KR101017448B1 - Colorectal health promoting composition containing Bifidobacterium longum HY8004 as an effective factor - Google Patents
Colorectal health promoting composition containing Bifidobacterium longum HY8004 as an effective factor Download PDFInfo
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- KR101017448B1 KR101017448B1 KR1020080091535A KR20080091535A KR101017448B1 KR 101017448 B1 KR101017448 B1 KR 101017448B1 KR 1020080091535 A KR1020080091535 A KR 1020080091535A KR 20080091535 A KR20080091535 A KR 20080091535A KR 101017448 B1 KR101017448 B1 KR 101017448B1
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- South Korea
- Prior art keywords
- bifidobacterium
- bifidobacterium longum
- health
- large intestine
- active ingredient
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Abstract
본 발명은 대장의 건강 증진 효능을 갖는 새로운 비피도박테리움 롱검 HY8004 및 이를 유효성분으로 함유하는 약학조성물, 발효유, 음료 및 건강기능식품에 관한 것으로서, 대장염 유발과 연관된 박테로이데스 불가투스의 억제, 전염증성 사이토카인 분비와 연관된 TLR4의 억제 및 항염증성 사이토카인인 IL-10 분비를 증가시키고, TNBS로 유도한 대장염 실험동물모델에서 대장길이 축소 억제, 미엘로퍼옥시다제(Myeloperoxidase) 활성 억제, 분변내 유해효소인 베타-글루쿠로니다아제, 베타-글루코시다아제 및 콘드로이티나아제 활성을 저해함으로써 대장내 유해균 억제 및 염증발생 억제를 통하여 대장의 건강을 증진하는 목적으로 새로운 비피도박테리움 롱검 HY8004를 유효성분으로 함유하는 약학 조성물, 발효유, 음료 및 건강기능식품으로 이용될 수 있다.The present invention relates to a novel Bifidobacterium longgum HY8004 having a health promoting effect of the large intestine, and a pharmaceutical composition, fermented milk, a beverage and a dietary supplement containing the same as an active ingredient, the inhibition of Bacteroides vulgaris associated with colitis induction, Inhibition of TLR4 associated with proinflammatory cytokine secretion and increased secretion of anti-inflammatory cytokine IL-10, inhibition of colon length reduction, inhibition of myeloperoxidase activity, intratracheal in TNBS-induced colitis experimental animal models New Bifidobacterium long gum for the purpose of improving the health of the colon by inhibiting harmful bacteria in the colon and suppressing inflammation by inhibiting the beta-glucuronidase, beta-glucosidase and chondroitinase activity It can be used as a pharmaceutical composition, fermented milk, beverage and health functional food containing HY8004 as an active ingredient.
대장염, 박테로이데스 불가투스(Bacteroides vulgatus), 비피도박테리움 롱검(Bifidobacterium longum) HY8004, 염증, TLR4, TNBS Colitis, Bacteroides vulgatus, Bifidobacterium longum HY8004, Inflammation, TLR4, TNBS
Description
본 발명은 대장의 건강 증진 효능을 갖는 새로운 비피도박테리움 롱검 HY8004 및 이를 유효성분으로 함유하는 약학조성물, 발효유, 음료 및 건강기능식품에 관한 것으로서, 보다 상세하게는 대장염 유발과 연관된 박테로이데스 불가투스의 억제, 전염증성 사이토카인 분비와 연관된 Toll Like Receptor4(이하 ‘TLR4’라 함)의 억제 및 항염증성 사이토카인 IL-10 분비를 증가시키고 트리니트로벤젠 설포닉 산(Trinitrobenzene Sulfonic Acid, 이하 ‘TNBS’라 함)으로 유도한 대장염 실험동물모델에서 대장길이 축소 억제, 미엘로퍼옥시다제(Myeloperoxidase) 활성 억제, 분변내 유해효소인 베타-글루쿠로니다아제, 베타-글루코시다아제 및 콘드로이티나아제 활성을 저해함으로써 대장내 유해균 억제 및 염증발생 억제 효능을 갖는 새로운 비피도박테리움 롱검 HY8004 및 이를 유효성분으로 함유하는 약학조성물, 발효유, 음료 및 건강기능식품에 관한 것이다.The present invention relates to a novel Bifidobacterium longgum HY8004 having a health promoting effect of the large intestine, and a pharmaceutical composition, fermented milk, a beverage and a dietary supplement containing the same as an active ingredient, and more particularly, no bacteroides associated with colitis induction. Inhibition of Tooth, inhibition of Toll Like Receptor4 (hereinafter referred to as 'TLR4') associated with pro-inflammatory cytokine secretion and increased secretion of anti-inflammatory cytokine IL-10, and Trinitrobenzene Sulfonic Acid (TNBS) Inhibition of colon length reduction, inhibition of myeloperoxidase activity, beta-glucuronidase, beta-glucosidase, and chondroitinase New Bifidobacterium longgum HY8004 with inhibitory activity against colon bacillus and inhibition of inflammation A pharmaceutical composition comprising a minute, the present invention relates to a fermented milk, beverages, and supplements.
최근에 서구형 식습관 등에 의해 대장질환이 지속적으로 늘어나고 있다. 대장항문 전문 대항병원 대장암센터는 1997년부터 2003년에 대장검사를 처음 받은 4만 764명을 분석한 결과 대장질환 이상증상이 42%로 나타났다고 밝혔다. 이중에는 대장용종이 36%로 가장 많아, 대장용종은 2001년 33%, 2002년 38.5%, 2003년에는 42.6%로 3년 전보다 약 10%의 증가했다. 그 다음으로 대장암이 2.8%, 대장염이 2.3%, 기타(만성염증, 궤양성대장염 등) 0.9% 순으로 나타났다. 이러한 대장질환이 증가하는 것은 고지방식과 지나친 육류 섭취나 인스턴트 음식 등 서구형 식생활과 유전적인 요인이 크게 작용하는 것으로 보고 되었다. 대장의 선종성 용종이 이형성의 과정을 거쳐 대장암으로 진행하는 데에는 여러 단계에서의 분자생물학적 변화가DNA에서 발생되는데 이러한 DNA의 변화는 암유전자(oncogene)의 활성화와 종양억제유전자(tumor suppressor gene) 기능의 손실이 축적되면서 대장 점막의 증식 양상을 변화시킴으로써 대장암의 발생에 기여하게 된다. 식이에 의한 발암 기전 중에서 동물성 지방은 담즙산의 분비를 증가시키는데 담즙산은 대장 상피 세포의 증식을 증가시키고 세포막의 변화를 가져오며 세포증식을 촉진하는 프로스타글란딘 E2(prostaglandin E2, PGE2)의 생성을 증가시킨다. 또한 대장 장내 세균총에서 2차 담즙산 생성을 촉진하는 혐기성 세균의 증식을 초래하는데 2차 담즙산은 발암 물질로 알려져 있다. 그리고 동물성 고단백질식도 대사과정에서 아미노산으로 분해된 후 부패미생물에 의하여 트립토판 대사산물(indole, skatole 등), 암모니아, 페놀, 아민, 니트로소(nitroso) 화합물로 변화되어 발암물질로 작용한다.Recently, colon diseases have been continuously increasing due to western eating habits. The Colorectal Cancer Center, a specialist in colon analgesics, analyzed 42,764 people who had undergone colorectal examinations for the first time in 1997-2003, and found 42% of colorectal disorders. Among them, colon polyps were the most common at 36%, and colon polyps were 33% in 2001, 38.5% in 2002, and 42.6% in 2003, an increase of about 10%. Next, colon cancer was 2.8%, colitis 2.3%, and others (chronic inflammation, ulcerative colitis, etc.) 0.9%. The increase in bowel disease has been reported to have a significant effect on Western diet and genetic factors such as high fat diet, excessive meat intake and instant food. There are several stages of molecular biological changes in DNA that cause adenomatous polyps of the colon to progress to colorectal cancer. These DNA changes result in the activation of oncogenes and tumor suppressor genes. Accumulation of loss of function alters the growth pattern of the colon mucosa and contributes to the development of colorectal cancer. Among the carcinogenic mechanisms of the diet, animal fats increase the secretion of bile acids, which increase the production of prostaglandin E2 (PGE2), which increases the proliferation of colonic epithelial cells, changes the cell membrane and promotes cell proliferation. In addition, intestinal bacterial flora causes the proliferation of anaerobic bacteria that promote the production of secondary bile acids. Secondary bile acids are known as carcinogens. In addition, animal high protein is broken down into amino acids in the esophageal metabolic process and then changed into tryptophan metabolites (indole, skatole, etc.), ammonia, phenol, amines, and nitroso compounds by rot microorganisms to act as carcinogens.
장내 세균총은 약 500여종의 박테리아로 이루어져 있으며, 회장과 대장에 많이 존재한다. 장내 세균총은 그람 양성균의 균체내독소(endotoxin)와 같은 독소와 세포독성물질 또는 발암물질을 생산하는 베타-글루쿠로니다아제(β-glucuronidase) 그리고 트립토판에이즈(tryptophanase)와 같은 유해한 효소를 생산한다. 세포독소(cytotoxin)와 균체내독소(endotoxin)는 선천성 면역반응(innate immune response)의 잠재적 자극원으로 대장 상피세포에서 전염증성 사이토카인(proinflammatory cytokines)을 분비시켜 염증성 장질환을 유발시킨다. 일반적으로 장내세균이 많은 위치에서 염증성 장질환이 주로 발생된다. 염증성 장질환은 무균동물(germ-free animals)에서는 쉽게 발생되지 않는다. 이것은 장내 세균총이 대장 염증의 발생과 지속에서 중요한 역할을 한다는 것을 의미한다. 특히 대장균(Escherichia coli), 클로스트리듐 디피실리균(Clostridium difficile), 박테로이데스 불가투스(Bacteroides vulgatus) 등이 궤양성 장질환의 발병과 관련이 있는 것으로 알려져 있다. 또한 대장에 존재하는 박테로이데스(Bacteroides)는 암모니아, 아민, 인돌 등을 생산하고 클로스트리듐(Clostridium), 유박테리움(Eubacterium)은 2차 담즙산 등의 해로운 물질을 생산하여 숙주에게 유해한 균으로 알려져 있다. 그리고 이 유해균들은 숙주가 섭취한 단백질, 지방, 배당체 등으로부터 암모니아, 황화수소, 아민, 인돌, 니트로소 아민, 페놀성 화합물 등의 유해물질을 생산하게 하는 효소를 생산하며, 발암과 관계있는 발암물질을 생산하는 베타-글루쿠로니다아제(β-glucuronidase) 그리고 트립토판에이즈(tryptophanase), 베타-글루코시다아제(β-glucosidase) 활성이 매우 높다.The intestinal flora is composed of about 500 kinds of bacteria and is present in the ileum and large intestine. The gut flora produces toxins such as Gram-positive bacteria, endotoxins, and harmful enzymes such as beta-glucuronidase and tryptophanase, which produce cytotoxic or carcinogens. . Cytotoxins and endotoxins are potential stimulators of the innate immune response and secrete proinflammatory cytokines in colonic epithelial cells, leading to inflammatory bowel disease. In general, inflammatory bowel disease occurs mainly at a large number of enterobacteriaceae. Inflammatory bowel disease does not occur easily in germ-free animals. This means that the gut flora plays an important role in the development and persistence of colorectal inflammation. In particular, Escherichia coli , Clostridium difficile , and Bacteroides vulgatus are known to be associated with the development of ulcerative bowel disease. In addition, Bacteroides in the large intestine produces ammonia, amines, indole, etc., and Clostridium and Eubacterium produce harmful substances such as secondary bile acids. Known. These harmful bacteria produce enzymes that produce harmful substances such as ammonia, hydrogen sulfide, amines, indole, nitrosoamines, and phenolic compounds from proteins, fats, and glycosides consumed by the host. The production of beta-glucuronidase (β-glucuronidase), tryptophanase, beta-glucosidase (β-glucosidase) activity is very high.
