TWI395813B - Manufacturing mmethod for obtaining polysaccharide from the solid fermentation product of antrodia camphorate - Google Patents
Manufacturing mmethod for obtaining polysaccharide from the solid fermentation product of antrodia camphorate Download PDFInfo
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Description
本發明係關於一種粗多醣體之製備方法，特別係關於一種萃取自牛樟芝(Antrodia camphorata )菌絲體固態醱酵產物之粗多醣體之製備方法。The present invention relates to a method for preparing a crude polysaccharide, and more particularly to a method for preparing a crude polysaccharide extracted from a solid fermentation product of Antrodia camphorata mycelium.
牛樟芝(Antrodia camphorata )，又稱樟芝或牛樟菇等，於分類學上屬於真菌界(Fungi)、擔子菌門(Basidiomycota)、擔子菌亞門(Basidiomycotina)、同擔子菌綱(Homobasidiomycetes)、無褶菌目(Aphyllophorales)、多孔菌科(Polyporaceae)、薄孔菌屬(Antrodia)，牛樟芝子實體生長於台灣保育類牛樟樹(Cinnamoum kanehirai Hay)之心材內壁，週邊向內彎曲，能分解木材之纖維素而造成木頭腐朽，為木頭腐朽菌。野生牛樟芝生長環境均屬於幽暗、潮濕且溫度較低之中海拔地區，特別是野生牛樟芝生長緩慢，因此產生子實體需要相當長的時間。由於牛樟樹為保育類樹種且分布數量極為稀少，再加上人為的盜採使得野生牛樟芝數量相當稀少，使得牛樟芝價格昂貴，所以具有「森林中的紅寶石」之美稱。 Antrodia camphorata , also known as Antrodia camphorata or Burdock mushroom, belongs to the fungi community (Fungi), Basidiomycota, Basidiomycotina, and Homobasidiomycetes. Aphyllophorales, Polyporaceae, Antrodia, Antrodia camphorata grown on the inner wall of the heartwood of Cinnamoum kanehirai Hay in Taiwan, the periphery is curved inward and can be decomposed The cellulose of wood causes the wood to decay and is a decaying wood. The growth environment of wild Antrodia camphorata belongs to the dark, humid and low temperature middle altitude areas, especially the wild Antrodia camphora grows slowly, so it takes a long time to produce fruiting bodies. Because the burdock tree is a kind of conservation tree species and the distribution is extremely rare, coupled with the artificial piracy, the number of wild burdock is quite rare, making the burdock an expensive price, so it has the reputation of "the ruby in the forest".
牛樟芝子實體為多年生，無柄且為木栓質至木質，具有濃厚的樟樹香氣，形態多變，有板狀、鐘狀、馬蹄狀或塔狀，緊貼於木材表面，菇體表面孔狀，初生時鮮紅色，隨著生長逐變為乳白色、淡紅褐色、淡褐色或淡黃褐色，週邊常呈放射狀，並往四週擴展生長，呈半圓狀或不規則形。The body of Ox sylvestris is perennial, sessile and cork-to-wood. It has a strong aroma of eucalyptus and has a variety of shapes. It has a plate shape, a bell shape, a horseshoe shape or a tower shape. It is close to the surface of the wood and has a pore shape on the surface of the mushroom. It is bright red at the beginning of life, and becomes milky white, reddish brown, light brown or yellowish brown as the growth progresses. The periphery is often radial and spreads to the periphery, showing a semicircular or irregular shape.
有些牛樟芝的種類被發現具有藥理活性，在台灣民俗醫學療法上，牛樟芝子實體被認為具有某些醫藥功效，將該子實體細磨成乾燥粉末或以熬煮方式口服，為民間流傳具療效的食藥菇類，具有解毒、抗發炎、抗腫瘤、抑制血管增生及治療肝臟疾病等活性。Some species of Antrodia camphorata have been found to have pharmacological activity. In Taiwan folk medicine therapy, the body of A. sylvestris is considered to have certain medical effects. The fruiting body is finely ground into a dry powder or taken orally in a boiled way, which is effective for folk circulation. The edible mushroom has the activities of detoxification, anti-inflammatory, anti-tumor, inhibition of vascular proliferation and treatment of liver diseases.
