The effect of collagen peptides (CPs) in intestinal mucosal protection has been approved in both cell and animal models. However, its structure–activity relationship and efficient peptide sequences are unclear, which hinders the in-depth study of its action mechanism and relative nutraceuticals and pharmaceuticals development. In this work, size exclusion chromatography, cation-exchange chromatography, and RP-HPLC were used to separate Alaska pollock skin-derived collagen hydrolysates based on their molecular weight, charge property, and hydrophobicity. The intestinal epithelial barrier function (IEBF) protective effect of separated peptide fractions were evaluated by tumor necrosis factor (TNF)-α-induced Caco-2 cell model. Results indicated that lower molecular weight (500–1000 Da) and higher hydrophilicity of CPs were related to better IEBF protective effect. Two high-efficiency IEBF protective peptide sequences, GPSGPQGSR and GPSGLLGPK with the corresponding molecular weights of 841.41 Da and 824.38 Da, were subsequently identified by UPLC-QToF-MS/MS. Their IEBF protective ability are comparable or even better than the currently used intestinal health supplements glutamine and arginine. The present findings suggested that the hydrophilic CPs, with molecular weight between 500 Da to 1000 Da, should be preferred in IEBF protective peptides preparation. GPSGPQGSR and GPSGLLGPK might have the potential of being IEBF protective ingredients used in intestinal health supplements and drugs.

The source and type of collagen are crucial to its application, and both play a decisive role. Collagen was prepared from both tilapia skin and bone and skate skin and cartilage, named as CI-TI-s, CI-TI-b, CI-SK-s, and CII-SK-c, respectively. Types, distributions, structures, and self-assembly of collagen were studied. It showed that yellow collagen fibers from skin arranged longitudinally, while collagen fibers from skate cartilages displayed varying colors. CI-TI-s, CI-TI-b, CI-SK-s, and CII-SK-c showed the typical amide A (3316–3336 cm⁻¹) and amide B (2929–2948 cm⁻¹) in FTIR spectra. CI-TI-b and CII-SK-c showed 218–229 nm of UV absorption, 11.56–12.20 Å of d values in XRD, and 0.12–0.14 of Rpn values in CD. The thermal denaturation temperatures of CI-TI-s and CI-SK-s were 30.7 and 20.6 °C, respectively. The self-assembly of CI-TI-s and CII-SK-c were maximum at pH 7.2 and 7.4–7.6, respectively. The unique collagen peptides of tilapia and skate were GPSGPQGAVGATGPK, PAMPVPGPMGPMGPR, SPAMPVPGPMGPMGPR, GESGPSGPAGPAGPAGVR, SSGPPVPGPIGPMGPR, GLTGPIGVPGPPGAQGEK, GLAGPQGPR, and GLSGDPGVQGIK, respectively. The unique peptides of type I and type II collagen were GPTGEIGATGLAGAR, GVLGLTGMR, LGLTGMR, GEPGAAGPAGPSGPMGPR, SSGPPVPGPIGPMGPR, and GLSGDPGVQGIK, respectively.