大豆亲脂性蛋白的界面吸附、乳化及输送特性研究
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摘要
商业大豆分离蛋白中除7S和11S储藏蛋白外，还含有一种含量较高（31%）的富含磷脂的蛋白―大豆亲脂性蛋白（lipophilic protein, LP）。LP蛋白是包含大豆油体蛋白的蛋白质―磷脂复合体。目前的研究表明，LP蛋白具有良好的生理保健功能。但由于在水中的溶解性很差，其作为功能性蛋白的研究还未见报道。本文系统研究了大豆亲脂性蛋白的功能特性，包括界面吸附、乳化特性及输送特性，并在此基础上提出了疏水蛋白/小分子表面活性剂复合界面的概念。结果如下：
1.本文发现利用简单超声处理LP蛋白，可制备具有良好分散能力的纳米颗粒，大大改善其溶解性，使其工业化应用成为可能。超声处理LP蛋白能够形成粒径为136±0.8nm，表面电荷为-20.0±0.3mV的纳米颗粒。通过表面疏水性分析以及磷脂酶处理，发现该纳米颗粒为磷脂覆盖的疏水蛋白聚集体。这种复合颗粒能够扩散至界面并在界面上形成由蛋白颗粒聚集物与磷脂组成的复合界面，该复合界面能够耐受小分子表面活性剂（Tween20）的竞争性取代。采用界面流变技术分析了磷脂酶和蛋白酶对颗粒的界面流变行为的影响，发现磷脂和疏水蛋白颗粒之间存在着协同作用。该协同作用使得大豆亲脂性蛋白纳米颗粒制备的乳液在长期存储（8weeks）、高温―高盐(90℃/200mM NaCl)处理以及添加小分子表面活性剂（4wt.%Tween20）等情况下具有良好的物理稳定性。
2.研究了油体界面组成对其稳定性的影响。通过不同有机溶剂（丙酮、正己烷、氯仿/甲醇）对大豆油体脱脂以控制重组油体界面的油体蛋白和磷脂比例。结果证实较高的磷脂含量有利于油体蛋白在油水界面的吸附并能获得较高的界面压和界面粘弹模量。油体蛋白和磷脂复合物形成的重组油体的物理稳定性和氧化稳定性随磷脂含量的提高而提高。
3.在上述研究的基础上，本文提出了疏水蛋白与小分子表面活性剂形成的复合界面的概念，并对此机制进行了验证。用疏水蛋白zein与小分子表面活性剂硬脂酸钠模拟天然复合界面制备乳液，并采用凝胶捕获技术观察了zein颗粒在油-水界面的形貌及三相接触角。发现小分子表面活性剂控制疏水蛋白颗粒在界面上的吸附。在硬脂酸钠浓度较低时（2.5mM），zein在界面上的吸附量较少，呈规则的球状，并且三相接触角远低于90°。当硬脂酸钠浓度较高时（10mM），zein颗粒部分展开且在界面上形成了致密的吸附层，三相接触角接近或略大于90°。利用zein和硬脂酸钠复合物成功制备了稳定的食品级Pickering乳液，并且该乳液能够在不使用饱和脂肪酸或反式脂肪酸的情况下经过冻干形成结构化油脂。
4.本文探索了LP蛋白纳米颗粒作为新类型高荷载的输送载体的可能性。以大豆亲脂性蛋白纳米颗粒作为疏水性活性物质的输送载体，通过超声诱导共组装技术成功荷载了共轭亚油酸，且荷载能力高达26.3±0.4wt.%，荷载共轭亚油酸的纳米颗粒粒径为170±0.63nm。采用红外光谱和X-光衍射证实了共轭亚油酸在颗粒内部的荷载。采用顶空氧气消耗量和过氧化物含量分析了共轭亚油酸的氧化稳定性，结果表明采用大豆亲脂性蛋白纳米颗粒荷载的共轭亚油酸相比酪元酸钠荷载的共轭亚油酸和共轭亚油酸的乙醇溶液具有更好的氧化稳定性。此外，大豆亲脂性蛋白纳米颗粒荷载的共轭亚油酸在模拟胃肠液消化过程中具有缓释特征。
5.本文提出了一种双功能胶体颗粒的概念，该颗粒既能作为界面稳定剂，又具有抗氧化性。通过将维生素E荷载至大豆亲脂性蛋白纳米颗粒中，制备了同时具有界面稳定性和抗氧化性的双功能纳米颗粒。将该颗粒制备的藻油乳液冻干处理后成功制备出结构化的藻油。采用激光共聚焦显微镜表征了该结构化油脂的内部结构，观察到大豆亲脂性蛋白纳米颗粒形成了立体网络结构，而油滴在该网络内部呈独立分散状态。剪切流变分析显示该结构化油脂具有明显的固体特征，其流变学特性可通过乳液中大豆亲脂性蛋白纳米颗粒的浓度调节。由于抗氧化剂在界面富集，采用该双功能纳米颗粒制备的结构化藻油比油相中添加维生素E的结构化藻油和未结构化处理的藻油具有更好的氧化稳定性。
Other than the storage protein glycinin and β-conglycinin, soy lipophilic protein (LP) isthe third principle fraction (content of31%) in commercial soy protein isolate, which is richin OBBP and soy phospholipids and presents as an amorphous aggregates in aqueous phase.Studies have shown that LP has a good physiological function. Due to their poor solubilityand diffusivity, few researches take notes on their functionality such as interfacial andemulsifying properties. This thesis has evaluated the functionality of LP systematically,including its interfacial adsorption, emulsifying properties and as a delivery device.Furthermore, a concept of “complex interface” composited with hydrophobic protein andsmall molecular weight surfactant was proposed based on the above studies. The main resultsinclude following.