由於上述得知牛樟芝具藥理活性，市面上已不乏牛樟芝系列之保健產品。目前市面上販售的牛樟芝相關保健產品，製程係由牛樟芝子實體得一菌絲體進行醱酵，主要以食用該菌絲體達保健功效。但是，牛樟芝係為一成份複雜之腐朽菌，有研究指出在人工培養篩選牛樟芝菌絲體時，會篩選到致癌的菌株，表示牛樟芝並非所有成份食用後皆有益於人體健康，也有可能含有某些毒素，若貿然食用反而會影響健康，產生無法預期的副作用，而喪失保健的功效。再者，若要破壞牛樟芝內非藥理活性物質對健康的危害，會選擇經一高溫加熱步驟以確保食用上的安全，然而該高溫加熱步驟亦可能使牛樟芝內有效成份之活性喪失，而失去牛樟芝藥理活性的價值，故找出且分離一具功效且安全的牛樟芝菌絲體菌株(分離株)是為一重要課題。As a result of the above-mentioned knowledge of the pharmacological activity of Antrodia camphorata, there is no shortage of health products of the Antrodia camphorata series. At present, the related health products of Niuzhizhi sold in the market are processed by a mycelium from the body of the burdock. The main purpose is to eat the mycelium for health care. However, Antrodia camphorata is a complex rot fungus. Some studies have indicated that when cultured and screened for the mycelium of Antrodia camphorata, the carcinogenic strains will be screened, indicating that not all ingredients are beneficial to human health after eating, and may also contain certain Toxins, if consumed rashly, can affect health, produce unpredictable side effects, and lose health care. Furthermore, in order to destroy the health hazard of non-pharmacological active substances in Antrodia camphorata, a high-temperature heating step is selected to ensure the safety of eating. However, the high-temperature heating step can also lose the activity of the active ingredients in Antrodia camphorata, and lose the Antrodia camphorata. The value of pharmacological activity, so finding and isolating an effective and safe strain of mycelium of A. angustifolia (isolated strain) is an important issue.
目前從許多關於牛樟芝的研究中，已知牛樟芝成份非常複雜，發現牛樟芝子實體中含有獨特且不同骨架形成之三萜類化合物，也有研究發現牛樟芝成份含有多醣體。關於多醣體與調節免疫反應的機制研究指出，多醣體之結構會快速活化T淋巴抗原呈現細胞(antigen-presenting cells)，而具有提升免疫力、抗腫瘤、抗發炎及抑菌等活性。若能從牛樟芝萃取物中找到具藥理活性的多醣體來促成提升免疫力，且能將該多醣體以人工培養方式大量培養獲得，能應用於提升人類免疫系統，將為人類健康帶來莫大的幫助。At present, from many studies on Antrodia camphorata, it is known that the composition of Antrodia camphorata is very complicated. It is found that the body complex of A. angustifolia contains unique and different skeletons of triterpenoids. It has also been found that the composition of A. annuum contains polysaccharides. The mechanism of the polysaccharide and the regulation of the immune response indicates that the structure of the polysaccharide rapidly activates the antigen-presenting cells, and has the functions of enhancing immunity, anti-tumor, anti-inflammatory and antibacterial activity. If a pharmacologically active polysaccharide can be found from the extract of Antrodia camphorata to promote immunity, and the polysaccharide can be cultured in large quantities by artificial culture, it can be applied to enhance the human immune system, which will bring great benefits to human health. help.
本發明之主要目的係提供一牛樟芝菌絲體固態醱酵產物粗多醣體之製備方法，分離出兼具安全性及藥理活性之牛樟芝菌絲體，進而生產牛樟芝菌絲體固態醱酵產物之粗多醣體。The main object of the present invention is to provide a method for preparing a crude polysaccharide of a solid fermentation product of the mycelium of Antrodia camphorata, and to isolate the mycelium of Antrodia camphorata which has both safety and pharmacological activity, thereby producing a crude solid fermentation product of Antrodia camphorata mycelium. Polysaccharide.
本發明的次一目的，係提供一種牛樟芝菌絲體固態醱酵產物粗多醣體之製備方法，該製備方法係可經由人工培養一牛樟芝菌絲體固態醱酵物，經萃取及分離純化大量獲得一粗多醣體。A second object of the present invention is to provide a method for preparing a crude polysaccharide of a solid fermentation product of Antrodia camphorata mycelium, which can be cultured by artificially cultivating a solid mash of Mycelium of Antrodia camphorata, and obtained by extraction, separation and purification. Crude polysaccharide.
本發明的再一目的，係提供一種牛樟芝菌絲體固態醱酵產物粗多醣體之製備方法，該粗多醣體能有效活化免疫細胞。A further object of the present invention is to provide a method for preparing a crude polysaccharide of a solid fermentation product of the mycelium of Antrodia camphorata, which can effectively activate immune cells.
根據本發明的一種牛樟芝菌絲體固態醱酵產物粗多醣體之製備方法，係關於建立一種適合從一牛樟芝菌絲體固態醱酵培養物中大規模地萃取出具藥理活性之粗多醣體的製備方法，特定言之，該製備方法係藉由培養期間的溫度調節、時間控制、培養基的選擇及粗多醣體的分離來建立，以獲得牛樟芝菌絲體固態醱酵產物的粗多醣體。The invention relates to a method for preparing a crude polysaccharide of a solid fermentation product of Antrodia camphorata mycelium according to the invention, relating to preparing a large-scale extraction of a pharmacologically active crude polysaccharide from a solid fermentation culture of A. angustifolia mycelium. The method, in particular, the preparation method is established by temperature regulation during culture, time control, selection of a medium, and separation of a crude polysaccharide to obtain a crude polysaccharide of a solid fermentation product of the mycelium of Antrodia camphorata.
