KR101823634B1 - Lactobacillus helveticus CBG-C23 strain producing conjugated linoleic acid and uses thereof - Google Patents
Lactobacillus helveticus CBG-C23 strain producing conjugated linoleic acid and uses thereof Download PDFInfo
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- KR101823634B1 KR101823634B1 KR1020160089185A KR20160089185A KR101823634B1 KR 101823634 B1 KR101823634 B1 KR 101823634B1 KR 1020160089185 A KR1020160089185 A KR 1020160089185A KR 20160089185 A KR20160089185 A KR 20160089185A KR 101823634 B1 KR101823634 B1 KR 101823634B1
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- lactobacillus helveticus
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Abstract
본 발명의 균주는 공액리놀레산을 우수한 효율로 생성할 수 있을 뿐만 아니라, 젖산 생성능, 내산성, 내담즙성 및 항생제 내성을 가지고 있으며, 유해세균인 살모넬라 티피뮤리움(Salmonella typhimurium)에 대한 우수한 항균 활성을 지닌다. 또한, 본 발명의 균주는 한국 성인의 분변으로부터 유래하여, 섭취시 장내 정착성이 우수하여 장기능 개선 및 배변활동을 향상시키는 조성물로서 이용될 수 있다.The strain of the present invention not only can produce conjugated linoleic acid with excellent efficiency, but also has excellent lactic acid production ability, acid resistance, biliary and antibiotic resistance, and excellent antimicrobial activity against harmful bacteria Salmonella typhimurium I have. Further, the strain of the present invention can be used as a composition derived from the feces of Korean adults and having excellent intestinal fixation upon ingestion, thereby improving bowel function and improving bowel activity.
Description
본 발명은 공액리놀레산을 생산하는 락토바실러스 헬베티커스 CBG-C23 균주 및 이의 용도에 관한 것이다.The present invention relates to Lactobacillus helveticus CBG-C23 strain producing conjugated linoleic acid and its use.
공액리놀레산(Conjugated Linoleic acid; CLA)은 필수지방산인 리놀레산(linoleic acid, 이하 LA'라 한다)의 공액화 이성체로, 반추동물의 젖이나 근육에서 미량으로 발견되는 천연 지방산 성분이다. CLA는 사슬 내부의 시스(cis)-9, 트랜스(trans)-11 또는 트랜스-10, 시스-12 위치에 공액화이중결합을 가지는데, 특히 시스-9, 트랜스-11 부위에 공액화이중결합을 갖는 이성체가 인체에 유용한 생리활성을 나타낸다.Conjugated linoleic acid (CLA) is a co-liquefied isomer of linoleic acid (LA), an essential fatty acid, and a natural fatty acid component found in trace amounts in milk or muscle of ruminants. CLA has a conjugated double bond in the cis-9, trans-11 or trans-10 and cis-12 positions in the chain, and particularly has covalent double bonds in the cis-9 and trans- Isomer exhibits a useful physiological activity in the human body.
CLA는 동맥경화증의 발생저하, 면역기능향상, 항암작용, 성장촉진 및 당뇨병 등의 질환에 대해 우수한 치료 효과를 나타내며, 체지방감소를 통해 비만을 억제한다고도 알려져 있다. 이러한 특성으로 인해 CLA는 식품 및 의약품의 유효성분으로 유용하게 이용될 수 있다.CLA is known to exert excellent therapeutic effects against diseases such as atherosclerosis development, immune function improvement, anticancer effect, growth promotion and diabetes, and to suppress obesity through reduction of body fat. Due to these properties, CLA can be usefully used as an active ingredient in 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 양이 확인된다. 인간의 일일 CLA 섭취량은 채식 위주인 동양인의 경우에는 0.1g/day이고, 육식을 많이 하는 서양인의 경우는 0.4g/day로 예상되고 있다.CLA is mainly contained in animal foods, and is known to exist in a large number of ruminants. The amount of CLA in beef is 2.9 ~ 4.3 mg CLA / fat, 5.6 mg CLA / fat in lamb and 0.3 ~ 0.6 mg CLA / fat in seafood. The daily intake of CLA in humans is estimated to be 0.1 g / day for Asian-dominated oriental populations and 0.4 g / day for Western-style predators.
현재까지 CLA의 대량 생산을 위해 여러 가지 방법이 개발되어 왔다. 기존의 방법은 요소부가법, 분자증류법, HPLC 방법 등 CLA를 LA로부터 화학적으로 합성하는 방법들이 있다. 그러나 화학적 합성법들은 고가의 장비가 필요하거나, 공정에 너무 많은 시간이 소요되는 등의 문제가 많다. 또한, 이들 방법은 단일 종류의 CLA뿐만 아니라, 다양한 종류의 이성체들을 함께 생성하기 때문에, 기존의 화학적 방법으로 단일 종류의 CLA 이성체만을 생산한다는 것은 매우 비효율적이다.To date, several methods have been developed for mass production of CLA. Conventional methods include chemically synthesizing CLA from LA such as urea addition method, molecular distillation method, and HPLC method. However, chemical synthesis methods require expensive equipment or take too much time to process. In addition, since these methods produce not only a single kind of CLA but also various kinds of isomers together, it is very inefficient to produce a single kind of CLA isomer by conventional chemical methods.
따라서, CLA를 가장 효율적으로 생산할 수 있는 방법은 CLA를 생성할 수 있는 미생물로부터 CLA를 생산 및 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 식품이나 의약품의 유효성분으로 유용하게 사용되고 있다.Thus, 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 intestinal microorganisms such as Lactobacillus, Propionibacterium, and Butyrivibrio fibrisolvens. In many countries, they are used as feeds to livestock probiotics, feed, and the like.
현재까지 알려진 바에 의하면 장내 미생물에 의해 합성된 CLA는 시스-9, 트랜스-11과 트랜스-10, 시스-12의 비율이 약 50%씩 생성되는 화학적인 합성법과는 달리, 시스-9, 트랜스-11만 특이적으로 생성된다. 반추동물 유래의 육유에서는 소량의 시스-9, 트랜스-11이 확인되지만, 탈지유를 이용한 발효 유제품에서는 매우 낮은 시스-9, 트랜스-11이 검출될 뿐이며, 특히 각종 유산균을 이용한 발효 김치에서도 시스-9, 트랜스-11이 존재하지 않는 것으로 알려지고 있다. 따라서 한국공개특허 제2001-0047292호에서는 락토바실러스 속 미생물을 이용하여 미생물에 의해 합성된 순수한 시스-9, 트랜스-11형의 CLA를 식품에 첨가시키는 방법을 개시한 바 있으나, 실제 식품에서는 극미량의 CLA 함량이 확인되었을 뿐이다.It is known that CLA synthesized by intestinal microorganisms has a cis-9, trans-11, trans-10 and cis-12 ratio of about 50% 11 < / RTI > In fermented dairy products using skim milk, very low cis-9 and trans-11 were detected only in small quantities of cis-9 and trans-11 in ruminant-derived fats, , It is known that trans-11 does not exist. Thus, Korean Laid-Open Patent Publication No. 2001-0047292 discloses a method of adding pure cis-9 or trans-11 type CLA synthesized by microorganisms to a food using a microorganism belonging to 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 장내에서 높은 생존율과 활성을 지녀야 한다. 또한 슈퍼박테리아(superbacteria)를 비롯한 장내의 유해한 균과 경쟁하여 우위를 점하기 위해서는 항생물질에 대해 우수한 내성을 갖추어야 한다. 그러나 현재 시판중인 제품의 유효성분으로 사용되는 미생물 중 많은 균주들은 저장 중에 제품뿐만 아니라 섭취 후 위를 통과하는 과정에서조차 잔존하지 못하며, 항생물질에 대한 내성도 미약한 것으로 나타났다. In addition, in order for the CLA-producing microorganism to be used as an active ingredient of food or medicine, the microorganisms must remain at a high rate similar to that during the production of the product throughout the distribution period of the manufactured product, And have high survival rate and activity. In addition, excellent resistance to antibiotics is required to compete with harmful bacteria in the intestines, including superbacteria. However, many of the microorganisms used as active ingredients in the currently marketed products were not able to remain in storage during the storage as well as in the product during storage, and the resistance to antibiotics was weak.
