Antifungal activity of nanoemulsions encapsulating oregano (Origanum vulgare) essential oil: in vitro study and application in Minas Padrão cheese

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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

Antifungal

activity

of

nanoemulsions

encapsulating

oregano

(Origanum

vulgare)

essential

oil:

in

vitro

study

and

application

in

Minas

Padrão

cheese

Carolina

M. Bedoya-Serna

a

_,

_Gustavo

_C.

_Dacanal

a

_,

_Andrezza

_M.

_Fernandes

b

_,

Samantha

C. Pinho

a,∗

a_Universidade_de_São_Paulo_(USP),_Faculdade_de_Zootecnia_e_Engenharia_de_Alimentos,_Departamento_de_Engenharia_de_Alimentos,

Pirassununga,SP,Brazil

b_Universidade_de_São_Paulo_(USP),_Faculdade_de_Zootecnia_e_Engenharia_de_Alimentos,_Departamento_de_Medicina_{Veterinária,}

Pirassununga,SP,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received2February2018

Accepted17May2018

Availableonline14August2018

AssociateEditor:ElaineDeMartinis

Keywords:

Hardcheese

Antifungalactivity

Oreganoessentialoil

Nanodispersions

a

b

s

t

r

a

c

t

Theobjectiveofthisstudywastoevaluatetheantifungalactivityofnanoemulsions

encap-sulatingessentialoiloforegano(Origanumvulgare),bothinvitroandafterapplicationon

Minas Padrãocheese. Nanodispersionswereobtained bythe phaseinversion

tempera-turemethod.Cladosporiumsp.,Fusariumsp.,andPenicilliumsp.generawereisolatedfrom

cheesesamplesandusedtoevaluateantifungalactivity.Minimalinhibitoryconcentrations

ofnon-encapsulatedandencapsulatedoreganoessentialoilweredetermined,andthey

wereinﬂuencedbytheencapsulationoftheessentialoildependingonthetypeof

fun-gus.Theantifungalactivityofthenanoencapsulatedoreganoessentialoilincheeseslices

showednoevidenceofaneffectoftheMICs,whenappliedinthematrix.Ontheotherhand,

aninﬂuenceofcontacttimeofthenanoemulsionwiththecheesewasobserved,duetothe

increaseinwateractivity.Itwasconcludedthatnanoencapsulatedoreganoessentialoil

presentedaninhibitoryeffectagainstthethreegeneraoffungievaluated.Ifenvironmental

parameters,suchasstoragetemperatureandwateractivity,werecontrolled,theinhibitory

effectofnanoemulsionsoforeganooilcouldpossiblybegreatlyimproved,andtheycould

bepresentedasapotentialalternativeforthepreservationofMinasPadrãocheeseagainst

fungalcontamination.

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

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

∗ _{Corresponding}_author_at:_Departamento_de_Engenharia_de_Alimentos,_Faculdade_de_Zootecnia_e_Engenharia_de_Alimentos_(FZEA),

Uni-versidadedeSãoPaulo(USP),Av.DuquedeCaxiasNorte225,Jd.Elite,Pirassununga,SP13635-900,Brazil.Tel.:+551935654288;

fax:+551935654284.

E-mail:samantha@usp.br(S.C.Pinho).

https://doi.org/10.1016/j.bjm.2018.05.004

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Introduction

Essential oils are aromatic compounds obtained from

veg-etable materials, characterized bytheir high volatility and

complexcomposition.1_Due_to_their_known_{antimicrobial}_and

antifungalactions,theyareconsideredextensivelyforusein

pharmaceutical,food,agricultural,andcosmeticproducts.1,2

Regardingantifungalactivity,severalstudieshave

demon-strated the potential of essential oils to inhibit the

prolif-erationof severaltypes offungiin food products,such as

Aspergillus, Microsporum,Mucor,Penicillium, Eurotium, Debary-omyces,Pichia,Zygosaccharomyces,andCandida.3–6

