3. 3
Josie Ochsner 11LONGAN 11BIO, 2012
1.0 Abstract:
In thisexperimentthe effectsof temperature onPectinase throughthe productionof apple juice
was investigated.Thiswasdone byplacingbeakers,containingPectinase andgratedapple,in
differenttemperaturesthensievingthemafter20minutestoreveal the amountof juice extracted
fromthe apple.A control wasset upfor eachtemperature,abeakercontainingonlygratedapple,to
reveal the difference Pectinase hadonthe productionof juice. The average amountsof juice
producedpertemperature were 2.5mLin25°C, 6mL in 37.5°C, 8.5mL in 50°C, 16mL in62.5°C and
15mL in75°C. With thisknowledge,applejuice canbe producedmore efficientlyonalarge scale in
industry.Witha catalystsuchas temperature,juice canbe producedquickerwithoutincreasingthe
cost.
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Josie Ochsner 11LONGAN 11BIO, 2012
2.0 Introduction:
If the optimumconditionsforextractingjuice fromapples are undertaken,industriesaroundthe
worldcan produce millionsof tonesof apple juice inthe mosteffectivewaysrelatedtotime,cost
and labour.
Pectinaseis an enzyme thatbreaksdown Pectins,whichare a polysaccharide foundinplantcell
wallsandabundantlyinfruits (Miller,1992). Polysaccharides,suchas Pectins, are usedinplantsfor
structural support,heldinCellulose (Spenceley,2004). Pectins holdparticlesinsuspensioninfruit
juicesandincrease viscosityof the juice (Miller,1992). Whenthe substrate (Pectin) comesinto
contact withthe enzyme (Pectinase) the cellsburstbecause the Pectinasedigeststhe Pectins,(which
are amongthe Cellulose and withinthe layersof the cell walls),causingthe cell toopenupand
reduce the structural support (Barber,2011). Pectinaseiscommonlyusedtoextractjuice from
applesbecause itcauses the juice tobe releasedfromthe damagedcells (Barber,2011). If the
Pectins getdigested,the particlesare nolongerheldinsuspensionandthe viscosityof the juice has
beenlessened sothe juice runsout (Williamson,2008).
Enzymaticoxidationalsocausesdamage toapplescells,therefore releasingjuice (Chang,2005).
Whenan apple iscut, enzymesare released,whichreactwithoxygeninthe airto formbrown
pigmentscalled Melanins (Australianinstitue of foodscience andtechnologyincorporated,2009).
Thiscausesthe applescellstosoftenandoccurs mostefficiently atwarmtemperatures of around
20°C or so (Chang,2005).
As concentrationincreases,more particles are containedperunitof area (Smith,2005). Asparticles
are alwaysmoving,thiscausesmore collisionstooccur,therefore afasterrate of reaction (Smith,
2005). Whena substance’stemperature increases,itcausesthe particlestomove around faster,also
causingmore collisionanda fasterrate of reaction (Spenceley,2004).Differentenzymes operate in
differenttemperature rangesandthe optimumtemperature forPectinaseisaround50°C (Gupta,
2007). In a chemical reaction, the substrate needstocollide withthe enzyme forthe reactionto
begin(Spenceley,2004).If the temperature istoolow (below approximately20°Cfor Pectinase)
(Gonzalez,2010), the particles won’thave enoughenergytocollide aneffective amountforthe
reactionto be catalysed.
If the temperature reachesover75°C,the Pectinasewill denature andbecome inactive (Gonzalez,
2010). Enzymesare all specificbecause theiractive site hasto‘fit’withthe substrate whenthey
come intocontact (Spenceley,2004).Whenan enzyme denatures,itsstructure changes, inhibiting
itsabilitytobreakdown the substrate (Spenceley,2004).Therefore,the reaction will be made
impossible if the enzyme’sshapeischanged (Spenceley,2004).
