Purification Of Peptides Research Articles

Heterologous Expression and Functional Characterization of CAP18 from Oryctolagus cuniculus.

Antimicrobial peptides belong to the innate defence system of creatures. These peptides attach to the bacterial membrane in order to die microorganisms by penetrating them. Hence, biotechnology researchers pay more attention to produce antimicrobial peptides for use in various fields. The studies showed that rabbit tissue with inflammation and skin ulcers would be producing CAP18 peptide, which belongs to the cathelicidin group. In this study, the optimized sequence of the cap18 gene was placed into the pPICZAα plasmid after the alpha-factor signal and transformed into Pichia pastoris (X-33 strain). Purification of the recombinant peptide was done based on its histidine tail at C-terminal, and western blotting method was used to demonstrate the purification of rCAP18. The antibacterial activity of the purified and desalted rCAP18 was investigated at different concentrations against pathogenic bacteria. The maximum expression level of rCAP18 (17.5 kDa) was seen 90 h after induction of alcohol oxidase I (AOX1) promoter with methanol. The concentration of rCAP18 was 33 mg/L after purification with Ni-NTA Sepharose column. The function of rCAP18 (4.3, 5.7, 7 µg/ml) was investigated against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Results showed that %CFU/cm2 reached 28% after P. aeruginosa cells treatment with 7 μg/ml of rCAP18. This study presented the findings related to heterologous expression of cap18 gene, and evaluation of rCAP18 antibacterial effects. Our results showed that rCAP18 plays a significant role in inhibiting bacterial growth, especially Gram-negative bacteria.

Isolation, purification and bioactivity of ACE inhibitory peptides from peach kernel protein enzymatic hydrolysate

Peaches mainly produced in China are a source of food-derived proteins, but much of it is wasted. To make full use of the peaches and peaches related resources, we extracted proteins, including albumin, globulin, gliadin, and glutelin, from defatted Tibet wild peach kernels using Osborne method to study their angiotensin I-converting enzyme (ACE) inhibiting activity, which is an active ingredient for hypertension treatment. The different enzymatic products of these extracts were further separated by ultrafiltration membranes and reversed phase high-performance liquid chromatography (RP-HPLC). The component K2 originated from globulin hydrolysate by alcalase treatment showed the highest ACE inhibiting activity (IC50 0.78 mg/mL). Through further analyzing the Mass spectrometry results of K2 and searching the BIOEP database, the specific dipeptide AH (consisted of Ala and His) has been identified as the ACE inhibitory peptide, providing a potential for using Tibet peach kernel-derived peptides as an ingredient in functional food to control hypertension.

Food-derived bioactive peptides-methods for purification and analysis

Bioactive peptides attract the attention of researchers thanks to their high potential to beneficially influence human health. Various activities are reported, and some of these peptides are commercialized as therapeutic agents. Food-related proteins represent an excellent source in this regard. However, the identification, purification, and characterization of bioactive peptides require а complex approach. The full range of analytical techniques is used in combination with the chemical and biological properties of the peptides. The emerging "omics" techniques and "in silico" methods have given a new direction to peptide analysis in recent years. Developing new methods, rapid and low-cost, for the identification, characterization and purification, is a challenging task because of the complexity of food samples. However, bioinformatics is a promising technique for their exploration. These new strategies can predict different types of peptides, their properties and represent a new horizon for releasing their potential. That is why, in this review, we summarize information about methods used for purification and analysis of food-derived bioactive peptides so far, as well as present our point of view about the role of bioinformatics in this process.

Isolation of the hemeoxygenase-1 inducer from rice-derived peptide.

Bioactive peptides with various health benefits have been reported from rice protein hydrolysates. We previously showed that rice-derived peptides (RP) increased intracellular glutathione levels and induced the expression of γ-glutamylcysteine synthetase, which is regulated by nuclear transcription factor-erythroid 2-related factor 2 (Nrf2). Heme oxygenase-1 (HO-1) is an important Nrf2 downstream antioxidant enzyme that protects against oxidative stress. This study aimed to investigate the protective effects of RP on hydrogen peroxide (H2O2)-induced oxidative stress in human hepatoblastoma cell line HepG2 and identified HO-1 induced peptides from RP. Pretreatment of cells with RP reduced the cytotoxicity caused by H2O2 in a dose-dependent manner. Moreover, RP induced HO-1 expression in a concentration- and time-dependent manner. Next, we attempted to isolate the HO-1 inducer from RP by bioactivity-guided fractionation. Purification of the active peptides using a Sep-Pak C18 cartridge and reversed-phase HPLC, followed by sequence analysis by mass spectrometry, led to the identification of the three peptides. These peptides effectively reduced H2O2-induced oxidative stress. Among them, only P3 (peptide sequence: RSAVLLSH) increased HO-1 protein expression. Additionally, the knockdown of Nrf2 suppressed the induction of HO-1 expression by P3. Our results indicated that P3 identified from RP induced HO-1 by activating the Nrf2 signaling pathway.

