Research Article Pepsin Digested Oat Bran Proteins ...downloads.hindawi.com/journals/jchem/2016/8216378.pdfResearch Article Pepsin Digested Oat Bran Proteins: Separation, Antioxidant

Research ArticlePepsin Digested Oat Bran Proteins SeparationAntioxidant Activity and Identification of New Peptides

Ariane Vanvi12 and Apollinaire Tsopmo13

1Food Science and Nutrition Program Carleton University 1125 Colonel By Drive Ottawa ON Canada K1S 5B62National Institute of Higher Education in Agronomy Food and Environmental Sciences 26 Boulevard Docteur-PetitjeanBP 87999 Dijon France3Institute of Biochemistry Carleton University 1125 Colonel By Drive Ottawa ON Canada K1S 5B6

Correspondence should be addressed to Apollinaire Tsopmo apollinaire tsopmocarletonca

Received 16 September 2015 Revised 31 December 2015 Accepted 3 January 2016

Academic Editor Somdet Srichairatanakool

Copyright copy 2016 A Vanvi and A Tsopmo This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The aim of this study was to determine pepsin hydrolysis conditions to produce digested oat bran proteins with higher radicalscavenging activities and separate and identify peptides Isolated proteins were then digested with different concentrations ofpepsin and incubation times Hydrolysates produced with 1 30 enzyme substrate (ES) ratio and 2 h possessed the highest peroxylradical scavenging activity 608 plusmn 17120583MTEg (compared to 456ndash474 120583MTEg for other digests) and was therefore subsequentlyfractionated into eight fractions (F1ndashF8) by high performance liquid chromatography (HPLC) F1 and F2 had little activity becauseof their low protein contents Activities of F3ndashF8 were 447ndash874 120583MTEg 20ndash36 and 10ndash14 in the peroxyl superoxide anionand hydroxyl radical tests respectively Liquid chromatography-tandem mass spectrometry (LC-MSMS) was used to identify atotal of fifty peptides that may have contributed to the activity of F3 a fraction that better scavenged radicals

1 Introduction

Oxidation is a well-known phenomenon that affects thequality of food products as it creates compounds responsiblefor the rancid odour and changes in color or texture ofsome foods Oxidised lipids can further react for exam-ple with proteins to form toxic compounds like pyridinepyrrole and other N-heterocyclic compounds [1] In vivoaerobic metabolism naturally produces free radicals thatare eliminated by enzymes such as superoxide dismutasecatalase and glutathione peroxidase and by molecules suchas glutathione uric acid and bilirubin [2] However withaging the decreased efficiency of the antioxidant systemcoupled to pollution fatigue or an unbalanced diet cangenerate free radicals in excess thereby promoting oxidativestress [3] The accumulation of free radicals manifests itselfby causing damage to cell membrane molecules whichmay then increase the risk of diseases such as cancerand cardiovascular and neurodegenerative diseases [4] Inaddition to synthetic antioxidants currently available to

limit oxidation researchers are actively looking for naturalantioxidants because of their ability to reduce oxidation offood and biological molecules and preference by consumers[5] Fruits and vegetables have well been studied because ofthe presence of exogenous antioxidants (vitamin C vitaminE carotenoids and polyphenols) that can contribute to thecellular redox balance [6] Attention has also been focussedon the role of other food molecules and in that respect pep-tides derived from hydrolysis of food proteins are currentlybeing investigated [7 8] Food peptides have then been shownto possess biological activities such as antioxidant antihyper-tensive and antimicrobial and modulation of the immunesystem [7 9] Antioxidant peptides in foods are generallyreleased only after enzymatic hydrolysis [10] or fermentationin the presence of microorganisms [11] Depending on theirmolecular weight peptides may be transported throughintestinal-expressed transporters across the enterocytes [7]Cereals are source of fibres and polyphenols that have beenextensively investigated for their effects in cardiovasculardiseases and diabetes [12] but their peptides have received

Hindawi Publishing CorporationJournal of ChemistryVolume 2016 Article ID 8216378 8 pageshttpdxdoiorg10115520168216378

2 Journal of Chemistry

less attention Oat proteins digested with alcalase and trypsinwere demonstrated to possess antioxidant activities [13 14]However the action of other proteases or the activity offractions from chromatography separation remains to beinvestigated The objective of this study was to optimizethe conditions for pepsin hydrolysis of oat bran proteinsperformHPLC separation determine the antioxidant activityof fractions and identify peptides

2 Materials and Methods

21 Chemicals Medium oat bran (id 112-001) was sup-plied by Richardson Milling (Portage La Prairie ManitobaMB) Viscozyme L (100 Fungal Beta Glucanase (FBG)g)pepsin from porcine gastric mucosa (367 unitsmg solid) L-glutathione Tris(hydroxymethyl)aminomethane hydrochlo-ride (Tris-HCl) pyrogallol 110-phenanthroline iron sulfatepentahydrate (FeSO

4sdot7H2O) sodium carbonate (Na

2CO3)

sodium hydroxide sodium tartrate cupric sulfate pentahy-drate (CuSO

4sdot5H2O) Folin-Ciocalteu reagent and bovine

serum albumin (BSA) were obtained from Sigma Aldrich(Oakville ON) Hydrogen peroxide (H

2O2) 6-hydroxy-2

578-tetramethylchromane-2-carboxylic acid (Trolox) fluo-rescein methanol mono- and di-basic potassium phosphateethylenediaminetetraacetic acid and sodium dodecyl sulfatewere purchased from Fisher Scientific (Nepean ON) 221015840-Azobis(2-amidinopropane) dihydrochloride (AAPH) wasfromWakoChemicals Spectrophotometric and fluorometricmeasurements were performed on the BioTek Epochtrade UV-Vis and Biotek FLx 800 microplate reader respectively bothcontrolled by Gen5trade data analysis software Incubations weredone on a MaxQtrade 5000 shaker model (Fisher ScientificNepean ON)

22 Protein Extraction The extraction procedure was per-formed based on a previousmethod [14] Samples of oat brans(4times 100 g) were defatted by stirring in hexane (1 3 wv) for 1 hat room temperature filtration on cheesecloth and overnightdrying under fume hoods Deionized water was added tothe defatted oat bran at the ratio 1 10 (wv) and adjusted topH 45 Viscozyme L and 3 FBGg of defatted bran was usedto breakdown carbohydrates and reduced the viscosity Themixture was then incubated at 45∘C 150 rpm and 15 h Atthe end of the incubation the pH was adjusted to 95 using2M NaOH solution and further incubated for 15 h in orderto carry out the alkaline extraction of proteins After coolingcentrifugation was performed at 2500 g for 20min at 4∘CSupernatants containing proteins were adjusted to pH 40and then centrifuged (10000 g 40min 4∘C) to obtain proteinisolates (ie pellets) that were suspended in pH 70 waterbefore being freeze-dried

