KR101371648B1 - Lactobacillus brevis with high alcohol dehydrogenase activity and dairy products, health functional food and food additives comprising the same - Google Patents

Lactobacillus brevis with high alcohol dehydrogenase activity and dairy products, health functional food and food additives comprising the same Download PDF

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KR101371648B1
KR101371648B1 KR1020120013753A KR20120013753A KR101371648B1 KR 101371648 B1 KR101371648 B1 KR 101371648B1 KR 1020120013753 A KR1020120013753 A KR 1020120013753A KR 20120013753 A KR20120013753 A KR 20120013753A KR 101371648 B1 KR101371648 B1 KR 101371648B1
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박민희
김주용
예현수
임광세
김완식
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Abstract

본 발명은 알코올 분해능이 우수한 락토바실러스 브레비스HD-01 및 그를 함유하는 유제품, 건강기능식품 및 식품첨가제에 관한 것으로, 본 발명의 락토바실러스 브레비스(Lactobacillus brevis) HD-01는 알코올 디하이드로게나아제의 활성이 높아 알코올 분해능이 우수하면서, 동시에 아세트알데하이드 디하이드로게나아제의 활성이 높아 숙취해소 활성이 뛰어나며, 내산성 및 내담즙성이 우수하기 때문에, 알코올성 간질환을 예방하거나 숙취해소를 위한 유제품, 건강기능식품 또는 식품첨가물로서 이용될 수 있다.The present invention relates to Lactobacillus brevis HD-01 having excellent alcohol resolution and dairy products, health functional foods and food additives containing the same, and Lactobacillus brevis of the present invention. brevis ) HD-01 has high activity of alcohol dehydrogenase, so it has high alcohol decomposability, and at the same time, acetaldehyde dehydrogenase has high activity of relieving hangover, and has excellent acid and bile resistance. It can be used as dairy products, nutraceuticals or food additives to prevent disease or hangover.

Description

알코올 분해능이 우수한 락토바실러스 브레비스 및 그를 함유하는 조성물{Lactobacillus brevis with high alcohol dehydrogenase activity and dairy products, health functional food and food additives comprising the same}Lactobacillus brevis with high alcohol dehydrogenase activity and dairy products, health functional food and food additives comprising the same}

본 발명은 알코올 분해능이 우수한 락토바실러스 브레비스 및 그를 함유하는 유제품, 건강기능식품 및 식품첨가제에 관한 것이다.
The present invention relates to Lactobacillus brevis excellent in alcohol resolution and dairy products, nutraceuticals and food additives containing the same.

간은 우리 몸에서 에너지 대사를 전체적으로 관리하며 빌리루빈, 담즙산, 콜레스테롤, 인지질 등의 각종 대사산물 및 합성물을 저장하거나 온몸에 분배하는 기능을 갖고, 독소를 해독하거나 주요한 면역기관으로서의 역할을 담당하는 매우 중요한 기관이다.The liver manages energy metabolism in our body as a whole and has the ability to store or distribute various metabolites and compounds such as bilirubin, bile acids, cholesterol, and phospholipids throughout the body, and to detoxify or serve as a major immune organ. It is an organ.

현재 국내에서 질병에 의한 사망원인을 보면, 간질환에 의해 사망하는 순위가 인구 10만명당 22.9명(2001년 기준)으로 4위에 올라와 있으며, 이중 알코올 섭취에 의한 간 질환자를 무시할 수 없다. 세계 보건기구가 96년 153개 국가를 대상으로 음주량을 조사한 결과, 우리나라 성인 1인당 알코올 섭취량은 연간 14,4ℓ로 세계 2위로 나타났으며, 해마다 음주로 인한 사고와 질병으로 2만 3천여명이 숨지며, 이로 인한 경제적 손실도 16조원에 달한다고 발표하였다. 또한 우리나라 성인 중 알코올 중독자 비율이 21.6%로 세계 최고 수준이며, 성인 5명 중 1명은 알코올 중독자로 심한 국민병에 다다르고 있다.Currently, the cause of mortality caused by disease in the country, the rank of death from liver disease is ranked 4th with 22.9 people per 100,000 population (as of 2001), and the liver disease due to alcohol consumption can not be ignored. According to the World Health Organization's alcohol consumption survey in 153 countries in 1996, Korea's adult alcohol consumption per capita was 14,4ℓ per year, ranking 2nd in the world, and 23,000 people died of accidents and diseases caused by alcohol every year. It also announced that the economic loss amounted to 16 trillion won. In addition, the proportion of alcoholics among Korean adults is 21.6%, the highest in the world, and one out of five adults is suffering from severe national illness as an alcoholic.

알코올성 간질환은 지속적이고 과다한 음주에 의해서 유발되는 간세포 손상을 동반하는 급/만성 간질환으로, 간에는 알코올을 분해하는 효소들이 존재하여 알코올을 알데하이드를 거쳐 분해하는데, 상기 아세트알데하이드가 독성을 나타내어 간세포에 손상을 입히게 된다. Alcoholic liver disease is acute / chronic liver disease with hepatocellular damage caused by persistent and excessive drinking. In the liver, there are enzymes that break down alcohol and decompose alcohol through aldehydes. Acetaldehyde is toxic to liver cells. It will be damaged.

이러한 알코올성 간질환은 크게 알코올성 지방간, 알코올성 간염, 알코올성 간경변으로 분류되는데 간 손상 기전은 알코올 자체에 의한 경우, 아세트알데하이드와 같은 대사산물에 의한 경우, 면역반응에 의한 경우 등이 있으며, 특히 아세트알데하이드는 지방의 페록시데이션(Peroxidation), 세포질과의 결합, 미토콘드리아의 전자전달계 교란, 마이크로터블(Microtuble)의 기능방해, 단백질과 결합물질 형성, 콜라겐 합성의 증가 등 간 독성의 주범으로 작용한다.These alcoholic liver diseases are classified into alcoholic fatty liver, alcoholic hepatitis, and alcoholic cirrhosis. The mechanism of liver damage may be due to alcohol itself, metabolites such as acetaldehyde, or immune response. Peroxidation of fat, binding to cytoplasm, disruption of electron transport systems in mitochondria, disruption of microtuble, formation of proteins and binding substances, increased collagen synthesis, etc.

알코올은 위에서 20% 정도가 흡수되고 나머지는 대부분 소장에서 흡수되어 이중 80~90%가 간에서 대사되므로 간에 의한 알코올 대사 중 많은 부분을 소장 및 대장에서 처리할 수 있다면 간의 부담을 덜 수 있고, 나아가 간 손상을 억제할 수 있을 것이다. Alcohol is absorbed by about 20% in the stomach and most of the rest is absorbed in the small intestine, 80-90% of the metabolism in the liver, so much of the liver's alcohol metabolism can be treated in the small and large intestine, less burden on the liver, further It may be able to suppress liver damage.

그러므로, 알코올은 체내에 저장되지 못하기 때문에 대사가 되어야 하는데, 이 과정은 대부분 간에서 이루어지며 이러한 대사는 간에 존재하는 알코올분해 효소들에 의해 이루어진다. 알코올은 이러한 효소들에 의해 아세트알데하이드란 물질을 거쳐 분해되는데, 상기 아세트알데하이드는 독성이 있어 간세포에 손상을 주게 된다. 그리고, 알코올 대사 결과 지방산이 많이 만들어져 간에 지방이 축적되는데 이를 알코올성 지방간이라고 한다. 상기 알코올성 지방간은 만성 간질환으로 진행되는 경우가 많은데, 알코올성 간염이 10~35%에서 간경변증이 8~20%에서 발생된다고 한다.Therefore, because alcohol is not stored in the body, it must be metabolized, which is mostly done in the liver, and this metabolism is done by alcohol-degrading enzymes present in the liver. Alcohol is broken down through acetaldehyde by these enzymes, which are toxic and damage liver cells. And, as a result of alcohol metabolism, many fatty acids are made and fat is accumulated in the liver, which is called alcoholic fatty liver. The alcoholic fatty liver is often progressed to chronic liver disease, alcoholic hepatitis is said to occur in 10 to 35% cirrhosis in 8 to 20%.

