Optimization of liquid fermentation conditions for biotransformation zein by Cordyceps militaris 202 and characterization of physicochemical and functional properties of fermentative hydrolysates

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Food

Microbiology

Optimization

of

liquid

fermentation

conditions

for

biotransformation

zein

by

Cordyceps

militaris

202

and

characterization

of

physicochemical

and

functional

properties

of

fermentative

hydrolysates

Shuang

Yang

a,b

_,

_Mingzhu

_Zheng

a

_,

_Yong

_Cao

a,b

_,

_Yanjiao

_Dong

a

_,

Sanabil

Yaqoob

a

_,

_Jingsheng

_Liu

a,∗

a_{JilinAgriculturalUniversity,CollegeofFoodScienceandEngineering,Changchun,China}

b_{JilinAgriculturalUniversity,NationalEngineeringLaboratoryforWheatandCornDeepProcessing,Changchun,China}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received5June2017

Accepted1December2017

Availableonline13February2018

AssociateEditor:EleniGomes

Keywords: Zein Degreeofhydrolysis Cordycepsmilitaris202 Functionality

a

b

s

t

r

a

c

t

Cordycepsmilitaris202isapotentialfungusforbiotransformationzein,duetoitsvarious

pro-teases,hightoleranceandviabilityinnature.Inthisarticle,singlefactorexperimentand

responsesurfacemethodologywereappliedtooptimizetheliquidfermentationconditions

andimprovetheabilityofbiotransformationzein.Theoptimizedfermentationconditions

wereasfollows:inoculumconcentrationof19%,volumeofliquorof130mL/500mLandpH

of4.7.Underthiscondition,thedegreeofhydrolysis(DH)was27.31%.Thezeinhydrolysates

fromfungifermentationmaintainedahighthermalstability.Comparedto theoriginal

zein,thezeinhydrolysateswerefoundtohavehighsolubility,whichmostlikelyresults

inimprovedfoamingandemulsifyingproperties.Overall,thisresearchdemonstratesthat

hydrolysis ofzein byC.militaris202isapotentialmethodforimprovingthefunctional

propertiesofzein,andthezeinhydrolysatescanbeusedasfunctionalingredientswithan

increasedantioxidanteffectinbothfoodandnon-foodapplications.

©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis

anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

Zein is the major proportion (65%) of corn gluten meal

(CGM), and CGM is major co-product of corn wet-milling

process.Chinaisthesecondlargest countryforproduction

and consumptionof corn.Zein isexcellent materials with

∗ _{Correspondingauthorat:DepartmentofFoodScience,JilinAgriculturalUniversity,2888XinchengStreet,Changchun,Jilin130118,China.}

E-mail:[email protected](J.Liu).

unique nature,and itspoorsolubility inwater,which

con-tributestozeindifficultinfoodindustryapplication.1_Protein

modification may becarried out toimprove its nutritional

and/or functional properties. Modification of protein

usu-allycontainphysical,2_chemical3_{andbiologicaltreatments,}4,5

which change the physicochemical and functional

proper-ties bychanging its conformation and structure. This has

https://doi.org/10.1016/j.bjm.2017.12.005

1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC

beenreviewedbyseveralresearchers.6–8_{Thebiological}

treat-mentisoneofthemostusedmethodsamongtheresearches

formodification, which consistsof enzymetreatment and

fermentation. The hydrolysates may be expected to have

increasedsolubility,foamingandemulsifyingproperties

com-paredwithnativeprotein,andtherearemanystudieshave

provedthese.9–11_{Comparedwiththesingleenzyme}

hydrol-ysis,thehydrolysisbyliquidfermentationhasadvantageon

severalaspects,suchaslowcost,andfoodsafety,thusmaking

itmoresuitableforproteinmodification.Zhang12_thoughtthat

theproductofpolypeptidebyliquidfermentationcan save

40–50%cost,comparedwithenzymehydrolysis.

Cordycepsmilitarisisconsideredasoneofthemost

expen-sivemushroomsinChina,becauseofitsattractivebeneficial

effectsandrarityinnature.13Thesubmergedculturefor

pro-ducingmycelium can beused asingredients infunctional

foods, which have conduct to meet the demand for

com-mercial applications.14–16 _Xiao17 _{found the C. militaris can}

degradechickpeaproteinandimprovewaterholding

capac-ity,fatabsorptioncapacityandemulsifyingpropertiesofflours

bysolid-statefermentation.Theutilizationofliquid

fermenta-tionofzeinwithC.militaris202cantransformzeinintosoluble

protein,thusimprovingtheutilizationandbioavailabilityin

theaqueousphase.However,theexistent researchesabout

C.militaris mostlyfocus on its metabolite,18–20 _{no available}

reportsaboutuseofenzymaticproteolysisbyC.militaris202

formodifyingthefunctionalityofzeincouldbefound.

Thus,oursubjectistooptimizethefermentationcondition

oftransformingzeintosolubleproteinbyliquidfermentation

withC.militaris202.Thedegreeofhydrolysis(DH)wasused

astheevaluationtoobtainoptimalfermentationconditions

byresponsesurfacemethodology(RSM).Moreover,we

investi-gatedtheeffectsoffermentationtimeonthephysicochemical

andfunctionalproperties(solubility,emulsification,foaming

properties and antioxidative activity) ofzein hydrolysates.

Theinformationisessentialinmodifyingzeinbyliquid

fer-mentation,whichcanprovideapotentialmethodtoimprove

functionpropertiesandbiologicalactivityofzein,andimprove

itsutilizationvalueandfield.

Materials

and

methods

Materials

ZeinwasobtainedfromShanghaiRyonBiologicalTechnology,

China(purity>92%).C.militaris202waspreservedinNational

EngineeringLaboratoryforWheatandCornDeepProcessing.

Allotherchemicalswereofanalyticalgrade

Media

Threemediawereusedinthisexperiment,asfollows:PDA,

fermentationseeding,fermentationmedia.Thecomposition

ofthreemediawasasfollows:

PDAmedium(g/L):potatoes200.0, glucose20.0andagar

18.0.

