KR100516377B1 - New strains capable of producing conjugated linoleic acid, capsulated composition comprising them, and the functional food using them - Google Patents

New strains capable of producing conjugated linoleic acid, capsulated composition comprising them, and the functional food using them Download PDF

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KR100516377B1
KR100516377B1 KR10-2003-0023164A KR20030023164A KR100516377B1 KR 100516377 B1 KR100516377 B1 KR 100516377B1 KR 20030023164 A KR20030023164 A KR 20030023164A KR 100516377 B1 KR100516377 B1 KR 100516377B1
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cla
strain
present
cbg
strains
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김소미
오덕근
백대헌
신홍식
박시호
이유진
엄수종
노영쇠
박종섭
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Abstract

본 발명은 공역화 리놀레인산(CLA)을 생산할 수 있는 신규한 균주에 관한 것이다.The present invention relates to a novel strain capable of producing conjugated linoleic acid (CLA).

본 발명의 균주에는 비피도박테리움 브레베(Bifidobacterium breve) CBG-C2 균주, 비피도박테리움 슈도카르테눌라툼(Bifidobacterium pseudocartenulatum) CBG-C4 균주 및 엔테로코커스 패시움(Enterococcus faecium) CBG-C5 균주가 포함된다. Bifidobacterium breve CBG-C2 strain, Bifidobacterium pseudocartenulatum CBG-C4 strain, and Enterococcus faecium CBG-C5 strain include strains of the present invention. Included.

본 발명의 균주는 CLA 생산 능력이 우수하며, CLA를 생성하여 배지로 분비하는 한편 균체 내에 축적할 수 있다. 또한 본 발명의 균주는 위산이나 담즙 등의 산과 항생물질에 대해 강한 내성을 지닌다. The strain of the present invention is excellent in CLA production capacity, can produce CLA and secrete it into the medium, and can accumulate in the cells. In addition, the strain of the present invention has a strong resistance to acid and antibiotics such as gastric acid or bile.

본 발명의 균주를 포함하는 조성물은 수용성 다당류로 이루어진 피복물질 내에 본 발명의 균주와 CLA를 포함하는 캡슐제 형태로 제조되어, 기능성 식품 및 의약품으로 사용될 수 있다.The composition comprising the strain of the present invention may be prepared in the form of a capsule containing the strain of the present invention and CLA in a coating material consisting of water-soluble polysaccharides, and thus may be used as functional foods and pharmaceuticals.

Description

공역화 리놀렌산을 생산하는 신규한 균주, 이를 함유하는 캡슐제 및 이를 이용한 기능성 식품 {New strains capable of producing conjugated linoleic acid, capsulated composition comprising them, and the functional food using them}New strains producing conjugated linolenic acid, capsules containing the same, and functional foods using them {New strains capable of producing conjugated linoleic acid, capsulated composition comprising them, and the functional food using them}

본 발명은 공역화 리놀레인산(conjugated linoleic acid, 이하 'CLA'라 한다)을 생산할 수 있는 신규한 균주에 관한 것이다.The present invention relates to a novel strain capable of producing conjugated linoleic acid (hereinafter referred to as "CLA").

CLA는 필수지방산인 리놀레인산(linoleic acid, 이하 'LA'라 한다)의 공역화 이성체로, 반추동물의 젖이나 근육에서 미량으로 발견되는 천연 지방산 성분이다. CLA is a conjugated isomer of linoleic acid (LA), an essential fatty acid, and is a natural fatty acid component found in trace amounts in the ruminant's milk and muscle.

CLA는 사슬 내부의 시스(cis)-9 및 트랜스(trans)-11, 또는 트랜스-10 및 시스-12 위치에 사슬 내부의 및 트랜스(trans) 배열에 공역화이중결합을 가지는데, 특히 시스-9 및 트랜스-11 부위의 공역화이중결합으로 인해 인체에 유용한 생리활성이 나타난다.CLAs have conjugated double bonds in the chain and in the trans arrangement at cis-9 and trans-11, or trans-10 and cis-12 positions, particularly cis- The conjugated double bonds at site 9 and trans-11 result in useful physiological activity in the human body.

CLA는 동맥경화증의 발생저하(Artery. 1997. 22:266-277), 면역기능향상(J. Nut. 1999. 129:32-38), 항암작용(Anticancer research. 1997. 17:969-973), 성장촉진(J. Nut. 2000. 130:2981-2989) 및 당뇨병 등의 질환에 대해 우수한 치료 효과를 나타내며, 체지방감소(Am. J. Physiol. 1998. 275:R667-R672)를 통해 비만을 억제한다고도 알려져 있다. 이러한 특성으로 인해 CLA는 기능성 식품 및 의약품의 유효성분으로 유용하게 이용될 수 있다.CLA decreases the incidence of atherosclerosis (Artery. 1997. 22: 266-277), improves immune function (J. Nut. 1999. 129: 32-38), and anticancer activity (Anticancer research. 1997. 17: 969-973) , Growth promotion (J. Nut. 2000. 130: 2981-2989) and excellent treatment effect for diseases such as diabetes, body fat reduction (Am. J. Physiol. 1998. 275: R667-R672) It is also known to suppress. Due to these characteristics, CLA can be usefully used as an active ingredient in functional foods and pharmaceuticals.

CLA는 주로 동물성 식품에 함유되어 있으며, 특히 반추동물에 많이 존재하는 것으로 알려져 있다. 쇠고기의 CLA 함량은 2.9∼4.3 ㎎ CLA/fat이며, 어린양에서는 5.6 ㎎ CLA/fat이고, 해산물에서는 0.3∼0.6 ㎎ CLA/fat의 소량이 존재하며, 유제품에서의 함량은 우유의 경우 5.5 ㎎ CLA/fat, 그리고 치즈에서는 3∼7 ㎎ CLA/fat의 양이 확인된다. 인간의 일일 CLA 섭취량은 채식 위주인 동양인의 경우에는 0.1 g/day이고, 육식을 많이 하는 서양인의 경우는 0.4 g/day로 예상되고 있다. CLA is found primarily in animal foods and is known to be present in many ruminants. The CLA content of beef is 2.9-4.3 mg CLA / fat, 5.6 mg CLA / fat in lambs, small amounts of 0.3-0.6 mg CLA / fat in seafood, and 5.5 mg CLA in dairy products. / fat, and in cheese the amount of 3-7 mg CLA / fat is identified. Human daily CLA intake is expected to be 0.1 g / day for vegetarian-based Asians and 0.4 g / day for Westerners who eat a lot of meat.

현재까지 CLA의 대량 생산을 위해 여러 가지 방법이 개발되어 왔다. 기존의 방법은 요소부가법, 분자증류법, HPLC 방법 등 CLA를 LA로부터 화학적으로 합성하는 방법들이 있으며, LA를 CLA로 전환시킬 수 있는 활성을 지닌 미생물이 다수 분리된 바 있다. To date, several methods have been developed for mass production of CLAs. Conventional methods include urea addition method, molecular distillation method, HPLC method and the like to chemically synthesize CLA from LA, and a large number of microorganisms having activity to convert LA to CLA have been isolated.

그러나, 화학적 합성법들은 고가의 장비를 필요로 하거나, 공정에 너무 많은 시간이 소요되는 등의 문제가 많다. 또한 이들 방법은 단일 종류의 CLA 뿐 아니라 다양한 종류의 이성체들을 함께 생성하기 때문에, 기존의 화학적 방법으로 단일 종류의 CLA 이성체만을 생산한다는 것은 매우 비효율적이다. However, chemical synthesis methods often require expensive equipment, or require too much time for the process. In addition, since these methods produce not only a single type of CLA but also various types of isomers, it is very inefficient to produce only a single type of CLA isomer by conventional chemical methods.

따라서 CLA를 가장 효율적으로 생산할 수 있는 방법은 CLA를 생성할 수 있는 미생물로부터 CLA를 생산, 분리하는 것이다. CLA를 생성하는 것으로 알려진 대표적인 미생물은 락토바실러스(Lactobacillus), 프로피오니박테리움 (Propionibacterium), 부티리비브리오 피브리솔벤스(Butyrivibrio fibrisolvens) 등의 장내 미생물이며, 많은 국가에서 이들은 가축에게 투여하는 생균제(probiotics), 사료 등의 기능성 식품이나 의약품의 유효성분으로 유용하게 사용되고 있다.Therefore, the most efficient way to produce CLA is to produce and isolate CLA from microorganisms capable of producing CLA. Representative microorganisms known to produce CLA are enteric microorganisms, such as Lactobacillus , Propionibacterium , and Butyrivibrio fibrisolvens , and in many countries they are probiotics that are administered to livestock ( It is usefully used as an active ingredient of functional foods and pharmaceuticals such as probiotics and feed.

그런데, 국내에서는 CLA를 식품이나 의약품의 유효성분으로서 직접 첨가하는 것이 아직 허가되지 않고 있다. 따라서 CLA를 식품 및 의약품의 유효성분으로 사용하려면 미생물로부터 CLA를 분리하여 첨가하는 방법보다는, CLA를 생성할 수 있는 미생물의 균체(菌體) 자체를 직접 첨가하여 CLA를 간접적으로 생성하도록 하는 방법을 사용해야 한다.However, in Korea, it is not yet permitted to add CLA directly as an active ingredient of food or medicine. Therefore, in order to use CLA as an active ingredient of food and medicines, rather than separating and adding CLA from microorganisms, a method of indirectly generating CLA by directly adding a cell of microorganisms capable of producing CLA is required. Should be used.

