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Food
Microbiology
Antibacterial
efficacy
of
nisin,
bacteriophage
P100
and
sodium
lactate
against
Listeria
monocytogenes
in
ready-to-eat
sliced
pork
ham
Ana
Cláudia
L.
Figueiredo
a,
Rogeria
C.C.
Almeida
b,∗aFaculdadedeFarmácia,UniversidadeFederaldaBahia,RuaBarãodeGeremoabo,s/n,Ondina,Salvador,BA,Brazil bDepartamentodeCiênciadosAlimentos,EscoladeNutric¸ão,UniversidadeFederaldaBahia,Salvador,BA,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received26January2016
Accepted11February2017
Availableonline3June2017
AssociateEditor:ElaineDeMartinis
Keywords: Antimicrobials Foodsafety Meatproducts Listeriamonocytogenes
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b
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TheeffectivenessofbacteriophageP100,nisinandsodiumlactate,individuallyandin
com-bination,ininhibitingListeriamonocytogenesinready-to-eatporkhamsliceswasassessed.
Theantimicrobialswereappliedtothesurfacesofready-to-eatporkhamslices,which
wereinoculatedwithamixtureofL.monocytogenes. Amongtheindividualantimicrobial
treatments,bacteriophage P100 was themost effective, decreasing L. monocytogenesto
undetectablelevelsatzeroand72hpost-infection.Sodiumlactatewastheleasteffective
treatment.Treatmentwithnisinatzerohsignificantlyreducedinitialcelldensity(p<0.05).
However,thispatternwasnotobservedat72hofstorage.Asignificantdifference(p<0.05)
existedbetweentheresultsofseparatebacteriophageandnisintreatmentsafter
refriger-atedstorage,butnotimmediatelyuponinoculationofthebacteria.Theresultsshowedthat
theuseofbacteriophageP100isthemethodofchoiceforthecontrolofbacteria.
©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis
anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
Introduction
ThefirstconclusiveconnectionofListeriamonocytogeneswitha
foodborneoutbreakoccurredin1981,stimulatingresearchers
todeterminethe ubiquityofthe organismandits modeof
transmission. Since then,when the mortality rate did not
seem to diminish over years, L. monocytogenes has gained
recognitionasanimportantfoodbornepathogen.1
Although L. monocytogenes is inactivated by thermal
treatments in processed food, post-processing
cross-contamination from equipment and the environment may
∗ Correspondingauthorat:EscoladeNutric¸ão,UniversidadeFederaldaBahia,Salvador,BA,Brazil.
E-mail:[email protected](R.C.Almeida).
occur duetothepathogen’spersistenceand abilitytoform
biofilms.2,3
Considering that L. monocytogenes iscapable of growing
atrefrigeratedtemperatures,antimicrobialstrategiesto
over-comethismicroorganism’stoleranceforlowtemperaturesare
essential,and the food industry hassought moreeffective
methodstocontrolthispathogen.4
Despiteadvancesinhurdletechnology,foodpreservation
techniquesarestillevolving,notonlyindevelopingcountries
but alsointhe industrializedworld.Theconcept ofhurdle
technology has been applied in the food industry
follow-ingtheobservationsthattherateofmicroorganismsurvival
http://dx.doi.org/10.1016/j.bjm.2017.02.010
1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC
decreases greatly when they are confronted with multiple
antimicrobialfactors,orhurdles.5
Currently,theapplicationofbacteriocinsaspartofhurdle
technologyhasgainedattention.Bacteriocinsare
antimicro-bialactivepeptidesorproteinssynthesizedintheribosomes
of bacterial cells and secreted by some bacteria against
microorganisms that are closely related to the producer
organism.6 They are bacteriostatic or bactericidal against
Gram-positive bacteria,7–10 but bacteriocins synthesized by
Gramnegativebacteria(e.g.colicins)areactiveagainstGram
negativebacteria.Theseantimicrobialpeptideshavebeen
tra-ditionallyusedasbio-preservativestoextendtheshelflives
offoodproductswithoutcompromisingtheirnutritionaland
organolepticproperties7,11,12andtheyhavealreadybeen
suc-cessfullyappliedinseveralfoodsystemstocontrolthegrowth
ofL.monocytogenes.13,14
Several bacteriocins show synergism when used in
combination with other antimicrobials, including chemical
preservatives,phenoliccompounds,andothernatural
antimi-crobialproteins.15,16Theeffectivenessofbacteriocinsisoften
dependentuponenvironmentalfactorssuchaspHand
tem-perature, interaction with food components, precipitation,
inactivation,orunevendistributionofbacteriocininthefood
matrix,andfoodmicrobiota.17
Nisin,thebacteriocinproducedbyLactococcuslactissubsp.
