Effects of hurdle technology on Monascus ruber growth in green table olives: a response surface methodology approach

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

Food

Microbiology

Effects

of

hurdle

technology

on

Monascus

ruber

growth

in

green

table

olives:

a

response

surface

methodology

approach

Leandro

P.

Cappato

a,∗

_,

_Amanda

_M.

_Dias

_Martins

a

_,

_Elisa

_H.R.

_Ferreira

a

_,

Amauri

Rosenthal

b

a_{FederalRuralUniversityofRiodeJaneiro,Seropédica,RJ,Brazil} b_{EmbrapaFoodTechnology,RiodeJaneiro,RJ,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received1July2016 Accepted9May2017

Availableonline13October2017 AssociateEditor:LuisAugustoNero

Keywords: Preservatives Monascusruber

Face-CenteredCentralComposite Design

Tableolives

a

b

s

t

r

a

c

t

AnascomycetesfunguswasisolatedfrombrinestorageofgreenolivesoftheAraucocultivar importedfromArgentinaandidentifiedasMonascusruber.Thecombinedeffectsofdifferent concentrationsofsodiumchloride(3.5–5.5%),sodiumbenzoate(0–0.1%),potassiumsorbate (0–0.05%)andtemperature(30–40◦C)wereinvestigatedonthegrowthofM.ruberinthebrine ofstoredtableolivesusingaresponsesurfacemethodology.Afull24_{factorialdesignwith}

threecentralpointswasfirstusedinordertoscreenfortheimportantfactors(significant andmarginallysignificantfactors)andthenaFace-CenteredCentralCompositeDesignwas applied.Bothpreservativespreventedfungalspoilage,butpotassiumsorbatewasthemost efficienttocontrolthefungigrowth.Thecombineduseofthesepreservativesdidnotshow asynergisticeffect.Theresultsshowedthattheuseofthesesaltsmaynotbesufficientto preventfungalspoilageandthegreatestfungalgrowthwasrecordedat30◦C.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

Introduction

Tableolivesareanimportantfermentedfruitproductofthe westernworld.Thefruitoftheolivetreeisunsuitableforfresh consumptionduetothepresenceofoleuropein,aglycoside, whichisresponsibleforthebittertasteofrawolives.1 _The

∗ _{Correspondingauthorat:DepartmentofFoodTechnology–InstituteofTechnology,FederalRuralUniversityofRiodeJaneiro,CEP:}

23890-000,Seropédica,RiodeJaneiro,Brazil. E-mail:leandrocappato@gmail.com(L.P.Cappato).

mainreasontoprocessolivesistoeliminateoleuropein.There are severaldifferentprocesses,butthemostimportantare: Californianstyle,GreekstyleandSpanishstyle.2

FungifromgenusMonascusareascomycetescharacterized bytheproductionofheat-resistantascospores,whichareable tosurvivethethermalpasteurization.M.ruberisfoundinsoil anditisrelatedtoapost-harvestfruitcontamination,andthis

https://doi.org/10.1016/j.bjm.2017.05.009

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

specieswasisolatedforthefirsttimefrompasteurizedgreen olives,inGreece.3

Intableolives,M.rubergrowthoccursduetoitsabilityto adaptatcertainconditions,likegrowingatverylowoxygen tensions,and survivingatlowpHandhigh salt concentra-tions;insuchconditions,M.rubercanproduceheat-resistant ascospores.3,4 _{Theseconditionsallowthemicroorganismto}

developinolivesafterharvest,whichcanhappenduring stor-ageandincannedtableolives.3_{TheM.ruberspoilageresults}

inthefruitsofteningandapHincreasinginthebrine, with-outnecessarilygrowinganyvisiblefungusmyceliumonthe brinesurface.3,4 Suchalterations affecttheproductquality, causingeconomiclosses.Furthermore,itpromotesariskto themicrobiologicalsafetyoftheproduct,sincetheincreaseof thepHinthebrinecanallowthegrowthofseveralpathogens, suchasEscherichiacoliO157:H7,SalmonellaEnteritidis,Listeria monocytogenes5–7_{andClostridiumbotulinum.}8,9

