Development and characterization of an innovative synbiotic fermented beverage based on vegetable soybean

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Food

Microbiology

Development

and

characterization

of

an

innovative

synbiotic

fermented

beverage

based

on

vegetable

soybean

Carolina

Battistini

_,

_Beatriz

_Gullón

_,

_Erica

_Sayuri

_Ichimura

_,

Ana

Maria

Pereira

Gomes

_,

_Eliana

_Paula

_Ribeiro

_,

_Leo

_Kunigk

_,

José

Ubirajara

Vieira

Moreira

_,

_Cynthia

_Jurkiewicz

a_{InstitutoMauádeTecnologia,SãoCaetanodoSul,SP,Brazil}

b_{UniversidadeCatólicaPortuguesa,EscolaSuperiordeBiotecnologia,Porto,Portugal} c_{EmbrapaSoja,Londrina,PR,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Received25January2017 Accepted9August2017 Availableonline18October2017 AssociateEditor:SolangeI. Mussatto Keywords: Prebiotic Probiotic Soymilk Oligosaccharides Vegetablesoybean

a

b

s

t

r

a

c

t

Soymilkwasproducedfromvegetablesoybeanandfermentedbyprobiotics(Lactobacillus

acidophilusLa-5,BifidobacteriumanimalisBb-12)inco-culturewithStreptococcusthermophilus.

Thecompositionofthefermentedbeverageandoligosaccharidescontentweredetermined. Theeffectoffructooligosaccharidesandinulinonthefermentationtimeandviabilityof pro-bioticmicroorganismsthroughout28daysofstorageat5◦Cwereevaluated.Thesoymilk fromvegetablesoybeanswasfermentedinjust3.2h,whenpHreached4.8.Fermentation reducedthecontentsofstachyoseandraffinoseinsoymilk.Prebioticshadnoeffecton acidi-ficationrateandonviabilityofB.animalisandS.thermophilusinthefermentedbeverage.The viablecountsofB.animalisBb-12remainedabove108_CFUmL−1_{inthefermentedsoymilk}

during28daysofstorageat5◦CwhileL.acidophilusLa-5wasdecreasedby1logCFUmL−1. Thefermentedsoymilkfromvegetablesoybeansshowedtobeagoodfoodmatrixtodeliver probioticbacteria,aswellasasoyproductwithalowercontentofnon-digestible oligosac-charides.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

Introduction

Soymilk hasreceived increasingattention from consumers asanalternativetodairyproductsduetoitsproteinquality, absenceofcholesterolandlactose,andfunctionalproperties.1

∗ _{Correspondingauthor.}

E-mail:[email protected](C.Jurkiewicz).

However,theconsumptionofsoymilkislimitedduetothe presenceofnon-digestibleoligosaccharides,suchasraffinose andstachyose,whicharenothydrolyzedinthesmallintestine andmaycauseabdominalcramps,diarrheaandbloating.2,3

Lactic acid bacteria and probiotic microorganisms, for example, Streptococcus thermophilus, Lactobacillus acidophilus,

https://doi.org/10.1016/j.bjm.2017.08.006

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

andBifidobacterium,cangrowinsoymilkandconsume non-digestible oligosaccharides,decreasing or eliminatingthese anti-nutritionalcompounds,resultinginahealthierproduct forconsumers.4–7_{Aprobioticfermentedsoymilkcombinesthe}

beneficialpropertiesofsoywiththehealthbenefitsof pro-bioticmicroorganisms.However,theacidificationrateofpure probioticculturesisusuallylow,andoftenoff-flavorsinthe finalproductare produced.Thesolutiontothis problemis tomixtheprobioticcultureswithyogurtculturestoreduce thefermentationtimeandimprovethesensory characteris-ticsoftheproduct.8,9_{Prebioticingredients,suchasinulinand}

fructooligosaccharides (FOS),mayalso improvethe activity andsurvivalofprobioticbacteriainfermentedsoymilk.7,10,11

Moreover,thecombinationofprebioticsandprobioticsresults inasynbioticeffectongutmicrobiota.12_{Non-dairysynbiotic}

beverage13,14_{isalsoanalternativetoconsumerthatare}

lac-toseintolerantorallergictomilkprotein.

