CN114948902B - 一种醇溶蛋白复合中空粒子及其制备方法与应用 - Google Patents
一种醇溶蛋白复合中空粒子及其制备方法与应用 Download PDFInfo
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- CN114948902B CN114948902B CN202210617995.0A CN202210617995A CN114948902B CN 114948902 B CN114948902 B CN 114948902B CN 202210617995 A CN202210617995 A CN 202210617995A CN 114948902 B CN114948902 B CN 114948902B
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- prolamin
- quercetin
- hydrophobic functional
- hollow particles
- composite hollow
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Abstract
本发明涉及一种醇溶蛋白复合中空粒子及其制备方法与应用,属于食品技术领域。本发明的制备方法包括以下步骤,先向模板剂三聚磷酸钠的醇溶液中加入疏水性功能因子和醇溶蛋白,转移至水相,得到负载疏水性功能因子的醇溶蛋白中空粒子;然后,将所述的负载疏水性功能因子的醇溶蛋白中空粒子加入到乳蛋白/壳聚糖混合液中,包覆得到所述醇溶蛋白复合中空粒子。本发明所述的醇溶蛋白复合中空粒子能有效提高疏水性功能因子的热稳定性、储藏稳定性,且具有更高的耐盐性。
Description
技术领域
本发明属于食品技术领域,尤其涉及一种醇溶蛋白复合中空粒子及其制备方法与应用。
背景技术
疏水性功能因子具有较差的水溶性,对环境敏感,不利于其作为营养补充剂或药物进行开发和应用。槲皮素是天然的黄酮醇类化合物,具有抗氧化、抗菌和抗炎等生理功能。然而,槲皮素存在水溶性差、化学不稳定性、生物利用度低和生物半衰期短等缺点,限制了它在食品和药品中的加工应用。研究表明,设计包埋载体是提高功能因子稳定性和生物利用度的有效途径。其中,纳米/微米粒子具有体积小、控释、保护药物分子和能够通过血脑屏障等多种优点。蛋白质粒子由于其高营养品质、结合能力和靶向细胞吸收率高等优势,在封装和递送功能因子方面有良好潜力。
谷物醇溶蛋白易溶于70%的乙醇或高碱性(pH>11)水溶液中,可通过反溶剂沉淀法自组装生成实心粒子以传递疏水功能因子。目前相关研究仍集中于醇溶蛋白实心粒子(Solid zein particle,SZ)。与实心粒子相比,采用“模板(Templating)”法制备的中空的微/纳米结构材料具有高比表面积、低密度、高载量等优势,其组装过程有利于特异性功能载体的设计,在药物和功能因子传递方面有巨大潜力。碳酸钠(Na2CO3)是制备中空玉米醇溶蛋白粒子(Hollow zein particle,HZ)常用模板,但其特性决定了体系呈碱性,而碱性环境会导致多酚类化合物的羟基离子化而迅速氧化降解,限制了HZ作为多酚化合物载体的应用。因此,亟需找到新的牺牲模板在中性条件下制备以中空粒子,以实现对槲皮素等弱疏水性功能因子的高效包埋。
发明内容
为此,本发明所要解决的技术问题在于克服现有技术中对弱疏水性功能因子的包埋率低、稳定性差等问题。
