Structural changes in casein aggregates under frozen conditions affect the entrapment of hydrophobic materials and the digestibility of aggregates Kyuya Nakagawa a,n , Teeraya Jarunglumlert b,c , Shuji Adachi a a Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan b Department of Mechanical and System Engineering, University of Hyogo, Himeji, Hyogo 671-2280, Japan c Vidyasirimedhi Institute of Science and Technology, Wangchan, Rayong 21210, Thailand HIGHLIGHTS  Freeze-drying a caseinate with β-carotene successfully entrapped this dispersion.  The entrapped β-carotenes were spatially distributed in the dried powder.  Freezing modified casein aggregate structures due to the interactions with ice.  The structural change during freezing affected the encapsulation efficiencies.  The structural change during digestion was dependent on the freezing conditions. article info Article history: Received 24 August 2015 Received in revised form 19 November 2015 Accepted 1 January 2016 Available online 15 January 2016 Keywords: Casein Nanoparticle Freezing Drying Digestion Small angle x-ray scattering abstract Freeze-dried casein nanoparticles that could entrap β-carotene were produced after aging under frozen conditions. Structural changes that occurred during aging and simulated digestion were investigated. Freeze-dried specimens of casein particles prepared from sodium caseinate solutions containing dis- persed β-carotene successfully entrapped this dispersion in the resulting freeze-dried powders. The entrapped β-carotenes were distributed between the surface (surface load) and interior (inner load) of these dried powders. Because of aging, the amount of the inner load decreased while the surface load simultaneously increased. The hydrophobicity of rehydrated casein particles indicated that a change, caused by the aging process, occurred in particle structure. These structural modifications increased the hydrophobicity in the dried specimens. When rehydrated, these hydrophobic surfaces reassociated with each other to cancel the net gain in hydrophobicity. SAXS measurements on freeze-thawed casein nanoparticles also suggested the formation of modified nano- and microstructures. These modified structures were formed by freezing and thawing, along with interactions between clusters or between clusters and ice. The kinetics of the proteolytic reactions of the freeze-thawed specimens in a simulated gastric fluid were measured. The degree of aggregation and processing conditions did not significantly affect the digestion kinetics of the casein clusters. The SAXS analyses, however, suggested that these conditions affected nano- and microstructure formation during digestion. When the aggregated casein clusters were exposed to the gastric conditions, the fractured clusters produced by the proteolytic reactions produced 2–3 times larger aggregates with hollow networks and rough surfaces created by the crosslinked casein clusters. Processing conditions, such as pH and aging time, likely affected these nano- and microstructure formations under gastric conditions. These results may provide exciting future research opportunities if the structural modifications can impact the bioavailability of entrapped materials. & 2016 Elsevier Ltd. All rights reserved. 1. Introduction Protein-based nanoparticles are being recognized as potential delivery vehicles for nutraceutical and pharmaceutical materials (Chen et al., 2006; Hawkins et al., 2008). Nanoparticles designed to Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/ces Chemical Engineering Science http://dx.doi.org/10.1016/j.ces.2016.01.001 0009-2509/& 2016 Elsevier Ltd. All rights reserved. n Corresponding author. Tel./fax: þ81 75 753 6287. E-mail address: kyuya@kais.kyoto-u.ac.jp (K. Nakagawa). Chemical Engineering Science 143 (2016) 287–296