KR20100109661A - Lactobacillus curvatus hy7601 having inhibitory activity against blood cholestrol and obesity, and product containing thereof as an effective factor - Google Patents

Lactobacillus curvatus hy7601 having inhibitory activity against blood cholestrol and obesity, and product containing thereof as an effective factor Download PDF

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KR20100109661A
KR20100109661A KR1020090028025A KR20090028025A KR20100109661A KR 20100109661 A KR20100109661 A KR 20100109661A KR 1020090028025 A KR1020090028025 A KR 1020090028025A KR 20090028025 A KR20090028025 A KR 20090028025A KR 20100109661 A KR20100109661 A KR 20100109661A
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lactobacillus curvatus
lactobacillus
obesity
cholesterol
blood
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KR100996577B1 (en
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박도영
이정희
안영태
허철성
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주식회사한국야쿠르트
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66Microorganisms or materials therefrom
    • A61K35/74Bacteria
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66Microorganisms or materials therefrom
    • A61K35/74Bacteria
    • A61K35/741Probiotics
    • A61K35/744Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747Lactobacilli, e.g. L. acidophilus or L. brevis
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20Bacteria; Culture media therefor
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12RINDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00Microorganisms ; Processes using microorganisms
    • C12R2001/01Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225Lactobacillus

Abstract

PURPOSE: A novel Lactobacillus curvatus HY7601 and composition containing the same are provided to increase inflow of cholesterol through LDL-receptor increase. CONSTITUTION: A novel Lactobacillus curvatus HY7601(deposit number: KCTC 11456BP) has efficiency of reducing cholesterol in blood and obesity. A pharmaceutical composition for reducing cholesterol in blood and obesity contains Lactobacillus curvatus HY7601 as an active ingredient. A fermentation milk for suppressing cholesterol in blood and obesity contains Lactobacillus curvatus HY7601 as an active ingredient.

Description

혈중 콜레스테롤 강하와 비만 억제 효능을 갖는 신규의 락토바실러스 커베터스 에이취와이7601 및 이를 유효성분으로 함유하는 제품{Lactobacillus curvatus HY7601 having inhibitory activity against blood cholestrol and obesity, and product containing thereof as an effective factor}Novel Lactobacillus curvatus HY7601 having inhibitory activity against blood cholestrol and obesity, and product containing describes as an effective factor

본 발명은 혈중 콜레스테롤 강하와 비만 억제 효능을 갖는 신규의 락토바실러스 커베터스(Lactobacillus curvatus) HY7601 및 이를 유효성분으로 함유하는 약학적 조성물, 발효유, 음료, 건강기능식품에 관한 것으로서, 보다 상세하게는 저밀도 지단백 수용체(LDL-receptor) 증가를 통해 콜레스테롤의 세포 내 유입을 증가시킴으로써 궁극적으로 혈중 콜레스테롤의 양을 저하 시키는 효능 및 비만 억제 효능을 갖는 신규의 락토바실러스 커베터스(Lactobacillus curvatus) HY7601 및 이를 유효성분으로 함유하는 약학적 조성물, 발효유, 음료, 건강기능식품에 관한 것이다.The present invention relates to a novel Lactobacillus curvatus HY7601 having a blood cholesterol lowering effect and an obesity inhibitory effect, and a pharmaceutical composition containing the same as an active ingredient, fermented milk, beverages, and health functional foods. A novel Lactobacillus curvatus HY7601 with the effect of lowering the amount of cholesterol in the blood by increasing intracellular intake of cholesterol through increasing LDL-receptor and the effect of inhibiting obesity. It relates to a pharmaceutical composition, fermented milk, beverages, health functional foods containing as an ingredient.

최근 한국인의 혈중 콜레스테롤 농도가 높아진 결과 뇌혈관장애, 심질환, 고혈압 등의 질병 발생 역시 매년 증가하고 있다. 이러한 변화는 동물성 지방과 단백 질 섭취량이 현저히 증가한 한국인의 식생활 변화에서 그 원인을 찾을 수 있다.As a result of the recent increase in blood cholesterol levels in Koreans, the incidence of diseases such as cerebrovascular disorders, heart disease, and high blood pressure is also increasing every year. This change may be attributable to changes in the diet of Koreans with a significant increase in animal fat and protein intake.

혈중 콜레스테롤의 대부분은 저밀도 지단백(low-density lipoprotein, 이하 'LDL'이라 함) 형태로 존재하고 이는 세포막의 저밀도 지단백 수용체(low-density lipoprotein receptor, 이하 'LDL-receptor'라 함)를 통해 세포 내로 이동된다. 세포 내 콜레스테롤의 농도가 높을 경우 LDL-receptor의 합성속도는 감소되며 혈액으로부터 세포 내로의 콜레스테롤의 흡수가 느려지고 그 결과 혈중 콜레스테롤이 축적된다.Most of the cholesterol in the blood is in the form of low-density lipoprotein (LDL), and it is introduced into the cell through the low-density lipoprotein receptor (LDL-receptor) of the cell membrane. Is moved. High concentrations of intracellular cholesterol decrease the synthesis rate of LDL-receptor and slow down the absorption of cholesterol from the blood into the cell, resulting in the accumulation of blood cholesterol.

현재 혈중 콜레스테롤의 수준을 낮추는 약물로서 니아신(niacin), 콜레스티라민(cholestyramine), 스타틴(statins) 등이 널리 사용되고 있다. 니아신은 간에서 초저밀도 지단백(very low-density lipoprotein)의 분비를 억제시키는 작용을 하며, 콜레스티라민과 같은 담즙산 격리제는 배출된 담즙산이 간으로 재흡수 되는 것을 막음으로써 자연스럽게 담즙산 생합성 원료인 콜레스테롤의 소모를 증가시킨다. 컴팩틴(compactin) 유도체인 로바스타틴(lovastatin), 프로바스타틴(pravastatin), 심바스타틴(simvastatin) 등은 가장 높은 시장 점유율을 확보하고 있는 약물로서 콜레스테롤 생합성 조절효소인 HMG-CoA 환원효소(3-hydroxy-methyl-glutaryl coenzyme A reductase)의 활성을 저해한다. 그 결과 LDL-receptor의 발현량이 증가되고 LDL의 세포 내 유입이 증가됨에 따라 혈중 콜레스테롤 양이 감소된다. 이외에도 콜레스테롤 전달효소인 acyl-CoA:cholesterol acyltransferase(ACAT)의 활성을 저해하거나 고밀도 지단백(high-density lipoprotein)에 있는 콜레스테롤을 LDL로 전달하는 효소인 콜레스테롤 에스테르 전이 단백질(cholesterol ester transfer protein; CETP)을 저해하는 등 혈중 콜레스테롤을 억제하고자 하는 시도가 많이 진행되고 있다.Currently, niacin, cholestyramine, and statins are widely used as drugs to lower blood cholesterol levels. Niacin acts to inhibit the secretion of very low-density lipoproteins in the liver, and bile acid sequestrants such as cholestyramine prevent cholesterol from being reabsorbed into the liver, which naturally leads to bile acid biosynthesis. To increase the consumption. Compactin derivatives, lovastatin, lovastatin, pravastatin, and simvastatin, have the highest market share and are HMG-CoA reductase (3-hydroxy-methyl), a cholesterol biosynthesis regulator. -glutaryl coenzyme A reductase) inhibits the activity. As a result, the amount of LDL-receptor is increased and the amount of cholesterol in the blood decreases as the influx of LDL increases. In addition, cholesterol ester transfer protein (CETP), an enzyme that inhibits the activity of cholesterol transfer enzyme acyl-CoA: cholesterol acyltransferase (ACAT) or transfers cholesterol in high-density lipoprotein to LDL, Attempts have been made to suppress blood cholesterol, including inhibition.