Vegetable soybean [Glycine max (L.) Merril] is a popular foodthatisconsumedinAsia,the UnitedStatesandother countries,mainlyasasnack,avegetableforsoupsorstews,or insalads.Itisasoybeanthatisharvestedwhiletheseedsare atapproximately80%ofmaturity,suchthatithasagreencolor andasofttexture.15,16_{Theseimmatureseedshaveadvantages}

overmaturesoybean,includingimprovedsensoryattributes andnutritionalvalue,suchassweeterflavorandlesscontents ofstachyoseandraffinose,resultinginbetterdigestibility.17,18

Similartomaturesoybean,vegetablesoybeanisrichingood qualityprotein,hasahighmineralcontentandhasthe poten-tialtopreventsomediseases,includingcancer,osteoporosis andmenopausalsymptomsduetoitscontentinisoflavones.19

Numerousstudieshavebeenperformedonthegrowthof probioticculturesinsoybeverage4,8,20_;however,no

informa-tionisavailableonthefermentationofsoymilkfromvegetable soybean.Thus,theaimofthisstudywastoinvestigatethe acidificationrateofsoymilkproducedwithvegetablesoybeans supplementedwithinulinandfructooligosaccharide(FOS),by amixedcultureofL.acidophilusLa-5,Bifidobacteriumanimalis

subsp.lactisBb-12andS.thermophilus.Inaddition,thesurvival ofmicroorganismsduring28daysofstorageat5◦Candthe contentsofstachyoseandraffinoseinsoymilkandfermented beverageswerealsoevaluated.

Materials

and

methods

Productionofsoymilk

Vegetable soybeans, cultivar BRS-232, were supplied by EmbrapaSoybean,Brazil.Theplantswereharvested mechani-callyandtakentothelaboratorywherethepodswereremoved and immediately bleached in boiling water for 3min and cooledat5◦C.Seedswereremovedfromthepods,packedin plasticbags,frozenat−18◦_{Candfreeze-driedinalabscale}

lyophilizer(EnterpriseI;TERRONI,SãoCarlos,SP,Brazil). Soymilk wasproduced bysoaking fifty grams of freeze-driedvegetablesoybeansin455gofwateratroom tempera-turefor10min.Themixturewasheatedat85◦Candblended for3min.Theslurrywasstirredat85◦Cfor5minandfiltered ina0.5mmconicalsievetoobtainthesoymilk.

Fermentationofsoymilk

Soymilkwassupplementedwith40gkg−1 fructooligosaccha-ride, FOS (Orafti® P 95, Beneo Latinoamericana), 40gkg−1 inulin (Orafti®GR, Beneo Latinoamericana) or a mixtureof 40gkg−1FOSand40gkg−1inulin.Controlfermentedsoymilk waspreparedwithouttheaddition oftheprebiotic ingredi-ents.Thefourformulationswereproducedintriplicate.After theadditionoftheprebioticingredients,soymilkswere pas-teurizedat75◦Cfor15s,cooledat37◦C,andinoculatedwith 0.02%ofafreeze-driedABT-4culture(ChristianHansen, Den-mark)containingL.acidophilusLa-5, B.animalissubsp. lactis

Bb-12andS.thermophilus.Next,thesoymilksweredistributed in50mLsterilebottlesand incubatedat37◦C untilthepH reached4.7–4.8.Thefermentedbeverageswerestoredat5◦C for28days.

