Inactivation of Listeria monocytogenes ATCC 7644 on fresh-cut tomato using nisin in combinations with organic salts

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

Food

Microbiology

Inactivation

of

Listeria

monocytogenes

ATCC

7644

on

fresh-cut

tomato

using

nisin

in

combinations

with

organic

salts

Adebola

O.

Oladunjoye

∗

,

Suren

Singh,

Oluwatosin

A.

Ijabadeniyi

DepartmentofBiotechnologyandFoodTechnology,DurbanUniversityofTechnology,Durban,SouthAfrica

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received26June2015

Accepted14January2016

Availableonline28April2016

AssociateEditor:ElaineCristina

PereiraDeMartinis Keywords: Listeriamonocytogenes Nisin Freshproduce Sodiumcitrate Sodiumacetate Qualityparameters

a

b

s

t

r

a

c

t

TheinhibitionofListeriamonocytogenesATCC7644onfresh-cuttomatowasinvestigated

using nisinalone, andin combinationswith organicsalts.Nisin ata concentrationof

5000UI/mLwasintroducedaloneorincombinationwithanorganicsalt(sodiumcitrate

orsodiumacetateeachat3and5g/100mLeach)onfresh-cuttomatopreviously

inocu-latedwith108_{CFU/mLofL.monocytogenesATCC7644.Chlorineat200ppmwasusedasa}

control.Theinoculatedsampleswereincubatedatdifferenttemperatures(4,10and25◦C)

andexaminedat0,24,48and72h.Theeffectsoftheantimicrobialtreatmentsonquality

parametersoftomato(pH,solublesolids,titratableacidityandvitaminC)werealso

evalu-ated,andcolourparameterswereobservedattheloweststoragetemperaturefor10days.

BothnisinandtheorganicsaltsinhibitedgrowthofL.monocytogenes,butthecombinations

oftwocompoundsweremoreeffective.Thenisin–sodiumcitrate(5%)combinationwas

sig-nificantly(p≤0.05)effective,whilechlorinewasleasteffectiveagainstL.monocytogenes.The

qualityparametersweresubstantiallyretained,especiallyat4◦C,suggestinggoodshelf

sta-bilityatalowtemperature.Theseresultssubstantiatetheuseofthecheapandeco-friendly

approachtoreducingthispathogenofhealthconcernincommonfreshproduce.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis

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

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

Introduction

Fresh produce is an essential component of human diet.

It contains micro-nutrients, vitamins and certain

phyto-chemicals which contribute significantly to human diet.1

∗ _{Correspondingauthor.}

E-mail:debbyoladunjoye@yahoo.com(A.O.Oladunjoye).

Growing demands for exotic freshproduce inrecent years

haveattractedlaudableattentionduetoitshealth-promoting

nature.2 _{It has been strongly advocated by relevant global}

agencies,that consumptionofapproximately 400goffresh

produceperday,whichequalsto“fiveservingsaday”asa

rec-ommendeddailyintakehasaprophylacticcapacitytostem

http://dx.doi.org/10.1016/j.bjm.2016.04.027

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC

758

brazilian journal of microbiology47(2016)757–763

thetideofcertainmaladies inhumans suchascarcinoma,

diabetesandcardiovasculardiseases.3,4

Despite its veritable contribution to human diet, the

benefitsoffreshproduceconsumptionhavebeengreatly

chal-lengedbytheemergenceofdiseaseoutbreakslinkedtocertain

pathogens,sincealargeproportionoffreshproduceisoften

usedraw, withlittle or no antimicrobialtreatment. Listeria

monocytogeneshasbeen reportedas oneofsuchpathogens

implicatedintheoutbreaks.4,5

L.monocytogenesisatenaciousmicro-organism thatcan

befound invarious locations inthe environment, such as

soil,waterandanimaldung,andinawidevarietyoffresh

andminimally-processedproduce.6_{Thisgram-positive}

oppor-tunistic bacterium is of very high public health concern

asthe cause ofa life-threateningdisease called listeriosis.

