Long cold storage influences the microbiological quality of raw goat milk

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ContentslistsavailableatSciVerseScienceDirect

Small

Ruminant

Research

j o urna l h o m e pa g e: www.e l s e v i e r . c o m / l o c a t e / s m a l l r u m r e s

Long

cold

storage

inﬂuences

the

microbiological

quality

of

raw

goat

milk

Anderson

Keizo

Yamazi,

Thalita

Scatamburlo

Moreira,

Valéria

Quintana

Cavicchioli,

Raquel

Cristina

Konrad

Burin,

Luís

Augusto

Nero

∗

UniversidadeFederaldeVic¸osa,DepartamentodeVeterinária,CampusUFV,36570-000Vic¸osa,MG,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received4February2013 Accepted7February2013 Available online 6 March 2013

Keywords: Goatmilk Coldstorage Psychrotrophics Proteolytics

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Storageofgoatmilkproductionincoldtemperaturesis acurrentBrazilianlegislation request,howeverthereisnospecificationofalimitperiodforthis.Thepresentworkaimed tocharacterizethemicrobiologicalcharacteristicsofrawgoatmilkproducedinaspecific regionofBrazil,aswellastheinfluenceofthestoragesystemandperiodonitsquality. Sixty-onesamplesfrom12goatfarmswerecollectedandsubjectedtoanalysistoenumerate hygieneindicatormicroorganisms,psychrotrophicsandproteolyticpsychrotrophics.The obtainedcountsweredescribedandcomparedconsideringthesystemandperiodof stor-age(ANOVA,Tukey).Despitepresentinglowcountsofmesophiles,thesamplespresented highcountsofothergroupsandarelevantpresenceofproteolytics.Samplescollectedfrom bulktankspresentedhighercountsofmesophilesandpsychrotrophicswhencompared toimmersiontanksandfreezers(p<0.05).Whenstoredforaperiodof48horlonger,the countsofmesophiles,coliforms,Escherichiacoliandpsychrotrophicswerealsosignificantly higherwhencomparedtoastorageperiodof24horless(p<0.05).Theresultsindicate spe-cificproblemsingoatmilkproductioninthestudiedareaandtheneedofestablishinga periodlimitforrawgoatmilkcollectioninBrazil.

1. Introduction

GoatmilkproductioninBrazilhasimprovedsincethe 1990s,anditisconsideredanimportantanimalproductto beexploitedasa commodity.Despitethisdevelopment, thegoatmilk industryin Brazilisstill characterizedby small scale farmers, withlow daily production (around 80L)that complicate thelogisticsof the dairy industry interms ofplanninga properroutinefor collectionand processing(Costaetal.,2010;Oliveiraetal.,2011).Asa con-sequence,thestorageofgoatmilkforalongperiodoftime iscommonplaceondairyfarms.Thisoccursatlow tem-peraturesusingavarietyofequipment,suchasimmersion

∗ Correspondingauthor.Tel.:+553138991463;fax:+553138991457. E-mailaddress:[email protected](L.A.Nero).

tanks(wherethecollectedmilkisstoredincans,immersed inwaterandkeptatalowtemperatureusinga conven-tionalrefrigerationsystem).Bulktanksarenotusuallyused despitebeingconsideredadequateequipmentforproper storage(Sierraetal.,2009).

The absence of standards and speciﬁcations for the coldstorageofrawgoatmilkhasanegativeeffectonits quality(Goetschetal.,2011).Storageforlongperiodsof time,evenatlowtemperatures,selectsaspeciﬁcgroupof microorganismscalledpsychrotrophics,whichare respon-siblefortheproductionofspoilageenzymeswithadirect impactonthequalityofrawgoatmilkanditsproducts (Sørhaug andStepaniak, 1997).Themain enzymes pro-ducedbypsychrotrophicsarelipasesandproteases,types ofthermo-stableproteinsthatkeeptheirspoilagepotential evenaftertheheattreatmentsusuallyadoptedinthedairy industry(Champagneetal.,1994;AgnihotriandPal,1996).

http://dx.doi.org/10.1016/j.smallrumres.2013.02.004

Open access under the Elsevier OA license.

