KR102251295B1 - A composition for preventing or relieving hangover of the comprising heat-killed lactobacillus salivarius v133 as an active ingredient - Google Patents
A composition for preventing or relieving hangover of the comprising heat-killed lactobacillus salivarius v133 as an active ingredient Download PDFInfo
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- KR102251295B1 KR102251295B1 KR1020200040386A KR20200040386A KR102251295B1 KR 102251295 B1 KR102251295 B1 KR 102251295B1 KR 1020200040386 A KR1020200040386 A KR 1020200040386A KR 20200040386 A KR20200040386 A KR 20200040386A KR 102251295 B1 KR102251295 B1 KR 102251295B1
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- Prior art keywords
- lactobacillus salivarius
- strain
- preventing
- dead cells
- relieving
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- 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
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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
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Abstract
Description
본 발명은 락토바실러스 살리바리우스 V133 균주의 사균체를 유효성분으로 함유하는 숙취 예방 또는 해소용 조성물에 관한 것으로, 보다 구체적으로는 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체를 유효성분으로 함유하는 숙취 예방 또는 해소용 약학적 조성물과, 숙취 예방 또는 해소용 식품첨가제 조성물에 관한 것이다.The present invention relates to a composition for preventing or relieving hangovers containing dead cells of Lactobacillus salivarius V133 strain as an active ingredient, and more specifically, Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P dead cells as an active ingredient It relates to a pharmaceutical composition for preventing or relieving hangovers, and a food additive composition for preventing or relieving hangovers.
식물성 유산균은 동물성 유산균에 비하여 영양분이 부족하고, 낮은 pH, 높은 염 농도 등의 척박한 환경에서도 강한 생존력을 보이기 때문에 장내에 안정적으로 도달할 수 있게 한다는 장점이 있다. 또한, 동물성 유산균은 배지 영양분으로 탄수화물, 단백질, 지방 등을 사용하여 장시간 발효된 이후 단백질과 지방에 의해 부패되는 균주의 수가 증가하지만, 식물성 유산균은 배지 영양분으로 탄수화물만을 사용하므로 발효 시 부패하지 않는다. 이로 인해, 국내에서는 김치 등의 식물성 발효 식품에서 분리한 유산균의 우수성을 평가한 연구결과를 지속적으로 발표하고 있다.Vegetable lactic acid bacteria have the advantage of being able to stably reach the intestines because they lack nutrients compared to animal lactic acid bacteria and show strong viability even in harsh environments such as low pH and high salt concentration. In addition, animal lactic acid bacteria use carbohydrates, proteins, and fats as medium nutrients, and after a long time fermentation, the number of strains that are spoiled by proteins and fats increases, but plant lactic acid bacteria do not spoil during fermentation because only carbohydrates are used as medium nutrients. For this reason, domestically, research results that evaluate the excellence of lactic acid bacteria isolated from plant fermented foods such as kimchi are continuously published.
여러 가지 유익한 작용을 하는 미생물을 프로바이오틱스(probiotics)라고 하는데, 프로바이오틱스의 정의는 점차 확대되어 1989년 Fuller는 프로바이오티스를 “장내 균총을 개선시켜 줌으로써 숙주에게 유익한 영향을 주는 생균제제”라고 정의하였고 살아있는 미생물로서의 정립이 이루어졌지만 1999년 Salminen 등은 “숙주에 유익한 작용을 갖는 미생물 또는 미생물의 성분”으로 정의함으로써 프로바이오틱스의 범위를 사균(死菌) 및 그 균체 성분으로까지 확대시켰다. 최근 들어서는 사균 및 그 균체 성분으로까지 확대되어 정의되던 프로바이오틱스 개념에서 새로운 포스트바이오틱스(postbiotics)라는 새로운 개념이 나타났다. 포스트바이오틱스는 생균과 대사(발효)산물들을 열처리 등에 의해 균의 성장이 일어나지 못하도록한 형태로 균체성분과 대사(발효)산물은 직접 또는 간접적으로 장 내 면역을 조절한다고 알려져 있다(Aguilar-Toal￡ JE et al., Trends Food Sci Technol, 2018). 균체성분으로 세포질(cytoplasm), 세포벽(cell wall), 리포테이코산(lipoteichoic acid), 테이코산(eichoic acid), 펩티도글리칸(peptidoglycan), DNA 등이 있으며, 대사(발효)산물은 유기산(organic acid), 단쇄지방산(short chain fatty acid), 다당류(polysaccharides) 등을 포함하고 있다. 대표적인 부산물인 유기산은 면역세포가 집결되어 있는 파이엘판을 자극하여 면역물질이 나오게 하며 주변의 유해물질을 억제하는 작용을 한다. 포스트바이오틱스는 여러 가지 사균화 방법이 보고되고 있는데 물리적 방법으로는 기계적 파괴(mechanical disruption), 열처리(heat treatment), 감마 또는 UV 조사(gamma- or UV irradiation), 고수압(High hydrostatic pressure), 동결건조(Freeze-drying), 음파처리(Sonication)가 있으며, 화학적 방법으로는 산성 탈수활성(Acid deactivation), 효소처리(enzymatic treatment), 용매추출(solvent extract)법이 있다. 포스트바이오틱스는 heat-treated probiotics, ghost probiotics, inactivated probiotics, paraprobiotics, 유산균 사균체(乳酸菌 死菌體), 열처리 유산균(熱處理 乳酸菌), 유산균 추출물(乳酸菌 抽出物) 등 다양한 용어로 불리고 있다.Microorganisms that have various beneficial actions are called probiotics. The definition of probiotics gradually expanded, and in 1989 Fuller defined probiotics as "probiotics that have a beneficial effect on the host by improving the intestinal flora". Although it was established as a microorganism, in 1999, Salminen et al. defined it as "a microorganism or a component of microorganisms that has a beneficial effect on the host", thereby expanding the scope of probiotics to dead bacteria and their microbial components. In recent years, a new concept of postbiotics emerged from the concept of probiotics, which was extended and defined to include dead cells and their microbial components. Postbiotics are known to control the intestinal immunity directly or indirectly in a form that prevents the growth of bacteria by heat treatment of live bacteria and metabolism (fermentation) products, etc. (Aguilar-Toal￡ JE et al., Trends Food Sci Technol, 2018). Cellular components include cytoplasm, cell wall, lipoteichoic acid, eichoic acid, peptidoglycan, and DNA, and metabolic (fermentation) products are organic acids ( organic acids), short chain fatty acids, and polysaccharides. Organic acids, which are representative by-products, stimulate PIELPAN, where immune cells are gathered, to release immune substances and inhibit harmful substances around them. Postbiotics have reported several killing methods. Physical methods include mechanical disruption, heat treatment, gamma- or UV irradiation, and high hydrostatic pressure There are freeze-drying and sonication, and chemical methods include acid deactivation, enzymatic treatment, and solvent extract. Postbiotics are referred to as various terms such as heat-treated probiotics, ghost probiotics, inactivated probiotics, paraprobiotics, lactic acid bacteria dead cells, heat-treated lactic acid bacteria, and lactic acid bacteria extract.
포스트바이오틱스(사균)는 프로바이오틱스(생균) 보다 다양한 장점을 가지고 있어 이미 일본, 미국 및 유럽에서는 다양한 형태로 상용화되고 있다. 프로바이오틱스는 위장관 내 물리, 화학적(위산, 담즙, 소화효소) 소화과정에 의해 대부분 사멸하여 외부환경에 의한 안정성이 떨어지고 열에 불안정하여 유통과정의 저온보관이 필요하고 이로 인한 저온보관 및 저온배송이 필요함에 따라 생산비용이 증가하게 된다. 또한 열에 불안정하여 살균처리 공정이 포함된 제품 등의 적용이 불가능하다는 단점이 발생한다. 포스트바이오틱스의 경우, 이미 균의 성장이 일어나지 못하도록 한 형태로 위장관 내 물리, 화학적 소화과정에 영향을 받지 않으며, 열에 안정하여 보관기간별 일정한 기능성을 나타내고, 저온보관 및 저온배송이 불필요함에 따라 생산비용을 절감할 수 있다. 또한 열 안정성이 있어 살균처리 공정이 포함된 제품에 적용이 가능하여 적용 제품의 다양성을 가진다. 이러한 특징으로 포스트바이오틱스는 프로바이오틱스 보다 소재 안정성이 있어 산업적 적용의 범위가 넓고, 유통과정에서 다루기가 쉽고, 일반식품에도 첨가되어 식품의 기능성을 강화하면서 면역조절 기능에서 프로바이오틱스와 동일한 효과로 건강기능식품, 식품첨가제, 의약품, 동물사료 등과 같은 기존의 유산균이 적용되어 온 분야 외에도 화장품 원료로도 적용되어 그 시장이 확대되고 있다. 또한 항생제 사용에 대한 규제가 강화되고 있기 때문에 대체제로서 활용성과 아직 사균체 제품 생산에 본격적으로 뛰어든 업체가 손에 꼽을 정도이기 때문에 시장성과 성장 가능성은 크다고 할 수 있다. 특히 일본에서는 이미 사균체 제품이 많이 출시되어 꽃가루 알러지 등 면역 관련 효능을 기반으로 홍보하고 있다. 하지만 국내는 아직 개별인정소재로 인정받은 소재가 없을 뿐 아니라 관련 제품 또한 수입에 의존하고 있다.Postbiotics (dead bacteria) have various advantages over probiotics (live bacteria) and are already commercialized in various forms in Japan, the United States, and Europe. Probiotics are mostly killed by the digestion process of physical and chemical (gastric acid, bile, digestive enzymes) in the gastrointestinal tract, resulting in poor stability due to the external environment and unstable to heat, requiring low-temperature storage during the distribution process, resulting in the need for low-temperature storage and low-temperature delivery. As a result, production costs increase. In addition, it is unstable to heat, causing the disadvantage that it is impossible to apply a product including a sterilization process. In the case of postbiotics, it is a form that prevents the growth of bacteria and is not affected by the physical and chemical digestion processes in the gastrointestinal tract, is stable against heat, and exhibits constant functionality for each storage period, and production costs due to the need for low-temperature storage and low-temperature delivery Can be saved. In addition, due to its thermal stability, it can be applied to products that include a sterilization process, so it has a variety of applied products. Due to these characteristics, postbiotics have a wider range of industrial applications because they have more material stability than probiotics, and are easy to handle during distribution, and are added to general foods to enhance the functionality of foods, while providing the same effect as probiotics in terms of immune regulation. In addition to the fields where existing lactic acid bacteria have been applied, such as food additives, pharmaceuticals, animal feeds, etc., the market is expanding as it is applied as a raw material for cosmetics. In addition, as the regulations on the use of antibiotics are tightening, it can be said that the market performance and growth potential are high as companies that have been able to use them as substitutes and are still in full swing in the production of dead cell products are among the best. In particular, a lot of dead cell products have already been released in Japan, and are being promoted based on immunity-related efficacy such as pollen allergy. However, in Korea, there are no materials that have been recognized as individually recognized materials, and related products are also dependent on imports.
