益生菌(probiotics)是一类通过调节宿主黏膜与系统免疫功能或调节肠道内菌群平衡、对宿主发挥健康作用的活性微生物[1],包括双歧杆菌属、乳杆菌属、链球菌属、乳球菌属、芽孢杆菌属、酵母菌属等。益生菌必须能够顺利到达肠道内且有足够活菌量(通常应达到至少106 CFU/g以上)才能达到有效递送效果,发挥其益生作用[2]。然而,大多数益生菌对加工和储存过程中的酸、氧气、热、光照、水分活度等外部环境,以及对人体消化道胃酸和胆盐的环境抵抗力差,致使活菌量大幅度降低,益生作用失效[3]。

为了提高益生菌的存活率,包埋技术是保护益生菌最有效的方法之一。近年来国内外都有益生菌包埋固态微胶囊的相关研究报道,但目前常见的固态包埋方法还存在不同缺点[4],比如挤压法和锐孔法的工业规模化生产难度较大;界面聚合法使用的有机溶剂对人体有害;喷雾干燥法的高温处理易使菌体存活率降低;喷雾冷凝法操作繁琐昂贵;挤流化床技术手段复杂等。乳液是不相溶的多相同时存在、以液滴(分散相)的形式分散在另一相(连续相)中的体系,具有与液体有高度兼容、分散液滴粒径小、易操作、适合大规模生产等优点[5]。其中,水包油包水(w/o/w)双重乳液相比其他乳液体系更能有效保护益生菌[6],益生菌可以包埋在双重乳液内层水相中,而益生菌在水中的溶解性普遍优于油;w/o/w双重乳液的水状体系能与更广泛的食品进行兼容,应用性更广;双重乳液的油相和外层水相都能保护在内层水相包埋的益生菌。故使用w/o/w双重乳液体系包埋益生菌对于提高益生菌的包埋和递送效果、促进其益生作用的充分发挥具有重要意义。

1 w/o/w双重乳液结构的优缺点

w/o/w体系是近年来研究最多、应用最广的双重乳液,其原因主要如下:(1)减脂:相对于o/w乳液,w/o/w双重乳液是脂肪减少的体系,并被应用于减脂食品和脂肪替代物的开发[7];(2)减糖[8]、减盐[9]:w/o/w双重乳液可以通过2种途径提高人体对味觉物质(糖、盐)的感知:一方面在外层水相中加入高浓度的味觉物质、在内层水相中加入低浓度的味觉物质,并保证体系中总浓度降低、对口腔处理稳定,从而达到口感没有减弱、但味觉物质总摄入量降低的效果;另一方面则是在外层水相中低浓度、在内层水相中高浓度且保证体系总浓度降低,经口腔处理后破乳释放出内层味觉物质;(3)生物活性物质递送体系:w/o/w体系的双层结构决定了其既可以包封亲水性生物活性物质在内层水相中,也可以包覆亲油性生物活性物质在油相中,甚至达到共包埋、协同增效的作用[10]。

双重乳液属于热力学不稳定体系,因此易受乳液结构组成、内外层水相之间的渗透压不平衡、制备方法及条件参数、加热、储存条件等物理化学因素的影响而失稳。常见的w/o/w双重乳液不稳定现象主要包括:(1)聚合:同类型液滴合并形成更大液滴的过程;相对于内层水滴之间的聚合,油滴-油滴聚合更容易出现在双重乳液中,且能改变双重乳液特征[11];(2)沉积:由于油水密度差导致,又称为重力分层,普遍存在于液体形式的双重乳液体系中;(3)分层:双重乳液液滴界面膜彻底破裂,最终形成w/o相浮于上方、水相沉于底部的分层现象;避免聚合、沉积等不稳定现象是减少双重乳液分层的最有效方法[12];(4)絮凝:当分散液滴之间的相互吸引力大于排斥力时出现的现象;乳化剂或增稠剂浓度过高都是促进双重乳液出现絮凝现象的重要因素[11];(5)奥氏熟化:小液滴扩散至大液滴内部形成更大液滴的现象;发生于分散相过大导致乳液黏度突然降低的情况或温度、离子强度等环境条件变化。斯托克定律明确提出了液滴粒径、连续相黏度、两相密度差是影响乳液分层的决定因素,即粒径越小、两相密度差越小、连续相黏度越高,液滴移动速度越慢,乳液越稳定[13]。因此,通过筛选制备方法和乳化剂以减小内层水滴和w/o油滴粒径并形成更稳定的液滴界面膜、添加加重剂提高油相密度、加入胶体提高内外层水相的黏度等方式都是常用于改善双重乳液结构稳定性的有效措施[14]。

