h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /
Medical
Microbiology
Lactobacillus
plantarum
BL011
cultivation
in
industrial
isolated
soybean
protein
acid
residue
Chaline
Caren
Coghetto
a,
Carolina
Bettker
Vasconcelos
a,
Graziela
Brusch
Brinques
b,
Marco
Antônio
Záchia
Ayub
a,∗aFederalUniversityofRioGrandedoSul,Biotechnology&BiochemicalEngineeringLaboratory(BiotecLab),PortoAlegre,
RS,Brazil
bFederalUniversityofHealthSciencesofPortoAlegre,NutritionDepartment,PortoAlegre,RS,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received2June2014 Accepted17February2016 Availableonline2July2016 AssociateEditor:RosaneFreitas Schwan
Keywords:
LactobacillusplantarumBL011 Lacticacid
Liquidacidproteinresidueof soybean
Plackett–Burmandesign Industrialproductionofbiomass
a
b
s
t
r
a
c
t
In this study,physiological aspects of Lactobacillus plantarum BL011 growing in a new, all-animalfree mediuminbioreactors wereevaluated aimingattheproductionofthis importantlacticacidbacterium.Cultivationswereperformedinsubmergedbatch biore-actorsusingthePlackett–Burmanmethodologytoevaluatetheinfluenceoftemperature, aerationrateandstirringspeedaswellastheconcentrationsofliquidacidproteinresidue ofsoybean,soypeptone,cornsteepliquor,andrawyeastextract.Theresultsshowedthat allvariables,exceptforcornsteepliquor,significantlyinfluencedbiomassproduction.The bestconditionwasappliedtobioreactorcultures,whichproducedamaximalbiomassof 17.87gL−1,whereaslacticacid,themostimportantlacticacidbacteriametabolite,peaked at37.59gL−1,correspondingtoaproductivityof1.46gL−1h−1.Thisisthefirstreportonthe useofliquidacidproteinresidueofsoybeanmediumforL.plantarumgrowth.Theseresults supporttheindustrialuseofthissystemasanalternativetoproduceprobioticswithout animal-derivedingredientstoobtainhighbiomassconcentrationsinbatchbioreactors.
©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Introduction
Inbioprocesses,variousconstituents canbeusedtomatch culturemediacompositionsforlacticacidbacteria(LAB) pro-duction;however,akeyissueisthecostinvolved.Theuseof
∗ Correspondingauthor.
E-mail:mazayub@ufrgs.br(M.A.Ayub).
agro-residuestoobtain biotechnology-derivedproductshas received great attention in recent years,1,2 and several
by-productsandrawmaterialsofthefoodindustry havebeen used in bioprocesses. Thelarge availability ofthese mate-rials, along with their low or even very low costs, makes themalternativesourcesofsubstratesthatcouldbeusedfor
http://dx.doi.org/10.1016/j.bjm.2016.06.003
1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
bacteria cultivations.3 Among common agro-residues are
whey, whey permeate,1 starchy wastewater,4 corn steep
liquor,2 and lignocellulosic materials.5,6 The production of
soyproteinisolateandsoyproteinconcentrate,bothinlarge demandintheworldmarket,generatesliquidacidstreams ofprotein-richresidueasaresultofthewashingstepsand separationoftheisolatesoyprotein.Thisliquidfraction com-prises sugars and proteins of low molecular weight. This agro-residueisdischargedasanindustrialeffluent,andithas neverbeentestedinbioprocessesbefore.
Fromthenutritionalpointofview,thereisagrowing inter-estofprofessionalsandconsumersinhealthierdietsthat,in additiontoprovidingbasicnutrients,willalsohavebeneficial effectsonhealth,andprobioticsareamongthemost impor-tantcomponentsofsuchdiets.7–9 Thecurrentdefinitionof
probiotics,givenbytheWorldHealthOrganization,statesthat probioticsarelivemicroorganismsthat,whenadministered inadequateamountsindiets,willconferhealthbenefitsfor thehost,humanoranimal.BacteriaofthegeneraLactobacillus
andBifidobacteriumareclassifiedamongthemainprobiotics consideredsafeforfoodandfeeduse,andtheyareproduced inindustrialscales.10Therefore, thereisagreatinterest in
screeningfornewpotentiallyprobioticstrainsofLABs,such asLactobacillusplantarum.