lactis, hasbeen successfully usedas anantibacterial agent
in various food products.8,16 Currently, nisin is the only
bacteriocin widely used as afood preservative, and it has
been acceptedbythe World HealthOrganization asafood
bio-preservative.6,15 However, its successful application in
meat systems has been limited due its interaction with
phospholipids,lowsolubilityandinactivationbyendogenous
meatenzymes.15
Anotherstrategytoensurefoodsafetyistheuseof
bac-teriophages,whichare virusesthatinfectandkill bacteria.
Phages are components of the natural microflora that are
present throughoutfood production, from the farm tothe
retail outlet.17 They are stable and recovered from soil,
sewage, water, farm and processing plant effluents, feces,
and retail foods.17,18 The USFDA has approved a
bacte-riophage preparation made from six individually purified
bacteriophagesforuseasanantimicrobialagentagainstL.
monocytogenesonready-to-eat meatandpoultryproducts.19
Thecommercial productnamedLISTEXP100wasapproved
as a food bio-preservative and granted GRAS (Generally
RecognizedasSafe)status.20
Organic acids with short chains and/or their salts are
frequentlyusedaschemicaldecontaminantsandhavealso
been granted GRAS status.21,22 Organic acids cross the
cell membrane in their undissociated form and
dissoci-ate in the cytoplasm, causing a decrease in intracellular
pH, which significantly impacts cell metabolism,
result-ing in reduced growth.23,24 They are extensively used in
the meat and poultry industries to enhance antimicrobial
benefits.25–27
Theantimicrobialeffectsofthesaltsoforganicacids,either
aloneorincombinationwithotherfoodadditives,onthe
sur-vivalandgrowthofL.monocytogeneshasbeenexaminedand
reported4,27–30;however,littleisknownabouttheireffectsin
combinationwithotherantimicrobialsubstances.Thegrowth
of L. monocytogenes in solutions containing sodium lactate
dependsmainlyonaproduct’swatercontent,storage
tem-peratureand,toalesserextent,theamountofnitritethatit
contains.29
The present study was undertaken to evaluate the
inhibitory effect of nisin, alone and in combination with
sodiumlactateandbacteriophageP100,onamixtureoftwo
strainsofL.monocytogenesinartificiallyinoculated
ready-to-eatslicedporkhambothimmediatelyand afterthreedays
underrefrigeratedtemperature.
Materials
and
methods
Pork hamproduct: Approximately 500g ofready-to-eat pork
hamsliceswerecollectedatalocalsupermarketofSalvador,
BA, Brazil, and transported in iced containers to the
lab-oratory foranalysis. Theproduct was sliced, weighed and
packagedimmediatelybeforepurchased(threedaysofshelf
life).
Nisin: Nisin from L. lactis was purchased from
Sigma–Aldrich (Sigma-Aldrich Brasil Ltda, São Paulo, SP,
Brazil,ref.n◦ 5764).Astocksolutionwaspreparedwith0.1g
of nisin and 10mL of0.02M HCl filter-sterilized through a
0.22-mmembrane(Millipore).Beforeeachexperiment,nisin
wasdilutedwith10mMphosphatebuffer(pH6.4)to50g/L.10
Sodiumlactate:Commercialsodiumdl-lactate(50%purity,
U.S.P.)wasobtainedfromSynth(SynthBrasilLtda,SãoPaulo,
SP,Brazil).Astocksolutionat2%(v/w)waspreparedand
ster-ilizedat121◦Cfor15min.
