Substrate For Enzymatic Hydrolysis Research Articles

Production Enhancement of Bacterial Cellulase Cocktail Using Potato Peels Waste Feedstock and Combination of Water Hyacinth Root and Pea Pod Extract as Natural Nutrient Media: Application in Bioconversion of Potato Peels.

Solid wastes are the major contributors in global environmental pollution and their management is the need of urgency towards development of sustainable world. In the present work, solid waste of potato peels has been used as feedstock for fermentation of bacterial cellulase production and substrate for enzymatic hydrolysis via this enzymes cocktail. Additionally, liquid extracts of pea pod and root of water hyacinth wastes have been used to complete nutritional requirements and moisture balance in SSF process during the course of enzyme production. At optimum feedstock concentration of 6.0g PPW and 10:40 extract-based moisture ratio of WHR and Ppw, Bacillus sp.produced 15 U/gds FP in 18h, whereas maximum 36 U/gds BGL and 42 U/gds EG have been recorded in 24h of SSF. Temperature 35°C and pH 5.5 were optimum for enzyme production while the produced enzyme was thermally stable upto 30h at 35°C with 100% pH stability upto 14h and 77% relative activity at 34h. The optimized bacterial enzymes have been used for bioconversion of PPW biomass and 26g/L glucose has been recorded at a hydrolytic temperature of 50°C and pH 5.0. The study may have feasible promising scope in cellulosic biorefineries and waste management.

Study of washing method of oil palm empty fruit bunch-treated with alkali explosion in lignocellulosic bioethanol production

The conventional process of lignocellulosic bioethanol production includes four steps: pretreatment, hydrolysis, fermentation, and purification. Pretreatment is the crucial way to enhance cellulose purity and remove lignin as well as other impurities compounds in biomass. Alkaline explosion is one of the most used pretreatment methods but pulp-produced is still in base condition. This pulp is not suitable as a substrate for enzymatic hydrolysis that needed a pH of 4.5-5.5. Therefore, pulp washing is required for separating pulp with black liquor and obtaining neutral pH pulp before going to hydrolysis. This study explored the washing method of oil palm empty fruit bunch (EFB)-treated with alkaline explosion in bioethanol production. The washing method using freshwater, acid solution, and a combination of freshwater and acid solution was investigated. As a result, the washing used freshwater needed the highest of water and wash cycles. The washing method using acid solution could obtain neutral pulp in two cycles wash step or more, thus, requiring higher acid consumption. The washing with combination method provided neutral pulp with fewer wash cycles, water, and acid. This method also provided the highest glucose and ethanol concentration. Therefore, the combination washing method is considered more efficient, economical, and environmentally for EFB pulp washing in bioethanol production.

Single-Stage Fractionation of Vine Shoots Using Microwave Heating

Vine shoots are agricultural residues that can be used as a raw material in agro-biorefineries, in which their main constituents can be individually converted into valuable bioproducts. The treatment of vine shoots in uncatalyzed media containing water and 1-butanol enabled the single-stage separation of the major vine shoots constituents in different phases: the aqueous phase from treatments contained hemicellulose-derived products (mainly in the form of oligosaccharides), the organic phase accumulated the dissolved lignin, and the cellulosic fraction was recovered in solid phase. The aqueous phase from treatments was refined using membranes and processed with enzymes to obtain a refined product (RP) containing 92.2 g of oligosaccharides/100 g of non-volatile compounds. The oligosaccharides were mainly composed of anhydroxylose units substituted by acetyl and uronic groups. Enzymatic hydrolysis of RP with endo-xylanases reduced the average degree of polymerization to 2–3, which are preferred for application as healthy food ingredients. The solid phase from treatments was used as a substrate for enzymatic hydrolysis, enabling the production of solutions containing 34.9 g glucose /L and 4.2 g xylose /L.