하지만 이러한 약제는 간독성, 위장장해, 발암성 등의 부작용이 있기 때문에 약물요법 보다도 지질 섭취량을 적정 범위로 제한하는 식사요법이 더욱 중시된다. 그러나 식사 제한 또한 정신적 고통을 수반함과 동시에 식생활의 즐거움을 빼앗기 때문에 엄격한 실시는 곤란하고 효과에 한계가 있는 것이 많다.However, since these drugs have side effects such as hepatotoxicity, gastrointestinal disorder, and carcinogenicity, diet therapy that limits the intake of lipids to an appropriate range is more important than drug therapy. However, since dietary restrictions also entail mental pain and deprivation of the enjoyment of eating, strict enforcement is difficult and often has limitations.

이에 반해 안전한 미생물로 여겨져 온 유산균을 이용하여 혈중 콜레스테롤 수준을 감소시키고자 하는 노력 역시 많이 이루어졌다. 유산균은 장내 정상균총의 유지, 장내 균총의 개선, 항당뇨 및 항고지혈증 효과, 발암 억제, 대장염 억제, 그리고 숙주의 면역체계의 비특이적 활성 등의 효과를 나타낸다고 보고 되고 있다. 그 중에서도 락토바실러스 속 균주는 인체의 장내에 서식하는 정상 미생물 군집의 주요 구성원으로서, 건강한 소화기관과 질내 환경을 유지하는 데 있어서 중요한 것으로 오래 전부터 알려져 왔고 미국의 공중건강 가이드라인(U.S. Public Health Service guidelines)에 의하면, 현재 미국 균주 기탁기관(ATCC)에 기탁된 락토바실러스 균주 모두 인체나 동물에 질병을 유발할 잠재적 위험에 대해서는 알려진 것이 없다고 인정되는 '안정수준(Bio-safty Level) 1'로 분류되어 있다.On the other hand, many efforts have been made to reduce blood cholesterol levels using lactic acid bacteria, which have been considered safe microorganisms. Lactobacillus has been reported to show the effects of maintaining normal intestinal flora, improving intestinal flora, antidiabetic and antihyperlipidemic effects, inhibiting carcinogenesis, inhibiting colitis, and nonspecific activity of the host's immune system. Among them, the strain Lactobacillus is a major member of the normal microbial community in the intestine of the human body, which has long been known to be important in maintaining a healthy digestive system and the vaginal environment, and the US Public Health Service guidelines. ), All of the Lactobacillus strains currently deposited with the American Strain Deposit Agency (ATCC) are classified as 'Bio-safty Level 1', which is not known for any potential risk of causing disease in humans or animals. .

락토바실러스 균주의 콜레스테롤 저하 기작으로서 잘 알려진 것은 콜레스티라민(cholestyramine)의 것과 동일하다. 즉, 배출된 담즙산이 락토바실러스 균주에 의해 탈접합(deconjugation)된 결과 간으로 재흡수 되지 못하게 되어, 결국 담즙산 생합성 원료인 콜레스테롤의 소모가 증가되고 자연스럽게 콜레스테롤의 양이 감소하게 되는 것이다. 하지만 이외의 기작에 대해서는 아직 잘 알려지지 않고 있다.The well known cholesterol lowering mechanism of Lactobacillus strains is the same as that of cholestyramine. That is, the discharged bile acid is not desorbed into the liver as a result of deconjugation by the Lactobacillus strain, resulting in increased consumption of cholesterol, a bile acid biosynthetic raw material, and naturally reduced amount of cholesterol. However, other mechanisms are still unknown.

이에 본 발명자들은 락토바실러스 커베터스(Lactobacillus curvatus)에 의해 세포막의 LDL-receptor 발현량이 증가하고 콜레스테롤의 세포 내 유입이 증가함에 따라 궁극적으로 혈중 콜레스테롤의 감소와 비만 억제 사실을 확인함으로써 본 발명을 완성하게 되었다.Accordingly, the present inventors completed the present invention by confirming the fact that the LDL-receptor expression of the cell membrane is increased by Lactobacillus curvatus and the cholesterol inflow is ultimately decreased and the inhibition of obesity is increased as Lactobacillus curvatus is increased. Was done.

따라서, 본 발명은 혈중 콜레스테롤 강하와 비만 억제 효능을 갖는 신규의 락토바실러스 커베터스(Lactobacillus curvatus) 및 이를 유효성분으로 함유하는 약학적 조성물, 발효유, 음료, 건강기능식품을 제공하는 것을 목적으로 한다.Accordingly, an object of the present invention is to provide a novel Lactobacillus curvatus ( Lactobacillus curvatus ) and a pharmaceutical composition containing the same as an active ingredient, fermented milk, beverages, health functional foods having a blood cholesterol lowering and obesity inhibitory effect .

상기한 목적을 달성하기 위하여, 본 발명은 세포막의 LDL-receptor 발현량과 세포 내 콜레스테롤 유입을 증가시킴으로써 혈중 콜레스테롤 수준을 저하 시키는 동시에 비만 억제 효능을 갖는 신규의 락토바실러스 커베터스(Lactobacillus curvatus) HY7601을 제공하는 것을 특징으로 한다.In order to achieve the above object, the present invention provides a novel Lactobacillus curvatus HY7601 which has an effect of inhibiting obesity while lowering blood cholesterol level by increasing LDL-receptor expression level and intracellular cholesterol influx of cell membranes. It is characterized by providing.

이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.

본 발명에 따른 균주를 분리하기 위하여 국내 가정에서 제조한 김치를 0.02% 소디움 아지드(sodium azide)가 포함된 엠알에스(MRS) 액체 배지에 넣고 37℃에서 24시간 배양한 후, 10㎕ 백금이를 사용하여 배양액을 취하여 다시 0.02% 소디움 아지드가 포함된 엠알에스 한천 평판배지에 도말하고 37%에서 48시간 동안 배양하였다. 이렇게 형성된 균락 중에서 Sterol regulatory element-binding protein 2(이하 'SREBP-2'라 함)의 활성을 시험하여 본 발명의 균주를 분리하였다.In order to isolate the strain according to the present invention, kimchi prepared in domestic homes in a liquid medium containing MRS (MRS) containing 0.02% sodium azide (MRS) incubated for 24 hours at 37 ℃, 10μl platinum The culture medium was taken using and smeared again on MLS agar plate medium containing 0.02% sodium azide and incubated for 48 hours at 37%. The strain of the present invention was isolated by testing the activity of Sterol regulatory element-binding protein 2 (hereinafter referred to as 'SREBP-2') among the fungi thus formed.

즉, 지질 대사 관련 유전자(HMGCR, LDL-receptor 등)들의 전사인자(transcriptional factor)인 SREBP-2는 세포 내 콜레스테롤 항상성에 기여하는 중요한 유전자로서 세포 내 콜레스테롤 양이 감소하면 SREBP-2의 활성이 증가하게 되어 SREBP-2의 하위 유전자인 LDL-receptor의 발현량 역시 증가함에 따라 혈액 내의 콜레스테롤이 세포 내로 유입됨으로써 혈중 콜레스테롤의 양이 줄어들게 되는 효과가 나타난다는 사실을 근거로 상기 균락 중에 있는 여러 유산균 중에서HepG2 세포 내에서 SREBP-2의 활성을 증가 시키는 활성을 갖는 균주를 분리하였다.In other words, SREBP-2, a transcription factor of lipid metabolism-related genes (HMGCR, LDL-receptor, etc.), is an important gene that contributes to intracellular cholesterol homeostasis. As the expression level of LDL-receptor, a subordinate gene of SREBP-2, also increased, cholesterol in the blood flowed into the cells, reducing the amount of cholesterol in the blood. Strains with activity that increase the activity of SREBP-2 in cells were isolated.

