Effect of acid-catalyzed methanolysis on the bioactive components of rice bran oil - ITS Repository

Effect

of

acid-catalyzed

methanolysis

on

the

bioactive

components

of

rice

bran

oil

Yi-Hsu

Ju

a

,

Siti

Zullaikah

b,

*

a_{DepartmentofChemicalEngineering,NationalTaiwanUniversityofScienceandTechnology,Taipei106-07,Taiwan}

b_{DepartmentofChemicalEngineering,InstitutTeknologiSepuluhNopember,KampusITSKeputihSukolilo,Surabaya60111,Indonesia}

1. Introduction

Biodieselis becomingan attractivealternativefuelfor petro diesel.Itsnon-toxic,biodegradable,haslowemissionprofiles,and thereforeisenvironmentallybenign[1,2].However,biodieselcost isstillsignificantlyhigherthanthatofpetrodiesel.Eventhough biodiesel can be prepared from a variety of sources including vegetableoils,animal fats,andwastegreases,mostcommercial biodieselproductionsuserefinededibleoil.About60–75%ofthe biodieselproductioncostisfromtherawmaterial[3–5].

Ricebranoil(RBO)isoneofthemostnutritiousoilbecauseofits favorable fatty acid composition and a unique combination of naturally occurring biologically active and antioxidant compo-nents.However,crudeRBOhasbeendifficulttorefinebecauseof itshighfreefattyacids(FFAs),acetone-insolublecontentanddark color[6].Sincericebranisalowvaluebyproductofricemilling withlipid contentof 15–20%, RBO is a potentialcandidate for biodieselproduction.

Biodiesel obtainedfrom ricebran oil yieldslarge amountof byproductsolid(defattedricebran)andliquid(biodieselresidue). Theutilizationofdefattedricebranfortheproductionofproteins

[7], carbohydrates [8], phytochemicals [9], and isolation and purificationofvalueaddednutraceuticalfrombiodieselresidue,

such assqualene, tocols, phytosterols, and

g

-oryzanol [10] are attractiveoptionstolowerfurtherthecostofbiodiesel.

Bioactivecomponentsaregainingimportanceinfood, pharma-ceuticals, and cosmetics due to its antioxidant activity. The antioxidantactivityof

g

-oryzanolfromricebranwasevaluatedat mildconditionunderairflow.

g

-Oryzanolfromricebranevidenced significantantioxidantactivitywhenitmixedwithlinolenicacidina certainmolarratio[11].

g

-Oryzanolalsohasapotentialapplication forthestabilizationoflipidicrawmaterial[12].

g

-Oryzanol,which degradesatalowerratethan

a

-tocopherol,isapromisingantioxidant forhightemperatureapplication[13].Phytosterolfattyacylesters havebeengrantedasararehealthclaimbytheUSFoodandDrug Administration(FDA)forloweringbothLDL-cholesterollevelsandthe riskofheartdisease. Squaleneand tocols,its applicationinfood, pharmaceuticals,andcosmeticsiswellknown.However,thereisstill veryfewinformationregardinghowmuchthosebioactive compo-nentsaredegraded/retainedandoxidationproductsproducedduring theproductionofbiodieselviaacidcatalysis.

Ourpreviousworkreportedthatintheisolationof

g

-oryzanol fromRBO,abyproductliquidphase(LP2)wasgenerated[14].This liquidphasecomprisesFFAs,acylglycerides(AGs),andbioactive componentssuchas

g

-oryzanol,phytosterols,squalene, tocopher-olsand tocotrienols.CompositionsofLP2similarassoybeanoil deodorizerdistillate(SODD),abyproductofthedeodorizationstep during the refining of soybean oil which is most bioactive components are concentrated. Therefore, they can be a good rawmaterialfortheproductionoftocopherols,phytosterolsand

ARTICLE INFO

Articlehistory: Received24July2012

Receivedinrevisedform6March2013 Accepted17March2013

Availableonline11May2013

Keywords: Biodiesel

Bioactivecomponents RBO

Acidmethanolysis Oxidation

ABSTRACT

Thechangeinbioactivecomponentsinoilderivedfromricebranoilafteracid-catalyzedmethanolysis wasinvestigatedinthisstudy.Theeffectsofcatalystamount,molarratioofmethanoltooil,reaction time,andnitrogenpurgingonacid-catalyzedmethanolysiswereinvestigatedtofind theoptimum conditioninconvertingallfreefatty acidsandacylglyceridesintobiodiesel withminimumlossof bioactivecomponents.

