TheEffectsofFruitBasedProteolyticEnzymesontheSetupTimeofGelatin

ByOksanaPopadiuk,EliShotola,andCesarRamos
ResearchProposal

Abstract
Inthisexperiment,westudiedtheeffectsofproteolyticenzymesonthestructural
integrityofgelatin.Totestthisenzymeaction,weinvestigatedaproteincalled
Bromelain,whichisfoundincertainfruits.Bromelainisatypeofproteolyticacid,which
wediscoveredhasanadverseaffectonthesettingofJellO.Wedecidedtoconstructan
experimenttotestthesolidityofJellOwhenmixedwithfruitsthatcontainBromelain
withthehypothesisthatthefruitwithhighestlevelsofthisproteolyticenzymeswould
havethemosteffectongelatins(JellOs)structuralintegrity(solidity).Thisparticular
experimentwasinterestingtousbecauseofitsdirectconnectiontoeverydaylife.JellO
isacommonlyprepareddishforparties,holidays,andotherspecialoccasions.Ifour
hypothesisiscorrect,perhapsthisexperimentwillbeacautionarytaleforthose
experimentingwithdifferentfruitsintheirJellOdesserts.
Wetestedthesoliditybycalculatinghowmuchweightittooktopuncturethe
surfaceofeachsample.Wepredictedthatitwouldtakelessweighttobreakthesurface
oftheJellOwithfruitcontaininghigherlevelsofbromelain,whileitwouldtakemore
weighttobreakthesurfaceofthejellOthatcontainedfruitwithlowerlevelsof
bromelain.Ourdataleadustoconcludethatourhypothesiswasnotonlycorrect,butthe
dixiecupscontainingthefruitswiththehighestlevelsofproteolyticaciddidnotsolidify

atall.WhilethefruitcontaininglowerlevelsofproteolyticacidallowedtheJellOtoset,
butwithstructuralintegritythatwascompromised.Ournumericaldataleadusto
concludethatwecouldrejectthenullhypothesis.

Introduction
Pineapplecanmakeyourmouthhurt,specificallyyourtongue,andinsomecases
evenbleed.Thisisbecausepineapplehasanenzymecalledbromelain.Bromelainiswhat
isknownasaproteolyticenzyme.Proteolyticenzymesbreakdownproteinmolecules
intoaminoacidsandsomearefoundnaturallyoccurringinthehumandigestivesystem.
Whatthismeansisthattheseenzymesactuallydigestyourtongue.Severalcommon
fruitshavenaturallyoccurringproteolyticenzymessuchasthekiwi,whichcontains
actinidinandthepapayathatcontainspapain.Whentheseenzymesarecombinedwith
collagen,theybegintoeatawayatthecollagen,breakingitdown.JellOcontains
collagen.Soforourexperiment,wearegoingtoinvestigatewhichofthesefruits(along
withpineapple)containsthehighestlevelsproteolyticenzymesandtodoso,wewillput
theminJellO.WewouldliketoseewhichfruitscausetheJellOtotakethelongestto
set,andiftherearefruits,orafruit,thatcontainlevelsofproteolyticenzymesthatdonot
allowtheJellOtosetatall.Wearegoingtomeasurethedensitybyweighingand
comparingeachJellOsamplewiththedifferentfruitsinthemtocollectourdata.Each
samplewillsetforthesameamountoftime:2andhalfhours.JellOisalsofragile
becauseitismadeofgelatin,awatersolubleproteinproducedbythehydrolysisof
animalcollagen.
TheJournalofAgriculturalandChemistry
providesthisdefinitionof

gelatinaswellasadescriptionofitsuniquefragility.Asthearticleillustrates,gelatins
poormechanicalstructureisexhibitedevenwhenexposedtothingslikewaterand
humidity.ThisiswhythesolidificationofJellOissosensitivetowhatyouadd.Inour
experiment,wewillbeabletoobservethefragilityofgelatinsmechanicalstructureina
veryrealandobvioussense.
Theeffectoftheseproteolyticenzymesarewellknownanddocumented.Inone
particularstudy,
Proteolyticdegradationofcoldwaterfishgelatinsolutionsandgels
,
researchersstatedintheirfindingsthatIfFG,oranytypeofgelatin,istobeusedasthe
gelformingmatrixinthepresenceofproteases,itmaybewisetoinactivatetheenzymes
priortoencapsulation(Solgaard,2008,p.196).Whatisbeingsaidhereisthatthis
particularsubsetofenzymesimpedestheabilityofthegelatintoformanditwouldbea
goodideatoinactivateordisablethoseenzymesifonewantedtoincludetheminthe
gelatin.Althoughthisexperimentdealtwithfishgelatin,theyextendtheirfindingstoany
typeofgelatin.
InastudydoneattheUniversityofZaragozacalledLimitedproteolysisof
myofibrillarproteinbybromelaindecreasestoughnessofcoarsedrysausage,headedby
J.A.Meleno,theresearchersaddedcommercialproteasebromelaintocoarselychopped
sausage.Theresearchersdividedtheirchorizomixintofourdifferentbatchesand
bromelainwasaddedtothreeofthematdifferentconcentrations(6,60,or600Uper
100g).Whattheyconcludedwasthatthebromelain,whenmixedwithdry,refrigerated
sausage,madethemeatsignificantlymoretender.Theyconcludedthatthistenderizing
effectwasobservablewithintheinitial48hourperiod.Alsointhisstudy,theymentioned

