Aminotransferase and glutamate dehydrogenase activities in lactobacilli and streptococci

(1)

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

Food

Microbiology

Aminotransferase

and

glutamate

dehydrogenase

activities

in

lactobacilli

and

streptococci

Guillermo

Hugo

Peralta

a

_,

_Carina

_Viviana

_Bergamini

a,b,∗

_,

_Erica

_Rut

_Hynes

a,b

a_Institute_of_Industrial_Lactology,_National_University_of_Litoral,_National_Council_of_Scientiﬁc_and_Technique_Research (INLAIN-UNL/CONICET),SantiagodelEstero,SantaFe,Argentina

b_Faculty_of_Chemical_Engineering,_National_University_of_Litoral_(FIQ-UNL),_Santiago_del_Estero,_Santa_Fe,_Argentina

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received16March2015

Accepted22December2015

Availableonline22April2016

AssociateEditor:MaricêNogueira

deOliveira

Keywords:

Aminotransferaseactivity

Glutamatedehydrogenaseactivity

Lactobacilli Streptococci

a

b

s

t

r

a

c

t

Aminotransferasesandglutamatedehydrogenasearetwomaintypesofenzymesinvolved

intheinitialstepsofaminoacidcatabolism,whichplaysakeyroleinthecheeseﬂavor

development.Inthepresentwork,glutamatedehydrogenaseandaminotransferase

activ-itieswerescreenedintwentyonestrainsoflacticacidbacteriaofdairyinterest,either

cheese-isolatedorcommercialstarters,includingﬁfteenmesophiliclactobacilli,four

ther-mophiliclactobacilli,andtwostreptococci.ThestrainsofStreptococcusthermophilusshowed

thehighestglutamatedehydrogenaseactivity,whichwassigniﬁcantlyelevatedcompared

withthelactobacilli.Aspartateaminotransferaseprevailedinmoststrainstested,whilethe

levelsandspeciﬁcityofotheraminotransferaseswerehighlystrain-andspecies-dependent.

Theknowledgeofenzymaticproﬁlesofthesestarterandcheese-isolatedculturesishelpful

inproposingappropriatecombinationsofstrainsforimprovedorincreasedcheeseﬂavor.

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

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

Introduction

Cheeseﬂavor developmentisa dynamicand complex

bio-chemical process, which results from catabolic reactions

involvingthemainmilkconstituents,i.e.,proteins,lipids,

lac-tose,and citrate.1_In_particular, _proteolysis_and_subsequent

aminoacidcatabolismplaysigniﬁcantrolesinthisprocess,

regardlessofthecheesevariety.Essentially,ithasbeen

estab-lishedthatmorethan50%ofvolatilecompoundsinvolvedin

thecheeseﬂavorareproducedviaaminoacid(AA)catabolism,

and lactic acid bacteria (LAB) are mainly responsible for

thesereactions in mostcheeses.1,2 _One _of_the _main

path-waysthatconvertAAsintoﬂavorcompoundsbeginswitha

∗ _{Corresponding}_author.

E-mail:bergaminicarina@gmail.com(C.V.Bergamini).

transamination reaction catalyzed by speciﬁc

aminotrans-ferases(ATs).1,2_In_this_reaction,_AAs_are_converted_into_their

corresponding␣-ketoacidsduetothetransferofthe␣-amino

groupofanAAtoasuitableacceptor,usually␣-ketoglutarate,

althoughpyruvateandoxaloacetatehavealsobeenreported

as possible acceptors.3,4 _The _{␣-ketoacids} _produced _during

transamination are intermediate compounds inthe aroma

development becausethey canbe metabolizedvia a range

ofenzymaticandchemicalreactionstoprovideseveral

com-pounds thatcan haveanimpact oncheese ﬂavor,suchas

alcohols,aldehydes,carboxylicacids,andesters.1_In_general,

ATsarespeciﬁcforanAAgroup,suchasaromatic(Ar-AT)or

branched-chain(Bc-AT)AAs,orforasingleAA,suchas

aspar-tate(Asp-AT),althoughoverlappingintheiractivitieshasbeen

http://dx.doi.org/10.1016/j.bjm.2016.04.005

(2)

reported.2,5–8_No_speciﬁc_AT_for_methionine_has_been_isolated

sofar,butotherATsshowactivityforthisaminoacid.6,7

Vari-ousATshavebeenidentiﬁedandcharacterizedinLactococcus

lactissubsp.lactisandL.lactissubsp.cremoris,5,7,9_as_well_as_in Lactobacillusspp.8_In_addition,_it_has_been_evidenced_that

trans-aminationistheﬁrststepofthecatabolismofseveralAAsin

strainsofLactobacilluscasei,L.helveticus, L.delbrueckiisubsp.

