Technology and safety assessment for lactic acid bacteria isolated from traditional Bulgarian fermented meat product "lukanka"

brazilian journal of microbiology48(2017)576–586

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

Food

Microbiology

Technology

and

safety

assessment

for

lactic

acid

bacteria

isolated

from

traditional

Bulgarian

fermented

meat

product

“lukanka”

Svetoslav

Dimitrov

Todorov

_,

_Saso

_Stojanovski

_,

_Ilia

_Iliev

_,

_Penka

_Moncheva

_,

Luis

Augusto

Nero

_,

_Iskra

_Vitanova

_Ivanova

a_{UniversidadeFederaldeVic¸osa,DepartamentodeVeterinária,CampusUFV,Vic¸osa,MinasGerais,Brazil}

b_{SofiaUniversity“St.KlimentOhridski”,FacultyofBiology,DepartmentofGeneralandIndustrialMicrobiology,Sofia,Bulgaria} c_{DepartmentofBiochemistryandMicrobiology,FacultyofBiology,PlovdivUniversity“PaisiiHilendarski”,Plovdiv,Bulgaria}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received4February2016 Accepted16February2017 Availableonline11May2017 AssociateEditor:SusanaSaad

Keywords:

Lukanka

Antibioticresistance Virulencefactors Lacticacidbacteria Bacteriocins

a

b

s

t

r

a

c

t

The present work discusses the technological and new selection criteria that should beincluded forselectinglactic acid bacteria forproductionof fermentedmeat. Lactic acidbacteriaisolatedfromBulgariantraditionalfermented“lulanka”salamiwasstudied regardingsomepositivetechnologicalparameters(growthatdifferenttemperature,pH,and proteolyticactivity).Thepresenceofgenesrelatedtothevirulencefactors,productionof biogenicamines,andvancomycinresistancewerepresentedinlowfrequencyinthestudied lacticacidbacteria.Ontheotherhand,productionofantimicrobialpeptidesandhighspread ofbacteriocingeneswerebroadlypresented.VerystrongactivityagainstL.monocytogenes

wasdetectedinsomeofthestudiedlacticacidbacteria.Inaddition,thestudiedstrainsdid notpresentanyantimicrobialactivityagainsttestedcloselyrelatedbacteriasuchas Lacto-bacillusspp.,Lactococcusspp.,Enterococcusspp.orPediococcusspp.Toourknowledgethisis thefirststudyonthesafetyandantimicrobialpropertiesoflacticacidbacteriaisolatedfrom Bulgarianlukankaobtainedbyspontaneousfermentation.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Introduction

Theproductionoffermentedmeatproductsispartofthe gas-tronomichabits ofhumanculture from all continentsand

∗ _{Correspondingauthorat:UniversidadeFederaldeVic¸osa,DepartamentodeVeterinária,CampusUFV,Vic¸osa,MinasGerais,Brazil.}

E-mail:slavi310570@abv.bg(S.D.Todorov).

1 _{Presentaddress:BitolaUniversity“St.KlimentOhridski”,FacultyofVeterinaryMedicine,Bitola,FormerYugoslavianRepublic}

Mace-donia.

canbetracedbacktoearliestrecordsdating from1500BC. Besidestheimprovedpreservationcharacteristicsandsafety ofthefinalproduct,fermentationprocessesplayanessential roleintheorganolepticpropertiesofmeatproducts.Thisrole

http://dx.doi.org/10.1016/j.bjm.2017.02.005

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

brazilian journal of microbiology48(2017)576–586

577

isrelatedtothepresenceandinteractionsbetweendifferent starterculturesandautochthonousmicrobiotafoundin prod-uctsofanimalorigin,differentadditivesofbothanorganicand inorganicnature,essentialoils,nondigestive supplements, fibres,andspecificmetaboliticproducts.Consumerdemands forhigh-quality,safe,nutritious,andconvenientmeat prod-uctshaveprovidedimportantinsightsintothedevelopment ofahostofprocessedmeat products,including fermented meats.Thisoffersuniqueperspectivesfortheapplicationof novelfunctionalmicrobialculturesandpromisingemerging technologies in the manufacture of fermented meats. Fer-mentedmeatproductsareuniqueandoftenrepresentedas anelementofculinaryheritageandidentity.

Differentgeographicalareasdevelopeduniquevarietiesof preservedmeatproductsthatvariedinsize,shape,texture, appearance,andflavour.1_{Therehasbeenarenewedinterestin}

traditionalfermentedmeatproducts,mainlyinEurope,where theyhaveagreatsignificanceandeconomicimpact.2_The

fer-mentationofmeatproductsisrelatedtoanumberofphysical, biochemicalandmicrobialchangesinthemuscle-based prod-uct,causedbyendogenousandmicrobialenzymaticactivities. Thetasteoffermentedmeatproductsisprimarilyduetothe presenceoflacticacidandtheproductionoflowmolecular weightflavourcompounds,suchaspeptides,freeaminoacids, aldehydes,organicacids,andamines,arisingfromthe proteo-lysisofmeat.3_{Naturalfermentationofmeatcanalsoaffectthe}

uniformityofproducts,whichdependonthespecific composi-tionoftheso-called‘houseflora’.Forallofthesereasons,the additionofstartercultureshasbeenrecommendedforthe manufactureoffermentedmeat.Theuseofstartercultures hasbecomeincreasinglynecessaryinordertoproduceamore consistentandstableproductbyimprovingquality,reducing variabilityandenhancingtheorganolepticcharacteristicsof fermentedmeatproducts.4,5_{Inadditionnovelstartercultures}

mayincludemicroorganismsthatgeneratehealth-promoting molecules,possessprobioticattributesorreducethelevelof biogenicamine andcholesterolcontentinfermentedmeat products.4 _{Inadditiontoallthese,the safetyofthestarter}

culturesusedisanessentialissue.Probioticsarelive microor-ganismswhich, wheningestedinsufficientquantities,can exertsomebeneficialhealtheffectsinthehost,andare pri-marilyderivedfromBifidobacteriumandLactobacillusspecies. Additionally,somespeciesofLactococcus,Enterococcus, Saccha-romyces,and Propionibacteriumwerealsoreportedtopresent probioticcharacteristics.6_{Severalstudieshavedemonstrated}

thefeasibilityofaddingprobioticculturestofermentedmeat productformulations.Erkkilaetal.7_{examinedtheabilityof}

threeprobioticLactobacillusrhamnosusstrainstobeinvolved in the production of fermented dry sausage; Pennacchia etal.8 _{reportedtheuse ofLactobacillus plantarumand}

Lacto-bacillusparacaseiasprobiotics inmeatproducts; Pediococcus acidilacticiand Lactobacillussakei also showed good survival characteristicsinfermentedsausages;bothareconsideredas beinggood probioticcandidates foruse inmeat products9_;

Todorovetal.10investigatedthesafetyaspectsofLb.sakei,Lb. plantarum,andEnterococcusfaeciumstrainsisolatedfrom Por-tuguesefermentedmeatproductsinordertoapplythemas abeneficial(starter,bacteriocinproducersorprobiotics) cul-ture.Thebacteriocinsproducedbystarterculturesmayhave anadvantageforthesestrainsincompetitiveinteractionswith

thepathogenicbacteriafromthefoodmatrixandcanbe con-sideredasbeing advantageousfortheprobioticculturesas well.

