CN106491662B - 一种从桑黄栽培废料中同时提取多糖和黄酮的方法 - Google Patents
一种从桑黄栽培废料中同时提取多糖和黄酮的方法 Download PDFInfo
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Classifications
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
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- C—CHEMISTRY; METALLURGY
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/17—Preparation or pretreatment of starting material involving drying, e.g. sun-drying or wilting
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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Abstract
本发明涉及一种废弃物处理方法,特别涉及一种从桑黄栽培废料中同时提取多糖和黄酮的方法,该方法包括如下步骤:将桑黄栽培废料烘干、粉碎得到桑黄栽培废料干粉,与碱性乙醇溶液混合;将混合液超声处理20~40分钟,过滤得滤液和残渣;向残渣中加入去离子水,混合均匀,调pH值4~6得到混合液,加入复合酶,复合酶为纤维素酶和葡萄糖氧化酶混合组成,45~55℃下酶解,酶解液经离心处理分离得上清液和沉淀物;得到的滤液经旋转蒸发后除去乙醇,加入乙酸乙酯进行萃取,分液,上层液经减压浓缩干燥得到桑黄黄酮;得到的上清液用旋转蒸发仪减压浓缩后进行醇沉,离心,得上清液和沉淀物,沉淀物即为桑黄多糖。
Description
技术领域
本发明涉及一种废弃物处理方法,特别涉及一种从桑黄栽培废料中同时提取多糖和黄酮的方法。
背景技术
桑黄主要寄生于桑、杨、暴马丁香、松、白桦等树干上,其实是一类药用真菌的总称。桑黄作为我国传统的中药材,最早记载于《神农百草经》和李时珍的《本草纲目》,主要用于止泻、崩漏带下、脾虚泄泻,此外还能利五脏、排毒、活血。桑黄的化学成分有多糖、黄酮、香豆素、麦角甾醇、落叶松覃酸、脂肪酸、三萜类、芳香酸和多种氨基酸,以及木糖氧化酶、脲酶、醋酶、过氧化氢酶、蔗糖酶、乳糖酶、纤维素酶等多种酶类。有研究统计,桑黄的药理学功能有20 多种,包括抑菌、消炎、抗氧化、抗肿瘤、增强机体免疫、保肝护肝、降血糖、降血脂、抗肺炎等,其有效成分主要是多糖、黄酮和三萜类物质。在桑黄的药用价值被广泛揭示以及市场前景看好的同时, 桑黄的产量已经成为抑制桑黄应用的主要限制因素。
目前,由于受生理状态的特殊性和复杂性以及外部环境的制约,桑黄在自然界中形成子实体困难,造成天然的桑黄数量非常稀少,自然界中可供药用的资源有限,加之国内外市场对桑黄的需求量越来越大,致使国内各产地药农掠夺式采集,子实体无法大量形成,难以成为稳定的工业品来源,对桑黄产业的发展十分不利,因此通过人工栽培来弥补野生资源的不足显得非常重要。
随着桑黄栽培的发展,随之产生大量的桑黄栽培废料,对这些废料的有效处理成为目前面临的一个重要课题。一方面,如将产后栽培废料在环境中丢弃,会产生大量危害桑黄生产的生物,如各种霉菌,蚊类,螨类等,还会污染环境。另一方面,这些废料中含有大量的可再利用的活性物质。因此,为了达到生物资源的高效利用,减少环境污染。有效提取桑黄栽培废料中活性物质,既可提高桑黄生产的附加值,还具有重要的市场价值和社会效益。
发明内容
本发明提供一种从桑黄栽培废料中同时提取多糖和黄酮的方法,该方法有效提取桑黄栽培废料中的活性物质桑黄黄酮和桑黄多糖,既可提高桑黄生产的附加值,还具有重要的市场价值和社会效益。
