KR20200126204A - Composition for improving intestinal health comprising red ginseng residue - Google Patents
Composition for improving intestinal health comprising red ginseng residue Download PDFInfo
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- KR20200126204A KR20200126204A KR1020190049897A KR20190049897A KR20200126204A KR 20200126204 A KR20200126204 A KR 20200126204A KR 1020190049897 A KR1020190049897 A KR 1020190049897A KR 20190049897 A KR20190049897 A KR 20190049897A KR 20200126204 A KR20200126204 A KR 20200126204A
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- KR
- South Korea
- Prior art keywords
- red ginseng
- composition
- intestinal
- bacteria
- indigestible
- Prior art date
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Abstract
Description
본 발명은 홍삼박을 포함하는 장 건강 개선용 조성물에 관한 것으로, 보다 구체적으로는 비피도박테리움(Bifidobacterium) 균의 증식을 촉진하면서 클로스트리듐(Clostridium)균의 증식은 억제하고, 더불어 장내 균총을 개선시키며(Firmicutes 문 및 Bacteroidetes 문의 비율 증가), 장내 단쇄지방산 (Short chain fatty acid, SCFA) 농도를 증가시키고 암모니아태 질소 및/또는 스카톨(skatole)의 농도를 감소시킴으로써 장내환경을 개선할 수 있는 홍삼박 또는 홍삼박 내 난소화성 물질을 유효성분으로 포함하는 장 건강 개선용 조성물에 관한 것이다.The present invention relates to a composition for improving intestinal health comprising red ginseng leaf, and more specifically, promoting the proliferation of Bifidobacterium bacteria while inhibiting the proliferation of Clostridium bacteria, and together with the intestinal flora The intestinal environment can be improved by increasing the concentration of short chain fatty acid (SCFA) in the intestine and reducing the concentration of ammonia-like nitrogen and/or skatole (increased the rate of Firmicutes and Bacteroidetes ). It relates to a composition for improving intestinal health comprising red ginseng leaf or indigestible material in red ginseng leaf as an active ingredient.
장내세균은 유익균과 유해균이 공생 또는 길항 관계를 유지하면서 섭취된 음식물 또는 소화관에서 분비된 점액을 영양분으로 이용하여 끊임없이 증식하고 배설된다. 이 과정 중에 건전하지 못한 식습관이 지속되면 장내세균 구성이 변화하여 장관 면역상태에 이상이 발생하고 장 질환 등 건강에 나쁜 영향을 주게 된다. 세계적으로 궤양성 대장염(ulcerative colitis)이나 크론병(Crohn disease) 등을 포함한 염증성 장 질환(inflammatory bowel disease, IBD) 발병률이 계속 증가하고 있으며 한국에서도 이러한 장 질환이 증가추세를 보이고 있다. 장내 유해균에 의해 생성되는 부패 대사 산물(암모니아, 황화수소, 아민, 페놀, 인돌 등), 세균독소, 발암물질(니트로소 화합물, 에폭시 등) 및 2차성 담즙산 등의 유해물질은 장관에 직접 장애를 일으키며, 일부는 흡수되어 장기간 여러 장기에 장애를 주어 이른바 생활습관병의 원인이 되기도 한다.Intestinal bacteria are constantly proliferating and excreted using ingested food or mucus secreted from the digestive tract as nutrients while maintaining a symbiotic or antagonistic relationship between beneficial and harmful bacteria. If unhealthy eating habits continue during this process, the composition of intestinal bacteria changes, causing abnormalities in the intestinal immunity and adversely affecting health such as intestinal diseases. The incidence of inflammatory bowel disease (IBD), including ulcerative colitis and Crohn disease, continues to increase around the world, and such intestinal diseases are showing an increasing trend in Korea. Hazardous substances such as decay metabolites (ammonia, hydrogen sulfide, amine, phenol, indole, etc.), bacterial toxins, carcinogens (nitroso compounds, epoxy, etc.) and secondary bile acids produced by harmful bacteria in the intestine directly cause disorders in the intestine. In some cases, some of them are absorbed, causing disorders in various organs for a long period of time, leading to so-called lifestyle-related diseases.
장 기능과 관련된 대표적 기능성 성분으로는 비전분성 다당류인 식이섬유(dietary fiber) 및 올리고당(oligosaccharides)이 있고, 전분성 다당류로는 난소화성 말토덱스트린 및 난소화성 전분(resistant starch, RS) 등이 있다. 이러한 물질들을 난소화성 물질(당질) 또는 기능성 식이섬유라고 부른다. 식이섬유는 인체의 소화효소에 의해 소화되지 않는 대표적인 비소화성 다당류로, 물에 대한 용해도에 따라 수용성과 불용성으로 분류되는데 전자에는 구아감(guar gum), 난소화성 말토덱스트린, 수용성 펙틴(pectin), 이눌린(inulin) 및 올리고당 등이 포함된다. 후자에는 셀룰로오스(cellulose), 헤미셀룰로오스(hemicellulose), 리그닌(lignin), 불용성 펙틴 및 키토산(chitosan) 등이 있다. 또한 난소화성 전분(RS)에는 4가지 형태가 존재한다. RS1은 물리적으로 소화가 되지 않는 전분을 말하고, RS2는 생감자 전분이나 고아밀로스 전분(high-amylose starch, HAS)처럼 겔화(gelatination)가 잘 되지 않는 전분을 지칭한다. RS3은 주로 노화전분을, 그리고 RS4는 화학적 변성전분(food modified starch)을 말한다. 이와 같이 난소화성 물질은 그 종류와 범위가 매우 넓다. 현재까지 알려진 난소화성 물질에 대한 정장기능(整腸機能)으로는, 결장 내 소화물 체류시간 단축 및 배변량을 증가시키는 배변활동 개선효과 및 장내 유익균이 늘어나는 프리바이오틱스(prebiotics) 효과 등이 알려져 있다. 장내 최종발효 성분인 단쇄지방산이 이러한 장 건강 증진에 관련되어 주요 생리 기능 요인으로 주목받고 있다. 난소화성 물질은 장 건강 증진 이외에도, 혈중 콜레스테롤 감소, 혈당조절, 장관 면역 증강 및 다이어트 효과 등의 다양한 생리활성을 가진 다기능성 건강식품 소재로도 인정받고 있다.Representative functional ingredients related to intestinal function include non-starchy polysaccharides such as dietary fiber and oligosaccharides, and starchy polysaccharides include indigestible maltodextrin and resistant starch (RS). These substances are called indigestible substances (saccharides) or functional dietary fiber. Dietary fiber is a representative non-digestible polysaccharide that is not digested by human digestive enzymes, and is classified into water-soluble and insoluble according to its solubility in water. The former includes guar gum, indigestible maltodextrin, water-soluble pectin, and These include inulin and oligosaccharides. The latter include cellulose, hemicellulose, lignin, insoluble pectin, and chitosan. There are also four forms of indigestible starch (RS). RS1 refers to starch that cannot be physically digested, and RS2 refers to starch that does not gelatinize well, such as raw potato starch or high-amylose starch (HAS). RS3 mainly refers to aged starch, and RS4 refers to food modified starch. As such, indigestible materials have a very wide variety and range. As for the intestinal function of indigestible substances known to date, the effect of improving bowel activity to shorten the residence time of digestive substances in the colon and increase the amount of bowel movement, and the effect of prebiotics to increase the beneficial bacteria in the intestine are known. Short-chain fatty acids, the final fermentation component in the intestine, are attracting attention as a major physiological function factor related to the improvement of intestinal health. Indigestible substances are recognized as a multifunctional health food material having various physiological activities such as reducing blood cholesterol, regulating blood sugar, enhancing intestinal immunity and dieting effects, in addition to promoting intestinal health.
현재 국내에서 사용되고 있는 대부분의 식이섬유는 전분 유래의 저분자량 수용성 식이섬유(폴리덱스트로오스, polydextrose)로 음료제조에 주로 이용되고 있으며, 밀기울, 귀리, 옥수수 껍질 등으로부터 얻는 불용성 식이섬유는 제과 및 제빵 등에 이용되고 있다.Most of the dietary fiber currently used in Korea is a low molecular weight water-soluble dietary fiber derived from starch (polydextrose, polydextrose), which is mainly used for beverage production.Insoluble dietary fiber obtained from bran, oats, corn husks, etc. is confectionery and It is used for baking.
유럽에서는 치커리(chicory) 뿌리에서 수용성 식이섬유인 이눌린(inulin)을 정제하여 대량 생산하고 있다. 현재 이눌린 형태의 프락탄(inulin-type fructan)을 이용한 지방대체 기술이 주목받고 있는데, 이눌린을 잘 섞으면 크림과 같은 물성을 형성하게 되고 형성된 크림 물성을 지방대체 용도로 사용하는 기술이다. 남미에서는 용설란(agave) 뿌리에서 가지형 이눌린을 추출하여 시럽 등 설탕의 대체 감미료로 사용하고 있고, 기능성을 감안한 기능성 식품 및 가공식품에 첨가한 보강식품 등의 개발이 증가하고 있다. 일본의 경우 10여종의 식이섬유 및 올리고당이 상품화되었다. 상품화된 제품들은 일부 미생물 효소공정에 의해 대량 생산되고 있지만, 대부분의 제품은 주제품 생산공정에서 나오는 부산물 또는 폐기물을 재이용할 목적으로 개발된 환경친화적 제품이 많다.In Europe, inulin, a water-soluble dietary fiber, is purified and mass-produced from chicory roots. Currently, a technology for replacing fat using inulin-type fructan is attracting attention. When inulin is well mixed, it forms a cream-like property, and the formed cream property is used for fat replacement. In South America, eggplant-type inulin is extracted from the root of agave and is used as an alternative sweetener for sugar such as syrup, and the development of functional foods considering functionality and supplementary foods added to processed foods is increasing. In the case of Japan, 10 kinds of dietary fiber and oligosaccharides have been commercialized. Commercialized products are mass-produced by some microbial enzyme processes, but most of them are environmentally friendly products developed for the purpose of reusing by-products or wastes from the main product production process.
홍삼에는 주로 원삼과 같이 진세노사이드(ginsenosides)라는 여러 종류의 사포닌 물질이 다량으로 함유되어 있으며, 그 외에 폴리아세틸렌계 화합물, 함질소성분, 미량원소, 유기산 및 아미노산 등이 함유되어 있다. 일반 성분으로는 탄수화물이 전체 60-70%를 차지한다. 홍삼 제품을 제조할 경우, 대부분 추출 및 농축 과정을 거쳐 농축액을 만든 후, 각 제품의 유형에 맞게 액상, 분말, 또는 음료 등의 형태로 제조하고 있다. 인삼 가공 업체에서는 농축액을 제조하기 위하여 홍삼의 원형을 열수 또는 주정(에탄올)을 이용하여 유효성분을 추출해 내고 있다. 하지만, 현재 대부분의 업체에서는 농축액을 추출한 후 남은 홍삼박의 처리나 활용에 많은 애로를 겪고 있다. 또한 유효성분뿐만 아니라 식이섬유 및 산성다당체 등이 홍삼박에 다량 잔류하고 있음에도 불구하고 기술적 한계로 인하여 홍삼박의 재활용이 매우 미흡한 실정이다.Like original ginseng, red ginseng contains a large amount of various types of saponin substances called ginsenosides, and in addition, polyacetylene-based compounds, nitrogen-containing components, trace elements, organic acids, and amino acids. As a general ingredient, carbohydrates account for 60-70% of the total. When manufacturing red ginseng products, most of them are extracted and concentrated to make a concentrate, and then they are manufactured in the form of liquid, powder, or beverage according to the type of each product. Ginseng processing companies use hot water or alcohol (ethanol) to extract the active ingredients from the original form of red ginseng in order to prepare a concentrate. However, at present, most companies are experiencing a lot of difficulties in processing or utilizing the red ginseng meal remaining after extracting the concentrate. In addition, although a large amount of dietary fiber and acidic polysaccharides, as well as active ingredients, remain in red ginseng leaf, recycling of red ginseng leaf is very insufficient due to technical limitations.
또한, 난소화성 물질은 그 종류와 범위가 매우 넓으며 종류에 따라 생리적인 특성이 차이가 있으므로, 홍삼박(또는 원삼박)과 같은 천연한방의 미이용 자원을 활용하여 차별화된 고부가가치 제품을 개발하고, 수요 창출을 위해 각각의 난소화성 물질의 생리기능에 관한 체계적인 연구가 시급히 필요한 실정이다.In addition, indigestible substances have a very wide type and range, and physiological characteristics are different according to the type, so we develop differentiated high value-added products by utilizing unused resources of natural oriental medicine such as red ginseng leaf (or raw ginseng leaf). In order to create demand, a systematic study on the physiological function of each indigestible substance is urgently needed.
이에 본 발명자들은 돼지 분변을 이용한 액침혐기배양(in vitro)시험에 있어서 홍삼박의 장내 발효 특성 및 장내 세균의 균형에 대한 연구를 진행한 결과, 홍삼박 유래 난소화성 물질이 장내 균총의 균형개선과 더불어 유익균(특히, Bifidobacterium균)의 증식을 촉진하고, 암모니아 생성에 관련된 유해균(특히, Clostridium균)의 상대적 비율을 억제하며, 장내 단쇄지방산(초산 및 프로피온산)의 농도를 증가시키고 암모니아태 질소 및 스카톨의 농도를 감소시킴으로써 장 건강 또는 장 기능을 개선할 수 있다는 사실을 발견함으로써 본 발명을 완성하였다.Therefore, the present inventors conducted a study on the intestinal fermentation characteristics of red ginseng meal and the balance of intestinal bacteria in an immersion anaerobic culture ( in vitro ) test using pig feces. As a result, the indigestible substance derived from red ginseng meal improved the balance of the intestinal flora and In addition, it promotes the proliferation of beneficial bacteria (especially Bifidobacterium bacteria), suppresses the relative ratio of harmful bacteria (especially Clostridium bacteria) related to ammonia production, increases the concentration of short-chain fatty acids (acetic acid and propionic acid) in the intestine, and increases the concentration of ammonia-like nitrogen and scar. The present invention has been completed by finding that reducing the concentration of toll can improve intestinal health or intestinal function.
