TurkJVetAnimSci 29(2005)873-879 ©TÜB‹TAK ResearchArticle IdentificationandDistributionofLacticAcidBacteriaDuringthe RipeningoffiavakTulumCheese GülsümÖKSÜZTEPE,BahriPATIR,MehmetÇALICIO⁄LU* DepartmentofFoodHygieneandTechnology,FacultyofVeterinaryMedicine,F›ratUniversity,Elaz›¤-TURKEY *E-mail:[email protected] Received:28.05.2004 Abstract: Thisstudywasundertakentodeterminechangesinthefloraoflacticacidbacteriaduringtheripeningoffiavaktulum cheeseandtoidentifythemajorspeciesplayinganimportantroleintheripeningprocess.Tulumcheesewasproducedfromraw sheepmilkusingtraditionalmethodsandripenedinplasticcontainersat4°Cfor90days.Samplesweretakenondays0,15,3 0, 60and90andanalyzedforisolationandidentificationoflacticacidbacteria.Atotalof783isolatesoflacticacidbacteri awere identified.Theresultsindicatedthat,ingeneral,lacticstreptococciwerepredominantwithinthefirstmonthofripeningand ,then, replacedbylactobacilli.Itisconcludedthat Lactobacilluscasei subsp.casei,Lactobacillusplantarum fromthefamilyLactobacillaceae andLactococcuslactis subsp.cremoris,Lactococcuslactis subsp.lactis,and Leuconostocmesenteroides subsp.cremoris fromthe familyStreptococcaceaewerethepredominantspeciesamongtheisolates,indicatingthatthesespeciesmayplayamajorroleinthe ripeningoftulumcheese. KeyWords: Tulumcheese,ripening,lacticacidbacteria. fiavakTulumPeynirininOlgunlaflmas›S›ras›ndaLaktikAsitBakterilerinin Da¤›l›m›ve‹dentifikasyonu Özet: Buçal›flmafiavaktulumpeynirininolgunlaflmas›ndaönemliroloynayantürleribelirlemekveolgunlaflmaesnas›ndalaktikasit bakterifloras›ndameydanagelende¤iflimleriaraflt›rmakamac›ylayap›ld›.Tulumpeynirigelenekselmetotlarkullan›larakçi¤ko yun sütündenüretildiveplastikbidonlarda4°C’de90günolgunlaflt›r›ld›.Örnekler,0,15,30,60ve90.günlerdeal›nd›velaktik asit bakterilerininizolasyonveidentifikasyonuiçinanalizedildi.Toplam783laktikasitbakteriizolat›identifiyeedildi.Sonuç lar gösterdiki;genelolarakolgunlaflman›nilkay›ndalaktikstreptekoklar,dahasonralaktobasillerbask›nbulundu.‹zolatlariçer isinde Lactobacillaceaefamilyas›ndanLactobacilluscasei subsp.casei veLactobacillusplantarum,Streptococcaceaefamilyas›ndanLactococcus lactis subsp. cremoris, Lactococcuslactis subsp. lactis,ve Leuconostocmesenteroides subsp. cremoris’inpredominantoldu¤uve tulumpeynirininolgunlaflmas›ndaönemlibirroloynayabilece¤isonucunavar›lm›flt›r. AnahtarSözcükler: Tulumpeyniri,olgunlaflma,laktikasitbakterisi. Introduction fiavaktulumcheeseisgenerallyproducedfromraw fiavaktulumcheeseisapopularsemi-hardcheese sheepmilkinruralareasinEasternTurkey.Villagersin (e.g.,43%moisture)inTurkeymanufacturedfrom thisregionproducetulumcheeseusingtraditional unpasteurizedsheepmilk.Thisparticulartypeofcheese methodsandmarketthemafter15to90daysof isthethirdmostproducedamongthecheesetypes ripening,dependingonthemarketdemand.Traditionally, manufacturedinTurkey.fiavaktulumcheeseusedtobe thepackingmaterialisacontainermadeofgoatskin. producedanddistributedlocally.Atpresent,demandfor However,intherecentyears,ripeningofthecheesein thischeesehasincreasedandtheannualproduction plasticcontainershasbecomewidespreadintheindustry. reached4000tduetoitshighnutritionalvalueand Microbiological,chemical,andphysicalaspectsofthe highlyrecognizedflavorandaroma.Since1987,tulum cheesemayvarydependingontheplant,experienceof cheesehasbeenamongtheexportproductsofTurkey(1- thepersonnel,andqualityofrawmilk.Asaresult,a 3). standardqualityofproductcannotbeproduced(4-8). 