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Abstract The aim of this study was to hydrolysis of rainbow trout viscera by application of flavourzyme, papain and pepsin enzymes and compare the functional and antioxidant properties of these three types of proteins. At the same time, the maximum degree of hydrolysis and nitrogen recovery was recorded for the hydrolysate produced by flavourzyme (23.12 ± 1.05% and 55.64 ± 0.68% respectively). In all pH values tested (apart pH 8 and 10), hydrolysate produced by flavourzyme showed the highest solubility compared to other proteins (p<0.05). In addition, emulsion activity (apart from pH 4) and emulsion stability index (apart from pH 8) in this protein were higher in comparison with two other proteins (p<0.05). To compare the antioxidant properties of hydrolysate, the inhibition capability of scavenging of 2,2 diphynyl-1-picrylhydrazyl (DPPH) free radicals and reduction capacity of iron (III), were measured. As a result, hydrolysate produced by pepsin showed highest DPPH scavenging power (83.59 ± 2.27 %) and iron (III) reduction power (0.886 ± 0.013 absorbtion in 700 nm).This study showed that the proteins produced from the substrate has favorable properties and various factors, including the type of enzyme used greatly affect these properties.

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Lipid oxidation is one of the major processes in deterioration of food quality and nutritional value. In this study, antioxidative activity of peptide was determined from hydrolysate of protein isolate from common kilka (Clupeonella cultriventris caspia) muscle using trypsin enzyme of pyloric caeca extraction. The optimum pH and temperature of trypsin enzyme for BAPNA (Nα –benzoyl -DL- argentine – ρ – nitroanilide -HCL) hydrolysis were measured 8.0 and 60 °C, respectively. The finding showed that antioxidative activities determined by DPPH, ABTS radical scavenging activities and ferric reducing antioxidant power (FRAP) increased significantly with variation of degree of hydrolysates from 20 to 40% (p<0.05). The results suggest that trypsin enzyme from pyloric caeca extraction could be a useful tool for peptide production from protein isolate with antioxidant activity and used as an alternative for commercial enzymes such as microbial enzymes in production of protein hydrolysates.

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In this experiment, head wastes were prepared and enzymatically hydrolyzed using alcalase (2.4 L) enzyme. The hydrolysate was fractionated by ultrafiltration with 10 kDa molecular weight cut-offs and the desired fraction was encapsulated following ion coagulation method (chitosan and triphosphate (TPP)) in nanochitosan capsules. Encapsulation process was optimized based on different ratios of chitosan:TPP and different concentrations (1, 5 and 10 mg/ml) of peptidic fraction. Finally, the degree of hydrolysis and the length of the peptides obtained from enzymatic hydrolysis were determined. The nanocapsules were examined for size, zeta potential and polydispersity index (PDI) using dynamic light scattering (Malvern, England). Structural and surface morphology studies including scanning electron microscopy (SEM) and infrared spectroscopy (FTIR) of capsules produced under favorable conditions were also performed. Particle size was measured in various concentrations and treatments in the range of 30 to 150 nm. The best results were obtained in the treatment of 2: 1 ratio of chitosan to polyphosphate and concentration of 10 mg / ml. The size, dispersion index, zeta potential and size of nanocapsules in the optimal conditions were 0.375, 2020 and 30.13 nm, respectively, and storage conditions at -20 °C had no effect on the quality of nanocapsules. Based on the efficiency study, it was found that fraction with a concentration of 10 mg/ml is well encapsulated by chitosan with an efficiency of 91.04 ± 0.18 percent. The results showed that chitosan-TPP could be used for nanocapsulation of bioactive peptides with an approximate molecular weight of less than 10 kDa.