1. This thesis find that a nanoparticle can be formed by simple ultrasonic treatment, whichhad an excellent diffusion capacity, improved functionality and be potential to be used in foodindustry. LP can be transformed into a sphere particle with a size of136±0.8nm and surfacecharge of-20.0±0.3mV by ultrasonic treatment. Though the surface hydrophobicity analysisand treated with phospholipase, the structure of the particle was confirmed which wasaggregates of the hydrophobic protein with phospholipids covered on the surface. LP particlewas able to diffuse to the interface and form a complex layer composited with thehydrophobic protein aggregates and phospholipids. The rigid interface was so stable that itwas resistant to be displaced by the small molecular surfactant Tween20. Interfacial rheologytechnology was used to investigate the effects of phospholipase and protease on the interfacialrheology behavior of LP particle, which found that there existed synergic effects between thephospholipids and the hydrophobic protein particles. The synergic effects also be beneficialfor the stability of emulsions which had an excellent physical stability even at long-termstorage (8weeks), heating-salting treatment (90℃/200mM NaCl) and the competitivedisplacement of SMW surfactant (4wt.%Tween20).
2. To evaluate the effects of composition of the complex on its stability, soy oil body wasdegreased by organic solvent and then used to form a recombiant oil body. Three differentorganic solvent (acetone, n-hexane, chloroform/methanol) was used to degrease the soy oilbody to control the ratio between OBBP and phospholipids on the surface of recombiant oilbody. Results showed that high content of phospholipids could improve the adsorption of theOBBP on oil-water interface and obtained a high surface pressure and dilatational module. The physical and oxidant stability of recombinant soy oil body formed by the complex ofOBBP and phospholipids was improved by the increasing of phospholipids content.