為讓本發明之上述及其他目的、特徵及優點能更明顯易懂，下文特舉本發明之較佳實施例，並配合所附圖式，作詳細說明如下：以下介紹為本發明較佳實施例，牛樟芝菌絲體固態醱酵產物粗多醣體係取自於一NPU-50菌株之固態醱酵物，首先介紹如何以人工培養篩選出該NPU-50菌株，進行鑑定該NPU-50菌株為一牛樟芝菌絲體菌株，再者，經萃取該牛樟芝菌絲體NPU-50菌株之固態醱酵物得一粗多醣體(ACP)，並利用免疫測試發現該粗多醣體具免疫活性，可增殖免疫細胞。The above and other objects, features and advantages of the present invention will become more <RTIgt; For example, the crude polysaccharide system of the mycelium of Astragalus membranaceus mycelium is obtained from the solid mash of a NPU-50 strain. First, how to screen the NPU-50 strain by artificial culture, and identify the NPU-50 strain as A. The mycelial strain, further, obtained a crude polysaccharide (ACP) by extracting the solid mash of the A. niger mycelium NPU-50 strain, and found that the crude polysaccharide has immunological activity and can proliferate immune cells by immunological tests.
一、牛樟芝菌絲體NPU-50菌株製備I. Preparation of N. gingivalis mycelium NPU-50 strain
首先取野生牛樟芝子實體磨碎成一子實體粉末，本發明較佳實施例係選擇將該子實體粉末懸浮於0.1%蛋白腖稀釋成一懸浮液，再取該懸浮液塗抹於一含有抗生素之MEA平板培養基上，該MEA平板培養基係包含一基材、一碳源及一氮源，該基材係選擇為洋菜，該碳源係選自由葡萄糖(glucose)、果糖(fructose)、半乳糖(galactose)、乳糖(lactose)、澱粉(starch)、纖維素(cellulose)及蔗糖(sucrose)所組成之族群，該氮源係選由蛋白腖(peptone)、麥芽抽出物(malt extract)、酵母抽出物(yeast extract)及三蛋白腖(tripeptone)所組成之族群，本發明之MEA平板培養基較佳係選擇洋菜作為基材，葡萄糖作為碳源，蛋白腖及麥芽抽出物作為氮源，該MEA平板培養基培養條件較佳係選擇為27℃，2-3天。於培養的過程中，見單一菌落即將該單一菌落接種至新MEA平板培養基，藉此方法分離出不同性狀之牛樟芝菌絲體菌株。本發明係依上述方法分離出一菌株具較鮮紅之外觀顏色、特殊香氣及生長速率較快之特性，該菌株命名為NPU-50。First, the wild A. sylvestris fruit body is ground into a fruit body powder. In a preferred embodiment of the present invention, the fruit body powder is suspended in 0.1% peptone and diluted into a suspension, and the suspension is applied to an MEA plate medium containing antibiotics. The MEA plate medium comprises a substrate, a carbon source and a nitrogen source, and the substrate is selected from a seaweed, the carbon source being selected from the group consisting of glucose, fructose, and galactose. a group consisting of lactose, starch, cellulose, and sucrose selected from the group consisting of peptone, malt extract, and yeast extract ( The MEA plate medium of the present invention preferably uses acai as a substrate, glucose as a carbon source, peptone and malt extract as a nitrogen source, and the MEA plate medium is cultured. The conditions are preferably selected to be 27 ° C for 2-3 days. During the cultivation process, a single colony was inoculated into the new MEA plate medium, and the strains of A. angustifolia mycelium having different traits were isolated by this method. The invention separates a strain with a bright red appearance color, a special aroma and a growth rate according to the above method, and the strain is named as NPU-50.
二、牛樟芝菌絲體NPU-50菌株鑑定2. Identification of NPU-50 strain of Mycelium of Antrodia camphorata
取得該NPU-50菌株後，需進行鑑定該菌株是否確為一牛樟芝菌絲體。本發明較佳實施例係選擇將該NPU-50菌株接種至一MEB液態培養基，該MEB液態培養基係包含一碳源及一氮源，該碳源係選自由葡萄糖(glucose)、果糖(fructose)、半乳糖(galactose)、乳糖(lactose)、澱粉(starch)、纖維素(cellulose)及蔗糖(sucrose)所組成之族群，該氮源係選由蛋白腖(peptone)、麥芽抽出物(malt extract)、酵母抽出物(yeast extract)及三蛋白腖(tripeptone)所組成之族群。本發明之MEB液態培養基較佳係選擇葡萄糖作為碳源，蛋白腖及麥芽抽出物作為氮源，該MEB液態培養基培養條件為震盪培養於25-30℃，5-12天，本發明較佳實施例係選擇為震盪培養27℃，7天，該震盪係設定在約 300 rpm，較佳係選擇為120 rpm之速率下，再將培養好的菌液係選擇利用超高速冷凍離心機進行離心10,000 rpm離心20分鐘，再以布氏漏斗過濾取得該NPU-50菌絲體，進行菌株鑑定。After obtaining the NPU-50 strain, it is necessary to identify whether the strain is indeed a mycelium of Antrodia camphorata. In a preferred embodiment of the present invention, the NPU-50 strain is selected to be inoculated into a MEB liquid medium comprising a carbon source and a nitrogen source selected from the group consisting of glucose and fructose. a group consisting of galactose, lactose, starch, cellulose, and sucrose, selected from peptone, malt extract ), a mixture of yeast extract and tripeptone. Preferably, the MEB liquid medium of the present invention selects glucose as a carbon source, peptone and malt extract as a nitrogen source, and the culture condition of the MEB liquid medium is shaking culture at 25-30 ° C for 5-12 days, preferably in the present invention. The system was selected to be shake culture at 27 ° C for 7 days. The shock system was set at about 300 rpm, preferably at a rate of 120 rpm, and the cultured bacteria system was selected for centrifugation using an ultra-high speed refrigerated centrifuge. The NPU-50 mycelium was obtained by centrifugation at rpm for 20 minutes, and then filtered by a Buchner funnel.
