Walnut Protein Hydrolysates Research Articles

Enhancing functionality and bioactivity of walnut protein through limited enzyme digestion.

Walnut protein (WP) is recognized as a valuable plant protein. However, the poor solubility and functional properties limit its application in the food industry. It is a great requirement to improve the physicochemical properties of WP. Following a 90 min restricted enzymatic hydrolysis period, the solubility of WP significantly increased from 3.24% to 54.54%, with the majority of WP hydrolysates (WPHs) possessing a molecular weight exceeding 50 kDa. Circular dichroism spectra showed that post-hydrolysis, the structure of the protein became more flexible, while the hydrolysis time did not significantly alter the protein's secondary structure. After hydrolysis, WP's surface hydrophobicity significantly increased from 2279 to 6100. Furthermore, WPHs exhibited a strong capacity for icariin loading and micelle formation with critical micelle concentration values of 0.71, 0.99 and 1.09 mg mL-1, respectively. Moreover, similar immuno-enhancement activities were observed in WPHs. After exposure to WPHs, the pinocytosis of RAW264.7 macrophages was significantly improved. WPH treatment also increased the production of nitric oxide, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in macrophages. Up-regulation of mRNA expressions of IL-6, inducible nitric oxide synthase (iNOS) and TNF-α was observed in a dose-dependent manner. The enhancement of functionality and bioactivity in WP can be achieved through the application of limited enzyme digestion with trypsin. This process effectively augments the nutritional value and utility of the protein, making it a valuable component in various dietary applications. © 2024 Society of Chemical Industry.

The walnut-derived peptide TW-7 improves mouse parthenogenetic embryo development of vitrified MII oocytes potentially by promoting histone lactylation

BackgroundPrevious studies have shown that the vitrification of metaphase II (MII) oocytes significantly represses their developmental potential. Abnormally increased oxidative stress is the probable factor; however, the underlying mechanism remains unclear. The walnut-derived peptide TW-7 was initially isolated and purified from walnut protein hydrolysate. Accumulating evidences implied that TW-7 was a powerful antioxidant, while its prospective application in oocyte cryopreservation has not been reported.ResultHere, we found that parthenogenetic activation (PA) zygotes derived from vitrified MII oocytes showed elevated ROS level and delayed progression of pronucleus formation. Addition of 25 μmol/L TW-7 in warming, recovery, PA, and embryo culture medium could alleviate oxidative stress in PA zygotes from vitrified mouse MII oocytes, furtherly increase proteins related to histone lactylation such as LDHA, LDHB, and EP300 and finally improve histone lactylation in PA zygotes. The elevated histone lactylation facilitated the expression of minor zygotic genome activation (ZGA) genes and preimplantation embryo development.ConclusionsOur findings revealed the mechanism of oxidative stress inducing repressed development of PA embryos from vitrified mouse MII oocytes and found a potent and easy-obtained short peptide that could significantly rescue the decreased developmental potential of vitrified oocytes, which would potentially contribute to reproductive medicine, animal protection, and breeding.

A novel angiotensin I-converting enzyme inhibitory peptide from walnut (Juglans sigillata) protein hydrolysates and its evaluation in Ang II-induced HUVECs and hypertensive rats

This study aims to seek angiotensin-I-converting enzyme inhibitory (ACEi) peptides from walnut using different enzymatic hydrolysis, and further to validate the potent ACEi peptides identified and screened via peptidomics and in silico analysis against hypertension in spontaneously hypertensive rats (SHRs). Results showed that walnut protein hydrolysate (WPH) prepared by combination of alcalase and simulated gastrointestinal digestion exhibited high ACEi activity. WPH was separated via Sephadex-G25, and four peptides were identified, screened and verified based on their PeptideRanker score, structural characteristic and ACE inhibition. Interestingly, FDWLR showed the highest ACEi activity with IC50 value of 8.02 μg/mL, which might be related to its close affinity with ACE observed in molecular docking. Subsequently, high absorption and non-toxicity of FDWLR was predicted via in silico absorption, distribution, metabolism, excretion and toxicity. Furthermore, FDWLR exhibited positively vasoregulation in Ang II-induced human umbilical vein endothelial cells, and great blood pressure lowering effect in SHRs.

Synergistic effects of tilapia head protein hydrolysate and walnut protein hydrolysate on the amelioration of cognitive impairment in mice.

