KR20100135612A - Method for production of conjugated linolenic acid using bifidobacterium breve lmc520 strain - Google Patents
Method for production of conjugated linolenic acid using bifidobacterium breve lmc520 strain Download PDFInfo
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- KR20100135612A KR20100135612A KR1020090054087A KR20090054087A KR20100135612A KR 20100135612 A KR20100135612 A KR 20100135612A KR 1020090054087 A KR1020090054087 A KR 1020090054087A KR 20090054087 A KR20090054087 A KR 20090054087A KR 20100135612 A KR20100135612 A KR 20100135612A
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- linolenic acid
- bifidobacterium breve
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- 241000186012 Bifidobacterium breve Species 0.000 title claims abstract description 52
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 41
- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 title claims description 49
- 235000020661 alpha-linolenic acid Nutrition 0.000 title claims description 41
- 229960004488 linolenic acid Drugs 0.000 title claims description 40
- KQQKGWQCNNTQJW-UHFFFAOYSA-N linolenic acid Natural products CC=CCCC=CCC=CCCCCCCCC(O)=O KQQKGWQCNNTQJW-UHFFFAOYSA-N 0.000 title claims description 31
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- 238000000034 method Methods 0.000 claims description 6
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 abstract description 20
- JBYXPOFIGCOSSB-GOJKSUSPSA-N 9-cis,11-trans-octadecadienoic acid Chemical compound CCCCCC\C=C\C=C/CCCCCCCC(O)=O JBYXPOFIGCOSSB-GOJKSUSPSA-N 0.000 abstract description 17
- 229940108924 conjugated linoleic acid Drugs 0.000 abstract description 17
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 abstract 4
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- LVGKNOAMLMIIKO-QXMHVHEDSA-N ethyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC LVGKNOAMLMIIKO-QXMHVHEDSA-N 0.000 description 1
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- 125000005481 linolenic acid group Chemical group 0.000 description 1
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/1203—Addition of, or treatment with, enzymes or microorganisms other than lactobacteriaceae
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- A23V2200/32—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract
- A23V2200/3204—Probiotics, living bacteria to be ingested for action in the digestive tract
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Abstract
Description
본 발명은 비피더스속 균주를 이용한 공액리놀렌산(CLN)의 생산방법에 관한 것으로, 보다 자세하게는 인체로부터 분리된 유산균인 비피도박테리움 브레베 LMC520 균주를 리놀렌산이 포함된 배지 내에서 배양함으로써, 자연발생적으로 공액리놀렌산(CLN)으로 전환하는 비피도박테리움 브레베 LMC520 균주를 이용한 공액리놀렌산의 생산방법에 관한 것이다.The present invention relates to a method for producing conjugated linolenic acid (CLN) using a bifidus strain, and more particularly, by culturing the Bifidobacterium breve LMC520 strain, a lactic acid bacterium isolated from the human body, in a medium containing linolenic acid, The present invention relates to a method for producing conjugated linolenic acid using Bifidobacterium breve LMC520 strain which is converted to conjugated linolenic acid (CLN).
프로바이오틱(probiotic)은 "살아있는 미생물 첨가물로써 호스트내 장내 미생물의 균형을 증진시켜 유익한 효과를 나타내는 것"이라고 정의된다. 이러한 프로바이오틱은 건강 증진에 있어서 그 유익성, 기능성, 특성들에 관한 과학적 근거들이 축적됨에 따라 영양적 측면에서 점점 더 주목받고 있다.Probiotic is defined as "a living microbial additive that has a beneficial effect by enhancing the balance of intestinal microflora in the host." These probiotics are getting more and more attention in terms of nutrition as scientific evidences about their benefits, functionality, and traits are promoted in health promotion.
이러한 프로바이오틱 중에서 비피도박테리아(Bifidobacteria)는 무기물 이용 도, 비타민 합성의 증진, 면역력 증강, 위장장애 및 암 발병의 감소 등과 같은 특유의 인체 유익성을 지니고 있다. 또한, 일부 비피도박테리움 브레베(Bifidobacterium breve)는 기능성 지방산인 공액리놀렌산(conjugated linoleic acid, CLA)을 생산한다고 보고되고 있다.Among these probiotics, Bifidobacteria have unique human benefits, such as mineral utilization, enhanced vitamin synthesis, enhanced immunity, reduced gastrointestinal disorders and cancer. In addition, some Bifidobacterium breves have been reported to produce conjugated linoleic acid (CLA), a functional fatty acid.
그러나 최근에는 CLA 뿐만 아니라 CLN(conjugated linolenic acid)에서도 여러가지 독특한 약리 효능이 발견되고 있다. CLN은 3개의 공액 이중결합을 지닌 불포화 지방산의 위치 이성질체 및 기하 이성질체로써, 일부 식물 종자유에서 많이 생성된다. 퓨니닌산(시스-9, 트랜스-11, 시스-13-CLN)은 석류 종자유에 약 72% 함유되어 있고, α-엘레오스테아린산(시스-9, 트랜스-11, 트랜스-13-CLN)은 비터 고드(bitter gourd)유와 퉁(tung) 종자유에 각각 60%와 70% 함유되어있다. 또한, 개오동(catalpa) 종자유는 카탈핀산(트랜스-9, 트랜스-11, 시스-13-CLN)을 약 31% 함유하고 있고, 금잔화(pot marigold) 종자유는 카렌딘산(트랜스-8, 트랜스-10, 시스-12-CLN)을 약 33% 함유하고 있다. 그리고 α-리놀렌산(α-LNA) 또는 종자유의 알칼리 이성질화에 의해 생성되는 CLN 이성질체들의 혼성물이 항암작용과 체지방 감소에 어떠한 약리적 기능을 한다는 연구결과들이 상당히 존재한다. 또한, 정제된 α-엘레오스테아린산과 α-엘레오스테아린산이 풍부한 비터 고드 종자유는 체내외 실험에서 항암작용을 보였고, 그 밖에 다른 연구에서 퓨닌산이 함유된 석류 종자유의 급여가 쥐의 지방조직 세포를 감소시킨다고 보고되었다.Recently, however, several unique pharmacological effects have been found in CLA as well as in conjugated linolenic acid (CLN). CLN is a positional isomer and geometric isomer of unsaturated fatty acids with three conjugated double bonds, which are produced a lot in some plant seed oils. Funninic acid (cis-9, trans-11, cis-13-CLN) is present in about 72% pomegranate seed oil, and α-eleostearic acid (cis-9, trans-11, trans-13-CLN) 60% and 70% of bitter gourd and tung seed oils, respectively. In addition, the catalpa seed oil contains about 31% of catalic acid (trans-9, trans-11, cis-13-CLN), and the pot marigold seed oil contains carrenic acid (trans-8, trans-10). Cis-12-CLN). There is considerable research showing that a mixture of α-linolenic acid (α-LNA) or CLN isomers produced by alkali isomerization of seed oil has some pharmacological function in anticancer activity and body fat reduction. In addition, Bitter God seed oil enriched with purified α-Eleostearic acid and α-Eleostearic acid showed anticancer activity in vitro and other studies showed that the feeding of pomegranate seed oil containing purinic acid in rat fat tissue cells Has been reported.
