Enzymes Alcalase Research Articles

Alkaline Proteases from Rose Snapper (Lutjanus guttatus): Evaluation of Their Stability to Chemical Denaturants and Potential Application to Hydrolyze Seafood Waste Proteins.

Large quantities of by-products are generated after processing of rose snapper (Lutjanus guttatus), such as viscera, head, tail, skin, and bones, which are considered a potential source of valuable molecules. Therefore, the aim of the present study was the biochemical characterization of alkaline proteases isolated from the intestines of L. guttatus and the evaluation of their stability against different chemical denaturants (salts, surfactants/reducing agents, organic solvents, and commercial detergent formulations). In addition, the efficiency to hydrolyze proteins from rose snapper wastes (head, tail, skin, and muscle trimmings) by Alcalase® and alkaline protease extract (APE) isolated from Lutjanus guttatus intestine was evaluated. The APE exhibited a maximum activity at pH 12 and 45°C and high stability at pH and temperature ranges from 9 to 12 and 10 to 40°C, respectively. Assays with specific protease inhibitors indicated that trypsin and chymotrypsin are the main types of proteases in APE. An 80% of the proteolytic activity was retained in the presence of 25% NaCl and was stable in the presence of the reducing agent DTT; however, it lost around 70% of proteolytic activity in the presence of 2-mercaptoethanol. The enzymatic activity of APE was maintained above 60% in methanol, ethanol, and propanol as well as in liquid commercial detergents. Alkaline proteases from rose snapper exhibited higher hydrolytic efficiency, compared to the microbial enzyme Alcalase when protein from L. guttatus wastes were hydrolyzed. According to these results, the integral exploitation of rose snapper could be reached by proper usage of its by-products, creating a baseline to promote circular economy.

Novel Peptides LFLLP and DFFL from Jack Bean Protein Hydrolysates Suppress the Inflammatory Response in Lipopolysaccharide-Stimulated RAW 264.7 Cells.

The production of inflammatory cytokines such as tumor necrosis factor (TNF)-α by activated macrophage cells plays an important role in the development of intestinal inflammation. The present study investigated the anti-inflammatory effect of the protein hydrolysates prepared from the jack bean (JBPHs), Canavalia ensiformis (L.) DC, using the enzyme Alcalase, in a murine macrophage model, RAW 264.7 cells, which were stimulated by lipopolysaccharides. JBPHs reduced the TNF-α expression at the protein and mRNA levels through the downregulation of cellular signaling pathways involved in nuclear factor kappa B (NF-κB), extracellular signal-regulated kinase (ERK), and p38. A combination of mass spectrometry and in silico approaches identified 10 potential anti-inflammatory peptides in the JBPHs, including LFLLP and DFFL. Interestingly, while LFLLP targeted the NF-κB pathway, DFFL targeted p38 and ERK to suppress the TNF-α production in the RAW 264.7 cells. In addition, LFLLP and DFFL were localized in the cytosol of the cells. These results demonstrated that LFLLP and DFFL were incorporated by RAW 264.7 cells and, at least in part, contributed to the reduction in TNF-α by JBPHs. These peptides isolated from JBPHs may well be utilized as new alternatives to alleviate intestinal inflammation.

Ultrasonication assisted enzymatic hydrolysis for generation of pulses protein hydrolysate having antioxidant and ACE-inhibitory activity