Chemicalandphysicochemicalanalyses

Thechemicalcompositionoflyophilizedvegetablesoybeans and soymilk without prebiotic ingredientswas determined accordingtoAOACmethods.21Themoistureofthevegetable soybeanswasdeterminedbydryingthesampleinanovenat 105◦Cuntilconstantmass,basedonAOACmethod925.09B, without vacuumutilization.Thetotalsolids contentofthe soymilkswasdeterminedaccordingtoAOACmethod990.20, andthemoisturewascalculatedsubtractingthisvaluefrom 100.Todeterminetheashcontent,thesamplewas inciner-atedinamuffleat550◦C(method923.03andmethod945.46 forgrains and soymilk,respectively,both fromAOAC).The proteincontentwascalculatedbythemeasurementoftotal nitrogenusingthemicroKjeldahlmethod,andtheconversion factorappliedwas6.25,basedonAOACmethod979.09.The fatcontentwasdeterminedusingtheSoxhlet,basedonAOAC method920.39,usinghexaneassolvent(boilingpoint=70◦C at101,325Pa).Thetotalcarbohydratecontentwascalculated bythedifference.

ThepHofthesoymilkandfermentedsoymilkwere mea-suredusingapHmeter(TEC-2;TECNAL,Pìracicaba,SP,Brazil), accordingtoAOACmethod981.12(AOAC21_).

Extraction of oligosaccharides (stachyose and raffinose) fromvegetablesoybeanswasperformedaccordingtoOliveira etal.22_{withadaptations.Thefreeze-driedvegetablesoybeans}

were groundtopass througha0.5mmsieve,andasample of2.50gwasmixed with50mLofan80% ethanolsolution andstirredfor2min.Themixturewascentrifugedat5000rpm for10min,andthecontentoftheoligosaccharideswas deter-minedinthesupernatant.

Toanalyzetheoligosaccharidesandorganicacidscontents in soymilk and fermented beverages, the samples were centrifugedat5000rpmfor5min.Thesupernatantwas cen-trifugedagainunderthesameconditions,dilutedindeionized water at a volumetric ratio of 50%, and neutralized with barium carbonate. Centrifugation was performed again at 5000rpmfor5min,andtheoligosaccharidescontentinthe supernatantwasdeterminedusingHPLC.

TheHPLCmethoddescribedbyRivasetal.23_wasusedto

determinethecontents ofoligosaccharides.Samplesofthe liquors were filtered using 0.20␮m celluloseacetate mem-branes,neutralizedwithbariumcarbonate,andassayedby

HPLCforstachyoseandraffinoseusinga1100series Hewlett-Packard chromatograph equipped with a refractive index detectoroperatedat50◦Canda300×7.8mmCARBOsepCHO 682column(Transgenomic,Glasgow,UK)operatingat80◦C. Distilled water was used as the mobile phase (flow rate 0.4mLmin−1).

LacticandaceticacidsweredeterminedaccordingtoRivas etal.23_{Samplesofliquorswerefilteredusing0.20␮m}

mem-branesandusedfordirectHPLCdeterminationoflacticand aceticacidusinganAgilent1200seriesHPLCinstrumentwith arefractiveindex(RI)detector(Agilent,Waldbronn,Germany) operatedat50◦C.Theotheranalysisconditionswereas fol-lows:AminexHPX-87Hcolumn(BioRad,Hercules,CA,USA); mobilephase,0.003MH2SO4;flow,0.6mLmin−1.

Microbiologicalanalysis

Todeterminethe survivalofmicroorganisms infermented vegetable soymilkduring thestorage period(1,4,7, 14,21 and28days),10gofeachsamplewereblendedwith90gofa 8.5gL−1sterilesalinesolutionfor1minat260rpmina Stom-acher(Seward,Worthing,UK)andsubjectedtoserialdecimal dilutionswiththesamediluent.Oneachsamplingday,anew bottlecontainingfermentedsoymilkfromthesamebatchwas usedfortheanalysis.

Viable cells numbers ofS. thermophilus was determined bysurface-plating20␮LofeachdilutioninM17agar supple-mentedwith5gL−1lactose.Plateswereincubatedat37◦Cfor 48hunderaerobicconditions.24

B.animalisBb-12viabilitywasmonitoredbysurface-plating 20␮LofeachdilutioninDeManRogosa Sharpe(MRS)agar containing0.2gL−1ofbilesalts,0.3gL−1ofsodiumpropionate, 0.5gL−1ofcysteine-HCland0.2gL−1oflithiumchloride.Plates wereincubatedat37◦Cfor48hunderanaerobicconditions (GasPakTM_,BDBBLTM_,EUA).25