LukankaisaBulgarian(sometimesspicy)salamiwhichis uniquetoBulgariancuisine.Lukankaissemi-dried,hasa flat-tenedcylindricalshape,andbrownish-redinteriorinaskin thatisnormallycoveredwithawhitefungus.Thefinal prod-uctischaracterizedashavinglowwateractivity,slightlyacid taste,andcanbestoredunderrefrigeration(duringsummer) orattheenvironmentaltemperatureinthewinterperiod.The mixofsmallpiecesofmeatandfatgivetheinterioragrainy structure.Traditionally,lukankasalamiismadeofpork,veal, andspices(blackpepper,cumin,salt),mincedtogether and stuffedintoalengthofdriedcow’sintestineascasing.After thestuffingprocess,thecylindricalsalamiishungtodryfor about40–50daysinawell-ventilatedlocation.Intheprocess ofdrying,thesalamiispressedtoacquireitstypicalflatform. Inthepreliminaryworks,morethan200isolatesofthe gen-eraLactobacilluswereisolatedduringthedifferentstagesofthe fermentationprocessofthenaturallyfermentedlukanka.12

Theisolatesweretaxonomicidentifiedby16SrRNAandthe majoritywasclassifiedasbeingrelatedtoLb.plantarum,Lb. brevisandLb.sakei.Researchintotheproductionoflacticacid, determinationofpHandscreeningforproducersof antimi-crobialswasperformed.Oneofthemostimportantrolesof

Lactobacillusstrainsinthisprocessistheiracidifying proper-tiesforthepreservationofthefinalproduct,dependingaswell onthetotalcellnumber,andoftheacidifyingpropertiesofthe particularstrains.12

Theaimofthis workwastoinvestigatebeneficial, tech-nologicalproperties,andsafetyaspectsoflacticacidbacteria (LAB)isolatedfrom “lukanka”,includinggrowthatdifferent temperature,pH,andproteolyticactivity;presenceofgenes relatedtovirulencefactors,productionofbiogenicamines, and vancomycin resistance;productionofbacteriocins and presenceofrelatedgenes.

Materials

and

methods

Strainsandmedia

Lb.plantarum(13strains:S2,S3,S5,S7,S9,S18,S20,S23,S29, S30,S35,S37,andS39),Lb.brevis(4strains:S8,S27,S36andS38) andLb.sakei(4strains:S11,S12,S13andS15)isolatedfromthe Bulgariandrysausagelukanka,producedbyspontaneous fer-mentation(withoutaddingstartercultures)werepreviously isolated,identifiedandsomeofthemcharacterizedas bac-teriocinproducersandculturedat30◦CinMRSbroth(Difco, LePontdeClaix,France).12_{Lactobacillusspp.,Lactococcusspp.,}

Enterococcusspp.,Pediococcusspp.andL.monocytogenesstrains usedinthebacteriocinproductiontestastestmicroorganisms (Table1

)were grownon MRS(Difco) at30◦C and BHI(Difco)at 37◦C,respectively,andstoredinthepresenceof20%glycerol at−80◦_{C.Beforeuse,thestrainswerecultivatedatleasttwice}

inMRSorBHImedia.

GrowthofLb.plantarum,Lb.brevisandLb.sakeistrainswas determinedinMRSbrothinthepresenceofNaCl(2%,4%,6.5%, 10%and18%(m/v)).Inaddition,strainswereculturedat15◦C,

578

Table1–AntimicrobialspectrumofactivityofselectedLABisolatedfrom“Lukanka”.

Testmicroorganisms Lb.plantarumS2 Lb.plantarumS3 Lb.plantarumS5 Lb.plantarumS7 Lb.brevisS8 Lb.plantarumS9 Lb.sakeiS11 Lb.sakeiS12 Lb.sakeiS13 Lb.sakeiS15

L.innocuaATCC33090 – – – 16 8 8 – – – – L.monocytogenes101 – – – – – – – – – – L.monocytogenes104,106, 506,520 – – – – – – – – – – L.monocytogenes211 – – – 15 8 9 – – – – L.monocytogenes302 – – – – – – – – – – L.monocytogenes409 – – – 15 – – – – – – L.monocytogenes422 – – – 12 10 10 – – – – L.monocytogenes426 – – – 7 – – – – – – L.monocytogenes603 – – – 17 – – – – – – L.monocytogenes607 – – – 13 – – – – – – L.monocytogenes703 – – – 14 – – – – – – L.monocytogenes711 – – – 16 8 9 – – – – L.monocytogenes724 – – – 14 – – – – – – L.monocytogenesATCC7644 – – – 16 8 10 – – – – L.monocytogenesScottA – – – 15 7 9 – – – –

E.faecium11en4,11en3, 13ba2,13en4,13lc23, 11en5,1lb8,S5,ST154Ch, S100,ST62BZ,ST211Ch, ET12,ET88,ET05

– – – – – – – – – –

EnterococcushiraeD105 – – – – – – – – – –

Lb.plantarumSt16Pa,ST8Sh – – – – – – – – – –

LactobacillusacidophilusLa14, Lac4,La5

– – – – – – – – – –

LactobacilluscurvatusET06, ET31,ET30

– – – – – – – – – –

LactobacillusdelbrueckiiB5, ET32

– – – – – – – – – –

LactobacillusfermentumET35 – – – – – – – – – –

LactobacillusrhamnosusLr34 – – – – – – – – – –

LactobacillussakeiATCC 15521

– – – – – – – – – –

Lactobacillussakeisubsp.

sakei2a,MK02R,D2

– – – – – – – – – –

Lactococcuslactissubsp.lactis

B1,D4,B2,B15,D3,D5, B17,R704 – – – – – – – – – – LactococcuslactisV94,V69, B16 – – – – – – – – – –

b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 8 (2 0 1 7) 576–586

579

Testmicroorganisms Lb.plantarumS18 Lb.plantarumS20 Lb.plantarumS23 Lb.brevisS27 Lb.plantarumS29 Lb.plantarumS30 Lb.plantarumS35 Lb.brevisS36 Lb.plantarumS37 Lb.brevisS38 Lb.plantarumS39

