CN111944868A - 一种蝉花甾醇的生物发酵制备方法 - Google Patents
一种蝉花甾醇的生物发酵制备方法 Download PDFInfo
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- CN111944868A CN111944868A CN202010728393.3A CN202010728393A CN111944868A CN 111944868 A CN111944868 A CN 111944868A CN 202010728393 A CN202010728393 A CN 202010728393A CN 111944868 A CN111944868 A CN 111944868A
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Abstract
本发明一种蝉花甾醇的生物发酵制备方法,涉及生物工程技术领域。以甘油、木糖和尿素为主要原料,通过蝉拟青霉菌体液态摇瓶培养、液体种子罐扩大培养获得含有蝉拟青霉发酵液。发酵液通过浓缩、乙醇提取、水饱和正丁醇萃取、重结晶获取蝉花甾醇。经试验研究,蝉拟青霉液体发酵及其从该菌发酵液中分离蝉花甾醇,该蝉花甾醇可以作为治疗慢性肾病和肾脏肿瘤的药物,为肾癌患者提供理想的药物。
Description
技术领域
本发明涉及生物工程技术领域,特指利用感染蝉形成蝉花的蝉拟青霉菌种采用液体发酵技术制备蝉花甾醇的方法。
背景技术
肾细胞癌起源于肾上皮细胞。它占肾脏癌症的90%以上,占所有致命,恶性肿瘤的3%。多达30%的肾细胞癌患者出现局部复发或远处转移,目前的五年生存率不理想。尽管使用一线靶向酪氨酸激酶抑制剂(例如索拉非尼或舒尼替尼)进行肾细胞癌治疗取得了显著进展,但在肾细胞癌治疗中仍需要具有优异效果和安全性的新食品或药物。天然食品和动植物是药物开发的非常宝贵的资源,活性产品的利用是一种抗击癌症的有希望的策略。传统中草药是治疗各种流行病包括癌症的常用药物。迄今为止,在中国传统中医学理论的指导下,许多有效的抗癌配方已经从这些草药中衍生。中药蝉花(Cordycep cicadae)是一种可食用的虫生真菌,由蝉拟青霉感染的蝉若虫形成,并被广泛用于预防和治疗各种疾病。在中国,它已被用于医药和食用超过1000年,经常被推荐用于治疗肺部疾病。最近,越来越多地发现了蝉花及蝉拟青霉菌丝体的抗肿瘤作用,例如妇科癌细胞中的抗增殖和促凋亡特性。甾醇是广泛存在于生物体内的一种重要的天然活性物质,按其原料来源分为动物性甾醇、植物性甾醇和菌类甾醇等三大类。其中麦角甾醇是最主要的菌类甾醇。麦角甾醇类化合物具有抗炎、抗肿瘤、抑菌、免疫调节、抗病毒和抗氧化等多种活性。麦角甾醇具有抑制血管生长、激活抑癌基因或者通过细胞毒性直接杀死肿瘤细胞的作用,是药食用真菌发挥抑癌作用的主要小分子成分。其他研究也表明麦角甾醇具有免疫调节作用,能够增强免疫力。除此之外,麦角甾醇作为维生素D的前体物质,在紫外光照射下会转化为维生素D2,因此食用菌也可以作为丰富的天然维生素D来源。蝉花甾醇是蝉拟青霉的主要活性成分之一。蝉花甾醇可以从蝉花子实体中术分离出来。蝉花甾醇具有激活免疫反应和发挥抗癌/病毒效果。最近,越来越多的证据已经发现蝉花甾醇对结肠直肠癌,肝细胞癌,前列腺癌,骨髓瘤和白血病的具有抗肿瘤活性。目前蝉花甾醇可通过色谱方法从毛癣菌,萤火虫,蝉虫草,灵芝等中分离出来。本发明是发明者通过反复实验研究发现,蝉拟青霉通过液体发酵产生菌丝体,从菌丝体中可以分离获得蝉花甾醇。该蝉花甾醇同样具备抑制人肾细胞癌的增殖,侵袭/迁移抑制,促肾癌细胞凋亡。