L.acidophilusLa-5populationwasdeterminedby surface-plating20␮LofeachdilutioninM-MRSagar(formulatedMRS agarcontaining20gL−1ofmaltoseinsteadofglucose).Plates wereincubatedat37◦Cfor48hunderaerobicconditions.25

Experimentaldesignandstatisticalanalysis

Theeffectsoftwo independentvariables,namely FOSand inulincontents, on vegetable soymilk physicaland chemi-calcharacteristics were studiedusing a22 _{factorial design}

replicated in three randomized blocks, which totaled four treatmentsand twelvetrials.These resultswereexpressed asthe mean±standarddeviation(SD).Differencesbetween meansduringtheexperimentalperiodwereanalyzedusing ANOVAfollowedbyTukeytestuponverificationofnormality andhomogeneity(p<0.05).Minitabsoftware,version16,was utilizedforthestatisticalanalysis.

Results

Chemicalcompositionofvegetablesoybeansandsoymilk

ThecompositionofvegetablesoybeancultivarBRS232usedin thisstudyandthesoymilkproducedwiththesamecultivaris

presentedinTable1.Thecontentsofraffinoseandstachyose indryseedsofvegetablesoybeanBRS232were2.8±0.5mgg−1 and4.8±0.1mgg−1,respectively.

The yield of the soymilk extraction process was 6.35±0.09kg of soymilk per kg of freeze-dried vegetable soybeans.Theproteincontentinsoymilkwas23±2mgg−1, andtheproteinrecoverywas37.8±0.5%.

ChangesinpH,organicacidsandoligosaccharidesduring fermentationandrefrigeratedstorageofsoymilk

Theadditionofprebiotics,i.e.,FOS,inulinortheircombination tosoymilkdidnotaffectthefermentationtime(p>0.05).The timerequiredtoreachapHof4.8rangedfrom3.13to3.33h, andthepHdecreasedatameanrateof0.39±0.01units(U)

perhour(Table2).

ThepH,lacticandaceticacidvaluesoffermentedsoymilks duringrefrigeratedstorageareshowninTable2.Allofthe bev-eragespresentedasmallbutsignificant(p<0.05)reductionin pHandanincreaseinlacticacidcontentduring28daysof storage.However,thegreatestreductioninpH(0.14U)andthe highestcontentoflacticacid(2.59gL−1)wereobservedin bev-erageswithinulinsupplementation.Theaceticacidcontent inall ofthe fermentedsoymilkhad nosignificant(p>0.05) increaseduringstorage.

Theestimationofmaineffect,thatwas obtainedbythe differenceinprocessperformancecausedbyachangefrom thelowtothehighlevelofthecorrespondingfactor,showed thatFOSadditiondecreasedthelacticacidcontentby0.1gL−1 (p<0.05),whileinulinadditionincreasedthecontentoflactic acidby0.25gL−1infermentedsoymilk.Nosignificant(p>0.05) interactionbetweenfactorswasobserved.

Acetic acidcontentinfermentedsoymilksupplemented withprebioticswas lower (p<0.05) than incontrolsoymilk however, thedifferences wereingeneralless than0.1gL−1

(Table2).

The concentration of oligosaccharides in soymilk with-outprebioticbeforefermentationwas0.63gL−1and0.38gL−1 forstachyoseand raffinose,respectively (Fig. 1).Due tothe reducedcontentofoligosaccharidesinvegetablesoybean,the soymilk obtainedinour studyalso presentedalower con-tentofstachyoseandraffinosecomparedwithsoymilkfrom maturesoybean.

Afterfermentation ofsoymilk,the contentofstachyose andraffinosesignificantlydecreased(p<0.05)to0.45gL−1and 0.23gL−1,respectively.

Table1–Chemicalcompositionofthevegetable soybeansfromcultivarBRS232andofsoymilk.

Parameter Soybean(mgg−1) Soymilk(mgg−1)

Moisture 696±3 927±1

Protein 127±3 23±2

Fat 65±2 13.0±0.6

Ash 16.0±0.4 5.3±0.1

Carbohydratesa _{96±4 32±5}

Table2–Fermentationtime,acidificationrate,pH,lacticandaceticacidcontentoffermentedsoymilkafter1and28days ofstorageat5◦C.

