Survival of Salmonella spp. in minced meat packaged under vacuum and modified atmosphere

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

Food

Microbiology

Survival

of

Salmonella

spp.

in

minced

meat

packaged

under

vacuum

and

modiﬁed

atmosphere

Jasna

Djordjevi´c

a,∗

_,

_Marija

_{Bo ˇskovi´c}

a

_,

_Marija

_{Starˇcevi´c}

a

_,

_Jelena

_Ivanovi´c

a

_,

Nedjeljko

Karabasil

a

_,

_Mirjana

_{Dimitrijevi´c}

a

_,

_Ivana

_Brankovi´c

_Lazi´c

b

_,

_{Milan ˇZ.}

_Balti´c

a

a_University_of_Belgrade,_Faculty_of_Veterinary_Medicine,_Department_of_Food_Hygiene_and_Technology_of_Animal_Origin,_Belgrade,_Serbia b_Institute_of_Meat_Hygiene_and_Technology,_Belgrade,_Serbia

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received8November2016

Accepted23September2017

Availableonline12February2018

AssociateEditor:LuisAugustoNero

Keywords:

Mincedmeat

Modiﬁedatmospherepackaging

Vacuum Salmonella pH

a

b

s

t

r

a

c

t

TheeffectofdifferentmodiﬁedatmospherepackagingregimesonthebehaviorofSalmonella

spp.onmincedmeatwasstudied.Mincedmeatwasexperimentallycontaminatedwith

a Salmonella spp. cocktail(S. Enteritidis, S. Typhimurium, S. Infantis and S. Arizonae),

packaged under vacuum or modiﬁed atmosphere with initial headspaces containing

20%O2/50%CO2/30%N2and20%O2/30%CO2/50%N2)andstoredat3±1◦Cfor12days.

Sam-pleswereanalyzedforSalmonellaspp.,viableandlacticacidbacteriacounteverythirdday.

Salmonellaspp.countsdecreasedduringstorageinallpackagingtypes,withreductionsof

about 1.5logCFU/g.A signiﬁcantdifference(p<0.01)wasnotedbetweenSalmonellaspp.

countsinmeatpackagedinvacuumandmodiﬁedatmospheres,althoughtherewasno

signiﬁcantdifferenceinSalmonellaspp.countbetweenmeatpackagedin50%CO2,andmeat

packagedin30%CO2.Attheendofthestudy,thereweresigniﬁcantdifferences(p<0.01;

p<0.05)intotalviableandlacticacidbacterialcountsbetweenmeatpackagedinvacuum

andmodiﬁedatmosphere,andthelowestcountswerenotedinmeatpackagedinmodiﬁed

atmospherewith50%CO2.

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

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

Introduction

PorkandbeefmincedmeatarewidelyconsumedinEurope.1

In Serbia as well as in other Balkan and some

Mediter-ranean countries, minced meat is an inseparable part of

traditionaldishes(e.g.moussaka,sarma),andmeatproducts

(e.g. sausages, ´cevap ˇci ´ci, hamburger).The mincing process

disrupts the meatcellular structure,releasing tissue ﬂuids

and making the minced meat a highly nutritious medium

∗ _{Corresponding}_author_at:_Faculty_of_Veterinary_Medicine,_University_of_Belgrade,_Bulevar_oslobodjenja_18,₁₁₀₀₀_Belgrade,_Serbia.

E-mail:jasnalonce@gmail.com(J.Djordjevi ´c).

supporting bacterialgrowth;mincingalsoallowsmigration

of surface bacteria throughout the product.2 _Therefore, _it

presentsahighlyperishableproductthatneedtobewrapped

orpackagedandchilledimmediatelytoaninternal

tempera-tureofnotmorethan2◦Corfrozento-18◦Cduringstorage

andtransport(Regulation(EC)853/2004).3

Despite measuresto control foodborne pathogens from

farm to fork the burden of diseases caused by foodborne

pathogensremainsimportanthealthandeconomicissue.4–8

Someofthesepathogens,suchasSalmonellaspp.,continue

https://doi.org/10.1016/j.bjm.2017.09.009

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tocausemajorhumanpublichealthandeconomicproblems

inbothdevelopedanddevelopingcountries.9_Salmonella_spp.