CLN은 식물 종자유에 풍부하다고 알려져 있지만, 그 유효성이 매우 제한적이다. 따라서, 극히 일부의 영양보충제로 이용되는 CLN은 유기합성을 통해 생산되고 있지만, 아직 상용화되고 있지 않다. 또한, 유기합성된 CLN 및 식물 종자유 내에 함유되어 있는 CLN은 동정되지 않은 여러 이성질체들이 존재하여 제약 또는 건강식으로 이용되기 위해서는 안전한 이성질체들의 선별이 요구된다.CLN is known to be abundant in plant seed oil, but its effectiveness is very limited. Thus, CLN, which is used as only a few nutritional supplements, is produced through organic synthesis, but has not been commercialized yet. In addition, CLN contained in organically synthesized CLN and plant seed oil has a variety of unidentified isomers and requires the selection of safe isomers for use in pharmaceutical or health foods.
상기와 같은 종래 기술의 문제점을 해결하기 위하여 안출된 본 발명은 공액리놀렌산(CLN)의 생산방법에 관한 것으로, 보다 자세하게는 리놀렌산이 포함된 배지 내에서 인체로부터 분리된 유산균인 비피도박테리움 브레베 LMC520 균주를 배양하여 자연발생적으로 공액리놀렌산(CLN)을 생산함으로써, 유제품 내에서 스타터로 사용시에 공액리놀렌산을 증진시켜 유제품의 기능성을 증진시킬 수 있는 비피도박테리움 브레베 LMC520 균주를 이용하여 공액리놀렌산을 생산하는 방법을 제공함에 본 발명의 목적이 있다.The present invention devised to solve the problems of the prior art relates to a method for producing conjugated linolenic acid (CLN), and more specifically, Bifidobacterium breve which is a lactic acid bacterium isolated from the human body in a medium containing linolenic acid. Conjugated linolenic acid using Bifidobacterium breve LMC520 strain which can enhance the functionality of dairy products by enhancing conjugated linolenic acid when used as starter in dairy products by culturing LMC520 strains naturally It is an object of the present invention to provide a method for producing the same.
하나의 양태로서, 본 발명은 리놀렌산을 포함하는 배지 내에서 비피도박테리움 브레베 LMC520 균주(KCTC 10455BP)를 배양함으로써 공액리놀렌산을 생산하는 방법을 제공한다.In one embodiment, the present invention provides a method for producing conjugated linolenic acid by culturing Bifidobacterium breve LMC520 strain (KCTC 10455BP) in a medium comprising linolenic acid.
본 발명에서, 상기 리놀렌산(linolenic acid)은 3개의 이중결합과 카르복실산을 가진 불포화 지방산으로, 식물성 건성유 속에 글리세린 에스테르로 존재하는 무색·무취의 액체이다. 상기 리놀렌산은 크게 α-리놀렌산과 γ-리놀렌산으로 구분된다. α-리놀렌산은 (Z,Z,Z)-9,12,15-옥타데카트리에노익산이라고 하며, 체내에서는 합성되지 않는 필수 지방산으로 식품으로 섭취해야하며, 아마씨유(flexed-seed oil)나 호두유와 같은 식물성 유지 등에 많이 들어 있다. 또한, γ-리놀렌산 은 (Z,Z,Z)-6,9,12-옥타데카트리에노익산이라고 하며, 천연에서는 달맞이꽃이나 블랙커런트씨유, 보리지 오일(borage oil) 등에 함유되어 있는데, 혈중 콜레스테롤의 수치를 낮추는 데 효과적인 프로스타글란딘(prostaglandin)의 생체 내 합성에 꼭 필요한 물질로 알려져 있다.In the present invention, the linolenic acid is an unsaturated fatty acid having three double bonds and a carboxylic acid, and is a colorless, odorless liquid present as a glycerin ester in vegetable dry oil. The linolenic acid is largely divided into α-linolenic acid and γ-linolenic acid. α-linolenic acid is called (Z, Z, Z) -9,12,15-octadecatrienoic acid, which is an essential fatty acid that is not synthesized in the body and should be consumed as a food. It is found in vegetable oils such as walnut oil. In addition, γ-linolenic acid is called (Z, Z, Z) -6,9,12-octadecatenoic acid, and is naturally contained in evening primrose, blackcurrant seed oil, borage oil, etc. It is known to be essential for in vivo synthesis of prostaglandin, which is effective in lowering blood cholesterol levels.
본 발명에서, 상기 리놀렌산으로는 α-리놀렌산 또는 γ-리놀렌산 등 모든 리놀렌산이 사용될 수 있지만, 시스-9, 시스-12, 시스-15 위치에 비공액 이중결합을 포함하는 α-리놀렌산을 사용하는 것이 가장 바람직하다.In the present invention, all linolenic acid such as α-linolenic acid or γ-linolenic acid may be used as the linolenic acid, but it is preferable to use α-linolenic acid having a non-conjugated double bond in the cis-9, cis-12, cis-15 positions. Most preferred.
또한, 본 발명에서, 공액리놀렌산(conjugated linolenic acid, CLN)은 시스(cis) 혹은 트랜스(trans) 배열에 공액이중결합을 가지고 있는 모든 리놀렌산의 일련의 위치적 및 형태적 이성질체를 의미한다. 이들 CLN은 9, 11, 15 위치, 9, 11, 13 위치 또는 8, 10, 12 위치에 시스 또는 트랜스 배열을 취하여, 이들 위치에서 공액화된 이중결합을 갖는다.In addition, in the present invention, conjugated linolenic acid (CLN) refers to a series of positional and conformational isomers of all linolenic acids having conjugated double bonds in cis or trans configuration. These CLNs take a cis or trans configuration at
본 발명의 일실시예에서는, 리놀렌산으로 α-리놀렌산을 사용하여 이를 시스-9, 트랜스-11, 시스-15 위치에 공액 이중결합을 포함하는 시스-9, 트랜스-11, 시스-15-CLN으로 전환한다. 이의 구조는 하기 화학식 1 또는 화학식 2로 나타낼 수 있다.In one embodiment of the present invention, using linolenic acid α- linolenic acid to cis-9, trans-11, cis-15-CLN containing a conjugated double bond in the cis-9, trans-11, cis-15 position Switch. Its structure can be represented by the following formula (1) or (2).
다른 하나의 양태로서, 본 발명은 리놀렌산을 포함하는 우유 또는 탈지유 내에서 비피도박테리움 브레베 LMC520 균주(KCTC 10455BP)를 배양함으로써 CLN을 함유하는 발효유를 제조하는 방법을 제공한다.As another aspect, the present invention provides a method for producing a fermented milk containing CLN by culturing the Bifidobacterium breve LMC520 strain (KCTC 10455BP) in milk or skim milk containing linolenic acid.
본 발명에서, 상기 발효유는 일반적인 발효유의 의미를 갖는 것으로서, 원유 또는 유가공품을 유산균, 효모 등으로 발효시킨 것을 의미하며, 발효유의 형태, 원료, 고형분의 함량, 미생물의 종류, 생산지역 등에 따라서 다양할 수 있다. 발효유의 종류는 액상 발효유, 농후 발효유로 구별되는데, 이는 무지유 고형분 함량에 따라서 3.0% 이상의 경우는 액상 발효유로, 8.0% 이상의 경우는 농후 발효유로 구분 된다. 또한, 농후 발효유는 과일을 넣어서 떠먹도록 만든 호상 타입의 요구르트와 과즙을 넣어 마실 수 있도록 만든 드링크 타입 요구르트로 세분화된다.In the present invention, the fermented milk has a meaning of general fermented milk, and means fermented crude milk or dairy products with lactic acid bacteria, yeast, etc. Can be. The types of fermented milk are classified into liquid fermented milk and rich fermented milk, which are classified into liquid fermented milk in the case of 3.0% or more and rich fermented milk in the case of more than 3.0% according to the non-fat solid content. In addition, rich fermented milk is subdivided into a staple-type yogurt made by putting fruit and a drink-type yogurt made by drinking juice.