Parameter Soymilkformulationa

C F I FI

TimetoreachpH4.8(h) 3.33a_{±0.22 3.22}a_{±0.13 3.19}a_{±0.10 3.13}a_±0.05

Acidificationrate(pHU/h) 0.41a_{±0.03 0.38}a_{±0.05 0.38}a_{±0.08 0.39}a_±0.01

pH(day1) 4.56a_{±0.12 4.56}a_{±0.11 4.54}a_{±0.11 4.53}a_±0.10

pH(day28) 4.48a_{±0.05 4.46}a_{±0.09 4.40}a_{±0.06 4.42}a_±0.04

Lacticacid,gL−1(day1) 2.09a_{±0.13 1.79}b_{±0.04 2.11}a_{±0.14 2.13}a_±0.06

Lacticacid,gL−1(day28) 2.24b_{±0.03 2.28}b_{±0.16 2.59}a_{±0.12 2.42}a_±0.07

Aceticacid,gL−1(day1) 0.54a_{±0.03 0.46}ab_{±0.05 0.43}b_{±0.01 0.47}ab_±0.03

Aceticacid,gL−1(day28) 0.58a_{±0.05 0.46}a_{±0.07 0.47}a_{±0.04 0.41}a_±0.07

a _{C(withoutprebiotic),F(with40gkg}−1_{offructooligosaccharide,FOS),I(with40gkg}−1_{ofinulin),FI(with40gkg}−1_{ofFOSand40gkg}−1_of

inulin).Mean±standarddeviationofthreeruns.Meansinthesamerowwithdifferentlettersaresignificantlydifferent(p<0.05).

NFS FB-T1 FB-T14 FB-T21 FB-T28 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 a a b b b b b b b b O ligos accharides content (g L -1)

Fig.1–Oligosaccharidescontent,stachyose(greybars)and raffinose(blackbars),inthenon-fermentedsoymilk(NFS) andinthesoymilkfermentedbeverages(FB)duringthe storageperiod(T1,T14,T21andT28).Foreach

oligosaccharide,meanswithdifferentlettersare significantlydifferent(p<0.05).

Changesinmicrobiologicalcountsduringthestorageof fermentedsoymilk

ThepopulationofS. thermophilus,L.acidophilus La-5and B. animalisBb-12infermentedsoymilksthroughoutstorageis showninTable3.TheadditionofFOSand/orinulininsoymilk hadnosignificant(p>0.05)effectonS.thermophilusandB. ani-malisviability infermentedbeverages.Althoughinulinhad increasedlacticacidcontentduringthestorageoffermented soymilk,nosignificantenhancementinS.thermophilus popu-lationwasobserved.Anosignificant(p>0.05)decrease,below 0.3logCFUmL−1,inS.thermophilusandB.animalispopulations wasobservedinalmostallthefermentedbeveragesduring28 daysofstorage.TheB.animalisBb-12viabilityinfermented vegetablesoymilkwasgreaterthanL.acidophilusLa-5.Atthe endof28daysofstorage,theviablecellnumberofB.animalis

washigherthan8logCFUmL−1inallofthefermented bever-ages,regardlessofthepresenceorabsenceofprebiotic,while theviablecellnumberofL.acidophiluswasreportedbetween 5.6and6.4logCFUmL−1.

The decrease in L. acidophilus population in fermented soymilk duringstoragewas significant(p<0.05)and higher than1logCFUmL−1foralltreatments.Ingeneral,beverages withinulinand/orFOSpresentedsignificantly(p<0.05)lower countscomparedtobeverageswithoutprebiotics.

