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Squalen Bulletin of Marine & Fisheries Postharvest & Biotechnology, 8 (2), 2013, 69-76
Department ofAquatic ProductTechnology, Faculty of Fisheries and Marine Sciences BogorAgricultural University,
Kampus IPB Darmaga, Jl.Agatis, Bogor 16680 WestJava, Indonesia
*Corresponding author: kustya@gmail.com
Article history:
Received: 4August 2012; Revised:10 May 2013;Accepted: 10 May 2013
ABSTRACT
Mangrove plants are known as source of traditional medicines. Endophytic fungi have an
important economic potential for antibacterial, enzyme and secondary metabolites that are useful
especially in pharmaceutical industry. The purpose of this research was to determine the
antibacterial activityof theendophytic fungal extractsfrom mangroveplant Rhizophora mucronata
against diarrhea causing bacteria. This research included isolation, characterization, cultivation,
extraction,andantibacterialactivitytest.Thestudyyielded5typesofendophyticfungiwithdifferent
morphology. Endophytic fungus DS1 was the selected isolate based on antagonism test. The
growth pattern showed the stationary phase of isolate DS1 was on the 15 th day. Culture broth
extract showed considerable inhibition on Pseudomonas aeruginosa with diameter of inhibition
zonewas 18.5mm 3.32.Extract ofmyceliuminhibited thegrowth ofStaphylococcus aureus (2
mm). The culture broth extract showed more potent antibacterial activity in comparison with the
mycelium extract.
Keywords: antibacterial, diarrhea, endophytic fungi, mangrove
ABSTRAK
Tumbuhan mangrove merupakan sumber bahan obat tradisional. Kapang endofit memiliki
potensi ekonomi yang penting sebagai sumber antibakteri, enzim dan metabolit sekunder lain
yangbermanfaatkhususnyapadaindustrifarmasi.Tujuanpenelitianiniuntukmenentukanaktivitas
antibakteri ekstrak kapang endofit dari mangrove Rhizophora mucronata terhadap bakteri
penyebab diare.Tahapan penelitian yang dilakukan meliputi isolasi kapang endofit, karakterisasi,
kultivasi, ekstraksi, dan pengujian aktivitas antibakteri. Studi ini menghasilkan 5 isolat kapang
dengan morfologi berbeda. Kapang DS1 merupakan isolat terpilih dari hasil uji antagonisme.
Kurva pertumbuhan kapang menunjukkan bahwa kapang DS1 mencapai fase stasioner pada
hari ke-15.Ekstrak media kultur menunjukkandaya hambat paling besarterhadap Pseudomonas
aeruginosa yaitu 18.5 mm 3.32. Zona hambat ekstrak miselium paling besar terhadap
Staphylococcus aureus yaitu sebesar 2 mm. Ekstrakmediakulturlebih efektif sebagaiantibakteri
dibandingkan dengan ekstrak miselium.
Kata Kunci: antibakteri, diare, kapang endofit, mangrove
Permalink/DOI: http://dx.doi.org/10.15578/squalen.v8i2.88
1. Introduction
Mangrove is one of the rare and distinctive
ecosystemsintheworld,duetotheextentofonly
2% oftheearthssurface.Thisecosystem hasan
importantroleintheecological,socio-economic,and
socio-culturalaspects(Setyawan&Winarno,2006).
Mangroveis asourceoftraditionalmedicinethat
contains bioactive compounds with antimicrobial,
antifungal, antiviral, antitumor, insecticide and
antileukemiaactivities(Soetarno,2000).Oneofthe
plantspossessingthesecharacteristicsisRhizophora
mucronata.Traditionally,coastalcommunityusesthis
plantfortreatinghematuria(bleedingintheurine).In
Japan and China this mangrove is used as an
antidiarrheal(Rahmanetal.,2011).
Plantswithahistoryoftraditionalusesbylocal
peopleinJapanandChinaweresuggestedtohave
specificsecondarymetabolites,andexpectedtobe
a host for endophytic fungi. Several studies on
endophyticmicrobesshowedthatendophyticfungi
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Squalen Bulletin of Marine & Fisheries Postharvest & Biotechnology, 8 (2), 2013, 69-76
havesignificanteconomicpotentialespeciallyinthe
pharmaceuticalindustry(Kumala&Fitri2008).The
results of Melliawati and Wulandari study (2008)
showedthattheendophyticfungiofHalimunMountain
NationalParkcouldinhibitthegrowthofmicrobial
pathogensSalmonella typhiandCandida albicans,
andadditionallyaccordingtoMelliawati&Harni(2009),
thoseendophytic fungi couldinhibit the growth of
Escherichia coliandStaphylococcus aureus.
