The effects of different thermal processing methods on the protein structure and digestibility of <i>Procambarus clarkii</i>

<i>Procambarus clarkii</i> is a commercially important freshwater crustacean species. This study examined the effects of different thermal processing methods (hot water steam treatment: HS, cold water steam treatment: CS, hot water boiling: HB, and cold water boiling: CB) on the protein structure and digestibility of <i>P. clarkii</i>. The results indicated that different thermal treatments had significant effects on the integrity and digestibility of the protein structure. Different thermal processing methods result in differential oxidation of <i>P. clarkii</i> proteins, with the carbonyl content in the HS group increasing by approximately 31.06% compared to the HB group. This change further leads to varying degrees of structural damage. For instance, the α-helix content in the CS group is 13.62%, whereas it is only 4.24% in the HB group. These structural alterations significantly affect the digestibility of <i>P. clarkii</i> proteins. Levels of Schiff bases, total amino acid content, and fluorescence intensity studies indicated the formation of advanced glycation end products (AGEs). Molecular docking and exogenous addition experiments demonstrated that Nε-(carboxymethyl)lysine (CML), Nε-(carboxyethyl)lysine (CEL), and pentosidine (PEN) significantly affect the protein structure of <i>P. clarkii</i> and inhibit digestive enzyme activity. Among them, PEN exhibits the most significant inhibitory effect on digestibility, followed by CML, which has a weaker inhibitory effect, while CEL has the least impact on digestibility. In conclusion, different thermal treatments influence protein aggregation by altering the protein structure and varying the levels of Schiff bases. Furthermore, AGEs can modify the structure and morphology of proteins, further promoting protein aggregation and reducing digestive enzyme activity, leading to decreased digestibility of <i>P. clarkii</i> protein.