Oregano essential oil is among the most investigated

essentialoilsduetoitsantifungalproperties,asitcanprevent

thegrowthofabroadspectrumoffungi,suchasAspergillus

sp., Fusarium sp., and Penicillium sp.7–11 Oregano essential

oilis mainly composed of carvacrol, thymol, cymene, and

terpinene.12,13_The_growing_interest_in_its_use_in_food

preserva-tionismainlyduetoconsumers’demandsforasubstitution

ofsyntheticpreservativesinfoodbynaturalsubstanceswith

asimilareffect.14,15

From a technological point of view, the application of

essential oils as food preservatives presents some

disad-vantages due to their strong odor and ﬂavor, which could

affecttheorganolepticpropertiesoftheproduct.16_Moreover,

essentialoilsareeasilydegradedbyoxidation,volatilization,

heating,andexposuretolight17 _and_difﬁcult_to_disperse_in

hydrophilicmedia.18 _In_order_to_overcome_these_drawbacks,

nanoencapsulationtechniqueshavebeenproposed,asthey

increasethedispersibilityofessentialoilsinaqueous

formu-lations,increasechemicalstabilityduringstorage,minimize

organolepticchanges,andinsomecases,evenimprovetheir

antimicrobialaction.19_Due_to_their_very_reduced_size,

nanos-tructurescanleadtoanincreaseinpassivecellularabsorption

mechanisms, thus reducing mass transfer resistance and

increasingantimicrobialactivity.20

Amongthenanostructureswhichcanbeproduced,there

are the nanoemulsions, which are emulsions with mean

dropletsizeof20–200nm.Nanoemulsionsexhibithighkinetic

stabilityandarethereforestableforalongperiodofstorage.21

Amongthe productionmethodsusedto obtain

nanoemul-sionswithaveragedropletsizessmallerthan50nm,thereare

low-energymethods,suchasthephaseinversion

tempera-ture(PIT) method. Suchmethodsare based onthe change

in solubility of non-ionic polyethoxylatedsurfactants with

temperature.22,23.

MinasPadrão isapressed,semi-hardcuredcheese

con-sumedextensivelyinBrazil.24_It_is_produced_with_pasteurized

milk and has characteristic color, odor, and ﬂavor.25,26 _It

isa product that requires refrigerationduringstorage and

commercialization,ashighertemperaturescandecreaseits

quality.27 _As _it _is _a _ripened _product, _it _is _susceptible _to

contaminationbypathogenicbacteria andfungiduringthe

maturationprocess.Thismaybeacauseforseriousconcern,

asthecurrentBrazilianlegislationdoesnotestablishlimits

forcontaminationbyfungi.

Therefore,consideringtheincreasingconsumerdemand

fornaturalingredientsandthepossibilityofusingessential

oilsinfoodpreservation, theobjectiveofthisstudy wasto

evaluate the antifungal actionof the oregano essential oil

encapsulated innanoemulsions, bothinvitro andinMinas

Padrãocheese.

Materials

and

methods

Nanoemulsions were produced using essential oil of

Orig-anum vulgare(Ferquima, Cotia,SP,Brazil)and sunﬂoweroil

(Cargill,Mairinque,SP,Brazil).Thesurfactantsusedwere

PEG-40hydroxylatedcastoroil(Ricinuscommunis)(CremophorRH

40,BASF,Ludwigshafen,Germany),polyoxyethylene4-lauryl

ether(Brij30,Sigma-Aldrich,StLouis,MO,EUA),and

sorbi-tanmonooleate(Span80,Sigma-Aldrich,St.Louis,MO,USA).

Ultrapurewater(DirectQ3,Millipore,Billerica,MA,USA)was

usedthroughoutthestudy.

Fortheisolationandidentiﬁcationofthefungifromthe

cheese samples, dichloranrose bengal agar

chlorampheni-col (DRBC) agar (Acumedia, Neogen, São Paulo, SP, Brazil),

dextrosepotatoagar(DPA,Acumedia,Neogen,SãoPaulo,SP,

Brazil),andchloramphenicol(HomeopatiaOuroPreto,

Piras-sununga,SP,Brazil)wereused.

Productionofnanoemulsions

NanoemulsionswereproducedbythePITmethodaccording

toMoraes-Lovisonetal.28_._The_components_of_the

nanoemul-sions(sunﬂoweroil,surfactants,deionizedwater,andoregano

essentialoil)weremixedandmagneticallystirredat1350rpm,

and this mixture was heated to 65◦C. The two

formula-tions ofnanoemulsions producedare described in Table1.

