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GALACTOMANNANASSAYPROCEDUREK-GALM09/19(100AssaysperKit)Megazyme2019www.
megazyme.
comINTRODUCTION:Galactomannansoccurinnatureasthereservepolysaccharidesintheendospermsofawiderangeoflegumeseeds.
Thesepolysaccharides,inpartiallypurifiedform,findwidespreadapplicationasthickeningandgelling(inthepresenceofotherpolysaccharides)agentsinthefoodindustry.
Partiallydegradedguargalactomannanisusedasanoveldietaryfibercomponent.
Galactomannaniscomposedofa1,4-β-linkedD-mannanbackbonetowhichsingleD-galactosylunitsareattachedtoC-6ofsomeoftheD-mannosylresidues.
ThemajordifferencebetweengalactomannansfromdifferentseedspeciesistheratioofD-galactosetoD-mannose.
However,somevariationinthemolecularweightofthegalactomannanshasalsobeenreported.
TheD-galactose:D-mannoseratioofgalactomannanfromdifferentvarietiesofagivenseedspeciesappearstoberemarkablyconstant.
Inadetailedstudyofgalactomannans,purifiedfromapprox.
50samplesofcarobflourfromdiversesourcesandfromarangeofcarobvarieties,itwasshownthattheD-galactose:D-mannoseratioisconstant,i.
e.
22±1%(w/w)D-galactoseand78±1%(w/w)D-mannose.
1Inaparallelstudyonthegalactomannanfromguarseedvarieties,aD-galactosecontentof38±1%(w/w)andD-mannosecontentof62%±1%(w/w)wasfound.
Thisobservationwouldalsoappeartoholdtrueforgalactomannanfromotherseeds,e.
g.
fenugreekseed,butinthesecasesthestudieshavenotbeenascomprehensive.
GalactomannansarequantitativelyhydrolysedtoD-galactoseandmanno-oligosaccharidesbyamixtureofα-galactosidaseplusβ-mannanase.
2Thus,aprocedurehasbeendevelopedforthemeasurementofcarobgalactomannanandguargalactomannan(or,infact,possiblyanyothergalactomannan)basedonthemeasurementoftheD-galactosecontent.
PRINCIPLE:ThemethoddescribedinthisbookletisbasedonthequantitativehydrolysisofgalactomannantoD-galactoseandmanno-oligosaccharidesusingacombinationofβ-mannanaseandα-galactosidase.
3ThereleasedD-galactoseisthenquantitativelydeterminedusingamixtureofβ-galactosedehydrogenase.
CalculationofgalactomannancontentisbasedonapriorknowledgeoftheD-galactose:D-mannoseratioofthegalactomannanbeinganalysed.
Inbroadterms,asampleoftestmaterial(approx.
100mg)isextractedwithaqueousethanol(80%v/v)toremovegalactosylsucroseoligosaccharides(which,likegalactomannan,containα-linkedD-galactose)andisthensuspendedinacetatebufferandincubatedinaboilingwaterbathtoobtaincompletehydrationofthegalactomannan.
1Theviscousslurryisthencooledto40°Candtreatedwithβ-mannanasetoeffectdepolymerisationandcompletesolubilisationofthegalactomannan,alongwithadramaticviscositydecrease.
Followingcentrifugation,aliquotsofthesupernatantsolutionaretreatedwithamixtureofpureα-galactosidaseandpureβ-mannanasetoeffectcompletehydrolysisofthegalactomannantoD-galactoseandmanno-oligosaccharides.
Thismethodcanbeappliedtothemeasurementofgalactomannaninwholemilledseed,millingfractions,puregalactomannansamplesorfoodproductscontaininggalactomannan.
TheD-galactose:D-mannosecontentsofsomecommerciallyavailable,orotherwisecommonlystudied,galactomannansareasfollows:CommonnameBotanicalnameGal:ManCassiaCassiatora19:81Carob(locustbean)Ceratoniasiliqua22:78GuarCyamopsistetragonoloba38:62Lucerne(alfalfa)Medicagosativa48:52FenugreekTrigonellafoenum-graecum48:52KITS:Kitssuitableforperforming100assaysareavailablefromMegazyme.
