KR102251294B1 - A composition for preventing, improving or treating alcoholic gastritis of the comprising heat-killed lactobacillus salivarius v133 as an active ingredient - Google Patents
A composition for preventing, improving or treating alcoholic gastritis of the comprising heat-killed lactobacillus salivarius v133 as an active ingredient Download PDFInfo
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- KR102251294B1 KR102251294B1 KR1020180134911A KR20180134911A KR102251294B1 KR 102251294 B1 KR102251294 B1 KR 102251294B1 KR 1020180134911 A KR1020180134911 A KR 1020180134911A KR 20180134911 A KR20180134911 A KR 20180134911A KR 102251294 B1 KR102251294 B1 KR 102251294B1
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- South Korea
- Prior art keywords
- lactobacillus salivarius
- strain
- dead cells
- alcoholic gastritis
- composition
- Prior art date
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Abstract
본 발명은 락토바실러스 살리바리우스 V133 균주의 사균체를 유효성분으로 함유하는 알코올성 위염의 예방, 개선 또는 치료용 조성물에 관한 것으로, 보다 구체적으로는 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체를 유효성분으로 함유하는 알코올성 위염의 예방, 개선 또는 치료용 약학적 조성물과 알코올성 위염의 예방 또는 개선용 식품첨가제 조성물에 관한 것이다.The present invention relates to a composition for the prevention, improvement or treatment of alcoholic gastritis containing dead cells of Lactobacillus salivarius V133 strain as an active ingredient, and more specifically, Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain. It relates to a pharmaceutical composition for preventing, improving or treating alcoholic gastritis containing cells as an active ingredient and a food additive composition for preventing or improving alcoholic gastritis.
Description
본 발명은 락토바실러스 살리바리우스 V133 균주의 사균체를 유효성분으로 함유하는 알코올성 위염의 예방, 개선 또는 치료용 조성물에 관한 것으로, 보다 구체적으로는 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체를 유효성분으로 함유하는 알코올성 위염의 예방, 개선 또는 치료용 약학적 조성물과 알코올성 위염의 예방 또는 개선용 식품첨가제 조성물에 관한 것이다.The present invention relates to a composition for the prevention, improvement or treatment of alcoholic gastritis containing dead cells of Lactobacillus salivarius V133 strain as an active ingredient, and more specifically, Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain. It relates to a pharmaceutical composition for preventing, improving or treating alcoholic gastritis containing cells as an active ingredient and a food additive composition for preventing or improving alcoholic gastritis.
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). 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). The various physiological activities of lactic acid bacteria dead cells are not affected by heat and pH due to the characteristics of dead cells, so they 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 알코올성 위염의 예방, 개선 또는 치료용 약학적 조성물과 알코올성 위염의 예방 또는 개선용 식품첨가제 조성물에 대해서는 밝혀진 바가 전혀 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 a strain of Lactobacillus salivarius CJLS1511 (KCCM11829P) or an animal containing dead cells thereof. Although the contents related to the feed additive composition are disclosed, a pharmaceutical composition for the prevention, improvement or treatment of alcoholic gastritis containing dead cells of Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain as an active ingredient and prevention of alcoholic gastritis or Nothing has been revealed about the improvement food additive composition.
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 알코올성 위염의 예방, 개선 또는 치료용 약학적 조성물을 제공함에 그 목적이 있다.An object of the present invention is to provide a pharmaceutical composition for the prevention, improvement or treatment of alcoholic gastritis containing the 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 알코올성 위염의 예방 또는 개선용 식품첨가제 조성물을 제공함에 다른 목적이 있다.In addition, the present invention has another object to provide a food additive composition for preventing or improving alcoholic gastritis 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 알코올성 위염의 예방, 개선 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for the prevention, improvement or treatment of alcoholic gastritis containing the dead cells of the Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain as an active ingredient.
이때, 상기 알코올성 위염의 예방, 개선 또는 치료용 약학적 조성물은, 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체가 조성물 1g당 1×105cfu 내지 1×1012cfu로 포함되는 것을 특징으로 할 수 있다.At this time, the pharmaceutical composition for the prevention, improvement or treatment of alcoholic gastritis, Lactobacillus salivarius V133 (Lactobacillus salivarius V133) 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. It can be characterized by that.