Thediseasehasthecapacitytoaffecttheentirevulnerable

population.Arecentmulti-stateincidenceofthisdisease

out-break,linkedtoacommerciallyproducedappleproduct,has

beenreportedintheUnitedStatesin35cases,ofwhich34

werehospitalisedandeventuallyleftsevenpatientsdead.7_In

anotherrelateddevelopment,Canadapublichealthagencies

arealsoonthealertastwosimilarcasesofcontamination

withL.monocytogenesofsamegeneticbackgroundhavebeen

reported.8 _{As controlmeasures,various interventions have}

beenendorsedtoreduceitscontaminationoffreshproduce

along the food chain and during processing. Over time, a

chlorinewashhasbeencommonlyemployedtoreduce

micro-bialloadsonfreshproducepriortosubsequentprocessing.

However,developmentofresistantmutants,and

accumula-tionofhalogen-basedresidues,whichcouldelicitanoxious

risktobothfoodandconsumershavereduceditscontinuous

utilisation.9,10_{Theseoutcomeshaveincitedaclamourtothe}

useofnaturalantimicrobialssuchasnisinandorganicacids

orsalts forreducing pathogens withahigh mortalityrate,

suchasL.monocytogenes,whereinthecompoundsserveas

bio-cidalagents,whilethenutritionalintegrityoffreshproduceis

substantiallyretained.11

Nisinisabacteriocinproducedbythelacticacid

bacterium-Lactococcuslactissubsp.lactiswithapreservativecapacityin

thefoodindustry.Ithasbeenreportedtoexhibitbactericidal

effectsongram-positivebacteriasuchasL.monocytogenes.12

Thisbiologicalantimicrobialagenthasbeengrantedthe

“gen-erallyregardedassafe”(GRAS)statusbytheUnitedStatesFood

andDrugAdministration,which hasmadeit commercially

available.Organicsaltshavealsobeenregardedasveritable

antimicrobialagents.TheybasicallyreducepHofthe

cellu-larenvironment,disrupttransportofmaterialsandincrease

permeabilityofthecytoplasmicmembrane.Advantageously,

theyareinexpensive,lessharmfultopersonnel’shealthand

theprocessingenvironmentandtheirGRASstatushasalso

beenendorsedforcommercialuse.

Nisininsynergywithsaltsoforganic acidsmaybe

con-sideredapromisinganti-listerialbarrierwhichcouldprolong

theshelfstabilityoffreshproducesuchastomato.This

hur-dleapproachhasbeenextensivelyutilisedforfoodsofanimal

origin withasuccessfullog reductionofthe pathogen.For

instance,nisinincombinationwithsodiumlactateappliedon

cold-smokedrainbowtroutreducedtheL.monocytogenes

pop-ulationfrom3.26to1.8logCFU/govertwoweeksofstorage

at8◦C,withnodeleteriousimpactonsensoryquality.13_Also,

Grosulescu14 _{reportedtheuse ofdifferentorganic salt}

con-centrationsagainstL.monocytogenesonsomemeatproducts,

with significant reductions in bacterial counts. This

natu-ralhurdlelookspromisingandmaysufficeinthecontrolof

L.monocytogenesinfreshproducesuchastomato.Therefore

this researchaimedtoinvestigate thesynergisticeffectsof

nisin and saltsoforganic acids onthe survivalofL.

mono-cytogenesinfresh-cutproduceandtheirimpactsonproduct quality.

Materials

and

methods

Preparationoffreshproduce

Freshtomatoes(Lycopersiconesculentum)wereobtainedfrom

a localgrocerysupermarket (Woolworthsupermarket,

Dur-ban,SouthAfrica).Priortoexperimentalstudies,theproduce

waswashedwithdistilledwater,surfacedisinfectedwith70%

ethanolandallowedtodryatroomtemperaturebeforebeing

cutinto10mmthickslices.