判斷牛樟芝的真偽方法主要有三種：(1)顏色外觀上的判斷，(2)型態學上可觀察是否有斷生孢子(arthrospores)的存在，(3)分子生物鑑定系統，則使用於物種分類鑑定上最常用之18S核醣體DNA基因序列，該18S核醣體DNA(18 rDNA)基因序列被作為真菌中屬之間的分類依據，具有高倍複製(high copy number)及演化中高保留性的特點。本發明係選擇分子生物鑑定系統，將該NPU-50之菌絲體利用液態氮冷凍磨碎，進行該NPU-50之DNA萃取，將該NPU-50 DNA做為模版，再加入用來鑑定18S rDNA基因序列之引子NS3(5’-GCAAGTCTGGTGCCAGCAGCC-3’)及NS6(5’-GCATCACAGACCTGTTATTGCCTC-3’)，進行聚合酶連鎖反應(Polymerase Chain Reaction，PCR)得一聚合酶連鎖反應產物，該反應條件為起始於93℃至99℃變性3至5分鐘後，再於93℃至99℃的變性溫度反應30至60秒、於50℃至57℃之煉合反應溫度反應30至60秒及於70℃至75℃的延展溫度反應1至3分鐘，且DNA變性、煉合及延展之循環數為10至50循環，再以70℃至75℃之延展溫度反應7至12分鐘，最後保存於4℃下；本發明較佳聚合酶連鎖反應條件係選擇為起始變性溫度98℃，2分鐘，變性溫度95℃，45秒，煉合溫度52℃，45秒，延展溫度72℃，2分鐘，共進行35個循環，最後延展溫度72℃，10分鐘。之後再以電泳分析判斷是否有該聚合酶連鎖反應產物，若於電泳分析中有見該聚合酶連鎖反應產物，再定序該聚合酶連鎖反應產物，最後將該定序結果與NCBI的資料庫比對，比對結果為牛樟芝(Antrodia camphorata )菌株且比對相似度達100%，確認本發明之該NPU-50菌株確為牛樟芝菌絲體，以下稱為牛樟芝菌絲體NPU-50，於是本發明將該牛樟芝菌絲體NPU-50菌株於中華民國99年3月19日將此菌株寄存於中華民國食品工業研究所之菌種保存及研究中心(BCRC)，該菌株寄存編號為BCRC 930127，命名為牛樟芝菌絲體NPU-50。There are three main methods for judging the authenticity of Antrodia camphorata: (1) judging the appearance of color, (2) observing the existence of arthrospores in type, and (3) molecular bioassay system. The most commonly used 18S ribosomal DNA gene sequence is identified by species classification. The 18S ribosomal DNA (18 rDNA) gene sequence is used as a classification basis between fungal genus, with high copy number and high retention in evolution. Features. The invention selects a molecular biological identification system, and the mycelium of the NPU-50 is freeze-ground by liquid nitrogen, and the DNA extraction of the NPU-50 is performed, and the NPU-50 DNA is used as a template, and then added to identify 18S. The primers NS3 (5'-GCAAGTCTGGTGCCAGCAGCC-3') and NS6 (5'-GCATCACAGACCTGTTATTGCCTC-3') of the rDNA gene sequence are subjected to a polymerase chain reaction (PCR) to obtain a polymerase chain reaction product. After denaturation at 93 ° C to 99 ° C for 3 to 5 minutes, reaction at a denaturation temperature of 93 ° C to 99 ° C for 30 to 60 seconds, reaction at a reaction temperature of 50 ° C to 57 ° C for 30 to 60 seconds and The reaction temperature of 70 ° C to 75 ° C is 1 to 3 minutes, and the number of cycles of DNA denaturation, refining and stretching is 10 to 50 cycles, and then reacted at an elongation temperature of 70 ° C to 75 ° C for 7 to 12 minutes, and finally stored in At 4 ° C; preferred polymerase chain reaction conditions of the present invention are selected as the initial denaturation temperature of 98 ° C, 2 minutes, denaturation temperature 95 ° C, 45 seconds, synthesis temperature 52 ° C, 45 seconds, extension temperature 72 ° C, 2 minutes A total of 35 cycles were performed, and the final extension temperature was 72 ° C for 10 minutes. Then, the electrophoresis analysis is used to determine whether there is a polymerase chain reaction product. If the polymerase chain reaction product is seen in the electrophoresis analysis, the polymerase chain reaction product is reordered, and finally the sequencing result is compared with the NCBI database. The alignment was compared with the Antrodia camphorata strain and the similarity was 100%. It was confirmed that the NPU-50 strain of the present invention was indeed the mycelium of Antrodia camphorata , which is hereinafter referred to as N. chinensis mycelium NPU-50. In the present invention, the strain of N. gingivalis NPU-50 was deposited in the Republic of China Food Industry Research Institute's Culture Collection and Research Center (BCRC) on March 19, 1999. The strain registration number is BCRC 930127. Named as Ophiopogon japonicus mycelium NPU-50.
