A non-toxic, biocompatible and generally recognized as safe (GRAS) chemical, citric acid, was employed for pH adjustment and cross-linking of whey proteins either before or after their microgelification by heat. A conventionally-made microgel sample was also fabricated by using hydrochloric acid for pH adjustment on 5.8 and subsequent heating at 85 °C. Size approximation of microgel particles from atomic force microscopy images indicated that chemical cross-linking resulted in smaller particles (80-130 nm and 100-150 nm for post-and pre-microgelification cross-linked samples, respectively) compared with conventional counterpart (150-300 nm). Based on Fourier transform infrared spectroscopy results, it was concluded that prolonged citric acid cross-linking of whey proteins prior to microgelification caused extensive cross-linking of protein units and preserved proteins α-helical structure. The pre-cross-linked microgels formed firmer (higher fracture stress and complex modulus) calcium-induced cold-set bulk gel with higher water-holding capacity and denser microstructure compared with conventional and postcross-linked microgels. These superiorities were attributed to existence of higher number of carboxyl residues in the structure of the pre-cross-linked microgels. The alteration in the properties of the final bulk hydrogels via cross-linking of the microgel building-units can be exploited for modulating and controlling the release behavior of loaded cargo within hydrogel, resulting in a more tuned application of gels as edible delivery systems.
Protein nanofibrils with 10-20 nm diameters were formed by heating whey protein solution at pH 2.0. Nanofibrils solution was deacidified slowly through dialysis followed by adding different amounts of CaCl2 (0-80 mM) into the dialysis water resulting in formation of a soft viscoelastic gel over time. The gel fabricated from the nanofibrils solution dialyzed against distilled water with 0 mM CaCl2 had zero ash content. Fourier transform infra-red spectroscopy revealed a change in the pattern of hydrogen bond formation in gel network by calcium chloride. The higher the ash content of gels, the lower was the storage modulus and fracture stress of samples. Gels with higher ash contents had a more porous microstructure which was attributed to the diminished hydrophobic interactions and hydrogen bonding among nanofibrils by the action of chloride. Higher ash contents also led to higher water holding capacity of gels which was attributed to the influence of the strongly hydrated calcium ions that interacted with the non-charged regions of proteins via site-specific interactions.
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