Acid-catalyzedesterificationat608Cusing5wt%ofsulphuricacidasthecatalystcanconvertallfree fattyacids(initialcontent=59.19%)andacylglycerides(initialcontent=19.31%)intofattyacidmethyl estersin5hwithamolarratioofmethanoltooil=40.Afterthereaction,thelossesofsqualene,a -tocopherol,g-tocotrienol,campesterol,stigmasterol,b-sitosterol,andg-oryzanolare50.07%,18.06%, 63.09%,21.68%,28.74%,25.42%,and35.43%,respectively.Whennitrogenpurgingwasappliedduringthe reaction,thelossesoftheaforementionedbioactivecomponentsbecame42.54%,0.00%,43.47%,23.47%, 26.66%,24.07%,and29.76%,respectively.Inaddition,oxidationproductswerenotdetectedbyGC–MS duringacid-catalyzedmethanolysis.Fromthepresentinvestigation,lossofbioactivecomponentscanbe mitigatedbycarriedoutthereactionundernitrogenatmosphere.

ß2013TaiwanInstituteofChemicalEngineers.PublishedbyElsevierB.V.Allrightsreserved.

*Correspondingauthor.Tel.:+62315946240;fax:+62315999282. E-mailaddress:szulle@chem-eng.its.ac.id(S.Zullaikah).

ContentslistsavailableatSciVerseScienceDirect

Journal

of

the

Taiwan

Institute

of

Chemical

Engineers

j o urn a lhom e pa g e :ww w . e l se v i e r. c om / l oca t e / j t i ce

1876-1070/$–seefrontmatterß2013TaiwanInstituteofChemicalEngineers.PublishedbyElsevierB.V.Allrightsreserved.

fattyacids[15].CrudeRBOusuallycomprisessaponifiablelipids (90–96%)andunsaponifiablelipids(4–5%)[16–18].Sincebioactive componentssuchasphytosterols,tocols,squaleneand

g

-oryzanol are unsaponifiable lipids, their change after acid-catalyzed methanolysisweredifficulttoidentifyusingHTGCduetotheir contentsarelow. Isolationof

g

-oryzanolfromresidueobtained duringtheproduction ofbiodieselfromRBOneededaseriesof stepsonlytoincrease

g

-oryzanolcontentto16%.Afterapplying solvent extraction, modified soxhlet extraction, and finally applyingsilicagelcolumnchromatography,theycanincrease

g

-oryzanolto83%[10].Probably,thepossibility ofre-isolationof bioactive components after acid-catalyzed methanolysis was higherifLP2usedasstartingmaterial,becauseafterdistillation the composition of residue was only bioactive components. Therefore,inthis study,LP2usedasa modeltoinvestigatethe effect of acid-catalyzed methanolysis on the loss of bioactive componentsonoil(LP2)derivedfromcrudeRBO.

2. Materialsandmethods

2.1. Materials

Rice bran was donated by a rice mill located in Kaoshiung County, Taiwan. Analytical thin layer chromatography (TLC) aluminum plate (20cm20cm250

m

m), silica gel 60F254,

waspurchasedfromMerck(Darmstadt,Germany).Advantecfilter paperwasobtainedfromToyoRoshi KaishaLtd.(Tokyo,Japan). Standardsofmethyllinoleate,squalene,oleicacid,

a

-tocopherol,

d

-tocopherol,

g

-tocopherol,

g

-tocotrienol, monooleylglycerol, dioleylglycerol, and trioleylglycerol were obtained from Sigma Chemicals Company(St.Louis,MO). Stigmasterolandsulphuric acid(H2SO4)weresuppliedbyAcrossOrganics(USA).

g

-Oryzanol

andpracticalgrade

b

-sitosterolwasobtainedfromWako(Japan) andMPBiomedicals,LLC(Aurora,OH),respectively.Allsolvents andreagentswereeitherofHPLCgradeorofanalyticalreagent gradeandwereobtainedfromcommercialsources.