현재까지 알려진 바에 의하면 장내 미생물에 의해 합성된 CLA는, 시스-9, 트랜스-11과 시스-12, 트랜스-10의 비율이 약 50%씩 생성되는 화학적인 합성법과는 달리, 시스-9와 트랜스-11만 특이적으로 생성된다. 반추동물 유래의 육유에서는 소량의 시스-9와 트랜스-11이 확인되지만, 탈지유를 이용한 발효 유제품에서는 매우 낮은 시스-9와 트랜스-11이 검출될 뿐이며, 특히 각종 유산균을 이용한 발효 김치에서도 시스-9와 트랜스-11이 존재하지 않는 것으로 알려지고 있다. 따라서, 대한민국 특허출원번호 2001-0047292에서는 락토바실러스 속 미생물을 이용하여 미생물에 의해 합성된 순수한 시스-9와 트랜스-11형의 CLA를 식품에 첨가시키는 방법을 개시한 바 있으나, 실제 식품에서는 극미량의 CLA 함량이 확인 되었을 뿐이다. To date, CLA synthesized by intestinal microorganisms, unlike chemical synthesis, produces about 50% of cis-9, trans-11, cis-12, and trans-10. Only -11 is specifically produced. Small amounts of cis-9 and trans-11 are found in meat derived from ruminants, but very low cis-9 and trans-11 are detected in fermented dairy products using skim milk, especially cis-9 in fermented kimchi using various lactic acid bacteria. And trans-11 are not known to exist. Therefore, Korean Patent Application No. 2001-0047292 discloses a method of adding pure cis-9 and trans-11 CLA synthesized by microorganisms to food using microorganisms of the genus Lactobacillus. Only the CLA content has been confirmed.

또한 CLA 생성 미생물이 식품 또는 의약품의 유효성분으로 사용될 수 있기 위해서는, 제조된 제품의 유통기간 내내 미생물이 제품 제조시와 비슷하게 높은 비율로 잔존해야 하며, 사람 등 동물의 체내로 섭취된 후에는 장(腸) 내에서 높은 생존율과 활성을 지녀야 한다. 또한 수퍼박테리아(superbacteria)를 비롯한 장 내의 유해한 균과 경쟁하여 우위를 점하기 위해서는 항생물질에 대해 우수한 내성을 갖추어야 한다.In addition, in order for the CLA-producing microorganism to be used as an active ingredient of food or medicine, the microorganism must remain at a high rate similar to that of the product during the distribution period of the manufactured product, and after ingestion into the body of animals such as humans, Vi) must have high survival and activity within In addition, to be able to compete with harmful bacteria in the gut, including superbacteria, it must have good resistance to antibiotics.

그러나 현재 시판 중인 제품의 유효성분으로 사용되는 미생물 중 많은 균주들은 저장 중의 제품 뿐 아니라 섭취 후 위를 통과하는 과정에서조차 잔존하지 못하며, 항생물질에 대한 내성도 미약한 것으로 나타났다. However, many strains of microorganisms that are used as active ingredients of commercially available products do not survive the passage of the stomach after ingestion as well as products in storage, and have a weak resistance to antibiotics.

따라서, 우수한 CLA 생산 능력과 함께, 동물의 체내에서 높은 생존력과 활성을 나타내며, 균체 자체를 식품 및 의약품의 유효성분으로 직접 첨가할 수 있는 균주에 대한 발명은 여전히 과제로 남아있다. Therefore, the invention of a strain that exhibits a high viability and activity in the body of the animal, with excellent CLA production capacity, and can be directly added as an active ingredient of food and pharmaceuticals remains a problem.

상기 과제를 달성하기 위해, 본 발명에서는 공역화 리놀레인산을 생산할 수 있고, 산과 항생물질에 대해 뛰어난 내성을 지니며, 균체 자체를 식품 및 의약품에 첨가할 때 CLA를 간접적으로 생성할 수 있는 신규한 균주를 제공하고자 한다. In order to achieve the above object, the present invention is capable of producing conjugated linoleic acid, having excellent resistance to acids and antibiotics, and indirectly producing CLA when adding the cells themselves to food and pharmaceuticals. One strain is to be provided.

또한 본 발명에서는 상기 균주와 CLA를 함유하는 캡슐제를 제공함으로써, 기능성 발효식품, 유제품 및 의약품을 제조할 수 있는 방법을 제공하고자 한다.In another aspect, the present invention provides a capsule containing the strain and CLA, to provide a method for producing a functional fermented food, dairy products and pharmaceuticals.

본 발명은 CLA를 생산할 수 있는 신규한 균주를 제공한다.The present invention provides novel strains capable of producing CLA.

본 발명의 균주는 한국인 영아의 분변으로부터 분리된 균주로, LA를 CLA로 전환시킬 수 있음을 특징으로 한다.example The strain of the invention is a strain isolated from feces of Korean infants, characterized by being able to convert LA to CLA.

본 발명의 균주에는 비피도박테리움 브레베(Bifidobacterium breve) CBG-C2 균주, 비피도박테리움 슈도카르테눌라툼(Bifidobacterium pseudocartenulatum) CBG-C4 균주 및 엔테로코커스 패시움(Enterococcus faecium) CBG-C5 균주가 포함된다. Bifidobacterium breve CBG-C2 strain, Bifidobacterium pseudocartenulatum CBG-C4 strain, and Enterococcus faecium CBG-C5 strain include strains of the present invention. Included.

본 발명의 균주를 분리하는 방법은 다음과 같다. Method for separating the strain of the present invention is as follows.

한국인 영아의 분변으로부터 균주들을 분리하여, 300개 정도의 균락(colony)들을 임의로 채취하여 기질인 LA가 첨가된 배지에서 배양한다. 배양액을 헥산(hexane)으로 추출한 후 흡광도를 측정하여 CLA 생성 능력이 우수한 균주들을 선별한다.Strains are isolated from feces of Korean infants, and about 300 colonies are arbitrarily collected and cultured in a medium containing LA as a substrate. After extracting the culture solution with hexane (hexane), the absorbance is measured to select strains excellent in CLA production capacity.

상기 과정에 의해 선별된 균주들에 대해, 각 균주들이 생성하는 지방산을 가스 크로마토그래피(Gas Chromatograph; 이하 GC라고 한다)법으로 분석하여, LA로부터 CLA를 생성할 수 있는지를 재차 확인한다. With respect to the strains selected by the above process, fatty acids produced by each strain are analyzed by gas chromatography (Gas Chromatograph; hereinafter referred to as GC) to again check whether CLA can be generated from LA.

이렇게 하여 선별된 본 발명의 균주는 세 종류로, 각각 CBG-C2, CBG-C4 및 CBG-C5로 명명되며, 2002년 4월 3일 농업생명공학연구원 농용미생물보존센터에 각각 기탁번호 KACC 91001, KACC 91003 및 KACC 91002으로 기탁된 상태이다. 또한 CBG-C2(KCTC 10462BP)와 CBG-C4(KCTC 10208BP)는 생명공학연구원 유전자원센터 유전자은행에도 각각 2003년 4월 10일과 2002년 3월 25일자로 기탁된 상태이다.Three strains of the present invention selected in this way, named CBG-C2, CBG-C4 and CBG-C5, respectively, on April 3, 2002, the Agricultural and Biotechnology Institute Agricultural Microorganisms Preservation Center, respectively Accession No. KACC 91001, Deposited as KACC 91003 and KACC 91002. In addition, CBG-C2 (KCTC 10462BP) and CBG-C4 (KCTC 10208BP) have been deposited on April 10, 2003 and March 25, 2002, respectively, to the Genetic Center Genetic Bank of the Biotechnology Research Institute.

상기와 같은 방법으로 얻은 본 발명의 균주는 CLA 생산 능력이 우수하며, 위산이나 담즙 등의 산과 항생물질에 대해 강한 내성을 지니고 있다.The strain of the present invention obtained by the above method is excellent in CLA production capacity, and has a strong resistance to acid and antibiotics such as gastric acid or bile.

본 발명의 미생물로부터 생산되는 CLA는 시스-9와 트랜스-11의 입체구조만을 포함하는데, 이들은 기존에 공지된 바와 같이 다양한 생리활성을 가지는 이성체이므로, 이들 구조물을 포함하는 지방산과 아실글리세롤은 각종 동물성 유래의 유제품, 식물 유래의 원료물질에 의한 유제품, 발효식품 및 건강성 기능성식품과 미생물제재(probiotics) 등의 개발을 위해 광범위하게 이용될 수 있다. The CLA produced from the microorganism of the present invention includes only the three-dimensional structure of cis-9 and trans-11, and since these are isomers having various physiological activities as known in the art, fatty acids and acylglycerol containing these structures are various animal species. It can be widely used for the development of dairy products derived from, dairy products derived from plant-derived raw materials, fermented foods and health functional foods and microbial products (probiotics).

따라서, 본 발명의 균주들은 균체 고정화 등의 생물학적 방법에 의해 CLA의 이성체들을 대량으로 생합성하는데 효율적으로 이용될 수 있다.Therefore, the strains of the present invention can be efficiently used to biosynthesize large amounts of isomers of CLA by biological methods such as cell immobilization.

본 발명의 균주는 생균체 뿐 아니라 사균체(死菌體) 형태로도 식의약품 첨가가 가능한데, 이는 본 발명의 균주가 CLA를 배양액 혹은 반응액에 분비하면서, 동시에 상당량의 CLA를 균체 내 축적할 수 있는 특성을 지니기 때문이다.The strains of the present invention can be added to food and pharmaceutical in the form of dead cells as well as live cells, which means that the strains of the present invention can secrete CLA in a culture medium or a reaction solution and at the same time accumulate a significant amount of CLA in the cells. This is because it has characteristics that can be.

