CN116948836A - 一种高产黄酮的灵芝菌株及其培养基、培养方法和用途 - Google Patents
一种高产黄酮的灵芝菌株及其培养基、培养方法和用途 Download PDFInfo
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Classifications
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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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- 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
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Abstract
本发明公开了一种白肉灵芝菌株GA82，于2023年05月29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.40597。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黄酮，为相关药理研究及保健品开发奠定重要基础。
Description
技术领域
本发明属于生物技术领域，具体涉及一种高产黄酮的灵芝菌株及其培养基、培养方法和用途。
背景技术
黄酮是高等真菌中广泛存在的一类活性次级代谢产物，其母核为2-苯基色原酮，兼具多个苯环和酚羟基结构，可成为电子供体，在抗氧化衰老、抗炎抑菌、降血糖血脂等方面发挥非常显著的药理功效。灵芝作为一类传统食药用真菌的统称，已被《中华人民共和国药典》收录，所分泌的次级代谢产物含抗氧化、抑肿瘤、抑菌、抗病毒、增强免疫力、降血糖血脂等多种生物活性。截至目前，世界范围内灵芝共有114个物种，其中中国有物种40个，包括树舌灵芝Ganoderma applanatum(Pers.)Pat.、南方灵芝G.australe(Fr.)Pat.、狭长孢灵芝G.boninense Pat.、有柄灵芝G.gibbosum(Blume&T.Nees)Pat.、白肉灵芝G.leucocontextum T.H.Li,W.Q.Deng,Sheng H.Wu,D.M.Wang&H.P.Hu、灵芝G.lingzhiSheng H.Wu,Y.Cao&Y.C.Dai、亮壳灵芝G.lucidum(Curtis.)P.Karst.、重盖灵芝G.multipileum Ding Hou、山西灵芝G.shanxiense L.Fan&H.Liu、热带灵芝G.tropicum(Jungh.)Bres.、松杉灵芝G.tsugae Murrill、韦伯灵芝G.weberianum(Bres.&Henn.exSacc.)Steyaert等。液体发酵是替代野外采集或人工栽培、克服灵芝生长周期长、条件苛刻、易受影响等限制的有效方式，且胞外次级代谢产物可有效分泌到发酵液中，易通过离心沉淀萃取等方法获得，从而满足市场对于灵芝次级代谢产物产品开发的需求。
发明内容
本发明以白肉灵芝Ganoderma leucocontextum为例，提供了一种高产黄酮的灵芝菌株及其培养基和培养方法，以期通过液体发酵高效获得黄酮，为相关药理研究及保健品开发奠定重要基础。
本发明是通过以下技术方案实现的：
一种白肉灵芝菌株Ganoderma leucocontextum GA82，于2023年05月29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.40597。
利用白肉灵芝菌株GA82制备黄酮发酵液的方法，包括以下步骤：
(1)将白肉灵芝菌株接种在固体培养基上，活化，24℃恒温培养箱培养7天；
(2)将活化后的菌种接种在基础液体培养基上，恒温摇床24℃、150r/min振荡培养7天，制成种子发酵悬液；
(3)将种子发酵悬液接种在液体培养基于上，恒温摇床24℃、150r/min振荡培养7天得到含食药用真菌灵芝胞外黄酮的溶液。
进一步地，固体培养基为：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。
进一步地，液体培养基为：玉米粉30.0g/L，葡萄糖10.0g/L，蛋白胨4.0g/L，磷酸二氢钾1.0g/L，七水合硫酸镁0.4g/L，黄豆粉5.0g/L，苯丙氨酸0.3g/L，吐温80 2.0mL/L，赤霉酸0.02g/L，醋酸钠0.3g/L，维生素B1 0.01g/L，pH 5.0。
本发明也公开了白肉灵芝菌株GA82的黄酮发酵液在制备保健品或食品中的应用。
本发明同时公开了一种食品或保健品，所述的食品或保健品中含有白肉灵芝菌株GA82的发酵液。
本发明还公开了白肉灵芝菌株GA82的发酵液在制备提高人体免疫力或者抗癌药品中的应用。
有益效果
本发明提供了新的白肉灵芝菌株，提供的培养基和培养方法，培养的白肉灵芝菌株液体发酵高效获得黄酮，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，从而为白肉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29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.40597；
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：白肉灵芝Ganoderma leucocontextum T.H.Li,W.Q.Deng,DongM.Wang&H.P.Hu。
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
本申请采用的菌种白肉灵芝菌株GA82是从采自云南省曲靖市沾益县，寄主为阔叶树倒木，通过分离和菌种纯化而得到，该菌株分类命名为白肉灵芝Ganodermaleucocontextum T.H.Li,W.Q.Deng,Dong M.Wang&H.P.Hu，已于2023年05月29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.40597。