Fermentationseeding (g/L):potatoes200.0,glucose 20.0,

MgSO41.5,KH2PO43.0,VB10.2.

Fermentation media (g/100mL): glucose 7.0, zein 6.0,

MgSO40.15,KH2PO40.3,VB10.2.

Methods

Preparationofliquidfermentation

C.militaris202wasincubatedintheagarmediumat24◦Cfor

about7d,untilthewhitemyceliacoveredonthesurfaceofthe

agarculturemedia.Threetruffleswereinoculatedin100mL

conicalflaskscontaining30mLliquidmedium.Mediumshad

beensterilizedat121◦Cfor20minbeforeinoculation

(YXQ-LS-SII,BoxunCorp,Shanghai,China).Theconicalflaskswere

keptat24±1◦Cfor3d,and thenwereusedastheC. mili-taris202seedingcultureforzeinfermentation.C.militaris202

seeding was brokenusinga sterilizedBlender, then

inocu-lated 10mLseeding[equivalentto10%ofthefermentation

medium(100mL)]to500mLconicalflasks,theconicalflasks

wereshakingwith160r/minat24◦Cfor8d.

Optimizationofzeinhydrolysatesfermentationconditions

The fermentation conditions were optimized to improve

degreeofhydrolysis(DH),includinginoculumconcentration

(v/v,5%,10%,15%,20%,25%),volumeofliquor(equivalentto

total volumeofthe fermentationmedium)(mL/mL,80/500,

100/500,120/500,140/500,and160/500),pH(2.0,4.0,6.0,8.0,

10.0).Thebiomassanddegreeofhydrolysisweredetermined.

Theexperimentswereconductedinthreereplicates.

Biomass

Themyceliainthefermentedbrothwerewashedfor4times

bydistilledwater,andthenwereobtainedbyfilteringusing

absorbentgauze.Themyceliawasdriedtoaconstantweight

inthehotairovenat105◦C(YLA-6000,Shanghai,China),then

measuringdryweightforbiomass.Theexperimentswere

con-ductedinthreereplicates.

Degreeofhydrolysis(DH)

TheDH wasmeasured accordingtothe methodofYang.21

Thesupernatantoffermentedbrothwasobtainedby

centrifu-gation,then,mixing15mL10%trichloroaceticacidsolution

(TCA)with15mLsupernatant,andthenthemixturewasput

at25◦Cfor30min.Thesedimentwasremovedby

centrifuga-tionat4000×g.Thesolublenitrogeninsupernatantandtotal

nitrogencontentofsubstrateweredeterminedby

microKjel-dahl.Theexperimentswereconductedinthreereplicates.The

DHwascalculatedasfollows:

DH= X−X0

S0−X0×100% (1)

DH:degreeofhydrolysis

X:solublenitrogencontentin10%TCAoffermentedliquor,

g

X0:solublenitrogencontentin10%TCAofunfermented

liquor,g

S0:totalnitrogencontentofsubstrate,g

Preparationofzeinhydrolysatesbyliquidfermentation

After fermentationunder the optimized fermentation

con-ditions(optimizedbyRSM),thereactionwasterminatedby

boilingfor5min.Inordertoextractthezeinhydrolysates,the

fermentedbrothwascentrifuged at4000×gfor20minand

withammoniumsulfateat50%saturation.Theprecipitations

weredialyzedagainstwaterat4◦Cfor24hinordertoremove

saltandthenwerefreeze-dried.Thepurifiedzeinhydrolysates

wereusedforcharacterizationoffunctionalproperties.

Functionalpropertiesofzeinandzeinhydrolysates

Thesolubilityofzeinanditshydrolysatesweredetermined

usingthe methods previouslyreported.22 _Thefoam

capac-ityand stabilityweredetermined byRuth andGroninger.23

Emulsifying activity and stability were determined by

Zayas.24 The experiments were conducted in three

repli-cates.

Thermalproperties

The thermal properties of zein and its fermentative

hydrolysates were measured using a differential scanning

calorimetry(DSC)(Q20DSC,TA,USA).Aprocedurepresented

byZhao,25_{withsomemodifications,wasutilized.The}

sam-ples(2mg)wereheatedfrom0◦Cto160◦Cataheatingrateof

10◦C/min.Anemptypanwasusedasareference.Thepeak

temperature(Tp)andtotalcalorimetricenthalpy(H)were

cal-culated.Theexperimentswereconductedinthreereplicates.

Determinationofantioxidantactivities

The scavenging effect of zein and its fermentative

hydrolysateson DPPHfreeradicalwasmeasuredaccording

tothemethodofXiaetal.26 _{Thedeterminationofhydroxyl}

radical scavenging activity, was presented by Halliwell,27

with some modifications. A procedure presented by Xia

et al.26 _{with some modification was applied to determine}

Fe2+-chelatingactivity.AprocedurepresentedbyLu etal.28

withsomemodificationwasappliedtomeasurethereducing

power.Theexperimentswereconductedinthreereplicates.

Statisticalanalysis

RSMwasusedtoperformtheexperimentaldesigns,

statisti-calanalysisandregressionmodelbyDesignExpertVersion

8.0.6.Box–Behnkendesigns(BBD)atthreelevelswasapplied

toevaluatetheircombinedeffect,asshowninTable1.The

rangeandcenterpointvaluesofthreeindependentvariables

wereselectedbasedontheresultsofpreliminaryexperiments

(Fig.1).Table3showstheexperimentaldesignconsistingof

17factorialpoints.Thepolynomialquadraticequation(1)for

Table1–Independentvariablesandtheirlevelsusedin theresponsesurfacedesign.

Factors Levels

−1 0 1

A 10 15 20

B 120 140 160

C 4 6 8

A,inoculumconcentration(%);B,volumeofliquor(mL/500mL);C, pH.

theoptimalpointwasfittedtoanalyzetheeffectofeach

inde-pendentvariabletotheresponse:

Y=b0+b1A+b2B+b3C+b12AB+b13AC+b23BC+b11A2+b22B2+b33C2 (2)

whereYistheDHcalculatedbyresponse.A,BandCare

the independent variables. Thecoefficients ofthe

polyno-mialwererepresentedbythefollowing:b0(constantterm);b1,

b2andb3 (lineareffects);b11,b22andb33(quadraticeffects);

and b12,b13and b23 (interactioneffects).Thestatistical

dif-ferencesinparameterswereevaluatedbyDuncan’smultiple

rangetestsinTable2.