유산균 사균체의 생리활성에 대해 보고된 연구들에 따르면 DSS(Dextran sulfate sodium)로 유도된 대장염을 앓고 있는 마우스의 경우 유산균 사균체를 섭취한 마우스에서 DSS 완화 효과를 보였으며 이식한 육종암세포(Sarcoma-180)의 증식이 감소되고 NK세포가 활성화됨이 보고되었다(Tadano et al., J. Japan Mibyou System association,2011). 또한 유해균의 억제 및 정장 작용에 관해서, 항생제를 투여한 마우스에서 장내 대조군에 비해 유익균은 빠르게 증식하고 유해균은 억제시키는 효능이 개시되어 있다(Simohashi et al., Medicine and biology, 2002). 아울러, 유산균 사균체의 다양한 생리활성은 사균체의 특성상 열과 pH에 영향을 받지 않아 다양한 형태의 제제로 가공이 가능한 장점이 있다(Kan, Food industry, 2001).According to studies reported on the physiological activity of lactic acid bacteria, mice suffering from DSS (Dextran sulfate sodium)-induced colitis showed a DSS alleviation effect in mice that ingested lactic acid bacteria dead cells, and transplanted sarcoma cancer cells (Sarcoma). -180) has been reported to decrease proliferation and activate NK cells (Tadano et al., J. Japan Mibyou System Association, 2011). In addition, with regard to the inhibition of harmful bacteria and intestinal action, the efficacy of inhibiting harmful bacteria and proliferating beneficial bacteria faster than in the intestinal control in mice administered with antibiotics has been disclosed (Simohashi et al., Medicine and biology, 2002). In addition, various physiological activities of lactic acid bacteria dead cells are not affected by heat and pH due to the characteristics of dead cells, so it has the advantage that it can be processed into various types of preparations (Kan, Food industry, 2001).
한편, 대한민국 등록특허 제10-1098946호에서는 내산성, 내담즙성 및 장부착능이 있고, 한국농업미생물지원센터(KACC)에 기탁번호 KACC91449P로 기탁된 균주인 락토바실러스 살리바리우스(Lactobacillus salivarius) G1-1 및 이를 유효성분으로 함유한 사료첨가제 조성물에 관련된 내용이 개시되어 있으며, 대한민국 등록특허 제10-1873899호에는 락토바실러스 살리바리우스 CJLS1511(Lactobacillus salivarius CJLS1511)(KCCM11829P)의 균주 또는 이의 사균체를 포함하는 동물 사료 첨가제 조성물에 관련된 내용이 개시되어 있으나, 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체를 유효성분으로 함유하는 숙취 예방 또는 해소용 약학적 조성물과 숙취 예방 또는 해소용 식품첨가제 조성물에 대해서는 밝혀진 바가 없다. On the other hand, in Korean Patent Registration No. 10-1098946, Lactobacillus salivarius , a strain that has acid resistance, bile resistance, and bowel attachment ability, and deposited with the Korean Agricultural Microbiology Support Center (KACC) under the deposit number KACC91449P (Lactobacillus salivarius) G1-1 And a feed additive composition containing the same as an active ingredient is disclosed, and Korean Patent No. 10-1873899 discloses an animal containing a strain of Lactobacillus salivarius CJLS1511 (KCCM11829P) or dead cells thereof. Although the contents related to the feed additive composition are disclosed, the pharmaceutical composition for preventing or relieving hangovers containing the dead cells of the Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain as an active ingredient and the food additive composition for preventing or relieving hangovers Nothing has been revealed about it.
본 발명은 상술한 것과 같은 문제점을 해결하고 필요한 기술을 제공하기 위하여 안출된 것으로서,The present invention has been devised to solve the above-described problems and provide necessary technologies,
본 발명은 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체를 유효성분으로 함유하는 숙취 예방 또는 해소용 약학적 조 성물 제공함에 그 목적이 있다.An object of the present invention is to provide a pharmaceutical composition for preventing or relieving hangovers containing dead cells of the Lactobacillus salivarius V133 KCTC18732P strain as an active ingredient.
또한, 본 발명은 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체를 유효성분으로 함유하는 숙취 예방 또는 해소용 식품첨가제 조성물을 제공함에 다른 목적이 있다.In addition, the present invention is to provide a food additive composition for preventing or relieving hangovers containing the dead cells of the Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain as an active ingredient.
상기와 같은 목적을 달성하기 위한 본 발명의 일 실시형태로서,As an embodiment of the present invention for achieving the above object,
본 발명은 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체를 유효성분으로 함유하는 숙취 예방 또는 해소용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or relieving hangovers containing dead cells of the Lactobacillus salivarius V133 KCTC18732P strain as an active ingredient.
이때, 상기 숙취 예방 또는 해소용 약학적 조성물에 함유된 균주의 사균체는 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주를 식용배지에 접종하여 배양액을 제조하고, 배양액을 80℃ 이상의 온도로 가열한 뒤 60℃ 이하의 온도가 되도록 급속 냉각시키는 공정을 1회 이상 반복 실시하여 열처리한 것임을 특징으로 할 수 있다.At this time, the dead cells of the strain contained in the pharmaceutical composition for preventing or relieving hangovers are Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain is inoculated into an edible medium to prepare a culture solution, and the culture solution is heated to a temperature of 80°C or higher. After that, it may be characterized in that the heat treatment is performed by repeating the process of rapid cooling to a temperature of 60°C or less once or more.
또한, 상기 숙취 예방 또는 해소용 약학적 조성물은 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체가 조성물 1g당 1×105cfu 내지 1×1012cfu로 포함되는 것을 특징으로 할 수 있다.In addition, the pharmaceutical composition for preventing or relieving a hangover may be characterized in that the dead cells of Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain are contained in 1×10 5 cfu to 1×10 12 cfu per 1 g of the composition. have.
본 발명은 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체를 유효성분으로 함유하는 숙취 예방 또는 해소용 식품첨가제 조성물을 제공한다.The present invention provides a food additive composition for preventing or relieving hangovers containing the dead cells of the Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain as an active ingredient.
이때, 상기 숙취 예방 또는 해소용 식품첨가제 조성물에 함유된 균주의 사균체는 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주를 식용배지에 접종하여 배양액을 제조하고, 배양액을 80℃ 이상의 온도로 가열한 뒤 60℃ 이하의 온도가 되도록 급속 냉각시키는 공정을 1회 이상 반복 실시하여 열처리한 것임을 특징으로 할 수 있다.At this time, the dead cells of the strain contained in the food additive composition for preventing or relieving hangovers are Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain is inoculated into an edible medium to prepare a culture solution, and the culture solution is heated to a temperature of 80°C or higher. After that, it may be characterized in that the heat treatment is performed by repeating the process of rapid cooling to a temperature of 60°C or less once or more.
또한, 상기 숙취 예방 또는 해소용 식품첨가제 조성물은 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체가 조성물 1g당 1×105cfu 내지 1×1012cfu로 포함되는 것을 특징으로 할 수 있다.In addition, the food additive composition for preventing or relieving a hangover may be characterized in that the dead cells of Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain are contained in 1×10 5 cfu to 1×10 12 cfu per 1 g of the composition. have.
본 발명의 일 실시형태에 따른 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체를 유효성분으로 함유하는 숙취 예방 또는 해소용 조성물은 열처리 공정을 수행함에 따라 세포 독성이 없고, 혈중 알코올 및 아세트알데히드 농도를 감소시키는 효과가 있으며, 간 조직의 디하이드로게나아제(ADH) 및 아세트알데하이드 디하이드로게나아제(ALDH) 효소 활성을 증가 시켜 알코올 섭취에 의한 숙취 예방 또는 해소의 효과를 나타내므로, 알코올성 숙취 예방 또는 해소용 조성물의 유효성분으로서 효과가 있어 약학적 조성물 또는 식품첨가제 조성물로 사용될 수 있다. The composition for preventing or relieving hangovers containing dead cells of the Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain according to an embodiment of the present invention as an active ingredient has no cytotoxicity as the heat treatment process is performed, and has no cytotoxicity, blood alcohol and It has the effect of reducing acetaldehyde concentration and increases the activity of dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) enzymes in liver tissue to prevent or relieve hangovers caused by alcohol intake. Since it is effective as an active ingredient of a composition for preventing or relieving hangovers, it can be used as a pharmaceutical composition or a food additive composition.
즉, 본 발명의 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주에 열처리를 통한 사균화는 생균의 단점(안정성, 제품 적용성 등)을 개선하여 안정하고 다양한 제품으로의 적용이 가능하면서 생균과의 동등 이상의 기능성을 보유한 소재를 제공할 수 있는 장점이 있기 때문에 알코올성 숙취 예방 또는 해소용 약학적 조성물 또는 식품첨가제 조성물로 사용될 수 있다.That is, killing of the Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain of the present invention through heat treatment improves the shortcomings of live bacteria (stability, product applicability, etc.), so that it is stable and can be applied to various products. It can be used as a pharmaceutical composition or food additive composition for preventing or relieving alcoholic hangover because it has the advantage of providing a material having the functionality equivalent to or higher than that of.