bulgaricus,andStreptococcusthermophilus.10–12_Glutamate

dehy-drogenase (GDH) also plays a key role in transamination

becauseitcatalyzestheoxidativedeaminationofglutamateto

␣-ketoglutarate,providinganaminogroupacceptor.2,6,12

Sev-eralspeciesofLABhavedemonstratedGDHactivity,including

Lactobacillusspp., namely, L.casei, L.paracasei,L.plantarum, L.rhamnosus, L.pentosus, and L.acidophilus,13–16 _Lactococcus lactis,13,14,17,18Leuconostocspp.,14,18andS.thermophilus.11

Thetransamination stepisconsidered thebottleneckin

theAAcatabolismandsubsequentproductionofﬂavor

com-poundsincheeses.Inparticular,ATactivitiesofstarterand

non-starter(NS)LAB,aswellastheavailabilityofanacceptor

ofthe aminogroup,suchas␣-ketoglutarate,are important

forthedevelopmentofﬂavorincheeses.2,13_Similarly,_Tanous

etal.13 _have_found_that_the_ability_of_LAB_to_produce_aroma

compoundsfromAAsiscloselyrelatedtotheirGDHactivity,

dueto the productionofanaminogroup acceptor insitu.

Onthe other hand,it has been proposedthat competition

exists between ATs for the available ␣-ketoglutarate, and

thereforeATactivityproﬁlesoflacticculturesmayaffectthe

volatilecompoundsproducedincheeses.19 _Thus,_screening

forenzymesplayingkeyrolesincheeseﬂavordevelopmentis

relevanttotheselectionofstrainsaspotentialﬂavor-forming

cultures.

Thiswork aimedto study the proﬁles of ATsand GDH

activitiesintwentyoneLABstrainsofdairyinterest,

includ-ingﬁfteenmesophiliclactobacillistrains(14ofNSoriginand

awildstrain),fourstrainsofthermophiliclactobacilli(three

strainsisolatedfromwheyculturesandonefroma

commer-cialsource),andtwocommercialstrainsofS.thermophilus.

Materials

and

methods

Chemicals

l-Methionine (Met), l-tryptophan (Trp), l-tyrosine (Tyr),

l-leucine(Leu),l-isoleucine(Ile),l-valine(Val),l-phenylalanine

(Phe), l-aspartic acid (Asp), l-glutamic acid (Glu),

␣-ketoglutaricaciddisodiumsaltdihydrate(␣-KGA),pyridoxal

5-phosphate (P5P), Tris–HCl, and Tris base were obtained

from Sigma Chemical Co. (St. Louis, MO, USA). Potassium

phosphate monobasic and potassium phosphate dibasic

were obtainedfrom Anedra(Buenos Aires,Argentina). The

Bradford protein estimation kit was from Pierce Chemical

Company (Rockford, IL, USA), and the l-Glu assay kit was

fromR-Biopharm/BoehringerMannheim(Germany).

Strains

Twenty-one strains of LAB were studied, including ﬁfteen

mesophiliclactobacilli,fourthermophiliclactobacilli,andtwo

streptococci. Oneof the mesophiliclactobacilli wasa wild

strain,L.caseiBL23,whosegenomehadbeensequenced.20_The

other14strainsofmesophiliclactobacilliwereisolatedfrom

two-month-oldgoodqualityTybocheeseandbelongedtothe

collectionofourInstitute(InstitutodeLactologíaIndustrial,

INLAIN).Theywereasfollows:L.plantarum29,33,87,89,and

91,L.rhamnosus73,75,77,and78,L.casei72,81,and90,andL. fermentum28and46.21_Three_of_the_strains_of_thermophilic

lac-tobacilliwereisolatedfromwheyculturesandalsobelonged

totheINLAINcollection,includingL.delbrueckii133andL.

hel-veticus138and209.22,23_Finally,_three_commercial_strains_were

studied,including S.thermophilus1and2andL.helveticus3

(suppliersarenotmentionedforconﬁdentialityreasons).

Stock cultures of all strains were maintained frozenat

−80◦_C _in _appropriate _broths _supplemented_with _15% _(v/v)

glycerolasacryoprotectiveagent.Ellikerbroth(Biokar

Diag-nostics,France)wasusedforthestreptococci,andMRSbroth

(BiokarDiagnostics)wasusedforthelactobacilli.Beforeuse,

the strains were grownovernight twice intheir respective

broths.

