Potential Antioxidant Peptides Research Articles

Identification and molecular mechanism of novel antioxidant peptide from fish sauce: A combined quantum chemistry and molecular simulation

Fish sauce, derived from fermented fish, exhibits a notable antioxidant effect after a six-month fermentation process, and we propose that potential antioxidant peptides were present in the fish sauce. We isolated, purified, and identified potential bioactive antioxidant peptides by using fish sauce fermented for 6 months. Additionally, molecular simulation was employed to investigate the antioxidant action mechanism of these bioactive peptides. The molecular docking results revealed that FS4-1 (MHQLSKK), FS4-2 (VLDNSPER), FS4-3 (MNPPAASIK), FS6-1(VLKQAAAGR), and FS6-2 (SPDVSPRR), could dock with the Keap1 receptor. The primary force (Van der Waals' force and hydrogen bonds) and key sites (GLY509 and ALA510) of Keap1 binding to peptides were determined. The active center was located in the side chain of amino acid Met at positions C7H78 and C7H79. We here identified antioxidant peptides in fish sauce and revealed the antioxidant mechanism through molecular simulations.

Evaluation of the antioxidant activity and identification of potential antioxidant peptides in commercially available probiotic Cheddar cheese

The aim of this study was to evaluate the antioxidant activity of two commercially available probiotic Cheddar cheeses (C-1 and C-2) and to preliminarily identify potential antioxidant peptides. The results revealed that the water-soluble extract and ultrafiltration fractions from both cheeses exhibited remarkable antioxidant effects, effectively protecting H2O2-induced cells by reducing ROS and MDA levels while increasing CAT and SOD activity. Notably, the <3 kDa fraction of C-1 cheese demonstrated the highest antioxidant activity. Seven fractions with significant antioxidant activity were identified by Sephadex G-25 gel filtration analysis of the < 3 kDa fraction of C-1, and fraction V had the highest activity at approximately 80%. Furthermore, four peptides were identified from fraction Ⅴ as αs2-CN f(204–212) (AMKPWIQPK), β-CN f(177–183) (AVPYPQR), β-CN f(108–113) (EMPFPK), and β-CN f(170–176) (VLPVPQK). These peptides possessed molecular weights of 1098.622 Da, 830.458 Da, 747.374 Da, and 779.502 Da, respectively. AVPYPQR and VLPVPQK have been previously reported, while EMPFPK and AMKPWIQPK may exhibit potential antioxidant properties due to their amino acid sequence characteristics such as proline and methionine residues. Overall, these findings indicate that commercial probiotic cheddar cheeses have great potential as a natural source of antioxidants.

Isolation of peptide mixtures with potential biological activities from the protein hydrolysate of Arsenura armida larval meal

Abstract This study explores using insects as a high-quality protein source rich in bioactive peptides for human nutrition. The objectives include establishing optimal conditions for peptide extraction, identifying their sequences, and assessing their potential as antioxidants and antimicrobial agents in Arsenura armida flour. The protein content in the A. armida flour extract was 54%. To obtain potential antioxidant and antimicrobial peptides, a 10:1 and 1:1 enzyme (pepsin)/substrate ratio was employed, with digestion times of 2 h (A2) and 1 h (A4), respectively. The IC50 value for the antioxidant assay DPPH (2,2-Difenil-1-Picrilhidrazilo) was 22.5 μg/μL for A2. In the antioxidant in vivo assay with Caenorhabditis elegans, A2 peptides at concentrations of 0.75 μg/μL and 1.5 μg/μL exhibited a survival percentage ranging from 32.6% to 35.3%. The A4 sample demonstrated the highest antimicrobial activity, inhibiting the growth of Gram-positive bacteria (Listeria monocytogenes and Staphylococcus aureus) with MIC (minimum inhibitory concentration) values of 0.11 μg/mL and 0.23 μg/mL and MBC (minimum bactericidal concentration) of 0.23 μg/mL for both. Peptide analysis revealed that A2 contained peptides with identified antihypertensive (37%) and antioxidant (58%) activities, while A4 comprised peptides with antihypertensive (62%), antioxidant (31%), and antimicrobial (2%) properties. The antimicrobial peptides identified through the BIOPEP database were LFGF and FLLF. This investigation underscores the potential of A. armida peptides as valuable components in functional foods or nutraceuticals, offering health benefits beyond basic nutrition.

