Food Sci Nutr. 2017;1–9.  | 1www.foodscience-nutrition.com

Received:24February2017  |  Accepted:12May2017DOI: 10.1002/fsn3.493

O R I G I N A L R E S E A R C H

Oxidative changes in lipids, proteins, and antioxidants in yogurt during the shelf life

Anna Citta1 | Alessandra Folda1 | Valeria Scalcon1 | Guido Scutari1 |  Alberto Bindoli2 | Marco Bellamio3 | Emiliano Feller3 | Maria Pia Rigobello1

ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium,providedtheoriginalworkisproperlycited.©2017TheAuthors.Food Science & NutritionpublishedbyWileyPeriodicals,Inc.

1DepartmentofBiomedicalSciences, UniversityofPadova,Padova,Italy2InstituteofNeuroscience(CNR),Padova,Italy3CentraledelLattediVicenzas.p.a.,Vicenza,Italy

CorrespondenceMariaPiaRigobello,DepartmentofBiomedicalSciences,UniversityofPadova,Padova,Italy.Email:mariapia.rigobello@unipd.it

Funding informationThisworkwassupportedbyFSE2013(RegioneVeneto)2105/1/8/1686/2012andFSE2014(RegioneVeneto)2105/201/12/1148/2013.

AbstractOxidationprocessesinmilkandyogurtduringtheshelflifecanresultinanalterationofproteinand lipidconstituents.Therefore, theantioxidantpropertiesofyogurt instandardconditionsofpreservationwereevaluated.Totalphenols,freeradicalscav-engeractivity,degreeoflipidperoxidation,andproteinoxidationweredeterminedinplainandskimyogurtswithorwithoutfruitpuree.Afterproduction,plain,skim,plainberries,andskimberriesyogurtswerecomparedduringtheshelflifeupto9weeks.Alltypesofyogurtsrevealedabasalantioxidantactivitythatwashigherwhenafruitpuree was present but gradually decreased during the shelf life. However, after5–8weeks,antioxidantactivityincreasedagain.Bothinplainandberriesyogurtslipidperoxidation increased until the seventh week of shelf life and after decreased,whereasproteinoxidationofallyogurtswassimilareitherintheabsenceorpresenceofberriesand increasedduringshelf life.During theshelf life,adifferentbehaviorbetween lipidandproteinoxidation takesplaceand thepresenceofberriesdeter-minesaprotectiononlyagainstlipidperoxidation.

K E Y W O R D S

antioxidants,lipidperoxidation,proteinoxidation,shelflife,yogurt

1  | INTRODUCTION

Freeradicalsandotherreactiveoxygenspecies(ROS)maycauseto living and food systems an oxidative damage that, however,can be prevented by several types of antioxidants (Halliwell &Gutteridge,2015;Gülçin,2012).Forinstance,inthehumandiet,fruitsarerelevantcomponentsandprovidenutrientssuchascar-bohydrates,minerals,andvitaminstogetherwithphytochemicalswhich include polyphenols and carotenoids, all endowed withpotent antioxidantproperties (Jacobetal., 2012). In addition totheantioxidantsofendogenousorexogenousorigin,acontribu-tiontotheantioxidantdefenseisalsogivenbyproteins,peptides,andaminoacids(Power,Jakeman,&Fitzgerald,2013;Sarmadi&

Ismail, 2010). For instance, the tripeptide glutathione is awell-knownantioxidantactingbothdirectlyandasasubstrateofglu-tathioneperoxidasewhichisabletoconverthydrogenperoxidetowater.Thedipeptidecarnosine (β-alanyl-L-histidine),particularlyabundant in skeletalmuscle andbrain,may act as a free radicalscavengerandmetalchelator(Decker,Livisay,&Zhou,2000;Bayeetal.,2016).