따라서, 본 발명의 균주는 LA 첨가 배지에서 배양한 균체 자체를 식품 및 의약품 등 다양한 조성물의 유효성분으로 첨가하여 CLA가 간접적으로 생성되도록 할 수 있으므로, CLA를 식품 및 의약품에 직접 첨가할 수 없었던 기존의 문제를 해결하여 CLA를 다량 함유한 기능성 제품의 개발을 촉진할 수 있다.Therefore, the strain of the present invention can be added indirectly to CLA by adding the cells themselves cultured in the LA-added medium as an active ingredient of various compositions such as foods and medicines, CLA can not be added directly to foods and medicines Solving the problem can accelerate the development of functional products containing large amounts of CLA.

본 발명은 또한 상기 균주를 함유하는 식품 및 약제학적 조성물을 제공한다.The present invention also provides food and pharmaceutical compositions containing the strains.

본 발명의 조성물은 본 발명의 비피도박테리움 브레베 CBG-C2 균주, 비피도박테리움 슈도카르테눌라툼 CBG-C4 균주 또는 엔테로코커스 패시움 CBG-C5 균주 중 선택된 하나 이상의 균체를 유효성분으로 함유한다.The composition of the present invention contains at least one selected from among the Bifidobacterium breve CBG-C2 strain, Bifidobacterium pseudocartenulum CBG-C4 strain, or Enterococcus fascium CBG-C5 strain as an active ingredient. do.

본 발명의 조성물은 CLA 생성률을 높이고 균주의 생육안정성을 증대시키기 위해, 유기산 등을 추가의 유효성분으로 함유할 수 있다. 본 발명의 조성물에서 유기산은 화학적으로 합성되거나, 또는 미생물로부터 정제되어 분리된 CLA가 될 수 있다.The composition of the present invention may contain an organic acid or the like as an additional effective ingredient in order to increase the CLA production rate and increase the growth stability of the strain. In the composition of the present invention, the organic acid may be chemically synthesized or may be purified CLA separated from the microorganism.

구체적으로 본 발명의 조성물은 본 발명의 균주 중 선택된 하나의 균체와 CLA를 함께 포함할 수 있다.Specifically, the composition of the present invention may include CLA and one of the cells selected from the strains of the present invention.

본 발명의 조성물은 상기 유효성분 외에도 필요에 따라 다양한 보조성분을 추가로 함유할 수 있다. The composition of the present invention may further contain various auxiliary ingredients as necessary in addition to the active ingredient.

본 발명의 식품 조성물의 경우, 비타민 A, 비타민 B1, 비타민 B2, 비타민 B3, 비타민 B6, 비타민 B12, 엽산(folic acid), 비타민 C, 비타민 D3, 비타민 E 등의 비타민 류와, 구리, 칼슘, 철, 마그네슘, 칼륨, 아연 등의 미네랄 또는 유산균 등을 함유할 수 있다.In the case of the food composition of the present invention, vitamins such as vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B12, folic acid, vitamin C, vitamin D3, vitamin E, copper, calcium, Minerals such as iron, magnesium, potassium, zinc or lactic acid bacteria, and the like.

또한 본 발명의 식품 조성물 중, 건강음료 조성물은 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 향미제로는 타우마틴, 스테비아 추출물과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 들 수 있다. 천연 탄수화물로는 포도당, 과당 등의 단당류, 말토스, 수크로오스 등의 이당류, 덱스트린, 사이클로덱스트린 등의 다당류, 자일리톨, 소르비톨, 에리트리톨 등의 당알코올류 등이 들 수 있다. In addition, in the food composition of the present invention, the health beverage composition may contain various flavors or natural carbohydrates and the like as an additional component, as in a general beverage. Flavoring agents include natural sweeteners such as taumartin and stevia extract, and synthetic sweeteners such as saccharin and aspartame. Natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol.

본 발명의 약제학적 조성물의 투여량 또는 섭취량은 60∼130 uM(Cancer Epidemiol. Biol. Prev. 2000. 9:689-696), 그리고 식품 조성물의 투여량 또는 섭취량은 3.4∼6 g/day(J. Nutr. 2000. 130:2943-2948)인 것이 바람직하나, 필요에 따라 가감할 수 있다. 본 발명의 조성물은 섭취량에 관계없이 체내에 안전하게 흡수된다. 투여량 또는 섭취량은 조성물에 함유된 유효성분 및 다른 성분의 종류 및 함량, 제형의 종류 및 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료 기간, 동시 사용되는 약물을 비롯한 다양한 인자에 따라 조절될 수 있다.The dosage or intake of the pharmaceutical composition of the present invention is 60 to 130 uM (Cancer Epidemiol. Biol. Prev. 2000. 9: 689-696), and the dosage or intake of the food composition is 3.4 to 6 g / day (J Nutr. 2000. 130: 2943-2948), but may be added or subtracted as necessary. The composition of the present invention is safely absorbed into the body regardless of the intake amount. Dosage or intake depends on the type and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the age, weight, general health, sex and diet of the patient, time of administration, route of administration and rate of composition, duration of treatment This can be adjusted according to a variety of factors, including drugs used concurrently.

본 발명의 조성물은 인체에 투약하는 경우, 화학적으로 합성된 CLA 함유 식품 및 의약품에 비해 부작용의 우려가 없다.When the composition of the present invention is administered to the human body, there is no concern of side effects compared to chemically synthesized CLA-containing foods and drugs.

본 발명의 조성물은 상기 유효성분 이외에도 약제학적 또는 식품에서 허용 가능한 담체를 1종 이상 포함하여 제제화할 수 있다.The composition of the present invention can be formulated to include one or more carriers that are acceptable in pharmaceutical or food in addition to the active ingredient.

약제학적 또는 식품에서 허용 가능한 담체는 식염수, 멸균수, 링거액, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 더 나아가 당분야의 적정한 방법으로, 또는 Remington's Pharmaceutical Science(최근판), Mack Publishing Company, Easton PA에 개시되어 있는 방법을 이용하여 각 질환에 따라, 또는 성분에 따라 바람직하게 제제화할 수 있다.Pharmaceutical or food acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, as necessary, including antioxidants, buffers, Other conventional additives such as bacteriostatic agents can be added. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

본 발명의 조성물의 제제 형태는 과립제, 산제, 과립제, 피복정, 정제, 캡슐제, 탕제, 엑기스제, 좌제, 시럽, 즙, 현탁제, 유제 및 활성 화합물의 서방출형 제제 등이 될 수 있다.Formulation forms of the compositions of the present invention may be granules, powders, granules, coated tablets, tablets, capsules, milk preparations, extracts, suppositories, syrups, juices, suspensions, emulsions and sustained release formulations of the active compounds, and the like. .

본 발명은 상기 조성물의 캡슐제를 제공한다.The present invention provides a capsule of the composition.

본 발명의 캡슐제는 피복물질(coating material)과, 피복되는 내부의 핵물질(core material)로 이루진다.The capsule of the present invention consists of a coating material and a core material therein to be coated.

본 발명의 캡슐제에서 핵물질은 본 발명의 균주 중 선택된 하나와 CLA를 함께 포함하는 것이 바람직하다.In the capsule of the present invention, the nuclear material preferably includes CLA and one selected from the strains of the present invention.

본 발명의 캡슐제에서 핵물질을 둘러싸는 피복물질은 흡수성, 분산성, 점착성이 우수한 수용성 다당류를 사용한다. In the capsule of the present invention, the coating material surrounding the nuclear material uses a water-soluble polysaccharide having excellent absorbency, dispersibility, and adhesion.

본 발명에서 사용가능한 수용성 다당류는 전분, 한천, 카라기난, 알긴산, 알긴산나트륨(sodium alginate), 폴리메틸메타크릴레이트(polymetacrylate), 소맥단백, 대두단백을 비롯하여, 메틸셀룰로오스(methylcellulose), 하이드록시프로필셀룰로오스(hydroxylpropylcellulose), 하이드록시프로필메틸셀룰로오스(hydroxyl-propylmethylcellulose) 등의 셀룰로오스 유도체, 잔탄검(xanthan gum), 아라비아검(arabic gum), 로커스트콩검(locust bean gum), 구아검(guar gum), 타마린드검(tamarind gum), 타라검(Tara gum), 카라야검(karaya gum), 트라가칸검(tragacanth gum), 가티검(ghatti gum) 등의 검류, 그리고 젤란(gellan), 잔탄(xanthan), 펙틴(LM, HM type), 덱스트란(dextran), 글루칸(glucan), 글루코만난(glucomannan), 아라비노갈락탄(arabino galactan), 퍼셀레란(furcelleran), 풀루란(pullulan), 글루코사민(glucosamine), 젤라틴(gelatin), 카제인(casein) 중 선택된 하나 이상이 될 수 있다.The water-soluble polysaccharides usable in the present invention include starch, agar, carrageenan, alginic acid, sodium alginate, polymethylacrylate, polymethylacrylate, wheat protein, soy protein, methyl cellulose, hydroxypropyl cellulose cellulose derivatives such as (hydroxylpropylcellulose) and hydroxyl-propylmethylcellulose, xanthan gum, arabic gum, locust bean gum, guar gum, tamarind Gums such as tamarind gum, tara gum, karaya gum, tragacanth gum, ghatti gum, and gellan, xanthan, pectin (LM, HM type), dextran, glucan, glucanannan, glucomannan, arabino galactan, furcelleran, pullulan, glucosamine, Gelatin, casein It may be one or more selected.

