Complex Phase Behaviors Of Protein/Polysaccharide Aqueous Mixtures

As the major structuring agents for food systems, protein and polysaccharide play key roles in modern food structure design. Although protein/polysaccharide aqueous mixtures have been extensively studied, the effect of conformation and conformational transition on their phase behaviors has been barely reported. Most proteins and polysaccharides undergo conformational transition and/or gelation during food processing and storage. The coupling of phase behaviors with conformational transition can be utilized to design food microstructures and mechanical and functional properties. By using different techniques including micro DSC, UV/Vis, NMR, DLS, CLSM, ITC, FTIR, XRD, XPS, rheometry, fluorescence and conductivity, the project aimed to establish the complex phase diagram of protein/polysaccharide mixtures, and to elucidate the conversion and coexisting of different phase behaviors. The coupling between phase separation and conformational transition was also investigated both experimentally and theoretically. Main conclusions are as follows:1. A detailed and complete phase diagram was established for type B gelatin/k-carrageenan（k-car） mixtures in the ionic strength-temperature coordinate, which comprised of seven distinct phase regions:（I） compatible region;（II） electrostatically induced associative phase separation（EIAPS） region;（III） hydrogen bonding induced associative phase separation（HBIAPS） region;（IV） coexistence of EIAPS and HBIAPS;（V） segregative phase separation（SPS） region;（VI） coexistence of HBIAPS and SPS;（VII） SPS trapped by gelation. The HBIAPS reported for the first time here was attributed to the extensive hydrogen bonding formation between gelatin and k-car above their conformational transition temperatures.2. The effect of conformational ordering on protein/polysaccharide electrostatic complexation was studied by using type B gelatin/k-car. The effect can be decomposed into ionic binding and chain stiffening. At the initial stage of conformational ordering, electrostatic complexation was either suppressed（in the presence of K+） or enhanced（in the presence of I–） due to ionic binding, which significantly altered the charge density of k-car. Beyond a certain stage of conformational ordering, i.e., helix content q > 0.30, the effect of chain stiffening became dominant and tended to dissociate the electrostatic complexation. The effect of chain stiffening was theoretically interpreted in terms of double helix association.3. The effect of protein/polysaccharide electrostatic complexation on the conformation transition of polysaccharide was studied both experimentally and theoretically by using k-car/b-lactoglobulin（b-lg） as an example. The effect was related to the molecular state of electrostatic complexes:（I） soluble electrostatic complex had subtle effect on the double helix formation of k-car upon cooling due to the limited binding of b-lg to k-car, where k-car molecules remained of high freedom;（II） insoluble electrostatic complex significantly suppressed the double helix formation, which was attributed to the high steric effect of the bound b-lg and the low freedom of k-car molecules. Based on the McGhee-Hippel theory, a quantitative model was developed to describe the effect of protein/polysaccharide electrostatic complexation on the conformation ordering of polysaccharide. Furthermore, it was found that the effect of electrostatic complexation on polysaccharide conformational ordering was related to protein molecular weight. Protein or polypeptide with molecular weight higher than 2000 Da hindered the conformational ordering of polysaccharide, while that with molecular weight lower than 1000 Da tended to stabilize the ordered conformation. The study opened up a new approach to control the gelation and gel properties of ionic polysaccharides.4. The interaction of l-carrageenan（l-car） with metal ions of different valency was studied. A specific binding of trivalent metal ions(Fe³⁺ and Al³⁺) to l-car was identified, which led to either a weak gel formation or precipitation. The specific binding was attributed to the valency and coordination effect of Fe³⁺ and Al³⁺ ions. Both Fe³⁺ and Al³⁺ had a binding stoichiometry of 1:1 with l-car（relative to repeating units）, while the former exhibited a higher binding constant.