Traditionally,probioticsaregenerallyaddedtoorare com-ponentsofdairyfoods. However,therehasbeenagrowing demandforprobioticsbynon-dairyconsumersdueto increas-ing vegetarianism as a dietary preference throughout the world,andalargepartoftheworldpopulationhasallergiesto dairyproductscausedeitherbylactoseormilkproteins.11–13
Theseissues,associatedwithconcernsoftheoccurrenceof bovinespongiformencephalopathybysomeconsumers, espe-ciallyinEurope,haveincreasedthedemandforallanimal-free foods,14andthereisastronggrowingdemandfornew
prod-uctsinthesemarkets.Inthecaseofprobiotics,itwouldbe highlydesirableiftechnologycoulddeliverbiosystemsforcell growthfreeofanyanimalcomponentstopreventthetransfer oftheseconstituentstothefinalproduct.15
Amongprobioticbacteriaorpotentiallyprobiotics depend-ing on the strain, L. plantarum is a versatile lactic acid bacteriumfoundinavarietyoffoodsandotherenvironments suchasthehumangastrointestinal-tract.16L.plantarumissafe
tobeusedinfoodproducts.17Thisprobiotichasbeenstudied
byseveralauthorsthatuseddifferentagro-residuesubstrates forbiomassproduction,andithasbeenstudiedforits metabo-litesofinterest,especiallylacticacid.Suchsubstratesinclude malt,wheat,barleyextracts,18 molassesstillage,sugarbeet
molasses,19coffeehusks,tamarindseedpowder,groundnut
oilcake,wheatbrans,ricebrans,bengalgrampowder,black gram flour,green gramflour, barley, millet,ragi, oats,corn flour,riceflour,20yorkcabbage,21quinoa,andwheatslurry.22
Inthiscontext,theaimsofthisresearchweretoinvestigate thegrowthandfermentationactivityofL.plantarumBL011,a strainisolatedfromSerranocheese,23 usinganew,
alterna-tiveculturemediumthatiscompletelyfreeofanimal-derived componentsandisinexpensive.Thegrowthandnutritional conditionsthatcouldhaveaninfluenceonbiomassformation wereinvestigatedusingthePlackett–Burmandesign method-ology.Wealsofollowedandanalyzedtheproductionoflactic acid along the process because it is the most important
metaboliteofLAB.Finally,theprocesswasscaled-upto sub-mergedculturesinbatchbioreactors.
Materials
and
methods
Microorganismandchemicals
TheL.plantarumBL011strain,whichwasisolatedbyourgroup fromSerranocheeseanddescribedelsewhere,23wasusedin
this study.ThisstrainwasidentifiedasL.plantarumBL011, anditismaintainedattheMicrobiologyCultureCollectionof BiotecLab(UFRGS,PortoAlegre,Brazil).Thestrainwas iden-tified atDSMA Laboratory (Mogi das Cruzes, SP, Brazil) by comparingthe16SrRNAampliconsequenceswithGenBank databases (accessnumberAB5989861),which showed100% homologywithL.plantarumgenusandspecies.Workingstocks ofculturesweremaintainedin20%(volumefraction)glycerol suspensionfrozenat−22◦C±1◦C.
Unlessotherwisestated,allchemicalsusedinthisresearch wereofanalyticalgradeandpurchasedfromSigma–Aldrich (St.Louis,USA).
Inoculumpreparation
Erlenmeyerflasks(2000mL)containing400mLofMRS24were
inoculatedwith1.5mLofglycerolstockculture,andthe cul-tureswereincubatedat37◦C±1◦Cinarotaryshaker(MA830, Marconi,Piracicaba,Brazil)at180rpmandgrowntoanoptical density(OD)of1.0at600nm.Thecellswereharvestedby cen-trifugation(Hitachi,HimacCR21E,Tokyo,Japan)at3500×g
for15minat4◦C±1◦C.Thecellpelletwaswashedand resus-pendeddirectlyinto thecultivationbroth(150mL),and the compositionofthebrothwasvariedaccordinglytothe exper-imentaldesigndescribedinTables1and2.Thisprocedure wasusedasthestandardinoculumpreparationforall exper-iments.