Consolidated bioprocessing for bioethanol production by metabolically engineered Bacillus subtilis strains

Bioethanol produced by fermentative microorganisms is regarded as an alternative to fossil fuel. Bioethanol to be used as a viable energy source must be produced cost-effectively by removing expense-intensive steps such as the enzymatic hydrolysis of substrate. Consolidated bioprocessing (CBP) is believed to be a practical solution combining saccharification and fermentation in a single step catalyzed by a microorganism. Bacillus subtills with innate ability to grow on a diversity of carbohydrates seems promising for affordable CBP bioethanol production using renewable plant biomass and wastes. In this study, the genes encoding alcohol dehydrogenase from Z. mobilis (adhZ) and S. cerevisiae (adhS) were each used with Z. mobilis pyruvate decarboxylase gene (pdcZ) to create ethanologenic operons in a lactate-deficient (Δldh) B. subtilis resulting in NZ and NZS strains, respectively. The S. cerevisiae adhS caused significantly more ethanol production by NZS and therefore was used to make two other operons including one with double copies of both pdcZ and adhS and the other with a single pdcZ but double adhS genes expressed in N(ZS)2 and NZS2 strains, respectively. In addition, two fusion genes were constructed with pdcZ and adhS in alternate orientations and used for ethanol production by the harboring strains namely NZ:S and NS:Z, respectively. While the increase of gene dosage was not associated with elevated carbon flow for ethanol production, the fusion gene adhS:pdcZ resulted in a more than two times increase of productivity by strain NS:Z as compared with NZS during 48 h fermentation. The CBP ethanol production by NZS and NS:Z using potatoes resulted in 16.3 g/L and 21.5 g/L ethanol during 96 h fermentation, respectively. For the first time in this study, B. subtilis was successfully used for CBP ethanol production with S. cerevisiae alcohol dehydrogenase. The results of the study provide insights on the potentials of B. subtilis for affordable bioethanol production from inexpensive plant biomass and wastes. However, the potentials need to be improved by metabolic and process engineering for higher yields of ethanol production and plant biomass utilization.

Effect of in vitro gastro‐pancreatic digestion on antioxidant activity of low‐molecular‐weight (<3.5 kDa) peptides from dry‐cured pork loins with probiotic strains of LAB

SummaryThe antioxidant properties of low‐molecular‐weight peptides (<3.5 kDa) obtained from the dry‐cured pork loins inoculated with probiotic and potentially probiotic lactic acid bacterial strains were tested after two‐stage enzymatic hydrolysis under in vitro conditions. The water soluble fraction (WSF) was found to be a better substrate for enzymatic hydrolysis by proteolytic enzymes, especially when they were extracted from the meat product after 12 months of ageing. Furthermore, protein hydrolysates extracted from the salt soluble fraction (SSF) exhibited better or similar ABTS free radical scavenging activity and Fe2+‐chelating activity than those extracted from WSF (despite their less quantity). Moreover, both SSF and WSF exhibited weak ferric‐reducing antioxidant activity after two‐step enzymatic hydrolysis (A700 averaged 0.138 for the WSF and 0.088 for the SSF). These results might be useful to the meat‐processing industry in developing innovative and functional meat products.

New insight into enzymatic hydrolysis of peptides with site-specific amino acid d-isomerization

The isomerization of l-amino acids in peptides and proteins into d-configuration under physiological conditions would affect the physiological dysfunction and caused protein conformational diseases. The presence of d-amino acids might change the higher-order structure of proteins and triggered abnormal aggregation. In order to better understand this phenomenon and promote degradation, we systematically studied the enzymatic hydrolysis of a series of peptides obtained by replacing l-amino acids in different positions of template peptide KYNETWRSED with d-amino acids under the action of Protease K. The results showed that, compared with normal peptide, isomerization of different amino acids had different effects on the anti-enzymatic hydrolysis of the peptides, especially d-tryptophan at position 6, which significantly inhibited enzymatic hydrolysis. The analysis of the peptide cleavage site revealed that the efficiency of enzymatic hydrolysis mainly depended on the isomerization of the amino acids at a specific site of the peptide cleavage. Further studies showed that the enzymatic hydrolysis of substrates could be facilitated by optimized reaction conditions such as temperature, pH, addition of metal ions, and change of buffer. In this way the accumulation of disease-associated d-amino acid containing polypeptides/proteins could be prevented.

