extraction and characterization of “batuan” garcinia …...their dietary proteins. “batuan”,...

Annals of Tropical Research 36[2]89-103(2014)© VSU, Leyte, Philippines

Correspondence : E.S. Quevedo Address: Department of Pure and Applied Chemistry, Visayas State University, Baybay City, Leyte 6521-A E-mail: [email protected]

Extraction and Characterization of “Batuan”[Garcinia binucao (Blco.) Choisy] Seed Protein

1 2Elizabeth S. Quevedo , Marivic S. Lacsamana and Antonio C. 3Laurena

1DepartmentofPureandAppliedChemistry,VisayasStateUniversity,BaybayCity,2Leyte6521-A, InstituteofChemistry,UniversityofthePhilippinesLosBaños3College,Laguna, InstituteofPlantBreedingCropScienceCluster,Universityofthe

PhilippinesLosBañosCollege,Laguna

ABSTRACT

“Batuan” [Garcinia binucao (Blco.) Choisy], an indigenous, lesserknown member of the Gutifferae family with export potential isunderutilizedandunderstudied. Thepresentstudywascarriedouttoextractandcharacterizetheproteinin“batuan”[Garciniabinucao(Blco.)Choisy] seeds for nutritional quality assessment. Protein content of“batuan”seedmealwas8.9±0.59%drybasis. Solubilityfractionationof“batuan” seed meal showed globulin and glutelin as the major seedproteins.SDS-PAGEresolvedtheglobulinandglutelinintothreegroupsofpolypeptideswithmolecularweightsofabout20–54kDa.Aminoacidanalysisrevealedthatseedproteincontainedalltheessentialaminoacidswithleucineasthemostabundantwhiletryptophan,theleast. “Batuan”seedproteinsweremostlymadeupofacidicandhydrophobicaminoacidswith glutamic acid (2.67%) as the highest. Nutritional assessmentsincludingE/T(38.4%),aminoacidscore(1.6%),predictedPER(3.2-3.7)andestimatedBV(98.3%)suggestedthattheseedproteinsareofgoodquality.Hence,“batuan”seedshasapromisingpotentialasanimportantsources of valuable proteins and amino acids for use as foodsupplement/enhancingingredient.

Keywords:aminoacid,“batuan”seedprotein,nutritional,tryptophan

[email protected]

Typewritten text

DOI: 10.32945/atr3625.2014

INTRODUCTION

Seed storage proteins which accumulate in high amounts in someplantseedsassourcesofnutrients forgerminatingseedsconstituteanimportant source of dietary proteins for human consumption. In thePhilippines,manyedible,indigenous,seed-bearingfruittreeswhichareknownonlybythelocalpeopleprovideincomeaswellascheapsourcesoftheirdietaryproteins.“Batuan”,Garciniabinucao(Blco.)Choisy,isoneofthe lesser-known, indigenous, seed-bearing fruit trees identified withexportpotential (de laCruz,2013). The fruitwhich ismainlyusedassouring agent in native dishes contains several (more or less 7) large,edibleseeds(Quevedo,2013;FloridoandCortiguerra,2003). However,thequalityandquantityoftheseedproteinof“batuan”fruit(Figure1)remainslittleifnoneatallhasbeenpublishedtodate. Quevedo(2013)revealedthattheproteincontentof“batuan”fruitsisgenerallylow(lessthan10%)withtheseedscontainingthehighest. Characterization of the seed storage proteins in “batuan” fruit isimportantsinceitisconsumedandconsideredasafavoritesouringagentbyFilipinosespeciallyinsomepartoftheprovincesofNegrosOccidental,Iloilo,Leyte,MasbateandinotherplacesinthePhilippines(FloridoandCortiguerra,2003;Cojuangco,2012;delaCruz,2013). Duringfruitingseason,thefruitsareprocessedintovariousproductssuchassaltedpuree,jam, jelly,prunes, and candies. Thus, information in termsofproteinquality and quantity are very valuable and may make significantcontributiontoitsnutrientqualityandprocessingpropertiesaswellasitspotentialapplicationinfoodprocessing.Furthermore,analysisoftheseedstorageproteinsin“batuan”mayalsoprovideinsightintocharacterizationof genetic diversity, improvement of nutritional quality, and seeddevelopmentfor“batuan”. Thisarticlepresentstheresultsontheextractionandcharacterizationoftheproteinin“batuan”seed.“Batuan”seedproteinswereevaluatedfortheirrelativeamountandaminoacidcomposition. Anattemptwasalsomadetopredictitsnutritionalquality.