Cognitive impairment (CI) is a significant public health concern, and bioactive peptides have shown potential as therapeutic agents. However, information about their synergistic effects on cognitive function is still limited. Here, we investigated the synergistic effects of tilapia head protein hydrolysate (THPH) and walnut protein hydrolysate (WPH) in mitigating CI induced by scopolamine in mice. The results showed that the combined supplementation of THPH and WPH (mass ratio, 1:1) was superior to either individual supplement in enhancing spatial memory and object recognition abilities in CI mice, and significantly lessened brain injury in CI mice by alleviating neuronal damage, reducing oxidative stress and stabilizing the cholinergic system. In addition, the combined supplementation was found to be more conducive to remodeling the gut microbiota structure in CI mice by not only remarkably reducing the ratio of Firmicutes to Bacteroidota, but also specifically enriching the genus Roseburia. On the other hand, the combined supplementation regulated the disorders of sphingolipid and amino acid metabolism in CI mice, particularly upregulating glutathione and histidine metabolism, and displayed a stronger ability to increase the expression of genes and proteins related to the brain-derived neurotrophic factor (BDNF)/TrkB/CrEB signaling pathway in the brain. These findings demonstrate that tilapia head and walnut-derived protein hydrolysates exerted synergistic effects in ameliorating CI, which was achieved through modulation of gut microbiota, serum metabolic pathways and BDNF signaling pathways. © 2024 Society of Chemical Industry.

The antioxidant peptides from walnut protein hydrolysates and their protective activity against alcoholic injury.

In this study, walnut protein was hydrolyzed, separated by ultrafiltration, purified by RP-HPLC, identified by LC-MS/MS, and screened by molecular docking to finally obtain three novel antioxidant peptides HGEPGQQQR (1189.584 Da), VAPFPEVFGK (1089.586 Da) and HNVADPQR (949.473 Da). These three peptides exhibited excellent cellular antioxidant activity (CAA) with EC50 values of 0.0120 mg mL-1, 0.0068 mg mL-1, and 0.0069 mg mL-1, respectively, which were superior to that of the positive control GSH (EC50: 0.0122 mg mL-1). In the ethanol injury model, three antioxidant peptides enhanced the survival of cells treated with ethanol from 47.36% to 62.69%, 57.06% and 71.64%, respectively. Molecular docking results showed that the three antioxidant peptides could effectively bind to Keap1, CYP2E1 and TLR4 proteins. These results suggested that walnut-derived antioxidant peptides could be potential antioxidants and hepatoprotective agents for application in functional foods.

Novel strategy to the characterization and enhance the glycemic control properties of walnut-derived peptides via zinc chelation

This study aimed to utilize zinc coordination to promote the hypoglycemic and antioxidant properties of walnut-derived peptides, such as walnut protein hydrolysate (WPH) and Leu-Pro-Leu-Leu-Arg (LPLLR, LP5), of which LP5 was previously identified from WPH. The optimal conditions for the chelation were a peptide-to-zinc ratio of 6:1, pH of 9, duration of 50 min, and temperature of 50 °C. The WPH-Zn and LP5-Zn complexes increased the α-glucosidase inhibition, α-amylase inhibition, and antioxidant activity more than WPH and LP5 (p < 0.05). In particular, the antioxidant activity of WPH-Zn was superior to LP5-Zn. This is attributable to the WPH containing more aromatic amino acids, carboxylate groups and the imidazole groups, which implies its capacity to potentially coordinate with Zn2+ to form the WPH-Zn complex. Moreover, particle size, zeta potential, and scanning electron microscope indicated that the chelation of Zn2+ by peptides led to intramolecular and intermolecular folding and aggregation.

Structure-activity relationship of walnut peptide in gastrointestinal digestion, absorption and antioxidant activity

The structure-activity relationship between digestion of walnut peptide (<3 kDa) and antioxidant activity were explored, and the inhibitory effect of DPP-IV on antioxidant peptide KGHLFPN (Lys-Gly-His-Leu-Phe-Pro-Asn) was selected. After simulated gastrointestinal digestion, the peptides’ antioxidant capacities were enhanced, tertiary structures were weaker, and secondary structures were random coiled. After the digestion, the peptide and C–H phylyl bonds were broken and macromolecular proteins were hydrolyzed. Antioxidant walnut peptide KGHLFPN was a good DPP-IV inhibitor. And 4–8 h may be the optimal time for oral absorption of KGHLFPN in vivo. Therefore, walnut protein hydrolysate (<3 kDa) can be used as an antioxidant precursor, and walnut peptides can be potential food-derived antioxidants.

