CN113599293A - 一种抗辐射型美白保湿面霜 - Google Patents
一种抗辐射型美白保湿面霜 Download PDFInfo
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- CN113599293A CN113599293A CN202110939066.7A CN202110939066A CN113599293A CN 113599293 A CN113599293 A CN 113599293A CN 202110939066 A CN202110939066 A CN 202110939066A CN 113599293 A CN113599293 A CN 113599293A
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- radiation
- melanin
- rehmannia
- curdlan
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Abstract
本发明公开了一种抗辐射型美白保湿面霜,包括毛蕊花糖苷、黑色素、凝胶多糖、甘油、透明质酸、精氨酸、谷胱甘肽、乳化剂、珍珠粉、维生素E、水。黑色素能够保护机体免受太阳光、紫外光、核辐射、电离辐射、X辐射以及γ射线等导致的损伤,凝胶多糖可以将黑色素牢牢的聚凝在一起,保护皮肤。凝胶多糖加热80℃以上可转化成不可逆的高强度凝胶,不但可以牢牢锁住水分和黑色素,还能避免黑色素进入皮肤内部,还不影响其他护肤成分对皮肤内部的渗透。毛蕊花糖苷具有超强的抗氧化活性,利用转基因技术使毛蕊花糖苷在地黄发状根大量表达。地黄发状根培养较单纯植物体生长快,产量稳定,也不受季节影响。
Description
技术领域
本发明涉及化妆品技术领域,具体为一种抗辐射型美白保湿面霜。
背景技术
随着辐射技术在生产、科研、医疗、军事等方面的广泛应用,人体受放射线辐射的机会越来越多,辐射对人体的危害也日趋明显和严重。
辐射是一种重要的氧化应激因素。电离辐射可以作用于机体内水分子,水分子被电离激发,产生大量具有强氧化性能的自由基,自由基与生物大分子作用,从而间接地引起组织细胞变性、坏死,导致机体辐射损伤的发生。因此,从抗氧化、清除自由基的角度寻找有效的辐射防护剂已成为现代医药研发的一个重要思路。
而皮肤作为身体第一道防线,受辐射后可引起细胞损伤、皮肤黑色素堆积、皮肤老化等问题。
因此,设计抗辐射和保湿效果好的一种抗辐射型美白保湿面霜及其制备方法是很有必要的。
发明内容
本发明的目的在于提供一种抗辐射型美白保湿面霜及其制备方法,以解决上述背景技术中提出的问题。
为了解决上述技术问题,本发明提供如下技术方案:一种抗辐射型美白保湿面霜,包括以下重量成分组成:毛蕊花糖苷3-10份、黑色素2-7份、凝胶多糖发酵液5-10份、甘油4-6份、透明质酸3-10份、谷胱甘肽0.8-3份、乳化剂3-5份、珍珠粉3-10份、维生素E0.6-1.5份、水8-20份。
一种抗辐射型美白保湿面霜的制备方法,包括以下具体步骤:
步骤一:取黑色素、1/2水、凝胶多糖发酵液,90℃下60-120rpm搅拌4h,获得A相;
步骤二:将毛蕊花糖苷、透明质酸、甘油、珍珠粉、精氨酸、谷胱甘肽、维生素E和1/2水70℃下100-200rpm搅拌1h,冷却至室温,获得B相;
步骤三:将乳化剂与A相、B相混合搅拌,40℃下80-200rpm搅拌3h制得面霜。
根据上述技术方案,所述黑色素提取自粒毛盘菌发酵液或黑芝麻中。
