Development
and
optimization
of
a
new
culture
media
using
extruded
bean
as
nitrogen
source
Karla
A.
Batista
*
,
Kátia
F.
Fernandes
LaboratóriodeQuímicadePolímeros,DepartamentodeBioquímicaeBiologiaMolecular,Universidade FederaldeGoiás,Goiânia,GO,Brazil
GRAPHICAL ABSTRACT
ABSTRACT
Thecompositionofaculturemediumisoneofthemostimportantparameterstobeanalyzedinbiotechnological
processeswith industrialpurposes,becausearound30–40% oftheproduction costswereestimatedtobe
accountedforthecostofthegrowthmedium[1].Sincemediumoptimizationusingaone-factor-at-a-time
approachistime-consuming,expensive,andoftenleadstomisinterpretationofresults,statisticalexperimental
designhasbeenappliedtomediumoptimizationforgrowthandmetaboliteproduction[2–5].Inthisscenario,the
useofmixturedesigntodevelopaculturemediumcontainingacheapernitrogensourceseemstobemore
appropriateandsimple.Inthissense,thefocusofthisworkistopresentadetaileddescriptionofthesteps
involvedinthedevelopmentofaoptimizedculturemediumcontainingextrudedbeanasnitrogensource.
InapreviousworkwetestedadevelopmentofnewculturemediabasedonthecompositionofYPDmedium,
aimingtoreducebioprocesscostsaswellastoimprovethebiomassproductionandheterologousexpression.
ThedevelopedmediumwastestedforgrowthofSaccharomycescerevisiaeandPichiapastoris(GS115).
Theuseofculturemediacontainingextrudedbeanassolenitrogensourceshowedbetterbiomassproduction
andproteinexpressionthanthoseobservedinthestandardYPDmedium.
ã2015TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense(http://
creativecommons.org/licenses/by/4.0/).
*Correspondingauthorat:PB131,Goiânia,GOCEP74001-970,Brazil.Tel.:+556235211492;fax:+556235211190. E-mailaddress:karla-batista@hotmail.com(K.A. Batista).
http://dx.doi.org/10.1016/j.mex.2015.03.001
2215-0161/ã2015TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense(http:// creativecommons.org/licenses/by/4.0/).
ContentslistsavailableatScienceDirect
MethodsX
ARTICLE INFO
Keywords:Extrudedbean,Mixturedesign,S.cerevisiae,P.pastoris,Mathematicalmodeling
Articlehistory:Received16December2014;Accepted6March2015;Availableonline12March2015
Methoddetails
Briefly,thedevelopmentof thenewculturemediacontainingextrudedbean assubstitute of traditionalnitrogensource(peptoneandyeastextract)involvedthefollowingsteps:
(1)Productionoftheextrudedbeanflour.
(2)Useofstatisticalapproachtooptimizethecompositionofnitrogensource.
(3)EvaluationoftheyeastgrowthprofileinthenewmediacomparedtoacommercialYPDmedia.
(4)Estimationofthepotentialuseofthenewculturemediaforheterologousexpression.
Productionofextrudedbeanflour
Hard-to-cookcommonbeans(Phaseolusvulgaris,cvcarioca)weregroundinahammermilland siftedinascreenof0.425mm.Then,themoistureofthebeanflourwasadjustedto25%andtheflour waskeptoutatrefrigerator(4C)overnightaimingtoreachawettingbalance.Beforeextrusion,the bean flour was keptout at roomtemperature for 2h and the moisturecontent was confirmed gravimetrically[6].TheextrusionprocesswascarriedoutusingaCerealtecInternationalsinglescrew extruder(CT-L15),withfourzonesofheating,compressionratioscrewof3:1,5mmdieandscrew speedof150rpm.Thetemperaturesusedineachheatingzoneoftheextruderwere80C,140C, 150Cand130C[7,8].Theextrudatesweredriedatroomtemperaturefor12h,milledandsiftedina screenof0.425mm.Thisextrudedbeanflourwasusedasnitrogensourceintheculturemedia. Mathematicalmodeldeterminationforoptimizationofculturemedia
TheinitialcompositionofculturemediawasbasedonthecommercialmediumYPD,widelyused foryeastsgrowth.Thismediumiscomposedby1%yeastextract,2%peptoneand2%dextrose.So,in ordertoevaluatetheeffectivenessofsubstitutingpeptoneandyeastextractforextrudedbean,the compositionoftheculturemediawasoptimizedusinga3-factorsimplex-latticedesign.Thismixture designwasusedtostudytherelationshipbetweentheproportionofthedifferentnitrogensources andtheirrespectiveresponsesintheopticaldensityofSaccharomycescerevisiaeandPichiapastoris (GS115).ThedesignwasimplementedusingStatistica7.0software(StatSoftInc.,Tulsa,OK,USA)and theproportionofeachnitrogensourcevariedfrom0to2%(Table1).
