KR102027029B1 - Natural liposome comprising lactic acid bacteria, process for the preparation thereof and food or pharmaceutical composition comprising the same - Google Patents
Natural liposome comprising lactic acid bacteria, process for the preparation thereof and food or pharmaceutical composition comprising the same Download PDFInfo
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- KR102027029B1 KR102027029B1 KR1020170064792A KR20170064792A KR102027029B1 KR 102027029 B1 KR102027029 B1 KR 102027029B1 KR 1020170064792 A KR1020170064792 A KR 1020170064792A KR 20170064792 A KR20170064792 A KR 20170064792A KR 102027029 B1 KR102027029 B1 KR 102027029B1
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- Prior art keywords
- lactic acid
- natural
- acid bacteria
- lactobacillus
- liposomes
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- A23V2300/48—Ultrasonic treatment
Abstract
본 발명은 (a) 코어로서 유산균; (b) 상기 코어를 둘러싸며, 천연 다당체를 포함하는 제1쉘층; (c) 상기 제1쉘층을 둘러싸며, 천연 유화제 및 천연 단백질을 포함하는 제2쉘층; 및 (d) 상기 제2쉘층을 둘러싸며, 천연 동결보호제를 포함하는 제3쉘층을 포함하는 유산균 함유 천연 리포좀, 그 제조방법 및 이를 포함하는 식품 또는 약학 조성물에 관한 것이다.The present invention (a) as a core lactic acid bacteria; (b) a first shell layer surrounding the core and comprising natural polysaccharides; (c) a second shell layer surrounding the first shell layer and comprising a natural emulsifier and a natural protein; And (d) a lactic acid bacterium-containing natural liposome surrounding the second shell layer, the third shell layer comprising a natural cryoprotectant, a method of preparing the same, and a food or pharmaceutical composition comprising the same.
Description
본 발명은 유산균 함유 천연 리포좀, 그 제조방법 및 이를 포함하는 식품 또는 약학 조성물에 관한 것이다.The present invention relates to lactic acid bacteria-containing natural liposomes, a method for producing the same, and a food or pharmaceutical composition comprising the same.
유산균(Lactic acid bacteria)은 락트산균 또는 젖산균이라고도 하며, 산소가 적은 환경에서 자라면서 주로 당류를 영양공급원으로 사용하고 젖산을 생성하는 세균을 모두 일컫는다. 이러한 유산균은 락토바실러스(Lactobacillus), 락토코커스(Lactococcus), 류코노스톡(Leuconostoc), 페디오코커스(Pediococcus), 비피도박테리움(Bifidobacterium) 등의 균속에 해당된다. 유산균은 오래 전부터 유제품(요구르트, 치즈 등), 김치, 양조식품(청주, 된장, 간장 등) 등의 발효식품 제조에 이용되어 왔으며, 포유류의 장내에서 유익균(프로바이오틱스, Probiotics)으로서 클로스트리디움(Clostridium), 대장균(Escherichia Coli), 박테로이데스(Bacteroides), 유박테리움(Eubacterium), 펩토스트렙토커스(Peptostreptococcus) 등과 같은 유해균의 이상발효를 억제하는 정장제(Digestive)로도 알려져 있다. 특히, 유산균이 장에 정착할 경우에는 유산균에 의한 장관운동 활성, 소화불량 개선, 배변활동 원활, 면역력 증강 등 다양한 생리효과를 기대할 수 있다. 이러한 장 기능 개선을 위해 유산균 제품을 섭취할 수 있으나, 일반적으로 섭취된 유산균은 소화기관인 위와 십이지장을 거치면서 위산, 담즙과 같은 소화효소에 의해 대부분 사멸되어 유산균에 의한 생리활성 효과를 얻지 못하게 된다.Lactic acid bacteria, also known as lactic acid bacteria or lactic acid bacteria, refers to all bacteria that grow in low-oxygen environments and use sugar as a nutrient source and produce lactic acid. Such lactic acid bacteria are Lactobacillus (Lactobacillus), Lactococcus (Lactococcus), Leukonostoc (Leuconostoc), Pediococcus (Pediococcus), Bifidobacterium (Bifidobacterium) and the like. Lactic acid bacteria have long been used in the manufacture of fermented food products such as dairy products (yogurt, cheese, etc.), kimchi, brewed foods (cheongju, soybean paste, soy sauce, etc.) and as a beneficial bacterium (probiotics, Probiotics) in mammalian intestines, Clostridium ), E. coli (Escherichia Coli), Bacteroides (Bacteroides), Eubacterium (Eubacterium), Pepttostreptococcus (Peptostreptococcus) is also known as a digestive (Digestive) that inhibits abnormal fermentation. In particular, when lactic acid bacteria settle in the intestine, various physiological effects such as intestinal exercise activity, improvement of indigestion, smooth bowel activity, and immunity enhancement can be expected. The lactic acid bacteria can be ingested to improve the intestinal function, but in general, the lactic acid bacteria ingested are mostly killed by digestive enzymes such as gastric acid and bile through the stomach and duodenum, which are digestive organs, thereby preventing the physiological activity effect of the lactic acid bacteria.
이러한 문제점을 해결하기 위해, 다양한 코팅 방법이 개발되었다. 한국등록특허 제0395722호에는 장에서만 녹을 수 있는 장용피막코팅 조성물이 기재되어 있다. 그러나, 장용피막코팅 조성물에는 점착방지제, 알칼리화제, 가소제 등이 함유되어 있어, 이러한 화학 물질에 대한 유해성 논란이 발생할 수 있다. 또한, 한국등록특허 제0373104호에는 유산균을 다공성 미립담체에 흡착시켜 다당체로 코팅하는 방법이 기재되어 있다. 그러나, 미립담체로 규조토, 활성탄 및/또는 제올라이트를 사용하여 유해성 논란이 일어날 수 있으며, 이러한 유산균이 흡착된 미립담체를 다당체로 코팅할 경우에는 미립담체의 표면을 완전히 코팅할 수 없어 소화효소로부터 유산균을 보호하기 어렵다.To solve this problem, various coating methods have been developed. Korean Patent No. 0395722 describes an enteric coating coating composition that can be dissolved only in the intestine. However, the enteric coating coating composition contains an anti-sticking agent, an alkalizing agent, a plasticizer, and the like, and thus, a controversy may be caused about such chemicals. In addition, Korean Patent No. 0373104 describes a method of adsorbing lactic acid bacteria to porous particulate carriers and coating them with polysaccharides. However, harmful controversy may arise using diatomaceous earth, activated carbon and / or zeolite as a microcarrier, and when the microcarriers to which these lactic acid bacteria are adsorbed are coated with polysaccharides, the surface of the microcarrier may not be completely coated. Hard to protect.
이와 같이, 종래 유산균 제품은 코팅 성분으로 사용된 (화학) 물질에 의해 유해성 문제가 제기되며, 형성된 코팅층은 충분한 내산성 및 내담즙성이 보장되지 않아 유산균에 의한 생리효과를 기대하기 어렵다. 따라서, 유해한 성분을 함유하지 않으면서 유산균을 장까지 안전하게 전달할 수 있는 유산균의 코팅 기술에 대해, 여전히 많은 연구개발이 필요한 실정이다.As such, the conventional lactic acid bacteria products pose a hazard problem by the (chemical) material used as the coating component, the formed coating layer is difficult to expect the physiological effect by the lactic acid bacteria because the sufficient acid resistance and bile resistance is not guaranteed. Therefore, a lot of research and development is still required for the coating technology of lactic acid bacteria that can safely deliver the lactic acid bacteria to the intestine without containing harmful ingredients.
상기한 문제점을 해결하기 위해, 본 발명은 인체에 무해한 천연 물질을 사용하여 유산균을 장까지 안전하게 전달할 수 있는 유산균 함유 천연 리포좀, 그 제조방법 및 이를 포함하는 식품 또는 약학 조성물을 제공하는 것을 목적으로 한다.In order to solve the above problems, an object of the present invention is to provide a lactic acid bacteria-containing natural liposomes that can safely deliver the lactic acid bacteria to the intestine using a natural material harmless to the human body, a method for producing the same and a food or pharmaceutical composition comprising the same. .
본 발명은 (a) 코어로서 유산균; (b) 상기 코어를 둘러싸며, 천연 다당체를 포함하는 제1쉘층; (c) 상기 제1쉘층을 둘러싸며, 천연 유화제 및 천연 단백질을 포함하는 제2쉘층; 및 (d) 상기 제2쉘층을 둘러싸며, 천연 동결보호제를 포함하는 제3쉘층을 포함하는 유산균 함유 천연 리포좀을 제공한다. 이때, 상기 제3쉘층은 천연 보존제를 더 포함할 수 있다.The present invention (a) as a core lactic acid bacteria; (b) a first shell layer surrounding the core and comprising natural polysaccharides; (c) a second shell layer surrounding the first shell layer and comprising a natural emulsifier and a natural protein; And (d) provides a lactic acid bacteria-containing natural liposome surrounding the second shell layer, comprising a third shell layer comprising a natural cryoprotectant. In this case, the third shell layer may further include a natural preservative.
또한, 본 발명은 (i) 40 내지 75℃의 용매에 천연 유화제를 투입하여 초음파 처리하는 제1단계; (ii) 40 내지 75℃의 용매에 천연 단백질, 천연 다당체를 투입하여 초음파 처리한 후 유산균을 투입하여 혼합하는 제2단계; (iii) 상기 제1단계에 얻어진 생성물, 상기 제2단계에서 얻어진 생성물, 천연 동결보호제를 혼합하는 제3단계; 및 (iv) 상기 제3단계에서 얻어진 생성물을 동결건조하는 제4단계를 포함하는 유산균 함유 천연 리포좀의 제조방법을 제공한다. 이때, 상기 제4단계에 천연 보존제를 더 투입하여 혼합할 수 있다.In addition, the present invention (i) a first step of ultrasonic treatment by adding a natural emulsifier to the solvent of 40 to 75 ℃; (ii) a second step of adding natural protein and natural polysaccharide to a solvent at 40 to 75 ° C., sonicating the same, and then adding and mixing the lactic acid bacteria; (iii) a third step of mixing the product obtained in the first step, the product obtained in the second step, and a natural cryoprotectant; And (iv) provides a method for producing a lactic acid bacteria-containing natural liposomes comprising a fourth step of lyophilizing the product obtained in the third step. At this time, the natural preservative may be further added and mixed in the fourth step.
또한, 본 발명은 (i) 40 내지 75℃의 용매에 천연 다당체를 투입하여 초음파 처리한 후 유산균을 투입하여 혼합하는 제1단계; (ii) 40 내지 75℃의 용매에 천연 유화제를 투입하여 초음파 처리하는 제2단계; (iii) 40 내지 75℃의 용매에 천연 단백질을 투입하여 초음파 처리하는 제3단계; (iv) 상기 제1단계에서 얻어진 생성물, 상기 제2단계에서 얻어진 생성물, 제3단계에서 얻어진 생성물, 천연 동결보호제를 혼합하는 제4단계; 및 (v) 상기 제4단계에서 얻어진 생성물을 동결건조하는 제5단계를 포함하는 유산균 함유 천연 리포좀의 제조방법을 제공한다. 이때, 상기 제5단계에 천연 보존제를 더 투입하여 혼합할 수 있다.In addition, the present invention (i) a first step of adding a natural polysaccharide to the solvent at 40 to 75 ℃ ultrasonic treatment and then adding and mixing the lactic acid bacteria; (ii) a second step of ultrasonically adding a natural emulsifier to a solvent at 40 to 75 ° C; (iii) a third step of sonicating natural protein in a solvent at 40 to 75 ° C; (iv) a fourth step of mixing the product obtained in the first step, the product obtained in the second step, the product obtained in the third step, and a natural cryoprotectant; And (v) provides a method for producing a lactic acid bacteria-containing natural liposomes comprising a fifth step of lyophilizing the product obtained in the fourth step. At this time, the natural preservative may be further added and mixed in the fifth step.
또한, 본 발명은 상기 유산균 함유 천연 리포좀을 유효성분으로 포함하는 식품 조성물, 장염 예방 또는 치료용 약학 조성물을 제공한다.In another aspect, the present invention provides a food composition, enteritis prevention or therapeutic pharmaceutical composition comprising the lactic acid bacteria-containing natural liposomes as an active ingredient.
본 발명에 따른 유산균 함유 천연 리포좀은 저장 및 유통 과정에서 유산균이 파괴되거나 유산균의 활성이 저하되는 것을 방지하고, 유산균을 위산과 담즙으로부터 보호하여 장까지 안전하게 전달할 수 있다. 이러한 유산균 함유 천연 리포좀을 포함하는 식품 또는 약학 조성물은 종래 유산균 제품에 비해 내산성 및 내담즙성이 우수하여 유산균의 장내 정착능을 향상시킨다. 상기 식품 또는 약학 조성물을 섭취할 경우에는 유산균이 장내 정착함으로써 유해균의 생장 및 증식을 저해하여 장관운동 및 배변활동을 원활하게 하고, 장내 면역세포를 증가시켜 손상된 장 세포를 빠르게 회복시킴으로써 장염, 대장암과 같은 장내 질환을 예방하고 치료하는데 도움이 될 수 있다.The lactic acid bacteria-containing natural liposomes according to the present invention prevent lactic acid bacteria from being destroyed or deteriorated in the activity of lactic acid bacteria during storage and distribution, and protect the lactic acid bacteria from gastric acid and bile, thereby safely delivering them to the intestine. The food or pharmaceutical composition comprising such lactic acid bacteria-containing natural liposomes has superior acid resistance and bile resistance compared to conventional lactic acid bacteria products to improve the intestinal fixation ability of lactic acid bacteria. When the food or pharmaceutical composition is ingested, the lactic acid bacteria settle in the intestine, inhibiting the growth and proliferation of harmful bacteria to facilitate bowel movement and bowel movements, and increase the intestinal immune cells to quickly recover damaged intestinal cells. It can help prevent and treat intestinal diseases such as
도 1은 본 발명의 일 예에 따른 유산균 함유 천연 리포좀의 제조방법의 모식도이다.
도 2는 본 발명의 일 예에 따른 유산균 함유 천연 리포좀의 제조방법의 모식도이다.
도 3은 본 발명의 실험예 5에 따른 베타-글루쿠로니다아제의 활성을 확인한 그래프이다.
도 4는 본 발명의 실험예 5에 따른 베타-글루코시다아제의 활성을 확인한 그래프이다.
도 5는 본 발명의 실험예 6에 따른 장 상피세포의 생존율을 확인한 그래프이다.
도 6은 본 발명의 실험예 6에 따른 장벽형성 단백질의 발현능을 확인한 그래프이다.
도 7은 본 발명의 실험예 6에 따른 물리적 손상 회복능을 확인한 이미지이다.
도 8은 본 발명의 실험예 7에 따른 대장암 세포에 형성된 콜로니를 확인한 그래프이다.
도 9는 본 발명의 실험예 8에 따른 장내 유익균과 유해균의 수를 확인한 그래프이다.
도 10은 본 발명의 실험예 8에 따른 IL-6의 분비량을 확인한 그래프이다.
도 11은 본 발명의 실험예 8에 따른 TNF-a의 분비량을 확인한 그래프이다.
도 12는 본 발명의 실험예 8에 따른 IgA의 분비량을 확인한 그래프이다.
도 13은 본 발명의 실험예 8에 따른 M 세포의 분포를 확인한 이미지이다.
도 14는 본 발명의 실험예 8에 따른 T 세포의 분포를 확인한 이미지이다.
도 15는 본 발명의 실험예 8에 따른 T 세포의 분포를 확인한 이미지이다.
도 16은 본 발명의 실험예 8에 따른 B 세포의 분포를 확인한 이미지이다.1 is a schematic diagram of a method for producing a lactic acid bacteria-containing natural liposome according to an embodiment of the present invention.
Figure 2 is a schematic diagram of the production method of lactic acid bacteria-containing natural liposomes according to an embodiment of the present invention.
3 is a graph confirming the activity of beta-glucuronidase according to Experimental Example 5 of the present invention.
4 is a graph confirming the activity of beta-glucosidase according to Experimental Example 5 of the present invention.
5 is a graph confirming the survival rate of intestinal epithelial cells according to Experimental Example 6 of the present invention.
6 is a graph confirming the expression ability of the barrier protein according to Experimental Example 6 of the present invention.
7 is an image confirming the physical damage recovery ability according to Experimental Example 6 of the present invention.
8 is a graph confirming colonies formed in the colorectal cancer cells according to Experimental Example 7 of the present invention.
9 is a graph confirming the number of enteric beneficial bacteria and harmful bacteria according to Experimental Example 8 of the present invention.
10 is a graph confirming the secretion amount of IL-6 according to Experimental Example 8 of the present invention.
11 is a graph confirming the secretion amount of TNF-a according to Experimental Example 8 of the present invention.
