CN117126745B - 药用真菌灵芝、适于药用真菌灵芝高产胞外多酚的培养基及其培养方法和用途 - Google Patents
药用真菌灵芝、适于药用真菌灵芝高产胞外多酚的培养基及其培养方法和用途 Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- 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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- 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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- A—HUMAN NECESSITIES
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- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
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Abstract
本发明公开了一种南方灵芝(Ganoderma australe)菌株GA79，于2023年05月15日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.40594。还公开了该菌株的培养基和培养条件。同时公开了该菌株在食品、保健品、药品中的用途。该菌株及其培养基可高效提高液体发酵过程中的多酚含量得率，从而为灵芝多酚等食品、保健品及药品的开发利用研究提供重要依据。
Description
技术领域
本发明属于生物技术领域，具体涉及一药用真菌灵芝、适于药用真菌灵芝高产胞外多酚的培养基及其培养方法和用途。
背景技术
“灵芝”，隶属于真菌界(Fungi)、担子菌门(Basidiomycota)、伞菌纲(Agaricomycetes)、多孔菌目(Polyporales)、灵芝科(Ganodermataceae)，是我国一类传统食药用真菌的统称，具有抑肿瘤、抗氧化、抑菌、抗炎、免疫调节等多种功效，目前已被《中华人民共和国药典》收录。基于形态学和分子系统学数据表明，目前世界共有灵芝物种114种，其中中国共有灵芝物种40种，包括灵芝(Ganoderma lingzhi Sheng H.Wu,Y.Cao&Y.C.Dai)、亮壳灵芝(Ganoderma lucidum(Curtis.)P.Karst.)、树舌灵芝(Ganodermaapplanatum(Pers.)Pat.)、南方灵芝(Ganoderma australe(Fr.)Pat.)、狭长孢灵芝(Ganoderma boninense Pat.)、木麻黄灵芝(Ganoderma casuarinicola J.H.Xing,B.K.Cui&Y.C.Dai)、窄孢灵芝(Ganoderma fornicatum J.H.Xing,B.K.Cui&Y.C.Dai)等。现代药理学研究表明，灵芝含包括多糖、灵芝酸、多酚、黄酮、甾醇、生物碱、蛋白质、油脂、有机锗、矿物元素等在内的多种生物活性成分，其中多酚是灵芝的关键药理成分之一，由一个或多个羟基组成，能够成为强有效的自由基清除剂和金属离子螯合剂。然而，以往通过野外采集或人工栽培灵芝，生长周期长、所需条件苛刻、成分比例不稳定、易受环境影响，使得提取灵芝多酚远不能满足市场需求。液体发酵过程中产生的灵芝多酚主要存在于发酵液中，通过离心、醇沉、萃取等方法易获取，得率也高。
发明内容
本发明以南方灵芝(Ganoderma australe)为例，提供了一种适于其高产胞外多酚的培养基及培养方法，可高效提高液体发酵过程中的多酚含量得率，从而为灵芝多酚等食品、保健品及药品的开发利用研究提供重要依据。
本发明是通过以下技术方案实现的：
本发明提供了一种南方灵芝(Ganoderma australe)菌株GA79，于2023年05月15日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.40594。
本发明还提供了南方灵芝菌株GA79的培养基和培养方法。
本发明同时提供了南方灵芝菌株GA79在制备保健品或食品中的应用。
本发明同时提供了一种食品或保健品，所述的食品或保健品中含有所述的南方灵芝菌株GA79。
本发明同时提供了南方灵芝菌株GA79在制备提高人体免疫力的药品中的应用。
有益效果
本发明提供了新的南方灵芝菌株，提供的培养基和培养方法，培养的南方灵芝菌株有效提高了液体发酵过程灵芝多酚的含量，药用价值高，可用于制备提高人体免疫力的食品或保健品作用的药物，从而为南方灵芝用途扩宽了途径，具有十分重要的意义。
菌种保藏信息
保藏时间：2023年05月15日；
保藏单位：中国微生物菌种保藏管理委员会普通微生物中心；
保藏编号：CGMCC No. 40594；
保藏单位地址：北京市朝阳区北辰西路1号院3号 中国科学院微生物研究所；
分类命名：南方灵芝Ganoderma australe。
具体实施方式
下面对本发明的实施例作详细说明，本实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施例。
实施例1菌种的筛选、鉴定、纯化
本申请采用的菌种南方灵芝菌株GA79是从广东省韶关市仁化县阔叶树倒木通过分离和菌种纯化而得到，该菌株分类命名为南方灵芝Ganoderma australe，已于2023年05月15日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.40594。
