physicochemical characterization of galactomannan from...

IntroductIonArenga sacchariferaLabill.isawellknownsugarpalminthehumidtropics.ItsgenericnameisderivedfromtheJavanesewordforsugarpalm(Arenga).Itsspecificname, from Latin saccharum and Greek saccharon,alsocomesfromsanskritsarkara,whichmeansugar.the plant belongs to the subfamilyAracoidae, tribecaryotaeofthePalmaefamily.Itisanuncultivatedplant

Physicochemical Characterization of Galactomannan from Sugar Palm (Arenga saccharifera Labill.) Endosperm

at Different Stages of Nut Maturity

A water-soluble polysaccharide was extracted from the endosperm of nuts of Arenga saccharifera Labill. at different stages (young, 8–12 mo old; mid mature, 16–18 mo old; and mature, 22–24 mo old) of maturity. The polysaccharide was extracted with water, precipitated with 95% ethanol and further purified with Fehling solution and then freeze dried. The Arenga gum samples were white and powder-like after drying; insoluble in organic solvents, slightly soluble in inorganic solvents, and soluble in hot and cold water. Specific rotation values [α]D27 of +32.97, +39.53 and +35.93 were obtained for the purified gum samples, young, mid mature and mature, respectively, before inversion; corresponding [α]D27 values after inversion were -35.58, -41.70, and -41.03. The specific gravity of all gum isolates at different stages of nut maturity and those of commercial gums were not significantly different and was equal to the density of water at standard room temperature (25° C). The water-holding capacity of the three gum isolates — 42.55%, 47.00%, and 47.28%, respectively, were not significantly different from the value for gum ghatti. The gelatinization temperature of the gum isolates were the same (30–70° C). Viscosity of the gum isolates increased with concentration and maturity. Total sugar and soluble protein slightly increased with maturity while total reducing sugar remained the same upon maturity. The Arenga gums had high molecular weights of >2M daltons. Analysis by gas chromatography showed that the gum was composed of mannose and galactose and can, therefore, be correctly termed as galactomannan. The mannose:galactose (M/G) ratio of the Arenga galactomannan increased with maturity (2:1, 3:1 and 5:1) for the young, mid mature, and mature samples, respectively.

thatgrowsinprimaryandsecondarygrowthforestsandinabandonedlandsatlowandmediumaltitudesinthePhilippines.Kaong,asArengaisknowninthePhilippines,isusedasasourceofsugar,wine,vinegar,andkulang-kaleng.the cooked endosperm of young sugar palmfruits,about12–18moold,areusedasingredientsinfruitmixesanddesserts.However,therearenoreportedusesfortheoldernuts(>18mo).HighvalueproductssuchasgumscanbeextractedfromtheArengaendospermsinceithasgelatinoustexture.Gumsobtainedfromplantsare

Philippine Journal of Science135(1):19-30,June2006ISSn0031-7683

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Mary Ann O. Torio*, Joydee Saez and Florinia E. Merca

Key Words:Arenga saccharifera,galactomannan,gums,physicochemicalcharacteristics

*correspondingauthor:[email protected]

Instituteofchemistry,collegeofArtsandSciencesuniversityofthePhilippinesLosBaños,college,Laguna

solidsthatconsistofmixturesofpolysaccharides,whichareeitherwater-solubleorabsorbswaterandswellsuptoformagelorjellywhenaddedtowater.Insolubleinoils or organic solvents, gums are often complex andon hydrolysis yield simple sugars such as arabinose,galactose,mannose,andglucuronicacid.

the immature unripe gelatinous endosperm ofArenga sacchariferahasbeenshowntocontainagumcomposed mainly of water-soluble polysaccharidegalactomannans that consists of repeating mannose(M) (linked β1→4) and galactose (G) (linked α1→6)unitwith2:1M/Gratio(Kooiman1971).nootherwork on Arenga galactomannan has been reported.Galactomannansarereservedpolysaccharidesofplantsthatformhighlyviscoussubstancescommonlyknownasgums.Gumsareeitherhydrophobicorhydrophilichighmolecularweightmoleculeswithcolloidalproperties.they produce gels in appropriate solvent or swellingagentsandformhighlyviscoussolutionwithlowdrysubstancecontent(WhistlerandBeMiller1958).theyalsoformstablesolutions,andhavetheabilitytoformgelatveryhightemperature.theyhavesomeimportantproperties such as strong water-binding capacity andstability in solution which is a characteristic of atexturalmodifyingagentfordifferenttypesofproducts.duetoitscolloidalproperties,theterm“gum”isalsoreferred to as hydrocolloid (Meer 1977).there is aninterplaybetweenviscosityandgellingcharacteristicsof any specific gum, and these factors must be takeninto consideration when gums are used. commercialgums such as gum arabic, gum ghatti, gum karaya,gum tragacanth, locust bean gum, and guar gum arewellknownduetotheirwideapplicationsinindustries,especiallyinfoodindustry,pharmaceuticals,cosmetics,paperproducts,paintsandplasters,welldrilling,miningandexplosives,andfirefighting.

