KR20120019311A - Composition for immunostimulation which comprises polysaccharide from fermented ginseng with phellinus linteus to enhance the immune function - Google Patents
Composition for immunostimulation which comprises polysaccharide from fermented ginseng with phellinus linteus to enhance the immune function Download PDFInfo
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- KR20120019311A KR20120019311A KR1020100082654A KR20100082654A KR20120019311A KR 20120019311 A KR20120019311 A KR 20120019311A KR 1020100082654 A KR1020100082654 A KR 1020100082654A KR 20100082654 A KR20100082654 A KR 20100082654A KR 20120019311 A KR20120019311 A KR 20120019311A
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- Prior art keywords
- ginseng
- mushroom mycelium
- mycelium
- fraction
- activity
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K36/06—Fungi, e.g. yeasts
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/324—Foods, ingredients or supplements having a functional effect on health having an effect on the immune system
Abstract
Description
본 발명은 면역기능을 증진시키기 위하여 조제한 인삼의 상황버섯 균사체 발효물로부터 분리된 다당류를 유효성분으로 포함하는 면역활성용 조성물에 대한 것이다.
The present invention relates to a composition for immunological activity comprising a polysaccharide isolated from the fermented product of the situation mushroom mycelium of ginseng prepared to enhance immune function.
인삼(Panax ginseng C.A. Meyer)은 한반도가 원산인 한국의 특산 약용식물로 2000여년 전부터 동북아시아에서 보원기제로 사용되어 온 중요한 한약 중의 하나이다. 동양에서 가장 오래된 본초서인 신농본초경에 인삼은 오장을 보하고, 원기를 보충한다고 기록되어 있다.
Panax ginseng ginseng CA Meyer) is a Korean specialty medicinal plant native to the Korean peninsula and is one of the important herbal medicines that has been used as a conservation mechanism in Northeast Asia for over 2000 years. Ginseng is written in the Xinongboncho, the oldest herbaceous book in the Orient, to supplement the five chapters.
인삼의 생리활성은 체계적인 약리학적 접근으로 심혈관계(Lee et al., 1981), 면역계(Jie et al., 1984), 신경계(Kim et al., 1998)에 대한 효능과 해독작용(Joo et al., 1977), 항암작용(Tahara et al., 1985) 그리고 항당뇨작용(Yokozawa et al., 1985) 등이 보고되었다. 인삼의 주요한 생리활성물질은 인삼사포닌(ginsenosides), polyacetylenes, 산성다당체, 인삼단백질, 폴리페놀물질 등이 알려져 있다(Park, 1996; Sanata et al., 1974; Kitagawa et al., 1987).
The physiological activity of ginseng is a systematic pharmacological approach that is effective and detoxifying to the cardiovascular system (Lee et al., 1981), the immune system (Jie et al., 1984), and the nervous system (Kim et al., 1998). , 1977), anticancer activity (Tahara et al., 1985) and antidiabetic activity (Yokozawa et al., 1985). Ginseng saponins (ginsenosides), polyacetylenes, acidic polysaccharides, ginseng proteins, polyphenols, etc. are known (Park, 1996; Sanata et al., 1974; Kitagawa et al., 1987).
그러나 우리나라의 인삼을 이용한 가공제품은 단순한 인삼추출물 또는 농축음료, 분말, 캡슐과 과립차 등이 주를 이루고 있어 기능성 유효성분이 증진된 고부가가치의 새로운 인삼 가공제품 개발 필요성에 관심이 모아지고 있다. 최근, 인삼의 생리활성을 증진시키기 위한 유효성분의 추출수율 향상과 특정 유용성분의 함량증대를 위한 방법으로 인삼을 효소처리하는 연구가 진행되고 있으며(Kim YC, Cho CW, Rhee YK, Yoo KM, Rho J. Antioxidant activity of ginseng extracts prepared by enzyme and heat treatment. J. Korean Soc. Food Sci. Nutr. 36: 1482-1485 (2007)), 고온처리법과 고압처리법 역시 인삼의 가공온도 및 압력을 높여 사포닌 전환에 의한 생리활성을 증대시키는 것이다(Yang SJ, Woo KS, Yoo JS, Kang TS, Noh YH, Lee J, Jeong HS. Changes of Korean ginseng components with high temperature and pressure treatment. Korean J. Food Sci. Technol. 38: 521-525 (2006)). 그러나 이러한 연구는 특정 사포닌 성분에 한정되어 있고 사포닌을 제외한 인삼의 비사포닌계 화합물, 특히 다당류에 대한 전환연구는 미미한 실정이다.
However, the processed products using ginseng in Korea is mainly made up of simple ginseng extract or concentrated drink, powder, capsules and granular teas, which is attracting attention to the need to develop new value-added processed ginseng products with enhanced functional and active ingredients. Recently, research is being conducted on enzyme treatment of ginseng as a method for improving the extraction yield of active ingredients and increasing the content of specific useful ingredients to enhance the physiological activity of ginseng (Kim YC, Cho CW, Rhee YK, Yoo KM, Rho J. Antioxidant activity of ginseng extracts prepared by enzyme and heat treatment.J. Korean Soc.Food Sci.Nutr. 36: 1482-1485 (2007)), and high-temperature and high-pressure treatments also increase the processing temperature and pressure of ginseng (Yang SJ, Woo KS, Yoo JS, Kang TS, Noh YH, Lee J, Jeong HS.Changes of Korean ginseng components with high temperature and pressure treatment.Korean J. Food Sci.Technol 38: 521-525 (2006). However, these studies are limited to specific saponin components, and studies on the conversion of non-saponin compounds, especially polysaccharides, of ginseng except saponins are insignificant.
진흙버섯류는 담자균문(Basidiomycotina), 민주름버섯목(Aphyllophorales), 소나무비늘과(Hymenochaetaceae)에 속하는 진흙버섯속(Phellinus)의 균류를 지칭하는 버섯으로 뽕나무 줄기에 자생하며 삿갓표면을 제외하고는 모두 황색이므로 상황으로 불리고 있는데, 특히 국내와 일본에서는 진흙버섯속 중 목질진흙버섯(Phellinus linteus)을 상황버섯으로 인정하고 있다. 그러나 최근 중국산 상황버섯이 대량 수입되면서 국산 상황버섯의 수요는 경제적 이유로 줄어들고 있는 것이 실정이다.
Mud mushrooms are fungi of the genus Phellinus belonging to the genus Basidiomycotina, Aphyllophorales, and Hymenochaetaceae. Since it is yellow, it is called a situation. Especially, in Korea and Japan, woody mud mushroom ( Phellinus linteus ) among mud mushrooms is recognized as a situation mushroom. However, with the recent import of Chinese situation mushrooms, the demand for domestic situation mushrooms is decreasing for economic reasons.
본 발명자는 인삼을 상황버섯 균사체로 배양하여 발효물을 조제하는 과정에서, 상황버섯 균사체의 생물학적 전환을 통하여 인삼 또는 균사체 자체보다도 발효물에서 면역활성이 증강되는 것과 함께 유효성분으로 다당류가 관여되어 있음을 발견하고 본 발명을 완성하였다.
The present inventors in the process of preparing a fermented product by cultivating ginseng to the situation mushroom mycelium, the polysaccharide is involved as an active ingredient with enhanced immune activity in the fermentation product than the ginseng or mycelium itself through the biological conversion of the situation mushroom mycelium And the present invention was completed.
본 발명의 목적은 다당류를 유효성분으로 포함하는 면역활성용 조성물을 제공하는 것이다.
It is an object of the present invention to provide a composition for immunoactivation comprising a polysaccharide as an active ingredient.
상기 목적을 달성하기 위하여 본 발명은 인삼의 상황버섯 균사체 발효물로부터 분리된 다당류를 유효성분으로 포함하는 면역활성용 조성물을 제공한다.
In order to achieve the above object, the present invention provides a composition for immunological activity comprising a polysaccharide isolated from the fermented product of the situation mushroom mycelium of ginseng as an active ingredient.
본 발명에서 인삼의 상황버섯 균사체 발효물로부터 분리된 다당류를 유효성분으로 포함하는 면역활성용 조성물은 체내 면역계를 구성하는 면역세포를 활성화시켜 바이러스, 세균 등으로부터의 감염되는 것을 방지하는 효과가 있다.
In the present invention, the immunologically active composition comprising a polysaccharide isolated from the fermented product of the situation mushroom mycelium of ginseng as an active ingredient has the effect of preventing the infection from viruses, bacteria, etc. by activating immune cells constituting the body's immune system.
도 1 내지 도 3은 인삼-상황버섯 균사체 발효물, 인삼 및 상황버섯 균사체의 조다당획분을 DEAE-Sepharose CL-6B를 이용하여 분획한 결과를 나타낸다.
도 4는 인삼-상황버섯 균사체 발효물, 인삼 및 상황버섯 균사체의 조다당획분을 DEAE-Sepharose CL-6B를 이용하여 분획한 획분의 마이토젠 활성을 나타낸다.
도 5은 인삼-상황버섯 균사체 발효물, 인삼 및 상황버섯 균사체의 조다당획분을 DEAE-Sepharose CL-6B를 이용하여 분획한 획분의 마크로파지 활성을 나타낸다.
도 6는 인삼-상황버섯 균사체 발효물, 인삼 및 상황버섯 균사체의 조다당획분을 DEAE-Sepharose CL-6B를 이용하여 분획한 획분의 Peyer's patch를 매개로 한 장관면역 활성을 나타낸다.1 to 3 show the results of fractionation of crude polysaccharides of ginseng-situation mushroom mycelium fermented product, ginseng and situation mushroom mycelium using DEAE-Sepharose CL-6B.
Figure 4 shows the mitogen activity of the fraction obtained by fractionation of crude polysaccharides of ginseng-situation mushroom mycelium fermented product, ginseng and situation mushroom mycelium using DEAE-Sepharose CL-6B.
Figure 5 shows the macrophage activity of the fraction obtained by fractionation of crude polysaccharides of ginseng-situation mushroom mycelium fermented product, ginseng and situation mushroom mycelium using DEAE-Sepharose CL-6B.
Figure 6 shows the intestinal immune activity through the Peyer's patch of the fraction obtained by fractionation of crude polysaccharides of ginseng-situation mushroom mycelium fermented product, ginseng and situation mushroom mycelium using DEAE-Sepharose CL-6B.
본 발명은 인삼의 상황버섯 균사체 발효물로부터 분리된 다당류를 유효성분으로 포함하는 면역활성용 조성물을 제공한다.