BacteriophageP100:LISTEXTMwaspurchasedfromEBIFood
Safety(NieuweKanaal7P,6709PAWageningen,The
Nether-lands). Before use, thetiter ofphageP100wasdetermined
accordingtoaprotocolsuggestedbyEBIFoodSafety(personal
communication).
Bacteria,phageandgrowthconditions
L. monocytogenes, serotype 1/2a (B7, AL48/15, institutional
strain collection) isolated from ready-to-eat turkey breast
slicesinapreviousstudyinourlaboratoryandL.monocytogenes
strainScottA–ATCC15313(serotype4b)wereusedto
inocu-latesamplesofready-to-eatporkhamslices.L.ivanoviiWSLC
3009(SLCC4769,institutionalstraincollection)wasusedasa
helperstraintodeterminethetiteroftheP100bacteriophage
(LISTEXTMP100),providedbyEBIFoodSafety.
TheculturesofL.monocytogenesandL.ivanoviiwerestored
inHognessmedium(1.3mMK2HPO4·3H2O,1.3mMKH2PO4,
2.0mMcitrate-Na·2H2O,1.0mMMgSO4·7H2O,and4.4%(v/v)
glycerol)andfrozenat−80◦C.32
Before use, the L.monocytogenescultures were activated
separatelyintrypticsoybroth(Himedia,SãoPaulo,SP,Brazil)
supplemented with 0.6% (w/v) of yeast extract (Himedia)
(TSB-YE)at35◦Covernightinashaker(CientecmodelCT712,
São Paulo, SP,Brazil)at150rev/min.L. ivanovii culturewas
grownovernightat30◦C inahalf-concentratedbrain-heart
infusionbroth(BHI1/2(v/v),DifcoCodeNo.237500)withthe
NaCl concentrationadjustedto5g/L.Following the
incuba-tion,thecultureswerecentrifugedat3600×gfor5minina
Labnet,SãoPaulo,SP,Brazil).Theresultantpelletwaswashed
twiceinPBS (pH7.0),suspendedinthesame solution,and
adjustedto0.5pointsontheMcFarlandscale(∼108CFU/mL)
withpeptonewater.33
Thecultureswerethencombinedanddilutedto
approx-imately 104CFU/mL before inoculating onto the food. The
presenceofviablecellsofL.monocytogeneswasconfirmedby
plating 100Lon Listeriaagar Ottaviani & Agosti(ALOATM,
Laborclin,Pinhais,PR,Brazil)andincubatingat37◦Cfor48h.
Bacterialsurvivalintreatedanduntreatedsampleswas
deter-minedbycountingcolony-formingunits(CFU/g)onALOAagar
platesincubatedat37◦Cfor24h.34
TitrationofP100bacteriophage
ThetiterofthephageP100wasdeterminedaccordingtoa
pro-tocolsuggestedbyEBIFoodSafety(personalcommunication).
Forthisprotocol,serialdilutionsofthebacteriophage
suspen-sionweremadeinalambdabuffer(6mmol/LTrisbuffer,pH
7.2;10mmol/LMg(SO4)2·7H2O;50g/mLgelatin)and100L
of each dilution was mixed into 3.5mL of molten overlay
agarcooledto45◦C,whichcontained150Lofalog-phaseL.
ivanoviiculturegrownovernightat30◦Cinahalf-concentrated
brain-heartinfusionbroth(BHI,Himedia)withtheNaCl
con-centrationadjusted to5g/L.This mixturewas pouredinto
BHIagar (1.2%,w/v, agar)plates andincubated at30◦C for
20–24h. Following incubation, the titerwas determined as
plaque-formingunits(PFU).