따라서, 본 발명에서 해결하고자 하는 기술적 과제는 장 건강 개선용 조성물을 제공하기 위한 것이다.Therefore, the technical problem to be solved in the present invention is to provide a composition for improving intestinal health.
상기한 기술적 과제를 해결하기 위하여, 본 발명은 홍삼박을 유효성분으로 포함하는 장 건강 개선용 식품 조성물을 제공한다.In order to solve the above technical problem, the present invention provides a food composition for improving intestinal health comprising red ginseng as an active ingredient.
본 발명의 하나의 구현예에 따르면, 본 발명은 홍삼박을 유효성분으로 포함하는 조성물이 장내 균총을 개선(파르미큐테스(Firmicutes) 문 및 박테리오이데테스(Bacteroidetes) 문의 비율 증가)시키는 것을 특징으로 하는 장 건강 개선용 식품 조성물을 제공한다.According to an embodiment of the invention, wherein the present invention is a composition comprising the red ginseng as the active ingredient to the intestinal microflora improvement (Parr vermiculite test (Firmicutes) door and bakteriohyi to test (Bacteroidetes) increased contact ratio) It provides a food composition for improving intestinal health.
본 발명의 다른 하나의 구현예에 따르면, 본 발명은 홍삼박을 유효성분으로 포함하는 조성물이 장내 유익균, 특히 비피도박테리움(Bifidobacterum) 균을 증식시키는 것을 특징으로 하는 장 건강 개선용 식품 조성물을 제공한다.According to another embodiment of the invention, the invention is a composition comprising the red ginseng as the active ingredient the intestinal yuikgyun, in particular Bifidobacterium (Bifidobacterum) Food composition sheet, comprising a step of proliferating the bacteria health improvement to provide.
여기서, 상기 증식은 장내 유익균, 특히 비피도박테리움(Bifidobacterum) 균 수를 1.2 내지 30배, 바람직하게는 1.5 내지 20배, 보다 바람직하게는 2 내지 20배, 보다 더 바람직하게는 2 내지 10배 증가시키는 것을 말한다.Here, the proliferation is intestinal yuikgyun, in particular Bifidobacterium (Bifidobacterum) 1.2 to 30 times the number of bacteria, preferably 1.5 to 20 times, more preferably 2 to 20 times, and more preferably, from 2 to 10 times It says to increase.
본 발명의 다른 하나의 구현예에 따르면, 본 발명은 홍삼박을 유효성분으로 포함하는 장내 암모니아 생산을 억제하여 혈중 암모니아 농도를 저하시키는 것을 특징으로 하는 장 건강 개선용 식품 조성물을 제공한다.According to another embodiment of the present invention, the present invention provides a food composition for improving intestinal health, characterized in that reducing the concentration of ammonia in blood by suppressing the production of ammonia in the intestine containing red ginseng as an active ingredient.
본 발명은 또한 홍삼박을 유효성분으로 포함하는 장 건강 개선용 사료 조성물을 제공한다.The present invention also provides a feed composition for improving intestinal health comprising red ginseng leaf as an active ingredient.
상기 장내 균총 개선이란 장내 유익균의 생장은 촉진하고, 장내 유해물질 생성균의 생장은 억제하여, 장내 유익균과 유해균의 균형을 개선시키는 것을 의미한다. 특히, 장내 균총 중 Firmicutes 문 및 Bacteroidetes 문의 비율을 증가시키는 것을 의미한다.The improvement of the intestinal flora means promoting the growth of beneficial bacteria in the intestine and inhibiting the growth of harmful substances producing bacteria in the intestine, thereby improving the balance between beneficial bacteria in the intestine and harmful bacteria. In particular, it is meant to increase the proportion of Firmicutes and Bacteroidetes in the intestinal flora.
프로바이오틱스(probiotics, 생균제)는 체내에서 건강에 좋은 영향을 미치는 미생물을 의미하는 것으로, 비피도박테리움 (Bifidobacterium) 균을 포함하는 미생물에 의해 이용되어 유익균의 생육이나 활성을 촉진함으로써 숙주의 건강에 좋은 효과를 나타내게 하는 성분이다.Probiotics (probiotics) refer to microorganisms that have a good effect on health in the body, and are used by microorganisms including Bifidobacterium to promote the growth or activity of beneficial bacteria, which is good for the health of the host. It is an ingredient that makes it effective.
본 발명의 구체적인 실시 양태에 따르면, 프로바이오틱스(probiotics, 생균제)의 먹이 역할을 하는 프리바이오틱스(prebiotics)로서 대표적 물질인 자일로올리고당을 사용하여 홍삼박의 장내 유산균(특히, Bifidobacterium균) 증식 및 유해물질 생성균(특히, Clostridium속 세균)의 상대적 비율의 억제효과가 있음을 확인하여 홍삼박의 프리바이오틱스로서의 가능성을 확인하였다.According to a specific embodiment of the present invention, probiotics (prebiotics) that serve as food for probiotics (probiotics) are used as a representative substance, xyloligosaccharide, to proliferate and harm intestinal lactic acid bacteria (in particular, Bifidobacterium bacteria) It was confirmed that there is an inhibitory effect of the relative ratio of substance-producing bacteria (especially, bacteria of the genus Clostridium ), and the possibility of red ginseng meal as a prebiotic was confirmed.
본 발명의 하나의 구현예에서, 상기 홍삼박은 홍삼박 또는 홍삼박 난소화성 물질일 수 있다.In one embodiment of the present invention, the red ginseng leaf may be a red ginseng leaf or red ginseng leaf indigestible material.
상기 홍삼박은 홍삼을 물 또는 알코올 등의 용매로 추출하고 남은 잔여물로서 홍삼을 수회 반복 추출하더라도 내부의 유효성분을 모두 추출할 수 없고, 또한 추출용매에 따라 물 또는 알코올에 용해되는 성분만 추출되고 용해되지 않은 사포닌, 폴리아세틸렌, 단백질 등의 유효성분이 대략 20 ~ 40% 정도 잔류하는 천연물질이다.The red ginseng leaf is a residue left after extracting red ginseng with a solvent such as water or alcohol. Even if the red ginseng is repeatedly extracted several times, it is not possible to extract all the active ingredients inside, and only components soluble in water or alcohol are extracted depending on the extraction solvent. It is a natural substance in which approximately 20 to 40% of active ingredients such as undissolved saponin, polyacetylene, and protein remain.
상기 홍삼박 난소화성 물질은 홍삼박이 체내에 들어가 소화효소인 판크레이틴(pancretin) 및 알파-아미로글리코시다아제로 가수분해될 때 가수분해되지 않는 물질을 의미하는 것으로, 체내에서 생성되는 물질이다.The red ginseng foil indigestible substance refers to a substance that does not hydrolyze when red ginseng meal enters the body and is hydrolyzed with digestive enzymes, pancretin and alpha-amiroglycosidase, and is a substance produced in the body. .
본 발명의 구체적인 실시태양에 따르면, 홍삼박을 인간의 장내세균총 구성과 유사한 돼지 분변물을 사용하여 혐기적 액침배양을 수행한 결과 pH는 음성대조군인 셀룰로오스 첨가구에 비해 유의하게 낮은 것을 확인할 수 있다.According to a specific embodiment of the present invention, as a result of performing anaerobic immersion culture of red ginseng meal using pig feces similar to the composition of human intestinal flora, it can be seen that the pH is significantly lower than that of the negative control group cellulose added. .
장내 pH가 낮은 상태에서는, 유해균은 산에 민감하여 생육 성장이 감소하지만 유익균은 능동적으로 기질을 발효시켜 산에 대한 내성을 갖기 때문에 성장이 유지된다. 따라서 낮은 pH 환경에서 유익균은 병원균 등의 유해균과 접착 경쟁에 우위를 차지함으로써 장관내 유해균의 침입을 저해하는 역할을 한다.In a state of low intestinal pH, harmful bacteria are sensitive to acid, so growth growth decreases, but beneficial bacteria actively ferment substrates to have resistance to acid, so growth is maintained. Therefore, in a low pH environment, beneficial bacteria play a role in inhibiting the invasion of harmful bacteria in the intestine by taking advantage of the competition for adhesion with harmful bacteria such as pathogens.
본 발명의 구체적인 실시 양태에 따르면, 홍삼박을 인간의 장내세균총 구성과 유사한 돼지 분변물을 사용하여 혐기적 액침배양을 수행한 결과 총 단쇄지방산 농도가 음성대조구인 셀룰로오스 첨가구보다 전반적으로 유의적으로 높은 것을 확인할 수 있다.According to a specific embodiment of the present invention, as a result of performing anaerobic immersion culture of red ginseng meal using pig feces similar to the composition of human intestinal flora, the total short-chain fatty acid concentration was significantly higher than that of the negative control cellulose-added group. Can be confirmed.
상기 단쇄 지방산이란 3개 이하의 탄소 원자를 포함하는 지방산을 의미하는 것으로, 초산(acetic acid), 프로피온산(propionic acid), 또는 낙산(butyric acid)일 수 있다.The short-chain fatty acid refers to a fatty acid containing 3 or less carbon atoms, and may be acetic acid, propionic acid, or butyric acid.
결장 안으로 흘러 들어간 발효성 식이섬유 또는 난소화성 전분(RS)은 장내 세균에 의해 단쇄지방산으로 대사된다. 생성된 단쇄지방산은 결장에서 중요한 음이온(anion)으로 작용하여 수분과 나트륨 흡수를 자극하고 장관 운동성을 조절하며, 대장암 위험을 감소시키는 등 결장 기능에 많은 영향을 미치고 있다. 또한 각각의 단쇄지방산은 서로 다른 생리 기능을 가지고 있다. 예를 들어, 초산은 대장에서 가장 많이 생성되는 단쇄지방산으로 프로피온산과 함께 대장 상피세포에서 흡수되어 전신적으로 다양하게 이용된다. 특히 초산은 간과 말초기관으로 이동하여 포도당 신생합성(gluconeogenesis)과 지방 합성(lipogenesis)을 위한 기질이 되고, 프로피온산은 반대로 주요 지질 기질의 대사를 억제하는 것으로 알려져 있다. 한편, 낙산은 대장 세포의 가장 중요한 에너지원이 된다.Fermentable dietary fiber or indigestible starch (RS) flowing into the colon is metabolized to short-chain fatty acids by intestinal bacteria. The produced short-chain fatty acid acts as an important anion in the colon, stimulating the absorption of water and sodium, regulating intestinal motility, and reducing the risk of colon cancer. In addition, each short-chain fatty acid has a different physiological function. For example, acetic acid is the most frequently produced short-chain fatty acid in the large intestine and is absorbed in colon epithelial cells along with propionic acid and is used in a variety of systemic ways. In particular, acetic acid moves to the liver and peripheral organs and becomes a substrate for glucose synthesis and lipogenesis, and propionic acid, on the contrary, is known to inhibit the metabolism of major lipid substrates. On the other hand, Naksan is the most important energy source for colon cells.
본 발명의 구체적인 실시 양태에 따르면, 홍삼박은 프로피온산 생성에 큰 영향을 주는 것을 확인할 수 있다. 일반적으로 사람의 대변 내 초산：프로피온산：낙산 분자량 비율(molar proportion)은 60：20：20을 이루고 있는데 홍삼박 첨가구의 경우 프로피온산이 증가하였고 낙산이 감소하는 것을 확인할 수 있다. 홍삼박 첨가구에서 생성된 단쇄지방산 및 각 단쇄지방산 분자량 비율 변화 차이는, 홍삼박 내 셀룰로오스와 같은 비발효성 식이섬유가 다량 함유되어 있는 점과 홍삼박에는 자일로올리고당과 다른 장내세균이 선호하는 발효성 고분자 다당류가 혼잡해 있을 가능성이 존재하기 때문이다.According to a specific embodiment of the present invention, it can be seen that red ginseng leaf has a great effect on the production of propionic acid. In general, the acetic acid: propionic acid: butyric acid molecular proportion in human feces is 60:20:20. In the case of adding red ginseng meal, propionic acid increased and butyric acid decreased. The difference in the ratio of the short-chain fatty acids and the molecular weight of each short-chain fatty acid produced in the red ginseng meal added is that it contains a large amount of non-fermentable dietary fiber such as cellulose in red ginseng meal, and the fermentation favored by xyloligosaccharide and other intestinal bacteria. This is because there is a possibility that there is a congestion of sex polymer polysaccharides.
본 발명의 구체적인 실시 양태에 따르면, 홍삼박을 인간의 장내세균총 구성과 유사한 돼지 분변물을 사용하여 혐기적 액침배양을 수행하여 장내세균 증식 및 마이크로바이옴을 분석한 결과 비피도박테리움 (Bifidobacterium) 속 균의 증식 및 전체 세균 중 비피도박테리움 속 균의 상대적 비율이 증가하는 것을 확인할 수 있다.According to a specific embodiment of the present invention, as a result of analyzing the growth of intestinal bacteria and microbiome by performing anaerobic immersion culture using pig feces similar to the composition of human intestinal flora of red ginseng leaf, Bifidobacterium It can be seen that the proliferation of bacteria of the genus ( Bifidobacterium ) and the relative proportion of bacteria of the genus Bifidobacterium among all bacteria increase.
본 발명의 다른 하나의 구현예에 따르면, 상기 홍삼박을 포함하는 조성물은 비피도박테리움(Bifidobacterum) 균을 1.2 내지 30배, 바람직하게는 1.5 내지 20배, 보다 바람직하게는 2 내지 20배, 보다 더 바람직하게는 2 내지 10배로 증식하는 것을 특징으로 한다.According to another embodiment of the present invention, the composition comprising the red ginseng leaf is 1.2 to 30 times, preferably 1.5 to 20 times, more preferably 2 to 20 times, Bifidobacterum bacteria, Even more preferably, it is characterized in that it proliferates 2 to 10 times.