23 Hydrolysis with Pepsin Freeze-dried oat bran proteinisolates (500mg each) were transferred into twelve differenttubes and rehydrated with water (1 12 wv) The pH wasadjusted to 40 with 1M HCl followed by addition of pepsinat 1 20 1 30 1 40 and 1 50 (ww) enzyme-substrate (ES)ratios Mixtures were homogenized adjusted to pH 20

and then incubated at 37∘C 150 rpm Four samples wereremoved at 1 2 and 3 h respectively At the end of eachincubation time the enzyme was inactivated by adjusting thepH to 70 with 1M NaHCO

3 Supernatants were collected

after centrifugation at 2500 g for 15min to remove denaturedpepsin and nondigested proteins The hydrolysates werefreeze-dried and used for oxygen radical absorbance capacity(ORAC) assay For HPLC separation oat proteins (3 times 100 g)were digested with pepsin (1 30 ES) for 2 h The hydrolysateproduced under these conditions had the highest ORACvalue

24 Oxygen Radical Absorbance Capacity (ORAC) Assay Theassay was performed using a Bio-Tek FLx800 instrumentaccording to literature [15] Reagents standards digestedproteinsHPLC fractions (01mgmL) and assay control(GSH 01mgmL) were prepared with potassium phosphatebuffer (75mM pH 74) Five concentrations of Trolox (625ndash100 120583M) were used to obtain the standard curve To performthe assay fluorescein (120120583L 0080 120583M) was transferred into96-well microplate followed by addition of 20 120583L of samplesstandards or buffer (blank) The plate was incubated for20min at 37∘C AAPH (15mM 60 120583L) was then added toeach well Data (triplicates) were collected every minute over50min and used to calculate ORAC values expressed as 120583MTrolox equivalent (TE) per gram of lyophilized hydrolysate offraction

25 Separation by RP-HPLC Digested bran proteins (2 h1 30 ES) with the most peroxyl radical scavenging activitywere selected for HPLC separation The system consisted of1525 binary pump 2998 photodiode array detector (set at220 nm) 2707 autosampler maintained at 8∘C and fractioncollector III from Waters (Montreal QC Canada) Thefreeze-dried oat bran protein hydrolysate was dissolved in01 acetic acid (100mgmL) and filtered through 045120583mnylonmembraneThe columnwasWaters Prep XBridge BEHC18 130 A 10 120583m 19 times 150mm and the injection volumewas 2mL A linear gradient (5 to 90 B) of 01 acetic acidin water (A) or in methanol (B) over 45min was used forseparation at a flow rate of 4mLmin Fractions (8mL each)were collected and pooled into eight fractions according tothe chromatogram 0ndash6min (F1) 6ndash10min (F2) 10ndash18min(F3) 18ndash24min (F4) 24ndash28min (F5) 28ndash32min (F6) 32ndash36min (F7) and 36ndash42min (F8) Solvent in each fractionwasremoved under vacuum at 45∘C using a Buchi Rotavapor R-215 reconstituted in water freeze-dried and stored at minus20∘CThe protein content of the hydrolysate and fractions weredetermined using a modified Lowry method [16]

26 Superoxide Anion and Hydroxyl Radicals ScavengingAssays The superoxide scavenging activity was measuredaccording to literature [17]Hydrolysates or peptides fractions(80 120583L 1mgmL) or glutathione (1mgmL positive control)was mixed with 80120583L of 50mM Tris-HCl buffer containing1mM EDTA (pH 83) into a 96-well clear microplate then40 120583L of Pyrogallol in 10mM HCl (15mM) were added toeach well Absorbances were measured at 420 nm every 20 s

Journal of Chemistry 3

for 4min at room temperatureThe reaction rateΔ119860minwasused to calculate the superoxide radical scavenging activity[18] For determination of the hydroxyl radical scavengingactivity 50 120583L of peptide fractions (1mgmL) in potassiumphosphate buffer (075mM pH 74) was transferred into a96-well clear microplate followed by 110-phenanthroline inbuffer (3mM 50 120583L) FeSO

4sdot7H2O in water (3mM 50120583L)

and 003 aqueous H2O2(50 120583L) The assay control con-

tained both phenanthroline and H2O2while the blank only

contained phenanthroline The plate was incubated for 1 h at37∘C 200 rpm Absorbances were then read at 536 nm andused to calculate the scavenging activity [10]

27 Mass Spectrometry The tandemmass spectrometry anal-ysis was performed using the Quebec Proteomics Platformavailable at the Quebec Genomics Center (Sainte-Foy QCCanada) Peptides were identified in F3 one of the mostactive fractions from semiprep HPLC Experiments wereperformed with an Agilent nanoscale capillary liquid chro-matography (nanoLC) coupled to a triple TOF5600plusmassspectrometer (AB Sciex USA) equipped with a nanoelectro-spray ion source in positivemode Peptides were separated onPicoFrit 15 120583m tip BioBasic C18 10 cm times 75 120583mcolumn (NewObjective USA) Mobile phases were 01 formic acid inwater (solvent A) and 01 acetic acid in acetonitrile (solventB) A linear gradient 5 to 80 of solvent B in 60min at300 nLmin was used for separation Spectra were acquiredusing a data-dependent acquisition mode (Analyst softwareversion 16) Each full scan mass spectrum (400 to 1250mz)was followed by collision-induced dissociation (MSMS) ofmultiple charged peaks (2+ to 5+) Dynamic exclusion was setfor a period of 3 s and a tolerance of 01 Da

28 Database Searching and Criteria for Peptide IdentificationMascot generic format (MGF) files containing MSMS peaklists were generated using Protein Pilot version 45 software(ABSciexUSA) Charge state deconvolution anddeisotopingwere not performed All MSMS samples were analyzedusing Mascot (Matrix Science London UK version 241)and X Tandem (The Global ProteomeMachine thegpmorgversion CYCLONE (201012011)) Mascot was set up tosearch the TAX Poeae 147387 20141216 database assumingthat the digestion enzyme is nonspecific X Tandem wasset up to search a subset of the TAX Poeae 147387 20141216database also assuming nonspecificity Mascot and X Tan-dem were searched with a fragment ion mass tolerance of0100Da and a parent ion tolerance of 0100Da Dehydra-tion of the n-terminus glu-gtpyro-Glu of the n-terminusammonia-loss of the n-terminus gln-gtpyro-Glu of the n-terminus deamidation of asparagine and glutamine andoxidation of methionine were specified as variable modifi-cations in X Tandem and Mascot to achieve a False Dis-covery Rate (FDR) less than 10 by the Scaffold Local FDRalgorithm

29 Statistical Analysis All results are presented as mean plusmnstandard deviation from replicates made in triplicate One-way ANOVA was used and performed by SPSS (version 21

2 31Time (h)

0

100

200

300

400

500

600

700

ORA

C (120583

M T

Eg

sam

ple)