또한, 상기 알코올성 위염의 예방, 개선 또는 치료용 약학적 조성물에 함유된 균주의 사균체는, 락토바실러스 살리바리우스 V133 KCTC18732P 균주를 식용배지에 접종하여 배양액을 제조하고, 배양액을 80℃ 이상의 온도에서 가열한 뒤 60℃ 이하의 온도가 되도록 급속 냉각시키는 공정을 1회 이상 반복 실시하여 열처리한 것임을 특징으로 할 수 있다.In addition, the dead cells of the strain contained in the pharmaceutical composition for the prevention, improvement or treatment of alcoholic gastritis are prepared by inoculating the strain Lactobacillus salivarius V133 KCTC18732P into an edible medium to prepare a culture solution, and heating the culture solution at a temperature of 80°C or higher. After that, it may be characterized in that heat treatment is performed by repeatedly performing a process of rapid cooling to a temperature of 60°C or less once or more.
본 발명은 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체를 유효성분으로 함유하는 알코올성 위염의 예방 또는 개선용 식품첨가제 조성물을 제공한다.The present invention provides a food additive composition for preventing or improving alcoholic gastritis containing the dead cells of the Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain as an active ingredient.
이때, 상기 알코올성 위염의 예방 또는 개선용 식품첨가제 조성물은, 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체가 조성물 1g당 1×105cfu 내지 1×1012cfu로 포함되는 것을 특징으로 할 수 있다.At this time, the food additive composition for preventing or improving alcoholic gastritis is 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. You can do it.
또한, 상기 알코올성 위염의 예방 또는 개선용 식품첨가제 조성물에 함유된 균주의 사균체는, 락토바실러스 살리바리우스 V133 KCTC18732P 균주를 식용배지에 접종하여 배양액을 제조한 뒤, 배양액을 80℃ 이상의 온도에서 가열한 뒤 60℃ 이하의 온도가 되도록 급속 냉각시키는 공정을 1회 이상 반복 실시하여 열처리한 것임을 특징으로 할 수 있다.In addition, the dead cells of the strain contained in the food additive composition for the prevention or improvement of alcoholic gastritis were prepared by inoculating the Lactobacillus salivarius V133 KCTC18732P strain in an edible medium to prepare a culture solution, and then heating the culture solution at a temperature of 80° C. or higher. 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 after one or more times.
본 발명의 일 실시형태에 따른 락토바실러스 살리바리우스 V133 균주의 사균체를 유효성분으로 함유하는 알코올성 위염의 예방, 개선 또는 치료용 조성물은 열처리 공정을 수행함에 따라 세포 독성이 없고 알코올성 위염에 의한 위 손상을 예방 및 개선하여 위염의 치료 효과를 나타내므로, 위염으로 인한 위 손상의 예방 또는 치료용 조성물의 유효성분으로서 효과가 있어 약학적 조성물 또는 식품첨가제 조성물로 사용될 수 있다.The composition for preventing, improving or treating alcoholic gastritis containing dead cells of Lactobacillus salivarius V133 strain as an active ingredient according to an embodiment of the present invention has no cytotoxicity and gastric damage due to alcoholic gastritis as the heat treatment process is performed. Since it exhibits the therapeutic effect of gastritis by preventing and improving gastritis, it is effective as an active ingredient of a composition for preventing or treating gastric damage due to gastritis, so it can be used as a pharmaceutical composition or a food additive composition.
즉, 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 other words, 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 functionality.
또한, 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주를 열처리한 사균체는 알코올성 위염에 대한 육안적 위 손상, 병리학적 변화 및 염증성 사이토카인 생성을 억제하여 위염 예방, 개선 또는 치료의 효과가 있다.In addition, the dead cells heat-treated strain Lactobacillus salivarius V133 KCTC18732P suppress gross gastric damage, pathological changes and inflammatory cytokine production for alcoholic gastritis, thereby preventing, improving or treating gastritis. .
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 투여로 위 손상을 유발한 쥐에서 락토바실러스 살리바리우스 V133 균주와 사균체에 의한 위 점막 손상 억제 효과를 나타낸 사진이다.
도 4는 알코올 투여로 위 손상을 유발한 쥐에서 락토바실러스 살리바리우스 V133 균주와 사균체에 의한 위 조직 병리학적 변화를 나타낸 사진이다.