are the second mostoften reported bacteria causing

food-borne disease in humans, followingCampylobacter spp.10,11

MeatcanbecontaminatedwithSalmonelladuringthe

slaugh-ter,dressing anddeboningprocesses,or duringprocessing,

transport,storage and householduse, as aresultof

cross-contamination.12,13_S._Enteritidis_and_S._Typhimurium_are_the

mostfrequentlyreportedserotypescausinghuman

salmonel-losisinboththeEUandtheUnitedStates,whiletheincidence

ofS.Infantisisincreasing.14–18 _This_highlights_the_need_for

improvedpreventionandcontrolofSalmonellaspp.infood.

Thefoodindustryhasdevelopednumerouspreservation

techniques in order to prevent and control Salmonella and

otherfood-bornepathogensandspoilagemicroorganismsin

freshmeat products, making the meatsafer for

consump-tionandextendingitsshelflife.Vacuumpackaging(VP)and

modiﬁed atmosphere packaging (MAP) are the most

com-monlyusedpackagingmethodsformeatandmeatproducts.

MAPisconsideredtobeaneffectivetechniqueforrawmeat

preservation.19–23_These_methods_are_based_on_removal_of_the

surrounding atmosphere(VP)or ﬂushingitout and

replac-ingitwithagasmixture(MAP)beforesealingingasbarrier

materials.20–23_Gases_most_often_used_in_MAP_are_carbon

diox-ide,whichinhibitsbacterialgrowth,oxygen,whichprevents

anaerobicgrowthandretainsmeatcolor,andnitrogen,which

avoidsoxidationoffatsandpackcollapse.Dependingonthe

typeoffoodoreffectdesired,thesegasescanbeused

sepa-ratelyorincombinationinvariousconcentrations.19,21

ConsideringtheprevalenceofSalmonellainmincedmeat

andthe frequencyofitsconsumptionvia many traditional

productsand,takingintoaccountthatpackagingofmeatis

the mostcommonmethod offoodpreservation, thereis a

needtoexploretheeffectofpackagingmethodsonSalmonella

spp.survival,especiallyinmixedmincedmeat(porkandbeef).

Therefore,theaimofthisstudywastocomparetheeffectsof

vacuumandtwoinitialheadspace-modiﬁedatmosphere

con-ditions(20%O2/50%CO2/30%N2and20%O2/30%CO2/50%N2)on

thesurvivalofSalmonellaspp.,totalviablebacteriaandlactic

acidbacteriainmincedmeatstoredat3±1◦C.

Materials

and

methods

Porkandbeefmusclesfrom legofdifferentcarcassesused

in the study were provided 48h post-slaughter by a local

slaughterhouse(Pe ´cinci-Subotiˇste,Serbia).Connectivetissues

andvisiblefatweretrimmedafterwhichthepiecesofmeat

weremincedseparatelyinasterilegrinder(4mmperforation

diameterinthemeatgrinderplate),mixedina50:50ratioof

pork:beefandtransportedunderrefrigerationtothe

labora-torywithinanhour.

Fourserovars ofS.enterica (S.EnteritidisATCC 13076,S.

Typhimurium ATCC 14028, S.Arizonae ATCC 13314 and S.

InfantisATCC51741)(www.atcc.org)wereusedinthisstudy.

TheserovarswerestoredinBrainHeartInfusion(BHI;Merck,

Germany) with 20% glycerol at −80◦_C _until _needed. _One

ml of each frozen Salmonella serovar was added to 10ml

ofBHI (Merck, Germany), incubated at37◦C for 24h, then

werestreakedonXyloseLysineTergitol-4Agar(XLT4)(Merck,

Germany)toverifytheircharacteristics.Inordertogeta

sec-ondsubcultureisolated,blackcolonieswerepickedfromthe

XLT4platesandinoculatedintoBHItubes(1colonypertube)

and furtherincubatedforanother24hat37◦C.After

incu-bation,thecultureswerecentrifugedat5000×g(Eppendorf,

Hamburg, Germany) for10minand suitable dilutions were

preparedinBHI.ASalmonellacocktailwaspreparedby

com-biningequalportionsofstandardizedcellsuspensionstoyield

approximately 8logCFU/ml ofeach serovarinthe mixture.