이러한 목적으로 장내 유익균인 유산간균(lactobacilli) 및 비피도박테리아(bifidobacteria)를 이용한 프로바이오틱스(Probioitcs) 제품들이 많이 개발되었다. 이러한 유산균 프로바이오틱스는 항생제 연관 설사, 여행자 설사 등의 질환에서부터 과민성 장증후군, 염증성 장질환의 치료효과가 있는 것으로 보고 되고 있다. 현재까지 알려져 있는 프로바이오틱스의 효능에 관한 기전은 다음과 같다. 투여된 프로바이오틱스가 장내에서 균락화해서 병원균의 활성과 생장, 장상피세포와의 접촉 등을 방해하는 것이다. 다음으로는 장상피세포나 점막 면역세포와의 관계를 통해서 숙주의 면역기능을 조절하거나 항진하고 장벽 기능(barrier function)을 강화시키는 것이다. 이러한 사실은 프로바이오틱스의 균주 자체가 장상피세포나 면역세포의 수용체를 통해 인식이 되거나 균주에서 분비된 효소나 단백질들을 통해서 세포 신호 전달 과정을 변화시키는 과정이 알려짐으로써 확인이 되었다.For this purpose, many probiotics have been developed using enterococci, lactobacilli and bifidobacteria. The lactic acid bacteria probiotics have been reported to be effective in treating diseases such as antibiotic-associated diarrhea and traveler's diarrhea, irritable bowel syndrome and inflammatory bowel disease. The mechanisms of the efficacy of probiotics known to date are as follows. Administered probiotics crack in the intestine and interfere with pathogen activity, growth and contact with intestinal epithelial cells. Next, it regulates or enhances the host's immune function and enhances barrier function through its relationship with intestinal epithelial or mucosal immune cells. This fact has been confirmed by the probiotic strain itself being recognized through receptors of intestinal epithelial cells or immune cells, or the process of altering cellular signal transduction through enzymes or proteins secreted from the strain.
선천성 면역계(innate immunity)에서 가장 중요한 역할을 하는 TLR(Toll Like Receptor)은 병원성 미생물이 갖는 특이적인 분자패턴(pathogen-associated molecular patterns)을 인지하는데, TLR4는 그람 음성균의 리포폴리사카라이드(lipopolysaccharide, 이하 'LPS'라고 함)를 인지함으로써 전염증성 유전자의 전사인자인 nuclear factor-γB(NF-γB) 경로를 통해 전염증성 사이토카인이 생산된다. 최근에 유산균이 TLR4를 억제시켜 유산균의 장내 면역증가와 관련하여 유산균이 대장염을 완화시켜 주는 것으로 보고 되었다. 사이토카인은 대표적인 인체 면역을 위한 호르몬이며 면역관련 세포의 분화와 기능에 직접적인 영향을 끼친다. 그 중에서 인터루킨-10(IL-10)은 항염증 작용을 나타내며, 인터루킨-12(IL-12), IFN- γ 등은 염증을 촉진하는 작용을 나타낸다.Toll Like Receptor (TLR), which plays the most important role in the innate immunity, recognizes pathogen-associated molecular patterns of pathogenic microorganisms, and TLR4 is a gram-negative lipopolysaccharide (lipopolysaccharide). By recognizing 'LPS' below, proinflammatory cytokines are produced through the nuclear factor-γB (NF-γB) pathway, which is a transcription factor of proinflammatory genes. Recently, lactobacillus has been reported to inhibit TLR4, which may help to relieve colitis associated with increased intestinal immunity. Cytokines are representative hormones for human immunity and directly affect the differentiation and function of immune-related cells. Among them, interleukin-10 (IL-10) exhibits anti-inflammatory action, and interleukin-12 (IL-12), IFN-γ, and the like promote inflammation.
본 발명은 대장염 유발과 연관된 박테로이데스 불가투스의 억제, 전염증성 사이토카인 분비와 연관된 TLR4의 억제 및 항염증성 사이토카인 IL-10 분비를 증가시키고 TNBS로 유도한 대장염 실험동물모델에서 대장길이 축소 억제, 미엘로퍼옥시다제 활성 억제, 분변내 유해효소인 베타-글루쿠로니다아제, 베타-글루코시다아제 및 콘드로이티나아제 활성을 저해함으로써 대장내 유해균 억제 및 염증발생 억제 효능을 갖는 새로운 비피도박테리움 롱검 HY8004 및 이를 유효성분으로 함유하는 약학조성물, 발효유, 음료 및 건강기능식품을 제공하는 것을 목적으로 한다.The present invention is directed to inhibition of Bacteroides vulgaris associated with colitis induction, inhibition of TLR4 associated with proinflammatory cytokine secretion, and inhibition of colon length reduction in TNBS-induced colitis experimental animal models with increased anti-inflammatory cytokine IL-10 secretion. Inhibits myeloperoxidase activity, inhibits fecal harmful enzymes beta-glucuronidase, beta-glucosidase and chondroitinase activity, thereby inhibiting colon bacillus and inhibiting inflammation It is an object of the present invention to provide a terium long gum HY8004 and a pharmaceutical composition, fermented milk, a beverage and a dietary supplement containing the same as an active ingredient.
상기와 같은 목적을 달성하기 위하여, 대장염 유발과 연관된 박테로이데스 불가투스의 억제, 전염증성 사이토카인 분비와 연관된 TLR4의 억제 및 항염증성 사이토카인 IL-10 분비를 증가시키고, TNBS로 유도한 대장염 실험동물모델에서 대장길이 축소 억제, 미엘로퍼옥시다제 활성 억제, 분변내 유해효소인 베타-글루쿠로니다아제, 베타-글루코시다아제 및 콘드로이티나아제 활성을 저해함으로써 대장내 유해균 억제 및 염증발생 억제 효능을 갖는 새로운 비피도박테리움 롱검 HY8004 및 이를 유효성분으로 함유하는 약학조성물, 발효유, 음료 및 건강기능식품을 제공하는 것을 특징으로 한다.In order to achieve the above objectives, the study of inhibition of Bacteroides buccus associated with colitis induction, inhibition of TLR4 associated with proinflammatory cytokine secretion and increased secretion of anti-inflammatory cytokine IL-10, and colitis induced by TNBS Inhibition of intestinal harmful bacteria and inflammation by inhibiting colon length reduction, inhibiting myeloperoxidase activity, inhibiting fecal harmful enzymes beta-glucuronidase, beta-glucosidase and chondroitinase activity in animal models It provides a novel Bifidobacterium long gum HY8004 having an inhibitory effect and a pharmaceutical composition containing the same as an active ingredient, fermented milk, a beverage and a health functional food.
이하, 본 발명을 좀 더 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명에 따른 신균주를 분리하기 위하여 건강한 한국인 성인의 분변을 비에스(BS) 용액이 첨가된 비엘(BL) 한천배지에 도말한 후 37℃에서 72시간 혐기 배양하였다. 비에스 용액은 다음과 같이 제조하였다. 100ml의 멸균 비이커에 30g의 소디움 프로피오네이트(sodium propionate)를 정량하여 약 80ml의 증류수에 용해하고 100㎎의 파로모마이신 설페이트(paromomycin sulfate), 400㎎의 네오마이신 설페이트(neomycin sulfate), 6g의 리튬 클로라이드(lithium chloride)를 첨가하여 용해한 다음, 증류수를 첨가하여 100ml로 부피를 조절하고 121℃에서 15분 멸균한 후 비엘 한천배지 1L당 50ml을 첨가하였다. 형성된 균락 중에서 장염 유발과 연관된 박테로이데스 불가투스의 억제, 전염증성 사이토카인 분비와 연관된 TLR4 억제 등을 시험하여 본 발명의 신균주를 분리하였다.In order to isolate the new strain according to the present invention, feces of healthy Korean adults were plated on agar (BL) agar medium to which a BS (BS) solution was added, followed by anaerobic culture for 72 hours at 37 ° C. The BC solution was prepared as follows. In a 100 ml sterile beaker, 30 g of sodium propionate is quantified and dissolved in about 80 ml of distilled water, 100 mg of paromomycin sulfate, 400 mg of neomycin sulfate, 6 g of Lithium chloride (lithium chloride) was added to dissolve, and then distilled water was added to adjust the volume to 100 ml, sterilized for 15 minutes at 121 ℃ and 50ml per 1L Biel agar medium was added. Among the fungi formed, the bacterial strains of the present invention were isolated by testing inhibition of Bacteroides vulgaris associated with enteritis and inhibition of TLR4 associated with proinflammatory cytokine secretion.
본 발명에 따른 신균주의 특성은 다음과 같다.The characteristics of the new strain according to the present invention are as follows.
1)균의 형태1) Form of bacteria
비엘 한천평판배지에서 37℃, 3일간 혐기 배양했을 때 균의 특성 Characteristics of Bacteria in Anaerobic Cultures at 37 ° C for 3 Days in Biel Agar Plates
① 세포의 형태: 간균, Y자형, 곤봉형 ① Cell type: Bacillus, Y-shaped, club-shaped
② 운동성: 없음 ② Mobility: None
③ 포자형성능: 없음 ③ Spore Formation Capacity: None
④ 그람(Gram) 염색: 양성 ④ Gram staining: positive
2)균락의 형태2) Form of the collapse
비엘 한천평판배지에서 37℃, 3일간 혐기 배양했을 때 균락의 형태 Morphology of the fungi in anaerobic culture at 37 ° C for 3 days in Biel agar plate
① 형상: 원형 ① Shape: Round
② 융기: 볼록 ② uplift: convex
③ 표면: 매끄러움(Smooth) ③ Surface: Smooth
3)생리적 성질3) physiological properties
① 최적 생육온도: 36~38℃ ① Optimal growth temperature: 36 ~ 38 ℃
② 최적 생육 pH: 6.0~7.0 ② optimum growth pH: 6.0 ~ 7.0
③ 산소에 대한 영향: 절대혐기성 ③ Effects on oxygen: absolute anaerobic
4)카탈라제: -Catalase:-
5)가스형성여부: -5) Whether gas is formed:-
6)15℃에서 생육: -6) Growth at 15 ℃:-
7)45℃에서 생육: +7) Growth at 45 ℃: +
8)인돌생산: -8) Indole Production:-
9)젖산생산: +9) lactic acid production: +
10)16S rDNA 분석10) 16S rDNA Analysis
16S rDNA 분석을 통한 분자유전학적인 방법을 실시하여 본 발명의 신균주를 동정하였다.Molecular genetic methods through 16S rDNA analysis were performed to identify new strains of the present invention.
그 결과를 표 1에 나타내었다.The results are shown in Table 1.
표 1에서 확인할 수 있는 바와 같이, 본 발명의 신균주의 16S rDNA 유전자는 비피도박테리움 롱검(Bifidobacterium longum)의 16S rRNA 유전자와 100% 일치하는 것으로 확인되었다.As can be seen in Table 1, the 16S rDNA gene of the new strain of the present invention was confirmed to be 100% identical to the 16S rRNA gene of Bifidobacterium longum .
따라서 본 발명자들은 본 발명의 신균주를 비피도박테리움 롱검(Bifidobacterium longum) HY8004로 명명하고, 한국생명공학연구원 생물자원센터에 2008년 4월 17일자로 기탁하였다(기탁번호: KCTC 11316BP).Therefore, the present inventors named the new strain Bifidobacterium longum HY8004, and deposited it on April 17, 2008 at the Korea Institute of Biotechnology and Biotechnology Center (Accession No .: KCTC 11316BP).