또 다른 하나의 양태로서, 본 발명은 리놀렌산을 포함하는 우유 또는 탈지유 내에서 비피도박테리움 브레베 LMC520 균주(KCTC 10455BP)를 배양함으로써 제조된 CLN을 함유하는 발효유를 제공한다.As another aspect, the present invention provides a fermented milk containing CLN prepared by culturing Bifidobacterium breve LMC520 strain (KCTC 10455BP) in milk or skim milk containing linolenic acid.
따라서, 본 발명의 비피도박테리움 브레베 LMC520 균주를 이용한 CLN의 생산방법은 인체로부터 분리된 유산균인 비피도박테리움 브레베 LMC520 균주를 이용함으로써, CLN을 자연발생적으로 생산할 수 있는 효과가 있다.Therefore, the method of producing CLN using the Bifidobacterium breve LMC520 strain of the present invention has the effect of naturally producing CLN by using the Bifidobacterium breve LMC520 strain, which is isolated from the human body.
또한, 본 발명의 비피도박테리움 브레베 LMC520 균주를 이용한 CLN의 생산방법은 인체로부터 분리된 유산균인 비피도박테리움 브레베 LMC520 균주를 이용하여 자연발생적으로 CLN을 생산함으로써, 유제품 내에서 스타터로 사용시에 CLN을 증진시켜 유제품의 기능성을 증진시킬 수 있는 효과가 있다.In addition, the production method of CLN using the Bifidobacterium breve LMC520 strain of the present invention by producing a CLN naturally using the Bifidobacterium breve LMC520 strain, which is isolated from the human body, as a starter in dairy products In use, it has the effect of promoting the functionality of dairy products by promoting CLN.
본 명세서 및 청구범위에 사용된 용어나 단어는 통상적이거나 사전적인 의미로 한정해서 해석되어서는 아니되며, 발명자는 그 자신의 발명을 가장 최선의 방법으로 설명하기 위해 용어의 개념을 적절하게 정의할 수 있다는 원칙에 입각하여 본 발명의 기술적 사상에 부합하는 의미와 개념으로 해석되어야만 한다.The terms or words used in this specification and claims are not to be construed as being limited to their ordinary or dictionary meanings, and the inventors may appropriately define the concept of terms in order to best describe their invention. It should be interpreted as meaning and concept corresponding to the technical idea of the present invention based on the principle that the present invention.
따라서, 본 명세서에 기재된 실시예와 도면에 도시된 구성은 본 발명의 가장 바람직한 일 실시예에 불과할 뿐이고 본 발명의 기술적 사상을 모두 대변하는 것은 아니므로, 본 출원시점에 있어서 이들을 대체할 수 있는 다양한 균등물과 변형예들이 있을 수 있음을 이해하여야 한다.Therefore, the embodiments described in the specification and the drawings shown in the drawings are only the most preferred embodiment of the present invention and do not represent all of the technical idea of the present invention, various modifications that can be replaced at the time of the present application It should be understood that there may be equivalents and variations.
실시예 1 : CLN의 미생물적 생산Example 1 Microbial Production of CLN
본 실시예에서는 스크린된 비피도박테리아 중에 가장 높은 CLN 생산 활성을 가지는 비피도박테리움 브레베 LMC520 균주(KCTC 10455BP)를 사용하였다.In this example, the Bifidobacterium breve LMC520 strain (KCTC 10455BP) having the highest CLN production activity among the screened Bifidobacteria was used.
CLN의 생산을 위하여, 상기 균주를 셉텀 스토퍼(septum stopper)(Bellco)와 알루미늄 실(seal)(Bellco)로 덮은 벨코 튜브(18×150mm; Bellco, Vineland, NJ, USA) 내, 0.05% L-시스테인·HCl이 포함된 MRS 배지(mMRS; Sigma)에서 O2가 존재하지 않는 CO2 하, 37℃로 18시간 동안 두차례 계대배양하였다. 상기 활성화된 배양물을 새로운 mMRS로 옮긴 다음, 다른 기질 농도 조건들, 즉 각각 1, 2, 4, 6, 8 및 10mM의 α-리놀렌산(α-linolenic acid, α-LNA) 농도 조건들 하에서 배양하였다. 성장률은 600nm에서 마이크로플레이터 리더(BIO-RAD, Hercules, CA, USA)를 이용하여 광학 밀도(O.D)를 측정함으로써 평가하였다.For the production of CLN, the strain was 0.05% L − in a Belco tube (18 × 150 mm; Bellco, Vineland, NJ, USA) covered with septum stopper (Bellco) and aluminum seal (Bellco). MRS medium containing cysteine-HCl (mMRS; Sigma) was subcultured twice for 18 hours at 37 ° C. under CO 2 without O 2 . The activated culture is transferred to new mMRS and then cultured under different substrate concentration conditions, i.e., α-linolenic acid (α-LNA) concentration conditions of 1, 2, 4, 6, 8 and 10 mM, respectively. It was. Growth rate was evaluated by measuring optical density (OD) using a microplate reader (BIO-RAD, Hercules, CA, USA) at 600 nm.
실시예 2 : 균주 배양물의 지방산 분석Example 2 Fatty Acid Analysis of Strain Cultures
CLN이 생산된 상기 실시예 1의 균주 배양물로부터 지방산을 추출하기 위해, 내부표준(IS) 물질로서 헵타데카노익산(C17:0)을 첨가한 1㎖의 배양물을 12㎖의 클로로포름/메탄올(1:1, v/v)로 추출하였다. 상기 추출물의 아래층을 2㎖의 0.88% KCl 용액과 격렬하게 혼합한 다음, 건조될 때까지 질소로 증발시켰다.In order to extract fatty acids from the strain culture of Example 1 in which CLN was produced, 1 ml of the culture to which heptadecanoic acid (C 17: 0 ) was added as an internal standard (IS) material was added to 12 ml of chloroform /. Extracted with methanol (1: 1, v / v). The bottom layer of the extract was vigorously mixed with 2 ml of 0.88% KCl solution and then evaporated to nitrogen until dry.
상기 추출된 지질을 80℃에서 60분 동안 10㎖의 에탄올 내 2% H2SO4를 이용하여 에틸 에스테르화하였다. 8㎖의 포화 NaCl 용액과 4㎖의 n-헥산을 첨가한 후, 지방산 에틸 에스터를 n-헥산층에서 획득하고, 불꽃 이온화 검출기(flame ionization detector)(Agilent Technologies)를 사용한 7890A 가스 크로마토그래피(gas chromatography, GC)를 이용하여 CLN 이성질체를 포함한 총 지방산을 분석하였다.The extracted lipids were ethyl esterified with 2% H 2 SO 4 in 10 ml of ethanol at 80 ° C. for 60 minutes. After addition of 8 ml of saturated NaCl solution and 4 ml of n-hexane, fatty acid ethyl ester was obtained in the n-hexane layer and 7890A gas chromatography using flame ionization detector (Agilent Technologies) Total fatty acids including CLN isomers were analyzed by chromatography (GC).
상기 지방산 에틸 에스터는 1.2㎖/분의 헬륨 기류로 슈펠코왁스-10 융합 실리카 모세관 컬럼(Supelcowax-10 fused silica capillary column, 100m×0.25mm i.d., 0.2㎛ 필름 두께; Supelco, Inc., Bellefonte, PA, USA)을 이용하여 분리하였다. 오븐의 온도는 4℃/분의 속도로 190℃로부터 240℃까지 증가시켰다. 주입기와 검출기의 온도는 둘 다 260℃였다. 1㎕의 시료를 스플릿 모드(50:1)로 컬럼에 주입하였다.The fatty acid ethyl ester was a Supelcoax-10 fused silica capillary column, 100 m × 0.25 mm id, 0.2 μm film thickness at 1.2 mL / min helium airflow; Supelco, Inc., Bellefonte, PA , USA). The temperature of the oven was increased from 190 ° C. to 240 ° C. at a rate of 4 ° C./min. The temperature of the injector and detector was both 260 ° C. 1 μl of sample was injected into the column in split mode (50: 1).
