fnhibition ot Bacillussubtilis and Escherichiacoli by
AntimicrobialStarch-BasedFilm incorporatedwith LauricAcid
and Chitosan
Eraricarsallehl*, lda ldayu Muhamadi and NozieanaKhairuddinr
lFacultyof ChemicalEngineering
Teknologi
and NaturalResourcesEngineering,
Universiti
8'1310
Skudai,
Johor,Malaysia.
Malaysia,
*Corresponding
Fax:+607-5581463
author.Phone:+607-5535543,
Email:eraricar@fkkksa.utm.my
Written for presentation at the
2007 CIGR Section Vl International Symposium on
FOODAND AGRICULTURALPRODUCTS:PROCESSINGAND INNOVATIONS
Naples,ltaly
24-26 Seotember 2007
Abstract. Food safetyls one of key issues of public health. One optionis to usepackagingto
provldean inqeased margin of safetyand quality.Active packagingtechnologiesare being
developedas a rcsuftof these cliving forces.Active packagingis an innovativeconceptthat can be
definedas a mode of packagingin which the package,the productand the environmentinteractto
prctong shelflife or enhancesafety ot sensorypropefties,whilemaintainingthe qualv ofthe product
(Suppakul,Miltz,Sonneveld& Bigget, 2003).Antimicrobial(AM)packagingis one ofthe most
prcmisingactivepackaging systems.Statch-basedfilm is consideredan economicalmateial for
antimicrobialpackaging.
Thisstudyaimedat the developmentof food packagingbased on wheatstarch incorporctedwith
lauic acid and chitosanas antimicrobialagents. The purposeis to restrainor inhibitthe grov,/thof
spoitageand/orpathogenicmicroorganismsthat are contaminatingfoods. The antimicrobialeffect
was fesfedon E. substilisand E. coli. lnhibitionof bacteial gro\uthwas examinedusing two
methods,i.e. zone of inhibitiontest on solid media and liquid culture test (opticaldensity
measurementsIThe controland AM films (incorporatedwith chitosanand lauric acid) were
producedby castingmethod.From the obsevations,onlyAM filmsexhibitedinhihitoryzones
Interestingly,a wide clear zone on solidmedia was obseNedfor B. substilisgrov'th inhibition
whereasinhibitionfor E. coli was only revealedundemeaththe film discs.From the liquid cufture
test,the AM filmsclearly demonstrateda more effectiveinhibitionagainstB. substilisthan E coli.
packaging,Lauricacid,Chitosan,B. subtilis,E. coli.
Kelrwords.Antimicrobial
Vl International
symposiumon
@ilon
PROCESSING
ANDINNOVATIONS
PRODUCTS:
FOODANDAGRICULTURAL
Naples,ltaly, 24-26Se mber2007
lntroduction
Antimicrobialpackaging(AM) is gaining interestfrom researchersand industrydue to its
potentialto providequalityand safety benefits.Interestin antimicrobialpackagingfilms has
increasedin recent years due to a concem over the risk of foodborneillness,desire for
extendedfood shelflife,and advancesin the technologyof film production.Slowingthe growth
of spoilagebacteriawill reducethe lossesof productto spoilageand extendshelflife.Reduction
of pathogengroMh will reducethe riskof foodborneillnesscausedby thoseproducts(Dawson,
of varioussolid
Carl,Acton& Han,2002).lt can effectivelycontrolthe microbialcontaminatlon
groMh
microorganisms
on
the
surface
of the food,
the
of
and semisolidfoodstuffsby inhibiting
packaging
Antimicrobial
functionof
material.
whichnormallycomesinto directcontactwith the
activesubsiancesinto the packaging
the packagingsystemcan be achievedby incorporating
systemby variousways (Han, 2003). The antimicrobialpackagingis conductedby (1) the
additionof antimicrobialcontainingsachets or pads into food packages;(2) the coating'
into food packagingmaterialsor (3) the
of antimicrobials
or direct incorporation
immobilization
(Appendini& Hotchkiss'2002).
antimicrobial
useof packagingmaterialsthat are inherently
Nowadays.about 150 milliontons of plasticare producedannuallyall over the world,and the
production
continueto increase(Parra,Tadini,Ponce& Lugao,2004).Mostof
and consumption
theseDlasticsare crudeoil based.In addition,handlingof plasticwasteassociatedwith serious
pollutionproblemdue to waste disposaland undegradedpolymers.Therefore,
environmental
the use of agriculturalbiopolymersthat are easily biodegradablenot only would solvethese
problems,bui wouldalso providea potentialnew usefor surplusfarm production(Okada,2002;
Pavlath & Robertson,1999; Scott, 2000). Because of the environmentalconcernsand
technologicalproblemssuch as denaturingeffectsof thermalpolymerprocessingmethods,
filmsis
intobiodegradable
of biopreservatives
extrusionand injectionmolding,the incorporation
moresuitablethan incorporationinto plasticfilms (Appendiniand Hotchkiss,2OO2;Han,2000i
al.,2003).
