Elucidation of larvicidal potential of metallic and environment friendly food-grade nanostructures against Aedes albopictus.

To combat health challenges associated with mosquito-borne diseases, the larvicidal activity of metallic nanoparticles, food-grade polymeric nano-capsules and insecticides was investigated against larvae of Aedes albopictus as an effective alternate control approach. The Ae. albopictus was identified using sequencing and phylogenetic analyses of COXI, CYTB and ITS2 genes. The characterization of synthesized nanostructures was performed through Zetasizer, UV-VIS spectroscopy, atomic force microscopy, scanning electron microscopy and energy dispersive X-ray spectroscopy. The mosquito larvae were exposed to varying concentration of nanostructures and insecticides, and their percentage mortality was evaluated at different time intervals of 24 h and 48 h exposure. The highest efficacy was observed in zinc oxide nanoparticles (ZnO-NPs) and polymeric nanocapsules FG-Cur E-III (LC50 = 0.24 mg/L, LC90 = 0.6 mg/L) and (LC50 = 3.8 mg/L, LC90 = 9.33 mg/L), respectively, after 24 h; while (LC50 = 0.18 mg/L, LC90 = 0.43 mg/L) and (LC50 = 1.95 mg/L, LC90 = 6.46 mg/L), respectively, after 48 h against fourth instar larvae of Ae. albopictus. Ag, CuO, NiTiO3 and CoTiO3 nanoparticles evaluated in this study also showed promising larvicidal activity. Although ZnO-NPs proved to be effective larvicides, their possible toxicity (producing ROS species) can limit their use. The curcumin nanostructures (FG-Cur E-III) stabilized by food-grade materials are thought to exert their larvicidal activity by binding to sterol carrier protein-2, and depriving the larvae from the essential dietary cholesterol, and bears effective larvicidal potential as safe alternative for chemical larvicides, due to their environment friendly, food-grade and easy biodegradability.

Antibiotics, Acid and Heat Tolerance of Honey adapted Escherichia coli, Salmonella Typhi and Klebsiella pneumoniae.

The medicinal importance of honey has been known for many decades due to its antimicrobial properties against life-threatening bacteria. However, previous studies revealed that microorganisms are able to develop adaptations after continuous exposure to antimicrobial compounds. The present study was conducted to explore the impact of subinhibitory concentrations of branded honey (Marhaba) and unbranded honey (extracted from Ziziphus mauritiana plant) locally available in Pakistan on Escherichia coli ATCC 10536, Salmonella Typhi and Klebsiella pneumoniae by investigating the development of self- or cross-resistance to antibiotics (gentamicin, kanamycin and imipenem). Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of autoclaved honeys were determined. The bacterial cells of E. coli ATCC 10536, S. Typhi and K. pneumoniae were subjected to honey adaptation by exposing to » × MIC (4 passages) and ½ × MIC (4 passages) of both honeys. Moreover, tolerance to low pH and high temperature was also studied in adapted and unadapted cells. The decreasing trend in growth pattern (OD600nm) of E. coli ATCC 10536, S. Typhi and K. pneumoniae was observed with increases in the concentration of honeys (6.25-50% v/v) respectively. Our results showed that continuous exposure of both honeys did not lead to the development of any self- or cross-resistance in tested bacteria. However, percent survival to low pH was found to be significantly higher in adapted cells as compared to unadapted cells. The results indicate that both branded honey (Marhaba) and unbranded honey (extracted from Ziziphus mauritiana plant) were effective in controlling the growth of tested pathogenic bacteria. However, the emergence of tolerance to adverse conditions (pH 2.5, temperature 60 °C) deserves further investigation before proposing honey as a better antibacterial agent in food fabrication/processing, where low pH and high temperatures are usually implemented.

Physicochemical, Sensorial and Microbiological Characterization of PoroCheese, an Artisanal Mexican Cheese Made from Raw Milk.

Poro cheese is a regional product originally from the area of Los Rios, Tabasco in Mexico. In the context of preserving the heritage of Poro cheese and protecting the specific characteristics that define its typicity through an origin designation, the present study was conducted to establish a general profile of Poro cheese by characterizing their physicochemical, textural, rheological, sensorial and microbiological characteristics. Differences in moisture, proteins, fats, NaCl, titrable acidity, pH, color texture and rheology amongst cheese factories were observed and ranges were established. Fifteen descriptors were generated to provide a descriptive analysis, eight of which were significantly different amongst the factories with no differences in the global acceptability of cheese. The favorite cheese had the highest scores for aroma attributes. Conventional and molecular methods were used to identify the main microorganisms, for which Lactobacillus plantarum, L. fermentum, L. farciminis and L. rhamnosus were the main microorganisms found in Porocheese. The obtained data constituted the parameters for characterizing Poro cheese, which will strongly help to support its origin appellation request process.