Milk and dairy products, basic foods for human nutrition, canalsocontributetothebodydefenseagainstoxidants.However,oxi-dationprocessesinmilkcanresultinsharpoff-flavorsandadeclineof its nutritional properties. Consequently, the oxidative stabilityofmilkanddairyproductshasgreat importanceespeciallyconsid-ering their shelf life.Theantioxidantactivity isdue to thenatural

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antioxidantspresentinmilk(Lindmark-Månsson&Akesson,2000);and is in part depending from the food supply to herbivores. Forinstance, phenolic compounds, products of the secondarymetab-olism in plants, are effective natural antioxidants and are presentin noticeable amounts in the ruminantmilk,mostly deriving fromthe feeding (O’Connell & Fox, 2001). In addition, both fermentedmilkandyogurtcontainbioactivepeptides,formedbyhydrolysisofmilk proteins during the fermentation process, and also endowedwithantioxidantactivity(Poweretal.,2013;Pihlanto,2006;Aloğlu&Oner,2001).However, there is still a lackofknowledgeon theantioxidantcapacityofdairyproductssuchasprobioticyogurtscon-tainingdifferenttypesoffruits.Inthiscase,theantioxidantpowerlargely depends on the presence of added fruit puree containingvarious bioactive compounds such as tocopherols, carotenoids,ascorbate,andespeciallyphenoliccompoundswhicharegoodcon-tributors to the total antioxidant capacity of yogurt (O’Connell &Fox,2001;Şengül,Erkaya,Şengül,&Yildiz,2012).

Thisstudyaimstodeterminehowtheantioxidantpowerofyo-gurtchangesduringshelflifeinstandardconditionsofpreservation.Therefore,weexaminedtheantioxidantcapacityofaplainorskimyogurtwithorwithoutaddedfruitpureeinrelationtothetotalcon-tent of phenols and free radical scavenging capacity. Furthermore,lipoperoxidation processes and the content of carbonyl groups, anindexofproteinoxidation,werealsoevaluated,showingadivergentbehavior.

2  | EXPERIMENTAL

2.1 | Materials

TroloxC,ABTS(2,2′-azinobis(3-ethylbenzothiazoline6-sulfonate))were purchased from Fluka-Sigma-Aldrich (St. Louis,MO, USA).DPPH (1,1-diphenyl-2-picrylhydrazyl) and Folin-Ciocalteau rea-gent were obtained from Sigma-Aldrich (St. Louis, MO, USA).All types of yogurts analyzed in this study were obtained fromCentraledelLattediVicenza(Vicenza,IT),Lactobacillus delbrueckii subs. bulgaricus and Streptococcus thermophilus from Danisco (Copenhagen,DK), and fruit puree fromZuegg (Verona, IT), andDarbo(Stans,A).

2.2 | Preparation of probiotic yogurt

The different types of yogurtswere collected on the day of theirmanufacturing.Briefly,pasteurizedmilkwassubjectedtoultrafiltra-tiontoachievethedesiredfatandsolidcontent,thentosterilizationat120°Cfor30secandhomogenization(APV,SPXFlowTechnology,Crawley,UnitedKingdom).Milkwasthenincubatedinamaturationtank at 38°C and inoculatedwith the starter culture (Lactobacillus delbrueckii subs. bulgaricus and Streptococcus thermophilus). At theendofthisprocess,whenthedesiredpHwasachieved,theclotwasbrokenandtheyogurtwascooledtostopthefermentationprocess.Fruitpureewasthenaddedandtheyogurtwaspackedandstoredat4°C.

2.3 | Lipid and protein composition of yogurt

Lipid fraction of plain yogurt (40mg/mL) is composed of 69.14%saturated fats (palmitic acid [32%], stearic acid [10.8%], andmyris-ticacid[10.7%]),27.7%monounsaturatedfats (oleicacid[24%]andpalmitoleicacid[1.7%]),and3.2%polyunsaturatedfats (linoleicacid[2.7%]andlinolenicacid[0.4%]).However,skimyogurtshowsaverylowconcentrationoffats (about1mg/mL) incomparisonwithplainyogurt,butmaintainsasimilarpercentageofsaturated,monounsatu-rated,andpolyunsaturatedfattyacids.Ofnote,skimyogurtexhibitsaproteincontentslightlyhigher(42mg/mL)thanplainyogurt(38mg/mL).Thereporteddataareprovidedbythemanufacturer.

