KR20240086743A - Novel Leuconostoc mesenteroides VITA-PB2, KCTC19046P and composition for relieving hangover comprising thereof - Google Patents
Novel Leuconostoc mesenteroides VITA-PB2, KCTC19046P and composition for relieving hangover comprising thereof Download PDFInfo
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- leuconostoc mesenteroides
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- lactic acid
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Classifications
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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
- C12N1/205—Bacterial isolates
-
- 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
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- 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
-
- 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
- A23V2200/334—Foods, ingredients or supplements having a functional effect on health treating the effects of consuming alcohol, narcotics or other addictive behavior, e.g. treating hangover or reducing blood alcohol levels
-
- 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
Abstract
본 발명의 신규한 유산균 류코노스톡 메센테로이데스 VITA-PB2 (Leuconostoc mesenteroides VITA-PB2, KCTC19046P)는 알코올 디하이드로게나아제 및 아세트알데하이드 디하이드로게나아제의 활성이 현저히 높아 아세트알데하이드로 인한 알코올성 간질환 예방 및 숙취해소 활성이 우수하기 때문에, 이를 위한 유제품, 건강기능식품 또는 식품첨가물로서 이용될 수 있다.The novel lactic acid bacterium Leuconostoc mesenteroides VITA-PB2 (KCTC19046P) of the present invention has significantly high alcohol dehydrogenase and acetaldehyde dehydrogenase activities, preventing alcoholic liver disease caused by acetaldehyde. And because it has excellent hangover relieving activity, it can be used as a dairy product, health functional food, or food additive for this purpose.
Description
본 발명은 알코올 및 아세트알데하이드 분해능이 우수한 신규한 유산균 류코노스톡 메센테로이데스 VITA-PB2(Leuconostoc mesenteroides VITA-PB2, KCTC19046P) 및 이를 포함하는 숙취 해소 조성물에 관한 것이다.The present invention is a novel lactic acid bacterium Leuconostoc mesenteroides VITA-PB2 ( Leuconostoc mesenteroides VITA-PB2, KCTC19046P) and a hangover relief composition containing it.
알코올 섭취량은 매해 증가하고 있으며 이에 따라 알코올성 질병의 발생률도 증가하는 추세이다. 알코올성 간 질환의 대표적인 원인은 알코올이 효소에 의해 분해되면서 생성되는 아세트알데하이드이며, 이에 의하여 간세포가 손상된다. 알코올성 간 질환은 크게 알코올성 지방간, 알코올성 간염, 알코올성 간 경변 등으로 분류되며 손상 기전은 알코올 자체에 의한 경우, 아세트알데하이드에 의한 경우, 그리고 면역반응에 의한 경우 등이 있다.Alcohol consumption is increasing every year, and the incidence of alcohol-related diseases is also increasing accordingly. The representative cause of alcoholic liver disease is acetaldehyde, which is produced when alcohol is broken down by enzymes, which damages liver cells. Alcoholic liver disease is largely classified into alcoholic fatty liver disease, alcoholic hepatitis, and alcoholic liver cirrhosis, and the damage mechanisms include alcohol itself, acetaldehyde, and immune response.
알코올은 섭취 시 위장에서 20% 정도 흡수되고 나머지는 대부분 소장에서 흡수된다. 이 중 흡수된 알코올의 80~90%가 간에서 효소에 의해 분해되며 소량이 대장 내에서 장내 미생물에 의해 분해가 된다. 이때, 알코올은 빠르게 분해되지만, 아세트알데하이드를 분해하는 데에는 비교적 오랜 시간이 걸린다. 분해되지 않은 아세트알데하이드는 장내에 머무르며 직접적인 장벽의 손상 및 일부 흡수되어 다양한 증상이 야기하는데, 표면상의 대표적인 증상이 ‘숙취’이다. 따라서 소장과 대장에서 더 많은 양의 알코올 및 아세트알데하이드를 처리할 수 있다면 숙취와 간의 부담을 줄이고, 나아가 간 손상과 간 질환을 줄일 수 있다.When alcohol is consumed, approximately 20% is absorbed from the stomach and most of the remainder is absorbed from the small intestine. Of these, 80-90% of absorbed alcohol is broken down by enzymes in the liver, and a small amount is broken down by intestinal microorganisms in the large intestine. At this time, alcohol is decomposed quickly, but it takes a relatively long time to decompose acetaldehyde. Undecomposed acetaldehyde stays in the intestines and directly damages the intestinal wall and is partially absorbed, causing various symptoms, the representative symptom on the surface being ‘hangover’. Therefore, if the small and large intestines can process larger amounts of alcohol and acetaldehyde, it can reduce hangovers and the burden on the liver, and further reduce liver damage and liver disease.
최근 숙취 해소 및 간 질환 예방을 위한 목적으로 장내 미생물에 관한 관심이 급증하면서 휴먼 마이크로바이옴 (Human Microbiome)이 주목받고 있다. 마이크로바이옴 즉, 균총이란, ‘인체에 사는 개체 수준의 세균, 바이러스 등의 각종 미생물’을 총칭하여 말하는 것으로, 70kg 성인 기준 약 38조의 미생물을 가지고 있다고 알려져 있다. 사람이 태어날 때부터 성장함에 따라 마이크로바이옴이 다양하게 변하면서 인간의 질병과 건강에 영향을 미친다고 보고되고 있으며. 이때, 건강한 장내 마이크로바이옴의 형성은 소화를 원활하게 하는 데 도움을 주거나 질병을 일으키는 박테리아가 장벽에 달라붙는 것을 방지하여 질병을 예방하는 등의 유익한 영항을 준다.Recently, the human microbiome has been attracting attention as interest in intestinal microorganisms has increased rapidly for the purpose of relieving hangovers and preventing liver disease. Microbiome, or flora, is a general term for ‘various microorganisms such as bacteria and viruses at the individual level living in the human body.’ It is known that a 70kg adult has approximately 38 trillion microorganisms. It has been reported that as a person grows from birth, the microbiome changes in various ways, affecting human disease and health. At this time, the formation of a healthy intestinal microbiome has beneficial effects such as helping to facilitate digestion or preventing disease by preventing disease-causing bacteria from sticking to the intestinal wall.