子实体形态特征：担子果一年生，有柄，木栓质，无气味、味苦，干后变轻；菌盖匙形，长17.7cm，宽7.4cm，厚2.5cm，表面具强烈漆状光泽，深棕褐色，表面具细微的同心沟纹，边缘锐；菌柄近圆柱形，侧生，红褐色，长16.2cm，宽1.6cm；孔口表面幼时白色，伤后浅褐色，干时奶油色，多角形，4个/mm，管壁薄；菌肉乳白色，无同心环纹，质地较软，长2.2cm，无黑色壳质线；菌管乳白色，硬木栓质，不分层，长可达0.3cm。
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1.3-4.5μm，缠绕菌丝0.9-2.5μm，菌管菌丝中，骨架菌丝2.2-4.2μm，生殖菌丝1.7-2.9μm，缠绕菌丝1.3-2.6μm；皮壳细胞棍棒状，顶端略粗于底部，亮黄色，厚壁，长24-50×宽6-11μm，成熟担子果中有强烈的拟糊精反应；担孢子卵圆形，脐状突起不明显，黄褐色，无拟糊精反应，有嗜蓝反应，双层壁，外壁平滑，内壁表面具中度粗糙的点状纹饰，长(7-)7.3-9.3(-9.7)×宽5.2-5.9μm，平均长度8.03μm，平均宽度5.48μm，长宽比1.47(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GA82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30μL：2×EasyTaq PCR SuperMix 15.0μL，引物ITS5 10μmol/L 1.0μL，引物ITS4 10μmol/L1.0μL，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，结果与白肉灵芝菌株的相似性最高，因此菌株GA82鉴定为白肉灵芝Ganoderma leucocontextum T.H.Li,W.Q.Deng,Dong M.Wang&H.P.Hu，GenBank登记号为OR079443。
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，24℃恒温培养箱培养7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10.0g/L，蛋白胨4.0g/L，磷酸二氢钾1.0g/L，七水合硫酸镁0.4g/L，黄豆粉5.0g/L，苯丙氨酸0.3g/L，吐温802.0mL/L，赤霉酸0.02g/L，醋酸钠0.3g/L，维生素B1 0.01g/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24℃、150r/min振荡培养7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24℃、150r/min振荡培养7天后含食药用真菌灵芝胞外黄酮的溶液。
黄酮的提取：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，再配制成灵芝胞外黄酮水溶液。
黄酮的测定：称取0.01g芦丁标准品溶于70％乙醇并定容至50.0mL，即得芦丁标准液；分别吸取0、1.0、2.0、3.0、4.0、5.0、6.0mL芦丁标准液与6.0mL 70％乙醇及1.0mL 5％(m/V)亚硝酸钠溶液混合，静置6min后加入1.0mL 10％(m/V)硝酸铝溶液，混匀后静置6min，再加入10.0mL 4％(m/V)氢氧化钠溶液，最后用70％乙醇定容至25.0mL，混匀后静置15min，于510nm处测定吸光值，设3个平行，求平均值；以反应体系中芦丁标准品的浓度为横坐标、吸光值为纵坐标作标准曲线，计算回归方程为：y＝0.1459x-0.1314，相关系数R2＝0.9926。按制作标准曲线的方法进行样品的黄酮含量测定，分别以70％乙醇替代上清液、芦丁的反应体系设为空白及对照，黄酮含量以培养液中芦丁当量计。
结果：
表1各因子水平及试验结果(其他成分如下：玉米粉30.0g/L，葡萄糖10.0g/L，蛋白胨4.0g/L，磷酸二氢钾1.0g/L，七水合硫酸镁0.4g/L，维生素B1 0.01g/L)
本发明白肉灵芝菌株GA82的发酵液的黄酮含量高，说明本发明白肉灵芝菌株GA82的药用价值高，可用于制备提高人体免疫力或抗癌的保健品，或用于制备提高人力免疫力或抗癌的药物组分，附加值高，经济效益高。
Claims (7)
1.一种白肉灵芝Ganoderma leucocontextum菌株GA82，于2023年05月29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.40597。
2.一种利用权利要求1所述的白肉灵芝菌株GA82制备黄酮发酵液的方法，其特征在于，包括以下步骤：
(1)将白肉灵芝菌株接种在固体培养基上，活化，24℃恒温培养箱培养7天；
(2)将活化后的菌种接种在基础液体培养基上，恒温摇床24℃、150r/min振荡培养7天，制成种子发酵悬液；
(3)将种子发酵悬液接种在液体培养基于上，恒温摇床24℃、150r/min振荡培养7天得到含食药用真菌灵芝胞外黄酮的溶液。
3.根据权利要求2所述的白肉灵芝菌株GA82制备黄酮发酵液的方法，其特征在于，固体培养基为：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，pH5.0。
4.根据权利要求2所述的白肉灵芝菌株GA82制备黄酮发酵液的方法，其特征在于，液体培养基为：玉米粉30.0g/L，葡萄糖10.0g/L，蛋白胨4.0g/L，磷酸二氢钾1.0g/L，七水合硫酸镁0.4g/L，黄豆粉5.0g/L，苯丙氨酸0.3g/L，吐温80 2.0mL/L，赤霉酸0.02g/L，醋酸钠0.3g/L，维生素B1 0.01g/L，pH 5.0。
5.权利要求1所述的白肉灵芝菌株GA82的黄酮发酵液在制备保健品或食品中的应用。
6.一种食品或保健品，其特征在于，所述的食品或保健品中含有权利要求1所述的白肉灵芝菌株GA82的发酵液。
7.权利要求1所述的白肉灵芝菌株GA82的发酵液在制备提高人体免疫力或者抗癌药品中的应用。
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