12 is a graph confirming the secretion amount of IgA according to Experimental Example 8 of the present invention.
13 is an image confirming the distribution of M cells according to Experimental Example 8 of the present invention.
14 is an image confirming the distribution of T cells according to Experimental Example 8 of the present invention.
15 is an image confirming the distribution of T cells according to Experimental Example 8 of the present invention.
16 is an image confirming the distribution of B cells according to Experimental Example 8 of the present invention.
이하, 첨부된 도면을 참조하여 본 발명에 따른 유산균 함유 천연 리포좀, 그 제조방법 및 이를 포함하는 식품 또는 약학 조성물을 보다 상세하게 설명한다. 그러나, 이러한 설명은 본 발명의 이해를 돕기 위하여 예시적으로 제시된 것일 뿐 본 발명의 범위가 이러한 예시적인 설명에 의하여 제한되는 것은 아니다.Hereinafter, with reference to the accompanying drawings will be described in detail the lactic acid bacteria-containing natural liposomes according to the present invention, a method for preparing the same and a food or pharmaceutical composition comprising the same. However, these descriptions are provided by way of example only to assist in understanding the present invention, and the scope of the present invention is not limited by these exemplary descriptions.
<유산균 함유 천연 <Lactobacillus-containing natural 리포좀Liposomes >>
본 발명은 (a) 코어로서 유산균; (b) 상기 코어를 둘러싸며, 천연 다당체를 포함하는 제1쉘층; (c) 상기 제1쉘층을 둘러싸며, 천연 유화제 및 천연 단백질을 포함하는 제2쉘층; 및 (d) 상기 제2쉘층을 둘러싸며, 천연 동결보호제를 포함하는 제3쉘층을 포함하는 유산균 함유 천연 리포좀을 제공한다. 이때, 상기 제3쉘층은 천연 보존제를 더 포함할 수 있다. 이러한 유산균 함유 천연 리포좀은 유산균을 다층으로 둘러싼 형태이기 때문에 장내 소화효소에 의해 유산균이 파괴되거나 유산균의 활성이 저하되는 것이 방지될 수 있어, 유산균을 장내로 안전하게 전달할 수 있다. The present invention (a) as a core lactic acid bacteria; (b) a first shell layer surrounding the core and comprising natural polysaccharides; (c) a second shell layer surrounding the first shell layer and comprising a natural emulsifier and a natural protein; And (d) provides a lactic acid bacteria-containing natural liposome surrounding the second shell layer, comprising a third shell layer comprising a natural cryoprotectant. In this case, the third shell layer may further include a natural preservative. Since the lactic acid bacteria-containing natural liposomes have a form surrounding the lactic acid bacteria in multiple layers, the lactic acid bacteria can be prevented from being destroyed by the intestinal digestive enzymes or the activity of the lactic acid bacteria can be prevented, so that the lactic acid bacteria can be safely delivered to the intestine.
이하, 상기 유산균 함유 천연 리포좀을 각 층별로 나누어 설명하면 다음과 같다.Hereinafter, the lactic acid bacteria-containing natural liposomes will be described by dividing each layer as follows.
(a) 코어: 유산균(a) Core: lactic acid bacteria
3층 구조의 유산균 함유 천연 리포좀은 코어(Core)에 유산균이 위치한다.Lactic acid bacteria-containing natural liposomes of the three-layer structure is the lactic acid bacteria are located in the core (Core).
상기 유산균은 일반적으로 알려진 락토바실러스(Lactobacillus), 락토코커스(Lactococcus), 류코노스톡(Leuconostoc), 페디오코커스(Pediococcus), 비피도박테리움(Bifidobacterium) 등의 균속에 해당되는 균주일 수 있다. 보다 구체적으로는, 락토바실러스 아시도필루스(Lactobacillus acidophilus), 락토바실러스 불가리쿠스(Lactobacillus bulgaricus), 락토바실러스 카제이(Lactobacillus casei), 락토바실러스 퍼멘텀(Lactobacillus fermentum), 락토바실러스 가세리(Lactobacillus gasseri), 락토바실러스 헬베티쿠스(Lactobacillus helveticus), 락토바실러스 파라카제이(Lactobacillus paracasei), 락토바실러스 플란타룸(Lactobacillus plantarum), 락토바실러스 람노서스(Lactobacillus rhamnosus), 락토바실러스 루테리(Lactobacillus reuteri), 락토바실러스 살리바리우스(Lactobacillus salivarius), 락토코커스 락티스(Lactococcus lactis), 스트렙토코커스 써모필러스(Streptococcus thermophilus), 비피도박테리움 애니멀리스 락티스(Bifidobacterium animalis ssp. lactis), 비피도박테리움 비피덤(Bifidobacterium bifidum), 비피도박테리움 브레브(Bifidobacterium breve), 비피도박테리움 롱검(Bifidobacterium longum) 및 엔테로코커스 페칼리스(Enterococcus faecalis)로 이루어진 군에서 선택되는 1종 이상일 수 있다. The lactic acid bacterium may be a strain corresponding to a bacterium such as Lactobacillus, Lactococcus, Lactococcus, Leuconostoc, Pediococcus, Bifidobacterium, and the like. More specifically, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus fermentum, Lactobacillus gaseri (Lactobacillus fermentum) ), Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus rhamnosus, Lactobacillus luteus reuteri Lactobacillus salivarius, Lactococcus lactis, Streptococcus thermophilus, Bifidobacterium animalis ssp.lactis, Bifidobacterium bifiderm Bifidobacterium bifidum, Bifidobacterium breve, Bifidogam One member selected from the group consisting of Solarium ronggeom (Bifidobacterium longum) and Enterococcus faecalis (Enterococcus faecalis) may be equal to or greater than.
본 발명의 일 예에 따르면, 상기 유산균은 락토바실러스 아시도필루스, 비피도박테리움 비피덤 및 비피도박테리움 롱검일 수 있다.According to one embodiment of the present invention, the lactic acid bacteria may be Lactobacillus asidophilus, Bifidobacterium bifidum and Bifidobacterium longgum.
본 발명의 다른 일 예에 따르면, 상기 유산균은 락토바실러스 아시도필루스, 락토바실러스 퍼멘텀, 락토바실러스 플란타룸, 락토바실러스 람노서스, 스트렙토코커스 써모필러스, 비피도박테리움 애니멀리스 락티스 및 비피도박테리움 롱검일 수 있다.According to another embodiment of the present invention, the lactic acid bacteria are Lactobacillus asidophilus, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus rhamnosus, Streptococcus thermophyllus, Bifidobacterium animal lactis and Bifidobacterium longgum.
이러한 유산균을 이용하여 코어 형성시, 균질기(Homogenizer) 및/또는 교반기를 사용하는 것이 바람직하다. 제1쉘층 및 제2쉘층 형성시 사용되는 초음파 파쇄기(Ultrasonicator)는 열과 진동을 발생시키므로, 이로 인해 유산균이 파괴되거나 손상될 수 있기 때문이다.In forming the core using such lactic acid bacteria, it is preferable to use a homogenizer and / or a stirrer. Ultrasonic crusher (Ultrasonicator) used when forming the first shell layer and the second shell layer because it generates heat and vibration, this is because the lactic acid bacteria can be destroyed or damaged.
(b) (b) 제1쉘층First shell layer : 천연 다당체Natural polysaccharide
제1쉘층은 상기 유산균을 둘러싼 층으로, 유산균을 위산 및 담즙으로부터 보호하기 위해 천연 다당체를 포함한다.The first shell layer is a layer surrounding the lactic acid bacteria, and contains natural polysaccharides to protect the lactic acid bacteria from gastric acid and bile.
상기 천연 다당체로는 천연 물질로부터 추출 및 정제된 것으로, 아라비아검(Arabic gum), 구아검(Guar gum), 로커스트빈검(Locust bean gum), 타마린드검(Tamarind gum), 타라검(Tara gum), 잔탄검(Xanthan gum) 및 카라기난(Carrageenan)으로 이루어진 군에서 선택되는 1종 이상을 사용할 수 있다. 바람직하게는, 로커스트빈검, 타마린드검 및 타라검으로 이루어진 군에서 선택되는 1종 이상을 사용할 수 있다.The natural polysaccharide is extracted and purified from a natural substance, Arabic gum, Guar gum, Locust bean gum, Tamarind gum, Tara gum At least one selected from the group consisting of Xanthan gum and Carrageenan may be used. Preferably, one or more selected from the group consisting of locust bean gum, tamarind gum, and tara gum can be used.
상기 천연 다당체는 장에서 분해되어, 장내 정착하는 유산균의 생장 및 증식을 위한 영양공급원(프리바이오틱스, Prebiotics)으로 이용될 수 있다.The natural polysaccharide is degraded in the intestine, and can be used as a nutrient source (prebiotics, Prebiotics) for growth and proliferation of lactic acid bacteria that enter the intestine.
이러한 천연 다당체를 이용하여 제1쉘층 형성시, 천연 다당체가 용매에 균일하게 분산될 수 있도록 초음파 파쇄기를 사용할 수 있다.When the first shell layer is formed using the natural polysaccharide, an ultrasonic crusher may be used to uniformly disperse the natural polysaccharide in the solvent.
(c) (c) 제2쉘층Second shell layer : 천연 유화제 및 천연 단백질: Natural Emulsifiers & Natural Proteins
제2쉘층은 상기 천연 다당체를 둘러싼 층으로, 유산균을 장에 안전하고 용이하게 전달하기 위해 천연 유화제 및 천연 단백질을 포함한다.The second shell layer is a layer surrounding the natural polysaccharide, and contains a natural emulsifier and a natural protein to safely and easily deliver the lactic acid bacteria to the intestine.
상기 천연 유화제는 수용성인 인산과 지용성인 지방산으로 구성된 인지질로, 지질 이중층을 형성한다. The natural emulsifier is a phospholipid consisting of water-soluble phosphoric acid and fat-soluble fatty acids, and forms a lipid bilayer.
상기 천연 유화제로는 포스파티딜콜린, 리소포스파티딜콜린, 포스파티딜에탄올아민, 포스파티딜산, 포스파티딜세린, 포스파티딜글리세롤, 포스파티딜이노시톨 및 이들의 수소첨가 생성물로 이루어진 군에서 선택되는 1종 이상을 사용할 수 있다. 이때, 산화안전성이 우수한 포스파티딜콜린의 수소첨가 생성물(하이드로제네이티드 포스파티딜콜린, Hydrogenated phospatidylcholine)을 사용하는 것이 바람직하다.As the natural emulsifier, at least one selected from the group consisting of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidyl acid, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and hydrogenated products thereof may be used. At this time, it is preferable to use a hydrogenated product (hydrogenated phospatidylcholine) of phosphatidylcholine having excellent oxidation safety.
상기 천연 단백질로는 천연 물질로부터 추출 및 정제된 것을 모두 사용할 수 있다. As the natural protein, both extracted and purified from natural materials can be used.
상기 천연 단백질은 장에서 분해되어, 장내 정착하는 유산균의 생장 및 증식을 위한 영양공급원으로 이용될 수 있다. 이때, 유산균의 영양공급원으로 적합한 우유 유래 단백질 및/또는 콩 유래 단백질을 사용하는 것이 바람직하며, 특히 유청단백질을 사용하는 것이 더욱 바람직하다.The natural protein is degraded in the intestine, and can be used as a nutrient source for growth and proliferation of lactic acid bacteria that enter the intestine. At this time, it is preferable to use a milk-derived protein and / or soy-derived protein suitable as a nutritional source of lactic acid bacteria, more preferably whey protein.
이러한 천연 유화제 및 천연 단백질을 이용하여 제2쉘층 형성시, 천연 유화제 및 천연 단백질이 용매에 균일하게 분산될 수 있도록 초음파 분쇄기를 사용할 수 있다.When the second shell layer is formed using the natural emulsifier and the natural protein, an ultrasonic grinder may be used to uniformly disperse the natural emulsifier and the natural protein in the solvent.
(d) (d) 제3쉘층3rd shell layer : 천연 동결보호제: Natural cryoprotectant
제3쉘층은 상기 천연 유화제 및 천연 단백질을 포함하는 제2쉘층을 둘러싼 최외각층으로, 동결건조시 유산균 또는 유산균을 둘러싼 다층 구조가 파괴되는 것을 방지하기 위해 천연 동결보호제를 포함한다.The third shell layer is an outermost layer surrounding the second shell layer including the natural emulsifier and natural protein, and includes a natural cryoprotectant to prevent the multilayer structure surrounding the lactic acid bacteria or the lactic acid bacteria from being lyophilized.
상기 천연 동결보호제로는 탈지분유, 말토덱스트린 및 글리세린으로 이루어진 군에서 선택되는 1종 이상을 사용할 수 있다. 이때, 동결보호 효과를 증대시키기 위해 2종 이상을 혼합하여 사용하는 것이 바람직하며, 탈지분유 및 글리세린 또는 말토덱스트린 및 글리세린을 사용할 수 있다.The natural cryoprotectant may be used at least one selected from the group consisting of skim milk powder, maltodextrin and glycerin. At this time, it is preferable to use a mixture of two or more in order to increase the cryoprotective effect, skim milk powder and glycerin or maltodextrin and glycerin can be used.
이러한 제3쉘층은 상기 천연 동결보호제와 함께 천연 보존제를 더 포함할 수 있다.The third shell layer may further include a natural preservative together with the natural cryoprotectant.
상기 천연 보존제는 제조된 유산균 함유 리포좀이 저장 및 유통 과정에서 부패되는 것을 방지할 뿐만 아니라, 장에서 분해되어 장내 정착하는 유산균의 생장 및 증식을 위한 영양공급원으로 이용될 수 있다.The natural preservative not only prevents the prepared lactic acid bacteria-containing liposomes from decaying in the storage and distribution process, but may be used as a nutrient source for growth and proliferation of lactic acid bacteria that are degraded in the intestine and settled in the intestine.
상기 천연 보존제로는 식품에 첨가할 수 있는 천연 보존제로서, 감귤 추출물, 고삼 추출물, 유자 추출물, 유칼립투스 추출물, 자몽 추출물, 정향 추출물 및 크랜베리 추출물로 이루어진 군에서 선택되는 1종 이상을 사용할 수 있다. 이때, 보존력을 증대시키기 위해 자몽 추출물을 사용하는 것이 바람직하며, 더욱 향상된 보존력을 나타내기 위해 자몽 추출물 및 크랜베리 추출물을 사용하는 것이 더욱 바람직하다. 상기 크랜베리 추출물은 장내 유해균의 생장을 제어하면서 유산균의 생존율을 향상시킬 수 있어, 유산균에 의한 생리활성 효과를 더욱 향상시킬 수 있다.As the natural preservative, one or more selected from the group consisting of citrus extract, red ginseng extract, citron extract, eucalyptus extract, grapefruit extract, cloves extract and cranberry extract can be used as a natural preservative that can be added to food. At this time, it is preferable to use a grapefruit extract to increase the preservation power, it is more preferable to use a grapefruit extract and cranberry extract to exhibit more improved preservation power. The cranberry extract can improve the survival rate of lactic acid bacteria while controlling the growth of harmful bacteria in the intestine, it can further improve the physiological activity effect by lactic acid bacteria.
이와 같은 유산균 함유 천연 리포좀은 유산균을 3층으로 둘러싼 캡슐(Capsule) 형태이다. Natural liposomes containing such lactic acid bacteria are in the form of a capsule (Capsule) surrounding the lactic acid bacteria in three layers.
상기 유산균 함유 천연 리포좀은 전체 중량을 기준으로, 유산균 1 내지 20 중량%, 천연 다당체 0.5 내지 5 중량%, 천연 유화제 5 내지 30 중량%, 천연 단백질 0.5 내지 10 중량% 및 천연 동결보호제 45 내지 75 중량%를 포함하거나, 또는 전체 중량을 기준으로, 유산균 1 내지 20 중량%, 천연 다당체 0.5 내지 5 중량%, 천연 유화제 5 내지 30 중량%, 천연 단백질 0.5 내지 10 중량%, 천연 동결보호제 40 내지 70 중량% 및 천연 보존제 5 내지 15 중량%를 포함할 수 있다.The lactic acid bacteria-containing natural liposomes, based on the total weight, 1 to 20% by weight of lactic acid bacteria, 0.5 to 5% by weight of natural polysaccharide, 5 to 30% by weight of natural emulsifier, 0.5 to 10% by weight of natural protein and 45 to 75% by weight of natural cryoprotectant % By weight or based on total weight, 1 to 20% by weight of lactic acid bacteria, 0.5 to 5% by weight of natural polysaccharide, 5 to 30% by weight of natural emulsifier, 0.5 to 10% by weight of natural protein, 40 to 70% by weight of natural cryoprotectant % And 5 to 15 weight percent natural preservative.