90Quevedo et al.

Figure1. A. The “batuan” [G.binucao (Blco.)Choisy] fruits.B. Cross-sectionof“batuan”fruitwithseeds.

MATERIALS AND METHODS

Materials

Seedsof“batuan”wereobtainedfromharvestedmaturefruitsatthePomologyProjectoftheVisayasStateUniversity,Visca,BaybayCity,Leyte.ThefruitswereauthenticatedfromtheUniversityofthePhilippinesLosBaños(UPLB)MuseumofNaturalHistory,College,Laguna,Philippines. Analyticalgradeacrylamide,ammoniumpersulfate(APS),bis-acrylamide,molecular weight markers 14,400-200,000 Da (BIORAD), bromphenolblue, Coomassie Brilliant Blue G-250, dihydrate hydrogen phosphate,glycine, potassium dihydrogen phosphate, sodium chloride, sodiumdodecyl sulfate (SDS), sodiumhydroxide, andTrizma base, acetic acid,Bradford reagent, ethanol, hydrochloric acid, beta-mercaptoethanol,N,N,N',N'-tetramethylenediamine (TEMED) were procured fromcommercialsuppliers.Thedialysistubingcellulosemembrane(MWCO12,000)waspurchasedfromSigmaChemicals.

PreparationoftheDefatted“Batuan”SeedMeal

Dried“batuan”seedendospermwasgroundinablender,thenpassedthrough a 40-mesh sieve and defatted using direct solvent (hexane)

91Extraction and Characterization of “Batuan” Seed Protein

A B

methodwithconstantstirringatroomtemperatureunderthefumehoodfor24hr. Themealwasstoredat5°Cafterremovalofhexaneandair-dryingunderthefumehood.

ProteinFractionation

Storageproteinsfromthedefatted“batuan”seedmealwereextractedwithstirringat4ºCfor1hrin1:20w/vwater,0.5MNaCl,70%ethanoland0.1 M NaOH according to the Osborne solubility procedure (Osborne,1924). Allextractionswerecarriedouttwice. Albumin,globulinandglutelinfractionsweredialyzed. Allproteinfractionswerefreeze-dried,thenthedriedsampleswerestoredat5°Cforsubsequentuse.

PreparationofTotal“Batuan”SeedProteinExtract

Total“batuan”seedproteinwasobtainedfromdefattedseedmealbydispersingat1:2w/vin20mMTris-HClbuffer. Themixturewasstirredfor2hat4C,centrifugedat10,000rpmfor10minat4ºCandfilteredthroughWhatmanNo.4filterpaper. Thesupernatantwasfreeze-driedandthedriedsampleextractwasstoredat4°Cforsubsequentuse.

ProteinContent

ProteincontentsintheextractandtheOsbornefractionswereassayedbyBradford'smethod(Bradford,1976)usingbovineserumalbumin(BSA)as protein standard. Protein concentrations of the samples werecalculated using the linear regression formula from the bovine serumalbumin(BSA)standardcurve.

SDS-PAGEAnalysis

Theestimatedmolecularweightsofthepolypeptidesinthedifferentprotein solutions (crude extract and Osborne protein fractions) weredetermined by the sodium dodecyl sulfate polyacrylamide gelelectrophoresis(SDS-PAGE)using12.5%polyacrylamidegelaccordingtoLaemmli (1970) and low molecular weight protein standard markers(BIORAD). ThelyophilizedcrudeextractandOsbornefractionsweredissolvedin1mLof20mMTris-HClbufferatpH8.8. Thesolutionwasthencentrifugedat10,000rpmfor2minat4ºC toobtain thepurified

92Quevedo et al.

sample. AliquotsoftheextractandOsbornefractionswereaddedwithsamplebuffer,heatedfor5minandcooledtoroomtemperaturebefore

-1loadingtothewellsataconcentrationof0.25mgµL .CoomassiebrilliantblueR-250wasusedforstaining.