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Diabetes mellitus is a major health problem in the worldwide. Inhibition of DPP-IV is one of the methods to control diabetes type 2. Inhibition of this enzyme may improve glycemic control in diabetics by preventing the rapid breakdown and there by prolonging the physiological action of incretin hormones. Furthermore, improving the antioxidant system in diabetic patients can prevent the occurrence of secondary disease caused by oxidative stress. Therefore, the head of skipjack tuna was hydrolyzed with alcalase enzyme (1/5% of raw material weight) for 4 hours, in order to produce a product with antidiabetic and antioxidant activities. The DPP-IV inhibition activity, DPPH radical scavenging activity and reducing power of hydrolysate were measured. The results showed that the skipjack tuna head protein hydrolysate possess bioactive properties in a concentration dependent manner and increasing the protein concentration leads to a significant increase in bioactive properties of hydrolyzed product (p≤0.05). The IC50 of protein hydrolysate in DPP-IV inhibition and DPPH radical scavenging activities were obtained 1.016±0.02 mg/ml and 0.297±0.015 mg/ml, respectively. Also the reducing power of hydrolysate was 0.176±0.002 in 2.5 mg/ml protein concentration. Overall, according to the obtained results, it can be concluded that protein hydrolysate of skipjack tuna head possess high antioxidant and antidiabetic activities in vitro, and can be used as a food additive to enhance health level if additional research be conducted.

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In this study, the orangefin ponyfish (Leiognathus bindus) was hydrolyzed by alcalase in an enzyme to substrate ratio of 1: 100 for 300 minutes, and the degree of hydrolysis was measured for 5 hours. Also, the hydrolysate was fractionated by centrifugal having molecular mass cutoffs of 3, 10, and 30 kDa, and four peptide fractions were obtained. Then, the antioxidant activity (DPPH and ABTS free radicals scavenging activity) of peptide fractions, as well as hydrolysate, were measured at different hydrolysis times. The degree of hydrolysis was the highest (55.43 ± 2.11%) at a hydrolysis time of 240 minutes. The hydrolysate had a high amount of hydrophobic amino acids (50.6%) which cause antioxidant properties. The results of DPPH radical scavenging activity showed that the highest scavenging activity was obtained at a hydrolysis time of 240 minutes (75.59 ± 1.46). Also, among all the fractions, the 3-10 kDa fraction exhibited the highest scavenging activity compared to other fractions (80.58 ± 2.96% at a concentration of 5mg /ml). Based on the result of ABTS radical scavenging, the highest activity was reported at 240 minutes after hydrolysis (50.54 ± 0.63). Also, among all peptide fractions, the 3-10 kDa fraction had significantly higher scavenging activity than other fractions (84.58 ± 0.44 at a concentration of 5 mg /ml). The results of this study showed that the peptides obtained by enzymatic hydrolysis of orangefin ponyfish are a good candidate for providing antioxidant properties.

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In this study, the effect of different hydrolysis times (45, 90, 135, 180, and 225 minutes) by alcalase enzyme on the yield and quality of oil extracted from common kilka fish (Clupeonella cultriventris caspia) was investigated. The results showed that the highest extraction yield (40.41%) was in the hydrolyzed treatment for 135 minutes, which was not significantly different from other treatments. The qualitative indices of TBA, FFA, and CD of the extracted oils increased by increasing hydrolysis time, so the highest value of the mentioned indices was at 225 minutes, while the highest value of the PV index was at 180 minutes. Because the treatment of oil extracted in 45 minutes had lower values in the investigated oxidative spoilage indicators, it was selected as the treatment. The composition of its fatty acids was investigated, and it was found that monounsaturated fatty acids were the predominant fatty acids. Also, the amounts of saturated and polyunsaturated fatty acids did not show significant differences (p < 0.05). According to the obtained results, it was found that different hydrolysis times have different effects on the yield and quality of the obtained oil. Therefore, more research and studies are needed to fully investigate the effect of hydrolysis time on the quality characteristics of the oil extracted from Kilka fish.
 

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Studies on the production of protein supplements for phenylketonuria (PKU) patients indicate that whey protein is one of the most important protein sources for production of these compounds, owning to its specific combination of amino acids.Whey is considered as one of the most polluting by-product in environment, otherwise, it is an excellent source of functional proteins that can be used in medicine and special diet products. In this study, three different concentrations of whey protein concentrate (WPC) were used as a substrate for three enzymes Flavourzyme^®, Neutrase^®and Protamex^®. Effect of substrate concentration and hydrolysis time were studied on the separation efficiency of phenylalanine using response surface methodology (RSM) design. Phenylalanine was removed from hydrolyzed samples using ultra filtration (UF) and its concentration was measured using HPLC technique. Results of this study showed that samples hydrolyzed by Flavourzyme^® contain the lowest level of phenylalanine. Also, process optimization was done in order to predict the best conditions of hydrolyzing .The best condition in order to remove the maximum phenylalanine from whey proteins was found for the minimum hydrolyzing time and the lowest substrate concentration for all three enzymes.