M.ruberspoilagecanbecontrolledwiththecombination ofintrinsicandextrinsicfactors.10_{Thistechniqueisknown}

ashurdletechnologyandinvolvesthemanipulationofseveral factorssuchas:pH,wateractivity,preservativesand tempera-tureincombination,whichmayactsynergisticallytoinhibitor retardmicrobialgrowth.11,12_{However,theantagonisticeffect}

canoccur,reducing theeffectivenessoftheantimicrobials. Thisfactismorefrequentwhenthe ‘hurdles’havesimilar mechanismsofaction.13,14

Theadditionofpreservativesisacommontechniqueused infoodpreservation.Foodsafetyisincreasedandensuredby theuseoftheseadditives.12,15,16 _{Theefficiencyof}

preserva-tives,likebenzoateandsorbateisstrictlydependentonthe pHandthepKaoftheacidgroups.AtlowpH,these preserva-tivesaremorelikelytobeintheundissociatedform,allowing themtopassfreelyacrossthelipidmembraneandtoinhibit themicrobialgrowth.15,17

Table olives, according to the Trade Standard For Table Olives,canbepreservedbyheattreatmentorbybrinewith apH≤4.0, withor without the use ofpreservatives.18 _The

preservativesthatareallowedincludebenzoicandsorbicacid, as well as their corresponding salts of sodium and potas-sium,allwithinthemaximumlimitsof1000and500ppm, respectively.18_{However,informationaboutthecombineduse}

ofthesepreservativesonthegrowthoffungiinthebrineof tableolivesisstillscarce.

Theaimofthisstudy wastoevaluatetheeffects ofthe combineduseofNaCl,temperature,potassiumsorbateand sodiumbenzoateatdifferentconcentrationsonthegrowthof M.ruberontableolivebrineafteraperiodof10,30and50days.

Material

and

methods

Fungalisolatesandidentification

Thefunguswasisolatedfromplasticdrumscontaining180kg ofolivesoftheAraucocultivarinstoragebrine(pH=3.8and 10% salt). Isolation was carried out byinoculating 1mL of contaminatedbrine inPotatoDextrose Agar(PDA)– (Fluka –Sigma–Aldrich®) acidifiedwith1% (v/v)ofsteriletartaric acid (10%) to lower the pH to 3.5, and incubated at 30◦C for7days.Identificationofmicroorganismswasmadefrom

the observation ofthe micro and macroscopic characteris-tics,accordingtothemethodofkeydifferentiationdescribed by Udagawa and Baba19 _{and modified by Stchigel et al.}20

Themicroscopiccharacteristicsthatwereevaluatedwerethe number ofascospores inside the asci,pigmentationof the ascomata,sizeand formoftheascosporesandpresenceor absenceofpatchesontheascomatawalls.Macroscopic eval-uated characteristicswere growth ornogrowth ofcolonies on theCzapeck AgarYeastExtract(CYA) –(Himedia®) and Malt ExtractAgar(MEA) –(Himedia®),presenceor absence ofsolublepigmentandthecolorofthepigmentformed.The measurements ofmicroscopic structures were taken using lactophenolasthemountingmedium.Thesecharacteristics wereevaluatedafterfungigrowthonCYAorMEAatdifferent temperatures(25and30◦Cfor7days).

Preparationofascosporessuspension

Thefungus wasgrown inaRouxbottle containing200mL ofnon-acidifiedPotatoDextroseAgar(PDA)for1monthat 30◦C.Ascosporeswereharvestedafterscrapingtheinoculated PotatoDextroseAgar(PDA)surfacewithasterilespatulausing 10mLofsteriledistilled watercontaining0.1%(v/v)Tween 80followedbyfiltrationthrough3layersofsterilegauzeto removethehyphalfragments.Theascosporessuspensionwas homogenized byvortex for3min toopen the cleistothecia andliberatetheascospores.Theprocedurewasfollowedby microscopicobservationtoassureahighproportion(>90%)of freeascospores.MicroscopiccountinginaNeubauerchamber resultedina105_{ascospores/mLsuspension.Theascospores} suspensionwasstoredat4◦Candservedastheinoculumfor allexperiments.

