CN113429288A - 一种同时提取牛蒡叶精油和绿原酸的方法与应用 - Google Patents
一种同时提取牛蒡叶精油和绿原酸的方法与应用 Download PDFInfo
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- CN113429288A CN113429288A CN202110621252.6A CN202110621252A CN113429288A CN 113429288 A CN113429288 A CN 113429288A CN 202110621252 A CN202110621252 A CN 202110621252A CN 113429288 A CN113429288 A CN 113429288A
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Abstract
本发明涉及一种同时提取牛蒡叶精油和绿原酸的方法,(1)获得牛蒡叶浆液;(2)超声波‑酶解;(3)CO2超临界流体萃取,收集的萃取物即为牛蒡叶精油;(4)将步骤(3)萃取之后的液体加入到D101B树脂吸附柱中进行吸附;(5)按照步骤(4)的方法进行大孔树脂吸附,收集目标产物的洗脱液,减压浓缩,浓缩液重结晶,获得绿原酸产品。本发明提供了一种同时提取牛蒡叶中精油和绿原酸的方法,而且,精油和绿原酸的得率均较高,实现了资源利用最大化。
Description
技术领域
本发明涉及一种同时提取牛蒡叶精油和绿原酸的方法与应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的一些理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
牛蒡为菊科两年生草本类植物,又称鼠黏草、牛鞭菜、蝙蝠刺、东洋萝卜等。牛蒡营养丰富,富含纤维素、菊糖、蛋白质、钙、铁、磷等矿物质和多种维生素以及多种具有生物活性的物质,而且具有很优异的保健功能,果实、根、茎和叶均可以使用。我国大面积种植牛蒡,主要利用果实和根,而对于牛蒡叶的利用和研究的较少,尤其是关于牛蒡叶精油鲜有报道,大部分的牛蒡叶被作为废弃物舍弃掉,造成了资源浪费和环境污染。
发明内容
针对以上背景技术,本发明提供一种从牛蒡叶中同时提取精油和绿原酸的方法与应用,实现资源利用最大化。
具体的,本发明采用以下技术方案:
在本发明的第一个方面,提供一种同时提取牛蒡叶精油和绿原酸的方法,该方法包括以下步骤:
(1)将牛蒡叶洗净、制浆,获得牛蒡叶浆液;
(2)超声波-酶解:将牛蒡叶浆液与水混合均匀,置于超声波仪器内,然后加入复合酶混合均匀,同步进行超声波和酶解处理;
其中,所述复合酶是由纤维素酶、半纤维素酶、糖化酶、酸性α-淀粉酶和酸性脂肪酶组成;
(3)CO2超临界流体萃取:超声波酶解后,采用CO2超临界流体萃取,夹带剂为乙烷,夹带剂含量:2~5mol%,萃取温度为25~50℃,萃取时间为1~3h,萃取压力为15~25MPa;收集的萃取物即为牛蒡叶精油;
(4)将步骤(3)萃取之后的液体加入到D101B大孔树脂吸附柱中进行吸附;用洗脱液洗脱吸附柱,收集目标产物的洗脱液;
(5)将步骤(4)中的浓缩液再次加入到D101B大孔树脂吸附柱中进行吸附;用洗脱液洗脱吸附柱,收集目标产物的洗脱液,减压浓缩,浓缩液重结晶,获得绿原酸产品。
优选的,步骤(2)中,所述牛蒡叶和水的质量比例为1:(2~4)。
