Original article

Comparison of rice bran oil and wax yields; a systematic approach

to wax ester analysis

Junghong Kim* & James Samuel Godber

Department of Food Science, Louisiana State University, South Campus Drive, Baton Rouge, LA 70803, USA

(Received 20 March 2013; Accepted in revised form 19 July 2013)

Summary Rice bran oil (RBO) comprises 2–4% of rice bran wax (RBW) which contains wax esters (WE), hydrocar-

bons and other minor constituents, and there is still a lack of information on the RBW yields by various

extraction methods and the WE contents in RBW. In this study, the yields of RBO and RBW extracted

with Soxhlet and microwave-assisted extraction (MAE) were compared with establish suitable RBW prep-

aration methods. HPLC methods were also developed to determine the WE contents in RBW. MAE with

hexane was the most efficient method for higher RBW yields as well as higher WE contents. The HPLC

analysis proved that around half of the refined RBW consists of WE. The results established an efficient

procedure for RBW preparation, which includes MAE with hexane, RBO refinement, winterisation, sol-

vent fractionation and HPLC separation.

Keywords Rice bran oil extraction, wax ester analysis, wax separation.

Introduction

Crude rice bran oil (RBO) contains 2–4% waxes(Ghosh, 2007). Rice bran wax (RBW) is composed ofalkyl esters, steryl esters, hydrocarbons and othersminor constituents (Ito et al., 1983), and when saponi-fied, it generates long-chain fatty acids, long-chainfatty alcohols and phytosterols (Gunawan et al.,2006). Phytosterols from edible oils have been foundto have blood cholesterol-lowering effects on humansubjects (Vissers et al., 2000). Long-chain fatty acidsand alcohols (policosanol) have attracted interest aspotential therapeutic agents (Hargrove et al., 2004;Singh et al., 2006).

Rice bran oil has been prepared by various extrac-tion methods in the laboratory; solvent extractions withmethanol:chloroform (2:1) (Ito et al., 1983), Soxhletextraction with hexane or petroleum ether (Belavadi &Bhowmick, 1988; Vali et al., 2005), supercritical car-bon dioxide extraction (Garcia et al.,1996; Xu &Godber, 2000), microwave-assisted extraction (MAE)(Zigoneanu et al., 2008). The RBO yields varied from12% to 20% in the studies mentioned above. There isstill lack of information on which oil extraction methodwould be most suitable for RBW preparation.

Wax can be removed from the crude oil by winterisa-tion, in which the oil is placed in a cold place and the

wax crystallises (Hermann et al., 1999). Morrison &Robertson (1975) performed a winterisation study ofsunflower seed oil in hexane:acetone (15:85) solution,suggesting that more wax can be removed at a lowercrystallisation temperature and at a lower concentra-tion of solvents, and that the more solvent is used, thelonger the clouding time is. Morrison & Thomas (1976)studied solvent winterisation and refining of sunflowerseed oil (50% in hexane) and found that degummingand then winterisation removed more wax. De & Bhat-tacharyya (1998) found that high-temperature degum-ming (65–70 °C) and low-temperature dewaxing(10 °C, centrifugation at 15 000 g) improved RBOquality. Ghosh & Bandyopadhyay (2005) performedwinterisation of RBO in solvent, indicating that themaximum size (2.5–4 lm) of wax crystal is achieved at10–15 °C, and after 1 h of incubation at 10 °C, no crys-tal growth was observed. Those researches indicate onedirection for the purpose of wax recovery; high-temper-ature degumming (65–70 °C) and low-temperature crys-tallisation (10 °C) by solvent winterisation in 50%hexane. The wax separated by winterisation containsnot only wax esters but also other lipid species, so toobtain purer forms of wax esters, defatting or washingwith organic solvents is necessary, which is called sol-vent fractionation (Cousins et al., 1953). Yoon & Rhee(1982) used methyl ethyl ketone to remove the oil andthen isopropanol to crystallise the wax. Vali et al.(2005) used hexane and isopropanol to separate polar*Correspondent: Fax: +82 51 629 5842; e-mail: noidkjh@lycos.co.kr

International Journal of Food Science and Technology 2014, 49, 410–415

doi:10.1111/ijfs.12314

© 2013 The Authors. International Journal of Food Science and Technology © 2013 Institute of Food Science and Technology

410

lipids from crude RBO. Gunawan et al. (2006) per-formed acetone fractionation to separate wax estersfrom other nonpolar lipids. Several studies have beenpublished on RBW separation by winterisation and sol-vent fractionation, but there is still a lack of informa-tion on the relationship between RBO extractionmethods and RBW yields by winterisation followed bysolvent fractionation.