한편, 한국등록특허 제10-1402028호에는 신규한 락토바실러스 헬베티커스 RMK 85 균주 및 이를 이용한 감마-아미노부티르산의 생산방법에 대해 개시하고 있으며, 한국등록특허 제10-1407231호에는 락토바실러스 헬베티커스 RMK85를 이용한 GABA 증진 발효 식물 추출물 제조방법에 대해 개시하고 있다. 하지만, 본 발명의 공액리놀레산을 생산하는 락토바실러스 헬베티커스 CBG-C23 균주 및 이의 용도에 대해 아직까지 개시된 바가 없다.Korean Patent No. 10-1402028 discloses a novel Lactobacillus helveticus RMK 85 strain and a method for producing gamma-aminobutyric acid using the same. In Korean Patent No. 10-1407231, Discloses a method for producing a GABA-enhanced fermentation plant extract using Curt RMK85. However, Lactobacillus helveticus CBG-C23 strain producing the conjugated linoleic acid of the present invention and its use have not yet been disclosed.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명에서는 사람의 분변으로부터 락토바실러스 헬베티커스(Lactobacillus helveticus) CBG-C23 균주를 분리하였다. 상기 균주는 기존에 알려진 동속 균주보다 공액리놀레산(conjugated linoleic acid) 생산능력이 우수할 뿐만 아니라, 젖산 생성능, 내산성, 내담즙성 및 항생제 내성을 가지고 있으며, 유해세균인 살모넬라 티피뮤리움(Salmonella typhimurium)에 대한 항균 활성을 나타내는 것을 확인함으로써, 본 발명을 완성하였다.The present invention has been made in view of the above-mentioned needs. In the present invention, Lactobacillus helveticus CBG-C23 strain was isolated from human feces. The strains have higher productivity than conjugate linoleic acid, and have lactic acid production ability, acid resistance, bile resistance and antibiotic resistance, and are resistant to harmful bacteria such as Salmonella typhimurium , , The present invention has been completed.
본 발명의 목적을 달성하기 위하여, 본 발명은 공액리놀레산을 생산하는 락토바실러스 헬베티커스(Lactobacillus helveticus) CBG-C23 균주(수탁번호 KACC 92085P)를 제공한다.In order to achieve the object of the present invention, the present invention provides Lactobacillus helveticus CBG-C23 strain (Accession No. KACC 92085P) producing conjugated linoleic acid.
또한, 본 발명은 상기 균주 또는 이의 배양액을 유효성분으로 포함하는 공액리놀레산 생산용 미생물 제제를 제공한다.The present invention also provides a microorganism preparation for producing a conjugated linoleic acid comprising the strain or a culture thereof as an active ingredient.
또한, 본 발명은 상기 균주를 유효성분으로 함유하며, 수용성 다당류를 피복물질로 함유하는 캡슐제를 제공한다.The present invention also provides a capsule containing the strain as an active ingredient and containing a water-soluble polysaccharide as a covering substance.
또한, 본 발명은 상기 균주 또는 이의 배양액을 유효성분으로 함유하는 식품을 제공한다.The present invention also provides a food containing the strain or a culture thereof as an active ingredient.
또한, 본 발명은 상기 균주 또는 이의 배양액을 유효성분으로 포함하는 동물 사료용 조성물을 제공한다.The present invention also provides a composition for animal feed comprising the strain or a culture thereof as an active ingredient.
또한, 본 발명은 상기 균주 또는 이의 배양액을 유효성분으로 포함하는 자양강장제를 제공한다.The present invention also provides a nutritional tonic agent comprising the strain or a culture solution thereof as an active ingredient.
또한, 본 발명은 상기 균주 또는 이의 배양액을 유효성분으로 포함하는 암, 동맥경화, 당뇨병 또는 비만의 예방 또는 치료용 약학 조성물을 제공한다.The present invention also provides a pharmaceutical composition for preventing or treating cancer, arteriosclerosis, diabetes or obesity comprising the strain or a culture thereof as an active ingredient.
또한, 본 발명은 상기 균주를 배양하는 단계를 포함하는 공액리놀레산을 생산하는 방법을 제공한다.The present invention also provides a method for producing a conjugated linoleic acid comprising culturing the strain.
본 발명의 균주는 공액리놀레산을 우수한 효율로 생성할 수 있을 뿐만 아니라, 젖산 생성능, 내산성, 내담즙성 및 항생제 내성을 가지고 있으며, 유해세균인 살모넬라 티피뮤리움(Salmonella typhimurium)에 대한 우수한 항균 활성을 지닌다. 또한, 본 발명의 균주는 한국 성인의 분변으로부터 유래하여, 섭취시 장내 정착성이 우수하여 장기능 개선 및 배변활동을 향상시키는 조성물로서 이용될 수 있다.The strain of the present invention not only can produce conjugated linoleic acid with excellent efficiency, but also has excellent lactic acid production ability, acid resistance, biliary and antibiotic resistance, and excellent antimicrobial activity against harmful bacteria Salmonella typhimurium I have. Further, the strain of the present invention can be used as a composition derived from the feces of Korean adults and having excellent intestinal fixation upon ingestion, thereby improving bowel function and improving bowel activity.
도 1은 본 발명의 락토바실러스 헬베티커스(Lactobacillus helveticus) CBG-C23 균주의 rRNA 염기서열 분석 결과를 나타낸 것이다.
도 2는 본 발명의 락토바실러스 헬베티커스(Lactobacillus helveticus) CBG-C23 균주가 리놀레산(LA)이 첨가된 배지에서 생성하는 지방산의 조성을 확인하기 위한 GC 분석 결과를 나타낸 것이다.
도 3은 리놀레산(LA)이 첨가된 배지에서 본 발명의 락토바실러스 헬베티커스(Lactobacillus helveticus) CBG-C23 균주의 생장상태, CLA 생성 정도 및 배양액의 pH 변화를 나타낸 것이다.
도 4는 L-시스테인이 첨가된 배지에서 본 발명의 락토바실러스 헬베티커스(Lactobacillus helveticus) CBG-C23 균주의 생장상태, 젖산 생성 정도 및 배양액의 pH 변화를 나타낸 것이다.
도 5는 본 발명의 락토바실러스 헬베티커스(Lactobacillus helveticus) CBG-C23 균주를 포도당(glucose), 과당(fructose), 젖당(Lactose) 및 설탕(sucrose)이 각각 탄소원으로 첨가된 배지에서 배양하여 생장 정도 및 CLA 생성량을 측정한 결과를 나타낸 것이다.
도 6은 본 발명의 락토바실러스 헬베티커스(Lactobacillus helveticus) CBG-C23 균주를 포도당(glucose), 과당(fructose), 젖당(Lactose) 및 설탕(sucrose)이 각각 탄소원으로 첨가된 배지에서 배양하여 생장 정도 및 젖산 생성량을 측정한 결과를 나타낸 것이다.FIG. 1 shows the results of rRNA sequencing of Lactobacillus helveticus CBG-C23 strain of the present invention.
FIG. 2 is a graph showing the results of GC analysis for confirming the composition of fatty acid produced in the medium to which Lactobacillus helveticus CBG-C23 strain of the present invention is added with linoleic acid (LA).
FIG. 3 shows the growth state, the degree of CLA production, and the pH of the culture medium of the Lactobacillus helveticus CBG-C23 strain of the present invention in the medium supplemented with linoleic acid (LA).
FIG. 4 shows the growth state, lactic acid production, and pH of the culture medium of the Lactobacillus helveticus CBG-C23 strain of the present invention in the medium supplemented with L-cysteine.
FIG. 5 is a graph showing the results of cultivating Lactobacillus helveticus CBG-C23 strain of the present invention in a medium supplemented with glucose, fructose, lactose and sucrose as carbon sources, And the amount of CLA produced.
FIG. 6 is a graph showing the results of cultivation of Lactobacillus helveticus CBG-C23 strain of the present invention in a medium supplemented with glucose, fructose, lactose and sucrose as carbon sources, And lactic acid production.
본 발명의 목적을 달성하기 위하여, 본 발명은 공액리놀레산을 생산하는 락토바실러스 헬베티커스(Lactobacillus helveticus) CBG-C23 균주를 제공한다.In order to accomplish the object of the present invention, the present invention provides a method for producing conjugated linoleic acid, which comprises contacting Lactobacillus helveticus CBG-C23 strain.
상기 락토바실러스 헬베티커스(Lactobacillus helveticus) CBG-C23 균주는 한국인 분변으로부터 분리하였으며, 공액리놀레산 생성 능력이 우수한 균주로 선발되었다. 상기 락토바실러스 헬베티커스(Lactobacillus helveticus) CBG-C23 균주를 농업생명공학연구원에 2015년 07월 10일자로 기탁하였다(기탁번호 : KACC92085P).The Lactobacillus helveticus CBG-C23 strain was isolated from Korean feces and was selected as a strain having excellent ability to produce conjugated linoleic acid. The Lactobacillus helveticus CBG-C23 strain was deposited at the Institute of Agricultural Biotechnology on Jul. 10, 2015 (Accession No .: KACC92085P).
본 발명의 일 구현 예에 있어서, 상기 균주는 젖산 생성능, 내산성 및 내담즙성을 가지는 것일 수 있으나, 이에 제한되지 않는다.In one embodiment of the present invention, the strain may have, but is not limited to, lactic acid producing ability, acid resistance and biliary cholesterol.