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In the present study Protein hydrolysate was prepared from the sheep visceral (stomach and intestine) using Alcalase 2.4 L. The effect of temperature (40, 45, 50 and 55 °C), time ( in six levels) and enzyme/substrate ratio (30, 60 and 90 Anson unit), on degree of hydrolysis and antioxidant activity were investigated using factorial experiment. The highest degree of hydrolysis was observed at 45 °C, after 180 min and enzyme/substrate ratio of 90 Anson unit/ Kg substrate (p< 0.05). Under these conditions, degree of hydrolysis was 36/92%. To study the antioxidant activity of protein hydrolysate, DPPH radical scavenging activity, ferric reducing power and effect of protein hydrolysate on stability of soybean oil were measured. All antioxidant activity experiments were performed at constant temperature (45°C). The highest DPPH radical scavenging activity and ferric reducing power were achieved after 150 min at enzyme/substrate ratio of 90 Anson unit/ Kg substrate (p< 0.05) and after 180 min at 90 Anson unit/ Kg substrate (p< 0.05), respectively. Results show that protein hydrolysate can be used as a natural antioxidant source instead of synthetic antioxidants.    

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Fenugreek seed (Graceum Trigonella foenum) is a rich source of lysine and tryptophan. In this study, the enzymatic hydrolysis of fenugreek seed protein with the pancreatin enzyme was performed using the response surface methodology with independent variable including: temperature 20 - 60 ° C, time 30-270 min, and enzyme to substrate ratio of 25.23 - 0.25%. Optimum conditions to achieve the highest DPPH radical scavenging capacity and reducing power were obtained at 46. 12 ° C, and enzyme: substrate ratio of 1.84% and time of 175.96 min. Then, the antioxidant properties of the optimum treatment were measured at different concentrations (10-20 mg/ml) and compare with antioxidant properties of non-hydrolyzed protein and vitamin C. The highest DPPH radical scavenging capacity and radical hydroxyl inhibition and Fe2+ chelating and total antioxidant activity were 52.98%, 70.99%, 72.63%, absorption 1.28 at wavelength of 695 nm and 0.80 at wavelength of 700 nm, respectively. The results showed that enzymatic hydrolysis significantly increased the antioxidant properties of the protein. Therefore, the hydrolyzed protein of fenugreek seed can be used in food as a substitute for synthetic antioxidants.

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Since cancer treatment has been done through chemotherapy and various drugs that are accompanied with severe side effects in addition to therapeutic properties, nowadays sscientists are investigating anticancer therapies using natural compounds such as hydrolyzed proteins and bioactive peptides with higher treatment properties and fewer side effects. In this study, for the first time, hydrolyzed proteins obtained from pepsin, proteinase k and alcalase enzymes were used to examine its cytotoxic effect on human lung adenocarcinoma cells (A549). Human lung carcinoma A549 cell lines were grown in 90% RPMI medium supplemented with 10% fetal bovine serum and 1% penstrep. Different concentrations of hydrolysates during times of 24, 48 and 72h were affected by A549 cell lines via XTT assay. Then, the cell survival ability was evaluated by XTT method. Results showed that hydrolysates produced from wheat germ protein affected the viability of cells and it depending on the enzyme applied, concentration and time. The results of IC₅₀ were evaluated for A549 cells in the case of pepsin, alcalase and proteinase k hydrolysates at 72 h, 11.17mg/mL, 12.94mg/mL and 11.27mg/mL. These results showed that wheat germ protein hydrolysates would be used as new source of anticancer peptides and could be a replace for common cancer therapy drugs in the near future.
 