본 발명에서 장내 유익균이란, 장내에 서식하고 있으면서 인체에 유익한 효능을 미치는 미생물을 총칭할 수 있다. 예컨대, 장내 유익균은 프로바이오틱스(Bifidobacterium, Lactobacillus)를 포함할 수 있다. 예컨대, 상기 장내 유익균은 이에 제한되지 않고, 락토코커스(Lactococcus), 스트렙토코커스(Streptococcus), 아커멘시아(Akkermansia), 패컬리박테리움(Faecalibacterium), 또는 엔테로코커스(Enterococcus)를 포함할 수 있다.In the present invention, beneficial bacteria in the intestine may collectively refer to microorganisms that live in the intestine and have beneficial effects on the human body. For example, the beneficial bacteria in the intestine may include probiotics ( Bifidobacterium , Lactobacillus ). For example, the intestinal yuikgyun is not limited to this, Lactococcus (Lactococcus), may include Streptococcus (Streptococcus), Acker men cyano (Akkermansia), L Curley tumefaciens (Faecalibacterium), or Enterococcus (Enterococcus).
상기 프로바이오틱스는 인체에 이로움을 주는 생균제를 말하고 대표적 프로바이오틱스로는 유산균이 있다. 하지만 모든 유산균이 프로바이오틱스를 지칭하는 것은 아니다. 그에 반해 프리바이오틱스는 프로바이오틱스 증식에 도움을 주는 식품 유래의 난소화성 성분을 말하며 장내 유해균을 제한하거나 유익균의 생육을 선택적으로 촉진시켜 숙주 장내환경 개선에 도움을 준다. 대표적 프리바이오틱스로는 저분자 물질인 올리고당(프락토올리고당, 갈락토올리고당, 이소말토올리고당, 자일로올리고당, 키틴올리고당 등)과 고분자 물질인 식이섬유(난소화성 덱스트린, 이눌린, 구아검 등) 등이 있다. 이러한 프리바이오틱스는 구성물질의 종류 및 분자간 결합형태, 연결사슬의 길이에 따라 유익균의 선호가 다르게 나타난다. 예를 들어, Ito 등은 중합도가 서로 다른 이눌린 형태의 프락탄(DP4, DP8, DP16, DP23, 평균 중합도)을 랫트에 투여한 결과 Lactobacillus 균은 짧은 사슬인 프락토올리고당(DP4, DP8)에서 생육이 증가했고, Bifidobacterium의 경우는 대체적으로 고분자 형태 이눌린(DP8, DP16, DP23) 투여군에서 증가했다고 보고하고 있다(Ito et al., J. Agri. Food Chem., doi: 10.1021/jf200859z).The probiotics refer to probiotics that benefit the human body, and representative probiotics include lactic acid bacteria. However, not all lactobacilli refer to probiotics. On the other hand, prebiotics refer to food-derived indigestible ingredients that help probiotic proliferation, and help improve the host intestinal environment by limiting harmful bacteria in the intestine or selectively promoting the growth of beneficial bacteria. Representative prebiotics include oligosaccharides (fructooligosaccharides, galactooligosaccharides, isomaltooligosaccharides, xyloligosaccharides, chitinoligosaccharides, etc.), which are low molecular weight substances, and dietary fibers (indigestible dextrin, inulin, guar gum, etc.), which are polymer substances. have. These prebiotics have different preferences for beneficial bacteria depending on the type of constituent material, the form of intermolecular bonding, and the length of the linking chain. For example, as a result of administration of inulin-type fructans (DP4, DP8, DP16, DP23, average degree of polymerization) with different polymerization degrees to rats, Lactobacillus bacteria grow from short chain fructooligosaccharides (DP4, DP8). It has been reported that Bifidobacterium was generally increased in the group administered with high molecular weight inulin (DP8, DP16, DP23) (Ito et al ., J. Agri. Food Chem., doi: 10.1021/jf200859z).
한편, 본 발명에서 장내 유해균이란, 장내에서 서식하고 유해물질을 생성하며 장염 등과 같이 인체에 해로운 효과를 미치는 미생물을 총칭할 수 있다. 예컨대, 상기 장내 유해균은, Clostridium균에 제한되지 않고, 푸소박테리움(Fusobacterium) 또는 포피로모나스 (Porphyromonas)를 포함할 수 있다.Meanwhile, in the present invention, the intestinal harmful bacteria may collectively refer to microorganisms that inhabit the intestine, generate harmful substances, and have harmful effects on the human body such as enteritis. For example, the intestinal harmful bacteria are not limited to Clostridium bacteria, and may include Fusobacterium or Porphyromonas .
본 발명의 하나의 구현예에 따르면, 본 발명의 조성물은 비피도박테리움(Bifidobacterium)의 증식 또는 생장을 촉진할 수 있고, 클로스트리듐(Clostridium)의 증식 또는 생장을 억제할 수 있다.According to one embodiment of the present invention, the composition of the present invention may promote the proliferation or growth of Bifidobacterium , and may inhibit the proliferation or growth of Clostridium .
본 발명의 구체적인 실시 양태에 따르면, 홍삼박 난소화성 물질을 인간의 장내세균총 구성과 유사한 돼지 분변물을 사용한 혐기적 액침배양액의 차세대 시퀀싱에 의한 메타 분석을 통하여 장내세균 종의 수에 유의적 차이가 나타났고, 주좌표분석 (베타-다양성)에서도 첨가구 사이 큰 차이를 확인할 수 있다.According to a specific embodiment of the present invention, there is a significant difference in the number of intestinal bacterial species through meta-analysis by next-generation sequencing of anaerobic immersion cultures using pig feces similar to the composition of human intestinal flora of red ginseng foil indigestible substances. Appeared, and the main coordinate analysis (beta-diversity) also showed a large difference between the added spheres.
본 발명의 구체적인 실시태양에 따르면, 홍삼박을 인간의 장내세균총 구성과 유사한 돼지 분변물을 사용하여 혐기적 액침배양을 수행한 결과 암모니아태 질소 생성 농도가 음성 대조군인 셀룰로오스 처리구에 비해 유의하게 억제되는 것을 확인할 수 있다.According to a specific embodiment of the present invention, as a result of performing anaerobic immersion culture of red ginseng meal using pig feces similar to the composition of human intestinal flora, the concentration of ammonia nitrogen production is significantly suppressed compared to the cellulose treatment group, which is a negative control. Can be confirmed.
일반적으로 음식물로부터 분해된 아미노산과 숙주로부터 유래된 단백질의 발효는 다양하고 광범위한 대사산물을 만들어낸다. 예를 들어, 대변의 이소낙산, 이소발레르산 및 2-메틸 낙산 등과 같은 분기 사슬 지방산(branched-chain fatty acid, BCFA)은 분기 사슬 아미노산(branched-chain amino acid) 발효의 지표이며, 이들의 대변 내 농도는 고단백 식사일수록 증가한다. 또한 대장의 환경에 따라, 유산, 호박산, 알파-케토산 등의 중간 대사 산물이 대장 내에 축적될 수도 있다. 단백질 발효 생성물에는 잠재적 독성을 갖는 것들이 있는데, 대표적인 예로 암모니아(NH3), N-니트로소 화합물, 아민류, p-크레솔 등을 들 수 있다. 특히, 대장 내 암모니아 생성은 음식물 유래의 아미노산이 세균에 의해 분해되거나 요소가 세균의 우레아제(urease)에 의해 분해될 때 생성된다. 대장 내 암모니아는 낮은 pH보다 높은 pH에서 더 많이 생성된다. 생체 내 암모니아는 단백질의 대사 과정에서 아미노산으로 탈아미노되어 생기며 간의 요소회로에서 요소로 변환되지만, 혈중 암모니아의 대부분은 소장 점막과 대장 내 세균에 의해 생산된 암모니아에서 유래한다. 혈중 암모니아 농도가 상승하게 되면 신경독성물질로 작용하여 간성뇌증(minimal hepatic encephalopathy)의 주요 원인이 될 수 있다. 장내 암모니아를 생성하는 세균으로는 Clostridium종, Peptostreptococcus, Fusobacterium, Actinomyces, Bacteroides, Megasphaera 및 Propionibacterium 등이 있으며 (Richardson et al., BMC Microbiol., doi: org/10.1186/1471-2180-13-6), 유산균은 암모니아를 매우 적게 형성한다(Vince & Burridge, J Med Microbiol, doi: 10.1099/00222615-13-2-177).In general, fermentation of amino acids degraded from food and proteins derived from the host produces a wide variety of metabolites. For example, branched-chain fatty acids (BCFAs), such as isobutyric acid, isovaleric acid and 2-methylbutyric acid in feces, are indicators of branched-chain amino acid fermentation, and their feces My concentration increases with high protein meals. Also, depending on the environment of the large intestine, intermediate metabolites such as lactic acid, succinic acid, and alpha-ketosan may accumulate in the large intestine. Protein fermentation products include those with potential toxicity. Representative examples include ammonia (NH 3 ), N -nitroso compounds, amines, and p -cresol. In particular, the production of ammonia in the large intestine is produced when amino acids derived from food are degraded by bacteria or when urea is decomposed by bacteria's urease. Ammonia in the large intestine is produced more at higher pH than at lower pH. In vivo, ammonia is deaminated to amino acids in the process of protein metabolism and is converted to urea in the liver urea circuit, but most of ammonia in blood comes from ammonia produced by bacteria in the small intestine mucosa and colon. When the concentration of ammonia in the blood rises, it acts as a neurotoxic substance and can be a major cause of minimal hepatic encephalopathy. Intestinal ammonia-producing bacteria include Clostridium species, Peptostreptococcus , Fusobacterium , Actinomyces , Bacteroides , Megasphaera, and Propionibacterium (Richardson et al ., BMC Microbiol., doi: org/10.1186/1471-2180-13-6), Lactobacillus forms very little ammonia (Vince & Burridge, J Med Microbiol, doi: 10.1099/00222615-13-2-177).
본 발명의 구체적인 실시태양에 따르면, 홍삼박을 인간의 장내세균총 구성과 유사한 돼지 분변물을 사용하여 혐기적 액침배양을 수행한 결과 스카톨(Skatole) 생성 농도가 셀룰로오스 첨가구보다 통계적으로 유의하게 낮은 것을 확인할 수 있다.According to a specific embodiment of the present invention, as a result of performing anaerobic immersion culture of red ginseng meal using pig feces similar to the composition of human intestinal flora, it was found that the concentration of Skatole production was statistically significantly lower than that of the cellulose-added group. I can confirm.
상기 스카톨은 소화관 내 미생물에 의해 트립토판에서 분해되어 생성된 인돌(indole) 유도체로 인돌에 메틸기가 붙어 있는 구조(3-methylindole)를 갖고 있으며, 돼지 웅취 또는 분변취의 원인이 된다.The scatol is an indole derivative produced by decomposition of tryptophan by microorganisms in the digestive tract and has a structure in which a methyl group is attached to the indole (3-methylindole) and causes boar odor or fecal odor in pigs.
따라서, 본 발명에 따른 홍삼박을 유효성분으로 포함하는 조성물은 장 건강 개선, 장내 균총 개선, 프로바이오틱스(probiotics, 생균제) 효능 증강용 조성물로 식품 또는 사료 조성물로 유용하게 사용할 수 있다.Therefore, the composition comprising the red ginseng leaf according to the present invention as an active ingredient can be usefully used as a food or feed composition as a composition for improving intestinal health, improving intestinal flora, and enhancing the efficacy of probiotics (probiotics).
본 발명의 조성물에 유효성분으로 포함되는 홍삼박은 전체 조성물의 총 중량을 기준으로 0.001 내지 99 중량%, 바람직하게는 0.01 내지 80 중량%, 보다 바람직하게는 0.01 내지 50 중량%의 함량으로 포함될 수 있다. 상기 홍삼박의 함량이 0.001 중량% 미만일 경우 장 건강 개선 효과가 미약한 문제가 있다.Red ginseng foil included as an active ingredient in the composition of the present invention may be included in an amount of 0.001 to 99% by weight, preferably 0.01 to 80% by weight, more preferably 0.01 to 50% by weight based on the total weight of the total composition. . When the content of the red ginseng foil is less than 0.001% by weight, there is a problem that the effect of improving intestinal health is weak.
본 명세서에서 사용되는 용어 "개선"은 개체에서 질환 또는 질병의 증세가 호전되는 모든 행위를 의미한다.As used herein, the term "improvement" means any action in which a disease or symptom of a disease is improved in an individual.
본 명세서에서 사용되는 용어 "개체"는 본 발명의 상기 조성물을 투여하여 증상이 호전될 수 있는 질환을 가진 인간을 포함한 침팬지, 원숭이, 돼지, 개, 고양이, 래트, 마우스 등의 포유동물을 의미한다.The term "individual" as used herein refers to mammals such as chimpanzees, monkeys, pigs, dogs, cats, rats, mice, etc., including humans with diseases capable of improving symptoms by administering the composition of the present invention. .
본 명세서에서 사용되는 용어 "난소화성 물질"은 포유류의 소화효소에 의해 분해되지 않은 이당류 이상의 탄수화물과 함질소성분으로, 식이섬유, 올리고당, 난소화성 전분, 난소화성 단백질 및 난소화성 펩타이드를 의미한다.The term "digestible substance" as used herein refers to a carbohydrate and nitrogen-containing component of more than disaccharides that are not degraded by the digestive enzymes of mammals, and refers to dietary fiber, oligosaccharides, indigestible starch, indigestible proteins, and indigestible peptides.
본 발명의 조성물을 식품 조성물로 사용할 경우, 상기 홍삼박을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용할 수 있고, 통상의 방법에 따라 적절하게 사용할 수 있다. 유효 성분의 혼합양은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있으며, 본 발명의 조성물은 친환경적이며 안정성 면에서 문제가 없기 때문에 혼합량에 큰 제한은 없다.When the composition of the present invention is used as a food composition, the red ginseng leaf may be added as it is or may be used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health, or therapeutic treatment), and the composition of the present invention is environmentally friendly and there is no problem in terms of stability, so there is no large limitation on the mixing amount.
본 발명의 식품 조성물은, 일상적으로 섭취하는 것이 가능하기 때문에 대장 기능 개선 효과를 기대할 수 있으므로, 건강 증진을 목적으로 하는 건강기능식품으로써 유용하게 사용될 수 있다.Since the food composition of the present invention can be consumed on a daily basis, an effect of improving large intestine function can be expected, and thus can be usefully used as a health functional food for the purpose of improving health.