2.2. Acid-catalyzedmethanolysisofLP2

Acid-catalyzedmethanolysiswascarriedoutina50ml screw-cappedvesselwith1gofLP2at608Candatmosphericpressure withmagneticstirrerat600rpm.Nitrogenpurgingwascarriedout inthescrew-cappedvesselbefore thereactionwasstarted.The molarratiosofmethanoltoLP2usedwere10and40.Fattyacid compositionofLP2wasfoundtobesimilartothatofRBOwith palmiticacid(C16:0,17.70%),stearicacid(C18:0,1.99%),oleicacid (C18:1,47.30%),andlinoleicacid(C18:2,31.26%)asthemajorfatty acids.Theaveragemolecularweightsoffattyacidandfattyacid methyl ester of LP2 were estimated to be 276.40 and 290.40, respectively.Sulphuricacid(0.01and0.05gor1and5wt%ofoil) wasdissolvedinmethanolandstirredat600rpmfor30minbefore added into thereaction vessel. At theend of thereaction, the solution was cooled to ambient temperature and 10ml of n -hexanewasadded.Themixturewasstirredat600rpmfor10min, thentransferredtoa separatorfunneland letaloneforanother 10min to separate water phase and n-hexane phase. The extractionstepswererepeateduntilnolipidswerefoundinthe waterphase.Alln-hexanephaseswerepooledandwashedwith 10ml508Cdistilledwaterunderstirringat100rpmfor10min. Afterwards,themixturewastransferredtoaseparatorfunneland letaloneforanother10mintoseparatewaterphaseandn-hexane phase.Theextractionstepswererepeateduntilnocatalystswere foundinthen-hexanephase.Afterwashing,n-hexanephasewas driedovermagnesiumsulfateandsubjectedtoarotaryevaporator. Therecoveredproductswerepurgedwithnitrogenandanalyzed byTLC,HTGCandUV-VISspectrophotometer.

2.3. AnalysisbyUV-spectrophotometer

g

-Oryzanol content in the sample was determined spectro-photometrically(V-550UV-VISSpectrophotometer,JASCO,Japan). Thesamplewasdissolvedinhotn-hexane(60₈C)ina1cmquartz cell and absorptions at multi wavelength (200–400nm) were measured[19,20].Theanalysiswasperformedunder100nm/min, bandwidth=1nm,and data pitch=1nm. The calibrationcurve wasobtainedwithpure

g

-oryzanolinaconcentrationrangeof4– 56mg/l.InthisconcentrationrangetheabsorptionobeysBeer’s law and thecalibrationcurve at

l

_{316 is a straight line passing}

through the origin (R2_{=0.9954) and the slope represents the}

specificextinctioncoefficient[E(316nm)=(36.840.28)g/l/cm].

2.4. AnalysisbyTLCandHTGC

CompoundsineachfractionwereidentifiedbyTLCandHTGC usingauthenticstandards.TLCplatesweredevelopedinamixture of n-hexane/ethylacetate/acetic acid(90/10/1, v/v/v). After air-drying, spots on each plate were visualized by exposing the chromatogram in iodine vapor. FASEs, phytosterols, and

g

-oryzanolspots weredetected byspraying withafreshsolution of50mgferricchlorideina mixtureof90mlwater,5mlacetic acid,and5mlsulphuricacid.Afterheatingat1008Cfor3–5min,it was indicated by a red-violet color. The contents of squalene, tocopherols,tocotrienols,phytosterols,FFAs,andacylglycerolsin each fraction were determined by HTGC. External standard calibration curves were obtained by using 0.02–20mg pure standard. In this concentration range, each external calibration curve is a straight line passing through the origin (R2_>0.99).