The present invention provides a composition for immunological activity comprising a polysaccharide isolated from the fermented product of the situation mushroom mycelium of ginseng as an active ingredient.
또한 본 발명은 인삼을 상황버섯 균사체로 발효시켜 발효물을 조제하는 단계를 포함하는 면역활성용 조성물의 제조 방법을 제공한다.
In another aspect, the present invention provides a method for producing an immunoactive composition comprising the step of preparing a fermented product by fermenting ginseng to the situation mushroom mycelium.
이하, 본 발명을 자세히 설명한다.
Hereinafter, the present invention will be described in detail.
본 발명의 인삼은 P. ginseng C.A. Meyer이다. 본 발명의 인삼은 수삼, 홍삼, 장뇌삼, 산삼 등이 될 수 있으나 이에 제한되는 것은 아니다. 온전한 인삼을 사용하여도 되지만, 경제적인 이유에서 인삼의 일부가 손상되거나 상업적 가치가 떨어지는 파삼을 사용하여도 무방하다. 상기 인삼은 인삼의 잎, 뿌리, 줄기 등이 될 수 있다. 바람직하게는 상기 인삼은 인삼의 뿌리이다. 상기 인삼은 채취 후 생삼의 형태로 이용할 수 있으며, 동결건조, 자연건조 등을 통하여 건조된 삼의 형태로 이용할 수도 있다. 또한 상기 인삼은 세절하여 이용하거나 분쇄하여 분말의 형태로 이용할 수도 있다. 당업자가 주변상황 및 조건에 따라 적절히 인삼의 형태를 선택하여 본 발명을 실시할 수 있다는 것은 자명하다.
Ginseng of the present invention is P. ginseng CA Meyer. Ginseng of the present invention may be, but is not limited to, ginseng, red ginseng, camphor ginseng, wild ginseng. Intact ginseng may be used, but for economic reasons, some ginseng may be damaged or it may be of low commercial value. The ginseng may be leaves, roots, stems, and the like of ginseng. Preferably the ginseng is the root of ginseng. The ginseng may be used in the form of fresh ginseng after harvesting, may also be used in the form of ginseng dried through freeze-drying, natural drying and the like. In addition, the ginseng may be used in the form of powder by cutting or grinding. It will be apparent to those skilled in the art that the present invention may be implemented by appropriately selecting the form of ginseng according to the surrounding conditions and conditions.
상기 상황버섯은 진흙버섯속(Phellinus)의 균류이며, 바람직하게는 목질진흙버섯(P. linteus)이다.
The situation mushroom is a fungus of the genus Phellinus , preferably P. linteus .
상기 상황버섯 균사체로 발효시킨 인삼발효물은 상황버섯 균사체로 인삼을 발효시킨 인삼 자체일 수도 있으나, 상기 발효된 인삼발효물을 열수 추출하거나 가수분해효소로 분해하거나, 탄소수 4 이하의 저급 알코올로 추출하여 이용할 수도 있다. 또한 상황버섯 균사체로 발효시킨 인삼발효물을 열수 추출한 후 에탄올로 침전시켜 이용할 수도 있으며, 상기 에탄올로 침전한 침전물을 이온교환 크로마토그래피, 겔 여과 크로마토그래피, 친화 크로마토그래피 등을 이용하여 분획하여 이용할 수도 있다.
The ginseng fermentation product fermented with the situation mushroom mycelium may be ginseng itself fermented ginseng with the situation mushroom mycelium, but the fermented ginseng fermentation product is hydrothermally extracted or hydrolyzed, or extracted with a lower alcohol having 4 or less carbon atoms. It can also be used. In addition, the ginseng fermented with the situation mushroom mycelium may be extracted by hot water and then precipitated with ethanol, and the precipitate precipitated with ethanol may be fractionated using ion exchange chromatography, gel filtration chromatography, affinity chromatography, and the like. have.
상기 면역활성용 조성물은 식품 조성물 또는 약학적 조성물이 될 수 있으며, 바람직하게는 상기 조성물은 식품 조성물이다. 상기 식품이란 건강보조식품, 건강기능식품, 기능성 식품 등이나 이에 제한되는 것은 아니며, 천연식품, 가공식품, 일반적인 식자재 등에 본 발명의 면역활성용 조성물을 첨가한 것도 포함된다.
The immunoactive composition may be a food composition or a pharmaceutical composition, preferably the composition is a food composition. The food is not limited to, but not limited to, health supplements, health functional foods, functional foods, and the like, natural foods, processed foods, and general food materials, including the addition of the composition for immunoactivation of the present invention.
본 발명의 면역활성용 식품 조성물은, 상기 조성물을 그대로 첨가하거나 다른 식품 또는 식품 조성물과 함께 사용될 수 있으며, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합양은 그의 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 본 발명의 면역활성용 조성물을 식품 또는 음료의 제조 시에 식품 또는 음료의 원료 100 중량부에 대하여 0.1 내지 70 중량부, 바람직하게는 2 내지 50 중량부 첨가될 수 있다. 상기 면역활성용 조성물의 유효용량은 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 범위 이하일 수 있으며, 유효성분은 안전성 면에서 아무런 문제가 없기 때문에 상기 범위 이상의 양으로도 사용될 수 있다.
The immunoactive food composition of the present invention may be added as it is or used with other food or food compositions, and may be suitably used according to a conventional method. The mixed amount of the active ingredient can be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, 0.1 to 70 parts by weight, preferably 2 to 50 parts by weight of the immunoactive composition of the present invention may be added to 100 parts by weight of the raw material of the food or beverage during the preparation of the food or beverage. The effective dose of the immunologically active composition may be less than the above range in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, and since the active ingredient has no problem in terms of safety, Can also be used as.
상기 식품의 종류에는 특별한 제한은 없다. 상기 면역활성용 식품 조성물은 정제, 경질 또는 연질 캅셀제, 액제, 현탁제 등과 같은 경구투여용 제제의 형태로 이용될 수 있으며, 이들 제제는 허용 가능한 통상의 담체, 예를 들어 경구투여용 제제의 경우에는 부형제, 결합제, 붕해제, 활택제, 가용화제, 현탁화제, 보존제 또는 증량제 등을 사용하여 조제할 수 있다. There is no particular limitation on the kind of food. The immunologically active food composition may be used in the form of oral preparations such as tablets, hard or soft capsules, solutions, suspensions, and the like, and these preparations are acceptable carriers, for example, oral preparations. It can be prepared using excipients, binders, disintegrants, lubricants, solubilizers, suspending agents, preservatives or extenders.
상기 면역활성용 조성물을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등을 들 수 있으나 이들 종류의 식품으로 제한되는 것은 아니다.
Examples of foods to which the immunoactive composition may be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea , Drinks, alcoholic beverages and vitamin complexes, but are not limited to these kinds of food.
이하, 본 발명을 다음의 실시예 및 실험예에 의해 보다 상세하게 설명한다. 단, 하기 실시예 및 실험예는 본 발명의 내용을 예시하는 것일 뿐 발명의 범위가 실시예 및 실험예에 의해 한정되는 것은 아니다.
Hereinafter, the present invention will be described in more detail by the following examples and experimental examples. However, the following examples and experimental examples are intended to illustrate the contents of the present invention, but the scope of the invention is not limited by the examples and the experimental examples.
<재료 및 방법> ≪ Materials and methods >
인삼Ginseng
인삼은 충북 증평군에서 2007년도에 수확한 4~5년 근 인삼을 증평영농조합인삼연구회(Jeungpyeong-gun, Chungbuk, Korea)로부터 구입한 후 품질저하를 방지하기 위하여 -5 ℃ 냉동실에 보관하면서 사용하였다.
Ginseng was purchased from Jeungpyeong-gun, Chungbuk, Korea for 4 ~ 5 years from Ginseng Research Group (Jeungpyeong-gun, Chungbuk, Korea) and stored in -5 ℃ freezer to prevent quality deterioration. .
상황버섯Situation Mushroom
상황버섯(P. linteus) 균주는 충청북도 농업기술원(Cheongwon-gun, Chungbuk, Korea)으로부터 분양 받았다. 고체배양의 상황버섯 종균을 조제하기 위하여 mushroom complete medium(MCM) 액체배지에 위에서 언급한 인삼의 열수추출로부터 조제된 인삼추출물(고형분 65%)이 5% 농도가 되도록 첨가하여 진탕배양기(SI-4100R, Jeio Tech, Daejeon, Korea)에서 30 ℃로 배양하였다.
P. linteus strains were distributed from Cheongwon-gun, Chungbuk, Korea. In order to prepare the spawn mushroom spawn in solid culture, ginseng extract (65% solids) prepared from the above-mentioned hot water extract of ginseng was added to the mushroom complete medium (MCM) liquid medium to give a shaking culture (SI-4100R). , Jeio Tech, Daejeon, Korea) was incubated at 30 ℃.
인삼-상황버섯 균사체 Ginseng-Situation Mushroom Mycelia 발효물Fermentation product
상황버섯 균사체의 고체배양에 의한 인삼발효물(인삼-상황버섯 균사체 발효물)은 인삼을 수세한 후 실온에서 3~4일간 건조시켜 수분함량을 조절하고 조직을 연화시킨 후 현미분말을 10% 첨가하여 121 ℃에서 20분 멸균시킨 인삼에 상황버섯 종균을 10% 접종하고 30일간 30 ℃에서 배양(Multi Room Incubator, Vision Scientific, Bucheon, Kyonggi-do, Korea)하고 동결건조하여 제조하였다.
The ginseng fermentation product (Ginseng-Situation mushroom mycelium fermentation product) by solid culture of the situation mushroom mycelium is washed with ginseng and dried at room temperature for 3 to 4 days to adjust the water content, soften the tissue and add 10% brown rice powder. Was prepared by inoculating 10% of the situation mushroom spawn in ginseng sterilized at 121 ° C. for 20 minutes and incubating at 30 ° C. for 30 days (Multi Room Incubator, Vision Scientific, Bucheon, Kyonggi-do, Korea) and lyophilizing.