이러한 유산균 함유 천연 리포좀을 식품 또는 의약품으로 섭취할 경우에는 침에 포함된 소화효소에 의해 제3쉘층 일부가 분해되며, 그 상태로 위장 및 십이지장을 통과하게 된다. 이때, 유산균은 제1쉘층, 제2쉘층 및 일부 제3쉘층으로 둘러싸여, 위산과 담즙으로부터 보호된다. 이후, 소장과 대장에서 유산균의 제1쉘층, 제2쉘층 및 제3쉘층이 분해됨으로써 유산균은 외부로 노출되며, 안전하게 장에 전달될 수 있다. 이때, 유산균은 제1쉘층, 제2쉘층 및 제3쉘층의 성분(예컨대, 천연 다당체, 천연 단백질, 천연 보존제)을 영양공급원으로 사용하여 생장 및 증식을 통해 장내 정착하면서 산성 환경을 만들어 유해균의 생장 및 증식을 저해하고, 장내 면역세포를 증가시키기 때문에 장관운동 및 배변활동을 원활하게 하고 장내 염증 발생을 억제시키는 등 다양한 생리활성 효과를 나타낼 수 있다.When the lactic acid bacteria-containing natural liposomes are ingested as food or medicine, part of the third shell layer is decomposed by digestive enzymes contained in saliva, and the stomach and duodenum pass as they are. At this time, the lactic acid bacteria are surrounded by the first shell layer, the second shell layer and some third shell layer, and are protected from gastric acid and bile. Thereafter, the first shell layer, the second shell layer and the third shell layer of the lactic acid bacteria in the small intestine and lactic acid bacteria are exposed to the outside and can be safely delivered to the intestine. At this time, lactic acid bacteria use the components of the first shell layer, the second shell layer and the third shell layer (eg, natural polysaccharides, natural proteins, and natural preservatives) as nutrient sources to create an acidic environment while growing in the intestine through growth and proliferation. Since it inhibits the growth and proliferation of harmful bacteria, and increases the intestinal immune cells, it can exhibit various physiological activities such as smooth bowel movements and bowel movements and suppress the intestinal inflammation.
<유산균 함유 천연 <Lactobacillus-containing natural 리포좀의Liposome 제조방법> Manufacturing Method>
본 발명은 초음파 파쇄기(Ultrasonicator), 균질기(Homogenizer) 등을 이용한 유산균 함유 천연 리포좀의 제조방법을 제공한다. 그러나, 이러한 제조방법에 의해서만 한정되는 것은 아니며, 필요에 따라 각 공정의 단계가 변형되거나 또는 선택적으로 혼용되어 수행될 수 있다.The present invention provides a method for producing lactic acid bacteria-containing natural liposomes using an ultrasonic crusher (Ultrasonicator), homogenizer (Homogenizer) and the like. However, it is not limited only by this manufacturing method, and the steps of each process may be modified or optionally mixed as necessary.
본 발명의 일 예에 따른 유산균 함유 천연 리포좀의 제조방법은 도 1에 도시된 바와 같이, (i) 40 내지 75℃의 용매에 천연 유화제를 투입하여 초음파 처리하는 제1단계; (ii) 40 내지 75℃의 용매에 천연 단백질, 천연 다당체를 투입하여 초음파 처리한 후 유산균을 투입하여 혼합하는 제2단계; (iii) 상기 제1단계에 얻어진 생성물, 상기 제2단계에서 얻어진 생성물, 천연 동결보호제를 혼합하는 제3단계; 및 (iv) 상기 제3단계에서 얻어진 생성물을 동결건조하는 제4단계를 포함할 수 있다. 이때, 도 2에 도시된 바와 같이, 상기 제4단계에 천연 보존제를 더 투입하여 혼합할 수 있다.As shown in FIG. 1, a method for preparing a lactic acid bacterium-containing natural liposome according to an embodiment of the present invention may include: (i) a first step of adding a natural emulsifier to a solvent at 40 to 75 ° C .; (ii) a second step of adding natural protein and natural polysaccharide to a solvent at 40 to 75 ° C., sonicating the same, and then adding and mixing the lactic acid bacteria; (iii) a third step of mixing the product obtained in the first step, the product obtained in the second step, and a natural cryoprotectant; And (iv) may comprise a fourth step of lyophilizing the product obtained in the third step. At this time, as shown in Figure 2, the natural preservative may be further added to the fourth step and mixed.
이하, 상기 제조방법을 각 공정 단계별로 나누어 설명하면 다음과 같다.Hereinafter, the manufacturing method will be described by dividing the process step by step.
제1단계: 40 내지 75℃의 용매에 천연 유화제를 투입하여 초음파 처리한다.First step: ultrasonic treatment by adding a natural emulsifier to a solvent of 40 to 75 ℃.
상기 용매로는 증류수, 천연 유래의 부틸렌글리콜, 프로판디올, 글리세린 및 발효주정으로 이루어진 군에서 선택되는 1종 이상을 사용할 수 있다.As the solvent, one or more selected from the group consisting of distilled water, butylene glycol, propanediol, glycerin, and fermentation spirits derived from nature may be used.
상기 천연 유화제로는 천연 물질에서 유래된 인지질과 지방산을 포함하는 1종 이상을 사용할 수 있다. 구체적으로는, 포스파티딜콜린, 리소포스파티딜콜린, 포스파티딜에탄올아민, 포스파티딜산, 포스파티딜세린, 포스파티딜글리세롤, 포스파티딜이노시톨 및 이들의 수소첨가 생성물로 이루어진 군에서 선택되는 1종 이상을 사용할 수 있다.As the natural emulsifier, one or more kinds including phospholipids and fatty acids derived from natural substances may be used. Specifically, at least one selected from the group consisting of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidyl acid, phosphatidylserine, phosphatidylglycerol, phosphatidyl inositol and hydrogenated products thereof can be used.
상기 초음파 처리는 용매에 천연 유화제를 충분히 분산시키기 위한 것으로, 일반적인 초음파 파쇄기를 사용할 수 있다. 보다 효율적으로 분산시키기 위해서는, 연속순환식 초음파분산기(Ultrasonic Liquid processor)를 사용하는 것이 바람직하다.The sonication is to sufficiently disperse the natural emulsifier in a solvent, and a general ultrasonic wave crusher may be used. In order to disperse more efficiently, it is preferable to use a continuous-cyclic ultrasonic liquid processor (Ultrasonic Liquid processor).
이러한 용매를 40 내지 75℃로 가온한 후 천연 유화제를 투입하여 초음파 처리함으로써, 용매 내 천연 유화제가 균일하게 분산될 수 있다. 이때, 교반기도 사용할 수 있으며, 1,500 내지 4,500 rpm에서 3 내지 30분 동안 교반할 수 있다.After heating the solvent to 40 to 75 ℃ by adding a natural emulsifier and sonication, the natural emulsifier in the solvent can be uniformly dispersed. At this time, a stirrer may also be used, and may be stirred for 3 to 30 minutes at 1,500 to 4,500 rpm.
제2단계: 40 내지 75℃의 용매에 천연 단백질, 천연 다당체를 투입하여 초음파 처리한 후 유산균을 투입하여 혼합한다.Second step: natural protein and natural polysaccharides are added to the solvent at 40 to 75 ° C., sonicated, and then lactic acid bacteria are added and mixed.
상기 용매로는 상기 제1단계에서 사용된 용매와 동일한 것을 사용할 수 있다. The solvent may be the same as the solvent used in the first step.
상기 천연 단백질은 천연 물질로부터 추출 및 정제된 것으로, 우유 유래 단백질 및/또는 콩 유래 단백질일 수 있다.The natural protein is extracted and purified from a natural substance, and may be a milk-derived protein and / or a soy-derived protein.
상기 천연 다당체는 천연 물질로부터 추출 및 정제된 것으로, 아라비아검(Arabic gum), 구아검(Guar gum), 로커스트빈검(Locust bean gum), 타마린드검(Tamarind gum), 타라검(Tara gum), 잔탄검(Xanthan gum) 및 카라기난(Carrageenan)으로 이루어진 군에서 선택되는 1종 이상을 사용할 수 있다.The natural polysaccharide is extracted and purified from a natural substance, Arabic gum, Guar gum, Locust bean gum, Tamarind gum, Tara gum, One or more selected from the group consisting of Xanthan gum and Carrageenan may be used.
상기 초음파 처리는 용매에 천연 단백질과 천연 다당체를 충분히 분산시키기 위한 것으로, 일반적인 초음파 파쇄기를 사용할 수 있다. 보다 효율적으로 분산시키기 위해서는, 연속순환식 초음파분산기(Ultrasonic Liquid processor)를 사용하는 것이 바람직하다.The sonication is to sufficiently disperse the natural protein and the natural polysaccharide in a solvent, and a general ultrasonic crusher may be used. In order to disperse more efficiently, it is preferable to use a continuous-cyclic ultrasonic liquid processor (Ultrasonic Liquid processor).
상기 유산균으로는 락토바실러스 아시도필루스(Lactobacillus acidophilus), 락토바실러스 불가리쿠스(Lactobacillus bulgaricus), 락토바실러스 카제이(Lactobacillus casei), 락토바실러스 퍼멘텀(Lactobacillus fermentum), 락토바실러스 가세리(Lactobacillus gasseri), 락토바실러스 헬베티쿠스(Lactobacillus helveticus), 락토바실러스 파라카제이(Lactobacillus paracasei), 락토바실러스 플란타룸(Lactobacillus plantarum), 락토바실러스 람노서스(Lactobacillus rhamnosus), 락토바실러스 루테리(Lactobacillus reuteri), 락토바실러스 살리바리우스(Lactobacillus salivarius), 락토코커스 락티스(Lactococcus lactis), 스트렙토코커스 써모필러스(Streptococcus thermophilus), 비피도박테리움 애니멀리스 락티스(Bifidobacterium animalis ssp. lactis), 비피도박테리움 비피덤(Bifidobacterium bifidum), 비피도박테리움 브레브(Bifidobacterium breve), 비피도박테리움 롱검(Bifidobacterium longum) 및 엔테로코커스 페칼리스(Enterococcus faecalis)로 이루어진 군에서 선택되는 1종 이상일 수 있다. The lactobacillus is Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus fermentum, Lactobacillus gaseri gasseri (Lactobacillus fermentum) , Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus rhamnosus, Lactobacillus luterus reacti Lactobacillus Salivacillus (Lactobacillus salivarius), Lactococcus lactis, Streptococcus thermophilus, Bifidobacterium animalis ssp.lactis, Bifidobacterium bacterium Bifido bifidum), Bifidobacterium breve, bifidobacter At least one selected from the group consisting of Bifidobacterium longum and Enterococcus faecalis.
이러한 용매를 40 내지 75℃로 가온한 후 천연 단백질, 천연 다당체를 투입하여 초음파 처리함으로써, 용매 내 천연 단백질과 천연 다당체가 균일하게 분산될 수 있다. After heating the solvent to 40 to 75 ℃ natural protein, by adding a natural polysaccharide and sonication, the natural protein and natural polysaccharide in the solvent can be uniformly dispersed.
이후, 천연 단백질과 천연 다당체가 분산된 용매의 온도가 50℃ 미만이 되면, 유산균을 투입하여 혼합한다. 유산균은 열에 약하므로, 용매의 온도가 유산균의 활성에 영향을 미치지 않는 상태, 즉 상기와 같이 용매의 온도가 50℃ 미만일 때 유산균을 투입하는 것이 바람직하다. 또한, 유산균 혼합시 초음파 파쇄기를 사용할 경우에는 열과 진동이 발생하여 유산균을 파괴하거나 손상시킬 수 있으므로, 균질기 및/또는 교반기를 사용하는 것이 바람직하다. 이때, 1,500 내지 4,500 rpm에서 3 내지 30분 동안 교반할 수 있다.Thereafter, when the temperature of the solvent in which the natural protein and the natural polysaccharide are dispersed is less than 50 ° C, lactic acid bacteria are added and mixed. Since the lactic acid bacteria are weak in heat, it is preferable to add the lactic acid bacteria when the temperature of the solvent does not affect the activity of the lactic acid bacteria, that is, when the temperature of the solvent is less than 50 ° C as described above. In addition, when using an ultrasonic crusher when mixing lactic acid bacteria, heat and vibration may occur to destroy or damage the lactic acid bacteria, it is preferable to use a homogenizer and / or a stirrer. At this time, it may be stirred for 3 to 30 minutes at 1,500 to 4,500 rpm.
제3단계: 제1단계의 생성물, 제2단계의 생성물, 천연 동결보호제를 혼합한다.Step 3: Mix the product of the first step, the product of the second step, and the natural cryoprotectant.
상기 천연 동결보호제는 다층으로 둘러싸인 유산균을 동결건조시, 유산균 또는 유산균을 둘러싼 다층 구조가 파괴되는 것을 방지할 수 있다.The natural lyoprotectant may prevent the lactic acid bacteria surrounded by the multilayer from being lyophilized, and the multilayer structure surrounding the lactic acid bacteria or lactic acid bacteria is destroyed.
상기 천연 동결보호제로는 탈지분유, 말토덱스트린 및 글리세린으로 이루어진 군에서 선택되는 1종 이상을 사용할 수 있으며, 동결보호제의 효과를 증대시키기 위해 2종 이상을 혼합하여 사용할 수 있다.The natural cryoprotectant may be used at least one selected from the group consisting of skim milk powder, maltodextrin and glycerin, and may be used by mixing two or more kinds to increase the effect of the cryoprotectant.
이러한 제1단계에서 얻어진 생성물과 제2단계에서 얻어진 생성물을 충분히 혼합한 후 천연 동결보호제를 투입하여 혼합한다. 이때, 균질기와 교반기를 사용할 수 있으며, 1,500 내지 4,500 rpm에서 3 내지 30분 동안 교반할 수 있다.The product obtained in the first step and the product obtained in the second step are sufficiently mixed, and then a natural cryoprotectant is added and mixed. At this time, a homogenizer and a stirrer may be used, and may be stirred for 3 to 30 minutes at 1,500 to 4,500 rpm.
제4단계: 제3단계의 생성물을 동결건조한다.Fourth step: The product of the third step is lyophilized.
제3단계에서 얻어진 생성물을 바로 동결건조하거나, 또는 천연 보존제를 더 투입한 후 동결건조한다.The product obtained in the third step is lyophilized immediately or lyophilized after further addition of a natural preservative.
상기 천연 보존제로는 식품에 첨가할 수 있는 천연 보존제로서, 감귤 추출물, 고삼 추출물, 유자 추출물, 유칼립투스 추출물, 자몽 추출물, 정향 추출물 및 크랜베리 추출물로 이루어진 군에서 선택되는 1종 이상을 사용할 수 있다.As the natural preservative, one or more selected from the group consisting of citrus extract, red ginseng extract, citron extract, eucalyptus extract, grapefruit extract, cloves extract and cranberry extract can be used as a natural preservative that can be added to food.
이러한 제3단계에서 얻어진 생성물에 천연 보존제를 투입하여 혼합함으로써 최종 생성물을 제조한다. 이때, 균질기와 교반기를 사용할 수 있으며, 1,500 내지 4,500 rpm에서 3 내지 30분 동안 교반할 수 있다. The final product is prepared by adding a natural preservative to the product obtained in this third step and mixing. At this time, a homogenizer and a stirrer may be used, and may be stirred for 3 to 30 minutes at 1,500 to 4,500 rpm.
상기 동결건조는 당업계에 알려진 동결건조 장치를 사용할 수 있다. 이때, 최종 생성물을 -10 내지 -30℃의 저온에서 건조시켜 분말화할 수 있다.The lyophilization may use a lyophilization device known in the art. At this time, the final product may be powdered by drying at a low temperature of -10 to -30 ℃.
본 발명의 다른 일 예에 따른 유산균 함유 천연 리포좀의 제조방법은 (i) 40 내지 75℃의 용매에 천연 다당체를 투입하여 초음파 처리한 후 유산균을 투입하여 혼합하는 제1단계; (ii) 40 내지 75℃의 용매에 천연 유화제를 투입하여 초음파 처리하는 제2단계; (iii) 40 내지 75℃의 용매에 천연 단백질을 투입하여 초음파 처리하는 제3단계; (iv) 상기 제1단계에서 얻어진 생성물, 상기 제2단계에서 얻어진 생성물, 제3단계에서 얻어진 생성물, 천연 동결보호제를 혼합하는 제4단계; 및 (v) 상기 제4단계에서 얻어진 생성물을 동결건조하는 제5단계를 포함할 수 있다. 이때, 상기 제5단계에 천연 보존제를 더 투입하여 혼합할 수 있다.According to another embodiment of the present invention, a method for preparing a lactic acid bacterium-containing natural liposome may include: (i) adding a lactic acid bacterium by adding natural polysaccharide to a solvent at 40 to 75 ° C., and then adding the lactic acid bacterium; (ii) a second step of ultrasonically adding a natural emulsifier to a solvent at 40 to 75 ° C; (iii) a third step of sonicating natural protein in a solvent at 40 to 75 ° C; (iv) a fourth step of mixing the product obtained in the first step, the product obtained in the second step, the product obtained in the third step, and a natural cryoprotectant; And (v) a fifth step of lyophilizing the product obtained in the fourth step. At this time, the natural preservative may be further added and mixed in the fifth step.