本发明解决其技术问题所采用的技术方案是:
一种从桑黄栽培废料中同时提取多糖和黄酮的方法,该方法包括如下步骤:
(1)将桑黄栽培废料烘干、粉碎得到桑黄栽培废料干粉;
(2)将桑黄栽培废料干粉与碱性乙醇溶液按质量比1:10~1:30混合;
(3)将步骤(2)中混合液超声处理20~40分钟,过滤得滤液和残渣;
(4)向步骤(3)中的残渣中加入残渣重量8~12倍的去离子水,混合均匀,调pH值4~6得到混合液,加入占混合液重量0.2~0.5%的复合酶,复合酶为纤维素酶和葡萄糖氧化酶混合组成, 45~55℃下酶解40~80分钟,酶解液经离心处理分离得上清液和沉淀物;
(5)步骤(3)中得到的滤液经旋转蒸发后除去乙醇,加入乙酸乙酯进行萃取,分液,上层液经减压浓缩干燥得到桑黄黄酮;
(6)步骤(4)中得到的上清液用旋转蒸发仪减压浓缩后进行醇沉,离心,得上清液和沉淀物,沉淀物即为桑黄多糖。
为提高提取率,本发明人采用了新的酶组合。本发明的创新性为首次采用葡萄糖氧化酶对桑黄栽培废料进行处理,发明人发现葡萄糖氧化酶与纤维素酶组合使用时,对桑黄栽培废料中的有效物质——桑黄黄酮和桑黄多糖的回收率较高,桑黄黄酮提取率可达0.5%,桑黄多糖提取率约为2%。
作为优选,步骤(1)中,桑黄栽培废料60±5℃烘干后进行超微粉碎。用超微粉碎机粉碎处理即可得到超微粉的桑黄栽培废料。
作为优选,步骤(2)中所述的碱性乙醇溶液中乙醇浓度80%~90%,利用氢氧化钠调pH=10~12得到碱性乙醇溶液。
作为优选,步骤(3)中,超声处理频率为100~500Hz,超声处理功率为100~1000W。
作为优选,在步骤(4)中,用醋酸或甲酸调节pH值。
作为优选,在步骤(4)中,复合酶为纤维素酶:葡萄糖氧化酶=2-3:1的混合物。
作为优选,步骤(4)中得到的上清液用旋转蒸发仪减压浓缩后加入3倍体积的95%乙醇进行醇沉后,在6000转/分下离心10分钟。
作为优选,步骤(4)中酶解液离心条件为6000转/分,10分钟。
碱液的使用更有利于超声能量的传递,超声和碱液的结合能够很大程度上破坏桑黄栽培基质的组织,乙醇的使用更有利于增加细胞膜的通透性,使多糖和黄酮的提取更为有效;纤维素酶和葡萄糖氧化酶的使用更有利于黄酮和多糖从桑黄栽培残渣中释放出来。在萃取的过程中,由于碱液的存在,更能提高萃取效率。采用此方法同时提取桑黄多糖和黄酮,提取效率高,提取的活性物质活性强。
本发明提供一种从桑黄栽培废料中同时提取多糖和黄酮的方法,包括提取准备步骤、超声辅助碱性乙醇提取步骤、残渣酶解步骤、乙酸乙酯萃取黄酮步骤和醇沉制备多糖步骤。本发明的有益效果是:本发明将超声辅助提取、酶解、萃取等操作有机结合,对桑黄栽培废料进行有效的回收再利用同时提取多糖和黄酮,简化了生产工艺,安全可靠,产率高,较好的保留了桑黄栽培废料中多糖和黄酮成分的生理和药理活性,具有较大的经济价值。
具体实施方式
下面通过具体实施例,对本发明的技术方案作进一步的具体说明。应当理解,本发明的实施并不局限于下面的实施例,对本发明所做的任何形式上的变通和/或改变都将落入本发明保护范围。
在本发明中,若非特指,所有的份、百分比均为重量单位,所采用的设备和原料等均可从市场购得或是本领域常用的。下述实施例中的方法,如无特别说明,均为本领域的常规方法。
纤维素酶:160000U/g, 湖州礼来生物科技有限公司;
葡萄糖氧化酶:50000 U/g, 河南华瑞生物科技有限公司。
桑黄栽培废料来源:淳安千岛湖桑都食用菌专业合作社。以下实施例以ZL201410477690.X“一株桑黄菌株及其应用”中实施例1的方法和培养基生产后得到的桑黄栽培废料为例来说明本发明效果。
实施例1
一种从桑黄栽培废料中同时提取多糖和黄酮的方法,具体包括如下步骤:
步骤(1):将桑黄栽培废料烘干和粉碎;
步骤(2):将桑黄栽培废料干粉与碱性乙醇溶液(体积分数80%的乙醇,利用氢氧化钠调pH=10)按质量比1:10混合;
