcctcccgta ggagtatgga ccgtgtctca 1140 gttccattgt ggccgataag tct 1163 <110> Industry University Cooperation Foundation of Chungbuk National University <120> Novel Lactobacillus reuteri DB2 strain and use of the same <130>NPDC83550 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1163 <212> DNA <213> artificial sequence <220> <223> Lactobacillus reuteri DB2 strain 16S rRNA sequence <400> 1 ctccctccat aaaggttagg ccaccgactt tgggcgttac aaactcccat ggtgtgacgg 60 gcggtgtgta caaggcccgg gaacgtattc accgcggcat gctgatccgc gattactagc 120 gattccgact tcgtgtaggc gagttgcagc ctacagtccg aactgagaac ggctttaaga 180 gattagctta ctctcgcgag cttgcgactc gttgtaccgt ccattgtagc acgtggtgtag 240 cccaggtcat aaggggcatg atgatctgac gtcgtcccca ccttcctccg gtttgtcacc 300 ggcagtctca ctagagtgcc caacttaatg ctggcaacta gtaacaaggg ttgcgctcgt 360 tgcgggactt aacccaacat ctcacgacac gagctgacga cgaccatgca ccacctgtca 420 ttgcgtcccc gaagggaacg ccttatctct aaggttagcg caagatgtca agacctggta 480 aggttcttcg cgtagcttcg aattaaacca catgctccac cgcttgtgcg ggcccccgtc 540 aattcctttg agtttcaacc ttgcggtcgt actccccagg cggagtgctt aatgcgttag 600 ctccggcact gaagggcgga aaccctccaa cacctagcac tcatcgttta cggcatggac 660 taccagggta tctaatcctg ttcgctaccc atgctttcga gcctcagcgt cagttgcaga 720 ccagacagcc gccttcgcca ctggtgttct tccatatatc tacgcattcc accgctacac 780 atggagttcc actgtcctct tctgcactca agtcgcccgg tttccgatgc acttcttcgg 840 ttaagccgaa ggctttcaca tcagacctaa gcaaccgcct gcgctcgctt tacgcccaat 900 aaatccggat aacgcttgcc acctacgtat taccgcggct gctggcacgt agttagccgt 960 gactttctgg ttggataccg tcactgcgtg aacagttact ctcacgcacg ttcttctcca 1020 acaacagagc tttacgagcc gaaacccttc ttcactcacg cggtgttgct ccatcaggct 1080 tgcgcccatt gtggaagatt ccctactgct gcctcccgta ggaggtatgga ccgtgtctca 1140 gttccattgt ggccgataag tct 1163
Claims (10)
상기 장기능 개선은 장내 균총을 개선하는 것이고,
상기 면역증진은 NK(Natural killer) 세포활성 증진; TNF-α, IFN-γ 또는 IL-6의 사이토카인 생성 증진; 또는 NF-kB 활성 증진을 통해 이루어지는 것을 특징으로 하는, 장기능 개선 및 면역증진 활성을 모두 갖는 반려 동물용 식품 조성물.According to claim 3,
The improvement of intestinal function is to improve intestinal flora,
The immune enhancement is to promote NK (Natural killer) cell activity; enhancing cytokine production of TNF-α, IFN-γ or IL-6; Or a food composition for companion animals having both intestinal function improvement and immunostimulating activity, characterized in that through enhancement of NF-kB activity.
상기 조성물은 비피도박테리움 롱검 유산균을 추가로 더 포함하는 것을 특징으로 하는, 장기능 개선 및 면역증진 활성을 모두 갖는 반려 동물용 식품 조성물.According to claim 3,
The composition further comprises Bifidobacterium longum lactic acid bacteria, a food composition for companion animals having both intestinal function improvement and immunity enhancing activity.
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