도 5는 알코올 투여로 위 손상을 유발한 쥐에서 락토바실러스 살리바리우스 V133 균주와 사균체에 의한 위 조직의 염증성 사이토카인 함량 변화를 나타낸 사진이다.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 photograph showing the effect of inhibiting gastric mucosa damage by Lactobacillus salivaryus V133 strain and dead cells in mice that caused gastric damage by alcohol administration.
FIG. 4 is a photograph showing gastric histopathological changes by Lactobacillus salivaryus V133 strain and dead cells in mice that caused gastric damage by alcohol administration.
5 is a photograph showing the change in the content of inflammatory cytokines in gastric tissues by Lactobacillus salivaryus V133 strain and dead cells in rats that caused gastric damage by alcohol administration.
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 알코올성 위염의 예방, 개선 또는 치료용 약학적 조성물(이하, ‘약학적 조성물’이라고도 함)을 제공한다.The present invention provides a pharmaceutical composition for the prevention, improvement or treatment of alcoholic gastritis containing the dead cells of Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain as an active ingredient (hereinafter, also referred to as'pharmaceutical composition'). .
또한, 본 발명은 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체를 유효성분으로 함유하는 알코올성 위염의 예방 또는 개선용 식품첨가제 조성물(이하, ‘식품첨가제 조성물’이라고도 함)을 제공한다.In addition, the present invention provides a food additive composition for preventing or improving alcoholic gastritis containing dead cells of Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain as an active ingredient (hereinafter, also referred to as'food additive composition'). .
본 발명에 따른 약학적 조성물 및 식품첨가제 조성물은 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체를 유효성분으로 함유하는 것으로, 알코올성 위염에 대한 육안적 위 손상, 병리학적 변화 및 염증성 사이토카인 생성을 억제하여 위염 예방, 개선 또는 치료의 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. It is expected that the effect of preventing, ameliorating or treating gastritis by inhibiting the production of kine will be high.
본 발명의 일 실시형태에 따르면, 상기 알코올성 위염의 예방, 개선 또는 치료용 약학적 조성물 및 상기 알코올성 위염의 예방 또는 개선용 식품첨가제 조성물은 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체가 조성물 1g당 1×105cfu 내지 1×1012cfu로 포함되는 것을 특징으로 할 수 있다. According to an embodiment of the present invention, the pharmaceutical composition for the prevention, improvement or treatment of alcoholic gastritis and the food additive composition for preventing or improving alcoholic gastritis are Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain of dead cells It may be characterized in that it is contained in 1×10 5 cfu to 1×10 12 cfu per 1 g of the composition.
이는, 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체가 조성물 1g당 1×105cfu 미만의 농도로 포함될 경우 알코올성 위염의 예방, 개선 또는 치료 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 there is a concern that it may not be possible to describe the effect of preventing, improving or treating alcoholic gastritis when the dead cells of the Lactobacillus salivarius V133 KCTC18732P strain are included at a concentration of less than 1×10 5 cfu per 1 g of the composition, 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).
또한, 본 발명의 일 실시형태에 따르면, 상기 알코올성 위염의 예방, 개선 또는 치료용 약학적 조성물 및 상기 알코올성 위염의 예방 또는 개선용 식품첨가제 조성물에 함유된 균주의 사균체는, 락토바실러스 살리바리우스 V133 KCTC18732P 균주를 식용배지에 접종하여 배양액을 제조한 뒤, 배양액을 미생물의 사멸을 유도 할 수 있는 80℃ 이상의 온도에서 가열한 뒤 60℃ 이하의 온도가 되도록 급속 냉각시키는 공정을 1회 이상 반복 실시하여 열처리한 것임을 특징으로 할 수 있다.In addition, according to an embodiment of the present invention, the dead cells of the strain contained in the pharmaceutical composition for the prevention, improvement or treatment of alcoholic gastritis and the food additive composition for prevention or improvement of alcoholic gastritis, Lactobacillus salivarius V133 After preparing the culture solution by inoculating the KCTC18732P strain in an edible medium, heating the culture solution at 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 is repeated one or more times. It may be characterized by heat treatment.