Solid-phase peptide synthesis on disulfide-linker resin followed by reductive release affords pure thiol-functionalized peptides.

Thiol groups are suitable handles for site-selectively modifying, immobilizing or cyclizing individual peptides or entire peptide libraries. A limiting step in producing the thiol-functionalized peptides is the chromatographic purification, which is particularly laborious and costly if many peptides or even large libraries are to be produced. Herein, we present a strategy in which thiol-functionalized peptides are obtained in >90% purity and free of reducing agent, without a single chromatographic purification step. In brief, peptides are synthesized on a solid support linked via a disulfide bridge, the side-chain protecting groups are eliminated and washed away while the peptides remain on resin, and rather pure peptides are released from the solid support by reductive cleavage of the disulfide linker. Application of a volatile reducing agent, 1,4-butanedithiol (BDT), enabled removal of the agent by evaporation. We demonstrate that the approach is suited for the parallel synthesis of many peptides and that peptides containing a second thiol group can directly be cyclized by bis-electrophilic alkylating reagents for producing libraries of cyclic peptides.

Combined thermal and carboxypeptidase Y stability assays for probing the threaded fold of lasso peptides.

Lasso peptides are natural products belonging to the superfamily of ribosomally synthesized and post-translationally modified peptides (RiPPs). The defining characteristic of lasso peptides is their threaded structure, which is reminiscent of a lariat knot. When working with lasso peptides, it is therefore of major importance to understand and evidence their threaded folds. While the full elucidation of their three-dimensional structures via NMR spectroscopy or crystallization remains the gold standard, these methods are time-consuming, require large quantities of highly pure lasso peptides, and therefore might not always be applicable. Instead, the unique properties of lasso peptides in context of their behavior at elevated temperatures and toward carboxypeptidase Y treatment can be leveraged as a tool to investigate and evidence the threaded lasso fold using only minute amounts of compound that does not need to be purified first. This chapter will provide insights into the thermal stability properties of lasso peptides and their behavior when treated with carboxypeptidase Y in comparison to a branched-cyclic peptide with the same amino acid sequence. Furthermore, it will be described in detail how to set up a combined thermal and carboxypeptidase Y stability assay and how to analyze its outcomes.

Protein Extraction and Sample Preparation Methods for Shotgun Proteomics with Central Nervous System Cells and Brain Tissue.

Shotgun proteomics based in mass spectrometry has been extensively utilized to investigate biological samples for basic and applied research in the clinical field to elucidate changes in the molecular mechanisms caused by diseases. There is still a great lack of information about the molecular mechanisms and the origins of most brain disorders, which makes shotgun proteomics an interesting tool in the study of these diseases. A wide range of samples can be used to study such diseases, such as cerebrospinal fluid, central nervous system cells, and brain tissue via postmortem analysis. As such, different protein extraction methods must be applied to achieve the best results for each sample type. The lysis buffer, digestion protocol, and peptide purification steps chosen prior to liquid chromatography-mass spectrometry analyses are essential to obtain reliable proteomic datasets. For this reason, the availability of a list with a variety of methods and a description of the pros and cons for each one has been compiled and elaborated upon. This review presents several methods for protein extraction, protein digestion, and sample cleanup with a focus on shotgun proteomics via mass spectrometry and a further focus on studying brain disorders.

