BromelainandCapsaicinWrittenbyKelseyBergandSaraCarey

WorkcompletedunderDr.BrucePattersonforuseinMCB181LMCB492,Spring2012

Researchwascompletedtolookintotheinhibitionofbromelainviacapsaicin.Sincebromelainismeasuredinmilkclottingunits(MCU)andgelatindissolvingunits(GDU),thisinhibitionwasstudiedthroughmilkclottingandjellogelatinhardening.Twoassaysweredevelopedinwhichpineappleisusedasthesourceofbromelain,andchiliesasthesourceofcapsaicin.

BROMELAIN

KEYPOINTS:- Optimaltemperature:50–60oC- Deactivationtemperature:>65oC- OptimalpH:4.5-5.5- Measuredin:

o MCU(milkclottingunits)o GDU(gelatindissolvingunits)

where1GDU=1.5MCU(“Bromelain”)- Potentproducts(suchaspineapple)thatcontainbromelainhaveapprox.2,000

MCU/- Canbuyitinpillsof3,000GDU(“Find”)- Highestconcentrationofbromelaininstemofpineapple(“Bromelain”)- Destroysproteinsthroughlysisofpeptidebonds(Wong)

o Tendstobreakpeptidebondsthatfollowtheaminoacidslysine,alanine,tyrosineandglycine-therefore,thedegreetowhichaproteinmaybebrokendownbybromelaindependsontheamountsoftheseaminoacidsintheprotein(“Bromelain”)

OTHERUSES:- Meattenderizer- Anti-in`lammatory(reducessymptomsofarthritis)- Helpsdigestiveproblems- Mayreduceilleffectsofsometypesofchemotherapy- Anticoagulantbybreakingdown`ibrin- Thinsmucus(maybene`itasthmaticstoreduceeffectsofchronicbronchitis)- Maytriggerchangesinwhitebloodcells(immunesystemimprovement)- Speedwoundhealing- Enhanceseffectofantibiotics(“Bromelain”)

OTHERPROTEASESFOUNDINFRUITSLIKEBROMELAIN:

*Pineappleistheonlysourceoftheabovelistedproteasethatisbothavailableandharvestedyear-round.(“Dole”,“CUESA”)

Protease FruitSource Availability PeakSeasonality

Bromelain Pineapple Allyear Allyear

Papain Papaya Allyear June-Sept

Actinidin Kiwi Sept-Mar Sept-Dec

Ficin Fig June-Nov June-Nov

(Namenotfound) Guava Nov-April Nov–April

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CAPSAICIN

KEYPOINTS:- Activecomponentinchilies(“Capsaicin”)- Colorlessandodorless- Hydrophobic(Erowid)- Measuredinscovilleheatunits(SHU)- Strongirritant,especiallytomucusmembranes- Mosthighlyconcentratedinthewhitepithofachili,whichislocatedintheinner

wall(“Capsaicin”)- Itisknownthatsomechemicalinchilidestroysproteolyticenzymesandthis

chemicalispresumedtobecapsaicin(Schwarcz).

OTHERUSES:- Relievespainassociatedwithshingles- Reliefofminorpainassociatedwitharthritis- Reliefofminorpainassociatedwithmusclepain- Reducesin`lammationanditchingassociatedwithpsoriasis- Decreasesinsulinreleaseinhumans,leadingtoreducedbloodsugar

o Affectsoninsulinarespeciesspeci`ic- Inhibitsgrowthofsometypesofcancercellsthroughpromotionofapoptosis- Aidsincoagulation(Eustice)

INTERESTINGFACTS:- Productionofcapsaicinisamechanismusedbychiliesthatprotectsitsseedsfrom

mammals,whicheat,crush,andruintheseedsbecauseoftheirtoughmolars- Birdsarenotaffectedbycapsaicin–thismakessensebecausebirdscaneatthe

seedswithoutruiningthem,`lylargedistances,andpoop.Thisdispersestheseedsandpromotesthegrowthofmorechilies(“Capsaicin”).