Bacteriumandpreparationofinoculum

AnL.monocytogenes:serovar1/2c(ATCC7644)frozenstock

cul-ture inglycerol (Merck,SouthAfrica) was allowedtothaw

inawaterbathat25◦Cfor3min.Thethawed culturewas

grown in 50mLof tryptone Fraser broth base(Oxoid, Ltd.,

England) supplemented with5g/Lof yeastextract, 1.35g/L

ofpotassiumdihydrogenphosphateand12g/Lofdi-sodium

hydrogen phosphate at37±1◦C for 20–24h. Bacterial cells

wereharvestedbycentrifugation(Eppendorfcentrifuge5810R)

at3000rpmfor15minat4±1◦C andre-suspendedin

ster-ilesalinepeptone(8.5%NaCland1%peptone).A108_CFU/mL

suspensionofthecellswasthenpreparedusingaMcFarland

standardsolution(0.05mLof1.175%BaCl2·2H2Oand9.95mL

of1%H2SO4).

Preparationofantimicrobialsandsampleinoculation

The procedures were carried out using modified method

describedbySamelisetal.15_{Nisin(N5764.Sigma–Aldrich,St.}

Louis,MO,USA)waspreparedat0.5g/100mL,whichisequal

to 5000IU/mL waspreparedand storedattherefrigeration

temperature, while organic salts – sodium citrate

(Associ-ated Chemical Enterprise, Johannesburg, SouthAfrica) and

sodiumacetate(Sigma–Aldrich)wereeachpreparedat3and

5g/100mL.Chlorine(SodiumhypochloriteMerckChemicals

Ltd.,Gauteng,SouthAfrica)whichservesascontrolwas

pre-paredat200ppm.Tomatosliceswereplacedonaluminiumfoil

inabiohazardhoodand1mLofinoculumwasspreadonboth

sidesofeachsliceusingasterilebentglassrod.Theinoculated

sliceswerelefttostandseparatelyat4◦Cfor15minandthen

dipped antimicrobialsolutions. Thesolutions were poured

aseptically into pre-sterilised stainlesssteel pots equipped

withstrainerstofacilitatedippinganddrainingfor1mineach.

Thesliceswerethenstoredinaperforatedstomacherbagat

differenttemperatures(4,10and25◦C)andfordifferentperiod

oftime(0,24,48and72h)toassesslogreductionlevelsofthe

Bacterialanalysis

RecoveryoftheL.monocytogenesATCC7644populationsfrom

thefresh-cuttomatowasperformedafter0,24,48and72hof

storageat4,10,and25◦C.Thetomatoslicesfromstomacher

bag were dilutedwith9mLofbuffered peptonewaterand

blendedinahomogeniserfor120satahighspeed.Aliquotsof

themixtureswereseriallydilutedinbufferedpeptonewater,

and100␮LofthedilutionswerespreadonListeriaagarbase

(Oxoid)withaListeriaselectivesupplement(Sigma–Aldrich).

Colonieswere countedafterincubationat37±1◦Cfor48h

using a Doc-itcolony counter (Analytik Coy Germany). All

treatmentswereconductedintriplicate.

Qualityparameters

Qualityparameters(pH,solublesolids,titratableacidity,

vita-minCandcolourvalues)ofthefresh-cuttomatosliceswere

evaluatedbeforeandaftertheexperiment.ThepHvalueswere

determinedusing apenetration-electrodepHmetre (Model

Basic20CrisonInstrument,Barcelona,Spain).Solublesolids

(SS)weredeterminedusingarefractometer(PR-201+,Atago,

Pty.,Ltd).Titratableacidity(TA)wasdeterminedbydiluting

10mLofahomogenisedsamplewith10mLofdistilledwater,

followedbytitrationofthemixturewith0.1NNaOHuptopH

8.1.VitaminCwasdeterminedbythemethodofMohammed

et al.,16 _{using a UV spectrophotometer (Jenway7305,Bibby}

Scientific, Ltd., Stone, Staffordshire, UK). TheInternational

CommissiononIlluminationCIEL*a*andb*valueswere

deter-minedat the refrigerated temperature using acolorimeter

(ColorFlexEZ,0840,HunterLab,Reston,USA)colorimeter.All

parameterswereevaluatedintriplicate.