본 발명의 일 구현 예에 있어서, 상기 균주는 항생제 내성 및 항균 활성을 가지는 것일 수 있으며, 바람직하게는 상기 항생제는 앰피실린(ampicillin), 테트라사이클린(tetracycline), 스트렙토마이신(streptomycin), 리파마이신(rifamycin), 카나마이신(kanamycin), 아미카신(amikacin), 겐타마이신(gentamicin), 네오마이신(neomycin), 메티실린(methicillin), 옥사실린(oxacillin), 리팜피신(rifampicin), 노보바이오신(novobiocin), 린코마이신(lincomycin) 또는 클로람페니콜(chloramphenicol)일 수 있으며, 상기 항균 활성은 살모넬라 티피뮤리움(Salmonella typhimurium)에 대한 항균 활성일 수 있으나, 이에 제한되지 않는다.In one embodiment of the present invention, the strain may have antibiotic resistance and antimicrobial activity, and preferably the antibiotic is selected from the group consisting of ampicillin, tetracycline, streptomycin, rifamycin rifamycin, kanamycin, amikacin, gentamicin, neomycin, methicillin, oxacillin, rifampicin, novobiocin, Lincomycin or chloramphenicol, and the antimicrobial activity may be an antibacterial activity against Salmonella typhimurium , but is not limited thereto.
본 발명의 상기 균주로부터 생산되는 CLA(conjugated linoleic acid)는 시스-9, 트랜스-11과 트랜스-10, 시스-12의 입체구조만 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 건강 기능성 식품과 미생물제제(probiotics) 등을 개발하기 위해 광범위하게 이용될 수 있을 뿐만 아니라 생물학적 방법에 의해 CLA의 이성체들을 대량으로 생합성하는데 효율적으로 이용될 수 있다.The conjugated linoleic acid (CLA) produced from the strain of the present invention contains only three-dimensional structures of cis-9, trans-11 and trans-10 and cis-12, , Fatty acids including these structures and acylglycerols can be widely used for developing dairy products derived from various animal species, dairy products derived from plant-derived raw materials, fermented foods, health functional foods, and probiotics In addition, it can be efficiently used for biosynthesis of large amounts of CLA isomers by biological methods.
본 발명의 미생물로부터 생산되는 젖산은 주로 L-(+)-젖산 및 D-(-)-젖산을 포함하는데, 이들 중 L-(+)-젖산은 기존에 공지된 바와 같이 사람을 포함한 동물에서 다양한 생리활성을 가지는 이성체이며, D-(-)-젖산은 생분해성 플라스틱의 생산이나 약리물질로 활용되고 있다. 따라서 이들 구조물은 각종 동물성 유제품, 식물 유래의 원료물질에 의한 유제품, 발효식품 및 건강 기능성식품과 미생물제재(probiotics) 등의 개발을 위해 광범위하게 이용될 수 있다. 따라서 본 발명의 균주들은 균체 고정화 등의 생물학적 방법에 의해 젖산을 대량으로 생산하는데 효율적으로 이용될 수 있다.The lactic acid produced from the microorganism of the present invention mainly includes L - (+) - lactic acid and D - (-) - lactic acid. Among them, L - D - (-) - lactic acid is an isomer having various physiological activities, and it is utilized as a biodegradable plastic production or pharmacological material. Therefore, these structures can be widely used for the development of various animal dairy products, dairy products made from plant-derived raw materials, fermented foods, health functional foods, and probiotics. Therefore, the strains of the present invention can be efficiently used for mass production of lactic acid by a biological method such as cell immobilization.
또한, 본 발명은 상기 균주 또는 이의 배양액을 유효성분으로 포함하는 공액리놀레산 생산용 미생물 제제를 제공한다. 상기 균주는 바람직하게는 락토바실러스 헬베티커스이며, 더욱 바람직하게는 락토바실러스 헬베티커스(Lactobacillus helveticus) CBG-C23 균주(KACC92085P)이다.The present invention also provides a microorganism preparation for producing a conjugated linoleic acid comprising the strain or a culture thereof as an active ingredient. The strain is preferably Lactobacillus helveticus , more preferably Lactobacillus helveticus CBG-C23 strain (KACC92085P).
또한, 본 발명은 상기 균주를 유효성분으로 함유하며, 수용성 다당류를 피복물질로 함유하는 캡슐제를 제공한다. 상기 균주는 바람직하게는 락토바실러스 헬베티커스이며, 더욱 바람직하게는 락토바실러스 헬베티커스 CBG-C23 균주(KACC92085P)이다. The present invention also provides a capsule containing the strain as an active ingredient and containing a water-soluble polysaccharide as a covering substance. The strain is preferably Lactobacillus helveticus, more preferably Lactobacillus helveticus CBG-C23 strain (KACC92085P).
본 발명의 캡슐제는 피복물질(coating material)과, 피복되는 내부의 핵물질(core material)로 이루어진다. 본 발명의 캡슐제에서 핵물질은 본 발명의 균주와 리놀레산을 함께 포함하는 것이 바람직하다. 본 발명의 캡슐제에서 핵물질을 둘러싸는 피복물질은 흡수성, 분산성, 점착성이 우수한 수용성 다당류를 사용한다.The capsules of the present invention are composed of a coating material and a core material to be coated. The nucleus material in the capsules of the present invention preferably contains the strain of the present invention together with linoleic acid. In the capsules of the present invention, the coating material surrounding the nuclear material uses a water-soluble polysaccharide having excellent water absorption, dispersibility, and stickiness.
본 발명에서 사용가능한 수용성 다당류는 전분, 한천, 카라기난, 알긴산, 알긴산나트륨(sodium alginate), 폴리메틸메타크릴레이트(polymethylmetacrylate), 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, polymethylmethacrylate, wheat protein, soy protein, methylcellulose, hydroxypropylcellulose cellulose derivatives such as hydroxypropylcellulose and hydroxypropylmethylcellulose, xanthan gum, arabic gum, locust bean gum, guar gum, tamarind A gum such as tamarind gum, Tara gum, karaya gum, tragacanth gum, ghatti gum, and gellan, xanthan, pectin, (LM, HM type), dextran, glucan, glucomannan, arabino galactan, furcelleran, pullulan, glucosamine, Gelatin, casein (c asein).
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 바람직하다. 또한, 본 발명의 캡슐제는 필요에 따라 유화제, 보호제 및 가소제를 추가로 포함할 수 있다. 본 발명의 캡슐제의 제조 방법은 통상적으로 사용되는 캡슐화 방법 중 적절한 방법을 택하여 사용할 수 있다. 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 amount of the coating material in the preparation of the capsules of the present invention may be appropriately adjusted depending on the application and purpose, and it is preferable to use the coating material in an amount ranging from 1 to 80% by weight, which is generally used on the basis of nuclei Do. In addition, the capsules of the present invention may further comprise an emulsifying agent, a protecting agent and a plasticizer, if necessary. The capsule preparation of the present invention may be prepared by using an appropriate one of the commonly used encapsulation methods. The composition containing the capsules of the present invention can be specifically used as dairy products (milk, soy milk, processed milk), fermented milk (liquid yogurt, yogurt yogurt), fermented foods (kimchi, .
또한, 본 발명은 상기 균주 또는 이의 배양액을 유효성분으로 포함하는 식품을 제공한다. 상기 균주는 바람직하게는 락토바실러스 헬베티커스이며, 더욱 바람직하게는 락토바실러스 헬베티커스 CBG-C23 균주(KACC92085P)이다. 본 발명의 식품은 액상요구르트, 호상요구르트, 치즈, 김치류, 장류 등일 수 있으나, 이에 제한되지 않는다.The present invention also provides a food comprising the strain or a culture thereof as an active ingredient. The strain is preferably Lactobacillus helveticus, more preferably Lactobacillus helveticus CBG-C23 strain (KACC92085P). The food of the present invention may be, but not limited to, liquid yogurt, batter yogurt, cheese, kimchi, soy sauce, and the like.
본 발명의 식품은 상기 유효성분 외에도 필요에 따라 다양한 보조성분을 추가로 함유할 수 있다. 본 발명의 식품의 경우, 비타민 A, 비타민 B1, 비타민 B2, 비타민 B3, 비타민 B6, 비타민 B12, 엽산(folic acid), 비타민 C, 비타민 D3, 비타민 E 등의 비타민류와, 구리, 칼슘, 철, 마그네슘, 칼륨, 아연 등의 미네랄 또는 유산균 등을 포함할 수 있다.In addition to the active ingredient, the food of the present invention may further contain various auxiliary ingredients as necessary. In the case of the food 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 and vitamin E and copper, , Minerals such as magnesium, potassium, and zinc, and lactic acid bacteria.