Growthcurves

Anovernight cultureofeach strainofthe streptococciand

lactobacilliwasusedtoinoculate(2%,v/v)100mLofElliker

orMRSbroth,respectively.Themesophiliclactobacilliwere

incubated at37◦C, whilethe thermophilic lactobacilli and

streptococci were incubated at42◦C.Bacterial growth was

monitored by assessing optical density (OD) at 560nm in

a spectrophotometer (UV/VIS Lambda 20, Perkin Elmer) at

appropriateintervalstoobtaingrowthcurvesanddetermine

thelateexponentialgrowthphaseforeachstrain.

Cell-freeextractpreparation

Cell-freeextracts(CFEs)were obtainedfrom culturesatthe

lateexponentialphasebymechanicaldisruptionofthecells

withglassbeads(106␮m,Sigma,G8893)inamini-beadbeater

8TMcelldisruptor(BiospecProducts,Bartlesville,IL,USA).Ina

preliminaryexperiment,weidentiﬁedthebestconditionsfor

thebeateroperationtoobtainaproperdegreeofcell

disrup-tion.Forthat,weassessedthreedifferentratiosofbeads(g)to

cells(mL)(1.2,0.7,and0.3g/mL)andusedthreeorﬁvecycles

ofdisruption,1mineach,atthehighestspeedofthedisruptor

forarandomlyselectedstrain,L.plantarum91.Theefﬁciency

ofcelldisruptionwasquantiﬁedastheratiobetweenplate

countsafterandbeforethedisruption.

Eachstrainwasinoculatedat2%(v/v)andgrowninthe

medium underthe conditions described previouslyuntil it

reachedthelateexponentialphase,determinedbythevalue

of OD560 associated with this growth stage according to

each growth curve. Cells were harvested bycentrifugation

(10,000×g,10min,4◦C),washedtwicewith50mMpotassium

phosphatebuffer(pH7.0),andsuspendedinanappropriate

volumeofthesamebuffertogivea30-foldconcentrationof

thecells.Thesesuspensionsweretransferredtomicrotubes

(2mL), which contained different quantities of previously

sterilized beads. CFEs were obtained under the following

conditionsestablishedinthepreliminaryexperiment:three

cyclesof1mineachatthemaximumspeedofthebeater,

(3)

of1.2g/mL.ACFEconsistedofthecelllysatecentrifugedat

16,000×g/10◦Cfor15mininordertoseparatethebeads,cells,

andcelldebrisandwasthenﬁlteredthrougha0.45-␮mpore

diametermembrane(Millipore,SaoPaulo, Brazil).TheCFEs

were storedat−20◦_C _until _used_for_analysis _of_enzymatic

activities.DuplicateCFEswere obtainedfrom two

indepen-dentculturesofeachstrainstudied.

Determinationofglutamatedehydrogenaseactivity

TheGDHactivitywasdeterminedcolorimetricallyby

measur-ingtheGlu-dependentreductionofNAD+_in_a_coupled_reaction

withdiaphoraseusingthecommerciall-Gluassaykit.24_A_CFE

(50–150␮L)was incubated inareactionmixturecontaining

potassiumphosphate/triethanolamine buffer,pH8.6,l-Glu,

NAD+_,_{iodonitrotetrazolium}_chloride,_and_diaphorase_(all

pro-vided in the l-Glu assay kit) for 1h at 37◦C. Endogenous

(Glu-independent)NAD+ _reduction_by_the_CFE_was_assessed

in control samples without Glu addition.15 _GDH _activity

wasexpressedpermilligramofproteinasthedifferencein

absorbanceat492nmbetweenthesampleanditsrespective

control.

Determinationofaminotransferaseactivities

ATactivitiesagainstAsp,Met,branched-chain(Leu,Val,and

Ile),andaromatic (Tyr,Trp,andPhe)AAswereanalyzedby

quantifyingtheGluderivedfromatransaminationreaction.

Forthispurpose,aCFE(15–50␮L)wasincubatedfor20min

at 37◦C in a reaction mixture (250␮L) containing 200mM

Tris buffer (pH 8.0), 1mM P5P, freshly prepared 50mM

␣-KGA,and15mMeachAAsubstrate.Inthisreaction,anAA

istransaminatedto the corresponding ␣-ketoacid, and the

␣-KGA,whichactsasanacceptoroftheaminogroup,is

con-verted to Glu. EndogenousGlu formation was assessed in

controlsampleswithouttheadditionofasubstrateAA.The

reactionwasstoppedbyheatingat80◦Cfor15min,andthe

leveloftheGluproducedwasquantiﬁedwiththesamel-Glu

kitdescribedforGDH.13,25,26_For_that,_an_aliquot_of_the

sam-plewasincubatedinareactionmixturecontainingpotassium

phosphate/triethanolaminebuffer,pH8.6,NAD+_,

iodonitrote-trazoliumchloride,diaphorase,andGDHfor45minat37◦C.26

SpeciﬁcATactivitywasexpressedasmicrogramsofGlu

pro-ducedperminutepermilligramofprotein.