이렇게 분리한 균주의 관찰은 MRS 한천평판배지에서 37℃로 48시간 배양한 후 그람염색(Cowan, 1974)을 통하여 분리 균주의 크기와 형태를 광학 현미경(SAMWON, CSB-FEI)으로 관찰하였으며, 분리한 균주의 생화학적 특성은 Cowan과 Steel(1984) 그리고 Macfaddin(1984)의 방법에 따라 균주의 특성을 조사한 후 Bergey's Manual of Systematic Bacteriology와 Bergey's Manual of Determinative Bacteriology에 준하여 분류 및 동정하였다. 카탈라제(Catalase) 테스트는 3% 과산화수소를 첨가한 후 산소 발생유무를 관찰하였고, 옥시다제(Oxydase)테스트는 1% dimethyl-p-phenylenediamine gydrochloride를 넣고 변색 여부를 관찰하였다.Observation of the isolated strains was incubated for 48 hours at 37 ℃ in MRS agar plate medium and the size and shape of the isolated strains were observed by light microscopy (SAMWON, CSB-FEI) through Gram staining (Cowan, 1974). The biochemical characteristics of a strain were classified and identified according to Bergey's Manual of Systematic Bacteriology and Bergey's Manual of Determinative Bacteriology after the characteristics of the strains were investigated according to the methods of Cowan, Steel (1984) and Macfaddin (1984). Catalase test was used to observe the presence of oxygen after adding 3% hydrogen peroxide. Oxidase test was carried out by adding 1% dimethyl-p-phenylenediamine gydrochloride to observe discoloration.

따라서, 본 발명에 따른 신규의 유산균의 특성은 다음과 같다.Therefore, the characteristics of the novel lactic acid bacteria according to the present invention are as follows.

1)균의 형태1) Form of bacteria

엠알에스(MRS) 한천평판배지에서 37℃, 2일간 배양했을 때 균의 특성Characteristics of Bacteria When incubated in MRS Agar Plate Medium at 37 ° C for 2 Days

①세포의 형태: 간균① Cell Type: Bacillus

②운동성: 없음② Mobility: None

③포자형성능: 없음③ Sporulation ability: None

④그람(Gram) 염색: 양성④Gram staining: positive

2)균락의 형태2) Form of the collapse

엠알에스(MRS) 한천평판배지에서 37℃, 2일간 배양했을 때 균락의 형태Form of fungi when incubated for 2 days at 37 ° C on MRS agar plate medium

①형상: 원형① Shape: Round

②융기: 볼록② bump: convex

③표면: 매끄러움(Smooth)③ Surface: Smooth

3)생리적 성질3) physiological properties

①생육온도: 생장가능 생육온도: 15~40℃① Growth temperature: Growth possible Growth temperature: 15 ~ 40 ℃

최적 생장온도: 36~38℃Optimum growth temperature: 36 ~ 38 ℃

②생육 pH: 생장가능 생육 pH: 5.0~7.5② Growth pH: Growth possible Growth pH: 5.0 ~ 7.5

최적 pH: 6.0~6.5Optimum pH: 6.0-6.5

③산소에 대한 영향: 통성혐기성③ Effects on oxygen: anaerobic 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)당 발효 실험 및 동정10) sugar fermentation experiment and identification

Biomerieux 사의 api 50 CH kit를 이용하여 당 발효 실험을 한 결과를 표 1과 도 1에 나타내었다.The results of the sugar fermentation experiment using the api 50 CH kit of Biomerieux are shown in Table 1 and FIG. 1.

Figure 112009019663053-PAT00001
Figure 112009019663053-PAT00001

위와 같은 균의 형태학적, 생리적 및 생장 특성에 근거하여 본 발명의 균주는 락토바실러스 커베터스(Lactobacillus curvatus)로 동정하였고, 본 발명자들은 이를 락토바실러스 커베터스(Lactobacillus curvatus) HY7601로 명명하고, 2009.01.22.자로 국제기탁기관인 한국생명공학연구원(기탁번호: KCTC 11456BP)에 기탁하였다.Based on the morphological, physiological and growth characteristics of the above bacteria, the strain of the present invention was identified as Lactobacillus curvatus ( Lactobacillus curvatus ), the inventors named it Lactobacillus curvatus ( HYacte ) HY7601, As of January 22, 2009, the fund was deposited with the Korea Biotechnology Research Institute (Accession No .: KCTC 11456BP).

한편, 본 발명의 혈중 콜레스테롤 강하 및 비만 억제 효능을 갖는 신규의 락토바실러스 커베터스 HY7601을 유효성분으로 함유하는 혈중 콜레스테롤 강하 및 비만 억제용 약학적 조성물은 단독 또는 약제학적으로 사용되는 부형제들과 함께 약제학적으로 통상으로 사용되는 방법에 따라 정제, 캡슐제 등과 같은 제재형태로 제재화하여 사용될 수 있다.Meanwhile, the pharmaceutical composition for inhibiting blood cholesterol drop and obesity, which contains a novel Lactobacillus catheterus HY7601 having an effect of inhibiting blood cholesterol drop and obesity of the present invention as an active ingredient, is used alone or in combination with excipients used pharmaceutically. It may be used by formulating the formulation in the form of tablets, capsules and the like according to the method commonly used pharmaceutically.

사람의 경우, 통상적인 1일 투여량은 1~30㎎/㎏ 체중의 범위일 수 있고, 1회 또는 수회로 나누어 투여할 수 있다. 그러나 실제 투여량은 투여경로, 환자의 연령, 성별, 체중, 건강상태 및 질환의 중증도 등의 여러 관련 인자에 비추어 결정되어야 한다.For humans, typical daily dosages can range from 1-30 mg / kg body weight, and can 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, age, sex, weight, health status and severity of the disease.

물론, 본 발명의 상기 약학적 조성물은 독성 및 부작용은 거의 없으므로 장기간 복용시에도 안심하고 사용할 수 있는 약제이다.Of course, the pharmaceutical composition of the present invention is a drug that can be used with confidence even when taken for a long time because there is little toxicity and side effects.

또한, 혈중 콜레스테롤 강하 및 비만 억제 효능을 갖는 신규의 락토바실러스 커베터스 HY7601을 유효성분으로 함유하는 발효유는 유산균 배양액, 락토바실러스 커베터스 HY7601 및 혼합과즙시럽을 일정비율로 조합하여 150bar에서 균질한 후 10℃ 이하로 냉각한 후 용기에 포장하여 제조한다.In addition, the fermented milk containing a novel Lactobacillus cabbetts HY7601 having an effect of lowering blood cholesterol and anti-obesity as an active ingredient is homogenized at 150 bar by combining a lactobacillus culture solution, Lactobacillus cabbetts HY7601 and mixed fruit syrup in a constant ratio. After cooling to 10 ℃ or less and then packaged in a container to prepare.

또한, 혈중 콜레스테롤 강하 및 비만 억제 효능을 갖는 신규의 락토바실러스 커베터스 HY7601을 유효성분으로 함유하는 음료는 혼합과즙시럽, 락토바실러스 커베터스 HY7601 및 물을 일정한 비율로 조합하여 150bar에서 균질한 후 10℃ 이하로 냉각한 후 유리병, 패트병 등 소포장 용기에 포장하여 제조한다.In addition, the beverage containing the novel Lactobacillus cabbetts HY7601 as an active ingredient having blood cholesterol lowering and obesity inhibitory effect was homogenized at 150 bar by combining a mixed juice syrup, Lactobacillus cabbetts HY7601 and water at a constant ratio. After cooling to 10 ° C. or less, the product is packaged in a small packaging container such as a glass bottle or a plastic bottle.