또한, 본 발명의 식품 중, 건강음료는 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 포함할 수 있다. 향미제로는 타우마틴, 스테비아 추출물과 같은 천연 감미제나, 사카린, 아스파탐과 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 등을 들 수 있다.In addition, among the foods of the present invention, the health drink may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Examples of the flavoring agent include natural sweetening agents such as tau Martin and stevia extract, and synthetic sweetening agents such as saccharin and aspartame. Examples of 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.
또한, 본 발명은 상기 균주 또는 이의 배양액을 유효성분으로 포함하는 동물 사료용 조성물을 제공한다. 상기 균주는 바람직하게는 락토바실러스 헬베티커스이며, 더욱 바람직하게는 락토바실러스 헬베티커스 CBG-C23 균주(KACC92085P)이다.The present invention also provides a composition for animal feed comprising the strain or a culture thereof as an active ingredient. The strain is preferably Lactobacillus helveticus, more preferably Lactobacillus helveticus CBG-C23 strain (KACC92085P).
본 발명의 사료용 조성물은 기초사료에 일정 비율로 첨가하는 것이다. 상기 기초사료는 주성분이 옥수수, 대두박, 유청, 어분, 당밀, 소금, 비타민 프리믹스 및 미네랄 프리믹스 등으로 이루어질 수 있다. 비타민 프리믹스는 비타민 A, 비타민 D, 비타민 E, 리보프라빈 및 나이아신으로 구성될 수 있으며, 미네랄 프리믹스는 망간, 철 아연, 칼슘, 구리, 코발트 및 셀레니늄 등으로 구성될 수 있다.The feed composition of the present invention is added to the base feed at a certain ratio. The basic diet may be composed of corn, soybean meal, whey, fish meal, molasses, salt, vitamin premix, and mineral premix. The vitamin premix can be composed of vitamin A, vitamin D, vitamin E, riboflavin and niacin, and the mineral premix can be composed of manganese, iron zinc, calcium, copper, cobalt and selenium.
또한, 본 발명은 상기 균주 또는 이의 배양액을 유효성분으로 포함하는 자양강장제를 제공한다. 상기 균주는 바람직하게는 락토바실러스 헬베티커스(Lactobacillus helveticus) CBG-C23 균주(KACC92085P)이다.The present invention also provides a nutritional tonic agent comprising the strain or a culture thereof as an active ingredient. The strain is preferably selected from the group consisting of Lactobacillus < RTI ID = 0.0 > helveticus CBG-C23 strain (KACC92085P).
본 발명은 또한, 본 발명의 균주 또는 이의 배양액을 유효성분으로 포함하는 암, 동맥경화, 당뇨병 또는 비만의 예방 또는 치료용 약학 조성물을 제공한다. 상기 약학 조성물은 공액리놀레산을 함유하고 있으므로 공액리놀레산에 의해 조절되는 질환을 예방 또는 치료할 수 있다. 본 발명의 균주는 CLA를 생산하며, 생산된 CLA는 동맥경화증의 발생저하(Artery. 1997. 22:266-277), 면역기능향상(J. Nut. 1999. 129:32-38), 항암작용(Anticancer research. 1997. 17:969-973), 당뇨병 등의 질환에 대해 우수한 치료 효과를 나타내며, 체지방감소(AM. J. Physiol. 1998. 275: R667-R672)를 통해 비만을 억제한다고도 알려져 있으므로, 본 발명의 균주 또는 이의 배양액은 암, 동맥경화, 당뇨병 또는 비만의 예방 또는 치료용 약학 조성물로 이용될 수 있는 것이다. 상기 균주는 바람직하게는 락토바실러스 헬베티커스(Lactobacillus helveticus) CBG-C23 균주(KACC92085P)이다.The present invention also provides a pharmaceutical composition for preventing or treating cancer, arteriosclerosis, diabetes, or obesity comprising the strain or the culture liquid of the present invention as an active ingredient. Since the pharmaceutical composition contains conjugated linoleic acid, it is possible to prevent or treat diseases controlled by conjugated linoleic acid. The strain of the present invention produces CLA, and the produced CLA exhibits a decrease in the incidence of arteriosclerosis (Artery, 1997. 22: 266-277), immune function improvement (J. Nut. 1999. 129: 32-38) (Anticancer research 1997. 17: 969-973), and it is also known that it exhibits excellent therapeutic effect against diseases such as diabetes and suppresses obesity through reduction of body fat (AM, J. Physiol., 1998. 275: R667-R672) Therefore, the strain of the present invention or a culture thereof can be used as a pharmaceutical composition for preventing or treating cancer, atherosclerosis, diabetes or obesity. The strain is preferably selected from the group consisting of Lactobacillus < RTI ID = 0.0 > helveticus CBG-C23 strain (KACC92085P).
본 발명의 약학 조성물의 투여량 또는 섭취량은 60∼130 uM(Cancer Epidemiol. Biol. Prev. 2000. 9:689-696), 그리고 기능성 식품의 투여량 또는 섭취량은 3.4∼6 g/day(J. Nutr. 2000. 130:2943-2948)인 것이 바람직하나, 필요에 따라 가감할 수 있다. 본 발명의 약학 조성물은 섭취량에 관계없이 체내에 안전하게 흡수된다. 투여량 또는 섭취량은 약학 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, 치료기간, 동시 사용되는 약물을 비롯한 다양한 인자에 따라 조절될 수 있다. 본 발명의 약학 조성물은 인체에 투약하는 경우, 화학적으로 합성된 CLA 함유 식품 및 의약품에 비해 부작용의 우려가 없다. The dosage or amount of the pharmaceutical composition of the present invention is 60-130 uM (Cancer Epidemiol. Biol. Prev. 2000. 9: 689-696), and the dosage or amount of functional food is 3.4-6 g / day (J. Nutr. 2000. 130: 2943-2948), but it can be added or subtracted as needed. The pharmaceutical composition of the present invention can be safely absorbed into the body regardless of the intake amount. The dosage or amount may vary depending on factors such as the type and amount of the active ingredient and the other ingredients contained in the pharmaceutical composition, the type of the formulation and the age, body weight, general health condition, sex and diet, time of administration, Duration of administration, drug used concurrently, and the like. The pharmaceutical composition of the present invention is free from side effects when administered to human body as compared to chemically synthesized CLA-containing foods and medicines.
본 발명의 약학 조성물은 상기 유효성분 이외에도 약제학적으로 허용 가능한 담체를 1종 이상 포함하여 제제화할 수 있다. 약제학적으로 허용 가능한 담체는 식염수, 멸균수, 링거액, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 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 당 분야의 적정한 방법으로, 또는 Remington's Pharmaceutical Science(최근판), Mack Publishing Company, Easton PA에 개시되어 있는 방법을 이용하여 각 질환에 따라, 또는 성분에 따라 바람직하게 제제화할 수 있다.The pharmaceutical composition of the present invention may contain one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. If necessary, an antioxidant, a buffer, Other conventional additives may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease, or according to each ingredient, using the method disclosed in the appropriate field in the art, or the method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA.
본 발명의 약학 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.Formulations of the pharmaceutical compositions of the present invention may be granules, powders, granules, coated tablets, tablets, capsules, sticks, extracts, suppositories, syrups, juices, suspensions, emulsions, have.
또한, 본 발명은 상기 균주를 배양하는 단계를 포함하는 공액리놀레산을 생산하는 방법을 제공한다. 상기 균주는 바람직하게는 락토바실러스 헬베티커스(Lactobacillus helveticus) CBG-C23 균주이다. 본 발명의 균주를 배양하는 방법은 당업계에 통상적으로 이용되는 방법을 이용할 수 있다. 본 발명의 균주 배양시 적합한 탄소원으로는 포도당, 과당, 젖당 및 설탕 등을 이용할 수 있으나, 젖당이 공액리놀레산을 최대로 생산하므로 바람직하다.The present invention also provides a method for producing a conjugated linoleic acid comprising culturing the strain. The strain is preferably a strain of Lactobacillus helveticus CBG-C23. As a method for culturing the strain of the present invention, a method commonly used in the art can be used. As a carbon source suitable for culturing the strain of the present invention, glucose, fructose, lactose, sugar and the like may be used, but lactose is preferable because it maximally produces conjugated linoleic acid.
이하, 실시예를 이용하여 본 발명을 더욱 상세하게 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로 본 발명의 범위가 이들에 의해 제한되지 않는다는 것은 당해 기술분야에서 통상의 지식을 가진 자에게 있어 자명한 것이다. Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited thereto.