이러한 유산균 함유 천연 리포좀의 제조방법에서, 각 단계에서 사용되는 물질은 상기 일 예에 따른 유산균 함유 천연 리포좀의 제조방법과 동일하다.In the production method of such lactic acid bacteria-containing natural liposomes, the material used in each step is the same as the production method of lactic acid bacteria-containing natural liposomes according to the above example.
이와 같은 제조방법을 통해, 3층 구조를 갖는 유산균 함유 천연 리포좀 입자를 형성할 수 있다. 이러한 유산균 함유 천연 리포좀은 분말화되어 저장 및 포장이 간편하며, 부패균의 증식이 저해되어 저온 및 상온에서도 장기간 보관할 수 있다. Through such a manufacturing method, it is possible to form lactic acid bacteria-containing natural liposome particles having a three-layer structure. Such lactic acid bacteria-containing natural liposomes are powdered, so they are easy to store and package, and proliferation of rot bacteria is inhibited, so that they can be stored for a long time even at low temperature and room temperature.
<유산균 함유 천연 <Lactobacillus-containing natural 리포좀을Liposomes 포함하는 식품 또는 약학 조성물> Food or pharmaceutical composition comprising>
본 발명의 유산균 함유 천연 리포좀은 내산성 및 내담즙성이 우수하여 유산균을 높은 생존율로 장까지 안전하게 전달하며, 장내 정착된 유산균으로부터 다양한 생리활성 효과를 나타낼 수 있다.The lactic acid bacteria-containing natural liposomes of the present invention are excellent in acid resistance and bile resistance, and safely transmit lactic acid bacteria to the intestine with high survival rate, and may exhibit various physiological activity effects from the intestinal lactic acid bacteria.
상기 유산균 함유 천연 리포좀은 유산균을 둘러싼 코팅 물질 모두 천연 물질 유래 성분으로, 섭취하더라도 인체에 부작용을 나타내지 않는다. 이러한 유산균 함유 천연 리포좀을 섭취할 경우에는 유산균이 소장과 대장에 정착하여 유해균의 생장 및 증식을 저해하고 장관운동 및 배변활동을 원활하게 하며, 장내 면역세포를 증가시켜 장내 질환을 예방할 수 있어, 유산균 함유 천연 리포좀을 유효성분으로 포함하는 식품 또는 약학 조성물로 이용될 수 있다. 특히, 유산균 함유 천연 리포좀을 포함하는 조성물은 세포실험 및 동물실험을 통해 장내 환경을 개선하고 염증을 억제하는 효과가 우수한 것으로 확인되어, 장염 예방 또는 치료용 약학 조성물로 이용될 수 있다.The lactic acid bacteria-containing natural liposome is a coating material surrounding the lactic acid bacteria as a component derived from natural materials, even if ingested does not show side effects to the human body. When ingesting these natural liposomes containing lactic acid bacteria, lactic acid bacteria settle in the small and large intestine, inhibiting the growth and proliferation of harmful bacteria, facilitating bowel movements and bowel movements, and increasing intestinal immune cells to prevent intestinal diseases. It can be used as a food or pharmaceutical composition containing a natural liposome containing as an active ingredient. In particular, the composition comprising the lactic acid bacteria-containing natural liposomes have been found to be excellent in improving the intestinal environment and inhibiting inflammation through cell experiments and animal experiments, it can be used as a pharmaceutical composition for preventing or treating enteritis.
상기 유산균 함유 천연 리포좀을 유효성분으로 포함하는 식품 조성물은 건강기능식품으로 제공될 수 있다. 상기 건강기능식품은 분말, 과립, 정제, 캡슐, 음료 등의 형태일 수 있고, 캔디, 초콜릿, 음료, 껌, 차, 비타민복합체, 건강보조식품 등으로 섭취할 수 있다. 이때, 건강기능식품은 당 업계에 알려진 식품 첨가제를 더 포함할 수 있다.The food composition comprising the lactic acid bacteria-containing natural liposomes as an active ingredient may be provided as a health functional food. The health functional food may be in the form of powder, granules, tablets, capsules, beverages, and the like, and may be ingested as candy, chocolate, beverages, gum, tea, vitamin complexes, health supplements, and the like. In this case, the health functional food may further include food additives known in the art.
상기 유산균 함유 천연 리포좀을 유효성분으로 포함하는 약학 조성물은 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구제; 외용제; 좌제; 주사제 등의 제형일 수 있다. 이때, 상기 약학 조성물은 당 업계에 알려진 담체, 부형제 및 희석제를 더 포함할 수 있다. 이때, 담체, 부형제 및 희석제로는 락토오스, 덱스트로오스, 수크로오스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트, 광물유 등일 수 있다. Pharmaceutical compositions comprising the lactic acid bacteria-containing natural liposomes as an active ingredient, oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols; External preparations; Suppositories; Formulations such as injections. In this case, the pharmaceutical composition may further include a carrier, excipient and diluent known in the art. At this time, the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose , Polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like.
이하, 본 발명을 실시예를 통해 구체적으로 설명하나, 하기 실시예는 본 발명의 한 형태를 예시하는 것일 뿐, 본 발명의 범위가 하기 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to Examples, but the following Examples are merely illustrative of one embodiment of the present invention, and the scope of the present invention is not limited by the following Examples.
[[ 실시예Example 1] 유산균 함유 천연 1] lactic acid bacteria-containing natural 리포좀Liposomes
하기 표 1의 조성에 따라 유산균 함유 천연 리포좀을 제조하였다.The lactic acid bacteria-containing natural liposomes were prepared according to the composition of Table 1 below.
먼저, 반응기에서 증류수를 65℃로 가온한 후 다당체 혼합물을 투입하여 3,000 rpm에서 5분 동안 분산시키고 초음파 처리하였다. 이들의 온도가 50℃ 미만이 될 때까지 기다린 후 유산균 혼합물을 투입하고 혼합하였다.First, the distilled water was warmed to 65 ℃ in the reactor, the polysaccharide mixture was added, dispersed for 5 minutes at 3,000 rpm and sonicated. After waiting for these temperatures to be less than 50 ° C., the lactic acid bacteria mixture was added and mixed.
다른 반응기에서 증류수를 65℃로 가온한 후 포스파티딜콜린을 천천히 투입하여 3,000 rpm에서 5분 동안 혼합한 후 초음파 처리하여 제2혼합물을 준비하였다. After distilled water was warmed to 65 ° C. in another reactor, phosphatidylcholine was slowly added thereto, mixed at 3,000 rpm for 5 minutes, and sonicated to prepare a second mixture.
다른 반응기에서 증류수를 65℃로 가온한 후 유청단백질을 투입하여 3,000 rpm에서 5분 동안 혼합한 후 초음파 처리하여 제3혼합물을 준비하였다. In another reactor, distilled water was warmed to 65 ° C., and whey protein was added thereto, mixed at 3,000 rpm for 5 minutes, and sonicated to prepare a third mixture.
상기 제2혼합물이 준비된 반응기에 제3혼합물을 투입하여 혼합한 후 제1혼합물을 투입하여 3,000 rpm에서 5분 동안 혼합하였다. 이후, 탈지분유, 글리세린을 투입하여 혼합하고, 이를 동결건조한 후 분쇄하여 유산균 함유 천연 리포좀을 제조하였다.The third mixture was added to the reactor in which the second mixture was prepared, followed by mixing. Then, the first mixture was added and mixed at 3,000 rpm for 5 minutes. Thereafter, skim milk powder and glycerin were added thereto, mixed, lyophilized and ground to prepare a lactic acid bacteria-containing natural liposome.
(로스트빈검, 카라기난, 타마린드검, 타라검)Polysaccharide mixture
Roast Bean Sword, Carrageenan, Tamarind Sword, Tara Gum
(락토바실러스 아시도필루스, 비피도박테리움 비피덤, 비피도박테리움 롱검)Lactobacillus mixture
(Lactobacillus asidophilus, Bifidobacterium Bifiderm, Bifidobacterium longgum)
[[ 실시예Example 2] 유산균 함유 천연 2] natural lactic acid-containing 리포좀Liposomes
하기 표 2의 조성에 따라, 유산균 함유 천연 리포좀을 제조하였다.According to the composition of Table 2 below, lactic acid bacteria-containing natural liposomes were prepared.
먼저, 반응기에서 증류수를 65℃로 가온한 후 포스파티딜콜린을 투입하여 3,000 rpm에서 5분 동안 분산시키고, 초음파 처리하여 제1혼합물을 준비하였다.First, distilled water was heated to 65 ° C. in a reactor, phosphatidylcholine was added thereto, dispersed at 3,000 rpm for 5 minutes, and sonicated to prepare a first mixture.
다른 반응기에서 증류수를 65℃로 가온한 후 유청단백질, 다당체 혼합물을 천천히 투입하여 3,000 rpm에서 5분 동안 혼합하고 초음파 처리하였다. 이후, 유청단백질과 다당체 혼합물이 혼합된 증류수의 온도가 50℃ 미만이 되면, 유산균 혼합물을 투입하여 혼합하여 제2혼합물을 준비하였다.In another reactor, distilled water was warmed to 65 ° C., and a whey protein and polysaccharide mixture was slowly added thereto, mixed at 3,000 rpm for 5 minutes, and sonicated. Subsequently, when the temperature of the distilled water mixed with the whey protein and the polysaccharide mixture is less than 50 ° C., the lactic acid bacteria mixture is added and mixed to prepare a second mixture.
상기 제1혼합물이 준비된 반응기에 제2혼합물을 투입한 후 말토덱스트린, 글리세린을 투입하여 3,000rpm에서 5분 동안 혼합하였다. 이후, 자몽종자 추출물을 투입하여 충분히 혼합하였고, 이를 동결건조한 후 분쇄하여 유산균 함유 천연 리포좀을 제조하였다.After the second mixture was added to the reactor in which the first mixture was prepared, maltodextrin and glycerin were added and mixed at 3,000 rpm for 5 minutes. Then, the grapefruit seed extract was added to the mixture sufficiently, lyophilized and ground to prepare a lactic acid bacteria-containing natural liposomes.
(락토바실러스 아시도필루스, 비피도박테리움 비피덤, 비피도박테리움 롱검)Lactobacillus mixture
(Lactobacillus asidophilus, Bifidobacterium Bifiderm, Bifidobacterium longgum)
(로스트빈검, 카라기난, 타마린드검, 타라검)Polysaccharide mixture
Roast Bean Sword, Carrageenan, Tamarind Sword, Tara Gum
[[ 실시예Example 3] 유산균 함유 천연 3] lactic acid bacteria-containing natural 리포좀Liposomes
하기 표 3의 조성에 따라, 유산균 함유 천연 리포좀을 제조하였다.Following the composition of Table 3, lactic acid bacteria-containing natural liposomes were prepared.
먼저, 반응기에서 증류수를 65℃로 가온한 후 포스파티딜콜린을 투입하여 3,000 rpm에서 15분 동안 분산시키고, 초음파 처리하여 제1혼합물을 준비하였다.First, distilled water was heated to 65 ° C. in the reactor, phosphatidylcholine was added thereto, dispersed at 3,000 rpm for 15 minutes, and sonicated to prepare a first mixture.
다른 반응기에서 증류수를 65℃로 가온한 후 유청단백질, 다당체 혼합물을 천천히 투입하여 3,000 rpm에서 20분 동안 혼합하고 초음파 처리하였다. 이후, 유청단백질과 다당체 혼합물이 혼합된 증류수의 온도가 50℃ 미만이 되면, 유산균 혼합물을 투입하여 혼합하여 제2혼합물을 준비하였다.In another reactor, distilled water was warmed to 65 ° C., and a whey protein and polysaccharide mixture was slowly added thereto, mixed at 3,000 rpm for 20 minutes, and sonicated. Subsequently, when the temperature of the distilled water mixed with the whey protein and the polysaccharide mixture is less than 50 ° C., the lactic acid bacteria mixture is added and mixed to prepare a second mixture.
상기 제1혼합물이 준비된 반응기에 제2혼합물을 투입한 후 말토덱스트린, 글리세린을 투입하여 3,000 rpm에서 20분 동안 혼합하였다. 이후 자몽종자 추출물, 크랜베리 추출물을 투입하여 충분히 혼합하였고, 이를 동결건조한 후 분쇄하여 유산균 함유 천연 리포좀을 제조하였다.After the second mixture was added to the reactor in which the first mixture was prepared, maltodextrin and glycerin were added and mixed at 3,000 rpm for 20 minutes. Thereafter, grapefruit seed extract and cranberry extract were mixed and sufficiently mixed, and then lyophilized and ground to prepare a lactic acid bacterium-containing natural liposome.
(락토바실러스 아시도필루스, 비피도박테리움 비피덤, 비피도박테리움 롱검)Lactobacillus mixture
(Lactobacillus asidophilus, Bifidobacterium Bifiderm, Bifidobacterium longgum)
(로스트빈검, 카라기난, 타마린드검, 타라검)Polysaccharide mixture
Roast Bean Sword, Carrageenan, Tamarind Sword, Tara Gum
[[ 실시예Example 4] 유산균 함유 천연 4] Natural lactic acid bacteria 리포좀Liposomes
유산균 혼합물로 락토바실러스 아시도필루스, 비피도박테리움 비피덤 및 비피도박테리움 롱검 대신 락토바실러스 아시도필루스, 락토바실러스 퍼맨텀, 락토바실러스 플란타룸, 락토바실러스 람노서스, 스트렙토코커스 써모필러스, 비피도박테리움 애니멀리스 락티스 및 비피도박테리움 롱검을 사용한 것을 제외하고는, 실시예 2와 동일한 방법으로 유산균 함유 천연 리포좀을 제조하였다.Lactobacillus acidophyllus, Bifidobacterium bifidum and Bifidobacterium long gum instead of Lactobacillus acidophyllus, Lactobacillus permantum, Lactobacillus plantarum, Lactobacillus rhamnosus, Streptococcus thermophiler Lactic acid bacteria-containing natural liposomes were prepared in the same manner as in Example 2, except for using S, Bifidobacterium animalis lactis and Bifidobacterium longgum.
[[ 실시예Example 5] 유산균 조성물 5] Lactic acid bacteria composition
하기 표 4의 조성에 따라, 실시예 4의 유산균 함유 천연 리포좀을 유효성분으로 함유하는 유산균 조성물을 제조하였다.According to the composition of Table 4, the lactic acid bacteria composition containing the lactic acid bacteria-containing natural liposome of Example 4 as an active ingredient was prepared.
[[ 비교예Comparative example 1] 유산균 1] Lactobacillus
실시예 1에서 사용된 동결 건조 상태의 유산균 혼합물을 사용하였다.The freeze-dried lactobacillus mixture used in Example 1 was used.
[[ 비교예Comparative example 2] 유산균 함유 천연 2] natural lactic acid-containing 리포좀Liposomes
유청단백질을 사용하지 않은 것을 제외하고는, 실시예 1과 동일한 방법으로 하기 표 5의 조성에 따라 유산균 함유 천연 리포좀을 제조하였다.Except not using whey protein, lactic acid bacteria-containing natural liposomes were prepared according to the composition of Table 5 in the same manner as in Example 1.
(로스트빈검, 카라기난, 타마린드검, 타라검)Polysaccharide mixture
Roast Bean Sword, Carrageenan, Tamarind Sword, Tara Gum
(락토바실러스 아시도필루스, 비피도박테리움 비피덤, 비피도박테리움 롱검)Lactobacillus mixture
(Lactobacillus asidophilus, Bifidobacterium Bifiderm, Bifidobacterium longgum)
[[ 비교예Comparative example 3] 유산균 제품 3] Lactobacillus Products
하기 표 6의 조성을 포함하는 시판중인 유산균 제품(셀바이오텍社, 듀오락 골드)을 사용하였다.A commercially available lactic acid bacterium product (Cellbiotech Co., Duolac Gold) including the composition of Table 6 was used.
[[ 실험예Experimental Example 1] 유산균 함유 천연 1] lactic acid bacteria-containing natural 리포좀의Liposome 내담즙성Bile resistance 측정 Measure
실시예 1의 유산균 함유 천연 리포좀, 비교예 1의 유산균, 비교예 2의 유산균 함유 천연 리포좀을 각각 0.3% Oxgal 용액과 1:1로 혼합한 후 24시간 동안 반응시켰다. 이후, PBS로 10-8배까지 희석하였고, 100 ul씩 BCP 한천 배지에 도말하였다. 상기 BCP 한천 배지를 37℃, 5% CO2 배양기에서 2일 동안 배양한 후 세포수를 세었고, 그 결과를 하기 표 7에 나타내었다.Lactic acid bacteria-containing natural liposomes of Example 1, lactic acid bacteria of Comparative Example 1, lactic acid bacteria-containing natural liposomes of Comparative Example 2 were mixed 1: 1 with 0.3% Oxgal solution and reacted for 24 hours. Thereafter, the mixture was diluted 10 -8 times with PBS and plated in 100 ul of BCP agar medium. The BCP agar medium was incubated for 2 days at 37 ° C., 5% CO 2 incubator, and the number of cells was counted. The results are shown in Table 7 below.