실험동물과 시약Laboratory Animals and Reagents
생후 6주령의 자성 C3H/He, BALB/c 또는 BKW 마우스를 (주)오리엔트바이오(Seongnam, Kyonggi-do, Korea) 및 (주)나라바이오텍(Pyeongtaek, Kyonggi-do, Korea)에서 구입한 후, 사육조에 5마리씩 넣고 정수된 물과 실험동물용 펠렛사료(Samyang Co., Incheon, Korea)를 자유공급하였으며 스트레스를 받지 않도록 주의하여 사육하였다. 면역활성 측정의 동물세포 배양을 위한 RPMI 1640 medium과 Hank's balanced salt solution(HBSS)은 Gibco-BRL Co.(Grand Island, NY, USA)으로부터 구입하였다. 또한 fetal bovine serum(FBS)은 Cell Culture Laboratories(Cleveland, OH, USA)에서 penicillin, streptomycin 및 amphotericin B는 Flow Laboratories(Irvine, Scotland)에서 입수하였다. 한편, 마이토젠 활성의 비장세포 및 장관면역 활성의 골수세포 증식을 측정하기 위한 Cell Counting Kit-8(CCK-8)은 Dojindo Laboratories(Kumamoto, Japan)로부터 구입하였다.
After 6 weeks of age, magnetic C3H / He, BALB / c or BKW mice were purchased from OrientBio (Seongnam, Kyonggi-do, Korea) and Nara Biotech (Pyeongtaek, Kyonggi-do, Korea). Five dogs were placed in the breeding tank and purified water and pellets (Samyang Co., Incheon, Korea) for experimental animals were supplied freely and were carefully bred to avoid stress. RPMI 1640 medium and Hank's balanced salt solution (HBSS) for animal cell culture for immunoactivity measurements were purchased from Gibco-BRL Co. (Grand Island, NY, USA). Fetal bovine serum (FBS) was obtained from Cell Culture Laboratories (Cleveland, OH, USA) and penicillin, streptomycin and amphotericin B were obtained from Flow Laboratories (Irvine, Scotland). Meanwhile, Cell Counting Kit-8 (CCK-8) for measuring mytogen-activated splenocytes and enteroimmune-activated bone marrow cell proliferation was purchased from Dojindo Laboratories (Kumamoto, Japan).
통계처리Statistical processing
실험결과들은 4개 군에 대한 실험을 통하여 평균치±SD(standard deviation)로 나타내었고 Student's t-test를 이용, p<0.05 수준에서 saline 대조군과 시료 및 시료대조군과 시료들간의 유의성을 검정함으로써 인삼-상황버섯 균사체 발효물로부터 분획한 시료의 면역활성으로 나타내었다.
The experimental results were expressed as mean ± SD (standard deviation) through experiments on four groups, and ginseng-tested by using Student's t-test to test the significance between saline control and sample and sample control and sample at p <0.05 level. Immune activity of the samples fractionated from the situation mushroom mycelium fermentation is shown.
<실시예 1-1>≪ Example 1-1 >
동결건조시킨 인삼-상황버섯 균사체 발효물의 열수추출물을 조제하기 위하여 발효물에 소량의 증류수를 첨가하여 homogenizer(Ultra-turrax T-50; Janke & Kunkel GmbH & Co., KG-IKA-Labortechnik, Staufen, Germany)로 5,000 rpm에서 10분간 분쇄한 후 20배의 증류수를 가하여 열수추출하였다. 추출액을 여과하여 여과액을 회수하고 농축, 원심분리(7,600×g, 30분) 및 동결건조하여 인삼-상황버섯 균사체 발효물의 열수추출물로 조제하였다(원료 대비 수율 45.0%).
In order to prepare the hot water extract of the lyophilized ginseng-situation mushroom mycelium fermentation, a small amount of distilled water was added to the fermentation product to prepare a homogenizer (Ultra-turrax T-50; Janke & Kunkel GmbH & Co., KG-IKA-Labortechnik, Staufen, Germany) was pulverized at 5,000 rpm for 10 minutes and hot water was extracted by adding 20 times distilled water. The extract was filtered to recover the filtrate, concentrated, centrifuged (7,600 × g, 30 minutes) and lyophilized to prepare a hot water extract of the ginseng-shang mushroom mycelium fermentation (yield 45.0% of raw material).
<실시예 1-2>≪ Example 1-2 >
실시예 1-1의 열수추출물을 회수하여 다시 소량의 증류수에 용해하고 5배의 에탄올을 가하여 침전물을 조제하였다. 상기 침전물을 투석(Spectra/Por 4, MWCO 12~14,000, Spectrum Laboratories Inc., Rancho Dominguez, CA, USA)하고 농축, 원심분리하여 인삼-상황버섯 균사체 발효물의 조다당획분(수율 8.80±0.03%)으로 조제하였다.
The hot water extract of Example 1-1 was recovered, dissolved in a small amount of distilled water, and 5 times ethanol was added thereto to prepare a precipitate. Crude fraction of ginseng-situation mushroom mycelium fermentation product (yield 8.80 ± 0.03%) by dialysis (Spectra / Por 4, MWCO 12 ~ 14,000, Spectrum Laboratories Inc., Rancho Dominguez, CA, USA), concentration and centrifugation Was prepared.
<실시예 2-1><Example 2-1>
실시예 1-1과 마찬가지로, 수세 후 동결건조시킨 인삼에 소량의 증류수를 첨가하여 homogenizer로 5,000 rpm에서 10분간 분쇄한 후 20배의 증류수를 가하여 열수추출하였다. 추출액을 여과하여 여과액을 회수하고 농축, 원심분리(7,600×g, 30분) 및 동결건조하여 인삼 열수추출물을 조제하였다(수율 46.3%).
As in Example 1-1, a small amount of distilled water was added to the lyophilized ginseng after washing with water, pulverized at 5,000 rpm for 10 minutes with a homogenizer, and hot water was extracted by adding 20 times distilled water. The extract was filtered to recover the filtrate, concentrated, centrifuged (7,600 × g, 30 minutes) and lyophilized to prepare a ginseng hot water extract (yield 46.3%).
<실시예 2-2><Example 2-2>
실시예 2-1의 인삼 열수추출물을 회수하여 다시 소량의 증류수에 용해하고 5배의 에탄올을 가하여 침전물을 조제하였다. 상기 침전물을 투석(Spectra/Por 4, MWCO 12~14,000, Spectrum Laboratories Inc., Rancho Dominguez, CA, USA)하고 농축, 원심분리하여 인삼의 조다당획분(수율 7.91±0.08%)으로 조제하였다.
The ginseng hot water extract of Example 2-1 was recovered, dissolved in a small amount of distilled water, and 5 times ethanol was added thereto to prepare a precipitate. The precipitate was dialyzed (Spectra / Por 4, MWCO 12-14,000, Spectrum Laboratories Inc., Rancho Dominguez, CA, USA), concentrated and centrifuged to prepare a crude polysaccharide fraction of ginseng (yield 7.91 ± 0.08%).
<실시예 3-1><Example 3-1>
실시예 1-1과 마찬가지로, 종균배양액에서 액체배양 후 동결건조시킨 상황버섯 균사체에 소량의 증류수를 첨가하여 homogenizer로 5,000 rpm에서 10분간 분쇄한 후 20배의 증류수를 가하여 열수추출하였다. 추출액을 여과하여 여과액을 회수하고 농축, 원심분리(7,600×g, 30분) 및 동결건조하여 상황버섯 균사체의 열수추출물을 조제하였다(수율 39.5%).
In the same manner as in Example 1-1, a small amount of distilled water was added to the lyophilized mycelium mycelium after liquid culture in the seed culture, and then pulverized at 5,000 rpm for 10 minutes with a homogenizer, and 20 times distilled water was added thereto. The extract was filtered to recover the filtrate, concentrated, centrifuged (7,600 × g, 30 minutes) and lyophilized to prepare a hot water extract of the situation mushroom mycelium (yield 39.5%).
<실시예 3-2><Example 3-2>
실시예 3-1의 상황버섯 균사체의 열수추출물을 회수하여 다시 소량의 증류수에 용해하고 5배의 에탄올을 가하여 침전물을 조제하였다. 상기 침전물을 투석(Spectra/Por 4, MWCO 12~14,000, Spectrum Laboratories Inc., Rancho Dominguez, CA, USA)하고 농축, 원심분리하여 상황버섯 균사체의 조다당획분(수율 5.33±0.05%)으로 조제하였다.
The hot water extract of the situation mushroom mycelium of Example 3-1 was recovered, dissolved in a small amount of distilled water, and ethanol was added five times to prepare a precipitate. The precipitate was dialyzed (Spectra / Por 4, MWCO 12-14,000, Spectrum Laboratories Inc., Rancho Dominguez, Calif., USA), concentrated and centrifuged to prepare a crude polysaccharide fraction of the situation mushroom mycelium (yield 5.33 ± 0.05%). .
<실험예 1> 비장세포의 마이토젠 활성Experimental Example 1 Mitogen Activity of Splenocytes
BALB/c 마우스(6~8주령, female)를 경구탈추시킨 후 멸균적으로 비장(spleen)을 적출하여 마쇄하고 0.2% NaCl과 금속망(# 200)을 이용하여 적혈구와 이물질을 제거시킨 후 비장세포(splenocyte)를 회수하였다. 회수한 비장세포는 1% penicillin-streptomycin, 0.5% fungizone amphotericin B와 0.3% Hepes(Sigma-Aldrich Co.)를 함유한 RPMI-1640으로 2~3회 세척하고 세포수를 5×106 cells/㎖로 조정하였다. 다음으로 비장세포 현탁액을 96-well plate(SPL Life Science, Bucheon, Kyonggi-do, Korea)의 각 well에 90 ㎕씩 분주하고 적당한 농도로 희석한 시료 10 ㎕를 첨가하여 46시간 동안 5%-CO2 incubator(Vision Scientific)에서 배양하였다. 시료의 마이토젠 활성은 CCK-8 kit 용액을 10 ㎕첨가하여 4시간 배양한 후 450 nm에서 흡광도(Sunrise, TECAN, Groedingen, Austria)를 측정하여 saline 대조군의 비장세포 증식도에 대한 상대적 활성(%)으로 나타내었다(Ishiyama M, Tominaga H, Shiga M, Sasamoto K, Ohkura Y, Ueno K. A combined assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt, neutral red and crystal violet. Biol. Pharm. Bull. 19: 1518-1520 (1996)).