L.innocuaATCC33090 – 10 – – – 16 – 17 14 – 17 L.monocytogenes101 – – – – – – – – 17 – 18 L.monocytogenes104,106, 506,520 – – – – – – – – – – – L.monocytogenes211 – – – – – 10 – 11 15 – 17 L.monocytogenes302 – 8 – – – 9 – 7 – – – L.monocytogenes409 – – – – – – – – 12 – 13 L.monocytogenes422 – – – – – 8 – 7 10 – 12 L.monocytogenes426 – – – – – – – – – – – L.monocytogenes603 – – – – – 8 – 9 16 – 17 L.monocytogenes607 – – – – – 11 – 12 10 – 8 L.monocytogenes703 – – – – – 7 – 7 – – – L.monocytogenes711 – – – – – 9 – – 11 – 14 L.monocytogenes724 – – – – – 13 – 12 10 – 11 L.monocytogenesATCC7644 – 9 – – – 14 – 15 17 – 18 L.monocytogenesScottA – – – – 17 – 16 16 – 17

E.faecium11en4,11en3, 13ba2,13en4,13lc23, 11en5,1lb8,S5,ST154Ch, S100,ST62BZ,ST211Ch, ET12,ET88,ET05

– – – – – – – – – – –

EnterococcushiraeD105 – – – – – – – – – – –

Lb.plantarumSt16Pa,ST8Sh – – – – – – – – – – –

LactobacillusacidophilusLa14, Lac4,La5

– – – – – – – – – – –

LactobacilluscurvatusET06, ET31,ET30

– – – – – – – – – – –

LactobacillusdelbrueckiiB5, ET32

– – – – – – – – – – –

LactobacillusfermentumET35 – – – – – – – – – – –

LactobacillusrhamnosusLr34 – – – – – – – – – – –

LactobacillussakeiATCC 15521

– – – – – – – – – – –

Lactobacillussakeisubsp.

sakei2a,MK02R,D2

– – – – – – – – – – –

Lactococcuslactissubsp.lactis

B1,D4,B2,B15,D3,D5, B17,R704 – – – – – – – – – – – LactococcuslactisV94,V69, B16 – – – – – – – – – – –

PediococcuspentosaceusET34 – – – – – – – – – – –

580

25◦C,37◦C,40◦Cand50◦CinMRSbroth.Theabilityofthe studiedstrainstodevelopinthemediumatdifferentinitial pH(pH3.0–pH5.0)wasmonitoredduringtheircultivationin MRS,aswellastheirgrowthcapabilityonacetateagarwith pH5.4(Sigma–Aldrich,Co,St.Luis,MO,USA).

Growthat37◦CinMRSbroth(recommendedoptimal con-ditionsforLAB)wasacceptedasbaselinecriteriaandreferred as“copious”.When growth parametersofinvestigatedLAB werelowerthan30%comparedtooptimal(copious),a bac-terialgrowthwasdefinedas“moderate”.Growthparameters lowerthan70%comparedtobaselinewerereferredas “abun-dant”.

Detectionofproteoliticactivity

SDS-PAGEwasusedfordeterminationoftheproteolitic activ-ityoftheinvestigatedstrains.

Productionofenzymes

Theenzymaticactivityofthestudiedwasdeterminedusing APIZYMstrips(bioMérieux,Marcy-l’Etoile,France)according tothemanufacturer’sinstructions.The20(1controland19 differentsubstrates,Table2)variantsfromtheAPIZYMstrips wereinoculated with24-h-oldculturesgrownonMRSagar (Difco),andthenincubatedat30◦Cfor4h.Theevaluationof theactivitywasgradedfrom0to5usingtheAPIZYMcolour reactionchartaccordingtotheintensityofcolourationand manufacturer’sinstructions.

Antibacterialactivity

Selectedmicroorganismswerescreenedforbacteriocin pro-ductionusingthe agar-spot-testmethodagainstapanelof testmicroorganisms,includingvariousstrainsofListeriaspp.,

Enterococcusspp.,andLactobacillusspp.(Table2).13_Strainswere

growninMRSbrothfor24hat30◦C,cell-freesupernatantwas obtainedaftercentrifugation(6000g,10min, 4◦C),followed bycorrectionofpH to6.0–6.5 with1MNaOH and thermal treatmentfor10minat80◦C.Tenmicrolitersofpreviously preparedmaterialwerespottedonthesurfaceofMRSorBHI containing1%agar,inseminatedwithoneofthepreviously mentionedtestmicroorganisms(Table1)atanapproximate concentrationof106_{CFU/ml,andincubatedfor24hatan}

opti-maltemperatureforthetestmicroorganism(30◦Cor37◦C).

Screeningforpresenceofbacteriocingenes

Total DNA was isolated from the selected microorganisms usingZRFungal/BacterialDNAKit(ZymoResearch,Irvine,CA, USA)accordingtothemanufacturer’sinstructions.DNAwas submittedto PCRreactionsto detect genesresponsible for codificationofthefollowingbacteriocins:nisin,pediocin PA-1,curvacinA, plantaricinS,plantaricin NC8,plantaricin W, sakacinG,sakacinT␣,sakacinT␤andsakacinX(Table3).14–19

ThePCR reaction was preparedusing primers at 10pM/␮L andconditionsasdescribedpreviously,adjustingthe anneal-ingtemperatureaccordingtothespecificationoftheprimers used.14–19 _{The amplified products were separated by}

elec-trophoresisonagarosegelsin0.5×TAEbuffer.Agarosegels

werestainedin0.5×TAEbuffercontaining0.5␮g/mlethidium bromide(Sigma).

Characterizationofvirulencepotential

Theselectedmicroorganismsweretestedfortheharbouring ofvirulencegenesgelE(gelatinase),hyl(hyaluronidase),asa1

(aggregation substance), esp (enterococcal surface protein),

cylA (cytolisin),efaA(endocarditisantigen),ace(adhesionof collagen),vanAandvanB(bothrelatedtovancomycin resis-tance),andgenesforaminoaciddecarboxylases:hdc1andhdc2

(bothrelatedtohistidinedecarboxylase),tdc(tyrosine decar-boxylase), andodc(ornithinedecarboxylase)(Table3),using the PCRprotocolsofMartin-Plateroet al.20_{, Vankerckhoven}

etal.21_{andRivasetal.}22_{.Theamplifiedproductswere}

sepa-ratedbyelectrophoresison0.8–2%(w/v)agarosegelsin0.5× TAEbuffer.Gelswerestainedin0.5×TAEbuffercontaining 0.5␮g/mlethidiumbromide.