WhiletheyoungArenga sacchariferanutendospermcontainsanediblegalactomannan,theolder(>18mo)nutsthatdonothaveanyreportedusesmightbeagoodindustrial sourceof ediblegum,whichcouldbeusedas stabilizers, thickening agent, and gelling agent inthefoodindustry.Itspossiblehealthusescouldalsobeexplored.

this study aimed to isolate and purify thegalactomannnans from the endosperm of Arenga sacchariferanutsatdifferentstagesofmaturity.thephysicochemicalpropertiesandchemicalcompositionof Arenga galactomannans or gums were alsodeterminedandcomparedwiththoseofcommerciallyavailablegums.

MAtErIALSAndMEtHodSSample Collection. Samples of the nuts of Arenga saccharifera Labill. at 3 different stages of maturitywerecollectedatBarangayBalagbag,cuenca,Batangas.Maturityofthefruitswasdeterminedbytaggingthenutsfrompollinationandbyconsideringthehardnessoftheendosperm.

Sample Preparation.Aftercollection,theendospermwas gathered by dividing the nut into halves and theendospermwastakenoutwithasmallknife.thecollectedendosperms were kept in a refrigerator at 2–4° cforatmost 24 h prior to isolation and purification.

Chemical Analysis.Moisturecontent,ash,crudefat,crudeprotein,crudefiber,andnitrogenfreeextract(totalcarbohydrate)weredeterminedusingstandardproceduresset by theAssociation of officialAnalytical chemists(AoAc1995).

Extraction and Isolation of the gum. Extractionandisolationofthewater-solublepolysaccharidesfromendospermofsugarpalmweredoneusingtheprocedureofKooiman (1971).theendosperm(~170g)of sugarpalmatdifferentstagesofmaturitywassuspendedin500mLdistilledwaterfor3dat0°candwashomogenizedusing a blender.the viscous mass was further stirredusingamagneticstirrerovernightatroomtemperatureandcentrifugedatx9000gat0° c.theclearsupernatantliquidwasseparatedfromtheresidueandanequalamountof95%ethanolwasaddedtothesupernatantliquidwithcontinuousstirring.theresultingwhiteprecipitatewasallowed to settle and separated by decantation. theprecipitatewaswashedwithethanolandfreezedried.

topurifythegum,thedriedgumwasredissolvedindistilledwaterwithcontinuousstirringuntilcompletelydissolved. Fehling’s solution was added to the gumsolutionresultingintheformationoflightblueprecipitate.the precipitate was separated by decantation, washedandsuspendedwithdistilledwaterand2Mhydrochloricacidsolution.theresultingmixturewasstirredandanequalamountof95%ethanolwasadded to regeneratethegum.thegumextractwaswashedagainwithethanolandfreezedried.

Qualitative Analysis of the Gum

SolubilitytestofGumExtractsFifty(50)mLeachofvariousorganicsolvents(petroleumether,acetone,chloroform,benzene,methanol,ethanol,isopropanol, and butanol) and inorganic solvents (5%Hcl,5%H2So4, 5%H3Po4, 5%naoH,5%naHco3,1Mand0.1Mnacl)wereaddedto0.50gofthedried

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Philippine Journal of ScienceVol. 135 No. 1, June 2006

Torio et al.: Galactomanna from SugarPalm Nut Endosperm

purifiedgumextract.Solubilityofthegumextractwasalso determined using hot (80–90°c) and cold water(10–15°c). dissolution of the samples in test solventswasobserved.

Benedict’stestforreducingSugars(davis1963)Eachofthegumisolates(0.025g)wasdissolvedin

10mLdistilledwater.one(1)mLofBenedict’sreagentwasmixedwith3mLsolution.thereactionmixturewasheated inhotwaterbathuntil thecolorchangedandabrickredprecipitateformed.

ninhydrintestforProtein(Holmes1968)thepurifiedgum(0.025g)wasdissolvedin10mL

distilledwater.ten(10)dropsof10%ninhydrinsolutionwasaddedto1–2mLgumsolutionandwasplacedinahotwaterbath.Formationoflightlavandercolorindicatesthepresenceofprotein.

Physicochemical Properties of the Gum

determinationofSpecificrotationofGums(dawberetal.1966)Eachgumsample(0.05gram)wasdissolvedin100mLwater.thegumsolutionswerefilteredandequilibratedin27° cconstanttemperaturebath.opticalrotationsweredeterminedbeforeadditionofHcl.twentyfive(25)mLof 4 N HCl was mixed with an equal volume of the gum solutionandequilibratedin27° cconstanttemperaturebath.opticalrotationswereobservedevery15minforthefirsthourofdetermination.Inversionofsugarspresentinthegumsolutionswasdeterminedafterstandingin27° cwaterbathfor2dwithopticalreadingsmadedaily.

SpecificGravitySpecific gravity of the gum isolates was determinedfollowingtheprocedureofSkoogandWest(1963)usingstandardizedpycnometer.