Liquidacidproteinresidueofsoybean(LAPRS)
preparationandconcentration
The LAPRS is the liquid fraction resulting of the washing and separationstepsduringtheisolatesoyprotein produc-tion. Thisliquid fractioncomprises sugars and proteinsof lowmolecularweight.TheLAPRSwaskindlydonatedbythe SolaeCompany(Esteio,RioGrandedoSul,Brazil).Thisresidue was collected in the industrial plant in the precipitation stage, which is the first unit operation in the wastewa-ter treatmentplant. The liquid was immediately storedin 5000mL polypropylene containers, sealed and transported underrefrigerationtothelaboratorywhereitwasimmediately vacuum-concentratedat45◦C±1◦Cfor4handsubsequently storedat−22◦C±1◦Cuntilexperiments.Thisoperationwas performedtoconcentratethesugarcontentofsamples.The LAPRS, ascollected intheindustry,hadthefollowing com-position(ingL−1):totalsugars,10;protein,2;lipids,1;ashes (mineralsalts),2;andpH,4.6.Theanalysesofprotein,lipids, ashes,andtotalsugarwerequantifiedaccordingtotheAOAC methods.25
Table1–IndependentvariablesstudiedinthePlackett–BurmandesignforthecultivationofL.plantarumBL011. Variables/level Temperature (◦C) Cornsteep liquor(gL−1) LAPRS(gL−1) Peptoneof soy(gL−1) Yeastextract (gL−1) Stirringspeed (rpm) Aerationrate (vvm) −1 25 5 5 2 2 200 2.5 0 34 12.5 12.5 8.5 8.5 300 3.5 +1 37 20 20 15 15 400 4.5
Table2–Plackett–BurmanexperimentaldesignmatrixforbiomassandlacticacidproductionofL.plantarumBL011.
Trialno. Variablesa/levelb Biomass(gL−1) Lacticacid(gL−1)
X1 X2 X3 X4 X5 X6 X7 D1 D2 D3 D4 D5 D6 D7 D8 1 − − − − + + + + + + − − − − + 4.39 7.06 2 − − − − − − − + + + + + + − − 0.44 4.74 3 − + − − − + + − − + + + − + − 2.21 9.46 4 + + − − + − − + − + − − + + + 2.31 13.63 5 − − + − + − + − + − + − + + − 7.74 21.09 6 + − + − − + − − + − − + − + + 1.46 12.06 7 − + + − − − + + − − − + + − + 3.07 15.88 8 + + + − + + − + − − + − − − − 4.61 20.77 9 − − − + + + − + − − − + + + − 0.90 6.29 10 + − − + − − + + − − + − − + + 0.58 5.69 11 − + − + − + − − + − + − + − + 0.70 8.10 12 + + − + + − + − + − − + − − − 2.16 14.62 13 − − + + + − − − − + + + − − + 4.39 10.01 14 + − + + − + + − − + − − + − − 1.99 11.50 15 − + + + − − − + + + − − − + − 5.36 16.21 16 + + + + + + + + + + + + + + + 2.56 19.61 17 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2.12 12.38 18 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2.14 13.66 19 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2.33 17.00 a X
1temperature,athighestlevelof37◦C,centrallevelof31◦C,andlowestlevelof25◦C;X2cornsteepliquorconcentrationathighestlevel
of20.0gL−1,centrallevelof12.5gL−1,andlowestlevelof5.0gL−1;X3totalsugars(LAPRS)athighestof20.0gL−1,centralconcentrationof
12.5gL−1,andlowestconcentrationof5.0gL−1;X4peptoneofsoyathighestconcentrationof15.0gL−1,centralconcentrationof8.5gL−1,
andlowestconcentrationof2.0gL−1;X5yeastextractathighestconcentrationof15.0gL−1,centralconcentrationof8.5gL−1,andlowest
concentrationof2.0gL−1;X6stirredagitationathighestlevelof400rpm,centrallevelof300rpm,andlowestlevelof200rpm;X7aerationrate
athighestlevelof4.5vvm,centrallevelof3.5vvm,andlowestlevelof2.5vvm;D1,D2,D3,D4,D5,D6,D7andD8aredummyvariables. b (+)Highestconcentrationofvariable;(−)lowerconcentrationofvariable;(0)centrallevelofvariable.Valuesofbiomassandlacticacidwere
measuredat48hofcultivation.
