Comparative enzymatic hydrolysis of porcine tracheal cartilage generates ACE/DPP-IV-inhibitory and antioxidant peptides with enhanced bioactivity following simulated gastrointestinal digestion and molecular docking analysis

Enhanced digestion and absorption kinetics of ultra–low molecular weight collagen containing di- and tripeptides: evidence from human and cell models

Across both in vitro and in vivo models, ultra-low molecular weight collagen (LMWCP), with more than 45% di- and tripeptides, exhibited faster early-phase absorption kinetics, thereby enhancing early-phase bioavailability.

Complex enzymolysis-derived peanut peptides: in silico selection of antioxidant peptides, emulsion-microcapsule encapsulation, and in vitro digestive performance evaluation.

Peanut peptides (PP) are promising plant-derived functional ingredients, but bitterness and gastrointestinal instability limit oral applications. This study integrated complex enzymolysis, LC-MS/MS-assisted in silico screening, molecular dynamics (MD), and W1/O/W2 double-emulsion microencapsulation to establish a laboratory-scale discovery-to-delivery workflow for antioxidant PP. Under optimized alkaline protease/flavor protease hydrolysis (5:5, w/w; 10% enzyme; 59 °C; 9 h), LC-MS/MS identified 763 peanut-derived peptides. Stepwise screening using PeptideRanker (>0.5), ToxinPred3.0 (<0.5), and AlgPred2.0 (<0.5) yielded 157 candidates. CDOCKER docking identified P152 (FEELNADLFR) and P142 (YFPTQALNFAFK) as the top docking-ranked Keap1-Kelch and MPO candidates, respectively, with CDOCKER energies of -163.791 and - 158.855 kcal/mol; P34 (SPNVDPPKTP) and P135 (IPVPFPDPDGDYT) showed the lowest calculated binding energies with ABTS+ and DPPH, respectively. MD/MM-PBSA further supported dynamic stability and favorable binding tendencies of P152-Keap1-Kelch and P142-MPO complexes. The optimized PP-loaded double-emulsion microcapsule (PP-M) showed high apparent encapsulation efficiency (89.43%, nitrogen-balance estimate). Human sensory evaluation and electronic-tongue analysis demonstrated that PP-M significantly reduced perceived bitterness and instrumental bitterness response compared with free PP hydrolysate at matched peptide-equivalent concentration. Static INFOGEST digestion indicated gastric structural retention followed by intestinal disintegration, accompanied by increased peptide-equivalent content and enhanced radical-scavenging activity in the digesta. Overall, PP-M improved the in vitro sensory acceptability and bioaccessibility of PP, supporting formulation-level bitterness alleviation rather than complete bitterness elimination. Targeted bitter-peptide identification, marker-peptide release profiling, epithelial transport, and in vivo validation remain necessary.

Ultrasonic-Assisted Enzyme Hydrolysis of Chicken By-Products: Optimization, Peptide Mapping and Antioxidative Efficacy by In-vitro and In-silico Approach

This study investigates the ultrasound-assisted enzymatic extraction of collagen hydrolysates (CH) from chicken skin, heads, and feet, aiming to generate bioactive peptides with potent antioxidant activity. Process optimization involved acetic acid pre-treatment, ultrasonication (39 kHz), and enzymatic hydrolysis using collagenase, followed by ultrafiltration (10 kDa). The optimized treatment (T2) yielded hydrolysates with a high degree of hydrolysis (85.31%) and protein content (11.73 g/dL). SDS-PAGE revealed predominantly low-molecular-weight peptides, indicative of effective collagen breakdown. Antioxidant capacity, measured via DPPH and ABTS+ assays, was significantly elevated in T2 (76.38% and 91.41%, respectively). Peptidomic profiling was conducted using LC-ESI-MS/MS on a QTOF mass spectrometer, enabling the identification of numerous peptide sequences. In-silico bioactivity prediction using the BIOPEP database and Peptide Ranker algorithm revealed multiple sequences with high antioxidant potential, particularly short-chain peptides rich in hydrophobic and aromatic residues. The integration of advanced proteomic tools and bioinformatic analyses confirmed the efficacy of ultrasound-assisted enzymatic hydrolysis in producingfunctionally relevant peptides. These findings support the valorization of poultry by-products as a sustainable source ofantioxidative peptides for potential applications in food and health sectors.