PRECAUTIONS:- Gloves

o Anybitofcontactcanirritatetheskinandcontactwithanymucusmembranessuchasintheeye,nose,andmouthwillcauseintenseburning

- Facemasko Preventsinhalationofanypowdercontainingcapsaicin

SOURCESOFCAPSAICINUSED:- DriedwholeHabaneroChiliPeppers

o Hadtocrushusingmortarandpestleo Approx.100,000-350,000SHU

- HotNewMexicoChiliPeppero Providedalreadyground

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FigureA:molecularstructureofcapsaicin(Erowid)

o Approx.3,500–8,000SHU(“ScovilleScale”)

MILK

Milkiscomposedofbutterfatglobulesinawater-basedsolution(“Milk”).Tounderstandthisstructure,itisbestto`irstlookatthebasicstructureofamicelle,asshowninFigureB.Micellesaretypicallymadeofphospholipids,whichhavepolarheadsandnonpolar,orgreasy,tails.Whenputintoanaqueoussolution,thepolarheadshavepartialchargesthatinteractwiththepartialchargesofwater.Thispushesthegreasytailstogether,formingabubble-likestructurewherethegreasypartsareshieldedfromthepartiallychargedaqueoussolutionontheoutside.Inmilk,micellesarecomprisedofbutterfattriglycerides(FigureC),phospholipids,andproteinsknownascaseins(“Milk”).

Therearefourtypesofcaseinsincludingalpha-s1,alpha-s2,beta,andkappa,whichwillbereferredtoask-caseinfromhereonout.Theimportantthingtoknowhereisthatthe`irstthreecaseinsarehydrophobic,andthereforereadilyprecipitateinaqueoussolutionsuchasthatofmilk.K-casein,however,issolubleinmilk(Bowen).Asaresult,hydrophobiccaseinsarewrappedink-casein,“shielding”themfromtheaqueoussurroundings.Thisstabilizesthemicelleandkeepsitsolubleinaqueoussolution.Theexactconformationofthecaseinproteinsinthesemicellesisunknown,butallproposedmodelsshowthemicellesthatareformedwithcaseinaggregatescoveredink-casein.TwooftheproposedmodelscanbeviewedbelowinFiguresDandE(Goff).

Nowthatthestructureofmilkisestablished,wecangointohowitclots.Rennetisthegeneralnameforanyenzymaticpreparationthatactstoclotmilk.Inmammals,rennetisproducedinthestomachcomprisedmainlyofpepsin,lipase,andchymosin,whichisalsoknownasrennin(“Rennet”).Chymosincleavesk-casein.Sincek-caseiniswhatkeepsthemicellestableinaqueoussolution,destructionofthisproteinleadstodestabilizationofthemicelle.Greasycaseinproteinsarenowabletoprecipitate,resultinginclottingofthemilk(Bowen).

Chymosinisanimportantenzymeformammalsbecauseofitsabilitytoclotmilkinthestomach.Clottinginthestomachmakesitpossibleformilktostickaroundlongerduetoitsincreasedthickness.Itmakessensethatthiswouldbebene`icialformammalsbecausethelongermilksticksaround,thelongerthebodyhasachancetoabsorbasmanynutrientsaspossible.Thisisespeciallytrueinthestomachsofyoungmammalswhosedietiscomprisedentirelyoftheirmother’smilk.Asaresult,chymosinispresentinthehighestquantitiesinveryyoungmammals.Consequently,traditionalcheese-makingmethodsinvolvetheuseofchymosinobtainedbytheslaughterofyoungcalves(“K-casein”).Forobviousreasons,thismethoddoesnotgooverwellwithmanyreligiousandactivistgroups.Inadditiontothisopposition,itisdif`iculttoobtainenoughcalfstomachstomakeallcheesethisway.Thisleadsustothediscoveryofotherproteasesthatcanbeusedasrennetincluding,butnotlimitedto,enzymesfoundin`ig,ivy,soybeans,andcertainmolds,bacteria,fungi,andyeasts.

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FigureB:Basicstructureofa“typical”micelle(“Rennet”)

Throughourownresearchonjellodissolvingandthebromelaininpineapple,itwasdiscoveredthatbromelainisnotonlymeasuredingelatindissolvingunitsbutalsoinmilkclottingunits.Thisledustothestudyoftheclottingofmilkviabromelainasrennet.Wehavebeenledtobelievethattheproteasebromelainactsjustliketheseotherproteasesbycuttingk-caseinandinterferingwithitsabilitytostabilizethemicelle.

