KR101809616B1 - A probiotic strain from kefir with anti-obesity effect - Google Patents
A probiotic strain from kefir with anti-obesity effect Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
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- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/332—Promoters of weight control and weight loss
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/155—Kefiri
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- A23Y2220/47—
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- C12R1/225—
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
Abstract
Description
본 발명은 항비만효과를 가지는 케피어 유래 프로바이오틱 균주에 관한 것이다.The present invention relates to a probiotic strain derived from kefir having an anti-obesity effect.
비만은 체내에 과도한 지방이 축적되는 복합적인 질환으로 전 세계적으로 그 유병률이 30%에 달하고 있다. 비만의 유병률이 증가하는 요인으로는 현대인의 활동량 및 운동 감소와 더불어 고지방식이 섭취 등의 식생활의 변화를 들 수 있다. 비만은 심혈관계 질환, 제 2형 당뇨 등의 대사성 질환 등 다양한 질환들의 발생 가능성을 높이는 기저질환이 될 수 있기 때문에 예방 및 치료가 시급하나, 현재까지 개발된 비만치료제는 효과가 적거나 부작용이 심하여 그 이용이 제한적이다.Obesity is a complex disease in which excess fat accumulates in the body and its prevalence reaches 30% worldwide. The increase in the prevalence of obesity can be attributed to changes in dietary habits such as high fat diet, as well as decreased activity and exercise. Obesity is a basic disease that increases the possibility of various diseases such as cardiovascular diseases and metabolic diseases such as
관련 특허로는 대한민국공개특허공보 제10-2012-0034482호에는 3T3-L1 지방세포 분화 억제 효능을 갖는 락토바실러스 플란타룸(Lactobacillus plantarum) KY1032(기탁번호 : KCCM-10430)이 개시되어 있고,대한민국등록특허공보 제10-0996577호에는 혈중 콜레스테롤 강하 및 비만 억제 효능을 갖는 신규의 락토바실러스 커베터스(Lactobacillus curvatus) HY7601(기탁번호 : KCTC 11456BP)이 개시되어 있고, 대한민국공개특허공보 제10-2013-0046896호에는 락토바실러스 플란타룸(Lactobacillus plantarum) DSR920(기탁번호 : KCCM11210P)을 유효성분으로 포함하는 비만 예방 및 치료용 조성물이 개시되어 있고, 대한민국공개특허공보 제10-2010-0010015호에는 혈중 콜레스테롤 저하 및 항비만 활성을 갖는 락토바실러스 존소니(Lactobacillus johnsonii) HF1 108 균주(KCTC 11356BP)가 개시되어 있다. 그러나, 상업적으로 성공할 만큼 항비만 효과가 우수한 유산균 관련 기술은 출현하지 않고 있는바, 항비만 효과가 우수한 새로운 균주의 스크리닝 및 이의 다양한 기능을 규명할 필요가 있다.As a related patent, Korean Patent Publication No. 10-2012-0034482 discloses Lactobacillus plantarum KY1032 (accession number: KCCM-10430) having an inhibitory effect on 3T3-L1 adipocyte differentiation, Patent Publication No. 10-0996577 discloses a novel Lactobacillus curvatus HY7601 (accession number: KCTC 11456BP) having blood cholesterol lowering and obesity inhibitory activity, Korean Patent Laid-Open Publication No. 10-2013 -0046896 discloses a composition for the prevention and treatment of obesity comprising Lactobacillus plantarum DSR920 (accession number: KCCM11210P) as an active ingredient, and Korean Patent Publication No. 10-2010-0010015 discloses a composition for preventing and treating obesity Lactobacillus johnsonii HF1 108 strain (KCTC 11356BP) having cholesterol lowering and anti-obesity activity is disclosed. However, there are no lactobacillus-related technologies that have an anti-obesity effect so as to be commercially successful. Therefore, screening of novel strains with excellent anti-obesity effect and their various functions need to be clarified.
본 발명은 상기의 필요성에 의하여 안출된 것으로서 본 발명의 목적은 항비만 활성을 갖는 신규 유산균을 제공하는 것이다.The present invention has been made in view of the above needs, and an object of the present invention is to provide novel lactic acid bacteria having anti-obesity activity.
본 발명의 다른 목적은 신규 유산균의 다양한 용도를 제공하는 것이다.Another object of the present invention is to provide various uses of novel lactic acid bacteria.
상기의 목적을 달성하기 위하여 본 발명은 항비만효과를 가지는 락토바실러스 케피리(Lactobacillus kefiri) DH5 균주(수탁번호 : KCCM 11837P)를 제공한다.In order to achieve the above object, the present invention provides a strain of Lactobacillus kefiri DH5 (accession number: KCCM 11837P) having an anti-obesity effect.
본 발명의 균주는 2016년 4월29일 대한민국 서울시 서대문구 홍제내2가길 45 (홍제동) 유림빌딩 3F 한국 미생물 보존센터에 수탁번호 : KCCM 11837P로 기탁하였다.The strain of the present invention was deposited on April 29, 2016 with the accession number: KCCM 11837P to the 3F Microorganism Conservation Center, Yuri Building, 2 Gagil 45 (Hongje-dong), Seodaemun-gu, Seoul, Korea.