The main objective of this work was to investigate the impact of ultrasonication assisted enzymatic treatment on the physicochemical and bioactive properties of broad bean (BBP), lentil bean (LBP), and mung bean (MBP) protein isolates. The protein was extracted using alkaline acid precipitation method, ultrasonicated at a frequency of 20 kHz, temperature 20–30 °C and then hydrolysed using alcalase enzyme (1 % w/w, pH 8.5, 30 min, 55 οC). The generated hydrolysates were characterized by degree of hydrolysis (DH), SDS, FTIR, surface hydrophobicity, amino acid composition, antioxidant and antihypertensive properties. Results showed that the degree of hydrolysis was found to increase in ultrasonicated protein hydrolysate (18.9 to 40.71 %) in comparison to non- ultrasonicated protein hydrolysate (11 to 16.3 %). SDS-PAGE results showed significant changes in protein molecular weight profiles (100–11kDa) in comparison to their natives. However, no substantial change was found in ultrasonicated and non-ultrasonicated protein hydrolysates. The FTIR spectrum showed structural alterations in ultrasonicated and non-ultrasonicated protein hydrolysates, suggesting modifications in secondary structure such as amide A, amide I and amide II regions. The essential amino acid content varied in the range of 60.09 mg/g to 73.77 mg/g and 28.73 to 50.26 mg/g in case of ultrasonicated and non-ultrasonicated protein hydrolysates, and non-essential content varied in the range of 49.42 to 65.93 mg/g and 43.12 to 47.12 mg/g. Both antioxidant and antihypertensive activities were found to increase significantly in ultrasonicated and non-ultrasonicated protein hydrolysates in comparison to their native counterparts, highlighting their potential as functional ingredients for management of hypertension. It was concluded that ultrasonication assisted enzymatic hydrolysis is an efficient approach for production of bioactive pulse protein hydrolysates with enhanced nutracutical properties, thus offering promising avenues for their utilization in the food industry and beyond.

Generation, Characterisation and Identification of Bioactive Peptides from Mesopelagic Fish Protein Hydrolysates Using In Silico and In Vitro Approaches.

This study generated bioactive hydrolysates using the enzyme Alcalase and autolysis from mesopelagic fish, including Maurolicus muelleri and Benthosema glaciale. Generated hydrolysates were investigated for their bioactivities using in vitro bioassays, and bioactive peptides were identified using mass spectrometry in active hydrolysates with cyclooxygenase, dipeptidyl peptidase IV and antioxidant activities. In silico analysis was employed to rank identified peptide sequences in terms of overall bioactivity using programmes including Peptide Ranker, PrepAIP, Umami-MRNN and AntiDMPpred. Seven peptides predicted to have anti-inflammatory, anti-type 2 diabetes or Umami potential using in silico strategies were chemically synthesised, and their anti-inflammatory activities were confirmed using in vitro bioassays with COX-1 and COX-2 enzymes. The peptide QCPLHRPWAL inhibited COX-1 and COX-2 by 82.90% (+/-0.54) and 53.84%, respectively, and had a selectivity index greater than 10. This peptide warrants further research as a novel anti-inflammatory/pain relief peptide. Other peptides with DPP-IV inhibitory and Umami flavours were identified. These offer potential for use as functional foods or topical agents to prevent pain and inflammation.

The Effect of Whey Protein Isolate Hydrolysate on Digestive Properties of Phytosterol.

Phytosterol (PS) is a steroid, and its bioavailability can be enhanced by interacting with protein in the C-24 hydroxyl group. The interaction between sterols and amino acid residues in proteins can be enhanced by enzymatic hydrolysis. Phytosterol and whey insulation hydrolysates (WPH1-4) fabricated by the Alcalase enzyme at different enzymatic hydrolysis times were selected as delivery systems to simulate sterol C-24 hydroxyl group interaction with protein. Increasing hydrolysis time can promote the production of β-Lg, which raises the ratio of β-turn in the secondary structure and promotes the formation of interaction between WPH and PS. The correlation coefficient between hydrogen bonds and encapsulation efficiency (EE) and bioaccessibility is 0.91 and 0.88 (P < 0.05), respectively, indicating that hydrogen bonds of two components significantly influenced the combination by concealing the hydrophobic amino acids and some residues, which improved PS EE and bioavailability by 3.03 and 2.84 times after PS was combined with the WPI hydrolysate. These findings are expected to enhance the absorption of PS and other macromolecules by protein enzymatic hydrolysis to broaden their applications for food.