Salmonellacountsweredeterminedbyserialdilutionand

sub-sequentenumerationonXLT4.ThisSalmonellacocktail(40ml

ofthecocktail)wasusedtoinoculate9kgofmincedmeatin

the sterilemixerintheexperimentallaboratoryofthe

Fac-ultyofVeterinaryMedicine,UniversityofBelgrade.According

tolegalrequirementfortheabsenceofSalmonellain25gof

raw meat, meat used in the present study was not

natu-rallycontaminatedwithSalmonella.Mincedmeatwasdivided

inportionsof100g,andpackaged inthreedifferent

condi-tions:VP,modiﬁedatmospherepackage1(MAP1,containing

20%O2/50%CO2/30%N2)andmodiﬁedatmospherepackage2

(MAP2, containing 20%O2/30%CO2/50%N2). MAP treatments

were conductedconsideringratio of 1:3(v/w) between the

volume of gasand weight ofthe minced meat (G/P ratio).

A Variovacpackaging machine(VariovacPrimus,Zarrentin,

Germany)wasusedforVPandMAP.Mincedmeatwas

pack-agedinaOPA/EVOH/PEfoil(orientedpolyamide/ethylenevinyl

alcohol/polyethylene Dynopack, POLIMOON, Kristiansand,

Norway), withlowgaspermeability (O2 –3.2cm3/m2/dayat

23◦C,N2–1cm3/m2/dayat23◦C,CO2–14cm3/m2/dayat23◦C,

watervapor–15g/m2_/day_at₃₈◦_C)._All_minced_meat_samples

weighed100±5gandwererefrigeratedat3±1◦C.

MincedmeatwasanalyzedforSalmonellaspp.,totalviable

count(TVC-mesophiles,30◦C),andlacticacidbacteria(LAB)

count immediately and on days 3, 6, 9 and 12 ofstorage.

Forbacterialenumeration,approximately10gofmeatwere

weighed asepticallyafterpackageopening,transferredinto

sterileStomacherbagsand90mlofBufferedPeptoneWater

(BPW)(Merck,Germany)wasaddedtoeachsample.Meat

sam-pleswerehomogenizedinaStomacherblender(Stomacher

400Circulator,Seward,UK)for2min.Serialdecimaldilutions

were preparedinbufferedpeptonewater(Merck,Germany)

and1mlor0.1mlofappropriatelydilutedhomogenizedmeat

wasinoculated directlyonthesurfaceofXLT4 (Merck,

Ger-many) forSalmonellaspp. enumeration24 _and_incubated _for

24hat37◦C,PlateCountAgar(PCA;Merck,Germany)for

TVC-mesophiles enumeration accordingto ISO 4833:2003,25 _and

incubated at30◦Cfor72hand MRSAgar(Merck,Germany)

forLABenumerationaccordingtoISO15214:1998,26_and

incu-batedat30◦Cfor72h.Afterincubation,plateswereexamined

visually fortypicalcoloniesand morphological

characteris-tics associated with each growth medium, the number of

colonies wascounted and results were recorded ascolony

formingunitsperg(CFU/g).SuspectcoloniesofSalmonellaspp.

weretestedusingAPI20e(BioMerieuxItalia-BagnoaRipoli,

Florence),whilesuspectcoloniesoflacticacidbacteriawere

stainedbyGramandcatalasetestwasdone.

The meat pHwas measured after 10min atroom

tem-perature usingahand-heldpH meter,Testo 205(Testo AG,

Lenzkirch,Germany),equippedwithapenetratingglass

(3)

Measurementofheadspacegascompositionintheminced

meat packaging was conductedusing the gascomposition

tester, Oxybaby (WITT Gasetechnik GmbH & Co. KG,

Wit-ten,Germany). Themeasurement range ofthe instrument

is0–100%byvolume(vol)foroxygen(O2)andcarbon

diox-ide(CO2).Thenitrogencontentiscalculatedasthedifference

from100%afterthemeasuredvaluesofoxygenandcarbon

dioxidearededucted.Theaccuracyofthedeviceis0.1%for

oxygenandcarbondioxide.