1 30 1 501 401 20

Figure 1 Effect of digestion time and enzyme substrate ratio (ES)on the peroxyl radical scavenging activity of oat bran proteinstreated with pepsin from the oxygen radical absorbance capacity(ORAC) assay

2012) Statistical significance was set to 119901 lt 005 Means wereseparated using Fisherrsquos protected least significant difference

3 Results and Discussion

31 Optimization of Extraction Conditions Four concentra-tions of pepsin in combination with three digestion timeswere used to produce twelve hydrolysates from oat branprotein isolates To determine which conditions producedsamples with highest radical scavenging activity hydrolysateswere screened for peroxyl radical scavenging propertiesusing the ORAC assay and results expressed as 120583MTEgof lyophilized hydrolysate (Figure 1) After 1 h hydrolysisproteins treated with the highest pepsin concentration (1 20ES) had higher peroxyl radical activity but the valuedecreased at 2 and 3 h possibly because of further degradationof the active peptides For other ES ratios (1 30ndash1 50)activities increased after 2 h digestion followed by a decreaseat 3 h (Figure 1) Of all the samples hydrolysates producedfrom bran proteins digested for 2 h with pepsin 1 30 ESpossessed the highest peroxyl radical scavenging activity(608 plusmn 17 120583MTEg) A large quality was then produced andsubjected to semipreparativeHPLC seperation Other studieshave shown that the ES ratio and the duration of digestionhad positive effect on ORAC values of hydrolyzed milkalpha-lactalbumin caseinomacropeptide and whey proteinconcentrate [19]

32 Separation by RP-HPLC Enzymatic hydrolysis of foodproteins is often used to produce mixtures of peptideswith stronger antioxidant activities than native proteins Tofurther enhance the activity of digested oat bran proteinsa separation based on hydrophobicity in which moleculesare eluted from the column by increasing the percentage oforganic solvent was chosen because previous investigations


A

E

G

F

D

F

D EC B

ORA

C (120583

M T

Eg

sam

ple)

OPH F2 F3 F4 F5 F6 F7 F8GSHFractions (1mgmL)

00

2000

4000

6000

8000

10000

12000

Figure 2 Oxygen radical absorbance capacity (ORAC) values of oatprotein hydrolysate (OPH) hydrolyzed for 2 h with pepsin 1 30 ESratio and its RP-HPLC fractions (F2ndashF8) GSH glutathione (con-trol) Bars with different letters on the same graph are significantlydifferent in the Fisher LSD test (119899 = 3 119901 lt 005) Data are means plusmnstandard deviations

have demonstrated the relationship between the degree ofhydrophobicity and the antioxidant activity of peptides fromfood like soy and milk [20 21] A C18 column was then usedto separate the pepsin hydrolyzed oat bran proteins into eightdifferent fractions (F1ndashF8) as indicated in the experimentalsection Less hydrophobic molecules have little interactionwith the stationary phase resulting in their rapid elutionwhile more hydrophobic molecules bind strongly to C18 andare therefore eluted later as methanol percentage increasedProtein contents (weightweight) of lyophilized hydrolysatesdetermined by a modified Lowry method were 635 plusmn 03nonfractionated oat protein hydrolysate (OPH) 00 plusmn 06(F1) 40 plusmn 03 (F2) 679 plusmn 06 (F3) 1022 plusmn 29 (F4)976 plusmn 06 (F5) 835 plusmn 09 (F6) 946 plusmn 06 (F7) and950 plusmn 25 (F8) F1 had no protein and was not furtherinvestigated F2 had very low protein content compared toF3ndashF8 This is because salts used in different pH adjustmentsand sugars are highly water soluble and therefore will beeluted early

33 Oxygen Radical Absorbance Capacity (ORAC) Assay ofHPLC Fractions The ability of antioxidant molecules to neu-tralize the peroxyl radical (ROO∙) a common reactive speciespresent in vivo and in food systems is often performed usingthe ORAC assay [22] The peroxyl radical quenching abilityof the hydrolysate and its peptide fractions are shown inFigure 2 ORAC values significantly increased (119901 lt 0005)with elution time from F5 to F8 (4770 plusmn 323 to 8244 plusmn58 120583MTEg) As organic solvent increases in the eluent sodo the degree of hydrophobicity when using a C18 column Itis therefore conceivable that there may be gradual increaseof hydrophobic amino acids from F5 to F8 Although F4had the highest protein content amongst the fractions itdid not possess the highest ORAC value demonstratingthat the peptide structure (ie sequence) was an importantfactor in scavenging radicals as suggested in other studies[7] The presence of amino acids like tryptophan histidinetyrosine or cysteine on the sequence is also important for

B BC C C

B

A

DC

OPH F2 F3 F4 F5 F6 F7 F8GSHFractions

0

20

40

60

80

100

120

O2∙minus

scav

engi

ng (

)

(a)

A

B B B B

CCC C

HO

∙sc

aven

ging

()


02468

101214161820

(b)

Figure 3 Superoxide (a) and hydroxyl (b) radical scavengingactivities of oat protein hydrolysate (OPH) hydrolyzed for 2 h withpepsin 1 30 ES ratio and its RP-HPLC fractions (F2ndashF8) GSHglutathione (control) Bars with different letters on the same graphare significantly different in the Fisher LSD test (119899 = 3 119901 lt 005)Data are means plusmn standard deviations

radical scavenging properties [14] although their contentswere not determined in this study Four of the fractionspossessed significantly higher scavenging power than theunfractionated oat bran protein hydrolysate (5819 120583MTEg)indicating that hydrophobic separation enhanced their per-oxyl radical scavenging activity The highest active fractionF8 (8244 plusmn 58 120583MTEg) was 08-fold the activity of controlglutathione The activity of fractions from this study is lowerthan that of fractions from HPLC separation of hempseedpepsinpancreatin protein hydrolysates [23] Hydrolysis withboth pepsin and pancreatin will result in smaller peptidescompared with pepsin alone

34 Superoxide Anion and Hydroxyl Radical ScavengingActivities Superoxide anion (O

2

∙minus) and hydroxyl radical(HO∙) are generally measured together because in additionto peroxyl radicals both are related to oxidation of foodsand biological molecules In fact many secondary ROS aregenerated after reactions of O

2

∙minus with nonradical molecules[24 25] Data on the O

2

∙minus activity of OPH and its fractionsare shown in Figure 3(a) Fractions F3 F4 and F8 possessedsignificantly higher (119901 lt 005) activities (355 plusmn 65 355 plusmn61 and 344plusmn19 resp) than the whole hydrolysate OPH(226 plusmn 15) The inhibitory activity of F5 F6 or F7 wassimilar to the activity of nonfractionated OPH Fraction F2with just 97 plusmn 10 protein had the least O

2

∙minus activity It


6083185

760396610135253

2571

2637

5033271628102471

2438 2907 4905

(a)

(b)