Results

Selectionoftheproperfermentationcondition

Theeffects ofinoculum concentrationon DHand biomass

were presentedinFig. 1A. TheDH and biomassincreased

withincreasinginoculumconcentrationandmaximumpoint

wasrecorded at15%.Then,the DHandbiomassdecreased

athigherinoculumconcentration.Thevolumeofliquorhad

significantinfluenceonDHandbiomass,asshowninFig.1B.

TheDHandbiomasswereachievedat140mL/500mL.With

higher orlower values, theDH and biomasswere

compar-atively reduced.Theeffects ofpHon theDH and biomass

(Fig.1C)showthatthehighestDHandbiomasswereachieved

attheinitialpH6.0.Withhigherorlowervalues,theDHand

biomasswerecomparativelyreduced.

OptimizationoffermentationparametersforDHbythe

RSManalysis

A total of17 experimentalpoints for optimizingthe three

individual parameters(the inoculumconcentration (A),the

volume of liquor (B) and pH (C)) were shown in Table 2.

Resultswerefittedwithasecond-orderpolynomialequation.

Thesecond-orderpolynomialequationwasusedtoanalyze

therationofproteintransform,anddescribetherelationship

betweenthevariableasfollows;

Ration of protein transform=25.55−0.79A−3.44B

−3.08C−1.86AB−1.7AC+3.37BC

−1.81A2_−8.69B2_−4.44C2 ₍₃₎

TheresultsofANOVAforRSMareshowninTable3.The

determination coefficient forthe model was 0.9852, which

indicatedthatthemodelfitwellwiththedata.Thelackof

fitwas0.1769,whichwasnotsignificantrelativetothepure

error.TheseresultsmeanthattheDHofC.militaris202could

beanalyzedandpredictedbythemodel.The3Dresponse

sur-faceplotsandthe2DcontourplotsoftheresponsesofDHare

showninFig.2.AtafixedpH,wheninoculumconcentration

wasatahigherlevel,DHincreasedandthendecreasedsharply

with increasingvolume of liquor,which could be ascribed

tothedissolvedoxygenrestrictionintheculturemedium.29

20 18 16 14 12 10 8 6 4 2 0 2 4 6 8 10 12 14 5 10

C

A

B

15 20 25 200 500 160 140 120 100 80 600 700 800 900 1000 1100 1200 1300 400 600 800 1000 1200 200 12 10 8 pH Volume of liquor (mL/500mL) Inoculum concentration (%) DH (%) DH (%) DH (%) Biomass (mg/100mL f e rmentation liquor) Biomass (mg/100mL f e rmentation liquor) Biomass (mg/100mL f e rmentation liquor) 6 4 2 4 6 8 10 12 14 16 DH Biomass DH Biomass DH Biomass 400 600 800 1000 1200

Fig.1–Singlefactorexperimentsoftheeffectofinoculumconcentration(A),volumeofliquor(B),pH(C)ontheDHand

biomass.

Table2–TheexperimentaldesignandRSMresults.

Theexperimentnumber A B C DH(%)

1 0 0 0 25.71 2 0 0 0 26.52 3 0 1 1 9.05 4 1 0 −1 23.69 5 0 −1 −1 22.52 6 1 1 0 11.34 7 −1 1 0 11.71 8 −1 0 −1 20.49 9 1 0 1 14.7 10 0 1 −1 9.05 11 −1 0 1 18.3 12 0 0 0 25.8 13 1 −1 0 22.09 14 0 0 0 24.01 15 −1 −1 0 15.01 16 0 0 0 25.68 17 0 −1 1 9.05

insignificantly.However,whenvolumeofliquorwasatalower

level,DHincreasedandthentendedtoconstantwith

increas-inginoculumconcentration.Itwasindicatedthatvolumeof

liquorhadsignificantlyeffectonproteintransform.Atafixed

volumeofliquor,theDHincreasedandthendecreasedwith

increasingpHandinoculumconcentration.Itwasnoticedthat

proteintransformofinoculumatlowerlevelincreasedquickly

thanthatathigherlevelwithincreasingpH.Atafixed

inocu-lum concentration, DH ofvolume ofliquor athigher level

increasedmarkedlythanthatatlowerlevelwithincreasing

ofpH.

Theoptimizedconditionswereasfollows:inoculum

con-centrationof18.6%,volumeofliquorof132.04mL/500mLand

pH of 4.73. Under the fermentation conditions, the DH in

theory was 27.49%.However,consideringthe operabilityin

actual production,theoptimalconditionswereadjustedas

follows:inoculumconcentrationof19%,volumeofliquorof

130mL/500mLandpHof4.7.Inordertoverifytheaccuracy

ofthismodel,verificationexperimentswerecarriedout.The

averageDHofstrainsreached27.31%.Therelativeerrorwas

0.65% in comparisonwith the predicted value. These data

provedthatthemodelwasaccurateandreliablefor

predict-ingtheexperimentalresults.Thus,thismodelhasapractical

guidingsignificance.Theseresultssuggestedthatthe

hydrol-ysisofzeinbyliquidfermentationwithC.militaris202was

effective.

Solubility

Inthepresentstudy,afoodgradefunguswasselectedfor

pro-ducingzeinhydrolysates,whichwasabletosecreteefficient

proteases forthehydrolysis oftheinsolubleprotein.Under

theoptimizedconditions,theDHreached27.31%,indicating

C.militaris202waseffectiveforzeinproteolysis.DHis

gen-erallyusedasaproteolysismonitoringparameter,indicating

theextentofhydrolysisdegradationofprotein.TheDHofzein

atfermentationtimeof2,4,6,8,10,12and14dayisshown

Table3–ANOVAforresponsesurfacequadraticmodel.