Statisticalanalysis

Thestudywasconductedinacompletelyrandomizeddesign,

sixrepetitions werecarriedout foreach treatmentandthe

treatmentswerearrangedina3×5factorialdesign(3

treat-ments,5samplingdays).Numbersofbacteria (CFU/g)were

transformedintologarithms(log)beforestatisticalanalysis.

Statisticalanalysesoftheresultswereconductedusingthe

softwareGraphPadPrismversion6.00forWindows

(Graph-PadSoftware,SanDiego,CA,USA,www.graphpad.com).The

resultswereexpressedasmean±standarderrorofthemean

andarereportedintables.Theeffectsofdifferenttreatments

duringthestorageperiodwereappraisedbyone-factor

analy-sisofvariance-ANOVAandTukey’smultiplecomparisontest

(p<0.05).

Results

Microbiologicalstatusofthemincedmeat

Theinitial Salmonellaspp. countin the inoculated minced

meat was 8.8±0.04logCFU/g (Table 1). The Salmonellaspp.

countdecreaseduntiltheday6inallgroups,withsigniﬁcantly

higher(p<0.05)countsinVPthaninMAP1mincedmeat. A

signiﬁcantreductionofSalmonellaspp.countwas foundon

day6(averagereductionof1.9logCFU/g).Fromday9,slightly

increasingSalmonellaspp.countswereobservedinall

packag-ingtypes,exceptinpackageswith50%CO2whereitdecrease

againfromday9today12.NumberofSalmonellaspp.inVP

meatwassigniﬁcantlyhigher(p<0.05)thaninbothMAPmeat

onday12 (Table1).Signiﬁcantdifference(p<0.05)alsowas

notedbetweentwoMAPpackaging,withlowercountinMAP

with50%CO2.

TheLABcountinmincedmeatincreasedduringstorage

inall packaging types(Table 1). On day 12,the maximum

detectedLABcountwas inVP meat, whilethe lowestLAB

count was in MAP1, i.e. in meat packaged with modiﬁed

atmospherewithahigher concentrationofcarbon dioxide.

Signiﬁcant differences (p<0.05) were detected betweenthe

LABcountinVP meatand MAP1,aswellasbetweenMAP1

andMAP2.

ThemaximumdetectedTVCwasintheVPonday12.The

lowest TVCatthe endof experimentwas recorded inthe

MAP1,andit was signiﬁcantlylower (p<0.05)than it other

groups.

Headspacegas

TheheadspacegasdataforthetwoMAPtypesareshownin

Table2.

MeatpH

Inallmeatsampled,thepHwas5.7atthebeginningofthe

studyandthenincreasedduringstorage(Table3).Asigniﬁcant

difference(p<0.05)inpHwasobservedbetweenVPandMAP1

onday9.

Discussion

ThedecreaseofSalmonellaspp.countuntilday6intheMAP

packaginginthepresentstudypartlycanbeattributedtothe

inhibitory actionofcarbondioxide, especially intheMAP1

withhighCO2content(50%),whichwasmoreeffectivethanVP

inthereducingSalmonellacount(Table1).Gram-negative

bac-teria,includingSalmonellaarehighlysensitivetoCO2because

its interactwith membrane lipids, causing changes in ion

membranetransport,penetratesthemembraneand causes

cytoplasmic acidiﬁcation, disordered synthesis of speciﬁc

enzymes,reducescellmetabolismextendedthelagphaseof

microbialgrowthandreducedthegrowthrateduringthe

loga-rithmicphase.22,27–30_More_sensitive_{Gram-negative}_Salmonella

comparetoLAB,Gram-positivebacteria,asisthecaseinthe

present study,canbeexplainedduetothedensercellwall

andhigherpeptidoglycancontentofGram-positivebacteria

comparedtoGram-negativebacteria.31,32

Carbon dioxide is highly soluble in high moisture and

fattyfoodssuchasmeat.30_In_addition_to_the_level_of

solubil-ityofcarbondioxideanimportantfactoraffectingbacterial

growth isstorage temperatureofthe packaged meat.