도 1은 본 발명에서 분리한 락토바실러스 살리바리우스 V133 균주의 플레이트 배양 사진이다.
도 2는 본 발명에서 분리한 락토바실러스 살리바리우스 V133 균주(a)와 사균체(b)의 Escherichia coli O157(A), Salmonella typhimurium(B), Staphylococcus aureus(C)에 대한 항균활성을 평가한 사진이다.
도 3은 알코올 투여 30분 전 락토바실러스 살리바리우스 V133 균주의 사균체를 투여한 실험동물의 혈중 알코올 및 아세트알데히드의 농도 감소 효과를 관찰한 결과를 나타내는 그래프이다.
도 4는 알코올 투여 30분 후 락토바실러스 살리바리우스 V133 균주의 사균체를 투여한 실험동물의 혈중 알코올 및 아세트알데히드의 농도 감소 효과를 관찰한 결과를 나타내는 그래프이다.
도 5는 알코올 투여 30분 전 락토바실러스 살리바리우스 V133 균주의 사균체를 투여한 실험동물 간조직의 알코올 디하이드로게나아제(ADH) 및 아세트알데하이드 디하이드로게나아제(ALDH) 효소 활성을 관찰한 결과를 나타내는 그래프이다.
도 6은 알코올 투여 30분 후 락토바실러스 살리바리우스 V133 균주의 사균체를 투여한 실험동물 간조직의 알코올 디하이드로게나아제(ADH) 및 아세트알데하이드 디하이드로게나아제(ALDH) 효소 활성을 관찰한 결과를 나타내는 그래프이다.1 is a photograph of plate culture of the Lactobacillus salivaryus V133 strain isolated in the present invention.
Figure 2 is a photograph of evaluating the antimicrobial activity against Escherichia coli O157 (A), Salmonella typhimurium (B), Staphylococcus aureus (C) of the Lactobacillus salivaryus V133 strain (a) and dead cells (b) isolated in the present invention to be.
3 is a graph showing the results of observing the effect of reducing the concentration of alcohol and acetaldehyde in the blood of an experimental animal to which the dead cells of Lactobacillus salivarian strain V133 were administered 30 minutes before alcohol administration.
FIG. 4 is a graph showing the results of observing the effect of reducing the concentration of alcohol and acetaldehyde in the blood of experimental animals to which the dead cells of the Lactobacillus salivarius V133 strain were administered 30 minutes after alcohol administration.
Figure 5 shows the results of observing the activity of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) enzymes in liver tissues of experimental animals to which the dead cells of Lactobacillus salivarius V133 strain were administered 30 minutes before alcohol administration. It is a graph showing.
Figure 6 shows the results of observing the activity of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) enzymes in the liver tissues of experimental animals to which the dead cells of Lactobacillus salivarius V133 strain were administered 30 minutes after alcohol administration. It is a graph showing.
이하, 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있도록 본 발명의 실시형태를 들어 상세히 설명한다. 본 발명의 실시형태는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 더욱 완전하게 설명하기 위해서 제공되는 것이다. 따라서, 본 발명의 실시형태는 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 이하 설명하는 실시형태로 한정되는 것은 아니다.Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. Embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art. Accordingly, embodiments of the present invention may be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below.
본 발명의 명세서 전체에서, 어떤 부분이 어떤 구성요소를 “포함”한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성요소를 더 포함하는 것을 의미한다.Throughout the specification of the present invention, when a certain part "includes" a certain component, it means that other components are not excluded but other components are further included unless otherwise stated.
본 발명의 명세서 전체에서 사용되는 정도의 용어 “약”, “실질적으로” 등은 언급된 의미에 고유한 제조 및 물질 허용 오차가 제시될 때 그 수치에서 또는 그 수치에 근접한 의미로 사용되고, 본 발명의 이해를 돕기 위해 정확하거나 절대적인 수치가 언급된 개시 내용을 비양심적인 침해자가 부당하게 이용하는 것을 방지하기 위해 사용된다.The terms "about", "substantially", etc. of the degree used throughout the specification of the present invention are used at or close to the numerical value when a manufacturing and material tolerance inherent to the stated meaning is presented, and the present invention To aid in understanding of, accurate or absolute figures are used to prevent unreasonable use of the stated disclosure by unscrupulous infringers.
본 발명은 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체를 유효성분으로 함유하는 숙취 예방 또는 해소용 약학적 조성물(이하, ‘약학적 조성물’이라고도 함)을 제공한다.The present invention provides a pharmaceutical composition for preventing or relieving hangovers (hereinafter, also referred to as'pharmaceutical composition') containing dead cells of the Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain as an active ingredient.
또한, 본 발명은 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체를 유효성분으로 함유하는 숙취 예방 또는 해소용 식품첨가제 조성물(이하, ‘식품첨가제 조성물’이라고도 함)을 제공한다.In addition, the present invention provides a food additive composition for preventing or relieving hangovers (hereinafter, also referred to as'food additive composition') containing dead cells of the Lactobacillus salivarius V133 KCTC18732P strain as an active ingredient.
본 발명에 따른 약학적 조성물 및 식품첨가제 조성물은 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체를 유효성분으로 함유하는 것으로, 혈중 알코올 및 아세트알데히드 농도를 감소시키고, 간 조직의 디하이드로게나아제(ADH) 및 아세트알데하이드 디하이드로게나아제(ALDH) 효소 활성을 증가 시켜 알코올 섭취에 의한 숙취 예방 또는 해소의 효과가 높을 것으로 기대된다.The pharmaceutical composition and food additive composition according to the present invention contains the dead cells of the Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain as an active ingredient, reducing the concentration of alcohol and acetaldehyde in blood, and reducing the concentration of liver tissue. It is expected that the effect of preventing or relieving hangovers caused by alcohol intake will be high by increasing the enzyme activity of genase (ADH) and acetaldehyde dehydrogenase (ALDH).
본 발명의 일 실시형태에 따르면, 상기 숙취 예방 또는 해소용 약학적 조성물 및 상기 숙취 예방 또는 해소용 식품첨가제 조성물에 함유된 균주의 사균체는 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주를 식용배지에 접종하여 배양액을 제조하고, 배양액을 미생물의 사멸을 유도 할 수 있는 80℃ 이상의 온도로 가열한 뒤 60℃ 이하의 온도가 되도록 급속 냉각시키는 공정을 1회 이상 반복 실시하여 열처리한 것임을 특징으로 할 수 있다.According to an embodiment of the present invention, the dead cells of the strain contained in the pharmaceutical composition for preventing or relieving hangover and the food additive composition for preventing or relieving hangover is Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain is edible. It is characterized by heat treatment by repeating the process of inoculating the medium to prepare a culture solution, heating the culture solution to a temperature of 80°C or higher to induce the death of microorganisms, and then rapidly cooling it to a temperature of 60°C or less. can do.
또한, 본 발명의 일 실시형태에 따르면, 상기 숙취 예방 또는 해소용 약학적 조성물 및 상기 숙취 예방 또는 해소용 식품첨가제 조성물은 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체가 조성물 1g당 1×105cfu 내지 1×1012cfu로 포함되는 것을 특징으로 할 수 있다. In addition, according to an embodiment of the present invention, the pharmaceutical composition for preventing or relieving a hangover and a food additive composition for preventing or relieving a hangover are Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P The dead cells of the strain KCTC18732P per 1 g of the composition It may be characterized in that it is included in 1×10 5 cfu to 1×10 12 cfu.
이는, 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체가 조성물 1g당 1×105cfu 미만의 농도로 포함될 경우 알코올 섭취로 인한 숙취 예방 또는 해소 효과를 기재하지 못할 우려가 있기 때문이며, 균 수 범위(농도)는 전임상 섭취농도를 인체적용시험을 위한 체표면적 대비 환산계와 성인체중(60㎏)을 계산하여 1×1012cfu 까지 범위로 설정하였다.This is because if the dead cells of Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain are included in a concentration of less than 1×10 5 cfu per 1 g of the composition, there is a possibility that the effect of preventing or relieving hangovers caused by alcohol intake may not be described. The range (concentration) of the number of bacteria was set to a range of 1×10 12 cfu by calculating the preclinical intake concentration compared to the body surface area for the human application test and the adult weight (60 kg).
본 발명의 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체를 유효성분으로 함유하는 숙취 예방 또는 해소용 약학적 조성물은 혈중 알코올 및 아세트알데히드 농도 감소, 간 조직의 디하이드로게나아제(ADH) 및 아세트알데하이드 디하이드로게나아제(ALDH) 효소 활성 증가를 유발하여 숙취를 예방하고 해소하기 위한 약학적 조성물로 사용될 수 있다. 본 발명의 약학적 조성물을 사용할 수 있는 질환 또는 질병은 이에 제한되는 것은 아니며, 알코올 섭취로 인해 야기된 숙취와 관련된 질환 또는 질병은 모두 포함될 수 있다. The pharmaceutical composition for preventing or relieving hangovers containing the dead cells of the Lactobacillus salivarius V133 KCTC18732P strain as an active ingredient of the present invention reduces the concentration of alcohol and acetaldehyde in blood, and dehydrogenase (ADH) in liver tissue. ) And acetaldehyde dehydrogenase (ALDH) by causing an increase in enzyme activity to prevent and relieve hangovers, it can be used as a pharmaceutical composition. Diseases or diseases in which the pharmaceutical composition of the present invention can be used are not limited thereto, and all diseases or diseases related to hangover caused by alcohol intake may be included.
본 발명의 약학적 조성물을 사용하는 경우 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다.When using the pharmaceutical composition of the present invention, it may further include an appropriate carrier, excipient, and diluent commonly used in the preparation of the pharmaceutical composition.
또한, 본 발명에 따른 약학적 조성물은 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다.In addition, the pharmaceutical composition according to the present invention is formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injectable solutions according to a conventional method. Can be used.