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milkduring cheese production, and alsodetermine the developmentofundesirablearomaandﬂavorinend prod-ucts, jeopardizing their quality (McPhee and Grifﬁths, 2011).

Considering these characteristics, the Brazilian Agri-cultureMinistryestablished specificrules for goat milk production only in 2000,aiming to improve itsquality basedonanimalhealth,milkingprocedures,storage, trans-port,and processingconditions(Brasil,2000).However, Brazilianlegislationdoesnotspecifythelimitperiodofcold storageofrawmilkonfarms,norwhichequipmentmust beusedforrawgoatmilkstorage,hamperingthe adop-tionofastandardizedsystemforstorageandjeopardizing theinitialqualityofthisproduct(Delgado-Perti ñezetal., 2003;Zweifeletal.,2005).A currentproblemin Brazil-iandairyproductionisdeficienciesatthebeginningofthis foodchain,mainlyrelatedtothehealthstatusofproducing animalsandhygienicpracticesduringmilkingandstorage (Neroetal.,2004,2009;Monteiroetal.,2007).Asa conse-quence,Brazilianauthoritiesadoptedapolicytoimprove thequalityofmilkproductionbasedmainlyon establish-ingnewmicrobiologicalparametersofqualityandsafety, aswellasspecificconditionsofcoldstorageandtransport ofmilkproductionfromdairyfarms(Brasil,2000,2011).

Evenbeingconsideredanimportantanimalproductfor Brazilianeconomy,thereisalackofscientiﬁcinformation aboutthemicrobiologicalqualityof goatmilk,and also concerningtheeffectofthestorageconditionsonits micro-biotadevelopment.Basedonthis,thepresentworkaimed tocharacterizethemicrobiologicalcharacteristicsofraw goatmilkproducedinaspeciﬁcregioninBrazil,aswellas toinvestigateoftheeffectsofalongperiodofstorageat lowtemperatureonitsmicrobiota.

2. Materialandmethods

2.1. Studyareaandrawmilksamples

Twelvegoatfarmslocatedintheregionofthecities Vic¸osaandMuriaé,MinasGeraisState,Brazil,wereselected forthepresentstudy,asrepresentativeofgoatmilk pro-ductioninthisregionandotherBrazilianregions.Rawgoat

fromallselectedfarms,fromstoredmilkproductionand withaminimumofthreesamplesperfarm.During sam-plecollection,thestoragetimeofgoatmilkateachspeciﬁc farmwasrecordedandcategorizedas(1)justaftermilking, (2)24hstorage,and(3)48hstorage(orlonger).Themain productioncharacteristicsoftheselectedgoatfarmsand thenumberofsamplesperfarmaredetailedinTable1. 2.2. Sampledilutionandmicrobiologicalanalysis

Rawgoatmilksamplesweretransportedunder refrig-erationuntilanalysisandten-folddilutedusing0.85%NaCl (w/v)accordingtoWehrandFrank(2004).Aliquotsof1mL of selecteddilutionswereplatedfor aerobicmesophilic countsusingPetrifilmTM_Aerobic_Count_plates₍₃_M Micro-biology,St.Paul,MN,USA)followedbyincubationat35◦C for24h,forEnterobacteriaceaeusingPetrifilmTM Entero-bacteriaceae(3MMicrobiology)followedbyincubationat 35◦Cfor24h,andfor coliformsandE.colicountsusing PetrifilmTM _E._coli_plates₍₃_M_{Microbiology)}_followed_by incubationat35◦Cfor24h(coliforms)and48h(E.coli). Afterincubation,alltypicalcolonieswereenumeratedand thefinalresultswereexpressedascolonyformingunitsper mL(cfu/L).

Psychrotrophicsandproteolyticpsychrotrophicswere enumeratedaccordingtoDownesandIto(2001).Aliquots of0.1mLofselecteddilutionsweresurfaceplatedinplate countagar(OxoidLtd.,Basingstoke,England)addedto ster-ile skimmilk (10%v/v,Molico®_,_Nestlé _Brasil _Ltda,_São Paulo, SP, Brazil) followed by incubation at 7◦C for 10 days.Allcolonieswereenumeratedafterincubation,and wererecordedaspsychrotrophics. Then,each platewas ﬂoodedwithanaceticacidsolution(8%v/v)andkeptat roomtemperaturefor1h,atwhichpointtheacidsolution wasdiscardedandtheremainingproteolytichaloswere recordedasproteolyticpsychrotrophics.Theﬁnalresults wereexpressedascfu/mL.

