KR20230073579A - Novel strain with serum uric acid level reducing effect and renal protective effect, and use of the same - Google Patents
Novel strain with serum uric acid level reducing effect and renal protective effect, and use of the same Download PDFInfo
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- KR20230073579A KR20230073579A KR1020210160118A KR20210160118A KR20230073579A KR 20230073579 A KR20230073579 A KR 20230073579A KR 1020210160118 A KR1020210160118 A KR 1020210160118A KR 20210160118 A KR20210160118 A KR 20210160118A KR 20230073579 A KR20230073579 A KR 20230073579A
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- South Korea
- Prior art keywords
- mjm60390
- lactobacillus brevis
- strain
- lactobacillus
- hyperuricemia
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Abstract
Description
본 발명은 신규 균주에 관한 것으로서, 더 상세하게는 혈중 요산 저감 효능 및 신장 보호 효능과 같은 다양한 기능성을 가진 신규 균주에 관한 것이다. 또한, 본 발명은 신규 균주의 다양한 용도에 관한 것으로서, 더 상세하게는 신규 균주의 고요산혈증, 통풍 또는 신장 손상에 대한 예방, 개선 또는 치료 용도에 관한 것이다.The present invention relates to a novel strain, and more particularly, to a novel strain having various functions such as blood uric acid lowering effect and renal protective effect. In addition, the present invention relates to various uses of the novel strain, and more particularly, to prevention, improvement, or treatment of hyperuricemia, gout, or kidney damage of the novel strain.
요산(C5H4N4O3)은 생체내 퓨린 대사과정 중 하이포잔틴(hypoxanthine; C5H4N4O) 또는 잔틴으로부터 잔틴산화효소(xanthine oxidase, XO) 또는 잔틴탈수효소(xanthine dehydrogenase, XDH)에 의해 생성되는 것으로서, 혈중 요산의 70% 가량은 세포의 턴오버(turn-over)에 따른 내인성 퓨린 대사에 의해 만들어지고, 30%는 음식으로 섭취된 퓨린으로부터 형성되어 대부분 신장을 통해 배설된다.Uric acid (C 5 H 4 N 4 O 3 ) is produced from hypoxanthine (C 5 H 4 N 4 O) or xanthine by xanthine oxidase (XO) or xanthine dehydrogenase during purine metabolism in vivo. , XDH), about 70% of blood uric acid is made by endogenous purine metabolism following cell turn-over, and 30% is formed from purine ingested with food, mostly through the kidneys. is excreted
고요산혈증(hyperuricemia)은 체내에서 요산이 과잉 생성되거나 잘 배출되지 않아 혈액 내에 요산 농도가 비정상적으로 높은 상태인 질환으로서, 혈중 요산 농도가 남성에서는 7.0 mg/dL 이상, 여성에서는 6.5 mg/dL 이상인 경우가 이에 해당한다. 요산 농도의 증가는 요산 결정(urate crystal)이 관절막에 침착되면서 염증반응을 유발하는 통풍(gout)의 원인이 되는 것으로 알려져 있으며, 최근 요산과 신장병, 고혈압을 포함한 심혈관계 질환 그리고 대사증후군 및 당뇨병의 발생/악화 사이의 연관에 대한 흥미로운 역학 또는 임상 연구가 발표되고 있다. 한편, 고요산혈증인 사람에게서 발생하는 대표적인 질환인 통풍(gout)은 여러 요인에 의해 생성된 요산 결정을 면역반응에 의해 백혈구가 탐식하면서 관절과 관절 주위 조직에 재발성 발작성 관절염을 일으키고, 요산의 과잉이 지속되는 경우에는 축적된 요산결절에 의해서 관절의 장애를 일으키는 만성 전신성 대사성 질환이다.Hyperuricemia is a disease in which uric acid concentration in the blood is abnormally high due to excessive production or poor excretion of uric acid in the body. When the blood uric acid concentration is 7.0 mg/dL or more in men and 6.5 mg/dL or more in women corresponds to this. An increase in uric acid concentration is known to cause gout, in which uric acid crystals are deposited in the joint membrane and cause an inflammatory reaction. Recently, uric acid and kidney disease, cardiovascular diseases including hypertension, metabolic syndrome and diabetes Interesting epidemiological or clinical studies of the association between onset/exacerbation are being published. On the other hand, gout, a representative disease that occurs in people with hyperuricemia, causes recurrent paroxysmal arthritis in joints and tissues around joints as leukocytes devour uric acid crystals generated by various factors by an immune response, resulting in excess uric acid. If this persists, it is a chronic systemic metabolic disease that causes joint disorders due to accumulated uric acid nodules.
이러한 고요산혈증 또는 통풍의 치료를 위한 치료제로 잔틴산화효소 억제제(xanthine oxidase inhibitors), 요산 배설 촉진제(Uricosuric agents), 및 요산 분해제(Uricolytic drugs, urozyme) 등이 사용되고 있다. 잔틴산화효소 억제제는 요산 합성을 억제하는 것으로서, 이를 사용할 경우 요산 대사가 억제되고 혈중 요산 농도가 감소되어 고요산혈증 및 통풍을 치료할 수 있다. 요산 배설 촉진제는 신장의 근위세뇨관에 작용하여 요산의 재흡수를 억제함으로써 소변으로의 배설을 촉진하는 기능을 한다. 요산 분해제는 요산을 알라토닌(allantoin)으로 산화시키는 효소이고, 알라토닌은 요산에 비해 용해도가 높아 신장으로 배설되기 용이하다. 예를 들어, 통풍의 치료를 위해 잔틴산화효소(xanthine oxidase, XO) 억제제인 알로퓨리놀, 페복소스타트 등의 약물이 사용되고 있다.As therapeutic agents for the treatment of hyperuricemia or gout, xanthine oxidase inhibitors, uricosuric agents, and uricolytic drugs (urozyme) have been used. Xanthine oxidase inhibitors inhibit uric acid synthesis, and when used, uric acid metabolism is suppressed and blood uric acid concentration is reduced, thereby treating hyperuricemia and gout. The uric acid excretion promoter acts on the proximal tubule of the kidney to inhibit reabsorption of uric acid, thereby promoting excretion into the urine. Uric acid decomposer is an enzyme that oxidizes uric acid to allantoin, and allantoin is more soluble than uric acid and is easily excreted by the kidneys. For example, drugs such as allopurinol and peboxostat, which are xanthine oxidase (XO) inhibitors, are used for the treatment of gout.
그러나, 고요산혈증 또는 고요산혈증 관련 대사 장애에 이용하고 있는 치료제들 중의 다수는 여러 가지 부작용들이 알려져 있다. 예를 들면, 알로퓨리놀과 같은 잔틴산화효소 억제제는 과민성 맥관염, 스티븐스-존슨 증후군, 탈락성 피부염, 재생불량성 빈혈 및 간 기능부전과 관련이 있다. 프로베네시드 및 벤즈브로마론과 같은 요산 배설 촉진제는 위장 장애, 요로 결석증 및 특이체질 환자에서의 전격성 간부전과 같은 부작용이 있다. 또한, 비스테로이드성 항염증 약물(NSAID)의 장기적 사용은 궤양성 천공 및 상부 위장관 출혈을 포함하는 부작용을 초래할 수 있다.However, many of the therapeutic agents used for hyperuricemia or hyperuricemia-related metabolic disorders are known to have various side effects. For example, xanthine oxidase inhibitors such as allopurinol have been associated with hypersensitivity vasculitis, Stevens-Johnson syndrome, exfoliative dermatitis, aplastic anemia and liver failure. Uric acid excretion promoters such as probenecid and benzbromarone have side effects such as gastrointestinal disorders, urolithiasis, and fulminant hepatic failure in patients with idiopathic constitution. In addition, long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) can lead to side effects including ulcerative perforation and upper gastrointestinal bleeding.
고요산혈증 또는 고요산혈증 관련 대사 장애인 통풍 치료와 관련하여, 최근 식이요법의 새로운 방향이 제시되고 기존에 사용하던 약물에 저항을 보이는 환자에 대하여 사용할 수 있는 새로운 요산 강하제가 개발되는 등의 변화를 보이고 있다. 이와 더불어, 천연물을 이용한 요산 저하 효과를 유발하는 물질에 대한 연구개발이 활발히 이루어지고 있다. 또한, 유산균을 이용하여 비만, 고지혈증과 같은 질환을 개선 또는 치료할 수 있는 건강기능식품 또는 의약품 개발이 이루어지고 있다.Regarding the treatment of gout, a metabolic disorder related to hyperuricemia or hyperuricemia, a new direction of dietary therapy has recently been suggested and new uric acid lowering agents that can be used for patients who show resistance to existing drugs have been developed. . In addition, research and development on materials that induce uric acid lowering effects using natural products are being actively conducted. In addition, health functional foods or medicines that can improve or treat diseases such as obesity and hyperlipidemia using lactic acid bacteria are being developed.
유산균을 이용한 고요산혈증 또는 통풍 치료와 관련하여 대한민국 등록특허공보 제10-1584380호에는 혈중 요산치 상승을 억제하고 고요산혈증을 치료하기 위한 용도로 사용될 수 있는 수탁번호:NITE BP-462로서 기탁되어 있는 락토바실러스 가세리(Lactobacillus gasseri) 유산균 OLL2922주가 개시되어 있다. 또한, 대한민국 등록특허공보 제10-1450511호에는 혈중 요산치 상승을 억제하고 고요산혈증을 치료하기 위한 용도로 사용될 수 있는 Lactobacillus oris OLL2779(수탁번호:NITE BP-223)가 개시되어 있다. 또한, 대한민국 등록특허공보 제10-1385864호에는 혈중 요산치 상승을 억제하고 고요산혈증을 치료하기 위한 용도로 사용될 수 있는 Lactobacillus gasseri OLL2959(수탁번호:NITE BP-224)가 개시되어 있다.Regarding the treatment of hyperuricemia or gout using lactic acid bacteria, Korean Patent Registration No. 10-1584380 discloses lactose deposited as Accession No.: NITE BP-462, which can be used for suppressing the rise in blood uric acid level and treating hyperuricemia. Bacillus gasseri ( Lactobacillus gasseri ) Lactobacillus OLL2922 strain is disclosed. In addition, Korean Patent Registration No. 10-1450511 discloses Lactobacillus oris OLL2779 (accession number: NITE BP-223), which can be used for suppressing an increase in blood uric acid level and treating hyperuricemia. In addition, Korean Patent Registration No. 10-1385864 discloses Lactobacillus gasseri OLL2959 (accession number: NITE BP-224), which can be used for suppressing an increase in blood uric acid level and treating hyperuricemia.
본 발명은 종래의 기술적 배경하에서 도출된 것으로서, 본 발명의 목적은 안전성이 높고 혈중 요산 저감 효능 및 신장 보호 효능과 같은 다양한 기능성을 가진 신규 유산균을 제공하는데에 있다. 또한, 본 발명의 목적은 신규 유산균의 다양한 용도를 제공하는데에 있다.The present invention has been derived under the conventional technical background, and an object of the present invention is to provide a novel lactic acid bacteria having various functionalities such as high safety, blood uric acid reducing effect and renal protective effect. In addition, an object of the present invention is to provide various uses of the novel lactic acid bacteria.
본 발명의 발명자들은 합성 화학물질에 비해 안전성이 높고 고요산혈증 또는 통풍 치료에 사용할 수 있는 신규 소재를 발굴하기 위해 100여종의 유산균으로부터 퓨린 분해 활성이 우수한 5개의 균주를 스크니링하고 프로바이오틱스 특성,고요산혈증 동물모델을 이용한 생체내 실험 등을 통해 특정 락토바실러스 브레비스 균주가 식품학적으로 안전하고 프로바이오틱스로서 필요한 활성을 가지며 동시에 고요산혈증, 고요산혈증 관련 대사 장애 및 신장 손상 개선 효능과 같은 다양한 기능성을 가진다는 점을 확인하고 본 발명을 완성하였다.The inventors of the present invention screened 5 strains with excellent purine decomposition activity from 100 kinds of lactic acid bacteria in order to discover new materials that are safer than synthetic chemicals and can be used for the treatment of hyperuricemia or gout, and have probiotics properties, hyperuricemia. Through in vivo experiments using animal models, certain Lactobacillus brevis strains are food-safe, have the necessary activity as probiotics, and at the same time have various functionalities such as hyperuricemia, hyperuricemia-related metabolic disorders and renal damage improvement effects. confirmed and completed the present invention.
본 발명의 일 목적을 해결하기 위하여, 본 발명의 일 예는 퓨린(purine) 분해 활성을 가지는 락토바실러스 브레비스(Lactobacillus brevis) MJM60390 균주(수탁번호 : KACC 81193BP)를 제공한다.In order to solve one object of the present invention, an example of the present invention provides a Lactobacillus brevis MJM60390 strain (accession number: KACC 81193BP) having a purine degrading activity.
본 발명의 일 목적을 해결하기 위하여, 본 발명의 일 예는 락토바실러스 브레비스(Lactobacillus brevis) MJM60390 균주(수탁번호 : KACC 81193BP), 이의 배양물, 이의 파쇄물 또는 이의 추출물에서 선택되는 1종 이상을 유효성분으로 포함하는 고요산혈증 예방, 개선 또는 치료용 조성물을 제공한다.In order to solve one object of the present invention, one example of the present invention is Lactobacillus brevis ( Lactobacillus brevis ) MJM60390 strain (accession number: KACC 81193BP), a culture thereof, a lysate thereof, or at least one selected from an extract effective It provides a composition for preventing, improving or treating hyperuricemia comprising as a component.