또한, 혈중 콜레스테롤 강하 및 비만 억제 효능을 갖는 신규의 락토바실러스 커베터스 HY7601을 유효성분으로 함유하는 건강기능식품은 상기 락토바실러스 커베터스 HY7601을 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, a health functional food containing a novel Lactobacillus corbetts HY7601 having an effect of lowering blood cholesterol and obesity as an active ingredient includes vitamins B1, B2, B5, B6, E and acetic acid esters, nicotinic acid amides, oligosaccharides and the like may be added and other food additives may be added.

이상에서 설명한 바와 같이, 본 발명에 따른 락토바실러스 커베터스(Lactobacillus curvatus) HY7601은 혈중 콜레스테롤과 중성지방의 양을 감소시킴은 물론 비만 억제 효과가 매우 뛰어나므로 락토바실러스 커베터스(Lactobacillus curvatus) HY7601을 이용한 지질대사에 영향을 미치는 기능성 식품 및 약학 조성물 등으로 이용될 수 있다.As described above, Lactobacillus curvatus HY7601 according to the present invention reduces the amount of cholesterol and triglycerides in the blood, as well as excellent anti-obesity effect, so Lactobacillus curvatus ( Lactobacillus curvatus ) HY7601 It can be used as a functional food and pharmaceutical composition affecting the lipid metabolism using.

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>&Lt; Example 1 >

락토바실러스 커베터스 HY7601을 포함한 동결건조 분말 제조Preparation of lyophilized powders, including Lactobacillus cavities HY7601

본 발명의 락토바실러스 커베터스 HY7601은 식품원료용 Proteose peptone #3, Yeast Extract, Beef Extract, 그리고 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 혈중 콜레스테롤 강하 소재로 사용하였다.The lactobacillus corbettus HY7601 of the present invention is a liquid medium containing Proteose peptone # 3 for food ingredients, Yeast Extract, Beef Extract, and glucose, incubated at 37 ° C. for about 16 hours, and then centrifuged and sterilized. It was washed with physiological saline and then dispersed in sterile oil. Freeze-drying again yielded about 10 11 cfu bacteria per gram (g) of lyophilized powder. This lyophilized powder was used as a blood cholesterol lowering material.

한편 본 발명의 락토바실러스 커베터스 HY7601은 상기와 같이 동결건조된 분말 형태 또는 배양물 형태로 제공될 수 있다.Meanwhile, the Lactobacillus corpus HY7601 of the present invention may be provided in the form of a lyophilized powder or culture.

<실시예 2><Example 2>

락토바실러스 커베터스 HY7601을 유효성분으로 함유하는 약학적 조성물의 제조Preparation of a pharmaceutical composition containing Lactobacillus cabbetts HY7601 as an active ingredient

본 발명의 혈중 콜레스테롤 강하 및 비만 억제 효능을 갖는 신규의 락토바실러스 커베터스 HY7601을 유효성분으로 함유하는 약학적 조성물의 제제 예를 설명하나, 본 발명은 이를 한정하고자 함이 아닌 단지 구체적으로 설명하고자 함이다.Formulation examples of the pharmaceutical composition containing a novel Lactobacillus cabbetts HY7601 having an effective effect of lowering blood cholesterol and obesity of the present invention as an active ingredient, but the present invention is not intended to limit the present invention but only to be described in detail. It is.

정제의 제조Manufacture of tablets

상기 실시예 1의 락토바실러스 커베터스 HY7601을 포함한 동결건조분말 100㎎, 옥수수전분 100㎎, 유당 100㎎ 및 폴리비닐피롤리돈 97㎎을 균질하게 혼합하여 습식과립법으로 과립화하고 스테아린산 마그네슘 2㎎을 가하여 혼합한 후 1정이 400㎎이 되도록 타정하였다.Lyophilized powder 100mg, Lactobacillus cabbetts HY7601 of Example 1, 100mg cornstarch, 100mg lactose and 97mg polyvinylpyrrolidone were homogeneously mixed and granulated by wet granulation method and magnesium stearate 2 MG was added and mixed, and then one tablet was compressed to 400 mg.

캡슐제의 제조Preparation of Capsules

상기 실시예 1의 락토바실러스 커베터스 HY7601을 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.Lyophilized powder 100mg, Lactobacillus cavities HY7601 of Example 1 100mg, corn starch 100mg, lactose 100mg, magnesium stearate 2mg thoroughly mixed in the hard gelatin capsules according to the manufacturing method of conventional capsules The capsule was prepared by filling.

<실시예 3><Example 3>

락토바실러스 커베터스 HY7601을 유효성분으로 함유하는 발효유의 제조Preparation of fermented milk containing Lactobacillus cabbetts HY7601 as an active ingredient

유산균 배양액과 본 발명의 혈중 콜레스테롤 강하 및 비만 억제 효능을 갖는 락토바실러스 커베터스 HY7601 및 혼합과즙시럽으로 구성된 발효유를 제조하는 방법은 다음과 같다.Method for producing a fermented milk consisting of Lactobacillus culture medium and Lactobacillus cabbetts HY7601 having a blood cholesterol lowering and obesity inhibitory effect of the present invention and mixed fruit juice syrup are as follows.

먼저, 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 브릭스(Brixo)는 16.3~16.5% 정도가 되도록 혼합하였다. 혼 합 후에 이를 UHT 열처리(135℃에서 2초간 살균)하고 40℃로 냉각한 뒤, 스트렙토코커스 써모필러스균과 유당분해효소(Valley laboratory, USA)를 각기 0.02중량%씩 첨가하고 6시간동안 배양하여 BCP 배지에서의 총 유산균수가 1.0×109 cfu/㎖ 이상, 적정산도가 0.89~0.91%, pH는 4.55~4.65가 되도록 하여 제조하였다.First, the lactic acid bacteria culture medium was stirred at 95.36% by weight of crude milk and 4.6% by weight of skim milk powder (or mixed milk powder), and the specific gravity at 15 ° C was 1.0473 to 1.0475, the titratable acidity was 0.200 to 0.220%, and the pH was 6.65 to 6.70, 20 ° C. Brix o was mixed to be 16.3 ~ 16.5%. After mixing, it was UHT heat-treated (sterilized at 135 ° C for 2 seconds) and cooled to 40 ° C. Then, Streptococcus thermophilus and Lactobacillus (Valley laboratory, USA) were added to each of 0.02% by weight and incubated for 6 hours. Total lactic acid bacteria in the BCP medium was prepared by 1.0 × 10 9 cfu / ㎖ or more, the titratable acidity is 0.89 ~ 0.91%, pH is 4.55 ~ 4.65.

그 다음, 혼합과즙시럽은 액상과당 13중량%, 백설탕 5중량%, 혼합과즙농축액 56Brixo 10.9중량%, 펙틴 1.0중량%, 후레쉬 후르츠 믹스 에센스 0.1중량% 및 정제수 70중량%를 30~35℃에서 교반하여 혼합한 후 UHT 열처리(135℃에서 2초간 살균)한 후 냉각하여 제조하였다.Next, the mixed fruit syrup is 13% by weight liquid fructose, 5% by weight white sugar, 10.9% by weight mixed juice concentrate 56Brix o , 1.0% by weight pectin, 0.1% by weight fresh fruit mix essence and 70% by weight purified water at 30 ~ 35 ℃ After mixing by stirring, UHT heat treatment (sterilized for 2 seconds at 135 ℃) and then prepared by cooling.