发明内容
针对目前甾醇类化合物主要来源于植物或药用真菌子实体,受到资源限制的问题、甾醇类化合物由于受到地质环境、气候等影响导致含量不稳定等问题,本发明的目的在于:提供一种通过液体发酵获得蝉拟青霉发酵液,蝉拟青霉发酵液通过浓缩、乙醇提取、水饱和正丁醇萃取、重结晶获得具有抑制肾细胞癌的蝉花甾醇方法。
技术方案
本发明人经过深入的研究,摸索出一种以甘油、木糖和尿素为主要原料,通过蝉拟青霉菌体液态摇瓶培养、液体种子罐扩大培养获得含有蝉拟青霉发酵液。发酵液通过浓缩、乙醇提取、水饱和正丁醇萃取、重结晶获取蝉花甾醇。具体方案如下:
一种蝉花甾醇的生物发酵制备方法,按照下述步骤进行:
步骤1、根据配合比和制造量选择适当量的麸皮、木糖、果糖、甘油、尿素、鱼粉蛋白胨、KH2PO4、ZnSO4、VB2、VC;所使用的的1L培养基中主要成分包括麸皮5-55g、木糖10-50g、果糖1-40g、甘油5-45g、尿素1-4g、鱼粉蛋白胨1-4g、KH2PO4 1-2.4g、ZnSO4 0.5-2g、VB2 0.01-0.4g、VC 0.1-0.6g。
步骤2、将上述麸皮、木糖、果糖、甘油、尿素、鱼粉蛋白胨、KH2PO4、ZnSO4、VB2、VC。溶解于适量的水中,装入发酵罐121℃灭菌30min;
步骤3、以蝉拟青霉(Paecilomyces cicadae)保藏的试管菌种为对象,将蝉拟青霉菌种接种于马铃薯葡萄糖培养基中,20-32℃下培养3-7天,放置在2-10℃环境下保藏,制得蝉拟青霉斜面菌种;
步骤4、将步骤3制得的蝉拟青霉斜面菌种切成3mm×3mm大小,挑取4-6块接种到装有液体摇瓶培养基的摇瓶中,在100-200rpm、20-32℃摇床里培养3-7天,制得液体摇瓶菌种;其中,每1L液体摇瓶培养基组成为:蛋白胨1-16g、葡萄糖10-50g、、玉米粉15-45g、麸皮12-48g;
步骤5、将步骤4制得的液体摇瓶菌种按5-20%(摇瓶菌种液体体积与种子罐培养基的液体体积比)接种量接种到种子罐培养基,在20-32℃,通气量0-1.2/1/min(v/v/min,即通气体积/种子罐中种子液液体体积/min),转速60-150转/min培养2-4天,制得种子罐菌种液体;其中,每1L种子罐培养基含有:蛋白胨1-16g、葡萄糖10-50g、、玉米粉15-45g、麸皮12-48g;
步骤6、采用液体发酵培养技术,将种子罐菌种按5-20%(种子罐菌种液体体积与发酵培养基的液体体积比)接种量无菌地转移到发酵培养基中,在20-32℃,通气量0-1.2/1/min(v/v/min,即通气体积/发酵罐中发酵液液体体积/min),发酵罐搅拌转速60-150转/min下培养24-96h,制得含有蝉花甾醇的蝉拟青霉发酵液;
步骤7、将步骤6中的发酵液4000转/分离心10min获得蝉拟青霉菌丝体。
所述蝉拟青霉菌丝体中蝉花甾醇的分离纯化的方法,按照下述步骤进行:
(a)步骤6制得的含有蝉花甾醇的蝉拟青霉发酵液,过滤,收集滤液;
(b)滤液减压浓缩至膏状,与30倍体积无水乙醇混合,85℃的回流提取4h,收集提取液;。