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In this study, the effect of enzyme type (alcalase and pepsin) and process time (50-300 minutes) on the degree of hydrolysis and antioxidant indices including free radical scavenging of DPPH, ABTS, hydroxyl, reducing power, and chelating activity of Iron and copper ions were evaluated for navy bean protein (Phaseolus vulgari L.). Also, the composition of amino acids (hydrophobic and antioxidants types) and structural properties (FTIR) of primary protein and hydrolysates were investigated. The results showed that enzymatic hydrolysis improves antioxidant properties. Also, the composition of amino acids has a significant effect on antioxidant activities. On the other hand, the type of enzyme and the time of the hydrolysis process affected the degree of hydrolysis and the antioxidant activity of the hydrolysates. Thus, the highest percentage of free radicals scavenging of DPPH (82.4%), ABTS (58.3%), reducing power (0.97), hydroxyl radical scavenging (57.5%), and chelation of Fe (53.7%) and Cu ions (12.1%) were affected by the type of enzyme and process time. Among different treatments, the highest value of these indices (except copper ion chelating) was related to hydrolysates with alcalase. Structural properties of white bean protein were evaluated and enzymatic hydrolysis caused changes in the amide regions (I and II) as well as exposure to some hydrophobic-buried groups. The results of this study indicated the positive effect of enzymatic hydrolysis on the production of antioxidant hydrolysates that can be used in the food industry.

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Today, special attention is paid to the use of bioactive peptides in the production of functional foods and drugs. In the present study, isolation and evaluation of bioactive properties of peptides derived from enzymatic hydrolysis of soy whey were investigated. For enzymatic hydrolysis of soy whey protein, two types of enzymes, ficin and trypsin, were used at temperatures of 37 °C and 45 °C and at times of 2 and 4 hours. Then, the obtained protein hydrolysates were separated by reverse phase- high performance liquid chromatography and the obtained fractions were collected for biological activity. Statistical analysis of hydrolysis degree results was conducted by factorial method. One-way analysis of variance was used to analyze the results of the bioactive properties of peptides. The means were compared by Duncan's multiple range test at 5% probability level. Based on the results, peptide fractions had good antioxidant activity and also showed antimicrobial effect against Escherichia coli and Staphylococcus aureus. The T4F7 fraction (the seventh fraction obtained from trypsin-prepared protein hydrolyzate at 45 °C for 4 hours) had the highest antioxidant and antimicrobial properties. Therefore, this fraction was considered as the superior fraction. Electrophoregram of separation of peptide components in the selected bioactive peptide fraction (T4F7) showd that the presence of peptides with a molecular mass often of about 5 to 10 kDa and Less than 50 kDa is the main cause of desirable antioxidant properties in this treatment. Therefore, soy whey peptide can be used as a functional ingredient and natural preservative in food products.
 

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Corn starch derivatives, including glucose syrups, are nowadays widely used in food industry. Glucose syrup is used in food industry, not only due to its sweetening power and nutritional value, but also for its functional properties (moisture stabilization, softening ability, improving texture and preventing sucrose crystallization). Floury (soft) corn is usually used to produce glucose syrup, but the most imported corn in Iran is flint or hard corn which is all the year round available and consequently, using both corn flour types would be inevitable. Therefore, the purpose of this study was to investigate the effect of using flint (hard) and floury (soft) corn flour to produce glucose syrup. Four treatments including hard flour + soft flour in four ratios of 30% + 70%, 50% + 50%, 70% + 30% and 100% soft flour as control were prepared and the physicochemical and organoleptic properties of the produced syrups were evaluated. According to the obtained results, using flint corn flour affected physicochemical and organoleptic properties of the samples. Increasing the ratio of flint corn flour had significantly decreased DE (Dextrose Equivalent), soluble solids and pH of glucose syrups. Also induced the increasing of color parameters and sulfated ash values of the produced syrups. However, cost estimates indicated a reduction in the cost of raw materials and consequently general reduction in production costs by replacing hard corn flour. It can be concluded from the results that hard flour can be used on all surfaces, but the best treatment was 50% replacement level or a bit more, in the production which had a good effect on the properties of glucose syrup and showed more similarity with the control sample. All of these, along with being cost effective, appeared this treatment to have the potential of supposing as a sugar substitute in food industry.