실시예Example 1. 본 발명의 균주 분리 및 동정 1. Strain isolation and identification of the present invention
본 발명의 균주를 분리하기 위해, 한국인 성인의 분변 10종을 채취하였으며, 이를 L-시스테인이 0.05% 첨가된 MRS(Man Rogosa Sharpe) 액상배지에 접종하였다. 멸균 생리식염수 0.85%(w/v)로 30~150개 정도의 균락(colony)이 생기도록 희석하여 MRS 한천배지에 도말한 후, 37℃에서 72시간 배양하였다. In order to isolate the strain of the present invention, 10 kinds of feces of Korean adults were collected and inoculated into a liquid medium of MRS (Man Rogosa Sharpe) supplemented with 0.05% of L-cysteine. Diluted with sterile physiological saline 0.85% (w / v) to give 30 ~ 150 colony, and plated on MRS agar medium and cultured at 37 ° C for 72 hours.
배양된 배지에서 300개 정도의 균 집락을 임의 선택하고, L-시스테인이 0.05% 첨가된 MRS 배지로 2회 계대배양한 후 L-시스테인 0.05%가 첨가된 MRS 배지를 함유한 20㎖ 시험관에 2×107 cfu/ml의 균을 접종하여 48시간 배양하였다. 이후 배양세포를 포함한 배양액의 pH를 측정하고, 이를 균체량, 즉 600nm에서의 흡광도 값으로 보정하여 상대적인 공액리놀레산 및 젖산 생성량을 비교하였다.Approximately 300 bacterial colonies were randomly selected from the cultured medium, followed by subculture twice with MRS medium supplemented with 0.05% L-cysteine and then cultured in a 20 ml test tube containing MRS medium supplemented with 0.05% L-
생균수 측정은 MRS 한천 배지를 이용하여 10배 희석법에 의해 평판 배양하고 72시간 경과 후 생성된 균집락 수를 세어 측정하였다. 이렇게 하여 공액리놀레산 및 젖산 생성 능력이 우수한 균주를 선발하고 CBG-C23로 명명하였으며, 본 발명의 균주에 대해 rRNA의 염기서열을 분석하여 그 속과 종을 동정하였다. 그 결과, CBG-C23 균주는 락토바실러스 헬베티커스(Bifidobacterium bifidum)에 속하는 것으로 판명되었다(도 1). The viable cell count was determined by counting the number of bacterial colonies formed after 72 hours of plate culture by 10 times dilution method using MRS agar medium. Thus, a strain having excellent ability to produce conjugated linoleic acid and lactic acid was selected and named CBG-C23. The nucleotide sequence of rRNA was analyzed for the strain of the present invention, and the genus and species thereof were identified. As a result, the CBG-C23 strain was identified as Lactobacillus helveticus ( Bifidobacterium bifidum ) (Fig. 1).
실시예Example 2. 본 발명의 균주가 생성하는 지방산의 조성 확인 2. Confirmation of the composition of the fatty acid produced by the strain of the present invention
본 발명의 균주가 생성하는 지방산의 조성을 확인하여, 본 발명의 균주가 CLA(conjugated linoleic acid)를 생성하는지 재차 확인하기 위해, 다음과 같은 GC를 수행하였다. 균체를 LA(500㎍/㎖)가 포함된 배지에서 42시간 동안 배양하였다. 균주에 의한 총 CLA 생성량을 분석하기 위해, 균체를 포함한 배양액에 2배 부피의 이소프로필 알콜(isopropyl alcohol)을 첨가하여 격렬히 혼합한 후, 여기에 1.5배 부피의 헥산을 가하여 3분 동안 흔들어 주면서 혼합하였다.In order to confirm the composition of the fatty acid produced by the strain of the present invention and to confirm again whether the strain of the present invention produces CLA (conjugated linoleic acid), the following GC was performed. The cells were cultured in a medium containing LA (500 / / ml) for 42 hours. In order to analyze the total amount of CLA produced by the strain, 2-fold volume of isopropyl alcohol was added to the culture solution containing the cells, vigorously mixed, 1.5 times as much hexane was added thereto, Respectively.
상기 혼합액을 상온에서 3000rpm으로 5분 동안 원심분리한 후, 234nm에서 상등액의 흡광도를 측정하였다. 추출한 지방산은 American Oil Chemists's Society에 기재된 방법(Official Method and Recommended Practoces pf AOCS, 4th. ed., 1989)에 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 the absorbance of the supernatant was measured at 234 nm. The extracted fatty acids were methylly esterified according to the method described in American Oil Chemists' Society (Official Method and Recommended Practice pf AOCS, 4th ed., 1989) to prepare samples for GC analysis.
GC 분석시 조건은 다음과 같다. 이때, FID가 부착된 GC DS-6200(DONAM)을 사용하였고, 컬럼은 HP-FFAP 캐필러리 컬럼(30m×0.25㎜ 및 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 표준물질의 면적과 CLA의 면적비에 의해 분석시료로 사용하였다. 도 2에 개시한 바와 같이, GC 분석 결과에서 LA 및 CLA 피크가 관찰되었으며, 본 발명의 균주가 LA를 기질로 하여 CLA를 생성할 수 있음을 확인하였다. The conditions for GC analysis are as follows. In this case, GC DS-6200 (DONAM) with FID was used. The column was an HP-FFAP capillary column (30 m × 0.25 mm and a thickness of 0.25 μm), the oven temperature was 210 ° C., The temperature was 250 占 폚, and the detector temperature was 270 占 폚. The carrier gas was helium, eluted at a flow rate of 1 ml / min, and a split ratio of 50: 1. The area of each peak was determined using an integrator (Model 3390A, Hewlett-Packard, USA) connected to the instrument. The identification of CLA was confirmed by comparing with the retention time of the reference material, and the content of CLA was used as the analytical sample by the area of the reference material and the area ratio of CLA. As shown in FIG. 2, LA and CLA peaks were observed in GC analysis results, and it was confirmed that the strain of the present invention can produce CLA using LA as a substrate.
실시예Example 3. 본 발명의 균주 배양시 3. In the culture of the strain of the present invention CLACLA 생성 최적 조건 확인 Identify optimal conditions for generation
본 발명의 균주에 대한 CLA 최적 생성조건을 확인하기 위해, 기질인 LA가 배지에 존재하는 경우와 존재하지 않는 경우의 두 조건에서 생육 특성을 관찰하였다. 우선, 전배양하여 활성화된 균주를 LA(500㎍/㎖)가 포함된 2ℓ의 MRS 배지에 1%가 되도록 접종한 후, 시간별로 배양액을 회수하여 균체 농도, CLA 생성량 및 배양액의 pH를 측정하였다. 그 결과, 도 3에 개시한 바와 같이 락토바실러스 헬베티커스는 대수기(exponential phase)에 접어들면서 CLA 생성량도 증가하였으며, 정지기(stationary phase)에 도달하기 직전에 CLA를 최대로 생성하였다. 배양액의 최초 pH는 6.5 정도였으나, 배양 시간이 지날수록 떨어져 약 24시간 발효 후에는 pH 4.1 정도를 나타내었다.In order to confirm the optimum conditions for CLA production of the strain of the present invention, growth characteristics were observed under two conditions of the presence or absence of LA as a substrate. First, the activated strain was inoculated to 2 L of MRS medium containing LA (500 μg / ml) at 1%, and the culture solution was recovered by time to measure the cell concentration, the amount of CLA and the pH of the culture solution . As a result, as shown in FIG. 3, Lactobacillus helveticus increased CLA production as it entered the exponential phase, and produced CLA just before reaching the stationary phase. The initial pH of the culture broth was about 6.5, but after fermentation, the pH of broth was about 4.1.
실시예Example 4. 본 발명의 균주가 생성하는 4. The strain produced by the strain of the present invention CLA의CLA 양 확인 및 전환율 분석 Volume Verification and Conversion Rate Analysis
본 발명의 균주 배양시, 각각의 균주가 생성하는 CLA의 양을 확인하기 위해, 다음과 같이 실험을 수행하였다. 전환율 비교를 위한 대조구로는 미생물자원센터(KCTC) 등록균주인 락토바실러스 헬베티커스 KCTC 3545 균주를 사용하였다. 우선, LA가 포함된 배양액에 균주를 접종한 후 24시간 동안 배양하면서 2시간 간격으로 CLA 생성 정도를 측정하였고, 균체와 배양액에 존재하는 총 CLA(시스-9 및 트랜스-11과 트랜스-10 및 시스-12의 합)의 농도를 분석하였다. 그 결과, 하기 표 1에 개시한 바와 같이 대조구인 락토바실러스 헬베티커스 KCTC 3545 균주보다 본 발명의 락토바실러스 헬베티커스 CBG-23 균주가 약 1.3배의 CLA를 생산하였으며, LA로부터 CLA 전환률이 더 우수한 것을 확인할 수 있었다.In order to confirm the amount of CLA produced by each strain at the time of culturing the strain of the present invention, the following experiment was conducted. Lactobacillus helveticus strain KCTC 3545, a microorganism resource center (KCTC) registered strain, was used as a control for the conversion rate comparison. First, CLA production was measured at intervals of 2 hours while culturing for 24 hours after inoculation with the strain containing LA. Total CLA (cis-9 and trans-11 and trans-10, Cis-12) was analyzed. As a result, as shown in Table 1 below, the Lactobacillus helveticus CBG-23 strain of the present invention produced about 1.3-fold more CLA than the Lactobacillus helveticus KCTC 3545 strain of the control, and the CLA conversion rate from LA I was able to confirm that it was excellent.