본 발명의 용어, "건강기능식품"이란, 특정보건용 식품(food for special health use, FoSHU)과 동일한 용어로, 영양 공급 외에도 생체조절기능이 효율적으로 나타나도록 가공된 의학, 의료효과가 높은 식품을 의미한다. 여기서 "기능"이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건용도에 유용한 효과를 얻는 것을 의미한다. 본 발명의 건강기능식품은 당 업계에서 통상적으로 사용되는 방법에 의하여 제조가능하며, 상기 제조시에는 당 업계에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있다. 또한, 상기 건강기능식품의 제형 또한 식품으로 인정되는 제형이면 제한 없이 제조될 수 있다. 본 발명의 건강기능식품은 다양한 형태의 제형으로 제조될 수 있으며, 일반 약품과는 달리 식품을 원료로 하여 약품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있고, 휴대성이 뛰어나므로, 본 발명의 건강기능식품은 장 기능 개선 효과를 증진시키기 위한 보조제로 섭취가 가능하다.The term "health functional food" of the present invention is the same term as a food for special health use (FOSHU), and is a food with high medical effects and medicine processed to effectively show a biological regulation function in addition to nutrition supply Means. Here, the term "function" means to control nutrients on the structure and function of the human body or to obtain useful effects for health purposes such as physiological actions. The health functional food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and ingredients commonly added in the art. In addition, the formulation of the health functional food may be prepared without limitation as long as it is a formulation recognized as a food. The health functional food of the present invention can be prepared in various forms of formulation, and unlike general drugs, it has the advantage of not having side effects that may occur when taking the drug for a long time using food as a raw material, and is excellent in portability. The health functional food of the present invention can be ingested as a supplement for enhancing the effect of improving intestinal function.
상기 건강기능식품은 일반식품에 비해 적극적인 건강유지나 증진효과를 가지는 식품을 의미하고, 건강보조식품(health supplement food)은 건강보조 목적의 식품을 의미한다. 경우에 따라, 건강기능식품, 건강식품, 건강보조식품의 용어는 혼용된다.The health functional food refers to a food having an active health maintenance or promoting effect compared to a general food, and a health supplement food refers to a food for health supplement purposes. In some cases, the terms health functional food, health food, and health supplement food are used interchangeably.
구체적으로, 상기 건강기능식품은 본 발명의 조성물을 음료, 차류, 향신료, 껌, 과자류 등의 식품 소재에 첨가하거나, 캡슐화, 분말화, 현탁액 등으로 제조한 식품으로, 이를 섭취할 경우 건강상 특정한 효과를 가져오는 것을 의미하나, 일반 약품과는 달리 식품을 원료로 하여 약품의 장기 복용 시 발생할 수 있는 부작용이 없는 장점이 있다.Specifically, the health functional food is a food prepared by adding the composition of the present invention to food materials such as beverages, teas, spices, gums, confectionery, or encapsulating, powdering, and suspending. It means bringing about an effect, but unlike general drugs, it has the advantage of having no side effects that may occur when taking the drug for a long time by using food as a raw material.
상기 건강기능식품은 생리학적으로 허용 가능한 담체를 추가로 포함할 수 있는데, 담체의 종류는 특별히 제한되지 않으며 당해 기술 분야에서 통상적으로 사용되는 담체라면 어느 것이든 사용할 수 있다.The health functional food may further include a physiologically acceptable carrier, and the kind of the carrier is not particularly limited, and any carrier commonly used in the art may be used.
또한, 상기 건강기능식품은 식품에 통상 사용되어 냄새, 맛, 시각 등을 향상시킬 수 있는 추가 성분을 포함할 수 있다. 예들 들어, 비타민 A, C, D, E, B1, B2, B6, B12, 니아신(niacin), 비오틴(biotin), 폴레이트(folate), 판토텐 산(panthotenic acid) 등을 포함할 수 있다. 또한, 아연(Zn), 철(Fe), 칼슘(Ca), 크롬(Cr), 마그네슘(Mg), 망간(Mn), 구리(Cu) 등의 미네랄; 및 라이신, 트립토판, 시스테인, 발린 등의 아미노산을 포함할 수 있다.In addition, the health functional food may include additional ingredients that are commonly used in food to improve odor, taste, and vision. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, and the like may be included. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), and copper (Cu); And amino acids such as lysine, tryptophan, cysteine, and valine.
또한, 상기 건강기능식품은 보존제(소르빈산 칼륨, 벤조산나트륨, 살리실산, 데히드로초산나트륨 등), 살균제(표백분과 고도 표백분, 차아염소산나트륨 등), 산화방지제(부틸히드록시아니졸(BHA), 부틸히드록시톨류엔(BHT) 등), 착색제(타르색소 등), 발색제(아질산 나트륨, 아초산 나트륨 등), 표백제(아황산나트륨), 조미료(MSG 글루타민산나트륨 등), 감미료(둘신, 사이클레메이트, 사카린, 나트륨 등), 향료(바닐린, 락톤류 등), 팽창제(명반, D-주석산수소칼륨 등), 강화제, 유화제, 증점제(호료), 피막제, 검기초제, 거품억제제, 용제, 개량제 등의 식품 첨가물(food additives)을 포함할 수 있다. 상기 첨가물은 식품의 종류에 따라 선별되고 적절한 양으로 사용될 수 있다.In addition, the health functional foods include preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), disinfectants (bleaching and highly bleaching, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butyl Hydroxytoleuene (BHT), etc.), colorants (tar colors, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasoning (MSG sodium glutamate, etc.), sweeteners (dulsin, cyclamate, etc.) Saccharin, sodium, etc.), fragrances (vanillin, lactones, etc.), expanding agents (alum, D-potassium hydrogen stannate, etc.), reinforcing agents, emulsifiers, thickeners (thickening agents), coating agents, gum base agents, foam inhibitors, solvents, improving agents It may contain food additives. The additive may be selected according to the type of food and used in an appropriate amount.
본 발명의 건강기능식품의 일 예로 건강음료 조성물로 사용될 수 있으며, 이 경우 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로 함유할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드; 말토스, 수크로스와 같은 디사카라이드; 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드; 자일리톨, 소르비톨, 에리트리톨 등의 당 알콜일 수 있다. 감미제는 타우마틴, 스테비아 추출물과 같은 천연 감미제; 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 건강음료 조성물 100 mL 당 일반적으로 약 0.01 내지 0.04 g, 구체적으로 약 0.02 내지 0.03 g이 될 수 있다.As an example of the health functional food of the present invention, it may be used as a health beverage composition, and in this case, it may contain various flavoring agents or natural carbohydrates as an additional component, such as a conventional beverage. The natural carbohydrates described above include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Polysaccharides such as dextrin and cyclodextrin; It may be a sugar alcohol such as xylitol, sorbitol, and erythritol. Sweeteners include natural sweeteners such as taumatin and stevia extract; Synthetic sweeteners such as saccharin and aspartame can be used. The ratio of the natural carbohydrate may be generally about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g per 100 mL of the health beverage composition of the present invention.
상기 외에 건강음료 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산, 펙트산의 염, 알긴산, 알긴산의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올 또는 탄산화제 등을 함유할 수 있다. 그 밖에 천연 과일주스, 과일주스 음료, 또는 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 혼합하여 사용할 수 있다. 이러한 첨가제의 비율은 크게 중요하진 않지만 본 발명의 건강음료 조성물 100 중량부당 0.01 내지 0.1 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, health beverage compositions include various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, It may contain alcohol or a carbonation agent. In addition, it may contain flesh for the manufacture of natural fruit juice, fruit juice beverage, or vegetable beverage. These ingredients may be used independently or in combination. The ratio of these additives is not very important, but it is generally selected from 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present invention.
또 다른 하나의 양태로서, 홍삼박을 포함하는 장 건강 개선용 사료 조성물을 제공한다.In another aspect, it provides a feed composition for improving intestinal health comprising red ginseng leaf.
본 발명의 용어 "사료"란, 가축 및 반려동물이 먹고, 섭취하며, 소화시키기 위한 또는 이에 적당한 임의의 천연 또는 인공 규정식, 한끼식 등 또는 상기 한끼식의 성분으로, 본 발명의 홍삼박을 유효성분으로 포함하는 사료는 당업계의 공지된 다양한 형태의 사료로 제조 가능하며, 구체적으로는 농후사료, 조사료 및/또는 특수사료가 포함될 수 있다.The term "feed" of the present invention is any natural or artificial diet for eating, ingesting, and digesting or suitable for livestock and companion animals, or as a component of the single meal, and the red ginseng leaf of the present invention The feed containing as an active ingredient can be prepared into various types of feed known in the art, and specifically, may include a thick feed, a roughage and/or a special feed.
본 발명의 사료 조성물에 포함되는 본 발명의 상기 조성물은 사료의 사용목적 및 사용조건에 따라 달라질 수 있으며, 일 예로 가축 사료 조성물의 총 중량에 대하여 0.01 내지 100 중량%, 보다 구체적으로는 5 내지 20 중량%로 포함될 수 있다.The composition of the present invention contained in the feed composition of the present invention may vary depending on the purpose of use and conditions of use of the feed, for example, 0.01 to 100% by weight, more specifically 5 to 20, based on the total weight of the livestock feed composition. It may be included in weight percent.
본 발명의 홍삼박을 유효성분으로 포함하는 조성물은 기타 다른 프로바이오틱스와 병용하여 사용하면 프로바이오틱스와의 시너지 효과를 수득할 수 있다.When the composition comprising the red ginseng leaf of the present invention as an active ingredient is used in combination with other probiotics, synergistic effects with probiotics can be obtained.
한편, 본 발명의 홍삼박은 세포독성이 없는 무해한 물질이다. 따라서, 본 발명의 홍삼박은 장기간 사용시에도 안심하고 사용할 수 있으며, 특히 상기한 바와 같은 식품 또는 사료 조성물에 안전하게 사용할 수 있다.On the other hand, the red ginseng meal of the present invention is a harmless substance without cytotoxicity. Therefore, the red ginseng leaf of the present invention can be safely used even when used for a long period of time, and can be used safely in a food or feed composition as described above.
이와 같이, 본 발명의 홍삼박을 유효성분으로 함유하는 조성물은 장내 유익균의 증식을 촉진하고, 유해균의 증식을 억제하고, 장내 단쇄 지방산의 함량을 증가시키며, 장내 균총을 개선하고, 암모니아태 질소 및 스카톨의 농도를 감소시킬 수 있다. 또한, 본 발명의 조성물을 이용하면, 장 건강 또는 장 기능을 개선할 수 있다. 한편, 본 발명의 홍삼박을 포함하는 조성물은 인체에 부작용이 없으며, 식품 또는 사료 조성물에 효과적으로 사용할 수 있다.Thus, the composition containing the red ginseng leaf of the present invention as an active ingredient promotes the proliferation of beneficial bacteria in the intestine, inhibits the proliferation of harmful bacteria, increases the content of short-chain fatty acids in the intestine, improves the intestinal flora, ammonia nitrogen and The concentration of scatol can be reduced. In addition, when the composition of the present invention is used, intestinal health or intestinal function can be improved. On the other hand, the composition containing the red ginseng leaf of the present invention has no side effects on the human body, and can be effectively used in food or feed compositions.
도 1은 홍삼박 난소화성 물질의 배양기간 중 pH 변화를 나타낸 그래프이다.
도 2는 홍삼박 난소화성 물질의 배양기간 중 단쇄지방산의 농도변화를 나타낸 그래프이다.
도 3은 홍삼박 난소화성 물질의 배양기간 중 단쇄지방산의 분자량 비율 변화를 나타낸 그래프이다.
도 4는 홍삼박 난소화성 물질의 배양기간 중 일반 혐기성균 및 대장균 총수 추이를 나타낸 것이다.
도 5는 홍삼박 난소화성 물질의 배양기간 중 유산균 수 추이를 나타낸 것이다.
도 6은 장내세균의 다양성을 분석한 것이다.
도 7은 군집분포를 나타낸 것이다.
도 8은 문(phylum) 단위에서 시료간 장내세균 상대 분포비를 나타낸 것이다.
도 9는 속(genus) 레벨에서 장내세균의 상대 구성비를 나타낸 것이다.
도 10은 속(genus) 소속에서 장내세균총의 상대 비율을 나타낸 것이다.
도 11은 홍삼박 난소화성 물질의 배양기간 중 암모니아태 질소 생성 변화를 나타낸 것이다.
도 12는 홍삼박 난소화성 물질의 배양기간 중 스카톨 생성 변화를 나타낸 것이다.1 is a graph showing the change in pH of red ginseng foil indigestible substances during the incubation period.
2 is a graph showing the change in the concentration of short-chain fatty acids during the incubation period of red ginseng foil indigestible material.
3 is a graph showing the change in the molecular weight ratio of short-chain fatty acids during the culture period of red ginseng foil indigestible material.
Figure 4 shows the trend of the total number of general anaerobic bacteria and E. coli during the cultivation period of red ginseng foil indigestible material.
Figure 5 shows the trend of the number of lactic acid bacteria during the culture period of the indigestible material of red ginseng.
6 is an analysis of the diversity of intestinal bacteria.
7 shows the cluster distribution.
8 shows the relative distribution ratio of intestinal bacteria between samples in a phylum unit.
9 shows the relative composition ratio of enterobacteriaceae at the genus level.
10 shows the relative ratio of the intestinal flora in the genus.
11 shows the change in ammonia-like nitrogen production during the incubation period of red ginseng foil indigestible material.
12 shows the change in scatol production during the culture period of the indigestible material of red ginseng meal.
이하, 본 발명의 이해를 돕기 위하여 실시예 등을 들어 상세하게 설명하기로 한다. 그러나, 본 발명에 따른 실시예들은 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 하기 실시예들에 한정되는 것으로 해석되어서는 안 된다. 본 발명의 실시예들은 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples, etc. will be described in detail to aid understanding of the present invention. However, the embodiments according to the present invention may be modified in various forms, and the scope of the present invention should not be construed as being limited to the following examples. The embodiments of the present invention are provided to more completely describe the present invention to those of ordinary skill in the art.