6515 20 25 30 35 40 45 50 55 60105Time (min)

93e6

11170713 (2+)

7450547 (3+)4476393 (5+)

5590444 (4+)50e515e625e635e645e655e665e675e685e6

Inte

nsity

(cps

)

00

20e3

40e3

60e3

80e3

10e4

12e4

14e4

16e4

18e4

20e4

22e4

Inte

nsity

(cps

)

450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250400mz (Da)

Figure 4 Mass spectrometry analysis of F3 total ions count over 65min (a) TOF MS at 256min (b) Full scan was performed from400 to 1250Da Signals at mz 44763 55904 74505 and 111707 are quintuple quadruple triple and double peaks of the same peptide(YRISRQEARNLKNNRGQE) Other peaks are for peptides with the same retention time

appears that RP-HPLC fractionation increased the O2

∙minus radi-cal activity of some fractions compared to the unfractionatedhydrolysates (OPH) None of the fraction was as active as thecontrol GSH In contrast with the peroxyl radical scavengingactivity therewas no correction between theO

2

∙minus activity andthe organic solvent content of the eluent suggesting that thesequence of peptides in the fractions played a more impor-tant role than hydrophobicity as reported in other studies[26 27]

Hydroxyl radical (HO∙) can be generated from O2

∙minus andis considered the most damaging species in the oxidativestress process due to its high oxidizing power [27] Asdisplayed in Figure 3(b) fractions F3 F6 F7 and F8 havesignificantly higher HO∙ inhibition percentages compared toOPH (90 plusmn 03) but they were not as active as GSH (180 plusmn07) Fractions F2 F4 andF5 had activities comparable (119901 gt005) to that of OPH Fraction F3 was obtained with an eluenthigh in water (60) and mass spectrometry analysis showedthat some of its peptides contained polar amino acids such astyrosine aspartic acid glutamic acid and serine (Table 1) thatmay have enhanced its HO∙ activity through metal-chelatingas reported in the literature for other peptides [28] This isconsistent with a previous study which found that later elutedHPLC (C18 column) peptide fractions were not the ones withhigher HO∙ scavenging activities [23]

The O2

∙minus activity of fractions from this study (226ndash355) is similar to that of pea peptide ultrafiltered fractions(25ndash32) [17] but lower than the activity of some fractionsfromHPLC separation of digested salmon proteins (27ndash56)

[23] The HO∙ scavenging activity of the hydrolysate and itsfractions are within the range (14ndash37) of literature data foroat bran proteins extracted with twelve different conditionsand hydrolyzed with alcalase [14] Chickpea peptide fractionshad 38 to 81 HO∙ activities [10] however the separationwasperformed based on size and cannot directly be comparedto the present data because of differences in fractionationtechniques

35 Identification of Peptides The TripleTOF 5600 plusis a hybrid quadrupole time-of-flight hybrid instrumentthat offers the opportunity to conduct multiple-charge ionscanning thereby facilitating the identification of relevantfunctional and biological peptides in digested proteinsor fractions The system was used here to characterizepeptides in one of the fractions (ie F3) with higher O

2

∙minus

and HO∙ radical scavenging activities Multiple charge (2+to 5+) scanning was performed over 65min on eluatesfrom the nanoLC system MSMS peak lists were analyzedusing Mascottrade and X Tandem as described in Section 2Search results from Mascot and X Tandem were statisticallyinterpreted by Scaffold a software used to validate MSMSdataThe software peptide prophet algorithm converts searchengine scores into combined probabilities that improve thesensitivity and the accuracy of the identification [29] In thetotal ion count chromatogram of F3 (Figure 4(a)) peptidepeaks appeared between 23 and 31min The MS scan at257min (Figure 4(b)) showed double triple quadruple andquintuple ions of the same peptide atmz 111707 (2+) 74505


Table 1 List of peptides identified in F3 a fraction with higher superoxide anion and hydroxyl radical scavenging activities from HPLCseparation of pepsin digested oat brans proteins on a C18 column Scaffold software was used to validate MSMS data Peptide identificationswere accepted if they could be established at greater than 95 probability

Peptide sequence Observed mass (charge) Actual mass Oat protein ID UniProt databaseRALPVDVL 44177 (+2) 88153 P12615 P14812 P27919SPYWNINA 48273 (+2) 96344 Q38781 P12615 P14812PQYHNAPGLV 54828 (+2) 109455 O49257 O49258 P12615 P14812HGQNFPILNL 57681 (+2) 115161 O49257 O49258NSKNFPILNI 58033 (+2) 115864 Q38779 Q38780VYILQGRGFTG 60583 (+2) 120965 Q38779 Q38780 P12615 P14812VYLLQGRGFTG 60583 (+2) 120965 O49257VIRRVIEPQGLL 46496 (+3) 139186 O49257 O49258 P12615 P14812IRRVIEPQGLLL 46963 (+3) 140588 O49257 O49258 P12615 P14812QQVFQPQQQAQF 73886 (+2) 147571 I4EP64 I4EP88 F2Q9W3 F4MJY2SVIRRVIEPQGLL 49397 (+3) 147890 O49257 P12615 P14812PAGIVHWGYNDGDAPVVA 91944 (+2) 183687 O49257 O49258QAAQRIQSQKEQRGEI 61833 (+3) 185196 O49257 O49258KTNPNSMVSHIAGKSSIL 62867 (+3) 188299 O49257 O49258 Q38780 P27919IQGHARVQVVNNNGQTVF 66102 (+3) 198003 O49257 Q38779 Q38780IQGRARVQVVNNHGQTVF 50653 (+4) 202209 Q38781 P12615 P14812DVNNNANQLEPRQKEFL 67701 (+3) 202800 O49257 O49258 P12615 P14812 Q38780AEGQSQSQNLKDEHQRVH 52350 (+4) 208999 P14812YRISRQESQNLKNNRGEE 55603 (+4) 222010 P12615 Q38781YRISRQEARNLKNNRGEE 111708 (+2) 223214 Q38779 Q38780YRISRQEARNLKNNRGQE 55904 (+4) 223215 O49258 P27919YRISRQEARNLKNNRGQES 58056 (+4) 231819 O49258 P27919YRISRQEARNLKNNRGQESG 59481 (+4) 237522 O49258 P27919NAYRISRQESQNLKNNRGEE 80273 (+3) 240518 P12615 Q38781NAYRISRQEARNLKNNRGQE 60531 (+4) 241723 O49258 P27919NAYRISRQEARNLKNNRGEE 60531 (+4) 241723 Q38779 Q38780YRISRQEARNLKNNRGQESGV 61958 (+4) 247428 O49258 P27919ANAYRISRQEARNLKNNRGQE 62307 (+4) 248826 O49258 P27919ANAYRISRQEARNLKNNRGEE 62307 (+4) 248826 Q38779 Q38780NAYRISRQEARNLKNNRGQES 62683 (+4) 250327 O49258 P27919NAYRISRQEARNLKNNRGQESG 85444 (+3) 256030 O49258 P27919ANAYRISRQEARNLKNNRGQES 64459 (+4) 257431 O49258 P27919RQNIENPKRADTYNPRAGRITH 65260 (+4) 260635 Q38781 P14812 P12615 O49257ANAYRISRQEARNLKNNRGQESG 65884 (+4) 263133 O49258 P27919ARQNIENPKRADTYNPRAGRITH 53649 (+5) 267739 Q38781 P14812 P12615RALPIDVL 44878 (+2) 89555 Q38779 Q38780IRRVIEPQGLL 43194 (+3) 129279 O49257 AVESAQQQFQPFDQAQ 68282 (+2) 136362 P14812 O49258LIVPQHY 43525 (+2) 86848 O49258LLLPQYH 44226 (+2) 88250 O49257 O49258 P14812 P12615LIVPQHF 42725 (+2) 85249 Q38779 Q38780HGQNFPIL 46325 (+2) 92448 O49257 AVESA O49258SPFWNINA 47473 (+2) 94745 O49257 O49258 Q38780 Q38779Q38779 11S globulin Q38780 11S globulin Q38781 oat storage 12S globulin O49257 12S globulin O49258 12S globulin P12615 12S seed storage globulin 1P14812 12S seed storage globulin 2 P27919 avenin I4EP88 avenin I4EP64 avenin F2Q9W3 avenin protein and F4MJY2 avenin protein