Statisticalanalysis

All experiments were replicated thrice for each treatment.

The data obtainedwere subjected to analysis of variance,

andthemeanswereseparatedusingDuncan’smultiple-range

test(p≤0.05).ResultswereanalysedusingtheSPSSsoftware

package.

Results

and

discussion

Effectofanti-microbialtreatmentsonListeria monocytogenespopulations

Theresults obtainedfrom the antimicrobialdipping

treat-ments showed a significant reductionin the survivalof L.

monocytogenes at 4◦C (Fig. 1). The fresh-cut tomato slices

dipped in chlorine for 1 and 3min showed significantly

(p≤0.05) smaller log reduction values of 1.90–2.81 and

1.91–2.85CFU/mL,respectivelydependingonthetimeof

incu-bation.Thenisin-aloneshowed alogreductionof∼2.05to

2.99CFU/mLdependingonthetimeofincubation.However,

nisininthecombinationswithsodiumcitrate,especiallyat

5%concentrationofthelatter,showedthehighestlog

reduc-tionof2.56–3.48CFU/mLdependingonthetimeofincubation

comparedtotheotherantimicrobialtreatments.

0 1 2 3 4 5 6 7 8 9 72h 48h 24h 0h Log(cfu/ml) Time (hour) Lm Lm+Nisin Lm+Ns+SC3% Lm+Ns+SC5% Lm+Ns+SA3% Lm+Ns+SA5% Lm+CL 1min Lm+CL 3min

Fig.1–Listeriamonocytogenespopulations(logCFU/mL)on

fresh-cuttomato(n=3)storedat4◦Cwithnisinaloneand

incombinationswiththeorganicsaltsaswellasinthe

chlorinecontrol(Lm−,L.monocytogenes;Lm+Nisin,L.

monocytogenesandnisin;Lm+Ns+SC3%,L.monocytogenes,

nisinand3%sodiumcitrate;Lm+Ns+SC5%,L.

monocytogenes,nisinand5%sodiumcitrate;Lm+Ns+SA3%,

L.monocytogenes,nisinand3%sodiumacetate;

Lm+Ns+SC5%,L.monocytogenes,nisinand5%sodium

acetate;atLm+CL−L.monocytogenesandchlorine).

TheantimicrobialtreatmentsagainsttheL.monocytogenes

populationonfresh-cuttomatoincubatedat10◦Cexhibited

similarpatterntothoseobservedat4◦C(Fig.2).Thefresh-cut

tomatoslicesdippedinchlorineforboth(1and3min)showed

significantly(p≤0.05)lowerlogreductionvaluesof∼1.32to

1.48and1.38to1.50CFU/mL,respectivelydependingontime

ofincubation.Thetreatmentwithnisinaloneresultedinan

about1.88–1.72CFU/mLlogreduction,dependingonstorage

temperature. Meanwhile,nisin inthe combinationwith5%

sodiumcitrateshowedhigherlogreductionvaluesof∼2.27

to2.28CFU/mLcomparedwithnisinalone.

Similar trend of log reduction of L. monocytogenes was

observed at elevated temperature of 25◦C (Fig. 3).

Chlo-rine treatment at both contact time significantly (p≤0.05)

retained the least log values of ∼0.26 to 0.39 and 0.37 to

0.54CFU/mLrespectively.Nisinalone resultedinlog

reduc-tion of1.60–1.76CFU/mL. Nisin–sodiumcitratecombination

at5%concentrationretainedthehighestlogreductionvalues

ofrecoverymeanvaluesof2.44–2.56logCFU/mL.