본 발명의 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체를 유효성분으로 함유하는 숙취 예방 또는 해소용 식품첨가제 조성물은 혈중 알코올 및 아세트알데히드 농도 감소, 간 조직의 디하이드로게나아제(ADH) 및 아세트알데하이드 디하이드로게나아제(ALDH) 효소 활성 증가를 유발하여 숙취를 예방하고 해소하는 효과를 가지는 것으로, 이를 목적으로 하는 식품에 첨가될 수 있다. 이에 제한되는 것은 아니나, 본 발명에 따른 식품첨가제 조성물은 식품의 주원료, 부원료, 식품 첨가제, 기능성 식품 또는 음료에 사용될 수 있다. The food additive composition for preventing or relieving hangovers containing the dead cells of the Lactobacillus salivarius V133 KCTC18732P strain as an active ingredient of the present invention reduces the concentration of alcohol and acetaldehyde in blood, and dehydrogenase (ADH) in liver tissue. ) And acetaldehyde dehydrogenase (ALDH) by causing an increase in enzyme activity to prevent and relieve hangover, it can be added to foods for the purpose. Although not limited thereto, the food additive composition according to the present invention may be used as a main raw material, an auxiliary raw material, a food additive, a functional food or a beverage.
본 발명에서 ‘식품’이라 함은 영양소를 한 가지 또는 그 이상 함유하고 있는 천연물 또는 가공품을 의미하며, 바람직하게는 어느 정도의 가공 공정을 거쳐 직접 먹을 수 있는 상태가 된 것을 의미하며, 통상적인 의미로서, 식품, 식품 첨가제, 건강 기능성 식품, 건강 보조식품 및 음료를 모두 포함한다.In the present invention, the term'food' refers to a natural product or processed product containing one or more nutrients, and preferably refers to a state that can be eaten directly through a certain amount of processing process. As such, it includes all foods, food additives, health functional foods, dietary supplements and beverages.
본 발명의 식품첨가제 조성물을 첨가할 수 있는 식품으로는 각종 식품류, 예를 들어, 음료, 껌, 차, 비타민 복합제, 건강보조 식품류 등이 있으며, 환제, 분말, 과립, 침제, 정제, 캡슐 또는 음료 형태로 제형화될 수 있다.Foods to which the food additive composition of the present invention can be added include various foods such as beverages, gums, teas, vitamin complexes, health supplement foods, and the like, pills, powders, granules, needles, tablets, capsules, or beverages. It can be formulated in a form.
이하, 본 발명을 구체적인 실시예에 따라 상세히 설명한다. 단, 하기의 실시예는 본 발명을 예시로 하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail according to specific examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.
락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133 ( Lactobacillus salivarius Lactobacillus salivarius V133) KCTC18732P 균주V133) KCTC18732P strain
1. 항균활성이 높은 유산균의 확보1. Securing lactic acid bacteria with high antibacterial activity
유산균 분리를 위해 김치 시료는 가정에서 담근 김치를 사용하였으며, 각종 김치를 무균적으로 잘게 다진 다음, 이 중 1g을 취하여 멸균 생리식염수(0.85% NaCl) 9㎖에 현탁하고 단계적으로 희석하여 혼합용액 1㎖을 취하여 십진희석법으로 희석한 후 MRS 고체배지(Difco, MI, USA)에 도말하여 37℃, 48시간 배양하였다. 각 시료별 배지에서 형성된 단일 콜로니는 bromocresol purple이 첨가된 MRS 고체배지에 도말하여 노란색을 보이는 콜로니를 최종 선별하였다. 선별된 균주의 플레이트 배양 사진은 도 1과 같다. 선별된 균주는 항균활성을 평가하여 항균활성이 가장 뛰어난 1종의 유산균을 선발하였으며, 보관은 20% glycerol stock으로 -70℃에서 보관하였다.For the separation of lactic acid bacteria, kimchi prepared at home was used. After aseptically chopping various kinds of kimchi, 1 g of the kimchi was taken and suspended in 9 ml of sterile physiological saline (0.85% NaCl), diluted stepwise, and
선발된 균주의 형태학적 및 생화학적 특징을 분석한 결과 그람 양성, 간균으로 포자형성능, 카탈라아제(catalase), 옥시다아제(oxidase) 반응은 음성으로 확인되었다.As a result of analyzing the morphological and biochemical characteristics of the selected strains, it was confirmed that Gram-positive, bacillus spore-forming ability, catalase, and oxidase reaction were negative.
2. 김치로부터 분리한 유산균의 분류학적 성질 유전학적 동정2. Genetic Identification of Taxonomic Properties of Lactobacillus Isolated from Kimchi
선발 유산균은 MRS 액체배지에서 24시간 배양한 균액을 CHL 배지에 접종하였다. API 50 CHL 키트(Biomerieux France)는 건조된 기질을 함유하고 있는 49개의 튜브로 되어있다. 각 캡슐에 균액을 접종하고 37℃에서 48시간 배양한 후, 색의 변화를 체크하였다. 49개 탄수화물이 유일한 탄소원으로 들어있는 키트의 캡슐에 배양한 후 균액이 당을 이용할 수 있는지 여부를 색의 변화로 판정할 수 있다. 보라색의 배지는 각각의 당을 이용하면서 노란색으로 변하게 되며, 이를 양성(+)으로 표시하였다. 결과 값은 API 웹사이트에서 있는 웹 소프트웨어에서 확인하여 락토바실러스 살리바리우스(Lactobacillus salivarius)로 확인되었으며, 그 결과는 하기 표 1에 나타내었다. The selected lactic acid bacteria were inoculated into CHL medium with a bacterial solution cultured for 24 hours in MRS liquid medium. The API 50 CHL kit (Biomerieux France) consists of 49 tubes containing the dried substrate. Each capsule was inoculated with a bacterial solution and cultured at 37° C. for 48 hours, and then the color change was checked. After incubation in the capsules of the kit containing 49 carbohydrates as the only carbon source, it can be determined by the change in color whether the bacterial fluid can use sugar. The purple medium turned yellow while using each sugar, and this was marked as positive (+). The result value was confirmed in the web software on the API website and confirmed as Lactobacillus salivarius , and the results are shown in Table 1 below.
또한, 분리 균주의 유전학적 동정을 위하여 MRS 액체배지에 접종한 다음 37℃에서 18시간 2차 계대 배양하여 배양액 2㎖을 4,000rpm에서 10분 동안 원심분리하였다. 원심분리하여 얻은 세포 침전물을 0.85% NaCl로 3회 세척하였다. 세척 후, 리소자임(lysozyme, 10㎎/㎖) 0.5㎖을 첨가하여 37℃, 1시간 동안 처리하였다. 단백질 분해효소인 프로테아제 K(Protease K) 20㎕와 10% SDS(sodium dodecyl sulfate) 25㎕를 첨가한 후, 60℃ 항온수조(water bath)에서 30분 처리한 후 RNA 분해효소인 RNase 1㎕를 첨가하여 37℃, 1시간 처리하였다. 동량의 페놀(Phenol)-클로로포름(Chloroform)-이소아밀 알콜(Isoamyl alcohol)을 25:24:1의 비율로 첨가하여 현탁한 후, 14,000rpm으로 5분 동안 4℃에서 원심분리하여 상층액만 취한 다음, 이와 같은 과정을 2회 반복하였다. 상층액 양의 1/2 양의 3M 암모늄 아세테이트(ammonium acetate, pH 4.8)와 2배 양의 100% alcohol을 첨가하고 -20℃에서 1시간 정치하였다. 3,000rpm으로 5분 동안 4℃에서 원심분리 하여 세포 침전물을 확인하고, 상등액을 완전히 제거한 후에 70% ethanol 1㎖/ℓ을 넣고 다시 3,000rpm으로 5분 동안 4℃에서 원심분리 하였다. 상층액 제거 후에 건조시키고 TE 버퍼나 증류수로 현탁하여 DNA를 분리하였다. 미생물의 16S rRNA를 증폭하기 위해 정방향 프라이머(27f) : (5'- AGA GTT TGA TCM TGG CTC AG -3' ; 서열번호 2)와 역방향 프라이머(1492r) : (5'- GGT TAC CTT TGT TAC GAC TT -3' ; 서열번호 3)를 사용하였다. PCR 프리믹스 (Bioneer, Cat No. K-2012)에 증류수 17㎕, 정방향 프라이머 1㎕, 역방향 프라이머 1㎕, DNA 1㎕를 첨가하여 혼합한 후 PCR을 수행하였다. PCR 조건은 94℃에서 5분간 처리 후, 94℃에서 1분, 62℃에서 40초, 72℃에서 40초로 30회를 반복하였으며, 72℃에서 7분으로 반응을 종료하였다. PCR 반응 산물을 0.8% 아가로스 겔(agarose gel)로 전기영동을 실시하여 확인하였다. 최종 염기서열을 분석하고 이를 데이터베이스와 비교하여 유산균주를 동정한 결과, 본 발명에 의해 분리된 균주는 락토바실러스 살리바리우스로 나타났으며, 상기 유산균주는 한국생명공학연구원 생물자원센터에 2018년 10월 30일자로 기탁하였다(KCTC18732P).In addition, for the genetic identification of the isolated strain, the strain was inoculated in MRS liquid medium, followed by subculture at 37° C. for 18 hours, and 2 ml of the culture solution was centrifuged at 4,000 rpm for 10 minutes. The cell precipitate obtained by centrifugation was washed 3 times with 0.85% NaCl. After washing, 0.5 ml of lysozyme (10 mg/ml) was added, followed by treatment at 37°C for 1 hour. After adding 20 µl of protease K (Protease K) and 25 µl of 10% sodium dodecyl sulfate (SDS), treatment in a water bath at 60°C for 30 minutes, and then 1 µl of RNA degrading enzyme RNase. It was added and treated at 37°C for 1 hour. After adding and suspending the same amount of phenol (Phenol)-chloroform-isoamyl alcohol in a ratio of 25:24:1, centrifugation at 4°C for 5 minutes at 14,000 rpm to take only the supernatant. Next, this process was repeated twice. Half of the amount of the supernatant was added with 3M ammonium acetate (ammonium acetate, pH 4.8) and twice the amount of 100% alcohol, and the mixture was allowed to stand at -20°C for 1 hour. Cell sediment was confirmed by centrifugation at 3,000 rpm for 5 minutes at 4° C., after completely removing the supernatant, 1 ml/ℓ of 70% ethanol was added and centrifugation was performed at 4° C. for 5 minutes at 3,000 rpm. After removing the supernatant, it was dried and suspended in TE buffer or distilled water to separate DNA. To amplify 16S rRNA of microorganisms, forward primer (27f): (5'- AGA GTT TGA TCM TGG CTC AG -3'; SEQ ID NO: 2) and reverse primer (1492r): (5'- GGT TAC CTT TGT TAC GAC TT-3'; SEQ ID NO: 3) was used. To a PCR premix (Bioneer, Cat No. K-2012), 17 µl of distilled water, 1 µl of forward primer, 1 µl of reverse primer, and 1 µl of DNA were added and mixed, followed by PCR. PCR conditions were treated at 94° C. for 5 minutes, then repeated 30 times at 94° C. for 1 minute, 62° C. for 40 seconds, and 72° C. for 40 seconds, and the reaction was terminated at 72° C. for 7 minutes. The PCR reaction product was confirmed by electrophoresis with 0.8% agarose gel. As a result of identifying the lactic acid strain by analyzing the final nucleotide sequence and comparing it with the database, the strain isolated by the present invention appeared as Lactobacillus salivarius, and the lactic acid strain was reported to the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center in October 2018. Deposited on the 30th day (KCTC18732P).
