Use of Different Proteases to Obtain Flaxseed Protein Hydrolysates with Antioxidant Activity (original) (raw)
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Iranian Food Science and Technology Research Journal, 2024
In this research, the effect of protease enzyme type (pepsin and pancreatin) and hydrolysis time (40-200 minutes) on the degree of hydrolysis and antioxidant properties (DPPH radical scavenging activity, Fe chelating activity, Fe reducing power and total antioxidant capacity) of flaxseed meal protein hydrolysates was investigated. The results showed that increasing the hydrolysis time increased the degree of hydrolysis, and the samples obtained from pancreatin had a higher degree of hydrolysis than pepsin. The highest activity of Fe2+ chelating (53.71 ± 0.45%) and Fe3+ reduction (1.32 ± 0.02, absorbance at 700 nm) was achieved by pancreatin after 200 minutes of hydrolysis. Pancreatin samples were more capable of inhibiting DPPH free radicals than pepsin, and their activity increased with increasing time up to 160 minutes. The highest total antioxidant capacity (1.36 ± 0.08 absorbance at 695 nm) among the samples was obtained after 160 minutes of hydrolysis with pancreatin. The antioxidant capacity of flax seed protein hydrolysates in inhibiting DPPH radical, Fe chelating activity, and total antioxidant capacity was lower than the antioxidant capacity of vitamin C at a concentration of 50 (mg/ml), but it had more Fe reducing power than vitamin C. Therefore, it can be concluded that compared to pepsin, pancreatin had a greater ability to produce flaxseed protein hydrolysates with significant antioxidant properties. According to the results, flaxseed protein hydrolysates from pancreatin enzyme and a hydrolysis time of 160 minutes have the ability to be used in food formulations to produce functional products.
Antioxidant Activity of Hydrolysates Prepared from Flaxseed Cake Proteins Using Pancreatin
Polish Journal of Food and Nutrition Sciences, 2014
Proteins were isolated from defatted flaxseed cake and hydrolysed with pancreatin. The hydrolysis process was conducted at a stable temperature of 50°C and pH 7.5, and monitored with the pH-stat method. The obtained hydrolysates with a degree of hydrolysis (DH) of 5, 10, 15, 20, 25% were investigated in terms of antioxidant properties. The radical scavenging activity was assayed against DPPH• and ABTS•+, the reducing ability – with FRAP assay, and the capability to bind Fe(II) – by reaction with ferrozine. SE-HPLC analysis was used to determine molecular weight distribution of hydrolysis products. The antiradical activity of pancreatin hydrolysates of flaxseed proteins was increasing along with an increasing DH and for the hydrolysate with DH 25% the EC50 value determined with the DPPH assay accounted for 0.083 mg/assay, and the ABTS•+ scavenging activity – for 0.218 mmol Trolox/g. This hydrolysate was constituted mainly by peptides with low molecular weights (MW) of 0.238-0.556 kDa. In turn, the Fe(II) binding capability increased from 44.5% to 64.9% in the case of hydrolysates with DH 5-20% and decreased in the case of the hydrolysate with DH 25%. A similar dependency was observed in the ability of pancreatin hydrolysates of flaxseed proteins to reduce Fe(III). The maximum value of reducing ability reached 0.25 mmol Fe(II)/g for the hydrolysate with DH 20% that was predominated by polypeptides and peptides with MW of 0.238-1.046 Da.
LWT - Food Science and Technology, 2016
In this study, the hydrolysis of a flaxseed protein isolate with Alcalase ® was performed as a strategy to generate antioxidant peptides. A chromatographic separation of the hydrolysate was conducted by RP-HPLC. Both hydrolysate and six collected fractions were subjected to ORAC and FRAP assays to evaluate their antioxidant capacity. The higher antioxidant values were shown by fractions containing predominantly low molecular weight peptides, as it was demonstrated by MALDI analysis. Four peptides were identified by LC-MS/MS and one by Edman degradation. The peptide with sequence GFPGRLDHWCASE was synthesised showing a notable ORAC activity, 3.20 µmol Trolox equivalents/µmol of peptide. This value was higher than that reported for butylated hydroxyanisole. Therefore, the contribution of this peptide to the activity of the fraction where it had been found was 61%. The identified sequences represent an advance in the molecular characterization of the flaxseed protein fraction.