Full24_{factorialdesign}

Afull24 _{factorialdesignwiththreecentralpointswas} car-riedouttoevaluatetheeffectintheconcentrationof:NaCl (X1)varyingbetween3.5and5.5%(w/v);sodiumbenzoate(X2), varyingbetween0.0and0.1%(w/v);potassiumsorbate(X3) varyingbetween0.0and0.05%(w/v);temperature(X4), vary-ingbetween30and40◦C,andtheirinteractionsonthefungal growth andspoilage.Accordingtofull24_{factorialdesign,4} negativecontrol(withoutpreservatives)wasperformed.

The concentrations of the two independent variables, sodium benzoate (X2) and potassium sorbate (X3) were selectedbasedontheTradeStandardsforTableOlives.11_The

concentrationsofNaCl (X1)werechosenbecausetheseare theonescommonlyusedbythetableoliveindustriesinBrazil, whilethetemperatures(X4)werechosenaccordingtoclimatic conditionsusedduringstorageoftableolivesinBrazil. Statis-ticalanalysesat95%confidenceintervalincludingAnalysisof Variance(ANOVA),ParetoChartofStandardizedEffects(Fig.1) andhistogramofnormalresidualdistributionwerecarriedout usingtheStatistica7.0softwarepackage(StatSoftInc,Tulsa, OK,USA).

Face-CenteredCentralCompositeDesigns(FCCCD)

Fromtheresultsanalyzedinthefull24_{factorialdesign,the} ParetochartwhichdisplaytheStandardizedEstimateEffect

X₃ X 3 X₂ X₁ X₄ X₄ X₄ X₂ X₁ X₁ X₃ X₂ X₁*X₃ X₁*X₂ X 1*X3 X₂*X₄ X 3*X4 X₃*X₄ X₂*X₄ X₁*X₂ X₂*X₃*X₄ X₁*X₃*X₄ X₁*X₂*X₃ X₁*X₂*X₄ X₂*X₃*X₄ X₂*X₃*X₄ X 1*X3*X4 X₁*X₂*X₄ X₁*X₂*X₃ X 1*X3*X4 X₁*X₂*X₃ X₁*X₂*X₄ X₁*X₄ X₃*X₄ X₂*X₄ X₁*X₄ X₁*X₃ X₁*X₂ X₁*X₄ X 2*X3 X₂*X₃ X 2*X3 Curvature Curvature Curvature -8.72546 -6.51076 3.466701 3.299017 2.889186 2.681062 -2.3016 1.473379 -1.07405 -.40382 -.234996 .1389195 -.007114 p=.05 p=.05

A

C

B

-3.62237 -1.78267 -.876215 -.572605 .5372885 .4635983 .4458118 -.440531 -.202976 .1838942 -.181752 .1432972 .1340411 p=.05 10.23449 -10.1791 -36,243 35,5709 13.50811 -11.4937 -10.7526 -6.53979 -5.41614 3.860204 2.885109 2.571707 2.387564 1.795551 1.656497 -1.27509 1.102012 .7608933 -.13359

Fig.1–ParetochartofabsolutestandardizedvaluesofMonascusrubergrowthontableolivebrine,at10days(A);30days(B) and50days(C)resultingfromthemodelobtainedbyafull24_{factorialdesignwiththreecentralpoints.Monascusruber} growthwasanalyzedwithdifferentconcentrationsofNaCl(X1)sodiumbenzoate(X2),potassiumsorbate(X3),temperature (X4)andtheirinteractions,performedbyfull24factorialdesign.

(AbsoluteValue)wasconstructedinordertoevaluatethe sig-nificanceofeachindependentfactor,theirinteractionsand tocheckthecurvature(Fig.1).Thedatafromthemicrobial growthwastransformedintomathematicaltermslikelog10, whichisstandardpractisefordeterminingmicrobialcolony counts.21–24

Thesignificanceof the curvaturesuggests that there is aquadraticterm inthemodel,soapolynomialofahigher degreemustbeused,suchasthesecond-ordermodel,25_so,a

FCCCDwasused(Table1).Theexperimentaldatawere ana-lyzedusingresponsesurfacemethodology(Fig.2)andfittedto apolynomialmodelofregressioncoefficients(Eq.(1)).To eval-uatethenormalityoftheresidualdistributionaShapiro–Wilk testwasperformed,21usingtheStatistica7.0softwarepackage (StatSoftInc,Tulsa,OK,USA).