<실시예 1> 홍삼박 유래 난소화성 물질<Example 1> Indigestible material derived from red ginseng meal 제조Produce
100 mM 말레인산 완충용액(pH6.0)에 알파-아미로글루코시다아제 (Sigma-Aldrich, St. Louis, USA) 300 U/100 mL 및 판크레이틴 (Sigma-Aldrich)을 0.75 g/100mL의 비율이 되도록 용해시켜 소화효소액을 제조하였다. 홍삼박을 일반 분쇄기로 분쇄한 후 분말화한 홍삼박 분말 20 g을 삼각 플라스크에 넣고 상기 소화효소액 100 mL를 첨가하였다. 그 후 37℃의 항온조(water bath)에서 16 시간 배양한 후 99% 에탄올을 첨가하여 3 시간 정치하였다. 가수분해된 단백질 및 전분을 제거하기 위해 삼각 플라스크 내 용액을 원심용 튜브에 분주하고 원심분리(3,000 rpm)를 실시하여 상층액을 제거하였다. 이어 99% 에탄올 적당량을 침전물에 첨가하여 재원심분리하여 침전물을 세정하였다. 마지막으로 아세톤 적당량을 첨가하여 잔사물을 세정한 후, 튜브 내 침전물을 채취하여 드래프트 내에서 하룻밤 자연 건조시켰다. 그 후 건조된 침전물 중량을 측정하고 최종적으로 홍삼박으로부터 난소화성 물질의 수율을 산출하였다. 홍삼박 난소화성 물질은 믹서를 사용하여 분쇄하였다.In 100 mM maleic acid buffer (pH6.0), alpha-amiroglucosidase (Sigma-Aldrich, St. Louis, USA) 300 U/100 mL and pancretin (Sigma-Aldrich) were added at a ratio of 0.75 g/100 mL. The digestion enzyme solution was prepared by dissolving it so as to be. After pulverizing red ginseng leaf with a general grinder, 20 g of powdered red ginseng leaf powder was put into an Erlenmeyer flask, and 100 mL of the digestive enzyme solution was added. Then, after 16 hours incubation in a 37 ℃ water bath (water bath), 99% ethanol was added and allowed to stand for 3 hours. To remove the hydrolyzed protein and starch, the solution in the Erlenmeyer flask was dispensed into a centrifugal tube and centrifuged (3,000 rpm) to remove the supernatant. Then, an appropriate amount of 99% ethanol was added to the precipitate, followed by re-centrifugation to wash the precipitate. Finally, after washing the residue by adding an appropriate amount of acetone, the precipitate in the tube was collected and naturally dried overnight in a draft. Thereafter, the weight of the dried precipitate was measured, and finally, the yield of the indigestible material was calculated from the red ginseng leaf. The red ginseng foil indigestible material was pulverized using a mixer.
<시험예 1> 홍삼박 난소화성 물질의 일반성분 분석<Test Example 1> Analysis of general components of red ginseng foil indigestible substances
홍삼박 분말 중의 단백질 함량은 켈달 질소 정량법 (AOAC 920.87 질소-단백질 환산 계수 6.25)에 의거하여 측정하였다. 홍삼박 분말 중의 총지질 함량은 속실렛(soxhlet) 추출법 (AOAC 920.85)에 준하여 측정하였다. 홍삼박 분말 중의 회분 함량은 550℃에서 직접회화법 (AOAC 923.03)에 준하여 측정하였다. 홍삼박 분말 중 수분 함량은 건조 감량을 통한 가열건조법(110℃에서 2시간 건조, AOAC 925.10)에 준하여 측정하였다. 홍삼박 내 식이 섬유 함량은 Prosky 변법 (AOAC 991.43)으로 측정하였다. 수용성 식이섬유(soluble dietary fiber) 및 불용성 식이섬유(insoluble dietary fiber)의 총계를 총 식이섬유 함량(total dietary fiber)으로 산출하였다. 홍삼박 내 난소화성 전분(RS) 함량은 시판되고 있는 RS 분석 측정 키트 (Megazyme, Wieklow, Ireland, AOAC 2002.02)를 사용하여 측정하였다. 본 키트분석을 통해 난소화성 전분 및 소화성 전분을 분석하였고, 두 합을 이용하여 총 전분량을 산출하였다.The protein content in the red ginseng meal powder was measured according to the Keldahl nitrogen determination method (AOAC 920.87 nitrogen-protein conversion factor 6.25). The total lipid content in the red ginseng foil powder was measured according to the soxhlet extraction method (AOAC 920.85). The ash content in the red ginseng foil powder was measured at 550° C. according to the direct conversation method (AOAC 923.03). The moisture content in the red ginseng foil powder was measured according to the heat drying method (dried at 110° C. for 2 hours, AOAC 925.10) through loss on drying. Dietary fiber content in red ginseng meal was measured by Prosky's variant (AOAC 991.43). The total amount of soluble dietary fiber and insoluble dietary fiber was calculated as total dietary fiber. The content of indigestible starch (RS) in red ginseng meal was measured using a commercially available RS assay kit (Megazyme, Wieklow, Ireland, AOAC 2002.02). Indigestible starch and digestible starch were analyzed through this kit analysis, and the total amount of starch was calculated using the two sums.
홍삼박 및 홍삼박 난소화성 물질의 성분분석 결과를 표 1에 나타냈다.Table 1 shows the results of component analysis of red ginseng leaf and red ginseng leaf indigestible substances.
난소화성 물질Red ginseng
Indigestible substances
건물(dry) 기준으로 보았을 때 홍삼박은 식이섬유 41.7%와 난소화성 전분(RS) 4.11%을 함유하였다. 건조 홍삼박 분말을 아밀로글리코시다아제 및 판크레아틴으로 가수 분해한 결과, 홍삼박 미가수분해물 수율은 건물기준으로 67.4%였다. 본 실험에서는 이러한 미가수분해물을 난소화성 물질로 규정하였다. 미가수분해물의 대부분은 탄수화물이었고(64.3%) 그 다음은 단백질이었다(17.9%). 수율(67.4%)을 이용한 각 난소화성 물질의 잔류량을 산출한 결과, 홍삼박 효소처리물 내 식이섬유 함량은 61.8%(41.7 g/0.674 = 61.8 g)로 나타났다. 난소화성 전분의 경우, 산출치는 6.1%(4.11 g/0.674 = 6.10 g)였고 실제 측정치는 7.3%이었다. 본 실험에서 발견된 특이한 점 한가지는 홍삼박을 효소처리해도 단백질은 대부분 분해되지 않았다는 점이다. 홍삼박 효소처리물 내 단백질 함량 산출치 17.4% (11.7 g/0.674 = 17.4 g)와 실제 홍삼박 효소처리물 내 측정치 17.9%를 비교해도 커다란 차이가 없었다. 이로부터 홍삼박 내 단백질 또한 난소화성물질로 사료된다.On a dry basis, red ginseng meal contained 41.7% dietary fiber and 4.11% indigestible starch (RS). As a result of hydrolyzing the dried red ginseng leaf powder with amiloglycosidase and pancreatin, the yield of unhydrolyzed red ginseng leaf was 67.4% on a dry matter basis. In this experiment, these unhydrolyzed products were defined as indigestible substances. Most of the unhydrolyzed products were carbohydrates (64.3%), followed by proteins (17.9%). As a result of calculating the residual amount of each indigestible material using the yield (67.4%), the dietary fiber content in the enzyme treatment product of red ginseng was 61.8% (41.7 g/0.674 = 61.8 g). For indigestible starch, the calculated value was 6.1% (4.11 g/0.674 = 6.10 g) and the actual measured value was 7.3%. One of the peculiarities found in this experiment is that most of the proteins were not degraded even when the red ginseng meal was enzymatically treated. There was no significant difference when comparing the calculated protein content of 17.4% (11.7 g/0.674 = 17.4 g) in the enzyme-treated red ginseng meal and 17.9% in the actual red ginseng-baked enzyme-treated product. From this, the protein in red ginseng meal is also considered to be an indigestible substance.
<시험예 2> 홍삼박 난소화성 물질의 장 환경 개선효과 측정<Test Example 2> Measurement of the intestinal environment improvement effect of red ginseng foil indigestible material
1) 자발효기(Jarfermentor, 액침혐기배양기)를 이용한 혐기성 발효1) Anaerobic fermentation using a jarfermentor (immersion anaerobic incubator)
자발효기(jarfermentor)는 발효조 내부를 혐기교반하면서 장내세균총 또는 단독 미생물을 배양하며 장내세균의 동태를 파악할 수 있도록 장내환경을 모델화한 에코시스템(ecosystem) 장치이다. 본체에는 미생물 접종구, 온도감응장치, pH 전극, 온도계, 알칼리 주입구, 시료채취구, 배지주입구, 가스배출구 등이 있다. 또한 발효조 안으로 이산화탄소를 공급함으로써 장내 환경인 혐기상태를 유지하고, 산 발효에 의한 비정상적인 장 환경을 알칼리 용액을 주입함으로써 장관 내 pH 조건에 맞는 환경에서 장내세균총(microbiota) 배양이 가능하다.The jarfermentor is an ecosystem device that models the intestinal environment so that the inside of the fermentation tank is anaerobicly stirred and the intestinal flora or single microorganism is cultivated, and the dynamics of the intestinal bacteria are identified. The main body includes a microbial inoculation port, a temperature sensitive device, a pH electrode, a thermometer, an alkali injection port, a sample collection port, a medium injection port, and a gas discharge port. In addition, by supplying carbon dioxide into the fermentation tank, the intestinal environment, which is anaerobic, is maintained, and the abnormal intestinal environment due to acid fermentation is injected with an alkaline solution, thereby cultivating the intestinal microbiota in an environment suitable for the intestinal pH conditions.
본 실험에서는 자발효기를 사용하여 홍삼박 난소화성 물질의 발효특성 등 장내 환경개선에 미치는 영향을 조사하였다. 초기 배양조 내 pH는 5.6으로 설정했고, 산 발효에 의해 pH가 산성쪽으로 기울어질 경우 멸균 수산화나트륨 용액(2N NaOH)이 자동적으로 배양조 내에 주입되어 비생리적 상황인 pH 5.5 미만으로 내려가지 못하도록 설정하였다. 배양조 온도는 37℃로 유지되었고 160 rpm의 속도로 배양액을 균질하게 교반하였으며, 이산화탄소(CO2) 가스를 분당 0.4 L 주입하여 혐기성 상태를 유지시켰다. 하기 표 2에 나타낸 실험군의 조성에서 보듯이, 전체 배양액(220 mL)의 2%에 해당하는 분변 배설물 44 mL와 3%에 해당하는 탄소원(시료) 및 0.83%에 해당하는 단백질원(nutrient broth)을 첨가 후 48시간 혐기상태에서 배양을 실시하였다.In this experiment, the effect on the improvement of the intestinal environment, such as the fermentation characteristics of red ginseng meal indigestible substances, was investigated using a self-fermentation machine. Initially, the pH in the culture tank was set to 5.6, and when the pH is tilted toward the acid side due to acid fermentation, a sterile sodium hydroxide solution (2N NaOH) is automatically injected into the culture tank to prevent it from falling below pH 5.5, which is an unphysiological condition. I did. The temperature of the culture bath was maintained at 37°C, and the culture solution was homogeneously stirred at a rate of 160 rpm, and 0.4 L of carbon dioxide (CO 2 ) gas was injected per minute to maintain an anaerobic state. As shown in the composition of the experimental group shown in Table 2 below, 44 mL of fecal excrement equivalent to 2% of the total culture medium (220 mL), a carbon source (sample) equivalent to 3%, and a protein source (nutrient broth) equivalent to 0.83% After addition, culture was carried out in an anaerobic condition for 48 hours.
(3.0% 수준)Carbon source
(3.0% level)
(0.83% 수준)Protein source
(0.83% level)
(2.0% 수준)Bacterial source
(2.0% level)
2) 분변 배설물 현탁액 조제2) Preparation of fecal excrement suspension
액침혐기배양을 위해 장내 세균원으로 돼지의 신선한 배설물을 이용하였다. 본 실험에서는 최소 1개월 내에 항균제 투여가 면제된 돼지(약 생후 3-5개월)를 대상으로 3마리의 돼지 항문에서 직접 분변을 채취하였다. 채취된 분변은 아네로팩 (AnaeroPack, 탈산소제, Mitsubishi Gas Chem., Tokyo, Japan)으로 혐기성 상태로 유지된 이중 지퍼백에 즉시 넣고 분석하기 전까지는 4℃에서 저장하였다. 분변채취는 총 5회 실시하였다. 각 회 채취된 3마리 분변에서 각각 2.3 g을 측량 후 투명 비닐봉투에 넣어 혼합하였다. 그 후 멸균 생리식염수(0.85%)를 10배 첨가하여 희석한 후 고형물을 제거하여 여과액을 준비하였다. 이 여과액을 분변 배설물 현탁액으로 사용하였다. 배양조(Able & Biott, Tokyo, Japan) 내 배양혼합액 용량(220 mL)의 2%(v/v)가 되도록 분변 현택액 44 mL을 넣었다. 장내 세균총의 안정화를 위해 초기 12시간은 분변 배설물 현탁액만 배양하였다.For immersion anaerobic culture, fresh pig excrement was used as an intestinal bacterial source. In this experiment, feces were collected directly from the anus of 3 pigs from pigs (about 3-5 months of age) exempted from antibiotic administration within at least 1 month. The collected feces were immediately placed in a double zipper bag maintained in an anaerobic state with AnaeroPack (AnaeroPack, deoxidant, Mitsubishi Gas Chem., Tokyo, Japan) and stored at 4°C until analysis. Fecal collection was conducted a total of 5 times. 2.3 g of each of the feces collected each time was weighed and mixed in a transparent plastic bag. Then, 10 times of sterilized physiological saline (0.85%) was added and diluted, and the solid was removed to prepare a filtrate. This filtrate was used as a fecal excrement suspension. 44 mL of fecal suspension was added to 2% (v/v) of the culture mixture volume (220 mL) in the culture tank (Able & Biott, Tokyo, Japan). For stabilization of the intestinal flora, only fecal excrement suspension was cultured for the first 12 hours.