Development of Pretreatment Strategies for the Fractionation of Hazelnut Shells in the Scope of Biorefinery

Hazelnut shells are an important waste from the hazelnut processing industry that could be valorized in a multi-product biorefinery. Individual or combined pretreatments may be integrated in processes enabling the integral fractionation of biomass. In this study, fractionation methods based on alkaline, alkaline-organosolv, organosolv, or acid-catalyzed organosolv treatments were applied to raw or autohydrolyzed hazelnut shells. A comparative analysis of results confirmed that the highest lignin removal was achieved with the acid-catalyzed organosolv delignification, which also allowed limited cellulose losses. When this treatment was applied to raw hazelnut shells, 65.3% of the lignin was removed, valuable hemicellulose-derived products were obtained, and the cellulose content of the processed solids increased up to 54%. Autohydrolysis of hazelnut shells resulted in the partial solubilization of hemicelluloses (mainly in the form of soluble oligosaccharides). Consecutive stages of autohydrolysis and acid-catalyzed organosolv delignification resulted in 47.9% lignin removal, yielding solids of increased cellulose content (55.4%) and very low content of residual hemicelluloses. The suitability of selected delignified and autohydrolyzed-delignified hazelnut shells as substrates for enzymatic hydrolysis was assessed in additional experiments. The most susceptible substrates (from acid-catalyzed organosolv treatments) reached 74.2% cellulose conversion into glucose, with a concentration of 28.52 g glucose/L.

Lipid Production by Yeast Trichosporon oleaginosus on the Enzymatic Hydrolysate of Alkaline Pretreated Corn Cobs for Biodiesel Production

Biodiesel is still mainly produced from different vegetable (e.g., rapeseed, palm, soybean, sunflower, and used cooking oils) oils and animal fats, and therefore it has a negative impact on food and feed prices. For biodiesel production as an alternative feedstock, lipids from oleaginous microorganisms could be used. One of the oleaginous microorganisms is yeast Trichosporon oleaginosus that has the capacity to grow and accumulate lipids on different lignocellulosic hydrolysates. In this study, corn cobs were pretreated by alkali at loading from 0.08 to 1.6 g/g dry weight of untreated (raw) corn cobs (g NaOH/gDW_UCC) at 121 °C for 30 min. In a further step, alkaline pretreated corn cobs were subjected to the enzymatic hydrolysis by using commercial multienzymes mixtures. The optimal alkali loading (0.16 g/gDW_UCC) was related to the highest glucose and xylose yields which were observed during enzymatic hydrolysis of substrate. In this study, T. oleaginosus was applied for lipid production on the enzymatic...

Lignocellulosic biomass to biofuels and biochemicals: A comprehensive review with a focus on ethanol organosolv pretreatment technology.

Lignocellulosic biomass is one of the potential feedstocks to produce second-generation cellulosic ethanol and biochemicals. To enhance the enzymatic digestibility of lignocellulosic biomass for efficient enzymatic saccharification, a variety of pretreatment methods have been studied. Among these, organosolv pretreatment using ethanol is a promising pretreatment method owing to its inherent advantages, such as low solvent cost, lack of toxicity, the ability to retain most cellulose fraction in substrates for enzymatic hydrolysis, coproduction of high-purity lignin and hemicellulosic sugars, easy solvent recovery, and reuse. In this review, the research progress regarding different types of ethanol organosolv pretreatment processes has been summarized in terms of methods, substrate properties, reaction mechanisms, delignification kinetic as well as the impact of pretreatment methods on the enzymatic digestibility. Attempts are also made to provide insights into the complete utilization of lignocellulosic biomass to achieve high potential revenues. Though some ethanol organosolv processes have been studied or are being developed towards commercialization, ethanol organosolv pretreatment is still facing some challenges. Finally, the direction for future work is given to develop a proper ethanol organosolv pretreatment for commercialization.