2 w/o/w双重乳液结构的制备方法

双重乳液主要通过两步法制成,即首先将含亲油性乳化剂的油相与水相混合制成w/o初乳,再将w/o初乳与含亲水性乳化剂外层水相混合制成w/o/w双重乳液。乳化过程通常采用高速剪切、高压均质、超声乳化等传统方法,但第二乳化步骤需要投入更低的机械力以免破坏双重乳液的双层结构[15]。虽然传统乳化方法具有制备液滴粒径小、稳定性高、适用于工业规模化生产等优势,但通常会引起温度升高、粒径呈多峰分布且耗能高。而低机械力的新型乳化方式能弥补传统乳化方法的缺陷,并具有粒径均一可控、适用于热或剪切力敏感的材料等优点[11]。膜乳化方法是通过一定压力将分散相压过大小均匀的微/纳孔膜,并在温和剪切力作用下使分散液滴从膜表面剥离进入连续相中进而形成乳液的过程[16]。笔者课题组对比发现膜乳化法制得的双乳不仅粒径均一性显著提高,且可通过理论模型预测乳液粒径及通过设备参数变化对粒径进行调控,并能实现矿物质[17]、香味物质[18]等生物活性物质缓释、控释的目的。除此之外,微流控技术也是制备双重乳液的新型乳化方式,主要通过微管道将分散相强制转化为不混溶的连续相,从而生成单分散性液滴,具有内液滴数量、液滴直径和界面膜厚度可精确控制的优点[19]。但制得的乳液粒径较大、通量较低等问题仍是目前限制新型乳化方式在食品工业中应用的主要原因。

双重乳液也可通过一步法制得[20]。相转变乳化法是将水添加到已经生成的w/o乳液中以刺激相浓度的变化,从而生成w/o/w乳液,这种方法也被称为突变相反转或乳液反转点[21]。在w/o乳液加热至层状相后,通过改变温度的方法可以制备过渡型w/o/w双重乳液,这个过程被称为相变温度技术[14]。双重乳液也可通过微流控技术一步法制得,即不同流速的三相流体同时在一个交叉口相遇,并由互不相溶的内层水相和油相组成的同轴射流破裂,在外层水相形成w/o液滴[19]。一步法虽然操作简易,但所制备的双重乳液通常稳定性较差。

3 w/o/w双重乳液的结构调控

3.1 液体

乳化剂的选择是成功构建w/o/w双重乳液的关键因素之一(图1)。聚甘油蓖麻醇酯(polyglycerol-6 polyricinester,PGPR)是最广泛用于稳定w/o乳液或w/o/w内层油水界面的亲油性表面活性剂,但存在安全食用限量(每日容许摄入量为25 mg/kg)、异味及消费者对人工合成添加剂抵触等缺陷[22],对双重乳液的可食用性也存在影响。故找到能替代甚至超越PGPR乳化性的亲油性乳化剂(特别是天然乳化剂)一直是研究关注的焦点。ZHANG等[23]评估了蔗糖油酸酯替代PGPR制备w/o/w双重乳液的效果,发现蔗糖酯稳定的w/o乳液中水滴出现聚集现象,且这种微观结构延续到双重乳液体系中,而PGPR稳定的w/o乳液无聚集现象且水滴粒径更小;两者对双重乳液盐包埋率的影响不相上下。GOIBIER等[24]以无水乳脂肪稳定内层水滴、以酪蛋白酸钠稳定w/o油滴制得w/o/w双重乳液;对比PGPR,脂肪晶体稳定的w/o/w双重乳液在延缓盐离子释放、抗渗透压、抗聚合、热响应释放等方面的性能得到提高。