It isexpected thatwhenapple isheatedto50°C the mostjuice will be released,followedby 62.5°C,
75°C, 37.5°C then25°C. At50°C (the optimumtemperature) Pectinasebreaks downPectins and
releases juice mostreadily. The apple in62.5°Cispredictednextbecause the higherthe
temperature causesmore collisionbetweenthe substrateandenzyme,catalysingthe extractionof
juice.75°C is where the Pectinasebeginstodenature soit’spredictednext (Gonzalez,2010).Next
shouldbe 37.5°C then25°C as the temperaturesare lower,causinglesscollisionbetweenparticles
and slowerextractionof juice.
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Josie Ochsner 11LONGAN 11BIO, 2012
3.0 Aim:To investigatethe effect of temperature onPectinase throughthe extractionof juice
fromapple.
6. 6
Josie Ochsner 11LONGAN 11BIO, 2012
4.0 Method:
4.1 Materials:
3 waterbaths
Hot plate
20 beakers
750mL Pectinase
Peeler
Grater
Apples(1kgafterpeelingandgrating)
Scales
Sieve
Funnel
20 measuringcylinders
6 stopwatches
Pestle
4.2 Procedure:
Three waterbathswere preheatedtotemperatures37.5,50 and 62.5 degreesCelsiusandahot
plate to 75°C. The roomwas setto 25°C for an experimenttotake place ona bench.Appleswere
peeledandgrateduntil 1kilogramwascollectedand1 beakerat a time wasplacedon the scalesand
50 grams of apple wasaddedtoeach. Fourbeakerswere placedineachtemperature anda
stopwatchstartedto time the controls for20 minutes.The otherthree beakersineachtemperature
were leftforone minute topreheat.50mL of Pectinase wasaddedtothe otherthree beakersonthe
hot plate anda stopwatchwas startedimmediately.Thiswasrepeatedwiththe other12beakers,
usinga separate stopwatchforeach temperature.A sievewasplacedoverafunnel,whichwason
top of a measuringcylinder.The beakerswere removedandsievedintoseparate measuring
cylindersassoonas theirstopwatchreached20 minutes.A pestle wasusedtolightlysqueezethe
apple sothe extractedjuice wouldflow throughthe funnel.The apple fromthe three trialsineach
temperature were placedintoone beakerandweighed.Toreveal the amountof juice extractedfor
each trial,50mL was subtractedfromthe liquid(the initial volume of Pectinase) andthe results
recorded.
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Josie Ochsner 11LONGAN 11BIO, 2012
5.0 RiskAssessment:
Substance Risk Control Measure
Water baths Burns Do not touchor put
away whenstill hot:
use warningsign
Do not tipover
Hot plate Burns Do not touch or put
away whenstill hot:
use warningsign
Keepoutof reach of
youngchildren
Glasswear Cuts/damage to skin
and clothing
Handle carefully
If broken,cleanup
immediatelyand
efficiently
Pectinase Eyes: May cause
irritation.
Skin: May cause
irritationtoskin.
Ingestion: Maycause
gastrointestinal
discomfort.
Inhalation: Maycause
irritationtorespiratory
tract.
EmergencyOverview:
Do not splashor create
dustor aerosolsof the
product
Eye Protection: Splash
proof chemical safety
gogglesshouldbe
worn.
OtherProtective
ClothingorEquipment:
Lab coat, apron,eye
wash,safetyshower
Eyes- In case of contact
witheyes,rinse
immediatelywith
plentyof waterand
seekmedical advice.
Skin- Aftercontact
withskin,wash
immediatelywith
plentyof water.
Ingestion - If
swallowed,rinse mouth
withwater(onlyif the
personisconscious).If
swallowed,donot
induce vomiting:seek
medical advice
immediatelyandshow
thiscontaineror label.