Optimization of cashew nut (Anacardium occidentale) oil extraction yield by the enzymatic method

This study aimed to extract as much cashew oil as possible by enzymatic means, an environmentally friendly method. To achieve this, cashew almond paste was reacted with the enzyme alcalase 2.4 L, taking into account the matrix provided by a Box-Behnken design. The factors highlighted were those of enzyme concentration, incubation temperature, incubation time, pH ratio of the medium, and substrate/water. These factors were optimized to obtain the maximum cashew oil yield. The results showed that the alcalase 2.4 L enzyme optimized the cashew oil yield. The optimum yield (38.20 %) was achieved with an enzyme concentration of 2.5 %, a temperature of 60 °C, an incubation time of 8 h, a pH of 8 and a substrate/water ratio of 1:5. Thus, enzymatic extraction using alcalase 2.4 L can be considered an effective extraction method for obtaining oil from cashew nuts.

Impact of Time and Enzyme Concentration on Sangyod Rice Bran Hydrolysate: Phytochemicals, Antioxidants, Amino Acids, and Cytotoxicity

This study investigated the production of Sangyod rice bran hydrolysate (SYRB) from Sangyod rice, focusing on incubation times (1, 3, and 5 h) and alcalase enzyme concentrations (0, 0.7, and 1% v/v). The results demonstrated a concentration-dependent relationship: higher alcalase concentrations increased hydrolysate yield. Prolonged incubation, especially with alcalase, enhanced substrate breakdown, further increasing hydrolysate production. The degree of hydrolysis, reflecting peptide bond cleavage, depended on both incubation time and enzyme concentration, emphasizing the role of enzyme activity in efficiency. Moreover, color analysis (L*, a*, b*) and color difference (∆E) revealed intricate changes from enzymatic hydrolysis. Proximate composition analysis showed higher protein and lipid content with increased enzyme concentration and longer incubation times, whereas ash content varied with both factors. Hydrolysate powders exhibited higher moisture content than raw rice bran, indicating the impact of the hydrolysis process. The study also explored SYRB's antioxidant properties and cytotoxicity, which were sensitive to incubation time and alcalase concentration. Longer incubation increased DPPH scavenging activity, with the highest efficacy at 3 h. Meanwhile, ABTS scavenging displayed a delicate balance with alcalase concentration. The cytotoxicity study of SYRB revealed that all concentrations of SYRB were non-toxic to C2C12 cells, with cell viability values exceeding 70%.

The Role of Ultrasound Cavitation Combined with Alcalase Enzyme in Improving the Structural and Antioxidant Attributes of Sulfated Polysaccharide from Yellowfin (Thunnus albacares) Skin

ABSTRACT Alcalase enzyme (1 mg) and ultrasound (400 W) at 0–20 min were applied to obtain sulfated polysaccharides from yellowfin skin. Applying 15 min ultrasound resulted in a 0.12% increase in the rate of recovery yield. The intact structure of polysaccharides was determined by Fourier transform infrared spectroscopy, X-ray diffractometry, and scanning electron microscope. Ultrasound improved the thermal behavior of sulfated polysaccharides by affecting their microstructure, as analyzed by DSC. The color characterization (L*, a*, and b*) of ultrasound-treated samples was found to be superior compared to the control. Applying 15 min led to 16% and 9% enhancing DPPH and ABTS radical scavenging activities along with improving ferric-reducing antioxidant power of the polysaccharide.

Characterisation and beneficial effects of a Lupinus angustifolius protein hydrolysate obtained by immobilisation of the enzyme alcalase®.

