KR20030021673A - Improved methods of mycelial culture of Phellinus linteus KCTC 10055BP for increasing anti-oxidanic effects - Google Patents
Improved methods of mycelial culture of Phellinus linteus KCTC 10055BP for increasing anti-oxidanic effects Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A01G18/00—Cultivation of mushrooms
- A01G18/10—Mycorrhiza; Mycorrhizal associations
Abstract
Description
본 발명은 상황버섯 균사체 배양물의 항산화 및 노화방지 효과를 상승시키기 위하여 천연물 을 이용하여 증강된 항산화 효과가 있는 상황버섯 균사체 제조에 그 목적을 둔다.The present invention aims to produce a situation mushroom mycelium having an enhanced antioxidant effect using natural products to increase the antioxidant and anti-aging effect of the situation mushroom mycelium culture.
상황버섯의 새로운 기능성New functionality of the situation mushroom
상황버섯(Phellinus linteus)은 대표적인 약용버섯 중의 하나로 항보체 활성(송치현 외 1998균학회지26:86-90), B-임파구 활성(Song et. al. 1995.Chem. Pharm. Bull.43:2105-2108), Phellinus linteus ( Phellinus linteus ) is one of the representative medicinal mushrooms. Anti-complement activity (Song Chi-hyun et al. 1998 Korean Journal of Culture Journal 26: 86-90), B-lymphocyte activity (Song et. Al. 1995. Chem. Pharm. Bull. 43: 2105- 2108),
체액 및 세포성 면역 활성(Kim et. al. 1996.Int. J. Immunopharmac. 18:295-303), 암 전이 방지효과(Han et. al. 1999.Immunopharmacol. 41:157-164) 등 상황버섯 균사체 및 균사배양액으로부터 생산된 다당체의 면역증강 및 암의 예방에 관련된 연구들이 주를 이뤄왔다. 최근 상황버섯의 다양한 기능성에 관한 연구가 진행되어 항혈당 효과(Kim et. al. 2001.J. Microbiol. Biotechnol. 11:167-171) 및 항산화 효과(특허출원번호 10-2000-0054321)등 면역증강 효과 이외의 다양한 기능성이 밝혀졌다.Humoral and cellular immune activity (Kim et. Al. 1996. Int. J. Immunopharmac . 18: 295-303), anticancer effect (Han et. Al. 1999. Immunopharmacol . 41: 157-164) Research has been focused on the immunity enhancement and the prevention of cancer of polysaccharides produced from mycelia and mycelium broth. Recently, studies on various functionalities of the situation mushrooms have been conducted, such as anti-glycemic effects (Kim et. Al. 2001. J. Microbiol. Biotechnol . 11: 167-171) and antioxidant effects (patent application No. 10-2000-0054321) Various functionalities other than enhancement effects have been found.
항산화 효과Antioxidative effect
생명유지에 절대적으로 필요한 산소가 체내 효소계, 환원대사, 화학약품, 공해물질, 광화학반응 등 각종 물리적 화학적요인에 의해 반응성이 매우 큰 활성산소(수퍼옥사이드 라디칼, 하이드록실 라디칼 및 과산화 수소)로 전환되는데 이것이 생체에 치명적인 산소독성을 일으킨다. 즉 활성산소는 세포구성 성분들인 지질, 단백질, 당, DNA 등에 파괴작용을 함으로써, 암을 비롯하여 각종 질환과 노화를 일으키는 것으로 알려져 있는데 이러한 활성산소를 소거하는 기작이 항산화 활성이다. 지금까지 개발되고 사용되고 있는 항산화제로는 토코페롤, 카로틴, 탄닌 등이 있으며, 천연물 추출물에는 산사 등이 알려져 있다. 이렇듯 개별적으로 보유하고 있는 항산화 및 노화방지효과를 생물공학적 배양법을 이용한다면 항산화 상승효과를 얻을 수 있는데 본 기술의 의의를 둔다Oxygen, which is absolutely necessary for life support, is converted into highly reactive reactive oxygen (superoxide radical, hydroxyl radical and hydrogen peroxide) by various physical chemical factors such as enzyme system, reducing metabolism, chemicals, pollutants and photochemical reaction. This causes deadly oxygen toxicity to the living body. That is, active oxygen is known to cause various diseases and aging including cancer by destroying lipids, proteins, sugars, DNA, and the like, which is an antioxidant activity. Antioxidants that have been developed and used so far include tocopherol, carotene, tannin, and the like. The anti-oxidation and anti-aging effects of these individual compounds can be obtained by using biotechnological cultivation methods.
본 발명자들은 상황버섯이 가지고 있는 항산화 및 노화방지능력을 배가시키기 위해 여러 가지 천연물 추출액을 대상으로 하여 연구하였다. 이 중 산사와 산사육를 배지에 첨가하여 상황버섯 균사체을 배양한 결과 각각 독립적인 형태에 비해 최대 21배의 항산화 및 노화방지 효과의 증가를 나타냈기에 본 발명을 완성하게 되었다.The present inventors studied various natural product extracts to double the antioxidant and anti-aging ability of the situation mushroom. Among them, the cultivated mycelium mushroom mycelium was added to the medium and mountain cultivation medium to increase the antioxidant and anti-aging effect of up to 21 times as compared to the independent form was completed the present invention.