Molecular Docking Revealed the Potential Anti-Oxidative Stress Mechanism of the Walnut Polypeptide on HT22 Cells.

The preparation of novel antioxidant peptides from food raw materials is one of the research focuses, but there are fewer studies on the preparation of antioxidant peptides from walnut meal, a by-product of processing walnuts. This study analyzed the antioxidant properties and protective effects of walnut protein hydrolyzed by alkaline protease and trypsin on the oxidative stress of HT22 cells. The peptides were identified by UPLC-MS/MS, and the anti-oxidative peptides were screened based on virtual computer tools. The potential anti-oxidative stress mechanism of the walnut polypeptide on HT22 cells was explored by molecular docking. The results revealed that walnut protein hydrolysates (WPH) with molecular weights of less than 1 kDa had good antioxidant properties and inhibited oxidative damage of HT22 cells by regulating the levels of reactive oxygen species (ROS) and antioxidant enzyme catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). Six of the ninety identified new peptides showed good solubility, non-toxicity, and bioactivity. The molecular docking results showed that the six peptides could dock with Keap1 successfully, and EYWNR and FQLPR (single-letter forms of peptide writing) could interact with the binding site of Nrf2 in the Keap1-Kelch structural domain through hydrogen bonds with strong binding forces. The results of this study provided important information on the antioxidant molecular mechanism of the walnut polypeptide and provided a basis for further development of walnut antioxidant polypeptide products.

Neuroprotective Effects of a Novel Tetrapeptide SGGY from Walnut against H2O2-Stimulated Oxidative Stress in SH-SY5Y Cells: Possible Involved JNK, p38 and Nrf2 Signaling Pathways.

SGGY, an antioxidant tetrapeptide identified from walnut protein hydrolysate in our previous study, has been suggested to possess the potential to alleviate oxidative stress in cells. In this paper, the neuroprotective effects of SGGY on H2O2-stimulated oxidative stress in SH-SY5Y cells and the underlying mechanisms were investigated. Results showed that SGGY alleviated H2O2-induced oxidative stress by decreasing the intracellular reactive oxygen species (ROS) level and altering the mitochondrial membrane potential (MMP), thereby inhibiting apoptosis and increasing cell viability. SGGY significantly restored antioxidant enzyme activities and reduced malondialdehyde (MDA) content accordingly. Moreover, SGGY promoted the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and suppressed the H2O2-induced activation of JNK and p38 mitogen-activated protein kinases (MAPKs). Taken together, these results suggested that SGGY protected SH-SY5Y cells from H2O2-provoked oxidative stress by enhancing the ability of cellular antioxidant defense, and the possible mechanism involved MAPKs and Nrf2 signaling pathways.

Purification, Identification and Molecular Docking of Novel Antioxidant Peptides from Walnut (Juglans regia L.) Protein Hydrolysates

Walnut protein isolate (WPI) was hydrolyzed using Alcalase for 0, 30, 60, 90, 120 and 150 min to investigate the effect of different hydrolysis times on the structure and antioxidant properties of walnut proteins. The identified peptides HADMVFY, NHCQYYL, NLFHKRP and PSYQPTP were used to investigate the structure-activity relationship by using LC-MS/MS and molecular docking. The kinetic equations DH = 3.72ln [1 + (6.68 E0/S0 + 0.08) t] were developed and validated to explore the mechanism of WIP hydrolysis by Alcalase. Structural characteristics showed that the UV fluorescence intensity and endogenous fluorescence intensity of the hydrolysates were significantly higher than those of the control. FTIR results suggested that the secondary structure gradually shifted from an ordered to a disordered structure. Enzymatic hydrolysis containing much smaller molecule peptides than WPI was observed by molecular weight distribution. In vitro, an antioxidant test indicated that Alcalase protease hydrolysis at 120 min showed more potent antioxidant activity than hydrolysates at other hydrolysis times. In addition, four new antioxidant peptides were identified by LC-MS/MS. Molecular docking indicated that these peptides could interact with ABTS through interactions such as hydrogen bonding and hydrophobic interactions. Thus, WPI hydrolysates could be used as potential antioxidants in the food and pharmaceutical industries.