Diarrhealdiseaseremainsapublichealthproblem
indevelopingcountriessuchasIndonesia.In2010,
anextraordinaryeventsofdiarrhea(KejadianLuar
Biasa, KLB) has ocured in which the number of
patientswas4204,and73peopleofthemdied(KKRI,
2011). Diarrhea usual ly occurs due to poor
environmentalsanitation.In1995to2001,diarrhea
patientsfromseveralprovincesinIndonesiasuchas
Jakarta, Padang, Medan, Denpasar, Pontianak,
MakassarandBatamhassufferedfromShigella sp.,
Salmonella sp., Vibrio parahaemolyticus, Salmonella
typhi, Campylobacter jejuni, and Salmonella paratyphi,
butthemostcommoncausewasVibrio cholerae(Zein
etal.,2004).
BacteriasuchasShigella flexneri,Staphylococcus
aureus,Escherichia coliandSalmonella typhi arealso
reported causing diarrhea (Umer et al., 2013).
Pseudomonas aeruginosacausesinfectiousdiarrhea
whichusuallyrepresentsanosocomialinfectionin
an immunocompromised host (Porco & Visconte,
1995;Kimetal., 2001).Diarrheaistheleadingcause
ofdeathinchildrenunder5years(Fagundes-Neto&
Scaletsky, 2000). In dev eloping countries
enteropathogenicEscherichia coli (EPEC) isamajor
causeofinfantilediarrhea.EPECposesasignificant
healththreattochildrenworldwide(Vallance&Finlay,
2000).
Abilityofendophyticmicrobestoproducebioactive
compounds is a challenging opportunity in the
provisionofrawmaterialsfordrugs(Strobeletal.,
2004).Theuseofendophyticmicrobesasasource
of rawmaterials fordrugs isexpectedtobemore
efficienteconomically than usingmedicinalplants
(Sinaga et al., 2009). This study aimed to obtain
isolates of endophytic fungi from mangrove plant
Rhizophora mucronata,tocharacterizetheendophytic
fungal isolates, andto determinethe antibacterial
activityofthefungalextractsagainstdiarrhea-causing
bacteria.
2. Material and Methods
Fungalisolatesusedinthisstudywereisolated
fromleavesofRhizophora mucronatacollectedat
TamanWisataAlamAngke Kapuk,North Jakarta.
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Chemicalsandculturemediausedweresolventsand
microbiologicalculturemedia.Reagentsusedwere
forphytochemicaltest.
The research stages included preparation of
Rhizophora mucronata mangroveleaves,isolationof
endophyticfungiusingsurfacesterilizationmethod
andthenleavesinoculatedintoPotatoDextroseAgar
(PDA)mediumandincubatedatroomtemperature
(27-29C)for2-14days(Tarman,2011).Subsequently,
thefungiwereselectedthroughantagonismtestto
getapotentialisolateforfurtherantibacterialactivity
assay. Select ed f ungal isolates t hen were
characterizedmacroscopicallyandmicroscopically.
Growthofthefunguswasdeterminedbasedon
thedryweightofthefungalbiomass(Tarman,2011)
for18daysofcultivationinwhichitwassampledfor
each3days.DuringcultivationthepHofthemedium
wasmeasured.Cultivationforbioactiveproductionwas
conductedfor15days.Harvestingwasperformedby
separatingculturemediumandthemycelia.Then,
themyceliawereextractedusingmethanolandthe
culturemediumextractedwithethylacetate(Tarman,
2011).Bothmethanolandethylacetateextractswere
testedfortheirantibacterialactivityagainstdiarrhea
causingbacteria(enteropathogenic Escherichia coli
(EPEC), Escherichia coli, Staphylococcus aureus,
Pseudomonas aeruginosa andSalmonella typhi)with
extractconcentrationusedwere0.5mg,1mgand2
mg,chloramphenicolasapositivecontrolandrelated
solventasanegativecontrol.Diameterofinhibition
zonewasmeasuredusingaruler.Antibacterialactivity
testwasperformedafterMoorthyetal.(2007)using
agar well diffusion method. The potential of the
antibacterialactivitywasclassifiedafterDavis&Stout
(1971).Theyclassifiedantibacterialactivityintofour
categoriesbasedondiameteroftheinhibitionzone,
namely(1)weakiflowerthan5mm,(2)mediumranges
5-10mm,(3)strongif10-20mm,and(4)verystrong
if 20 mm or more. Phytochemical test of mycelia
extractswas conductedforalkaloids andphenolic
compoundsanalyses(Harborne,1987).