AminoAcidAnalysis

Dried,powdered,defattedseedendospermmealsamplewassentforamino acid content analysis at the SGS Thailand, Inc. and analyzedfollowing their in-housemethod based on the protocol for amino acidanalysis in the Handbook of Food Analysis Vol. 1 and using GasChromatography-MassSpectrophotometry.

EvaluationofNutritionalParameters

Theaminoacidcompositionwasusedtoestimatethenutritionalvalueof theproteinbasedon theprovisional aminoacid scoringpattern forpreschoolchildren(WHO,2007).Theparameterssuchastheproportionofessentialaminoacidswithtotalaminoacids intheprotein(E/T%),amino acid score or chemical score, predicted protein efficiency ratio(PER) and estimated biological value (BV) were used for nutritionalquality assessment. PER values were estimated using the regressionequationsproposedearlierbyAlsmeyeretal.(1974)whichareoutlinedasfollows:

PERI=-0.684+0.456(Leu)-0.047(Pro) PERII=-0.468+0.454(Leu)-0.105(Tyr) PERIII=-1.816+0.435(Met)+0.780(Leu)+0.211(His)-0.944(Tyr)

BVofproteinin“batuan”seedwasestimatedusingtheequationdevelopedbyMørupandOlesen(1976)andisshownbelow:

where:

or

ai=mgofaminoacidpergoftotalessentialaminoacids.


Statistical Analysis

Protein content and SDS-PAGE analyses were done in triplicates while amino acid analysis in duplicate. Results on protein content were reported as mean ± SD.

RESULTSANDDISCUSSION

Fractionationof“Batuan”SeedProteins

Thecrudeproteinof“batuan”seedendospermonmoisturefreebasiswas 8.9±0.52%. Osborne's protein fractionation procedure extracted92.82% of the proteins (Table 1) and contained albumin, globulin,prolaminandglutelin. Globulinsarethemostabundant,accountingforabout67.49%ofthetotalsolubleproteinsfollowedbyglutelin(20.40%),albumin(3.81%)andprolamin(1.12%).ThisfindingissimilartothoseofMoringaseedswhereglobulinsandglutelinsconstitutethemajorstorageproteins(Reyes,2010). Furthermore, therearenopreviouslyreporteddataontheseedstorageproteinsof“batuan”inthePhilippinesandfromothercountriestocomparetheresultsofthispresentwork.

Table 1. Seed storage proteins (%) of "batuan" fractionated using the Osborne solubility scheme.

PROTEIN CONTENT (% db)1 % OF TOTAL PROTEIN CONTENT

Initial Protein 8.92 ± 0.59 Total soluble protein 8.28 ± 0.61 92.82

Albumin 0.34 ± 0.02c 3.81

Globulin 6.02 ± 0.05c 67.49

Prolamin 0.10 ± 0.00a 1.12

Glutelin 1.82 ± 0.54b 20.4

Nonsolubilized residue 0.025 ± 0.00 0.28

Total crude protein recovered 8.305 ± 0.61 93.1

MolecularWeightDeterminationbySDS-PAGE

ThedifferentOsbornefractionsfromthedefatted“batuan”seedmealwerefurtherresolvedonSDS-PAGEandtheelectrophoretogramisshown

94Quevedo et al.

1Averageof3trialsMeanswithinacolumnfollowedbyacommonsuperscriptarenotsignificantlydifferentat5%level,LSD.

inFigure2. Globulin fractionsrevealedpolypeptidesof lowmolecularweights at approximately 51.85, 27.52 and 20.97 kDa. The glutelinfractions also exhibited three prominent polypeptide bands withmolecularweightsofabout53.92,27.92and21.06kDa.

Figure2.SDS-PAGEoftheseedstorageproteinfractionsof“batuan”.Lane:1-prolamine;Lane2–albumin;Lanes3&4–glutelins;Lanes5&6–globulin;Lanes7&8–totalprotein;Lane9–proteinstandard

Meanwhile, no bandswere observed in the albumin, and prolaminfractions.Thisisprobablyduetothelowproteincontentinthealbuminorprolaminfractions.However,itisalsopossiblethattheproteincontentforalbuminandprolaminobtainedusingBradfordassaywasanestimateforothercompoundssuchaspolyphenolsandsomealkaloidsthatwerealsoextractedbythesolventsfromtheseedmatrix. TheseimpuritieswhenpresentinhighconcentrationintheproteinsamplemayhaveinterferedwiththeassaybyassociatingwiththeCoomassiedye,givingaresponseat595nm.ComptonandJones(1985)pointedoutthatbases,detergentsandothernon-proteincompoundswhenpresentathighconcentrationintheprotein sample interfere with Bradford assay, resulting in an overestimationoftheproteincontent.