Priortouseincultivations,theLAPRSwasconcentratedto 50or25%ofitsoriginalvolume,resultinginfinalsugar con-centrationsof20gL−1 (LAPRS20,forthescreeningvariables design)and 40gL−1 (LAPRS40,usedinthe allother experi-ments),respectively.
Cultivationprocedures
Screeningofvariables
APlackett–Burman(PB)designwasusedtodeterminewhich nutrientsandconditionshadasignificanteffectonL. plan-tarum BL011 biomass formation, the dependent variable, whichispresentedinTable1.26Theconcentrationsand
val-ues fortheindependent variables were fixedbased onthe generalvaluesfoundontheliteratureforstudieswiththis microorganismandonapreviousresearchfromourgroup.1
Lacticacidproductionwasalsofollowedintheseexperiments becauseitisthemostrelevantmetaboliteofthisbacterium (Table2).Sevenrealandeightdummyvariableswerescreened in19trialswithtriplicatesatthecentralpoint.Theminimal andmaximal rangesselectedforthe sevenparametersare presentedinTable 2,in which each columnrepresents an
independentvariable,andeachrowrepresentsatrial. Vari-ables withconfidencelevels>95% wereconsidered tohave significantinfluenceonbiomassproduction.
Inthe investigationofvariableswithasignificant effect on biomass production, cultivations were performed with LAPRS20mediumcontainingthefollowingadditives(ingL−1): MgSO4·7H2O,0.2;andMnSO4·H2O,0.04.Thismineralsolution wasalsousedatthesameconcentrationsforallcultivations in this study. The other components of the mediumwere soypeptone,cornsteepliquor,andrawyeastextract (auto-lysednon-purifiedyeast extract;Prodesa, SP,Brazil), which isaninexpensivesourceofmineralandvitamins.Avolume of150mLofinoculumwastransferredtoabioreactor filled with 1350mLof medium, and the compositions were var-ied inaccordancewith thespecificassay ofthePB design. Theseexperimentswereperformedusinga2000mLBiostat Bbioreactor(B.BraunBiotechInternational,Melsungen, Ger-many),andthepHwassetat5.5±0.21andwascontrolledby
theautomaticadditionof10MNaOHor1MH3PO4.The dis-solvedoxygenconcentration(DOC)ofcultureswasmeasured usingapolarographicO2probe(Mettler-Toledo,Germany).The volumetric oxygen mass transferrate (kLa) was calculated
Table3–EstimatedeffectsforbiomassproductioncalculatedfromtheresultsofthePlackett–Burmandesign.
Variables Parameters Effect p-Value
X1 Temperature* −1.58 0.0013
X2 Cornsteepliquor 0.14 0.1431
X3 LAPRS* 2.19 0.0007
X4 Peptoneofsoy* −0.95 0.0037
X5 Yeastextract* 1.66 0.0012
X6 Stirringspeed* −0.90 0.0041
X7 Aerationrate* 0.57 0.0103
Standarderror=0.58;p-values≤0.5;R2:0.98. ∗ Statisticallysignificantat95%confidencelevel.
usingthedynamicgassingoutmethod.27Intwoexperiments,
the DOC was keptat a minimum of30% ofsaturation by controllingthe agitationspeed throughacontrol-loop. The temperature,aerationrate,andstirringspeedwereset accord-ing to the PB design (Table 2). A confirmation experiment usingthe best conditions foundinthe PB designwas per-formedinthesamebioreactordescribedabove,exceptforthe LAPRSconcentration, whichwas doubled.Themediumfor thisexperimentwascomposedof15gL−1 rawyeastextract and1500mLofLAPRS40,andtheconditionswere25◦C,pH 5.5±0.2,200rpmagitation,and4.5vvmaeration.
Saccharificationandco-saccharificationinthebioreactor
TohydrolysethepolymericsugarspresentinLAPRS, saccha-rification reactionsof this substrate were performedusing commercial invertase through the following two different approaches:(1)saccharificationfollowedbyfermentationas independent and sequential reactions (separate hydrolysis followed by fermentation); and (2) co-saccharification and fermentationperformedas asinglereaction (simultaneous hydrolysisandfermentationinthesamevessel).