为提高精油出油率和绿原酸的提取率,本发明选择了适宜的超声波处理条件,适宜的超声条件可提高酶解效率,提高酶活性;而不合适的超声条件会降低酶解反应效率或可能使酶失去活性。经过试验验证,该超声波处理可以同时提高精油和绿原酸的得率。优选的,步骤(2)中,超声波条件为:超声频率为20~30kHz,功率为100~300W,温度30~50℃,超声时间为30~90min。
优选的,步骤(2)中,超声波-酶解的反应体系pH为5~6。酸性体系可以分解牛蒡叶中挥发性化合物与糖配键结合形成的糖苷化合物,分解出挥发性化合物,提高了牛蒡叶精油得率。
优选的,步骤(2)中,复合酶的用量为物料质量的0.5~2%。
优选的,步骤(2)中,所述复合酶是由以下重量百分比的成分组成:纤维素酶30~50%、半纤维素酶10~20%、糖化酶5~10%,酸性α-淀粉酶5~10%,剩余为酸性脂肪酶;其中,纤维素酶的酶活性为50万U/g,半纤维素酶的酶活性为50万U/g,糖化酶的酶活性为10万U/g,酸性α-淀粉酶的酶活性为10万U/g,酸性脂肪酶的酶活性为10万U/g。以上酶均可通过市售获得。
本发明选择了有利于萃取精油成分的CO2超临界流体萃取条件,而降低对绿原酸的选择和萃取,通过该萃取条件,提高了牛蒡叶精油得率。优选的,步骤(3)中,采用CO2超临界流体萃取,夹带剂为乙烷,夹带剂含量:3mol%,萃取温度为35℃,萃取时间为2h,萃取压力为20MPa。试验中发明人发现,本发明采用乙烷作为非极性夹带剂,可提高牛蒡叶出油率,同时对绿原酸的选择性较低。
本发明选择D101B大孔吸附树脂,相比于其他大孔吸附树脂,对绿原酸的吸附力更强,吸附率和洗脱率更高。通过两次大孔树脂的吸附处理,能够获得高纯度的绿原酸。
优选的,步骤(4)和(5)中,吸附参数:吸附液pH为4~5,流速2.0~4.0mL/min;解析参数为:解吸剂为50~60v/v%乙醇溶液,洗脱速度为1.5~3mL/min,pH值为2.5~3.5。
在本发明的第二个方面,提供采用上述方法制备得到的牛蒡叶精油和/或绿原酸产品。
在本发明的第三个方面,提供所述牛蒡叶精油在制备烟草增香剂、化妆品添加剂、食品添加剂和医疗保健品中的应用。
与本发明人知晓的相关技术相比,本发明其中的一个技术方案具有如下有益效果:
本发明提供了一种同时提取牛蒡叶中精油和绿原酸的方法,而且,精油和绿原酸的得率均较高,实现了资源利用最大化。
本发明采用超声波辅助酶解的方法,可提高牛蒡叶的出油率和绿原酸的渗出率,复合酶的酶解体系为酸性,可以分解牛蒡叶中挥发性化合物与糖配键结合形成的糖苷化合物,分解出挥发性化合物,提高了牛蒡叶精油得率,此外,酶解作用使得绿原酸提取物中的糖分等大分子物质含量降低,利于后续工艺的过滤和大孔树脂分离,无需单独进行脱色、脱脂处理,进一步提高了绿原酸的得率。
附图说明
构成本发明一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1为实施例1中牛蒡叶精油的总离子流图。
具体实施方式
应该指出,以下详细说明都是示例性的,旨在对本发明提供进一步的说明。除非另有指明,本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案,以下将结合具体的实施例详细说明本发明的技术方案。
实施例1
一种同时提取牛蒡叶精油和绿原酸的方法,该方法包括以下步骤:
(1)将采摘自徐州牛蒡种植基地的1kg新鲜牛蒡叶清洗干净,将清洗干净的牛蒡叶在常温下进行制浆,获得牛蒡叶浆液;