3) 배지 첨가3) medium addition
세균배양에 필요한 배지는 뉴트리엔트 브로스(Nutrient broth, Difco, Sparks, MD, USA)를 사용하였다. 뉴트리엔트 브로스는 쇠고기 추출물 및 펩톤이 3대 5의 비율로 구성되어 있는 배지이다. 배양액 중 단백질원이 전체 0.83%(1.83 g)가 되도록 배양조에 첨가하였다. 사전에 뉴트리엔트 브로스 배지를 증류수(1.83 g/50 mL)에 녹인 후 121℃, 15분간 멸균처리하였다. 충분히 방냉한 배지를 혐기상태에서 배양조 안으로 50 mL를 주입하였다.As a medium required for bacterial culture, Nutrient broth (Difco, Sparks, MD, USA) was used. Nutrient broth is a medium consisting of beef extract and peptone in a ratio of 3 to 5. It was added to the culture tank so that the total protein source in the culture solution was 0.83% (1.83 g). In advance, the Nutrient broth medium was dissolved in distilled water (1.83 g/50 mL) and sterilized at 121° C. for 15 minutes. 50 mL of the sufficiently cooled medium was injected into the culture tank in an anaerobic condition.
4) 탄소원 첨가4) carbon source addition
탄소원은 셀룰로오스, 자일로올리고당, 홍삼박 난소화성 분말을 사용하였다. 배양조 내 탄수화물 첨가량은 전체 중 3%가 되도록 멸균처리된 셀룰로오스 및 자일로올리고당 6.60 g을 멸균 생리식염수 126 mL에 섞은 후 각각의 배양조 안에 주입하였다. 또한 멸균처리된 홍삼박 난소화성 분말 10.26 g(126 mL)을 주입하였다. 홍삼박 난소화성 물질의 첨가량은 홍삼박 난소화성 물질 내 탄소화물 및 단백질 농도를 분석하여 배양액에 첨가량을 결정하였다.As a carbon source, cellulose, xylo-oligosaccharide, and red ginseng foil indigestible powder were used. 6.60 g of sterilized cellulose and xylo-oligosaccharide were mixed with 126 mL of sterile physiological saline so that the amount of carbohydrate added in the culture tank was 3% of the total, and then injected into each culture tank. In addition, 10.26 g (126 mL) of sterilized red ginseng foil indigestible powder was injected. The addition amount of the red ginseng foil indigestible material was determined by analyzing the concentration of carbohydrates and proteins in the red ginseng foil indigestible material.
5) pH 분석5) pH analysis
배양 48 시간 동안 실시간 배양액 내 pH를 관찰하고 기록하였다. 배양 0, 6, 12, 24, 48시간 마다 멸균 튜브에 배양액을 채취하였다. 1.5 mL용 에펜 튜브에 채취한 배양액은 차세대 염기서열 분석(NGS분석용)으로 사용하기 위해 분석 전까지 -80℃에 저장했고, 2.0 mL용 에펜 튜브에 채취한 배양액은 채취 즉시 세균 검사에 사용되었으며 나머지는 각 실험 분석 전까지 -30℃ 냉동고에 저장하였다.During 48 hours of incubation, the pH in the real-time culture was observed and recorded. Cultures were collected in sterilized tubes every 0, 6, 12, 24, and 48 hours of culture. The culture solution collected in the 1.5 mL eppen tube was stored at -80°C until analysis to be used for next-generation sequencing (for NGS analysis), and the culture solution collected in the 2.0 mL eppen tube was immediately used for bacterial testing. Was stored in the -30 ℃ freezer until each experimental analysis.
도 1은 홍삼박 난소화성 물질의 배양기간 중 pH 변화를 나타낸 그래프이다. 여기에서 보듯이, 셀룰로오스 첨가구는 배양 시작 pH 5.6에서 배양 종료시 pH 6.5까지 상승하였다. 이에 비해 자일로올리고당 및 홍삼박 난소화성 물질 첨가구는 전체 배양기간 중 pH 변화가 거의 없었으며, 두 실험구 모두 셀룰로오스 첨가구 pH보다 통계적으로 유의하게 낮았다(p<0.05).1 is a graph showing the change in pH of red ginseng foil indigestible substances during the incubation period. As shown here, the cellulose-added sphere increased from pH 5.6 at the beginning of the culture to 6.5 at the end of the culture. On the other hand, the pH of the group with xylo-oligosaccharide and red ginseng meal indigestible material had little change during the entire incubation period, and both experimental groups were statistically significantly lower than the pH of the group with cellulose added (p<0.05).
5) 단쇄지방산 농도의 측정5) Measurement of short-chain fatty acid concentration
배양액 중의 단쇄지방산인 초산(acetic acid), 프로피온산(propionic acid) 및 낙산(n-butyric acid) 농도를 고성능 액체 크로마토 그래피(LC-10AD, Shimadzu, Kyoto, Japan)을 이용하여 측정하였다. 단쇄지방산용 시료를 준비하기 위하여, 배양액을 원심분리(16,000rpm, 4℃, 15분)하여 상층액을 취하고 상층액 내 단백질을 제거하기 위해 0.5N HClO4을 첨가하여 실온에서 5분간 정치하였다. 다시 원심분리(16,000rpm, 4℃, 10분)를 실시하고 HPLC용 일회용 필터(0.45 μm, Advantec, Tokyo, Japan)를 사용하여 여과된 시료를 HPLC에 공여하였다. HPLC 설정조건은 RSpak KC-811 (8.0 mm × 300 mm, Shodex, Tokyo, Japan) post 컬럼에 이동상은 2 mM 과염소산을 분당 1.0 mL가 되도록 47℃의 칼럼을 통과한 후, 혼합기에서 유속 0.5 mL/min의 ST3-R(Shodex, Tokyo, Japan) 용액과 반응하여 UV-검출기(Shimadzu)를 이용하여 파장 430 nm에서 각각의 SCFA를 검출하였다. 각종 단쇄지방산의 식별은 크로마토팩 (C-R6A, Shimadzu)에서 얻어진 시료와 표준 용액의 유지 시간(retention time, RT)의 비교에 의해 정성하였고 피크 면적을 비교하여 정량분석하였다. 또한 3종의 SCFA 농도 합계를 총 SCFA 농도로 산출하였다.The concentrations of short-chain fatty acids, such as acetic acid, propionic acid, and n-butyric acid, in the culture medium were measured using high-performance liquid chromatography (LC-10AD, Shimadzu, Kyoto, Japan). In order to prepare a sample for short-chain fatty acid, the culture solution was centrifuged (16,000 rpm, 4° C., 15 minutes) to take a supernatant, and 0.5N HClO 4 was added to remove the protein in the supernatant, followed by standing at room temperature for 5 minutes. Centrifugation (16,000 rpm, 4° C., 10 minutes) was performed again, and a filtered sample was donated to HPLC using a disposable filter for HPLC (0.45 μm, Advantec, Tokyo, Japan). The HPLC setting conditions are RSpak KC-811 (8.0 mm × 300 mm, Shodex, Tokyo, Japan) post column, and the mobile phase passes through a column at 47° C. to 1.0 mL per minute of 2 mM perchloric acid, and flow rate 0.5 mL/min in a mixer. Each SCFA was detected at a wavelength of 430 nm using a UV-detector (Shimadzu) by reacting with a min ST3-R (Shodex, Tokyo, Japan) solution. The identification of various short-chain fatty acids was qualitative by comparing the retention time (RT) of the sample obtained in the chromatographic pack (C-R6A, Shimadzu) and the standard solution, and quantitative analysis was performed by comparing the peak area. In addition, the sum of the concentrations of the three SCFAs was calculated as the total SCFA concentration.
도 2는 홍삼박 난소화성 물질의 배양기간 중 단쇄지방산의 농도변화를 나타낸 그래프이다. 여기에서 보듯이, 초산 농도는 배양 6시간째 자일로올리고당 및 홍삼박 난소화성 물질 첨가구에서 셀룰로오스 첨가구보다 유의하게 증가하였다(p<0.05). 배양 12시간째부터 자일로올리고당 첨가구는 셀룰로오스 및 홍삼박 난소화성 물질 첨가구보다 유의하게 증가하였다(p<0.05). 홍삼박 난소화성 물질 첨가구는 배양 24시간 이후부터 셀룰로오스 첨가구보다 유의하게 (p<0.05) 증가하였다. 전반적으로 자일로올리고당 첨가구가 홍삼박 난소화성 물질 첨가구보다 초산 발효가 우위를 차지하였다. 프로피온산 농도는 배양 개시 6시간부터 자일로올리고당 첨가구가 셀룰로오스 첨가구보다 유의하게 증가하였다(p<0.05). 홍삼박 난소화성 물질 첨가구의 경우 배양 12시간째 이후 셀룰로오스 첨가구에 비해 유의하게 증가했지만(p<0.05) 48시간째 자일로올리고당 첨가구와 통계적 유의차는 없었다. 낙산(butyric acid) 농도는 배양 6시간부터 자일로올리고당 첨가구가 셀룰로오스 및 홍삼박 난소화성 물질 첨가구보다 유의하게 증가하였다(p<0.05). 홍삼박의 경우 셀룰로오스 첨가구와 유사한 농도를 나타냈다. 전체 배양 기간을 통해 홍삼박 난소화성 물질 첨가구 내 총 단쇄지방산 농도는 자일로올리고당보다는 낮았다. 이러한 차이는 홍삼박 내 비발효성 식이섬유인 셀룰로오스 및 헤미셀룰로오스가 높게 함유되어 있기 때문이라 판단된다.2 is a graph showing the change in the concentration of short-chain fatty acids during the incubation period of red ginseng foil indigestible material. As shown here, the acetic acid concentration was significantly increased in the group with xylo-oligosaccharide and red ginseng meal indigestible substances added at 6 hours compared to the group with cellulose added ( p <0.05). From the 12th hour incubation, the addition of xylo-oligosaccharide significantly increased compared to the addition of cellulose and red ginseng meal indigestible substances (p<0.05). The addition of red ginseng foil indigestible substances increased significantly ( p <0.05) from the addition of cellulose after 24 hours of culture. In general, acetic acid fermentation took the lead in xylo-oligosaccharide-added group compared to red ginseng meal indigestible substances-added group. Propionic acid concentration was significantly increased in the group added with xylo-oligosaccharide compared to the group added with cellulose from 6 hours of initiation of culture ( p <0.05). In the case of the indigestible substance-added group of red ginseng, it increased significantly compared to the cellulose-added group after 12 hours of culture ( p <0.05), but there was no statistically significant difference from the group added with xylooligosaccharide at 48 hours. The concentration of butyric acid increased significantly from 6 hours of incubation in the group added with xyloligosaccharide compared to the group added with cellulose and red ginseng meal indigestible substances ( p <0.05). In the case of red ginseng leaf, the concentration was similar to that of the cellulose-added group. Throughout the entire cultivation period, the total short-chain fatty acid concentration in the red ginseng meal indigestible substances was lower than that of xyloligosaccharide. This difference is believed to be due to the high content of cellulose and hemicellulose, which are non-fermentable dietary fibers in red ginseng foil.
도 3은 홍삼박 난소화성 물질의 배양기간 중 단쇄지방산의 분자량 비율 변화를 나타낸 그래프이다. 여기에서 보듯이 배양시간대별 배양조 내 단쇄지방산 분자량 비율은 각 시료에 따라 서로 다른 양상을 보였다. 홍삼박 난소화성 물질은 셀룰로오스와 유사하게 낙산 생성에 영향을 거의 주지 못하는 것을 확인할 수 있었다. 일반적으로 사람의 대변 내 초산：프로피온산：낙산 분자량 비율(molar proportion)은 60：20：20을 이루고 있다. 본 실험에서는 사용한 돼지분변의 배양초기 경우 54：28：17로 나타났다. 도 3에서 보면, 셀룰로오스 첨가구의 경우 초산이 증가했고 프로피온산이 감소했으며, 자일로올리고당 첨가구의 경우 초산이 감소했고 낙산이 증가하였다. 반면 홍삼박 난소화성 물질 첨가구의 경우 프로피온산이 증가하였고 낙산이 감소하였다. 홍삼박 난소화성 물질 첨가구에서 생성된 단쇄지방산 및 각 단쇄지방산 분자량 비율 변화 차이는, 홍삼박 내 셀룰로오스와 같은 비발효성 식이섬유가 다량 함유되어 있는 점과 홍삼박에는 자일로올리고당과 다른 장내세균이 선호하는 발효성 고분자 다당류가 혼잡해 있을 가능성이 존재하기 때문이라 판단된다.3 is a graph showing the change in the molecular weight ratio of short-chain fatty acids during the culture period of red ginseng foil indigestible material. As shown here, the molecular weight ratio of short-chain fatty acids in the culture tank for each culture time period showed different patterns for each sample. It was confirmed that the indigestible material of red ginseng foil had little effect on the production of butyric acid, similar to cellulose. In general, the molecular proportion of acetic acid: propionic acid: butyric acid in human feces is 60:20:20. In this experiment, the initial culture of used pig feces was 54:28:17. 3, in the case of the cellulose-added group, acetic acid increased and propionic acid decreased, and in the case of the xyloligosaccharide-added group, acetic acid decreased and butyric acid increased. On the other hand, propionic acid increased and butyric acid decreased in the case of adding red ginseng meal indigestible substances. The difference in the ratio of short-chain fatty acids and the molecular weight of each short-chain fatty acid produced in the addition of red ginseng foil indigestible substances is that it contains a large amount of non-fermentable dietary fibers such as cellulose in red ginseng foil, and xylooligosaccharide and other intestinal bacteria This is believed to be due to the possibility that the preferred fermentable polymer polysaccharide is congested.