본 발명의 캡슐제 제조시 코팅물질의 양은 용도 및 목적에 따라 그 양을 적절하게 가감할 수 있으며, 핵을 기준으로 하여 일반적으로 사용되는 범위인 1∼80 중량% 범위의 양으로 사용하는 것이 좋다.In the preparation of the capsule of the present invention, the amount of coating material may be appropriately added or subtracted according to the use and purpose, and it is preferable to use the amount in the range of 1 to 80 wt%, which is generally used based on the nucleus. .

본 발명의 캡슐제는 상기 수용성 다당류 외에도, 방출제어효과를 높이거나 용해도를 높이기 위해 일반적으로 사용되는 코팅 물질들을 추가로 포함할 수 있다.In addition to the water-soluble polysaccharide, the capsule of the present invention may further include coating materials generally used to increase the release control effect or increase the solubility.

또한 본 발명의 캡슐제는 필요에 따라 유화제, 보호제 및 가소제를 추가로 포함할 수 있다.In addition, the capsule of the present invention may further include an emulsifier, a protective agent and a plasticizer as necessary.

본 발명의 캡슐제의 제조 방법은 통상적으로 사용되는 캡슐화 방법 중 적절한 방법을 택하여 사용할 수 있다. 예를 들면, 본 발명의 캡슐제를 제조하는 방법은 유화안정성을 지닌 분산매에 본 발명의 균주의 배양균체, 유기산 및 캡슐 피복물질을 분산시키는 유화공정, 유화분산액을 교반시켜 캡슐막을 제조하는 캡슐막 생성공정, 그리고 경화제와 반응제를 첨가하여 캡슐막을 경화시키는 경화공정을 포함하는 일반적 캡슐화 제조공정으로 이루어진다.The preparation method of the capsule of the present invention can be used by selecting an appropriate method from the commonly used encapsulation method. For example, the method for producing a capsule of the present invention is an emulsion process for dispersing the culture cells, organic acid and capsule coating material of the strain of the present invention in a dispersion medium having an emulsion stability, a capsule membrane for preparing a capsule membrane by stirring the emulsion dispersion General encapsulation manufacturing process including a production step and a curing step of curing the capsule film by adding a curing agent and a reactant.

이와 같이 제조된 본 발명의 캡슐제는, 산화방지를 통해 외부환경으로부터 균주를 보호하므로 저온에서 장기간 안정하게 저장이 가능하다.The capsule of the present invention prepared as described above can be stably stored at a low temperature for a long time since it protects the strain from the external environment through oxidation prevention.

CLA와 같은 지방산은 원래 열, 효소, 산, 알카리, 미생물 등에 대해 불안정하나, 적절한 피복물질에 의해 미세캡슐화(microencapsulation)되면, 저장안정성 및 복용상의 편의성이 증가된다.Fatty acids such as CLA are inherently unstable against heat, enzymes, acids, alkalis, microorganisms, etc., but microencapsulation with appropriate coatings increases storage stability and ease of taking.

본 발명의 캡슐제는 산성 조건인 장내에서도 일정한 방출 조절에 의해 일정 수준의 생균수를 유지하게 한다.The capsule of the present invention allows to maintain a certain level of viable cell number by constant release control even in the intestinal acidic condition.

또한 본 발명의 캡슐제는 미세캡슐화에 의해 체내 수용계에서의 비중 및 분산성을 증가시키므로, 생체 이용률이 더욱 높아져 저장 식품 및 유음료 및 유제품에 대한 적응성을 향상시킬 수 있다. In addition, since the capsule of the present invention increases the specific gravity and dispersibility in the body water receiving system by microencapsulation, the bioavailability is further increased to improve the adaptability to stored food and milk beverages and dairy products.

따라서 본 발명의 캡슐제는 장내에서 본 발명의 균주가 안정하게 생육할 수 있게 한다. 장내에서 증식한 본 발명의 균주는 지속적으로 CLA를 생산하므로 동맥경화증의 발생저하, 체지방감소, 면역기능향상, 항암효과, 성장촉진 효과 등을 얻을 수 있다. 또한 본 발명의 균주가 장내에서 우점종의 위치를 차지함에 따라, 장내에서 유해한 미생물의 성장을 저해하는 생물제재(probiotics)로서 작용하게 되므로, 지속적으로 장기능을 활성화하고 개선시킬 수 있다.Therefore, the capsule of the present invention allows the strain of the present invention to stably grow. Since the strain of the present invention propagated in the intestine continuously produces CLA, it is possible to reduce the incidence of atherosclerosis, reduce body fat, improve immune function, anticancer effect, growth promoting effect and the like. In addition, as the strain of the present invention occupies the position of the dominant species in the intestine, it acts as a probiotic (probiotics) inhibiting the growth of harmful microorganisms in the intestine, it is possible to continuously activate and improve intestinal function.

본 발명의 캡슐제를 포함하는 조성물은 구체적으로 유제품(우유, 두유, 가공우유), 발효유(액상 요구르트, 호상 요구르트), 발효성식품(김치류, 장류), 가축용 사료, 건강보조식품 등이 될 수 있다.The composition comprising the capsule of the present invention may be specifically a dairy product (milk, soy milk, processed milk), fermented milk (liquid yogurt, staple yogurt), fermentable food (kimchi, entertain), livestock feed, health supplements, etc. Can be.

본 발명의 균주를 유효성분으로 함유하는 식품 조성물은 가축용 사료를 비롯하여 각종 김치류, 장류 등의 발효 식품 및 요구르트, 치즈 등의 발효유제품으로, 항암, 면역증강, 체지방 감소 등의 효과를 기대할 수 있다.The food composition containing the strain of the present invention as an active ingredient is a fermented food product such as livestock feed, various kimchi and soy sauce, and fermented milk products such as yogurt and cheese, and can be expected to have effects such as anticancer, immune enhancement, and body fat reduction. .

본 발명의 균주를 유효성분으로 함유하는 약제학적 조성물은 암, 동맥경화, 당뇨병 및 비만 등 CLA에 의해 억제되는 질환의 예방 또는 치료에 사용될 수 있다.Pharmaceutical compositions containing the strain of the present invention as an active ingredient can be used for the prevention or treatment of diseases inhibited by CLA, such as cancer, arteriosclerosis, diabetes and obesity.

이하, 본 발명의 균주들을 하기 실시예에서 보다 상세히 설명하되, 실시예에 의해 본 발명이 제한되는 것은 아니다.Hereinafter, the strains of the present invention are described in more detail in the following examples, but the present invention is not limited by the examples.

[실시예 1] 본 발명의 균주 분리, 동정 및 특성 확인 Example 1 Isolation, Identification and Characterization of Strains of the Invention

1) 본 발명의 균주의 분리 및 동정1) Isolation and Identification of Strains of the Invention

본 발명의 균주를 분리하기 위해, 영아의 분변을 채취하였다.In order to isolate the strain of the present invention, feces of infants were collected.

CLA를 생성하는 미생물을 분리하기 위해 모유, 혼합, 이유영양아의 분변 10종을 채취하여, 멸균 유동파라핀이 중층된 MRS 배지에 접종하였다. 멸균 생리식염수(0.5%w/v)로 30∼150개 정도의 균락(colony)이 생기도록 희석하여 L-cysteine이 0.05% 첨가된 MRS(Man Rogosa Sharpe) 배지에 도말한 후, 혐기조에 가스 팩(Gas pack, MGC, mitsubishi)과 함께 넣어 37 에서 72시간 배양하였다. 이때 LA를 0.5 %(w/v) 트윈 80(tween 80)에 잘 유화시켜 제면여과한 후 MRS 배지에 첨가하였으며, 균 배양시 혐기 조건을 조성하기 위해, 멸균된 배양용기에 MRS 배지를 빈공간이 없도록 가득 분주하였다. In order to isolate CLA-producing microorganisms, 10 feces of mother's milk, mixed milk and nutrients were collected and inoculated in sterile liquid paraffin-layered MRS medium. Dilute to 30 to 150 colonies with sterile saline solution (0.5% w / v), smear it on MRS (Man Rogosa Sharpe) medium containing 0.05% L-cysteine, and then pack the gas into the anaerobic tank. (Gas pack, MGC, mitsubishi) and incubated at 37 to 72 hours. At this time, LA was emulsified well in 0.5% (w / v) Tween 80 and filtration was added to MRS medium.In order to form anaerobic conditions in culture of bacteria, MRS medium was vacated in a sterile culture vessel. It was full so that there was no.

배양된 배지에서 300개 정도의 균락을 임의 선택하여 각 균주를 MRS 배지에 2회 계대배양한 후 20 ㎖ 시험관에 1% 접종하여 48시간 배양하였다. 배양액을 헥산(hexane)으로 추출한 후 233 nm에서 흡광도를 측정하여 CLA 생성량을 얻고, 이를 균체량, 즉 600 nm에서의 흡광도 값으로 보정하여 상대적인 CLA 생성량을 비교하였다. About 300 colonies were randomly selected in the cultured medium, and each strain was passaged twice in MRS medium, and then cultured for 48 hours by inoculating 1% in a 20 ml test tube. After extracting the culture solution with hexane, the absorbance was measured at 233 nm to obtain the amount of CLA produced, and the relative amount of CLA was compared by correcting the cell mass, that is, the absorbance at 600 nm.

