CN115491313B - 一株蛹虫草白色菌株及其应用 - Google Patents
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Abstract
本发明公开了一种蛹虫草白色菌株MU001、选育过程、培养方法及其应用，属于食用菌育种领域。所述蛹虫草白色菌株MU001于2022年3月17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:40113。其选育方法为：将黄色蛹虫草出发菌株经活化、固体培养获得菌丝体；将菌丝体液体培养获得分生孢子；将分生孢子利用60Co‑γ诱变，高温初筛，复筛，突变体栽培筛选得到蛹虫草白色突变菌株MU001。本发明公开的白色蛹虫草相较于黄色蛹虫草具有更高的蛋白质含量，具有鲜美独特的口味，因此更受消费者的青睐，也是化妆品更好的原料。
Description
技术领域
本发明属于食用菌育种领域，具体涉及一种蛹虫草白色菌株MU001、选育过程、培养方法及其应用。
背景技术
蛹虫草（Cordyceps militaris(L.ex Fr.) Link.），又名北冬虫夏草、北虫草，与冬虫夏草同属异种。含有丰富的蛋白质、脂肪、虫草酸、虫草素、虫草多糖、SOD酶、多酚、类黄酮以及一些微量元素。具有与冬虫夏草极相似的有效成分和药用功效，如益精气、补虚损、抑菌、降压、镇静、降温、免疫作用等。虫草多糖、多酚、类黄酮等物质可高效抗氧化和清除自由基，以及延缓皮肤衰老作用，是多种化妆品的原料。人工栽培蛹虫草为橙黄色子实体，白色蛹虫草市场未见有销售。白色食用菌具有鲜美独特的口味，深受消费者的青睐，因此白色蛹虫草具有较大的市场前景。
发明内容
针对上述问题，本发明提供一种蛹虫草白色菌株及其应用。所述蛹虫草白色菌株MU001于2022年3月17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:40113。
为了实现上述目的，本发明提供了如下技术方案：
本发明提供了一种蛹虫草诱变白色菌株（Cordyceps militaris(L.ex Fr.)Link）MU001，其特征在于，所述蛹虫草诱变白色菌株MU001于2022年3月17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:40113。
进一步地，所述蛹虫草诱变白色菌株（Cordyceps militaris(L.ex Fr.) Link）MU001在制备具有抗氧化功能的化妆品中的应用。
进一步地，所述白色菌株MU001的培养方法具体步骤如下：将白色菌株MU001的菌块接种至PD培养基中，静置培养24h后置于摇床上振荡暗光培养；菌种长满培养基后进行稀释，接种到小麦培养基中，置于暗光培养，菌种吃透整个培养基后见光转色，温差刺激促进草芽形成。
更进一步地，所述培养方法步骤中摇床震荡培养温度为23℃，摇床转数为150 r·min-1。
更进一步地，所述培养方法步骤中菌种长满培养基后进行稀释的倍数为4倍，置于暗光培养的温度为20℃。
本发明还提供一种保健食品，其特征在于，包括上述任一项所述的白色蛹虫草菌株MU001。
本发明还提供一种药品，其特征在于，包括上述任一项所述的白色蛹虫草菌株MU001。
本发明还提供一种化妆品，其特征在于，包括上述任一项所述的白色蛹虫草菌株MU001。
与现有技术相比本发明的有益效果。
（1）目前人工栽培蛹虫草为橙黄色子实体，白色蛹虫草市场未见到。由于传统蛹虫草为橙黄色子实体，用作化妆品原料时需要高成本的脱色步骤，而本发明公开的蛹虫草为白色子实体，可减少化妆品制备过程中脱色步骤，节约成本。
（2）经过试验证实，本发明公开的白色蛹虫草MU001的提取物具有高的总蛋白含量和多肽。多肽是指氨基酸残基肽键的形式连接形成的化合物，蛹虫草蛋白和多肽对维持生物体的生命活动有益或针对某些疾病或病毒具有治疗或预防的作用，在保健和医药上有着广泛的应用，这表明本发明的新蛹虫草MU001在食品及医药领域具有重要的开发利用的前景。
（3）蛹虫草的鲜味与其含有丰富的蛋白质及氨基酸有关，本发明公开的白色蛹虫草MU001相较于黄色蛹虫草具有鲜美独特的口味，因此更受消费者的青睐。
附图说明
图1 白色蛹虫草MU001与黄色蛹虫草出发菌株WT见光后菌落对比。左图为白色蛹虫草MU001见光后的菌落。右图为黄色蛹虫草出发菌株WT见光后的菌落。出发的黄色菌株见光2天后，菌落变黄。白色突变株见光后不变色。
图2白色蛹虫草MU001与黄色蛹虫草出发菌株WT子实体比较。
图3白色突变菌株突变位点测序鉴定图。
具体实施方式
下面结合具体实施例对本发明做详细的说明。以下实施例将有助于对本发明的了解，但这些实施例仅为了对本发明加以说明，本发明并不限于这些内容。在实施例中的操作方法均为本技术领域常规操作方法。
实施例1白色蛹虫草MU001的选育。
1. 蛹虫草分生孢子液体培养基的配制。
玉米粉20g，麦麸100g，用纱布包好煮沸35min，取滤液加入20g葡萄糖，3g磷酸二氢钾，1.5g硫酸镁，定容至1000ml，PH自然，分装到250mL锥形瓶中。
2. 蛹虫草液体菌种的生产。
首先活化黄色蛹虫草出发菌株WT，将试管冷藏保存的蛹虫草WT取出后接种到PDA平皿中进行活化，培养条件23℃暗光培养，15天后见光培养。见光3天后用无菌打孔器沿虫草菌落的边缘打取直径9 mm的菌种块，每个锥形瓶中接入1块。置于23℃的摇床中以200r.min-1的速度恒温暗光培养5天，培养后液体菌种浑浊，显微镜下观察无菌丝，全是分生孢子。
3. 蛹虫草分生孢子的60Co-γ射线诱变。
将孢子悬液分装在2mL保藏管中，每个保藏管分装1mL孢子悬液，置于辐射台面中进行60Co-γ射线照射，累计辐射量为600Gy，剂量率为3Gy.min-1。将辐照后的孢子悬液稀释104倍，以出发菌株为对照，诱变致死率是95.2％。
4. 高温胁迫诱变分生孢子。
将辐射后的分生孢子保藏管置于60℃水浴锅中水浴1.5min。以未水浴的保藏管分生孢子做对照，60℃水浴锅中水浴1.5min分生孢子致死率52%。
5. 小麦培养基的制备。
750 mL栽培瓶装40g小麦（干料），60mL水，采用高压灭菌法，温度121~126℃，灭菌40min。
6. 处理后的分生孢子白色突变体筛选.