건강한 장내 마이크로바이옴 균형이 중요하게 강조되는 가운데, 유산균은 유익균으로서 유해균의 침입을 막는 역할을 할 뿐만 아니라, 소화를 돕거나, 지방 축적을 억제하거나, 에탄올 및 아세트알데하이드를 분해하는 등 다양한 기능성을 갖는 것으로 보고되고 있어 다양한 분야로의 활용을 위한 연구가 활발하게 진행 중이다.As the importance of a healthy intestinal microbiome balance is emphasized, lactic acid bacteria not only serve as beneficial bacteria to prevent the invasion of harmful bacteria, but also provide various functions such as aiding digestion, suppressing fat accumulation, and decomposing ethanol and acetaldehyde. It is reported that it has the potential to be used in various fields, and research is actively underway for its use in various fields.
장내 유익균으로 알려진 유산균 중 일부는 알코올 탈수소효소 (ADH) 및 아세트알데하이드 탈수소 효소 (ALDH)를 생산하여 소장 내의 알코올(alcohol) 대사에 도움을 줄 수 있으며, ADH 및 ALDH의 활성을 증가시킬 수 있는 조효소를 생산한다고 알려져 있다.Some of the lactic acid bacteria known as beneficial intestinal bacteria can help metabolize alcohol in the small intestine by producing alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH), and coenzymes that can increase the activity of ADH and ALDH. It is known to produce .
특히 유산균은 주로 발효식품으로부터 분리되어 안전성이 탁월한 소재이므로, 알코올 및 아세트알데하이드를 분해하는 유산균 소재를 활용한다면, 알코올 뿐만 아니라 숙취의 원인 물질인 아세트알데하이드를 빠르게 분해하여 근본적인 숙취 해소의 효능과 간에 대한 부담을 줄이는 효과를 기대할 수 있을 뿐만 아니라 더 나아가 장내 정착함으로써 안전하면서도 일회성이 아닌 지속 가능한 효과를 기대할 수 있다.In particular, lactic acid bacteria are mainly isolated from fermented foods and are a material with excellent safety, so if you use lactic acid bacteria material that decomposes alcohol and acetaldehyde, it quickly decomposes not only alcohol but also acetaldehyde, the cause of hangovers, and has the effect of fundamentally relieving hangovers and improving liver health. Not only can we expect the effect of reducing the burden, but furthermore, by settling in the intestines, we can expect a safe and sustainable effect rather than a one-off effect.
본 발명은 알코올 및 아세트알데하이드 분해 활성이 높아 아세트알데하이드로 인한 알코올성 간질환 예방과 숙취해소 활성이 우수한 유산균을 제공하는 것을 목적으로 한다. 본 발명의 목적은 신규한 유산균 류코노스톡 메센테로이데스 VITA-PB2 (Leuconostoc mesenteroides VITA-PB2, KCTC19046P) 및 이를 포함하는 숙취해소 조성물을 제공하는 데 있다.The purpose of the present invention is to provide lactic acid bacteria with high alcohol and acetaldehyde decomposition activity and excellent hangover relieving activity and preventing alcoholic liver disease caused by acetaldehyde. The object of the present invention is to develop a novel lactic acid bacterium Leuconostoc mesenteroides VITA-PB2 ( Leuconostoc mesenteroides VITA-PB2, KCTC19046P) and a hangover relief composition containing it.
본 발명은 우수한 알코올 및 아세트알데하이드 제거 활성을 갖는 류코노스톡 메센테로이데스 VITA-PB2(Leuconostoc mesenteroides VITA-PB2, KCTC19046P)를 제공한다. 상기 균주는 도 1과 같은 16S rRNA 유전자 염기서열을 가진 류코노스톡 메센테로이데스의 신규 스트레인이다.The present invention relates to Leuconostoc mesenteroides VITA-PB2 ( Leuconostoc mesenteroid es VITA-PB2, KCTC19046P). The strain is a new strain of Leuconostoc mesenteroides with the 16S rRNA gene base sequence as shown in Figure 1 .
본 발명은 신균주 류코노스톡 메센테로이데스 VITA-PB2(Leuconostoc mesenteroides VITA-PB2, KCTC19046P) 또는 이의 배양액을 포함하는 숙취해소용 조성물을 제공한다.The present invention provides a composition for relieving hangovers containing the new strain Leuconostoc mesenteroides VITA-PB2 (KCTC19046P) or its culture solution.
상기 약학 조성물은 우수한 아세트알데하이드 분해능을 통하여 숙취해소 효과를 가질 수 있다.The pharmaceutical composition can have a hangover relieving effect through its excellent acetaldehyde decomposition ability.
상기 신균주 류코노스톡 메센테로이데스 VITA-PB2(Leuconostoc mesenteroides VITA-PB2, KCTC19046P) 또는 이의 배양액은 전체 약학 조성물 총 중량에 대하여 바람직하게는 0.001~99중량%, 더 바람직하게는 0.001~60중량%, 가장 바람직하게는 0.001~40중량%로 하여 첨가될 수 있다.The new strain Leuconostoc mesenteroides VITA-PB2 (KCTC19046P) or its culture medium is preferably 0.001 to 99% by weight, more preferably 0.001 to 60% by weight, based on the total weight of the entire pharmaceutical composition. %, most preferably 0.001 to 40% by weight.
상기 약학 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 액제, 에어로졸 등의 제형으로 제형화하여 사용될 수 있으나 이에 제한되는 것은 아니다. 상기 약학 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제, 감미제, 산미제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형제제는 하나 이상의 부형제, 예를 들면, 전분, 탄산칼슘, 수크로스 또는 락토즈, 젤라틴 등을 섞어 조제될 수 있다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당될 수 있으며, 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제, 산미제 등이 포함될 수 있다.The pharmaceutical composition may be formulated and used in dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, liquids, aerosols, etc. according to conventional methods, but is not limited thereto. Carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, and cellulose. , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, sweeteners, and acidulants. Solid preparations for oral administration may be prepared by mixing one or more excipients, such as starch, calcium carbonate, sucrose or lactose, and gelatin. Liquid preparations for oral use may include suspensions, oral solutions, emulsions, and syrups. In addition to simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, preservatives, and acidulants are included. may be included.
본 발명의 약학적 조성물의 투여량은 치료받을 대상의 연령, 성별, 체중과, 치료할 특정 질환 또는 병리 상태, 질환 또는 병리 상태의 심각도, 투여 경로 및 처방자의 판단에 따라 달라질 것이다. 이러한 인자에 기초한 투여량 결정은 당업자의 수준 내에 있으며, 일반적으로 투여량은 0.01㎎/㎏/일 내지 대략 500㎎/㎏/일의 범위이다. 바람직한 투여량은 0.1㎎/㎏/일 내지 200㎎/㎏/일이며, 더 바람직한 투여량은 1㎎/㎏/일 내지 200㎎/㎏/일이다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The dosage of the pharmaceutical composition of the present invention will vary depending on the age, gender, and weight of the subject to be treated, the specific disease or pathological state to be treated, the severity of the disease or pathological state, the route of administration, and the judgment of the prescriber. Dosage determinations based on these factors are within the level of one skilled in the art, and dosages generally range from 0.01 mg/kg/day to approximately 500 mg/kg/day. A preferred dosage is 0.1 mg/kg/day to 200 mg/kg/day, and a more preferred dosage is 1 mg/kg/day to 200 mg/kg/day. Administration may be administered once a day, or may be administered several times. The above dosage does not limit the scope of the present invention in any way.