생균수 측정은 MRS 한천 배지를 이용하여 10배 희석법에 의해 평판 배양하고 이를 혐기조(Difco, USA)에 넣어 가스 팩과 함께 48시간 경과 후 생성된 균락의 수를 세어 측정하였다. The viable cell count was measured by plate culture by 10-fold dilution using MRS agar medium and placed in an anaerobic tank (Difco, USA) to count the number of fungi generated after 48 hours with the gas pack.

이렇게 하여 CLA 생성 능력이 우수한 세 균주를 선발하고 CBG-C2, CBG-C4 및 CBG-C5로 각각 명명하였다. 본 발명의 균주들에 대해 rRNA의 염기서열을 분석하여 그 속과 종을 동정하였다.In this way, three strains with excellent CLA production ability were selected and named as CBG-C2, CBG-C4 and CBG-C5, respectively. For the strains of the present invention, the genome and species were identified by analyzing the nucleotide sequence of rRNA.

그 결과, CBG-C2 균주는 비피도박테리움 브레베, CBG-C4 균주는 비피도박테리움 슈도카르테눌라툼, CBG-C5 균주는 엔테로코커스 패시움에 속하는 것으로 판명되었다.As a result, the CBG-C2 strain was found to belong to the Bifidobacterium breve, the CBG-C4 strain to the Bifidobacterium pseudocartenulatum, and the CBG-C5 strain belonged to the enterococcus fascium.

각 균주는 2002년 4월 3일 농업생명공학연구원 농용미생물보존센터에 CBG-C2(KACC 91001), CBG-C4(KACC 91003), CBG-C5 균주는 기탁번호 KACC 91002으로 기탁되었다. 또한 CBG-C2(KCTC 10462BP)와 CBG-C4(KCTC 10208BP)는 생명공학연구원 유전자원센터 유전자은행에도 각각 2003년 4월 10일과 2002년 3월 25일자로 기탁된 상태이다.Each strain was deposited on April 3, 2002 at the Agricultural Biotechnology Conservation Center, Center for Agricultural Microbiology, CBG-C2 (KACC 91001), CBG-C4 (KACC 91003), and CBG-C5 strains with accession number KACC 91002. In addition, CBG-C2 (KCTC 10462BP) and CBG-C4 (KCTC 10208BP) have been deposited on April 10, 2003 and March 25, 2002, respectively, to the Genetic Center Genetic Bank of the Biotechnology Research Institute.

2) 본 발명의 균주가 생성하는 지방산의 조성 확인2) Confirmation of the composition of fatty acids produced by the strain of the present invention

본 발명의 균주가 생성하는 지방산의 조성을 확인하여, 본 발명의 균주가 CLA를 생성하는지 재차 확인하기 위해, 다음과 같이 GC를 수행하였다. In order to confirm the composition of the fatty acid produced by the strain of the present invention, and to check again whether the strain of the present invention produces CLA, GC was performed as follows.

균체를 LA(500 ㎍/㎖)가 포함된 배지에서 48시간 동안 배양한 후 원심분리하였다. The cells were incubated for 48 hours in a medium containing LA (500 μg / ml) and centrifuged.

배양액 혹은 증류수에 현탁한 균체에 2배 부피의 이소프로필 알콜(isopropyl alcohol)을 첨가하여 격렬히 혼합한 후, 여기에 1.5배 부피의 헥산을 가하여 3분 동안 흔들어 주면서 혼합하였다. Two-volume isopropyl alcohol was added to the cells suspended in the culture solution or distilled water and mixed vigorously, and 1.5-volume hexane was added thereto, followed by shaking for 3 minutes.

상기 혼합액을 상온에서 3000 rpm으로 5분 동안 원심분리한 후, 233nm에서 상청액의 흡광도를 측정하였다. 추출한 지방산은 American Oil Chemists's Society : Official Method and Recommended Practoces pf AOCS, 4th. ed.(1989) 방법에 따라 메틸 에스테르(methly ester)화하여 GC 분석에 필요한 시료를 준비하였다.The mixture was centrifuged at 3000 rpm for 5 minutes at room temperature, and then the absorbance of the supernatant was measured at 233 nm. The extracted fatty acid is American Oil Chemists's Society: Official Method and Recommended Practoces pf AOCS, 4th. Methyl ester was prepared according to the method of ed. (1989) to prepare a sample for GC analysis.

GC 분석시 조건은 다음과 같다. 이때, FID가 부착된 GC DS-6200 (DONAM)을 사용하였고, 컬럼은 HP-FFAP capillary column (30m×0.25㎜, 두께 0.25㎛)을 사용하였으며, 오븐 온도는 210 ℃, 인젝터(injector) 온도는 250 ℃, 디텍터(detector) 온도는 270 ℃ 이었다. 운반기체는 헬륨을 사용하였으며 1 ㎖/min 유속으로 용출시켰고, 분해비(split ratio)는 50:1로 하였다. 각 피크의 면적은 기기에 연결된 적분계(model 3390A, Hewlett-packard, USA)를 이용하여 구하였다. CLA의 동정은 표준 물질의 머무름 시간과 비교하여 확인하였으며, CLA의 함량은 표준 물질의 면적과 CLA의 면적비에 의해 분석시료로 사용하고, 그 결과를 도 1에 나타내었다.The conditions for GC analysis are as follows. At this time, FID-attached GC DS-6200 (DONAM) was used, and the column was HP-FFAP capillary column (30m × 0.25㎜, thickness 0.25㎛), oven temperature is 210 ℃, injector (injector) temperature 250 degreeC and the detector temperature were 270 degreeC. The carrier gas was helium and eluted at a flow rate of 1 ml / min, and the split ratio was 50: 1. The area of each peak was determined using an integrating system (model 3390A, Hewlett-packard, USA) connected to the instrument. Identification of the CLA was confirmed by comparison with the retention time of the standard material, the content of the CLA was used as an analytical sample by the area ratio of the area of the standard material and the CLA, the results are shown in FIG.

도 1에 나타난 바와 같이, 모든 균주에서 LA 피크와 CLA 피크가 함께 관찰되었다. As shown in FIG. 1, the LA peak and the CLA peak were observed together in all strains.

따라서 본 발명의 균주들은 LA를 기질로 하여 CLA를 생성할 수 있음을 알 수 있다.Therefore, it can be seen that the strains of the present invention can produce CLA using LA as a substrate.

3) 본 발명의 균주에 있어서, CLA 생성의 최적조건 확인3) In the strain of the present invention, confirming the optimal conditions of CLA production

본 발명의 균주에 있어서, CLA 생성의 최적조건을 확인하기 위해, 기질인 LA가 배지에 존재하는 경우와 존재하지 않는 경우의 두 조건에서 각 균주의 생육 특성을 관찰하였다. In the strains of the present invention, in order to confirm the optimum conditions for CLA production, the growth characteristics of each strain were observed under two conditions, when the substrate LA is present in the medium and when it is not present.

우선, 전배양하여 활성화된 균주를 LA(500 ㎍/㎖)가 포함된 MRS배지 2ℓ에 1%가 되도록 접종한 후, 시간 별로 배양액을 회수하여 균체 농도, CLA 생성량 및 배양액의 pH를 측정하여 그 결과를 도 2에 나타내었다.First, inoculate the activated strain to 2% MRS medium containing LA (500 ㎍ / ㎖) to 1%, and then recovered the culture medium by time to measure the cell concentration, CLA production and pH of the culture medium The results are shown in FIG.

도 2에 나타난 바와 같이, 각 균주들은 대수기(log phase)에 접어듬에 따라 CLA 생성량도 증가하였으며, 정지기(stationary phase)에 도달하기 직전에 CLA를 최대로 생성하였다. 배양액의 최초 pH는 6.5 정도였으나, 배양 시간이 지날수록 떨어져 약 50시간 발효 후에는 pH 4.2 정도를 나타냈다. As shown in FIG. 2, each strain also increased the amount of CLA produced by folding into the log phase and maximized CLA immediately before reaching the stationary phase. The initial pH of the culture medium was about 6.5, but after the incubation time, the pH was about 4.2 after 50 hours of fermentation.

한편, 전배양한 균주를 LA가 포함되지 않은 MRS 배지에 1%가 되도록 접종한 후, 20 ㎖ 시험관에 분주하고 배양하여 생장 곡선을 측정하고, 18시간 배양 후부터 6∼12시간마다 배양액의 pH를 측정하였다. 균체를 회수하여 균체량의 10배 부피의 트리스(Tris) 완충액에 현탁시킨 후, 효소 반응을 수행하여 생장곡선의 변화에 따른 CLA 이소머라제(isomerase)의 역가 변화를 관찰하였다. 이 때 효소 반응에 첨가한 LA의 농도는 100 ㎍/㎖였으며, 그 결과를 도 3에 나타내었다. On the other hand, after pre-cultivated strains were inoculated to 1% in the MRS medium containing no LA, it was dispensed in 20 ml test tube and cultured to measure the growth curve, the pH of the culture solution every 6 to 12 hours after 18 hours incubation Measured. After the cells were recovered and suspended in 10 times the volume of Tris buffer, the enzyme reaction was performed to observe the change in the titer of CLA isomerase according to the growth curve. At this time, the concentration of LA added to the enzyme reaction was 100 ㎍ / ㎖, the results are shown in FIG.

