CN116850094A - 一种海藻多酚提取方法 - Google Patents
一种海藻多酚提取方法 Download PDFInfo
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- CN116850094A CN116850094A CN202310836067.8A CN202310836067A CN116850094A CN 116850094 A CN116850094 A CN 116850094A CN 202310836067 A CN202310836067 A CN 202310836067A CN 116850094 A CN116850094 A CN 116850094A
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Abstract
本发明属于植物提取物技术领域。本发明公开了一种海藻多酚提取方法。本发明藻类多酚的高效提取流程包括:藻类烘干粉碎、发酵、酶解、高温处理、超声萃取、离心、乙醇浸提、溶剂蒸发、冻干步骤。通过本发明的提取方法使藻类中多酚物质大量溶出,大幅度提高了海藻多酚的提取量,为以后海藻多酚的规模化生产提供了一定的理论基础,且本发明在不破坏海藻多酚其抗氧化活性的条件下尽可能提高海藻的总酚含量。
Description
技术领域
本发明涉及植物提取物技术领域,尤其涉及一种海藻多酚提取方法。
背景技术
我国海域辽阔,具有丰富的海洋生物资源,如鱼类、贝类、虾类、藻类及海洋微生物等。其中,藻类作为一大类重要的海洋生物资源,藻类含有大量的高活性生物质成分,海藻富含海藻多糖、蛋白质、酚类、萜类、岩藻黄素、氨基酸、甘露醇、激素等多种活性物质,海藻多酚是从海藻中提取出的一类多羟基酚类化合物,也被认为是间苯三酚的衍生物,具有抗氧化、抗菌、抗病毒、抗肿瘤、抗心血管疾病、抗糖尿病综合症等广泛的生物活性,未来将在海洋药物、功能食品和日化领域得到广泛应用。尤其藻类中抗氧化活性成分的研究已经成为海洋生物活性物质研究的热点,藻类多酚分子中的酚性羟基能够切断氧化过程中产生的过氧化物自由基、羟基自由基的链式反应,从而达到抑制和减缓氧化的效果,在食品、化妆品和生物医药领域具有重要的开发应用前景。
藻类多酚类化合物是藻类的次级代谢产物,是我国海域常见藻类的活性成分,藻类多酚大多以间苯三酚的衍生物和聚合物的形式存在。但是,藻类中的多酚不易于直接提取,目前藻类多酚的提取方法主要是有机溶剂萃取,用这种方法提取效率低且提取量少,由于藻类多酚一般位于大多数藻类细胞内部,导致常规有机溶剂提取的多酚量较少的同时得到的藻类多酚的抗氧化活性会受到一些影响。而近期较为先进的藻类多酚提取方法有离心提取法、超临界流体萃取法、液压萃取法、酶辅助提取法,离心分区提取法和超临界流体萃取法等,离心分区提取法和超临界流体萃取法提取出来的海藻多酚虽然纯度比较高,但是,提取的时间比较长,而且,提取量比较少;液压萃取法和酶辅助提取法虽然在多酚的提取量上略有增加,但是,这种提取量的增加还是不明显,而且,工艺复杂,成本高,难以实现工业化生产。
发明内容
有鉴于此,针对现有技术存在的缺陷,本发明提供了一种海藻多酚提取方法,既充分保证了藻类多酚的抗氧活性,又提高了藻类多酚的提取量。
为了达到上述目的,本发明采用如下技术方案:
一种海藻多酚提取方法,包括以下步骤:
⑴将藻类洗净、烘干、粉碎,备用;