根据上述技术方案,所述凝胶多糖发酵液是利用假单胞菌发酵获得的:蔗糖30g/L,酵母粉8g/L,碳酸钙3g/L,发酵液初始pH值7.5-8.5,发酵周期为3-5天,发酵温度为30-35℃;取发酵完成的发酵液,5000-8000rpm离心30分钟,取上清液,低温蒸发浓缩得凝胶多糖发酵液。
根据上述技术方案,所述于毛蕊花糖苷是从地黄发状根中提取的。
根据上述技术方案,所述地黄发状根,用发根农杆菌A4对地黄叶片进行侵染,外植体龄为7-14D。
根据上述技术方案,所述用发根农杆菌对面积为0.5*0.5cm的地黄叶片进行侵染,侵染时间为5-10min后,然后在无菌滤纸上吸干菌液,将叶片倒置于共培养固体培养基中,每个培养皿中放置10个叶片,于26℃,黑暗条件下共培养,共培养培养基为MS基,添加100μmol/L的AS;共培养2d后用无菌水冲洗3次,无菌滤纸吸干液体后,将叶片接种于筛选培养基中,26℃暗培养,筛选培养基为MS+0.4g/L的Cef+100μmol/L的AS。
根据上述技术方案,所述发根农杆菌其Ri质粒中含有拟南芥21R1R2R3-MYB基因,通过PCR扩增MYB基因,经酶双切后,连接到Ri质粒。
根据上述技术方案,所述筛选培养基存活的地黄发状根进行PCR检测:根据设计的一对MYB基因引物,电泳检测出现了与基因引物相同的条带,而未转化的地黄发状根则没有扩增出条带,证明重组基因己经整合进地黄发状根的基因组中。
根据上述技术方案,所述地黄发状根转接到液体培养基中扩大增殖培养:用YEB培养基,牛肉浸膏5g/L,酵母膏1g/L,蛋白胨5g/L,七水硫酸镁0.5g/L,琼脂15g/L,培养20D后,添加终浓度为25μmol/L的SA,12h后取地黄发状根备用。
黑色素能够保护机体免受太阳光、紫外光、核辐射、电离辐射、X辐射以及γ射线等导致的损伤,是一种内源性的抗辐射剂。黑色素本身能够清除OH、O2ˉ和DPPH等自由基、螯合Fe2+、防止脂质过氧化等功能,对温度、光照、PH等条件下的稳定性较好。
粒毛盘菌胞内黑色素C、H、N、O和S的百分比分别为60.71%、7.61%、6.19%、24.94%和0.55%,有羰基和-NH2或者-OH的存在;黑芝麻黑色素的降解产物硅烷化衍生物经过气相色谱-质谱分析,分析发现其降解产物中含儿茶酸、儿茶酚以及对苯二酚,这一结果说明黑芝麻黑色素属儿茶酚型黑色素。
凝胶多糖提取自假单胞菌发酵胞外产物,较纯的凝胶多糖是白色粉末状固体,能够溶于水。凝胶多糖的分子量为618KDa,是一种直链的β-1,3-葡聚糖,表面多孔,比表面积较大,是一种微生物凝胶、絮凝剂。
因凝胶多糖和黑色素的结构特点,凝胶多糖可以将黑色素牢牢的聚凝在一起,用来对抗外界辐射损伤,达到保护皮肤的作用;还能避免黑色素进入皮肤内部,增加皮肤黑色素含量。
凝胶多糖还有一个特点,加热80℃以上可转化成不可逆的高强度凝胶,可以牢牢锁住水分,还不影响毛蕊花糖苷、珍珠粉、甘油、珍珠粉、精氨酸、谷胱甘肽、维生素E对皮肤内部的渗透。
通过离心去除假单胞菌发酵液中菌体,通过高温不但可以杀死残存的菌体,还可以将发酵液中残存的蔗糖、酵母粉和碳酸钙聚凝留在凝胶多糖胶体内部,节省了提取步骤;而且酵母粉也具有美白作用。
毛蕊花糖苷属于双糖苷,是一种对苯乙醇苷,含有多个共轭不饱和体系及酚羟基结构单元,具有较强的抗氧化活性。能够结合体内的自由基,避免自由基在体内大量蓄积,还可促进DNA、RNA合成,有效对抗了电离辐射引发的生物效应。而且毛蕊花糖苷空间位阻较小,利于与自由基接近,其结构内还含有邻二酚羟基,是具有抗氧化活性的重要基团。