본 발명에 사용되는 상황버섯 균사체는 발명자들이 경주지역에서 분리하여 배양하고 부산대학교 미생물학과에 의뢰하여Phellinus linteus로 동정 받았으며(정지원 등. 1999.Kor. J. Mycol. 27:124-131) 생명공학연구소에 유전자은행(KCTC)에 기탁한Phellinus linteusKCTC 10055BP이다. 상황버섯Phellinus linteusKCTC 10055BP의 최적 배양 온도 및 pH는 각각 30℃와 pH 5.0∼6.0 이며, 상황 균사체 배양에 사용한 배지는 YMG이다(조성 : 효모추출물 0.4%, 맥아추출물 1%, 포도당 0.4%).The situation mushroom mycelium used in the present invention was identified as Phellinus linteus by the inventors, isolated from the Gyeongju area, and commissioned by the Department of Microbiology, Pusan National University (Jung Ji-won et al. 1999. Kor. J. Mycol . 27: 124-131) Phellinus linteus KCTC 10055BP, which was deposited in the KCTC at the Institute. The optimum culture temperature and pH of the Phellinus linteus KCTC 10055BP were 30 ° C. and pH 5.0-6.0, respectively. The medium used for cultivating the mycelia was YMG (composition: 0.4% yeast extract, 1% malt extract, 0.4% glucose).
본 발명은 발명자들이 분리한 상황버섯Phellinus linteusKCTC 10055BP가 산사 및 산사육이 첨가된 새로운 배지에서 나타내는 증가된 항산화 및 노화방지 효능에 관한 것이다.The present invention relates to the increased antioxidant and anti-aging effects of the Phellinus linteus KCTC 10055BP isolated from the inventors in new medium supplemented with hawthorn and hawthorn.
본 발명의 배지에 첨가되는 산사 및 산사육는 일반 한약상에서 건조체를 입수하여 사용하였다. 이를 통상적인 열수추출법을 이용하여 1-2 시간동안 열수추출하여 추출물을 제조하였다. 이렇게 제조된 추출물은 여과지로 걸러 여과한 후, 감압농축기를 이용하여 10배 감압 농축하여 농축액을 제조하였다. 이를 24시간 냉동시킨 후 동결건조기를 이용하여 시료분말을 제조하였다. 이렇게 제조된 산사 및 산사육 시료분말은 YMG배지에 0.1% 첨가하여 상황버섯 균사체 배양용 배지를 제조하였다.Sansa and mountain breeding added to the medium of the present invention was obtained by using a dry body in a general Chinese medicine. The extract was prepared by hot water extraction for 1-2 hours using a conventional hot water extraction method. The extract thus prepared was filtered through a filter paper, and then concentrated under reduced pressure using a vacuum condenser 10 times to prepare a concentrate. After freezing for 24 hours, a sample powder was prepared using a lyophilizer. Thus prepared hawthorn and mountain cultivation sample powder was added to the YMG medium 0.1% to prepare a culture medium for mushroom mycelium.
제조된 배지를 121℃, 1.5기압하에서 20분간 멸균시킨 후 하온시킨다. 하온 후 미리 배양하여 준비해둔 상황버섯 균사체를 약 5%(v/v) 좁정하여 15일간 상기 최적 조건에서 액체 심부 배양한다. 배양된 상황버섯 균사체는 균질기(Homogenizer)를 이용하여 약 5분간 균질화 한 후 원심분리하여 상등액을 취한다. 얻어진 상등액으로 항산화 및 노화방지작용 시험을 수행하였으며, 수행한 샘플의 종류는 표 1과 같다.The prepared medium is sterilized at 121 ° C. under 1.5 atm for 20 minutes and then cooled. After warming, the situation mushroom mycelium prepared by incubating in advance is narrowed to about 5% (v / v) and cultured deep in the liquid at the optimum conditions for 15 days. The cultured mushroom mycelium is homogenized for about 5 minutes using a homogenizer and centrifuged to take a supernatant. Antioxidant and anti-aging tests were performed with the obtained supernatant, and the types of samples performed are shown in Table 1.