子实体形态特征：担子果多年生，无柄，新鲜时硬木栓质，干后硬木质；菌盖贝壳形，长14.6cm，宽22.7cm，厚4.3cm，表面无漆状光泽，浅黄褐色，具明显同心环痕，在近边缘处更加明显，中部具较细的辐射状沟纹，边缘锐；孔口表面幼时白色，成熟时硫磺色，伤后褐色，干时草黄色；管口圆形，多数全缘，4个/mm，管壁稍厚；菌肉上层浅褐色，下层深褐色，木栓质，无同心环纹，长1.3cm，成熟担子果具黑色壳质线；菌管褐色，硬木栓质，长2.5cm。
显微结构特征：菌丝三体系；骨架菌丝占绝大多数，棕黄色，厚壁，多二分枝；生殖菌丝具锁状联合，无色，薄壁；缠绕菌丝浅棕色，厚壁，内腔窄；无拟糊精反应，有嗜蓝反应，在氢氧化钾中变黑；菌肉菌丝中，骨架菌丝3−4.5μm，生殖菌丝2−3.5μm，缠绕菌丝1.5−3μm；菌管菌丝中，骨架菌丝2.5−4μm，生殖菌丝2−3.5μm，缠绕菌丝1−2.8μm；担孢子卵圆形，脐状突起不明显，脱落后平截，浅黄褐色，无拟糊精反应，有嗜蓝反应，双层壁，外壁平滑，内壁表面具轻度粗糙的点状纹饰，长(6−)7−9.5(−10)×宽4−5.5(−6)μm，平均长度8.06μm，平均宽度5.05μm，长宽比1.60（按孢子顶端无脐突时计算，1个标本测量30个孢子）。
菌种分子鉴定：使用德曼特尔公司的快速无毒植物DNA提取试剂盒（FH Plant DNAKit）对分离得到的菌株GA79进行基因组DNA的提取。将其作为模板，对其内转录间隔区（internal transcribed spacer，ITS）进行PCR扩增，以ITS5（5’−GGAAGTAAAAGTCGTAACAAGG−3’）/ITS4（5’−TCCTCCGCTTATTGATATGC−3’）基因为引物，体系共30.0μL：2×EasyTaq PCRSuperMix 15.0μL，引物ITS5 10μmol/L 1.0μL，引物ITS4 10μmol/L 1.0μL，基因组DNA 1.0μL，去离子水12.0μL。程序为：95℃预变性3min；94℃变性40s，54℃退火45s，72℃延长1min，重复34个循环；最后72℃延长10min；4℃保温。PCR产物送至北京六合华大基因科技有限公司进行测序。
比对：将测序结果在NCBI（National Center for Biotechnology Information）数据库（https://www.ncbi.nlm.nih.gov/）中进行Blast比对，结果与南方灵芝菌株的相似性最高，因此菌株GA79鉴定为南方灵芝Ganoderma australe，GenBank登记号为OQ975944。
实施例2菌种的培养
配制固体培养基：去皮马铃薯300.0g/L，葡萄糖20.0g/L，琼脂20.0g/L，磷酸二氢钾1.0g/L，pH 5.0，1×105Pa高压灭菌30min。
固体培养：于固体培养基上活化，28℃恒温培养箱培养6天备用。
配制液体培养基成分(具体成分见表1)：玉米粉30.0g/L，葡萄糖5.0g/L，蛋白胨3.5g/L，磷酸二氢钾1.0g/L，七水合硫酸锌0.3g/L，七水合硫酸镁0.5g/L，六水合氯化钴0.02g/L，油酸1.0mL/L，吐温40 1.5mL/L，花生油1.0mL/L，丙酮1.5mL/L，维生素B10.01g/L，pH 5.0，1×105Pa高压灭菌30min。
液体培养：取250mL三角瓶分装100mL基础液体培养基，接种5个直径1.0cm菌饼，于恒温摇床28℃、150r/min振荡培养6天。利用内切式匀浆机以5000r/min、1min将培养物制成种子发酵悬液，充分振荡备用。将种子发酵悬液以10.0％(V/V)的接种量加入含100mL液体培养基的250mL三角瓶中，于恒温摇床28℃、150r/min振荡培养6天后含食药用真菌灵芝胞外多酚的溶液。
多酚的提取：将发酵液经12000r/min离心20min，得上清液真空浓缩至原体积的1/3；加入3倍体积的预冷95％乙醇，4℃过夜后去沉淀(除多糖)得上清液；后加入3倍体积的氯仿萃取，去上层(除杂质)得下层水溶液；再加入3倍体积的乙酸乙酯萃取，浓缩后冻干，再配制成灵芝胞外多酚水溶液。
多酚的测定：分别吸取0、0.1、0.2、0.3、0.4、0.5、0.6、0.7mL 0.08g/L邻苯三酚标准液与0.3mL 1.0mol/L福林酚试剂混合，避光放置8min，再加入0.6mL 10％(m/V)碳酸钠溶液，混匀后25℃避光反应30min，用去离子水定容至5.0mL，于750nm处测定吸光值；设3个平行，求平均值；以反应体系中邻苯三酚的浓度为横坐标、吸光值为纵坐标作标准曲线，计算回归方程为：y＝0.2350x-0.1830，相关系数R2＝0.9968。按制作标准曲线的方法进行样品的多酚含量测定，分别以去离子水替代上清液、福林酚试剂的反应体系设为空白及对照，多酚含量以培养液中邻苯三酚当量计。
结果：
表1各因子水平及试验结果(其他成分如下：葡萄糖5.0g/L，磷酸二氢钾1.0g/L，七水合硫酸锌0.3g/L，七水合硫酸镁0.5g/L，六水合氯化钴0.02g/L，花生油1.0mL/L，维生素B10.01g/L)
本发明南方灵芝菌株GA79的发酵液的多酚含量高，说明本发明南方灵芝菌株GA79的药用价值高，可用于制备提高人体免疫力的保健品，或用于制备提高人力免疫力的药物组分，附加值高，经济效益高。
Claims (2)
1.一种南方灵芝（Ganoderma australe）菌株GA79，于2023年05月15日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO. 40594。
2.权利要求1所述的南方灵芝菌株GA79在制备提高人体免疫力的药品中的应用。
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