BALB / c mice (6 ~ 8 weeks old, female) are orally infested and sterilized spleens are pulverized, and red blood cells and debris are removed using 0.2% NaCl and metal mesh (# 200). Splenocytes were harvested. The recovered splenocytes were washed 2-3 times with RPMI-1640 containing 1% penicillin-streptomycin, 0.5% fungizone amphotericin B and 0.3% Hepes (Sigma-Aldrich Co.), and the number of cells was 5 × 10 6 cells / ml. Adjusted. Next, spleen cell suspension was dispensed into 90 wells of each well of a 96-well plate (SPL Life Science, Bucheon, Kyonggi-do, Korea), and 10 μl of the diluted sample was added to 5% -CO for 46 hours. Incubated in 2 incubator (Vision Scientific). The mitogen activity of the sample was measured by absorbance (Sunrise, TECAN, Groedingen, Austria) at 450 nm after 4 hours of incubation for 10 hours with 10 μl of CCK-8 kit solution. (Ishiyama M, Tominaga H, Shiga M, Sasamoto K, Ohkura Y, Ueno K. A combined assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt, neutral red and crystal violet. Pharm. Bull. 19: 1518-1520 (1996)).
그 결과, 실시예 1-1(saline 대조군의 1.09배)은 실시예 2-1(1.05배)과는 유의적인 활성의 차이를 보이지 않았으나, 실시예 3-1(1.03배)보다는 유의적으로 증가된 활성을 나타내었다.
As a result, Example 1-1 (1.09-fold of the saline control group) did not show a significant difference in activity from Example 2-1 (1.05-fold), but significantly increased than Example 3-1 (1.03-fold). Activity was shown.
열수추출물로부터 분획된 조다당획분의 경우에서도 실시예 1-2의 경우에는 saline 대조군의 1.40배의 활성을 나타내어 실시예 2-2(1.35배)와는 유의차가 없었으나 실시예 3-3(1.27배)보다는 유의적으로 증가된 활성을 나타내었다(표 1).
In the case of the crude polysaccharide fraction fractionated from the hot water extract, Example 1-2 showed 1.40 times the activity of the saline control group, but there was no significant difference from Example 2-2 (1.35 times), but Example 3-3 (1.27 times) ) Showed significantly increased activity (Table 1).
1)Control: 시료를 첨가하지 않은 saline 대조군 1) Control: saline control without sample
2)LPS: lipopolysaccharide (1 ㎍/㎖, positive control) 2) LPS: lipopolysaccharide (1 ㎍ / ml, positive control)
*, p<0.05; significant difference between the control and Example, a, p<0.05; significant difference between Example 1-1 and the other hot-water Example in the column, and b, p<0.05; significant difference between Example 1-2 and the other crude polysaccharide Example in the column by Student t-test.
*, p <0.05; significant difference between the control and Example, a, p <0.05; significant difference between Example 1-1 and the other hot-water Example in the column, and b, p <0.05; significant difference between Example 1-2 and the other crude polysaccharide Example in the column by Student t-test.
<실험예 2> 마크로파지 활성Experimental Example 2 Macrophage Activity
마크로파지 lysosomal phosphatase의 활성도로 마크로파지의 활성을 측정하였다. BKW mouse(6주령, female) 복강에 3% thioglycollate medium(Sigma-Aldrich Co.)을 2 ㎖ 주입하고 48~72시간이 경과된 후에 유도된 복강 마크로파지를 회수하여 사용하였다. 회수한 마크로파지는 RPMI-1640 medium으로 세척하고 세포수가 1×106 cells/㎖이 되도록 RPMI-1640 medium에 재분산시킨 후 96-well plate의 각 well에 200 ㎕씩 분주하고 2시간 동안 배양하여 well의 기벽에 macrophage monolayer를 형성시켰다(Conrad RE. Induction and collection of peritoneal exudates macrophages. pp. 5-11. In: Manual of Macrophage Methodology. Herscowitz BH, Holden HT, Bellanti JA, Ghaffar A (eds). Marcel Dekker Incorporation, New York, NY, USA (1981)). 다음으로 상등액을 제거하고 10% FBS가 함유된 RPMI 1640-FBS로 3회 세척하여 non-adherent cell을 제거시킨 후 세척된 각 well에 RPMI 1640-FBS 180 ㎕와 20 ㎕의 시료를 분주하여 37 ℃, 5% CO2 배양기에서 24시간 배양하면서 마크로파지를 자극하였다. 상등액을 제거한 후 시료와 배양한 마크로파지에 0.1% Triton X-100(Sigma-Aldrich Co.) 25 ㎕를 가하여 세포막을 용해시켜 lysosomal phosphatase를 분비시키고 기질로서 100 mM p-nitrophenyl phosphate(Sigma-Aldrich Co., 150 ㎕)와 0.1 M citrate buffer(50 ㎕)를 같이 넣어주어 산성상태에서 반응시켰다. 시료의 마크로파지 활성은 반응 1시간 후 0.2 M borate buffer를 가하여 반응을 정지시키고 405 nm에서 ELISA reader로 흡광도를 측정하여(Suzuki I, Tanaka H, Konoshita A, Oikawa S, Osawa M, Yadomae T. Effect of orally administered beta-glucan on macrophage function in mice. Int. J. Immunopharmacol. 12: 675-684 (1990)) saline 대조군에 대한 phosphatase의 활성을 상대적인 활성(%)으로 나타내었다.
Macrophage activity was measured by the activity of lysosomal phosphatase. After injection of 2 ml of 3% thioglycollate medium (Sigma-Aldrich Co.) into the abdominal cavity of BKW mouse (6 weeks old, female), 48-72 hours later, the induced peritoneal macrophages were collected and used. The recovered macrophages were washed with RPMI-1640 medium, redispersed in RPMI-1640 medium so that the number of cells was 1 × 10 6 cells / ml, and then 200 μl of each well of the 96-well plate was incubated for 2 hours. Macrophage monolayers were formed on the base wall of (Conrad RE. Induction and collection of peritoneal exudates macrophages.pp. 5-11.In: Manual of Macrophage Methodology.Herscowitz BH, Holden HT, Bellanti JA, Ghaffar A (eds). Incorporation, New York, NY, USA (1981)). Next, the supernatant was removed and washed three times with RPMI 1640-FBS containing 10% FBS to remove non-adherent cells. Then, 180 μl and 20 μl of RPMI 1640-FBS were dispensed into each washed well at 37 ° C. , Macrophages were stimulated for 24 hours in a 5% CO 2 incubator. After removing the supernatant, 25 μl of 0.1% Triton X-100 (Sigma-Aldrich Co.) was added to the cultured macrophage and the cell membrane was dissolved to secrete lysosomal phosphatase and 100 mM p-nitrophenyl phosphate (Sigma-Aldrich Co. , 150 μl) and 0.1 M citrate buffer (50 μl) were added together and reacted in an acidic state. Macrophage activity of the sample was stopped by adding 0.2 M borate buffer after 1 hour of reaction and measuring the absorbance with an ELISA reader at 405 nm (Suzuki I, Tanaka H, Konoshita A, Oikawa S, Osawa M, Yadomae T. Effect of orally administered beta-glucan on macrophage function in mice.Int. J. Immunopharmacol. 12: 675-684 (1990)) The phosphatase activity of the saline control group was expressed as relative activity (%).
그 결과, 표 2와 같이 실시예 2-1과 실시예 3-1은 각각 saline 대조군의 1.55배와 1.54배의 활성을 보였으나, 실시예 1-1은 1.77배의 유의적으로 증가된 활성을 나타내었다. 조다당획분에서도 실시예 1-2가 1.83배의 활성을 나타내어 실시예 2-1(1.69배)과 실시예 3-3(1.62배)보다 인삼-상황버섯 균사체 발효물 조다당획분의 활성이 유의적으로 높다는 것이 확인되었다(표 2).
As a result, as shown in Table 2, Example 2-1 and Example 3-1 showed 1.55-fold and 1.54-fold activity of the saline control, but Example 1-1 showed 1.77-fold significantly increased activity. Indicated. In crude polysaccharides, Example 1-2 showed 1.83 times of activity, and thus, the activity of crude polysaccharides of ginseng-situation mushroom mycelium fermented product was significantly higher than that of Example 2-1 (1.69 times) and Example 3-3 (1.62 times). It was confirmed that it is high as a target (Table 2).
1)Control: 시료를 첨가하지 않은 saline 대조군 1) Control: saline control without sample
2)LPS: lipopolysaccharide (10 ㎍/㎖, positive control) 2) LPS: lipopolysaccharide (10 ㎍ / ml, positive control)
*, p<0.05; significant difference between the control and Example, a, p<0.05; significant difference between Example 1-1 and the other hot-water Example in the column, and b, p<0.05; significant difference between Example 1-2 and the other crude polysaccharide Example in the column by Student t-test.
*, p <0.05; significant difference between the control and Example, a, p <0.05; significant difference between Example 1-1 and the other hot-water Example in the column, and b, p <0.05; significant difference between Example 1-2 and the other crude polysaccharide Example in the column by Student t-test.
<실험예 3> Peyer's patch를 경유한 장관면역 활성Experimental Example 3 Intestinal Immunity Via Peyer's Patch
C3H/He 마우스(6~8주령, female) 복부를 절개하여 소장벽 위에 존재하는 Peyer's patch를 조심스럽게 적출한 후 HBSS 용액이 담긴 페트리디쉬에 옮겨 마쇄하고 0.2% NaCl과 금속망(# 200)을 이용하여 적혈구와 이물질을 제거시킨 후 Peyer's patch 세포액으로 조제하였다. 세포현탁액은 RPMI 1640-FBS(5% FBS 함유)로 세척하여 2×106 cells/㎖의 세포농도로 조정한 후 96 well plate에 180 ㎕씩 분주하고 적당한 농도로 희석한 시료를 20 ㎕씩 첨가하여 37 ℃, 5% CO2 배양기에서 5일간 배양하고 상등액만을 회수하여 골수세포 증식실험에 사용하였다. 한편, 골수세포는 동일 마우스의 대퇴부 뼈로부터 회수한 뒤 여과, 세척하고 세포수를 2.5×105 cells/㎖ RPMI 1640-FBS로 조정하여 100 ㎕씩 96-well plate의 각 well에 분주하고 시료와 Peyer's patch 세포의 배양에서 회수한 상등액과 RPMI 1640-FBS를 각각 50 ㎕씩 첨가하여 37 ℃, 5%-CO2 incubator에서 6일간 재배양하였다. 시료의 Peyer's patch를 경유한 장관면역 활성은 6일간 배양된 배양액에 CCK-8 kit 용액 20 ㎕를 첨가하고 4시간 반응시킨 후 450 nm에서 흡광도를 측정하여 saline 대조군의 골수세포의 증식도에 대한 상대적 활성(%)으로 나타내었다.