Food Research International, 2012
The antioxidant capacity of maize proteins, wheat gluten, pea protein isolates and hydrolysates and melanoproteins (MRPs) was evaluated as a radical scavenging activity against ABTS (2,2-azino-bis/3-ethil-benothiazoline-6-sulfonic acid) reagent by the direct procedure. In addition, the effect of high temperature and water during thermal treatments on changes of the antioxidant capacity of proteins was investigated. The concentration of total cysteine, free sulfhydryl groups (−SH) and disulfide bonds (−S-S) were determined using Ellman's reagent, 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) and disodium 2-nitro-5-thiosulfobenzoate (NTSB 2−). Wheat gluten, as the most complex protein, had higher concentration of disulfide bonds (45.37 nmol/mg) and total cysteine (93.88 nmol/mg) compared to other tested proteins and showed the highest ABTS radical scavenging activity (74.39 mmol Trolox/kg). The antioxidative capacity of tested proteins was correlated negatively with free-SH group concentration and positively with total cysteine and-S-S bond concentration indicating a high effect of physical structure of a protein on its antioxidative properties. The antioxidant activity of melanoproteins formed in pea protein isolate/glucose model system during heating was threefold higher than that of initial pea protein. However, the highest amount of furosine was also formed in this model system under the same heating conditions.
International Journal of Molecular Sciences, 2012
The aim of this study was to produce a valuable protein hydrolysate from palm kernel cake (PKC) for the development of natural antioxidants. Extracted PKC protein was hydrolyzed using different proteases (alcalase, chymotrypsin, papain, pepsin, trypsin, flavourzyme, and bromelain). Subsequently, antioxidant activity and degree of hydrolysis (DH) of each hydrolysate were evaluated using DPPH• radical scavenging activity and O-phthaldialdehyde spectrophotometric assay, respectively. The results revealed a strong correlation between DH and radical scavenging activity of the hydrolysates, where among these, protein hydrolysates produced by papain after 38 h hydrolysis exhibited the highest DH (91 ± 0.1%) and DPPH• radical scavenging activity (73.5 ± 0.25%) compared to the other hydrolysates. In addition, fractionation of the most effective (potent) hydrolysate by reverse phase high performance liquid chromatography indicated a direct association between hydrophobicity and radical scavenging activity of the hydrolysates. Isoelectric focusing tests also revealed that protein hydrolysates with basic and neutral isoelectric point (pI) have the highest radical scavenging activity, although few fractions in the acidic range also exhibited good antioxidant potential.
Journal of Agricultural and Food Chemistry, 2017
The impact of the naturally-present phenolic compounds and/or proteins on the antioxidant capacity of flaxseed products-phenolic fraction, protein concentrates and hydrolysates-before and after simulated gastrointestinal digestion was studied. For that, whole and phenolic reduced products were assessed. Four glycosylated phenolic compoundssecoisolariciresinol and ferulic, p-coumaric and caffeic acids-were identified in flaxseed products. Phenolic fraction exerts the highest antioxidant capacity that increased by alkaline hydrolysis and by simulated gastrointestinal digestion. The action of Alcalase ® and digestive enzymes resulted in an increase of the antioxidant capacity of whole and phenolic reduced products. Principal component analysis showed that proteinaceous samples act as antioxidant is by H+ transfer, while those samples containing phenolic compounds exert their effects by both electron donation and H+ transfer mechanisms. Protein/peptide-phenolic complexation, confirmed by fluorescence spectra, exerted a positive effect on the antioxidant capacity, mainly in protein concentrates.