Formulation, characterization and antimicrobial properties of black cumin essential oil nanoemulsions stabilized by OSA starch.

Preparation of oil-in-water nanoemulsions has emerged as a subject of interest for the encapsulation of lipophilic functional ingredients to increase their stability and activity. In this study, black cumin essential oil nanoemulsions (BCO-NE) using different ratios of essential oil with canola and flax seed oils (ripening inhibitors) were formulated and stabilized with octenyl succinic anhydride (OSA) modified waxy maize starch. The nanoemulsions exhibited monomodal size distributions with mean droplet diameter below 200 nm and zeta potential above -30, indicating a strong electrostatic repulsion between the dispersed oil droplets. Further, during storage (4 weeks at 25 °C ± 2) emulsions showed shear thinning phenomena and stability towards coalescence. Antimicrobial properties of nanoemulsions were determined by minimum inhibitory concentration and time-kill method against two Gram-positive bacterial (GPB) strains (Bacillus cereus and Listeria monocytogenes). Negatively charged BCO-NE showed prolonged bactericidal activities as compared to pure BCO due to better stability, controlled release and self-assembly with GPB cell membrane followed by destruction of cellular constituents. Our results suggest the application of BCO-NE may be exploited in aqueous food systems for extending the shelf life and other functional properties.

Microencapsulation of Oils: A Comprehensive Review of Benefits, Techniques, and Applications.

Microencapsulation is a process of building a functional barrier between the core and wall material to avoid chemical and physical reactions and to maintain the biological, functional, and physicochemical properties of core materials. Microencapsulation of marine, vegetable, and essential oils has been conducted and commercialized by employing different methods including emulsification, spray-drying, coaxial electrospray system, freeze-drying, coacervation, in situ polymerization, melt-extrusion, supercritical fluid technology, and fluidized-bed-coating. Spray-drying and coacervation are the most commonly used techniques for the microencapsulation of oils. The choice of an appropriate microencapsulation technique and wall material depends upon the end use of the product and the processing conditions involved. Microencapsulation has the ability to enhance the oxidative stability, thermostability, shelf-life, and biological activity of oils. In addition, it can also be helpful in controlling the volatility and release properties of essential oils. Microencapsulated marine, vegetable, and essential oils have found broad applications in various fields. This review describes the recognized benefits and functional properties of various oils, microencapsulation techniques, and application of encapsulated oils in various food, pharmaceutical, and even textile products. Moreover, this review may provide information to researchers working in the field of food, pharmacy, agronomy, engineering, and nutrition who are interested in microencapsulation of oils.

Insights into chitosan multiple functional properties: the role of chitosan conformation in the behavior of liposomal membrane.

Interactions between chitosan and the liposomal membrane are relevant to the physiological functionality of chitosan, including dietary fiber, antimicrobial action, and fabrication of a delivery system for bioactives. To elucidate the multiple functions of chitosan, the dependence of liposomal membrane properties on the biopolymer conformation was investigated. The concentration dependence of chitosan conformation in aqueous solution was quantified by fluorescence and viscosity measurements. As the concentration increased, the extended chains of chitosan (0-1.0 mg mL(-1)) partially crimped (1.0-1.5 mg mL(-1)), and then self-aggregated forming irregular coils (>1.5 mg mL(-1)). Adsorption of chitosan linear chains on the liposomal membrane surface tended to maintain the morphology of liposomes, decrease the membrane interior micropolarity and rigidify the liposomal membrane. However, these effects were negligible or even opposite in the case of chitosan coils. Analysis on the membrane fluidity revealed that the microviscosity of liposomes decorated by 1.5 mg mL(-1) concentration of chitosan decreased by 17% after being heated at 80 °C for 10 min, in contrast to the decreased percentage of 55 at 4 mg mL(-1). Additionally, compared with the poor oxidative stability of liposomes decorated by chitosan coils, those decorated by chitosan linear chains exhibited slight lipid peroxidation with the TBARS inhibition of around 10% and 6% against oxygen and ferric ions, respectively. These findings suggest that the conformational effects of chitosan on the liposomal membrane are responsible for its multiple functional properties.

Biopolymer-Lipid Bilayer Interaction Modulates the Physical Properties of Liposomes: Mechanism and Structure.