최근 장내 유익균으로 각광을 받고 있는 프로바이오틱스(Probiotics)는 질병의 예방 및 치료에 다양하게 적용되고 있으며, 특히 장내 부패균과 병원균에 대한 증식과 부패활동을 억제하는 효과가 있다고 널리 알려져 있다(Schrezenmeir J, De Vrese M: Am J Clin Nutr. 73(2001), P 361S-4S). 프로바이오틱스는 암모니아나 아민 등을 생성하는 유해균의 증식을 억제하여 중독성 물질의 생성량을 억제시킴으로서 혈중 암모니아 및 알파 아미노 질소량이 감소하여 간 질환자의 증상을 개선시킨다는 보고도 발표되었다. 또한 이러한 유용한 균들 중에 알코올로부터 간을 보호하고 간의 특이 기능을 향상시켜 준다는 결과가 보고되었다(Raibaud, P: The 3rd International Synposium on Lactic Acid Bacteria and Human Health. (1983), P116-126). 최근에 프로바이오틱스 락토바실러스(Probiotics lactobacillus)와 비피도박테리움(Bifidobacterium)과 같은 유용한 유산균을 이용하여 알코올 대사를 활발하게 촉진시켜 알코올을 신속하게 분해하고 이러한 과정에서 생성되는 인체에 유해한 아세트알데하이드를 대사하여 무독화시키는 가능성의 연구 결과가 보고되었다(Nosava. T. et al: Alcohol and Alcoholism. 35(2000), P561-568).Recently, probiotics, which have been in the spotlight as enteric beneficial bacteria, have been widely applied to the prevention and treatment of diseases. In particular, it is widely known that they have the effect of inhibiting proliferation and decay activity against intestinal rot and pathogens (Schrezenmeir J, De Vrese M: Am J Clin Nutr. 73 (2001), P 361S-4S). Probiotics have been reported to reduce the amount of toxic substances by inhibiting the growth of harmful bacteria that produce ammonia and amines, thereby reducing the amount of ammonia and alpha amino nitrogen in the blood and improving symptoms of liver disease. In addition, it has been reported to protect the liver from alcohol and improve the specific function of the liver among these useful bacteria (Raibaud, P: The 3rd International Synposium on Lactic Acid Bacteria and Human Health. (1983), P116-126). Recently, useful lactic acid bacteria such as Probiotics lactobacillus and Bifidobacterium actively promote alcohol metabolism to rapidly break down alcohol and metabolize acetaldehyde harmful to human A study of the possibility of detoxification has been reported (Nosava. T. et al: Alcohol and Alcoholism. 35 (2000), P561-568).

한국특허 제0543115호는 한국 성인의 분변에서 분리한 것으로써, 에탄올과 아세트알데하이드 분해효소 활성과 감소능이 우수한 젖산균인 락토바실러스 브레비스 HY7401 균주에 대해 개시하고 있고, 본 출원인이 출원하여 등록받은 한국특허 제0769299호에서 알코올에 대한 내성을 가지며, 알코올 분해효소를 가지고 있어 알코올을 분해할 수 있는 능력을 보유한 락토바실러스 퍼멘텀 KCTC 10713BP를 개시하고 있다.Korean Patent No. 0543115 discloses a strain of Lactobacillus brevis HY7401, which is isolated from feces of Korean adults and which is excellent in ethanol and acetaldehyde degrading enzyme activity and reducing ability, and has been filed and registered by the present applicant. 0769299 discloses Lactobacillus permanent KCTC 10713BP, which is resistant to alcohol and possesses an alcohol degrading enzyme and thus the ability to degrade alcohol.

그러나 상기 균주들은 알코올 분해 기능성 유제품 또는 건강기능식품의 제조에 적합한 알코올 분해능을 달성할 정도로 충분한 알코올 디하이드로게나아제 활성이 뛰어나지 않았고, 또한 알코올 디하이드로게나아제 활성과 더불어 알코올 분해 생성물로서 숙취와 급성 간독성의 원인이 되는 아세트알데하이드 디하이드로게나아제의 활성이 높지 않은 한계가 있었다.
However, these strains did not have sufficient alcohol dehydrogenase activity to achieve alcohol degradability suitable for the manufacture of alcohol-degradable dairy products or health foods, and also with alcohol dehydrogenase activity as hangover and acute hepatotoxicity. There was a limit in which the activity of acetaldehyde dehydrogenase, which is a cause of, was not high.

본 발명은 알코올 디하이드로게나아제의 활성이 높아 알코올 분해능이 우수하면서, 동시에 아세트알데하이드 디하이드로게나아제의 활성이 높아 숙취해소 활성이 뛰어나며, 내산성 및 내담즙성이 우수하고, gas forming이 약해서 유제품에서 저장 중 품질과 맛에 영향이 적은 락토바실러스 브레비스를 제공하는 것을 목적으로 한다.
The present invention has a high alcohol dehydrogenase activity and excellent alcohol decomposing ability, and at the same time, acetaldehyde dehydrogenase activity has high hangover relieving activity, excellent acid resistance and bile resistance, and gas forming is weak in dairy products. It is an object to provide Lactobacillus brevis with little impact on quality and taste during storage.

본 발명은 알코올 분해능이 우수한 락토바실러스 브레비스(Lactobacillus brevis) HD-01[기탁번호: KACC91701P]을 제공한다.The present invention provides Lactobacillus brevis HD-01 [Accession Number: KACC91701P] excellent in alcohol resolution.

본 발명의 락토바실러스 브레비스(Lactobacillus brevis) HD-01[기탁번호: KACC91701P]은 알코올 디하이드로게나아제 활성 및 아세트알데하이드 디하이드로게나아제의 활성이 동시에 우수한 것을 특징으로 한다. Lactobacillus brevis of the present invention brevis ) HD-01 [Accession No .: KACC91701P] is characterized by superior alcohol dehydrogenase activity and acetaldehyde dehydrogenase activity at the same time.

본 발명은 알코올 분해능이 우수한 락토바실러스 브레비스(Lactobacillus brevis) HD-01[기탁번호: KACC91701P]을 함유하는 유제품을 제공한다.The present invention provides a dairy product containing Lactobacillus brevis HD-01 [Accession No. KACC91701P] excellent in alcohol resolution.

본 발명의 유제품은 발효유, 버터, 치즈 또는 분유인 것을 특징으로 한다.The dairy product of the present invention is characterized in that the fermented milk, butter, cheese or milk powder.

본 발명은 알코올 분해능이 우수한 락토바실러스 브레비스(Lactobacillus brevis) HD-01[기탁번호: KACC91701P]을 함유하는 알코올성 간질환 예방용 또는 숙취해소용 건강기능식품을 제공한다.The present invention provides a dietary supplement for preventing or hangover of alcoholic liver disease containing Lactobacillus brevis HD-01 [Accession Number: KACC91701P] excellent in alcohol resolution.

본 발명의 숙취해소용 건강기능식품은 분말, 과립, 정제 또는 캅셀 형태인 것을 특징으로 한다.Hangover health functional food of the present invention is characterized in that the powder, granules, tablets or capsule form.