LAPRShydrolysiswasperformedusinginvertasefrom S. cerevisiae(MaxinvertL10000,batch409200451,DSMFood Spe-cialties,TheNetherlands.Enzymeactivity(1U)wasdefined as 1mol glucose released from sucrose per minute at 50◦C±1◦Cand42%sucrose(massfraction)).Forcondition1, 1500mLof LAPRS40 wasadded (18.21U) ina2-L flaskand incubated at50◦C and pH 4.6for3h underagitation. The othernutrientswerethenaddedtothehydrolysedsolution andusedforfermentationwithoutfurthermodifications.For condition2consistingoftheco-saccharificationand fermen-tation,theenzymewasdirectlyaddedtothefinalmediumin thebioreactorinthepresenceofcellsundertheconditionsof thefermentation.Alltrialswereperformedinduplicates.
All cultivations were performed in the bioreactors as describedaboveunderthesameconditions(25◦C,pH5.5±0.2, 200rpmagitation,and4.5vvmaeration).
Analyticalmethods
Samples(10mL)ofculturebrothwerecentrifugedat3500×g
for15minat4◦C±1◦C.Thecell-freesupernatantwasused forthequantificationofsugarsandlacticacid.Biomasswas measuredgravimetricallyasdryweightofcells.Sampleswere centrifuged,washedtwicewithcolddistilledwater,anddried
inpre-weighedplastictubesat80◦C±1◦Ctoconstantweight invacuumovens.Totalsugarconcentrationwasdetermined by the Duboismethod using glucose as astandard.28
Lac-ticandaceticacidconcentrationsweredeterminedbyHPLC (Shimadzu, Kyoto, Japan) equipped with arefractive index detector and Bio-Rad HPX-87H column (300mm×7.8mm) using5mMsulphuricacidastheeluentat65◦C±1◦C,flow rate of0.8mLmin−1 and sample volumeof20L.Samples were filteredthrough celluloseacetate0.22mmembranes priortoHPLCinjection.
Dataanalysis
Allexperimentaldesignsandresultsanalyseswereperformed usingStatistica10.0(Statsoft,Tulsa,USA).
Results
and
discussion
Experimentaldesign
ThePlackett–Burmandesignwasusedtoevaluatetheeffects ofconcentrationsofLAPRS,soypeptone,cornsteepliquor,and rawyeastextractasmediumcomponentsaswellas tempera-ture,stirringspeed,andaerationrateascultivationconditions on L. plantarumBL011 biomassformation (dependent vari-able)andlacticacidproduction(importantmetabolite).Table2
presents the PB experimentaldesignfor19trials withtwo study levels for each variable as well as the correspond-ing biomass productionafter48h ofbioreactor cultivation.
Table3presentsthestatisticalanalysesofthestudied vari-ables. Exceptforthe concentrationofcornsteep liquor,all othervariableswerefoundtobesignificantatthe95%level forbiomassproduction.Thehighestvaluesofbiomass for-mation(7.74gL−1)andlacticacidproduction(21.09gL−1)were obtainedintest 5.ThevalueofkLameasured underthese conditionswas30h−1(200rpm,4.5vvm,andmid-exponential growthphase),indicatingthatthisaerationconditioncould beadoptedforscaling-uptheprocesstoindependentlyallow testingofothervariables.
Temperatureandsteeringspeedhadsignificantnegative effects.Therefore,forthenextsetofexperimentsinthe biore-actor,thesevariablesweredecreasedandset at25◦C±1◦C and200rpm,respectively.Theaerationrateat4.5vvmshowed significant positive effects. Corn steep liquor, which was the only variable that did not have a significant effect, was excludedfrom thenext experiments. Finally,although
10 40 30 20 10 0 0.1 Biomass (g L –1 )
Lactic acid, acetic acid, total sugars (g L
–1 ) 1 0 10 20 30 Time (h) 40 50
Fig.1–TimecourseofbatchcultivationsofL.plantarum BL011inmediumcontaining(gL−1):MgSO4·7H2O,0.2;
MnSO4·H2O,0.04;LAPRS,40(totalsugars);yeastextract,15.
Cultureconditions:25◦C±1;4.5vvm;200rpm,pH5.5±0.2; ()drycellweight;(䊉)lacticacidconcentration;()acetic acidconcentration;()totalsugarsconcentration.The resultsarethemeanofduplicates.
statisticallysignificant,thesoy peptoneshoweda negative effect,andbecauseithasahighcost,itwasremovedfrom themediumcompositionforthenextexperimentalstep.