3. Based on the above studies, a concept of “complex interface” composited withhydrophobic protein and small molecular weight surfactant was proposed and comfirmed.Zein and sodium stearate (SS) were used to simulate the natural complex interface. The geltrapping technology (GTT) was applied to observe the morphology and interfacial contactangle of zein particles on oil-water interface. Results showed that SS controlled the adsorptionof zein on oil-water interface. At a low content of SS (2.5mM), the amount of zein adsorptedon interface was low and they presented as regular spherical particles, their contact angle wasfar less than90°. When at a higher content of SS, the particially unfolded zein particleaccumulated on the interface formed a condensed packed layer, their contact angle wasassumed to be closed to or slightly higher than90°according to the SEM. The complex ofzein and SS could be used to form a stable food grade Pickering emulsion, and such stableemulsion could be further freeze-dried to form a structured fat without the usage of trans-orsaturated fatty acids.
4. The possibility of LP nanoparticle act as a novel delivery vehicle with hight loadingcapacity for the hydrophobic bioactives was explored. LP nanoparticles as a delivery devicefor the hydrophobic bioactives can load conjugated linoleic acids (CLA) successfully byultrasonic induced assembly, and the loading capacity of this system are as high as26.3±0.4wt.%. The CLA-loaded particle has a size of170±0.63nm. Infrared spectrum and X-raydiffraction were used to confirm the entrapment of CLA in the particles. The oxidationstability of CLA was evaluated by the headspace oxygen consumption and peroxide value.Results showed that CLA loaded in soy lipophilic protein nanoparticles have improvedoxidation stability than it loaded in sodium caseinate micelle or in ethanol. Moreover, theCLA loaded in soy lipophilic protein particles had resistant release characteristics whensubjected to the simulated gastrointestinal digestion.
5. The thesis had developed a concept of “double-functional colloidal particle” which couldact as interfacial stabilizer and antioxidants simultaneously. A double-functional nanoparticlecould be formed by entrapping the VEin LP nanoparticles via ultrasonic induced co-assembly.Algae oil emulsions stabilized by this double-functional nanoparticles could form an oil gel(structured fat) when subjected to freeze-drying. Laser scan confocal microscope was appliedto characterize the internal structure of this oil gels. It could be observed that a3-D networkwas formed by soy lipophilic protein nanoparticles and the oil droplets entrapped in thisnetwork separately. Oscillating rheology