2.4 | Preparation of yogurt aqueous extracts

Forthepreparationofthesamples,aliquotsof0.25mLofyogurtwereextracted for18hrwith5mLofdistilledwaterat4°C inanorbitalshaker. The samples were then centrifuged at 20,000g for 20 min at 15°C. The supernatants were collected and filtrated throughWhatmanChr.1.

2.5 | Determination of total phenolic content

Phenolic compoundswere determined using themethod describedbyGülçin, (2012)withsomemodifications.Briefly,1mLofaqueousextractofyogurt,obtainedasdescribedabove,wasaddedto1mLofFolin-Ciocalteaureagentdiluted1:2withwater.After3min,2mLof10%Na2CO3wasaddedandthesampleswereincubatedfor15minat room temperature. At the end of this step, the absorbancewasmeasured at 750nm. A calibration curve was performed with gal-licacidand the resultswereexpressedasmicrogramsofgallicacidequivalentsper100mLofsample(GAE).

2.6 | Antioxidant activity of yogurt aqueous extracts estimated with the DPPH method

DPPH(1,1-diphenyl-2-picrylhydrazyl)isastable-freeradicalwhosere-actionwithantioxidantmoleculescanbefollowedasdecrease inab-sorbance.TheevaluationoftheantioxidantactivitywasperformedwiththemethoddescribedbyŞengületal.(2012)withsomemodifications.Yogurtextracts (0.02mL)werediluted in0.08mLofwaterand thentreatedwith0.1mLof0.16mmol/LDPPHdissolvedinethanol.Thede-creaseinabsorbancewasmeasuredspectrophotometricallyat517nm.Thepercentageofantioxidantactivityinhibitionwascalculatedas:

2.7 | Antioxidant activity of yogurt aqueous extracts estimated with the ABTS method

ABTS (2, 2′-azinobis(3-ethylbenzothiazoline 6-sulfonate)) can beoxidizedtoastableradicalcation(ABTS•+)whichisdecolorizedafter

%DPPH scavenging = (Abs control - Abs sample)/(Abs control)×100

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reactionwithantioxidants.ABTS•+wasgeneratedbyreacting7mmol/LABTSwith2.46mmol/Lpotassiumpersulfate(finalconcentration)andthemixturewasmaintainedatroomtemperature,inthedark,for16hrbeforeuse(Perna,Intaglietta,Simonetti,&Gambacorta,2013).Thenitwasaddedtoyogurtextractstodeterminetheirantioxidantcapacity.Briefly,0.01mLofwaterextractsofeachtypeofyogurtwasdilutedin0.09mLofwaterandthentreatedwith0.1mLofABTS•+. The de-creaseinabsorbancewasmeasuredat415nmwithaplatereader.AcalibrationcurvewasperformedwithTroloxCandtheresultsareex-pressedasTroloxCequivalentantioxidantcapacity(TEAC).

2.8 | Determination of lipid peroxidation

Lipidperoxidationwasassessedasmalondialdehyde(MDA)forma-tionusingthe2-thiobarbituricacidassayasdescribedbyYagi(1984)

withmodifications(Rigobelloetal.,2008).Aliquotsof0.1mLofyo-gurtweretreatedwith3.9mLof0.3mol/Lsulfuricacidand0.5mLof10%phosphotungsticacid.After10minat25°C, sampleswerecentrifugedat4,000gfor10min.Pelletswereresuspendedin2mLof0.3mol/Lsulfuricacidand0.3mLof10%phosphotungsticacidandcentrifugedagainatthesamespeed.Then,samplesweresus-pendedwith1mLof0.67%thiobarbituricacid(TBA),0.2mLof5%Nonidet,0.04mLof1%BHT,and2.8mLofwaterandincubatedfor1hrat95°C.Afterheattreatment,3mlofn-butanolwasaddedtothesampleswhichwerevigorouslymixedandcentrifugedat6,000g for5min.Thefluorescenceoftheupperphasewasmeasured(Exat530nmandEmat540nm).Acalibrationcurvewith1,1,3,3-tetraeth-oxypropanestandardsolutionwasusedtoquantitativelydeterminethe concentrationof thiobarbituric reactive substances (TBARS) inthesamples.