ChromatographicanalyseswereperformedonaTLCplateanda Shimadzu GC-17A (Kyoto, Japan) gas chromatograph equipped withaflameionizationdetector.Separationswerecarriedoutona DB-5HT (5(-phenyl) – methylpolysiloxane non-polar column (15m0.32mmi.d.;AgilentTech.PaloAlto,CA).The tempera-turesofinjectoranddetectorwerebothsetat3708C.Temperature ofthecolumnwasstartedat808C,increasedto3658Cat158C/ min,andmaintainedat3658Cfor8min.Thesplitratiowas1:50 andthecarriergaswasnitrogen.Twentymilligramssamplewas dissolvedin 1ml ethyl acetate, heateduntil clearsolutionwas observed,and1

m

lsamplewasinjectedintotheHTGC.

2.5. Analysisbylowtemperaturegaschromatography(LTGC)

The content of FAMEs, which is the same as fatty acid composition, in each fraction was determined by LTGC. External standard calibration curves were obtained by using 0.02–20mg pure standard.Chromatographic analysis of fatty acid composition was performed on a China Chromatography

8700F (Taiwan) gas chromatograph equipped with a flame ionization detector.Separations were carried out onan Rtx-233010%cyanopropylphenyl-90%biscyanopropylpolysiloxane column (30m0.25mm i.d.; Restek, Bellefonte, PA). The temperatures of injector and detector were both set at 2508C.Temperatureofthe columnwasstartedat 1508Cand heldfor2min,increasedto2458Cat58C/min,andmaintained at 245₈Cfor14min. Capillary headpressure, purge velocity, and vent velocity were 150kg/cm2_{, 2–3ml/min, and100ml/}

min,respectively. Twentymilligramssamplewasdissolvedin 1mln-hexane, heated untilclear solutionwasobserved, and 1

m

lsamplewasinjectedintotheGC.

2.6. AnalysisbyGC–MS

The fragmentations of compounds were determined by a

selective detector, and a 5990A MS Chemstation (HP-UX) (Hewlett-Packard,NorthHollywood, CA,USA).Separationswere carried out on a DB-5HT (5(-phenyl)-methylpolysiloxane non-polarcolumnusingthesametemperatureprogramasdescribedin HTGCanalysis.Allmassspectrawereacquiredusingtheelectron impact(EI)modeat70eV,withanioncurrentof50

m

A,andanion sourcetemperatureof2008C.TheMSscannedintherangeofm/z

10–490at2.25scan/s.

2.7. Statisticalanalysis

Reliabilityoftheresultswascheckedbystatisticalanalysis.The excelspreadsheetprogramwasusedtocalculatemean,standard deviation,andvariance.Thesignificancelevelwasp<0.05.The

experimental datarepresent meanstandarddeviationofthree independentexperiments.

3. Resultsanddiscussion

3.1. CompositionofLP2

LP2wascollectedduringisolationof

g

-oryzanolfromRBO.The compositionofLP2usedinthisstudy(Table1)showsthatFFAsis the major component (ca. 59.19%). Acylglycerides (ca. 19.31%) contain mainly diacylglycerides (ca. 16.43%). Bioactive compo-nents(ca.21.5%)comprisedsqualene(ca.1.06%),

a

-tocopherol(ca.

0.43%),

g

-tocotrienol (ca. 0.60%), phytosterols (campesterol, stigmasterol and

b

-sitosterol) (ca. 8.24%), and

g

-oryzanol (ca.

9.11%).

The composition of LP2 is similar to that of soybean oil deodorizerdistillate(SODD) whichcontains about45.38%FFAs, 23.30%AGs,and17.50%bioactivecomponents[15]andcanbeused asfeedstockforbiodieselproduction.