Suppakulet
films are edibleand their film formationoccursunder mild conditions
Most of biodegradable
Different edibie films incorporatedwith biopreservativesincludes cellulose derivatives,
carrageenan,alginate,proteinfilms includecasein,collagen,corn zein' gelatin'soy protein,
whey proteinsa;d wheat gluten (Padgett,Han & Dawson,'1998;Cha, Choi, Chinnan& Pa|k
et al.'2003).
& Vicini,2002;Suppakul
2002:Han,2OO0;
Quintavalla
ln the food packagingsector, biodegradablepolymersbased on naturalpolysacaccharides'
particularly
starchhas gain more attentionowingto its availabilityas agriculturalsurplusraw
material,abundant, can be produced at low cost and at large scale' nonallergicand
Several studies are concentratedon the developmentof starch-based
thermoorocessable.
reasons.
materialsfor the above-mentioned
Starch based materials reduce nonrenewableresources use and environmentalimpact
associatedwith increasingemlssionsas CO2and other products Starchesare polymerthat
naturallyoccursin a varietyof botanicalsourcessuch as wheat,sago,corn,yam, potatoesand
taoioca.Starchescan interactwith many additivesor componentsof the food (Fame,Rojas'
2004).Howeverstarchpresentssome majordrawbackssuch as the
Goyanes& Gerschenson,
stronghydrophilicbehaviour(poormoisturebarrier)and poorermechanicalpropertiesthan the
plasticfilmsusedin the foodpackagingindustries
conv;ntionalnon-biodegradable
In the lastyearsmuchresearchhas beendoneconcerningthe use of biodegradable
filmsas a
in food products.Severalresearchers
wayof supportingantimicrobials
havepreviouslyreported
compounds.Nisinand lauricacid are two
on coatingfood contactsurfaceswith antimicrobial
shown to be effectivein food applications.Dawsonet al. (2002),
food-gradeantimicrobials
incorporated
lauricacid and nisinsinglyand togetherintothermallycompactedsoy films.Nisin
and lauricacidfilmswere equallyeffec{ivein reducingL. monocytogenes
in 1% peptonewater
of nisinand lauricacid in corn zein cast films
after48h exposure. However,the combination
in peptonewaterthanwheneach
wasfoundto be moreeffectivein rcducingL. monocylogenes
(Hoffman,
The
Han& Dawson,2001). advantagein havinga film materialcarying a
usedsingly
biocideis that continuedinhibitioncan occurduringstorageor distribution
of the foodproduct.
A widespreadtrend worldwideis the movementtowards"natural"food products.In effortto
meetthis demand,therehas beenincreasedinterestin the food industryin usingantimicrobial
Dreservatives
that are Derceivedas more "natural".Future work will focus on the use of
biologicallyactive derived antimicrobialcompoundsbound to biopolymers.Howevermany
have a limitedspectrumof activityand are effectiveonly at very high
naturalantimicrobials
withwidespectrumactivityand lowtoxicitywill
The needfor new antimicrobials
concentrations.
possible
(Sofos,Beuchat,
solution
may
be
using
combinations
of antimicrobials
increase.A
on developmentof new antimicrobial,
Davidson& Johnson,1998).lnsteadof concentrating
it
agentsthat alreadybeingresearched.
couldbe morepracticalto combinethe antimicrobial
Lauricacid,a mediumlength-longchainfattyacidis foundin the formof glyceridesin a number
oil. lt offersadvantages
in food processing
as it acts
of naturalfats,coconutoil and palm-kernel
stavingoff oxidationand spoilage.Lauricacid has been shownto
as a kind of preservative,
effectagainstgrampositivebacteriaand yeasts(Beuchat& Golden1989;
havean antimicrobial
and may
Kabara1993).Beuchat& Golden(1989)suggestedthatfattyacidswere bacteriostatic
potential
inhibitors
in
foods
using
a
systematic
approach
with
other
antimicrobials.