한편, 본 발명의 대장염 유발과 연관된 박테로이데스 불가투스의 억제, 전염증성 사이토카인 분비와 연관된 TLR4의 억제 및 항염증성 사이토카인 IL-10 분비를 증가시키고, TNBS로 유도한 대장염 실험동물모델에서 대장길이 축소 억제, 미엘로퍼옥시다제 활성 억제, 분변내 유해효소인 베타-글루쿠로니다아제, 베타-글루코시다아제 및 콘드로이티나아제 활성을 저해함으로써 대장내 유해균 억제 및 염증발생 억제 효능을 갖는 새로운 비피도박테리움 롱검 HY8004를 유효성분으로 함유하는 대장의 건강 증진용 약학 조성물은 단독 또는 약제학적으로 사용되는 부형제들과 함께 약제학적으로 통상으로 사용되는 방법에 따라 정제, 캡술제 등과 같은 제재형태로 제제화하여 사용될 수 있다.On the other hand, inhibition of Bacteroides vulgaris associated with colitis induction of the present invention, inhibition of TLR4 associated with proinflammatory cytokine secretion and increased secretion of anti-inflammatory cytokine IL-10, and colonization in TNBS-induced colitis experimental animal model Inhibition of length reduction, inhibition of myeloperoxidase activity, inhibition of fecal harmful enzymes beta-glucuronidase, beta-glucosidase and chondroitinase activity, thereby inhibiting intestinal harmful bacteria and inflammation The large intestine health-promoting pharmaceutical composition containing the new Bifidobacterium longgum HY8004 as an active ingredient may be prepared in the form of a preparation such as tablets, capsules, etc. according to the methods commonly used pharmaceutically or in combination with excipients used pharmaceutically. Can be formulated and used.
사람의 경우, 통상적인 1일 투여량은 1~30㎎/kg 체중의 범위일 수 있고, 1회 또는 수회로 나누어 투여할 수 있다. 그러나 실제 투여량은 투여경로, 환자의 연령, 성별 및 체중, 건강상태 및 질환의 중증도 등의 여러 관련 인자에 비추어 결정되어야 한다.For humans, a typical daily dose may range from 1-30 mg / kg body weight and may be administered once or in divided doses. However, the actual dosage should be determined in light of several relevant factors such as the route of administration, the age, sex and weight of the patient, health condition and severity of the disease.
물론, 상기 대장의 건강 증진용 약학조성물은 독성 및 부작용은 거의 없으므로 예방 목적으로 장기간 복용시에도 안심하고 사용할 수 있는 약제이다.Of course, the pharmaceutical composition for health promotion of the large intestine is toxic and side effects, so it is a drug that can be used with confidence even for long-term administration for the purpose of prevention.
또한, 본 발명의 대장염 유발과 연관된 박테로이데스 불가투스의 억제, 전염증성 사이토카인 분비와 연관된 TLR4의 억제 및 항염증성 사이토카인 IL-10 분비를 증가시키고, TNBS로 유도한 대장염 실험동물모델에서 대장길이 축소 억제, 미엘로퍼옥시다제 활성 억제, 분변내 유해효소인 베타-글루쿠로니다아제, 베타-글루코시다아제 및 콘드로이티나아제 활성을 저해함으로써 대장내 유해균 억제 및 염증발생 억제 효능을 갖는 새로운 비피도박테리움 롱검 HY8004를 유효성분으로 함유하는 대장의 건강 증진용 발효유는 유산균배양액과 비피도박테리움 롱검 HY8004 및 혼합과즙시럽을 일정비율로 조합하여 150bar에서 균질한 후 10℃ 이하로 냉각한 후 용기에 포장하여 발효유를 제조한다.In addition, the inhibition of Bacteroides vulgaris associated with colitis induction of the present invention, the inhibition of TLR4 associated with proinflammatory cytokine secretion and the increase of anti-inflammatory cytokine IL-10 secretion, and the colon in TNBS-induced colitis experimental animal model Inhibition of length reduction, inhibition of myeloperoxidase activity, inhibition of fecal harmful enzymes beta-glucuronidase, beta-glucosidase and chondroitinase activity, thereby inhibiting intestinal harmful bacteria and inflammation Fermented milk for health promotion of large intestine containing new Bifidobacterium longgum HY8004 as an active ingredient is a homogeneous combination of lactic acid bacteria culture medium, Bifidobacterium longgum HY8004 and mixed fruit juice syrup at 150 bar and then cooled to below 10 ℃ After packaging in a container to prepare fermented milk.
또한, 본 발명의 대장염 유발과 연관된 박테로이데스 불가투스의 억제, 전염증성 사이토카인 분비와 연관된 TLR4의 억제 및 항염증성 사이토카인 IL-10 분비를 증가시키고, TNBS로 유도한 대장염 실험동물모델에서 대장길이 축소 억제, 미엘로퍼옥시다제 활성 억제, 분변내 유해효소인 베타-글루쿠로니다아제, 베타-글루코시다아제 및 콘드로이티나아제 활성을 저해함으로써 대장내 유해균 억제 및 염증발생 억제 효능을 갖는 새로운 비피도박테리움 롱검 HY8004를 유효성분으로 함유하는 대장의 건강 증진용 음료는 혼합과즙시럽, 비피도박테리움 롱검 HY8004 및 물을 일정한 비율로 조합하여 150bar에서 균질한 후 10℃ 이하로 냉각한 후 유리병, 패트병 등 소포장 용기에 포장하여 기능성음료를 제조한다.In addition, the inhibition of Bacteroides vulgaris associated with colitis induction of the present invention, the inhibition of TLR4 associated with proinflammatory cytokine secretion and the increase of anti-inflammatory cytokine IL-10 secretion, and the colon in TNBS-induced colitis experimental animal model Inhibition of length reduction, inhibition of myeloperoxidase activity, inhibition of fecal harmful enzymes beta-glucuronidase, beta-glucosidase and chondroitinase activity, thereby inhibiting intestinal harmful bacteria and inflammation The large intestinal health drink containing the new Bifidobacterium longgum HY8004 as an active ingredient is a mixture of fruit juice syrup, Bifidobacterium longgum HY8004 and water at a constant ratio, homogenized at 150 bar and then cooled to 10 ° C or lower. Functional drinks are prepared by packing them in small packaging containers such as glass bottles and plastic bottles.
또한, 본 발명의 대장염 유발과 연관된 박테로이데스 불가투스의 억제, 전염증성 사이토카인 분비와 연관된 TLR4의 억제 및 항염증성 사이토카인 IL-10 분비를 증가시키고, TNBS로 유도한 대장염 실험동물모델에서 대장길이 축소 억제, 미엘로퍼옥시다제 활성 억제, 분변내 유해효소인 베타-글루쿠로니다아제, 베타-글루코시다아제 및 콘드로이티나아제 활성을 저해함으로써 대장내 유해균 억제 및 염증발생 억제 효능을 갖는 새로운 비피도박테리움 롱검 HY8004를 유효성분으로 함유하는 대장의 건강 증진용 건강기능식품은 상기 비피도박테리움 롱검 HY8004를 포함하는 것 이외에 영양보조성분으로서 비타민 B1, B2, B5, B6, E 및 초산에스테르, 니코틴산 아미드, 올리고당 등이 첨가될 수 있으며 여타의 식품 첨가물이 첨가되어도 무방하다.In addition, the inhibition of Bacteroides vulgaris associated with colitis induction of the present invention, the inhibition of TLR4 associated with proinflammatory cytokine secretion and the increase of anti-inflammatory cytokine IL-10 secretion, and the colon in TNBS-induced colitis experimental animal model Inhibition of length reduction, inhibition of myeloperoxidase activity, inhibition of fecal harmful enzymes beta-glucuronidase, beta-glucosidase and chondroitinase activity, thereby inhibiting intestinal harmful bacteria and inflammation The health supplement food for the large intestine containing the new Bifidobacterium longgum HY8004 as an active ingredient includes vitamin B1, B2, B5, B6, E and acetic acid as a nutritional supplement in addition to the Bifidobacterium longgum HY8004. Esters, nicotinic acid amides, oligosaccharides and the like may be added and other food additives may be added.
이상에서 살펴 본 바와 같이, 본 발명의 비피도박테리움 롱검 HY8004는 대장염 유발과 연관된 박테로이데스 불가투스의 억제, 전염증성 사이토카인 분비와 연관된 TLR4의 억제 및 항염증성 사이토카인 IL-10 분비를 증가시키고, TNBS로 유도한 대장염 실험동물모델에서 대장길이 축소 억제, 미엘로퍼옥시다제 활성 억제, 분변내 유해효소인 베타-글루쿠로니다아제, 베타-글루코시다아제 및 콘드로이티나아제 활성을 저해함으로써 대장내 유해균 억제 및 염증발생을 억제함으로써 유해균 억제 및 염증발생 억제 효능을 통하여 대장의 건강을 증진하는 목적으로 이용될 수 있다.As described above, the Bifidobacterium longgum HY8004 of the present invention increases the inhibition of Bacteroides buccus associated with colitis induction, the inhibition of TLR4 associated with proinflammatory cytokine secretion and the increase of anti-inflammatory cytokine IL-10 secretion. Inhibition of colon length reduction, inhibition of myeloperoxidase activity, inhibition of fecal beta-glucuronidase, beta-glucosidase and chondroitinase activity in experimental animal models induced by TNBS. It can be used for the purpose of promoting the health of the colon through the suppression of harmful bacteria and inflammation by inhibiting harmful bacteria in the colon by inhibiting the occurrence of inflammation.
이하 실시예를 통하여 본 발명을 보다 상세하게 설명한다. 그러나 다음의 실시예는 본 발명의 범위를 한정하는 것은 아니며, 본 발명의 기술적 사상의 범위 내에서 당업자에 의한 통상적인 변화가 가능하다.Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are not intended to limit the scope of the present invention, and ordinary changes by those skilled in the art are possible within the scope of the technical idea of the present invention.
<실시예 1>≪ Example 1 >
비피도박테리움 롱검 HY8004를 포함한 동결건조분말 제조Preparation of lyophilized powder including Bifidobacterium long gum HY8004
본 발명의 비피도박테리움 롱검 HY8004는 식품원료용 Proteose peptone #3, Yeast Extract, Beef Extract 및 포도당을 첨가한 액체배지를 제조하여 37℃에서 약 16시간 배양한 후 배양액을 원심분리하고 멸균된 생리식염수로 세척한 다음 멸균유에 분산하였다. 다시 동결 건조하여 동결건조 분말 그램(g)당 약 1011cfu 균수를 얻었다. 이 동결건조 분말을 대장의 건강 증진 소재로 사용하였다.Bifidobacterium longgum HY8004 of the present invention is a liquid medium containing
한편, 본 발명의 비피도박테리움 롱검 HY8004는 상기와 같이 동결건조된 분말 형태 또는 배양물 형태로 제공될 수 있다.Meanwhile, the Bifidobacterium long gum HY8004 of the present invention may be provided in the form of a lyophilized powder or culture as described above.
<실시예 2><Example 2>
비피도박테리움 롱검 HY8004를 유효성분으로 함유하는 약학적 조성물의 제조Preparation of pharmaceutical composition containing Bifidobacterium long gum HY8004 as an active ingredient
본 발명의 비피도박테리움 롱검 HY8004를 유효성분으로 함유하는 약학 조성물의 제제예를 설명하나, 본 발명은 이를 한정하고자 함이 아닌 단지 구체적으로 설명하고자 함이다.Although the formulation example of the pharmaceutical composition containing Bifidobacterium longgum HY8004 of the present invention as an active ingredient is described, the present invention is not intended to limit the present invention but is intended to be described in detail only.
정제의 제조Manufacture of tablets
상기 실시예 1의 비피도박테리움 롱검 HY8004를 포함한 동결건조분말 100㎎, 옥수수전분 100㎎, 유당 100㎎ 및 폴리비닐피롤리돈 97㎎을 균질하게 혼합하여 습 식과립법으로 과립화하고, 스테아린산 마그네슘 2㎎을 가하여 혼합한 후, 1정이 400㎎이 되도록 타정하였다.Lyophilized powder 100mg, corn starch 100mg, lactose 100mg and polyvinylpyrrolidone 97mg, including Bifidobacterium longgum HY8004 of Example 1 were homogeneously mixed, granulated by wet granulation,
캡슐제의 제조Preparation of Capsules
상기 실시예 1의 비피도박테리움 롱검 HY8004를 포함한 동결건조분말 100㎎, 옥수수 전분 100㎎, 유당 100㎎, 스테아린산 마그네슘 2㎎을 완전히 혼합한 후, 통상의 캡슐제의 제조 방법에 따라서 경질 젤라틴 캡슐에 충전하여 캡슐제를 제조하였다.After completely mixing the lyophilized powder 100mg, corn starch 100mg, lactose 100mg, magnesium stearate 2mg, including Bifidobacterium longgum HY8004 of Example 1, hard gelatine capsules according to the manufacturing method of conventional capsules It was filled in to prepare a capsule.
