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1 Abstract Lacticacidbacteriaareknowntohavepotentialinproducingantimicrobial compounds, including antifungal agents. This study aims to determine whetherthelacticacidbacteriafromhumanoralmucosahavethepotential to produce the secondary metabolite to inhibit the candidiasis, which caused by pathogen fungi, Candida albicans. Lactic acid bacteria were isolatedfromthreehealthyrespondentsbasedonagedifferencethatisan infant, child, and adult. Lactic acid bacteria isolation using the demann RogosaSharpeBroth(MRSB)Jliquidmedium,followedbypurificationstage withthequadrantscratchmethodusingthedemannrogosasharpeagar (MRSA)Jsolid medium. The purified isolates obtained were characterized and identified by Gram staining, biochemical tests, and inhibition zone diameter measurement toward pathogenic fungi Candida albicans. The resultswereobtainedfromeachofthetwolacticacidbacteriaisolatesfrom eachrespondentwithcharacteristicsincludinggramjpositivebacteriawith shortstemcellshape,possiblyaslactobacillussp.itisconcludethatthe lactic acid bacteria of oral mucosa from adult had greater anticandidal activitythaninfantandchild. ISSN:2580J2410 eissn:2580j2119 StudyofLacticAcidBacteriaActivitiesfromHumanOral MucosaforCandidaalbicansInhibition EviLauw 1,KhusnulHatimahIlham 1,FeblianiTarukPalinggi 1,DesySetiady 2 andsartini 1 1 FacultyofPharmacy,HasanuddinUniversity,Makassar,Indonesia 2 FacultyofDentalMedicine,HasanuddinUniversity,Makassar,Indonesia OPENACCESS InternationalJournalofAppliedBiology International Journal of Applied Biology is licensed under a Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ArticleHistory Received23May2017 Accepted10November2017 Keyword LacticAcidBacteria HumanOralMucosa AntifungalActivity Candidaalbicans Introduction During the last one decade, the prevalence of infectious diseases caused by pathogenicfungi,whichiscandidiasis,hasincreasedsharply(fortúnetal.,2012).candidiasis is a mucosal fungal infection on humans of all ages that affect the skin and mucous membranesoforopharynx,oesophagus,andvagina(fortúnetal.,2012;gajeretal.,2012). Thereareatleast15distinctCandidaspeciesthatcausehumandisease,butmorethan90% ofinvasivediseaseiscausedbythefivemostcommonpathogens,c.albicans,c.glabrata,c. tropicalis,c.parapsilosis,andc.krusei(silvajdiasetal.,2014).candidaalbicanscontinuesto bethemostprevalentandproblematicofallcandidaspecies(pereraetal.,2015;pauletal., 2014).ThepathogenicityofCandidaspeciesisattributedtocertainvirulencefactors,suchas theabilitytoevadehostdefences,adhesionandbiofilmformation(onhosttissuesandoron medical devices), and the production of tissuejdamaging hydrolytic enzymes, such as proteases,phospholipasesandhaemolysins(pauletal.,2014).moreover,themorbidityand CONTACT:EVILAUWevi.lauw09@gmail.com InternationalJournalofAppliedBiology 58

2 InternationalJournalofAppliedBiology,1(2),2017 mortalityassociatedwithcandidiasisarestillveryhigh,evenusingtheactualantifungaldrugs. Annually, 50% of adults and up to 30% of children die of candidiasis (Paul et al., 2016; Martinezetal.,2015). ThemajorclassesofantifungaldrugsusedfortreatmentofCandidaspeciesinfections are azoles, polyenes, and echinocandins (Martinez et al., 2015). According to previous experimentalresults,irrationalusemayleadtocolonizationofcandidasp.thatareresistant tothesedrugs.ithasbeenreportedthatconventionaltreatmentfortreatingcandidiasiswith