실험예 1 : 항산화 효과 검정 시험Experimental Example 1 Antioxidant Effect Assay
본 발명의 항산화 효과 검정 시험은 Winterbourn(1975.J. Lab. Clin. Med.85:337)의 방법에 준하여 실시하였다. 3 ㎖ 시험관에 0.1 M EDTA(0.3 mM cyanide 포함) 0.2 ㎖, 1.5 mM NBT 0.1 ㎖, 40 mM Tris 완충액(buffer) 2.6 ㎖ 및 샘플 0.1 ㎖을 넣고 약 5분간 안정화 시킨 다음 0.12 mM 리보플라빈 0.05 ㎖을 넣고 1,000 lux의 백열등을 조사한다. 1분에 1번씩 560 ㎚에서 흡광도 값을 10회 측정하고 평균값을 구하여 1분간 변화하는 흡광도 값을 구한다. 이에 이용한 공식은 다음과 같으며, 실험결과는 그림 1과 2에 나타낸 바와 같다.The antioxidant effect assay test of the present invention was carried out according to the method of Winterbourn (1975. J. Lab. Clin. Med. 85: 337). Add 0.2 ml of 0.1 M EDTA (with 0.3 mM cyanide), 0.1 ml of 1.5 mM NBT, 2.6 ml of 40 mM Tris buffer and 0.1 ml of sample in a 3 ml test tube, stabilize for about 5 minutes, add 0.05 ml of 0.12 mM riboflavin Incandescent lamp of 1,000 lux. Once per minute, the absorbance value is measured 10 times at 560 nm, and the average value is obtained to determine the absorbance value that changes for 1 minute. The formula used is as follows, and the experimental results are shown in Figure 1 and 2.
Unit = (대조군의 1분간 Δ흡광도 560 ㎚ /시료의 1분간 Δ흡광도 560 ㎚)-1Unit = (Δabsorbance 560 nm for 1 min. Of control / Δ absorbance 560 nm for 1 min. Of sample) -1
그림 1. 상황(PL-C), 산사(CP01) 및 산사가 첨가된 상황버섯 균사체추출액(PL-CP01)의 항산화 효과Figure 1.Antioxidative Effect of Phellinus linteus Mycelium Extract (PL-CP01)
그림 2. 상황(PL-C), 산사육(CP02) 및 산사육이 첨가된 상황버섯 균사체 추출액(PL-CP02)의 항산화 효과Figure 2. Antioxidative Effect of Pseudomonas Mushroom Mycelium Extract (PL-CP02) with Phenomena (PL-C), Acid Breeding (CP02) and Acid Breeding
산사 및 산사육 추출물 과 기존의 방법으로 배양된 상황균사체 추출물에서도 항산화 및 노화방지 효과를 관찰 할 수 있었으나, 산사 및 산사육 추출물이 첨가된 배지에서 생장된 상황버섯 균사체는 최소 3배에서 최대 21배까지 항산화 및 노화방지 상승효과를 관찰 할 수 있었다. 따라서 상황버섯의 항산화 및 노화방지 효과를 극대화시키기 위해서는 산사 및 산사육 추출물을 첨가하는 것이 본 발명의 효과이다.Antioxidant and anti-aging effects were observed in hawthorn and hawthorn extracts and cultivated mycelium mycelium cultured by conventional methods. And anti-aging synergistic effect could be observed. Therefore, in order to maximize the antioxidant and anti-aging effect of the situation mushrooms, the addition of the hawthorn and mountain breeding extract is the effect of the present invention.
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| KR101599697B1 (en) * | 2015-09-15 | 2016-03-04 | 주식회사 일심바이오 | Method for manufacturing culture medium of sparassis crispa using antioxidant and amino acid |
| KR101599699B1 (en) * | 2015-09-15 | 2016-03-04 | 주식회사 일심바이오 | Method for manufacturing culture medium of sparassis crispa using antioxidantm, amino acid and vitamin |
| KR20160043241A (en) * | 2014-10-13 | 2016-04-21 | 농업회사법인자연생명대체의학(주) | Agricultural products and the mycelium mixtures are fermented food composition for anticancer, antidiabetic, and immune function and method of preparing the same |
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| DE4325532A1 (en) * | 1993-07-29 | 1995-02-02 | Schwabe Willmar | Preparations of Crataegus species, medicinal products and their use for the prevention of sudden cardiac death and reperfusion-related cardiovascular damage |
| KR0152692B1 (en) * | 1994-10-27 | 1998-09-15 | 안영남 | Method of cultivating phellinus linteus |
| KR100570120B1 (en) * | 1998-12-30 | 2006-07-27 | 주식회사 코리아나화장품 | Free radical scavenging cosmetics containing hawthorn extract |
| KR100338318B1 (en) * | 1999-09-15 | 2002-05-27 | 김천환 | Culturing method of cordyceps spp |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20160043241A (en) * | 2014-10-13 | 2016-04-21 | 농업회사법인자연생명대체의학(주) | Agricultural products and the mycelium mixtures are fermented food composition for anticancer, antidiabetic, and immune function and method of preparing the same |
| KR101855421B1 (en) * | 2014-10-13 | 2018-07-20 | 농업회사법인자연생명대체의학(주) | Agricultural products with a pharmacological action and the mycelium mixtures are fermented food composition method of preparing the same |
| KR101599697B1 (en) * | 2015-09-15 | 2016-03-04 | 주식회사 일심바이오 | Method for manufacturing culture medium of sparassis crispa using antioxidant and amino acid |
| KR101599699B1 (en) * | 2015-09-15 | 2016-03-04 | 주식회사 일심바이오 | Method for manufacturing culture medium of sparassis crispa using antioxidantm, amino acid and vitamin |
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