<실시예 3><Example 3>
비피도박테리움 롱검 HY8004를 유효성분으로 함유하는 발효유의 제조Preparation of fermented milk containing Bifidobacterium long gum HY8004 as an active ingredient
유산균 배양액과 상기 실시예 1의 비피도박테리움 롱검 HY8004를 포함한 동결건조분말 및 혼합과즙시럽으로 구성된 발효유를 제조하는 방법은 다음과 같다.Method for producing a fermented milk consisting of a freeze-dried powder and mixed juice syrup including lactic acid bacteria culture medium and Bifidobacterium long gum HY8004 of Example 1 is as follows.
유산균 배양액은 원유 95.36중량%와 탈지분유(또는 혼합분유) 4.6중량%를 교반하여 15℃에서의 비중은 1.0473~1.0475, 적정산도는 0.200~0.220%, pH는 6.65~6.70, 20℃에서의 Brixo는 16.3~16.5%정도가 되도록 혼합하였다. 그런 다음, 이를 UHT 열처리(135℃에서 2초간 살균)하고 40℃로 냉각한 뒤, 스트렙토코커스 써모필러스균과 유당분해효소(Valley laboratory에서 구입, USA)를 각기 0.02중량%씩 첨가하고 6시간동안 배양하여, BCP 배지에서의 총 유산균수가 1.0 × 109cfu/㎖ 이 상, 적정산도가 0.89~0.91%, pH는 4.55~4.65가 되도록 하여 제조하였다.The lactic acid bacteria culture medium was stirred with 95.36% of crude milk and 4.6% by weight of skim milk powder (or mixed milk powder). o was mixed so as to be about 16.3-16.5%. Then, it was UHT heat-treated (sterilized at 135 ° C for 2 seconds) and cooled to 40 ° C. Then, Streptococcus thermophilus and lactose (available from Valley laboratory, USA) were added at 0.02% by weight, respectively, for 6 hours. The total lactic acid bacteria count in the BCP medium was 1.0 × 10 9 cfu / ㎖ or more, the titratable acidity was 0.89 ~ 0.91%, pH was prepared to be 4.55 ~ 4.65.
혼합과즙시럽은 백설탕 3~5중량%, 갈색설탕 3~5중량%, 혼합과즙농축액 56 Brixo 10~15중량%, 펙틴 0.1~1.0중량%, 후레쉬 후르츠 믹스 에센스 0.05~0.15중량% 및 정제수 68.85~88.85중량%를 30~35℃에서 교반하여 혼합한 후 UHT 열처리(135℃에서 2초간 살균)한 후 냉각하여 제조하였다.Mixed fruit syrup is 3 ~ 5% by weight of white sugar, 3-5% by weight of brown sugar, 10 ~ 15% by weight of mixed juice concentrate 56 Brix o , 0.1 ~ 1.0% by weight pectin, 0.05 ~ 0.15% by weight fresh fruit mix essence and purified water 68.85 After stirring at ~ 88.85% by weight at 30 ~ 35 ℃ mixed UHT heat treatment (sterilization for 2 seconds at 135 ℃) was prepared by cooling.
상기의 방법으로 제조된 유산균 배양액 65~75중량%와 상기 실시예 1의 비피도박테리움 롱검 HY8004를 포함한 동결건조분말 0.001~0.1중량% 및 상기 방법으로 제조된 혼합과즙시럽 24.9~34.999중량%를 조합하여 150bar에서 균질한 후 10℃ 이하로 냉각하여 대장내 유해균 억제 및 대장염발생 억제 효능을 갖는 비피도박테리움 롱검 HY8004를 유효성분으로 함유하는 발효유를 제조하였다.65-75% by weight of the lactic acid bacteria culture medium prepared by the above method and 0.001-0.1% by weight of the lyophilized powder including the Bifidobacterium long gum HY8004 of Example 1 and 24.9-34.999% by weight of the mixed fruit juice syrup prepared by the above method. The mixture was homogenized at 150 bar and then cooled to 10 ° C. or lower to prepare fermented milk containing Bifidobacterium long gum HY8004 having the effect of inhibiting harmful bacteria in colon and inhibiting colitis.
<실시예 4><Example 4>
비피도박테리움 롱검 HY8004를 유효성분으로 함유하는 하는 기능성 음료Functional drink containing Bifidobacterium long gum HY8004 as an active ingredient
상기 실시예 1의 비피도박테리움 롱검 HY8004를 포함한 동결건조분말과 혼합과즙시럽으로 구성된 기능성 음료를 제조하는 방법은 다음과 같다.Method for producing a functional beverage consisting of lyophilized powder and mixed fruit syrup, including Bifidobacterium long gum HY8004 of Example 1 is as follows.
혼합과즙시럽은 액상과당 10~15중량%, 백설탕 3~5중량%, 갈색설탕 3~5중량%, 혼합과즙농축액 56Brixo 10~15중량%, 펙틴 0.1~1.0중량%, 후레쉬 후르츠 믹스 에센스 0.05~0.15중량% 및 정제수 68.85~88.85중량%를 30~35℃에서 교반하여 혼합한 후 UHT 열처리(135℃에서 2초간 살균)한 후 냉각하여 제조하였다.Mixed fruit syrup consists of 10-15% by weight of liquid fructose, 3-5% by weight of white sugar, 3-5% by weight of brown sugar, 10-15% by weight of mixed juice concentrate 56Brix o 10-15% by weight, pectin 0.1-1.0% by weight, fresh fruit mix essence 0.05 ~ 0.15% by weight and purified water 68.85 ~ 88.85% by mixing at 30 ~ 35 ℃ stirred and mixed, and then prepared by cooling UHT heat treatment (sterilization for 2 seconds at 135 ℃).
상기의 방법으로 제조된 혼합과즙시럽 29.9~39.999중량%와 상기 실시예 1의 비피도박테리움 롱검 HY8004를 포함한 동결건조분말 0.001~0.1중량% 및 멸균 정제수 60~70중량%를 조합하여 150bar에서 균질한 후 10℃ 이하로 냉각한 후 이를 유리병, 패트병 등 소포장 용기에 포장하여 대장내 유해균 억제 및 대장염발생 억제 효능을 갖는 비피도박테리움 롱검 HY8004를 유효성분으로 함유하는 기능성 음료를 제조하였다.29.9 ~ 39.999% by weight of the mixed juice syrup prepared by the above method and 0.001 ~ 0.1% by weight of lyophilized powder and 60 ~ 70% by weight of sterile purified water, including Bifidobacterium long gum HY8004 of Example 1 homogeneous at 150 bar After cooling to 10 ° C or less, and then packaged in a small packaging container such as glass bottles, plastic bottles to prepare a functional beverage containing Bifidobacterium long gum HY8004 having the effect of inhibiting harmful bacteria in colon and inhibiting colitis.
<실시예 5>Example 5
비피도박테리움 롱검 HY8004를 유효성분으로 함유하는 건강기능식품Health functional food containing Bifidobacterium long gum HY8004 as an active ingredient
상기 실시예 1의 비피도박테리움 롱검 HY8004를 포함한 동결건조분말 0.001~0.1중량%에 영양보조성분(비타민 B1, B2, B5, B6, E 및 초산에스테르, 니코틴산 아미드), 올리고당을 상기의 실시예 1의 비피도박테리움 롱검 HY8004를 포함한 동결건조분말 100중량%에 대하여 5~10중량%가 되도록 첨가하여 고속회전 혼합기에서 혼합하였다. 상기 혼합물에 멸균 정제수 10중량%를 첨가, 혼합하고 직경 1~2mm의 과립상으로 성형하였다. 상기 성형된 과립은 40~50℃의 진공건조기에서 건조시킨 후, 12~14 메쉬(mesh)를 통과시켜 균일하게 과립을 제조하였다. 상기와 같이 제조된 과립은 적당량씩 압출 성형되어 정제 또는 분말로 되거나 경질캡슐에 충전되어 경질캡슐제품으로 제조하였다.The lyophilized powder (vitamin B1, B2, B5, B6, E and acetate ester, nicotinic acid amide) and the oligosaccharide in 0.001 to 0.1% by weight of the lyophilized powder including Bifidobacterium long gum HY8004 of Example 1 1 was added to 5 to 10% by weight relative to 100% by weight of the lyophilized powder including Bifidobacterium longgum HY8004 and mixed in a high speed rotary mixer. 10% by weight of sterile purified water was added to the mixture, mixed, and molded into granules having a diameter of 1 to 2 mm. The molded granules were dried in a vacuum dryer at 40 to 50 ° C., and then passed through 12 to 14 meshes to uniformly prepare granules. The granules prepared as described above were extruded by appropriate amounts into tablets or powders or filled into hard capsules to produce hard capsule products.
<시험예 1><Test Example 1>
비피도박테리움 롱검 HY8004의 유해균 억제활성 측정Determination of Harmful Bacteria Inhibitory Activity of Bifidobacterium Longgum HY8004
본 발명의 비피도박테리움 롱검 HY8004의 대장염 유발과 연관된 박테로이데스 불가투스(Bacteroides vulgatus), 대장 점막의 염증 유발과 연관된 콘드로이티나제 활성을 갖는 박테로이드 물티아시더스(Bacteroides multiacidus)의 억제효과를 시험하였다.Foil teroyi Death not associated with colitis induction of Bifidobacterium ronggeom HY8004 of the invention tooth inhibition of (Bacteroides vulgatus), foil having proinflammatory and corned Roy proteinase activity associated with the colorectal mucosal steroid water thiazol when Ranges (Bacteroides multiacidus) The effect was tested.
1-1 비피도박테리움 롱검 HY8004의 박테로이데스 불가투스의 억제활성 측정1-1 Bifidobacterium long gum HY8004 inhibitory activity of bacteroides vulgaris
(1)비엘(BL) 액체배지에서 약 20시간 배양된 비피도박테리움 롱검 HY8004와 갬(GAM) 액체배지에서 약 24시간 배양된 박테로이데스 불가투스(Bacteroides vulgatus) KCCM11423을 갬 액체배지(Nissui)에 1%씩 접종한 다음, 37℃에서 약 20시간 혐기적 조건에서 혼합 배양하였다. 그런 다음, 박테로이데스(Bacteroides) 선택배지(Nissui)를 사용하여 박테로이데스 불가투스 KCCM11423의 생균수를 측정하였다.(1) Bifidobacterium longgum HY8004 incubated in BL liquid medium and Bacteroides vulgatus KCCM11423 incubated in GAM liquid medium for 24 hours. ) Was inoculated at 1% and mixed cultured at 37 ° C. for about 20 hours in anaerobic conditions. Then, the viable cell count of Bacteroide vulgarus KCCM11423 was measured using Bacteroides selection medium (Nissui).
(2)비피도박테리움(Bifidobacterium) sp. KJ-8, 비피도박테리움(Bifidobacterium) sp. KE-3, 비피도박테리움 롱검(Bifidobacterium longum) B10J4-1, 비피도박테리움(Bifidobacterium) sp. KJ-1 각각에 대하여도 상기 (1)의 비피도박테리움 롱검 HY8004와 박테로이데스 불가투스 KCCM11423의 혼합배양방법과 동일한 방법으로 혼합배양을 하여 박테로이데스 불가투스 KCCM11423의 생균수를 측정하였다.(2) Bifidobacterium (Bifidobacterium) sp. KJ-8, Bifidobacterium (Bifidobacterium) sp. KE-3, ronggeom Bifidobacterium (Bifidobacterium longum) B10J4-1, Bifidobacterium (Bifidobacterium) sp. The KJ-1 was also cultured in the same manner as the mixed culture method of Bifidobacterium longgum HY8004 and Bacteroides vulgarus KCCM11423 of (1), and the viable bacterial count of Bacteroides vulgarus KCCM11423 was measured.