Results

and

discussion

Acidificationability,growthatdifferenttemperatures,pH andNaCllevels

TheabilityofLABtoacidifyisoneofthemostimportant tech-nologicalcharacteristicsforpotentialstarterculturesinthe meatindustry.TheloweringofthepHoftheLABcaninhibit the growthofalargenumber ofpathogenic orundesirable microorganismsthatcausespoilageoffermentedmeat prod-ucts,andcanimprovethehygienicpropertiesandstorageof thefinalproducts.Ontheotherhand,apHof5.1–5.3hasa positiveeffectonthehumidityofthefermentedmeat prod-uctsand accelerates the maturation process.Theobtained resultsshowthatalltestedstrains,exceptforLb.plantarum

S23andLb.brevisS36,havegoodacidformationability.The range of the acidifying ability for the studied strains was foundtobefrompH4.03topH5.2.Foracorrect fermenta-tionprocessintheproductpHmustreachvaluesneartothe isoelectricpointoffibrilarproteins.Proteolyticreactionsthat occurduringfermentationofrawsausageshavebeenstudied extensively.Primarily,meatproteinsare degradedinto pep-tidesbyendogenous enzymesduringfermentationofmeat products.Sincemostbacteria(e.g.,lactobacilli)grownin fer-mented meat productshaveonlyweak proteolyticactivity, thedegradationofproteinsisnotgreatlyaffectedby bacte-ria.However,LABinfluenceproteindegradationbycausinga decreaseinpH,whichresultsinincreasedactivityofmuscle proteases.Moreimportantly,thepeptidesgeneratedby mus-cleproteolysiscanbetakenupbybacteriathatfurthersplit themintracellularlyintoaminoacidsandmayconvertthem toaromacomponents.

The pH value and acidity are typical indicators of fer-mented meat products.23 _{Beforedrying, mostdrysausages}

have similar acid activity. In somespontaneous fermenta-tionprocessespHvaluesfrom5.1to5.5andaconcentration of lactic acid of 0.3%to 0.9% have been reported.24

Natu-ral LAB microbiota, particularly well adapted to these dry sausages,ischaracterizedbyahighacidifyingpotential.For the properfunctioningoffermentation,thepHatthetime

b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 8 (2 0 1 7) 576–586

581

Table2–APIZYMenzymaticprofileofLABisolatedfrom“Lukanka”.

Enzymes Lb.plantarumS2 Lb.plantarumS3 Lb.plantarumS5 Lb.plantarumS7 Lb.brevisS8 Lb.plantarumS9 Lb.sakeiS11 Lb.sakeiS12 Lb.sakeiS13 Lb.sakeiS15

Alkalinephosphatase 2 2 0 0 2.5 0 0 0 0 0 Esterase(C4) 2 1 0 0 0.5 0 0 2 2 2 Esteraselipase(C8) 1.5 0.5 2 0 0 0 0 0 0 0 Lipase(C14) 0.5 0 2 2 0 2 0 0 0 0 Leucinearylamidase 0 5 0 0 5 2 0 2 2 2 Valinearylamidase 5 1 5 5 1 5 5 5 5 5 Cystinearylamidase 1 5 4 4 5 4 3 4 5 5 Trypsin 5 0 0 3 0 3 0 0 2 2 ␣-Chymotrypsin 0 0 0 0 0 0 0 0 0 0 Acidphosphatase 0 3.5 0 0 4 0 0 0 0 0 Naphitol-AS-BI-phosphohydrolase 4 2 3 4 3 4 3 3 3 3 ␣-Galactosidase 2 1 4 4 0.5 4 4 4 4 4 ␤-Galactosidase 0.5 4 0 0 4 0 0 0 0 0 ␤-Glucuronidase 4 0 5 4.5 0 5 4 4 4 2 ␣-Glucosidase 0 4 0 0 4.5 0 0 0 0 0 ␤-Glucosidase 4.5 3.5 3 3 5 4 3 2 0 0 N-acetyl-␤-glucosaminidase 3.5 5 5 5 5 5 5 0 0 0 ␣-Mannosidase 5 0 5 5 0 5 5 0 0 0 ␣-Fucosidase 0 0 0 0 0 0 0 0 0 0

Enzymes Lb.plantarumS18 Lb.plantarumS20 Lb.plantarumS23 Lb.brevisS27 Lb.plantarumS29 Lb.plantarumS30 Lb.plantarumS35 Lb.brevisS36 Lb.plantarumS37 Lb.brevisS38 Lb.plantarumS39

Alkalinephosphatase 1 0 0 3 0 0 0 1 2 3.5 0 Esterase(C4) 1 2 2 2 1 2 2 2 1 1 1 Esteraselipase(C8) 1 2 2 1.5 2 2 2 2 2 0.5 0.5 Lipase(C14) 1 2 2 0 2 2 2 1 0 0 0 Leucinearylamidase 1 2 2 5 2 2.5 2 2 1 5 1 Valinearylamidase 5 5 5 2.5 5 5 5 5 5 1 5 Cystinearylamidase 4 4 4 5 4 4 4 4 4 5 4 Trypsin 3 3 3 3 0 3 3 3 3 0 3 ␣-Chymotrypsin 0 0 0 0 0 0 0 0 0 0 0 Acidphosphatase 0 0 0 4 0 0 0 0 0 3.5 0 Naphitol-AS-BI-phosphohydrolase 4 4 4 4 4 4 4 4 4 2 3 ␣-Galactosidase 4 5 5 3 4 5 4 4 4 2 3 ␤-Galactosidase 0 0 0 4 0 0 0 0 0 4 0 ␤-Glucuronidase 4 5 5 4.5 4 5 5 4 4 0 2 ␣-Glucosidase 0 0 0 3.5 0 0 0 0 0 4 0 ␤-Glucosidase 4 4 4 3 5 4 3 3 3 5 3 N-acetyl-␤-glucosaminidase 5 5 5 4 5 5 5 5 5 5 5 ␣-Mannosidase 5 5 5 0 5 5 5 5 5 0 0 ␣-Fucosidase 0 0 0 0 0 0 0 0 0 0 0

582

Table3–Presenceofbacteriocingenesandgenesrelatedtovirulencefactors,productionofbiogenicaminesandvancomycinresistanceinLABisolatedfrom “Lukanka”.

Lb.plantarumS2 Lb.plantarumS3 Lb.plantarumS5 Lb.plantarumS7 Lb.brevisS8 Lb.plantarumS9 Lb.sakeiS11 Lb.sakeiS12 Lb.sakeiS13 Lb.sakeiS15

Bacteriocingenes CurvacinA – – – – – – – – – – Nisin + – – – – – – + – – PediocinPA1 – – – – – – – – – – PlantaricinW – – – – – – – – – – PlantaricinNC8 – – – – – – – + – – PlantaricinS – – – – – – – – – – SakacinG – – – – – – – – – – SakacinT␣ – – – – – – – – – – SakacinT␤ + + + + + + + + + + SakacinX + + + + – + + + + +

Genesrelatedtovirulencefactors,productionofbiogenicaminesandvancomycinresistance

VancomicinB – + – – – – – – + –

VancomicinA – – – – – – – – – –

Gelatinase

Enterococcocalsurfaceprotein – – – – – – – – – –

Hystidinedecarboxilase(2setsof primers) – – – – – – – – – – Hyaluronidase – – + – – – – – – + Aggregationsubstance – – – – – – – – – – Cytolisin – – – – – – – – – – Endocarditisantigen – – – – – – – – – – Tyrosinedecarboxylase + + + + + + + + + +