Purification, identification and molecular docking studies of antioxidant and anti-inflammatory peptides from Edible Bird's Nest

This study investigated antioxidant and anti-inflammatory peptides from Edible Bird's Nest (EBN). The prepared EBN peptides were sequentially separated, purified, and successively identified by ultrafiltration, gel filtration and mass spectrometry techniques. Four potential antioxidant and anti-inflammatory peptides were identified as Peptide 1 (LFWSPSVYLK), Peptide 2 (GWPHLEDNYLDW), Peptide 3 (NPPADLHK) and Peptide 4 (GDLAYLDQGHR). Molecular docking analysis revealed that Peptide 1 and Peptide 2 can competitively interrupt the formation of Keap1-Nrf2 due to the presence of hydrophobic and antioxidant amino acids in their peptide sequences. Peptide 3 and Peptide 4 have a strong effect on interacting with the binding site of IKK-β due to the interaction of anti-inflammatory amino acids and C-terminal arginine/lysine. The four peptides were synthesised and validated for their antioxidant and anti-inflammatory activities. The results suggest that the four peptides may serve as promising bioactive peptides for preventing oxidative stress and inflammation-related diseases.

Changes in physicochemical and digestibility properties of foxtail millet prolamin after cooking, and novel antioxidant peptide identification from cooked foxtail millet prolamin

The presence of antioxidant peptides in cooked foxtail millet prolamin (CFMP) is pivotal for its ability to mitigate oxidative stress and subsequently exert an anti-diabetic effect. However, the alterations in structural characteristics and antioxidant activities of foxtail millet prolamin following cooking remain unclear. Furthermore, the identification of antioxidant peptides in CFMP targeting myeloperoxidase (MPO), a key enzyme implicated in oxidative stress, has yet to be achieved. This study aims to examine the changes in physicochemical, digestibility, and antioxidant properties of foxtail millet prolamin post-cooking, and to identify potential antioxidant peptides from CFMP targeting MPO. The findings revealed significant reductions in water and oil holding capacities, foaming and emulsifying capacities, surface hydrophobicity, and zeta potential of foxtail millet prolamin after cooking, along with a notable increase in average particle size, indicative of protein molecule aggregation and increased resistance to digestion. Three potential antioxidant peptides (IFVFPSGAG, MMFPM, and VLIPMP) were identified in CFMP hydrolysate. Molecular dynamic simulation demonstrated favorable binding interactions between all three peptides and MPO, primarily mediated by hydrogen bonds. In vitro antioxidant activity assays confirmed IFVFPSGAG as the most potent antioxidant peptide. This study sheds light on the properties of CFMP and the antioxidant potential of its derived peptides.

Exploring the potential of high hydrostatic pressure and ionic polysaccharides in improving interfacial stability of sweet potato protein hydrolysate emulsions

SummaryThis study aims to explore the potential of high hydrostatic pressure (HHP, 300–500 MPa) and ionic polysaccharides (sodium alginate, SA; chitosan, CH) in improving interfacial stability of sweet potato protein hydrolysate (SPPH) emulsions. HHP markedly improved emulsifying performance of SPPH with SA and CH at different concentrations (0.02% and 0.06%, w/v) and enhanced the apparent viscosities of their emulsions. SPPH‐CH0.02% at 400 MPa showed the highest emulsifying stability index (ESI, 255.96 min) and zeta potential absolute value (54.13 mV), and its emulsion exhibited the highest adsorbed peptide and CH at interface (31.72% and 1.51%, respectively) and oxygen radical absorbance capacity (ORAC) value of interfacial layer (264.56 μg TE mL−1). Additionally, multiple potential antioxidant peptides were obtained from interfacial layer of emulsion stabilised with SPPH‐CH0.02% at 400 MPa through LC‐MS/MS, and the relationship between their structure and emulsion stability was analysed. Therefore, HHP has great application potential in production of SPPH‐CH emulsion with enhanced stability.

Isolation and characterization of a new potential source of bioactive peptides: White mulberry (Morus alba) fruits and its leaves

In this study, white mulberry (WM; Morus alba L.) and its leaf (WML) were hydrolyzed using enzymes at different preheating temperatures (50, 60, 70, 80, and 90 °C) with distilled water at a ratio of 1:10 (w/v). Pepsin, trypsin, chymotrypsin, and alcalase were employed for enzymatic hydrolysis, aiming to investigate the impact of heat treatment and enzymatic hydrolysis on bioactive properties; in particular, angiotensin-converting enzyme-inhibitory (ACE-i) and antioxidant activities. The highest hydrolysis efficiency was observed when using pepsin. The smaller peptides with <3 kDa had better ACE-i and antioxidant activities in both WM and WML samples. Regarding antioxidant activity, the highest level was identified at a preheating temperature of 90 °C in the WM sample, and at preheating temperatures of 50 °C and without heating in the WML samples. In conclusion, heat treatment and enzymatic hydrolysis were found to be a positive impact on ACE-i and antioxidant activities under various conditions. Moreover, the proteins derived from WM and WML were rich in the sources of potential ACE-i and antioxidant peptides.