873 IdentificationandDistributionofLacticAcidBacteriaDuringtheRipeningoffiavakTulumCheese Sincetulumcheeseisproducedbytraditionalmethods usefuldatatodevelopastartercultureblendthatcanbe insmall-scaleestablishments,starterculturesareusually usedbythemanufacturersofthisparticulartypeof notusedinproductionandfermentationdependson cheese. indigenousbacterialflora.Thereareverylimiteddataon floraoflacticacidbacteriaduringthemanufactureand ripeningoftulumcheese.Forexample,Karasoy(9) MaterialsandMethods reportedthat Streptococcuslactis playedaroleinthe RawSheepMilk initialripeningwhile Streptococcuslactis , RawsheepmilkwasobtainedfromtheDairySheep Thermobacteriumbulgaricum,andOidium lactis together UnitoftheResearchandTrainingFarmoftheFacultyof weretheessentialfloraoftheactualripening.Inanother VeterinaryMedicine,F›ratUniversity,Elaz›¤,Turkey.The study,Bostanetal.(10)experimentallyproducedtulum milkwastransferredtothemilk-processinglaboratory cheesefromrawcow’smilkandisolatedatotalof684 withinapproximately1haftermorningmilking.A200 strainsoflacticacidbacteriaatvariousstagesofripening. mlportionwastakenformicrobiologicalandchemical Theirresultsshowedthat Streptococcuslactis and analysisoftherawmilk.Briefly,numbersoftotalaerobic Streptococcusfaecalis werethepredominantfloraduring mesophilebacteria,Lactobacillus-Leuconostoc- theinitialphasewhereas Streptococcusfaecium , Pediococcus,lactococci,enterococci,Staphylococcus- Streptococcuslactis,Lactobacilluscasei,andLactobacillus Micrococcus,coliform,andmold-yeastweredetermined plantarum predominatedinlaterstagesoftheripening bypour-platemethodontoappropriatemediaas period.Inthestudy,itwasnotedthatLeuconostocand describedpreviously(12-14).Thesampleswereanalyzed Pediococcusspeciesexistedbutatlowlevels.Insome fortitrableacidity(%lacticacid)andlevelsoffatanddry studies,tulumcheesewasproducedusingvariousstarter matter(15).Theremainingvolumeofmilkwasusedfor culturecombinations.Forexample,Bostan(4)reported makingfiavaktulumcheese. thatthecombinationof Lactobacilluscasei and Streptococcuslactis wasthemostappropriateamong DeterminationofInhibitorySubstancesinRaw manyotherculturestested.Inanotherstudy,Pat›retal. Milk (8)reportedthat Streptococcuscremoris,Streptococcus Inhibitorsubstancesinthemilkwasdetectedusing lactis, Lactobacilluscasei , Lactobacillusplantarum , fermentationtest(16).Milkthatcontainedanyinhibitor Lactobacilluscurvatus , Leuconostoc cremoris, wasrejected. Streptococcusfaecium,and Streptococcusfaecalis might ManufactureofTraditionalTulumCheese playanimportantroleinripeningoftulumcheese.Bostan etal.