Serial fractionation of spent brewer's yeast protein hydrolysate by ultrafiltration: A peptide-rich product with low RNA content

Spent brewer's yeast (SBY) application as a new source of peptides depends on the development of proper downstream processes. This work aimed to separate yeast hydrolysate peptides from sugars and ribonucleic acids (RNA) by a 3-step designed fractionation process using ceramic ultra and nanofiltration following two sequences: (1) 50, 8 and 1 kDa and (2) 15, 8 and 1 kDa molecular weight cut-off (MWCO) membranes. In step one, 15 kDa MWCO membrane retained more components and increased performance of subsequent steps, when compared to 50 kDa. Peptide composition of the initial hydrolysate was gradually changed until 90% of the peptides smaller than 1 kg mol −1 were found in the final permeate. Fractionation increased peptide purity regarding RNA and total sugars up to 1.7 and 2.7-fold, respectively. Innovative protein-rich ingredients from SBY residue with an RNA content as low as 1.4% were produced by membrane separation technology. High quality ingredients presented different peptide profiles and great potential in food and pharmaceutical industries applications. • Efficient RNA reduction was achieved using membrane separation technology. • RNA content in protein-rich fraction was as low as 1.4% (dry weight). • Membranes of 15–1 kDa range were adapted to spent yeast ultrafiltration. • Protein purity of obtained fractions increased 1.7 and 2.7-fold for RNA and sugars. • A fraction with bioactive potential (65% of the peptides < 1 kg mol −1 ) was produced.

Synthesis and evaluation of chloro SPIRO and amino SPIRO supports: Novel hydrophilic cross-linked polystyrene polymers for solid phase peptide synthesis

Synthesis of a novel, beaded, hydrophobic/hydrophilic balanced and chemically robust chloro SPIRO polymer matrix consisting of styrene and 4-Chloromethyl styrene (VBC) as backbone and 3,9-Divinyl-2,4,8,10-tetraoxaspiro[5.5]undecane as cross-linking units is demonstrated here. The suspension polymerisation technique was preferred to achieve complete monomer to polymer conversion as well as to attain spherical beaded form. Two different cross-linking densities (2 and 4 mol.%) of polymer supports were synthesized and the composition of the functional bearing site VBC was fixed as 3 mol.% for both supports. The integrity of chloro SPIRO polymer formed was verified by Fourier transform infrared spectroscopy and scanning electron microscopy analysis. Chloro SPIRO resin was subjected to Gabriel Phthalimide reaction to form amino SPIRO resin. Chemical integrity of chloro SPIRO resin was tested in different standard solid phase peptide synthetic conditions and swelling capacity was studied using amino SPIRO resin in different polar solvent conditions. The synthetic capacity of novel amino SPIRO polymer matrix was evaluated by synthesising classically challenging acyl carrier protein peptide by Fmoc strategy. The purity of peptide was checked by high performance liquid chromatography and mass by matrix assisted laser desorption.

Soybean-Derived Peptides Attenuate Hyperlipidemia by Regulating Trans-Intestinal Cholesterol Excretion and Bile Acid Synthesis.

Increased triglyceride, cholesterol, and low-density lipoprotein (LDL) levels cause hyperlipidemia. Despite the availability of statin-based drugs to reduce LDL levels, additional effective treatments for reducing blood lipid concentrations are required. Herein, soybean hydrolysate prepared via peptic and tryptic hydrolysis promoted trans-intestinal cholesterol excretion (TICE) by increasing ATP-binding cassette subfamily G member 5 (ABCG5) and ABCG8 expression. The peptide sequence capable of promoting TICE was determined via HPLC and LC-MS/MS. Based on this, pure artificial peptides were synthesized, and the efficacy of the selected peptides was verified using cellular and hyperlipidemic mouse models. Soybean hydrolysates, including two bioactive peptides (ALEPDHRVESEGGL and SLVNNDDRDSYRLQSGDAL), promoted TICE via the expression of ABCG5 and ABCG8 in enterocytes. They downregulated expression of hepatic cytochrome P450 family 7 subfamily A member 1 (CYP7A1) and CYP8B1 via expression of fibroblast growth factor 19 (FGF19) in a liver X receptor α (LXRa)-dependent pathway. Administration of bioactive peptides to hyperlipidemic mouse models by oral gavage reduced cholesterol levels in serum via upregulation of ABCG5 and ABCG8 expression in the proximal intestine and through fecal cholesterol excretion, upregulated FGF 15/19 expression, and suppressed hepatic bile acid synthesis. Oral administration of soybean-derived bioactive peptides elicited hypolipidemic effects by increasing TICE and decreasing hepatic cholesterol synthesis.