각각의 CLN 이성질체 및 다른 지방산의 피크는 각각의 지방산 표준 물질의 머무름 시간(retention time) 및 피크 면적과 비교함으로써 확인 및 정량되었다. 내부표준 물질은 각 시료 내 지방산의 회수를 측정하기 위하여 추출 전에 내부기준 물질로서 첨가되었다.Peaks of each CLN isomer and other fatty acids were identified and quantified by comparing the retention time and peak area of each fatty acid standard. Internal standards were added as internal reference materials prior to extraction to determine the recovery of fatty acids in each sample.
상기 GC로 분석한 스펙트럼 결과를 도 1a 및 도 1b에 나타내었다.The spectral results analyzed by the GC are shown in FIGS. 1A and 1B.
도 1a는 균주 배양액에 α-LNA를 첨가하고 배양하기 전의 스펙트럼 결과를 나타내며, 가장 오른쪽 피크가 α-LNA의 피크이었다.Figure 1a shows the spectral results before addition and incubation of the α-LNA to the strain culture, the rightmost peak was the peak of the α-LNA.
도 1b에는 본 발명에 따른 비피도박테리움 브레베 LMC520 균주(KCTC 10455BP) 배양물에 α-LNA를 첨가하여 배양함으로써, CLN이 생산된 후의 스펙트럼 결과를 나타내었다. CLN 생산이 시작된지 3시간 후에 측정한 결과로, 아직 전환능이 최대가 아니기 때문에 일부 α-LNA가 남아 있었으며, 가장 오른쪽 피크가 CLN 피크이었다.Figure 1b shows the spectral results after the production of CLN by culturing by adding α-LNA to the Bifidobacterium breve LMC520 strain (KCTC 10455BP) culture according to the present invention. Three hours after CLN production began, some α-LNA remained because the conversion was not maximum yet, and the rightmost peak was the CLN peak.
상기 GC 측정 후, 얻어진 지방산 에틸 에스터에 대하여 GC/MS를 통하여 분자량을 분석하였다.After the GC measurement, the obtained fatty acid ethyl ester was analyzed for molecular weight through GC / MS.
분석시에 1.0㎖/분의 헬륨 기류로 DB-23 컬럼(60m×0.25mm i.d., 0.25㎛)을 이용하였으며, 오븐의 온도는 1분 동안 150℃로 유지하고, 이후 15℃/분의 속도로 150℃로부터 200℃까지 증가시킨 다음, 2℃/분의 속도로 200℃로부터 230℃까지 증가시키고, 5분 동안 230℃에서 유지시켰다. 주입기 온도는 250℃였고, 검출 온도(Aux temp.)는 280℃이었다. 스플릿 비율은 2:1이었다.In the analysis, a DB-23 column (60 m × 0.25 mm id, 0.25 μm) was used as the helium stream at 1.0 mL / min. It was increased from 150 ° C. to 200 ° C., then from 200 ° C. to 230 ° C. at a rate of 2 ° C./min and held at 230 ° C. for 5 minutes. The injector temperature was 250 ° C. and the detection temperature (Aux temp.) Was 280 ° C. The split ratio was 2: 1.
도 2에 본 발명에 따른 비피도박테리움 브레베 LMC520 균주(KCTC 10455BP)로부터 생산된 CLN을 GC/MS로 분석한 스펙트럼 결과를 나타내었다.2 shows the spectral results of the CLN produced from the Bifidobacterium breve LMC520 strain (KCTC 10455BP) according to the present invention by GC / MS.
도 2에서, 1번 피크는 9, 11, 15-옥타데카트리에노익산 에틸 에스터(9, 11, 15-Octadecatrienoic acid ethyl ester)(리놀렌산 에틸 에스터, C18:3, Mw 306, 9, 11, 15-CLN)의 피크에 해당하고, 2번 및 3번 피크는 9, 12-옥타데카디에노익산 에틸 에스터(9, 12-Octadecadienoic acid ethyl ester)(리놀레인산 에틸 에스터, C18:2, Mw 308)의 피크에 해당하며, 4번 피크는 9-옥타데세노익산 에틸 에스터(9-Octadecenoic acid ethyl ester)(올레인산 에틸 에스터, C18:1, Mw 310)의 피크에 해당하고, 5번 피크는 옥타데카노익산 에틸 에스터(Octadecanoic acid ethyl ester)(스테아릭산 에틸 에스터, C18:0, Mw 312)의 피크에 해당하며, 6번 피크는 헥사데칸산 에틸 에스터(Hexadecanoic acid ethyl ester)(C16:0, Mw 284)의 피크에 해당한다.In Figure 2,
따라서, 상기 도 2의 결과를 통해 알 수 있는 바와 같이, 본 발명에 따른 비피도박테리움 브레베 LMC520 균주(KCTC 10455BP)로부터 생산된 CLN는, CLN 이성질체 중에서 9, 11, 15-CLN 이성질체가 주요 이성질체(> 90%)였으며, 그 밖에 다른 CLN 이성질체는 생산되지 않았다.Therefore, as can be seen from the results of FIG. 2, CLN produced from the Bifidobacterium breve LMC520 strain (KCTC 10455BP) according to the present invention, 9, 11, 15-CLN isomers of the CLN isomers are the main Isomers (> 90%) and no other CLN isomers were produced.
실시예 3 : 균주 배양물로부터 분리한 지방산의 구조 분석Example 3 Structural Analysis of Fatty Acids Isolated from Strain Cultures
상기 실시예 1의 균주 배양물로부터 얻은 지방산의 구조를 명확히 하기 위해, 핵자기공명 분광기(400MHz NMR, Inova AS400, Varian, CA, USA)로 1H-NMR, 13C-NMR, gCOSY 및 gHMBC를 측정하였다. NMR 용매로는 CDCl3를 사용하였다. 그 결과를 하기 표 1에 나타내었다.In order to clarify the structure of the fatty acid obtained from the strain culture of Example 1, 1 H-NMR, 13 C-NMR, gCOSY and gHMBC with a nuclear magnetic resonance spectrometer (400 MHz NMR, Inova AS400, Varian, CA, USA) Measured. CDCl 3 was used as the NMR solvent. The results are shown in Table 1 below.
1.20~1.38(m, 8H)
1.20-1.38 (m, 8H)
H9↔C10(J 2)H9↔C11 ( J 3 ), H9↔C8 ( J 2 ),
H9↔C10 ( J 2 )
H10↔C11(J 2), H10↔C9(J 2)H10↔C12 ( J 3 ), H10↔C8 ( J 3 ),
H10↔C11 ( J 2 ), H10↔C9 ( J 2 )
H11↔C12(J 2), H11↔C10(J 2)H11↔C13 ( J 3 ), H11↔C9 ( J 3 ),
H11↔C12 ( J 2 ), H11↔C10 ( J 2 )
H12↔C13(J 2), H12↔C11(J 2)H12↔C14 ( J 3 ), H12↔C10 ( J 3 ),
H12↔C13 ( J 2 ), H12↔C11 ( J 2 )
[주] a 1H-NMR(400MHz) A 1 H-NMR (400MHz)
b 13C-NMR(100MHz) b 13 C-NMR (100 MHz)
c 1H-13C gradient heteronuclear multiple bonding connectivity : 주로, J3 상호관계 c 1 H- 13 C gradient heteronuclear multiple bonding connectivity: mainly, J 3 correlation
1H-NMR 및 13C-NMR 데이터를 분석한 결과, 상기 지방산이 불포화 지방산이며, 여러 가지 지방산을 포함하는 것으로 추정되었다. 그러나 지방산의 구조는 일차적으로 주요 구성성분의 시그날을 통해 확인하였다.Analysis of 1 H-NMR and 13 C-NMR data indicated that the fatty acid was an unsaturated fatty acid and contained various fatty acids. However, the structure of fatty acids was primarily confirmed through the signal of the major constituents.