본 발명의 락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체를 유효성분으로 함유하는 알코올성 위염의 예방, 개선 또는 치료용 약학적 조성물은 알코올성 위염에 대한 육안적 위 손상, 병리학적 변화 및 염증성 사이토카인 생성을 억제하여 위염을 예방, 개선 또는 치료하기 위한 약학적 조성물로 사용될 수 있다.The pharmaceutical composition for the prevention, improvement or treatment of alcoholic gastritis containing the dead cells of the Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain as an active ingredient of the present invention includes gross gastric damage, pathological changes and It can be used as a pharmaceutical composition for preventing, ameliorating or treating gastritis by inhibiting the production of inflammatory cytokines.
본 발명의 약학적 조성물을 사용할 수 있는 질환 또는 질병은 이에 제한되는 것은 아니며, 알코올성 위염으로 야기된 질환 또는 질병은 모두 포함될 수 있다.Diseases or diseases in which the pharmaceutical composition of the present invention can be used are not limited thereto, and all diseases or diseases caused by alcoholic gastritis 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 알코올성 위염의 예방 또는 개선용 식품첨가제 조성물은 알코올성 위염에 대한 육안적 위 손상, 병리학적 변화 및 염증성 사이토카인 생성을 억제하여 위염을 예방 및 개선 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 the prevention or improvement of alcoholic gastritis containing the dead cells of the Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain as an active ingredient of the present invention is the gross gastric damage, pathological changes and inflammatory cytopathy against alcoholic gastritis. It has the effect of preventing and improving gastritis by inhibiting the production of kine, and can be added to foods for this 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 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 mixed solution 1 After taking ㎖, diluted by decimal dilution method, plated on MRS solid medium (Difco, MI, USA) and cultured at 37°C for 48 hours. Single colonies formed in the medium for each sample were spread on MRS solid medium to which bromocresol purple was added, and colonies showing yellow were finally selected. The selected strain was evaluated for antimicrobial activity, and one type of lactic acid bacteria having the best antimicrobial activity was selected, and stored at -70°C with 20% glycerol stock.
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.
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 계산식으로 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 co-aggregation ability was calculated by the following calculation formula according to the method of Kos et al. (2003), and the results are shown in Table 4 below.
[계산식][formula]
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 균주의 사균체의 제조방법은 하기와 같다.The preparation method of the dead cells of the Lactobacillus salivarius 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) KCTC18732P 균주 및 상기의 사균체의 약학적 조성물로서의 활용 특성 분석Analysis of Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain and its utilization characteristics as a pharmaceutical composition of the dead cells
상기 실시예 3에서 제조에 사용된 락토바실러스 살리바리우스 V133 균주와 사균체에 대한 유해균에 대한 항균활성과 알코올성 위염 개선 효과를 평가하였다.The antibacterial activity against harmful bacteria and the effect of improving alcoholic gastritis against the Lactobacillus salivarius V133 strain and dead cells used in the preparation 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 항균황성의 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 liquid state 1×10 6 cfu/ After making it to a concentration of ml, pour it into a petri dish to prepare a base medium, sterilize the MRS liquid medium containing 0.75% agar, and then pour it onto the base medium at 45 to 50 ℃ to make a medium layer. . Then, a well was made using a sterile biopsy punch (Stiefel Biopsy Punch; Stiefel Laboratories, Research Triangle Park, NC, USA). 50 µl of Lactobacillus salivarius V133 strain and dead cells were dispensed into each well, and cultured at 37°C for 24 hours. After incubation, the presence or absence of antimicrobial sulfur properties was confirmed by checking the inhibitory ring formed around the well, and the results are shown in FIG. 2 below.
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 알코올성 위염 개선 효과2. Effect of Lactobacillus salivarius V133 Dead Cells on Alcoholic Gastritis Improvement
< 실험군 설정 및 시료 투여 ><Experimental group setup and sample administration>
(주)오리엔트바이오(Seongnam, Korea)로부터 공급 받은 5주령의 수컷 ICR 마우스를 일주일 동안 설치류 사육실에서 적응시킨 후 적응기간 중 일반 상태를 관찰하여 건강한 개체만 실험에 사용하였다. 사육환경은 온도 22±3℃, 습도 50±5%에서 명/암 주기는 12시간으로 유지하고 식이와 물을 자유롭게 공급하여 1주일간 기본식이로 적응시켰다. 순화기간 중 건강하다고 판정된 마우스에 한하여 무작위법을 이용하여 군 분리하였다.Five-week-old male ICR mice supplied from Orient Bio Co., Ltd. (Seongnam, Korea) were acclimated in a rodent breeding room for a week, and then the general condition was observed during the adaptation period, and only healthy individuals were used for the experiment. The breeding environment was maintained at 22±3℃ and the humidity 50±5%, the light/dark cycle was 12 hours, and diet and water were supplied freely and adapted to the basic diet for 1 week. Only mice judged to be healthy during the acclimatization period were grouped using a random method.