This study was conducted to elucidate the conformational dependence of the modulating ability of chitosan, a positively charged biopolymer, on a new type of liposome composed of mixed lipids including egg yolk phosphatidylcholine (EYPC) and nonionic surfactant (Tween 80). Analysis of the dynamic and structure of bilayer membrane upon interaction with chitosan by fluorescence and electron paramagnetic resonance techniques demonstrated that, in addition to providing a physical barrier for the membrane surface, the adsorption of chitosan extended and crimped chains rigidified the lipid membrane. However, the decrease in relative microviscosity and order parameter suggested that the presence of chitosan coils disturbed the membrane organization. It was also noted that the increase of fluidity in the lipid bilayer center was not pronounced, indicating the shallow penetration of coils into the hydrophobic interior of bilayer. Microscopic observations revealed that chitosan adsorption not only affected the morphology of liposomes but also modulated the particle aggregation and fusion. Especially, a number of very heterogeneous particles were visualized, which tended to confirm the role of chitosan coils as a "polymeric surfactant". In addition to particle deformation, the membrane permeability was also tuned. These findings may provide a new perspective to understand the physiological functionality of biopolymer and design biopolymer-liposome composite structures as delivery systems for bioactive components.

Stable nanoparticles prepared by heating electrostatic complexes of whey protein isolate-dextran conjugate and chondroitin sulfate.

A simple and green method was developed for preparing the stable biopolymer nanoparticles with pH and salt resistance. The method involved the macromolecular crowding Maillard process and heat-induced gelation process. The conjugates of whey protein isolate (WPI) and dextran were produced by Maillard reaction. The nanoparticles were fabricated by heating electrostatic complexes of WPI-dextran conjugate and chondroitin sulfate (ChS) above the denaturation temperature and near the isoelectric point of WPI. Then, the nanoparticles were characterized by spectrophotometry, dynamic laser scattering, zeta potential, transmission electron microscopy, atomic force microscopy, and scanning electron microscopy. Results showed that the nanoparticles were stable in the pH range from 1.0 to 8.0 and in the presence of high salt concentration of 200 mM NaCl. WPI-dextran conjugate, WPI, and ChS were assembled into the nanoparticles with dextran conjugated to WPI/ChS shell and WPI/ChS core. The repulsive steric interactions, from both dextran covalently conjugated to WPI and ChS electrostatically interacted with WPI, were the major formation mechanism of the stable nanoparticles. As a nutrient model, lutein could be effectively encapsulated into the nanoparticles. Additionally, the nanoparticles exhibited a spherical shape and homogeneous size distribution regardless of lutein loading. The results suggested that the stable nanoparticles from proteins and strong polyelectrolyte polysaccharides would be used as a promising target delivery system for hydrophobic nutrients and drugs at physiological pH and salt conditions.

Modulating effect of lipid bilayer-carotenoid interactions on the property of liposome encapsulation.

Liposomes have become an attractive alternative to encapsulate carotenoids to improve their solubility, stability and bioavailability. The interaction mechanism of carotenoid with lipid bilayer is one of the major concerns in improving the delivery efficiency of liposomes. In this study, the microstructure and carotenoid encapsulation efficiency of liposomes composed of native phospholipid (egg yolk phosphatidylcholine, EYPC) and nonionic surfactant Tween 80 were investigated by atomic force microscopy, dynamic light scattering, and Raman spectroscopy, respectively. Subsequently, the effects of carotenoid incorporation on the physical properties of liposomal membrane were performed by Raman spectroscopy, fluorescence polarization, and electron paramagnetic resonance. Results showed that the incorporation of carotenoids affected the liposomes morphology, size and size distribution to various extents. Analysis on the Raman characteristic peaks of carotenoids revealed that lutein exhibited the strongest incorporating ability into liposomes, followed by ß-carotene, lycopene, and canthaxanthin. Furthermore, it was demonstrated that carotenoids modulated the dynamics, structure and hydrophobicity of liposomal membrane, highly depending on their molecular structures and incorporated concentration. These modulations were closely correlated with the stabilization of liposomes, including mediating particle aggregation and fusion. These findings should guide the rationale designing for liposomal encapsulation technology to efficiently deliver carotenoids in pharmaceutics, nutraceuticals and functional foods.

Fabrication of polymeric nanocapsules from curcumin-loaded nanoemulsion templates by self-assembly.