(c)提取液5000r/min离心10min,收集上清液;上清液真空浓缩至膏状;
(d)膏状物用2倍体积的去离子水混匀,分别5倍体积水饱和正丁醇萃取3次,收集正丁醇项,合并,减压浓缩至干;
(e)干燥物用2被体积无水乙醇,加入5倍乙醇体积的去离子水,混合均匀,4℃沉淀18h,抽滤获得粗蝉花甾醇;
(f)粗皂苷,用2倍体积无水乙醇溶解,-20℃结晶,得到蝉花甾醇粗品。
一种蝉花甾醇的拮抗肾癌的片剂制备方法,按照下述步骤进行:
(a)将蝉花甾醇的粉状物86份,混合均匀,加入75份玉米淀粉、45份麦芽糊精、0.5份羟丙甲基纤维素混合均匀;
(b)称取4.5份羟丙甲基纤维素,然后加入90%乙醇制成10%的羟丙甲基纤维素溶液,备用;
(c)将(a)中混合粉末加入(b)中粘合剂,14目筛网制粒;
(d)烘箱温度调至55℃,干燥3h;
(e)干燥颗粒于14目筛网进行整粒,后外加2份硬脂酸镁和2份二氧化硅,进行总混;
(f)颗粒进行压片,调整片重为0.6g/片,进行薄膜包衣,铝塑包装,即得到可以用于治疗慢性肾病和肾脏肿瘤的片剂。
有益效果:本发明利用蝉拟青霉菌种通过液体发酵生产蝉花甾醇;本发明的蝉花甾醇是一种纯天然药物,与现有化学合成的治疗慢性肾病和肾脏肿瘤的药物相比,无任何毒副作用;蝉花甾醇作为治疗慢性肾病和肾脏肿瘤的药物,为慢性肾病和肾脏肿瘤患者提供理想的药物。
附图说明:
图1为齐墩果酸标准品与蝉花甾醇在氯仿:甲醇:乙酸乙酯:水(25:10:5:2)下的薄层色谱图。
注:1:齐墩果酸标准品;2,3:实施例1和3蝉花皂苷。
具体实施方式
下面结合具体实施例对本发明作进一步说明。
实施例1蝉花甾醇的制备
a.以蝉拟青霉(Paecilomyces cicadae江南大学馈赠,编号bio-33088)保藏的试管菌种为对象,将蝉拟青霉菌种接种于马铃薯葡萄糖培养基中,25℃下培养7天,放置在10℃环境下保藏,制得蝉拟青霉斜面菌种;
b.将步骤a制得的蝉拟青霉斜面菌种切成3mm×3mm大小,挑取4块接种到装有液体摇瓶培养基的摇瓶中,在100rpm、20℃下培养7天,制得液体摇瓶菌种;其中,每1L液体摇瓶培养基组成为:蛋白胨1g、葡萄糖10g、、玉米粉45g、麸皮48g;
c.将步骤b制得的蝉拟青霉液体摇瓶菌种按5%(摇瓶菌种液体体积与种子罐培养基的液体体积比)接种量接种到种子罐培养基,在28℃,通气量0.6(v/v/min,即通气体积/种子罐中发酵液体积/min),转速95转/min培养3天,制得种子罐液体菌种;其中,每1L种子罐培养基含有:蛋白胨9g、葡萄糖31g、、玉米粉29g、麸皮30g;
d.液体发酵培养:将种子罐菌种按20%接种量(种子罐菌种液体体积与发酵培养基的液体体积比)无菌地转移到发酵培养基中,在20℃,通气量1.2/1/min(v/v/min,即通气体积/发酵罐中发酵液体积/min)培养,发酵罐搅拌转速60转/min下培养96h,制得含有蝉花甾醇的蝉拟青霉发酵液;所使用的1L发酵培养基主要成分包括麸皮55g、木糖10g、果糖1g、甘油45g、尿素1g、鱼粉蛋白胨4g、KH2PO4 1-2.4g、ZnSO4 2g、VB2 0.4g、VC 0.1g。
实施例2蝉花甾醇的制备
a.以蝉拟青霉(Paecilomyces cicadae,从北京北纳创联生物技术研究所购买,编号:BNCC114807)保藏的试管菌种为对象,将蝉拟青霉菌种接种于马铃薯葡萄糖培养基中,25℃下培养3天,放置在2℃环境下保藏,制得蝉拟青霉斜面菌种;
