摘要
如何有效利用蛋白质与多糖的相互作用来构建（或设计）具有不同结构功能特性的新食品及食品配料是目前食品研究领域的热点问题。壳聚糖是世界上产量仅次于纤维素的天然多糖，其在医药和天然高聚物领域有一定的研究。但是，壳聚糖在食品领域的研究较少，尤其利用壳聚糖与食物蛋白的相互作用构建食品结构及配料的系统研究更是空白。本文系统研究了不同pH、蛋白与多糖复合比、离子强度和温度等条件下的食物蛋白与壳聚糖的相互作用机制，并深入探讨了食物蛋白-壳聚糖复合物在乳液稳定性、油脂抗氧化及凝胶结构修饰方面的应用前景，主要研究结果如下：
（1）本文系统研究了β-伴大豆球蛋白（7S）和大豆球蛋白（11S）与壳聚糖（CS）的相互作用及复合物稳定性。通过调节pH值（pH3-8）、复合比（0.05、0.1和0.2g g-1）、壳聚糖分子量（150、350和500kDa）、离子强度（100mM）和温度（95°C）的方式对比研究了7S-CS和11S-CS所形成的复合体系的相行为，微结构及稳定性。结果显示7S-CS和11S-CS的相互作用是静电相互作用，两种体系均在体系电荷接近等电点时发生静电凝聚现象（Coacervation），在体系ζ-电位为+20~+30mV时形成可溶性复合物（Soluble complex），说明体系电荷量是体系稳定性的决定因素。壳聚糖的分子量由于对体系的电荷影响较小，从而对复合体系稳定性的影响较小；pH、复合比、热处理和离子强度由于对体系的电荷影响较大，所以对复合体系稳定性的影响较大。
（2）本文研究了11S-CS可溶性复合物的形成、界面吸附及其与乳液稳定性之间的关系。利用等温量热滴定（ITC）、浊度滴定和粒度分布的方法研究了11S-CS可溶性复合物的形成过程，发现在pH4.5，复合比0.1g g-1，无离子添加时形成的可溶性复合物为稳定状态，此时ζ-电位值为+27.95mV。利用动态界面吸附测定技术发现可溶性复合物的形成有助于提升复合物在油-水界面上的吸附效果。通过可溶性复合物形成的乳液稳定性的研究发现，复合乳液在pH4.5，复合比0.1-0.2g g-1时具有最好的乳化稳定性和长期储藏稳定性。研究表明，利用蛋白多糖可溶性复合物在酸性条件下的形成可以制备一种具有良好酸性pH稳定的大豆蛋白-壳聚糖复合乳液。
（3）在上述研究基础上，本文研究了7S-CS复合乳液的抗菌稳定性，制备获得一种在酸性条件下既保证物理稳定性又具有抗菌性的乳液。首先乳液稳定性的研究发现复合乳液在低pH值（pH3.0-4.0）具有较好的物理稳定性。抑菌环实验说明复合乳液对金黄色葡萄球菌（S. aureus，革兰氏阳性菌），枯草芽孢杆菌（B. subtilis，革兰氏阳性菌），大肠杆菌（E. coli，革兰氏阴性菌），沙门氏菌（Salmonella，革兰氏阴性菌）均有一定的抑制作用。抗真菌（面包酵母，S. cerevisiae）储藏稳定性实验体现出7S-CS复合乳液较好的抗菌储藏稳定性。本文结合可溶性复合物界面性质的观点提出将复合抗菌剂用于油-水界面，使其起到稳定乳液及抗菌的双重功效的新概念。
（4）本文初步探索了SPI-CS的凝聚物（Coacervate）的形成及在油脂的包裹和抑制氧化方面的效果。凝聚物产率、流变学的结构学研究说明，凝聚物通过SPI带负电荷的羧基基团与CS带正点的氨基基团结合形成。pH6.5-7.0、复合比0.1-0.2g g-1条件下形成的SPI-CS凝聚物表现出较高的粘度，并且这类凝聚物具有弹性模量（G'）大于粘性模量（G"）的弱凝胶型的流变学特性。对于凝聚物用于油脂包裹的研究发现，这种方法可有效的提升包裹效率，减少被包裹油脂与外界的接触从而明显的抑制了油脂的氧化。
（5）本文系统研究了壳聚糖对乳清分离蛋白（WPI）在酸性条件下形成的热置凝胶的结构与质构修饰作用。微结构显示在pH3.5和4.0时形成了完全不同的复合凝胶类型，pH3.5时形成了相分离型的（Phase-separated）复合凝胶，而pH4.0形成了结合型的（Coupled）复合凝胶，这两者之间的区别在于体系中蛋白与多糖的相互作用方式的不同，并且后者更易受到离子强度的影响。结合凝胶微结构、质构学及物理性质的结果发现，凝胶的微结构直接决定了凝胶的质构及保水性，并不依赖于pH是酸性或碱性。WPI-CS复合凝胶具有蛋白连续型（Protein continuous）的微结构时具有好的凝胶强度及较高的保水性，但具有粗线状（Coarse stranded）的微结构会导致凝胶强度和保水性的降低。
（6）本文研究了甲壳素微纤维材料（Chitin microfiber，CMF）与大豆7S蛋白复合凝胶的流变学及微结构特性。利用微射流高压均质的方式制备了一种甲壳素微纤维材料，这种材料具有较高的体系电荷（+30mV）并长期稳定的悬浮在水相中（2个月）。红外光谱的分析说明微纤维的化学结构在处理前后并未受到破坏。流变学研究发现，甲壳素微纤维材料能显著的提高TG酶诱导7S冷置凝胶的凝胶强度，并且提高程度赖于微纤维材料的添加量。凝胶网络结构的结果显示7S-CMF复合凝胶形成了蜂窝型的微结构，并且结构孔径明显小于7S凝胶。分析CMF对凝胶的增强机理可能是由于其参与了凝胶的形成，增加了蛋白网络承受外力的能力并有效的防止凝胶内部结构的破裂出现。
The exploitation of protein-polysaccharide interactions offers opportunities for thedesign of new food structures with different functional properties and it has been of greatconcern in food research field. As the second largest abundant natural polysaccharide in theworld, chitosan (CS) and its derivatives shows wide application prospect due to its cost andfunctional properties. So far, the investigations regarding using food protein-chitosaninteraction to tailor the food structure are limited, especially in soy protein-based foodsystem.Consequently, this thesis systematically studied the effect of pH, mixing ratio, ionicstrength and temperature on the mechanism of interaction between food protein and chitosan.The formed complex and coacervate with different functional properties were theninvestigated in food emulsion and food gel to show their application. The main results are asfollows:
(1) This study explored the interaction between the β-conglycinin (7S) and glycinin (11S)and chitosan (CS) and investigated the influence of pH (pH3-8), mixing ratio (0.05、0.1and0.2g g-1), chitosan molecular weight (150、350and500kDa), ionic strength (100mM) andheat treatment (95°C). The turbidity versus ζ-potential pattern showed that, independent ofprotein, the coacervation of7S-CS and11S-CS mixtures were all obtained at chargeneutralization pH while soluble complex were all obtained atζ-potential of+20to+30mV,indicating the7S-CS and11S-CS mixtures were electrostatically driven. The molecularweight of chitosan showed less effect on the stability of mixtures. Mixing ratio as well as heattreatment and ionic strength, however, showed great effect on the stability of mixtures.