본 발명의 일 목적을 해결하기 위하여, 본 발명의 일 예는 락토바실러스 브레비스(Lactobacillus brevis) MJM60390 균주(수탁번호 : KACC 81193BP), 이의 배양물, 이의 파쇄물 또는 이의 추출물에서 선택되는 1종 이상을 유효성분으로 포함하는 고요산혈증 관련 대사 장애 예방, 개선 또는 치료용 조성물을 제공한다.In order to solve one object of the present invention, one example of the present invention is Lactobacillus brevis ( Lactobacillus brevis ) MJM60390 strain (accession number: KACC 81193BP), a culture thereof, a lysate thereof, or at least one selected from an extract effective It provides a composition for preventing, improving or treating hyperuricemia-related metabolic disorders comprising as components.
본 발명의 일 목적을 해결하기 위하여, 본 발명의 일 예는 락토바실러스 브레비스(Lactobacillus brevis) MJM60390 균주(수탁번호 : KACC 81193BP), 이의 배양물, 이의 파쇄물 또는 이의 추출물에서 선택되는 1종 이상을 유효성분으로 포함하는 신장 손상 예방, 개선 또는 치료용 조성물을 제공한다.In order to solve one object of the present invention, one example of the present invention is Lactobacillus brevis ( Lactobacillus brevis ) MJM60390 strain (accession number: KACC 81193BP), a culture thereof, a lysate thereof, or at least one selected from an extract effective It provides a composition for preventing, improving or treating kidney damage comprising as a component.
본 발명에 따른 락토바실러스 브레비스(Lactobacillus brevis) MJM60390 균주(수탁번호 : KACC 81193BP)는 프로바이오틱 특성을 만족하기 때문에 인체에 안전하고, 혈중 요소 저감 활성, 잔틴산화효소(xanthine oxidase) 억제 활성 및/또는 신장 손상 억제 활성과 같은 다양한 기능성을 가지기 때문에 고요산혈증, 고요산혈증 관련 대사 장애 또는 신장 손상을 예방, 개선 또는 치료하기 위한 의약품 내지 건강기능식품의 유용한 소재로 사용될 수 있다. Lactobacillus brevis MJM60390 strain (accession number: KACC 81193BP) according to the present invention is safe for humans because it satisfies probiotic properties, blood urea reducing activity, xanthine oxidase inhibitory activity and / Or, since it has various functionalities such as renal damage inhibitory activity, it can be used as a useful material for pharmaceuticals or health functional foods for preventing, improving, or treating hyperuricemia, hyperuricemia-related metabolic disorders or renal damage.
도 1은 본 발명의 실시예에서 선발된 4종의 균주(Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, Lactobacillus gasseri PA-3)를 가지고 구강-위장관 통과 조건에서의 내성 평가 실험을 진행한 결과이다.
도 2는 본 발명의 실시예에서 선발된 4종의 균주(Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, Lactobacillus gasseri PA-3)에 대해 Caco-2 세포주를 이용하여 장내 부착능 평가 실험을 진행한 결과이다.
도 3은 본 발명의 실시예에서 선발된 4종의 균주(Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, Lactobacillus gasseri PA-3)를 퓨린 또는 뉴클레오사이드가 존재하는 배지에서 배양하였을 때의 증식 특성을 나타낸 그래프이다.
도 4는 본 발명의 실시예에서 고요산혈증 동물모델을 이용하여 선발된 2종의 균주(Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3)의 고요산혈증 개선 효능 평가 실험(In-vivo test)을 진행하였을 때 실험군별 마우스 체중 변화를 나나탠 것이다.
도 5는 본 발명의 실시예에서 고요산혈증 동물모델을 이용하여 선발된 2종의 균주(Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3)의 고요산혈증 개선 효능 평가 실험(In-vivo test)을 진행하였을 때 0일차 및 15일차 실험군별 혈중 요산 함량을 분석한 결과이다.
도 6은 본 발명의 실시예에서 고요산혈증 동물모델을 이용하여 선발된 2종의 균주(Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3)의 고요산혈증 개선 효능 평가 실험(In-vivo test)을 진행하였을 때 15일차 실험군별 혈중 크레아티닌 함량 및 혈액요소질소(BUN)의 함량을 분석한 결과이다.
도 7은 본 발명의 실시예에서 고요산혈증 동물모델을 이용하여 선발된 2종의 균주(Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3)의 고요산혈증 개선 효능 평가 실험(In-vivo test)을 진행하였을 때 15일차 실험군별 혈중 잔틴산화효소(xanthine oxidase, XO) 활성을 분석한 결과이다.
도 8은 본 발명의 실시예에서 고요산혈증 동물모델을 이용하여 선발된 2종의 균주(Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3)의 고요산혈증 개선 효능 평가 실험(In-vivo test)을 진행하였을 때 15일차 실험군별 혈중 URAT1 단백질 함량을 분석한 결과이다.
도 9는 본 발명의 실시예에서 고요산혈증 동물모델을 이용하여 선발된 2종의 균주(Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3)의 고요산혈증 개선 효능 평가 실험(In-vivo test)을 진행하였을 때 15일차 실험군별 대변 내 단쇄지방산(SCFA) 함량을 측정한 결과이다.
도 10은 본 발명의 실시예에서 고요산혈증 동물모델을 이용하여 선발된 2종의 균주(Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3)의 고요산혈증 개선 효능 평가 실험(In-vivo test)을 진행하였을 때 15일차 실험군별 장내 미생물 군집을 분석한 결과이다.
도 11은 본 발명의 실시예에서 고요산혈증 동물모델을 이용하여 선발된 2종의 균주(Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3)의 고요산혈증 개선 효능 평가 실험(In-vivo test)을 진행하였을 때 15일차 실험군별 적출한 신장의 조직을 Hematoxylin & eosin(H&E) 염색한 후 관찰한 사진이다.
도 12는 본 발명의 실시예에서 스크리닝한 Lactobacillus brevis MJM60390 균주의 계통관계도(phylogenetic tree)를 나타낸 것이다.Figure 1 shows the results of the resistance evaluation experiment under oral-gastrointestinal passage conditions with four strains ( Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, Lactobacillus gasseri PA-3) selected in Examples of the present invention. am.
2 shows four strains selected in the examples of the present invention ( Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, Lactobacillus gasseri PA-3) using Caco-2 cell line to evaluate intestinal adhesion ability. is a result
Figure 3 shows the growth of the four strains ( Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, Lactobacillus gasseri PA-3) selected in Examples of the present invention when cultured in a medium containing purines or nucleosides. It is a graph showing the characteristics.
Figure 4 is a hyperuricemia improvement efficacy evaluation experiment (In-vivo test) of two strains ( Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3) selected using an animal model for hyperuricemia in an embodiment of the present invention. It shows the change in body weight of mice by experimental group.
Figure 5 is a hyperuricemia improvement efficacy evaluation experiment (In-vivo test) of two strains ( Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3) selected using an animal model for hyperuricemia in an embodiment of the present invention. These are the results of analyzing the blood uric acid content for each experimental group on
Figure 6 is a hyperuricemia improvement efficacy evaluation experiment (In-vivo test) of two strains ( Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3) selected using an animal model for hyperuricemia in an embodiment of the present invention. These are the results of analyzing the blood creatinine content and blood urea nitrogen (BUN) content for each experimental group on the 15th day.
Figure 7 is when the hyperuricemia improvement efficacy evaluation experiment (In-vivo test) of two strains ( Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3) selected using an animal model for hyperuricemia in an embodiment of the present invention was conducted This is the result of analyzing the activity of xanthine oxidase (XO) in the blood of each experimental group on the 15th day.
8 is when an experiment (in-vivo test) was conducted to evaluate the hyperuricemia improvement efficacy of two strains ( Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3) selected using an animal model for hyperuricemia in an embodiment of the present invention. This is the result of analyzing the URAT1 protein content in the blood of each experimental group on the 15th day.
Figure 9 is a hyperuricemia improvement efficacy evaluation experiment (In-vivo test) of two strains ( Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3) selected using an animal model for hyperuricemia in an embodiment of the present invention. This is the result of measuring the content of short-chain fatty acids (SCFA) in the feces of each experimental group on the 15th day.
Figure 10 is when the hyperuricemia improvement efficacy evaluation experiment (In-vivo test) of two strains ( Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3) selected using an animal model for hyperuricemia in an embodiment of the present invention was conducted This is the result of analyzing the intestinal microbial community for each experimental group on the 15th day.
11 is when an experiment (in-vivo test) was conducted to evaluate the hyperuricemia improvement efficacy of two strains ( Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3) selected using an animal model for hyperuricemia in an embodiment of the present invention. This is a photograph observed after hematoxylin & eosin (H&E) staining of kidney tissues extracted from each experimental group on the 15th day.
12 shows a phylogenetic tree of Lactobacillus brevis MJM60390 strains screened in Examples of the present invention.
이하, 본 발명에서 사용한 용어를 설명한다.Hereinafter, terms used in the present invention will be described.
본 발명에서 사용되는 용어 "배양물"은 미생물을 공지의 액체 배지 또는 고체 배지에서 배양시켜 수득한 산물을 의미하여, 배양된 미생물이 포함되는 개념이이다. 구체적으로 상기 배양물은 미생물 배양액일 수도 있고, 미생물 배양액을 고형화한 고체상일 수도 있다.The term "culture" used in the present invention refers to a product obtained by culturing a microorganism in a known liquid medium or solid medium, and is a concept including the cultured microorganism. Specifically, the culture may be a microbial culture medium, or may be a solid phase in which the microbial culture medium is solidified.
본 발명에서 사용되는 용어 "조성물"은 2가지 이상의 성분이 균일하게 혼합되어 있는 상태의 물질을 의미하며, 완제품뿐만 아니라 완제품 제조를 위한 중간 소재를 포함하는 개념이다.The term "composition" used in the present invention refers to a material in which two or more components are uniformly mixed, and is a concept including an intermediate material for manufacturing a finished product as well as a finished product.
본 발명에서 사용되는 용어 "약학적으로 허용가능한" 및 "식품학적으로 허용가능한"이란 생물체를 상당히 자극하지 않고 투여 활성 물질의 생물학적 활성 및 특성을 저해하지 않는 것을 의미한다.As used herein, the terms "pharmaceutically acceptable" and "food acceptable" mean that they do not significantly stimulate organisms and do not inhibit the biological activity and properties of the active substance administered.
본 발명에서 사용되는 용어 "예방"은 본 발명의 조성물의 투여로 특정 질환의 증상을 억제하거나 진행을 지연시키는 모든 행위를 의미한다.As used herein, the term "prevention" refers to any action that suppresses symptoms or delays the progression of a specific disease by administering the composition of the present invention.
본 발명에서 사용되는 용어 "치료"는 본 발명의 조성물의 투여로 특정 질환의 증상을 호전 또는 이롭게 변경시키는 모든 행위를 의미한다.The term "treatment" used in the present invention refers to all activities that improve or beneficially change the symptoms of a specific disease by administration of the composition of the present invention.
본 발명에서 사용되는 용어 "개선"은 치료되는 상태와 관련된 파라미터, 예를 들면 증상의 정도를 적어도 감소 또는 완화시키는 모든 행위를 의미한다.As used herein, the term "improvement" refers to any action that at least reduces or alleviates a parameter associated with the condition being treated, eg, the severity of a symptom.
본 발명에서 사용되는 용어 "투여"는 임의의 적절한 방법으로 개체에 소정의 본 발명의 조성물을 제공하는 것을 의미한다. 이때, 개체는 본 발명의 조성물을 투여하여 특정 질환의 증상이 호전될 수 있는 질환을 가진 인간, 원숭이, 개, 염소, 돼지 또는 쥐 등 모든 동물을 의미한다.As used herein, the term "administering" means providing a given composition of the present invention to a subject by any suitable method. In this case, the subject refers to all animals such as humans, monkeys, dogs, goats, pigs, or rats having a disease in which symptoms of a specific disease can be improved by administering the composition of the present invention.
본 발명에서 용어 "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜 또는 위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 이는 개체의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출비율, 치료기간, 동시에 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다.In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit or risk ratio applicable to medical treatment, which is a subject's disease type, severity, drug activity, drug It may be determined according to factors including sensitivity, time of administration, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field.
이하, 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 일 측면은 프로바이오틱스로서의 필요한 특성과 함께 인체에 유익한 다양한 기능성을 가지면서 식품학적으로 안전한 신규 유산균에 관한 것이다.One aspect of the present invention relates to a novel food-safe lactic acid bacteria having various functionalities beneficial to the human body along with necessary properties as probiotics.