Bioactive peptides have been considered potential components for the future functional foods and nutraceuticals generation. The enzymatic method of hydrolysis has several advantages compared to those of chemical hydrolysis and fermentation. Despite this fact, the high cost of natural and commercial proteases limits the commercialization of hydrolysates in the food and pharmacological industries. For this reason, more efficient and economically interesting techniques, such as the immobilisation of the enzyme, are gaining attention. In the present study, a new protein hydrolysate from Lupinus angustifolius was generated by enzymatic hydrolysis through the immobilisation of the enzyme alcalase® (imLPH). After the chemical and nutritional characterization of the imLPH, an in vivo study was carried out in order to evaluate the effect of 12 weeks treatment with imLPH on the plasmatic lipid profile and antioxidant status in western-diet-fed apolipoprotein E knockout mice. The immobilisation of alcalase® generated an imLPH with a degree of hydrolysis of 29.71 ± 2.11%. The imLPH was mainly composed of protein (82.50 ± 0.88%) with a high content of glycine/glutamine, arginine, and aspartic acid/asparagine. The imLPH-treatment reduced the amount of abdominal white adipose tissue, total plasma cholesterol, LDL-C, and triglycerides, as well as the cardiovascular risk indexes (CRI) -I, CRI-II, and atherogenic index of plasma. The imLPH-treated mice also showed an increase in the plasma antioxidant capacity. For the first time, this study demonstrates the beneficial in vivo effect of a lupin protein hydrolysate obtained with the alcalase® immobilised and points out this approach as a possible cost-effective solution at the expensive generation of the hydrolysate through the traditional batch conditions with soluble enzymes.

Antioxidant activity of palm kernel meal protein hydrolysate and characterization of its peptide profile

Palm kernel meal (PKM) is a major by-product of the palm oil industry, and its high protein content is a potential source of value-added functional food or feed. In this study, the total protein from PKM was isolated and hydrolyzed with alcalase enzyme (pH 7.5, 55oC) to obtain palm kernel protein hydrolysate. The results showed that PKM protein hydrolysate after 60 min of hydrolysis exhibited strong radical scavenging activity with IC50 values of 5.73±0.23 and 7.84±0.90 μg/mL as determined by 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), respectively. Furthermore, PKM protein hydrolysate was not toxic to mouse L929 fibroblast cells while protecting cells from H2O2-induced oxidative damage. Analysis of its peptide profile by liquid chromatography-mass spectrometry (LC-MS/MS) revealed nine peptide sequences with hydrophobic and negatively-charged amino acids with molecular weights ranging from 1,085.13 to 1,292.32 Da. Taken together, alcalase hydrolysate of PKM was found to have potent antioxidant and cytoprotective properties justifying further study for potential development as a functional food/feed.

Development of lipopeptide surfactants from silk sericin and evaluation of their surface active properties

The synthesis of lipopeptides from silk sericin was realized in two steps: enzymatic hydrolysis of protein with Alcalase enzyme and the grafting of lipid chain (C10, C12 or C14) following the Schotten-Baumann reaction. The influence of experimental conditions on the degree of hydrolysis (DH) and the acylation rate (AR) was investigated. Then, equilibrium surface adsorption properties of lipopeptides at air/water interface were studied. The effect of lipopeptide structure, in particular the size of peptide and of the hydrophobic chain, was characterized. Lipopeptides obtained with DH from 20 % to 45 % and AR from 27 % to 41 % were successfully produced. The obtained results demonstrated that sericin peptides have poor amphiphilic properties, whereas lipopeptides efficiently decreased air/water surface tension up to 30.9 mN/m. The surface activity of lipopeptides seem to be closely related to their structure. Sericin lipopeptides show surface properties comparable to those of commercial surfactants, which emphasize the interest of these biomass-derived amphiphilic molecules for potential replacement of petroleum-based surfactants.