본 발명은 알코올 분해능이 우수한 락토바실러스 브레비스(Lactobacillus brevis) HD-01[기탁번호: KACC91701P]을 함유하는 식품첨가제를 제공한다.
The present invention provides a food additive containing Lactobacillus brevis HD-01 [Accession Number: KACC91701P] excellent in alcohol resolution.

본 발명의 락토바실러스 브레비스(Lactobacillus brevis) HD-01[기탁번호: KACC91701P]은 알코올 디하이드로게나아제의 활성이 높아 알코올 분해능이 우수하면서, 동시에 아세트알데하이드 디하이드로게나아제의 활성이 높아 숙취해소 활성이 뛰어나며, 내산성 및 내담즙성이 우수하기 때문에, 알코올성 간질환을 예방하거나 숙취해소를 위한 유제품, 건강기능식품 또는 식품첨가물로서 이용될 수 있다.
Lactobacillus brevis of the present invention brevis ) HD-01 [Accession No .: KACC91701P] has high alcohol dehydrogenase activity and excellent alcohol decomposability, and at the same time, acetaldehyde dehydrogenase has high activity to relieve hangover, and is resistant to acid and bile. Since it is excellent, it can be used as a dairy product, dietary supplement or food additive to prevent alcoholic liver disease or to relieve hangover.

도 1은 실험예 6에서 본 발명의 락토바실러스 브레비스 HD-01를 섭취시킨 군의 알코올 섭취 후 분해 효과를 확인한 것으로, 시간에 따른 혈중 알코올 농도 변화를 나타낸 그래프이다.1 is a graph showing the degradation effect after the alcohol intake of the group ingested Lactobacillus brevis HD-01 of the present invention in Experimental Example 6, it is a graph showing the change in blood alcohol concentration over time.

본 발명의 락토바실러스 브레비스 HD-01은 김치로부터 분리되어 알코올 디하이드로게나아제의 활성이 높아 알코올 분해능이 우수하면서, 동시에 아세트알데하이드 디하이드로게나아제의 활성이 높아 숙취해소 활성이 뛰어나며, 내산성 및 내담즙성이 우수하다.The lactobacillus brevis HD-01 of the present invention is separated from kimchi and has high alcohol dehydrogenase activity, and thus has high alcohol decomposability, and at the same time, high acetaldehyde dehydrogenase activity, which is excellent in hangover resolution, acid resistance and bile resistance. Excellent in sex

또한 본 발명의 락토바실러스 브레비스HD-01은 탈지유에 배양했을 때 유산균의 증식이 활발하고, 가스를 생성하지 않으면서, pH, 산도, 커드 생성에 영향을 주지 않기 때문에 유제품, 특히 발효유에 첨가되었을 때 알코올 분해능과 숙취해소능과 같은 기능성을 부여하면서도 제품의 물성이나 보존기간에 거의 영향을 주지 않는다.In addition, when the Lactobacillus brevis HD-01 of the present invention is cultured in skim milk, the proliferation of lactic acid bacteria is active, and when it is added to dairy products, especially fermented milk, because it does not affect pH, acidity and curd production without generating gas. Functionality such as alcohol degradability and hangover ability is imparted but little effect on product properties or shelf life.

본 발명의 락토바실러스 브레비스 HD-01은 알코올성 간질환 예방용 또는 숙취해소용 유제품, 건강기능식품, 식품첨가제의 유효성분으로 사용될 수 있다.Lactobacillus brevis HD-01 of the present invention can be used as an active ingredient in dairy products, health functional foods, food additives for preventing or hangover alcoholic liver disease.

본 발명의 락토바실러스 브레비스 HD-01은 생균, 파쇄된 세포벽 분획, 사균, 건조균 또는 배양물을 유효성분으로 포함할 수 있으며, 부형제 또는 담체를 추가로 포함할 수 있다. 상기 배양물은 액체 배지에서 배양한 배양액 자체, 상기 배양액을 여과 또는 원심분리하여 균주를 제거한 여액(원심분리한 상등액)등을 포함한다. The Lactobacillus brevis HD-01 of the present invention may include live bacteria, crushed cell wall fractions, dead bacteria, dried bacteria or cultures as an active ingredient, and may further include an excipient or a carrier. The culture includes a culture solution itself cultured in a liquid medium, a filtrate (centrifuged supernatant) from which the strain is removed by filtration or centrifugation of the culture solution.

본 발명에 따른 유제품은 락토바실러스 브레비스HD-01를 포함하는 발효유, 버터, 치즈 또는 분유이고, 건강기능식품은 락토바실러스 브레비스HD-01를 포함하는 분말, 과립, 정제 또는 캅셀 형태인 것이며, 식품첨가제는 다양한 식품 예를 들어, 음료류, 과자류, 다이어트바, 초코렛, 피자, 라면, 기타 면류, 껌류, 아이스크림류 등이다.The dairy product according to the present invention is fermented milk, butter, cheese or milk powder containing Lactobacillus brevis HD-01, and the health functional food is in the form of powder, granules, tablets or capsules containing Lactobacillus brevis HD-01, and a food additive Is a variety of foods, for example, beverages, sweets, diet bars, chocolate, pizza, ramen, other noodles, gums, ice cream and the like.

상기 유제품, 건강기능식품 또는 식품첨가제를 제조하기 위하여 유효성분으로 락토바실러스 브레비스 HD-01 뿐만 아니라, 식품 제조 시에 통상적으로 첨가되는 성분을 포함할 수 있으며, 예를 들어, 단백질, 탄수화물, 지방, 영양소, 조미제 및 향미제를 포함한다. In order to prepare the dairy products, dietary supplements or food additives, the active ingredient may include not only Lactobacillus brevis HD-01, but also commonly added ingredients in food preparation, for example, proteins, carbohydrates, fats, Nutrients, seasonings and flavoring agents.

식품제조에 사용되는 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스, 올리고당 등; 및 폴리사카라이드, 예를 들어 덱스트린, 사이클로덱스트린 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 또한 향미제로서 천연 향미제 [타우마틴, 스테비아 추출물 (예를 들어 레바우디오시드 A, 글리시르히진 등]) 및 합성 향미제(사카린, 아스파르탐 등)를 사용할 수 있다. Examples of carbohydrates used in food production include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. As flavoring agents, natural flavoring agents [tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.

건강기능식품이란, 섭취할 경우 건강상 특정한 효과를 가져오는 것을 의미하나, 일반 약품과는 달리 식품을 원료로 하여 약품의 장기 복용시 발생할 수 있는 부작용 등이 없는 장점이 있다. 이와 같이 하여 얻어지는 본 발명의 건강기능식품은, 일상적으로 섭취하는 것이 가능하기 때문에 매우 유용하다. Healthy functional food means that it brings about a certain effect on health when consumed, but unlike general medicine, there is an advantage that there is no side effect that can occur when a medicine is taken for a long time by using food as a raw material. The health functional food of the present invention thus obtained is very useful because it can be ingested routinely.

건강기능식품을 분말, 과립, 정제 또는 캅셀 형태로 제형화 할 경우, 식품제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다. 상기 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 상기 부형제 이외에 마그네슘 스티레이트, 탈크 같은 윤활제들도 사용된다. When formulating the health functional food in the form of powder, granules, tablets or capsules, it may further comprise suitable carriers, excipients and diluents commonly used in food preparation. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. In addition to the above excipients, lubricants such as magnesium styrate and talc are also used.

