Enzymatic Hydrolysis Research Articles

Bromelain Immobilized onto Clay-Carboxymethylcellulose Composites for Improving Nutritive Value of Soybean Meal.

Improvement of nutritional value and reduction of antinutritional factors (ANFs) of soybean meal (SBM) for animal feed applications could be achieved by using bromelain immobilized onto bentonite (Bt)-carboxymethylcellulose (CMC) composites. The composite with mass ratio between CMC to calcium ion (Ca2+) at 1:20 provided the highest enzyme activity, immobilization yield higher than 95%, with superior thermal and storage stabilities. Performance of the immobilized bromelain for soybean protein hydrolysis was further studied. The results showed that at 60 °C, the immobilized bromelain exhibited the highest efficiency in enzymatic hydrolysis to release free alpha amino nitrogen (FAN) as a product with high selectivity and to effectively reduce SBM allergenic proteins within 30 min. In conclusion, immobilization of bromelain onto Bt-CMC composites leads to stability enhancement of the enzyme, enabling effective improvement in SBM quality in a short treatment time and showing great potential for application in animal feed industries.

Enhanced reducing sugar production by blending hydrolytic enzymes from Aspergillus fumigatus to improve sugarcane bagasse hydrolysis.

Biomass pretreatment for the production of second-generation (2G) ethanol and biochemical products is a challenging process. The present study investigated the synergistic efficiency of purified carboxymethyl cellulase (CMCase), β-glucosidase, and xylanase from Aspergillus fumigatus JCM 10253 in the hydrolysis of alkaline-pretreated sugarcane bagasse (SCB). The saccharification of pretreated SCB was optimised using a combination of CMCase and β-glucosidase (C + β; 1:1) and addition of xylanase (C + β + xyl; 1:1:1). Independent and dependent variables influencing enzymatic hydrolysis were investigated using response surface methodology (RSM). Hydrolysis using purified CMCase and β-glucosidase achieved yields of 18.72mg/mL glucose and 6.98mg/mL xylose. Incorporation of xylanase in saccharification increased the titres of glucose (22.83mg/mL) and xylose (9.54mg/mL). Furthermore, characterisation of SCB biomass by scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy respectively confirmed efficient structural disintegration and revealed the degree of crystallinity and spectral characteristics. Therefore, depolymerisation of lignin to produce high-value chemicals is essential for sustainable and competitive biorefinery development.

Bioactive Peptides Derived from Whey Proteins for Health and Functional Beverages

Milk serves as a crucial source of natural bioactive compounds essential for human nutrition and health. The increased production of high-protein dairy products is a source of whey—a valuable secondary product that, along with other biologically valuable substances, contains significant amounts of whey proteins and is often irrationally used or not utilized at all. Acid whey, containing almost all whey proteins and approximately one-quarter of casein, presents a valuable raw material for generating peptides with potential health benefits. These peptides exhibit properties such as antioxidant, antimicrobial, anti-inflammatory, anticarcinogenic, antihypertensive, antithrombotic, opioid, mineral-binding, and growth-stimulating activities, contributing to improved human immunity and the treatment of chronic diseases. Bioactive peptides can be produced by enzymatic hydrolysis using a variety of proteolytic enzymes, plant extracts, and microbial fermentation. With the participation of plant enzymes, peptides that inhibit angiotensin-converting enzyme are most often obtained. The use of enzymatic hydrolysis and microbial fermentation by lactic acid bacteria (LAB) produces more diverse peptides from different whey proteins with α-lactalbumin and β-lactoglobulin as the main targets. The resulting peptides of varying lengths often have antimicrobial, antioxidant, antihypertensive, and antidiabetic characteristics. Peptides produced by LAB are promising for use in medicine and the food industry as antioxidants and biopreservatives. Other beneficial properties of LAB-produced, whey-derived peptides have not yet been fully explored and remain to be studied. The development of whey drinks rich in bioactive peptides and based on the LAB proteolytic activity is underway. The strain specificity of LAB proteases opens up broad prospects for combining microorganisms to obtain products with the widest range of beneficial properties.