본 발명의 약학 조성물은 쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 본 발명의 약학 조성물은 독성 및 부작용이 거의 없으므로 예방 목적으로 장기간 복용시에도 안심하고 사용할 수 있다.The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, and humans through various routes. The pharmaceutical composition of the present invention has almost no toxicity and side effects, so it can be safely used even when taken for a long period of time for preventive purposes.
본 발명은 신균주 류코노스톡 메센테로이데스 VITA-PB2(Leuconostoc mesenteroides VITA-PB2, KCTC19046P) 또는 이의 배양액을 포함하는 숙취해소용 식품을 제공한다. 상기 식품은 발효유, 홍삼 발효 식품, 콩 발효식품, 치즈, 유제품, 두유, 두부, 음료, 분유, 이유식, 생식, 음료, 선식 또는 건강보조식품 중의 어느 하나일 수 있으나 이에 제한되는 것은 아니다. 상기 식품은 식품학적으로 허용 가능한 식품 보조 첨가제를 포함할 수 있다.The present invention provides a food for relieving hangovers containing the new strain Leuconostoc mesenteroides VITA-PB2 (KCTC19046P) or its culture solution. The food may be any one of fermented milk, fermented red ginseng food, fermented soybean food, cheese, dairy products, soy milk, tofu, beverage, powdered milk, baby food, raw food, beverage, sun food, or health supplement, but is not limited thereto. The food may contain food auxiliary additives that are foodologically acceptable.
상기 식품은 류코노스톡 메센테로이데스 VITA-PB2(Leuconostoc mesenteroides VITA-PB2, KCTC19046P) 또는 이의 배양액이 전체 식품 총 중량에 대하여 바람직하게는 0.001~99중량%, 더 바람직하게는 0.001~60중량%, 가장 바람직하게는 0.001~40중량%로 첨가될 수 있다.The food contains Leuconostoc mesenteroides VITA-PB2 (KCTC19046P) or its culture solution in an amount of preferably 0.001 to 99% by weight, more preferably 0.001 to 60% by weight, based on the total weight of the entire food. , most preferably 0.001 to 40% by weight.
상기 식품은 정제, 캡슐제, 환제 또는 액제 등의 형태를 포함하며, 본 발명의 추출물을 첨가할 수 있는 식품으로는, 예를 들어, 각종 식품류, 음료, 껌, 차, 비타민 복합제, 건강기능성식품류 등일 수 있다.The food includes tablets, capsules, pills, or liquid forms. Foods to which the extract of the present invention can be added include, for example, various foods, beverages, gum, tea, vitamin complexes, and health functional foods. It may be, etc.
본 발명의 신규한 유산균 류코노스톡 메센테로이데스 VITA-PB2(Leuconostoc mesenteroides VITA-PB2, KCTC19046P)는 알코올 디하이드로게나아제 및 아세트알데하이드 디하이드로게나아제의 활성이 현저히 높아 알코올성 간질환 예방 및 숙취 해소 활성이 뛰어나기 때문에, 이를 위한 유제품, 건강기능식품 또는 식품첨가물로서 이용될 수 있다.The novel lactic acid bacterium Leuconostoc mesenteroides VITA-PB2 (KCTC19046P) of the present invention has significantly high alcohol dehydrogenase and acetaldehyde dehydrogenase activities, preventing alcoholic liver disease and relieving hangovers. Because it has excellent activity, it can be used as a dairy product, health functional food, or food additive.
도 1은 본 발명의 류코노스톡 메센테로이데스 VITA-PB2(Leuconostoc mesenteroides VITA-PB2, KCTC19046P)의 16S rRNA 유전자의 염기서열을 나타낸 것이다.
도 2는 본 발명의 류코노스톡 메센테로이데스 VITA-PB2(Leuconostoc mesenteroides VITA-PB2, KCTC19046P)의 알코올 탈수소효소(Alcohol Dehydrogenase, ADH) 활성 결과를 나타낸 그래프이다.
도 3은 본 발명의 류코노스톡 메센테로이데스 VITA-PB2(Leuconostoc mesenteroides VITA-PB2, KCTC19046P)의 알데하이드 탈수소효소(Aldehyde Dehydrogenase, ALDH) 활성 결과를 나타낸 그래프이다.
도 4는 본 발명의 류코노스톡 메센테로이데스 VITA-PB2(Leuconostoc mesenteroides VITA-PB2, KCTC19046P)의 용혈 안정성 실험 결과를 나타낸 사진이다.
도 5는 본 발명의 류코노스톡 메센테로이데스 VITA-PB2(Leuconostoc mesenteroides VITA-PB2, KCTC19046P)의 세포 독성 실험 결과를 나타낸 그래프이다.
도 6은 본 발명의 류코노스톡 메센테로이데스 VITA-PB2(Leuconostoc mesenteroides VITA-PB2, KCTC19046P)의 담즙산염 탈접합 안정성 결과를 나타낸 결과이다.Figure 1 shows the base sequence of the 16S rRNA gene of Leuconostoc mesenteroides VITA-PB2 (KCTC19046P) of the present invention.
Figure 2 is a graph showing the alcohol dehydrogenase (ADH) activity results of Leuconostoc mesenteroides VITA-PB2 (KCTC19046P) of the present invention.
Figure 3 is a graph showing the results of aldehyde dehydrogenase (ALDH) activity of Leuconostoc mesenteroides VITA-PB2 (KCTC19046P) of the present invention.
Figure 4 is a photograph showing the results of a hemolysis stability test of Leuconostoc mesenteroides VITA-PB2 (KCTC19046P) of the present invention.
Figure 5 is a graph showing the results of a cytotoxicity test of Leuconostoc mesenteroides VITA-PB2 (KCTC19046P) of the present invention.
Figure 6 is a result showing the bile salt deconjugation stability results of Leuconostoc mesenteroides VITA-PB2 (KCTC19046P) of the present invention.
