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Box－Behnken_响应面法优化超声提取皱皮木瓜总黄酮工艺

Ｂｏｘ－Ｂｅｈｎｋｅｎ响应面法优化超声提取皱皮木瓜总黄酮工艺刘岩，吕宗凯，刘连芬，钱关泽∗㊀（聊城大学生命科学学院，山东聊城２５２０５９）摘要㊀以蔷薇科中的皱皮木瓜为研究对象，以其叶片为试验材料，分别进行料液比㊁提取时间㊁提取温度㊁乙醇浓度单因素试验探究总黄酮提取最适范围，通过响应面法（ＲＳＭ）和Ｂｏｘ－Ｂｅｈｎｋｅｎ试验设计结合二次回流优化超声辅助提取总黄酮工艺㊂结果表明，不同因素对木瓜叶片总黄酮提取率的影响顺序为提取时间＞乙醇浓度＞料液比；最佳工艺条件为提取时间５２ｍｉｎ㊁乙醇浓度７４％㊁料液比１ʒ４１（ｇʒｍＬ）时，总黄酮为１５６．６５ｍｇ／ｇ，提取率达到１５．６７％㊂关键词㊀皱皮木瓜；总黄酮；响应面法；提取工艺优化中图分类号㊀Ｒ２８４．２㊀㊀文献标识码㊀Ａ㊀㊀文章编号㊀０５１７－６６１１（２０２３）１０－０１４４－０５ｄｏｉ：１０．３９６９／ｊ．ｉｓｓｎ．０５１７－６６１１．２０２３．１０．０３２㊀㊀㊀㊀㊀开放科学（资源服务）标识码（ＯＳＩＤ）：ＯｐｔｉｍｉｚａｔｉｏｎｏｆＵｌｔｒａｓｏｎｉｃＥｘｔｒａｃｔｉｏｎｏｆＴｏｔａｌＦｌａｖｏｎｏｉｄｓｆｒｏｍＣｈａｅｎｏｍｅｌｅｓｓｐｅｃｉｏｓａｂｙＢｏｘ⁃ＢｅｈｎｋｅｎＲｅｓｐｏｎｓｅＳｕｒｆａｃｅＭｅｔｈｏｄｏｌｏｇｙＬＩＵＹａｎ，ＬÜＺｏｎｇ⁃ｋａｉ，ＬＩＵＬｉａｎ⁃ｆｅｎｅｔａｌ㊀（ＳｃｈｏｏｌｏｆＬｉｆｅＳｃｉｅｎｃｅ，ＬｉａｏｃｈｅｎｇＵｎｉｖｅｒｓｉｔｙ，Ｌｉａｏｃｈｅｎｇ，Ｓｈａｎｄｏｎｇ２５２０５９）Ａｂｓｔｒａｃｔ㊀ＴａｋｉｎｇＣｈａｅｎｏｍｅｌｅｓｓｐｅｃｉｏｓａｉｎｔｈｅＲｏｓａｃｅａｅａｓｔｈｅｒｅｓｅａｒｃｈｏｂｊｅｃｔａｎｄｉｔｓｌｅａｖｅｓａｓｔｈｅｅｘｐｅｒｉｍｅｎｔａｌｍａｔｅｒｉａｌ，ｔｈｅｓｉｎｇｌｅｆａｃｔｏｒｅｘｐｅｒ⁃ｉｍｅｎｔｓｗｅｒｅｃｏｎｄｕｃｔｅｄｔｏｅｘｐｌｏｒｅｔｈｅｏｐｔｉｍａｌｒａｎｇｅｏｆｔｏｔａｌｆｌａｖｏｎｏｉｄｓｅｘｔｒａｃｔｉｏｎ，ｉｎｃｌｕｄｉｎｇｓｏｌｉｄ⁃ｌｉｑｕｉｄｒａｔｉｏ，ｅｘｔｒａｃｔｉｏｎｔｉｍｅ，ｅｘｔｒａｃｔｉｏｎｔｅｍｐｅｒａ⁃ｔｕｒｅａｎｄｅｔｈａｎｏｌｃｏｎｃｅｎｔｒａｔｉｏｎ．Ｒｅｓｐｏｎｓｅｓｕｒｆａｃｅｍｅｔｈｏｄｏｌｏｇｙ（ＲＳＭ）ａｎｄＢｏｘ⁃Ｂｅｈｎｋｅｎｅｘｐｅｒｉｍｅｎｔａｌｄｅｓｉｇｎｗｅｒｅｃｏｍｂｉｎｅｄｗｉｔｈｓｅｃｏｎｄａｒｙｒｅ⁃ｆｌｕｘｔｏｏｐｔｉｍｉｚｅｔｈｅｕｌｔｒａｓｏｎｉｃａｓｓｉｓｔｅｄｅｘｔｒａｃｔｉｏｎｐｒｏｃｅｓｓｏｆｔｏｔａｌｆｌａｖｏｎｏｉｄｓ．Ｔｈｅｒｅｓｕｌｔｓｓｈｏｗｅｄｔｈａｔｔｈｅｏｒｄｅｒｏｆｉｎｆｌｕｅｎｃｅｏｆｄｉｆｆｅｒｅｎｔｆａｃｔｏｒｓｏｎｔｈｅｅｘｔｒａｃｔｉｏｎｒａｔｅｏｆｔｏｔａｌｆｌａｖｏｎｏｉｄｓｆｒｏｍｐａｐａｙａｌｅａｖｅｓｗａｓｅｘｔｒａｃｔｉｏｎｔｉｍｅ＞ｅｔｈａｎｏｌｃｏｎｃｅｎｔｒａｔｉｏｎ＞ｓｏｌｉｄ⁃ｌｉｑｕｉｄｒａｔｉｏ；ｔｈｅｏｐｔｉｍａｌｐｒｏｃｅｓｓｃｏｎｄｉｔｉｏｎｓｗｅｒｅｅｘｔｒａｃｔｉｏｎｔｉｍｅ５２ｍｉｎｕｔｅｓ，ｅｔｈａｎｏｌｃｏｎｃｅｎｔｒａｔｉｏｎ７４％ａｎｄｓｏｌｉｄ⁃ｌｉｑｕｉｄｒａｔｉｏ１ʒ４１（ｇʒｍＬ），ｔｈｅｔｏｔａｌｆｌａｖｏｎｏｉｄｓｗｅｒｅ１５６．６５ｍｇ／ｇ，ａｎｄｔｈｅｅｘｔｒａｃｔｉｏｎｒａｔｅｒｅａｃｈｅｄ１５．６７％．Ｋｅｙｗｏｒｄｓ㊀Ｃｈａｅｎｏｍｅｌｅｓｓｐｅｃｉｏｓａ；Ｔｏｔａｌｆｌａｖｏｎｏｉｄｓ；Ｒｅｓｐｏｎｓｅｓｕｒｆａｃｅｍｅｔｈｏｄｏｌｏｇｙ（ＲＳＭ）；Ｏｐｔｉｍｉｚａｔｉｏｎｏｆｅｘｔｒａｃｔｉｏｎｐｒｏｃｅｓｓ基金项目㊀国家自然科学基金项目（３１０７０６１９，３１１７０１７８）；山东省自然科学基金项目（ＺＲ２０１１ＣＭ０４５）㊂作者简介㊀刘岩（１９９８），女，河北唐山人，硕士研究生，研究方向：植物学㊂∗通信作者，教授，博士，硕士生导师，从事种子植物分类及资源利用研究㊂收稿日期㊀２０２２－０７－０６；修回日期㊀２０２２－０７－１８㊀㊀皱皮木瓜（Ｃｈａｅｎｏｍｅｌｅｓｓｐｅｃｉｏｓａ（ｓｗｅｅｔ）Ｎａｋａｉ）又称楙㊁贴梗木瓜㊁贴梗海棠㊁铁脚梨等，是蔷薇科木瓜属植物，在安徽㊁浙江㊁陕西㊁甘肃㊁广东㊁云贵川及缅甸等均有分布，为常见的栽培及药用植物，花色有乳白色㊁粉红色㊁大红色且有重瓣及半重瓣品种，早春先花后叶［１］㊂皱皮木瓜含有大量的有机酸㊁三萜类㊁黄酮类化合物㊁熊果酸㊁多糖及超氧化物歧化酶（ＳＯＤ）等成分，被称为百益之果，是一种药食同源的植物［２］㊂中医认为木瓜味酸性温，入肝㊁脾经，有健脾开胃㊁去湿舒筋之功效，药理学上认为这些物质具有抗癌㊁抑制肿瘤㊁抗炎杀菌㊁抗氧化等功效［３］㊂目前对于皱皮木瓜的研究多集中在栽培技术［４］和药用价值［５］方面，对其活性物质的研究包括对皂苷㊁熊果酸㊁齐墩果酸及有机酸等物质的提纯技术及功能效果方面［６－７］；仅少量学者报道了皱皮木瓜中黄酮类物质的提纯方法及总黄酮含量，指出了由于皱皮木瓜分布区的差异，其含有的总黄酮含量也不尽相同［８－９］，对木瓜中黄酮类物质的提取工艺优化的研究还鲜见报道㊂黄酮类化合物的传统提取方法主要包括水提法㊁溶剂萃取法㊁树脂吸附法等，近年来国内外新开发的提取方法有超声辅助提取法［１０］㊁超临界流体萃取法［１１］㊁微波萃取法［１２］㊁酶提取法［１３］等，其中超声辅助提取法具有用时短㊁成本低㊁提取率高㊁无试剂残留污染环境等优点［１４］，它亦可结合其他提取方式共同使用，是一种广泛应用的有极大发展前景的物质提取方式㊂为发掘皱皮木瓜叶片的潜在利用价值，减少枯枝败叶对环境造成的污染和压力，该试验采用Ｂｏｘ－Ｂｅｈｎｋｅｎ响应面法研究料液比㊁乙醇浓度㊁提取温度㊁提取时间４个因素对总黄酮提取效果的影响，优化超声冷凝回流法提取皱皮木瓜叶片中总黄酮的工艺参数，以期为皱皮木瓜叶片的深度开发利用及其中活性物质的研究提供理论和技术支持㊂１㊀材料与方法１．１㊀试验材料１．１．１㊀试材㊂皱皮木瓜春季４月新鲜幼嫩叶片，采自山东省聊城大学植物园，挑选大小㊁幼嫩程度相似㊁无病虫害㊁完整新鲜的叶片㊂１．１．２㊀试剂㊂亚硝酸钠㊁氢氧化钠㊁九水硝酸铝㊁芦丁标准品（纯度ȡ９８％），购自天津市大茂化学试剂厂；无水乙醇购自国药集团化学试剂有限公司；所有试剂均为分析纯（ＡＲ）㊂１．１．３㊀仪器与设备㊂ＤＧＸ－９０５３Ｂ－１型电热鼓风干燥箱，上海优浦科学仪器有限公司；仙桃ｘｔ－２００型高速多功能粉碎机，浙江省永康市红太阳机电有限公司；ＦＡ１００４电子分析天平，上海越平科学仪器有限公司；ＳＢ－４２００ＤＴＤ数控加热超声波清洗机，宁波新芝生物科技股份有限公司；ＳＨＫ－Ⅲ循环水式多用真空泵，郑州科泰实验设备有限公司；ＵＶ紫外可见分光光度计，上海佑科仪器仪表有限公司㊂１．２㊀试验方法１．２．１㊀试验材料的处理㊂将采摘的新鲜皱皮木瓜叶片洗净晾干，放于７０ħ烘干箱中恒温烘干至恒重，后用粉碎机粉碎㊁过筛得粉末，储存于干燥袋中于４ħ冰箱密封保存备用㊂１．２．２㊀芦丁标准曲线建立㊂根据卞京军等［１５］的方法稍作改良建立标准曲线㊂以芦丁作为标准品，精确称取１ｇ样品，６０％乙醇５００ｍＬ溶解，并用６０％乙醇定容至１０００ｍＬ，得浓㊀㊀㊀安徽农业科学，Ｊ．ＡｎｈｕｉＡｇｒｉｃ．Ｓｃｉ．２０２３，５１（１０）：１４４－１４８度为１ｍｇ／ｍＬ的芦丁标准溶液㊂取６支试管，分别加入０㊁０．２㊁０．４㊁０．６㊁０．８㊁１．０ｍＬ标准溶液，之后加入６０％乙醇至体积为１ｍＬ，先加入１０％亚硝酸钠溶液０．５ｍＬ，振荡摇匀并静置５ｍｉｎ，之后加入１０％硝酸铝溶液０．５ｍＬ，振荡并摇匀，静置５ｍｉｎ，最后加入４％氢氧化钠溶液４ｍＬ，振荡并摇匀，静置１０ｍｉｎ，以第一管为空白对照，测定５１０ｎｍ处的吸光度，以吸光度为纵坐标（ｙ）㊁芦丁质量浓度为横坐标（ｘ）建立芦丁标准曲线㊂１．２．３㊀总黄酮提取及含量测定㊂准确称量木瓜粉末１ｇ于圆底烧瓶中，分别在不同料液比㊁乙醇浓度㊁提取时间㊁提取温度条件下，遵循单一变量原则进行超声冷凝回流提取㊂考虑到高温溶剂易挥发导致提取不充分等问题，该研究根据宋璇等［１６］的方法稍作改良，在第一次提取结束后，另加入同等体积乙醇进行二次回流提取，合并提取液，定容于１００ｍＬ容量瓶，后转移至１２５ｍＬ棕色广口瓶保存㊂采用硝酸铝比色法对总黄酮含量进行测定㊂取２００μＬ样品至试管中，对照中加入同体积蒸馏水，各加入８００μＬ对应体积的乙醇，加１０％ＮａＮＯ２溶液１ｍＬ，振荡摇匀后反应５ｍｉｎ；加１０％Ａｌ（ＮＯ３）３溶液１ｍＬ，振荡摇匀反应５ｍｉｎ；加入４％ＮａＯＨ溶液５ｍＬ，振荡摇匀反应１０ｍｉｎ，测５１０ｎｍ处的ＯＤ值㊂参照芦丁标准品计算皱皮木瓜中总黄酮含量，求得总黄酮的提取率，计算公式如下：Ｅ＝ＣˑＶˑｎｍˑ１００％（１）式中，Ｅ为总黄酮提取率（％）；Ｃ为含有的总黄酮质量浓度（ｇ／ｍＬ）；Ｖ为加入的提取液体积（ｍＬ）；ｎ为稀释倍数；ｍ为木瓜粉末的质量（ｇ）㊂１．２．４㊀单因素试验㊂１．２．４．１㊀提取温度对总黄酮提取率的影响㊂精确称取皱皮木瓜叶片粉末１ｇ，以料液比１ʒ５０（ｇʒｍＬ）㊁乙醇浓度６０％㊁提取时间４０ｍｉｎ条件下，提取温度分别为室温（３０）㊁４０㊁５０㊁６０㊁７０ħ进行回流提取，计算总黄酮提取率，重复３次㊂１．２．４．２㊀提取时间对总黄酮提取率的影响㊂精确称取皱皮木瓜叶片粉末１ｇ，以料液比１ʒ５０㊁乙醇浓度６０％㊁提取温度５０ħ条件下，提取时间分别为２０㊁３０㊁４０㊁５０㊁６０ｍｉｎ进行回流提取，计算总黄酮提取率，重复３次㊂１．２．４．３㊀料液比对总黄酮提取率的影响㊂精确称取皱皮木瓜叶片粉末１ｇ，以乙醇浓度６０％㊁提取时间４０ｍｉｎ㊁提取温度５０ħ条件下，料液比分别为１ʒ３０㊁１ʒ４０㊁１ʒ５０㊁１ʒ６０㊁１ʒ７０进行回流提取，计算总黄酮提取率，重复３次㊂１．２．４．４㊀乙醇浓度对总黄酮提取率的影响㊂精确称取皱皮木瓜叶片粉末１ｇ，以料液比１ʒ５０㊁提取时间４０ｍｉｎ㊁提取温度５０ħ条件下，乙醇浓度分别为５０％㊁６０％㊁７０％㊁８０％㊁９０％进行回流提取，计算总黄酮提取率，重复３次㊂１．２．５㊀响应面优化试验设计㊂根据单因素试验的结果，且由于试验材料采集时间原因，选取３因素３水平的响应面分析法对超声辅助提取工艺进行优化，以料液比㊁乙醇浓度㊁提取时间３个因素为自变量，以总黄酮提取率为响应值，将获得数据导入软件Ｄｅｓｉｇｎ－Ｅｘｐｅｒｔ，以其中的Ｂｏｘ－Ｂｅｈｎｋｅｎ设计原理得到１７组试验设计，分析多因素交互作用，优化提取条件，建立回归模型，最后确定最佳提取参数及验证试验分析㊂２㊀结果与分析２．１㊀建立芦丁标准曲线㊀以吸光度为纵坐标（ｙ）㊁芦丁质量浓度（ｍｇ／ｍＬ）为横坐标（ｘ）建立芦丁标准曲线（图１），得出芦丁标准曲线的回归方程为ｙ＝１．４４５ｘ－０．０１７６（Ｒ２＝０．９９９９），表明芦丁在０１．５ｍｇ／ｍＬ表现出良好的线性关系㊂图１㊀芦丁标准曲线Ｆｉｇ．１㊀Ｒｕｔｉｎｓｔａｎｄａｒｄｃｕｒｖｅ２．２㊀单因素试验２．２．１㊀乙醇浓度对总黄酮提取率的影响㊂由图２可知，在提取温度㊁提取时间㊁料液比一定的条件下，乙醇浓度在５０％７０％总黄酮提取率呈上升趋势，在乙醇浓度７０％时提取率达到最大值（１５．１６％）；乙醇浓度大于７０％，总黄酮提取率呈显著下降趋势，到９０％时总黄酮提取率为１３．７３％，同比最大值下降了９．４３％，说明皱皮木瓜总黄酮在７０％乙醇中溶解度最大，由此选取７０％为皱皮木瓜叶片总黄酮提取的乙醇浓度㊂图２㊀乙醇浓度对总黄酮提取率的影响Ｆｉｇ．２㊀Ｅｆｆｅｃｔｏｆｅｔｈａｎｏｌｃｏｎｃｅｎｔｒａｔｉｏｎｏｎｔｈｅｅｘｔｒａｃｔｉｏｎｒａｔｅｏｆｔｏｔａｌｆｌａｖｏｎｏｉｄｓ２．２．２㊀料液比对总黄酮提取率的影响㊂由图３可知，在提取温度㊁提取时间㊁乙醇浓度一定的条件下，总黄酮提取率随着料液比减少呈先上升后下降的趋势，料液比１ʒ３０时总黄酮提取率为１３．０２％，１ʒ４０时总黄酮提取率为１４．２９％，提取率增长了９．７５％，达到最大值；之后总黄酮提取率呈下降趋势㊂因此选择１ʒ４０为皱皮木瓜叶片总黄酮提取的料液比㊂２．２．３㊀提取时间对总黄酮提取率的影响㊂由图４可知，在提取温度㊁乙醇浓度㊁料液比一定的条件下，提取时间在２０５４１５１卷１０期㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀刘岩等㊀Ｂｏｘ－Ｂｅｈｎｋｅｎ响应面法优化超声提取皱皮木瓜总黄酮工艺图３㊀料液比对总黄酮提取率的影响Ｆｉｇ．３㊀Ｅｆｆｅｃｔｏｆｓｏｌｉｄ⁃ｌｉｑｕｉｄｒａｔｉｏｏｎｔｈｅｅｘｔｒａｃｔｉｏｎｒａｔｅｏｆｔｏｔａｌｆｌａｖｏｎｏｉｄｓ５０ｍｉｎ，总黄酮提取率先平缓后急剧增加，提取时间５０ｍｉｎ时总黄酮提取率达到最高值，为１５．５５％；随着提取时间延长，总黄酮提取率急剧下降，６０ｍｉｎ时，总黄酮提取率为１４．