RuBisCO-Polyphenol Interactions Drive Tea Cream Formation via Synergistic Noncovalent Bridging and Covalent Quinone-Thiol Coupling

Cold-induced tea creaming is a persistent bottleneck in the ready-to-drink tea industry. Current mechanistic models mainly rely on exogenous proteins, limiting their physiological relevance. In this study, we definitively identified ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) as an important protein component in tea cream. Using purified RuBisCO and a multiscale biophysical approach, we elucidated the structural drivers of precipitation. DLS and SAXS revealed that EGCG drives massive macroscopic aggregation and RuBisCO unfolding, significantly outperforming epicatechin, caffeine, α-d-galacturonic acid, and l-theanine. NMR and CD demonstrated that catechin aromatic rings actively drive complexation with RuBisCO, inducing a profound α-helix to β-sheet transition. Crucially, residue and quinone-trapping assays unveiled a synergistic dual mechanism: alongside multidentate noncovalent bridging, catechins spontaneously auto-oxidize into electrophilic quinones, driving covalent cross-linking with protein cysteine thiols. Ultimately, this authentic physiological model establishes the RuBisCO-polyphenol complex as the dominant driver of tea creaming, providing a theoretical foundation for future beverage turbidity mitigation.