Extraction optimization and antioxidant activity evaluation of se‐enriched walnut proteins

In this study, the proteins of selenium-enriched walnuts produced in three seleniferous areas of Enshi (Yesanguan (YS), Lichuan (LC), and Sancha (SC)) were extracted by the alkaline method. Response surface methodology (RSM) was used for the optimization of protein extraction. The total selenium (Se) content of walnut proteins was 1.3345 mg/kg in YS, 0.6219 mg/kg in LC, and 0.4219 mg/kg in SC, and about 82–85% was organic Se. The major Se speciation was selenocystine and selenomethionine. All walnut proteins showed antioxidant activities in a Se-concentration-dependent manner. The antioxidant activity of YS walnut was stronger than that of other proteins, which exhibited high DPPH radical (69.5%) and moderate hydroxyl radical (46.5%) scavenging activities at 1.0 mg/ml. After enzymatic hydrolysis, protein hydrolysates displayed significantly higher antioxidant capacities than undigested proteins at all tested concentrations. The overall results indicated that Se-enriched walnuts have great potential as functional food additives for a dietary supplement. Novelty impact statement Organic selenium accounts for more than 82% of the total Se in walnut proteins, in which SeCys2 and SeMet are major organic forms of selenium. The Se content in walnut proteins is positively correlated with the antioxidant ability. Walnut protein hydrolysates exhibit better antioxidant activities than proteins.

A Two-Stage Enzymolysis Method and Its Application in Exerting Antioxidant Activity of Walnut Protein.

Traditional enzymolysis method for producing bioactive peptides does not consider the utilization of digestive enzymes in the human gastrointestinal tract, leading to the possibility of excessive hydrolysis and higher production cost. Therefore, a two-stage enzymolysis method was established in this study based on in vitro limited enzymolysis and gastrointestinal digestion, and applied it to the research of walnut protein (WP) in exerting antioxidant activity. Results showed that WP could be well-digested by pepsin and pancreatin. WP with limited enzymolysis degree of 0% could achieve high antioxidant activity after the simulated gastrointestinal digestion, and the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) scavenging activity and reducing power were 66.53% and 8.55 μmoL TE/mL, respectively. In vivo experimental results also exhibited that both WP and WP hydrolysate (WPH) could alleviate the oxidative damage induced by D-galactose in SD rats to some extent. Considering the digestive function of human body, in vitro limited enzymolysis, in vitro simulated gastrointestinal digestion and in vivo validation are necessary processes for the production of bioactive peptides.

Structure identification of walnut peptides and evaluation of cellular antioxidant activity

Walnut peptides are widely accepted as nutraceuticals with multiple health benefits. However, information about the structure and biological activity of walnut peptides is limited. In this work, walnut protein hydrolysate (WPH) was prepared by enzymatic hydrolysis. The total and free amino acids compositions in WPH were determined. The peptides sequence of WPH was analyzed by UPLC-MS/MS, and forty eight bioactive peptides were identified. Antioxidant activities were detected for WPH in both extracellular and intercellular assays. QGRPWG, PSRADIY and AYNIPVNIAR were the peptides that accounted for the antioxidant activity of WPH. Among them, QGRPWG showed the highest ORAC value (2801 µmol TE/g), and AYNIPVNIAR possessed the strongest protective effect against H2O2-induced oxidative stress in PC-12 cells in a dose-dependent manner. The results can aid in the recognition of peptides from walnut as nutraceuticals.

Walnut protein hydrolysates ameliorate alcohol-induced cognitive impairment (AICI) by alleviating oxidative stress and inflammation in the brain and improving hippocampal synaptic plasticity in Sprague-Dawley rats.