(2)按照1:2的质量比例向牛蒡叶浆液中加入水,混合均匀,置于超声波仪器中,然后加入物料(牛蒡浆液和水)质量的1%的复合酶,用醋酸调节pH至5.5,混合均匀,开启超声波仪器,调节超声波条件为:超声频率为25kHz,功率为150W,温度35℃,同步进行超声波和酶解处理;处理45min后,过滤,获得酶解液;
其中,所述复合酶由以下重量百分比的成分组成:纤维素酶45%、半纤维素酶15%、糖化酶8%,酸性α-淀粉酶8%,剩余为酸性脂肪酶;其中,纤维素酶的酶活性为50万U/g,半纤维素酶的酶活性为50万U/g,糖化酶的酶活性为10万U/g,酸性α-淀粉酶的酶活性为10万U/g,酸性脂肪酶的酶活性为10万U/g,以上酶均可通过市售获得;
(3)将酶解液投入萃取釜中,对萃取釜、分离釜分别加热或冷却,当系统各部分达到设定温度后,开启CO2钢瓶,夹带剂为乙烷,含量为3mol%,向萃取釜中通入CO2,升压至20MPa,温度为35℃,CO2流速为20L/h,对萃取釜中的物料进行萃取,含有萃取物的CO2流入分离釜中,经分离釜减压与萃取物分离后循环使用,分离釜中的压力为10MPa,温度为30℃;萃取2h后,收集分离釜中的萃取物,除去水分,得到4.56g牛蒡叶精油,得率为0.46%。
本发明采用气相色谱-质谱对获得的牛蒡叶精油进行化合物定性和定量检测。气相色谱条件为:
GC-MS分析:
色谱分析条件:检测器为TIC,色谱柱为DB-5ms(30.0m×0.25mm×0.25um)。起始柱温50℃,保持5min,10℃/min升温到250℃,保持15min;进样口温度为250℃,进样量:lμL,流速:1.0mL/min;柱温:50℃;载气为氦气;不分流。
质谱条件:EI离子源,电子能量70eV,离子源温度:250℃,溶剂延迟:5min,全扫描范围为30-650amu;成分定性定量:参照NIST98谱库,根据保留时间、峰面积及样品匹配度。
定性定量分析结果见表1和图1,如表1和图1所示,本发明制备的牛蒡叶精油中呈香化合物和萜类成分相对含量较高,长链脂肪酸含量相对较低,精油品质较高。
表1牛蒡叶精油主要成分及相对含量
(4)将残余液采用4倍质量的蒸馏水进行稀释,稀释后缓慢倒入D101B大孔树脂吸附柱中进行吸附,吸附参数:吸附液pH为4.5,流速3mL/min;解析参数为:解吸剂为60v/v%乙醇溶液,洗脱速度为1.5mL/min,pH值为2.5;收集含有绿原酸的洗脱液;
(5)将收集到的洗脱液缓慢倒入D101B大孔树脂吸附柱中进行吸附,吸附参数:吸附液pH为4.5,流速4.0mL/min;解析参数为:解吸剂为60v/v%乙醇溶液,洗脱速度为2mL/min,pH值为2.5;收集含有绿原酸的洗脱液,45℃减压浓缩,浓缩液重结晶,即可获得高纯度的绿原酸产品,经过测定,绿原酸产品的纯度为99.5%,得率为2.35%。
实施例2
一种同时提取牛蒡叶精油和绿原酸的方法,该方法包括以下步骤:
(1)将采摘自徐州牛蒡种植基地的1kg新鲜牛蒡叶清洗干净,将清洗干净的牛蒡叶在常温下进行制浆,获得牛蒡叶浆液;
(2)按照1:2.5的质量比例向牛蒡叶浆液中加入水,混合均匀,置于超声波仪器中,然后加入物料(牛蒡浆液和水)质量的0.8%的复合酶,用醋酸调节pH至5,混合均匀,开启超声波仪器,调节超声波条件为:超声频率为30kHz,功率为200W,温度35℃,同步进行超声波和酶解处理;处理60min后,过滤,获得酶解液;
