Qianru Chen

Marine-derived collagen peptides exhibit potent intestinal barrier protection; however, their gastrointestinal fate and molecular targets remain unclear, limiting their practical applications. This study investigated the digestive stability and transepithelial transport of GPSGPQGSR, a mucoprotective peptide from Alaska pollock (Gadus chalcogrammus) skin, using simulated gastrointestinal digestion, a Caco-2 cell transport model, and an UPLC-QTOF-MS/MS. The results showed that GPSGPQGSR was a digestion-resistant peptide that reached the intestinal epithelium intact. Although brush border membrane enzymes partially hydrolysed the peptide, 42.16% of intact GPSGPQGSR remained in the luminal compartment after 2 h of incubation. No intact peptide was detected in the basolateral compartment. Molecular docking and 100 ns molecular dynamics simulations identified TLR2 (−14.936 kcal/mol) and PAR2 (−10.154 kcal/mol) as high-affinity extracellular targets of GPSGPQGSR, with stable peptide–receptor interactions and extensive hydrogen bonding networks between the peptide and each receptor (RMSD of 1.8 Å and 2.2 Å, respectively). Pharmacological blockade of TLR2 or PAR2 abolished the protective effects of GPSGPQGSR. These findings demonstrate that GPSGPQGSR acts as a digestion-resistant extracellular signalling peptide that reaches the intestinal epithelium intact and protects barrier function through apical TLR2 and PAR2, providing a mechanistic basis for the rational development of marine collagen peptides for improving intestinal health.

Microbial fermentation is an effective strategy to enhance the functional value of plant-derived ingredients. In this study, Stevia rebaudiana leaves were subjected to microbial fermentation to improve their antioxidant potential and functional properties. A composite fermentation system composed of Bacillus subtilis and Candida utilis was established through strain screening, and fermentation conditions were optimized using single-factor and orthogonal experiments, with chlorogenic acid (CA) content and antioxidant activity as evaluation indices. The optimal conditions were determined to be a fermentation temperature of 34 °C, a duration of 36 h, a microbial ratio (Bs:Cu) of 2:1, a moisture content of 55%, and an inoculum level of 3%. Under these optimal conditions, fermentation significantly increased CA content, total phenolic and flavonoid levels, and antioxidant capacity compared with unfermented material. Untargeted metabolomic analysis revealed extensive fermentation-induced remodeling of secondary metabolites, particularly phenolic acids, flavonoids, and terpenoids, including the generation of multiple newly formed bioactive compounds. Functional validation using a laying hen model demonstrated that fermented S. rebaudiana exhibited enhanced antioxidant and anti-inflammatory status and favorable modulation of physiological indicators compared with unfermented samples. Overall, this study demonstrates that microbial consortium fermentation effectively transforms S. rebaudiana from a sweetener-oriented plant into a multifunctional, fermentation-derived functional ingredient. This research is significant as it provides a dual-purpose strategy for developing antioxidant-enriched functional foods for humans and health-promoting natural feed additives for the livestock industry.

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.

Calcium binding peptides from Pacific cod (Gadus macrocephalus) bone have attracted attention due to their potential effects on bone health. In this study, calcium binding peptides (CBP) were prepared from Pacific cod bone by trypsin and neutral protease. Ultraviolet spectra, circular dichroism (CD), and Fourier transform infrared spectroscopy (FTIR) revealed that carboxyl and amino groups in CBP could bind to Ca²⁺, and form the peptide-calcium complex (CBP-Ca). Single-pass intestinal perfusion (SPIP) experiments indicated that the intestinal calcium absorption was significantly enhanced (p < 0.01) in CBP-Ca treated Wistar rats. The anti-osteoporosis activity of CBP-Ca was investigated in the ovariectomized (OVX) Wistar rat model. The administration of CBP-Ca significantly (p < 0.01) improved the calcium bioavailability, trabecular bone structure, bone biomechanical properties, bone mineral density, and bone mineralization degree. CBP-Ca notably (p < 0.01) increased serum calcium, however, it remarkably (p < 0.01) reduced the levels of osteocalcin (OCN), bone alkaline phosphatase (BALP), tartrate-resistant acid phosphatase isoform 5b (TRAP5b), and C-telopeptide of type I collagen (CTX-1) in serum. Results suggested that the cod bone derived CBP could bind with calcium, improve the intestinal calcium absorption, calcium bioavailability, and serum calcium, then reduce the bone turnover rate, and thus ameliorate osteoporosis.