군 분리는 하기 표 5에서와 같이, 정상대조군(G1), 알코올성 위염 유발 시험대조군(G2), 양성대조군(omeprazole 20 mg/kg)(G3), 락토바실러스 살리바리우스 V133 균주 저농도(1×109cfu/g) 50 mg/mice 투여군(G4), 락토바실러스 살리바리우스 V133 균주 고농도(1×109cfu/g) 100 mg/mice 투여군(G5), 락토바실러스 살리바리우스 V133 사균체 저농도(5×108cfu/g) 100 mg/mice 투여군(G6) 및 락토바실러스 살리바리우스 V133 사균체 고농도(5×108cfu/g) 200 mg/mice 투여군(G7), 총 7군으로 군당 6마리씩 구성하여 실험을 진행하였다. G1과 G2군은 DW를 G3, G4, G5, G6, G7군은 시험물질을 각각 매일 1회씩 총 5일 동안 경구투여 한 후, 마지막 시험물질 투여 2시간 후 G2, G3, G4, G5, G6, G7군에 100% 알코올을 1㎖씩 경구투여 하여 알코올성 위염을 유발시켰다. 알코올 투여 4시간 뒤 희생시켜 심장채혈 및 위조직을 적출하였다.Group separation as shown in Table 5 below, normal control (G1), alcoholic gastritis inducing test control (G2), positive control (omeprazole 20 mg / kg) (G3), Lactobacillus salivaryus V133 strain low concentration (1 × 10 9 cfu/g) 50 mg/mice administration group (G4), Lactobacillus salivarius V133 strain high concentration (1×10 9 cfu/g) 100 mg/mice administration group (G5), Lactobacillus salivarius V133 low concentration of dead cells (5×10 8 cfu/g) 100 mg/mice administration group (G6) and Lactobacillus salivarius V133 dead cells high concentration (5×10 8 cfu/g) 200 mg/mice administration group (G7), a total of 7 groups consisting of 6 animals per group. Proceeded. G1 and G2 groups received DW and G3, G4, G5, G6, and G7 groups administered the test substance once daily for a total of 5 days, followed by G2, G3, G4, G5, G6 2 hours after the last test substance administration. , Alcoholic gastritis was induced by oral administration of 1 ml of 100% alcohol to the G7 group. After 4 hours of alcohol administration, a heart blood sample and gastric tissue were extracted by sacrificing.
< 락토바실러스 살리바리우스 V133 사균체의 알코올에 의한 위 점막 손상 억제 효과 ><Effect of Lactobacillus salivarius V133 on dead cells against alcohol-induced gastric mucosa damage>
알코올성 위염 유발 4시간 뒤 희생시킨 후 개복하여 방혈한 다음 위를 십이지장부위와 식도부위에서 절제하여 적출하였다. 적출한 위는 분문부에서 시작하여 대만곡 부위를 따라 정중 절개한 후 내용물을 제거하고 cold PBS로 세척하고 남아있는 PBS를 paper towel로 제거한다. 육안적 소견 평가를 위해 위를 5각형 형태로 고정하여 디지털 카메라로 촬영 하였다. 육안적 소견평가는 하기 표 6과 같이 La Casa et al. (J. Ethnopharmacol. 2000; 71:45-53)의 방법을 통해 모든 개체의 위 병변을 점수화 하였다.After 4 hours of induction of alcoholic gastritis, the victim was sacrificed, bled, and the stomach was excised from the duodenum and esophagus and excised. The extracted stomach starts at the cardiac area, and after a median incision is made along the large portion of the cornea, the contents are removed, washed with cold PBS, and the remaining PBS is removed with a paper towel. To evaluate the visual findings, the stomach was fixed in a pentagonal shape and photographed with a digital camera. The gross findings were evaluated by La Casa et al. Gastric lesions of all subjects were scored by the method of (J. Ethnopharmacol. 2000; 71:45-53).