fluconazolehasacaseofresistance(martinezetal.,2015;costaetal.,2015).theoccurrence of Candida species infections becoming more difficult to treat due to the growth of immunogenic diseases, endocrine disorders, malnutrition, the disproportionate use of immunosuppressivedrugs,andbroadspectrumantibiotics(costaetal.,2015).itisbelieved thattheincreaseofc.albicansinfectionsisduetoitsintrinsicallylowsusceptibilitytoazoles, including the imidazole and the oraljparenteral triazoles (e.g., fluconazole, voriconazole) (Gayford JJ Harskell R, 2010). Additionally, it is known that the acquired resistance is resultedofraremutationsthatareselectedbydrugpressure(samaranayake,2012). The recent efforts of various excellent researches tremendously broadened on the complex mechanisms of normal flora(scatassa et al., 2015). They were shown that living creatureswithoutnormalfloraaremoresusceptibletopathogenicmicrobes(kanmanietal., 2013; Amadou et al., 2013; Amenu, 2013). In this sense, the authors highlighted the importance of normal flora and that the addition of secondary metabolite to improve the efficacyofcandidasis.theresultofastudyshowedthattherewasanincreaseinoraland dentalhealth,aswellasadecreaseinthenumberofcandidaalbicansfungiinschoolchildren whoconsumedmilkorcheesecontaininglactobacillusrhamnosusstraingg.3whichbelongs to the lactic acid bacteria group(canabarro et al., 2012). According to the authors, some strains of lactic acid bacteria have a positive activity in reducing the amount of Candida albicansinthesalivaofthehumanoralcavity(amenu,2013;canabarroetal.,2012). Recent studies revealed the involvement lactic acid bacteria, especially the genus Lactobacillus, can be isolated from animal and human digestive tracts, including upper gastrointestinal organs such as the oral cavity (Corona et al., 2016). It has obtained Lactobacillusgasseriisolatesidentifiedusing16SJrRNAsequencingfromoralmucosaofa4J monthjoldinfant(settannietal.,2013).however,thisarticleshowedantijfungalactivityof lacticacidbacteriaisolatesfromthreemostpotentsourcesasproducersofanticandidiasis metabolitecompoundsthatareaninfant,child,andadult.thesignificanceofthisstudyis duetoitscontributiontoknowledgeonthedeterminationoftheabilityoflacticacidbacteria isolatesfromtheoralcavityofhumansasanticandidiasisagents. MaterialsandMethods PreparationofToolsandMaterials ThetoolsusedinthisresearchwereAutoclave(AllAmerican),Incubator(Memmert), LaminaryAirFlow,Microscope(Olympus),Oven,Caliper,AnalyticalScales(Ohauss),Sterile Swab (OneMed), petri disc, Chemical Glass (Pyrex), Bunsen, Cotton, Measuring cylinder (Pyrex),handscoon,electricstove,clothmask,roundloop,straightloop,spoon,testtube,vial tube,etc. Thematerialsusedinthisresearchwere70%alcohol,aquadistillate,pureCandida albicansculture(obtainedfrommicrobiologylaboratory,facultyofpharmacy,hasanuddin 59