3.2. Effectofcatalystamount

The acid-catalyzed methanolysis does not enjoy the same popularity in commercial biodiesel production as the base-catalyzed methanolysis because acid-catalyzed methanolysis is slower.However,itholdsanimportantadvantageoverthe base-catalyzed one in that its performance is not affected by the presence of FFAs in the reactant. In fact, acid catalyst can simultaneouslycatalyzebothesterificationandtransesterification. Thus,agreat-advantagewithacid-catalyzedmethanolysisisthatit candirectlyproducebiodieselfromlow-costfeedstocks,generally associatedwithhighFFAscontent.Typically,H2SO4isusedasthe

catalystintheamountof1–5wt%[3].Therefore,1and5wt%of H2SO4wereusedinthisworktostudytheeffectofcatalystamount

onthelossofbioactivecomponentsafteracid-catalyzed metha-nolysisandtheresultsareshowninFig.1.

Almost all bioactive components contents decrease with increasingcatalystamount.However,thedifferenceofphytosterol contentbetween using 1 and 5wt% ofH2SO4 is not significant

(p>0.05). Acid-catalyzed methanolysis of LP2 using 5wt% of

H2SO4 canconvertedallFFAs and AGsintoFAMEsin 5hwhile

unreactedAGs(ca.3%)wasdetectedwhen1wt%H2SO4wasused.

ThisisbecausethereactionrateofAGswasmuchslowerthanthat of FFAs in acid catalyzed reaction. Increasing the amount of catalyst did increase the reaction rate of AGs; however more bioactivecomponentswillbedestroyed.

Fig. 2 shows the effect of H2SO4 amount on bioactive

components recovery. By increasing H2SO4 amount from 1 to

Table1

CompositionofLP2.

Compound wt%

FFAs 59.190.19

MGs 1.880.31

DGs 16.430.61

TGs 1.000.08

Squalene 1.060.03

a-Tocopherol 0.430.06

g-Tocotrienol 0.600.05

Campesterol 1.750.05

Stigmasterol 1.870.08

b-Sitosterol 4.620.09

Oryzanol 9.110.14

Othersa

2.061.01

a_{Tocols,phytosterolsandfattyacidsterylesters(FASEs).}

Fig.1.Effectofcatalystamountonthecontentofbioactivecomponents.Reaction conditions: methanol/LP2=40/1 (mol/mol), T=608C, stirrer speed=600rpm, reactiontime=5handunderairatmosphere.

0 10 20 30 40 50 60 70 80 90 100

Squalene

Alfa-Tocopherol Gama-Tocotrienol

Campesterol Stigmasterol

Beta-Sitosterol_Gama-Oryza nol

Reco

v

e

ry

(

%

)

1 wt% of H2SO4

5 wt% of H2SO4

5wt%, squalene recovery decreased from 84 to 50% and

g

-tocotrienolfrom49 to37%,whiletherecoveryof

a

-tocopherol increasedfrom66%to81%.Therecoveriesofphytosteroland

g

-oryzanoldecreasedslightly.

3.3. EffectofmolarratioofmethanoltoLP2

LP2containsabout20%AGs.Itisknownthatesterformation increased with increasing molar ratio of methanol to oil [3]. Therefore,inthisstudyhighmethanoltooilmolarratiosof10and 40 and its effect on the content and recovery of bioactive componentsisshowninTable2.Itcanbeseenthatthecontent andrecoveryofbioactivecomponentsdecreaseswithincreasing molarratioofmethanoltooil.Table2alsoshowsthatdegradation ofbioactivecomponentsincreasedasmoremethanolwasusedin thereaction.However,atalowermolarratioofmethanoltooilof 10,allFFAsandAGscannotbeconvertedintobiodiesel(datanot shown).As this ratio is raised to40, all FFAs and AGs can be completelyreactedwithin5h.