락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133 ( Lactobacillus salivarius Lactobacillus salivarius V133) KCTC18732P 균주의 식품첨가제 조성물로서 활용 특성 분석V133) Analysis of the properties of KCTC18732P strain as a food additive composition
1. 락토바실러스 살리바리우스 V133 균주의 내산성 평가1. Evaluation of acid resistance of Lactobacillus salivarius V133 strain
유산균이 장내 도착하기 위해서는 섭취 후 pH 2인 위를 통과하여야 한다. 이러한 극한의 조건을 견뎌내는 내산성 조사하기 위하여 락토바실러스 살리바리우스 V133 균주를 MRS 액체배지(MRS broth medium; Difco, USA)에 접종한 후 37℃에서 24시간 동안 배양하였다. 원심분리(4,000×g, 4℃, 5min)하여 Phosphate-buffered saline(PBS, pH7)에 2번 세척하였고, PBS로 균을 OD600 = 1.0(108~109cfu/㎖)으로 조정하여 사용하였다. 유산균 희석액 1㎖을 9㎖ PBS(pH 2와 7)에 첨가 후, 진탕한 뒤 37℃에서 배양하였다. 배양 초기부터 시간별(0hour, 30min, 1hour, 3hour)로 샘플링한 후 MRS 액체배지로 희석하고 MRS 고체배지에 도말한 후 37℃에서 48시간 배양 후 평판배지 위의 집락 수를 계수하여 생균수를 측정하였으며 그 결과는 하기 표 2와 같다.In order for lactic acid bacteria to arrive in the intestine, it must pass through the stomach at pH 2 after ingestion. In order to investigate the acid resistance to withstand these extreme conditions, Lactobacillus salivaryus V133 strain was inoculated in MRS broth medium (Difco, USA) and cultured at 37°C for 24 hours. Centrifuged (4,000×g, 4℃, 5min) and washed twice in Phosphate-buffered saline (PBS, pH7), and the bacteria were adjusted to OD 600 = 1.0 (10 8 ~ 10 9 cfu/ml) with PBS. I did. 1 ml of the diluted solution of lactic acid bacteria was added to 9 ml PBS (pH 2 and 7), shaken, and incubated at 37°C. After sampling by time (0hour, 30min, 1hour, 3hour) from the beginning of cultivation, dilute with MRS liquid medium, spread on MRS solid medium, incubate at 37℃ for 48 hours, and count the number of colonies on the plate medium to measure the number of viable cells. And the results are shown in Table 2 below.
하기 표 2에 나타나 바와 같이, 락토바실러스 살리바리우스 V133 균주는 생리적 활성보다 낮은 pH 2 에서도 생존 균수의 감소가 적어 내산성이 우수함을 확인하였다.As shown in Table 2 below, it was confirmed that the Lactobacillus salivarius V133 strain had less reduction in the number of viable bacteria even at pH 2 lower than its physiological activity, and thus excellent acid resistance.
2. 락토바실러스 살리바리우스 V133 균주의 내담즙성 평가2. Evaluation of bile resistance of Lactobacillus salivarius V133 strain
본 발명에 따른 락토바실러스 살리바리우스 V133 균주의 내담즙성을 평가하였다. 장관 내 담즙산염 농도가 0.1% 내외임을 감안하여 담즙산염(Porcine bile salts)농도가 각각 0, 1, 3%가 되도록 MRS 액체배지를 제조하였다. 본 발명에 따른 락토바실러스 살리바리우스 V133 균주를 멸균된 MRS 액체배지에 접종하여 37℃에서 24시간 동안 배양한 후 담즙산염이 함유된 MRS 액체배지에 0.1%(v/v)씩 접종하였다. 접종 후 37℃에서 배양하면서 30분 단위로 시료를 채취한 후에 멸균된 MRS 액체배지에 희석하고 MRS 평판배지에 도말한 다음 37℃에서 48시간동안 배양한 후, 평판배지 위의 집락 수를 계수하여 생균수를 측정하였다. 그 결과는 하기 표 3에 나타내었으며, 고농도의 담즙산염(3%)에서는 대조군(0%)와 비교 했을 때 만에 75% 급감하였으나, 장내 실제 농도와 유사한 1% 뿐만 아니라 더 높은 3%에서도 적정 균수를 유지하였기 때문에 락토바실러스 살리바리우스 V133 균주는 인체나 동물의 장 내에서도 충분히 생존할 수 있음을 확인 할 수 있는 근거가 될 수 있다.The bile resistance of the Lactobacillus salivarian V133 strain according to the present invention was evaluated. Considering that the concentration of bile salts in the intestine was around 0.1%, MRS liquid medium was prepared so that the concentrations of Porcine bile salts were 0, 1, and 3%, respectively. The Lactobacillus salivarius V133 strain according to the present invention was inoculated into a sterilized MRS liquid medium, cultured at 37° C. for 24 hours, and then 0.1% (v/v) into MRS liquid medium containing bile salts. After inoculation, samples were collected every 30 minutes while incubating at 37°C, diluted in sterilized MRS liquid medium, plated on MRS plate medium, incubated at 37°C for 48 hours, and counted the number of colonies on the plate medium. The number of viable cells was measured. The results are shown in Table 3 below, and in the high concentration of bile salts (3%), when compared with the control group (0%), a sharp decrease of 75% was achieved, but not only 1% similar to the actual concentration in the intestine, but also at a higher 3% Since the number of bacteria was maintained, the Lactobacillus salivarius V133 strain can be a basis for confirming that it can sufficiently survive in the intestine of humans or animals.
Viable cell count (cfu/ml)
3. 락토바실러스 살리바리우스 V133 균주의 공응집능 평가3. Evaluation of coagulation ability of Lactobacillus salivarian strain V133
유산균의 항염 작용에 있어 유산균이 장내에 점착하면서 장내 병원성 유해세균과의 응집력이 매우 중요한 요인이다. 본 발명에서는 지시균으로 대장균(Escherichia coli), 대장균(Escherichia coli O157), 황색포도상구균(Staphylococcus aureus), 장티푸스(Salmonella typhimurium) 균주를 사용하였다. 배양 배지로 락토바실러스 살리바리우스 V133 균주와 대장균을 각각 MRS 액체배지와 Nutrient 액체배지(Difco; USA)를 사용하였다. 락토바실러스 살리바리우스 V133 균주를 전 배양한 배양액을 OD600 = 1.0(108~109cfu/㎖)으로 조정하여 새로운 5㎖ MRS 배지에 1% 접종한 뒤, 24시간 배양한 후. 원심분리(4,000×g, 4℃, 5min)하여 세포를 회수하였다. 병원성 균은 배양배지로 전배양한 후 OD600 = 0.1으로 조정하여 접종하고 37℃에서 24시간 동안 배양하여 사용하였다. 이를 PBS 완충용액에 다시 현탁하여 생균수를 1.0×108cfu/㎖로 조정하였다. 락토바실러스 살리바리우스 V133 균주와 유해균 현탁액 각각 2㎖씩 혼합하고 10초간 교반한 후 37℃에서 5시간 동안 정치 보관하면서 각 시간별(0hour, 5hour)로 공응집능을 조사하였다. 공응집능은 Kos 등의 방법(2003)에 따라 하기 [계산식 1]로 계산하였으며, 그 결과는 하기 표 4와 같다.In the anti-inflammatory action of lactic acid bacteria, the cohesive strength with the intestinal pathogenic harmful bacteria is a very important factor as the lactic acid bacteria adhere to the intestine. In the present invention, E. coli ( Escherichia coli ), E. coli ( Escherichia coli O157), Staphylococcus aureus , typhoid ( Salmonella typhimurium ) strains were used as indicator bacteria. MRS liquid medium and Nutrient liquid medium (Difco; USA) were used as culture medium for Lactobacillus salivarius V133 strain and E. coli, respectively. Lactobacillus salivarius V133 strain was pre-cultured, the culture solution was adjusted to OD 600 = 1.0 (10 8 ~ 10 9 cfu/ml), 1% inoculated in a new 5ml MRS medium, and cultured for 24 hours. Cells were recovered by centrifugation (4,000×g, 4° C., 5 min). Pathogenic bacteria were pre-cultured with a culture medium, adjusted to OD 600 = 0.1, inoculated, and cultured at 37° C. for 24 hours to be used. This was re-suspended in a PBS buffer solution to adjust the number of viable cells to 1.0×10 8 cfu/ml. 2 ml of each of the Lactobacillus salivaryus V133 strain and the harmful bacteria suspension were mixed, stirred for 10 seconds, and stored at 37° C. for 5 hours, and coaggregation capacity was investigated for each hour (0 hours, 5 hours). The coagulation ability was calculated by the following [Calculation 1] according to the method of Kos et al. (2003), and the results are shown in Table 4 below.