Proteindetermination

TheproteinconcentrationoftheCFEwasdeterminedbythe

Bradfordmethod27withtheCoomassieproteinassayreagent

providedbyPierceChemicalCompany.Bovineserumalbumin

wasusedasanexternalstandard.Allproteindeterminations

wereperformedinduplicate.

Statisticalanalysis

Analysisofvariance(ANOVA)wasappliedtodetectsigniﬁcant

differencesinthelevelsofATactivitiesagainstdifferentAAs

(p<0.05).Whendifferenceswerefound,homogenousgroups

ofmeanswereidentiﬁed(Duncantest).Principalcomponent

analysis(PCA),withstandardizationtoameanofzeroand

toastandarddeviationofone(correlationmatrix),was

per-formedfortheATdatainordertomapculturesbytheirAA

transformation abilities. BesidesPCAmapping,hierarchical

clusteranalysis(HCA)wasappliedtotheﬁrsttwoprincipal

componentstogroupthestrainsobjectivelyinascoreplot.

IBMSPSSStatistics20.0(SPSS,Inc.,Chicago,IL,USA)was

usedforthestatisticalanalysis.

Results

and

discussion

Cell-freeextractpreparation

Inthepresentwork,weobtainedCFEsbyamechanical

dis-ruptioninabeadmillusingthebeadsof106␮m,whichare

optimalforbacterialcelldisruption.28_We_performed

prelimi-naryexperimentstooptimizethebeateroperationconditions

andfoundthattheamountofdisruptedcellsincreased

signif-icantlywhenthebead/cellratioincreased,whichisattributed

toanincreasedbead-to-beadinteraction.28_Contrarily,_no

dif-ferenceindisruptionwasfounddependingonwhetherthree

orﬁvecycleswereapplied.Basedontheseresults,weuseda

bead/cellratioof1.2g/mLforthesubsequentexperiments.

All cultures reachedthe end of the exponentialgrowth

phasebetween8and12hofincubation(datanotshown),with

valuesofOD560from1.6to2.1(Table1).Thecellloadsat

har-vestingweresimilarforallthestrainstested,withthetotal

averageof1.94×109_{colony-forming}_units_mL−1_._The_cell

dis-ruptiondegreeswerevariableamongtheculturesandranged

between74.4and99.8%,withameanof93%(Table1).In

addi-tion,the proteincontentinthe CFEswas6.6±1.7mgmL−1

(datanotshown).

Glutamatedehydrogenaseandaminotransferaseactivities

DifferentlevelsofNAD-dependentGDHweredetectedinthe

strainsassayed(Fig.1).ThetwostrainsofS.thermophilushad

thehighestlevelsofGDHandwereseparatedfromtherestof

thestrains,especiallyS.thermophilus2.TheGDHlevelsinthe

otherstrainswerelowerandsimilarbetweenthestrains;the

lowestactivitiesweredetectedinthethermophiliclactobacilli

andL.caseistrains.Overall,thestrainsofL.plantarum,L. rham-nosus,andL.fermentumhadintermediatelevelsofGDH.The

GDHenzymecatalyzestheinterconversionof␣-ketoglutarate

andGlu,withNADorNADPasacofactor.Ithasbeenreported

thatthecofactorfortheanabolicenzymeduringGlu

produc-tion isthe reduced form,NADP, whileNADis thecofactor

forthecatabolicenzymeinvolvedintheGlubreakdownand

subsequentproductionof␣-ketoglutarate.15_Helinck_et_al.11

reported the presence ofNADP-dependent GDH activity in

ﬁveofsixstrainsofS.thermophilus,whileonlyonestrainhad

NAD-dependentGDHactivity.ThepresenceandlevelsofGDH

activity anditscofactordependencehavebeenfoundtobe

species-and strain-dependent inlactobacilli.In agroupof

isolatesofNSorigin, thehighest frequencyandactivity of

GDHweredetectedinL.plantarumstrains.15_Similarly,_Tanous

etal.13_found_higher_levels_of_{NADP-dependent}_GDH_activity_in

L.plantarumstrainsincomparisonwithL.caseiandLactococcus.

Wefoundasimilartrend,i.e.,thehighestlevelsofGDHwere

(4)

Table1–Cellloadsforculturesinthebrothbeforeharvesting,inthebufferbeforeandafterthedisruptiontreatment, andtheefﬁciencyofthedisruptionforeachstraintested.