그 결과는 하기 도 3과 같으며, 알코올성 위염 유발 시험대조군의 위 병변을 점수화한 결과 Grade 6.5의 출혈성 궤양병변이 관찰되었다. 락토바실러스 살리바리우스 V133 균주 저농도와 고농도 투여군은 각각 Grade 4.8, 2.7 정도의 병변이 관찰되어 알코올성 위염 유발 시험대조군 대비 병변의 감소효과를 보였으며 락토바실러스 살리바리우스 V133 사균체 저농도 투여군과 고농도 투여군은 각각 Grade 3.7, 1.7 정도의 병변이 관찰되었다. 이는 락토바실러스 살리바리우스 V133 사균체가 균주 보다 알코올에 의한 위 점막의 손상을 억제함을 알 수 있다.The results are shown in FIG. 3 below, and as a result of scoring the gastric lesions of the alcoholic gastritis-induced test control group, a grade 6.5 hemorrhagic ulcer lesion was observed. Lactobacillus salivarius V133 strain low concentration and high concentration group showed a lesion reduction effect of grade 4.8 and 2.7, respectively, compared to the alcoholic gastritis-inducing test control group. Lesions of 3.7 and 1.7 were observed. It can be seen that the Lactobacillus salivaryus V133 dead cells suppress the damage to the gastric mucosa by alcohol than the strain.
< 락토바실러스 살리바리우스 V133 사균체의 알코올에 의한 위 조직의 병리학적 변화 관찰 ><Observation of pathological changes in gastric tissue of Lactobacillus salivarius V133 dead cells by alcohol>
위 조직을 좌우로 절반으로 절개하고 좌측 중 절반을 10% 중성 포르말린에 고정한 후 3일 뒤 일반적인 조직처리 과정을 거쳐 파라핀 포매하여 조직절편기를 이용하여 4㎛의 두께로 절단한 다음 polylysine으로 coating된 slide에 붙였다. 조직절편은 xylene을 이용하여 파라핀을 제거하고 알코올과 증류수로 10분간 함수시켜 증류수로 세척한 후, 위조직의 변화양상을 관찰하기 위해 H&E(Hematoxylin & Eosin) 염색을 실시하여 광학현미경으로 관찰하였다. 조직학적 소견평가는 하기 표 7과 같이 Kim et al.(Am. J. Physiol. Gastrointest. Liver Physiol. 2012; 304:G193-G202)의 방법을 통해 점수화하였다. The upper tissue is cut in half to the left and right, and the left half is fixed in 10% neutral formalin. After 3 days, after 3 days, paraffin embedding is carried out through a general tissue treatment process, and then cut to a thickness of 4 μm using a tissue sectioning machine, and then a slide coated with polylysine. Attached to Tissue sections were washed with distilled water after removing paraffin with xylene, hydrating with alcohol and distilled water for 10 minutes, and then H&E (Hematoxylin & Eosin) staining was performed and observed with an optical microscope to observe changes in gastric tissue. Histological findings were scored through the method of Kim et al. (Am. J. Physiol. Gastrointest. Liver Physiol. 2012; 304:G193-G202) as shown in Table 7 below.
thickness of gastric mucosaDamage greater than that o score 1 and involving <50% of the
thickness of gastric mucosa
그 결과는 하기 도 4와 같으며, 알코올성 위염 유발 시험대조군은 위점막상피층 전반에 손상을 유발하였으며 현저한 출혈소견과 점막하조직의 부종 및 상피세포의 탈락(또는 괴사) 소견이 관찰됨에 따라 평균 Grade 2.6로 산출되었다. 락토바실러스 살리바리우스 V133 균주 저농도와 고농도 투여군은 각각 Grade 2.2, 1.6 정도의 병변이 관찰되어 알코올에 의한 위점막의 조직학적 손상에 대하여 미미한 보호효과를 보였으나 락토바실러스 살리바리우스 V133 사균체 저농도와 고농도 투여군은 각각 Grade 1.7, 1.0로 알코올성 위염 유발 시험대조군 대비 현저한 감소를 보여 이는 락토바실러스 살리바리우스 V133 사균체가 균주 보다 알코올에 의한 위 점막의 손상을 억제함을 알 수 있다.The results are shown in Fig. 4 below, and the alcoholic gastritis-induced test control induces damage to the entire gastric mucosal epithelium, and remarkable bleeding findings, swelling of the submucosal tissue, and dropout (or necrosis) of epithelial cells were observed. Was calculated as. Lactobacillus salivarius V133 strain low concentration and high concentration group showed a slight protective effect against histologic damage to the gastric mucosa due to grade 2.2 and 1.6 lesions, respectively, but Lactobacillus salivarius V133 dead cells at low and high concentration groups Showed a remarkable decrease compared to the alcoholic gastritis-inducing test control group at Grades 1.7 and 1.0, respectively, indicating that the Lactobacillus salivaryus V133 dead cells suppressed the damage to the gastric mucosa by alcohol than the strain.
