果胶是从植物细胞壁中提取的一种天然无毒、结构多样的阴离子多糖,在苹果、柑橘和甜菜加工过程中,产生的大量残渣废粕是果胶的主要来源[1-3]。对果胶进行深加工不仅能提高其利用率和附加值,还能避免资源浪费和环境污染等问题[4]。果胶具备一些特殊的界面性质,如空间位阻、静电相互作用和流变学特性等,是开发或构建乳液的理想材料[5-7]。然而,天然果胶中极性基团与非极性基团的比例未达到平衡,使其易从油-水界面脱离,造成乳液体系的失稳,这极大地限制了其在营养递送等领域的应用[8-9]。
纳米乳液是一种由表面活性剂稳定的胶体分散体系,其平均粒径一般在20~200 nm,少量文献将其定义为20~500 nm[10]。与传统乳液相比,纳米乳液的粒径小,界面性能好,具有良好的透明性和动力学稳定性[11-12],将生物活性成分包封在纳米乳液中,有助于改善活性成分的理化稳定性和生物学功效[13-14]。果胶可以减少油-水界面的张力,常被用作纳米乳液的乳化剂或稳定剂。有研究报道,对天然果胶进行甲酯化改性,或者将其与小分子表面活性剂(如吐温、司盘等)或大分子表面活性剂(如蛋白质等)联合使用,能够有效提高纳米乳液的稳定性,以及提高生物活性物质的包封效果[15-18]。系统研究果胶衍生物和复合物构建的纳米乳液传递体系具有重要意义。本文综述果胶基纳米乳液的制备方法、表征手段,以及果胶衍生物和复合物在稳定纳米乳液方面发挥的作用,旨在为果胶基纳米乳液体系的构建及其在生物活性物质包埋或释放中的应用提供理论依据。
高 能乳化法(High-energy emulsification methods)是利用机械装置产生的强大破坏力来生产果胶基纳米乳液的方法,包括高压均质法、高速剪切法、超声波乳化法和微射流乳化法等[19]。图1为高能乳化法生产果胶基纳米乳液的示意图,这些方法经常联合使用,以获得粒径更小、分散更均匀的乳液液滴。Trujillo-Ramírez 等[20]利用高速剪切机预处理得到粗乳液,再将粗乳液进行高压均质处理,制备出果胶基纳米乳液。Gharehbeglou 等[18]使用超声波乳化装置破碎液滴,制备出W1/O/W2型果胶基纳米乳液,其最小粒径为191 nm。微射流乳化法是将粗乳液在超高压作用下,通过微小孔径形成高速的流体,这些流体相互碰撞,产生强烈的破坏力,使液滴破裂[21]。Guerra-Rosas 等[22]通过在150 MPa 下进行5 个循环的微流化来获得果胶基纳米乳液,结果显示经过微流化处理后,乳液的平均粒径小于50 nm。高能乳化法的工艺简单,所需的表面活性剂较少,然而它对机械设备的要求较高,在进行工业化生产时应该充分考虑成本和能耗等问题[23]。
图1 高能乳化法制备果胶基纳米乳液的示意图
Fig.1 Schematic diagram of preparing pectin-based nanoemulsions by high energy emulsification
低能乳化法(Low-energy emulsification methods)主要依赖体系的组分和系统内部的化学能来诱导纳米乳液的自发形成,包括相转变法和自发乳化法等,其中相转变法已成功应用于果胶基纳米乳液的制备[24-25]。如图2所示,将改性果胶均匀分散在油相中,然后向该体系中缓慢持续地加水,逐渐改变油水比例,在达到体系的相转变点时,W/O 型乳液转变为O/W 型纳米乳液[15,26]。Hua等[15]通过相转变法制备的果胶基纳米乳液具有良好的贮藏稳定性和pH 稳定性。与高能乳化法相比,低能乳化法不需要特定的仪器设备,节约了能源成本[27],然而低能乳化法对原料的要求高,需要大量的表面活性剂,形成的乳液在高温下通常不稳定[28]。
图2 相转变法制备果胶基纳米乳液的示意图