For phage control, the sample was diluted in lambda
buffer,andanaliquotof100Lwasincorporatedinto3.5mL
of molten overlay agar cooled to 45◦C, which contained
150LofL.ivanovii(helper strain). Asdescribed above,the
mixture was poured into BHI agar (1.2%, v/w, agar) plates
and incubated at 30◦C for 20–24h. Plaques were counted,
and the titer was determined as plaque forming units
(PFU/g).18
Inoculationofthesamples
Ready-to-eat pork ham slices were divided into 12
por-tions,weighed (30±0.5g)inalaminarflowhood(Labconco
Purifier Class IIb, Total Exhaust, model 36210-04, certified
ISO 9002, Labconco Corporation, Kansas City, MO, USA).
Sixofthe weighed samples were individuallytreated with
700Lofnisin(0.0012g/g),311000LofP100bacteriophage
(5×105UFP/g),35 and 700L of sodium lactate (0.5g/g).31
Two samples were individually treated with nisin
com-binedwithphageP100, andanothertwowere treatedwith
nisin combined with sodium lactate. The remaining two
samples were treated with 700L sterile water to act as
a control. The solutions were absorbed on the food
sur-face by using a sterile glass spreader and were air-dried
under a class II biosafety cabinet for 15min, after which
the samples were each inoculated on the one-side with
1mL of the mixture of L. monocytogenes 1/2a and Scott A
(approximately104CFU/mL).Theinoculatedsampleswere
air-driedunder aclass II biosafety cabinetfor 15min at21◦C
to allow for the attachment of bacteria to the pork ham
slices.
EnumerationofListeriamonocytogenesinready-to-eat slicedporkham
For enumeration ofL.monocytogenes,each 30gsamplewas
addedto270mLof0.1%peptonewaterandhomogenizedin
astomacher(240bpm;ITRmodel1204,series126,Esteio,RS,
Brazil)for2min.Aliquotsof100Lofthehomogenatewere
spread onto duplicate ALOA plates, which were incubated
at37◦Cfor24h.Bacterialsurvivalintreatedanduntreated
(control)sampleswasdeterminedbycountingcolony-forming
units(CFU).
Half of the samples were immediately subjected to L.
monocytogenesenumerationonALOAagar,andtheremaining
sampleswerestoredat6–8◦Cfor72h(porkhamslicesshelf
life)inaerobicconditionbeforeenumeration.
Dataanalysis
WheninvestigatingtheeffectivenessofbacteriophageP100,
nisin and sodium lactate in the inhibition ofthe L.
mono-cytogenes1/2aandScottAmix,bacterialcountswerealways
determinedinduplicate,andalloftheexperimentsdescribed
here wereindependently performedfivetimes.All bacteria
counts(CFU/g)recoveredfromready-to-eatslicedporkham
wereconvertedtologarithmsbeforecomputingtheirmeans
and standarddeviations. Foreach treatmentcondition, the
decimalreduction(DR)ofthepopulationofthebacteriawas
calculatedrelativetothecountsobtainedinthecontrol.The
data were subjected to Analysis of Variance (ANOVA) and
Tukey’smultiplerange test(SoftwareASSISTAT,version7.6
beta,2011)todetermineifsignificantdifferences(p<0.05)in
the mean log valuesofthepopulations ofL.monocytogenes
existedbetweenthetreatmentgroups.
Results
Theinsituantilisterialactivitiesofnisin,bacteriophageP100
(LISTEXTMP100)andsodiumlactateagainsttwostrainsofL.
monocytogeneswerestudiedusingporkhamslicesasamodel substrate.
Theaverage L.monocytogenespopulation recovered from
porkhamslicesafterinoculationwas2.83±0.11logCFU/g.L.
monocytogeneswasnotdetectedinsamplesthatwerenot inoc-ulated.
The effect ofthe treatments containing nisin,
bacterio-phageP100and sodiumlactate,eitherindividually,ornisin
combinedwitheitherbacteriophageP100orsodiumlactate,
on the survivalofthe L.monocytogenes strainsattwo time
pointsisshowninTable1.