A dried and clean pycnometer was filled with 1%gumsolutionthatwaspreviouslyequilibratedat20°ctotheindicatedlevel,cappedandplacedin25° cconstanttemperaturebathfor30min.Afterwards,thepycnometerwasdriedandweighed.Specificgravityofthesolutionwasobtainedbydividingtheweightofthegumsolutionbytheweightofwaterat25° cdeterminedpreviously.

Water-Holdingcapacity(Jaurigue1981)thegum(0.5g)wasdissolvedin30mLwaterandstoredovernight at 2–4° c.thesolutionwasthencentrifugedfor30minatx2000gat30° c.thesupernatantwasdrained

for15min.thevolumeofwaterdecantedwasmeasuredandpercentwaterretentionwascalculated.

ViscosityandGelatinizationtemperature(Stoloff1958)Four (4) different concentrations (0.5, 0.75, 1.0, and 2%) of gum solutions were prepared by heating andcontinuous stirring using a magnetic stirrer until thesolutiongelatinized.thegelatinizationtemperaturewasrecorded.theactualviscosityofthegelwasmeasuredat 30° c at 2.5 rpm by aWell Brookfield cone/PlateMicroviscometer model rVt.the viscometer readingincentipoisewasmultipliedbyafactorequivalenttothespindlenumberthathasbeenused.

Chemical Properties

totalSugartotal sugar content was determined according to theprocedure of dubois et al. (1956). Each gum sample(0.005 g) was dissolved in 100 mL distilled water.one(1)mLof5%phenolsolutionwasaddedto1mLgum solution in acid-washed test tube. Five (5) mLconcentratedH2So4(reagentgrade98.5%withspecificgravity of 1.84) was directly and rapidly added to the test tubes.thesolutionwasallowedtostandfor10minandwas shaken. Absorbance of the solution was read at 490 nmusingaBioradModel3550uVmicroplatereader.

Standardcalibrationsolutionsrangingfrom0.01to0.60mg/mLwaspreparedintriplicatesfrom10mg/mLstockgalactosesolution.

totalreducingSugarthereducingsugarofthegumsampleswasdeterminedfollowingtheprocedureofMiller(1959).

one(1)gramofeachgumsampleswasdissolvedin100mLdistilledwater.three(3)mLdnSreagent(preparedas:1gdinitrosalicylicacid,0.2gphenol,1gnaoHand20grochellesaltdissolvedindistilledwaterupto100mL)wasaddedto1mLgumsolution.thesolutionwasheatedinhotbathfor15min.thesolutionwascooledanddilutedto20mLwithdistilledwater.Absorbancewasreadat550nminamicroplatereader.Blankswerepreparedbysubstitutingdistilledwaterforthegumsolution.

Standardcalibrationsolutionsrangingfrom0.1to1mg/mLwerepreparedintriplicatesfrom1mg/mLstockgalactosesolution.

totalSolubleProtein(Lowryetal.1951)one1%(w/v)solutionofeachgumsamplewasprepared.Five (5) mL of freshly prepared reducing agent was

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added to 0.5 mL of gum solution and immediatelymixedusingvortexmixer.Folin reagent (0.5mL)wasadded to the solution after standing for 10 min.Aftermixing, the solution was allowed to stand for 30 min,anditsabsorbancewasmeasuredat660nminauV-Visspectrophotometer.

Standard BSA solution ranging from 110 to 210µg/mLwaspreparedintriplicateandtreatedinthesamewayasthesample.Blankwasalsopreparedusingdistilledwater.

Monosaccharidecompositionthe monosaccharide composition of the gum isolatesat different stages of maturity was determined by gaschromatographyasalditolacetatesusingthemethodofBlakeneyetal.(1983).

Samples were digested in 72% H2So4 and furtherhydrolyzed in 6M H2So4. the hydrolysates wereneutralized with nH3 and reduced with naBH4. thereduced sugars were acetylated with acetic anhydrideandthealditolacetatederivativeswereanalyzedbygaschromatography.

the monosaccharides were identified based onthe retention timesof the individual standards such asrhamnose, arabinose, xylose, mannose, glucose, andgalactose,whichwerereducedandacetylatedunderthesameconditionsasthesamples.Mixedstandards(2mgeach)werealsopreparedtoquantifytheamountofsugarsinthesamplewithmyo-inositolasinternalstandard.

Physical Characterization

MolecularWeightdeterminationGelfiltrationusingaSephadexG-200columnwasusedformolecularweightdetermination.thepolysaccharidesusedasstandardswerebluedextran(2000Kda),gumtragacanth (840 KDa), gum arabic (580 KDa), and guar gum(220Kda).

two (2) mL of 1% solution of each sample (gumisolates and standards) were eluted in the column (30cmx2.5cm)ataflowrateof0.5mL/min.theelutionprofilewasfollowedbydeterminingthetotalsugarineachfractioncollectedfromgelchromatography.

degreeofBranching(Whistler1973)Gum isolates (200 mg) were dissolved in a 50-mLvolumetric flask containing 25 mL co2-free distilledwater.ten(10)mLof0.3MnaIo4andsufficientco2-freedistilledwaterwasaddedtotheresultingsolutiontobringthevolumeto50mL.Ablankwasalsoprepared

usingdistilledwaterinsteadofgumsolution.theflaskwascappedandwrappedwithusedcarbonpaperandwaskeptinadarkplace.