C3H / He mice (6-8 weeks old, female) were incised and carefully removed the Peyer's patch on the small intestine wall, then transferred to a Petri dish containing HBSS solution and ground and 0.2% NaCl and metal mesh (# 200) Red blood cells and foreign substances were removed using the Peyer's patch cell solution. The cell suspension was washed with RPMI 1640-FBS (containing 5% FBS), adjusted to a cell concentration of 2 × 10 6 cells / mL, and 180 μl were dispensed into a 96 well plate and 20 μl of the diluted sample was added. Incubated for 5 days at 37 ℃, 5% CO 2 incubator, and recovered only the supernatant was used for myeloid cell proliferation experiment. On the other hand, bone marrow cells were recovered from the femoral bones of the same mouse, filtered, washed, and adjusted to 2.5 × 10 5 cells / mL RPMI 1640-FBS, and 100 μl were dispensed into each well of a 96-well plate. 50 μl of the supernatant and RPMI 1640-FBS recovered from the culture of Peyer's patch cells were added and cultured at 37 ° C. in a 5% -CO 2 incubator for 6 days. Intestinal immune activity through Peyer's patch of the sample was added to 20 μl of CCK-8 kit solution in culture medium cultured for 6 days and reacted for 4 hours, and then absorbance was measured at 450 nm. Expressed as% activity.
Peyer's patch를 경유한 장관면역 활성에서는 실시예 2-1과 실시예 3-1은 각각 saline 대조군의 1.05배와 1.09배를, 실시예 1-1의 경우에도 1.12배의 낮은 활성을 나타내었으나, 조다당획분의 경우에는 실시예 1-2가 1.19배의 활성으로 실시예 2-2(1.10배)보다 유의적으로 증가된 활성을 보였다(표 3).
In intestinal immune activity via Peyer's patch, Example 2-1 and Example 3-1 showed 1.05-fold and 1.09-fold lower activity than saline control, and 1.12-fold lower activity in Example 1-1, In the case of polysaccharide fraction, Example 1-2 showed 1.19 times more activity than Example 2-2 (1.10 times) (Table 3).
1)Control: 시료를 첨가하지 않은 saline 대조군 1) Control: saline control without sample
2)LPS: lipopolysaccharide (10 ㎍/㎖, positive control) 2) LPS: lipopolysaccharide (10 ㎍ / ml, positive control)
*, p<0.05; significant difference between the control and Example, a, p<0.05; significant difference between Example 1-2 and the other crude polysaccharide Example in the column by Student t-test.
*, p <0.05; significant difference between the control and Example, a, p <0.05; significant difference between Example 1-2 and the other crude polysaccharide Example in the column by Student t-test.
<실시예 4 및 실시예 5-1 내지 실시예 5-6><Example 4 and Example 5-1 to Example 5-6>
인삼 또는 상황버섯 균사체 자체의 조다당획분에 비하여, 인삼-상황버섯 균사체 발효물의 조다당획분의 면역기능 증강 활성이 강한 것으로 확인되었는바, 고체배양으로 조제한 인삼-상황버섯 균사체 발효물의 조다당획분(실시예 1-2)을 물에 녹여 증류수로 평형화시킨 DEAE-Sepharose CL-6B(Amersham Biosciences, Uppsala, Sweden)가 충진된 column(Cl- form, 4.0×30.0 cm)에 loading한 후 증류수와 0.1, 0.2, 0.3, 0.4, 0.5, 1.0 및 2.0 M NaCl 용액으로 차례로 용출시키고 각 획분을 투석, 농축 및 동결건조하여 증류수 용출획분(실시예 4, DEAE loading 양 대비 수율 16.4%)과 0.1~1.0 M NaCl 농도별 용출획분인 실시예 5-1(0.1 M, 수율 13.6%), 실시예 5-2(0.2 M, 수율 13.0%), 실시예 5-3(0.3 M, 수율 12.0%), 실시예 5-4(0.4 M, 수율 9.6%), 실시예 5-5(0.5 M, 수율 5.0%) 및 실시예 5-6(1.0 M, 수율 4.2%)의 7개의 획분으로 분획하였다(도 1).
Compared to the crude polysaccharide of ginseng or situation mushroom mycelium itself, it was confirmed that the crude polysaccharide fraction of ginseng-situation mushroom mycelium fermentation had stronger immune function enhancement activity, and the crude polysaccharide fraction of ginseng-sulfur mushroom mycelium fermentation prepared by solid culture ( Example 1-2) was loaded in a column (Cl-form, 4.0 × 30.0 cm) filled with DEAE-Sepharose CL-6B (Amersham Biosciences, Uppsala, Sweden), which was dissolved in water and equilibrated with distilled water, followed by distilled water and 0.1, Elution with 0.2, 0.3, 0.4, 0.5, 1.0, and 2.0 M NaCl solutions in turn, and each fraction was dialyzed, concentrated and lyophilized to yield distilled water elution fraction (Example 4, yield 16.4% compared to DEAE loading) and 0.1 to 1.0 M NaCl. Example 5-1 (0.1 M, yield 13.6%), Example 5-2 (0.2 M, yield 13.0%), Example 5-3 (0.3 M, yield 12.0%), and Example 5 which are the elution fractions by concentration. Fractionated into seven fractions of -4 (0.4 M, yield 9.6%), Example 5-5 (0.5 M, yield 5.0%) and Example 5-6 (1.0 M, yield 4.2%) ( 1).
<실시예 6 및 실시예 7-1 내지 실시예 7-3><Example 6 and Example 7-1 to Example 7-3>
인삼 조다당획분(실시예 2-2)을 상기 실시예 4 및 실시예 5-1 내지 실시예 5-6과 동일한 칼럼에 분획하여 증류수(실시예 6, 수율 21.2%), 0.1 M NaCl(실시예 7-1, 수율 4.7%), 0.2 M(실시예 7-2, 수율 2.4%)과 0.3 M NaCl(실시예 7-3, 수율 4.4%)의 4개의 획분으로 분획하였다(도 2).
Ginseng crude polysaccharide fraction (Example 2-2) was fractionated in the same column as Example 4 and Examples 5-1 to Example 5-6, and distilled water (Example 6, yield 21.2%), 0.1 M NaCl (Example Four fractions of Example 7-1, yield 4.7%), 0.2 M (Example 7-2, yield 2.4%) and 0.3 M NaCl (Example 7-3, yield 4.4%) (FIG. 2).
<실시예 8 및 실시예 9-1 내지 실시예 9-5><Example 8 and Example 9-1 to Example 9-5>
상황버섯 균사체의 조다당획분(실시예 3-2)을 상기 실시예 4 및 실시예 5-1 내지 실시예 5-6과 와 동일한 칼럼에 분획하여 실시예 8(증류수 용출)과 0.1~0.5 M NaCl 농도별 용출획분인 실시예 9-1(0.1 M), 실시예 9-2(0.2 M), 실시예 9-3(0.3 M), 실시예 9-4(0.4 M), 실시예 9-5(0.5 M)의 6개의 획분으로 분획하였다(수율: 1.1~16.8%)(도 3).
The crude polysaccharide fraction of the situation mushroom mycelium (Example 3-2) was fractionated into the same columns as in Example 4 and Examples 5-1 to 5-6, and Example 8 (distilled water eluted) and 0.1 to 0.5 M Example 9-1 (0.1 M), Example 9-2 (0.2 M), Example 9-3 (0.3 M), Example 9-4 (0.4 M), and Example 9- Fractions were made into 6 fractions of 5 (0.5 M) (yield: 1.1-16.8%) (FIG. 3).
<실험예 4> 인삼-상황버섯 균사체 발효물 조다당획분의 분획에 따른 면역활성 변화Experimental Example 4 Changes in Immune Activity by Fractions of Crude Polysaccharide from Ginseng-Situation Mushroom Mycelia
DEAE-Sepharose CL-6B column에 의한 인삼 및 상황버섯 균사체의 조다당획분과 인삼-상황버섯 균사체 발효물 조다당획분을 분획한 결과, 인삼-상황버섯 균사체 발효물의 0.2 M과 0.3 M NaCl에서 용출된 획분인 실시예 5-2 및 실시예 5-3은 동일 농도에서 분획된 인삼의 실시예 7-2 및 실시예 7-3 또는 균사체의 실시예 9-2 및 실시예 9-3보다 산성당 검출이 많은 profile을 보여주고 있으며, 1.0 M NaCl 농도(실시예 5-6)에서도 단백질이 검출되는 peak가 용출되어 인삼 및 상황버섯 균사체의 NaCl 농도별 분획물과는 차이가 있는 DEAE profile의 물질로 구성되어 있는 특징을 보여주었다(도 1 내지 도 3). 이러한 column profile의 차이는 고체배양 중 균사체로부터 유래된 고분자 물질(다당류 또는 단백다당류 등)에 기인할 수도 있으나 발효과정 중 배양된 균사체로부터 생산된 효소 등의 다양한 수식에 의해 인삼의 고분자 물질이 전환될 수 있는 가능성도 제시하고 있기 때문에, 분획된 획분의 활성비교와 함께 구성분 및 구성당을 분석하여 고체배양을 통한 인삼-상황버섯 균사체 발효물이 인삼에서의 상황버섯 균사체의 단순배양인지 또는 본 발명에서 추구하는 균사체의 생물학적 전환에 의한 것인지의 여부를 확인하고자 하였다.
The crude polysaccharide fraction of ginseng and situation mushroom mycelium and the ginseng-condition mushroom mycelium mycelium fermented by DEAE-Sepharose CL-6B column were fractionated, and eluted from 0.2 M and 0.3 M NaCl of the ginseng-sulfur mushroom mycelia. Example 5-2 and Example 5-3, which are fractions, detected acidic sugars from Examples 7-2 and 7-3 or Mycelium of Mycelium, which were fractionated at the same concentration. It shows many profiles, and the peak of protein detection is eluted even at 1.0 M NaCl concentration (Example 5-6), which is composed of DEAE profile substance which is different from the fractions of NaCl concentrations of ginseng and mushroom mushroom mycelium. Showed a characteristic (FIGS. 1 to 3). The difference in column profile may be due to the polymer material (polysaccharide or protein polysaccharide) derived from the mycelium during solid culture, but the polymer material of ginseng may be converted by various formulas such as enzymes produced from the mycelium cultured during fermentation. The present invention also suggests that the fermented ginseng-situation mushroom mycelium is a simple culture of the situation mushroom mycelium in ginseng by analyzing the components and sugars together with the active comparison of fractions. The purpose of this study was to determine whether the mycelia were pursued by biological conversion.
