Antimicrobial effect of essential oils of Laurus nobilis L. and Rosmarinus officinallis L. on shelf-life of minced “Maronesa¿? beef stored under different packaging conditions

Antimicrobial

effect

of

essential

oils

of

Laurus

nobilis

L.

and

Rosmarinus

of

ficinallis

L.

on

shelf-life

of

minced

“Maronesa”

beef

stored

under

different

packaging

conditions

$

Joana

Vilela

a

,

Dirce

Martins

a

,

Filipe

Monteiro-Silva

b

,

Gerardo

González-Aguilar

b

,

José

M.M.M.

de

Almeida

b,c

,

Cristina

Saraiva

a,

*

a

SchoolofAgrarianandVeterinarySciences,CECAV,UniversityofTrás-os-MonteseAltoDouro,Apartado1013,P2,5001-801 VilaReal,Portugal

b_{CentreforAppliedPhotonics,INESCTEC,FacultyofSciencesofUniversityofPorto,4169-007Porto,Portugal,Portugal}

c_{DepartmentofPhysics,SchoolofSciencesandTechnology,UniversityofTrás-os-MonteseAltoDouro,Apartado1013,5001-801VilaReal,Portugal}

ARTICLE INFO Articlehistory:

Received30October2015

Receivedinrevisedform25March2016 Accepted25April2016 Availableonlinexxx Keywords: Essentialoils Spoilagemicroorganisms Foodsafety Shelf-life Gaschromatography ABSTRACT

Theaimofthisstudywastoevaluatetheeffectofessentialoils(EOs)ofplantsnaturallyoccurringin

northernPortugalonthespoilageoffreshMaronesabeefburgersstoredat2and8Cunderdifferent

packagingconditions.

EOswereobtainedfromdriedleavesoflaurel(LaurusNobilisL.)androsemary(RosmarinusofficinallisL.)

byhydro-distillationusingaClevenger-typeapparatus.Analysisofvolatilecompositionofessentialoils

ofrosemaryandlaurelwasachievedbyGasChromatography–MassSpectrometry(GC–MS)andGas

Chromatography-ThermalConductivityDetection(GC-TCD)resultinginthedetectionof95.8%and89.4%

ofitscompounds,respectively.

Fresh beef (semitendinosus and semimembranosus) of DOP-Maronesa breed (males; n=4) were

obtainedfromlocalmarketandtransportedtothelaboratory.Sampleswerestoredat2and8_Cintwo

differentconditions:aerobiosis(A)andvacuum(V)andanalyzedat0, 1,2,3,5,7, 10, 14,21and28daysfor

Lactic acid bacteria (LAB), Enterobacteriaceae,Pseudomonas spp., Fungi, Total mesophilic (TM) and

psychrotrophic(TP),color(L*a*b*)andpH.

LaurelwasthemosteffectiveEOkeepingpHfromincreasing.CoordinatesL*anda*werehigheron

samplescontaininglaurelEOforbothAandVpackaging.Laurelalsoshowedbettereffectinreducing

microbiologiccountsinsamplespackedinAatboth2and8CandpackedinVat8C.Rosemarywas

effectiveinreducingmicrobialcountsonallVsamplesstoredat2C.

ThisstudyallowstoconcludethatLaurelEOhassignificanteffectinshelf-life,maintainingfreshbeef

color.

ã2016ElsevierLtd.Allrightsreserved.

1.Introduction

Meatisamajorsourceofproteinofhighbiologicalvaluefor humans(Lund,Heinonen,Baron,&Estévez,2011), beingalsoa sourceofotherimportantnutrients.Itisanidealsubstrateforthe

development of deteriorative and pathogenic microorganisms,

thereforeitisimportanttoensurethesafetyofitsconsumption (Kodogiannis,Pachidis,&Kontogianni,2014).

Mincedmeathasareducedshelflifewhencomparedtowhole meat,sincethesurfaceareaexposedtotheexternalenvironmentis increased(Limbo,Torri,Sinelli,Franzetti,&Casiraghi,2010).The

best way to improve food safety and shelf life is minimizing

contamination and slowing or even inhibiting the growth of

deteriorativeandpathogenicmicroorganisms(Sallam&Samejima, 2004).

Deteriorationoffreshmeatcanbesubjectivesinceitdepends ontheculture,economiccapacity,levelofeducationandsensory

acuity of consumers. Although deterioration is not always

apparent,thefollowingaspectsarecommonlyconsideredasthe maincriteriaforrejection:discoloration,off-odorsandoff-flavors and slime appearance (Ellis, Broadhurst, Kell, Rowland, & Goodacre,2002).

$ _{Themanuscriptwaspresentedat‘InnovationsinFoodPackaging,ShelfLifeand}

FoodSafety’heldon15–17thSeptember2015,inErding,Germany.http://www. foodpackconference.com/.

* Correspondingauthor.

E-mailaddress:crisarai@utad.pt(C.Saraiva).

http://dx.doi.org/10.1016/j.fpsl.2016.04.002

2214-2894/ã2016ElsevierLtd.Allrightsreserved.