Preparationoftableolivesamples

All thesamples ofbrinedtableolives werepreparedusing fifteen green olives of the Arauco cultivar (graded-size 320/360olives/kg),with200mLofacidifiedbrine,toanalyze thefungalgrowthunderrealconditions.Theformulationwas preparedaccordingtotheexperimentaldesign.ThefinalpH of3.5wasreachedbytheadditionoflacticacid85%.

Initially,thesampleswerepasteurizedat100◦Cfor10min, in order to eliminate the natural microbiota and thus be abletoevaluateexclusivelythedeteriorationresultingfrom

contaminationbyM.ruber.Preliminaryexperimentsshowed thatthetime×temperatureusedwerethemostappropriate thermaltreatmentconditions.Theexperimentswerecarried out inquintuplicatewhere,itwasnotobservedanygrowth afterplating with1mLofsamplesinPotatoDextroseAgar (PDA)–(Fluka–Sigma–Aldrich®)andPlatecountagar(PCA) –(Himedia®)after3–5daysofincubationat30–35◦C.

Pasteurized samples (100◦C/10min) were inoculated with 0.2mL of the ascospores suspension (“Preparation of ascosporessuspension”section),toreachaconcentrationof around102_{ascospores/mLofbrine.Thesewerethesamples} incubated at temperatures set in the experimentaldesign (30, 35, 40◦C). Inoculum concentrations between 102 and 103_{CFU/mLarecommonlyusedandrecommendedinstudies} involvingmicrobialbehavior.26

MicrobiologicalanalysesandpHmeasurements

Forenumerationofmicrobialgrowth,1mLofthebrine sam-ple aliquotwas addedtodilution tubes containing9mLof steriledistilledwater.Aftersubsequentdecimaldilutions,the inoculationwascarriedoutusingthespreadplatetechnique withnon-acidifiedPDA(Fluka–Sigma–Aldrich)induplicate. Allplateswereincubatedat30◦Cfor48–72h.Foreach sam-ple,theresultsweretheaverageoftwoplatesandexpressed

Table1–AFCCCDwiththreecentralpoints:codes,levels,originalsvariablesandresults(log10N/N0)ofcombinedfactors

effectsofMonascusrubergrowthontableolivebrineat10days.

Factors Variables Unit Low(−1) Medium(0) High(+1)

X1 NaCl % 3.5 4.5 5.5

X2 Sodiumbenzoate % 0.0 0.05 0.1

X3 Potassiumsorbate % 0.0 0.025 0.05

X4 Temperature ◦C 30 35 40

Standardorder X1 X2 X3 X4 log10N/N0(10days)

1 3.5 0.0 0.0 30 1.53 2 3.5 0.0 0.0 40 1.09 3 3.5 0.0 0.05 30 −0.74 4 3.5 0.0 0.05 40 −1.12 5 3.5 0.1 0.0 30 0.26 6 3.5 0.1 0.0 40 0.16 7 3.5 0.1 0.05 30 0.37 8 3.5 0.1 0.05 40 0.04 9 5.5 0.0 0.0 30 1.35 10 5.5 0.0 0.0 40 1.05 11 5.5 0.0 0.05 30 −1.03 12 5.5 0.0 0.05 40 −1.09 13 5.5 0.1 0.0 30 0.27 14 5.5 0.1 0.0 40 0.28 15 5.5 0.1 0.05 30 0.26 16 5.5 0.1 0.05 40 0.23 17 3.5 0.05 0.025 35 0.25 18 5.5 0.05 0.025 35 0.42 19 4.5 0.0 0.025 35 0.46 20 4.5 0.1 0.025 35 0.67 21 4.5 0.05 0.0 35 0.61 22 4.5 0.05 0.05 35 0.10 23 4.5 0.05 0.025 30 0.00 24 4.5 0.05 0.025 40 0.16 25(C) 4.5 0.05 0.025 35 −0.14 26(C) 4.5 0.05 0.025 35 −0.11 27(C) 4.5 0.05 0.025 35 −0.23 2.00 1.50 1.00 0.50 0.00 -0.50 -1.00 44.00 40.00 38.00 32.00 28.00 3.20 3.60 4.00 4.40 4.60 5.20 5.60 Te m per ature °C -0.2 -0.3 -0.4 -0.5 -0.6 -0.7 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 -1.0 -1.5 -2.0 0.06 0.05 0.04 0.03 0.02 0.01 0.00 -0.01 -0.02 0.00 0.02 0.04 0.06 0.08 0.10 0.12 Sodium Benz oate % P o_{tassium sorbate %} log 10 (N/N 0_{) 10 da} ys log 10 (N/N 0_{) -10 da} ys 2 1 0 1.5 0.5 -0.5 -1