In this study, biodegradable polymeric nanocapsules were prepared by sequential deposition of food-grade polyelectrolytes through the self-assembling process onto the oil (medium chain triglycerides) droplets enriched with curcumin (lipophilic bioactive compound). Optimum conditions were used to prepare ultrasound-assisted nanoemulsions stabilized by octenyl-succinic-anhydride (OSA)-modified starch. Negatively charged droplets (-39.4 ± 1.84 mV) of these nanoemulsions, having a diameter of 142.7 ± 0.85 nm were used as templates for the fabrication of nanocapsules. Concentrations of layer-forming cationic (chitosan) and anionic (carboxymethylcellulose) biopolymers were optimized based on the mean droplet/particle diameter (MDD/MPD), polydispersity index (PDI) and net charge on the droplets/capsules. Prepared core-shell structures or nanocapsules, having MPD of 159.85 ± 0.92 nm, were characterized by laser diffraction (DLS), ζ-potential (ZP), atomic force microscopy (AFM), transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). Furthermore, physical stability of curcumin-loaded nanocapsules in suspension was determined and compared at different storage temperatures. This study may provide information regarding the formation of ultrasound-assisted polymeric nanocapsules from the nanoemulsion templates which could be helpful in the development of delivery systems for lipophilic food bioactives.

Modulation of the carotenoid bioaccessibility through liposomal encapsulation.

The low bioaccessibility of carotenoids is currently a challenge to their incorporation in pharmaceutics, nutraceuticals and functional foods. The aim of this study was to evaluate the modulating effects of liposome encapsulation on the bioaccessibility, and its relationship with carotenoid structure and incorporated concentration. The physical stability of liposomes, lipid digestibility, carotenoids release and bioaccessibility were investigated during incubation in a simulated gastrointestinal tract. Analysis on the liposome size and morphology showed that after digestion, the majority of particles maintained spherical shape with only an increase of size in liposomes loading ß-carotene or lutein. However, a large proportion of heterogeneous particles were visible in the micelle phase of liposomes loading lycopene or canthaxanthin. It was also found that the release of lutein and ß-carotene from liposomes was inhibited in a simulated gastric fluid, while was slow and sustained in a simulated intestinal fluid. By contrast, lycopene and canthaxanthin exhibited fast and considerable release in the gastrointestinal media. Both carotenoid bioaccessibility and micellization content decreased with the increase of incorporated concentration. Anyway, the bioaccessibility of carotenoids after encapsulated in liposomes was in the following order: lutein>ß-carotene>lycopene>canthaxanthin. Bivariate correlation analysis revealed that carotenoid bioaccessibility depended strongly on the incorporating ability of carotenoids into a lipid bilayer, loading content, and nature of the system.

The effect of soy protein structural modification on emulsion properties and oxidative stability of fish oil microcapsules.

Hydrolysates of soy protein isolate-maltodextrin (SPI-Md) conjugate were used as wall material to prepare fish oil microcapsules by freeze-drying method. Effects of the protein structural modifications on the physicochemical properties of the emulsion and the oxidative stability of the microcapsules were characterized. Compared with emulsions of SPI-Md conjugates or soy protein isolate/maltodextrin (SPI/Md) mixture, lower droplet size (212.5-329.3nm) and polydispersity index (PDI) (0.091-0.193) were obtained in the fish oil emulsions prepared by SPI-Md conjugate hydrolysates. The improved amphiphilic property of SPI-Md conjugate hydrolysates was supported by the results of surface and interfacial tension, and further confirmed by the improved emulsion stability during the storage period. Although the microencapsulation efficiency (MEE) of SPI-Md conjugate hydrolysates slightly decreased from 97.84% to 91.47% with the increasing degree of hydrolysis (DH), their oxidative stabilities (peroxide value and headspace propanal) were apparently improved compared with native SPI/Md mixture or SPI-Md conjugates system. Moreover, favorable thermal stability as well as a porous and uniform surface structure of the microcapsules coated by SPI-Md conjugate hydrolysates (DH 2.9%) was observed via the thermal analysis and scanning electron microscope (SEM) micrographs, respectively.

Liposome as a delivery system for carotenoids: comparative antioxidant activity of carotenoids as measured by ferric reducing antioxidant power, DPPH assay and lipid peroxidation.

