物理相变耦联机械胁迫的大米蛋白溶解机制及自乳化载体的控释行为

发布时间：2018-05-02 16:57

本文选题：大米蛋白 + MG转变　；参考：《江南大学》2017年博士论文

【摘要】：大米蛋白是一种廉价的高营养蛋白,也是一种低致敏性的蛋白资源,可作为婴儿及敏感人群食品配方。但是低溶解性成为限制大米蛋白作为食品功能性成分及商业化应用的主要瓶颈。尽管目前已有多种溶解性改良手段,但是大米蛋白的改性存在着分子修饰不合理、产物破坏严重、处理效率差等多方面问题。针对上述问题,本研究从相变态水分子及蛋白质拓扑结构入手,构建一种全新的物理处理方法—物理相变耦联机械胁迫(PTMS),全面分析大米蛋白结构改性的分子学机理,并从纳米科学及界面科学角度开发改性大米蛋白纳米载体,拓展大米蛋白及其他谷物蛋白应用的新领域。基于溶融态(MG)的PTMS大米蛋白的分子学溶解机制研究。结构学表征表明,大米蛋白在MG转变条件下,三级结构解体而维持原有的一、二级结构;溶剂相变使得水分子氢键发生重排而产生自由水,并在相变温度下渗透到蛋白质内部结构,进一步形成冰晶体;机械胁迫借冰晶体媒介作用改变大米蛋白的二级及高级结构,导致其聚集度下降、蛋白分子展开、极性基团暴露,形成20~80 nm的极性纳米颗粒。此外,大米蛋白在PTMS过程中可维持原有的一级结构。在pH12.5的MG条件下,经三次PTMS,大米蛋白溶解度达到50.4%。因此,基于MG的PTMS溶解大米蛋白的分子学机制是,在维持大米蛋白一级机构的基础上实现二级结构的折叠-转角转变,最终形成聚集度较低的极性蛋白体颗粒。基于优化PTMS的改性大米蛋白(MRPs)理化及功能特性研究。采用中心组合实验,在pH12.5的MG转变条件下,分析料液比、相变时间及相变温度对MRPs得率的影响。响应面分析表明,在料液比1:40、相变时间40 h及相变温度-13°C条件下,大米蛋白经PTMS后,MRPs得率达到40.50%,并在此条件下制备MRPs并喷雾干燥和冷冻干燥分别得到SD-RP和FD-RP。SDS-PAGE实验表明,SD-RP和FD-RP与对照具有相似度95%以上的一级结构。水溶性实验表明,SD-RP和FD-RP在pH≤6.0时溶解度均10%,而当pH≥7.0时溶解度均90%。由于乳化性及起泡性的发挥依赖于蛋白质的水合能力,在中性条件下(pH7.0),SD-RP和FD-RP乳化性从25.18 m2/g(对照组)分别提高至29.43 m2/g和46.99 m2/g;起泡性从7.65 cm3(对照组)分别提高至18.2 cm3和16.55cm3。此外,形态学研究结果表明,对照组结构致密,FD-RP结构疏松,而SD-RP呈分散的颗粒状结构;SD-RP和FD-RP在溶剂中聚集程度的降低是功能性质提高的原因。基于MRPs-油乳液载体的β-胡萝卜素结构学控释机制研究。在pH从9.0至6.2~7.0降低过程中,MRPs发生反溶剂沉淀并在油滴表面结构化形成滴粒径为300~400 nm的乳滴(Em6.2~Em7.0)。利用乳液壳层(载体)的对pH的高敏感度,导致其结构密度可控,实现在模拟胃肠道消化过程中载体中β-胡萝卜素的控释。结构分析及释放动力学结果表明,Em6.2之间通过蛋白质交联发生疏水聚集,致使MRPs与油的结合程度减弱。因而,Em6.2在胃肠道模拟消化过程中,β-胡萝卜素释放速率最高。Em6.4~Em7.0在模拟胃液消化过程中β-胡萝卜素释放量均10%(即释放过程被抑制),在模拟肠道消化过程中β-胡萝卜素释放过程均符合零级动力学且释放速率随pH升高而增加。此结果与乳滴表面蛋白质的堆积密度随pH升高而降低有关。因此,MRPs可应用于制备具有控释功能新型乳液运载系统。同时,这项研究也大大提高了MRPs乳液稳定性,为MRPs作为功能性成分提供新的解决方案。基于MRPs-精油自乳化行为的控释机理研究。发现溶解在油相中的活性物质可完成自乳化,这不仅是一种有趣的现象,且对功能活性物质及药物的运载具有重要的意义。为此,瞄准MRPs-精油内源性结合反应,开发具有控释功能的油核-蛋白壳复合材料。丁子香酚在与1%MRPs低速搅拌过程中去质子化,并通过电荷转移降低体系pH,造成MRPs结构改变而堆积在油滴表面形成复合乳滴。蛋白质-精油反应导致油-水界面张力发生极显著下降,相同剪切速率下油滴表面Laplace张力大大减小,最终油滴平均粒径250 nm。同时,扫描透射电镜及聚焦电子束扫描电镜实验表明,丁子香酚浓度的变化造成蛋白质在油滴表面堆积强度发生改变。乳液在体外透析阶段精油呈现一级动力学释放且释放速率常数k随蛋白质-精油比的增加而降低。因此,作者利用这种现象进行生物活性物质的载运及控释研究。结果表明,在0.2μg/mL及2μg/mL剂量浓度下,装载于蛋白质-精油比为1:4载体内的咖啡酸苯乙酯(CAPE)与溶解在DMSO的CAPE对HCT-116癌细胞抑制率相似,两种浓度下抑制率分别为5%和35%。而在0.2μg/mL及2μg/mL剂量浓度下,装载于蛋白质-精油比为1:20载体内的CAPE对HCT-116的抑癌率分别达到50%和70%。以上实验表明,具有自乳化特性的MRPs不仅能够大大提高生产效率,还能提高生物活性物质的生物利用度,因而在脂溶活性物质载运实践中具有可观的应用前景。
[Abstract]:Rice protein is a cheap high nutrient protein and also a kind of low sensitizing protein resource, which can be used as food formula for infants and sensitive people. But low solubility becomes the main bottleneck for limiting rice protein as a functional component and commercial application of food. Although there are many methods of dissolved improvement, rice protein has been found at present. There are many problems in the modification, such as irrational molecular modification, serious damage and poor treatment efficiency. In this study, a new physical treatment method, physical transformation coupled mechanical stress (PTMS), is constructed to analyze the structural modification of rice protein. The mechanism is to develop the modified rice protein nanoscale from the perspective of nanoscience and Interface Science, and expand the new field of rice protein and other grain protein applications. The molecular dissolution mechanism of the PTMS rice protein based on melting state (MG) is studied. The structural characterization shows that the rice protein