본 발명의 일 구현예에 있어서, 상기 균주는 서열번호 1의 16s rDNA 서열을 가지는 것이 바람직하나 이에 한정되지 아니한다.In one embodiment of the present invention, the strain preferably has the 16s rDNA sequence of SEQ ID NO: 1, but is not limited thereto.
또 본 발명은 상기 본 발명의 균주, 이의 배양물, 이의 파쇄물 또는 이의 추출물을 유효성분으로 포함하는 비만 또는 비만 관련 질환의 예방 또는 치료용 약학 조성물을 제공한다.The present invention also provides a pharmaceutical composition for preventing or treating obesity or obesity-related diseases comprising the above-mentioned strain, a culture thereof, a crushed product thereof or an extract thereof as an active ingredient.
또한 본 발명은 상기 본 발명의 균주, 이의 배양물, 이의 파쇄물 또는 이의 추출물을 유효성분으로 포함하는 비만 또는 비만 관련 질환의 예방 또는 개선용 식품 조성물을 제공한다. The present invention also provides a food composition for preventing or ameliorating an obesity or obesity-related disease comprising the strain of the present invention, a culture thereof, a crushed product thereof or an extract thereof as an active ingredient.
이하, 본 발명에서 사용한 용어를 설명한다.Hereinafter, terms used in the present invention will be described.
본 발명에서 사용되는 용어 "배양물"이란 미생물을 공지의 액체 배지 또는 고체 배지에서 배양시켜 수득한 산물을 의미하여, 미생물이 포함되는 개념이다.The term "culture product " used in the present invention means a product obtained by culturing a microorganism in a known liquid medium or solid medium, and includes a microorganism.
본 발명에서 "약학적으로 허용가능한" 및 "식품학적으로 허용가능한"이란 생물체를 상당히 자극하지 않고 투여 활성 물질의 생물학적 활성 및 특성을 저해하지 않는 것을 의미한다.The terms " pharmaceutically acceptable "and" pharmaceutically acceptable "in the present invention are intended to mean not significantly irritating the organism and not interfering with the biological activity and properties of the administered active substance.
본 발명에서 사용되는 용어 "예방"은 본 발명의 조성물의 투여로 특정 질환(예를 들어, 비만)의 증상을 억제시키거나 진행을 지연시키는 모든 행위를 의미한다.As used herein, the term "prophylactic " refers to any act that inhibits the symptoms of, or delays the progression of, a particular disease (e.g., obesity) upon administration of the composition of the present invention.
본 발명에서 사용되는 용어 "치료"는 본 발명의 조성물의 투여로 특정 질환(예를 들어, 비만)의 증상을 호전 또는 이롭게 변경시키는 모든 행위를 의미한다.The term "treatment" as used herein refers to any action that improves or alleviates the symptoms of a particular disease (e. G., Obesity) upon administration of the composition of the present invention.
본 발명에서 사용되는 용어 "개선"은 치료되는 상태와 관련된 파라미터, 예를 들면 증상의 정도를 적어도 감소시키는 모든 행위를 의미한다.The term "improvement" as used in the present invention means all actions that at least reduce the degree of symptom associated with the condition being treated.
본 발명에서 사용되는 용어 "투여"는 임의의 적절한 방법으로 개체에 소정의 본 발명의 조성물을 제공하는 것을 의미한다. 이때, 개체는 본 발명의 조성물을 투여하여 특정 질환의 증상이 호전될 수 있는 질환을 가진 인간,원숭이, 개, 염소, 돼지 또는 쥐 등 모든 동물을 의미한다.The term "administering" as used herein is meant to provide any desired composition of the invention to an individual by any suitable method. The term " individual " means any animal such as a human, a monkey, a dog, a goat, a pig, or a mouse having a disease in which symptoms of a specific disease can be improved by administering the composition of the present invention.
본 발명에서 용어 "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜 또는 위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 이는 개체의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출비율, 치료기간, 동시에 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다.The term "pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit or risk ratio applicable to medical treatment, including the type of disease, severity, activity of the drug, Sensitivity, the time of administration, the route of administration and the rate of excretion, the duration of the treatment, factors including the drugs used concurrently, and other factors well known in the medical arts.
본 발명의 항비만제는 상기의 본 발명 유산균을 경구투여 가능하고 살아 있는 그대로 장관에 도원(到遠)시킬 수 있는 생균제로 함으로써 조제된다. 이 제형에 대해서는 특별히 제약은 없고, 예를 들면 분말제, 과립, 정제, 캡슐제 등의 고형, 젤리, 페이스트 등의 반고형, 현탁액, 시럽 등의 액상이어도 된다. 이들의 각 제형은 제약분야에서 공지의 방법으로 제조할 수 있다.The anti-obesity agent of the present invention is prepared by allowing the above-mentioned lactic acid bacteria of the present invention to be orally administrable, and to be a probiotic agent capable of being passed on to the intestinal tract as it is alive. The formulation is not particularly limited, and may be, for example, a solid such as a powder, granule, tablet, or capsule, a semi-solid such as jelly or paste, or a liquid such as a suspension or a syrup. Each of these formulations can be prepared by methods known in the pharmaceutical arts.