Basedon the resultsobtained inthe PB design, experi-mentswereperformedtoconfirmthebestconditionsforL. plantarumBL011cultivation.Theconditionswerefixedas fol-lows:kLaof30h−1(obtainedusingthecombinationof200rpm and 4.5vvm),25◦C,pHcontrolledat5.5, 15gL−1 raw yeast extract,and 1500mLofLAPRS40.Thedoubling ofthetotal amount sugars ofLAPRS from 20 to40gL−1 was proposed becausethisvariableproducedapositiveandsignificanteffect onthePB.Fig.1depictsthekineticsofthesecultivations.The maximalbiomassobtainedwas10.85±0.03gL−1,whichwas 28%higherthan thebest conditionfoundinthePB design duetothehighersugarconcentration.Thehighestlacticacid concentrationwas achievedat12h ofcultivation, reaching 19.56±0.73gL−1 withaproductivityof1.54gL−1h−1,which wassimilartovaluesofthePBdesign.Theaceticacid concen-trationobtainedwas18.43±0.25gL−1in48hofcultivation.
Theremainingsugarleftincultivationmightbea conse-quenceoftheinability ofL.plantarumBL011tocompletely hydrolyse all sugars present in the LAPRS because this vegetablesourcecontainssomeoligosaccharides,suchas raf-finoseand stachyose,whichrequire enzymatic breakdown. Themainenzymes involvedinthehydrolysisofthese sug-arsarealpha-galactosidaseandbeta-fructosidase(invertase), whichshowedtohavenegligibleactivitiesforthisstrain grow-ingundertheconditionsofthePBinthisstudy(resultsnot shown).
Saccharificationandco-saccharificationinthebioreactor
Because the absence of invertase activity for L. plantarum
BL011wasconfirmed,twosetsofenzyme-mediated sacchari-ficationexperimentsweredevisedintendingtoincreasethe hydrolysis ofsugars of LAPRS, consequently offering more sugarsforbacterialmetabolism.ThehydrolysisofLAPRSwas
10 40 30 20 10 0 0.1 Biomass (g L –1 )
Lactic acid, acetic acid, total sugars (g L
–1 ) 1 0 20 40 Time (h) 60 80
Fig.2–TimecourseofbatchcultivationofL.plantarum BL011inhydrolysedLAPRSusinginvertase.Medium composition(gL−1):MgSO4·7H2O,0.2;MnSO4·H2O,0.04;
LAPRS,40(totalsugars);yeastextract,15.Culture
conditions:25◦C±1;4.5vvm;200rpm,pH5.5±0.2;()dry cellweight;(䊉)lacticacidconcentration;()aceticacid concentration;()totalsugarsconcentration.Theresults arethemeanofduplicates.
doneusinginvertasebecausethisenzymecleavesthe-1.2 bondsin raffinose andstachyose,producing melibiose and fructose,respectively.Inthefirstexperiment,saccharification wasperformedindependentlywithenzymatichydrolysis fol-lowed by fermentation, and the results of the cultivation kinetics are presentedinFig. 2.In the second experiment, saccharificationand fermentationwereperformed simulta-neously,andtheresultsofthecultivationkineticspresented in Fig. 3. Although the growth kinetics were similar, both showing faster consumption of sugars when compared to the PB design, the hydrolysis of LAPRS before cultivation producedbetterresultsthanco-saccharificationand fermen-tation because conditions for hydrolysis and fermentation
10 40 30 20 10 0 0.1 Biomass (g L –1 )
Lactic acid, acetic acid, total sugars (g L
–1 ) 1 0 20 40 Time (h) 60 80
Fig.3–Timecourseofsimultaneoussaccharificationand cultivationofL.plantarumBL011.Mediumcontaining (gL−1):MgSO4·7H2O,0.2;MnSO4·H2O,0.04;LAPRS,40(total
sugars);yeastextract,15.Cultureconditions:25◦C±1; 4.5vvm;200rpm,pH5.5±0.2()drycellweight;(䊉)lactic acidconcentration;()aceticacidconcentration;()total sugarsconcentration.Theresultsarethemeanof duplicates.