Species Strain Medium(MRSorElliker) Buffer

Opticaldensitya _Cell_counts

(logCFUmL−1₎b

Cellcounts(logCFUmL−1₎ _Disruption_(%)d

Initialc _Finalc L.plantarum 33 2.010 8.73 10.24 8.93 94.3 91 2.020 9.25 9.30 7.18 99.2 89 2.071 8.95 10.46 7.00 99.9 29 1.971 8.51 10.50 9.02 94.3 87 1.963 9.28 10.64 9.72 89.2 L.casei 81 1.960 9.17 10.56 9.93 74.4 72 2.007 9.84 10.31 8.87 96.3 BL23 2.080 9.65 9.45 7.70 99.0 L.paracasei 90 1.980 9.40 10.57 9.27 96.1 L.rhamnosus 73 1.847 9.46 10.15 8.04 99.2 77 2.003 9.54 10.58 9.79 89.4 78 1.958 9.22 10.74 8.08 99.8 75 2.030 9.75 10.08 9.24 85.5 L.fermentum 28 1.985 8.99 9.51 8.00 96.7 46 2.005 9.05 10.13 7.00 99.9 L.delbrueckii 133 2.056 8.93 10.33 8.24 99.1 L.helveticus 138 1.949 8.75 10.35 9.04 95.5 209 2.059 8.79 10.15 8.08 99.2 3 2.112 8.81 10.06 9.06 90.0 S.thermophilus 1 1.604 8.45 10.04 8.94 92.4 2 1.793 9.57 10.65 9.88 75.6

Thevaluesrepresenttheaverageoftwoindependentexperiments.

a _Optical_density_of_the_cultures_at_harvesting. b _Cell_counts_in_cultures_at_harvesting.

c _Cell_counts_of_the_suspensions_in_potassium_phosphate_buffer_before_and_after_the_disruption_treatment_with_the_bead_mill.

d_Percentages_of_cell_disruption_expressed_as_[(cell_count_before_treatment₋_cell_count_after_{treatment)/cell}_count_before_treatment]_×_100.

strains,whileL.caseiandL.helveticusshowedloweractivities.

DeAngelisetal.16_also_found_the_highest_levels_of_GDH_in_L.

plantarumstrains,buttheL.caseiandL.rhamnosusstrainsthat

theycharacterizedhadsimilarlevels.

On the other hand, the strains described in this study

showed different levels of AT activity against Asp, Met,

branched-chain,andaromaticAAswith␣-ketoglutarateasan

aminogroupacceptor(Table2).ATspeciﬁcitytowardAspwas

themainATactivityamongtheculturesstudied,anditslevels

were3–6timeshigherthanthoseofanyotherATsspeciﬁcfor

theAAstested.TheexceptionswereL.rhamnosus78,L.casei

72,andthefourtestedstrainsofthermophiliclactobacilli,in

whichactivitiesofallATsweresimilarorshowedlowabsolute

differences,withoutaprevalenceofAsp-AT.Overall,strains

thatshowedpreferentialATactivityagainstAsphadsimilarly

lowATactivitiesagainsttheremainingAAs,withfew

excep-tions.Thus,thesecondpreferentialATactivityafterAsp-AT

wasagainstTyrinL.plantarum89(p<0.05).ForL.casei81,the

secondAAbeneﬁtingfromATactivitywasVal,whileTrp,Phe,

andMetwerelesstransformed,andintermediatevalueswere

foundfortheremainingAAs.Finally,forS.thermophilus2the

ATactivityagainstLeuandTyrwasthesecondafterAsp-AT.

Similartootherauthors,2,8,29_we_conﬁrmed_species_and_strain

variabilityinATproﬁles.

InthePCAofthelevelsofATactivities,twoprincipal

com-ponentsexplained91.1%ofthetotalvariance.Fig.2showsthe

scoreandloadingplots;theellipsesinthescoreplotenclose

strainsaccordingtoHCA.Intheﬁrstgroup,threestrainsofL.

casei(72,81,andBL23),onestrainofS.thermophilus(2),andone

replicaofL.rhamnosus73weregroupedinthepositive

hemi-planeofPC1.Thesestrainswerecharacterizedbythehighest

activityofATs.Amongthestrainsclusteredinthisgroup,L.

casei72hadthelowestAsp-ATactivity,whichwasevidenced

byanegativescoreonPC2.

In thesecond group, locatedinthe positivehemi-plane

ofPC2,therewerethreestrainsofL.plantarum(89,33,and

91),andonereplicaofeachoneofthefollowingfourstrains:

L.rhamnosus73and75,L.paracasei90,andL.plantarum87.

ThesestrainswerecharacterizedbytheprevalenceofAsp-AT

activity.

