Influence of ultrasound and enzymatic cross-linking on freeze-thaw stability and release properties of whey protein isolate hydrogel

This study investigated the effect of ultrasound and enzymatic cross-linking on the freeze-thaw (FT) stabil-ity and release properties of whey protein isolate hy-drogels. We evaluated the FT stability by the changes in the microstructure, riboflavin retention, syneresis, water holding capacity (WHC), and texture of gels sub-jected to 3 FT cycles. High-intensity ultrasound (HUS) and transglutaminase (TGase)-mediated cross-linking improved the FT stability of whey protein isolate hy-drogels loaded with riboflavin (WPISAR), as demon-strated by a more uniform and denser porous structure, significantly higher riboflavin retention, WHC, and textural properties, and lower syneresis after 3 FT cycles than those of untreated hydrogels. Furthermore, HUS-and TGase-mediated cross-linking decreased protein erosion and swelling ratio of WPISAR in simu-lated gastrointestinal fluids (SGIF) and reduced the riboflavin release rate in SGIF both with and without the addition of digestive enzymes. After 3 FT cycles, faster riboflavin release occurred due to a more porous structure induced by ice crystal formation compared with their unfrozen counterparts as detected by con -focal laser scanning microscopy. High-intensity ultra- sound-and TGase-mediated cross-linking alleviated the FT-induced faster riboflavin release rate in SGIF. High-intensity ultrasound-and TGase-treated gel sam-ples showed that both diffusion and network erosion were responsible for riboflavin release regardless of FT. These results suggest that HUS-and TGase-mediated cross-linking improved the FT stability of WPISAR with a high riboflavin retention, and might be a good candidate as a controlled-release vehicle for riboflavin delivery to overcome undesired FT processing.