Wax esters (WE), the main components of crystalli-sable wax in edible vegetable oils, have been measuredby HPLC methods. Hamilton (1995) indicated thatHPLC methodology for wax analysis has been develop-ing slowly because of the need to find a suitable detec-tor, as waxes have no useful UV chromophore.Evaporative light scattering detector (ELSD) is a robustchoice especially for substances that lack UV-detectablechemical structures because it detects everything that isnot volatile and blocks the light path (Megoulas &Koupparis, 2005). Nordback & Lundberg (1999) usedan ELSD and normal-phase alumina columns for theirwax analysis using HPLC. Moreau et al. (2002) testedseveral kinds of columns and detectors for ester separa-tion, and the method using alumina column at 75 °Cwith an ELSD achieved the highest resolution. Theincreased temperature not only avoided solidification ofwax in HPLC lines and columns but also enhanced thecolumn polarity. In both studies, Aluspher alumina col-umns were used, but unfortunately, its manufacturer,Merck, no longer commercially produces the column(Personal communication to Merck representative inthe US, 2007).

The specific objectives of this study were to developefficient methods for extraction of oil and wax fromfull-fat rice bran (FFRB) and defatted rice bran(DFRB), to establish preparation methods for WEfrom RBW and to obtain more accurate analyticalmethods for WE using HPLC.

Materials and methods

Sample preparation and rice bran oil extraction

Full-fat rice bran (extruded collets) and DFRB (pow-der) were provided by Riceland Foods (Stuttgart,Arkansas). FFRB was ground with a food grinder. TheRB was filtered through a 20-mesh sieve and stored at�20 °C. For Soxhlet extraction, 20 g of RB and200 mL of hexane were used, and the extraction wasperformed at 62–63 °C for 7 h. After the solvent wasremoved, the crude RBO was weighed and stored at�20 °C. For MAE (Zigoneanu et al., 2008), 40 g ofRB and 150 mL of hexane or isopropanol were mixedin each of three extraction vessels, and the vessels wereplaced in an MAE device (Ethos E apparatus,Milestone, Monroe, CT, USA). The extraction wasconducted at 80 °C with hexane (HEX, 80) and 80 °C

or 120 °C for isopropanol (ISO, 80 or ISO, 120) for30 min. After extraction, the mixture was filtered, andthe filtered solution was evaporated to recover theextract. Hexane (100 mL) was added to the extract andheated at 60 °C until the solution became clear. It wasfiltered when still hot, and the crude RBO was obtainedafter hexane evaporation. The crude RBO was weighedafter hexane evaporation and stored at �20 °C.

Refinement of crude rice bran oil

The refining process of crude RBO was modified fromthe method of De & Bhattacharyya (1998). CrudeRBO (about 2 mL) was weighed into a clean test tube,and degummed with 1 mL of hot water, 1 mL of 0.2%(w/w) aqueous phosphoric acid, 1 mL of hot watercontaining 50 lL of 10% CaO and then 1 mL of hotwater. The degummed RBO was transferred to a cen-trifuge tube with a conical bottom and bleached with100 mg of activated carbon (100–200 mesh; Eastmanorganic chemicals, Rochester, NY, USA) and 100 mgof Fuller’s earth (100–200 mesh, Sigma, St. Louis, MO,USA). The bleached RBO was dehydrated with 100 mgof sodium sulphate. Each steps in the processes aboveaccompanied heating at 80 °C, vortexing and centrifu-gation (80 °C, 1500 g, 20 min) (Vacuum centrifugeevaporator; CentriVap Console Labconco, KansasCity, MO, USA). The dehydrated RBO was transferredto a clean test tube and dissolved in 4 mL of hexane.The hexane solution was heated and filtered through asyringe filter (0.2 lm, 25-mm PTFE, Whatman,Madstone, UK). The processed RBO was weighed afterhexane evaporation and stored at �20 °C.