이하 본 발명의 바람직한 실시예를 상세히 설명한다. 그러나, 본 발명은 여기서 설명되는 실시예에 한정되지 않고 다른 형태로 구체화될 수도 있으며, 여기서 소개되는 내용은 본 발명의 사상을 충분히 전달하기 위해 제공하는 것이다.Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms, and the content introduced here is provided to sufficiently convey the spirit of the present invention.
<< 실시예Example 1. 유산균의 분리> 1. Isolation of lactic acid bacteria>
청주, 대전 및 경기도 화성 지역에서 수득 된 김치로부터 유산균을 분리하였다. 유산균을 분리하기 위해 각 샘플들을 0.85% 생리식염수에 현탁시켰고, 그 현탁액을 반코마이신이 포함된 MRS agar 배지에 도말하여 37℃에서 24시간 동안 배양하였다. 배양 후 MRS agar 위에 형성된 집락의 morphology가 상이한 것 위주로 선발하여, MRS broth 배지에 계대 하여 37℃에서 24시간 동안 배양하였다. 분리된 균주 중에서 배양액의 pH가 5.0 이하인 205종을 선발하였고, 선발된 균주들은 BCP agar 배지를 통해 유산을 생성하는 균주라는 것을 확인하였다.Lactic acid bacteria were isolated from kimchi obtained from Cheongju, Daejeon, and Hwaseong, Gyeonggi-do. To isolate lactic acid bacteria, each sample was suspended in 0.85% physiological saline, and the suspension was spread on MRS agar medium containing vancomycin and cultured at 37°C for 24 hours. After culturing, colonies formed on MRS agar with different morphology were selected, passaged into MRS broth medium, and cultured at 37°C for 24 hours. Among the isolated strains, 205 strains with a culture medium pH of 5.0 or less were selected, and the selected strains were confirmed to produce lactic acid through BCP agar medium.
<< 실시예Example 2. 알코올 내성 및 2. Alcohol tolerance and 아세트알데하이드Acetaldehyde 분해능을 갖는 유산균의 선발> Selection of lactic acid bacteria with degrading ability>
2.1 유산균의 배양2.1 Culture of lactic acid bacteria
실시예 1에서 분리한 유산균은 MRS broth 배지에서 37℃에서 6시간 동안 전 배양 후, MRS brtoh 배지에 2% 농도가 되도록 접종하여 37℃에서 16시간 동안 계대 배양하였다. 배양된 균은 10℃, 6,000 rpm에서 5 분간 원심분리하여 균체를 회수하고, 이를 phosphate buffer (PBS buffer, pH 7.4)를 이용하여 3회 washing 하였다. 최종적으로 얻어진 균 현탁액을 실험에 사용하였다.The lactic acid bacteria isolated in Example 1 were pre-cultured in MRS broth medium at 37°C for 6 hours, then inoculated into MRS brtoh medium at a concentration of 2% and subcultured at 37°C for 16 hours. The cultured bacteria were centrifuged at 10°C and 6,000 rpm for 5 minutes to recover the cells, and washed three times using phosphate buffer (PBS buffer, pH 7.4). The finally obtained bacterial suspension was used in the experiment.
2.2 알코올 내성 유산균 선발2.2 Selection of alcohol-resistant lactic acid bacteria
실시예 1에서 분리한 205종 균주에 대해 10% 알코올 내성 실험을 실시하였다. 알코올 내성을 갖는 균주는 ADH 또는 ALDH 활성을 가지고 있어 알코올이 존재하는 환경에서 이를 분해하여 탄소원으로의 이용이 가능하다는 것을 의미한다. 따라서 알코올 내성을 갖는 균주는 알코올 및 아세트알데하이드 분해능이 높은 균주일 수 있다.A 10% alcohol tolerance test was performed on the 205 strains isolated in Example 1. Strains with alcohol tolerance have ADH or ALDH activity, meaning that they can be decomposed in an environment where alcohol is present and used as a carbon source. Therefore, a strain with alcohol tolerance may be a strain with a high ability to decompose alcohol and acetaldehyde.
MRS broth 배지에 최종 농도가 10%가 되도록 에탄올을 첨가하여 반응 용액을 제조하고 이를 1 ml 씩 1.5 ml 에펜도르프 튜브에 분주하였다. 이후 상기 실시예 1에서 선발하고 상기 실시예 2.1의 방법대로 배양한 205종 균주 현탁액을 에탄올을 함유한 반응 용액에 1:9의 비율로 반응시켜, 37℃에서 6시간 동안 배양하였다. 반응시간 2시간 간격으로 균주 현탁액의 흡광도를 600 nm 파장에서 optical density (O.D)를 비교하였다. 반응 전,후 O.D 값을 비교하여 흡광도 값이 약 20% 이상 증가한 균주를 10% 농도의 알코올에서 내성을 갖는 균주로 판단하고, 상기 방법으로 92종의 알코올 내성 균주를 선발하였다.A reaction solution was prepared by adding ethanol to the MRS broth medium to a final concentration of 10%, and 1 ml of this solution was dispensed into 1.5 ml Eppendorf tubes. Thereafter, the suspension of 205 strains selected in Example 1 and cultured according to the method in Example 2.1 was reacted in a reaction solution containing ethanol at a ratio of 1:9 and cultured at 37°C for 6 hours. The optical density (O.D) of the strain suspension was compared at a wavelength of 600 nm at 2-hour reaction time intervals. By comparing the O.D. values before and after the reaction, strains whose absorbance value increased by about 20% or more were judged to be resistant to alcohol at a concentration of 10%, and 92 alcohol-resistant strains were selected using the above method.
