Topographical and nanomechanical characterization of casein nanogel particles using atomic force microscopy

Casein micelle (CM), porous colloidal phosphoprotein-mineral complex, naturally present in milk to deliver minerals, also has several features, which could ensure its use as nanocarrier for bioactives. CM structure being not steady according to the physico-chemical conditions, its stability can be improved by intra-micellar cross-linking using transglutaminase (TGase) inducing a strengthened structure called casein nanogel. The aim of this research was to investigate the morphology and nanomechanics of casein nanogel particles cross-linked by TGase (TG-CM) using atomic force microscopy (AFM) in native-like liquid environment (lactose-free simulated milk ultrafiltrate, SMUF). Prior to AFM, TG-CM were captured by anti-phospho-Ser/Thr/Tyr monoclonal antibodies covalently bound to a gold-coated slide via carbodiimide chemistry. Surface topography and size properties evaluation revealed an increase in size of TG-CM compared to native CM, TG-CM being characterized by a mean width of 264 ± 7 nm and a mean height of 111 ± 5 nm. TG-CM displayed a relatively high contact angle (62°) indicating a limited flattening of these particles after adsorption on the substrate. The TG-CM elasticity was then evaluated applying low indentation forces on single TG-CM. The TGase treatment led to a significant modification of CM nanomechanics attributed to intramolecular rearrangements within the micellar structure. The elasticity distribution of TG-CM revealed three elasticity peaks centered at 219 ± 14 kPa, 536 ± 14 kPa and 711 ± 11 kPa. The lower elasticity peak is related to the native CM nanomechanical signature and the two stiffer peaks were attributed to the substantial changes in the TG-CM structure.