Altering the substrate specificity of a myofibril‐bound serine proteinase from Crucian carp by site‐directed mutagenesis

AbstractBACKGROUNDMyofibril‐bound serine proteinase (MBSP) comprises a class of trypsin‐type serine proteinases that can specifically cleave the carboxyl side of peptide bonds of arginine or lysine residues. To obtain higher specificity MBSPs, mutants were designed based on the substrate‐binding pocket and constructed through site‐directed mutagenesis.RESULTSThe wild‐type (WT) and mutants were expressed in Pichia pastoris, and its enzymatic properties indicated that the mutants D170S, D170T, D170K and D170T/G203A were not biologically active. However, S171A specifically cleaved arginine residues and G203A preferably hydrolyzed lysine residues. The secondary structures of mutants were approximately similar to that of the WT according to the results of circular dichroism spectroscopy. Additionally, molecular docking showed that substrates were able to combine with S171A within the critical areas through hydrophobic interaction, hydrogen bonds and van der Waals electrostatic force.CONCLUSIONThe structural stability of MBSP was consistent using amino acid substitution. Due to the interaction pattern of substrates and mutant enzyme changes, only S171A was selective in cutting Arg residues, which illustrates how the catalytic triad and substrate‐binding pocket of MBSP plays an important role during enzymatic hydrolysis of substrates. © 2018 Society of Chemical Industry

Enhanced hydrolysis of cellulose hydrogels by morphological modification.

Cellulose is one of the most abundant bio-renewable materials on earth, yet the potential of cellulosic bio-fuels is not fully exploited, primarily due to the high costs of conversion. Hydrogel particles of regenerated cellulose constitute a useful substrate for enzymatic hydrolysis, due to their porous and amorphous structure. This article describes the influence of several structural aspects of the cellulose hydrogel on its hydrolysis. The hydrogel density was shown to be directly proportional to the cellulose concentration in the initial solution, thus affecting its hydrolysis rate. Using high-resolution scanning electron microscopy, we show that the hydrogel particles in aqueous suspension exhibit a dense external surface layer and a more porous internal network. Elimination of the external surface layer accelerated the hydrolysis rate by up to sixfold and rendered the process nearly independent of cellulose concentration. These findings may be of practical relevance to saccharification processing costs, by reducing required solvent quantities and enzyme load.

In planta production and characterization of a hyperthermostable GH10 xylanase in transgenic sugarcane.

Sugarcane (Saccharum sp. hybrids) is one of the most efficient and sustainable feedstocks for commercial production of fuel ethanol. Recent efforts focus on the integration of first and second generation bioethanol conversion technologies for sugarcane to increase biofuel yields. This integrated process will utilize both the cell wall bound sugars of the abundant lignocellulosic sugarcane residues in addition to the sucrose from stem internodes. Enzymatic hydrolysis of lignocellulosic biomass into its component sugars requires significant amounts of cell wall degrading enzymes. In planta production of xylanases has the potential to reduce costs associated with enzymatic hydrolysis but has been reported to compromise plant growth and development. To address this problem, we expressed a hyperthermostable GH10 xylanase, xyl10B in transgenic sugarcane which displays optimal catalytic activity at 105 °C and only residual catalytic activity at temperatures below 70 °C. Transgene integration and expression in sugarcane were confirmed by Southern blot, RT-PCR, ELISA and western blot following biolistic co-transfer of minimal expression cassettes of xyl10B and the selectable neomycin phosphotransferase II. Xylanase activity was detected in 17 transgenic lines with a fluorogenic xylanase activity assay. Up to 1.2% of the total soluble protein fraction of vegetative progenies with integration of chloroplast targeted expression represented the recombinant Xyl10B protein. Xyl10B activity was stable in vegetative progenies. Tissues retained 75% of the xylanase activity after drying of leaves at 35 °C and a 2 month storage period. Transgenic sugarcane plants producing Xyl10B did not differ from non-transgenic sugarcane in growth and development under greenhouse conditions. Sugarcane xylan and bagasse were used as substrate for enzymatic hydrolysis with the in planta produced Xyl10B. TLC and HPLC analysis of hydrolysis products confirmed the superior catalytic activity and stability of the in planta produced Xyl10B with xylobiose as a prominent degradation product. These findings will contribute to advancing consolidated processing of lignocellulosic sugarcane biomass.