A

B

NaCl %

Fig.2–ResponsesurfaceplotsofinteractionfactorsonMonascusrubergrowth(log10(N/N0))ontableolivebrineafter10 days;(A)interactionsoftemperatureandNaCl;(B)interactionsofpotassiumsorbateandsodiumbenzoate.Othervariables areconstantattheircenterpoints.ThenumbersinsidethecontourplotsindicatetheMonascusrubergrowth.

aslog10values.ThepHwasmeasuredinsamplesincubated accordingtotheFCCCDdesign,toanalyzethespoilagefungi.

Results

Fungusidentification

Thefunguswasisolatedfrombrinestorageofgreenolivesof theAraucocultivarimportedfromArgentina.Thismaybedue tothefewstudiesinvolvingdeteriorationofolivesinBrazil. ThefunguswasidentifiedasM.ruber(IOC4667)andregistered attheCultureCollectionofFilamentousFungiofOswaldoCruz Foundation(CCFF/IOC).

Effectofindividualhurdlesonmicrobialgrowth

The Pareto chart (Fig. 1) shows that the interaction effect betweenbenzoateandsorbatewassignificant (p<0.05)and showedthesame responsetrendobtainedat10,30and50 daysofgrowth;thustheresultsfor10dayscanbeusedto describethetrendofgrowthandspoilagefor30and50days. Theresultsofthetemperatureandsalineconcentration inter-actionsdidnotshowanysignificanteffect,consequentlythese parameterswerenotconsideredimportanttocontrolfungus growth.

Furthermore,astheParetochart(Fig.1)showedthatthe curvature only had a significant influence for 10 days, a second-order model was applied. Based on the results of growthandtheinfluenceofthecurvature,asurfacemodel designwasusedtofindtheoptimalconditionstoreduceor eliminatethefungalgrowthandtheresultingdeteriorationof thetableolives.

ResponsesurfacemethodologyofFCCCD

Afterevaluatingtheimportanceofthefactorsonthefungal growthat10,30and50daysusingthefull24_{factorialdesign,a} FCCCDwiththreecentralpointswasusedtofindthebestset ofvalues,whichwouldgivetheoptimalconditiontocontrol thefungalgrowthandtheresultingdeterioration.TheFCCCD wascarriedoutonlyconsideringtheresultfromthe10days ofgrowthofthepreliminaryanalysisfromthefull24_factorial design,whichpresentedasignificantcurvature,asshownin

Fig.1A.Thefactorsthatshowedastrongandweakeffecton thefungalgrowth,inotherwordsthesignificant(p<0.05)and themarginallysignificant(0.05>p<0.1)factors,werechosen tobeanalyzedinFCCCDalthoughtheNaClconcentrationwas notsignificant,thisfactorwasanalyzedbyFCCCD,becausethe interactionwithtemperature(X4)wasmarginallysignificant (Fig.1A).