Summary: Gly‐Pro‐Ala‐Gly‐Pro‐His‐Gly‐Pro‐Pro‐Gly (GPAGPHGPPG), a chelating peptide derived from Alaska pollock skin, has been approved with outstanding ability to chelate calcium, zinc and iron ions. In this study, the stability of GPAGPHGPPG during in vitro gastrointestinal enzymatic digestion, the potential binding site between peptide and metal ions as well as its effects on mineral transport in Caco‐2 cells were investigated. Results showed that approximately 75% of GPAGPHGPPG remained intact at the end of the in vitro gastrointestinal enzymatic digestion. The formation of peptide‐metal complex was potentially related to histidine and cyclic structure in terms of dehydration. Moreover, GPAGPHGPPG revealed significant promotional effects on calcium (112.7%, P < 0.01), zinc (32.3%, P < 0.01) and iron (27.7%, P < 0.01) transport in Caco‐2 cell monolayer. In conclusion, GPAGPHGPPG could be developed as a potential functional ingredient to prevent mineral deficiency.

The effect of early enteral nutrition (EN) supplemented with Alaska pollock skin-derived collagen peptides (CPs) on post-burn inflammatory responses was investigated in a mouse model. Male blab/c mice were randomly assigned to four groups: a sham burn (SB) group, a control group (burn + EN + glycine, BE), a positive control group (burn + EN + glutamine, BEG) and a treatment group (burn + EN + CPs, BEC). Burn-induced increases of serum endotoxin level, and systemic and intestinal concentration of TNF-α and IL-6 were attenuated in BEG and BEC at post-burn day (PBD) 1, 3 and 7 (p < 0.05 vs. BE). Notably, BEC revealed a prominent decrease of the serum endotoxin level, TNF-α and IL-6 as compared to BEG at PBD 7 (p < 0.05). Furthermore, EN supplemented with CPs diminished the phosphorylation of intestinal NF-κB p65 and simultaneously down-regulated the mRNA expression of TNF-α and IL-6 in small intestine (p < 0.05 vs. BE). Also, it demonstrated a comparable effect with glutamine in ameliorating post-burn inflammatory responses in mice with burns. Therefore, CPs could be considered as a potential immunonutrient supplement in EN to improve post-burn outcomes in burn patients.

Dysfunction of the intestinal barrier plays a key role in the pathogenesis of inflammatory bowel disease (IBD) and multiple organ failure. The effect of Alaska pollock skin-derived collagen and its 3 tryptic hydrolytic fractions, HCP (6 kDa retentate), MCP (3 kDa retentate) and LCP (3 kDa permeate) on TNF-α induced barrier dysfunction was investigated in Caco-2 cell monolayers. TNF-α induced barrier dysfunction was significantly attenuated by the collagen and its peptide fractions, especially LCP, compared to TNF-α treated controls (P < 0.05). Compared to a negative control, 24 h pre-incubation with 2 mg mL⁻¹ LCP significantly alleviated the TNF-α induced breakdown of the tight junction protein ZO-1 and occludin and inhibited MLC phosphorylation and MLCK expression. The activation of NFκB and Elk-1 was suppressed by LCP. Thus, collagen peptides may attenuate TNF-α induced barrier dysfunction of Caco-2 cells by inhibiting the NFκB and ERK1/2-mediated MLCK pathway with associated decreases in ZO-1 and occludin protein expression.