비장세포의Splenocyte 마이토젠Maitozen 활성 activation
실시예 1-2, 실시예 2-2 및 실시예 3-2로부터 DEAE에 의해 분획된 모든 시료획분의 비장세포를 이용한 마이토젠 활성을 검토하였다. 도 4에서 나타낸 바와 같이 실시예 1-2로부터 분획된 0.2 M NaCl 용출획분인 실시예 5-2는 saline 대조군보다 유의적으로 가장 높은 1.98배의 마이토젠 활성을 나타내었으며, 실시예 2-2 또는 실시예 3-2에서 동일 농도로 분획된 실시예 7-2(1.60배)와 실시예 9-2(1.65배) 및 실시예 2-2 및 실시예 3-2의 DEAE로 분획된 획분 중 가장 높은 실시예 9-3(1.71배)보다도 유의적으로 높은 활성을 나타내었다. 이로써, 고체배양을 통한 인삼-상황버섯 균사체 발효물의 0.2 M NaCl 획분(실시예 5-2)은 인삼 또는 상황버섯 균사체의 동일 획분보다 마이토젠 활성이 유의적으로 증가되었음이 확인되었다(도 4).
The mitogen activity using splenocytes of all sample fractions fractionated by DEAE from Example 1-2, Example 2-2 and Example 3-2 was examined. As shown in FIG. 4, Example 5-2, which is a 0.2 M NaCl elution fraction fractionated from Example 1-2, showed significantly higher 1.98-fold mitogen activity than the saline control group, Example 2-2 or The fractions of the fractions fractionated by DEAE of Example 7-2 (1.60-fold) and Example 9-2 (1.65-fold) and Example 2-2 and Example 3-2 fractionated at the same concentration in Example 3-2 It showed significantly higher activity than the high Example 9-3 (1.71 fold). As a result, 0.2 M NaCl fraction (Example 5-2) of the ginseng-situation mushroom mycelium fermentation broth through solid culture was found to significantly increase the mitogen activity than the same fraction of the ginseng or situation mushroom mycelium (Fig. 4). .
마크로파지Macrophage 활성 activation
마크로파지 활성에서도 실시예 1-2로부터 DEAE-Sepharose CL-6B를 통하여 분획한 7개 획분 중에서 실시예 5-1(0.1 M NaCl 용출획분)과 실시예 5-2(0.2 M NaCl 용출획분)는 saline 대조군보다 유의적으로 높은 2.01배와 1.94배의 마크로파지 활성을 보여 모든 획분 중에서 가장 높은 마크로파지 활성을 보였다(도 5).In macrophage activity, Example 5-1 (0.1 M NaCl eluted fraction) and Example 5-2 (0.2 M NaCl eluted fraction) were saline out of seven fractions fractionated from DE 1-2 using DEAE-Sepharose CL-6B. Significantly higher macrophage activity of 2.01 fold and 1.94 fold than the control group showed the highest macrophage activity among all fractions (FIG. 5).
또한 실시예 5-1 및 실시예 5-2는 NaCl 동일농도에서 분획된 인삼의 실시예 7-1(1.73배)과 실시예 7-2(1.66배) 또는 균사체의 실시예 9-1(1.79배, 실시예 2-2 및 실시예 3-2의 획분 중 가장 높음)과 실시예 9-2(1.72배)보다 유의적으로 증가된 마크로파지 활성을 보였는바, 인삼-상황버섯 균사체 발효물의 활성 다당류와 인삼 또는 상황버섯 균사체의 활성 다당류가 구조적인 차이를 가지고 있을 것으로 추정되었다(도 5).
In addition, Example 5-1 and Example 5-2 are Example 7-1 (1.73-fold) and Example 7-2 (1.66-fold) or Example 9-1 (1.79) of mycelia of ginseng fractionated at the same concentration of NaCl. Active polysaccharides of ginseng-situation mushroom mycelium fermentation, which showed significantly increased macrophage activity than that of pear, Example 2-2 and Example 3-2) and Example 9-2 (1.72 fold). It was estimated that the active polysaccharides of the ginseng or the situation mushroom mycelium had a structural difference (FIG. 5).
Peyer'sPeyer's patchpatch 를 매개로 한 장관면역 활성Intestinal Immunity via Mediation
Peyer's patch를 매개로 한 장관면역 활성을 측정한 결과에서는 인삼-상황버섯 균사체 발효물의 DEAE 획분 중 0.3 M NaCl에서 분획된 실시예 5-3이 saline 대조군의 1.56배로, 인삼 또는 상황버섯 균사체의 DEAE 획분을 포함한 모든 획분들 중에서 가장 높은 활성을 나타내었다. 이러한 실시예 5-3의 장관면역 활성은 동일한 농도(0.3 M NaCl)에서 분획된 인삼의 실시예 7-3(1.12배) 및 균사체의 실시예 9-3(1.23배)보다 유의적으로 현저히 높아, 고체배양으로 조제된 인삼-상황버섯 균사체 발효물로부터 분획된 면역활성 다당획분이 인삼 또는 상황버섯 균사체로부터 분획한 획분보다 활성이 증진되었음을 나타내었다(도 6).
As a result of measuring the intestinal immune activity through the Peyer's patch, Example 5-3 fractionated from 0.3 M NaCl in the DEAE fraction of the ginseng-situation mushroom mycelium fermentation was 1.56 times the saline control, and the DEAE fraction of the ginseng or situation mushroom mycelium. It showed the highest activity among all fractions, including. The intestinal immune activity of this Example 5-3 was significantly higher than Example 7-3 (1.12 fold) of ginseng fractionated at the same concentration (0.3 M NaCl) and Example 9-3 (1.23 fold) of mycelium. It was shown that the immunoactive polysaccharide fraction fractionated from the ginseng-situation mushroom mycelium fermented product prepared by the solid culture was enhanced than the fraction fractionated from the ginseng or situation mushroom mycelium (FIG. 6).
따라서 이러한 다양한 면역활성의 결과로부터 고체배양을 통한 인삼-상황버섯 균사체 발효물 및 조다당획분의 조제와 음이온교환수지를 통한 분획은 동일조건에서 인삼 또는 상황버섯 균사체로부터 조제된 조다당 및 이로부터 분획된 동일농도의 NaCl 용출획분이나 그 외 활성 획분들보다도 면역활성이 증강된 획분을 얻을 수 있음을 나타내는 것으로 면역활성에 미치는 인삼에 대한 균사체 고체배양의 중요한 의의를 확인할 수 있었다.
Therefore, from the results of these various immune activities, preparation of ginseng-situation mushroom mycelium fermentation product and crude polysaccharide fraction through solid culture and an anion exchange resin fraction were prepared from ginseng or situation mushroom mycelium under the same conditions. Significant implications of the mycelial solid culture for ginseng on the immune activity were obtained by indicating that the fraction of NaCl eluting at the same concentration or other active fractions was obtained with enhanced immune activity.
<실험예 5> 인삼-상황버섯 균사체 발효물 조다당획분으로부터 분리된 면역활성 다당체 획분의 특성
Experimental Example 5 Characteristics of Immune Active Polysaccharide Fraction Isolated from Ginseng-Sitling Mushroom Mycelia Ferment Polysaccharide
인삼-상황버섯 균사체 발효물 조다당획분의 음이온교환수지로 분획된 다양한 획분 중 실시예 5-1, 실시예 5-2 및 실시예 5-3은 10% 이상의 높은 수율과 함께 측정한 면역활성의 종류에 따라 인삼 또는 상황버섯 균사체의 동일획분뿐만 아니라 모든 획분들과의 비교에서도 가장 높은 활성을 가지고 있음을 확인할 수 있었다. 이러한 원인이 인삼의 상황버섯 균사체 단순배양이 아닌 발효과정에서의 생물학적 전환에 의한 것임을 명백히 하기 위하여 인삼-상황버섯 균사체 발효물과 인삼 및 상황버섯 균사체 동일획분(0.2M NaCl 및 0.3M NaCl 분획) 간의 구성분과 구성당 특성을 비교분석하였다.
Among various fractions fractionated with anion exchange resins of ginseng-situation mushroom mycelium fermentation crude polysaccharide fractions, Example 5-1, Example 5-2 and Example 5-3 showed the immunological activity measured with a high yield of 10% or more. Depending on the type, it could be confirmed that it has the highest activity in comparison with all the fractions as well as the same fraction of ginseng or situation mushroom mycelium. To clarify that the cause is due to the biological conversion of the ginseng mushroom mushroom mycelium, not the simple culture of ginseng, the same fraction (0.2M NaCl and 0.3M NaCl fraction) of ginseng and S. mushroom mycelium. The composition and the properties per composition were analyzed.
획분의 구성분 분석Composition Analysis of Fractions
중성당, 산성당 및 단백질은 glucose, galacturonic acid와 bovine serum albumin(BSA)을 각각 표준물질로 사용하여 phenol-sulfuric acid법(Dubois M, Gilles KA, Hamiltonm JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28: 350-356 (1956)), m-hydroxydiphenyl법(Blumenkrantz N, Asboe-Hansen G. New method for quantitative determination of uronic acid. Anal. Biochem. 54: 484-489 (1973))과 Bio-Rad dye(Bio-Rad Laboratories Inc., Hercules, CA, USA)를 이용한 Bradford법(Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254 (1976))으로 정량하였다.
Neutral sugars, acidic sugars, and proteins are used as standard for glucose, galacturonic acid and bovine serum albumin (BSA), respectively, using the phenol-sulfuric acid method (Dubois M, Gilles KA, Hamiltonm JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances.Anal.Chem. 28: 350-356 (1956)), m-hydroxydiphenyl method (Blumenkrantz N, Asboe-Hansen G. New method for quantitative determination of uronic acid.Anal. Biochem. 54: 484 -489 (1973)) and the Bradford method (Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of) using Bio-Rad dye (Bio-Rad Laboratories Inc., Hercules, CA, USA) protein-dye binding.Anal. Biochem. 72: 248-254 (1976)).