Inhalation - Incase of
accidentbyinhalation:
remove casualtyto
freshairand keepat
9. 9
Josie Ochsner 11LONGAN 11BIO, 2012
0
2
4
6
8
10
12
14
16
18
20
25°C 37.5°C 50°C 62.5°C 75°C
Trial 1
Trial 2
Trial 3
6.0 Results:
6.1 Figure 1: Juice Production
Temperature
(°C)
Trial 1 Trial 2 Trial 3 Average Control Final mass
of apple
(g)
25 1mL 4mL 12mL* 2.5mL 9mL 127.7
37.5 9mL 2mL 7mL 6mL 16mL 120.4
50 8mL 9mL 19mL* 8.5mL 7mL 108.5
62.5 17mL 15mL 16mL 16mL 13mL 86.0
75 15mL 18mL 12mL 15mL 16mL 95.0
6.2 Figure 2: Juice Production in Each Trial
Juice
produced
(mL)
Temperature
* Thistrial was an anomalysothe resultwasexcludedwhencalculatingthe average
10. 10
Josie Ochsner 11LONGAN 11BIO, 2012
0
2
4
6
8
10
12
14
16
18
25°C 37.5°C 50°C 62.5°C 75°C
Average volume of juice
produced
Control
6.3 Figure 3: Average Volume of Juice Vs. Control
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Josie Ochsner 11LONGAN 11BIO, 2012
7.0 Discussion:
The beakersin temperature 25°Cgave 1mL, 4mL and12mL of juice,havinganaverage of 2.5mL as
the thirdtrial was an anomalysoit was excludedfromthe calculationsandthe control gave 9mL. In
37.5°C there was9mL, 2mL and 7mL of juice produced;havinganaverage of 6mL andthe control
gave 16mL. In 50°C the trialsproduced8mL, 9mL and 19mL of juice,givinganaverage of 8.5mL as
the thirdtrial was an anomalysoit was excludedfromthe calculations andthe control gave 7mL of
juice.In62.5°C the trialsgave 17mL, 15mL and 16mL, givinganaverage of 16mL and the control
produced 13mL. The trialsin75°C gave 15mL, 18mL and 12mL of juice,havinganaverage of 15mL
and the control produced16mL.
As seeninfigure 1,the more juice producedthe lighterthe final apple weightisbecause the cells
have excretedthe juice mass. Asseeninfigure 2,the trialsall have similarvolumesof juice foreach
temperature,exceptforthe thirdtrialsin25°C and 50°C as theywere anomalies. Basically,the
productionof juice increasesasthe temperature does, whichpeaksat62.5°C and dropsslightlyat
75°C. Asdemonstratedinfigure 3,the controlshave similarvolumesof juice asthe average of the
trialsin50°C, 62.5°C and 75°C because oxidationand Pectinasewere damagingthe apple cells,
therefore releasingjuice, atbasicallythe same rate.Oxidationoccursreadilyinwarmtemperatures
so the applescells in25°C and 32.5°C softened,causingjuice tobe released (Chang,2005).These
temperaturesweren’tclose enoughto Pectinase’soptimumtemperature (50°C) so the average of
those trialsdidn’tproduce asmuch juice as the controlsusing oxidation.
Grating wasan effective methodof cuttingasitallowedthe apple tobe cut intosmall piecessothe
Pectinase(andoxygeninthe controls) couldreachmore of the apple cells,therefore releasingmore
juice. The Pectinase(whichwasrefrigerated) wasnotpreheatedbefore beingaddedtothe apple so
the reactionstartedat a temperature lowerthanrequired.Thisallowedforthe resultstobe
recordedincorrectlysonexttime the Pectinaseshouldbe preheatedandaddedonlywhenthe
desiredtemperature isreached. The thirdtrial in50°C, producing19mL of juice,wasananomaly
causedwhenthe Pectinase was spilledwhen beingaddedtothe beaker.The contentswere quickly
pouredintoa sieve todrainoff the Pectinase anda new 50mL lotof Pectinase wasadded.Notall of
the Pectinase wassievedoff asitwasabsorbed, sothere was a higherconcentration inthatbeaker
than inthe othertwo, allowingthe breakdownof Pectins more quicklyandtherefore more juice
released (Smith,2005).This resultwastherefore discardedwhencalculatingthe average because
had an advantage overthe othertwotrials. Nexttime the Pectinase shouldbe addedwithmore care
to ensure itisnot spilled sothe methodiscarriedoutequally.The thirdtrial in25°C wasan anomaly
because the Pectinasewasleftwiththe apple forlongerthan intended,allowingitmore time to
breakdown Pectins andrelease more juice sonexttime the trialsshouldall be drainedatthe same
time. Whendrainingthe juice apestle wasusedtoslightlysqueezethe extractedjuice throughthe
sieve butif differentpressure wasapplieditcould’ve causedjuice tobe extractedthroughforce,
changingthe results.Insteadaweightshouldbe placedontopof the apple sothe same amountof
pressure isappliedforeachtrial. Infuture investigations there shouldbe more controlssoan
average can be calculated,thereforedecreasingthe riskof aninaccurate result.