Althoughallrennetshavethesameactivityofcuttingk-casein,eachmaydosoinitsownway.Inotherwords,differentproteasesmaycutthek-caseinatdifferentspots,butallresultinthecoagulationofmilk(“K-casein”).Thesedifferencesmayleadtodifferencesinclottingtimeswherethebreakdownofk-caseinistherate-limitingstep.Timesmayalsovarydependingonthetypeofmilk,pH,andtemperature(“K-casein”).Becauseoftheseothervariables,itisdif`iculttodeterminereproducibleclottingtimesforbromelain.Thismakesithardforustodevelopanassayinwhichclottingtimesareconsistentwitheverytrial.Asasidenote,methodshavebeenusedtodeterminetherateofclottingthroughk-caseindegradationtracking,asawaytoquantifymilkclottingbywayofrennet.Onecommonwaythishasbeendoneisthroughlabelingk-caseininmicelleswitha`luorochromecompoundknownas`luoresceinisothiocyanate,orFITC.Thisallowsresearcherstodetectclottingthroughproteaseactivityalone.Italsoallowsresearcherstoseeiflowlevelsofclottingareoccurringthatmaynotbevisibletothenakedeye(“K-casein”).

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FigureC:butterfattriglyceride(“Milk”)

FigureD:Proposedstructureofcaseinconformationinmicellesinwhichmicelleisformedbysubmicellesthatarecoveredink-casein(Goff)

FigureE:Proposedstructureofcaseinconformationinmicellesinwhichcaseinsareinterlinked(Goff)

JELLOGELATIN

Gelatinisderivedfromthecollageninanimalbonesandskin(“Gelatin”).CollagenhasahelicalstructuresimilartothatofDNA,withweakhydrogenbondsholdingtogetheraminoacidchains.However,collagen`ibersarecomprisedofatriplehelix,incontrasttothedoublehelixseeninDNA(“Collagen”).Placingjellogelatinpowderinhotwaterdissolvesthepowder,andweakhydrogenbondsholdingtogethertheproteinchainsarebroken.Additionofcoldwatertothedissolvedgelatin,followedbyfurthercoolingcausesthebrokenchainstocomebacktogether,butthistimewaterisincorporatedintothestructure(“Gelatin”).Thisgives`inishedgelatinitswiggly-jigglyform.

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FigureF:Collagentriplehelix(“Collagen”)

FigureG:Materialsusedingelatinproduction(“Gelatin”)

- Mortarandpestle- Cheesecloth- Micropipette- Pipettips

MilkAssayMATERIALS:

- Wholemilk- Freshlycutpineapple- Five20mLbeakers- GroundHotNewMexicoChiliPowder- Hotplate

PROPOSEDMETHOD(studentswilldevelopthisthemselves):(1) Obtainfour20mLbeakersandlabelasnecessary(2) Grindpineappleusingmortarandpestle(3) Placecheeseclothoverextrabeakerandpourmashedpineappleintopitmadewith

theclothto`ilter.Squeezetheclothtogetoutanyextrajuice.(4) Filleachbeakerwiththematerialsasseenbelow.Usemicropipettetomeasurethe

smallamountofjuice.

(5) Immediatelyplacebeakersonhotplatesettolowestsetting(6)Watchbeakersandrecordanyobservationsmadeincludingtimeofclotting

BEAKERPUPOSES:(1) Control–Doesmilkclotonitsownwithheat?(2) Control–Doesgroundchilicausemilktoclotwithheat?(3) Experiment–Doesbromelaininpineapplecausemilktoclotwithheat?(4) Experiment–Doescapsaiciningroundchilipepperinhibittheeffectsofbromelain

inpineappleontheclottingofmilk?

TYPICALRESULTS:

Beaker# Contents Observations

1 20mLmilk Didnotclot

2 20mLmilk2.0mLgroundchilipepper Didnotclot

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1

20mLmilk

2

20mLmilk2.0mLchilipepper

4

20mLmilk2.0mLchilipepper0.5mLjuice

3

20mLmilk0.5mLJuice

SOMETECHNICALASPECTSABOUTTHEMILKASSAY

POSITIVETHINGSTOKNOW:- AddinggroundHotNewMexicoChiliPepperdirectlytomilkshowedslowedeffects

ofbromelain.- AddingmoregroundHotNewMexicoChiliPepperdirectlytomilkresultsinslower

clottingtimes.- Addingsmalleramountsofbromelainandlargeramountsofcapsaicineachtrialhas

leadusclosertodevelopinganeffectivenumberofeachtousesothattheexperimentisconsistentlyreproducible.