Simulated in vitro gastrointestinal digestion of β-lactoglobulin treated by ultrasound: Detection of peptides profile and the antioxidant activity

The influence of ultrasonic pretreatment on the release and antioxidant activity of potential antioxidant peptides after in-vitro simulated gastrointestinal digestion of β-lactoglobulin (BLG) were measured by HPLC-MS/MS, chemical and cellular-based assays. The gastrointestinal digest was fractionated into four fractions by Sephadex G-25 gel filtration column, and fractions showed a considerable ABTS·+ scavenging ability. The fraction with the strongest antioxidant activity was produced by ultrasonicated BLG after gastrointestinal digestion, which relies on ultrasonic-promoted proteolysis to produce many small-molecule antioxidant peptides. The best active fraction has better cellular antioxidant activity and protection of H2O2-induced oxidative HepG2 cell model, which significantly increases the activities of antioxidant enzyme, and is concentration-dependent. HPLC-MS/MS analysis showed that there were more potential antioxidant peptides in the best active fraction. This research will provide a basis for the further application of ultrasonic in dairy products, which can promote the release of more potential antioxidant peptides-derived from gastrointestinal digestion.

Novel antioxidant peptides in fermented pork sausage: Purification, characterization, and cytoprotective functions on Caco-2 cells.

In this study, crude peptides from fermented sausages inoculated with Lactobacillus plantarum CD101 and Staphylococcus simulans NJ201 were initially separated by ultrafiltration and molecular-sieve chromatography. The obtained fractions with high 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and ferric-reducing antioxidant power values (MWCO-1 and fraction A) were used in Caco-2 cells to evaluate their cytoprotective effect on oxidative damage triggered by H2O2. MWCO-1 and A showed slight cytotoxicity. Increased glutathione peroxidase, catalase, and superoxide dismutase activities and decreased malondialdehyde content were observed in the peptide-treated groups. Fraction A was further purified using reversed high-performance liquid chromatography. Eighty potential antioxidant peptides were identified by liquid chromatography with tandem mass spectrometry, and fourteen antioxidant peptides were synthesized. SDEEVEH and FAGDDAPR showed strong DPPH radical scavenging activity, whereas ALELDSNLYR and QEYDESGPSIVHR presented strong ABTS+· scavenging activity. These peptides might have great potential for food and pharmacological applications.

Identification of potential antioxidant peptides from protein hydrolysates of pearl oil apricot almonds: Combination of in vitro and molecular docking studies

AbstractPearl oil apricot almonds are rich in proteins, being a valuable raw material for preparing antioxidant peptides. In this study, almond protein hydrolysates (APHs) were obtained by enzymatic hydrolysis and divided into APHs‐1 (&lt;3 kDa), APHs‐2 (3–10 kDa), and APHs‐3 (&gt;10 kDa). APHs‐1‐c, purified from APHs‐1, was proved to show the strongest antioxidant effect through activity tracking experiments. APHs‐1‐c also can protect HepG2 cells against oxidative damage by reducing intracellular reactive oxygen species and MDA levels, elevating glutathione levels as well as increasing superoxide dismutase and catalase activities, and its protective effect is related to the Keap1–Nrf2 signaling pathway. Forty bioactive peptide sequences were identified from APHs‐1‐c, and Thr‐Glu‐Asp‐Asp‐Trp‐Arg‐Trp‐His (TEDDWRWH; P1), Trp‐Tyr‐Asp‐Asn‐Glu‐Trp‐Gly‐Tyr‐Arg (WYDNEWGYR; P2), and Ala‐Glu‐Asp‐His‐Glu‐Trp‐Trp‐Arg (AEDHEWWWR; P3) had the most potential for antioxidant activity by molecular docking studies. P1, P2, and P3 showed desirable 1,1‐diphenyl‐2‐picrylhydrazyl radical scavenging activity (IC50 = 0.634–3.381 mmol/L), 2,2′‐azino‐bis (3‐ethylbenzothiazoline‐6‐sulfonic acid) diammonium salt radical scavenging activity (IC50 = 0.221–0.309 mmol/L), and ferric‐reducing antioxidant power. These results suggested that almonds protein hydrolysates and its derived antioxidant peptides could serve as potential ingredients applied in functional foods.