analysis showed that the formed oil gel in this research had an apparent solid characteristic, and its rheology properties were tunableaccording the concentration of nanoparticles in emulsions. Due to the enrichment ofanti-oxidants on the interface, the algae oil in the double functional particles formed oil gelwas performed to have improved oxidation stability as compared to the case that VEin oilphase or fluid algae oil.
1.本文发现利用简单超声处理LP蛋白，可制备具有良好分散能力的纳米颗粒，大大改善其溶解性，使其工业化应用成为可能。超声处理LP蛋白能够形成粒径为136±0.8nm，表面电荷为-20.0±0.3mV的纳米颗粒。通过表面疏水性分析以及磷脂酶处理，发现该纳米颗粒为磷脂覆盖的疏水蛋白聚集体。这种复合颗粒能够扩散至界面并在界面上形成由蛋白颗粒聚集物与磷脂组成的复合界面，该复合界面能够耐受小分子表面活性剂（Tween20）的竞争性取代。采用界面流变技术分析了磷脂酶和蛋白酶对颗粒的界面流变行为的影响，发现磷脂和疏水蛋白颗粒之间存在着协同作用。该协同作用使得大豆亲脂性蛋白纳米颗粒制备的乳液在长期存储（8weeks）、高温―高盐(90℃/200mM NaCl)处理以及添加小分子表面活性剂（4wt.%Tween20）等情况下具有良好的物理稳定性。
2.研究了油体界面组成对其稳定性的影响。通过不同有机溶剂（丙酮、正己烷、氯仿/甲醇）对大豆油体脱脂以控制重组油体界面的油体蛋白和磷脂比例。结果证实较高的磷脂含量有利于油体蛋白在油水界面的吸附并能获得较高的界面压和界面粘弹模量。油体蛋白和磷脂复合物形成的重组油体的物理稳定性和氧化稳定性随磷脂含量的提高而提高。
3.在上述研究的基础上，本文提出了疏水蛋白与小分子表面活性剂形成的复合界面的概念，并对此机制进行了验证。用疏水蛋白zein与小分子表面活性剂硬脂酸钠模拟天然复合界面制备乳液，并采用凝胶捕获技术观察了zein颗粒在油-水界面的形貌及三相接触角。发现小分子表面活性剂控制疏水蛋白颗粒在界面上的吸附。在硬脂酸钠浓度较低时（2.5mM），zein在界面上的吸附量较少，呈规则的球状，并且三相接触角远低于90°。当硬脂酸钠浓度较高时（10mM），zein颗粒部分展开且在界面上形成了致密的吸附层，三相接触角接近或略大于90°。利用zein和硬脂酸钠复合物成功制备了稳定的食品级Pickering乳液，并且该乳液能够在不使用饱和脂肪酸或反式脂肪酸的情况下经过冻干形成结构化油脂。
4.本文探索了LP蛋白纳米颗粒作为新类型高荷载的输送载体的可能性。以大豆亲脂性蛋白纳米颗粒作为疏水性活性物质的输送载体，通过超声诱导共组装技术成功荷载了共轭亚油酸，且荷载能力高达26.3±0.4wt.%，荷载共轭亚油酸的纳米颗粒粒径为170±0.63nm。采用红外光谱和X-光衍射证实了共轭亚油酸在颗粒内部的荷载。采用顶空氧气消耗量和过氧化物含量分析了共轭亚油酸的氧化稳定性，结果表明采用大豆亲脂性蛋白纳米颗粒荷载的共轭亚油酸相比酪元酸钠荷载的共轭亚油酸和共轭亚油酸的乙醇溶液具有更好的氧化稳定性。此外，大豆亲脂性蛋白纳米颗粒荷载的共轭亚油酸在模拟胃肠液消化过程中具有缓释特征。
5.本文提出了一种双功能胶体颗粒的概念，该颗粒既能作为界面稳定剂，又具有抗氧化性。通过将维生素E荷载至大豆亲脂性蛋白纳米颗粒中，制备了同时具有界面稳定性和抗氧化性的双功能纳米颗粒。将该颗粒制备的藻油乳液冻干处理后成功制备出结构化的藻油。采用激光共聚焦显微镜表征了该结构化油脂的内部结构，观察到大豆亲脂性蛋白纳米颗粒形成了立体网络结构，而油滴在该网络内部呈独立分散状态。剪切流变分析显示该结构化油脂具有明显的固体特征，其流变学特性可通过乳液中大豆亲脂性蛋白纳米颗粒的浓度调节。由于抗氧化剂在界面富集，采用该双功能纳米颗粒制备的结构化藻油比油相中添加维生素E的结构化藻油和未结构化处理的藻油具有更好的氧化稳定性。
Other than the storage protein glycinin and β-conglycinin, soy lipophilic protein (LP) isthe third principle fraction (content of31%) in commercial soy protein isolate, which is richin OBBP and soy phospholipids and presents as an amorphous aggregates in aqueous phase.Studies have shown that LP has a good physiological function. Due to their poor solubilityand diffusivity, few researches take notes on their functionality such as interfacial andemulsifying properties. This thesis has evaluated the functionality of LP systematically,including its interfacial adsorption, emulsifying properties and as a delivery device.Furthermore, a concept of “complex interface” composited with hydrophobic protein andsmall molecular weight surfactant was proposed based on the above studies. The main resultsinclude following.