“NEGATIVE”THINGSTOKNOW:- Crushingdriedwholehabaneropeppersandaddingdirectlytomilkdidnotshow

anyinhibitionofbromelain.- Capsaicinshouldnotbeextractedintooilforuseinthisassaybecauseoilandmilk

donotmix.- Capsaicinextractioninalcoholdidnotshowasmuchinhibitionofbromelainasdid

addingchilipepperdirectlytothemilk,andwassetasideasamethod.

OTHERNOTES:- pHofmilkis6.4–6.8(“Milk”).NotethatthisisnottheoptimalpHofbromelain.- 1grennetcancoagulate2–4Lmilk(“Rennet”).

NEXTSTEPS:Themilkassayendedwitha`inalexperimentwhereclearinhibitionofbromelainbycapsaicinwasobserved,andspeci`icdetailswerequanti`iedsothattheassaycouldberepeated(Table8).Therewas,however,someclottingseeninthecontrolbeakerswhennobromelainwaspresent.ItisbelievedthatthisclottingcouldbeattributedbothtoprolongedheataswellasapossibleunknownsubstanceinthechilipepperthatcouldalterthepHofthemilk.Nextstepsshouldincludecompletingtheexperimentwherenohotplateisused(keptatroomtemperature),andabufferisaddedtoeachbeakertoeliminatepHasaconfoundvariable.Furtherresearchshouldbecompletedtodetermineifitisindeed

3 20mLmilk0.5mLpineapplejuice

Thick,heavyclottingafterapprox.4–6min.

420mLmilk0.5mLpineapplejuice2.0mLgroundchilipepper

Smallerclottingafter17–22min.

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capsaicinthataffectsbromelain,orifitissomeotherchemicalinchilies.Fromtheliteraturewefoundsofar,ithasonlybeenpresumedthatcapsaicinisthechemical(Schwarcz).

TABLESFROMOURMILKCLOTTINGSTUDIES

TABLE1:MilkClottingwithGroundHOTNEWMEXICOCHILIPEPPERAddedDirectlytoMilk

TABLE2:MilkclottingusingdriedHABANEROAddedDirectlytoMilk

Milk Pineapple GroundChiliPepper Outcome

25mL

Approx.4cm3 1.5mL Clotting(grain-like)–12min.

25mL Approx.4cm3 1.0mL Clotting(grain-like)–10min.

25mL

Approx.4cm3 0.5mL Clotting(smallchunks)–9min.

25mL

Approx.4cm3 0.1mL Heavyclotting(thickchunks)–5

min.

25mL

Approx.4cm3 - Heavyclotting(thickchunks)–5

min.

25mL - 1.5mL Noclottingafter12min.

25mL - - Noclottingafter12min.

Milk Pineapple Habanero Outcome

25mL

Approx.2cm3 1.5mL Clottedat7min

25mL

Approx.2cm3 1.0mL Clottedat7min

25mL

Approx.2cm3 0.5mL Clottedat7min

25mL

Approx.2cm3 0.1mL Clottedat7min

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TABLE3:4/12/2012–MilkExperimentwithCHILIPEPPERExtractAftera2-DayExtractioninOil

*Ratiosarewrittenintheformatgroundchilipepper:oil.

TABLE4:MilkExperimentwithHABANEROExtractAftera2-DayExtractioninAlcohol

25mL

Approx.2cm3 - Clottedat6min

25mL - 1.5mL Didnotclot

25mL - - Didnotclot

Cup#

Pineapple

Milk(mL)

Ratioof

Extract

Amountof

ExtractOutcome

1 Approx.2cm3 20 1:1 1mL Clottedat9min.

2 Approx.2cm3 20 1:2 1mL Clottedat6min.

3 Approx.2cm3 20 1:5 1mL Clottedat9min.

4 Approx.2cm3 20 1:10 1mL Clottedat9min.

5 Approx.2cm3 20 1:15 1mL Clottedat8min.

6 pprox.2cm3 20 1:20 1mL Clottedat8min.

Cup#

Pineapple

(cm3)

Milk(mL)

Ratioof

Extract

AmountofExtract(mL)

Outcome

- - - 1:1 (none) -

- - - 1:2 (none) -

1 Approx.2 25 1:5 1 Clottedat9min

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*Ratiosarewrittenintheformatgroundchilipepper:alcohol.

TABLE5:MilkExperimentwithCHILIPEPPERExtractaftera2-DayExtractioninAlcohol

*Ratiosarewrittenintheformatgroundchilipepper:alcohol.