ContentslistsavailableatScienceDirect

Food

Packaging

and

Shelf

Life

Thereareseveralfactorsthatcaninfluenceshelf-lifeofmeat: temperature, atmospheric oxygen, water activity (aw), light,

endogenous enzymes and microbiological development. All of

these factors cause changes in color, odor, flavor and texture. Althoughthedeteriorationofmeatcanbeduetoprocessessuchas proteolysis,lipolysisandoxidation,microbialgrowthisthemost importantfactor(Nychas,Marshall,&Sofos,2007).Microbialloads from107_CFUcm 2_{areassociatedwiththeoccurrenceofoff-odors.}

Thoseoff-odorscanbecomefruitywhenthemicrobialcountsrise andbecome putrid,asa result ofamino acidconsumption, for microbial counts greater than 109_CFUcm 2_{. Whenthe glucose}

present in the aqueous phase is used, other substrates are

sequentiallyconsumedwith thereleased of odors of ammonia

andnitrogenouscompounds,suchasdimethyl-1sulfide(Ercolini, Russo,Torrieri,Masi,&Villani,2006).AerobicbacteriaandGram negativefacultativeanaerobicareconsideredthegroupwiththe greatestspoilagepotential.Manymembersof the

Enterobacter-iaceae family contribute to the meat spoilage. However, in

refrigeratedmeatstoredunderaerobicconditions,Pseudomonas, Acinetobacter, Psychrobacter and Moraxella present high growth rates(Ercolinietal.,2006)andthegenusPseudomonasisgenerally dominant,activelycontributingtothedeterioration,duetotheir abilitytodegrade glucoseand amino acids atreduced temper-atures (Mohareb et al., 2015). Although Acinetobacter would competewithPseudomonasforaminoacidsandlacticacid,those havelittleaffinityforoxygen,whichfavorsPseudomonas(Ercolini etal.,2006).Eventhoughthedominantspoilagemicroflorainthe freshmeatisgenerallyGramnegative,theinitialpopulationcan includeGrampositivegenerasuchasLacticAcidBacteria(LAB)and Brochothrixthermosphacta (Mohareb et al., 2015).LAB playsan importantroleinthespoilageofrefrigeratedfreshmeatandare important competitors of other groups of deteriorative micro-organisms.Brochothrixthermosphactaisamicroorganismthatmay developunderaerobicandanaerobicconditions,resultinginthe releaseofoff-odors(Ellisetal.,2002).

Recentlyithasbeenobservedagrowinginterestinthesearch fornaturalproductswithantimicrobialandantioxidantproperties inordertoreplacechemicalandsyntheticadditivescurrentlyused inthefoodindustry(Wang,Wu,Zu,&Fu,2008).

Since ancient times,spices and herbs are usednot only for medicinalpurposes butalsotoimproveorganoleptic character-isticsoffood(Calo,Crandall,O’Bryan,&Ricke,2015).EOscanbe usedasfoodpreservativestoimprovefoodhygiene,reducingthe microbiotadevelopmentandenhancingshelf-lifeofmeat.Theyare aromaticoilyliquidsobtainedfromvariousorgansofplantssuchas flowers,leaves, seeds, roots,fruitsand others (Korifi, LeDréau, Antinelli,Valls,&Dupuy,2013).Theyaresecondarymetabolites synthesizedbyherbsasaformofprotectionfrombacteria,virus, fungi,insects,herbivoresandtheclimate.Ontheotherhandthey canattractsomeinsectstopromotethedispersal ofpollenand seeds(Burt,2004).

The antibacterial, antiviral, anti-parasitic, anti-mycotic, anti-toxicogenicactivity andinsecticidal propertiesofsomeEOs are wellknown,hencetheinterestinthestudyofitsapplicabilityin foodstuffpreservation(Burt,2004).

Thequality,quantityandchemicalcompositionoftheEOsmay varyaccordingtotheweatherandcompositionofthesoil,plant organ from it is extracted, age and stage of the growthcycle (Bakkali,Averbeck,Averbeck,&Idaomar,2008).Thus,inorderto

obtain EOs of constant composition, plants must be collected

underthesameconditions.

Invitrophysicalandchemicalstudiescharacterizemostofthe

compounds present in EOs as antioxidants. Depending on the

concentration, these can be cytotoxic but are generally

non-genotoxic(Bakkali et al.,2008).The characteristicsof thefood matrixsuchaslipidcomposition,proteins,aw,pHandenzymescan

diminishor enhancethe effectivenessof theEOs. According to Bajpai,Baek,andKang(2012),lowpHmayincreasethesolubility andstabilityoftheEOincreasingtheantimicrobialcapacity.

AccordingtoFisherandPhillips(2008),thecompoundspresent intheEOpenetratetheproteinstructureofthecellwallcausing proteindenaturationanddestructionofthecellmembrane.Thus, theoperationofthecellularcomponents,includingthecore,are reducedbythepresenceofcompoundsintheEOsduetochanges incellmembranepermeability(Fisher&Phillips,2008).

SomestudiessuggestthatgenerallyGrampositivebacteriaare

more sensitive to compounds of the EOs than Gram negative

bacteria; this is thought to be related to the Gram negative

impermeableoutermembrane(Fisher&Phillips,2008).However, accordingtoSmith-Palmer,Stewart,andFyfe(2001),overtimethe EOsultimatelyhavethesameeffectonbothtypes.