由以上所述，得知該NPU-50菌株為一牛樟芝菌絲體，以下稱為一牛樟芝菌絲體NPU-50菌株，接下來係取該牛樟芝菌絲體NPU-50菌株進行液態培養及固態醱酵成一固態醱酵物，再取該固態醱酵物進行萃取獲得牛樟芝菌絲體固態醱酵產物粗多醣體(ACP)，並研究該牛樟芝菌絲體固態醱酵產物粗多醣體的功效。From the above, it is known that the NPU-50 strain is a mycelium of Antrodia camphorata, which is hereinafter referred to as a strain of N. gingivalis NPU-50, and then the strain of N. gingivalis NPU-50 is taken for liquid culture and solid state. The yeast is fermented into a solid mash, and the solid mash is extracted to obtain the crude polysaccharide (ACP) of the solid fermentation product of the mycelium of Antrodia camphorata, and the effect of the crude polysaccharide of the solid fermentation product of the mycelium of Astragalus membranaceus is studied.
三、本發明之牛樟芝菌絲體固態醱酵產物粗多醣體(ACP)之製備方法3. Method for preparing crude polysaccharide (ACP) of the solid fermentation product of Antrodia camphorata mycelium of the present invention
接下來係研究該牛樟芝菌絲體NPU-50菌株之固態醱酵物內，是否具有藥理活性之成份，首先要液態培養及固態醱酵該牛樟芝菌絲體NPU-50菌株成一固態醱酵物，再進行萃取該固態醱酵物，以進一步研究該固態醱酵物。Next, it is studied whether the solid lysate of the A. niger mycelium NPU-50 strain has pharmacologically active ingredients. First, the liquid culture and solid fermentation of the A. sinensis mycelium NPU-50 strain into a solid mash is carried out. The solid mash is extracted to further study the solid mash.
該液態培養及固態醱酵步驟，係可取該牛樟芝菌絲體NPU-50菌株於液態培養基及固態培養基上進行液態培養及固態醱酵，本發明較佳實施例，係選擇將該牛樟芝菌絲體NPU-50菌株於液態培養基做初步醱酵，以形成一醱酵液，再將該醱酵液接種至固態培養基，完成一固態醱酵物。該固態培養基含有較佳生長成份，供給營養長成為該固態醱酵物，該固態醱酵物具有強烈的牛樟芝香氣，且該固態醱酵物內部形成類似海綿狀的結構，該類似海綿狀的結構係如野生牛樟芝生長時之結構，且該固態醱酵物內係含有一粗多醣體。The liquid culture and the solid fermentation step can be carried out by liquid culture and solid state fermentation on the liquid medium and the solid medium. The preferred embodiment of the present invention selects the mycelium of Antrodia camphorata. The NPU-50 strain is initially fermented in a liquid medium to form a mash, which is then inoculated into a solid medium to complete a solid mash. The solid medium contains a preferred growth component, and the nutrient is supplied to form the solid mash. The solid mash has a strong aroma of burdock, and the solid mash has a sponge-like structure inside, and the sponge-like structure is wild. The structure of the growth of Antrodia camphorata, and the solid yeast contains a crude polysaccharide.