０９％㊂结果表明随着提取时间的增加，总黄酮提取率提高，当时间超过一定限值后总黄酮提取率下降，在工业生产中延长提取时间会增加生产成本和消耗，为节约时间和经济成本，选择５０ｍｉｎ为皱皮木瓜叶片总黄酮提取时间㊂图４㊀提取时间对总黄酮提取率的影响Ｆｉｇ．４㊀Ｅｆｆｅｃｔｏｆｅｘｔｒａｃｔｉｏｎｔｉｍｅｏｎｔｈｅｅｘｔｒａｃｔｉｏｎｒａｔｅｏｆｔｏｔａｌｆｌａｖｏｎｏｉｄｓ２．２．４㊀提取温度对总黄酮提取率的影响㊂由图５可知，在乙醇浓度㊁提取时间㊁料液比一定的条件下，提取温度低于４０ħ，随着提取温度的增加总黄酮提取率逐渐增加；提取温度４０ħ时总黄酮提取率达到峰值，为１６．０１％；高于４０ħ后总黄酮提取率呈急速下降趋势㊂结果表明随着提取温度的增加，总黄酮提取率提高，而当温度超过一定限值后总黄酮提取率下降，可能是由于温度过高导致部分黄酮类化合物结构遭到破坏或是达到溶剂沸点后溶剂挥发损失，最终导致总黄酮提取率降低，因此选取４０ħ为皱皮木瓜叶片总黄酮提取温度㊂２．３㊀响应面试验㊀根据单因素试验结果，对影响皱皮木瓜叶片总黄酮提取率的不同因素（料液比㊁提取时间㊁乙醇浓度）进行Ｂｏｘ－Ｂｅｈｎｋｅｎ试验设计，表１为不同因素及水平组合条件下皱皮木瓜叶片总黄酮提取率，结果表明，提取时间５０ｍｉｎ㊁乙醇浓度７０％㊁料液比１ʒ４０时，皱皮木瓜叶片总黄酮提取率最高，为１５．７７％㊂㊀㊀以ＤｅｓｉｇｎＥｘｐｅｒｔ８．０５软件对表１数据进行统计分析，建图５㊀提取温度对总黄酮提取率的影响Ｆｉｇ．５㊀Ｅｆｆｅｃｔｏｆｅｘｔｒａｃｔｉｏｎｔｅｍｐｅｒａｔｕｒｅｏｎｔｈｅｅｘｔｒａｃｔｉｏｎｒａｔｅｏｆｔｏｔａｌｆｌａｖｏｎｏｉｄｓ立料液比（Ａ）㊁提取时间（Ｂ）㊁乙醇浓度（Ｃ）３个因素与皱皮木瓜叶片总黄酮提取率（Ｙ）的二次回归方程：Ｙ＝１５．７４＋０．０３６Ａ＋０．０５９Ｂ＋０．０４５Ｃ－０．０１０ＡＢ＋０．０１８ＡＣ－０．０１２ＢＣ－０．２２０Ａ２－０．１３０Ｂ２－０．０６０Ｃ２（Ｒ２＝０．９９９４）㊂方差分析（表２）显示，模型显著而失拟项不显著，说明试验误差小，具有统计学意义；决定系数（Ｒ２）大于０．９，说明模型具有较高的拟合度，可用于皱皮木瓜叶片总黄酮提取的条件优化㊂表１㊀响应面试验设计与结果Ｔａｂｌｅ１㊀Ｒｅｓｐｏｎｓｅｓｕｒｆａｃｅｔｅｓｔｄｅｓｉｇｎａｎｄｒｅｓｕｌｔｓ试验序号ＴｅｓｔＮｏ．Ａ（料液比Ｓｏｌｉｄ⁃ｌｉｑｕｉｄｒａｔｉｏ）Ｂ（提取时间Ｅｘｔｒａｃｔｉｏｎｔｉｍｅʊｍｉｎ）Ｃ（乙醇浓度Ｅｔｈａｎｏｌｃｏｎｃｅｎｔｒａｔｉｏｎ％）总黄酮提取率Ｅｘｔｒａｃｔｉｏｎｒａｔｅｏｆｔｏｔａｌｆｌａｖｏｎｏｉｄｓʊ％１１ʒ４０６０６０１５．５４２１ʒ３０５０８０１５．４６３１ʒ３０４０７０１５．２４４１ʒ４０５０７０１５．６９５１ʒ５０４０７０１５．３３６１ʒ４０４０８０１５．５７７１ʒ４０４０６０１５．４８８１ʒ４０５０７０１５．７４９１ʒ４０５０７０１５．７７１０１ʒ４０６０８０１５．５８１１１ʒ４０５０７０１５．７６１２１ʒ５０６０７０１５．５１１３１ʒ５０５０８０１５．５７１４１ʒ３０６０７０１５．４６１５１ʒ３０５０６０１５．３８１６１ʒ４０５０７０１５．７２１７１ʒ５０５０６０１５．４２㊀㊀回归模型显著性检验结果（表２）表明，模型中料液比（Ａ）不显著（Ｐ＞０．０５），提取时间（Ｂ）㊁乙醇浓度（Ｃ）均显著（Ｐ＜０．０５），表明料液比对皱皮木瓜叶片总黄酮提取率的影响不显著，提取时间㊁乙醇浓度对提取率的影响显著；Ａ２㊁Ｂ２表现为极显著（Ｐ＜０．０１），Ｃ２表现为显著（Ｐ＜０．０５），说明乙醇浓度㊁料液比㊁提取时间对皱皮木瓜叶片总黄酮提取率的影响是较为复杂的二次关系，影响顺序为提取时间＞乙醇浓度＞料液比㊂６４１㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀安徽农业科学㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀２０２３年表２㊀响应面回归模型方差分析Ｔａｂｌｅ２㊀Ａｎａｌｙｓｉｓｏｆｖａｒｉａｎｃｅｏｆｒｅｓｐｏｎｓｅｓｕｒｆａｃｅｒｅｇｒｅｓｓｉｏｎｍｏｄｅｌ方差来源Ｓｏｕｒｃｅｏｆｖａｒｉａｔｉｏｎ平方和ＳＳ自由度ｄｆ均方ＭＳＦ值ＦｖａｌｕｅＰ值Ｐｖａｌｕｅ模型Ｍｏｄｅｌ０．３７９０．０４１１５．２３０．０００８Ａ０．０１１１０．０１１３．８７０．０８９７Ｂ０．０２８１０．０２８１０．１８０．０１５３Ｃ０．０１６１０．０１６５．９７０．０４４５ＡＢ４．０００Ｅ－００４１４．０００Ｅ－００４０．１５０．７１２４ＡＣ１．２２５Ｅ－００３１１．２２５Ｅ－００３０．４５０．５２３２ＢＣ６．２５０Ｅ－００４１６．２５０Ｅ－００４０．２３０．６４５９Ａ２０．２００１０．２００７３．７４＜０．０００１Ｂ２０．０７４１０．０７４２７．４５０．００１２Ｃ２０．０１５１０．０１５５．６８０．０４８７残差Ｒｅｓｉｄｕａｌ０．０１９７２．７１４Ｅ－００３失拟项Ｍｉｓｆｉｔｔｉｎｇｔｅｒｍ０．０１５３４．９５８Ｅ－００３４．８１０．０８１５纯误差Ｐｕｒｅｅｒｒｏｒ４．１２０Ｅ－００３４１．０３０Ｅ－００３总误差Ｔｏｔａｌｅｒｒｏｒ０．３９０１６２．４㊀响应面多因素交互作用分析㊀由图６８可知，在料液比㊁提取时间㊁乙醇浓度两两因素一定的条件下，总黄酮提取率都随着第３个因素的增大而先上升后下降㊂料液比和提取时间的等高线形状偏圆形，说明两者交互作用较缓和；乙醇浓度与料液比的等高线呈椭圆形，表明两者交互作用显著；乙醇浓度和提取时间的等高线呈椭圆形，表明两者交互作用显著㊂从响应面的３Ｄ图可知，料液比和提取时间㊁料液比和乙醇浓度㊁乙醇浓度和提取时间的曲线均较陡，说明两图６㊀料液比与提取时间对木瓜叶片总黄酮提取率交互影响的等高线和响应面Ｆｉｇ．６㊀Ｃｏｎｔｏｕｒａｎｄｒｅｓｐｏｎｓｅｓｕｒｆａｃｅｏｆｔｈｅｉｎｔｅｒａｃｔｉｏｎｂｅｔｗｅｅｎｓｏｌｉｄ⁃ｌｉｑｕｉｄｒａｔｉｏａｎｄｅｘｔｒａｃｔｉｏｎｔｉｍｅｏｎｔｈｅｅｘｔｒａｃｔｉｏｎｒａｔｅｏｆｔｏｔａｌｆｌａｖｏｎｏｉｄｓｆｒｏｍｐａｐａｙａｌｅａｖｅｓ图７㊀料液比与乙醇浓度对木瓜叶片总黄酮提取率交互影响的等高线和响应面Ｆｉｇ．７㊀Ｃｏｎｔｏｕｒａｎｄｒｅｓｐｏｎｓｅｓｕｒｆａｃｅｏｆｔｈｅｉｎｔｅｒａｃｔｉｏｎｂｅｔｗｅｅｎｓｏｌｉｄ⁃ｌｉｑｕｉｄｒａｔｉｏａｎｄｅｔｈａｎｏｌｃｏｎｃｅｎｔｒａｔｉｏｎｏｎｔｈｅｅｘｔｒａｃｔｉｏｎｒａｔｅｏｆｔｏｔａｌｆｌａ⁃ｖｏｎｏｉｄｓｆｒｏｍｐａｐａｙａｌｅａｖｅｓ７４１５１卷１０期㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀刘岩等㊀Ｂｏｘ－Ｂｅｈｎｋｅｎ响应面法优化超声提取皱皮木瓜总黄酮工艺图８㊀乙醇浓度与提取时间对木瓜叶片总黄酮提取率交互影响的等高线和响应面Ｆｉｇ．８㊀Ｃｏｎｔｏｕｒａｎｄｒｅｓｐｏｎｓｅｓｕｒｆａｃｅｏｆｔｈｅｉｎｔｅｒａｃｔｉｏｎｂｅｔｗｅｅｎｅｔｈａｎｏｌｃｏｎｃｅｎｔｒａｔｉｏｎａｎｄｅｘｔｒａｃｔｉｏｎｔｉｍｅｏｎｔｈｅｅｘｔｒａｃｔｉｏｎｒａｔｅｏｆｔｏｔａｌｆｌａ⁃ｖｏｎｏｉｄｓｆｒｏｍｐａｐａｙａｌｅａｖｅｓ者的交互作用对总黄酮提取率的影响较大㊂２．５㊀总黄酮最佳提取参数的确定及验证性试验㊀经过分析回归方程，选择Ｍａｘｉｍｉｚｅ模式对总黄酮提取工艺进行参数优化，得到最优提取总黄酮的条件为提取时间５２ｍｉｎ㊁乙醇浓度７３．６４％㊁料液比１ʒ４０．９４，此时，皱皮木瓜叶片总黄酮提取率的理论值为１５．７５％㊂为验证回归方程，以提取时间５２ｍｉｎ㊁乙醇浓度７４％㊁料液比１ʒ４１进行皱皮木瓜叶片总黄酮提取率验证试验，进行３组平行试验，回流提取２次，结果发现总黄酮提取率实际均值为１５．６７％，ＲＳＤ小于５％，与理论值基本一致，说明Ｂｏｘ－Ｂｅｈｎｋｅｎ响应面优化设计得到的各因素水平条件组合比较可靠，可以用于实际操作以及优化提取参数㊂３㊀结论与讨论前人研究报道，一般皱皮木瓜果实总黄酮含量在６４０ｍｇ／ｇ［１７］，皱皮木瓜皮渣总黄酮得率为０．２％０．５％［１５］㊂唐浩国［１８］研究表明，超声波具有空化作用，该作用可加速植物有效成分溶解出来，进而提高活性成分的提取率㊂该研究利用超声冷凝回流提取法提取皱皮木瓜叶片中总黄酮，结合二次回流提取并利用Ｂｏｘ－Ｂｅｈｎｋｅｎ响应面试验设计对提取条件进行优化，结果表明在提取时间５２ｍｉｎ㊁乙醇浓度７４％㊁料液比１ʒ４１条件下总黄酮得率最高，总黄酮提取率达到１５．６７％，总黄酮含量为１５６．６５ｍｇ／ｇ，比文献报道的总黄酮得率显著提高［１９－２２］㊂究其原因，或是由于采样时间处于春季，叶片较嫩，总黄酮含量更高，且一次提取后又加入溶剂进行了二次回流，补充了溶剂，保证总黄酮的大部分能够提取出来㊂由此可见，Ｂｏｘ－Ｂｅｈｎｋｅｎ响应面法优化及二次回流的应用使得总黄酮提取率更高㊂该研究结果最大程度提高了皱皮木瓜叶片总黄酮得率，证明了以响应面法优化提取皱皮木瓜叶片总黄酮工艺的可行性，充分挖掘出木瓜的潜在利用价值，在控制成本㊁提高效率㊁保护环境㊁减少污染方面有着巨大的优势，从而实现了皱皮木瓜原料更为高效利用，为扩大皱皮木瓜工业生产及产品的开发利用提供了可靠依据㊂参考文献［１］中国科学院中国植物志编辑委员会．中国植物志：第３６卷［Ｍ］．北京：科学出版社，１９７４：４００－４０２．［２］陈红，王关祥，郑林，等．木瓜属（贴梗海棠）品种分类的研究历史与现状［Ｊ］．山东林业科技，２００６，３６（５）：７０－７１，７８．［３］国家中医药管理局‘中华本草“编辑委员会．中华本草：第４卷［Ｍ］．上海：上海科学技术出版社，１９９９：１１１．［４］郭建全，刘春华，黄金铭，等．皱皮木瓜栽培技术要点［Ｊ］．江西农业，２０１９（１２）：１５．［５］程翔．皱皮木瓜均一多糖的分离纯化㊁结构鉴定及抗肿瘤活性研究［Ｄ］．上海：上海中医药大学，２０１９［６］刘世尧．不同产区皱皮木瓜有机酸组成及主要活性成分分离纯化研究［Ｄ］．重庆：西南大学，２０１２．［７］王志芳．皱皮木瓜中齐墩果酸和熊果酸测定㊁提取及抗肿瘤活性研究［Ｄ］．武汉：华中农业大学，２００６．［８］王文平，蒋朝晖．木瓜中总黄酮的提取分离及含量测定［Ｊ］．食品工业科技，２００４，２５（３）：８１－８２．［９］李娜，姜洪芳，金敬宏，等．不同采收期的宣木瓜总黄酮含量分析［Ｊ］．食品研究与开发，２０１１，３２（２）：１１２－１１４．［１０］周胜男，褚翠翠，陆宁．食用仙人掌中黄酮类物质的提取研究［Ｊ］．食品工业科技，２００８，２９（２）：２２８－２３０．［１１］ＤＵＧＯＰ，ＭＯＮＤＥＬＬＯＬ，ＤＵＧＯＧ，ｅｔａｌ．Ｒａｐｉｄａｎａｌｙｓｉｓｏｆｐｏｌｙｍｅｔｈｏｘｙ⁃ｌａｔｅｄｆｌａｖｏｎｅｓｆｒｏｍｃｉｔｒｕｓｏｉｌｓｂｙｓｕｐｅｒｃｒｉｔｉｃａｌｆｌｕｉｄｃｈｒｏｍａｔｏｇｒａｐｈｙ［Ｊ］．Ｊｏｕｒｎａｌｏｆａｇｒｉｃｕｌｔｕｒａｌａｎｄｆｏｏｄｃｈｅｍｉｓｒｙ，１９９６，４４（１２）：３９００－３９０５．［１２］孙萍，李艳，成玉怀．甘草总黄酮的微波提取及含量测定［Ｊ］．时珍国医国药，２００３，１４（５）：２６６－２６７．［１３］ＷＵＭＬ，ＺＨＯＵＣＳ，ＣＨＥＮＬＳ，ｅｔａｌ．Ｓｔｕｄｙｏｎｔｈｅｅｘｔｒａｃｔｉｏｎｏｆｔｏｔａｌｆｌａ⁃ｖｏｎｏｉｄｓｆｒｏｍＧｉｎｇｏｌｅａｖｅｓｂｙｅｎｚｙｍｅｈｙｄｒｏｌｙｓｉｓ［Ｊ］．Ｎａｔｕｒａｌｐｒｏｄｕｃｔｒｅ⁃ｓｅａｒｃｈａｎｄｄｅｖｅｌｏｐｍｅｎｔ，２００４，１６（６）：５５７－５６０．［１４］代彩玲，王萍，王静，等．籽瓜瓜皮果胶提取方法的优化与评价［Ｊ］．中国瓜菜，２０１８，３１（１０）：１３－１８．［１５］卞京军，程密密，刘世尧，等．皱皮木瓜皮渣齐墩果酸㊁熊果酸和总黄酮连续提取工艺研究［Ｊ］．西南大学学报（自然科学版），２０１５，３７（３）：１５８－１６５．［１６］宋璇，王汝华，于建丽，等．山楂叶黄酮分离纯化及抗氧化活性［Ｊ］．食品研究与开发，２０２２，４３（４）：５７－６３．［１７］郑璇，申国明，高林，等．不同产区皱皮木瓜总黄酮含量与土壤主要化学指标的关系［Ｊ］．江苏农业科学，２０１８，４６（１７）：２０２－２０５．［１８］唐浩国．黄酮类化合物研究［Ｍ］．北京：科学出版社，２００９：６４－６５．［１９］王有为，何敬胜，范建伟，等．木瓜道地起源与道地产区形成研究［Ｃ］／／中国中西医结合学会中药专业委员会．２００９年全国中药学术研讨会论文集．北京：中国中西医结合学会，２００９：１６３－１６８．［２０］郭锡勇，唐修静，郭莉莉．木瓜不同炮制品中总黄酮含量测定［Ｊ］．贵阳中医学院学报，２０００，２２（４）：６１－６２．［２１］陈翠，熊德琴，李春晖．木瓜中总黄酮提取最佳工艺的研究［Ｊ］．广东石油化工学院学报，２０１２，２２（１）：１５－１７，２５．［２２］严睿文，丁毅．宣木瓜中黄酮的提取分离及含量的测定［Ｊ］．生物学杂志，２００８，２５（３）：６２－６４．８４１㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀安徽农业科学㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀２０２３年。