Thekitscontainthefullassaymethodplus:Bottle1:Buffer(25mL,pH8.
6)plussodiumazide(0.
02%w/v)asapreservative(BufferA).
Stablefor>2yearsat4°C.
Bottle2:NAD+.
Stablefor>5yearsbelow-10°C.
Bottle3:β-Mannanasesuspension(A.
niger;1.
1mL).
Stablefor>4yearsat4°C.
Bottle4:α-Galactosidase(guarseed)plusβ-mannanase(A.
niger)suspension,2.
2mL.
Stablefor>4yearsat4°C.
Bottle5:β-Galactosedehydrogenase(2.
4mL).
Stablefor>4yearsat4°C.
Bottle6:D-Galactosestandardsolution(5mL,0.
4mg/mLin0.
02%sodiumazide).
Stablefor>4years;storesealedat4°C.
Bottle7:Controlcarobgalactomannanfloursample(withgalactomannancontentasstatedontheviallabel).
Stablefor>5yearsatroomtemperature.
2PREPARATIONOFREAGENTSOLUTIONS/SUSPENSIONS:1.
Usethecontentsofbottle1assupplied.
Stablefor>2yearsat4°C.
2.
Dissolvethecontentsofbottle2in11mLofdistilledwater.
Stablefor>1yearat4°Corstablefor>2yearsbelow-10°C(toavoidrepetitivefreeze/thawcycles,divideintoappropriatelysizedaliquotsandstoreinpolypropylenetubes).
3,4Usethecontentsofbottles3,4and5assupplied.
Before&5.
openingforthefirsttime,shakethebottlestoremoveanyenzymethatmayhavesettledontherubberstopper.
Subsequently,storethebottlesinanuprightposition.
Stablefor>4yearsat4°C.
6.
Usethecontentsofbottle6assupplied.
Stablefor>4years;storesealedat4°C.
7.
Usethecontentsofbottle7assupplied.
Stablefor>5yearsatroomtemperature.
SOLUTIONSNOTSUPPLIED:1.
Sodiumacetatebuffer(BufferB)(100mM,pH4.
5).
Add5.
8mLofglacialaceticacid(1.
05g/mL)to900mLofdistilledwater.
AdjustthepHto4.
5bycarefuladditionof2M(8g/100mL)sodiumhydroxidesolution.
Adjustthevolumeto1Lwithdistilledwater.
Stablefor>3monthsat4°C.
2.
Aqueousethanol(~80%v/v)Add20mLofdistilledwaterto80mLofethanolandmix.
Stablefor>5yearsatroomtemperatureinawell-sealedbottle.
EQUIPMENT(RECOMMENDED):1.
Volumetricflasks(50mL,100mLand500mL).
2.
Disposableplasticcuvettes(1cmlightpath,3.
0mL).
3.
Micro-pipettors,e.
g.
GilsonPipetman(20μLand200μL).
4.
Positivedisplacementpipettor,e.
g.
EppendorfMultipette-with5.
0mLCombitip(todispense0.
2mLaliquotsofBuffer1and0.
1mLaliquotsofNAD+solution).
-with25mLCombitip(todispense2.
0mLaliquotsofdistilledwater).
5.
Analyticalbalance.
6.
Spectrophotometersetat340nm.
37.
Vortexmixer(e.
g.
IKAYellowlineTestTubeShakerTTS2).
8.
Thermostatedhot-blockheaterorwaterbath(setat40°C).
9.
Stopclock.
10.
WhatmanNo.
1(9cm)filterpapers.
METHOD:1.
Millseedsampleorseedfraction(e.
g.
carobseed)topassa0.
5mmscreenusingaTecatorCyclotecmill(orequivalent).
Commercialgalactomannanfloursdonotrequiremilling.
2.
Accuratelyweighapprox.
100mgofmilledsampleintoaglass,round-bottomedtesttube(16x120mm).