2.3. Statisticalanalysis

Allmicrobiologicalcountswereconvertedtolog10and meanvalueswerecalculatedforeachgoatfarm.Samples

Table1

Mainproductioncharacteristicsfrom12goatfarmslocatedintheregionofVic¸osaandMuriaé,MinasGeraisState,Brazil,andrawgoatmilksamples collectedforthepresentstudy.

Farm Goat breed Lactating animals(n) Dailymilk production(l)a Milking system

Storagesystem Numberof samples(n)

Storageperiodb

1 Saanen 72 300 Automatic Bulktank 4 Upto2h,and24h

2 Saanen 42 120 Automatic Freezing 4 Upto2h

3 Saanen 48 170 Handmade Immersiontank 10 Upto2h,24h,and48h

4 Saanen 75 200 Handmade Bulktank 3 Upto2h

5 Saanen 45 150 Handmade Immersiontank 9 Upto2h,24h,and48h 6 Saanen 19 40 Handmade Immersiontank 5 Upto2h

7 Saanen 9 20 Handmade Immersiontank 5 Upto2h,and24h 8 Saanen 39 90 Automatic Bulktank 5 Upto2h,24h,and5d 9 Saanen 44 150 Handmade Bulktank 5 Upto2h,24h,and48h 10 Saanen 24 50 Handmade Immersiontank 5 Upto2h

11 Saanen 47 150 Automatic Bulktank 3 Upto2h,24h,and4d 12 Alpine 21 80 Automatic Immersiontank 3 Upto2h,and6d

a_Approximate_value,_based_on_records_during_the_sampling. b _Storage_periods_of_milk_production,_recorded_during_sampling.

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Table2

Meancounts±standarddeviationofhygieneindicatormicroorganismsfromrawgoatmilksamplescollectedin12dairyfarmslocatedinVic¸osaand Muriaé,MinasGerais,Brazil(valuesinlog10colonyformingunitspermL).

Farm Mesophilicaerobes Enterobacteriaceae Coliforms Escherichiacoli Psychrotrophics Proteolyticpsychrotrophics

1 5.1±0.3 2.9±1.4 2.8±1.8 2.2 4.1±0.7 3.8±1.1 2 4.9±0.7 3.9±0.5 3.6±0.4 – 3.9±0.3 3.9±0.4 3 4.9±0.6 3.9±1.4 3.4±1.1 1.5 4.3±0.8 3.7±1.1 4 5.5±0.2 4.0±0.7 2.9±0.3 1.3±0.5 4.5±0.7 4.5±0.0 5 5.0±0.3 3.8±0.6 3.0±0.6 2.8±0.6 4.2±0.5 4.1±0.6 6 4.5±0.6 2.4±1.0 2.5±1.0 – 3.6±1.0 2.4±0.3 7 4.1±0.6 2.0±1.6 1.6±0.6 1.0 2.9±1.0 2.3±0.5 8 6.1±1.4 5.2±2.3 3.5±2.7 1.0 5.0±0.9 5.3±0.8 9 4.6±0.6 3.2±0.9 3.0±0.9 1.0 4.3±0.4 3.9±0.5 10 4.7±0.5 2.8±0.6 2.5±0.7 – 3.2±1.1 3.4±1.7 11 5.4±0.3 4.0±0.6 2.8±0.6 – 4.8±0.9 4.3±1.5 12 5.8±0.6 4.4±0.7 3.8±0.4 – 3.9±0.4 3.5±0.7 meanvalue 5.0±0.8 3.5±1.3 3.0±1.1 1.7±0.8 4.1±0.9 3.8±1.0

werealsogroupedaccordingtheirmicrobiologicalcounts

and reference values from Brazilian legislation (Brasil,

2000) and scientiﬁc data (Chambers, 2002). The mean countsofrawmilksampleswerealsocalculated consid-eringtheirstoragesystemandstorageperiodingoatfarms andcomparedbyANOVAandTukeytest(p<0.05). 3. Resultsanddiscussion