(3)대조구로 박테로이데스 불가투스 KCCM11423을 단독으로 갬 액체배지에 1% 접종한 다음 37℃에서 약 20시간 혐기적 조건에서 단독 배양한 다음, 박테로이데스(Bacteroides) 선택배지를 사용하여 박테로이데스 불가투스 KCCM11423의 생균수를 측정하였다.(3) 1% inoculation of Bacteroides Bulgus KCCM11423 alone as a control and incubated alone at 37 ° C for about 20 hours under anaerobic conditions, followed by using Bacteroides selective medium. The viable cell count of Terroides vulgarus KCCM11423 was measured.
그 결과를 도 1에 나타내었다.The results are shown in FIG.
도 1의 가로축에는 박테로이데스 불가투스 KCCM11423을 단독 배양한 군을 '11423'으로 표시하고, 박테로이데스 불가투스 KCCM11423과 비피도박테리움 롱검 HY8004를 혼합 배양한 군을 'HY8004'로 표시하고, 박테로이데스 불가투스 KCCM11423과 비피도박테리움(Bifidobacterium) sp. KJ-8을 혼합 배양한 군을 'KJ-8'로 표시하고, 박테로이데스 불가투스 KCCM11423과 비피도박테리움(Bifidobacterium) sp. KE-3을 혼합 배양한 군을 'KE-3'으로 표시하고, 박테로이데스 불가투스 KCCM11423과 비피도박테리움 롱검(Bifidobacterium longum) B10J4-1을 혼합 배양한 군을 ‘B10J4-1’로 표시하고, 박테로이데스 불가투스 KCCM11423과 비피도박테리움(Bifidobacterium) sp. KJ-1을 혼합 배양한 군을 ‘KJ-1’으로 표시하였다.In the horizontal axis of FIG. 1, the group incubated with Bacteroides vulgarus KCCM11423 alone is labeled '11423', and the group incubated with the culture of Bacteroides buccus KCCM11423 and Bifidobacterium long gum HY8004 is labeled as 'HY8004', night teroyi death Not KCCM11423 tooth and Bifidobacterium (Bifidobacterium) sp. Display the mixed culture of the group KJ-8 to 'KJ-8' and night teroyi Deaths No Bluetooth KCCM11423 and Bifidobacterium (Bifidobacterium) sp. The group cultured with KE-3 was labeled as 'KE-3', and the group cultured with Bacteroides buccus KCCM11423 and Bifidobacterium longum B10J4-1 was labeled as 'B10J4-1'. and night teroyi Deaths No Bluetooth KCCM11423 and Bifidobacterium (Bifidobacterium) sp. The group cultured with KJ-1 was designated as 'KJ-1'.
도 1에서 확인할 수 있는 바와 같이, 박테로이데스 불가투스 KCCM11423을 단독 배양한 경우의 박테로이데스 불가투스 KCCM11423의 생균수와 본 발명의 비피도박테리움 롱검 HY8004를 포함한 상기 비피도박테리아 균주들과 박테로이데스 불가투스 KCCM11423을 혼합 배양한 경우의 박테로이데스 불가투스 KCCM11423의 생균수 를 비교하였을 때, 본 발명의 비피도박테리움 롱검 HY8004를 포함한 상기 비피도박테리아 균주들과 혼합 배양한 경우 박테로이데스 불가투스 KCCM11423의 생균수가 감소하는 것을 알 수 있었다.As can be seen in Figure 1, the Bifidobacteria strains and the bacterium containing Bifidobacterium longgum HY8004 of the present invention Bacteroides buccus KCCM11423 and the Bifidobacterium long gum HY8004 of the present invention cultured alone Bacteroides when mixed with the Bifidobacteria strains, including Bifidobacterium longgum HY8004 of the present invention, when comparing the viable cell number of Bacteroides buccus KCCM11423 in the case of mixed culture of Terroides buccus KCCM11423 It was found that viable cell number of KCCM11423 decreased.
특히, 본 발명의 비피도박테리움 롱검 HY8004와 혼합 배양하였을 때 박테로이데스 불가투스 KCCM11423의 생균수가 7.0 Log10 cfu/㎖로, 감소효과가 가장 크게 나타나, 본 발명의 비피도박테리움 롱검 HY8004가 박테로이데스 불가투스의 증식을 효과적으로 억제하는 것을 알 수 있었다.In particular, when mixed and cultured with Bifidobacterium longgum HY8004 of the present invention, the viable cell count of Bacteroides vulgaris KCCM11423 was 7.0 Log 10 cfu / ml, showing the greatest reduction effect, Bifidobacterium longgum HY8004 of the present invention It was found that the growth of Bacteroides vulgarus is effectively suppressed.
1-2 비피도박테리움 롱검 HY8004의 박테로이드 물티아시더스의 억제활성 측정1-2 Inhibitory Activity of Bifidobacterium Long Gum HY8004 on Bacterial Multhiasides
(1)비엘 액체배지에서 약 20시간 배양된 비피도박테리움 롱검 HY8004와 갬(GAM) 액체배지에서 약 24시간 배양된 박테로이드 물티아시더스(Bacteroides multiacidus) BJY6을 갬 액체배지에 1%씩 접종한 다음, 37℃에서 약 20시간 혐기적 조건에서 혼합 배양하였다. 그런 다음, 박테로이데스(Bacteroides) 선택배지(Nissui)를 사용하여 박테로이드 물티아시더스 BJY6의 생균수를 측정하였다.(1) 1% of Bifidobacterium longgum HY8004 incubated in Biel liquid medium and Bacteroides multiacidus BJY6 incubated in GAM liquid medium for about 24 hours After inoculation, the mixtures were cultured in anaerobic conditions at 37 ° C. for about 20 hours. Then, the viable cell count of bacteroid multhiasides BJY6 was measured using Bacteroides selective medium (Nissui).
(2)비피도박테리움(Bifidobacterium) sp. KJ-8, 비피도박테리움(Bifidobacterium) sp. KE-3, 비피도박테리움 롱검(Bifidobacterium longum) B10J4-1, 비피도박테리움(Bifidobacterium) sp. KJ-1 각각에 대하여도 상기 (1)의 비피도박테리움 롱검 HY8004와 박테로이드 물티아시더스 BJY6의 혼합 배양방법과 동일한 방법으로 혼합배양을 하여 박테로이드 물티아시더스 BJY6의 생균수를 측정 하였다.(2) Bifidobacterium (Bifidobacterium) sp. KJ-8, Bifidobacterium (Bifidobacterium) sp. KE-3, ronggeom Bifidobacterium (Bifidobacterium longum) B10J4-1, Bifidobacterium (Bifidobacterium) sp. For each of KJ-1, the number of viable bacteria of Bacterium multhiasiders BJY6 was measured by mixing culture in the same manner as in the mixed culture method of Bifidobacterium long gum HY8004 and Bacterium multhiasiders BJY6. It was.
(3)대조구로 박테로이드 물티아시더스 BJY6을 단독으로 갬 액체배지에 1% 접종한 다음 37℃에서 약 20시간 혐기적 조건에서 단독 배양한 다음, 박테로이데스(Bacteroides) 선택배지를 사용하여 박테로이드 물티아시더스 BJY6의 생균수를 측정하였다.(3) 1% inoculation of bacteroid thiathiacis BJY6 alone as a control, incubated in anaerobic conditions for about 20 hours at 37 ℃, and then using a bacteroides selection medium The number of viable bacteria of bacteroid thiathiacis BJY6 was measured.
그 결과를 도 2에 나타내었다.The results are shown in FIG.
도 2의 가로축에는 박테로이드 물티아시더스 BJY6을 단독 배양한 군을 ‘BJY6'로 표시하고, 박테로이드 물티아시더스 BJY6과 비피도박테리움 롱검 HY8004를 혼합 배양한 군을 ‘HY8004'로 표시하고, 박테로이드 물티아시더스 BJY6과 비피도박테리움(Bifidobacterium) sp. KJ-8을 혼합 배양한 군을 ‘KJ-8'로 표시하고, 박테로이드 물티아시더스 BJY6과 비피도박테리움(Bifidobacterium) sp. KE-3을 혼합 배양한 군을 ‘KE-3’으로 표시하고, 박테로이드 물티아시더스 BJY6과 비피도박테리움 롱검(Bifidobacterium longum) B10J4-1을 혼합 배양한 군을 ‘B10J4-1’로 표시하고, 박테로이드 물티아시더스 BJY6과 비피도박테리움(Bifidobacterium) sp. KJ-1을 혼합 배양한 군을 ‘KJ-1’로 표시하였다. In the horizontal axis of FIG. 2, the group incubated with Bacterium thiathiacis BJY6 alone is labeled 'BJY6', and the group incubated with the culture of bacteroids thiatiacis BJY6 and Bifidobacterium long gum HY8004 is labeled as 'HY8004'. and steroid night when water Tia Saunders BJY6 and Bifidobacterium (Bifidobacterium) sp. Display a mixed culture of the group KJ-8 as 'KJ-8', and the foil steroid water thiazol when Ranges BJY6 and Bifidobacterium (Bifidobacterium) sp. The group in which the culture of KE-3 was mixed was labeled as 'KE-3', and the group in which the culture of the mixed bacteroid thiatiacis BJY6 and Bifidobacterium longum B10J4-1 was referred to as 'B10J4-1'. display and steroid night when water Tia Saunders BJY6 and Bifidobacterium (Bifidobacterium) sp. The group cultured with KJ-1 was labeled as 'KJ-1'.
도 2에서 확인할 수 있는 바와 같이, 박테로이드 물티아시더스 BJY6을 단독배양한 경우의 박테로이드 물티아시더스 BJY6의 생균수와 본 발명의 비피도박테리움 롱검 HY8004를 포함한 상기 비피도박테리아 균주들과 박테로이드 물티아시더스 BJY6을 혼합 배양한 경우의 박테로이드 물티아시더스 BJY6의 생균수를 비교하였을 때, 본 발명의 비피도박테리움 롱검 HY8004를 포함한 상기 비피도박테리움 균주들과 혼합 배양한 경우 박테로이드 물티아시더스 BJY6의 생균수가 감소하는 것을 알 수 있었다.As can be seen in Figure 2, the Bifidobacteria strains including the viable bacteroid thiathiacis BJY6 viable number and Bifidobacterium long gum HY8004 of the present invention in the case of culture alone BJY6 BJY6 When comparing the viable cell numbers of the bacteroid thiathiacis BJY6 with the mixed culture of the bacteroid thiathiacis BJY6, the mixed culture with the above-mentioned Bifidobacterium strains including the Bifidobacterium long gum HY8004 of the present invention In one case, it was found that the bacterial count of bacteroid thiatiacis BJY6 decreased.
특히, 본 발명의 비피도박테리움 롱검 HY8004와 혼합 배양하였을 때 박테로이드 물티아시더스 BJY6의 생균수가 7.0 Log10 cfu/㎖로, 감소효과가 가장 크게 나타나, 본 발명의 비피도박테리움 롱검 HY8004가 박테로이드 물티아시더스 BJY6의 증식을 효과적으로 억제하는 것을 알 수 있었다.In particular, when mixed and cultured with Bifidobacterium longgum HY8004 of the present invention, the number of viable bacteroids thiatiacis BJY6 viable bacteria was 7.0 Log 10 cfu / ㎖, showing the greatest reduction effect, Bifidobacterium longgum HY8004 of the present invention Was found to effectively inhibit the proliferation of bacteroid thiathiacis BJY6.