0800In

tens

ity (c

ps)

100 300 700 900 1100500 1500 17001300

mz (Da)

11509

13608

14806

(Y1

)22517

30315

(B2

-NH

3)

32017

(B2

)33410

(Y3

)43326

(B3

)46725

52029

(B4

)55904

60427

(Y5

)62932

71831

(Y6

)

84641

(Y7

)91651

(B7

-NH

3)

95953

(Y8

)

107348

(Y9

)116046

(B9

)

12747

(B10

)

138779

(B11

)

15703516e3

24e332e340e348e3

Figure 5 TOF product of the peak atmz 55904The sequence waselucidated as YRISRQEARNLKNNRGQE The main peaks B- andY-ions are indicated in parenthesis TOF products of peaks at mz111708 (2+) and 44743 (5+) 745 (3+) afforded the same sequence

(3+) 55904 (4+) and 44763 (5+) respectively Productsspectra (MSMS) of each of these ions and database searchesall correspond to the sequence YRISRQEARNLKNNRGQE(Tyr-Arg-Ile-Ser-Arg-Gln-Glu-Ala-Arg-Asn-Leu-Lys-Asn-Asn-Arg-Gly-Gln-Glu) with 100 Scaffold probability Theproduct ion spectrum (MSMS) of the quadruple chargedpeak at mz 55904 can be seen in Figure 5 The list of allidentified peptides in this study is shown in Table 1 Theyare derived from 12S globulin 11S globulin 12S seed storageglobulin avenin and gliadin like avenin proteins Most ofthe peptides are from 12S globulin with minor variation insequences Amino acids like tyrosine cysteine methionineand arginine present in many identified peptides mayhave contributed to the activity of fraction F3 as they areknown to contribute to the antioxidant activity of peptides[30] The contribution of these amino acids is howevernot known at this time because they were not individuallytested and also because MSMS data are only quantitativePeptides reported here are different from Tyr-His-Asn-Ala-Pro-Gly-Leu-Val-Tyr-Ile-Leu Asp-Val-Asn-Asn-Asn-Ala-Asn-Gln-Leu-Glu-Pro-Arg Gly-Gln-Thr-Val-Phe-Asn-Asp-Arg-Leu-Arg-Gln-Gly-Gln-Leu-Leu and Val-Val-Asn-Asn-Asn-Gly-Gln-Thr-Val-Phe-Asn-Asp-Arg-Leu-Arg-Gln-Gly-Gln-Leu-Leu recently identified in oats [14] Two of thepeptides Ser-Pro-Phe-Trp-Asn-Ile-Asn-Ala (SPFWNINA)and Gln-Gln-Pro-Ile-Pro-Gln-Gln-Pro-Gln (QQPIPQQPQ)have been predicted as potential antihypertensive andceliac toxic from barley hordein and wheat omega-gliadinrespectively [31] Other peptides present in the literaturefrom oats are mainly made of two or three amino acids[32]

4 Conclusion

Results from this study showed that optimum conditions toproduce digested proteins with the highest peroxyl radicalactivity were treatment with pepsin 1 30 ES ration followedby 2 h incubation RP-HPLC fractionation of hydrolysedproteins under the above conditions enhanced peroxylsuperoxide anion and hydroxyl radical scavenging activitiesof some fractions New peptides were identified by tandem

mass spectrometry In future studies chemometric methodswill be used to identify those peptideswith potential bioactiveactivity for synthesis and evaluation of their capacity toprevent oxidation in vitro and in cell culture models

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This work was supported by a grant from National Scienceand Engineering Research Council of Canada (Grant no371908) The authors thank Mr Daniel Defoy from the Que-bec Proteomics Platform available at the Quebec GenomicsCenter for technical assistance

References

[1] S Kubow ldquoRoutes of formation and toxic consequences of lipidoxidation products in foodsrdquo Free Radical Biology amp Medicinevol 12 no 1 pp 63ndash81 1992

[2] AM Rizzo P Berselli S Zava et al ldquoEndogenous antioxidantsand radical scavengersrdquoAdvances in ExperimentalMedicine andBiology vol 698 pp 52ndash67 2010

[3] A G P Samaranayaka and E C Y Li-Chan ldquoFood-derivedpeptidic antioxidants a review of their production assessmentand potential applicationsrdquo Journal of Functional Foods vol 3no 4 pp 229ndash254 2011

[4] M Valko D Leibfritz J Moncol M T D Cronin M Mazurand J Telser ldquoFree radicals and antioxidants in normal physi-ological functions and human diseaserdquo International Journal ofBiochemistry amp Cell Biology vol 39 no 1 pp 44ndash84 2007

[5] D Dziki R Rozyło U Gawlik-Dziki and M Swieca ldquoCurrenttrends in the enhancement of antioxidant activity of wheatbread by the addition of plant materials rich in phenoliccompoundsrdquoTrends in Food Science and Technology vol 40 no1 pp 48ndash61 2014

[6] DM Peterson ldquoOat antioxidantsrdquo Journal of Cereal Science vol33 no 2 pp 115ndash129 2001

[7] C C Udenigwe and R E Aluko ldquoFood protein-derivedbioactive peptides production processing and potential healthbenefitsrdquo Journal of Food Science vol 77 no 1 pp R11ndashR24 2012