is disintegrated and maintains the original three stage structure under the condition of MG transformation. The first, two stage structure, the solvent phase change causes the hydrogen bond of water molecules to rearrange and produce free water, and permeate into the internal structure of protein at the temperature of phase change, and further form the ice crystal. The mechanical stress changes the two grade and the advanced structure of rice protein by the action of ice crystal medium, and leads to the decrease of the aggregation degree, the expansion of protein molecules and the polar group storm. In addition, the rice protein can maintain the 20~80 nm polar nanoparticles. In addition, the rice protein can maintain the original first order structure in the PTMS process. Under the MG condition of pH12.5, the solubility of rice protein reaches 50.4%. after three times PTMS, so the fractional mechanism of the PTMS dissolved rice protein based on MG is to realize the two level knot on the basis of maintaining the first grade mechanism of rice protein. The folding and turning angle of the structure was changed to form the polar protein body particles with low aggregation. The physicochemical and functional properties of the modified rice protein (MRPs) based on the optimized PTMS were studied. The effect of the ratio of material to liquid, the time of phase change and the temperature of phase change on the yield of MRPs was analyzed by the central combination experiment under the MG transformation condition of pH12.5. When the liquid was at 1:40, the phase transition time was 40 h and the phase transition temperature was -13 C, the yield of MRPs was 40.50% after PTMS, and the preparation of MRPs, spray drying and freeze-drying under the conditions of SD-RP and FD-RP.SDS-PAGE, respectively, showed that SD-RP and FD-RP had a first grade structure with similar degree similar to 95%. Water solubility experiment showed SD-RP The solubility of FD-RP and FD-RP at pH < 6 is 10%, while the solubility of the solubility of 90%. depends on the hydration ability of the protein when the emulsification and foaming ability is equal to 7. Under neutral conditions (pH7.0), the emulsification of SD-RP and FD-RP increases from 25.18 m2/g (control group) to 29.43 m2/g and 46.99 m2/g respectively, and the foaming property is increased to 1 from 7.65 cm3 (control group), respectively. 8.2 cm3 and 16.55cm3. in addition, the morphological study showed that the structure of the control group was dense, the FD-RP structure was loose, and the SD-RP was dispersed in granular structure. The decrease of the aggregation degree of SD-RP and FD-RP in the solvent was the reason for the improvement of the functional properties. The study on the structural release mechanism of beta Hu Luo based on MRPs- oil emulsion carrier was studied in pH from 9 to 6.2~7.. During the 0 reduction, MRPs precipitated and formed a droplet of 300~400 nm on the surface of the oil droplet (Em6.2~Em7.0). Using the high sensitivity of the emulsion shell (carrier) to pH, the structure density was controlled, and the controlled release of beta carotene in the carrier of simulated gastrointestinal tract, structure analysis and release power were realized. The results showed that the binding of Em6.2 through