Discussion

The resultsobtainedforthe composition ofvegetable

soy-bean BRS 232 (Table 1) were consistent with previous

reports for other cultivars. Song et al.18 _{and the USDA}26

reported 655mgg−1 and 675mgg−1 ofmoisture,153mgg−1 and 130mgg−1 ofprotein, 44.9mgg−1 and 68mgg−1 offat, 20mgg−1and17mgg−1ofash,and127mgg−1and110mgg−1 of carbohydrates in immature soybeans. The differences observedbetweenthosestudiesandtheresultsreportedin thisstudymayberelatedtodifferentvegetablesoybean geno-types.

Formatureseedsfromthesamecultivar,Oliveiraetal.22

reported three-fold and eight-fold higher concentrations, namely,8.5mgg−1and37.0mgg−1inraffinoseandstachyose, respectively. This difference in sugar content between the different maturation stages of soybeans has been previ-ously reported byother studies.16,27 _{Withinone week, the}

contentsofstachyoseandraffinosemightduplicateor trip-licatedependingonthecultivar.15

Theproteinrecovery(37.8%)invegetablesoymilkwaslow compared with 67.4% to 78.8% reported by Vishwanathan et al.28 _{forsoymilk extractedfrommaturesoybeans.These}

authorsemployedhotextraction,withthesametemperature usedinthepresentstudy(between80and95◦C),andthus,the discrepancybetweenourstudiesmayberelatedtothe soy-beanparticlesize,29_thegenotype30_{andwiththematuration}

stageoftheseeds.

Fermentationofsoymilkfromvegetablesoybeanwasvery fast, in 3.2h pH reached 4.8 and no significant difference (p>0.05)wasobservedwiththeadditionofinulinorFOS.Our resultsdifferedfromthoseofRinaldonietal.,11_whoreported

that ultrafiltrated soymilk concentrate fermented by Lacto-bacillusdelbrueckiisubsp.bulgaricusandStreptococcussalivarius

subsp.thermophiluswithoutinulindidnotreachapHlower than 4.9, and soyyogurt was not obtained.These authors alsoobservedthatindependentoftheconcentrationofinulin

Table3–ViablecountsofS.thermophilus,L.acidophilusLa-5andB.animalisBb-12infermentedsoymilkduringstorage periodfor28daysat5◦C.

Microorganism Time(days) LogCFUmL−1a

C F I FI S.thermophilus 1 8.22±0.13Aa _8.16±0.06Aa _8.13±0.05Aa _8.18±0.03Aa 4 8.23±0.04Aa _8.21±0.06Aa _8.26±0.14Aa _8.18±0.14Aa 7 8.16±0.04Aa _8.11±0.10Aa _8.15±0.08Aa _8.22±0.10Aa 14 8.16±0.04Aa _8.26±0.11Aa _8.16±0.20Aa _8.20±0.19Aa 21 8.13±0.07Aa _8.12±0.10Aa _8.07±0.19Aa _8.06±0.04Aa 28 7.90±0.10Aa _7.90±0.13Aa _8.04±0.13Aa _8.03±0.18Aa L.acidophilus 1 7.56±0.26Aa _7.38±0.19Aa _7.35±0.19Aa _7.30±0.16Aa 4 7.55±0.24Aa _7.39±0.13Aa _7.38±0.09Aa _7.30±0.16Aa 7 7.37±0.09Aa _7.29±0.18Aa _7.32±0.15Aa _7.28±0.07Aa 14 7.17±0.14Aa _6.98±0.27Aa _6.94±0.16Aa _6.85±0.18ABa 21 7.02±0.18Aa _6.24±0.07Bb _6.20±0.02Bb _6.20±0.47BCb

28 6.18±0.28Bab _6.05±0.35Bab _5.63±0.29Cb _6.42±0.08BCa

B.animalis 1 8.58±0.17Aa _8.50±0.09Aa _8.50±0.08Aa _8.49±0.08Aa 4 8.48±0.01ABa _8.49±0.07Aa _8.31±0.27Aa _8.39±0.03ABa 7 8.34±0.13ABa _8.39±0.07Aa _8.44±0.01Aa _8.51±0.07Aa 14 8.13±0.06Ba _8.28±0.12Aa _8.47±0.05Aa _8.47±0.09Aa 21 8.34±0.03ABa _8.25±0.19Aa _8.25±0.23Aa _8.21±0.24ABa 28 8.30±0.05ABa _8.33±0.14Aa _8.24±0.35Aa _8.00±0.15Ba

a _{C(withoutprebiotic),F(with40gkg}−1_{offructooligosaccharide,FOS),I(with40gkg}−1_{ofinulin),FI(with40gkg}−1_{ofFOSand40gkg}−1_of

inulin).Mean±standarddeviationofthreeruns.Meansinthesamerowwithdifferentlowercaselettersaresignificantlydifferent(p<0.05).