[계산식 1][Calculation 1]
colicoli
coli O157 Escherichia
coli O157
typhimuriumtyphimurium
aureusaureus
상기 표 4에 나타난 바와 같이 실험에 사용한 유해균 모두 배양시간이 경과할수록 락토바실러스 살리바리우스 V133 균주와의 응집률이 높았다. 특히, 락토바실러스 살리바리우스 V133 유산균과 살모넬라의 응집률이 63%로 장관 흡착능의 우수성을 보였다.As shown in Table 4, the aggregation rate with the Lactobacillus salivarius V133 strain was higher as the cultivation time elapsed for all harmful bacteria used in the experiment. In particular, the aggregation rate of Lactobacillus salivarius V133 lactic acid bacteria and Salmonella was 63%, showing excellent intestinal adsorption ability.
락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133 ( Lactobacillus salivarius Lactobacillus salivarius V133) KCTC18732P 균주의 사균체V133) dead cells of KCTC18732P strain
락토바실러스 살리바리우스 V133 균주 사균체의 제조방법은 하기와 같다.The preparation method of the dead cells of Lactobacillus salivaryus V133 strain is as follows.
락토바실러스 살리바리우스 V133 균주를 식용배지에 접종하고 37℃에서 24시간 배양하여 배양액을 제조하였다. 정지기(Stationary phase)로 세포 증식이 최고로 도달하고 다량의 대사산물을 포함하고 있는 시기에 상기 배양액을 100℃로 1차 가열(살균)하고 50℃ 이하로 급속냉각하였다. 완전한 사균화를 위하여 다시 100℃로 2차 가열(살균)하고 50℃ 이하로 급속냉각하여 열처리 유산균(이하, ‘사균체’이라고 함)을 제조하였다. 이후 상기 사균체에 부형제로 덱스트린, 말토덱스트린, 포도당 등의 부형제(또는 보호제)를 혼합하여 분무건조하여 분말화 하였다. 이때, 분무건조의 입구 온도는 180℃, 출구온도는 100℃ 이하로 하여 분무건조하였다.Lactobacillus salivaryus V133 strain was inoculated into an edible medium and cultured at 37° C. for 24 hours to prepare a culture solution. At the time when cell proliferation reached the highest level in the stationary phase and contained a large amount of metabolites, the culture solution was first heated (sterilized) to 100° C. and rapidly cooled to 50° C. or less. For complete killing, heat-treated lactic acid bacteria (hereinafter referred to as “dead cells”) were prepared by secondary heating (sterilization) to 100°C again and rapid cooling to 50°C or less. Thereafter, an excipient (or protective agent) such as dextrin, maltodextrin, and glucose as an excipient was mixed with the dead cells, spray-dried and powdered. At this time, the spray drying was performed at an inlet temperature of 180°C and an outlet temperature of 100°C or less.
락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133 ( Lactobacillus salivarius Lactobacillus salivarius V133) KCTC18732P 균주 및 상기의 사균체의 약학적 조성물로서의 활용 특성 분석V133) Analysis of the utilization characteristics of the KCTC18732P strain and the dead cells as a pharmaceutical composition
락토바실러스 살리바리우스 V133 균주와 상기 실시예 3에서 제조된 사균체에 대한 유해균 항균활성 및 숙취 예방 또는 해소 효과를 평가하였다.The antibacterial activity of harmful bacteria and the effect of preventing or relieving hangovers against the Lactobacillus salivarius V133 strain and the dead cells prepared in Example 3 were evaluated.
1. 락토바실러스 살리바리우스 V133 사균체의 항균활성1. Antibacterial activity of Lactobacillus salivarius V133 dead cells
신규의 락토바실러스 살리바리우스 V133 균주의 사균체에 대한 항균 활성은 agar well diffusion assay를 이용하여 확인하였다. 식품 유해균인 Escherichia coli O157, Salmonella typhimurium, Staphylococcus aureus를 대상으로 TSB(Tryptic soy broth) 배지에서 16시간 이상 배양한 후 균액을 액체상태로 유지된 1.5% 한천을 포함하는 배지에 1×1O6cfu/㎖의 농도가 되게 만든 후, 페트리 디쉬(petri dish)에 부어 기층 배지를 만들어 준비하고 한천 0.75%를 넣은 MRS 액체배지를 멸균한 후 45~50 ℃가 되었을 때 기층배지 위에 부어 굳혀 중층배지를 만들었다. 이후 멸균 상태의 biopsy punch(Stiefel Biopsy Punch; Stiefel Laboratories, Research Triangle Park, NC, USA)를 이용하여 well을 만들었다. 각 well에 락토바실러스 살리바리우스 V133 균주와 사균체를 각 well당 50㎕씩 분주하고 37℃에서 24시간 배양하였다. 배양 후, well 주위에 형성된 저해환을 확인하여 항균 활성의 유무를 확인하였으며, 그 결과는 하기 도 2와 같다.The antimicrobial activity of the novel Lactobacillus salivarius V133 strain against dead cells was confirmed using an agar well diffusion assay. Food harmful bacteria Escherichia coli O157, Salmonella typhimurium , Staphylococcus aureus were incubated in TSB (Tryptic soy broth) medium for 16 hours or longer, and then the bacterial solution was placed in a medium containing 1.5% agar in a
도 2에 도시된 바와 같이, 락토바실러스 살리바리우스 V133 균주는 Escherichia coli O157, Salmonella typhimurium 및 Staphylococcus aureus에 대한 높은 항균활성을 보였으며 락토바실러스 살리바리우스 V133 균주의 사균체 또한 3종의 균주에 대한 항균활성이 높은 것으로 확인되었다. 이러한 결과는 락토바실러스 살리바리우스 V133 균주의 사균체는 균주와 동일한 항균활성을 나타내면서 균주가 가지는 단점인 소재의 안전성, 외부환경(내산성, 내담즙성) 및 열에 대한 안정성 부분을 보완한 사균체의 식품으로서의 활용가능성을 보이는 결과이다.As shown in Figure 2, Lactobacillus salivarius V133 strain showed high antibacterial activity against Escherichia coli O157, Salmonella typhimurium, and Staphylococcus aureus. It was found to be high. These results indicate that the dead cells of the Lactobacillus salivarian strain V133 exhibit the same antibacterial activity as the strain, while supplementing the safety of the material, the external environment (acid resistance, bile resistance) and heat stability, which are the disadvantages of the strain. It is a result showing the possibility of being used as
2. 락토바실러스 살리바리우스 V133 사균체의 숙취 예방 또는 해소 효과 측정2. Lactobacillus salivarius V133 dead cell hangover prevention or relieving effect measurement
< 혈중 알코올 및 아세트알데히드 농도 측정 실험><Blood alcohol and acetaldehyde concentration measurement experiment>
락토바실러스 살리바리우스 V133 사균체를 실험동물에 경구 투여하여 혈중 알코올 농도 및 아세트알데히드 농도를 측정하는 실험을 실시하였다.Lactobacillus salivarius V133 dead cells were orally administered to experimental animals to measure blood alcohol concentration and acetaldehyde concentration.
실험군 설정 및 시료 투여Experimental group setting and sample administration
본 발명은 (주)오리엔트바이오(Seongnam, Korea)로부터 공급 받은 7주령의 수 컷 Sprague-Dawley rat를 일주일 동안 설치류 사육실에서 적응시킨 후, 적응기간 중 일반 상태를 관찰하여 건강한 개체만 실험에 사용하였다. 사육환경은 온도 22±2℃, 습도 55±5% 및 명/암 주기는 12시간으로 유지하고, 식이와 물을 자유롭게 공급하여 일주일간 기본식이로 적응시켰다. 순화기간 중 건강하다고 판정된 마우스에 한하여 무작위법을 이용하여 군을 분리하였다.In the present invention, 7-week-old male Sprague-Dawley rats supplied from Orient Bio Co., Ltd. (Seongnam, Korea) were acclimated in a rodent breeding room for a week, and then only healthy individuals were used in the experiment by observing the general condition during the adaptation period. . The breeding environment was maintained at 22±2℃, humidity 55±5%, and the light/dark cycle at 12 hours, and diet and water were supplied freely and adapted to the basic diet for one week. Groups were separated using a random method only for mice judged to be healthy during the acclimatization period.
락토바실러스 살리바리우스 V133 사균체에 대하여 혈중 알코올성 숙취에 미치는 영향을 알아보기 위해 하기 표 5에서와 같이, 정상대조군(NC), 에탄올군(EtOH), 양성대조군(milk thistle 200㎎/㎏/day for 10days, p.o.)(PC), 락토바실러스 살리바리우스 V133 균주의 사균체(2×1010cfu/g)(V133) 200㎎/㎏/day 투여군 총 3군으로 군당 동일한 체중을 지닌 개체 8마리로 구성하여 실험을 진행하였다.In order to investigate the effect of Lactobacillus salivarius V133 dead cells on blood alcoholic hangovers, as shown in Table 5 below, the normal control group (NC), the ethanol group (EtOH), and the positive control group (milk thistle 200 mg/kg/day for 10days, po)(PC), dead cells of Lactobacillus salivarius V133 strain (2×10 10 cfu/g) (V133) 200 mg/kg/day administration group total 3 groups, consisting of 8 individuals with the same weight per group The experiment was carried out.
숙취 예방 활성 관찰 실험 방법Hangover prevention activity observation experiment method
락토바실러스 살리바리우스 V133 사균체의 숙취 예방 활성을 평가하기 위해서, 알코올 섭취 30분 전에 락토바실러스 살리바리우스 V133 사균체를 포함한 배양물을 섭취하게 한 뒤 숙취 예방 활성을 관찰하는 실험을 실시하였다.In order to evaluate the hangover preventing activity of Lactobacillus salivarius V133 dead cells, an experiment was conducted to observe hangover prevention activity after ingesting a culture containing Lactobacillus salivaryus V133 dead cells 30 minutes before alcohol intake.