3.4. Effectofreactiontime

Ashorterreactiontimeispreferableduetolesslossofbioactive components.However,withtooshortreactiontime,AGscannotbe completelyremovedevenusinghigheramountofcatalyst(5wt%) andhighermolarratioofmethanoltooil(40).Inthiswork,the completeconversionofFFAsandAGsintoBDandhighcontentand recoveryofbioactivecomponentsintheresiduewerechosenas criteriafordeterminingtheoptimalreactiontime.

Theeffectsofreactiontimeonthecontentsandrecoveriesof bioactive components are given in Table 3. The contents of squalene,

g

-tocotrienol,and

g

-oryzanoldecreasedwhilethoseof

a

-tocopherolandphytosterols(campestreol,stigmasterol,and

b

-sitosterol)increasedslightlyasthereactionproceeded.Table3also shows thatthe contentsof mostbioactive componentsarenot significantly different (p>0.05) when reaction time increased

from1 to 3hand from 3 to 5h. However, the differencesare significantfrom1to5h(p<0.05).

Asreactionproceededfrom1to5h,recoveriesofsqualene,

g

-tocotrienol,and

g

-oryzanoldecreasedwhilethoseof

a

-tocopherol and phytosterols (campestreol, stigmasterol, and

b

-sitosterol) increasedslightly.Phytosterolsincreasedslightlydueto degrada-tionof

g

-oryzanol.

g

-Oryzanolisamixturecontainingferulate (4-hydroxy-3-methoxycinnamic acid) esters of triterpene alcohols andplantsterols.Thereare5majorcomponentsof

g

-oryzanolin RBOwhichwereidentifiedasferulateestersofcycloartenol, 24-methylenecycloartanol, campesterol,

b

-sitosterol,and cycloarta-nol.Degradationof

g

-oryzanolintheacid-catalyzedmethanolysis resultedin phytosterols.Therefore, thecontentand recoveryof phytosterolsincreasedslightlywithreactiontime.Therecoveryof

a

-tocopherolwashigherthanthatof

g

-tocotrienolinthereaction due to their structure difference.

g

-Tocotrienol contains more double bonds in their side chain than their counterpart

a

-tocopherol.Thedegradationrateof

g

-tocotrienolis higherthan that of squalene due tothe fact that

g

-tocotrienol is a potent antioxidant.Hence,therecoveryof

g

-tocotrienolisthelowestone. In addition, the recovery of

g

-oryzanol is higher than that of squalene.

3.5. Effectofnitrogen

Mostacid-catalyzedmethanolysisofoilreportedinliteratures were conducted under air atmosphere at high temperature (608C)and withmedium tohighamount ofH2SO4 (2wt%) [3,4].Dependingonthecompositionofoil,reactioncantakeupto 8htoreach 98% conversion[5].Under suchconditions,loss of bioactivecomponentsisinevitable.Inthisstudy,acid-catalyzed methanolysiswascarriedoutundernitrogenatmosphereandits effectsonthelossofbioactivecomponentsaregiveninTable4.As canbeseen,bothcontentandrecoveryofbioactivecomponents increasedwhen thereaction wascarried undernitrogen atmo-sphere.Theeffectismorepronouncedespeciallyfortocopherols. After acid-catalyzed methanolysis, the reaction product (crude biodiesel)usuallyisbrownish[5,10].Thesamereactioncarriedout undernitrogenatmosphereyieldyellowishproduct.

Compositions ofcrude biodieselobtainedwere biodieseland bioactivecomponents.Isolationofbioactivecomponentsfromcrude biodieselcanbedonesimplybyvacuumdistillation.Morethan95% low-boiling pointcomponents, suchasFFAsand biodiesel, were obtainedasthedistillate[10].Therefore,theresidueobtainedwas mainlybioactivecomponents.Theycanbeagoodrawmaterialfor theproductionofsqualene,tocols,phytosterols,and

g

-oryzanol.