1H-NMR 스펙트럼 분석 결과, 6개의 올레핀 메틴(methine) 프로톤[δH 6.29(dd, J=14.8, 10.8Hz, H-11), δH 5.92(dd, J=10.8, 10.8Hz, H-10), δH 5.64(dt, J=14.8, 7.2Hz, H-12), δH 5.38(td, J=7.2, 10.8Hz, H-16), δH 5.33(ddd, J=6.8, 14.8, 10.8Hz, H-9) 및 δH 5.29(td, J=7.2, 10.8Hz, H-15)], 8개의 알릴 메틸렌 프로톤[δH 2.32(t, J=7.2Hz, H-2), δH 2.14(tdd, J=7.2, 14.8, 10.8Hz, H-8b), δH 2.13(tdd, J=7.2, 14.8, 10.8Hz, H-8a), δH 2.12(td, J=7.2, 7.2Hz, H-13), δH 2.03(td, J=7.2, 7.2Hz, H-14)], 12개의 메틸렌 프로톤[δH 1.61(tt, J=7.2, 7.2Hz, H-3), δH 1.20~1.38(m, H3~H7, H17)], 그리고 δH 0.86(t, J=7.6Hz, H-18)에서 지방산의 말단 메틸이 관찰되었다. 이로부터 측정 화합물이 3개의 올레핀기을 포함하는 지방산임을 확인하였다. 1 H-NMR spectrum analysis, six olefinic methine (methine) Proton [δ H 6.29 (dd, J = 14.8, 10.8Hz, H-11), δ H 5.92 (dd, J = 10.8, 10.8Hz, H- 10), δ H 5.64 (dt, J = 14.8, 7.2 Hz, H-12), δ H 5.38 (td, J = 7.2, 10.8 Hz, H-16), δ H 5.33 (ddd, J = 6.8, 14.8 , 10.8 Hz, H-9) and δ H 5.29 (td, J = 7.2, 10.8 Hz, H-15)], 8 allyl methylene protons [δ H 2.32 (t, J = 7.2 Hz, H-2), δ H 2.14 (tdd, J = 7.2, 14.8, 10.8 Hz, H-8b), δ H 2.13 (tdd, J = 7.2, 14.8, 10.8 Hz, H-8a), δ H 2.12 (td, J = 7.2, 7.2 Hz, H-13), δ H 2.03 (td, J = 7.2, 7.2 Hz, H-14)], 12 methylene protons (δ H 1.61 (tt, J = 7.2, 7.2 Hz, H-3), δ H 1.20-1.38 (m, H3 ~ H7, H17)], and δ H 0.86 (t, J = 7.6 Hz, H-18), the terminal methyl of fatty acids was observed. From this, it was confirmed that the measurement compound is a fatty acid including three olefin groups.
13C-NMR 스펙트럼은 δC 179.9(C-1)에서 카르보닐 카본 시그날, 6개의 올레핀 메탄 카본 시그날[δC 133.8(C-9), δC 132.0(C-16), δC 130.1(C-12), δC 128.5(C-10), δC 128.1(C-15) 및 δC 125.8(C-11)], 10개의 메틸렌 카본 시그날[δC 34.1(C-2), δC 33.0(C-13), δC 31.7(C-7), δC 29.7(C-6), δC 29.2(C-5), δC 29.1(C-4), δC 27.7(C-8), δC 27.1(C-14), δC 24.7(C-3) 및 δC 22.7(C-17)], 그리고 δC 14.2(C-18)에서 말단 메틸 카본 시그날을 산출하였다. 전체적으로, 이러한 18개의 카본 시그날은 3개의 올레핀기를 포함하는 불포화 옥타데카노익산임을 나타내었다. The 13 C-NMR spectra are carbonyl carbon signals at δ C 179.9 (C-1), six olefin methane carbon signals [δ C 133.8 (C-9), δ C 132.0 (C-16), δ C 130.1 (C -12), δ C 128.5 (C-10), δ C 128.1 (C-15) and δ C 125.8 (C-11)], 10 methylene carbon signals [δ C 34.1 (C-2), δ C 33.0 (C-13), δ C 31.7 (C-7), δ C 29.7 (C-6), δ C 29.2 (C-5), δ C 29.1 (C-4), δ C 27.7 (C-8) , δ C 27.1 (C-14), δ C 24.7 (C-3) and δ C 22.7 (C-17)], and δ C 14.2 (C-18). In total, these 18 carbon signals were shown to be unsaturated octadecanoic acid comprising three olefin groups.
추가적으로, 3개의 올레핀기의 위치는 gCOSY 및 gHMBC 스펙트럼으로 정확하게 확인하였다. gCOSY 스펙트럼에서, 올레핀 메틴 프로톤 시그날 H-9(δH 5.33)는 H-8a(δH 2.13)/8b(δH 2.14)와의 J 3 상호관계 피크 및 10.8Hz의 화학상수를 가진 H-10(δH 5.92)와의 J 3 상호관계 피크를 보였으며, 이로부터 상기 올레핀 메틴 프로톤이 (Z)-배열, 즉 시스 배열을 나타냄을 알 수 있었다. H-10은 10.8Hz의 화학상수를 가진 H-11(δH 6.29)과의 J 3 상호관계 피크를 보였으며, 이로부터 H-10이 (Z)-배열, 즉 시스 배열을 나타냄을 알 수 있었다. 그리고 H-11(δH 6.29)은 14.8Hz의 화학상수를 가진 H-12(δH 5.64)와의 J 3 상호관계 피크를 보였으며, 이로부터 H-11이 (E)-배열, 즉 트랜스 배열을 나타냄을 알 수 있었다. 따라서, 2개의 올레핀기가 Z-Z-E와 같이 순서대로 연결되었다. H-16(δH 5.38)은 10.8Hz의 화학상수로 연결된 H-15(δH 5.29)와의 J 3 상호관계 피크를 보였으며, 이로부터 H-16이 (Z)-배열, 즉 시스 배열을 나타냄을 알 수 있었다. 3개의 올레핀기의 위치를 포함하는 지방산의 최종 구조를 gCOSY 및 gHMBC로 확인한 결과, 본 발명에 따라 비피도박테리움 브레베 LMC520 균주에 의해 생산된 지방산은 시스-9, 트랜스-11, 시스-15-CLN인 9Z, 11E, 15Z-옥타데카-9, 11, 15-트리에노익산으로 확인되었다(화학식 1 및 화학식 2 참조).In addition, the positions of the three olefin groups were accurately identified by the gCOSY and gHMBC spectra. In the gCOSY spectrum, the olefin methine proton signal H-9 (δ H 5.33) is H-10 with a J 3 correlation peak with H-8a (δ H 2.13) / 8b (δ H 2.14) and a chemical constant of 10.8 Hz. J 3 correlation peak with δ H 5.92), indicating that the olefin methine proton exhibits a ( Z ) -configuration, ie a cis configuration. H-10 showed a J 3 correlation peak with H-11 (δ H 6.29) with a chemical constant of 10.8 Hz, indicating that H-10 represents the ( Z ) -array, or cis-array. there was. And H-11 (δ H 6.29) showed a J 3 correlation peak with H-12 (δ H 5.64) with a chemical constant of 14.8 Hz, from which H-11 was ( E ) -arranged It could be seen that. Thus, two olefin groups were connected in order like ZZE. H-16 (δ H 5.38) showed a J 3 correlation peak with H-15 (δ H 5.29) linked to a chemical constant of 10.8 Hz, from which H-16 resolved the ( Z ) -array, the cis array. It can be seen that. The final structures of the fatty acids comprising the positions of the three olefin groups were confirmed by gCOSY and gHMBC. As a result, the fatty acids produced by the Bifidobacterium breve LMC520 strain according to the present invention were cis-9, trans-11, cis-15. It was identified as 9Z, 11E, 15Z-octadeca-9, 11, 15-trienoic acid which is -CLN (see
실시예 4 : 배양 시간에 따른 비피도박테리움 브레베 LMC520 균주의 성장률과 이로부터 생산된 CLN의 생산량 조사Example 4 Investigation of the growth rate of Bifidobacterium breve LMC520 strain and the amount of CLN produced therefrom
상기 비피도박테리움 브레베 LMC520 균주(2%, v/v)의 배양물을 2mM α-LNA가 포함된 mMRS 내에 접종하였다. 그리고 이것을 혐기조건에서 37℃로 48시간 동안 배양하였다.Cultures of the Bifidobacterium breve LMC520 strain (2%, v / v) were inoculated in mMRS containing 2 mM α-LNA. And it was incubated for 48 hours at 37 ℃ under anaerobic conditions.