Maximizing hypocholesterolemic peptides from an olive byproduct by enzymatic hydrolysis

Novel food proteases (Maxipro A, Maxipro B, Maxipro C, and Maxipro D) with broad specificity and different effective pH range (from 2.5 to 9.0) were evaluated to produce hypocholesterolemic peptides from olive seeds proteins. Box-Behnken experimental design was employed to evaluate the effect of the hydrolysis time, pH, temperature, and enzyme:substrate ratio on the amount of released peptides. The production of peptides, under optimal conditions, was higher with Maxipro C and lower with Maxipro B. The capability of extracts to reduce the absorption of exogenous cholesterol and the production of endogenous cholesterol was also evaluated. All hydrolysates showed a higher capacity to bind bile acids and inhibit cholesterol esterase enzyme than the obtained with usual Alcalase enzyme. Proteomic analysis using high resolution RP-HPLC-ESI-QTOF with database searching and de novo identification was employed to identify peptides in hydrolysates. Some sequences with hypocholesterolemic features were detected among the 45 peptides identified in Maxipro A hydrolysate, the 56 peptides in that of Maxipro C, and the 50 in the Maxipro B one. This work demonstrates that Maxipro A enzyme can hydrolyze olive seeds proteins, under acidic pH, releasing peptides with multifunctional and higher hypocholesterolemic capacity than the obtained with Alcalase, under alkaline conditions.

Improving functional properties of rice protein isolates by hydrolysis using alcalase enzyme and assessing bioactivities of hydrolysates

AbstractBackgroundDeveloping value‐added products from rice grains is becoming increasingly significant to enhance productivity from excess rice grains. Therefore, the objective of this study was to improve the functional properties of rice protein from four rice varieties, namely, RRF‐127, RRF‐105, Swarna, and Protezin by enzymatic hydrolysis, and also to evaluate bioactivities like antioxidant, ACE, and DPP‐IV inhibitory activities of the hydrolysates.ResultsThe alcalase hydrolysis increased protein solubility to &gt;80% in the pH range of 4–10. The foaming capacity and stability of rice protein hydrolysates (RPHs) improved with the increase in pH. The hydrolysis significantly improved the emulsification activity index (EAI) in the pH range of 5–9. The hydrolysates showed antioxidant activity, namely, ABTS•+radical scavenging, reducing power, and metal ion chelation. RRF‐105 was found to possess the most potent antioxidant capacity among the hydrolysates. RPH could alleviate oxidative damage in erythrocytes, and their protective effects were concentration‐dependent. The hydrolysates had stronger inhibition of ACE and DPP‐IV enzymes. Protezin exhibited the highest ACE inhibition (63%), while Swarna demonstrated the most effective DPP‐IV inhibition (66%).ConclusionHydrolysis with alcalase improved the functional and bioactive properties of rice proteins, indicating that RPH could serve as a source of bioactive peptides or as a functional ingredient with potential properties.

Effects of Enzyme Concentrations and Digestion Time on Degree of Hydrolysis and Chemical Properties of Protein Hydrolysate from Black Soybean Using Alcalase Enzyme

Effects of Enzyme Concentrations and Digestion Time on Degree of Hydrolysis and Chemical Properties of Protein Hydrolysate from Black Soybean Using Alcalase Enzyme

Ultrasonication and microwave pre-treated locust protein hydrolysates enhanced the storage stability of meat emulsion

Locust protein hydrolysates (LoProHs) pre-processed with microwave and ultrasonication were developed and evaluated for their potential for enhancing the quality of the stored meat emulsion (MEmul). Locust protein (LoPro) samples pre-processed with ultrasonication (Ult) or microwave (Mic) or with no treatment (Not) were hydrolysed with alcalase enzyme (3%). The microwave pre-processed (Mic-LoProHs) and ultrasonicated (Ult-LoProHs) hydrolysates showed significantly (P < 0.05) higher antioxidant [FRAP (ferric reducing antioxidant power) and ABTS and DPPH radical scavenging activities] and antimicrobial [minimum inhibitory concentration (MIC) and inhibitory halos (mm)] potential. The MEmul samples incorporated with Mic-LoProHs and Ult-LoProHs at the maximum level of 1.5% exhibited significantly (P < 0.05) improved results for all the quality parameters such as antioxidant potential (FRAP, ABTS and DPPH), protein oxidation (total carbonyl content), lipid stability, and microbial quality during refrigerated storage (4 ± 1 °C) of two-weeks compared to the control MEmul without any LoProHs. A positive (P < 0.05) impact of the LoProHs was found on the sensory quality of MEmul samples after one week of storage. The digestion simulation improved (P < 0.05) the antioxidant potential of the MEmul samples.

