CN112707881A - 一种蓝莓花青素的制备方法 - Google Patents
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Abstract
本发明公开了一种蓝莓花青素的制备方法:(1)选材;(2)原料预处理;(3)蓝莓花青素提取;(4)除杂;(5)柱层析纯化;(6)浓缩与干燥。本发明以生产果酱果汁等蓝莓衍生物的废料蓝莓果皮为原料,利用超声辅助亚临界萃取,提高蓝莓花青素的提取效率,并减少蓝莓花青素在提取纯化过程中的降解,所制备的蓝莓花青素纯度高、工艺简单,有利于大规模生产应用。
Description
技术领域
本发明涉及植物活性产物,具体涉及一种从蓝莓果皮中提取、纯化花青素的方法。
背景技术
花青素又称花色素,是一种广泛存在于自然界中的水溶性天然植物色素。作为植物花瓣和果实中主要的呈色物质,花青素的颜色会因细胞液中pH值的改变而发生变化。蓝莓中含有丰富的花青素,蓝莓花青素具有抗氧化、清除自由基、抑制肿瘤细胞增生、防止细胞衰老、改善视力、增加皮肤弹性、降低血压和胆固醇含量、抗炎、预防心血管疾病、改善身体微循环、维护泌尿系统健康等功效,在化妆品、食品、临床医药领域具有重要的作用。
蓝莓的种植面积和产量逐年增加,但蓝莓成熟时间集中,贮藏能力低,鲜销市场能力有限,来不及销售就会腐烂变质,因此,通常加工成果汁、果酒、果酱、乳酸菌饮料、果醋等初级产品。随着蓝莓衍生加工产业的迅速发展,生产过程中生成了大量果渣。作为副产品的蓝莓果渣中保留了多酚类物质和膳食纤维等有效活性成分,其中,当前研究较多的多酚类物质主要存在于蓝莓果皮中。对蓝莓果皮进行合理再利用,不仅可以节约资源,还可避免环境污染,但目前利用率不高是亟待解决的问题。
近年来,随着人们对食品添加剂、功能性食品和可持续食品生产的认识不断提高,消费者对食品更自然、更安全、更具功能性的要求也越来越强烈,蓝莓花青素的提取纯化在在国内外得到了更多的研究。蓝莓花青素的提取有多种方法可供选择,但传统的提取方法(如煎煮法、消化法、浸提法、浸渍法、渗滤法、热连续提取法和逆流提取法)往往费时费力,产生稀释的提取物,导致提取物的降解,并涉及大量溶剂的处理问题。微波辅助萃取、超临界流体萃取、加速溶剂萃取、酶辅助萃取和超声波辅助萃取(USAE)等方法的提取率低。在纯化方面,蓝莓花青素通常采用大孔树脂纯化法,但在纯化过程中花青素容易发生降解,降低了花青素的产率、纯度与活性。另外,在对蓝莓果皮预处理中,干粉是通过通风烘箱获得,与氧气接触后多酚迅速氧化、发生降解,导致花青素得率下降。
中国专利201210278875.9公开了一种“蓝莓果皮提取物的制备方法”,该专利结合使用了酸化乙醇水溶液超声辅助提取、大孔树脂纯化、浓缩及冷冻干燥,但所得提取物中总花青素含量仅达到40%左右。
发明内容
本发明的目的在于提供一种蓝莓花青素的制备方法,可以提高蓝莓花青素的提取、纯化效率。
为达到上述目的,本发明采用了以下技术方案:
1)将冻干的蓝莓果皮粉碎、过筛,得到蓝莓果皮干粉;
2)将蓝莓果皮干粉经脱脂后对蓝莓果皮所含花青素进行超声辅助亚临界萃取(即先进行亚临界萃取,再对萃取残渣进行超声辅助乙醇浸提,合并提取物),得萃取产物;
3)将萃取产物中含有的花青素通过柱层析分离,然后浓缩、干燥,得到蓝莓花青素。