b.将步骤a制得的蝉拟青霉斜面菌种切成3mm×3mm大小,挑取5块接种到装有液体摇瓶培养基的摇瓶中,在150rpm、26℃下培养5天,制得液体摇瓶菌种;其中,每1L液体摇瓶培养基组成为:蛋白胨8g、葡萄糖31g、、玉米粉29g、麸皮26g;
c.将步骤b制得的蝉拟青霉液体摇瓶菌种按20%(摇瓶菌种液体体积与种子罐培养基的液体体积比)接种量接种到种子罐培养基,在32℃,通气量0/1/min(v/v/min,即通气体积/发酵罐中发酵液体积/min),转速150转/min培养4天,制得种子罐液体菌种;其中,每1L种子罐培养基含有:蛋白胨16g、葡萄糖50g、、玉米粉15g、麸皮12g;;
d.液体发酵培养:将种子罐菌种按14%接种量(种子罐菌种液体体积与发酵培养基的液体体积比)无菌地转移到发酵培养基中,在32℃,通气量0/1/min(v/v/min,即通气体积/发酵罐中发酵液体积/min)培养,发酵罐搅拌转速90转/min下培养73h,制得含有蝉花甾醇的蝉拟青霉发酵液;所使用的1L发酵培养基主要成分包括麸皮5g、木糖50g、果糖40g、甘油5g、尿素4g、鱼粉蛋白胨1g、KH2PO4 1g、ZnSO4 0.5g、VB2 0.01g、VC 0.6g。
实施例3蝉花甾醇的制备
a.以蝉拟青霉(Paecilomyces cicadae江南大学馈赠,编号bio-33088)保藏的试管菌种为对象,将蝉拟青霉菌种接种于马铃薯葡萄糖培养基中,30℃下培养5天,放置在5℃环境下保藏,制得蝉拟青霉斜面菌种;
b.将步骤a制得的蝉拟青霉斜面菌种切成3mm×3mm大小,挑取6块接种到装有液体摇瓶培养基的摇瓶中,在200rpm、32℃下培养3天,制得液体摇瓶菌种;其中,每1L液体摇瓶培养基组成为:蛋白胨16g、葡萄糖50g、、玉米粉15g、麸皮12g;
c.将步骤b制得的蝉拟青霉液体摇瓶菌种按13%(摇瓶菌种液体体积与种子罐培养基的液体体积比)接种量接种到种子罐培养基,在20℃,通气量1.2/1/min(v/v/min,即通气体积/发酵罐中发酵液体积/min),转速60转/min培养2天,制得种子罐液体菌种;其中,每1L种子罐培养基含有:蛋白胨1g、葡萄糖10g、玉米粉45g、麸皮48g;
d.液体发酵培养:将种子罐菌种按12%接种量(种子罐菌种液体体积与发酵培养基的液体体积比)无菌地转移到发酵培养基中,在26℃,通气量0.6/1/min(v/v/min,即通气体积/发酵罐中发酵液体积/min)培养,发酵罐搅拌转速150转/min下培养24h,制得含有麦角甾醇过氧化物的蝉拟青霉发酵液;所使用的1L发酵培养基主要成分包括麸皮30g、木糖31g、果糖22g、甘油26g、尿素2.4g、鱼粉蛋白胨2.5g、KH2PO4 1.7g、ZnSO4 1.3g、VB2 0.2g、VC0.3g。
实施例4蝉花甾醇的提取
(a)步骤6制得的含有蝉花甾醇的蝉拟青霉发酵液,过滤,收集滤液;
(b)滤液减压浓缩至膏状,与30倍体积无水乙醇混合,85℃的回流提取4h,收集提取液;。
(c)提取液5000r/min离心10min,收集上清液;上清液真空浓缩至膏状;
(d)膏状物用2倍体积的去离子水混匀,分别5倍体积水饱和正丁醇萃取3次,收集正丁醇项,合并,减压浓缩至干;
(e)干燥物用2被体积无水乙醇,加入5倍乙醇体积的去离子水,混合均匀,4℃沉淀18h,抽滤获得粗蝉花甾醇;