상기 유제품, 건강기능식품, 식품첨가제에 있어서 락토바실러스 브레비스 HD-01의 첨가량은, 유제품, 건강기능식품, 식품첨가제에 대하여 통상 0.01 내지 50 중량%, 바람직하기로는 0.1 내지 20 중량%의 범위이고, 최종 소비되는 제형을 기준으로 1×105 내지 1×109 CFU/g, 바람직하게는 1×105 내지 1×108 CFU/g이다. The amount of Lactobacillus brevis HD-01 added in the dairy products, health functional foods and food additives is usually in the range of 0.01 to 50% by weight, preferably 0.1 to 20% by weight relative to dairy products, health functional foods and food additives. 1 × 10 5 to 1 × 10 9 CFU / g, preferably 1 × 10 5 to 1 × 10 8 CFU / g, based on the final consumed formulation.

상기 유제품, 건강기능식품, 식품첨가제를 통한 본 발명의 락토바실러스 브레비스HD-01의 1일 섭취량은 1×105 내지 1×108 CFU/kg, 바람직하게는 1×105 내지 1×107 CFU/kg이고, 섭취는 하루에 한 번 섭취할 수도 있고, 수회 나누어 섭취할 수도 있다.
The daily intake of Lactobacillus brevis HD-01 of the present invention through the dairy products, health functional foods, food additives is 1 × 10 5 to 1 × 10 8 CFU / kg, preferably 1 × 10 5 to 1 × 10 7 CFU / kg, intake can be taken once a day or divided several times.

이하, 바람직한 실시예를 들어 본 발명을 더욱 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이에 의하여 제한되지 않는다는 것은 당업계의 통상의 지식을 가진 자에게 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. It will be apparent, however, to those skilled in the art that these embodiments are for further explanation of the present invention and that the scope of the present invention is not limited thereby.

실험예Experimental Example 1: 김치에서 유산균의 분리 1: Isolation of Lactic Acid Bacteria from Kimchi

가정에서 수득된 13가지의 김치를 통해 유산균을 분리하였다. 유산균을 분리하기 위해 각 김치를 생리식염수에 현탁시켰고, 그 현탁액을 MRS agar 배지에 도말하여 37℃에서 48시간 동안 배양하였다. MRS agar 위에 형성된 유산균을 morphology가 상이한 것 위주로 선발하여 MRS broth에 계대 하여 37℃에서 24시간 동안 배양하였다. 분리된 균주들 중에서 배양 중 가스 형성이 이루어지는 것을 위주로 49 종을 선발하였고, 선발된 균주들은 BCP agar를 통해 모두 유산을 생성하는 균주라는 것을 확인하였다.
Lactic acid bacteria were isolated through 13 kinds of kimchi obtained at home. To separate the lactic acid bacteria, each kimchi was suspended in saline, and the suspension was plated in MRS agar medium and incubated at 37 ° C. for 48 hours. The lactic acid bacteria formed on the MRS agar were selected based on the different morphology and passaged at 37 ° C. for 24 hours at MRS broth. Among the isolated strains, 49 species were selected based on the formation of gas during cultivation, and the selected strains were confirmed to be all strains that generate lactic acid through BCP agar.

실험예Experimental Example 2: 유산균의 알코올 분해능 측정 2: Determination of Alcohol Resolution of Lactic Acid Bacteria

실험예 1에서 선발한 49 종의 유산균과 매일유업에서 보유하고 있는 65 종의 유산균, 총 114 종의 유산균에 대하여 다음의 방법으로 알코올 분해능을 측정하였다.For 49 kinds of lactic acid bacteria selected in Experimental Example 1, 65 kinds of lactic acid bacteria possessed by Maeil Dairy, and a total of 114 kinds of lactic acid bacteria, alcohol resolution was measured by the following method.

1. 유산균의 조효소액 제조1. Preparation of coenzyme solution of lactic acid bacteria

균주를 MRS broth 배지에 37℃에서 24시간 동안 배양시킨 후, 균체를 회수하여 멸균된 100mM potassium phosphate buffer (PBS, pH 7.4)로 washing하였다. 회수된 균체는 100mM potassium phosphate buffer에 현탁하여 McFarland No. 3 정도의 수준으로 맞춰주었다. 준비된 균현탁액을 과열되지 않도록 ice에 천천히 냉각시키면서 30초씩 5회 가량 정도 sonification을 시켜주었다. 이 때 얻어진 세포파쇄액을 13,000rpm으로 1시간 동안 원심 분리시킨다. 상등액을 취하여 조효소액을 분리하고, -80℃인 초저온 냉동고에 저장시키며 사용하였다.
After culturing the strain in MRS broth medium at 37 ℃ for 24 hours, the cells were recovered and washed with sterile 100mM potassium phosphate buffer (PBS, pH 7.4). The recovered cells were suspended in 100 mM potassium phosphate buffer and McFarland No. 3 levels were set. The solubilization of the prepared bacteria suspension was performed 5 times for 30 seconds while slowly cooling on ice to prevent overheating. The cell lysate obtained at this time is centrifuged at 13,000 rpm for 1 hour. The supernatant was taken and the crude enzyme solution was separated and used in a cryogenic freezer at -80 ° C.

2. 알코올 디하이드로게나아제(ADH) 활성 측정2. Determination of Alcohol Dehydrogenase (ADH) Activity

각 유산균의 알코올 분해능을 알아보기 위해 조효소액을 이용하여 ADH 활성을 측정하였다. 2.5mM NAD+를 함유시킨 0.1M Glycine buffer(pH 9.6)에 에탄올을 첨가시킨 용액 1ml에 각 유산균의 조효소액 100㎕를 water bath에서25℃, 5분간 반응시켰다. 반응이 완료된 후, 반응시킨 용액 내의 생성된 NADH 함량을 340nm, 25℃에서 흡광도를 측정하였다. 조효소액의 단백질 함량은 단백질 정량 키트(Bio-Rad)를 통해 측정되었다. 측정된 결과치를 통하여 조효소액의 단백질 mg당 반응시간 동안 생성된 NADH량을 통해 ADH의 활성을 비교하였다. In order to determine the alcohol degradability of each lactic acid bacteria, the ADH activity was measured using the coenzyme solution. To 1 ml of a solution in which ethanol was added to 0.1 M Glycine buffer (pH 9.6) containing 2.5 mM NAD + , 100 μl of the coenzyme solution of each lactic acid bacterium was reacted for 5 minutes at 25 ° C. in a water bath. After the reaction was completed, the absorbance was measured at 340 nm, 25 ° C. in the resulting NADH content in the reacted solution. Protein content of the coenzyme solution was measured through a protein quantification kit (Bio-Rad). The measured results were used to compare the activity of ADH through the amount of NADH produced during the reaction time per mg of protein in the crude enzyme solution.