본 발명의 일 예에 따른 신규 유산균은 퓨린(purine) 분해 활성을 가지는 락토바실러스 브레비스(Lactobacillus brevis) MJM60390 균주(수탁번호 : KACC 81193BP)이다. 상기 락토바실러스 브레비스(Lactobacillus brevis) MJM60390 균주(수탁번호 : KACC 81193BP)는 서열번호 1의 염기서열로 이루어진 16S rDNA를 갖는다. 또한, 상기 락토바실러스 브레비스(Lactobacillus brevis) MJM60390 균주(수탁번호 : KACC 81193BP)는 퓨린(purine) 분해 활성 외에 혈중 요소 저감 활성, 잔틴산화효소(xanthine oxidase) 억제 활성 또는 신장 손상 억제 활성에서 선택되는 적어도 2개 이상의 활성을 더 갖는다. 또한, 상기 락토바실러스 브레비스(Lactobacillus brevis) MJM60390 균주(수탁번호 : KACC 81193BP)는 퓨린(purine)는 장내 미생물 군집 중에서 박테로이데테스(Bacteroidetes) 문에 속하는 세균을 증가시키고 피르미쿠테스(Firmicutes) 문 및 프로테오박테리아(Proteobacteria) 문에 속하는 세균을 감소시킨다. 특히, 상기 락토바실러스 브레비스(Lactobacillus brevis) MJM60390 균주(수탁번호 : KACC 81193BP)는 박테로이데테스(Bacteroidetes) 문의 리케넬라세아에(Rikenellaceae) 과에 속하는 세균을 증가시켜 요산 생성을 억제하고 요산의 배출을 원활하게 한다.The novel lactic acid bacteria according to an example of the present invention is Lactobacillus brevis MJM60390 strain (accession number: KACC 81193BP) having purine decomposition activity. The Lactobacillus brevis strain MJM60390 (accession number: KACC 81193BP) has 16S rDNA consisting of the nucleotide sequence of SEQ ID NO: 1. In addition, the Lactobacillus brevis strain MJM60390 (accession number: KACC 81193BP) is at least selected from blood urea reducing activity, xanthine oxidase inhibitory activity or renal damage inhibitory activity in addition to purine decomposition activity. It has two or more activities. In addition, the Lactobacillus brevis strain MJM60390 (accession number: KACC 81193BP) has purine, which increases bacteria belonging to the phylum Bacteroidetes among the intestinal microbial community, and increases the phylum Firmicutes. and bacteria belonging to the phylum Proteobacteria. In particular, the Lactobacillus brevis MJM60390 strain (accession number: KACC 81193BP) inhibits uric acid production and excretion of uric acid by increasing bacteria belonging to the Rikenellaceae family of the Bacteroidetes phylum. make it smooth
본 발명의 다른 측면은 신규 유산균인 락토바실러스 브레비스(Lactobacillus brevis) MJM60390 균주(수탁번호 : KACC 81193BP)의 다양한 용도에 관한 것이다.Another aspect of the present invention relates to various uses of a novel lactic acid bacterium, Lactobacillus brevis ( Lactobacillus brevis ) strain MJM60390 (accession number: KACC 81193BP).
본 발명의 일 예는 락토바실러스 브레비스(Lactobacillus brevis) MJM60390 균주, 이의 배양물, 이의 파쇄물 또는 이의 추출물에서 선택되는 1종 이상을 유효성분으로 포함하는 고요산혈증 예방, 개선 또는 치료용 조성물을 제공한다.One example of the present invention provides a composition for preventing, improving or treating hyperuricemia comprising at least one selected from Lactobacillus brevis MJM60390 strain, its culture, its lysate or its extract as an active ingredient.
또한, 본 발명의 일 예는 락토바실러스 브레비스(Lactobacillus brevis) MJM60390 균주, 이의 배양물, 이의 파쇄물 또는 이의 추출물에서 선택되는 1종 이상을 유효성분으로 포함하는 고요산혈증 관련 대사 장애 예방, 개선 또는 치료용 조성물을 제공한다. 상기 고요산혈증 관련 대사 장애는 급성 또는 만성통풍, 통풍성 발적, 통풍성 관절염, 통풍성 신결석증 및 통풍성 신병증 중에서 선택된 어느 하나인 것이 바람직하지만 이에 한정하는 것은 아니다. 상기 통풍성 발적은 통풍에 의한 염증 등으로 붉어지는 증상을 의미한다.In addition, one example of the present invention is Lactobacillus brevis ( Lactobacillus brevis ) MJM60390 strain, a culture thereof, a lysate thereof, or a hyperuricemia-related metabolic disorder containing at least one selected from an extract as an active ingredient For prevention, improvement, or treatment composition is provided. The hyperuricemia-related metabolic disorder is preferably any one selected from acute or chronic gout, gouty flare-ups, gouty arthritis, gouty nephrolithiasis and gouty nephropathy, but is not limited thereto. The gouty redness means a symptom of redness due to inflammation or the like caused by gout.
또한, 본 발명의 일 예는 락토바실러스 브레비스(Lactobacillus brevis) MJM60390 균주, 이의 배양물, 이의 파쇄물 또는 이의 추출물에서 선택되는 1종 이상을 유효성분으로 포함하는 신장 손상 예방, 개선 또는 치료용 조성물을 제공한다. 상기 신장 손상은 요산에 의해 유발되는 것으로서, 구체적으로 혈중 요산 수준의 증가로 인해 유발되고, 혈중 요산 수치의 증가로 인한 보우만 캡슐 공간(bowman's capsule space)의 감소, 근위 세관 세포(proximal tubule's cell)의 팽창, 사구체의 비대화 등을 포함한다.In addition, one embodiment of the present invention is Lactobacillus brevis ( Lactobacillus brevis ) MJM60390 strain, its culture, its lysate or its extract containing at least one selected from the active ingredient Provides a composition for preventing, improving or treating kidney damage do. The renal damage is caused by uric acid, specifically caused by an increase in the level of uric acid in the blood, a decrease in Bowman's capsule space due to an increase in the level of uric acid in the blood, a decrease in proximal tubule's cells dilatation, glomerular hypertrophy, etc.
본 발명에서 배양물은 미생물을 배지에서 배양시켜 수득한 산물로서, 상기 배지는 공지의 액체 배지 또는 고체 배지에서 선택될 수 있으며, 예를 들어 MRS 액체 배지, MRS 한천 배지, BL 한천 배지일 수 있다. In the present invention, the culture is a product obtained by culturing microorganisms in a medium, and the medium may be selected from known liquid medium or solid medium, and may be, for example, MRS liquid medium, MRS agar medium, or BL agar medium. .
또한, 본 발명에서 상기 조성물은 사용 목적 내지 양상에 따라 약학 조성물, 건강기능식품 조성물 등으로 구체화될 수 있고, 조성물 내에서 유효성분인 특정 락토바실러스속 균주 등의 함량도 조성물의 구체적인 형태, 사용 목적 내지 양상에 따라 다양한 범위에서 조정될 수 있다.In addition, in the present invention, the composition may be embodied as a pharmaceutical composition, a health functional food composition, etc. according to the purpose or aspect of use, and the content of a specific Lactobacillus sp. It can be adjusted in various ranges depending on the aspect.
본 발명에 따른 약학 조성물에서 유효성분인 신규 유산균, 이의 배양물, 이의 파쇄물 또는 이의 추출물의 함량은 크게 제한되지 않으며, 예를 들어 조성물 총 중량을 기준으로 0.01~99 중량%, 바람직하게는 0.5~50 중량%, 더 바람직하게는 1~30 중량%일 수 있다. 또한, 본 발명에 따른 약학 조성물은 유효성분 외에 약학적으로 허용가능한 담체, 부형제 또는 희석제와 같은 첨가제를 더 포함할 수 있다. 본 발명의 약학 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 또한, 본 발명의 약학 조성물은 신규 유산균, 이의 배양물, 이의 파쇄물 또는 이의 추출물 외에 고요산혈증 또는 통풍 예방 또는 치료 효과를 갖는 공지의 유효성분을 1종 이상 더 함유할 수 있다. 본 발명의 약학 조성물은 통상의 방법에 의해 경구 투여를 위한 제형 또는 비경구 투여를 위한 제형으로 제제화될 수 있고, 제제화할 경우 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구 투여를 위한 고형 제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형 제제는 유효성분에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(Calcium carbonate), 수크로스(Sucrose), 락토오스(Lactose) 또는 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용될 수 있다. 경구 투여를 위한 액상 제제로는 현탁제, 내용액제, 유제 및 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함될 수 있다. 비수성용제, 현탁용제로는 프로필렌글리콜(Propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다. 더 나아가 당 분야의 적정한 방법으로 또는 Remington's Pharmaceutical Science(최근판), Mack Publishing Company, Easton PA에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다. 본 발명의 약학 조성물은 목적하는 방법에 따라 인간을 포함한 포유류에 경구 투여되거나 비경구 투여될 수 있으며, 비경구 투여 방식으로는 피부 외용, 복강내주사, 직장내주사, 피하주사, 정맥주사, 근육내 주사 또는 흉부내 주사 주입방식 등이 있다. 본 발명의 약학 조성물의 투여량은 약학적으로 유효한 양이라면 크게 제한되지 않으며, 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도에 따라 그 범위가 다양하다. 본 발명의 약학 조성물의 통상적인 1일 투여량은 크게 제한되지 않으나 바람직하게는 유효성분을 기준으로 할 때 0.01 내지 3000 ㎎/㎏ B.W.이고, 더 바람직하게는 0.1 내지 2000 ㎎/㎏ B.W.이며, 하루 1회 또는 수회로 나누어 투여될 수 있다. 본 발명의 약학 조성물의 통상적인 1일 투여량은 락토바실러스 브레비스(Lactobacillus brevis) MJM60390 균주(수탁번호 : KACC 81193BP)를 기준으로 할 때 1×106 CFU/kg B.W. 내지 1×1012 CFU/kg B.W.이고 바람직하게는 1×108 CFU/kg B.W. 내지 1×1010 CFU/kg B.W.이다.In the pharmaceutical composition according to the present invention, the content of the novel lactic acid bacteria, culture thereof, lysate thereof, or extract thereof as an active ingredient is not significantly limited, for example, 0.01 to 99% by weight, preferably 0.5 to 99% by weight, based on the total weight of the composition. 50% by weight, more preferably 1 to 30% by weight. In addition, the pharmaceutical composition according to the present invention may further include additives such as pharmaceutically acceptable carriers, excipients or diluents in addition to active ingredients. Carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate , cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, the pharmaceutical composition of the present invention may further contain at least one known active ingredient having an effect of preventing or treating hyperuricemia or gout, in addition to the novel lactic acid bacteria, cultures thereof, lysates thereof, or extracts thereof. The pharmaceutical composition of the present invention may be formulated into a dosage form for oral administration or a dosage form for parenteral administration by a conventional method, and when formulated, commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. It may be formulated using diluents or excipients. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose, etc. ), lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium styrate and talc may also be used. Liquid formulations for oral administration include suspensions, internal solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used. Furthermore, it can be preferably formulated according to each disease or component by using an appropriate method in the art or by using a method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA. The pharmaceutical composition of the present invention can be administered orally or parenterally to mammals, including humans, depending on the desired method, and parenteral administration methods include external skin application, intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, and intramuscular injection. Intrathoracic or intrathoracic injections are available. The dosage of the pharmaceutical composition of the present invention is not significantly limited as long as it is a pharmacologically effective amount, and the range depends on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease. Varies. A typical daily dose of the pharmaceutical composition of the present invention is not particularly limited, but is preferably 0.01 to 3000 mg/kg BW based on the active ingredient, more preferably 0.1 to 2000 mg/kg BW, and It may be administered once or divided into several times. A typical daily dose of the pharmaceutical composition of the present invention is 1 × 10 6 CFU / kg BW to 1 × 10 12 CFU / kg based on the Lactobacillus brevis MJM60390 strain (Accession Number: KACC 81193BP) BW, preferably 1×10 8 CFU/kg BW to 1×10 10 CFU/kg BW.
또한, 본 발명에 따른 건강기능식품 조성물에서 유효성분인 신규 유산균, 이의 배양물, 이의 파쇄물 또는 이의 추출물의 함량은 조성물 총 중량을 기준으로 0.01~50 중량%, 바람직하게는 0.1~25 중량%, 더 바람직하게는 0.5~10 중량%이나, 이에 한정되는 것은 아니다. 본 발명의 건강기능식품 조성물은 환제, 분말, 과립, 침제, 정제, 캡슐, 또는 액제 등의 형태를 포함하며, 구체적인 식품의 예로는 육류, 소시지, 빵, 초콜릿, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 기능수, 드링크제, 알코올음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강기능식품(health functional food) 외에 이와 유사한 개념을 갖는 식이보충제(dietary supplement) 및 식품보충제(food supplement)를 모두 포함한다. 본 발명의 건강기능식품 조성물은 유효성분 외에 식품학적으로 허용 가능한 식품 보조 첨가제를 더 포함할 수 있으며, 상기 보조 첨가제에는 통상적으로 사용되는 적절한 담체, 부형제 또는 희석제 등이 있다. 본 발명의 건강기능식품 조성물은 유효성분 외에 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 또한, 본 발명의 식품 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 건강기능식품 조성물은 천연 과일주스, 과일주스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분들은 독립적으로 또는 혼합하여 사용할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토스, 슈크로스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨 등의 당알코올이다. 향미제로는 타우마틴, 스테비아 추출물과 같은 천연 향미제나 사카린, 아스파르탐과 같은 합성 향미제 등을 사용할 수 있다.In addition, in the health functional food composition according to the present invention, the content of the new lactic acid bacteria, culture thereof, lysate thereof, or extract thereof as an active ingredient is 0.01 to 50% by weight, preferably 0.1 to 25% by weight, based on the total weight of the composition. More preferably 0.5 to 10% by weight, but not limited thereto. The health functional food composition of the present invention includes forms such as pills, powders, granules, precipitates, tablets, capsules, or liquids, and examples of specific foods include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, Ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, functional water, drinks, alcoholic beverages, and vitamin complexes. Includes both dietary supplements and food supplements with a similar concept. The health functional food composition of the present invention may further include a food-acceptable food auxiliary additive in addition to the active ingredient, and the auxiliary additive includes a suitable carrier, excipient, or diluent that is commonly used. The health functional food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients in addition to active ingredients. In addition, the food composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol , carbonation agents used in carbonated beverages, and the like. In addition, the health functional food composition of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The aforementioned natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agent, natural flavoring agents such as thaumatin and stevia extract or synthetic flavoring agents such as saccharin and aspartame may be used.