用于稳定w/o油滴的亲水性乳化剂类型众多(表1),主要包括表面活性剂和蛋白质、多糖、多酚及这三者之间通过不同添加顺序(比如逐层覆盖、共混合等)、以共价(包括美拉德反应、酶促交联、化学交联)或非共价结合作用(比如静电、氢键、疏水作用等)形成的二元或三元复合物等[25-26](图1)。WANG等[27]利用细菌纤维素纳米颗粒稳定的w/o/w双重乳液可提高嗜酸乳杆菌(Lactobacillus acidophilus)在酸性(pH 1.5,120 min)、贮存(4 ℃,14 d)和模拟胃肠消化环境中的存活率(分别约达93.45%、59.62%和15.76%)及结肠黏附率(43.27%),而未被包埋的益生菌在贮存后存活率为49.65%、在酸性和模拟胃肠消化环境中全部死亡且结肠黏附率为14.2%。LIANG等[28]制备的乳清浓缩蛋白和果胶静电稳定w/o/w双重乳液能显著提高鼠李糖乳杆菌(Lacticaseibacillus rhamnosus)经贮存(4 ℃,28 d)、巴氏杀菌(63 ℃,30 min)和模拟胃肠消化后的存活率(分别损失约1、4和3.6 lg CFU/mL),而未被包埋的益生菌在贮存后损失3.08 lg CFU/mL、在杀菌和模拟消化后全部死亡。这可能归功于蛋白质和多糖可以协同形成更稳定、更致密、更高机械强度的凝胶网状结构,从而提高双重乳液对外部环境的稳定性及对包埋物的保护效果[29]。另外,蛋白质-多糖共价交联复合物[30]和蛋白质-多糖-多酚三元结合物[26]均展现出优异的w/o/w双重乳液制备稳定性和生物活性物质包埋递送效果。

3.2 水凝胶

水凝胶(hydrogel)是通过调节pH值、加热、冷却、盐离子、酶促交联、高压、3D打印等方式诱导形成、可保持大量水不溶解的生物聚合物分子三维交联网络体系[37](表1)。w/o/w双重乳液的水凝胶结构主要有3种类型(图1):内层水相凝胶化[38]、外层水相凝胶化[39]和内外双层水相凝胶化[40]。LI等[41]对比了内层和外层水相分别凝胶化处理对w/o/w乳液稳定性的影响,发现2种凝胶化处理均能降低油水界面张力,但内层水相胶凝的乳液有更小的粒径、更高的维生素B12包埋率、能更好保护内层水相抵抗温度和pH值的变化以及更高的生物活性物质生物利用度,而外层水相胶凝的乳液有更高的净ζ电位值、更稳定的胶体结构和更强的抗氧化效果。FRAKOLAKI等[42]发现在4和-18 ℃贮存4周、模拟胃肠消化及pH 4环境中,外层水相凝胶化的w/o/w乳液凝胶珠中乳双歧杆菌(Bifidobacterium subsp.lactis)活性分别为6 lg CFU/g以上、68.6%～86.1%和80%以上,且藻朊酸盐-菊粉-壳聚糖三层覆盖的双重乳液凝胶珠能达到最好的保护效果,而藻朊酸盐凝胶珠中益生菌活性分别仅能保持15 d、46.8%和67.32%。以上研究表明,通过内外层水相的凝胶化处理形成双重乳液水凝胶结构,是提高双重乳液稳定性、改善双重乳液物理化学性质及改善生物活性物质递送效果的有效途径。