상기 114종의 균주의 ADH 활성을 측정한 결과 중에서 ADH 활성이 우수한 8 종의 유산균의 분리원 및 학명을 ADH 활성이 높은 순서대로 표 1에 나타내고, 각 유산균의 ADH 활성값이 높은 순서대로 표 2에 나타내었다.Among the results of measuring the ADH activity of the 114 strains, the isolates and scientific names of the 8 lactic acid bacteria having excellent ADH activity are shown in Table 1 in the order of high ADH activity, and the order of high ADH activity values of each lactic acid bacterium is shown in Table 2 Shown in

균주번호Strain number 균주명Strain name 학명Scientific name 분리원Separation source 1One 김치분리유산균 11Kimchi isolated lactic acid bacteria 11 LactobacillusLactobacillus brevisbrevis 김치Kimchi 22 김치분리유산균 24Kimchi Isolated Lactobacillus 24 LactobacillusLactobacillus brevisbrevis 김치Kimchi 33 발효유 분리 균주 1Fermented milk isolated strain 1 LactobacillusLactobacillus brevisbrevis 국내 발효유Domestic fermented milk 44 분리균주 019Isolated strain 019 LactobacillusLactobacillus fermentumfermentum 유아분변Infant feces 55 락토바실러스 퍼멘텀 KCTC 10713BPLactobacillus Percentum KCTC 10713BP LactobacillusLactobacillus fermentumfermentum 자사기탁균주Privately Owned Strains 66 락토바실러스 루테리 LC40Lactobacillus ruteri LC40 LactobacillusLactobacillus reuterireuteri 상용균주Commercial strain 77 김치분리유산균 30Kimchi Isolated Lactobacillus 30 LactobacillusLactobacillus collinoidescollinoides 김치Kimchi 88 분리균주 D4-1Separated strain D4-1 LactobacillusLactobacillus fermentumfermentum 자사보유균주Owned strains

균주번호Strain number ADH활성(NADH μM/min/mg protein)ADH activity (NADH μM / min / mg protein) 1One 297297 22 282282 33 209209 44 159159 55 132132 66 8686 77 8585 88 5858

대체로 상용균주보다 분리균주에서 ADH 활성값이 높게 나타났고, ADH 활성값은 김치분리유산균 11, 김치분리유산균 24, 발효유 분리 균주 1, 분리균주 019 순이었다. 상용균주 중에서 ADH활성이 가장 높았던 발효유 분리 균주 1(3번 균주)에 비하여 김치분리유산균 11(1번 균주) 및 24(2번 균주)는 각각 42% 및 35% 높은 ADH 활성을 나타내었다.
In general, ADH activity values were higher in isolates than commercial strains, followed by kimchi lactic acid bacteria 11, kimchi lactic acid bacteria 24, fermented milk strain 1, and strain 019. Kimchi isolated lactic acid bacteria 11 (strain 1) and 24 (strain 2) showed 42% and 35% higher ADH activities than fermented milk isolated strain 1 (strain 3), which had the highest ADH activity among commercial strains.

실험예Experimental Example 3: 아세트알데하이드 분해능 측정 3: Determination of acetaldehyde resolution

알코올이 아세트알데하이드로 분해되더라도 아세트알데하이드가 분해되지 못하고 축적되면 급성 간독성을 일으킬 수 있고, 이는 숙취의 원인이 될 수 있으므로, 알코올의 분해와 동시에 아세트알데하이드의 분해가 신속히 이루어지는 것이 중요하므로, 실험예 2의 알코올 분해능이 높았던 8 종의 균주의 아세트알데하이드 분해능을 확인하기 위하여 실험예 2의 유산균 조효소액을 이용하여 ALDH활성을 측정하였다. Even if the alcohol is decomposed into acetaldehyde, acetaldehyde cannot be decomposed and accumulated, which may cause acute hepatotoxicity, which may cause a hangover. Therefore, it is important to rapidly decompose the acetaldehyde simultaneously with the decomposition of the alcohol, Experimental Example 2 ALDH activity was measured using the lactic acid bacteria coenzyme solution of Experimental Example 2 in order to confirm the acetaldehyde degradability of the eight strains with high alcohol degradability.

1mM NADP+가 함유된 60mM Sodium pyrophosphate buffer(pH 8.8)에 최종 농도가 100μM이 되도록 acetaldehyde와 4-methylpyrazole을 첨가시켰다(4-methylpyrazole은 ADH의 inhibitor). 위의 혼합액 1ml과 각 유산균 조효소액 100㎕를 혼합하여 water bath에서 25℃, 5분간 반응시켰다. 반응이 완료된 후, 반응시킨 용액 내의 생성된 NADPH 함량을 340nm, 25℃에서 흡광도를 측정하였다. 조효소액의 단백질 함량은 단백질 정량 키트(Bio-Rad)를 통해 측정되었다. 측정된 결과치를 통하여 조효소액의 단백질 mg당 반응시간 동안 생성된 NADPH량을 통해 ALDH의 활성을 비교하여 표 3에 나타내었다.Acetaldehyde and 4-methylpyrazole were added to 60 mM Sodium pyrophosphate buffer (pH 8.8) containing 1 mM NADP + to a final concentration of 100 μM (4-methylpyrazole is an inhibitor of ADH). 1 ml of the mixed solution and 100 μl of each lactic acid coenzyme solution were mixed and reacted at 25 ° C. for 5 minutes in a water bath. After the reaction was completed, the resulting NADPH content in the reacted solution was measured for absorbance at 340nm, 25 ℃. Protein content of the coenzyme solution was measured through a protein quantification kit (Bio-Rad). The measured results are shown in Table 3 comparing the activity of ALDH through the amount of NADPH produced during the reaction time per mg of protein of the crude enzyme solution.

균주번호Strain number ALDH활성(NADPH μM/min/mg protein)ALDH activity (NADPH μM / min / mg protein) 1One 6060 22 2727 33 1717 44 1515 55 2626 66 1515 77 66 88 3434

실험예Experimental Example 4:  4: 내산성Acid resistance  And 내담즙성My bile 측정 Measure

상기 실험예 2 및 3에서 ADH 활성 및 ALDH 활성이 동시에 가장 우수했던 김치분리유산균 11(1번 균주)이 상용균주와 비교했을 때 내산성 및 내담즙성을 확보하고 있는 지를 확인하는 실험을 실시하였다.In Experimental Examples 2 and 3, the kimchi isolated lactic acid bacteria 11 (strain No. 1), which had the best ADH activity and ALDH activity at the same time, were compared with the commercial strain to confirm whether the acid resistance and the bile resistance were secured.

내산성 실험은 50mM, phosphate buffer를 만들어 1N HCL 용액으로 pH 3.0으로 맞추어 멸균한 용액을 사용하였다. MRS broth 배지에 24시간 배양시킨 균을 내산용액에 1% 접종하여 초기 접종 수와 2시간 동안 37℃에 배양한 생균수를 측정하여 표 4에 나타내었다. 측정된 생균수는 LogCFU/ml로 나타내어 그 차이가 적은 것이 내산성이 있는 균주로 판단하였다.For acid resistance experiment, 50mM, a phosphate buffer was prepared, and the sterilized solution was adjusted to pH 3.0 with 1N HCL solution. Inoculated with 1% of the bacteria incubated in MRS broth medium for 24 hours in the acid solution was measured in the initial inoculation and the number of viable cells incubated at 37 ℃ for 2 hours is shown in Table 4. The measured number of live bacteria was expressed as LogCFU / ml, and it was judged that the difference was less acid resistant.

균주번호
Strain number
Log(CFU/ml)Log (CFU / ml)
0시간0 hours 2시간2 hours 차이Difference 1One 8.168.16 7.087.08 1.081.08 33 8.108.10 6.966.96 1.141.14 55 8.248.24 7.137.13 1.111.11 66 8.458.45 7.237.23 1.211.21

김치분리유산균 11(1번 균주)은 상용균주와 비교했을 때 이들보다 우수한 내산성을 지니고 있음을 확인하였다.Kimchi isolated lactic acid bacteria 11 (strain No. 1) was confirmed to have better acid resistance than those compared to the commercial strain.