拟南芥21R1R2R3-MYB基因是一种促进毛蕊花糖苷多糖表达的调控基因,利用发根农杆菌对地黄外植体进行侵染,可获得高毛蕊花糖苷表达量的地黄发状根。地黄发状根培养较单纯植物体生长快,产量稳定,也不受季节影响。利用水杨酸(SA)可促进毛蕊花糖苷表达的特点,用SA刺激地黄发状根12h,可获得高浓度的毛蕊花糖苷。
与现有技术相比,本发明所达到的有益效果是:
(1)黑色素能够保护机体免受太阳光、紫外光、核辐射、电离辐射、X辐射以及γ射线等导致的损伤,凝胶多糖可以将黑色素牢牢的聚凝在一起,用来对抗外界辐射损伤,达到保护皮肤的作用;还能避免黑色素进入皮肤内部,增加皮肤黑色素含量;
(2)凝胶多糖加热80℃以上可转化成不可逆的高强度凝胶,可以牢牢锁住水分,还不影响毛蕊花糖苷、透明质酸、珍珠粉、甘油、珍珠粉、精氨酸、谷胱甘肽、维生素E对皮肤内部的渗透;通过高温不但可以杀死残存的菌体,还可以将发酵液中残存的蔗糖、酵母粉和碳酸钙聚凝留在凝胶多糖胶体内部,节省了提取步骤;残存的酵母粉也具有美白作用;
(3)毛蕊花糖苷具有超强的抗氧化活性,利用转基因技术,促进毛蕊花糖苷在地黄发状根中大量表达;地黄发状根培养较单纯植物体生长快,产量稳定,也不受季节影响;利用水杨酸(SA)可促进毛蕊花糖苷表达的特点,用SA刺激地黄发状根12h,可获得高浓度的毛蕊花糖苷。
具体实施方式
下面将结合本发明中各实施例,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明提供技术方案:一种抗辐射型美白保湿面霜,包括以下重量成分组成:毛蕊花糖苷3-10份、黑色素2-7份、凝胶多糖发酵液5-10份、甘油4-6份、透明质酸3-10份、谷胱甘肽0.8-3份、乳化剂3-5份、珍珠粉3-10份、维生素E0.6-1.5份、水8-20份。
一种抗辐射型美白保湿面霜的制备方法,包括以下具体步骤:
步骤一:取黑色素、1/2水、凝胶多糖发酵液,90℃下60-120rpm搅拌4h,获得A相;
步骤二:将毛蕊花糖苷、透明质酸、甘油、珍珠粉、精氨酸、谷胱甘肽、维生素E和1/2水70℃下100-200rpm搅拌1h,冷却至室温,获得B相;
步骤三:将乳化剂与A相、B相混合搅拌,40℃下80-200rpm搅拌3h制得面霜。
根据上述技术方案,所述黑色素提取自粒毛盘菌发酵液或黑芝麻中。
根据上述技术方案,所述凝胶多糖发酵液是利用假单胞菌发酵获得的:蔗糖30g/L,酵母粉8g/L,碳酸钙3g/L,发酵液初始pH值7.5-8.5,发酵周期为3-5天,发酵温度为30-35℃;取发酵完成的发酵液,5000-8000rpm离心30分钟,取上清液,低温蒸发浓缩得凝胶多糖发酵液。
根据上述技术方案,所述于毛蕊花糖苷是从地黄发状根中提取的。
根据上述技术方案,所述地黄发状根,用发根农杆菌A4对地黄叶片进行侵染,外植体龄为7-14D。
根据上述技术方案,所述用发根农杆菌对面积为0.5*0.5cm的地黄叶片进行侵染,侵染时间为5-10min后,然后在无菌滤纸上吸干菌液,将叶片倒置于共培养固体培养基中,每个培养皿中放置10个叶片,于26℃,黑暗条件下共培养,共培养培养基为MS基,添加100μmol/L的AS;共培养2d后用无菌水冲洗3次,无菌滤纸吸干液体后,将叶片接种于筛选培养基中,26℃暗培养,筛选培养基为MS+0.4g/L的Cef+100μmol/L的AS。
根据上述技术方案,所述发根农杆菌其Ri质粒中含有拟南芥21R1R2R3-MYB基因,通过PCR扩增MYB基因,经酶双切后,连接到Ri质粒。