Thetotalproteinextractexhibitedlessprominentpolypeptidebandofapproximately21.32kDaidenticaltotheglobulinandglutelinbands.The


resultimpliesthattheseparationgelbuffer(20mMTris-HCl)usedintheextraction of the total proteins in defatted “batuan” seedmeal did notefficiently solubilize most of the proteins present. Probably, theconcentration of Tris-HCL in the separation buffer (20 mM) was notenoughtoisolatethesolubleproteinsintheendosperm.Tris-HClwasusedastheextractantofthetotalproteinsinthedefatted“batuan”seedmealsinceitisacomponentoftheseparationgeloftheSDS-PAGE,andhasbeenoneofthewidelyusedextractionbufferinproteinpurificationaswellasintheextractionofnucleicacids. Globulins,themostwidelydistributedgroupsofstorageproteinsareoligomericproteinsconsistingof2ormoresubunitsthatcanbeseparatedundermilddissociatingconditions.BewlyandBlack(1994)pointedoutthat these subunits are composed of a number of polypeptide chainscontainingvariableaminoacidsequencebetweenhomologoussubunits. Basedontheirsedimentationcoefficients,globulinsareclassifiedasbeingthe7Svicilin-typeandthe11Slegumin-typewhichexhibitconsiderablevariationintheirstructureresultingfromposttranslationalprocessing. In thisstudy,both theglobulinand theglutelin fractionscontainedthree groups of polypeptides with almost similar molecular weights. However,the53.92kDaglutelinbandwasnotveryvisiblecomparedtothe51.85kDaglobulinbandwhichmaybeduetoalowerconcentrationofglutelincomparedtoglobulin. Analysisofthe21.06kDaoftheglutelinbandshowedsimilarityinthemolecularweightwiththe19–22kDabasicsubunitofriceglutelins(Robertetal.,1985;Kagawaetal.,1998).Severalearlierstudiesreportedthatglutelinsshowingvariationsinstructuresandmolecularweightsarealsostructurallysimilartothe11Sglobulins(Zhaoetal.,1983;WenandLuthe,1985). Theseobservationsmayalsoexplainthesimilarityofthenumberofglutelinandglobulinpolypeptidesobtainedin this study. The identitiesaswellas thecharacteristicsof these lowmolecular weight polypeptides in “batuan” seeds have never beenpublished to date. Thus,more detailed studies to give insights on itscharacteristicsandpropertiesareneeded.

Amino Acids Profile

The amino acids composition of the “batuan” seed endosperm isshowninTable2.Glutamicacid(2.67%drybasis)wasfoundasthemostabundantaminoacidofthe“batuan”seedproteinsfollowedbyasparticacid (0.93%) and leucine (0.90%). This finding is similar with those

96Quevedo et al.

obtained in its close relative, mangosteen,G. mangostana (Ajayi et al.,2013)aswellastootherfruitseedssuchastamarind(Glewetal.(1997),andpigeonproteins(Etonihuetal.,2009),withglutamicacidcontentsof

-1 -1 -110.938g100g ,2.82mgg ,14.64g100g (dryweight),respectively. Moreover,thetotalcombinedamount(3.6%)ofacidicaminoacidswashigher than thoseof theunchargedpolaraminoacids(1.36%)and thebasic amino acids (0.48%) and is almost the same as that of thehydrophobic amino acids (3.5%). This suggests that the proteins in“batuan” seeds and their protein fractions may be of acidic nature.Moreover,theacidicandhydrophobicaminoacidsin“batuan”seedsarealmostequalinproportion. Theresultssuggestthatthelowermolecularweightpolypeptidesin“batuan”seedsmayhaveamphiphaticcharacter,havingbothahydrophobicregioninteractingwithlipidsandapositivelychargedhydrophilic region interactingwithwaterandothernegativelychargedresidues.