Winterisation and solvent fractionation

The winterisation method was modified from themethods elsewhere (see Introduction). One gram of theprocessed RBO was mixed with the same volume ofhexane and heated at 60 °C until the solution becametransparent. It was cooled in an incubator at 20 °C,which was reduced to 18, 16, 14 and 12 °C at 1-hintervals, and left at 10 °C overnight for the comple-tion of wax crystallisation. Yellowish crude rice branwax (RBW) was recovered after the solution was cen-trifuged (10 000 g, 10 °C, 20 min). After the superna-tant (dewaxed RBO) was carefully removed, theprecipitate (Crude RBW) was dried in a centrifugalevaporator and weighed.The solvent fractionation method was modified from

Vali et al. (2005). The same volume of hexane wasadded into the crude RBW and heated at 60 °C untilthe solution became clear. The same winterisation pro-cedure as above was followed, and the supernatant wasadded to the dewaxed RBO (RBO raffinate). To the hex-ane-washed RBW, the same volume of isopropanol was

© 2013 The Authors

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Rice bran wax ester analysis J. Kim and J. S. Godber 411

added and heated at 80 °C. After winterisation and cen-trifugation, the supernatant (isopropanol-soluble frac-tion) was set aside. The isopropanol-washed RBW wasdistilled in 1 mL of isopropanol containing a droplet of10% aqueous sodium borohydride (Sigma) and filteredthrough a 0.2-lm membrane filter, while the solutionwas still hot. The filtered reddish particles were added tothe isopropanol-soluble fraction. After solvent evapora-tion from the filtered solution, the remaining materialwas washed with 1 mL of distilled water. The precipi-tate (refined RBW) was dried at 80 °C and weighed.The water used for the washing was added to the isopro-panol-soluble fraction. After the washed-out fractionwas dried in the centrifugal evaporator, 2 mL of hexanewas added to the dried matter, and the mixture washeated at 60 °C with vigorous agitation. The hexanelayer was removed into a clean test tube. This lipidextraction was carried out twice. After hexane evapora-tion, the remnant red precipitate and the extracted lipid(isopropanol-soluble RBW, IS RBW) were weighed. Allthe final products, RBO raffinate, refined RBW and ISRBW were stored at �20 °C for HPLC analysis.

Column chromatography for the preparation of waxesters-rich fractions

For low-pressure column chromatography, 30 g of sil-ica gel (60–200 mesh; EM Science, Gibbstown, NJ,USA) was packed into a glass column (2 9 25 cm)with a 300-mL solvent reservoir. Five hundred milli-grams of the processed RBO, RBO raffinate, IS RBWor refined RBW from FFRBO or DFRBO extracted bythe Soxhlet method was dissolved in 10 mL of hexane,heated until it became transparent and loaded onto theglass column. The portion of the column just above thesilica gel surface was wrapped with heating tape to pre-vent crystallisation. Slight air pressure was applied toincrease the flow rate. The WE-rich fraction was elutedwith hexane:diethyl ether (95:5, v/v). After solventevaporation, the WE-rich fraction was weighed.

HPLC system

HPLC analysis of RBO samples was performed with aWaters Delta Prep 4000 preparative chromatographysystem (Waters Corporation, Milford, MA, USA)and an ELSD (ELSD-LT2; Shimadzu, Kyoto, Japan).A 2-mL sample loop was connected with the prepara-tive HPLC system. The data were processed with aShimadzu GC solution program (Shimadzu). OnlyHPLC-grade solvents were used for all analysis. Thestandard materials for HPLC analysis, octacosane(C28), stearyl stearate (SS), stearate methyl ester(SME), cholesteryl stearate (CS) and tristearine (TS)were all purchased from Sigma-Aldrich (St. Louis,MO, USA).