⑵将步骤⑴制得的藻类粉末加入复配菌悬液,发酵,即得藻类发酵液,备用;
(3)采用高静压协同酶液对藻类发酵液进行处理,即得藻类菌酶混合液,备用;
⑷将步骤(3)制得的藻类菌酶混合液加入金属无机盐进行高温处理,即得藻类提取液Ⅰ,备用;
⑸向步骤⑷制得的藻类提取液中加入萃取溶液和表面活性剂,超声处理,即得藻类提取液Ⅱ,备用;
⑹将步骤⑸制得的藻类提取液Ⅱ离心,弃去沉淀、取上清液,即得藻类多酚提取液,备用;
⑺将步骤⑹制得的藻类多酚提取液用乙醇溶液进行浸提,即得藻类多酚醇提液,备用;
⑻将步骤⑺制得的藻类多酚醇提液旋蒸回收、冻干即得藻类多酚成品。进一步,本发明所述的藻类多酚的高效提取方法,具体为以下步骤:
⑴将藻类洗净、放入在60℃电热鼓风干燥箱中烘干,然后用粉碎机将其粉碎,并过60目筛网制得粉末,备用;
⑵将复配菌悬液与藻类粉末按重量比2:1混合,用无菌水调节混合物含水量为70~80%,30℃恒温发酵7d;即得藻类发酵液;
⑶将藻类发酵液密封于双层聚乙烯塑料袋中,抽真空密封后,置于高压设备中,于室温下400Mpa高静压保压处理20~25min;
⑷将步骤⑶高静压处理后藻类发酵液加柠檬酸,调节溶液pH至4.0~5.2再加入复合植物水解酶升温至45~55℃,酶解3~5h,即得藻类菌酶混合液;
⑸将藻类菌酶混合液加入金属无机盐溶液后加热至沸腾,保持沸腾10~15min,冷却至室温,得到藻类提取液Ⅰ;
⑹向藻类提取液Ⅰ液中加入萃取溶液和表面活性剂,于45~55℃以功率494W超声萃取20~30min,即得藻类提取液Ⅱ;
⑺将藻类提取液Ⅱ在室温下以4000~8000r/min,离心10~15min,取上清液,即得藻类多酚粗提取液;
⑻将藻类多酚提取液按体积比1:6加体积浓度为40~50%的乙醇水溶液置于50℃~80℃水浴摇床中,转速100r/min,避光浸提1h~6h,即得藻类多酚醇提液;
⑼将藻类多酚醇提液放入40℃,40rpm/min的旋转蒸发仪中蒸去溶剂,在冻干机中进行冻干后成冻干粉,即得海带多酚成品,放入-20℃冰箱中保存。
进一步,所述的复配菌悬液制备方法:分别将乳酸菌细胞悬液、毕赤酵母细胞悬液、德氏乳杆菌细胞悬液在30℃、180r/min培养24h,10000r/min、4℃离心10min,分别收集上清液,再分别加入无菌浓度为0.9%的NaCl溶液,分别配成1.0×107cfu/mL的菌悬液,按重量比1:2:2比例混合即得复配菌悬液。
进一步,所述萃取溶液由氯化胆碱、1,2丁二醇按摩尔比1:4混合,在80℃恒磁搅拌下,直至形成透明均匀的液体,然后在50℃真空烘箱中干燥24h,再与蒸馏水按照质量比为7:3的比例混合,充分拌匀即可得到萃取溶液。
进一步,所述的金属无机盐选择FeSO4·7H2O、ZnCl2、CaCl2·2H2O、MnCl2·2H2O中的任意一种或多种组合。
进一步,优选的,金属无机盐选择多种组合,具体为FeSO4·7H2O和MnCl2·2H2O进行组合,其中FeSO4·7H2O浓度为0.001~0.002mmol/L,MnCl2·2H2O浓度为0.004~0.006mmol/L。
进一步,所述的复合植物水解酶为ViscozymeL。
进一步,所述的复合植物水解酶浓度为1%(V/V)。
进一步,所述的表面活性剂为月桂酰基谷氨酸钠。
进一步,所述的月桂酰基谷氨酸钠浓度为0.024~0.036mol/L。