상기 표 7과 같이, 실시예 1의 유산균 함유 천연 리포좀은 비교예 1의 유산균, 비교예 2의 유산균 함유 천연 리포좀에 비해 유산균의 생존율이 높은 것으로 나타났다.As shown in Table 7, the lactic acid bacteria-containing natural liposomes of Example 1 were found to have a higher survival rate of lactic acid bacteria than lactic acid bacteria of Comparative Example 1 and lactic acid bacteria-containing natural liposomes of Comparative Example 2.
따라서, 실시예 1의 유산균 함유 천연 리포좀은 담즙으로부터 유산균을 보호할 수 있다.Therefore, the lactic acid bacteria-containing natural liposomes of Example 1 can protect the lactic acid bacteria from bile.
[[ 실험예Experimental Example 2] 천연 보존제에 따른 유산균 함유 천연 2] Natural lactic acid bacteria containing natural preservatives 리포좀의Liposome 내저온성Low temperature resistance , , 내산성Acid resistance 및 And 내담즙성Bile resistance 측정 Measure
(1) 내저온성 측정(1) low temperature resistance measurement
실시예 2의 유산균 함유 천연 리포좀, 실시예 3의 유산균 함유 천연 리포좀을 4℃에서 10일 동안 보관하였다. 이후, 각각 PBS로 10-8배까지 희석하였고, 이를 100 ul씩 BCP 한천 배지에 도말하였다. 상기 BCP 한천 배지를 37℃, 5% CO2 배양기에서 2일 동안 배양한 후 세포수를 세었고, 그 결과를 하기 표 8에 나타내었다.Lactic acid bacteria-containing natural liposomes of Example 2, lactic acid bacteria-containing natural liposomes of Example 3 were stored at 4 ° C for 10 days. Thereafter, each of them was diluted 10 -8 times with PBS, which was then plated in 100 ul of BCP agar medium. The BCP agar medium was incubated for 2 days at 37 ° C., 5% CO 2 incubator, and the number of cells was counted. The results are shown in Table 8 below.
상기 표 8과 같이, 실시예 2의 유산균 함유 천연 리포좀, 실시예 3의 유산균 함유 천연 리포좀은 유산균의 생존율이 모두 우수하며, 특히 실시예 3의 유산균 함유 천연 리포좀이 높은 유산균의 생존율을 가지는 것으로 나타났다.As shown in Table 8, the lactic acid bacteria-containing natural liposomes of Example 2, lactic acid bacteria-containing natural liposomes of Example 3 are all excellent in the survival rate of the lactic acid bacteria, in particular, the lactic acid bacteria-containing natural liposomes of Example 3 was found to have a high survival rate of lactic acid bacteria .
(2) 내산성 측정(2) acid resistance measurement
실시예 2의 유산균 함유 천연 리포좀, 실시예 3의 유산균 함유 천연 리포좀을 각각 NaH2PO4(pH 2)와 1:1로 혼합한 후 5시간 동안 반응시켰다. 이후, PBS로 10-8배까지 희석하였고, 100 ul씩 BCP 한천 배지에 도말하였다. 상기 BCP 한천 배지를 37℃, 5% CO2 배양기에서 2일 동안 배양한 후 세포수를 세었고, 그 결과를 하기 표 9에 나타내었다.Lactic acid bacteria-containing natural liposomes of Example 2, lactic acid bacteria-containing natural liposomes of Example 3 were mixed 1: 1 with NaH 2 PO 4 (pH 2) and reacted for 5 hours. Thereafter, the mixture was diluted 10 -8 times with PBS and plated in 100 ul of BCP agar medium. The BCP agar medium was incubated for 2 days at 37 ° C. in a 5% CO 2 incubator, and the number of cells was counted. The results are shown in Table 9 below.
상기 표 9와 같이, 실시예 2의 유산균 함유 천연 리포좀, 실시예 3의 유산균 함유 천연 리포좀은 유산균의 생존율이 모두 우수하며, 특히 실시예 3의 유산균 함유 천연 리포좀이 높은 유산균의 생존율을 가지는 것으로 나타났다.As shown in Table 9, the lactic acid bacteria-containing natural liposomes of Example 2, lactic acid bacteria-containing natural liposomes of Example 3 are all excellent in the survival rate of lactic acid bacteria, in particular, the lactic acid bacteria-containing natural liposomes of Example 3 was found to have a high survival rate of lactic acid bacteria .
(3) 내담즙성 측정(3) measurement of bile resistance
실시예 2의 유산균 함유 천연 리포좀, 실시예 3의 유산균 함유 천연 리포좀에 대해 상기 실험예 1과 동일한 방법으로 실험하였고, 그 결과를 하기 표 10에 나타내었다.The lactic acid bacteria-containing natural liposomes of Example 2 were tested in the same manner as in Experimental Example 1 for the lactic acid-containing natural liposomes of Example 3, and the results are shown in Table 10 below.
상기 표 10과 같이, 실시예 2의 유산균 함유 천연 리포좀, 실시예 3의 유산균 함유 천연 리포좀은 유산균의 생존율이 모두 우수하며, 특히 실시예 3의 유산균 함유 천연 리포좀이 높은 유산균의 생존율을 가지는 것으로 나타났다.As shown in Table 10, the lactic acid bacteria-containing natural liposomes of Example 2, the lactic acid bacteria-containing natural liposomes of Example 3 are all excellent in the survival rate of the lactic acid bacteria, in particular, the lactic acid bacteria-containing natural liposomes of Example 3 was found to have a high survival rate of lactic acid bacteria .
따라서, 천연 보존제를 더 포함하는 유산균 함유 천연 리포좀은 유산균의 내저온성, 내산성 및 내담즙성이 우수한 것을 알 수 있었다.Therefore, it was found that the lactic acid bacteria-containing natural liposome further comprising a natural preservative is excellent in the low temperature resistance, acid resistance and bile resistance of the lactic acid bacteria.
[ [ 실험예Experimental Example 3] 유산균 함유 천연 3] lactic acid bacteria-containing natural 리포좀의Liposome 온도별 생존율 측정 Temperature survival rate measurement
실시예 2의 유산균 함유 천연 리포좀을 각각 -20℃, 4℃, 24℃, 37℃, 45℃에서 0일, 5일 동안 보관하였다. 이후, 상기 실험예 2의 (1)과 동일한 방법으로 실험하였고, 그 결과를 하기 표 11에 나타내었다.Lactic acid bacteria-containing natural liposomes of Example 2 were stored at -20 ° C, 4 ° C, 24 ° C, 37 ° C, and 45 ° C for 0 days and 5 days, respectively. Then, the experiment in the same manner as in (1) of Experimental Example 2, the results are shown in Table 11 below.
상기 표 11과 같이, 실시예 2의 유산균 함유 천연 리포좀은 -20 내지 24℃에서 보관하더라도 유산균의 생존율이 크게 감소하지 않는 것으로 나타났다.As shown in Table 11, the lactic acid bacteria-containing natural liposomes of Example 2 did not significantly reduce the survival rate of lactic acid bacteria even when stored at -20 to 24 ℃.
[[ 실험예Experimental Example 4] 유산균의 4] lactic acid bacteria 내저온성Low temperature resistance , , 내산성Acid resistance 및 And 내담즙성Bile resistance 측정 Measure
(1) 내저온성 측정(1) low temperature resistance measurement
실시예 2의 유산균 함유 천연 리포좀, 비교예 1의 유산균, 비교예 3의 유산균 제품을 4℃에서 각각 0일, 10일 동안 보관하였다. 이후, 상기 실험예 2의 (1)과 동일한 방법으로 실험하였고, 그 결과를 하기 표 12에 나타내었다.Lactobacillus-containing natural liposomes of Example 2, lactic acid bacteria of Comparative Example 1, lactic acid bacteria products of Comparative Example 3 were stored at 4 ° C for 0 days and 10 days, respectively. Then, the experiment in the same manner as in (1) of Experimental Example 2, the results are shown in Table 12 below.
상기 표 12와 같이, 보관일이 늘어날수록 실시예 2의 유산균 함유 천연 리포좀, 비교예 1의 유산균, 비교예 3의 유산균 제품은 생균수가 감소하는 것으로 나타났다. 이때, 실시예 2의 유산균 함유 천연 리포좀의 생균수는 약 1/2로 감소되는 반면, 비교예 1의 유산균의 생균수는 약 1/100, 비교예 3의 종래 유산균 제품의 생균수는 약 1/10로 감소되는 것으로 나타났다.As shown in Table 12, as the storage days increased, the lactic acid bacteria-containing natural liposomes of Example 2, the lactic acid bacteria of Comparative Example 1, the lactic acid bacteria products of Comparative Example 3 was found to decrease the number of live bacteria. At this time, the number of viable bacteria of the lactic acid bacteria-containing natural liposome of Example 2 is reduced to about 1/2, while the viable bacterial count of the lactic acid bacteria of Comparative Example 1 is about 1/100, and the viable bacterial count of the conventional lactic acid bacteria product of Comparative Example 3 is about 1 / 10.
보관 10일째, 실시예 2의 유산균 함유 천연 리포좀은 비교예 1의 유산균, 비교예 3의 종래 유산균 제품에 비해 생균수가 많은 것으로 나타났다. On the 10th day of storage, the lactic acid bacteria-containing natural liposomes of Example 2 were found to have a greater number of viable cells than the lactic acid bacteria of Comparative Example 1 and the conventional lactic acid bacteria products of Comparative Example 3.
(2) 내산성 측정(2) acid resistance measurement
실시예 2의 유산균 함유 천연 리포좀, 비교예 1의 유산균, 비교예 3의 유산균 제품을 각각 NaH2PO4(pH 2)와 1:1로 혼합한 후 각각 0시간, 5시간 동안 반응시켰다. 이후, 상기 실험예 2의 (2)와 동일한 방법으로 실험하였고, 그 결과를 하기 표 13에 나타내었다.The lactic acid bacteria-containing natural liposomes of Example 2, the lactic acid bacteria of Comparative Example 1, and the lactic acid bacteria products of Comparative Example 3 were mixed 1: 1 with NaH 2 PO 4 (pH 2), respectively, and reacted for 0 hours and 5 hours, respectively. Then, the experiment in the same manner as in (2) of Experimental Example 2, the results are shown in Table 13 below.
상기 표 13과 같이, 산성 용액과의 반응 시간이 늘어날수록 실시예 2의 유산균 함유 천연 리포좀, 비교예 1의 유산균, 비교예 3의 유산균 제품은 생균수가 감소하는 것으로 나타났다. 이때, 실시예 2의 유산균 함유 천연 리포좀의 생균수는 약 1/4로 감소되는 반면, 비교예 3의 종래 유산균 제품의 생균수는 약 1/450으로 감소되는 것으로 나타났다.As shown in Table 13, as the reaction time with the acidic solution increases, the number of live bacteria was reduced in the lactic acid bacteria-containing natural liposome of Example 2, lactic acid bacteria of Comparative Example 1, lactic acid bacteria products of Comparative Example 3. At this time, the number of live bacteria of the lactic acid bacteria-containing natural liposomes of Example 2 was reduced to about 1/4, while the number of live bacteria of the conventional lactic acid bacteria product of Comparative Example 3 was reduced to about 1/450.
반응 5시간째, 비교예 1의 유산균은 모두 사멸하였고, 실시예 2의 유산균 함유 천연 리포좀은 비교예 3의 종래 유산균 제품에 비해 생균수가 많은 것으로 나타났다. After 5 hours of reaction, the lactic acid bacteria of Comparative Example 1 were all killed, and the lactic acid bacteria-containing natural liposomes of Example 2 were found to have more viable bacteria than the conventional lactic acid bacteria products of Comparative Example 3.
(3) 내담즙성 측정(3) measurement of bile resistance
실시예 2의 유산균 함유 천연 리포좀, 비교예 1의 유산균, 비교예 3의 유산균 제품을 각각 0.3% Oxgal 용액과 1:1로 혼합한 후 각각 0시간, 24시간 동안 반응시켰다. 이후, 상기 실험예 1과 동일한 방법으로 실험하였고, 그 결과를 하기 표 14에 나타내었다.Lactic acid bacteria-containing natural liposomes of Example 2, lactic acid bacteria of Comparative Example 1, lactic acid bacteria products of Comparative Example 3 were mixed 1: 1 with 0.3% Oxgal solution, respectively, and reacted for 0 hours and 24 hours, respectively. Then, the experiment in the same manner as in Experimental Example 1, the results are shown in Table 14 below.
상기 표 14와 같이, 담즙과의 반응 시간이 늘어날수록 비교예 1의 유산균, 비교예 3의 유산균 제품은 생균수가 감소하는 반면, 실시예 2의 유산균 함유 천연 리포좀은 오히려 생균수가 증가하는 것으로 나타났다.As shown in Table 14, as the reaction time with the bile increases, the lactic acid bacteria of Comparative Example 1, the lactic acid bacteria products of Comparative Example 3 decreased the number of viable cells, while the lactic acid bacteria-containing natural liposomes of Example 2 was found to increase the viable bacteria.
반응 24시간째, 실시예 2의 유산균 함유 천연 리포좀의 생균수는 약 12배가 증가되는 반면, 비교예 1의 유산균의 생균수는 약 1/300,000, 비교예 3의 종래 유산균 제품의 생균수는 약 1/1,000로 감소되는 것으로 나타났다.At 24 hours after the reaction, the number of viable cells of the lactic acid bacteria-containing natural liposomes of Example 2 was increased by about 12 times, whereas the number of viable bacteria of Comparative Example 1 was about 1 / 300,000, and that of the conventional lactic acid bacteria product of Comparative Example 3 was about It was found to decrease to 1 / 1,000.
따라서, 유산균 함유 천연 리포좀은 코팅되지 않은 유산균, 종래 유산균 제품에 비해 유산균의 내저온성, 내산성 및 내담즙성이 우수한 것을 알 수 있었다.Therefore, it was found that lactic acid bacteria-containing natural liposomes were superior in low temperature resistance, acid resistance and bile resistance of lactic acid bacteria as compared to non-coated lactic acid bacteria and conventional lactic acid bacteria products.
[[ 실험예Experimental Example 5] 장내 유해균 활성의 측정 5] Determination of intestinal harmful bacterium activity
장에는 유해균과 유익균이 공존하며, 유산균과 같은 유익균에 비해 유해균의 균수가 많거나 활성이 높을 경우, 장 건강이 좋지 않을 수 있다. 장내 베타-글루쿠로니다아제와 베타-글루코시다아제의 활성을 측정하여 유해균의 활성 정도를 확인할 수 있다.Harmful bacteria and beneficial bacteria coexist in the intestine, and when the number of harmful bacteria is higher or higher in activity than beneficial bacteria such as lactic acid, intestinal health may be poor. Intestinal beta-glucuronidase and beta-glucosidase activity can be measured to determine the activity of harmful bacteria.
(1) 베타-글루쿠로니다아제(β-glucuronidase)(1) beta-glucuronidase (β-glucuronidase)
베타-글루쿠로니다아제는 장내 세균에 의해 생성되는 효소로, 장내 지용성 유독 물질과 결합된 글루쿠론산의 결합을 끊어 장세포에 독성 물질을 제공하게 된다. 이러한 베타-글루쿠로니다아제의 활성에 의해 장내 염증 및 암이 발생되는 것으로 알려져 있다. 베타-글루쿠로니다아제의 활성은 주로 가스괴저균(Clostridium perfringens), 대장균, 박테로이데스, 유박테리움, 펩토스트렙토커스 등의 장내 유해균에 의해 생성되는 것으로 알려져 있다.Beta-glucuronidase is an enzyme produced by intestinal bacteria that breaks down the binding of glucuronic acid combined with intestinal fat-soluble toxic substances to provide toxic substances to enterocytes. It is known that intestinal inflammation and cancer are caused by the activity of such beta-glucuronidase. Beta-glucuronidase activity is known to be produced mainly by intestinal harmful bacteria such as Clostridium perfringens, Escherichia coli, Bacteroides, Eubacterium, Peptostreptococcus.