为解决上述技术问题,本发明提供了一种醇溶蛋白中空粒子及其制备方法与应用。本发明选用醇溶蛋白为载体,选用对槲皮素等疏水性功能因子破坏较小的三聚磷酸钠(Sodium tripolyphosphate,STP)作为牺牲模板,通过反溶剂沉淀法制备得到负载疏水性功能因子的醇溶蛋白中空粒子。在此基础上,通过乳蛋白/壳聚糖复合涂层,得到醇溶蛋白复合中空粒子,从而提高醇溶蛋白的物理稳定性,实现对疏水性功能因子的高效包埋和保护。
本发明的第一个目的是提供一种醇溶蛋白复合中空粒子的制备方法,包括以下步骤,
S1、向模板剂三聚磷酸钠的醇溶液中加入疏水性功能因子和醇溶蛋白,转移至水相,得到负载疏水性功能因子的醇溶蛋白中空粒子;
S2、将S1所述的负载疏水性功能因子的醇溶蛋白中空粒子加入到乳蛋白/壳聚糖混合液中,包覆得到所述醇溶蛋白复合中空粒子。
在本发明的一个实施例中,在S1中,所述转移至水相是通过反溶剂沉淀法实现的。
在本发明的一个实施例中,在S1中,所述疏水性功能因子为槲皮素、姜黄素、α-生育酚和视黄醇中的一种或多种。
在本发明的一个实施例中,在S1中,所述醇溶蛋白为玉米醇溶蛋白、高粱醇溶蛋白、小麦醇溶蛋白和大麦醇溶蛋白中的一种或多种。
在本发明的一个实施例中,在S1中,所述模板剂三聚磷酸钠、疏水性功能因子和醇溶蛋白的质量比为1-8:2-20:50-200。
在本发明的一个实施例中,在S1中,所述醇溶液中的溶剂为乙醇和/或甲醇。
在本发明的一个实施例中,在S2中,所述乳蛋白为乳清蛋白和/或酪蛋白。乳蛋白选用两亲性乳蛋白,能够能吸附在粒子表面的疏水区域,提高醇溶蛋白粒子的静电和空间稳定,提高粒子的耐盐性。
在本发明的一个实施例中,在S2中,所述壳聚糖作为作为一种无毒、生物相容性的多阳离子多糖聚合物,具有良好的粘附性,能通过静电相互作用提高醇溶蛋白纳米粒子稳定性。
在本发明的一个实施例中,所述酪蛋白为酪蛋白酸钠。
在本发明的一个实施例中,在S2中,所述乳蛋白/壳聚糖混合液中乳蛋白和壳聚糖质量比0.5-1.5:1。
本发明的第二个目的是提供一种所述的方法制备的醇溶蛋白复合中空粒子。
本发明的第三个目的是提供一种所述的醇溶蛋白复合中空粒子在制备化妆品中的应用。
本发明的技术方案相比现有技术具有以下优点:
(1)本发明所述的方法以三聚磷酸钠(STP)为牺牲模板制备的醇溶蛋白中空粒子不会引起疏水性功能因子氧化降解。
(2)本发明所述的醇溶蛋白复合中空粒子中醇溶蛋白与两亲性的乳蛋白形成的复合粒子,比单独醇溶蛋白粒子更稳定。其中,具有两亲性的乳蛋白能吸附在粒子表面的疏水区域,提高醇溶蛋白粒子的静电和空间稳定。而且,壳聚糖作为一种无毒、生物相容性的多阳离子多糖聚合物,具有良好的粘附性,能通过静电相互作用提高复合醇溶蛋白纳米粒子稳定性。
(3)本发明所述的醇溶蛋白复合中空粒子能有效提高疏水性功能因子的热稳定性。在90℃水浴条件下孵育60min后,疏水性功能因子保留率为98.89%。
(4)本发明所述的醇溶蛋白中空粒子能有效提高疏水性功能因子的储藏稳定性。在25℃和45℃的培养箱内保存28天后,疏水性功能因子保留率分别为86.76%和80.06%。
(5)本发明所述的醇溶蛋白复合中空粒子具有更高的耐盐性,能在500mM的NaCl溶液中保持粒子稳定。
(6)本发明所述的醇溶蛋白复合中空粒子能够有效包埋疏水性功能因子,对200和400μg/mL的α-生育酚的包埋率分别为95.75%和98.57%。醇溶蛋白复合中空粒子还能对两种疏水性功能因子进行共包埋,当槲皮素浓度为100μg/mL,α-生育酚浓度为200μg/mL时,槲皮素和α-生育酚的包埋率分别是92.28%和98.63%;当槲皮素浓度为100μg/mL,α-生育酚浓度为400μg/mL时,槲皮素和α-生育酚的包埋率分别是90.55%和99.60%。