About 10 mL aliquots were removed from bothblank and sample at 1, 4, 8, and 24 h after initiation of thereaction.toeachaliquotwasadded0.5mLethyleneglycol. the samples were incubated for 10 min afterwhich2dropsof 1%phenolphthaleinwas added.thesampleswerethentitratedwithstandardizedco2-free5mMnaoHsolution.

Statistical AnalysisAnalysisofvariance(AnoVA)wasdonebyrandomizedcompleteBlockdesign(rcBd)andduncan’sMultiplerangetest(dMrt).

rESuLtSAnddIScuSSIon

Proximate composition of Arenga endospermsProximate analyses show that the endosperm of sugarpalm nuts at 8–12, 16–18, and 22–24 mo old, respectively, contained 90.23–92.28% water, 1.57–3.11% protein,3.42–4.09% carbohydrates, 1.59–2.50 crude fiber, and 0.27–0.67crudefat(table1).

Table 1. Proximate analysis (dry basis) of endosperms of Arenga sacchariferaLabill.atdifferentstagesofnutmaturity

Samples(montshold)

%moisturecontent

%ash %crudefat

%crudeprotein

%crudefiber %nFE

8-12(young) 92.28b 0.12a 0.27a 1.42a 2.50b 3.42a

16-18(mid-ma-

ture)92.09b 0.29b 0.42b 1.57a 2.06ab 3.57a

22-24(mature) 90.23a 0.30b 0.67c 3.11b 1.59a 4.09b

Meansfollowedbyacommonletterinacolumnarenotsignificantlydifferentat5%leveldMrt

the samples at 8–12 and 16–18 mo old were notsignificantlydifferentintermsofmoisture,crudeprotein,crudefiber,andcarbohydrateswhilesamplesat16–18and22–24 mo old were not significantly different in terms of ash and crude fiber. crude fat content significantlyincreased during maturation of the nut. Significantincreases in crude protein and carbohydrate contentsof the endosperm of the maturing nut were observed,while moisture and crude fiber contents significantlydecreased.

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Isolation of gums from sugar palm endospermtheendospermsofsugarpalm(A. saccharifera)nutsdifferedintextureandweightastheymatured(table2).theyoungsugarpalmnutendosperm(8–12moold)wastransparent,verysoftand,lightestwithanaverageweightof0.82g/nut(Figure1).the16–18mooldnuthaswhiteendosperm,hardcenter,andwasheavierwithanaverageweightof1.35g/nut.The 22–24 mo old mature nut has white hard endosperm and washeaviestat2.50g/nut.thedifferentsamplescanthusbedescribedasyoungforthe8–12moold,midmatureforthe16–18 mo old, and mature for the 22–24 mo old nuts.

thepurifiedgumatdifferentstagesofnutmaturityischaracterizedbywhite,thread-likeprecipitate,whichupondryingproducedapowder-likesubstance(Figure2).themost mature sample (22–24 mo old) has the highest gum contentwith3.72%,followedbythemid-maturesample(16–18 mo old) with 2.71%, and the youngest sample(8–12moold)with1.27%.

thesugarpalmendospermgumisolateswereinsolubleinorganicsolvents;mostlysolubleininorganicsolventssuchassalts,acidsandbases,and;slightlysolubleincoldandhotwatersimilartothecharacteristicsofcommerciallyavailablegums,exceptforgumarabic(table3).

Table 2.PhysicalcharacteristicsandgumcontentofArenga sacchariferaLabill.nutmaturity

Samples(montshold)

colorofnut

textureofendosperms

Ave.weightofendosperm/nut(g) Gum(%)1

8-12(young)

darkgreen Soft 0.82 1.27a

16-18(mid-mature) Green Softw/hard

center 1.35 2.71b

22-24(mature) Green Hard 2.50 3.72c

1Meansfollowedbyacommonletterinacolumnarenotsignificantlydifferentat5%leveldMrt

Table 3.Solubilitypropertiesofgumisolatesandsomecommerciallyavailablegumsat30°c

testSolvent

GumIsolates2

GuarGum

Gumtragacanth

Gumarabic

Gumghatti

organicsolvents3 - - - - -

5%nacl +/- + + + +

5% +/- + + + +

naHco3

5%naoH + + + + +

5%Hcl + + + + +

5%H2So4 + + + + +

coldH2o +/- +/- +/- + +/-

HotH2o +/- +/- +/- + +/-1Variousorganicsolvents(petroleumether,acetone,chloroform,benzene,methanol,ethanol,isopropanolandbutanol),inorganicsolvents(5%Hcl,5%H2So4,5%H3Po4,5%naoH,5%naHco3,1Mand0.1Mnacl),andhotandcoldwaterwereaddedtothedriedpurifiedgumextract.dissolutionofsamplesintestsolventswasobserved.2Similarresultswereobtainedforthethreesamples,young(8–12moold),mid-mature (16–18 mo old) and mature (22–24 mo old).3organicsolvents:petroleumether,acetone,chloroform,benzene,methanol,ethanol,isopropanolandbutanol.Legend:+,soluble;+/-,partiallysoluble;-,insoluble

thedegreeofbranchingorgalactosecontentof thegumscouldexplainthesolubilityofgumssincetheextensionofmannanchainsbygalactose sidechainsprevents theformationofhydrogen-bondedintermolecularassociation.

