Enzymatic hydrolysis of crayfish shell protein: Unveiling peptide structures for superior prebiotic promotion of Bifidobacterium animalis

Crayfish shell protein is an abundant but underutilized resource for developing peptide-based prebiotics to support gut health. However, systematic evidence linking protease selection to peptide structure and subsequent probiotic responses remains limited. This study evaluated five hydrolysis treatments (papain, trypsin, pepsin, alkaline protease, and neutral protease) to identify structure–function determinants for promoting Bifidobacterium animalis subsp. lactis 3296. Papain and trypsin hydrolysates (CSPe-P and CSPe-T) achieved moderate hydrolysis (16.87–22.24% and 17.14–20.39%), balanced molecular weights (7409–7445 Da), semicrystalline organization (48.12–50.60%), and relatively high β-turn and random-coil proportions. These hydrolysates also contained higher total amino acid contents (610.39–625.34 mg/g) and were enriched in Glu, Asp, Arg, and Lys. Functionally, CSPe-P and CSPe-T increased Bifidobacterium animalis subsp. lactis 3296 with final OD 600 values of approximately 1.8–1.9 and maximum specific growth rates at 12 h of 5.14 h −1 and 4.83 h −1 compared with 4.00 h −1 in control. At 12 h, viable counts in the fructooligosaccharides (FOS), CSPe-P, and CSPe-T reached 59.94–64.19 × 10 8  CFU/mL, whereas the control reached 50.0 × 10 8  CFU/mL. CSPe-P and CSPe-T performed comparably to commercial FOS in this in vitro growth assay. In contrast, alkaline and pepsin hydrolysates presented disordered structures and inferior probiotic promotion. Overall, this study provides preliminary in vitro evidence that structure-guided enzyme selection can identify crayfish shell peptides with probiotic-supporting potential.