CLA(㎍)/배양액(ml)Production rate
CLA (㎍) / culture (ml)
실시예Example 5. 본 발명의 균주가 생성하는 유기산의 조성 확인 5. Confirmation of the composition of the organic acid produced by the strain of the present invention
본 발명의 균주가 생성하는 유기산의 조성을 확인하여, 본 발명의 균주가 젖산을 생성하는지 재차 확인하기 위해, 다음과 같이 HPLC를 수행하였다. 균체를 L-시스테인 0.05%가 포함된 배지에서 42시간 동안 배양하였다. 균주에 의한 총 젖산 생성량을 분석하기 위해, 균체를 포함한 배양액을 상온에서 3000rpm으로 5분 동안 원심분리한 후, 상층액을 100㎕ 취한 후, 3차 증류수로 10배 희석을 하여 0.22μm 멤브레인 필터로 여과한 후 HPLC(Ultimate 3000, Dionex, Sunnyvale, CA, USA) 분석의 시료로 사용하였다. 이때 이용된 HPLC의 칼럼은 Aminex 87H 칼럼(300×7.8mm, Bio-Rad, Hercules, CA, USA)을 사용하였으며 이동상은 0.01N H2SO4를 이용하여 0.5ml/min으로 흘려보냈다. 시료의 1회 주입량은 10㎕였으며, 검출기는 RI(Shodex RI-101, Tokyo, Japan) 및 UV(210nm)를 사용하여 검출하였다. 젖산의 동정은 표준물질의 머무름 시간과 비교하여 확인하였으며, 젖산의 함량은 표준물질의 면적과 배양액의 면적비에 의해 분석시료로 사용하였다.In order to confirm the composition of the organic acid produced by the strain of the present invention and to confirm again whether the strain of the present invention produced lactic acid, HPLC was performed as follows. The cells were cultured in a medium containing 0.05% L-cysteine for 42 hours. To analyze the total lactic acid production by the strain, the culture solution containing the cells was centrifuged at 3000 rpm for 5 minutes at room temperature, then 100 μl of the supernatant was diluted 10 times with 3 rd distilled water and filtered through a 0.22 μm membrane filter After filtration, it was used as a sample for HPLC (Ultimate 3000, Dionex, Sunnyvale, CA, USA) analysis. The HPLC column used was an Aminex 87H column (300 × 7.8 mm, Bio-Rad, Hercules, CA, USA) and the mobile phase was flowed at 0.5 ml / min using 0.01NH 2 SO 4 . One injection amount of the sample was 10 μl, and the detector was detected using RI (Shodex RI-101, Tokyo, Japan) and UV (210 nm). Identification of lactic acid was confirmed by comparing with the retention time of the reference material, and the content of lactic acid was used as the analytical sample by the area of the reference material and the area ratio of the culture solution.
본 발명의 균주 배양시, 각각의 균주가 생성하는 젖산의 양을 확인하기 위해, 다음과 같이 실험을 수행하였다. 생성률 비교를 위한 대조구로는 미생물자원센터(KCTC) 등록균주인 락토바실러스 헬베티커스 KCTC 3545 균주를 사용하였다. 우선, L-시스테인 0.05%가 포함된 배양액에 균주를 접종한 후 24시간 동안 배양하면서 2시간 간격으로 젖산 생성 정도를 측정하였다. 그 결과, 하기 표 2에 개시한 바와 같이 두 균주의 젖산 생성량을 비교해 보면, 대조군인 락토바실러스 헬베티커스 KCTC 3545 균주는 배양 24시간 후에 생균수가 2.5×109 cfu/ml에서 62.9g/L의 젖산을 생산하는데 반해, 본 발명에서 선발한 락토바실러스 헬베티커스 CBG-C23 균주는 2.2×109 cfu/ml에서 65.0g/L의 젖산을 생산하였으며, 배양시간별 젖산의 생성량에서도 비교 균주보다 두드러지는 생산성을 보이는 것을 확인할 수 있었다.In order to confirm the amount of lactic acid produced by each strain during the strain culture of the present invention, the following experiment was conducted. Lactobacillus helveticus strain KCTC 3545, a microorganism resource center (KCTC) registered strain, was used as a control for comparison of the production rates. First, the strains were inoculated into the culture medium containing 0.05% L-cysteine, and the lactic acid production was measured at intervals of 2 hours while culturing for 24 hours. As a result, as shown in Table 2 below, when the lactic acid production amounts of the two strains were compared, Lactobacillus helveticus coarse KCTC 3545 strains, whereas the number of live bacteria produce lactic acid of 62.9g / L at 2.5 × 10 9 cfu /
또한, 본 발명의 균주에 대한 젖산의 최적 생성조건을 확인하기 위해, L-시스테인 0.05%가 포함된 배지에서 24시간 동안 배양하면서 생육 특성을 관찰하였다. 그 결과, 도 4에 개시한 바와 같이 락토바실러스 헬베티커스는 대수기(exponential phase)에 접어들면서 젖산 생성량도 증가하였으며, 정지기(stationary phase)에 도달하기 직전에 CLA를 최대로 생성하였다. 배양액의 최초 pH는 6.0 정도였으나, 배양 시간이 지날수록 떨어져 약 24시간 발효 후에는 pH 3.5 정도를 나타내었다.Also, In order to confirm the optimal production conditions of lactic acid against the strain of the present invention, growth characteristics were observed while culturing in a medium containing 0.05% L-cysteine for 24 hours. As a result, as shown in FIG. 4, Lactobacillus helveticus increased lactic acid production as it entered the exponential phase, and produced CLA just before reaching the stationary phase. The initial pH of the culture was about 6.0, but after fermentation for about 24 hours, the pH was about 3.5.
실시예Example 6. 본 발명의 균주의 pH, 담즙 및 6. pH of the strain of the present invention, bile and 항생물질에On antibiotics 대한 내성 확인 Confirm resistance to
본 발명에서 분리 및 동정한 균주를 발효 제품 또는 식품 첨가제로 사용하기 위해 pH 내성, 담즙 내성 및 항생물질 내성 등의 생육특성을 알아보았다.In order to use the strains isolated and identified in the present invention as fermented products or food additives, growth characteristics such as pH tolerance, bile resistance and antibiotic resistance were examined.
1) pH 내성1) pH tolerance
본 발명의 균주의 pH별 내성을 확인하기 위해, 24시간 정도 배양한 각각의 균주를 멸균한 0.5%(w/v) 염화나트륨 용액으로 희석하였다(105cfu/ml). 산에 대한 내성 비교를 위한 대조구로는 미생물자원센터(KCTC) 등록균주인 락토바실러스 헬베티커스 KCTC 3545 균주를 사용하였다. 산 내성 분석을 위한 인공위액은 빙초산, 염화나트륨 및 증류수와 pH 미터를 이용하여 pH 2, 3 및 4로 맞추었다. 배양 세포 희석액 0.2ml 및 0.3ml의 0.5%(w/v) 멸균 염화나트륨 용액, 그리고 1ml의 인공위액을 멸균 에펜도르프 튜브에서 혼합하고, 3시간 동안 방치한 후 MRS 고체배지에 접종하여 출현한 유산균을 계수(cfu/g)하였다. 그 결과, 하기 표 3에 개시한 바와 같이 본 발명의 균주는 pH 3~6의 범위에서 모두 생육이 가능하였고, 대조구에 비해 우수한 pH 내성을 보였다. 따라서 본 발명의 균주는 약알칼리와 약산성에 걸치는 넓은 범위에서 모두 생육이 가능하며, 특히 위산이 분비되어 산성 환경이 조성되는 위에서 충분히 생존할 수 있음을 확인할 수 있었다.To confirm the resistance of the strain of the present invention to pH, each strain cultured for about 24 hours was diluted with sterilized 0.5% (w / v) sodium chloride solution (10 5 cfu / ml). Lactobacillus helveticus KCTC 3545 strain, a microorganism resource center (KCTC) registered strain, was used as a control for the comparison of tolerance to acids. The artificial gastric juice for acid tolerance analysis was adjusted to
단위 : cfu/gUnit: cfu / g
2) 담즙 내성2) bile resistance
본 발명의 균주의 담즙 내성을 확인하기 위해, 담즙의 성분인 소듐 디옥시콜레이트(sodium deoxycholate)가 0, 100, 300, 500, 800 및 1,000㎍/㎖으로 다양하게 첨가된 MRS 배지를 준비하였다. 각 배지에 균주를 접종하여 37℃에서 48시간 동안 배양하면서 생육 여부를 검토하였으며, 담즙 내성 비교를 위한 대조구로는 미생물자원센터(KCTC) 등록 균주인 락토바실러스 헬베티커스(Lactobacillus helveticus) KCTC 3545 균주를 사용하였다. 그 결과, 표 4에 개시된 바와 같이 본 발명의 균주는 담즙 1,000㎍/㎖까지도 우수하게 생육하는 것을 확인하였다. 따라서 본 발명의 균주는 담즙이 분비되는 위장관에서 안정적으로 생육할 수 있음을 확인할 수 있었다.In order to confirm the bile resistance of the strain of the present invention, MRS medium supplemented with sodium deoxycholate (0, 100, 300, 500, 800, and 1,000 μg / ml), which is a component of bile, was prepared. Each strain was inoculated with the strains and cultured at 37 ° C for 48 hours. Growth of the strain was investigated. Lactobacillus helveticus strain KCTC 3545, a microorganism resource center (KCTC) registered strain, Were used. As a result, as shown in Table 4, the strain of the present invention was found to grow well up to 1,000 μg / ml of bile. Therefore, it was confirmed that the strain of the present invention can stably grow in the bile secreted gastrointestinal tract.