[8] S K Kim IWijesekara E Y Park YMatsumura YNakamuraand S Kenji ldquoBioactive proteins and peptidesrdquo in BioactiveFood Proteins and Peptides Applications in Human Health NS Hettiarachchy S Sato M R Marshall and A Kannan Edspp 97ndash116 CRC Press Boca Raton Fla USA 2011

[9] L Zhu C Jie X Tang and Y L Xiong ldquoReducing radicalscavenging and chelation properties of in vitro digests ofalcalase-treated zein hydrolysaterdquo Journal of Agricultural andFood Chemistry vol 56 no 8 pp 2714ndash2721 2008

[10] Y H Li B Jiang T Zhang W M Mu and J Liu ldquoAntioxi-dant and free radical-scavenging activities of chickpea proteinhydrolysate (CPH)rdquo Food Chemistry vol 106 no 2 pp 444ndash450 2008

[11] M C Garcıa P Puchalska C Esteve and M L MarinaldquoVegetable foods a cheap source of proteins and peptideswith antihypertensive antioxidant and other less occurrencebioactivitiesrdquo Talanta vol 106 pp 328ndash349 2013


[12] T Madhujith and F Shahidi ldquoAntioxidative and antiprolif-erative properties of selected barley (Hordeum vulgarae L)cultivars and their potential for inhibition of Low-DensityLipoprotein (LDL) cholesterol oxidationrdquo Journal of Agricul-tural and Food Chemistry vol 55 no 13 pp 5018ndash5024 2007

[13] A Tsopmo A Cooper and S Jodayree ldquoEnzymatic hydrolysisof oat flour protein isolates to enhance antioxidative propertiesrdquoAdvance Journal of Food Science and Technology vol 4 no 4 pp206ndash212 2010

[14] S Jodayree J C Smith and A Tsopmo ldquoUse of carbohydraseto enhance protein extraction efficiency and antioxidativeproperties of oat bran protein hydrolysatesrdquo Food ResearchInternational vol 46 no 1 pp 69ndash75 2012

[15] D Huang B Ou M Hampsch-Woodill J A Flanagan andE K Deemer ldquoDevelopment and validation of oxygen radicalabsorbance capacity assay for lipophilic antioxidants using ran-domly methylated 120573-cyclodextrin as the solubility enhancerrdquoJournal of Agricultural and Food Chemistry vol 50 no 7 pp1815ndash1821 2002

[16] M A K Markwell S M Haas L L Bieber and N ETolbert ldquoA modification of the Lowry procedure to simplifyprotein determination in membrane and lipoprotein samplesrdquoAnalytical Biochemistry vol 87 no 1 pp 206ndash210 1978

[17] T L Pownall C C Udenigwe and R E Aluko ldquoAmino acidcomposition and antioxidant properties of pea seed (Pisumsativum L) Enzymatic protein hydrolysate fractionsrdquo Journal ofAgricultural and Food Chemistry vol 58 no 8 pp 4712ndash47182010

[18] R Alrahmany and A Tsopmo ldquoRole of carbohydrases on therelease of reducing sugar total phenolics and on antioxidantproperties of oat branrdquo Food Chemistry vol 132 no 1 pp 413ndash418 2012

[19] T G Tavares M M Contreras M Amorim et al ldquoOptimisa-tion by response surface methodology of degree of hydrolysisand antioxidant and ACE-inhibitory activities of whey pro-tein hydrolysates obtained with cardoon extractrdquo InternationalDairy Journal vol 21 no 12 pp 926ndash933 2011

[20] H-M Chen K Muramoto and F Yamauchi ldquoStructuralanalysis of antioxidative peptides from soybean 120573-conglycininrdquoJournal of Agricultural and Food Chemistry vol 43 no 3 pp574ndash578 1995

[21] B Hernandez-Ledesma A Davalos B Bartolome and LAmigo ldquoPreparation of antioxidant enzymatic hydrolysatesfrom 120572-lactalbumin and 120573-lactoglobulin Identification ofactive peptides by HPLC-MSMSrdquo Journal of Agricultural andFood Chemistry vol 53 no 3 pp 588ndash593 2005

[22] R L Prior X Wu and K Schaich ldquoStandardized methodsfor the determination of antioxidant capacity and phenolicsin foods and dietary supplementsrdquo Journal of Agricultural andFood Chemistry vol 53 no 10 pp 4290ndash4302 2005

[23] A T Girgih C C Udenigwe and R E Aluko ldquoReverse-phase HPLC separation of hemp seed (Cannabis sativa L)protein hydrolysate produced peptide fractions with enhancedantioxidant capacityrdquo Plant Foods for Human Nutrition vol 68no 1 pp 39ndash46 2013

[24] L Magnani E M Gaydou and J C Hubaud ldquoSpectrophoto-metric measurement of antioxidant properties of flavones andflavonols against superoxide anionrdquo Analytica Chimica Actavol 411 no 1-2 pp 209ndash216 2000

[25] C F Ajibola J B Fashakin T N Fagbemi and R E AlukoldquoEffect of peptide size on antioxidant properties of Africanyam bean seed (Sphenostylis stenocarpa) protein hydrolysate

fractionsrdquo International Journal of Molecular Sciences vol 12no 10 pp 6685ndash6702 2011

[26] X Li ldquoImproved pyrogallol autoxidation method a reliableand cheap superoxide-scavenging assay suitable for all antioxi-dantsrdquo Journal of Agricultural and Food Chemistry vol 60 no25 pp 6418ndash6424 2012

[27] M Laguerre J Lecomte and P Villeneuve ldquoEvaluation of theability of antioxidants to counteract lipid oxidation existingmethods new trends and challengesrdquoProgress in Lipid Researchvol 46 no 5 pp 244ndash282 2007

[28] K M Chan and E A Decker ldquoEndogenous skeletal muscleantioxidantsrdquo Critical Reviews in Food Science and Nutritionvol 34 no 4 pp 403ndash426 1994

[29] B C Searle ldquoScaffold a bioinformatic tool for validatingMSMS-based proteomic studiesrdquo Proteomics vol 10 no 6 pp1265ndash1269 2010

[30] A McDermott Bioactive Peptides Springer Science amp BusinessMedia New York NY USA 2009

[31] A Cavazos and E Gonzalez de Mejia ldquoIdentification of bioac-tive peptides from cereal storage proteins and their potentialrole in prevention of chronic diseasesrdquo Comprehensive Reviewsin Food Science and Food Safety vol 12 no 4 pp 364ndash380 2013

[32] I W Y Cheung S Nakayama M N K Hsu A G PSamaranayaka and E C Y Li-Chan ldquoAngiotensin-I convertingenzyme inhibitory activity of hydrolysates from oat (Avenasativa) proteins by in silico and in vitro analysesrdquo Journal ofAgricultural and Food Chemistry vol 57 no 19 pp 9234ndash92422009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


less attention Oat proteins digested with alcalase and trypsinwere demonstrated to possess antioxidant activities [13 14]However the action of other proteases or the activity offractions from chromatography separation remains to beinvestigated The objective of this study was to optimizethe conditions for pepsin hydrolysis of oat bran proteinsperformHPLC separation determine the antioxidant activityof fractions and identify peptides