Adhesionofcollagenprotein – – – – – – – – – –

Ornithinedecarboxilase + + + + + + – + + +

Lb.plantarumS18 Lb.plantarumS20 Lb.plantarumS23 Lb.brevisS27 Lb.plantarumS29 Lb.plantarumS30 Lb.plantarumS35 Lb.brevisS36 Lb.plantarumS37 Lb.brevisS38 Lb.plantarumS39

Bacteriocingenes CurvacinA – – – – – – – – – – – Nisin + – – – – – – – – – – PediocinPA1 – – – – – – – – – – – PlantaricinW – – – – – – – – – – – PlantaricinNC8 – – – – – – – – – – – PlantaricinS – – – – – – – – – – – SakacinG – – – – – – – – – – – SakacinT␣ – – – – – – – – – – – SakacinT␤ + + + + + + + + + + + SakacinX + – + + – – + + + – –

Genesrelatedtovirulencefactors,productionofbiogenicaminesandvancomycinresistance

VancomicinB – – – – + – – – – – –

VancomicinA – – – – – – – – – – –

Gelatinase +

Enterococcocalsurfaceprotein – – – – – – – – – – –

Hystidinedecarboxilase(2setsofprimers) – – – – – – – – – – –

Hyaluronidase – – – – – – – – – – –

Aggregationsubstance – – – – – – – – – – –

Cytolisin – – – – – – – – – – –

Endocarditisantigen – – – – – – – – – – –

Tyrosinedecarboxylase + + + + + + + + + + +

Adhesionofcollagenprotein + – – – + – – – – – –

brazilian journal of microbiology48(2017)576–586

583

offilling shouldreach 4.6–5.1,avalueclose tothe isoelec-tricpointoffibrillarproteins.31_{Positivetechnologicalaspects}

ofacidification,besidestheinhibitionofpathogenic microor-ganismsandfasterdryingoftheproduct,are theimproved texturethroughthedenaturationandcoagulationofproteins, theactivationofmuscleproteasesandtheformationofthered colourofthesausagebystimulatingtheformationofnitrite andnitrozomioglobulin.25

Theabilityofstudiedstrainstodevelopinthemediumat differentinitialpH(pH3.0–pH5.0)wasmonitoredduringtheir cultivationinMRSmedium,alongwiththeirgrowthcapability onacetateagar.Thelargemajorityofthestrainsisolatedfrom thedrylukankasausagewereabletogrowonacetateagar. FromthestudiedstrainsonlyLb.plantarumS7andLb.brevis

S8havegoodgrowthunderacidicconditions(pH3.3)(datanot shown).Formost(Lb.plantarumS7,S18andS29,Lb.brevisS8, S36andS38)pH4.0wasmorefavourable.Allstrainsgrewvery wellintheneutralandweaklyalkalineregion,andwiththe exceptionofstrainsLb.plantarumS23andLb.brevisS38,have verylowgrowthinhighalkalineconditions(pH9.6).Someof thestrainswerecharacterizedbyaverynarrowpHrangeof growth(Lb.plantarumS2,S3andS7growwellonlyataninitial pHof7.0–7.5).Lb.plantarumS7haveawidepHrange,growing intherangeof3.3–8.5).Lb.brevisS38hastheabilitytogrow intheentirepHrangeinvestigated(pH4.0–9.6).Manyofthe strainsinvestigated(withthe exceptionofLb.plantarumS3, S18andS37)werenotabletogrowatpH8.5and9.6.

Theabilityoftheculturestogrowinaparticular temper-aturerangeisanimportantphysiologicalcharacteristicused fortheidentificationofLAB.Forthispurpose,isolatesgrown attemperaturesof15◦C,25◦C,37◦C,40◦C,and 50◦C were studied.Almostallstrainstestedhadgood(copious)growthat 37◦C(withtheexceptionofLb.plantarumS3andLb.brevisS36), moderategrowthat40◦C(withtheexceptionofthestrainsLb. plantarumS2andS3andLb.brevisS36),andweakgrowthat 50◦C(excludingLb.plantarumS3).ThegrowthofLb.plantarum

S37wasabundantathightemperatures.At25◦Cmoststrains haveabundantgrowth.Approximatelyhalfofthestrainsdid notgrowat15◦C.Lb.plantarumS29 andS39had verygood growthcharacteristicsoverawidetemperaturerange.

The resistance of LAB to the concentration of NaCl in themiddlevariesandisanimportantcharacteristicoftheir speciesdifferentiation.Furthermore,stabilityandgrowthin increasingconcentrationsofsaltareimportanttechnological characteristicsoftheLABoffermentedmeat(s).Inthisstudy, theabilityoftheLABtogrowhasbeenexaminedinthegrowth mediumsupplementedwith2%,4%,6.5%,10%,and18%NaCl. Thegrowthofmoststrainswasabundantintheconcentration ofNaClintherangeof2–6.5%.At10%concentrationofNaCl, manystrainshadmoderateandabundantgrowth,butsome (Lb.brevis S27and Lb.plantarumS29)grew poorly,and only

Lb.brevisS36didnotdevelop.Inthepresenceof18%NaClall strainspresentedpoorgrowth.

Technologicalpropertiesofsodiumchloridearerelatedto itseffectontheflavour,textureandshelflifeofmeat prod-ucts.Insearchofhealthierfermentedfoodproducts,inmost cases a salt content of over 2% can be markedly lowered without substantial sensory deterioration or technological problemsleadingtoeconomicallosses.Sodiumchloride con-tentdownto1.4%incookedsausagesand1.75%inleanmeat

productsisconsideredtobeenoughtoproduceaheat-stable gelwithacceptableperceived saltinessaswell asfirmness, water-bindingandfatretention.However,aparticular prob-lemwithlow-saltmeatproductsisthatnotonlytheperceived saltiness,butalsotheintensityofthecharacteristicflavour canbeaffected.Increasedmeatproteincontent(i.e.leanmeat content) inmeat products reducesthe perceived saltiness. Fromthetechnologicalpointofview,therequiredsalt con-tentforacceptablegelstrengthdependsontheformulation oftheproduct.However,whenphosphatesareaddedorthe fatcontentishigh,lowersaltadditionsprovideamorestable gelthaninnon-phosphateandinlow-fatproducts.Small dif-ferencesinsaltcontentatthe2%leveldonothaveamarked effectontheshelflifeoftheproducts.Byusingsaltmixtures, usuallyNaCl/KCl,theintakeofsodium(NaCl)canbefurther reduced.11

