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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
� 10
*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.
� 15
� 16
TABLESFROMOURJELLOHARDENINGSTUDIES
TABLE9:DeterminingHowMuchTritonx100CanbeUsedWithoutInterferingWithJelloHardening
� 17
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.
� 18
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
� 19
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)
� 23
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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