Rosemary(RosmarinusofficinalisL.)isashrubthatgrowsinall Mediterraneancountries.Ofthedifferentspecies(R.officinalis,R. eriocalyx,R.lavandulaceusandR.laxiflorus),onlyR.officinalisgrows naturally in theMediterranean (Angioniet al., 2004; Tassou & Nychas,1995).Besidesbeingusedforcenturiesasafoodflavoring,it is alsoveryimportant intraditional medicineand it is usedto

combat cramping and relievethe symptoms of diseases of the

nervoussystem(Miresmailli,Bradbury,&Isman,2006;Wangetal., 2008).R.officinalisL.isoneofthespiceswithanti-inflammatory (Steiner et al., 2001) and high antioxidant properties (Bozin, Mimica-Dukic, Samojlik, & Jovin, 2007), attributed to phenolic compoundssuchascarnosol,carnosicacid,rosmanol,rosmadial, epirosmanol,rosmadiferolandrosmarinicacid.Thesecompounds promotethemaintenanceofthenervoustissues(Offord, Aescg-bach, Loliger, & Pfeifer, 1997). Carnosic acid has anticancer properties, inhibitingthe proliferationof abnormal cells (Chan, Ho, & Huang, 1995; Kosaka & Yokoi, 2003). It is effective in controllingvariouspathogenicanddeteriorativemicroorganisms (Chanetal.,1995).

Laurel(LaurusNobilisL.)isanevergreenshrubortreenativeof thesouthernMediterraneanregion(Sellamietal.,2011).Itsdried leavesandEOareusedinthefoodindustryasaspicyforflavoring andfoodpreservativeandareusedinfolkmedicine(Ali-Shtayeh etal.,2000Ali-Shtayeh,Yaniv,&Mahajna,2000)(Kilic,Hafizoglu, Kollmannsberger,&Nitz,2004;Ramosetal.,2012).Accordingto Sellamietal.(2011),1,8-cineoleisthemajorlaurelEOcomponent withpercentagesrangingbetween31.4and56%.Othercompounds

were present in appreciable amounts include linalool,

trans-sabinene hydrate,

a

-terpinyl-acetate, methyl eugenol, sabinene andeugenol.Benzenecompoundspresentinpercentagesranging between1 and12%, are responsiblefor thespicyaroma of the leavesandareextremelyimportantfactorsdeterminingitssensory quality.

ThepurposeofthisworkwastoassesstheeffectofEOsoflaurel (LaurusNobilisL.)androsemary(RosmarinusofficinallisL.)naturally occurringinTrás-os-MonteseAltoDouro,Portugal,onthespoilage offreshMaronesabeefburgersstoredat2and8Cunderdifferent packagingconditions.

2.Materialandmethods

2.1.Extractionofessentialoils

FortheextractionofEOs,leavesoflaurel(LaurusNobilisL.)and rosemary (Rosmarinus officinallis L.) were obtained from wild regionsinnorthernPortugal.Thematerialwasweighedanddriedin anovenatapproximately40Cuntilnoweightchangewasfound andsealedundervacuumuntilused.InordertoobtaintheEOs,the material wassubmittedto hydro-distillationusingaClevenger-type apparatusfor3h.Inthismethod,thedriedsampleisgroundandput inavolumetricflaskwithdistilledwater(1:10),placedinaheating

mantleandundertheClevenger-typeapparatuswhichisconnected toacondenser.Aswaterboilstheformedvaporcarriesthevolatile

compoundsretainedin thesample,whichcondensesincontact

withthecondenserfallingintheClevengercolumnwhiletheEO staysatthesurfaceduetoitslowerdensity.WithaPasteurpipette theEOwascollectedandstoredinsmalltubes,at4Candprotected fromlight.Theyieldwasexpressedin%v/w.

2.2.VolatilecompositionofEOsbygaschromatography-mass spectrometryandgaschromatography-thermalconductivity detection

Theanalysisbygaschromatography(GC)wasperformedusing aThermoScientificTM_TRACETM_{1300gaschromatographcoupled}

toanISQTMSeriesSingleQuadrupoleMSSystemsmass spectrom-eter(MS).

The analytes separation was performed with a Thermo

ScientificTG-5MScolumn(60m0.25mm0.25

m

m).Theoven temperatureprogramwasasfollows:initialtemperatureof60C heldfor2min,increasingto280Catarateof10.00C/minand

held for 5min. Samplesand standards were preparedprior to

analysisusingn-hexane(Merck)atconcentrationsof1.0and0.2% (v/v),respectively,andavolumeof1.0

m

Lwasinjectedusingan auto-sampler.Theinjectorwassettosplitmode(1:5),operatingat 250_{Cand165kPa.Themassspectrometer’stransferlineandthe}

ionsourcetemperaturewassetto280and250,respectively,with thelastoperatingunderelectronimpactmode(70eV,massscan range30–400amu).

Analysis of the same samples was also carried out using a

ShimadzuTM_{GC-2010Plus(ShimadzuCorporation,Japan).}

Sepa-ration of analytes was performed with a Zebron ZB-5 column

(30m0.25mm0.25

m

m) using a similar oven temperature

programandinjection/injectorparametersexceptforcarriergas

flow which was set to82.5kPa. The detectortemperature and

currentwasprogrammedto300Cand75mA,respectively,witha

make-upflowof5.0mL/min.