Inhibition of 2-AG hydrolysis alleviates posttraumatic headache attributed to mild traumatic brain injury

BackgroundPosttraumatic headache (PTH) is a common and debilitating symptom following repetitive mild traumatic brain injury (rmTBI), and it mainly resembles a migraine-like phenotype. While modulation of the endocannabinoid system (ECS) is effective in treating TBI and various types of pain including migraine, the role of augmentation of endocannabinoids in treating PTH has not been investigated.MethodsRepetitive mild TBI was induced in male C57BL/6J mice using the non-invasive close-head impact model of engineered rotational acceleration (CHIMERA). Periorbital allodynia was assessed using von Frey filaments and determined by the “Up-Down” method. Immunofluorescence staining was employed to investigate glial cell activation and calcitonin gene-related peptide (CGRP) expression in the trigeminal ganglion (TG) and trigeminal nucleus caudalis (TNC) of the rmTBI mice. Levels of 2-arachidonoyl glycerol (2-AG), anandamide (AEA), and arachidonic acid (AA) in the TG, medulla (including TNC), and periaqueductal gray (PAG) were measured by mass spectrometry. The therapeutic effect of endocannabinoid modulation on PTH was also assessed.ResultsThe rmTBI mice exhibited significantly increased cephalic pain hypersensitivity compared to the sham controls. MJN110, a potent and selective inhibitor of the 2-AG hydrolytic enzyme monoacylglycerol lipase (MAGL), dose-dependently attenuated periorbital allodynia in the rmTBI animals. Administration of CGRP at 0.01 mg/kg reinstated periorbital allodynia in the rmTBI animals on days 33 and 45 post-injury but had no effect in the sham and MJN110 treatment groups. Activation of glial cells along with increased production of CGRP in the TG and TNC at 7 and 14 days post-rmTBI were attenuated by MJN110 treatment. The anti-inflammatory and anti-nociceptive effects of MJN110 were partially mediated by cannabinoid receptor activation, and the pain-suppressive effect of MJN110 was completely blocked by co-administration of DO34, an inhibitor of 2-AG synthase. The levels of 2-AG in TG, TNC and PAG were decreased in TBI animals, significantly elevated and further reduced by the selective inhibitors of 2-AG hydrolytic and synthetic enzymes, respectively.ConclusionEnhancing endogenous levels of 2-AG appears to be an effective strategy for the treatment of PTH by attenuating pain initiation and transmission in the trigeminal pathway and facilitating descending pain inhibitory modulation.

Implementation of Spore Display in Paenibacillus polymyxa with Different Hydrolytic Enzymes

Biotechnological processes are essential for producing climate-friendly high-value chemicals or pharmaceutical compounds, which can include steps catalyzed by enzymes. Therefore, establishing new, robust, and cheap enzyme production processes is desirable. One possible way to enhance processes is through the use of the spore display method. Spore display can present heterologous proteins on the surface of bacterial spores, offering numerous advantages in a range of biotechnological applications. This study demonstrates the implementation of the spore display method in Paenibacillus polymyxa, achieved by modifying the spore surface, incorporating an anchoring protein, and attaching green fluorescent protein to it, allowing the visualization of fluorescent spores. Following the initial experiment, a native lipase (Lip3), a heterologous lipase (LipA) from Bacillus subtilis, a native esterase (PnbA) from P. polymyxa, and a lipoyl synthase were expressed during sporulation and displayed on the spore surface. The activity profiles were determined in the temperature range from 4 °C to 70 °C. The PnbA reached its optimum at 4 °C, whereas the LipA from B. subtilis showed 4.4-fold higher activity at 42 °C compared to the control. Furthermore, we explored a possible new technique for the purification of enzymes with the TEV cleavage site between the anchor and the protein of interest. Finally, we showed a not-yet-described side activity of the lipoyl synthase over a wide temperature range.