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Figure 1.thesugarpalmfruitsat8–12montholdofmaturity

A B

cFigure 2.GumisolatesfromtheendospermofArenga sacchariferaat

differentstagesofnutmaturity:(A)Young(8-12monthsold), (B)midmature (16-18monthsold) and (c)mature(22-24 months old).

Forthegumisolates,sincetheycontaingalactosesidechainsintheirstructureorarehighlybranched[asshownlaterinthepaperandalsobyKooiman (1971)], theybehaveaspartiallysolubleinhotandcoldwater.thesamebehaviorwasobservedwiththeothercommercialgumssuchasguargum,gumtragacanth,andgumghatti. Guargumformsviscoussolutioninhotandcoldwaterduetoitshighdegreeofbranchingorhighgalactosecontentalongthemannanchain.Forgumtragacanth,itssolubilitybehaviorisduetoitssolublefraction,tragacanthin,andinsolublefraction,bassorin(60–70%ofitsstructure)(rowson1937).Forgumghatti,italsoformsaviscoussolutionbecauseithasasolublefraction(almost90%)andinsolublefractioninitsstructure(Stoloff1958).Gumarabicisexceptionalsinceitistotallysolubleinwaterduetothepresenceofneutralorslightlyacidicsaltofacomplexsaccharidecontainingcalcium,magnesium,andpotassiumionsthatarehighlywater-soluble.

the gum isolates and the commercial gums gavepositiveresultsusingtheBenedict’stest.thisconfirmsthepresenceofreducingsugarintheirstructurethatisusefulinverificationofthegum’sstructure.

thedifferentgumisolatesandcommercialgumswerealsopositivetotheninhydrintestindicatingthatthesamplesalso containedproteins.theseproteins likely are fromcytoplasmicproteins,whichco-precipitatedduringisolationandpurificationof theguminaddition to intrinsicwallprotein.theproteincanalsobelinkedtogalactomannanchainindicatingthatthegummayalsobeaglycoprotein(tizardetal.1989;ValentineandSalyers1992).

Qualitativeanalysisprovesthatthegumisolatescontainnotonlycarbohydratesbutalsoproteins.thepresenceofproteinsalsocontributestothephysicochemicalpropertiesofthegum.Also,thestudyontheattractiveinteractionsbetweenproteinsandcarbohydratesisimportantinmanybiologicalsystemsandinpharmaceuticalproductsandprocessedfood(e.g.purificationofmacromolecules,microencapsulationofingredientsorcosmetics,fatsubstitutes,meatanalogues,films, coating,packagingandothers) (dickinson1998;doublieretal.2000;deKruifandtuinier2001;SanchezandPaquin1997;tolstoguzov1996).

Physicochemical characteristicstheArengagumisolatesaswellastheothergumstestedwere found to be optically active (Table 4). Optical rotationofguargumbeforeinversionwaslowest,whilethatofthemid-mature(sampleB)Arengawasthehighest.Afterinversion,gumtragacanthhasthelowestwhereasguar gum has the highest. Before inversion, the gumsweredextrorotatory.AfteradditionofHcl,theybecamelevorotatory.thechangeinopticalrotationofthegumsolutionssuggeststhatthesugarsunderwentmutarotationandindicatesthattheycanbedigestedoracteduponbyenzymes in thehumanbody if theycouldformthed-enantiomers,thustheyaresafeasfoodadditives.AdditionofHclorpartialacidhydrolysiscouldhavecleavedthegalactosesidechainsthatisα-linkedalongthemannanchain.thealpha(α)linkagewaseasilycleavedbecausetheβ-linkage has relatively greater stability (MorrisonandBoyd1987).

Table 4. Physicochemicalcharacteristicsof thegumisolatesatdifferentstagesofnutmaturityandsomecommerciallyavailablegums

PhysicochemicalProperties

Samples

A(young)

B(mid-mature)

c(mature)

Guargum

Gumtragacanth

Gumarabic

Gumghatti

Specificrotation

Before +32.97 +39.53 +35.93 +24.93 +26.05 +31.98 +33.72

After -35.68 -41.70 -41.03 -44.18 -31.95 -38.47 -37.43

SpecificGravity 1.0035a 1.0055a 1.0055a 1.0065a 1.0065a 1.0045a 1.0045a

Gravitywaterholdingcapacity 42.55d 47.00c 47.28c 100.0a 53.68b 7.49e 44.90cd

Gelatinization 30-70 30-70 30-70 30-60 30-62 45-90 30-65

temperature(c)ActualViscosity(cPS)

0.5% 100b 115b 120b 1400a 320b 95b 310b

0.75% 130b 150b 160b 2000a 360b 110b 340b

1.00% 370d 440c 630bc 3400a 820b 200d 800b

2.00% 980d 1160d 1550cd 18700a 3400b 250e 2000c

1Meansfollowedbyacommonletterarenotsignificantlydifferentat5%leveldMrt.