Conservation and Analysis of Textiles Recovered From Exhumed Human Remains After More Than a Century of Burial

An 1891 indigent Chinese burial in an East Perth cemetery was exhumed in 2020 with textiles covering the upper part of the body. The project was to conserve the textiles for future exhibitions and to relocate the bones at a new ossuary. The ethical issues pertaining to the treatment of the human remains were approved by the Chung Wah Association. The conservation process recovered remnants of a singlet, vest, jacket, belt, a scarf, and fragments of a purse. Surface pH and chloride readings established the decay process while the influence of the timber coffin on the microenvironment was also noted. The rock-solid sand and textile matrix was softened with the protein dissolving Alcalase enzyme, which facilitated removal of a complex matrix of plant roots along the bones. Rehumidification of the partially cleaned textiles with aqueous glycerol solutions improved the strength and viability of the very degraded woollen fabrics. Analysis of the degraded wool by x-ray fluorescence and FTIR showed that chromium dyes were responsible for their preservation while phosphate from dissolving bones accelerated decay. The XRF data also enabled identification of the mordants used in the dying process. It took over seven months of work to complete the project.

Improving microbial and lipid oxidative stability of cheddar cheese using cricket protein hydrolysates pre-treated with microwave and ultrasonication

The study was carried out to investigate cricket protein hydrolysates’ (CPH) potential to enhance the storage stability of cheddar cheese. The cricket protein (CP) samples pre-processed with microwave (T1), ultrasonication (T2) or without pre-treatment (T0) were used for developing the CPH using alcalase enzyme (3%). Freeze-dried CPH were incorporated in the cheese samples (CPH-T1, CPH-T2 and CPH-T0) at the maximum level of 1.5% and were analysed for quality during 3 months of storage (4 ± 1 °C) compared to the control samples without CPH. The pre-treatments significantly improved the antimicrobial and antioxidant potential of the CPH. The CPH exhibited a significant positive effect on antioxidant potential, lipid stability, protein oxidation, microbial growth, and sensory quality of the cheddar cheese during storage. Digestion simulation showed a significant positive impact on the antioxidant activity of the cheddar cheese. Our results indicate the potential of CPH to enhance the quality of fat-rich foods during storage.

Obtaining and Study of Peptide Compositions Based on Hydrolysates of Collagen-Containing Fish Raw Materials

Experimental studies of fish cutting waste—scales and skin were carried out, their general biochemical composition was studied, a high content of collagen was established, and elastin was noted, which accounted for 76–86% of the protein mass. Processes for the hydrolysis of secondary fish raw materials have been developed: fish scales and skin. Technological schemes have been developed and the influence of the conditions of thermal, enzymatic, enzymatic-thermal, electrochemical hydrolysis on the amino acid composition and molecular weight distribution (MWD) and antioxidant activity of peptides and proteins in the obtained hydrolysates has been studied. It has been established that the enzymatic and enzymatic-thermal method of hydrolysis of fish scales using the enzyme Alcalase 2.5 L and the electrochemical hydrolysis of the skin of cod, trout and herring made it possible to obtain protein hydrolysates with a protein content of 80–90%. At the same time, 91–98% of enzymatic hydrolysates from scales and 62%, 74% and 82.5% of electrochemically obtained hydrolysates from the skin of trout, herring, cod, respectively, account for the share of low-molecular peptides with a molecular weight of less than 10 kDa. The prospects of their use in functional foods and oil-containing products are noted.