상기 항비만제에 배합되는 본 발명 유산균은 공지의 유산균의 배양방법을 적용하여 배양을 행할 수 있다. 이 배양방법으로서는 통상의 방법에 따라 본 발명 유산균을 액체 배양하고, 얻어진 배양물을 그대로 이용해도, 또한 이 배양물로부터 원심분리 등의 수단으로 균체를 모아 사용해도, 또는 배양물을 동결건조하여 분말상으로 한 것을 사용해도 된다.The lactic acid bacteria of the present invention to be blended with the anti-obesity agent can be cultured by applying a known method for culturing lactic acid bacteria. As the culture method, the lactic acid bacteria of the present invention may be cultured by liquid culture according to a conventional method, and the obtained culture may be used as it is, or the cells may be collected from the culture using centrifugal separation or the like, or the lyophilized product may be lyophilized May be used.
고형상인 본 발명 비만제의 일반적인 제법으로서는 본 발명 유산균을 물, 전분, 미세결정 셀룰로오스, 밀가루,설탕 등의 담체와 함께 배합하여, 목적하는 형태로 하는 방법을 들 수 있다. 상기 담체도 공지로서, 사용형태에 맞추어 적절히 선택하여 사용할 수 있다. 보다 구체적으로는 통상의 방법으로 배양하여 얻어진 본 발명 유산균의 균체를 동결건조 분말로 하고, 이 설탕과 혼합함으로써 분말제를 조제해도 된다. 또한, 본 발명 유산균의 균체를 적절한 정제용(錠劑用) 담체와 함께 혼합하고, 이것을 통상의 방법에 따라서 타정하여 정제를 얻는 것도 가능하다. 또한, 본 발명 유산균의 습(濕)균체를 시럽 중에 현탁하여 시럽제로 해도 된다. 본 발명의 항비만제의 조제에 있어서는 필요에 따라서 다른 성분, 예를 들면 다른 미생물이나 유효성분, 또는 감미료, 향료, 착색제 등을 함유하고 있어도 된다.As a general method for producing the obesity agent of the present invention, there may be mentioned a method in which the lactic acid bacteria of the present invention are blended with a carrier such as water, starch, microcrystalline cellulose, wheat flour, sugar, etc. to obtain a desired form. The carrier may also be appropriately selected and used in accordance with the mode of use as well known in the art. More specifically, the powder of the lactic acid bacteria of the present invention obtained by culturing by a conventional method may be prepared as a lyophilized powder and mixed with the sugar. It is also possible to mix the microbial cells of the lactic acid bacteria of the present invention with a suitable carrier for tablets and to obtain tablets by tableting them according to a conventional method. In addition, wet microbes of the lactic acid bacteria of the present invention may be suspended in syrup to make a syrup. In the preparation of the anti-obesity agent of the present invention, other components such as other microorganisms and effective ingredients, or sweeteners, flavors, coloring agents and the like may be contained as necessary.
상기와 같이 하여 얻어지는 항비만제의 투여량은 대상의 신체적 상황, 예를 들면 건강상태, 체중, 연령, 사용되는 다른 성분 등을 고려하여 적절히 결정할 수 있는데, 일반적으로는 본 발명 유산균의 균수로서, 어른 1인당 대략 108부터 109 CFU/일 정도 전후이다.The dose of the anti-obesity agent thus obtained can be suitably determined in consideration of the physical condition of the subject, for example, the health condition, body weight, age, other components to be used, etc. Generally, It is around 10 8 to 10 9 CFU / day for each adult.
또한, 본 발명 유산균을 사용하여 항비만 음식물을 조제하기 위해서는, 종래 공지의 유산균의 배양방법을 이용하여 경구섭취 가능한 발효식품으로 하면 된다. 구체적으로는, 요구르트 등의 발효유, 유산균 음료, 발효 소시지 등의 식품으로 할 수 있고, 이들 식품의 제조는 사용 유산균의 일부 또는 전부를 본 발명 유산균으로 함으로써 행할 수 있다. 또한, 본 발명 유산균을 보다 많이 포함하는 형태로 하여, 건강식품이나 기능성 식품으로 하는 것도 가능하다. 이 항비만 식품의 조제에 있어서는 본 발명 유산균 단독이 아니라 다른 유산균을 포함하고 있어도 되는 것은 물론이며, 또한 식품첨가물 또는 조미료 등을 첨가해도 된다.In addition, in order to prepare an anti-obesity food using the lactic acid bacteria of the present invention, a fermented food which can be ingested orally by using a conventionally known culture method of lactic acid bacteria may be used. Specifically, it can be a food such as a fermented milk such as yogurt, a lactic acid bacterium drink, a fermented sausage, etc. These foods can be produced by partially or entirely using the lactic acid bacteria of the present invention. In addition, it can be a health food or a functional food in a form containing more of the lactic acid bacteria of the present invention. In the preparation of the anti-obesity food, not only the lactic acid bacteria of the present invention but also other lactic acid bacteria may be contained, and food additives or seasoning may be added.