도 3에 나타난 바와 같이, LA를 첨가하지 않고 배양한 경우는 LA를 첨가하고 배양한 경우보다 정지기에 이르는 시간이 더 길었다. 배양액의 최종 pH는 4.2 정도였다. As shown in FIG. 3, the incubation without the addition of LA took longer to reach a stationary phase than the addition and incubation with LA. The final pH of the culture was about 4.2.

따라서, 본 발명의 균주들은 대수기에서 정지기로 넘어가기 직전에 최대로 CLA를 생성함을 알 수 있다. Therefore, it can be seen that the strains of the present invention produce the maximum CLA just before the transition from the log phase to the stationary phase.

또한 상기 결과 중, LA가 존재할 때 얻은 실험 결과는 기질 존재 하에 균을 발효 반응시켜 제품을 개발할 때, 그리고 LA가 존재하지 않을 때 얻은 실험 결과는 균 자체를 제품의 유효 성분으로 첨가하여 CLA를 간접적으로 생성하고자 할 경우에 단위 균체당 최대 역가를 나타내는 균체의 성장시기를 파악하는데 유용하게 사용될 수 있다.In addition, the experimental results obtained in the presence of LA is a fermentation reaction of the bacteria in the presence of the substrate in the development of the product, and the experimental results obtained in the absence of LA is the indirect CLA by adding the bacteria itself as an active ingredient of the product When it is to be generated as a cell can be useful to determine the growth time of the cells showing the maximum titer per cell.

4) 본 발명의 균주 배양시, 배양액과 균체에 각각 분포되는 CLA의 양 확인4) In the culture of the strain of the present invention, the amount of CLA distributed in the culture medium and cells

본 발명의 균주 배양시, 배양액과 균체에 각각 분포되는 CLA의 양을 확인하기 위해, 다음과 같이 실험을 수행하였다.When culturing the strain of the present invention, to confirm the amount of CLA distributed in the culture medium and cells, respectively, the experiment was carried out as follows.

우선, LA가 포함된 배양액에 각 균주를 접종한 후, 18, 24, 36, 48시간마다 균체 농도 및 CLA 생성 정도를 측정하여, 그 결과를 도 4에 나타내었다. First, after inoculating each strain into the culture medium containing LA, the cell concentration and the degree of CLA production were measured every 18, 24, 36, 48 hours, and the results are shown in FIG.

도 4에 나타난 바와 같이, 균체와 배양액에 각각 존재하는 CLA의 비율이 초기, 즉 18시간 후에는 CBG-C2의 경우 1:1.85, CBG-C4의 경우 1:2.1, CBG-C5의 경우 1:1.54 이었다. 점차 생육 시간이 지날수록 배지 내의 CLA 양이 증가하여, 48시간 후에는 하기 표 1에 나타난 바와 같이 CBG-C2의 경우 1:3.5, CBG-C4의 경우 1:2.9, CBG-C5의 경우 1:2의 비율을 나타내었다.As shown in Figure 4, the ratio of CLA present in the cells and the culture medium, respectively, that is, after 18 hours, 1: 1.85 for CBG-C2, 1: 2.1 for CBG-C4, 1: 1: for CBG-C5 1.54. Gradually, the amount of CLA in the medium increased over time, and after 48 hours, as shown in Table 1, 1: 3.5 for CBG-C2, 1: 2.9 for CBG-C4, and 1: 2.9 for CBG-C5. A ratio of two is shown.

생산률CLA(㎍)/건조세포량(㎎)Production rate CLA (㎍) / dry cell mass (mg) CLA 전환률(%)% CLA conversion rate 분포비 (%)Distribution ratio (%) CBG-C2CBG-C2 배양액Culture 213.68213.68 54.754.7 7878 균체Cell 59.9759.97 2222 CBG-C4CBG-C4 배양액Culture 145.96145.96 38.838.8 7575 균체Cell 48.2048.20 2525 CBG-C5CBG-C5 배양액Culture 126.72126.72 37.6337.63 6767 균체Cell 61.7061.70 3333

한편, LA를 첨가하지 않은 배양액에 각 균주를 접종한 후, 18, 24, 36, 48시간 동안 배양한 균체를 회수하여 LA가 함유된 완충액과 혼합한 후 1시간 동안 반응시킨 후, 생성된 CLA의 분포를 측정하여 그 결과를 도 5에 나타내었다. On the other hand, after inoculating each strain in the culture medium without the LA, the cells were incubated for 18, 24, 36, 48 hours, recovered and mixed with the buffer containing LA and reacted for 1 hour, and then produced CLA The distribution of is measured and the result is shown in FIG. 5.

도 5에 나타난 바와 같이, 생육 시간이 점차 지날수록 배지 내의 CLA 양이 증가하였다. 배양 36시간 후에는 표 2에 나타난 바와 같이, 균체에 의해 1시간 동안 생성된 CLA의 분포는 균체와 배지의 비가 CBG-C2, CBG-C4, CBG-C5가 각각 1.3:1, 1.6:1, 0.6:1이었다.As shown in Figure 5, as the growth time gradually increased the amount of CLA in the medium. After 36 hours of culture, as shown in Table 2, the distribution of CLA produced by the cells for 1 hour was 1.3: 1, 1.6: 1, CBG-C2, CBG-C4, CBG-C5, respectively. 0.6: 1.

생산률CLA(㎍)/건조세포량(㎎)Production rate CLA (㎍) / dry cell mass (mg) CLA 전환률(%)% CLA conversion rate 분포비 (%)Distribution ratio (%) CBG-C2CBG-C2 배양액Culture 63.2063.20 100100 4343 균체Cell 82.9082.90 5757 CBG-C4CBG-C4 배양액Culture 49.2149.21 8080 3939 균체Cell 75.0275.02 6161 CBG-C5CBG-C5 배양액Culture 38.638.6 52.5352.53 6060 균체Cell 25.3725.37 4040

따라서, 본 발명의 균주는 CLA를 생성하여 배지로 분비하는 한편, 균체 내에도 축적할 수 있다. Therefore, the strain of the present invention can produce and secrete CLA into the medium, and can also accumulate in the cells.

본 발명의 균주의 이러한 특성은 균체 자체를 식품 및 의약품의 유효성분으로 첨가하여 CLA가 생성되도록 하는데 사용될 수 있다. This property of the strains of the present invention can be used to add the cells themselves as active ingredients of foods and pharmaceuticals to produce CLA.

5) 본 발명의 균주에 있어서, 탄소원의 종류에 따른 균의 생장 및 CLA 생성량의 변화 확인5) In the strain of the present invention, the growth of the bacteria and the change in the amount of CLA production according to the type of carbon source confirmed

본 발명의 균주에 있어서, 당원의 종류에 따른 생장 및 CLA 생성량의 변화를 확인하기 위해, 포도당(glucose), 과당(fructose), 젖당(lactose) 및 설탕(sucrose) 등 다양한 탄소원이 첨가된 배지에서 균주를 배양하여 생장 정도와 CLA 생성량을 측정하고, 그 결과를 도 6에 나타내었다. In the strain of the present invention, in order to confirm the change in growth and CLA production according to the type of sugar source, in a medium to which various carbon sources such as glucose (glucose), fructose (fructose), lactose and sugar (sucrose) are added The strains were cultured to measure the extent of growth and the amount of CLA produced, and the results are shown in FIG. 6.

각 균주의 생장에 가장 적합한 탄소원은 도 6a에 나타난 바와 같이, 배지에 LA가 존재하는 경우 CBG-C2는 포도당, CBG-C4는 젖당과 설탕, CBG-C5는 젖당이었으며, 배지에 LA가 존재하지 않는 경우 CBG-C2는 젖당, CBG-C4는 젖당과 설탕이었다. As shown in FIG. 6A, the most suitable carbon source for the growth of each strain was CBG-C2 for glucose, CBG-C4 for lactose and sugar, CBG-C5 for lactose, and no LA in the medium. CBG-C2 was lactose and CBG-C4 was lactose and sugar.

한편 LA 첨가 배지에서 48시간 동안 배양한 후의 CLA 생성량은 도 6b에 나타난 바와 같이, CBG-C2와 CBG-C4는 포도당을 탄소원을 사용하였을 때 최대값을 나타낸 반면, CBG-C5는 탄소원에 의해 거의 영향을 받지 않았다.On the other hand, CLA production after 48 hours in LA addition medium, as shown in Figure 6b, CBG-C2 and CBG-C4 showed the maximum value when using a carbon source of glucose, while CBG-C5 was almost by the carbon source It was not affected.

6) 본 발명의 균주의 pH, 담즙 및 항생물질에 대한 내성 확인6) Confirmation of pH, bile and antibiotic resistance of the strain of the present invention

본 발명의 균주의 pH 내성, 담즙 내성 및 항생물질 내성 등의 특성을 다음과 같이 확인하였다.The characteristics of the pH resistance, bile resistance and antibiotic resistance of the strain of the present invention was confirmed as follows.

분리, 동정된 신균주를 이용하여 CLA 생산 유산균을 접종하여 발효시킨 제품이나 혹은 균체내 함유되어 있는 CLA를 이용한 첨가제로의 용도를 위해 각 균주의 생육특성을 비교하였다. The growth characteristics of each strain were compared for use as a product fermented by inoculating CLA-producing lactic acid bacteria using isolated and identified new strains or as an additive using CLA contained in cells.

