CN104642142A - 一种灵芝菌种的诱变及选育方法 - Google Patents
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Abstract
一种灵芝菌种的诱变及选育方法，使用该方法来处理从自然界分离到的野生梅灵芝菌种，可得到满足适合灵芝沉没发酵工程技术的生产菌种，利用这些生产菌种可大量生产多种有效成分稳定的富含天然灵芝多醣、灵芝有机锗、三萜烯化合物和甘露醇等有效成分的天然有机灵芝口服液，从而实现灵芝制品的规模化生产。服用该灵芝口服液对调整血压，降低血粘度，净化血液，促进细胞活化，防止动脉硬化，提高代谢水平，增强免疫功能，改善睡眠，提高睡眠质量，补肾壮阳，益智安神，调节自由基，抗癌、防癌、抗衰老等方面均有良好的作用，市场前景非常广阔。
Description
技术领域
本发明属于生物育种技术的菌种选育技术领域，特别涉及一种灵芝沉没发酵菌种的诱变育种方法。
背景技术
灵芝制品含有丰富的多种灵芝多糖、灵芝有机锗以及C27以下的灵芝三萜烯化合物。其有效成份通常包括灵芝多糖、灵芝多肽、三萜类、16种氨基酸(其中含有七种人体必需氨基酸)、蛋白质、甾类、甘露醇、香豆精苷、生物碱、有机酸(主含延胡索酸)，以及微量元素Ge、P、Fe、Ca、Mn、Zn、等。在抗肿瘤、保肝解毒、防治心血管疾病、防治高血脂、防治中风、抗衰老、抗神经衰弱的各个方面都有明显的医疗功效。然而，从自然界采集的野生灵芝菌种，虽然也能进行人工栽培，但却不能满足工业化大规模生产灵芝制品对菌种的特殊要求，包括：发酵条件、培养基、温度、湿度，以及它在发酵过程中对基质的合成、转化、降解等的生理生化变化。因此必须采用特殊的诱变育种技术来处理野生灵芝菌种，以求获得优良的生产菌种来满足规模化生产要求。
发明内容
本发明的目的是提供一种新的高科技生物技术方法(包括高科技生物遗传育种技术、诱变育种技术及菌种选育技术等)，来处理自然界中分离到的野生灵芝菌种，从而得到满足适合灵芝沉没发酵工程技术的菌种，实现灵芝制品的规模化生产。
该方法包括以下步骤：
步骤一，利用原生质体分离技术，在从梅树下采集到的梅灵芝野生菌子实体的菌盖和菌柄上进行原生质体分离纯化，接种到基础培养基进行培养，经过培养15～20天后，对菌丝体进行反复分离、纯化，获得野生梅灵芝菌种(菌丝体)。分离梅灵芝子实体菌盖和菌柄进行原生质体分离纯化的基础培养基为：
步骤二，对分离到的野生梅灵芝菌丝体进行单因子和多因子正交试验；单因子正交试验对基础培养基进行调整，获得满意的培养基；多因子正交试验对沉没发酵中的培养基，包括氮源、碳源、微量元素、培养温度、需氧量等，进行最佳发酵条件的选择，获得野生梅灵芝菌丝体的最佳培养基、最佳培养温度和需氧量。
步骤三，采用紫外线灯照射诱变、叠氮化钠(NaN3)化学诱变、80℃～90℃的无菌水瞬间加热对野生灵芝菌种进行处理，最终获得规模化灵芝沉没发酵生产菌种。利用这些生产菌种可大量生产多种有效成分稳定的富含天然灵芝多醣、灵芝有机锗、三萜烯化合物和甘露醇等有效成分的天然有机灵芝口服液。服用该灵芝口服液对调整血压，降低血粘度，净化血液，促进细胞活化，防止动脉硬化，提高代谢水平，增强免疫功能，改善睡眠，提高睡眠质量，补肾壮阳，益智安神，调节自由基，抗癌、防癌、抗衰老等方面均有良好的作用。尤其是对抗癌、防癌、提高抗癌免疫功能、结合放、化疗治疗，能有效增强治疗效果，改善睡眠，并还具有深度减轻放化疗后的毒副作用的功效。还对避免自由基对机体的危害以及避免氧自由基反应和脂质过氧化反应产生的代谢紊乱失调，能有效补充能量、增强体质、恢复体能、改善供氧、缺氧状况，增强耐力，改善睡眠，提高睡眠质量、补肾壮阳、调整电解质平衡，排出体内废物及毒素等，发挥最佳体能状态。市场前景非常广阔。
具体实施方式
下面对本发明的实施例进行具体说明：
第一步，先利用原生质体分离技术，在从梅树下采集到的梅灵芝野生菌子实体的菌盖和菌柄上进行原生质体分离纯化，接种到基础培养基进行培养，经过培养15天后，对菌丝体进行反复分离、纯化，获得野生梅灵芝菌种(菌丝体)。
其中，分离梅灵芝子实体菌盖和菌柄进行原生质体分离纯化的基础培养基为：
第二步，第一步完成之后，对分离到的野生梅灵芝菌丝体进行单因子和多因子正交试验：
(1)单因子正交试验
该实验目的是对基础培养基进行调整，以求获得满意的培养基。
先称取梅树下的土壤50g，梅树叶和梅树枝各100g，用纯净水5000ml煮沸20分钟，过滤后加足到5000m1并加入上述基础培养基的不同比例的其他成分，溶解后分装在500ml的三角瓶中，每瓶200ml，并编号接种野生梅灵芝菌丝体；培养10天后，将三角瓶中的菌丝体用滤纸过滤并在105±1℃温度中烘烤24小时，进行称量、比较，获得满意的培养基如下：
(2)多因子正交试验
该实验是对沉没发酵中的培养基，包括氮源、碳源、微量元素、培养温度、需氧量等，进行最佳发酵条件的选择。
最后获得的最佳发酵条件为：
最佳培养基：
最佳培养温度：32±1℃
需氧量：每分钟220～240转。
