Ability of casein hydrolysate-carboxymethyl chitosan conjugates to stabilize a nanoemulsion: Improved freeze-thaw and pH stability

An oil-in-water (O/W) nanoemulsion stabilized by casein hydrolysates (CHs) and carboxymethyl chitosan (OCC) (CHs-OCC) conjugates was prepared and its stability characteristics were evaluated under environmental conditions. The conjugated reaction between CHs and OCC was easier with a maximum degree of glycosylation (DG) of 40.7% and a browning index (BI) of 1.47 were obtained on the 20th day. This result was confirmed by the bathochromic-shift (350–358 nm) of the conjugates determined by intrinsic tryptophan fluorescence spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Transmission electron microscopy (TEM) revealed that the particles of the nanoemulsion prepared with CHs-OCC (0.5–3.0%, w/v) had a smaller size (227–833 nm) and a spherical structure. In addition, due to the strong steric effect provided by the conjugated polysaccharide chains, the CHs-OCC nanoemulsion exhibited the best stability characteristics; the droplet particle size increased from 227 nm (fresh, 2.5%, w/v) to 427 nm (pH 2.0–7.4 at room temperature, 12 h), 488 nm (63 °C/30 min), 389 nm (95 °C/10 min), 236 nm (0–200 mM NaCl), and 255 nm (1–15-d storage, 4 °C). In particular, the CHs-OCC considerably enhanced the freeze-thaw stability of the nanoemulsion: the particle size after 3 freeze-thaw cycles showed a narrow distribution and did not exhibit the bimodal (0.1–1 μm) appearance, compared with casein. CHs-OCC may be suitable as a natural emulsifier in O/W nanoemulsions.