Treatmentwithnisinatzerohsignificantlyreducedinitial
celldensity(p<0.05),andintwoassaysthebacteriocinledto
amorethantwo-foldinhibitionofthegrowth.However,this
patternwasnotobservedat72hofstorageat6–8◦C,anda
meanDRvalueof1.66logcycleswasfound.Indeed,theinitial
L.monocytogenespopulation(2.83logCFU/g)increasedtoalevel
of3.23logCFU/gfortheuntreatedgroup(inoculatedwithonly
Table1–ReductionintheviablecountsofL.monocytogenesinready-to-eatslicedporkhambybacteriophageP100 (1.5×107UFP/mL),nisin(50g/L)andsodiumlactate(2%,v/w)aloneornisincombinedwitheitherbacteriophageP100
orsodiumlactate,atzeroand72h(6–8◦C)(mean±standarddeviation,n=5).
Listeriamonocytogenes(Log10UFC/g)
Control Zerohour
2.83±0.08a1,x2 72h 3.27±0.05a,x Treatment DR Treatment DR P100 0.0b,x 2.83±0.08 0.0b,x 3.27±0.05 Nisin 0.8±0.98b,x 2.03±1.2 1.57±1.13a,y 1.67±1.11 Sodiumlactate 2.47±0.17a,x 0.37±0.17 2.10±0.22a,x 1.13±0.26 Nisin+P100 2.27±0.40a,x 0.57±0.31 0.33±0.47b,y 2.9±0.42 Nisin+sodiumlactate 2.40±0.24a,x 0.43±0.26 1.83±0.24a,x 1.40±0.22 DR,decimalreduction.
Superscriptnumbers:1,treatmentmeanswithdifferent(aorb)lettersaredifferentatthe0.05significancelevel(Tukey’stest);2,timemeans withintreatmentwithdifferent(xory)lettersaredifferentatthe0.05significancelevel(Tukey’stest).
Amongtheindividualantimicrobialtreatments, bacterio-phageP100wasthemosteffective,decreasingL.monocytogenes toundetectablelevelsatzeroand72hpost-infection(6–8◦C). Sodiumlactatewastheleasteffectivetreatment,reducing L.monocytogenescountsby0.2–0.6logCFU/gon day0. Addi-tionally,although abetter DRwas achieved, no significant reductioninthecountsofL.monocytogeneswasobservedfor theslicedporkhamstoredat72hat6–8◦C(Table1).
To evaluate additive and synergistic effects, inoculated
porkhamsliceswastreatedwithnisincombinedwitheither
bacteriophageP100orsodiumlactate.Theresultsshowedthat
thecombinationofnisinand P100phagehadasmall
anti-listeriaeffectatzeroh,indicatingantagonismbetweenthese
agents.However,at72h,almost3logcyclesofreductionwere
observedinthenumberofviablebacterialcells(Table1).In
contrast,thetreatmentusingsodiumlactateincombination
withnisindemonstratednoimprovementovertheefficacyof
thebacteriocinalone.
Amongthefiveantimicrobialtreatments,attwodifferent
timepoints,ANOVAshowednosignificantdifferencebetween
samples treated with sodium lactate and samples treated
withsodiumlactateandnisin(p>0.05)relativetothecontrol.
Bycontrast,theindividualtreatmentswithphageP100and
nisinatzerohshowedsignificantdifference fromthe
con-trol(p<0.05).At72h,phageP100andthecombinationphage
P100andnisintreatmentsweresignificantlydifferent(p<0.05)
whencomparedtothecontrol(Table1).
Discussion
TheoccurrenceofL.monocytogenesinRTEfoods,especiallyin
meatproducts,presentsasignificanthealthproblem.