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the specificgravityofguargum,gum tragacanth,gum arabic, and gum ghatti from literature were 0.9,1.384, 1.487, and 1.08, respectively (Neukom 1989), whichwerealmostthesamewiththeexperimentalvaluesforcommerciallyavailablegums(1.0065forguargumand gum tragacanth, and 1.0045 for gum ghatti and gum arabic).thedifferencesinvalues(rangedfrom1.0035to1.0065)werenotsignificantbetweenthegumsamples(Table 4) since specific gravity is a constant that varies slightlyforanygivenkindofcarbohydrates.Itisoflittlevalueindeterminingthepurityofthesampleanditcanonlybeusedinthecharacterizationofagivensample.

Water-holdingcapacityistheabilityofthegumtoholdwaterandisequaltothemoisturecontentofthegumafterequilibriumhasbeenestablishedunderagivencondition.thewater-holdingcapacityofthegumisolatesincreased with nut maturity from 42.55% to 47.28%, with more mature samples (16–18 mo old and 22–24 mo old)beingsignificantlydifferentfromtheyoungsample(8–12moold).However,theA. sacchariferagumhadabout50%lowercapacitytoholdwatercomparedtoguargumbuthadaboutseven(7)timesgreatercapacitytoholdwaterthangumArabic.Itswaterholdingcapacityisalsocomparabletothatofgumghatti,whichisalsoa galactomannan (Hirst and Jones 1948; Tischer et al. 2002).thewaterbindingcapacityofgumsdoesnotonlydependonthefunctionalgroupofcarbohydratesthatarehydrophilicbutalsototheproteinspresentinthegumssincetheyalsocontainfunctionalgroupsthatareabletobindwithwatermolecules(Jaurigue1981).

Gelatinizationtemperatureisarangeoftemperaturewithinwhichagumstartstoswellirreversiblyinhotwateruntilthesolvent(liquid)hasbeenabsorbedduetoswellingofthegumandfinallybecomesimmobilized(Glicksman1969). Table 4 shows the gelatinization temperatures of Arenga gum isolates and some commercial gums.the3Arengagums(at2%solution)hadgelatinizationtemperature from 30–70° c and formed gels at roomtemperature. Similarly, the commercial samples (2%solution)except forgumarabicalsohadgelatinizationtemperaturestartingat30° cupto60to65° cand,thus,also formed gel at room temperature. Gelatinizationtemperature is influenced by the structure of gumsas well as its solubility behavior. the formation ofviscoussolutionsofvariousgumsamplesevenatroomtemperaturecouldbeduetothedegreeofbranchingandmonomerunitsthatmakeupthesegums(Stoloff1958).Gumarabicistotallysolubleorisnotviscousevenupto2%concentrationofgumsolutionduetosomeneutralsaltspresentinitsstructurewhicharehighlysoluble(Meer1977). This gum can form viscous solution at 40–50% concentration.

Viscosityvaluesofgum isolates,gum tragacanth,gum Arabic, and gum ghatti at 0.5% and 0.75%concentration of gum solution are not statisticallysignificantfromeachother,withtheexceptionofguargum (Table 4). Viscosity values at 1% concentration showsthatArengagumfromthemostmaturenutsample,gum ghatti, and gum tragacanth are not significantlydifferentfromeachother,whiletheviscosityvalueofsampleA is not significantlydifferent to gumarabic.Guargumhadthehighestviscosityamongthesamples.therewasnosignificantdifferenceinviscositybetweenandamongthe3Arengasamplesat2%concentration.However,thevaluesaresignificantlydifferentfromtheothergumsamples,withguargumasthehighestandgumarabicthelowest.Itcanbeobservedthatviscosityof the gum isolates increases with concentration anduponmaturity.

thephysicochemicalpropertiesofthegumsuchasspecific gravity, water-holding capacity or hydration,andviscosity canbeattributed to thegum’s structuresuchas the typeandnumberofmonosaccharidesandtheirconfigurationand the type,numberand locationof the linkedgroup (Kuntz1999).Specificgravityofthe solution is nearly equal to 1 which is the densityof water that controls the distribution of particles inthe solution (Kuntz 1999). thus, addition of othersubstances to thegumsolutioncanbeaccommodatedandmayalterthecharacteristicbyimprovingthetextureofaddedmaterial.Viscosityandwater-bindingcapacityplay important roles in food processing. Gums withhighwater-bindingcapacityareusedasfoodadditivesthatcaninfluenceprocessingconditionssuchaswaterretention, reductionofevaporation rates,alterationoffreezingrates,andmodificationoficecrystalsformationasinthemanufactureoficecream.Gumsareusedtoimprove texture by modifying ice crystals and waterretention. In the preparation of soups and sauces,gums are used to improve body and its consistency.the presence of several —oH groups in guar gumcreatesanintermolecularinteractionwithneighboringhydrocolloids,which leads to the increasingviscosityof the solution. these properties are attributes of agoodcoatingagentfortextileandpaperduetostronggumabsorptionpower(WhistlerandBeMiller1958).thesepropertiesarealsoresponsibleinsuspendingandstabilizingfoodsystem.