This study was conducted to understand how carotenoids exerted antioxidant activity after encapsulation in a liposome delivery system, for food application. Three assays were selected to achieve a wide range of technical principles, including 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging, ferric reducing antioxidant powder (FRAP), and lipid peroxidation inhibition capacity (LPIC) during liposome preparation, auto-oxidation, or when induced by ferric iron/ascorbate. The antioxidant activity of carotenoids was measured either after they were mixed with preformed liposomes or after their incorporation into the liposomal system. Whatever the antioxidant model was, carotenoids displayed different antioxidant activities in suspension and in liposomes. The encapsulation could enhance the DPPH scavenging and FRAP activities of carotenoids. The strongest antioxidant activity was observed with lutein, followed by ß-carotene, lycopene, and canthaxanthin. Furthermore, lipid peroxidation assay revealed a mutually protective relationship: the incorporation of either lutein or ß-carotene not only exerts strong LPIC, but also protects them against pro-oxidation elements; however, the LPIC of lycopene and canthaxanthin on liposomes was weak or a pro-oxidation effect even appeared, concomitantly leading to the considerable depletion of these encapsulated carotenoids. The antioxidant activity of carotenoids after liposome encapsulation was not only related to their chemical reactivity, but also to their incorporation efficiencies into liposomal membrane and modulating effects on the membrane properties.

Liposomes as delivery systems for carotenoids: comparative studies of loading ability, storage stability and in vitro release.

This study compared the loading ability of various carotenoids into liposomal membrane, lipid peroxidation inhibition capacity, storage stability and in vitro release behavior in simulated gastrointestinal (GI) media. It was found that carotenoids exhibited various incorporating abilities into liposomes ranging from the strongest to the weakest: lutein > ß-carotene > lycopene > canthaxanthin. A similar trend was also observed in their antioxidant activities against lipid peroxidation during preparation. Storage measurements demonstrated that a liposomal membrane can strongly retain ß-carotene and lutein, whereas this effect was not pronounced for lycopene and canthaxanthin. In vitro release experiments showed that lutein and ß-carotene were hardly released in a simulated gastric fluid, while displaying a slow and sustained release in a simulated intestinal fluid. By contrast, lycopene and canthaxanthin underwent fast and considerable release in GI media. Dynamic light scattering indicated that carotenoid incorporation strongly affected the particle stability and dispersion during preparation and GI incubation. The differences in molecular release may be attributed to the different modulating effects of carotenoids. Our results may guide the potential application of liposomes as carriers for the controlled delivery of carotenoids in nutraceutical and functional foods.

Combined of ultrasound irradiation with high hydrostatic pressure (US/HHP) as a new method to improve immobilization of dextranase onto alginate gel.

In this research work, dextranase was immobilized onto calcium alginate beads by the combination of ultrasonic irradiation and high hydrostatic pressure (US/HHP) treatments. Effects of US/HHP treatments on loading efficiency and immobilization yield of dextranase enzyme onto calcium alginate beads were investigated. Furthermore, the activities of immobilized enzymes prepared with and without US/HHP treatments and that prepared with ultrasonic irradiation (US) and high hydrostatic pressure (HHP), as a function of pH, temperature, recyclability and enzyme kinetic parameters, were compared with that for free enzyme. The maximum loading efficiency and the immobilization yield were observed when the immobilized dextranase was prepared with US (40 W at 25 kHz for 15 min) combined with HHP (400 MPa for 15 min), under which the loading efficiency and the immobilization yield increased by 88.92% and 80.86%, respectively, compared to immobilized enzymes prepared without US/HHP treatment. On the other hand, immobilized enzyme prepared with US/HHP treatment showed Vmax, KM, catalytic and specificity constants values higher than that for the immobilized enzyme prepared with HHP treatment, indicated that, this new US/HHP method improved the catalytic kinetics activity of immobilized dextranase at all the reaction conditions studied. Compared to immobilized enzyme prepared either with US or HHP, the immobilized enzymes prepared with US/HHP method exhibited a higher: pH optimum, optimal reaction temperature, thermal stability and recyclability, and lower activation energy, which, illustrating the effectiveness of the US/HHP method. These results indicated that, the combination of US and HHP treatments could be an effective method for improving the immobilization of enzymes in polymers.

Process optimization of ultrasound-assisted curcumin nanoemulsions stabilized by OSA-modified starch.