알코올 섭취 30분 전 락토바실러스 살리바리우스 V133 사균체 섭취 시 락토바실러스 살리바리우스 V133 사균체의 숙취 예방 활성을 평가하기 위해서 NC와 EtOH군은 멸균한 증류수를 경구 투여 하였고, 양성대조군(PC), 락토바실러스 살리바리우스 V133 사균체(V133)군은 멸균한 증류수에 각각의 시험물질(200㎎/㎏ body weight/d)을 준비하여 경구 투여하였다. 멸균 증류수와 각각의 시험물질을 경구 투여하고 30분이 경과한 후, 정상대조군(NC)은 멸균 증류수를 경구 투여하였고, 에탄올군(EtOH), 양성대조군(PC), 락토바실러스 살리바리우스 V133 균주 사균체(V133)군은 25% 에탄올을 10㎖/㎏으로 경구 투여하였다. 에탄올을 경구 투여 한 후 1시간, 3시간, 5시간 경과 후, 각각 미동맥에서 채혈을 하였고, 에탄올 투여 5시간 뒤 마우스를 희생시켜 간 조직을 적출하여 락토바실러스 살리바리우스 V133 사균체의 숙취 예방 활성을 관찰하였다.To evaluate the hangover preventing activity of Lactobacillus salivarius V133 dead cells 30 minutes before alcohol intake, NC and EtOH groups were orally administered sterilized distilled water, and positive control (PC), Lactobacillus Salivarius V133 dead cells (V133) group prepared each test substance (200 mg/kg body weight/d) in sterilized distilled water and administered orally. After 30 minutes of oral administration of sterile distilled water and each test substance, the normal control group (NC) was orally administered sterile distilled water, and the ethanol group (EtOH), positive control group (PC), and Lactobacillus salivarius V133 strain dead cells were administered orally. Group (V133) was orally administered 25% ethanol at 10 ml/kg. 1 hour, 3 hours, and 5 hours after oral administration of ethanol, blood was collected from the caudal artery, and 5 hours after ethanol administration, the mice were sacrificed to extract liver tissue to prevent hangover of Lactobacillus salivarius V133 dead cells. Was observed.
숙취 해소 활성 관찰 실험 방법Hangover Relief Activity Observation Experiment Method
락토바실러스 살리바리우스 V133 사균체의 숙취 해소 활성을 평가하기 위해서, 알코올 섭취 30분 후에 락토바실러스 살리바리우스 V133 사균체를 포함한 배양물을 섭취하게 한 뒤 숙취 해소 활성을 관찰하는 실험을 실시하였다.In order to evaluate the hangover-relieving activity of the Lactobacillus salivarius V133 dead cells, an experiment was conducted to observe the hangover-relieving activity after ingesting a culture containing Lactobacillus salivarius V133 dead cells 30 minutes after alcohol intake.
알코올 섭취 30분 후 락토바실러스 살리바리우스 V133 사균체 섭취 시 락토바실러스 살리바리우스 V133 사균체의 숙취 해소 활성을 평가하기 위해서 정상대조군(NC)은 멸균 증류수를 경구 투여하고, 에탄올군(EtOH), 양성대조군(PC), 락토바실러스 살리바리우스 V133 균주 사균체(V133)군은 25% 에탄올을 10㎖/㎏으로 경구 투여하고 30분이 경과한 후, NC와 EtOH군은 멸균한 증류수를 경구 투여 하였고, 양성대조군(PC), 락토바실러스 살리바리우스 V133 사균체(V133)군은 멸균한 증류수에 각각의 시험물질(200㎎/㎏ body weight/d)을 준비하여 경구 투여하였다. 에탄올을 경구 투여 한 후 1시간, 3시간, 5시간 경과 후, 각각 미동맥에서 채혈을 하였고, 에탄올 투여 5시간 뒤 마우스를 희생시켜 간 조직을 적출하여 락토바실러스 살리바리우스 V133 사균체의 숙취 해소 활성을 관찰하였다.In order to evaluate the hangover-relieving activity of Lactobacillus salivarius V133 dead cells after 30 minutes of alcohol intake, the normal control group (NC) was orally administered sterile distilled water, ethanol group (EtOH), positive control group. (PC), Lactobacillus salivarius V133 strain dead cells (V133) group were orally administered 25% ethanol at 10 ml/kg and 30 minutes later, NC and EtOH groups were orally administered sterilized distilled water, and the positive control group (PC), Lactobacillus salivarius V133 dead cells (V133) group prepared each test substance (200 mg/kg body weight/d) in sterilized distilled water and administered orally. 1 hour, 3 hours, 5 hours after oral administration of ethanol, blood was collected from the caudal artery, respectively, and 5 hours after ethanol administration, the mice were sacrificed to extract liver tissue to relieve hangover of Lactobacillus salivarius V133 dead cells. Was observed.
혈중 알코올 및 아세트알데히드 농도 측정 방법Blood alcohol and acetaldehyde concentration measurement method
에탄올 투여 후 시간별(1시간, 3시간 및 5시간)로 채취 한 혈액은 4℃, 3000rpm에서 10분간 원심분리하여 혈청을 얻었다. 채취한 혈청의 에탄올 함량을 측정하기 위해서 제조된 에탄올 및 아세트알데히드 측정용 assay kit(Germany, Roche Co., Darmstadt)를 이용하였다. Photassium phosphate buffer(pH 9)와 NAD+를 혼합한 후 혈액 상등액 샘플을 첨가 하여 20℃에서 5분간 반응 시킨 후 micro reader를 이용 하여 340㎚에서 흡광도를 측정하였다(A1). 또한 혼합액의 ADH와 ALDH 50㎕를 첨가하여 20℃에서 5분간 반응시킨 후 340㎚에서 흡광도를 측정하였다(A2). 이를 바탕으로 시간별 혈중 알코올의 농도는 하기의 [계산식 2]에 대입하여 정량하였으며, 혈중 아세트알데히드의 농도는 하기의 [계산식 3]에 대입하여 정량하였다.Blood collected hourly (1 hour, 3 hours and 5 hours) after ethanol administration was centrifuged at 4°C and 3000 rpm for 10 minutes to obtain serum. In order to measure the ethanol content of the collected serum, an assay kit for measuring ethanol and acetaldehyde (Germany, Roche Co., Darmstadt) was used. Photassium phosphate buffer (pH 9) and NAD+ were mixed, and a blood supernatant sample was added and reacted at 20° C. for 5 minutes, and the absorbance was measured at 340 nm using a micro reader (A1). In addition, 50 µl of ADH and ALDH of the mixed solution were added and reacted at 20° C. for 5 minutes, and the absorbance was measured at 340 nm (A2). Based on this, the concentration of blood alcohol per hour was quantified by substituting in [Calculation 2] below, and the concentration of acetaldehyde in blood was quantified by substituting in [Calculation 3] below.
[계산식 2][Calculation 2]
Concentration = 0.7259/3.6×△AConcentration = 0.7259/3.6×△A
△A = sample(A2-A1)-blank(A2-A1)△A = sample(A2-A1)-blank(A2-A1)
[계산식 3][Equation 3]
Concentration = 0.7158/3.6×△AConcentration = 0.7158/3.6×△A
△A = sample(A2-A1)-blank(A2-A1)△A = sample(A2-A1)-blank(A2-A1)
숙취 예방 또는 해소 활성 관찰 실험 결과Observation of Hangover Prevention or Relief Activity Results
실험동물에 에탄올 투여 30분 전 각각의 시료를 투여하여 혈액의 알코올(에탄올) 및 아세트알데히드 함량 변화를 관찰한 실험 결과는 도 3과 같다. 도 3에 도시된 바와 같이, 에탄올 투여 후 1시간까지는 알코올 및 아세트알데히드 함량이 급격히 증가 하였다가 그 이후 감소하였으며, 락토바실러스 살리바리우스 V133 사균체(V133) 투여군은 5시간에서 유의적으로 혈중 알코올 및 아세트알데히드 함량이 감소되는 것으로 확인되었다.The experimental results of observing changes in the alcohol (ethanol) and acetaldehyde content of blood by administering each sample 30 minutes before ethanol administration to the experimental animals are shown in FIG. 3. As shown in FIG. 3, the alcohol and acetaldehyde content rapidly increased until 1 hour after ethanol administration and then decreased thereafter, and the Lactobacillus salivarius V133 dead cells (V133) administration group significantly increased blood alcohol and It was found that the acetaldehyde content was reduced.
실험동물에 에탄올 투여 30분 후 각각의 시료를 투여하여 혈액의 알코올(에탄올) 및 아세트알데히드 함량 변화를 관찰한 실험 결과는 도 4와 같다. 도 4에 도시된 바와 같이, 에탄올 투여 후 1시간까지는 알코올 및 아세트알데히드 함량이 급격히 증가하였지만 그 이후 감소하였으며, 양성대조군(PC)과 락토바실러스 살리바리우스 V133 사균체(V133) 투여군은 5시간에서 모두 유의적으로 혈중 알코올 및 아세트알데히드 함량이 감소되는 것으로 확인되었다.The experimental results of observing changes in the alcohol (ethanol) and acetaldehyde content of blood by administering each sample 30 minutes after ethanol administration to the experimental animals are shown in FIG. 4. As shown in Figure 4, alcohol and acetaldehyde content rapidly increased until 1 hour after ethanol administration, but decreased thereafter, and the positive control group (PC) and Lactobacillus salivarius V133 dead cells (V133) administration group were all at 5 hours. It was found that the blood alcohol and acetaldehyde contents were significantly reduced.