(7)本发明所述的方法制备的醇溶蛋白中空粒子和乳蛋白/壳聚糖复合的醇溶蛋白中空粒子能够有效包埋疏水性功能因子,包埋率分别达到84.89%和85.87%,显著高于实心粒子。
附图说明
为了使本发明的内容更容易被清楚地理解,下面根据本发明的具体实施例并结合附图,对本发明作进一步详细的说明,其中:
图1为本发明实施例1中不同STP浓度下,HZ和HZ-Cas/Chi的pH(A)和ζ-电位(B),不同粒子的pH(C)和ζ-电位(D);
图2为本发明实施例2中HZ(A)和HZ-Cas/Chi(B)的透射电子显微镜图;
图3为本发明实施例3在1.5mg/ml STP和Na2CO3为牺牲模板的中空粒子中槲皮素含量随静置时间变化;
图4为本发明实施例5在90℃加热60min后槲皮素的保留率;
图5为本发明实施例6在25℃和45℃储藏28天后槲皮素的保留率。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
在本发明中,除非另有说明,三聚磷酸钠(STP)的浓度为1.5mg/mL。
实施例1:玉米醇溶蛋白中空粒子及其制备
以0.5、1、1.5、2和4mg/mL三聚磷酸钠(STP)为牺牲模板制备玉米醇溶蛋白中空粒子(HZ),具体步骤如下:按体积比将7:3将无水乙醇加入浓度为3.3-26.6mg/mL的STP水溶液,然后按体积比1:1与溶于70%乙醇的玉米醇溶蛋白(5%,w/v)混合,再按体积比1:4将STP-Zein分散液加入到超纯水中,在1000rpm搅拌30min,获得中空玉米醇溶蛋白粒子。按1:1的体积比,将HZ分散液添加到0.32%酪蛋白酸钠、0.32%壳聚糖或两者等体积混合的水溶液中,分别得到酪蛋白涂层的玉米醇溶蛋白中空粒子(HZ-Cas)、壳聚糖涂层的玉米醇溶蛋白中空粒子(HZ-Chi)和酪蛋白/壳聚糖复合涂层的玉米醇溶蛋白中空粒子(HZ-Cas/Chi)。采用旋转蒸发仪去除乙醇,用水补足损失的体积后,在4℃、2000g条件下离心15min,去除已聚集的和不稳定的粒子。
作为对比,参考中空粒子的制备方法,不同的是:将玉米醇溶蛋白的70%乙醇溶液加入STP水溶液中,制备玉米醇溶蛋白实心粒子(SZ)和酪蛋白/壳聚糖涂层的玉米醇溶蛋白实心粒子(SZ-Cas/Chi)。
利用pH计测定样品的pH值;使用Nanobook Omini粒度分析仪上的相位分析光散射(PALS)功能,测定粒子的ζ-电位,结果如图1所示:
从图1(A)和(B)可以看出,随着STP浓度增加,HZ溶液的pH从6.3升高至8.8,HZ的ζ-电位从-7.9mV改变至-48.1mV。由于两亲性的酪蛋白能吸附在HZ表面的疏水区域,从而降低疏水吸引力,增加静电和空间斥力;带有正电荷胺基的壳聚糖通过静电吸引在粒子表面形成阳离子壳层,最终形成带正电荷的HZ-Cas/Chi,在0.5mg/mL STP条件下,ζ-电位为+37.8mV,随着STP浓度增加,ζ -电位约为+42.0mV;pH降低约4.0,且不受到STP浓度的影响。
从图1(C)和(D)可以看出,STP浓度为1.5mg/mL时,溶液pH为7.6,玉米醇溶蛋白中空粒子所带的净负电位低于实心粒子;HZ-Chi比HZ-Cas/Chi具有更高的正电位值和更低pH,表明了壳聚糖阳离子壳层以及酪蛋白/壳聚糖复合壳层的形成。酪蛋白和壳聚糖涂层的玉米醇溶蛋白中空粒子和实心粒子具有相似的正电位值和pH。HZ-Cas在pH4.0左右不稳定,因为酪蛋白在等电点pH4.6附近易于发生聚集和失稳。
实施例2:透射电子显微镜观测复合粒子的形貌和尺寸