Consideringthegeneralprofileofgoatmilkproduction inBrazilandtheregion,theselectedgoatfarmswerean adequaterepresentativeofthisactivity,sincetheyfulfilled themaincharacteristicsofBrazilian andlocalgoatmilk production(Costaetal.,2010;Oliveiraetal.,2011).Based onthesecharacteristics(Table1),thegoatmilkproduction inthespecificstudyareaincludedinthisstudy,andalsoin Brazil,canbeconsideredasapromisingruralactivitywhich isundergoingsignificantdevelopment.Basedonthis,itis relevanttoidentifythekeypointsofgoatmilkproduction toimprovethequalityandsafetyofthisfarmproduct,thus avoidingthecurrentproblemsincowmilkproductionin Brazil(Neroetal.,2004,2009;Monteiroetal.,2007;Mattos etal.,2010;Ortolanietal.,2010;Yamazietal.,2010).

Evenconsideringtheabsenceofproperstandardization of storagesystems ongoatfarmsin Brazil,theanalysis ofthemeanvaluesofhygienicindicatormicroorganisms presentedbythegoatmilksamplesdemonstratespecific problems(Table2).Onlyonefarmpresentedameancount ofmesophilicaerobeshigherthan6logcfu/mL,a refer-encevalueusuallyconsideredasindicativeofpoorhygienic practicesduringproduction(Chambers,2002).However, themeanvaluesofenterobacteriaceae,coliforms,andE.coli were substantially higher when compared to reference values (Chambers, 2002); similarresultswerefoundfor psychrotrophics and proteolytic psychrotrophics. These resultsindicatespecific hygienicdeficiencies duringthe firststepsofgoatmilkproductionandproblemsduringthe coldstorageofthisproductongoatfarms.Thisanalysis was confirmedwhen the datawere analyzed consider-ing thelevelsofcontamination(Fig.1);themajority of rawgoatmilksamplespresentedmesophilicaerobecounts lower than6.0 logcfu/mL(Chambers, 2002), and lower than5.7logcfu/mL,thereferenceparameterofthe cur-rentBrazilianlegislation(Brasil,2000).Theseresultsare

alsoinaccordancewithsimilarstudiesonrawgoatmilk fromdifferentregions(Fonsecaetal.,2006;Kondylietal., 2012).However,whentheothergroupsofhygiene indi-catormicroorganismsareconsidered,higherfrequencies ofsampleswithcountshigherthanreferenceorliterature values werefound(Chambers,2002; Picoliet al.,2006; Kondylietal.,2012).

Currently,thelogisticsofgoatmilkcollectioninBrazil arehighlyinfluencedbythecharacteristicsofgoatfarms; asthedaily productionis low, thedairyindustry tends to establish collecting routes with intervals of several days between collections, supportedby theabsence of specification in the current Brazilian legislation (Brasil, 2000).Asresult,goatmilkis storedfor severaldayson dairyfarms,allowingthedevelopmentofpsychrotrophic microorganisms(Table2,Fig.1).Thedataalsorevealedthe relevantproteolyticactivityofthepsychrotrophiccolonies enumerated; 22 samples presented more than 50% of thepsychrotrophiccountscomprisedofproteolytics, con-firmingthespoilagepotentialofthisgroupasaconstituent oftherawgoatmilkmicrobiota.Psychrotrophicsarehighly inducedtogrowandproduceproteolyticenzymeswhen presentinmilkstoredunderrefrigerationforlongperiods oratinappropriatetemperatures(Champagneetal.,1994; SørhaugandStepaniak,1997;McPheeandGriffiths,2011). Theimpact of suchvariables in thecomposition of the raw milk microbiota and spoilage activity has already beendescribed,mainlyincowmilk(Martinsetal.,2006; Wijnandset al., 2006;Ercolini etal., 2009;Perin et al., 2012);however,consideringtheproductioncharacteristics ofgoatmilk,therelevanceofthisgroupcanbeconsidered similar.