<시험예 2><Test Example 2>
비피도박테리움 롱검 HY8004의 세포생존율(Cell viability) 측정Cell viability of Bifidobacterium long gum HY8004
돌연변이원은 Caco-2 세포에 DNA 손상을 입혀 세포생존율(cell viability)을 감소시킨다.Mutants cause DNA damage to Caco-2 cells, reducing cell viability.
(1)본 발명의 비피도박테리움 롱검 HY8004를 비엘 액체배지에 접종하고 37℃에서 overnight 혐기 배양한 후 균체를 회수하여 세척한 후 10㎖의 생리식염수에 다시 분산시켰다. 상기 비피도박테리움 롱검 HY8004 현탁액에 돌연변이원 4-NQO stock solution(1㎎/㎖)을 0.1mM 농도가 되도록 첨가한 다음, 37℃에서 150분간 100rpm으로 반응시켰다. 반응 완료 후 원심분리한 다음 상등액을 취하여 제균하고 돌연변이원 변형시험 반응물로 사용하였다. Caco-2 cell은 10% foetal calf serum 과 2% 항생제를 첨가한 DMEM 배지에서 배양하였다. 그런 다음, 상기 Caco-2 cell 약 1 × 106 cells를 24 well plate에 분주하여 37℃에서 18시간 동안 배양한 후, 본 발명의 비피도박테리움 롱검 HY8004와 돌연변이원 4-NQO를 반응시킨 돌연변이원 변형시험 반응물 100μl를 첨가하여 다시 37℃에서 18시간 동안 반응시켰다. 그 다음, 상기 Caco-2 cell을 DMEM 배지로 두 번 세척하고 100μl씩 opaque-walled 96 well에 분주하고 30분간 상온에 방치한 다음, CellTiter-Glo Luminescent cell viability assay kit(Promega)를 이용하여 세포생존율을 측정하였다.(1) Bifidobacterium longgum HY8004 of the present invention was inoculated in a Biel liquid medium and anaerobicly cultured at 37 ° C. overnight, and then the cells were recovered and washed, and then dispersed again in 10 ml of physiological saline. Mutagen 4-NQO stock solution (1 mg / ml) was added to the Bifidobacterium longgum HY8004 suspension at a concentration of 0.1 mM, followed by reaction at 100 rpm for 150 minutes at 37 ° C. After completion of the reaction, centrifuged, the supernatant was removed, sterilized, and used as a mutagen modification test reaction. Caco-2 cells were cultured in DMEM medium with 10% foetal calf serum and 2% antibiotics. Then, after dispensing the caco-2 cells about 1 × 10 6 cells in a 24 well plate and incubated for 18 hours at 37 ℃, the mutation of the reaction of the Bifidobacterium longgum HY8004 of the present invention and the mutant 4-
(2)비피도박테리움(Bifidobacterium) sp. KJ-8, 비피도박테리움(Bifidobacterium) sp. KE-3, 비피도박테리움 롱검(Bifidobacterium longum) B10J4-1, 비피도박테리움(Bifidobacterium) sp. KJ-1 각각에 대하여도 상기 (1)의 비피도박테리움 롱검 HY8004와 동일한 방법으로 세포생존율을 측정하였다.(2) Bifidobacterium (Bifidobacterium) sp. KJ-8, Bifidobacterium (Bifidobacterium) sp. KE-3, ronggeom Bifidobacterium (Bifidobacterium longum) B10J4-1, Bifidobacterium (Bifidobacterium) sp. For each KJ-1, cell viability was measured in the same manner as in the Bifidobacterium long gum HY8004.
그 결과를 도 3에 나타내었다.The results are shown in Fig.
도 3의 가로축에는 비피도박테리움 롱검 HY8004, 비피도박테리움(Bifidobacterium) sp. KJ-8, 비피도박테리움(Bifidobacterium) sp. KE-3, 비피도박테리움 롱검(Bifidobacterium longum) B10J4-1, 비피도박테리움(Bifidobacterium) sp. KJ-1과 돌연변이원 4-NQO 반응물에 의한 Caco-2 세포의 생존율 측정결과를 각각 ‘HY8004', ‘KJ-8', ‘KE-3', ‘B10J4-1', ‘KJ-1'로 하 여 표시하였다.The horizontal axis of FIG. 3, Bifidobacterium ronggeom HY8004, Bifidobacterium (Bifidobacterium) sp. KJ-8, Bifidobacterium (Bifidobacterium) sp. KE-3, ronggeom Bifidobacterium (Bifidobacterium longum) B10J4-1, Bifidobacterium (Bifidobacterium) sp. The survival rate of Caco-2 cells by KJ-1 and the mutant 4-NQO reactant was measured as 'HY8004', 'KJ-8', 'KE-3', 'B10J4-1' and 'KJ-1', respectively. It was indicated by.
도 3에서 확인할 수 있는 바와 같이, 본 발명의 비피도박테리움 롱검 HY8004와 돌연변이원 4-NQO을 반응시킨 돌연변이원 변형시험 반응물에서 Caco-2 세포의 생존율이 약 99%로 가장 높게 나타났다. As can be seen in Figure 3, in the mutagen modified test reacted with the Bifidobacterium longgum HY8004 and mutant 4-NQO of the present invention, the survival rate of Caco-2 cells was the highest 99%.
<시험예 3><Test Example 3>
비피도박테리움 롱검 HY8004의 TLR4 저해 측정Determination of TLR4 Inhibition of Bifidobacterium Longgum HY8004
비피도박테리움Bifidobacterium 롱검Longgum HY8004HY8004 시료 준비 Sample Preparation
(1)TLR4 조절 능력을 측정하기 위하여, 본 발명의 비피도박테리움 롱검 HY8004를 비엘 액체배지에 접종하여 37℃에서 20시간 배양하였다. 상기 비피도박테리움 롱검 HY8004 배양액을 3,000rpm, 4℃에서 10분간 원심 분리하여 균체를 얻은 후 멸균된 PBS 완충용액을 이용하여 3회 세척하였다. 그런 다음, 균체를 다시 멸균된 PBS 완충용액에 현탁한 후 흡광도를 측정하여 OD600=1.0으로 조절한 다음 현탁액을 100℃에서 20분간 살균처리 하였다. 이렇게 사멸된 비피도박테리움 롱검 HY8004 균을 3,000rpm, 4℃에서 10분간 원심 분리하여 균체를 얻은 후 멸균된 PBS 완충용액 1ml에 현탁하였다. 이렇게 사멸된 비피도박테리움 롱검 HY8004 균 현탁액을 -20℃에 보관하면서 시험에 사용하였다.(1) In order to measure the ability to control TTR4, Bifidobacterium longgum HY8004 of the present invention was inoculated in a Biel liquid medium and incubated at 37 ° C for 20 hours. The Bifidobacterium long gum HY8004 culture was centrifuged at 3,000 rpm for 10 minutes at 4 ° C. to obtain cells, and then washed three times using sterile PBS buffer solution. Then, the cells were suspended in sterile PBS buffer again, the absorbance was measured and adjusted to OD 600 = 1.0, and then the suspension was sterilized at 100 ° C. for 20 minutes. The killed Bifidobacterium longgum HY8004 was centrifuged at 3,000 rpm for 10 minutes at 4 ° C. to obtain cells, and then suspended in 1 ml of sterile PBS buffer solution. This killed Bifidobacterium longgum HY8004 bacterial suspension was used in the test while stored at -20 ° C.
(2)락토바실러스 플란타럼 BS1, 락토바실러스 sp. BR0285, 락토바실러스 sp. BR0559, 락토바실러스 sp. BR0262, 비피도박테리움 sp. CS-1, 비피도박테리움 sp. KK-1 각각에 대하여도 상기 (1)의 비피도박테리움 롱검 HY8004와 동일한 방법으로 시료를 준비하였다.(2) Lactobacillus plantarum BS1, Lactobacillus sp. BR0285, Lactobacillus sp. BR0559, Lactobacillus sp. BR0262, Bifidobacterium sp. CS-1, Bifidobacterium sp. Samples were also prepared for each of KK-1 in the same manner as in the Bifidobacterium long gum HY8004.
세포 준비Cell preparation
293-hTLR4A-HA cell(Invivogen)을 DMEM(GIBCO), 10% FBS(GIBCO), 1X Antibiotic-Antimycotic(GIBCO), 1X Blasticidin(Invivogen)을 혼합한 배지를 이용하여 5% CO2, 37℃에서 배양하였다.293-hTLR4A-HA cell (Invivogen) at 5% CO 2, 37 ℃ using a medium containing DMEM (GIBCO), 10% FBS (GIBCO), 1X Antibiotic-Antimycotic (GIBCO), 1X Blasticidin (Invivogen) Incubated.
비피도박테리움 롱검 HY8004의 NF-κB 활성 억제Inhibition of NF-κB Activity of Bifidobacterium Longgum HY8004
(1)TLR4 신호 전달에 의한 NF-kB의 핵내 도입 및 전사활성을 간접적으로 측정하였다. 상기 293-hTLR4A-HA 세포를 10㎠ 세포배양접시에서 배양하였다. pNiFty-Luc(Invivogen)라는 reporter DNA를 배양된 293-hTLR4A-HA 세포에 transfection시켰다. 6시간 후 배지를 갈아주고 세포를 균일하게 96-well plate(Corning Incorporated 3603)로 옮긴 후, 18시간을 배양하였다. 1μg/ml의 TLR4 agonist(E. coli LPS, invivogen)와 상기 준비된 비피도박테리움 롱검 HY8004(105 cfu/μl) 1μl를 같이 상기 293-hTLR4A-HA 세포에 처리하여 6시간 동안 배양하였다. 발현되는 NF-kB의 양을 측정하기 위하여, Bright-GloTM Luciferase Assay System(Promega)을 이용하였다. 96-well plate에 100μl의 루시페라아제(luciferase) 완충용액을 넣어 주고 2분간 상온에서 배양하였다. 배양이 완료된 후 Luminometer(Synergy HT, Bio-Tek)를 통하여 발광(Luminoscence) 양을 측정 하였다.(1) Indirect nuclear transduction and transcriptional activity of NF-kB by TLR4 signal transduction were measured. The 293-hTLR4A-HA cells were cultured in a 10
(2)락토바실러스 플란타럼 BS1, 락토바실러스 sp. BR0285, 락토바실러스 sp. BR0559, 락토바실러스 sp. BR0262, 비피도박테리움 sp. CS-1, 비피도박테리움 sp. KK-1 각각에 대하여도 상기 (1)의 비피도박테리움 롱검 HY8004와 동일한 방법으로 hTLR4의 활성억제 정도를 측정하였다.(2) Lactobacillus plantarum BS1, Lactobacillus sp. BR0285, Lactobacillus sp. BR0559, Lactobacillus sp. BR0262, Bifidobacterium sp. CS-1, Bifidobacterium sp. For each KK-1, the degree of inhibition of hTLR4 activity was measured by the same method as Bifidobacterium long gum HY8004.
그 결과를 도 4에 나타내었다.The results are shown in FIG.
도 4의 가로축에는 비피도박테리움 롱검 HY8004, 락토바실러스 플란타럼 BS1, 락토바실러스 sp. BR0285, 락토바실러스 sp. BR0559, 락토바실러스 sp. BR0262, 비피도박테리움 sp. CS-1, 비피도박테리움 sp. KK-1을 각각 ‘HY8004', ‘BS1', ‘BR0285', 'BR0559', ‘BR0262', ‘CS-1’, ‘KK-1’로 하여 표시하였다.On the horizontal axis of Figure 4 Bifidobacterium long gum HY8004, Lactobacillus plantarum BS1, Lactobacillus sp. BR0285, Lactobacillus sp. BR0559, Lactobacillus sp. BR0262, Bifidobacterium sp. CS-1, Bifidobacterium sp. KK-1 is represented as HY8004, BS1, BR0285, BR0559, BR0262, CS-1, and KK-1, respectively.