Isolation, Characterization, and Functional Properties of Antioxidant Peptides from Mulberry Leaf Enzymatic Hydrolysates

Recent evidence suggests that mulberry leaves have good antioxidant activity. However, what the antioxidant ingredient is and how the ingredient works are still not well understood. In this study, we enzymatically hydrolyze mulberry leaf proteins (MLPs) using neutral protease and find that the mulberry leaf protein hydrolysates (MLPHs) have stronger antioxidant activity compared to MLPs. We separate the core antioxidant components in MLPHs by ion-exchange columns and molecular sieves and identify 798 antioxidant peptides by LC-MS/MS. Through bioinformatics analysis and biochemical assays, we screen two previously unreported peptides, P6 and P7, with excellent antioxidant activities. P6 and P7 not only significantly reduce ROS in cells but also improve the activities of the antioxidant enzymes SOD and CAT. In addition, both peptides are found to exert protective effects against H2O2-induced chromatin damage and cell apoptosis. Collectively, these results provide support for the application of mulberry leaf peptides as antioxidants in the medical, food and livestock industries.

Mechanisms of Cell Wall Degrading Enzymes from Bacillus methylotrophicus and Bacillus subtilis in Suppressing Foliar Blight Pathogens

Bacillus strains are potent Biocontrol agents (BCAs) and have been identified as an effective way to control the growth of phytopathogens of wheat. Through a direct inhibition mechanism which involves the production of cell wall degrading enzymes (proteases, glucanase, and chitinases) and siderophores they suppress the foliar blight disease. Both the strains of Bacillus P10 and UP11 shows growth inhibition Alternaria triticina (77.56%, 67.83%) and Bipolaris sorokiniana (73.97%, 62.16%) through a dual culture assay. These strains were subsequently examined for their ability to produce cell wall-degrading enzymes i.e., chitinase, protease, and β-1,3-glucanases and antifungal metabolites i.e., siderophores. It was found that when the antagonist’s bacteria were Co-cultured with fungal pathogens then maximum production of hydrolytic enzymes and siderophore was achieved at 96 hrs, but when both strains P10 and UP11 were alone maximum production was found at 48 hrs i.e., the exponential phase. The current study determined that Bacillus methylotrophicus (P10) and Bacillus subtilis (UP11) are highly effective strain for controlling Foliar Blight disease. Direct or Antibiosis is the main mechanism involved in this study. Further research on the interaction mechanisms between Bacillus-derived compounds and host plants is necessary.

Crude glycerol-water organosolv delignification of sugarcane bagasse to produce fermentable sugars

This study evaluated the organosolv pretreatment with crude glycerol as a cosolvent for the delignification of raw sugarcane bagasse and the release of fermentable sugars from enriched-cellulose fraction by enzymatic hydrolysis. The pretreated solid fractions were characterized by pulp yield, weight loss, cellulose and hemicellulose content, lignin content, and delignification extent. Condition 9 (210 ºC, 60 min, 8 mL g-1) led to the highest delignification (~79%) with high total reducing sugars (TRS) concentration (~300 g L-1) and lower carbohydrate preservation (< 41%). While condition 1 (180 ºC, 150 min, 8 mL g-1) led to the highest cellulose preservation (~70%) and TRS (> 412 g L-1), even with a lower delignification extent (> 69%). This work contributed to an effective and optimized strategy to reuse crude glycerol in organosolv pretreatment as a facilitator of lignocellulose delignification, in addition to validating new protocols for the biorefinery production chain, providing sustainable biomaterials with added value.

Digested casein phosphopeptides impact intestinal calcium transport in vitro.

Calcium is the most abundant mineral in the human body and is involved in critical physiological and cellular processes. It is essential for the development, maintenance, and integrity of bone tissue throughout life. Identifying new natural food-grade chelating agents to improve calcium uptake is of increasing interest. Casein phosphopeptides (CPPs), highly phosphorylated peptides obtained after enzymatic hydrolysis of caseins, represent promising calcium-chelating candidates. The aim of this study was to investigate, using cell culture models, the ability of a digested milk matrix enriched in CPPs to regulate calcium transport through the intestinal barrier and elucidate the involved mechanisms. To this end, a CPP-preparation underwent in vitro static digestion and was subsequently incubated with an intestinal barrier model to monitor calcium uptake and transport. Our results demonstrated that the digested CPP preparation enhanced the trans-epithelial calcium transport via paracellular pathways and that CPPs, identified by peptidomics, crossed the intestinal barrier in the same time.