Fractionation and purification of antioxidant peptides from abalone viscera by a combination of Sephadex G‐15 and Toyopearl HW‐40F chromatography

SummaryAbalone viscera, a protein‐rich by‐products from the abalone processing industry, are normally discarded as wastes. In the present study, four different proteases were used to hydrolyse abalone viscera to prepare high‐activity antioxidant peptides, and their hydrolysis effects were compared. Although the hydrolytic abilities of papain and trypsin were inferior to the alcalase and neutrase, their use resulted in significantly (P &lt; 0.05) higher scavenging activities for DPPH. The hydrolysates mainly consisted of peptides with a molecular weight &lt;3 kDa, and the fractionation was achieved using a combination of Sephadex G‐15 and Toyopearl HW‐40F gel filtration chromatography, which overcomes the disadvantages of traditional membrane separation technology in fractionating peptides with relatively similar molecular weight distribution. The DPPH scavenging activities of the components containing peptides &lt;1 kDa (fractions E) were significantly (P &lt; 0.01) higher than those containing peptides &gt;1 kDa. The different fractions E were further purified by reversed‐phase high‐performance liquid chromatography, and a total of 16 target peptides, containing 4‐8 amino acids, enriched in hydrophobic amino acids and C‐terminal Arg, with molecular weights ranging from 500 to 850 Da, were identified.

Optimization of Enzymatic Preparation Process of Hypoglycemic Peptide from Moringa oleifera Seeds by Response Surface Methodology and Its Hypoglycemic Activities Evaluation in Vitro

In order to improve the resource utilization of Moringa oleifera seeds powder, enzymatic method was used to prepare hypoglycemic peptide from Moringa oleifera seeds. Taking the the degree of proteolysis and α-glucosidase inhibition rate as the response value, single-factor experimental screening and response surface method was used to optimize the optimum enzymatic hydrolysis time, liquid-to-material ratio, hydrolysis pH, enzyme addition, and enzymatic hydrolysis temperature. The hypoglycemic peptide with a molecular weight of <3 kDa were prepared by coarse separation by ultrafiltration, and their inhibitory effects on human liver cancer cells (HepG2 cells) were analyzed by MTT method. The results showed that the optimal conditions of enzymatic hydrolysis were as follows: Time 4.6 h, ratio of liquid to material 40.5:1, pH8.3, enzyme dosage 5.5%, temperature 55 ℃, and the inhibition rate of α-glucosidase was 23.62%±0.14%. The α-glucosidase inhibition rate of the ultrafiltration fraction with a molecular weight of less than 3 kDa had an IC50 value of 5.56 mg/mL. When the concentration of ultrafiltration fraction was 300 μg/mL, the proliferation of HepG2 cells was significantly inhibited after 48 h(P<0.05). The research can lay the foundation for the further separation and purification of hypoglycemic peptide from Moringa oleifera seeds.

A rapid one-step process for the isolation of antibacterial peptides by silica-decorated Fe3O4nanoparticles

Antimicrobial peptides (AMPs), which are widely used as food preservatives, were purified to control the spoilage microorganism Alicyclobacillus acidoterrestris. The AMPs were produced by Pediococcus acidilactici CICC10344 and purified by silica-decorated Fe3O4 nanoparticles (SMNPs). The SMNPs were synthesized and characterized by transmission electron microscopy (TEM), vibrating sample magnetometry (VSM), X-ray diffraction (XRD), and Fourier-transform infrared spectroscopy (FT-IR). The diameter of the SMNPs was 31 ± 7 nm. The VSM measurements indicated that the saturation magnetization value (Ms) of the SMNPs was 30 emu/g. We monitored AMP binding to the surface of Fe3O4 nanoparticles by FT-IR, showing that the amide had been successfully formed. The optimal condition for purification by SMNPs was an AMP concentration of 0.5 mg/mL for 30 min of exposure. The AMP purified by SMNPs retained good antimicrobial activity in pH 4–8 and temperature of 80 °C and 100 °C for 20 min. Scanning electron microscope (SEM) images indicated that a likely antibacterial mechanism is pore formation in the cell membrane. The one-step process proposed in the present work is convenient and effective for the purification of antibacterial peptides that may be widely used for the purification of other peptides.