All analytical separations were made using helium with

99.999%purityascarriergas.

Identificationofanalyteswasperformedbycomparisonofthe

Kovats and Linear Retention Indices, using NIST/EPA/NIH Mass

SpectralLibrary(2011)andotherlibraries,andbycomparisonof authenticstandards.

2.3.Sampling

Fresh beef (semitendinosus and semimembranosus) of

DOP-Maronesabreed(males;n=4)wereobtainedfromlocalmarket and transported to the laboratory. After cut and minced, 20g sampleswereindividually packed in duplicate in two different conditions:aerobiosis(A)andvacuum(V)withandwithoutEO (control).Sampleswerestoredat2and8_{Candanalyzedat1,2,3,}

5,7,10,14and21daysforAand1,7,14,21,28and35daysforVfor microbiological, pH and color (L*a*b*) in one hour after open package.ThemicroorganismsanalyzedwereLacticacidbacteria (LAB), Enterobacteriaceae, Pseudomonas spp., Fungi, Total Meso-philic(TM)andPsychrotrophic(TP).

2.4.Microbiologicalanalysis

Thecollectionandweighingofthesampleswasaccomplished byremoving,aseptically, 10gofeachhamburgerwhichwasdiluted in90mLoftryptonesaltsolution(0.3%tryptoneandNaClat0.85%, sterilizedat121Cfor15min)andhomogenizedin“stomacher” for30s.Successivedecimaldilutionswereperformedintesttubes containing9mLsteriletryptonesalt.Afterwards,theyweresown byincorporationoratthesurfacedependingonthemicroorganism

andtheculturemedium.Colonycountingresultswereexpressed aslogCFU/g.

For TM (ISO4833, 1991) and TP (PortugueseStandard2307, 1987),spreadingwasmadebyincorporationof1mLoftheoriginal suspensionandtherespectivedilutionsonPCA(PlateCountAgar), spreadplateswereincubatedat30Cfor72hfortheTMand7C for10daysforTP.

ForEnterobacterieceae(ISO5552,1997)spreadingwasdoneby addition of 1mLof theoriginal suspension and the respective dilutionsonVRBGselectivemedium(VioletRedBileGlucoseAgar) (Scharlau01-295-500)withdoublelayer.Theplateswereplacedat 37_{Candafter24h,typicalcolonycountingwasperformed(color}

pinktored,withorwithoutprecipitationhalosormucoidcolonies undefinedcolor). Accordingto(ISO5552,1997), 5colonieswere peakedandtransferredtonutrientagarandplacedat30Cfor24h tomaketheoxidasetest(Biochemicalconfirmation)andglucose fermentationcapacityamongGlucoseAgar(Harrigan&McCance, 1979). It was considered truewhen the result was positive to oxidaseandglucoseaswell.

Regarding Pseudomonas spp. (FrenchStandardV04-504

(AFNOR),1998)spreadingwas donebyincorporationof1mLof

the original suspension and the respective dilutions through

selective culture CFC (Cetrimide, Fucidin, Cephaloridine) with

Pseudomonas Agar base (OXOID CM0559) and CFC selective

supplement(OXOIDSR0103).Thespreadplateswereincubatedat 25C for 72h. After colony counting, 5 were transplanted to nutrient agarand incubatedat 30C for24h, thensubjectedto biochemicalcharacterizationofthetestoxidase(positive)andby growthaerobicallyinthemiddleofKLIGLER(KLIGLERIronAgar)

(OXOIDCM0033).

LAB (FrenchStandardV04-503(AFNOR), 1988) spreading was

done by addition of 1mL of the original suspension and the

respective dilutionindouble layerselectivemediumMRS(Man

Rogosa Sharpe Agar) (Oxoid CM0361). The seeded plates were

incubatedat30Cfor72h.

Forfungi(ISO13681, 1995)spreadingwasdoneonthesurfaceof 0.1mL of the original suspension and the respective dilutions

through selective culture GCA (GlucoseChloramphenicol Agar)

(VWR84604.0500).

2.5.pHandcolor

ThepHvaluewasobtainedbythearithmeticaverageofthree measurementsmadewithapH330iWTWpHmeterplaceddirectly

inabodysamplehamburger.

The color measurement was carried out on a hamburger

sampleswithabout1–1.5cmthickness.Threemeasurementswere

performed oneach sample 60minafter openingtheindividual

package.The colormeasurementwasperformedusing are

flec-tometerMinoltaChromoMeterCR-310(Minolta,Japan)throughthe CIELABcolorsystemofL*a*b*(D65illuminant).