F IGURE  1 Determinationoftotalphenoliccontent(TPC)indifferenttypesofyogurt.(A)yogurtaqueousextracts(1mL)weretestedandTPCwasdeterminedbytheFolin-Ciocalteaumethod.(a)plainyogurt;(b)skimyogurt;(c)plainberriesyogurt;(d)skimberriesyogurt;(e)plainstrawberriesyogurt;(f)plainblueberriesyogurt;(g)plaincherryyogurt;(h)plainpineappleandorangeyogurt;(i)plainplumyogurt;(j)plainapricotyogurt;(k)skimappleyogurt;(l)skimbananayogurt;m:skimpeachyogurt.(B)Determinationoftotalphenoliccontent(TPC)of(a–d),duringtheshelflife.Theexperimentswereperformedonceaweekfor9weeks.Totalphenoliccontentwasexpressedasmgofgallicacidequivalents(GAE)per100mLofyogurt

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2.9 | Protein carbonyl content estimation

Protein oxidation was determined with the method describedby Fenaille etal., (2006) with some modifications. Briefly, 2mgproteins of the original yogurt were treated with 10mmol/L2,4-dinitrophenylhydrazine (DNPH) dissolved in 2mol/L HCl, for30min in thedark. Proteinswere thenprecipitatedwith10%TCAandcentrifugedfor5minat10,000g.ToremoveunreactedDNPH,theobtainedpelletswerewashedthreetimeswith1mLofethanol/ethylacetate(50:50),anddissolvedwith8mol/Lguanidine(pH2.3).Absorbancewasestimatedat370nmandproteinconcentrationwasdeterminedbytheBradfordassay,(Bradford,1976)using0.01mLofsamplesdissolvedwithguanidine.

2.10 | Statistical analysis

Eachanalysisof thesampleswasperformedonceaweek fromtheproduction to 2weeks after the shelf life. All the experiments re-portedarethemeanwithrespectiveSDof,atleast,fourexperiments.The statistical analysis of variance (ANOVA) was performed usingTukeytestwithINSTAT3.3(GraphPad)software.

3  | RESULTS AND DISCUSSION

3.1 | Total phenolic content of different types of yogurt

Theconcentrationof totalphenolicgroups (TPC) inyogurt samplestreatedwithdifferentfruitpureeswasanalyzed.Figure1AreportstheTPCcontentatthebeginningofthestorage,estimatedasmicrogramsofgallicacidequivalents(GAE)per100mLofsample.Theadditionoffruitpureedeterminedanincreaseintotalphenolicgroups,particu-larlywhenberries(c,d),cherry(g),andpeach(m)pureeswerepresent.Testswere repeatedduring the shelf lifebychoosing two typesofyogurt,plainandskimwithandwithoutberries,andtheresultsarereportedinFigure1B.Thedurationofstoragedeterminedaslightde-clineofTPCmostlyinskimyogurttreatedwithpureeofberries(d),whereasforplain (a),skim(b),andplainwithberries (c)yogurtsthecontentdidnot change.Therefore, data analysis shows thatduringtheshelflife,theproductsarenotsignificantlyalteredregardingtheirTPC.