参照实施例1方法制备HZ和HZ-Cas/Chi,将稀释的样品用1%磷钨酸染色,在120kV电子加速电压下用透射电子显微镜(TEM)对粒子的微观形貌和尺寸进行观测。
如图2(A)所示,TEM图显示HZ具有颜色更浅的核和颜色更深的壳,说明粒子具有空心结构,HZ粒径约为80nm左右。如图2(B)所示,HZ-Cas/Chi具有比HZ颜色更深的壳,说明酪蛋白和壳聚糖在HZ表面形成了更厚的复合涂层;HZ-Cas/Chi粒径增加至100nm左右。
实施例3:以STP和Na2CO3为牺牲模板对槲皮素稳定性的影响
参照实施例1方法制备负载槲皮素的HZ粒子,STP的浓度为1.5mg/mL,即STP@HZ,不同的是在乙醇和STP水溶液混合前,将2mg槲皮素溶于0.7mL无水乙醇中。作为对比,在相同的条件下,将STP替换为Na2CO3,制备负载槲皮素的Na2CO3@HZ。常温静置0,10,30,60,120和180min后,测定样品中槲皮素的含量。
如图3所示,由于槲皮素对碱的敏感性,Na2CO3@HZ溶液的pH约为11.0,在Na2CO3@HZ中槲皮素含量相当于加入量的62%,且随着静置时间延长降低至19%,表明Na2CO3@HZ制备和静置过程中槲皮素均明显发生降解。在STP@HZ中槲皮素的含量保持100%,说明槲皮素是稳定的。因此,相比于Na2CO3,STP更适合作为牺牲模板制备HZ,扩展其作为多酚化合物载体的应用。
实施例4:比较粒子实心和中空结构对槲皮素包埋率的影响
采用实施例1和3制备的包埋槲皮素的玉米醇溶蛋白实心(SZ)和空心(HZ)粒子及它们的酪蛋白和壳聚糖涂层粒子(SZ-Cas/Chi、HZ-Cas/Chi)。将样品在2,000离心除去沉淀的未包埋槲皮素,然后在100,000g离心,用紫外分光光度计在λmax为373nm测定整体溶液和上清液中槲皮素含量,计算SZ、SZ-Cas/Chi、HZ和HZ-Cas/Chi对槲皮素的包埋率,结果如表1所示,按以下公式计算包埋率:
表1 槲皮素包埋率
注:不同字母表示统计分析差异显著(p<0.05)。
由表1可知,在玉米醇溶蛋白实心粒子中槲皮素的保留率为55.83%,酪蛋白/壳聚糖涂层增加槲皮素包埋率至62.34%。一方面,酪蛋白/壳聚糖涂层阻止了槲皮素从玉米醇溶蛋白粒子中释放;另一方面,槲皮素也可能通过静电和氢键等与酪蛋白/壳聚糖发生作用,提高包埋率。在玉米醇溶蛋白中空粒子及其酪蛋白/壳聚糖涂层粒子中,槲皮素的包埋率约为85%。与实心粒子相比,中空粒子对槲皮素具有更好的包埋作用。
实施例5:包埋对槲皮素热稳定性的影响
采用实施例4方法制备包埋槲皮素的HZ和HZ-Cas/Chi,在90℃水浴加热60min,以热处理后槲皮素含量除以初时含量计算槲皮素的保留率。同时,以在pH7.6和4.0时未包埋的槲皮素为空白对照。
如图4所示,在pH为7.6时,未包埋槲皮素热处理60min后保留率降低至27.79%,在玉米醇溶蛋白中空粒子中包埋将槲皮素的保留率提高至为78.75%。在pH为4.0时,热处理后未包埋槲皮素的保留率降低至40.21%,在酪蛋白/壳聚糖涂层中空粒子中包埋将槲皮素的保留率提高至98.88%。在加热条件下,槲皮素在水溶液中的自氧化作用是其降解的主要原因。这些结果说明玉米醇溶蛋白及复合中空粒子可以有效地改善槲皮素的热稳定性。
实施例6:包埋对槲皮素储藏稳定性的影响
参照实施例1方法制备包埋槲皮素的HZ和HZ-Cas/Chi,在25℃和45℃储藏28天,以储藏后槲皮素含量除以初时含量计算槲皮素的保留率。同时,以在pH7.4和4.1时未包埋的槲皮素为空白对照。