3 InternationalJournalofAppliedBiology,1(2),2017 University),ketoconazole,deMannRogosaSharpeAgarandBroth(MRSAandMRSB)media, andpotatodextroseagar(pda)medium. ExperimentalMethods IsolationofLacticAcidBacteria Oralmucoussamplesweretakenfromeachofthethreesamplesindividually,infant, child,andadult.samplingisdonebyrubbingintotheinsidecheeksinsamplesusingasterile swabsticker.theswabresultistheninsertedintoavialcontainingthemrsbmediumasa transportmedium,thenimmediatelytakentothelaboratory. Samplesoftheoralmucosaweredilutedby1mLpelletedandfedinto9mLofsterile distilledwater,thendilutingthedilutionsfrom10 J1 to10 J5.Atotalof1mLofliquidfromeach dilutionwasintroducedintopetridishesand10mlofmrsaandcaco 3 1%wereaddedand homogenized,thenincubatedat37 Cfor1x24hours.Whenaclearzoneisformedaround thecolony,thenthecolonyissuspectedaslacticacidbacteria. PurificationofLacticAcidBacteriaIsolates LactateAcidBacteriaisolatesweretheninoculatedbyscrapingthemontothemedium inapetridishthenincubatedfor1x24hoursat37 Cattheaerobicincubator.Thenpurified byinoculatingthemrsamediuminclinedinthetesttube,afterwhichincubatedfor1 24 hoursat37 Cintheaerobicincubator. CharacterizationsandIdentificationsofLacticAcidBacteria MacroscopicTest PurelacticacidbacteriaisolateswereinoculatedonMRSAmediuminpetridishes, afterwhichincubatedfor1x24hoursat37 Cinaerobicincubators.Then,observedcolonies. MicroscopicTest GramstainingmethodwasusedbyisolategrowingonMRSBmediumplacedonthe preparation. Then, fixed and added 2J3 drops of gram A(crystal violet), left for 1 minute. Washwithrunningwater,driedwithfilterpaper.AddgramB(mordant).Left1minute.Spilled with gram C(95% ethanol) for 10J20 seconds. Wash with running water, dried with filter paper.addgramd(safranin)for1minute.washwithrunningwater,driedwithfilterpaper. Itwasobservedunderamicroscopewith100xwideningbyusingimmersionoil.Grampositive bacteriawillbepurpleandgramnegativewillbered(ventimigliaetal.,2015). BiochemicalTest Biochemicaltestsinthisstudyconsistoftheseveraltestsasfollowing; CarbohydrateFermentationTest CulturesoflacticacidbacteriainoculatedintoLactoseBroth,SucroseBroth,Glucose Broth,andMannitolBrothmediawereincubatedfor1x24hoursat37 C.Thus,observed changesinmediumcolorandgasformation. MotilityTest ThecultureoflacticacidbacteriawasinoculatedpunctureonanerectSIMmedium andincubatedfor1x24hours.ifisolategrowthspreadsfarfrompunctureindicatesmotile isolate,whereasifgrowthonlyoccurspuncturedischarge,itindicatesnomotileisolate. 60

4 InternationalJournalofAppliedBiology,1(2),2017 CatalaseTest Suspensionisolatesdropsasmuchastwodropsonglassobject,andthenspilled3% H 2 S 2 solution.thepositivecatalasereactionisshownbythepresenceoffoamorfoamthat occursaftertheadditionofthesolutionfor1min. TestofOptimumGrowthTemperature A total of 1 ml of pure culture suspension were each inoculated into 4 tubes containing 9 ml MRSB media on which the purple bromjkresol indicator was split. The mediumisincubatedatatemperatureof10 C,15 C,37 Cand45 Cfor1J5x24hoursunder suitable conditions. The media color change from purple to yellow signifies the growth of culture. TestofOptimumGrowthpH Atotalof1loopofpureisolateswaseachinoculatedinto4tubescontainingMRSB mediaonwhichthepurplebromjkresolindicatorwassplit.themediumwasincubatedatph 2,4,6and8for1J5x24hoursat37 C.Themediacolorchangefrompurpletoyellowsignifies thegrowthofculture. TestofIsolateActivityagainstCandidaalbicans TheisolatedlacticacidbacteriaweregrowninMRSBrothmediafor24J48hours.The result of the fermentation will be precipitated its cell mass. Then the supernatant fluid containing the bioactive component was tested for its anticandidal activity of Candida albicansbythepaperdiscjdiffusionmethod.theisolateswerepreparedforthetestwithtwo replicationsofeachsample. Resultsanddiscussion LacticAcidBacteriaIsolation This research began with sterilization as the most important stage in the