이하, 본 발명을 실시예를 통하여 보다 구체적으로 설명한다. 다만, 하기 실시예는 본 발명의 기술적 특징을 명확하게 예시하기 위한 것 일뿐, 본 발명의 보호범위를 한정하는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. However, the following examples are only for clearly illustrating the technical features of the present invention, but do not limit the protection scope of the present invention.
1. 퓨린 분해 활성을 가진 유산균의 스크리닝1. Screening of lactic acid bacteria with purinergic activity
명지대학교 자연캠퍼스 내에 위치하고 있는 유용미생물추출물은행(ECUM; 경기도 용인시 처인구 남동 산 38-2 행정동 1층 17102호)에 보관되어 있는 100여종의 유산균으로부터 퓨린 분해 활성을 가진 유산균을 스크리닝하였다. 상기 100여종의 유산균은 전통 발효 음식으로부터 분리한 것이다.Lactic acid bacteria with purine decomposition activity were screened from about 100 types of lactic acid bacteria stored in the Useful Microbial Extract Bank (ECUM; 17102, 1st Floor, Administrative Building, San 38-2 Namdong, Cheoin-gu, Yongin-si, Gyeonggi-do) located on the natural campus of Myongji University. The above 100 kinds of lactic acid bacteria are isolated from traditional fermented foods.
먼저, 이노신(inosine) 33.7㎎ 및 구아노신(guanosine) 35.7 ㎎을 인산칼륨(K3PO4) 용액 100 ㎖에 첨가하고 용해시켜 이노신 농도 및 구아노신 농도가 각각 1 mM인 이노신-구아노신 용액을 제조하고 멸균하였다. 100여종의 유산균을 각각 MRS 배지에 접종하고 37℃에서 48 hr 동안 무산소 조건으로 배양한 후, 배양액 2 ㎖를 4000×g의 조건에서 10분 동안 원심분리하여 배양된 균체를 수득하였다. 이후, 수득한 균체를 0.85% 염화나트륨(NaCl) 용액 1 ㎖로 3번 세척한 후 균체를 이노신-구아노신 용액 750 ㎕에 현탁하고 37℃에서 120분 동안 배양하였다. 이후, 배양액에서 상등액 270 ㎕를 취한 후 여기에 0.1M 농도의 HClO4 용액 30 ㎕를 첨가하여 뉴클레오사이드 분해 반응을 중단시키고 0.2 ㎛ 필터로 여과한 후 여과액을 HPLC로 분석하여 유산균의 뉴클레오사이드 분해능을 확인하였다.First, 33.7 mg of inosine and 35.7 mg of guanosine were added to and dissolved in 100 ml of a potassium phosphate (K 3 PO 4 ) solution to obtain an inosine-guanosine solution having an inosine concentration and guanosine concentration of 1 mM, respectively. prepared and sterilized. Each of 100 kinds of lactic acid bacteria was inoculated into the MRS medium and cultured at 37° C. for 48 hr under anoxic conditions, and then 2 ml of the culture medium was centrifuged at 4000×g for 10 minutes to obtain cultured cells. Thereafter, the obtained cells were washed three times with 1 ml of a 0.85% sodium chloride (NaCl) solution, and then the cells were suspended in 750 μl of an inosine-guanosine solution and incubated at 37° C. for 120 minutes. Thereafter, after taking 270 μl of the supernatant from the culture medium, 30 μl of a 0.1 M HClO 4 solution was added thereto to stop the nucleoside decomposition reaction, and after filtering through a 0.2 μm filter, the filtrate was analyzed by HPLC to analyze the nucleosome of lactic acid bacteria. Side resolution was confirmed.
HPLC 분석에 사용된 조건은 다음과 같다.Conditions used for HPLC analysis were as follows.
* 컬럼 : YMC-Pack ODS-A 컬럼(AA12S05-2546WT S-5 ㎛, 12 ㎜, 250×4.6 ㎜ column); 컬럼 온도 : 37℃; 유속 : 1 ㎖/min; 이동상 : NaClO4-H3PO4 용액(dH2O 중 0.1 mM NaClO4 및 0.187 mM H3PO4); 검출파장 : 254 nm* Column: YMC-Pack ODS-A column (AA12S05-2546WT S-5 μm, 12 mm, 250 × 4.6 mm column); column temperature: 37°C; flow rate: 1 ml/min; Mobile phase: NaClO 4 -H 3 PO 4 solution (0.1 mM NaClO 4 and 0.187 mM H 3 PO 4 in dH 2 O); Detection wavelength: 254 nm
총 57개의 균에서 구아노신 및 이노신 분해능을 보였고, HPLC 피크 크기로 정량분석을 진행하여 구아노닌 및 이노신 분해율이 99.9%에 가까운 총 5개의 균주를 선발하였다. 선발된 균주는 Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60384, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, Lactobacillus gasseri PA-3 이다. 선발된 균주 중 4종의 균주(Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, Lactobacillus gasseri PA-3)에 대해 이후의 실험을 진행하였다.A total of 57 strains showed guanosine and inosine decomposition ability, and a total of 5 strains with guanonine and inosine decomposition rates close to 99.9% were selected through quantitative analysis based on HPLC peak size. The strains selected were Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60384, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, and Lactobacillus gasseri PA-3. Subsequent experiments were conducted on four strains ( Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, and Lactobacillus gasseri PA-3) among the selected strains.
2. 선발된 균주의 프로바이오틱스 특성 규명2. Characterization of probiotics of selected strains
(1) 구강-위장관 통과 조건에서의 내성 평가(1) Tolerance evaluation under oral-gastrointestinal transit conditions
선발된 4종의 균주(Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, Lactobacillus gasseri PA-3)에 대해 [Bove, P., Gallone, A., Russo, P., Capozzi, V., Albenzio, M., Spano, G., and Fiocco, D. 2012. Probiotic features of Lactobacillus plantarum mutant strains. Applied microbiology and biotechnology 96(2): 431-441. doi: 10.1007/s00253-012-4031-2]에 기술된 방법을 약간 수정하여 구강-위장관 통과 내성 어세이(Oro-gastrointestinal transit tolerance assay)를 수행하였다.[Bove , P. , Gallone, A., Russo, P., Capozzi, V. , Albenzio , M., Spano, G., and Fiocco, D. 2012. Probiotic features of Lactobacillus plantarum mutant strains. Applied microbiology and biotechnology 96(2): 431-441. doi: 10.1007/s00253-012-4031-2] was slightly modified to perform an oral-gastrointestinal transit tolerance assay.
먼저, 구강 통과 스트레스를 주기 위해, 150 ㎎/ℓ의 라이소자임(lysozyme)을 포함하는 전해질 용액(NaCl, 6.2g/ℓ; KCl, 2.2g/ℓ; CaCl2, 0.22g/ℓ; NaHCO3, 1.2g/ℓ)에 선발된 4종의 균주(Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, Lactobacillus gasseri PA-3)을 각각 1×109 CFU/㎖씩 접종하고 37℃에서 5분 동안 배양하였다. 배양이 완료된 후 배양액을 연속희석법을 이용하여 MRS 한천 배지에 도포한 후 계수하였다.First, to give oral passage stress, an electrolyte solution (NaCl, 6.2 g/L; KCl, 2.2 g/L; CaCl 2 , 0.22 g/L; NaHCO 3 , 1.2 g/L) containing 150 mg/L lysozyme g / ℓ) was inoculated with 1 × 10 9 CFU / ml of each of the four selected strains ( Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, Lactobacillus gasseri PA-3) and incubated for 5 minutes at 37 ° C. . After the culture was completed, the culture solution was applied to MRS agar medium using a serial dilution method, and then counted.
이후, 구강 통과 스트레스 조건에서 배양된 균체를 원심분리(1,800 × g, 5분) 하여 구강 스트레스 용액으로부터 분리하고, 위 통과 스트레스를 주기 위해 분리된 균체를 0.3% 펩신(pepsin)을 포함하고 pH가 3인 전해질 용액에 접종한 후 37℃에서 1 hr 동안 배양하였다. 배양이 완료된 후 배양액을 연속희석법을 이용하여 MRS 한천 배지에 도포한 후 계수하였다.Thereafter, the cells cultured under oral passage stress conditions were separated from the oral stress solution by centrifugation (1,800 × g, 5 minutes), and the cells isolated to give gastric passage stress contained 0.3% pepsin and the pH was After inoculation in the 3-person electrolyte solution, the cells were incubated at 37° C. for 1 hr. After the culture was completed, the culture solution was applied to MRS agar medium using a serial dilution method, and then counted.
이후, 위 통과 스트레스 조건에서 배양된 균체를 원심분리(1,800 × g, 5분) 하여 위 스트레스 용액으로부터 분리하고, 소장 통과 스트레스를 주기 위해 분리된 균체를 0.1% 판크레아틴(pancreatin) 및 0.3% 담즙염(bile oxgall)을 포함하고 pH가 7인 전해질 용액(NaCl, 5g/ℓ; KCl, 0.6g/ℓ; CaCl2, 0.25g/ℓ)에 접종하고 37℃에서 2 hr 동안 배양하였다. 배양이 완료된 후 배양액을 연속희석법을 이용하여 MRS 한천 배지에 도포한 후 계수하였다.Thereafter, the cells cultured under gastric transit stress conditions were separated from the gastric stress solution by centrifugation (1,800 × g, 5 minutes), and the separated cells were treated with 0.1% pancreatin and 0.3% bile to give small intestine transit stress. It was inoculated into an electrolyte solution (NaCl, 5 g/L; KCl, 0.6 g/L; CaCl 2 , 0.25 g/L) containing salt (bile oxgall) and having a pH of 7, and incubated at 37° C. for 2 hr. After the culture was completed, the culture solution was applied to MRS agar medium using a serial dilution method, and then counted.
도 1은 본 발명의 실시예에서 선발된 4종의 균주(Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, Lactobacillus gasseri PA-3)를 가지고 구강-위장관 통과 조건에서의 내성 평가 실험을 진행한 결과이다. 도 1에서 보이는 바와 같이 구강 스트레스에서의 저항력은 Lactobacillus fermentum MJM60393 균주가 가장 높았고 위 스트레스에서의 저항력은 Lactobacillus brevis MJM60390이 가장 높았고 소장 스트레스에서의 저항력은 Lactobacillus paracasei MJM60396이 가장 높은 것으로 나타났다.Figure 1 shows the results of the resistance evaluation experiment under oral-gastrointestinal passage conditions with four strains ( Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, Lactobacillus gasseri PA-3) selected in Examples of the present invention. am. As shown in Figure 1, the resistance in oral stress was the highest in Lactobacillus fermentum MJM60393 strain, the resistance in gastric stress was the highest in Lactobacillus brevis MJM60390, and the resistance in small intestine stress was found to be the highest in Lactobacillus paracasei MJM60396.
(2) 항균 활성 평가(2) Evaluation of antibacterial activity
선발된 4종의 균주(Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, Lactobacillus gasseri PA-3)에 대한 항균 활성 평가는 인간, 동물 또는 음식에서 기인되는 유해 미생물인 Staphylococcus aureus, Listeria monocytogenes KACC 10764, Bacilus cereus KACC 11240, Bacilus subtilis KSP12, Salmonella typhi KCTC 2514, Salmonella chloeraesuis KCTC 2932, Pseudomonas aeruginosa KCCM 11802, Salmonella gallinarum KCTC 2931, Escherichia coli O1 KCTC 2441을 사용하여 수행하였다. Antimicrobial activity against the four selected strains ( Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, and Lactobacillus gasseri PA-3) was evaluated against harmful microorganisms such as Staphylococcus aureus , Listeria monocytogenes KACC 10764, Bacillus cereus KACC 11240, Bacilus subtilis KSP12, Salmonella typhi KCTC 2514, Salmonella chloeraesuis KCTC 2932, Pseudomonas aeruginosa KCCM 11802, Salmonella gallinarum KCTC 2931, Escherichia coli O1 KCTC 2441 were used.