protein crosslinking resulted in a decrease in the binding of MRPs to oil. Thus, the highest beta carotene release rate of.Em6.4~Em7.0 in the simulated gastrointestinal digestion process was 10% (the release process was inhibited) during the digestive process of the simulated gastric juice (i.e. the release process was suppressed), and in the simulated intestinal tract. In the process of digestion, the release process of beta carotene conforms to zero order kinetics and the release rate increases with the increase of pH. The accumulation density of the protein on the milk drop surface decreases with the increase of pH. Therefore, MRPs can be applied to the preparation of a new emulsion delivery system with controlled release function. This study also greatly improved the stability of MRPs emulsion. Qualitatively, it provides a new solution for MRPs as a functional component. Based on the study of the controlled release mechanism of the self emulsifying behavior of MRPs- essential oil, it is found that the active substances dissolved in the oil phase can be self emulsified, which is not only an interesting phenomenon, but also is of great significance to the delivery of functional active substances and drugs. To this end, aim at the essential oil in the MRPs- oil. The source binding reaction was used to develop the oil core protein shell composite with controlled release function. The butylphenols were protonated during the low speed stirring process with 1%MRPs and reduced the system pH by charge transfer, resulting in the structure change of the MRPs and accumulated on the surface of the oil droplets. The protein fine oil reaction resulted in a very significant lower oil water interfacial tension. Under the same shear rate, the Laplace tension on the surface of oil droplets decreased greatly, and the average particle size of the oil droplets was 250 nm.. The scanning transmission electron microscopy and focused electron beam scanning electron microscopy showed that the change of the concentration of Ding Zixiang phenol resulted in the change of the protein accumulation strength on the surface of the oil droplet. The release rate constant K decreases with the increase of the protein essential oil ratio. Therefore, the author uses this phenomenon to carry out the study on the carrier and controlled release of bioactive substances. The results show that at the dose concentration of 0.2 and 2 mu g/mL, the protein - essential oil is compared to the 1:4 - loaded caffeic acid benzol ethyl (CAPE) and the CAPE against the DMSO in DMSO. The inhibition rates of -116 cancer cells were similar, and the inhibition rates under two concentrations were 5% and 35%. respectively, while at 0.2 and 2 g/mL doses, the inhibition rate of CAPE against HCT-116 in protein essential oil compared to the 1:20 carrier was 50% and 70%., respectively. The results showed that MRPs with self emulsifying property could not only greatly improve the production efficiency, but also could improve the production efficiency. Improving the bioavailability of bioactive substances has great potential for application in the transportation of liposoluble active substances.

【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS201.21

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