be
microbial
killsL. p/arfarumcellswhereas
Basedon Padgett,Han& Dawson(2000),nisininstantaneously
of lipid compounds
lauricinhibitsmore slowly but steadyinhibitoryeffect.The incorporation
such as fatty acid to a starchfilm decreasesthe moisturetransferdue to their hydrophobic
properties(Coma,Sebti,Pardon,Deschamps& Pichavant,2001).Fattyacids,such as lauric
acid werefoundto be effectivein limitingwatervaportransferthroughediblefilm (Gennadios,
Weller& Testin,1993;Greener& Fennema1989 a, b; Kamper& Fennema1984;Kester&
Fennema
1989).
spectrumwouldbe necessaryand desirablefor
A packagingmaterialwith a wide antimicrobial
universaluse to improvethe storage stabilityof variety of foods. For this purpose,the
incorporation
of anotherantimicrobialagent into the packagingmaterialswould be useful.
packagingfllmswas basedon the
Besides,the choiceof chitosanin preparingthe antimlcrobial
& Lucia(2005),reported
fact that it has goodfilm formingproperties.Ban,Song,Argyropoulos
of starch-based
filmstrength.
thatchitosancan alsoplayan importantrolein the enhancement
of 91, 4 linkagesand a deacetylated
form of chitin,appearsas a
Chitosan,a polysaccharide
candidate
for
the
incorporation
because
it
can
inhibitthe groMh of a wide
natufalantimicrobial
(Rhoades
& Rastall, 2000;No, Park,Lee & Meyers,2003;
varietyof fungi,yeastsand bacteria
& Roller,2002).
Tsai,Su,Chen& Pan,2002;Sagoo,Board
of lauricacid and chitosanas
The objectiveof the researchwas to determinethe effectiveness
(8.
into starch-based
film againsttest stralnof Gram-positive
antimicrobial
agentincorporated
bacteria(E cor).
subsfl/,s)
and Gram-negative
2.
MATERIALS
AND METHODS
2. 1
Materials
Wheatstarchand aceticacid (glacial100%)that usedto dissolvechitosanwas purchasedfrom
fi/ersk(Malaysia).
Mediummolecularweightchitosanwasfrom Sigma-Aldrich
(Malaysia).Lauric
acidwas 99% pure purchasedfrom FlukaChemika(Malaysia)and glycerolas a plastisizerwas
boughtfromHmbc chemicals(Malaysia).
2-2
Film preparation
A starchbasedfilm was formedusingcastingprocessfollowingpreviouswork by Fameet al.
(2004).A controlfilm,withoutlauricacidor chitosanwasformedusingmixturesof starch(5.09),
glycerol(2.59)and water(92.59).
Chitosanwas djspersedin 400m1of distilledwater to which 20 ml of glacialaceticacid was
addedto dissolvethe chitosan.The solutionof starchand chitosanwith differentmixinoratios
(Ww)lwereprepared
5:5,4:6,3:7,2:8,1:9starch/chitosan
by addingg'tycerol
lgt1,8:2,7:3,614,
(halfamountof the starch)and 8% lauricacid(wasaddedbasedon the percentageof starch(g
fattyacid per g starch).The solutionwas mixedby gentlestirringwith a magneticstir bar until
starchdissolved.The solutionwas then homogenized
for about 15 min with additionof slow
heating.Stirringand heatingwere endedwhenthe solutionreachestemperature
of 80-86oC.
The 10 ml of the film formingsolutionwas pipetteand spreadevenlyinto a petri dish bottom
(100x15 mm)and allowedto air-dryat roomtemperature
overnight.
After casting,5 measurementswere made on each sample using an electronicmicrometer
(modelMitutoyo)and the meanthicknesswas calculatedto the nearest0.002mm.
2.3 Testing Antimicrobial
Effectiveness of AM Starch-Based Film
2.3.1 Agar Diffusion Method (Zone lnhibition Assay)
Antimicrobial
activitytest was carriedout usingagar diffusjonmethod.lndicatorcultureswere
Bacillussubtilisand Escheicha cor, representing
Gram-positive
and Gram-negative
bacteria.
Onehundredmicrolitersof the inoculumsolutionwas addedto 5 ml of the appropriate
softagar,
whichwasoverlaidonto hardagar plates.