Stor-agetemperaturesbelow5◦C,asinthecurrentstudywhere

meatsampleswerestoredat3±1◦C,increasethesolubility

ofcarbondioxide,whoseantimicrobialactivityincreases,but

alsoincreasethesensitivityofbacterialcellstotheeffectsof

carbondioxide.27,30,33,34_In_the_present_study,_the_CO

2

concen-trationwas50%(MAP1)and30%(MAP2)atthebeginningofthe

experiment.DecreaseofCO2duringﬁrsttreedaysofstorage

isattributedtoitsabsorptioninmeatandwasfallowedbyits

increaseuntiltheendofthestorageperiod.IncreaseofCO2

wascausedbybacterialactivity.Similarresultswerereported

byGoulas.35 _The_decrease _of_O

2 concentration observedin

bothMAPcanbeattributedtothegrowthofaerobicbacteria

andmicrobialrespiration,whichutilizeO2andproduceCO2

thatcontributestospoilage.

Competitivemicrobiota,aswellastheinitialconcentration

ofbacterialcells,hasaninﬂuenceonthegrowthofSalmonella

spp.AlthoughSalmonellaspp.areabletogrowandcompete

withothermicroorganisms,36_these_bacteria_are_described_in

theliteratureasarelativelyweakcompetitor.30,37–40_Because

ofthesereasonitissupposedthatthisgroupofbacteriaare

inhibitedinMAPinthepresentstudyuntilday6(Table1)by

LAB,dominantbacteriainpackagedmeatstoredat

(4)

Table1–ChangeinSalmonellaspp.count,LABcountandTVC(logCFU/g)inpackagedmincedmeatsamplesduring storageat3±1◦C(mean±SEM).

Parameter Dayofstorage

0 3 6 9 12 F df p Salmonellaspp.count VP 8.8Aa_±_0.04 _8.3Aa_±_0.01 _6.9Ab_±_0.00 _7.1Ab_±_0.01 _7.4Ac_±_0.08 _212.0 ₄ _<0.0001 MAP1 8.8Aa_±_0.04 _8.0Ab_±_0.02 _6.7Bc_±_0.01 _7.1Ad_±_0.00 _6.9Be_±_0.01 _569.6 ₄ _<0.0001 MAP2 8.8Aa_±_0.04 _8.0Ab_±_0.02 _6.8ABc_±_0.02 _7.1Ad_±_0.00 _7.2Cd_±_0.02 _344.6 ₄ _<0.0001 F – 3.003 6.558 0.3535 57.68 df – 2 2 2 2 p – 0.0800 0.0090 0.7079 <0.0001 LABcount VP 3.1Aa_±_0.02 _3.9Ab_±_0.01 _4.0Abc_±_0.02 _4.4Ac_±_0.09 _5.0Ad_±_0.09 _50.86 ₄ _<0.0001 MAP1 3.1Aa_±_0.02 _4.0Abd_±_0.05 _3.7Bc_±_0.04 _3.9Bb_±_0.03 _4.0Bd_±_0.03 _556.4 ₄ _<0.0001 MAP2 3.1Aa_±_0.02 _3.6Bb_±_0.03 _3.9Cc_±_0.02 _4.1ABd_±_0.05 _4.9Ac_±_0.03 _514.9 ₄ _<0.0001 F – 25.45 155.9 4.529 66.67 df – 2 2 2 2 p – <0.0001 <0.0001 0.0289 <0.0001 TVC VP 7.0Aa_±_0.02 _8.1Ab_±_0.01 _7.9Ab_±_0.04 _9.2Ac_±_0.03 _9.5Ac_±_0.08 _73.77 ₄ _<0.0001

MAP1 7.0Aa_±_0.02 _7.7Abc_±_0.07 _7.2Ba_±_0.00 _7.3Bab_±_0.00 _7.9Bc_±_0.01 _12.24 ₄ _<0.0001