implementationofresearchrelatedtomicroorganisms.furthermore,themanufactureofa specialmediumforthegrowthoflacticacidbacteriaismrsb(demannrogosasharpebroth) as the transport medium. The next stage is the oral mucosal sampling of three people samples; infant, child, and adult from three volunteers with healthy dental and mouth conditions.samplingisdonebyusingsterileswabswabsandimmediatelyinsertedintothe transport medium immediately after taking to minimize the occurrence of contamination. The purpose of this early stage is to obtain lactic acid bacteria from the oral mucosa and increasethenumberofcells(cultivation)beforethenextstage. ThisprocessusedMRSA(deMannRogosaSharpeAgar)mediuminapetridishand calciumcarbonate(caco 3 1%)addedtothemediuminordertoidentifydifferencesoflactic acidbacteriacomparedtotheothertypesofbacteria.thevalidationofthelacticacidbacteria wasperformedbythemuddinessofmediumcolour(vestmanetal.,2013).ifitcontained lacticacidbacteria,thecalciumfromcaco 3 inmrsacanreactwithlacticacidproducedby lacticacidbacteriatoformsolublecalciumlactate,whichmakesthecolourofthemedium becomestransparent(vestmanetal.,2013). 61

5 InternationalJournalofAppliedBiology,1(2),2017 LacticAcidBacteriaPurification Thenextstagewasthepurificationofisolates.Thisisolationwascarriedouteachinto 2petridishesforeachsample.Theobservationofcolonygrowthwillbedoneondays1,3,5, and7.onthe5theday,atransparentzonehasbeenformedonthemrsamediumthatis thoughttobeacolonyoflacticacidbacteria.thepurificationresultsarerepeatedtwicewith theaimofobtainingmorepureisolatesandincreasinglyfreefrommixturesofothertypesof bacterialcolonies. Subsequently, the isolates were moved into the test tube containing the inclined MRSAinthetesttubeforthefurtherstages,i.e.identificationofisolates.Isolateswerenamed bycode,i.e.infant smouthisolates(mb,mb2),child smouthisolates(ma,ma),andadult s mouthisolates(md,md2). LacticAcidBacteriaCharacterizationsandIdentifications Inthisstudy,theidentificationoflacticacidbacteriamicroscopicallywasdonebythe gram staining method. The staining results for the three types of isolates (MB, MA, MD) showedpurplebacterialcells,sotheisolatesinthisstudyincludedgramjpositivebacteria. Thissupportsthetheorythatlacticacidbacteria,oneofwhichisfromthegenusLactobacillus, isatypeofgramjpositivebacteriathatingramstainingwillhaveapurpleappearanceunder anopticalmicroscope(ventimigliaetal.,2015;norderetal.,2013). Inaddition,besidesthemicroscopictestsandgeneidentificationabove,theisolates weretestedfortheirantimicrobialactivitybythediffusionmethodinordertousepaperdiscs. Itwasmeasuredfromhorizontal,vertical,anddiagonal,thenthecalculatedfortheaverage diameter.basedonantimicrobialactivitytestfromoralmucosaisolatetocandidaalbicans fungicausinginfectiousdiseaseofcandidiasis,andobtainedtheresultofthezonediameter asfollows. Kontrol1 MB1 MA1 MD12 MB2 Kontrol2 MA2 MD2 Figure1.Inhibitionzonediameteroflacticacidbacteriaisolatesfromhumanoralmucosa 62