Fig.2 Schematic diagram of preparing pectin-based nanoemulsions by phase change method
果胶基纳米乳液的液滴特性主要包括粒径大小、微观结构、粒子电荷和界面特征等(图3),这些特性决定了纳米乳液的理化性质,例如:光学性质、流变性、稳定性、包埋和释放特性等。常用的表征手段有光散射技术(如动态光散射技术、静态光散射技术、小角X 射线散射技术等)、显微技术(如激光共聚焦显微镜、扫描电子显微镜、透射电子显微镜等)或其它技术(如电位仪、界面张力测定仪等),这些技术通常组合使用,从而获得更加全面的液滴特性信息。Gharehbeglou 等[29]采用扫描电子显微镜(SEM)对由果胶和乳清蛋白制备的纳米乳液的颗粒形态进行分析,发现乳液中存在一些团簇状颗粒,形状近似球形且表面光滑,粒径为100~200 nm。
图3 果胶基纳米乳液的液滴特性
Fig.3 Drop characteristics of pectin-based nanoemulsions
果胶等生物大分子及其衍生物或复合物是构建纳米乳液的理想材料。一般来说,果胶基纳米乳液大致可以分为果胶衍生物纳米乳液和果胶复合物纳米乳液,分别通过果胶衍生物乳化剂和果胶复合物乳化剂来形成或稳定,以下将对这些果胶基乳化剂在纳米乳液油水界面的吸附机制及乳化性质进行介绍。
果胶分子中同时含有极性基团(如羟基)和非极性基团(如甲氧基),然而天然果胶中非极性基团的数量较少,导致其疏水性不足,因此不易在两相界面吸附[8-9]。有研究报道,果胶的乳化性能与其甲氧基的数量和分布密切相关,甲氧基的存在会增加果胶分子的疏水性,使其具有更高的表面活性[6,30]。果胶的甲酯化反应是指主链半乳糖醛酸单元上的游离羧基与甲醇发生的酯化反应,如图4所示,最常见的反应方法是用硫酸或盐酸酸化的甲醇处理果胶[31]。在反应过程中,果胶的主链和侧链可能会发生水解和断裂,使其分子间相互作用减弱,分子质量降低,溶解度增大[32-33]。
图4 果胶甲酯化衍生物的合成途径
Fig.4 Schematic diagram of the synthesis of pectin methylated derivatives
超高甲氧基果胶(DM>90%)是天然果胶的甲酯化产物,它具有较低的分子质量,较高的分子柔性,合适的极性基团和非极性基团比例,以及均匀的基团分布,因此能够更好地在两相界面伸展并降低界面张力。分子质量的减小和疏水性的增强,降低了其凝胶和增稠性能,却显著提高了其界面活性,因此超高甲氧基果胶能够形成粒径更小且更稳定的乳液,可作为一种新型的纳米乳液的乳化剂使用[15,32]。Hua 等[15]将柑桔果胶在无水甲醇中酯化制备出超高甲氧基果胶,其酯化度提高到91.2%,溶解度增加至5%(质量分数),分子质量降低至15 000 g/mol。研究还发现这种改性果胶在纳米乳液油水界面的吸附速度比未改性的果胶快,且具有更好地降低界面张力的能力。
2.2.1 果胶-小分子复合纳米乳液 果胶与一些小分子表面活性剂(如吐温、司盘等)联用时,小分子表面活性剂一般作为乳化剂,而果胶主要起到增稠或稳定乳液的作用[16,34-35]。吐温是一种低分子质量的表面活性剂,摩尔质量为1.310 g/mol,具有典型的极性头部和非极性尾部,在乳化过程中能够迅速涂覆在所形成的油水界面的表面。尽管吐温是非离子表面活性剂,但当乳液的pH 值高于4时,吸附在液滴表面的吐温仍带有少量的负电荷,这可能是由于油相或表面活性剂中存在一些阴离子杂质(如游离脂肪酸等)[36-37]。果胶是一种阴离子多糖,在该pH 值下由于羧基解离而带负电,因此果胶在油水界面的吸附可能会增大油滴表面的负电荷。