Thecomparativeineffectivenessofthechlorinecontrolas

demonstrated by lower the log reduction valuescompared

to the other antimicrobial treatmentscan be explainedby

thedevelopmentofchlorineresistancebythepathogen.For

example,L.monocytogenesisabletoformbiofilms,whichare

microbialaggregatesinanexo-polysaccharidematrix.

Bacte-rialcellsinbiofilmsresistcommonsanitiserssuchaschlorine,

causinganimmenseconcerninthefreshproduceindustry.17

However,Ijabadeniyi,18_{reportedtheinactivationofpathogen}

760

brazilian journal of microbiology47(2016)757–763 0 1 2 3 4 5 6 7 8 72h 48h 24h 0h Lo g( cfu/ ml) Time (hour) Lm Lm+Nisin Lm+Ns+SC3% Lm+Ns+SC5% Lm+Ns+SA3% Lm+Ns+SA5% Lm+CL 1min Lm+CL 3min

Fig.2–L.monocytogenespopulations(logCFU/mL)on

fresh-cuttomato(n=3)storedat10◦Cwithnisinaloneand

incombinationswiththeorganicsaltsaswellasinthe

chlorinecontrol(Lm−,L.monocytogenes;Lm+Nisin,L.

monocytogenesandnisin;Lm+Ns+SC3%,L.monocytogenes,

nisinand3%sodiumcitrate;Lm+Ns+SC5%,L.

monocytogenes,nisinand5%sodiumcitrate;Lm+Ns+SA3%,

L.monocytogenes,nisinand3%sodiumacetate;

Lm+Ns+SC5%,L.monocytogenes,nisinand5%sodium

acetate;atLm+CL−L.monocytogenesandchlorine).

0 1 2 3 4 5 6 7 8 9 72h 48h 24h 0h Lo g( cfu/m) Time (hour) Lm Nisin Ns+SC3% Ns+SC5% Ns+SA3% Ns+SA5% CL 1min CL 3min

Fig.3–L.monocytogenespopulations(logCFU/mL)on

fresh-cuttomato(n=3)storedat25◦Cwithnisinaloneand

incombinationswiththeorganicsaltsaswellasinthe

chlorinecontrol(Lm−L.monocytogenes;Lm+Nisin,L.

monocytogenesandnisin;Lm+Ns+SC3%,L.monocytogenes,

nisinand3%sodiumcitrate;Lm+Ns+SC5%,L.

monocytogenes,nisinand5%sodiumcitrate;Lm+Ns+SA3%,

L.monocytogenes,nisinand3%sodiumacetate;

Lm+Ns+SC5%,L.monocytogenes,nisinand5%sodium

acetate;atLm+CL−L.monocytogenesandchlorine).

via pore formationin thecytoplasmicmembrane of

gram-positivebacteriasuchasL.monocytogenes,whichenhancesthe

bacterialinactivationonfresh-cutproduce.Similarfindingsby

Hoelzer19_{indicated1.33and2.64log}

10CFUreductionsofthe

pathogenbynisinonbroccoliandcabbage,respectively.The

higherratesofreductionsofthepathogenonfresh-cuttomato

bynisinincombinationswiththesaltsoforganicacidsonthe

fresh-cuttomatocanbeassociatedprimarilywiththe

reduc-tion ofpHand disruptionofcellularmaterials.Therelative

efficacyofsodiumcitratecanbelinkedtoitsstrongerbiocidal

andmetal-chelatingcapabilitiescomparedtomonocarboxylic

saltssuchasacetates.20,21Increasedinstoragetemperatures

tendstofavourL.monocytogenessurvivalonfreshproduceas

reflectedbythelogreductionrates.10

Effectofanti-microbialtreatmentonqualityparameterof thefresh-cuttomato

Theresultsofthequalityparameterassays(pH,TA,SSand

vitaminC) forfresh-cuttomato are representedinTable1.