Based onthe results(Table 2, Fig.1), a direct effect ofthestorageroutineofrawgoatmilkonits microbio-logical quality was observed; the analysis presented in Tables3and4conﬁrms thisobservation.Althoughbulk tanks are considered as adequate for raw milkstorage (Sierra etal.,2009), goatmilksamplesstoredusingthis equipmentpresentedhighercountsofthemajorityofthe researchedhygieneindicatormicroorganismswhen com-paredtosamplesstoredinimmersiontanksandfreezers (p<0.05,Table3).Immersiontanksarewidelyusedinmilk production in Brazil,and areconsidered inadequatefor propercoolingofthisproduct,astemperaturecontroland

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Fig.1. Frequenciesofrawgoatmilksamplescollectedin12dairyfarmsfromVic¸osaandMuriaéregions,MinasGeraisState,Brazil,presentingdistinctlevels ofcontaminationbymesophilicaerobes(I),enterobacteriaceae(II),coliforms(III),Escherichiacoli(IV),psychrotrophics(V)andproteolyticpsychrotrophics (VI).

coldtransferaredeﬁcient(Tebaldietal.,2008;Neroetal., 2009;Perinetal.,2012);consideringthis,currentBrazilian legislationallowstheadoptionofimmersiontanksonlyon farmswithlowdailymilkproduction(Brasil,2011).Asgoat milkproductioninBrazilischaracterizedbylowdaily pro-duction(Table1),thissystemwouldbeanalternativefor properstorageofthis product onfarms,as observedin

theanalyzedsamples(Table3).Theobtainedcountsfor rawmilkstoredinbulktankswerenotexpected,asthis systemis considered adequatefor propermilk storage; theseresultswereaconsequenceofthedatafromfarm 8, asit presentedhighcounts forall researchedgroups (Table 2) and production characteristics that suggest seriousdeﬁcienciesingoatmilkproduction.Despitethis

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Table3

Meancounts±standarddeviationofhygieneindicatormicroorganismsfromrawgoatmilksamplescollectedin12dairyfarmslocatedinVic¸osaand Muriaé,MinasGerais,Brazil,consideringtheirstoragesystem(valuesinlog10colonyformingunitspermL).

Storagesystem n Mesophilic aerobes

Enterobacteriaceae Coliforms Escherichiacoli Psychrotrophics Proteolytic psychrotrophics bulktank 20 5.3±0.9a _3.9_±_1.5a _3.0_±_1.4a _1.4_±_0.5a _4.6_±_0.7a _4.3_±_0.9a

immersiontank 37 4.8±0.7b _3.3_±_1.3a _2.9_±_1.0a _2.0_±_0.9a _3.9_±_0.9b _3.5_±_0.9b

freezing 4 4.9±0.7a,b _3.9_±_0.5a _3.6_±_0.4a _– _3.9_±_0.3a,b _3.9_±_0.4a,b

ANOVA F(2,58)=3.38; p=0.041 F(2,55)=1.05; p=0.358 F(2,56)=0.84; p=0.436 F(1,7)=1.50; p=0.260 F(2,51)=4.10; p=0.022 F(2,39)=3.15; p=0.054 ANOVA(F):Analysisofvariance,p:levelofsigniﬁcance.MeanvalueswithdistinctlettersinasamecolumnaresigniﬁcantlydifferentbyTukeyHDStest (p<0.05)

Table4

Meancounts±standarddeviationofhygieneindicatormicroorganismsfromrawgoatmilksamplescollectedin12dairyfarmslocatedinVic¸osaand Muriaé,MinasGerais,Brazil,consideringtheirstorageperiod(valuesinlog10colonyformingunitspermL).

Storageperiod n Mesophilic aerobes

Enterobacteriaceae Coliforms Escherichiacoli Psychrotrophics Proteolytic psychrotrophics Justaftermilking 36 4.9±0.7b _3.1_±_1.0a _2.7_±_0.9b _1.4_±_0.5b _3.9_±_0.7b _3.6_±_0.9a