Preparative HPLC

Silica gel (SilicAR TLC-7, 35–60 lm, Mallinckrodt,Phillipsburg, NJ, USA) was packed into a stainlesssteel column (5 9 20 cm). The evaporation tempera-ture for ELSD was set to 50 °C, and nitrogen wasused for solvent evaporation at the pressure of2.067 bar (30 psi). The sensitivity of ELSD was set toone (lowest), and ELSD data acquisition started 5 minafter injection. The column, the stainless steel tubingand the sample loop were wrapped with heating tapesto maintain the temperature of the solvent and the col-umn at 40 °C. One millilitre of the hexane solutioncontaining about 250 lg of the WE-rich fraction wasinjected into the preparative HPLC and eluted withhexane (Solvent A), hexane:diethyl ether (10:1, v/v,Solvent B) and chloroform (Solvent C); SolventA100% for the first 5 min, A50%B50% for the next15 min and C100% for the rest of the time. The flowrate was fixed at 40 mL min�1, and the eluent was dis-tributed to the ELSD and the fraction collector at theratio of 2.5:37.5. The WE fraction was collected andweighed after solvent evaporation. The WE was usedas the weight standard for analytical HPLC.

Analytical HPLC

Two analytical silica columns (Supelcosil LC-SI col-umn, 250 9 4.6 mm, 5 lm; Supelco, Bellefonte, PA,USA) were connected consecutively with a 5-cm stain-less steel tubing (2SI column method). The column,the stainless steel tubing and the sample loop werewrapped with heating tapes to maintain the tempera-ture of the solvent and the column at 50 °C. TheELSD sensitivity was set to three (middle), and theELSD data acquisition started 2 min after injection.The injected lipids were eluted with Hexane (SolventA) for the first 5 min and hexane:diethyl ether (20:1,v/v, Solvent B) for the next 10 min. The columns werewashed with methanol:acetone (50:50, v/v, Solvent C)for 10 min. During operation, the flow rate was fixedat 2.5 mL min�1. Each WE sample from preparativeHPLC was prepared in hexane at the concentration of1.0 mg mL�1, and 1–50 lL was injected to draw thestandard curves. The processed RBO, RBO raffinate,IS RBW or refined RBW was made into 10 mg mL�1

solutions with hexane, and 10 lL of each sample wasinjected. The amounts of WE in the samples werecalculated by the regression equations.

Data analysis

For statistical data analysis, four sets of RBO sampleswere prepared in each of four extraction conditionsfor FFRB and DFRB. The data were analysedwith SAS one-way ANOVA program (P < 0.05) (SAS

© 2013 The Authors

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Rice bran wax ester analysis J. Kim and J. S. Godber412

Institute, Cary, NC, USA), and Tukey’s Studentisedrange test was used for the comparison of any signifi-cant differences (P < 0.05).

Results and discussion

Rice bran oil yields

The crude RBO yields from DFRB were about 3.5%,and there is no published data comparable with theyield from DFRB. The crude RBO yields from FFRBwere about 20%, which is at the upper level of therange found in other studies (see Introduction)(Table S1). There was no significant difference betweenthe oil yields (~20%) from FFRB due to the extractionmethods. On the other hand, for DFRB, MAE withisopropanol at 120 °C showed significantly highercrude oil yields (4.39–4.67%) than the other methods(3.14–3.81%). Isopropanol is more polar than hexane,possibly extracting more polar substances from DFRBand resulting in higher RBO yields.

The extracted RBO was almost solid at room temper-ature, which made an increased temperature necessaryfor all experimental procedures. The degumming pro-cess of crude RBO was necessary to remove the phos-pholipids which can significantly reduce wax crystalsize, possibly due to their emulsifying ability (Morrison& Thomas, 1976), resulting in diminished filtration rates(Leibovitz & Ruckenstein, 1984). Table S1 shows theyields of the processed RBO from the crude RBO. Thecrude FFRBO samples were found to contain less mate-rial (i.e. higher yields) which could be removed duringoil processing. The crude RBO prepared by MAE withisopropanol at 80 °C presented the lowest yields of theprocessed FFRBO (67.18–68.69%) and DFRBO(31.37–36.00%), and the other methods showed higheryields with FFRB (72.36–76.30%) and DFRB (53.36–60.42%), which means that higher extraction tempera-ture with isopropanol significantly contributed to theprocessed RBO yields.