Source Sumofsquares df Meansquare Fvalue p-value

Prob>F Model 693.0657 9 77.0073 51.6 <0.0001 Significant A 4.969443 1 4.969443 3.329856 0.1108 N.S. B 94.64816 1 94.64816 63.42054 <0.0001 b C 75.97746 1 75.97746 50.90994 0.0002 b AB 13.8909 1 13.8909 9.307825 0.0186 a AC 11.5481 1 11.5481 7.737994 0.0272 a BC 45.37841 1 45.37841 30.40654 0.0009 b A2 13.83777 1 13.83777 9.272223 0.0187 a B2 318.0952 1 318.0952 213.1449 <0.0001 b C2 82.9266 1 82.9266 55.56632 0.0001 b Residual 10.44673 7 1.49239

Lackoffit 7.032887 3 2.344296 2.746813 0.1769 Notsignificant

Pureerror 3.413841 4 0.85346

Cortotal 703.5125 16

R2_=0.9852

N.S.,nosignificant(p>0.05);R2_{,correlationcoefficient.} a _{Significant(p<0.05).}

b _{Significant(p<0.01).}

increased,increasingrapidlywithintheinitial8d.Thehighest

DHwas29.66%,atfermentationtimeof14day.SincetheDH

ofzeincorrespondedtoitsfermentationtimeunderfixed

fer-mentationcondition,thefermentationtimewastakenasthe

factorforevaluatingthephysicochemicalpropertiesofzein

hydrolysates.

Solubilityisoneof importantprerequisiteforthe

func-tional properties of proteins and directly influences their

applicationsinthefoodandnon-foodindustry.25_The

solu-bilities ofzein and fermentative hydrolysateare shown in

Table4. ThezeinshowedpoorsolubilityinpH7.0distilled

water,andthesolubilitywas1.84%.Thepoorsolubilitywas

duetotherigidstructureofzeinwhichcontainedhigher

inter-molecular,intramoleculardisulphidebondsandhydrophobic

interactions. However,the liquid fermentation ofzein can

significantlyimprovesolubility(p<0.05).Themaximum

solu-bilitywasatfermentationfor8d(86.18%).Comparedwiththe

originalzein,theimprovedsolubilityofhydrolysatecouldbe

attributedtotheunfoldingofthepeptidechains,thedecrease

ofsizecausedbyprotease hydrolysis,and the exposureof

morechargedresiduesandpolar groupsintothe

surround-ingaqueousphase.Thesestructuralchangesresultedinan

improvementof protein–water interaction, thus increasing

thesolubility.30

Thermalproperties

Thermal property is closely related to the structure and

functionalpropertiesofproteins.31_{Variationsinthethermal}

behaviorofzeinanditsfermentativehydrolysatesobtainedby

differentfermentationtimeweremeasured,andareshownin

Fig.4.Thepeaktemperatures(Tp)ofzeinwas87.25◦C,which

issimilarwiththeresultbyLi,32 _{who alsofoundtheT}

p of

zeinwas90◦C.TheTpofzeinfermentativehydrolysatewere

foundtobesignificantlydifferentfromthecontrol(p<0.05).

Withincreasingfermentationtime(8d–14d),therewereno

significantdifferencesamongthehydrolysates(p>0.05).The

high Tp valueusually implies high thermalstability. Thus,

the highTp valueofzeinhydrolysatesindicatedgood

ther-mal stability. As shown in Table 2, the thermal enthalpy

(H) of the zein (135.43◦C) was significantly higher than

thatofzeinhydrolysates(p<0.05),whichindicatedthat

fer-mentationcould markedly decrease theordered structures

and aggregation tendency of the protein were decreased

duringthefermentationperiod.Manyfactorscaninfluence

the thermal properties of proteins, such as, hydrophobic

interactions,–SH/–SS–,hydrogenbondsandhydration.33_The

synergism among these factors resulted in the complex

changesobservedinTpandH.

Foamingpropertiesandfoamingstability

Thefoamcapacity(FC)andfoamstability(FS)ofzeinandits

hydrolysatesatpH7.0areshowninFig.5A.TheFCandFS

ofzeinhydrolysateswerehigher(p<0.05)thanthoseofzein,

andtheFCandFSofzeinhydrolysateswerealsoaffectedby

fermentationtime.TheirmaximumFCandFSwereobtained

at8dand14d,respectively(122.56%and85.54%).Thus,

con-trollingofthefermentationtimeshouldbeconductasitleads

toundesirablepropertychanges.

Emulsifyingactivityandemulsifyingstability

Theemulsifyingactivity index(EAI) andemulsifying

stabil-ity index(ESI)ofthezeinandits hydrolysateatpH7.0are

showninFig.5B.TheEAIofhydrolysateswasimprovedand

wassignificanthigher(p<0.05)thanthatofzein.TheEAIwas

increasedfrom4.5%to84.82%,whenthefermentationtime

wasincreasedfrom0dto8d.TheESIofthefermentative

hydrolysateshowedthesimilartrend.TheincreaseofEAIand

solubilityreflectedtheenhancedsolubilityofproteinafter

fer-mentation,enablingproteindiffusiontotheoil/watersurface

easily.However,furtherincreasingthefermentationtimefrom

10dto14d,theEAIoffermentativehydrolysateswere

main-tainedataconstantvalue(morethan60%).Thisphenomenon

30

A

DH 15 20 25 20 20 22 24 22 26 20 15 25 10 15 18 DH DH B: Volume of liquor B: Volume of liquor B: Volume of liquor A: Inoculum concentration A: Inoculum concentration A: Inoculum concentration A: Inoculum concentration B: V olume of liquor C: pH C: pH DH DH DH C: pH C: pH

B

C

25 20 15 10 5 30 25 20 15 10 5 30 25 20 15 10 5 1.00 1.00 0.50 0.00 0.50 0.00 0.00 0.50 1.00 –0.50 –0.50 –0.50 –0.50 0.00 0.50 1.00 –1.00 1.00 0.50 0.00 –0.50 –1.00 1.00 0.50 0.00 –0.50 –1.00 –1.00 –0.50 0.00 0.50 1.00 –1.00 –0.50 0.00 0.50 1.00 –1.00 –1.00 1.00 0.50 0.00 –0.50 –1.00 1.00 0.50 0.00 –0.50 –1.00 –1.00 0.00 0.50 1.00 –0.50 –1.00 0.00 0.50 1.00 –0.50 –1.00