도 4에서 확인할 수 있는 바와 같이, 본 발명의 비피도박테리움 롱검 HY8004는 hTLR4의 억제율이 85%로 나타나, TLR4 저해능이 우수함을 알 수 있었다.As can be seen in Figure 4, Bifidobacterium longgum HY8004 of the present invention showed an inhibition rate of 85% of hTLR4, it can be seen that the TLR4 inhibitory ability is excellent.
<시험예 4><Test Example 4>
비피도박테리움 롱검 HY8004의 IL-10 농도 증가 측정Measurement of IL-10 Concentration of Bifidobacterium Longgum HY8004
세포 준비Cell preparation
Raw264.7 cell을 DMEM(GIBCO), 10% FBS(GIBCO), 1X Antibiotic- Antimycotic(GIBCO)을 혼합한 배지를 이용하여 5% CO2, 37℃에서 배양하였다. 그리고 시험에 사용하는 세포는 3 × 105 cell/㎖로 맞추어 주었다.Raw264.7 cells were cultured at 5% CO 2, 37 ℃ using a medium mixed with DMEM (GIBCO), 10% FBS (GIBCO), 1X Antibiotic- Antimycotic (GIBCO). And the cells used for the test was adjusted to 3 × 10 5 cell / ㎖.
비피도박테리움 롱검 HY8004의 IL-10 증가 측정Measurement of IL-10 Increase in Bifidobacterium Longgum HY8004
상기의 배양한 RAW264.7 세포에 상기 시험예 3의 비피도박테리움 롱검 HY8004 세포 조성물 시료를 agonist인 LPS와 같이 넣어주어 18시간 동안 배양하였다. 배양 후 IL-10 Cytokine을 측정하기 위하여 배지를 수거하였고, 이를 96 well IL-10 ELISA plate(Pierce)에 각각 50μl의 표준물질과 수거한 배지를 각각 넣어주고 플레이트(plate)를 덮은 다음, 2시간 동안 상온에서 반응하였다. TBS-T 완충용액으로 플레이트를 4회 세척하였다. 그 다음 50μl의 Biotinylated Antibody 용액을 넣어주고, 30분 동안 반응하였다. 다시 TBS-T 완충용액으로 플레이트를 4회 세척하였다. 다시 100μl의 Streptavidin-HRP 용액을 넣어주고, 30분 동안 반응한 다음 TBS-T 완충용액으로 플레이트를 4회 세척하였다. 그 다음 100μl의 TMB Substrate를 넣어주고, 암실에서 30분간 반응하였다. 반응 완료 후 100μl의 TMB-Stop 용액을 넣어 반응을 종료시킨 다음, ELISA reader를 이용하여 450nm와 550nm에서 흡광도를 측정하였다.The Bifidobacterium longgum HY8004 cell composition sample of Test Example 3 was added to the cultured RAW264.7 cells with LPS as an agonist and cultured for 18 hours. After incubation, the medium was collected to measure the IL-10 cytokine, and 50 μl of the standard material and the collected medium were respectively put into 96 well IL-10 ELISA plate (Pierce), and the plate was covered, followed by 2 hours. Reacted at room temperature. Plates were washed four times with TBS-T buffer. Then 50μl of Biotinylated Antibody solution was added and reacted for 30 minutes. Again the plates were washed four times with TBS-T buffer. 100 μl of Streptavidin-HRP solution was added again, reacted for 30 minutes, and the plate was washed four times with TBS-T buffer. Then 100μl of TMB Substrate was added and reacted in the dark for 30 minutes. After the reaction was completed, the reaction was terminated by adding 100 μl of TMB-Stop solution, and the absorbance was measured at 450 nm and 550 nm using an ELISA reader.
그 결과를 도 5에 나타내었다.The results are shown in Fig.
도 5에서 확인할 수 있는 바와 같이, LPS 만을 처리했을 때의 IL-10의 농도가 814pg/ml인데 반하여, 본 발명의 비피도박테리움 롱검 HY8004를 LPS에 처리 했을 때 IL-10의 농도가 약 1309pg/ml로 증가하였다. As can be seen in Figure 5, the concentration of IL-10 when only LPS treatment was 814pg / ml, whereas the concentration of IL-10 when LPS treatment of Bifidobacterium longgum HY8004 of the present invention is about 1309pg increased to / ml.
따라서, 본 발명의 비피도박테리움 롱검 HY8004는 항염증성 사이토카인인 IL-10의 농도를 증가시켜 염증억제 효과가 우수함을 알 수 있었다.Therefore, the Bifidobacterium long gum HY8004 of the present invention was found to have an excellent anti-inflammatory effect by increasing the concentration of the anti-inflammatory cytokine IL-10.
<시험예 5><Test Example 5>
TNBS로 유도한 대장염 동물모델에서 비피도박테리움 롱검 HY8004 투여에 의한 대장길이 축소저해Inhibition of colon length reduction by administration of Bifidobacterium longgum HY8004 in TNBS-induced colitis animal model
먼저, 비피도박테리움 롱검 HY8004 균주를 비엘 액체배지에 접종하여 37℃에서 20시간 배양하였다. 이 배양액을 3,000rpm, 4℃에서 10분간 원심분리하여 균체를 얻은 후 생리식염수로 2회 세척하고 다시 원심분리한 균체를 시험에 사용하였다.First, Bifidobacterium longgum HY8004 strain was inoculated in Biel liquid medium and incubated at 37 ° C. for 20 hours. The culture solution was centrifuged at 3,000 rpm for 10 minutes at 4 ° C. to obtain cells. The cells were washed twice with physiological saline, and centrifuged cells were used for the test.
실험동물은 (주)중앙실험동물에서 ICR mouse 4주령의 수컷 쥐를 사용하였다. 3일간 순화시킨 후 5일간 상기의 비피도박테리움 롱검 HY8004 균체를 각각 실험동물에 마우스 무게(kg)당 습중량 50㎎/kg을 경구투여 하였으며, 비피도박테리움 롱검 HY8004 경구투여 3일 후 실험동물을 에테르(ether)에 가볍게 마취하고, 끝이 둥근 1ml 주사기를 항문을 통해 3.5~4cm 정도 깊이로 대장내 TNBS(Trinitrobenzene Sulfonic Acid)를 투여하였다. TNBS는 2.5㎎을 50% 에탄올 용액에 용해한 후 100μl를 천천히 투여한 후 대장내 잘 퍼질 수 있도록 수직으로 30~60초간 유지시켰다. TNBS를 투여한 후 2일째 되는 날 해부를 시행하였다.As the experimental animals, 4 weeks old male rats of ICR mouse were used in the central experimental animals. After 3 days of purification, the Bifidobacterium longgum HY8004 cells were orally administered to the experimental animals at 50 mg / kg of wet weight per kg (kg), respectively, and 3 days after the oral administration of Bifidobacterium longgum HY8004. Animals were lightly anesthetized with ether, and 1 ml syringes with round tips were administered intestine TNBS (Trinitrobenzene Sulfonic Acid) to a depth of 3.5-4 cm through the anus. TNBS was dissolved 2.5 mg in 50% ethanol solution, and then slowly administered 100 μl and maintained vertically for 30 to 60 seconds to spread well in the large intestine. Dissection was performed on
실험동물을 에테르 마취하여 맹장아래 1cm되는 지점부터 대장 끝까지를 적출하고 길이를 측정하였다. Experimental animals were anesthetized with ether and extracted from the
그 결과를 도 6에 나타내었다.The results are shown in FIG.
도 6의 가로축에는 TNBS를 처리하지 하지 않은 실험 동물군을 'N', TNBS만 처리된 실험 동물군을 'C', TNBS가 처리된 실험동물에게 비피도박테리움 롱검 HY8004를 투여한 실험 동물군은 '8004' 및 TNBS가 처리된 실험동물에게 궤양성대장염 치료제인 설파살라진(sulfasalazine)을 투여한 실험 동물군은 'S'로 하여 표시하였다.In the horizontal axis of Figure 6 the experimental animal group not treated with TNBS 'N', the experimental animal group treated with only TNBS 'C', the experimental animal group administered Bifidobacterium longgum HY8004 to the TNBS-treated experimental animals '8004' and the experimental animal group treated with sulfasalazine (sulfasalazine) for treating ulcerative colitis to the experimental animals treated with TNBS is indicated as 'S'.
도 6에서 확인할 수 있는 바와 같이, TNBS 투여에 의해 실험동물의 대장길이가 축소되었으나, 비피도박테리움 롱검 HY8004 투여에 의해 대장길이 축소가 억제되어 대조구(S)의 대장길이와 유사한 수준인 것을 확인할 수 있었다(p<0.05).As can be seen in Figure 6, the colon length of the experimental animal was reduced by the TNBS administration, the colon length reduction is suppressed by the administration of Bifidobacterium longgum HY8004 to confirm that the level similar to the colon length of the control (S) (P <0.05).
<시험예 6><Test Example 6>
TNBS로 유도한 대장염 동물모델에서 비피도박테리움 롱검 HY8004 투여에 의한 대장조직의 Myeloperoxidase(MPO) 활성저해 측정Determination of Myeloperoxidase (MPO) Inhibition of Colon Tissue by Bifidobacterium Longgum HY8004 in TNBS-induced Colitis Animal Model
상기 시험예 5와 동일한 방법 및 조건으로 채취한 대장점막 조직에 0.5% hexa-decyl-trimethyl-ammonium bromide를 함유한 10mM 포타슘포스페이트 완충용액(pH 7.0)에 넣고 균질한 후 8000rpm에서 30분간 원심분리를 하였다. 이렇게 원심 분리된 상등액 100μl에 1.6mM tetra-methyl benzidine 100μl, 0.1% H2O2 5μl, 증류수 795μl를 넣고 650nm에서 time course로 미엘로퍼옥시다제(Myeloperoxidase)의 활성 변화를 측정하였다. 효소활성은 37℃에서 효소가 분해하는 과산화수소(H2O2)의 양을 말하여, μUnit/㎎ protein으로 표시하였다. 단백질량 측정은 Bradford 방법에 준하여 시행하였다. The colonic mucosal tissues obtained by the same method and conditions as in Test Example 5 were placed in 10 mM potassium phosphate buffer solution (pH 7.0) containing 0.5% hexa-decyl-trimethyl-ammonium bromide and homogenized, followed by centrifugation at 8000 rpm for 30 minutes. It was. 100 μl of the supernatant was centrifuged and 1.6 μM tetra-
그 결과를 도 7에 나타내었다.The results are shown in FIG.
도 7의 가로축에는 TNBS를 처리하지 하지 않은 실험 동물군을 'N', TNBS만 처리된 실험 동물군을 'C', TNBS가 처리된 실험동물에게 비피도박테리움 롱검 HY8004를 투여한 실험 동물군은 '8004' 및 TNBS가 처리된 실험동물에게 설파살라진을 투여한 실험 동물군은 'S'로 하여 표시하였다.In the horizontal axis of Figure 7 the experimental animal group not treated with TNBS 'N', the experimental animal group treated with only TNBS 'C', the experimental animal group administered Bifidobacterium longgum HY8004 to the TNBS-treated experimental animals The '8004' and the experimental animal group in which sulfasalazine was administered to the TNBS-treated experimental animals were designated as 'S'.
도 7에서 확인할 수 있는 바와 같이, TNBS 투여에 의해 실험동물의 대장 조직내 미엘로퍼옥시다제(MPO) 활성이 증가되었으나, 비피도박테리움 롱검 HY8004 투여에 의해 미엘로퍼옥시다제(MPO) 활성이 TNBS를 처리하지 않은 대조구(N)와 거의 비슷한 수준으로 감소되었음을 알 수 있었다. As can be seen in FIG. 7, myeloperoxidase (MPO) activity was increased in colon tissue of experimental animals by TNBS administration, but myeloperoxidase (MPO) activity was increased by administration of Bifidobacterium longgum HY8004. It can be seen that the reduced to almost the same level as the control (N) was not treated.