2.3 2.3 아세트알데하이드Acetaldehyde 분해능을 갖는 유산균 선발 Selection of lactic acid bacteria with degrading ability
상기 실시예 2.2에서 선발된 92종 균주가 숙취의 원인 물질인 아세트알데하이드를 효과적으로 분해하는지 확인하기 위해 각 균주의 아세트알데하이드 분해능을 분석하였다. 92종 균주의 균 현탁액은 상기 실시예 2.1과 같은 방법으로 제조하였으며, MRS broth 배지에 아세트알데하이드를 200 μg/ml의 농도가 되도록 첨가하여 반응 용액을 제조하였다. 제조된 반응 용액과의 비율이 1:15가 되도록 균주 현탁액을 접종하고, 진탕 배양기를 이용하여 37℃에서 4시간 동안 반응시켰다. 이후 반응액을 회수하여 10℃, 13,000 rpm 조건하에 3분간 원심분리하여 최종적으로 얻어진 각 유산균 배양 상등액의 잔여 아세트알데하이드를 Megazyme acetaldehyde assay kit(Megazyme)를 이용하여 분석하였고, 아세트알데하이드 분해도가 50% 이상인 균주 7종의 분해도를 표 1에 나타내었다. 대조군으로는 반응 용액에 1:15 비율로 3차 증류수를 첨가한 시료를 사용하였다.To determine whether the 92 strains selected in Example 2.2 effectively decompose acetaldehyde, the causative agent of hangovers, the acetaldehyde decomposition ability of each strain was analyzed. A bacterial suspension of 92 strains was prepared in the same manner as in Example 2.1, and a reaction solution was prepared by adding acetaldehyde to MRS broth medium to a concentration of 200 μg/ml. The strain suspension was inoculated at a ratio of 1:15 with the prepared reaction solution, and reacted at 37°C for 4 hours using a shaking incubator. Afterwards, the reaction solution was recovered and centrifuged for 3 minutes at 10°C and 13,000 rpm, and the remaining acetaldehyde in each lactic acid bacteria culture supernatant was analyzed using the Megazyme acetaldehyde assay kit (Megazyme). The decomposition degree of 7 strains is shown in Table 1. As a control, a sample in which tertiary distilled water was added to the reaction solution at a ratio of 1:15 was used.
상기 표 1에서 보는 바와 같이 선발된 알코올 내성 균주 92종 중 7종 균주에서 50%이상의 아세트알데하이드 분해능을 나타내었다. 이 중 아세트알데하이드 분해능이 95%이상으로 뛰어난 분해능력을 나타낸 VITA-PB2, VITA-PB8, VITA-PY1 3종 균주를 선발하였다.As shown in Table 1, 7 of the 92 selected alcohol-resistant strains showed acetaldehyde decomposition ability of more than 50%. Among these, three strains, VITA-PB2, VITA-PB8, and VITA-PY1, which showed excellent acetaldehyde decomposition ability of over 95%, were selected.
2.4 선발된 균주 동정2.4 Identification of selected strains
상기에서 선별된 알코올 내성 및 아세트알데하이드 분해능을 갖는 균주 3종을 포함하여, 시중에서 판매되고 있는 숙취 해소 제품에서 분리된 양성 대조군 2종의 동정 결과를 표 2에 나타내었다. 동정 결과 상기 실시예 2.3에서 선발된 3종 균주 모두 류코노스톡 메센테로이드(Leuconostoc mesenteroides)인 것으로 확인되었다. The identification results of two positive control strains isolated from commercially available hangover relief products, including the three strains with alcohol tolerance and acetaldehyde decomposition ability selected above, are shown in Table 2. As a result of identification, all three strains selected in Example 2.3 were Leuconostoc mesenteroids ( Leuconostoc mesenteroides ).
<< 실시예Example 3. 류코노스톡 3. Leuconostoc 메센테로이드mesenteroid VITA-PB2의 알코올 대사 활성 확인> Confirmation of alcohol metabolic activity of VITA-PB2>
3.1 균주의 3.1 Strains of 조추출액crude extract 제조 manufacturing
상기 표 2의 7종의 균주를 상기 실시예 2.1과 같은 방법으로 배양하여 균 현탁액을 제조하였다. 7종 균주의 현탁액은 흡광도(optical densitiy, O.D)가 1.0이 되도록 0.85% 생리식염수를 이용하여 조정하였다. 그 다음, 초음파 세포파쇄기(Ultrasonic Processor VCX-130, Sonics)를 이용하여 5분 동안 파쇄를 진행하였다. 파쇄가 완료된 세포 파쇄액은 4℃, 13,000 rpm 조건하에 30분간 원심분리하였고, 최종적으로 얻어진 균주의 조추출액은 -75℃에서 보관하여 실험에 사용하였다. 각 균주의 조추출액 단백질 함량은 브래드포드 단백질 정량법(Bradford assay)을 이용하여 분석하였다. 이후 알코올 분해 효소 (ADH), 아세트알데하이드 분해 효소 (ALDH) 활성은 각 균주의 조추출액의 NADH 환원 정도를 분광광도법을 이용하여 측정하여, 단백질 (mg protein) 및 시간 당 생성되는 NADH 양으로 계산하였다.In Table 2 above Seven types of strains were cultured in the same manner as Example 2.1 to prepare a bacterial suspension. The suspension of the seven strains was adjusted using 0.85% physiological saline so that the optical density (OD) was 1.0. Next, disruption was performed for 5 minutes using an ultrasonic cell disruptor (Ultrasonic Processor VCX-130, Sonics). The cell homogenate after complete disruption was centrifuged for 30 minutes at 4°C and 13,000 rpm, and the crude extract of the strain finally obtained was stored at -75°C and used in experiments. The protein content of the crude extract of each strain was analyzed using the Bradford assay. Afterwards, alcohol degrading enzyme (ADH) and acetaldehyde degrading enzyme (ALDH) activities were measured using spectrophotometry to measure the degree of NADH reduction in the crude extract of each strain, and calculated based on protein (mg protein) and the amount of NADH produced per hour. .
3.2 균주의 알코올 탈수소 효소(Alcohol 3.2 Alcohol dehydrogenase of the strain (Alcohol dehydrogenasedehydrogenase , , ADHADH ) 활성 측정) activity measurement
상기 표 2의 7종의 균주의 알코올 분해 효소 활성을 측정하였다. 먼저, 0.1M Glycine buffer (pH 9.6)에 최종 농도가 2.5 mM NAD와 25 mM 에탄올이 되도록 반응 용액을 제조하였다. 상기 실시예 3.1에서 제조한 각 균주의 조추출액과 반응 용액을 1:9 비율로 25°C에서 5분간 반응시켰다. 반응이 완료된 후 생성된 NADH의 양을 분광광도계를 이용하여 340 nm에서 측정하고 그 결과를 도 2에 나타내었다.In Table 2 above The alcohol-degrading enzyme activity of seven strains was measured. First, a reaction solution was prepared so that the final concentration was 2.5mM NAD and 25mM ethanol in 0.1M Glycine buffer (pH 9.6). The crude extract of each strain prepared in Example 3.1 and the reaction solution were reacted at 25°C for 5 minutes at a ratio of 1:9. After the reaction was completed, the amount of NADH produced was measured at 340 nm using a spectrophotometer, and the results are shown in Figure 2.