3.Experimentalresultsanddiscussion 3.1.Essentialoilsyield

Theyieldofessentialoils(EOs)fromdriedleavesofRosmarinus officinalisL.andLaurusNobilisL.were,respectively,0.61and0.58% (v/w).AsreferredbySerrano,Palma,Tinoco,Venâncio,andMartins (2002) andAngionietal.(2004),theessentialoilyieldpresents variationswiththeplacewheretheleaveswerecollected,withthe seasonand thevegetativestate.Nevertheless,inthis study,the yield attainedfor rosemarywas similartotheaveragevalueof 0.57%reportedbySerranoetal.(2002)forRosmarinusofficinalis L. collected in different Alentejo’s zones (Portugal). Regarding Laurusnobilis L. essentialoil, theyieldattained in thisstudy is

greater(0.58%)thantheoneobtainedatSellamietal.(2011),study inthesameconditions(0.35%) whichcanbeexplainedbythe geographicaldifferencewheretheleaveswerecollected.

3.2.VolatilecompositionofEOs

GC–MS analysisof rosemary and laurelEOs resulted in the detection of95.75% and 89.42% ofits compounds,respectively. Table1

Gaschromatography–massspectrometryanalysis(GC–MS)ofrosemaryandlaurelessentialoils. Kovats

index

Retention time

IdentifiedCompound %peak

Rosmarinusofficinalis L. LaurusNobilisL. 600 6.97 c n-Hexane 0.24 851 7.39 a1_{2-Hexenal,(E)- 0.73} 858 7.40 b1 3-Hexen-1-ol 0.38 900 8.24 c Nonane 0.33 0.35 933 8.89 a2_a -Thujene 0.21 940 9.08 a2_a -Pinene,(D)- 7.44 2.12 952 9.42 a2 Camphene 3.29 957 9.49 a2 2,4(10)-Thujadiene 0.68 977 9.83 a2_{Sabinene 4.10} 979 9.96 a2 ( )-b-Pinene 2.95 2.91 981 9.99 a2_b -Myrcene 3.35 2.87 990 10.03 a2_()-b -Pinene 3.3 1.12 1007 10.42 a2_b -Thujene 0.2 1012 10.55 a2 3-Carene 0.69 0.84 1019 10.65 a2_{a-Terpinene 0.49} 1025 10.8 a2_{p-Cymene 0.33} 1030 10.92 a2 d-Limonene 3.12 1033 10.99 a1 1,8cineole(Eucalyptol) 15.86 18.47 1058 11.42 a2_g -Terpinene 0.87 0.65 1163 11.61 a1

trans-b-Terpineol(p-Menth-1-en-8-ol) 0.57

1073 11.85 d

Diallyldisulphide(4,5-dithia-1,7-octadiene) 0.27 0.81

1076 11.92 a1_{cis-b-Terpineol(p-Menth-8-en-1-ol) 0.25}

1084 12.00 a2_{a-Terpinolene 0.8} 1098 12.09 a1_b -Linalool 3.7 19.97 1108 12.30 a1 2-Pinen-7-one(Crisantenone) 0.36 1145 13.20 a1 (+)-2-Bornanone(Camphor) 9.32 1169 13.56 a1 endo-Borneol 8.79 1179 13.66 a1 Terpinen-4-ol 4.26 3.01 1191 13.86 a1_{a-Terpineol 3.64 4.52} 1200 14.00 a1_{Myrtenol 2.24} 1218 14.33 a1 L-Verbenone 9.36 1228 14.54 a1 Nerol 0.62 1.02 1241 14.72 b2 Linalylo-aminobenzoate 1.57 1248 14.85 e

Ethanol,2-(3,3-dimethylcyclohexylidene)-,(Z)-(5-Caranol) 0.53

1272 15.25 a1_{p-Mentha-1,8-dien-3-one 0.39} 1290 15.35 a1_{Thymol 0,31} 1291 15.45 a1_{L-a-bornylacetate 3.87} 1297 15.63 a1 ( )-trans-Pinocarvylacetate 0.27 1325 16.02 a1 Myrtenylacetate 1.75 1343 16.37 a1_a -Terpineolacetate 11.74 1345 16.40 a1 Nerolacetate 0.52 1351 16.54 e_{Eugenol 1.62} 1383 16.69 a1_{Geranylacetate 0.81} 1327 16.98 e 3,5-Heptadienal,2-ethylidene-6-methyl- 0.27 1401 17.09 e Methyleugenol 2.25 1427 17.72 b2 Caryophyllene 2.44 1476 18.69 b2_b -Selinene 0.71 1556 19.19 e Elemicin 0.31 1601 19.95 b1_{( )-Spathulenol 1.17} 1609 20.07 b1_{Caryophylleneoxide 0.75 0.50} 1621 20.19 b1 Viridiflorol 0.31 1640 20.34 b1 Ledol 0.28 1642 20.69 b1_t -Muurolol 0.26 1652 20.87 b1_a -Cadinol 0.28 1654 20.93 b1_b

-Eudesmol;2-Naphthalenemethanol,decahydro-a,a,4a-trimethyl-8-methylene-, [2R-(2a,4aa,8ab )]-0.55 Total 95.75 89.42 a Monoterpenes 90.92 77.82 a1 Oxygenatedmonoterpenes 63.41 62.94 a2 Monoterpenehydrocarbons 27.51 14.82 b Sesquiterpenes 3.19 6.01 b1 Oxygenatedsesquiterpenes 0.75 3.73 b2_{Sesquiterpenehydrocarbons 2.44 2.28} c Aliphaticcompounds 0.57 0.35 d

Nonisoprenoidscompounds 0.27 0.81

e

TheseareshowninTable1withitspercentage,retentiontimeand Kovatsindex.Itisnotoriousthattheidentifiedcompoundsbelong

majorly to the monoterpenes class, particularly oxygenated

monoterpenes representing63.41% and 62.94% of the total for

rosemaryEOandlaurelEO,respectively.