먼저, 인삼-상황버섯 균사체 발효물로부터 분획된 활성 다당획분의 구성분 분석결과, 실시예 5-2 및 실시예 5-3은 중성당이 73.5%와 67.3%, 산성당이 23.2%와 24.6% 및 소량의 단백질(3.3%와 8.1%)이 함유되어 있어 중성당과 산성당이 주성분임을 확인할 수 있었다(표 4). 한편, 동일 NaCl 농도에서 분획된 인삼의 실시예 7-2 및 실시예 7-3은 중성당(실시예 7-2; 65.5%, 실시예 7-3; 56.2%)과 산성당(실시예 7-2; 26.0%, 실시예 7-3; 34.8%) 및 단백질(실시예 7-2; 8.5%, 실시예 7-3; 9.0%)을, 균사체의 실시예 9-2 및 실시예 9-3은 각각 중성당 77.5%와 64.9%, 산성당 14.5%와 19.6% 및 단백질 8.0%와 15.5%를 포함하는 획분임을 알 수 있었다(표 4).
First, as a result of component analysis of active polysaccharide fraction fractionated from ginseng-situation mushroom mycelium fermentation, Example 5-2 and Example 5-3 are neutral sugars 73.5% and 67.3%, acidic sugars 23.2% and 24.6% And a small amount of protein (3.3% and 8.1%) contained a neutral sugar and acidic sugar was confirmed that the main components (Table 4). On the other hand, Example 7-2 and Example 7-3 of ginseng fractionated at the same NaCl concentration are neutral sugar (Example 7-2; 65.5%, Example 7-3; 56.2%) and acidic sugar (Example 7 -2; 26.0%, Example 7-3; 34.8%) and protein (Example 7-2; 8.5%, Example 7-3; 9.0%), Example 9-2 and Example 9- of the mycelium 3 showed fractions containing 77.5% and 64.9% of neutral sugar, 14.5% and 19.6% of acidic sugar, and 8.0% and 15.5% of protein, respectively (Table 4).
특히, 다당류의 주 구성분인 중성당에서 실시예 5-2 및 실시예 5-3은 실시예 9-2 및 실시예 9-3과는 차이가 크지 않았으나, 실시예 7-2 및 실시예 7-3과는 각각 유의적으로 차이를 보였으며, 산성당에서는 0.2 M 획분(실시예 5-2)은 실시예 9-2와 유의적인 차이를 보였고, 0.3 M 분획획분(실시예 5-3)은 실시예 7-3 및 실시예 9-3 모두와 유의적인 차이를 보여주었다.
In particular, in the neutral sugar which is the main component of the polysaccharide, Example 5-2 and Example 5-3 were not significantly different from Examples 9-2 and 9-3, but Example 7-2 and Example 7 -3 and 3, respectively, in the acidic sugar 0.2 M fraction (Example 5-2) was significantly different from Example 9-2, 0.3 M fraction fraction (Example 5-3) Showed significant differences from both Example 7-3 and Example 9-3.
또한 단백질 구성 비율에 있어서도 동일획분간에 유의적인 차이를 확인할 수 있었는데 이러한 결과는 인삼의 상황버섯 균사체 발효과정에서 원료의 구성분에 전환이 일어나고 있음을 제시하는 것으로 추정하고 인삼-상황버섯 균사체 발효물의 활성 다당획분과 인삼 또는 균사체 등의 시료대조군 동일획분간의 구조적 특성을 살펴보기 위하여 시료의 alditol acetate 유도체를 조제한 후 GC로 구성당을 분석하였다.
In addition, significant differences were observed in the proportion of protein in the same fraction, and these results suggest that the conversion of the raw material components during the fermentation of the situation mushroom mycelium of ginseng was estimated. To examine the structural characteristics of the active polysaccharide fraction and the same fraction of the sample control group such as ginseng or mycelium, the alditol acetate derivatives of the samples were prepared and the constituent sugars were analyzed by GC.
*, p<0.05; significant difference between Example 1-2 and the other crude polysaccharide Example in the column, a, p<0.05; significant difference between Example 5-2 and Example 7-2/9-2, and b, p<0.05; significant difference between Example 5-3 and Example 7-3/9-3 in the column.
*, p <0.05; significant difference between Example 1-2 and the other crude polysaccharide Example in the column, a, p <0.05; significant difference between Example 5-2 and Example 7-2 / 9-2, and b, p <0.05; significant difference between Example 5-3 and Example 7-3 / 9-3 in the column.
획분의 Fractional 구성당Per composition 분석 analysis
다당획분의 구성당은 Jones와 Albersheim(Jones TM, Albersheim P. A gas chromatographic method for the determination of aldose and uronic acid constituents of plant cell wall polysaccharides. Plant Physiol. 49: 926-936 (1972))의 방법으로 분석하였는데, 먼저 시료를 2.0 M TFA(trifluoroacetic acid, Sigma-Aldrich Co.)로 121 ℃에서 1.5시간 가열하여 가수분해시킨 후 NaBH4(Sigma-Aldrich Co.)를 이용하여 각각의 구성당을 alditol로 환원시켰다. 다음으로 (CH2CO)2O(Sigma-Aldrich Co.)를 첨가하여 alditol acetate 유도체로 전환시킨 후 SP-2380 column(0.2 ㎛ film, 0.25 mm i.d×30 m, Supelco, Bellefonte, PA, USA)이 장착된 gas chromatography(Young Lin Co., Ltd., Anyang, Kyonggi-do)로 구성당을 분석하였다. GC 온도조건은 injector 250 ℃, detector 250 ℃이었고, column은 60 ℃에서 1분경과 후 30 ℃/분의 속도로 220 ℃까지 상승시켜 12분간 유지하고 다시 8 ℃/분의 속도로 250 ℃까지 상승시켜 15분을 유지하였다. 시료의 구성당은 표준당의 retention time과 비교하여 분석하였고 구성당 mol.%는 각 peak들의 면적비와 구성당 alditol acetate 유도체의 분자량으로 계산한 후 정량화 하고 구성당 비율은 molar ratio로 표시하였다(Zhao JF, Kiyohara H, Yamada H, Takemoto N, Kawamura H. Heterogeneity and characterization of mitogenic and anti-complementary pectic polysaccharides from the roots of Glycyrrhiza uralensis Fisch et DC. Carbohydr. Res. 219: 149-172 (1991)).
Polysaccharide fractions were prepared by Jones and Albersheim (Jones TM, Albersheim P. A gas chromatographic method for the determination of aldose and uronic acid constituents of plant cell wall polysaccharides.Plant Physiol. 49: 926-936 (1972)). First, the sample was hydrolyzed by heating at 1.2 ° C. for 1.5 hours with 2.0 M trifluoroacetic acid (Sigma-Aldrich Co.) and then reducing each constituent sugar to alditol using NaBH4 (Sigma-Aldrich Co.). I was. Next, (
실시예 5-2의 경우에는 uronic acid(GalA와 GlcA 등의 산성당)와 Glc, Ara, Gal 및 Rha 등의 중성당이 주요 구성당으로 함유되어 있었다(molar ratio; uronic acid : Glc : Ara : Gal : Rha = 0.81 : 1.00 : 0.49 : 0.42 : 0.28, 표 5). 반면 실시예 5-2와 동일한 0.2 M NaCl 농도에서 분획된 실시예 7-2는 실시예 5-2와 산성당의 구성 비율은 유사하였으나 중성당의 주요 구성당 분포와 그 molar ratio에 있어서는 크게 차이를 보였으며(molar ratio; uronic acid : Ara : Glc : Gal : Rha = 0.73 : 1.00 : 0.34 : 0.31 : 0.22), 실시예 9-2와는 전혀 다른 구성당 분포(molar ratio; uronic acid : Glc : Man : Xyl = 0.26 : 1.00 : 0.30 : 0.19)를 나타내고 있음을 확인할 수 있었다(표 5).
In Example 5-2, uronic acid (acidic sugars such as GalA and GlcA) and neutral sugars such as Glc, Ara, Gal, and Rha were included as the major constituent sugar (molar ratio; uronic acid: Glc: Ara: Gal: Rha = 0.81: 1.00: 0.49: 0.42: 0.28, Table 5). On the other hand, Example 7-2 fractionated at the same concentration of 0.2 M NaCl as Example 5-2 had a similar composition of acidic sugars as Example 5-2, but showed a large difference in the distribution of the major constituents of neutral sugar and its molar ratio. (Molar ratio; uronic acid: Ara: Glc: Gal: Rha = 0.73: 1.00: 0.34: 0.31: 0.22), and the molar ratio (uronic acid: Glc: Man: Xyl) was completely different from Example 9-2. = 0.26: 1.00: 0.30: 0.19) was confirmed (Table 5).
그러므로 실시예 5-2는 uronic acid와 Rha가 주요 구성당으로 함유되어 있고 Ara와 Gal의 중성당이 분포하고 있는 결과로부터, 인삼 유래의 pectic polysaccharide인 것으로 생각되며 arabinan, galactan 또는 arabinogalactan 등이 펙틴 주쇄에 측쇄로 결합되어 있을 가능성을 보여주는 것으로 생각된다. 또한 실시예 5-2는 중성당인 Glc가 주성분을 이루고 있고(28.80 mol.%) Gal과 Man(12.21과 6.07 mol.%)도 상당량 포함되어 있으며, 상황버섯 균사체 일반발효물의 실시예 9-2 획분에서도 Glc와 Man(54.81 및 16.65 mol.%)가 주요 구성당으로 분포하고 있음을 알 수 있었는데(표 5), 이러한 결과는 실시예 5-2의 구성 다당류로서 위에서 언급한 펙틴 다당류 이외에도 중성다당류가 관여할 수 있는 가능성을 보여주는 것으로 생각된다. 즉, 이러한 중성다당류는 인삼에서의 균사체 발효과정 중 인삼의 전분-유사 다당류 또는 인삼에 생육한 균사체에서 생산되는 glucan 또는 glycan 다당류로부터 그 자체로 또는 균사체의 계속적인 변화과정을 통하여 기인된 다당류로 생각된다. 한편, 인삼에는 일반적으로 상당한 양의 전분이 함유되어 있는 것으로 보고되고 있으나, 요오드시험 및 glucan분석을 통하여 실시예 5-2에 함유된 Glc가 전분 유래가 아님은 확인할 수 있었다(결과는 도시하지 않음).Therefore, Example 5-2 is considered to be a pectic polysaccharide derived from ginseng, containing uronic acid and Rha as major constituent sugars, and the distribution of neutral sugars of Ara and Gal, and arabinan, galactan, or arabinogalactan, etc. It is thought to show the possibility of being bound side by side. In addition, Example 5-2 is a main component of the neutral sugar Glc (28.80 mol.%) And contains a considerable amount of Gal and Man (12.21 and 6.07 mol.%), Example 9-2 fractions of the common mushroom mycelium mycelium It was also found that Glc and Man (54.81 and 16.65 mol.%) Were distributed as the major constituent sugars (Table 5). It seems to show the possibility of involvement. In other words, these neutral polysaccharides are thought to be polysaccharides derived from the glucan or glycan polysaccharides produced by the starch-like polysaccharides of ginseng or the mycelium grown in ginseng during the mycelial fermentation process, or through the continuous change of the mycelium. do. On the other hand, ginseng is generally reported to contain a significant amount of starch, it was confirmed through the iodine test and glucan analysis that Glc contained in Example 5-2 was not derived from starch (results not shown) ).