1. This thesis find that a nanoparticle can be formed by simple ultrasonic treatment, whichhad an excellent diffusion capacity, improved functionality and be potential to be used in foodindustry. LP can be transformed into a sphere particle with a size of136±0.8nm and surfacecharge of-20.0±0.3mV by ultrasonic treatment. Though the surface hydrophobicity analysisand treated with phospholipase, the structure of the particle was confirmed which wasaggregates of the hydrophobic protein with phospholipids covered on the surface. LP particlewas able to diffuse to the interface and form a complex layer composited with thehydrophobic protein aggregates and phospholipids. The rigid interface was so stable that itwas resistant to be displaced by the small molecular surfactant Tween20. Interfacial rheologytechnology was used to investigate the effects of phospholipase and protease on the interfacialrheology behavior of LP particle, which found that there existed synergic effects between thephospholipids and the hydrophobic protein particles. The synergic effects also be beneficialfor the stability of emulsions which had an excellent physical stability even at long-termstorage (8weeks), heating-salting treatment (90℃/200mM NaCl) and the competitivedisplacement of SMW surfactant (4wt.%Tween20).
2. To evaluate the effects of composition of the complex on its stability, soy oil body wasdegreased by organic solvent and then used to form a recombiant oil body. Three differentorganic solvent (acetone, n-hexane, chloroform/methanol) was used to degrease the soy oilbody to control the ratio between OBBP and phospholipids on the surface of recombiant oilbody. Results showed that high content of phospholipids could improve the adsorption of theOBBP on oil-water interface and obtained a high surface pressure and dilatational module. The physical and oxidant stability of recombinant soy oil body formed by the complex ofOBBP and phospholipids was improved by the increasing of phospholipids content.
3. Based on the above studies, a concept of “complex interface” composited withhydrophobic protein and small molecular weight surfactant was proposed and comfirmed.Zein and sodium stearate (SS) were used to simulate the natural complex interface. The geltrapping technology (GTT) was applied to observe the morphology and interfacial contactangle of zein particles on oil-water interface. Results showed that SS controlled the adsorptionof zein on oil-water interface. At a low content of SS (2.5mM), the amount of zein adsorptedon interface was low and they presented as regular spherical particles, their contact angle wasfar less than90°. When at a higher content of SS, the particially unfolded zein particleaccumulated on the interface formed a condensed packed layer, their contact angle wasassumed to be closed to or slightly higher than90°according to the SEM. The complex ofzein and SS could be used to form a stable food grade Pickering emulsion, and such stableemulsion could be further freeze-dried to form a structured fat without the usage of trans-orsaturated fatty acids.
4. The possibility of LP nanoparticle act as a novel delivery vehicle with hight loadingcapacity for the hydrophobic bioactives was explored. LP nanoparticles as a delivery devicefor the hydrophobic bioactives can load conjugated linoleic acids (CLA) successfully byultrasonic induced assembly, and the loading capacity of this system are as high as26.3±0.4wt.%. The CLA-loaded particle has a size of170±0.63nm. Infrared spectrum and X-raydiffraction were used to confirm the entrapment of CLA in the particles. The oxidationstability of CLA was evaluated by the headspace oxygen consumption and peroxide value.Results showed that CLA loaded in soy lipophilic protein nanoparticles have improvedoxidation stability than it loaded in sodium caseinate micelle or in ethanol. Moreover, theCLA loaded in soy lipophilic protein particles had resistant release characteristics whensubjected to the simulated gastrointestinal digestion.
5. The thesis had developed a concept of “double-functional colloidal particle” which couldact as interfacial stabilizer and antioxidants simultaneously. A double-functional nanoparticlecould be formed by entrapping the VEin LP nanoparticles via ultrasonic induced co-assembly.Algae oil emulsions stabilized by this double-functional nanoparticles could form an oil gel(structured fat) when subjected to freeze-drying. Laser scan confocal microscope was appliedto characterize the internal structure of this oil gels. It could be observed that a3-D networkwas formed by soy lipophilic protein nanoparticles and the oil droplets entrapped in thisnetwork separately. Oscillating rheology analysis showed that the formed oil gel in this research had an apparent solid characteristic, and its rheology properties were tunableaccording the concentration of nanoparticles in emulsions. Due to the enrichment ofanti-oxidants on the interface, the algae oil in the double functional particles formed oil gelwas performed to have improved oxidation stability as compared to the case that VEin oilphase or fluid algae oil.
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