24h 16 4.8±0.9b _3.6_±_1.6a _3.1_±_1.2a,b _1.2_±_0.3b _4.0_±_1.0a,b _3.7_±_1.2a 48horlonger 9 5.6±0.8a _4.9_±_1.2a _3.7_±_1.5a _2.8_±_0.6a _4.8_±_0.6a _4.5_±_0.8a ANOVA F(2,58)=3.93; p=0.025 F(2,55)=7.21; p=0.002 F(2,56)=3.04; p=0.056 F(2,6)=5.59; p=0.043 F(2,51)=4.10; p=0.022 F(2,39)=2.93; p=0.065 ANOVA(F):analysisofvariance,p:levelofsigniﬁcance.MeanvalueswithdistinctlettersinasamecolumnaresigniﬁcantlydifferentbyTukeyHDStest (p<0.05).

farmpresentsautomaticutensilsandequipmentsfordairy

production, suchasautomatic milkingsystemand bulk

tank,italsopresentedsomecharacteristicsthatjeopardize

the milkquality, suchas low dailyproduction withan

averagenumberoflactatinganimals,suggestingmastitis,

andlongperiodofstorage,sinceoneofthesampleswas

collectedafter5daysinthebulktank(Table1).

Indepen-dentofthestoragesystem,thepoorqualityofstoredraw goatmilkmustbeconsidered,duetothehighcountsof hygieneindicatormicroorganismsandmesophilicaerobes (Tables2and3,Fig.1).

Incontrast,adirectinfluenceofthestorageperiodon themicrobiologicalqualityofrawgoatmilkwas demon-strated(Table4).Rawgoatmilksamplesstoredforperiods greaterthan48hpresentedhighercountsofmesophilic aerobes,coliforms,E.coli,andpsychrotrophicswhen com-paredtocountsfromsamplesobtainedjustaftermilking andafter24hstorage(p<0.05,Table4).Thesedata con-firm therelevance ofthe storageperiod onthegrowth oftherawgoatmilkmicrobiota,enhancingtheconcern forthespoilagepotentialofspecificgroups(Grametal., 2002;Perinetal.,2012).Theinadequacyoflongperiods ofstorageusingimmersiontanksasacoolingsystemwas already demonstrated by Perin et al. (2012), who also demonstratedtherelevanceofinitialmicrobial contami-nationonthegrowthofrelevantspoilagemicroorganisms, suchaspsychrotrophics.Althoughthepresentdata indi-catebetterperformanceofimmersiontanksforrawgoat milkstoragerelatedtomesophilicaerobecounts(Table3), thelongperiodofstorageofthisproductongoatfarms mustbeconsidered.Independentofmilkstorage condi-tions,alongperiodallowsthegrowthofpsychrotrophic microorganismsevenatappropriatetemperatures(upto 4◦C)(Champagneet al.,1994).Duringgrowth,theyare abletoproduceheatstableproteasesandlipasesthatwill beactiveevenaftertheheattreatmentsemployedinthe

dairyindustryforrawgoatmilk,jeopardizingthequalityof theprocessedproducts(Grifﬁthsetal.,1981;Fairbairnand Law,1986;PatelandBartlett,1988;Owusuetal.,1991). Themainpsychrotrophicmicroorganismsusuallypresent inrawgoatmilkhavenotbeenpreviouslydescribed,but consideringitsproductionscharacteristics,thismicrobiota isbasicallysimilartocowmilk,whichiscomposedmainly byPseudomonasspp.andBacillusspp.(PatelandBartlett, 1988;Francisetal.,1998;Ercolinietal.,2009).

Inconclusion,thesedatademonstratespecificfailures in the goat milk production chain in Brazil, leading to thepoor microbiological quality of this product identi-fiedbyhighcountsofhygieneindicatormicroorganisms other than mesophilic aerobes, such as coliforms and psychrotrophics.Inaddition,arelevantinfluenceoflong storageperiodsonthemicrobiologicalqualityofthis prod-uctwasobserved,whichisaconsequenceofthecurrent logisticsadoptedbythedairyindustryallowablebycurrent Brazilianlegislation. Basedontheseresults, itis impor-tanttohighlightthenecessitythattheBrazilianMinistryof Agricultureestablishthespecificequipmenttobeadopted ongoatfarmsaswellasaperiodlimitforcollection. Acknowledgments

The authors are supported by CAPES, CNPq, and FAPEMIG.ThepresentresearchwassupportedbyCNPq. References

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