3. Result and Discussion
3.1. Fungal Endophytes from Rhizophora
mucronata
Plantsandmicroorganismsinteractcloselywith
oneanother,suchasmicrogranismslivinginsidethe
plants tissue (endophytic microbes). Endophytic
microbesi.e.,microbesthatgrowinthetissuesof
plantcanbefoundinalmostallplantsonearth(Strobel
etal.2004).Inthisstudy,5endophyticfungiwere
successfully isolated from leaves of Rhizophora
mucronata.Theisolatesweremorphologicallydifferent
ascanbeseeninTable1.
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Squalen Bulletin of Marine & Fisheries Postharvest & Biotechnology, 8 (2), 2013, 69-76
Table1.FungalisolatesfromRhizophora mucronata
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Squalen Bulletin of Marine & Fisheries Postharvest & Biotechnology, 8 (2), 2013, 69-76
DS1
DS1
DS3
DS2
DS2
DS3
(a)
DS1
DS2
DS2
DS3
DS1
DS3
(b)
Figure1.Antagonismtestofthefungalisolatesfor11days(a)topview,(b)bottomsideview.
conidia
conidiofor
(a)(b)
Figure2.DS1Isolate(a)11daysold,(b)hyphae(400x).
ofthefungusarebrowntoblackwithwavyedgesand
formconcentricconfiguration.Microscopically,the
fungushasseptatehyphae,2-septateconidia,and
in chain form. The isolate was further observed
morphologically and according to Gandjar et al.
(2000), it was proposed to belong to the genus
Alternaria.PreviousstudyonAlternariashowedthat
theextractsexhibitedhighactivityininhibitingthe
growth of pat hogenic mi croorganisms,
Staphylococcus aureusandCandida albicanswith
correspondinginhibitionzonewas27mmand16mm
(extractconcentrationof1mg/ml)(Fernandesetal.,
2009).
3.4. Fungal Growth
ThegrowthoffungusDS1wasdeterminedbythe
weightofdriedfungalbiomass.ThepHofthemedium
wasmeasuredduringfungalcultivation.Thegrowth
curveofthefungusispresentedinFigure3.ThepH
changesofthefermentationmediumarecausedby
themetabolicactivityofthefungi.Inthisstudythe
pHvalueofthefungusduringcultivationwas4(Figure
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3).Commonly,theoptimumpHofthefungalgrowth
rangesfrom4to7(Srikandaceetal.,2007).
Thefungalgrowthcurveshowedthatthelagphase
ofthefunguswasuntilday6.Theexponentialgrowth
phasewasonday6to9,ascanbeseenfromthe
considerableincreaseofthebiomass.Afterday9to
day15,thegrowthcurveshowedthatthefunguswas
instationaryphase.Generally,secondarymetabolites
areproducedduringthisphase(Srikandaceetal.,
2007).Inthisphasethefungalcellsaremoreresistant
to extreme conditions. Synthesis of secondary
metabolitesbeginswiththenutrientdepletionofthe
growthmedium.Thiscanleadtotheproductionof
substances which are the result of catabolism of
secondarymetabolites.Sinceday18thefunguswas
indeathphase.
3.5. Antibacterial Activity against Diarrhea-causing Bacteria
Thepreliminarystudyshowedthatthebioactive
compounds were highly produced on day 15.
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Squalen Bulletin of Marine & Fisheries Postharvest & Biotechnology, 8 (2), 2013, 69-76
driedbiomass
pHofculturemedia
Figure3.GrowthofDS1isolatefor18days.
Additionally,theextractsexhibitedhighestactivity;
therefore the fungus was cultured for 15 days.
Antidiarrhealpotentialoftheextractswasdetermined
byantibacterialactivitytestagainstdiarrhea-causing
bacteria.
3.6. Antibacterial Activity of Culture Broth Extracts
Ethylacetateextractofculturebrothobtainedwas
29.38%(w/v).Subsequently,theextractsweretested
againstthetestbacteria.Resultoftheantibacterial
activitytestsofculturebrothextractsispresentedin
Figure4.