Afterwards, this mixturewas cooledrapidly byplacingthe

dispersioninjacketedvesselscooledbywaterat3◦C,under

magneticstirring, untilit reachedroom temperature

(cool-ingrate:10◦C/min).Theheating/coolingcycleswererepeated

twice.

IsolationandidentiﬁcationoffungiinMinasPadrão cheesesamples

Eighteensamplesoffungi-contaminatedMinasPadrãocheese

were obtained. Thefungiwere isolated from boththe

sur-face and interior of the cheese. A mass of 10g of each

contaminated cheese sample (surface and interior),

previ-ously homogenizedand diluted, were inoculated on DRBC

agar and incubated at 25◦C for 7 days. The colonies of

different morphological typeswere isolated inpotato

dex-trose+chloramphenicol(PDA+C)agar,andafteridentiﬁcation

bythe microculturetechnique,thefungiwere classiﬁedby

genera.29

Evaluationoftheinvitroantifungalactivityof nanoemulsions

Toevaluatetheinvitroinhibitoryactivity,non-encapsulated

oreganoessentialoilandnanoemulsionsoforeganoessential

oilweretested. Theconcentrationsoforeganoessentialoil

usedareshowninTable2.

A volume of 0.1ml of spore suspension of each

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Table1–Formulationofnanoemulsionsencapsulatingoreganoessentialoil.

Formulation Surfactant Lipidphase Continuousphase

A 13% –CremophorRH40 (9.75%) –Brij30(3.25%) 6.5% –SOa_(3.25%) –OOa_(3.25%) 80.5%–Deionizedwater B 20% –CremophorRH40 (12%) –Span80(8%) 10% –SO(5%) –OO(5%) 70%–Deionizedwater

a _SO₌_sunﬂower_oil;_OO₌_oregano_oil.

Table2–Concentrationsofnon-encapsulatedandnanoemulsiﬁedoreganoessentialoilusedintheinvitroassays.

Fungigenera Oreganoessentialoil(␮gessentialoil/mLnanoemulsion)

Non-encapsulated NanoemulsionsAandB

Cladosporiumsp. 0.05–0.3 0.16–0.5

Fusariumsp. 0.1–0.5 0.03–0.5

Penicilliumsp. 0.1–0.5 0.16–2.5

containing between 104 _and ₁₀5 _spores/ml _in _potato

dextrose agar+chloramphenicol (PDA+C) containing each non-encapsulatedessentialoilornanoemulsionswasspread onPetridishes.Theinoculateddisheswereincubatedat25◦C for7days.30_Inhibitory_activity_was_determined_by_the

pres-enceorabsenceoffungal growth.Theminimuminhibitory

concentration(MIC)wasdeterminedasthelowest

concentra-tionofnon-encapsulatedoilornanoemulsionatwhichthere

wasnofungalgrowth.30_All_experiments_were_performed_in

duplicate.

Evaluationofantifungalactivityofnanoemulsionsin cheeseslices

SlicesofMinasPadrãocheese(2mmthickand50mmin

diam-eter,previouslyexposedtoUVlightfor60min)wereimmersed

inthe dispersions ofnanoemulsions. Thetreatmentsused

forimmersionwere:control,non-dilutednanoemulsion,MIC,

nanoemulsionformulationA,andnanoemulsionformulation

B.ThetreatmentsaredescribedinTable3.

Thecheesesliceswere submittedtodifferentperiodsof

immersion(immediateremovalorremovalafter30min),then

placedin Petri dishes and inoculated with fungal

suspen-sioncontainingbetween104_and₁₀5_spores_of_fungus._Finally,

thesliceswerestoredatroomtemperature(25◦C)orunder

refrigeration(4◦C±1◦C).Allexperimentswereperformedin

duplicate. The described methodology was modiﬁed from

Taniwakietal.31

Theantifungaleffectofnanoemulsiﬁedoreganoessential

oilwasevaluatedbymeasuringthecontaminatedareadue

tothefungalgrowthofeachgenusonthecheeseslices.The

contaminatedareaofthesliceswasdeterminedusingimage

processingsoftware,wherebytotal(cheeseslices) and

con-taminatedareaswereestimatedautomaticallybypixelcount,

andthepercentageofcontaminationwascalculatedby

com-paringthetwoareas.