3.6. Oxidationandhydrolysisproducts

g

-OryzanolandphytosterolscontentinLP2arehigherthanthe otherbioactivecomponentsascanbeseeninTable1.Therefore,both compenentswereinvestigatedfortheirdegradationproductsafter theyweresubjectedtoacid-catalyzedmethanolysis.Inthemodel experiment,

g

-oryzanol (0.1g) was subjected to acid-catalyzed

Table2

EffectofmolarratioofmethanoltoLP2onlossofbioactivecomponents.a

Compound Methanol/LP2=10/1 Methanol/LP2=40/1

Content(%) Recovery(%) Content(%) Recovery(%)

Squalene 0.630.02 90.903.49 0.340.02 49.931.78

a-Tocopherol 0.290.03 100.0010.00 0.240.00 81.943.28

g-Tocotrienol 0.110.01 59.312.92 0.070.00 36.911.66

Campesterol 0.960.05 80.596.32 0.920.07 78.327.72 Stigmasterol 1.070.02 84.183.32 0.900.04 71.263.95

b-Sitosterol 2.620.15 82.806.84 2.330.09 74.684.81

g-Oryzanol 7.640.12 70.072.79 6.970.31 64.571.94

a

Reactionconditions:H2SO4=5wt%,T=608C,stirrerspeed=600rpm,reaction

time=5h.

Table3

Effectofreactiontimeonthecontentandrecoveryofbioactivecomponents.a

Compound Content(%) Recovery(%)

1h 3h 5h 1h 3h 5h

Squalene 0.460.04 0.400.02 0.340.02 69.665.87 57.782.23 49.931.78

a-Tocopherol 0.130.01 0.240.02 0.240.00 46.693.65 81.827.39 81.943.28

g-Tocotrienol 0.090.00 0.080.01 0.070.00 50.282.06 43.494.53 36.911.66

Campesterol 0.740.02 0.860.07 0.920.07 64.071.47 72.736.38 78.327.72

Stigmasterol 0.720.04 0.820.05 0.900.04 58.843.28 65.134.79 71.263.95

b-Sitosterol 1.890.12 2.210.15 2.330.09 61.853.78 70.455.22 74.684.81

g-Oryzanol 7.190.20 6.790.06 6.970.31 68.332.17 62.900.09 64.571.94

a

methanolysis (reaction conditions were the same as LP2) to determineitsdegradationproductsbyGC–MS.Oxidationproducts werenotdetected, whereascampesteroland

b

-sitosterolwerefound as hydrolysis products. Meanwhile, oxidation and hydrolysis productswerenotdetectedbyGC–MSwhenphytosterols(0.1g) wassubjectedtothesameacid-catalyzedmethanolysis.WhenLP2 wassubjectedtothesameacid-catalyzedmethanolysis,nooxidation productsweredetected.Therefore,degradationproductsofminor componentsduringacid-catalyzedmethanolysisweretheproducts ofhydrolysis.

4. Conclusion

Lossofbioactivecomponentsinacid-catalyzedmethanolysisof vegetableoilisinevitable.Increasingtheamountofcatalystand molarratioofmethanoltooilcanconvertallFFAsandAGsinto FAMEs;however,lossofbioactivecomponentsalsoincreases.Loss of bioactive components can be mitigated by carried out the reactionundernitrogenatmosphere.

Acknowledgments

FinancialsupportsfromTaiwan’sMinistryofEducation,Amia EnterpriseCo.Ltd.andNationalTaiwanUniversityofScienceand Technologythroughproject94DI062aregreatlyappreciated.

References

[1]ChenYH,HuangYH,LinRH,ShangNC,ChangCY,ChangCC,etal.Biodiesel productioninarotatingpackedbedusingK/g-Al2O3solidcatalyst.JTaiwan

InstChemEng2011;42:937–44.

[2]ShangNC,LiuRZ,ChenYH,ChangCY,LinRH.Characterizationoffattyacid methylestersinbiodieselusinghigh-performanceliquidchromatography.J TaiwanInstChemEng2012;43:353–9.

[3]LotereE,LiuY,LopezDE,SuwannakarnK,BruceDA,GoodwinJG.Synthesisof biodieselviaacidcatalysis.IndEngChemRes2005;44:5353–63.