Upcycling soybean meal through enzymatic conversion of insoluble fiber into soluble dietary fiber enhanced by ball milling.

Insoluble dietary fiber (IDF) in soybean meal, due to the insolubility, is one of the major impediments to upcycle the soybean meal for its value-added use. This study converted IDF to soluble dietary fiber (SDF) using ball milling and enzymatic hydrolysis of the IDF. The impact of ball milling and enzymatic hydrolysis on the physicochemical and functional properties of SDF was evaluated. Cellulase, hemicellulase, xylanase, galacturonase, and arabinofuranosidase were employed for hydrolyzing IDF. The results showed that ball milling significantly reduced the particle size of IDF, facilitating enhanced enzymatic hydrolysis and resulting in SDF with lower molecular weight and varied monosaccharide composition. The synergistic effect of ball milling and enzymatic processes with combination of cellulase-xylanase-galacturonase was evident by the improved conversion rates (69.8%) and altered weight-averaged molecular weight (<5900 Da) of the resulting SDF. Rheological and microstructural analyses of the SDF gel indicated that specific enzyme combinations led to SDF gels with distinct viscoelastic properties, pore sizes, and functional capabilities, suitable for varied applications in the food and pharmaceutical sectors. This comprehensive evaluation demonstrates the potential of optimized physical bioprocessing techniques in developing functional ingredients with tailored properties for industrial use.

Production of cellulose nano/microfibers through simultaneous milling and enzymatic hydrolysis with an optimized cocktail of cellulase/xylanase/LPMO

Cellulose nanofibers (CNF) and microfibrillated cellulose (MFC) are biodegradable micro and nanomaterials derived from plant cellulose. The properties of CNF, influenced by fibril length, impact the strength of cellulose-based nanomaterials. This study uses enzymatic milling to explore pretreatments, enzymatic optimization, and CNF/MFC production. The study found that hammer milling and lamination are effective pretreatments for preserving fibers. The optimal enzymatic mixture contained 75 % cellulase and 25 % xylanase, yielding 68.7 % and 2.86 %/h productivity, respectively. The addition of the LPMO enzyme increased the yield by 10–13 %, depending on its concentration. Enzymatic milling was introduced as an innovative approach to CNF production, reducing hydrolysis time and creating milder processing conditions. The combined process of ball milling and enzymatic hydrolysis on pre-ground pulp in the hammer mill achieved a maximum CNF yield of 45.83 % and productivity of 13.10 %/h. X-ray diffraction showed that the lamination-pre-treated pulp had a high crystallinity index of 84.34 %. Scanning electron microscopy revealed fibers with diameters ranging from 52 nm to 296 nm. These findings support the search for sustainable and eco-friendly alternatives to non-renewable resources, enabling sustainable CNF production at room temperature with reduced processing time.

Bioconversion of Apple Pomace to Meyerozyma guilliermondii and Scheffersomyces stipitis Biomass

Poland is one of the leading apple-producing countries, both in Europe and around the world. One of the main byproducts of apple processing is pomace, which can account for 20–35% of the harvested apples. Pomace is a potential source of many valuable bioactive components and can also serve as a food ingredient, either directly or indirectly (after bioconversion with fodder yeast). This study aimed to evaluate the possibility of converting polysaccharides contained in apple pomace to yeast biomass. Meyerozyma guilliermondii and Scheffersomyces stipitis yeasts were grown in a medium prepared by pretreatment of the raw material with water or 2% sulphuric acid at 120 °C. Subsequently, enzymatic hydrolysis was performed using a Cellic CTec2 preparation at 30 °C or 50 °C. The resulting hydrolysates were enriched with ammonium salts, and shaken yeast cultures were incubated at 30 °C for 72 h. Based on the results, it can be concluded that acid pretreatment of apple pomace is more effective than water pretreatment under the same time and temperature conditions. The Meyerozyma guilliermondii strain grows in apple pomace hydrolysates more efficiently (16.29 g/L) than Scheffersomyces stipitis cells do (14.63 g/L).