Fig.2–RSMcurveoftheinteractionsbetweendifferentfactors.Interactioneffectofinoculumconcentrationandvolumeof

liquorontheDH(A),interactioneffectofinoculumconcentrationandpHontheDH(B),interactioneffectofvolumeofliquor

andpHontheDH(C).

byprotease,inwhichextensivehydrolysisdidnotincreasethe

proteinemulsifyingactivity.34_{Thiswasduetotheextensive}

hydrolysispreventstheformationofacontinuousproteinfilm

attheoil/waterinterfacetostabilizetheemulsions.26

Antioxidantpropertiesofzeinhydrolysates

Theantioxidant activity ofzein was not tested because it

barelydissolvedinpH7.0distilledwater.TheDPPHradical

scavengingcapacities ofzeinhydrolysatesarepresentedin

Fig.6A.Allofthesamplestestedexhibitedgoodscavenging

abilityagainstDPPH (31.60–56.98%).In particular,the

activ-ityofhydrolysates(56.98%)atfermentationtimeof8dwas

higherthanthatofothers(p<0.05).TheDPPHradical

scav-engingofzeinhydrolysatesbyalkalineproteaseandpepsin

hydrolysateswere12–28%and22–59%,respectively.35,36_Thus,

theactivityoffermentativehydrolysateswascomparableto

thehydrolysatesreportedintheseliteratures.Theresult

indi-cated that fermentativehydrolysates containedsubstances

thatwereelectrondonorsandcouldreactwithfreeradicals

toconvertthemtostableproductsandterminatetheradical

chainreaction.

Hydroxylradicalcanactivatelipidperoxidationandreact

with almost any adjacent biomoleculessuch asDNA,

pro-teinsandaminoacids.26_{Sincethisdamagecancauseseveral}

diseases, its removal may be one of the most effective

method to prevent putrefaction offood and occurrenceof

somediseases.37_{Thehydroxylradicalscavengingactivitiesof}

fermentativehydrolysatesareshowninFig.6B.The

fermen-tativehydrolysatesexhibitedstronginhibitoryeffectagainst

40 35 30 25 20 15 10 5 0 2 4 6 8 10 12 Fermentation time (d) Deg ree of h ydrolysis (%) 14

Fig.3–Degreeofhydrolysisofzeinhydrolysatesat

differentfermentationtime.

fermentativehydrolysateshavethepotentialtoprotectfood

againsthydroxylradical-induceddamages.

The Fe2+_{-chelating ability of zein fermentative}

hydrolysatesisshowninFig.6C.TheFe2+_{-chelatingcapacity}

ofthe hydrolysates increased from 15.98% to 20.02% with

increasingoffermentationtime, andthenreduced slightly.

Liuetal.34 _{reportedasimilarFe}2+_{-chelating abilityfor}

pro-teasehydrolysates(about10–30%)derivedfromcornglutelin.

Kongpreviouslyreportedthatthetypeofproteasesusedand

theDHaffecttheantioxidantactivity.38_{Thus,thedifference}

ofFe2+_{-chelatingcapacityinourresultsmaybeascribedto}

the typeofproteases or/and DH comparedwith literature.

Comparedwiththenativezein,theimprovementofthemetal

ionbindingpowerofthefermentativehydrolysatesmightbe

ascribedtoanincreasedconcentrationofcarboxylicgroups

inthesidechainoftheacidicandbasicaminoacids.39

Thereducing powerassay isoften usedtoevaluatethe

potential antioxidant activity of natural products. Fig. 6D

showsthereducingpoweroffermentativehydrolysates.The

fermentative hydrolysates exhibited good reducing power

(0.15–0.19%), which were more effective reducing capacity

thanthoseofbarleyglutelinhydrolysates(0.121at2mg/mL).26

0.2 Zein 2d 4d 6d 8d 10d 12d 14d 0.0 –0.2 –0.4 –0.6 0 20 40 60 80 Temperature (°C) Heatflo w (m W/g) 100 120 140 160

Fig.4–Thermalpropertiesofzeinanditshydrolysates.

TheseresultsrevealedthatzeinhydrolysatesbyC.militaris202

havebetterreducingpower.

Discussion

Toimprovethe utilizationandbioavailability ofzeininthe

aqueousphase,weutilizedliquidfermentationofzeinwith

C. militaris 202 to transform zein into soluble protein. In

thisstudy,theeffectsofinoculumconcentration,volumeof

liquorandpHontheDHandbiomasswereinvestigated.The

DH increasedwith increasinginoculum concentration and

maximumpointwasrecordedat15%,whileitdecreasedat

higherinoculum concentration.Thehydrolysateinoculated

withhigherinoculumconcentrationhadloweryield,which

wassimilarwiththereportbyFerchichi.40_{Thisphenomenon}

maybeexplainedbythatthetransformationofzeininto

prod-uctswasaccompaniedbysomemetabolicactivities,andthis

actionresultsin negativeimpacton thefermentation

pro-cess.Besides,thismayresultfromcellwereunabletoattain

completegrowthduetonutritionalinhibitionbefore

secret-ingprotease. Thus,itwasobservedthattheDHwaslowat

higherinoculumconcentration.C.militaris202exhibited

bet-terabilitytohydrolyzezeininanacidcondition.Asshown

inFig.1C,theDH increasedwithincreaseintheinitialpH

Table4–Solubilityandthermalpropertiesofzeinanditshydrolysates.

Timeoffermentation(d) Solubility(%) Thermalproperties

Maximum(◦_{C) Area(J/g)} Zein 1.84±0.25a _87.26±9.08a _{135.433±9.09}d 2 80.45±0.78b _102.76±0.28c _89.99±7.82a 4 81.74±1.01b _100.76±0.44c _96.60±9.05a 6 83.14±0.51c _107.13±0.54d _92.15±8.68a 8 86.18±0.86d _92.75±1.05b _90.46±0.49a 10 84.47±1.38c _92.26±1.52b _97.36±4.87b 12 84.21±0.90c _92.38±0.12b _107.57±0.84c 14 83.96±0.73c _93.65±1.00b _108.78±0.56c

Foaming capacity

A

140 120 100 80 60 40 20 0 zein 2 4 6 8 10 12 14 100 80 60 40 20 0

B

Foaming stability EAI ESI zein 2 Fermentation time (d) F oam capacity/F oaming stability EAI/ESI Fermentation time (d) 4 6 8 10 12 14

Fig.5–Foamingpropertiesandfoamingstability(A),andemulsifyingactivityandemulsifyingstability(B)ofzeinandits

hydrolysates.