Sea Bass Side Streams Extracts Obtained by Pulsed Electric Fields: Nutritional Characterization and Effect on SH-SY5Y Cells.

Fish side streams are an environmental and economic problem. In this work, pulsed electric fields (PEF) extraction was optimized and used as a new method for their valorization. Sea bass head, skin, viscera, and backbone were used. PEF technology (123-300 kJ/kg, 1-3 kV/cm) improved the extraction of proteins and antioxidant compounds from head and skin, but was not successful for viscera. SDS-PAGE showed that the protein molecular weight distribution was affected by the extraction process, revealing differences between the control and PEF extraction conditions. In addition, the extraction of macro-minerals and micro-minerals were also evaluated. The effect of PEF differed according to the matrix and the mineral studied. Heavy metals were also taken into account, studying the presence of As, Cd, Hg, and Pb in the extracts. PEF pre-treatment reduced the presence of As in skin, viscera, and backbone, ranging from 18.25 to 28.48% according to the matrix evaluated. The analysis of potential antioxidant bioactive peptides showed that the treatment of the sample directly influenced their variety. Additionally, the extracts obtained from the head were found to increase cell viability when tested on SH-SY5Y cells. In conclusion, PEF extraction can be a useful tool for the valorization of fish side streams.

Protective Effect of Peptides from Pinctada Martensii Meat on the H2O2-Induced Oxidative Injured HepG2 Cells

Pinctada martensii is a major marine pearl cultured species in southern China, and its meat is rich in protein, which is an excellent material for the preparation of bioactive peptides. In this study, the peptides from Pinctada martensii meat were prepared by simulated gastrointestinal hydrolysis, and after multistep purification, the structures of the peptides were identified, followed by the solid-phase synthesis of the potential antioxidant peptides. Finally, the antioxidant activities of the peptides were verified using HepG2 cells, whose oxidative stress was induced by hydrogen peroxide (H2O2). It was shown that the antioxidant peptide (S4) obtained from Pinctada martensii meat could significantly increase the cell viability of HepG2 cells. S4 could also scavenge reactive oxygen species (ROS) and reduce the lactate dehydrogenase (LDH) level. In addition, it could enhance the production of glutathione (GSH) and catalase (CAT) in HepG2 cells, as well as the expression of key genes in the Nrf2 signaling pathway. Three novel antioxidant peptides, arginine-leucine (RL), arginine-glycine-leucine (RGL), and proline-arginine (PR), were also identified. In conclusion, peptides from Pinctada martensii meat and three synthetic peptides (RGL, RL, PR) showed antioxidant activity and could have the potential to be used as antioxidant candidates in functional foods.

Desirable characteristics of casein peptides with simultaneously enhanced emulsion forming ability and antioxidative capacity in O/W emulsion

This study aimed to explore the desirable characteristics of casein hydrolysates with simultaneously enhanced emulsion forming and antioxidative ability in oil-in-water (O/W) emulsion. Effects of physicochemical properties of casein hydrolysates prepared by trypsin, alcalase, and neutrase on physical and oxidative stability of O/W emulsion were studied, and potential antioxidant peptides were characterized by UPLC-MS/MS and bioinformatics analysis. Results showed that casein hydrolysates at a lower degree of hydrolysis (DH) produced by trypsin displayed higher zeta potential absolute values (−31.7 mV), smaller particle size (170.75 nm) and improved antioxidant properties compared to alcalase and neutrase, and could form more stable emulsion with d4,3 of 0.39–0.41 μm at a broader range of DH. Additionally, compared with sodium caseinate (NaCas), the oxidation of emulsion was significantly retarded during the storage progress prepared by the trypsin hydrolysates at DH of 4.49% alone or with Tween 20. Peptidomics indicated that the trypsin hydrolysates contained more peptides with basic amino acids at C-terminal and aromatic amino acids at N-terminus, which exhibited cationic amphiphilic properties with grand average of hydrophilicity values (GRAVY) of −0.5 ∼ +0.5 and pI > pH 7.0. This could contribute to their better emulsion formation and inhibition of lipid oxidation ability by producing dense interfacial layers with peptides interactions or excluding metal ions in O/W emulsions. These results suggested that casein hydrolysates at lower DH prepared by trypsin owned potential amphiphilic antioxidant peptides, which were desired to enhance the stability and functional activity of O/W emulsion.