For rosemary EO the main constituents detected were 1.8

cineole (15.86%), L-verbenene (9.36%), endo-Borneol (8.79%),

camphor (9.32%) and

a

-Pinene (7.44%). According to (Flamini, Cioni,Morelli,Macchia,&Ceccarini,2002)rosemaryEOscanbe dividedintwochemicalgroups:onewhichismainlycomposedby

a

-pinene(20.6%) followedby1.8cineole(6.6%)and another whosepredominantcompoundis1,8cineole(_40.2%)followedby

a

-pinene(13.2%).Takingthisintoconsideration,itcanbesaid thattherosemaryEOobtainedinthisstudyisclosertothesecond group,beinghowever theconcentrations lowerthan the

refer-enced(1.8cineole 15.86%;

a

-pinene 7.44%).Othercommon

compoundsonrosemaryEOareD-limonene,

b

-pineneandL

a

bornylacetate(Nowak,Kalemba,Krala,Piotrowska,&

Czyzow-ska, 2012), these compounds are also found in our sample in

amountsof3.12%,6.25%and3.87%,respectively.

According to Santoyo et al. (2005) and Jiang et al. (2011),

a

-pinene,1.8cineole,L-verbenene,camphorandendo-Borneolare

themaincompoundsthatexhibitantimicrobialeffectinrosemary

EO.InthisstudytheextractedOEpresentsrelevantconcentrations of thesecompounds(7.44;15.86; 9.36;9.32and8.79%, respec-tively). In Ojeda-Sana, van Baren,Elechosa,Juárez,and Moreno (2013)’sstudymyrcene(3,35%inthisstudy)wasshowntohave highantioxidantactivity.

RegardinglaurelEO,35compoundsweredetermined,

repre-senting89.42%ofthetotalcontent.The6maincompoundswere

b

-linalool (19.97%), 1,8 cineole (18.47%),

a

-Terpineol acetate (11.74%),sabinene(4.10%),

a

-terpineol(4.52%)andterpinen-4-ol (3.10%),representing61.90%ofthetotaldeterminedcompounds. Someauthors(Cherratetal.,2014;HadjibagherKandi&Sefidkon, 2011;Sellamietal.,2011;Silveiraetal.,2014)havereportedhigher

levels of sabinene and 1,8 cineole whereas for linalool and

terpinen-4-ol the contents obtained by the authorsmentioned

abovewerelower.

InthestudyperformedbySilveiraetal.(2014),similarresults forlaurelEOcompoundswereachieved;thisessentialoilshowed notonlyrelevantantimicrobialandantioxidanteffectbutwasalso veryeffectivemaintainingmeat’s(sausages)pHlevels.

Nevertheless,itisimportanttokeepinmindthataccordingto

various authors not only the main compounds that exhibit

bactericidal and antioxidanteffect but the interactionbetween theseandthecompoundsinlowerconcentrationthatalsoplayan

Fig.1.Timeevolutionoftotalmesophilic(TM)forsamplesstoredat2and8_Cina)

aerobiosis(A)andinb)vacuum(V).

Fig.2.Timeevolutionoftotalpsychrotrophic(TP)forsamplesstoredat2and8_C

importantrole(Burt,2004; Hyldgaard,Mygind,&Meyer,2012; Ojeda-Sanaetal.,2013).

3.3.Microbiologicalparameters

Figs. 1–5 illustrate the time evolution of TM, TP, LAB, Pseudomonasspp. and Enterobacteriaceae for samples stored at 2 and 8C in A and V packages, respectively. Allsamples in V presentedlesscountscomparedwiththosepackedinA.Intermsof temperatureallsamplesstoredat 2C presentless countsthan

samples stored at 8C. Therefore, as expected, the better

combinationforbeefstoragerevelstobetheVpackagingunder 2C.

FromFig.1a)itcanbeseenthatforstorageinApackageunder 2Cabove48hEOsreducedTMcountsbyalmost1logCFU/gwhile under8CtheEOsshownorelevanteffect.

Fig.1b)shows,forsamplesstoredinVunder2C,adecreaseof TMcountsforsampleswithEOsbeingthemosteffectiverosemary

EOwith counts beingin average 1.5 logUFC/g lowerthan the

control samples.Below 84h of storage both EOs presented no

visibleeffect. Furthermore,after 240hof storage, rosemaryEO presentsinhibitoryeffectonTM.

RegardingTP,fromFig.2a)(A)under2C,itcanbenoticedthat after48hofstoragebothlaurelandrosemaryEOpresentpositive effectreducingcounts.At8C,EOseffectcanonlybeseenafter 168hbeingrosemaryEOthemosteffective.