acharacteristicofbromelainthatiswhatourresearchprojectisbasedon.Inadditionto
itshighactivityonmyofibrillarproteins,bromelainpossessesanoticeablecollagenolytic
activity(Melendo,1995,p.429).Ourresearchisontheeffectofenzymessuchas,
bromelain,oncollagencontainsubstances(JellO).Therefore,wehavereasontobelieve
thatbromelainwillinfacthaveaneffectonthesettlingtimeofJellO
InanAustralianstudy:
TheEffectofaKiwiFruitBasedSolutiononMea
t,
researchersdemonstratedthattheenzymesinkiwihaveanoticeabletenderizingeffecton
notonlyporkasdemonstratedinthestudyabove,butinbeefaswellstating
Itcanbe
concludedthatinjectionofakiwifruitbasedsolutionwillconfertendernessbenefitsbut
Despiteimprovementsinmeattendernessgainedbythekiwifruitbasedsolutionthe
increaseddiscolourationwouldlimittheuseofthetreatedproductbytheretailsector
(Toohey,2008,p.470).Withthisresearchweknowthatkiwihasthepropertiesto
degradetheconnectivetissueinbeef,whichiswhyitwillbeincludedinthistest,tosee
howwellitworksinaJellOsolution.
AnotherdriveforourresearchprojectwasastudydonebyReighin1976,which
showedthatenzymes,specificallyproteases,showedthattheywerecapableofcleaving
thepeptidelinkagesofproteins(Reigh,1976,p.386).Itcontinuedtostatethatgelatin
wasaproteinpreparedfromcollagenandthatitwasreadilyhydrolyzedbyproteolytic
enzymessuchasbromelain(Reigh,1976,p.386).Thisshowshowthebromelainwill
provetoelongate,ifnotcompletelystop,thesettlingofJellO.thisexperimentconcluded
thatheatdestroysbromelainactivity.Furthermore,theexperimentconcludedthat
freezingtemperatureshadtheoppositeeffectonbromelainactivity.Toputitsimply,we

canconcludethatfrozenpineappleisunsuitablefordesertswhereascanned(notfresh)
pineapplewillgiveyoubetterresults.Thisdirectlyconnectstoourexperimentsincewe
willbeworkingwithfreshpineapple,whichcontainshighlevelsofbromelain.
Weconsiderourexperimenttobecloselyrelatedtothepreviouslymentioned
experimentsbecausewewillbestudyingthesame(orverysimilar)effectsofproteolytic
acidsinslightlymoremodifiedandspecializedconditions.Weconsiderourexperiment
tobealittlemorerelatable,since,asmentionedbefore,jellOdessertsarecommonly
madeineverydaylife.

Methods
Wefoundthatkiwi,pineappleandpapayawerethefruitsthatcontainedthe
highestlevelsofproteolyticacid.ThesearethefruitsweaddedtoJellOinorderto
observetheeffectsofproteolyticacidongelatin.Inordertoachieveeffectiveresults,we
willbeusedfreshfruit,notcanned,becausewhenthesefruitsarefresh,theycontaintheir
highestamountofproteolyticacid.Wecuteachfruitintosmallsquaresandpreparingthe
jellOintodixiecups.Wepreparedfourrowsof20dixiecups:onerowforthejellO
containingkiwi,onerowcontainingpapaya,onerowcontainingpineappleandonerow
forourcontrolgroup,containingnofruit.Thedixiecupswerefilledhalfway,withsix
piecesoffruitmixedintoeachone(apartfromthecontrolgroup,whichdidnotcontain
fruit).OncewemadeourjellOmixtures,weletthemsetatarefrigeratedtemperaturefor
3hourseach.After3hours,weremovedoursamplesfromtherefrigeratorandtestedthe
solidityofeachdixiecup.Totestthesolidity,weusedastrawastheinstrumentwhich