首先取上述該牛樟芝菌絲體NPU-50菌株於該MEB液態培養基進行震盪培養於27℃，培養7天之醱酵液，利用均質機均質該醱酵液，再將該均質過後之醱酵液接種至一固態培養基上。該固態培養基係包含一基材、一碳源及一氮源，該基材為米，係選自於糙米、胚芽米、糯米、蓬萊米、在來米或茭白之一，該碳源係選自由葡萄糖(glucose)、果糖(fructose)、半乳糖(galactose)、乳糖(lactose)、澱粉(starch)、纖維素(cellulose)及蔗糖(sucrose)所組成之族群，該氮源係選由蛋白腖(peptone)、麥芽抽出物(malt extract)、酵母抽出物(yeast extract)及三蛋白腖(tripeptone)所組成之族群。本發明之固態培養基較佳係選擇胚芽米作為基材，葡萄糖作為碳源，蛋白腖及麥芽抽出物作為氮源，該固態培養醱酵條件為培養於25℃至30℃，2至6個月，本發明較佳係選擇於27℃醱酵3個月形成一固態醱酵物。First, the above-mentioned A. sinensis mycelium NPU-50 strain is cultured in the MEB liquid medium at 27 ° C, cultured for 7 days, and the homogenizer is used to homogenize the broth, and then the homogenized mash is used. Inoculate onto a solid medium. The solid medium comprises a substrate, a carbon source and a nitrogen source, and the substrate is rice, which is selected from the group consisting of brown rice, germ rice, glutinous rice, Penglai rice, rice or glutinous rice, and the carbon source is selected. a group of free glucose, fructose, galactose, lactose, starch, cellulose, and sucrose, which is selected from peptone ( A group consisting of peptone), malt extract, yeast extract, and tripeptone. Preferably, the solid medium of the present invention selects germ rice as a substrate, glucose as a carbon source, peptone and malt extract as a nitrogen source, and the solid culture fermentation conditions are cultured at 25 ° C to 30 ° C for 2 to 6 months. Preferably, the present invention is selected to be fermented at 27 ° C for 3 months to form a solid mash.
接著，將該固態醱酵物以水或有機溶劑進行萃取，藉以取得牛樟芝菌絲體固態醱酵產物水萃取物或有機溶劑萃取物，並從該萃取物中獲得該粗多醣體，其中該有機溶劑係選自醇類、酯類、烷類或鹵烷所組成之族群，但並不受此限制。本發明較佳實施例係選擇使用水萃取法，首先將該固態醱酵物係選擇加入蒸餾水，利用均質機均質後，再於75℃至90℃之環境下以超音波震盪，本發明係選擇於80℃震盪2小時，待冷卻後，選擇以超高速冷凍離心機10,000 rpm離心20分鐘得一上清液，選擇以布氏漏斗過濾該上清液，將該過濾後之上清液以95%酒精於4℃沉澱一個晚上得一沉澱物，利用抽氣過濾收集該沉澱物，並利用70%酒精清洗該沉澱物兩次，該沉澱物係為該牛樟芝菌絲體NPU-50固態醱酵物之粗多醣體，稱為牛樟芝菌絲體固態醱酵產物粗多醣體(Antrodia camphorata polysaccharides，ACP)，以下簡稱為ACP，請參照第1圖所示，其係該牛樟芝菌絲體固態醱酵產物粗多醣ACP在HPLC-RI色層層析所產生波鋒滯留時間圖譜。Next, the solid mash is extracted with water or an organic solvent to obtain an aqueous extract or an organic solvent extract of the solid lyophilized product of the genus Antrodia camphorata, and the crude polysaccharide is obtained from the extract, wherein the organic solvent It is selected from the group consisting of alcohols, esters, alkanes or molars, but is not limited thereto. In a preferred embodiment of the present invention, the water extraction method is selected. First, the solid mash system is selectively added to distilled water, and after homogenization, the ultrasonic wave is oscillated in an environment of 75 ° C to 90 ° C. After shaking at 80 ° C for 2 hours, after cooling, a supernatant was selected by centrifugation at 10,000 rpm for 20 minutes in an ultra-high speed refrigerated centrifuge, and the supernatant was selected to be filtered by a Buchner funnel, and the supernatant was filtered to 95%. The alcohol was precipitated at 4 ° C for one night to obtain a precipitate, and the precipitate was collected by suction filtration, and the precipitate was washed twice with 70% alcohol, and the precipitate was the NPU-50 solid mash of the Astragalus membranaceus mycelium. The crude polysaccharide is called Antrodia camphorata polysaccharides (ACP), hereinafter referred to as ACP. Please refer to Figure 1 for the crude fermentation product of the mycelium of Astragalus membranaceus. Polysaccharide ACP produced a wave front retention time map by HPLC-RI chromatography.
目前關於多醣體的研究指出與調節免疫反應的機制有關，可能為多醣體上的結構活化抗原呈現細胞(antigen-presenting cells，APC)的TLR-4、Denctin 1、MR或MRL的接受器，使APC快速將訊息傳給未活化的T淋巴細胞，活化後的T淋巴細胞會透過特殊途徑進行免疫力的提升、抗腫瘤、抗發炎及抑菌等活性，以下將進行測試，以證明利用本發明之方法所醱酵萃取出的牛樟芝菌絲體固態醱酵產物粗多醣體(ACP)具有刺激免疫細胞增殖的能力。At present, studies on polysaccharides indicate that it is related to the mechanism of modulating the immune response, and may be a receptor for TLR-4, Dentin 1, MR or MRL of the antigen-presenting cells (APC) on the polysaccharide. APC quickly transmits the message to the unactivated T lymphocytes, and the activated T lymphocytes will carry out immunity, anti-tumor, anti-inflammatory and antibacterial activities through special routes, and will be tested below to prove the use of the present invention. The crude polysaccharide (ACP) of the crude extract of Astragalus membranaceus mycelium extracted by the method has the ability to stimulate the proliferation of immune cells.