3.3 油凝胶

油凝胶是利用凝胶剂(如单硬脂酸甘油、硬脂酸甘油、可可脂、单双酰甘油、天然蜡、植物甾醇、乙基纤维素、氢化油等)自组装作用使液态油脂凝固成为晶体,进而形成一个三维网络结构的油脂混合体系(表1)。w/o/w双重乳液中的油相可形成油凝胶结构(图1),其作用不仅可以限制内层水相及其内部包埋生物活性物质的移动和与外部环境因子的接触,同时可以提高油凝胶中亲脂性生物活性物质的活性、以实现功能最大化[43]。含油凝胶结构的双重乳液在开发健康油脂产品、改善生物活性物质功能活性等方面具有重要的应用前景[44-45],但其在益生菌递送方面的研究应用尚未见报道。w/o乳液油凝胶结构已被证实可通过降低脂肪消化程度而提高益生菌经模拟消化后的存活率[46],而关于w/o/w双重乳液油凝胶结构对益生菌的影响机制还有待深入研究。

3.4 微胶囊

微胶囊是一种以乳液为制备基础,利用物理(喷雾干燥、冷冻干燥、静电喷涂、挤出技术、分子微囊化技术等)或化学方法(复合凝聚、复合沉淀、原位聚合等)进行固化处理制成的微型胶囊或微粒(表1)。从实际应用的角度来看,粉状或固体微胶囊由于易操作性和较长的保质期而受到食品工业的青睐。对于w/o/w乳液基微胶囊(图1),亲水性生物活性物质被固定在固体w/o微胶囊的内层基质中,固体油脂层和壁材层形成双层物理屏障,因此被包裹的生物活性物质与外界因素的相互作用受到限制,其理化稳定性能得到显著提高[47]。喷雾干燥和冷冻干燥是在食品工业中常用于将双乳转化为固体产品的物理技术,前者具备低成本、快速加工、高产量等优点,后者适用于蛋白质、肽、不饱和脂肪酸、香精、益生菌等热敏性成分的包覆。然而,这些传统干燥方法还存在一定缺陷,比如喷雾干燥的高温过程、冷冻干燥的耗时耗能均能对益生菌的活性造成较大损伤。而近年来基于电流体动力学的静电喷涂、静电纺丝等技术展现出可观的应用潜力。BELDARRAIN-IZNAGA等[48]采用静电喷涂法将藻朊酸钙和壳聚糖依次喷涂在酪蛋白酸钠稳定的w/o液滴表面,再利用冷冻干燥法制得粒径为8.5 μm的三层覆盖w/o微胶囊;经冷冻干燥和模拟胃肠消化后,酪蛋白酸钠-藻朊酸钙-壳聚糖微胶囊中干酪乳杆菌(L.casei)活菌数仅损失0.1和3.1 lg CFU/g,而冷冻干燥后未被包埋、酪蛋白酸钠单层微胶囊、酪蛋白酸钠-藻朊酸钙双层微胶囊分别损失约2、0.8和0.4 lg CFU/g,模拟胃肠消化后分别全部死亡、损失7.3和5.5 lg CFU/g。