willpuncturethesurfaceofthejellO.Wetapedadixiecuptothetopofthestraw,which
actedasaflatsurfaceforustoaddweight.Oncethiscontraptionwassetonthesurface
ofthejellO,weaddedgrainsofriceuntilthestrawpuncturesthesurface,atwhichpoint
weusedascaletomeasurehowmanygramsofriceittook.Thiswashowwedetermined
theproteolyticstrengthoftheenzymeineachfruitandthereforehaveevidenceto
supportwhichisstrongestandweakest.Sincethecontraptionwassittingonthesurface
ofthejellObeforeweaddedrice,theweightofthestrawanddixiecupwastakeninto
accountandaddedtotheweightoftherice.Theweightofthecontraptionplustheweight
inricewastheweightrecorded.
Forthisresearchprojectourexplanatoryvariablewastheproteolyticenzymein
eachfruit(bromelain)andourresponsevariablewastheweightmeasuredthatisrequired
topiercetheouterportionofthegelatinsample.Becauseofthis,ourstudyisan
experimentaloneinthatweexposedthesameamountofgelatintodifferentproteolytic
enzymes.
Byexecutingthisexperiment,determinedwhichfruithasastrongerproteolytic
effectandthereforewhichfruitslowsdownthesettlingprocessofgelatinthemost.

Data and Results

Duetothenatureofourexperimentonlytwosetsofsamplesyieldedusabledata.
Asshowninthetables,thetwosampleswhichwecoulddrawcomparabledatafrom
werethatofourcontrolgroupandthegroupcontainingpapaya.Thisisbecausethe
enzymesinfreshpineappleandfreshkiwicompromisedtheintegrityoftheJellOso

muchthatwewereunabletotestthemastheywereentirelyliquid.Therefore,boththe
pineappleandthekiwirenderedtheJellOuntestable.
Belowisatablewhichrepresentsthedatacollectedfromthecontrolgroupand
thegroupcontainingpapaya.Asyoucansee,thetwocolumnsofdataaresignificantly
andconsistentlydifferent.Theinformationshownclearlyleadsustoconcludethatthe
groupofjellOmixedwithpapayadidnotsetasthoroughlyasthecontrolgroup,which
didnotcontainanyfruitswithproteolyticacids.Thenumbersinthecolumnsrepresent
theweightofriceingramsthatittooktopenatratethesurfaceofthejellO.

Trials
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

Control
Papaya
(L1)
(L2)
13.7
8
12.4
6.2
7.2
3.5
15.4
7.5
12.9
4.5
13
6.8
13
8.2
12.2
9.1
12.8
4.8
8.2
5.1
7.8
3.6
11.6
7.8
12.2
5.6
7
3.8
10.9
6.7
7.5
8.3
13.4
4.1
14.2
3.7
9.6
5.5
12.7
2.9

Next,werantheconfidenceintervalsonthedataavailabletousandtheyareas
follows:

95%CI

L1
10.2912.4
8

L2
4.976.6

Thisshowswith95%certaintythatthemeansdistinctlydiffer,thusconfirming
thatthejellOcontainingpapayadidnotsetassolidlyasthecontrolgroup.
Basedonthesefindings,wewereonlyabletodoasinglehypothesistest
comparingthesetsagainstoneanother.

H0

H1

L1=L2

L1>L2

ZScore

PValue

8.02 <.001

Theresultsshownarehighlystatisticallysignificantinshowingthatthedatasets
differgreatlyandthatthecontrolismuchstrongeronaveragethantheJellOwith
Papayainit,nottomentiontheJellOwithPineappleandKiwiinit.

Discussion
TBA

REFERENCES

Agric,J.(2010).ChemicalModificationofGelatinbyaNaturalPhenolicCrosslinger,
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Melendo,J.A.Beltrn,I.Jaime,R.Sancho,P.Roncals,Limitedproteolysis
ofmyofibrillarproteinsbybromelaindecreasestoughnessofcoarsedrysausage,
FoodChemistry,Volume57,Issue3,November1996,Pages429433

Reigh,D.L.,(1976).Bromolain.Experimentsillustratingproteolyticenzymeaction.
JournalofChemicalEducation53(6),386.DOI:10.1021/ed053p386

Solgaard,G.IngvildJ.Haug,KurtI.Draget,Proteolyticdegradationofcoldwaterfish
gelatinsolutionsandgels,InternationalJournalofBiologicalMacromolecules,
Volume43,Issue2,15August2008,Pages192197
(http://www.sciencedirect.com/science/article/pii/S0141813008000913)

Toohey,E.,Kerr,M.,Ven,R.,&Hopkins,D.(2011).Theeffectofakiwifruitbased
solutiononmeattraitsinbeefm.semimembranosus(topside).
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