Finally,thethirdgroupcontainedallstrainsofthermophilic

lactobacilli(L.delbrueckii133andL.helveticus138,209,and3),

thetwostrainsofL.fermentum(28and46),L.rhamnosus77,and

78,S.thermophilus1,L.plantarum29,andonereplicaofeachone

ofthefollowingthreestrains:L.plantarum87,L.paracasei90,

andL.rhamnosus75.Overall,thisgroupwasplacedonthe

neg-ativesidesofPC1andPC2,whichindicatesthatthesestrains

(5)

S. thermophilus 2

Str

ain

Glutamate dehydrogenase activity

0 1 2 (ΔAbs/mg proteins) S. thermophilus 1 Lb. helveticus 3 Lb. helveticus 209 Lb. helveticus 138 Lb. delbruekii 133 Lb. fermentum 46 Lb. fermentum 28 Lb. rhamnosus 75 Lb. rhamnosus 78 Lb. rhamnosus 77 Lb. rhamnosus 73 Lb. paracasei 90 Lb. casei BL23 Lb. casei 72 Lb. casei 81 Lb. plantarum 87 Lb. plantarum 29 Lb. plantarum 89 Lb. plantarum 91 Lb. plantarum 33

Fig.1–GlutamatedehydrogenaseactivityintheCFEsofthe21testedstrains.Valuesaremeans±standarddeviationofthe resultsoftwoindependentexperiments.

Table2–Aminotransferaseactivitiesforeightaminoacids.

Species Strain Asp BcAA ArAA Met

Leu Val Ile Tyr Trp Phe

L.plantarum 33* _7.46_±_0.35a _0.81_±_0.11b _0.84_±_0.11b _0.69_±_0.06b _0.74_±_0.05b _0.80_±_0.05b _0.85_±_0.11b _0.79_±_0.17b 91* _7.36_±_1.42a _0.78_±_0.04b _1.08_±_0.07b _0.30_±_0.07b _1.36_±_0.07b _1.25_±_0.07b _1.25_±_0.07b _1.17_±_0.07b 89* _9.46_±_0.16a _0.19_±_0.01b,c _0.08_±_0.06c _0.02_±_0.01c _0.69_±_0.42b _0.49_±_0.21b,c _0.54_±_0.36b,c _0.05_±_0.01c 29* _3.12_±_0.34a _1.00_±_0.62b _1.02_±_0.66b _0.84_±_0.65b _1.12_±_0.69b _1.13_±_0.64b _1.06_±_0.69b _0.93_±_0.70b 87* _6.53_±_2.80a _1.06_±_0.36b _0.83_±_0.26b _0.83_±_0.25b _1.16_±_0.46b _1.35_±_0.65b _1.28_±_0.56b _1.02_±_0.51b L.casei 81* _7.18_±_0.36a _3.03_±_0.18b,c _3.31_±_0.11b _2.74_±_0.06c,d _2.31_±_0.20d,e _2.18_±_0.07e _1.92_±_0.03e _1.92_±_0.35e 72 3.34±0.52 2.61±0.45 2.96±0.67 2.73±0.61 3.02±0.52 2.90±0.56 2.58±0.56 2.07±0.55 BL23* _5.83_±_0.71a _1.95_±_0.18b _1.95_±_0.23b _1.34_±_0.15b _1.70_±_0.36b _1.53_±_0.08b _1.39_±_0.05b _1.65_±_0.19b L.paracasei 90* _5.76_±_2.34a _1.34_±_0.07b _1.26_±_0.26b _1.89_±_1.07b _0.99_±_0.49b _1.86_±_0.07b _0.64_±_0.37b _0.86_±_0.51b L.rhamnosus 73* _6.97_±_1.75a _2.50_±_0.28b _1.80_±_0.12b _1.65_±_0.03b _1.84_±_0.09b _2.43_±_0.47b _2.39_±_0.41b _2.25_±_0.31b 77* _3.97_±_0.21a _1.19_±_0.20b,c _1.06_±_0.17b,c _0.89_±_0.01c _1.21_±_0.11b,c _1.19_±_0.14b,c _1.35_±_0.25b _0.88_±_0.01c 78 1.51±1.03 1.26±0.52 0.96±0.23 0.85±0.10 1.17±0.66 1.62±0.75 1.70±0.98 1.19±0.04 75* _4.33_±_2.21a _0.53_±_0.32b _0.57_±_0.51b _0.55_±_0.40b _0.53_±_0.60b _0.60_±_0.43b _0.62_±_0.43b _0.61_±_0.26b L.fermentum 28* _3.42_±_0.17a _0.32_±_0.10b _0.30_±_0.01b _0.24_±_0.03b _0.38_±_0.01b _0.35_±_0.04b _0.34_±_0.01b _0.24_±_0.02b 46* _3.36_±_0.21a _0.23_±_0.14b _0.21_±_0.11b _0.23_±_0.01b _0.21_±_0.16b _0.21_±_0.13b _0.23_±_0.04b _0.21_±_0.01b L.delbrueckii 133* _0.81_±_0.07b,c _1.11_±_0.03a _0.99_±_0.03a,b _0.97_±_0.07b _0.89_±_0.05b,c _0.87_±_0.04b,c _0.92_±_0.07b,c _0.86_±_0.07b,c