处理后的分生孢子稀释104倍，涂到PDA平皿中23℃暗光培养，3天后可见点状菌落并见光培养，6天后挑起不转色菌落接种到PD的锥形瓶中，静置培养24 h，置于摇床上进行振荡暗光培养，摇床转数150 r·min-1，温度23℃，8天后菌丝布满液体培养基中稀释4倍接种到小麦培养基中。置于20℃暗光培养，菌种吃透整个培养基后见光，5度温差刺激促进草芽形成。变异的蛹虫草在出白色草芽并草芽长到2cm时，连续组织分离继代培养3代，白色突变菌株稳定后组织分离接种到试管中保存，定名为MU001。
实施例2白色蛹虫草MU001与黄色蛹虫草出发菌株WT的比较。
1. 白色蛹虫草MU001与黄色蛹虫草出发菌株WT子实体比较：两种菌株均为接种50天后的子实体（见图2），出发菌株橙黄色，白色突变菌株白色，子实体高度明显低于出发菌株。
2. 白色蛹虫草MU001与黄色蛹虫草出发菌株WT鲜重和干重对比：分别取出发菌株和白色突变菌株菌块接种放入PD培养基中。静置培养24h，置于摇床上进行振荡暗光培养，摇床转数150 r·min-1，温度23℃，培养时间5d。菌种长满培养基后稀释4倍接种到小麦培养基中，置于20℃暗光培养，菌种吃透整个培养基后见光转色，温差刺激促进草芽形成。70d后黄色蛹虫草子实体不再增粗增长，头部膨大，白化突变体MU001生长健壮，子实体继续增粗增长。此阶段采收后称取鲜重、干重，白色蛹虫草鲜重、干重都低于黄色原始菌株，见下表。
。
3. 白色突变菌株突变位点：
通过基因组分析，蛹虫草白化突变体MU001基因组中polyketide synthase聚酮合酶PKS（基因ID：CM_09042）核酸序列第2029位核苷酸由出发菌株WT的C突变为T，导致该位置编码的氨基酸由出发菌株WT密码子CGC编码精氨酸R突变为TGC编码半胱氨酸C。精氨酸R为碱性氨基酸，分子量：174.188，等电点PI：10.76；而半胱氨酸C为含硫类氨基酸，分子量：121.145，等电点PI：5.02。同时，该位点位于聚酮合酶PKS酮基合成酶PKI结构域上，氨基酸性质的改变将引起蛋白空间构象发生改变，从而影响蛋白功能。
4．白色蛹虫草MU001与黄色蛹虫草出发菌株WT总蛋白含量。
采收的蛹虫草通过冷冻干燥后，依照南京建成生物工程研究所的蛋白定量（TP）测定试剂盒进行检测。白色蛹虫草MU001总蛋白含量38.7%，黄色蛹虫草出发菌株WT总蛋白含量34.6%。
5. 白色蛹虫草MU001与黄色蛹虫草出发菌株WT的SOD活性测定。
采收的蛹虫草通过冷冻干燥后，依照南京建成生物工程研究所的总超氧化物歧化酶（T-SOD）测试盒进行检测。白色蛹虫草MU001的SOD含量326U／g，黄色蛹虫草出发菌株WT的SOD含量212U／g。
Claims (5)
1.一种蛹虫草诱变白色菌株（Cordyceps militaris）MU001，其特征在于，所述蛹虫草诱变白色菌株MU001于2022年3月17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:40113；
所述白色菌株MU001的培养方法具体步骤如下：将白色菌株MU001的菌块接种至PD培养基中，静置培养24h后置于温度为23℃，摇床转数为150 r·min-1的摇床上振荡暗光培养；菌种长满培养基后以倍数为4倍进行稀释，接种到小麦培养基中，置于暗光20℃培养，菌种吃透整个培养基后见光转色，温差刺激促进草芽形成。
2.如权利要求1所述的一种蛹虫草诱变白色菌株（Cordyceps militaris）MU001在制备具有抗氧化功能的化妆品中的应用。
3.一种保健食品，其特征在于，包括权利要求1所述的白色蛹虫草菌株MU001。
4.一种药品，其特征在于，包括权利要求1所述的白色蛹虫草菌株MU001。
5.一种化妆品，其特征在于，包括权利要求1所述的白色蛹虫草菌株MU001。
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