Impact of Chitosan Molecular Weight and Attached Non-Interactive Chains on the Formation of α-Lactalbumin Nanogel Particles

Abstract

Thermal treatment of protein-polysaccharide complexes will form nanogel particles, wherein the polysaccharide controls nanogel formation by limiting protein aggregation. To determine the impact of the chitosan molecular weight and non-interactive chains on the formation of nanogels, mixtures of α-lactalbumin were prepared with selectively-hydrolyzed chitosan containing covalently-attached polyethylene glycol chains (PEG) and heated near the protein's isoelectric point to induce formation of nanogels. Turbidity of heated mixtures indicated the formation of suspended aggregates, with greater values observed at higher pH, without attached PEG, and among samples with 8.9 kDa chitosan. Mixtures containing 113 kDa chitosan-PEG formed precipitating aggregates above pH 5, coinciding with a low-magnitude colloidal charge and average hydrodynamic radii > 400 nm. All other tested mixtures were stable to precipitation and possessed average hydrodynamic radii~100 nm, with atomic force microscopy showing homogeneous distributions of spherical nanogel aggregates. Over all of the tested conditions, attached PEG led to no additional significant changes in the size or morphology of nanogels formed from the protein and chitosan. While PEG may have interfered with the interactions between protein and the 113 kDa chitosan, prompting greater aggregation and precipitation, PEG did not indicate any such interference for shorter chitosan chains.

Characterization of the Structural and Colloidal Properties of α-Lactalbumin/Chitosan Complexes as a Function of Heating

“…25,30 Therefore, the change of ζ potential from negative to positive demonstrated that α-la molecules were surrounded by chitosan molecules. Similar results have been observed in the research of Du et al 31 For the sample with an α-la/chitosan ratio of 5:1, α-la/chitosan complexes had an average ζ potential of +21 ± 2 mV. With a further increase of chitosan addition to a ratio of 2:1, the formed complexes had a much higher ζ potential value of +33 ± 2 mV, which was similar to pure chitosan, indicating that the surface of α-la molecules was fully surrounded by chitosan molecules.…”

“…The addition of chitosan significantly increased the size to a maximum value of 810 ± 33 nm at a ratio of 5:1, which was due to the bridging effect of chitosan. It is important to note that the α-la/chitosan complexes formed at pH > 5.0 were reported to be insoluble, , which could be confirmed by results from optical appearance and turbidity. The decreased size with an increasing temperature indicated that heating promoted the formation of α-la/chitosan aggregates.…”