도 2에서 보는 바와 같이, 상기에서 선별된 3종 VITA-PB2, VITA-PB8, VITA-PY1를 포함한 7종 균주의 알코올 탈수소 효소 활성 분석 결과, VITA-PB2 균주가 가장 높은 알코올 탈수소 효소 활성을 나타내었다. 특히, 동일 종 표준 균주인 류코노스톡 메센테로이데스 KCTC 3505 (508.3 nmol NADH/min/mg protein) 보다도 본 발명의 VITA-PB2 균주가 1714.9 nmol NADH/min/mg protein으로, 알코올 탈수소 효소 활성이 약 3배 이상 높은 것을 확인하였으며, 양성 대조군인 P.C1 (Lactobacillus brevis) 또는 P.C2 (Lactobacillus fermentum)과 비교했을 때에도 약 2~3배 가량 높은 알코올 탈수소 효소 활성을 보이며 뛰어난 알코올 분해 능력을 갖고 있음을 확인하였다.As shown in Figure 2, as a result of alcohol dehydrogenase activity analysis of the 7 strains including the 3 strains selected above, VITA-PB2, VITA-PB8, and VITA-PY1, the VITA-PB2 strain showed the highest alcohol dehydrogenase activity. It was. In particular, the VITA-PB2 strain of the present invention has an alcohol dehydrogenase activity of 1714.9 nmol NADH/min/mg protein compared to the same species standard strain, Leuconostoc mesenteroides KCTC 3505 (508.3 nmol NADH/min/mg protein). It was confirmed to be more than 3 times higher, and compared to the positive controls P.C1 ( Lactobacillus brevis ) or P.C2 ( Lactobacillus fermentum ), it shows about 2 to 3 times higher alcohol dehydrogenase activity and has an excellent alcohol decomposition ability. was confirmed.
3.3 균주의 3.3 Strains of 아세트알데하이드Acetaldehyde 탈수소 효소(Acetaldehyde Dehydrogenase (Acetaldehyde) dehydrogenasedehydrogenase , , ALDHALDH ) 활성 측정) activity measurement
상기 표 2의 7종의 균주의 아세트알데하이드 분해 효소 활성을 측정하였다. 먼저, 60mM sodium pyrophosphate buffer (pH 8.8)에 최종 농도가 1mM NAD. 0.2mM 아세트알데하이드 그리고 0.1mM 4-methylpyrazole가 되도록 첨가하여 반응 용액을 제조하였다. 상기에서 제조한 각 균주의 조추출액을 1:9 비율로 반응 용액과 25°C에서 5분간 반응시켰다. 반응이 완료된 후 생성된 NADH의 양을 분광광도계를 이용하여 340 nm에서 측정하고 그 결과를 도 3에 나타내었다.In Table 2 above Acetaldehyde-decomposing enzyme activity of seven strains was measured. First, the final concentration of 1mM NAD was added to 60mM sodium pyrophosphate buffer (pH 8.8). A reaction solution was prepared by adding 0.2mM acetaldehyde and 0.1mM 4-methylpyrazole. The crude extract of each strain prepared above was reacted with the reaction solution at a ratio of 1:9 for 5 minutes at 25°C. After the reaction was completed, the amount of NADH produced was measured at 340 nm using a spectrophotometer, and the results are shown in Figure 3.
도 3에서 보는 바와 같이, 상기에서 선별된 3종 VITA-PB2, VITA-PB8, VITA-PY1 균주를 포함한 7종 균주의 아세트알데하이드 탈수소효소 활성 분석 결과, VITA-PB2 균주가 가장 높은 아세트알데하이드 탈수소효소 활성을 나타내었다. 특히, 동일 종 표준 균주인 류코노스톡 메센테로이데스 KCTC 3505 (205.5 nmol NADH/min/mg protein)와 시중에서 판매되고 있는 숙취해소 제품에서 분리된 균주 양성 대조군 PC1 (171.8 nmol NADH/min/mg protein) 및 PC2 (504.5 nmol NADH/min/mg protein)보다도 본 발명의 VITA-PB2 균주가 1113 nmol NADH/min/mg protein으로 약 2~5배 높은 아세트알데하이드 분해 효소 활성을 보이는 것을 확인하였다.As shown in Figure 3, as a result of analyzing the acetaldehyde dehydrogenase activity of the 7 strains including the 3 strains selected above, VITA-PB2, VITA-PB8, and VITA-PY1, the VITA-PB2 strain had the highest acetaldehyde dehydrogenase activity. showed activity. In particular, Leuconostoc mesenteroides KCTC 3505 (205.5 nmol NADH/min/mg protein), a standard strain of the same species, and PC1 (171.8 nmol NADH/min/mg protein), a positive control strain isolated from a commercially available hangover relief product. ) and PC2 (504.5 nmol NADH/min/mg protein), it was confirmed that the VITA-PB2 strain of the present invention exhibits about 2 to 5 times higher acetaldehyde decomposition enzyme activity at 1113 nmol NADH/min/mg protein.
<< 실시예Example 4. 4. 프로바이오틱스probiotics 특성 측정> Characteristic measurements>
4.1 용혈 안전성4.1 Hemolysis Safety
상기에서 매우 높은 알코올 분해 능력을 확인한 류코노스톡 메센테로이데스 VITA-PB2의 용혈 안정성을 확인하였다. 양성 대조군으로는 바실러스 세레우스 (Bacillus cereus)를 이용하여 균주의 용혈성 실험을 수행하였다. 7% Sheep Blood Defibrinated (MB-S1876) 가 포함되어 있는 Blood agar에서 배양된 균주들의 양상은 크게 α-hemolysis 용혈능, β-hemolysis 용혈능, γ-hemolysis 용혈능 세 가지로 나뉘어진다. α-hemolysis 용혈능을 가진 균주의 경우, 적혈구를 부분적으로 용혈시키는 부분 용혈능으로 형성된 집락이 일부 녹색으로 변하지만 배지의 변화가 없다. β-hemolysis 용혈능을 가진 균주의 경우 적혈구를 완전히 파괴시키는 완전 용혈능으로 집락 주변으로 투명한 환(clear zone)이 생성시키나, γ-hemolysis의 용혈능을 가지고 있는 균주는 적혈구를 분해하지 못해 콜로니는 형성되지만 아무런 배지의 변화가 없다.The hemolytic stability of Leuconostoc mesenteroides VITA-PB2, which was confirmed to have a very high alcohol decomposition ability above, was confirmed. As a positive control, a hemolytic test was performed using Bacillus cereus. The aspects of strains cultured on blood agar containing 7% Sheep Blood Defibrinated (MB-S1876) are largely divided into three categories: α-hemolysis hemolytic activity, β-hemolysis hemolytic activity, and γ-hemolysis hemolytic activity. In the case of strains with α-hemolysis hemolytic ability, the colonies formed by partially hemolyzing red blood cells turn green, but there is no change in the medium. In the case of strains with the hemolytic ability of β-hemolysis, a clear zone is created around the colony due to the complete hemolytic ability that completely destroys red blood cells, but strains with the hemolytic ability of γ-hemolysis cannot decompose red blood cells and colonies form. is formed, but there is no change in the medium.