이하 본 발명을 설명한다.Hereinafter, the present invention will be described.
본 발명에서는 장수마을인 코카서스 지방의 천연 복합 발효유인 케피어로부터 유산균을 분리하여 비만 예방 및 치료용 프로바이오틱스 제제로서 Lactobacillus kefiri DH5 균주를 선발하였다. In the present invention, Lactobacillus was isolated from kefir which is a natural complex fermented milk in the longevity village of Caucasus, and Lactobacillus kefiri DH5 strain was selected as a probiotic preparation for the prevention and treatment of obesity.
본 발명에서는 케피어 발효유에서 분리된 균주을 활용하여 in vitro 실험을 통한 생존능 확인 및 비만 모델 동물을 활용한 in vivo 실험에서 체중 감소효과 및 체지방 감소 효과를 분석하여 최종적으로 Lactobacillus kefiri DH5 균주를 항비만 프로바이오틱스로 선발하였다.In the present invention, by observing in vitro viability using a strain isolated from Kefir fermented milk and analyzing the effect of body weight reduction and body fat reduction in an in vivo experiment using an animal model of obesity, Lactobacillus kefiri DH5 strain was used as an anti-obesity probiotic Respectively.
본 발명의 케피어에서 분리된 L. kefiri DH5 균주는 인공위액 및 담즙액에서 뛰어난 생존능을 나타냈으며, 콜레스테롤 저하능 또한 매우 우수한 것으로 나타났다. 또한 Diet-induced obesity mouse model을 이용한 in vivo test에서도 6주간의 투여 결과 본 균을 경구투여한 실험군에서 유의적인 체중 감소 및 체지방 무게 감소, 지방세포 크기 감소가 나타나는 것으로 보아 본 균이 비만 예방 효과가 있음을 알 수 있었고, 따라서 본 발명의 새롭게 선발된 L. kefiri DH5 균주는 비만 치료용 probiotics 제제로 유용하게 사용될 수 있다. The L. kefiri DH5 strain isolated from the kefir of the present invention showed excellent survival ability in artificial gastric juice and bile juice, and showed excellent cholesterol lowering ability. In addition, in the in vivo test using a diet-induced obesity mouse model, the results of the 6-week administration showed that significant reductions in body weight, body fat weight and fat cell size were observed in the oral administration group of the present invention, Therefore, the newly selected L. kefiri DH5 strain of the present invention can be usefully used as a probiotics preparation for obesity treatment.
도 1은 Lactobacillus kefiri DH5의 16S rRNA 시퀀싱 결과의 neighbor-joining tree. DH5번 균주는 Lactobacillus kefiri와 가장 높은 상동성을 나타내었음 (>99.5%)
도 2는 Diet-유도된 비만 마우스 모델에서의 대조군, L. kefir DH5 투여군, Leuconostoc mesenteroides KU50404 투여군의 체중 변화, * 대조군과 비교시 통계학적 유의차를 의미함 (p<0.05, student t test)
도 3은 L. kefiri DH5를 섭취한 비만 마우스의 부고환지방 무게 감소. * 대조군과 비교시 통계학적 유의차를 의미함 (p<0.05, student t test)
도 4는 L. kefiri DH5를 섭취한 비만 마우스의 지방 세포 크기 감소. HE 염색사진의 bar는 50μM를 의미함. * 대조군과 비교시 통계학적 유의차를 의미함 (p<0.05, student t test)Figure 1 shows the neighbor-joining tree of 16S rRNA sequencing results of Lactobacillus kefiri DH5. DH5 strain showed the highest homology with Lactobacillus kefiri (> 99.5%)
Figure 2 shows the statistical significance (p < 0.05, Student t test) in comparison with the control group, L. kefir DH5 treated group and Leuconostoc mesenteroides KU50404 treated group in the Diet-induced obese mouse model,
Figure 3 shows the epididome fat weight loss of obese mice ingested with L. kefiri DH5. * Statistically significant difference compared to control group (p <0.05, Student t test)
Figure 4 shows the reduction in fat cell size in obese mice fed L. kefiri DH5. Bar of HE staining image means 50 μM. * Statistically significant difference compared to control group (p <0.05, Student t test)
이하 비한정적인 실시예를 통하여 본 발명을 더욱 상세하게 설명한다. 단 하기 실시예는 본 발명을 예시하기 위한 의도로 기재된 것으로서 본 발명의 범위는 하기 실시예에 의하여 제한되는 것으로 해석되지 아니한다.The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.
