CN114403449B - 一种蛋白-甾醇共价复合物及其制备方法和应用 - Google Patents
一种蛋白-甾醇共价复合物及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种蛋白‑甾醇共价复合物及其制备方法和应用,属于食品加工技术领域。所述蛋白‑甾醇共价复合物为羧基活化的蛋白质与植物甾醇通过共价聚合制得。本发明通过化学改性的方法,在几乎不破坏植物甾醇C‑3羟基的前提下,利用植物甾醇的C‑3位羟基与碳二亚胺(EDC)活化的蛋白质羧基发生酯化反应,形成结构可控的蛋白‑甾醇共价复合物。该复合物具有两亲性能,使甾醇的水溶性得以提升,增强甾醇的口服生物可及性。该共价复合物在水溶性介质中可自组装为稳定的亲水壳‑疏水核结构,作为如甾醇等疏水性生物活性物的较好载体。该复合物作为添加剂可应用于食品、保健品、药品、生物制品以及化妆品等行业。
Description
技术领域
本发明涉及食品加工技术领域,具体涉及一种蛋白-甾醇共价复合物及其制备方法和应用。
背景技术
植物甾醇是一种具有环戊烷多氢菲结构的植物源性化合物,具有多种对人体有益的生理功能,研究发现植物甾醇能够竞争性地抑制胆固醇在肠道的吸收,显著降低人体血浆中胆固醇含量,减少血浆低密度脂蛋白(LDL)合成,降低密度脂蛋白胆固醇,防治心血管疾病,具有免疫调节,抗炎症,抑制癌细胞增殖等功效。由于动物不能内源性地合成植物甾醇,人体只能通过摄取植物来源的食物来获得,因此开发植物甾醇产品有实际意义。
由于其疏水性和水不溶性,植物甾醇难以形成稳定的分散体,也难以均匀添加到食品中,这一缺点限制了植物甾醇在水溶性食品中的应用。当食用膳食补充剂形式的植物甾醇时,溶解速率可能会影响其降低低密度脂蛋白胆固醇的能力,从而引发植物甾醇在以补充形式输送时有效性低的问题。
以乳液形式摄入有利于甾醇的消化吸收,乳液可以通过改变它们的组成和结构来改善活性物质的生物利用度,因此这也是人体从食品中摄取甾醇的主要形式之一。但是传统的O/W乳液形式,植物甾醇存在于乳液的油相中,整体乳液的水相与油相不易于混合且乳化效果并不理想。且有文献报道指出乳液中的植物甾醇会在乳液界面发生氧化反应劣变为甾醇氧化物,对人体产生危害作用。
因此,如何增强植物甾醇的水溶性及提升植物甾醇生物可及性是研究人员需要解决的技术问题。
蛋白质作为一种重要的营养物质,具有多种特性,由于其侧链具有游离的羧基,因此也具有良好的改性性能,侧链经修饰后可获得两亲性的蛋白衍生物。近年来,有文献报道蛋白质与多糖或多酚之间的共价复合物制备方法,如专利文献CN 105601701 A公开利用蛋白质与多酚的自由基接枝聚合反应或碱诱导反应,制备蛋白质-多酚共价复合物,以该复合物作为乳化剂可显著提升β-胡萝卜素乳状液的化学稳定性和生物利用率。
目前并无针对脂质与蛋白质共价复合物制备的研究报道。如何通过蛋白与植物甾醇相互作用安全而高效地提高植物甾醇溶解性,提高生物可及性具有广泛的研究意义。
发明内容
本发明的目的在于提供一种蛋白-甾醇共价复合物,通过优化植物甾醇的分子结构提升植物甾醇生物可及性,以解决现有技术中植物甾醇不易于添加到食品、生物可及性低的问题。
为实现上述目的,本发明采用如下技术方案:
一种蛋白-甾醇共价复合物,所述共价复合物为羧基活化的蛋白质与植物甾醇通过共价聚合制得。
本发明利用植物甾醇C-3位的酚羟基活性,与蛋白质游离羧基共价结合,形成新型两亲性植物甾醇基蛋白衍生物。一方面,该衍生物具有两亲性,增强植物甾醇的水溶性,促进植物甾醇在摄入后肠腔内胶束化率进而提高生物可及性。另一方面,该两亲性复合物可在水溶性介质中可自组装为亲水壳-疏水核结构,可作为疏水性生物活性物质的载体。
进一步的,所述蛋白质为乳清分离蛋白、大豆分离蛋白、玉米醇溶蛋白、酪蛋白酸钠中的一种或多种;所述甾醇为β-谷甾醇、豆甾醇、菜油甾醇、菜籽甾醇中的一种或者多种。