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The present work aimed to study the effect of enzymatic hydrolysis of sarcoplastic and myofibrillar proteins from pangasius sutchi fish on the chemical compositions, the solubility, degree of hydrolysis (DH), peptide content and amino acid compositions was evaluated and their molecular weight recorded. The fish sarcoplasmic protein hydrolysates (SPHs) and myofibrillar protein hydrolysates (MPHs) were produced using three types of proteases: papain, alcalase and flavourzyme. Physicochemical properties of proteins and molecular weight were investigated. Results indicated that type of protease affected the degree of hydrolysis (DH), where all of the enzymes showed high rate of hydrolysis during the first hour, and then gradually decreased. The type of enzyme and the extent of the DH greatly influenced the amino acid residue composition and the molecular weight of the protein hydrolysates. Different amino acid composition of proteins and their hydrolysates was observed. The soluble protein and peptide content of hydrolysates significantly increased by the increase in time of incubation. The high amount of hydrophobic and aromatic amino acids in the SPH and MPH can enhance the biological activities of the peptides. Results suggest that the protein hydrolysates derived from patin may be used in functional food and supplements.
 

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Bioactive peptides are actually specific protein parts that, in addition to nutritional value, have positive effects on body function and conditions that lead to health effects. The combined use of pretreatment and enzymatic hydrolysis causes changes in the physical and chemical properties of hydrolyzed proteins. Microwave pretreatment is a well-known strategy to increase the accessibility of hydrolysis-sensitive bonds, facilitating enzyme cleavage sites and exposure to proteases. The aim of this research was to investigate the effect of hydrolysis time and also the effect of microwave pretreatment on enzymatic hydrolysis of edible mushroom (Agaricus bisporus) protein by trypsin enzyme to produce antioxidant peptides. To conduct this research, first, edible mushrooms were turned into powder after purchase from the market and conducting related processes. In this research, hydrolyzed edible mushroom was produced using trypsin enzyme without pretreatment and with microwave pretreatment at different powers of 120, 200, and 280W and time intervals of 30, 60, 90, 120, 150, 180 and 210 minutes. The antioxidant properties of the hydrolysed samples (DPPH radical scavenging activity, iron chelating power, iron ion reduction power and total antioxidant activity) were evaluated and compared. The results showed that in most of the experiments, pretreatment with microwaves decreased the time of obtaining and increased the antioxidant capacity of the samples, so that the samples pretreated with microwave power of 120W showed higher antioxidant power compared to the other treated samples. In all antioxidant tests, the sample pre-treated with microwave with power 120W and hydrolysed at 90minutes showed higher antioxidant performance, and therefore it is considered as suitable treatment.
 

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In recent years, researchers have identified and extracted bioactive peptides with acceptable antioxidant properties from different protein sources. Flaxseed meal, which is the main by-product of the linseed oil extraction process, contains a large amount of protein, which can be obtained by enzymatic hydrolysis to peptides with antioxidant properties. In this research, the effect of hydrolysis conditions (enzyme concentration 2.7-1% and time 30-183.64 minutes), type of protease (pancreatin and alcalase) and ultrasound pretreatment on the degree of hydrolysis and antioxidant properties (DPPH free radical inhibition, Total antioxidant activity, and iron ion chelation) of hydrolyzed protein obtained from flaxseed meal were evaluated using the response surface method. The optimal conditions for the production of hydrolyzed protein with the most antioxidant properties with alcalase enzyme with and without pretreatment and pancreatin enzyme with and without pretreatment, respectively, were hydrolysis time 79, 146.79, 111.77 and 97.21 minutes and enzyme concentration 2.29%, 1.46%, 2.26% and 1.38%; According to the obtained results, by examining the degree of hydrolysis and antioxidant properties of the hydrolyzed proteins of flaxseed meal, hydrolyzed protein with pancreatin enzyme was suggested as the optimal treatment. Hydrolyzed protein with pancreatin enzyme with ultrasound pretreatment was reported to have 75.66% DPPH free radical inhibition, 70.39% iron ion chelating activity, total antioxidant activity with absorbance of 0.86 nm and 80.69% hydrolysis degree. Therefore, it can be stated that the hydrolyzed protein of flax seed meal with strong antioxidant capacity is a bioactive compound for use in food formulations and the production of beneficial products.