상기 실시예 2.1의 방법대로 배양된 균을 Sheep blood가 7% 함유된 Blood agar 배지에 획선 도말하여 24시간 동안 37℃에서 배양하고, 최종적으로 배양된 양상을 통해 각 균주의 용혈성을 확인하고 그 결과를 도 4에 나타내었다. 도 4에서 보는 바와 같이, 본 발명에 따른 VITA-PB2 균주는 양성 대조군인 바실러스 세레우스(Bacillus cereus)와 비교하였을 때, γ-hemolysis인 것으로 확인되었다.The bacteria cultured according to the method of Example 2.1 above were smeared on a blood agar medium containing 7% sheep blood, cultured at 37°C for 24 hours, and the hemolytic property of each strain was confirmed through the final culture pattern. is shown in Figure 4. As shown in Figure 4, the VITA-PB2 strain according to the present invention was confirmed to be γ-hemolytic when compared to Bacillus cereus, a positive control.
4.2 세포 독성 확인4.2 Confirmation of cytotoxicity
마우스 대식세포에서 본 발명 VITA-PB2의 독성 유무를 확인하기 위해서 MTT assay를 실시했다.MTT assay was performed to confirm the toxicity of VITA-PB2 of the present invention in mouse macrophages.
MTT(2-2(4,5-dimethyl-thiazol)-2,5-diphenyltetrazolium bromide) assay는 생존 세포의 효소작용에 의해 MTT가 환원되어 formazan crystal로 침전되는 정도를 흡광도로 측정하여 이로부터 첨가한 유산균 및 배양액에 의해 세포가 사멸 또는 증식 억제되는 정도를 결정하는 실험법이다. 마우스 대식세포를 96 well plate에 세포 밀도가 1×105 cells/well이 되도록 하여 10% FBS, 1% antibiotic-antimycotic가 첨가된 배지에 24시간 동안 배양 후, VITA-PB2를 농도별로 희석하여 처리하였다. 이후 MTT solution을 처리하고, 아무것도 처리하지 않은 세포를 대조군으로 하여 도 5에 나타내었다. 도 5에서 보는 바와 같이, VITA-PB2을 처리하였을 때 세포 독성을 보이지 않는 것으로 확인하였다.The MTT (2-2(4,5-dimethyl-thiazol)-2,5-diphenyltetrazolium bromide) assay measures the degree to which MTT is reduced and precipitated into formazan crystals by the enzymatic action of viable cells by absorbance, and then added from this. This is an experimental method to determine the extent to which cells are killed or proliferation is inhibited by lactic acid bacteria and culture media. Mouse macrophages were cultured in a 96 well plate at a cell density of 1×10 5 cells/well for 24 hours in medium supplemented with 10% FBS and 1% antibiotic-antimycotic, then treated with VITA-PB2 diluted by concentration. did. Afterwards, cells were treated with MTT solution, and untreated cells were used as a control group and are shown in Figure 5. As shown in Figure 5, it was confirmed that no cytotoxicity was observed when treated with VITA-PB2.
4.3 4.3 담즙산염bile salts 탈접합Deconjugation 안전성 (Bile-salt coagulation) Safety (Bile-salt coagulation)
담즙산염의 경우 지방을 유화시켜 지방 분해 효소 (lipase)의 작용을 촉진하여 지방산 분해에 도움을 준다. 프로바이오틱스에 존재하는 담즙산염 가수분해 효소 (bile salt hydrolase)는 담즙산염으로부터 프로바이오틱스를 보호하여 장내 정착을 늘리기도 하지만, 담즙산염을 탈접합시켜 담즙산염의 유화기능을 없애며 지방산 분해를 어렵게 하기도 한다.In the case of bile salts, they help decompose fatty acids by emulsifying fat and promoting the action of lipase. Bile salt hydrolase present in probiotics protects probiotics from bile salts and increases their colonization in the intestines, but it also deconjugates bile salts, eliminating the emulsification function of bile salts and making it difficult to decompose fatty acids.
이에 따라 상기 선별된 VITA-PB2 균주의 담즘산염 탈접합 안전성을 확인하였다. 0.5% taurodeoxycholic acid (TDCA; TRC INC,)를 함유 또는 함유하지 않은 MRS agar 배지에 균주를 획선 도말하여 37°C에서 24 시간에서 48 시간 동안 혐기 배양 하여 자란 균주의 콜로니(집락) 양상을 확인하고 그 결과를 도 6에 나타내었다. 도 6에서 보는 바와 같이, 0.5% TDCA를 함유한 MRS 배지에서 자란 균주의 콜로니 양상을 0.5% TDCA를 함유하지 않은 대조군 MRS 배지의 균주와 비교하였을 때, 균주가 자란 양상이 크게 차이가 없었으며 콜로니 주위에 작은 입자가 형성되거나, 콜로니의 경계가 흐려지는 현상이 발견되지 않았다. 따라서 상기 VITA-PB2 균주의 경우, 탈접합 담즙산염 생성에서 음성으로 확인되었다.Accordingly, the safety of damjeum salt deconjugation of the selected VITA-PB2 strain was confirmed. The strain was smeared on MRS agar medium containing or not containing 0.5% taurodeoxycholic acid (TDCA; TRC INC,), cultured anaerobically at 37°C for 24 to 48 hours, and the colony pattern of the grown strain was confirmed. The results are shown in Figure 6. As shown in Figure 6, when the colony pattern of the strain grown in MRS medium containing 0.5% TDCA was compared with the strain in control MRS medium without 0.5% TDCA, there was no significant difference in the growth pattern of the strain, and the colony growth pattern was not significantly different. No small particles were formed around the area or the boundaries of the colonies were blurred. Therefore, in the case of the VITA-PB2 strain, it was confirmed to be negative in the production of deconjugated bile salts.