그런 다음, 상기 유산균배양액 69.5중량%와 실시예 1의 락토바실러스 커베터스 HY7601을 포함한 동결건조분말 0.1중량% 및 상기 혼합과즙시럽 30.4중량%를 조합하여 150bar에서 균질한 후 10℃ 이하로 냉각하여 혈중 콜레스테롤 강하 및 비만 억제 효능을 갖는 락토바실러스 커베터스 HY7601을 유효성분으로 함유한 발효유를 제조하였다.Then, 69.5% by weight of the lactic acid bacteria culture medium and 0.1% by weight of the lyophilized powder including Lactobacillus cavities HY7601 of Example 1 and 30.4% by weight of the mixed fruit juice syrup were homogenized at 150 bar and then cooled to 10 ° C. or less. Fermented milk containing Lactobacillus cabbetts HY7601 having blood cholesterol lowering and obesity inhibitory efficacy was prepared.

<실시예 4><Example 4>

락토바실러스 커베터스 HY7601을 유효성분으로 함유하는 기능성 음료의 제조Preparation of Functional Drinks Containing Lactobacillus Cabbetts HY7601 as Active Ingredient

상기의 혈중 콜레스테롤 강하 및 비만 억제 효능을 갖는 락토바실러스 커베터스 HY7601과 혼합과즙시럽으로 구성된 기능성 음료를 제조하는 방법은 다음과 같다.Method for producing a functional beverage consisting of Lactobacillus Corbettus HY7601 and mixed juice syrup having a blood cholesterol lowering and obesity inhibitory effect is as follows.

먼저, 혼합과즙시럽은 액상과당 13중량%, 백설탕 2.5중량%, 갈색설탕 2.5중량%, 혼합과즙농축액 56Brixo 10.9중량%, 펙틴 1.0중량%, 후레쉬 후르츠 믹스 에센스 0.1중량% 및 정제수 70중량%를 30~35℃에서 교반하여 혼합한 후 UHT 열처리(135℃에서 2초간 살균)한 후 냉각하여 제조하였다.First, mixed fruit syrup is 13% by weight of liquid fructose, 2.5% by weight of white sugar, 2.5% by weight of brown sugar, 56% by weight of mixed juice concentrate 56Brix o , 1.0% by weight pectin, 0.1% by weight of fresh fruit mix essence and 70% by weight of purified water After mixing by stirring at 30 ~ 35 ℃ UHT heat treatment (sterilized for 2 seconds at 135 ℃) was prepared by cooling.

그리고 상기의 방법으로 제조된 혼합과즙시럽 30.4중량%와 실시예 1의 락토바실러스 커베터스 HY7601을 포함한 동결건조분말 0.1중량% 및 나머지 정제수 69.5중량%를 조합하여 150bar에서 균질한 후 10℃ 이하로 냉각한 후 이를 유리병, 패트병 등 소포장 용기에 포장하여 혈중 콜레스테롤 강하 및 비만 억제 효능을 갖는 락토바실러스 커베터스 HY7601을 유효성분으로 함유하는 기능성 음료를 제조하였다.And a mixture of 30.4% by weight of the mixed juice syrup prepared by the above method, 0.1% by weight of lyophilized powder including Lactobacillus cavities HY7601 of Example 1 and 69.5% by weight of the remaining purified water were homogenized at 150 bar to 10 ° C or less. After cooling, the resultant was packaged in a small packaging container such as a glass bottle or a plastic bottle to prepare a functional beverage containing Lactobacillus corpus HY7601 as an active ingredient having blood cholesterol lowering and anti-obesity efficacy.

<실시예 5><Example 5>

락토바실러스 커베터스 HY7601을 유효성분으로 함유하는 건강기능식품의 제조Preparation of dietary supplement containing Lactobacillus cabbetts HY7601 as an active ingredient

상기 실시예 1의 락토바실러스 커베터스 HY7601을 포함한 동결건조분말 0.1중량%에 영양보조성분(비타민 B1, B2, B5, B6, E 및 초산에스테르, 니코틴산 아미드) 및 올리고당을 상기 실시예 1의 락토바실러스 커베터스 HY7601을 포함한 동결건조분말 100중량부에 대하여 10중량부가 되도록 첨가하여 고속회전 혼합기에서 혼합하였다. 상기 혼합물 100중량부에 대하여 멸균 정제수 10중량부를 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.Lactobacillus HY7601 lactobacillus HY7601 of Example 1 in the lyophilized powder (vitamin B1, B2, B5, B6, E and acetate esters, nicotinic acid amide) and oligosaccharides in the lactose of Example 1 10 parts by weight based on 100 parts by weight of the lyophilized powder including Bacillus cathetus HY7601 was added and mixed in a high speed rotary mixer. 10 parts by weight of sterile purified water was added and mixed with respect to 100 parts by weight of the mixture, 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 uniformly prepared granules were passed through 12 to 14 mesh. 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>

락토바실러스 커베터스 HY7601에 의한 SREBP-2 활성증가 능력측정Determination of ability to increase SREBP-2 activity by Lactobacillus cavities HY7601

1-1. pGL3-SRE 루시퍼라제 플라스미드 제작1-1. pGL3-SRE Luciferase Plasmid Construction

Genomic DNA 상에서 SREBP-2가 결합하여 전사를 개시하는 부위인 sterol-response element, 이하 'SRE'라 한다)를 pGL3 루시퍼라제 플라스미드(Promega, E1751)에 삽입하여 pGL3-SRE 루시퍼라제 플라스미드를 제작하였다. SRE를 포함하는 부위로서 HMG-CoA 합성효소 1(Homo sapiens 3 hydroxy 3 methylglutaryl Coenzyme A synthase 1) 유전자 상위 -488~+37 부분을 선택하고 중합효소 연쇄반응(polymerase chain reaction, 이하 'PCR'이라 함)을 통해 이를 증폭하였다. PCR을 위한 주형(template)으로 HeLa 세포의 genomic DNA를 이용하고 프라이머(primer)는 표 2와 같이 고안하였다. 프라이머 염기 서열에서 밑줄 친 부분은 제한 효소 염기 서열을 나타낸 것이다.A sterol-response element (SRE), which is a site where SREBP-2 binds to genomic DNA and initiates transcription, was inserted into pGL3 luciferase plasmid (Promega, E1751) to prepare pGL3-SRE luciferase plasmid. The site containing SRE is selected from -488 to +37 of the HMG-CoA synthase 1 gene (Homo sapiens 3 hydroxy 3 methylglutaryl Coenzyme A synthase 1) and the polymerase chain reaction (PCR). Amplified by Genomic DNA of HeLa cells was used as a template for PCR, and primers were designed as shown in Table 2. The underlined portion of the primer base sequence represents the restriction enzyme base sequence.

Figure 112009019663053-PAT00002
Figure 112009019663053-PAT00002

증폭된 SRE 부위를 pGL3 루시퍼라제 플라스미드 내의 XhoⅠ/ HindⅢ 제한 효소 자리에 삽입하여 pGL3-SRE 루시퍼라제 플라스미드(luciferase plasmid)를 획득한 후 최종적으로 Solgent 사에 염기 서열 분석을 의뢰하여 정확하게 삽입되었는지를 확인하였다. 도 2에서 붉은 색으로 표기된 부분이 삽입된 염기서열이다.The amplified SRE site was inserted into the XhoI / HindIII restriction enzyme site in the pGL3 luciferase plasmid to obtain the pGL3-SRE luciferase plasmid, and finally the sequencing was performed by Solgent to confirm that the insertion was correct. . In FIG. 2, the nucleotide sequence into which the part marked in red is inserted is inserted.

1-2. HepG2 세포 배양 1-2. HepG2 cell culture

HepG2 세포는 한국세포주은행에서 분양받았으며, 10% fetal bovine serum과 L-글루타민(glutamine)이 함유된 DMEM 배지를 이용하여 5% CO2, 37℃에서 배양하였다. 그리고 일주일에 2~3번 정도 계대하였다.HepG2 cells were obtained from Korea Cell Line Bank and cultured at 37% with 5% CO 2 using DMEM medium containing 10% fetal bovine serum and L-glutamine (glutamine). And they were passaged two or three times a week.