상기와 같이 배양된 배양물을 600nm에서 마이크로플레이트 리더(BIO-RAD, Hercules, CA, USA)로 광학 밀도(optical density, O.D)를 측정함으로써 비피도박테리움 브레베 LMC520 균주로부터 생산된 CLN의 생산량 및 성장률을 확인하였다.Production of CLN produced from Bifidobacterium breve LMC520 strain by measuring the optical density (OD) with a microplate reader (BIO-RAD, Hercules, CA, USA) at 600 nm in cultured as described above And growth rate was confirmed.
도 3에 본 발명에 따른 비피도박테리움 브레베 LMC520 균주(KCTC 10455BP)로부터 생산된 CLN의 생산량 및 성장률에 관한 그래프를 나타내었다(여기서 a, b, c 및 d는 서로 다른 값을 나타냄, p<0.05).Figure 3 shows a graph of the yield and growth rate of CLN produced from Bifidobacterium breve LMC520 strain (KCTC 10455BP) according to the present invention (where a, b, c and d represent different values, p <0.05).
도 3에서, CLN 생산량은 로그대수기(logarithmic growth phase) 중간인 배양을 시작한지 6시간이 지난 시점에서 상당히 증가하였으며, 초기 성장 정체기(stationary growth phase)에서 최대 수준에 도달하였다. 그리고 CLN 생산량은 배양을 시작한지 24시간이 지난 후부터 약간씩 감소하였다. 또한, CLN이 점차 생산됨에 따라 배양물 내의 pH가 감소하는 것을 확인할 수 있었다.In FIG. 3, CLN production increased significantly 6 hours after initiation of the middle of the logarithmic growth phase, reaching a maximum level in the initial stationary growth phase. CLN production decreased slightly after 24 hours of incubation. In addition, as the CLN is gradually produced it was confirmed that the pH in the culture decreases.
한편, 비피도박테리움 브레베 LMC520 균주(2%, v/v)의 배양물을 2mM LA가 포함된 mMRS 내에 접종하여, 혐기조건에서 37℃로 48시간 동안 배양하면서 시간에 따른 상기 균주의 성장률과 CLA의 생산량을 조사하였다.Meanwhile, the incubation of Bifidobacterium breve LMC520 strain (2%, v / v) in mMRS containing 2mM LA, the growth rate of the strain with time while incubating for 48 hours at 37 ℃ in anaerobic conditions And CLA production were investigated.
그 결과를 도 4에 나타내었다. 도 4를 통해 알 수 있듯이, CLA 생산량은 성장과 함께 상당히 증가하였다(여기서 a, b 및 c는 서로 다른 값을 나타냄).The results are shown in FIG. As can be seen from FIG. 4, CLA production increased significantly with growth, where a, b and c represent different values.
상기 도 3과 도 4의 결과를 통해 알 수 있는 바와 같이, 종래기술에 따른 CLA 생산량은 균주의 성장과 함께 상당히 증가하였으나, 본 발명에 따른 CLN 생산량은 초기 로그대수기(logarithmic growth phase, 0시부터 6시)에서 상당히 증가함을 확인하였다.As can be seen from the results of Figures 3 and 4, the CLA production according to the prior art significantly increased with the growth of the strain, CLN production according to the present invention is the logarithmic growth phase (0 o'clock) From 6 o'clock).
실시예 5 : α-LNA 농도에 따른 비피도박테리움 브레베 LMC520 균주로부터 생산된 CLN의 생산량 및 전환률 조사Example 5 Investigation of Production and Conversion Rate of CLN Produced from Bifidobacterium breve LMC520 Strain According to α-LNA Concentration
상기 비피도박테리움 브레베 LMC520 균주(KCTC 10455BP)를 각기 다른 농도의 α-LNA(1mM 내지 10mM)가 포함된 mMRS 내에서 배양하였다. 배양은 혐기조건에서 37℃로 24시간 동안 진행되었다.The Bifidobacterium breve LMC520 strain (KCTC 10455BP) was incubated in mMRS containing different concentrations of α-LNA (1 mM to 10 mM). The culture was carried out for 24 hours at 37 ℃ in anaerobic conditions.
도 5에 각각 다른 α-LNA 농도에서 본 발명에 따른 비피도박테리움 브레베 LMC520 균주로부터 생산된 CLN의 생산량 및 전환률에 관한 그래프를 나타내었다(각 그래프 내에 기재된 a, b, c, d 및 e는 서로 다른 값을 나타냄, <0.05).Figure 5 shows a graph of the yield and conversion of CLN produced from Bifidobacterium breve LMC520 strain according to the present invention at different α-LNA concentrations (a, b, c, d and e described in each graph). Indicates different values, <0.05).
도 5에서, 상기 비피도박테리움 브레베 LMC520 균주를 α-LNA가 1mM 첨가된 배지에서 배양하여 생산된 CLN의 생산량은 약 0.2mg/㎖이고, 8mM가 첨가된 배지에서 배양하였을 때 생산된 CLN의 생산량은 약 1.4mg/㎖로 그 생산량이 약 7배 증가함을 알 수 있었다.In FIG. 5, the production of CLN produced by culturing the Bifidobacterium breve LMC520 strain in a medium containing 1 mM of α-LNA was about 0.2 mg / ml, and was produced when cultured in a medium containing 8 mM added thereto. The yield of about 1.4mg / ㎖ was found to increase about 7 times.