步骤(3):将步骤(2)中混合液超声处理20分钟(100 Hz,1000W),过滤得滤液I和残渣;
步骤(4):向步骤(3)中的残渣中加入残渣重量8倍的去离子水,混合均匀,调节pH值至4,加入占混合液重量0.2%的复合酶(复合酶为纤维素酶:葡萄糖氧化酶=2:1的混合物,下同), 45℃下酶解40分钟,酶解液离心(6000转/分, 10分钟),分离得上清液II和沉淀物;
步骤(5):步骤(3)中滤液I,旋转蒸发后除去乙醇,加入乙酸乙酯进行萃取,分液,上层液经减压浓缩干燥获得桑黄黄酮,桑黄黄酮得率为0.24%;
步骤(6),上清液II,用旋转蒸发仪减压浓缩后加入3倍体积的95%乙醇进行醇沉后,在6000转/分下离心10分钟,得上清液和沉淀物,沉淀物即为桑黄多糖,桑黄多糖得率为1.26%。
实施例2
一种从桑黄栽培废料中同时提取多糖和黄酮的方法,具体包括如下步骤:
步骤(1):将桑黄栽培废料烘干和粉碎;
步骤(2):将桑黄栽培废料干粉与碱性乙醇溶液(体积分数85%乙醇, pH=11)按质量比1:20混合;
步骤(3):将步骤(2)中混合液超声处理30分钟(200 Hz,800 W),过滤得滤液I和残渣;
步骤(4):向步骤(3)中的残渣中加入残渣重量10倍的去离子水,混合均匀,调pH值4.5,加入占混合液重量0.3%的复合酶, 45℃下酶解50分钟,酶解液离心(6000转/分, 10分钟),分离得上清液II和沉淀物;
步骤(5):步骤(3)中滤液I,旋转蒸发后除去乙醇,加入乙酸乙酯进行萃取,分液,上层液经减压浓缩干燥获得桑黄黄酮,桑黄黄酮得率为0.39%;
步骤(6),上清液II,用旋转蒸发仪减压浓缩后加入3倍体积的95%乙醇进行醇沉后,在6000转/分下离心10分钟,得上清液和沉淀物,沉淀物即为桑黄多糖,桑黄多糖得率为1.81%。
实施例3
一种从桑黄栽培废料中同时提取多糖和黄酮的方法,具体包括如下步骤:
步骤(1):将桑黄栽培废料烘干和粉碎;
步骤(2):将桑黄栽培废料干粉与碱性乙醇溶液(体积分数90%乙醇, pH=12)按质量比1:30混合;
步骤(3):将步骤(2)中混合液超声处理30分钟(400 Hz, 200 W),过滤得滤液I和残渣;
步骤(4):向步骤(3)中的残渣中加入残渣重量12倍的去离子水,混合均匀,调pH值5.0,加入占混合液重量0.4%的复合酶(复合酶为纤维素酶:葡萄糖氧化酶=2.5:1的混合物), 50℃下酶解60分钟,酶解液离心(6000转/分, 10分钟),分离得上清液II和沉淀物;
步骤(5):步骤(3)中滤液I,旋转蒸发后除去乙醇,加入乙酸乙酯进行萃取,分液,上层液经减压浓缩干燥获得桑黄黄酮,桑黄黄酮得率为0.44 %;
步骤(6),上清液II,用旋转蒸发仪减压浓缩后加入3倍体积的95%乙醇进行醇沉后,在6000转/分下离心10分钟,得上清液和沉淀物,沉淀物即为桑黄多糖,桑黄多糖得率为1.60%。
实施例4
一种从桑黄栽培废料中同时提取多糖和黄酮的方法,具体包括如下步骤:
步骤(1):将桑黄栽培废料烘干和粉碎;
步骤(2):将桑黄栽培废料干粉与碱性乙醇溶液(体积分数90%乙醇, pH=12)按质量比1:30混合;
步骤(3):将步骤(2)中混合液超声处理30分钟 (500 Hz, 100 W),过滤得滤液I和残渣;
步骤(4):向步骤(3)中的残渣中加入残渣重量10倍的去离子水,混合均匀,调pH值5.5,加入占混合液重量0.5%的复合酶(复合酶为纤维素酶:葡萄糖氧化酶=3:1的混合物),55℃下酶解80分钟,酶解液离心(6000转/分, 10分钟),分离得上清液II和沉淀物;
步骤(5):步骤(3)中滤液I,旋转蒸发后除去乙醇,加入乙酸乙酯进行萃取,分液,上层液经减压浓缩干燥获得桑黄黄酮,桑黄黄酮得率为0.49%;
步骤(6),上清液II,用旋转蒸发仪减压浓缩后加入3倍体积的95%乙醇进行醇沉后,在6000转/分下离心10分钟,得上清液和沉淀物,沉淀物即为桑黄多糖,桑黄多糖得率为1.94%。
实施例5