먼저, GAM(General anaerobic medium) 배지 5 ml에 음성대조군으로 아무것도 처리하지 않고, 실시예 2의 유산균 함유 천연 리포좀, 비교예 1의 유산균, 비교예 3의 유산균 제품을 농도 0.5, 1%로 각각 처리한 후 pH 7.2로 맞춘 후 멸균하여 배지를 준비하였다.First, 5 ml of GAM (General anaerobic medium) medium was treated with no negative control group, and the lactic acid bacteria-containing natural liposomes of Example 2, lactic acid bacteria of Comparative Example 1, and lactic acid bacteria products of Comparative Example 3 were treated at concentrations of 0.5 and 1%, respectively. After the pH was adjusted to 7.2 and sterilized to prepare a medium.
건강한 한국인의 신선한 분변을 GAM 배지로 10배 희석한 후 5 ul를 준비된 GAM 배지에 접종하였다. 이후, 37℃, 혐기적 배양기에서 12시간 이상 배양하여 효소액 시료로 사용하였다.Fresh feces of healthy Koreans were diluted 10-fold with GAM medium and then 5 ul were inoculated into the prepared GAM medium. Then, incubated for at least 12 hours at 37 ℃, anaerobic incubator was used as an enzyme solution sample.
효소 반응액으로 0.1 M 칼륨인산염 완충액(KH2PO4, pH 7.0) 380 ul에 10 mM 니트로페닐 β-D-글루쿠로니드(nitrophenyl β-D-glucuronide) 20 ul를 첨가하였고, 여기에 효소액 시료를 100 ul씩 첨가하였다. 이후, 37℃에서 10~60분 동안 반응시킨 후 0.5 N NaOH 500 ul를 첨가하여 반응을 종결시켰다. 이를 3,000 rpm에서 10분 동안 원심분리한 후 상층액을 이용하여 405 nm에서 흡광도를 측정하였고, 그 결과를 도 3에 나타내었다. 이때, 흡광도로 유리된 페놀프탈레인의 양을 측정하며, 측정된 페놀프탈레인의 양이 많을수록 베타-글루쿠로니다아제의 활성이 높은 것을 의미한다.As enzyme reaction solution, 20 ul of 10 mM nitrophenyl β-D-glucuronide was added to 380 ul of 0.1 M potassium phosphate buffer (KH 2 PO 4 , pH 7.0), and the enzyme solution was added thereto. Samples were added in 100 ul portions. Then, the reaction was terminated by adding 500 Nul of 0.5 N NaOH after reacting for 10 to 60 minutes at 37 ℃. After centrifugation at 3,000 rpm for 10 minutes, the absorbance was measured at 405 nm using the supernatant, and the results are shown in FIG. 3. In this case, the amount of phenolphthalein liberated by absorbance is measured, and the greater the amount of phenolphthalein measured, the higher the activity of beta-glucuronidase.
도 3과 같이, 실시예 2의 유산균 함유 천연 리포좀, 비교예 1의 유산균을 처리한 경우에는 처리 농도가 증가할수록 페놀프탈레인 양이 감소된 반면, 비교예 3의 유산균 제품을 처리한 경우는 처리 농도가 증가할수록 페놀프탈레인 양이 오히려 증가된 것으로 나타났다.As shown in FIG. 3, when the lactic acid bacteria-containing natural liposome of Example 2 and the lactic acid bacteria of Comparative Example 1 were treated, the amount of phenolphthalein was decreased as the treatment concentration was increased, whereas the treatment concentration was increased when the lactic acid bacteria product of Comparative Example 3 was treated. It was found that the amount of phenolphthalein increased with increasing.
또한, 실시예 2의 유산균 함유 천연 리포좀을 처리한 경우에는 페놀프탈레인 양이 비교예 1의 유산균, 비교예 3의 유산균 제품을 처리한 경우에 비해 적은 것으로 나타났다.In addition, when the lactic acid bacteria-containing natural liposomes of Example 2 were treated, the amount of phenolphthalein was found to be less than that of the lactic acid bacteria of Comparative Example 1 and the lactic acid bacteria product of Comparative Example 3.
(2) 베타-글루코시다아제(β-glucosidase)(2) beta-glucosidase
베타-글루코시다아제는 주로 미생물과 동물의 소장점막에 분포하는 효소로, 배당체(Glycoside)의 베타-글루코시드 결합을 가수분해하여 글루코스(Glucose)와 비배당체를 생성한다. 식품으로 섭취된 배당체 중 청산 배당체, 쿠마린 배당체 및 페놀성 배당체는 체내에서 분해되어 이들의 독성기가 유리되는데, 이러한 독성기가 많이 생성되면 장 건강에 좋지 않은 것으로 알려져 있다.Beta-glucosidase is an enzyme mainly distributed in the intestinal mucosa of microorganisms and animals, and hydrolyzes beta-glucoside bonds of glycosides to produce glucose and nonglycosides. Among glycosides ingested as food, the clearing glycosides, coumarin glycosides and phenolic glycosides are broken down in the body to release their toxic groups.
배지와 효소액 시료는 상기 실험예 5의 (1)과 동일한 방법으로 준비하였다.The medium and the enzyme solution samples were prepared in the same manner as in (1) of Experimental Example 5.
효소 반응액으로 0.1 M 인산 완충액(phosphate, pH 6.0) 300 ul에 2 mM p-니트로페닐 β-D-글루코시드(p-nitrophenyl β-D-glucoside) 200 ul를 첨가하였고, 여기에 상기 효소액 시료를 100 ul씩 첨가하였다. 이후, 37℃에서 10~60분 동안 반응한 후 0.5 N NaOH 400 ul를 첨가하여 반응을 종결시키고, 증류수 1 ml을 첨가하였다. 이를 3,000 rpm에서 20분 동안 원심분리한 후 상층액을 이용하여 405 nm에서 흡광도(optical density)를 측정하였고, 그 결과를 도 4에 나타내었다. 이때, 흡광도로 유리된 p-니트로페닐의 양을 측정하며, 측정된 p-니트로페닐의 양이 많을수록 베타-글루코시다아제의 활성이 높은 것을 의미한다.As the enzyme reaction solution, 200 ul of 2 mM p-nitrophenyl β-D-glucoside was added to 300 ul of 0.1 M phosphate buffer (phosphate, pH 6.0). 100 ul was added. Thereafter, the reaction was completed at 37 ° C. for 10 to 60 minutes to terminate the reaction by adding 400 ul of 0.5 N NaOH, and 1 ml of distilled water was added thereto. After centrifugation at 3,000 rpm for 20 minutes, the optical density was measured at 405 nm using the supernatant, and the results are shown in FIG. 4. In this case, the amount of p-nitrophenyl liberated by absorbance is measured, and the higher the amount of p-nitrophenyl measured, the higher the activity of beta-glucosidase.
도 4와 같이, 비교예 1의 유산균을 처리한 경우에는 처리 농도가 증가할수록 p-니트로페닐 양이 감소된 반면, 실시예 2의 유산균 함유 천연 리포좀, 비교예 3의 유산균 제품을 처리한 경우에는 처리 농도가 증가하더라도 p-니트로페닐 양이 큰 변화 없는 것으로 나타났다.As shown in FIG. 4, when the lactic acid bacteria of Comparative Example 1 were treated, the amount of p-nitrophenyl was decreased as the treatment concentration was increased, whereas when the lactic acid bacteria-containing natural liposome of Example 2 and the lactic acid bacteria product of Comparative Example 3 were treated There was no significant change in the amount of p-nitrophenyl even with increasing treatment concentration.
또한, 실시예 2의 유산균 함유 천연 리포좀한 경우에는 p-니트로페닐 양이 비교예 1의 유산균, 비교예 3의 유산균 제품을 처리한 경우에 비해 적은 것으로 나타났다.In addition, in the case of lactic acid-containing natural liposomes of Example 2, the amount of p-nitrophenyl was found to be smaller than that of the lactic acid bacteria of Comparative Example 1 and the lactic acid bacteria product of Comparative Example 3.
따라서, 유산균 함유 천연 리포좀은 코팅되지 않은 유산균, 종래 유산균 제품에 비해 장내 유해균의 활성을 억제하여 독성 물질의 생성을 저해하는 효과가 우수한 것을 알 수 있었다.Therefore, it was found that the lactic acid bacteria-containing natural liposomes had an excellent effect of inhibiting the production of toxic substances by inhibiting the activity of harmful bacteria in the intestine compared to the uncoated lactic acid bacteria and conventional lactic acid products.
[[ 실험예Experimental Example 6] 장 상피세포를 이용한 실험(in vitro) 6] Experiment using intestinal epithelial cells (in vitro)
인간 유래 장 상피세포(Caco-2)를 이용하여 유산균에 대한 세포 생존율, 장벽 형성 단백질의 발현능, 물리적 손상 회복능, 유산균과 유해균의 부착능을 측정하였다.Human-derived intestinal epithelial cells (Caco-2) were used to measure cell viability, expression of barrier-forming proteins, repair of physical damage, and adhesion of lactic acid bacteria and harmful bacteria to lactic acid bacteria.
(1) 세포 생존율(1) cell viability
혈청배지(EMEM + 10% FBS + 1% Pen/strep)를 이용하여 마이크로플레이트(96 well)에 Caco-2 세포를 105 cells/well씩 분주한 후 37℃, 5% CO2 배양기에서 24시간 동안 배양하였다. Disperse Caco-2 cells in microplates (96 wells) by 10 5 cells / well using serum medium (EMEM + 10% FBS + 1% Pen / strep) for 24 hours at 37 ℃, 5% CO 2 incubator Incubated for
각 세포의 배지를 제거한 후 정상군으로 혈청배지를 처리하고, 음성대조군, 양성대조군, 실험군으로 장염 유발 물질인 덱스트란설페이트소듐(Dextran sulfate sodium; DSS)을 처리하였다. 이때, 양성대조군에 장염치료제인 설파진(sulfazine)을 처리하고, 실험군에 각각 실시예 2의 유산균 함유 천연 리포좀, 실시예 4의 유산균 함유 천연 리포좀, 실시예 5의 유산균 조성물, 비교예 3의 유산균 제품을 처리한 후 24시간 동안 배양하였다. MTT 분석을 하기 위해, 기존 배지를 제거한 후 PBS에 5 mg/ml의 농도로 용해한 MTT 용액을 배지의 10%가 되도록 희석하여 분주하고 4시간 배양하였다. 이후, 배지를 제거한 후 DMSO 1 ml을 넣고 10분 동안 혼합하여 540 nm에서 흡광도를 측정하였고, 그 결과를 도 5에 나타내었다.After removing the medium of each cell, the serum medium was treated with the normal group, and the dextran sulfate sodium (DSS), which is an enteritis causing substance, was treated with the negative control group, the positive control group, and the experimental group. At this time, the positive control group was treated with sulfazine (sulfazine), which is an enteritis treatment agent, and the experimental group contained lactic acid bacteria-containing natural liposomes of Example 2, lactic acid-containing natural liposomes of Example 4, lactic acid bacteria composition of Example 5, and lactic acid bacteria of Comparative Example 3, respectively. The product was incubated for 24 hours after treatment. For MTT analysis, after removing the existing medium, the MTT solution dissolved in PBS at a concentration of 5 mg / ml was diluted to 10% of the medium and incubated for 4 hours. Thereafter, after removing the medium, 1 ml of DMSO was added, mixed for 10 minutes, and the absorbance was measured at 540 nm. The results are shown in FIG. 5.
도 5와 같이, 실시예 2의 유산균 함유 천연 리포좀, 실시예 4의 유산균 함유 천연 리포좀, 실시예 5의 유산균 조성물을 처리한 경우에는 비교예 3의 종래 유산균 제품을 처리한 경우에 비해 세포 생존율이 높고, 종래 장염치료제를 처리한 경우(양성대조군)와 유사한 세포 생존율을 가지는 것으로 나타났다.As shown in Figure 5, when the lactic acid bacteria-containing natural liposomes of Example 2, the lactic acid bacteria-containing natural liposomes of Example 4, the lactic acid bacteria composition of Example 5 was treated compared to the case of treating the conventional lactic acid bacteria product of Comparative Example 3 It was found to have high cell viability similar to that of the conventional enteritis treatment (positive control).
(2) 장벽형성 단백질의 발현능(2) Expression of barrier protein
장내 세포는 접착 단백질을 통해 세포간의 기계적 결합을 형성하고 있다. 이러한 접착 단백질로는 오클루딘(Occludin)이 있다.Intestinal cells form mechanical bonds between cells through adhesion proteins. One such adhesion protein is Occludin.
먼저, 혈청배지(EMEM + 10% FBS + 1% Pen/strep)를 이용하여 마이크로플레이트(24 well)에 Caco-2 세포를 5ⅹ105 cells/well씩 분주한 후 37℃, 5% CO2 배양기에서 24시간 동안 배양하였다. First, disperse Caco-2 cells by 5ⅹ10 5 cells / well in a microplate (24 wells) using serum medium (EMEM + 10% FBS + 1% Pen / strep), and then incubate at 37 ° C and 5% CO 2 . Incubated for 24 hours.
각 세포의 배지를 제거한 후 정상군으로 혈청배지를 처리하고, 음성대조군, 양성대조군, 실험군으로 장염 유발 물질인 덱스트란설페이트소듐을 처리하였다. 이때, 양성대조군에 장염치료제인 설파진을 처리하고, 실험군에 각각 실시예 2의 유산균 함유 천연 리포좀, 실시예 4의 유산균 함유 천연 리포좀, 실시예 5의 유산균 조성물, 비교예 3의 유산균 제품을 처리한 후 24시간 동안 배양하였다. 각 배지를 제거한 후 세포 용해액(Lysis buffer)을 이용하여 세포액을 모았다. 세포액 내 단백질을 정량하기 위해, 세포액을 5분 동안 끓인 후 15분 동안 얼음에서 냉각하여 정량 시료를 준비하였다. 상기 정량 시료를 10% SDS-PAGE 젤에 로딩한 후 NC 막(membrane)으로 옮겼다(transfer). 상기 NC 막을 5% 스킴 밀크-TBST 용액에 2시간 반응시킨 후 5% 스킴 밀크-TBST 용액에 희석된 오클루딘 항체에 4 하룻밤 동안 반응시켰다. TBST 용액에 30분 동안 흔들어서 세척한 후 2차 항원을 붙여주었다. 암실에서 2차 항원과 ECL 용액을 반응시켜 웨스턴 블롯 필름(western blot film)을 이용하여 오클루딘의 발현을 확인하였고, 그 결과를 도 6에 나타내었다.After removing the medium of each cell, the serum medium was treated with the normal group, and dextran sulfate sodium, which is a enteritis-causing substance, was treated with the negative control group, the positive control group, and the experimental group. At this time, the positive control group was treated with sulfazine, a enteritis treatment agent, and the experimental group was treated with the lactic acid bacteria-containing natural liposomes of Example 2, the lactic acid bacteria-containing natural liposomes of Example 4, the lactic acid bacteria composition of Example 5, and the lactic acid bacteria products of Comparative Example 3, respectively. Then incubated for 24 hours. After removing each medium, the cell solution was collected using a cell lysis solution (Lysis buffer). To quantify the protein in the cell solution, the cell solution was boiled for 5 minutes and then cooled on ice for 15 minutes to prepare a quantitative sample. The quantitative sample was loaded onto a 10% SDS-PAGE gel and then transferred to an NC membrane. The NC membrane was reacted with a 5% Scheme Milk-TBST solution for 2 hours and then reacted for 4 nights with an oculin antibody diluted in 5% Scheme Milk-TBST solution. The TBST solution was shaken for 30 minutes, and then attached with a secondary antigen. Reaction of the second antigen with the ECL solution in the dark was confirmed the expression of occlusin using a western blot film (western blot film), the results are shown in FIG.
도 6과 같이, 실시예 2의 유산균 함유 천연 리포좀, 실시예 4의 유산균 함유 천연 리포좀, 실시예 5의 유산균 조성물을 처리한 경우에는 종래 장염치료제를 처리한 경우(양성대조군)에 비해 오클루딘의 발현량이 많은 것으로 나타났다.As shown in Figure 6, when the lactic acid bacteria-containing natural liposomes of Example 2, the lactic acid bacteria-containing natural liposomes of Example 4, the lactic acid bacteria composition of Example 5, compared to the conventional treatment of enteritis treatment (positive control group) The amount of expression was found to be high.
특히, 실시예 5의 유산균 조성물을 처리한 경우가 오클루딘의 발현양이 가장 많은 것으로 나타났다.In particular, when the lactic acid bacteria composition of Example 5 was treated, the expression amount of occlusin was found to be the highest.
(3) 물리적 손상 회복능(3) physical damage recovery
혈청배지(EMEM + 10% FBS + 1% Pen/strep)를 이용하여 배양접시(35 mm dish)에 Caco-2 세포를 106 cells/well씩 분주한 후 37℃, 5% CO2 배양기에서 24시간 동안 배양하였다. Dispense Caco-2 cells by 10 6 cells / well in a 35 mm dish using serum medium (EMEM + 10% FBS + 1% Pen / strep), and then incubate at 37 ° C and 5% CO 2. Incubated for hours.