Preparation and Characterization of a Multicomponent Arthrospira platensis Biomass Hydrolysate with Superior Anti-Hypertensive, Anti-Hyperlipidemic and Antioxidant Activities via Selective Proteolysis.

Arthrospira platensis biomass is a sustainable source of bioactive products for the food, cosmetic, and medicine industries. As well as primary metabolites, different secondary metabolites can be obtained via distinct enzymatic degradation of biomass. In this work, different hydrophilic extracts were obtained after treating the biomass with: (i) a serine endo-peptidase (Alcalase®), (ii) a mixture of amino-, dipeptidyl-, and endo-peptidases (Flavourzyme®), (iii) a mixture of endo-1,3(4)-β-glucanase and an endo-1,4-xylanase, and β-glucanase (Ultraflo®), and (iv) an exo-1,3-glucanase (Vinoflow®) (all the enzymes from Novozymes A/S (bagsvaerd, Denmark)); with subsequent extraction of the biocomponents with an isopropanol/hexane mixture. The composition of each aqueous phase extract (in terms of amino acids, peptides, oligo-elements, carbohydrates, and phenols) and their in vitro functional properties were compared. The conditions described in this work using the enzyme Alcalase® permits the extraction of eight distinctive peptides. This extract is 7.3 times more anti-hypertensive, 106 times more anti-hypertriglyceridemic, 26 times more hypocholesterolemic, has 4.4 times more antioxidant activities, and has 2.3 times more phenols, than the extract obtained without any prior enzyme biomass digestion. Alcalase® extract is an advantageous product with potential application in functional food, pharmaceutics, and cosmetics.

Antioxidant potential of gamma ray irradiated winged bean (Psophocarpus tetragonolobus) seed protein hydrolysate

Background: Winged bean or Psophocarpus tetragonolobus (WB) seeds have high protein content and could be applied as a source of antioxidant proteins and peptides. The utilization of gamma rays in plant protein extraction provides consumers with a safe and harmless technology.Objective: To determine the efficiency of gamma rays in the protein extraction process of WB seeds and investigate the antioxidant activity, cytotoxicity, cytoprotective effect of the gamma ray irradiated WB (WB-G) seed protein hydrolysate.Methods: WB seeds were irradiated with gamma ray (WB-G) and total protein were extracted by acid-base extraction method. The total protein was further hydrolyzed with alcalase enzyme to obtain the protein hydrolysate. The antioxidant activity was evaluated by DPPH and ABTS assay. The cytotoxicity and cytoprotective effect were determined by MTT assay. The cellular reactive oxygen species was further analyzed by ACFDA assay.Results: The protein extracted from WB-G seeds showed higher protein yield and antioxidant activity than that of non-irradiated seeds. After hydrolysis with alcalase enzyme at 55°C for 6 hours, WB-G protein hydrolysate with degree of hydrolysis of 90.00±0.91% exhibited strong antioxidant activity with IC50 value of 10.30±0.02 µg/ml and 3.10±0.06 µg/ml as measured by DPPH and ABTS assay, respectively. Moreover, it showed no toxicity toward L929 mouse fibroblast cells and IPEC-J2 cell lines and can reduce the damage caused by H2O2. As determined in cellular reactive oxygen species by H2-DCFDA assay, the WB-G protein hydrolysate can reduce the level of ROS and subsequently the damage of cell by H2O2. Conclusions: This study demonstrated that gamma ray irradiation on the WB seeds can increase the protein yield and its protein hydrolysate exhibited promising antioxidant activity and cytoprotective effect on cells against H2O2 damage. It might be developed as an alternative food or feed supplement.Keywords:Psophocarpus tetragonolobus; Gamma rays; Protein hydrolysate; Antioxidant activity; Cytoprotective effect