선발된 4종의 균주를 각각 MRS 배지에 접종하고 37℃에서 24 hr 동안 배양하였다. 이후, 배양액 50 ㎕를 8 ㎜ 직경의 필터 페이퍼 디스크(filter paper disk)에 로딩하고 건조하였다. 이후, 필터 페이퍼 디스크를 유해 미생물이 1×106 CFU/㎖ 농도로 도포된 MRS 한천 플레이트 상에 위치시키고 37℃에서 배양한 후 저해 구역을 확인하여 항균 활성을 평가하였다. 하기 표 1에 선발된 4종의 균주(Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, Lactobacillus gasseri PA-3)에 대한 항균 활성 평가 결과를 요약하였다.Each of the four selected strains was inoculated into MRS medium and cultured at 37° C. for 24 hr. Thereafter, 50 μl of the culture was loaded onto an 8 mm diameter filter paper disk and dried. Thereafter, the filter paper disk was placed on an MRS agar plate coated with harmful microorganisms at a concentration of 1×10 6 CFU/ml, incubated at 37° C., and the inhibition zone was evaluated to evaluate the antibacterial activity. Table 1 summarizes the antibacterial activity evaluation results for the four selected strains ( Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, and Lactobacillus gasseri PA-3).
* A : Staphylococcus aureus * A: Staphylococcus aureus
* B : Listeria monocytogenes KACC 10764* B: Listeria monocytogenes KACC 10764
* C : Bacilus cereus KACC 11240*C: Bacilus cereus KACC 11240
* D : Bacilus subtilis KSP12*D: Bacillus subtilis KSP12
* E : Salmonella typhi KCTC 2514*E: Salmonella typhi KCTC 2514
* F : Salmonella chloeraesuis KCTC 2932* F : Salmonella chloeraesuis KCTC 2932
* G : Pseudomonas aeruginosa KCCM 11802* G: Pseudomonas aeruginosa KCCM 11802
* H : Salmonella gallinarum KCTC 2931* H: Salmonella gallinarum KCTC 2931
* I : Escherichia coli O1 KCTC 2441* I : Escherichia coli O1 KCTC 2441
상기 표 1에서 보이는 바와 같이 L. brevis MJM60390,균주는 Listeria monocytogenes KACC 10764, Salmonella typhi KCTC 2514, Pseudomonas aeruginosa KCCM 11802, Salmonella gallinarum KCTC 2931, Escherichia coli O1 KCTC 2441에 대해 항균 활성을 보였고 이중 Listeria monocytogenes KACC 10764 와 Salmonella gallinarum KCTC 2931에 대해서는 강력한 항균 활성을 보였다. L. fermentum MJM60393 균주는 Listeria monocytogenes KACC 10764, Pseudomonas aeruginosa KCCM 11802, Salmonella gallinarum KCTC 2931, Escherichia coli O1 KCTC 2441에 대해 항균 활성을 보였고 이중 Pseudomonas aeruginosa KCCM 11802에 대해서는 강한 항균 활성을 보였다. L. paracasei MJM60396 균주는 Pseudomonas aeruginosa KCCM 11802에 대해서만 강한 항균 활성을 보였다. L. gasseri PA-3균주는 Listeria monocytogenes KACC 10764, Salmonella typhi KCTC 2514, Pseudomonas aeruginosa KCCM 11802, Salmonella gallinarum KCTC 2931, Escherichia coli O1 KCTC 2441에 대해 항균 활성을 보였고 Pseudomonas aeruginosa KCCM 11802에 대해서는 강력한 항균 활성을 보였다.As shown in Table 1, the L. brevis MJM60390 strain showed antibacterial activity against Listeria monocytogenes KACC 10764, Salmonella typhi KCTC 2514, Pseudomonas aeruginosa KCCM 11802, Salmonella gallinarum KCTC 2931, Escherichia coli O1 KCTC 2441, and Listeria monocytogenes KACC 10764 and Salmonella gallinarum KCTC 2931 showed strong antibacterial activity. L. fermentum MJM60393 showed antibacterial activity against Listeria monocytogenes KACC 10764, Pseudomonas aeruginosa KCCM 11802, Salmonella gallinarum KCTC 2931, Escherichia coli O1 KCTC 2441, and showed strong antibacterial activity against Pseudomonas aeruginosa KCCM 11802. L. paracasei strain MJM60396 showed strong antibacterial activity only against Pseudomonas aeruginosa KCCM 11802. L. gasseri PA-3 strain showed antibacterial activity against Listeria monocytogenes KACC 10764, Salmonella typhi KCTC 2514, Pseudomonas aeruginosa KCCM 11802, Salmonella gallinarum KCTC 2931, Escherichia coli O1 KCTC 2441 and strong antibacterial activity against Pseudomonas aeruginosa KCCM 11802. seemed .
(3) 항생제 감수성 평가(3) Antibiotic susceptibility evaluation
선발된 4종의 균주(Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, Lactobacillus gasseri PA-3)에 대한 항생제 감수성 평가는 유럽식품안전청(European Food Safety Authority, EFSA)에서 권장하는 항생제(Chloramphenicol, Ampicillin, Tetracycline, Gentamycin, Kanamycin, Streptomycin, Erythromycin, Clindamycin, Vancomycin)을 사용하여 진행하였다.The antibiotic susceptibility evaluation for the selected 4 strains ( Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, Lactobacillus gasseri PA-3) was performed using antibiotics (Chloramphenicol, Ampicillin) recommended by the European Food Safety Authority (EFSA). , Tetracycline, Gentamycin, Kanamycin, Streptomycin, Erythromycin, Clindamycin, Vancomycin) were used.
항생제를 8 ㎜ 직경의 필터 페이퍼 디스크(filter paper disk)에 디스크 당 100 ㎍의 농도가 되게 로딩하였다. 이후, 항생제가 로딩된 디스크를 선발된 4종의 균주가 각각 도말된 MRS 한천 플레이트 상에 위치시키고 37℃에서 48 hr 동안 배양하였다. 이후, 필터 페이퍼 디스크 주변 환의 크기를 통해 항생제 감수성을 평가하였다. 항생제 감수성 평가 기준은 유럽식품안정청(EFSA)이 명시한 2011~2012년 기준을 참고로 하였다. 하기 표 2에 선발된 4종의 균주(Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, Lactobacillus gasseri PA-3)에 대한 항생제 감수성 평가 결과를 요약하였다.Antibiotics were loaded onto 8 mm diameter filter paper disks at a concentration of 100 μg per disk. Thereafter, the antibiotic-loaded disk was placed on an MRS agar plate on which the four selected strains were respectively smeared, and incubated at 37° C. for 48 hr. Then, antibiotic susceptibility was evaluated through the size of the ring around the filter paper disk. The antibiotic susceptibility evaluation criteria referred to the 2011-2012 criteria specified by the European Food Safety Authority (EFSA). The antibiotic susceptibility evaluation results for the four selected strains ( Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, and Lactobacillus gasseri PA-3) are summarized in Table 2 below.
* S : sensitive; R : resistant* S : sensitive; R: resistant
상기 표 2에서 보이는 바와 같이 4종의 균주(Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, Lactobacillus gasseri PA-3)는 모두 kanamycin과 vancomycin에 대해 저항성을 가지는 것을 확인할 수 있었다. 또한, Lactobacillus fermentum MJM60393을 제외한 나머지 3개의 균주는 Streptomycin에 대해서도 저항성을 가졌다. kanamycin, vancomycin 및 Streptomycin을 제외한 나머지 항생제에 대해서는 4종의 균주 모두 감수성을 나타내었다.As shown in Table 2, it was confirmed that all four strains ( Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, and Lactobacillus gasseri PA-3) had resistance to kanamycin and vancomycin. In addition, the remaining three strains except for Lactobacillus fermentum MJM60393 were also resistant to Streptomycin. All four strains showed susceptibility to the remaining antibiotics except for kanamycin, vancomycin and streptomycin.
(4) Caco-2 세포주를 이용한 장내 부착능 평가(4) Evaluation of intestinal adhesion using Caco-2 cell line
인간 상피조직 유사 Caco-2 세포주를 미생물자원센터(KCTC)에서 분양받아 상피세포 부착능 실험에 사용하였으며, [Tuo et al., 2013]에 기재된 방법을 참조하여 실험을 수행하였다.The human epithelial tissue-like Caco-2 cell line was distributed from the Microbial Resource Center (KCTC) and used for epithelial cell adhesion test, and the experiment was performed by referring to the method described in [Tuo et al., 2013].
먼저, 20%(v/v)의 비활성화 FBS(fetal bovine serum), 100 U/㎖의 페니실린 및 100 ㎎/㎖의 스트렙토마이신 항생제가 첨가된 DMEM 배지(Gibco)에 Caco-2 세포를 접종하고 37℃의 온도 및 5%의 CO2 농도 환경을 가진 배양기에서 배양하였다. 이후, 잘 분화된 Caco-2 세포를 12-웰 플레이트(Well Plates)에 웰당 2×105 cells의 양으로 분주하고 매일 배지를 교환하면서 24 hr 동안 배양하여 80%가 채워진 단일층(monolayer)을 형성시켰다. 이후, Caco-2 세포 단일층을 PBS로 세번 세척하고 항생제가 없는 DMEM 배지로 교환한 후, 시험 균주를 최종 농도가 1×108 CFU/㎖가 되게 접종하고 37℃, 5% CO2 환경에서 2 hr 동안 배양하였다. 이후, 단일층을 PBS로 세번 세척하여 부착되지 않은 시험 균주를 제거하고 1% Triton-X 100(Sigma-Aldrich) 200 ㎕를 첨가하여 시험 균주가 부착된 단일층을 파쇄하였다. 이후, 파쇄액 1 ㎖를 연속희석법을 이용하여 MRS 한천 배지에 도포한 후 37℃에서 48 hr 동안 배양한 후 균수를 확인하였다. 시험 균주의 부착능은 부착된 시험 균주의 콜로니 형성 단위(colony forming units, CFU)를 계산하여 평가하였다. 시험 균주의 Caco-2 세포주에 대한 부착 %는 아래의 식을 사용하였다.First, Caco-2 cells were inoculated in DMEM medium (Gibco) supplemented with 20% (v/v) inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin antibiotics, and It was cultured in an incubator having a temperature of °C and a CO 2 concentration environment of 5%. Thereafter, the well-differentiated Caco-2 cells were dispensed in an amount of 2×10 5 cells per well in 12-well plates and cultured for 24 hr while exchanging the medium every day to form a monolayer filled with 80%. formed Thereafter, the Caco-2 cell monolayer was washed three times with PBS and exchanged with antibiotic-free DMEM medium, and the test strain was inoculated to a final concentration of 1×10 8 CFU/mL and inoculated at 37° C. in a 5% CO 2 environment. Incubated for 2 hr. Thereafter, the monolayer was washed three times with PBS to remove non-adhered test strains, and 200 μl of 1% Triton-X 100 (Sigma-Aldrich) was added to break up the test strain-attached monolayer. Thereafter, 1 ml of the lysate was applied to the MRS agar medium using the serial dilution method, and then incubated at 37° C. for 48 hr, and the number of bacteria was confirmed. The adhesion ability of the test strain was evaluated by calculating the colony forming units (CFU) of the attached test strain. The following formula was used for the percent attachment of the test strain to the Caco-2 cell line.
도 2는 본 발명의 실시예에서 선발된 4종의 균주(Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, Lactobacillus gasseri PA-3)에 대해 Caco-2 세포주를 이용하여 장내 부착능 평가 실험을 진행한 결과이다. 도 2에서 보이는 바와 같이 Caco-2 세포주를 이용하여 시험 균주의 세포부착능을 확인한 결과 Lactobacillus brevis MJM60390과 Lactobacillus gasseri PA-3이 Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396보다 상대적으로 높은 수치를 나타내었다.2 shows four strains selected in the examples of the present invention ( Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, Lactobacillus gasseri PA-3) using Caco-2 cell line to evaluate intestinal adhesion ability. is a result As shown in Figure 2, as a result of confirming the cell adhesion ability of the test strains using the Caco-2 cell line , Lactobacillus brevis MJM60390 and Lactobacillus gasseri PA-3 showed relatively higher values than Lactobacillus fermentum MJM60393 and Lactobacillus paracasei MJM60396.
(5) 생체 유래 아민(Biogenic amine) 생성 평가(5) Assessment of biogenic amine generation
L-tyrosine, L-histidine, L-ornithine, L-phenylalanine, L-lysine과 같은 아미노산을 각각 첨가한 decarboxylase 배지(Tryptone : 0.5 wt%; yeast extract : 0.5 wt%; beef extract : 0.5 wt%; sodium chloride : 0.25 wt%; glucose : 0.05 wt%; tween 80 : 0.1 wt%, MgSO4 : 0.02 wt%; MnSO4 : 0.005 wt%; FeSO4 : 0.004 wt%; Ammonium citrate : 0.2 wt%; Thiamine : 0.001 wt%; dipotassium phosphate : 0.2 wt%; calcium carbonate : 0.01 wt%; pyridoxal 5-phosphate : 0.005 wt%; Amino acid(one of the amino acid listed above) : 1 wt%; bromocresol purple : 0.006 wt%; agar 2 wt%; pH : 5.3)에 시험 균주를 접종하고 37℃에서 16~24 hr 동안 배양한 후, Bover-Cid와 Holzapfel의 방법(Bover-Cid S, Hozapfel WH. Improved screening procedure for biogenic amine production by lactic acid bacteria. International Journal of Food Microbiology, 1999)에 따라 생체 유래 아민 생성 여부를 확인하였다.Decarboxylase medium (Tryptone: 0.5 wt%; yeast extract: 0.5 wt%; beef extract: 0.5 wt%; sodium chloride: 0.25 wt%; glucose: 0.05 wt%; tween 80: 0.1 wt%; MgSO 4 : 0.02 wt%; MnSO 4 : 0.005 wt%; FeSO4: 0.004 wt%; Ammonium citrate: 0.2 wt%; Thiamine: 0.001 wt% %; dipotassium phosphate : 0.2 wt%; calcium carbonate : 0.01 wt%; pyridoxal 5-phosphate : 0.005 wt%; Amino acid (one of the amino acids listed above) : 1 wt%; bromocresol purple : 0.006 wt%;
하기 표 3에 선발된 4종의 균주(Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, Lactobacillus gasseri PA-3)에 대한 생체 유래 아민 생성 평가를 요약하였다.Table 3 summarizes the evaluation of bio-derived amine production for the four selected strains ( Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, and Lactobacillus gasseri PA-3).