Table2. Aminoacidcomposition(%drybasis)of"batuan"[G.binucao(Blco.)Choisy]seedsandthepercentage(%)essentialaminoacidrequirementsforpreschoolchildren(3-10years)(WHO,2007)

.

AMINO ACID BATUAN SEED PROTEIN

(%)1 WHO (2007)

(%)

Valine 0.52 2.9

Leucine 0.90 4.4

Isoleucine 0.43 2.3

Threonine 0.37 1.8

Methionine 0.15 1.8

Phenylalanine 0.47 3.0

Lysine 0.27 3.5

Histidine 0.21 1.2

Tryptophan 0.11 4.8

Total essential amino acids (E) 3.43 20.9

Alanine 0.43 Glycine 0.43 Serine 0.25 Proline 0.48 Aspartic acid 0.93 Glutamic acid 2.67 Tyrosine 0.27 Cystine 0.04 Total nonessential amino acids (N) 5.50

Total amino acids 8.18

Classified distribution of amino acids 1. Hydrophobic 3.51

2. Uncharged polar 1.36 3. Basic 0.48 4. Acidic 3.60

1Average of 2 measurements


MurrayandRoxburgh(1984)asreportedbyTounkaraetal.(2013)alsorevealedthathigh levelsofalbuminelevatedthesulfur-containingaminoacids in chickpeaproteins. In thepresent study, theamountofsulfur-containing amino acids which are most likely associated withalbumins were low, supporting the observation of the absence or thepresenceoftraceamountofalbuminin“batuan”seedproteins.

Table2 further shows that “batuan” seedendospermcontainsnineessentialaminoacidswithatotalcombinedamountof3.43%drybasis. Leucine(0.90%)wasthemostabundantfollowedbyvaline(0.52%)andphenylalanine (0.47%). However, the total essential amino acids in“batuan”seedprotein(3.43%)waslowerthanthe20.9%total essentialaminoacidrequirementforpreschoolchildren(WHO,2007)buthighercomparedtothereported0.58%essentialaminoacidsintamarindseed(Glewetal.,1997).Nonetheless,“batuan”seedscouldsupply16.4%totalessentialaminoacids,10.6%totalS-containingaminoacidsand24.72%totalaromaticaminoacidsofthereferencevaluesasreportedbyWHO(2007). AspointedoutbyKrzysciak(2011),thearomaticaminoacids,beside their structural function in proteins, are precursors of manyimportant biological compounds including neurotransmitters andhormonesinthehumanbody. Atmaon(2004)alsoreportedthattheS-containingaminoacids cysteineandmethionineareknown topossessantioxidantpropertywhichisinfluencedbythetypeofoxidantstressandthephysiologicalconditions. Inaddition,leucine,theonlydietaryaminoacidwith the capability of stimulatingmuscle synthesis (Etzel, 2004),having the highest value (0.9%) among the essential amino acids in“batuan” seed, couldalsodeliver20.4%of required leucine. Thus, theamountof essential aminoacidspresent in “batuan” seedswouldbe asignificantvalueadditionorcontributiontoitsnutritionalqualityaswellasagoodcomplementarysourceofdietaryproteins.Atpresent,therearenopreviouslyreporteddataontheaminoacidcontentof“batuan”seedproteinsinthePhilippinesandfromothercountrieswithwhichtheresultsofthispresentworkcanbecompared.

EstimationofNutritionalQualityandQuantity

The nutritional quality of protein is principally determined by itsaminoacidscomposition.Certainnutritionalparameterswerequantifiedusingtheaminoacidcomposition(Table2).

In 1973, the World Health Organizations had suggested the

98Quevedo et al.