根据上述技术方案,所述筛选培养基存活的地黄发状根进行PCR检测:根据设计的一对MYB基因引物,电泳检测出现了与基因引物相同的条带,而未转化的地黄发状根则没有扩增出条带,证明重组基因己经整合进地黄发状根的基因组中。
根据上述技术方案,所述地黄发状根转接到液体培养基中扩大增殖培养:用YEB培养基,牛肉浸膏5g/L,酵母膏1g/L,蛋白胨5g/L,七水硫酸镁0.5g/L,琼脂15g/L,培养20D后,添加终浓度为25μmol/L的SA,12h后取地黄发状根备用。
实施例1
一种抗辐射型美白保湿面霜,包括以下重量成分组成:毛蕊花糖苷5份、黑色素4份、凝胶多糖8份、甘油5份、透明质酸6份、谷胱甘肽1.8份、乳化剂4份、珍珠粉9份、维生素E0.9份、水15份。
一种抗辐射型美白保湿面霜的制备方法,包括以下具体步骤:
步骤一:取黑色素、1/2水、凝胶多糖发酵液,90℃下100rpm搅拌4h,获得A相;
步骤二:将毛蕊花糖苷、透明质酸、甘油、珍珠粉、精氨酸、谷胱甘肽、维生素E和1/2水70℃下120rpm搅拌1h,冷却至室温,获得B相;
步骤三:将乳化剂与A相、B相混合搅拌,40℃下160rpm搅拌3h制得面霜。
根据上述技术方案,所述黑色素提取自粒毛盘菌发酵液或黑芝麻中。
根据上述技术方案,所述凝胶多糖发酵液是利用假单胞菌发酵获得的:蔗糖30g/L,酵母粉8g/L,碳酸钙3g/L,发酵液初始pH值7.5-8.5,发酵周期为3-5天,发酵温度为30-35℃;取发酵完成的发酵液,8000rpm离心30分钟,取上清液,低温蒸发浓缩得凝胶多糖发酵液。
根据上述技术方案,所述于毛蕊花糖苷是从地黄发状根中提取的。
根据上述技术方案,所述地黄发状根,用发根农杆菌A4对地黄叶片进行侵染,外植体龄为7-14D。
根据上述技术方案,所述用发根农杆菌对面积为0.5*0.5cm的地黄叶片进行侵染,侵染时间为5-10min后,然后在无菌滤纸上吸干菌液,将叶片倒置于共培养固体培养基中,每个培养皿中放置10个叶片,于26℃,黑暗条件下共培养,共培养培养基为MS基,添加100μmol/L的AS;共培养2d后用无菌水冲洗3次,无菌滤纸吸干液体后,将叶片接种于筛选培养基中,26℃暗培养,筛选培养基为MS+0.4g/L的Cef+100μmol/L的AS。
根据上述技术方案,所述发根农杆菌其Ri质粒中含有拟南芥21R1R2R3-MYB基因,通过PCR扩增MYB基因,经酶双切后,连接到Ri质粒。
根据上述技术方案,所述筛选培养基存活的地黄发状根进行PCR检测:根据设计的一对MYB基因引物,电泳检测出现了与基因引物相同的条带,而未转化的地黄发状根则没有扩增出条带,证明重组基因己经整合进地黄发状根的基因组中。
根据上述技术方案,所述地黄发状根转接到液体培养基中扩大增殖培养:用YEB培养基,牛肉浸膏5g/L,酵母膏1g/L,蛋白胨5g/L,七水硫酸镁0.5g/L,琼脂15g/L,培养20D后,添加终浓度为25μmol/L的SA,12h后取地黄发状根备用。
实施例2
一种抗辐射型美白保湿面霜,包括以下重量成分组成:毛蕊花糖苷4份、黑色素5份、凝胶多糖6份、甘油4份、透明质酸4份、谷胱甘肽1份、乳化剂3份、珍珠粉6份、维生素E1.2份、水10份。
一种抗辐射型美白保湿面霜的制备方法,包括以下具体步骤:
步骤一:取黑色素、1/2水、凝胶多糖发酵液,90℃下60rpm搅拌4h,获得A相;
步骤二:将毛蕊花糖苷、透明质酸、甘油、珍珠粉、精氨酸、谷胱甘肽、维生素E和1/2水70℃下180rpm搅拌1h,冷却至室温,获得B相;
步骤三:将乳化剂与A相、B相混合搅拌,40℃下120rpm搅拌3h制得面霜。
根据上述技术方案,所述黑色素提取自粒毛盘菌发酵液或黑芝麻中。