一些研究发现,吐温等小分子表面活性剂与带负电的生物聚合物在油水界面上可能存在竞争性吸附,即小分子表面活性剂会把生物聚合物从油水界面上部分或完全置换掉。然而如果小分子表面活性剂的数量不足以覆盖油水界面,则可能会导致生物聚合物分子的吸附,使油滴带有更大的负电荷[37-39]。Artiga-Artigas 等[40]阐明了果胶和小分子表面活性剂(吐温)在纳米乳液界面的相互作用机制。结果发现,吐温优先在油滴表面吸附,导致果胶在界面的吸附较弱。随着果胶浓度的增加,油滴表面的负电荷增加,这说明果胶也开始逐渐吸附到油水界面上。Guerra-Rosas 等[22]以果胶和吐温作为复合乳化剂制备了添加几种精油的纳米乳液。结果发现,牛至或百里香精油纳米乳液在贮藏过程中很不稳定,原因是果胶没有吸附到油水界面上,导致液滴之间的静电排斥力较弱,容易发生聚集或絮凝。然而柑橘或柠檬草精油纳米乳液表现出良好的贮藏稳定性,这可能是因为带负电的果胶在油水界面上吸附,使液滴之间产生较强的静电排斥作用。
2.2.2 果胶-蛋白质复合纳米乳液 蛋白质与多糖的相互作用在纳米乳液运载体系设计中起着重要作用[41]。与单独的生物大分子相比,果胶-蛋白质复合物具有更强的乳化性能和乳液稳定性,它们可以在油水界面形成更加致密的层,增加液滴之间的静电或空间排斥力,从而抑制液滴的聚结或絮凝[42]。根据相互作用的方式,果胶-蛋白质复合物主要包括非共价复合物和共价复合物,分别通过物理共混和美拉德反应的方式产生。
2.2.2.1 果胶-蛋白质非共价复合物 果胶与蛋白质之间的非共价相互作用可能包括静电相互作用、氢键、范德华力和疏水相互作用,其中静电相互作用是主要的驱动力[43-44]。果胶与蛋白质的静电相互作用受到许多参数的影响,例如:溶剂条件(如pH 值、离子强度)、生物聚合物的特性(如浓度、比例、电荷密度)和加工条件(如温度、机械应力、压力)等。这些参数决定了果胶-蛋白静电复合物的理化性质,例如溶解性等[45-47]。由于一些阴离子官能团,如羧基、磷酸基或硫酸基的存在,大多数天然果胶在pH>pKa 时带负电荷,而蛋白质在低pH 值时带正电荷,在高pH 值时带负电荷,在等电点(pI)处电荷为零。因而可以预测在pH 值低于蛋白质的pI 值时,带正电的蛋白质可以与带负电的果胶之间产生足够强的静电吸引力,从而彼此缔合形成果胶-蛋白静电复合物[48-49]。因此,果胶和蛋白质可通过这种静电作用的方式,构成纳米乳液的界面层,其中复合物中的蛋白质吸附在油滴的表面,决定了界面的流变学特性,而果胶链则位于复合物的外围并朝向外部水相,决定了液滴的带电特性[17]。
一般情况下,果胶-蛋白静电复合物稳定的纳米乳液可通过以下两种方法形成。第1 种方法是以蛋白质为乳化剂,果胶为稳定剂形成纳米乳液,即在乳化过程中,首先将蛋白质引入油滴表面形成第1 吸附层,然后将带相反电荷的果胶在蛋白质表面进行静电沉积,形成第2 吸附层[50];第2 种方法是以果胶-蛋白静电复合物为乳化剂形成纳米乳液,即果胶与蛋白质在特定的pH 值下发生静电络合,形成的生物聚合物可直接用于制备纳米乳液[17,20]。
2.2.2.2 果胶-蛋白质共价复合物 果胶-蛋白共价复合物可通过美拉德(Maillard)反应制备,即果胶还原端的羰基与蛋白质中氨基酸侧链的氨基相互作用形成共价键[51]。其中,果胶与蛋白质的比例、相对湿度以及反应时间和温度等因素均会影响该反应的效果[8]。果胶-蛋白共价复合物在稳定O/W 型纳米乳液中发挥着重要作用,因为蛋白质分子可以在油水界面吸附,并形成较厚的界面层,而果胶的亲水基则延伸到水相,通过空间排斥力阻止液滴的聚集,同时果胶的增稠效果还可以提高乳液体系的稳定性[52]。此外,美拉德反应的产物具有较强的抗氧化能力,可以阻止自由基的链式反应,进而抑制纳米乳液中油滴的氧化[53]。