6 InternationalJournalofAppliedBiology,1(2),2017 Table1.Inhibitionzonediametermeasurementoflacticacidbacteriaisolatesfromhuman oralmucosa TypesofIsolate Replication I II Diameter±StandardDeviation(mm) Ketoconazole(Control+) Average ± Infant(MB) Average ± Child(MA) Average ± Adult(MD) Average ±0.09 Fromthetableabove,itcanbeseenthattheaverageinhibitoryzonediameterofthe oralmucosaofinfants(mbandmb2)is7.23mmand7.20mm(average7.22mm±0.02mm), lacticacidbacteriaisolatefromtheoralmucosaofchildren(ma)is9.30mmand10.13mm (average9.72mm±0.58mm),andlacticacidbacteriaisolatefromadultmouthmucosa(md) were16.17mmand16.03mm(average16.10mm±0.09mm).whiletheresultsofinhibitory powerusingketoconazoleantifungaldrugsasacomparisonwere19.07mmand19.30mm (average19.19mm±0.16mm).itisshowedthatlacticacidbacteriaisolatesfromadultmouth mucosa(md)hadthebestinhibitorypowerseenfromtheirlargerinhibitorydiameterzones comparedtobothinfantisolate(mb)andchildisolate(ma). Figure2.Thecollectionofpotentlacticacidbacteriaisolatesasanticandidiasis 63

7 InternationalJournalofAppliedBiology,1(2),2017 Foradditionaldata,biochemicaltestsconsistingofcarbohydratefermentationtest, motilitytest,catalasetest,optimumphtest,andoptimumtemperaturetestwereconducted. Theaimofthesefivebiochemicaltestsistoconfirmthattheisolatesobtainedfromtheoral mucosa of humans in this study can satisfy the characteristics of lactic acid bacteria with specificphysiologicalandmetabolicactivitiesspecifically. Theresultsofcarbohydratefermentationtestonthethreeisolates(MB,MA,MD)with medium LB, GB, and SB showed the three results of each positive able to ferment carbohydrate.thepositiveresultismarkedbythecolourchangeofthemediumtoyellow andtheformationofgasinthedurhamtube.thisisconsistentwiththereferencethatlactic acidbacteriaincludeheterofermentativebacteria,whicharecapableoffermentingsugars (monosaccharaidesanddisaccharides)toproduceacidicandco 2 gases.theacidicconditions inthemediumresultinachangeofmediumcolourandbubbleformation(minervinietal., 2012;Pepeetal.,2013). The result of the motility test of all three isolates showed the growth of colonies perpendicularlyinthedirectionofinoculationusingaloop,andwithoutspreadingaroundthe inoculants. If the bacterial cell is motile, its colonies will spread throughout the medium. Therefore,thethreeisolates(MB,MA,MD)arenonJmotilebacteria(Minervinietal.,2012; Pepeetal.,2013). Theresultofcatalasetestoflacticacidbacteriaisolatedfromoralmucosaofthethree samples(mb,ma,md)wasnegativecatalase.thisisinaccordancewiththereferencetothe fact that lactic acid bacteria do not produce catalase enzymes so that the test results are negativeasaresultoftheabsenceofh 2 O 2 degradationbycatalaseenzymes(minervinietal., 2012;Pepeetal.,2013). TheoptimumpHtestresultsinthisstudywerepH4.AtpH4,themostturbidvisible mediumwasthoughttobethehighestgrowthrateoflacticacidbacteriainthisstudy.the previous study stated that lactic acid compounds capable of inhibiting the growth of pathogenic microbes by lowering the ph of the environment to 3 to 4.5 so that other microbialgrowthwillbehampered(minervinietal.,2012;pepeetal.,2013). Theoptimumtemperaturetestresultsareat45 C,whichisthehighesttemperature ofthistest.thehighestgrowthofcoloniesintheformofthecloudyjlookingmediumoccurred at45 Ccomparedto25 Cand37 C.Basedonthereference,lacticacidbacteriacangrow atatemperatureof20j50 C(Minervinietal.,2012;Pepeetal.,2013). Basedonthefivebiochemicaltests,thethreetypesofisolatesarecatalasenegative, nonmotile,andincludegrampositive(+)bacteriawithbasil(stem)morphology.inthisstudy, itcanbesaidthattheisolatesobtainedfromthemucosaofthehumanmouth(mb,ma,md) belong to the lactic acid bacteria group, were from the genus Lactobacillus based on the biochemicaltestresults. Conclusions Theanticandidiasisactivityoflacticacidbacteriaisolatesfromtheadultoralmucosa (MD) most potentially inhibited the growth of Candida albicans that is mm, when comparedtolacticacidbacteriaisolatesfromtheinfant(mb)andthechild(ma) Acknowledgment TheauthorsaregratefultoMinistryofResearch,Technology,andHigherEducation IndonesiawhichhasfundedthisresearchthroughStudentCreativityProgramJExactResearch 64