Table1
Mixturedesignandassignedconcentrationsofeachcompoundatdifferentlevelsofthemixturedesign.
Experiment Peptone(X1) Yeastextract(X2) Extrudedbean(X3)
1 2% 0 0 2 0 2% 0 3 0 0 2 4 1% 1% 0 5 1% 0 1% 6 0 1% 1%
Mediapreparation
Thepreparationofculturemediacontainingextrudedbeanasnitrogensourcewascarriedout consideringthepercentageofsubstitutiondeterminedbythemathematicaldesign.Consideringthat extrudedbeanisacomplexmaterialnotcompletelysoluble,afterdissolutionofallcomponentsand beforeautoclaving,themediumwasfiltered throughacellulosic membrane(gauze)toavoidthe presenceoffibrousinsolublematerials.
Effectivenessofculturemediaforgrowthofyeasts
ThedifferentculturemediaproposedbymixturedesignweretestedforthegrowthofS.cerevisiae andP.pastoris(GS115).Priortotestspre-inoculumsweredoneasfollows:5mLofYPDmediumwas prepared and 20
m
L of a culture containing 2104 cells was inoculated. The pre-inoculum of S.cerevisiaewasincubatedat30Cfor24hundershakingat150rpm.RegardingtoP.pastoris(GS115), thepre-inoculumwasincubatedat37Cfor48hundershakingat150rpm.Afterincubation,cells werecentrifugedat4Cfor10minat5000gandthepelletwaswashedwith1mLofsterilesaline solution(0.15molL1).Then,theabsorbanceofcellsuspensionwasadjustedto0.500(l
600nm), usingsterilesalinesolution(0.15molL1)and250m
LofcellsuspensionofS.cerevisiaeorP.pastoris (GS115)strainsweretransferredtoflaskscontaining250mLofthedifferentmedia.AimingtoevaluatethegrowthprofileofS.cerevisiae,theculturemediawereincubatedat37Cand 150rpmandaliquotsof1mLwerewithdrawevery2huntilthestabilizationoftheopticaldensityat 600nm. For P. pastoris (GS 115), which presents a slower growth rate, theculture media were incubatedat30Cand150rpmandthealiquotswerewithdrawevery4h.Aliquotswerecentrifugedat 4C for 10min at 5000g and the cell mass was re-suspended in 1mL sterile saline solution (0.15molL1).Theopticaldensityofcellsuspensionwasdeterminedat600nmbyusingaUV–vis spectrophotometer.Consideringthatculturemediacontainingextrudedbeanarecoloredmixtures (Fig.1),blanksweremadewithrespectiveculturemediawithoutinoculum.
TheresultsofgrowthevidencedthatthebestmediumcompositionforgrowthofS.cerevisiaewas thatcontaining1%extrudedbeanflourand1%peptone(experiment5)or1%extrudedbeanflourand 1%yeastextract(experiment6)plus2%dextrose.Intheotherhand,forP.pastoris(GS115),thebest compositionwasfoundas2%ofextrudedbeanflour(experiment3)plus2%dextrose.