根据上述技术方案,所述凝胶多糖发酵液是利用假单胞菌发酵获得的:蔗糖30g/L,酵母粉8g/L,碳酸钙3g/L,发酵液初始pH值7.5-8.5,发酵周期为3-5天,发酵温度为30-35℃;取发酵完成的发酵液,6000rpm离心30分钟,取上清液,低温蒸发浓缩得凝胶多糖发酵液。
根据上述技术方案,所述于毛蕊花糖苷是从地黄发状根中提取的。
根据上述技术方案,所述地黄发状根,用发根农杆菌A4对地黄叶片进行侵染,外植体龄为7-14D。
根据上述技术方案,所述用发根农杆菌对面积为0.5*0.5cm的地黄叶片进行侵染,侵染时间为5-10min后,然后在无菌滤纸上吸干菌液,将叶片倒置于共培养固体培养基中,每个培养皿中放置10个叶片,于26℃,黑暗条件下共培养,共培养培养基为MS基,添加100μmol/L的AS;共培养2d后用无菌水冲洗3次,无菌滤纸吸干液体后,将叶片接种于筛选培养基中,26℃暗培养,筛选培养基为MS+0.4g/L的Cef+100μmol/L的AS。
根据上述技术方案,所述发根农杆菌其Ri质粒中含有拟南芥21R1R2R3-MYB基因,通过PCR扩增MYB基因,经酶双切后,连接到Ri质粒。
根据上述技术方案,所述筛选培养基存活的地黄发状根进行PCR检测:根据设计的一对MYB基因引物,电泳检测出现了与基因引物相同的条带,而未转化的地黄发状根则没有扩增出条带,证明重组基因己经整合进地黄发状根的基因组中。
根据上述技术方案,所述地黄发状根转接到液体培养基中扩大增殖培养:用YEB培养基,牛肉浸膏5g/L,酵母膏1g/L,蛋白胨5g/L,七水硫酸镁0.5g/L,琼脂15g/L,培养20D后,添加终浓度为25μmol/L的SA,12h后取地黄发状根备用。
试验1.毛蕊花糖苷产量试验
测试样品:取长约2-3cm的转基因与非转基因发状根分别接种于MS液体培养基中,于26℃,130r/min,黑暗条件下摇床培养72h,洗净备用。
色谱:色谱柱为AthenaC18,(4.6×250mm,5μm);以乙腈-0.1%醋酸(16:84)为流动相;检测波长为334nm;流速为1.0mL·min-1;柱温为30℃;进样量为20μL;理论塔板数按毛蕊花糖苷色谱峰计算不低于5000;上述条件下,色谱峰之间分离度良好。
供试品溶液的制备:精密称定0.4g地黄毛状根粉末,置具塞锥形瓶中,精密加入甲醇25ml,称定重量,65℃回流提取1.5h,放冷至室温,补足失重;摇匀,滤过,精密量取续滤液10ml,浓缩至近干;残渣用流动相溶解,转移至5ml量瓶中,用流动相定容,摇匀,经0.22μm微孔滤膜过滤,即得。
对照品溶液的制备:精密称定毛蕊花糖苷标准品0.08mg,置5ml容量瓶中,用流动相定容制成0.016mg/ml的对照品溶液,即得。
平行称取两组毛状根85g,各6份,制备供试品溶液,按照上述色谱条件进样,分别测定毛蕊花糖苷的含量,计算RSD值。
取对照品溶液,按照上述色谱条件重复进样6次,测定毛蕊花糖苷的峰面积,计算RSD值。
表1RSD值计算结果
由表1可知,转基因的产毛蕊花糖苷量高于非转基因组,产量提高了34%。
试验2.防辐射效果试验:
细胞培养:用含10%胎牛血清的DMEM培养液,在5%CO2、37℃孵箱恒温培养人皮肤成纤维细胞(HSF)。
建立HSF辐射损伤模型:以直线加速器造辐射损伤模型。收集对数生长期HSF,调整细胞悬液浓度至2.5×104/mL。实验分为3组:阴性对照组不辐射1组、16Gy辐射组、16Gy添加美白保湿面霜保护组,收集对数生长期HSF,调整细胞悬液浓度至2.5×104/mL接种于96孔板。5%CO2、37℃孵箱恒温培养48h后,除阴性对照组外用6MVX射线一次性辐射,辐射剂量率为3Gy/min,辐射野大小为25cm×25cm,辐射源距靶中心距离为100cm。辐射时,96孔板上面放置1cm厚等效散射板,下面放置3cm厚等效散射板。分别于辐射后2,12,24,48,采用MTT法检测各组细胞的OD570,每组6个复孔。