(아미노산)biogenic amines
(amino acid)
상기 표 3에서 보이는 바와 같이 선발된 4종의 균주는 모두 L-tyrosine, L-histidine, L-ornithine, L-phenylalanine, L-lysine을 첨가한 배지에서 생체 유래 아민을 생성하지 않은 것으로 확인되었다.As shown in Table 3, it was confirmed that all of the four selected strains did not produce bio-derived amine in a medium supplemented with L-tyrosine, L-histidine, L-ornithine, L-phenylalanine, and L-lysine.
3. 선발된 균주의 세포증식 실험3. Cell proliferation experiment of selected strains
DM 배지에 AMP(adenine mono phosphate), Adenosine, Adenine을 각각 400 μM 농도로 첨가하여 세포증식 실험용 배지를 준비하였다. 또한, 퓨린, 뉴클레오사이드 및 뉴클레오타이드가 첨가되지 않은 기본 DM 배지를 대조군(control)으로 사용하였다. 이후, 세포증식 실험용 배지에 시험 균주를 2×108 CFU/㎖의 양으로 접종하고 37℃에서 혐기 조건을 유지하면서 배양하였다. 배양 시간에 따른 시험 균주의 증식 여부를 스펙트로포토미터를 이용하여 OD 650 nm에서 측정하였다.A medium for cell proliferation experiments was prepared by adding AMP (adenine mono phosphate), Adenosine, and Adenine at a concentration of 400 μM, respectively, to the DM medium. In addition, a basic DM medium to which purines, nucleosides and nucleotides were not added was used as a control. Thereafter, the test strain was inoculated in an amount of 2×10 8 CFU/ml in a medium for cell proliferation experiments, and cultured while maintaining anaerobic conditions at 37°C. Proliferation of the test strain according to the incubation time was measured at OD 650 nm using a spectrophotometer.
도 3은 본 발명의 실시예에서 선발된 4종의 균주(Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, Lactobacillus gasseri PA-3)를 퓨린 또는 뉴클레오사이드가 존재하는 배지에서 배양하였을 때의 증식 특성을 나타낸 그래프이다. 도 3에서 보이는 바와 같이Figure 3 shows the growth of the four strains ( Lactobacillus brevis MJM60390, Lactobacillus fermentum MJM60393, Lactobacillus paracasei MJM60396, Lactobacillus gasseri PA-3) selected in Examples of the present invention when cultured in a medium containing purines or nucleosides. It is a graph showing the characteristics. As shown in Figure 3
Lactobacillus brevis MJM60390 균주와 Lactobacillus gasseri PA-3 균주는 기본 DM 배지보다 AMP, Adenosine 또는 Adenine이 첨가된 배지에서 균의 성장이 증가하였다.The growth of Lactobacillus brevis MJM60390 strain and Lactobacillus gasseri PA-3 strain was increased in the medium supplemented with AMP, Adenosine or Adenine than in the basic DM medium.
4. 고요산혈증 동물모델을 이용한 선발된 균주의 고요산혈증 개선 효능 평가(In-vivo test)4. Evaluation of hyperuricemia improvement efficacy of selected strains using hyperuricemia animal model (In-vivo test)
(1) 실험 방법(1) Experiment method
7주령 된 C57BL/6 수컷 마우스(공급사 : 라온바이오)에 물과 사료를 자유롭게 공급하고 22±2℃, 습도 55±5% 및 명암 주기(light-dark cycle) 12 hr 단위의 사육실 환경하에서 1주일 동안 적응시켰다. 1주일 동안의 적응기를 거친 마우스들을 정상군, 음성대조군, Allopurinol 처리군, Lactobacillus gasseri PA-3 처리군, Lactobacillus brevis MJM60390 처리군과 같이 총 5개의 군(n=8)으로 나누고, 정상군을 제외한 나머지 군의 마우스들에게 고퓨린함량 식이를 14일 동안 제공하여 고요산혈증을 유도하였다. 기본적으로 마우스 같은 설치류들은 요산을 분해할 수 있는 유리케이즈(uricase)라는 효소를 만들어 혈액 내에 요산 수치를 감소시킨다. 이를 막기 위해 음성대조군, Allopurinol 처리군, Lactobacillus gasseri PA-3 처리군, Lactobacillus brevis MJM60390 처리군에 potassium oxonate 용액을 potassium oxonate 기준 300 mg/kg B.W. 용량으로 14일 동안 매일 1회 복강주사 투여하였다. 상기 고퓨린함량 식이는 기본식이(제품명 : AIN-93M; 공급사 : 라온바이오)에 효소 추출물을 첨가한 것으로서 고퓨린함량 식이 1 ㎏ 기준으로 효소 추출물 함량은 570 g이다. 또한, 정상군에게는 고퓨린함량 식이 대신 기본 식이를 14일 동안 제공하였다. 또한, Allopurinol 처리군에는 Allopurinol을 비히클(vehicle)인 식염수에 용해시켜 제조한 약물 시료를 Allopurinol 기준 5 mg/kg B.W. 용량으로 14일 동안 매일 1회 경구투여하였다. 또한, Lactobacillus gasseri PA-3 처리군에는 Lactobacillus gasseri PA-3 균체를 식염수에 현탁하여 제조한 약물 시료를 3×109 CFU/mouse 용량으로 14일 동안 매일 1회 경구투여하였다. 또한, Lactobacillus brevis MJM60390 처리군에는 Lactobacillus brevis MJM60390 균체를 식염수에 현탁하여 제조한 약물 시료를 3×109 CFU/mouse 용량으로 14일 동안 매일 1회 경구투여하였다. 한편, 정상군 및 음성대조군에는 약물 시료 대신 비히클(vehicle)로 사용한 식염수를 경구투여 하였다.7-week-old C57BL/6 male mice (Supplier: Raon Bio) were freely supplied with water and feed, and maintained at 22 ± 2 ° C, humidity 55 ± 5%, and a light-dark cycle of 12 hr in a breeding room environment for 1 week. adapted during Mice that had undergone a 1-week adaptation period were divided into 5 groups (n=8): normal group, negative control group, Allopurinol treatment group, Lactobacillus gasseri PA-3 treatment group, and Lactobacillus brevis MJM60390 treatment group. Hyperuricemia was induced by feeding the mice of the remaining group with a high purine content diet for 14 days. Basically, rodents like mice make an enzyme called uricase that can break down uric acid, reducing the level of uric acid in the blood. To prevent this, a potassium oxonate solution was administered intraperitoneally once daily for 14 days at a dose of 300 mg/kg BW based on potassium oxonate to the negative control group, Allopurinol treatment group, Lactobacillus gasseri PA-3 treatment group, and Lactobacillus brevis MJM60390 treatment group. The high purine content diet is a basic diet (product name: AIN-93M; supplier: Raon Bio) with enzyme extract added, and the enzyme extract content is 570 g based on 1 kg of the high purine content diet. In addition, the normal group was provided with a basic diet instead of a high purine content diet for 14 days. In addition, to the Allopurinol-treated group, a drug sample prepared by dissolving Allopurinol in saline as a vehicle was orally administered once daily for 14 days at a dose of 5 mg/kg BW based on Allopurinol. In addition, to the group treated with Lactobacillus gasseri PA-3, a drug sample prepared by suspending Lactobacillus gasseri PA-3 cells in saline was orally administered once daily for 14 days at a dose of 3×10 9 CFU/mouse. In addition, to the Lactobacillus brevis MJM60390 treatment group, a drug sample prepared by suspending Lactobacillus brevis MJM60390 cells in saline was orally administered once daily for 14 days at a dose of 3×10 9 CFU/mouse. On the other hand, saline used as a vehicle was orally administered to the normal group and the negative control group instead of the drug sample.
(2) 체중 변화 분석(2) Weight change analysis
고퓨린함량 식이가 마우스의 체중에도 영향을 미치는지 알아보기 위해 마우스의 체중을 실험군별로 측정하였다. 도 4는 본 발명의 실시예에서 고요산혈증 동물모델을 이용하여 선발된 2종의 균주(Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3)의 고요산혈증 개선 효능 평가 실험(In-vivo test)을 진행하였을 때 실험군별 마우스 체중 변화를 나나탠 것이다. 도 4에서 "ND"는 정상군을 나타내고, "(-)con"은 음성대조군을 나타내고, "Allopurinol"은 Allopurinol 처리군을 나타내고, "PA-3"은 Lactobacillus gasseri PA-3 처리군을 나타내고, "60930"은 Lactobacillus brevis MJM60390 처리군을 나타낸다. 도 4에서 보이는 바와 같이 고요산혈증을 유도하기 위해 고퓨린함량 식이를 공급한 실험군과 그렇지 않은 실험군에서 마우스 체중 차이는 크지 않음을 확인할 수 있다.In order to determine whether a high-purine content diet also affects the body weight of mice, the body weights of the mice were measured for each experimental group. Figure 4 is a hyperuricemia improvement efficacy evaluation experiment (In-vivo test) of two strains ( Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3) selected using an animal model for hyperuricemia in an embodiment of the present invention. It shows the change in body weight of mice by experimental group. In FIG. 4, "ND" represents a normal group, "(-)con" represents a negative control group, "Allopurinol" represents an Allopurinol treatment group, "PA-3" represents a Lactobacillus gasseri PA-3 treatment group, "60930" represents the Lactobacillus brevis MJM60390 treatment group. As shown in Figure 4, it can be seen that there is no significant difference in body weight between the experimental group supplied with the high purine content diet and the experimental group without it in order to induce hyperuricemia.
(3) 혈장 분석(3) Plasma analysis
0일차 및 약물 시료를 마지막으로 투여한 다음날인 15일차에 마우스를 이소플로렌으로 마취시킨 후 심장에서 300 ㎕(0일차) 및 700 ㎕(15일차)을 채혈하였다. 채혈된 샘플을 원심분리하여 혈장을 수득하고 수득한 혈장을 -80℃에서 보관하면서 이후의 분석에 사용하였다. 혈액분석기(FUJIFILM DRI-CHEM NX500i)를 이용하여 요산, 크레아티닌 및 혈액요소질소(BUN)의 함량을 측정하였다. 또한, 잔틴산화효소 활성 분석 키트(MAK078-1 KIT; Sigma-Aldrich)를 이용하여 잔틴산화효소(xanthine oxidase, XO) 수준을 분석하였다. 또한, SLC22A12 elisa kit(MyBioSource)를 사용하여 혈액에 있는 URAT1(urate transporter 1) 단백질의 양을 측정하였다.On
도 5는 본 발명의 실시예에서 고요산혈증 동물모델을 이용하여 선발된 2종의 균주(Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3)의 고요산혈증 개선 효능 평가 실험(In-vivo test)을 진행하였을 때 0일차 및 15일차 실험군별 혈중 요산 함량을 분석한 결과이다. 도 5에서 보이는 바와 같이 Lactobacillus brevis MJM60390 처리군과 Lactobacillus gasseri PA-3 처리군에서 요산 수치가 현저하게 감소하는 것을 확인할 수 있었고 Lactobacillus brevis MJM60390 처리군에서 요산 수치 감소가 더 현저하였다.Figure 5 is a hyperuricemia improvement efficacy evaluation experiment (In-vivo test) of two strains ( Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3) selected using an animal model for hyperuricemia in an embodiment of the present invention. These are the results of analyzing the blood uric acid content for each experimental group on
도 6은 본 발명의 실시예에서 고요산혈증 동물모델을 이용하여 선발된 2종의 균주(Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3)의 고요산혈증 개선 효능 평가 실험(In-vivo test)을 진행하였을 때 15일차 실험군별 혈중 크레아티닌 함량 및 혈액요소질소(BUN)의 함량을 분석한 결과이다. 혈중 크레아티닌(Creatinine)과 혈액요소질소(BUN)는 신장 독성을 확인하는 표적물질로서 그 수치가 높을 경우 신장에 독성을 일으킬 수 있다. 도 6에서 보이는 바와 같이 Lactobacillus brevis MJM60390 처리군과 Lactobacillus gasseri PA-3 처리군은 모두 정상군과 비슷하거나 낮은 혈중 크레아티닌(Creatinine)과 혈액요소질소(BUN) 수치를 보여 상기 유산균들이 체내에 미치는 신장 독성은 없는 것으로 확인되었다.Figure 6 is a hyperuricemia improvement efficacy evaluation experiment (In-vivo test) of two strains ( Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3) selected using an animal model for hyperuricemia in an embodiment of the present invention. These are the results of analyzing the blood creatinine content and blood urea nitrogen (BUN) content for each experimental group on the 15th day. Blood creatinine (Creatinine) and blood urea nitrogen (BUN) are target substances that confirm kidney toxicity, and high levels can cause toxicity to the kidney. As shown in Figure 6, both the Lactobacillus brevis MJM60390 treatment group and the Lactobacillus gasseri PA-3 treatment group showed blood creatinine and blood urea nitrogen (BUN) levels similar to or lower than those of the normal group, showing renal toxicity of the lactic acid bacteria in the body. was confirmed to be absent.