1-3. 트랜스팩션(transfection)1-3. Transfection

pGL3-SRE 루시퍼라제 플라스미드를 HepG2 세포에 트랜스팩션(transfection)하면 세포 내에 존재하는 SREBP-2가 플라스미드 상의 SRE 부위에 결합하여 전사를 개시함에 따라 SRE 부위 뒤에 연결되어 있는 루시퍼라제 발광 유전자도 함께 발현된다. 이 발광 세기를 검출기(Synergy 2, BIO-TEK)로 측정하여 SREBP-2의 활성 정도를 판단하였다. Transfection of the pGL3-SRE luciferase plasmid to HepG2 cells results in expression of the luciferase luminescent gene linked behind the SRE site as SREBP-2 present in the cell binds to the SRE site on the plasmid to initiate transcription. . This emission intensity was measured by a detector (Synergy 2, BIO-TEK) to determine the activity of SREBP-2.

트랜스팩션(transfection)을 하기 위해 우선 EndoFree 플라스미드 Maxi Kit(Qiagen, 12362)을 이용해서 pGL3-SRE 루시퍼라제 플라스미드를 1㎍/㎕ 농도로 대량 생산하였다. 75㎠ 플라스크 기준으로 할 때 Opti-MEM Ⅰ Reduced serum 배지 1.2㎖에 pGL3-SRE 루시퍼라제 플라스미드 24㎍을 희석하고 동일한 배지 1.2㎖에 48㎕의 리포펙타민 2000(Lipofectamine 2000; invitrogen, 11668-027)을 희석한 후 이 두 희석 액을 섞어 20분간 방치하였다. 75㎠ 플라스크에서 70~80% 정도 자란 HepG2 세포를 트립신(trypsin)으로 떼어낸 후 상기 혼합물과 함께 37℃, 5% CO2 세포 배양기에서 배양시켰다.For transfection, the mass of pGL3-SRE luciferase plasmid was first mass-produced using EndoFree plasmid Maxi Kit (Qiagen, 12362). Dilute 24 μg of pGL3-SRE luciferase plasmid in 1.2 mL of Opti-MEM I Reduced serum medium on a 75 cm 2 flask and 48 μL of Lipofectamine 2000 (Invitrogen, 11668-027) in 1.2 mL of the same medium. After dilution, the two dilutions were mixed and left for 20 minutes. HepG2 cells grown at about 70-80% in 75 cm 2 flasks were separated with trypsin and incubated with the mixture at 37 ° C. in a 5% CO 2 cell incubator.

1-4. HepG2 세포 내에서 락토바실러스 커베터스 HY7601에 의한 SREBP-2 활성증가 능력측정1-4. Determination of ability to increase SREBP-2 activity by Lactobacillus catheter HY7601 in HepG2 cells

락토바실러스 커베터스 HY7601에 의한 SREBP-2 활성증가 능력을 측정하기에 앞서 먼저 제작된 pGL3-SRE 루시퍼라제 플라스미드가 SREBP-2의 활성을 측정하기에 적당한지 여부를 확인하였다.Prior to measuring the ability to increase SREBP-2 activity by Lactobacillus cavities HY7601, it was confirmed whether the previously prepared pGL3-SRE luciferase plasmid was suitable for measuring the activity of SREBP-2.

그 결과를 도 3에 나타내었다.The results are shown in Fig.

도 3에서 A는 무(none)처리, B는 25-하이드록시콜레스테롤(25-Hydroxycholesterol; Sigma, H1015) 1㎍/㎖와 콜레스테롤(Sigma C8667) 10㎍/㎖, C는 팔미토일글리세로포스포콜린(Rac-1-Palmitoyl-glycero-3-phosphocholine, 이하'LPC'라함; Fluka, 76186) 20μM, D는 U18666A(Sigma, U3633) 2㎍/㎖를 처리한 군으로서 A에 대한 B, C, D 각각의 상대적인 pGL3-SRE 루시퍼라제 세기를 나타낸다.In Figure 3, A is non-treated, B is 25-hydroxycholesterol (25-Hydroxycholesterol; Sigma, H1015) 1 µg / ml and cholesterol (Sigma C8667) 10 µg / ml, C is palmitoylglycerophospho Choline (Rac-1-Palmitoyl-glycero-3-phosphocholine, hereinafter referred to as 'LPC'; Fluka, 76186) 20 μM, D was treated with U18666A (Sigma, U3633) 2 μg / ml, B, C, D represents the relative pGL3-SRE luciferase intensity of each.

도 3에서 확인할 수 있는 바와 같이, B의 경우는 상기 시약들에 의해 세포 내 콜레스테롤 양이 증가되어 그 결과 SREBP-2의 활성이 줄어들었기 때문에 pGL3-SRE 루시퍼라제 세기가 줄어들었음을 나타내고, C는 상기 시약에 의해 세포 내 콜레스테롤이 세포 밖으로 배출된 결과 세포 내 콜레스테롤의 양이 적어짐에 따라 SREBP-2의 활성이 증가하기 때문에 pGL3-SRE 루시퍼라제 세기가 증가하였음을 나타내며, D는 상기 시약에 의해 콜레스테롤 합성이 저해됨에 따라 세포 내 콜레스테롤 양이 줄어든 결과 SREBP-2의 활성이 증가됨으로써 pGL3-SRE 루시퍼라제 세기가 증가하였음을 나타낸다. 이상의 결과를 종합하여 보면 상기의 pGL3-SRE 루시퍼라제 플라스미드가 SREBP-2의 활성을 측정하기에 적당하다고 결론을 내릴 수 있었다.As can be seen in Figure 3, the case of B indicates that the amount of intracellular cholesterol is increased by the reagents as a result of which the activity of SREBP-2 is reduced, indicating that pGL3-SRE luciferase intensity is reduced, C is The concentration of pGL3-SRE luciferase increased because the activity of SREBP-2 increased as the amount of intracellular cholesterol decreased as a result of the excretion of intracellular cholesterol by the reagent, and D represents cholesterol by the reagent. As the synthesis was inhibited, the amount of intracellular cholesterol decreased, indicating that the activity of SREBP-2 was increased, indicating that pGL3-SRE luciferase intensity was increased. Taken together, it was concluded that the pGL3-SRE luciferase plasmid was suitable for measuring the activity of SREBP-2.

상기와 같이, pGL3-SRE 루시퍼라제 플라스미드가 SREBP-2의 활성을 측정하기에 적당하므로, 락토바실러스 커베터스 HY7601에 의한 SREBP-2 활성증가 능력을 측정하였다.As described above, since the pGL3-SRE luciferase plasmid was suitable for measuring the activity of SREBP-2, the ability to increase SREBP-2 activity by Lactobacillus corpus HY7601 was measured.

먼저, 국내 가정에서 제조한 김치를 0.02% 소디움 아지드(sodium azide)가 포함된 엠알에스(MRS) 액체 배지에 넣고 37℃에서 24시간 배양한 후, 10㎕ 백금이를 사용하여 배양액을 취하여 다시 0.02% 소디움 아지드가 포함된 엠알에스 한천 평판배지에 도말하고 37%에서 48시간 동안 배양하였다. 이렇게 형성된 균락 중의 여러 유산균을 HepG2 세포에 처리한 후 pGL3-SRE 루시퍼라제 세기 변화를 측정한 결과 본 발명의 락토바실러스 커베터스 HY7601에 의해 pGL3-SRE 루시퍼라제 세기가 증가됨을 발견하였다.First, the domestically prepared kimchi in MRS liquid medium containing 0.02% sodium azide (MRS) liquid medium and incubated for 24 hours at 37 ℃, take the culture medium using 10μl platinum yi again Plated in agar plate agar containing 0.02% sodium azide and incubated for 48 hours at 37%. As a result of measuring the pGL3-SRE luciferase intensity change after treatment of various lactic acid bacteria in the thus-formed HepG2 cells, it was found that the pGL3-SRE luciferase intensity was increased by the Lactobacillus catheterus HY7601 of the present invention.