基于层次分析-熵权法优化刺五加多组分超声提取工艺

林亚美，支红欣，孙霁骧，等. 基于层次分析-熵权法优化刺五加多组分超声提取工艺[J]. 食品工业科技，2023，44（20）：239−249.doi: 10.13386/j.issn1002-0306.2022120137LIN Yamei, ZHI Hongxin, SUN Jixiang, et al. Optimization of the Ultrasonic Extraction Process of Acanthopanax senticosus Multiple Components Based on a Coupling Methodology of Analytic Hierarchy Process and Entropy Weight Method[J]. Science and Technology of Food Industry, 2023, 44(20): 239−249. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022120137· 工艺技术 ·基于层次分析-熵权法优化刺五加多组分超声提取工艺林亚美1,2，支红欣1,2，孙霁骧1,2，陈博宇1,2，刘胜凯1,2，刘志国1,2,*（1.东北林业大学化学化工与资源利用学院，黑龙江哈尔滨 150040；2.东北林业大学森林植物生态学教育部重点实验室，黑龙江哈尔滨 150040）摘　要：基于超高效液相色谱-质谱（ultra-high performance liquid chromatography-mass spectrometry ，UPLC-MS/MS ）定量方法以及多指标综合评价方法，对刺五加多组分超声提取工艺进行优化。

开发UPLC-MS/MS 定量方法，同时测定刺五加多组分（绿原酸、紫丁香苷、刺五加苷E 、异嗪皮啶、咖啡酸、芝麻素）含量；再由层次分析法（analytic hierarchy process ，AHP ）及熵权法（entropy weight method ，EWM ）组建多指标综合评价方法—层次分析-熵权法（AHP-EWM ）；最终由单因素结合基于Box-Behnken 设计（Box-Behnken design ，BBD ）的响应面法（response surface methodology ，RSM ）优化刺五加多组分超声提取工艺。

山银花绿原酸提取工艺的优化及不同时期绿原酸的含量

贵州农业科学 2019,47(5):128〜131 Guizhou Agricultural Sciences
［文章编号］1001-3601(2019)05-0173-0128-04
山银花绿原酸提取工艺的优化及不同时期绿原酸的含量
赵佳利，张晓娜，黄娟，邓娇**
(贵州师范大学养麦产业技术研究中心，贵州贵阳550001)
(Research Center of Buckwheat Industry Technology , Guizhou Normal University , Guiyang, Guizhou 550001, China)
Abstract: To provide a theoretical basis for the comprehensive development and utilization of Lonicerae resources 9 orthogonal experiment and ultraviolet spectrophotometry were used to optimize the ultrasonic extraction process of Lonicerae and to investigate the content of bioactive component chlorogenic acid in different growth stages. Results: The optimum extraction process of ultrasonic extraction of Lonicerae was solid-liquid ratio 1 ： 40, extraction time 20 min, ethanol concentration 75%. Under these conditions? the chlorogenic acid extraction rate of young leaves of Lonicerae was 1. 042%. The content of chlorogenic acid was the highest in the cyan period, which was 10. 415 mg/g FW. The second was the buds and the mature leaves. The chlorogenic acid content was 9. 074 mg/g FW and 8. 520 mg/g FW, respectively. The contents of chlorogenic acid in Lonicerae in green-white stage, silver florescence stage and young leaves were similar 9 respectively 2. 033 mg/g FW, 2. 252 mg/g FW and 2. 432 mg/g FW.

211251888_响应面优化藏羊皮胶原蛋白肽超声辅助提取工艺及其体内抗氧化活性分析

牛金鸽，吴海玥，马世科，等. 响应面优化藏羊皮胶原蛋白肽超声辅助提取工艺及其体内抗氧化活性分析[J]. 食品工业科技，2023，44（11）：163−170. doi: 10.13386/j.issn1002-0306.2022060009NIU Jinge, WU Haiyue, MA Shike, et al. Optimization of Ultrasonic Assisted Extraction of Tibetan Sheep Skin Collagen Peptide by Response Surface Methodology and Its Antioxidant Activity in Vivo [J]. Science and Technology of Food Industry, 2023, 44(11):163−170. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022060009· 工艺技术 ·响应面优化藏羊皮胶原蛋白肽超声辅助提取工艺及其体内抗氧化活性分析牛金鸽1，吴海玥2，马世科2，闫忠心2, *，王学江3，李　婧3，胡　蓉2，祁全青3（1.青海大学农牧学院，青海西宁 810016；2.青海大学畜牧兽医科学院，青海西宁 810016；3.青海省乡村产业发展指导中心，青海西宁 810000）摘　要：为明确超声辅助提取对藏羊皮胶原蛋白肽含量及功能活性的影响。

试验采用响应面法研究了料液比、超声功率和超声时间3个因素，确定了胶原蛋白肽最佳超声辅助提取工艺；并对藏羊皮胶原蛋白肽进行了抗氧化能力分析，模拟消化过程中的还原力和·OH 清除能力研究。

结果表明：超声辅助处理能明显提高胶原蛋白肽含量，最佳超声提取工艺为料液比1:18、超声功率220 W ，超声时间27 min ，藏羊皮胶原蛋白肽含量为30.21%±1.67%。

超声波辅助提取黄芪中总黄酮最佳工艺

（1）芦丁标准曲线的绘制。精确称取 20 mg 芦丁标准品，120 ℃干燥至恒重，选取 70% 浓度的乙醇进行溶解，并用 100 mL 容量瓶定容，摇匀即制成质量浓度为 0.20 mg·mL-1 的芦丁标准溶液。准备 6 个 25 mL 的容量瓶，各加入 0.00、2.00、4.00、6.00、8.00 mL 和 10.00 mL 芦丁标准液，再分别加入 1 mL 质量分数为 5% 的 NaNO2 溶液，摇匀静置 5 min；继续各加入 1 mL 质量分数为 10% 的 Al（NO3）3 溶液，摇匀静置 5 min；再分别加入 4 mL 质量分数为 4%NaOH 溶液，用 70% 浓度的乙醇溶液定容至 25 mL，摇匀放置 10 min；将 70% 乙醇溶液作为空白对照，测定其在 510 nm 波长的吸光度。得到线性回归方程为 y=0.507 7x+0.041 1，R2=0.995 9。其中 y 为吸光度值，x 为黄酮的质量浓度（μg·mL-1）。
◎ 李泽民，秦晓芳，李霞，田舒媛，张妍，王鑫（天津天狮学院食品工程学院，天津 301700）
Li Zemin, Qin Xiaofang, Li Xia, Tian Shuyuan, Zhang Yan, Wang Xin (School of Food Engineering, Tianshi College, Tianjin 301700, China)
关键词：黄芪；黄酮；超声波法；正交法 Abstract：Ultrasonic assisted extraction of total flavonoids in Astragalus membranaceus was optimized by orthogonal experimental design. Methods： Based on the single factor experiment, the factors selected were ethanol concentration, extraction temperature, material-liquid ratio, extraction time, etc. The investigation indicators were based on the total flavonoid extraction rate. The orthogonal extraction method was used to optimize the optimal extraction conditions. Results： The best process conditions were： etanol volume fraction 60%, extraction temperature 60 ℃ , material-liquid ratio 1：25（g ∶ mL）, extraction time 40 minutes, and total flavonoid extraction rate 0.446%. Key words：Radix astragali; Flavonoids; Ultrasonic method; Orthogonal test 中图分类号：R284.2