Isolation and Purification of a Hydrophobic Non-Ribosomal Peptide from an Escherichia coli Fermentation Broth

Non-ribosomal peptide synthases (NRPSs) generate versatile bioactive peptides by incorporating non-proteinogenic amino acids and catalyzing diverse modifications. Here, we developed an efficient downstream process for the capture, intermediate purification and polishing of a rhabdopeptide (RXP) produced by the NRPS VietABC. Many typical unit operations were unsuitable due to the similar physical and chemical properties of the RXP and related byproducts. However, we were able to capture the RXP from a fermentation broth using a hydrophobic resin (XAD-16N), resulting in a 14-fold increase in concentration while removing salts as well as polar and weak non-polar impurities. We then used ultra-high-performance liquid chromatography (UHPLC) for intermediate purification, with optimized parameters determined using statistical experimental designs, resulting in the complete removal of hydrophobic impurities. Finally, the UHPLC eluents were removed by evaporation. Our three-step downstream process achieved an overall product recovery of 81.7 ± 8.4%.

Purification and identification of novel antibacterial peptides isolated from Tualang honey

SummaryThis study aimed to determine the role of bioactive peptides in the antibacterial activity of honey. The peptides were extracted, and the concentration was determined. The peptides were fractionated and evaluated for their antibacterial activity towards selected bacteria. The results showed high peptide content in Tualang honey 1.01 mg g−1. A total of seven essential amino acids were present in Tualang honey. The peptide mixture extracted from Tualang honey showed high antibacterial activity towards Staphylococcus aureus, Salmonella enterica serovar Typhimurium, Escherichia coli and Pseudomonas aeruginosa. A total of 33 peptides were identified in the fractions 9 and 10 obtained from RP‐HPLC. The identified peptides belonged to different sources including novel peptides that have no match to the protein database. The results of this first study revealed the role of the peptides generated by honeybees, nectar and pollen, and novel peptides of the natural microflora in the antibacterial activity of honey.

Ultrasonication Improves Solid Phase Synthesis of Peptides Specific for Fibroblast Growth Factor Receptor and for the Protein-Protein Interface RANK-TRAF6.

Considering our interest in the use of peptides as potential target-specific drugs or as delivery vectors of metallodrugs for various biomedical applications, it is crucial to explore improved synthetic methodologies to accomplish the highest peptide crude purity in the shortest time possible. Therefore, we compared “classical” fluorenylmethoxycarbonyl (Fmoc)-solid phase peptide synthesis (SPPS) with ultrasound(US)-assisted SPPS based on the preparation of three peptides, namely the fibroblast growth factor receptor 3(FGFR3)-specific peptide Pep1 (VSPPLTLGQLLS-NH2) and the novel peptides Pep2 (RQMATADEA-NH2) and Pep3 (AAVALLPAVLLALLAPRQMATADEA-NH2), which are being developed aimed at interfering with the intracellular protein-protein interaction(PPI) RANK-TRAF6. Our results demonstrated that US-assisted SPPS led to a 14-fold (Pep1) and 4-fold time reduction (Pep2) in peptide assembly compared to the “classical” method. Interestingly, US-assisted SPPS yielded Pep1 in higher purity (82%) than the “classical” SPPS (73%). The significant time reduction combined with high crude peptide purity attained prompted use to apply US-assisted SPPS to the large peptide Pep3, which displays a high number of hydrophobic amino acids and homooligo-sequences. Remarkably, the synthesis of this 25-mer peptide was attained during a “working day” (347 min) in moderate purity (approx. 49%). In conclusion, we have reinforced the importance of using US-SPPS towards facilitating the production of peptides in shorter time with increased efficacy in moderate to high crude purity. This is of special importance for long peptides such as the case of Pep3.

A hemolysin secretion pathway-based novel secretory expression platform for efficient manufacturing of tag peptides and anti-microbial peptides in Escherichia coli

