CN107519162A - 一种利用凹土提高蓝莓花青素稳定性的方法 - Google Patents
一种利用凹土提高蓝莓花青素稳定性的方法 Download PDFInfo
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Abstract
本发明公开了一种利用凹土提高蓝莓花青素稳定性的方法,高速打浆促进凹土分散,增大凹土的比表面积,再利用湿法研磨实现花青素与凹土的充分结合,得到蓝莓花青素‑凹土混合物。本发明操作简单,成本低,且最大程度保护花青素不受破坏,提高了蓝莓花青素的稳定性和抗氧化活性,具有较好的应用价值和广阔的开发前景。
Description
技术领域
本发明涉及一种提高蓝莓花青素稳定性的方法,具体涉及一种利用凹土吸附性能以提高蓝莓花青素稳定性的方法。
技术背景
蓝莓花青素作为一种天然食用色素,安全、无毒、资源丰富,且具有一定的药理作用和营养价值,在医药、食品、化妆品等方面有着巨大的应用潜力。另外,花青素是目前为止所知晓的最有效的天然自由基清除剂,它可以清除体内过多的自由基,对一系列由自由基过多引起的疾病,如炎症疾病、心血管疾病以及与衰老有关的疾病等均具有预防和改善的作用。但从化学结构来看,花青素具有缺少一个电子的特性,易受到活性氧基团或自由电子的攻击,因此花青素具有较大的不稳定性,易发生降解。
目前,为改善蓝莓花青素的稳定性,多采用改变储存条件、对色素精制提纯、添加辅色素等方法。其中,避光、密封、低温等条件下存储,可改善蓝莓花青素的稳定性,但同时也限制了蓝莓花青素的应用范围;由于蓝莓花青素的不稳定性,很难通过分离提取得到高纯度的蓝莓花青素,且化学合成蓝莓花青素也存在一定的难度;辅色素可以是黄酮、生物碱、有机酸、多糖等,但辅色素的添加在一定程度上也影响蓝莓花青素本身的稳定性。
凹土,全称凹凸棒石黏土,被称为“万土之王”,具有良好的吸附、净化和凝固等性能。作为药物辅料,凹土的显微结构单元为棒晶,其较大的比表面积、可观的孔隙结构和良好的热稳定性使其具有吸附性、悬浮性、分散性、粘结性和触变性,是一种极具开发潜质的药用功能材料。
发明内容:
本发明要解决的技术问题是提供一种利用凹土提高蓝莓花青素稳定性的方法,针对花青素缺少一个电子的特性,利用凹土表面结合位点的原理来提高蓝莓花青素稳定性,以克服花青素不稳定、易降解的缺点。
本发明通过以下技术方案实现:
一种利用凹土提高蓝莓花青素稳定性的方法,以凹土为辅料,利用高速打浆和湿法混合得到蓝莓花青素-凹土混合物,其操作步骤如下:
A: 将凹土粉碎,过200目筛,以水浸润后对辊挤压,再加入去离子水配制成浆料后,在10000~12000 rpm下打浆30~60 min以分散凹土,离心取上层悬液,继续离心上层悬液30min后得到膏体,放入电热鼓风干燥箱中烘干成粉体,备用;
B:将蓝莓花青素与步骤A得到的凹土粉体混合均匀后,加入超纯水充分研磨至水分完全蒸发,即得蓝莓花青素-凹土混合物;
优选地,步骤A中离心转速为1500~3000 rpm;
优选地,步骤A中凹土的粒径大小为60~80µm;
优选地,步骤B中蓝莓花青素与凹土粉体的质量比为1:0.2~2;
更优选地,步骤B中蓝莓花青素与凹土粉体的质量比为1:0.5;
优选地,步骤B中,每1g的蓝莓花青素与凹土混合物与0.2~0.5ml的超纯水混合;
优选地,步骤B中研磨时间为15~30 min。
本发明具有以下优点:
1. 本发明首次利用蓝莓花青素缺少一个电子的特性,与表面带负电荷的凹土形成正负电荷引力,来提高蓝莓花青素的稳定性和抗氧化活性,由此扩大了蓝莓花青素的应用范围。
2. 本发明采用高速打浆促进凹土分散,增大比表面积,采用湿法研磨混合蓝莓花青素与凹土,不仅加大了蓝莓花青素在凹土表面的吸附,使花青素与凹土得到充分结合,提高蓝莓花青素的稳定性,还避免了高温操作对蓝莓花青素的结构破坏,减少蓝莓花青素的损失。
3. 本发明工艺简单,对操作环境要求低,成本低,且环境友好,绿色环保。
4.本发明得到的蓝莓花青素-凹土混合物可直接用于药品、保健品、食品等,与现有技术相比,在添加相同单位的情况下,提高了药品、保健品、食品的有效价值,降低了药品、保健品、食品的制造成本。
附图说明
图1为凹土样品的红外光谱图
图2为蓝莓花青素的红外光谱图
图3为蓝莓花青素-凹土混合物的红外光谱图。
具体实施方式
为使本领域技术人员充分理解本发明的技术方案和有益效果,以下结合附图及具体实施例进一步说明,此处实施例仅用于解释本发明,并不用于限定本发明。