Kinetics of the enzymatic hydrolysis of lignocellulosic materials at different concentrations of the substrate

The kinetics of the enzymatic hydrolysis of two substrates—lignocellulosic materials from Miscanthus and oat hulls—in an acetate buffer is studied at different concentrations of the substrates. The substrates are obtained via single-step treatment with a dilute solution of nitric acid. The content of a nonhydrolyzable component—acid-insoluble lignin—for Miscanthus and oat hulls was 11 and 14%, respectively. A multi-enzyme composition of commercially available enzyme preparations CelloLux-A and BrewZyme BGX was used as a catalyst. It is shown that treatment with the nitric acid solution produces reactive substrates for the enzymatic hydrolysis. The innovative science of the results is confirmed by Russian patent 2533921. Kinetics of the enzymatic hydrolysis of these substrates in an acetate buffer can be described by a mathematical model based on a modified Michaelis–Menten equation. The main kinetic constants for both substrates are determined from the experimental data. The equilibrium concentrations of reducing substances (RSes) for the substrates are calculated from the initial substrate concentrations. It is found that within the studied range of substrate concentrations (33.3–120.0 g/L), the initial rate of enzymatic hydrolysis for the lignocellulosic material from oat hulls is higher than that for the lignocellulosic material from Miscanthus by 1 g/(L h). It is shown that the yield of RS depends of the initial concentration of the substrates: as the concentration rises from 33.3 to 120 g/L, the yield of RS falls 1.5–2.0 times, due to substrate inhibition. At low initial concentrations, the yields of RS are similar for the substrates from Miscanthus and oat hulls. When the initial concentration of the substrate reaches 120 g/L, the yield of reducing substances for the lignocellulosic material from Miscanthus is approximately 20% higher than that for the lignocellulosic material from oat hulls. The established dependences and the proposed mathematical model allow us to optimize the initial concentration of the substrate for efficient enzymatic hydrolysis.

Autohydrolysis Pretreatment and Delignification of Silver Fir Wood to Obtain Fermentable Sugars for Bioethanol Production

Fir wood biomass, non-pretreated and pretreated by autohydrolysis under selective conditions followed by chlorite delignification, was used as a substrate for enzymatic hydrolysis. The influence of solid loading on the enzymatic hydrolysis yield was studied. Compared with non-pretreated wood, autohydrolysis pretreatments at all temperatures tested enhanced enzymatic hydrolysis yields. A total of 33.0 g sugars can be generated from 100 g silver fir wood. The results show that elimination of hemicellulose by autohydrolysis pretreatment and lignin by delignification, before enzymatic hydrolysis, enhances enzymatic overall process yield.

Environmentally friendly technologies for obtaining high sugars concentrations from invasive woody species

The efficient utilization and conversion of inexpensive invasive raw materials into bioethanol following a biorefinery approach is a priority in the research field of renewable fuel. With this purpose, Acacia dealbata wood samples were pretreated with 1-ethyl-3-methylimidazolium acetate under optimized conditions, and the resulting solids were employed as a substrate for enzymatic hydrolysis. Enzymatic assays were performed according to a complete factorial experimental design, in which the effects of two independent variables (liquor to solid ratio and enzyme to substrate ratio) on the kinetics and yields of the xylan and cellulose saccharification were assessed. The Response Surface Methodology was employed for optimizing the experimental conditions. High sugar concentrations (around 80 g/L), and favorable polysaccharide conversions (CCG = 79.4% and XnCX = 77.9%). were predicted by the model under the selected operational conditions (6 g liquor/g substrate, 22 FPU/g). The results reported in this work compare well with other studies dealing with either other ionic liquids or classical pretreatments, using the same raw material or other woody substrates.