Optimization of ultrasonic-assisted extraction of water-soluble polysaccharides

Carbohydrate Polymers 87 (2012) 614–619Contents lists available at ScienceDirectCarbohydratePolymersj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c a r b p olOptimization of ultrasonic-assisted extraction of water-soluble polysaccharides from Boletus edulis mycelia using response surface methodologyWei Chen a ,b ,Wei-Ping Wang a ,b ,∗,Hua-Shan Zhang a ,b ,Qin Huang a ,ba College of Bioengineering,Hubei University of Technology,Wuhan 430068,ChinabKey Laboratory of Fermentation Engineering (Ministry of Education),Hubei University of Technology,Wuhan 430068,Chinaa r t i c l ei n f oArticle history:Received 27May 2011Received in revised form 3August 2011Accepted 12August 2011Available online 19 August 2011Keywords:Ultrasonic extractionResponse surface methodology Boletus edulis PolysaccharidesOptimization of extractiona b s t r a c tAn ultrasound-assisted procedure for the extraction of water-soluble polysaccharides from the submerged-cultured mycelia of Boletus edulis was investigated using response surface methodology (RSM).Three independent variables were ratio of dried mycelia to water (X 1:1:40–1:60),extraction time (X 2:6–10min),and ultrasonic temperature (X 3:50–70◦C).The statistical analysis indicated theindependent variables (X 2,X 3),the quadratic terms (X 22and X 23)and the interaction between X 3and X 1had signiﬁcant effects on the yield of polysaccharides (p <.05).The optimized conditions were 56◦C,1:55of ratio of dried mycelia to water,and a time of contact of 8.4min.Under these conditions,the experi-mental yield of polysaccharides was 15.48%,which was well matched with the predictive yield of 15.53%.© 2011 Elsevier Ltd. All rights reserved.1.IntroductionBoletus edulis is one of the most well-known edible mushroom collected especially in the Northern Hemisphere across Europe,Asia and North America.Polysaccharides extracted from lis have been reported to have many biological functions such as anti-cancer,antioxidant and anti-inﬂammatory effects (Cengiz,Bektas,&Mustafa,2008;Dentinger,Ammirati,&Both,2010).However,fruit bodies of lis are precious due to their rareness and difﬁ-culty in cultivation (Salerni &Perini,2004)lis is a mycorrhizal fungus.Its mycelia but not fruit bodies can be easily cultivated.So lis can be obtained in the form of mycelia from sub-merged culture and bioactive polysaccharides can be extracted from lis mycelia.A literature survey indicated that there was no investigation on the extraction of water-soluble polysaccha-rides from lis mycelia.Heating or boiling was conventionally used to extract water-soluble polysaccharides.However,during the extraction,many bioactive compounds due to ionization,hydroly-sis and oxidation are easily lost.Recently,various novel extraction techniques have been developed for the extraction of bioactive compounds such as ultrasound-assisted extraction,microwave-assisted extraction and supercritical ﬂuid extraction (Wang &∗Corresponding author at:College of Bioengineering,Hubei University of Tech-nology,China.Tel.:+862787210399;fax:+862788032320.E-mail addresses:wang1wei1ping1@ ,wangwp@ (W.-P.Wang).Weller,2006).Among these,ultrasound-assisted extraction is one of the most inexpensive,simple and efﬁcient techniques (Chen et al.,2010;Huang,Xue,Niu,Jia,&Wang,2009;Yan et al.,2011;Zhang,Yang,Zhao,&Wang,2009;Zhong &Wang,2010),which can increase the yield of extracted components,reduce extraction time and make higher processing throughput.It is very useful for the extraction of thermolabile and unstable compounds,presum-ably by avoiding degradation reactions (Vilkhu,Mawson,Simons,&Bates,2008).In this study,we investigated the ultrasound-assisted extraction condition of polysaccharides from submerged-cultured lis mycelia.To improve the yield of polysaccharides,response surface methodology (RSM)was designed to systematically analyze the effects of extraction parameters on the yields of polysaccharides from lis mycelia and their interactions.RSM is an effective statistical technique for optimizing complex processes,which is widely used in optimizing the extraction process variables (Ebru &Ozgul,2010;Guan &Yao,2008;Guo,Zou,&Sun,2010;Hou &Chen,2008;Pompeu,Silva,&Rogez,2009;Silva,Rogez,&Larondelle,2007).The main advantage of RSM is the reduced number of exper-imental trials needed to evaluate multiple parameters and their interactions,which is more efﬁcient and easier to arrange and inter-pret experiments in comparison with others.Box–Behnken design (BBD)(Ferreira,Bruns,Ferreira,&Matos,2007),one of RSM,based on a 3levels and 3variables central composite,was employed to obtain the best possible combination of extraction temperature,extraction time and ratio of water to dried mycelia for maximum polysaccharides production.0144-8617/$–see front matter © 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.carbpol.2011.08.029W.Chen et al./Carbohydrate Polymers87 (2012) 614–619615 Table1Factors and levels for RSM,and Box–Behnken experimental design with the independent variables.Run Coded and uncoded variable levels Yield of polysaccharide(%)X1/ratio of dried mycelia to water(g/mL)X2/extractiontime(min)X3/ultrasonictemperature(◦C)ActualvaluesPredictedvalues1−1(1:40)−1(6)0(60)14.2714.28 2−1(1:40)0(8)−1(50)14.3114.45 3−1(1:40)1(10)0(60)14.4014.28 4−1(1:40)0(8)1(70)13.4313.41 51(1:60)1(10)0(60)14.7714.77 61(1:60)0(8)−1(50)15.0515.07 71(1:60)−1(6)0(60)13.7713.90 81(1:60)0(8)1(70)13.0412.90 90(1:50)1(10)−1(50)14.3614.34 100(1:50)1(10)1(70)12.5812.73 110(1:50)−1(6)−1(50)14.0513.90 120(1:50)−1(6)1(70)12.2812.30 130(1:50)0(8)0(60)15.2915.34 140(1:50)0(8)0(60)15.5015.34 150(1:50)0(8)0(60)15.2315.342.Materials and methods2.1.Microorganism and culture conditionslis ACCC50559was from Agricultural Culture Collection of China(ACCC).Agar slants containing potato–dextrose–agar were inoculated with mycelia and incubated at25◦C for6days and then used as inoculums for seed culture.The seed culture was grown in250mL bafﬂedﬂasks on a rotary shaker for60h at natural pH,25◦C and120rpm with a medium(g/L)containing: glucose20,potato200(put200g potato into the water,boiled for30min,andﬁltration,metered volume to1L),MgSO4·7H2O 0.5,KH2PO4 1.0,ZnSO4·7H2O0.1,yeast extract14,peptone6. Fermentation was carried out in the medium of following com-position(g/L):potato100(put100g potato into the water,boiled for30min,andﬁltration,metered volume to1L),sucrose20, peptone6,MgSO4·7H2O2,KH2PO43,CaCO32.All media were sterilized at115◦C for30min.The fermentation cultivation was inoculated at10%(v/v)of the above seed culture medium and kept at28◦C and200rpm in250mL bafﬂedﬂasks on a rotary shaker for 5days.2.2.Extraction of crude polysaccharides from lis myceliawith ultrasound-assisted treatmentlis mycelia from submerged culture were washed by distilled water.The mycelia were dried at30◦C.Then,it was grinded and stored in desiccators at room temperature(15–20◦C) until used.The process of polysaccharides extraction from B. edulis mycelia by ultrasound-assisted treatment was performed in an ultrasonic processor(SY-360,Shanghai Ninson Inc.,Shanghai, China).One-tenth of dried mycelia powders were extracted with distilled water in a50mL centrifuge tube.The centrifuge tube was held in the ultrasonic processor and exposed to extract polysac-charides for different time at varied ultrasonic temperatures in different ratios of dried mycelia to water.2.3.Determination of the yield of polysaccharides from lis myceliaAfter ultrasonic treatment,the extracted slurry was centrifuged at7000rpm for10min to collect the supernatant and the polysac-charides were determined by phenol–sulfuric acid method(Dubois, Gilles,Hamilton,Rebers,&Smith,1956).2.4.Experimental design and statistical analysesSingle-factor-test was employed to determine the preliminary range of the extraction variables including X1(ratio of dried mycelia to water),X2(extraction time)and X3(ultrasonic temperature). Then,a three-level-three-factor BBD was employed to determine the best combination of extraction variables for the yields of lis mycelia water-soluble polysaccharides.Table1represents the coded and non-coded values of the experimental variables and 15experimental points.Three replicates(13–15)were used to evaluate the pure error.Experimental data shown that response variables wereﬁtted to a quadratic polynomial model.The general form of the quadratic polynomial model was as follows:Y=ˇ0+3i=1ˇi X i+3i=1ˇii X2i+2i=13j=i+1ˇij X i X j(1)where Y is the measured response associated with each factor lever combination;ˇ0,ˇi,ˇii andˇij are the regression coefﬁcients for intercept,linearity,square and interaction,respectively;X i and X j are the independent variables.Design Expert software(Trial Version7.1.6.)was used to estimate the response of each set of experimental design and optimized conditions.Theﬁtness of the quadratic polynomial model was inspected by the regression coef-ﬁcient R2.F-value and p-value were used to check the signiﬁcances of the regression coefﬁcient.3.Results and discussion3.1.Selection of ratio of dried mycelia to water for extractionyield of polysaccharidesPreliminary studies were performed in order to determine the required ratio of dried mycelia to water for the extraction yields of polysaccharides from lis.Extraction was carried out at differ-ent ratios of dried mycelia to water(1:20,1:30,1:40,1:50and1:60), while other extraction parameters were as following:extraction time2min,ultrasonic temperature40◦C.The results showed that the extraction of the polysaccharides was dependent on the solid-to-liquid ratio(Fig.1(A)).The yield of polysaccharides increased with the increase of the solid-to-liquid ratio.A plateau in the mass transfer was reached at the solid-to-liquid ratio of1:50.The max-imum yield(14.1%)was also achieved.Thus,ratio of dried mycelia to water1:40–1:60was favorable for producing polysaccharides.616W.Chen et al./Carbohydrate Polymers 87 (2012) 614–619Fig.1.Effect of different ratio of dried mycelia to water (A),extraction time (B)andultrasonic temperature (C)on extraction yield of polysaccharides.3.2.Selection of time on extraction yield of polysaccharidesExtraction time is another factor that would inﬂuence the extraction efﬁciency.Extraction process was carried out using the time of 2,4,6,8and 10min,when other extraction parameters were as following:ultrasonic temperature 40◦C,ratio of dried mycelia to water 1:20.The effect of different time on extraction yield of polysaccharides was shown in Fig.1(B).When extraction time var-ied from 6to 10min,the variance of extraction yield was relatively rapid and polysaccharides production reached a maximum at 8min (15.22%),and then decreased as the extraction proceeded.This indi-cated that extraction time of 6–10min was sufﬁcient to obtain polysaccharides,which was less than the conventional heating or boiling extraction time (Guo et al.,2010;Hou &Chen,2008).3.3.Selection of temperature on extraction yield ofpolysaccharidesTo study the effect of different temperature on extraction yield of polysaccharides,extraction process was carried out using the dif-ferent extraction temperature of 40,50,60,70,80◦C,when other extraction condition was as following:extraction time 2min,ratio of dried mycelia to water 1:20.The extraction yield of polysaccha-rides had been increasing when ultrasonic temperature increased from 40to 60◦C.As shown in Fig.1(C),the maximum yield (15.01%)of polysaccharides was observed when extraction temperature was 60◦C,and then decreased as the extraction proceeded.Therefore,Table 2Analysis of variance for the ﬁtted quadratic polynomial model of extraction of polysaccharides.SourceSS a DF b MS c F -valuep -Value Model 13.349 1.4847.120.0003Residual 0.1650.031Lack of ﬁt 0.1230.039 1.940.3578Pure error 0.04020.020Cor.total13.5014R 2=0.9883;R 2adj =0.9674;C.V.%=1.25.a Sums of squares.b Degree freedom.cMean square.extraction temperature range of 50–70◦C was considered to be optimal in the present experiment.Because conventional heating extraction temperature was more than 70◦C (Guo et al.,2010;Hou &Chen,2008),ultrasound-assisted extraction was lower.3.4.Optimization of extraction conditions of polysaccharides3.4.1.Predicted model and statistical analysisThe design matrix and the corresponding results of RSM exper-iments to determine the effects of the three independent variables including ratio of dried mycelia to water (X 1),extraction time (X 2)and ultrasonic temperature (X 3)were shown in Table 1.Through multiple regression analysis on the experimental data,the model for the predicted response Y could be expressed by the following quadratic polynomial equation (in the form of coded values):Y =15.34+0.027X 1+0.22X 2−0.81X 3−0.2X 21−0.84X 22−1.18X 23+0.22X 1X 2−0.28X 1X 3−2.5×10−3X 2X 3(2)where Y is the yield of polysaccharides,X 1,X 2and X 3are the coded variables for the ratio of dried mycelia to water,extraction time and ultrasonic temperature,respectively.Statistical testing of the model was performed in the form of analysis of variance (ANOVA).The ANOVA for the ﬁtted quadratic polynomial model of extraction of polysaccharides were shown in Table 2.The quadratic regression model showed the value of the determination coefﬁcient (R 2)was 0.9883,which implied that 98.83%of the variations could be explained by the ﬁtted model.Fora good statistical model,R 2adjshould be close to R 2.As shown in Table 2,R 2adjwas 0.9674,which implied that only less 4.0%of the total variations were not explained by the model.It also indicated that a high degree of correlation between the observed and pre-dicted values.A relatively low value of C.V.(coefﬁcient of variation)(1.25%)indicated a better reliability of the experiments values.The corresponding variables would be more signiﬁcant if the F -value becomes greater and the p -value becomes smaller (Atkinson and Donev,1992).Values of p -value less than 0.05showed model terms were signiﬁcant.SO the F -value (F =47.12)and p -value (p =0003)implied this model was signiﬁcant.Signiﬁcance of the model was also judged by lack-of-ﬁt test.As shown in Table 2,F -value and p -value of the lack of ﬁt were 1.94and 0.3578,respectively,which implied that it was not signiﬁcant and a 35.78%chance could occur due to noise.The signiﬁcance of each coefﬁcient was determined using F -value and p -value.The results were given in Table 3.It could be seen that two independent variables (X 2,X 3)and twoquadratic terms (X 22and X 23)signiﬁcantly affected the yield of polysaccharides,and the interaction between X 1and X 3was signiﬁ-cant too (p <.05).Results also showed that the independent variable X 3was the most signiﬁcant factor on the experimental yield of polysaccharides.W.Chen et al./Carbohydrate Polymers87 (2012) 614–619617 Table3Estimated regression model of relationship between response variables(yield ofpolysaccharides)and independent variables(X1,X2,X3).Variables DF a SS b MS c F-value p-ValueX11 6.05×10−3 6.05×10−30.190.6793X210.380.3812.030.0179X31 5.18 5.18164.84<0.0001X2 110.150.15 4.640.0839X2 21 2.60 2.6082.590.0003X2 31 5.17 5.17164.51<0.0001X1X210.190.19 6.020.0577 X1X310.320.3210.150.0244 X2X31 2.50×10−5 2.50×10−57.949×10−40.9786a Degree freedom.b Sums of squares.c Mean square.3.4.2.Analysis of response surfaceThe relationship between independent and dependent variables was illustrated by the three-dimensional representation of the response surfaces and the two-dimensional contours generated by the model(seen in Figs.2–4).Different shapes of the contour plots indicated different interactions between the variables,an ellipti-cal contour plot indicated the interactions between the variables were signiﬁcant while a circular contour plot means otherwise.In these three variables(ratio of dried mycelia to water,extraction time and ultrasonic temperature),when two variables within the experimental range were depicted in three-dimensional surface plots,the third variable was kept constant at zero level.As shown in Fig.2,when ultrasonic temperature(X3)wasﬁxed at0level, extraction time(X2)demonstrated quadratic effects on the extrac-tion yields.When ratio of dried mycelia to water kept at lower level, the yield increased atﬁrst and then decreased with the increase of extraction time(X2).As shown in Fig.3,when extraction time (X2)wasﬁxed at0level,ultrasonic temperature(X3)displayed a quadratic effect on the response yield.The elliptical contour plot shown in Fig.3indicated the mutual interactions between ratio of dried mycelia to water and extraction temperature were signiﬁ-cant.The results of Fig.4showed that when ratio of dried mycelia to water(X1)wasﬁxed at0level,the ultrasonic temperature(X3) was increased with increases in extraction time(X2).And then,the ultrasonic temperature(X3)was decreased with further increase in extraction time.Extraction time and ultrasonic temperature demonstrated quadratic effects on the response.3.5.Optimization of extracting parameters and validation of the modelThrough these three-dimensional plots and their respective contour plots,the suitability of the model equation for predicting the optimum response values were tested using the selected opti-mal conditions.The results(Table4)showed that theoptimized Fig.2.Response surface plot and contour plot of ratio of raw material to water and extraction time,and their mutual interactions on the yield ofpolysaccharides. Fig.3.Response surface plot and contour plot of ratio of raw material to water and ultrasonic temperature,and their mutual interactions on the yield of polysaccharides.618W.Chen et al./Carbohydrate Polymers 87 (2012) 614–619Fig.4.Response surface plot and contour plot of extraction time and ultrasonic temperature,and their mutual interactions on the yield of polysaccharides.Table 4Optimum conditions,and the predicted and experimental value of response at the optimum conditions.Ultrasonic temperature (◦C)Extraction time (min)Ratio of raw material to waterYield of polysaccharide (%)Optimum conditions (predicted)56.068.381:54.5315.53Modiﬁed conditions (actual)568.41:5515.48conditions were ultrasonic temperature of 56.06◦C,extraction time of 8.38min,and ratio of dried mycelia to water 1:54.53.Under the conditions,the extraction yield of polysaccharides was 15.53%.However,considering the operability in actual production,the optimal conditions can be modiﬁed as follows:ultrasonic tem-perature of 56◦C,extraction time of 8.4min,and ratio of dried mycelia to water 1:55.Under the modiﬁed conditions,the exper-imental yield of polysaccharides was 15.48%(N =3),which was close to the predicted value.Generally,the extraction yields of polysaccharides from fungal mycelia or mushrooms were below 10%.Hou &Chen (2008)investigated that the extraction efﬁciency of polysaccharides from wild edible BaChu mushroom was 8.75%.Yan et al.(2011)reported the extraction yields of polysaccharides from Tremella mesenterica was 8.26%.Guo et al.(2010)found that the extraction yields of polysaccharides from Phellinus igniarius was 5.04%.So the extraction efﬁciency of 15.48%was very high.4.ConclusionIn this study,we had investigated an ultrasonic-assisted method to extract polysaccharides from the lis mycelia using RSM.The results showed that the independent variables (ultrasonic temperature and extraction time),and quadratic terms of ultra-sonic temperature and extraction time,and the interaction effects between ultrasonic temperature and ratio of dried mycelia to water had signiﬁcant effects on the yield of polysaccharides.Ultrasonic temperature was the most signiﬁcant factor on the experimen-tal yield of polysaccharides.A second-order polynomial model was employed to optimize polysaccharides extraction from lis mycelia by ultrasonic technology.The optimal extraction conditions for the polysaccharides were as follows:ultrasonic tem-perature 56◦C,extraction time of 8.4min,and ratio of dried mycelia to water 1:55.Under these conditions,the experimental yield of polysaccharides was 15.48%,which was agreed closely with the predicted yield value of 15.53%.The study provided a new and efﬁ-cient method for the extraction of water-soluble polysaccharides from lis mycelia.Further studies on the chemical structures and the bioactive function of polysaccharides from lis mycelia are under process.AcknowledgementWe gratefully acknowledge the ﬁnancial support received from the Doctoral Scientiﬁc Research Foundation of Hubei University of Technology (Contract No.BSQD0911).ReferencesAtkinson,A.C.,&Donev,A.N.(1992).Optimum experimental designs .Oxford:OxfordUniversity Press.(pp.132–189).Cengiz,S.,Bektas,T.,&Mustafa,Y.(2008).Evaluation of the antioxidant activity offour edible mushrooms from the Central Anatolia,Eskisehir –Turkey:Lactarius deterrimus ,Suillus collitinus ,Boletus edulis ,Xerocomus chrysenteron .Bioresource Technology ,99,6651–6655.Chen,X.P.,Wang,W.X.,Li,S.B.,Xue,J.L.,Fan,L.J.,Sheng,Z.J.,et al.(2010).Optimization of ultrasound-assisted extraction of Lingzhi polysaccharides using response surface methodology and its inhibitory effect on cervical cancer cells.Carbohydrate Polymers ,80,944–948.Dentinger,B.T.M.,Ammirati,J.F.,&Both,E.E.(2010).Molecular phylogenetics ofporcini mushrooms (Boletus section).Molecular Phylogenetics and Evolution ,57,1276–1292.Dubois,M.,Gilles,K.A.,Hamilton,J.K.,Rebers,P.A.,&Smith,F.(1956).Colorimetricmethod for determination of sugars and related substances.Analytical Chemistry ,28,350–356.Ebru,F.D.,&Ozgul,E.(2010).Response surface methodology for protein extrac-tion optimization of red pepper seed (Capsicum frutescens ).Food Science and Technology ,43,226–231.Ferreira,S.L.C.,Bruns,R.E.,Ferreira,H.S.,&Matos,G.D.(2007).Box–Behnkendesign:An alternative for the optimization of analytical methods.Analytica Chemical Acta ,597,179–186.Guan,X.,&Yao,H.Y.(2008).Optimization of viscozyme l -assisted extraction of oatbran protein using response surface methodology.Food Chemistry ,106,345–351.Guo,X.,Zou,X.,&Sun,M.(2010).Optimization of extraction process by responsesurface methodology and preliminary characterization of polysaccharides from Phellinus igniarius .Carbohydrate Polymers ,80,344–349.Hou,X.J.,&Chen,W.(2008).Optimization of extraction process of crude polysac-charides from wild edible BaChu mushroom by response surface methodology.Carbohydrate Polymers ,72,67–74.Huang,W.,Xue,A.,Niu,H.,Jia,Z.,&Wang,J.W.(2009).Optimised ultrasonic-assistedextraction of ﬂavonoids from Folium eucommiae and evaluation of antioxidant activity in multi-test systems in vitro.Food Chemistry ,114,1147–1154.Pompeu,D.R.,Silva,E.M.,&Rogez,H.(2009).Optimisation of the solvent extractionof phenolic antioxidants from fruits of Euterpe oleracea using response surface methodology.Bioresource Technology ,100,6076–6082.Salerni,E.,&Perini,C.(2004).Experimental study for increasing productivity ofBoletus edulis s.l.in Italy.Forest Ecology and Management ,201,161–170.Silva,E.M.,Rogez,H.,&Larondelle,Y.(2007).Optimization of extraction of pheno-lics from Inga edulis leaves using response surface methodology.Separation and Puriﬁcation Technology ,55,318–387.W.Chen et al./Carbohydrate Polymers87 (2012) 614–619619Vilkhu,K.,Mawson,R.,Simons,L.,&Bates,D.(2008).Applications and opportunities for ultrasound assisted extraction in the food industry–A review.Innovative Food Science and Emerging Technologies,9,161–169.Wang,L.,&Weller,C.L.(2006).Recent advances in extraction of nutraceuticals from plants.Trends in Food Science and Technology,17,300–312.Yan,Y.L.,Yu,C.H.,Chen,J.,Li,X.X.,Wang,W.,&Li,S.Q.(2011).Ultrasonic-assisted extraction optimized by response surface methodology,chemical composition and antioxidant activity of polysaccharides from Tremella mesenterica.Carbohy-drate Polymers,83,217–224.Zhang,H.F.,Yang,X.H.,Zhao,L.D.,&Wang,Y.(2009).Ultrasonic-assisted extrac-tion of epimedin C from fresh leaves of Epimedium and extraction mechanism.Innovative Food Science and Emerging Technologies,10,54–60.Zhong,K.,&Wang,Q.(2010).Optimization of ultrasonic extraction of polysaccha-rides from dried longan pulp using response surface methodology.Carbohydrate Polymers,80,19–25.。