2 Materials and Methods

21 Chemicals Medium oat bran (id 112-001) was sup-plied by Richardson Milling (Portage La Prairie ManitobaMB) Viscozyme L (100 Fungal Beta Glucanase (FBG)g)pepsin from porcine gastric mucosa (367 unitsmg solid) L-glutathione Tris(hydroxymethyl)aminomethane hydrochlo-ride (Tris-HCl) pyrogallol 110-phenanthroline iron sulfatepentahydrate (FeSO

4sdot7H2O) sodium carbonate (Na

2CO3)

sodium hydroxide sodium tartrate cupric sulfate pentahy-drate (CuSO

4sdot5H2O) Folin-Ciocalteu reagent and bovine

serum albumin (BSA) were obtained from Sigma Aldrich(Oakville ON) Hydrogen peroxide (H

2O2) 6-hydroxy-2

578-tetramethylchromane-2-carboxylic acid (Trolox) fluo-rescein methanol mono- and di-basic potassium phosphateethylenediaminetetraacetic acid and sodium dodecyl sulfatewere purchased from Fisher Scientific (Nepean ON) 221015840-Azobis(2-amidinopropane) dihydrochloride (AAPH) wasfromWakoChemicals Spectrophotometric and fluorometricmeasurements were performed on the BioTek Epochtrade UV-Vis and Biotek FLx 800 microplate reader respectively bothcontrolled by Gen5trade data analysis software Incubations weredone on a MaxQtrade 5000 shaker model (Fisher ScientificNepean ON)

22 Protein Extraction The extraction procedure was per-formed based on a previousmethod [14] Samples of oat brans(4times 100 g) were defatted by stirring in hexane (1 3 wv) for 1 hat room temperature filtration on cheesecloth and overnightdrying under fume hoods Deionized water was added tothe defatted oat bran at the ratio 1 10 (wv) and adjusted topH 45 Viscozyme L and 3 FBGg of defatted bran was usedto breakdown carbohydrates and reduced the viscosity Themixture was then incubated at 45∘C 150 rpm and 15 h Atthe end of the incubation the pH was adjusted to 95 using2M NaOH solution and further incubated for 15 h in orderto carry out the alkaline extraction of proteins After coolingcentrifugation was performed at 2500 g for 20min at 4∘CSupernatants containing proteins were adjusted to pH 40and then centrifuged (10000 g 40min 4∘C) to obtain proteinisolates (ie pellets) that were suspended in pH 70 waterbefore being freeze-dried

23 Hydrolysis with Pepsin Freeze-dried oat bran proteinisolates (500mg each) were transferred into twelve differenttubes and rehydrated with water (1 12 wv) The pH wasadjusted to 40 with 1M HCl followed by addition of pepsinat 1 20 1 30 1 40 and 1 50 (ww) enzyme-substrate (ES)ratios Mixtures were homogenized adjusted to pH 20

and then incubated at 37∘C 150 rpm Four samples wereremoved at 1 2 and 3 h respectively At the end of eachincubation time the enzyme was inactivated by adjusting thepH to 70 with 1M NaHCO

3 Supernatants were collected

after centrifugation at 2500 g for 15min to remove denaturedpepsin and nondigested proteins The hydrolysates werefreeze-dried and used for oxygen radical absorbance capacity(ORAC) assay For HPLC separation oat proteins (3 times 100 g)were digested with pepsin (1 30 ES) for 2 h The hydrolysateproduced under these conditions had the highest ORACvalue

24 Oxygen Radical Absorbance Capacity (ORAC) Assay Theassay was performed using a Bio-Tek FLx800 instrumentaccording to literature [15] Reagents standards digestedproteinsHPLC fractions (01mgmL) and assay control(GSH 01mgmL) were prepared with potassium phosphatebuffer (75mM pH 74) Five concentrations of Trolox (625ndash100 120583M) were used to obtain the standard curve To performthe assay fluorescein (120120583L 0080 120583M) was transferred into96-well microplate followed by addition of 20 120583L of samplesstandards or buffer (blank) The plate was incubated for20min at 37∘C AAPH (15mM 60 120583L) was then added toeach well Data (triplicates) were collected every minute over50min and used to calculate ORAC values expressed as 120583MTrolox equivalent (TE) per gram of lyophilized hydrolysate offraction

25 Separation by RP-HPLC Digested bran proteins (2 h1 30 ES) with the most peroxyl radical scavenging activitywere selected for HPLC separation The system consisted of1525 binary pump 2998 photodiode array detector (set at220 nm) 2707 autosampler maintained at 8∘C and fractioncollector III from Waters (Montreal QC Canada) Thefreeze-dried oat bran protein hydrolysate was dissolved in01 acetic acid (100mgmL) and filtered through 045120583mnylonmembraneThe columnwasWaters Prep XBridge BEHC18 130 A 10 120583m 19 times 150mm and the injection volumewas 2mL A linear gradient (5 to 90 B) of 01 acetic acidin water (A) or in methanol (B) over 45min was used forseparation at a flow rate of 4mLmin Fractions (8mL each)were collected and pooled into eight fractions according tothe chromatogram 0ndash6min (F1) 6ndash10min (F2) 10ndash18min(F3) 18ndash24min (F4) 24ndash28min (F5) 28ndash32min (F6) 32ndash36min (F7) and 36ndash42min (F8) Solvent in each fractionwasremoved under vacuum at 45∘C using a Buchi Rotavapor R-215 reconstituted in water freeze-dried and stored at minus20∘CThe protein content of the hydrolysate and fractions weredetermined using a modified Lowry method [16]

26 Superoxide Anion and Hydroxyl Radicals ScavengingAssays The superoxide scavenging activity was measuredaccording to literature [17]Hydrolysates or peptides fractions(80 120583L 1mgmL) or glutathione (1mgmL positive control)was mixed with 80120583L of 50mM Tris-HCl buffer containing1mM EDTA (pH 83) into a 96-well clear microplate then40 120583L of Pyrogallol in 10mM HCl (15mM) were added toeach well Absorbances were measured at 420 nm every 20 s










2 31Time (h)

0

100

200

300

400

500

600

700

ORA

C (120583

M T

Eg

sam

ple)

1 30 1 501 401 20







A

E

G

F

D

F

D EC B

ORA

C (120583

M T

Eg

sam

ple)


00

2000

4000

6000

8000

10000

12000




B BC C C

B

A

DC


0

20

40

60

80

100

120

O2∙minus

scav

engi

ng (

)

(a)

A

B B B B

CCC C

HO

∙sc

aven

ging

()


02468

101214161820

(b)




2


2


2


2



6083185

760396610135253

2571

2637

5033271628102471

2438 2907 4905

(a)

(b)