were more ideal than the combined process with the fol-lowingresults:theobtainedbiomasseswere13.39±0.10gL−1 and 11.4±0.48gL−1, respectively; the lactic acid peaked at concentrationsof42.04±0.08gL−1(24h)and37.36±0.81gL−1 (24h),respectively;andtheaceticacidpeakedat concentra-tions of32.35±0.79gL−1 (72h) and 22.71±0.19gL−1 (72h), respectively.Theproductivitiesoflacticacidmeasuredat24h of cultivation were 1.71gL−1h−1 and 1.46gL−1h−1 for the saccharification and co-saccharification, respectively. Com-paratively,Sikder etal.29 reportedalactic acidproductivity
of1.24gL−1h−1byL.plantarumNCIM2912growingin sugar-canejuicemediumcontaining140gL−1totalsugars(sucrose, 126;glucose,8;andfructose,6).Laopaiboonetal.30obtained
alacticacidproductivityofonly0.36gL−1h−1inculturesofL. lactisIO-1(JCM7638)usinghydrolysedsugarcanebagasseasa substrate(30gL−1).
The results obtained using LAPRS40 for the growth of
L.plantarumBL011 resultedinthe highestbiomass produc-tioncomparedtotheliterature.Forinstance,Brinquesetal.1
reportedamaximalbiomassof10.2gL−1 L.plantarumusing cheesewhey(equivalentto140gL−1oflactose)asthemedium carbonsource.Gaoetal.31reportedafinalbiomassproduction
of6.6gL−1 L.rhamnosusNBRC3863whengrowingthisLAB inhydrolysedfishwaste.Muetal.32obtainedamaximalcell
densityof3.34gL−1Lactobacillussp.SK007inamedium con-sistingofglucose(30gL−1),yeastextract(30gL−1),andcorn stepliquor (47gL−1). Hwanget al.33 formulated acomplex
mediumcomposedofdifferentsugarsandnitrogensources forbiomassproductionofL.acidophilusDGK,andtheseauthors reportedmaximalbiomassproductionof4.54gL−1under aer-obicconditions.Finally,S.boulardiiMYA-769wasproducedina mediumconsistingofgrassjuicefeedstockextractedfrom rye-grassLoliumperenne,anditreachedabiomassof4.96gL−1.34
TheresultsobtainedusingtheLAPRSasthesolecarbon sourcedemonstrated that this industrial residue is a suit-ablesubstrateforthegrowthofL.plantarumBL011,whichhas notbeenreportedintheliterature.Anindustrialplantofsoy proteinisolatecanproducealargevolumeofacidresidue, suchasthecompanythatsuppliedthematerialforthisstudy, whichhasamonthlydischargeofapproximately5×104m3 ofLAPRS (dataprovidedbythecompany).When compared tootherresiduesreportedintheliteratureorevenwith syn-theticmedia,LAPRSallowsforasignificantimprovementof biomassformation.Moreover,theothercomponentofthe cul-turemedium,thenon-purifiedrawyeastextractobtainedas thewasteofbeerproduction,hasalowcostcomparedtothe traditional,purifiedyeastextractusedforcellgrowth,further supportingthehypothesisthattheculturemediumusedin thisworkisinexpensiveandsuitableforLactobacillus cultiva-tions.
Figs.2and3showtheprofileoflacticacidformation,which isthemostimportantmetaboliteoflacticacidbacteria,andits subsequentconversionintoaceticacidbyL.plantarumBL011. Lacticacidsupportedasmalldiauxicgrowthtowardstheend ofcultivation, butit wasmainlyconvertedintoacetic acid.
L.plantarumshowedflexibleandadaptivebehaviourforthe first time in the chromosome of the WCFS1 strain, which encodes a variety ofproteins related to sugar uptake and utilization,35thusallowingL.plantarumtogrowonseveral
car-bonsources.Thegenesencodingtransportersaregenerally
10 40 50 30 20 10 0 0.1 Biomass (g L –1)
Lactic acid, acetic acid, total sugars (g L
–1)
1
0 10 20 30 40
Time (h)
50
Fig.4–TimecourseofbatchcultivationofL.plantarum BL011underDOCof30%saturationorhigher.Medium composition(gL−1):MgSO4·7H2O,0.2;MnSO4·H2O,0.04;
LAPRS,40(totalsugars);yeastextract,15.Culture
conditions:25◦C±1;4.5vvm;200rpm,pH5.5±0.2()dry cellweight;(䊉)lacticacidconcentration;()aceticacid concentration;()totalsugarsconcentration.Theresults arethemeanofduplicates.