Tapthetubetoensureallofthesamplefallstothebottomofthetube.
3.
Add5mLof80%(v/v)aqueousethanoltoeachtube,stironavortexmixerandincubateat85-90°Cforapprox.
5min.
Addafurther5mLofaqueousethanol,againstironavortexmixerandcentrifugeat1,500gfor10min.
Carefullydecantthesupernatantsolution(discardthissolution).
4.
Resuspendthepelletin5mLofaqueousethanolasinStep3,andrepeattheoperationsinStep3.
Again,discardthesupernatantaftercentrifugationandallowexcessliquidtodrainfromthetubesbyinvertingonabsorbentpaper.
5.
Resuspendthepelletin8mLof100mMsodiumacetatebuffer(pH4.
5)(BufferB)andstirvigorouslyonatest-tubestirrertoeffectcompletedispersion.
Immediatelyplacethetubeinaboilingwaterbath.
After30sec,removethetubeandstiritvigorouslyonavortexmixer.
Returnthetubetotheboilingwaterbathforafurther30secandagainremoveitandstironavortexmixer.
Returnthetubetotheboilingwaterbathfor4min.
Removethetube,stiritvigorouslyonavortexmixerandplaceitinawaterbathat40°C.
4NOTE:Thissolutioncontainsgalactosyl-sucroseoligosaccharides.
NOTE:Theseoperationsareperformedtoensurethoroughsuspensionandevenhydrationofthepolysaccharide.
Formationof"lumps"ofpartiallyhydratedandswollengalactomannanwillhindertheabilityofβ-mannanasetopenetrateanddepolymerisethepolymer.
6.
After5minat40°C,add10μLofβ-mannanasesuspension(suspension3)andstirthetubevigorouslyonavortexmixerfor30sec.
Incubatethetubesat40°Cfor60minwithintermittentvigorousstirring(about2-3timesonavortexmixer).
7.
Quantitativelytransferthesolutiontoa25mLvolumetricflaskusingawaterwashbottleandadjusttovolumewithdistilledwater.
Mixtheflaskcontentsthoroughly.
8.
Filteranaliquotofthesolution(WhatmanNo.
1filterpaper)orcentrifugeat1,500gfor10min.
9.
Usethissolutionintheassaydescribedbelow.
5NOTE:Atthisstage,thesolutioniseitheranalyseddirectly,ordilutedbasedonthepredictedgalactomannancontentaswellastheD-galactose/D-mannoseratioofthegalactomannan,e.
g.
forasampleofcarobflourcontainingapprox30%(w/w)galactomannanwithaD-galactose/D-mannoseratioof22:78,thesolutionisanalyseddirectly;howeverforaguarfloursamplewithasimilargalactomannancontent,butwithaD-galactose/D-mannoseratioof38:62,thesolutionisdiluted2-foldwithwaterbeforeanalysis.
PROCEDURE:Wavelength:340nmCuvette:1cmlightpath(glassorplastic)Temperature:Optimally40°Cinadryhot-blockheaterorinthespectrophotometerFinalvolume:2.
94mLSamplesolution:4-80μgofD-galactoseReadagainstair(withoutacuvetteinthelightpath)oragainstwater.
PipetteintocuvettesBlank*Samplesample0.
20mL0.
20mLbufferB(acetatebuffer)0.
10mL0.
10mLsolution4(α-galactos.
/β-mannanase)-0.
02mLMix**,capthecuvetteandincubateat40°Cfor60min.
Add:distilledwater2.
32mL2.
30mLsolution1(buffer)0.
20mL0.
20mLsolution2(NAD+)0.
10mL0.
10mLMix**,readtheabsorbancesofthesolutions(A1)afterapprox.
3minandstartthereactionsbyadditionof:suspension5(β-GalDH)0.
02mL0.
02mLMix**andreadtheabsorbancesofthesolutions(A2)attheendofthereaction(approx.
30min)(Reactionwilltake60minat25°C).
*performasingleblankassaywitheachsetofdeterminations.