각 세포에 팁(tip)을 이용하여 십자 모양으로 긁어 상처를 낸 후 PBS로 2회 세척하였다.Each cell was scratched in a cross shape using a tip, and then washed twice with PBS.
이후, 정상군으로 혈청배지를 처리하고, 음성대조군, 양성대조군, 실험군으로 장염 유발 물질인 덱스트란설페이트소듐을 처리하였다. 이때, 양성대조군에 장염치료제인 설파진을 처리하고, 실험군에 각각 실시예 2의 유산균 함유 천연 리포좀, 실시예 4의 유산균 함유 천연 리포좀, 실시예 5의 유산균 조성물을 처리한 후 24시간 동안 배양하였다. 세포의 회복 정도를 현미경을 관찰하였고, 그 결과를 도 7에 나타내었다.Subsequently, the serum medium was treated with the normal group, and dextran sulfate sodium, which was a enteritis causing substance, was treated with the negative control group, the positive control group, and the experimental group. At this time, the positive control group was treated with sulfazin, a enteritis treatment agent, and treated with the lactic acid bacteria-containing natural liposomes of Example 2, the lactic acid bacteria-containing natural liposomes of Example 4, and the lactic acid bacteria composition of Example 5, respectively, and incubated for 24 hours. . The degree of recovery of the cells was observed under a microscope, and the results are shown in FIG.
도 7과 같이, 실시예 2의 유산균 함유 천연 리포좀, 실시예 4의 유산균 함유 천연 리포좀, 실시예 5의 유산균 조성물을 처리한 경우에는 종래 장염치료제를 처리한 경우(양성대조군), 비교예 3의 유산균 제품을 처리한 경우에 비해 상처난 부위의 세포수가 증가한 것으로 나타났다.As shown in Fig. 7, when the lactic acid bacteria-containing natural liposomes of Example 2, the lactic acid bacteria-containing natural liposomes of Example 4, the lactic acid bacteria composition of Example 5 was treated with a conventional enteritis treatment (positive control group), Comparative Example 3 The number of cells in the wounded area was increased compared with the treatment with the lactic acid bacteria product.
특히, 실시예 5의 유산균 조성물을 처리한 경우에는 상처난 부위의 세포수가 가장 많이 증가한 것으로 나타났다.In particular, when the lactic acid bacteria composition of Example 5 was treated, the number of cells in the wounded area was found to increase the most.
(4) 유산균 및 대장균의 부착능(4) adhesion of lactic acid bacteria and E. coli
혈청배지(EMEM + 10% FBS + 1% Pen/strep)를 이용하여 마이크로플레이트(24 well)에 Caco-2 세포를 105 cells/well씩 분주한 후 37℃, 5% CO2 배양기에서 세포가 단층(monolayer)을 형성할 때까지 배양하였다. The serum-free medium (EMEM + 10% FBS + 1 % Pen / strep) to cells in microtiter plates (24 well) in Caco-2 cells 10 5 cells / well one by one division after 37 ℃, 5% CO 2 incubator using Incubation was made until a monolayer was formed.
락토바실러스 퍼멘텀(Lactobacillus fermentum) Miev L1106(KCTC 12082BP)은 유산균으로, MRS 배지를 사용하여 3회 계대 배양하여 준비하였다. Lactobacillus fermentum (Mactobacillus fermentum) Miev L1106 (KCTC 12082BP) is a lactic acid bacterium, prepared by passage three times using MRS medium.
대장균(Escherichia coli, KCTC 1041)은 유해균으로, TSB(Trypticase soy broth) 배지를 사용하여 3회 계대 배양하여 준비하였다.E. coli (Escherichia coli, KCTC 1041) is a harmful bacterium, prepared by passage 3 times using TSB (Trypticase soy broth) medium.
각 세포의 배지를 제거한 후 PBS 500 ul로 4회 세척하였다. 각 세포에 음성대조군으로 무항생제배지(EMEM + 10% FBS)를 처리하였고, 실험군으로 각각 실시예 2의 유산균 함유 천연 리포좀, 실시예 4의 유산균 함유 천연 리포좀, 실시예 5의 유산균 조성물, 비교예 3의 유산균 제품을 무항생제배지(EMEM + 10% FBS)에 희석하여 처리하였다. 이후, 각 세포에 락토바실러스 퍼멘텀을 1ⅹ109 cfu/ml로 혈청배지에 희석하거나, 또는 대장균을 1ⅹ109 cfu/ml로 PBS에 희석하여 처리하고 37℃, 5% CO2 배양기에서 2시간 동안 배양하였다. 각 세포의 배지를 제거한 후 PBS로 3분씩 3회 세척하였다. 각 세포를 0.2% 트립신-EDTA로 떼어내고 펩톤수를 이용하여 연속희석법으로 MRS 한천 배지(락토바실러스 퍼멘텀 처리시) 또는 Mac Conkey 한천 배지(대장균 처리시)에 도말을 하였다. 이를 37℃, 5% CO2 배양기에서 24시간 동안 배양한 후 균수를 측정하였고, 그 결과를 하기 표 15에 나타내었다.After removing the medium of each cell was washed four times with 500 ul PBS. Each cell was treated with a non-antibiotic medium (EMEM + 10% FBS) as a negative control group, the lactic acid bacteria-containing natural liposomes of Example 2, lactic acid bacteria-containing natural liposomes of Example 4, respectively, as an experimental group, lactic acid bacteria composition of Example 5, Comparative Example The lactic acid bacteria product of 3 was treated by diluting in antibiotic free medium (EMEM + 10% FBS). Then, each cell was treated with 1 × 10 9 cfu / ml of lactobacillus fermentum in serum medium or E. coli diluted with PBS at 1 × 10 9 cfu / ml and treated for 2 hours at 37 ° C. in a 5% CO 2 incubator. It was. After removing the medium of each cell was washed three times with PBS for 3 minutes. Each cell was detached with 0.2% trypsin-EDTA and plated on MRS agar medium (when treated with Lactobacillus permanent) or Mac Conkey agar medium (when treated with E. coli) by continuous dilution using peptone water. This was incubated for 24 hours at 37 ℃, 5% CO 2 incubator and the number of bacteria was measured, the results are shown in Table 15 below.
(cfu/ml)Lactobacillus
(cfu / ml)
(cfu/ml)Escherichia coli
(cfu / ml)
상기 표 15와 같이, 실시예 2의 유산균 함유 천연 리포좀, 실시예 4의 유산균 함유 천연 리포좀, 실시예 5의 유산균 조성물을 처리한 경우에는 비교예 3의 종래 유산균 제품을 처리한 경우에 비해 세포에 부착된 유산균 수가 많고, 대장균 수가 적은 것으로 나타났다.When the lactic acid bacteria-containing natural liposomes of Example 2, the lactic acid bacteria-containing natural liposomes of Example 4, and the lactic acid bacteria composition of Example 5 were treated as shown in Table 15 above, The number of lactic acid bacteria attached and the number of Escherichia coli were small.
특히, 실시예 5의 유산균 조성물을 처리한 경우에는 부착된 유산균 수가 가장 많고, 실시예 4의 유산균 함유 천연 리포좀을 처리한 경우에는 부착된 대장균 수가 가장 적은 것으로 나타났다.In particular, when the lactic acid bacterium composition of Example 5 was treated, the number of lactic acid bacteria attached was the highest, and when the lactic acid bacteria-containing natural liposomes of Example 4 were treated, the smallest number of E. coli was attached.
[[ 실험예Experimental Example 7] 대장암 세포를 이용한 실험(in vitro) 7] Experiment using colon cancer cells (in vitro)
인간 유래 대장 선암(Colorectal adenocarcinoma) 세포인 HT-29 세포를 이용하여 유산균의 대장암 발생 억제 효과를 측정하였다.HT-29 cells, colorectal adenocarcinoma cells, were used to measure the inhibitory effect of lactic acid bacteria on colorectal cancer.
혈청배지(EMEM + 10% FBS + 1% Pen/strep)를 이용하여 마이크로플레이트(24 well)에 HT-29 세포를 0.8ⅹ104 cells/well씩 분주한 후 37℃, 5% CO2 배양기에서 4시간 동안 배양하였다. 각 세포에 음성대조군으로 무혈청배지를 처리하고, 양성대조군으로 대장암 치료제인 5-플루오로우라실(5-Fluorouracil)을 처리하고, 실험군으로 각각 실시예 2의 유산균 함유 천연 리포좀, 실시예 4의 유산균 함유 천연 리포좀, 실시예 5의 유산균 조성물, 비교예 3의 유산균 제품을 처리하였고, 37℃, 5% CO2 배양기에서 7일 동안 배양하였다. 이후, 각 세포에 6% 글루타르알데히드, 0.1% 크리스탈 바이올렛을 첨가하여 15분 동안 혼합한 후 PBS로 세척하였다. 각 세포를 현미경으로 관찰하였고, 그 결과를 도 8에 나타내었다.Using serum medium (EMEM + 10% FBS + 1% Pen / strep), HT-29 cells were aliquoted into microplates (24 wells) at 0.8 cells10 4 cells / well, and then incubated at 37 ° C and 5% CO 2 incubator. Incubated for hours. Each cell was treated with a serum-free medium with a negative control group, 5-Fluorouracil, a colorectal cancer therapeutic agent, with a positive control group, and the lactic acid bacteria-containing natural liposomes of Example 2 were treated as experimental groups, respectively. Lactic acid bacteria-containing natural liposomes, the lactic acid bacteria composition of Example 5, the lactic acid bacteria products of Comparative Example 3 were treated, and incubated for 7 days at 37 ℃, 5% CO 2 incubator. Thereafter, 6% glutaraldehyde, 0.1% crystal violet was added to each cell, mixed for 15 minutes, and washed with PBS. Each cell was observed under a microscope, and the results are shown in FIG.
도 8과 같이, 실시예 2의 유산균 함유 천연 리포좀, 실시예 4의 유산균 함유 천연 리포좀, 실시예 5의 유산균 조성물을 처리한 경우에는 비교예 3의 종래 유산균 제품을 처리한 경우에 비해 콜로니가 적게 형성되며, 이는 종래 대장암 치료제를 처리한 경우(양성 대조군)와 유사한 것으로 나타났다.As shown in FIG. 8, when the lactic acid bacteria-containing natural liposomes of Example 2, the lactic acid bacteria-containing natural liposomes of Example 4, and the lactic acid bacteria composition of Example 5 were treated, there was less colony than when the conventional lactic acid bacteria products of Comparative Example 3 were treated. It was formed, which appeared to be similar to that when treated with a conventional colorectal cancer treatment (positive control).
특히, 실시예 4의 유산균 함유 천연 리포좀을 처리한 경우에는 콜로니가 가장 적게 형성된 것으로 나타났다.In particular, when treated with the lactic acid bacteria-containing natural liposomes of Example 4, it was found that the least colony was formed.
[[ 실험예Experimental Example 8] 8] 랫트(Rat)를Rat 이용한 동물실험(in Animal testing using vivovivo ))
5주령 SD 랫트를 정상군, 음성대조군, 양성대조군, 실험군으로 나누어 실험하였다.Five-week-old SD rats were divided into two groups: normal, negative control, positive control, and experimental groups.
정상군과 음성대조군에는 일반사료를 30일 동안 제공하고, 양성대조군에는 일반사료와 장염치료제인 설파진을 30일 동안 제공하고, 실험군에는 일반사료와 각각 실시예 2의 유산균 함유 천연 리포좀, 실시예 4의 유산균 함유 천연 리포좀, 실시예 5의 유산균 조성물, 비교예 3의 유산균 제품을 30일 동안 제공하였다. 이후, 정상군에는 별도로 제공하지 않고, 음성대조군, 양성대조군, 실험군에는 덱스트란설페이트소듐을 5-7일 동안 음용수의 3~5%로 제공하여 장염을 유발시켰다.The normal group and the negative control group was provided with the general feed for 30 days, the positive control group was given the general feed and enteritis treatment sulfaphine for 30 days, and the experimental group with the normal feed and lactic acid bacteria-containing natural liposomes of Example 2, Example Lactic acid bacteria-containing natural liposome of 4, lactic acid bacteria composition of Example 5, lactic acid bacteria products of Comparative Example 3 were provided for 30 days. Thereafter, not provided to the normal group, the negative control group, the positive control group, dextran sulfate sodium in the experimental group was provided for 3-7% of drinking water for 5-7 days to induce enteritis.
(1) 장내 유익균과 유해균의 증식(1) Proliferation of enterococci and harmful bacteria
각 랫트를 희생하여 즉시 장 내용물을 채취한 후 멸균된 0.1% 펩톤수(Peptone water)에 균질화하여 10배 희석하였다. 이를 PBS로 10-8배까지 희석하여 분석 시료를 준비하였다.Intestinal contents were immediately taken at the expense of each rat and homogenized in sterile 0.1% peptone water and diluted 10-fold. This was diluted 10 -8 times with PBS to prepare analytical samples.
상기 분석 시료를 BS 한천 배지, Mac Conkey 한천 배지에 도말하였다. BS 한천 배지는 37℃, CO2 배양기에서 2일 동안 배양하고, Mac Conkey 한천 배지는 37℃, 혐기적 배양기(10% H2, 10% CO2, 80% N2)에서 2일 동안 배양한 후 세포수를 세었고, 그 결과를 도 9에 나타내었다. 이때, BS 한천 배지에서는 유익균으로 비피도박테리움을 확인할 수 있고, Mac Conkey 한천 배지에서는 유해균으로 대장균을 확인할 수 있다.The assay samples were plated in BS agar medium, Mac Conkey agar medium. BS agar medium was incubated for 2 days at 37 ℃, CO 2 incubator, Mac Conkey agar medium was incubated for 2 days at 37 ℃, anaerobic incubator (10% H 2 , 10% CO 2 , 80% N 2 ) After counting the cells, the results are shown in FIG. At this time, Bifidobacterium can be identified as beneficial bacteria in BS agar medium, and E. coli can be identified as harmful bacteria in Mac Conkey agar medium.
도 9와 같이, 장염이 유발될 경우(음성대조군)에는 장내 유익균에 비해 유해균이 증가하는 것으로 나타났다. 이때, 장염치료제(양성대조군), 실시예 2의 유산균 함유 천연 리포좀, 실시예 4의 유산균 함유 천연 리포좀, 실시예 5의 유산균 조성물, 비교예 3의 유산균 제품을 제공할 경우에는 유익균이 증가하고, 유해균이 감소하는 것으로 나타났다.As shown in FIG. 9, when enteritis is induced (negative control group), harmful bacteria are increased as compared to enteric beneficial bacteria. At this time, when providing enteritis treatment agent (positive control), lactic acid bacteria-containing natural liposomes of Example 2, lactic acid bacteria-containing natural liposomes of Example 4, lactic acid bacteria composition of Example 5, lactic acid bacteria products of Comparative Example 3, the beneficial bacteria increases, The harmful bacteria were found to decrease.
특히, 실시예 2의 유산균 함유 천연 리포좀을 제공한 경우에는 유해균이 현저하게 감소하고, 실시예 5의 유산균 조성물을 제공한 경우에는 유익균이 가장 많이 증가하는 것으로 나타났다.In particular, when the lactic acid bacteria-containing natural liposomes of Example 2 were provided, the harmful bacteria were significantly reduced, and when the lactic acid bacteria composition of Example 5 was provided, the beneficial bacteria were most increased.
(2) 장내 염증인자 측정(2) Intestinal Inflammation Factor Measurement
각 랫트를 희생한 후 대장 말단 부위를 적출하여 PBS로 세척하였다. 이를 균질기(homogenizer)로 균질화한 후 20,000 xg, 4에서 30분 동안 원심분리하여 상층액을 분석 시료로 사용하였다.After sacrifice of each rat, the colonic distal area was extracted and washed with PBS. This was homogenized with a homogenizer and centrifuged at 20,000 xg, 4 for 30 minutes to use the supernatant as an analytical sample.
상기 분석 시료를 이용하여 염증인자 IL-6, TNF-a를 측정하기 위해, IL-6 ELISA 키트(고마바이오텍社, Rat IL-6), TNF-a ELISA 키트(고마바이오텍社, Rat TNF-a)를 사용하였고, 그 결과를 도 10 및 도 11에 나타내었다.In order to measure inflammatory factors IL-6 and TNF-a using the assay sample, IL-6 ELISA kit (Rama IL-6, Goma Biotech Co., Ltd.), TNF-a ELISA kit (Rat TNF-a, Goma Biotech Co., Ltd.) ), And the results are shown in FIGS. 10 and 11.