Proteolyticactivity

The strains cultured in milk and analyzed by SDS-PAGE, showed differentprofiles related tothe proteolyticactivity. Theperformed analysisdetermined that thetested strains hydrolysedhighmolecularweightproteinsofthemilkwith varyingcapacity.Theresidueamountsofk-caseinand␣, ␤-casein were below50% withinthe majority ofthe strains. Lowmolecularweightproteinsofthemilkwerealso hydrol-ysedbymostofthestudiedstrains.Itshouldbenotedthat somestrainshydrolysed␤-lactoglobulincompletely. Accord-ingtopreviouslyreporteddata,theLABhavearelativelylow proteolyticactivity againstmyofibrillar proteins.23_However,

somestrainsofLb.casei, Lb.plantarum,Lb.curvatus,andLb. sakeicontributedsignificantlytothehydrolysisof sarcoplas-micproteins,aswellastothesubsequentdecompositionof thepeptidestofreeaminoacids.23_{Furthermore,somestrains}

ofLb.sakei,Lb.curvatus,andLb.plantarumshowedthe pres-enceofleucineandvalineaminopeptidases,whichcontribute tothebreakdownofproteinsand peptides.Inthiscontext, aminoacid release, attributedtoprecursors,is responsible fortheflavourofthe finalproduct.11_{All theseresults}

indi-cate thatmilkproteinsmaybeusedas amodelsystemto establishtheproteolyticactivityofstrains.Theanalysisof pro-teolysisinthemanufactureoffermentedsausages,screening forproteinases,peptidases,andaminopeptidasesduringthe selectionofstartersshouldberecommendedaspartofthe screeningfornewstartercultureswithbeneficialproperties.

Enzymeactivities

InTable2theactivitiesoftheenzymescorrelatedwith car-bohydratecatabolismareshownandtheydifferconsiderably. ␤-GalactosidaseactivitywasexhibitedbyonestrainofLb. plan-tarum(S3)andthreestrainsofLb.brevis(S8,S27andS38).In contrast,themajorityofthestrainsexhibit␣-galactosidase activity (24 strains). Almost all strains have ␤-glucosidase activity (except forLb. sakei S13 and S15), whilethe num-ber ofstrains with ␣-glucosidase activity was considerably lower. The results for ␣-galactosidase, ␤-glucuronidase, ␣-mannosidase,andN-acetyl-␤-glucosaminidaseactivity,found inthemajorproportionofstrains,confirmedthoseobtained fromPapamanolietal.11_{.Theacidtasteoffermentedmeat}

584

products,whichiscorrelated totheacid content,isoneof thecomponentsoftheoveralltaste.26_{Metabolismduring}

fer-menteddrysausageripeningisacomplexinteractionbetween residualenzymeactivityofthemuscleand/orfattissueand bacterialmetabolism.

Withrespectto lipolyticactivity for the strainsisolated fromlukanka,ourstudieshaveshownthatallstrainshavea verylowactivity.Thesedataareconsistentwiththereferred tointhe work byMontel et al.26 _{According toVestergaard}

etal.,27_{thelipolysisofmeatproductsismainlyduetomuscle}

lipaseandphospholipase,whilebacteriallipasesshowvery littleactivityintermsoffermentedproducts.28

The degradation of amino acids to volatile molecules playedan importantrole in determiningthe characteristic tasteofdrysausages.StudiesbyMonteletal.26_showedthat

thealdehydes,alcoholsandacidsderivedfromthebreakdown ofleucine,valine,phenylalanineandmethionine havevery lowvalues.Theexaminedactivityofvalinearylamidaseand cysteine arylamidase detectedwhenusing APIZYM forthe majorityofstrainsinourstudywashigh(Table2).Theactivity ofleucinearylamidaseisrelativelylowformoststrains,while forsomestrainsofLb.plantarum(S2,S5,andS7)andL.sakei

(S11)wasnotdetected.Acidphosphataseandalkaline phos-phataseactivitieswere notexhibitedbymostofthetested strains,butallfourLb.brevishavegoodalkalineactivity.

Spectrumofantimicrobialactivity

ThestudiedLABwereevaluatedfortheirantimicrobial activ-ityusingtheagar-spot-testagainstapaneloftestorganisms (Table1).Cell-freesupernatantsobtainedfromLb.plantarum

(BS7, BS9, BS20, BS30, BS37 and BS39) and Lb. brevis (BS8, BS36)(adjustedtopH6.0–6.5)inhibitedthegrowthofseveral strainsbelongingtoL.monocytogenes.However,noneofthe otherbacteriaincludedinthemicroorganismpaneltestwas affected(Table1).Thisnarrowspectrumofbacteriocinactivity wasrevealedasbeinguniqueforLactobacillusspp.Infact,most ofthebacteriocinsdescribedforLactobacillusspp.werefound tobeactiveagainstamuchbroaderrangeofmicrobial gen-eraandspecies.19_{However,itisimportanttoemphasizethat}

theverystrongactivityagainstL.monocytogenesexhibitedby BS7,BS30,BS36,BS37andBS39strainscouldhavesignificant applicationinthebiopreservationoffermentedfoodproducts. However,tobenotactiveagainstvariousLABandtoinhibit variousL.monocytogenessuggestsaverygoodpotentialforthis bacteriocin(s)orappropriateLABtobeusedinthe biopreser-vationofvariousfoodproductswhileatthesametimenot affectingtheappliedstartercultures.

Screeningforpresenceofbacteriocingenes

OnthebasisofthePCRreactionsperformedtargetingnisin, pediocinPA-1,curvacinA,plantaricinS,plantaricinNC8, plan-taricinW,sakacinG,sakacinT␣,sakacinT␤andsakacinX, someofthetestedstrainsgeneratedpositiveresults(Table3). Thesequenceofthegeneratedampliconswas100% identi-caltothetargetedbacteriocingenes.Allthestrainsproduced positiveresultsforthepresenceofsakacinT␤,howevernone ofthemgeneratedevidencesforthepresenceofsakacinT␣. ItisimportanttorefertothefactthatsakacinT␣andsakacin

T␤arepartofthetwo-bacteriocincomplex,andthepresence ofbothpeptideisrequiredfortheactivityofamaturepeptide system.17 _{Carryingonlyoneoftherequiredgenesfor}

two-peptidebacteriocinsismostprobablyrelatedtothefactthat primarysolicitationofsakacinT␤inthelifecircleofthetested strainsisnotanantimicrobialanddefenceprotein,but prob-ablyhasadifferentrole.Recentlyraisedthehypothesisthat someofthepolypeptidesproducedbytheLABhasaregulatory function,butbecauseofthesharedsimilaritytosome antimi-crobialpeptidestheyexhibitsomeantimicrobialactivityas well.29,30

Weneedtopointoutanotherfact:someofthetestedLAB hasgeneratedantibacterialactivity basedonthe agar-spot-testmethod,butwecannotdetectthepresenceofthetested bacteriocingenes(Tables1and3)andviceversa.Ithasbeen previouslyreportedthatspecificLABstrainscancarrymore thanonebacteriocingene,butcannotexpresssomeofthem.31