Theresultsshowedthattheextractoftheculture
brothcouldinhibitthegrowthofalltestbacteriafrom
aconcentrationof0.5mg.Theethylacetateextracts
were considerably inhibited the growth of
Pseudomonas aeruginosa andEscherichia coli,while
Salmonella typhiiwasthemostresistantbacterium
to the extracts tested. From this result, it can be
roughlyconcludedthattheethylacetateculture-broth
extractsweremoreeffectivetoinhibitthegrowthof
Gram-negativebacteria.Thisresultwassimilartothe
studyofMaria et al. (2005)onendophyticfungiof
mangrove. Extracts of the fungal culture broth
exhibited highest activity against Pseudomonas
control +,
2 mg,
1 mg,
0.5mg)
Figure4.Antibacterialactivityofculturebrothextracts.
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Squalen Bulletin of Marine & Fisheries Postharvest & Biotechnology, 8 (2), 2013, 69-76
(control+,2mg,1mg)
Figure5.Antibacterialactivityofmycelialextracts.
aeruginosa,ontheotherhandSalmonella typhiiwas
themostresistant.
3.7. Antibacterial Activity of Mycelia Extracts
Yieldofthemycelialextractswas26.34%(w/w).
Intracellularmetaboliteswhicharecontainedinthe
mycelia,namelypolysaccharides,proteins,lipidsand
nucleicacidsarethemainconstituentoftheprimary
metabolites. Mycelial extracts were also tested
against the test bacteria. Figure 5 shows the
antibacterialactivityofmycelialextracts.
Antibacterialtestresultsindicatedthatthemycelia
extractshowedthehighestinhibitionactivityagainst
Staphylococcus aureus,whilethelowestactivitywas
against Pseudomonas aeruginosa. Antibacterial
activityofthemycelialextractswasnoticeablylower
incomparisonwiththeactivityofculturebrothextracts.
Theresultsshowedthatthediameterofinhibition
zoneindicatingantibacterialactivityoftheextracts
wasdiverse.Thisis duetothesensitivity ofeach
bacteriumtoextractstestedwasalsovaried.Study
of Dali et al. (2011)reported that each bacterium
possessesdifferentbiologicalcapabilitiesinresponse
toantibacterialagents.Oneofthemostinfluential
factorstotheantibacterialactivityisthestructural
differencesbetweenthebacterialcellwallofGramnegativeandGram-positivebacteria.
Somefactorsaffecttheantibacterialactivityofthe
compounds,suchastheconcentrationorintensityof
the anti microbial agents, t he number of
microorganismstested,speciesofmicroorganisms,
temperature,thepresentoforganicmatter,acidityand
alkalinity(pH)(Pelczar&Chan,2008).
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Theresultsshowedthattheextractsoftheculture
mediumexhibitedmuchhigherantibacterialactivity
comparedtothemycelialextracts.Thisissimilarto
theresultsofotherstudiesonbiologicalactivitiesof
mycelialandculturebrothextractsofthefungi(Jung
etal.,2002;Nursidetal.,2010;Tarmanetal.,2011).
Kusumaetal.(2011)reportedthatthemethanol
extractofthemangroveR. mucronataleavescould
inhibit the growth of B. subtilis, S. aureus, and P.
aeruginosa with an inhibition zone diameter
correspondinglywas18mm,16mmand14mm.By
comparingKusumasstudywiththisexperiment,it
couldberoughlysumupthattheextractsofthefungal
endophytesofR. mucronata exhibitedhigheractivity
thanthoseoftheleaves.Strobeletal.(2004)also
reportedthattheextractsofendophyticfungifromR.
mucronatashowedhigheractivitythanthoseofthe
hostplant.
3.8. Bioactive Components
Bioactive components of the extracts can be
determinedusingphytochemicaltest.Phytochemical
testisaqualitativetestusedtodeterminethecontent
of secondary metabolites present in the sample
(Copriyadietal.,2005).Inthisstudy,phytochemical
testwasperformedtoidentifythepresentofalkaloids
and phenol hydroquinone in mycelia extracts.
Lumyong et al., (2004) stated that the fungal
endophyteproducesalkaloidsandmycotoxinsthat
maybeusedtoimproveplantresistancetodisease.
Phenoliccompoundisoneofthecompoundsthatcan
inhibitthemetabolismofcells.Phytochemicaltest
results showed that the alkaloids and phenolic
hydroquinonecompoundswerenotdetectedinthe
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Squalen Bulletin of Marine & Fisheries Postharvest & Biotechnology, 8 (2), 2013, 69-76
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Squalen Bulletin of Marine & Fisheries Postharvest & Biotechnology, 8 (2), 2013, 69-76
Setyawan,A.D.&Winarno,K.(2006).Directutilizationof
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