进一步的,甾醇与蛋白质量比为1:1~15。
本发明还提供了所述的蛋白-甾醇共价复合物的制备方法,包括以下步骤:
(1)利用活化剂处理蛋白质,得到羧基活化的蛋白溶液;
(2)结合超声处理,将植物甾醇溶液加入到活化的蛋白溶液中,20~40℃条件下搅拌聚合反应,制得所述的蛋白-甾醇共价复合物。
步骤(1)中,利用活化剂活化蛋白质游离羧基,具体的,首先将蛋白质溶于水中,得到蛋白储备液,再将蛋白储备液与活化剂混合,4℃条件下搅拌,获得活化的蛋白溶液。
作为优选,所述蛋白溶液的质量百分比浓度为0.2-0.5%。
作为优选,所述活化剂为碳二亚胺(EDC)、抗坏血酸或过氧化氢。
步骤(2)中,将植物甾醇溶于二甲亚砜溶液(DMSO)中,再将甾醇溶液缓慢加入蛋白溶液中,添加过程中结合超声辅助混合,以降低复合物粒径。
作为优选,所述超声处理的条件为超声功率为200W~800W,超声时间为30~120分钟。所述超声处理模式为1s脉冲模式。
作为优选,甾醇与蛋白质量比为1:5~10。
更为优选,甾醇与蛋白质量比为1:10,超声条件为功率200W,超声60分钟。
作为优选,聚合反应温度为30℃,反应时间为20~24h。
步骤(2)中,反应结束后,将反应产物依次经有机溶剂沉淀、复溶透析、冷冻干燥制得所述蛋白-甾醇共价复合物。
作为优选,所述有机溶剂沉淀包括:首先加入2倍体积的丙酮,离心后收集沉淀,再依次用乙醇、乙醚洗涤沉淀。
作为优选,选取12000-14000Da分子截留量透析袋,透析时间为70~72h。
本发明还提供了所述的蛋白-甾醇共价复合物在制备食品、保健品、药品、生物制品或化妆品添加剂中的应用。
本发明利用蛋白的羧基与甾醇的羟基共价聚合制备两亲性复合物,使得甾醇水溶解性能以提升,易于形成乳糜胶束,提高了甾醇的生物可及性。同时蛋白-甾醇共价复合物在溶液的表面为乳液提供了空间排斥力降低界面张力增加乳液稳定性,可作乳化稳定剂用于稳定乳状液,提高乳液的稳定性,稳定剂与乳液达到共同提升生物可及性的效果,提高乳液的品质,制得的乳液可应用于食品、保健品、药品、生物制品以及化妆品行业。
本发明具备的有益效果:
(1)针对植物甾醇不溶于水,不易添加于水溶性介质中,在食品递送系统中生物可及性低的特点,本发明通过化学改性的方法,在几乎不破坏植物甾醇C-3羟基的前提下,利用植物甾醇的C-3位羟基与碳二亚胺(EDC)活化的蛋白质羧基发生酯化反应,形成结构可控的蛋白-甾醇共价复合物。
(2)本发明将植物甾醇、蛋白质的功能特性进行整合,合成具有优良性能的生物大分子,并减少副产物的生成。该共价复合物具有更好的水溶性,在水溶性介质中可自组装为稳定的亲水壳-疏水核结构,作为如甾醇等疏水性生物活性物的较好载体。
(3)本发明提供了适用于提高甾醇生物可及性的载体材料,复合物具有两亲性能,使甾醇的水溶性得以提升,增强甾醇的口服生物可及性。
(4)本发明制备蛋白-甾醇共价复合物的原料大众接受度高,而且材料组成简单,可操作性强,效果良好,节约成本。
附图说明
图1为乳清分离蛋白与植物甾醇共价复合物(WPI-PS)、植物甾醇(PS)、乳清分离蛋白(WPI)和乳清分离蛋白与植物甾醇混合物(WPI+PS)的红外光谱图。
图2为不同比例乳清分离蛋白-甾醇共价复合物外观图。
具体实施方式
下面结合具体实施例对本发明做进一步说明。以下实施例仅用于说明本发明,不用来限制本发明的适用范围。在不背离本发明精神和本质的情况下,对本发明方法、步骤或条件所做的修改或替换,均属于本发明的范围。
下述实施例中所使用的试验方法如无特殊说明,均为常规方法;所使用的材料、试剂等,如无特殊说明,为可从商业途径得到的试剂和材料。
实施例1
一、蛋白-甾醇共价聚合物制备
1、将具有游离羧基的乳清分离蛋白(WPI,CAS:84082-51-9)加入到去离子水中充分溶解,4℃搅拌1.5h,得到蛋白储备液;然后按照蛋白每摩尔羧基添加0.7moL 1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC),将蛋白储备液中与EDC混合,4℃搅拌2h,获得羧基活化的蛋白溶液。
