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

16 Casein micelle (CM), porous colloidal phosphoprotei n-mineral complex, naturally present in 17 milk to deliver minerals, also has several features , which could ensure its use as nanocarrier 18 for bioactives. CM structure being not steady accor ding to the physico-chemical conditions, 19 its stability can be improved by intra-micellar cro ss-linking using transglutaminase (TGase) 20 inducing a strengthened structure called casein nan ogel. The aim of this research was to 21 investigate the morphology and nanomechanics of cas ein nanogel particles cross-linked by 22 TGase (TG-CM) using atomic force microscopy (AFM) i n native-like liquid environment 23 (lactose-free simulated milk ultrafiltrate, SMUF). Prior to AFM, TG-CM were captured by 24 anti-phospho-Ser/Thr/Tyr monoclonal antibodies cova lently bound to a gold-coated slide via 25 V er si on p os tp rin t Comment citer ce document : Bahri, A., Martin, M., Gergely, Marchesseau, S., Chevalier-Lucia, D. (2018). Topographical and nanomechanical characterization of casein nanogel particles using atomic force microscopy. Food Hydrocolloids, 83, 53-60. , DOI : 10.1016/j.foodhyd.2018.03.029 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT 2 carbodiimide chemistry. Surface topography and size properties evaluation revealed an 26 increase in size of TG-CM compared to native CM, TG -CM being characterized by a mean 27 width of 264 ± 7 nm and a mean height of 111 ± 5 nm . TG-CM displayed a relatively high 28 contact angle (62°) indicating a limited flattening of these particles after adsorption on the 29 substrate. The TG-CM elasticity was then evaluated pplying low indentation forces on single 30 TG-CM. The TGase treatment led to a significant mod ification of CM nanomechanics 31 attributed to intramolecular rearrangements within the micellar structure. The elasticity 32 distribution of TG-CM revealed three elasticity pea ks centered at 219 ± 14 kPa, 536 ± 14 kPa 33 and 711 ± 11 kPa. The lower elasticity peak is rela ted to the native CM elasticity 34 characteristic and the two stiffer peaks were attri bu ed to the substantial changes in the TG35 CM structure. 36 37

increase in size of TG-CM compared to native CM, TG-CM being characterized by a mean 27 width of 264 ± 7 nm and a mean height of 111 ± 5 nm. TG-CM displayed a relatively high 28 contact angle (62°) indicating a limited flattening of these particles after adsorption on the 29 substrate. The TG-CM elasticity was then evaluated applying low indentation forces on single 30 TG-CM. The TGase treatment led to a significant modification of CM nanomechanics 31 attributed to intramolecular rearrangements within the micellar structure. The elasticity 32 distribution of TG-CM revealed three elasticity peaks centered at 219 ± 14 kPa, 536 ± 14 kPa 33 and 711 ± 11 kPa. The lower elasticity peak is related to the native CM elasticity 34 characteristic and the two stiffer peaks were attributed to the substantial changes in the TG-35 CM structure.   According to the 2D and 3D AFM heights (Fig. 1a, c, e, g, h, j, l), it appeared that TG-CM are 228 higher and wider than native CM. The surface coverage of TG-CM (11 ± 4 micelles/µm 2 ) was 229 significantly (p < 0.05) lower than the native CM surface coverage (20 ± 2 micelles/µm 2 ), this 230 lower density being attributed to the smaller size of native CM compared to TG-CM. At the 231 same time, the hydrodynamic diameter distribution curves of native CM and TG-CM 232 measured by PCS have been compared. The both size distribution curves in intensity exhibit 233 monomodal and polydisperse populations (Fig. 3). TG-CM have however a significantly (p < 234 0.05) higher average hydrodynamic diameter of 214 ± 8 nm compared to 192 ± 8 nm for 235 native CM. 236 This result was confirmed by the height and width distributions (Fig. 4a, b)  considering the total collected values of samples. A multi-peak fitting was then applied using 240 OriginPro 8 software to calculate the mean width and height. As depicted in Fig. 4a Height and width distributions (Fig. 4a, b) show that TG-CM captured on SAM-gold substrate 269 via MAH-PSer/Thr/Tyr antibody were definitely larger than higher, as it was also the case for This deformation can also be displayed by plotting the height against the width for each single 281 object (Fig. 4c, d). The comparison with the dotted line representing a perfect sphere points 282 towards the fact that the TG-CM (Fig. 4c) are less flattened compared to native CM (Fig. 4d) 283 once captured on the gold substrate. This phenomenon clearly highlights a structural 284 strengthening of CM due to the molecular rearrangements induced by TGase. 285 The CM contact angle was deduced from the AFM-measured (h and w) of each CM. The 286 contact angle (θ) corresponding to the interior angle formed by the substrate and the tangent 287 to the drop interface at the apparent intersection of these interfaces describes the object 288 deformation upon adsorption (Brown, 1999;Russel, 2009

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13 when the particle spreads on the surface, while a large contact angle is observed when the 290 liquid beads on the surface. More specifically, a perfect spherical particle would exhibit a 291 contact angle greater than 90° (Yuehua & Randall, 2013). The equilibrium shape of a given 292 particle does not depend only on surface forces but is also greatly affected by the elastic 293 modulus of the droplet, the droplet deformation depending of material elastic nature resulting 294 from stress development across the bulk in opposition to that deformation (Brown, 1999 The histogram of TG-CM stiffness values (Fig. 5) reveals a multimodal stiffness distribution 324 with three prominent peaks at 218 ± 14 kPa, 536 ± 10 kPa and 711 ± 11 kPa, as identified by 325 the peak analyzing software. The intensity of the three Gaussian distributions used to fit the 326 TG-CM histogram is similar suggesting that the three populations have the same weight (Fig.  327   5). In comparison, a broad unimodal stiffness distribution with a peak centered at 269 ± 14 328 kPa was observed for native immobilized CM (Fig. 5) images reveal a spherical-cap shape with a wider (264 ± 7 nm) and higher (111 ± 5 nm) 375 structure than native CM. Moreover, TG-CM shows a more resistant structure upon 376 adsorption on gold substrate owing to a high contact angle of 62°. 377 The TG-CM nanomechanical properties highlight a low elasticity peak at 218 ± 14 kPa that 378 could correspond to the mechanical signature of native CM and also two stiffer elasticity 379 peaks observed at 536 ± 10 kPa and 711 ± 11 kPa, most likely directly related to substantial 380 changes in the shape and structure of CM induced by TGase and responsible for the 381 modification of their functional properties. 382 These results support the improved stability of TG-CM suggesting that these nanogel particles 383 can be an excellent matrix for bioactives encapsulation.

Highlights
• TG cross-linked CM topography and nanomechanics were evaluated by AFM in liquid. • TG cross-linked CM are significantly wider and higher than native CM.
• TG-CM are less flattened once captured on gold substrate compared to native CM.
• TG-CM stiffness distribution is multimodal with stiffer peaks compared to native CM.