또한, 비피도박테리움 브레베 LMC520 균주를 α-LNA가 첨가된 배지에서 배양하였을 때, 상기 배양물이 CLN으로 전환되는 전환률(conversion rate)은 거의 100%였으며, 심지어 성장이 최대 수준에 도달하기 이전에 CLN을 고수율로 얻을 수 있었다. CLN으로의 전환률은 α-LNA를 8mM 첨가하였을 때까지 90% 이상을 유지하였다.In addition, when the Bifidobacterium breve LMC520 strain was cultured in a medium supplemented with α-LNA, the conversion rate at which the culture was converted to CLN was almost 100%, and even growth reached its maximum level. CLN was previously obtained with high yield. The conversion to CLN was maintained at 90% or more until 8 mM of α-LNA was added.
한편, 비피도박테리움 브레베 LMC520 균주(KCTC 10455BP)를 각기 다른 농도의 LA(1mM 내지 8mM)가 포함된 mMRS 내 접종하여, 혐기조건에서 37℃로 24시간 동안 배양한 후, 각각의 LA 농도에서 상기 균주로부터 생산된 CLA 생산량 및 전환률을 조사하였다.Meanwhile, Bifidobacterium breve LMC520 strain (KCTC 10455BP) was inoculated in mMRS containing different concentrations of LA (1 mM to 8 mM), incubated for 24 hours at 37 ° C. under anaerobic conditions, and then each concentration of LA The production and conversion rate of CLA produced from the above strain was investigated.
그 결과를 도 6에 나타내었다. 도 6을 통해 알 수 있듯이, CLA를 생산하는데 있어서 최적의 LA 농도는 1mM이었다(각 그래프 내에 기재된 a, b, c, d 및 e는 서로 다른 값을 나타냄).The results are shown in FIG. As can be seen from FIG. 6, the optimal LA concentration in producing CLA was 1 mM (a, b, c, d and e described in each graph represent different values).
상기 도 5와 도 6의 결과를 통해 알 수 있는 바와 같이, 종래 CLA를 생산하는데 있어서 최적의 LA 농도는 1mM인 반면, 본 발명에 따른 CLN 생산에 있어서 α-LNA의 최적의 농도는 2mM이었다.As can be seen through the results of FIGS. 5 and 6, the optimal concentration of LA in producing CLA is 1 mM, whereas the optimal concentration of α-LNA in CLN production according to the present invention was 2 mM.
또한, 본 발명에 따른 CLN 생산량은 2mM LA에서의 종래의 CLA 생산량보다 30% 더 높았으며, 전환률도 더 높았다. 이러한 결과는 종래의 CLA 생산량과 비교하였을 때, 본 발명에 따른 비피도박테리움 브레베 LMC520 균주(KCTC 10455BP)가 이성화 효소(isomerase)를 보다 더 많이 가지고 있다는 것을 나타낸다.In addition, the CLN production according to the present invention was 30% higher than the conventional CLA production at 2 mM LA, and the conversion was higher. These results indicate that the Bifidobacterium breve LMC520 strain (KCTC 10455BP) according to the present invention has more isomerase compared to conventional CLA production.
실시예 6 : pH에 따른 비피도박테리움 브레베 LMC520 균주의 CLN 생산량 조사Example 6 CLN Production of Bifidobacterium breve LMC520 Strains According to pH
pH는 CLN 생산에 있어서 균의 생육 활성 뿐만 아니라, CLN 생산 효소의 활성에 있어서도 중요한 인자이다.pH is an important factor not only in the growth activity of bacteria in CLN production but also in the activity of CLN producing enzyme.
pH를 3.5 내지 9.0으로 달리 하면서, 각 pH 조건에서 비피도박테리움 브레베 LMC520 균주(KCTC 10455BP)가 생산한 CLN의 생산량을 조사하였다. 배양은 혐기조건에서 37℃로 24시간 동안 진행하였다. 그 결과를 도 7에 나타내었다.By varying the pH from 3.5 to 9.0, the yield of CLN produced by the Bifidobacterium breve LMC520 strain (KCTC 10455BP) at each pH condition was investigated. The culture was carried out for 24 hours at 37 ℃ in anaerobic conditions. The results are shown in FIG.
한편, pH를 4.0 내지 10.0으로 달리하면서 각 pH 조건에서 비피도박테리움 브레베 LMC250 균주(KCTC 10455BP)가 생산한 CLA 생산량을 조사하여, 그 결과를 도 8에 나타내었다(그래프 내에 기재된 a, b, c, d, e, f, g, h, i 및 j는 서로 다른 값을 나타냄).On the other hand, while varying the pH from 4.0 to 10.0, the CLA production amount produced by the Bifidobacterium breve LMC250 strain (KCTC 10455BP) under each pH condition was investigated, and the results are shown in FIG. 8 (a, b described in the graph). , c, d, e, f, g, h, i and j represent different values).
pH 5와 pH 5.5는 유제품 생산에 있어서 대부분의 스타터 균주들의 최적 활성 범위인데, 도 7을 통해 상기 범위에서 CLN으로의 전환률이 상당히 높음을 알 수 있었다. 그리고 CLN으로의 전환률은 염기성 조건에서도 상당히 높았으며, 도 7과 도 8을 비교함으로써 산성 조건에서도 CLN으로의 전환률이 CLA로의 전환률보다 상대적으로 높았음을 알 수 있었다.
전반적으로 비피도박테리움 브레베 LMC520 균주를 이용한 CLN 생산능은 같은 pH 조건에서의 CLA 생산능보다 높았으며, pH에 크게 영향을 받지 않음을 알 수 있었다.Overall, CLN production capacity using Bifidobacterium breve LMC520 strain was higher than CLA production capacity at the same pH condition, and it was found that pH was not significantly affected.
실시예 7 : 산소 조건 따른 비피도박테리움 브레베 LMC520 균주의 CLN 전환률 조사Example 7: CLN conversion of Bifidobacterium breve LMC520 strain according to oxygen conditions
호기 또는 혐기적 조건에서 각각 본 발명에 따른 비피도박테리움 브레베 LMC520 균주(KCTC 10455BP)를 배양하여 이로부터 생산된 CLN의 양을 비교하였다. 배양은 37℃로 48시간 동안 수행하였다.Bifidobacterium breve LMC520 strain (KCTC 10455BP) according to the present invention was cultured in aerobic or anaerobic conditions, respectively, and the amount of CLN produced therefrom was compared. Cultivation was performed at 37 ° C. for 48 hours.
그 결과를 도 9에 나타내었다. 도 9를 통해 알 수 있듯이, 6시간까지는 혐기적 조건에서 전환능이 더 높았으나, 12시간부터는 호기적 조건과 혐기적 조건에서의 전환능이 유사하였다.The results are shown in FIG. As can be seen from Figure 9, the conversion was higher in anaerobic conditions up to 6 hours, the conversion was similar in aerobic and anaerobic conditions from 12 hours.
일반적으로 비피도박테리움 균주들은 호기적 조건에서 활성의 저해를 받지만, 도 9의 결과를 통해, 비피도박테리움 브레베 LMC520 균주(KCTC 10455BP)의 CLN 전환에 있어서는 크게 영향을 받지 않는 것을 알 수 있었다. 따라서, 비피도박테리움 브레베 LMC520 균주를 이용하여 CLN이 함유된 유제품을 산업적으로 생산할 시에 산소의 유무와 관계없이 CLN 함유 유제품을 생산해 낼 수 있음을 알 수 있었다.In general, Bifidobacterium strains are inhibited in activity under aerobic conditions, but the results of FIG. 9 indicate that Bifidobacterium breve LMC520 strain (KCTC 10455BP) is not significantly affected by the CLN conversion. there was. Therefore, it was found that when the industrial production of CLN-containing dairy products using Bifidobacterium breve LMC520 strain can produce CLN-containing dairy products with or without oxygen.