2、随后加入等体积的含植物甾醇(β-Sitosterol,CAS:83-46-5)的二甲亚砜溶液(DMSO),使甾醇溶液与活化的蛋白溶液混合,该过程中结合超声处理,功率为200W,1s脉冲模式下,将植物甾醇溶液缓慢加入蛋白溶液中,得到1:10(w/w%)的甾醇:蛋白(w/w混合)溶液;30℃下磁力搅拌反应24h。
3、随后倾入2倍体积的丙酮,4500rpm离心1h,并收集沉淀;再依次用乙醇、乙醚洗涤沉淀;采用去离子水复溶后透析72h;最后将复合物冷冻干燥48h,得到蛋白-甾醇共价复合物。
二、性能检测
1、共价键测定
将乳清分离蛋白、蛋白-甾醇混合物(乳清分离蛋白与甾醇以相同质量比均匀混合得到)、上述步骤一制备的蛋白-甾醇共价复合物三组冷冻干燥处理,利用FTIR光谱仪通过KBr片剂法测定,所有谱图均在4000到400cm-1范围内平均扫描32次,分辨率为4cm-1。
结果如图1所示,对比蛋白与复合物,发现芳烃C-H伸缩振动峰变大且向高波数变化,主要原因为甾醇的苯环使该峰发生蓝移。蛋白在1744cm-1羧基的C=O伸缩振动特征峰在复合后消失,表明羧基发生反应。氨基特征峰发生高波数位移,由1311变为1319;酰胺Ⅰ带N-H变形震动特征峰、C=O伸缩振动峰发生低波数移动,由1640变为1647。此外,指纹区羟基C-O伸缩振动峰由1072变为1053发生低波数移动,蛋白分子中酰胺键发生变化,同时氢键O-H键特征峰未发生变化,证明蛋白与甾醇形成新键,蛋白-甾醇共价复合物形成。
2、粒径、电位测定
通过动态光散射粒度仪对共价复合物的粒径、Zeta电位等进行测定。
3、生物可及性测定
通过动态体外模拟胃肠消化来测量植物甾醇生物可及性的影响。
模拟胃液(SGF)含有2mg/mL氯化钠,3.2mg/mL胃蛋白酶。然后用盐酸将pH调节至1.3。将样品溶于10mL去离子水中,与10mL SGF混合,然后在37℃的水浴振动器中连续摇动2小时(100rpm)。
模拟肠液(SIF)含有6.8mg/mL磷酸二氢钾、0.2mol/L氢氧化钠、10mg/mL胰蛋白酶。然后用氢氧化钠将pH调节至7.0。然后将等体积小肠消化液(SIF)添加到上述悬浮液中。将制备好的混合物在37℃的水浴振动器中连续摇动2小时(100转/分)。
消化过程后,所有混合物在10℃下以12000rpm的转速离心30分钟。收集中间胶束层并测定样品的植物甾醇含量。
取0.5mL消化液胶束层溶液利用乙醇萃取甾醇,利用真空离心浓缩仪浓缩干燥,乙醇复溶,加入1/2体积的磷硫铁显色剂,颜色反应15min,25℃测定520nm吸光值。利用甾醇标曲定量消化液胶束层甾醇含量。结果如表1所示。
表1共价复合物稳定性及植物甾醇生物可及性
实施例2
一、乳清分离蛋白-植物甾醇共价复合物稳定的O/W乳状液的制备
将甾醇-乳清分离蛋白共价复合物溶解于pH为7.0的磷酸盐缓冲液(10mM)中,得到蛋白-甾醇共价复合物溶液,使复合物溶液中甾醇浓度为2mg/mL。中链甘油三酸酯(MCT)作为油相材料,加热至140℃。利用高速剪切机的剪切均质作用,缓慢将油相加入到水相中,油相与水相比例为1:9(w/w)。高速均质机均质剪切2分钟,形成粗乳状液。利用高压均质机进一步均质得到含有甾醇的乳状液。然后使用高压微流化器在70MPa的压力下进一步减小乳液的粒径5次,最终甾醇含量与实施例1中相同。
二、性能检测
粒径、电位、生物可及性测定,方法同实施例1,结果如表2所示。
对比例1
一、含有植物甾醇的O/W乳状液
将植物甾醇溶解在中链甘油三酸酯(MCT)中浓度为(0.1,w/w%)作为油相,并在50℃下搅拌20分钟以使其完全溶解。将乳清分离蛋白(WPI)分散在pH为7.0的磷酸盐缓冲液(10mM)中浓度为(2w/w%)作为水相。将蛋白溶液搅拌3小时以确保完全溶解和水合。