第三步，第二步完成之后，采用紫外线灯照射诱变、叠氮化钠(NaN3)化学诱变、80℃～90℃的无菌水瞬间加热对野生灵芝菌种进行处理，最终获得规模化灵芝沉没发酵生产菌种。
在该步骤中，紫外线灯照射诱变是用40瓦的紫外线灯在20cm距离反复对野生梅灵芝菌丝体进行诱变育种处理，时间设置为下表所示：
| 编号1处理时间 | 编号2处理时间 | 编号3处理时间 |
| 5分钟 | 10分钟 | 20分钟 |
在该步骤中，叠氮化钠(NaN3)化学诱变是先用DMF二甲基甲酰胺和DMSO二甲基亚砜各5ml，将1g1.2当量的叠氮化钠(NaN3)放入其中溶解，作为化学诱变剂，再将编号1～3处理的样品放入其中进行处理，处理时间为下表所示：
| 编号1处理时间 | 编号2处理时间 | 编号3处理时间 |
| 5分钟 | 10分钟 | 20分钟 |
处理后编号1～3样品用无菌水反复洗涤3遍；85℃±1℃的无菌水瞬间加热，处理时间为下表所示：
| 编号1处理时间 | 编号2处理时间 | 编号3处理时间 |
| 1秒 | 5秒 | 10秒 |
在本发明实施例中，按上述方法处理野生梅灵芝菌种，并反复对野生梅灵芝菌丝体进行诱变育种，成功得到了满意的规模化生产菌种，用于生产多功效“灵宝康口服液”。该口服液为富含天然灵芝多醣、灵芝有机锗、三萜烯化合物等有效成分的天然有机灵芝口服液，成分稳定，对调整血压、降低血粘度、净化血液，促进细胞活化、防止动脉硬化、提高代谢水平、增强免疫功能、改善睡眠、提高睡眠质量、补肾壮阳、益智安神、调节自由基、抗癌、防癌、抗衰老等方面均有良好的作用。
Claims (5)
1.一种灵芝菌种的诱变及选育方法，其特征在于该方法包括以下步骤：
步骤一，利用原生质体分离技术，在从梅树下采集到的梅灵芝野生菌子实体的菌盖和菌柄上进行原生质体分离纯化，接种到基础培养基进行培养，经过培养15～20天后，对菌丝体进行反复分离、纯化，获得野生梅灵芝菌种(菌丝体)；
步骤二，对分离到的野生梅灵芝菌丝体进行单因子和多因子正交试验；单因子正交试验对基础培养基进行调整，获得满意的培养基；多因子正交试验对沉没发酵中的培养基(包括氮源、碳源、微量元素)，培养温度、需氧量等，进行最佳沉没发酵条件的选择，获得野生梅灵芝菌丝体的最佳培养基、最佳培养温度和最佳需氧量；
步骤三，采用紫外线灯照射诱变、叠氮化钠(NaN3)化学诱变、80℃～90℃的无菌水瞬间加热对野生灵芝菌种进行联合诱变育种处理，最终获得规模化灵芝沉没发酵生产菌种。
2.根据权利要求1所述的一种灵芝菌种的诱变及选育方法，其特征在于：步骤一所述的分离梅灵芝子实体菌盖和菌柄进行原生质体分离纯化的基础培养基为：
3.根据权利要求1所述的一种灵芝菌种的诱变及选育方法，其特征在于：步骤二所述的对分离到的野生梅灵芝菌丝体进行单因子正交试验时，先称取梅树下的土壤50g，梅树叶和梅树枝各100g，用纯净水5000ml煮沸20分钟，过滤后加足到5000ml并加入上述基础培养基的不同比例的 其他成分，溶解后分装在500ml的三角瓶中，每瓶200ml，并编号接种野生梅灵芝菌丝体；培养10～12天后，将三角瓶中的菌丝体用滤纸过滤并在105±1℃温度中烘烤24小时，进行称量、比较，获得满意的培养基如下：
。
4.根据权利要求1所述的一种灵芝菌种的诱变及选育方法，其特征在于：步骤二在对分离到的野生梅灵芝菌丝体进行多因子正交试验时，最后获得的发酵条件为：
最佳培养基：
最佳培养温度：32±1℃
最佳需氧量：每分钟220～240转。
5.根据权利要求1所述的一种灵芝菌种的诱变及选育方法，其特征在于：步骤三所述的紫外线灯照射诱变是用40瓦的紫外线灯在20cm距离反复对野生梅灵芝菌丝体进行诱变处理，时间设置为下表所示：
；所述的叠氮化钠(NaN3)化学诱变是先用DMF二甲基甲酰胺和DMSO二甲基亚砜各5ml，将1g1.2当量的叠氮化钠(NaN3)放入其中溶解，作为化学诱变剂，再将编号1～3处理的样品放入其中进行处理，处理时间为下表所示：
处理后编号1～3样品用无菌水反复洗涤3遍；所述的80℃～90℃的无菌水瞬间加热，处理时间为下表所示：
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| CN107384809A (zh) * | 2017-09-05 | 2017-11-24 | 云南明侍达生物科技有限责任公司 | 一株灵芝菌及其应用 |
| CN109479634A (zh) * | 2018-12-07 | 2019-03-19 | 淄博市农业科学研究院 | 一种番茄育种装置 |
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| CN110616152A (zh) * | 2019-09-20 | 2019-12-27 | 广东省微生物研究所（广东省微生物分析检测中心） | 一种改良的真菌原生质体的制备方法 |
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