优选的,所述步骤1)中,冻干的蓝莓果皮是通过将蓝莓清洗后剥离果皮,并将果皮经匀浆后真空冷冻干燥而得到的。
优选的,所述步骤1)中,冻干的蓝莓果皮经粉碎后过60~80目筛。
优选的,所述步骤2)中,脱脂采用索氏提取法,具体脱脂条件为:蓝莓果皮干粉采用85%~95%乙醇溶液于50~65℃回流1.5~2.5h。
优选的,所述步骤2)中,超声辅助亚临界萃取具体包括以下步骤:
2.1)将经过脱脂的蓝莓果皮干粉进行2~3次亚临界萃取,得到提取液I,其中,萃取时间为40~60min,萃取温度为50~60℃,料液比为1:20~30;
2.2)在超声辅助下以50%~65%乙醇溶液对步骤2.1)中亚临界萃取的残渣进行浸提,得到提取液II,其中,浸提时间为3~5h,温度为35℃~50℃,料液比为1:13~18;
2.3)将提取液I与提取液II混合后经浓缩、过滤,得到蓝莓花青素粗提液。
优选的,所述步3)具体包括以下步骤:将萃取产物(例如,蓝莓花青素粗提液)上样于大孔吸附树脂层析柱,然后经水洗除杂后洗脱花色苷,将所得洗脱液浓缩,得到花青素浓缩液,将花青素浓缩液真空冷冻干燥,得到蓝莓花青素。
优选的,所述上样的条件为:蓝莓花青素粗提液以每小时1~3倍柱体积的速度流过大孔吸附树脂;所述水洗的条件为:用2~5倍柱体积的经0.02%~0.04%酒石酸溶液酸化的双蒸水以每小时1~3倍柱体积的速度流过大孔吸附树脂,其中,0.02%~0.04%酒石酸溶液与双蒸水的体积比为1:4000~6000;所述洗脱的条件为:用1~3柱体积的经0.02%~0.04%酒石酸溶液酸化的40%~45%乙醇溶液以每小时1~3倍柱体积的速度流过大孔吸附树脂,其中,0.02%~0.04%酒石酸溶液与40%~45%乙醇溶液的体积比为1:4000~6000。
优选的,所述步骤2)及步骤3)中,浓缩的条件为:于50~55℃减压蒸发。
优选的,所述步骤1)及步骤3)中,真空冷冻干燥的条件为:于-20~-25℃预冻12~14h,然后于-55℃~-50℃、0.08~0.09MPa(真空度)条件下冷冻干燥24~48h。
本发明的有益效果体现在:
本发明利用亚临界萃取法保证提取环境低温无氧,可以防止花青素氧化,提高花青素质量,同时,加速萃取剂的渗透,从而减少花青素的降解且提高花青素的提取速率,既提高了经济效益,又减少资源浪费。
进一步的,本发明将蓝莓果皮匀浆后冷冻干燥,温度低且不与氧气接触,减少花青素降解。
进一步的,本发明采用超声辅助亚临界萃取法提取蓝莓果皮中的花青素,显著降低了花青素提取时间,同时,超声辅助提取亚临界萃取的残渣,提高了蓝莓果皮的利用率。
进一步的,本发明在大孔树脂纯化过程中采用酒石酸酸化溶剂(例如,乙醇溶液),避免酰化花青素的分解,维持花青素的稳定,解决了蓝莓花青素纯度低及易降解失活的问题。
具体实施方式
下面结合实施例对本发明作进一步详细说明。
(一)蓝莓果皮中花青素的提取、纯化实例
1.原料选材
采用的原料为工厂加工果酱果汁等蓝莓衍生物的废料蓝莓果皮,通过对蓝莓果皮进行废物利用,提高经济效益、减少资源浪费。
选择表皮完整无缺,且无腐烂现象的蓝莓,清水清洗后用去皮机处理(剥离果皮),得到蓝莓果皮。
2.预处理
将蓝莓果皮在匀浆机中匀浆,倒盘后放入-20℃冰箱中,预冻12h,再于冷冻干燥机中真空冻干48h(真空度0.09MPa,温度-50℃)。将干燥后的蓝莓果皮用中药粉碎机进行粉碎,过80目筛,制成蓝莓果皮干粉。蓝莓果皮干粉中加入3倍体积95%(体积分数)乙醇溶液,50℃水浴下回流脱脂2次,每次2h,脱脂棉过滤,挥干溶剂至恒重,即为预处理样品,室温避光密闭保存,备用。
