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This research underlines the potential of alginate multilayered gel microspheres for the layered encapsulation and the simultaneous delivery of vitamin B2 (VB) and β-carotene (BC). Chitosan was used to improve the stability and controlled release ability of alginate-based gel microspheres. It was shown that a clear multilayered structure possessed the characteristics of pH response, and excellent thermal stability. The sodium alginate concentration and the number of layers had notable effects on mechanical properties and particle size of gel microspheres. Fourier-transform infrared spectroscopy and X-ray diffraction analyses further proved that VB and BC were encapsulated within the gel microspheres. Compared with the three-layer VB-loaded gel microspheres, the total release of VB from the three-layer VB and BC-loaded gel decreased from 93.23 to 85.58%. The total release of BC from the three-layer VB and BC-loaded gel increased from 66.11 to 69.24% compared with three-layer BC-loaded gel. The simultaneous encapsulation of VB and BC in multilayered gel microspheres can markedly improve their bioaccessibility and bioavailability. These results showed the multilayer gel microspheres synthesized herein have potential for applications in the layered encapsulation and simultaneous delivery of various bioactive substances to the intestinal tract.

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