However,additionalexperimentsonbacteriocinpurification, andtheamino-acidsequenceoftheexpressedbacteriocin(s) isrequiredinordertoconfirmtheexpressionofthedetected bacteriocingene(s).Fromthisperspective,itseemsimportant topointoutthattheapplicationofdifferentapproachesfor thecharacterizationofabacteriociniscrucial.Veryfrequently authorsreportonnewbacteriocinidentificationmerelybased onthedeterminationofthepresenceofgene(s)for bacterio-cin(s)production.32,33_Albanoetal.34_{verifiedthepresenceof}

pediocin PA-1 genesintwodifferentstrains ofP. acidilactici

isolatedfrom“Alheira.”However,theevidencesforthe expres-sionofthesebacteriocinswerenotreportedandthestudied bacteriocinshavebeenonlypresumedtobeexpressed.32–34

Thepurificationofthebacteriocinfollowedbymass spectrom-etryandpartialorcompleteaminoacidsequencingisproof thatthegenesareactuallybeingexpressed.35_{Moreover,Poeta}

et al. showed that some bacteriocin-producer strains may carryseveralgenesforbacteriocinproductionand, depend-ingongrowthconditions,theycouldexpressoneoranother gene.31_{Asaconsequence,reportsbasedonlyonthedetection}

oridentification ofbacteriocingenesneedtobeconsidered with high scepticism and results should be accepted only whenadditionalresearchandsufficientevidenceof bacterio-cinproductionandexpressionismadeavailable.

Screeningforphysiologicalandgenetictraitrelatedto virulencefactor,biogenicaminesandantibioticresistance

Despitebeingveryrelevantinfoodisolatesandwellaccepted asGRASmicroorganisms,theverificationofvirulencefactors inLactobacillusspp.bybio-molecularproceduresisnecessary duetotheriskofgenetictransfer,sincethesegenesare usu-allylocatedinconjugativeplasmids.36_{Interestingly,fromall}

the screenedgeneticsequencesrelatedtoseveralvirulence factors,biogenic aminesand antibioticresistance(Table3), onlyPCRtargetingvancomycinB(in3from21testedisolates), gelatinase(in1from21testedisolates),hyaluronidase(in2 from21testedisolates),tyrosinedecarboxylase(in21from21 testedisolates),ornithinedecarboxilase(in2from21tested isolates)andadhesionofcollagenprotein(in2from21tested isolates)genesgeneratedpositiveresults.VanBgenesconfer resistancetovariousconcentrationsofvancomycinonlyand havebeendescribedinvariousLAB.37

brazilian journal of microbiology48(2017)576–586

585

Thepresenceofthevirulencefactorsisanimportantpoint inthesafetyevaluationofLABwithapotentialapplicationas functionalstrains(probioticorstartercultures).Evenifthese virulencefactorshavebeendescribedasplayingaroleinthe pathogenicityofthe Enterococcusspp., we needtoconsider theirpresenceinfunctionalLABwithhighprecision.The sce-narioofhorizontalgenetransferisaveryhighpossibilityin themixedmicrobialpopulationandweneedtobeawareof thisfact.

Conclusions

FactorssuchasthephysiologicalstateofLABasstarters,the physicalconditionsofripeningandstorage(e.g.temperature, pH, sodium chloride), chemical composition of the matrix (acidity,availablecarbohydratecontent,wateractivity,etc.) andthepossibleinteractionofthestartercultureswith probio-ticand/orothermicroorganismsoccurringnaturallyoradded tothesystemofthestudiedmeatproducthavetobe consid-eredwhendevelopnewfunctionalfermentedfoodproducts.

Somemetabolitessuchasantimicrobialpeptidescanplaya roleinLABperformancesandmetabolism,affectingnotonly microbialsafety,butalsothetotalpopulationandthe ecol-ogyoffermentedmeatproducts.Verystrongactivityagainst

L.monocytogeneswasdetectedforsomeofthestudiedLAB. Inaddition, thestudiedstrainsdidnotpresentany antimi-crobial activity against tested closely related bacteria, and frequentlyusedasstartercultures,suchasLactobacillusspp.,

Lactococcusspp.,Enterococcusspp.orPediococcusspp.Studied LAB presented low levels of presenceof virulence factors, includingantibiotic resistanceandbiogenic aminesrelated genesandmaybeconsideredassafe,inadditiontopositive technologicalcharacteristicsforproductionoffermentedfood productssuchasacidificationproperties,survivalatincreased NaClconcentrations,productionoftechnologicallyimportant enzymesandproteoliticactivity.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorswouldliketoacknowledgeCNPq313852/2013-8 andCNPq401140/2014-8projectsforprovidingfinancial sup-portinadditiontosupportfromCAPESandFAPEMIG.

r

e

f

e

r

e

n

c

e

s

1.MooreJE.Gastrointestinaloutbreaksassociatedwith fermentedmeats.MeatSci.2004;67:565–568.

2.VignoloG,FontanaC,FaddaS.Semidryanddryfermented sausages.HandbookMeatProc.2010;5:379–398.

3.ClaeysE,DeSmetS,BalcaenA,RaesK,DemeyerD. QuantificationoffreshmeatpeptidesbySDS-PAGEin relationtoageingtimeandtasteintensity.MeatSci. 2004;67:281–288.

4.LeroyF,VerluytenJ,deVuystL.Functionalmeatstarter culturesforimprovedsausagesfermentation.IntJFood Microbiol.2006;106:270–285.

5.AndrighettoC,ZampeseL,LombardiA.RAPD-PCR

characterizationoflactobacilliisolatedfromartisanalmeat plantsandtraditionalfermentedsausagesofVenetoregion (Italy).LettApplMicrobiol.2001;33:26–30.

6.ZhangY,LiuJ-Z,HuangJ-S,MaoZ-W.Genomeshufflingof PropionibacteriumshermaniiforimprovingvitaminB12 productionandcomparativeproteomeanalysis.JBiotechnol. 2010;148:139–143.

7.ErkkilaS,PetejaE,EerolaS,LillebergL,Mattila-SandholmT, SuihkoM-L.Flavourprofilesofdrysausagesfermentedby selectednovelmeatstartercultures.MeatSci.

2001;58:111–116.

8.PennacchiaC,ErcoliniD,BlaiottaG,PepeO,MaurielloG, VillaniF.SelectionofLactobacillusstrainsfromfermented sausagesfortheirpotentialuseasprobiotics.MeatSci. 2004;67:309–317.

9.RavytsF,VuystLD,LeroyF.Bacterialdiversityand functionalitiesinfoodfermentations.EnginLifeSci. 2012;12:356–367.

10.TodorovSD,FrancoBDGM,WiidIJ.Invitrostudyofbeneficial propertiesandsafetyoflacticacidbacteriaisolatedfrom Portuguesefermentedmeatproducts.BenefMicrob. 2013;5:351–366.