四、本發明牛樟芝菌絲體固態醱酵產物粗多醣體之增殖免疫細胞效果4. The effect of the proliferation of the crude polysaccharides of the solid fermentation product of the mycelium of Antrodia camphorata in the present invention
有關食用真菌研究中，已知靈芝多醣體可抑制腫瘤生長及刺激T淋巴細胞及B淋巴細胞活化，以及巴西蘑菇多醣體可抗腫瘤及提升免疫力活性，於是本發明較佳實施例係取本發明之牛樟芝菌絲體固態醱酵產物粗多醣體(ACP)進行免疫細胞增殖試驗。In the study of edible fungi, it is known that Ganoderma lucidum polysaccharide can inhibit tumor growth and stimulate T lymphocyte and B lymphocyte activation, and Brazilian mushroom polysaccharide can resist tumor and enhance immunity activity, so the preferred embodiment of the present invention is taken The invented Astragalus membranaceus mycelium solid fermentation product crude polysaccharide (ACP) was subjected to an immunocyte proliferation test.
首先分離SD大鼠的周邊血液單核球細胞及脾臟中的免疫細胞，本發明較佳實施例係選擇於分離出大鼠之周邊血液單核球細胞及脾臟中的免疫細胞分別加入96孔盤中，每孔係選擇加入50 μL 2×106 cells/mL細胞，再分別加入劑量為1 μg/mL lipopolysaccharides(LPS)、10 μg/mL concanalin A(ConA)、ACP含1 μg/mL LPS及ACP含10 μg/mL ConA，係選擇置於5% CO2 的培養箱37℃，培養細胞72小時後，再於每孔加入細胞增殖檢測試劑Alamar BlueTM 10 μL，於5% CO2 37℃之培養箱中反應四小時後，選擇利用ELISA reader於570 nm及600 nm分別測吸光值，再經計算公式得到免疫細胞之增值百分比。在老鼠周邊血液單核球細胞增殖結果如第2圖所示，該ACP與LPS共同刺激增值效果約為控制組的2倍，該ACP與ConA共同刺激增值效果約為控制組的5.8倍；在老鼠脾臟中的免疫細胞增殖結果，如第3圖所示，該ACP與LPS或ConA共同刺激增殖效果皆可達約為控制組的2倍。First, the peripheral blood mononuclear cells of the SD rats and the immune cells in the spleen are isolated. In the preferred embodiment of the present invention, the immune cells in the peripheral blood mononuclear cells and the spleen of the rat are separately added to the 96-well plate. In each well, 50 μL of 2×10 6 cells/mL cells were added to each well, and a dose of 1 μg/mL lipopolysaccharides (LPS), 10 μg/mL concanalin A (ConA), ACP containing 1 μg/mL LPS, and ACP containing 10 μg/mL ConA was selected in a 5% CO 2 incubator at 37 ° C. After incubating the cells for 72 hours, add cell proliferation assay reagent Alamar Blue TM 10 μL per well at 5% CO 2 37 °C. After reacting for four hours in the incubator, the absorbance values were separately measured at 570 nm and 600 nm using an ELISA reader, and the percentage increase of the immune cells was obtained by the calculation formula. As shown in Figure 2, the proliferation of mononuclear cells in the peripheral blood of mice is as shown in Fig. 2. The value-added effect of ACP and LPS is about twice that of the control group. The effect of co-stimulation of ACP and ConA is about 5.8 times that of the control group. As a result of the proliferation of immune cells in the spleen of mice, as shown in Fig. 3, the proliferation effect of the ACP and LPS or ConA was about twice as high as that of the control group.
由上述免疫試驗結果得知，該牛樟芝菌絲體固態醱酵產物粗多醣體ACP與LPS混合，或者將該牛樟芝菌絲體固態醱酵產物粗多醣體ACP與ConA混合，以共同刺激誘導週邊血液單核球細胞及脾臟免疫細胞增殖的效果佳，可藉由多醣體上的結構活化抗原呈現細胞，快速將訊息傳給未活化的T淋巴細胞，進而活化T淋巴細胞提升免疫力；由於加入ACP會加強LPS或ConA的功效，而達到共同提升免疫細胞增殖，因此能有效引發產生免疫反應，且可達到刺激免疫細胞增殖及活化者。可驗證經由本發明較佳實施例之製備方法所篩選到該牛樟芝菌絲體NPU-50菌株，為一富含藥理功效且安全的牛樟芝菌絲體菌株，且將該牛樟芝菌絲體NPU-50菌株經培養固態醱酵成一固態醱酵物，再經由分離純化獲得一粗多醣體，該粗多醣體具有藥理活性，能有效增殖免疫細胞。According to the results of the above immunoassay, the crude polysaccharide ACP of the Astragalus membranaceus mycelium is mixed with LPS, or the crude polysaccharide ACP of the A. sinensis mycelium is mixed with ConA to stimulate the peripheral blood. The proliferation of mononuclear cells and spleen immune cells is good. The cells can be activated by the activation of the antigen on the structure of the polysaccharide, and the message can be quickly transmitted to the unactivated T lymphocytes, thereby activating T lymphocytes to enhance immunity; It will strengthen the efficacy of LPS or ConA, and achieve the common promotion of immune cell proliferation, so it can effectively trigger the immune response, and can stimulate the proliferation and activation of immune cells. It can be verified that the strain of N. gingivalis NPU-50 is screened by the preparation method of the preferred embodiment of the present invention, which is a pharmacologically effective and safe strain of Mycelium of Astragalus membranaceus, and the Mycelium of Asteris sinensis NPU-50 The strain is fermented into a solid mash by solid culture, and then a crude polysaccharide is obtained by separation and purification. The crude polysaccharide has pharmacological activity and can effectively proliferate immune cells.