6) 배양액 내 각종 세균 수 측정6) Measurement of the number of various bacteria in the culture medium
장내 세균은 선택적 배지를 사용한 평판 도말법으로 배양 후 콜로니(colony) 수를 시각적으로 측정하였다. 일반 혐기성균 배양에는 BL 한천 배지 (Eiken, Tokyo, Japan), 대장균 배양에는 EMB 한천 배지 (Eiken), 유산균은 MRS 한천 배지(Oxoid, UK), Lactobacillus 배양에는 Rogosa 한천 배지 (Kanto Chemical, Tokyo, Japan), Bifidobacterium 배양에는 TOS 프로피온산 한천 배지 (Yakult Pharmaceutocal Industry, Tokyo, Japan)를 사용하였다. 각 시간대별로 채취한 배양액을 멸균 생리식염수로 10배 단계적 희석한 후(10-7까지) 100 μL를 한천 배지에 도말하여 배양하였다. 배양기 37℃에서 GasPak 법에 준한 혐기상태에서 배양하였다. 대장균군은 24시간 후, 일반 혐기성균, 총 유산균 및 Lactobacillus은 48시간 후, Bifidobacterium은 72시간 후 콜로니 수를 측정하였다. 각 배지에서 형성된 콜로니 수(Colony Forming Unit)는 CFU로 표기하였고 최종적으로 로그(log10)로 산출하였다.Intestinal bacteria were visually measured for colonies after culture by plate smearing using a selective medium. BL agar medium (Eiken, Tokyo, Japan) for general anaerobic bacteria culture, EMB agar medium (Eiken) for E. coli culture, MRS agar medium (Oxoid, UK) for lactic acid bacteria, and Rogosa agar medium for Lactobacillus culture (Kanto Chemical, Tokyo, Japan) ), Bifidobacterium culture was used TOS propionic acid agar medium (Yakult Pharmaceutocal Industry, Tokyo, Japan). The culture solution collected for each time period was diluted 10-fold with sterile physiological saline (up to 10 -7 ), and then 100 μL was plated on agar and cultured. It was cultured in an anaerobic condition according to the GasPak method at 37°C in an incubator. Colonies were measured after 24 hours for Coliform group, 48 hours for general anaerobic bacteria, total lactic acid bacteria and Lactobacillus , and 72 hours for Bifidobacterium . The number of colonies formed in each medium (Colony Forming Unit) was expressed as CFU and was finally calculated as a log (log 10 ).
도 4는 홍삼박 난소화성 물질의 배양기간 중 일반 혐기성균 및 대장균 총수 추이를 나타낸 것이다. 여기에서 보듯이, 일반 혐기성 세균수는 배양 12시간 이후, 자일로올리고당 및 홍삼박 난소화성 물질 첨가구에서 셀룰로오스 첨가구에 비해 유의하게 증가하였다(p<0.05). 배양 24시간째 대장균 총수는 자일로올리고당 첨가구에서 셀룰로오스 첨가구에 비해 유의하게 감소했고, 배양 48시간째 자일로올리고당 첨가구에서 셀룰로오스 첨가구 및 홍삼박 난소화성 물질 첨가구에 비해 유의하게 생육이 감소하였다(p<0.05). 전체 배양기간 중 홍삼박 난소화성 물질 첨가구의 대장균군 생균수는 셀룰로오스 첨가구와 거의 유사하였다.Figure 4 shows the trend of the total number of general anaerobic bacteria and E. coli during the cultivation period of red ginseng foil indigestible material. As shown here, the number of general anaerobic bacteria significantly increased after 12 hours of culture, compared to the cellulose-added group in the group with xylo-oligosaccharide and red ginseng meal indigestible substances added ( p <0.05). The total number of E. coli at 24 hours of cultivation was significantly decreased in the group added with xylo-oligosaccharide compared to the group added with cellulose. Decreased ( p <0.05). During the entire cultivation period, the number of viable coliforms of the red ginseng foil-added indigestible material was almost similar to that of the cellulose-added group.
도 5는 홍삼박 난소화성 물질의 배양기간 중 유산균 수 추이를 나타낸 것이다. 여기에서 보듯이, 총 유산균(유산균 간균 및 유산균 구균 포함) 수는 배양 6시간 및 12시간째 홍삼박 난소화성 물질 첨가구에서 셀룰로오스 및 자일로올리고당 첨가구에 비해 유의하게 증가하였다(p<0.05). 홍삼박 난소화성 물질 첨가구 총 유산균 수는 배양 12시간 이후로 48시간까지 지속적으로 감소하면서 배양 24시간째 자일로올리고당 첨가구와 함께 셀룰로오스 첨가구에 비해 통계학적으로 높은 생균수를 나타냈고(p<0.05), 배양 48시간째에는 더욱 감소하여 셀룰로오스 첨가구와 비슷한 균수가 되었다. 자일로올리고당 첨가구는 배양 24시간째까지 최대 생육수를 보이다 48시간째 감소했지만 여전히 셀룰로오스 첨가구에 비해 유의하게 높았다(p<0.05). 전체 배양 기간을 통해, 자일로올리고당 및 홍삼박 난소화성 물질 첨가구 Lactobacillus 균수는 셀룰로오스 첨가구의 것에 비해 유의하게 많았다(p<0.05). 홍삼박 난소화성 물질 첨가구 Lactobacillus 균수는 배양 6시간에서 12시간까지 자일로올리고당 첨가구보다 유의하게 증가했지만(p<0.05) 배양 24시간을 기점으로 두 첨가구 사이 유사한 수치를 보이다가 48시간째에는 역전되어 자일로올리고당 첨가구보다 유의하게 감소하였다(p<0.05). 하지만 셀룰로오스 첨가구 생균수보다는 유의하게 많았다(p<0.05). Bifidobacterium 균수는 배양 24시간째를 제외하고는 전 배양기간을 통해 홍삼박 난소화성 물질 첨가구 및 자일로올리고당 첨가구에서 셀룰로오스 첨가구에 비하여 유의하게 증식하였다(p<0.05). 홍삼박은 자일로올리고당보다 Bifidobacterium 생육에 더 친화적이었다.Figure 5 shows the trend of the number of lactic acid bacteria during the culture period of the indigestible material of red ginseng. As shown here, the total number of lactic acid bacteria (including Lactobacillus bacillus and Lactobacillus aureus) was significantly increased at 6 and 12 hours of cultivation compared to those with cellulose and xylooligosaccharides in the red ginseng meal indigestible substances added ( p <0.05) . The total number of lactic acid bacteria in the group with the addition of red ginseng foil indigestible substances continued to decrease from 12 hours to 48 hours after incubation, showing a statistically higher number of viable cells compared to the group with the addition of cellulose at 24 hours of culture ( p < 0.05), at 48 hours of incubation, the number of bacteria decreased further and the number of bacteria was similar to that of the cellulose-added group. The group added with xylo-oligosaccharide showed the maximum number of growth until 24 hours of incubation and then decreased after 48 hours, but it was still significantly higher than that of the group added with cellulose ( p <0.05). Throughout the entire cultivation period, the number of Lactobacillus bacteria added with xylo-oligosaccharide and red ginseng meal indigestible substance was significantly higher than that of the cellulose-added group ( p <0.05). The number of Lactobacillus bacteria in the group with the indigestible substance added to the red ginseng meal increased significantly from the group added with xylooligosaccharide from 6 to 12 hours of incubation ( p <0.05), but showed a similar value between the two added groups starting from 24 hours of incubation. It was reversed and significantly decreased ( p <0.05) than in the group with xylooligosaccharide added. However, it was significantly higher than the number of viable cells added with cellulose ( p <0.05). The number of Bifidobacterium bacteria proliferated significantly compared to the cellulose-added group in the red ginseng meal indigestible substance-added group and the xylo-oligosaccharide-added group throughout the entire culture period except at 24 hours of culture ( p <0.05). Red ginseng meal was more friendly to the growth of Bifidobacterium than xyloligosaccharide.
홍삼박에는 다양한 난소화성 당류를 함유하고 있기 때문에 홍삼박 내 저분자 난소화성 당류는 배양 초기부터 24시간까지 Lactobacillus 균 영양원으로 이용되다가 24시간 이후 거의 소화되었을 것이라 추측된다. 한편 전체 배양기간을 통해 홍삼박에 의한 Bifidobacterium 균 생육이 자일로올리고당 첨가구보다 유의적으로 높은 생균수를 보인 것은 홍삼박 내 난소화성 고분자 다당류는 올리고당에 비해 Bifidobacterium이 선호하는 영양원일 가능성이 추론되며 Bifidobacterium 생육에 관해서는 자일로올리고당보다 유용한 프리바이오틱스일 가능성을 시사한다.Since red ginseng meal contains various indigestible saccharides, it is estimated that the low-molecular indigestible saccharides in red ginseng meal were used as a nutrient for Lactobacillus bacteria from the beginning of culture to 24 hours, and then almost digested after 24 hours. The whole thing is Bifidobacterium bacteria growth by red ginseng through the incubation period showed a high number of viable cells oligosaccharides added guboda significant as xylene red ginseng in indigestible polymeric polysaccharides and speculation is likely to be a nutrient that Bifidobacterium preferred compared to oligosaccharides Bifidobacterium In terms of growth, this suggests that it may be a more useful prebiotic than xylooligosaccharide.
7) 차세대 시컨싱(next geneartion sequencing, NGS) 분석7) Analysis of next geneartion sequencing (NGS)
In vitro 배양 48시간째 각 실험구의 배양액으로부터 QIAamp® DNA Stool Mini Kit(QIAGEN®)를 사용하여 Microbial DNA를 추출하였다. 추출된 배양액 Microbial DNA는 멸균 증류수를 사용하여 ５ng/μl의 농도로 희석하였다. 이 후 장내세균총(microbiota) 염기서열 분석을 위하여 amplicon는 Illumina 16S Metagenomic sequencing library protocols을 따라 준비하였다. PCR 증폭은 PCR forward primer 5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3’(서열번호 1)와 reverse primer 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3’(서열번호 2)을 사용하여 각각 95℃로 3분간 DNA를 변성 후, 95℃-30초, 55℃-30초, 72℃-30초 주기로 총 25 cycle 반복하였다. 최종적으로 72℃ 5분간 조건으로 박테리아 16S rDNA의 V1-9 중 V3-4 가변영역을 증폭한 후 4℃에 보관하였다 (Klindworth et al., Nucleic Acids Res., doi: org/10.1093/nar/한808). V3-4 primer를 사용하여 증폭된 PCR은 동일한 양으로 혼합되어 전기영동으로 증폭된 amplicon (약 200 bp)을 확인 후, AMPure XP beads (Beckman Coulter, High Wycombe, UK)을 이용하여 정제하였으며, 이후 multiplex index들과 Illumina sequencing adapter 추가를 위한 index PCR을 95℃로 3분간 DNA를 변성 후, 95℃-30초, 55℃-30초, 72℃-30초로 8 cycle, 최종적으로 72℃ 5분간 실시하였다. 최종 생성물에서 DNA농도를 구하고 normalization 및 pooling을 거친 후 MiSeqTM platform (Illumina inc., San Diego, CA, USA)을 이용해 염기서열을 분석하였다. 시료 안에 장내세균총 종류와 그 비율을 알기 위해 시료에서 얻은 염기서열 간의 유사도(97%)에 근거하여 염기서열들을 묶고, 이를 한 단위로 하여 시료 내의 미생물 다양성을 평가하였다. 이러한 조작상분류단위(Operational taxonomic unit, 이하 OTU) 및 장내세균총 조성은 QIIME(Quantitative Insights Into Microbial Ecology)로 분석하였고 데이터의 시각화를 위해 Calypso 소프트웨어를 사용하였다.The Microbial DNA was extracted using the QIAamp ® DNA Stool Mini Kit (QIAGEN ®) from the In vitro
각 처리구 사이 단쇄지방산 농도차가 선명하게 관찰된 48시간째 배양액을 채취하여 차세대 시퀀싱 수법에 의한 장내세균총 메타분석을 실시하였다.The culture medium was collected at 48 hours in which the difference in the concentration of short-chain fatty acids was clearly observed between the treatment groups, and meta-analysis of the intestinal flora by the next generation sequencing method was performed.
도 6은 장내세균의 다양성을 분석한 것이다. 메타 16S 해석에 의한 시컨싱 데이터의 다변량 해석을 적용했고 각출된 OTU의 종류와 수로부터 주좌표분석(PCoA)을 실시한 다양성 분석을 나타냈다. 그 결과, 각 시료 내 장내세균총 종(species)수는 알파-다양성 분석을 통해 통계학적 유의차가 나타났다(Kruskal-Wallis test, p<0.05, 도 6(a)). 또한 주좌표분석을 통해 셀룰로오스, 자일로올리고당 및 홍삼박 난소화성 물질 첨가구 사이 분포(plot) 거리가 서로 멀리 떨어져 있음을 관찰하였다. 이러한 결과는 3종류 시료가 장내세균 형성(OTU의 종류 및 수)에 서로 다른 영향을 주는 것을 의미한다(베타-다양성, 도 6(b)).6 is an analysis of the diversity of intestinal bacteria. A multivariate analysis of sequencing data by meta 16S analysis was applied, and a diversity analysis was shown that performed a principal coordinate analysis (PCoA) from the type and number of OTUs revealed. As a result, there was a statistically significant difference in the total number of intestinal bacteria in each sample through alpha-diversity analysis (Kruskal-Wallis test, p <0.05, FIG. 6(a)). In addition, through the principal coordinate analysis, it was observed that the plot distances between the cellulose, xylooligosaccharide and red ginseng foil indigestible substances added are far from each other. These results mean that the three samples have different effects on the formation of intestinal bacteria (type and number of OTUs) (beta-diversity, Fig. 6(b)).
또한, 16S rRNA 유전자 시컨싱 데이타를 기존의 데이터베이스와 비교하여, 문(phylum) 레벨에서 장내세균을 규명하였다. 그 결과 발견된 문은 총 8개였으며 상위 4개 문의 상대 분포비는 Actinobacterium(약 3%, 3%, 6%, 셀룰로오스, 자일로올리고당 및 홍삼박 순), Bacteroidetes(약 20%, 3%, 38%, 셀룰로오스, 자일로올리고당 및 홍삼박 순), Firmicutes(약 73%, 90%, 54%, 셀룰로오스, 자일로올리고당 및 홍삼박 순) 및 Proteobacterium(약 2.2%, 0.3%, 0.8%, 셀룰로오스, 자일로올리고당 및 홍삼박 순)에서 전체 비중의 약 97% 이상으로 나타났다. 또한 총 15개 시료에서 각각 관찰된 장내세균총의 상대 구성 비율에 따른 클러스터 분석을 실시하였다.In addition, 16S rRNA gene sequencing data were compared with existing databases to identify enterobacteriaceae at the phylum level. As a result, there were a total of 8 phylums, and the relative distribution ratios of the top four were Actinobacterium (about 3%, 3%, 6%, cellulose, xyloligosaccharide and red ginseng leaf in order), Bacteroidetes (about 20%, 3%, 38%, cellulose, xylooligosaccharide and red ginseng leaf in order), Firmicutes (about 73%, 90%, 54%, cellulose, xyloligosaccharide and red ginseng leaf in order) and Proteobacterium (about 2.2%, 0.3%, 0.8%, cellulose , Xyloligosaccharide and red ginseng (in that order), it was found to be over 97% of the total proportion. In addition, cluster analysis according to the relative composition ratio of the intestinal flora observed in each of a total of 15 samples was performed.