그 결과를 도 4에 나타내었다.The results are shown in FIG.

도 4에서 A는 무(none)처리, B는 LPC 20μM, C는 락토바실러스 커베터스 H7601 104개(HepG2 세포 개수와 동일함)를 처리한 군으로서 A에 대한 B, C 각각의 상대적인 pGL3-SRE 루시퍼라제 세기를 나타낸다.In FIG. 4, A is treated with none, B is 20 μM of LPC, and C is a group treated with 10 4 lactobacillus catheterus H7601 (same as the number of HepG2 cells), and the relative pGL3 of B and C with respect to A is shown. SRE Luciferase intensity.

도 4에서 확인할 수 있는 바와 같이, 락토바실러스 커베터스 HY7601에 의해서 pGL3-SRE 루시퍼라제 세기가 A에 비하여 30% 이상 증가하는 것을 발견하였다. 이 결과로 미루어 보아 락토바실러스 커베터스 HY7601에 의해 HepG2 세포 내 콜레스테롤의 양이 감소하는 것을 알 수 있었다.As can be seen in FIG. 4, it was found that the pGL3-SRE luciferase intensity was increased by 30% or more compared to A by Lactobacillus catheterus HY7601. As a result, it was found that the amount of cholesterol in HepG2 cells was reduced by Lactobacillus catheterus HY7601.

<시험예 2><Test Example 2>

락토바실러스 커베터스 HY7601에 의한 콜레스테롤 강하 및 비만 억제 능력 측정Determination of Cholesterol Lowering and Obesity Inhibition by Lactobacillus catheter HY7601

2-1. 비만 억제 능력 측정2-1. Obesity Inhibition

실험동물은 (주)중앙실험동물에서 ICR 3주령 수컷 마우스를 구입하여 1주일 동안 고지방 식이를 공급하면서 적응시킨 후 이용하였다. 모든 실험군의 마우스는 10마리씩 임의로 배치하였다. 하기 표 3과 같이 실험군은 일반 식이군, 고지방 식이군, 락토바실러스 커베터스 HY7601군(고지방 식이 + 락토바실러스 커베터스 HY7601)의 세 군으로 하였고 일반 식이와 고지방 식이는 Research diets사에서 각각 D12450B(10% 지방)와 D12451(45% 지방)을 구입하여 이용하였다. 락토바실러스 커베터스 HY7601 균주는 엠알에스 액체배지에 접종하여 37℃에서 20시간 배양하였다. 이 배양액을 3,000rpm, 4℃에서 10분간 원심 분리하여 얻은 균체를 식이와 혼합하여 마우스에 투여하였다.The experimental animals were used after adapting to a high-fat diet for 1 week by purchasing three-week-old male mice from ICR. Mice in all experimental groups were randomly assigned to 10 mice. As shown in Table 3, the experimental group was composed of three groups of the general diet group, high fat diet group, Lactobacillus cabbetts HY7601 group (high fat diet + Lactobacillus cabbetts HY7601), and the general diet and high fat diet were D12450B respectively from Research diets. (10% fat) and D12451 (45% fat) were used and used. Lactobacillus catheter HY7601 strain was inoculated in MS liquid medium and incubated at 37 ° C. for 20 hours. The cultured cells were centrifuged at 3,000 rpm at 4 ° C. for 10 minutes to mix with the diet and administered to mice.

총 8주 동안 실험 식이를 공급하며 1주일에 2회 간격으로 체중과 사료 섭취량을 측정하였다. The diet was fed for a total of eight weeks, and body weight and feed intake were measured twice a week.

그 결과를 도 5 및 표 4에 나타내었다.The results are shown in FIG. 5 and Table 4.

도 5에서 확인 할 수 있는 바와 같이, 락토바실러스 커베터스 HY7601을 섭취한 군은 고지방 식이만 섭취한 군과 비교하여 체중 증가가 현저히 억제되었음을 알 수 있었다. 또한, 하기의 표 4에 나타난 바와 같이 8주간의 체중 증가량을 보면, 락토바실러스 커베터스 HY7601군은 9.42±1.65이고, 고지방식이군은 16.58±3.94로서 락토바실러스 커베터스 HY7601에 의해 체중 증가가 대략 45% 정도 억제되었음을 확인하였다.As can be seen in Figure 5, the group ingesting Lactobacillus corbetters HY7601 was found that the weight gain was significantly suppressed compared to the group ingested only a high fat diet. In addition, as shown in Table 4, the weight gain of the 8-week weight, Lactobacillus cavities HY7601 group is 9.42 ± 1.65, high-fat diet group is 16.58 ± 3.94, the weight gain is increased by Lactobacillus Corbetts HY7601 It was confirmed that approximately 45% inhibition.

Figure 112009019663053-PAT00003
Figure 112009019663053-PAT00003

Figure 112009019663053-PAT00004
Figure 112009019663053-PAT00004

2-2. 혈중 콜레스테롤 및 중성지방 측정2-2. Blood Cholesterol and Triglyceride Measurement

혈중 생화학 지표의 측정을 위해 상기 각군의 마우스를 12시간 절식시키고 심장채혈을 한 후 혈장을 분리하였다. 아산제약으로부터 구입한 kit를 이용하여 혈중 콜레스테롤 및 중성지방의 농도를 측정하였다.In order to measure blood biochemical markers, the mice of each group were fasted for 12 hours, and blood samples were separated from the plasma. Blood concentrations of cholesterol and triglycerides were measured using a kit purchased from Asan Pharmaceutical.

효소시약 1병과 완충액 1병을 섞어 효소시액을 만들고 효소시액 1.5㎖와 혈청 10㎕ 또는 표준액 10㎕와 섞어 반응을 비교하였다. 37℃에서 5분간 반응시킨 후 500nm에서 흡광도를 측정하였다. 샘플(sample)의 콜레스테롤 및 중성지방 농도는 다음과 같이 계산하였다.An enzyme reagent was prepared by mixing one bottle of enzyme reagent and one bottle of buffer solution, and the reaction was compared by mixing 1.5 ml of enzyme solution with 10 μl of serum or 10 μl of standard solution. After reacting for 5 minutes at 37 ℃ absorbance was measured at 500nm. The cholesterol and triglyceride concentrations of the sample were calculated as follows.

샘플의 농도(㎎/㎗)= 샘플의 흡광도/표준액의 흡광도 × 표준액의 농도Concentration of sample (mg / dl) = absorbance of sample / absorbance of standard solution x concentration of standard solution

그 결과를 표 5에 나타내었다. The results are shown in Table 5.

표 5에서 확인할 수 있는 바와 같이, 락토바실러스 커베터스 HY7601군의 혈중 콜레스테롤과 중성지방 농도 모두 고지방식이만 섭취한 군(고지방식이군)에 비해 현저하게 감소된 것을 확인할 수 있었다. 콜레스테롤의 경우 17% 정도 감소하였고, 중성지방의 경우 32% 정도로 상당한 감소 효과를 관찰할 수 있었다.As can be seen in Table 5, the blood cholesterol and triglyceride concentrations of the Lactobacillus catheterus HY7601 group was found to be significantly reduced compared to the high-fat diet only group (high-fat diet group). The cholesterol decreased by 17% and triglyceride by 32%.

