AFM imaging of milk casein micelles: evidence for structural
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1 Supporting Information AFM imaging of milk casein micelles: evidence for structural rearrangement upon acidification Mustapha Ouanezar,, Fanny Guyomarc'h,,,*, Antoine Bouchoux,,** INRA, UMR 1253 Science et Technologie du Lait et de l'œuf, F-3542 Rennes, France AGROCAMPUS OUEST, UMR1253 Science et Technologie du Lait et de l'œuf, F-3542 Rennes, France * Corresponding author 1. Tel: ; Fax: ; fanny.guyomarch@rennes.inra.fr ** Corresponding author 2. Tel: ; Fax: ; antoine.bouchoux@rennes.inra.fr; Present address: CNRS, Laboratoire de Génie Chimique, 118 Route de Narbonne, F-3162 Toulouse, France.
2 I. Size distributions 1. Casein micelles at ph 6.7 A 1. Casein micelles at ph 6.7 B P(h).5 P(w) Height, h (nm) Width, w (nm) 1. Casein micelles at ph 5 C 1. Casein micelles at ph 5 D P(h).5 P(w) Height, h (nm) Width, w (nm) Figure S1. Size properties of casein micelles imaged in liquid through AFM in different conditions of ph. (A) and (C) are height distributions in close-to-native (ph 6.7) and in acidified (ph 5) conditions, respectively. (B) and (D) are the corresponding width distributions. The full lines are log-normal distributions that best fits the data. For comparison, the intensity size distribution obtained through DLS with the same casein micelle dispersion at ph 6.7 is given in (A) and (B) as solid squares. The particle size distribution of the dialyzed casein micelle dispersions was determined at ~1g.kg -1 in SMUF at 25 C (refractive index = 1.333, viscosity =.89 mpa.s) using a Zetasizer nanozs dynamic light scattering (DLS) equipment (λ = 633 nm, Malvern Instruments, Orsay, France). Figure S1 compares the DLS distribution with those of the height and width of individual casein micelles as measured by particle analysis of AFM images obtained in liquid,
3 at ph 6.7 (top; brown colour) or 5. (bottom, blue colour). The results show that in all experimental conditions or methods, the casein micelles show polydisperse dimensions with log-normal distributions. The mean DLS diameter is ~175 nm, in agreement with previous reports. 1,2 At ph 6.7, the mean height and width of the casein micelles imaged by the AFM are ~1 and 3 nm, respectively; as a result of their deformation when covalently immobilized onto the gold surface. II. Extra AFM images in air When performing AFM observations in air, the topography of the dehydrated sample can reveal internal features. 3 Different authors have used this approach to comment on the structure of casein micelles layers, but not individuals. 4-6 Images of casein micelles covalently bound on gold surfaces then dehydrated are shown and discussed in the manuscript. As an alternative to chemical immobilization, physical adsorption onto mica surfaces was also attempted. Two µl of a 25 g/l NMC dispersion (in MUF) were spread onto freshly cleaved mica and dried as described in the experimental methods. Using such physisorption on mica, no satisfying images could be obtained in liquid, probably due to desorption of the casein micelles by the SMUF. On the other hand, imaging in air shows closely packed particles with the rough aspect of cauliflowers (Figure S2A). Calculations indicate that several layers of casein micelles were deposited onto the mica. For these reasons, neither the exact contours nor the sizes of the casein micelles can be defined using these images. However, topographical heterogeneity is visible at about the same length-scale, i.e. ~5 nm, as on the images of covalently-bound, individualized micelles (Figure 3). Dilution yielded images of disintegrated structures (Figures 4C and 4D). It suggests that close-packing the casein micelles is a requirement for maintaining their structure on hydrophobic surfaces. 7,8 Providing these conditions, the
4 improved resolution brings further detail to the previous reports by Régnault et al.,6 MüllerBuschbaum et al.,5 or Gebhardt et al.,4 who rather reported casein micelles with smooth surfaces. A 8 B 7 C 7 D 6 Figure S2. AFM height images of casein micelles simply deposited onto mica and subsequently dried. All images were taken in air. (A) to (C) are samples obtained from a 25 g/l dispersion of casein micelles at ph 6.7. (D) was obtained from a 2.5 g/l dispersion. The white scale bar is 2 nm. III. AFM cross-section profiles The following figure provides cross-sections of the individual casein micelles at ph 6.7 (Figure 3C in the manuscript) and 5. (Figure 4C in the manuscript) imaged in air. The cross-
5 sections are taken at the top of the casein micelle, where lateral effects on the tip are minimal, to evidence topographical differences between the two samples. A. Casein micelle at ph 6.7 Height 54nm nm 15 2 B. Casein micelle at ph 5. 26nm Height nm Figure S3. AFM height images and cross sections of individual casein micelles immobilized on gold surfaces at ph 6.7, immersed in SMUF at ph 6.7 (A) or 5. (B) then scanned in air after dessication. The red bars on the left locate the cross sections shown on the right. The white bar is 1 nm. The cross-section at ph 6.7 clearly evidences the roughness of the surface of the dried casein micelle (Figure S3A), while that of the acidified micelle is smooth (Figure S3B). Figure S3A also illustrates that the typical length-scale of the bumps is 3-5 nm, as discussed in the manuscript.
6 References [1] De Kruif, C. G. Supra-Aggregates of Casein Micelles As a Prelude to Coagulation. J. Dairy Sci. 1998, 81, [2] Holt, C. Structure and Stability of Bovine Casein Micelles. Adv. Protein Chem. 1992, 43, [3] Morris, V.J.; Kirby, A.R.; Gunning, A.P. In Atomic Force Microscopy for Biologists; Morris, V.J., Kirby, A.R., Gunning, A.P., Eds.; Imperial College Press: London, UK, 28; pp [4] Gebhardt, R. Structural Characterization of Casein Micelles: Shape Changes During Film Formation. Journal of Physics: Condensed Matter 211, 23, [5] Müller-Buschbaum, P.; Gebhardt, R.; Roth, S. V.; Metwalli, E.; Doster, W. Effect of Calcium Concentration on the Structure of Casein Micelles in Thin Films. Biophys. J. 27, 93, [6] Regnault, S.; Thiebaud, M.; Dumay, E.; Cheftel, J. C. Pressurisation of Raw Skim Milk and of a Dispersion of Phosphocaseinate at 9 C or 2 C: Effects on Casein Micelle Size Distribution. Int. Dairy J. 24, 14, [7] Dalgleish, D. G.; Spagnuolo, P. A.; Goff, H. D. A Possible Structure of the Casein Micelle Based on High-Resolution Field-Emission Scanning Electron Microscopy. Int. Dairy J. 24, 14, [8] Helstad, K.; Rayner, M.; van Vliet, T.; Paulsson, M.; Dejmek, P. Liquid Droplet-Like Behaviour of Whole Casein Aggregates Adsorbed on Graphite Studied by Nanoindentation With AFM. Food Hydrocolloids 27, 21,
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