3.2 | Antioxidant capacity of different types of yogurts

3.2.1 | DPPH radical scavenging during the shelf life of yogurts

Highlyreactivefreeradicalsandotherreactiveoxygenspeciesgene-rated from a wide variety of sources occur in biological systems.However, these species are efficiently recognized by antioxidants.DPPHisarelativelystableorganic-freeradicalwhichhasbeenwidelyused to test the free radical scavenging ability of various samples

containingantioxidantmolecules.Themethodisbasedonthereduc-tion in alcoholicDPPH solutions in the presenceof an antioxidant,asDPPH solutions show a deep violet colorwith a strong absorp-tionbandat517nmwhichdecreasesinthepresenceofantioxidantmolecules.TheresultingbleachingofDPPHisstoichiometricwiththeantioxidantmoleculesexaminedandtheestimationoftheconsumedDPPHradicalsisameasureoftheradicalscavengingactivity(Kulisic,Radonic, Katalinic,&Milos, 2004). The results reported in Figure2refertoplain(a),skim(b),plainberries(c),andskimberries(d)yogurtextracts.TheDPPHscavengingactivityofyogurtsamplesincreasedinthepresenceofberriespuree,however,berriespureealonedoesnotshowthesamepercentageofscavengingactivity (insetofFigure2,columnp)respecttothecontrolofplainberriesyogurt. Inaddition,theantioxidantpowerdecreasedduringtheshelflifeuntiltheeighthweekandafterincreasedtovaluescomparabletothoseobservedatthebeginning.Asdiscussedinthenextparagraph(3.2.2),thisbiphasicbehaviorisprobablyduetothelateformationofpeptideswithanti-oxidantproperties,derivingfromthefragmentationofmilkproteins(Pernaetal.,2013;Dziuba&Dziuba,2014).

3.2.2 | Measurement of ABTS radical scavenging activity

Asacomplement tothepreviousestimation, theantioxidantcapac-ityofaqueousextractsofyogurt samplesusing theABTS test,wasalsodetermined.Firstofall,acalibrationstandardcurvewithTroloxCwaspreparedand,thedataareexpressedasequivalentsoftrans-formedTroloxC (TEAC).During theshelf lifesignificantdifferenceswereobserved,mainlyforplainandskimyogurt,comparedwiththesameyogurtswithberries.Asreported inTable1, in thefirstweek,theantioxidantcapacity ranges from835μmol/LTroloxequivalentsforplainyogurtto1,853μmol/LTroloxequivalentsforplainberriesyogurtandfrom1,044μmol/LTroloxequivalentsforskimyogurtto1,925μmol/LTroloxequivalents forskimberriesyogurt,onceagainhighlightingthatthepresenceofpolyphenolsisrelevantfortheanti-oxidantpower.Inaddition,duringtheshelflife,inthethirdandfifthweek, we can observe a marked decrease in antioxidant capacity,reaching50%,forplainandskimyogurtsinthefifthweek.However,intheseventhweek,theantioxidantcapacityincreasesinaccordancewiththeantioxidantactivitypatternseenwiththeDPPHmethod(seeabove).Again,likepreviouslyobserved,thisbehaviorseemstodependonthegenerationofbioactivepeptidesderivingfromtheproteolyticcleavageofcaseinsandothermilkproteinsendowedwithantioxidantproperties(Pihlanto,2006;Pernaetal.,2013;Dziuba&Dziuba,2014).Ofnote,theproteolyticactivitycandeterminethereleaseofantioxi-dantsnaturallypresentintheyogurtsamplesandentrappedinglobu-larproteinssuchascaseins(Trigueros,Wojdyło,&Sendra,2014).

3.2.3 | Estimation of lipid peroxidation in basal or stimulated conditions

Toassessthespontaneouslipoperoxidationofyogurtoccurringdur-ingtheshelflife,atestbasedonmalondialdehyde(MDA)production