provisionalaminoacidscoringpatternforanidealprotein. Thepatternrecommendsthatforagoodprotein,theE/Tratioshouldbeatleast36%.“Batuan”seedproteinexceededthiscriterionsuggestingthattheproteinsof“batuan”seedareofgoodquality.Moreover,theobtainedE/Tratiofor“batuan” seed proteins is higher compared to the 30.54% for cassavatuber-associatedproteinsobtainedbyAzucena(2005). ComputationofthepredictedPERandestimatedBVfor“batuan”seedproteinbasedontheiraminoacidcompositionwasverysatisfactory.ItisclearfromthecalculatedvaluesinTable3thattheequationsdevelopedbyAlsmeyeretal.(1974)forpredictingPERandbyMørupandOlesen(1976)forestimatingBV,originallydesignedformeatproducts,wereapplicableto“batuan”seedproteins. Ingeneral, theproteinefficiencyratio(PER)below1.5impliesaproteinofloworpoorquality,whilebetween1.5and2.0indicatesanintermediateproteinqualityandabove2.0meansofhighqualityprotein(Friedman,1996). ThepredictedPERvaluesfor“batuan”seedproteincalculatedusingPERequationsItoIII(3.2to3.7)asshowninTable3,wereintherangeofhighqualityproteinandrelativelycomparablewiththereportedPERof3.1forwholeegg,&higherthanthe2.9forbeef,2.5 forcasein&cow'smilk,2.2 forsoyprotein,&0.8 forwheatgluten(Hoffman&Falvo,2004).Thisimpliesthatthe“batuan”seedproteinisofbetterquality.

Table 3. Nutritional evaluation of "batuan" seed proteins.

NUTRITIONAL PARAMETER TOTAL "BATUAN"

SEED PROTEIN

E/T, %1 38.4

Amino acids score (%)

Tryptophan 1.7

Lysine 5.6

Methionine + Cysteine 6.2

Phenylalanine + Tyrosine 11.5

Leucine 14.7

Valine 13.0

Isoleucine 18.7 Histidine 32.5

Predicted Protein Efficiency Ratio

PER I 3.2

PER II 3.3

PER II 3.7

Estimated Biological Value 98.23

1E/T, % = proportion of essential amino acids and total amino acids. Amino acid scores were evaluated using the reported essential amino acid pattern as reference (WHO, 2007).


Theestimatedbiologicalvalue(BV)of“batuan”seedproteinwaslowercomparedtothereported100%ofegg,buthigherthanthe93%ofmilk,75%of beef&fish,72%ofcorn(Whitney&Flores,2010),74%ofsoyprotein, & 64% of wheat gluten (Hoffman & Falvo, 2004). The highestimatedBVof theproteinof “batuan” seed suggests that theproteinwouldbeefficientlyutilizedbythebody.However,nocomparisoncouldbemadeonthepredictedBVofthe“batuan”seedproteininotherplacesinthePhilippinesandothercountriesduetolackofpublishedstudiesonnutrientevaluation.

CONCLUSION

Globulins (67.49%) and glutelins (20.40%) were the major seedsstorageproteinsof“batuan”basedonOsbornefractionationschemeandBradfordassay. ThreegroupsofpolypeptidesintheOsborne'sglobulinandglutelinfractionswithmolecularweightsrangingfrom20–54kDawere resolved on SDS-PAGE. Glutamic acid (2.67%) was the mostabundantaminoacidin“batuan”seedproteinfollowedbyasparticacid(0.93%)andleucine(0.9%).“Batuan”seedproteinscontainedmoreoftheacidic amino acid groups (3.60%) and the hydrophobic amino acids(3.51%)thantheunchargedpolar(1.36%)andbasicaminoacids(0.48%).Furthermore, all essential amino acids were present in “batuan” seedproteinandleucine(0.90%)wasthemostabundant.Ratioofessentialtototal amino acids (38.4%) exceeded the 36% criterion suggested byFAO/WHO/UNO(1975)forgoodqualityprotein. Basedonaminoacidscore,tryptophan(1.67%)wasthemostdeficientessentialaminoacid. PredictedPER(3.2-3.7)of“batuan”seedproteiniswithintherangeforefficientlyabsorbedandutilizedproteins.EstimatedBV(98.3%)wasalsohigh. Nutritionalassessmentssuggestedthat“batuan”seedproteinsareof goodquality and couldbea cheapnatural sourceof supplementarydietaryprotein.

ACKNOWLEDGMENT

TheauthorswouldliketothanktheDA-BIOTECHResearchFellowshipProgram, and the Visayas State University for funding this research;faculty, staffs and students of the Analytical Service and BiochemistryLaboratories,IPB,UPLBandtheQuevedofamilyfortheirextraordinaryandgeneroushelpinthisstudy.

100Quevedo et al.

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extraction and characterization of “batuan” garcinia …...their dietary proteins. “batuan”,...

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