Table3–Treatmentsusedincheeseslices.

Treatments Emulsionconcentrationa

Control Noemulsion Non-diluted nanoemulsion A1:42.1 B1:64.8 MICA

MICACla:0.26(Cladosporiumsp.)

MICAFu:0.11(Fusariumsp.)

MICAPe:1.62(Penicilliumsp.)

MICB

MICBCla:0.32(Cladosporiumsp.)

MICBFu:0.10(Fusariumsp.)

MICBPe:0.80(Penicilliumsp.)

FormulationA A2:3.25 A3:6.5 A4:11.7 FormulationB B2:5 B3:10 B4:18

a _Emulsion _{concentration:} _␮g _oregano _oil _{nanoemulsiﬁed/mL}

immersionliquid.

Results

Isolationandidentiﬁcationoffungifromcontaminated MinasPadrãocheese

Asexpected,fungalgrowth occurredmainlyonthesurface

ofthe cheesesamples(56% ofthe contaminatedsamples),

asMinasPadrãocheeseisoftenexposedtotheenvironment

when it issubmitted to the maturation process. The

gen-eraoffungiwiththehighestoccurrenceonthesurfacewere

Penicilliumsp.,followedbyCladosporiumsp.andScopulariopsis

sp.,with percentagesof67%,18%,and 5%oftotal,

respec-tively.However,intheinteriorofthecheesesCladosporiumsp.

occurred atahigherpercentage(33%)intermsof

contami-natedarea,followedbyFusariumsp.andScopulariopsissp.(both

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Table4–Minimuminhibitoryconcentrationvaluesforthefungitested,fornon-encapsulatedoreganoessentialoiland

nanoemulsionsencapsulatingessentialoil.

Fungalgenera Minimalinhibitoryconcentration(␮gessentialoil/mlnanoemulsion)

Oreganooil NanoemulsionA NanoemulsionB

Cladosporiumsp. – 0.26 0.32

Fusariumsp. 0.20 0.11 0.10

Penicilliumsp. 0.30 1.62 0.80

Invitroevaluationofantifungalactivityofpureand emulsiﬁedoreganoessentialoil

TheMICofnon-encapsulatedoreganoessentialoilwas deter-minedforeachfungus.ForFusariumsp.andPenicilliumsp.,the MICvalueswere0.2␮g/mland0.3␮g/ml,respectively.Inthe caseofCladosporiumsp.,itwasnoticedthatthefungusdidnot growatall,evenatthelowestconcentrationoforegano essen-tialoiltested.Sucharesultmayindicatethatthisgenusof funguswasverysusceptibletotheinhibitoryeffectoforegano oilwhencomparedwiththeinhibitoryeffectoforegano essen-tialoilonFusariumsp.andPenicilliumsp.(Table4).

Regardingtheantifungalactivityofnanoemulsions,for

for-mulationAtheMICsobtainedwere0.26␮g/mlforCladosporium

sp.,0.113␮g/mlforFusariumsp.,and1.62␮g/mlforPenicillium

sp.AsforformulationB,thevaluesofMICwere0.32␮g/ml,

0.1␮g/ml,and0.8␮g/mlforCladosporiumsp.,Fusariumsp.,and

Penicilliumsp.,respectively.TheMICsfoundfornanoemulsion

Awerethushigherthan thosefornanoemulsionB,forthe

threeevaluatedfungi.Sucharesultwasprobablyduetothe

higheroiloforeganocontentinformulationB(5%)compared

withformulationA(3.25%).

Therefore,itwasconcludedfromtheMICsobtainedthat

nanoemulsionsencapsulatingoreganoessentialoil,obtained

bythePITmethod,showedantifungalactivitysimilartothat

oforeganooilappliedinitsfreeform,justifyingthe

contin-uationofresearchtoverify theantifungalactivityinMinas

Padrãocheese.

Evaluationofantifungalactivityofnanoemulsionsin MinasPadrãocheeseslices

Threefactorswereconsideredintheevaluationofantifungal

activityofnanoemulsionsencapsulatingoreganoessentialoil:

theamountoforeganoessentialoil,theperiodofimmersion

ofthe cheeseslicesinthe nanoemulsions,andthe storage

temperature.