Fluorescence-Based Enzyme Activity Assay: Ascertaining the Activity and Inhibition of Endocannabinoid Hydrolytic Enzymes

The endocannabinoid system, known for its regulatory role in various physiological processes, relies on the activities of several hydrolytic enzymes, such as fatty acid amide hydrolase (FAAH), N-acylethanolamine-hydrolyzing acid amidase (NAAA), monoacylglycerol lipase (MAGL), and α/β-hydrolase domains 6 (ABHD6) and 12 (ABHD12), to maintain homeostasis. Accurate measurement of these enzymes’ activities is crucial for understanding their function and for the development of potential therapeutic agents. Fluorometric assays, which offer high sensitivity, specificity, and real-time monitoring capabilities, have become essential tools in enzymatic studies. This review provides a comprehensive overview of the principles behind these assays, the various substrates and fluorophores used, and advances in assay techniques used not only for the determination of the kinetic mechanisms of enzyme reactions but also for setting up kinetic assays for the high-throughput screening of each critical enzyme involved in endocannabinoid degradation. Through this comprehensive review, we aim to highlight the strengths and limitations of current fluorometric assays and suggest future directions for improving the measurement of enzyme activity in the endocannabinoid system.

Study on efficient extraction of saponins from Polygonatum sibiricum by enzyme assisted cold isostatic pressing technology

Saponin is one of the main active ingredients of Polygonatum sibiricum. In this study, the method of enzyme-assisted cold isostatic pressure extraction (EACIP) of Polygonatum sibiricum saponins was established. The effects of different cold isostatic extraction pressures, pressure-maintaining time times, enzymatic temperatures and enzymatic pH on the yield of saponin were investigated, and the factors were simplified by single factor experiments and Box-benhken design (BBD). Under the best extraction conditions, the EACIP method achieved the best possible results and extraction rate was 11.45 % ± 0.017. In addition, SEM-Mapping, XRD analysis and FTIR analysis proved that EACIP treatment increased the extent of cell deformation and improved the mass transfer system between solvent and active substance. Furthermore, by comparing different extraction methods, EACIP was found to have a higher extraction rate due to the synergistic effect of cold isostatic pressure extraction (CIP) and enzymatic hydrolysis extraction (EH). In vitro antioxidant and anti-inflammatory assays demonstrated that EACIP extracts have good antioxidant and anti-inflammatory capacity. Therefore, EACIP is a method to extract natural products from plants with increased extraction rate.

Apple Blossom Agricultural Residues as a Sustainable Source of Bioactive Peptides through Microbial Fermentation Bioprocessing

This study explored the impact of starter-assisted fermentation on apple blossoms to enhance their potential as a source of antioxidant and antifungal molecules. Fructobacillus fructosus PL22 and Wickerhamomyces anomalus GY1 were chosen as starters owing to their origin and promising ability to modify plant secondary metabolites. An initial assessment through microbiological and physicochemical analyses showed superior outcomes for starter-assisted fermentation compared to the spontaneous process. Enzymatic hydrolysis of proteins, primarily controlled by starters, orchestrated the generation of new low-molecular-weight peptides. W. anomalus GY1 also induced modifications in the phenolic profile, generating a diverse array of bioactive metabolites. These metabolic changes, particularly the release of potentially bioactive peptides, were associated with significant antioxidant activity and marked antifungal efficacy against three common mold species. Our results shed light on the potential of microbial starters to valorize agricultural wastes and convert them into a valuable resource for industry.

Dissolved Organic Phosphorus Bond-Class Utilization by Synechococcus.