inclusteredgenecassettesencodingenzymesandregulatory proteinsimplicatedinsugarmetabolism.16
Theconversionoflacticacidintoaceticacidunderaerobic conditionsresultsinoneATPgeneratedvialactate dehydro-genase,pyruvateoxidase,pyruvatedehydrogenase,pyruvate formatelyase,phosphotransacetylase,andacetatekinase.36
Thispathwayalsoproduceshydrogen peroxideand carbon dioxide.Hydrogenperoxideisgeneratedbytheconversionof oxygenthroughthemanganese-dependentprocess.Thefinal accumulationofaceticacid,insteadoflacticacid,causescell pHhomeostasis.36
ExperimentswithDOCataminimumof30%saturation
Althoughaerationandagitationspeedofturbinesinthe biore-actorwereappliedtoguaranteeadequateoxygensupplyto cultures,theDOCmayeventuallyfallbelowthecritical oxy-genconcentration,whichwouldcauseashiftfromaerobiosis to anaerobiosis. Therefore, wedecided toperformcultures under the best conditions, except for aeration, which was alwaysmaintainedabove30% ofDOCusingacascade loop intheaerationspeed.Theresultsoftheseexperimentsusing the best conditions obtainedforthe PB design(Fig. 1) and for thesaccharification (Fig. 2)are shown inFigs. 4and 5, respectively.Fig.4showsamaximumbiomassproductionof 14.86±0.19gL−1,whichwasapproximately37%higherthan inconditionswithoutDOCcontrol.Lacticacidalsoincreased upto22.34±0.3gL−1(comparedto19.56gL−1).Inthe exper-iments wherethe LAPRS washydrolysed(saccharification), thebiomasswas17.87±0.25gL−1,whichwasanincreaseof approximately 33.5%relativetothe conditionwithout DOC control,whereaslacticacidpeakedat37.59±0.4gL−1(Fig.5). Under the condition of DOC controlled at 30% of satura-tion,lacticacidwascompletelyconsumedtoformbiomass and acetic acid, whichare the twoproducts atthe endof cultivation. The resulting biomass in the saccharification
10 40 50 30 20 10 0 0.1 Biomass (g L –1)
Lactic acid, acetic acid, total sugars (g L
–1)
1
0 15 30 45 60
Time (h)
75
Fig.5–TimecourseofbatchcultivationofL.plantarum BL011inhydrolysedLAPRSunderDOCof30%saturationor higher.Mediumcomposition(gL−1):MgSO4·7H2O,0.2;
MnSO4·H2O,0.04;LAPRS,40(totalsugars);yeastextract,15.
Cultureconditions:25◦C±1;4.5vvm;200rpm,pH5.5±0.2; ()drycellweight;(䊉)lacticacidconcentration;()acetic acidconcentration;()totalsugarsconcentration.The resultsarethemeanofduplicates.
experimentisthe highestamountthus far reportedinthe literature(Fig.5).
Conclusions
Theliquid acid protein residue ofsoybean (LAPRS), a veg-etable carbon substrate, was successfully used to produce biomass of L. plantarum BL011 as a source of a potential probioticcultureforfoodapplications.Lactic acidwas also producedinlargeamounts,whichshouldbeexploredifits productionisofinterest.Theproductionlevels ofbiomass andlacticacidwerehighwhencomparedtootherreportsin theliterature.ThePlackett–Burmandesignprovidedan effi-cientmethodforthescreeningoftheimportantcultivation variablesofaeration,agitation speed,DOC,andtheoxygen volumetricmasstransfer,thusallowingforfuturescaling-up experiments.Aninnovativeculturemediumlackinglactoseor anyanimal-derivedsourcesfortheproductionof microorgan-ismscouldhelpthedevelopmentofnewindustrialprocesses wherelactose-freeandanimal-freeingredientsarerequired. Furtherresearchshouldbeperformedtotestdifferentbacteria oryeastsinthissubstrate.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
TheauthorswishtothankCNPqandCAPES(Brazil)forthe financialsupportandscholarshipsforthiswork.
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