L.helveticus 138* _1.20_±_0.24a,b _1.29_±_0.15a,b _1.12_±_0.09b _1.29_±_0.29a,b _1.77_±_0.39a _1.23_±_0.24a,b _1.25_±_0.28a,b _1.16_±_0.22a,b

209 1.21±0.15 1.71±0.21 1.44±0.32 1.52±0.18 1.45±0.32 1.14±0.09 1.14±0.29 1.18±0.10 3 1.02±0.45 0.99±0.38 1.06±0.41 1.17±0.54 1.37±0.51 1.04±0.48 0.98±0.44 1.14±0.51

S.thermophilus 1* _3.04_±_0.01a _0.90_±_0.07b _0.79_±_0.10b _0.74_±_0.14b _0.91_±_0.06b _0.81_±_0.02b _0.89_±_0.06b _0.72_±_0.10b

2* _5.78_±_0.73a _4.37_±_0.41b,c _2.90_±_0.33d _2.53_±_0.07d _4.70_±_0.66b _3.00_±_0.07d _3.40_±_0.50c,d _3.03_±_0.48d

Asp,branched-chainaminoacids(BcAA:Leu,Val,Ile),aromaticaminoacids(ArAA:Tyr,TrpPhe),andMet,inCFEofthe21strainstested. Activitiesareexpressedas␮gofglutamicproducedfrom␣-ketoglutarateperminuteandmilligramofprotein.

Valuesarethemeans±standarddeviationoftheresultsoftwoindependentexperiments.

∗ _Strains_that_showed_signiﬁcant_differences_in_ATs_activity_toward_different_AA_(p_<_0.05).

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1.0 0.5 0.0 –0.5 –1.0 3 2 1 0 –1 –2 –2 –1 0 133 138 138 78 3 133 209 3 209 78 77 87 77 11 29 75 28 4628 46 29 90 75 91 90 87 91 33 89 89 33 73 Lb. casei Lb. delbrueckii Lb. fermentum Lb. helveticus Lb. paracasei Lb. plantarum Lb. rhamnosus S. thermophilus 72 72 73 2 2 81 81 BL23 BL23 1 2 3 –1.0 –0.5 0.0 0.5 AspAT

A

B

TrpAT PheAT MetAT TyrAT ValAT lleAT PC1 (78.7%) PC1 (78.7%) PC2 (12.4%) PC2 (12.4%) LeuAT 1.0

Fig.2–Principalcomponentanalysis(PCA)ofATactivitiestowardeightAAsforthe21strains:A–Loadingplot(PC1vs. PC2),B–Scoreplot(PC1vs.PC2).Ellipsesenclosesamplesgroupedaccordingtohierarchicalclusteranalysis(HCA).

thefourstrainsofthermophiliclactobacilliandL.rhamnosus

78werecharacterizedbythelowestlevelsofATforAsp.

In agreement with our results, other researchers have

reportedhighAsp-ATactivitiesinL.paracaseiandL.plantarum

strains.8,19 _This_AT_proﬁle_is_interesting_for_adjunct_cultures

whendiacetylandacetoinarethedesiredﬂavorcompounds

produced via an alternative or complementary metabolic

pathway for the catabolism of citrate by citrate-positive

strains.2,30_Diacetyl_and_acetoin_formation_from_Asp_has_been

demonstratedinreactionmixtureswith␣-ketoglutarate,30_as

wellasincheesemodels.19,31

Ontheotherhand,whilescreeningcheese-relatedcultures

for AT activity, Smit et al.32 _found _that _Leu-AT

predomi-natedinLactococcusstrains, followedbyS.thermophilusand

L.acidophilus;thelowestlevelswerefoundinL.casei,L. helveti-cus,andPropionibacterium,Leuconostoc,andBiﬁdobacteriumspp.

Also,Kieronczyk etal.19 _reported _higher_levels_of_AT

activ-ityagainstbranched-chainAAsinLactococcuslactiscompared

withL.paracasei.In thepresent work,thehighestlevelsof

Leu-ATwerefoundinS.thermophilus2,followedbytheL.casei

strains,whilethelowestlevelswererevealedbytheL.