TABLE6:MilkclottingwithGroundHOTNEWMEXICOCHILIPEPPERAddedDirectlytoMilk

2 Approx.2 25 1:10 1 Clottedat9min

3 Approx.2 25 1:15 1 Clottedat8min

4 Approx.2 25 1:20 1 Clottedat7min

5 Approx.2 25 - - Clottedat7min

Cup#

Pineapple

(cm3)

Milk(mL)

Ratioof

Extract

AmountofExtract(mL) Outcome

- - - 1:1 (none) -

1 Approx.2 20 1:2 1 Clottedat8min.

2 Approx.2 20 1:5 1 Clottedat7min.

3 Approx.2 20 1:10 1 Clottedat6min.

4 Approx.2 20 1:15 1 Clottedat6min.

5 Approx.2 20 1:20 1 Clottedat6min.

Milk(mL)

Pineapple(cm3)

Groundchilipepper(mL)

Outcome

25 Approx.2 1.5 Clottedat8min.

25 Approx.2 1.0 Clottedat7min.

25 Approx.2 0.5 Clottedat6min.

25 Approx.2 0.1 Clottedat6min.

25 Approx.2 - Clottedat6min.

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TABLE7:MilkClottingWithFreshPineappleJuicetoDetermineMinimumAmountNeededforObservableClotting

TABLE8:MilkClottingWithMinimumFreshPineappleJuicetoDetermineAmountofGroundNewMexicoChiliPowderNeededforObservableInhibitionofClotting

Beaker#

AmountPineapple

AmountMilk Outcome

1 5.0mL 20mL Startedshowingsignsof`ineclottingat1min.,heavyclottingat4.5min.

2 1.0mL 20mL Heavilyclottedat5min.

3 0.5mL 20mL Heavilyclottedat5min.

4 0.25mL 20mLStartedshowingsignofclottingat8min.,clottedat9min.,butwasnotasdramaticas

beaker#3

5 0.05mL 20mL Startedshowingsignsofclottingat9min.,lightclottingby12min.

6 0.01mL 20mL Startedshowingsignsofclottingat10min.,lightclottingby12min.

7 - 20mL Begantobubbleat12min.

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PURPOSE:Canweusefreshpineapplejuicetoeffectivelyclotmilk,andifso,whatistheminimumamountneededtodoso?

OUTCOME:Clottingwasachievedwithjuice.Atleast0.25mLwereneededforclotting,butclottingwasmuchmoredramaticandtookhalfthetimewith0.5mLjuice.

Beaker#

AmountPineapple

AmountMilk

AmountChili

PowderOutcome

1 0.5mL 20mL 2.0mL Clottingafter22min.

2 0.5mL 20mL 1.5mL Clottingat14min.

3 0.5mL 20mL 1.0mL Clottingat13min.

4 0.5mL 20mL 0.5mL Clottingat12min.

5 0.5mL 20mL 0.1mL Clottingat5min.

6 0.5mL 20mL - Clottingat4min.

7 - 20mL 2.0mL Clottingafter22min.

8 - 20mL - Thickeningafter22min.

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JelloAssay

MATERIALS:- GelatinPackages- Water- HotPlate- Ice- Freshlycutpineapple- Disposableplasticcups

2XRECIPE:(1) Obtain89mLboilingwater.(2) Obtain60mLwateratroomtemperatureandaddiceuntil volumeincreasesto

150mL.(3) Addonestandardpackageofjellogelatinpowderto boilingwaterandmix

untildissolved.(4) Addicewatertojellomixture.Thoroughlymixandremove anyunmeltedice.

àRecipewasderivedfrombasic“quicksetting”recipeonJello box.TwicetheamountofJellopowderwasfoundtobebetterduetoshortened hardeningtime.

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PURPOSE:Howmuchgroundchilipepperisneededtoinhibit0.5mLpineapplejuicefromclottingmilk?

OUTCOME:Ittook18minuteslongertoclotinthepresenceof2.0mLgroundchilipepperthanwithnogroundchilipepperpresent.

SOMETECHNICALASPECTSABOUTTHEJELLOASSAY

POSITIVETHINGSTOKNOW:- Heatingthepineappleinboilingwaterfor2-3minutesallowedthegelatintohardenwhenputinthefridge

- Themorethoroughlymixedthejuiceisinthegelatin,themoreliquidthegelatinwillappearafterhardeningtime.

- Usinga2xconcentrationofgelatinmixtureallowsforthehardeningofthegelatintobetteroccurina30–60minutetimeperiod.