经由上述的技术方案可知,本发明公开提供了一种海藻多酚提取方法,现在的藻类多酚,一般只采用传统的有机溶剂浸提法进行提取,效率差、抗氧活性无法保证。而本发明与传统的有机溶剂浸提法相比,通过本发明的提取方法使藻类中多酚物质大量溶出,大幅度提高了海藻多酚的提取量,为以后海藻多酚的规模化生产提供了一定的理论基础,且本发明在不破坏海藻多酚其抗氧化活性的条件下尽可能提高海藻的总酚含量,而提取的多酚是一种小分子的物质,更容易被人体所吸收。
1、本发明采用的高效提取流程包括:藻类烘干粉碎、发酵、酶解、高温处理、超声萃取、离心、乙醇浸提、溶剂蒸发、冻干步骤:
2、与现有技术相比,本发明在酶解前,将藻类粉末进行了发酵处理,发酵采用乳酸菌、毕赤酵母、德氏乳杆菌混合的菌悬液进行了发酵,通过发酵使得使得藻类多酚含量从源头上显著增加,为提取打下了良好的基础。
3、与现有技术相比,本发明在发酵后进行酶解反应,与传统的单一酶解相比,一方面采用了复合植物水解酶进行处理,相比于传统的果胶酶和纤维素酶,采用的ViscozymeL酶,活性更高、酶解效率更高、酶解效果更好,为以下的多酚物质萃取更进一步的打下良好的基础,另一方面,在配合高静压处理,藻类细胞物质更容易溶出,使得此步骤的酶促反应,反应时间缩短,反应效率显著提升。
4、与现有技术相比,传统进行灭酶灭菌处理时,只是单纯的进行高温灭菌,藻类中的多酚物质容易受温度影响,温度在升高的过程中,藻类多酚氧化酶活性会升高,使得藻类多酚含量下降,抗氧化活性降低,本发明为了解决在升温环境过程中,藻类多酚含量损失抗氧活性降低的问题,在进行高温处理时提前加入了金属无机盐溶液,比如:FeSO4·7H2O、ZnCl2、CaCl2·2H2O、MnCl2·2H2O中的任意一种或多种组合,加入的金属离子如:Fe2+、Mn2+协同作用,在升温过程中能够钝化藻类多酚氧化酶,从而降低该酶的活性,使得藻类多酚的含量流失降低。
5、与现有相比,本发明在进行超声萃取时,由于萃取前的发酵、酶解以及金属离子的加入从而保证了进入超声萃取步骤的藻类多酚含量增多,进而再加至超声萃取步骤,采用了氯化胆碱、1,2丁二醇混合制备的共溶溶剂进行萃取,萃取效率显著提高,而且在萃取的过程中,还加入了氨基酸表面活性剂月桂酰基谷氨酸钠,进一步减少了藻类多酚在此步骤中的消耗量,保证了藻类多酚的抗氧活性,使得提取效率显著提高,最终使得得到的藻类多酚含量显著提高。
具体实施方式
下面将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。
实施例1
本实施例提供的高效的藻类多酚制备方法,其制备方法如下
⑴海带处理:将采集的新鲜海带(褐藻)洗净、放入在60℃电热鼓风干燥箱中烘干,然后用粉碎机将其粉碎,并过60目筛网制得粉末,备用;
⑵复配菌悬液制备:分别将乳酸菌细胞悬液、毕赤酵母细胞悬液、德氏乳杆菌细胞悬液在30℃、180r/min培养24h,10000r/min、4℃离心10min,分别收集上清液,再分别加入无菌浓度为0.9%的NaCl溶液,分别配成1.0×107cfu/mL的菌悬液,按重量比1:2:2比例混合即得复配菌悬液;
⑶发酵:将复配菌悬液与海带粉末按重量比2:1混合,用无菌水调节混合物含水量为70%,30℃恒温发酵7d;即得海带发酵液;
⑷高静压处理:将海带发酵液密封于双层聚乙烯塑料袋中,抽真空密封后,置于高压设备中,于室温下400Mpa高静压保压处理20min;
⑸酶解:将步骤⑷高静压处理后海带发酵液加柠檬酸,调节溶液pH至4.0再加入酶浓度为1%(V/V)的ViscozymeL升温至45℃,酶解3h,即得海带菌酶混合液;