<시험예 7><Test Example 7>
TNBS로 유도한 대장염 동물모델에서 비피도박테리움 롱검 HY8004 투여에 의한 분변 베타-글루쿠로니다아제, 베타-글루코시다아제, 콘드로이티나아제 활성 저해의 측정Determination of Fecal Beta-glucuronidase, Beta-glucosidase, and Chondroitinase Activity Inhibition by Bifidobacterium Longgum HY8004 Administration in TNBS-Induced Colitis Animal Model
분변처리 Fecal Treatment
각 실험군의 마우스의 분변을 cold saline에 현탁 후 500rpm에서 5분간 원심분리하여 섬유질과 기타 불순물을 제거한 후 4℃, 8000rpm에서 30분간 원심분리하여 침전시켰다. 상등액은 제거하고 침전물을 분변 효소 활성 측정에 사용하였다. Mouse feces of each experimental group were suspended in cold saline and centrifuged at 500 rpm for 5 minutes to remove fibers and other impurities, and then precipitated by centrifugation at 4 ° C and 8000 rpm for 30 minutes. The supernatant was removed and the precipitate used for measuring fecal enzyme activity.
7-1 베타-글루쿠로니다아제 활성 측정 7-1 Beta-glucuronidase Activity Determination
분변 침천물을 0.1M 포타슘 포스페이트 완충용액(pH 7.0)으로 현탁한 후 10배 희석한 것을 조효소액으로 사용하였다. 조효소액 200μl, 0.1M 포타슘 포스페이트 완충용액(pH 7.0) 760μl, 그리고 기질(2mM p-nitrophenyl-β-glucuronide) 40μl를 60분간 37℃에서 반응시킨 후 0.5N NaOH 500μl를 가하여 반응을 정지시킨 다음 3000rpm에서 10분간 원심분리하여 얻은 상등액에 대해 405nm에서 흡광도를 측정하였다. Fecal precipitate was suspended in 0.1 M potassium phosphate buffer (pH 7.0) and diluted 10-fold as a coenzyme solution. After 200 μl of coenzyme solution, 760 μl of 0.1 M potassium phosphate buffer (pH 7.0), and 40 μl of substrate (2 mM p-nitrophenyl-β-glucuronide) were reacted at 37 ° C. for 60 minutes, 0.5 N NaOH 500 μl was added to stop the reaction, followed by 3000 rpm The absorbance at 405 nm was measured for the supernatant obtained by centrifugation for 10 minutes at.
그 결과를 도 8에 나타내었다.The results are shown in FIG.
도 8의 가로축에는 TNBS를 처리하지 하지 않은 실험 동물군을 'N', TNBS만 처리된 실험 동물군을 'C', TNBS가 처리된 실험동물에게 비피도박테리움 롱검 HY8004를 투여한 실험 동물군은 '8004' 및 TNBS가 처리된 실험동물에게 설파살라진을 투여한 실험 동물군은 'S'로 하여 표시하였다.In the horizontal axis of Figure 8 the experimental animal group not treated with TNBS 'N', the experimental animal group treated with only TNBS 'C', the experimental animal group administered Bifidobacterium long gum HY8004 to the TNBS-treated experimental animals The '8004' and the experimental animal group in which sulfasalazine was administered to the TNBS-treated experimental animals were designated as 'S'.
도 8에서 확인할 수 있는 바와 같이, TNBS 투여에 의해 실험동물의 분변내 베타-글루쿠로니다아제 활성이 증가되었으나, 비피도박테리움 롱검 HY8004 투여에 의해 베타-글루쿠로니다아제 활성이 감소되었음을 알 수 있었다. As can be seen in FIG. 8, the fecal beta-glucuronidase activity was increased by administration of TNBS, but beta-glucuronidase activity was decreased by administration of Bifidobacterium longgum HY8004. Could know.
7-2 베타-글루코시다아제 활성 측정7-2 Beta-glucosidase Activity Measurement
분변 침전물을 100mM 포타슘 포스페이트 완충용액(pH 7.0)으로 현탁한 후 10배 희석한 것을 조효소액으로 사용하였다. 조효소액 50μl, 0.1M phosphate buffer 350μl, 기질(20mM p-nitrophenyl-β-glucopyranoside) 100μl를 60분간 37℃에서 반응시킨 후 0.5N NaOH 400μl를 가하여 반응을 정지시킨 다음 3000rpm에서 10분간 원심분리하여 얻은 상등액에 대해 405nm에서 흡광도를 측정하였다. Fecal precipitate was suspended in 100 mM potassium phosphate buffer (pH 7.0) and diluted 10-fold as a coenzyme solution. 50 μl of crude enzyme solution, 350 μl of 0.1 M phosphate buffer, and 100 μl of substrate (20 mM p-nitrophenyl-β-glucopyranoside) were reacted at 37 ° C. for 60 minutes, and then 400 μl of 0.5N NaOH was added to stop the reaction, followed by centrifugation at 3000 rpm for 10 minutes. The absorbance was measured at 405 nm for the supernatant.
그 결과를 도 9에 나타내었다.The results are shown in FIG.
도 9의 가로축에는 TNBS를 처리하지 하지 않은 실험 동물군을 'N', TNBS만 처리된 실험 동물군을 'C', TNBS가 처리된 실험동물에게 비피도박테리움 롱검 HY8004를 투여한 실험 동물군은 '8004' 및 TNBS가 처리된 실험동물에게 설파살라진을 투여한 실험 동물군은 'S'로 하여 표시하였다.In the horizontal axis of Figure 9 the experimental animal group not treated with TNBS 'N', the experimental animal group treated with only TNBS 'C', experimental animal group administered Bifidobacterium longgum HY8004 to the TNBS-treated experimental animals The '8004' and the experimental animal group in which sulfasalazine was administered to the TNBS-treated experimental animals were designated as 'S'.
도 9에서 확인할 수 있는 바와 같이, TNBS 투여에 의해 실험동물의 분변내 베타-글루코시다아제 활성이 증가되었으나, 비피도박테리움 롱검 HY8004 투여에 의해 베타-글루코시다아제 활성이 감소되었음을 알 수 있었다.As can be seen in FIG. 9, the fecal beta-glucosidase activity was increased by TNBS administration, but it was found that beta-glucosidase activity was decreased by the administration of Bifidobacterium longgum HY8004.
7-3 콘드로이티나아제 활성 측정 7-3 Measurement of Chondroitinase Activity
분변 침전물을 100mM 포타슘 포스페이트 완충용액(pH 7.0)으로 현탁한 후 10배 희석한 것을 조효소액으로 사용하였다. 조효소액 600μl, chondroitin sulfate A(0.1mg/ml) 200μl를 60분간 37℃에서 반응시킨 후 3000rpm, 4℃에서 원심분리하였다. 상등액 500μl를 취하여 0.4M NaOH 100μl, 0.4M potassium borate 100μl와 혼합한 후 5분간 끓였다. 실온으로 냉각한 다음 67mM ρ-dimethylaminobenzaldehyde 3ml을 가한 후 다시 37℃에서 20분간 반응시키고 585nm에서 흡광도를 측정하였다. Fecal precipitate was suspended in 100 mM potassium phosphate buffer (pH 7.0) and diluted 10-fold as a coenzyme solution. 600 μl of crude enzyme solution and 200 μl of chondroitin sulfate A (0.1mg / ml) were reacted at 37 ° C. for 60 minutes, and then centrifuged at 3000 rpm and 4 ° C. 500 μl of the supernatant was mixed with 100 μl of 0.4M NaOH and 100 μl of 0.4M potassium borate and then boiled for 5 minutes. After cooling to room temperature, 3 ml of 67 mM ρ-dimethylaminobenzaldehyde was added thereto, followed by reaction at 37 ° C. for 20 minutes, and absorbance at 585 nm was measured.
그 결과를 도 10에 나타내었다.The results are shown in FIG.
도 10의 가로축에는 TNBS를 처리하지 하지 않은 실험 동물군을 'N', TNBS만 처리된 실험 동물군을 'C', TNBS가 처리된 실험동물에게 비피도박테리움 롱검 HY8004를 투여한 실험 동물군은 '8004' 및 TNBS가 처리된 실험동물에게 설파살라진을 투여한 실험 동물군은 'S'로 하여 표시하였다.In the horizontal axis of FIG. 10, the experimental animal group not treated with TNBS 'N', the experimental animal group treated with only TNBS 'C', and the experimental animal group administered Bifidobacterium long gum HY8004 to the TNBS-treated experimental animal. The '8004' and the experimental animal group in which sulfasalazine was administered to the TNBS-treated experimental animals were designated as 'S'.
도 10에서 확인할 수 있는 바와 같이, TNBS 투여에 의해 실험동물의 분변내 베타-글루코시다아제 활성이 증가되었으나, 비피도박테리움 롱검 HY8004 투여에 의해 베타-글루코시다아제 활성이 감소 되었음을 알 수 있었다.As can be seen in FIG. 10, the fecal beta-glucosidase activity of the experimental animals was increased by TNBS administration, but beta-glucosidase activity was decreased by the administration of Bifidobacterium longgum HY8004.
도 1은 비피도박테리움 롱검 HY8004의 박테로이데스 불가투스 억제 효과를 나타낸 그래프이다.1 is a graph showing the inhibitory effect of Bacteroides vulgarus of Bifidobacterium longgum HY8004.
도 2는 비피도박테리움 롱검 HY8004의 박테로이드 물티아시더스 억제 효과를 나타낸 그래프이다.Figure 2 is a graph showing the inhibitory effect of Bifidobacterium longgum HY8004 bacteroid thiathiacis.
도 3은 비피도박테리움 롱검 HY8004의 세포 생존율 증가 효과를 나타낸 그래프이다.Figure 3 is a graph showing the effect of increasing the cell viability of Bifidobacterium longgum HY8004.
도 4는 비피도박테리움 롱검 HY8004의 hTLR4 억제 효과를 나타낸 그래프이다.4 is a graph showing the hTLR4 inhibitory effect of Bifidobacterium long gum HY8004.
도 5는 비피도박테리움 롱검 HY8004의 IL-10 증가 효과를 나타낸 그래프이다.5 is a graph showing the IL-10 increase effect of Bifidobacterium long gum HY8004.
도 6은 TNBS로 유도한 대장염 실험동물에서 비피도박테리움 롱검 HY8004 처리에 의한 대장길이 축소 저해 효과를 측정한 결과를 나타낸 그래프이다.Figure 6 is a graph showing the results of measuring the inhibition of colon length reduction by treatment with Bifidobacterium longgum HY8004 in TNBS-induced colitis experimental animals.
도 7은 TNBS로 유도한 대장염 실험동물에서 비피도박테리움 롱검 HY8004 처리에 의한 미엘로퍼옥시다제(Myeloperoxidase) 활성 저해 효과를 측정한 결과를 나타낸 그래프이다.7 is a graph showing the results of measuring the inhibitory effect of myeloperoxidase activity by treatment with Bifidobacterium longgum HY8004 in TNBS-induced colitis test animals.
도 8은 TNBS로 유도한 대장염 실험동물에서 비피도박테리움 롱검 HY8004 처리에 의한 베타-글루쿠로니다아제 활성 저해 효과를 측정한 결과를 나타낸 그래프이다.8 is a graph showing the results of measuring the inhibitory effect of beta-glucuronidase activity by treatment with Bifidobacterium longgum HY8004 in TNBS-induced colitis test animals.
도 9는 TNBS로 유도한 대장염 실험동물에서 비피도박테리움 롱검 HY8004 처리에 의한 베타-글루코시다아제 활성 저해 효과를 측정한 결과를 나타낸 그래프이다.9 is a graph showing the results of measuring the inhibitory effect of beta-glucosidase activity by treatment with Bifidobacterium longgum HY8004 in TNBS-induced colitis test animals.
도 10은 TNBS로 유도한 대장염 실험동물에서 비피도박테리움 롱검 HY8004 처 리에 의한 콘드로이티나아제 활성 저해 효과를 측정한 결과를 나타낸 그래프이다.10 is a graph showing the results of measuring the inhibitory effect of chondroitinase activity by treatment with Bifidobacterium longgum HY8004 in TNBS-induced colitis test animals.
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