In vitro and in silico analysis of potential antioxidant peptides obtained from chicken hydrolysate produced using Alcalase

Chicken hydrolysates (CHs) have been reported to protect mice against alcoholic liver injury possibly through oxidative stress reduction. In this study, the antioxidant activity of CHs was studied. Results showed that CHs exhibited significant antioxidant activity (around 600 and 400 μM TEAC/g in ORAC and ABTS assay, respectively) and could resist simulated gastrointestinal digestion. A total of 22 peptides were identified after antioxidant activity-oriented isolation using size-exclusion chromatography and high-performance liquid chromatography. Further in silico analysis and the validation of antioxidant activity revealed that novel peptides (RWGG and YYCQ) exhibited strong antioxidant activity. The most active peptide YYCQ displayed a TEAC value of 3.54 and 4.28 μM TEAC/μM in ORAC and ABTS assay, respectively. These peptides could contribute to reduce oxidative stress and protect against alcohol-induced liver injury. However, further studies understanding the bioactivity of such peptides in vivo are necessary before further applying them as functional food ingredient.

Development of a machine learning-based predictor for identifying and discovering antioxidant peptides based on a new strategy

It is necessary to solve the problem of food corruption and oxidation to improve food quality. Peptides are a good candidate to solve the above problems. In this paper, a machine learning method was used to construct an antioxidant peptide classification model based on the pseudo-amino acid composition and motifs of peptides as input features. The AUC of PseAAC-dipeptide-motif hybrid model is 0.939 and the average precision score is 0.947, which is the best among all models in this paper. Besides, the classification threshold has been increased to make the model precision above 0.95. Then, the model was used as predictor to discover potential antioxidant peptides from a random peptide dataset. According to the predicted results, 5 potential antioxidant peptides (PSGK, LKPQ, GRP, QCQ, QGM) were synthesized to determine their DPPH radical-scavenging activity and the total antioxidant capacity (T-AOC). The experimental results show that QCQ has strong antioxidant properties, its T-AOC value is 9.59U/mg prot, and the DPPH scavenging activity is 95.52% at concentration of 125 μg/mL. Meantime, the predictor can be used to mine multifunctional peptides with antioxidant function. In general, the predictor is an effective tool for discovering peptides with antioxidant function. • A machine learning-based predictor was developed for virtual screening antioxidant. • Peptides of unknown function can be quickly identified whether they have antioxidant properties. • Multifunctional peptides with antioxidant function were quickly discovered based on a new strategy. • A low-cost and not time-consuming method for discovering antioxidant peptides.

Effect of Sous-vide cooking on the quality and digestion characteristics of braised pork

This study aimed to evaluate the effect of Sous-vide (SV) cooking on the quality, flavor and digestion characteristics of braised pork. Traditional (TD) sample had the highest fat content and malondialdehyde (MDA) value, but the lowest protein content and total sulfhydryl (SH) content (P < 0.05). The SH content in SV samples decreased with the heating time, but MDA content increased (P < 0.05). In addition, α-helix content of TD samples was higher but β-sheet content was lower than SV samples. LC-MS/MS indicated that SV samples at 65 °C for 8 h had potential bioactive and antioxidant peptides. GC–MS mainly identified 395 volatile components and SV samples at 70 °C for 8 h and 75 °C for 8 h had similar flavor compounds to TD samples. Thus, Sous-vide cooking produced better quality and digestion characteristics of braised pork. These findings give a new insight into the associations of processing methods with meat quality.

Structure-function relationship of small peptides generated during the ripening of Spanish dry-cured ham: Peptidome, molecular stability and computational modelling

A systematic insight into the structure–function properties of small bioactive peptides is of great importance. Herein, peptidomics and computational methodology were adopted to investigate the stabilization patterns and building blocks of antioxidant peptides resulting from proteolysis during the ripening of Spanish dry-cured ham (9–24 months of processing). The results showed that native peptides underwent manufacture-induced steric/redox stress, while homogeneous/heterogeneous p-π/π-π interaction significantly improved the ABTS+ inhibition activity of hydrophobic peptides. However, for more hydrophilic peptides, the intrinsic π-interaction system (i.e., cation-π and π-π packing) substantially interfered with ABTS+/DPPH scavenging events when compared to the aromatic residues. Semi-quantitative peptidomics and molecular simulation/docking revealed that VFSSQGQSELILLQK and LCPSPDGLYL are two potential antioxidant peptides at the late ripening-drying. They had distinctive self-folding destinies following solvation owing to varying charged/hydrophobic properties of termini and hydrogen atom donor, allowing different flexibility of backbone and interactive surface towards free radicals ex situ followed by electron/proton transfer.