A

B

D

C

70 70 60 60 50 50 40 40 30 30 20 20 10 10 0 0 Ascorbic acid

Ascorbic acid Ascorbic acid

Ascorbic acid 2 2 2 2 4 4 4 4 6 6 6 6 8 8 8 8 10 10 10 10 12 12 12 12 14 14 14 14 Fermentation time (d)

Fermentation time (d) Fermentation time (d)

Fermentation time (d) DPPH r adical sca v enging (%) Hydro xyl r adial sca v enging activity (%) 25 20 15 10 5 0 0.30 0.25 0.20 0.15 0.10 0.05 0.00 Reducing po w e r (absorbance) Fe 2+ chelating activity (%)

Fig.6–DPPHradicalscavengingactivity(A),hydroxylradicalscavengingactivity(B),Fe2+_{-chelatingactivity(C)andreducing}

power(D)ofzeinhydrolysates.

from4.0to6.0andmaximumpointwasrecordedatpH6.0.

Then,itdecreasedathigherpHvalues.Previousstudies

eval-uatingtheeffectofpHonthehydrolysisofzeinreportedthe

highDHmostlyatalkalinecondition.36_{Nevertheless,acidic}

fermentation mediuminduced elevation in the DH in our

study.Suchsimilar acidic pHvalues(5.0–8.0)were

demon-stratedto maximizebiomassfrom entomopathogens, such

asPaecilomycesfarinosus,Beauveriabassiana,andMetarhizium

anisopliae.41_{SincethemaximumbiomassofC.militaris202is}

achievedatacidiccondition,42_{therefore,acidicconditioncan}

promotethegrowthandenzymeproductioninthemedium,

which resultsinenhancementofzeintransform. Highcell

density,isalsorequiredforefficientproteasebiosynthesis.43,44

Thedeterminationcoefficientforthemodelwas0.9852,which

indicatedthatthemodelfitwellwiththedata.Thelackof

error.TheRSMcanbeaccuratelyusedforpredictionand

opti-mizationofzeinhydrolysisbyfungiundertheexperimental

conditionsemployedinthisstudy.

Thereareseveralreportsonmodifyingthe functionality

ofisolated glutelin from barley and corn.31,34 _However,no

available reports about use of enzymatic proteolysis by C.

militaris202formodifyingthefunctionalityofzeincouldbe

found. Thus,we evaluated the feasibilityofimproving the

functional properties of corn zein in an aqueous solution

usingofenzymaticproteolysisbyC.militaris202.Underthe

optimizedconditions, theDH reached27.31%,indicatingC.

militaris202waseffectiveforzeinproteolysis. SincetheDH

ofzeincorrespondstoitsfermentationtimeunderfixed

fer-mentationcondition,thefermentationtimewastakenasthe

factorforevaluatingthephysicochemicalpropertiesofzein

hydrolysates,thusprovidingusefulinformationonpotential

commercial applications. The fermentativehydrolysates of

zeinshowedsignificantlyimprovedsolubility(p<0.05)inpH

7.0aqueousphase.Evenlimitedtimehydrolysisby

fermen-tationwas ableto significantlyimprove thezein solubility.

Solubilityisimportantprerequisiteforthefunctional

prop-erties of protein; the increased solubility can affect their

applicationsinthe food industry.TheFCofzeinwas 45%,

whichwaslower(p<0.05)thanthatofriceprotein(118.8%).25

ThismightbeduetothepoorsolubilityofzeininpH7.0water.

TheFCandFSofzeinhydrolysateproteinwerehigher(p<0.05)

thanthoseofzein,accompanywiththeincreasingsolubility.

Zheng45alsoreportedthattheFCandFSofproteinincreased

afterenzymatichydrolysis.Thehydrolysis byfermentation

significantlyimprovedproteinsolubility(p<0.05),which

facil-itatesthe formationofan interfacialmembrane and foam

production.Nevertheless,theFCandFSofzeinhydrolysates

fermentedfor2–6daychangednotsignificantly.Accordingto

theresults,zeinhydrolysatesexhibitedsolubilityof80.45%but

poorfoamcapacityandstabilityatpH7.0.Thisshowedthat

solubilityisnottheonlyrequirementtoimprovefoam

proper-tiesorotherfunctionalproperties.Thisphenomenonmaybe

ascribedtothelowDH.AsshowninFig.5A,theFSandFCof

proteinsamplesweresignificantlychangedwiththeincrease

offermentationtime.Enzymatichydrolysishasshowntobe

asuitable methodformodifying thefoamingproperties of

proteinasafunctionofthemolecularweightprofileand

com-positionofthedifferentfractionswhich,inturn,dependson

theDH.46

The results (Fig. 6) indicated that zein hydrolysates

prepared by fermentation possessed excellent antioxidant

activity,andtherewassignificantdifferencedetectedamong

differentfermentationtime.Liuet al.47_{discoveredthatthe}

DHofporcineplasma hydrolysatesincreasedwith

increas-ing hydrolysistime, observedfrom 0.5to5hofhydrolysis.

The study of the hydrolysis of peanut peptides thought

there was correlation between DH and antioxidant.48 _In

general, researchers thought the antioxidant activities of

hydrolysatesincreasedwithincreasingDH.Ourresearchalso

showed the similar results, which the antioxidant

activ-ities of hydrolysates (2–8 day) increased with increasing

fermentation time. Nevertheless, the antioxidant

activi-tiesofhydrolysates(10–14day)decreased, whichindicated

there may other factors affect the antioxidant activities

ofhydrolysates,suchas molecularweightdistribution and

aminoacidcomposition.48 _{Inourstudy,the resultrevealed}

thatfermentativehydrolysatescanbeusedforminimizingor

preventinglipid oxidationinfoodproducts,andprolonging

theshelflifeoffoods.