Crude rice bran wax yields

Crude RBW yields (4.81–17.57 mg g�1 RB)from FFRB were higher than those from DFRB(1.12–7.60 mg g�1 RB) (Table S1). Hexane extractedtwo or three times more crude RBW than isopropanolfrom RB. Soxhlet and MAE with hexane extractedmore crude RBW from FFRB and from DFRB,respectively. The increased extraction temperatureyielded more crude RBW from DFRB, but not fromFFRB. The processed DFRBO had a higher concen-tration of crude RBW (9.40–39.44%) than that fromthe processed FFRBO (3.67–11.24%), and the hexane-extracted RBOs showed higher crude RBW yields thanthe isopropanol-extracted RBOs. The RBO raffinate

yields, after hexane winterisations, were higher in theisopropanol-extracted RBO than the hexane extracts,and the differences were smaller in the FFRBOraffinate.The higher wax yields from DFRBO were expected

by the fact that Riceland Foods company alreadyextracted the lipid from the RB in a way that most ofthe crystallisable components in the RBO wouldremain in the RB. MAE with hexane was found moreefficient than the Soxhlet method for RBW extractionfrom DFRB. The mixture in the extraction vessel inthe MAE device can be vigorously agitated by a mag-netic stirrer. The extraction temperature can also beincreased with MAE, which accompanies higher pres-sures and more radical extraction conditions. Theagitation and higher temperature explain why MAEachieved higher wax yields from DFRBO.

Refined rice bran wax yields

The refined RBW yields from crude RBW indicatethat the solvent fractionation removed a large part ofcrude RBW and left a relatively small amount ofrefined RBW (Table S1). The refined RBW yields indi-cated that hexane is more efficient for wax extractionthan isopropanol. The refined wax yields from FFRBwere similar to those from DFRB. Soxhlet extractionand MAE with hexane yielded 1.68% and 1.54% fromthe processed FFRBO and 9.45% and 11.78% fromthe processed DFRBO, respectively, while the MAEwith isopropanol achieved <0.15% from the processedFFRBO and <1.52% from the processed DFRBO.The yields from Crude RBW were three or four timeshigher with hexane extraction than isopropanol extrac-tion from both RBs. The extraction temperature withMAE appeared to affect the refined RBW yields, butthe difference was very small, compared with the dif-ference from hexane extractions (Table S1).Vali et al. (2005) used sodium borohydride to

remove resinous matter in crude RBW during solventfractionation. They observed a reddish brown solid inthe RBW-isopropanol solution. In our study, therewas a similar occurrence just after the addition ofsodium borohydride; the reddish precipitate wasremoved by filtration and water washing. The weightpercentages of IS RBW as well as red precipitates fromthe crude RBW also appear in Table S1, which showsthat the solvent fractionation process removed a greatportion of the crude RBW. Hexane-extracted RBOyielded relatively smaller percentages of IS RBW.

Wax esters contents in the final products

The signal intensity of ELSD depends not only on thenumber of detectable molecules but also the size of themolecules (Megoulas & Koupparis, 2005). Based on

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Rice bran wax ester analysis J. Kim and J. S. Godber 413

the crystallising nature of the wax esters, even thoughthe same amount of WE samples is injected, the signalintensity may vary when the WE compositions are dif-ferent. For the reason, WE for quantification standardswere separated from the processed RBO, the RBO raff-inate, IS RBW and the refined RBW before the analyti-cal steps. All the standard curves showed differentsignal intensities at the same amount of samples anddifferent standard regression curves (data not shown).

The WE-rich fraction from the processed FFRBOor DFRBO was injected to the preparative HPLC toprepare the weight standards for analytical HPLC(Fig. S1). The chromatograms of WE-rich fractionsfrom the RBO raffinates, the IS RBWs and the refinedRBWs appeared with similar peak patterns (chroma-tograms not shown). The WE appeared as a sharppeak near 6 min in the chromatograms of the analyti-cal method (Fig. S2). The WE standards, stearyl stea-rate (SS), stearate methyl ester (SME) and cholesterylstearate (CS) were detected at the same positions. Allstepwise samples were analysed by HPLC (Fig. S3).The WE standards were analysed through the 2SImethod, which were found efficient for total WE mea-surements.