도 7은 본 발명의 실시예에서 고요산혈증 동물모델을 이용하여 선발된 2종의 균주(Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3)의 고요산혈증 개선 효능 평가 실험(In-vivo test)을 진행하였을 때 15일차 실험군별 혈중 잔틴산화효소(xanthine oxidase, XO) 활성을 분석한 결과이다. 잔틴산화효소(xanthine oxidase, XO)는 Hypoxantine을 xanthine으로, xanthine을 uric acid로 바꾸어 주는 효소로서 혈중 XO 활성이 높은 경우 요산의 양이 증가하게 된다. 도 7에서 보이는 바와 같이 Lactobacillus brevis MJM60390 처리군과 Lactobacillus gasseri PA-3 처리군 모두에서 정상군보다 혈중 XO 활성이 낮게 나타났다. 특히, Lactobacillus brevis MJM60390 처리군에서 Lactobacillus gasseri PA-3 처리군보다 혈중 XO 활성이 1 U/L이상 낮은 것을 확인할 수 있다. Figure 7 is when the hyperuricemia improvement efficacy evaluation experiment (In-vivo test) of two strains ( Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3) selected using an animal model for hyperuricemia in an embodiment of the present invention was conducted This is the result of analyzing the activity of xanthine oxidase (XO) in the blood of each experimental group on the 15th day. Xanthine oxidase (XO) is an enzyme that converts hypoxantine to xanthine and xanthine to uric acid. When the activity of XO in the blood is high, the amount of uric acid increases. As shown in FIG. 7, both the Lactobacillus brevis MJM60390-treated group and the Lactobacillus gasseri PA-3-treated group showed lower blood XO activity than the normal group. In particular, it can be confirmed that the Lactobacillus brevis MJM60390-treated group had a lower blood XO activity by 1 U/L or more than the Lactobacillus gasseri PA-3-treated group.
도 8은 본 발명의 실시예에서 고요산혈증 동물모델을 이용하여 선발된 2종의 균주(Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3)의 고요산혈증 개선 효능 평가 실험(In-vivo test)을 진행하였을 때 15일차 실험군별 혈중 URAT1 단백질 함량을 분석한 결과이다. URAT1 단백질은 신장에서 요산의 재흡수를 돕는 단백질이다. 도 8에서 보이는 바와 같이 Lactobacillus brevis MJM60390 처리군(3.26 ng/ml)과 Lactobacillus gasseri PA-3 처리군(3.31 ng/ml) 모두에서 정상군 및 Allopurinol 처리군에 비해 URAT1 단백질 발현이 감소되었다. Lactobacillus brevis MJM60390 균주 및 Lactobacillus gasseri PA-3 균주는 모두 요산 생성을 억제하고 요산의 배출을 원활하게 하는 것으로 확인되었고, Lactobacillus brevis MJM60390 균주가 Lactobacillus gasseri PA-3 균주에 비해 좀더 나은 효과를 가지는 것으로 확인되었다.8 is when an experiment (in-vivo test) was conducted to evaluate the hyperuricemia improvement efficacy of two strains ( Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3) selected using an animal model for hyperuricemia in an embodiment of the present invention. This is the result of analyzing the URAT1 protein content in the blood of each experimental group on the 15th day. URAT1 protein is a protein that helps reabsorption of uric acid in the kidney. As shown in FIG. 8, URAT1 protein expression was decreased in both the Lactobacillus brevis MJM60390-treated group (3.26 ng/ml) and the Lactobacillus gasseri PA-3-treated group (3.31 ng/ml) compared to the normal group and the Allopurinol-treated group. Both the Lactobacillus brevis MJM60390 strain and the Lactobacillus gasseri PA-3 strain were confirmed to inhibit uric acid production and facilitate the excretion of uric acid, and the Lactobacillus brevis MJM60390 strain was confirmed to have a better effect than the Lactobacillus gasseri PA-3 strain. .
(4) 대변 분석(4) stool analysis
약물 시료를 마지막으로 투여한 다음날인 15일차에 채혈을 마치고 난 후 경추탈골로 마우스를 희생시키고 장을 분리한 후 장내에 있는 대변을 튜브에 모은 후 -80℃에서 보관하면서 이후의 분석에 사용하였다.After blood collection was completed on the 15th day, the day after the last drug sample was administered, the mouse was sacrificed by cervical dislocation, the intestine was separated, and the feces in the intestine were collected in a tube and stored at -80 ° C for subsequent analysis. .
대변에 함유된 단쇄지방산(Short Chain Fatty Acid, SCFA)의 종류 및 함량을 분석하기 위해 대변 샘플에서 단쇄지방산을 추출하고, GC-MS 분석을 통해 Acetic acid, Propionic acid, butyric acid 등의 양을 측정하였다. 또한, 장내 미생물 군집을 분석하기 위해 Exgen Stool DNA Mini Kit(GeneAll)를 이용하여 대변 샘플에서 DNA를 추출하고 16S rRNA 유전자의 V3-V4 영역을 증폭시킨 후 16S-기반 Microbial Taxonomic Profiling platform인 EzBioCloud Apps(ChunLab Inc., Republic of Korea)를 이용하여 Microbiome profiling을 수행하였다.In order to analyze the type and content of short-chain fatty acids (SCFA) contained in feces, short-chain fatty acids are extracted from stool samples and the amounts of acetic acid, propionic acid, butyric acid, etc. are measured through GC-MS analysis. did In addition, to analyze the intestinal microbial community, DNA was extracted from feces samples using the Exgen Stool DNA Mini Kit (GeneAll), and the V3-V4 region of the 16S rRNA gene was amplified, followed by EzBioCloud Apps (a 16S-based Microbial Taxonomic Profiling platform). Microbiome profiling was performed using ChunLab Inc., Republic of Korea).
도 9는 본 발명의 실시예에서 고요산혈증 동물모델을 이용하여 선발된 2종의 균주(Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3)의 고요산혈증 개선 효능 평가 실험(In-vivo test)을 진행하였을 때 15일차 실험군별 대변 내 단쇄지방산(SCFA) 함량을 측정한 결과이다. Acetic acid, Propionic acid, butyric acid, Isovaleric acid, valeric acid 등과 같은 단쇄지방산(SCFA)은 포스트바이오틱스로 크게 주목받고 있다. 도 9에서 보이는 바와 같이 Lactobacillus brevis MJM60390 처리군에서 Lactobacillus gasseri PA-3 처리군보다 더 많은 단쇄지방산(SCFA)이 검출되었다.Figure 9 is a hyperuricemia improvement efficacy evaluation experiment (In-vivo test) of two strains ( Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3) selected using an animal model for hyperuricemia in an embodiment of the present invention. This is the result of measuring the content of short-chain fatty acids (SCFA) in the feces of each experimental group on the 15th day. Short-chain fatty acids (SCFA), such as acetic acid, propionic acid, butyric acid, isovaleric acid, and valeric acid, are attracting great attention as postbiotics. As shown in FIG. 9, more short-chain fatty acids (SCFA) were detected in the Lactobacillus brevis MJM60390-treated group than in the Lactobacillus gasseri PA-3-treated group.
도 10은 본 발명의 실시예에서 고요산혈증 동물모델을 이용하여 선발된 2종의 균주(Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3)의 고요산혈증 개선 효능 평가 실험(In-vivo test)을 진행하였을 때 15일차 실험군별 장내 미생물 군집을 분석한 결과이다. 도 10에서 보이는 바와 같이 Lactobacillus brevis MJM60390 처리군에서 장내 미생물 중 Rikenellaceae의 비중이 다른 실험군에 비해 10% 이상 높게 나왔다. Rikenellaceae은 Bacteroidetes에 속한 균주중 Butyrate를 생산하는 소수의 균주 중 하나로 다른 실험군들보다 Laactobacillus brevis MJM60390 처리군에서 butyrate 수치가 높은 이유는 장내 미생물 군집에서 Rikenellaceae의 비중이 큰 것에서 기인하는 것으로 판단된다.Figure 10 is when the hyperuricemia improvement efficacy evaluation experiment (In-vivo test) of two strains ( Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3) selected using an animal model for hyperuricemia in an embodiment of the present invention was conducted This is the result of analyzing the intestinal microbial community for each experimental group on the 15th day. As shown in FIG. 10, the ratio of Rikenellaceae among intestinal microorganisms in the Lactobacillus brevis MJM60390 treatment group was higher than that of other experimental groups by 10% or more. Rikenellaceae is one of the few strains that produce butyrate among strains belonging to Bacteroidetes .
(5) 신장 분석(5) kidney analysis
약물 시료를 마지막으로 투여한 다음날인 15일차에 채혈을 마치고 난 후 경추탈골로 마우스를 희생시키고 장을 분리한 후 바로 신장을 적출하였다. 적출한 신장을 세척하고 4% 파라포름알데히드 용액에 고정시키고 이후 paraffin 포매 및 Hematoxylin & eosin(H&E) 염색한 후 조직을 관찰하였다. 또한, 신장 사구체(glomerulus) 형태 변화와 비대 수준을 정량화하기 위해 신장 사구체 이미지를 20개의 이미지로 분할하고 CaseViewer(3DHISTECH)의 draw closed polygon tool을 이용하여 면적을 계산하였다.After blood collection was completed on the 15th day, the day after the last drug sample was administered, the mice were sacrificed by cervical dislocation, the intestines were separated, and the kidneys were removed immediately. The extracted kidney was washed, fixed in 4% paraformaldehyde solution, paraffin embedded, and stained with Hematoxylin & eosin (H&E), and the tissue was observed. In addition, to quantify the change in the shape of the glomerulus and the level of hypertrophy, the image of the glomerulus was divided into 20 images and the area was calculated using the draw closed polygon tool of CaseViewer (3DHISTECH).
도 11은 본 발명의 실시예에서 고요산혈증 동물모델을 이용하여 선발된 2종의 균주(Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3)의 고요산혈증 개선 효능 평가 실험(In-vivo test)을 진행하였을 때 15일차 실험군별 적출한 신장의 조직을 Hematoxylin & eosin(H&E) 염색한 후 관찰한 사진이다. 도 11에서 보이는 바와 같이 고요산혈증이 유도된 음성대조군에서는 신장 사구체의 모양이 찌글어지고 경계가 불규칙적으로 변하는 등 신장 손상이 발생하였다. 반면, Lactobacillus brevis MJM60390 처리군은 정상군과 유사한 형태의 신장 이미지를 보였다.11 is when an experiment (in-vivo test) was conducted to evaluate the hyperuricemia improvement efficacy of two strains ( Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3) selected using an animal model for hyperuricemia in an embodiment of the present invention. This is a photograph observed after hematoxylin & eosin (H&E) staining of kidney tissues extracted from each experimental group on the 15th day. As shown in FIG. 11, in the negative control group in which hyperuricemia was induced, renal damage occurred such that the shape of the kidney glomerulus was distorted and the border was irregularly changed. On the other hand, the Lactobacillus brevis MJM60390-treated group showed kidney images similar to those of the normal group.
하기 표 4에 본 발명의 실시예에서 고요산혈증 동물모델을 이용하여 선발된 2종의 균주(Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3)의 고요산혈증 개선 효능 평가 실험(In-vivo test)을 진행하였을 때 15일차 실험군별 적출한 신장 사구체 다발 면적을 정리하였다.In Table 4 below, the hyperuricemia improvement efficacy evaluation experiment (In-vivo test) of the two strains ( Lactobacillus brevis MJM60390, Lactobacillus gasseri PA-3) selected using the hyperuricemia animal model in the examples of the present invention was conducted. At the 15th day, the area of the renal glomerular bundle excised for each experimental group was summarized.
* ND : 정상군* ND: normal group
* (-)con : 음성대조군* (-)con: negative control group
* Allopurinol : Allopurinol 처리군* Allopurinol: Allopurinol treatment group
* PA-3 : Lactobacillus gasseri PA-3 처리군* PA-3 : Lactobacillus gasseri PA-3 treatment group
* 60390 : Lactobacillus brevis MJM60390 처리군* 60390 : Lactobacillus brevis MJM60390 treatment group
상기 표 4에서 보이는 바와 같이 Lactobacillus brevis MJM60390 처리군에서 신장 사구체 다발 면적은 정상군과 거의 같은 반면, 나머지 군에서는 신장 사구체 다발 면적이 정상군보다 높게 나타났고 이러한 결과는 나머지 군에서 고요산혈증 유도로 인해 신장 조직이 크게 손상되었음을 의미한다. 결론적으로 Lactobacillus brevis MJM60390은 마우스에 고요산혈증을 유도할 때 신장의 형태학적 변화를 최소화하고 신장의 기능을 정상적으로 유지시키는 효과를 가진다고 볼 수 있다.As shown in Table 4, the renal glomerular bundle area in the Lactobacillus brevis MJM60390-treated group was almost the same as that of the normal group, whereas in the other groups, the renal glomerular bundle area was higher than that of the normal group. This means that the kidney tissue has been severely damaged. In conclusion, when Lactobacillus brevis MJM60390 induces hyperuricemia in mice, it can be seen that it has the effect of minimizing the morphological changes of the kidney and maintaining normal kidney function.