将含有植物甾醇的油相和蛋白质溶液以1:9(w/w)的比例混合形成粗乳液。使用高速均质机均质2分钟。然后使用高压微流化器在70MPa的压力下进一步减小乳液的粒径5次,最终甾醇含量与实施例1中相同。
二、性能检测
粒径、电位、生物可及性测定,方法同实施例1,结果如表2所示。
对比例2
一、植物甾醇自组装复合物
将植物甾醇在45℃溶于乙醇溶液浓度为2mg/mL,蛋白溶液浓度为5mg/mL,5mL甾醇溶液溶解于45mL蛋白溶液中。超声波处理,功率为200W,1s脉冲模式,控制甾醇与蛋白的比例为1:10(w/w%)。复合物在45℃条件下用真空旋蒸器蒸发,除去有机溶剂,加入去离子水,以保持分散的初始体积,最终甾醇含量与实施例1中相同。
二、性能检测
粒径、电位、生物可及性测定,方法同实施例1,结果如表2所示。
对比例3
一、甾醇分散体系
将植物甾醇(0.2%,w/v)首先溶解在无水乙醇中,然后于45℃保持5分钟以完全溶解。然后,使用高速分散装置将有机相与水相的比例为1:10(v/v)混合。充分混合后进行2分钟均质化处理。最后,使用旋转蒸发器除去乙醇和部分水,最终甾醇含量与实施例1中相同。
二、性能检测
粒径、电位、生物可及性测定,方法同实施例1,结果如表2所示。
表2不同类型乳状液稳定性及植物甾醇生物可及性
分析表1、表2数据可知,实施例1中制备的共价复合物、实施例2中将共价复合物应用于乳液体系会增加植物甾醇的生物可及性,这证明共价形式及乳液递送系统均对增加生物可及性产生积极影响。
平均粒度可作为衡量乳液稳定性的标度,植物甾醇与蛋白形成的共价复合物稳定的乳液(实施例2)粒径小于对比例1、对比例2、对比例3,证明共价复合物稳定的乳液分散均匀稳定,蛋白-甾醇共价复合物稳定的O/W乳液(实施例2)相较于蛋白稳定的O/W乳液、蛋白-甾醇自组装胶束、植物甾醇水分散体(对比例1-3)乳液的稳定性有所提高。
此外,乳液的Zeta电位绝对值越大表明乳液稳定效果越好,实施例2Zeta电位绝对值>对比例1>对比例2>对比例3,证明实施例2稳定性高于其他三种对比例。
同时,对比发现植物甾醇的生物可及性在实施例2中比例为80.17%,远高于对比例1生物可及性22.31%,远高于对比例2生物可及性15.92%,远高于对比例3生物可及性9.23%,说明共价复合物形成的O/W乳液稳定性和生物可及性均得到了较好的改善。
实施例3
不同种类蛋白-甾醇共价复合物
1、配制蛋白储备液时,蛋白选取大豆分离蛋白(SPI)、玉米蛋白(Zein)、酪蛋白酸钠(NaCAS),其余同实施例1。
将SPI、NaCAS于去离子水中充分溶解,Zein充分溶于乙醇中,4℃搅拌1.5h,得到浓度为5mg/mL蛋白溶液;然后将蛋白储备液与蛋白每摩尔羧基0.7moL的1-(3-二甲氨基丙基)-3-乙基碳二亚胺EDC混合,4℃搅拌2h,获得羧基活化的蛋白溶液。
2、随后加入等体积的含植物甾醇的二甲亚砜溶液(DMSO),使甾醇溶液与活化的蛋白溶液混合;超声处理,功率为200W,1s脉冲模式下,将植物甾醇溶液缓慢加入蛋白溶液中,得到1:1,1:5,1:10,1:15的甾醇:蛋白(w/w)溶液;30℃下磁力搅拌反应24h。
3、随后倾入2倍体积的丙酮,4500rpm离心1h,并收集沉淀;再依次用乙醇、乙醚洗涤沉淀;采用去离子水复溶后透析72h;最后将复合物冷冻干燥48h,得到蛋白-甾醇共价复合物。
4、粒径、生物可及性测定,方法同实施例1,结果如表3所示。
表3不同蛋白与甾醇形成的共价复合物中甾醇接枝率及生物可及性