A biorefinery approach based on fractionation with a cheap industrial by-product for getting value from an invasive woody species

Acacia dealbata wood (an invasive species) was subjected to fractionation with glycerol (a cheap industrial by-product), and the resulting solid phase was used as a substrate for enzymatic hydrolysis. Glycerol fractionation allowed an extensive delignification while preserving cellulose in solid phase. The solids from the fractionation stage showed high susceptibility to enzymatic hydrolysis. Solids obtained under selected fractionation conditions (glycerol content of media, 80 wt%; duration, 1h; liquid to solid ratio, 6g/g; alkaline and neutral washing stages) were subjected to enzymatic saccharification to achieve glucose concentrations up to 85.40g/L, with almost complete cellulose conversion into glucose. The results confirmed the potential of glycerol as a fractionation agent for biorefineries.

Valorization of olive stones for xylitol and ethanol production from dilute acid pretreatment via enzymatic hydrolysis and fermentation by Pachysolen tannophilus

Olive stones are an agro-industrial by-product abundant in the Mediterranean area that is regarded as a potential lignocellulosic feedstock for sugar production. Statistical modeling of dilute-sulphuric acid hydrolysis of olive stones has been performed using a response surface methodology, with treatment temperature and process time as factors, to optimize the hydrolysis conditions aiming to attain maximum d-xylose extraction from hemicelluloses. Thus, solid yield and composition of solid and liquid phases were assessed by empirical modeling. The highest yield of d-xylose was found at a temperature of 195°C for 5min. Under these conditions, 89.7% of the total d-xylose was recovered from raw material. The resulting solids from optimal conditions were assayed as substrate for enzymatic hydrolysis, while fermentability of hemicellulosic hydrolysates was tested using the d-xylose-fermenting yeast Pachysolen tannophilus. Both bioprocesses were considerably influenced by enzyme loading and inoculum size. In the enzymatic hydrolysis step, about 56% of cellulose was converted into d-glucose by using an enzyme/solid ratio of 40FPUg−1, while in the fermentation carried out with a cell concentration of 2gL−1 a yield of 0.44g xylitol/g d-xylose and a global volumetric productivity of 0.11gL−1h−1 were achieved.

Combination of ultrasonic irradiation with ionic liquid pretreatment for enzymatic hydrolysis of rice straw

The application of ultrasonic irradiation and ionic liquids (ILs) in the degradation of rice straw under different processes of pretreatment and enzymatic hydrolysis was investigated. Various substrates for enzymatic hydrolysis by cellulase with and without ultrasound were as follows: untreated rice-straw powder (RS); RS treated by ILs of 1-ethyl-3-methylimidazolium ethylsulfate and trihexyl (tetradecyl) phosphonium decanoate with ultrasound at 300 W/(40 kHz, 28 kHz); RS treated by IL of choline hydroxide ([Ch][OH]) with ultrasound at 300 W/40 kHz (CHRS). In ultrasound-mediated enzymatic hydrolysis, the yield of total reducing sugar (TRS) converted from CHRS was up to 96.22% at 240 min and was greater than that from the other substrates; the TRS yield for CHRS with ultrasound was 19.5% greater than that without irradiation. Lignocellulosic biomass pretreated with [Ch][OH] showed the highest efficiency among the tested ILs, and ultrasound can be applied effectively in rice-straw pretreatment and enzymatic hydrolysis.