实施例1
A:将凹土粉碎,过200目筛,以水浸润后对辊挤压三次,加入去离子水配制成浆料,在10000 rpm下打浆30 min以分散凹土,1500 rpm下离心15 min,取上层悬液,将上层悬液在3000 rpm下继续离心30 min后得到膏体,放入电热鼓风干燥箱中烘干成粉体,备用。
B:取0.5g步骤A得到的凹土(粒径为70µm)与1g蓝莓花青素混合均匀,加入0.6ml的超纯水充分研磨至水分完全蒸发(约需15 min),得蓝莓花青素-凹土混合物。
实施例2
A:将凹土粉碎,过200目筛,以水浸润后对辊挤压三次,加入去离子水配制成浆料,在10000 rpm下打浆30 min以分散凹土,1500 rpm下离心15 min,取上层悬液,将上层悬液在3000 rpm下继续离心30 min后得到膏体,放入电热鼓风干燥箱中烘干成粉体,备用。
B:取1g步骤A得到的凹土(粒径为70µm)与1g蓝莓花青素混合均匀,加入0.8ml的超纯水充分研磨至水分完全蒸发(约需15 min),得蓝莓花青素-凹土混合物。
实施例3
A:将凹土粉碎,过200目筛,以水浸润后对辊挤压三次,加入去离子水配制成浆料,在10000 rpm下打浆30 min以分散凹土,1500 rpm下离心15 min,取上层悬液,将上层悬液在3000 rpm下继续离心30 min后得到膏体,放入电热鼓风干燥箱中烘干成粉体,备用。
B:取2g步骤A得到的凹土(粒径为70µm)与1g蓝莓花青素混合均匀,加入1.2ml的超纯水充分研磨至水分完全蒸发(约需15 min),得蓝莓花青素-凹土混合物。
性能测定
(1)蓝莓花青素-凹土混合物与蓝莓花青素在酸性溶液中稳定性比较
取醋酸钠5.1g,加冰醋酸20 mL混合后,加水稀释至250mL,即得pH为3.6的缓冲液。将实施例1至实施例3制得的蓝莓花青素-凹土混合物分别溶解于pH3.6的缓冲溶液中,配制成起始最大吸光度均为1.0的溶液,放置于25℃的水浴中,2h、4h、6h、12h、24h、48h、72h后用紫外分光光度计对蓝莓花青素、蓝莓花青素-凹土混合物的溶液进行测定,根据吸光度值保存率A存%比较其稳定性。
注:A0为起始时520 nm下的吸光度;At为在t时520 nm下的吸光度。
表1花青素及花青素-凹土混合样品在酸性溶液中稳定性比较
由表1可知,蓝莓花青素在酸性溶液中72h保存率下降至33.75%,而蓝莓花青素与凹土质量比为1:0.5(实施例1)的混合物保存率仅下降至54.61%,较蓝莓花青素保存率提升20%,显著增强了蓝莓花青素在酸性溶液中的稳定性。另外,从表1可知,当凹土的比例增大后,出现蓝莓花青素与凹土吸附饱和的状态,过剩的凹土会吸附空气中的水分,从而影响了蓝莓花青素的稳定性,出现了蓝莓花青素在酸性溶液中保存率下降的情况,影响了花青素的保存,综合考虑生产成本,蓝莓花青素与凹土的最佳质量比为1:0.5。以下所有性能测定及比较均用实施例1得到的混合物。
(2)蓝莓花青素-凹土混合物的红外光谱测定
对凹土、蓝莓花青素以及蓝莓花青素-凹土混合物分别采用红外光谱表征,由图1可知,凹土的3614 cm-1吸收峰为Al-OH的伸缩振动,1657 cm-1吸收峰为表面吸附水H-O-H的弯曲振动,1034 cm-1吸收峰为Si-O-Si的伸缩振动,985 cm-1吸收峰为Si-OH的弯曲振动。由图2可知,蓝莓花青素的3363 cm-1吸收峰为-OH伸缩振动,1639-1455 cm-1吸收峰为苯环骨架伸缩振动峰,1338-1069 cm-1吸收峰为C-O-C的伸缩振动。图3中,蓝莓花青素-凹土混合物除了含有图1、图2中的所有吸收峰外,无明显新吸收峰出现,但蓝莓花青素特征峰发生明显蓝移,说明蓝莓花青素、凹土表面的电荷影响了混合后蓝莓花青素的微环境。
(3)花青素-凹土混合物与蓝莓花青素抗氧化活性比较
3.1 清除超氧阴离子自由基能力的测定
取5mLEP管若干支,设两组对照;每管中分别加入50mM磷酸缓冲液(pH 7.0)1.5mL,130mM甲硫氨酸溶液0.3mL,750μMNBT溶液0.3mL,20μM EDTA-2Na溶液0.3mL,20μM核黄素溶液0.3 mL,各浓度样液(花青素、混合物)0.2 mL,双蒸水0.5mL,两组对照管以溶剂(纯水)代替提取液。将反应体系中各试剂混匀后,一组对照管用铝箔包裹完全遮光,另一组对照管及反应管同时置于日光灯下反应30min,反应温度为25°C。反应结束后,迅速将全部EP管用铝箔完全遮光。在560nm下,以遮光对照组作为空白调零管,测定各管的吸光度。