단위 : cfu/gUnit: cfu / g
3) 3) 항생물질Antibiotic 내성 tolerance
본 발명의 균주의 항생물질 내성을 확인하기 위해, 20㎖의 시험관에 밤새 배양한 균주를 MRS 배지에 희석하여 고체 배지를 제조하였다. 대조구로는 미생물자원센터(KCTC) 등록 균주인 락토바실러스 헬베티커스(Lactobacillus helveticus) KCTC 3545 균주를 사용하였다. 본 실험에 사용한 항생물질은 앰피실린(ampicillin), 테트라사이클린(tetracycline), 스트렙토마이신(streptomycin), 리파마이신(rifamycin), 카나마이신(kanamycin), 아미카신(amikacin), 겐타마이신(gentamicin), 네오마이신(neomycin), 메티실린(methicillin), 옥사실린(oxacillin), 리팜피신(rifampicin), 노보바이오신(novobiocin), 린코마이신(lincomycin) 및 클로람페니콜(chloramphenicol)이 첨가된 배지를 준비하였으며, 시험용 균주는 MRS 액체 배지에서 1×109 cfu/㎖ 정도로 진탕배양하여 준비하였다.In order to confirm antibiotic resistance of the strain of the present invention, a strain cultivated overnight in a 20 ml test tube was diluted in MRS medium to prepare a solid medium. As a control, Lactobacillus ( Lactobacillus ), a microorganism resource center (KCTC) registered strain, helveticus KCTC 3545 strain was used. The antibiotics used in this study were selected from the group consisting of ampicillin, tetracycline, streptomycin, rifamycin, kanamycin, amikacin, gentamicin, A medium supplemented with neomycin, methicillin, oxacillin, rifampicin, novobiocin, lincomycin, and chloramphenicol was prepared, and the test strain MRS liquid medium at a concentration of 1 x 10 < 9 > cfu / ml.
상기와 같이 준비된 항생물질을 포함한 배지를 각각 15㎖의 시험관에 5㎖씩 분주하고, 배양하여 준비한 시험용 균주를 1×105 cfu/㎖이 되도록 첨가하고, 37℃에서 15시간 동안 배양하면서 생육이 저해되는 최소농도를 측정하였다. 그 결과, 표 5에 개시된 바와 같이 대조구 대비 본 발명의 균주는 앰피실린, 스트렙토마이신, 카나마이신, 겐타마이신, 옥사실린, 리팜피신, 노보바이오신 및 클로람페니콜에 대해 우수한 내성을 보였으며, 테트라사이클린, 리파마이신, 아미카신, 네오마이신 및 린코마이신에 대해서는 유사한 항생물질 내성을 나타내었다. 따라서 본 발명의 균주는 광범위한 종류와 농도 범위의 항생물질에 대해 내성을 지니고 있음을 확인할 수 있었다.5 ml of each of the prepared antibiotic-containing media was dispensed into each 15 ml test tube, and the test strain prepared by culturing was added at a concentration of 1 × 10 5 cfu / ml. After incubation at 37 ° C. for 15 hours, The minimum concentration to be inhibited was measured. As a result, as shown in Table 5, the strain of the present invention as compared with the control showed excellent resistance to ampicillin, streptomycin, kanamycin, gentamicin, oxacillin, rifampicin, novobiocin and chloramphenicol, and tetracycline, rifamycin , Amikacin, neomycin, and lincomycin. Therefore, it was confirmed that the strain of the present invention is resistant to a wide range of species and concentrations of antibiotics.
실시예Example 7. 본 발명의 균주의 살모넬라 7. Salmonella of the strain of the present invention 티피뮤리움Tiffy myrium (Salmonella (Salmonella entericaenterica serovar typhimurium) serovar typhimurium) 의 장세포 침투에 대한 저지 활성 측정Of inhibition of intestinal cell infiltration
락테올 표준 동결건조물에 의한 살모넬라 티피뮤리움(Salmonella enterica serovar typhimurium)의 Caco-2 내부 침투 저해에 관한 약리활성 확인을 위한 시험으로, 검체 준비는 원심분리를 통해 본 발명의 균주 균체와 배양액을 분리하였다. 본 발명의 균주 균체는 인산완충식염수에 희석하여 처리하고, 배양액은 그대로 사용하였다. Caco-2 세포배양 준비물로는 플라스틱 배양 용기, DMEM(Dulbeco's Modified Eagle's Medium; 배지), 100배 농축 비필수 아미노산 및 우태혈청을 준비하였다.In order to confirm the pharmacological activity of Salmonella enterica serovar typhimurium on the inhibition of Caco-2 internal infiltration by the lactate standard freeze-dried product, the preparation of the sample was carried out by centrifugation to isolate the strain of the present invention and the culture broth Respectively. The strain bacterium of the present invention was diluted with phosphate buffered saline and treated, and the culture broth was used as it was. As a preparation for Caco-2 cell culture, a plastic culture container, DMEM (Dulbeco's Modified Eagle's Medium; medium), 100-fold concentrated non-essential amino acid and folate serum were prepared.
Caco-2 배양을 위한 배지는 DMEM에 100배 농축 비필수 아미노산 1%와 우태혈청 15%를 첨가하였다. 인산완충식염수 10배 농축액(PBS 10X)을 정제수로 10배로 희석하고 121℃에서 15분 동안 고압 멸균하였다. 트립신-EDTA 용액 A는 트립신을 인산완충식염수에 0.25%로 섞었으며, 0.22μm의 멤브레인 필터로 여과를 한 후 -20℃에 보관하였다. 트립신-EDTA 용액 B는 8ml의 용액 A를 0.1% EDTA를 포함한 인산완충식염수 100ml에 넣어주었으며(트립신의 최종 농도는 0.02% 및 EDTA의 최종농도는 3mM), 0.22μm의 멤브레인 필터로 여과를 한 후 사용하였다.The medium for Caco-2 culture was supplemented with 1% non-essential amino acid and 15% fetal calf serum in 100-fold concentrated DMEM. Phosphate-buffered saline 10-fold concentrated (PBS 10X) was diluted 10-fold with purified water and autoclaved at 121 캜 for 15 minutes. Trypsin-EDTA solution A was mixed with 0.25% trypsin in phosphate-buffered saline, filtered through a 0.22 μm membrane filter, and stored at -20 ° C. To trypsin-EDTA solution B, 8 ml of solution A was added to 100 ml of phosphate buffered saline containing 0.1% EDTA (final concentration of trypsin was 0.02% and final concentration of EDTA was 3 mM), followed by filtration with a 0.22-μm membrane filter Respectively.