The hypothesiswaspartiallycorrectasitwas expectedthatthe temperaturesinorderfrommostto
leastjuice givenonaverage wouldbe 50°C,62.5°C, 75°C, 37.5°C then25°C andthe actual orderwas
62.5°C, 75°C, 50°C, 37.5°C then25°C. The trialsin 50°C producedlessjuice thanexpectedasthe
12. 12
Josie Ochsner 11LONGAN 11BIO, 2012
temperature inside the beakerswaslowerthanintendedsothe particles weren’tmovingasfast,
causinglesscollisiontotake place.Thismeansthe substrate andthe enzyme didn’tmake asmuch
contact as intended,causingaslowerrate of reactionforthe Pectinaseto breakdownthe Pectins.
The other temperatureswereinthe correctorder.The apple in62.5°C excretedthe mostjuice asits
actual temperature waslowerthan62.5°C (thereforeclosertothe critical temperature of 50°C than
normal) sothe Pectinase wasmost activelydigestingthe Pectins, causingthe juice tobe released
mostreadily.The Pectinasein75°C slightlydenatured,causing the appletoexcrete lessjuice thanin
62.5°C. The apple in37.5°C came nextthen,lastly,was 25°C because asthe temperatures getlower,
there is lesscollisionbetweenthe enzyme andsubstrate andtherefore the breakdownof Pectins
was catalysedless,producinglessjuice.
Thisinvestigationcouldbe takenfurtherbytesting the optimumpHandconcentration foryielding
the most amountof juice ina settime.
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Josie Ochsner 11LONGAN 11BIO, 2012
8.0 Conclusion:
The effectof temperature onPectinase throughthe productionof apple juice wasinvestigated.The
orderof temperaturesfromleasttomostamountof juice producedwas25°C, 32.5°C, 50°C, 75°C
then62.5°C. It waspredictedthatthe orderwouldbe 25°C, 32.5°C, 75°C, 62.5°C then50°C but due
to the inaccurate carry outof the procedure the temperature waslowerso the trialsin50°C didnot
yieldthe highestvolume of juice. If the optimumconditionsforextractingjuice fromapples are
undertaken,industriesaroundthe worldcanproduce millionsof tonesof apple juice inthe most
effectivewaysrelatedtotime,costand labour.
14. 14
Josie Ochsner 11LONGAN 11BIO, 2012
9.0 Bibliography:
Australianinstitueof foodscience andtechnologyincorporated.(2009).EnzymaticBrowning of
Apples.RetrievedMarch13, 2012, fromCurriculumSupport:
http://www.curriculumsupport.education.nsw.gov.au/secondary/science/assets/aifst/Experiments/
apple_browning.pdf
Thissource is an online experimental writeupbythe AustralianInstitue of FoodScienceand
TechnologyIncorporated.The source is publishedby anAustraliangovernmentcurriculumsupport
website.Thisdocumentwaswrittenin2009 andthe informationretrieved(on13th
March, 2012)
has beenfoundinothersourcesaswell.Thissource hasnoevidence of biasasit ispurelymade
available foreducationbythe Australiangovernment.
Barber,B. (2011, June 6). Investigating TheEffectof Temperatureon The EnzymePectinase.