Nisin inthecombination withsodiumcitrateata3%

con-centrationgavethelowestpHvalue(p≤0.05)at4and10◦C,

whilenisin–sodiumacetateata5%concentrationshowedthe

highestpHvaluesatbothtemperatures. At25◦C,however,

nisin–sodiumacetate(5%)showedthelowestpHvalue,while

that incontrol wassignificantly (p≤0.05) highercompared

with the other anti-microbial treatments. Anti-microbial

treatmentsshowedhigher(p≤0.05)TAvaluesthanthosein

controlsamplesat4◦Candlowervaluesthanthoseincontrol

sampleat10◦Cexceptnisin–sodium acetate(5%).However,

at 25◦C,all the antimicrobial treatments were lower than

thatofthecontrolsample.Theamountofsolublesolidsin

thenisin–sodiumacetateat(5%)weresignificantly(p≤0.05)

higherthanthoseinthecontrolandotherantimicrobial

treat-mentsat4and10◦C,however,areversetrendwasobserved

at25◦C.ThevitaminCvalueinthenisin–sodiumacetate(5%)

treatmentoffresh-cutproducewashigherthanthevaluesin

thecontrolandotherantimicrobialtreatmentsatall

temper-atureregimes,whilethetreatmentwithnisinaloneshowed

thelowestvitaminCvalue.Nisininthecombinationwiththe

organic salts resultedinfurtherpHlowering,whichagrees

withtheirbasicantimicrobialmodeofaction.Similarfindings

reported by22 _{corroborate the above conclusion. Lower pH}

representsagoodkeepingqualityinfreshproducesuchas

tomatosincemostspoilagebacteriafinditdifficulttothrive

insuchamedium.ThedecreaseinTAwiththeincreasing

storagetemperature,increasescanbelinkedtoalossofcitric

acid.23 _{TAin fresh produce representsthe indexof}

matu-rity. Soluble solids also decreased with increasing storage

temperature,whichcorrelateswiththesugarlevel, anddry

mattercontentintomatoandisalsoinverselyproportionalto

theproducesize.Beckles24 _{discussedinhisreviewprevious}

studies showing that an increased temperature enhance

the rates rate ofevaporation and transpirationwhich

ulti-mately affects sugar levels intomato. Theincrease in the

vitaminC content couldbedueto theorganic compounds

oftheseantimicrobialsactingasprecursors.Ayala-Zavala25

reportedanincreaseintheascorbicacidcontentoffresh–cut

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 7 (2 0 1 6) 757–763

761

Treatment Temperature 4◦_{C 10}◦_{C 25}◦_C pH TA(gcitric acid/10g) SS(◦Brix) VitC (mg/10g) pH TA(gcitric acid/10g) SS(◦Brix) VitC (mg/10g) pH TA(gcitric acid/10g) SS(◦Brix) VitC (mg/10g) Control 4.40c_{±0.01 6.00}a_{±0.20 6.10}b_{±0.20 1.18}a_{±0.02 4.42}d_{±0.02 6.00}c_{±0.20 5.90}d_{±0.20 0.88}a_{±0.02 4.43}e_{±0.02 5.70}c_{±0.20 5.40}c_{±0.20 0.43}a_±0.01 Nisin 3.67b_{±0.02 6.20}ab_{±0.10 5.80}ab_{±0.20 1.17}a_{±0.01 3.79}c_{±0.02 5.70}bc_{±0.20 5.40}c_{±0.20 0.83}a_{±0.02 3.82}d_{±0.01 5.00}b_{±0.50 4.90}c_{±0.20 0.45}a_±0.00 Ns+SC3% 3.46a_{±0.02 6.30}ab_{±0.20 5.80}ab_{±0.20 1.19}a_{±0.02 3.51}a_{±0.02 5.60}b_{±0.20 4.70}ab_{±0.20 0.93}a_{±0.02 3.70}c_{±0.02 4.70}ab_{±0.20 4.30}ab_{±0.10 0.49}b_±0.02 Ns+SC5% 3.47ab_{±0.01 6.40}b_{±0.10 5.60}a_{±0.20 1.23}b_{±0.02 3.62}b_{±0.01 5.20}a_{±0.20 4.93}b_{±0.15 0.97}a_{±0.01 3.58}a_{±0.02 4.83}b_{±0.25 4.37}ab_{±0.06 0.50}b_±0.02 Ns+SA3% 3.48a_{±0.04 6.20}ab_{±0.10 5.50}a_{±0.20 1.24}b_{±0.01 3.56}ab_{±0.01 5.70}bc_{±0.10 4.60}a_{±0.20 0.96}a_{±0.02 3.64}b_{±0.01 4.30}a_{±0.10 4.50}b_{±0.10 0.63}c_±0.02 Ns+SA5% 3.51d_{±0.01 7.10}c_{±0.20 6.60}c_{±0.20 1.30}c_{±0.01 6.30}e_{±0.10 6.30}d_{±0.10 6.23}e_{±0.06 1.04}b_{±0.20 3.67}bc_{±0.02 4.70}ab_{±0.20 4.20}a_{±0.10 0.70}d_±0.02