<< 실시예Example 5. 최종 선별된 균주 동정> 5. Identification of final selected strains>
상기와 같은 실험을 통해 뛰어난 알코올 및 아세트알데하이드 분해능이 우수한 균주 VITA-PB2를 최종적으로 선정하였다. VITA-PB2 균주의 16S rRNA 유전자 동정 결과, 류코노스톡 메센테로이데스 (Leuconostoc mesenteroides)로 확인되었으며 이를 도 1에 나타내었다. 상기 균주는 2022년 11월 7일자로 한국생명공학연구원 생물자원센터에 기탁되었다. (미생물 기탁번호 : KCTC19046P)Through the above experiments, strain VITA-PB2, which had excellent alcohol and acetaldehyde decomposition ability, was finally selected. As a result of 16S rRNA gene identification of VITA-PB2 strain, Leuconostoc mesenteroides mesenteroides ) and is shown in Figure 1. The above strain is effective as of November 7, 2022 It was deposited at the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center. (Microorganism accession number: KCTC19046P)
상기와 같은 실험을 통하여 높은 알코올 내성, 알코올 및 아세트알데하이드 분해능을 가지며, 용혈 안전성을 갖고 세포 독성이 없으며, 담즙산염 탈접합 안전성을 나타내는 신규한 유산균 류코노스톡 메센테로이데스 VITA-PB2(Leuconostoc mesenteroides VITA-PB2, KCTC19046P)를 최종 발굴하였으며, 본 발명의 신규한 유산균 류코노스톡 메센테로이데스 VITA-PB2(Leuconostoc mesenteroides VITA-PB2, KCTC19046P)를 숙취해소용 약학 조성물, 건강기능식품 등에 이용할 수 있을 것으로 기대된다.Through the above experiments, a novel lactic acid bacterium, Leuconostoc mesenteroides VITA-PB2 ( Leuconostoc mesenteroides VITA), has high alcohol tolerance, alcohol and acetaldehyde decomposition ability, hemolytic safety, no cytotoxicity, and bile salt deconjugation safety. -PB2, KCTC19046P) was finally discovered, and it is expected that the novel lactic acid bacterium Leuconostoc mesenteroides VITA-PB2 ( Leuconostoc mesenteroides VITA-PB2, KCTC19046P) of the present invention can be used in pharmaceutical compositions for hangover relief, health functional foods, etc. do.
<< 제제예Example 1. 약학적 제제> 1. Pharmaceutical preparations>
본 발명의 신규한 유산균 류코노스톡 메센테로이데스 VITA-PB2(Leuconostoc mesenteroides VITA-PB2, KCTC19046P) 또는 이의 배양액 농축 분말 400g을 락토즈 175.9g, 감자전분 180g 및 콜로이드성 규산 32g과 혼합하였다. 이 혼합물에 10% 젤라틴 용액을 첨가시킨 후, 분쇄하여 14 메쉬체를 통과시켰다. 이것을 건조시키고 여기에 감자전분 160g, 활성 50g 및 스테아린산 마그네슘 5g을 첨가해서 얻은 혼합물을 정제로 만들었다.400 g of the novel lactic acid bacterium Leuconostoc mesenteroides VITA-PB2 (KCTC19046P) of the present invention or its culture concentrate powder was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. A 10% gelatin solution was added to this mixture, then pulverized and passed through a 14 mesh sieve. This was dried, and 160 g of potato starch, 50 g of activator, and 5 g of magnesium stearate were added thereto, and the resulting mixture was made into tablets.
<< 제제예Example 2. 식품의 제조> 2. Manufacturing of food>
제제예Example 2-1. 건강기능식품의 제조 2-1. Manufacturing of health functional foods
본 발명의 신규한 유산균 류코노스톡 메센테로이데스 VITA-PB2(Leuconostoc mesenteroides VITA-PB2, KCTC19046P) 또는 이의 배양액 농축 분말 2g, 비타민 혼합물 적량, 비타민 A 아세테이트 70㎍, 비타민 E 1.0㎎, 비타민 B1 0.13㎎, 비타민 B2 0.15㎎, 비타민 B6 0.5㎎, 비타민 B12 0.2㎍, 비타민 C 10㎎, 비오틴 10㎍, 니코틴산아미드 1.7㎎, 엽산 50㎍, 판토텐산 칼슘 0.5㎎, 무기질 혼합물 적량, 황산제1철 1.75㎎, 산화아연 0.82㎎, 탄산 마그네슘 25.3㎎, 제1인산칼륨 15㎎, 제2인산칼슘 55㎎, 구연산칼륨 90㎎, 탄산칼슘 100㎎, 염화마그네슘 24.8㎎을 섞어 과립으로 제조하였다.2 g of the novel lactic acid bacterium Leuconostoc mesenteroides VITA-PB2 (KCTC19046P) of the present invention or its culture concentrate powder, appropriate amount of vitamin mixture, 70 μg of vitamin A acetate, 1.0 mg of vitamin E, 0.13 mg of vitamin B1 , Vitamin B2 0.15㎎, Vitamin B6 0.5㎎, Vitamin B12 0.2㎍, Vitamin C 10㎎, Biotin 10㎍, Nicotinamide 1.7㎎, Folic Acid 50㎍, Calcium Pantothenate 0.5㎎, mineral mixture, ferrous sulfate 1.75㎎, 0.82 mg of zinc oxide, 25.3 mg of magnesium carbonate, 15 mg of potassium phosphate monobasic, 55 mg of calcium phosphate dibasic, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride were mixed to prepare granules.
서열목록 전자파일 첨부Sequence list electronic file attached
Claims (6)
상기 약학 조성물은 아세트알데하이드 분해능을 갖는 것을 특징으로 하는 숙취해소용 약학 조성물.According to paragraph 2,
A pharmaceutical composition for relieving a hangover, characterized in that the pharmaceutical composition has the ability to decompose acetaldehyde.
상기 약학 조성물은 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽 및 액제로 이루어진 군으로부터 선택되는 하나로 제제화 되는 것을 특징으로 하는 숙취해소용 약학 조성물.According to paragraph 2,
The pharmaceutical composition is a pharmaceutical composition for relieving a hangover, characterized in that it is formulated with one selected from the group consisting of powders, granules, tablets, capsules, suspensions, emulsions, syrups and solutions.
상기 식품은 발효유, 홍삼 발효 식품, 콩 발효식품, 치즈, 두유, 두부, 음료, 분유, 이유식, 생식, 음료, 선식 또는 건강보조식품 중의 어느 하나인 것을 특징으로 하는 숙취해소용 식품.According to clause 5,
The food is a food for relieving a hangover, characterized in that it is one of fermented milk, fermented red ginseng food, fermented soybean food, cheese, soy milk, tofu, beverage, powdered milk, baby food, raw food, beverage, sun food, or health supplement.
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