⑹灭菌酶:将海带菌酶混合液加入浓度为0.001mmol/LFeSO4·7H2O和浓度为0.004mmol/LMnCl2·2H2O后加热至沸腾,保持沸腾10min,冷却至室温,得到海带提取液Ⅰ;
⑺萃取溶液制备:将氯化胆碱、1,2丁二醇按摩尔比1:4混合,在80℃恒磁搅拌下,直至形成透明均匀的液体,然后在50℃真空烘箱中干燥24h,再与蒸馏水按照质量比为7:3的比例混合,充分拌匀即可得到萃取溶液;
⑻超声萃取:向海带提取液Ⅰ液中加入萃取溶液和浓度为0.024mol/L月桂酰基谷氨酸钠,于45℃以功率494W超声萃取20min,即得藻类提取液Ⅱ;
⑼将海带提取液Ⅱ在室温下以4000r/min,离心10min,取上清液,即得海带多酚提取液;
⑽将海带多酚提取液按体积比1:6加体积浓度为40%的乙醇水溶液置于50℃水浴摇床中,转速100r/min,避光浸提1h,即得海带多酚醇提液;
⑾将海带多酚醇提液放入40℃,40rpm/min的旋转蒸发仪中蒸去溶剂,在冻干机中进行冻干后成冻干粉,即得海带多酚成品,放入-20℃冰箱中保存。
按照本领域技术人员熟知的酚类物质以及体外抗氧活性的测定方法,最后得海藻多酚的总酚含量为19.47mg/g(以干的海带粉计,以没食子酸为当量),纯度为30.31%(总酚含量/总酚干物质的量),其DPPH、羟自由基均有较好的体外清除活性,IC50值分别为4.496mg/L、13.115mg/L,总抗氧化能力为68.76U/mg。
实施例2
本实施例提供的高效的藻类多酚制备方法,其制备方法如下
⑴鼠尾藻处理:将采集的新鲜鼠尾藻洗净、放入在60℃电热鼓风干燥箱中烘干,然后用粉碎机将其粉碎,并过60目筛网制得粉末,备用;
⑵复配菌悬液制备:分别将乳酸菌细胞悬液、毕赤酵母细胞悬液、德氏乳杆菌细胞悬液在30℃、180r/min培养24h,10000r/min、4℃离心10min,分别收集上清液,再分别加入无菌浓度为0.9%的NaCl溶液,分别配成1.0×107cfu/mL的菌悬液,按重量比1:2:2比例混合即得复配菌悬液;
⑶发酵:将复配菌悬液与鼠尾藻粉末按重量比2:1混合,用无菌水调节混合物含水量为80%,30℃恒温发酵7d;即得鼠尾藻发酵液;
⑷高静压处理:将鼠尾藻发酵液密封于双层聚乙烯塑料袋中,抽真空密封后,置于高压设备中,于室温下400Mpa高静压保压处理25min;
⑸酶解:将步骤⑷高静压处理后鼠尾藻发酵液加柠檬酸,调节溶液pH至5.2再加入酶浓度为1%(V/V)的ViscozymeL升温至55℃,酶解5h,即得鼠尾藻菌酶混合液;
⑹灭菌酶:将海带菌酶混合液加入浓度为0.002mmol/LFeSO4·7H2O和浓度为0.006mmol/LMnCl2·2H2O后加热至沸腾,保持沸腾10min,冷却至室温,得到海带提取液Ⅰ;
⑺萃取溶液制备:将氯化胆碱、1,2丁二醇按摩尔比1:4混合,在80℃恒磁搅拌下,直至形成透明均匀的液体,然后在50℃真空烘箱中干燥24h,再与蒸馏水按照质量比为7:3的比例混合,充分拌匀即可得到萃取溶液;
⑻超声萃取:向鼠尾藻提取液Ⅰ液中加入萃取溶液和浓度为0.036mol/L月桂酰基谷氨酸钠,于55℃以功率494W超声萃取30min,即得鼠尾藻提取液Ⅱ;
⑼将鼠尾藻提取液Ⅱ在室温下以8000r/min,离心15min,取上清液,即得鼠尾藻多酚提取液;