如图5所示,在25℃条件下,在pH7.4和4.1条件下槲皮素发生氧化降解具有较低的保留率。在HZ和SZ都能一定程度上提高槲皮素保留率;HZ-Cas/Chi具有最高的保留率,为86.76%。45°C条件下,HZ的槲皮素保留率显著降低,为1.23%;HZ-Cas/Chi的槲皮素保留率只发生轻微的降低,为80.06%。上述结果说明,酪蛋白/壳聚糖复合涂层能够显著提高槲皮素储藏稳定性,一方面,致密的蛋白质/多糖复合物能以制槲皮素的释放,并减少槲皮素与以导致功能因子降解的环境因素相互作用,延长其贮藏寿命;另一方面,玉米醇溶蛋白、酪蛋白和壳聚糖自身具有固有的自由基清除特性,还能通过疏水作用和氢键作用抑制黄酮类化合物的自氧化作用。
实施例7:多种疏水性功能因子共包埋研究
参考实施例1的方法,在乙醇和STP水溶液混合前,将α-生育酚或槲皮素和α-生育酚共同溶于0.7mL无水乙醇中,获得单包埋α-生育酚以及共包埋槲皮素和α-生育酚的HZ-Cas/Chi。槲皮素浓度为100μg/mL,α-生育酚的终浓度为200和400μg/mL。参考实施例5测定槲皮素包埋率。采用离心联合高效液相色谱法测定α-生育酚(α-tocopherol,α-Toc)的包埋率和负载量。α-生育酚采用HPLC(Waters,MA,USA)测定,采用2695分离模块、2998PDA检测器和C18色谱柱(5μm,4.6mm×250mm),流动相为甲醇,流速1mL/min,柱温为35℃,洗脱并检测α-生育酚含量。通过100,000g离心1h分离未包埋的槲皮素和α-生育酚。在0.4mL样品中添加4mL正己烷,在3,500g、4℃条件下离心5min,以提取α-生育酚。用氮气吹干上清中的正己烷,并用甲醇复溶后,经0.22μm滤膜过滤后注入高效液相色谱***。计算HZ-Cas/Chi单独包埋α-生育酚,以及共包埋槲皮素和α-生育酚时功能因子的包埋率,结果如表2所示,按以下公式计算包埋率:
表2
如表2所示,HZ-Cas/Chi单独包埋200和400μg/mL的α-生育酚的包埋率为95.73%和95.57%。HZ-Cas/Chi对槲皮素和α-生育酚共包埋。当槲皮素浓度为100μg/mL,α-生育酚浓度为200μg/mL时,槲皮素和α-生育酚的包埋率分别是92.28%和95.63%;当α-生育酚浓度为400μg/mL时,槲皮素和α-生育酚的包埋率分别是90.56%和99.60%。上述结果说明,酪蛋白-壳聚糖涂层玉米醇溶蛋白中空粒子还能高效包埋和保护一种或多种疏水性功能因子。
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (3)
1.一种醇溶蛋白复合中空粒子的制备方法,其特征在于,包括以下步骤,
S1、向模板剂三聚磷酸钠的醇溶液中加入疏水性功能因子和醇溶蛋白,转移至水相,得到负载疏水性功能因子的醇溶蛋白中空粒子;所述疏水性功能因子为槲皮素、姜黄素、α-生育酚和视黄醇中的一种或多种;所述醇溶蛋白为玉米醇溶蛋白、高粱醇溶蛋白、小麦醇溶蛋白和大麦醇溶蛋白中的一种或多种;所述模板剂三聚磷酸钠、疏水性功能因子和醇溶蛋白的质量比为1-8:2-20:50-200;
S2、将S1所述的负载疏水性功能因子的醇溶蛋白中空粒子加入到乳蛋白/壳聚糖混合液中,包覆得到所述醇溶蛋白复合中空粒子;所述乳蛋白为乳清蛋白和/或酪蛋白;所述乳蛋白/壳聚糖混合液中乳蛋白和壳聚糖质量比为0.5-1.5:1。
2.权利要求1所述的方法制备的醇溶蛋白复合中空粒子。
3.权利要求2所述的醇溶蛋白复合中空粒子在制备化妆品中的应用。
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