“NEGATIVE”THINGSTOKNOW:- Ifthegelatinisnotstirredbetweeneachpourintothedisposablecups,thegelatinwillnotbeevenlydispersedandgelatinwillnotharden.

- Ifextraiceisnotremovedfromthemixtureafterthoroughmixing,gelatinwillnothardenorwilltakethreetimesaslongtoharden.

- Capsaicininhibitstheeffectsofbromelaintoaverymildextent.Insteadofmakingitcontinuetohardentofullextent,itmerelyallowsthegelatintoreachahigherviscosity.

OTHERNOTES:- The1:2extractionofcapsaicinand1:1extractionofcapsaicinwerethebest

candidatesforfurtherstudy.- Ifgivenenoughtime,freshpineapplewilleatthroughalreadyhardenedgelatin.- Ittakesatleast3mLoffreshpineappletoinhibitjellohardening.

NEXTSTEPS:Thejelloassaywashaltedwitha`inalexperimenttoseeifcapsaicincouldbeextractedintoalcoholinordertobothmakeitmoreconcentratedaswellassolubleingelatin.Theexperimentshowedpromisewithhigherconcentrationsofcapsaicin,butdramaticinhibitionwasneverobserved.Ifoneweretoworktocontinuedevelopmentofthisassay,startingpointsmaybeto`indamoreconcentratedsourceofcapsaicin.Heatingpineappletoinhibittheeffectsofbromelainshouldalsobeconsideredasaprospect.Aswiththemilkassay,furtherresearchshouldbecompletedtodetermineifcapsaicinisindeedthechemicalinchiliesthatactsonbromelain,orifissomeothercomponentofthechili.Thisresearchshouldalsolookto`indthemechanismthroughwhichthisinhibitionacts.

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TABLESFROMOURJELLOHARDENINGSTUDIES

TABLE9:DeterminingHowMuchTritonx100CanbeUsedWithoutInterferingWithJelloHardening

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PURPOSE:Isitpossibletousedetergenttoextractcapsaicinwithoutitselfinterferingwiththehardeningofjello?

OUTCOME:Themaximumdetergentthatcanbeusedistoosmalltoholdaneffectiveamountofcapsaicin.

TABLE10:BasicJelloExperimentwith25mLof2xJelloRecipe

Tritonx100 Jello Outcome

1.0mL 20mLThick`ilmontopofcup,butliquidbeneath

0.9mL 20mL

0.8mL 20mLVerysoftgelwithabout1mLliquidstillpresent–gel

fallsapartuponmovingthecup0.7mL 20mL

0.6mL 20mL

0.5mL 20mL Noliquidpresent,butjellopullsawayfromsidesofthecupuponmoving-verylittleliquidpresent0.4mL 20mL

0.3mL 20mL

Noliquidpresent,butjelloisverysoftformed0.2mL 20mL

0.1mL 20mL

0.0mL 20mL Completelyhardenedjellothatstayssolidincupevenwhentippedupsidedown

Cup# Pineapple Outcome

1 Approx.4cm3Fresh

After1hour–completeliquid2

3 Approx.4cm3

Cooked

After25minutes–hardenedto`irmconsistency.Stayedinsidecupwhenheldupsidedown.4

5(None) After25minutes–hardenedto`irmconsistency.Stayed

insidecupwhenheldupsidedown.

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PURPOSE:Doesusingtwicetheamountofgelatinpowdermakeforashorterhardeningtime?

OUTCOME:Comparedtoprevioustrials,usingtwicethegelatinmakesthejellohardenfaster.

TABLE11:JelloExperimentwith25mLof2xJelloandGroundHOTNEWMEXICOCHILIPOWDERAddedDirectlytoJello

TABLE12:JelloExperimentUsingHABANEROExtractafter1-DayExtractioninAlcoholand25mLof2xJello

6(None) After25minutes–hardenedto`irmconsistency.Stayed

insidecupwhenheldupsidedown.

Cup# Pineapple Chili

powder Outcome

1Approx.4cm3Fresh

1mL After1hour–completeliquid2

3

4

(None) 1mLAfter25minutes–hardenedto`irm

consistency.Stayedinsidecupwhenheldupsidedown.

5

6

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PURPOSE:DoesaddinggroundHotNewMexicoChiliPowdertogelatininhibitaffectsofbromelainonthegelatin?