Conclusion

SinglefactorexperimentandRSMwereappliedtooptimize

the liquid-state fermentation conditions and improve the

ability of zein transform ability. The optimized

fermenta-tion conditionswere asfollows:inoculumconcentrationof

19%,volumeofliquorof130mL/500mLandpHof4.7.Under

this condition,theDH was27.31%.Thehydrolysates

main-tained ahighthermalstability. Thezeinhydrolysateswere

foundtohaveahighersolubilitywhichmostlikelyresulted

inimprovedfoamingandemulsifyingpropertiescomparedto

theoriginalzein.Theresultsindicatedthatzeinfermentative

hydrolysatespreparedby fermentationpossessed excellent

antioxidantactivity.Overall,thisresearchindicatedthatzein

hydrolysatescanbepotentiallyusedasfunctionalingredients

withanincreasedantioxidanteffectinbothfoodandnon-food

application.

Conflicts

of

interest

Theauthorsreportnoconflictsofinterestinthiswork.

Acknowledgement

This work was supported by the National Key R&D

Pro-gram of China (Grant Number: 2016YFD0400702), and Jilin

Province “Double Ten Project” Major Scientific and

Tech-nological Projects – Key Technology Research and Product

DevelopmentofHealthStapleFoodofCoarseGrains(Grant

Number:20150201010NY).

r

e

f

e

r

e

n

c

e

s

1.SellingGW,HamakerSAH,SessaDJ.Effectofsolventand

temperatureonsecondaryandtertiarystructureofzeinby

circulardichroism.CerealChem.2007;84(3):265–270.

2.MirmoghtadaieL,AliabadiSS,HosseiniSM.Recent

approachesinphysicalmodificationofproteinfunctionality.

FoodChem.2016;199:619–627.

3.ZhangM,HuangG,JiangJ.Effectsofchemicalmodification

andmolecularweightdistributiononironbindingabilityof

phytate-removalsoybeanproteinisolatehydrolysate.AdvJ

FoodSciTechnol.2012;4(2).

4.LiT,ZhaoC,WuH,etal.Physical-enzymaticmodification

methodsimprovingemulsifyingpropertiesofsoybean

proteinisolateunderacidicconditions.TransChinSocAgric

Eng.2016;18:111.

5.YalcinE,CelikS.Solubilitypropertiesofbarleyflour,protein

isolatesandhydrolysates.FoodChem.2007;104(4):1641–1647.

6.WangXB,ZhangYH,JiangLZ.Improvementofemulsifying

propertiesofsoybeanproteinisolatethroughglycosylation

modification.AdvMaterRes.2013;781–784:1495–1499.

7.CapritaA.Modificationofthesolubleproteincontentof

8. Hai-PingLI,Ju-ZhenYI.Modificationstudyofsoybean

proteinisolate.PolymBull.2009;35(2):58–63.

9. SunXD.Enzymatichydrolysisofsoyproteinsandthe

hydrolysatesutilisation.IntJFoodSciTechnol.

2011;46(12):2447–2459.

10.KonradG,KleinschmidtT,RohenkohlH,ReimerdesEH.

Pepticpartialhydrolysisofwheyproteinconcentratefor

modifyingthesurfacepropertiesofwheyprotein.II.Effects

ontheemulsifyingandfoamingproperties.MilchwissenscSci

Int.2005;60(2):195–198.

11.PanyamD,KilaraA.Enhancingthefunctionalityoffood

proteinsbyenzymaticmodification.TrendsFoodSciTechnol.

1996;7(4):120–125.

12.ZhangYP.Industrializedproductionofbioactivepeptides

fromsoybeanproteinbyfermentation.SoybeanTechnol.

2003;(3):26–27.

13.XiaoY,XingG,RuiX,etal.Enhancementoftheantioxidant

capacityofchickpeasbysolidstatefermentationwith

CordycepsmilitarisSN-18.JFunctFoods.2014;10:210–222.

14.WenTC,LiGR,KangJC,KangC,HydeKD.Optimizationof

solid-statefermentationforfruitingbodygrowthand

cordycepinproductionbyCordycepsmilitaris.ChiangMaiJSci.

2014;41(4):858–872.

15.XieCY,GuZX,FanGJ,GuFR,HanYB,ChenZG.Productionof

cordycepinandmyceliabysubmergedfermentationof

Cordycepsmilitarisinmixturenaturalculture.ApplBiochem Biotechnol.2009;158(2):483–492.

16.KimSW,HwangHJ,XuCP,SungJM,ChoiJW,YunJW.

Optimizationofsubmergedcultureprocessfortheproductionof mycelialbiomassandexo-polysaccharidesbyCordycepsmilitaris C738.2003;94(1):120–126.

17.XiaoY,XingG,RuiX,etal.Effectofsolid-statefermentation

withCordycepsmilitarisSN-18onphysicochemicaland

functionalpropertiesofchickpea(CicerarietinumL.)flour.

Lebensmittel-WissenscTechnol.2015;63(2):1317–1324.

18.KangC,WenTC,KangJC,MengZB,LiGR,HydeKD.

Optimizationoflarge-scalecultureconditionsforthe

productionofcordycepinwithCordycepsmilitarisbyliquid

staticculture.SciWorldJ.2014;2014(8):510627.

19.CuiJD,ZhangBZ.Comparisonofculturemethodson

exopolysaccharideproductioninthesubmergedcultureof

Cordycepsmilitarisandprocessoptimization.LettAppl Microbiol.2011;52(2):123–128.

20.KimHO,YunJW.Acomparativestudyontheproductionof

exopolysaccharidesbetweentwoentomopathogenicfungi

CordycepsmilitarisandCordycepssinensisinsubmerged

mycelialcultures.JApplMicrobiol.2005;99(4):728–738.

21.YangQW,ZhenC,ChenS,etal.Optimizationofenzymatic

hydrolysisforproducingferrousbindingpeptidesfromhorse

mackerel(Trachurusjaponicus)processingbyproducts.