Regardingtheinﬂuenceoftemperatureonantifungal

activ-ity,fungalgrowthwasnotobservedoncheeseslicesstored

underrefrigeration,neitherforslicesimmediatelyremoved

norforthoseimmersedfor30mininthenanoemulsions.No

fungalgrowthwasdetectedafter30daysofstorageoncheese

slicesimmediatelyremovedfromthenanoemulsions

contain-ingoreganoessentialoilandmaintainedat25◦C.

Theonlyconditionsunderwhich theantifungalactivity

ofnanoemulsionscouldbeevaluatedwasstorageofcheese

slicesat25◦Cfollowing30minofimmersion.Whenthe

gen-eral effects of both nanoemulsion formulations (A and B)

on the growth of the three evaluated fungal genera were

compared, thenanoemulsionspresentedsigniﬁcant growth

inhibitoryeffects.

TreatmentsA1andB1inhibitedthegrowthofCladosporium

sp.andFusariumsp.for30daysduringstorage.TreatmentsA3,

A4,B3,andB4ofnanoemulsiﬁedoreganooildelayedtheonset

ofgrowthuntiltheseventhdayofstorageforthesamefungal

genera.InthecaseofPenicilliumsp.,onlythehigher

concen-trationsofnanoemulsion(treatmentsA1andB1)inhibitedits

growthuntiltheseventhdayofstorage,indicatingthatthis

funguspresentedhigherresistancetotheantifungaleffectof

nanoemulsiﬁedoreganoessentialoilthanfungiofthegenera

Cladosporiumsp.andFusariumsp.(Fig.1).

Regarding the antifungalactivity ofthe two

nanoemul-sionformulationsattheMIC,theydidnotdemonstratethe

expected effects (withrespect tothe in vitroassays) when

appliedoncheeseslices,asfungalgrowthwashigherthanon

thecontrolslices.Sucharesultmaybeduetothehighwater

activity,assliceswereimmersedfor30mininthe

nanoemul-sions.

Althoughtheantifungalactionofnanoemulsiﬁedoregano

essential oilatdifferentconcentrationswasveriﬁed, itwas

noted that higher concentrations of nanoemulsions

(treat-mentsA1andB1)wererequiredtosigniﬁcantlyinhibitfungal

growthoncheeseslices.

Discussion

The present results agree with those of Kure and Skaal32

and Kure et al.33_, _who _isolated ₁₃ _species _of _Penicillium,

Alternaria, Aureobasidium,Cladosporium,Epicocum,Geotrichum, Mucor, Phoma, Trichoderma, and Ulocladium from semi-hard

cheeses. In thesame way, DeMorais34 _isolated_the _genera

Penicillium,Aspergillusniger,Rhizopus,Aspergillusochraceus,and

Mucorfrombuttercheese.Therefore,consideringthefungal

genera found inthe contaminated cheese samples,

subse-quentstepsofthestudywereperformedwithCladosporium

sp.,Fusariumsp.,andPenicilliumsp.

InthecaseofPenicilliumsp.,thevaluesofMICwereclose

to those reported by Stupar et al.35_, _who _obtained _MICs

in the range of 0.2–1.0␮g/ml for the same fungal genus.

For Cladosporium sp., the obtained MICs were higher than

thosefoundbyZabkaetal.36_for_Cladosporium_{cladosporoides}

(0.028–0.066␮g/ml). For Fusarium sp., Daferera et al.37 _and

Stevi ´cetal.38_found_MICs_of₁₅₀_␮g/ml_and_70–1160_␮g/ml_of

oreganoessentialoil,respectively,valuesmuchhigherthan

thosepresentedinthisstudy.

Nanoencapsulation can lead to a positive or negative

impactontheantimicrobialactivityofessentialoilsrelative

tothatofnon-encapsulatedessentialoils.18,39,40_In_this_study,

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0 5 10 15 20 25 30 0 5 10 15 20 25 30 35 Inf ec te d a rea ( % ) Time (days)

CONTROL MIC

3,25 6,5 11,7 42,1 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 In fec te d a rea ( % ) Time (days)

CONTROL MIC

5 10 18 64,8 0 20 40 60 80 100 120 0 5 10 15 20 25 30 35 In fe ct e d ar e a (% ) Time (days) CONTROL MIC 3,25 6,5 11,7 42,1 0 20 40 60 80 100 120 0 5 10 15 20 25 30 35 In fe cted a re a (% ) Time (days)

CONTROL MIC

5 10 18 64,8 0 10 20 30 40 50 60 70 80 0 5 10 15 20 25 30 35 In fe c te d a re a (% ) Time (days)

CONTROL MIC

3,25 6,5 11,7 42,1 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 In fe c te d a re a (% ) Time (days)

CONTROL MIC

5 10

18 64,8

Nanoemulsion A Nanoemulsion B

Cladosporium sp.