실시예Example 1: One: 케피어Kefir 발효유에서 유산균의 분리 및 동정 Isolation and Identification of Lactic Acid Bacteria from Fermented Milk
케피어 그레인 50g을 멸균 증류수를 이용하여 무균적으로 세척해준 후 멸균우유 1L에 접종하여 고르게 분포시킨 후 25°C에서 24시간 동안 정치 배양하여 얻은 케피어 발효유를 멸균된 loop를 이용하여 de Man, Rogosa, and Sharpe (MRS) agar (Difco)에 획선도말하였다. 이후 8시간 동안 37°C, 혐기 조건 하에서 배양하였다. 형성된 단일집락을 취하여 MRS 배지를 이용하여 상기 조건 하에서 2회 계대배양하였다. 배양된 집락에서 genomic DNA를 추출하여 16S ribosomal DNA sequencing 후 GenBank를 database로 하여 BLAST를 실시하여 총 13주의 유산균을 동정하였다.50 g of kefir grains were aseptically cleaned with sterile distilled water, and then inoculated into 1 L of sterilized milk, uniformly distributed, and cultured for 24 hours at 25 ° C. The kefir fermented milk was sterilized using a sterilized loop de Man, Rogosa, and Sharpe (MRS) agar (Difco). And then cultured at 37 ° C under anaerobic conditions for 8 hours. A single colonies formed were taken and subcultured 2 times under the above conditions using MRS medium. Genomic DNA was extracted from cultured colonies, 16S ribosomal DNA sequencing was performed, and GenBank was used as BLAST database to identify 13 lactic acid bacteria.
실시예Example 2: 인공 위액 및 담즙에서의 2: in artificial gastric juice and bile 생존성Survivability 확인 시험 Confirmation test
유산균 균주의 활력 회복을 위해 MRS agar에서 37°C, 48시간 동안 혐기 조건 하에서 2번 계대배양하였다. 인공 위액에서의 생존성 확인 시험은 pH를 2.5으로 맞추고 1,000 units/mL pepsin을 첨가한 MRS broth (pepsin-MRS broth) 를 이용하여 실시하였다. L. kefiri DH5 생균을 3ml의 pepsin-MRS broth에 첨가하여 37°C에서 배양하며 0시간, 2시간째 생균수를 계수하여 생존률을 확인하였다.Lactobacillus In order to restore the viability of the strain, subculture was carried out on MRS agar at 37 ° C for 48 hours under anaerobic conditions. The viability test in artificial gastric juice was performed using MRS broth (pepsin-MRS broth) supplemented with 1,000 units / mL pepsin at pH 2.5. L. kefiri DH5 live cells were added to 3 ml of pepsin-MRS broth, incubated at 37 ° C, and viable counts were obtained by counting viable cells for 0 hours and 2 hours.
또한, 담즙에서의 생존성을 확인하기 위하여 pH를 8으로 맞추고 0.3%의 oxgall 을 첨가한 MRS broth (oxgall-MRS broth)를 사용하였다. 케피어 분리 유산균을 3ml의 oxgall-MRS broth에 첨가하여 37°C에서 배양하며 0시간, 24시간째 생균수를 계수하여 생존률을 확인하였다. 생존률 시험에서 우수한 특성을 나타낸 2 균주를 선정하여 in vivo test에 활용하였다.MRS broth (oxgall-MRS broth) supplemented with 0.3% oxgall was used to adjust the pH to 8 to confirm viability in bile. Kefir isolated lactic acid bacteria were added to 3 ml of oxgall-MRS broth and cultured at 37 ° C. Cell viability was counted by counting viable cells at 0 hours and 24 hours. Two strains with excellent survival rate were selected for in vivo testing.
실시예Example 3: 3: 고지방식이를Highlander 투여한 동물에서의 체중 감소 효과 입증 Prove weight loss effect in administered animals
4주령 수컷 C57BL/6 mouse에 60% 고지방식이를 섭취시켜 비만을 유도하는 diet-induced obesity model을 사용하여 고지방식이를 투여한 동물에서의 체중 감소 효과를 확인하였다. 실험에 들어가기 전에 1주일 동안 동물을 실험동물 시설에 순응시켰다. 실험군은 대조군 (일반식이), 대조군 (고지방식이), Lactobacillus kefiri DH5 투여군, Leuconostoc mesenteroides DH4 투여군으로 설정하였으며, 각 실험군별로 8마리의 마우스를 사용하였다. 모든 실험군에는 음수와 60% high fat diet를 자율공급하였다. 투여물질로 대조군에는 멸균된 생리식염수를, L. kefiri DH5 투여군에는 L. kefiri DH5 균주를 108 CFU씩, 대조유산균 투여군에는 Leuconostoc mesenteroides DH4를 108 CFU씩 매일 15시~17시 사이에 1회 경구투여하였다. 체중과 식이량을 매주 측정하며 총 6주동안 실험을 실시하였다. 동물실험은 건국대학교 동물실험윤리위원회의 승인하에 이루어졌다 (KU16046).A diet-induced obesity model that induces obesity by ingesting 60% high-fat diets in 4-week-old male C57BL / 6 mice was used to confirm the weight loss effect in animals fed high fat diet. The animals were acclimated to the laboratory animal facility for one week before entering the experiment. The experimental group consisted of control (normal diet), control (high fat diet), Lactobacillus kefiri DH5, Leuconostoc mesenteroides DH4 group, and 8 mice were used for each experimental group. All groups were autonomously fed negative and 60% high fat diets. Saline solution are sterilized in the control group administered substance, L. kefiri DH5 group has once between L. kefiri DH5 strain to 10 8 CFU each, the control group, the lactic acid bacterium Leuconostoc mesenteroides
실시예Example 4:부고환지방4: epididymis fat 무게 및 지방세포 크기 측정 Measure weight and fat cell size
실험동물을 안락사 후 복강 내 부고환 지방을 채취하여 무게를 측정하였다. 10% neutral formalin에서 fixation 한 뒤 HE 염색하였다. 이후 광학현미경을 이용하여 400X 시야에서 부고환조직의 지방세포의 크기를 측정하였다.Experimental animals were euthanized and epididymal fat was collected and weighed. After fixation in 10% neutral formalin, HE stained. The size of adipose tissue adipose tissue was measured by using optical microscope in 400X field of view.