⑽将藻类多酚提取液按体积比1:6加体积浓度为50%的乙醇水溶液置于80℃水浴摇床中,转速100r/min,避光浸提6h,即得鼠尾藻藻类多酚醇提液;
⑾将鼠尾藻多酚醇提液放入40℃,40rpm/min的旋转蒸发仪中蒸去溶剂,在冻干机中进行冻干后成冻干粉,即得海带多酚成品,放入-20℃冰箱中保存。
按照本领域技术人员熟知的酚类物质以及体外抗氧活性的测定方法,最后得海藻多酚的总酚含量为19.15mg/g(以干的海带粉计,以没食子酸为当量),纯度为30.06%(总酚含量/总酚干物质的量),其DPPH、羟自由基均有较好的体外清除活性,IC50值分别为4.428mg/L、13.102mg/L,总抗氧化能力为68.14U/mg。
实施例3
本实施例提供的高效的藻类多酚制备方法,其制备方法如下
⑴蛋白核小球藻处理:将采集的新鲜蛋白核小球藻洗净、放入在60℃电热鼓风干燥箱中烘干,然后用粉碎机将其粉碎,并过60目筛网制得粉末,备用;
⑵复配菌悬液制备:分别将乳酸菌细胞悬液、毕赤酵母细胞悬液、德氏乳杆菌细胞悬液在30℃、180r/min培养24h,10000r/min、4℃离心10min,分别收集上清液,再分别加入无菌浓度为0.9%的NaCl溶液,分别配成1.0×107cfu/mL的菌悬液,按重量比1:2:2比例混合即得复配菌悬液;
⑶发酵:将复配菌悬液与蛋白核小球藻粉末按重量比2:1混合,用无菌水调节混合物含水量为75%,30℃恒温发酵7d;即得蛋白核小球藻发酵液;
⑷高静压处理:将蛋白核小球藻发酵液密封于双层聚乙烯塑料袋中,抽真空密封后,置于高压设备中,于室温下400Mpa高静压保压处理22min;
⑸酶解:将步骤⑷高静压处理后蛋白核小球藻发酵液加柠檬酸,调节溶液pH至5.0再加入酶浓度为1%(V/V)的ViscozymeL升温至50℃,酶解4h,即得蛋白核小球藻菌酶混合液;
⑹灭菌酶:将海带菌酶混合液加入浓度为0.0015mmol/LFeSO4·7H2O和浓度为0.005mmol/LMnCl2·2H2O后加热至沸腾,保持沸腾10min,冷却至室温,得到海带提取液Ⅰ;
⑺萃取溶液制备:将氯化胆碱、1,2丁二醇按摩尔比1:4混合,在80℃恒磁搅拌下,直至形成透明均匀的液体,然后在50℃真空烘箱中干燥24h,再与蒸馏水按照质量比为7:3的比例混合,充分拌匀即可得到萃取溶液;
⑻超声萃取:向蛋白核小球藻提取液Ⅰ液中加入萃取溶液和浓度为0.030mol/L月桂酰基谷氨酸钠,于50℃以功率494W超声萃取25min,即得蛋白核小球藻提取液Ⅱ;
⑼将蛋白核小球藻提取液Ⅱ在室温下以6000r/min,离心12min,取上清液,即得蛋白核小球藻多酚提取液;
⑽将藻类多酚提取液按体积比1:6加体积浓度为45%的乙醇水溶液置于65℃水浴摇床中,转速100r/min,避光浸提3.5h,即得蛋白核小球藻藻类多酚醇提液;
⑾将蛋白核小球藻多酚醇提液放入40℃,40rpm/min的旋转蒸发仪中蒸去溶剂,在冻干机中进行冻干后成冻干粉,即得海带多酚成品,放入-20℃冰箱中保存。
按照本领域技术人员熟知的酚类物质以及体外抗氧活性的测定方法,最后得海藻多酚的总酚含量为20.08mg/g(以干的海带粉计,以没食子酸为当量),纯度为32.15%(总酚含量/总酚干物质的量),其DPPH、羟自由基均有较好的体外清除活性,IC50值分别为4.579mg/L、13.689mg/L,总抗氧化能力为69.69U/mg。