Table1liststhecodes,concentrationsandoriginalvalues

ofthevariablesused,aswellastheresultsobtainedafter10 daysoffungalgrowthresultingfromtheFCCCD.FromANOVA, apolynomialmodel was obtainedbased onthe regression analysisoftheoriginalsignificantandmarginallysignificant values.Themodel resultingfromtheregression analysisis showninEq.(1):

Y10days=−9.03−20.33X2+

130.24X2

2−43.70X3−0.62X4+446.65X2X3 (1)

BasedonANOVAofthedatafromtheFCCCD,thequality offitoftheresponsemodelwasevaluated.Thusinorderto determinetheaccuracyofthequadraticresponsesurfacetobe abletopredictthefungalgrowthintableolivestheR2_(0.9223), theadjusted-R2(0.8937)andthedistributionoftheresiduals wereobserved.

Thenormaldistributionofresidualswasanalyzedusing

the normality method of Shapiro–Wilk, because

numeri-cal approaches are the best way to test for the normality of data.21,27 _{The Shapiro–Wilk test proved to be the most}

powerful and efficient in comparison to other tests, like: Anderson–Darling,Kolmogorov–Smirnov,andLilliefors.27

Fig. 2 presents the response surface plot of significant andmarginallysignificantinteractionfactorsandrevealsthat theuseofpreservativeshasanimportantinfluenceon fun-gal growth. In Fig. 2A the interaction of temperature and concentrationofNaClforall theconditionstestedwasnot sufficienttopreventthefungal spoilage.Fig.2Bshowsthat sorbate was moreefficient than benzoate toinhibit fungal growth. An increasein the concentration ofsorbate hasa greatereffectonthepreventionofmicrobialgrowthcompared to benzoate. Theinitial concentrationof the fungus could onlybe reduced withsorbate,demonstratingits fungicidal effectiveness.InFig.2Btheregionthatprovidedsatisfactory

(log10(N/N0)<0.5)26,28 results inthe fungal growth,involved

concentrationsofpotassiumsorbatehigherthan0.025%.

Fig.3showsthedevelopmentofamycelialmatonthebrine surfaceofthetableolives.Inadditiontovisualgrowth,thepH wasmeasuredtoevaluatedeterioration.Theresultsshowed thatinthesampleswithoutpreservativestherewasagreater pHincreasewhencomparedtosampleswithpreservatives. Thelargestdevelopmentofamycelialmatandthegreatest increaseinpH(pH=4.0)occurredinthesampleincubatedat 30◦Cwith3.5%salt(Fig.3A).

Discussion

ThefirstresearchinvolvingM.rubercontaminationintable oliveswascarriedoutbyPanagouetal.3_{whoisolatedthis}

fun-gusfromthermallyprocessedgreenolivesoftheConservolea cultivar;whereithadshownrelativeresistancetoheat.The authorsreportedthatthefungalspoilageresultedinfruit soft-eningandincreasedpHofthebrine,andtheyreportedthatM. ruberwasanimportantspoilagefungusintableolives.

Inthepresentwork,M.ruberwasisolatedfromthespoiled brineofgreenolivesofthe Araucocultivar,and theeffects ofsalt(NaCl),potassiumsorbate,sodiumbenzoateand tem-perature onthe fungusgrowth were studied.Basedon the results,onecanconcludethatthefungusissalttolerantand thedeterioration(visiblegrowthandpHincreased)occurred insampleswithoutpreservatives.Theseresultsshowedthat the use of saltmay not besufficient tocontrol the fungal spoilage.ThisconclusionisconsistentwithPanagouetal.29

whoreportedthatthisfunguscouldevengrowintableolive brinecontaining9%NaCl,provingitstolerancetosalt.

Fig.3–ThedevelopmentofMonascusrubermycelialmatonthebrinesurfaceofgreentableolives(Araucocultivar).Letters (A)and(B)refertosampleswithoutpreservatives(sodiumbenzoateandpotassiumsorbate),incubatedat30◦Candwith 3.5%and5.5%ofNaCl,respectively.Letters(C)and(D)correspondtosampleswithoutpreservatives(sodiumbenzoateand potassiumsorbate),incubatedat40◦Candwith3.5%and5.5%ofNaCl,respectively.

Influenceoftemperatureonfungalgrowthwasforemost whenincubatedat30◦C,but thegrowth occursatall tem-peraturesevaluated.Thisresultisalsoinagreementwiththe previouslymentioned studybyPanagouetal.29 _who

evalu-atedthefungalgrowthbythegradientplatetechniqueat20, 30and35◦Candobservedthattheoptimalconditionswere alsoobtainedwhenincubatedat30◦C.