도 7은 군집분포를 나타낸 것이다. 각각의 배양액은 동일한 단위로 구성되어 시료별 3군집을 형성하였다. 이러한 군집 형성은 셀룰로오스와 홍삼박이 가까운 반면 자일로올리고당과는 먼 거리 형태를 보였다. 홍삼박에는 셀룰로오스가 함유되어 있기 때문에 자일로올리고당보다 셀룰로오스와 비슷한 패턴이 나타난 것으로 판단된다.7 shows the cluster distribution. Each culture medium was composed of the same unit to form three clusters for each sample. These cluster formations were close to cellulose and red ginseng, while far from xylo-oligosaccharide. Since red ginseng leaf contains cellulose, it is believed that a pattern similar to cellulose appeared than xyloligosaccharide.
도 8은 문(phylum) 단위에서 시료간 장내세균 상대 분포비를 나타낸 것이다. 여기에서 보듯이, 각 시료 간의 문 단위 상대 비율은 서로 다른 양상으로 나타냈다(Kruskal-Wallis test, p<0.05). 예를 들어, 식이섬유 섭취에 의해 대장에서 보편적으로 증가하는 것으로 보고되고 있는 Bacteroidetes문 세균과 감소하는 것으로 보고되고 있는 Firmicutes문 세균은 홍삼박 처리구에서 다른 처리구에 비해 각각 유의적으로 증가 및 감소하였다. 또한 Actinobacterium문 및 proteobacterium문 세균에서도 각 시료별 상대 분포비가 현저한 차이를 나타내었다.8 shows the relative distribution ratio of intestinal bacteria between samples in a phylum unit. As shown here, the relative ratio of phylum units between each sample was shown in different patterns (Kruskal-Wallis test, p <0.05). For example, the Bacteroidetes bacterium, which is reported to increase universally in the colon by dietary fiber intake, and the Firmicutes bacterium, which are reported to decrease, were significantly increased and decreased in the red ginseng foil treatment compared to other treatments. In addition, the relative distribution ratio of each sample was markedly different in Actinobacterium and proteobacterium bacterium.
도 9는 속(genus) 레벨에서 장내세균의 상대 구성비를 나타낸 것이다. 16S rRNA유전자 시컨싱 데이타를 기존의 데이터 베이스와 비교하여, 속(genus) 레벨에서 장내세균의 상대 구성비를 관찰하였다. 특히, 속 레벨에서 장내세균총의 상대 구성 비율에 따른 상동성을 비교한 결과, 58개 속 장내세균이 서로 다른 색으로 표현되었듯이 장내세균 구성비는 시료별로 다르게 나타났다. 본 실험에서는 41개 속 장내세균은 분류가 가능했고 17속 세균은 분류되지 못하였다.9 shows the relative composition ratio of enterobacteriaceae at the genus level. The 16S rRNA gene sequencing data was compared with the existing database to observe the relative composition ratio of enterobacteriaceae at the genus level. In particular, as a result of comparing the homology according to the relative composition ratio of the intestinal flora at the genus level, the composition ratio of intestinal bacteria was different for each sample, as 58 genera were expressed in different colors. In this experiment, 41 genera intestinal bacteria were classified, and 17 genera were not.
속 레벨에서 장내세균총의 상대 구성 비율에 따른 차이를 비교한 결과, 장내세균의 상대 분포는 문 레벨에서의 차이점과 비슷한 양상으로 나타났다.As a result of comparing the difference according to the relative composition ratio of the intestinal flora at the genus level, the relative distribution of the intestinal flora showed a similar pattern to the difference at the phylum level.
도 10은 속(genus) 소속에서 장내세균총의 상대 비율을 나타낸 것이다. 특히, 속 단위에서 특징적인 세균을 선별하여 각 시료 첨가구 사이 차이를 분석한 결과, 홍삼박 난소화성 물질 투여군은 다른 두 투여군에 비해 Bacteroidetes문 계열의 Prevotella속 세균 및 Actinoacteria문 계열의 Bifidobacterium속 세균의 상대적 비율이 다른 투여군에 비해 유의하게 증가하였다. 흥미롭게도 메타분석에서 Bifidobacterium속 결과는 선택배지에서 관찰된 Bifidobacterium균총 수 결과와 유사하였다. 하지만 Firmicutes문 계열 Lactobacillus속 세균의 상대적 비율은 자일로올리고당 처리구에서만 유의적으로 높았다. 한편, Prevotella속 세균은 주로 인간의 구강 및 장내, 반추위 또는 토양 등에서 발견되며, 아프리카 또는 동남아시아 지역에 사는 사람들 장내에 많이 존재하는 것으로 알려져 있다. 탄수화물, 특히 섬유질을 섭취하는 사람들에게는 Prevotella속 세균이 우세하다고 보고되고 있다(Wu et al., Science, doi:10.1126/science.1208344). 특히, Prevotella copri종은 식이섬유를 분해하는 능력이 높고, 주요 대사 산물로서 호박산이나 초산을 만드는 것으로 알려져 있다. 본 실험에서도 Prevotella copri종은 홍삼박 난소화성 물질 투여군에서 유의적으로 상대분포비가 다른 2종류 시료에 비해 높았다.10 shows the relative ratio of the intestinal flora in the genus. In particular, as a result of analyzing the difference between each sample addition group by selecting characteristic bacteria in the genus unit, the red ginseng leaf indigestible substance administration group showed that the bacteria of the genus Prevotella of the Bacteroidetes family and the bacteria of the Bifidobacterium family of the Actinoacteria family were The relative ratio increased significantly compared to other administration groups. Interestingly, the results of the genus Bifidobacterium in the meta-analysis were similar to the results of the number of Bifidobacterium flora observed in the selective medium. However, the relative proportion of bacteria in the genus Lactobacillus of the Firmicutes phylum family was significantly higher only in the xyloligosaccharide treatment group. On the other hand, the bacteria of the genus Prevotella are mainly found in the oral cavity and intestines of humans, rumen or soil, and are known to exist in the intestines of people living in Africa or Southeast Asia. Prevotella bacteria are reported to be dominant among those who consume carbohydrates, especially fiber (Wu et al., Science, doi:10.1126/science.1208344). Specifically, Prevotella copri species It has a high ability to break down dietary fiber and is known to make succinic acid or acetic acid as major metabolites. In this experiment, Prevotella copri species was significantly higher in the group administered with red ginseng foil indigestible substances than in the other two samples.
8) 암모니아태 질소 측정8) Measurement of ammonia nitrogen
배양액 중의 암모니아태 질소 농도는 암모니아 분석 키트 (Wako, Osaka, Japan)에 의해 측정하였다. 분석을 위해 배양액을 원심분리(16,000 rpm, 4℃, 15분)하여 상층액을 취한 후 상층액을 0.1 M 인산완충액(pH5.5)으로 적당히 희석하여 사용하였다. 최종적으로 마이크로 플레이트 리더 (Multiscan FC, ThermoFisher)를 사용하여 파장 620 nm에서 흡광도를 측정하였다. 표준 용액에서 제작한 검량선을 이용하여 암모니아태 질소 농도를 산출하였다.The concentration of ammonia nitrogen in the culture medium was measured by an ammonia assay kit (Wako, Osaka, Japan). For analysis, the culture solution was centrifuged (16,000 rpm, 4° C., 15 minutes) to take the supernatant, and the supernatant was appropriately diluted with 0.1 M phosphate buffer (pH5.5) and used. Finally, absorbance was measured at a wavelength of 620 nm using a microplate reader (Multiscan FC, ThermoFisher). Ammonia nitrogen concentration was calculated using a calibration curve prepared from a standard solution.
도 11은 홍삼박 난소화성 물질의 배양기간 중 암모니아태 질소 생성 변화를 나타낸 것이다. 여기에서 보듯이, 셀룰로오스 첨가구의 암모니아태 질소 농도는 배양개시부터 배양종료까지 계속 상승하였다. 배양개시 6시간째 홍삼박 난소화성 물질 첨가구의 암모니아태 질소 농도는 셀룰로오스 첨가구 및 자일로올리고당 첨가구의 암모니아태 질소 농도보다 유의하게 (p<0.05) 억제되었다. 자일로올리고당 첨가구의 암모니아태 질소 농도는 배양 6시간째 피크가 되었다가 그 후 감소하여 홍삼박 난소화성 물질 첨가구와 더불어 셀룰로오스 첨가구보다 유의하게 (p<0.05) 감소하였다.11 shows the change in ammonia-like nitrogen production during the incubation period of red ginseng foil indigestible material. As shown here, the concentration of ammonia nitrogen in the cellulose-added group continued to rise from the start of the culture to the end of the culture. At 6 hours from the initiation of culture, the ammonia nitrogen concentration of the indigestible substance-added group of red ginseng meal was significantly (p<0.05) suppressed than that of the cellulose-added group and the xylooligosaccharide-added group. The concentration of ammonia nitrogen in the xylooligosaccharide-added group peaked at 6 hours of incubation and then decreased, and significantly decreased (p<0.05) compared to the cellulose-added group with the red ginseng meal indigestible substance added group.
본 실험에서의 발효조 내 홍삼박 난소화성 물질 첨가구의 암모니아태 질소의 생성 억제는 유기산 생성에 의한 낮은 pH 상태에서 암모니아 생성이 저하되었을 가능성이 높다.In this experiment, the suppression of the formation of ammonia nitrogen in the fermenter with the addition of indigestible substances of red ginseng meal is likely to result in a decrease in ammonia production in the low pH state due to the production of organic acids.
9) 스카톨(skatole) 측정9) Measurement of skatole
이전에 보고된 분석 방법(Walstra et al., Livest. Prod. Sci., doi: org/10.1016/S0301-6226(99)00054-8)을 조금 변경하여 측정하였다. 구체적으로, 표준품은 스카톨 (3-메틸 인돌) 10 mg을 소량의 99% 에탄올로 용해시키고 0.1 M Tris-HCl 완충용액(pH7.5) 100 mL에 녹였다(최종 100 μg/mL). 발색액은 p-디메틸 아미노 벤젠알데히드 0.5 g을 황산을 함유한 알코올 50 mL (황산 20 mL + 95% 에탄올 30 mL)에 용해시켰다. 분석용 시료는 배양액 상층액 380 μL에 2 M Tris-HCl 완충용액(pH7.5)을 20 μL 첨가하여 사용하였다. 96 well plate의 각 웰(well)에 표준액 또는 시료액을 넣고, 발색액을 첨가하여 잘 혼합한 후 실온에서 20 분간 정치 상태에서 반응을 시켰다. 그 다음 마이크로 플레이트 리더 (Multiscan FC)를 사용하여 파장 570 nm에서 흡광도를 측정하였다.The previously reported analysis method (Walstra et al ., Livest. Prod. Sci., doi: org/10.1016/S0301-6226(99)00054-8) was measured with slight changes. Specifically, the standard was dissolved in 10 mg of scatol (3-methyl indole) in a small amount of 99% ethanol and dissolved in 100 mL of 0.1 M Tris-HCl buffer solution (pH7.5) (final 100 μg/mL). As a color developing solution, 0.5 g of p -dimethylaminobenzenealdehyde was dissolved in 50 mL of alcohol containing sulfuric acid (20 mL of sulfuric acid + 30 mL of 95% ethanol). The sample for analysis was used by adding 20 μL of 2 M Tris-HCl buffer (pH7.5) to 380 μL of the culture supernatant. A standard solution or a sample solution was added to each well of a 96 well plate, a color developing solution was added, mixed well, and the reaction was allowed to stand at room temperature for 20 minutes. Then, the absorbance was measured at a wavelength of 570 nm using a micro plate reader (Multiscan FC).
도 12는 홍삼박 난소화성 물질의 배양기간 중 스카톨 생성 변화를 나타낸 것이다. 본 실험에서는 자일로올리고당 첨가구가 스카톨 생성을 가장 억제하였다. 특히 배양시간 6시간째 피크가 되어 12시간 이후로는 셀룰로오스 첨가구에 비해 통계적으로 유의하게 (p<0.05) 감소하였다. 이에 비해 홍삼박 난소화성 물질 첨가구는 배양시간 12시간째 피크가 되었고 이후 완화한 감소를 보였으며 배양시간 48시간째에는 자일로올리고당 첨가구보다는 통계적으로 높았지만 셀룰로오스 첨가구보다 통계적으로 유의하게 (p<0.05) 낮은 생성 농도를 보였다.12 shows the change in scatol production during the culture period of the indigestible material of red ginseng meal. In this experiment, the addition of xylo-oligosaccharide inhibited the formation of scatol most. In particular, the incubation time peaked at 6 hours and decreased statistically significantly (p<0.05) compared to the cellulose-added group after 12 hours. In contrast, the group with red ginseng meal indigestible material peaked at 12 hours of incubation time and then showed a moderate decrease. At 48 hours of incubation time, it was statistically higher than the group with xylo-oligosaccharide, but statistically significant (p< 0.05) showed a low production concentration.
Claims (6)
상기 조성물이 장내 균총에서 피르미큐테스( F irmicutes) 문 및 박테리오이데테스(Bacteroidetes) 문의 비율을 개선시키는 것을 특징으로 하는 장 건강 개선용 식품 조성물.The method of claim 1,
The composition of the microflora in the intestines pireu vermiculite test (F irmicutes) bakteriohyi to test and doors (Bacteroidetes) section, comprising a contact rate improving health improved food composition.
상기 조성물이 비피도박테리움(Bifidobacterum) 균을 증식하는 것을 특징으로 하는 장 건강 개선용 식품 조성물.The method of claim 1,
Food composition for improving intestinal health, characterized in that the composition proliferates Bifidobacterum bacteria.
상기 조성물이 장내 암모니아 생산을 억제하여 혈중 암모니아 농도를 저하시키는 것을 특징으로 하는 장 건강 개선용 식품 조성물.The method of claim 1,
Food composition for improving intestinal health, characterized in that the composition reduces the concentration of ammonia in the blood by inhibiting the production of ammonia in the intestine.
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