鱼皮中胶原蛋白的提取

2
2.1
新鲜鲫鱼（曲靖沃尔玛超市）、乙酸（天津市恒兴化学试剂制造有限公司）、氯化钠（天津市鼎盛鑫化工有限公司）、盐酸（福州痕量分析生物科技有限公司）、透析袋（南京乐进生物实验器材商城）。
2.2
DK-8AB电热恒温水浴锅（上海-恒科学仪器有限公司）、CP214电子天平（奥豪斯仪器上海有限公司）、DHG9123型恒温烘箱（上海精密科学仪器有限公司）、80-2台式低速离心机(上海医疗器械（集团）有限公司手术器械厂)、SK3200LHC超声波清洗仪(上海科导超声仪器有限公司)，其他为实验室常用仪器设备。
鱼皮中胶原蛋白的提取
摘要
胶原蛋白是构成动物支持组织的结构蛋白质,其提取制品已广泛应用于医药、保健、食品加工、化妆品等众多领域,迄今其制品主要是从一些陆生哺乳动物如牛、鱼等的皮肤提取。但近年来，科学家对鱼皮中胶原蛋白的提取也没有止步，“鱼皮”含有丰富的蛋白质和多种微量元素，其蛋白质主要是大分子的胶原蛋白及粘多糖的成分，是女士养颜护肤美容保健佳品，也是滋补佳品。提取方法主要有聚能式脉冲逆流超声辅助法、中性盐萃取法、酶法、酸法、碱法，而这些方法都尚未获得较高的提取率，但其中酶法提取对胶原蛋白破坏性小，能更好、更完全地得到活性胶原蛋白，具有反应速度快、时间短、无环境污染等优点。超声波提取因其空穴效应、污染小、节能、环保等优越性能在当今的生产、生活和科学研究中被广泛使用。所以本论文以新鲜鲫鱼皮为原料，以胶原蛋白提取率为目标参数，采用超声波提取法对鱼皮中胶原蛋白提取，从而进一步提高鱼皮的药用、食用价值。选取提取时间、料液比、提取功率3个影响因子进行单因素实验。在单因素实验设计的基础上利用正交试验，得鱼皮中胶原蛋白提取的最佳工艺条件。结果表明，当提取时间为55min、料液比为1:45、提取功率为100%时候提取率最高为

流化床颗粒包衣法制备替米沙坦氢氯噻嗪片

China) ABSTRACT: OBJECTIVE To prepare Telmisartan Hydrochlorothiazide tablets by the methods of fluidized bed particles coating and assess its stability. METHODS Telmisartan particles were prepared by the fluidized bed one step granulation. Telmisartan particles of different coating weight, hydrochlorothiazide and pharmaceutical excipients were mixed. Then Telmisartan Hydrochlorothiazide tablets were prepared by rotary tablet machine. The optimization formulation were selected by orthogonal experiment. The assay and impurty were assayed by HPLC. The stability and dissolution study were conducted in an accelerated testing and long-term testing. RESULTS The optimal method of preparing Telmisartan Hydrochlorothiazide tablets showed a good stability. The formulation contained external additional sodium carboxymethyl starch 20.4 mg and sodium hydroxide 8.5 mg showed the best dissolution. The final optimized prescription had similar dissolution behavior to brand drug. CONCLUSION The Telmisartan Hydrochlorothiazide tablets produced by fluidized bed particles coating show a good quality, and the process is more simple than two-layered tablets, the optimal method is feasible. KEY WORDS: particles coating; coating weight; telmisartan hydrochlorothiazide tablets; one-layered tablets; two-layered tablets; stability

黑豆中大豆异黄酮超声波法提取工艺优化

第37卷第2期齐齐哈尔大学学报（自然科学版） Vol.37,No.2 2021年3月 Journal of Qiqihar University(Natural Science Edition) March,2021黑豆中大豆异黄酮超声波法提取工艺优化王双侠，苏适，柴宝丽（绥化学院食品与制药工程学院，黑龙江绥化 152061）摘要：为了提高黑豆中大豆异黄酮的提取率，采用超声提取法，以乙醇水溶液为提取溶剂，选择乙醇浓度、提取时间、提取温度及物料粉碎度进行四因素三水平的正交实验，考察各提取因素对黑豆中大豆异黄酮提取率的影响，以优选出黑豆中大豆异黄酮的最佳提取工艺。

结果表明，物料粉碎后过20目筛，以60%乙醇水溶液为溶剂，温度为50℃，超声提取50min，大豆异黄酮的提取率为1.91%，为最优提取工艺，工艺重复性良好。

关键词：黑豆；大豆异黄酮；超声提取法；提取率中图分类号：TS201.2 文献标志码：A 文章编号：1007-984X(2021)02-0070-03大豆异黄酮是具有雌激素样作用的天然活性物质，故又叫植物雌激素，因其多样而突出的生物学活性引起了国内外研究人员的广泛关注[1]。

大豆异黄酮主要来源于大豆，近年来黑龙江省黑豆的种植量在逐步增大，因此针对黑豆的综合开发利用前景更为广阔。

目前，大豆异黄酮的提取主要有溶剂提取法、超声波提取法、微波提取法、超临界流体萃取法、连续逆流提取法等，其中超声波法与其它提取法相比，具有提取时间短、提取率高、成本低等优点[2]。

为此本实验以黑豆为原料，采用超声波法提取，考察提取因素对大豆异黄酮提取率的影响，以确定其最佳提取工艺。

1 实验部分1.1 实验材料及仪器高效液相色谱仪（美国Waters公司）；超声波清洗器（上海皆准仪器设备有限公司）；电热恒温鼓风干燥箱（浙江恒威电子科技有限公司）；黑豆（产自黑龙江省绥化）；金雀异黄酮对照品（酷尔化学科技有限公司）；其他试剂均为分析纯。

超声波辅助酶解法提取文冠果籽粕蛋白工艺优化及抗氧化活性研究

中国果菜China Fruit &Vegetable第43卷,第9期2023年9月专家论坛Experts Forum 超声波辅助酶解法提取文冠果籽粕蛋白工艺优化及抗氧化活性研究宋健平1，郭欣奕1，刘欣雨1，路昌1，王雨萌1，王磊2，于梅1*（1.山东农业工程学院食品科学与工程学院，山东济南250100；2.山东省林草种质资源中心暖温带林草种质资源保存与利用国家林业和草原局重点实验室，山东济南250102）摘要:以文冠果籽粕作为原料，研究超声波辅助碱性蛋白酶酶解文冠果籽粕蛋白的工艺条件。

通过单因素和响应面试验，以水解度为指标，对文冠果籽粕蛋白酶解工艺进行优化并对其抗氧化活性进行研究。

结果表明，超声辅助酶解工艺的最佳条件为料液比1∶12（g/mL ），酶添加量0.15%，酶解时间6h ，酶解pH 8.5，超声波功率300W ，温度37℃、超声时间20min ，在此条件下的酶解液水解度为22.57%，与水酶法相比，增加了9.36%。

当酶解物质量浓度为17.5mg/mL 时，对超氧阴离子自由基、DPPH 自由基、ABTS 自由基的清除率分别为90.90%、86.90%、90.65%。

文冠果籽粕蛋白酶解物的抗氧化活性在三种体系中的50值分别为10.66、14.36、5.21mg/mL ，与同浓度下的VC 作用效果接近。

关键词:文冠果籽粕；酶解；超声处理；抗氧化活性中图分类号：TS201.2文献标志码：A文章编号：1008-1038(2023)09-0011-08DOI：10.19590/ki.1008-1038.2023.09.003Optimization of Ultrasound-assisted Enzymatic Hydrolysis Extraction of Protein fromSeed Meal and Study on ItsAntioxidant ActivitySONG Jianping 1,GUO Xinyi 1,LIU Xinyu 1,LU Chang 1,WANG Yumeng 1,WANG Lei 2,YU Mei 1*(1.School of Food Science and Engineering,Shandong Agriculture and Engineering University,Jinan 250100,China;2.Key Laboratory of National Forestry and Grassland Administration Conservation and Utilization of WarmTemperate Zone Forest and Grass Germplasm Resources,Shandong Provincial Center of Forest and GrassGermplasm Resources,Jinan 250102,China)Abstract:seed meal protein was used as raw material,the technological conditions of enzymatic hydrolysis ofseed meal protein with ultrasonic-assisted alkaline protease were studied.The收稿日期:2023-02-12基金项目:山东省农业良种工程项目（2020LZGC009）第一作者简介:宋健平（2002—），男，在读本科，专业为食品科学与工程*通信作者简介:于梅（1982—），女，副教授，硕士，主要从事功能食品开发方面工作文冠果（Bunge ）是无患子科文冠果属落叶灌木或小乔木,是我国北方特有的珍稀木本油料植物[1]，种仁可以榨油，制得的文冠果油是高档食用油，具有抗炎、清除自由基、抗肿瘤活性和修复脑神经等功能[2]。

Response surface methodology for optimization of the ultrasonic extraction of polysaccharides from