Lowgelatinizationtemperatureofgumisusefulforproductsthatdonotrequireheatingoraresensitivetohightemperatureortextureimprovement.Asconcentrationof gum solution increases, heating time is prolongedforgelatinizationtooccur.However,prolongedheatingat high temperature leads to degradation of gums

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(Goldstein andAlter 1959). Formation of gel is animportantcharacteristicofhydrocolloidsinfoodindustrysuchastextureimprovementandstabilizationoffoodproducts.

Chemical propertiesPercent totalsugarcontentof thegumisolatesslightlyincreased with increasing maturity of the nut: young(8–12 mo old), 59.6%; mid mature (16–18 mo old),

Table 5.chemicalpropertiesof thedifferentgum isolates and somecommerciallyavailablegums

Samples totalsugars(%)

totalreducingsugars(%)

totalprotein(%)

A(young) 59.6a 3.69b 1.36c

B(mid-mature) 65.6b 3.87b 1.67bc

c(mature) 68.6b 3.94b 2.11b

Guargum 98.2d 1.89a 3.69a

Gumtragacanth 88.4c 3.79b 4.38a

GumArabic 70.4b 1.88a 1.03d

GumGhatti 85.2c 1.34a 1.64bc

1Meansfollowedbyacommonletterarenotsignificantlydifferentat5%levelbydMrt

65.6%; and mature (22–24 mo old), 68.6%, and were significantlydifferentfromcommercialgumsexceptforgumArabic (table5).Guargumhad thehighest totalsugars (98.2%) followed by gum tragacanth (88.4%), gum ghatti(85.2%),whilegumarabichadthelowestpercentsugar (70.4%).

thepercentreducingsugarofgumisolates(young,3.69%; mid mature, 3.87%; and mature, 3.94%) did not change significantly with maturity (table 5). Forcommercial gums, gum ghatti (1.43%) had the lowest reducing sugar while gum tragacanth (3.79%) had thehighest but was not significantly different from gumisolates.

ProteincontentofArengagumsrangedfrom1.36to2.11%, lower than the 3.69% and 4.38% of guar gum and gumtragacanth,respectively,andsimilartothe1.03%and 1.64% of gum Arabic and gum ghatti.

Elution profile of the mixed standard containingrhamnose,arabinose,xylose,mannose,galactose,glucose,andmyo-inositolisshowninFigure3.retentiontimesof individual standards are 3.734 (rhamnose), 4.561 (arabinose), 5.394 (xylose), 9.672 (mannose), 10.511 (galactose),and11.257(glucose).

Monosaccharide composition of the gum isolatesatdifferentstagesofmaturityisshownintable6.All

Table 6.Physicalandchemicalpropertiesofthegumisolates

PropertiesSamples

A(young)

B(mid-mature)

c(mature)

Molecularweight >2M >2M >2M

%Branching1 2.31b 1.61a 1.13a

Monosaccharidecomposition

Gal (29.94%)Man (64.08%)

Gal(21.99%)Man(66.87%)

Gal (15.04%)Man (74.08%)

totalnumberofmonomerunits1 7013b 7720a 8072a

Mannose:Galactose(M/G)

ratio2:1 3:1 5:1

1Meansfollowedbyacommonletterarenotsignificantlydifferentat5%levelbydMrt

26



Figure 3. Gas chromatographic separation of the alditol acetatederivativesofamixtureofstandardsusingBP-225capillarycolumn with FIddetector. (1) rhamnose; (2)Arabinose;(3) Xylose; (4) Mannose; (5) Galactose; (6) Glucose; and (7)Myo-inositol.

gumisolatesregardlessofageshowed2peaksasshownin Figure 4 corresponding to mannose (64.08% for the young sample; 66.87% for mid mature sample; and74.08% for mature sample) and galactose (29.94% for theyoungsample;21.99%formidmaturesample;and15.04% for mature sample) and myo-inositol (internal standard).Highvaluesofpercenthydrolysisindicatethatthesamplesundergocompletehydrolysis.Mannosewasfoundtobethepredominantsugar,whichimpliesthatthe backbone of the carbohydrate is a mannan chain (1,4-linked)andgalactosemoleculesareprobablypresentassidechains(1,6-linked).theseresultsindicatethatthepolysaccharideinArengagumisgalactomannan,similar

Physical characteristicsthe 3 gum isolates of Arenga eluted starting at thevoidvolumeofSephadexG-200, indicating that theirmolecularweightsweregreaterthanorequalto2milliondaltonsandcouldconsistofmoleculesofvaryingsizes(Figure 5). Gums are reported to be high molecularweight molecules (e.g. 840 kDa for gum tragacanth, 580 kdaforgumArabic,and220kdaguargum)(WhistlerandMiller1958).