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wascarriedout.Yogurtswerecollectedattheendofthepackagingandstoredat4°Covertheperiodoftheshelflife.Theanalyseswerecarried out at the indicatedweeks until the end of the establishedshelflife(50days)and,inaddition,upto20daysaftertheexpirationdate.AsreportedinFigure3,theassayswerecarriedoutfor9weeksfortheplain(panelA),skim(panelB),plainberries(panelC),andskimberries (panelD)yogurts,respectively,using0.1mLofsample.Firstofall,skimandskimberriesyogurtsshowlowerlevelsofMDA,espe-ciallyinstimulatedconditionsbyadditionofcumenehydroperoxide/hemin(Figure4,seebelow),duetoaminorcontentoflipids.Inaddi-tion,weobservedaslightincreaseinlipoperoxidationparticularlyinthefifthorsixthweekespeciallyinplainyogurt.However,thelevelsoflipidperoxidationwererelativelylowreachingamaximumofabout21nmoles/mL(Figure3aandc).Inthepresenceofberries,MDAlevelwaseven lowerboth inplain and skimyogurt samples.Ofnote, atlongertimes(VIItoIXweeks)adecreaseinMDAformation,especiallyinplainandplainwithberriesyogurts,wasobserved.

However,when lipid peroxidationwas stimulated by addition ofcumene hydroperoxide/hemin, a strong increase in MDA formation

wasobserved.Asapparent inFigure4,arapid increase inMDApro-duction, especially forplainyogurt (Figure4a) compared to skimyo-gurt(Figure4b),wasdetected,inagreementwiththelowerfatcontentpresent in the skim yogurt. In addition, during the shelf life, a con-stant increase inMDAformationuntil thesixthweekwasobserved.Thisbehaviorwasfoundboth inthepresenceand intheabsenceofberries.However,inthepresenceofberriesalowerMDAproductionwithrespecttoplainyogurtaloneisapparentbycomparingFigure4a(400nmoles/mLatthesixthweek)withFigure4c(150nmoles/mLatthesixthweek).Similarly,thepresenceofberriesdeterminesadecreasein lipoperoxidation of skimyogurt compared to skimyogurtwithoutberries, butMDAvalues result far lower (Figure4b and d).Notably,MDAformationbothinbasalconditionandinthepresenceofcumenehydroperoxide/hemin was also estimated in a home-made yogurt,however,veryhighvaluesof lipidperoxidationalreadyat48hrafterproductionwerefound(datanotshown),indicatingthatcontrolledcon-ditionsofmanufacturingensureabetterproduct.AccordingtoSerra,Trujillo,Pereda,Guamis,&Ferragut(2008)thelowpH,thetemperatureofmaintenanceoftheproductduringtheshelflifeandthematerialof

F IGURE  2 Determination of the antioxidantactivitywithDPPHinvarioustypesofyogurtduringtheshelflife.Aliquots(20μL)ofyogurtextractsweretreatedwith0.08mmol/LDPPHandthedecrease in absorbance was estimated at517nm.Theanalysiswasrepeatedonceaweekfor9weeks.Theactivitywasexpressedas%ofDPPHradicalscavenging.(a)plainyogurt;(b)skimyogurt;(c)plainberriesyogurt,and(d)skimberriesyogurt.Theinsetreportstheantioxidantactivityofplainyogurt(a),yogurtaddedwithberriespuree(c),andberriespureealone(p)treatedinthesameconditions.***p < 0.001

Type of yogurt

Trolox equivalent antioxidant capacity (μmol/L Trolox/mL of yogurt)

Weeks

I III V VII

A 835.2±24.2 720.9±105.3 409.2±74.5 941.2±84.3

B 1044.3±64.3 947.7±109.6 441.2±63.1 912.1±80.7

C 1853.8±104.1 1372.0±198.3 1457.1±251.3 1756.9±341.9

D 1925.2±208.6 1582.2±240.3 1568.3±170.3 1811.7±298.1

Aliquots(10μL)ofaqueousyogurtextractsweretreatedwith0.04mmol/LABTS,andthedecreaseinabsorbancewasestimatedat415nmwithaplatereader.Thevaluesobtainedwerecomparedwithastandard curve of TroloxC and expressed as TEACor Trolox equivalent antioxidant activity (μmol/LTroloxC/mLofyogurt).(A)plainyogurt;(B)skimyogurt;(C)plainberriesyogurt;(D)skimberriesyogurt.