Carbohydrate Polymers 84 (2011) 503–508Contents lists available at ScienceDirectCarbohydratePolymersj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c a r b p olResponse surface methodology for optimization of the ultrasonic extraction of polysaccharides from Codonopsis pilosula Nannf.var.modesta L.T.ShenYuanfeng Zou,Xingfu Chen ∗,Wenyu Yang,Sha LiuCollege of Agronomy,Sichuan Agricultural University,Ya’an 625014,PR Chinaa r t i c l e i n f o Article history:Received 14October 2010Received in revised form 2December 2010Accepted 3December 2010Available online 10 December 2010Keywords:Polysaccharides from Codonopsis pilosula Response surface methodology Ultrasonic extraction Optimizationa b s t r a c tThe best ultrasonic extractions of polysaccharides with water from Codonopsis pilosula were investigated based on a Box–Behnken design.Three independent and main variables,including extraction time (min),ratio of water to raw material and ultrasonic power (W),which were of signiﬁcance for the yields of polysaccharides were studied and the Box–Behnken design was based on the results of a single-factors test.The experimental data were ﬁtted to a second-order polynomial equation using multiple regression analysis and also examined using the appropriate statistical methods.The best extraction conditions are as follows:extraction time 44min,ratio of water to raw material 56and ultrasonic power 320W.Under the optimization conditions,the experimental yield was 36.264%,which was well matched with the predictive yield.© 2010 Elsevier Ltd. All rights reserved.1.IntroductionRadix Codononpsis is one of the traditional Chinese herbal medicines (TCM),which was the root of Codonopsis pilosula (Franch.)Nannf., C.pilosula Nannf.var.modesta L.T.Shen and C.tangshen Oliv.It was used in TCM to lower blood pressure and increase white blood cell counts,cure appetite loss,strengthen the immunize system (China Pharmacopoeia Committee,2010).Radix Codononpsis was utilized primarily as a substitution for gin-seng (Panax ginseng ).Recently,several investigators reported that the polysaccharides extracted from Radix Codonopsis had several bioactivities such as the immune function and improving the com-pensatory hematopoiesis of spleen (Yang,Li,Liu,&Xian,2005;Zhang,Zhu,Hu,Lai,&Mo,2003)and anti-active oxygen free radicals (Li &Yang,2001).Meanwhile,reports about the reﬂux extrac-tion,puriﬁcation and structural elucidation of polysaccharides from Radix Codonopsis appeared too (Han,Cheng,&Chen,2005;Sun &Liu,2008;Zhang,Zhang,Yang,&Liang,2010).Hot-water reﬂux extraction technology is the main extraction method of polysac-charides from Radix Codononpsis in recent researches,which is a classical extraction method of polysaccharides.It usually requires long extraction time,high temperature,but the extraction efﬁ-ciency is low (Sun,Liu,&Kennedy,2010).Therefore,it is essential and desirable to ﬁnd an economical and high efﬁcient extraction method of polysaccharides from Radix Codonopsis .∗Corresponding author.Tel.:+868352882612;fax:+868352882612.E-mail address:chenxf64@ (X.Chen).Recently,ultrasonic extraction was tested as an alternative method for isolating polysaccharides from different plant mate-rials (Hromadkova and Ebringerova,2003;Wang,Cheng,Mao,Fan,&Wu,2009).It is reported that extraction efﬁciency is greatly enhanced by ultrasonic treatments.The ability of ultrasonic treatment to improve the recovery of polysaccharides is mainly attributed to its facilitation of mass transfer during immiscible phases through agitation,especially at low frequency (Vinatoru et al.,1997).Compared with classical methods,the ultrasonic treat-ment can reduce extraction time and energy,and enhance the extraction quotiety.Response surface methodology (RSM)is an effective statistical technique for optimizing complex processes.The main advantage of RSM is the reduced number of experimental trials needed to evaluate multiple parameters and their interactions.Therefore,it is less laborious and time-consuming than other approaches required to optimize a process (Zhong &Wang,2010).It was widely used in optimizing the polysaccharide extraction process variables (Gan,Abdul Manaf,&Latiff,2010;Guo,Zou,&Sun,2010;Zhu,Heo,&Row,2010).Box–Behnken design (BBD)is a type of response surface design.It is an independent quadratic design in that it does not contain an embedded factorial or fractional factorial design.In this design the treatment combinations are at the midpoints of edges of the process space and at the center.These designs are rotatable (or near rotatable)and require 3levels of each factor.It is more efﬁcient and easier to arrange and interpret experiments in comparison with others.It is widely used in many researches (Sun,Li,Yan,&Liu,2010;Zhao,Wang,&Lu,2009).Even the ultrasonic treatment has so many advantages,there is no information published about the optimization of ultrasonic0144-8617/$–see front matter © 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.carbpol.2010.12.013504Y.Zou et al./Carbohydrate Polymers84 (2011) 503–508extraction conditions for polysaccharides from C.pilosula.So,in the present experiments,we optimized the extraction parameters of polysaccharides from C.pilosula by employing a Box–Behnken design.We wish to optimize the ultrasonic extraction conditions through a BBD(3factors and3levels),to study the effects of extrac-tion time,ratio of water to raw material and ultrasonic power on the yield of polysaccharides.2.Materials and methods2.1.Materials and equipmentsThe roots of C.pilosula were collected from Jiuzhaigou County, Sichuan Province,China,and identiﬁed by Xingfu Chen professor of Sichuan Agriculture University.Ethanol,phenol,sulphuric acid and glucose were obtained from the Chengdu Kelong Chemical Factory(Chengdu,China).KQ-400KDZ ultrasonic generator(Kun-shan Ultrasonic Instruments Co.,LTD,Jiangsu,China)was used for extraction.All other chemicals were of analytical grade.2.2.Extraction methodsC.pilosula(2.0g)was ultrasonically extracted with80mL of80% ethanol for30min.Then,theﬁltered residues without ethanol were ultrasonically extracted with distilled water for different extrac-tion times,twice.Finally,mixed theﬁlter liquor of two extraction times together,added distilled water to250mL,prepared as sample liquor.2.3.Determination of polysaccharides yieldThe total sugar content of polysaccharide was quantiﬁed by the phenol–sulphuric acid method(Masuko et al.,2005),glucose was used as standard,and the results were then expressed as glucose equivalents(Hou&Chen,2008).2.4.Design of statistical experimentsAfter determining the preliminary range of extraction variables through single-factor test,a three-level-three-factor,Box–Behnken factorial design(BBD)was adopted in this optimization study. Extraction time(X1),ratio of water to raw material(X2),ultra-sonic power(X3)were the independent variables selected to be optimized for the extraction of polysaccharides.The range of inde-pendent variables and their levels were presented in Table1. Extraction yield(Y)was taken as the response for the combination of the independent variables given in Table2.Experimental runs were randomized to minimize the effects of unexpected variability Table1Independent variables and their levels used in the response surface design. Independent variables Levels−101Extraction time(X1)(min)304050 Ratio of water to raw material(X2)304050 Ultrasonic power(X3)(W)280320360in the observed responses.The variables were coded according to the equationXi=X i−X0X(1) where X i is the(dimensionless)coded value of the variable X i,X0is the value of X i at the center point,and X is the step change.The behavior of the system was explained by the following quadratic equation:Y=A0+3i=1A i X i+3i=1A ii X2i+2i=13j=i+1A ij X ij(2)where Y is the dependent variable,A0is constant,and A i,A ii,and A ij are coefﬁcients estimated by the model.X i,X j are levels of the independent variables.They represent the linear,quadratic, and cross-product effects of the X1,X2,and X3factors on the response,respectively.The model evaluated the effect of each independent variable to a response.Analysis of the experimen-tal design and calculation of predicted data were carried out by using SAS JMP Software(version8.0)to estimate the response of the independent variables.Subsequently,three additional conﬁr-mation experiments were conducted to verify the validity of the statistical experimental strategies.3.Results and discussion3.1.Effect of extraction time on extraction yield of polysaccharidesThe effect of extraction time on extraction yield of polysaccha-rides from C.pilosula was shown in Fig.1.Firstly,the extraction time was set at10,20,30,40,50and60min while other extrac-tion parameters were given as the followings:water volume80mL, ultrasonic power360W.It could be found that the extraction yield increased as extraction time ascended from10to40min,peaked at40min,and then no longer increased when the extraction time exceeded40min(see Fig.1a).Table2Box–Behnken experimental design and results for extraction yield.No.X1(extraction time,min)X2(ratio of water to raw material)X3(ultrasonic power,W)Extraction yield(%)11(50)1(50)0(320)32.5020(40)−1(30)1(360)28.643−1(30)−1(30)0(320)18.3041(50)0(40)1(360)35.2451(50)−1(30)0(320)26.5060(40)1(50)1(360)34.207−1(30)1(50)0(320)26.4580(40)1(50)−1(280)31.4090(40)0(40)0(320)32.16100(40)0(40)0(320)32.22111(50)0(40)−1(280)25.4612−1(30)0(40)1(360)21.35130(40)0(40)0(320)32.1514−1(30)0(40)−1(280)16.85150(40)−1(30)−1(280)22.36Y.Zou et al./Carbohydrate Polymers 84 (2011) 503–50850520.0023.0026.0029.0032.0035.00Ratio of water to raw materialE x t r a c t i o n y i e l d (%)20.0023.0026.0029.0032.0035.00Extraction time (min)E x t r a c t i o n y i e l d (%)20.0023.0026.0029.0032.0035.00Extraction power (W)E x t r a c t i o n y i e l d (%)Fig.1.Effect of different extraction parameters on extraction yield of polysaccha-rides from C.pilosula (extraction time,min;ratio of water to raw material;ultrasonicpower,W).3.2.Effect of ratio of water to raw material on yield of polysaccharidesDifferent ratio of water to raw material could signiﬁcantly affect the extraction yield.If ratio of water to raw material is too small,polysaccharides in raw material cannot be completely extracted up.If the ratio of water to raw material is too high,it will cause high process cost (Govender et al.,2005).In this study,the effect of ratio of water to raw material on extraction yield of polysaccha-rides from C.pilosula was investigated,and the results were listed in Fig.1b.The ratio of water to raw material was set at 10,20,30,40,50and 60while other extraction parameters were given as the followings:ultrasonic power 360W,extraction time 30min.It could be founded that the extraction yield of polysaccharides from C.pilosula continued to increase evidently with the increasing ratio of water to raw material.But the extraction yield of polysaccharides from C.pilosula started to increase slowly after the ratio of water to raw material exceeded 40(Fig.1b).3.3.Effect of ultrasonic power on yield of polysaccharidesIn this experiment,the efﬁciencies of different ultrasonic power on the yield of polysaccharides from C.pilosula were investigated,and the results were listed in Fig.1c.Firstly,the other extraction conditions such as water volume,extraction time,were ﬁxed atTable 3Regression coefﬁcients of the predicted quadratic polynomial model.Parameter Estimate Standard error t ratio P -value Prob.>F X 1 4.593750.5458028.420.0004a X 2 3.593750.545802 6.580.0012a X 32.920.545802 5.350.0031a X 1*X 2−0.53750.771881−0.700.5172X 1*X 3 1.320.771881 1.710.1479X 2*X 3−0.870.771881−1.130.3109X 1*X 1−5.3320830.803399−6.640.0012a X 2*X 2−0.9070830.803399−1.130.3101X 3*X 3−2.1195830.803399−2.640.0461aaMeans signiﬁcance (values of “Prob.>F ”<0.0500).80mL,30min respectively and only ultrasonic power was changed.As shown in Fig.1c,the extraction yield of polysaccharides from C.pilosula continued to increase with the increasing of ultrasonic power and reached the peak value (33.30%)when ultrasonic power was 320W.The extraction yield of polysaccharides from C.pilosula no longer increased when the ultrasonic power exceeded 320W.3.4.Optimization of the extraction parameters of polysaccharides3.4.1.Statistical analysis and the model ﬁttingTable 2shows the process variables and experimental data.The results of the analysis of variance,goodness-of-ﬁt and the adequacy of the models were summarized.The percentage yield ranged from 16.85%to 35.24%.The maximum yield of polysaccharides (35.24%)was recorded extraction time 50min,ratio of water to raw material 40and ultrasonic power 360W.The application of RSM suggested,based on parameter estimates,an empirical relationship between the response variable (extraction yield of polysaccharides)and the test variables under consideration.By applying multiple regression analysis on the experimental data,the response variable and the test variables are related by the following second-order polynomial equation:Y =32.1776+4.5938X 1+3.5938X 2+2.92X 3−0.5375X 1X 2+1.32X 1X 3−0.87X 2X 3−5.3221X 21−0.9071X 22−2.1196X 23(3)The determination coefﬁcient (R 2=0.9751)was showed by ANOVA of the quadratic regression model,indicating that only 2.49%of the total variations were not explained by the model.The value of the adjusted determination coefﬁcient (adjusted R 2=0.9304)also conﬁrmed that the model was highly signiﬁcant.At the same time,a very low value 5.57of coefﬁcient of the vari-ation (CV)clearly indicated a very high degree of precision and a good deal of reliability of the experimental values.The model was found to be adequate for prediction within the range of experi-mental variables.The regression coefﬁcient values of Eq.(3)were listed in Table 3.The P -values were used as a tool to check the signiﬁcance of each coefﬁcient,which in turn might indicate the pattern of the interactions between the variables.The smaller the value of P was,the more signiﬁcant the corresponding coefﬁcient was.It can be seen from this table that the linear coefﬁcients (X 1,X 2,X 3),a quadratic term coefﬁcient (X 21,X 23)were signiﬁcant,with very small P -values (P <0.05).The other term coefﬁcients were not signiﬁcant (P >0.05).The full model ﬁlled Eq.(3)was made three-dimensional and contour plots to predict the relationships between the independent variables and the dependent variables.3.4.2.Optimization of extraction conditionsResponse surfaces were plotted by using SAS JMP (version 8.0)software to study the effects of parameters and their interac-506Y.Zou et al./Carbohydrate Polymers84 (2011) 503–508Fig.2.Contour plot and response surface plot of Y =f 1(X 1,X 2).tions on polysaccharides yield.Three-dimensional response surface plots and two-dimensional contour plots,as presented in Figs.2–4,were very useful to judge interaction effects of the factors on the responses.These types of plots show effects of two factors on the response at a time.In all the presented ﬁgures,the other one factor was kept at level zero.The 3-D plot and the contour plot in Fig.2,which gives the ultrasonic power (0level),shows that extraction yield of polysaccharides increased with increasing of ratio of water to raw material,extraction time from 30to 44.1min,but beyond 44.1min,extraction yield of polysaccharides decreased with increasing extraction time.Fig.3shows the 3-D plot and the contour plot at varying extraction time and ultrasonic power at ﬁxed ratio of water to raw material (0level).From Fig.3,it can be seen that maximum extraction yield of polysaccharides can be achieved when extraction time and ultrasonic power were 44.1min and 339.2W,respectively.The extraction yield of polysaccharides increased evidently with increasing of extraction time from 30to 44.1min,but beyond 44.1min,extraction yield of polysaccha-rides decreased as extraction time ascended.The extraction yield of polysaccharides increased evidently with increasing of extraction time from 320to 339.2W,but beyond 339.2W,the extraction yield of polysaccharides descended with increasing ultrasonic power.The 3-D plot and the contour plot based on independent variables ratio of water to raw material and ultrasonic power were shown in Fig.4,while the extraction time was kept at a zero level.An increase in the extraction yield of polysaccharides could be signiﬁcantly achieved with the increases of ratio of water to raw material.It was obvious that the extraction yield of polysaccharides was increased with the increasing ultrasonic power from 320to 339.2W,mean-ing that further increases of ultrasonic power would not increase the extraction yield of polysaccharides.According to Figs.2–4,and above single parameter study,it can be concluded that optimal extraction condition of polysaccha-rides from C.pilosula were extraction time 44.1min,ratio of water to raw material 56,ultrasonic power 339.2W.Among the three extraction parameters studied,the extraction time was themostFig.3.Contour plot and response surface plot of Y =f 2(X 1,X 3).Y.Zou et al./Carbohydrate Polymers84 (2011) 503–508507Fig.4.Contour plot and response surface plot of Y=f3(X2,X3).Table4Predicted and experimental values of the responses at optimum and modiﬁedconditions.Extraction time(min)Ratio ofwater tomaterialUltrasonicpower(W)Yield ofpolysaccharidesOptimum conditions44.156.3339.236.745(predicted)Modiﬁed conditions445632036.264±0.067%(actual)signiﬁcant factor to affect the extraction yield of polysaccharides from C.pilosula,followed by ultrasonic power and ratio of water to raw material according to the regression coefﬁcients signiﬁcance of the quadratic polynomial model(Table3)and gradient of slope in the3-D response surface plot(Figs.2–4).3.5.Veriﬁcation of the modelsThe suitability of the model equation for predicting the opti-mum response values was tested by using the selected optimal conditions.The maximum predicted yield and experimental yield of C.pilosula polysaccharides were given in Table4.Additional experiments by using the predicted optimum conditions for polysaccharides extraction were carried out:extraction time of 44.1min,ratio of water to material56.3mL/g,ultrasonic power of 339.2W,and the model predicted a maximum response of36.745%. To ensure the predicted result was not biased toward the practi-cal value,experiment rechecking was performed by using these modiﬁed optimal conditions:extraction time of44min,ratio of water to material56mL/g,ultrasonic power of320W.A mean value of36.264±0.067%(N=3)was gained,obtained from real experi-ments,demonstrated the validation of the RSM model.The results of analysis conﬁrmed that the response model was adequate for reﬂecting the expected optimization(Table4),and the model of Eq.(3)was satisfactory and accurate.4.ConclusionsUltrasonic technology was performed for the polysaccharides extraction from C.pilosula in order to increase the yield extraction. Based on the single-factor experiments,Response surface method-ology(RSM)was used to estimate and optimize the experimental variables:extraction time(min),ratio of water to raw material and ultrasonic power(W).All the independent variables,quadratic of extraction time and ultrasonic power had highly signiﬁcant effects on the response values.The optimal extraction conditions for the polysaccharides were determined as follows:extraction time of 44min,ratio of water to material56,ultrasonic power of320W. Under these conditions,the experimental yield of polysaccharides was36.264±0.067%,which was closed with the predicted yield value.AcknowledgementWe gratefully acknowledge theﬁnancial support by Excellent Master Thesis Training Foundation by Sichuan Agriculture Univer-sity of P.R.China.ReferencesChina Pharmacopoeia Committee.(2010).Pharmacopoeia of People’s Republic of China.Beijing:Chemical Industry Press.,pp.264–265.Gan,C.-Y.,Abdul Manaf,N.H.,&Latiff,A.A.(2010).Optimization of alcohol insoluble polysaccharides(AIPS)extraction from the Parkia speciosa pod using response surface methodology(RSM).Carbohydrate Polymers,79,825–831.Govender,S.,Pillay,V.,Chetty,D.J.,Essack,S.Y.,Dangor,C.M.,&Govender,T.(2005).Optimization and characterization of bioadhesive controlled release tetracycline micro spheres.International Pharmacy Journal,306,24–40.Guo,X.,Zou,X.,&Sun,M.(2010).Optimization of extraction process by response surface methodology and preliminary characterization of polysaccharides from Phellinus igniarius.Carbohydrate Polymers,80,344–349.Han,F.,Cheng,L.,&Chen,Y.(2005).Study on isolation and composition of Codonopsis tangshen polysaccharides.Chinese Pharmaceutical Journal,40,1381–1383. Hou,X.,&Chen,W.(2008).Optimization of extraction process of crude polysac-charides from wild edible BaChu mushroom by response surface methodology.Carbohydrate Polymers,72,67–74.Hromadkova,Z.,&Ebringerova,A.(2003).Ultrasonic extraction of plant materi-als investigation of hemicellulose release from buck wheat hulls.Ultrasonics Sonochemistry,10,127–133.Li,G.,&Yang,S.(2001).Extraction of Codonopsis pilosula polysaccharide and its effects of anti-active oxygen free radicals.Chemical World,8,421–423. Masuko,T.,Minami,A.,Iwasaki,N.,Majima,T.,Nishimura,S.-I.,&Lee,Y.C.(2005).Carbohydrate analysis by a phenol–sulfuric acid method in a microplate format.Analytical Biochemistry,339,69–72.Sun,Y.,Li,T.,Yan,J.,&Liu,J.(2010).Technology optimization for polysaccharides (POP)extraction from the fruiting bodies of Pleurotus ostreatus by Box–Behnken statistical design.Carbohydrate Polymers,80,242–247.Sun,Y.,&Liu,J.(2008).Structural characterization of a water-soluble polysaccharide from the roots of Codonopsis pilosula and its immunity activity.International Journal of Biological Macromolecules,43,279–282.Sun,Y.,Liu,J.,&Kennedy,J.F.(2010).Application of response surface methodol-ogy for optimization of polysccharides production parameters from the roots508Y.Zou et al./Carbohydrate Polymers84 (2011) 503–508of Codonopsis pilosula by a central composite design.Carbohydrate Polymers,80, 949–953.Vinatoru,M.,Toma,M.,Radu,O.,Filip,P.I.,Lazurca,D.,&Mason,T.J.(1997).The use of ultrasonic for the extraction of bioactive principles from plant materials.Ultrasonics Sonochemistry,4,135–139.Wang,Y.,Cheng,Z.,Mao,J.,Fan,M.,&Wu,X.(2009).Optimization of ultrasonic-assisted extraction process of Poria cocos polysaccharides by response surface methodology.Carbohydrate Polymers,77,713–717.Yang,G.,Li,F.,Liu,H.,&Xian,F.(2005).Effects of polysaccharides from Radix Codonopsis on immune function in mice.Pharmacology and Clinics of Chinese Material Medical,21,39–40.Zhao,H.,Wang,J.,&Lu,Z.(2009).Optimization of process parameters of the Pho-liota squarrosa extra cellular polysaccharide by Box–Behnken statistical design.Carbohydrate Polymers,77,677–680.Zhong,K.,&Wang,Q.(2010).Optimization of ultrasonic extraction of polysaccha-rides from dried longan pulp using response surface methodology.Carbohydrate Polymers,80,19–25.Zhang,Y.,Zhang,L.,Yang,J.,&Liang,Z.(2010).Structure analysis of water-soluble polysaccharide CPPS3isolated from Codonopsis pilosula.Fitoterapia,81, 157–161.Zhang,X.,Zhu, C.,Hu,L.,Lai,X.,&Mo,J.(2003).Pharmacological action of polysaccharides from Radix Codonopsis on immune function and hematopoiesis in mice.Traditional Chinese Drug Research Clinical Pharmacology,14,174–176.Zhu,T.,Heo,H.J.,&Row,K.H.(2010).Optimization of crude polysaccharides extrac-tion from Hizikia fusiformis using response surface methodology.Carbohydrate Polymers,82,106–110.。