一种从桑黄栽培废料中同时提取多糖和黄酮的方法,具体包括如下步骤:
步骤(1):将桑黄栽培废料烘干和粉碎;
步骤(2):将桑黄栽培废料干粉与碱性乙醇溶液(体积分数90%乙醇, 利用氢氧化钠调pH=12)按质量比1:15混合;
步骤(3):将步骤(2)中混合液超声处理30分钟 (500 Hz, 100 W),过滤得滤液I和残渣;
步骤(4):向步骤(3)中的残渣中加入残渣重量10倍的去离子水,混合均匀,调pH值5.5,加入占混合液重量0.5%的复合酶(复合酶为纤维素酶:葡萄糖氧化酶=2.2:1的混合物), 55℃下酶解80分钟,酶解液离心(6000转/分, 10分钟),分离得上清液II和沉淀物;
步骤(5):步骤(3)中滤液I,旋转蒸发后除去乙醇,加入乙酸乙酯进行萃取,分液,上层液经减压浓缩干燥获得桑黄黄酮,桑黄黄酮得率为0.51%;
步骤(6),上清液II,用旋转蒸发仪减压浓缩后加入3倍体积的95%乙醇进行醇沉后,在6000转/分下离心10分钟,得上清液和沉淀物,沉淀物即为桑黄多糖,桑黄多糖得率为2.06%。
以上所述的实施例只是本发明的一种较佳的方案,并非对本发明作任何形式上的限制,在不超出权利要求所记载的技术方案的前提下还有其它的变体及改型。
Claims (6)
1.一种从桑黄栽培废料中同时提取多糖和黄酮的方法,其特征在于该方法包括如下步骤:
(1)将桑黄栽培废料烘干、粉碎得到桑黄栽培废料干粉;
(2)将桑黄栽培废料干粉与碱性乙醇溶液按质量比1:10~1:30混合;
(3)将步骤(2)中混合液超声处理20~40分钟,过滤得滤液和残渣;
(4)向步骤(3)中的残渣中加入残渣重量8~12倍的去离子水,混合均匀,调pH值4~6得到混合液,加入占混合液重量0.2~0.5%的复合酶,复合酶为纤维素酶和葡萄糖氧化酶混合组成,45~55℃下酶解40~80分钟,酶解液经离心处理分离得上清液和沉淀物;复合酶为纤维素酶:葡萄糖氧化酶=2-3:1的混合物;
(5)步骤(3)中得到的滤液经旋转蒸发后除去乙醇,加入乙酸乙酯进行萃取,分液,上层液经减压浓缩干燥得到桑黄黄酮;
(6)步骤(4)中得到的上清液用旋转蒸发仪减压浓缩后进行醇沉,离心,得上清液和沉淀物,沉淀物即为桑黄多糖。
2.根据权利要求1所述的方法,其特征在于:步骤(1)中,桑黄栽培废料60±5℃烘干后进行超微粉碎。
3.根据权利要求1所述的方法,其特征在于:步骤(2)中所述的碱性乙醇溶液中乙醇浓度80%~90%,利用氢氧化钠调pH=10~12得到碱性乙醇溶液。
4.根据权利要求1所述的方法,其特征在于:步骤(3)中,超声处理频率为100~500Hz,超声处理功率为100~1000W。
5.根据权利要求1所述的方法,其特征在于:在步骤(4)中,用醋酸或甲酸调节pH值。
6.根据权利要求1所述的方法,其特征在于:步骤(4)中得到的上清液用旋转蒸发仪减压浓缩后加入3倍体积的95%乙醇进行醇沉后,在6000转/分下离心10分钟。
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Denomination of invention: A method for simultaneously extracting polysaccharide and flavone from Mulberry Yellow cultivation waste Effective date of registration: 20211111 Granted publication date: 20190412 Pledgee: Industrial and Commercial Bank of China Limited Chun'an sub branch Pledgor: CHUN'AN QIANDAO LAKE SANGDU EDIBLE FUNGUS PROFESSIONAL COOPERATIVES Registration number: Y2021330002226 |