Antioxidant Activity of Oat Bran Hydrolyzed Proteins in Vitro and in Vivo
Oats contain molecules with well-known health benefits. Its fibers are used in different preparations to improve gastro-intestinal health and to reduce blood cholesterol. Avenanthramides, a group of phenolic antioxidants only present in oats have been demonstrated to be bioavailable in animals and humans. In recent years, proteins from foods have been viewed as not just sources of essential amino acids but also as sources of biologically active peptides (e.g. antioxidant, antihypertensive, antitumor). Antioxidant activities have been reported for hydrolyzed proteins from wheat, rice and corn byproducts but not for those from oats. The objective of this thesis was to investigate the antioxidant properties of hydrolyzed oat proteins in three different conditions (in vitro and in vivo). The first part of this thesis focusses on the activity of proteins extracted in the presence of salt and digested with trypsin (TPH) and alcalase (APH). The digests TPH and APH were ultra-filtered using 2kDa, and 10kDa molecular cutoff membranes. The radical scavenging properties were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and oxygen radical absorbance assay (ORAC). It was found that APH 2kDa was the strongest inhibitor (35%) of DPPH radicals and also TPH had a better peroxyl radical scavenging activity (ORAC) 434 ±16 µmol trolox equivalents (TE)/g) compared to APH (269±4 µmol TE/g) and TPH>2kDa (345±15 µmol TE/g). In the metal chelating assay, trypsin digest (TPH) better chelated Fe 2+ ions compared to APH and other fractions. The inhibition of linoleic acid autoxidation, showed that both trypsin and alcalase digests equally lowered the formation of lipid peroxides after five days of incubation. It appeared that trypsin hydrolysate and its ≤2kDa fraction possessed the strongest activities and may have application in food products.
International Journal of Food Science & Technology, 2008
Crude rice bran protein (CRBP) was prepared by alkaline extraction and then treated with 0.6 m HCl to remove phytic acid. The phytate-free rice bran protein (PFRBP) was hydrolysed with proteases M, N, S, P and pepsin under optimal conditions. Hydrolysates obtained from various hydrolysis periods were subjected to analysis for their degree of hydrolysis (DH) and functional properties. The hydrolysates were fractionated by reversed-phase column chromatography on Kaseigel ODS resin (120-140 lm) using a stepwise gradient of aqueous ethanol, and their activities were measured. The 40% ethanol fraction of protease P 4 h-hydrolysate was separated by successive reversed-phase high-performance liquid chromatography and the amino acid sequences of isolated antioxidative peptides were determined by a protein sequencer and matrix-assisted laser desorption ionisation-time of flight mass spectrometry. Crude rice bran protein had higher antioxidative activity than PFRBP, due to the presence of phytic acid. Phytate contents of rice bran, CRBP and PFRBP were 2.5%, 1.42% and 0%, respectively. The activity of PFRBP increased upon protease digestion. Protease M hydrolysates showed the highest DH, but the lowest antioxidative activity. Hydrolysates with DH below 10% had higher antioxidative activity than those above 20%. This result indicates that the antioxidative activity of the hydrolysates is inherent to their characteristics amino acid sequences of peptides depending on the protease specificities.
Antioxidants
Flaxseed proteins exhibit functionalities interesting for the food industry, including antioxidant capacity. Antioxidant activity depends on the protein composition and the presence of phenolic compounds extracted with them from the matrix. The research focused on the effect of subsequent protein extractions (water, salt and alkaline) of flaxseed meals (of three cultivars) on the protein fraction composition and its relations to antioxidant capacity. The protein and phenolic profiles and antioxidant functionalities (in antiradical ORAC and emulsion assays) were analysed. Spectroscopic characteristics of the fractions (fluorometric and FT-IR analysis) were also included. Our study has shown the effect of fractionation on the share of proteins at MW from 56–38 kDa (globulin-like) and <15 kDa (albumin-like) in the protein profiles. The highest globulin share was in the alkaline-extracted fractions (AEF) and albumin in the salt-extracted (SEF) ones. SDG (secoisolariciresinol diglucos...
Antioxidant activity of peptides isolated from alfalfa leaf protein hydrolysate
Food Chemistry, 2008
Alfalfa leaf protein, a potential source of high quality protein for human consumption, was hydrolyzed with protease. Alfalfa leaf protein hydrolysate was fractionated by ultrafiltration and the obtained peptides were purified by dynamical adsorption. The antioxidant activity of alfalfa leaf peptides (ALPs) was investigated and compared with that of a native antioxidant, reduced glutathione (GSH), which was used as a reference. The reducing power of ALPs was 0.69 at 2.00 mg/mL. ALPs at 1.60 mg/mL and 0.90 mg/mL exhibited 79.71% and 67.00% of scavenging activities on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and superoxide radical, respectively. In addition, ALPs showed 65.15% chelating effect on ferrous ion at 0.50 mg/mL. The molecular weight of the peptides was determined and 67.86% of the total amount was below 1000 Da. Combined with the result of the amino acid profiles, ALPs was believed to have high nutritive value in addition to antioxidant activity.