板栗壳中提取总黄酮的论文

本科毕业论文（实验）( 2016 届)题目：响应面优化超声法提取板栗壳中总黄酮及抗氧化性应用学院：化学化工学院专业：化学工程与工艺学生姓名：孙素英学号：21207052044指导教师：潘乐职称（学位）：讲师（博士）合作导师：职称（学位）：完成时间：2016 年3月20日成绩：黄山学院教务处制学位论文原创性声明兹呈交的学位论文，是本人在指导老师指导下独立完成的研究成果。

本人在论文写作中参考的其他个人或集体的研究成果，均在文中以明确方式标明。

本人依法享有和承担由此论文而产生的权利和责任。

声明人（签名）：年月日目录响应面优化超声法提取板栗壳中总黄酮及抗氧化性作用化学化工学院化学工程与工艺孙素英（21207052044）指导老师：潘乐（讲师）摘要：摘要利用响应面分析法优化板栗壳总黄酮的提取工艺。

方法:固定料液比为i:20以乙醇浓度、提取温度及提取时间为响应因子，总黄酮提取得率为响应值，实施3因素3水平的响应面分析，建立数学模型，并得出最佳工艺条件。

最终利用响应面分析法获得的提取板栗壳总黄酮的最佳工艺条件为:乙醇浓度为46%，提取温度为50，提取时间为3h该条件下提取2次，板栗壳总黄酮的得率可达19.66%。

关键词：响应面分析法;板栗壳;总黄酮The response surface optimization method of ultrasonic extraction of flavonoids in the chestnut shell andoxidation resistanceSun Suying Director: Pan Le(School of Chemistry and Chemical Engineering, Huangshan University) Abstract: By using the response surface analysis to optimize extraction technology of total flavonoids chestnut shell. Methods: the fixed ratio of material liquid I: 20 for ethanol concentration, extraction temperature and extraction time response factor, total flavonoids obtained rate in response to a value, the implementation of 3 factors 3 levels of response surface analysis, mathematical model is established, and optimum technological conditions are obtained. Eventually using response surface analysis method for the extraction of the optimum process conditions of total flavonoids of Chinese chestnut shell: 46% ethanol concentration, extraction temperature is 50, the extraction time is 3 h under the condition of the extraction twice, chestnut shell yield could reach 19.66% of total flavonoids.Keywords: The response surface method; Chestnut shell; general flavone1 引言1.1板栗1.1.1板栗的介绍板栗（学名：Castaneamollissima），又名栗、板栗、栗子、风栗，是壳斗科栗属的植物，原产于中国，分布于越南、台湾以及中国大陆地区，生长于海拔370－2800米的地区，多见于山地，已由人工广泛栽培。

超声波-微波辅助提取杜仲叶多糖工艺优化及其体外抗凝血活性分析

陈艳萍，贺菊萍，刘意，等. 超声波-微波辅助提取杜仲叶多糖工艺优化及其体外抗凝血活性分析[J]. 食品工业科技，2023，44（17）：202−211. doi: 10.13386/j.issn1002-0306.2022100189CHEN Yanping, HE Juping, LIU Yi, et al. Optimization of Ultrasonic-Microwave Assisted Extraction of Polysaccharides from Eucommia ulmoides Leaves and Its Anticoagulant Activity in Vitro [J]. Science and Technology of Food Industry, 2023, 44(17):202−211. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022100189· 工艺技术 ·超声波-微波辅助提取杜仲叶多糖工艺优化及其体外抗凝血活性分析陈艳萍1，贺菊萍2，刘　意3，杨万根1,3,*（1.吉首大学林产化工工程湖南省重点实验室，湖南张家界 427000；2.徐州工程学院江苏省食品资源开发与质量安全重点建设实验室，江苏徐州 221018；3.吉首大学食药两用资源研究与高值化利用湖南省重点实验室，湖南吉首 416000）摘　要：为开发我国丰富的杜仲叶资源，研究超声波-微波辅助提取杜仲叶多糖的最优工艺条件及其理化性质和体外抗凝血活性。

首先通过单因素实验确定超声波功率、提取温度、微波功率、料液比、提取时间等影响多糖得率因素的范围，然后通过Plackett-Burman 试验筛选关键影响因素，再采用Box-Behnken 试验对工艺条件进行优化，分析了所得杜仲叶精制多糖的分子量、单糖组成等理化性质及活化部分凝血酶原时间（APTT ）、凝血酶原时间（PT ）、凝血酶时间（TT ）等抗凝血指标。

Optimization of ultrasonic-assisted extraction process of Poria cocos polysaccharides by

Optimization of ultrasonic-assisted extraction process of Poria cocos polysaccharides by response surface methodologyWang Yongjiang a,*,Cheng Zhong a ,Mao Jianwei a ,Fan Minger a ,Wu Xueqian b,*aKey Laboratory of Agricultural Products Chemical and Biological Processing Technology of Zhejiang Province,School of Biological and Chemical Engineering,Zhejiang University of Science and Technology,Hangzhou 310023,PR China bZhejiang Province Academy of Forestry,Hangzhou 310023,PR Chinaa r t i c l e i n f o Article history:Received 20October 2008Received in revised form 5February 2009Accepted 17February 2009Available online 21February 2009Keywords:Poria cocos polysaccharides ExtractionCentral composite design OptimizationResponse surface methodology Correlationa b s t r a c tPolysaccharides production from Poria cocos was carried out using aqueous NaOH with the assistance of ultrasonic.Experimental design was used to investigate the effect of three parameters (extraction time,extraction concentration of NaOH,and ratio of aqueous NaOH to raw material)on polysaccharides yields.The ranges of the factors investigated were 1–3min for extraction time (X 1),0.5–1.0mol/L for extraction concentration of NaOH (X 2),and 30–50for ratio of aqueous NaOH to raw material (X 3).The statistical analysis of the experiment indicated that extraction concentration of NaOH had signiﬁcant effect on P.cocos polysaccharides yields.The central composite design showed that polynomial regression models were in good agreement with the experimental results with the coefﬁcients of determination of 0.9935for P.cocos polysaccharides yield.The optimal condition for P .cocos polysaccharides yield within the experimental range of the variables studied was at 2.44min,0.789mol/L,and 53.0.At this condition,the predicted yield of polysaccharides extracted was 82.3%.Ó2009Elsevier Ltd.All rights reserved.1.IntroductionPoria cocos (Fuling),a fungus that grows on the roots of pine trees,is one of the most important traditional medicines in China and other Asian countries,and has many culinary and medical uses such as anti-inﬂammatory,antitumor,complement activating,and immune stimulating activities (Kanayama,Adachi,&Togami,1983;Lee &Jeon,2003;Yasukawa et al.,1998;Yu &Tseng,1996).P.cocos sclerotium contains 93%dry weight of polysaccha-rides in which the largest chemical component is D -glucose (Wang et al.,2004).Poria cocos polysaccharides (PCP)have attracted con-siderably attention in the ﬁelds of biochemistry and pharmacology because of their biological activities (Chihara,Hamuro,Maeda,Aria,&Fukuoka,1970;Kanayama et al.,1983;Lee et al.,2004).Conventional techniques for solvent extraction of PCP have usu-ally been based on correct choice of solvents and use of heat and/or agitation to increase the solubility of the compounds and the rate of mass transfer (Wang et al.,2004).They usually require long extraction times and extraction efﬁciency is low.Ultrasonic-as-sisted extraction (UAE)has been widely used to isolate bioactive substances from different parts of plants.Ultrasonic enhancement of extraction is attributed to disruption of cell walls,particle-size reduction,and enhanced mass transfer of the cell contents as a re-sult of cavitation bubble collapse (Ma,Chen,&Hui,1989;Vinatoru et al.,1997).Recent studies have shown that UAE with organic sol-vents or water is an alternative means of increasing the speed of sample extraction (Gimeno,Marcé,&Borrull,2004;Schmeck &Wenclawiak,2005).The objective of this study was to investigate the signiﬁcant variables (extraction time,concentration of NaOH,and ratio of aqueous NaOH to raw material),and further to optimize the levels of the extraction variables,for PCP production by employ-ing response surface methodology (RSM).To the best of our knowledge,there were no reports of systematic studies of ultra-sonic-assisted extraction process of PCP in the media of aque-ous alkali.2.Materials and methods 2.1.Materials and equipmentsPoria cocos was purchased from Zhenan farm produce market,then ground to pass through 60mesh screen and stored at 60°C in loft drier.Acetone and ethylacetate were obtained from Quzhou Chemical Co.(Quzhou,China).Ethanol was obtained from Hangz-hou Shuanglin Reagent Co.(Hangzhou,China).All other chemicals used in the experiment were of analytical grade.The extraction procedure was carried out in the ultrasonic washing equipment (LABUY-10LHT Hangzhou LABUY Company,Hangzhou,China).0144-8617/$-see front matter Ó2009Elsevier Ltd.All rights reserved.doi:10.1016/j.carbpol.2009.02.011*Corresponding authors.Tel.:+8657185070375;fax:+8657185070370(Y.J.Wang).E-mail address:river0301@ (Y.J.Wang).Carbohydrate Polymers 77(2009)713–717Contents lists available at ScienceDirectCarbohydrate Polymersj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c a r b p ol2.2.Preparation of P.cocos polysaccharidesThe procedure for P.cocos polysaccharides extracts was carried out consulting the scientiﬁc literature on this subject(Wang et al., 2004).Fresh sclerotium of P.cocos cultivated in Lishui(Zhejiang,Chi-na)was peeled,and the white body of the sclerotium was dried and powdered.The powder was defatted by Soxhlet extraction with EtOAc for6h and acetone for6h and extracted in water to re-move soluble polysaccharides.Then the resulting residue was im-mersed in aqueous NaOH and extracted with the assistance of ultrasonic for some time.The extracted liquid fraction was col-lected,concentrated,decolorized with30%H2O2,and deproteinat-ed by the Sevage method10times and then dialyzed(regenerated cellulose tubing;Mw cut-off8000,USA)against tap water for5 days and distilled water for4days.The polysaccharide examined by UV spectroscopy(UV-160,Shimadzu,Japan)showed a main peak at200nm for polysaccharide,no absorption peaks at 280nm for protein and600nm for pigment.The polysaccharides wereﬁnally lyophilized(Christ Alpha1-2,Germany)to obtain a white powder and weighted.2.3.Experimental designThe extraction parameters were optimized using RSM.The cen-tral composite design(CCD)was employed in this regard.The range and center point values of three independent variables pre-sented in Table1were based on the results of preliminary D in the experimental design consists of twelve factorial points and three replicates of the central point(Table2).Extraction time(X1),concentration of NaOH(X2),and ratio of aqueous NaOH to raw material(X3)were chosen for independent variables.Yield of polysaccharides was selected as the response for the combina-tion of the independent variables given in Table2.Experimental runs were randomized to minimize the effects of unexpected var-iability in the observed responses.The variables were coded according to the equationx i¼ðX iÀX0Þ=D Xð1Þwhere x i is the(dimensionless)coded value of the variable X i,X0is the value of X i at the center point,and D X is the step change.The behavior of the system was explained by the following quadratic equation:Y¼A0þX3i¼1A i X iþX3i¼1A ii X2iþX2i¼1X3j¼1þ1A ij X i X jð2Þwhere Y is the dependent variable(yield of polysaccharides in real value),A0is constant,and A i,A ii,and A ij are coefﬁcients estimated by the model.X i,X j are levels of the independent variables.They repre-sent the linear,quadratic,and cross-product effects of the X1,X2, and X3factors on the response,respectively.The model evaluated the effect of each independent variable to a response.Analysis of the experimental design and calculation of predicted data were car-ried out using MATLAB Software(version7.0)to estimate the re-sponse of the independent variables.Subsequently,three additional conﬁrmation experiments were conducted to verify the validity of the statistical experimental strategies.3.Result and discussion3.1.Effect of different time on extraction yield of PCPExtraction time is a factor that would inﬂuence the extraction efﬁciency and selectivity of theﬂuid.A longer extraction time also presents a positive effect on the yield of polysaccharides.It was re-ported that a long extraction time favors the production of polysac-charides(Hou&Chen,2008;Liang,2008).The effect of different time on extraction yield of polysaccharides is shown in Fig.1. Extraction was carried out at different time conditions while other extraction parameters were same to ones.When extraction time varied from1to4min,the variance of extraction yield was rela-tively rapid,and polysaccharides production reached a maximum at4min,and then no longer changed as the extraction proceeded. This indicated that4min was sufﬁcient to obtain the polysaccha-rides production.Thus,4min was favorable for producing the polysaccharides.3.2.Effect of concentration of NaOH on extraction yield of PCPThe concentration of NaOH on extraction yield of polysaccha-rides is shown in Fig.2.Extraction was carried out at different con-centration of NaOH(0.25–1.25mol/L)conditions while other extraction parameters were same to ones described in Section 3.1.The extraction yields of the polysaccharides signiﬁcantly in-creased from32%to78.8%as the concentration of NaOH increased from0.25to0.75mol/L.However,when the concentration contin-ued to increase,the extraction yields of the polysaccharides de-creased,due to partly degradation of the polysaccharides(Luo, Xiao,&Wang,2007).3.3.Effect of different ratio of aqueous alkali to raw material on extraction yield of PCPThe effect of different ratio of aqueous alkali to raw material on extraction yield of polysaccharides is shown in Fig.3.Extraction was carried out at different ratio of aqueous alkali to raw material (10–50)conditions while other extraction parameters were same to ones described in Section3.1.The extraction yields of the poly-saccharides signiﬁcantly increased from31%to78.8%as the ratio increased from10to40shown in Fig.3,due to the increase of the driving force for the mass transfer of the polysaccharides (Bendahou,Dufresne,Kaddami,&Habibi,2007).However,when the ratio continued to increase,the extraction yields no longer changed.Experimental results also showed that most PCP were extracted when the number of extraction was only one.Besides,the inﬂu-ence of ultrasonic power to the yield of polysaccharides was not obvious,but the yield of polysaccharides achieved maximum using the ultrasonic power300W(100%).The extraction temperature was kept at room temperature.3.4.Optimization of the yield of the polysaccharides extractTable2shows the process variables and experimental data.The results of the analysis of variance,goodness-of-ﬁt and the ade-quacy of the models are summarized.The percentage yield ranged from41.3%to82.3%.The maximum yield of polysaccharides (82.3%)was recorded under the experimental conditions of ratio of aqueous alkali to raw material50,extraction time3.0min and concentration of NaOH0.75mol/L.The application of RSM offers,Table1Independent variables and their levels used for central composite rotatable design. Independent variables Levels aÀ101Extraction time(X1) 1.0 2.0 3.0 Concentration of NaOH(X2)0.50.75 1.0 Ratio of aqueous NaOH to raw material(X3)304050a x1=(X1À2)/1;x2=(X2À0.75)/0.25;x3=(X3À40)/10.714Y.J.Wang et al./Carbohydrate Polymers77(2009)713–717based on parameter estimates,an empirical relationship between the response variable(extraction yield of polysaccharides)and the test variables under consideration.By applying multiple regression analysis on the experimental data,the response variable and the test variables are related by the following second-order polynomial equation:Y¼À2:2391þ0:1517X1þ6:4345X2þ0:0128X3À0:02300X21À3:9560X22À0:00008X23À0:0050X1X2À0:00067X1X3À0:0034X2X3ð3ÞThe correlation measure for testing the goodness-of-ﬁt of the regression equation is the adjusted determination coefﬁcient (R2Adj).The value of R2Adj(0.98)for Eq.(2)is reasonably close to1,and indicates a high degree of correlation between the ob-served and predicted values.A very low value of coefﬁcient of the variation(C.V.)(0.0681)clearly indicated a very high degree of precision and a good deal of reliability of the experimental values.Statistical testing of the model was performed in the form of analysis of variance(ANOVA),which is required to test the signiﬁcance and adequacy of the model.The data showed a goodﬁt with the Eq.(3),which were statistically acceptable at P<0.05level and adequate with satisfactory R2value (R2=0.99).Response surfaces were plotted using METLAB version7.0soft-ware to study the effects of parameters and their interactions on polysaccharides yield.Three-dimensional response surface plots and two-dimensional contour plots,as presented in Figs.4–6,areTable2Central composite arrangement for independent variables and their response.No.X1(extraction time,min)X2(concentration of NaOH,mol/L)X3(ratio of aqueous NaOH to raw material)Extraction yield(%)1À1(1.0)À1(0.5)0(40:1)41.32À1(1.0)0(0.75)À1(30:1)67.73À1(1.0)0(0.75)1(50:1)75.24À1(1.0) 1.0(1)0(40:1)59.150(2.0)À1(0.5)À1(30:1)42.060(2.0)À1(0.5)1(50:1)46.670(2.0) 1.0(1)À1(30:1)61.680(2.0) 1.0(1)1(50:1)62.891(3.0)À1(0.5)0(40:1)44.7101(3.0)0(0.75)À1(30:1)77.5111(3.0)0(0.75)1(50:1)82.3121(3.0) 1.0(1)0(40:1)62.0130(2.0)0(0.75)0(40:1)78.8140(2.0)0(0.75)0(40:1)78.8150(2.0)0(0.75)0(40:1)78.8Y.J.Wang et al./Carbohydrate Polymers77(2009)713–717715Fig.4.Contour plot and response surface plot of Y =f 1(X 1,X 2).Fig.5.Contour plot and response surface plot of Y =f 2(X 1,X 3).Fig.6.Contour plot and response surface plot of Y =f 3(X 2,X 3).716Y.J.Wang et al./Carbohydrate Polymers 77(2009)713–717very useful to see interaction effects of the factors on the re-sponses.These types of plots show effects of two factors on the re-sponse at a time.In all the presentedﬁgures,the other one factor was kept at level zero.As expected,a greater increase in polysaccharides yield resulted when the extraction concentration of NaOH(X2)was increased in the range from0.25to0.75mol/L,then the concentration of NaOH curve started to go down at0.75mol/L,which may indicate that a concentration of NaOH of0.75mol/L(X2)is required to achieve maximum increase(Fig.4).Likewise,an increase in polysaccha-rides yield resulted when the extraction time(X1)was increased in the range from1to3min.The time curve did not level off at 3min,which may indicate that a slightly more time is required to achieve maximum increase(Fig.4).As in the case of polysaccharides extract,extraction time(X1) and ratio of aqueous NaOH to raw material(X3)used both had a positive impact on the polysaccharides production.There was a linear increase in the yield of polysaccharides with increase in the extraction time(X1)and ratio of aqueous NaOH to raw material (X3)(Fig.5).The contours were slightly inclined to the horizontal showing that there was a signiﬁcant interaction between the two parameters.Thus,it may be said that a higher level of extraction time(X1)and ratio of aqueous NaOH to raw material(X3)is re-quired to achieve maximum increase of polysaccharides yield (Fig.5).The3D response surface based on independent variables extrac-tion concentration of NaOH(X2)and ratio of aqueous NaOH to raw material(X3)was shown in Fig.6,while the extraction time(X1) was kept at a zero level.An increase in yield was observed with in-crease in concentration of NaOH(X2)from0.5to0.75mol/L,which was in good agreement to the above mention single-factor test (Fig.2).A single parameter study would overlook this entity.An interaction of extraction concentration of NaOH(X2)and ratio of aqueous NaOH to raw material(X3)was no obvious as time was a factor that inﬂuenced the yield of polysaccharides.It was obvious that the yield of polysaccharides was increase with the increase in extraction ratio of aqueous NaOH to raw material(X3)from30to 50,meaning that a larger extraction ratio of aqueous NaOH to raw material(X3)is required to achieve maximum increase of the yield of polysaccharides(Fig.6).3.5.Validation of the modelsIn order to validate the adequacy of the model equations(Eq.(3)),a veriﬁcation experiment was carried out under the optimal conditions(within the experimental range):extraction time 2.44min,extraction concentration of NaOH0.789mol/L,ratio of aqueous NaOH to raw material53.0.Under the optimal conditions, the model predicted a maximum response of82.3(%).To ensure the predicted result was not biased toward the practical value, experimental rechecking was performed using this deduced opti-mal condition.A mean value of81.4±1.09(%)(N=3),obtained from real experiments,demonstrated the validation of the RSM model.The good correlation between these results conﬁrmed that the response model was adequate for reﬂecting the expected opti-mization.The results of analysis indicated that the two groups of experimental values were in good agreement with the predicted one,and also suggested that the model of Eq.(3)are satisfactory and accurate.4.ConclusionThe performance of the ultrasonic-assisted extraction of poly-saccharides from P.cocos using aqueous NaOH was studied with a statistical method based on the response surface methodology in order to identify and quantify the variables which may maxi-mize the yield of polysaccharides.The three variables chosen, namely extraction time,extraction concentration of NaOH,and ra-tio of aqueous NaOH to raw material all have inﬂuence on the yield of polysaccharides using the extraction method.The optimal con-ditions obtained by RSM for production of polysaccharides include the following parameters:extraction time2.44min,concentration of NaOH0.789mol/L,and ratio of aqueous NaOH to raw material 53.0.AcknowledgementThis project isﬁnancially supported by the Science and Tech-nolody Department of Zhejiang Province of the People’s Republic of China(NO.2008C32003).ReferencesBendahou,A.,Dufresne,A.,Kaddami,H.,&Habibi,Y.(2007).Isolation and structural characterization of hemicelluloses from palm of Phoenix dactylifera L..Carbohydrate Polymers,68,601–608.Chihara,G.,Hamuro,J.,Maeda,Y.,Aria,Y.,&Fukuoka, F.(1970).Antitumor polysaccharide derived chemically from natural glucan.Nature,225,943–944. Gimeno,R.A.,Marcé,R.M.,&Borrull,F.(2004).Determination of plasticizers by high-performance liquid chromatography and atmospheric pressure chemical ionization mass spectrometry in water and sediment samples.Chromatographia, 58,37–41.Hou,X.J.,&Chen,W.(2008).Optimization of extraction process of crude polysaccharides from wild edible BaChu mushroom by response surface methodology.Carbohydrate Polymers,72,67–74.Kanayama,H.,Adachi,N.,&Togami,M.(1983).A new antitumor polysaccharide from the mycelia of Poria cocos Wolf.Chemical and Pharmaceutical Bulletin,31, 1115–1118.Lee,K.Y.,&Jeon,Y.J.(2003).Polysaccharide isolated from Poria cocos sclertium induces NF-kB/Rel activation and iNOS expression in murine macrophages.International Immunopharmacology,3,1353–1362.Lee,K.Y.,You,H.J.,Jeong,H.G.,Kang,J.S.,Kim,H.M.,&Dal Rhee,S.(2004).Polysaccharide isolated from Poria cocos sclerotium induces Nfkappa B/Rel activation and iNOS expression through the activation of p38kinase in murine macrophages.International Immunopharmacology,4,1029–1038.Liang,R.J.(2008).Optimization of extraction process of glycyrrhiza glabra polysaccharides by response surface methodology.Carbohydrate Polymers,74, 858–861.Luo,Y.J.,Xiao,X.F.,&Wang,Z.L.(2007).Study on extraction technics of alkaline-soluble polysaccharides in Stenoloma chusana(L.)Ching.Chemical Research and Application,19,195–198(in Chinese).Ma,X.Y.,Chen,Y.Z.,&Hui,R.H.(1989).Analysis of anthraquinones in rheum franzenbachii Münt(rhubarb)by thin-layer chromatography.Chromatographia, 27,465–466.Schmeck,T.,&Wenclawiak, B.W.(2005).Sediment matrix induced response enhancement in the gas chromatographic–mass spectrometric quantiﬁcation of insecticides in four different solvent extracts from ultrasonic and soxhlet extraction.Chromatographia,62,159–165.Vinatoru,M.,Toma,M.,Radu,O.,Filip,P.I.,Lazurca,D.,&Mason,T.J.(1997).The use of ultrasound for the extraction of bioactive principles from plant materials.Ultrasonics Sonochemistry,4,135–139.Wang,Y.F.,Zhang,M.,Ruan,D.,Shashkov,A.S.,Kilcoyne,M.,Savage,A.V.,et al.(2004).Chemical components and molecular mass of six polysaccharides isolated from the sclerotium of Poria cocos.Carbohydrate Research,339, 327–334.Yasukawa,K.,Kaminaga,T.,Kitanaka,S.,Tai,T.,Nunoura,Y.,&Natori,S.(1998).3b-p-Hydroxybenzoyl-dehydrotumulosic acid from Poria cocos,and its anti-inﬂammatory effect.Phytochemistry,48,1357–1360.Yu,S.J.,&Tseng,J.(1996).Fu-Ling,a Chinese herbal drug,modulates cytokine secretion by human peripheral blood monocytes.International Journal of Immunopharmacology,18,37–44.Y.J.Wang et al./Carbohydrate Polymers77(2009)713–717717。