Eachfilm was cut into squares(1cmx 1cm)and was placedon the bacteriallawns.Duplicate
agar plateswere preparedfor each type of film and controlfilm.Theplateswere incubatedfor
48 h ai 37 "C in the appropriateincubationchamber(aerobicchamberior Ecor). The plates
were visuallyexaminedfor zones of inhibitionaroundthe film disc, and the size of the zone
diameterwas measued at tlvo cross sectionalpoints and the averagewas taken as the
inhibition
zone.This methodwas slightlymodifiedlrom Padgettet al. (1998).
2.3.2 Liquid Culture Test (Optical Density Measurements)
For the liquidculturetest (Chung,Papadakis& Yam, 2002),each film was cut into squares
(1cmx '1cm).Threesamplesquareswereimmersedin 20 ml nutrientbroth(Merck,Germany)in
a 25 ml universalbottle.The mediumwas inoculatedwith200p1of Escheichacolil B. subtilisin
phase,and thentransferred
its lateexponential
to an orbitalshakerand rotatedat 37oCat 2OO
(0, 2, 4, 8, 12, 24 houts\duringthe incubationto
r.p.m.The culturewas sampledperiodically
obtainmicrobialgrowthprofiles.The sameprocedurewas repeatedfor the controlstarch-based
film. The opticaldensity(O.D. 600) was measuredat A = 600nmusing a spectrophotometer
(ModelUV-160,Shimadzu,Japan).
3. RESULTSAI{D DISCUSSION
3.1.Antimicrobial Starch-BasedFilm Formation
film incorporated
In generala translucentstarch-based
with lauricacidandchitosanpresented
goodflexibilitythan purelystarch-based
filmwasformulatedandformedas canbe seenin fig.
1. Filmthicknessrangedfrom0.03to 0.04mm,withan average0.03461 0.002mm.
film incorporated
Figure1: A translucentstarch-based
withlauricacidand chitosan
3.2. Antimicrobial
Effectiveness of AM Starch-Based Film
3.2.1.lnhibition of E, coli and B, subtilis on Agar Plate Test
The detailsof antimicrobial
effectiveness
of starch-based
film incorporated
with chitosanand
lauricacidare shownin fig. 2- 5 andtable 1. The inhibitoryactivitywas measuredbasedon the
averagediameterof the clear inhibitionzone. lf there was no clear zone surroundingas
revealedin fig. 2, it was assumedthat therewas no inhibitoryeffect,and was assignedas Nl
witha valueof 0.00.
After24 hoursincubationat 37oCfor starchfilm only (S), therewas no inhibitionoccurredfor
both 8. subtirs and E. coli. Bacteriacoloniesalso occurredat the top of film sample.On the
contrary,chitosanRlm only (C), showeda better inhibitionthan starchfilm only for both B.
srbt//bthan E. coli inhibition.Howeverthe effectwas not as good as combinationof chitosan
and_lauric
acidas shownin fig. 2.
ab
Figure2: Comparisonof inhibitionareaof (a) controlfilmand (b)AM incorporated
film
Starchand chiiosandifferentmixingratio (S:C)8:2, revealedthe best inhibitionon B. subt//s
whichis Gram-positivebacteriacomparedto otherS:C ratios.In contrast,S:C ratio9:.1showed
a verygoodinhibitionon E coli (Gram-negative
bacteria).Fromfig. 3, the resultsindicatedthat
S:C ratio8:2 is the bestformulationto inhibitboth B. subt/ls and E coli effectivelvfollowedbv
S:C ratio9:'1.S:C ratiofrom 1i9-3:7obviouslymore effectivetowardsinhibitiono-fE coli tha;
S:C4:6-7:3.
-"{})'ili:.{:":"-":.'#;r:,"1""'
*-;.c/
o"
o"
e!
?,Y kv ete ae 4J 2l
Fig.3: lnhibitionof A. subtlisand E coliOn Agar PlatesFigureBasedOn AverageZone
DiameterExpressedAs An Area (cm)of lnhibitionZone
of B. subtlisand E coliOn Agar PlatesBasedOn AverageZoneDiameter
Table1: Inhibition
ExpressedAs An Area(cm)of InhibitionZone
Film
B. subtilis
E. coli
Remarks
Starchonly(S)
NI
NI
Nl =No
inhibitory
effect (all
afea
on
plates and
film covered
by bacteria)
Chitosanonly(C)
1.7
2.O
S:Cratio1:9
2.423
2.425
S:C ratio2:8
2.55
2.8
S:Cratio3:7
2.444
2.95
SiC ratio4:6
2.5
1.7
S:Cratio5:5
2.631
1.7
SrCratio6i4
2.7
1.7
S:Cratlo7:3
2.825
1.7
S:Cratio8:2
3.125
3.00
S:Cratio9:1
3.075
3.05
3.2.2 Liquid Culture Test (O-D6ss,^Measurement)
S:C ratio 8:2 is the most effectiveformulationto inhibitL subf,Jisas can be seen in fig. 4.