(f)粗皂苷,用2倍体积无水乙醇溶解,-20℃结晶,得到蝉花甾醇粗品。
实施例5:用比色法测定蝉拟青霉发酵液中蝉花甾醇含量
(a)样品制备:发酵液用8层纱布过滤,吸取25mL滤液于50mL的三角瓶中,放入60℃烘箱中烘至剩余三分之一,加入25mL 75%乙醇,在75℃水浴锅中水浴4小时,倒入50mL的离心管中,5000r/min离心10min,上清液用于测定。
(b)标准曲线制作:准确称取105℃干燥至恒重的齐墩果酸标品25mg于25mL的烧杯瓶中,加入少量无水乙醇溶解,然后倒入25mL容量瓶中定容至刻度,摇匀、备用。分别吸取齐墩果酸标准液0、0.2、0.4、0.6、0.8、1.0mL于10mL的容量瓶中,用无水乙醇定容刻度,吸取1mL于试管中,依次滴加0.2mL 5%的香草醛-冰乙酸溶液和0.8mL的高氯酸,在60℃度的水浴锅中水浴15min,冷却至室温,滴加3mL冰醋酸反应,以无水乙醇代替样品为空白参比,用紫外分光光度计在570nm测定吸光值。以OD值作为横坐标,齐墩果酸浓度作为纵坐标,作齐墩果酸的标准曲线,建立回归方程。
(c)吸取预处理后的上清液1mL于10mL试管中,依次加入香草醛-冰乙酸0.2mL,高氯酸0.8mL,60℃下水浴15min,冷却至室温,加入3mL冰醋酸,混匀,在571nm下测吸光值,根据标准曲线回归方程计算甾醇含量。d.利用比色法测得实施例1、实施例2和实施例3发酵液中蝉花甾醇的含量见表1。
表1:实施例发酵液中蝉花甾醇含量
样品 | 实施例1 | 实施例2 | 实施例3 |
蝉花甾醇含量(g/L) | 2.499 | 0.501 | 1.432 |
实施例6分离产物蝉花甾醇薄层层析定性鉴定
(a)样品的制备:称取0.05g经实施例4分离纯化得到的样品或0.01g齐墩果酸溶于25mL甲醇,备用;
(b)薄层层析:5cm×10cm硅胶G薄板于110℃条件下活化30min后,用毛细管点样,点样量为1μL,点样后,硅胶板分别在氯仿:甲醇:乙酸乙酯:水(25:10:5:2)展开剂预饱和15min的展开缸里展开,展距7cm,取出,晾干,以10%硫酸乙醇溶液喷雾显色后,105℃加热至斑点清晰,在365nm的紫外光灯下检视皂苷的荧光斑点。结果见附图。根据此颜色反应和比色测定的颜色反应和中药化学,可以断定提取物含有蝉花甾醇。
实施例7蝉花甾醇片剂的制备方法
一种蝉花甾醇的拮抗肾癌的片剂制备方法:
(a)将蝉花甾醇86份,加入75份玉米淀粉、45份麦芽糊精、0.5份羟丙甲基纤维素混合均匀;
(b)称取4.5份羟丙甲基纤维素,然后加入90%乙醇制成10%的羟丙甲基纤维素溶液,备用;
(c)将(a)中混合粉末加入(b)中粘合剂,14目筛网制粒;
(d)烘箱温度调至55℃,干燥3h;
(e)干燥颗粒于14目筛网进行整粒,后外加2份硬脂酸镁和2份二氧化硅,进行总混;
(f)颗粒进行压片,调整片重为0.6g/片,进行薄膜包衣,铝塑包装,即得得到可以用于治疗慢性肾病和肾脏肿瘤的片剂。
Claims (2)
1.一种蝉花甾醇的生物发酵制备方法,其特征在于按照下述步骤进行:
步骤1、根据配合比和制造量选择适当量的麸皮、木糖、果糖、甘油、尿素、鱼粉蛋白胨、KH2PO4、ZnSO4、VB2、VC;所使用的的1L培养基中主要成分包括麸皮5-55g、木糖10-50g、果糖1-40g、甘油5-45g、尿素1-4g、鱼粉蛋白胨1-4g、KH2PO4 1-2.4g、ZnSO4 0.5-2g、VB2 0.01-0.4g、VC 0.1-0.6g;