Theemergenceofpathogenic bacteriaresistant tomost
availableantimicrobialsalongwiththeconsumerdemandfor
morenaturalfoodswithoutchemicalpreservativesandwith
longshelfliveshasbecomeacriticalissueforthefood
indus-try.Thisreality hasopened frontiersfortheapplication of
GRASsubstances, suchasbacteriocins,bacteriophagesand
saltsoforganicacids.
Weinvestigatedtheeffectsoftheantimicrobialsnisin,
bac-teriophageP100,andsodiumlactate,individually,andnisin
combinedwitheitherbacteriophageP100orsodiumlactate,at
twodifferenttimepoints(zeroand72h,6–8◦C),onthegrowth
ofamixtureoftwostrainsofL.monocytogenesonready-to-eat
porkhamslices.Statisticalanalysisshowedthatatzeroh
indi-vidualphageP100andnisintreatmentshadgreatereffectson
inhibitingthegrowthofL.monocytogenescomparedtothe
con-trol.Althoughtreatmentswithnisineffectivelyreducedthe
initialcontaminationlevelofL.monocytogenes,the
combina-tionofnisinwithphageP100wasmosteffectiveoninhibiting
thegrowthofL.monocytogenesafterrefrigeratedstorage.
Similar results were found in catfish fillets,36 fresh
sausage,37andsoftcheeses,4showingthatbacteriophageP100
reducesthe cell viability ofL.monocytogenes.However,
Ver-meirenet al.38 observedthattreatmentwithbacteriophage
P100ledtoasmallreductionofonly1.6logunitsinL.
mono-cytogenescountsoninoculatedfishfillets.
Onewaytolowerthenecessaryconcentrationsof
antimi-crobialsagentswithoutcompromisingactivityistouse the
synergistic effects that are observed between two
antimi-crobials that feature different mechanisms of action. For
example,astudyperformedbyLeverentzetal.39showedthat
theuseofnisinandthephagemixturesLM-103andLMP-102,
whichcontained14and6distinctlyticphages,respectively,
reducedL.monocytogenespopulationsbyupto5.7logunitson
honeydewmelonslicescomparedtothecontrol.
Recently,astudyusingtwoListeriabacteriophagesFWLLm1
and FWLLm3,incombinationwiththebacteriocincoagulin
C23 to inhibit L. monocytogenes in milk under refrigerated
conditions, showed that when usedalone the phages and
coagulin C23 kept L. monocytogenes counts lower than the
untreated control throughoutstorage. When used in
com-bination, L.monocytogenescounts wereunder the detection
limits(lessthan10CFU/mL)fromday4untiltheendofthe
experiment.3
Relatedtosodiumlactate,theresultsinthepresentstudy
were similar toa study performed byLung and Johnson40
that showed the addition ofsalt toturkey breast alone or
in combination withnisin did notinhibit the growth ofL.
monocytogenes during storage under refrigeration. Also, the
additionoforganicaciddidnotincreasethebactericidaleffect
ofnisin.However,study havebeenreportedforthe
monocytogeneson cold-smokedsalmonpaté,41 cold-smoked
rainbowtrout42andcookedham.43
Conclusion
PhageP100,whenappliedasanantimicrobialtreatment,was
foundtobeveryeffectiveininactivatingandinhibitingthe
growth ofL.monocytogeneson slicedporkham.Overall, we
havedemonstrated thatinslicedporkham under
refriger-atedconditions,thecombinationofbacteriophageP100and
thebacteriocinnisin ismoreeffectiveasabio-preservative
againstL.monocytogenesthannisinalone. Thiscombination
couldbeasmartstrategytoensureslicedporkhamsafety
duringstorageconditions.
Conflicts
of
interest
Thecontentofthisreportsolelyreflectstheopinionsofthe
authors,andwereportnoconflictsofinterest.
Acknowledgements
This researchwas supportedin part by Coordenac¸ão para
Aperfeic¸oamentodePessoaldeNível Superior(CAPES).The
authorsthankFundac¸ãodeAmparoàPesquisadoEstadoda
Bahia(FAPESB)forscholarshipsupport.
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