Marine-derived bioactive peptides have attracted increasing attention as value-added functional ingredients. In this study, peptides (<3 kDa) were prepared from yellowfin tuna processing by-products and further fractionated by Sephadex G-25 gel filtration. The major fraction (TBP-MF) exhibited markedly improved compositional homogeneity compared with the unfractionated hydrolysate (TBP), providing a well-defined peptide system for subsequent characterization and biological evaluation. Physicochemical analyses demonstrated that TBP-MF possessed enhanced thermal stability and a more ordered secondary structure, characterized by pronounced β-sheet enrichment, as revealed by TGA/DSC, FTIR, and circular dichroism analyses. Morphological and colloidal characterization further showed that TBP-MF formed relatively uniform lamellar and fibrous assemblies with a narrower particle size distribution and reduced electrostatic stabilization, indicating a higher tendency toward ordered self-association. Peptidomic profiling combined with in silico analysis revealed that TBP-MF was enriched in short peptides with relatively higher PeptideRanker scores and a functional motif distribution containing relatively more neuro-related annotations, although angiotensin-converting enzyme (ACE)- and dipeptidyl peptidase IV (DPP-IV)-related motifs remained predominant in both groups. In differentiated PC12 cells, TBP-MF exhibited excellent cytocompatibility and induced a stable, concentration-dependent increase in the Cell Counting Kit-8 (CCK-8) readout (OD₄₅₀), indicating enhanced cellular metabolic activity and/or increased cell number. In addition, TBP-MF significantly increased intracellular levels of key neurochemical factors associated with sleep-related regulation, including tetrahydrobiopterin (BH4), serotonin (5-HT), and γ-aminobutyric acid (GABA). Overall, this study highlights yellowfin tuna by-products as a promising marine resource for bioactive peptides and suggests that fractionation-driven structural refinement is associated with neuro-related biological activity in differentiated PC12 cells. These findings support the potential application of marine by-product-derived peptides as functional ingredients in health-related fields.

The design and optimization of immobilized metal affinity chromatography (IMAC) media are crucial to enhancing the purification efficiency of recombinant proteins. In this study, the agarose-based microspheres are prepared by using a three-factorial Box–Behnken design followed by NTA-Ni²⁺ agarose-based microspheres (ABM) preparation by the “one-step” crosslinking of epichlorohydrin (ECH)–nitrilotriacetic acid (NTA) to efficiently couple the NTA ligand to the surface of the matrix. After preparation, various sophisticated techniques, including SEM, AFM, DSC, FTIR, and SDS-PAGE, were used to analyze the morphological structure, thermal stability, and chemical composition of NTA-Ni²⁺ ABM. The optimal conditions are identified as an emulsifier PP concentration of 8.12 wt%, a stirring speed of 1624.46 rpm, and an oil-phase temperature of 53.86 °C, giving a span value (Y) of 0.50684. SEM, AFM, DSC, and FTIR results showed that the fabricated NTA-Ni²⁺ ABM were structurally stable and had a uniform cross-linking network for up to 8 h of coupling reaction time. The performance results showed that the beads had a high binding capacity for His-tagged proteins (15.2 ± 0.8 mg/mL), and SDS-PAGE results demonstrated the efficient purification ability for target proteins. These findings provide the theoretical basis and a practical solution for the rational design and application of IMAC medium.