11.PapamanoliE,TzanetakisN,LitopoulouE,KotzekidouP. CharacterizationoflacticacidbacteriaisolatedfromGreek dry-fermentedsausageinrespectoftheirtechnologicaland probioticproperties.MeatSci.2013;65:859–867.

12.StojanovskiS,DimovSG,IvanovaI,VassilevD.Assessment ofcelldensityandacidifyingpropertiesvariationof forty-twoLactobacillusstrainsisolatedfrom“Lukanka” sausage.BiotechnolBiotechnolEq.2010;24:708–711.

13.TodorovSD.BacteriocinproductionbyLactobacillusplantarum AMA-KisolatedfromAmasi,aZimbabweanfermentedmilk productandstudyofadsorptionofbacteriocinAMA-Kto Listeriaspp.BrazJMicrobiol.2008;38:178–187.

14.RemingerA,EhrmannMA,VogelRF.Identificationof bacteriocin-encodinggenesinLactobacillibyPolymerase ChainReaction(PCR).SystemApplMicrobiol.1996;19:28–34.

15.TodorovSD,WachsmanM,ToméE,etal.Characterisationof anantiviralpediocin-likebacteriocinproducedby

Enterococcusfaecium.FoodMicrobiol.2010;27:869–879.

16.TodorovSD,RachmanC,FourrierA,etal.Characterizationof abacteriocinproducedbyLactobacillussakeiR1333isolated fromsmokedsalmon.Anaerobe.2011;17:23–31.

17.MacwanaSJ,MurianaPM.A“bacteriocinPCRarray”for identificationofbacteriocin-relatedstructuralgenesinlactic acidbacteria.JMicrobiolMeth.2012;88:197–204.

18.KrugerMF,BarbosaMS,MirandaA,etal.Isolationof bacteriocinogenicstrainofLactococcuslactissubsp.lactisfrom Rocketsalad(ErucasativaMill.)andevidencesofproduction ofavariantofnisinwithmodificationintheleader–peptide. FoodControl.2013;33:467–476.

19.MuruaA,TodorovSD,VieiraADS,MartinezRCR,Cenci ˇcA, FrancoBDGM.Isolationandidentificationof

bacteriocinogenicstrainofLactobacillusplantarumwith potentialbeneficialpropertiesfromdonkeymilk.JAppl Microbiol.2013;114:1793–1809.

20.Martin-PlateroAM,ValdiviaE,MaquedaM,Martinez-Bueno M.Characterizationandsafetyevaluationofenterococci isolatedfromSpanishgoats’milkcheeses.IntJFoodMicrobiol. 2009;132:24–32.

21.VankerckhovenV,VanAutgaerdenT,VaelC,etal. DevelopmentofamultiplexPCRforthedetectionofasa1, gelE,cylA,esp,andhylgenesinenterococciandsurveyfor virulencedeterminantsamongEuropeanhospitalisolatesof Enterococcusfaecium.JClinMicrobiol.2004;42:4473–4479.

586

22.RivasP,AlonsoJ,MoyaJ,deGorgolasM,MartinellJ,Guerrero MLF.Theimpactofhospital-acquiredinfectionsonthe microbialetiologyandprognosisoflate-onsetprosthetic valveendocarditis.Chest.2005;128:764–771.

23.SanzY,FaddaS,VignoloG,AristoyM-C,OliverG,ToldraF. HydrolysisofmusclemyofibrillarproteinsbyLactobacillus curvatusandLactobacillussake.IntJFoodMicrobiol. 1999;53:115–125.

24.CapitaR,Llorente-MarigomezS,PrietoM,Alonso-CallejaC. Microbiologicalprofiles,pHandtitratableacidityofchorizo andsalchichon(TwoSpanishdryfermentedsausages) manufacturedwithostrich,deer,orporkmeat.JFoodProtect. 2006;69:1183–1189.

25.HugasM,MonfortJM.Bacterialstarterculturesformeat fermentation.FoodChem.1997;59:545–554.

26.MontelMC,MassonF,TalonR.Bacterialroleinflavour development.MeatSci.1998;49:111–123.

27.VestergaardCS,SchivazappaC,VirgiliR.Lipolysisin dry-curedhammaturation.MeatSci.2000;55:1–5.

28.DemeyerD,RaemaekersM,RizzoA,etal.Controlof bioflavourandsafetyinfermentedsausages:firstresultsofa Europeanproject.FoodResInt.2000;33:171–180.

29.RodaliVP,LingalaVK,KarlapudiAP,IndiraM,

VenkateswaruluTC,JohnBabuD.Biosynthesisandpotential applicationofbacteriocins.JPureApplMicrobiol.

2013;7:2933–2945.

30.KjosM,OppegardC,DiepDB,etal.Sensitivityoftwo-peptide bacteriocinlactocinGindependentonUppP,anenzyme involvedincell-wallsynthesis.MolMicrobiol.

2014;92:1177–1187.

31.PoetaP,CostaD,Rojo-BezaresB,etal.Detectionof antimicrobialactivitiesandbacteriocinstructuralgenesin faecalenterococciofwildanimals.MicrobiolRes.

2007;162:257–263.

32.DeKwaadstenietM,FraserT,vanReenenCA,DicksLMT. BacteriocinT8,anovelclassIIasec-dependentbacteriocin producedbyEnterococcusfaeciumT8,isolatedfromvaginal secretionsofchildreninfectedwithhuman

immunodeficiencyvirus.ApplEnvironMicrobiol. 2006;72:4761–4766.

33.DeKwaadstenietM,tenDoeschateK,DicksLMT.

CharacterizationofthestructuralgeneencodingnisinF,a newlantibioticproducedbyaLactococcuslactissubsp.lactis isolatedfromfreshwatercatfish(Clariasgariepinus).Appl EnvironMicrobiol.2008;74:547–549.

34.AlbanoH,TodorovSD,vanReenenCA,HoggT,DicksLMT, TeixeiraP.Characterizationofabacteriocinproducedby Pediococcusacidilacticiisolatedfrom“Alheira”,afermented sausagetraditionallyproducedinPortugal.IntJFood Microbiol.2007;116:239–247.

35.ZendoT,NakayamaJ,FujitaK,SonomotoK.Bacteriocin detectionbyliquidchromatography/massspectrometryfor rapididentification.JApplMicrobiol.2008;104:499–507.

36.EatonTJ,GassonMJ.MolecularscreeningofEnterococcus virulencedeterminantsandpotentialforgeneticexchange betweenfoodandmedicalisolates.ApplEnvironMicrobiol. 2001;67:1628–1635.

37.KilicA,DemirayT,SaracliMA,etal.Bacteriological characterizationofvancomycin-resistantenterococciin pediatrichospitalinTurkey.AnnMicrobiol.2004;54:543–551.