또한 내담즙성 실험은 50mM phosphate buffer(pH 7)에 Oxgall(Difco)을 1% 첨가한 용액을 내담즙성 테스트용 배지로 사용하였다. MRS broth 배지에 24시간 배양시킨 균을 내담즙 배지에 1% 접종하여 접종하여 초기 접종 수와 6시간 동안 37℃에 배양한 생균수를 측정하여 표 5에 나타내었다. 측정된 생균수는 LogCFU/ml로 나타내어 그 차이가 적은 것이 내담즙성이 있는 균주로 판단하였다.In addition, in the test of the bile resistance, a solution in which 1% of Oxgall (Difco) was added to 50mM phosphate buffer (pH 7) was used as a medium for the test for bile resistance. Inoculated by inoculating 1% in the biliary medium cultured bacteria in MRS broth medium for 24 hours to measure the initial number of inoculation and the number of viable cells incubated at 37 ℃ for 6 hours is shown in Table 5. The measured viable cell number was expressed as LogCFU / ml, and it was judged that the difference was small and the bile-resistant strain.

균주번호
Strain number
Log(CFU/ml)Log (CFU / ml)
0시간0 hours 6시간6 hours 차이Difference 1One 8.198.19 7.157.15 1.041.04 33 8.198.19 6.936.93 1.261.26 55 8.318.31 7.227.22 1.091.09 66 8.428.42 6.976.97 1.451.45

김치분리유산균 11(1번 균주)은 상용균주와 비교했을 때 이들보다 우수한 내담즙성을 지니고 있음을 확인하였다.
Kimchi isolated lactic acid bacteria 11 (strain No. 1) was found to have superior bile resistance than those compared to commercial strains.

실험예Experimental Example 5: 선별된 유산균의 동정 5: Identification of Selected Lactic Acid Bacteria

상기 실험예 1 내지 4를 통해 선발된 김치분리유산균 11(1번 균주)을 단일 콜로니로 분리한 후, 버지스의 분류 세균학 매뉴얼(Bergy's manual of systematic bacteriology)에 준하여 형태학적 및 생화학적 특성을 조사하였고, 그람 염색을 수행하였다. 그 결과, 상기 분리된 균주는 그람 양성 균주이며 간균의 형태를 갖고 있음을 확인할 수 있었다.After separating the kimchi lactic acid bacteria 11 (strain No. 1) selected through Experimental Examples 1 to 4 into a single colony, the morphological and biochemical characteristics were investigated in accordance with Burgess's manual of systematic bacteriology. Gram staining was performed. As a result, it was confirmed that the isolated strain is a Gram-positive strain and has a form of bacilli.

또한 상기 균주는 API 시스템(API system; La Balme-les-Grottes, France)으로 동정하였다. 우선, 멸균 백금이로 균 콜로니를 각각 취한 후 멸균 증류수 2 ㎖에 부유시켰다. 다시 상기 균액을 멸균 증류수 5 ㎖에 API 50CH 키트(BioMerieux, France)에서 제공되는 맥팔란드 표준 용액 No.2(MccFaland Standard Solution No.2)의 농도로 균액을 부유시켰다. 상기 부유액을 API 50CH 키트의 액체배지에 첨가하여 균질화시킨 후, API 50CH 키트의 50개 각 튜브에 200 ㎕씩 접종하였다. 미네랄 오일로 튜브 위를 엎어준 후, 30 ℃에서 48 시간 동안 배양시켰다. 상기 배양액을 API 시스템으로 분석하여 49종의 탄수화물 발효패턴을 확인한 후, 이 결과를 ATB 동정 컴퓨터 시스템에 입력하여 동정하였다.In addition, the strain was identified by the API system (API) (La Balme-les-Grottes, France). First, the sterilized platinum microbial colonies were each taken and suspended in 2 ml of sterile distilled water. Again, the bacteria solution was suspended in 5 ml of sterile distilled water at a concentration of McFalland Standard Solution No. 2 (MccFaland Standard Solution No. 2) provided by the API 50CH kit (BioMerieux, France). The supernatant was added to the liquid medium of the API 50CH kit and homogenized, and 200 μl of each of the 50 tubes of the API 50CH kit was inoculated. The tube was overlaid with mineral oil and then cultured at 30 DEG C for 48 hours. The culture solution was analyzed by an API system to identify 49 types of carbohydrate fermentation patterns, and the results were entered into an ATB identification computer system.

그 결과, 상기 김치분리유산균 11은 하기 표 6의 탄수화물 발효패턴을 갖고 있는 락토바실러스 속에 속하는 균주인 것으로 판명되었다.As a result, the kimchi isolated lactic acid bacteria 11 was found to be a strain belonging to the genus Lactobacillus having a carbohydrate fermentation pattern of Table 6.

대조군Control group -- D-만노즈D-Manno -- 살리신Salincin -- 겐티오비오즈Gentio Vioz -- 글리세롤Glycerol -- L-솔보즈L-Solbos -- 셀로비즈Cellobiz -- D-튜라노즈D-Turanz __ 에리쓰리톨Erythritol -- 람노즈Rannoz -- 말토즈Malts ++ D-리조즈D-rings -- D-아라비노즈D-arabinose -- 둘시톨Dissytol -- 락토즈Laktos -- D-타가토즈D-Tagatoz -- L-아라비노즈L-arabinose ++ 이노시톨Inositol -- 멜리비오즈Melibiose ++ D-퓨코즈D-fucose -- 리보오즈Riboze ++ 만니톨Mannitol -- 사카로즈Saka Rose -- L-퓨코즈L-fucose -- D-자일로즈D-xylose ++ 솔비톨Sorbitol -- 트레할로즈Trehalose -- D-아라비톨D-arabitol -- L-자일로즈L-xylose -- D-만노시드D-mannoside -- 이눌린Inulin -- L-아라비톨L-arabitol -- 아도니톨Adonitol -- D-글루코시드D-glucoside ++ 멜레지토즈Melrietto -- 글루코네이트Gluconate ++ 자일로시드Xyloside -- 글루코사민Glucosamine -- D-라피노즈D-Rapinoz -- 2-세토-글루코네이트2-Setto-gluconate -- 갈락토즈Galactos ++ 아미그달린Amigalline -- 아미돈Amidon -- 5-세토-글루코네이트5-Seto-gluconate ++ D-글루코즈D-glucose ++ 알부틴Arbutin -- 글리코겐Glycogen -- D-프록토즈D-Prostose ++ 에스큘린Esculin ++ 자일리톨Xylitol --

또한 당업계에 공지된 통상적인 방법에 따라 상기 김치분리유산균 11의 16S rDNA의 염기서열을 분석하였다. 그 결과, 상기 김치분리유산균 11의 16S rDNA 염기서열(서열번호 1)은 락토바실러스 브레비스의 16S rDNA 염기서열과 99 % 동일함을 알 수 있었다.In addition, the nucleotide sequence of 16S rDNA of the kimchi isolated lactic acid bacteria 11 was analyzed according to a conventional method known in the art. As a result, the 16S rDNA nucleotide sequence of the kimchi isolated lactic acid bacteria 11 (SEQ ID NO: 1) was found to be 99% identical to the 16S rDNA nucleotide sequence of Lactobacillus brevis.

따라서, 본 발명자들은 김치분리유산균 11을 "락토바실러스 브레비스 HD-01"로 명명하고 이를 국립농업과학원 농업유전자원센터에에 2012년 1월 4일자로 기탁하였다[수탁번호: KACC91701P].
Accordingly, the present inventors named Kimchi Lactobacillus 11 as "Lactobacillus brevis HD-01" and deposited it on January 4, 2012 at the National Institute of Agricultural Science, Agricultural Genetic Resources Center [Accession Number: KACC91701P].

실험예Experimental Example 6: 알코올 분해능 확인을 위한 동물시험 6: Animal testing for alcohol resolution

상기 락토바실러스 브레비스 HD-01이 생체내에서 알코올 분해능을 나타내는지를 확인하기 위하여 동물시험을 실시하였다.Animal tests were performed to determine whether the Lactobacillus brevis HD-01 exhibited alcohol degradability in vivo.