MAP2 7.0Aa_±_0.02 _7.8Aab_±_0.00 _8.8Cbc_±_0.02 _8.2ABbc_±_0.03 _9.1Ac_±_0.02 _8.973 ₄ _0.0001

F – 2.364 118.9 11.65 9.907

df – 2 2 2 2

p – 0.1281 <0.0001 0.0009 0.0018

Differentlowercaselettera–e_within_lines_indicate_signiﬁcant_difference_p_<_0.05_between_different_days_of_same_treatment;_different_uppercase

letterA–C_within_column,_within_same_bacterial_group,_indicate_signiﬁcant_difference_p_<_0.05_between_treatments_of_the_same_day.

Table2–ConcentrationsofCO2,O2andN2inheadspacegasofpackagedmincedmeatsamplesduringstorageat3±1◦C

(%).

Parameter Dayofstorage

0 3 6 9 12 F df p MAP1 O2 20.0a±0.0 15.5b±0.2 12.0c±0.4 8.5d±0.2 7.4e±0.2 417.5 4 <0.0001 CO2 50.0a±0.0 40.2b±0.2 42.1c±0.5 44.1d±0.7 53.5e±0.4 156.7 4 <0.0001 N2 30.0a±0.0 44.3b±0.3 45.7bc±0.4 47.3c±0.8 39.1d±0.5 190.2 4 <0.0001 MAP2 O2 20.0a±0.0 19.4a±0.6 14.5b±0.3 10.8c±0.4 10.5c±0.5 120.3 4 <0.0001 CO2 30.0a±0.0 19.1b±0.5 22.4c±0.3 25.9d±0.6 26.0d±0.5 79.91 4 <0.0001 N2 50.0a±0.0 61.4b±1.0 63.1b±0.3 63.3b±0.7 63.5b±0.7 77.16 4 <0.0001

Differentlowercaselettera–e_within_lines_indicate_signiﬁcant_difference_p_<_0.05_between_different_days_of_the_same_headspace_gas.

Table3–ChangeofpHinpackagedmincedmeatsamplesduringstorageat3±1◦C(mean±SEM).

Groupofsample Dayofstorage

0 3 6 9 12 F df p VP MAP1 MAP2 5.7Aa_±_0.004 _5.8Ab_±_0.003 _5.8Abc_±_0.003 _5.9Acd_±_0.003 _5.9Ad_±_0.009 _28.18 ₄ _<0.0001 5.7Aa_±_0.004 _5.8Aab_±_0.002 _5.8Abc_±_0.008 _5.8Bcd_±_0.006 _5.9Bd_±_0.006 _13.04 ₄ _<0.0001

5.7Aa_±_0.004 _5.8Aab_±_0.005 _5.8Abc_±_0.009 _5.9Bc_±_0.005 _5.9ABc_±_0.009 _15.46 ₄ _<0.0001

F – 1.98 2.58 7.51 3.99

df – 2 2 2 2

p – 0.1728 0.1091 0.0055 0.0409

Differentlowercaselettera–d_within_lines_indicate_signiﬁcant_difference_p_<_0.05_between_different_days_of_same_treatment;_different_uppercase

(5)