This study reports on the process optimization of ultrasound-assisted, food-grade oil-water nanoemulsions stabilized by modified starches. In this work, effects of major emulsification process variables including applied power in terms of power density and sonication time, and formulation parameters, that is, surfactant type and concentration, bioactive concentration and dispersed-phase volume fraction were investigated on the mean droplet diameter, polydispersity index and charge on the emulsion droplets. Emulsifying properties of octenyl succinic anhydride modified starches, that is, Purity Gum 2000, Hi-Cap 100 and Purity Gum Ultra, and the size stability of corresponding emulsion droplets during the 1 month storage period were also investigated. Results revealed that the smallest and more stable nanoemulsion droplets were obtained when coarse emulsions treated at 40% of applied power (power density: 1.36 W/mL) for 7 min, stabilized by 1.5% (w/v) Purity Gum Ultra. Optimum volume fraction of oil (medium chain triglycerides) and the concentration of bioactive compound (curcumin) dispersed were 0.05 and 6 mg/mL oil, respectively. These results indicated that the ultrasound-assisted emulsification could be successfully used for the preparation of starch-stabilized nanoemulsions at lower temperatures (40-45 °C) and reduced energy consumption.

Effect of ultrasound and high hydrostatic pressure (US/HHP) on the degradation of dextran catalyzed by dextranase.

In our current research work, the effect of combination of ultrasonic irradiation and high hydrostatic pressure (US/HHP) on the enzymatic activity and enzymatic hydrolysis kinetic parameters of dextran catalytic by dextranase were investigated. Furthermore, the effects of US/HHP on the structure of dextranase were also discussed with the aid of fluorescence spectroscopy and circular dichroism (CD) spectroscopy. The maximum hydrolysis of dextran was observed under US (40 W at 25 kHz for 15 min) combined with HHP (400 MPa for 25 min), in which the hydrolysis of dextran increased by 163.79% compared with the routine thermal incubation at 50 °C. Results also showed that, Vmax and KM values, as well as, kcat of dextranase under US/HHP treatment were higher than that under US, HHP and thermal incubation at 50 °C, indicated that, the substrate is converted into the product at an increased rate when compared with the incubation at 50 °C. Compared to the enzymatic reaction under US, HHP, and routine thermal incubation, dextranase enzymatic reaction under US/HHP treatment showed decreases in Ea, ΔG and ΔH, however small increase in ΔS value was observed. In addition, fluorescence and CD spectra reflected that US/HHP treatment had increased the number of tryptophan on dextranase surface with increased α-helix by 19.80% and reduced random coil by 6.94% upon US/HHP-treated dextranase protein compared to the control, which were helpful for the improvement of its activity. These results indicated that, the combination of US and HHP treatments could be an effective method for improving the hydrolysis of dextran in many industrial applications including sugar manufacturing processes.

Influence of enzymatic hydrolysis and enzyme type on the nutritional and antioxidant properties of pumpkin meal hydrolysates.

Nutritional and antioxidant properties of pumpkin meal and their hydrolysates prepared by hydrolysis with alcalase, flavourzyme, protamex or neutrase were evaluated. The hydrolysis process significantly increased protein content from 67.07% to 92.22%. All the essential amino acids met the Food and Agriculture Organization of United Nations/World Health Organization (WHO/FAO) suggested requirements for children and adults. The amino acid score (AAS) of meal was increased from 65.59 to 73.00 except for flavourzyme (62.97) and protamex (62.50). The Biological Value (BV) was increased from 53.18 to 83.44 except for protamex (40.97). However hydrolysis decreased the Essential Amino Acid/Total Amino Acid ratio (EAA/TAA) from 32.98% to 29.43%. Protein Efficiency Ratio (PER) was comparable to that of good quality protein (1.5) except for flavourzyme hydrolysate which had PER1 = 0.92, PER2 = 1.03, PER3 = 0.38. The in vitro protein digestibility (IVPD) increased from 71.32% to 77.96%. Antioxidant activity increased in a dose-dependent manner. At 10 mg mL(-1), the hydrolysates had increased 1,1-diphenyl-2-picrylhydrazy (DPPH) radical scavenging activities from 21.89% to 85.27%, the reducing power increased from Abs(700nm) 0.21 to 0.48. Metal (Iron) chelating ability was improved from 30.50% to 80.03% at 1 mg mL(-1). Hydrolysates also showed better capabilities to suppress or delay lipid peroxidation in a linoleic acid model system. Different proteases lead to different Degrees of Hydrolysis (DH), molecular weight (MW) distribution, amino acid composition and sequence, which influenced the nutritional properties and antioxidant activities of the hydrolysates. Alcalase was the most promising protease in production of pumpkin protein hydrolysates with improved nutritional quality, while flavourzyme was best in production of hydrolysates with improved antioxidative activity among various assays. These results showed that hydrolysates from by-products of pumpkin oil-processing might serve as alternative sources of dietary proteins with good nutritional quality, and protection against oxidative damage.