如表3所示,本发明将植物甾醇与蛋白以1:1、1:5、1:10、1:15(w/w)制备共价复合物,对比发现在同一蛋白组中不同比例下甾醇接枝率存在较大差异,图2外观也发现溶液均一性也存在较大不同。不同蛋白与甾醇形成共价复合物接枝率最高配比均为1:10(w/w)。在同等摄入量下甾醇的生物可及性顺序为WPI>SPI>NaCAS>Zein,主要原因为WPI相较于其他蛋白促进脂肪酶进入脂质核心,导致酯键分解,使甾醇胶束化率得以提升,生物可及性得到增加。而通过计算生物可及性发现不同甾醇蛋白比例下差异并不明显,说明选取接枝率较高组别对于节约原料具有较好的意义。
实施例4
复合物加工处理超声频率对复合物制备的影响
1、将乳清分离蛋白加入到去离子水中充分溶解,使蛋白浓度为5mg/mL,4℃搅拌1.5h,得到蛋白储备液;然后按照蛋白每摩尔羧基添加0.7moL的1-(3-二甲氨基丙基)-3-乙基碳二亚胺EDC混合,4℃搅拌2h,获得羧基活化的蛋白溶液。
2、随后加入等体积的含植物甾醇的二甲亚砜溶液(DMSO),使甾醇溶液与活化的蛋白溶液混合;超声处理30min,60min,超声功率为0,200W(22.2%),400W(44.4%),600W(66.7%),800W(88.9%)。将植物甾醇溶液缓慢加入蛋白溶液中,得到1:10(w/w%)的甾醇:蛋白(w/w%)溶液,30℃下磁力搅拌反应24h。
3、随后倾入2倍体积的丙酮,4500rpm离心1h,并收集沉淀;再依次用乙醇、乙醚洗涤沉淀;采用去离子水复溶后透析72h;最后将复合物冷冻干燥48h,得到蛋白-甾醇共价复合物。
4、粒径、电位、生物可及性测定,方法同实施例1,结果如表4所示。
表4不同超声条件对共价复合物中植物甾醇生物可及性的影响
如表4所示,由于植物甾醇-蛋白共价复合物粒径大小对于甾醇微胶束的形成有显著影响,通过超声的加工手段可降低复合物的粒子直径,因此本发明通过对蛋白甾醇共价复合物的制备方式进行比较,以取得最佳的粒子分散效果和生物可及性。超声处理22.2%为超声功率的优选值,此时共价复合物粒径较低,甾醇生物可及性呈现最高比值。当功率由0增大至22.2%时,粒径减小,生物可及性增加,当功率由22.2%增大至44.4%、66.7%、88.9%时粒径逐渐减小,说明过度超声不利于共价复合物的形成。同时,超声时间为60分钟时会与较优超声功率起到协同增效的作用,粒径最小且生物可及性最好。作为优选,选择乳清分离蛋白,超声条件为功率22.2%,超声60分钟时得到的聚合物生物可及性最好。
上述实施例为本发明较佳的实施方式,但本发明的各实施例中的技术方案也可以经适当组合,不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围内。
Claims (4)
1.一种蛋白-甾醇共价复合物,其特征在于,所述共价复合物为羧基活化的蛋白质与植物甾醇通过共价聚合制得;所述的蛋白-甾醇共价复合物的制备方法包括以下步骤:
(1)利用活化剂处理蛋白质,得到羧基活化的蛋白溶液;
(2)结合超声处理,将植物甾醇溶液加入到活化的蛋白溶液中,20~40℃条件下搅拌聚合反应,制得所述的蛋白-甾醇共价复合物;
所述蛋白质为乳清分离蛋白、大豆分离蛋白、玉米醇溶蛋白、酪蛋白酸钠中的一种或多种;所述甾醇为β-谷甾醇、豆甾醇、菜油甾醇、菜籽甾醇中的一种或者多种;甾醇与蛋白质量比为1:1~15;
步骤(1)中,所述活化剂为碳二亚胺;所述蛋白溶液的质量百分比浓度为0.2~0.5%;
步骤(2)中,所述超声处理的条件为超声功率200W~800W,超声时间为30~120分钟。
2.如权利要求1所述的蛋白-甾醇共价复合物,其特征在于,步骤(2)中,甾醇与蛋白质量比为1:10,超声条件为功率200W,超声60分钟。
3.如权利要求1所述的蛋白-甾醇共价复合物,其特征在于,步骤(2)中,反应结束后,将反应产物依次经有机溶剂沉淀、复溶透析、冷冻干燥制得所述蛋白-甾醇共价复合物。
4.如权利要求1-3任一项所述的蛋白-甾醇共价复合物在制备食品、药品或化妆品添加剂中的应用。
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