步骤2、将上述麸皮、木糖、果糖、甘油、尿素、鱼粉蛋白胨、KH2PO4、ZnSO4、VB2、VC;溶解于适量的水中,装入发酵罐121℃灭菌30min;
步骤3、以蝉拟青霉(Paecilomyces cicadae)保藏的试管菌种为对象,将蝉拟青霉菌种接种于马铃薯葡萄糖培养基中,20-32℃下培养3-7天,放置在2-10℃环境下保藏,制得蝉拟青霉斜面菌种;
步骤4、将步骤3制得的蝉拟青霉斜面菌种切成3mm×3mm大小,挑取4-6块接种到装有液体摇瓶培养基的摇瓶中,在100-200rpm、20-32℃摇床里培养3-7天,制得液体摇瓶菌种;其中,每1L液体摇瓶培养基组成为:蛋白胨1-16g、葡萄糖10-50g、、玉米粉15-45g、麸皮12-48g;
步骤5、将步骤4制得的液体摇瓶菌种按5-20%(摇瓶菌种液体体积与种子罐培养基的液体体积比)接种量接种到种子罐培养基,在20-32℃,通气量0-1.2/1/min(v/v/min,即通气体积/种子罐中种子液液体体积/min),转速60-150转/min培养2-4天,制得种子罐菌种液体;其中,每1L种子罐培养基含有:蛋白胨1-16g、葡萄糖10-50g、、玉米粉15-45g、麸皮12-48g;
步骤6、采用液体发酵培养技术,将种子罐菌种按5-20%(种子罐菌种液体体积与发酵培养基的液体体积比)接种量无菌地转移到发酵培养基中,在20-32℃,通气量0-1.2/1/min(v/v/min,即通气体积/发酵罐中发酵液液体体积/min),发酵罐搅拌转速60-150转/min下培养24-96h,制得含有蝉花甾醇的蝉拟青霉发酵液;
步骤7、将步骤6中的发酵液4000转/分离心10min获得蝉拟青霉菌丝体;
步骤8、蝉拟青霉菌丝体中蝉花甾醇的分离纯化的方法,按照下述步骤进行:
(a)步骤6制得的含有蝉花甾醇的蝉拟青霉发酵液,过滤,收集滤液;
(b)滤液减压浓缩至膏状,与30倍体积无水乙醇混合,85℃的回流提取4h,收集提取液;
(c)提取液5000r/min离心10min,收集上清液;上清液真空浓缩至膏状;
(d)膏状物用2倍体积的去离子水混匀,分别5倍体积水饱和正丁醇萃取3次,收集正丁醇项,合并,减压浓缩至干;
(e)干燥物用2被体积无水乙醇,加入5倍乙醇体积的去离子水,混合均匀,4℃沉淀18h,抽滤获得粗蝉花甾醇;
(f)粗皂苷,用2倍体积无水乙醇溶解,-20℃结晶,得到蝉花甾醇粗品。
2.一种蝉花甾醇的拮抗肾癌的片剂制备方法,其特征在于按照下述步骤进行:
(a)将蝉花甾醇的粉状物86份,混合均匀,加入75份玉米淀粉、45份麦芽糊精、0.5份羟丙甲基纤维素混合均匀;
(b)称取4.5份羟丙甲基纤维素,然后加入90%乙醇制成10%的羟丙甲基纤维素溶液,备用;
(c)将(a)中混合粉末加入(b)中粘合剂,14目筛网制粒;
(d)烘箱温度调至55℃,干燥3h;
(e)干燥颗粒于14目筛网进行整粒,后外加2份硬脂酸镁和2份二氧化硅,进行总混;
(f)颗粒进行压片,调整片重为0.6g/片,进行薄膜包衣,铝塑包装,即得到可以用于治疗慢性肾病和肾脏肿瘤的片剂。
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