Fusarium sp.

Penicillium sp.

Figure1–FungalgrowthonMinasPadrãocheeseslicesimmersedfor30mininnanoemulsions,storedat25◦C. Concentrationmeans␮goreganoessentialoil/mlnanoemulsion.

essentialoilwascomparedwiththoseofthetwo

nanoemul-sionformulations(AandB),itwasfoundthatforFusariumsp.,

theencapsulationoftheessentialoilimproveditsantifungal

effect,whereasforPenicilliumsp.,theuseoforeganoessential

oilencapsulationdiminisheditsinhibitoryeffectagainstthis

genus.

Manystudieshavereportedthe invitroantifungaleffect

of oregano essential oil, obtaining different degrees of

inhibition.7,41–43_The_inhibitory_{effectiveness}_of_oregano_oil_is

strictlyrelatedtofactors suchastaxonomy ofthespecies,

agronomicpractice,climate,extractionmethodofessential

oil,concentrationoftheactivecompounds,fungalgenus,and

theconcentrationofinoculatedspores.44,45 _In_addition,_the

resistanceoffungitothe inhibitoryeffectoforeganooilis

dependentonitschemicalcomposition.46

Inthecaseofnanoemulsionsincorporatingessentialoils,

the formulation, the average particle size, and the surface

charge inﬂuence the antimicrobialactivity of the essential

oil.47

Several factors can inﬂuence the antifungal efﬁciency

of nanoemulsions of essential oils. Depending on the

encapsulated essential oil, nanoemulsions may target one

microorganismmorethananother,resultinginaverybroad

ranges of activities of these antimicrobial systems.47 _The

active compoundsthat constitute the essentialoils canbe

easily degradedby interacting withthe nutrients infoods,

suchasproteins,lipids,orminerals;theycanalsobeabsorbed

into the variouslayersoffood, resultinginanuneven

dis-tribution,allowingsomepartsofthefoodtobeexposedto

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Consideringthe dataobtainedforfungal growth onthe

refrigeratedslices,asthestudiedfungicangrowatveryacid

orverybasicpH,attemperaturesfrom 0◦Cto37◦C,andat

highorlowsubstratemoisturelevels,theresultsindicatethe

nanoemulsionsencapsulatingoreganoessentialoilpresented

verygoodantifungalactivity,especiallyunderconditionsof

lowtemperature,duetowhichtherewasnogrowthduring

storageforanylengthoftime.

Conclusion

Thedataobtainedinthis studyprovedtheinvitro

antifun-galactivityofnanoemulsionsencapsulatingoreganoessential

oil,againstthegrowthofCladosporiumsp.,Fusariumsp.,and

Penicilliumsp.Theantifungalactivity wasveriﬁedonMinas

Padrãocheeseslicesinoculatedwiththesamefungalgenera.

In both typesof assays (in vitro and cheese slices),

Penicil-liumsp.presentedhigherresistancetotheantifungaleffect

oforegano essentialoilthan Cladosporiumsp.andFusarium

sp.

The antifungal effectiveness of the essential oil of

nanoemulsiﬁedoreganoinMinasPadrãocheesewashighly

dependentontheamountofnanoemulsion,immersiontime

ofslices,andstoragetemperature.Theseresultscanhelpto

establishoptimizedconditionsforthecontinuationof

stud-ies of antifungal activity of nanoemulsions encapsulating

oreganoessentialoilinmorerealisticenvironments,suchas

incheesematurationchambers.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

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TheauthorsthankCAPES(ComissãodeFormac¸ãodePessoal

deNívelSuperior,Brazil)fortheMasterfellowshipofCarolina

Bedoya-SernaandLaticínioImperialJureia(MonteBelo,MG,

Brazil)forthedonationofcheesesamples.

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