도 10 및 도 11과 같이, 장염이 유발될 경우(음성대조군)에는 정상군에 비해 IL-6, TNF-a의 분비량이 증가하는 것으로 나타났다. 이때, 실시예 2의 유산균 함유 천연 리포좀, 실시예 4의 유산균 함유 천연 리포좀, 실시예 5의 유산균 조성물을 제공한 경우에는 종래 장염치료제를 제공한 경우(양성대조군)와 IL-6의 분비량이 유사한 반면, TNF-a의 분비량이 감소하는 것으로 나타났다. 그러나, 비교예 3의 종래 유산균 제품을 제공한 경우에는 IL-6, TNF-a의 분비량이 감소하지 않는 것으로 나타났다.10 and 11, when enteritis is induced (negative control group), the secretion amount of IL-6 and TNF-a is increased compared to the normal group. In this case, when the lactic acid bacteria-containing natural liposomes of Example 2, the lactic acid bacteria-containing natural liposomes of Example 4, and the lactic acid bacteria composition of Example 5 were provided, the secretion amount of IL-6 was similar to that of the conventional enteritis treatment (positive control group). In contrast, the secretion of TNF-a was found to decrease. However, when the conventional lactic acid bacteria product of Comparative Example 3 was provided, the secretion amount of IL-6 and TNF-a did not decrease.
(3) 항체 농도 측정(3) antibody concentration measurement
소장의 면역기관인 페이에르판(peyer's patch)에는 면역세포인 T 세포, B 세포, M 세포가 많이 분포하며, 이러한 면역세포에서 분비된 IgA는 장내 점막면역에서 중요한 역할을 한다.Peyer's patch, an immune organ of the small intestine, contains a large number of immune cells, T cells, B cells, and M cells. IgA secreted from these immune cells plays an important role in intestinal mucosal immunity.
먼저, 상기 실험예 8의 (2)와 동일한 방법으로 분석 시료를 준비하였다. First, an analytical sample was prepared in the same manner as in (2) of Experimental Example 8.
상기 분석 시료를 이용하여 IgA를 측정하기 위해, IgA ELISA 키트(고마바이오텍社, Rat IgA)를 사용하였고, 그 결과를 도 12에 나타내었다.In order to measure IgA using the assay sample, an IgA ELISA kit (Roma IgA, Goma Biotech Co., Ltd.) was used, and the results are shown in FIG. 12.
도 12와 같이, 장염이 유발될 경우(음성대조군)에는 정상군에 비해 IgA의 분비량이 감소하는 것으로 나타났다. 이때, 실시예 2의 유산균 함유 천연 리포좀, 실시예 4의 유산균 함유 천연 리포좀, 실시예 5의 유산균 조성물을 제공한 경우에는 종래 장염치료제를 제공한 경우(양성대조군)에 비해 IgA의 분비량이 증가하는 것으로 나타났다. 특히, 실시예 5의 유산균 조성물을 제공한 경우에는 IgA의 분비량이 가장 많은 것으로 나타났다.As shown in Figure 12, when enteritis is induced (negative control group) it was shown that the amount of IgA secretion compared to the normal group. In this case, when the lactic acid bacteria-containing natural liposomes of Example 2, the lactic acid bacteria-containing natural liposomes of Example 4, and the lactic acid bacteria composition of Example 5 were provided, the amount of IgA secretion increased compared to the case of providing a conventional enteritis treatment agent (positive control group). Appeared. In particular, when the lactic acid bacterium composition of Example 5 was provided, the highest amount of IgA was found.
(4) 면역세포 관찰(4) Immune cell observation
소장의 면역기관인 페이에르판(peyer's patch)에 존재하는 T 세포, B 세포, M 세포를 면역조직화학법(Immunohistochemistry)을 이용하여 관찰하였다. 이때, T 세포의 마커(Marker)로는 CD3와 CD68, B 세포의 마커로는 CD79a, M 세포의 마커로는 CK8을 이용하였다.T cells, B cells, and M cells present in the Peyer's patch, the immune organs of the small intestine, were observed using immunohistochemistry. In this case, CD3 and CD68 as markers of T cells, CD79a as markers of B cells, and CK8 as markers of M cells were used.
먼저, 각 랫트를 희생한 후 소장에서 페이에르판이 함유된 조직을 적출하여 10% 포름알데히드에 담근 후 파라핀으로 고정시켜 조직을 높이 70 mm로 절단하였다. 절단된 조직을 슬라이드 위에 올려놓고 자일렌(Xylene)으로 20분 동안 3회 세척한다. 이후, 100% 에탄올에서 5분 동안, 90% 에탄올에서 5분 동안, 80% 에탄올에서 5분 동안 순차적으로 세척한 후 증류수로 세척한다. 상기 조직을 항원 노출 버퍼(Antigen retrieval buffer, 10 mM 시트르산나트륨+0.05% 트윈-20, pH 6.0)에 10분 동안 담가 끓인 후 증류수를 이용하여 식혀주었다. 상기 조직에 각 1차 항체(CK8, CD3, CD68, CD79a)를 처리하여 4℃에서 하룻밤 동안 반응시킨 후 2차 항체를 처리하여 상온에서 1시간 동안 반응시켰다. 이후, 100% 에탄올에서 5분 동안, 90% 에탄올에서 5분 동안, 80% 에탄올에서 5분 동안 순차적으로 세척한 후 자일렌으로 20분 동안 세척하였다. 각 조직에 DAPI가 첨가된 봉입제(Vector Laboratories社, Vectashield)를 올려 현미경으로 관찰하였고, 그 결과를 도 13 내지 도 16에 나타내었다.First, after sacrifice of each rat, Peyer's tissues were extracted from the small intestine, soaked in 10% formaldehyde and fixed with paraffin to cut the tissue to 70 mm in height. The cut tissue is placed on a slide and washed three times with xylene for 20 minutes. After 5 minutes in 100% ethanol, 5 minutes in 90% ethanol, 5 minutes in 80% ethanol and then sequentially washed with distilled water. The tissue was immersed in an antigen exposure buffer (Antigen retrieval buffer, 10 mM sodium citrate + 0.05% Tween-20, pH 6.0) for 10 minutes and then cooled using distilled water. Each tissue was treated with primary antibodies (CK8, CD3, CD68, CD79a) and reacted overnight at 4 ° C., followed by reaction with secondary antibodies for 1 hour at room temperature. Then washed sequentially for 5 minutes in 100% ethanol, 5 minutes in 90% ethanol, 5 minutes in 80% ethanol and then 20 minutes with xylene. DAPI was added to each tissue was added to observe the microscope (Vector Laboratories, Vectashield), and the results are shown in Figures 13 to 16.
도 13 내지 도 16과 같이, 장염이 유발될 경우(음성대조군)에는 정상군에 비해 페이에르판 주변에 면역세포의 분포가 감소하는 것으로 나타났다. 이때, 실시예 2의 유산균 함유 천연 리포좀, 실시예 4의 유산균 함유 천연 리포좀, 실시예 5의 유산균 조성물을 제공한 경우에는 종래 장염치료제를 제공한 경우(양성대조군), 비교예 3의 종래 유산균 제품을 제공한 경우에 비해 T 세포, B 세포, M 세포의 분포가 증가하는 것으로 나타났다.As shown in Figures 13 to 16, when enteritis is induced (negative control group) it was found that the distribution of immune cells around the Payerphane compared to the normal group. In this case, when the lactic acid bacteria-containing natural liposomes of Example 2, the lactic acid bacteria-containing natural liposomes of Example 4, and the lactic acid bacteria composition of Example 5 were provided, the conventional enteritis treatment agent (positive control group), the conventional lactic acid bacteria products of Comparative Example 3 The distribution of T cells, B cells, M cells was increased compared to the case provided.
따라서, 본 발명의 유산균 함유 천연 리포좀 및 이를 유효성분으로 함유하는 유산균 조성물은 장내 유익균과 유해균의 균총을 조절하여 장내 환경을 개선시켜 면역세포 증식과 항체 생성을 유도함으로써 장을 건강하게 유지하는데 도움을 줄 수 있다.Therefore, the lactic acid bacteria-containing natural liposomes of the present invention and the lactic acid bacteria composition containing the same as an active ingredient improve the intestinal environment by regulating the intestinal beneficial bacteria and harmful bacteria, thereby inducing immune cell proliferation and antibody production to help maintain the intestine healthy. Can give
Claims (18)
(b) 상기 코어를 둘러싸며, 천연 다당체를 포함하는 제1쉘층;
(c) 상기 제1쉘층을 둘러싸며, 천연 유화제 및 천연 단백질을 포함하는 제2쉘층; 및
(d) 상기 제2쉘층을 둘러싸며, 천연 동결보호제 및 천연 보존제를 포함하는 제3쉘층
을 포함하는 유산균 함유 천연 리포좀으로서,
상기 천연 다당체는 아라비아검(Arabic gum), 구아검(Guar gum), 로커스트빈검(Locust bean gum), 타마린드검(Tamarind gum), 타라검(Tara gum), 잔탄검(Xanthan gum) 및 카라기난(Carrageenan)으로 이루어진 군에서 선택되는 1종 이상이고,
상기 천연 유화제는 포스파티딜콜린, 리소포스파티딜콜린, 포스파티딜에탄올아민, 포스파티딜산, 포스파티딜세린, 포스파티딜글리세롤, 포스파티딜이노시톨 및 이들의 수소첨가 생성물로 이루어진 군에서 선택되는 1종 이상이며,
상기 천연 동결보호제는 탈지분유, 말토덱스트린 및 글리세린으로 이루어진 군에서 선택되는 1종 이상이며,
상기 천연 보존제는 감귤 추출물, 고삼 추출물, 유자 추출물, 유칼립투스 추출물, 자몽 추출물, 정향 추출물 및 크랜베리 추출물로 이루어진 군에서 선택되는 1종 이상이며,
상기 유산균 함유 천연 리포좀은 전체 중량을 기준으로, 상기 유산균 1 내지 20 중량%, 상기 천연 다당체 0.5 내지 5 중량%, 상기 천연 유화제 5 내지 30 중량%, 상기 천연 단백질 0.5 내지 10 중량%, 상기 천연 동결보호제 40 내지 70 중량% 및 천연 보호제 5 내지 15 중량%를 포함하는 것인 유산균 함유 천연 리포좀.(a) lactic acid bacteria as a core;
(b) a first shell layer surrounding the core and comprising natural polysaccharides;
(c) a second shell layer surrounding the first shell layer and comprising a natural emulsifier and a natural protein; And
(d) a third shell layer surrounding the second shell layer and comprising a natural cryoprotectant and a natural preservative
As lactic acid bacteria-containing natural liposomes containing,
The natural polysaccharides include arabic gum, guar gum, locust bean gum, tamarind gum, tara gum, xanthan gum and carrageenan. Carrageenan) is one or more selected from the group consisting of
The natural emulsifier is at least one selected from the group consisting of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidyl acid, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol and hydrogenated products thereof,
The natural cryoprotectant is at least one selected from the group consisting of skim milk powder, maltodextrin and glycerin,
The natural preservative is at least one member selected from the group consisting of citrus extract, red ginseng extract, citron extract, eucalyptus extract, grapefruit extract, clove extract and cranberry extract,
The lactic acid bacteria-containing natural liposomes, based on the total weight, 1 to 20% by weight of the lactic acid bacteria, 0.5 to 5% by weight of the natural polysaccharide, 5 to 30% by weight of the natural emulsifier, 0.5 to 10% by weight of the natural protein, the natural freezing Lactic acid bacteria-containing natural liposomes comprising 40 to 70% by weight of the protective agent and 5 to 15% by weight of the natural protective agent.
상기 유산균은 락토바실러스 아시도필루스(Lactobacillus acidophilus), 락토바실러스 불가리쿠스(Lactobacillus bulgaricus), 락토바실러스 카제이(Lactobacillus casei), 락토바실러스 퍼멘텀(Lactobacillus fermentum), 락토바실러스 가세리(Lactobacillus gasseri), 락토바실러스 헬베티쿠스(Lactobacillus helveticus), 락토바실러스 파라카제이(Lactobacillus paracasei), 락토바실러스 플란타룸(Lactobacillus plantarum), 락토바실러스 람노서스(Lactobacillus rhamnosus), 락토바실러스 루테리(Lactobacillus reuteri), 락토바실러스 살리바리우스(Lactobacillus salivarius), 락토코커스 락티스(Lactococcus lactis), 스트렙토코커스 써모필러스(Streptococcus thermophilus), 비피도박테리움 애니멀리스 락티스(Bifidobacterium animalis ssp. lactis), 비피도박테리움 비피덤(Bifidobacterium bifidum), 비피도박테리움 브레브(Bifidobacterium breve), 비피도박테리움 롱검(Bifidobacterium longum) 및 엔테로코커스 페칼리스(Enterococcus faecalis)로 이루어진 군에서 선택되는 1종 이상인 유산균 함유 천연 리포좀.The method of claim 1,
The lactobacillus is Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus fermentum, Lactobacillus fertilizer, L gasobacillus gasseri Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus rhamnosus, Lactobacillus lutobacilli Barius (Lactobacillus salivarius), Lactococcus lactis, Streptococcus thermophilus, Bifidobacterium animalis ssp.lactis, Bifidobacterium bifidum ), Bifidobacterium breve, Bifidobacterium Lactic acid bacteria-containing natural liposomes selected from the group consisting of long gum (Bifidobacterium longum) and Enterococcus faecalis.
상기 천연 단백질은 우유 유래 단백질이거나, 또는 콩 유래 단백질이거나, 또는 우유 유래 단백질과 콩 유래 단백질의 혼합물인 유산균 함유 천연 리포좀.The method of claim 1,
The natural protein is a milk-derived protein, or a soybean-derived protein, or a lactobacillus-containing natural liposome that is a mixture of milk-derived protein and soy-derived protein.
(ii) 40 내지 75℃의 용매에 천연 단백질, 천연 다당체를 투입하여 초음파 처리한 후 유산균을 투입하고 혼합하는 제2단계;
(iii) 상기 제1단계에 얻어진 생성물, 상기 제2단계에서 얻어진 생성물, 천연 동결보호제를 혼합하는 제3단계; 및
(iv) 상기 제3단계에서 얻어진 생성물에 천연 보존제를 더 투입하여 혼합하고 이를 동결건조하는 제4단계
를 포함하는 제1항, 제3항, 제6항 중 어느 한 항의 유산균 함유 천연 리포좀의 제조방법.(i) a first step of ultrasonically adding a natural emulsifier to a solvent at 40 to 75 ° C;
(ii) a second step of adding a natural protein and a natural polysaccharide to a solvent at 40 to 75 ° C., sonicating and then adding and mixing the lactic acid bacteria;
(iii) a third step of mixing the product obtained in the first step, the product obtained in the second step, and a natural cryoprotectant; And
(iv) adding a natural preservative to the product obtained in the third step, mixing and lyophilizing the same;
A method for producing a lactic acid bacterium-containing natural liposome according to any one of claims 1, 3 and 6.
상기 용매는 증류수, 천연 유래의 부틸렌글리콜, 프로판디올, 글리세린 및 발효주정으로 이루어진 군에서 선택되는 1종 이상인 유산균 함유 천연 리포좀의 제조방법.The method of claim 11,
The solvent is a method for producing a natural liposome containing lactic acid bacteria is at least one selected from the group consisting of distilled water, butylene glycol, propanediol, glycerin and fermentation alcohol derived from nature.
상기 제2단계는 50℃ 미만의 용매에 유산균을 투입하는 것인 유산균 함유 천연 리포좀의 제조방법.The method of claim 11,
The second step is a method for producing a lactic acid bacteria-containing natural liposomes that is added to the lactic acid bacteria in a solvent of less than 50 ℃.
(ii) 40 내지 75℃의 용매에 천연 유화제를 투입하여 초음파 처리하는 제2단계;
(iii) 40 내지 75℃의 용매에 천연 단백질을 투입하여 초음파 처리하는 제3단계;
(iv) 상기 제1단계에서 얻어진 생성물, 상기 제2단계에서 얻어진 생성물, 제3단계에서 얻어진 생성물, 천연 동결보호제를 혼합하는 제4단계; 및
(v) 상기 제4단계에서 얻어진 생성물에 천연 보존제를 더 투입하여 혼합하고 이를 동결건조하는 제5단계
를 포함하는 제1항, 제3항, 제6항 중 어느 한 항의 유산균 함유 천연 리포좀의 제조방법.(i) a first step of adding natural polysaccharides to a solvent at 40 to 75 ° C. and performing ultrasonic treatment followed by adding and mixing lactic acid bacteria;
(ii) a second step of ultrasonically adding a natural emulsifier to a solvent at 40 to 75 ° C;
(iii) a third step of sonicating natural protein in a solvent at 40 to 75 ° C;
(iv) a fourth step of mixing the product obtained in the first step, the product obtained in the second step, the product obtained in the third step, and a natural cryoprotectant; And
(v) a fifth step of adding a natural preservative to the product obtained in the fourth step, mixing and lyophilizing it;
A method for producing a lactic acid bacterium-containing natural liposome according to any one of claims 1, 3 and 6.
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