5주령의 rat으로부터 락토바실러스 브레비스 HD-01 섭취 시 알코올 분해 속도에 영향을 주는지를 측정하였다. 실시예는 락토바실러스 브레비스 HD-01 (1X109 CFU/ml)의 유산균 농축액을 제조하여 1회당 1ml 씩 제공하였고, 대조군으로 비교예는 유산균 현탁액 대신 물을 제공하였다. 실험은 유산균 현탁액 또는 물을 쥐에게 2주간 1일 2회씩 강제 경구 투여하는 것으로 진행되었다. 사료와 물은 자유급식으로 이루어졌으나 유산균 현탁액을 투여하기 1-2시간 전에 절수시켰다. 투여가 시작된 지 14일 째에 채혈 후 40% ethanol 용액 1.5ml을 강제 경구 투여하였고, 투여 후 1-6시간까지 1시간 간격으로 채혈한 뒤 GC (Gas Chromatography)를 이용하여 혈중 에탄올 분석을 진행하였다. 혈중 알코올 농도에 대한 결과는 도 1에 나타내었다.Lactobacillus brevis HD-01 from rat of 5 weeks old It was determined whether ingestion affects the rate of alcohol degradation. Example Lactobacillus brevis HD-01 Lactobacillus concentrate (1 × 10 9 CFU / ml) was prepared and provided in 1 ml each time. As a control, the comparative example provided water instead of the lactic acid bacteria suspension. The experiment proceeded with forced oral administration of lactobacillus suspension or water twice daily for two weeks to the rats. Feed and water were free-fed but water-saved 1-2 hours prior to administration of the lactobacillus suspension. After 14 days of administration, 1.5 ml of 40% ethanol solution was forcibly administered orally, and blood was collected at 1 hour intervals for 1-6 hours after administration, and then ethanol analysis was performed using GC (Gas Chromatography). . Results for blood alcohol concentration are shown in FIG. 1.

2주간 본 발명의 락토바실러스 브레비스 HD-01를 섭취시킨 실시예는 알코올 섭취 후 빠르게 알코올이 분해되는 것을 확인할 수 있었다. 특히 비교예의 경우 혈중 알코올 농도가 1시간째 232 mg/dl까지 상승했으나, 실시예의 경우는 그 보다 56 % 낮은 102 mg/dl까지 감소시켜, 락토바실러스 브레비스 HD-01의 섭취는 알코올 분해능을 현저히 증진시키는 것을 확인할 수 있었다.
Example in which Lactobacillus brevis HD-01 of the present invention was ingested for two weeks was confirmed that alcohol was rapidly decomposed after ingesting alcohol. In particular, in the comparative example, the blood alcohol concentration was increased to 232 mg / dl at 1 hour, but in the example, it was reduced to 102 mg / dl, which is 56% lower than that in the case, and ingestion of Lactobacillus brevis HD-01 markedly improved alcohol resolution. I could confirm that.

제조예Manufacturing example 1: 발효유의 제조 1: Preparation of Fermented Milk

본 발명의 락토바실러스 브레비스 HD-01를 이용하여 발효유를 제조하였다.Fermented milk was prepared using Lactobacillus brevis HD-01 of the present invention.

원유(74.41%)와 탈지분유(6.45%) 등을 혼합하여 배양 종균과 Lactobacillus brevis를 넣어 배양시켜 배양액(Lactobacillus brevis : 1X107 CFU/ml)을 제조하였다. 비교 대상으로는 동일한 혼합액 조성에서 배양 종균 만을 넣어 배양시킨 배양액을 제조하였다. 당액을 제조하여 앞에서 만들어진 각각의 배양액을 섞어 발효유 제품을 제조하였다.
Crude milk (74.41%) and skim milk powder (6.45%) are mixed and cultured by adding culture seed and Lactobacillus brevis to culture medium ( Lactobacillus brevis : 1 × 10 7 CFU / ml) was prepared. As a comparison target, a culture medium in which only the culture seed was added and cultured in the same mixed solution composition was prepared. The sugar solution was prepared, and the fermented milk product was prepared by mixing the respective culture solutions prepared above.

실험예Experimental Example 7: 발효유의 보존테스트 7: Preservation test of fermented milk

상기 제조예 1의 발효유를 130ml 공병에 담아 공기가 통하지 않게 입구를 막은 뒤 각각 5 ℃ 및 15 ℃에 보존테스트를 진행하였다. 2주간 4℃, 15℃에서 보관하여 2가지 제품의 관능을 비교하였으며, 알코올 분해능 선별 균주가 들어있는 제품의 경우에는 저장 기간 동안의 Lactobacillus brevis의 생균수와 T.A., pH, 가스 생성 여부가 측정되었다. The fermented milk of Preparation Example 1 was placed in a 130ml empty bottle to block the inlet through air, and then the storage test was performed at 5 ° C. and 15 ° C., respectively. The sensory properties of the two products were compared by storing them at 4 ℃ and 15 ℃ for 2 weeks, and Lactobacillus during the storage period for products containing alcohol-degrading strains. The viable counts of brevis , TA, pH and gas production were measured.

관능의 경우, 저장 기간 동안의 비교 대상 제품과 큰 차이가 없었으며, 본 발명의 락토바실러스 브레비스 HD-01가 들어있는 제품의 보존 검사 결과는 아래의 표 7에 나타내었다.In the case of sensory function, there was no significant difference from the comparison product during the storage period, and the result of preservation test of the product containing the Lactobacillus brevis HD-01 of the present invention is shown in Table 7 below.

저장일수Days saved 산도Acidity pHpH 가스생성여부Gas generation L. brevis (CFU/ml) L. brevis (CFU / ml) 0일차0 day 5℃5 ℃ 0.540.54 4.35 (25.0℃)4.35 (25.0 ℃) 1.30E+071.30E + 07 3일차Day 3 5℃5 ℃ 0.560.56 4.32 (26.7℃)4.32 (26.7 ℃) XX 5.40E+075.40E + 07 15℃15 ℃ 0.6250.625 4.16 (28.1℃)4.16 (28.1 ℃) XX 6.12E+076.12E + 07 6일차Day 6 5℃5 ℃ 0.5750.575 4.30 (15.3℃)4.30 (15.3 ℃) XX 2.95E+072.95E + 07 15℃15 ℃ 0.670.67 4.14 (18.7℃)4.14 (18.7 ℃) XX 7.80E+077.80E + 07 10일차10th day 5℃5 ℃ 0.720.72 4.04 (26.1℃)4.04 (26.1 ℃) XX 3.20E+073.20E + 07 15℃15 ℃ 0.780.78 3.95 (25.1℃)3.95 (25.1 ℃) XX 8.20E+078.20E + 07 14일차Day 14 5℃5 ℃ 0.750.75 3.92 (24.5℃)3.92 (24.5 ℃) XX 5.80E+075.80E + 07 15℃15 ℃ 0.860.86 3.84 (24.4℃)3.84 (24.4 ℃) XX 8.95E+078.95E + 07

보존 테스트 결과, 15℃ 저장 시에는 다소 산미 진행이 빨리 되는 것 같으나 5℃에서 저장 시에는 산 진행 등이 천천히 진행되는 것으로 보였다. 기존의 락토바실러스 브레비스는 배양 시 gas forming이 강한 균주이나 본 발명의 락토바실러스 브레비스 HD-01의 균수 측정 결과를 통해 제품 내에서 배양이 급속하게 진행되지 않았으며, 밀봉된 제품 내에서 가스 생성 등의 문제가 없었기에 본 균주는 유제품에 활용할 수 있을 것이라 사료되었다.
As a result of preservation test, acidity progressed to a little faster when stored at 15 ° C, but acid progressed slowly when stored at 5 ° C. Conventional Lactobacillus brevis has a strong gas-forming strain during cultivation, but the culture of the Lactobacillus brevis HD-01 of the present invention did not progress rapidly in the product, the production of gas in a sealed product It was thought that this strain could be used in dairy products because there was no problem.