OUTCOME:Nosignsofinhibitionwereobserved,andthismethodwasruledout.

PURPOSE:Canwemakethecapsaicinfromhabaneromoreconcentratedbyextractinginalcohol(foroneday)andthereforemoreeffectiveintheinhibitionofbromelain?

OUTCOME:experimentshowedsomepromisebutshouldberepeatedwithlesspineappleandmoreconcentratedextract(atlease2:1)

*Ratiosarewrittenintheformatgroundchilipepper:alcohol.

TABLE13:BasicJelloExperimentUsingBoiledPineappleand25mLof2xJello

Cup#

Ratioof

ExtractPineapple

Amountof

ExtractOutcome

1 1:1 Approx2cm3 1mL After5hours–stillliquid

Slightlymoregelatinousthanothers

2 1:2 Approx2cm3 1mL After5hours–stillliquid

Slightlylessgelatinousthan1:1

3 1:5 Approx2cm3 1mL After5hours–stillcompleteliquid

4 1:10 Approx2cm3 1mL After5hours–stillcompleteliquid

5 1:15 Approx2cm3 1mL After5hours–stillcompleteliquid

6 1:20 Approx2cm3 1mL After5hours–stillcompleteliquid

Cup# Pineapple Amountof

PineappleAmountofJello Outcome

1 Approx.4cm3Fresh

1.5mL 25mL After4hours–liquid

2 3.0mL 25mL After4hours–liquid

3 Approx.4cm3Boiled

(mashed)

2.5mL 25mLAfter4hours–completesolidDoesnotpourwhenheldupside

down

4 3.0mL 25mLAfter4hours–completesolidDoesnotpourwhenheldupside

down

5

Approx.4cm3Boiled(chunks)

2–2cm3chunks 25mL

After4hours–completesolidDoesnotpourwhenheldupside

down

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TABLE14:JelloExperimentUsingHABANEROExtractAfter2-DayExtractioninAlcoholand25mLof2xJello

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PURPOSE:Aretheresultsofthepreviousexperimentofinhibitingtheaffectsofbromelainviaheatreproducible?

OUTCOME:Yes,ithasconsistentlybeenmodeledthatheatingpineapplestopstheaffectsofbromelainongelatin.

*Ratiosarewrittenintheformatgroundchilipepper:alcohol.

TABLE15:JelloExperimentUsingHOTNEWMEXICOCHILIPEPPERExtractaftera2-DayExtractioninAlcoholand25mLof2xJello

*Ratiosarewrittenintheformatgroundchilipepper:alcohol.

Cup#

RatioofExtract Pineapple

Amountof

ExtractOutcome

1 1:1 Approx.2cm3 1mL After6hours–slightlygelled

2 1:2 Approx.2cm3 1mL After6hours–liquid

3 1:5 Approx.2cm3 1mL After6hours–liquid

4 1:10 Approx.2cm3 1mL After6hours–liquid

5 1:15 Approx.2cm3 1mL After6hours–liquid

6 1:20 Approx.2cm3 1mL After6hours–liquid

Cup#

RatioofExtract Pineapple

Amountof

ExtractOutcome

1 1:1 Approx.2cm3 1mL After6hours–slightlygelled

2 1:2 Approx.2cm3 1mL After6hours–liquid

3 1:5 Approx.2cm3 1mL After6hours–liquid

4 1:10 Approx.2cm3 1mL After6hours–liquid

5 1:15 Approx.2cm3 1mL After6hours–liquid

6 1:20 Approx.2cm3 1mL After6hours–liquid

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TABLE16:JelloExperimentUsingHOTNEWMEXICOCHILIPEPPERExtractAftera7-DayExtractioninAlcoholina1:1Ratio(chilipepper:alcohol)

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PURPOSE:Canwemakethe

PURPOSE:Canwemakethecapsaicinfromchilipeppermoreconcentratedbyextractinginalcohol(forsevendays)andthereforemoreeffectiveintheinhibitionofbromelain?

OUTCOME:Experimentshowedpromise,butnotcompleteinhibition.

Cup#

AmountJello

AmountPineapple

AmountExtract Outcome

1 20mL 3mL None Completelyliquidafter1.5hours

2 20mL 3mL 5mL Completelyliquidafter1.5hours

3 20mL 3mL 8mL Slightlymoreviscousthancontrolafter1.5hours

4 20mL 3mL 10mL Consistencyofhairgelafter1.5hours–mixofgelledandliquidparts

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