表2各组测试细胞的OD570值
表3人皮肤成纤维细胞生长率
由表3可知,添加美白保湿面霜的人皮肤成纤维细胞相比无保护辐射组生长率有大幅度提高,说明本发明的一种抗辐射型美白保湿面霜有很强的抗辐射能力。
添加美白保湿面霜的人皮肤成纤维细胞相比无辐射阴性对照组也有所提高,说明发明的一种抗辐射型美白保湿面霜有促进人皮肤成纤维细胞的功能。
需要说明的是,在本文中,诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来,而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程、方法、物品或者设备所固有的要素。
最后应说明的是:以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (1)
1.一种抗辐射型美白保湿面霜,其特征在于:包括以下重量成分组成:毛蕊花糖苷5份、黑色素4份、凝胶多糖8份、甘油5份、透明质酸6份、谷胱甘肽1.8份、乳化剂4份、珍珠粉9份、维生素E0.9份、水15份;
所述抗辐射型美白保湿面霜的制备方法包括以下具体步骤:
步骤一:取黑色素、1/2水、凝胶多糖发酵液,90℃下100rpm搅拌4h,获得A相;
步骤二:将毛蕊花糖苷、透明质酸、甘油、珍珠粉、精氨酸、谷胱甘肽、维生素E和1/2水70℃下120rpm搅拌1h,冷却至室温,获得B相;
步骤三:将乳化剂与A相、B相混合搅拌,40℃下160rpm搅拌3h制得面霜;
所述黑色素提取自粒毛盘菌发酵液或黑芝麻中;
所述凝胶多糖发酵液是利用假单胞菌发酵获得的:蔗糖30g/L,酵母粉8g/L,碳酸钙3g/L,发酵液初始pH值7.5-8.5,发酵周期为3-5天,发酵温度为30-35℃;取发酵完成的发酵液,8000rpm离心30分钟,取上清液,低温蒸发浓缩得凝胶多糖发酵液;
所述于毛蕊花糖苷是从地黄发状根中提取的;所述地黄发状根,用发根农杆菌A4对地黄叶片进行侵染,外植体龄为7-14D;所述用发根农杆菌对面积为0.5*0.5cm的地黄叶片进行侵染,侵染时间为5-10min后,然后在无菌滤纸上吸干菌液,将叶片倒置于共培养固体培养基中,每个培养皿中放置10个叶片,于26℃,黑暗条件下共培养,共培养培养基为MS基,添加100μmol/L的AS;共培养2d后用无菌水冲洗3次,无菌滤纸吸干液体后,将叶片接种于筛选培养基中,26℃暗培养,筛选培养基为MS+0.4g/L的Cef+100μmol/L的AS;
所述发根农杆菌其Ri质粒中含有拟南芥21R1R2R3-MYB基因,通过PCR扩增MYB基因,经酶双切后,连接到Ri质粒;
所述筛选培养基存活的地黄发状根进行PCR检测:根据设计的一对MYB基因引物,电泳检测出现了与基因引物相同的条带,而未转化的地黄发状根则没有扩增出条带,证明重组基因己经整合进地黄发状根的基因组中;
所述地黄发状根转接到液体培养基中扩大增殖培养:用YEB培养基,牛肉浸膏5g/L,酵母膏1g/L,蛋白胨5g/L,七水硫酸镁0.5g/L,琼脂15g/L,培养20D后,添加终浓度为25μmol/L的SA,12h后取地黄发状根备用。
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