In the case of gum isolates, the water-bindingcapacity or hydration and viscosity increases uponmaturitypossiblydue to the fact thatmaturesample(22–24 month old nut) is a longer molecule and

to theobservationofKooiman (1971).themannose:galactose(MG)ratio(2:1fortheyoungsample;3:1formidmature;and5:1formature)increaseduponmaturity,which implies that thedegreeof branchingdecreasesas nuts of sugar palm matures. this also indicatesthatthegalactomannaninmorematurenutswouldbelesswater-solublethanthegalactomannaninyoungerendosperm.Inmutantandnormalcoconut,theMGratioofgalactomannanwas3atdifferentstagesofmaturity(Samonteetal.1987)theseresultsagreewiththeresultasthedegreeofbranching(seebelow).theaboveresultsalsosuggestthatgumfromolderArenganutsmayhavedifferentfoodandpharmaceuticaluses.

less substituted as indicated by results in periodateoxidation.AlthoughArenga gum isolateshadhighermolecular weight (~2,000 kda) than commerciallyavailablegumsas statedabove, theirviscosities andwater-holding capacity were lower possibly becausecommerciallyavailablegumsaremorehighlybranchedthanthegumisolates.

the degree of branching was determined byanalyzingthenumberofterminalresidues,whichareoxidizedbyperiodatetoformicacid(HcooH)whichismeasuredbytitrationwithsodiumhydroxide(naoH).the Arenga samples had 2.31%, 1.61%, and 1.13%,

27



Figure 4.Gaschromatographicseparationofthealditolacetatederivativesof(a)sampleA(8-12monthsold);(b)sampleB(16-18monthsold);and (c) sample C (22-24 months old) using BP-225 capillary column with FID detector (peak 1- mannose, peak 2- galactose and peak 3-myo-inositol)

respectively, for the young, mid mature, and maturesamples,respectively(table6).

thetotalnumberofmonomerunits(averagechainlength)didnotdiffersignificantlyamongthe3samples(table6).thisisinagreementwiththeapproximatemolecular weight value and viscosity values (2%concentration)obtainedfor3gumsamples.Ingeneral,asthegumincreasedinsize,viscosityincreased,too.the degree of branching, on the other hand, had anoppositeeffectonviscosity.Whenthemolecularsizeincreased(longermolecules)withdecreaseindegreeofbranchingdecreases,viscosityalsoincreased.

results of this study showed that the variousphysicochemicalpropertiesofArengagummakesitasuitablereplacementforotherwidelyusedgumssuchasgumghattiinfood,drug,andcosmeticapplicationsprimarily as stabilizer for oil-in-water emulsion(Glicksman1969).Gumghatti isalsoused toreplacegumarabic as emulsification agent inpharmaceuticalformulations (Mantell 1947). It has been used in the preparationofdry, stable,oil-solublevitaminproductmade by combining the active ingredients with gumghattianddryingandpulverizingtheblend(dunn1959).Infoods,ghattiiseffectivelyutilizedastheemulsifierandstabilizerinmaplesyrupscontainingbutterforuseinpancakesandwaffles(topalianandElsesser1966).Also,theArengagumisolateshaveanadvantageovergumghattibecauseArengagumsarewhiteandthusdonotimpartcoloredcomponentstofood.

the health uses of Arenga gum isolates shouldbe studied because recent studies on commerciallyavailablegumsshowedbiomedical importanceasidefromfooduses.Someofthereportedbeneficialhealtheffects of gums are: anticancer and high antioxidantactivities[guargum(Gamal-Eldeenetal.2006);Gumtragacanth(Pierreficheetal.1993];GumArabic(Aliet al. 2004); probiotic properties [ guar gum (Giannini etal.2006);gumghatti(Salyersetal.1977).

thus,thefoodandhealthusesofArengagumsorgalactomannansfromtheyoungandmatureendospermsshould be studied to widen the applications of thisunderutilizedplantresource.

AcKnoWLEdGMEntStheauthorswouldliketoacknowledgedr.EvelynMaetecson-Mendoza for her valuable contribution in thecompletionofthismanuscript.

rEFErEncESALIBH,ALGAWArIAAandALunELIH. 2004. Does

treatmentwithgumArabicaffectexperimentalchronicrenal failure in rats? Funda clin Pharmacol 18(3):327−329.

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0

0.5

1

1.5

2

2.5

3

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

Test tube number

Abs

orba

nce

(490

nm)

Sample ASample BSample C

Figure 5.ElutionprofilesofthegumisolatesatdifferentstagesofmaturityonSephadexG-200.(voidvolume)

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