超声波微波双辅助提取荔枝壳总黄酮工艺优化

超声波微波双辅助提取荔枝壳总黄酮工艺优化摘要：以购自泸州市合江县的荔枝（Litchi chinensis）壳为原料，为了优化超声波微波双辅助提取荔枝壳总黄酮工艺，以总黄酮得率为评价指标，在单因素试验基础上，采用正交试验优化提取工艺。

结果表明，采用超声波微波双辅助提取荔枝壳总黄酮的最佳工艺条件为，以60%乙醇作提取剂、料液比为1∶30（m/V，g∶mL）、超声波提取时间为40 min、微波辐射时间为30 s、微波功率为500 W、微波间歇辐射2次，在此工艺条件下，总黄酮得率为38.59 mg/g。

关键词：荔枝（Litchi chinensis）壳；总黄酮；微波；超声波Optimization of Ultrasonic-Microwave Assisted Extraction Process of Total Flavonoids from Litchi Pericarp TU Hua，CHEN Bi-qiong，ZHOU Xi-lanAbstract：Ultrasonic-microwave assisted method were applied to extract total flavonoids from Luzhou litchi（Litchi chinensis）pericarp. The extraction process was optimized by orthogonal design on the basis of single factor tests using the yield of total flavonoids as indicator. Results showed that the optimal extraction conditions were，60% alcohol as extract solution，solid to liquid ratio at 1∶30（m/V，g∶mL），ultrasonic treatment time for 40 min，microwave irradiation time for 30 s，microwave power at 500 W，and microwave irradiation for twice. The extraction yield of total flavonoids from litchi pericarp was 38.59 mg/g.Key words：litchi（Litchi chinensis）pericarp；total flavonoids；ultrasonic extraction；microwave extraction荔枝（Litchi chinensis）盛产于中国广东、广西、福建、四川、台湾等亚热带地区。

超声-酶辅助低共熔溶剂提取桑叶总黄酮的工艺优化及其抗氧化活性研究

吴均，吴俊葶，杨碧文，等. 超声-酶辅助低共熔溶剂提取桑叶总黄酮的工艺优化及其抗氧化活性研究[J]. 食品工业科技，2024，45（3）：31−39. doi: 10.13386/j.issn1002-0306.2023070033WU Jun, WU Junting, YANG Biwen, et al. Optimization of Ultrasonic-Enzyme-Assisted Deep Eutectic Solvents Extraction Process of Total Flavonoids from Mulberry Leaves and Its Antioxidant Activity[J]. Science and Technology of Food Industry, 2024, 45(3): 31−39.(in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2023070033· 特邀主编专栏—食品中天然产物提取分离、结构表征和生物活性（客座主编：杨栩、彭鑫） ·超声-酶辅助低共熔溶剂提取桑叶总黄酮的工艺优化及其抗氧化活性研究吴　均1，吴俊葶2，杨碧文1，王　梅1，赵　珮1，马婧秋1，黄　越1, *，黄传书1,*（1.重庆市蚕业科学技术研究院，重庆 400700；2.中国人民解放军陆军勤务学院，重庆 401331）摘　要：为建立一种绿色高效的桑叶总黄酮提取方法，本研究采用了超声-酶辅助低共熔溶剂法对桑叶总黄酮进行提取。

在单因素实验的基础上，以桑叶总黄酮提取量为响应值，采用Box-Behnken 响应面法对提取工艺进行优化，并研究桑叶总黄酮对 ABTS +和DPPH 自由基的清除能力。

结果表明：在氯化胆碱/果糖/乙醇摩尔比为1:1:3、含水量为30%、液料比为40 mL/g 、超声功率为360 W 、超声温度为40 ℃、超声时间为40 min 、酶添加量为4%条件下，桑叶总黄酮提取量为46.58 mg/g ；当总黄酮质量浓度为0.08 mg/mL 时，对DPPH 自由基清除率为98.36%，当总黄酮质量浓度为0.2 mg/mL 时，对ABTS +自由基清除率为72.12%。

响应面法优化土人参根中总黄酮提取及其抗氧化活性研究

Products Chemical Engineering of Hunan Province, Jishou University, Zhangjiajie 427000, China; 2.College of A&F Engineering and Planning Tongren University, Tongren 554300, China) ABSTRACT: OBJECTIVE To compare in vitro antioxidant activity of flavonoids from the Talinnum paniculatum by different extract methods. METHODS On the basis of the single factors, the effects of liquid-solid ratio, ethanol volume fraction and their interaction on extraction of flavonoids were investigated using Box-behnken design response surface method. The antioxidant activities of three methods used to extract the flavoniods: ultrasonic-assisted, water bath and enzymic methods were compared by the experiments, scavenging capabilities of DPPH·free radical, hydroxyl free radical and superoxide aninoic free radical. RESULTS The optimal extraction conditions were as follows: liquid-solid ratio was 26︰1, ethanol volume fraction was 65% and extraction temperature was 70 ℃. Under the above conditions, the extraction yield of flavonoids were 2.620%, which was in accord with the predicted values 2.603%. The total flavonoids of Talinnum paniculatum extracted by the enzymic method had the best antioxidant activity and better antioxidant activity than Vc. CONCLUSION The target of extracting flavonoids compound efficiently from Talinum has been achieved by the optimized technology. The flavonids extracted using different methods has the significantly different antioxidant activity. KEY WORDS: traditional miao herbs; Talinum paniculatum; flavonoids; extraction; response surface methodology; antioxidant activity

响应面法优化茯苓总三萜超声波提取工艺-方毅

0.727%、0.822%、0.708%、0.510%、0.396%，因此选择
超声时间为 35 min。
2.2.5 提取温度
取茯苓粉末 2 g，精密称定 5 份。分别加入乙酸
乙酯 50 mL，依次在 25、35、45、55 ℃超声 30 min。按
“2.1.2”和“2.1.3”项下方法进行检测，计算总三萜
0.519%。因此选择料液比为 1∶30 作为提取条件。
2.2.4 提取时间
取茯苓粉末 2 g，精密称定 8 份。分别加入乙
酸乙酯 50 mL，依次超声 10、20、25、30、35、40、45、
50 min。按“2.1.2”和“2.1.3”项下方法进行检测，
计算总三萜的得率分别为 0.459%、0.564%、0.645%、
方毅，许凤清，吴德玲，刘梦迪，黄琪，金传山
（安徽中医药大学，安徽合肥 230012）
摘要：目的：优选茯苓总三萜的超声波提取工艺。方法：在单因素考察的基础上，以茯苓中总三萜的得率为
评价指标，采用响应面分析法（选取乙酸乙酯为溶剂，料液比、提取温度、提取时间）确定最佳的超声波辅助提取工
艺。结果：最佳工艺为 30 倍体积的乙酸乙酯，超声时间 32 min，超声温度 35 ℃，茯苓总三萜的得率为 0.685%。验证
取茯苓粉末 2 g，精密称定 8 份，依次加入乙酸乙酯 50 mL，分别用功率为 100、200、300、400、500 W 的超声清洗仪超声 30 min，按“2.1.2”和“2.1.3” 项下方法进行检测，计算总三萜的得率分别为 0.481%、0.485%、0.515%、0.497%、0.483%。因此选择超声功率 300 W 为最佳提取功率。

长心卡帕藻多糖的超声提取工艺优化及其抗过敏活性

吴天翔，李振兴，吴燕燕，等. 长心卡帕藻多糖的超声提取工艺优化及其抗过敏活性[J]. 食品工业科技，2023，44（19）：208−216.doi: 10.13386/j.issn1002-0306.2022110034WU Tianxiang, LI Zhenxing, WU Yanyan, et al. Optimization of Ultrasonic Extraction Process and Anti-allergic Activity of Sulfated Polysaccharides from Kappaphycus alvarezii [J]. Science and Technology of Food Industry, 2023, 44(19): 208−216. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022110034· 工艺技术 ·长心卡帕藻多糖的超声提取工艺优化及其抗过敏活性吴天翔1,2，李振兴1，吴燕燕2,3，杨贤庆2,3，李来好2,3，陈胜军2,3，戚　勃2,3，赵永强2,3,*（1.中国海洋大学食品科学与工程学院，山东青岛 266003；2.中国水产科学研究院南海水产研究所，农业农村部水产品加工重点实验室，广东广州 510300；3.三亚热带水产研究院，海南省深远海渔业资源高效利用与加工重点实验室，海南三亚 572018）摘　要：为获得一种长心卡帕藻多糖的最佳提取工艺并测定抗过敏活性，该研究在单因素实验的基础上，以长心卡帕藻的多糖得率作为考察指标，选择料液比、超声波预处理时间、提取时间进行响应面优化实验，确定最佳工艺条件，并通过DEAE-52纤维素层析柱对提取的粗多糖进行纯化。

随后利用RBL-2H3细胞模型，分别测定纯化后的长心卡帕藻多糖对RBL-2H3细胞活力、细胞脱颗粒抑制及细胞组胺释放水平调节的影响。