6515 20 25 30 35 40 45 50 55 60105Time (min)

93e6

11170713 (2+)

7450547 (3+)4476393 (5+)

5590444 (4+)50e515e625e635e645e655e665e675e685e6

Inte

nsity

(cps

)

00

20e3

40e3

60e3

80e3

10e4

12e4

14e4

16e4

18e4

20e4

22e4

Inte

nsity

(cps

)

450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250400mz (Da)




2




The O2




2

∙minus






0800In

tens

ity (c

ps)

100 300 700 900 1100500 1500 17001300

mz (Da)

11509

13608

14806

(Y1

)22517

30315

(B2

-NH

3)

32017

(B2

)33410

(Y3

)43326

(B3

)46725

52029

(B4

)55904

60427

(Y5

)62932

71831

(Y6

)

84641

(Y7

)91651

(B7

-NH

3)

95953

(Y8

)

107348

(Y9

)116046

(B9

)

12747

(B10

)

138779

(B11

)

15703516e3

24e332e340e348e3



4 Conclusion





Acknowledgments


References












































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










2 31Time (h)

0

100

200

300

400

500

600

700

ORA

C (120583

M T

Eg

sam

ple)

1 30 1 501 401 20







A

E

G

F

D

F

D EC B

ORA

C (120583

M T

Eg

sam

ple)


00

2000

4000

6000

8000

10000

12000




B BC C C

B

A

DC


0

20

40

60

80

100

120

O2∙minus

scav

engi

ng (

)

(a)

A

B B B B

CCC C

HO

∙sc

aven

ging

()


02468

101214161820

(b)




2


2


2


2



6083185

760396610135253

2571

2637

5033271628102471

2438 2907 4905

(a)

(b)

6515 20 25 30 35 40 45 50 55 60105Time (min)

93e6

11170713 (2+)

7450547 (3+)4476393 (5+)

5590444 (4+)50e515e625e635e645e655e665e675e685e6

Inte

nsity

(cps

)

00

20e3

40e3

60e3

80e3

10e4

12e4

14e4

16e4

18e4

20e4

22e4

Inte

nsity

(cps

)

450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250400mz (Da)




2




The O2




2

∙minus






0800In

tens

ity (c

ps)

100 300 700 900 1100500 1500 17001300

mz (Da)

11509

13608

14806

(Y1

)22517

30315

(B2

-NH

3)

32017

(B2

)33410

(Y3

)43326

(B3

)46725

52029

(B4

)55904

60427

(Y5

)62932

71831

(Y6

)

84641

(Y7

)91651

(B7

-NH

3)

95953

(Y8

)

107348

(Y9

)116046

(B9

)

12747

(B10

)

138779

(B11

)

15703516e3

24e332e340e348e3



4 Conclusion





Acknowledgments


References












































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


A

E

G

F

D

F

D EC B

ORA

C (120583

M T

Eg

sam

ple)


00

2000

4000

6000

8000

10000

12000




B BC C C

B

A

DC


0

20

40

60

80

100

120

O2∙minus

scav

engi

ng (

)

(a)

A

B B B B

CCC C

HO

∙sc

aven

ging

()


02468

101214161820

(b)




2


2


2


2



6083185

760396610135253

2571

2637

5033271628102471

2438 2907 4905

(a)

(b)

6515 20 25 30 35 40 45 50 55 60105Time (min)

93e6

11170713 (2+)

7450547 (3+)4476393 (5+)

5590444 (4+)50e515e625e635e645e655e665e675e685e6

Inte

nsity

(cps

)

00

20e3

40e3

60e3

80e3

10e4

12e4

14e4

16e4

18e4

20e4

22e4

Inte

nsity

(cps

)

450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250400mz (Da)




2




The O2




2

∙minus






0800In

tens

ity (c

ps)

100 300 700 900 1100500 1500 17001300

mz (Da)

11509

13608

14806

(Y1

)22517

30315

(B2

-NH

3)

32017

(B2

)33410

(Y3

)43326

(B3

)46725

52029

(B4

)55904

60427

(Y5

)62932

71831

(Y6

)

84641

(Y7

)91651

(B7

-NH

3)

95953

(Y8

)

107348

(Y9

)116046

(B9

)

12747

(B10

)

138779

(B11

)

15703516e3

24e332e340e348e3



4 Conclusion





Acknowledgments


References












































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


6083185

760396610135253

2571

2637

5033271628102471

2438 2907 4905

(a)

(b)

6515 20 25 30 35 40 45 50 55 60105Time (min)

93e6

11170713 (2+)

7450547 (3+)4476393 (5+)

5590444 (4+)50e515e625e635e645e655e665e675e685e6

Inte

nsity

(cps

)

00

20e3

40e3

60e3

80e3

10e4

12e4

14e4

16e4

18e4

20e4

22e4

Inte

nsity

(cps

)

450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250400mz (Da)




2




The O2




2

∙minus






0800In

tens

ity (c

ps)

100 300 700 900 1100500 1500 17001300

mz (Da)

11509

13608

14806

(Y1

)22517

30315

(B2

-NH

3)

32017

(B2

)33410

(Y3

)43326

(B3

)46725

52029

(B4

)55904

60427

(Y5

)62932

71831

(Y6

)

84641

(Y7

)91651

(B7

-NH

3)

95953

(Y8

)

107348

(Y9

)116046

(B9

)

12747

(B10

)

138779

(B11

)

15703516e3

24e332e340e348e3



4 Conclusion





Acknowledgments


References












































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





0800In

tens

ity (c

ps)

100 300 700 900 1100500 1500 17001300

mz (Da)

11509

13608

14806

(Y1

)22517

30315

(B2

-NH

3)

32017

(B2

)33410

(Y3

)43326

(B3

)46725

52029

(B4

)55904

60427

(Y5

)62932

71831

(Y6

)

84641

(Y7

)91651

(B7

-NH

3)

95953

(Y8

)

107348

(Y9

)116046

(B9

)

12747

(B10

)

138779

(B11

)

15703516e3

24e332e340e348e3



4 Conclusion





Acknowledgments


References












































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


0800In

tens

ity (c

ps)

100 300 700 900 1100500 1500 17001300

mz (Da)

11509

13608

14806

(Y1

)22517

30315

(B2

-NH

3)

32017

(B2

)33410

(Y3

)43326

(B3

)46725

52029

(B4

)55904

60427

(Y5

)62932

71831

(Y6

)

84641

(Y7

)91651

(B7

-NH

3)

95953

(Y8

)

107348

(Y9

)116046

(B9

)

12747

(B10

)

138779

(B11

)

15703516e3

24e332e340e348e3



4 Conclusion





Acknowledgments


References












































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

Documents

Research Article Pepsin Digested Oat Bran Proteins ...downloads.hindawi.com/journals/jchem/2016/8216378.pdfResearch Article Pepsin Digested Oat Bran Proteins: Separation, Antioxidant