한편, Peyer's patch를 경유한 장관면역 활성에서 가장 높은 활성을 나타내었으며 마이토젠 활성도 높았던 인삼-상황버섯 균사체 발효물 실시예 5-3의 구성당을 분석한 결과(molar ratio; uronic acid : Ara : Rha : Gal : Xyl : Glc = 1.00 : 0.75 : 0.69 : 0.63 : 0.42 : 0.34, 표 5), 위에서 언급한 대부분 면역활성에서 공통적으로 높았던 실시예 5-2(uronic acid : Ara : Rha : Gal : Xyl : Glc = 0.81 : 0.49 : 0.28 : 0.42 : 0.20 : 1.00)와 중성당 구성 비율(특히 Glc, Rha와 Xyl)에서 큰 차이를 나타냄으로써 2개의 활성획분이 다른 다당류일 가능성을 보여주었다. 0.3 M NaCl의 동일 농도에서 분획된 인삼의 실시예 7-3은(uronic acid : Ara : Rha : Gal = 1.00 : 0.91 : 0.45 : 0.36) 활성획분인 실시예 5-3보다 산성당 및 중성당인 Ara의 비율이 매우 높았고 Xyl와 Glc는 감소한 구성당 비율을 나타내었으며, 상황버섯 균사체의 실시예 9-3(uronic acid : Glc : Gal = 0.37 : 1.00 : 0.28)과는 전혀 다른 구성당 분포를 나타내었다(표 5). 구성당 분석결과, 실시예 5-3의 경우에도 동일 NaCl 농도에서 분획된 시료대조군 획분과는 다당류 구성당 분포와 비율이 다름을 알 수 있었으며, 실시예 5-2와는 달리 실시예 5-3(Glc 8.44 mol.% 함유)의 경우에는 glucan-유사 다당류보다는 pectic polysaccharide가 면역활성에 주로 관여하고 있는 것으로 생각된다. 또한 실시예 5-3의 pectic polysaccharide는 실시예 5-2와는 다른 구성당 비율을 나타내었는데, 산성당과 Rha의 비율이 높은 것으로부터 주쇄인 rhamnogalacturonan 영역(RG-I)이 더 많이 존재하는 것으로 보이며 중성당 측쇄에서도 Ara, Gal 이외에 Xyl 등의 비율이 증가하고 있음을 알 수 있었다(표 5).
On the other hand, the result of analysis of the constituent sugar of Example 5-3 of ginseng-situation mushroom mycelium fermented product showed the highest activity in the intestinal immunity through Peyer's patch and high mitogen activity (molar ratio; uronic acid: Ara: Rha : Gal: Xyl: Glc = 1.00: 0.75: 0.69: 0.63: 0.42: 0.34, Table 5), Example 5-2 (uronic acid: Ara: Rha: Gal: Xyl: Glc = 0.81: 0.49: 0.28: 0.42: 0.20: 1.00) and neutral sugar composition ratios (particularly Glc, Rha and Xyl) showed that the two active fractions were different polysaccharides. Example 7-3 of ginseng fractionated at the same concentration of 0.3 M NaCl (uronic acid: Ara: Rha: Gal = 1.00: 0.91: 0.45: 0.36) was an acidic and neutral sugar of Ara than active fraction of Example 5-3 The ratio of was very high and the ratio of Xyl and Glc was decreased per composition, and the distribution per composition was completely different from that of Example 9-3 (uronic acid: Glc: Gal = 0.37: 1.00: 0.28) of the situation mushroom mycelium. (Table 5). As a result of the composition analysis, it was found that even in Example 5-3, the distribution and the ratio of the polysaccharide composition were different from those of the sample control fraction fractionated at the same NaCl concentration, and, unlike Example 5-2, Example 5-3 ( In the case of Glc 8.44 mol.%), Pectic polysaccharides rather than glucan-like polysaccharides are thought to be mainly involved in immune activity. In addition, the pectic polysaccharide of Example 5-3 exhibited a different constituent sugar ratio than that of Example 5-2, and since the ratio of acidic sugar and Rha was high, it appeared that there was more rhamnogalacturonan region (RG-I). In the neutral sugar side chain, the ratio of Xyl and the like in addition to Ara and Gal was increased (Table 5).
이와 같이 인삼-상황버섯 균사체 발효물부터 음이온교환수지를 이용하여 분획된 인삼-상황버섯 균사체 발효물의 활성 획분과 NaCl 동일 농도에서 분획된 인삼 또는 상황버섯 균사체 획분의 구성분 및 구성당을 비교분석한 결과, 인삼에 대한 상황버섯 균사체의 발효과정은 인삼에 상황균사체가 단순하게 배양되는 것이 아닌 인삼에 대한 균사체의 생물학적 전환이 유도되고 있는 것으로 확인되었다.
Thus, the active fractions of the ginseng-situation mushroom mycelium fermentation fraction and the ginseng or sapling mycelium mycelium fraction fractionated at the same concentration of NaCl were compared and analyzed. As a result, it was confirmed that the fermentation process of the situation mushroom mycelium on ginseng induced a biological conversion of the mycelium on ginseng, rather than the culture of the situation mycelium on ginseng.
본 발명은 면역기능을 증진시키기 위하여 조제한 인삼의 상황버섯 균사체 발효물로부터 분리된 다당류를 유효성분으로 포함하는 면역활성용 조성물에 대한 것이다. 본 발명의 조성물은 체내 면역을 증진시키는바, 건강증진에 유용하며 건강기능식품, 건강보조식품 등으로 이용될 수 있다.
The present invention relates to a composition for immunological activity comprising a polysaccharide isolated from the fermented product of the situation mushroom mycelium of ginseng prepared to enhance immune function. The composition of the present invention is to improve the body immunity, useful for health promotion and can be used as a health functional food, health supplements and the like.
Claims (9)
Immune active food composition comprising a mycelium mushroom mycelium fermented product of ginseng as an active ingredient.
상기 인삼의 상황버섯 균사체 발효물은 다당류를 포함하는 것을 특징으로 하는 면역활성용 식품 조성물.
The method of claim 1,
The situation mushroom mycelium fermented product of the ginseng is an immunoactive food composition comprising a polysaccharide.
상기 인삼은 수삼, 홍삼, 장뇌삼 및 산삼으로 구성되는 군으로부터 선택되는 것을 특징으로 하는 면역활성용 식품 조성물.
The method of claim 1,
The ginseng is immune active food composition, characterized in that selected from the group consisting of ginseng, red ginseng, camphor ginseng and wild ginseng.
상기 인삼의 상황버섯 균사체 발효물은, 인삼을 상황버섯 균사체로 배양한 발효물의 열수추출물로부터 분리한 획분인 것을 특징으로 하는 면역활성용 식품 조성물.
The method of claim 1,
The ginseng mushroom mycelium fermentation product of the ginseng is a fraction separated from the hot water extract of the fermentation cultured with ginseng mushroom mycelium ginseng.
상기 인삼의 상황버섯 균사체 발효물은, 인삼을 상황버섯 균사체로 배양한 발효물의 열수추출물의 조다당획분으로부터 분리한 획분인 것을 특징으로 하는 면역활성용 식품 조성물.
The method of claim 1,
The ginseng mushroom mycelium fermentation product of the ginseng is an immunoactive food composition, characterized in that the ginseng is separated from the crude polysaccharide of the hot water extract of the fermentation cultured with the situation mushroom mycelium.
상기 인삼의 상황버섯 균사체 발효물은, 인삼을 상황버섯 균사체로 배양한 발효물의 조다당 획분을 음이온 교환수지로 분획한 획분인 것을 면역활성용 식품 조성물.
The method of claim 1,
The ginseng mushroom mycelium fermentation product of the ginseng is a fraction obtained by dividing the crude polysaccharide fraction of the fermentation product cultured with ginseng mushroom mycelium into an anion exchange resin.
상기 식품 조성물은 정제, 경질 또는 연질 캅셀제, 액제 또는 현탁제의 형태인 것을 특징으로 하는 면역활성용 식품 조성물.
The method of claim 1,
The food composition is an immunoactive food composition, characterized in that the form of tablets, hard or soft capsules, solutions or suspensions.
상기 식품은 건강기능식품, 건강보조식품, 기능성식품 또는 영양제인 것을 특징으로 하는 면역활성용 식품 조성물.
The method of claim 1,
The food is an immunologically active food composition, characterized in that the health functional food, health supplement food, functional food or nutrients.
A method for preparing an immunoactive food composition comprising fermenting ginseng into a situation mushroom mycelium to prepare a fermented product.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104256606A (en) * | 2014-10-11 | 2015-01-07 | 哈尔滨艾博雅食品科技开发有限公司 | Cordyceps militaris chewable tablet and manufacturing method thereof |
| KR101488434B1 (en) * | 2012-06-29 | 2015-02-04 | 한국교통대학교산학협력단 | Immune-enhancing composition including inonotus obliquus extract |
| KR20160046206A (en) * | 2014-10-20 | 2016-04-28 | 주식회사 어람 | The fermenting method of Codonopsis lanceolata for enhancing recognition ability improvement activity of Codonopsis lanceolata |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR101488434B1 (en) * | 2012-06-29 | 2015-02-04 | 한국교통대학교산학협력단 | Immune-enhancing composition including inonotus obliquus extract |
| CN104256606A (en) * | 2014-10-11 | 2015-01-07 | 哈尔滨艾博雅食品科技开发有限公司 | Cordyceps militaris chewable tablet and manufacturing method thereof |
| KR20160046206A (en) * | 2014-10-20 | 2016-04-28 | 주식회사 어람 | The fermenting method of Codonopsis lanceolata for enhancing recognition ability improvement activity of Codonopsis lanceolata |
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