BackgroundAlthough Escherichia coli has been widely used for the expression of exogenous proteins, the secretory expression in this system is still a big obstacle. As one of the most important secretion pathways, hemolysin A (HlyA) system of E. coli can transport substrates directly from the cytoplasm to extracellular medium without the formation of any periplasmic intermediate, making it an ideal candidate for the development of the secretory production platform for exogenous proteins.ResultsIn this work, we developed a novel production platform, THHly, based on the HlyA secretion system, and explored its applications in the efficient preparation and quick detection of tag peptides and anti-microbial peptides. In this novel platform the signal sequence of HlyA is fused to the C-terminal of target peptide, with Tobacco Etch Virus (TEV) protease cleavage site and 6*His tag between them. Five tag peptides displayed good secretory properties in E. coli BL21 (DE3), among which T7 tag and S tag were obtained by two rounds of purification steps and TEV cleavage, and maintained their intrinsic immunogenicity. Furthermore, Cecropin A and Melittin, two different types of widely explored anti-microbial peptides, were produced likewise and verified to possess anti-microbial/anti-tumor bioactivities. No significant bacterial growth inhibition was observed during the fusion protein expression, indicating that the fusion form not only mediated the secretion but also decreased the toxicity of anti-microbial peptides (AMPs) to the host bacteria. To the best of our knowledge, this is the first report to achieve the secretory expression of these two AMPs in E. coli with considerable potential for manufacturing and industrialization purposes.ConclusionsThe results demonstrate that the HlyA based novel production platform of E. coli allowed the efficient secretory production and purification of peptides, thus suggesting a promising strategy for the industrialized production of peptide pharmaceuticals or reagents.Graphical

Quick Access to High-Purity Peptide Drugs Bradykinin, Leuprolide Analogue, 2(PZ-128), and Rapastinel with Minimal Reagents

Peptide drugs bradykinin, a leuprolide analogue, 2(PZ-128), and rapastinel are synthesized in 56-77% yield using heating-assisted liquid-phase peptide synthesis on a soluble polynorbornene support. These drugs of commercial utility and complex structures are obtained in 2-5.5 h with no epimerization and >95% purity using only 1.2 equivalents of amino acids and coupling reagents. The peptide yield and purity are comparable or superior to the reported methods.

Separation and identification of ACE inhibitory peptides from lizard fish proteins hydrolysates by metal affinity-immobilized magnetic liposome

Purification of peptides responsible for angiotensin I-converting enzyme (ACE) inhibitory activity from highly complex protein hydrolysates is difficult. Affinity chromatography is a powerful method for purification of peptides. In this study, a metal affinity-immobilized magnetic liposome (MA-IML) was prepared using lipid, N-hexadecyl iminodiacetic acid (HIDA) and magnetic nanoparticles made of FeCl3·6H2O and FeCl2·4H2O as main materials. MA-IML was used to adsorb ACE inhibitory peptides from lizard fish proteins hydrolysates. The optimal pH of adsorption solution was 8.5. The peptide sample adsorbed by MA-IML was separated by reverse phase-high performance liquid chromatography (RP-HPLC). Upon amino acid sequence analysis and verification, an ACE inhibitory peptide with IC50 value of 108 μM was identified to be VYP. Molecular docking results indicated that VYP bound to ACE via multiple binding sites. The present study demonstrated that MA-IML might be a useful tool for separating ACE inhibitory peptides from proteins hydrolysates.

Purification, Identification and Characterization of Antioxidant Peptides from Corn Silk Tryptic Hydrolysate: An Integrated In Vitro-In Silico Approach

Corn silk (CS) is an agro-by-product from corn cultivation. It is used in folk medicines in some countries, besides being commercialized as health-promoting supplements and beverages. Unlike CS-derived natural products, their bioactive peptides, particularly antioxidant peptides, are understudied. This study aimed to purify, identify and characterize antioxidant peptides from trypsin-hydrolyzed CS proteins. Purification was accomplished by membrane ultrafiltration, gel filtration chromatography, and strong-cation-exchange solid-phase extraction, guided by 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation (ABTS•+) scavenging, hydrogen peroxide scavenging, and lipid peroxidation inhibition assays. De novo sequencing identified 29 peptides (6–14 residues; 633–1518 Da). The peptides consisted of 33–86% hydrophobic and 10–67% basic residues. Molecular docking found MCFHHHFHK, VHFNKGKKR, and PVVWAAKR having the strongest affinity (−4.7 to −4.8 kcal/mol) to ABTS•+, via hydrogen bonds and hydrophobic interactions. Potential cellular mechanisms of the peptides were supported by their interactions with modulators of intracellular oxidant status: Kelch-like ECH-associated protein 1, myeloperoxidase, and xanthine oxidase. NDGPSR (Asn-Asp-Gly-Pro-Ser-Arg), the most promising peptide, showed stable binding to all three cellular targets, besides exhibiting low toxicity, low allergenicity, and cell-penetrating potential. Overall, CS peptides have potential application as natural antioxidant additives and functional food ingredients.