Biotechnology.2013.

22.ZhengXQ,LiLT,LiuXL,WangXJ,LinJ,LiD.Productionof

hydrolysatewithantioxidativeactivitybyenzymatic

hydrolysisofextrudedcorngluten.ApplMicrobiolBiotechnol.

2006;73(4):763–770.

23.RuthM,GroningerHS.Functionalpropertiesof

enzyme-modifiedacylatedfishproteinderivatives.JFoodSci.

2010;41(2):268–272.

24.ZayasJF.EmulsifyingPropertiesofProteins.SpringerBerlin

Heidelberg;1997:134–227.

25.ZhaoQ,SelomulyaC,XiongH,etal.Comparisonof

functionalandstructuralpropertiesofnativeandindustrial

process-modifiedproteinsfromlong-grainindicarice.J

CerealSci.2012;56(3):568–575.

26.XiaY,BamdadF,GänzleM,ChenL.Fractionationand

characterizationofantioxidantpeptidesderivedfrombarley

glutelinbyenzymatichydrolysis.FoodChem.

2012;134(3):1509.

27.HalliwellB.Superoxide-dependentformationofhydroxyl

radicalsinthepresenceofironchelates:isitamechanism

forhydroxylradicalproductioninbiochemicalsystems?

FEBSLett.1978;92:321.

28.LuRR,PingQ,ZhenS,etal.Hempseedproteinderived

antioxidativepeptides:purification,identificationand

protectionfromhydrogenperoxide-inducedapoptosisin

PC12cells.FoodChem.2010;123(4):1210–1218.

29.ArcamoneF,ChainEB,FerrettiA,etal.Productionofanew

lysergicacidderivativeinsubmergedculturebyastrainof

ClavicepspaspaliStevens&Hall.ProcRSocLondon.

1961;155(958):26.

30.ChenC,ChiYJ,ZhaoMY,XuW.Influenceofdegreeof

hydrolysisonfunctionalproperties,antioxidantandACE

inhibitoryactivitiesofeggwhiteproteinhydrolysate.FoodSci

Biotechnol.2012;21(1):27–34.

31.ZhaoJ,TianZ,ChenL.Effectsofdeamidationonstructure

andfunctionalpropertiesofbarleyhordein.JAgricFood

Chem.2010;58(21):11448–11455.

32.LiHM.GraftingModificationofZeinandItsApplication.Zhejiang

University;2016.

33.BouaouinaH,DesrumauxA,LoiselC,LegrandJ.Functional

propertiesofwheyproteinsasaffectedbydynamic

high-pressuretreatment.IntDairyJ.2006;16(4):275–284.

34.ZhengXQ,WangJT,LiuXL,etal.Effectofhydrolysistimeon

thephysicochemicalandfunctionalpropertiesofcorn

glutelinbyProtamexhydrolysis.FoodChem.

2015;172:407–415.

35.CuiZ,LiS.Preparationofbioactivepeptidesbyprotease

hydrolysisofzeininthewater–ethanolsolution.GrainSci

TechnolEcon.2015.

36.TangN,ZhuangH.Evaluationofantioxidantactivitiesof

zeinproteinfractions.JFoodSci.2014;79(11):2174–2184.

37.PanM,JiangTS,PanJL.Antioxidantactivitiesofrapeseed

proteinhydrolysates.FoodBioprocessTechnol.

2011;4(7):1144–1152.

38.KongB,XiongYL.Antioxidantactivityofzeinhydrolysates

inaliposomesystemandthepossiblemodeofaction.JAgric

FoodChem.2006;54(16):6059.

39.ZhangJ,HuiZ,LiW,GuoX,WangX,YaoH.Isolationand

identificationofantioxidativepeptidesfromriceendosperm

proteinenzymatichydrolysatebyconsecutive

chromatographyandMALDI-TOF/TOFMS/MS.FoodChem.

2010;119(1):226–234.

40.FerchichiM,CrabbeE,HintzW,GilGH,AlmadidyA.

Influenceofcultureparametersonbiologicalhydrogen

productionbyClostridiumsaccharoperbutylacetonicumATCC

27021.WorldJMicrobiolBiotechnol.2005;21(6):855.

41.HallsworthJE,MaganN.Cultureage,temperature,andpH

affectthepolyolandtrehalosecontentsoffungal

propagules.ApplEnvironMicrobiol.1996;62(7):2435–2442.

42.ShihIL,TsaiKL,HsiehC.Effectsofcultureconditionsonthe

mycelialgrowthandbioactivemetaboliteproductionin

submergedcultureofCordycepsmilitaris.BiochemEngJ.

2007;33(3):193–201.

43.KirschLS,PintoAC,PortoTS,PortoAL,TeixeiraMF.The

influenceofdifferentsubmergedcultivationconditionson

mycelialbiomassandproteaseproductionbyLentinus

citrinusWalleynetRammelooDPUA1535

(Agaricomycetideae).IntJMedMushrooms.2011;13(2):185–192.

44.HelmoK,Winther-NielsenM,EmborgC.Protease

productivityinchemostatfermentationswithretentionof

biomassonsuspendedparticles.EnzymeMicrobTechnol.

1985;7(9):443–444.

45.ZhengXQ,WangJT,LiuXL,etal.Effectofhydrolysistimeon

thephysicochemicalandfunctionalpropertiesofcorn

glutelinbyProtamexhydrolysis.FoodChem.

46.DavisJP,DoucetD,FoegedingEA.Foamingandinterfacial

propertiesofhydrolyzedbeta-lactoglobulin.JColloidInterface

Sci.2005;288(2):412–422.

47.LiuQ,KongB,XiongYL,XiaX.Antioxidantactivityand

functionalpropertiesofporcineplasmaproteinhydrolysate

asinfluencedbythedegreeofhydrolysis.FoodChem.

2010;118(2):403–410.

48.ZhangY,HuiZ,LiW,GuoX,QiX,QianH.Influenceofthe

degreeofhydrolysis(DH)onantioxidantpropertiesand

radical-scavengingactivitiesofpeanutpeptidesprepared

fromfermentedpeanutmeal.EurFoodResTechnol.