(10)surveyedsomecommercialtulumcheese Milkthatwasfreeofanyinhibitorysubstancewas samplesofpremiumqualityforisolationand filteredandsufficientamountofrennet(approximately identificationoflacticacidbacteriaandreportedthat,of 1.85mlofrennetatthestrengthof1/6000per25kg the426isolates,themajoritywasStreptococcusfaecium, ofmilk)wasaddedaccordingtothemanufacturer’s Lactobacilluscasei,Streptococcusfaecalis,Streptococcus recommendation.Themilkcoagulatedwithin lactis,Lactobacillusplantarum,Leuconostoc cremoris,and approximately90min.Theresultingcurdwascutinto5 Lactobacilluscurvatus.InasimilarstudybySürmelietal. x5cmpiecesandpouredontocheeseclothonacolander (11),itwasindicatedthatbacterialspeciesfoundin fordrainingofwheyandthenpressed(firstpress)using commercialtulumcheesewasintheorderofenterococci, metalweights.After24hunderpress,curdwasmanually lacticacidbacteria,pyogenicandviridiansstreptococci brokentopiecesassmallasachickpeaand2%(w/w)salt andLeuconostocspp. wasaddedandmixedfollowedbyasecondpressingin cheeseclothfor24hatambienttemperature.Stepsat Resultsoftheabovestudiesonlacticacidbacterial firstpresswererepeatedforathirdpress.Attheendof floraarequitediverseandtherearesomeconflicting thethirdpress,curdswerebrokentosmallpiecesone data.Theobjectiveofthepresentstudywastodetermine moretimeandair-driedforapproximately24hatroom thespeciesoflacticacidbacteriamostimportantfor temperature.Theresultingcurdthenwasfilledinto flavorandaromadevelopmentduringtheripeningof plasticcontainers(750g)andsqueezedtightlytoremove traditionallyproducedfiavaktulumcheeseforproviding excessiveairusingawoodenstick.Theplasticcontainers 874 G.ÖKSÜZTEPE,B. PATIR,M.ÇALICIO⁄LU Rawsheepmilk (25kg,freeofantibioticanddetergentresidue) Addingrennet (strengthof1/6000,1.74mlper25kgofmilk) Cuttingthecoagulum (ca.90minafteradditionofrennet,atthesizeof5x5cm) Processingthecurd (curdpiecesweretakenontowheyclothsfordrainingtheexcessive wheyandheldatroomtemperaturefor24h) Pressingthecurd (1stpress,atroomtemperaturefor24h) 2ndpress(thecurdisbrokenintopieces,andthen2%saltisaddedfollowedbypressingandholdingfor24h. 3rdpress (breakingthecurdagainandaddinganother2%saltandthenpressingfor24h) Drying (manuallybrokencurdisheldatroomtemperaturefor24hwithoutpress) Packing(saltedanddriedcurdsarefilledintoplasticcontainersandsqueezed byawoodenstickandthenclosedtightly) Ripening (at4±1°Cforatleast90days) Figure.FlowchartformanufactureofSavaktulumcheese. werecloseduptightlywithaluminumfoilandstoredat4 thejarpriortoweighinga10gportionandtransferring ±1°Cfor90daysforripening(15,17,18).Thesame toasterilecontainerofhomogenizer(Buhler51800/00). cheesecontainerwasusedforsamplingatdifferent A90mlvolumeofsterile2%sodiumcitratewasadded intervals.Thestudywasconductedin5replicates. tothe10gsampleandhomogenizedfor1/10dilution. PreparationofSamples ThishomogenatewasthendecimallydilutedusingRinger solutionupto10-8.Onemlfromeachdilutionwaspour- Samplesweretakenformicrobiologicalanalysison platedinduplicateontoM17agar(Merck-15108)and days0,15,30,60,and90duringtheripeningperiod. ontoRogosa’sacetateagar(Oxoid–CM627).Bothtypes Portionsof150-200gweretakenoneachsampling ofplateswereincubatedat30±1°Cfor72hand5days intervalusingasterileknifeandtransferredintoasterile forM17platesandRogosa’sacetateagarplates, jar.Thesampleswerehomogenizedbyasterilespatulain respectively(12,13,16). 875 IdentificationandDistributionofLacticAcidBacteriaDuringtheRipeningoffiavakTulumCheese IdentificationofLacticAcidBacteria Discussion ColoniesfromM17orRogosa’aacetateagarplates Theuseofrawmilkwithhighcoliformcountfor wereselectedfromplateswithcolonynumbersbetween