果胶-蛋白共价复合物稳定的纳米乳液可以通过以下途径形成:首先将果胶与蛋白质按照一定的比例均匀混合,通过干热法使两者发生美拉德反应,得到果胶-蛋白共价复合物。然后再以该复合物作为乳化剂或稳定剂,将油相添加到含有共价复合物的水相中,均质后形成纳米乳液[54]。
生物活性物质是通过调节生理或细胞活动而对健康产生有益影响的物质,具有消炎、抗癌、抗氧化、神经保护、提高免疫力等生理活性[55]。然而一些生物活性物质对氧、光、热或湿度比较敏感,生物利用度较低,在食品和药物制剂中的应用非常有限[56-59]。近年来,利用改性果胶构建的纳米乳液体系,在包埋生物活性物质方面受到越来越多的关注。图6显示了果胶基乳化剂在单一或多重纳米乳液中封装生物活性物质的截面图。通常情况下,疏水性或亲水性生物活性物质被截留在分散相的液滴中,而连续相则为负载生物活性物质的分散相提供保护[60-61]。
图5 果胶衍生物和复合物在纳米乳液油水界面的吸附机制
Fig.5 Adsorption mechanism of pectin derivatives and complexes at the oil-water interface of nanoemulsions
图6 果胶基纳米乳液封装生物活性物质的截面图[2]
Fig.6 Cross-sectional view of pectin-based nanoemulsion encapsulating bioactive substances[2]
目前,果胶-小分子复合乳化剂和果胶-蛋白质复合乳化剂已经广泛应用于开发负载生物活性物质的纳米乳液。这可能是因为果胶复合物乳化剂具有更加复杂、多层次的界面结构,不易从两相界面解除吸附。表1总结了果胶复合物纳米乳液在生物活性物质包埋和释放中的应用,研究发现果胶复合物乳化剂可以提高纳米乳液在不同环境条件(如光、热、pH 值)下的理化稳定性,减少液滴聚集或重力沉降等现象的发生,提高生物活性物质的包封或保留效果。因此,果胶复合物纳米乳液可以作为维生素、天然色素或精油等活性成分的有效递送载体,在食品行业和纳米封装领域具有广阔的应用前景。
献文考[22][62][17][50][63][64][65][18][29][66][67]参稳外散溶稳稳盘苦剂埋散用应的中埋包质物性活物生在液乳米纳物合复胶果1表Application of pectin composite nanoemulsion in the encapsulation of bioactive substances现发要主率/%留保或率封包成构面界藏贮的液乳米纳强增会能可附吸的面界水油在胶果温吐+胶果油性定素因要重最的性定稳理物液乳米纳响影是胶果温吐+胶果的物合复于位链胶,果面表的滴油在附吸分部质白蛋白蛋清乳+胶果相水部外向朝并围),分数分量0.2%(质白蛋清),乳数分量0.5%(质胶果:88.9率留保白蛋清+乳胶果性定稳理物的高最有具系,体5%时数分积体相脂和素生维性溶水装封时同够能系体送递米纳种这钠酸白蛋酪+胶果素生维性热光的素卜萝β-胡中液乳米纳使物合复元三价共非:91.5率留保素黄+姜白蛋豆+豌胶果高提著显性定的好良有下度浓盐和理处热同不在液乳米纳油香丁:88率封包钠酸白蛋酪+胶果定稳不时pH 3.0~5.0在而,然性定),司数分量),WPC 8%(质数分量1.97%(质胶果:91率封包蛋清+乳胶/O);果Span(W1榄,橄1∶4,pH 6.1 时为相∶外相),内数分量8.74%(质)(O/W2白91%为率封包的苷化乳的当相温吐与种一为作可物合复白蛋清-乳胶果:85.8率封包蛋清+乳胶/O);果PGPR(W1液乳米纳重多定稳来)(O/W2白包是液乳米纳重多的定稳同共胶果与白蛋清乳过通:96.7率封包蛋清乳+胶);果/O(W1 Span体载效有的素花红藏)(O/W2白),分数分量4.0%(质白蛋清),乳数分量1.0%(质胶果:99率封包+胶/O);果Span/PGPR(W1 99%为率封包酸,叶pH 6 时15%,在数分积体相)(O/W2白蛋清乳Table 1 质物性活物生精等桔、柑草檬柠油精参姜烷癸素红茄番素生维素卜萝β-胡油香丁苷苦榄橄酸子食没素花红臧酸叶米纳合米纳合重多合型类复子分小-胶液复质白蛋-胶液复质白-蛋胶液乳米果乳果乳果纳