< 락토바실러스 살리바리우스 V133 사균체의 알코올에 의한 위 조직의 염증성 사이토카인 함량 관찰 ><Observation of Inflammatory Cytokine Content in Gastric Tissue by Alcohol of Lactobacillus salivarius V133 Dead Cells>
위 조직을 좌우로 절반으로 절개하고 좌측 중 나머지 절반을 200㎎ 정량한 후, 완충액 (100mM Tris-HCl, 250mM sucrose pH 7.4)를 첨가하여 균질화하였다. 균질화 후, 새로운 튜브에 담아 3,500rpm에 15분간 원심분리하였다. 상층액을 분리하여 샘플로 사용하였다. 준비된 샘플에서 interleukin-1β(IL-1β), IL-6, tumor necrosis factor-α(TNF-α) 함량은 IL-1β, IL-6, TNF-α kit (R&D Systems, Minneapolis, MN, USA)를 사용하여 enzyme-linked immunosorbent assay (ELISA)법으로 측정하였다. 반응액의 발색정도는 ELISA reader로 각 분석 kit별 최대흡수파장에서 흡광도를 측정한 후 표준곡선을 이용하여 사이토카인 농도를 정량화하였다.The stomach tissue was cut in half left and right, and the other half of the left was quantified 200 mg, and then a buffer solution (100mM Tris-HCl, 250mM sucrose pH 7.4) was added to homogenize. After homogenization, it was placed in a new tube and centrifuged for 15 minutes at 3,500 rpm. The supernatant was separated and used as a sample. The content of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) in the prepared sample is IL-1β, IL-6, TNF-α kit (R&D Systems, Minneapolis, MN, USA) Was measured by enzyme-linked immunosorbent assay (ELISA). The degree of color development of the reaction solution was measured using an ELISA reader at the maximum absorption wavelength for each analysis kit, and then the cytokine concentration was quantified using a standard curve.
그 결과는 하기 도 5와 같으며, 알코올성 위염 유발 시험대조군은 위 조직 내 IL-1β, IL-6, TNF-α함량이 정상대조군에 비해 각각 100%, 58%, 148% 증가하였다. 락토바실러스 살리바리우스 V133 균주 저농도 투여군은 알코올성 위염 유발 시험대조군 대비 IL-1β, IL-6, TNF-α함량이 각 23%, 17%, 16% 낮은 함량을 나타내었으며 고농도 투여군은 각각 20%, 18%, 25% 낮은 함량이 관찰되었다. 락토바실러스 살리바리우스 V133 사균체 저농도 투여군은 알코올성 위염 유발 시험대조군 대비 각각 13%, 15%, 14% 낮게 관찰되었으며 고농도 투여군에서는 각각 22%, 21%, 20% 낮은 함량을 보였다. 그 결과, 락토바실러스 살리바리우스 V133 사균체 저농도의 투여는 균주 보다 알코올에 의한 위 조직 내 염증성 사이토카인의 생성을 억제함을 알 수 있다.The results are shown in FIG. 5 below, and the alcoholic gastritis-induced test control increased the contents of IL-1β, IL-6, and TNF-α in the gastric tissue by 100%, 58%, and 148%, respectively, compared to the normal control group. Lactobacillus salivarius V133 strain in the low-dose group showed 23%, 17%, and 16% lower levels of IL-1β, IL-6, and TNF-α, respectively, compared to the alcoholic gastritis-inducing test control group. %, 25% lower content was observed. The low concentration of Lactobacillus salivarius V133 was observed to be 13%, 15%, and 14% lower than that of the alcoholic gastritis-inducing test control group, respectively, and 22%, 21%, and 20% lower contents in the high concentration group, respectively. As a result, it can be seen that administration of a low concentration of Lactobacillus salivarius V133 dead cells suppresses the production of inflammatory cytokines in gastric tissues by alcohol than the strain.