ProteinexpressionusingP.pastoris(GS115)
Considering thathighvaluesofgrowth profiledonotnecessarily meana proportionalprotein expression,theeffectivenessofheterologousexpressionbyP.pastoris(GS115)wastestedinmedium containing2%ofextrudedbeanflour.Thesetupoftestsforrecombinantproteinexpressionusing P.pastoris(GS115)wascarriedoutasrecommendedbythesupplier(Invitrogen,Carlsbad,CA).Starter cultureswerepreparedthroughinoculationof1mLofP.pastoris(GS115)culture(106cells)in100mL ofYPDmedium,incubatedat30Cand150rpmfor24h.Afterthisinitialgrowthphase,20mLofthe recombinantculturewasharvestedbycentrifugation(10,000g,5min)andcellpelletwaswashed twicewith 10mL of 100mmolL1 sodium phosphate buffer(pH 6.0). Thewashed recombinant
P.pastoris(GS115)cellswerere-suspendedin20mLoftheexpressionmediumsupplementedwith 1mLof20%(v/v)methanol,preparedwithsterilemilliQwater.
TheproteinexpressionbyP.pastoris(GS115)wastestedusingthestandardmedium(1%yeast extract, 2% peptone, 2% dextrose and 3% glycerol (UtraPureTM, Invitrogen)), and the medium containingextrudedbean(2%extrudedbean,2%dextroseand3%glycerol).Theexpressionsystems wereincubatedat30Cfor96hat150rpm,withtheadditionof1mLof20%methanol(preparedwith sterilemilliQwater)every24h.
Aliquots of 1mL of culture were collected every 24h and analyzed for the production of extracellular protein. The cell-free supernatant from the expression medium was recovered by centrifugationat4Cfor10minat5000g.Totalproteinsweresubsequentlyprecipitatedwithcold acetone(1:2v/v), incubated at 80C for 2h, centrifugedfor 5min(4C and 5000g) and re-suspendedin100
m
Lof100mmolL1sodiumphosphatebuffer(pH6.0).Totalproteinconcentration wasdeterminedfollowingthemethoddescribedbyBradford[9].Theprecipitatedproteinswereanalyzedforpuritybyusing10%denaturizingpolyacrylamidegel electrophoresis(SDS-PAGE)[10].Theproteinsampleswerepreparedbymixing2(doublestrength) gelloadingbuffer(10mMTris-HClpH6.8,4%SDS,0.2%bromophenolblue,20%glycerol)and10%
b
-mercaptoethanolmixedintheproportionof1:1,boiledfor5min,centrifuged(10,000g)for2min, andthenloadedontogel.Thetotalproteinwascalculatedtobeapproximately20m
g.Electrophoresis wasrunat25–40mAfor2hatroomtemperature.GelswerestainedwithComassieBlueR-250. Pre-stainedmolecularweightmarkers(NewEnglandBiolabs,MA)rangingfrom7to175kDawereusedas arunningstandard.TheimageanalysisofthegelwasperformedbyusingtheprogramImageJ1.46r (WayneRasband,NationalInstituteofHealth,USA).Theparametersforobtaining/recordingthegel imagewere300dpi-resolution,100%-zoom,and16bitsperpixel-depth.Statisticalanalysis
Thestatisticalanalysisoftheexperimentalmixturedesignwasperformedbymultivariateanalysis. Themodelwassimplifiedtoexcludetermsthatwerenotconsideredstatisticallysignificant(p>0.05) byanalysisofvariance(ANOVA).Thequalityofthepolynomialmodelequationwasjudgedbyusing thecoefficientofdeterminationr2.
Theresponsefunction(Y)ofthemixturemodelwasexplainedbythefollowingequation: y¼X q i¼1
b
ixiþ XXq i<jb
ijxixj (1)Geometrically,inEq.(1)theparameter
b
irepresentstheexpectedresponseofthepuremixture xi=1,xj=0,j6¼i.The termgivenbyy¼Xq
i¼1
b
ixi represents theresponsewhen blendingis strictly additivesandtherearenointeractionsbetweenthecomponentsofthemixture.Thequadraticterm
b
ijxixj represents the excess response over the linear model due to interaction between two components[11].The mixture design and all subsequent tests were conducted in triplicate and the level of significancewas95%.AllanalyseswerecarriedoutbyusingthesoftwareStatistica7.0(StatSoftInc., Tulsa,OK,USA).
Acknowledgments
MethodsXthanksthereviewersofthisarticlefortakingthetimetoprovidevaluablefeedback.The authorsacknowledgefinancialsupportfromtheCNPq(Universal479649/2010-3).
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