1) pH 내성1) pH tolerance

본 발명의 균주들의 pH 내성을 확인하기 위해, 배지에 LA가 포함되어 있을 경우와 포함되지 않을 경우의 두 조건 하에서 균을 배양하면서 배양액의 pH 변화를 관찰하였으며, 그 결과를 도 2와 도 3에 나타내었다. In order to confirm the pH resistance of the strains of the present invention, the pH change of the culture solution was observed while culturing the bacteria under two conditions when the medium contained LA and not included, and the results are shown in FIGS. 2 and 3. Indicated.

도 2와 도 3에 나타난 바와 같이, 배지에 LA가 포함되었는지의 여부에 상관없이 균 생육이 정지기에 접어들면서, 배양액의 pH가 급격히 감소하였다. 최종 pH가 4.2 정도까지 떨어지는 것으로 보아 전형적인 유산균의 발효 양상을 지니고 있다. As shown in Figures 2 and 3, regardless of whether or not LA contained in the medium, the growth of the fungi, the pH of the culture solution was drastically reduced. The final pH is lowered to 4.2, which is typical for the fermentation of lactic acid bacteria.

상기 결과를 바탕으로, 배양액의 pH 변화에 따른 균의 생육 여부를 확인하기 위해, 염산을 사용하여 MRS 배지를 pH 2∼6까지 다양하게 준비하였다. 각 배지에 각 균주를 접종하고 37 ℃에서 48시간 동안 배양하면서 균의 생육 여부를 관찰하고, 그 결과를 표 3에 나타내었다. Based on the results, in order to confirm the growth of the bacteria according to the pH change of the culture medium, MRS medium was prepared variously to pH 2-6 using hydrochloric acid. Inoculating each strain into each medium and incubating for 48 hours at 37 ℃ to observe the growth of the bacteria, the results are shown in Table 3.

pH 2pH 2 pH 3pH 3 pH 4pH 4 pH 5pH 5 pH 6pH 6 CBG-C2CBG-C2 -- ++ ++ ++ ++++ CBG-C4CBG-C4 -- ++ ++ ++++ ++++ CBG-C5CBG-C5 -- ++ ++ ++ ++++

(-: 전혀 증식하지 않음, +: 약간 증식, ++: 많이 증식)(-: No growth at all, +: slight growth, ++: growth)

표 3에 의하면, 본 발명의 균주들은 pH 3.0∼6.0의 범위에서 모두 생육이 가능하였으며, 특히 CBG-C4 균주가 가장 우수한 pH 내성을 보였다. According to Table 3, the strains of the present invention were able to grow all in the range of pH 3.0 to 6.0, in particular CBG-C4 strain showed the best pH resistance.

따라서 본 발명의 균주들은 약알칼리와 약산성에 걸치는 넓은 범위에서 모두 생육이 가능하며, 특히 위산이 분비되어 산성 환경이 조성되는 위에서 충분히 생존할 수 있음을 알 수 있다. Therefore, the strains of the present invention can be grown in a wide range over weak alkali and weak acidity, and in particular, it can be seen that the gastric acid can be sufficiently survived in the acidic environment.

2) 담즙 내성2) bile resistance

본 발명의 균주들의 담즙 내성을 확인하기 위해, 담즙의 성분인 소듐 디옥시콜레이트(sodium deoxycholate)가 0, 100, 300, 500, 800 및 1000 ㎍/㎖으로 다양하게 첨가된 MRS 배지를 준비하였다. 각 배지에 균주들을 접종하여 37℃에서 48시간 동안 배양하면서 생육 여부를 검토하였으며, 이때 대조 균주로는 담즙 내성을 전혀 지니지 않은 바실러스 서브틸리스(Bacillus subtilis)를 사용하여, 그 결과를 표 4에 나타내었다. In order to confirm the bile resistance of the strains of the present invention, MRS medium in which bile sodium deoxycholate (sodium deoxycholate) was variously added at 0, 100, 300, 500, 800 and 1000 μg / ml was prepared. Strains were inoculated into each medium and cultured for 48 hours at 37 ° C., and growth was examined. At this time, as a control strain, Bacillus subtilis having no bile resistance was used. Indicated.

CBG-C2CBG-C2 CBG-C4CBG-C4 CBG-C5CBG-C5 바실러스 서브틸리스Bacillus subtilis 0 ㎍/㎖0 μg / ml ++++ ++++ ++++ ++++ 100 ㎍/㎖100 μg / ml ++++ ++++ ++++ -- 300 ㎍/㎖300 μg / ml ++++ ++++ ++++ -- 500 ㎍/㎖500 μg / ml ++++ ++++ ++++ -- 800 ㎍/㎖800 μg / ml ++++ ++++ ++++ -- 1000 ㎍/㎖1000 μg / ml ++ ++++ ++ --

표 4에 나타난 바와 같이, 본 발명의 균주는 모두 담즙 농도 1000 ㎍/㎖까지도 양호하게 생육한 반면, 대조 균주인 바실러스 서브틸리스의 경우에는 100 ㎍/㎖에서도 생육이 불가능하였다. As shown in Table 4, all of the strains of the present invention grew well even at a bile concentration of 1000 μg / ml, whereas in the case of the control strain Bacillus subtilis, growth was not possible even at 100 μg / ml.

따라서 본 발명의 균주들은 담즙이 분비되는 위장관에서 안정적으로 생육할 수 있음을 알 수 있다.Therefore, it can be seen that the strains of the present invention can stably grow in the gastrointestinal tract in which bile is secreted.

3) 항생물질 내성3) antibiotic resistance

본 발명의 균주들의 항생물질 내성을 확인하기 위해, 20 ㎖ 시험관에 밤새 배양한 균주를 MRS 배지에 희석하여 고체 배지를 제조하였다. 대조 균주로는 락토바실러스 루테리(Lactobacillus reuteri)를 사용하였다.In order to confirm the antibiotic resistance of the strains of the present invention, a solid culture was prepared by diluting the strain cultured overnight in a 20 ml test tube in MRS medium. As a control strain, Lactobacillus reuteri was used.

항생물질로 본 실험에 사용한 항생제는 암피실린(ampicillin), 테트라사이클린(tetracycline), 스트렙토마이신(streptomycin), 리파마이신 sv(rifamycin sv) 및 카나마이신(kanamycin)으로, 각각 50 ㎍/㎖ ,1000 ㎍/㎖ 및 10000 ㎍/㎖의 세 농도를 준비하였다. Antibiotics used in this experiment were ampicillin, tetratracycline, streptomycin, rifamycin sv and kanamycin, 50 μg / ml and 1000 μg / ml, respectively. And three concentrations of 10000 μg / ml were prepared.

상기와 같이 준비된 항생물질들을 각각 종이 디스크(paper disc)에 40 ㎕씩 떨어뜨린 후, 디스크를 건조시켰다. 디스크를 균이 혼합된 배지 위에 올려놓고, 37 ℃에서 48시간 동안 배양하면서 생육 여부를 관찰하고 그 결과를 표 5에 나타내었다.The antibiotics prepared as described above were dropped by 40 μl onto paper discs, respectively, and the discs were dried. The disk was placed on the medium in which the bacteria were mixed and incubated at 37 ° C. for 48 hours to observe the growth and the results are shown in Table 5.

항생제 종류Antibiotic Class 항생제 농도(㎍/㎖)Antibiotic Concentration (µg / mL) CBG-C2CBG-C2 CBG-C4CBG-C4 CBG-C5CBG-C5 락토바실러스루테리Lactobacillus ruteri 암피실린Ampicillin 5050 -- -- -- -- 10001000 -- -- -- -- 1000010000 -- -- -- -- 테트라사이클린Tetracycline 5050 -- -- ++ ++ 10001000 -- -- ++ -- 1000010000 -- -- ++ -- 스트렙토마이신Streptomycin 5050 ++ ++ ++ ++ 10001000 -- -- -- -- 1000010000 -- -- -- -- 리파마이신 svRifamycin sv 5050 -- ++ -- -- 10001000 -- -- -- -- 1000010000 -- -- -- -- 카나마이신Kanamycin 5050 ++ ++ ++ ++ 10001000 -- ++ ++ -- 1000010000 -- -- -- --

표 5에 나타난 바와 같이, 본 발명의 균주들은 모두 2∼3 개의 항생물질에 대해 내성을 나타내었다. 특히 CBG-C4 균주는 1000 ㎍/㎖의 카나마이신 및 10000 ㎍/㎖의 테트라사이클린 등 높은 농도로 처리된 항생물질에 대해서도 내성을 나타내어, 항생물질에 대한 내성이 가장 우수하였다.As shown in Table 5, all of the strains of the present invention were resistant to 2-3 antibiotics. In particular, the CBG-C4 strain exhibited resistance to antibiotics treated at high concentrations, such as kanamycin of 1000 µg / ml and tetracycline of 10000 µg / ml, and showed the best resistance to antibiotics.

따라서, 본 발명의 균주는 광범위한 종류와 농도 범위의 항생물질에 대해 내성을 지니고 있음을 알 수 있다. Therefore, it can be seen that the strain of the present invention is resistant to antibiotics of a wide range of types and concentrations.

[실시예 2] 본 발명의 균주 및 CLA를 함유하는 캡슐제 및 유제품의 제조Example 2 Preparation of Capsules and Dairy Products Containing Strains and CLA of the Present Invention

본 발명의 균주 및 CLA를 함유하는 캡슐제 및 유제품을 다음과 같이 제조하였다.Capsules and dairy products containing the strains and CLA of the present invention were prepared as follows.