8 InternationalJournalofAppliedBiology,1(2),2017 withcontractnumber0547/b3.1/km/2017.wealsogratefultothelecturerdr.sartini,m.si., Apt.,forhervaluablehelpandtoHasanuddinUniversity,whichhasfacilitatedus,sothatthe researchcouldsuccessfullyconduct. References Amadou,I.,Le,G.W.Shi,Y.H.2013.Evaluationofantimicrobial,AntioxidantActivities,and NutritionalValuesofFermentedFoxtailMilletExtractsbyLactobacillusparacaseiFn032. Int.J.FoodProp,16(6):1179J1190. Amenu,D.2013.AntimicrobialActivityofLacticAcidBacteriaIsolatedfrom Ergo,Ethiopian TraditionalFermentedMilk.Curr.Res.Microbiol.Biotechnol,1(6):278J284. Canabarro,A.,Valle,C.,Farias,M.R.,Santos,F.B.,Lazera,M.,Wanke,B.2012.Associationof SubgingivalColonizationofCandidaalbicansandotherYeastswithSeverityofChronic Periodontitis.J.PeriodontalRes,48: Corona,O.,Alfonzo,A.,Ventimiglia,G.,Nasca,A.,Francesca,N.,Martorana,A.,Moschetti,G. Settanni,L.2016.IndustrialApplicationofselectedlacticacidBacteriaIsolatedfrom LocalSemolinasforTypicalSourdoughBreadProduction.FoodMicrobiol,59: Costa,C.,Pais,P.Teixeira,M.C.2015.Multidrugresistanceinpathogenicyeasts:Emphasis on the role of ABC and MFS multidrug transporters. In The Battle Against Microbial Pathogens:BasicScience,TechnologicalAdvancesandEducationalPrograms,Formatex: Badajoz,Spain. Fortún,J.,MartínJDávila,P.,GómezJGarcíadelaPedrosa,E.,Pintado,V.,Cobo,J.,Fresco,G., Meije, Y., Ros, L., Alvarez, M.E. Luengo, J Emerging Trends in Candidemia: A HigherIncidencebutaSimilarOutcome.J.Infect,65: Gajer,P.,Brotman,R.M.,Bai,G.,Sakamoto,J.,Schütte,U.M.,Zhong,X.,Koenig,S.S.,Ma,Z., Zhou, X. Abdo, Z Temporal Dynamics of the Human Vaginal Microbiota. Sci. Transl.Med,4: Gayford,J.J.HarskellR.2010.OralDiseases:ClinicalOralMedicine.Translatedby:Lilian Yuwono2 nd Edition.Jakarta:EGC. Kanmani, P., Satish Kumar, R. Yuvaraj, N Probiotics and its Functionally Valuable Products areview.crit.rev.foodsci.nutr,53(6):641j658. Martinez,L.R.,Nimrichter,L.,Nosanchuk,J.D.,Baltazar,L.M.,Ray,A.,Santos,D.A.,Cisalpino, P.S.Friedman,A.J.2015.AntimicrobialPhotodynamicTherapy:anEffectiveAlternative ApproachtoControlFungalInfections.Front.Microbiol,6:202. Minervini, F., Lattanzi, A., De Angelis, M., Di Cagno, R. Gobbetti, M Influence of Artisan BakeryJor LaboratoryJPropagated Sourdoughs on the Diversity of Lactic Acid BacteriumandYeastMicrobiotas.Appl.Environ.Microbiol,78(15): Norder,G.E.,Dahlén,G.,Ruth,M.,Ny,L.,GuidingJJärbrink,M.,Bergquist,H.Bove,M BacterialFloraofTheHumanOralCavity,andtheUpperandLowerEsophagus.Diseases oftheesophagus,26:84j90. Paul,S.MoyeJRowley,W.S.2014.MultidrugResistanceinFungi:RegulationofTransporterJ EncodingGeneExpression.Front.Physiol,5:143. Pepe,O.,Ventorino,V.,Cavella,S.,Fagnano,M.Brugno,R.2013.PrebioticcontentofBread PreparedwithflourfromimmaturewheatgrainandSelectedDextranJProducingLactic AcidBacteria.Appl.Environ.Microbiol,79(12): Perera, J., Weerasekera, M. Kottegoda, N Slow Release AntiJFungal Skin FormulationsBasedonCitricAcidIntercalatedLayeredDoubleHydroxidesNanohybrids. Chem.Cent.J,9:27. 65

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