ForsamplesstoredinVunder2C asitcanbeobservedin Fig.2b) bothEOhavesimilareffect.After72hthereisanotorious increaseofTPcountsofcontrolsamplesascomparedtobothEOs samples.Itmustbenoticedadifferenceofmorethan1logUFC/g betweencontrolandEOssamples.Forsamplesstoredunder8_C,

TPcountsarenotaffectedbythepresenceofEOsalthoughthereis asignificantdecreaseofTPcountsfrom168handforward.

ConcerningLAB,asitisshownonFig.3a)forsamplesstoredin Aunder2C,after72hbothEOshavepositiveeffectoninhibiting growth.Namely,thecontrolcountsaremorethan1.5logUFC/g higherthanEOscounts.Forsamplesstoredunder8Cafter84h EOs showvery similar effect being more efficient onreducing

countswhencomparedtocontrolsamples.

AsillustratedinFig.3b)forVpackedsamplesunderboth2and 8CthereisnoevidenteffectshowedbylaurelnorrosemaryEOs. Fig.4a)representsthetimeevolutionofPseudomonasspp.for

samples stored at 2 and 8C in A, it may be seen for both

temperaturesthatthetwoEOsweresuccessfullyoninhibitingits

growthbeinglaurelEOthemosteffectiveforsamplesunder2C androsemaryEObetterunder8C.Itis notoriousthat samples withEOs present a constantevolution at bothtemperatures in oppositiontocontrolsampleswhichpresenta growthincrease. Also,thefinalcountsaremuchapproximatedtotheinitialcounts onsampleswithEOs whereascontrol samples’final countsare about4logCFU/ghigherthaninitialcounts.

ForV asitmaybeseeninFig.4b) thetwoEOsareeffective

reducing Pseudomonas spp. counts at both temperatures being

theireffectverysimilar.Inthispackagingconditionthereisnota detachmentashighasobservedinA,butsampleswithEOsstill presentlesscountsthancontrolsamples.Forallsamplesunder 2Cfinalcountsarelowerthaninitialcounts,samplesunder8C haveaminorincreaseincountswithexceptionofcontrolsamples

that presentsa differenceof 2 more logCFU/g compared with

initialcounts.Itisimportanttoreferthatcontrolsamplesstoredin Vpackagepresentlowervariationofcountscomparedwithcontrol

samples stored in A package. This can be explained by the

atmospheremuchmorefavorabletothedevelopmentofspecific spoilage microorganisms,such asPseudomonasspp. in air than undervacuumsystem(Labadie,1999;Nychas,Skandamis,Tassou, & Koutsoumanis, 2008). According to Varnam and Sutherland (1995),Pseudomonasspp.canmultiplyinatmosphereswithlow

concentrationofO2,henceitsbehaviorinvacuumwhoseamount

ofresidualO2maybesufficienttosupportitsdevelopment.

InFig.5a)itcanbenoticedthatforsamplesstoredunderboth temperatureEOspresentedverysimilareffectmaintaining Enter-obacteriaceaecountsbelowcontrolsamplescounts.Nevertheless, under2ClaurelEOwas theonewithlessercountsin general.

However, Enterobacteriaceae are not consideredthe main

com-petitors inairandundervacuum,comparativelyforexampleto Pseudomonasspp.inairandLABinvacuumpackage(Doulgeraki, Ercolini,Villani,&Nychas,2012;Labadie,1999).

ForVasitisshowninFig.5b)theeffectofEOsismoreevident andalthoughfor2_{CthetwoEOshavesimilareffect,under8}_C

laurel EO demonstrates the best effect obtaining lower counts

comparedwithbothrosemarysamplesandcontrolsampleswith

differenceofalmost1logCFU/gand2logCFU/grespectively. In both packagingconditions,Enterobacteriaceae final counts aremuchhigherthaninitialcountswhichcanbeexplainedbythe factthatthismicroorganismcanmultiplyinbothaerobiosisand vacuumpackage(Brightwell,Clemens,Urlich,&Boerema,2007). 3.4.pHanalysis

Table2presentstheinitialandfinalpHvaluesforsamplesstored at2and8CunderAandVatmospheres.TheinitialpHofsamples

Fig.4.TimeevolutionofPseudomonasspp.forsamplesstoredat2and8_Cina)

aerobiosis(A)andinb)vacuum(V).

Fig.5.TimeevolutionofEnterobactereaceaeforsamplesstoredat2and8Cina) aerobiosis(A)andinb)vacuum(V).

was5.7,avaluethatfallsintheexpectedrangeof5.6–5.9forbeef afterslaughter.Nosignificantdifferenceswereobservedbetween thetwoEOssamplesbutareductionoffinalpHvalue,withexception ofLaurelat2C,comparativelytocontrolsamplescanbeobserved. Therewerenosignificantdifferencesbetweensamplesstoredat 2and8C,howeveritcanbeseenthatinAthepHvaluesforthetwo temperaturesareclearlydifferentwhileinVtheyareverysimilar. SamplesstoredunderVpackagepresentedlowerpHvaluesandhad

thebestresultin minimizingpHvariations(min. 5.3;max.5.6) comparedtoApackagedsamples(min.6.6;max.7.7).Thesehighest pHvalues observed inApacked samplescanbedue to theproduction ofvolatilesaminesbyspecificmicroorganismsofspoilage,suchas