Figure 112009019663053-PAT00005
Figure 112009019663053-PAT00005

(*P<0.05, **P<0.01)(* P <0.05, ** P <0.01)

<시험예 3><Test Example 3>

마우스 간조직에서 락토바실러스 커베터스 HY7601에 의한 마커 유전자 유의적 발현 차이 입증Significant difference in marker gene expression by Lactobacillus catheter HY7601 in mouse liver tissue

상기 시험예 2의 마우스로부터 적출한 간조직의 SREBP-2, LDL-receptor, GAPDH(실험결과를 보정하기 위한 house-keeping 유전자) 유전자들의 발현 차이를 리얼타임 PCR을 통해 측정하였다. 우선 RNA queous kit(Ambion, AM1912)를 이용하여 간조직으로부터 RNA를 추출하고 High Capacity RNA-to-cDNA kit(Applied Biosystems, 4387406)를 이용하여 RNA에 대한 상보적인 DNA(complimentary DNA, 이하 'cDNA'라 함)를 확보하였다. 하기의 표 6과 같이 구입한 택맨프로브(Taqman probes, Applied Biosystems 사에서 구입)와 상기 cDNA를 이용하여 리얼타임 RCR(Applied Biosystems, 7500 Real time PCR)을 시행하였다.Expression differences of SREBP-2, LDL-receptor, and GAPDH (house-keeping gene for correcting experimental results) genes of liver tissues extracted from the mouse of Test Example 2 were measured by real-time PCR. First, RNA is extracted from liver tissue using RNA queous kit (Ambion, AM1912), and complementary DNA ('cDNA') is complementary to RNA using High Capacity RNA-to-cDNA kit (Applied Biosystems, 4387406). Secured). Real-time RCR (Applied Biosystems, 7500 Real time PCR) was performed using Taqman probes (Taqman probes, purchased from Applied Biosystems) and the cDNA, as shown in Table 6 below.

그 결과를 도 6에 나타내었다.The results are shown in FIG.

도 6에서 확인할 수 있는 바와 같이 SREBP-2와 LDL-receptor 발현 양상이 일반 식이군(C)보다 고지방 식이군(A)에서 예상대로 더 낮게 관찰되었다. 하지만 락토바실러스 커베터스 HY7601군(B)에서는 이들 유전자의 발현 정도가 다시 회복되는 것을 관찰하였다. 즉 락토바실러스 커베터스 HY7601에 의해 세포막의 LDL-receptor 발현이 증가됨으로써 콜레스테롤 유입이 증가되어 혈중 콜레스테롤 양이 감소된 것으로 해석할 수 있다.As can be seen in Figure 6 SREBP-2 and LDL-receptor expression was observed lower than expected in the high-fat diet group (A) than the normal diet group (C). However, in the Lactobacillus catheterus HY7601 group (B), the expression level of these genes was observed to recover again. In other words, the LDL-receptor expression of the cell membrane is increased by Lactobacillus catheter HY7601, which can be interpreted as a decrease in the amount of cholesterol in the blood due to increased cholesterol inflow.

Figure 112009019663053-PAT00006
Figure 112009019663053-PAT00006

도 1은 Biomerieux 사의 api 50 CH kit를 이용하여 락토바실러스 커베터스 HY7601의 당 발효 실험을 한 결과를 나타낸 그림이다.1 is a diagram showing the results of a sugar fermentation experiment of Lactobacillus corbettus HY7601 using api 50 CH kit of Biomerieux.

도 2는 pGL3-SRE 루시퍼라제 플라스미드에 삽입되어 있는 HMG-CoA 합성효소 1 유전자 상위 -488~+37 부분을 나타낸 그림이다.Figure 2 is a diagram showing the upper portion of the HMG-CoA synthase 1 gene -488 ~ +37 inserted in the pGL3-SRE luciferase plasmid.

도 3은 HepG2 세포 내에서 pGL3-SRE 루시퍼라제 세기를 측정한 결과를 나타낸 그래프이다.Figure 3 is a graph showing the results of measuring pGL3-SRE luciferase intensity in HepG2 cells.

도 4는 HepG2 세포 내에서 락토바실러스 커베터스 HY7601에 의한 pGL3-SRE 루시퍼라제 세기를 측정한 결과를 나타낸 그래프이다.4 is a graph showing the results of measuring pGL3-SRE luciferase intensity by Lactobacillus catheter HY7601 in HepG2 cells.

도 5는 마우스의 8주간 평균 체중변화를 나타낸 그래프이다.5 is a graph showing the change in the average weight of the mice for 8 weeks.

도 6은 리얼타임 PCR을 이용하여 마우스 간조직 내 SREBP-2와 LDL-receptor 유전자의 상대적인 RNA 양을 조사한 그래프이다.6 is a graph illustrating the relative RNA amounts of SREBP-2 and LDL-receptor genes in mouse liver tissue using real-time PCR.

Claims (7)

혈중 콜레스테롤 강하 및 비만 억제 효능을 갖는 신규의 락토바실러스 커베터스(Lactobacillus curvatus) HY7601(기탁번호: KCTC 11456BP).Novel Lactobacillus curvatus HY7601 (Accession No .: KCTC 11456BP) with blood cholesterol lowering and obesity inhibition efficacy. 제1항에 있어서,The method of claim 1, 상기 락토바실러스 커베터스(Lactobacillus curvatus) HY7601은 간조직 세포의 LDL-receptor의 발현량을 증가시킴으로써 과도한 혈중 콜레스테롤을 세포 내로 유입시킴으로써 혈중 콜레스테롤을 감소시키는 것을 특징으로 하는 락토바실러스 커베터스(Lactobacillus curvatus) HY7601. Lactobacillus curvatus ( Lactobacillus curvatus ) HY7601 by increasing the amount of LDL-receptor expression of liver tissue cells to reduce the blood cholesterol by introducing excess blood cholesterol into the cells, characterized in that Lactobacillus curvatus ( Lactobacillus curvatus) ) HY7601. 제1항에 있어서,The method of claim 1, 상기 락토바실러스 커베터스(Lactobacillus curvatus) HY7601은 중성지방 감소 및 비만억제 효능을 가지는 것을 특징으로 하는 락토바실러스 커베터스(Lactobacillus curvatus) HY7601.The Lactobacillus curvatus HY7601 is characterized by having a triglyceride-reducing and obesity inhibitory effect ( Lactobacillus curvatus ) HY7601. 상기 제1항의 락토바실러스 커베터스(Lactobacillus curvatus) HY7601을 유효 성분으로 함유하는 것을 특징으로 하는 혈중 콜레스테롤 강하 및 비만 억제용 약학적 조성물. Lactobacillus curvatus of claim 1 ( Lactobacillus curvatus ) A pharmaceutical composition for lowering blood cholesterol and obesity, characterized in that it contains as an active ingredient. 상기 제1항의 락토바실러스 커베터스(Lactobacillus curvatus) HY7601을 유효성분으로 함유하는 것을 특징으로 하는 혈중 콜레스테롤 강하 및 비만억제용 발효유. Lactobacillus curvatus of claim 1 ( Lactobacillus curvatus ) Fermented milk for lowering blood cholesterol and obesity, characterized in that it contains HY7601 as an active ingredient. 상기 제1항의 락토바실러스 커베터스(Lactobacillus curvatus) HY7601을 유효성분으로 함유하는 것을 특징으로 하는 혈중 콜레스테롤 강하 및 비만억제용 음료. Lactobacillus curvatus ( Lactobacillus curvatus ) HY7601 of claim 1 characterized in that the blood cholesterol lowering and anti-obesity beverages containing as an active ingredient. 상기 제1항의 락토바실러스 커베터스(Lactobacillus curvatus) HY7601을 유효성분으로 함유하는 것을 특징으로 하는 혈중 콜레스테롤 강하 및 비만억제용 건강기능식품. Lactobacillus curvatus ( Lactobacillus curvatus ) HY7601 of claim 1, characterized in that it contains a healthy functional food for lowering blood cholesterol and obesity.
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