RetrievedFebruary23,2012, from Probiotic Superfood:
http://www.probioticsuperfood.com/investigating-the-effect-of-temperature-on-the-enzyme-
pectinase-when-used-to-digest-pectin-in-apple-pulp/
Chang,S. K. (2005). Apple Browning.RetrievedMarch13, 2012, fromResources:
http://resources.edb.gov.hk/~science/hkcho/1s/LSC.pdf
Gonzalez,S.L. (2010, February14). Determination of pectin methylesteraseactivity in commercial
pectinases.RetrievedFebruary23,2012, fromSciELO: http://www.scielo.br/scielo.php?pid=S0101-
20612011000200020&script=sci_arttext#nta
SamanthaGonzalezhasreceivedamaster'sdegree inFoodScience andTechnology.She isa
researcherinthe chemistrydepartmentatthe State Universityof Ponta,Brazil.Thissource isa
researcharticle ona website from 2007. The informationtakenfromthissite (on23rd
February,
2012) wasalsofoundon othersitesanda publishedbiologytextbook.There isnobiasapparentas
there are no productsbeingsoldonthe site andthisarticle ispart of the author’soccupationas a
researcher.
Gupta, M. N. (2007, April 10). A multipurposeimmobilized biocatalystwith pectinase.Retrieved
February23, 2012, from ChemistryCentral:
http://journal.chemistrycentral.com/content/1/1/16#B17
Miller,S.(1992). Simple EnzymeExperiments.RetrievedFebruary22,2012, fromableweb.org:
http://www.ableweb.org/volumes/vol-6/10-miller.pdf
SharonMillerhas receivedaB.A.inbiology,anM.S.in plantpathologyanda Ph.D. in
botany/mycology.She wasalaboratorycoordinatorforbiologyatClemsonUniversityfor2years
and taughtmycologyandbiology for15 yearsat college/highschool level.She thenbecame a
professoratuniversityandapart-time biologyinstructor.The source wasan experimentwriteupon
a website (accessed22nd
February,2012). It was writtenin1992 and althoughnotrecent,the
informationtakenfromthe source wasalsofoundonothersitesthatare recentand reliable.There
isno evidence of biasasthe experiment waswrittenforeducational purposesandnottosell
products.
15. 15
Josie Ochsner 11LONGAN 11BIO, 2012
Smith,D.(2005). Chemistry in Use. Cengage LearningAustralia.
DebSmithis Headof Departmentinalarge school in Brisbane andhashad much experience asa
teacher.She isa best-sellingauthorandhelpeddevelopthe new seniorChemistryprogramasthe
convenerof the QSA sub-committee.MarkGouldisalsoa memberof thiscommittee andisthe
Headof Department.He hasworkedforMcGraw Hill Australiaonmanychemistrypublications.Sue
Moneathis a chemistryteacherandalsohelpeddevelopthe seniorchemistryprogram.Roland
Smith’stextshave beenusedinschool educationfortwodecades.Forover25 years he has beena
science teacher,CSIROscientistanda researcherandlectureratuniversity.Thistextbookwas
publishedin2005 but isstill beingusedtoteachinschool (accessedon24th
February,2012). This
textbookisatrusteddocumentforschool educationandtherefore doesnotpresentbiasattitudes.
Spenceley,M.(2004). Biology A ContextualApproach. PortMelbourne:MalcolmParsons.
Maggie SpenceleyisanexperiencedauthorforHeinemannpublishing.She wonanawardas an
author inthe AustralianAwardsforExcellence inEducational Publishingin2007. This textbookwas
published in2004 but isstill beingusedtoteachinschool (accessedon25th
February,2012). This
textbookdoesn’tpresentbiasasitisa trusteddocumentforschool education.
Williamson,K.H.(2008, April 2). The Effectof EnzymeConcentration on AppleJuiceProduction.
RetrievedFebruary23,2012, from USC: http://www.usc.edu/CSSF/History/2008/Projects/J0416.pdf