利用果胶-蛋白质复合乳化剂制备的纳米乳液,在包封生物活性物质方面比单独的生物大分子更有效,未来有望将其添加到饮料、化妆品和药物制剂等产品中,以延缓活性成分的氧化降解。Wang 等[63]用果胶涂覆酪蛋白酸钠制备纳米乳液,并开发了一款能够同时封装水溶性和脂溶性维生素的新型功能性饮料。结果表明,果胶-酪蛋白酸钠复合物具有较好的包埋效果和控释能力,能够很好地保持维生素的抗氧化活性。Yi 等[64]发现果胶、豌豆蛋白和姜黄素的三元复合物可以显著改善纳米乳液中β-胡萝卜素的化学稳定性,原因是果胶可以与蛋白质组装成更坚硬、更厚的界面层,限制了β-胡萝卜素与助氧化剂(金属离子、自由基、溶解氧)之间的相互作用,从而保护β-胡萝卜素免受光热降解。
此外,利用果胶-蛋白质复合物构建的多重纳米乳液,在活性物质的递送方面比单一纳米乳液更具优势。它们可以更好地控制活性成分的释放,或者掩盖一些不良的风味和感官特性等[66-69]。在果胶-蛋白质复合多重纳米乳液中,最常见的类型是以非离子表面活性剂(如司盘Span 或聚甘油蓖麻醇酸酯PGPR)为W1/O 相的乳化剂,以果胶-蛋白质复合物为O/W2 相的乳化剂构建的W1/O/W2 型纳米乳液。在O/W2 相中使用果胶-蛋白质复合物可以显著提高多重纳米乳液中生物活性物质的包封率,因为果胶-蛋白质复合物可以在O/W2 相界面形成弹性的聚合层,从而提高生物活性物质对环境应力的稳定性[66-67,70]。Esfanjani 等[66]以果胶-乳清蛋白复合物为O/W2 相的稳定剂制备了W1/O/W2 型多重纳米乳液,该体系使藏红花素的包封率得到显著提高。Assadpour 等[67]对含有果胶-乳清蛋白复合物的多重纳米乳液进行喷雾干燥处理,并对叶酸的封装工艺进行优化,结果发现叶酸的包封率高达99%。
由果胶-乳清蛋白复合乳化剂制备的纳米乳液具有稳定性高,封装效果好等特点,在营养因子及生物活性物质递送等领域发展迅速。而果胶与其它蛋白质(如大豆蛋白等)的复合物在纳米乳液中的应用研究还很少,因此可作为进一步探索或尝试的新方向。
果胶基纳米乳液是提高生物活性成分整体运载功效的新型递送系统。以果胶衍生物和复合物作为乳化剂或稳定剂构建的纳米乳液,由于其界面结构的复杂性和多层化,在增强体系的稳定性以及提高生物活性物质的生物利用度等方面表现出更好的效果。然而其设计和应用还面临一些挑战,例如:生物活性成分的负载量低,蛋白质等生物大分子对环境胁迫较为敏感,以及纳米乳液的安全性需要进行全面系统的评估等[71-74]。因此,今后对于果胶基纳米乳液递送体系的研究可重点关注以下几个方面:(1) 优化工艺参数和技术条件,提高生物活性物质的装载量和封装率;(2)构建高效的纳米乳液体内递送系统,实现靶向释放和定点吸收;(3)结合其它纳米包封技术,设计生物活性物质的共同递送或多重递送系统;(4)完善风险评估体系及制度框架,加强纳米乳液产品的安全控制与监管工作。
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Research Progress on Encapsulation of Bioactive Substances in Nanoemulsions Based on Modified Pectin