Valuesaremeans±standarddeviationsofthreereplicates.Meanvaluesa,b,c,dandeinthesamecolumnwithsamesuperscriptarenotsignificantlydifferent(p≤0.05). TA,titratableacidityingcitricacid/10g;SS,solublesolidinobrix;VitC,vitaminC;SC,sodiumcitrate;SA,sodiumacetate.

Table2a–Colourparametersoffresh-cutdippedinnisinandincombinationwithotherantimicrobialsat4◦Cfortendays.

Treatment Days

Day0 Day2 Day4

L* a* b* L* a* b* L* a* b* Control 52.20f_{±0.02 23.72}d_{±0.00 26.71}c_{±0.02 54.02}e_{±0.01 23.37}c_{±0.01 27.36}e_{±0.02 52.30}f_{±0.02 23.04}b_{±0.02 26.90}e_±0.20 Nisin 41.41c_{±0.02 22.87}c_{±0.02 22.43}b_{±0.02 42.08}c_{±0.02 25.54}c_{±0.02 23.78}c_{±0.00 39.24}c_{±0.02 24.97}c_{±0.02 23.44}b_±0.01 Ns+SC3% 44.85d_{±0.01 26.43e±0.01 26.74}f_{±0.01 44.86}d_{±0.01 27.87}e_{±0.02 28.77}f_{±0.02 43.36}e_{±0.02 27.42}e_{±0.01 29.08}f_±0.02 Ns+SC5% 37.03a_{±0.02 20.46}a_{±0.02 17.99}a_{±0.02 38.59}a_{±0.00 30.21}f_{±0.00 22.46}a_{±0.00 35.72}a_{±0.02 30.63}f_{±0.02 23.85}c_±0.02 Ns+SA3% 51.13e_{±0.02 21.44}b_{±0.02 24.84}d_{±0.00 45.29}e_{±0.02 22.65}a_{±0.02 24.97}d_{±0.02 41.34}d_{±0.02 22.72}a_{±0.02 25.11}d_±0.00 Ns+SA5% 40.42b_{±0.01 28.32}d_{±0.02 23.41}c_{±0.00 43.67}c_{±0.01 26.88}d_{±0.02 22.91}a_{±0.01 38.23}b_{±0.02 26.43}d_{±0.02 22.58}a_±0.02

Valuesaremeans±standarddeviationsofthreereplicates.

Meanvaluesa,b,c,d,eandfinthesamecolumnwiththesamesuperscriptarenotsignificantlydifferent(p≤0.05).