5. 5. Lactobacillus brevisLactobacillus brevis MJM60390 균주의 계통관계도(phylogenetic tree) 및 기탁 정보 Phylogenetic tree and deposit information of strain MJM60390
Lactobacillus brevis MJM60390 균주의 16S rDNA 서열을 분석하여 공시균과 상동성을 비교하고 계통관계도를 완성하였다.The 16S rDNA sequence of the Lactobacillus brevis MJM60390 strain was analyzed to compare the homology with the strain to be tested and a phylogenetic relationship diagram was completed.
먼저, 유전체 DNA 분리 키트(genomic DNA isolation kit; GeneALL, Seoul, Republic of Korea)를 이용하여 제조자의 사용설명서에 따라 균주의 유전체 DNA를 분리하였다. 이후, 균주의 16S rDNA을 증폭시키기 위해 순방향 프라이머인 27F((5'-AGAGTTTGATCCTGGCTCAG-3')와 역방향 프라이머인 1492R(5'-GGTTACCT TGTTACGACTT-3')를 이용하여 PCR을 수행하였다. 이후, 염기서열 분석기를 이용하여 16S rDNA 염기서열을 분석하였다. 그 결과, Lactobacillus brevis MJM60390 균주의 16S rDNA는 서열번호 1의 염기서열을 갖는 것으로 나타났다. 또한, Lactobacillus brevis MJM60390 균주의 16S rDNA 염기서열을 Genebank (http://www.ncbi.nlm.nih.gov/)의 BLAST 검색으로 동정한 결과, 100% 동일한 16S rDNA 염기서열을 갖는 락토바실러스 브레비스(Lactobacillus brevis) 공시균은 검색되지 않았다. First, genomic DNA of the strain was isolated using a genomic DNA isolation kit (GeneALL, Seoul, Republic of Korea) according to the manufacturer's instructions. Subsequently, PCR was performed using forward primer 27F ((5'-AGAGTTTGATCCTGGCTCAG-3') and reverse primer 1492R (5'-GGTTACCT TGTTACGACTT-3') to amplify the 16S rDNA of the strain. Then, the base The 16S rDNA base sequence was analyzed using a sequence analyzer. As a result, it was found that the 16S rDNA of Lactobacillus brevis MJM60390 strain had the base sequence of SEQ ID NO: 1. In addition, the 16S rDNA base sequence of Lactobacillus brevis MJM60390 strain was obtained from Genebank (http As a result of identification by BLAST search of ://www.ncbi.nlm.nih.gov/), Lactobacillus brevis having a 100% identical 16S rDNA sequence was not found.
이후, Lactobacillus brevis MJM60390 균주의 16S rDNA 염기서열을 BLAST 프로그램을 이용하여 EzTaxon 비교하고, MEGA 6 소프트웨어(Tamura et al. 2013)를 이용하여 Lactobacillus brevis MJM60390 균주의 계통관계도(phylogenetic tree)를 완성하였다. 도 12는 본 발명의 실시예에서 스크리닝한 Lactobacillus brevis MJM60390 균주의 계통관계도(phylogenetic tree)를 나타낸 것이다.Then, the 16S rDNA sequence of the Lactobacillus brevis MJM60390 strain was compared with EzTaxon using the BLAST program, and the phylogenetic tree of the Lactobacillus brevis MJM60390 strain was completed using
또한, 본 발명의 발명자들은 Lactobacillus brevis MJM60390 균주를 2021년 11월 05일에 공인기탁기관인 국립농업과학원 농업미생물은행(Korean Agricultural Culture Collection, KACC; 주소 : 대한민국 전라북도 완주군 이서면 농생명로 166)에 부다페스트 조약에 의거한 국제특허기탁을 진행하여 수탁번호 KACC 81193BP를 부여받았다.In addition, the inventors of the present invention Lactobacillus brevis MJM60390 strain on November 05, 2021, the National Institute of Agricultural Science Agricultural Microbiology Bank (Korean Agricultural Culture Collection, KACC; Address: 166 Agricultural Life-ro, Iseo-myeon, Wanju-gun, Jeollabuk-do, Korea), which is an authorized depository institution, according to the Budapest Treaty Based on the international patent deposit, the accession number KACC 81193BP was granted.
이상에서와 같이 본 발명을 상기의 실시예를 통해 설명하였지만 본 발명이 반드시 여기에만 한정되는 것은 아니며 본 발명의 범주와 사상을 벗어나지 않는 범위 내에서 다양한 변형실시가 가능함은 물론이다. 따라서, 본 발명의 보호범위는 본 발명에 첨부된 특허청구의 범위에 속하는 모든 실시 형태를 포함하는 것으로 해석되어야 한다.As described above, the present invention has been described through the above embodiments, but the present invention is not necessarily limited thereto, and various modifications are possible without departing from the scope and spirit of the present invention. Accordingly, the scope of protection of the present invention should be construed to include all embodiments falling within the scope of the claims appended hereto.
<110> Myongji University Industry and Academia Cooperation Foundation <120> Novel strain with serum uric acid level reducing effect and renal protective effect, and use of the same <130> ZDP-21-0347 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1508 <212> DNA <213> Unknown <220> <223> 16S rDNA of Lactobacillus brevis MJM60390 <400> 1 aggctcagga cgaacgctgg cggcatgcct aatacatgca agtcgaacga gcttccgttg 60 aatgacgtgc ttgcactgat ttcaacaatg aagcgagtgg cgaactggtg agtaacacgt 120 gggaaatctg cccagaagca ggggataaca cttggaaaca ggtgctaata ccgtataaca 180 acaaaatccg catggatttt gtttgaaagg tggcttcggc tatcacttct ggatgatccc 240 gcggcgtatt agttagttgg tgaggtaaag gcccaccaag acgatgatac gtagccgacc 300 tgagagggta atcggccaca ttgggactga gacacggccc aaactcctac gggaggcagc 360 agtagggaat cttccacaat ggacgaaagt ctgatggagc aatgccgcgt gagtgaagaa 420 gggtttcggc tcgtaaaact ctgttgttaa agaagaacac ctttgagagt aactgttcaa 480 gggttgacgg tatttaacca gaaagccacg gctaactacg tgccagcagc cgcggtaata 540 cgtaggtggc aagcgttgtc cggatttatt gggcgtaaag cgagcgcagg cggtttttta 600 agtctgatgt gaaagccttc ggcttaaccg gagaagtgca tcggaaactg ggagacttga 660 gtgcagaaga ggacagtgga actccatgtg tagcggtgga atgcgtagat atatggaaga 720 acaccagtgg cgaaggcggc tgtctagtct gtaactgacg ctgaggctcg aaagcatggg 780 tagcgaacag gattagatac cctggtagtc catgccgtaa acgatgagtg ctaagtgttg 840 gagggtttcc gcccttcagt gctgcagcta acgcattaag cactccgcct ggggagtacg 900 accgcaaggt tgaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg 960 tttaattcga agctacgcga agaaccttac caggtcttga catcttctgc caatcttaga 1020 gataagacgt tcccttcggg gacagaatga caggtggtgc atggttgtcg tcagctcgtg 1080 tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttattatcag ttgccagcat 1140 tcagttgggc actctggtga gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc 1200 aaatcatcat gccccttatg acctgggcta cacacgtgct acaatggacg gtacaacgag 1260 ttgcgaagtc gtgaggctaa gctaatctct taaagccgtt ctcagttcgg attgtaggct 1320 gcaactcgcc tacatgaagt tggaatcgct agtaatcgcg gatcagcatg ccgcggtgaa 1380 tacgttcccg ggccttgtac acaccgcccg tcacaccatg agagtttgta acacccaaag 1440 ccggtgagat aaccttcggg agtcagccgt ctaaggtggg acagatgatt agggtgaagt 1500 cgtaacaa 1508 <110> Myongji University Industry and Academia Cooperation Foundation <120> Novel strain with serum uric acid level reducing effect and renal protective effect, and use of the same <130> ZDP-21-0347 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1508 <212> DNA <213> unknown <220> <223> 16S rDNA of Lactobacillus brevis MJM60390 <400> 1 aggctcagga cgaacgctgg cggcatgcct aatacatgca agtcgaacga gcttccgttg 60 aatgacgtgc ttgcactgat ttcaacaatg aagcgagtgg cgaactggtg agtaacacgt 120 gggaaatctg cccagaagca ggggataaca cttggaaaca ggtgctaata ccgtataaca 180 acaaaatccg catggatttt gtttgaaagg tggcttcggc tatcacttct ggatgatccc 240 gcggcgtatt agttagttgg tgaggtaaag gcccaccaag acgatgatac gtagccgacc 300 tgagagggta atcggccaca ttgggactga gacacggccc aaactcctac gggaggcagc 360 agtagggaat cttccacaat ggacgaaagt ctgatggagc aatgccgcgt gagtgaagaa 420 gggtttcggc tcgtaaaact ctgttgttaa agaagaacac ctttgagagt aactgttcaa 480 gggttgacgg tatttaacca gaaagccacg gctaactacg tgccagcagc cgcggtaata 540 cgtaggtggc aagcgttgtc cggatttatt gggcgtaaag cgagcgcagg cggtttttta 600 agtctgatgt gaaagccttc ggcttaaccg gagaagtgca tcggaaactg ggagacttga 660 gtgcagaaga ggacagtgga actccatgtg tagcggtgga atgcgtagat atatggaaga 720 acaccagtgg cgaaggcggc tgtctagtct gtaactgacg ctgaggctcg aaagcatggg 780 tagcgaacag gattagatac cctggtagtc catgccgtaa acgatgagtg ctaagtgttg 840 gagggtttcc gcccttcagt gctgcagcta acgcattaag cactccgcct gggggagtacg 900 accgcaaggt tgaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg 960 tttaattcga agctacgcga agaaccttac caggtcttga catcttctgc caatcttaga 1020 gataagacgt tcccttcggg gacagaatga caggtggtgc atggttgtcg tcagctcgtg 1080 tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttattatcag ttgccagcat 1140 tcagttgggc actctggtga gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc 1200 aaatcatcat gccccttatg acctgggcta cacacgtgct acaatggacg gtacaacgag 1260 ttgcgaagtc gtgaggctaa gctaatctct taaagccgtt ctcagttcgg attgtaggct 1320 gcaactcgcc tacatgaagt tggaatcgct agtaatcgcg gatcagcatg ccgcggtgaa 1380 tacgttcccg ggccttgtac acaccgcccg tcacaccatg agagtttgta acacccaaag 1440 ccggtgagat aaccttcggg agtcagccgt ctaaggtggg acagatgatt agggtgaagt 1500 cgtaacaa 1508
Claims (13)
Lactobacillus brevis having purine degradation activity ( Lactobacillus brevis ) strain MJM60390 (accession number: KACC 81193BP).
The Lactobacillus brevis strain MJM60390 (accession number: KACC 81193BP) according to claim 1, characterized in that it has 16S rDNA consisting of the nucleotide sequence of SEQ ID NO: 1.
According to claim 1, Lactobacillus brevis MJM60390 strain ( Accession number: KACC 81193BP).
Lactobacillus brevis ( Lactobacillus brevis ) MJM60390 strain (accession number: KACC 81193BP), a culture thereof, a lysate thereof, or a pharmaceutical composition for preventing or treating hyperuricemia comprising at least one selected from an extract thereof as an active ingredient.
Lactobacillus brevis ( Lactobacillus brevis ) MJM60390 strain (accession number: KACC 81193BP), a culture thereof, a lysate thereof, or a health functional food composition for preventing or improving hyperuricemia containing at least one selected from an extract thereof as an active ingredient.
Lactobacillus brevis ( Lactobacillus brevis ) MJM60390 strain (accession number: KACC 81193BP), a culture thereof, a lysate thereof, or a pharmaceutical composition for preventing or treating hyperuricemia-related metabolic disorders comprising at least one selected from an extract thereof as an active ingredient.
The pharmaceutical composition according to claim 6, wherein the hyperuricemia-related metabolic disorder is any one selected from gout, gouty flare-up, gouty arthritis, gouty nephrolithiasis or gouty nephropathy.
Lactobacillus brevis MJM60390 strain (accession number: KACC 81193BP), a culture thereof, a lysate thereof, or a health functional food for preventing or improving hyperuricemia-related metabolic disorders containing at least one selected from a lysate thereof or an extract thereof as an active ingredient composition.
[9] The functional food composition according to claim 8, wherein the hyperuricemia-related metabolic disorder is any one selected from gout, gouty flare-ups, gouty arthritis, gouty nephrolithiasis or gouty nephropathy.
Lactobacillus brevis ( Lactobacillus brevis ) MJM60390 strain (accession number: KACC 81193BP), a culture thereof, a lysate thereof, or a pharmaceutical composition for preventing or treating kidney damage comprising at least one selected from an extract thereof as an active ingredient.
11. The pharmaceutical composition according to claim 10, wherein the kidney damage is caused by uric acid.
Lactobacillus brevis ( Lactobacillus brevis ) MJM60390 strain (accession number: KACC 81193BP), a culture thereof, a lysate thereof, or a health functional food composition for preventing or improving kidney damage containing at least one selected from an extract thereof as an active ingredient.
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