제조예Manufacturing example 2: 건강기능식품( 2: health functional food ( 분말제Powder )의 제조)

락토바실러스 브레비스 HD-01 동결건조분말(1×109 CFU/g)20 mgLactobacillus brevis HD-01 lyophilized powder (1 × 10 9 CFU / g) 20 mg

유당 100 mgLactose 100 mg

탈크 10 mgTalc 10 mg

상기의 성분들을 혼합하고 기밀포에 충진하여 분말제를 제조한다.
The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

제조예Manufacturing example 3: 건강기능식품(정제)의 제조 3: Preparation of health functional food (tablet)

락토바실러스 브레비스 HD-01 동결건조분말(1×109 CFU/g)10 mgLactobacillus brevis HD-01 lyophilized powder (1 × 10 9 CFU / g) 10 mg

옥수수전분 100 mgCorn starch 100 mg

유당 100 mgLactose 100 mg

스테아린산 마그네슘 2 mgMagnesium stearate 2 mg

상기의 성분들을 혼합한 후 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조한다.
After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

제조예Manufacturing example 4: 건강기능식품( 4: Health functional food ( 캅셀제Capsule )의 제조)

락토바실러스 브레비스 HD-01 동결건조분말(1×109 CFU/g)10 mgLactobacillus brevis HD-01 lyophilized powder (1 × 10 9 CFU / g) 10 mg

결정성 셀룰로오스 3 mg3 mg of crystalline cellulose

락토오스 14.8 mgLactose 14.8 mg

마그네슘 스테아레이트 0.2 mgMagnesium Stearate 0.2 mg

통상의 캡슐제 제조방법에 따라 상기의 성분을 혼합하고 젤라틴 캡슐에 충전하여 캡슐제를 제조한다.
The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

제조예Manufacturing example 6: 건강기능식품( 6: Health functional food 혼합 분말제)의Mixed powder) 제조 Produce

락토바실러스 브레비스 HD-01 동결건조분말(1×109 CFU/g)1 gLactobacillus brevis HD-01 lyophilized powder (1 × 10 9 CFU / g) 1 g

비타민 혼합물 적량Vitamin mixture quantity

비타민 A 아세테이트 70 ㎍70 [mu] g of vitamin A acetate

비타민 E 1.0 mgVitamin E 1.0 mg

비타민 B1 0.13 mgVitamin B1 0.13 mg

비타민 B2 0.15 mg0.15 mg of vitamin B2

비타민 B6 0.5 mgVitamin B6 0.5 mg

비타민 B12 0.2 ㎍0.2 [mu] g vitamin B12

비타민 C 10 mgVitamin C 10 mg

비오틴 10 ㎍Biotin 10 μg

니코틴산아미드 1.7 mgNicotinic acid amide 1.7 mg

엽산 50 ㎍50 ㎍ of folic acid

판토텐산 칼슘 0.5 mgCalcium pantothenate 0.5 mg

무기질 혼합물 적량Mineral mixture quantity

황산제1철 1.75 mg1.75 mg of ferrous sulfate

산화아연 0.82 mg0.82 mg of zinc oxide

탄산마그네슘 25.3 mgMagnesium carbonate 25.3 mg

제1인산칼륨 15 mgPotassium monophosphate 15 mg

제2인산칼슘 55 mgSecondary calcium phosphate 55 mg

구연산칼륨 90 mgPotassium citrate 90 mg

탄산칼슘 100 mgCalcium carbonate 100 mg

염화마그네슘 24.8 mgMagnesium chloride 24.8 mg

상기의 비타민 및 미네랄 혼합물의 조성비는 비교적 건강기능식품에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만, 그 배합비를 임의로 변형 실시하여도 무방하며, 통상의 건강기능식품 제조방법에 따라 상기의 성분을 혼합한 다음, 과립을 제조하고, 통상의 방법에 따라 건강기능식품 조성물 제조에 사용할 수 있다.
Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a component suitable for a health functional food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above components may be mixed , Granules may be prepared and used in the manufacture of a health functional food composition according to a conventional method.

국립농업과학원 농업유전자원센터National Institute of Agricultural Science KACC91701PKACC91701P 2012010420120104

서열목록 전자파일 첨부Attach an electronic file to a sequence list

Claims (7)

알코올 디하이드로게나아제 및 아세트알데하이드 디하이드로게나아제의 활성이 우수한 락토바실러스 브레비스(Lactobacillus brevis) HD-01[기탁번호: KACC91701P].
Lactobacillus brevis HD-01 with excellent activity of alcohol dehydrogenase and acetaldehyde dehydrogenase (Accession No. KACC91701P).
삭제delete 제 1 항의 락토바실러스 브레비스(Lactobacillus brevis) HD-01[기탁번호: KACC91701P]을 함유하는 유제품.
A dairy product containing Lactobacillus brevis HD-01 (Accession No. KACC91701P) according to claim 1.
제 3 항에 있어서, 상기 유제품은 발효유, 버터, 치즈 또는 분유인 것을 특징으로 하는 유제품.
4. The dairy product of claim 3 wherein the dairy product is fermented milk, butter, cheese or milk powder.
제 1 항의 락토바실러스 브레비스(Lactobacillus brevis) HD-01[기탁번호: KACC91701P]을 함유하는 알코올성 간질환 예방용 또는 숙취해소용 건강기능식품.
Claim 1, Lactobacillus brevis ( Lactobacillus brevis ) HD-01 [Accession Number: KACC91701P] containing a dietary supplement for preventing or hangover alcoholic liver disease.
제 5 항에 있어서, 상기 건강기능식품은 분말, 과립, 정제 또는 캅셀 형태인 것을 특징으로 하는 알코올성 간질환 예방용 또는 숙취해소용 건강기능식품.
According to claim 5, wherein the health functional food is an alcoholic liver disease prevention or hangover health functional food, characterized in that in the form of powder, granules, tablets or capsules.
제 1 항의 락토바실러스 브레비스(Lactobacillus brevis) HD-01[기탁번호: KACC91701P]을 함유하는 식품첨가제.A food additive containing Lactobacillus brevis HD-01 [Accession No .: KACC91701P] of claim 1.
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WO2016068612A1 (en) * 2014-10-29 2016-05-06 중앙대학교 산학협력단 Food composition, containing lactococcus strain as active ingredient, for alleviating hangover
KR20160052953A (en) * 2014-10-29 2016-05-13 중앙대학교 산학협력단 Food composition for relieving hangover comprising Lactococcus fujiensis
KR101718590B1 (en) * 2014-10-29 2017-03-22 중앙대학교 산학협력단 Food composition for relieving hangover comprising Lactococcus fujiensis
CN107105685A (en) * 2014-10-29 2017-08-29 中央大学产学合作团 That is still drank after a night for alleviating contains Lactococcus strain as the food compositions of active component
US10092605B2 (en) * 2014-10-29 2018-10-09 Chung-Ang University Industry-Academy Cooperation Foundation Food composition, containing Lactococcus strain as active ingredient, for alleviating hangover
KR101670555B1 (en) * 2014-12-16 2016-10-31 중앙대학교 산학협력단 Food composition for relieving hangover comprising Lactococcus chungangensis

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