注:ODsample、ODblank分别是样液与空白样品(以溶剂代替样品提取液)的吸光值。IC50值通过线性回归得到。
表2蓝莓花青素及蓝莓花青素-凹土混合物清除超氧阴离子自由基能力的比较
由表2可知,蓝莓花青素清除超氧阴离子自由基的能力随时间延长而减弱,72h时蓝莓花青素的IC50值为342.14μg/mL,而蓝莓花青素-凹土混合物的IC50值为146.47μg/mL,约为蓝莓花青素的1/3,清除超氧阴离子自由基能力显著提高,说明蓝莓花青素与凹土混合后稳定性及抗氧化活性得到了显著的提高。
3.2 清除ABTS.+自由基能力的测定
称取48.01mgABTS(7mM)溶于12.5mLRO水,8.27mg过硫酸钾(2.45mM) 溶于12.5mLRO水,二者混匀后于室温条件下避光放置12 h;后用磷酸缓冲液溶液(pH7.4,0.05 M)稀释50倍成ABTS工作液。量取不同浓度的样液(蓝莓花青素、实施例1得到的混合物)各0.2 mL,后加入3mL的ABTS工作液,充分混匀后,避光放置30 min后于734 nm处测定吸光度;根据吸光值计算样品对ABTS.+自由基的清除作用。
注:OD样品:样液加ABTS工作液;OD空白:样液加磷酸缓冲液;OD对照:ABTS工作液加磷酸缓冲液;IC50通过线性回归得到。
表3蓝莓花青素及蓝莓花青素-凹土混合物清除ABTS.+自由基能力的比较
由表3可知,蓝莓花青素清除ABTS.+自由基的能力随时间延长而减弱,72h时蓝莓花青素的IC50值为71.25μg/mL,而混合物的IC50值为54.48μg/mL,清除ABTS.+自由基能力得到提高,说明蓝莓花青素与凹土混合后稳定性及抗氧化活性均显著提高。
3.3 亚铁离子螯合作用比较
分别称取菲啰嗪61.56mg溶于25mLRO水中、10.02 mg FeCl2·4H2O样品加RO水定溶至25mL、18.57mgMEDTA-Na2溶于25mLRO水中。量取不同浓度的样液(花青素、混合物)各400µL,分别先后加入50µLFeCl2溶液(2mM),5mM菲啰嗪溶液100 µL,2 mL甲醇溶液,充分摇匀后于室温条件下反应10min,于562 nm测定吸光值,以2mMEDTA-Na2溶液作空白对照,根据吸光值计算样品对亚铁离子螯合作用。
注:OD样品:样液加反应试剂;OD空白:EDTA-Na2溶液加反应试剂;OD对照:溶剂加反应试剂;IC50通过线性回归得到。
表4蓝莓花青素及蓝莓花青素-凹土混合物螯合亚铁离子能力的比较
由表4可知,蓝莓花青素螯合亚铁离子的能力随时间延长而减弱,72h时蓝莓花青素的IC50值为683.47μg/mL,而混合物的IC50值为328.44μg/mL,约为蓝莓花青素的1/2,螯合亚铁离子能力显著提高,说明蓝莓花青素与凹土混合后稳定性及抗氧化活性均显著提高。
Claims (6)
1.一种利用凹土提高蓝莓花青素稳定性的方法,其特征在于:以凹土为辅料,利用高速打浆和湿法混合得到蓝莓花青素-凹土混合物,操作步骤如下:
A: 将凹土粉碎,过200目筛,以水浸润后对辊挤压,再加入去离子水配制成浆料后,在10000~12000 rpm下打浆30~60 min以分散凹土,离心后取上层悬浮液,继续离心上层悬浮液得到膏体,膏体烘干成粉体后备用;
B:将蓝莓花青素与步骤A得到的凹土粉体混合均匀后,加入超纯水充分研磨至水分完全蒸发,得蓝莓花青素-凹土混合物。
2.根据权利要求1所述的一种利用凹土提高蓝莓花青素稳定性的方法,其特征在于:步骤A中离心转速为1500~3000 rpm。
3.根据权利要求1所述的一种利用凹土提高蓝莓花青素稳定性的方法,其特征在于:步骤A中凹土的粒径为60~80µm。
4.根据权利要求1所述的一种利用凹土提高蓝莓花青素稳定性的方法,其特征在于:步骤B中蓝莓花青素与凹土粉体的质量比为1:0.2~2。
5.根据权利要求4所述的一种利用凹土提高蓝莓花青素稳定性的方法,其特征在于:步骤B中蓝莓花青素与凹土粉体的质量比为1:0.5。
6.根据权利要求1所述的一种利用凹土提高蓝莓花青素稳定性的方法,其特征在于:步骤B中,蓝莓花青素-凹土混合物与超纯水混合比为1g/0.2~0.5ml。
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