**forexamplewithaplasticspatulaorbygentleinversionaftersealingthecuvettewithacuvettecaporParafilm.
CALCULATIONS:Determinetheabsorbancedifference(A2-A1)forbothblankandsample.
Subtracttheabsorbancedifferenceoftheblankfromtheabsorbancedifferenceofthesample,therebyobtainingΔAD-galactose.
TheconcentrationofD-galactosecanbecalculatedasfollows:c=VxMWxΔAD-galactose[g/L]εxdxv6where:V=finalvolume[mL]MW=molecularweightofthesubstancetobeassayed[g/mol]ε=extinctioncoefficientofNADHat340nm=6300[lxmol-1xcm-1]d=lightpath[cm]v=samplevolume[mL]ItfollowsforD-galactose:c=2.
94x180.
16xΔAD-galactose[g/L]6300x1.
0x0.
2=0.
4204xΔAD-galactoseThus,forgalactomannan:c=0.
4204xΔAD-galactosex100/Gx162/180[g/L]=37.
836xΔAD-galactosex1/Gwhere:100/G=conversionfromD-galactoseasdeterminedtogalactomannanconcentration.
G=galactosecontentofthegalactomannanasapercentage,e.
g.
22forcarobgalactomannan38forguargalactomannan48forfenugreekandalfalfagalactomannans.
162/180=AdjustmentfromfreeD-galactosetoanhydro-D-galactose(asoccursingalactomannan).
Ifthesamplehasbeendilutedduringpreparation,theresultmustbemultipliedbythedilutionfactor,F.
Whenanalysingsolidandsemi-solidsampleswhichareweighedoutforsamplepreparation,thecontent(g/100g)iscalculatedfromtheamountweighedasfollows:Contentofgalactomannan=cgalactomannan[g/Lsamplesolution]x100[g/100g]weightsample[g/Lsamplesolution]7Thus,fortheexampledetailedonpages4and5where100mgofsamplewasextractedandadjustedtoafinalvolumeof25mL,theweightofsampleasg/Lis0.
1g/25mLor4g/L.
Thus,thecontentofgalactomannan=37.
836xΔAD-galactosex1/Gx1/4x100[g/100g]Forcarobgalactomannan[with22%(w/w)D-galactose]=37.
836xΔAD-galactosex1/22x1/4x100[g/100g]=43.
00xΔAD-galactose[g/100g]8NOTE:TheD-galactosestandardsolutionisonlyassayedwherethereissomedoubtabouttheaccuracyofthespectrophotometerbeingusedorwhereitissuspectedthatinhibitionisbeingcausedbysubstancesinthesample.
TheconcentrationofD-galactoseisdetermineddirectlyfromtheextinctioncoefficientofNADH(page7).
REFERENCES:1.
McCleary,B.
V.
,Dea,I.
C.
M.
,Clark,A.
H.
&Rees,D.
A.
(1985).
Thefine-structuresofguarandcarobgalactomannans.
CarbohydrateResearch,139,237-260.
2.
McCleary,B.
V.
&Matheson,N.
K.
(1983).
Actionpatternsandsubstratebindingrequirementsofβ-mannanasewithmannosaccharidesandmannan-typepolysaccharides.
CarbohydrateResearch,119,191-219.
3.
McCleary,B.
V.
(1981).
Galactomannanquantitationinguarvarietiesandseedfractions.
Lebens.
WissenschaftTechnology,14,188-191.
NOTES:9NOTES:1011BrayBusinessPark,Bray,Co.
Wicklow,A98YV29,IRELAND.
Telephone:(353.
1)2861220Facsimile:(353.
1)2861264Internet:www.
megazyme.
comE-Mail:info@megazyme.
comWITHOUTGUARANTEETheinformationcontainedinthisbookletis,tothebestofourknowledge,trueandaccurate,butsincetheconditionsofusearebeyondourcontrol,nowarrantyisgivenorisimpliedinrespectofanyrecommendationorsuggestionswhichmaybemadeorthatanyusewillnotinfringeanypatents.