N4eanwhile,
S:C ratio 9:1 is the best formulationto inhibitE.coli (ng.5t. Althoughtherewere
with
for
inhibition both B. subfilisand E.coli,the antimicrobialstarch-basedfilm incorporated
Gram-positive
bacteria
than
the
Grameffective
against
were
more
lauricacid and chitosan
negativebacteriastudied.
Lauric acid alone only has antimicrobialeffect against Gram-positivebacteriaand yeasts
of lipidcomPounds
suchas fattyacid
(Beuchat& Golden1989;Kabara1993).The incorporation
to a starchfilm decreasesthe moisturetransferdue to their hydrophobicproperties(Comaet
propertiesof the
al.,2001).Thiswillbe observedfor the futureworkon physicaland mechanical
of chitosan,besidesinhibitE cot and
fllm. As well as incorporation
starch-based
antimicrobial
starch-based
it helpsto enhancethe antimicrobial
increasethe film effecton B.subli/isinhibition,
(Ban
et.a|.,2005).
filmstrength
In fact, one of the reasonsfor the antimicrobialcharacterof chitosanit's positivelycharged
aminogroupwhichinteractswith negativelychargedmicrobialcell membranes,leadingto the
(Shahidi,
constituentsof the microorganisms
and other intracellular
leakageof proteinaceous
bacteria,the majorconstituentof its cell wall is
Arachchi& Jeon, 1999).In the Gram-positive
protein.
peptidoglycan
is
very
little
The
cellwallof Gram-negative
andthere
bacteriaalso hasan
outer membrane,which consfitutesthe outer surfaceof the wall (Zheng& Zhu, 2003).Study
for Gramfrom (Jiang,Bi, Wang, Xu & Jiang,1997),observedthat from electronmicrographs
positiveand Gram-negative
bacteriain the presenceof chitosanshow the cell membraneof
bacteriawas weakenedor even broken,whilethe cytoplasmaof Gram-negative
Gram-positive
bacteriawas concentratedand the intersticeof the cell were cleady enlarged.This study
activityof chitosanwere differentbetween
indicatedthat the mechanismsof the antimicrobial
cram-positiveand Gram-negativebacteria.Additlonally,the antimicrobialmechanismof
becausethereare positiveschargeson
chitosanmightdifferfrom that of otherpolysaccharides
the surfaceof chitosan(Jianget al., 1997).
1 .2
o.2
Fig.4: Inhibition
of Controls(starchonlyand chitosanonly)and Starch(S):Chitosan(C)on B.
subtrlisin LiouidCultureTest
0.2
0
+a:zE
of Controls(starchonlyand chitosanonly)and Starch(S):Chitosan(C)on E.
Fig.5: Inhibition
collin LiquidCultureTest
Conclusions
Incorporating
chitosanand lauricacid into starchbasedfilm showedobviouseffectstowards
antimicrobial
effectwhen
and E coliindicatedthatthe film hadsynergistic
inhibitionof B. subliTis
starch-based
film
demonstrates
more
chitosanand lauricacidwere combined.The antimicrobial
abilityagainstB. subfrs thanE cor'.The solutionof starchand chitosan
effectiveantimicrobial
with differentmixing ratio (w/w) 8:2 and 9:1 were the most effectivemixingratio which had
greaterinhibitionon both B. subtirsand E coli than otherssolutionas revealedin agar plate
test and liquid culture test. Thus, the antimicrobialstarch-basedfilm may have potential
for bothfluid and semisolidfoods by inhibitingbacterialgrowthand extendedthe
applications
shelf-lifeand improvethe foodsafety.
Acknowkedgements
(MOSTI)Malaysia,
the
The authorswouldliketo thankthe Ministryof Scienceand Innovation
Teknologi
(MOHE)
Research
Management
Centre,
Universiti
and
Education
Ministryof Higher
Malaysiafor financialsupportof this project.
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