其中,所述复合酶由以下重量百分比的成分组成:纤维素酶40%、半纤维素酶10%、糖化酶10%,酸性α-淀粉酶5%,剩余为酸性脂肪酶;其中,纤维素酶的酶活性为50万U/g,半纤维素酶的酶活性为50万U/g,糖化酶的酶活性为10万U/g,酸性α-淀粉酶的酶活性为10万U/g,酸性脂肪酶的酶活性为10万U/g,以上酶均可通过市售获得;
(3)将酶解液投入萃取釜中,对萃取釜、分离釜分别加热或冷却,当系统各部分达到设定温度后,开启CO2钢瓶,夹带剂为乙烷,含量为2.5mol%,向萃取釜中通入CO2,升压至15MPa,温度为35℃,CO2流速为20L/h,对萃取釜中的物料进行萃取,含有萃取物的CO2流入分离釜中,经分离釜减压与萃取物分离后循环使用,分离釜中的压力为10MPa,温度为30℃;萃取1h后,收集分离釜中的萃取物,除去水分,得到4.42g牛蒡叶精油,得率为0.44%;
(4)将残余液采用5倍质量的蒸馏水进行稀释,稀释后缓慢倒入D101B大孔树脂吸附柱中进行吸附,吸附参数:吸附液pH为5,流速4mL/min;解析参数为:解吸剂为55v/v%乙醇溶液,洗脱速度为2mL/min,pH值为3;收集含有绿原酸的洗脱液。
(5)将收集到的洗脱液缓慢倒入D101B大孔树脂吸附柱中进行吸附,吸附参数:吸附液pH为5,流速4.0mL/min;解析参数为:解吸剂为55v/v%乙醇溶液,洗脱速度为2mL/min,pH值为3;收集含有绿原酸的洗脱液,45℃减压浓缩,浓缩液重结晶,即可获得高纯度的绿原酸产品,经过测定,绿原酸产品的纯度为99.2%,得率为2.24%。
实施例3
一种同时提取牛蒡叶精油和绿原酸的方法,该方法包括以下步骤:
(1)将采摘自徐州牛蒡种植基地的1kg新鲜牛蒡叶清洗干净,将清洗干净的牛蒡叶在常温下进行制浆,获得牛蒡叶浆液;
(2)按照1:3的质量比例向牛蒡叶浆液中加入水,混合均匀,置于超声波仪器中,然后加入物料(牛蒡浆液和水)质量的1.5%的复合酶,用醋酸调节pH至6,混合均匀,开启超声波仪器,调节超声波条件为:超声频率为20kHz,功率为300W,温度35℃,同步进行超声波和酶解处理;处理30min后,过滤,获得酶解液;
其中,所述复合酶由以下重量百分比的成分组成:纤维素酶35%、半纤维素酶15%、糖化酶10%,酸性α-淀粉酶10%,剩余为酸性脂肪酶;其中,纤维素酶的酶活性为50万U/g,半纤维素酶的酶活性为50万U/g,糖化酶的酶活性为10万U/g,酸性α-淀粉酶的酶活性为10万U/g,酸性脂肪酶的酶活性为10万U/g,以上酶均可通过市售获得;
(3)将酶解液投入萃取釜中,对萃取釜、分离釜分别加热或冷却,当系统各部分达到设定温度后,开启CO2钢瓶,夹带剂为乙烷,含量为4mol%,向萃取釜中通入CO2,升压至20MPa,温度为40℃,CO2流速为20L/h,对萃取釜中的物料进行萃取,含有萃取物的CO2流入分离釜中,经分离釜减压与萃取物分离后循环使用,分离釜中的压力为10MPa,温度为30℃;萃取1h后,收集分离釜中的萃取物,除去水分,得到4.13g牛蒡叶精油,得率为0.41%。
(4)将残余液采用5倍质量的蒸馏水进行稀释,稀释后缓慢倒入D101B大孔树脂吸附柱中进行吸附,吸附参数:吸附液pH为5,流速3.5mL/min;解析参数为:解吸剂为50v/v%乙醇溶液,洗脱速度为2mL/min,pH值为3.5;收集含有绿原酸的洗脱液;
(5)将收集到的洗脱液缓慢倒入D101B大孔树脂吸附柱中进行吸附,吸附参数:吸附液pH为5,流速3.5mL/min;解析参数为:解吸剂为50v/v%乙醇溶液,洗脱速度为2mL/min,pH值为3.5;收集含有绿原酸的洗脱液,45℃减压浓缩,浓缩液重结晶,即可获得高纯度的绿原酸产品,经过测定,绿原酸产品的纯度为99.3%,得率为2.13%。