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Table3–EndogenousaminotransferaseactivityinCFE ofthe21testedstrains.

Species Strain EndogenousATactivity

L.plantarum 33 0.67±0.07 91 1.23±0.04 89 0.03±0.03 29 1.20±0.04 87 0.79±0.38 L.casei 81 1.51±0.03 72 2.18±0.55 BL23 0.93±0.17 L.paracasei 90 1.13±0.04 L.rhamnosus 73 1.19±0.15 77 0.82±0.05 78 0.84±0.17 75 0.45±0.33 L.fermentum 28 0.29±0.01 46 0.20±0.01 L.delbrueckii 133 0.83±0.08 L.helveticus 138 1.27±0.27 209 1.11±0.14 3 0.84±0.19 S.thermophilus 1 0.60±0.09 2 2.46±0.32

Activities are expressed as ␮g of glutamic produced from ␣-ketoglutarate per min and mg protein. Values are the means±standard deviation of the results of two independent experiments.

Thermophiliclactobacillicultureshavebeenless charac-terizedfortheirATproﬁlesthanotherlactobacilli.Jensenand

Ardö26_reported_AT_speciﬁcity_toward_aromatic_and

branched-chainAAsinsixstrainsofL.helveticus,whileAsp-ATwasvery

loworundetectable.Weconﬁrmedthesametrendfor

Asp-AT,buttheotherATsshowedequallylowlevelsinthefour

thermophiliclactobacillistrainstested.

EventhoughnoATspeciﬁcforMethasyetbeenisolated,

activitytowardthisAAcanbequantiﬁedinlacticcultures,

and it is crucial for the production of important volatile

compounds suchasdimethyl disulﬁde,dimethyl trisulﬁde,

methanethial,andmethanethiol.33_Among_our_strains,

trans-formationofMetwasmainlyevidencedforS.thermophilus2,

thethreestrains ofL.casei, and L.rhamnosus73,whilethe

lowestlevelswere foundinthetwostrainsofL.fermentum.

Hanniffyetal.18_found_high_levels_of_AT_activity_for_Met_in_all

Lactococcusstrainsstudied,whilelowerorundetectablelevels

werefoundinstrainsofL.casei,L.plantarum,andL.fermentum.

Otherresearchershavealsoreportedthisactivityinstrainsof

L.caseiandL.plantarum,3 _as_well_as_in_Lactococcus_strains.34

Metmetabolismisprobablyaresultofnon-speciﬁcactivityof

otherATs,suchasAr-ATorBc-AT,asoverlappinghasbeen

suggested.7

Finally,allstrainsshowedendogenousATactivityranging

fromaminimallevelof0.03forL.plantarum89tolevelshigher

than2.0forS.thermophilus2andL.casei72(Table3).Forsome

strains,includingL.plantarum29and91andL.fermentum28

and46,thisactivitywasrelevantasitwasatthelevelsof

Ar-AT,Bc-AT, andMet-AT.However,evenforthesestrains,the

Asp-AT activity wasalwaysmuchhigher than the

endoge-nousATactivity.EndogenousATactivityhasbeenexplained

byahighintracellularpoolofGluornon-speciﬁc

transamin-ationofdiverseAAsinCFEs.29_Williams_et_al.29_detected_this

activityin60%ofthe29strainsoflactococciandmesophilic

lactobacilli;insomecases,theleveloftheendogenousactivity

wascomparabletothatofAr-ATorMet-AT.

Conclusions

Knowledge of GDH activity and AT proﬁles may be useful

asselectioncriteriaforstarterandadjunctculturesinorder

toenhanceordiversifycheeseﬂavors.Inthepresentwork,

we identiﬁedseveral strains ofpotentialinterest as

ﬂavor-formingcultures.Thus,bothS.thermophilusstrainsshowed

the potential of producing ␣-ketoglutarate from glutamic

acid duetotheirhighGDHactivity.AsfortheATdiversity,

most mesophilic lactobacilli seem appropriate to promote

the productionofAsp-related ﬂavorcompounds, especially

L.plantarumstrains89,33,and 91,whichmay beproposed

asadjunctculturesforthispurpose.Branched-chainamino

acid-andaromaticaminoacid-derivedcompoundsarealso

expectablefromtheuseofL.casei81,72,andBL23,L.

rhamno-sus73,andS.thermophilus2,basedontheirproﬁlesandlevels

ofATs.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgments

ThisworkwassupportedbyAgenciaNacionaldePromoción

CientíﬁcayTecnológica(AGENCIA)andConsejoNacionalde

InvestigacionesCientíﬁcasyTécnica(CONICET).

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