本發明分離出一安全且具藥理活性之牛樟芝菌絲體NPU-50菌株，使用牛樟芝菌絲體固態醱酵產物粗多醣體之製備方法，從該牛樟芝菌絲體NPU-50菌株固態醱酵物經萃取可取得一具藥理活性之粗多醣體。The invention separates a safe and pharmacologically active strain of N. gingivalis NPU-50, and uses the preparation method of the crude polysaccharide of the solid fermentation product of the mycelium of Astragalus membranaceus, from the solid lysate of the mycelium NPU-50 strain Extraction can obtain a pharmacologically active crude polysaccharide.
本發明之牛樟芝菌絲體固態醱酵產物粗多醣體之製備方法，利用人工大量的培養出該牛樟芝菌絲體NPU-50成一固態醱酵物取代野生牛樟芝，且該固態醱酵物直接經萃取及分離純化，就可大量地取得該牛樟芝菌絲體固態醱酵產物粗多醣體。The method for preparing the crude polysaccharide of the solid fermentation product of the mycelium of Antrodia camphorata according to the invention, the artificially mass-cultivating the mycelium NPU-50 into a solid mash to replace the wild Antrodia camphorata, and the solid mash is directly extracted and separated After purification, the crude polysaccharide of the solid fermentation product of the mycelium of A. angustifolia can be obtained in a large amount.
本發明之牛樟芝菌絲體固態醱酵產物粗多醣體之製備方法，該牛樟芝菌絲體固態醱酵產品粗多醣體誘導週邊血液單核球細胞及脾臟免疫細胞增殖的效果佳，與能刺激免疫細胞活化的LPS或ConA共同刺激，能有效提升免疫細胞活化，有效引發免疫反應，且達到刺激免疫系統細胞增殖的功效。The preparation method of the crude polysaccharide of the solid fermentation product of the mycelium of Antrodia camphorata according to the invention, the crude polysaccharide of the mycelium of the mycelium of the mycelium of the Antrodia camphorata induces the proliferation of peripheral blood mononuclear cells and the immune cells of the spleen, and stimulates immunity Cell-activated LPS or ConA co-stimulation can effectively enhance the activation of immune cells, effectively elicit an immune response, and achieve the effect of stimulating the proliferation of immune system cells.
雖然本發明已利用上述較佳實施例揭示，然其並非用以限定本發明，任何熟習此技藝者在不脫離本發明之精神和範圍之內，相對上述實施例進行各種更動與修改仍屬本發明所保護之技術範疇，因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。While the invention has been described in connection with the preferred embodiments described above, it is not intended to limit the scope of the invention. The technical scope of the invention is protected, and therefore the scope of the invention is defined by the scope of the appended claims.
第1圖：本發明之牛樟芝菌絲體固態醱酵產物粗多醣體ACP萃取後之HPLC-RI圖譜。Fig. 1 is a HPLC-RI spectrum of the crude polysaccharide of Astragalus membranaceus mycelium solid-state fermentation product ACP after extraction.
第2圖：本發明之牛樟芝菌絲體固態醱酵產物粗多醣體ACP與LPS或與ConA共同刺激大鼠的周邊血液單核球細胞增殖反應圖。Fig. 2 is a diagram showing the proliferation reaction of the peripheral blood mononuclear cells of the rat with the crude polysaccharide ACP of the Astragalus membranaceus mycelium of the present invention and LPS or with ConA.
第3圖：本發明之牛樟芝菌絲體固態醱酵產物粗多醣體ACP與LPS或與ConA共同刺激大鼠的脾臟免疫細胞增殖反應圖。Fig. 3 is a diagram showing the spleen immune cell proliferation reaction of the crude polysaccharide ACP of the Astragalus membranaceus mycelium of the present invention with LPS or with ConA.
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| TW200638867A (en) * | 2005-05-06 | 2006-11-16 | Golden Biotechnology Corp | Incubation and application methods for the culture of antrodia camphorata |
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| TWI236480B (en) * | 2000-09-25 | 2005-07-21 | Grape King Inc | A method for producing biological active material with antrodia camphorata and composition thereof |
| TW200638867A (en) * | 2005-05-06 | 2006-11-16 | Golden Biotechnology Corp | Incubation and application methods for the culture of antrodia camphorata |
| TW200835791A (en) * | 2007-02-26 | 2008-09-01 | Pei-Jung Li | Method for artificially propagating fruit body stroma of Antrodia Camphorate |
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