CN110387333A - 一种用兰坪虫草菌粉培养牛樟芝的方法 - Google Patents
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Abstract
本发明属于微生物技术领域,具体涉及一种用兰坪虫草菌粉培养牛樟芝的方法。主要操作步骤如下:牛樟芝的菌种接种于改良的PDA固体培养基,进行菌种活化;然后将活化的菌种接种在液体培养基上制成种子液;以兰坪虫草菌粉作为添加剂,配制发酵培养基,灭菌备用;将牛樟芝种子液接种于发酵培养基进行摇床培养;培养结束后,发酵液过滤,滤渣即为所需的牛樟芝菌丝体。本发明为首创,利用该方法可以稳定、连续、批量地培养药用价值较高的牛樟芝菌丝体。
Description
技术领域
本发明属于微生物技术领域,具体涉及一种用兰坪虫草菌粉培养牛樟芝的方法。
背景技术
牛樟芝(Antrodia cinnamomea)又名樟芝、樟菇、牛樟菇、红樟芝、血灵芝,是一种生长在牛樟树上的珍稀药用真菌。现代药理研究表明,牛樟芝具有抗肿瘤、保肝、降血压、降胆固醇、抑制血小板凝集等生理活性,自1990 年开始逐渐成医疗、保健品研发的热点,目前已用于治疗多种疾病。虽然牛樟芝药效显著,但生长过程极为缓慢,药用成分也不稳定,要想以普通栽培方法获得药用价值较高的牛樟芝菌体十分困难。
兰坪虫草(Ophiocordyceps lanpingensis)是本实验室于2013年发表的线虫草属新种,与冬虫夏草有较近的亲缘关系。研究表明,人工培养的兰坪虫草菌粉与西藏野生冬虫夏草的15种活性成分含量极为相似,可部分代替冬虫夏草入药;从兰坪虫草菌粉中提取的粗多糖对果蝇延缓衰老效果显著,0.2%的粗多糖能明显延缓雌、雄果蝇的平均寿命、最高寿命和半数死亡时间;兰坪虫草具有较强的镇痛作用,它的发酵提取物中石油醚部分、乙酸乙酯部分、无水乙醇部分和水提部分均有外周性镇痛效果。本发明将人工培养的兰坪虫草菌粉作为添加剂,用于牛樟芝的液体发酵,再通过严格的发酵条件控制,最终获得药用价值较高的牛樟芝菌丝体。
发明内容
本发明的目的在于提供一种用兰坪虫草菌粉培养牛樟芝的方法,这种方法培养的牛樟芝菌丝体药用价值较高。
为实现上述目的,本发明采用如下技术方案:
将牛樟芝菌株接种于改良的PDA固体培养基进行活化(该菌株已于2017年3月1日保藏于云南省微生物研究所菌种保藏中心,保藏号为YIM Yu201702)。然后将活化的菌种接种在液体培养基上制成种子液。选取生长良好的兰坪虫草子实体,进行冻干、粉碎处理,制成兰坪虫草菌粉。按兰坪虫草菌粉5-15g、蛋白胨10-20g、马铃薯浸粉20-30g、葡萄糖20-30g,水1000mL配置发酵培养基,121℃灭菌20分钟后自然冷却、备用。以5-10%的接种量接种牛樟芝种子液,在温度为25-30℃、转速为100-150r/min的条件下进行摇床培养。待20-30天长出大量菌丝体后将发酵液过滤。菌丝体沉淀经蒸馏水清洗 3 次、65℃烘干,密封保存。
具体实施方式
以下实施例可以对本发明作进一步说明:
1. PDA固体培养基配制:取没有发芽、变青的马铃薯,洗净去皮,称取200g,切成1厘米左右见方的小块。将切好的马铃薯小块放入约1000mL水中,煮沸后用温火保持30分钟。结束后用纱布过滤,得到滤液为马铃薯汁,将滤液补足至1000mL。然后加入葡萄糖20g,蛋白胨5g,琼脂20g,搅拌均匀后分别倒入试管,在121℃灭菌20分钟,取出后倾斜放置,待冷却凝固后可用于转种。
2. 液体培养基配制:取没有发芽、变青的马铃薯,洗净去皮,称取200g,切成1厘米左右见方的小块。将切好的马铃薯小块放入约1000mL水中,煮沸后用温火保持30分钟。结束后用纱布过滤,得到滤液为马铃薯汁,将滤液补足至1000mL后分装成500mL/瓶(三角瓶为1000mL规格)。每瓶加入葡萄糖10g,玉米粉5g,蛋白胨5g,酵母浸粉3g,硫酸镁1.0g,磷酸二氢钾0.5g,搅拌均匀,塞上棉塞,在121℃灭菌20分钟后自然冷却、备用。