[4]JuYH,ValiSR.Ricebranoilasapotentialresourceforbiodiesel.JSciIndRes 2005;64:866–82.

[5]ZullaikahS,LaiCC,ValiSR,JuYH.Atwo-stepacid-catalyzedprocessforthe production of biodiesel from rice bran oil. Bioresour Technol 2005;96: 1889–96.

[6]GhoshM.Reviewonrecenttrendsinricebranoilprocessing.JAmOilChem Soc2007;84:315–24.

[7]ZhangHJ,ZhangJ,WangL,GuoXN.Preparationandfunctionalpropertiesof rice bran proteinsfrom heat-stabilized defatted rice bran. Food Res Int 2012;47:359–63.

[8]FabianC,AyucitraA,IsmadjiS,JuYH.Isolationandcharacterizationofstarch fromdefattedricebran.JTaiwanInstChemEng2011;42:86–91.

[9]DeviRR,ArumughanC.Phytochemicalcharacterizationofdefattedricebran andoptimizationofaprocessfortheirextractionandenrichment.Bioresour Technol2007;98:3037–43.

[10]KasimNS,ChenH,JuYH.Recoveryofg-oryzanolfrombiodieselresidue.JChin InstChemEng2007;38:229–34.

[11]XuZ,GodberJS.Antioxidantactivitiesofmajorcomponentsofg-oryzanol fromricebranusingalinoleicacidmodel.JAmOilChemSoc2001;78:645–9.

[12]JulianoC,CossoM,AlamanniMC,PiuL.Antioxidantactivityofg-oryzanol: mechanismofactionanditseffectonoxidativestabilityofpharmaceutical oils.IntJPharm2005;299:146–54.

[13]NystromL,AchreniusT,LampiAM,MoreauRA,PiironenV.Acomparisonof theantioxidantpropertiesofsterylferulateswithtocopherolathigh tem-peratures.FoodChem2007;101:947–54.

[14]ZullaikahS,MelwitaE,JuYH.Isolationoforyzanolfromcrudericebranoil. BioresourTechnol2009;100:299–302.

[15]GunawanS,KasimNS,JuYH.Separationandpurificationofsqualenefrom soybeanoildeodorizerdistillate.SepPurifTechnol2008;60:128–35.

[16]DeDeckerEAM,KorverO.Minorconstituentofricebranoilasfunctional foods.NutrRev1996;54:S120–6.

[17]GhoshM.Reviewonrecenttrendsinricebranoilprocessing.JAmOilChem Soc2007;84:315–24.

[18]VanHoedV,DepaemelaereG,AyalaJV,SantiwattanaP,VerheR,DeGreytW. Influenceofchemicalrefiningonthemajorandminorcomponentsofricebran oil.JAmOilChemSoc2006;83:315–21.

[19]KrishnaAGG,KhatoonS,ShielaPM,SarmandalCV,IndiraTN,MishraA.Effect ofrefiningofcrudericebranoilontheretentionoforyzanolintherefinedoil.J AmOilChemSoc2001;78:127–31.

[20]BucciR,MagriAD,MagriAL,MariniF.Comparisonofthree spectrophotomet-ricmethodsforthedeterminationofg-oryzanolinricebranoil.AnalBioanal Chem2003;375:1254–9.

Table4

Effectofnitrogenonlossofbioactivecomponents.a

Compound Underair UnderN2 Underair UnderN2

Content(%) Recovery(%)

Squalene 0.34a _0.39a _49.93c _57.46d

a-Tocopherol 0.24a

0.31a

81.94c

106.29d

g-Tocotrienol 0.07a

0.10b

36.91 56.60d

Campesterol 0.92a

0.96a

78.32c

76.53c

Stigmasterol 0.90a

0.92a

71.26c

73.34c

b-Sitosterol 2.33a _2.36a _74.58c _75.93c

g-Oryzanol 6.97a _7.51a _64.57c _70.24c

a

Reactionconditions:methanol/LP2=40/1(mol/mol),H2SO4=5wt%,T=608C,