Dissolved organic phosphorus (DOP) contains compounds with phosphoester, phosphoanhydride, and phosphorus-carbon bonds. While DOP holds significant nutritional value for marine microorganisms, the bioavailability of each bond-class to the widespread cyanobacterium Synechococcus remains largely unknown. This study evaluates bond-class specific DOP utilization by Synechococcus strains from open and coastal oceans. Both strains exhibited comparable growth rates when provided phosphate, a phosphoanhydride (3-polyphosphate, 45-polyphosphate), or a DOP compound with both phosphoanhydride and phosphoester bonds (adenosine 5'-triphosphate). Growth rates on phosphoesters (glucose-6-phosphate, adenosine 5'-monophosphate, bis(4-methylumbelliferyl) phosphate) were variable, and neither strain grew on selected phosphorus-carbon compounds. Both strains hydrolyzed 3-polyphosphate, then adenosine 5'-triphosphate, and lastly adenosine 5'-monophosphate, exhibiting preferential enzymatic hydrolysis of phosphoanhydride bonds. The strains' exoproteomes contained phosphorus hydrolases, which combined with enhanced cell-free hydrolysis of 3-polyphosphate and adenosine 5'-triphosphate under phosphate deficiency, suggests active mineralization of phosphoanhydride bonds by these exoproteins. Synechococcus alkaline phosphatases presented broad substrate specificities, including activity towards the phosphoanhydride 3-polyphosphate, with varying affinities between strains. Collectively, these findings underscore the potentially significant role of compounds with phosphoanhydride bonds in Synechococcus phosphorus nutrition and highlight varied growth and enzymatic responses to molecular diversity within DOP bond-classes, thereby expanding our understanding of microbially-mediated DOP cycling in marine ecosystems.

Co-production of high-concentration fermentable sugar and lignin-based bio-adhesive from corncob residue via an enhanced enzymatic hydrolysis

Xylose plants (produce xylose from corncob through dilute acid treatment) generate a large amount of corncob residue (CCR), most of which are burned and lacked of valorization. Herein, to address this issue, CCR was directly used as starting material for high-solid loading enzymatic hydrolysis via a simple strategy by combining PFI homogenization (for sufficient mixing) with batch-feeding. A maximum glucose concentration of 187.1 g/L was achieved after the saccharification with a solid loading of 25 wt% and enzyme dosage of 10 FPU/g-CCR. Furthermore, the residue of enzymatic hydrolysis (REH) was directly used as a bio-adhesive for plywood production with both high dry (1.7 MPa) and wet (1.1 MPa) surface bonding strength (higher than the standard (0.7 MPa)), and the excellent adhesion was due to the interfacial crosslinking between the REH adhesive (containing lignin, free glucose, and nanosized fibers) and cell wall of woods. Compared with traditional reported adhesives, the REH bio-adhesive has advantages of formaldehyde-free, good moisture resistance, green process, relatively low cost and easy realization. This study presents a simple and effective strategy for better utilization of CCR, which also provides beneficial reference for the valorization of other kinds of lignocellulosic biomass.

Treatment with walnut peptide ameliorates memory impairment in zebrafish and rats: promoting the expression of neurotrophic factors and suppressing oxidative stress.

Walnut peptide, a low molecular weight peptide separated from walnuts by enzymatic hydrolysis, is considered as a potential nutraceutical with a variety of biological activities. In this study, we characterized the walnut peptide prepared by alkaline protease hydrolysis and evaluated its neuroprotective effect in zebrafish and rat models of memory disorders. Series of concentrations of the walnut peptide were orally administered to zebrafish and rats to examine its impact on the behavior and biochemical indicators. The results showed that the oral administration of walnut peptide significantly ameliorated the behavioral performance in zebrafish exposed to bisphenol AF (1 μg mL-1) and rats exposed to alcohol (30% ethanol, 10 mL kg-1). Furthermore, the walnut peptide upregulated the expression of neurotrophic-related molecules in zebrafish, such as the brain-derived neurotrophic factor (BDNF) and the glial cell-derived neurotrophic factor (GDNF). In the rat brain, the walnut peptide increased the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), while dramatically reduced malondialdehyde (MDA) level. Together, these findings elucidated that the walnut peptide might partially offset the declarative memory deficits via regulation of neurotrophic-related molecule expression and promotion of the antioxidant defense ability. Therefore, walnut peptide holds the potential for development into functional foods as a nutritional supplement for the management of certain neurodegenerative disorders.