Peptidomic analysis of whey protein hydrolysates and prediction of their antioxidant peptides

Enzymatic hydrolysis of proteins is a breakdown process of peptide bond in proteins, releasing some peptides with potential biological functions. Previous studies on enzymatic hydrolysis of whey proteins have not identified the complete peptide profiles after hydrolysis. In this study, we reconstructed a profile of peptides from whey hydrolysates with two enzymes and different processing conditions. We also developed an ensemble machine learning predictor to classify peptides obtained from whey hydrolysis. A total of 2572 peptides were identified over three process conditions with two enzymes in duplicate. 499 peptides were classified and chosen as potential antioxidant peptides from whey proteins. The peptides classified as antioxidants in the hydrolysates had a proportion of 13.1%–24.5% regarding all peptides identified. These results facilitate the selection of promising peptides involved in the antioxidant properties during the enzymatic hydrolysis of whey proteins, aiding the discovery of novel antioxidant peptides.

Antioxidant Activity of Novel Casein-Derived Peptides with Microbial Proteases as Characterized via Keap1-Nrf2 Pathway in HepG2 Cells.

Casein-derived antioxidant peptides by using microbial proteases have gained increasing attention. Combination of two microbial proteases, Protin SD-NY10 and Protease A "Amano" 2SD, was employed to hydrolyze casein to obtain potential antioxidant peptides that were identified by LCMS/ MS, chemically synthesized and characterized in a oxidatively damaged HepG2 cell model. Four peptides, YQLD, FSDIPNPIGSEN, FSDIPNPIGSE, YFYP were found to possess high 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging ability. Evaluation with HepG2 cells showed that the 4 peptides at low concentrations (< 1.0 mg/ml) protected the cells against oxidative damage. The 4 peptides exhibited different levels of antioxidant activity by stimulating mRNA and protein expression of the antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), as well as nuclear factor erythroid-2-related factor 2 (Nrf2), but decreasing the mRNA expression of Kelch-like ECH-associated protein 1 (Keap1). Furthermore, these peptides decreased production of reactive oxygen species (ROS) and malondialdehyde (MDA), but increased glutathione (GSH) production in HepG2 cells. Therefore, the 4 casein-derived peptides obtained by using microbial proteases exhibited different antioxidant activity by activating the Keap1-Nrf2 signaling pathway, and they could serve as potential antioxidant agents in functional foods or pharmaceutic preparation.

들깨박 단백질 가수분해물 유래 항산화 펩타이드의 항산화 활성에 대한 pH, 열 및 장내 프로테아제의 영향

Perilla seed meal (PSM) is a by-product of perilla seed oil extraction and a good source of bioactive peptides. In this study, the effects of heat, pH, and intestinal protease treatments on the antioxidant activity and stability of two antioxidant peptides (PAP1 and PAP2) were assessed and quantified; the stability was evaluated by measuring the 1,1-diphenyl-2-picrylhydrazine (DPPH) radical scavenging activity, 2,2'-azinobis-(3-ethylbenzothiazoline-6-sul- fonic acid (ABTS) radical scavenging activity, and reducing power. The strength of the antioxidant effects of PAP1 and PAP2 was found to be reasonably well preserved relative to that of positive controls. Specifically, PAP1 and PAP2 maintained their antioxidant activity or exhibited only slightly decreased activity after heat treatment, with &gt;76% activity retention. The antioxidant activities of PAP1 and PAP2 were lower than those of the controls under acidic and basic conditions but remained above 70% at all pH values except pH 12. Additionally, the antioxidant activity of both peptides remained above 66%, even after treatments of pepsin, chymotrypsin, and trypsin. Thus, the results indicate that antioxidant peptides from PSM protein hydrolysate are reasonably resistant to heat, pH, and intestinal proteases. Furthermore, these results demonstrate that antioxidant peptides derived from PSM hydrolysate exhibit high potential as natural antioxidant peptides for food and/or nutraceutical applications.