The WE contents in the processed RBO and thefinal products are shown in Table S2. The WE con-tents in the hexane-extracted processed FFRBOs andDFRBOs were almost two and three times larger,respectively, than the isopropanol-extracted processedRBOs. This trend was also shown in the WE contentsof the other final products. MAE with hexaneappeared more efficient in RBWE extraction thanSoxhlet. The WE contents in the RBO raffinate sam-ples reached 10% and 11% in hexane-extracted FFR-BOs and DFRBOs, respectively. Comparing the WEcontent in the processed RBO and the RBO raffinateextracted with hexane, only about 10% of WE wasremoved by winterisation and solvent fractionationfrom the processed FFRBO, and 34–37% of WE fromthe processed DFRBO. In the case of MAE-extractedRBO, about three and 15% of WE was removed fromthe processed FFRBO and DFRBO. IS RBWs alsohad significant WE contents, which were higher inMAE with hexane. Nearly half of the refined RBWwas found to be WE, and the WE contents in therefined RBW samples from FFRB and DFRB werenot significantly different (P = 0.12).

Wax esters distributions in the final products

The WE distributions among the final products areshown in Table S3. More than 80% of the total WEwas contained in the RBO raffinate, except for theDFRBO raffinates that had <65% of WE contents. ISRBWs contained <5% of the total WE. The refinedDFRBWs were found to have significantly larger con-

tents of WE than the refined FFRBWs, and especiallythe refined DFRBW from hexane-extraction methodscontained more than 30% of total WE. The WE con-tents, <1.45% in the refined FFRBW and <10.42% inthe refined DFRBW from isopropanol extractionmethods, were much lower than those in the refinedRBW from hexane-extraction methods. The WE con-tents in the final products from MAE with hexane andSoxhlet extraction did not show significant differencesexcept those in IS RBW.In the HPLC analysis, it is evident that not all the

crystallisable material in RBO is WE and not all theWEs are crystallisable. RBW and RBWE are definitelydifferent entities as much as they are different materi-als. The WE contents in the refined RBW samplesappeared to be similar, which indicates that the WEand the acylglycerides in the refined RBW may serveas a seed for RBO crystallisation. They can be called‘crystallisable wax’ at room or even higher tempera-ture. The WE in the RBO raffinate can be considered‘noncrystallisable’, which are still crystallisable atroom or lower temperature (<10 °C).

Conclusion

Hexane was more efficient for wax extraction than iso-propanol, and MAE with hexane rather than conven-tional Soxhlet extraction method achieved higher WEcontents in the processed RBO and in the refinedRBW. The higher extraction temperature of MAEwith isopropanol increased the yields of DFRBO, butit did not improve the FFRBO yield and the RBWyields. MAE with hexane is the most efficient methodfor generation of higher amounts of RBW as well ashigher WE contents. DFRB contains significantamounts of RBW, and the refined RBW contents werenot significantly different from FFRB. The HPLCanalysis proved that not all RBW is WE and not allRBWE is crystallisable.The results established an efficient procedure of

RBW preparation, which includes MAE with hexane,refinement, winterisation, solvent fractionation andHPLC separation. This method can be used not onlyfor RBW analysis but also for analysis of WE fromother edible oils. The next studies will focus on theWE composition analysis using GC or GC-MS andthe potential applications of the components ofRBWE in biomedical fields.

Acknowledgments

This dissertation research was supported by LouisianaState University Agricultural Center, Baton Rouge,Louisiana, USA. We also deeply appreciate the gener-ous donation of rice bran samples from RicelandFoods, Stuttgart, Arkansas, USA.

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Supporting Information

Additional Supporting Information may be found inthe online version of this article:Figure S1. Chromatograms of preparative HPLC.Figure S2. Chromatograms of analytical HPLC with

injections of FFRBO (upper), the standard mixture(middle) and individual five standards (bottom).Figure S3. Chromatograms of analytical HPLC with

injections of processed rice bran oil (RBO), RBO raffi-nate and refined rice bran wax (RBW) from full-fatrice bran (FFRB) and DFRB.Table S1. Stepwise yieldsa of rice bran oil (RBO),

Crude RBW and Refined RBW.Table S2. Wax esters (WE) contentsa (%) in the pro-

cessed rice bran oil (RBO) and final products.Table S3. Wax esters (WE) distribution dataa (%) in

the final products.

© 2013 The Authors

International Journal of Food Science and Technology © 2013 Institute of Food Science and Technology

International Journal of Food Science and Technology 2014

Rice bran wax ester analysis J. Kim and J. S. Godber 415