본 발명의 데이터는 SPSS (version 18.0, SPSS Inc.)를 이용하여 평균 ± 표준편차로 제시하였다. 또한 정량치간의 통계학적 유의차는 analysis of variance (ANOVA; Tukey's method)으로 분석하였으며 P값이 0.05이하인 경우를 통계학적으로 유의차가 있는 것으로 간주하였다.Data of the present invention are presented as mean + standard deviation using SPSS (version 18.0, SPSS Inc.). Statistical significance between the quantitative values was analyzed by analysis of variance (ANOVA; Tukey's method). P value <0.05 was considered statistically significant.
이하 상기 실시예의 결과를 기재한다.Hereinafter, the results of the above embodiments will be described.
유산균 분리 및 동정Isolation and Identification of Lactic Acid Bacteria
케피어 발효유에서 총 13주의 유산균을 분리하였으며, 16S rDNA sequencing 결과 2주의 Lactobacillus kefiri, 5주의 Leuconostoc mesenteroides, 4주의 Lactococcus lactis, 2주의 Enterococcus durans를 분리하였다. 그 중에서 본 발명에서 최종 선발된 Lactobacillus kefiri DH5의 16S rDNA 서열(서열번호 1)을 neighbor-joining method로 taxonomic tree를 작성하여 도 1에 나타내었다.A total of 13 lactic acid bacteria were isolated from Kefir fermented milk. 16S rDNA sequencing showed 2 Lactobacillus kefiri , 5 Leuconostoc mesenteroides , 4 weeks of Lactococcus lactis , 2 weeks of Enterococcus durans . Among them, the taxonomic tree of the 16S rDNA sequence (SEQ ID NO: 1) of Lactobacillus kefiri DH5 finally selected in the present invention was created by a neighbor-joining method and shown in FIG.
인공 위액 및 담즙 생존능 Artificial gastric juice and bile survival ability
케피어 분리 유산균의 인공위액 및 담즙 생존능 분석 결과를 표 1에 제시하였다. 인공위액에서 2시간동안 배양 후 생존을 관찰한 결과, Lactobacillus kefiri DH5 및 Leuconostoc mesenteroides KU4의 경우 초기 접종량에서 1 LogCFU/ml 이내로 감소하는 것을 확인하였다. Table 1 shows the results of analysis of artificial gastric juice and bile viability of lactic acid bacteria isolated from kefir. The survival rate of Lactobacillus kefiri DH5 and Leuconostoc mesenteroides KU4 was found to be less than 1 LogCFU / ml in the initial inoculation amount after 2 hours of incubation in artificial gastric juice.
또한, 인공담즙에서는 Lactobacillus kefiri DH5의 경우 1 LogCFU/ml 이내로 감소하였으며, Lactobacillus kefiri DN3, Leuconostoc mesenteroides KU1, Leuconostoc mesenteroides KU2, Leuconostoc mesenteroides KU3, Leuconostoc mesenteroides KU4, Leuconostoc mesenteroides KU5, Lactococcus lactis VPH3, Enterococcus durans KY1, Enterococcus durans KY2의 경우 2LogCFU/ml 이내로 균이 감소하는 것을 확인할 수 있었다. 본 생존률 시험 결과 종합하였을 때, Lactobacillus kefiri DH5 및 Leuconostoc mesenteroides KU4 가 소화관 생존능이 가장 뛰어난 것으로 판단되었기 때문에 이 두 균주를 in vivo test에 사용하기로 결정하였다. In the case of artificial bile, Lactobacillus kefiri DH5 decreased to 1 LogCFU / ml, and Lactobacillus kefiri DN3, Leuconostoc mesenteroides KU1, Leuconostoc mesenteroides KU2, Leuconostoc mesenteroides KU3, Leuconostoc mesenteroides KU4, Leuconostoc mesenteroides KU5, Lactococcus lactis VPH3, Enterococcus durans KY1, Enterococcus In case of durans KY2, it was confirmed that the number of bacteria decreased within 2LogCFU / ml. In the present survival rate test results, when Lactobacillus kefiri DH5 and Leuconostoc Since mesenteroides KU4 was considered to have the best digestive tract viability, we decided to use these two strains for in vivo testing.