공역화 리놀렌산의 미세캡슐화를 위한 코팅물질을 제조하기 위해, 식물성 다당류인 젤란, 잔탄, 전분, 한천이 1∼5%(w/v)의 농도로 포함된 혼합물을 준비하였다. 상기 혼합물에 유화제로서 HLB(hydrophilic lipophilic balance)의 수치가 4.7인 솔비탄 모노스티아레이트를 첨가한 후 60℃에서 가열하여 완전 용해한 후, 가열 살균하고 40℃로 냉각하여 혼합 수용액을 제조하였다. 이때 솔비탄 모노스티아레이트의 농도는 0.01 - 1% (w/v)가 되도록 처리하였고, 코팅제와 균체의 혼합비율은 7:3(w/w)로 하여 균질화하였다.In order to prepare a coating material for microencapsulation of conjugated linolenic acid, a mixture containing vegetable polysaccharides of gellan, xanthan, starch and agar at a concentration of 1 to 5% (w / v) was prepared. To the mixture was added sorbitan monostiarate having a hydrophilic lipophilic balance (HLB) of 4.7 as an emulsifier and then heated at 60 ° C. for complete dissolution, followed by heat sterilization and cooling to 40 ° C. to prepare a mixed aqueous solution. At this time, the concentration of sorbitan monostiarate was treated to be 0.01-1% (w / v), and the mixing ratio of the coating agent and the cells was homogenized to be 7: 3 (w / w).

균질화된 균체현탁액을 10℃의 냉각수에 분무기로 분무하여 미세캡슐의 현탁 균체액을 제조하였다. The homogenized cell suspension was sprayed with a nebulizer at 10 ° C. to prepare a suspension cell solution of microcapsules.

제조된 미세캡슐은 저온 발효제품인 김치 제조시 1%(w/v)를 가하였다. 제품별 캡슐첨가량은, 우유, 두유, 액상 요쿠르트의 경우 4.2%(w/v)를 첨가하였으며, 호상 요쿠르트와 두유발효유에는 7.3%(w/v)를 첨가하여 각 유제품을 제조하였다. 유제품들은 4℃ 또는 10℃로 저온상태가 유지되는 냉장고에서 보관되었다.The prepared microcapsules were added 1% (w / v) when preparing kimchi, a low-temperature fermentation product. For the amount of capsules added by product, 4.2% (w / v) was added to milk, soy milk, and liquid yogurt, and 7.3% (w / v) was added to fermented yogurt and soy milk fermented milk to prepare each dairy product. Dairy products were stored in refrigerators kept at 4 ° C or 10 ° C.

상기와 같이 제조된 유제품에 대해 미세캡슐의 보존성을 실험한 결과, 4℃ 및 10℃의 냉장온도에서 7~14일간 보관된 경우, 101~2개/㎖ 정도의 생균수가 감소하는 경향을 나타내었다. 이러한 정도의 감소율은 통상적인 기준으로 볼 때 매우 미약한 수치이다.As a result of experiments on the preservation of the microcapsules for the dairy product prepared as described above, when stored for 7 to 14 days at 4 ℃ and 10 ℃ refrigeration temperature, the number of viable bacteria of about 1 to 2 / / ml shows a tendency to decrease It was. This rate of decline is very low on a regular basis.

따라서, 본 발명의 캡슐제는 장기간 경과시에도 생균수가 안정적으로 유지되며, 저장안정성이 우수함을 알 수 있다.Therefore, it can be seen that the capsule of the present invention maintains viable cell numbers stably even after a long period of time and has excellent storage stability.

본 발명의 균주들은 LA로부터 CLA를 우수한 효율로 생성할 수 있을 뿐 아니라, 위산과 담즙산, 그리고 항생물질에 대해 뛰어난 내성을 지닌다. Strains of the present invention can not only produce CLA from LA with excellent efficiency, but also have excellent resistance to gastric acid, bile acid, and antibiotics.

본 발명의 균주들은 CLA를 생성한 후 균체 내에 축적할 수 있기 때문에, CLA가 간접적으로 생성되도록 하는 효과가 있다.Since the strains of the present invention can accumulate in cells after producing CLA, there is an effect that CLA is indirectly generated.

따라서, 본 발명의 균주를 포함하는 조성물은 수용성 다당류로 이루어진 피복물질 내에 본 발명의 균주와 CLA를 포함하는 캡슐제 형태 등으로 제조되어, 기능성 식품 및 의약품으로 유용하게 사용될 수 있다.Therefore, the composition comprising the strain of the present invention is prepared in the form of a capsule containing the strain and the CLA of the present invention in a coating material consisting of water-soluble polysaccharides, and can be usefully used as a functional food and pharmaceuticals.

또한 본 발명의 균주들은 균체 고정화 등의 생물학적 방법에 의해 CLA 외에도 CLA의 이성체들을 대량으로 생합성하는데 효율적으로 이용될 수 있다.In addition, the strains of the present invention can be efficiently used to biosynthesize large amounts of isomers of CLA in addition to CLA by biological methods such as cell immobilization.

도 1은 본 발명의 균주들이 생성하는 지방산의 조성을 확인하기 위한 HPLC 분석 결과를 나타낸 도이다.1 is a diagram showing the results of HPLC analysis for confirming the composition of fatty acids produced by the strains of the present invention.

도 2은 리놀레인산(LA)이 첨가된 배지에서 본 발명의 균주들의 생장 상태, CLA 생성 및 배양액의 pH 변화를 나타낸 도이다. Figure 2 is a diagram showing the growth state, CLA production and pH change of the culture of the strains of the present invention in the medium added linoleic acid (LA).

도 3은 리놀레인산(LA)이 첨가되지 않은 배지에서 본 발명의 균주들의 생장 상태 및 CLA 생성을 나타낸 도이다.Figure 3 is a diagram showing the growth status and CLA production of the strains of the present invention in medium without linoleic acid (LA) added.

도 4는 리놀레인산(LA)이 첨가된 배지에서 본 발명의 균주들을 배양하였을 때, 배양액과 균체에 각각 분포되는 CLA의 양을 비교하여 나타낸 도이다.Figure 4 is a view showing a comparison of the amount of CLA distributed in the culture medium and the cells when the strains of the present invention were cultured in a medium to which linoleic acid (LA) is added.

도 5는 리놀레인산(LA)이 첨가되지 않은 배지에서 본 발명의 균주들을 배양하였을 때, 배양액과 균체에 각각 분포되는 CLA의 양을 비교하여 나타낸 도이다.5 is a view showing the comparison of the amount of CLA distributed in the culture medium and cells when the strains of the present invention were cultured in a medium without linoleic acid (LA) added.

도 6은 본 발명의 균주들을 포도당, 과당, 젖당 및 설탕이 각각 탄소원으로 첨가된 배지에서 배양하여 생장 정도(a)와 CLA 생성량(b)을 측정한 결과를 나타낸 도이다.6 is a diagram showing the results of measuring the growth degree (a) and the amount of CLA production (b) by culturing the strains of the present invention in a medium in which glucose, fructose, lactose and sugar were added as carbon sources, respectively.

Claims (17)

삭제delete LA(linoleic acid)를 CLA(conjugated linoleic acid)로 전환시킬 수 있는 신균주 비피도박테리움 브레베(Bifidobacterium breve) CBG-C2 균주 Bifidobacterium breve CBG-C2 strain capable of converting LA (linoleic acid) into conjugated linoleic acid (CLA) 제 2항에 있어서, 기탁번호 KACC 91001으로 기탁된 균주The strain according to claim 2, deposited with accession number KACC 91001. 제 2항에 있어서, 기탁번호 KCTC 10462BP로 기탁된 균주The strain according to claim 2, deposited with accession number KCTC 10462BP. 삭제delete 삭제delete 삭제delete 삭제delete 삭제delete 제 2항 내지 제 4항 중 어느 한 항의 균주의 균체를 유효성분으로 하는 CLA 생산용 조성물The composition for producing CLA using the cells of the strain of any one of claims 2 to 4 as an active ingredient 제 10항에 있어서, 암, 동맥경화, 당뇨병 및 비만으로 이루어진 CLA에 의해 억제되는 질환의 예방 또는 치료용 조성물The composition for preventing or treating a disease according to claim 10, which is inhibited by CLA consisting of cancer, arteriosclerosis, diabetes and obesity. 제 2항 내지 제 4항 중 어느 한 항의 균주의 균체와 CLA를 핵물질로 함유하며, 수용성 다당류를 피복물질로 함유하는 캡슐제A capsule comprising a cell of the strain of any one of claims 2 to 4 and CLA as a nuclear material, and a water-soluble polysaccharide as a coating material 제 2항 내지 제 4항 중 어느 한 항의 균주를 이용하여 LA로부터 CLA를 생산하는 방법Method for producing CLA from LA using the strain of any one of claims 2 to 4 제 2항 내지 제 4항 중 어느 한 항의 균주의 균체를 식품 또는 의약품의 유효성분으로 첨가하여 CLA를 생성하는 방법Method for producing CLA by adding the cells of the strain of any one of claims 2 to 4 as an active ingredient of food or medicine 제12항의 캡슐제를 포함하는 기능성 식품Functional food containing the capsule of claim 12 제 15항에 있어서, 상기 식품은 발효식품인 것인 기능성 식품The functional food according to claim 15, wherein the food is fermented food. 제 16항에 있어서, 상기 발효식품은 액상요구르트, 호상요구르트, 치즈, 김치류, 장류로 이루어진 군에서 선택된 하나인 것인 기능성 식품The functional food of claim 16, wherein the fermented food is one selected from the group consisting of liquid yogurt, staple yogurt, cheese, kimchi, and jang.
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