Enzymatic Generation,Purification And Identification Of Antioxidative Peptides From White Button Mushroom(Agaricus Bisporus)Protein Isolate

The growing evidence from scientific research linking diet quality and quality health well-being has led to the development of new food concepts of functional foods and nutraceuticals.Nowadays,food is not only considered as a source of nutrients for nourishment and energy requirements,but as a source of bioactive components which play specific biological functions in the body.Bioactive peptides are among the biological components that have been recognized as dietary ingredients that beyond the basic nutritional benefits,exert diverse biological functions when ingested.Antioxidant peptides from food protein sources,in addition to being alternatives to synthetic antioxidants in the food industry,can be used in functional foods formulation to ameliorate oxidative stress and its associated effects in human health.Mushrooms have been utilized by humans for centuries as a source of food and along this history of use,is the belief in their curative abilities.Researchers have identified various bioactive components in mushrooms including polysaccharides,proteins and peptides,lipids and secondary metabolites which have been shown to exhibit numerous biological functions.White button mushroom(Agaricus bisporus),is the world's leading cultivated and consumed mushroom.Although several bioactivities from A.bisporus have been demonstrated and reported in literature,there is a paucity of information regarding antioxidant peptides generated from its proteins.The aim of this study was therefore to enzymatically generate,purify and identify antioxidative peptides from A.bisporus protein isolate.Acetic acid and ammonium sulphate precipitation method was used to extract soluble proteins from the fruiting bodies of A.bisporus.Three commercial food-grade proteases viz Alcalase(an endo-peptidase),Pancreatin and Flavourzyme(both with exo-and endo-peptidase properties)were used singly or in sequence to hydrolyze the extracted mushroom protein isolate(MPI).The obtained hydrolysates(MPHs)were characterized by high hydrolysate yields(5795%)and hydrolysate protein recoveries(PR%)(44-82%)which were indicative of the efficiency of the enzymatic processes and the susceptibility of the MPI to the proteases'hydrolytic capacities.This susceptibility was further attested by the gel electrophoretic profiles which revealed that the high molecular weight(MW)polypeptides in the MPI were hydrolyzed into low MW peptides,and this could possibly increase the antioxidant activity of the generated peptides.Flavourzyme hydrolysis was characterized by significantly(p<0.05)low degree of hydrolysis(DH%)compared to the other hydrolyses indicating that the MPI was least susceptible to this enzyme's proteolysis.Pancreatin and Alcalase-Pancreatin hydrolyses completely digested all the high MW polypeptides as indicated by the disappearance of prominent protein bands.An analysis of the hydrolysates amino acid composition showed they were characterized by high levels of negatively charged amino acids(NCAA)which are known to confer antioxidant activity to peptides.Except the Alcalase-Flavourzyme hydrolysate,the hydrolysates exhibited significantly(p<0.05)high levels of hydrophobic amino acids(HAA),which enhance peptide solubility in lipids thus increasing their interaction with free radicals,than the MPI.The generated MPHs were ultrafiltered to concentrate peptides into ultrafiltration fractions(UFs)with<1,1-3,3-5,and 5-10 kDa MW ranges.High UF yields(>78%)and UF PR%(>80%)comprising of low MW fraction(<3 kDa)was indicative of the high efficiency and the hydrolytic breakdown susceptibility of the MPI to enzymatic processes.DPPH radical scavenging activity(DRSA),metal chelating activity,ferric reducing power activity(FRAP)and linoleic acid oxidation inhibition were used to screen for antioxidant activity of the hydrolysates and their UFs.The hydrolysates exhibited higher metal chelating activity than their UFs and this may have been attributed to the synergistic effects of the constituent peptides responsible for the activities.The 1-3 kDa fractions from all the hydrolysates displayed the strongest metal chelating activity compared to the other peptide fractions.This suggested an abundance of negatively charged peptides in the 1-3 kDa fractions.The DRSA for the MPHs depended on the enzymatic process used and the highest activity was exhibited by Pancreatin hydrolysate(PH)(EC50 0.25 mg/mL).For the UFs,the highest potency of DRSA was displayed by 1-3 kDa fraction from PH.All the MPHs and their UFs exhibited concentration-dependent increase in FRAP.The highest FRAP values for the MPHs 0.16 and 0.41 absorbance units at 1 and 3 mg/mL,respectively)was displayed by PH and the predigestion of MPI with Alcalase before Pancreatin and Flavourzyme did not significantly alter the FRAP activity.For the UFs,the highest FRAP was achieved with 1-3 kDa from PH with an absorbance of 0.62 at 3 mg/ml.At 1 mg/mL,the GSH had a significantly(p<0.05)higher FRAP ability(absorbance value 2.23)compared to all MPHs and their UFs.The MPHs were able to maintain linoleic acid oxidation inhibition for up to 4 days compared to 2 days for GSH and on day 5,AFH,APH and FH still retained>50%inhibition activity compared to the uninhibited control.PH 1-3 kDa fraction showed significant protection on pUC19 plasmid DNA against hydroxyl radical-induced damage with the highest DNA damage protection at 68.9%by 10 mg/mL compared to the negative control.The Pancreatin's 1-3 kDa UF exhibited the strongest antioxidant activity and was selected for further purification.Using gel chromatography,six fractions were separated from the 1-3 kDa UF and a middle-sized MW peptide fraction(F2)exhibited significantly(p<0.05)higher DRSA than the smaller-and the larger-sized MW peptide fractions.F2 yielded four fractions on ion exchange chromatography and fraction C(weakly positively charged)and fraction D(with net negative charge)exhibited potent(p<0.05)DRSA compared to others.C and D were finally purified on reversed-phase high performance liquid chromatography(RPHPLC)and homogeneous target fractions were obtained whose DRSA were 2-fold higher compared to the MPHs.Fractions C and D were used to evaluate their cytoprotective ability against H2O2-induced oxidative stress in HepG2 and Caco-2 cells.The cell based-bioassays which included cell viability,intracellular redox status,cellular reactive oxygen species(ROS)accumulation as well as lipid peroxidation and lipid dehydrogenase LDH leakage were carried out.The results demonstrated that C and D attenuated H2O2-induced oxidative stress by preserving cell membrane integrity through decreasing LDH leakage possibly by inhibiting membrane lipid peroxidation as attested by suppression of malondialdehyde(MDA)increase.These cytoprotective effects were further reinforced by the enhancement of superoxide dismutase(SOD)and catalase(CAT)activities and increase in GSH levels,with fraction C displaying better protection than D at all tested concentrations for each assessed index,giving scientific evidence that these peptide fractions could be potential natural antioxidants for consideration for use in functional foods formulation to lower health risks associated with oxidative stress.To further elucidate the underlying antioxidant mechanism by which the peptide fractions confer cytoprotection against oxidative stress,it was demonstrated that the fractions could activate the nuclear factor erythroid 2-related factor-antioxidant response elements(Nrf2ARE)signaling pathway via Nrf2 nuclear translocation.This pathway activation was shown to up-regulate dose-dependently the expression levels of phase ? detoxifying enzymes including heme oxygenase-1(HO-1),NAD(P)H quinone oxidoreductase-1(NQO1)and glutathione Stransferase(GST).Nano liquid chromatography-electron spray ionization-tandem mass spectrometry(nano LC-ESI-MS/MS)and de novo sequencing identified four peptides each from C and D.The peptides were characterized by a high percentage of hydrophobic amino acids,a major hallmark of antioxidant peptides.The identified fractions revealed a 100%homology with hypothetical parent proteins from two varieties of A.bisporus after searching from the National Center for Biotechnology Information(NCBI)data base.