A biorefinery concept for simultaneous recovery of cellulosic ethanol and phenolic compounds from oil palm fronds: Process optimization

In this study, process optimization of an ultrasonic-assisted organosolv/liquid oxidative pretreatment (SOP) of oil palm fronds (OPFs) for the simultaneous recovery of cellulose, bioethanol and biochemicals (i.e. phenolic compounds) in a biorefinery concept was carried out. The effects of time (30–60min.), temperature (40–80°C), NaOH concentration (1–5%) and sample:solvent ratio (1:10–1:50g/ml) on cellulose content, bioethanol yield and total phenolics contents (TPC) after SOP were investigated. At optimum conditions of pretreatment (i.e. 60°C, 40min, 3% w/v aq. NaOH and 1:20g/ml sample to solvent ratio), the recovered cellulose (55.30%) which served as substrate for enzymatic hydrolysis and subsequent fermentation yielded about 20.1g/l glucose, 11.3g/l xylose and 9.3g/l bioethanol (yield of 0.769g/g). The pretreatment liquor (mostly regarded as wastes) obtained at the optimum pretreatment conditions contained about 4.691mg gallic acid equivalent (GAE)/g OPFs of TPC, 0.297mg vanillic acid (VA)/g OPFs, 1.591mg gallic acid (GA)/g OPFs and 0.331mg quercetin (QU)/g OPFs. The pretreatment liquor was again analyzed to possess high antiradical scavenging activity (about 97.2%) compared to the synthetic antioxidant, 3,5-di-tert-butyl-4-hydroxytoluene (BHT) (80.7%) at 100ppm. Thus one sustainable way of managing wastes in biorefinery is the recovery of multi-bioproducts (e.g. bioethanol and biochemicals) during the pretreatment process.

Housefly larvae hydrolysate: orthogonal optimization of hydrolysis, antioxidant activity, amino acid composition and functional properties

BackgroundAntioxidant, one of the most important food additives, is widely used in food industry. At present, antioxidant is mostly produced by chemical synthesis, which would accumulate to be pathogenic. Therefore, a great interest has been developed to identify and use natural antioxidants. It was showed that there are a lot of antioxidative peptides in protein hydrolysates, possessing strong capacity of inhibiting peroxidation of macro-biomolecular and scavenging free redicals in vivo. Enzymatic hydrolysis used for preparation of antioxidative peptides is a new hot-spot in the field of natural antioxidants. It reacts under mild conditions, with accurate site-specific degradation, good repeatability and few damages to biological activity of protein. Substrates for enzymatic hydrolysis are usually plants and aqua-animals. Insects are also gaining attention because of their rich protein and resource. Antioxidative peptides are potential to be exploited as new natural antioxidant and functional food. There is a huge potential market in medical and cosmetic field as well.ResultProtein hydrolysate with antioxidant activity was prepared from housefly larvae, by a two-step hydrolysis. Through orthogonal optimization of the hydrolysis conditions, the degree of hydrolysis was determined to be approximately 60%. Fractionated hydrolysate at 25 mg/mL, 2.5 mg/mL and 1 mg/mL exhibited approximately 50%, 60% and 50% of scavenging capacity on superoxide radicals, 1, 1-Diphenyl-2-picrylhydrazyl radicals and hydroxyl radicals, respectively. Hydrolysate did not exhibit substantial ion chelation. Using a linoneic peroxidation system, the inhibition activity of hydrolysate at 20 mg/mL was close to that of 20 μg/mL tertiary butylhydroquinone, suggesting a potential application of hydrolysate in the oil industry as an efficient antioxidant. The lyophilized hydrolysate presented almost 100% solubility at pH 3-pH 9, and maintained nearly 100% activity at pH 5-pH 8 at 0°C- 4°C and room temperature during the first 6 months of storage. Essential amino acids in the hydrolysate accounted for 43% of the total amino acids.ConclusionsThe results suggesting that hydrolysate could be added to food oils as an efficient antioxidant. It might be useful for food additives, diet nutrients and pharmaceutical agents.