Meansinthesamecolumn,forthesamemicroorganisms,withdifferentuppercaselettersaresignificantlydifferent(p<0.05).

addedtothesoymilk(20–70gL−1),thepHreached4.65within 4hoffermentation.Sincetheyusedasfoodmatrixa concen-tratedsoymilk,withproteincontentbetween40and58gL−1, almost twice compared with the soymilk from vegetable soymilk,thehigherproteincontentincreasedthe buffering capacity, and thus, reduced the fermentation rate. On the otherhand,PandeyandMishra31 _{observedthathigher FOS}

concentrations(20–100gL−1)insoymilkresultedinashorter fermentationprocess.Theeffectofinulinonthereductionin fermentationtimeinmilkhasalsobeenreportedbyprevious studies.12,32,33

Theacidificationrateofsoymilkfromvegetablesoybeanby probioticsstrainsinco-culturewithS.thermophilus(Table2) wasfasterthaninmilkwhencomparedwithresultsreported byCasarottietal.34_{Inmilkfermentedbythecombinationof} S.thermophilusandL.acidophilusLa-5,acidificationratewas 0.34pHU/h,whereasinthepresentstudy,valuesrangedfrom 0.38to0.41pHU/h.Thisdifferenceinacidificationratemay beexplainedduetothelowerbufferingcapacityofsoymilk as reported byChampagne et al.8 _{who alsoobserved that}

acidificationofsoybeveragebyS.thermophilusaloneorin com-binationwithLactobacillushelveticusorBifidobacteriumlongum

wasfasterthaninmilk.

DataintheliteratureshowsthatS.thermophiluscangrow wellinsoybeverages;however,theacidificationrateisa func-tionoftheselectedstrainandthepresenceofothercultures, suchasprobiotics.6,20_{OurresultsdemonstratedthatS.} ther-mophilusinthepresenceofL.acidophilusLa-5andB.animalis

Bb-12wasabletofermentsoymilkfromvegetablesoybeanin ashorttimeperiod.

The production of lactic acid during fermentation of soymilk can be attributedmainly to S.thermophilus, which isahomofermentativespeciesthatproduceslacticacidtoa higherextentthanprobioticbacteriainsoymilk.6_As

bever-ageswithinulinpresentedahighercontentoflacticacid,the presenceofthisingredientmayhaveenhancedS.thermophilus

metabolism.

Aceticacidisproducedmainlybybifidobacteriainwhich carbohydratemetabolismresultsintheproductionofacetic and lactic acids in a molar ratio 3–2. Thus, our results showed that the addition ofprebiotics to soymilk did not enhance bifidobacteria metabolism in vegetable soymilk beverage.

Duetothereducedcontentofoligosaccharidesinvegetable soybean,the soymilk obtainedinourstudy alsopresented a lower content ofstachyose and raffinose comparedwith soymilkfrommaturesoybean(Fig.1).Theliteraturedatashow thatinsoymilkextractedfrommaturesoybean,thestachyose contentcanvaryfrom2.1to8.8gL−1andthatraffinosevaried from1.44to2.2gL−1.35,36

The hydrolysis of these ␣-galacto-oligosaccharides requires the enzyme ␣-galactosidase, which hydrolyses ␣-galactosidebonds.37_{Bifidobacteriumspp.,L.acidophilusand} S. thermophilus have ␣-galactosidase activity; however, the utilization of these sugars varies with the culture species and strains employed.6,20 _{Donkor et al.}4 _{reported that S.} thermophilusreducedraffinoseinsoymilkby64.5%,whereas stachyose was metabolized by over 40% for most of the strains evaluated. Hou et al.5 _{observed that Bifidobacterium}

fermentationofsoymilk.Inthepresentstudy,thereduction of raffinose and stachyose was 39.5% and 28.5%, respec-tively (Fig. 1). The short fermentation time in our study mayhaveresultedinalower reductioninoligosaccharides content.