Caco-2 배양은 계대 횟수 20~32번째인 것을 사용하였으며, 세포는 일반적으로 15% 우태혈청 및 1% 비필수 아미노산을 함유한 DMEM 배지에서 배양하였다. 유지를 위해, 세포는 1주 간격으로 0.02% 트립신-3mM EDTA를 사용해 계대해주었다. Caco-2 세포를 24-웰 조직 배양용기에 2.5×104 cfu/ml으로 분주하고 배양하였다. 세포 실험 및 유지는 37℃의 10% 이산화탄소 배양기에서 수행하였고 배지는 매일 교환해 주었으며, 세포는 배양 15일 이후 완전히 가득 찼을 때 사용하였다. 세포 보관을 위해, 세포는 15% 우태혈청 및 1% 비필수 아미노산이 포함된 DMEM에 3×106 cfu/ml이 되도록 준비하고 이 현탁액 1~2ml에 0.15ml의 우태혈청 및 0.15ml의 글리세롤을 첨가하고 동결용 튜브에 옮겨 담았으며, 계대 횟수 18~20번째의 세포를 액체질소에 보관하였다.Caco-2 cultures were used in the 20th to 32nd passages. Cells were generally cultured in DMEM medium containing 15% fetal calf serum and 1% non-essential amino acid. For maintenance, the cells were incubated with 0.02% trypsin-3 mM EDTA at intervals of one week. Caco-2 cells were cultured in a 24-well tissue culture vessel at a density of 2.5 x 10 < 4 > cfu / ml. Cell experiments and maintenance were carried out in a 10
Caco-2/TC7 세포 내로 살모넬라 티피뮤리움(Salmonella enterica serovar Typhimurium)의 침투 저해효과 확인을 위해, 균주 살모넬라 티피뮤리움 ATCC14028(Salmonella enterica serovar Typhimurium ATCC14028) 균주를 표지 균주로서 사용하였으며, 균주는 LB 액체배지와 20% 글리세롤에 담겨 -80℃에 보관하였다. 살모넬라의 분주는 보관 균주를 해동하지 않고 멸균된 면봉으로 채취하여 LB 고체배지에 37℃에서 18시간 배양한 후 4℃에서 15일간 보관하였다. Caco-2 / TC7 cells into Salmonella enterica serovar Typhimurium ), the strain Salmonella enterica ATCC14028 ( Salmonella enterica serovar Typhimurium ATCC14028) was used as a marker strain. The strain was stored in LB liquid medium and 20% glycerol at -80 ° C. Salmonella strains were harvested by sterilized cotton swabs without thawing and cultured in LB solid medium at 37 ° C for 18 hours and stored at 4 ° C for 15 days.
실험재료로 겐타마이신 용액(50mg/ml) 및 카제인 대두소화 한천배지(casein soybean digest agar)를 준비하였다. 분석방법은 Caco-2 세포를 감염시키기 위해 살모넬라 균주는 LB 액체배지에 37℃에서 18시간 동안 배양한 후 새로운 LB 액체배지에 접종하여 37℃에서 3시간 동안 추가 배양하였다. 감염시키기 전 Caco-2 세포는 인산완충식염수로 2회 세척해주고 웰당 500㎕의 DMEM을 첨가하였다. 5000g에서 5분간 원심분리하여 LB 액체배지를 제거한 후 박테리아를 DMEM에 4×108 cfu/ml이 되도록 희석하였다. 각각의 웰에 250㎕의 희석 현탁액을 넣고 250㎕의 표본을 넣어주었다. 감염시킨 후 플레이트는 37℃ 및 10% 이산화탄소 조건에서 1시간 동안 배양하였으며, 이후에 세포는 멸균된 인산완충식염수로 3회 세척하고 100㎍/ml의 겐타마이신을 포함한 배지로 1시간을 이어 배양하였다. 살모넬라의 내부 침투 분석은 아미노글리코시드계 항생제에 대한 감수성을 사용해 감염된 세포에 있는 박테리아의 양을 측정하여 결정하였다. 세포는 인산완충식염수로 2회 세척하고 1ml의 멸균된 물로 파쇄하였으며, 세포 내 살아있는 박테리아의 수를 측정하기 위하여 대두소화 한천배지에 희석을 하여 배양하고 박테리아 콜로니를 계수하였다. 하기 표 6 내지 7에 개시한 바와 같이 검액, 대조군 및 실험군을 준비하였다. Gentamicin solution (50 mg / ml) and casein soybean digest agar were prepared as experimental materials. In order to infect Caco-2 cells, Salmonella strains were cultured in LB liquid medium at 37 ° C for 18 hours, then inoculated into fresh LB liquid medium and further cultured at 37 ° C for 3 hours. Prior to infection, Caco-2 cells were washed twice with phosphate-buffered saline and added with 500 μl DMEM per well. After centrifugation at 5000 g for 5 min, the LB liquid medium was removed and the bacteria were diluted to 4 x 10 8 cfu / ml in DMEM. A 250 μl dilution suspension was added to each well and 250 μl of sample was added. After the infection, the plate was incubated at 37 ° C and 10% carbon dioxide for 1 hour, after which the cells were washed three times with sterilized phosphate buffered saline and cultured for 1 hour in a medium containing 100 μg / ml of gentamycin . Internal penetration analysis of Salmonella was determined by measuring the amount of bacteria in infected cells using susceptibility to aminoglycoside antibiotics. The cells were washed twice with phosphate-buffered saline and disrupted with 1 ml of sterilized water. To measure the number of living bacteria in the cells, the cells were diluted in soybean digest agar medium and cultured and bacterial colonies were counted. Test solutions, control groups and experimental groups were prepared as described in Tables 6 to 7 below.
(cfu/ml)Number of cells per well
(cfu / ml)
그 결과, 표 8에 개시한 바와 같이 본 발명의 균주는 살모넬라 티피뮤리움의 장세포 침투에 대한 저지 활성이 양성대조군과 유사하거나 그 이상의 효능을 보유한 것으로 확인되었다. As a result, as shown in Table 8, it was confirmed that the strain of the present invention had an inhibitory activity against intestinal cell infiltration of Salmonella typhimurium having a similar or greater efficacy than the positive control.
따라서, 상기 결과들을 통해 본 발명의 락토바실러스 헬베티커스 CBG-C23 균주는 공액리놀레산 생성능, 젖산 생성능, 내산성, 내담즙성, 항생재 내성 및 살모넬라 티피뮤리움의 장세포 침투에 대한 저지 활성을 가진 우수한 프로바이오틱스 균주인 것을 확인할 수 있었다. Therefore, the Lactobacillus helveticus CBG-C23 strain of the present invention has the ability to inhibit intestinal cell infiltration of conjugated linoleic acid, lactic acid production ability, acid resistance, bile resistance, antibiotic resistance and Salmonella typhimurium It was confirmed that the strain was a probiotic strain.
실시예Example 8. 본 발명의 균주 배양시 8. When culturing the strain of the present invention 탄소원의Carbonic 종류에 따른 균의 생장 및 Growth and growth of microorganisms CLACLA 생성량 변화 Change in yield
본 발명의 균주 배양시, 당원의 종류에 따른 생장 및 CLA 생성량의 변화를 확인하기 위해, 포도당(glucose), 과당(fructose), 젖당(lactose) 및 설탕(sucrose)의 탄소원이 첨가된 배지에서 균주를 배양하여 생장 정도와 CLA 생성량을 측정하였다. 그 결과, 도 5에 개시한 바와 같이 본 발명의 락토바실러스 헬베티커스 CBG-23 균주의 생장에 가장 적합한 탄소원은 젖당(lactose)이었으며, LA 첨가 배지에서 24시간 동안 배양한 후의 CLA 생성량은 젖당을 탄소원으로 사용하였을 때 최대값을 나타내는 것을 확인할 수 있었다.In order to confirm the growth of CLA and the amount of CLA produced according to the type of glycogen in the culture of the strain of the present invention, the culture medium containing glucose, fructose, lactose and sucrose carbon source Were cultured and the degree of growth and the amount of CLA produced were measured. As a result, as shown in FIG. 5, the most suitable carbon source for the growth of the Lactobacillus helveticus CBG-23 strain of the present invention was lactose, and the amount of CLA produced after culturing in LA supplemented medium for 24 hours was lactose And it was confirmed that the maximum value was obtained when it was used as a carbon source.
실시예Example 9. 본 발명의 균주 배양시 9. In culturing the strain of the present invention 탄소원의Carbonic 종류에 따른 균의 생장 및 젖산 생성량 변화 Variation of Bacterial Growth and Lactic Acid Production by Different Types
본 발명의 균주 배양시, 당원의 종류에 따른 생장 및 젖산 생성량의 변화를 확인하기 위해, 포도당(glucose), 과당(fructose), 젖당(lactose) 및 설탕(sucrose)의 탄소원이 첨가된 배지에서 균주를 배양하여 생장 정도 및 젖산 생성량을 측정하였다. 그 결과, 도 6에 개시된 바와 같이 본 발명의 락토바실러스 헬베티커스(Lactobacillus helveticus) CBG-C23 균주의 생장에 가장 적합한 탄소원은 젖당(lactose)이었으며, 젖산 생성량은 젖당을 탄소원으로 사용하였을 때 최대값을 나타내는 것을 확인할 수 있었다.In the culture of the strain of the present invention, in order to examine the changes in the growth and lactic acid production depending on the species of the glycogen, the strains were cultured in a medium containing glucose, fructose, lactose and sucrose, And the degree of growth and lactic acid production were measured. As a result, as shown in FIG. 6, the best carbon source for the growth of the Lactobacillus helveticus CBG-C23 strain of the present invention was lactose and the lactic acid production was the maximum value when lactose was used as a carbon source . ≪ / RTI >
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