3. 制种:取牛樟芝菌种,无菌条件下接入步骤1制作的固体斜面培养基,28℃避光培养。菌丝覆盖表面约70%时,可将菌丝接入液体培养基制种。制种过程如下:从斜面培养基上取1小块(大小约3-5mm×3-5mm)菌丝放入装有液体培养基的三角瓶内,在120r/min、28℃下培养约10天,待菌球长至适宜大小,即可用于接种。
4. 兰坪虫草的培养与处理:以大米和蚕蛹粉为培养基原料,培养兰坪虫草子实体。在40天左右选取生长良好的兰坪虫草子实体,进行冻干、粉碎处理,制成兰坪虫草菌粉。用于培养兰坪虫草子实体的菌株保藏于云南省微生物研究所菌种保藏中心,保藏号为YIMYu201301。
5. 发酵培养基配制:按配方配制发酵培养基,分装成200mL/瓶(三角瓶为500mL规格),121℃灭菌20分钟后自然冷却、备用。发酵培养基配方为:兰坪虫草菌粉5g、蛋白胨10g、马铃薯浸粉20g、葡萄糖25g,水1000mL,pH自然。
6. 接种和培养:以10%的接种量接种牛樟芝种子液于发酵培养基,在温度为28℃、转速为120r/min的条件下进行摇床培养。
7. 采收:待三角瓶中长出大量牛樟芝菌丝体后将发酵液过滤。菌丝体沉淀经蒸馏水清洗 3 次、65℃烘干,密封保存。
8. 兰坪虫草菌粉培养的牛樟芝有效成分分析
经检测,上述干燥、粉碎的牛樟芝菌丝体中,每1g含多糖、D-甘露醇、总三萜、麦角甾醇分别为30.48mg、70.15mg、34.63mg、4.72μg,明显优于兰坪虫草菌粉和普通牛樟芝(见表1)。
按照上述方法进行多批次培养,结果稳定。
表1 兰坪虫草菌粉培养的牛樟芝有效成分比较(n=3)
样品 | 多糖mg/g | D-甘露醇mg/g | 总三萜mg/g | 麦角甾醇μg/g |
兰坪虫草菌粉培养的牛樟芝 | 30.48±2.67 | 70.15±2.08 | 34.63±1.49 | 4.72±0.12 |
兰坪虫草菌粉 | 23.79±4.47 | 51.39±3.33 | 28.57±1.53 | 1.51±0.02 |
普通牛樟芝 | 3.36±0.27 | 32.96±3.29 | 16.04±0.32 | 1.06±0.02 |
注:普通牛樟芝,指不添加兰坪虫草菌粉的发酵培养基培养的牛樟芝菌丝体。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (5)
1.一种用兰坪虫草菌粉培养牛樟芝的方法,其特征在于:
a、将牛樟芝菌种接种于改良的PDA固体培养基,进行菌种活化;
b、将活化的菌种接种在液体培养基上进行种子液的配制;
c、取500mL三角瓶装入160-200mL发酵培养基,湿热灭菌并冷却后,按5-10%(w/w)的接种量加入牛樟芝种子液,在温度为25-30℃、转速为100-150r/min的条件下进行摇床培养。
2.根据权利要求1所述的方法,其特征在于a所述改良PDA固体培养基原料配比为:马铃薯汁1000mL,葡萄糖5-25g,蛋白胨5-15g,琼脂15-20g。
这里的马铃薯汁由下述步骤获得:将切成小块的去皮马铃薯放入水中,马铃薯与水的重量比为1:5-1:6,煮沸后温火保持25-40min,滤去渣,滤液即为马铃薯汁,以下马铃薯汁与此相同。
3.根据权利要求1所述的方法,其特征在于b所述液体培养基原料配比为:马铃薯汁1000mL,葡萄糖5-25g,玉米粉5-25g,蛋白胨5-15g,酵母浸粉5-10g,硫酸镁1.0-3.5g,磷酸二氢钾1.0-2.0g。
4.根据权利要求1所述的方法,其特征在于c所述发酵培养基的配方为:兰坪虫草菌粉5-15g、蛋白胨10-20g、马铃薯浸粉20-30g、葡萄糖20-30g,水1000mL,pH自然。
5.根据权利要求4所述的兰坪虫草菌粉,其特征在于菌粉是由兰坪虫草菌株固体发酵,再经冻干和粉碎处理精制而成。
这里的兰坪虫草菌株保藏于云南省微生物研究所菌种保藏中心,保藏号为YIMYu201301。
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