Pseudomonasspp. whichfoundinthisatmospheretheadequate

conditionsfor theirgrowth (Labadie,1999).TheadvantageofV packagingisthatthegrowthoftheseaerobicmicroorganismsis inhibited,howeverthedeteriorationeventuallystartsasaresultof

the multiplication of organisms able to tolerate anaerobic

conditions(Balamurugan,Ahmed,&Chambers,2013). 3.5.Coloranalysis

TheL*parametercorrespondstothebrightnessofthemeat.The

analysis of Fig. 5 shows that the package influences the

L * parameter. In the case of A package there was a steady

decrease of the L* coordinate which may be due to the

transformationofoxymyoglobin(red)inmetamyoglobin(brown) throughoxidationandarapidreductioncanbeobservedat8C.On

V package it can be seen in all samples a steady increase of

brightnessovertimewithvalueshigherthaninAsamples.These valueswereexpectedsinceinthisconditionoccurstheformation ofoxymyoglobin(red)fromdeoxymyoglobin(purplish)afteropen Table2

InitialandfinalpHvaluesforsamplesstoredat2and8Cunderdifferentpackaging conditions,aerobiosis(A)andvacuum(V).

Samples Time pH T(C) A V Control t=0 5.7 5.7 Final 2 7.3 5.3 8 7.6 5.4 LaurusnobilisL. t=0 5.7 5.7 Final 2 6.6 5.5 8 7.6 5.5 RosmarinusofficiallisL. t=0 5.7 5.7 Final 2 7.2 5.5 8 7.7 5.6

Fig.6. EvolutionofL*coordinateovertimeforstorageindifferenttypesof packagingaerobiosis(A)andvacuum(V))ata)2Candb)8C.

Fig.7. Evolutionof a* coordinateover timefor storage indifferenttypes of packagingaerobiosis(A)andvacuum(V))ata)2Candb)8C.

package meat samples and exposition to the air (Pennacchia, Ercolini,&Villani,2011).Inaddition,therearenotgreatdifferences between2and8_C(Fig.6).

Fig.7displaysthemeasuredvaluesofthea*coordinate.The higherthea*coordinatethegreaterwillbetheredcolor.Inthis casethetypeofpackagingalsohadeffectonmeatcolor.Inboth packagesthe a*valuedecreased overtime. This reductionwill occurinmeatonanypackagingaftersometime.TheVpackagewas foundtobetheonewithgreaterpreservationofredcoloruntil about240handconcerningtothetemperaturefactor,2Cshowed betterresultscompared to8C samples,eventhoughboth had verysimilarvalues.Forthis packagingtherewasa considerably higherriseinthefirstfewdayswhich canbeexplainedbythe

contactofthemeatsamplewithoxygenwhenremovedfromthe

package(Strydom&Hope-Jones,2014).ForApackagetherewasa relativelyconstantlossofredcolorandthevariationwasnotso obviousatanearlystage,whileinthelastdaysofstoragethevalues

decreased considerably which indicates the metamyoglobin

formationonthesamplesurface.

Fig.8displaysthemeasuredvaluesoftheb*coordinate.Itcanbe

seen that it had approximately the same value for the two

temperatures. Moreover, it can beobserved a slight rise at the beginningofthestoragefollowedbyadecrease(3–7daysat2Cand

5–10daysat8C)andthenthiscoordinateseemtostabilize.There wasaslightdecreaseofb*valuesonthepackageunderAconditions andaslightincreaseforthepackingundervacuum,whichmeans that thepackaging method that bestretains thecolor is theVpackage becausethehighertheb*valuethelesseristhelossofredcolor.

Ingeneral,EOspresentedapositiveeffectonthecolorofthe meat.LaurelEOpresentedbettereffectonL*anda*values,yielding abetterredcolorpreservation.BothEOallowedhigherb*values. 4.Conclusions

ThisstudyallowstoconcludethatEOsfromLaurusnobilisL.and RosmarinusofficinalisL.showsomeeffectmaintainingfreshbeef color,whichisanimportantindexofqualityforconsumersandcan beusedasfoodpreservativestoimprovefoodhygienereducing spoilagemicrobiotadevelopmentandenhancingshelf-lifeofbeef. Concerningstoreconditions,asexpected,vacuumpackageand 2Cwerethecombinationwithbetterresults.

ThetwoEOs demonstratedsomepositiveresultsnotjust by reducingspoilagemicrobiotacounts.LaurusnobilisL.isbetterin maintainingtheredmeatcolorandcontrollingpHinsamplesat 2_{Cwhichisanimportantfactorformeatdeterioration.}

In a future investigation the influence of these two EOs

combinedshouldbestudied. Acknowledgements

This work is financed by National Funds through the

FCT – Fundação para a Ciência e a Tecnologia (Portuguese

Foundationfor ScienceandTechnology)withinUID/CVT/00772/

2013. This workis financed by the ERDF – European Regional

Development Fund through the Operational Programme for

Competitiveness and Internationalisation-COMPETE 2020

Pro-gramme,andbyNationalFundsthroughtheFCT–Fundaçãoparaa

Ciência e a Tecnologia (Portuguese Foundtion for Science and

Technology)withinproject«POCI-01-0145-FEDER-006961». References

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