A Comparative Study on the Biological Activities of a Synthetic Peptide (Acetyl Hexapeptide-3) and a Natural Bee Product (Apilarnil): Antioxidant Capacity, Enzyme Inhibitory Activities, and Cytotoxicity

Skin aging involves the progressive dysregulation of extracellular matrix-degrading enzymes and oxidative stress pathways, driving demand for biologically characterized cosmeceutical ingredients. This study provides the first direct comparative evaluation of apilarnil , a lyophilized drone larvae-derived bee product (Apis mellifera) and acetyl hexapeptide-3 (Argireline®; Ac-Glu-Glu-Met-Gln-Arg-Arg-NH₂), a synthetic neurotransmitter inhibitor peptide, with respect to total antioxidant capacity, elastase, collagenase, and human tyrosinase inhibitory activities, and cytotoxicity toward HaCaT human keratinocytes. Compositional characterization of apilarnil revealed a protein-rich profile (42.67 g/100 g) containing 16 amino acids, multiple vitamins, and mineral constituents. Acetyl hexapeptide-3 exhibited higher total antioxidant capacity (5304.7 ± 103.9 µM Trolox equivalents) compared to apilarnil (645.3 ± 23.3 µM TE), attributable to the abundance of electron-donating residues in its defined sequence. Acetyl hexapeptide-3 also produced potent, concentration-dependent inhibition of elastase (IC₅₀ = 1.534 mg/mL) and collagenase (IC₅₀ = 0.00811 mg/mL), whereas apilarnil displayed a biphasic hormetic dose–response for both enzymes, with low-dose activation followed by high-dose inhibition. Neither compound showed an apparent inhibitory effect on human tyrosinase in a single preliminary experiment (n = 1); given the lack of statistical replication, this finding should be considered exploratory and requires confirmatory replication. Cytotoxicity assessment demonstrated that apilarnil was non-toxic across all tested concentrations (0.3125–20 mg/mL; viability >90%), while acetyl hexapeptide-3 exhibited concentration-dependent cytotoxicity (CC₅₀ = 5.34% v/v). These findings reveal complementary bioactivity profiles between the two compounds, suggesting potential value in further investigating their combined use in cosmeceutical formulations.

Multi-Enzyme Hydrolysis and Ultrafiltration-Based Optimization, Identification, and Characterization of Buttermilk Peptides and Myricetin for Enhancing Antioxidant Synergy.

Dairy peptides and plant polyphenols attract significant attention for their bioactive properties; however, the synergistic antioxidant mechanism of buttermilk peptides and myricetin remains unclear. In this study, buttermilk proteins from Lacprodan MFGM-10, a milk fat globule membrane (MFGM)-enriched buttermilk powder, were subjected to multi-enzyme hydrolysis using papain (Pap), alkaline protease (AP), and neutral protease (NP) at an enzyme concentration of 6000 U/g in different combinations. The results showed that the synergistic application of enzymes significantly increased the degree of hydrolysis (DH), with the AP group reaching a peak DH of approximately 42% at 150 min. Two candidate peptides, WGSPP and SWPWQ, were selected from the screened buttermilk peptide fractions. Through Caco-2 transmembrane transport validation and molecular docking, it is confirmed that these peptides can non-competitively bind to acetylcholinesterase (AChE) via molecular docking, with binding energies of -12.7 and -11.1 kcal/mol, respectively. Additionally, myricetin exhibits a strong binding energy, with a binding energy of -9.5 kcal/mol. In an L6 myoblast injury model induced by rotenone, combined treatment significantly enhances AChE inhibitory rate, reduces reactive oxygen species (ROS) and malondialdehyde (MDA) levels, restores superoxide dismutase (SOD) activity and the NAD+/NADH ratio, and improves mitochondrial membrane potential and ATP levels, without demonstrating cytotoxicity. Buttermilk-derived peptides and myricetin exert synergistic antioxidant effects by improving redox homeostasis and mitochondrial function, thereby alleviating oxidative stress-induced injury in rotenone-induced L6 cells. These findings provide preliminary evidence for the potential role of these compounds in oxidative stress-associated skeletal muscle health; however, direct sarcopenia-related endpoints were not evaluated in the present study.

Identification of VAYPQCV, a Gastrointestinally Stable Competitive DPP-IV Inhibitory Peptide from Bitter Leaf (Vernonia amygdalina)