本说明书中各个实施例采用递进的方式描述,每个实施例重点说明的都是与其他实施例的不同之处,各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (9)
1.一种海藻多酚提取方法,其特征在于,包括以下步骤:
⑴将藻类洗净、放入在60℃电热鼓风干燥箱中烘干,然后用粉碎机将其粉碎,并过60目筛网制得粉末,备用;
⑵将复配菌悬液与藻类粉末按重量比2:1混合,用无菌水调节混合物含水量为70~80%,30℃恒温发酵7d;即得藻类发酵液;
⑶将藻类发酵液密封于双层聚乙烯塑料袋中,抽真空密封后,置于高压设备中,于室温下400Mpa高静压保压处理20~25min;
⑷将步骤⑶高静压处理后藻类发酵液加柠檬酸,调节溶液pH至4.0~5.2再加入复合植物水解酶升温至45~55℃,酶解3~5h,即得藻类菌酶混合液;
⑸将藻类菌酶混合液加入金属无机盐后加热至沸腾,保持沸腾10~15min,冷却至室温,得到藻类提取液Ⅰ;
⑹向藻类提取液Ⅰ液中加入萃取溶液和表面活性剂,于45~55℃以功率494W超声萃取20~30min,即得藻类提取液Ⅱ;
⑺将藻类提取液Ⅱ在室温下以4000~8000r/min,离心10~15min,取上清液,即得藻类多酚粗提取液;
⑻将藻类多酚提取液按体积比1:6加体积浓度为40~50%的乙醇水溶液置于50℃~80℃水浴摇床中,转速100r/min,避光浸提1h~6h,即得藻类多酚醇提液;
⑼将藻类多酚醇提液放入40℃,40rpm/min的旋转蒸发仪中蒸去溶剂,在冻干机中进行冻干后成冻干粉,即得海带多酚成品,放入-20℃冰箱中保存。
2.根据权利要求1所述的一种海藻多酚提取方法,其特征在于,所述的复配菌悬液制备方法:分别将乳酸菌细胞悬液、毕赤酵母细胞悬液、德氏乳杆菌细胞悬液在30℃、180r/min培养24h,10000r/min、4℃离心10min,分别收集上清液,再分别加入无菌浓度为0.9%的NaCl溶液,分别配成1.0×107cfu/mL的菌悬液,按重量比1:2:2比例混合即得复配菌悬液。
3.根据权利要求1所述的一种海藻多酚提取方法,其特征在于:
所述萃取溶液由氯化胆碱、1,2丁二醇按摩尔比1:4混合,在80℃恒磁搅拌下,直至形成透明均匀的液体,然后在50℃真空烘箱中干燥24h,再与蒸馏水按照质量比为7:3的比例混合,充分拌匀即可得到萃取溶液。
4.根据权利要求1所述的一种海藻多酚提取方法,其特征在于:所述的金属无机盐选择FeSO4·7H2O、ZnCl2、CaCl2·2H2O、MnCl2·2H2O、CuSO4·5H2O中的任意一种或多种组合。
5.根据权利要求4所述的一种海藻多酚提取方法,其特征在于:金属无机盐选择多种组合,具体为FeSO4·7H2O和MnCl2·2H2O进行组合,其中FeSO4·7H2O浓度为0.001~0.002mmol/L,MnCl2·2H2O浓度为0.004~0.006mmol/L。
6.根据权利要求1所述的一种海藻多酚提取方法,其特征在于:所述的复合植物水解酶为ViscozymeL。
7.根据权利要求6所述的一种海藻多酚提取方法,其特征在于:
所述的复合植物水解酶浓度为1%(V/V)。
8.根据权利要求1所述的一种海藻多酚提取方法,其特征在于:
所述的表面活性剂为月桂酰基谷氨酸钠。
9.根据权利要求1所述的一种海藻多酚提取方法,其特征在于:
所述的月桂酰基谷氨酸钠浓度为0.024~0.036mol/L。
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