(2) The formation and interfacial adsorption of11S-CS soluble complex wereinvestigated at acidic pH. The stability of the mixed emulsion stabilized by the complex wasalso evaluated at pH4.5. Turbidimetric analysis, isothermal titration calorimetry (ITC) anddynamic light scattering were used to characterize the dynamic formation of the complex. Theresults showed that soluble complexes were formed at pH4.5and saturated at mixing ratio of0.1g g-1, showing theζ-potential of+27.95mV. It also can be found that the soluble complexshowed improved interfacial adsorption. The droplet size and confocal observation of themixed emulsion fabricated with11S-CS soluble complex displayed improved stability at mixing ratios of0.1-0.2g g-1, suggesting the synergistic effect of the two molecules. Weconcluded that interfacial and emulsifying properties of glycinin could be improved byformation of11S-CS soluble complex at acidic pH.
(3)To fabricate a soy protein emulsion with good storage stability against microorganism,we investigated the stability and antimicrobial activity of7S-CSmixed emulsion at acidicpHs.Results of droplet size and microstructure showed7S/CS mixed emulsions were stable atacidic pHs. Agar well diffusion experiment suggested that the mixed emulsions inhibited allthe microorganisms (S. aureus, B. subtilis, E. coli andSalmonella).As for the storage test,results showed that7S-CS mixed emulsion displayed an significantly (p <0.05) improvedstorage stability than control both under un-inoculated and inoculated condition because thepresence of chitosan. The fabrication of7S-CS mixed emulsion illuminated a new idea thatantibacterial agent can be used on the oil-water interface and can play a multifunctional rolein increasing acidic stability and antimicrobial activity of emulsion in order to extendshelf-life.
(4) The SPI-CS coacervation and its application in inhibition of lipid oxidation wereinvestigated. Results showed that the coacervate formed between the negatively chargedcarboxyl group on SPI and the positively charged amino group on CS. The coacervates withhighest viscosity were formed at pH6.5-7.0, mixing ratio0.1-0.2g g-1, showing a weakgel-like rheological properties (G'> G"). A coated emulsion with high encapsulationefficiency can be fabricated in this way thereby reducing the lipid-air contact and inhibition oflipid oxidation.
(5)This study explored the microstructure, textural and physical properties of WPI-CSmixed gels at pH3.5and4.0. The results of microstructure showed that WPI-CS mixed gelsdisplayed a total different gel structure at different pH. Phase-separated gel structure wasobtained at pH3.5while coupled structure was obtained at pH4.0because differentinteraction occurred. Ionic strength showed greater effect on coupled gel. The results in thispaper suggested that segregative phase separation can be used to alter the fracture and waterholding properties of WPI gels formed below their pI, in a way similar to that seen above theirpI, indicating that the microstructure decide the textural and physical properties. WPI-CSmixed gels with protein continuous network showed higher gel strength and water holding value while mixed gels with coarse stranded network showed poor gel strength and waterholding value.
(6) A stable positively charged (+30mV) chitin microfiber (CMF) suspension wasfabricated by a facile Microfluidizer approach without changing its chemical structure. Theobtained CMFwere then developed in a transglutaminase cross-linked7S gel.Themorphological and rheological characterizations of the7S-CMF gels were done as a functionof the protein and CMF concentrations. Results showed that the presence of CMF networkimproved the gel strength significantly. This effect was CMF content dependent and wasrelated to the formation of sponge-like porous microstructure. We inferred that the CMFprovided an initial framework for gel formation and added structural rigidity to the protein gel.The role of protein was to participate in network development as an electrostatic coating andgelation component.
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