对比例1
与实施例1相同,不同的是夹带剂替换为乙酸乙酯。步骤(3)获得4.32g牛蒡叶精油,得率是0.43%。步骤(5)中绿原酸产品的得率为0.94%。由此可见,不同夹带剂对绿原酸的得率具有较大影响,本发明选择乙烷作为夹带剂,同时可获得高收率的牛蒡叶精油和绿原酸产品。
对比例2
与实施例1相同,不采用复合酶酶解处理。步骤(3)获得1.65g牛蒡叶精油,得率为0.17%。步骤(5)中绿原酸产品的得率为0.73%。
对比例3
与实施例1相同,不采用超声波处理。步骤(3)获得1.24g牛蒡叶精油,得率为0.12%。步骤(5)中绿原酸产品的得率为0.84%。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
1.一种同时提取牛蒡叶精油和绿原酸的方法,其特征是,该方法包括以下步骤:
(1)将牛蒡叶洗净、制浆,获得牛蒡叶浆液;
(2)超声波-酶解:将牛蒡叶浆液与水混合均匀,置于超声波仪器内,然后加入复合酶混合均匀,同步进行超声波和酶解处理;
其中,所述复合酶是由纤维素酶、半纤维素酶、糖化酶、酸性α-淀粉酶和酸性脂肪酶组成;
(3)CO2超临界流体萃取:超声波酶解后,采用CO2超临界流体萃取,夹带剂为乙烷,夹带剂含量:2~5mol%,萃取温度为25~50℃,萃取时间为1~3h,萃取压力为15~25MPa;收集的萃取物即为牛蒡叶精油;
(4)将步骤(3)萃取之后的液体加入到D101B大孔树脂吸附柱中进行吸附;用洗脱液洗脱吸附柱,收集目标产物的洗脱液;
(5)将步骤(4)中的浓缩液再次加入到D101B大孔树脂吸附柱中进行吸附;用洗脱液洗脱吸附柱,收集目标产物的洗脱液,减压浓缩,浓缩液重结晶,获得绿原酸产品。
2.如权利要求1所述的方法,其特征是,步骤(2)中,所述牛蒡叶和水的质量比例为1:(2~4)。
3.如权利要求1所述的方法,其特征是,步骤(2)中,超声波条件为:超声频率为20~30kHz,功率为100~300W,温度30~50℃,超声时间为30~90min。
4.如权利要求1所述的方法,其特征是,步骤(2)中,超声波-酶解的反应体系pH为5~6。
5.如权利要求1所述的方法,其特征是,步骤(2)中,复合酶的用量为物料质量的0.5~2%。
6.如权利要求1所述的方法,其特征是,步骤(2)中,所述复合酶是由以下重量百分比的成分组成:纤维素酶30~50%、半纤维素酶10~20%、糖化酶5~10%,酸性α-淀粉酶5~10%,剩余为酸性脂肪酶;其中,纤维素酶的酶活性为50万U/g,半纤维素酶的酶活性为50万U/g,糖化酶的酶活性为10万U/g,酸性α-淀粉酶的酶活性为10万U/g,酸性脂肪酶的酶活性为10万U/g。
7.如权利要求1所述的方法,其特征是,步骤(3)中,采用CO2超临界流体萃取,夹带剂为乙烷,夹带剂含量为3mol%,萃取温度为35℃,萃取时间为2h,萃取压力为20MPa。
8.如权利要求1所述的方法,其特征是,步骤(4)和(5)中,吸附参数:吸附液pH为4~5,流速2.0~4.0mL/min;解析参数为:解吸剂为50~60v/v%乙醇溶液,洗脱速度为1.5~3mL/min,pH值为2.5~3.5。
9.采用权利要求1~8中任一项所述方法制备得到的牛蒡叶精油或绿原酸产品。
10.权利要求9所述的牛蒡叶精油在制备烟草增香剂、化妆品添加剂、食品添加剂和医疗保健品中的应用。
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