< < 알코올에 의한 간 조직의 알코올 디하이드로게나아제(ADH) 및 아세트알데하이드 디하이드로게나아제(ALDH) 효소 활성 측정 실험 >Alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) enzyme activity measurement experiment in liver tissue>
알코올 섭취에 따른 에탄올 대사효소에 락토바실러스 살리바리우스 V133 사균체의 섭취가 미치는 영향을 확인하기 위하여, 간 조직의 디하이드로게나아제(ADH) 및 아세트알데하이드 디하이드로게나아제(ALDH) 효소 활성을 다음의 방법으로 측정하였다. -70℃에 넣어 급속 동결시켜 보관한 간 조직을 해동하여 D-PBS로 3회 세척한 후, 조직 무게(g) 10배의 0.1M Tris-HCl buffer(pH 7.4)를 가하고, 빙냉하에서 균질화하여 준비하였다. 간 조직 균질액의 ADH와 ALDH 활성은 NAD+ 환원에 따른 NADH 생성량을 비색적량하여 측정하는 방법으로 제조된 assay kit(Biovision, Mountain View, CA, USA)를 사용하여 측정하였다. 각각의 kit에 제시한 계산식을 이용 하여 nmol/㎖로 제시하였다.In order to confirm the effect of the intake of Lactobacillus salivarius V133 dead cells on ethanol metabolism enzymes according to alcohol intake, the activities of dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) enzymes in liver tissue were evaluated as follows. It was measured by the method. After thawing the liver tissue stored at -70°C for quick freezing and washing three times with D-PBS, 0.1M Tris-HCl buffer (pH 7.4) of 10 times the tissue weight (g) was added and homogenized under ice cooling. Ready. ADH and ALDH activities of the liver tissue homogenate were measured using an assay kit (Biovision, Mountain View, CA, USA) prepared by measuring the amount of NADH produced by NAD+ reduction by colorimetric quantity. It was presented in nmol/ml using the calculation formula presented in each kit.
실험동물에 에탄올 투여 30분 전 각각의 시료를 투여하여 ADH 및 ALDH 효소 활성을 측정한 실험 결과는 도 5와 같다. 도 5에 도시된 바와 같이, 알코올을 투여 30분 전 각각의 시료를 투여한 경우 에탄올(EtOH)의 ADH 및 ALDH 활성은 정상대조군(NC) 보다 통계적으로 낮은 활성을 나타내는 것으로 확인되었다. 락토바실러스 살리바리우스 V133 사균체(V133) 투여군의 경우 에탄올(EtOH)군 보다 ADH 및 ALDH의 활성이 유의적으로 높은 활성을 보이는 것으로 확인되었으며, 양성대조군(PC) 보다도 뛰어난 효소 활성을 보이는 것으로 확인되었다.The experimental results of measuring ADH and ALDH enzyme activities by administering each sample 30 minutes before ethanol administration to the experimental animals are shown in FIG. 5. As shown in FIG. 5, when each sample was administered 30 minutes before administration of alcohol, it was confirmed that the ADH and ALDH activities of ethanol (EtOH) showed statistically lower activity than that of the normal control group (NC). In the case of the Lactobacillus salivarius V133 dead cells (V133) administration group, it was confirmed that the activity of ADH and ALDH was significantly higher than that of the ethanol (EtOH) group, and it was confirmed to exhibit superior enzyme activity than that of the positive control group (PC). .
실험동물에 에탄올 투여 30분 후 각각의 시료를 투여하여 ADH 및 ALDH 효소 활성을 측정한 실험 결과는 도 6과 같다. 도 6에 도시된 바와 같이, 알코올을 투여 30분 후 각각의 시료를 투여한 경우에도 마찬가지로 에탄올(EtOH)의 ADH 및 ALDH 활성은 정상대조군(NC) 보다 통계적으로 낮은 활성을 나타내는 것으로 확인되었다. 락토바실러스 살리바리우스 V133 사균체(V133) 투여군의 경우에도 에탄올(EtOH)군 보다 ADH 및 ALDH 의 활성이 유의적으로 높은 활성을 보이는 것으로 확인되었으며, 양성대조군(PC) 보다도 뛰어난 효소 활성을 보이는 것으로 확인되었다.The experimental results of measuring ADH and ALDH enzyme activities by administering each sample 30 minutes after ethanol administration to the experimental animals are shown in FIG. 6. As shown in FIG. 6, it was confirmed that even when each sample was administered 30 minutes after alcohol administration, the ADH and ALDH activities of ethanol (EtOH) were statistically lower than those of the normal control group (NC). In the case of the Lactobacillus salivarius V133 dead cells (V133) administration group, it was confirmed that ADH and ALDH activities were significantly higher than those in the ethanol (EtOH) group, and it was confirmed that the enzyme activity was superior to that of the positive control group (PC). Became.
이러한 결과를 종합할 때 락토바실러스 살리바리우스 V133 사균체가 알코올성 숙취 예방 및 해소에 효과적임을 설명할 수 있다.When these results are summarized, it can be explained that Lactobacillus salivarius V133 dead cells are effective in preventing and relieving alcoholic hangovers.
결론적으로, 상기 실시예 1 내지 4의 실험 결과를 통해, 락토바실러스 살리바리우스 V133 균주의 사균체는 혈중 알코올 및 아세트알데히드 농도를 감소시키는 효과가 있으며, 간 조직의 디하이드로게나아제(ADH) 및 아세트알데하이드 디하이드로게나아제(ALDH) 효소 활성을 증가 시켜 알코올 섭취에 의한 숙취 예방 또는 해소의 효과를 나타내므로, 알코올성 숙취 예방 또는 해소용 약학적 조성물 또는 알코올성 숙취 예방 또는 해소용 식품첨가제 조성물로 사용될 수 있는 장점이 있으며, 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주에 열처리를 통한 사균화는 생균의 단점(안정성, 제품 적용성 등)을 개선하여 안정하고 다양한 제품으로의 적용이 가능하면서 생균과의 동등 이상의 기능성을 보유한 소재를 제공할 수 있는 장점이 있다.In conclusion, through the experimental results of Examples 1 to 4, the dead cells of Lactobacillus salivaryus V133 strain have the effect of reducing blood alcohol and acetaldehyde concentrations, and dehydrogenase (ADH) and acetate in liver tissue. As it increases aldehyde dehydrogenase (ALDH) enzyme activity to prevent or relieve hangovers caused by alcohol intake, it can be used as a pharmaceutical composition for preventing or relieving alcoholic hangovers or as a food additive composition for preventing or relieving alcoholic hangovers. There is an advantage, and the killing of Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain through heat treatment improves the shortcomings of live bacteria (stability, product applicability, etc.). There is an advantage of being able to provide a material with equivalent or higher functionality.
이상, 실시예를 들어 본 발명을 상세하게 설명하였으나, 본 발명은 상기 실시예에 한정되지 않으며, 여러 가지 다양한 형태로 변형될 수 있고, 본 발명의 기술적 사상 내에서 당 분야에서 통상의 지식을 가진 자에 의하여 여러 가지 많은 변형이 가능함이 명백하다. 또한, 청구범위의 기재된 본 발명의 기술적 사상을 벗어나지 않는 범위 내에서 당 기술분야의 통상의 지식을 가진 자에 의해 다양한 형태의 치환, 변형 및 변경이 가능할 것이며, 이 또한 본 발명의 범위에 속한다고 할 것이다.Above, although the present invention has been described in detail by way of examples, the present invention is not limited to the above embodiments, and can be modified in various forms, and those skilled in the art within the technical scope of the present invention. It is clear that many variations are possible by the ruler. In addition, various types of substitutions, modifications and changes will be possible by those of ordinary skill in the art within the scope not departing from the technical idea of the present invention described in the claims, and this also belongs to the scope of the present invention. something to do.
Claims (6)
상기 균주의 사균체는,
락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주를 식용배지에 접종하여 배양액을 제조하고, 배양액을 80℃ 이상의 온도로 가열한 뒤 60℃ 이하의 온도가 되도록 급속 냉각시키는 공정을 1회 이상 반복 실시하여 열처리한 것임을 특징으로 하는 숙취 예방 또는 해소용 약학적 조성물.The method according to claim 1,
Dead cells of the strain,
Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain was inoculated into an edible medium to prepare a culture solution, and the process of rapid cooling to a temperature of 60°C or less after heating the culture solution to a temperature of 80℃ or higher was repeated once or more. A pharmaceutical composition for preventing or relieving a hangover, characterized in that it is heat-treated.
상기 숙취 예방 또는 해소용 약학적 조성물은,
락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체가 조성물 1g당 1×105cfu 내지 1×1012cfu로 포함되는 것을 특징으로 하는 숙취 예방 또는 해소용 약학적 조성물.The method according to claim 1,
The pharmaceutical composition for preventing or relieving hangover,
Lactobacillus salivarius V133 (Lactobacillus salivarius V133) A pharmaceutical composition for preventing or relieving hangover, characterized in that the dead cells of the KCTC18732P strain are contained in 1×10 5 cfu to 1×10 12 cfu per 1 g of the composition.
상기 균주의 사균체는,
락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주를 식용배지에 접종하여 배양액을 제조하고, 배양액을 80℃ 이상의 온도로 가열한 뒤 60℃ 이하의 온도가 되도록 급속 냉각시키는 공정을 1회 이상 반복 실시하여 열처리한 것임을 특징으로 하는 숙취 예방 또는 해소용 식품첨가제 조성물.The method of claim 4,
Dead cells of the strain,
Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain was inoculated into an edible medium to prepare a culture solution, and the process of rapid cooling to a temperature of 60°C or less after heating the culture solution to a temperature of 80℃ or higher was repeated once or more. A food additive composition for preventing or relieving a hangover, characterized in that the heat treatment is performed.
상기 숙취 예방 또는 해소용 식품첨가제 조성물은,
락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체가 조성물 1g당 1×105cfu 내지 1×1012cfu로 포함되는 것을 특징으로 하는 숙취 예방 또는 해소용 식품첨가제 조성물.The method of claim 4,
The food additive composition for preventing or relieving a hangover,
Lactobacillus salivarius V133 (Lactobacillus salivarius V133) A food additive composition for preventing or relieving hangover, characterized in that the dead cells of the KCTC18732P strain are contained in 1×10 5 cfu to 1×10 12 cfu per 1 g of the composition.
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| CN115029271A (en) * | 2022-06-21 | 2022-09-09 | 江南大学 | Lactobacillus salivarius capable of reducing activity of serum glutamyl transpeptidase after drinking and application thereof |
| KR20230049138A (en) | 2021-10-05 | 2023-04-13 | 주식회사 블루존 | Method of manufacturing seasoned laver using red ginseng and postbiotics |
| KR102586000B1 (en) | 2023-04-04 | 2023-10-06 | 주식회사 현대바이오랜드 | Lactobacillus fermentum HDB1098 that selectively degrades acetaldehyde and composition for removing hangover containing the same as an active ingredient |
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