ofthenaturalmicrobiotaofmeat.46_During_present_study,_the

LABincreaseinthemincedmeatduring12daysat3±1◦C

wasgreaterthanincreasesoftheotherbacterialgroups

stud-ied,which couldbeduetothe betteradaptationofLAB to

theseconditions.Duringrefrigerationofpackagedrawmeat,

themostlyusedmethodinordertoextendshelflife,thereis

apronouncedgrowthofpsychrotrophicandstrictlyor

facul-tativeanaerobicmicrobeslikeLAB.46_Although_were_expected

increasednumberofLABunderhighconcentrationsofCO2,

LABcountswere higher inVP (Table1),which isin

agree-mentwiththeresultsofLietal.,40_who_reported_higher_LAB

countsforraw porkpackaged undervacuum than in MAP

(40%O2/40%CO2/20%N2).InthepresentstudytheLABcount

reachedaround5logCFU/g(Table1),whichwaslessthanthe

usuallimitofacceptabilityatlevels6logCFU/g.45,47–49

Increas-ingLABcounts,asthedominantmicroorganismsinpackaged

meatstoredatrefrigerationtemperatureswere reportedby

Pexaraet al.,50 _Santos_et _al.,51 _Martinez _et _al.,52 _and

Ruiz-Capillasand Jimenez-Colmenero.53 _Based_on _the _results_of

presentstudywhichshowedthatCO2iseffectiveagainsthigh

Salmonellacount(8logCFU/g),itissupposedthatwillbe

effec-tiveagainstlowerlevelsofnaturally-occurringSalmonellaas

well.

TVCis a parameter which also determines meat

shelf-life.Theinitialmicrobialloadisoneofthemostimportant

parametersdeterminingtheshelflifeofmeat.39_The_biggest

proportions of the initial microbiota on fresh meat are

mesophilicandpsychrotrophicbacteria,andthislattergroup

ofbacteriaismainlyresponsibleformeatspoilage.Forthese

reasonsTVCisusedasanimportantmicrobiological

quanti-tativeindicatorofproductionprocesshygiene,andforsafety

evaluation,aswellasaspoilageindicatorofrawmeat.2,54,55

Basedonnumerousinvestigations,aTVCvalueof107_CFU/g

inmeatis consideredas acritical valueforassessmentof

spoilage.56–59 _The_number_of_{microorganisms}_including

bac-teriadependsontheintrinsicandextrinsicfactorsincluding

pH,meatsurfacemorphology,O2availability,temperatureand

thepresenceanddevelopmentofotherbacteria.60_Changes

in these factors and bacterial competition could inﬂuence

thechangesintheTVCinthepresent study.Attheendof

experiment,lowerTVCwaspresent inmeatsamples

pack-agedwithmodiﬁedatmosphere,especiallyMAPwith50%CO2.

ResultsfromthepresentstudyshowedlowervaluesofTVCin

meatpackagedinthehigherconcentrationofcarbon

diox-ide,whichcanbeattributedtoantibacterialeffectofmodiﬁed

atmosphere,especiallycarbondioxideareconsistentwiththe

resultsofotherauthors.27,61

ManyfactorscanaffectthepHofpackagedmeat,butitis

consideredthatamajorfactorresponsibleforitsdeclineisthe

LABpopulation.62Microorganisms’growthaswellaschemical

reactionsoccurringduringproteolyticprocessesthroughout

storage cause increases in meat pH (Table 3). These

pro-cessescreatealkalicompounds (ammonia,trimethylamine,

dimethylamine)responsibleforpHincreases.63_The

increas-ingpHinallmeatpackagingregimescanbeexplaineddueto

thehighconcentrationofbacteria,resultinginproductionof

alkalicompounds.Furthermore,thepHincreasescouldalso

beduetoproteolysis, causingtheproductionoffreeamino

acidsandleadingtotheformationofNH3andamines.64The

resultsobtainedinthepresentstudyareconsistentwiththose

obtainedbyMilijaˇsevi ´c,65_Bozec_et_al.,66_and_Cachaldora_et_al.45

whileintheresultsobtainedbySchirmerandLangsrud44_and

Babi ´cetal.,67_meat_pH_remained_consistent_during_the_storage

period.

Conclusions

All types of packaging used in present study decreased

the Salmonella spp.count duringﬁrst days ofstorage.This

pathogen wasbest inhibitedbyMAPcontaininghigherCO2

concentration(50%),followedbyMAPwith30%CO2andVP.

Furthermore,MAPwithahigherCO2levelexhibitedgreater

antibacterialactivityagainstTVCandLAB.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgments

ThispaperwassupportedbytheMinistryofEducation,

Sci-ence and Technological Development, Republic of Serbia,

throughthefundingoftheprojectSelectedbiologicalhazards

tothesafety/qualityoffoodofanimaloriginandthecontrol

measuresfromfarmtoconsumer(No.31034).

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