TABLE  1 ABTSradicalscavengingactivityofwater-solubleyogurtextracts

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packaginggenerallywith lowpermeabilitytooxygen,make lipidper-oxidationasecondaryproblem.However, thequalityofmilkusedtoobtaintheyogurtandsomefortifications,forexample,withiron,de-termineadramaticincreaseinlipidperoxidation(Hekmat&McMahon,1997).Therefore,theresultsofFigure4clearlyshowthatwhenMDAwasstimulatedinthepresenceofcumenehydroperoxide/hemin,anetincreaseinMDAformationuntilatleast7weekstakesplace,indicatingapotentialsusceptibilityofyogurttoperoxidativeprocesses.

Asapparent inFigures3and4,after6weeksofshelf life,ade-crease inMDA formationwasobserved in all the samples.Thisoc-currence isprobablyassociatedwiththeactivityofbacteriapresentintheyogurtandproducingbioactivepeptides.Apreliminaryanalysisofthesepeptidesindicatesthatsomeofthemareendowedwithan-tioxidantproperties.Wehaveperformedsomepreliminarystudiesontheproteolyticprocess,and,usingtheo-phthalaldehyde(OPA)basedfluorescentassay,wenoticedthattherewasaneffectiveincreaseinpeptidesformationduringtheshelflife,strictlydependentonthetem-peratureofconservationof theproduct (datanotshown).Bioactive

peptides,inadditiontootherfunctions,exertalsoanantioxidantrolebehaving both as free radical scavengers or chelators of transitionmetal ions (Sarmadi& Ismail,2010).Usually, theyare inactivewhenpresentinthesequenceoftheoriginalproteinsbuttheygainactivityonce releasedbyenzymatichydrolysisof theparentprotein (Poweretal.,2013;Pihlanto,2006).Inaddition,theyarealsoabletostimu-latetheexpressionofgenescodingforantioxidantenzymessuchasheme oxygenase, glutathione peroxidase, catalase, and superoxidedismutase(Sarmadi&Ismail,2010).

3.3 | Estimation of protein carbonyl group formation

Duringthefermentationprocessofyogurt,proteinsarepartiallyhy-drolyzed into peptides and free amino acids (Germani etal., 2014).Theyogurtsamples(plain,skim,plainberries,andskimberries)werealsotestedforproteincarbonylformationduringtheshelflife,atthesameweeks inwhich lipidperoxidationwasmeasured.Theamountofproteincarbonylgroups(Figure5)wasverysimilarintheabsence

F IGURE  3 Estimationofmalondialdehydeduringtheshelflifeofyogurtsamples.Lipidperoxidationwasevaluatedduringyogurtstorage(weeks).Aliquotsof0.1mLofyogurtweresubjectedtoMDAdeterminationasdescribedinExperimental.Fluorometricanalysiswasperformedafterextractionofmalondialdehydewithn-butanol(λEx-530nm,λEm-540nm).ForthequantificationofMDA,astandardcurvewith1,1,3,3-tetraethoxypropanewasperformed.(a)plainyogurt;(b)skimyogurt;(c)plainberriesyogurt;(d)skimberriesyogurt.*p < 0.05;**p < 0.01; ***p < 0.001

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(Figure5A,a,b)orinthepresence(Figure5B,c,d)ofberries,indicat-ingthatberrieswereunabletopreventproteinoxidationandinaddi-tion,skimyogurt,whichhasahigherproteincontent,showedsimilarvaluesofcarbonylcontentincomparisontoplainyogurt.ThisresultisquitedifferentfromthatobtainedforMDAformation(Figures3and4)indicatingthattheantioxidantsofyogurtaremorepronetoprotectagainstlipidperoxidation,butseemscarcelyabletoinfluenceproteinoxidation.Duringtheshelflifeof45days,theproteolyticactivityofbacteria inyogurtproceeds (Germanietal.,2014)anddeterminesafragmentation and an oxidation particularly in plain yogurt. Proteinoxidationishighercomparedwithskimproductattheseventhweek(Figure5A), but this does not occur for the samples (plain or skim)containingpureeofberries(Figure5B).Inaddition,carbonylcontentafter the incubation of yogurts with cumene hydroperoxide/heminwasestimated(Figure5C).Alsointhiscase,thepresenceofberriesdonotreducetheextentofproteinoxidation.