Many studies have shown that soymilk isa good vehi-cle for probiotic bacteria1,4,6,10,33_{; however, no information}

isavailableforsoymilkfromvegetablesoybean.Ourresults showedthatviabilityofB.animalisBb-12remainedhigh,above 108_CFUmL−1_{during28daysofstorageoffermentedvegetable}

soymilk.However,atthesametime,cellcountsofL.acidophilus

La-5reducedmorethan1log.Apparently,thelowpHandthe accumulationoforganicacidswasnotresponsiblefortheloss ofprobiotic viability, sinceBedani et al.10 _{verifiedthat cell}

numberofL.acidophilusLa-5remainedabove108_CFUmL−1_in

afermentedsoyproduct,withasimilarpHreportedherein. Nevertheless, intheirstudy, lactose, sucroseand skimmed milkpowderwasaddedtothesoymilkbeforefermentation. Thus,theincreaseinsugarthatiseasilyfermentedand in aminoacidscontentmayhavecontributedtoahighercountof

Lacidophilusduringthestoragewhencomparedtothesoymilk fromvegetablesoybeans.10

Therehavebeensomecontradictoryreportsregardingthe useofFOSand inulintostimulatethegrowth andsurvival ofprobioticstrains.YeoandLiong7_{verifiedthatsoymilk}

sup-plementedwithinulinorFOSdidnotaffectthepopulation ofL.acidophilusATCC4356,B.longumFTDC8643and Bifidobac-teriumFTDC8943,butitdidenhancethegrowthofL.acidophilus

FTDC8033,suggestingthattheeffectwasstrain-dependent. Thus,thefactthattheadditionofFOSandinulintovegetable soymilkdidnotenhancedthesurvivalofL.acidophilus dur-ingrefrigeratedstoragemightbeattributedtoprobioticstrain response.

Thepresent study demonstrated that aserving portion of100mLoffermentedsoymilkfromvegetablesoybeanhas morethan1010_{CFUofprobiotics.Consideringthataprobiotic}

doseof108_{CFUperdayisneededforbeneficialeffectsinthe}

gut,38_{thefermentedsoymilkfromvegetablesoybeanisagood}

potentialvector,duringashelf-lifeof28days,forviable pro-bioticmicroorganismstopromotehealthbenefitstothehost coupledwithbetternutritionalquality.

Conclusions

Thesoymilkfromvegetablesoybeans(cultivarBRS232)was fermentedinashorttime,3.2h,byamixedculturecontaining

L.acidophilusLa5,B.animalisBb12andS.thermophilus, demon-strating an important technological property forindustrial purpose. Fermentationdecreased the content of stachyose andraffinose.TheviabilityofB.animalisinfermentedsoymilk remainedstableover28daysofstorageatabove108_CFUmL−1_,

whileL.acidophilusdecreasedby1loginthesameperiodto valuescloseto106_CFUmL−1_{.TheadditionofFOSor inulin}

hadnoeffectonacidification rateandalsoontheviability ofB.animalisandS.thermophilus.Takentogether,theseresults demonstratedthatfermentedsoymilkfromvegetablesoybean isaverypromisingproductasavehicleforprobioticbacteria, aswellassoyproductwithalowercontentofnondigestible oligosaccharides.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

ThisresearchwasfinanciallysupportedbySãoPauloResearch Foundation(FAPESP,grantnumber2013/12138-7),Maua Insti-tute of Technology (IMT), Center for Biotechnology and Fine Chemistry (CBQF) of Catholic University of Portugal, undertheFCTprojectUID/Multi/50016/2013.Fellowshipswere supported by Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES) and SantanderUniversities. The authors thankEmbrapaSoybean,Chr. Hansenand Clariant forprovidingpartofthematerialresources.

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