결론적으로, 상기 실시예 1 내지 4의 실험 결과를 통해, 락토바실러스 살리바리우스 V133 균주의 사균체는 알코올성 위염에 대한 육안적 위 손상, 병리학적 변화 및 염증성 사이토카인 생성을 억제하여 위염 예방, 개선 또는 치료의 효과가 있기 때문에 알코올성 위염의 예방, 개선 또는 치료용 약학적 조성물 또는 알코올성 위염의 예방 또는 개선용 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 salivarius V133 strain inhibited gross gastric damage, pathological changes, and inflammatory cytokine production for alcoholic gastritis to prevent, improve, or Because of its therapeutic effect, it has the advantage that it can be used as a pharmaceutical composition for preventing, ameliorating or treating alcoholic gastritis or a food additive composition for preventing or ameliorating alcoholic gastritis, and Lactobacillus salivarius V133 (Lactobacillus salivarius V133) KCTC18732P strain Killing through heat treatment has the advantage of improving the shortcomings of live bacteria (stability, product applicability, etc.) to provide a material that is stable and can be applied to a variety of products and has functionality equal to or higher than that of live bacteria.
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 알코올성 위염의 예방, 개선 또는 치료용 약학적 조성물은,
락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체가 조성물 1g당 1×105cfu 내지 1×1012cfu로 포함되는 것을 특징으로 하는 알코올성 위염의 예방, 개선 또는 치료용 약학적 조성물.The method according to claim 1,
The pharmaceutical composition for the prevention, improvement or treatment of alcoholic gastritis,
Lactobacillus salivarius V133 (Lactobacillus salivarius V133) A pharmaceutical composition for the prevention, improvement or treatment of alcoholic gastritis, 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 KCTC18732P 균주를 식용배지에 접종하여 배양액을 제조하고, 배양액을 80℃ 이상의 온도에서 가열한 뒤 60℃ 이하의 온도가 되도록 급속 냉각시키는 공정을 1회 이상 반복 실시하여 열처리한 것임을 특징으로 하는 알코올성 위염의 예방, 개선 또는 치료용 약학적 조성물.The method according to claim 1,
Dead cells of the strain,
A culture solution was prepared by inoculating the Lactobacillus salivarian strain V133 KCTC18732P in an edible medium, and the culture solution was heated at a temperature of 80°C or higher and then rapidly cooled to a temperature of 60°C or less. A pharmaceutical composition for the prevention, improvement or treatment of alcoholic gastritis.
상기 알코올성 위염의 예방 또는 개선용 식품첨가제 조성물은,
락토바실러스 살리바리우스 V133(Lactobacillus salivarius V133) KCTC18732P 균주의 사균체가 조성물 1g당 1×105cfu 내지 1×1012cfu로 포함되는 것을 특징으로 하는 알코올성 위염의 예방 또는 개선용 식품첨가제 조성물.The method of claim 4,
The food additive composition for preventing or improving alcoholic gastritis,
Lactobacillus salivarius V133 (Lactobacillus salivarius V133) A food additive composition for the prevention or improvement of alcoholic gastritis, 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 KCTC18732P 균주를 식용배지에 접종하여 배양액을 제조한 뒤, 배양액을 80℃ 이상의 온도에서 가열한 뒤 60℃ 이하의 온도가 되도록 급속 냉각시키는 공정을 1회 이상 반복 실시하여 열처리한 것임을 특징으로 하는 알코올성 위염의 예방 또는 개선용 식품첨가제 조성물.The method of claim 4,
Dead cells of the strain,
After preparing a culture solution by inoculating Lactobacillus salivarius V133 KCTC18732P strain in an edible medium, heating the culture solution at a temperature of 80°C or higher and then rapidly cooling it to a temperature of 60°C or less was repeated at least once to heat treatment. Food additive composition for preventing or improving alcoholic gastritis, characterized in that.
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