Alcohol-induced cognitive impairment (AICI) has become a public health concern and thus effective intervention is urgently needed. The present study aims to assess the amelioration effect of walnut protein hydrolysate (WPH) in AICI rats and elucidate the associated mechanism. The Morris water maze task verified the significant amelioration effect of WPH against learning and memory deficits. Moreover, a reduction of oxidative stress and inflammation in brain tissues was observed and the significant enhancement of brain-derived neurotrophic factor (BDNF), postsynaptic density protein (PSD95) and cAMP-response element binding protein (p-CREB) expression (P < 0.05) indicated the rescue of damaged hippocampal synaptic plasticity. In addition, after WPH administration, disorders of ERK and caspase-3 signal pathways, abnormality in the release of the neurotransmitters acetylcholine (ACh) and gamma-aminobutyric acid (GABA), and histopathological impairment in the hippocampus of AICI rats were ameliorated in a dose-dependent manner. Overall, the amelioration of AICI by WPH might result from the alleviation of oxidative stress and inflammation in brain tissue and the improvement of hippocampal synaptic plasticity.

Attenuation of UV-induced skin photoaging in rats by walnut protein hydrolysates is linked to the modulation of MAPK/AP-1 and TGF-β/Smad signaling pathways

There is growing evidence that prevention of skin photoaging by oral administration of food-derived proteins hydrolysates is intricately linked to its alleviation against oxidative stress through modulation of the signaling pathway. Previously, walnut protein hydrolysates (WPHs) were prepared by enzymatic hydrolysis by our group and exhibited excellent anti-photoaging effect through regulation of extracellular matrix metabolism and the NF-κB signaling pathway. However, its response to oxidative stress and cascade mechanism remain unknown. In the present study, Sprague-Dawley rats were exposed periodically to UV irradiation and orally administered with WPHs to further examine the effects of WPHs on the redox state, MAPK/AP-1 and TGF-β/Smad signaling pathways, type I procollagen synthesis, and histopathological impairments in photoaging skin. Intervention with WPHs for 18 weeks significantly alleviated the photoaging morphology, enhanced the antioxidant components, and downregulated the phosphorylation levels of extracellular signal-regulated kinase (ERK) and C-Jun N-terminal kinase (JNK and p38 proteins) in photoaging tissues, while significant alterations on the gene expression levels of ERK, JNK and p38 were not observed. Meanwhile, WPHs significantly activated the TGF-β/Smad signaling pathway and type I procollagen production. Furthermore, histopathological analysis illustrated that WPHs predominately attenuated epidermal hyperplasia, reduced inflammatory filtration, and promoted the deposition of collagen fibers in photoaging skin. Altogether, the underlying mechanism of WPHs attenuating skin photoaging might lie in the synergistic modulation by increasing the antioxidant capacity, modulating the MAPK/AP-1/MMP-1 and TGF-β/Smad signaling pathways, stimulating the synthesis of type I procollagen, and restoring the impaired architecture structure. Our findings suggest that WPHs are promising agents for preventing skin photoaging.

Characterization and Exploration of Potential Neuroprotective Peptides in Walnut (Juglans regia) Protein Hydrolysate against Cholinergic System Damage and Oxidative Stress in Scopolamine-Induced Cognitive and Memory Impairment Mice and Zebrafish.

The purpose of this study was to determine the neuroprotective effect and mechanism of walnut protein hydrolysates (WPH) against memory deficits induced by scopolamine in mice and further to validate the potent neuroprotective peptides identified by integrated approaches of in silico analysis and peptidomics in scopolamine-induced zebrafish. Results showed that a remarkable amelioration on behavioral performance was observed for oral administration of WPH, and disorders of cholinergic system and oxidative stress were normalized in the brains of mice also. Unfortunately, no obvious inflammatory response and anti-inflammatory effect were observed. Additionally, WPH significantly upregulated the expressions of antioxidant defense-related protein (Nrf2) and neurotrophic-related protein (BDNF and CREB). Furthermore, 20 peptides with relatively higher abundance and PeptideRanker scores were predicted by docking to AchE and Keap1. Among them, FY and SGFDAE with the highest binding affinities, -9.8 and -8.0 kcal/mol, were considered as the promising AchE and Keap1 inhibitors, respectively. They were further validated to have neuroprotective capacity in scopolamine-induced zebrafish, indicating that peptidomics and in silico prediction might be the effective approaches to screen neuroprotective peptides.