4 w/o/w双重乳液在益生菌递送中的应用

w/o/w双重乳液体系的结构调控对于益生菌在多项食品领域中的应用起到了推动作用,包括结肠靶向递送,应用于酸奶、奶酪、冰淇淋、饮料、果蔬、肉制品等。

4.1 结肠靶向递送

在设计结肠靶向递送体系时,重要的是要确保递送体系中的益生菌不会在口腔、胃或小肠中释放或吸收。通过w/o/w双重乳液包封益生菌可以提高益生菌经模拟胃肠消化后活性和结肠黏附效果,从而帮助益生菌在结肠中定植、充分发挥其益生作用。然而,大多数生物聚合物如淀粉、蛋白质和脂类会在上消化道被消化,不适用于制备结肠靶向递送载体,而膳食纤维(如藻朊酸盐和纤维素)在胃肠道上部不易消化、且可在结肠内被水解或发酵,因此膳食纤维的添加有助于实现双重乳液结肠靶向递送益生菌[49]。DING等[50]发现经模拟消化后,1%和1.5%藻朊酸盐的w/o/w双重乳液水凝胶珠在模拟肠液中具有持续释放能力,且消化6 h时罗伊氏乳杆菌(L.reuteri)活菌数超过107 CFU/mL;在4 ℃保存90 d后,1.5%藻朊酸盐水凝胶珠的罗伊氏乳杆菌活菌数为8.4×107 CFU/mL。QIN等[51]以乳清分离蛋白-表没食子儿茶素没食子酸酯共轭物和藻朊酸盐为亲水性乳化剂制备了包埋植物乳杆菌(L.plantarum)的pH响应性w/o/w双重乳液;对比未被包埋的植物乳杆菌(仅损失1.75 lg CFU/mL),双重乳液能有效提高益生菌经模拟胃肠消化后的活菌数(仅损失0.01 lg CFU/mL)。以上研究表明w/o/w双重乳液是一种可用作益生菌补充配方、帮助益生菌肠道定植的有效策略。

4.2 酸奶

应用于酸奶中的w/o/w双重乳液具有分离发酵益生菌的作用、以避免对发酵剂干扰,以及保护益生菌免受不良加工和消化条件的影响[52]。质构是酸奶的基本感官特性之一,但双重乳液的添加可能会引起凝胶硬度差、脱水等质构变化,影响消费者的接受度,因此构建能保护益生菌、同时不影响酸奶品质变化的双重乳液结构是关键。EL KADRI等[53]发现用w/o/w双重乳液包封的副干酪乳杆菌(Lactobacillus paracasei)对储存期间和模拟胃肠道条件抵抗力增强,活菌数高于普遍接受的最低浓度,且含有副干酪乳杆菌w/o/w双重乳液的凝固型酸奶在形态自由、质地稳定、细菌存活率高等方面与含有副干酪乳杆菌的酸奶具有相当的理化特性。LALOU等[54]发现益生菌w/o/w双重乳液的添加可降低凝固型酸奶的酸化速率、黏度和保水能力,发酵过程和结构性能均在可接受范围内发生变化。这些结果证明w/o/w双重乳液可用于酸奶益生菌活性保持,可以在不干扰发酵剂培养和发酵的情况下提高益生菌的功能性。

4.3 奶酪

与酸奶等其他发酵乳制品相比,奶酪被认为是一种优秀的功能性成分输送工具,因为它具有相对较高的pH值、脂肪含量、固体稠度和高缓冲能力[55]。但奶酪加工过程中的凝乳方式、成熟程度、长期贮存过程及食用消化环境等因素不仅会极大降低益生菌活菌量,而且会较大程度影响双重乳液结构的稳定性,因此高度稳定的双重乳液结构对于益生菌在奶酪中的递送至关重要。RODRGUEZ-HUEZO等[56]将植物乳杆菌和龙舌兰汁或甜乳清包裹在双重乳液内层水相中用于制造奶酪,加工后对照游离组、甜乳清奶酪和龙舌兰汁奶酪中益生菌活菌数分别为6.81、8.15和8.22 lg CFU/g;奶酪熔解后,对照游离组、甜乳清奶酪和龙舌兰汁奶酪中益生菌的存活率分别下降2.18、1.4和1.9 lg CFU/g;暴露于模拟胃肠道条件(pH 2.3,胆盐)后,甜乳清奶酪和龙舌兰汁奶酪中益生菌活性没有显著变化(活菌数分别为6.32和6.02 lg CFU/g),而对照游离组活性显著降低至3.36 lg CFU/g。故w/o/w双重乳液可以保护益生菌对奶酪加工过程及储存条件的抵抗力、保证益生菌活性,并有利于减脂食品的开发。