3.蓝莓花青素提取
称量蓝莓果皮干粉预处理样品并装入筛绢袋内,置于亚临界萃取设备内,设置萃取温度50℃、萃取时间45min,及料液比1:23(g/mL),萃取2次后得到提取液I。然后采用60%(体积分数)的食用乙醇超声辅助浸提亚临界萃取的残渣,其中,料液比为1:15(g/mL),第一次浸提时间为4h(提取温度为35℃),第二次浸提时间4h(提取温度50℃)。浸提后经压滤,合并两次滤液,作为提取液II,合并提取液I、II,再经脱溶塔回收乙醇后于50℃下减压浓缩,制成含花青素的混合浓缩液。
4.除杂
将步骤3所得混合浓缩液在沃特曼415号纸上过滤,弃去滤渣,留下的滤液即为蓝莓花青素粗提液。
5.柱层析纯化
取蓝莓花青素粗提液15~20mL上样AB-8大孔吸附树脂层析柱,其中,蓝莓花青素粗提液以每小时2倍柱体积的速度流过大孔吸附树脂;然后用2倍柱体积的0.02%(质量分数)酒石酸溶液酸化的双蒸水以相同的速度流过大孔吸附树脂,洗去大部分杂质,其中,0.02%酒石酸溶液与双蒸水的体积比为1:5000;再用2倍柱体积的0.02%(质量分数)酒石酸溶液酸化的40%(体积分数)乙醇溶液以相同速度流过大孔吸附树脂,将大孔吸附树脂上吸附的花色苷洗脱下来,其中,0.02%酒石酸溶液与40%乙醇溶液的体积比为1:5000;收集洗脱液。
6.浓缩与干燥
将洗脱液在50℃下减压蒸发以除去乙醇及酒石酸,得到花青素浓缩液,将花青素浓缩液冷冻干燥(在-20℃的冰箱中预冻12h;然后在真空度0.09MPa、温度-50℃的条件下,真空冻干48h),得花青素成品。
(二)对照工艺
对照例1
采用超声辅助乙醇浸提蓝莓果皮花青素:准确称取1.00g蓝莓果皮干粉预处理样品置于提取瓶中,加入一定量的60%乙醇溶液,然后在最佳提取条件下浸提花青素,其中,料液比1:30(g/mL)、提取温度55℃、提取时间30min、pH 1。将所得提取液离心后取上清液,测定花青素含量。其他与以上实例相同。
对照例2
大孔吸附树脂纯化采用盐酸酸化溶剂(盐酸酸化乙醇溶液至pH值为3)。其他与以上实例相同。
(三)蓝莓花青素的检验和比较
在对照例1中,以蓝莓果皮为原料,利用超声辅助乙醇浸提其中的花青素,在最佳条件下花青素的提取率为4.67mg/g,而本发明最佳条件下(实例)花青素提取率为9.96mg/g,提取率显著提高。
当对照例2采用盐酸酸化洗脱液时,采用大孔吸附树脂在最佳工艺条件下纯化花青素,纯化后花青素的质量分数为82.17%,而本发明中采用酒石酸酸化洗脱液,减少了花青素的降解,纯化后花青素质量分数为93.79%,花青素纯度显著提高。
(四)蓝莓花青素的活性
称取清洗干净的蓝莓鲜果,利用组织捣碎机将其捣碎成蓝莓浆,利用超声辅助乙醇浸提其中的花青素。将所得提取液在10000r/min、4℃条件下离心10min,取上清液浓缩、冻干,测定其花青素抗氧化活性。结果表明,当花青素浓度为29.74%时,对·OH的清除作用为92.68%,对DPPH·的清除作用为90.87%,对H2O2的清除作用为85.38%,对超氧阴离子自由基的清除作用为35.40%。