4  | CONCLUSION

Theantioxidantactivityofyogurtsamplesdependsontheendogenousantioxidants,onaddedantioxidantscontained in fruitpureeandontheformationofbioactivepeptides.Theantioxidantpropertiesofbio-activepeptidesdependontheirspecificaminoacidcompositionandTyr,Cys,andHisresiduesareparticularlyrelevant.Inaddition,somepeptidesmayalsobeabletoexertamarkedsynergisticeffectwithphenolic antioxidants (Saito etal., 2003). Theproductionof endog-enousantioxidantpeptides,andtheincidenceofaddedantioxidantspresentinthepureeundoubtedlyconstituteagoodprotectionagainstalipidperoxidation,andthereforereducetheperoxidizabilityofyo-gurt.Onthecontrary,theoxidationoftheproteinsisnotpreventedbytheantioxidantspresentinyogurt.Inaddition,DPPHandABTSas-saysshowthatthetotalantioxidantcapacityslowlydecreasesduring

F IGURE  4 Estimationofmalondialdehydeaftertreatmentofyogurtsampleswithcumenehydroperoxide/hemin.Lipidperoxidationwasevaluatedduringyogurtsamplestorageforaperiodof9weeks.Briefly,aliquotsof0.1mLofyogurtweretreatedfor30minwith25mmol/Lcumenehydroperoxide/0.05mmol/Lhemin,andthensubjectedtoMDAdeterminationasdescribedinExperimental.Afterextractionofmalondialdehydewithbutanol,fluorometricanalysiswasperformed(λEx-530nm;λEm-540nm).(a)plainyogurt;(b)skimyogurt;(c)plainberriesyogurt;(d)skimberriesyogurt.ForquantificationofMDA,acalibrationcurvewith1,1,3,3-tetraethoxypropaneasreferencestandardwasusedtodeterminetheconcentrationsofTBARSinthesamples.*p < 0.05;**p < 0.01; ***p < 0.001

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theshelf life.However,afterarelativelylongstorageanincreaseinoxidantspeciesscavengingcapabilitywasobservedandattributedtotheproductionofbioactivepeptides.Probably,hydrolyzedproteinsmayalsoactas“sacrificial”antioxidants(Halliwell&Gutteridge,2015)andthismightexplaintheobserveddifferentbehaviorbetweenlipidandproteinoxidationtowardoxidants.Inconclusion,thespecificpre-ventionoflipidperoxidationproductsformationbyendogenousandaddedantioxidantsimprovesthenutritionalqualityofyogurt.

CONFLICT OF INTEREST

Authorsdeclarenoconflictofinterest.

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F IGURE   5 Proteincarbonylscontentduringtheshelflife.Aliquotsofyogurt(2mg)weretreatedwith20mmol/LDNPHfor30mininthedark,thenprecipitatedwith10%TCAandwashedpelletswereresuspendedwith8Mguanidine(pH2.3).Thecontentofcarbonylwasestimatedspectrophotometricallyat370nm.(A)plain(a)andskim(b)yogurt;(B)plainberries(c),andskimberries(d)yogurt;(C)carbonylscontentintheabsence(a),(b),(c),(d)andinthepresence(a’),(b’),(c’)(d’)of25mmol/Lcumenehydroperoxide/0.05mmol/Lhemin,atthefifthweekofshelflife

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How to cite this article:CittaA,FoldaA,ScalconV,etal.Oxidativechangesinlipids,proteins,andantioxidantsinyogurtduringtheshelflife.Food Sci Nutr. 2017;00:1–9. https://doi.org/10.1002/fsn3.493