(MRS with 1000 IU of pepsin/ml, pH 2.5, 2h)Gastric environment
(MRS with 1000 IU of pepsin / ml, pH 2.5, 2h)
(MRS with 0.3% oxgall, pH8.0, 24h)Intestinal environment
(MRS with 0.3% oxgall, pH 8.0, 24h)
표 1은 인공위액 및 담즙에서의 생존 결과. 실험은 3 반복하였음.+++, < 1 log CFU reduction; ++, < 2 Log CFU reduction; +, > 2 Log CFU reduction; -, no survivalTable 1 shows survival results in artificial gastric juice and bile. The experiment was repeated three times. +++, <1 log CFU reduction; ++, < 2 Log CFU reduction; +,> 2 Log CFU reduction; -, no survival
고지방식이를Highlander 투여한 동물에서의 체중 감소 효과 입증 Prove weight loss effect in administered animals
고지방식이로 비만을 유도한 동물에서 L. kefiri DH5의 비만 예방 효과를 도 2에 나타내었다. 6주간 고지방식이를 제공하며 멸균생리식염수, L. kefiri DH5, Leuc. mesenteroides DH4를 경구투여한 결과, L. kefiri DH5를 경구투여한 그룹에서 실험 5주차부터 대조군(HFD)에 비하여 체중이 유의적으로 감소함을 확인할 수 있었다 (p<0.05, student t test). 대조군으로 사용한 Leuc . mesenteroides DH4의 경우 체중 감소가 나타나긴 하였으나 유의적인 차이를 보이지는 않았다 (p > 0.05). 실험 종료 시점에서 대조군(HFD)과 비교하였을 때, L. kefiri DH5의 경우 15.6%, Leuc. mesenteroides KU4의 경우 5.8%가 감소하였다. 본 결과로 미루어 보아, L. kefiri DH5를 정기적으로 경구투여하면 비만을 예방 및 치료할 수 있을 것으로 판단된다.The preventive effect of L. kefiri DH5 on obesity-induced obesity is shown in Fig. A six-week high-fat diet is provided and sterile saline, L. kefiri DH5, Leuc . As a result of oral administration of mesenteroides DH4, the body weight of L. kefiri DH5 was significantly decreased compared to the control group (HFD) at 5th week in the group administered with L. kefiri DH5 (p <0.05, student t test). As a control, Leuc . Mesenteroides DH4 showed weight loss but no significant difference ( p > 0.05). When compared with the control group (HFD) in the end of the experiment, 15.6% of L. kefiri DH5, Leuc. and 5.8% in mesenteroides KU4. These results suggest that regular oral administration of L. kefiri DH5 can prevent and treat obesity.
부고환지방Epididymis 무게 감소 및 Weight reduction and 지방세포내Adipocyte 지방구Provincial district 크기 size
비만에서는 체지방의 무게 및 지방조직 내 지방세포의 크기 증가가 특징적으로 나타나게 된다. 따라서, 본 발명에서는 케피어 유산균의 투여가 체지방의 무게 및 지방세포의 크기를 감소시킬 수 있는지 확인하였다. L. kefiri DH5 섭취 마우스의 경우 고지방식이 섭취 마우스와 비교하였을 때 부고환지방의 무게가 유의적으로 감소된 것을 확인할 수 있었다 (도 3, p < 0.05). 또한, 부고환지방에서의 HE염색을 실시 한 결과 고지방식이군(HFD)에서 현저한 부고환 지방 세포의 비대가 관찰되었으며, L. kefiri DH5의 투여로 그 크기가 유의적으로 감소하는 것을 확인할 수 있었다 (도 4, p < 0.05). 하지만, Leuc . mesenteroides DH4 투여군에서는 이러한 현상을 관찰할 수 없었다. 결론적으로 L. kefiri DH5의 투여가 지방조직의 축적을 억제한 것으로서 저장 지방의 감소한 것을 의미한다.In obesity, the weight of body fat and the size of fat cells in adipose tissue are characteristic. Therefore, in the present invention, it was confirmed whether administration of Kefir lactic acid bacteria can reduce the body fat mass and fat cell size. In the case of L. kefiri DH5-fed mice, the weight of epididymal fat was significantly reduced when compared to the high-fat diet-fed mice (FIG. 3, p <0.05). As a result of HE staining in epididymal fat, hypertrophic adipocyte hypercellular hyperplasia was observed in high fat diet (HFD) and it was confirmed that L. kefiri DH5 significantly decreased in
<110> Sensorgen Inc. <120> A probiotic strain from kefir with anti-obesity effect <130> P16-0252HS <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 1344 <212> DNA <213> Lactobacillus kefiri DH5 <400> 1 gtttcatgat ttaacacgaa acgagtggcg aactggtgag taacacgtgg gtaacctgcc 60 cttgaagtag gggataacac ttggaaacag gtgctaatac cgtataacaa ccaaaaccac 120 atggttttgg tttaaaagat ggcttcggct atcactttag gatggacccg cggcgtatta 180 gcttgttggt aaggtaatgg cctaccaagg caatgatacg tagccgacct gagagggtaa 240 tcggccacat tgggactgag acacggccca aactcctacg ggaggcagca gtagggaatc 300 ttccacaatg gacgaaagtc tgatggagca acgccgcgtg agtgatgaag ggtttcggct 