Separation, identification and molecular binding mechanism of dipeptidyl peptidase IV inhibitory peptides derived from walnut (Juglans regia L.) protein

Walnut protein was hydrolyzed with different proteases to evaluate the hydrolytic efficiency and dipeptidyl peptidase IV (DPP-IV) inhibitory activity in vitro. All of walnut protein hydrolysates (WPHs) exhibited DPP-IV inhibitory activity and Alcalase-derived hydrolysate (WPH-Alc) with better DPP-IV inhibitory activity of 33.90% (at 0.50 mg/mL) was subsequently separated by ultrafiltration and cation exchange chromatography on a SP Sephadex C-25 column. The results showed that fractions with lower molecular weight and higher basic amino acid residues possessed stronger DPP-IV inhibitory activity. Comparably, the obtained fraction B with the yield of 19.80% had the highest DPP-IV inhibitory activity of 76.19% at 0.25 mg/mL. Moreover, nine novel DPP-IV inhibitory peptides were identified using MALDI-TOF/TOF-MS. Molecular docking revealed the peptides could interact with DPP-IV through hydrogen bonds, salt bridges, hydrophobic interactions, π-cation bonds and π-π bonds. The walnut DPP-IV inhibitory peptides showed better stability with heating treatment, pH treatment, or in vitro gastrointestinal digestion.

Anti-osteoporosis effect and purification of peptides with high calcium-binding capacity from walnut protein hydrolysates.

The walnut protein hydrolysate (WPH) was prepared via simulated gastrointestinal digestion. The degree of hydrolysis (DH), amino acid composition, and relative molecular weight distribution of WPH were analyzed. The results showed that the DH of WPH was 11.6%, WPH was rich in Glu and Pro, and the relative average molecular weight of 572 Da accounted for 59.78%. The effects of WPH on osteoporosis were evaluated using a model of retinoic acid-induced osteoporosis rat. Treatment with WPH effectively increased the serum calcium and phosphorus contents, alleviated calcium loss, and reduced tartrate-resistant acid phosphate and alkaline phosphatase activities and bone gla protein content. WPH treatment significantly improved the biomechanical properties of the bone and increased the value of bone mineral density. In addition, WPH treatment improved the bone microstructure. WPH was isolated and purified by Sephadex G-25 gel filtration chromatography and semi-preparative reversed-phase high-performance liquid chromatography. A fraction with high calcium-binding activity was obtained and 15 peptides were identified.

Purification and identification of dual-enzyme hydrolysates obtained from defatted walnut and its antioxidant effects on d-galactose-induced aging mice

The purpose of this study was to explore the antioxidant activity in vivo of dual-enzyme hydrolysates obtained from defatted walnut, and to purify and identify it. The walnut protein hydrolysates (WPH) were obtained through two-step enzymatic hydrolysis of alkaline protease and trypsin. The effects of WPH on the oxidative stress-related biochemical indexes of d-galactose-treated mice were investigated. Next, WPH was passed through a Sephadex G-25 gel chromatography, anion exchange chromatography and Sephadex G-15 gel chromatography. Subsequently, peptides in the high antioxidant activity fraction were identified by RP-nano-HPLC–MS/MS. Finally, 14 potential peptides of length 11 AAs to 21 AAs with antioxidant activity were identified from WPH.

Physicochemical and bio-functional properties of walnut proteins as affected by trypsin-mediated hydrolysis

The walnut proteins were trypsin-hydrolyzed for 0, 15, 30, 60 and 120 min and the effects of hydrolysis on the structural, functional, antioxidant, bioactive-loading, and angiotensin converting enzyme (ACE)-inhibitory properties of walnut protein were studied. The trypsin-mediated hydrolysis of walnut proteins was monitored using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an O-phthaldialdehyde spectrophotometric assay. The unfolding of walnut protein structure during the hydrolysis process was confirmed using intrinsic fluorescence spectroscopy which increased the surface hydrophobicity of protein. The solubility of walnut protein was increased from 17 to 63% after 2 h of trypsin-mediated hydrolysis. The walnut protein hydrolysates showed higher surface activity compared to the native non-hydrolyzed protein, which significantly improved their foaming and emulsifying properties. The hydrolysates also showed significant enhanced antioxidant activity, namely, ABTS radical quenching activity and reducing power due to the release of active antioxidant sequences during the enzymatic hydrolysis. The ability of walnut proteins to inhibit the activity of ACE and their capacity to load hydrophobic bioactive molecules including curcumin and quercetin were also considerably improved by treating with trypsin. The results of this study suggested that the trypsin-mediated hydrolysis could be used as an efficient technology to produce bioactive multi-functional hydrolysates from walnut proteins.