본 발명은 이상에서 살펴본 바와 같이 바람직한 실시예를 들어 도시하고 설명하였으나, 상기한 실시예에 한정되지 아니하며 본 발명의 정신을 벗어나지 않는 범위 내에서 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자에 의해 다양한 변경과 수정이 가능할 것이다.Although the present invention has been shown and described with reference to the preferred embodiments as described above, it is not limited to the above embodiments and those skilled in the art without departing from the spirit of the present invention. Various changes and modifications will be possible.
본 발명은 비피도박테리움 브레베 LMC520 균주 이용하여 자연발생적으로 CLN을 생산함으로써, 미생물적으로 CLN를 생산할 수 있는 방법을 제공함은 물론, 상기 균주를 유제품의 스타터로 이용함으로써 유제품 내에 CLN을 증진시킬 수 있어 기능성이 증대된 유제품을 제공할 수 있으므로 식품 산업상 유용한 발명이다.The present invention provides a method for producing CLN microbially by naturally producing CLN using Bifidobacterium breve LMC520 strain, as well as using the strain as a starter of dairy products to enhance CLN in dairy products. It is an invention useful in the food industry because it can provide dairy products with increased functionality.
도 1a는 본 발명에 따른 균주 배양액에 α-LNA를 첨가하고 배양하기 전, 이를 GC로 분석한 결과이다.Figure 1a is the result of analysis by GC before adding and culturing α-LNA in the strain culture according to the present invention.
도 1b는 본 발명에 따른 비피도박테리움 브레베 LMC520 균주(KCTC 10455BP) 배양물에 α-LNA를 첨가하여 배양한 후 CLN이 생산되었을 때, 이를 GC로 분석한 결과이다.1B is a result of analyzing the GN when CLN was produced after culturing by adding α-LNA to the Bifidobacterium breve LMC520 strain (KCTC 10455BP) culture according to the present invention.
도 2는 본 발명에 따른 비피도박테리움 브레베 LMC520 균주(KCTC 10455BP)로부터 생산된 CLN을 GC/MS로 분석한 결과이다.Figure 2 shows the results of analyzing the CLN produced from Bifidobacterium breve LMC520 strain (KCTC 10455BP) according to the present invention by GC / MS.
도 3은 배양시간에 따른 비피도박테리움 브레베 LMC520 균주(KCTC 10455BP)의 성장률과 이로부터 생산된 CLN의 생산량에 관한 그래프이다.3 is a graph showing the growth rate of Bifidobacterium breve LMC520 strain (KCTC 10455BP) and the amount of CLN produced therefrom according to incubation time.
도 4는 비피도박테리움 브레베 LMC520 균주(KCTC 10455BP)의 성장률과 이로부터 생산된 CLA의 생산량에 관한 그래프이다.4 is a graph showing the growth rate of Bifidobacterium breve LMC520 strain (KCTC 10455BP) and the amount of CLA produced therefrom.
도 5는 각각 다른 α-LNA 농도에서 본 발명에 따른 비피도박테리움 브레베 LMC520 균주(KCTC 10455BP)로부터 생산된 CLN의 생산량 및 전환률에 관한 그래프이다.5 is a graph showing the yield and conversion rate of CLN produced from Bifidobacterium breve LMC520 strain (KCTC 10455BP) according to the present invention at different α-LNA concentrations.
도 6은 각각 다른 LA 농도에서 비피도박테리움 브레베 LMC520 균주(KCTC 10455BP)로부터 생산된 CLA의 생산량 및 전환률에 관한 그래프이다.Figure 6 is a graph of the yield and conversion of CLA produced from Bifidobacterium breve LMC520 strain (KCTC 10455BP) at different LA concentrations.
도 7은 각각의 pH에서 본 발명에 따른 비피도박테리움 브레베 LMC520 균주(KCTC 10455BP)로부터 생산된 CLN의 생산량에 관한 그래프이다.Figure 7 is a graph of the production of CLN produced from Bifidobacterium breve LMC520 strain (KCTC 10455BP) according to the present invention at each pH.
도 8은 각각의 pH에서 비피도박테리움 브레베 LMC520 균주(KCTC 10455BP)로 부터 생산된 CLA의 생산량에 관한 그래프이다.FIG. 8 is a graph of the yield of CLA produced from Bifidobacterium breve LMC520 strain (KCTC 10455BP) at each pH.
도 9는 호기 또는 혐기적 조건에서 본 발명에 따른 비피도박테리움 브레베 LMC520 균주(KCTC 10455BP)로부터 생산된 CLN의 생산량에 관한 그래프이다.Figure 9 is a graph of the production of CLN produced from Bifidobacterium breve LMC520 strain (KCTC 10455BP) according to the present invention in aerobic or anaerobic conditions.
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Cited By (6)
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EP2746398A1 (en) * | 2012-12-21 | 2014-06-25 | Laboratorios Ordesa, S.L | Process for producing conjugated linolenic acid from linolenic acid employing Bifidobacterium breve, Bifidobacterium bifidum, or Lactobacillus oris strains. |
KR20150112065A (en) * | 2014-03-26 | 2015-10-07 | 고려대학교 산학협력단 | Feed additive composition comprising Bifidobacterium breve LMC520 |
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CN105925514A (en) * | 2016-07-12 | 2016-09-07 | 江南大学 | Bifidobacterium breve and application thereof in preparing conjugated linoleic acid or conjugated linolenic acid |
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KR102454496B1 (en) * | 2022-06-29 | 2022-10-14 | 우석대학교 산학협력단 | Novel Bifidobacterium breve JKL2022 strain and method for producing conjugated linoleic acid thereof |
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EP1264893A1 (en) | 2001-06-08 | 2002-12-11 | Teagasc Dairy Products Research Centre | CLA biosynthesis by bifidobacteria |
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WO2003087344A1 (en) * | 2002-04-12 | 2003-10-23 | Chebigen Inc. | New strains capable of producing conjugated linoleic acid, capsulated composition comprising them, and the preparation methods thereof |
KR100515850B1 (en) * | 2003-06-28 | 2005-09-21 | 윤칠석 | A Bifidobacterium breve LMC520 strain containing a plasmid pBC520, a method for preparation of conjugated fatty acids and fermented milks containing such fatty acids using the same strain, and use of a plasmid pBC520 |
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EP2746398A1 (en) * | 2012-12-21 | 2014-06-25 | Laboratorios Ordesa, S.L | Process for producing conjugated linolenic acid from linolenic acid employing Bifidobacterium breve, Bifidobacterium bifidum, or Lactobacillus oris strains. |
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CN105211295A (en) * | 2015-09-22 | 2016-01-06 | 江西人之初乳品营养有限公司 | A kind of formula nutritional dairy produce slim for women's skin makeup |
CN105925514A (en) * | 2016-07-12 | 2016-09-07 | 江南大学 | Bifidobacterium breve and application thereof in preparing conjugated linoleic acid or conjugated linolenic acid |
CN105925514B (en) * | 2016-07-12 | 2019-04-09 | 江南大学 | The application of one plant of bifidobacterium breve and its preparation conjugated linoleic acid or conjugate linolenic acid |
WO2020035623A1 (en) | 2018-08-17 | 2020-02-20 | 4D Pharma Research Limited | Compositions comprising bacterial strains |
KR102454496B1 (en) * | 2022-06-29 | 2022-10-14 | 우석대학교 산학협력단 | Novel Bifidobacterium breve JKL2022 strain and method for producing conjugated linoleic acid thereof |
WO2024005573A1 (en) * | 2022-06-29 | 2024-01-04 | Woosuk University | Novel bifidobacterium breve jkl2022 strain and method for producing conjugated linoleic acid using thereof |
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