Preservativesareusedinvariousproductstocontrol fun-gal growth and spoilage. Organic acids suchas sorbic and benzoicacidsarethemostcommonpreservativesinfoods.30

The effectiveness of these compounds depends on many

environment factors, like: pH, temperature, aw, the physi-calmatrixofthefoodandtheirconcentrations.31Theaction ofthepreservativesisextremelypH-dependentbecausethe microbialinhibitionisduetotheirundissociatedacidicform,

and is influenced by the acid pKa. This form allows the

preservativestopenetrateinto thecell andtopromotethe acidification of the cytoplasm, thus inhibiting the fungal growth.32,33

Ingeneral,sorbicacid(pKa=4.76)tendstobemoreeffective thanbenzoicacid(pKa=4.20)duetothehigherpKa. Conse-quently,therewillalways beahigherconcentrationofthe undissociatedforminsorbicacidcomparedtobenzoicacid.16

Inourwork,theresultsshowedthatpotassiumsorbatehad greater efficiency in inhibiting fungal growth compared to

sodiumbenzoate.Themaximumefficientwaswhensorbate

wasusedatonly0.05%,whichallowsthedestructionofthe initialfungus concentration. Similar resultswere obtained

whenevaluatingtheeffectofthesepreservativesininhibiting acocktailofnativeyeastsisolatedfromtableolives.34

Lópezetal.35_{evaluatedtheinfluenceofpotassiumsorbate}

(0.05%)on the resistanceofIssatchenkia occidentalisisolated from tableolivebrine.Theresultsshowedthatthisfungus wasveryresistanttopreservatives,andwasinhibitedby0.03% potassiumsorbate.Theseresultsaresomewhatsimilartothe onesfoundforM.ruberinourworkthatshowedinhibitionof thefungusatpotassiumsorbateconcentrationshigherthan 0.025%.Turantas¸etal.24_{reportedthatinadditiontothe}

anti-fungaleffectofpotassiumsorbate,theuseofthispreservative at0.05%enhancedthe productionoflactic acid duringthe fermentationofblackolives.

Thecombineduseoftwoantimicrobialagentscanproduce differenteffectsonthemicrobial populationsuchas syner-gism,antagonism,andadditiveeffects.36 _{Theresultsofthis}

researchshowedthat thecombineduse ofpreservativesin tableolivesdidnotresultinsynergisticeffect,asexpected. However,whenusedincombination,benzoateinhibitedthe actionofsorbateonthefungus.Thiseffectcanbeclearlyseen inFig.2.Theseresultsweresimilartothosefoundby Arroyo-Lópezetal.34_{whoevaluatedtheeffectofusingsorbateand}

benzoateonanativeyeastcocktailintableolivesusingthe FICmethodology.

Otherresearchsuggeststhatthisphenomenonisdueto thefactthatthesepreservativeshavethesamemechanism ofactiononthemicrobialcell.Thesynergisticeffectgenerally occurswhenseveralantimicrobialfactorsthathavedifferent

mechanismsofactionareusedincombination,thus promot-ingagreaterassociatedimpactthantheadditiveorindividual effect.14,37

Conclusions

Theresultsofthisresearchshowedthatthe combineduse ofpotassiumsorbateandsodiumbenzoatedidnotpresenta synergiceffect,asexpected.Potassiumsorbatewasthemost efficientpreservativetopreventtableolivedeteriorationbyM. ruber.Basedontheresponsesurface,theminimuminhibitory concentrationwasaround0.03%ofpotassiumsorbate.These resultscouldbeusedinthetableoliveindustrytoassistin preventingM.ruberspoilage.Itssurvivalandgrowthcanlead toolivedeteriorationincludinganincreaseinpHup tothe dangerouslevelsfavorableforpathogengrowth.Aknowledge ofthecombinedtechnologieswiththeappropriate concen-trationsofthe‘hurdles’incombinationcancontributetothe designandcontroloftheprocessinordertoassurethesafety andqualityoftheproductsdemandedbytheconsumer mar-ket.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

r

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