762

brazilian journal of microbiology47(2016)757–763 T able 2b – Colour par ameters of toma to slices dipped in nisin, in combina tion with other antimicr obials at 4 ◦C for ten da ys. T reatment Da y 6 Da y 8 Da y 10 L * a * b * L * a * b * L * a * b * Contr ol 53.58 e± 0.00 23.05 b± 0.00 27.00 c± 0.00 33.91 a± 0.00 23.72 b± 0.00 18.97 a± 0.00 38.10 c± 0.00 23.73 f± 0.00 24.39 d± 0.00 Nisin 43.69 d± 0.04 26.72 e± 0.10 28.45 d± 0.13 48.97 d± 0.04 28.30 e± 0.05 33.00 d± 0.04 37.57 a± 0.03 25.66 e± 0.03 22.49 b± 0.02 Ns + SC3% 54.11 f± 0.07 17.94 a± 0.22 25.51 b± 0.15 54.72 e± 0.02 21.80 a± 0.03 28.73 b± 0.06 39.29 e± 0.08 24.02 d± 0.02 25.73 e± 0.02 Ns + SC5% 43.16 c± 0.02 25.79 d± 0.02 28.84 e± 0.12 48.32 d± 0.08 28.57 f± 0.15 33.73 d± 0.16 43.54 f± 0.12 20.34 a± 0.02 23.53 c± 0.02 Ns + SA3% 39.71 b± 0.04 24.70 c± 0.38 31.01 f± 0.06 41.35 b± 0.34 27.74 d± 0.23 36.71 e± 0.52 37.92 b± 0.01 23.43 c± 0.02 20.06 a± 0.03 Ns + SA5% 39.63 a± 0.36 22.82 b± 0.10 25.30 a± 0.06 42.17 c± 1.10 27.05 c± 0.11 31.60 c± 0.14 38.72 d± 0.02 22.79 b± 0.02 32.26 f± 0.02 V alues ar e means ± standar d de viations of thr ee re plicates. Mean v alues a, b, c, d, e and f in the same column with the same superscript ar e not significantl y differ ent (p ≤ 0.05). Ns, nisin; SC, sodium citr ate; SA, sodium acetate .

the vitaminCvaluesdecreasedasthestoragetemperature

increasedwhichisquiteexpected.26,27

Effectofantimicrobialtreatmentoncolourparameterof fresh-cuttomato

The results obtained for the colour parameters L*

(bright-ness), a* (red), and b* (yellow) of the fresh-cut tomato

(Tables 2a and 2b) showed that nisin alone increased the

values of redness (p≤0.05) from day 2 to day 10

com-paredwiththecontrol.Thedegreeofrednessincreasedwith

nisin–sodiumcitrate(3%)comparedtothecontrolfromday

0today8.Nisin–sodiumcitrate(5%)increasedtheredness

valuesthroughoutthe10days,exceptday0,comparedtothe

control.Nisin–sodiumacetate(3%)didnotincreaseredness

onmoststoragedays,exceptday8,whilethesame

combina-tionata5%concentrationofsodiumacetatedid,excepton

day10.Theincreaseinthedegreeofrednesscanbelinkedto

theincreaseinripeningviaethylenebiosynthesisand

chloro-phylldegradation.ApreviousworkbyYanuriati28_showedan

inhibitoryeffectontomatocolourofsomenatural

antimicro-bials.Rednessoftomatoisanimportantqualityattributefor

consumerappealandacceptability.

Conclusions

Theuseofnaturalantimicrobialssuchasnisinandsaltsof

organic acids remains an economicalmeans ofaddressing

microbialcontamination,especiallywithpathogensofpublic

healthconcern.Nisinandsodiumcitrateata5%concentration

canbeadoptedtosignificantlyreduceL.monocytogenes

con-taminationinfreshproducewithoutcompromisingitsquality.

Furtherresearchtoevaluatethissynergyusingawiderrange

numberoffreshproduceandchallengingpathogenswillbea

welcomedevelopment.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

WewouldliketothankspeciallytoCentreforResearchand

DevelopmentofDurbanUniversityofTechnologyforfunding

this researchwork,andtothestaffoftheFoodTechnology

ResearchGroupfortheirtechnicalsupport.

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