Jackfruit Leaf Protein Hydrolysates Obtained by Enzymatic Hydrolysis of Leaf Protein Concentrate with Pepsin and Pancreatin: Molecular Weight, Cytotoxicity, Antiproliferative Activity, and Oxidative Stress.

Jackfruit leaf protein hydrolysates obtained from the enzymatic hydrolysis of leaf protein concentrate with gastrointestinal enzymes have shown good techno-functional properties and high antioxidant capacity. However, molecular weight, antiproliferative activity, cytotoxicity and the ability to reduce reactive oxygen species (ROS) are still unknown. Therefore, this study aimed to evaluate the effect of jackfruit leaf protein hydrolysates obtained by enzymatic hydrolysis with pepsin and pancreatin at different hydrolysis times (30-240min) on molecular weights, cytotoxicity, antiproliferation of cancer cells, and the reduction of reactive oxygen species in H2O2-induced HaCaT cells. The electrophoretic profile indicated that H-Pep contains peptides with molecular weights between 25 - 20kDa. Meanwhile, H-Pan is composed of molecular weight products between 25 - 20kDa and < 20kDa. H-Pan and H-Pep (125-500µg/mL) did not show significant cytotoxicity on HaCaT (human keratinocytes) and J774A.1 (murine macrophage cells). Antiproliferative activity was achieved in human cervical, ovarian, and liver cancer cells. H-Pan-240min (1000µg/mL) reduced the cell viability of cervical cancer cells by 23% while H-Pan-60min significantly reduced cell viability of ovarian and liver cancer cells by 14.5 (500µg/mL) and 17% (1000µg/mL), respectively (P < 0.05). The protective effect against oxidative stress on H2O2-stressed HaCaT cells was obtained with H-Pep-60min, which reduced 25% of ROS at 250µg/mL (P < 0.05). The findings demonstrate the safe use of green biomass as a source of plant protein hydrolysates.

Jackfruit Leaf Protein Hydrolysates Obtained by Enzymatic Hydrolysis of Leaf Protein Concentrate with Pepsin and Pancreatin: Molecular Weight, Cytotoxicity, Antiproliferative Activity, and Oxidative Stress.

Jackfruit leaf protein hydrolysates obtained from the enzymatic hydrolysis of leaf protein concentrate with gastrointestinal enzymes have shown good techno-functional properties and high antioxidant capacity. However, molecular weight, antiproliferative activity, cytotoxicity and the ability to reduce reactive oxygen species (ROS) are still unknown. Therefore, this study aimed to evaluate the effect of jackfruit leaf protein hydrolysates obtained by enzymatic hydrolysis with pepsin and pancreatin at different hydrolysis times (30-240min) on molecular weights, cytotoxicity, antiproliferation of cancer cells, and the reduction of reactive oxygen species in H2O2-induced HaCaT cells. The electrophoretic profile indicated that H-Pep contains peptides with molecular weights between 25 - 20kDa. Meanwhile, H-Pan is composed of molecular weight products between 25 - 20kDa and < 20kDa. H-Pan and H-Pep (125-500µg/mL) did not show significant cytotoxicity on HaCaT (human keratinocytes) and J774A.1 (murine macrophage cells). Antiproliferative activity was achieved in human cervical, ovarian, and liver cancer cells. H-Pan-240min (1000µg/mL) reduced the cell viability of cervical cancer cells by 23% while H-Pan-60min significantly reduced cell viability of ovarian and liver cancer cells by 14.5 (500µg/mL) and 17% (1000µg/mL), respectively (P < 0.05). The protective effect against oxidative stress on H2O2-stressed HaCaT cells was obtained with H-Pep-60min, which reduced 25% of ROS at 250µg/mL (P < 0.05). The findings demonstrate the safe use of green biomass as a source of plant protein hydrolysates.