360 cgtaaaactc tgttgttgga gaagaacagg tgtcagagta actgttgaca tcttgacggt 420 atccaaccag aaagccacgg ctaactacgt gccagcagcc gcggtaatac gtaggtggca 480 agcgttgtcc ggatttattg ggcgtaaagc gagcgcaggc ggtttcttag gtctgatgtg 540 aaagccttcg gcttaaccgg agaagtgcat cggaaaccag gagacttgag tgcagaagag 600 gacagtggaa ctccatgtgt agcggtgaaa tgcgtagata tatggaagaa caccagtggc 660 gaaggcggct gtctggtctg taactgacgc tgaggctcga aagcatgggt agcgaacagg 720 attagatacc ctggtagtcc atgccgtaaa cgatgagtgc taagtgttgg agggtttccg 780 cccttcagtg ctgcagctaa cgcattaagc actccgcctg gggagtacga ccgcaaggtt 840 gaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgat 900 gctacgcgaa gaaccttacc aggtcttgac atcttctgcc aacctaagag attaggcgtt 960 cccttcgggg acagaatgac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt 1020 tgggttaagt cccgcaacga gcgcaaccct aattgttagt tgccagcatt cagttgggca 1080 ctctagcaag actgccggtg acaaaccgga ggaaggtggg gatgacgtca aatcatcatg 1140 ccccttatga cctgggctac acacgtgcta caatggacgg tacaacgagt cgcgaaaccg 1200 cgaggtcaag ctaatctctt aaagccgttc tcagttcgga ttgcaggctg caactcgcct 1260 gcatgaagtt ggaatcgcta gtaatcgtgg atcagcatgc cacggtgaat acgttcccgg 1320 gccttgtaca caccgcccgt cacg 1344 <110> Sensorgen Inc. <120> A probiotic strain from kefir with anti-obesity effect <130> P16-0252HS <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1344 <212> DNA <213> Lactobacillus kefiri DH5 <400> 1 gtttcatgat ttaacacgaa acgagtggcg aactggtgag taacacgtgg gtaacctgcc 60 cttgaagtag gggataacac ttggaaacag gtgctaatac cgtataacaa ccaaaaccac 120 atggttttgg tttaaaagat ggcttcggct atcactttag gatggacccg cggcgtatta 180 gcttgttggt aaggtaatgg cctaccaagg caatgatacg tagccgacct gagagggtaa 240 tcggccacat tgggactgag acacggccca aactcctacg ggaggcagca gtagggaatc 300 ttccacaatg gacgaaagtc tgatggagca acgccgcgtg agtgatgaag ggtttcggct 360 cgtaaaactc tgttgttgga gaagaacagg tgtcagagta actgttgaca tcttgacggt 420 atccaaccag aaagccacgg ctaactacgt gccagcagcc gcggtaatac gtaggtggca 480 agcgttgtcc ggatttattg ggcgtaaagc gagcgcaggc ggtttcttag gtctgatgtg 540 aaagccttcg gcttaaccgg agaagtgcat cggaaaccag gagacttgag tgcagaagag 600 gacagtggaa ctccatgtgt agcggtgaaa tgcgtagata tatggaagaa caccagtggc 660 gaaggcggct gtctggtctg taactgacgc tgaggctcga aagcatgggt agcgaacagg 720 attagatacc ctggtagtcc atgccgtaaa cgatgagtgc taagtgttgg agggtttccg 780 cccttcagtg ctgcagctaa cgcattaagc actccgcctg gggagtacga ccgcaaggtt 840 gaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgat 900 gctacgcgaa gaaccttacc aggtcttgac atcttctgcc aacctaagag attaggcgtt 960 cccttcgggg acagaatgac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt 1020 tgggttaagt cccgcaacga gcgcaaccct aattgttagt tgccagcatt cagttgggca 1080 ctctagcaag actgccggtg acaaaccgga ggaaggtggg gatgacgtca aatcatcatg 1140 ccccttatga cctgggctac acacgtgcta caatggacgg tacaacgagt cgcgaaaccg 1200 cgaggtcaag ctaatctctt aaagccgttc tcagttcgga ttgcaggctg caactcgcct 1260 gcatgaagtt ggaatcgcta gtaatcgtgg atcagcatgc cacggtgaat acgttcccgg 1320 gccttgtaca caccgcccgt cacg 1344
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| KR20190122636A (en) * | 2019-10-24 | 2019-10-30 | 건국대학교 산학협력단 | Animal Food Composition and Feed Additive comprising Kefir |
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| KR101896275B1 (en) | 2018-07-30 | 2018-09-07 | (주)지에프씨생명과학 | Lactobacillus kefiri kr-12 and Fermented Product Manufactured Using Thereof |
| KR20190122636A (en) * | 2019-10-24 | 2019-10-30 | 건국대학교 산학협력단 | Animal Food Composition and Feed Additive comprising Kefir |
| KR102136559B1 (en) * | 2019-10-24 | 2020-07-22 | 건국대학교 산학협력단 | Animal Food Composition and Feed Additive comprising Kefir |
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