Characterisation and screening analysis of haze-active proteins in thawed cloudy huyou (Citrus changshanensis) juice using a data-independent acquisition proteomics technique.

Turbidity loss is a significant issue in thawed huyou (Citrus changshanensis) juice, and understanding the composition of the precipitate is essential for effective control. In the present study, data-independent acquisition proteomics was used to investigate precipitation-related proteins in thawed cloudy huyou juice (TCHJ). A total of 1268 precipitation-related proteins were identified, the majority of which were localised in the chloroplast and predominantly contained P-loop NTPase domains. By cross-referencing various bioinformatics algorithms, 33 pivotal haze-active proteins were identified. The three-dimensional structures of haze-active proteins were constructed using artificial intelligence algorithms, and molecular docking simulations revealed that these proteins interact with haze-active small molecules primarily through van der Waals forces and hydrogen bonds, leading to flocculation and precipitation in the TCHJ. Fluorescence spectroscopy analysis revealed that the primary static quenching mode exists between the precipitated protein and the haze-activated small molecules; the binding ability and stability between the precipitated protein with the haze-activated small molecules were clarified; and the non-covalent binding mechanism was verified. The findings of this study provide a deeper understanding of the precipitated protein system in TCHJ, providing technical support for future theoretical and process studies on the protein stability of TCHJ.