本发明以蓝莓果皮为原料,经提取、纯化所制备的花青素活性更高,当花青素浓度为29.74%时,对·OH的清除作用为95.82%,对DPPH·的清除作用为96.95%,对H2O2的清除作用为95.79%,对超氧阴离子自由基的清除作用为58.37%。
总之,本发明不仅操作简便,而且还使蓝莓果皮细胞释放更多的花青素,提高了蓝莓花青素的提取效率的同时抑制了多酚氧化酶的活性,减少了蓝莓花青素在提取纯化过程中的降解,提高了蓝莓花青素的纯度。本发明通过提取纯化制备蓝莓花青素的工艺具有可操作性强、原料廉价易得等优势,可显著提高企业经济效益,利于产业化。
Claims (9)
1.一种蓝莓花青素的制备方法,其特征在于:包括以下步骤:
1)将冻干的蓝莓果皮粉碎、过筛,得到蓝莓果皮干粉;
2)将蓝莓果皮干粉经脱脂后对所含花青素进行超声辅助亚临界萃取;
3)将萃取产物中含有的花青素通过柱层析分离,然后浓缩、干燥,得到蓝莓花青素。
2.根据权利要求1所述一种蓝莓花青素的制备方法,其特征在于:所述步骤1)中,冻干的蓝莓果皮是通过将蓝莓清洗后剥离果皮,并将果皮经匀浆后真空冷冻干燥而得到的。
3.根据权利要求1所述一种蓝莓花青素的制备方法,其特征在于:所述步骤1)中,冻干的蓝莓果皮经粉碎后过60~80目筛。
4.根据权利要求1所述一种蓝莓花青素的制备方法,其特征在于:所述步骤2)中,脱脂采用索氏提取法。
5.根据权利要求1所述一种蓝莓花青素的制备方法,其特征在于:所述步骤2)中,超声辅助亚临界萃取具体包括以下步骤:
2.1)将经过脱脂的蓝莓果皮干粉进行亚临界萃取,得到提取液I,其中,萃取时间为40~60min,萃取温度为50~60℃,料液比为1:20~30;
2.2)在超声辅助下以50%~65%乙醇溶液对步骤2.1)中亚临界萃取的残渣进行浸提,得到提取液II,其中,浸提时间为3~5h,温度为35℃~50℃,料液比为1:13~18;
2.3)将提取液I与提取液II混合后经浓缩、过滤,得到蓝莓花青素粗提液。
6.根据权利要求1所述一种蓝莓花青素的制备方法,其特征在于:所述步3)具体包括以下步骤:将萃取产物上样于大孔吸附树脂层析柱,然后经水洗除杂后洗脱花色苷,将所得洗脱液浓缩,得到花青素浓缩液,将花青素浓缩液真空冷冻干燥,得到蓝莓花青素。
7.根据权利要求6所述一种蓝莓花青素的制备方法,其特征在于:所述上样的条件为:萃取产物以每小时1~3倍柱体积的速度流过大孔吸附树脂;所述水洗的条件为:用2~5倍柱体积的经0.02%~0.04%酒石酸溶液酸化的双蒸水以每小时1~3倍柱体积的速度流过大孔吸附树脂,其中,0.02%~0.04%酒石酸溶液与双蒸水的体积比为1:4000~6000;所述洗脱的条件为:用1~3柱体积的经0.02%~0.04%酒石酸溶液酸化的40%~45%乙醇溶液以每小时1~3倍柱体积的速度流过大孔吸附树脂,其中,0.02%~0.04%酒石酸溶液与40%~45%乙醇溶液的体积比为1:4000~6000。
8.根据权利要求6所述一种蓝莓花青素的制备方法,其特征在于:所述洗脱液浓缩的条件为:于50~55℃减压蒸发。
9.根据权利要求6所述一种蓝莓花青素的制备方法,其特征在于:所述步骤3)中,真空冷冻干燥的条件为:于-20~-25℃预冻12~14h,然后于-55℃~-50℃、0.08~0.09MPa真空度下冷冻干燥24~48h。
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