4.4 果蔬

在调控食品微观结构特征的加工条件下,w/o/w双重乳液可用于植物基质的益生菌富集,以改善食品的功能特性和营养价值。HUERTA-VERA等[57]利用渗透脱水技术,以w/o/w双重乳液包裹鼠李糖乳杆菌(L.rhamnosus)富集香蕉片,发现包裹的鼠李糖乳杆菌在高渗溶液和渗透脱水香蕉中活菌数达107 CFU/g以上,且黏附在香蕉表面。FLORES-ANDRADE等[58]采用渗透脱水法和真空渗透脱水法将新鲜苹果组织在m(w/o/w双重乳液)∶m(蔗糖溶液)=1∶9的渗透溶液中浸泡,发现真空处理促进了鼠李糖乳杆菌的更高浸渍率,渗透脱水后苹果组织中益生菌活菌量为106～108 CFU/g。因而w/o/w双重乳液可赋予果蔬制品益生菌的功能特性、同时保证益生菌活性。

5 结语

利用现代加工技术和结构组成设计调控w/o/w双重乳液体系多元化结构、以提高其稳定性及益生菌递送效果一直是学术和工业界研究的焦点。目前针对w/o/w双重乳液体系的结构调控已出现了一系列有效的策略,包括但不限于:开发替代PGPR的亲油性乳化剂以避免人工合成表面活性剂的使用,优化亲水性乳化剂以改善w/o液滴稳定性,油相、内层水相和/或外层水相凝胶化以加强双重乳液稳定性,开发固化w/o微胶囊以提高产品稳定性和包埋递送效果等。但双重乳液油凝胶结构的设计及其对益生菌递送的影响尚有待深入探索。针对益生菌对加工、贮存和消化等环境抵抗力差的问题,w/o/w双重乳液可将益生菌包覆在内层水相内部,实现益生菌在肠道定植、酸奶、奶酪、果蔬等方面的应用,具备有效递送益生菌、促进其益生作用发挥的应用前景。未来,w/o/w双重乳液益生菌递送系统的研究工作可在如下方面进一步探索:(1)利用对益生菌友好的新型制备技术(比如静电纺丝、静电喷涂、微流控、膜乳化等),开发保证益生菌活性的w/o/w双重乳液体系;(2)开展益生菌与其他生物活性物质共包埋的w/o/w双重乳液递送体系的构建机制及性能研究,实现多种生物活性功能共同递送的效果;(3)结合w/o/w双重乳液的减脂、减糖、减盐等优点,开发含活性益生菌的低脂食物、脂肪替代物等对人体健康的新型食品。

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A review in the structure manipulation of w/o/w double emulsions and applications in probiotics delivery

PU Xiaolu*

(College of Food Science and Biology, Hebei University of Science and Technology, Shijiazhuang 050018,China)

ABSTRACT To enhance the stability of double emulsions and the delivery performance of bioactives, water-in-oil-in-water (w/o/w) emulsions can form diverse structure through modern processing technologies and structural composition design, such as liquid, hydrogel, oleogel, microcapsule, and so on. The structure manipulation of w/o/w double emulsions can incorporate probiotics into the inner water phase and improve the resistance ability of probiotics against undesirable environmental conditions, which assists the implementation of its application in colon-targeted delivery, yogurt, cheese, fruit, and vegetables. With the advantages, disadvantages and manufacturing methods of w/o/w double emulsion structure as a starting point, this article comprehensively introduces the latest research on the diverse structure of w/o/w double emulsion system and the application progress in probiotics delivery. It can provide references for the design and development of novel fat-reduced food within encapsulated viable probiotics.

Key words w/o/w double emulsions; structure manipulation; gel; probiotics delivery; colon-targeted delivery; fat reduce

第一作者：博士,讲师(通信作者,E-mail:xiaolu.pu@hebust.edu.cn)

基金项目：河北省自然科学青年基金项目(C2020208017);河北省2020年引进留学人员资助项目(C20200326,C20200503);河北省重点研发计划项目(22321501D)

收稿日期：2023-01-26,改回日期：2023-02-17

DOI：10.13995/j.cnki.11-1802/ts.034948

引用格式：蒲晓璐.w/o/w双乳结构调控及在益生菌递送中的应用进展[J].食品与发酵工业,2023,49(11):280-287.PU Xiaolu.A review in the structure manipulation of w/o/w double emulsions and applications in probiotics delivery[J].Food and Fermentation Industries,2023,49(11):280-287.